soybean event detection method pdab9582.814.19.1

专利摘要:
METHOD OF DETECTION OF THE SOY EVENT pDAB9582.814.19.1 The present invention relates to a Soy Event pDAB9582.814.19.1 which comprises genes that encode Cry1F, Cry1Ac (synpro), and PAT, conferring insect resistance and tolerance to insects. herbicides to the soybean crops that contain the event, and allowing obtaining methods for crop protection and protection of stored products. The invention provides methods of detecting the event by PCR.
公开号:BR102012019436B1
申请号:R102012019436-8
申请日:2012-07-25
公开日:2021-02-02
发明作者:Lauren Clark；Kelley Ann Smith；Yang Wang；Ning Zhou
申请人:Dow Agrosciences Llc；
IPC主号:

专利说明:

BACKGROUND OF THE INVENTION
[0001] The genes encoding Cry1F and Cry1Ac synpro (Cry1Ac) are able to confer resistance to insects, for example, resistance to lepidopteran insects, to transgenic plants; and the gene encoding PAT (phosphinothricin acetyltransferase) is able to confer tolerance to the herbicide phosphinothricin (glufosinate) to transgenic plants. PAT has been successfully expressed in soy for use as a selectable marker when producing insect resistant transgenic crops, and to confer commercial levels of tolerance to the herbicide glufosinate in transgenic crops.
[0002] The expression of foreign genes in plants is known to be influenced by their location in the plant's genome, perhaps due to the chromatin structure (eg, heterochromatin) or the proximity of transcriptional regulatory elements (eg, enhancers) near the site of integration (Weising et al., Ann. Rev. Genet 22: 421-477, 1988). At the same time, the presence of the transgene at different locations in the genome will influence the global phenotype of the plant in different ways. For this reason, it is often necessary to screen a large number of events to identify an event characterized by optimal expression of a gene of interest introduced. For example, it has been observed in plants and other organisms that there can be a wide variation in the levels of expression of a gene introduced between events. There may also be differences in spatial or temporal patterns of expression, for example, differences in the relative expression of a transgene in various plant tissues, which may not correspond to the expected patterns of transcriptional regulatory elements present in the construction of the introduced gene. For this reason, it is common to produce hundreds of thousands of different events and screen those events for a single event that has the desired levels of transgene expression and standards for commercial purposes. An event that has the desired levels or patterns of transgene expression is useful for introgressing the transgene into genetic ancestors by sexual exogamy using conventional generation methods. The progenies of these crosses maintain the transgene expression characteristics of the original transformant. This strategy is used to ensure reliable gene expression in countless varieties that are well adapted to local breeding conditions.
[0003] It is desirable to be able to detect the presence of a specific event to determine whether the progenies of a sexual cross contain a transgene or group of transgenes of interest. In addition, a method for detecting a specific event would be useful in meeting regulations that require approval before launching on the market and the labeling of foods derived from recombinant crop plants, for example, or for use in environmental monitoring, to monitor traces in crops in the field, or to monitor products derived from a crop harvest, as well as for use in ensuring third party compliance with regulatory and contractual terms.
[0004] It is possible to detect the presence of a transgenic event by any nucleic acid detection method known in these techniques, including, but not limited to, polymerase chain reaction (PCR) or DNA hybridization using nucleic acid probes. These detection methods generally focus on frequently used genetic elements, such as promoters, terminators, marker genes, etc., because many DNA constructs, the coding region is interchangeable. As a result, these methods may not be useful for discriminating between different events, particularly those produced using the same DNA construct or very similar constructs unless the DNA sequence of the flanking DNA adjacent to the inserted heterologous DNA is known. For example, an event-specific PCR analysis is described in US patent application 2006/0070139 for the DAS-59122-7 event. It would be desirable to have a simple and discriminative method for identifying the Soy Event pDAB9582.814.19.1. SUMMARY OF THE INVENTION
[0005] The present invention relates to a method to detect a new transformation event of transgenic soybeans resistant to insects and tolerant to herbicides, called Soy Event pDAB9582.814.19.1. As part of this invention at least 2,500 seeds from a soybean strain comprising event soybean 9582.814.19.1 were deposited at the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA, 20110. The deposit, Deposit Designation of ATCC Patent PTA-12006, was received by the ATCC on July 21, 2011. This deposit has been made and will be maintained in accordance with and under the terms of the Budapest Treaty with respect to seed deposits for the purpose of patent procedure.
[0006] The DNA of soybean plants that contain this event includes the junction / flanking sequences described here that characterize the location of the DNA inserted within the soybean genome. SEQ ID NO: 1 and SEQ ID NO: 2 are diagnostics for the Soy Event pDAB9582.814.19.1. More particularly, the sequences surrounding the junctions at bp 1400/1401 and bp 1536/1537 of SEQ ID NO: 1, and bp 152/153 of SEQ ID NO: 2 are diagnostic for the Soy Event pDAB9582.814.19.1. Examples of sequences comprising these junctions that are characteristic of the DNA of soybeans that contain the Soy Event pDAB9582.814.19.1 are described below.
[0007] The invention provides a method for detecting the Soy Event pDAB9582.814.19.1 in a sample comprising soybean DNA, said method comprising: (a) putting said sample in contact with a first initiator that has a length of at least 10 bp that selectively binds to a flanking sequence within bp 1-1400 of SEQ ID NO: 1 or a complement thereof, and a second primer that has a length of at least 10 bp that binds selectively an insertion sequence within bp 1401-1836 of SEQ ID NO: 1 or a complement thereto; and (b) testing for an amplicon generated between said primers; or placing said sample in contact with a first primer having a length of at least 10 bp, which selectively binds to an insertion sequence within bp 1-152 of SEQ ID NO: 2 or its complement, and a second primer having a length of at least 10 bp, which selectively binds to the flanking sequence within bp 153-1550 of SEQ ID NO: 2 or its complement; and (c) testing for an amplicon generated between said primers.
[0008] In another embodiment, the invention provides a method for detecting the Soy Event pDAB9582.814.19.1, comprising: a) putting said sample in contact with a first initiator that selectively binds to a flanking sequence selected in the group that consists of bp 1-1400 of SEQ ID NO: 1 and bp 153-1550 of SEQ ID NO: 2, and their complements; and a second primer that selectively binds to SEQ ID NO: 3, or complement to them; b) subjecting said sample to a polymerase chain reaction; and c) testing for an amplicon generated between said primers.
[0009] In another embodiment, the invention provides an isolated DNA molecule that is diagnostic for the Soy Event pDAB9582.814.19.1. Such molecules include, in addition to SEQ ID NOS: 1 and 2, molecules of at least 25 bp in length comprising 1400 bp of SEQ ID NO: 1 and at least 10 bp of SEQ ID NO: 1 in each direction from junction bp 1400/1401; amplicons with a length of at least 25 bp comprising 152153 of SEQ ID NO: 2 and at least 10 bp of SEQ ID NO: 2 in each direction from the 152/153 bp junction. Examples are bp 1385-1415 of SEQ ID NO: 1; mp 1350-1450 of SEQ ID NO: 1; mp 1300-1500 of SEQ ID NO: 1; bp 1200-1600 of SEQ ID NO: 1; bp 137-168 of SEQ ID NO: 2; bp 103-203 of SEQ ID NO: 2; and bp 3-303 of SEQ ID NO: 2, and their complements.
[00010] Additionally, the present invention provides assays to detect the presence of the event in question in a sample (of soybeans, for example). The assays can be based on the DNA sequence of the recombinant construct, inserted in the soybean genome, and on the genomic sequences that flick the insertion site. Kits and useful conditions for conducting the tests are also provided.
[00011] The present invention relates in part to the cloning and analysis of the DNA sequences of the end regions resulting from the insertion of the pDAB9582 T-DNA in transgenic soybean lines. These strings are unique. Based on the insertion and joining sequences, specific event initiators can be and have been generated. A PCR analysis demonstrated that these events can be identified by analyzing the PCR amplicons generated with these sets of specific event primers. Accordingly, these and other related procedures can be used to uniquely identify the soybean strains that comprise the event of the present invention. BRIEF DESCRIPTION OF THE SEQUENCES
[00012] SEQ ID NO: 1 is the DNA sequence flanking the 5 'end for soybean event 9582.814.19.1. Nucleotides 1-1400 are the genomic sequence. Nucleotides 1401-1535 are a rearranged sequence of pDAB9582. Nucleotides 1536-1836 are the insertion sequence.
[00013] SEQ ID NO: 2 is the DNA sequence flanking the 3 'end for soybean event 9582.814.19.1. Nucleotides 1-152 are the insertion sequence. Nucleotides 153-1550 are the genomic sequence.
[00014] SEQ ID NO: 3 is the DNA sequence of pDAB9582, which is noted below in Table 1.
[00015] SEQ ID NO: 4 is the 81419_FW3 primer oligonucleotide for confirmation of the genomic DNA of the 5 'end.
[00016] SEQ ID NO: 5 is the 81419_RV1 primer oligonucleotide for confirmation of the 3 'end genomic DNA.
[00017] SEQ ID NO: 6 is the 81419_RV2 primer oligonucleotide for confirmation of the 3 'end genomic DNA.
[00018] SEQ ID NO: 7 is the 81419_RV3 primer oligonucleotide for confirming the 3 'end genomic DNA.
[00019] SEQ ID NO: 8 is the 5'IREnd-01 primer oligonucleotide for confirmation of the 5 'end genomic DNA.
[00020] SEQ ID NO: 9 is the 5'IREnd-02 primer oligonucleotide for confirmation of the 5 'end genomic DNA.
[00021] SEQ ID NO: 10 is the AtUbi10RV1 primer oligonucleotide for confirmation of the 5 'end genomic DNA.
[00022] SEQ ID NO: 11 is the AtUbi10RV2 primer oligonucleotide for confirmation of the 5 'end genomic DNA.
[00023] SEQ ID NO: 12 is the 3'PATEnd05 primer oligonucleotide for confirmation of the 3 'end genomic DNA.
[00024] SEQ ID NO: 13 is the 3'PATEnd06 primer oligonucleotide for confirmation of the 3 'end genomic DNA.
[00025] SEQ ID NO: 14 is the confirmed sequence of soybean event 9582.814.19.1, including the 5 'genomic flanking sequence, insertion of the DAB9582 T filament, and the 3' genomic flanking sequence.
[00026] SEQ ID NO: 15 is the 81419_3'F primer oligonucleotide that was used for the TAQMAN assay to detect the 3 'end soy event 9582.814.19.1.
[00027] SEQ ID NO: 16 is the 81419_3'R primer oligonucleotide that was used for the TAQMAN assay to detect the soybean event of the 3 'end 9582.814.19.1.
[00028] SEQ ID NO: 17 is the 81419_3'P oligonucleotide probe that was used for the TAQMAN assay to detect the soybean event of the 3 'end 9582.814.19.1. This probe had a fluorescent FAM cluster added to the 5 'end and an MGB extinguisher added to the 3' end.
[00029] SEQ ID NO: 18 is the GMS116 F primer oligonucleotide that was used for the TAQMAN assay to detect the endogenous reference gene, GMFL01-25-J19 (GenBank: AK286292.1).
[00030] SEQ ID NO: 19 is the GMS116 R primer oligonucleotide that was used for the TAQMAN assay to detect the endogenous reference gene, GMFL01-25-J19 (GenBank: AK286292.1).
[00031] SEQ ID NO: 20 is the oligonucleotide probe GMS116 that was used for the TAQMAN assay to detect the endogenous reference gene, GMFL01-25-J19 (GenBank: AK286292.1). This probe had a HEX fluorescent cluster added to the 5 'end and a BHQ extinguisher added to the 3' end. BRIEF DESCRIPTION OF THE FIGURES
[00032] Figure 1 is a plasmid map of pDAB9582 containing the expression cassette cry1F v3, cry1Ac and Pat v6.
[00033] Figure 2 represents the locations of the primers to confirm the sequence of the 5 'and 3' ends of the soybean event pDAB9582.814.19.1.
[00034] Figure 3 represents the arrangement of the genomic sequence in the soybean event pDAB9582.814.19.1.
[00035] Figure 4 represents the locations of the primers and probes for the TAQMAN test of the soybean event pDAB9582.814.19.1. DETAILED DESCRIPTION OF THE INVENTION
[00036] Both ends of the insertion of the Soy Event 9582.814.19.1 have been sequenced and characterized. Specific tests for the event were developed. It was also mapped on chromosome 02 of the soybean genome. The event can be introgressed into other select strains.
[00037] As alluded to above in the Background of the Invention Background, the introduction and integration of a transgene into a plant genome involves some random events (thus, the term "event" for a given insertion that is expressed). That is, with many transformation techniques such as Agrobacterium transformation, biolistic transformation (ie, gene gun), and silicon carbide-mediated transformation (ie, WHISKERS), it is unpredictable where a transgene will be inserted into the genome. Therefore, identifying the flanking plant genomic DNA on both sides of the insertion can be important to identify a plant that has a given insertion event. For example, PCR primers can be designed that generate a PCR amplicon through the insertion junction region and the host genome. This PCR amplicon can be used to identify a unique or distinct type of insertion event.
[00038] Definitions and examples are provided here to help describe the present invention and guide those skilled in the art to practice the invention. Unless otherwise noted, the terms must be understood according to the use by those skilled in the relevant techniques. The nomenclature for DNA bases as stated in 37 CFR § 1.822 is used.
[00039] As used here, the term "progeny" denotes the offspring of any generation of a parent plant that comprises the Soy Event pDAB9582.814.19.1.
[00040] A transgenic "event" is produced by transforming plant cells with a heterologous DNA, that is, a nucleic acid construct that includes the transgenes of interest, regeneration of a plant population resulting from the insertion of the transgene into the genome of the plant, and selection of a specific plant characterized by insertion at a specific location in the genome. The term "event" refers to the original transformant and the progeny of the transformant that includes the heterologous DNA. The term "event" also refers to the progeny produced by sexual exogamy between the transformant and another variety that includes the genomic DNA of the transgene. Even after repeated backcrosses with a recurrent parent, the DNA of the inserted transgene and the flanking genomic DNA (genomic DNA of the transgene) from the transformed parent is present in the progeny of the crossing at the same chromosome site. The term "event" also refers to the DNA of the original transformant and its progeny that comprises the inserted DNA and the flanking genomic sequence immediately adjacent to the inserted DNA that would be expected to be transferred to a progeny that receives the inserted DNA, including the transgene from interest as a result of a sexual crossing of a parental line that includes the inserted DNA (for example, the original transformant and the progeny resulting from autogamy) and a parental line that does not contain the inserted DNA.
[00041] A "junction sequence" or "end sequence" covers the point at which the DNA inserted within the genome is linked to the DNA of the native soy genome that flanks the insertion point, the identification or detection of one or more junction sequences in a genetic material of the plant being sufficient to be diagnostic for the event. Included are the DNA sequences that cover insertions in the soy events described here and similar lengths of flanking DNA. Specific examples of these diagnostic sequences are provided here; however, other sequences that overlap the insertion junctions, or the insertion junctions and the genomic sequence, are also diagnostic and could be used in accordance with the present invention.
[00042] The present invention relates to the identification of events using such flanking, joining, and insertion sequences. Related PCR primers and amplicons are included in the invention. In accordance with the present invention, PCR analysis methods using amplicons that cover through the inserted DNA and its ends can be used to detect or identify commercialized transgenic soybean varieties or lines derived from the patented transgenic soybean lines in question.
[00043] Flanking / junction sequences are diagnostic for the Soy Event pDAB9582.814.19.1. Based on these sequences, specific event initiators were generated. PCR analysis demonstrated that these soybean strains can be identified in different soybean genotypes by analyzing the PCR amplicons generated with these sets of specific event primers. Therefore, these and other related procedures can be used to uniquely identify these soybean strains. The strings identified here are unique.
[00044] The detection techniques of the present invention are especially useful in conjunction with plant breeding, to determine which progeny plants comprise a given event, after a parent plant comprising an event of interest is crossed with another lineage of the plant in question. an effort to confer one or more additional traits of interest in the progeny. These PCR analysis methods would benefit soybean breeding programs as well as quality control, especially for commercialized GM soybean seeds. PCR detection kits for these transgenic soy strains are now also manufactured and used. This will also benefit product registration and product management.
[00045] In addition, soybean flanking genomic sequences can be used to specifically identify the genomic location of each insert. This information can be used to produce specific molecular marker systems for each event. They can be used for accelerated creation strategies and to establish linkage data.
[00046] In addition, flanking sequence information can be used to study and characterize transgenic integration processes, characteristics of the genomic integration site, event selection, stability of transgenes and their flanking sequences, and gene expression (especially related to gene silencing, transgenic methylation patterns, effects of positions, and elements related to potential expression such as MARS [matrix attachment regions], and the like).
[00047] In light of the description in question, it should be clear that the present invention includes seeds available under the ATCC Deposit No. identified above. The present invention also includes a herbicide-tolerant plant developed from a seed deposited with the ATCC under the Deposit No. identified above. The present invention further includes parts of said plant, such as leaves, tissue samples, seeds produced by said plant, pollen, and the like (in which they comprise cry1F, cry1Ac, pat, and SEQ ID NOS: 1 and 2).
[00048] As used here, the term "soy" means Glycine max and includes all its varieties that can be created with a soy plant.
[00049] The DNA molecules of the present invention can be used as molecular markers in a marker-assisted creation (MAB) method. The DNA molecules of the present invention can be used in methods (such as AFLP markers, RFLP markers, RAPD markers, SNPs, and SSRs) that identify genetically linked agronomically useful traits, as is known in those techniques. Insect resistance and herbicide tolerance can be traced to the progeny of a cross with a soybean plant of the present invention (or its progeny and any other cultivar or variety of soybeans) using MAB methods. DNA molecules are markers for this trait, and MAB methods that are well known in these techniques can be used to trace the trait or traits of herbicide resistance in soybean plants, where at least one soybean strain of the present invention, or their progeny, was a parent or ancestor. The methods of the present invention can be used to identify any variety of soybeans that have the event in question.
[00050] As used herein, the term "lineage" is a group of plants that have little or no genetic variation between individuals as regards at least one trait. Such strains can be created by several generations of self-pollination and selection, or vegetative propagation from a single parent using tissue or cell culture techniques.
[00051] As used herein, the terms "cultivar" and "variety" are synonymous and refer to a strain that is used for commercial production.
[00052] The term "stability" or "stable" means with respect to a given component, that the component is maintained from generation to generation and, preferably, at least three generations.
[00053] The term "commercial utility" is defined as having good plant vigor and high fertility, such that the crop can be produced by farmers using conventional agricultural equipment, and the oil with the components described can be extracted from the seed using conventional crushing and extraction equipment.
[00054] The term "agronomically elite" means that a strain has desirable agronomic characteristics such as yield, maturity, resistance to diseases, and the like, in addition to resistance to insects and tolerance to herbicides due to the event or events in question. Any and all of these agronomic characteristics and data points can be used to identify these plants, either as a point or at either of the two ends or both of a range of characteristics used to define these plants.
[00055] As those skilled in the art should recognize in light of this teaching, preferred modalities of detection kits, for example, may include probes and / or primers targeted to and / or comprising "junction sequences" or "transition sequences" (where the soybean flanking genomic sequence meets the insertion sequence). For example, this includes polynucleotide probes, primers and / or amplicons designed to identify one or both of the junction sequences (where the insert meets the flanking sequence), as indicated in the Table above. A common design is to have an initiator that hybridizes in the flanking region, and an initiator that hybridizes on insertion. Such initiators often each have a length of at least about ~ 15 residues. With this arrangement, the primers can be used to generate / amplify a detectable amplicon that indicates the presence of an event of the present invention. These primers can be used to generate an amplicon that covers (and includes) a splicing sequence as indicated above.
[00056] The primer or primers that "land" in the flanking sequence are not typically designed to hybridize beyond about 1,200 bases or similar number beyond the junction. Therefore, typical flanking primers would be designed to comprise at least 15 residues of either of the two filaments within 1,200 bases in the flanking sequences from the beginning of the insertion. That is, primers that comprise a sequence of an appropriate size from (or that hybridize to) base pairs 800 to 1.00 of SEQ ID NO: 14 and / or base pairs 13,897 to 14,497 of SEQ ID NO: 14 are within the scope of the present invention. The insertion primers can be similarly designed anywhere on the insertion, but base pairs 1,400 to 2,000 of SEQ ID NO: 14 and / or base pairs 13,297 to 13,896 of SEQ ID NO: 14, can be used, for example. example, not exclusively for that initiator design.
[00057] Those skilled in the art should also recognize that primers and probes can be designed to hybridize, under a series of usual hybridization and / or PCR conditions, in which the primer or probe is not perfectly complementary to the exemplified sequence. That is, some degree of inequality can be tolerated. For a primer with approximately 20 nucleotides, for example, typically one or two or a similar number of nucleotides do not need to bind to the opposite strand of the unequal base is internal or at the end of the primer that is opposite the amplicon. Several appropriate hybridization conditions are provided below. Analogs of synthetic nucleotides, such as inosine, can also be used in probes. Peptide nucleic acid (PNA) probes, as well as DNA and RNA probes, can also be used. What is important is that these probes and primers are diagnostic for (able to uniquely identify and distinguish) the presence of an event of the present invention.
[00058] It should be noted that errors in PCR amplification can occur, which could result in minimal sequencing errors, for example. That is, unless otherwise indicated, the sequences listed here were determined by generating long amplicons from genomic soybean DNAs, and then cloning and sequencing the amplicons. It is not uncommon to find slight differences and minimal discrepancies in sequences generated and determined in this way, given the many rounds of amplification necessary to generate amplicon for sequencing from genomic DNAs. Those skilled in these techniques should recognize and be aware that any necessary adjustments due to these types of common sequencing errors or discrepancies are within the scope of the present invention.
[00059] It should be noted that it is unusual for any genomic sequence to be deleted, for example, when a sequence is inserted during the creation of an event. Therefore, some differences may also appear between the present flanking sequences and the genomic sequences listed in GENBANK, for example.
[00060] The components of the DNA sequence "insertion" are illustrated in the figures and are discussed in more detail below in the Examples. The DNA polynucleotide sequences of these components, or fragments thereof, can be used as DNA primers or probes in the methods of the present invention.
[00061] In some embodiments of the invention, compositions and methods are provided for detecting the presence of the genomic insertion region of the transgene, in plants and seeds and the like, from a soybean plant. DNA sequences are provided that comprise the sequence of the junction of the genomic insertion region of the 5 'transgene provided here (between base pairs -800 - 1,400 of SEQ ID NO: 14), segments thereof, and complements to the exemplified sequences and any segments of them. DNA sequences are provided that comprise the present junction sequence of the genomic insertion region of the 3 'transgene provided here (between base pairs 13,897 - 14,497 of SEQ ID NO: 14), segments thereof, and complements to the exemplified sequences and any segments of them. The sequence of the junction of the insertion region covers the junction between the heterologous DNA inserted into the genome and the DNA of the soybean cell that flanks the insertion site. Such sequences can be diagnostic for the given event.
[00062] Based on these insertion and end sequences, specific event initiators can be generated. A PCR analysis demonstrated that the soybean strains of the present invention can be identified in different soybean genotypes by analyzing the PCR amplicons generated with these sets of event specific primers. These and other related procedures can be used to uniquely identify these soybean strains. Therefore, PCR amplicons derived from these primer pairs are unique and can be used to identify these soybean strains.
[00063] In some embodiments, DNA sequences that comprise an unusual contiguous fragment from the genomic insertion region of the transgene are an aspect of this invention. Included are DNA sequences that comprise a sufficient length of polynucleotides from the transgene insertion sequence and a sufficient length of polynucleotides from the genomic soybean sequence of one or more of the above three plants and / or soybean sequences that are useful as primer sequences for the production of a diagnostic amplicon product for one or more of these soybean plants.
[00064] Related modalities belong to DNA sequences that comprise at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more contiguous nucleotides a portion of the transgene of a DNA sequence identified herein (such as SEQ ID NO: 1 and its segments), or their complements, and a similar length of the flanking soybean DNA sequence from these sequences, or their complements. Such sequences are useful as DNA primers in DNA amplification methods. The amplicons produced using these primers are diagnostic for any of the soy events referred to here. Therefore, the invention also includes the amplicons produced by such DNA primers and homologous primers.
[00065] This invention also includes methods to detect the presence of DNA in a sample, which corresponds to the soy event referred to here. Such methods may comprise: (a) bringing the sample comprising DNA into contact with a set of primers that, when used in a nucleic acid amplification reaction with the DNA of at least one of these soy events, produces an amplicon that is diagnosis for said event (s); (b) carrying out a nucleic acid amplification reaction, thereby producing amplicon; and (c) detecting the amplicon.
[00066] Other detection methods of the present invention include a method for detecting the presence of a DNA, in a sample, corresponding to said event, wherein said method comprises: (a) putting the sample comprising the DNA in contact with a probe that hybridizes under stringent hybridization conditions with the DNA of at least one of said soybean events and that does not hybridize under stringent hybridization conditions with a control soybean plant (DNA not belonging to the event of interest); (b) subjecting the sample and probe to stringent hybridization conditions; and (c) detecting probe hybridization to DNA.
[00067] DNA detection kits can be developed using the compositions described herein and methods well known in the techniques of DNA detection. The kits are useful for identifying the DNA of the soy event in question in a sample and can be applied to methods for generating soy plants that contain this DNA. The kits contain DNA sequences homologous or complementary to amplicons, for example, described herein, or to DNA sequences homologous or complementary to the DNA contained in the genetic elements of the transgene of the present events. These DNA sequences can be used in DNA amplification reactions or as probes in a DNA hybridization method. The kits can also contain the reagents and materials needed to carry out the detection method.
[00068] A "probe" is an isolated nucleic acid molecule to which is attached a conventional detectable marker or reporter molecule (such as a radioactive isotope, ligand, chemiluminescent agent, or enzyme). Such probe is complementary to a strand of a target nucleic acid, in the case of the present invention, to a strand of genomic DNA from one of said soybean events, either from a soybean plant or from a sample that includes the DNA of the event. The probes according to the present invention include not only deoxyribucleic or ribonucleic acids, but also polyamides and other probe materials that specifically bind to a target DNA sequence and can be used to detect the presence of that target DNA sequence.
[00069] "Primers" are isolated / synthesized nucleic acids that are annealed for a complementary target DNA strand hybridization to form a hybrid between the primer and the target DNA strand, and then extended along the target DNA strand by a polymerase, for example, a DNA polymerase. The primer pairs of the present invention refer to their use for amplifying a target nucleic acid sequence, for example, by polymerase chain reaction (PCR) or other conventional nucleic acid amplification methods.
[00070] The probes and primers have a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 , 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 , 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 , 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124 , 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 , 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174 , 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199 , 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 2 14, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 4 14, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, or 500 or more polynucleotides. Such probes and primers hybridize specifically to a target sequence under highly stringent hybridization conditions. Preferably, the probes and primers according to the present invention have complete sequence similarity to the target sequence, although probes that differ from the target sequence and that retain the ability to hybridize to the target sequences can be designed by methods conventional.
[00071] Methods for preparing and using probes and primers are described, for example, in "Molecular Cloning: A Laboratory Manual", 2nd edition, volumes 1-3, editors Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. PCR primer pairs can be derived from a known sequence, for example, using computer programs intended for this purpose.
[00072] Primers and probes based on the flanking DNA and insert sequences described herein can be used to confirm (and, if necessary, correct) the sequences described by conventional methods, for example, by cloning again and sequencing those sequences.
[00073] The nucleic acid probes and primers of the present invention hybridize under stringent conditions to a target DNA sequence. Any conventional hybridization or amplification method for nucleic acids can be used to identify the presence of DNA from a transgenic event in a sample. The nucleic acid molecules or fragments thereof are able to hybridize specifically to other nucleic acid molecules under certain circumstances. As used herein, two nucleic acid molecules are said to be able to specifically hybridize to each other if the two molecules are capable of forming an antiparallel double-stranded nucleic acid structure. A nucleic acid molecule is said to be the "complement" of another nucleic acid molecule if they are completely complementary. As used herein, the molecules are said to have "complete complementarity" when each nucleotide of one of the molecules is complementary to a nucleotide of the other. Two molecules are said to be "minimally complementary" if they hybridize to each other with sufficient stability to allow them to remain ringed together under at least conventional "low stringency" conditions. Similarly, molecules are said to be "complementary" if they hybridize to each other with sufficient stability to allow them to remain ringed together under conventionally "high stringency" conditions. Conventional conditions of rigor are described by Sambrook et al., 1989. Deviations from complete complementarity are therefore permissible, as long as these deviations do not completely impede the ability of the molecules to form a double-stranded structure. For a nucleic acid molecule to serve as a primer or probe it only needs to be sufficiently complementary in sequence to be able to form a stable double-stranded structure under the specific solvent and salt concentrations employed.
[00074] As used herein, a substantially homologous sequence is a nucleic acid sequence that will hybridize specifically to complement the nucleic acid sequence with which it is being compared under highly stringent conditions. The term "stringent conditions" is functionally defined as the hybridization of a nucleic acid probe to a target nucleic acid (i.e., to a specific nucleic acid sequence of interest) by the specific hybridization procedure discussed in Sambrook et al., 1989 , on 9.52-9.55. See also Sambrook et al., 1989 in 9.47-9.52 and 9.56-9.58. Consequently, the nucleotide sequences of the invention can be used because of their ability to selectively form duplex molecules with complementary extensions of DNA fragments.
[00075] Depending on the intended application, varied hybridization conditions can be used to achieve varying degrees of selectivity of the probe in the direction of the target sequence. For applications that require high selectivity, relatively stringent conditions will typically be used to form the hybrids, for example, conditions of low salt concentration and / or high temperature will be selected, such as those provided by NaCl at about 0.02 M at about 0.15 M at temperatures of about 50 ° C to about 70 ° C. Strict conditions, for example, could involve washing the hybridization filter at least twice with high stringency wash buffer (0.2X SSC, 0.1% SDS, 65 ° C). The appropriate stringency conditions that promote DNA hybridization, for example, 6.0X sodium chloride / sodium citrate (SSC) at about 45 ° C, then a 2.0X SSC wash at 50 ° C, they are known to those skilled in these techniques. For example, the concentration of salts in the washing step can be selected from a low stringency of about 2.0X SSC at 50 ° C to a high stringency of about 0.2X SSC at 50 ° C. In addition, the temperature in the washing step can be increased from low stringency conditions to about room temperature, about 22 ° C, to high stringency conditions at about 65 ° C. The temperature and salt can be varied, or the temperature or salt concentration can be kept constant while the other variable is changed. These selective conditions tolerate little or no inequality between the probe and the target model or filament. The detection of DNA sequences by means of hybridization is well known to those skilled in these techniques, and the teachings of the patents in US 4,965,188 and 5,176,995 are exemplary of the methods of hybridization analyzes.
[00076] In a particularly preferred embodiment, a nucleic acid of the present invention will hybridize specifically to one or more of the primers (or amplicons or other sequences) exemplified or suggested herein, including their complements and fragments, under highly stringent conditions. In one aspect of the present invention, a marker nucleic acid molecule of the present invention has the nucleic acid sequence set forth herein in one of the exemplified sequences, or their complements and / or fragments.
[00077] In another aspect of the present invention, a marker nucleic acid molecule of the present invention shares between 80% and 100% or 90% and 100% sequence identity with such nucleic acid sequences. In another aspect of the present invention, a marker nucleic acid molecule of the present invention shares between 95% and 100% sequence identity with that sequence. Such sequences can be used as markers in plant breeding methods to identify the progeny of genetic crosses. Hybridization of the probe to the target DNA molecule can be detected by a number of methods known to those skilled in the art, and they may include, but are not limited to, fluorescent markers, radioactive markers, antibody-based markers, and chemiluminescent markers.
[00078] As for the amplification of a target nucleic acid sequence (for example, by PCR) using a pair of specific amplification primers, "stringent conditions" are the conditions that allow the pair of primers to hybridize only to the sequence of target nucleic acids to which a primer that has the corresponding wild-type sequence (or its complement) would bind to and, preferably, will produce a unique amplification product, amplicon.
[00079] The term "specific for (a target sequence)" indicates that a probe or primer hybridizes under stringent hybridization conditions only to the target sequence in a sample comprising the target sequence.
[00080] As used herein, "amplified DNA" or "amplicon" refers to the nucleic acid amplification product of a target nucleic acid sequence that is part of a model nucleic acid. For example, to determine whether the soybean plant resulting from a sexual crossover contains the genomic DNA from the transgenic soybean event of the present invention, the DNA extracted from a tissue sample from the soybean plant can be subjected to an amplification method nucleic acid using a primer pair that includes a primer derived from the flanking sequence in the plant genome adjacent to the inserted heterologous DNA insertion site, and a second primer derived from the inserted heterologous DNA, to produce an amplicon that is diagnostic for the presence of the event's DNA. The amplicon has a length and a sequence that are also diagnostic for the event. The amplicon can have a length in the range between the combined length of the primer pairs plus a pair of nucleotide bases, and / or the combined length of the primer pairs plus about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, or 500, 750, 1000, 1250, 1500, 1750, 2000, or more pairs of nucleotide bases (more or less any of the increments listed above). Alternatively, a pair of primers can be derived from the flanking sequence on both sides of the inserted DNA in order to produce an amplicon that includes the entire nucleotide sequence of the insert. A member of a pair of primers derived from the plant's genomic sequence may be located at a distance from the inserted DNA sequence. This distance can be in the range between a pair of nucleotide bases and about twenty thousand pairs of nucleotide bases. The use of the term "amplicon" specifically excludes primer dimers that can be formed in the thermal amplification reaction of DNA.
[00081] Nucleic acid amplification can be performed by any of the various nucleic acid amplification methods known in these techniques, including polymerase chain reaction (PCR). Various amplification methods are known in these techniques and are described, inter alia, in the patents in US 4,683,195 and US 4,683,202. PCR amplification methods were developed to amplify up to 22 kb of genomic DNA. These methods as well as other methods known in these DNA amplification techniques can be used in the practice of the present invention. The sequence of the insertion of heterologous transgenic DNA or flanking genomic sequence of the soy event in question can be verified (and corrected, if necessary) by amplifying these event sequences using primers derived from the sequences provided here, and then sequencing the standard DNA of the event. amplicon from PCR or cloned DNA.
[00082] The amplicon produced by these methods can be detected by a plurality of techniques. Agarose gel electrophoresis and ethidium bromide staining is a well-known method for detecting DNA amplicons. Another method is Genetic Bit Analysis in which a DNA oligonucleotide is drawn that overlaps the adjacent flanking genomic DNA sequence and also the inserted DNA sequence. The oligonucleotide is immobilized in wells of a microwell plate. After PCR of the region of interest (using a primer in the inserted sequence and an adjacent flanking genomic sequence), a double-stranded PCR product can be hybridized to the immobilized oligonucleotide and serve as a model for the extension of a single base using a DNA polymerase and specific labeled ddNTPs for the next expected base. The reading can be fluorescent or ELISA-based. A signal indicates the presence of the flanking sequence of the insertion due to successful amplification, hybridization, and extension of a single base.
[00083] Another method is the pyrosequencing technique, as described by Winge (Innov. Pharma. Tech. 00: 18-24, 2000). In this method, an oligonucleotide is drawn that overlaps the adjacent genomic DNA and joins the insertion DNA. The oligonucleotide is hybridized to the single-stranded PCR product from the region of interest (one primer in the inserted sequence and one in the flanking genomic sequence) and incubated in the presence of a DNA polymerase, ATP, sulfurylase, luciferase, apyrase, 5- adenosine phosphosulfate and luciferin. DNTPs are added individually and the result of incorporation into a light signal is measured. A light signal indicates the presence of the flanking sequence of the insertion of the transgene due to the successful amplification, hybridization, and single-base or multiple-base intent.
[00084] Fluorescence polarization is another method that can be used to detect an amplicon of the present invention. Following this method, an oligonucleotide is drawn that overlaps the genomic glanking junction and inserted DNA. The oligonucleotide is hybridized to a single-stranded PCR product from the region of interest (a primer in the inserted DNA and one in the flanking genomic DNA sequence) and incubated in the presence of a DNA polymerase and a fluorescence-labeled ddNTP. The single base extension results in the incorporation of ddNTP. Incorporation can be measured as a change in polarization using a fluorometer. A change in polarization indicates the presence of the flanking sequence of the insertion of the transgene due to the successful amplification, hybridization and single base extension.
[00085] TAQMAN (PE Applied Biosystems, Foster City, CA) is a method for detecting and quantifying the presence of a DNA sequence. Briefly, a FRET oligonucleotide probe is designed and it overlaps the genomic and DNA Flanking junction of the insert. The FRET probe and PCR primers (one primer in the insert DNA sequence and one in the flanking genomic sequence) are cycled in the presence of a thermostable polymerase and dNTPs. During specific amplification, Taq DNA polymerase purifies and releases the fluorescent cluster out of the extinguishing cluster in the FRET probe. A fluorescent signal indicates the presence of the flanking sequence of the insertion of the transgene due to the successful amplification and hybridization.
[00086] "Molecular Beacons" have been described for use in the detection of sequences. Briefly, a FRET oligonucleotide probe is designed that overlaps the flanking genomic junction and DNA of the insert. The unique structure of the FRET probe results in it containing a secondary structure that keeps the fluorescent and extinguisher groups in close proximity. The FRET probe and PCR primers (one primer in the insert DNA sequence and one in the flanking genomic sequence) are cycled in the presence of a thermostable polymerase and dNTPs. Following successful PCR amplification, and hybridization of the FRET probe to the target sequence results in the removal of the secondary structure of the probe and spatial separation of the fluorescent groups and extinguishers, and results in a fluorescent signal. A fluorescent signal indicates the presence of the flanking genomic sequence from the insertion of the transgene due to the successful amplification of hybridization.
[00087] Having revealed a location in the soy genome that is excellent for an insertion, the present invention also comprises a soy seed and / or a soy plant that comprises at least one Soy Event insert other than 9582.814.19.1 in the general vicinity of this genomic location. One option is to put a different insert in place of one of the pDAB9582.814.19.1 shown here. In these generic aspects, the homologous recombination, for example, can be used in accordance with the present invention. This type of technology is the subject, for example, of the document in WO 03/080809 A2 and the corresponding published patent application in US 2003/0232410). Accordingly, the present invention includes plants and plant cells that comprise a heterologous insert (in place of or with multiple copies of the cry1F, cry1Ac, or pat genes), flanked by all or a recognizable part of the flanking sequences identified herein (bp 1 -1400 SEQ ID NO: 1 and bp 1531550 SEQ ID NO: 2). An additional copy (or additional copies) of a cry1F, cry1Ac, or pat could also be attached for insertion in these ways.
[00088] All patents, patent applications, provisional patent applications and publications referred to or cited herein are incorporated by reference in their entirety to the extent that they are not inconsistent with the explicit teachings of this specification.
[00089] The following examples are included to illustrate the procedures for practicing the invention and to demonstrate certain preferred embodiments of the invention. These examples are not to be construed as limiting. Those skilled in these techniques should appreciate that the techniques described in the examples that follow represent specific approaches used to illustrate the preferred modes for this practice. However, those versed in these techniques must, in the light of this specification, evaluate that many changes can be made in these specific modalities and at the same time still obtaining the same or similar results without departing from the spirit and scope of the invention. Unless otherwise indicated, all percentages are by weight in all proportions of solvent mixtures are by volume unless otherwise noted. The following abbreviations are used, and unless otherwise indicated: bp for bases ° C degrees Celcius DNA deoxyribonucleic acid EDTA ethylenediaminetetraacetic acid kb kilobase μg microgram μL microliter mL milliliter M molar mass PCR polymerase chain reaction PTU plant transcription unit SDS SDS plant transcription unit sodium dodecyl sulfate SSC a buffer solution containing a mixture of sodium chloride and sodium citrate, pH 7.0 TBE a buffer solution containing a mixture of Tris base, boric acid and EDTA, pH 8.3
[00090] The modalities of the present invention are further defined by the following examples. It should be understood that these examples are provided for illustrative purposes only. From the discussion above and these examples, those versed in these techniques can ascertain the essential characteristics of this invention, and without departing from its spirit and scope, can make several changes and modifications of the modalities of the invention to adapt it to various uses and conditions. Therefore, several modifications to the modalities of the invention, in addition to those illustrated and described herein, should be evident to those skilled in these techniques from the preceding description. Such modifications are intended to fall within the scope of the appended claims.
[00091] The discussion of each reference stated here is incorporated here as a reference in its entirety. EXAMPLES Example 1: Transformation and Selection of the Soy Event PDAB9582.814.19.1 CrylF and CrylAc
[00092] Transgenic soybeans (Glycine max) containing the soybean event pDAB9582.814.19.1 was generated through Agrobacterium-mediated transformation of explants from cotyledonous soybean nodes. The unarmed strain of Agrobacterium EHA101 (Hood et al., 1993), carrying the binary vector pDAB9582 (figure 1), containing the selectable marker, pat v6, and the genes of interest, cry1F v3 and cry1 Ac, within the filament region DNA T, was used to initiate the transformation. The DNA sequence for pDAB9582 is provided in SEQ ID NO: 3, which is shown below in Table 1. Table 1. Gene elements located in pDAB9582

[00093] Agrobacterium-mediated transformation was conducted using a modified procedure by Zeng et al. (2004). Briefly, soybean seeds (cv Maverick) were germinated on basal medium and cotyledon nodes were isolated and infected with Agrobacterium. The initiation of shoots, the stretching of shoots, and the rooting medium were supplemented with cefotaxime, timentin and vancomycin to remove Agrobacterium. The selection for glufosinate was used to inhibit the growth of untransformed shoots. The selected shoots were transferred to the rooting medium for root development, and then transferred to a soil mixture to acclimatize the plants.
[00094] The terminal leaflets of selected plants received leaf coverage with glufosinate to screen for putative transformants. The screened plants were transferred to the greenhouse, allowed to acclimatize and then received leaf cover with glufosinate to reconfirm tolerance and judged to be putative transformants. The screened plants were sampled and molecular analyzes were performed to confirm the selectable marker gene and / or the gene of interest. The T0 plants were allowed to self-fertilize in the greenhouse to generate T1 seed.
[00095] This event, soybean event pDAB9582.814.19.1, was generated from an independent transformed isolate. T1 plants were backcrossed and introgressed into elite varieties for subsequent generations. The event was selected based on its unique characteristics such as unique insertion site, normal Mendelian segregation, stable expression, and an excellent combination of efficacy, including herbicide tolerance and agronomic performance. The following examples contain the data that were used to characterize the soybean event pDAB9582.814.19.1. Example 2: Characterization of Protein Expression in the Soy Event pDAB9582.814.19.1
[00096] The biochemical properties of recombinant proteins Cry1F, Cry1Ac, and PAT expressed in soybean events 9582.814.19.1 have been characterized. The enzyme linked immunosorbent analysis (ELISA) is a biochemical analysis known in these techniques, which can be used to characterize the biochemical properties of proteins and confirm the expression of these proteins in the soybean event 9582.814.19.1. Example 2.1: Expression of PAT, CrylF, and CrylAc Protein in Plant Tissues
[00097] Soy tissue samples were isolated from test plants and prepared for expression analysis. PAT protein was extracted from soybean plant tissues with a phosphate-buffered saline solution, containing the Tween-20 detergent (PBST) containing 0.5% Bovine Serum Albumin (BSA). The vegetable tissue was centrifuged; the aqueous supernatant was collected, diluted with an appropriate buffer, as needed, and analyzed using a PAT ELISA kit in a sandwich format. The kit was used following the manufacturer's suggested protocol (Envirologix, Portland, ME). This analysis measured the expressed PAT protein.
[00098] The Cry1F protein was extracted from soybean plant tissues with a phosphate buffered saline solution, containing the Tween-20 detergent (PBST). The vegetable tissue was centrifuged; the aqueous supernatant was collected, diluted with an appropriate buffer, as needed, and analyzed using a Cry1F ELISA kit in a sandwich format. The kit was used following the manufacturer's suggested protocol (Strategic Diagnostics Inc., Newark, DE). This analysis measured the expressed Cry1F protein.
[00099] Cry1Ac protein was extracted from soybean plant tissues with a phosphate buffered saline solution, containing the Tween-20 detergent (PBST) containing 0.5% Bovine Serum Albumin (BSA). The vegetable tissue was centrifuged; the aqueous supernatant was collected, diluted with an appropriate buffer, as needed, and analyzed using a Cry1Ac ELISA kit in a sandwich format. The kit followed the manufacturer's suggested protocol (Strategic Diagnostics Inc., Newark, DE). This analysis measured the expressed Cry1Ac protein.
[000100] Detection analysis was performed to investigate the stability of expression vertically (between generations) and horizontally (between strains within a generation) in the soybean event pDAB9582.814.19.1. Example 2.2: Expression of the CrylF, CrylAc, and Pat Protein Protein in Plant Tissues
[000101] The levels of proteins Cry1F, Cry1Ac and PAT were determined in Soy Event 9582.814.19.1. The soluble extractable proteins were measured using an enzyme-linked immunosorbent analysis method (ELISA) from soybean leaf tissue. From the Soy Events 9582.814.19.1 of the T2 to T6 generations, the expression was stable (non-segregating) and consistent in all strains. Table 2 lists the average level of expression of transgenic proteins in the soybean event 9582.814.19.1. Table 2. Average level of expression of different transgenic proteins in the soybean event pDAB9582.814.19.1
Example 3: Cloning and Characterization of the DNA Sequence in the Insertion and Flanking End Regions of the Soy Event pDAB9582.814.19.1
[000102] To characterize and describe the genomic insertion site, the sequence of the flanking end regions of the genomic T-DNA from the soybean event pDAB9582.814.19.1 were determined. The genomic sequence of the soybean event pDAB9582.814.19.1 was confirmed, comprising 1400 bp of the 5 'flanking end sequence (SEQ ID NO: 1) and 1398 bp of the 3' flanking end sequence (SEQ ID NO: 2). PCR amplification based on the end sequences of the soybean event pDAB9582.814.19.1 validated that the end regions were of soybean origin and that the junction regions are unique sequences for the soybean event pDAB9582.814.19.1. Junction regions could be used for event specific identification of the soybean event pDAB9582.814.19.1. In addition, the T-filament insertion site was characterized by amplifying a genomic fragment corresponding to the region of the flanking end sequences from the unprocessed soybean genome. A comparison of the soybean event pDAB9582.814.19.1 with the non-transformed genomic sequence revealed that a deletion of about 57 bp from the original locus resulted during the integration of filament T. In total, the characterization of the insertion sequence and the end of the soybean event pDAB9582.814.19.1 indicated that an intact copy of the T filament of pDAB9582 was present in the soybean genome. Table 3. List of primers and their sequences used in confirming soybean genomic DNA in the soybean event pDAB9582.814.19.1





Example 3.1: Confirmation of Soy Genomic Sequences
[000103] The 5 'and 3' flanking ends aligned to the shotgun sequence of the entire Glycine max genome from chromosome 02, indicating that the soybean event pDAB9582.814.19.1 transgene was inserted into chromosome 02 of the soybean genome. To confirm the insertion site of the soybean event pDAB9582.814.19.1 from the soybean genome, a PCR was conducted with different pairs of primers (figure 2, Table 3, Table 4, and Table 5). The genomic DNA from the soybean event pDAB9582.814.19.1 and other transgenic or non-transgenic soybean strains was used as a model. To confirm that the 5 'end sequences are correct, a primer was designed to bind to the At Ubi10 promoter gene element, for example, AtUbi10RV1, and a primer designed to bind to the 5' end cloned on chromosome 02 of the soy genome. , the primer designated 81419_FW3, were used to amplify the segment of DNA covering the At Ubi10 promoter gene element for the 5 'end sequence. Similarly, for confirmation of the cloned 3 'end sequence, a specific pat primer, for example 3'PATEnd05, and three primers designed according to the 3' end sequence, designated 81419_RV1, 81419_RV2 and 81419_RV3, were used to amplify the DNA segments that cover the pat gene for the 3 'end sequence. DNA fragments of the expected sizes were amplified only from the genomic DNA of the soybean event pDAB9582.814.19.1 with each pair of primers, but not from DNA samples from other transgenic soybean strains or the non-transgenic control . The results indicate that the sequences of the cloned 5 'and 3' ends are the flanking sequences of the insertion of the T filament for the soybean event pDAB9582.814.19.1.
[000104] To further confirm the insertion of DNA into the soybean genome, a PCR amplification covering the sequences of the soybean ends was completed in the genomic DNA that did not contain the insertion of the T filament for the Soy Event pDAB9582.814.19.1 . The 81419_FW3 primer, designed according to the 5 'end sequence, and an 81419-RV3 primer, designed for the 3' end sequence, were used to amplify the DNA segments that contained the locus where the T strand of pDAB9582 integrated. As expected, PCR amplification completed with the 81419_FW3 and 81419_RV3 primer pair produced a DNA fragment of approximately 1.5 kb from all other control soybean strains, but not pDAB9582.814.19.1. Aligning the sequences of the 5 'and 3' ends identified from the soybean event pDAB9582.814.19.1 with a shotgun sequence of the entire genome of Glycine max from chromosome 02 revealed a deletion of about 57 bp from the original locus (figure 3) . These results demonstrated that the soybean event transgene pDAB8294 was inserted into the chromosome 02 site of the soybean genome. Example 4: Characterization of the Soy Event pDAB9582.814.19.1 by Southern Blot
[000105] A Southern blot analysis was used to establish the integration pattern of the soybean event pDAB9582.814.19.1. These experiments generated data that demonstrated the integration and integrity of the cry1Ac and cry1F transgenes within the soybean genome. The soybean event pDAB9582.814.19.1 was characterized as a single full-length integration event, containing a single copy of cry1Ac and cry1F PTU from plasmid pDAB9582.
[000106] Southern blot data suggested that a fragment of T filament inserted into the genome of the soybean event pDAB9582.814.19.1. Detailed Southern blot analysis was conducted using specific probes for the cry1Ac and cry1F genes, contained in the T filament integration region of pDAB9582.814.19.1, and descriptive restriction enzymes that have cleavage sites located within the plasmid and produce fragments of hybridization inside the plasmid or fragments that cover the junction of the plasmid with the genomic DNA of soybeans (fragments of the ends). The molecular weights indicated from the Southern hybridization for the combination of the restriction enzyme and the probe were unique for the event, and established their identification standards. These analyzes also indicated that the plasmid fragment had been inserted in the genomic DNA of soy without rearrangements of cry1Ac and cry1F PTU. Example 4.1: Collection of Leaf Soy Samples and Isolation of Genomic DNA (gDNA)
[000107] Genomic DNA was extracted from leaf tissue harvested from individual soybean plants that contain the soybean event pDAB9582.814.19.1. In addition, the gDNA was isolated from a conventional soybean plant, Maverick, which contains the genetic background that is representative of the material lineage, without the cry1Ac and cry1F genes. The individual genomic DNA was extracted from lyophilized leaf tissue following the standard CTAB method (Sambrook et al. (1989)). After extraction, the DNA was quantified by spectrophotometry using the PICO GREEN reagent (Invitrogen, Carlsbad, CA). The DNA was then visualized on an agarose gel to confirm the values from the PICO GREEN analysis and to determine the quality of the DNA. Example 4.2: Digestion and DNA Separation
[000108] For the Southern blot molecular characterization of the soybean event pDAB9582.814.19.1, ten micrograms (10 μg) of genomic DNA are digested. The genomic DNA from the soybean event pDAB9582.814.19.1 and from the Maverick non-transgenic soybean strain was digested by adding approximately five units of the selected restriction enzyme per μg of DNA and the corresponding reaction buffer for each DNA sample. Each sample was incubated at approximately 37 ° C overnight. Restriction enzymes AseI, HindIII, NsiI, and NdeI were used individually for single digestions (New England Biolabs, Ipswich, MA). Restriction enzymes NotI and ApaLI were used together for double digestion (New England Biolabs, Ipswich, MA). In addition, a positive hybridization control sample was prepared by combining plasmid DNA, pDAB9582 with the genomic DNA of the non-transgenic soybean variety, Maverick. The cocktail of plasmidial DNA / genomic DNA was digested using the same procedures and restriction enzyme as the test samples.
[000109] After that, the digestions were incubated overnight, 25μL of QUICK-PRECIP PLUS SOLUTION (Edge Biosystems, Gaithersburg, MD) was added and the digested DNA samples were precipitated with isopropanol. The precipitated DNA pellet was resuspended in 15 μL of 1X loading buffer (0.01% bromophenol blue, 10.0 mM EDTA, 10.0% glycerin, 1.0 mM Tris, pH 7.5 ). The DNA samples and molecular size markers were then electrophoresed through 0.85% agarose gels with 0.4X TAE buffer (Fisher Scientific, Pittsburgh, PA) at 35 volts for approximately 18-22 hours to achieve fragment separation. The gels were stained with ethidium bromide (Invitrogen, Carlsbad, CA) and the DNA was visualized under ultraviolet (UV) light. Example 4.3: Southern Transfer and Membrane Treatment
[000110] A Southern blot analysis was performed essentially as described by Memelink, et al. (1994). Briefly, after electrophoresis separation and visualization of DNA fragments, the gels were depurinized with 0.25 M HCl for approximately 20 minutes, and then exposed to a denaturing solution (0.4 M NaOH, 1.5 M NaCl) for approximately 30 minutes, and then a neutralizing solution (1.5 M NaCl, 0.5 M Tris, pH 7.5) for at least 30 minutes. The Southern transfer was performed overnight on nylon membranes using a 10X SSC wick system. After transfer, the DNA was attached to the membrane by crosslinking with UV and then washing the membrane with a 2X SSC solution. This process produced Southern blot membranes ready for hybridization. Example 4.4: DNA Probe Labeling and Hybridization
[000111] DNA fragments bound to the nylon membrane were detected using a labeled probe (Table 6). The probes were generated by a PCR-based incorporation of a digoxigenin-tagged nucleotide (DIG), [DIG-11] -dUTP, into the DNA fragment amplified from plasmid pDAB9582 using specific primers for gene elements. The generation of DNA probes by PCR synthesis was conducted using a PCR DIG Synthesis Kit (Roche Diagnostics, Indianapolis, IN) following the procedures recommended by the manufacturer.
[000112] The labeled probes were analyzed by agarose gel electrophoresis to determine their quality and quantity. A desired amount of labeled probe was then used for hybridization to the target DNA on nylon membranes for detection of specific fragments using the procedures essentially as described for DIG EASY HYB SOLUTION (Roche Diagnostics, Indianapolis, IN). Briefly, blots on the nylon membrane containing affixed DNA were washed briefly with 2X SSC and prehybridized with 20-25 mL of DIG EASY HYB SOLUTION preheated in hybridization flasks at approximately 45-55 ° C for about 2 hours in an oven hybridization. The prehybridization solution was then decanted and replaced with ~ 15 mL of preheated DIG EASY HYB SOLUTION containing a desired amount of specific denatured probes boiling in a water bath for approximately five minutes. The hybridization step was then carried out at approximately 45-55 ° C overnight in the hybridization oven.
[000113] At the end of the probe hybridization, DIG EASY HYB SOLUTIONS containing the probes were decanted into clean tubes and stored at approximately -20 ° C. These probes could be reused twice according to the procedure recommended by the manufacturer. The membrane blots were briefly rinsed and washed twice in clean plastic containers with low-stringency washing buffer (2X SSC, 0.1% SDS) for approximately five minutes at room temperature, and then washing twice with high rigor wash buffer (0.1X SSC, 0.1% SDS) for 15 minutes each time at approximately 65 ° C. The membrane blots are washed briefly with 1X of maleic acid buffer from the DIG WASH AND BLOCK BUFFER SET (Roche Diagnostics, Indianapolis, IN) for approximately 5 minutes. After that, blocking in 1X of blocking buffer is performed for 2 hours and an incubation with anti-DIG-AP (alkaline phosphatase) antibody (Roche Diagnostics, Indianapolis, IN) in 1X blocking buffer also for a minimum of 30 minutes. After 2-3 washes with 1X wash buffer, specific DNA probes remain attached to membrane blots and DIG-labeled DNA patterns were visualized using CDP-STAR CHEMILUMINESCENT NUCLEIC ACID DETECTION SYSTEM (Roche Diagnostics, Indianapolis, IN) following the manufacturer's recommendation. The blots were exposed to a chemiluminescent film for one or more points in time, to detect the hybridization fragments, to visualize molecular size patterns. The films were developed with an ALL-PRO 100 PLUS film developer (Konica Minolta, Osaka, Japan) and the images were scanned. The number and sizes of the detectable bands were documented for each probe. DIG-LABELED DNA MOLECULAR DNA WEIGHT MARKER II (DIG MWM II) AND DIG-LABELED DNA MOLECULAR WEIGHT MARKER VII (DIG MWM VII), visible after detection with DIG as described, were used to determine the size of the fragment in hybridization in Southern blots. Table 6. Location of length of probes used in the Southern analysis.

Example 4.5: Southern blot results
[000114] The expected and observed sizes of fragments with a specific digest and probe, based on the known restriction enzyme sites of cry1Ac and cry1F PTU, are provided in Table 7. Two types of fragments were identified from these digested and hybridizations: internal fragments in which the known enzyme sites flank the probe region and are completely contained within the cry1Ac and cry1F PTU insertion region, and end segments where a known enzyme site is located at one end of the probe region and a second site is expected in the soybean genome. The sizes of the end fragments vary by event because, in most cases, the integration sites of DNA fragments are unique for each event. The end fragments provide a means for locating a restriction enzyme site in relation to the integrated DNA and for assessing the number of DNA inserts. Southern blot analyzes completed on multiple generations of soybeans containing the pDAB9582.814.19.1 event produced data that suggested that cry1Ac and cry1F PTU intact with low copy number of plasmid pDAB9582 was inserted into the soybean genome of the soybean event pDAB9582.814.19 .1. Table 7. Predicted and observed fragments in hybridization in the Southern blot analysis. 1. The expected fragment sizes are based on the plasmid map of pDAB9582. 2. The observed sizes of the fragments are considered approximately from these analyzes and are based on the indicated sizes of the DIG-LABELED DNA MOLECULAR DNA fragment markers WEIGHT MARKER II and MARK VII.


[000115] The restriction enzymes AseI and NsiI bind and cleave unique restriction sites on plasmid pDAB9582. Subsequently, these enzymes were selected to characterize the insertion of the cry1Ac gene in the soybean event pDAB9582.814.19.1. End fragments with> 7286 bp or> 9479 bp were predicted to hybridize to the probe after digestions of AseI and NsiI, respectively (Table 7). The single hybridization bands with about 7400 and> 10000 bp cry1Ac were observed when digestions of AseI and NsiI were used, respectively. Hybridization of the probe for bands of this size suggests the presence of a single insertion site for the cry1Ac gene in the soybean genome of the soybean event pDAB9582.814.19.1. The restriction enzymes NotI and ApaLI were selected to perform double digestion and to release a fragment containing the plant transcription unit with cry1Ac (PTU; promoter / gene / terminator) (Table 7). The predicted fragments with 4550 bp were observed with the probe after double digestion with NotI and ApaLI. The results obtained with enzyme digestion of pDAB9582.814.19.1 samples and then probe hybridization indicated that an intact cry1Ac PTU from plasmid pDAB9582 was inserted into the soybean genome of the soybean event pDAB9582.814.19.1.
[000116] The restriction enzymes NdeI and NsiI bind and cleave restriction sites on plasmid pDAB9582. Subsequently, these enzymes were selected to characterize the insertion of the cry1F gene in the soybean event pDAB9582.814.19.1. End fragments with> 5569 bp and> 9479 bp were predicted to hybridize with the probe after digested with NdeI and NsiIs, respectively (Table 7). The single hybridization bands with cry1F of ~ 7500 bp and> 10000 bp were observed when NdeI and NsiI were used, respectively. Hybridization of sona for bands of this size suggests the presence of a single insertion site for the cry1F gene in the soybean genome of the soybean event pDAB9582.814.19.1. The restriction enzyme, HindIII, was selected to release a fragment that contains the cry1F plant transcription unit (PTU; promoter / gene / terminator) (Table 7). The predicted fragment with 7732 bp was observed with the probe after digestions with HindIII. The results obtained with the enzyme digestion of the pDAB9582.814.19.1 samples and then hybridization of the probe indicated that an intact cry1F PTU of the plasmid pDAB9582 was inserted into the soybean genome of the soybean event pDAB9582.814.19.1. Example 4.6: Absence of Framework Sequences
[000117] A Southern blot analysis was also conducted to verify the absence of the spectinomycin resistance gene (specR), Ori Rep element and trfA replication initiation protein (trf A element) in the soybean event pDAB9582.814.19.1. No specific hybridization for spectinomycin resistance, Ori Rep element or trf A element is expected when appropriate positive (pDAB9582 added to Maverick genomic DNA) and negative (Maverick genomic DNA) controls are included for Southern analysis. After NsiI digestion and hybridization with the specific specR probe, a band of the expected size of 15320 bp was expected to be observed in the positive control sample (pDAB9582 added to Maverick's genomic DNA). The specR probe did not hybridize to negative control samples and the soybean event pDAB9582.814.19.1. Similarly, a band with the expected size of 15320 bp was expected to be detected in the positive control sample (pDAB9582 plus Maverick), but absent in the samples of the negative control and soybean event pDAB9582.814.19.1 after digestion with NsiI and hybridization with the trfA probe. Another band with an expected size of 5329 bp was detected in the positive control sample (pDAB9582 added to Maverick genomic DNA), but absent in the negative control samples and in the soybean event pDAB9582.814.19.1 after digestion with NdeI and hybridization with the specific OriRep probe. These data indicate the absence of the spectinomycin resistance gene, Ori Rep element and replication initiation protein trfA in the soybean event pDAB9582.814.19.1. Example 5: Agronomic and Field Yield Testing and Tolerance to Herbicides
[000118] To test the agronomic characteristics and the effectiveness of the soybean event pDAB9582.814.19.1, the event was planted in an effectiveness test in Santa Isabel, Puerto Rico in October 2010 and February 2011. The cultivar Maverick, which was originally transformed to produce the pDAB9582.814.19.1 event, it was planted in each nursery and included as a control in the experiments. The seed for nursery T3 was derived from selections of single plant in stage T2 and the seed for nursery T4 was derived from selections of single plant in stage T3. Four strains of the event were tested each generation. Each strain was planted in a bed that was 4 rows wide and 2.30 meters long. The row spacing was 9 meters. The nurseries were created under lights for approximately 2.5 weeks to make up for the short duration of days in Puerto Rico. Each nursery was sprayed with glufosinate at a rate of 411 grams of active ingredient (pa) per hectare. One control plant nursery, Maverick, was sprayed with the same rate of glufosinate and a second nursery was not sprayed and used as a control comparison for the event.
[000119] Data were collected on emergence, general appearance, vigor, height, accommodation, and maturity. Tolerance to the herbicide was assessed visually by looking for chlorosis, leaf necrosis and plant death (Table 8).
[000120] For comparisons of the soybean event pDAB9582.814.19.1 with Maverick, only data from the Maverick non-sprayed block were used. For comparison of the sprayed and non-sprayed treatments, the data from the soybean event pDAB9582.814.19.1 sprayed with a given treatment were compared with the data from the Maverick non-sprayed control block. The soybean event pDAB9582.814.19.1 showed tolerance to the application of the herbicide glufosinate. In contrast, none of the Maverick plants were tolerant to herbicide treatments. Table 8. Comparison of the soybean event pDAB9582.814.19.1 with Maverick. Values are averages for nurseries T3 and T4. Each nursery of the soybean event pDAB9582.814.19.1 was sprayed with glufosinate in stage V3 at a rate of 411 g pa / ha.
Example 6: Characterization of the Insecticidal Activity for the Soy Event 9582.814.19.1
[000121] Field and greenhouse evaluations were conducted to characterize the activity of Cry1Ac and Cry1F in the soybean event pDAB9582.814.19.1 against laboratory-grown soybean pests, including Anticarsia gemmatalis (soybean caterpillar), Pseudoplusia includens ( soybean larva) and Spodoptera frugiperda (cartridge caterpillar). The soybean event pDAB9582.814.19.1 was compared with the Maverick untransformed soybean variety, to determine the level of plant protection provided by the proteins Cry1F and Cry1 Ac.
[000122] The greenhouse tests were conducted on plants aged approximately four weeks. Fifteen plants were used to evaluate the soybean event pDAB9582.814.19.1 and the Maverick control. For each insect species tested (Anticarsia gemmatalis, Pseudoplusia includes, and Spodoptera frugiperda) 3 leaf punctures made on each plant for a total of 45 leaf discs / plant / insect species. The 1.4 cm (1.54 cm2) leaf punctures were placed on a test platform on top of 2% water on agar, infested with a neonate larva and sealed with a perforated plastic cover. Mortality and leaf consumption were classified 4 days after infestation. Larvae that did not respond to a soft probe were considered dead. Leaf damage was assessed visually by scoring the percentage of leaf puncture consumed by the insect.
[000123] Field evaluations were conducted by collecting leaf samples from nursery seed beds in Santa Isabel, Puerto Rico and sending these leaves to Indianapolis, IN for testing. The nursery bed for the soybean event pDAB9582.814.19.1 was planted in February 2011 and consisted of approximately 180 plants arranged in four rows. Each row was 2.3 m long and the rows were spaced 76.2 cm apart from each other; the individual plants were spaced 5.1 cm apart within each row. In March 2011, a completely expanded trophy leaf from the main stem, located approximately four knots below the meristem, was excised from 10 plants from the soybean event pDAB9582.814.19.1 and 10 'Maverick' plants. The sheets were placed in labeled plastic bags, (one per bag) and sealed. The bagged leaves were compacted and transferred to the laboratory. In the laboratory, one or two leaf discs with a diameter of 3.33 cm (1.31 in) were punctured from each trifoliate leaf to provide a total of 16 leaf discs. Each leaf disc was placed on a test platform on 2% agar, infested with a neonate S. frugiperda larva, and sealed with a perforated plastic lid. The leaf discs were kept in a chamber with a controlled environment for 7 days, and at this time mortality and leaf consumption were scored. Larvae unresponsive to gentle probing were considered dead. Leaf damage was assessed visually by scoring the percentage of leaf puncture consumed by the insect.
[000124] The results obtained from these replicated experiments indicated that the soybean event pDAB9582.814.19.1 suffered substantially lower damage than the Maverick control plants for all tested insects. Therefore, the soybean event pDAB9582.814.19.1 has insecticidal activity on this broad spectrum of hosts. Example 7: Soy Event Sequence pDAB9582.814.19.1
[000125] SEQ ID NO: 14 provides the soybean event sequence pDAB9582.814.19.1. This sequence contains the 5 'genomic flanking sequence, the insertion of the T filament of pDAB9582 and the 3' genomic flanking sequences. With respect to SEQ ID NO: 14, residues 1-1400 are 5 'genomic flanking frequency, residues 1401 - 1536 are residues of a rearrangement of the pDAB9582 plamid and residues 1537 - 13896 are residues of the T filament insertion of pDAB9582 , and residues 13897 - 15294 are the 3 'flanking sequence. The sequence of the junction or transition in relation to the 5 'end of the insert thus occurs at residues 1400-1401 of SEQ ID NO: 14. The sequence of the junction or transition in relation to the 3 'end of the insert thus occurs at residues 13896-13897 of SEQ ID NO: 14.
[000126] It should be noted that the progeny of the soybean event pDAB9582.814.19.1 may have sequences that deviate slightly from SEQ ID NO: 14. During the introgression creation process to introduce the pDAB9582.814.19.1 soy event into the plant cell genome, it is not uncommon for some deletions or other insertion changes to occur. In addition, errors in PCR amplification can occur, which could result in minimal sequencing errors. For example, the flanking sequences listed here were determined by generating amplicons from the genomic soybean DNAs, and then cloning and sequencing the amplicons. It is not uncommon to find slight differences and small discrepancies in the sequences generated and determined in this way, given the many rounds of amplification that are necessary to generate enough amplicon for sequencing from genomic DNAs. Those skilled in these techniques should recognize and be warned that any adjustments necessary due to these types of common sequencing errors or discrepancies are within the scope of the present invention. Therefore, the relevant segment of the plasmid sequence provided here could comprise some minor variations. Accordingly, a plant comprising a polynucleotide that has some identity range with the insertion sequence in question is within the scope of the present invention. The sequence identity of SEQ ID NO: 14 can be a polynucleotide sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence exemplified or described herein. Therefore, some differences between SEQ ID NO: 14 and plants from the progeny of the soybean event pDAB9582.814.19.1 can be identified and are within the scope of the present invention. Example 8: Event-specific TaqMan Assay
[000127] An event-specific TAQMAN assay was developed to detect the presence of the soybean event pDAB9582.814.19.1 and to determine the zygous state of plants in breeding populations. The soybean event pDAB9582.814.19.1 contains the T filament of the binary vector pDAB9582 (figure 1). For the specific detection of the soybean event pDAB9582.814.19.1, specific TAQMAN primers and probes were designed according to the DNA sequences located at the junction insert to the plant 5 '(SEQ ID NO: 1) or 3' (SEQ ID NO : 2) (figure 4). An event-specific assay for the soybean event pDAB9582.814.19.1 was designed to specifically detect a 229 bp fragment that covers the 3 'integration junction using two primers from a target-specific MGB probe synthesized by Applied Biosystems (ABI) containing the FAM reporter at its 5 'end. The specificity of this TAQMAN detection method for the soybean event pDAB9582.814.19.1 was tested against 7 different events containing Cry1Ac and Cry1F PTUs and a variety of non-transgenic control soybean (Maverick) in duplex format with the endogenous reference gene specific soybean, GMFL01- 25-J19 (Glycine max cDNA, GenBank: AK286292.1). Example 8.1: Isolation of gDNA
[000128] Samples of gDNA from 7 different soy events and non-GM soy varieties were tested in this study. Genomic DNA was extracted using the modified QIAGEN MAGATTRACT PLANT DNA KIT (Qiagen, Valencia, CA). Fresh soybean leaf discs, 8 per sample, were used for gDNA extraction. The samples were diluted with DNase-free water, resulting in a concentration of approximately 10 ng / μL for the purpose of this study. Example 8.2: TaqMan Assay and Results
[000129] Specific TAQMAN primers and probe were designed for a specific TAQMAN assay for the soybean event pDAB9582.814.19.1. These reagents can be used with the conditions listed below to detect the transgene within the soybean event pDAB9582.814.19.1. Table 9 lists the primer and probe sequences that were developed specifically for the detection of the soybean event pDAB9582.814.19.1. Table 9. TAQMAN PCR primers and probes

[000130] The conditions of the multiplex PCR for amplification are as follows: Roche 1X PCR buffer, 0.4 μM event specific direct initiator, 0.4 μM event specific reverse initiator, 0.4 μM Initiator GMS116 F, 0, 4 μM GMS116 R initiator, 0.2 μM event specific probe, 0.2 μM GMS116 probe, 0.1% PVP, 6-20 ng gDNA in a total reaction of 10 μL. The cocktail was amplified using the following conditions: i) 95 ° C for 10 min., Ii) 95 ° C for 10 s, iii) 60 ° C for 40 s, iv) repeat steps ii-iii for 40 cycles, v) maintain 40 ° C. Real-time PCR was conducted on the ROCHE LIGHTCICLOR 480. Data analysis was based on the measurement of the crossing point (Cp value) determined by the LIGHTCICLOR 480 software, which is the number of PCR cycles at which the rate of change in fluorescence it reaches its maximum.
[000131] The TAQMAN detection method for the soybean event pDAB9582.814.19.1 has been tested against 7 different events containing Cry1Ac and Cry1F PTUs and a variety of non-transgenic soybean in duplex format with the specific endogenous soybean gene, GMFL01-25-J19 (GenBank: AK286292.1). The assay specifically detected the pDAB9582.814.19.1 soy event and did not produce or amplify any false positive results from the controls (ie, events containing Cry1Ac and Cry1F PTUs and a variety of non-GM soy). Event specific primers and probes can be used to detect the soybean event pDAB9582.814.19.1 and these conditions and reagents are applicable for zygosity assays.
[000132] Having illustrated and described the principles of the present invention, it should be evident to those skilled in these techniques that the invention can be modified in arrangement and detail without departing from these principles. The claimant claims all modifications that are within the spirit and scope of the attached claims.
[000133] All publications and published patent documents cited in this specification are hereby incorporated by reference to the same degree as if each publication or patent application had been specifically and individually indicated to be incorporated as a reference.

权利要求:
Claims (1)
[0001]
1. Method for detecting the Soy Event pDAB9582.814.19.1 in a sample comprising soybean DNA, characterized by the fact that it comprises: (a) putting said sample in contact with a first initiator that has a hair length minus 10 bp that binds selectively to a flanking sequence within bp 1-1400 of SEQ ID NO: 1 or the complement thereof, and a second primer that is at least 10 bp in length that binds selectively to a sequence of insertion within bp 1401-1836 of SEQ ID NO: 1 or complement thereof; and testing for an amplicon generated between said primers, wherein said amplicon comprises nucleotides 1390 to 1410 of SEQ ID NO: 1; or (b) placing said sample in contact with a first primer that is at least 10 bp in length, which selectively binds to an insertion sequence within bp 1-152 of SEQ ID NO: 2 or its complement, and a second primer having a length of at least 10 bp, which selectively binds to the flanking sequence within bp 153-1550 of SEQ ID NO: 2 or its complement; and testing for an amplicon generated between said primers, wherein said amplicon comprises nucleotides 142 to 163 of SEQ ID NO: 2; and where the DNA comprising the Soy Event pDAB9582.814.19.1 is obtainable from seeds deposited in the American Type Culture Collection under Accession No. PTA-12006.

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法律状态:
2015-04-28| B03A| Publication of a patent application or of a certificate of addition of invention [chapter 3.1 patent gazette]|

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2018-03-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-09-10| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|

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2020-04-14| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|

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2020-12-01| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-02-02| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 25/07/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201161511658P| true| 2011-07-26|2011-07-26|

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US61511658|2011-07-26|

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