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    • 专利摘要:
    The invention relates to a GhCAL-D07 gene and its application in promoting flowering of plants, which belongs to the technical field of plant genetic engineering. The GhCAL-D07 gene has the nucleotide sequence shown in SEQ ID NO: 1 and can encode the amino acid sequence shown in SEQ ID NO: 2. The increase in expression of the GhCAL-D07 gene in plants has been observed. obtained either by increasing the expression of the endogenous GhCAL-D07 gene in plants, or by overexpressing the exogenous GhCAL-D07 gene in plants, where overexpression of the exogenous GhCAL-D07 gene means that said GhCAL-D07 gene is expressed in plants using a plant expression vector, through the transformation of agrobacteria. The present invention makes it possible to considerably advance the flowering of Arabidopsis thaliana by obtaining the Arabidopsis thaliana plant modified by the GhCAL-D07 gene by transgenesis. By further reducing the GhCAL-D07 gene in cotton to silence, the result shows that the GhCAL-D07 gene plays a key role in promoting cotton flowering. The present invention provides a genetic resource favorable to the cultivation of short-season cotton.
    公开号:FR3096054A1
    申请号:FR2004887
    申请日:2020-05-16
    公开日:2020-11-20
    发明作者:Hengling Wei；Shuxun Yu；Shuaishuai Cheng；Hantao Wang；Liang Ma；Zhengzheng Su
    申请人:Institute of Cotton Research of Chinese Academy of Agricultural Sciences；
    IPC主号:
    专利说明:

    [0001] The invention relates to the field of plant genetic engineering technology, in particular the application of the GhCAL-D07 gene to promote the flowering of plants.
    [0002] context
    [0003] Food and clothing are people's livelihood, and our country is facing the basic situation of more people and less land, so there is a serious conflict between grain and cotton for plowed land , in order to mitigate this conflict, breeders are committed to the selection of short-season cotton, to achieve an agricultural production system of two crops of grain and cotton in one year. The shortcomings of conventional breeding techniques with long cycles have made molecular breeding a trend, transgenic methods being a proven method of breeding new varieties, so inventors are dedicated to developing new genes.
    [0004] Short-season cotton is one of the land cotton varieties with a short fertile period, and has been gradually formed and developed with the changes of the ecological environment and the agricultural cultivation system, adapting to certain social and economic conditions. , the level of production development and the level of scientific and technological development. In our country, short-season cotton is divided into ultra-early Northern ecotype, Yellow River basin ecotype and Yangtze River basin ecotype. The Yangtze River basin ecotype is mainly transplanted cotton after wheat (rapeseed) or directly sown cotton, but transplanted cotton dominates; the Yellow River basin ecotype is mainly harvested twice, usually from May 25 until early June (also called summer cotton); the most premature cotton area in the North is spring cotton in one crop. Short season cotton is generally grown in areas of greater latitude and heat deficit.
    [0005] The CAL gene of Arabidopsis thaliana has been shown to belong to the MADS-box gene family, and CAULIFLOWER (CAL) and AP1 of Arabidopsis thaliana are highly homologous in sequence and have partially overlapping functions. CAL is expressed in the flower meristem during early flower development and participates in determining the properties of the flower meristem. CAL-overexpressing transgenic Arabidopsis thaliana plants flowered much earlier than the wild type. The overexpression CAL gene in Arabidopsis thaliana can promote significantly earlier flowering in Arabidopsis thaliana.
    [0006] Description of the invention
    [0007] In order to solve the above problems, the intervention provide the application of GhCAL-D07 gene to promote plant flowering. The invention is implemented by the following technical solution.
    [0008] The inventors cloned the cotton GhCAL-D07 gene from Gossypium hirsutum, this gene has a high homology with the MADS61 and MADS69 genes in Gossypium hirsutum, and is homologous to CAL in Arabidopsis thaliana. The expression profile shows that this gene is primarily expressed in the terminal buds of young cotton plants, its level of expression is enhanced during the three-leaf period (period of differentiation from bud to flower) of cotton, and the level expression in early species is significantly higher than late species; By constructing the subcellular localization vector, it was found that this gene was localized to the nucleus; By constructing the overexpression vector of this gene and transforming it into Arabidopsis thaliana by floral-dip, the bolting and flowering of overexpressed Arabidopsis thaliana plants was significantly advanced, and the main stem of overexpressed plants was experienced a bifurcation; Virus-induced gene silencing (VIGS) technology further verified the function of GhCAL-D07 in cotton, and it was found that the silenced plants exhibited significantly late flowering compared to CK in the same plants. growing conditions. Therefore, it is believed that the GhCAL-D07 gene plays an important role in advancing cotton flowering, and may serve as a favorable genetic resource for short-season cotton cultivation. The present invention is thus achieved.
    [0009] The present achievement presented the application of GhCAL-D07 gene to promote plant flowering, the said GhCAL-D07 gene has the nucleotide sequence shown in SEQ ID NO:1. The open reading frame of this gene is 714bp. The invention therefore relates to the GhCAL-D07 gene which comprises a nucleotide sequence presented in SEQ ID NO: 1.
    [0010] In certain embodiments of the present invention, the nucleotide sequence presented in SEQ ID NO: 1 can code for an amino acid sequence presented in SEQ ID NO: 2. This amino acid sequence contains 237 amino acids, the The relative molecular mass of the protein is 27.07kDa, with an isoelectric point of 9.72.
    [0011] The present invention also relates to the application of GhCAL-D07 gene to promote flowering of plants, comprising increasing the expression amount of GhCAL-D07 gene in a plant to promote flowering of plants.
    [0012] Thus, in certain modes of application of the present invention, the level of expression of the GhCAL-D07 gene is increased, in order to promote the flowering of the plants.
    [0013] In certain specific embodiments of the present invention, the increase in the amount of expression of the GhCAL-D07 gene in a plant is obtained by the following method: increasing the expression of the endogenous GhCAL-D07 gene of the plant or overexpress the exogenous GhCAL-D07 gene in the plant.
    [0014] Overexpression of the exogenous GhCAL-D07 gene described in one embodiment of a specific claim refers to the fact that the GhCAL-D07 gene is transformed into a plant for Agrobacterium-mediated expression using a expression vector in plants.
    [0015] Further, the GhCAL-D07 gene is transformed into a plant cell, tissue or organ through the plant expression vector.
    [0016] Further, the plant expression vector drives the expression of the GhCAL-D07 gene through a constitutive promoter or an inducible promoter.
    [0017] More precisely, said constitutive promoter is the 35S promoter.
    [0018] For the present invention, the promotion of flowering corresponds to advancing the flowering period of the plants.
    [0019] In the present invention, the plant is the cotton plant, corn, rice, wheat or Arabidopsis, in particular Arabidopsis thaliana.
    [0020] Positive effects of the present invention:
    [0021] The present invention makes it possible to considerably advance the flowering of Arabidopsis thaliana by obtaining the Arabidopsis thaliana plant modified by the GhCAL-D07 gene by transgenesis. By further silencing the GhCAL-D07 gene in cotton, the result shows that the GhCAL-D07 gene plays a key role in promoting cotton flowering.
    [0022] The present invention provides a favorable genetic resource for the cultivation of short-season cotton.
    [0023] Description of figures
    [0024] , in particular sub-figure 1A shows the observation of the phenotype of wild-type Arabidopsis thaliana (Wild-type); sub-figures 1B, C show the observations of the phenotype of the genetically modified Arabidopsis thaliana plant 35S::GhCAL-D07; sub-figure 1D shows the level of expression of the GhCAL gene in the genetically modified plants and in the control plants;
    [0025] shows the GhCAL-overexpressing Arabidopsis thaliana phenotype;
    [0026] , in particular sub-figure 3A shows the expression levels of GhCAL-D07 in different tissues; Sub-Figure 3B shows the expression levels of GhCAL-D07 in materials of different growth stages;
    [0027] shows the expression of GhCAL in different tissues of Gossypium hirsutum;
    [0028] shows the growth comparison between wild-type cotton plants and 3 T3 transgenic cotton lines that have the GhCAL gene silenced, where a is the morphological comparison between the GhCAL-RNAi and ZM24(WT) transgenic cotton lines ), b corresponds to the relative transcription level of GhCAL and GhCAL missense fragments in WT and T3 transgenic cotton lines; c corresponds to paraffin sections of stem cusps at the 3-leaf stage of ZM24 (WT) and GhCAL-RNAi transgenic cotton lines; d corresponds to the relative transcription level of GhAGL6-D09, GhAP1-A04 and GhSEP4 in WT transgenic cotton lines T3;
    [0029] shows virus-induced GhCAL-D07 gene silencing and expression analysis.
    [0030] Process
    [0031] Technical modes are described in more detail and complete below in combination with the attached figures and specific embodiments.
    [0032] The following examples serve herein to demonstrate preferred embodiments of the present invention. Those skilled in this field know that the technologies disclosed in the following examples represent the technologies found by the inventors and can be used for the application of the present invention, and they can therefore be considered as the preferred modes of application of the present invention. Nevertheless, those skilled in the art should be aware, consistent with this specification, that the specific embodiments published herein may be subject to several modifications which will still achieve the same or similar results without departing from spirit or scope of the present invention.
    [0033] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those understood by those skilled in the art in the field to which this invention pertains, public quotations and the documents from which they are cited will be incorporated as references.
    [0034] Those skilled in the art will recognize or know by conventional testing many equivalent technologies of the specific embodiments of the invention described herein. These equivalents will be included in the claims.
    [0035] Application example 1
    [0036] 1. Cotton materials
    [0037] The cotton materials selected for this application example are Zhong 50, Zhongmiansuo 74, Guoxinmian 11, Bomian 1 species of Gossypium hirsutum, which are planted in the trial field of the National Biological Key Laboratory of the Research Institute of Cotton from China Academy of Agricultural Sciences (CAAS) (in Baibi of Anyang city), it is normal field management.
    [0038] 2. Reagents and Consumables
    [0039] 2.1 Enzymes and kits: The high-fidelity enzyme GXL DNA Polymerase, the gel recovery kit and the PCR product purification kit are purchased from Takara; RNA reverse transcription kit, KOD FX Neo enzyme (Code.No.KFX-201) are purchased from Toyobo company; the Ultra One Step Cloning Kit is purchased from Vazyme; the mini plasmid extraction kit is purchased from the Magen company; restriction endonucleases (BamH I, Sac I) are purchased from the company NEB; DNAMarker III and Plant Total RNA Extraction Kit were purchased from TIANGEN Company; TransStart Top GreenqPCR SuperMix for Quantitative Fluorescent PCR is purchased from TransGen Company.
    [0040] 2.2 Other agents: agarose is an original Spanish product, peptone, yeast extract, chloroform, isoamyl alcohol, ethanol, isopropanol, sodium chloride, sucrose, silwet L-77, resorcinol, etc. are of analytical purity and made in China, Kanamycin, Streptomycin Sulfate, Ampicillin, etc. are purchased from Takara Biotechnology(Dalian) Co., Ltd., E.coli Trans5α competent cells are purchased from TransGen Biotech, and Agrobacterium LBA4404 competent cells are purchased from Shanghai Weidi Biotechnology Co. , ltd.
    [0041] 2.3 Culture media: LB liquid medium: tryptone dosed at 10 g/L, yeast extract (Yeastextract) dosed at 5 g/L, sodium chloride (NaCl) dosed at 10 g/L; LB solid medium: tryptone dosed at 10 g/L, yeast extract (Yeastextract) dosed at 5 g/L, sodium chloride (NaCl) dosed at 10 g/L, Agar dosed at 15 g/L, with a constant volume of 1 L; LB selection medium: before LB plating, antibiotic of corresponding concentration is added as soon as the medium has been pressure sterilized using the autoclave and cooled to 55°C, and shaken well before plating. The solutions of various reagents mentioned but not listed in this article are all prepared by the methods given in the third edition of the Molecular Cloning: A Laboratory Manual, and the biochemical reagents are of analytical cleanliness or more superior.
    [0042] 2.4 Essential instruments: PCR amplifier (Eppendorf), high-speed centrifuge (Eppendorf 5427R), electrophoresis apparatus (Beijing LIUYI), gel imaging system (BIO-RAD), quantitative fluorescence PCR instrument (ABI7500), microscope with fluorescence (OlympusBX43), constant temperature culture oscillator (Shanghai Zhicheng), phytotron (Saifu), etc.
    [0043] Test method and results
    [0044] 1 Bioinformatics analysis and cloning of the cotton GhCAL-D07 gene
    [0045] 1.1 Gene sequence of GhCAL-D07 is obtained from NCBI, primers are designed using Primer Premier 5.0 software, PCR (Polymerase Chain Reaction) method is used for amplification on Gossypium Zhong 50 species hirsutum, the open reading frame is 714bp, it has 237 coded amino acids, the relative molecular mass of the protein is 27.07kDa, with an isoelectric point of 9.72. The cds sequence of the gene is as follows (SEQ ID NO:1):
    [0046] ATGGGTAGAGGTAGGGTTCAACTAAGACGGATCGAGAACAATATTAGCAGACAAGTAACATTCTCAAAGAGACGAAGTGGCTTATTAAAGAAAGCTCATGAGATCTCAGTTTTATGCGATGCTGATGTTGCTTTGATTGTTTTCTCTAACAAAGGAAAGCTCTTTGAGTTCTCTTCTGATCCCAGCATGGAGAGGATCCTAGAACGGTACGAACGACAAATATATGCCCCAACTGGTTCTGAATCACAGGCAAATTGGTCTTTGGAATCTTCCAAACTCATGTCAACTATTGAAGTCTTGCAAAGGAACTTGAGGAACTTTCGTGGAGAAGAGCTTGAACCCTTGAGTTTAAGGGACCTGCAACTTTTGGAACAACAAATTGGTAATTCTCTGAAGCGAATACGAACTAGAAAGAACAAACTCATGAATGAATCCATTTCAGTGCTGCAGAAGAGAGAAAAGACATTGCAAGACCAGAACAACATGCTAGCTAAAAAGCTTAAAGAAAAACAGCAGACACCGACGGAACATGCACAACATGAAGTGCAACAAAAATTTGTCCAAAACTCACCACCATCAACATCCGTACAACCACCAACACCACCACCGGCTGCAATACAGTTTCCTTGTTTGACTATTGGAGGGAGTTACGAAGCCATGAAAGGGACAAACAAGGAAGCTGAGCTCAATCTCAACCTAGTACCAAATCAGTGA
    [0047] The amino acid sequence e is as follows (SEQ ID NO:2):
    [0048] MGRGRVQLRRIENNISRQVTFSKRRSGLLKKAHEISVLCDADVALIVFSNKGKLFEFSSDPSMERILERYERQIYAPTGSESQANWSLESSKLMSTIEVLQRNLRNFRGEELEPLSLRDLQLLEQQIGNSLKRIRTRKNKLMNESISVLQKREKTLQDQNNMLAKKLKEKQQTPTEHAQHEVQQKFVQNSPPSTSVPNQPPTPPPAAIQFPCLTIGGSYEAMKGTNLNKNL
    [0049] 1.2 The specific process of genetic cloning is as follows:
    [0050] (1) Test materials consisting of early species Zhong 50, Zhongmiansuo 74 and late species Guoxinmian 11, Bomian 1 of Gossypium hirsutum are planted in the Anyang experimental base of the Cotton Research Institute of the Academy of China Agricultural Sciences(CAAS), with normal field management. The sampling method is to take the cotton plant tip from the time of cotyledon flattening, a sample is taken each time a true leaf is flattened, with three biological replicates each time the material taken is quickly placed in liquid nitrogen for freezing and stored in the refrigerator at -80°C. The flattening of the cotyledon is marked as 0TLS (0 true-leaf stage), the flattening of a true leaf is marked as 1TLS (1 true-leaf stage), and so on, until the fifth true leaf is fully extended. The extraction of total RNA from plants is carried out using the kit manufactured by the company TIANGEN.
    [0051] (2) The RNA extraction steps are as follows:
    [0052] 1) Homogenization: Take an appropriate amount of fiber sample for rapid grinding into powder in liquid nitrogen, shake the sample vigorously immediately after adding 700μL of SL(add β-Mercaptoethanol before use), to mix the sample evenly;
    [0053] 2) Centrifugation at 12000rpm for 2min;
    [0054] 3) Transfer the supernatant liquid to the CS filtration column, for centrifugation at 12000rpm for 2min, then carefully pipette supernatant liquid into the collection tube for transfer to a new RNase-Free centrifuge tube, the tip of the pipette should not touch cell debris in the collection tube;
    [0055] 4) Add absolute ethanol whose volume is 4 times the volume of the supernatant, and mix them well, transfer the mixture to the CR3 adsorption column, for centrifugation at 12000rpm for 15 seconds, throw liquid waste into the tube column, and put the CR3 adsorption column back into the collection tube;
    [0056] 5) Add 350μL of RW1 deproteinization solution to the CR3 adsorption column, for centrifugation at 12000rpm for 15 seconds, discard liquid waste into the collection tube, and put the CR3 adsorption column back into the collection tube;
    [0057] 6) DNase I working solution: take 10μL of DNase I stock solution and 70μL of RDD solution and mix them gently;
    [0058] 7) Add 80μL of DNase I working solution in the CR3, for a rest at room temperature for 15 minutes;
    [0059] 8) After standing, add 350μL of RW1 deproteinization solution to the CR3 adsorption column, for centrifugation at 12000rpm for 15 seconds, discard liquid waste into the collection tube, and put the CR3 adsorption column back into the collection tube;
    [0060] 9) Add 500μL of RW rinsing solution (add ethanol before use) to the CR3 adsorption column, for centrifugation at 12000rpm for 15 seconds, discard liquid waste into the collection tube, and put the adsorption column CR3 in the collection tube;
    [0061] 10) Repeat step 9;
    [0062] 11) Spin at 12000rpm (~13400×g) for 2 minutes, put the CR3 adsorption column in a new RNase-Free centrifuge tube, instill 30-50μL of RNase-Free ddH2O in the central part of the membrane adsorption, let it stand for 2 minutes, do a centrifugation at 12000rpm (~13400×g) for 1 minute, to obtain the RNA solution. Note: The volume of the elution buffer solution should not be less than 30μL, too small volume will affect the recovery efficiency. RNA samples should be stored at -70°C. If the expected RNA yield is greater than 30 μg, the RNA solution obtained at the end of the centrifugation in step 11 can be poured again into the CR3 adsorption column, let it stand at room temperature for 2 minutes, and centrifugation at 12000rpm (~13400×g) for 1 minute, to obtain an RNA solution.
    [0063] (3) cDNA synthesis. Reverse transcribe 500ng of RNA into cDNA, using Toyobo's FSQ-201 reverse transcription kit, the reverse transcription system is as follows:
    [0064] Prepare the RT reaction solution according to the following ingredients (the reaction solution is prepared on ice):
    [0065] 5×PrimeScript Buffer (for Real Time) 2μl
    [0066] PrimeScript RT Enzyme Mix I 0.5 μl
    [0067] Oligo dT Primer (50 μM) 0.5 μl
    [0068] Random 6 mers (100 μM) 0.5 μl
    [0069] Total RNA 1μg
    [0070] RNase Free dH2O up to 10 μL
    [0071] The reverse transcription reaction conditions are as follows:
    [0072] 15min at 37°C (reverse transcription reaction),
    [0073] 5s at 98°C (reverse transcriptase inactivation reaction);
    [0074] Dilute the reverse transcription product cDNA solution 6-fold to serve as the PCR reaction template.
    [0075] (4) PCR amplification of the target gene
    [0076] The following system is prepared on ice, and the target gene GhCAL-D07 is amplified using TM-1 cDNA as template. According to the TaKaRa GXL DNA Polymerase high fidelity enzyme instruction manual, the PCR reaction system is as follows:
    [0077] 5× PrimeSTAR GXL Buffer 10μl
    [0078] dNTP Mixture (2.5 mM each) 4 μl
    [0079] Primer 1 2 μl
    [0080] Primer 2 4μl
    [0081] 2 μl cDNA template
    [0082] PrimeSTAR GXL DNA Polymerase 1μl
    [0083] Sterilized distilled water up to 50 μl
    [0084] The PCR amplification procedures are as follows:
    [0085] Reaction conditions
    [0086] 98°C3min
    [0087] 98°C10s
    [0088] 56°C15s
    [0089] 1 min at 68°C 35 cycles
    [0090] 68°C10min
    [0091] Primer sequences:
    [0092] GhCAL-D07-F: 5′-ATGGGTAGAGGTAGGGTTCAA-3′ (SEQ ID NO:3)
    [0093] GhCAL-D07-R:5′-TCACTGATTTGGTACTAGGTT-3′ (SEQ ID NO:4)
    [0094] The product will be stored at 4°C after the end of the reaction, and will be detected by electrophoresis at 1% agarose, the result will be considered valid if the size of the band meets the expected size.
    [0095] (5) Use the gel retrieval kit for gel extraction of target fragments.
    [0096] (6) Connect, according to the Ultra One Step Cloning Kit, the product from the above gel extraction to the T vector to construct and transform E.coli.
    [0097] (7) Regarding the culture at 37°C daytime night, select monoclones on the resistant LB culture medium for rocking and culture at 37°C.
    [0098] (8) For the PCR verification of the bacterial solution, samples of positive clones are taken and sent to GENEWIZ company for sequencing, add certain amount of glycerin in the bacterial solution which sequence is correct, so that that the final concentration of glycerine is approximately 20%, with storage at -70°C.
    [0099] 2 Construction of the plant overexpression vector pBI121-35S::GhCAL-D07
    [0100] 2.1 Plasmid extraction and enzyme digestion
    [0101] The extraction of plasmids is carried out using the mini plasmid extraction kit from Magen, to extract cloning vector plasmids containing fragments of the target gene GhCAL-D07, the concentration of plasmids detected is 200ng/μL, the agarose gel electrophoresis test showed no protein contamination, it meets the test requirements; at the same time, the overexpression vector pBI121 is extracted for a double digestion with the endonucleases BamH I and Sac I, the purification after the digestion makes it possible to obtain the linearized vector pBI121.
    [0102] 2.2 Construction of the overexpression vector pBI121-35S::GhCAL-D07
    [0103] The vector for this assay is constructed using the Ultra One Step Cloning Kit which adapts to the connection between any vector and any fragment of the gene, the reaction time is only 15 minutes, it requires the spacer fragment and the linearized vector have an overlapping area of 15bp at the 5′ end and the 3′ end respectively.
    [0104] The amplification primers are as follows:
    [0105] OE-GhCAL-D07F(SEQ ID NO:5):
    [0106] 5′-GGACTCTAGAGGATCCATGGGTAGAGGTAGGGTTCAA-3′
    [0107] OE-GhCAL-D07R(SEQ ID NO:6):
    [0108] 5′-GATCGGGGAAATTCGAGCTCTCACTGATTTGGTACTAGGTT-3′
    [0109] The operation procedures are as follows: taking the target gene cloning vector GhCAL-D07 as a template, the spacer fragment is purified after amplification; the spacer fragment obtained GhCAL-D07 and the linearized vector pBI12 constitute a system with a 2:1 molar ratio.
    [0110] Mix this solution well, for a reaction at 50°C for 10 minutes, then place the solution on ice, to transform Trans5α competent cells, select monoclones and send samples for sequencing, in order to obtain the vector overexpression containing the correct target gene.
    [0111] The plate used for this process is kanamycin resistant LB culture medium, kanamycin is dosed at 50mg/mL, and is diluted 1000 times when using, i.e. 100μL of 50mg kanamycin /mL is added to a 100ml culture medium.
    [0112] 3 Construction and infection of the cotton VIGS vector with the GhCAL-D07 gene
    [0113] Virus induced gene silencing (VIGS) is an RNA-mediated post-transcriptional gene silencing dependent on the defense mechanism of plants against viruses. A 322bp fragment of the target gene GhCAL-D07 is connected to the shuttle plasmid pCLCrV, a vector (pCLCrV-GhCAL-D07) is constructed to transform E.coli (Escherichia Coli), monoclones are selected and sent to be sequenced (GENEWIZ, Suzhou). Monoclones that pass sequencing are amplified and scaled to extract plasmids. The positive control vector (pCLCrV-VA), the negative control (pCLCrV), the helper plasmid (pCLCrV-VB) as well as the constructed vector containing the fragment of the target gene GhCAL-D07 (pCLCrV-GhCAL-D07) are respectively used to transforming the Agrobacterium strain (Agrobacteriumtumefaciens) LBA4404. During the stage when the cotyledons are flattened and the true leaves have not yet sprouted, the injection of Agrobacterium is done to infect the cotton cotyledons, the detailed operation steps are as given in the silencing report induced gene silencing established by Gao et al.(2013) (Gaoetal., Functional genomic analysis of cotton genes with agrobacterium-mediated virus-induced gene silencing.Methods Mol Biol(2013),975157-65; Gu etal., A versatile system for functional analysis of genes and microRNAs incotton, Plant Biotechnology Journal(2014)12,pp.638–649, text is incorporated herein by citation). Young infected cotton plants are well protected from light for 24 hours, total RNA from cotton leaves is extracted after 40 days, to detect gene silencing by qRT-PCR technology.
    [0114] The amplification primers are as follows:
    [0115] pCLCrV-GhCAL-D07F (SEQ ID NO:7):
    [0116] 5′-ATGCCTGCAG ACTAGT GAAGCGAATACGAACTAGAAA-3′
    [0117] spI
    [0118] pCLCrV-GhCAL-D07R (SEQ ID NO:8):
    [0119] 5′-AGACCTAGGGG CGCGCC TCACTGATTTGGTACTAGGTT-3′
    [0120] Asci
    [0121] 4 Agrobacterium-mediated transformation of the GhCAL-D07 gene in Arabidopsis thaliana
    [0122] 4.1 Agrobacterium tumefaciens LBA4404 competent cells are transformed by freeze-thawing, the specific transformation process is as follows:
    [0123] (1) Add 1μg (2-10μL) of target gene overexpression vector plasmid already constructed into 100μL of competent cells of Agrobacterium tumefaciens LBA4404 from Shanghai Weidi Biotechnology Co., Ltd., mix the solution well and cool in an ice bath for 30 minutes; perform rapid freezing with liquid nitrogen for 2-3 minutes, followed by thermal excitation at 37°C for 90s;
    [0124] (2) Cool in an ice bath for 5 minutes, then add 800μL of LB liquid medium;
    [0125] (3) After culturing at 190rpm at 28°C for 4h, do centrifugation at 4000rpm for 5 minutes, pipette the supernatant to a remaining volume of 400-500μL, after pipetting and mixing repeatedly, take 200μL of bacterial solution to apply it on the selective medium with three antibiotics containing kanamycin, streptomycin sulphate and rifampicin, for a culture at 28°C for 36-48h, resistant colonies are visible;
    [0126] (4) Select single colonies for culture in 1ml LB liquid medium with three antibiotics for approximately 16 h, until the medium is cloudy;
    [0127] (5) Colonies are subjected to PCR and enzymatic digestion, for detection of positive strains of Agrobacterium, store the bacterial solution at 20% glycerol at -80°C.
    [0128] 4.2 Transformation of Arabidopsis thaliana by floral immersion
    [0129] (1) Inoculate 20μL of bacterial solution stored at -80°C into 1ml of LB liquid medium, for rock culture at 180rpm at 28°C overnight, then take 200μL of activated bacterial solution to add into 20ml LB liquid medium for oscillation culture at 180 rpm at 28° C.;
    [0130] (2) As soon as the OD value of the bacterial solution is between 1.2-1.6, centrifuge the bacterial solution at 3000rpm to collect bacteria;
    [0131] (3) The composition of the transformation medium: 5% sucrose, 0.03% silwet L-77 (Steven J, 1998);
    [0132] (4) Suspend bacteria in the above transformation medium, set OD 600＝0.8 to start infection;
    [0133] (5) Place the inflorescence of Arabidopsis thaliana in the transformation medium for 30-50s, after infection, wrap the Arabidopsis thaliana with food film for dark culture for 24h followed by culture in normal conditions, harvest seeds after maturation.
    [0134] 5 Identification and detection of transgenic Arabidopsis thaliana plants
    [0135] 5.1 The harvested seeds are sterilized with a 0.1% HgCl solution, and then purified at 4°C for 3-4 days, then these seeds are planted on 1/2MS containing kanamycin (agar concentration of 0 .6%) , after about 10 days, the difference between positive plants and negative plants can be observed, those which can grow normally would be positive plants, transplant the Arabidopsis thaliana plants which can grow normally to a culture chamber.
    [0136] 5.2 The enzyme used for the selection of transgenic plants is the KOD FX Neo PCR enzyme which is characterized by the fact that it can be directly used for PCR on living leaves without the need to extract DNA from Arabidopsis thaliana. During identification, Agrobacterium GhCAL-D07 solution serves as the positive control, and a PCR system with sterile water as a matrix serves as the negative control. The primers used for detection are as follows:
    [0137] Upstream Primer F1 (SEQ ID NO:9): 5'-GACGCACAATCCCACTATCC-3'
    [0138] Downstream primer R1 (SEQ ID NO:10): 5'-TGTCGTTCGTACCGTTCTAGGA-3'
    [0139] PCR reaction system:
    [0140] Autoclaved, distilled water up to 50μl
    [0141] 2× PCR Buffer for KOD FX Neo 25 μl
    [0142] 2mM dNTPs 10μl
    [0143] Primers (10μM each) 1.5μL
    [0144] Matrix crude extract 5μl
    [0145] KOD FX Neo (1U/μl) 1μl
    [0146] PCR amplification process:
    [0147] 94°C, 2 mins
    [0148] 98°C, 10 sec.
    [0149] 68°C, 60 sec./ kb 30 cycles
    [0150] 5.3 Separately take an appropriate amount of amplified products for electrophoretic detection on 1% agarose gel.
    [0151] 5.4 Select a total of 7 35S::GhCAL-D07 positive plants based on electrophoresis results, to harvest T0 generation seeds.
    [0152] 6 Identification, observation of the phenotype and statistical data for transgenic plants
    [0153] 6.1 After being sterilized, the harvested seeds are planted on 1/2MS containing kanamycin and then purified at 4°C for 3 days, then these seeds are transferred to a phytotron, after about 10 days, the plants positive plants grow normally, while negative plants experience cotyledon etiolation and no longer grow.
    [0154] 6.2 Positive Arabidopsis thaliana plants are transplanted into small pots, and subjected to DNA extraction and PCR detection after one month of growth. Each generation of plants must be tested to identify positive plants and negative plants, up to the T3 generation, to obtain homozygous transgenic Arabidopsis thaliana plants.
    [0155] 6.3 T3 generation homozygous transgenic plants are intended for phenotype observation and data statistics, as shown in Figure 1;
    [0156] By observing the phenotype of the T3 generation transgenic plants and the wild-type Arabidopsis thaliana plants, the inventors find that there is a certain proportion of plants with 35S::GhCAL-D07 overexpression which present a branching phenotype or a stem bifurcation, then wild-type plants do not show this phenotype (sub-figure 1, C), so the inventors presume that GhCAL-D07 can regulate the growth and development of the apex meristem of Arabidopsis plants thaliana; the overexpression of the GhCAL-D07 gene makes it possible to significantly advance the flowering of Arabidopsis thaliana plants (sub-figures 1A and B), to significantly reduce the number of rosette leaves and to increase the number of cauline leaves (like the shown in Figure 2). Genetic analyzes of the transgenic plants revealed: after the overexpression of the GhCAL-D07 gene, the level of expression of the GhCAL-D07 gene in Arabidopsis thaliana plants increases significantly.
    [0157] 7 Analysis on the expression patterns of GhCAL-D07 in materials of different growth stages
    [0158] 7.1 Test materials consisting of early species Zhong 50, Zhongmiansuo 74 and late species Guoxinmian 11, Bomian 1 of Gossypium hirsutum are planted in the Anyang experimental base of the Cotton Research Institute of the Academy of Agricultural Sciences of China (CAAS), with normal field management. The sampling method is to take the cotton plant tip from the time of cotyledon flattening, a sample is taken each time a true leaf is flattened, with three biological replicates each time the material taken is quickly placed in liquid nitrogen for freezing and stored in the refrigerator at -80°C. The flattening of the cotyledon is marked as 0TLS (0 true-leaf stage), the flattening of a true leaf is marked as 1TLS (1 true-leaf stage), and so on, until the fifth true leaf is fully extended.
    [0159] 7.2 Take from the different samples above, to extract the RNA which is then reverse transcribed into cDNA, analyze the level of expression of the GhCAL-D07 gene in different materials, while taking GhActin as an internal reference gene, the primers for quantitative fluorescence PCR are as follows:
    [0160] F1 upstream primer (SEQ ID NO:11): 5'- GGGACCTGCAACTTTTGGAAC-3'
    [0161] Downstream primer R1 (SEQ ID NO:12): 5'- GTCGGTGTCTGCTGTTTTTCT-3'
    [0162] Prepare the qRT-PCR reaction system on ice, to perform the quantitative fluorescence PCR reaction.
    [0163] The qRT-PCR reaction system is as follows:
    [0164] SYBR® Premix Ex Taq (Tli RNaseH Plus) (2×) 10.0μL
    [0165] PCR Forward Primer (10μM) 0.4μL
    [0166] PCR Reverse Primer (10μM) 0.4μL
    [0167] ROX Reference Dye II (50×) 0.4μL
    [0168] 2.0μL DNA template
    [0169] dH2O (sterilized distilled water) 6.8μL
    [0170] Total 20.0μL
    [0171] qRT-PCR reaction procedures:
    [0172] Pre-denaturation: 95°C 10 minutes,
    [0173] 95°C，5sec；
    [0174] 60°C, 34 seconds, 40 cycles.
    [0175] Collect fluorescent signals during the annealing period (60°C, 34 seconds).
    [0176] The terminal meristem of plants is similar to the stem cells of animals, it constantly differentiates into different tissues and organs above ground, if the internal and external conditions are suitable, the terminal meristem will differentiate into flower buds. According to the results of the qRT-PCR reaction, the GhCAL-D07 gene is mainly expressed in the leaves, terminal buds and small buds, and the expression level in the leaves is highest (subfigure 3.A). With the development of the terminal bud, the expression level of the GhCAL-D07 gene in materials of different growth stages is increasingly high, from the stage when the third true leaf unfolds, the expression level in two early species Zhong 50 and Yanzao 2 is significantly higher than that in late species (sub-figure 3, B). This shows that the GhCAL-D07 gene is possibly related to flower bud differentiation or even flower organ development in Gossypium hirsutum.
    [0177] 8. Role of GhCAL in regulating the transition from vegetative growth to reproductive growth
    [0178] GhCAL has a low level of expression at the root level, and a highest level of expression at the level of the stem cusp. GhCAL expression can also be detected in leaves, stems and buds (Figure 4). In order to address the role of GhCAL in flowering regulation, 3 lines of Arabidopsis thaliana genetically modified by 35S::GhCAL were constructed. qRT-PCR analysis with specific primers shows that the expression level of GhCAL in these transgenic lines is significantly higher compared to wild-type plants. Under long-day conditions, GhCAL-modified Arabidopsis thaliana plants flower 3-5 days earlier than wild type. Part of transgenic plants experience a bifurcation phenomenon during growth, which modifies the structure of Arabidopsis thaliana plants. These results show that overexpression of GhCAL promotes the transition of Arabidopsis thaliana plants from vegetative growth to reproductive growth.
    [0179] In order to further confirm the function of GhCAL in cotton, the countersense expression vector containing the coding region over the entire length of the GhCAL countersense sequence driven by the 35S promoter is constructed and transforms the early species of ZM24 cotton. 3 T3 transgenic cotton lines whose GhCAL gene is silenced are obtained, namely GhCAL-RNAi-1, GhCAL-RNAi-2 and GhCAL-RNAi-3. qRT-PCR analysis with specific primers proves that the expression level of GhCAL countersense fragments in transgenic cotton plants is high, and the transcription level of GhCAL is significantly lower compared to type plants. Savage. The flower buds of GhCAL-RNAi transgenic cotton plants later differentiate. Compared to wild-type plants, the buttoning time of 3 T3 transgenic lines is delayed by 14 days, 15 days and 12 days respectively, their flowering time is delayed by 19 days, 21 days and 14 days respectively. The first fruiting branches of wild-type plants usually appear at the sixth segment or the seventh segment of the main stem, while the first fruiting branches of transgenic cotton plants appear at the 11th segment, 12th segment, and 12th segment, respectively. 10th segment. In terms of plant height, the 3 transgenic cotton lines are significantly shorter than the wild type plants. These results show that the decrease in the expression level of GhCAL delays the transition of cotton from vegetative growth to reproductive growth (as shown in Figure 5).
    [0180] The level of expression of GhCAL decreases significantly in plants with silencing of the GhCAL-RNAi gene. In addition, the expression levels of GhAGL6 and GhAP1 decrease significantly, the GhCAL gene can directly regulate their expression. Specific data is shown in Table 1.
    [0181] Table 1 Comparison of early maturity traits between transgenic cotton and wild-type cotton
    [0182]
    [0183] 9. Virus-induced GhCAL-D07 gene silencing.
    [0184] Agrobacterium bacterial solutions of positive control virus vector (pCLCrVA), negative control virus vector (pCLCrV) and GhCAL-D07 virus vector (pCLCrV-GhCAL-D07) are respectively injected into the cotyledons of seedlings of cotton. The level of expression of the GhCAL-D07 gene in the injected plants is detected four weeks after the injection. As shown in Figure 6, after being infected with the vector containing the pCLCrV-GhCAL-D07 virus, the expression level of the GhCAL-D07 gene decreases significantly compared to the empty vector control. Compared to the pCLCrVA empty vector plant, the pCLCrV-GhCAL-D07 plant whose GhCAL-D07 gene expression level exhibits a late flowering phenotype and has a short stature (Fig. 6, A). Studies show that the GhCAL-D07 gene may play an important role in promoting cotton flowering, and may serve as a favorable genetic resource for short-season cotton cultivation.
    [0185] Obviously, the described embodiments relate only to certain embodiments within the scope of the present invention, and do not cover all embodiments. All other achievements obtained by any ordinary technician in this field without making creative efforts based on the embodiments in the present invention shall be covered by the scope of protection of the present invention.
    权利要求:
    Claims (9)
    [0001]
    GhCAL-D07 gene, wherein the GhCAL-D07 gene comprises a nucleotide sequence shown in SEQ ID NO: 1.
    [0002]
    GhCAL-D07 gene, wherein the nucleotide sequence shown in SEQ ID NO: 1 may code for an amino acid sequence shown in SEQ ID NO: 2.
    [0003]
    Application of the GhCAL-D07 gene according to claim 1 or 2 to promote flowering of plants, comprising increasing the amount of expression of the GhCAL-D07 gene in a plant to promote flowering of plants.
    [0004]
    Application of the GhCAL-D07 gene to promote flowering of plants according to claim 3, wherein increasing the expression amount of the GhCAL-D07 gene in a plant is achieved by the following method: increasing the expression of the GhCAL-D07 endogenous to the plant or overexpressing the exogenous GhCAL-D07 gene in the plant.
    [0005]
    Application of the GhCAL-D07 gene to promote flowering in plants according to claim 4, wherein overexpression of the exogenous GhCAL-D07 gene refers to the fact that the GhCAL-D07 gene is transformed into a plant for expression mediated by Agrobacterium using a plant expression vector.
    [0006]
    Application of the GhCAL-D07 gene to promote flowering in plants according to claim 5, wherein the GhCAL-D07 gene is transformed into a plant cell, tissue or organ.
    [0007]
    Application of the GhCAL-D07 gene to promote flowering in plants according to claim 6, wherein the plant expression vector drives expression of the GhCAL-D07 gene through a constitutive promoter or an inducible promoter.
    [0008]
    Application of the GhCAL-D07 gene to promote flowering in plants according to claim 7, wherein the constitutive promoter is the 35S promoter.
    [0009]
    Application of the GhCAL-D07 gene to promote flowering in plants according to any one of claims 5 to 8, wherein the plant is cotton plant, maize, rice, wheat or Arabidopsis.
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    同族专利:
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    CN110117320A|2019-08-13|
    引用文献:
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    CN106834338B|2016-12-23|2020-04-07|青岛农业大学|Expression vector of arabidopsis gene REM16 and application thereof in regulating and controlling plant flowering period|CN110373419B|2019-08-28|2022-02-11|中国农业科学院棉花研究所|Application of cotton GhMADS44-A03 gene in promoting plant flowering|
    CN110551735B|2019-09-17|2022-01-25|中国农业科学院棉花研究所|Application of cotton GhMADS45-D09 gene in promoting plant flowering|
    CN111607604B|2020-03-27|2021-12-14|中国农业科学院棉花研究所|Application of cotton GHPSAT2 gene in promoting flowering of plants|
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    2021-05-27| PLFP| Fee payment|Year of fee payment: 2 |
    2022-03-04| PLSC| Publication of the preliminary search report|Effective date: 20220304 |
    优先权:
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    CN201910411726.7A|CN110117320A|2019-05-16|2019-05-16|Cotton GhCAL-D07 gene is promoting the application in flowering of plant|
    CN201910411726.7|2019-05-16|
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