methods of modulating the alkaloid content and the content of a tobacco-specific nitrosamine (tsna),

专利摘要:
The present invention provides a method of modulating the alkaloid content of a plant (e.g., a tobacco plant), the method comprising modifying said plant by modulating the activity or expression of at least one Nic1 ERF gene. The present invention also provides for the use of at least one Nic1 ERF gene for modulating the alkaloid content of a plant, as well as tobacco cells, plants, plant propagating materials, harvested leaves, processed tobacco or tobacco products obtainable according to with the invention.
公开号:BR112019027710A2
申请号:R112019027710-2
申请日:2018-06-21
公开日:2020-08-18
发明作者:Matthew Edward HUMPHRY；Shengming Yang；Qiulin Qin
申请人:University Of Kentucky Research Foundation；
IPC主号:

专利说明:

[0001] [0001] The present invention relates to methods of modulating the alkaloid content, for example, nicotine content of a plant or part of it. The invention also extends to methods for modulating the expression and / or activity of polypeptides encoded by genes that modulate the content of alkaloids in plants. Alternatively, the invention provides methods for modulating the expression and / or activity of genes encoding polypeptides that modulate the content of alkaloids in plants. The invention also extends to constructs that can be used to modulate polypeptides, plant cells transformed with such constructions and to the transgenic plants themselves. The invention also relates to the use of leaves harvested from such transgenic plants that have been transformed with a genetic construct to modulate the content of alkaloids and tobacco articles (e.g. combustible tobacco articles) comprising these leaves. TECHNICAL STATUS
[0002] [0002] Alkaloids are a group of natural compounds that contain mainly basic nitrogen atoms and are produced by a wide variety of organisms, including bacteria, fungi, plants and animals. Alkaloids can be classified according to the similarity of the carbon skeleton, for example, indole-, isoquinoline- and pyridine-like. Pyridine derivatives are a class of monomeric alkaloids; this class includes simple pyridine derivatives, condensed and non-condensed polycyclic pyridine derivatives and sesquiterpene and pyridine derivatives. Examples are nicotine, nornicotine, anabasine, myosmin and anatabine. Most of the known biological functions of alkaloids are related to protection. Tobacco alkaloids enhance the smoke's sensory attributes.
[0003] [0003] Nicotine occurs naturally in several varieties of plants, but is found at a higher level in the tobacco plant. It is produced in wild and cultivated species of Nicotiana and plays an important role in the defense of plants against herbivores and insects (Voelckel et al. 2001, incorporated by reference), representing - 90% of the total content of alkaloids. The remaining 10% of the alkaloids variety is mainly made up of nornicotine, anatabine, myosmin and anabasine.
[0004] [0004] The regulation of the content of alkaloids in tobacco is complex. Several factors, including genotype, environment, fertilization and agronomic practices (eg cover), affect the levels of alkaloids in tobacco plants.
[0005] [0005] In the 1930s, certain types of Cuban cigar tobacco (Nicotiana tabacum) were identified as having a very low alkaloid content, and this characteristic was introduced in the US tobacco breeding lines (Valleau 1949, incorporated by reference) . The low alkaloid attribute was subsequently incorporated into the genetic structure of the cultivar Burley 21 (B21) through several generations of backcrosses (Legg et al. 1970 incorporated here by reference).
[0006] [0006] Genetic studies using low-alkali Burley 21 (LA-B21) suggested that two non-linked loci, initially referred to as locus A and B (Legg et al. 1969 incorporated by reference), but later known as MNicl and Nic2 , contribute to nicotine levels in the tobacco leaf as a regulating locus of nicotine biosynthesis (Legg and Collins 1971 incorporated by reference; Hibi et al.1994, incorporated by reference). LA B21 has been reported to be more susceptible to insect damage, according to the role of alkaloids in plant defense. It has also been reported that isogenic strains of tobacco cured in a smokehouse with low total alkaloids (about 0.2%) have a lower yield. By means of haploid duplication of progeny F, the crossing between wild type or B21 with high alkaloid (HA-B21, AABB) x LA-B21 (aabb), Collins et al. (1974 incorporated by reference) developed two other isogenic strains (NILs) of B21 with high intermediate alkali (HI-B21, AAbb) and low intermediate alkali (LI-B21, aaBB), which were later registered as varieties in 1988 (Nielsen et al., 1988 incorporated by reference). Quasi-isogenic strains (NILs) are referred to here as Burley 21 (B21, NiclNic2), High Intermediate (HI,
[0007] [0007] Subsequent studies have shown that these two loci also control the expression of numerous genes unrelated to nicotine biosynthesis, such as stress response genes (Kidd et al. 2006 incorporated by reference). The Nic2 locus was characterized based on the identification of a large exclusion (Shoji et al. 2010 incorporated here by reference), but elucidating the location of the Nicl locus in tobacco proved to be difficult, due to the complex nature of quantitative characteristics, such as the approaches map-based cloning.
[0008] [0008] Modifying the content of alkaloids in plants (for example, tobacco) can have several commercial advantages. For example, decreasing the total content of alkaloids in plants can increase the value of that plant as a biomass resource. For example, modifying the alkaloid content may comprise reducing the alkaloid content, for example, the nicotine content of tobacco plants. Tobacco plants and products with reduced nicotine may be desirable, in view of the potential regulation of the “maximum nicotine limits”, that is, the average upper nicotine limits in tobacco products. Alternatively, increasing the content of alkaloids in plants, for example, tobacco plants, can help protect plants against insects and herbivores. There is still a need for plants with modification of the alkaloid content, for example, with modulated nicotine content, with improved commercially desirable properties and methods for producing them.
[0009] [0009] Tobacco pyridine alkaloids are precursors to tobacco-specific nitrosamines (TSNAs) that form during leaf curing after harvest. The four main TSNAs found in cured tobacco leaves are N'-nitrosonornicotine (NNN), N'-nitrosoanatabine (NAT), N'- nitrosoanabasine (NAB) and 4- (methyl nitrosamino) -l- (3-pyridyl) - 11-butanone (NNK).
[0010] [0010] TSNAs form when nitrous oxide species (eg, NO, NO> z, N203; and N704) react with tobacco alkaloids. NAT and NAB are formed through the nitrosation of the secondary alkaloids anatabine and anabasine, respectively. Although early studies have stated that NNN originates from nicotine and nornicotine, more recent reports have shown that the occurrence of NNN in cured tobacco leaves is correlated with nornicotin content, not nicotine (Bush et al., Rec Adv. Tob. Sci. 27 23-46 (2001); Lewis et al., Plant Biotech J. 6: 346-354 (2008)). Nornicotine is the demethylated derivative of nicotine, the main alkaloid in tobacco responsible for 90% of the total content of alkaloids (Saitoh et al., 1985 Phytochemistry, 24 pp. 477-480). The precursor / product relationship of NNK formation is less clear. Some studies claim that NNK is a nicotine nitrosation product, but due to the slow reaction rate of nicotine nitrosation, it is likely that an oxidized nicotine derivative, instead of nicotine itself, will serve as a direct precursor to NNK (Caldwell et al., Ann. NY Acad. Sci. 686, 213-228 (1993)). The identification of the genes responsible for the production and regulation of TSNA precursors is of great importance.
[0011] [0011] Although nornicotine typically represents only 2 to 4% of the total content of pyridine alkaloids in tobacco plants, the genetic instability that leads to the spontaneous appearance of converting plants with a high content of nornicotine is a chronic problem in tobacco production. Maintaining low levels of nornicotine can prevent the unpleasant taste and aroma associated with this alkaloid, in addition to reducing the formation of N-nitrosonornicotine (NNN) in tobacco industry products, of which nornicotine is the direct precursor.
[0012] [0012] The gene responsible for the majority of nicotine to nornicotin conversion is a CYP82E4 nicotine demethylase gene, which encodes a cytochrome P450 monooxygenase (Siminszky et al., Proc. Natl. Acad. Scad. USA, 102 (2005), pp. 14919-14924; Xu et al., Physiol. Plantarum, 129 (2007), pp. 307-319). The nicotine demethylase gene family in tobacco is widely characterized, but little is known about other cellular processes that can influence nornicotin levels. There is still a great need to develop methodologies that can further reduce the levels of TSNAs in tobacco plants and products produced from tobacco plants.
[0013] [0013] As described in the Examples, the inventors sought to investigate the genes responsible for the synthesis of alkaloids, with the aim of modulating the content of alkaloids in plants, for example, decreasing the nicotine content in tobacco plants. Their research led them to create crosses of Burley 21 (B21), B21 with normal / high alkaloid (HA) x B21 with low intermediate alkaloid (LI), B21 with normal / high alkaloid (HA) x B21 with low alkaloid (LA) and B21 with high intermediate (HI) x tobacco plants with low alkali B21 (LA). The alkaloid content of the resulting plants F> was analyzed. SNP genotyping was performed in F; s with the highest or lowest alkaloids content of the F, HA x LA and HA x LI populations to identify polymorphic markers for further analysis. An HI x LA population was also generated for accurate mapping. The inventors developed markers that secrete with the locus Nicl. The nine genes of the ethylene response factor (ERF) subfamily have been identified as potential regulators of the alkaloid synthesis transcription factor. SUMMARY OF THE INVENTION
[0014] [0014] It has been surprisingly found that by modulating the activity or expression of a Nicl ERF gene, as taught here, the alkaloid content of plants can be modulated. Thus, tobacco products with modulated alkali content and commercially desirable properties sought by consumers of tobacco products can be produced. In some cases, consumers may want a product with low levels of alkaloid content, for example, low levels of nicotine content.
[0015] [0015] The present invention may be particularly useful in the field of molecular plant agriculture, where plants (such as tobacco and other Nicotiana spp.) Are used for the production of proteins, peptides and metabolites, for example, for the production of therapeutic products and pharmaceuticals, such as antibiotics, virus-like particles or neutraceuticals or small molecules. Tobacco was used for the development of an HIV neutralizing antibody in an EU-funded project called PharmPlant and Medicago Inc., Canada, worked on a tobacco-based platform for the production of virus-like particles for the manufacture of vaccines against the flu.
[0016] [0016] Thus, a plant according to the present invention can be used for molecular agriculture to reduce or eliminate the presence of nicotine and / or other nicotinic alkaloids. The use of a plant with a low nicotine or root content is beneficial in molecular agriculture and would reduce downstream processing costs associated with purification.
[0017] [0017] In other cases, it may be desirable to produce plants with high levels of alkaloids, for example, high levels of nicotine content, so that nicotine can be purified from the tobacco plant to produce a pure nicotine product, for example , for use in devices that use nicotine-containing liquids (for example, and cigarettes) or within tobacco heating devices. For example, the production of leafy plants containing high levels of nicotine could reduce nicotine extraction costs for the production of electronic liquids for electronic cigarettes.
[0018] [0018] The present inventors investigated the regulation of nicotine biosynthesis in tobacco plants. They investigated the regulatory loci Nicl and Nic2 which are believed to control the expression of nicotine-related structural genes and other unrelated genes. An aim of the inventors was to provide an altered alkali content. Nine ERF genes have been identified in the Nicl region, which unexpectedly modulated the alkaloid content in modified tobacco plants, compared to their counterparts in wild-type plants grown under the same conditions.
[0019] [0019] the present inventors surprisingly have determined a method for modulating the alkaloid content, for example, nicotine content of a tobacco plant by modulating the activity or expression of an ERF gene. The nicotine content in a tobacco plant can be decreased by inhibiting the activity or expression of an ERF gene. Prior to the present invention, it was not known that modulation of the activity or expression of a Nicl ERF gene as described here could be used to modulate the content of alkaloids.
[0020] [0020] The present inventors have determined that the modulation of a Nicl ERF gene can reduce the alkaloid content of the modified plant to a surprisingly low level.
[0021] [0021] The LI strain (low intermediate, aaBB) normally produces about half the content of alkaloids compared to the HA strain (high alkali, AABB). However, in Example 13, the EMS strains were produced and are equivalent to the LI strain. EMS strains with Nitab4.5 0003090g0030.1 (ERF199) mutations were produced. These mutant EMS strains produced about a third of the alkaloid content compared to the HA strain - much less than would be expected for an LI strain.
[0022] [0022] In another example, when Nicl ERF Nitab4.5 0003090g90030.1 (ERFI99) was eliminated by editing genes from the HI strain (AAbb), the alkaloid content of the knockout strain was much less than the alkaloid content of the equivalent LA strain (aabb) (see Example 14). These data surprisingly suggest that modulating the activity or expression (for example, knocking down or eliminating the activity or expression) of a Nicl ERF gene (for example, Nitab4.5 0003090g0030.1 or ERF199) alone is sufficient to modulate (for example, example, to reduce) the alkaloid content of a plant or part of it.
[0023] [0023] According to a first aspect, the present invention provides a method for modulating the alkaloid content of a plant or part of it or cell culture, the method comprising modifying said plant or cell culture by modulating activity or expression at least one Nicl ERF gene: wherein the at least one Nicl ERF gene encodes a polypeptide comprising an amino acid sequence as set out in: SEQ ID No. 8; or SEQ ID No. 12; or SEQ ID No. 16; or SEQ ID No. 20; or SEQ ID No. 24; or SEQ ID No. 28; or SEQ ID No. 32; or a functional variant or functional or orthologous fragment thereof; or wherein the ERF gene comprises a nucleotide sequence as set out in: SEQ ID No. 3; or SEQ ID No. 5; or SEQ ID No. 9; or SEQ ID No. 13; or SEQ ID No. 17; or SEQ ID No. 21; or SEQ ID No. 25; or SEQ ID No. 29 or a functional variant or functional or orthologous fragment thereof.
[0024] [0024] In another aspect, a method is provided for modulating the content of a tobacco specific nitrosamine (TSNA) or a precursor of a TSNA in a tobacco plant or part of the same plant, the method comprising modifying said plant or a cell culture modulating the activity or expression of at least one Nicl ERF gene: wherein the at least one Nicl ERF gene encodes a polypeptide comprising an amino acid sequence as set out in: SEQ ID No. 8; or SEQ ID
[0025] [0025] In another aspect, the use of a Nicl ERF gene is provided to modulate the alkaloid content of a cell (for example, a tobacco cell) or plant or part of it or a cell culture in which: at least one gene Nicl ERF encodes a polypeptide that comprises an amino acid sequence as set out in: SEQ ID No. 8; or SEQ ID No. 12; or SEQ ID No. 16; or SEQ ID No. 20; or SEQ ID No. 24; or SEQ ID No. 28; or SEQ ID No. 32; or a functional variant or functional or orthologous fragment thereof, or wherein the ERF gene comprises a nucleotide sequence as set out in: SEQ ID No. 3; or SEQ ID No. 5; or SEQ ID No. 9; or SEQ ID No. 13; or SEQ ID No. 17; or SEQ ID No. 21; or SEQ ID No. 25; or SEQ ID No. 29 or a functional variant or functional or orthologous fragment thereof.
[0026] [0026] The present invention provides in a further aspect a method for producing a plant or part of it, a cell culture, a plant propagating material, a tobacco leaf, a cut tobacco leaf, a processed tobacco leaf or a cut and processed tobacco leaf having modulated alkali content, the method comprising modifying said plant or cell culture to modulate the activity or expression of at least one Nicl ERF gene in which: at least one Nicl ERF gene encodes a polypeptide comprising an amino acid sequence as set out in: SEQ ID No. 8; or SEQ ID No. 12; or SEQ ID No. 16; or SEQ ID No. 20; or SEQ ID No. 24; or SEQ ID No. 28; or SEQ ID No. 32 or a functional variant or functional or orthologous fragment thereof, or wherein the ERF gene comprises a nucleotide sequence as set forth in: SEQ ID No. 3; or SEQ ID No. 5; or SEQ ID No. 9; or SEQ ID No. 13; or SEQ ID No. 17; or SEQ ID No. 21; or SEQ ID No. 25; or SEQ ID No. 29 or a functional variant or functional or orthologous fragment thereof.
[0027] [0027] Suitably, at least one Nicl ERF gene for use in accordance with the present invention can encode a polypeptide comprising an amino acid sequence as set out in SEQ ID No. 8 or a functional variant or functional or ortholog fragment thereof.
[0028] [0028] Suitably, at least one Nicl ERF gene for use in accordance with the present invention can comprise a nucleotide sequence as set out in SEQ ID No. 5 or a functional variant or functional or orthologous fragment thereof.
[0029] [0029] In one aspect, a method or use of the invention is provided, in which the alkaloid content is modulated compared to a plant or cell culture that has not been modified to modulate the activity or expression of at least one Nicl ERF gene .
[0030] [0030] In another aspect, a plant or part of it or a cell culture is provided that has been modified to achieve a modulation in the alkaloid content compared to an unmodified plant or unmodified cell culture, where the modification is modulating the activity or expression of at least one Nicl ERF gene.
[0031] [0031] In another aspect, plant propagation material is provided which can be obtained from a plant or cell culture according to the invention or from a plant produced by the method of the invention.
[0032] [0032] In one aspect, a method or use of the invention, or a plant or part of it, or a plant propagating material of the invention is provided, in which the alkaloid content of the plant decreases compared to a plant that has not been modified to modulate the activity or expression of at least one Nicl ERF gene.
[0033] [0033] In one aspect, a method or use of the invention, a plant or part of it, or a plant propagating material of the invention is provided, in which the activity or expression of at least one Nicl ERF gene is decreased.
[0034] [0034] In another aspect, a method or use of the invention, or a plant or part of it, or a plant propagating material of the invention is provided, in which the alkaloid content of the plant is increased compared to a plant that has not been modified to modulate the activity or expression of at least one Nicl ERF gene.
[0035] [0035] In one aspect, a method or use of the invention is provided, a plant or part of it, or a plant propagation material, wherein the plant is modified to increase the activity or expression of at least one Nicl ERF gene and the plant exhibits an increased alkaloid content compared to a plant that has not been modified to modulate the activity or expression of at least one Nicl ERF gene.
[0036] [0036] In one aspect, a method or use of the invention, a plant or part of it, or a plant propagating material of the invention, is provided, in which the total alkaloid content of the plant is modulated.
[0037] [0037] In one aspect, a method or use of the invention, a plant or part of it, of the invention, or of a plant propagating material of the invention is provided, wherein the content of one or more alkaloids selected from nicotine , nornicotine, anabasin, myosmin and anatabine are modulated. In one aspect, the nicotine content is modulated.
[0038] [0038] In one aspect, a method or use of the invention is provided, a plant or part of it, or a plant propagation material of the invention, wherein the plant is of the Solanaceae family.
[0039] [0039] In one aspect, a method or use of the invention, a plant or part of it, or a plant propagating material of the invention, in which the plant is of the genus Solanum, is provided.
[0040] [0040] In another aspect, a method or use of the invention, a plant or part of it, or a plant propagating material of the invention, in which the plant is of the genus Nicotiana, is provided.
[0041] [0041] In one aspect, a method or use of the invention is provided, a tobacco plant or part of the invention, or a plant propagating material of the invention, in which the nicotine content is modulated.
[0042] [0042] In one aspect, a method or use of the invention is provided, a tobacco plant or part thereof, or a plant propagating material of the invention, in which the nicotine content is decreased.
[0043] [0043] In one aspect, a method or use of the invention, a plant or part of it, of the invention, or of a plant propagating material of the invention is provided, wherein the at least one Nicl ERF gene encodes a polypeptide that comprises an amino acid sequence as set out in: SEQ ID No. 8 or a functional variant or functional or ortholog fragment thereof; or at least one Nicl ERF gene comprises a nucleotide sequence as set out in: SEQ ID No. 5 or a functional variant or functional or ortholog fragment thereof.
[0044] [0044] In one aspect, a method or use of the invention, a plant or part of it, or a plant propagation material of the invention is provided, wherein an additional ERF gene is modulated in which: the additional ERF gene is a gene Nic2 ERF and encodes a polypeptide comprising an amino acid sequence as set out in: SEQ ID No. 40; or SEQ ID No. 44; or SEQ ID No. 48; or SEQ ID No. 52; or SEQ ID No. 56; or SEQ ID No. 60; or SEQ ID No. 64; or SEQ ID No. 68; or SEQ ID No. 72 or a functional variant or functional or orthologous fragment thereof; or the additional ERF gene is a Nic2 ERF gene and comprises a nucleotide sequence as set out in: SEQ ID No. 37; or SEQ ID No. 41; or SEQ ID No. 45; or SEQ ID No. 49; or SEQ ID No. 53; or SEQ ID No. 57; or SEQ ID No. 61; or SEQ ID No. 65; or SEQ ID No. 69 or a functional variant or functional or orthologous fragment thereof.
[0045] [0045] In one aspect, a method or use of the invention, a plant or part of it, or a plant propagation material of the invention is provided, wherein an additional ERF gene is modulated wherein the additional ERF gene is a Nic2 gene ERF and comprises a nucleotide sequence as set out in SEQ ID No. 69 or a functional variant or functional or ortholog fragment thereof or encodes a polypeptide comprising an amino acid sequence as set out in SEQ ID No.72 or a functional variant or fragment functional or orthologous.
[0046] [0046] In one aspect, a method or use of the invention, a plant or part of it, or a plant propagating material of the invention is provided in which at least one Nicl ERF gene encodes a polypeptide comprising a sequence of amino acids such as established in: SEQ ID No. 8 or a functional variant or functional or orthologous fragment thereof; or at least one Nicl ERF gene comprises a nucleotide sequence as set out in: SEQ ID No. 5 or a functional variant or functional or orthologous fragment thereof and wherein an additional ERF gene is modulated in which: the additional ERF gene is at at least one Nic2 ERF gene, for example a Nic2 ERF gene that encodes a polypeptide comprising an amino acid sequence as set out in: SEQ ID No. 40; or SEQ ID No. 44; or SEQ ID No. 48; or SEQ ID No. 52; or SEQ ID No. 56; or SEQ ID No. 60; or SEQ ID No. 64; or SEQ ID No. 68; or SEQ ID No. 72 or a functional variant or functional or orthologous fragment thereof; or additional ERF gene is a Nic2 ERF gene, for example, a Nic2 ERF gene that comprises a nucleotide sequence, as set out in: SEQ ID No. 37; or SEQ ID No. 41; or SEQ ID No. 45; or SEQ ID No. 49; or SEQ ID No. 53; or SEQ ID No. 57; or SEQ
[0047] [0047] In one aspect, a method or use of the invention, a plant or part of it, or a plant propagating material of the invention is provided, wherein at least one Nicl ERF gene encodes a polypeptide comprising a sequence of amino acids such as established in: SEQ ID No. 8 or a functional variant or functional or orthologous fragment thereof; or at least one Nicl ERF gene comprises a nucleotide sequence as set out in: SEQ ID No. 5 or a functional variant or functional or ortholog fragment thereof and in which an additional ERF gene is modulated, where the additional ERF gene is a Nic2 ERF gene that comprises a nucleotide sequence as set out in SEQ ID No. 69 or a functional variant or functional or ortholog fragment thereof or encodes a polypeptide that comprises an amino acid sequence as set out in SEQ ID No. 72 or a functional variant or functional or orthologous fragment thereof.
[0048] [0048] In another aspect, the use of a plant or part of it of the invention is provided, or of a plant produced by the method of the invention to produce a plant.
[0049] [0049] In another aspect, the use of a plant or part of it of the invention is provided, or of a plant produced by the method of the invention for the production of a product (for example, a product of the tobacco industry).
[0050] [0050] In another aspect, it is provided the use of a plant or part of it of the invention, or of a plant produced by the method of the invention to grow a crop.
[0051] [0051] In one aspect, the invention provides cured tobacco material made from a plant or a part of it according to the invention or an extract thereof or a tobacco cell culture according to the invention.
[0052] [0052] In another aspect, a tobacco blend comprising said cured tobacco material according to the invention is provided.
[0053] [0053] In another aspect, it is provided to use a plant or part of the invention, or a plant produced by the method of the invention to produce a leaf.
[0054] [0054] In one aspect, a leaf taken from a plant of the invention is provided, or obtained from a plant propagated from a propagation material of the invention, or obtained from a plant obtained by a use of the invention, or obtained from a plant produced by the method of the invention.
[0055] [0055] In one aspect, a leaf harvested from a plant of the invention is provided, wherein the leaf harvested from a plant is a harvested leaf cut.
[0056] [0056] The invention provides in another aspect processed leaf, preferably a processed non-viable leaf: obtainable from a plant obtainable from a use of the invention; obtainable by processing a plant of the invention; obtainable from a plant propagated from a plant propagation material of the invention; or obtainable by processing a leaf harvested from a plant of the invention; or obtainable from a plant produced by the method of the invention.
[0057] [0057] In one aspect, a processed sheet of the invention is provided, wherein the sheet is processed by curing, fermenting, pasteurizing or a combination thereof.
[0058] [0058] In one aspect, a processed sheet of the invention is provided, wherein the processed sheet is a cut processed sheet.
[0059] [0059] The invention provides in another aspect a tobacco product prepared from: a tobacco plant of the invention or a part thereof; a tobacco plant or part thereof propagated from a tobacco plant propagation material of the invention; a leaf taken from a tobacco plant of the invention; a processed tobacco leaf of the invention; or a tobacco plant produced by the method of the invention.
[0060] [0060] The invention provides in another aspect a product of the tobacco industry prepared from: i) a tobacco plant of the invention or a part thereof or a cultivation of tobacco cells of the invention; ii) a tobacco plant or part thereof propagated from a tobacco plant propagation material of the invention; iii) a leaf harvested from a tobacco plant of the invention; iv) a processed tobacco leaf of the invention.
[0061] [0061] In one aspect, a tobacco product of the invention is provided, wherein the tobacco product is a combustible smoke article.
[0062] [0062] In another aspect, a tobacco industry product of the invention is provided, wherein the tobacco product is a smokeless tobacco product.
[0063] [0063] In one aspect, a tobacco industry product of the invention is provided, wherein the tobacco industry product is a non-combustible aerosol delivery system, such as a tobacco heating device or an aerosol generating device.
[0064] [0064] In one aspect, the use of a cell (e.g., tobacco cell) of the invention is provided to modulate the content of alkaloids in cell culture.
[0065] [0065] In one aspect, an article of smoke, smokeless tobacco product or tobacco heating device is provided comprising a plant or a part thereof according to the invention or an extract (for example, a tobacco extract) or a tobacco cell culture according to the invention; Or a cured tobacco material according to the invention; or a tobacco blend according to the invention.
[0066] [0066] In one aspect, the present invention provides the use of a nucleotide sequence of at least one Nicl ERF gene selected from: SEQ ID No. 3, or SEQ ID No. 5; or SEQ ID No. 9; or SEQ ID No. 13; or SEQ ID No. 17; or SEQ ID No. 21; or SEQ ID No. 25; or SEQ ID No. 29; or a functional variant or functional or ortholog fragment thereof, to select a plant with modulated alkali content (eg reduced) and / or modulated content (eg reduced) of tobacco specific nitrosamine (TSNA) or a precursor to a TSNA.
[0067] [0067] Suitably, the use may comprise determining the presence of a modification in at least one Nicl ERF gene selected from: SEQ ID No. 3, or SEQ ID No. 5; or SEQ ID No. 9; or SEQ ID No. 13; or SEQ ID No. 17; or SEQ ID No. 21; or SEQ ID No. 25; or SEQ ID No. 29; or a functional variant or functional or orthologous fragment thereof, in said plant, wherein said modulation modulates (for example, decreases) the activity or expression of at least one MNicl ERF gene. Suitably, the modification can reduce or eliminate the expression or function of the Nicl ERF gene so that the protein expression or function of the Nicl ERF gene in said plant is not detectable.
[0068] [0068] In one aspect, the present invention provides a mutant of a plant carrying an inherited mutation in a nucleotide sequence of at least one Nicl ERF gene, wherein the Nicl ERF gene is selected from: SEQ ID No. 3 or SEQ ID No. 5; or SEQ ID No. 9; or SEQ ID No. 13; or SEQ ID No. 17; or SEQ ID No. 21; or SEQ ID No. 25; or SEQ ID No. 29; or a functional variant or functional or orthologous fragment thereof; wherein said inheritable mutation modulates (for example, decreases) the activity or expression of at least one Nicl ERF gene and where the mutant plant modulates (for example, decreases) the content of alkaloids and / or the modulated content of a nitrosamine tobacco-specific (TSNA) or a precursor to a TSNA in relation to a comparable plant that does not carry the said hereditary mutation.
[0069] [0069] Hereditary mutation is performed by technical means, that is, the hereditary mutation is projected and is not a naturally occurring mutation. Any technical means can be used to induce the hereditary mutation. Suitably, hereditary mutation can be performed by chemical mutagenesis, such as treatment with ethyl methanesulfonate (EMS), physical irradiation and insertion agents, including T-DNAs; UV mutagenesis or gene editing techniques (such as CRISPR / Cas). In another aspect, the present invention provides a progeny or seed of a mutant plant that carries the inherited mutation according to the present invention.
[0070] [0070] In one aspect, the present invention provides a harvested leaf, processed leaf or cured tobacco material produced from a plant that comprises a modification in a nucleotide sequence of at least one Nicl ERF gene, wherein the at least one Nicl ERF gene is selected from: SEQ ID No. 3, or SEQ ID No. 5; or SEQ ID No. 9; or SEQ ID No. 13; or SEQ ID No. 17; or SEQ ID No. 21; or SEQ ID No. 25; or SEQ ID No. 29; or a functional variant or functional or orthologous fragment thereof; wherein said modulation modulates (for example, decreases) the activity or expression of at least one Nicl ERF gene and where said plant modulates (for example, decreased) the content of alkaloids and / or the modulated content of a specific nitrosamine tobacco (TSNA) or a precursor of a TSNA in relation to a comparable plant that does not carry said modification in at least one Nicl ERF gene.
[0071] [0071] The invention also provides a method, a leaf, a plant, a plant propagating material, a harvested leaf, processed tobacco, a tobacco product, a use or a combination thereof, as described here with reference to description and drawings. BRIEF DESCRIPTION OF THE DRAWINGS
[0072] [0072] Embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which:
[0073] [0073] Figure 1 Panel A shows the level of total alkaloids for parental and Fx lines; derive from the HA x LI crosses. Panel B shows nicotine levels for parental and Fx lines; derived from HA x LA crosses. Thirty individual plants for each parental line and 200 F; xs were selected for each population for chemical analysis of alkaloid levels.
[0074] [0074] Figure 2 shows a comparison of genetic maps of F7 individuals selected from the HA x LI and HA x LA crosses with the N. tabacum 30k Infinium HD 2015 consensus map. Dashed lines indicate markers identified between F7 maps , and the consensus map, dotted lines indicate markers identified between the two F> 2 maps. Bold font indicates markers common to more than one map. Only markers that have been identified on the HA x LA or HA x LI maps are shown on the consensus map, other marker positions are shown as horizontal black lines.
[0075] [0075] Figure 3 Panel A and B show genotyping of the selected F> individuals from the HA (AABB) X LI (aaBB) crosses with SNP4 CAPS marker. Panel A shows the 20 F2s with the lowest alkali levels. Panel B shows the 24 F> s with the highest alkali levels. Panels C and D show the genotyping of F> individuals selected from the HA (AABB) X LA (aabb) crosses with the SNP4 CAPS marker. Panel C shows the 24 F> xs with the lowest alkaloid levels. Panel D shows the 20 F2s with the highest alkali levels. HA, HI and LI and LA were used as controls. Potential recombinants are indicated with arrows.
[0076] [0076] Figure 4 Panel A shows a comparison between the phenotype values of total alkali content for LA, LI, HI and HA. The average values for 30 plants are LA = 0.4%, LI = 1.69%, HI = 2.89%, HA = 3.06%. Panel B shows a comparison of nicotine content phenotype values for L A, LI, HI and HA. The average values for 30 plants are LA = 0.39%, LI = 1.61%, HI = 2.76%, HA = 2.92%.
[0077] [0077] Figure 5 Panel A shows the levels of total alkaloids for the parental lines HI and LA, and F7 plants genotyped as AA (F; - AA) and aa (F; -aa). Panel B shows the nicotine levels for the parental lines HI and LA, and F plants, genotyped as AA (F; - AA) and aa (F; -aa). The plant genotyped as AA, but with the lowest alkaloid or nicotine levels, indicated by the arrow on the panels, was bagged to collect F3 seeds.
[0078] [0078] Figure 6 Panel A shows the content of total alkaloids for the parental lines HI and LA and F3s derived from plant F bagged. Panel B shows the nicotine levels for the parental HI and LA and F3 strains derived from the bagged F> plant. No phenotypic segregation was observed in 40 F3 plants.
[0079] [0079] Figure 7 shows the genetic map for Nicl. The number of recombinants observed with the respective marker is given. The genetic distance (cm) for each marker is indicated on the left side of the chromosome. The locus Nicl, co-secreting with SNP4, is flanked by SNP2 / SNP3 and SNP5. A deletion region greater than 500 Kb (INDEL1) has been reported around the locus Nicl by Adams et al. (US20160374387Al incorporated here by reference). Together with the upstream and downstream SNPs (13 and 14), the genomic deletion is outside the delimited region of the Nicl locus.
[0080] [0080] Figure 8 shows the genetic map for the Nic2 locus. The number of recombinants observed with the respective marker is given. The genetic distance (cM) for each marker was indicated on the left side of the chromosome. The Nic2 locus co-secretes with SNP17 and SNP 18.
[0081] [0081] Figure 9 shows the alkaloid analysis of hairy roots (LA background) transformed by Nicl ERFs. Hairy roots of HI and LA transformed with empty vector were used as controls.
[0082] [0082] Figure 10 to Figure 81 shows SEQ ID NO. 1 to SEQ ID No. 72 as described below.
[0083] [0083] Figure 82 shows a partially complete physical map of the Nicl genomic region, showing the locations of scaffolding and markers (not drawn to scale). Thin, black lines indicate BioNano hybrid scaffolds with dark gray scaffolding aligned below them, according to the pseudochromosomes of Edwards et al. (2017 incorporated here by reference). Dotted sections on the black line indicate non-continuous sequence regions; small gaps indicate breakpoints between BioNano super scaffolds (see Edwards et al. 2017 for details). Light gray indicates the scaffolding locations estimated based on reciprocal BLAST analysis for the genome of Sierro et al. (2014 Nature Communications 5, 3833 incorporated here by reference). The pale scaffolding indicates the estimated location based on the position of the associated marker on one of the genetic maps. Approximate locations of the SNP / INDEL marker shown below the scaffolding. Dotted gray boxes indicate the region of the sequence identified by Adams et al. (2016).
[0084] [0084] Figure 83 shows a complete physical map of the Nic2 genomic region, showing the locations of scaffolding and markers (not drawn to scale). Thin, black lines indicate BioNano hybrid scaffolds with dark gray scaffolding aligned below them, according to the pseudochromosomes of Edwards et al. (2017). Small gaps in the black line indicate breakpoints between BioNano's superandanimes (see Edwards et al. 2017 for details). Light gray indicates the scaffolding locations estimated based on reciprocal BLAST analysis for the genome of Sierro et al. (2014). The pale scaffolding indicates the estimated location based on the position of the associated marker on one of the genetic maps. The approximate locations of the SNP markers are shown below the scaffolding. Dotted gray boxes indicate the region of the sequence identified by Adams et al. (2016).
[0085] [0085] Figure 84 shows the levels of total alkaloids (nicotine, nornicotine, anabasin and anatabine) in a segregated population of EMS mutant plants, grouped by the state of the mutation in Nitab4.5 0003090g0030.1 (ERF199). The change from amino acids to mutant plants is shown in parentheses after the gene identifier. Significant differences in the respective wild-type plants with p <0.01 or p <0.05 (Student's t test) are indicated by a double or single asterisk, respectively.
[0086] [0086] Figure 85 shows analysis of Nitab4.5 editor-mediator knockout gene sequences of 0003090g0030.1 (ERF199) in the heterozygous mutant Ll line. Ll contained a mutant allele with an “A” insert, which led to a premature stop codon. PCR products amplified with the primer pair (SEQ ID No. 111 and SEQ ID No. 112) were cloned into pGEM-T Easy Vector, and at least 10 colonies were selected for sequencing.
[0087] [0087] Figure 86 shows the alkaloid analysis of anatabine, anabasine, nornicotine and nicotine with T1 plants of the L1 mutant edited by genes. The knockout of Nitab4.5 0003090g0030.1 significantly decreased the alkaloid content in the HI (A) plant, and the alkaloid levels in homozygous mutants were almost 1/10 than in LA (B) plants. WI-T1, Het-T1 and Mut-T1 were used to indicate the three genotypes of T1 plants; wild type, heterozygous and homozygous mutants, respectively. At least 15 individual plants were measured for each genotype. HI and LA plants were used as controls.
[0088] [0088] Figure 87 shows the RT-PCR analysis of the level of relative expression for Nitab4.5 0003090g0030.1 (ERF199) alleles between HI and LA. The expression Nitab4.5 0003090g0030.1 is root specific and is downregulated in LA plants. Actin was used as an internal control. The pair of primers used to amplify Nitab4.5 /0003090g0030.1 was 5'AGTCCTAGCTCAAGTTTTAGCAGCTTCGA3 '
[0089] [0089] A summary of the sequence identifiers used throughout the subject specification and the corresponding sequence listing are provided where:
[0090] [0090] SEQ ID No. 1 corresponds to the nucleotide sequence encoding the gene known as Nitab4.5 0003090g0020.1 or also known as ERF17L34AN.
[0091] [0091] SEQ ID No. 2 corresponds to the Nitab4.5 0003090g0020.1 cDNA sequence (ERFI7L34AN).
[0092] [0092] SEQ ID No. 3 corresponds to the Nitab4.5 0003090g0020.1 (ERFI7L3AN) CDs.
[0093] [0093] SEQ ID No. 4 corresponds to the amino acid sequence of the Nitab4.5 polypeptide 0003090g0020.1 (ERF17L34AN).
[0094] [0094] SEQ ID No. 5 corresponds to the nucleotide sequence encoding the gene known as Nitab4.5 0003090g0030.1 or also known as ERFI199.
[0095] [0095] SEQ ID No. 6 corresponds to the Nitab4.5 cDNA sequence 0003090g0030.1 (ERF199).
[0096] [0096] SEQ ID No. 7 corresponds to the Nitab4.5 0003090g0030.1 (ERF199) CDs.
[0097] [0097] SEQ ID No. 8 corresponds to the amino acid sequence of the Nitab4.5 polypeptide 0003090g0030.1 (ERF199).
[0098] [0098] SEQ ID No. 9 corresponds to the nucleotide sequence encoding the gene known as Nitab4.5 0003665g0040.1 or also known as (JRES5SL2).
[0099] [0099] SEQ ID No. 10 corresponds to the Nitab4.5 cDNA sequence 00036659g90040.1 (JRESL2).
[0100] [0100] SEQ ID No. 11 corresponds to the codes of Nitab4.5 000366590040.1 (JRESL2).
[0101] [0101] SEQ ID No. 12 corresponds to the amino acid sequence of the Nitab4.5 polypeptide 0003665g90040.1 (JRESL2).
[0102] [0102] SEQ ID No. 13 corresponds to the nucleotide sequence encoding the gene known as Nitab4.5 0004620g0010.1 or also known as ERF210.
[0103] [0103] SEQ ID No. 14 corresponds to the Nitab4.5 cDNA sequence 0004620g0010.1 (ERF210).
[0104] [0104] SEQ ID No. 15 corresponds to Nitab4.5 0004620g0010.1 (ERF210) CDs.
[0105] [0105] SEQ ID No. 16 corresponds to the amino acid sequence of the Nitab4.5 polypeptide 0004620g0010.1 (ERF210).
[0106] [0106] SEQ ID No. 17 corresponds to the nucleotide sequence encoding the gene known as Nitab4.5 0004620g0030.1 or also known as ERF91.
[0107] [0107] SEQ ID No. 18 corresponds to the Nitab4.5 0004620g0030.1 cDNA sequence (ERF91).
[0108] [0108] SEQ ID No. 19 corresponds to the codes of Nitab4.5 0004620g0030.1 (ERF91).
[0109] [0109] SEQ ID No. 20 corresponds to the amino acid sequence of the Nitab4.5 polypeptide 0004620g0030.1 (ERF91).
[0110] [0110] SEQ ID No. 21 corresponds to the nucleotide sequence encoding the gene known as Nitab4.5 0004620g0080.1 or also known as ERF29.
[0111] [0111] SEQ ID No. 22 corresponds to the Nitab4.5 0004620g90080.1 cDNA sequence (ERF29).
[0112] [0112] SEQ ID No. 23 corresponds to Nitab4.5 0004620g0080.1 (ERF29) CDs.
[0113] [0113] SEQ ID No. 24 corresponds to the amino acid sequence of the Nitab4.5 polypeptide 0004620g90080.1 (ERF29).
[0114] [0114] SEQ ID No. 25 corresponds to the nucleotide sequence encoding the gene known as Nitab4.5 0004620g0090.3, also known as ERF130.
[0115] [0115] SEQ ID No. 26 corresponds to the Nitab4.5 cDNA sequence 0004620g90090.3 (ERF130).
[0116] [0116] SEQ ID No. 27 corresponds to the codes of Nitab4.5 0004620g90090.3 (ERF130).
[0117] [0117] SEQ ID No. 28 corresponds to the amino acid sequence of the Nitab4.5 polypeptide 0004620g0090.3 (ERF130).
[0118] [0118] SEQ ID No. 29 corresponds to the nucleotide sequence encoding the gene known as Nitab4.5 0004620g0095.1, also known as ERF16.
[0119] [0119] SEQ ID No. 30 corresponds to the Nitab4.5 0004620g0095.1 cDNA sequence (ERFI16).
[0120] [0120] SEQ ID No. 31 corresponds to Nitab4.5 0004620g90095.1 (ERFI16) CDs.
[0121] [0121] SEQ ID No. 32 corresponds to the amino acid sequence of the Nitab4.5 polypeptide 0004620g90095.1 (ERFI16).
[0122] [0122] SEQ ID No. 33 corresponds to the nucleotide sequence encoding the gene known as Nitab4.5 0006382g0040.1, also known as ERF110.
[0123] [0123] SEQ ID No. 34 corresponds to the Nitab4.5 0006382g90040.1 cDNA sequence (ERF110).
[0124] [0124] SEQ ID No. 35 corresponds to the codes of Nitab4.5 0006382g90040.1 (ERF110).
[0125] [0125] SEQ ID No. 36 corresponds to the amino acid sequence of the Nitab4.5 polypeptide 0006382g90040.1 (ERF110).
[0126] [0126] SEQ ID No. 37 corresponds to the nucleotide sequence encoding the gene known as Nitab4.5 0002924g0010.1, also known as ERF17LI.
[0127] [0127] SEQ ID No. 38 corresponds to the Nitab4.5 cDNA sequence 0002924g90010.1 (ERFI17LI).
[0128] [0128] SEQ ID No. 39 corresponds to the Nitab4.5 0002924g90010.1 (ERF17LI) CDs.
[0129] [0129] SEQ ID No. 40 corresponds to the amino acid sequence of the Nitab4.5 polypeptide 000292490010.1 (ERF17LI).
[0130] [0130] SEQ ID No. 41 corresponds to the nucleotide sequence encoding the gene known as Nitab4.5 0002924g0020.2, also known as ERF179.
[0131] [0131] SEQ ID No. 42 corresponds to the Nitab4.5 cDNA sequence 000292490020.2 (ERF179).
[0132] [0132] SEQ ID No. 43 corresponds to the codes of Nitab4.5 000292490020.2 (ERF179).
[0133] [0133] SEQ ID No. 44 corresponds to the amino acid sequence of the Nitab4.5 polypeptide 000292490020.2 (ERF179).
[0134] [0134] SEQ ID No. 45 corresponds to the nucleotide sequence encoding the gene known as Nitab4.5 0002924g0040.2, also known as ERF17.
[0135] [0135] SEQ ID No. 46 corresponds to the Nitab4.5 cDNA sequence 000292490040.2 (ERF17).
[0136] [0136] SEQ ID No. 47 corresponds to the Nitab4.5 0002924g90040.2 (ERF17) CDs.
[0137] [0137] SEQ ID No. 48 corresponds to the amino acid sequence of the Nitab4.5 polypeptide 0002924g90040.2 (ERF17).
[0138] [0138] SEQ ID No. 49 corresponds to the nucleotide sequence encoding the gene known as Nitab4.5 0002924g0045.1, also known as ERFI168.
[0139] [0139] SEQ ID No. 50 corresponds to the Nitab4.5 cDNA sequence 00029249g90045.1 (ERF168).
[0140] [0140] SEQ ID No. 51 corresponds to the codes of Nitab4.5 0002924g90045.1 (ERF168).
[0141] [0141] SEQ ID No. 52 corresponds to the amino acid sequence of the Nitab4.5 polypeptide 0002924g90045.1 (ERF168).
[0142] [0142] SEQ ID No. 53 corresponds to the nucleotide sequence encoding the gene known as Nitab4.5 00029249g0050.2, also known as ERF115.
[0143] [0143] SEQ ID No. 54 corresponds to the Nitab4.5 cDNA sequence 000292490050.2 (ERF115).
[0144] [0144] SEQ ID No. 55 corresponds to Nitab4.5 000292490050.2 (ERF115) CDs.
[0145] [0145] SEQ ID No. 56 corresponds to the amino acid sequence of the Nitab4.5 polypeptide 0002924g90050.2 (ERF115).
[0146] [0146] SEQ ID No. 57 corresponds to the nucleotide sequence encoding the gene known as Nitab4.5 0006499g0010.1, also known as ERF104.
[0147] [0147] SEQ ID No. 58 corresponds to the Nitab4.5 cDNA sequence 0006499g90010.1 (ERF1O04).
[0148] [0148] SEQ ID No. 59 corresponds to the codes of Nitab4.5 0006499g90010.1 (ERF104).
[0149] [0149] SEQ ID No. 60 corresponds to the amino acid sequence of the Nitab4.5 polypeptide 0006499g90010.1 (ERF104).
[0150] [0150] SEQ ID No. 61 corresponds to the nucleotide sequence encoding the gene known as Nitab4.5 0006499g0020.2 also known as ERF221.
[0151] [0151] SEQ ID No. 62 corresponds to the Nitab4.5 cDNA sequence 0006499g0020.2 (ERF221).
[0152] [0152] SEQ ID No. 63 corresponds to the codes of Nitab4.5 0006499g90020.2 (ERF221).
[0153] [0153] SEQ ID No. 64 corresponds to the amino acid sequence of the Nitab4.5 polypeptide 0006499g0020.2 (ERF221).
[0154] [0154] SEQ ID No. 65 corresponds to the nucleotide sequence encoding the gene known as Nitab4.5 0012667g90020.2 (ERF91L1).
[0155] [0155] SEQ ID No. 66 corresponds to the Nitab4.5 cDNA sequence 001266790020.2 (ERF91LI1).
[0156] [0156] SEQ ID No. 67 corresponds to the codes of Nitab4.5 001266790020.2 (ERF91LI1).
[0157] [0157] SEQ ID No. 68 corresponds to the amino acid sequence of the Nitab4.5 polypeptide 001266790020.2 (ERF91LI1).
[0158] [0158] SEQ ID No. 69 corresponds to the nucleotide sequence encoding the gene known as Nitab4.5 00150559g0010.2, also known as ERF189.
[0159] [0159] SEQ ID No. 70 corresponds to the Nitab4.5 cDNA sequence 0015055g90010.2 (ERF189).
[0160] [0160] SEQ ID No. 71 corresponds to the codes of Nitab4.5 0015055g90010.2 (ERF189).
[0161] [0161] SEQ ID No. 72 corresponds to the amino acid sequence of the Nitab4.5 polypeptide 0015055g90010.2 (ERF189).
[0162] [0162] Some sequences disclosed here contain "N" in nucleotide sequences. "N" can be any nucleotide or an exclusion or insertion of one or more nucleotides. For example, in some cases, a sequence of "N" s is shown. The number of "N" s does not necessarily correlate with the actual number of nucleotides in that position. There may be more or less nucleotides than shown as "N" in the sequence. DETAILED DESCRIPTION
[0163] [0163] For the first time, the present inventors have shown that by modulating the activity or expression of at least one Nicl ERF gene in a plant (for example, a tobacco plant), the alkaloid and / or TSNA content of the plant can be modulated.
[0164] [0164] At least one Nicl ERF gene is selected from the group comprising: a gene encoding a polypeptide comprising an amino acid sequence as set out in: SEQ ID No. 8; or SEQ ID No. 12; or SEQ ID No. 16; or SEQ ID No. 20; or SEQ ID No. 24; or S EQ ID No. 28; or SEQ ID No. 32 or a functional variant or functional or orthologous fragment thereof; or wherein the ERF gene comprises a nucleotide sequence as set out in: SEQ ID No. 5; or SEQ ID No. 9; or SEQ ID No. 13; or SEQ ID No. 17; or SEQ ID No. 21; or SEQ ID No. 25; or SEQ ID No. 29; or a functional variant or functional or orthologous fragment thereof.
[0165] [0165] Suitably, at least one Nicl ERF gene can be one, or two, or three, or four, or five, or six or seven genes selected from the group comprising: a gene encoding a polypeptide comprising an amino acid sequence as defined in: SEQ ID No. 8; or SEQ ID No. 12; or SEQ ID No. 16; or SEQ ID No. 20; or SEQ ID No. 24; or SEQ ID No. 28; or SEQ ID No. 32 or a functional variant or functional or orthologous fragment thereof; or wherein the ERF gene comprises a nucleotide sequence as set out in: SEQ ID No. 5; or SEQ ID No. 9; or SEQ ID No. 13; or SEQ ID No. 17; or SEQ ID No. 21; or
[0166] [0166] In one aspect, at least one Nicl ERF gene encodes a polypeptide comprising an amino acid sequence as set out in: SEQ ID No. 8 or a functional variant or functional or ortholog fragment thereof; or wherein the Nicl ERF gene comprises a nucleotide sequence as defined in SEQ ID No. 5 or a functional variant or functional or ortholog fragment thereof.
[0167] [0167] In one aspect, the activity or expression of at least one additional Nicl ERF is modulated. Suitably, at least two, at least three, at least four, at least five, at least six, at least seven or at least eight additional Nicl ERFs selected from Table 1 can also be modulated.
[0168] [0168] In one aspect, at least one Nicl ERF gene encodes a polypeptide comprising an amino acid sequence as set out in: SEQ ID No. 8 or a functional variant or functional or ortholog fragment thereof; or at least one Nicl ERF gene comprises a nucleotide sequence as set out in SEQ ID No. 5 or a functional variant or functional or orthologous fragment thereof is modulated; and the activity or expression of at least one additional Nicl ERF is modulated. Suitably, at least one additional Nicl ERF can be selected from: a Nicl ERF gene that encodes a polypeptide comprising an amino acid sequence as set out in: SEQ ID No. 4; or SEQ ID No. 12; or SEQ ID No. 16; or SEQ ID No. 20; or SEQ ID No. 24; or SEQ ID No. 28; or SEQ ID No. 32; or SEQ ID No. 36 or a functional variant or functional or orthologous fragment thereof; or wherein the ERF gene comprises a nucleotide sequence as set out in: SEQ ID No. 1; SEQ ID No. 3, or SEQ ID No. 9; or SEQ ID No. 13; or SEQ ID No. 17; or SEQ ID No. 21; or SEQ ID No. 25; or SEQ ID No. 29; or SEQ ID No. 33 or a functional variant or functional or orthologous fragment thereof. Suitably, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight additional Nicl ERFs can be modulated.
[0169] [0169] In one embodiment, the present invention provides a method for modulating the alkaloid content of a plant (for example, a tobacco plant) or a part thereof, comprising the method of modifying said plant by modulating the activity or expression of at least one ERF gene.
[0170] [0170] The term “modulation” is used here to mean an increase or decrease.
[0171] [0171] The term "increased alkaloid content" is used here to mean that the concentration and / or the total alkaloid content in the product of the present invention (eg plant, part of it (eg leaf), leaf processed or a product made from the plant (e.g., a tobacco product) is greater compared to a similar product that has not been modified according to the present invention.
[0172] [0172] The term "decrease in alkaloids content" is used here to mean that the concentration and / or the total alkaloids content in the product of the present invention (eg plant, part of it (eg leaf), leaf processed or a product made from the plant (e.g., tobacco product) is less compared to a comparable product that has not been modified according to the present invention.
[0173] [0173] In one embodiment, the present invention provides a method of modulating (i.e., increasing or reducing) the tobacco specific nitrosamine (TSNA) content or a precursor of a TSNA in a plant (for example, a tobacco plant ) or a part thereof, the method comprising modifying said plant by modulating the activity or expression of at least one Nicl ERF gene.
[0174] [0174] In one embodiment, TSNA is N'nitrosonornicotine (NNN) and / or the precursor is nornicotin.
[0175] [0175] In one embodiment, TSNA can be one or more of the groups selected from: N'-nitrosonornicotine (NNN), N'nitrosoanatabina (NAT), N'-nitrosoanabasine (NAB) and 4 (methylnitrosamino) -1- ( 3-pyridyl) -1-butanone (NNK).
[0176] [0176] In a preferred embodiment, TSNA is N'-nitrosonornicotine (NNN).
[0177] [0177] TSNA can be measured in processed tobacco, for example, cured tobacco or reconstituted tobacco. In one embodiment, the TSNA content is measured and / or modified (for example, reduced) in a cured tobacco plant or in part of it (for example, cured tobacco leaf).
[0178] [0178] The term "tobacco-specific nitrosamine" or "TSNA", as used here, has its usual meaning in the state of the art, that is, a nitrosamine that is found only in tobacco products or other products that contain nicotine. Suitably, at least one tobacco-specific nitrosamine can be 4- (methylnitrosamino) -1- (3-pyridyl) -1-butanone (NNK), N'-nitrosonornicotine (NNN), N'-nitrosoanatabine (NAT) or N- nitrosoanabasine (NAB).
[0179] [0179] More appropriately, at least one tobacco-specific nitrosamine can be NNK or NNN.
[0180] [0180] In one embodiment, the tobacco-specific nitrosamine is NNN.
[0181] [0181] The term "precursor to it", when used in relation to at least one tobacco-specific nitrosamine, refers to one or more chemicals or compounds from a tobacco plant that give rise to the formation of a specific nitrosamine from tobacco tobacco or are involved in the nitrosation reaction that leads to the production of tobacco-specific nitrosamine. Suitably, the term "precursor to it" can refer to nitrate, nitrite or nitric oxide.
[0182] [0182] In one embodiment, the TSNA precursor is one or more of the selected group of nornicotine, anabasine, anatabine and an oxidized nicotine derivative, such as pseudooxynicotin (PON).
[0183] [0183] In a preferred embodiment, the precursor to TSNA is nornicotine.
[0184] [0184] In one embodiment, the precursor to TSNA can be PON. The precursor of TSNA (for example, NNN, NNK, NAB and / or NAT) can be measured on green tobacco leaves, for example, before processing, for example, before curing. In one embodiment, the TSNA precursor (for example, NNN, NNK, NAB and / or NAT) is measured and / or modified (for example, reduced) on a green tobacco leaf, for example, before processing, for example , before curing.
[0185] [0185] In one embodiment of carrying out a method and / or use of the invention, it results in a reduction of at least one TSNA or a precursor of the same in the modified tobacco plant (or part of it) when compared to a tobacco plant (or part thereof) that has not been modified in accordance with the present invention.
[0186] [0186] The terms "reducing at least one TSNA or precursor to it" or "reducing at least one TSNA or precursor to it" are used here to mean that the concentration and / or total content of at least one TSNA or precursor to it even in the product, method or use of the invention it is less in relation to a comparable product, method or use. For example, a comparable tobacco product would be derived from a tobacco plant that has not been modified in accordance with the present invention, but in which all other relevant characteristics are the same (for example, plant species, growing conditions , tobacco processing method, etc.).
[0187] [0187] Any method known in the art to determine the concentration and / or levels of at least one TSNA or precursor to it can be used. In particular, a method that can comprise the addition of deuterium-labeled internal standard, aqueous extraction and filtration can be used, followed by analysis using reverse phase high-performance liquid chromatography with tandem mass spectrometry (LC-MS / MS ). Other examples for determining the concentration and / or level of a tobacco specific nitrosamine precursor include a method such as that detailed in the method recommended by CORESTA CRM-72: Determination of tobacco-specific nitrosamines in tobacco and tobacco products by LC- MS / MS; CRM being developed at ISO / DIS 21766 or Wagner et al. Analytical Chemistry (2005), 77 (4), 1001-1006, which are incorporated herein by reference.
[0188] [0188] Suitably, the concentration and / or the total content of at least one nitrosamine or precursor thereof specific for tobacco can be reduced by carrying out a method and / or use of the present invention. Suitably, the concentration and / or level of at least one nitrosamine or precursor thereof specific to tobacco can be reduced in a tobacco plant of the invention (for example, obtainable or obtained by a method and / or use of the invention) when compared the concentration and / or level of at least one specific tobacco nitrosamine (s) or precursor thereof in a tobacco plant that has not been modified according to the present invention.
[0189] [0189] The concentration and / or the total content of at least one specific tobacco nitrosamine or precursor thereof can be reduced in a tobacco leaf, harvested leaf, processed tobacco leaf, tobacco industry product or combinations thereof or obtained from a tobacco plant (or part of a tobacco plant or a tobacco cell culture) of the invention, when compared to a tobacco leaf, harvested leaf, processed tobacco leaf, tobacco industry product or combinations donate the same obtainable or obtained from a tobacco plant (or part of a tobacco plant or a tobacco cell culture) that has not been modified according to the present invention.
[0190] [0190] Suitably, the concentration and / or total content of at least one specific tobacco nitrosamine or precursor thereof can be reduced in a processed tobacco leaf.
[0191] [0191] Suitably, the concentration and / or level of at least one specific tobacco nitrosamine or precursor to it can be reduced in a tobacco industry product.
[0192] [0192] In one embodiment, at least one tobacco specific nitrosamine or precursor thereof can be reduced by at least about 1%, at least about 3%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least least about 90%. In some embodiments, at least one tobacco-specific nitrosamine or precursor thereof can be reduced between about 5% and about 95%, between about 10% and about 90%, between 20% and about 80%, between 30% and about 70%, or between about 40% and 60%.
[0193] [0193] In relation to processed (for example, cured) tobacco leaves (for example, cured or reconstituted), at least one tobacco specific nitrosamine or precursor to it can be reduced between about 5000 ng / g and about 50 n9 / 9, between about 4000 n9g / g and about 100 ng / g, between about 3000 ng / g and 500 ng / g or between 2000 ng / g and 1000 ng / g. In some embodiments, at least one tobacco specific nitrosamine or precursor thereof can be reduced by at least about 5000 ng / g, at least about 4000 ng / g, at least about 3000 ng / g, at least about 2000 ng / g, at least about
[0194] [0194] The term "a comparable product", as defined here, would be a derivative of a plant (for example, a tobacco plant) that has not been modified according to the present invention, but in which all other relevant characteristics were the same (for example, plant species, growing conditions, plant processing method, for example, tobacco etc.). The comparable product according to the present invention can mean a plant (for example, a tobacco plant) or a part of it, such as a leaf (for example, a tobacco leaf), a harvested leaf (for example, a leaf harvested tobacco leaf), cut leaf (for example, cut harvested tobacco leaf), processed leaf (for example, processed tobacco leaf) or plant propagating material (for example, tobacco plant propagating material ) or a product comprising said plant or part, for example, a tobacco product or its combinations obtainable or obtained from a plant that has not been modified according to the present invention, for example, to modulate the activity or expression of a gene Nicl ERF (or one or more Nicl ERF genes in combination with one or more Nic2 ERF genes). Comparable products can also be known as controls or wild type. In one embodiment, a comparable product is one that does not comprise a Nicl ERF gene whose activity or expression has been modulated. In one embodiment, a comparable product is one that does not comprise a Nicl ERF gene whose activity or expression has been modulated or a Nic2 ERF gene whose activity or expression has been modulated.
[0195] [0195] The term "unmodified plant", as defined here, would be a plant (for example, a tobacco plant) that has not been modified according to the present invention, to modulate the activity or expression of a Nicl ERF gene and in which all other relevant characteristics were the same (for example, plant species, cultivation conditions, tobacco processing method, etc.). In one embodiment, an unmodified plant is one that does not comprise a Nicl ERF gene whose activity or expression has been modulated. In one embodiment, a comparable product is one that does not comprise a Nicl ERF gene whose activity or expression has been modulated or a Nic2 ERF gene whose activity or expression has been modulated.
[0196] [0196] Suitable plants according to the invention include plants of the Solanaceae family, which include, for example, tobacco, tomatoes, datura, eggplant, mandrake, belladonna, peppers (paprika, pepper) and potatoes. In one embodiment, a suitable genus of Solanaceae is Solanum, for example, Solanum lycopersicum or Solanum tuberosum. In one embodiment, a possible genus of Solanaceae is Nicotiana. Suitably, Nicotiana can be a species of Nicotiana tabacum. An appropriate species of Nicotiana can be designated here as a tobacco plant, or simply tobacco.
[0197] [0197] The "activity or expression" of a Nicl ERF gene (or a Nic2 ERF gene) can refer to the level of transcription, translation, that is, protein expression, or the activity of the protein encoded by the Nicl ERF gene (or the Nic2 ERF gene, respectively). The activity of a Nicl ERF gene (or a Nic2 ERF gene) is related to its ability to function as a transcription factor in alkali biosynthesis. The activity of a Nicl ERF gene (or a Nic2 ERF gene) can be determined by measuring the products of the synthesis of alkaloids, that is, measuring the content of alkaloids.
[0198] [0198] According to one aspect of the invention, gene expression can be decreased (or inhibited) by inhibiting transcription and / or translation. In one embodiment, the activity or expression of a gene can refer to the level of transcription, that is, the amount of mRNA produced, or translation, that is, the level or amount of protein produced.
[0199] [0199] In some embodiments, modulation of the alkaloid content refers to an increase in the alkaloid content, in which the activity or expression of at least one Nicl ERF gene is increased.
[0200] [0200] In some embodiments, modulation of the alkaloid content refers to a decrease in the alkaloid content, in which the activity or expression of at least one Nicl ERF gene is decreased (or inhibited).
[0201] [0201] In some embodiments, modulation of the alkaloid content refers to an increase in the alkaloid content, where the activity or expression of at least one Nicl ERF gene and the activity or expression of at least one of Nic2 ERF is increased in combination.
[0202] [0202] In some embodiments, modulation of the alkaloid content refers to a decrease in the alkaloid content in which the activity or expression of at least one Nicl ERF gene and the activity or expression of at least one Nic2 ERF is decreased (or inhibited ) in combination.
[0203] [0203] In an additional aspect, the content of alkaloids is measured from the leaves. In one aspect, the content of alkaloids is measured from green leaves. In an additional aspect, the content of alkaloids is measured from cured leaves, for example, air-cured, combustion-cured, fire-cured or sun-cured leaves. In an additional aspect, the alkaloid content is measured from combustion-cured leaves. In an additional aspect, the content of alkaloids is measured from air-cured leaves.
[0204] [0204] The term "alkaloid content" is used here to mean the concentration and / or total amount of the entire group of compounds classified as alkaloids. Alkaloids normally present in tobacco include nicotine, anatabine, anabasin, myosmin and nornicotine. In one embodiment, the content of one or more alkaloids selected from nicotine, anatabine, anabasine, myosmin and nornicotine is modulated. In one embodiment, the content of one or more alkaloids selected from nicotine, anatabine, anabasine, myosmin and nornicotine is reduced. In one embodiment, the content of one or more alkaloids selected from nicotine, anatabine, anabasine and nornicotine is increased. Adequate nicotine content is modulated. In one embodiment, the nicotine content is reduced.
[0205] [0205] Any method known in the art to determine the concentration and / or the total content of alkaloids can be used. A preferred method for analyzing the alkaloid content involves analysis by the gas ion chromatography (GC-FID) flame ionization detection method.
[0206] [0206] In one embodiment, a method is provided for producing a plant (for example, a tobacco plant) or part of it, a plant propagating material (for example, a tobacco plant propagating material), a cell (for example, a tobacco cell), a leaf (for example, a tobacco leaf), a harvested leaf (for example, a harvested tobacco leaf), a cut leaf (for example, a cut tobacco leaf), a processed leaf (for example, a processed tobacco leaf), a cut and processed leaf (for example, a cut and processed tobacco leaf), a product comprising said plant or part of it (for example, a tobacco product) or their combinations obtainable or obtained by a plant of the invention having a modulated alkaloid content, the method comprising modifying said tobacco to modulate the activity or expression of a Nicl ERF gene. The content of modulated alkaloids can be determined by comparing the content of alkaloids in the plant (eg tobacco plant) or part of it, plant propagation material (eg tobacco plant propagation material), a cell ( for example a tobacco cell), leaf (for example, tobacco leaf) harvested leaves (for example, harvested tobacco leaves), harvested leaves (for example, harvested tobacco leaves), processed leaves (for example, tobacco leaves processed), cut and processed leaves (e.g., cut and processed tobacco leaves), a product comprising a plant or part thereof, of the present invention, for example, a tobacco product, or combinations thereof with a comparable product.
[0207] [0207] Suitably, the content of alkaloids can be modulated in a plant, for example, a tobacco plant, for example, modified tobacco plant.
[0208] [0208] In one embodiment, the content of one or more alkaloids selected from nicotine, anatabine, anabasine, myosmin and nornicotine is decreased.
[0209] [0209] Suitably, the modulation of the alkaloid content described above may be a decrease in the nicotine content.
[0210] [0210] In one embodiment, the nicotine content of a modified plant (eg, tobacco plant), plant propagating material (eg, tobacco plant propagating material), leaf (eg, tobacco leaf ), harvested leaf (for example, harvested tobacco leaf), cut leaf (for example, harvested cut tobacco leaves), processed leaf (for example, processed tobacco leaf), and processed and cut leaf (for example, cut leaf) processed and cut tobacco) or tobacco product from a modified tobacco plant is decreased.
[0211] [0211] In one embodiment, the alkaloid content of a plant (e.g. tobacco plant) or part of it can be modulated by at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times when compared to the alkaloid content of a plant (eg tobacco plant) or part of it, respectively, that has not been modified to modulate the activity or expression of at least one Nicl ERF gene (or at least one Nicl ERF gene in combination with at least one Nic2 ERF gene) that was grown under similar growth conditions. Suitably, the content of alkaloids can be modulated about twice to about 10 times, preferably about 3 times to about 10 times, suitably about 3 times to about 5 times. Suitably, the modification can be an increase or a decrease in the content of alkaloids. Suitably, the modulation may be of one or more alkaloids selected from nicotine, anatabine, anabasine, myosmin and nornicotine. Suitably, the change is in the nicotine content.
[0212] [0212] In one embodiment of the invention, the alkaloid content of a plant (for example, a tobacco plant) or part of it can be modulated by 1%, 2%, 5%, 8%, 10%, 12% , 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% or 90% compared to a plant (for example, a tobacco plant) or part of it that does not has been modified in accordance with the present invention. Modulation can be an increase or a decrease in the content of alkaloids when compared to an unmodified plant (for example, a tobacco plant) or part of it. Suitably, the modulation may be of a total alkali content. Suitably, the modulation may be of one or more alkaloids selected from nicotine, anatabine, anabasine, myosmin and nornicotine. Suitably, the change is in the nicotine content.
[0213] [0213] In one embodiment, the method or use results in modulated alkaloid content compared to a plant (for example, a tobacco plant) or part of it that has not been modified to modulate the activity or expression of a Nicl ERF gene ( or a Nicl ERF gene and a Nic2 ERF gene) and, more particularly, in comparison with, or in relation to the expression of a plant (e.g., a tobacco plant) in the absence of the modulations introduced.
[0214] [0214] In one embodiment, a plant (for example, a tobacco plant) or part of it has been modified to achieve a modulation in the content of alkaloids compared to a plant (for example, a tobacco plant) or part of it , respectively, that has not been modified to modulate the activity or expression of at least one Nicl ERF gene (or at least one Nicl ERF gene and at least one Nic2 ERF gene).
[0215] [0215] The term "modifying" or "modified" as used here, means a plant (for example, a tobacco plant) that has been altered or modified. The present invention comprises the modification of plants using techniques of genetic modification of plants or non-genetic modification of plants. Such methods are well known in the art and examples of genetic modification techniques include transformation methods, transgenic, cisgenic and gene editing methods. Examples of non-genetic modification techniques include fast neutron mutagenesis, chemical mutagenesis, for example, ethyl methanesulfanate (EMS) mutagenesis and modern population analysis approaches.
[0216] [0216] In one embodiment, a natural variant that has a modified Nicl ERF gene is selected and that trait or gene is produced in a second plant that has commercially desirable traits.
[0217] [0217] In one embodiment, the plant (for example, a tobacco plant) according to the invention can be a transgenic plant.
[0218] [0218] In another embodiment, the plant (for example, a tobacco plant) according to the invention can be a non-transgenic plant.
[0219] [0219] Suitably, the modulation of at least one Nicl ERF gene (or at least one Nicl ERF gene and at least one Nic2 ERF gene in combination) is not present in LA Burley 21.
[0220] [0220] Suitably, modulation of at least one Nicl ERF gene (or at least one Nicl ERF gene and at least one Nic2 ERF gene in combination) is not present in Burley 21.
[0221] [0221] In some embodiments, a modification that decreases the activity or expression of at least one Nicl ERF gene (or at least one Nic2 ERF gene) and therefore decreases the alkaloid content that is selected from the group consisting of: decrease, prevent or attenuate the transcription, translation or expression of at least one Nicl ERF gene (or both, at least one Nicl ERF gene and at least one Nic2 ERF gene); inhibiting the synthesis of the encoded polypeptide by at least one Nicl ERF gene (or both, at least one Nicl ERF gene and at least one Nic2 ERF gene in combination), or its release from intracellular storage; or increasing the rate of degradation of the polypeptide encoded by at least one Nicl ERF gene (or at least one Nicl ERF gene and at least one Nic2 ERF gene in combination).
[0222] [0222] In one embodiment, the modification that decreases the activity or expression of at least one Nicl ERF gene (or one or more modifications that decrease the activity of at least one Nicl ERF gene and at least one Nic2 ERF gene in combination) comprises a mutation of one or more ERF genes.
[0223] [0223] In one embodiment, the mutation excludes one or more entire ERF genes.
[0224] [0224] In one embodiment, one or more EFERF genes can comprise one or more mutations in the genes. Suitably, one or more mutations result in reduced or eliminated gene activity in the mutant gene. In one embodiment, one or more mutations result in an inactive gene. In one embodiment, the mutation can be a deletion. In one embodiment, the mutation can be an insertion. In one embodiment, the mutation can introduce an early stop codon. In one embodiment, the target site is unique to the target ERF gene and does not exist in other ERF genes. In one embodiment, the mutation targets the 5'-end of the protein coding region.
[0225] [0225] In one embodiment, the mutation is a meaningless mutation.
[0226] [0226] In one embodiment, the mutants have reduced levels of total alkaloid and / or reduced nicotine.
[0227] [0227] In one embodiment, the present invention provides one or more mutations in a Nicl ERF gene encoding a polypeptide comprising (or consisting of) the amino acid sequence SEQ ID No. 4 or a sequence of at least 90%, preferably at least 96% identity with it.
[0228] [0228] In one embodiment, the present invention provides one or more mutations in a Nicl ERF gene that encodes a polypeptide comprising (or consisting of) amino acid sequence SEQ ID No. 8 or a sequence of at least 90%, preferably at least 96% identity with the same.
[0229] [0229] In one embodiment, the present invention provides one or more mutations in a Nicl ERF gene that encodes a polypeptide comprising (or consisting of) the amino acid sequence SEQ ID No. 12 or a sequence of at least 90%, preferably at least least 96% identity with it.
[0230] [0230] In one embodiment, the present invention provides one or more mutations in a Nicl ERF gene that encodes a polypeptide comprising (or consisting of) the amino acid sequence SEQ ID No. 16 or a sequence of at least 90%, preferably at least least 96% identity with it.
[0231] [0231] In one embodiment, the present invention provides one or more mutations in a Nicl ERF gene that encodes a polypeptide comprising (or consisting of) the amino acid sequence SEQ ID No. 20 or a sequence of at least 90%, preferably at least least 96% identity with it.
[0232] [0232] In one embodiment, the present invention provides one or more mutations in a Nicl ERF gene that encodes a polypeptide comprising (or consisting of) the amino acid sequence SEQ ID No. 24 or a sequence of at least 90%, preferably at least least 96% identity with it.
[0233] [0233] In one embodiment, the present invention provides one or more mutations in a Nicl ERF gene that encodes a polypeptide comprising (or consisting of) the amino acid sequence SEQ ID No. 28 or a sequence of at least 90%, preferably at least least 96% identity with it.
[0234] [0234] In one embodiment, the present invention provides one or more mutations in a Nicl ERF gene that encodes a polypeptide comprising (or consisting of) the amino acid sequence SEQ ID No. 32 or a sequence of at least 90%, preferably at least least 96% identity with it.
[0235] [0235] In one embodiment, the present invention provides one or more mutations in a Nicl ERF gene that encodes a polypeptide comprising (or consisting of) the amino acid sequence SEQ ID No. 36 or a sequence of at least 90%, preferably at least least 96% identity with it.
[0236] [0236] In one embodiment, the present invention provides one or more mutations in a Nicl ERF gene comprising (or consisting of) the nucleotide sequence as set out in: SEQ ID No. 1 or a sequence of at least 90%, preferably at least least 96% identity with it.
[0237] [0237] In one embodiment, the present invention provides one or more mutations in a Nicl ERF gene comprising (or consisting of) the coding sequence set forth in: SEQ ID No. 3 or a sequence of at least 90%, preferably at least 96% identity with the same.
[0238] [0238] In one embodiment, the present invention provides one or more mutations in a Nicl ERF gene comprising (or consisting of) the nucleotide sequence set forth in: SEQ ID No. 5 or a sequence of at least 90%, preferably at least 96% identity with the same.
[0239] [0239] In one embodiment, the present invention provides one or more mutations in a Nicl ERF gene comprising (or consisting of) the nucleotide sequence set forth in: SEQ
[0240] [0240] In one embodiment, the present invention provides one or more mutations in a Nicl ERF gene comprising (or consisting of) the nucleotide sequence set forth in: SEQ ID No. 13 or a sequence of at least 90%, preferably at least 96% identity with the same.
[0241] [0241] In one embodiment, the present invention provides one or more mutations in a Nicl ERF gene comprising (or consisting of) the nucleotide sequence set forth in: SEQ ID No. 17 or a sequence of at least 90%, preferably at least 96% identity with the same.
[0242] [0242] In one embodiment, the present invention provides one or more mutations in a Nicl ERF gene comprising (or consisting of) the nucleotide sequence set forth in: SEQ ID No. 21 or a sequence of at least 90%, preferably at least 96% identity with the same.
[0243] [0243] In one embodiment, the present invention provides one or more mutations in a Nicl ERF gene comprising (or consisting of) the nucleotide sequence set forth in: SEQ ID No. 25 or a sequence of at least 90%, preferably at least 96% identity with the same.
[0244] [0244] In one embodiment, the present invention provides one or more mutations in a Nicl ERF gene comprising (or consisting of) the nucleotide sequence set forth in: SEQ ID No. 29 or a sequence of at least 90%, preferably at least 96% identity with the same.
[0245] [0245] In one embodiment, the present invention provides one or more mutations in a Nicl ERF gene comprising (or consisting of) the nucleotide sequence set forth in: SEQ ID No. 33 or a sequence of at least 90%, preferably at least 96% identity with the same.
[0246] [0246] By way of example, the present method may comprise: º providing a mutation in a nucleic acid sequence encoding a protein comprising the amino acid sequence shown as SEQ ID No. 4; or SEQ ID No. 8; or SEQ ID No. 12; or SEQ ID No. 16; or SEQ ID No. 20; or SEQ ID No. 24; or SEQ ID No. 28; or SEQ ID No. 32; or SEQ ID No. 36 or an amino acid sequence that has at least 70% (preferably at least 80%, preferably at least 90%, preferably at least 96%, preferably at least 98%) sequence identity therewith; Provision of a mutation in a promoter for a nucleic acid sequence encoding a protein comprising the amino acid sequence shown as SEQ ID No. 4; or SEQ ID No. 8; or SEQ ID No. 12; or SEQ ID No. 16; or SEQ ID No. 20; or SEQ ID No. 24; or SEQ ID No. 28; or SEQ ID No. 32; or SEQ ID No. 36 or an amino acid sequence that has at least 70% (preferably at least 80%, preferably at least 90%, preferably at least 296%, preferably at least 98%) sequence identity therewith;
[0247] [0247] In one embodiment, as well as the modulation (for example, mutation) of one or more Nicl ERF genes, one or more Nic2 ERF genes are also modulated (for example, mutated).
[0248] [0248] Suitably, any of the modifications of the Nicl ERF gene (for example, mutations) taught here can be used in combination with one or more modifications of a Nic2 ERF gene in which the Nic2 ERF gene encodes a polypeptide comprising a sequence of amino acids, as set out in: SEQ ID No. 40; or SEQ ID No. 44; or SEQ ID No. 48; or SEQ ID No. 52; or SEQ ID No. 56; or SEQ ID No. 60; or SEQ ID No. 64; or SEQ ID No. 68; or SEQ ID No. 72 or a functional variant or functional or orthologous fragment thereof; or the Nic2 ERF gene comprises a nucleotide sequence as set out in: SEQ ID No. 37; or SEQ ID No. 41; or SEQ ID No. 45; or SEQ ID No. 49; or SEQ ID No. 53; or SEQ ID No. 57; or SEQ ID No. 61; or SEQ ID No. 65;
[0249] [0249] By way of example, the present method may comprise: providing a mutation in a nucleic acid sequence encoding a protein comprising the amino acid sequence shown as SEQ ID No. 40; or SEQ ID No. 44; or SEQ ID No. 48; or SEQ ID No. 52; or SEQ ID No. 56; or SEQ ID No. 60; or SEQ ID No. 64; or SEQ ID No. 68; or SEQ ID No. 72 or an amino acid sequence that has at least 70% (preferably at least 80% preferably at least 90%, preferably at least 296%, preferably at least 98%) sequence identity therewith; Provision of a mutation in a promoter for a nucleic acid sequence encoding a protein comprising the amino acid sequence shown as SEQ ID No. 40; or SEQ ID No. 44; or SEQ ID No. 48; or SEQ ID No. 52; or SEQ ID No. 56; or SEQ ID No. 60; or SEQ ID No. 64; or SEQ ID No. 68; or SEQ ID No. 72 or an amino acid sequence that has at least 70% (preferably at least 80%, preferably at least 90%, preferably at least 296%, preferably at least 98%) sequence identity with the same with the same; º provision of a mutation in a nucleic acid sequence of an ERF gene comprising SEQ ID No. 37; or SEQ ID No. 41; or SEQ ID No. 45; or SEQ ID No. 49; or SEQ ID No. 53; or SEQ ID No. 57; or SEQ ID No. 61; or SEQ ID No. 65;
[0250] [0250] In one embodiment, a mutation in at least one Nicl ERF gene is used in combination, selected from the group consisting of one or more mutations in a nucleic acid sequence that encodes a protein comprising the amino acid sequence shown as SEQ ID No. 4; or SEQ ID No. 8; or SEQ ID No. 12; or SEQ ID No. 16; or SEQ ID No. 20; or SEQ ID No. 24; or SEQ ID No. 28; or SEQ ID No. 32; or SEQ ID No. 36 or an amino acid sequence that has at least 70% (preferably at least 80%, preferably at least 90%, preferably at least 96%, preferably at least 98%) with sequence identity thereto with the same, or one or more mutations in a nucleic acid sequence of an ERF gene comprising SEQ ID No. 1; or SEQ ID No. 3; or SEQ ID No. 5; or SEQ ID No. 9; or SEQ ID No. 13; or SEQ ID No. 17; or SEQ ID No. 21; or SEQ ID No. 25; or SEQ ID No. 29; or SEQ ID No. 33 or a nucleotide sequence that has at least 70% (preferably at least 80%, preferably at least 90%, preferably at least 96%, preferably at least 98%) sequence identity therewith; and a mutation in at least one Nic2 ERF gene, particularly one or mutations in the nucleotide sequence that encodes the amino acid sequence SEQ ID No. 40, SEQ ID No. 44, SEQ ID No. 48, SEQ ID No. 52 or SEQ ID No. 56, SEQ ID No. 64, SEQ ID No. 68 or SEQ
[0251] [0251] In one embodiment, a mutation in at least one Nicl ERF gene that consists of one or more mutations in a nucleic acid sequence encoding a protein comprising the amino acid sequence shown as SEQ ID No. 8; or an amino acid sequence that is at least 70% (preferably at least 80%, preferably at least 290%, preferably at least 296%,
[0252] [0252] In one embodiment, a mutation in at least one Nicl ERF gene which consists of one or more mutations in a nucleic acid sequence encoding a protein comprising the amino acid sequence shown as SEQ ID No. 8; or an amino acid sequence that has at least 70% (preferably at least 80%, preferably at least 290%, preferably at least 296%, preferably at least 98%) sequence identity to the same, or one or more mutations in an ERF gene nucleic acid sequence comprising SEQ ID No. 5; or a nucleotide sequence that has at least 70% (preferably at least 80%, preferably at least 90%, preferably at least 96%, preferably at least 98%) sequence identity therewith; and a mutation in at least one Nic2 ERF gene that consists of one or more mutations in a nucleotide sequence that encodes the amino acid sequence as shown in SEQ ID No. 72 or an amino acid sequence that is at least 70% (preferably at least 80%, preferably at least 290%, preferably at least 96%, preferably at least 98%) sequence identity to the same, or one or more mutations in a nucleotide sequence as shown in SEQ ID No. 69 or a nucleotide sequence that has at least 70% (preferably at least 80%, preferably at least 90%, preferably at least 96%, preferably at least 98%) sequence identity therewith.
[0253] [0253] One or more Nic2 ERF genes can be one, or two, or three, or four, or five, or six, or seven, eight, or nine, Nic2 ERF genes selected in Table 2.
[0254] [0254] In some embodiments, a modification that decreases the activity or expression of at least one Nicl ERF gene (or at least one Nicl ERF gene and at least one Nic2 ERF gene in combination) and thus decreases the content of alkaloids is one or more selected from the group consisting of a point mutation, an exclusion, an insertion, a duplication and an inversion in one or more ERF genes. Suitably, the modification is introduced by a selected method of random mutagenesis and targeted mutagenesis. Suitably, the modification can be introduced by a method of targeted mutagenesis selected from meganuclease, zinc finger nuclease, TALEN, gene editing and CRISPR, for example.
[0255] [0255] As used here, the term "mutation" encompasses a natural genetic variant or a manipulated variant.
[0256] [0256] In particular, the term "mutation" refers to a variation in the amino acid sequence compared to the sequence shown as SEQ ID No. 4; or SEQ ID No. 8; or SEQ ID No. 12; or SEQ ID No. 16; or SEQ ID No. 20; or SEQ ID No. 24; or SEQ ID No. 28; or SEQ ID No. 32; or SEQ ID No. 36 or an amino acid sequence that has at least 70% sequence identity, which reduces the expression or function of the protein.
[0257] [0257] The term "mutation" can refer to a variation in the nucleotide sequence compared to the sequence shown as in SEQ ID No. 1; or SEQ ID No. 3; or SEQ ID No. 5; or SEQ ID No. 9; or SEQ ID No. 13; or SEQ ID No. 17; or SEQ ID No. 21; or SEQ ID No. 25; or SEQ ID No. 29; or SEQ ID No. 33 or in a nucleotide sequence that has at least 70% sequence identity thereto.
[0258] [0258] In a preferred embodiment, each copy of a nucleic acid sequence as shown in SEQ ID No. 1; or SEQ ID No. 3; SEQ ID No. 5; or SEQ ID No. 9; or SEQ ID No. 13; or SEQ ID No. 17; or SEQ ID No. 21; or SEQ ID No. 25; or SEQ ID No. 29; or SEQ ID No. 33 or in a sequence of at least 70%
[0259] [0259] In a preferred embodiment, the plant or plant cell according to the present invention is homozygous for the mutation.
[0260] [0260] In one embodiment, preferably the plant or plant cell according to the present invention expresses only the mutated nucleic acid. In other words, in some embodiments, no endogenous (or endogenous and functional) protein is present in the plants according to the present invention. In other words, if any endogenous protein is present, it is preferably in an inactive and / or truncated form.
[0261] [0261] The mutation can disrupt the nucleic acid sequence encoding a protein as detailed here.
[0262] [0262] Interruption can cause the nucleic acid sequence to not be transcribed and / or translated.
[0263] [0263] The nucleic acid sequence can be interrupted, for example, by deleting or modifying the ATG start codon of the nucleic acid sequence, so that protein translation is reduced or impeded.
[0264] [0264] The nucleic acid sequence may comprise one or more nucleotide changes that reduce or prevent protein expression or affect protein transport. For example, protein expression can be reduced or prevented by introducing one or more premature stop codons, a phase change, a splice mutation or an amino acid substitution not tolerated in the open reading phase.
[0265] [0265] A premature stop codon refers to a mutation that introduces a stop codon into the open reading phase and prevents the translation of the entire amino acid sequence. The premature stop codon can be a TAG (“amber”), TAA (“ocher”) or TGA (“opal” or “umber”) codon. Suitably, the premature stop codon can be introduced in Nitab4.5 0003090g0030.1 (ERF199); as shown in any of SEQ ID No. 5-7. Suitably, the premature stop codon in Nitab4.5 0003090g0030.1 (ERF199); as shown in any of SEQ ID No. 5-7 can be a TGA premature stop codon ("opal" or "umber").
[0266] [0266] A phase change mutation (also called a framing error or reading phase change) is a mutation caused by indels (insertions or deletions) of a number of nucleotides in a nucleic acid sequence that is not divisible by three. Due to the triple nature of gene expression by codons, insertion or exclusion can alter the reading frame, resulting in a completely different translation from the original. A phase change mutation will usually cause the codon reading after the mutation to encode different amino acids. The phase change mutation will generally result in the introduction of a premature stop codon.
[0267] [0267] A splice mutant inserts, deletes or changes a number of nucleotides at the specific site at which the splice occurs during the processing of the precursor messenger RNA into the mature messenger RNA. Deletion of the splicing site results in one or more introns remaining in the mature mRNA and can lead to the production of abnormal proteins.
[0268] [0268] An unsupported amino acid substitution refers to a mutation that causes a non-synonymous amino acid substitution in the protein that results in reduced or decreased protein function.
[0269] [0269] Any method known in the art to provide a mutation in a nucleic acid sequence can be used in the present method. For example, homologous recombination can be used, in which a vector is created in which the relevant nucleic acid sequences are mutated and used to transform plants or plant cells. Recombinant plants or plant cells that express the mutated sequence can then be selected.
[0270] [0270] The nucleic acid sequence can be totally or partially deleted. The deletion can be continuous or it can comprise a plurality of sequence sections. The deletion preferably removes a sufficient amount of the nucleotide sequence so that the nucleic acid sequence no longer encodes a functional protein. The deletion can, for example, remove at least 50, 60, 70, 80 or 90% of the coding portion of the nucleic acid sequence.
[0271] [0271] The deletion can be total, in the case where 100% of the coding portion of the nucleic acid sequence is absent, when compared with the corresponding genome a comparable unmodified plant.
[0272] [0272] Methods for deleting nucleic acid sequences in plants are known in the art. For example, homologous recombination can be used, in which a vector is created in which the relevant nucleic acid sequences are absent and used to transform plants or plant cells. Recombinant plants or plant cells that express the new portion of the sequence can then be selected.
[0273] [0273] Plant cells transformed with a vector as described above can be grown and maintained according to well-known tissue culture methods, such as culturing the cells in a suitable culture medium, provided with the necessary growth factors, such as amino acids , plant hormones, vitamins, etc.
[0274] [0274] Modification of the nucleic acid sequence can be performed using methods of targeted mutagenesis (also called targeted nucleotide exchange (TND) or oligo-directed mutagenesis (MOD)). Targeted mutagenesis methods include, without limitation, those employing zinc finger nucleases, TALENsS (see documents MWO2011 / 072246 and WO2010 / 079430), Cas9-like, Cas9 / crRNA / tracrRNA or Cas9 / gRNA CRISPR systems (see WO 2014/071006 and WO2014 / 093622),
[0275] [0275] Alternatively, mutagenesis systems such as TILLING (Targeting Induced Local Lesions IN Genomics; McCallum et al., 2000, Nat Biotech 18: 455 and McCallum et al.2000, Plant Physiol. 123, 439-442, both incorporated here by reference) can be used to generate plant strains that comprise a gene that encodes a protein with a mutation. The TILLING method uses traditional chemical mutagenesis (for example, ethyl methanesulfonate (EMS) mutagenesis) followed by high-throughput screening for mutations. Thus, plants, seeds and tissues comprising a gene with the desired mutation can be obtained.
[0276] [0276] The method may comprise the stages of plant seed mutagenesis (eg EMS mutagenesis), grouping of plants or individual DNA, PCR amplification of a region of interest, formation of heteroduplex and high yield detection, identification of mutant plant, sequencing of mutant PCR product. It is understood that other methods of mutagenesis and selection can also be used to generate such modified plants. The seeds can, for example, be irradiated or chemically treated and the plants can be screened for a modified phenotype.
[0277] [0277] Modified plants can be differentiated from unmodified plants, that is, wild type plants, by molecular methods, such as the mutations present in the DNA, and by the modified phenotypic characteristics. The modified plants can be homozygous or heterozygous for the mutation.
[0278] [0278] Suitably, the method may comprise the transformation of a plant cell (for example, a tobacco plant) with a genetic construct capable of inhibiting the activity or expression of at least one Nicl ERF gene (or a construct capable of inhibit the activity or expression of at least one Nicl ERF gene and at least one Nic2 ERF gene in combination).
[0279] [0279] In some embodiments, a modification that increases the activity or expression of at least one Nicl ERF gene (or at least one Nicl ERF gene and at least one Nic2 ERF gene in combination) and thus increases the content of alkaloids is selected from the group consisting of: increasing, promoting or increasing the transcription, translation or expression of at least one Nicl ERF gene (or at least one Nicl ERF gene and at least one Nic2 ERF gene in combination); increasing the synthesis of the polypeptide encoded by at least one Nicl ERF gene (or at least one Nicl ERF gene and at least one Nic2 ERF gene in combination), or their releases from intracellular reserves; or decreased rate of degradation of the polypeptide encoded by at least one Nicl ERF gene (or at least one Nicl ERF gene and at least one Nic2 ERF gene in combination).
[0280] [0280] Suitably, the method may comprise transforming a plant cell (for example, a tobacco plant) with a genetic construct that encodes at least one exogenous Nicl ERF gene (or that encodes at least one Nicl ERF gene and at least one Nic2 ERF gene in combination), or which comprises a nucleotide sequence encoding a protein that is capable of promoting or augmenting at least one endogenous Nicl ERF gene (or at least one endogenous Nicl ERF gene and at least one endogenous gene Nic2 ERF in combination). It will be appreciated that each of these options would result in increased activity and expression of the polypeptide encoded by at least one Nicl ERF gene (or at least one Nicl ERF gene and at least one Nic2 ERF gene in combination). The method can comprise the regeneration of the plant from the transformed cell.
[0281] [0281] Thus, the use of genetic construction is provided which is capable of increasing the activity and / or expression of a polypeptide encoded by at least one Nicl ERF gene (or at least one Nicl ERF gene and at least one Nic2 ERF gene in combination), to increase the alkaloid content in a plant transformed with construction.
[0282] [0282] The genetic construct can encode a polypeptide comprising the amino acid sequence as set out in: SEQ ID No. 4, SEQ ID No. 8, SEQ ID No. 12, SEQ ID No. 16 SEQ ID No. 20, SEQ ID No. 20, SEQ ID No. 24, SEQ ID No. 28, SEQ ID No. 32 or SEQ ID No. 36, or a functional variant or functional or orthologous fragment thereof. The construct can comprise the nucleotide sequence, as set out in: SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 9, SEQ ID No. 13, SEQ ID No. 17, SEQ ID No. 21, SEQ ID No. 25, SEQ ID No. 29 or SEQ ID
[0283] [0283] In some embodiments, a method or use according to the present invention comprises increasing the alkaloid content of a plant (for example, a tobacco plant) by increasing the activity or expression of a Nicl ERF gene and increasing the activity or expression of a Nic2 ERF gene.
[0284] [0284] Suitably, the method or use for increasing the alkaloid content comprises increasing the activity or expression of at least one Nicl ERF gene, wherein the at least one Nicl ERF gene encodes a polypeptide comprising an amino acid sequence substantially as established in: SEQ ID No. 4; or SEQ ID No. 8; or SEQ ID No. 12; or SEQ ID No. 16; or SEQ ID No. 20; or SEQ ID No. 24; or SEQ ID No. 28; or SEQ ID No. 32; or SEQ ID No. 36 or a functional variant or functional or orthologous fragment thereof; or wherein the ERF gene comprises a nucleotide sequence substantially as set forth in: SEQ ID No. 1; or SEQ ID No. 3; or SEQ ID No. 5; or SEQ ID No. 9; or SEQ ID No. 13; or SEQ ID No. 17; or SEQ ID No. 21; or SEQ ID No. 25; or SEQ ID No. 29; or SEQ ID No. 33 or a functional variant or functional or ortholog fragment thereof and a Nic2 ERF gene wherein the Nic2 ERF gene encodes a polypeptide comprising an amino acid sequence substantially as set forth in: SEQ ID No. 40; or SEQ ID No. 44; or SEQ ID No. 48; or SEQ ID No. 52; or SEQ ID No. 56; or SEQ ID No. 60; or SEQ ID No. 64; or SEQ ID No. 68; or SEQ ID No. 72 or a functional variant or functional or orthologous fragment thereof; or wherein the Nic2 ERF gene comprises a nucleotide sequence substantially as set forth in: SEQ ID No. 37; or SEQ ID No. 41; or SEQ ID No. 45; or SEQ ID No. 49; or SEQ ID No. 53; or SEQ ID No. 57; or SEQ ID No. 61; or SEQ ID No. 65; or SEQ ID No.
[0285] [0285] Suitably, the method or use for increasing the alkaloid content comprises increasing the activity or expression of at least one Nicl ERF gene, wherein the at least one Nicl ERF gene encodes a polypeptide comprising an amino acid sequence as set out in : SEQ ID No. 4; or SEQ ID No. 8; or SEQ ID No. 12; or SEQ ID No. 16; or SEQ ID No. 20; or SEQ ID No. 24; or SEQ ID No. 28; or SEQ ID No. 32; or SEQ ID No. 36 or a functional variant or functional or orthologous fragment thereof; or wherein the ERF gene comprises a nucleotide sequence as set out in: SEQ ID No. 1; or SEQ ID No. 3; or SEQ ID No. 5; or SEQ ID No. 9; or SEQ ID No. 13; or SEQ ID No. 17; or SEQ ID No. 21; or SEQ ID No. 25; or SEQ ID No. 29; or SEQ ID No. 33 or a functional variant or functional or ortholog fragment thereof and a Nic2 ERF gene, wherein the Nic2 ERF gene encodes a polypeptide comprising an amino acid sequence as set out in: SEQ ID No. 72 or a variant functional or functional or orthologous fragment thereof; or where the Nic2 ERF gene comprises a nucleotide sequence as set out in: SEQ ID No. 69.
[0286] [0286] The term "inhibition" (for example, inhibition of the activity or expression of a Nicl ERF gene), as used here, means that the activity or expression of the Nicl ERF gene is less or less compared to the activity or expression of the gene in a comparable product or the amount or activity of a protein produced by the Nicl ERF gene is less.
[0287] [0287] In one embodiment, the term "inhibition" (for example, inhibition of the activity or expression of a Nicl ERF gene), as used here, means that the activity or expression of the Nicl ERF gene is less compared to gene activity or expression of the gene in a comparable product.
[0288] [0288] The activity of specific Nicl ERF genes can be measured by measuring the transcription of the gene. Methods for measuring transcription are well known in the art and include, among others, Northern Blot, RNA-Segq, in situ hybridization, DNA sequencing and RT-PCR. Alternatively, the activity of a gene can be measured indirectly by measuring the level of the gene's product, for example, the protein encoded by that gene.
[0289] [0289] In some embodiments, the activity or expression of a Nicl ERF gene can be modulated, that is, increased or decreased by at least 10% 20% 30% or 40%, suitably at least about 50%, 60%, 70%, more appropriately at least about 80%, 90%, 95% or 100% when compared to the activity or expression of a Nicl ERF gene in a plant (for example, a tobacco plant) that has not been modified accordingly with the present invention.
[0290] [0290] Suitably, the expression or function of the Nicl ERF gene can be reduced, partially inactivated, inhibited, eliminated, eliminated or lost, so that the Nicl ERF protein expression or function is not detectable.
[0291] [0291] In one aspect, at least one MNicl ERF gene is knocked out. In other words, the Nicl ERF gene was completely inoperative.
[0292] [0292] In one aspect, a Nicl ERF gene encoding a polypeptide comprising an amino acid sequence as set out in SEQ ID No. 8 or a functional variant or functional or ortholog fragment thereof; or comprises a nucleotide sequence as set out in SEQ ID No. 5 or a functional variant or functional or orthologous fragment thereof is knocked out.
[0293] [0293] In a preferred embodiment, the Nicl ERF gene may have substantially no activity or expression, meaning that the plant may comprise less than about 1% (suitably less than about 0.1%) of activity or expression, preferably when compared to a plant that has not been modified to inhibit the activity or expression of a Nicl ERF gene.
[0294] [0294] In some embodiments, the activity or expression of a Nic2 ERF gene can be modulated, that is, increased or decreased by at least 10% 20% 30% or 40%, suitably at least about 50%, 60%, 70%, more appropriately at least about 80%, 90%, 95% or 100% when compared to the activity or expression of a Nic2 ERF gene in a plant (for example, a tobacco plant) that has not been modified accordingly with the present invention.
[0295] [0295] In a preferred embodiment, the Nic2 ERF gene may have substantially no activity or expression, meaning that the plant may comprise less than about 1%
[0296] [0296] An "ERF gene", as used here, refers to a transcription factor gene that belongs to the ethylene response factor (ERF) subfamily.
[0297] [0297] A "Nicl ERF gene", as used here, refers to an ERF gene that the present inventors have identified in the Examples here as mapping to the Nicl region. Nicl ERF genes, as used here are listed in Table 1 below, together with their corresponding nucleotide, cDNA, cds and amino acid sequence identifiers. Table 1. Nicl ERF strings.
[0298] [0298] Suitably, at least one Nicl ERF gene for use in the present invention is any of those listed in Table 1.
[0299] [0299] The genomic sequences of each of the Nicl ERFs and Nic genes ERFs listed in the tables above are identical to their corresponding coding strings, with the exception of Nicl ERF ERF17L3. The genomic sequence of ERF17L3 (SEQ ID No. 1) is not identical to the coding sequence of ERF17L3 (SEQ ID No. 3).
[0300] [0300] A "Nic2 ERF gene" ", as used here, refers to an ERF gene that the present inventors identified in the Examples here as mapping to the Nic2 region. The Nic2 ERF genes, as used here are listed in the Table 2 below together with their corresponding nucleotide, cDNA, cds and amino acid sequence identifiers Table 2. Nic2 ERF sequences.
[0301] [0301] Suitably, the Nic2 ERF gene for use in the present invention is any of those listed in Table 2.
[0302] [0302] In one embodiment, at least one Nicl ERF gene referred to herein can be encoded by a polynucleotide sequence comprising: i) a polynucleotide sequence shown here as SEQ ID No. 1, SEQ ID No. 3; SEQ ID No. 5, SEQ ID No. 9, SEQ ID No. 13, SEQ ID No. 17, SEQ ID No. 21, SEQ ID No. 25, SEQ ID No. 29 or SEQ ID No. 33; or ii) a functional fragment of the polynucleotide sequence shown in i) whose functional fragment encodes a Nicl ERF synthesis gene, or iii) a polynucleotide that encodes a polypeptide comprising the amino acid sequence shown here as SEQ ID No. 4, SEQ ID No. 8, SEQ ID No. 12, SEQ ID No. 12, SEQ ID No. 16, SEQ ID No. 20, SEQ ID No. 24, SEQ ID No. 28, SEQ ID No. 32 or SEQ ID No. 36, or iv) a polynucleotide sequence that can hybridize to the polynucleotide taught in i), ii) or iii) above under conditions of high restriction, or v) a polynucleotide sequence that has at least 70% (preferably 80%, preferably 85% preferably 290%, preferably 295%, more preferably 96%, more preferably 97%, more preferably 98%) identity with the polynucleotide shown in i), ii) or iii) above, or vi) a polynucleotide sequence which differs from the polynucleotide shown in i), ii) or iii) due to the degeneration of the g code enetic.
[0303] [0303] In one embodiment, at least one Nic2 ERF gene referred to here can be encoded by a polynucleotide sequence comprising:
[0304] [0304] In one embodiment, at least one Nicl ERF gene for use in accordance with the present invention can be endogenous to the plant (e.g., a tobacco plant).
[0305] [0305] In one embodiment, at least one Nic2 ERF gene for use in accordance with the present invention can be endogenous to the plant (e.g., a tobacco plant).
[0306] [0306] The reference mentioned here to an “endogenous” gene not only refers to the gene in question as found in a plant in its natural form (that is, without human intervention), but also refers to the same gene (or to a substantially homologous nucleic acid / gene) in an isolated form subsequently (re) introduced into a plant (a transgene) or a plant cell. For example, a transgenic plant containing that transgene may encounter a substantial reduction in the expression of the transgene and / or a substantial reduction in the expression of the endogenous gene. The isolated gene can be isolated from an organism or it can be made by man, for example, by chemical synthesis.
[0307] [0307] In another embodiment, at least one Nicl ERF gene for use in accordance with the present invention may be exogenous to the plant (e.g., a tobacco plant).
[0308] [0308] In another embodiment, at least one Nic2 ERF gene for use in accordance with the present invention may be exogenous to the plant (e.g., a tobacco plant).
[0309] [0309] The term "exogenous gene" may mean that the gene that is transformed into the unmodified plant is from an external source, that is, from a different species from the one that is being transformed. The exogenous ERF gene can comprise a nucleic acid sequence substantially the same or different from an endogenous ERF gene in the unmodified plant. The exogenous gene can be derived from a genomic or cDNA sequence corresponding to the ERF gene of any species. The exogenous gene can form a chimeric gene. The exogenous gene can encode a polypeptide comprising the amino acid sequence as set out in Table 11, or a functional variant or functional or orthologous fragment thereof. The exogenous gene can comprise the nucleotide sequence as shown in Table 2, or a functional variant or functional or orthologous fragment thereof.
[0310] [0310] The present invention also provides the use of a Nicl ERF gene to modulate a plant's alkaloid content.
[0311] [0311] In one embodiment, the invention also provides for the use of an additional ERF gene, wherein the additional ERF gene is a Nic2 ERF gene, as described here in Table 2.
[0312] [0312] Methods for decreasing the expression of genes or gene products are well documented in the state of the art. Any method described herein to modulate the activity or expression of a Nicl ERF gene can be used to modify the activity or expression of a Nicl ERF gene and a Nic2 ERF gene.
[0313] [0313] In one embodiment, the activity or expression of a Nicl ERF gene or the activity or expression of both Nicl ERF genes can be inhibited by any method known in the art. In another embodiment, the activity or expression of a Nicl ERF gene and the activity or expression of a Nic2 ERF gene can be inhibited by any method known in the art.
[0314] [0314] Methods to inhibit the activity or expression of a Nicl ERF gene or the activity or expression of a Nicl ERF gene and a Nic2 ERF gene can include gene editing, targeted mutagenesis, RNA interference, antisense cosense or sense (see Wang and Wagner 2003, Planta Volume 216, Edition 4, p. 686-691, which is incorporated here by reference). In one embodiment, inhibiting the activity or expression of a gene can be achieved by using gene editing. Gene editing can be performed using any method known in the art. Some non-limiting examples are presented here.
[0315] [0315] In one embodiment, inhibiting the activity or expression of a Nicl ERF gene or the activity or expression of both a Nicl ERF gene and a Nic2 ERF gene can be achieved using gene editing methods, including CRISPR, including the use of CRISPR / Cas9 system. CRISPR / Cas9 genomic editing tools are commercially available, such as Clontech's “Guide-it” (Avenida du President Kennedy 78100 Saint-Germain-en-Laye, France).
[0316] [0316] Suitably, to generate a PRGEB-M24 gene editing vector, the rice U3 snoRNA promoter in the pRGEB31 vector can be replaced by the M24 promoter amplified from pSiM24 (Sahoo et al., 2014 incorporated here by reference) via of cloning by assisted infusion with HindIII and Bsal. To maintain the integrity of the SsgRNA sequence and the BsaII recognition site in the pRGEB-M24 vector, the M24 promoter can first be amplified with primers linked to the B salt recognition site (HindIII EcoRI M24F: GATTACGCCAAGCTTTCCCGTATACCCCGGGGAATTCGT (SEQ ID No. 139);
[0317] [0317] The bases underlined in Table 3 above represent the antisense sense or sequences of target sites.
[0318] [0318] The oligo pairs are first ringed to produce a double stranded fragment with protrusions of 4 nucleotides 5 'at both ends and then ligated into the Bsal-digested PpRGEB-M24 vector.
[0319] [0319] Another gene editing method includes the use of TALEN (nuclease transcription activator effector) technology with commercially available kits (for example, from Addgene, lKendall Sq. Ste. B7102, Cambridge, MA 02139, USA). In one embodiment, inhibition of the activity or expression of at least one Nicl ERF gene or the activity or expression of both a Nicl ERF gene and at least one Nic2 ERF gene can be achieved using TALEN.
[0320] [0320] In another embodiment, the method may comprise the use of zinc finger nucleases, such as CompozrO zinc finger nuclease technology, available from Sigma-Aldrich. Another embodiment may comprise the use of meganucleases (or another method) described in Silva et al. Curr Gene Ther. Feb 2011; 11 (1): 11-27 (whose teaching is incorporated by reference).
[0321] [0321] In one embodiment, the method for inhibiting the activity or expression of a Nicl ERF gene or a Nic2 ERF gene can be the target of mutagenesis. Any method of targeted mutagenesis can be used. In one embodiment, the method can be oligonucleotide-directed mutagenesis (MDO), such as KeyBaseO available from Keygene (Agro Business Park 90, 6708 PW Wageningen, Netherlands). In another embodiment, inhibition of the activity or expression of a Nicl ERF gene or the activity or expression of a Nicl ERF gene and a Nic2 ERF gene can be achieved by using a construct or vector (for example, a plasmid).
[0322] [0322] The genetic constructs of the invention may be in the form of an expression cassette, which may be suitable for inhibiting the activity or expression of a Nicl ERF gene or the activity or expression of a Nicl ERF gene and a Nic2 ERF gene in a host cell or to increase the activity or expression of a Nicl ERF gene or the activity or expression of a Nicl ERF gene and a Nic2 ERF gene in a host cell. The genetic construct can be introduced into a host cell without being incorporated into a vector. For example, the genetic construct, which can be a nucleic acid molecule, can be incorporated into a liposome or virus particle. Alternatively, a purified nucleic acid molecule (for example, histone-free DNA or naked DNA) can be inserted directly into a host cell by suitable means, for example, uptake by direct endocytosis. The genetic construct can be introduced directly into the cells of an individual host (for example, a plant) by transfection, infection, microinjection, cell fusion, protoplast fusion or ballistic bombardment. Alternatively, the genetic constructs of the invention can be introduced directly into a host cell using a particle gun.
[0323] [0323] Alternatively, the genetic construct can comprise or be housed within a recombinant vector, for expression in a suitable host cell. The recombinant vector can be a plasmid, cosmid or phage. Such recombinant vectors are highly useful for transforming host cells with the genetic construct of the invention and for replicating the expression cassette therein. The skilled person will appreciate that the genetic constructs of the invention can be combined with many types of vector structure for expression. The structure of a vector can be a binary vector, for example, one that can replicate in E. coli and Agrobacterium tumefaciens. For example, a suitable vector can be a PBIN plasmid, such as pBIN19 (Bevan M., 1984, Nucleic Acids Research 12: 8711-21).
[0324] [0324] Recombinant vectors can include a variety of other functional elements, in addition to the sequence that inhibits the activity or expression of at least one Nicl ERF gene or the activity or expression of both, at least one Nicl ERF gene and at least one Nic2 ERF gene. For example, the vector can comprise a promoter. In addition, the recombinant vector can be designed so that it replicates autonomously in the cytosol of the host cell. In this case, elements that induce or regulate DNA replication may be needed in the recombinant vector. Alternatively, the recombinant vector can be designed in such a way that it integrates into the genome of a host cell. In this case, DNA sequences are envisaged that favor targeted integration (for example, by homologous recombination).
[0325] [0325] The recombinant vector can also comprise DNA encoding a gene that can be used as a selectable marker in the cloning process, that is, to allow the selection of cells that have been transfected or transformed and to allow the selection of cells that host vectors incorporating heterologous DNA. The vector can also comprise DNA involved in regulating the expression of the coding sequence or to target the expressed polypeptide to a particular part of the host cell, for example, glandular trichomes or trichomes. Therefore, the vector can comprise at least one additional element selected from a group consisting of: a selectable marker gene (for example, an antibiotic resistance gene); a polypeptide termination signal; and a protein targeting sequence (for example, a transit peptide).
[0326] [0326] In one embodiment, the method or use may comprise inhibiting the activity or expression of a Nicl ERF gene or the activity or expression of a Nicl ERF gene and a Nic2 ERF gene using an interfering oligonucleotide. In one embodiment, the oligonucleotide is based on RNA. In one embodiment, the oligonucleotide is the interfering RNA (RNAi), for example, dsRNAi. In one embodiment, the method may comprise transforming a cell in a plant (for example, a tobacco plant) with an RNAi molecule, for example, dsRNAi, which inhibits the activity or expression of a Nicl ERF gene or activity or expression of a Nicl ERF gene and a Nic2 ERF gene.
[0327] [0327] In one embodiment, the activity or expression of at least one Nicl ERF gene decreases by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75 %, 80%, 85%, 90%, 95% or more, or 100% compared to the activity or expression of the polypeptide in the wild type plant.
[0328] [0328] In one embodiment, the activity or expression of at least one Nic2 ERF gene is decreased by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, or 100% compared to the activity or expression of the polypeptide in the wild type plant.
[0329] [0329] In one embodiment, the activity or expression of both at least one Nicl ERF gene and at least one Nic2 ERF gene is reduced by at least 30%, 35%, 40%, 45%, 50%, 55%, 60 %, 65% 70%, 75%, 80%, 85%, 90%, 95% or more, or 100% compared to the activity or expression of the polypeptide in the wild type plant.
[0330] [0330] The activity or expression of at least one Nicl ERF gene or the activity or expression of both, at least one Nicl ERF gene and at least one Nic2 ERF gene, can be inhibited by any method known in the art. In any of the foregoing embodiments, the activity or expression of at least one Nicl ERF gene or the activity or expression of both, at least one Nicl ERF gene and at least one Nic2 ERF gene, can be inhibited by any method, including methods of gene editing including CRISPR, including the use of the CRISPR-Cas9 system, RNA interference (RNAi), antisense or sense cosuppression, gene editing or targeted mutagenesis. In any of the previous embodiments, the activity or expression of at least one Nicl ERF gene or the activity or expression of at least one Nicl ERF gene and at least one Nic2 ERF gene can be inhibited using an RNAi method, for example, using miRNA , siRNA, dsRNA or sSshRNA.
[0331] [0331] In one embodiment, the construct that modulates the activity or expression of the Nicl ERF gene or the activity or expression of at least one Nicl ERF gene and at least one Nic2 ERF gene can be comprised in a vector. Suitably, the vector can be a plasmid.
[0332] [0332] In one embodiment, the vector for use in the present invention is the Agrobacterium-based plasmid.
[0333] [0333] Consequently, in one embodiment plants (for example, a tobacco plant) and plant propagation materials (for example, a tobacco plant propagation material), the leaves (for example, tobacco leaves), harvested cut leaves, processed leaves (for example, processed tobacco leaves) or processed and cut leaves (for example, cut and processed tobacco leaves) are provided in which the expression of a Nicl ERF gene or the activity or expression of a gene Nicl ERF and a Nic2 ERF gene is modulated.
[0334] [0334] In another embodiment, the cell (for example, tobacco cell), plant (for example, a tobacco plant) or part of it and / or plant propagating material may apprehend a construction that modulates activity or expression of a Nicl ERF gene or the activity or expression of a Nicl ERF gene and a Nic2 ERF gene. In one embodiment, the construction decreases the activity or expression of a Nicl ERF gene or the activity or expression of both a Nicl ERF gene and a Nic2 ERF gene. In another embodiment, the construct increases the activity or expression of a Nicl ERF gene or the activity or expression of a Nicl ERF gene and a Nic2 ERF gene. In a further embodiment, the cell (e.g., tobacco cell), plant (e.g., a tobacco plant) or part of it and / or plant propagating material according to the invention may comprise: i) a sequence polynucleotides shown here as SEQ ID No. 1, SEQ ID No. 3; SEQ ID No. 5, SEQ ID No. 9, SEQ ID No.
[0335] [0335] In one embodiment, the cell (e.g., tobacco cell) is grown in a cell culture.
[0336] [0336] In one embodiment, at least one Nicl ERF gene (or at least one Nicl ERF gene and at least one Nic2 ERF gene) is used to modulate the content of alkaloids (eg, nicotine content) in a cell or culture cells (for example, a tobacco cell culture).
[0337] [0337] In an advantageous embodiment, inhibiting the activity or expression of at least one Nicl ERF gene or the activity or expression of both, at least one Nicl ERF gene and a minimum of one Nic2 ERF gene, can result in a decrease in the content of alkaloids. Adequate inhibition of the activity or expression of at least one Nicl ERF gene or the activity or expression of at least one Nicl ERF gene and at least one Nic2 ERF gene can result in a decrease in nicotine content.
[0338] [0338] In another embodiment, increasing the activity or expression of a Nicl ERF gene or the activity or expression of a Nicl ERF gene and a Nic2 ERF gene can result in an increase in the content of alkaloids. Properly increasing the activity or expression of a Nicl ERF gene or the activity or expression of a Nicl ERF gene and a Nic2 ERF gene can result in an increase in nicotine content.
[0339] [0339] In one embodiment, the plant or part of it is a tobacco plant. In one embodiment, the tobacco plant or part of it according to the present invention is a modified Burley or smoke cured plant according to the present invention. In one embodiment, the present invention relates to a modified smoke-cured or Burley plant according to the present invention. In one embodiment, the tobacco plant (e.g., modified tobacco plant) according to the present invention is an oriental or Turkish tobacco plant.
[0340] [0340] In one embodiment, the tobacco plant or part of it is cured. In one embodiment, the tobacco plant or part of it is cured, for example, air-cured, smoke-cured, fire-cured or sun-cured. In an additional aspect, the tobacco plant or part of it is cured in a smokehouse. In an additional aspect, the tobacco plant or part of it is air-cured.
[0341] [0341] Smoke curing is well known in the art and refers to the process of curing tobacco with smoke that is powered by fire boxes or gas powered systems. This process cures tobacco by heat without exposing it to smoke, slowly increasing the temperature during the curing. This method produces tobacco with a high sugar content and with medium to high levels of nicotine. The Smith Tobacco Barn is an example of a traditional smoke-cured tobacco barn.
[0342] [0342] Air-cured tobacco includes Burley, Maryland and dark tobacco. The common factor is that the cure is mostly without artificial sources of heat and moisture. Burley tobacco is light to dark brown in color, rich in oil and low in sugar. Burley tobacco is air-cured in barns. Burley's main growing countries are Argentina, Brazil, Italy, Malawi and the US Burley tobacco plants include, for example, Clay 402, Clay 403, Clay 502, Ky 14, Ky 14, Ky 907, Ky 910, Ky 8959, NC 2, NC 3, NC 4, NC 5, NC 2000, TN 86, TN 90, TN 97, R 610, R 630, R711, R 712, NCBH 129, Bu 21xKy 10, HBO4P, Ky 14xL 8, Kt 200, Newton 98, Pedigo 561, Pf561 and Va 509.
[0343] [0343] Maryland tobacco has good burning properties, low nicotine and a neutral aroma. Maryland's top growing countries include the USA and Italy. Dark air-cured tobacco is differentiated from other types mainly by the fermentation process, which gives air-cured tobacco its medium brown to dark brown color and distinct aroma. Its leaves are low in sugar, but high in nicotine. Dark air-cured tobacco is mainly used in the production of chewing and snuff tobacco. The main regions for growing dark tobacco with fire curing are Tennessee, Kentucky and Virginia, USA.
[0344] [0344] The term "functional fragment", as used here, refers to a portion of a polynucleotide that is capable of functioning in the same way as the polynucleotide. For example, if the polynucleotide is an ERF gene, then the functional fragment must be able to function as an ERF gene, for example, the functional fragment retains the activity of the ERF gene. The functional fragment can have an activity level that is equal to or greater than the activity level of a full-length polynucleotide.
[0345] [0345] In one embodiment, a functional fragment may be a portion of a Nicl ERF gene, as discussed here comprising at least 50, 75, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 continuous nucleotides. In some embodiments, the functional fragment can comprise at least 150 nucleotides from a Nicl ERF discussed here.
[0346] [0346] In one embodiment, a functional fragment of a Nic2 ERF gene can be a portion of a Nic2 ERF gene, as discussed here, comprising at least 50, 75, 100, 150, 200, 300, 300, 400, 500, 600, 700, 800, 900 or 1000 continuous nucleotides. In some embodiments, the functional fragment can comprise at least 150 nucleotides from a Nic2 ERF discussed here.
[0347] [0347] The term “functional variant”, as used here, refers to the variability that can arise in genomic sequences without significant loss of activity in gene function and / or protein function. For example, some amino acids present in a polypeptide (or some nucleotides present in a polynucleotide) can be replaced without significant loss of activity. The functional variant may have an activity level that is equal to, or greater than, the activity level of the non-variant polynucleotide and / or polypeptide. Sequences that differ from the ERF genes disclosed here due to the degeneracy of the genetic code are functional variants. A variant may differ from the sequence of interest by as few as 10, as few as 9, as few as 8, as few as 7 as few as 6, as few as 5, as few as 4, as few as 3, as few as 2 or as few as 1 amino acid.
[0348] [0348] The term “degeneration of the genetic code”, as used here, refers to the redundancy in codons that encode polypeptide sequences displayed as the multiplicity of combinations of three codons that specify an amino acid. For example, in an mRNA molecule that encodes a polypeptide that has an isoleucine amino acid, isoleucine can be encoded by AUU, AUC or AVA. This means that a DNA molecule encoding RNA can have multiple sequences, but the resulting polypeptide will have the same sequence. In other words, the polymorphic nucleotide sequences can encode the same polypeptide product. This means that a nucleic acid sequence can comprise a sequence with very low sequence identity with a second sequence while encoding the same polypeptide sequence.
[0349] [0349] Sequences that have a degree of sequence identity or sequence homology to the amino acid sequences of a polypeptide that has the specific properties described here or any nucleotide sequence described here can be functional variants.
[0350] [0350] The term "orthologist", as used here, refers to genes that are derived from a common ancestral gene and that are found in different species as a result of speciation. Orthologists can share at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97%, 98%, 99% or larger sequence identity in the nucleotide sequence and or at the amino acid sequence level. Orthologous genes generally share the same or similar functions, that is, they have a conserved function.
[0351] [0351] In some embodiments of the present invention, a promoter can be provided. The promoter for use in the present invention can be one or more selected from the group consisting of: a constitutive promoter, a senescence-specific promoter, a tissue-specific promoter, a development-regulated promoter and an inducible promoter. In one embodiment, the promoter can be a constitutive promoter.
[0352] [0352] A constitutive promoter directs the expression of a gene through the various parts of a plant continuously during the development of the plant, although the gene cannot be expressed at the same level in all cell types. Examples of known constitutive promoters include those associated with the 35S cauliflower mosaic virus transcript (odell JT, Nagy F, Chua NH (1985). Identification of DNA sequences required for activity of the cauliflower mosaic virus 358 promoter, Nature 313 810 -2), the rice actin 1 gene (Zhang W, McElroy D, Wu R. (1991) Analysis of rice Actl 5 'region activity in transgenic rice plants (Plant Cell 3 1155-65)) and the ubiquitin gene 1 of corn (Cornejo MJ, Luth D, Blankenship KM, Anderson OD, Blechl AE. (1993) Activity of à maize ubiquitin promoter in transgenic rice, Plant Molec. Biol. 23 567-81), which are incorporated by reference. constitutive promoters include the blackhead ring spot virus (CERV) promoter. The DNA sequence of the blackhead ring virus: comparison with the cauliflower mosaic virus and retrovirus ((Hull R, Sadler J, Longstaff M 1986 EMBO Journal, 5 (2): 3083-3090), which is incorporated here by reference).
[0353] [0353] The constitutive promoter can be selected from: a carnation ring spot virus (CERV) promoter, a cauliflower mosaic virus (CaMV 35S promoter), a promoter from the rice actin 1 gene or the corn ubiquitin 1 gene. Suitably, the promoter may be a CERV promoter.
[0354] [0354] Alternatively, in some embodiments, the promoter may not be a cauliflower mosaic virus (CaMV 35S promoter). In one embodiment, the promoter can be a specific senescence promoter. A "specific senescence promoter" (SAG) can be a mechanism that is associated with controlling the expression of a gene associated with senescence. In this way, the promoter can restrict the expression of a coding sequence (i.e., a gene) to which it is operationally linked substantially exclusively to the senescent tissue. Therefore, a specific senescence promoter may be a promoter capable of preferentially promoting gene expression in a plant tissue in a manner regulated by development, so that the expression of a 3'-protein coding region occurs substantially only when the plant tissue is going through senescence. It will be appreciated that senescence tends to occur in the older parts of the plant, such as the older leaves, and not in the younger parts of plants, such as the seeds.
[0355] [0355] An example of a plant that is known to express numerous genes associated with senescence is Arabidopsis Therefore, the promoter can be isolated from a gene associated with senescence in Arabidopsis. Gepstein et al. (The Plant Journal, 2003, 36, 629-642), incorporated herein by reference, conducted a detailed study of the SAGs and their promoters using Arabidopsis as a model. The genetic construct can comprise a promoter for any of the SAGs disclosed in this article. For example, a suitable promoter can be selected from a group consisting of SAG12, SAG13, SAG101, SAG21 and SAG18, or a functional variant or functional fragment thereof.
[0356] [0356] In one embodiment, the promoter can be a SAG12 or SAG13 promoter. In one embodiment, the promoter may be a SAG12 promoter, which will be known to the skilled person, or a functional variant or a functional fragment thereof (Gan & Amasino, 1997, Plant Physiology, 113: 313-319, incorporated by reference) . Suitable promoters and their sequences can be found in WO2010 / 097623 (incorporated herein by reference).
[0357] [0357] In another embodiment, the promoter can be a tissue-specific promoter. A tissue-specific promoter is one that directs the expression of a gene in one (or some) parts of a plant, usually throughout the useful life of those parts of the plant. The category of tissue-specific promoter also generally includes promoters whose specificity is not absolute, that is, they can also target expression at a lower level in tissues other than the preferred tissue. Several tissue-specific promoters are known in the art and include those associated with the patatin gene expressed in the potato tuber and the high molecular weight glutenin gene expressed in the endosperm of wheat, barley or corn. Any of these promoters can be used in the present invention.
[0358] [0358] Suitably, the tissue specific promoter can be a leaf specific promoter. Leaf specific promoters may suitably include ASYMMETRIC LEAVES 1 (AS1).
[0359] [0359] In a particularly preferred embodiment, the tissue specific promoter is a root specific promoter.
[0360] [0360] In another embodiment, the promoter may be a development regulated promoter. A development-regulated promoter directs a change in the expression of a gene in one or more parts of a plant at a specific time during the development of the plant. The gene can be expressed in that part of the plant at other times at a different level (usually lower) and can also be expressed in other parts of the plant.
[0361] [0361] In one embodiment, the promoter can be an inducible promoter. An inducible promoter is able to direct the expression of a gene in response to an inducer. In the absence of the inducer, the gene will not be expressed. The inducer can act directly on the promoter sequence or it can neutralize the effect of a repressor molecule. The inducer can be a chemical agent, such as a metabolite, a protein, a growth regulator or a toxic element, a physiological stress, such as heat, injury or osmotic pressure, or an indirect consequence of the action of a pathogen or pest. A development-regulated promoter can be described as a specific type of inducible promoter that responds to an endogenous inducer produced by the plant or to an environmental stimulus at a specific point in the plant's life cycle. Examples of known inducible promoters include those associated with wound response, as described by Warner SA, Scott R, Draper J. (1993) (Isolation of an asparagus intracellular PR gene (AOPRI) wound-responsive promoter by the inverse polymerase chain reaction and its characterization in transgenic tobacco. Plant J. 3 191-201.) incorporated here by reference, temperature response as disclosed by Benfey & Chua (1989) (Benfey, PN and Chua, NH. (1989) Regulated genes in transgenic plants. Science 244 174-181) incorporated herein by reference and chemically induced response, as described by Gatz (1995) (Gatz, C. (1995) Novel inducible / repressible gene expression systems. Methods in Cell Biol. 50 411-424), incorporated here by reference.
[0362] [0362] Thus, in one embodiment, the promoter can be selected from the group consisting of: the CERV promoter, the cauliflower mosaic virus 358 promoter (complete or truncated), the rubisco promoter, the plastocyanin promoter pea, the nopaline synthase promoter, the r / b chlorophyll binding promoter, high molecular weight glutenin promoter, a promoter, B-gliadin, hordein promoter and patatin promoter.
[0363] [0363] In one embodiment, the promoter may be the CaMV 358 promoter or a 35S promoter modified with a duplicated enhancer region or dual enhancer region (R. Kay et al. Science. 1987 jun. 5; 236 (4806): 1299- 302, which is incorporated by reference).
[0364] [0364] In one embodiment, the promoter can be the native promoter.
[0365] [0365] As used here, "native promoter" refers to the promoter that is endogenous to the gene, that is, that is operationally linked to the gene in nature.
[0366] [0366] The recombinant vector can also comprise DNA encoding a gene that can be used as a selectable marker in the cloning process, that is, to allow the selection of cells that have been transfected or transformed and to allow the selection of cells that host vectors incorporating heterologous DNA. The vector may also comprise DNA involved in regulating the expression of the coding sequence or directing the expressed polypeptide to a particular part of the host cell, for example, the chloroplast. Therefore, the vector can comprise at least one additional element selected from a group consisting of: a selectable marker gene (for example, an antibiotic resistance gene); a polypeptide termination signal; and a protein targeting sequence (for example, a chloroplast transit peptide).
[0367] [0367] Examples of suitable marker genes include antibiotic resistance genes, such as those that confer resistance to kanamycin, Geneticin (G418) and hygromycin (npt-II, hyg-B); herbicide resistance genes, such as those that confer resistance to phosphinothricin and sulfonamide herbicides (bar and south, respectively; EP-A-242246, EP-A-0249637), incorporated herein by reference; and screened markers, such as beta-glucuronidase (GB2197653), incorporated herein by reference, luciferase and green fluorescent protein (GFP). The marker gene can be controlled by a second promoter, which allows expression in cells, which may or may not be in the seed, thus allowing the selection of cells or tissues that contain the marker at any stage of plant development. The second suitable promoters are the promoter of the Agrobacterium nopaline synthase gene and the promoter derived from the gene encoding the 35S cauliflower mosaic virus (CaMV) transcript. However, any other suitable second promoter can be used. COMMERCIALLY DESIRABLE CHARACTERISTICS
[0368] [0368] In one embodiment, the plants of the present invention have a reduced total alkaloid content and / or the content of one or more alkaloids selected from nicotine, nornicotine, anabasine, myosmin and reduced anatabine and / or reduced nicotine, while the flavor characteristics and / or other commercially desirable characteristics are at least maintained. In one embodiment, the plants of the present invention produce leaves of similar quality and / or quality to plants that have not been modified according to the invention.
[0369] [0369] In one embodiment, the plants of the present invention have a reduced nicotine content without a significant change in the taste characteristics of the plant (for example, compared to the same plant that has not been modified according to the present invention).
[0370] [0370] In one embodiment, the plants of the present invention have a reduced nicotine content without a significant change (for example, decrease) in other commercially desirable characteristics of the plant (for example, compared to the same plant that has not been modified accordingly) with the present invention). In particular, the yield of the modified plant is preferably not reduced compared to the same plant that has not been modified according to the present invention.
[0371] [0371] Therefore, in one embodiment, the methods and uses of the present invention refer to the reduction of the total content of alkaloids and / or reduction of one or more selected alkaloids of nicotine, nornicotine, anabasin and anatabine and / or reduction of nicotine , maintaining flavor and / or other commercially desirable characteristics (for example, yield).
[0372] [0372] The term “commercially desirable characteristics” will include characteristics such as yield, height of mature plants, number of harvestable leaves, average length of nodes, length of the cut leaf, width of the cut leaf, quality, tolerance to abiotic stress (eg example, drought),
[0373] [0373] The term “commercially desirable characteristics” as taught here will include characteristics such as drought resistance, pest resistance, height of mature plants, the number of harvestable leaves, average node length, the length of the cut leaf, the width of the cut leaf, and yield, which are comparable to the aforementioned characteristics in the smoke-cured parent of a comparable plant when grown under similar field conditions.
[0374] [0374] Unless otherwise specified, used here, tobacco yield refers to the yield of cured leaves that is calculated based on the weight of the tobacco leaves cured per acre under standard field conditions, following agronomic practices and standard cure.
[0375] [0375] In one aspect, a plant (for example, a tobacco plant) of the present invention has a yield between 50% and 150%, between 55% and 145%, between 60% and 140%, between 65% and 135 %, between 70% and 130%, between 75% and 125%, between 80% and 120%, between 85% and 115% between 90% and 110%, between 90% and 110%, between 95% and 105%, between 50% and 100%, between 55% and 100%, between 60% and 100%, between 65% and 100% between 70% and 100%, between 75% and 100%, between 75% and 100%, between 80 % and 100%, between 85% and 100%, between 90% and 100%, between 95% and 100%, between 100% and 150%, between 105% and 150%, between 110% and 150%, between 110% and 150%, between 115% and 150%, between 120% and 150%, between 125% and 150%, between 130% and 150%, between 135% and 150%, between 140% and 150% or between 145% and 150% of the yield of a comparable plant when grown under similar field conditions.
[0376] [0376] In another aspect, the yield of the plant (for example, a tobacco plant) of the present invention is approximately 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2/7, 2.8, 2.9 or 3.0 times the yield of a comparable plant when grown under conditions similar field
[0377] [0377] In another aspect, the yield of a tobacco plant of the present invention is comparable to the yield of the comparable combustion-cured plant when grown under similar field conditions.
[0378] [0378] In one aspect, a tobacco plant of the present invention provides a yield selected from the group consisting of between about 1200 and 3500, between 1300 and 3400, between 1400 and 3300, between 1500 and 3200, between 1500 and 3200, between 1600 and 3100, between 1700 and 3000, between 1800 and 2900, between 1900 and 2800, between 2000 and 2700, between 2100 and 2600, between 2200 and 2500 and between 2300 and 2400 pounds / acre.
[0379] [0379] In another aspect, a tobacco plant of the present invention provides a yield selected from the group consisting of between about 1200 and 3500, between 1300 and 3500, between 1400 and 3500, between 1500 and 3500, between 1500 and 3500, between 1600 and 3500, between 1700 and 3500, between 1800 and 3500, between 1900 and 3500, between 2000 and 3500, between 2100 and 3500, between 2200 and 3500, between 2300 and 3500, between 2400 and 3500, between 2400 and 3500, between 2500 and 3500, between 2600 and 3500, between 2700 and 3500, between 2800 and 3500, between 2900 and 3500, between 3000 and 3500 and between 3100 and 3500 pounds / acre.
[0380] [0380] In a further aspect, a tobacco plant of the present invention provides a yield selected from the group consisting of between about 1200 and 3500, between 1200 and 3400, between 1200 and 3300, between 1200 and 3200, between 1200 and 3100, between 1200 and 3000, between 1200 and 2900, between 1200 and 2800, between 1200 and 2700, between 1200 and 2600, between 1200 and 2500, between 1200 and 2400, between 1200 and 2300, between 1200 and 2300, between 1200 and 2200, between 1200 and 2100, between 1200 and 2000, between 1200 and 1900, between 1200 and 1800, between 1200 and 1700, between 1200 and 1600, between 1200 and 1500 and between 1200 and 1400 pounds / acre. PLANTS
[0381] [0381] Suitable plants according to the invention include the Solanaceae plant family, which includes, for example, jimson weed, eggplant, mandrake, belladonna, peppers (paprika, pepper), potatoes and tobacco.
[0382] [0382] In one embodiment, a suitable genus of Solanaceae is Solanum, for example, Solanum lycopersicum.
[0383] [0383] In one embodiment, a suitable genus of Solanaceae is Nicotiana, for example, Nicotiana tabacum or MNicotiana rustica.
[0384] [0384] A suitable species of Nicotiana may be Nicotiana tabacum. Nicotiana species can be designated here as a tobacco plant, or simply tobacco. TOBACCO PLANTS
[0385] [0385] The present invention provides methods, uses directed to plants (for example, tobacco plants), as well as a cell (for example, a tobacco cell), a plant
[0386] [0386] The term "tobacco", as used here, refers to a plant of the Nicotiana genus that is used in the production of tobacco products. Non-limiting examples of suitable "tobacco" plants include N. tabacum and N. rustica (for example, N. tabacum L., LA B21, LN KYl171, Tl 1406, Basma, Galpao, Perique, Beinhart 1000-1 and Petico) .
[0387] [0387] In an embodiment of a suitable tobacco plant it can be any germplasm, strain or variety of N. tabacum.
[0388] [0388] In another embodiment, a suitable tobacco plant may be a different species of tabacum.
[0389] [0389] Tobacco material can be derived from or obtained from varieties of types of Nicotiana tabacum, commonly known as Burley varieties, smoke or bright varieties and dark varieties. In some embodiments, the tobacco material is derived from a Burley plant, or Virginia a dark tobacco plant. The tobacco plant can be selected from Burley tobacco, rare tobacco, specialty tobacco, expanded tobacco or the like.
[0390] [0390] The use of tobacco cultivars and elite tobacco cultivars is also contemplated here. The tobacco plant for use here may therefore be a tobacco variety or elite tobacco cultivar. Particularly useful varieties of Nicotiana tabacum include the smoked-cured Virginia type, the Burley type and the Oriental type.
[0391] [0391] In some embodiments, the tobacco plant can, for example, be selected from one or more of the following varieties: L. cultivar TI 1068, AA 37-1, B 13P, Xanthi (Mitchell-Mor), KT DiH3 Hybrid 107, Bel-W3, 79-615, Samsun Holmes NN, F4 from crossing BU21 x Hoja Parado, lineage 97, KTRDCt + 2 Hybrid 49, KTRDC * 4 Hybrid 110, Burley 21, PMO016, KTRDCH + 5 KY 160 SI, KTRDCH7 FCA, KTRDCH + 6 TN 86 SI, PMO21, K 149, K 326, K 346, K 358, K 394, K 399, K 730, KY 10, KY 14, KY 160, KY 17, KY 8959, KY 9, KY 907, MD 609, McNair 373, NC 2000, PG 01, PG 04, POl, PO2, PO3, RG 11, RG 17, RG 8, Speight G-28, TN 86, TN 90, TN 50, VA 509, AS44, PG 04, POl, PO2, PO3, RG 11, RG 17, RG 8, Speight G-28, TN 86, TN 90, VA 509, AS44, Banket Al, Basma Drama B84 / 31, Basma I Zichna ZP4 / B, Basma Xanthi BX 2A, Batek, Besuki Jember, C104, Coker 319, Coker 347, Criollo Misionero, PMO092, Delcrest, Djebel 81, DVH 405, Common Shed, HBO4P, Hicks Broadleaf, Kabakulak Elassona, PM102, Kutsage El, KY 14 x L8, K Y 171, LA BU 21, McNair 944, NC 2326, NF 71, NC 297, NF 3, PVH 03, PVH 09, PVH 19, PVH 21 10, Red Russian, Samsun, Saplak, Simmaba, Talgar 28, PM132, Wislica , Yayaldag, NC 4, TR Madole, Prilep HC-72, Prilep P23, Prilep PB 156/1, Prilep P12-2 / 1, Yaka JK-48, Yaka JB 125/3, TT -1068, KDH-960, TI -1070, TW136, PM204, PM205, Basma, TKF 4028, L8, TKF 2002, TN 90, GR141, Basma xanthi, GR149, GR153 and Petit Havana.
[0392] [0392] Non-limiting examples of varieties or cultivars are: BD 64, CC 101, CC 200, CC 27, CC 301, CC 400, CC 500, CC 600, CC 700, CC 700, CC 800, CC 900, Coker 176 , Coker 319, Coker 371 Gold, Coker 48, CD 263, DF91 1, DT 538 LC, Tobacco Galpao, GL 26H, GL 350, GL 600, GL 737, GL 939, GL 973, HB 04P, HB 04P LC, HB3307PLC , 403LC Hybrid, 404LC Hybrid, 501 LC Hybrid, K 149,
[0393] [0393] The tobacco plant can be a Burley, Virginia smoke cured or Oriental.
[0394] [0394] In one embodiment, plant propagation material can be obtained from a plant (for example, a tobacco plant) of the invention. A "plant propagating material", as used here, refers to any plant material taken from a plant from which other plants can be produced. Suitably, the plant's propagating material may be a seed. Suitably, the plant's propagating material may be pollen.
[0395] [0395] In one embodiment, the cell (for example, a tobacco cell), the plant (for example, a tobacco plant) and / or the plant propagating material of the invention may comprise activity or modulated expression of a gene Nicl ERF or a Nicl ERF gene and a Nic2 ERF gene. In another embodiment, the cell (e.g., tobacco cell), plant (e.g., tobacco plant) and / or plant propagating material may comprise a construction or vector according to the invention. In another embodiment, the cell (for example, tobacco cell), plant (for example, tobacco plant) and / or plant propagating material can be obtained (for example, obtained) by a method according to the invention.
[0396] [0396] Suitably, a plant (e.g., a tobacco plant) or part of it according to the present invention can comprise activity or modulated expression of a Nicl ERF gene (or a Nicl ERF gene and a Nic2 ERF gene ), when compared to a plant (for example, a tobacco plant) or part of it that has not been modified to modulate the activity or expression of a Nicl ERF gene (or both Nicl ERF gene and a Nic2 ERF gene).
[0397] [0397] In one embodiment, the plant (e.g., tobacco plant) or part thereof according to the present invention comprises a cell (e.g., a tobacco cell) of the invention. In another embodiment, the plant propagation material may be obtainable (for example, obtained) from a plant (for example, a tobacco plant) of the invention.
[0398] [0398] In one embodiment, the use of a cell (for example, a tobacco cell) is provided, as provided for in the previous embodiments for the production of a product (for example, a tobacco product). In addition, the use of a plant (for example, a tobacco plant) is provided, as described herein to produce a plant (for example, a tobacco plant).
[0399] [0399] The present invention also provides in another embodiment the use of a plant (for example, a tobacco plant) from the previous embodiments for the production of a product (for example, a tobacco product). In another embodiment, the use of a plant (e.g., a tobacco plant) of the invention is provided to grow a crop. In one embodiment, the use of a Nicl ERF gene or both the Nicl ERF and Nic2 ERF genes according to the present invention results in the modulation of the alkaloid content of a plant (e.g., a tobacco plant).
[0400] [0400] In one embodiment, the method or use of a Nicl ERF gene or a Nicl ERF gene and a Nic2 ERF gene according to the present invention can result in modulation of the alkaloid content.
[0401] [0401] In another embodiment, the method or use of a Nicl ERF gene or both Nicl ERF and Nic2 ERF genes, (for example, increased activity or expression thereof) may result in an increase in the content of one or more more alkaloids. Suitably, the content of one or more of anatabine, anabasine, nornicotine or nicotine can be increased. Suitably, the nicotine content is reduced. Suitably, this can be seen when the activity or expression of the Nicl ERF gene is increased compared to wild type plants.
[0402] [0402] In a suitably embodiment, the plant (for example, tobacco plant) or part thereof, for example, the harvested leaf or leaf or harvested processed leaf, or products (for example, tobacco products) comprising the plant comprise a modified (for example, mutated or deleted) Nicl ERF gene of the present invention (or a modified (for example, mutated or deleted) Nicl ERF gene in combination with a modified (for example, mutated or deleted) Nic2 ERF gene according to the present invention).
[0403] [0403] In one embodiment, the present invention provides a cultivation of tobacco cells (e.g., in vitro cultivation) The cultivation of tobacco cells can be a suspension culture of tobacco cells. These in vitro grown tobacco cells can be incorporated into a tobacco product, for example, as a substitute for conventional tobacco particles, fragments, fine-cut or long-cut tobacco blade as an additive ingredient or as a substitute and additive.
[0404] [0404] In one embodiment, the use of a tobacco cell culture, for example, a harvested and / or processed tobacco cell culture, or an extract thereof according to the present invention, is provided for the production of a tobacco product. tobacco.
[0405] [0405] Tobacco cells harvested from an in vitro culture can be dried, for example, lyophilized, for example, to produce a powder.
[0406] [0406] The specialist in the field will have knowledge of known methods for establishing in vitro cultures of tobacco cells. As an example, only the following method can be used: collecting seeds to form a tobacco plant of interest and sterilizing its exterior to eliminate unwanted organisms, planting said seeds to grow a tobacco plant of interest, removing tissue tobacco plant (for example, tobacco stem) for use as an explant, establishment of a callus cultivation from the tobacco explant, establishment of a cell suspension cultivation of the callus cultivation and harvest of the cultivation material ( for example, including tobacco cells) to produce a tobacco cell culture.
[0407] [0407] Tobacco cells can be harvested by various methods, including filtration, for example, vacuum filtration. The sample can be washed on the filter by adding water and the remaining liquid removed with filtration, for example, vacuum filtration.
[0408] [0408] The cultivation of harvested tobacco cells can be further processed, for example, drying, as air drying and / or lyophilisate. The cultivation of harvested tobacco cells or the cultivation of dry harvested tobacco cells or an extract thereof can be incorporated into the tobacco products according to the present invention.
[0409] [0409] In one embodiment, the present invention provides a tobacco plant or part of it for use in molecular agriculture. Suitably, a plant or part of it modified according to the present invention can be used in the manufacture of proteins as therapeutics, for example, antibiotics, virus-like particles, neutraceuticals or small molecules.
[0410] [0410] In one embodiment, the present invention provides a method for producing proteins (for example, therapeutic proteins); the method comprising modifying a plant or part of it capable of producing said protein (for example, therapeutic protein) by modulating the activity or expression of at least one Nicl ERF gene: wherein the at least one Nicl ERF gene encodes a polypeptide that comprises an amino acid sequence as set out in: SEQ ID No. 4; or SEQ ID No. 8; or SEQ ID No. 12; or SEQ ID No. 16; or SEQ ID No. 20; or SEQ ID No. 24; or SEQ ID No. 28; or SEQ ID No. 32; or
[0411] [0411] The present invention also provides products obtainable or obtained from tobacco according to the present invention. The products are supplied in such a way that they can be obtained or obtained from a tobacco plant in which the activity or expression of the Nicl ERF gene or both Nicl ERF and Nic2 ERF genes have been modulated and which comprises content of modulated alkaloids.
[0412] [0412] As used here, the term “tobacco industry product” is intended to include combustible smoking articles, such as cigarettes, cigarillos, cigars, pipe tobacco or for roll cigarettes (based on tobacco, tobacco derivatives, expanded tobacco, reconstituted tobacco, tobacco substitutes or other smoking material), non-combustible aerosol delivery systems, such as heating products that release compounds from substrate materials without burning, such as electronic cigarettes, tobacco heating products and hybrid systems to generate aerosol from a combination of substrate materials, for example, hybrid systems containing a liquid or gel or solid substrate, as well as aerosol forming substrate materials used within those aerosol delivery systems; and aerosol-free delivery items, such as lozenges, gums, adhesives, items comprising breathable powders and smokeless tobacco products, such as snus and snuff, whose aerosol-free delivery items may or may not deliver nicotine.
[0413] [0413] Suitably, the tobacco industry product can be prepared from (for example, it can comprise) a tobacco plant or a part thereof according to the present invention.
[0414] [0414] Suitably, the tobacco industry product can be prepared from a tobacco cell culture according to the present invention.
[0415] [0415] Suitably, the tobacco industry product can be prepared from (for example, it can comprise) a tobacco plant or part thereof propagated from a tobacco plant propagation material according to the present invention .
[0416] [0416] Suitably, the tobacco industry product can be prepared from (for example, it can comprise) a leaf harvested from a tobacco plant according to the present invention.
[0417] [0417] Suitably, the tobacco industry product can be prepared from (for example, it can comprise) a tobacco leaf processed in accordance with the present invention.
[0418] [0418] Suitably, the tobacco industry product can be prepared from (for example, it can comprise) a cured tobacco material according to the present invention.
[0419] [0419] Suitably, the tobacco industry product can be prepared from (for example, it can comprise) a tobacco blend according to the present invention.
[0420] [0420] In one embodiment, the tobacco industry product is a fuel smoking article, selected from the group consisting of a cigarette, a cigarillo and a cigar.
[0421] [0421] In one embodiment, the tobacco industry product comprises one or more components of a combustible smoke article, such as a filter, a filter rod, filter rod segments, tobacco, a tobacco rod, a tobacco segment tobacco rod, a spill, an additive release component such as a capsule, a thread, beads, a paper such as a wrapper, a filter paper or a cigarette paper.
[0422] [0422] In one embodiment, the tobacco industry's product is a non-combustible aerosol delivery system.
[0423] [0423] In one embodiment, the tobacco industry product comprises one or more components of a non-combustible aerosol supply system, such as a heater and an aerosol-forming substrate.
[0424] [0424] In one embodiment, the aerosol delivery system is an electronic cigarette also known as a vaporizer device.
[0425] [0425] In one embodiment, the electronic cigarette comprises a heater, a power source capable of supplying energy to the heater, an aerosol-forming substrate, such as a liquid or gel, a housing and optionally a mouthpiece.
[0426] [0426] In one embodiment, the aerosol-forming substrate is contained in a substrate container. In one embodiment, the substrate container is combined with or comprises the heater.
[0427] [0427] In one embodiment, the tobacco industry product is a heating product that releases one or more compounds by heating, but not burning, a substrate material. The substrate material is an aerosol-forming material that can be, for example, tobacco or other products other than tobacco, which may or may not contain nicotine. In one embodiment, the heating product is a tobacco heating product.
[0428] [0428] In one embodiment, the heating product is an electronic device.
[0429] [0429] In one embodiment, the tobacco heating product comprises a heater, an energy source capable of supplying energy to the heater, an aerosol-forming substrate, such as a solid material or gel.
[0430] [0430] In one embodiment, the heating product is a non-electronic item.
[0431] [0431] In one embodiment, the heating product comprises an aerosol-forming substrate, such as a solid material or gel, and a heat source that is capable of providing thermal energy to the aerosol-forming substrate without any electronic means, such as by burning a combustion material, such as coal.
[0432] [0432] In one embodiment, the heating product also comprises a filter capable of filtering the aerosol generated by heating the substrate that forms the aerosol.
[0433] [0433] In some embodiments, the aerosol forming substrate material may comprise a vapor or aerosol generating agent or a humectant, such as glycerol, propylene glycol, triacetin or diethylene glycol.
[0434] [0434] In one embodiment, the tobacco industry product is a hybrid system for generating aerosol by heating, but not burning, a combination of substrate materials. The substrate materials can comprise, for example, solid, liquid or gel that may or may not contain nicotine. In one embodiment, the hybrid system comprises a liquid or gel substrate and a solid substrate. The solid substrate can be, for example, tobacco or other non-tobacco products, which may or may not contain nicotine. In one embodiment, the hybrid system comprises a liquid or gel and tobacco substrate.
[0435] [0435] In another embodiment, the product may comprise a construction of the invention that modulates the activity or expression of the Nicl ERF gene and the decreased alkaloid content.
[0436] [0436] In another embodiment, the product may comprise one or more constructs of the invention that modulate both the activity or expression of the Nicl ERF gene and the activity or expression of the Nic2 ERF gene in which said product has the content of modulated alkaloids.
[0437] [0437] In one embodiment, the use of a plant of the invention (for example, a tobacco plant) is provided to produce leaves (for example, tobacco leaf). Suitably, the sheet (e.g., tobacco leaf) can be subjected to downstream applications, such as processing. In this way, in one embodiment the use of the previous embodiment can provide a processed sheet (for example a processed tobacco sheet). Suitably, the tobacco leaf can be subjected to curing, fermentation, pasteurization or combinations thereof.
[0438] [0438] In another embodiment, the leaf (e.g., tobacco leaf) can be cut. In some embodiments, the leaf (e.g., tobacco leaves) can be cut before or after being subjected to curing, fermentation, pasteurization or combinations thereof.
[0439] [0439] In one embodiment, the present invention provides a leaf harvested from a plant of the invention (for example, a tobacco plant). In one embodiment, the harvested leaf can be obtained from a plant (e.g., a tobacco plant) that has modulated the activity or expression of the Nicl ERF gene or that has modulated both the activities or expressions of the Nicl ERF and Nic2 ERF genes. Suitably, the harvested leaf has a modulated alkali content. In another embodiment, the harvested leaf may be obtainable (for example, obtained) from a plant (for example, a tobacco plant) propagated from a propagation material of the present invention. In another embodiment, a harvested leaf obtainable from a method or use of the present invention is provided. Suitably, the harvested leaf can be a cut harvested leaf. In some embodiments, the harvested leaf may comprise viable cells (e.g., viable tobacco cells). In some embodiments, the harvested leaf does not comprise viable cells (e.g., viable tobacco cells). In other embodiments, the harvested leaf can be subjected to further processing.
[0440] [0440] Some tobacco plants can be harvested by cutting the stems and harvesting all the leaves simultaneously (for example, as in Burley tobacco). Other tobacco plants (for example, smoke-cured tobacco) can be harvested in stages in a process such as conditioning, in which individual leaves are removed from the stem as they mature.
[0441] [0441] As used here, "conditioning" refers to the removal of leaves from tobacco plants. This may refer to the removal of mature or fit leaves from smoked cured plants.
[0442] [0442] A processed leaf (for example a processed tobacco leaf) is also provided. The processed leaf (e.g., processed tobacco leaf) can be obtained from a plant of the invention (e.g., tobacco plant). Suitably, the processed leaf can be obtained from a plant obtained according to any of the methods and / or uses of the present invention. In one embodiment, the processed leaf (e.g., processed tobacco leaf) can be obtained from a plant (e.g., tobacco plant) that has the activity or expression of the modulated Nicl ERF gene or both Nicl ERF genes and Nic2 ERF and the content of modulated alkaloids, preferably when compared to a control leaf, that is, compared to a leaf of a plant (e.g., tobacco plant) that has not been modified according to the invention. The processed leaf (e.g., processed tobacco leaf) can comprise a modulation in the activity or expression of the Nicl ERF gene or a Nicl ERF gene and a Nic2 ERF gene and modified alkali content.
[0443] [0443] In another embodiment, the processed leaf (for example, processed tobacco leaf) may be obtainable from a plant (for example, tobacco plant) propagated from a plant (for example, tobacco plant) propagating material from according to the present invention. The processed leaf (e.g., processed tobacco leaf) of the present invention is obtained by processing a harvested leaf of the invention.
[0444] [0444] The term "processed sheet", as used here, refers to a sheet that has undergone one or more processing steps to which the sheets are subjected in the state of the art. A "processed sheet" comprises none or practically no viable cells.
[0445] [0445] The term "processed tobacco leaf", as used here, refers to a tobacco leaf that has undergone one or more processing steps to which the tobacco is subjected in the prior art. A "processed tobacco leaf" comprises none or practically no viable cells.
[0446] [0446] The term "viable cells" refers to cells that are capable of growing and / or are metabolically active. Therefore, if a cell is considered non-viable, also called "non-viable", the cell does not exhibit the characteristics of a viable cell.
[0447] [0447] The term "substantially non-viable cells" means that less than about 5% of the total cells are viable. Preferably, less than about 3%, more preferably less than about 1%, even more preferably less than about 0.1% of the total cells are viable.
[0448] [0448] In one embodiment, the processed tobacco leaf can be processed by one or more of: curing, fermentation and / or pasteurization. Suitably, the processed tobacco leaf can be cured. The tobacco leaf can be cured by any method known in the art. In one embodiment, the tobacco leaf can be cured by one or more of the curing methods selected from the group consisting of: air curing, fire curing, smoke curing and sun curing. Suitably, the tobacco leaf can be air-cured. Suitably, the tobacco leaf can be cured in a smokehouse.
[0449] [0449] Air curing is usually achieved by hanging tobacco leaves in well-ventilated granaries and allowing drying. This is usually done over a period of four to eight weeks. Air curing is especially suitable for Burley tobacco.
[0450] [0450] Suitably, the tobacco leaf can be cured by fire. Fire curing is usually achieved by hanging tobacco leaves in large barns, where wood fires are kept over a continuous or intermittent low heat and usually take between three days and ten weeks, depending on the process and the tobacco.
[0451] [0451] In another embodiment, the tobacco leaf can be cured in a smokehouse. Smoke curing may comprise tying tobacco leaves onto tobacco sticks and hanging them on bars in curing barns. The granaries usually have a smokehouse that comes from externally powered fire boxes. This usually results in tobacco that has been cured by heat without being exposed to smoke. Normally, the temperature slowly increases during the curing, with the entire process taking approximately 1 week.
[0452] [0452] Suitably, the tobacco leaf can be cured in the sun. This method usually involves exposing tobacco discovered in the sun.
[0453] [0453] Suitably, the processed tobacco leaf can be processed by fermentation. Fermentation can be carried out in any manner known in the art. Normally, during fermentation, tobacco leaves are stacked in piles (the mass) of cured tobacco, covered, for example, with burlap to retain moisture. The combination of the remaining water inside the leaf and the weight of the tobacco generates a natural heat that ripens the tobacco. The temperature at the center of the volume is monitored daily. In some methods, every week the entire volume is opened. The leaves are then removed to be shaken and moistened and the dough is rotated so that the inner leaves come out and the lower leaves are placed on top of the dough. This ensures uniform fermentation over the entire volume. The additional moisture in the leaves, in addition to the actual rotation of the leaves themselves, generates heat, releasing the natural ammonia from the tobacco and reducing nicotine, in addition to attenuating the color and improving the aroma of the tobacco. Typically, the fermentation process continues for up to 6 months, depending on the variety of tobacco, the position of the bar on the leaf, the thickness and the intended use of the leaf.
[0454] [0454] Suitably, the processed tobacco leaf can be processed by pasteurization. Pasteurization can be particularly preferred when the tobacco leaf will be used to produce a smokeless tobacco product, more preferably snus. Pasteurization of tobacco leaves can be carried out by any method known in the art. For example, pasteurization can be performed as detailed in J. Foulds, L. Ramstrom, M. Burke, K. Fagerstrom. Effect of smokeless tobacco (snus) on smoking and public health in Sweden. Tobacco Control (2003) 12: 349-359, the teaching of which is incorporated herein by reference.
[0455] [0455] During the production of snus, pasteurization is usually carried out by a process in which the tobacco is heat treated with steam for 24 to 36 hours (reaching temperatures of approximately 100 ºC). This results in an almost sterile product and, without wishing to be bound by theory, it is believed that one of the consequences of this is a limitation of additional TSNA formation.
[0456] [0456] In one embodiment, pasteurization can be steam pasteurization.
[0457] [0457] In some embodiments, the processed tobacco leaf can be cut. The processed tobacco leaf can be cut before or after processing. Suitably, the processed tobacco leaf can be cut after processing.
[0458] [0458] In some embodiments, the tobacco plant, the leaf harvested from a tobacco plant and / or the processed tobacco leaf can be used to extract nicotine. Nicotine extraction can be achieved using any method known in the art. For example, a method for extracting nicotine from tobacco is taught in US 2,162,738 which is incorporated herein by reference.
[0459] [0459] In one aspect, the present invention provides cured tobacco material made from or part of a tobacco plant according to the invention.
[0460] [0460] In another aspect, the present invention provides a tobacco blend comprising tobacco material made from or part of a tobacco plant according to the present invention. In one aspect, the present invention provides a tobacco blend comprising cured tobacco material in accordance with the present invention.
[0461] [0461] Suitably, the tobacco blend according to the present invention can comprise approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% tobacco from a tobacco plant tobacco or part of it according to the present invention. Suitably, the tobacco blend can comprise approximately 10% tobacco from a tobacco plant or part of it according to the present invention. Suitably, the tobacco blend may comprise approximately 20% tobacco from a tobacco plant or part thereof according to the present invention. Suitably, the tobacco blend may comprise approximately 30% tobacco from a tobacco plant or part thereof according to the present invention. Suitably, the tobacco blend may comprise approximately 40% tobacco from a tobacco plant or part of it according to the present invention. Suitably, the tobacco blend may comprise approximately 50% tobacco from a tobacco plant or part thereof according to the present invention. Suitably, the tobacco blend may comprise approximately 60% tobacco from a tobacco plant or part thereof according to the present invention. Suitably, the tobacco blend may comprise approximately 70% tobacco from a tobacco plant or part thereof according to the present invention. Suitably, the tobacco blend may comprise approximately 80% tobacco from a tobacco plant or part of it according to the present invention. Suitably, the tobacco blend may comprise approximately 90% tobacco from a tobacco plant or part thereof according to the present invention.
[0462] [0462] In one aspect, a tobacco blend product of the present invention comprises at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 95 weight percent dry cured tobacco from a tobacco plant or part thereof according to the present invention.
[0463] [0463] Suitably, cured tobacco material can be air-cured. Suitably, the cured tobacco material can be cured in a smokehouse. Suitably, the cured tobacco material can be cured in the sun.
[0464] [0464] A tobacco product or tobacco article according to the present invention can comprise the tobacco material (e.g., cured tobacco material) according to the present invention.
[0465] [0465] In another aspect, the present invention provides a tobacco product. Suitably, a tobacco product can be a mixed tobacco product. In one embodiment, the tobacco product can be prepared from a tobacco plant of the invention or a part thereof. In one embodiment, the tobacco product can be prepared from a tobacco plant that has the activity of or expression of the Nicl ERF gene or the activity or expression of both the Nicl ERF and Nic2 ERF genes. The tobacco product may comprise a reduction in the activity or expression of the Nicl ERF gene and a reduced alkaloid content. Suitably, the tobacco plant or part thereof can be propagated from a tobacco plant propagation material according to the present invention.
[0466] [0466] The term "part of it" as used here in the context of a plant (for example, a tobacco plant) refers to a portion of the plant (for example, a tobacco plant). Preferably, the "part of it" is a leaf from a plant (for example, a tobacco plant).
[0467] [0467] In another embodiment, the tobacco product can be prepared from a harvested leaf of the invention. In another embodiment, the tobacco product can be prepared from a processed tobacco leaf of the invention. Suitably, the tobacco product can be prepared from a tobacco leaf processed by one or more of the following: curing, fermentation and / or pasteurization. Suitably, the tobacco product may comprise - a cut tobacco leaf, optionally processed according to the previous embodiment.
[0468] [0468] In one embodiment, the tobacco product can be a smoking article. As used herein, the term "smoking article" may include and smokable products, such as cigarette smoke, cigars and cigarillos, whether based on tobacco, tobacco products, expanded tobacco, reconstituted tobacco or tobacco substitutes. tobacco.
[0469] [0469] In another embodiment, the tobacco product can be a smokeless tobacco product. The term "smokeless tobacco product", as used here, refers to a tobacco product that is not intended to be smoked and / or subject to combustion. In one embodiment, a smokeless tobacco product can include snus, snuff, chewing tobacco or the like.
[0470] [0470] In a further embodiment, the tobacco product can be a tobacco heating device or hybrid device or electronic cigarettes or the like. Typically, in heating devices or hybrid devices, an aerosol is generated by transferring heat from a heat source to a physically separate aerosol-forming substrate or material, which can be located inside, around, or downstream the heat source. During smoking, volatile compounds are released from the aerosol-forming substrate by transferring heat from the heat source and entrained by the air aspirated through the smoke article. As the released compounds cool, they condense to form an aerosol that is inhaled by the user.
[0471] [0471] Aerosol-generating articles and devices for consumption or smoking tobacco heating devices are known in the art. They may include, for example, electrically heated aerosol generating devices in which an aerosol is generated by the heat transfer of one or more electrical heating elements from the aerosol generating device to the aerosol forming substrate of a tobacco heating device. . Suitably, the tobacco heating device can be an aerosol generating device.
[0472] [0472] Preferably, the tobacco heating device can be a heating device and without burning. Non-burning heating devices are known in the art and release compounds by heating, but not burning, tobacco. An example of a suitable non-burning heating device can be an example from WO2013 / 034459 or GB2515502, which are incorporated herein by reference.
[0473] [0473] In one embodiment, the aerosol forming substrate of a tobacco heating device can be a tobacco product according to the present invention.
[0474] [0474] In one embodiment, the tobacco heating device can be a hybrid device. POLYNUCLEOTIDS / POLYPEEPTIDS / CONSTRUCTIONS
[0475] [0475] In certain embodiments of the present invention, constructs that modulate the activity or expression of at least one Nicl ERF gene (or at least one Nicl ERF gene and at least one Nic2 ERF gene) can be transformed into plant cells properly under the direction of a prosecutor.
[0476] [0476] In certain embodiments of the present invention, constructs that decrease (i.e., inhibit) the activity or expression of a Nicl ERF gene (or a Nicl ERF gene and a Nic2 ERF gene) can be transformed into plant cells under the direction of a promoter. The genetic construct may be a gene editing construct or may comprise an RNAi molecule, which may comprise a small interfering RNA (siRNA) molecule or a short hairpin (shRNA) loop molecule.
[0477] [0477] In certain embodiments of the present invention, constructs that increase the activity or expression of a Nicl ERF gene (or a Nicl ERF gene and a Nic2 ERF gene) can be transformed into plant cells under the direction of a promoter, for example example, constructs that encode an endogenous Nicl ERF gene.
[0478] [0478] Constructions can be introduced into the plants according to the present invention by means of a suitable vector, for example, plant transformation vectors. A plant transformation vector may comprise an expression cassette comprising, in the direction of 5'-3 'transcription, a promoter sequence, a construction sequence targeting a Nicl ERF gene (or targeting both Nicl ERF and Nic2 ERF genes) and, optionally, a 3 'untranslated terminator sequence, including a stop signal for RNA polymerase and a polyadenylation signal for polyadenylase. The promoter sequence can be present in one or more copies, and those copies can be identical or variants of a promoter sequence as described above. The terminator sequence can be obtained from plant, bacterial or viral genes. Suitable termination sequences are the pea rbcS E9 terminator sequence, the terminator sequence derived from the nopaline synthase gene of Agrobacterium tumefaciens and the terminator sequence of the 35S cauliflower mosaic virus, for example. One skilled in the art will be readily aware of other suitable terminator sequences.
[0479] [0479] The construction of the present invention may also comprise a mechanism for improving gene expression to increase the strength of the promoter. An example of such an enhancer element is a derivative of a promoter portion of the pea plastocyanin gene, which is the subject of International Patent Application No. WO 97/20056, which is incorporated herein by reference. Suitable potentiating elements can be the potentiating element derived from the nopaline synthase gene of Agrobacterium tumefaciens and the potentiating element 35S of cauliflower mosaic virus, for example.
[0480] [0480] These regulatory regions can be derived from the same gene as the promoter DNA sequence or derived from different genes, from Nicotiana tabacum or other organisms, For example, from a plant in the Solanaceae family or the subfamily Cestroideae. All regulatory regions must be able to operate on the cells of the tissue to be transformed.
[0481] [0481] The DNA sequence of the promoter can be derived from the same gene as the gene of interest, for example, the gene that The promoter will target, for example, a gene encoding a Nicl ERF of the invention, a coding sequence used in present invention or can be derived from a different gene, from Nicotiana tabacum or from another organism, for example, from a plant of the family Solanaceae or from the subfamily Cestroideae.
[0482] [0482] The expression cassette can be incorporated into a basic plant transformation vector, such as pBIN 19 Plus, pBI 101, PKYLX71: 35S82, pCcCAMBIAZ300 or other suitable plant transformation vectors known in the art. In addition to the expression cassette, the plant transformation vector will contain the necessary sequences for the transformation process. These may include the Agrobacterium vir genes, one or more T-DNA border sequences and as a selectable marker or other means of identifying transgenic plant cells.
[0483] [0483] The term "plant transformation vector" means a construct capable of expression in vivo or in vitro. Preferably, the expression vector is incorporated into the organism's genome. The term "incorporated" preferably encompasses stable incorporation into the genome.
[0484] [0484] Techniques for transforming plants are well known in the art and include transformation mediated by Agrobacterium, for example. The basic principle in the construction of genetically modified plants is to insert genetic information into the plant's genome, in order to obtain a stable maintenance of the inserted genetic material. A review of general techniques can be found in the articles by Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991] 42: 205-225) and Christon (AgroFood-Industry Hi-Tech March / April 1994 17-27), which are incorporated here by reference.
[0485] [0485] Typically, in Agrobacterium-mediated transformation, a binary vector carrying a foreign DNA of interest, that is, a Nicl ERF construct, is transferred from an appropriate Agrobacterium strain to a target plant by co-cultivating Agrobacterium with explants of the target plant. The transformed plant tissue is then regenerated in a selection medium, which comprises a selectable marker and plant growth hormones. An alternative is the floral diving method (Clough & Bent, 1998 Plant J. Dez., 1998; 16 (6): 735-43, which is incorporated here by reference), whereby flower buds from an intact plant are placed in contact with a suspension of Agrobacterium strain containing the chimeric gene and then there is the establishment of the seeds, the transformed individuals are germinated and identified by growth in a selective medium. Direct infection of plant tissues by Agrobacterium is a simple technique that has been widely used and described in Butcher D.N. et al., (1980), Tissue Culture Methods for Plant Pathologists, ed .: D.S. Ingrams and J.P. Helgeson, 203-208. which is incorporated here by reference.
[0486] [0486] Other suitable transformation methods include direct gene transfer to protoplasts using polyethylene glycol or electroporation techniques, particle bombardment, microinjection and the use of silicon carbide fibers, for example. Transforming plants using ballistic transformation, including the silicon carbide mustache technique, are taught in Frame BR, Drayton PR, Bagnaall SV, Lewnau C JJ, Bullock WP, Wilson HM, Dunwell JM, Thompson JA and Wang K (1994), which is incorporated here by reference. The production of fertile transgenic corn plants by silicon carbide-mediated transformation is taught in The Plant Journal 6: 941-948, which is incorporated here by reference) and viral transformation techniques are taught, for example, in Meyer P , Heidmann I & Niedenhof I (1992), which is incorporated by reference. The use of the cassava mosaic virus as a vector system for plants is taught in Gene 110: 213-217, which is incorporated here by reference. Further teachings on plant transformation can be found in EP-A-0449375, incorporated here by reference.
[0487] [0487] In a further aspect, the present invention relates to a vector system that carries a construct and introduces it into the genome of an organism, such as a plant, suitably a tobacco plant. The vector system can comprise one vector, but it can comprise two vectors. In the case of two vectors, the vector system is usually called a binary vector system. Binary vector systems are described in more detail in Gynheung Anetal, (1980), Binary Vectors, Plant Molecular Biology Manual A3, 1-19, which is incorporated here by reference.
[0488] [0488] An extensively employed system for transforming plant cells uses the Ti plasmid from Agrobacterium tumefaciens or a Ri plasmid from Agrobacterium rhizogenes described by An et al. (1986), Plant Physiol. 81, 301-305 and Butcher D.N. et al., (1980), Tissue Culture Methods for Plant Pathologists, eds .: D.S. Ingrams and J.P. Helgeson, 203-208, which are incorporated by reference. After each method of introducing the desired exogenous gene according to the present invention into plants, the presence and / or insertion of other DNA sequences may be necessary. The use of T-DNA for the transformation of plant cells has been extensively studied and is described in EP-A-120516; Hoekema, in: The Binary Plant Vector System Offset-drukkerij Kanters B.B., Amsterdam, 1985, chapter V; Fraley et al., Crit. Rev. Plant Sci., 4: 1- 46; and Anetal., EMBO J (1985) 4: 277-284, incorporated herein by reference.
[0489] [0489] Plant cells transformed with constructs that modulate the activity or expression of a Nicl ERF gene or a Nicl ERF gene and a Nic2 ERF gene can be grown and maintained according to well-known tissue culture methods, such as cells in a suitable culture medium provided with the necessary growth factors, such as amino acids, plant hormones, vitamins, etc.
[0490] [0490] The term "transgenic plant" referring to the present invention includes any plant that comprises a construct that modulates the activity or expression of a Nicl ERF gene (or a Nicl ERF gene and a Nic2 ERF gene) according to the invention . Therefore, a transgenic plant is a plant that has been transformed with a construction according to the invention. Preferably, the transgenic plant exhibits modulated Nicl ERF activity or expression (or the activity or expression of both the Nicl ERF gene and the Nic2 ERF gene) and modulated alkali content in accordance with the present invention. The term "transgenic plant" does not include coding sequences for native nucleotides in their natural environment when they are under the control of their native promoter who is also in their natural environment.
[0491] [0491] In one aspect, a Nicl ERF gene, a construct, a plant or plant cell transformation vector according to the present invention is in an isolated form. The term "isolated" means that the sequence is at least substantially free of at least one other component with which the sequence is naturally associated in nature and as found in nature.
[0492] [0492] In one aspect, a Nicl ERF gene, a construction, plant or plant cell transformation vector according to the invention is in a purified form. The term "purified" means in a relatively pure state, for example, at least about 90% pure, or at least about 95% pure or at least about 98% pure.
[0493] [0493] The term "nucleotide sequence", as used here, refers to an oligonucleotide sequence or polynucleotide sequence and a variant, homolog, fragments and their derivatives (as portions thereof). The nucleotide sequence can be of genomic origin Or synthetic or recombinant, which can be double-stranded or single-stranded, representing the sense or antisense strand.
[0494] [0494] The term "nucleotide sequence" in relation to the present invention includes genomic DNA, cDNA, synthetic DNA and RNA. Preferably it means DNA, more preferably the cDNA sequence encoding the present invention.
[0495] [0495] In a preferred embodiment, the nucleotide sequence when related to and when covered by the scope of the present invention, that is, the Nicl ERF gene, includes the native nucleotide sequence when in its natural environment and when it is linked to (s) its naturally associated sequence (s) that are also / are in their natural environment. To facilitate the reference, we will call this preferred embodiment the "native nucleotide sequence". In this regard, the term "native nucleotide sequence" means an entire nucleotide sequence that is in its native environment and when operationally linked to an entire promoter to which it is naturally associated, whose promoter is also in its native environment.
[0496] [0496] A nucleotide sequence that encodes a protein that has specific properties like a Nicl gene
[0497] [0497] In a further alternative, the nucleotide sequence encoding the ERF transcription factor can be prepared synthetically by established standard methods, for example, the phosphoramidite method described by Beucage SL et al., (1981) Tetrahedron Letters 22, p 1859-1869, which is incorporated herein by reference, or the method described by Matthes et al., (1984) EMBO J. 3, p 801-805, which is incorporated here by reference. In the phosphoramidite method, oligonucleotides are synthesized, for example, in an automatic DNA synthesizer, purified, ringed, ligated and cloned into appropriate vectors.
[0498] [0498] As used here, the term "amino acid sequence" is synonymous with the term "polypeptide" and / or the term "protein". In some cases, the term “nucleotide sequence, that is, the ERF gene encoding that polypeptide (hereinafter referred to as a“ sequence (s homologous (s) ”). Here, the term“ homologous ”means an entity with a certain homology with the amino acid sequences in question and the nucleotide sequences in question. Here, the term "homology" can be equated with "identity".
[0500] [0500] The homologous amino acid sequence and / or sequence of nucleotides and / or fragments must provide and / or encode a polypeptide that retains functional activity and / or improves the activity of the ERF gene. Typically, homologous sequences will comprise the same active sites, etc., as the amino acid sequence in question, for example, or will encode the same active sites. Although homology can also be considered in terms of similarity (i.e., amino acid residues with “similar chemical properties / functions), in the context of the present invention, it is preferable to express homology in terms of sequence identity. Homologous sequences typically retain domains or functional patterns.
[0501] [0501] In one embodiment, a homologous sequence is taken to include an amino acid sequence or nucleotide sequence that has one, two or more additions, deletions and / or substitutions compared to the sequence in question.
[0502] [0502] Homology or identity comparisons can be performed by the eye, or more generally, with the help of readily available sequence comparison programs. These commercially available computer programs can calculate% homology between two or more sequences. The% homology or identity percentage can be calculated through continuous sequences, that is, one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called “gap-free” alignment. Typically, these alignments without gaps are performed only on a relatively short number of residues.
[0503] [0503] Although this is a very simple and consistent method, it does not take into account that, for example, in an identical pair of sequences, an insertion or deletion will cause the following amino acid residues to be misaligned, potentially resulting in a large reduction in% homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into account possible insertions and deletions without unduly penalizing the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximize local homology.
[0504] [0504] However, these more complex methods assign “gap penalties” to each gap that occurs in the alignment, so that, for the same number of identical amino acids, a sequence alignment with the fewest gaps possible - reflecting in greater relationship between the two sequences compared - will reach a higher score than one with many gaps. The “related gap costs” are usually used which charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent waste in the gap. This is the most widely used scoring system. High gap penalties are certain to produce optimized alignments with fewer gaps. Most alignment programs allow gap penalties to be modified. However, it is preferable to use the default values when using this software for sequence comparisons.
[0505] [0505] Therefore, the calculation of the maximum% of homology requires, first, the production of an ideal alignment, taking into account the gap penalties. A suitable computer program to perform this alignment is Vector NTI (Invitrogen Corp.). Examples of software that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al.1999 Short Protocols in Molecular Biology, 4th Ed - Chapter 18), BLAST 2 (see FEMS Microbiol Lett 1999 174 (2): 247-50; FEMS Microbiol Lett 1999 177 (1): 187-8 and tatiana & ncbi.nlm.nih.gov), FASTA (Altschul et al.1990 JJ. Mol. Biol. 403-410) and AlignX for example. At least BLAST, BLAST 2 and FASTA are available for offline and online research (see Ausubel et al.1999, pages 7-58 to 7-60).
[0506] [0506] Although% final homology can be measured in terms of identity, the alignment process itself is usually not based on a comparison of all or nothing pairs. Instead, a scale similarity scoring matrix is generally used that assigns scores to each paired comparison based on chemical similarity or evolutionary distance. An example of this commonly used matrix is the BLOSUM62 matrix - the standard matrix for the BLAST suite of programs. Vector NTI programs generally use public default values or a custom symbol comparison table, if provided
[0507] [0507] Alternatively, the percentages of homologies can be calculated using the multiple alignment feature in Vector NTI (Invitrogen Corp.), based on an algorithm, analogous to CLUSTAL (Higgins DG & Sharp PM (1988), Gene 73 (1 ), 237-244). After the software produces an optimal alignment, it is possible to calculate% homology, preferably% t of sequence identity. The software normally does this as part of the sequence comparison and generates a numerical result.
[0508] [0508] Gap penalties should be used for determining sequence identity, then preferably, the following parameters are used for alignment in pairs: mere TO
[0509] [0509] In one embodiment, CLUSTAL can be used with the gap penalty and gap length defined as defined above. In some embodiments, the gap penalties used for the BLAST or CLUSTAL alignment may differ from those detailed above. The specialist will assess that the standard parameters for the execution of the BLAST and CLUSTAL alignments may change periodically and will be able to select appropriate parameters based on the detailed standard parameters for the currently BLAST or CLUSTAL alignment algorithms.
[0510] [0510] Suitably, the degree of identity to a sequence of nucleotides is determined on at least 50 continuous nucleotides, preferably on at least 60 continuous nucleotides, preferably on at least 70 continuous nucleotides, preferably on at least 80 continuous nucleotides, preferably about at least 90 continuous nucleotides, preferably about at least 100 continuous nucleotides, preferably about at least 150 continuous nucleotides, preferably about at least 200 continuous nucleotides, preferably about at least 250 continuous nucleotides, preferably about at least 300 continuous nucleotides, preferably about at least at least 350 continuous nucleotides, preferably over at least 400 continuous nucleotides, preferably over at least 450 continuous nucleotides, preferably over at least 500 continuous nucleotides, preferably over at least 550 nucleotides continuous otides, preferably over at least 600 continuous nucleotides, preferably over at least 650 continuous nucleotides, or preferably over at least 700 continuous nucleotides.
[0511] [0511] Suitably, the degree of identity with respect to a sequence of nucleotides, cDNA, cds or amino acids can be determined throughout the sequence.
[0512] [0512] Sequences can also have deletions, insertions or substitutions of amino acid residues that produce a silent change and result in a functionally equivalent substance. The deliberate amino acid substitutions can be made based on the similarity of polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathic nature of the residues, as long as the secondary binding activity of the substance is maintained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with polar head groups not charged with similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine and tyrosine.
[0513] [0513] Conservative substitutions can be made, for example, according to the Table below. The amino acids in the same block in the second column and preferably in the same row in the third column can be replaced by each other:
[0514] [0514] The present invention also encompasses homologous substitution (substitution and replacement are used here to mean the exchange of an existing amino acid residue with an alternative residue) that can occur, that is, identical substitution, as basic to basic , acid to acid, polar to polar, etc. Non-homologous substitution may also occur, that is, from one class of residue to another or, alternatively, involving the inclusion of unnatural amino acids such as ornithine (hereinafter Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as O), pyrialalanine, thienylalanine, naphthylalanine and phenylglycine.
[0515] [0515] Substitutions can also be made for unnatural amino acids include; alpha * and alpha-disubstituted * amino acids, N-alkyl * amino acids, lactic acid *, halogenated derivatives of natural amino acids such as trifluorotyrosine *, p-Cl-phenylalanine *, p-Br-phenylalanine *, pI-phenylalanine *, L-allyl -glycine *, B-alanine *, La-amino butyric acid *, L-a-amino butyric acid *, L-a-amino isobutyric acid *, La-amino caprócot acid, 7-amino heptanoic acid *, L-methionine sulfone ”, L-norleucine *, L-norvaline *, p-nitro-L-phenylalanine *, L-hydroxyprolinat, L-thioproline *, methyl phenylalanine (Phe) derivatives, such as 4-methyl-Phe *, pentamethyl- Phe *, L-Phe (4-amino) * t, L-Tyr (methyl) *, L-Phe (4-isopropyl) *, L-Tic (1,2,3,4-tetrahydroisoquinoline-3 acid - carboxylic) *, L-diaminopropionic acid! and L-Phe (4-benzyl) *. The notation * was used for the purposes of the above discussion (related to homologous or non-homologous substitution), to indicate the hydrophobic nature of the derivative, while td was used to indicate the hydrophilic nature of the derivative, tf * indicates amphipathic characteristics.
[0516] [0516] Amino acid sequence variants may include suitable spacer groups that can be inserted between any two amino acid residues in the sequence including alkyl groups such as methyl, ethyl or propyl groups in addition to amino acid spacers such as glycine or B- residues alanine. Another form of variation involves the presence of one or more amino acid residues in the peptide form, which will be well understood by those skilled in the art. For the avoidance of doubt, "the peptide form" is used to refer to variant amino acid residues where the a-carbon substituent group is on the residue's nitrogen atom instead of a-carbon. Processes for preparing peptides in peptide form are known in the art, for example, Simon RJ et al., PNAS (1992) 89 (20), 9367-9371 and Horwell DC, Trends Biotechnol. (1995) 13 (4), 132-134.
[0517] [0517] Nucleotide sequences for use in the present invention can include synthetic or modified nucleotides therein. A number of different types of oligonucleotide modification are known in the art. These include the methylphosphonate and phosphorothioate backbones and / or the addition of acridine or polylysine chains at the 3 'and / or 5' ends of the molecule. For the purposes of the present invention, it is to be understood that the nucleotide sequences described herein can be modified by any method available in the art. Such modifications can be made in order to increase the in vivo activity or the useful life of the nucleotide sequences of the present invention.
[0518] [0518] The present invention also encompasses sequences that are complementary to the nucleic acid sequences of the present invention or sequences that are capable of hybridizing to the sequences of the present invention or to sequences that are complementary to them. The term "hybridization", as used here, should include "the process by which a nucleic acid strand joins a complementary strand through base pairing", as well as the amplification process carried out in polymerase chain reaction technologies (PCR).
[0519] [0519] The present invention also relates to nucleotide sequences that can hybridize to the nucleotide sequences of the present invention (including sequences complementary to those shown here). Preferably, hybridization is determined according to stringent conditions (for example 50 “* C and 0.2xSSC (1xSSC = 0.15 M NaCl, 0.015 M Na; pH 7.0 citrate)). More preferably, hybridization is determined according to high stringency of conditions (for example 65 ° C and 0.1xSSC (1xSSC = 0.15 M NaCl, 0.015 M PH 7.0 Nasxcitrate)).
[0520] [0520] In one aspect, the sequence for use in the present invention is a synthetic sequence - that is, a sequence that was prepared by chemical or enzymatic synthesis in vitro. It includes, but is not limited to, sequences made with the ideal use of codons for host organisms.
[0521] [0521] The term "expression vector" means a construct capable of expression in vivo or in vitro. In one embodiment, The vector of the present invention expresses a Nicl ERF gene as described here. In one embodiment, the vector of the present invention also expresses a Nic2 ERF gene as described herein. Preferably, the expression vector is incorporated into the genome of a suitable host organism. The term "incorporated" preferably encompasses stable incorporation into the genome.
[0522] [0522] The nucleotide sequence for use in the present invention can be present in a vector in which the nucleotide sequence is operably linked to regulatory sequences capable of providing expression of the nucleotide sequence by a suitable host organism. The constructs for use in the present invention can be transformed into a suitable host cell as described herein to provide for the expression of a polypeptide of the present invention. The choice of the vector, for example, a plasmid, cosmid or phage vector will often depend on the host cell into which it will be introduced. The vectors can be used in vitro, for example, for the production of RNA or used to transfect, transform, transduce or infect a host cell.
[0523] [0523] In some applications, the nucleotide sequence for use in the present invention is operationally linked to a regulatory sequence that is capable of providing expression of the nucleotide sequence, such as by the chosen host cell. By way of example, the present invention encompasses a vector comprising the nucleotide sequence of a Nicl ERF gene as described herein operably linked to such a regulatory sequence, i.e., the vector is an expression vector.
[0524] [0524] The term “operationally linked” refers to a juxtaposition in which the components described are in a relationship that allows them to function as intended. A regulatory sequence "operationally linked" to a coding sequence is linked in such a way that the expression of the coding sequence is achieved under conditions compatible with the control sequences.
[0525] [0525] The term "regulatory sequences" includes promoters and enhancers and other signs of expression regulation. The term "promoter" is used in the normal sense of the prior art, for example, an RNA polymerase binding site. The nucleotide sequence within a construct that encodes a Nicl ERF gene or a Nicl ERF gene and a Nic2 ERF gene can be operably linked to at least one promoter.
[0526] [0526] The term "construction" - which is synonymous with terms like "cassette" or "vector" - includes a sequence of nucleotides for use in accordance with the present invention, directly or indirectly linked to a promoter.
[0527] [0527] An example of indirect bonding is the provision of a suitable spacer group, such as an intron sequence, such as the Shl11 intron or the ADH intron, which is intermediate to the promoter and the nucleotide sequence of the present invention. The same goes for the term "fused" in relation to the present invention, which includes direct or indirect linkage. In some cases, the terms do not cover the natural combination of the nucleotide sequence that encodes the protein normally associated with the wild-type gene promoter and when both are in their natural environment. The construct may even contain or express a marker, which allows selection of the genetic construct.
[0528] [0528] A review of the general techniques used to transform plants can be found in the articles by Potrykus (Annu Rev Physiol Plant Mol Biol [1991] 42: 205-225) and Christou (Agro-Food-Industry Hi-Tech March / April 1994 17-27), which are incorporated by reference. Further teachings on plant transformation can be found in EP-A-0449375, incorporated here by reference.
[0529] [0529] In one embodiment provided here are SNPs for use in genotyping the locus Nicl in plants (e.g., tobacco plants).
[0530] [0530] In one embodiment, markers for use in genotyping the locus Nicl in plants (e.g., tobacco plants) are provided here. In one embodiment, pairs of primers for use in genotyping the locus Nicl in plants (e.g., tobacco plants) are provided here. In one embodiment, primers for genotyping the Nicl locus in tobacco plants are provided in Table 4.
[0531] [0531] In one embodiment, markers for use in genotyping the Nic2 locus in plants (e.g., tobacco plants) are provided here. In one embodiment, pairs of primers for use in genotyping the Nic2 locus in plants (e.g., tobacco plants) are provided here. In one embodiment, primers for genotyping the Nic2 locus in tobacco plants are provided in Tables 5 and 6.
[0532] [0532] As used here, "SNP" or "single nucleotide polymorphism" means a sequence variation that occurs when a single nucleotide (A, T, C or G) in the genome sequence is altered or variable in relation to a sequence of reference. "SNP markers" exist when SNPs are mapped to sites in the genome.
[0533] [0533] As used here, "marker" or "SNP marker" means a sequence of nucleic acids or amino acids that is sufficiently unique to characterize a specific locus in the genome. A polymorphic characteristic can be used as a marker if it is differentially inherited and exhibits a link imbalance with a phenotypic characteristic of interest. When a characteristic is said to be linked to a particular marker, it will be understood that the current DNA segment whose sequence affects the characteristic usually co-secretes with the marker.
[0534] [0534] In one embodiment, any SNP identified in Table 4 can be used in a plant genotyping method (for example, tobacco plants) to identify the presence, absence or modification of the locus Nicl. In one embodiment, any pair of primers identified in Table 4 can be used in a plant genotyping method (for example, tobacco plants) to identify the presence or absence of the locus Nicl.
[0535] [0535] In one embodiment, any SNP identified in Table 5 or Table 6 for use in a plant genotyping method (for example, tobacco plants) to identify the presence or absence of the Nic2 locus. In one embodiment,
[0536] [0536] Unless otherwise defined, all technical and scientific terms used in this document have the same meaning as is generally understood by a technician in the subject to which this disclosure belongs. Singleton, et al., DICIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide one of skills with a general dictionary of many of the terms used in this disclosure.
[0537] [0537] This disclosure is not limited by the exemplary methods and materials disclosed in this document, and any methods and materials similar or equivalent to those described here may be used in the practice or testing of embodiments of the present disclosure. Numeric ranges include the numbers that define the range. Unless otherwise defined, any nucleic acid sequences are written from left to right in the 5 'to 3' orientation; amino acid sequences are written from left to right in amino orientation for carboxy, respectively.
[0538] [0538] The titles provided here are not limitations on the various aspects or embodiments of this disclosure that can be obtained by reference to the specification as a whole. Therefore, the terms defined immediately below are more fully defined by reference to the specification as a whole.
[0539] [0539] Amino acids are referred to here using the name of the amino acid, the three letter abbreviation or the one letter abbreviation. The term "protein", as used here, includes polypeptide proteins and peptides. As used here, the term "amino acid sequence" is synonymous with the term "polypeptide" and / or the term "protein". In some cases, the term "amino acid sequence" is synonymous with the term "peptide". In some cases, the term "amino acid sequence" is synonymous with the term "enzyme".
[0540] [0540] In the present disclosure and claims, conventional one-letter and three-letter codes for amino acid residues can be used. The three-letter code for amino acids, defined in accordance with the Joint Biochemical Nomenclature Commission of IUPACIUB (JCBN). It is also understood that a polypeptide can be encoded by more than one nucleotide sequence due to the degeneration of the genetic code.
[0541] [0541] Other definitions of terms may appear throughout the specification. Before the exemplary embodiments are described in more detail, it is to be understood that this disclosure is not limited to the particular embodiments described, as these may, of course, vary. It should also be understood that the terminology used here is intended to describe only particular embodiments, and is not intended to be limiting, since the scope of this disclosure will be limited only by the appended claims.
[0542] [0542] When a range of values is provided, it is understood that each intermediate value, up to the tenth of the lower limit unit, unless the context clearly indicates otherwise, between the upper and lower limits of that range is also specifically disclosed . Each minor interval between any declared value or intermediate value in a declared interval and any other declared or intervening value in that declared interval is covered in this disclosure. The upper and lower limits of these minor ranges can be included or excluded in the range independently, and each range in which one, none or both of the limits is included in the smaller ranges is also covered in this disclosure, subject to any limit excluded specifically in the stated range. When the stated range includes one or both of the limits, ranges excluding one or both of the included limits are also included in this disclosure.
[0543] [0543] It should be noted that as used here and in the appended claims, the singular forms "one", "one", and "o / a" include said plurals unless the context clearly dictates otherwise. Thus, for example, the reference to "an enzyme" or "a nitrate reductase" includes a plurality of such candidate agents and equivalents of those known to those skilled in the art and so on. BENEFITS
[0544] [0544] It has surprisingly been found that by modulating the activity or expression of a Nicl ERF gene, as taught here, the alkaloid content and / or TSNA content of plants can be modulated. Thus, tobacco products with modulated alkali content and / or TSNA content and commercially desirable characteristics sought by consumers of tobacco products can be produced.
[0545] [0545] the present inventors surprisingly have determined a method for modulating the content of alkaloids, for example, nicotine content, and / or TSNA content of a tobacco plant by modulating the activity or expression of a Nicl ERF gene. The nicotine or TSNA content of a tobacco plant can be decreased by inhibiting the activity or expression of a Nicl ERF gene. Prior to the present invention, it was not known that modulation of the activity or expression of a Nicl ERF gene, as described here, could be used to modulate the content of alkaloids and / or TSNA.
[0546] [0546] The present inventors have determined that modulation of a Nicl ERF gene can reduce the alkaloid content of the modified plant to a surprisingly low level.
[0547] [0547] The LI strain (low intermediate, aaBB) normally produces about half the content of alkaloids compared to the HA strain (high alkali, AABB). However, in Example 13, EMS lines were produced that are equivalent to the LI line. EMS strains with Nitab4.5 0003090g0030.1 (ERF199) mutations were produced. These EMS mutant strains produced about a third of the alkaloid content compared to the HA line - much less than would be expected for an LI line.
[0548] [0548] Nicl ERF Nitab4.5 0003090g0030.1 (ERF199) was eliminated by editing HI lineage genes (AAbb), the alkaloid content of the eliminable lineage was much lower than the alkaloid content of the equivalent LA lineage (aabb) (see Example 14). These data surprisingly indicate that modulating the activity or expression (for example, knocking down or knocking out the activity or expression) of a Nicl ERF gene (for example Nitab4.5 0003090g0030.1 or ERF199) alone is sufficient to modulate (for example , reduce) the alkaloid content of a plant or part of it. º The reduction of alkaloids when the Nic2 mutation in HI (AAbDDb) is combined with the ERF199 mutation in a single plant is much greater than its combined additive effects, which suggests epistasis.
[0549] [0549] The publications discussed in this document are provided for publication only before the date of submission of this application. Nothing in this document is to be construed as an admission that such publications constitute state of the art to the appended claims.
[0550] [0550] The inventors sought to identify the Nicl genes responsible for the altered alkaloid content in plants. The four Burley NILS 21 (B21) - normal / high alkali B21 (HA-B21), high intermediate alkali B21 (HI-B21), low intermediate alkali B21 (LI-B21) and low alkali B21 (LA-B21) ( hereinafter referred to as HA, HI, LI and LA) were grown in the greenhouse in pots 6.5 inches deep x 6.5 inches in diameter, with 5 drain holes in PRO-MIX from the soil until they reached two months of age and root samples were collected for RNA-seq analysis. Three different populations of F> segregation were derived independently from the crossings of HA (AABB) x LI (aaBB), HA (AABB) x LA (aabb) and HI (AAbb) x LA (aabb). 200 F7 individuals from each of the HA x LA and HA x LI crosses were cultured in the field and alkaloid levels were measured in all strains at three months of age. Six hundred F> r HI x LA individuals were grown in the field until three months of age and tested for alkaloid content, as above. Results - Phenotyping of F> xs derived from the crosses HA (AABB) x LI (aaBB) and HA (AABB) x LA (aabb)
[0551] [0551] It was found that the total levels of alkaloids for the F 2 plants derived from the crosses of HA x LI and HA x LA are continuous (Figure 1, panel A shows the total levels of alkaloids for parental and F; xs derived of the HA x LI crosses. Panel B shows the nicotine levels for the parental lines and the F; xs derived from the HA x LA (B) crosses, so that the Nicl genotype for both populations could not be inferred unequivocally based on the value of the phenotype. Is this particularly the case in the F> population of HA x LI, where the range of phenotypic values for parental lines overlaps (Figure 1A). To map to the Nicl locus, the lowest 20 and 24 strains and the highest F> 24 and 20 strains of the HA x LI and HA x LA population, respectively, were selected. F> plants (total 88) with the lowest or highest levels of alkaloids could be genotyped as recessive (aa) or dominant (2A) homozygotes. EXAMPLE 2 Development of marker and linkage analysis
[0552] [0552] To determine the candidate genes responsible for the altered alkaloid content, Forty-four F; s from Example 1 with the most extreme high or low levels of alkaloids from the F, HA x LA and HA x LI populations were selected for SNP genotyping with a custom 30K Infinium iSelect HD BeadChip tobacco (Illumina Inc., San Diego, CA), with their respective parents. The SNP clusters were generated using GenomeStudio version 2011.1 (Illumina Inc., San Diego, CA) and all identified polymorphic markers were used for further analysis. Genetic linkage maps for both populations were constructed using Joinmap software version 3.0 (Stam, 1993) using the regression mapping function, with default settings. In order to treat the map of the Nicl and Nic2 loci, the interval mapping was performed in the two populations using MapoTL version 6.0 (Van Ooijen, 2009 incorporated here by reference) with the selected selective genotyping option and step size of 1 cM.
[0553] [0553] SNPs identified from both RNA-seq and the use of custom BeadChip 30K Infinium iSelect HD in the two populations above were validated by sequencing the respective genotypes and comparing with the HA sequence. All confirmed SNPs were converted to CAPS or dCAPS markers (Neff et al., 2002 incorporated here by reference) and used to generate a map for the F> HI x LA population. A genetic map for this population was built using Joinmap, using the settings above. The DNA used for genotyping was extracted from leaf samples from all strains using the CTAB method (Doyle, JJ and JL Doyle. 1987. The rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytoochemical Bulletin 19: 11-15 incorporated here by reference). RNA isolation and sequencing
[0554] [0554] Total RNA was isolated using the QIAGEN Plant RNeasy mini kit (QIAGEN) and treated with DNase I to remove residual DNA contamination, according to the manufacturer's manual. The quantity and quality of the RNA samples were assessed with BioAnalyzer 2100 (Agilent Genomics). The RNA-seq libraries were built using the Illumina TruSeq RNA Sample Prep kit (Illumina). All libraries were sequenced on the Illumina Hiseq Rapid Mode 150-Cycle platform. The base call and demultiplexing of the sample were performed using the Illumina HiSeg Control Software and CASAVA pipeline software.
[0555] [0555] Sample separation and cutting of the adapter / barcode were performed using the standard Illumina software and the quality of the cut readings was verified with FastQC. Two reference transcriptomes were used for mining RNA sequencing data. One contains 239 ERF genes noted by Rushton et al. (2008 incorporated here by reference). The other was generated by gene prediction in the TN 90 genome draft (Sierro et al., 2014 incorporated here by reference). The RNA-segq readings were aligned to the tobacco reference transcriptomes with the general resource format file (GFF) using TopHat v2.0.9 calling Bowtie2 v2.1.0 (Langmead and Salzberg, 2012 incorporated here by reference). Unified genotyper - genome analysis tool (GATK; version 2.8-1-g932cdôa) was used to call SNPs in the four accessions, resulting in a multiple sample variant (VCF) format file (McKenna et al., 2010 incorporated here by reference). Variant calls with a quality lower than 20 were later removed with the VCF filter. Physical mapping and identification of candidate genes
[0556] [0556] SNP markers found to be closely genetically linked with either Nicl or Nic2 locus were aligned against a high density consensus genetic map for tobacco (Nicotiana tabacum consensus 30k Infinium HD 2015; https://solgenomics.net/cview /map.pl map version id = 178 incorporated here by reference). Markers within the regions of interest around the Nicl and Nic2 loci that were able to be uniquely anchored to an improved tobacco genome assembly (Edwards et al., 2017 incorporated here by reference) were used to identify BioNano hybrid scaffolds (ie, pseudochromosome regions) subtending the two regions. The gaps in the sequences were subsequently filled by the identification of equivalent super-scaffolds of the tobacco variety TN 90 (Sierro et al., 2014 incorporated here by reference) containing correspondences identical to the sequences of gene models (coding regions) in the two regions of interest of the genome de Edwards et al. (supra 2017) using the megablast option with the default settings in the Basic Local Alignment Search Tool (BLAST) version
[0557] [0557] To compare the region identified in this study with that of Adams et al. (2016 incorporated here by reference), the BLAST analysis was performed as above, using genomic sequences by Adams et al. (supra 2016) against the genome of Edwards et al. (supra 2017). Sequence hits showing a minimum 99% identity in at least 1 kb were considered as evidence of matches. Results iSelect HD BeadChip genotyping of selected F; s derived from HA x LA and HA x LI
[0558] [0558] Using custom 30K Infinium iSelect HD BeadChip, several other unbound groups were identified in both HA x LI and HA x LA populations (data not shown), which indicates that more than the Nicl and Nic regions are segregating between HA and LA. This suggests that it would be unlikely to find the Nicl region just from mapping polymorphic markers between the two parents. The QTL analysis with the selective genotyping method MapoTL identified a linker group containing several markers common to the F, HA x LI and HA x LA populations that were significantly associated with the total alkaloids content (maximum LOD scores of 31.13 and 26 , 95, respectively), explaining the variation of 51.2% and 46.2% in this characteristic, respectively. These markers co-located with markers in link group 7 (Pseudochromosome 7 of the genome of Edwards et al. (Supra 2017)) of the consensus map of N. tabacum 30k Infinium HD 2015.
[0559] [0559] Figure 2 shows a comparison of genetic maps of F7 individuals selected from the HA x LI and HA x LA crosses with the N. tabacum 30k Infinium HD 2015 consensus map. Dashed lines indicate markers identified between F maps ; 7 and the consensus map, dotted lines indicate markers identified between the two F> 2 maps. The bold font indicates markers common to more than one map. Only the markers that have been identified on the HA x LA or HA x LI map are shown on the consensus map, other marker positions are shown as horizontal lines in black.
[0560] [0560] In addition to this group, another group was also identified that had a significant association with the total alkaloid content in the Fr HA x LA population (maximum LOD score of 14.01), but explained only about half of the variation for this characteristic as markers in link group 7 (maximum variation of 27.6% explained). This group also contained a specific marker of the dominant gene for Nic2 (Qin et al., 2015; Nic2 marked in Figure 2). The markers of this color group located with the markers in link group 19 of the consensus map (Pseudochromosome 19 of the genome of Edwards et al. (2017)), which is consistent with the proposed genomic region of the Nic2 locus (Kajikawa et al., 2017). SNP identification for ERF genes based on RNA sequencing
[0561] [0561] The annotation of transcription factors with the tobacco genome revealed 239 ERF genes in the tobacco genome (Rushton et al.2008 incorporated here by reference). The identification of SNP based on the RNA-seq analysis of HA, HI, LI and LA revealed an SNP in ERF110 (Nitab4.5 0006382g90040.1; Table 1). Assisted with the restriction enzyme Bstz17I, the SNP in ERF110 was converted into a CAPS marker, designated SNP4 (Table 7). The linkage analysis with the 88 F individuals selected from the HA x LI and HA x LA crosses demonstrated that SNP4 was linked to the Nicl locus, with 6 recombinants detected from 44 F> s with the lowest levels of alkaloids (Figure 3 ).
[0562] [0562] In addition, SNP4 binding analysis with 30K Infinium iSelect HD BeadChip markers indicated that it was closely linked to markers in binding group 7 that were significantly associated with the total alkaloid content in the two populations (Figure 2). However, due to the fact that the genotype of an individual F2> plant cannot be accurately determined from its phenotype data, as mentioned earlier, the observed recombination may be due to phenotyping errors. Thus, a more adequate segregated population is needed to map the locus Nicl. EXAMPLE 3 Comparison of the phenotype for B21 NILs
[0563] [0563] Thirty plants for each B21 NIL were then selected to measure their alkaloid content (Figure 4). The total levels of alkaloid or nicotine for different strains can be easily identified phenotypically, on an average basis. However, the identification in a single plant is not as consistent, because there is considerable phenotypic overlap between individual plants of different strains, such as LI and HI, LI andHAeHIeHA (Figure 4).
[0564] [0564] Even within the same strain, total alkaloid or nicotine levels can vary dramatically between individual plants, with the exception of the LA strain. Therefore, we generate an F population; derived from the cross between HI and LA and we performed analyzes of total alkaloids of this population (Figure 5).
[0565] [0565] Based on the results of the genotyping of HA x LI and HA x LA populations, we decided to test the diagnostic capacity of the SNP4 CAPS marker in a larger population.
[0566] [0566] Genotyping of 600 F> plants derived from HI x LA with SNP4 revealed a segregation rate of 150: 289: 161 for AA: Aa: aa, which fits the expected 1: 2: 1 (2 = 1, 21, df = 2, P = 0.55). 150 dominant homozygous predicted plants (AA) and 161 F> predicted recessive homozygous plants (aa) were selected for alkaloids analysis. The two groups F> were discrete, without overlapping. All individuals with the predicted aa genotype had low levels of alkaloids (0.17-0.41%), within the range of the LA parent (0.18-0.38% (Figure 5).
[0567] [0567] Similarly, alkaloid levels were high for predicted AA plants, and none of them fell within the range of the LA parent. However, some of the predicted AA plants were observed outside the HI parent range. To confirm the Nicl genotype in the plant that was genotyped as AA, but that contained less nicotine content (indicated by the arrow in Figure 5), we checked the genotypes and segregation of the alkaloid phenotype in the next generation for this individual. All F3 strains; maintained the predicted AA genotype when analyzed with the SNP4 marker.
[0568] [0568] The 40 F; s chemical analysis derived from this strain revealed that all plants contained levels comparable to HI alkaloid (Figure 6) and no plants fell within the range of LA alkaloids, substantiating the hypothesis that F7 selected was homozygous and the SNP marker we developed is co-segregated with the locus Nicl.
[0569] [0569] We performed the DNA sequencing of the four NILsS B21 to confirm the sequence polymorphisms that were identified as being closely linked to the Nicl locus using the Infinium iSelect HD BeadChip 30K (Figure 3). Six of these SNPs could be converted to PCR-based CAPS or dCAPS markers (Table 4 shows converted SNPs and markers identified based on the customized 30K Infinium iSelect HD BeadChip analysis for the Nicl region). To identify more markers for the genetic mapping of the Nicl locus, we carried out the identification of SNP based on RNA sequencing using the published TN 90 genome reference sequence (Sierro et al., 2014 incorporated by reference). Six more SNPs were detected among the B21l NILS segregating to Nicl, all converted to CAPS or dCAPS markers (Table 7). We also generated CAPS markers for two SNPs from a previous study, analyzing the potential Nicl deletion region, marked with SNP13 and SNPl4, in Figure 7 and Table 7 [SEQ. ID 133 and 136 in Adams et al. (2016 incorporated here by reference), respectively]. In addition, we used primers used to characterize a 500kb exclusion from the same publication [annotated as SEQ. IDs 3 and 4 in Adams et al. (2016))]), in order to develop a dominant marker for this deletion region (marked as INDEL1 in Figure 7). Genetic mapping of the locus Nicl was performed with the markers in Table 4 and Table 7 using 161 F recessive (aa) derived from HI x LA (Figure 7). As can be seen from the genetic map, the locus Nicl, co-secreting with SNP4, is flanked by SNP3 and SNP5 (Figure 7), and is genetically different from the region identified by Adams et al. (2016 incorporated here by reference).
[0570] [0570] To develop PCR-based markers in the region around MNic2, we performed the above sequencing on the SNP chip markers that are linked to that locus, based on the F2 population of HA x LA. Six of these SNPs could be converted to PCR-based CAPS or dCAPS markers (Table 5).
[0571] [0571] According to the Nicl locus, we used RNA-seq to identify more SNPs linked to the Nic2 locus. Three more SNPs were identified that could be converted to dCAPS markers (Table 6). Genetic mapping of the Nic2 locus was performed with the markers in Table 5 and Table 6 using 188 Frs of HA x LA (Figure 8). As can be seen in the genetic map, the Nic2 locus (defined by the NIC1l marker from Qin et al. (2015)) co-secretes with SNP17 and 18 (Figure 8).
[0572] [0572] Using the markers identified as being closely linked by comparing the N. tabacum 30k Infinium HD 2015 consensus map with the genetic maps F, HA x LI and HA x LA, we identified a genomic fusion region that encompasses the Nicl locus subtended by the Nt1AB6591 and Nt1AA9777 markers. Using the BioNano hybrid set by Edwards et al. (2017 incorporated here by reference), we were able to identify the scaffolding mapped to the pseudochromosomes that covered most of this region (see Figure 82, column 'Evidence' in Table 8). Reciprocal analysis of BLAST in TN 90 super-seals from Sierro et al. (2014 incorporated here by reference) from the genome set was able to identify more scaffolding by Edwards et al. (2017) in the region to fill possible gaps in the sequence set (see Figure 82, column 'TN 90 Superandaime'! In Table 8). Finally, markers that could not be integrated using any of the above methods, but could be mapped exclusively to scaffolding in Edwards et al. (2017), were integrated based on their position in the consensus genetic maps, HA x LA or HA x LI (Figure 82). In the case of contradictory evidence for the location of a scaffold, the location of the scaffold in the BioNano hybrid set was used.
[0573] [0573] To map the Nicl region, we use the genetic map of the population F>, derived from HI x LA (Figure 7). The SNP markers generated from both the 30K Infinium iSelect HD BeadChip and RNA-sec (Tables 4 and 5) were mapped to the fusion genomic region subtended by the markers identified above. Scaffolds containing new SNP markers within this region have been added to the genomic fusion region, as per the consensus genetic maps of HA x LA and HA x LI above. Based on the recombinants identified in the F> population, of HI x LA (Figure 7), we limit our region of interest to the genomic fusion region defined by the scaffolds Nitab4.5 0003553 and Nitab4.5 0007027 (Figure 82). This region notably contains a cluster of nine genes noted as ERF transcription factors (Table 8). We then used RNA-seq information from Edwards et al. (2017 incorporated here by reference), in order to improve the gene models for genes within this region identified in Table 8; Sequence IDs shown in Table 1).
[0574] [0574] Next, we compare the Nicl region identified with a scaffold that is deleted in the LI lines, identified by Adams et al. (2016 incorporated here by reference) [SEQ. ID 85]. This was accomplished through the BLAST analysis of this sequence for the genomic scaffolding by Edwards et al. (2017 incorporated here by reference). According to the results of the genetic mapping (Figure 7), the region previously identified by Adams et al. (2016) is located in a physical region upstream of our identified Nicl region (see Figure 82).
[0575] [0575] To better characterize the Nic2 region, we performed the same analysis as above for the genomic region subtended by the Ntl1AA9370 and NtlAC6499 markers (Figure 2). This region was also delimited by the SNP15 and SNP18 / 19 markers used in Nic2 fine mapping (Figure 8). The evaluation of the genes in the region indicated that they contained a cluster of nine genes noted as ERF transcription factors (Table 9), including many previously identified genes (Shoji et al., 2010; Kajikawa et al., 2017). We used RNA-seq information from Edwards et al. (2017), in order to improve the gene models for the ERF genes in that identified region (Table 9; sequence IDs shown in Table 2). This region was also compared by BLAST analysis to a sequence identified by Adams et al. (2016 incorporated here by reference), according to the Nicl region [SEQ.
[0576] [0576] Given the incidence of clusters of ERF transcription factors in both regions of interest, we were interested in the possibility that they may represent homologous genes (ie, equivalent genes from the ancestral genomes N. sylvestris and N. tomentosiformis ). BLAST analysis (using the blastn option with default settings) of the coding sequences of the current gene models for each of the ERF genes against the genome of Edwards et al. (2017 incorporated here by reference) indicated that, for most genes, the greatest similarity achieved was with the ERF in the equivalent reciprocal location (Table 10). This suggests that many ERFs in the two regions represent homologous genes.
[0577] [0577] In order to extend the analysis of the two regions of interest delimited by the scaffolding in Table 8 and Table 9, we were interested in the possibility that the entire regions could represent homologous chromosomal segments. Examination of scaffolding in the putative Nicl and Nic2 regions indicated that they were largely of N. sylvestris or N. tomentosiformis origin (data not shown), supporting the hypothesis that they are from homologous chromosomes.
[0578] [0578] BLAST analysis (as above) of each of the genes in the two regions of interest indicated that the best occurrence for a large number of genes was in the reciprocal region. In addition, the order of genes for both regions has been largely maintained, suggesting that very little rearrangement of the genome has occurred in these two regions since the formation of N. tabacum.
[0579] [0579] To generate overexpression vectors for Nicl candidate genes, the cDNA fragments of the protein coding regions were amplified and inserted into the pSITE-4NB vector using gateway cloning technology (Chakrabarty et al., 2007 Artificial Artificial Bromial Chromosomes incorporated here by reference). The primers with gateway recombination sequences used for the amplification of the sequences coding for Nicl candidates are listed in Table 11. Attb refers to a gateway recombination sequence.
[0580] [0580] To construct vectors for Nicl candidates under the control of their native promoters, the genomic sequences including coding regions, promoters and 3 "'- RTUs, were amplified with primers (Table 12) attached with an adapter from the HindIII recognition site Sixteen bases homologous to the ends of the linearized vector were indicated in lower case, while upper case indicated genetic sequences of tobacco.After digestion with HindIII, the amplified products were cloned into the vector pCAMBIALl305.1 by means of infusion cloning (Zhu et al., 2007 incorporated here by reference.) Table 12. Gene name synonymous | Direct primer Reverse primer of the gene Nitab4.5 000 gcaggcatgcaagettGAA | ggecagtgecaagettecTTT 3665 00270 7 | IRESL2 ACAACCCTGTGGTGCAGCG | GGGAATAGTTATTGGATTTTGGTTTTGGTTTTG 126) gcaggcatagcaagetteTc Nitab4.5 000 TTCACGGTTTCCACTTTCC | IFOCagtgccaaget ACCTT 3090g0030.1 | ERFL99 TET (SEQ ID No GACTTCCCTCATGGTTGAGG g ': (SEQ ID No. 128) 127) gcaggcatgcaagcttATA io = ggccagtgccaagettoGGAA dra ERF210 Grao ne neo TTGATTTGACGTCCGGTTGT 9Eoa: : (SEQ ID No. 130) 129) Nitab4.5 000 gcaggcatgcaagcttGTG | gaccagtaccaagettcATTG 1620 0030 7 | ERF91L2 | GCATATTTTATCTGAGGTA | TAGGTGACGTAGCATGGCAT 22: GA (SEQ ID No. 131) | (SEQ ID No. 132) gcaggcatgcaagcttTTG. if. Nitab4.5 000 TAAATTTGTGTATCATCTT | IISCagtaccaagetEGTGCA a620g0080.1 | ERF2º CARA (SEQ ID No TTGAACATATTGAATGTGGG 9,: 133): (SEQ ID No. 134)
[0581] [0581] The sequence tags of the complete genome profile (WGP) are aligned with the genome scaffolds by Edwards et al. (2017) to identify the bacterial artificial chromosomes (BACs) that correspond "consistently to scaffolding within the Nicl region of interest subtended by SNP markers to further assist the scaffold assembly. DNA is extracted from BAC clones using a QIAGEN Plasmid Midi kit , as instructed by the manufacturer (QIAGEN), linearized and sequenced using an Oxford Nanopore MinION device as instructed by the manufacturer (Oxford Nanopore Technologies) to provide long readings on the map. Final sequence readings paired with Illumina provide accurate short readings of the clones BAC The scaffolding of the tobacco genome identified as located in the Nicl region is remapped to the combined readings of the Oxford Nanopore / Illumina sequence of the overlapping BAC clones to create a continuous sequence Gene models are developed for the improved Nicl region, as Edwards et al. (2017).
[0582] [0582] The Nicl ERF candidate genes were overexpressed in tobacco plants by transforming the root into hair. The tobacco plants used for transforming roots into wigs were grown from magenta boxes and the leaves were cut into 0.5 cm x 0.5 cm pieces. The Aqual strain of Agrobacterium rhizogenes containing a binary vector was used to infect the leaf pieces. Disinfection and drug resistance selection were performed in Murashige and Skoog solidified medium containing 150 mg / L of kanamycin sulfate and 250 mg / L of ticarcillin. The transgenic roots were distinguished with a red fluorescence reporter using a confocal microscope. The selected root lines were maintained by subculture every 2 weeks in 10 ml of liquid Gamborg B5 medium with constant agitation at 80 rpm in the dark. Tobacco transformation Stable transgenic plants were generated by transformation mediated by Agrobacterium tumefaciens (Schardl et al., 1987 Gene, Volume 61, Edition 1, 1987, pages 1-11, which is incorporated here by reference). The Agrobacterium strain GV3101 was used to infect sterile leaf discs. The growth chambers used for the transformation of tobacco were programmed for 16 hours of light at 23 ºC and 8 hours of darkness at 20 "ºC. Briefly, tobacco leaves were excised from plants grown in magenta boxes and were cut into 0.4 cm disks. The leaf discs were incubated for 30 minutes in suspension of Agrobacterium tumefaciens (ODçsoo = 0.3-1) containing the target plasmid. After three days of co-cultivation in MS medium, the leaf discs were transferred to the TOM medium for regeneration or selection (MS medium with 20 g / L of sucrose; 1 mg / L of IAA, 2.5 mg / L of BAP). Ticarcillin (250 mg / L) was used to kill excess Agrobacteria. Kanamycin sulfate (300 mg / 1) for the pSITE-4NB or hygromycin B (40 mg / L) constructs for pCAMBIAI305.1 and the gene editing constructs were used as transformation selection, respectively. The regenerated shoots were removed from the leaf discs and transferred to the MS medium containing ticarcillin (250 mg / L) for rooting. After the development of 3 or 4 roots (at least 2 cm), the seedling was transferred to the soil.
[0583] [0583] We use root-to-hair transformation to assess the function of the gene for Nicl ERFs. Chemical analysis revealed that all constructions increased the levels of alkaloids in the roots in LA hair (Figure 9). However, we were unable to harvest roots in hair for Nitab4.5 0004620g90090.3. The hair roots transformed with this gene gradually darkened during the subculture and could not survive, although they were developed normally at the beginning. The lethal effects can be triggered by high levels of nicotinic acid in these hair roots, which need to be further investigated. Based on the transformation of the root into hair, at least six ERFs in the locus Nicl are able to regulate the biosynthesis of alkaloids with differential effects: Nitab4.5 0003090g0030.1 (ERF199);
[0584] [0584] We also validate Nicl ERFs function with stable transformation assays. At least twelve transgenic plants were obtained for each overexpression of Nicl ERFs under the control of the 35S promoter. Chemical analysis revealed that overexpression of Nitab4.5 0003090g0030.1, Nitab4.5 0004620g0080.1 and Nitab4.5 000462g0090.3 in LA plants significantly increased the content of alkaloids. In particular, the overexpression of Nitab4.5 0003090g0030.1 resulted in alkaloid levels comparable to HI plants.
[0585] [0585] We transferred genomic sequences including the native promoter, and coding region 3'-UTR (untranslated region) to Nitab4.5 0003090g0030.1 for LA plants. Chemical analysis with TO plants showed that the production of alkaloids was significantly improved by the genetic transformation of Nitab4.5 0003090g0030.1. It is notable that the alkaloid levels in transformants containing the Nitab4.5 0003090g0030.1 genomic construct were slightly lower than those of HI, which can be caused by TO plant hemizigosidase. EXAMPLE 13 Induced EMS Nicl Mutants
[0586] [0586] Complementation tests via stable transformation of Nicl ERFs indicated that it is possible to restore the Nicl phenotype. However, we wanted to determine whether knocking out any of these genes could generate a phenocopy of the Nicl phenotype by itself.
[0587] [0587] Therefore, approximately 2000 EMS mutant strains were developed from the TI 1068 variety for the purpose of identifying tobacco plants containing mutated genes. The mutations in the ERF genes in the Nicl region of interest in Table 1 above, as well as the mutations in the ERF189 (Nitab4.5 0015055g0010.2) of the Nic2 region were identified by DNA sequencing using clustered M2 lines from this population, according to Rigola et al. (2009 incorporated here by reference).
[0588] [0588] M2 mutant strains from seed clusters identified as containing Nitab4.5 0003090g0030.1 mutations were grown in a greenhouse and at 11 weeks of age which are analyzed for alkali content as above.
[0589] [0589] Homozygous and heterozygous M2 mutant strains for this gene that is identified by DNA sequencing; null segregants were used as control plants for each mutant strain. The average levels of alkaloids were determined for each class of genotype; Significant differences between the wild type and mutant / heterozygous groups were determined by the Student's t test (Figure 84). A minimum of four individuals in each genotype class were used for this analysis. Chemical analysis indicated that a homozygous mutation in Nitab4.5 0003090g0030.1 caused a premature stop codon (amino acid change (Q25 *) resulting in plants with approximately one third of the alkaloid content compared to the wild type control. EXAMPLE 14 Genetic editing
[0590] [0590] Gene editing was used to mutate a Nicl ERF gene in an HI state to determine whether plants equivalent to LA strains could be generated.
[0591] [0591] A heterozygous mutant strain, called Ll, caused by a bp insertion of A, has been identified for Nitab4.5 0003090g0030.1 in TO.
[0592] [0592] An early stop codon was introduced into the mutated Nitab4.5 0003090g90030.1 allele at L1 and disrupted the function of the gene accordingly (Figure 85). To better characterize the phenotype effect contributed by Nitab4.5 0003090g0030.1, we harvest L1 seeds and perform chemical analyzes on the Tl generation. The genotypes of the Tl plants, wild type (WTI-T1), heterozygous mutant (Het-Tl) and homozygous mutant (Mut-Tl), were determined by DNA sequencing. The levels of total WI-Tl alkaloids were comparable to those of HI plants (Figure 86A. Although the reduction in alkaloid levels in Het-Tl plants was not significant, on average, their phenotype values were reduced by half the levels of WT-Tl plants. For homozygous mutants, Mut-Tl, the alkaloid content was barely discernible, which was lower than that of LA plants (Figure 86A). As can be seen from Figure 86B, the knockout of Nitab4.5 0003090g0030.1 in the HI strain resulted in almost 1/10 of the levels of LA plant alkaloids.
[0593] [0593] To understand why the levels of Mut-Tl alkaloids were much lower than those of LA plants, we investigated the polymorphisms at the level of sequence and expression for Nitab4.5 0003090g0030.1 alleles between HI and LA alleles.
[0594] [0594] We sequenced approximately 2.2 kb upstream of the initiation codon, the coding sequence, and about 1.2 kb downstream of the termination codon, but no SNP was identified between these two alleles. Real-time PCR (RT) analysis revealed that Nitab4.5 0003090g0030.1 is specifically expressed at the root, which explains why nicotine biosynthesis is root-specific.
[0595] [0595] Compared to the HI strain, the expression of Nitab4.5 0003090g0030.1 is down-regulated in the LA strain (Figure 87). Therefore, the phenotypic difference between HI and LA is introduced by the differential expression of the Nicl gene.
[0596] [0596] Although the weak expression of Nicl leads to low levels of alkaloids in LA, it is predicted that an ultra-low level of alkaloids can be achieved by a complete interruption of Nicl, which is in line with our observation in Mut-T1 plants (Figure 86B).
[0597] [0597] All publications mentioned in the specification above are incorporated by reference. Various modifications and variations of the described methods and systems of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it is to be understood that the invention as claimed should not be unduly limited to those specific embodiments. In fact, several modifications of the modes described for carrying out the invention that are obvious to experts in biochemistry and biotechnology or related fields, must be within the scope of the following claims.
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权利要求:
Claims (42)
[1]
1. Method of modulating the alkaloid content of a plant or part of it, or a cell culture, characterized by the fact that the method comprises the modification of said plant or cell culture by modulating the activity or expression of at least one Nicl ERF gene: wherein the at least one Nicl ERF gene encodes a polypeptide comprising an amino acid sequence as set out in: SEQ ID NO: 8; or SEQ ID NO: 12; or SEQ ID NO: 16; or SEQ ID NO: 20; or SEQ ID NO: 24; or SEQ ID NO: 28; or SEQ ID NO: 32; or a functional or orthologous variant thereof; or wherein the ERF gene comprises a nucleotide sequence as set out in: SEQ ID NO: 3, or SEQ ID NO: 5; or SEQ ID NO: 9; or SEQ ID NO: 13; or SEQ ID NO: 17; or SEQ ID NO: 21; or SEQ ID NO: 25; or SEQ ID NO: 29; or a functional or orthologous variant thereof.
[2]
2. Method of modulating the content of a tobacco-specific nitrosamine (TSNA) or a precursor of a TSNA in a tobacco plant or plant part thereof characterized by the fact that the method comprises the modification of said plant or a cultivation of cells by modulating the activity or expression of at least one Nicl ERF gene: wherein the at least one Nicl ERF gene encodes a polypeptide comprising an amino acid sequence as set out in: SEQ ID NO: 8; or SEQ ID NO: 12; or SEQ ID NO: 16; or SEQ ID NO: 20; or SEQ ID NO: 24; or SEQ ID NO: 28; or SEQ ID NO: 32; or a functional or orthologous variant thereof; or where the ERF gene comprises a nucleotide sequence as set out in: SEQ ID NO: 3, or SEQ ID NO:
5; or SEQ ID NO: 9; or SEQ ID NO: 13; or SEQ ID NO: 17; or SEQ ID NO: 21; or SEQ ID NO: 25; or SEQ ID NO: 29; or a functional or orthologous variant thereof.
[3]
3. Use of at least one Nicl ERF gene characterized by the fact that it modulates the alkaloid content of a cell or plant or part of it or a cell culture in which: the at least one Nicl ERF gene encodes a polypeptide that comprises an amino acid sequence as set out in: SEQ ID NO: 8; or SEQ ID NO: 12; or SEQ ID NO: 16; or SEQ ID NO: 20; or SEQ ID NO: 24; or SEQ ID NO: 28; or SEQ ID NO: 32; or a functional or orthologous variant thereof, or wherein the ERF gene comprises a nucleotide sequence as set out in: SEQ ID NO: 3, SEQ ID NO: 5; or SEQ ID NO: 9; or SEQ ID NO: 13; or SEQ ID NO: 17; or SEQ ID NO: 21; or SEQ ID NO: 25; or SEQ ID NO: 29; or a functional or orthologous variant thereof.
[4]
4, Method to produce a plant or part of it, a cell culture, a plant propagation material, a leaf, a harvested cut leaf, a processed leaf or a cut and processed leaf that has modulated alkaloid content, characterized by the fact that that the method comprises modifying said plant or cell culture to modulate the activity or expression of at least one Nicl ERF gene in which: the at least one Nicl ERF gene encodes a polypeptide comprising an amino acid sequence as established in : SEQ ID NO: 8; or SEQ ID NO: 12; or SEQ ID NO: 16; or SEQ ID NO: 20; or SEQ ID NO: 24; or SEQ ID NO: 28; or SEQ ID NO: 32; or a functional or orthologous variant thereof, or wherein the ERF gene comprises a nucleotide sequence as set out in: or SEQ ID NO: 3, SEQ ID NO: 5; or SEQ ID NO: 9; or SEQ ID NO: 13; or SEQ ID NO: 17; or SEQ ID NO: 21; or SEQ ID NO: 25; or SEQ ID NO: 29; or a functional or orthologous variant thereof.
[5]
5. Method or use according to any one of claims 1 to 4, characterized by the fact that the alkaloid content is modulated in comparison to a cell or plant culture that has not been modified to modulate hair activity or expression least one Nicl ERF gene.
[6]
6. Modified plant or part of it or a modified cell culture characterized by the fact that it is to modulate the alkaloid content compared to an unmodified plant or an unmodified cell culture, where the modification is the modulation of the activity or expression of at least one Nicl ERF gene, wherein the at least one Nicl ERF gene encodes a polypeptide comprising an amino acid sequence as set out in: SEQ ID NO: 8; or SEQ ID NO: 12; or SEQ ID NO: 16; or SEQ ID NO: 20; or SEQ ID NO: 24; or SEQ ID NO: 28; or SEQ ID NO: 32; or a functional or orthologous variant thereof.
[7]
7. Plant propagation material characterized by the fact that it is obtainable from a plant according to claim 6 or from a cell or plant cultivation produced by the method of either of claims 4 or 5.
[8]
Method or use according to any one of claims 1 to 4, or a plant or part of it or cell culture according to claim 6, or a plant propagation material according to claim 7, characterized because the alkaloid content of the plant is decreased compared to a plant or cell culture that has not been modified to modulate the activity or expression of at least one Nicl ERF gene.
[9]
9. Method or use according to claim 8, a plant or part of it or cell culture according to claim 8, or a plant propagation material according to claim 8, characterized by the fact that the activity or expression of at least one Nicl ERF gene is decreased.
[10]
A method or use according to any one of claims 1 to 5, or a plant or part of it or a cell culture according to claim 6, or a plant propagating material, according to claim 7 , characterized by the fact that the alkaloid content of the plant or cell culture is increased compared to a plant that has not been modified to modulate the activity or expression of at least one Nicl ERF gene.
[11]
A method or use according to any one of claims 1 to 5 or 10, a plant or part of it or a cell culture according to claim 6 or 10, or a plant propagating material according to claims 7 or 10, characterized by the fact that the plant is modified to increase the activity or expression of at least one Nicl ERF gene and the cultivation of cells or plants exhibits an increase in the content of alkaloids compared to a cultivation of plants or cells that do not has been modified to modulate the activity or expression of at least one Nicl ERF gene.
[12]
A method or use according to any one of claims 1 to 5 or 5 to 11, a plant or part of it or a cell culture according to claims 6 or 8 to 11, or a plant propagating material according to claims 7 to 11, characterized by the fact that the total alkaloid content of the plant or cell culture is modulated.
[13]
13. Method or use according to any one of claims 1 to 5 or 8 to 12, a plant or part of it or cell culture according to claims 6 or 8 to 12, or a plant propagating material according to claims 7 to 12, characterized by the fact that the content of one or more alkaloids selected from nicotine, nornicotine, anabasine, myosmin and anatabine is modulated, preferably the nicotine content is modulated.
[14]
14. Method or use according to any one of claims 1 to 5 or 8 to 13, a plant or part of it or a cell culture according to claims 6 or 8 to 13, or a propagating material plant according to claims 7 to 13, characterized by the fact that the plant or plant cell belongs to the Solanaceae family.
[15]
A method or use according to any one of claims 1 to 5 or 8 to 14, a plant or part of it or a cell culture according to claims 6 or 8 to 14, or a plant propagating material according to claims 7 to 14, characterized by the fact that the plant or plant cell is of the genus Solanum.
[16]
16. Method or use according to any one of claims 1 to 5 or 8 to 14, a plant or part of it or cell culture according to claims 6 or 8 to 14, or a plant propagating material according to claims 7 to 14, characterized by the fact that the plant or plant cell is of the genus Nicotiana.
[17]
17. Method or use according to claim 16, a tobacco plant or part of it or cultivation of tobacco cells according to claim 16, or a plant propagating material according to claim 16, characterized by the fact that that the nicotine content is modulated.
[18]
18. A method or use according to claim 16, a tobacco plant or part of it or a tobacco cell cultivation according to claim 16, or a plant propagating material according to claim 16, characterized by the fact that that the nicotine content is decreased.
[19]
19. A method or use according to any one of claims 1 to 5 or 8 to 18, a plant or part of it or a cell culture according to claims 6 or 8 or 11 to 18,
or a plant propagating material according to claims 7 or 11 to 18 characterized by the fact that the at least one Nicl ERF gene encodes a polypeptide comprising an amino acid sequence as set out in SEQ ID NO: 8 or a functional variant or orthologist of the same; Or at least one Nicl ERF gene comprises a nucleotide sequence as set out in SEQ ID NO: 5 or a functional or orthologous variant thereof.
[20]
A method or use according to any one of claims 1 to 5 or 8 to 19, a plant or part of it or a cell culture according to claims 6 or 8 or 11 to 19, or a material of plant propagation according to claims 7 or 11 to 19 characterized by the fact that an additional ERF gene is modulated in which: the additional ERF gene is at least a Nic2 ERF gene and encodes a polypeptide comprising an amino acid sequence as established in: SEQ ID NO: 40; or SEQ ID NO: 44; or SEQ ID NO: 48; or SEQ ID NO: 52; or SEQ ID NO: 56; or SEQ ID NO: 60; or SEQ ID NO: 64; or SEQ ID NO: 68; or SEQ ID NO: 72 or a functional or orthologous variant thereof; or the additional ERF gene is at least one Nic2 ERF gene and comprises a nucleotide sequence as set out in: SEQ ID NO: 37; or SEQ ID NO: 41; or SEQ ID NO: 45; or SEQ ID NO: 49; or SEQ ID NO: 53; or SEQ ID NO: 57; or SEQ ID NO: 61; or SEQ ID NO: 65; or SEQ ID NO: 69.
[21]
21. Method or use according to any one of claims 1 to 5 or 8 to 20, a plant or part of it or a cell culture according to claims 6 or 8 or 11 to 20, or a propagating material plant according to claims 7 or 11 to 20, characterized in that an additional ERF gene is modulated in which the additional ERF gene is at least a Nic2 ERF gene and comprises a nucleotide sequence as set out in SEQ ID NO: 69 or a functional or orthologous variant thereof or encodes a polypeptide comprising an amino acid sequence as set out in SEQ ID NO: 72 or a functional or orthologous variant thereof.
[22]
22. Use of a plant or part thereof or cell cultivation characterized by the fact that it is according to any one of claims 6 or 8 to 21, or a plant produced by the method of any one of claims 4 or 5, or 8 to 20, to produce a plant.
[23]
23. Use of a plant or part thereof or a cell culture characterized in that it is in accordance with any one of claims 6 or 8 to 21, or a plant produced by the method of any one of claims 4 or 5 or 8 to 21 for the production of a product.
[24]
24. Use of a plant or part of it characterized in that it is in accordance with any one of claims 6 or 8 to 21, or a plant produced by the method of any one of claims 4 or 5 or 8 to 21 for the cultivation of a crop.
[25]
25. Use of a plant or part thereof characterized by the fact that it is according to any one of claims 6 or 8 to 21, or a plant produced by the method of any one of claims 4 or 5 or 8 to 21 for produce a leaf.
[26]
26. Leaf harvested from a plant characterized by the fact that it is in accordance with any of claims 6 or 8 to 21, or obtainable from a plant propagated from a propagation material according to any of claims 6 to 21, or obtainable from a plant obtainable by a use according to any one of claims 2 or 22 to 24, or obtainable from a plant produced by the method of any one of claims 4 or 5 or from 8 to 21.
[27]
27. Leaf harvested from a plant according to claim 26, characterized in that the leaf harvested from a plant is a harvested cut leaf.
[28]
28. Processed leaf, preferably a processed tobacco leaf, preferably a non-viable processed tobacco leaf characterized by the fact that it is: obtainable from a plant obtainable from a use according to any one of claims 2 or 22 to 24; obtainable by processing a plant according to any one of claims 5 or 7 to 21; obtainable from a plant propagated from a plant propagating material, according to any one of claims 6 to 21; or obtainable by processing a leaf harvested from a plant according to claims 26 or 27; or obtainable from a plant produced by the method of any one of claims 4 or 5 or 8 to 21.
[29]
29. Processed sheet according to claim 28, characterized by the fact that the sheet is processed by curing fermentation, pasteurization or a combination thereof.
[30]
30. Processed sheet according to claim 28 or 29, characterized in that the processed sheet is a cut processed sheet.
[31]
31. Cured tobacco material characterized by the fact that it is manufactured from a plant or part of it, according to any one of claims 16 to 21 or an extract thereof.
[32]
32. Tobacco blend characterized by the fact that it comprises said cured tobacco material, according to claim 31.
[33]
33. Product of the tobacco industry characterized by the fact that it is prepared from: a tobacco plant according to any one of claims 16 to 21, or a part of it or a cultivation of tobacco cells according to any one claims 16 to 21;
a tobacco plant or part thereof propagated from a tobacco plant propagation material according to any one of claims 16 to 21; a leaf harvested from a plant according to claim 25 or 26, wherein the plant is tobacco; processed leaf according to any one of claims 27 to 29, wherein the plant is tobacco; or a plant produced by the method of claim 16.
[34]
34. Product of the tobacco industry according to claim 33, characterized by the fact that the tobacco product is an article of combustible smoke.
[35]
35. The tobacco industry product according to claim 33, characterized by the fact that the tobacco product is a smokeless tobacco product.
[36]
36. Tobacco product according to claim 33, characterized in that the tobacco product is a non-combustible aerosol delivery system, such as a tobacco heating device or an aerosol generating device.
[37]
37. Use of a tobacco cell, according to claim 16, characterized by the fact that it is to modulate the content of alkaloids in cell culture.
[38]
38. Combustible smoke article, non-combustible aerosol supply system, smokeless tobacco product or tobacco heating device characterized by the fact that it comprises a plant or part of it according to any one of claims 6 to 21 or an extract (for example, a tobacco extract) of the same or a tobacco cell culture according to any one of claims 16 to 21; or a cured tobacco material according to claim 31; or a tobacco blend according to claim 32.
[39]
39. Use of a nucleotide sequence characterized by the fact that it is at least one Nicl ERF gene selected from: SEQ ID NO: 3 or SEQ ID NO: 5; or SEQ ID NO: 9; or SEQ ID NO: 13; or SEQ ID NO: 17; or SEQ ID NO: 21; or SEQ ID NO: 25; or SEQ ID NO: 29; or a functional or orthologous variant thereof, to select a plant with modulated alkali content (eg reduced) and / or modulated content (eg reduced) of tobacco specific nitrosamine (TSNA) or a precursor to a TSNA.
[40]
40. Mutant of a plant carrying an inherited mutation characterized by the fact that it is in a nucleotide sequence of at least one Nicl ERF gene, in which the Nicl ERF gene is selected from: SEQ ID NO: 3, or SEQ ID NO: 5; or SEQ ID NO: 9; or SEQ ID NO: 13; or SEQ ID NO: 17; or SEQ ID NO: 21; or SEQ ID NO: 25; or SEQ ID NO: 29; or a functional or orthologous variant thereof; wherein said hereditary mutation modulates (for example, decreases) the activity or expression of at least one Nicl ERF gene and where the mutant plant modulates (for example, decreases) the content of alkaloids and / or the modulated content of a nitrosamine specific tobacco (TSNA) or a precursor of TSNA in relation to a comparable plant that does not carry the said hereditary mutation.
[41]
41. Progeny or seed of a mutant plant characterized by the fact that it carries the hereditary mutation according to claim 40.
[42]
42. Harvested leaf, processed leaf or cured tobacco material characterized by the fact that it is produced from a plant that comprises a modification in a nucleotide sequence of at least one Nicl ERF gene, where the at least one Nicl ERF gene is selected from: SEQ ID NO: 3, or SEQ ID NO: 5; or SEQ ID NO: 9; or SEQ ID NO: 13; or SEQ ID NO: 17; or SEQ ID NO: 21; or SEQ ID NO: 25; or SEQ ID NO: 29; or a functional or orthologous variant thereof; in which said modulation modulates (for example, decreases) the activity or expression of at least one Nicl ERF gene and in which said plant modulates (for example, decreased) the content of alkaloids and / or modulated content of a nitrosamine specific to tobacco (TSNA) or a precursor of TSNA in relation to a comparable plant that does not carry said modification in at least one Nicl ERF gene.

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同族专利:

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法律状态:
2021-11-03| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US201762524216P| true| 2017-06-23|2017-06-23|

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US62/524,216|2017-06-23|

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PCT/US2018/038679|WO2018237107A1|2017-06-23|2018-06-21|Method|

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