Transgenic glycine max event ka-6g2Ø16-7 and detection method therefor

Abstract

The present invention relates to a transgenic Glycine max event, formula I, or a derivative line thereof, and also relates to a nucleic acid sequence for detecting a Glycine max plant, formula II, or a derivative line thereof, and a detection method therefor. Nucleic acid sequences derived from a transgenic Glycine max event, formula III, or a derivative thereof comprise SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11 and / or SEQ ID NO: 12, or nucleic acid sequences complementary thereto. The Glycine max plant of the present invention, formula IV, or a derivative line thereof has a good herbicide tolerance without affecting the yield. Moreover, the detection method can accurately and quickly identify whether a biological sample contains a DNA molecule of a transgenic Glycine max event KA-6G2Ø16-7 or a derivative line thereof.

Description

Genetically modified soybean incident KA-6G2Ø16-7 and its detection method Technical Field

[0001] This invention relates to genetically modified soybean events. Or its derivatives, and also involve a method for detecting soybean plants Nucleic acid sequences or their derivatives and their detection methods. Background Technology

[0002] Soybean (Glycine max (L.) Merr.) is one of the world's most important food crops, with a cultivation history of five thousand years. Its seeds are rich in plant protein. Currently, biotechnology is widely used in soybeans to improve their agronomic traits and quality.

[0003] Herbicide tolerance is an important agronomic trait in soybean production, especially tolerance to glufosinate and PPO inhibitor herbicides. Soybean tolerance to glufosinate and PPO inhibitor herbicides can be acquired through transgenic methods by expressing glufosinate resistance genes (such as PAT) and PPO inhibitor resistance genes in soybean plants.

[0004] Besides the functional genes themselves, the selection and sequential arrangement of regulatory elements are crucial for obtaining successful transformation events, and their technical effects are unpredictable. Furthermore, it is known that the expression of exogenous genes in plants is influenced by their location within chromosomes, such as chromatin structure (e.g., heterochromatin) or the proximity of transcriptional regulatory elements (e.g., enhancers) to integration sites. Therefore, screening a large number of events is usually required to identify commercially viable events (i.e., events where the introduced target gene is optimally expressed). Identical genes in the same type of transgenic plant (or other organisms) can exhibit wide variations in expression levels across different events, and may also show differences in spatial or temporal expression patterns. Summary of the Invention

[0005] The purpose of this invention is to provide a genetically modified soybean event. And for detecting genetically modified soybean events Nucleic acid sequences and detection methods.

[0006] The technical solution adopted in this invention is as follows:

[0007] This invention provides a nucleic acid sequence, the nucleic acid sequence comprising:

[0008] (a) SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11 and / or SEQ ID NO:12; or

[0009] (b) A nucleic acid sequence complementary to (a).

[0010] In one specific implementation, the nucleic acid sequence is derived from a genetically modified soybean event. Or its derivatives.

[0011] In another specific embodiment, the nucleic acid sequence is used to diagnose soybean events. Amplicones present in or derived from it.

[0012] The present invention also provides a DNA construct comprising two expression cassettes, wherein,

[0013] a) The first expression cassette contains, in an operable link, a promoter P-CsVMV with nucleic acid sequence as shown in SEQ ID NO:21, a pat gene coding region with nucleic acid sequence as shown in SEQ ID NO:22, and a terminator T-E9 with nucleic acid sequence as shown in SEQ ID NO:23 to terminate the expression of the gene.

[0014] b) The second expression cassette contains, in an operable linker, a promoter P-CLSV as shown in SEQ ID NO:24, a TEV protease leader sequence L-TEV as shown in SEQ ID NO:25, an OsPPO2-k1 gene coding region as shown in SEQ ID NO:26, and a terminator T-NOS as shown in SEQ ID NO:27 to terminate the expression of the gene.

[0015] In one specific embodiment, the nucleic acid sequence of the DNA construct includes SEQ ID NO:9.

[0016] This invention also provides a diagnostic method for genetically modified soybean events. A DNA probe is present, wherein the DNA probe is of sufficient length to bind the said nucleic acid sequence, and the DNA probe hybridizes to the said nucleic acid sequence under strict hybridization conditions and does not hybridize to other nucleic acid sequences.

[0017] The present invention also provides a DNA primer pair, which consists of a first DNA primer and a second DNA primer different from the first DNA primer, said DNA primer pair being associated with events involving transgenic soybeans. The samples were used together in an amplification reaction to generate a diagnostic tool for genetically modified soybean events in the samples. Existing amplicon.

[0018] In one specific embodiment, the amplicon comprises the aforementioned nucleic acid sequence.

[0019] In another specific embodiment, the primer pair includes SEQ ID NO:13 and SEQ ID NO:14, or SEQ ID NO:15 and SEQ ID NO:16.

[0020] This invention also provides a method for detecting genetically modified soybeans in samples. The kit contains at least one of the DNA probes or the DNA primer pair described herein.

[0021] This invention also provides a method for detecting genetically modified soybeans in samples. Methods for the presence of DNA include:

[0022] a) Contact the sample to be tested with at least one of the DNA primer pairs described above;

[0023] b) Perform an amplification reaction sufficient to produce DNA amplicones; and,

[0024] c) Detect the presence of the DNA amplicon in the reaction;

[0025] The amplicon sequence originated from a genetically modified soybean event.

[0026] Or the method may include:

[0027] a) Bring the sample to be tested into contact with the probe;

[0028] b) Hybridize the sample to be tested and the probe under stringent hybridization conditions; and

[0029] c) Detect the hybridization between the sample to be tested and the probe;

[0030] The aforementioned detection can diagnose genetically modified soybean events in samples. The DNA is present.

[0031] In one specific embodiment, the DNA amplicon comprises the aforementioned nucleic acid sequence.

[0032] The present invention also provides a method for protecting soybean plants from damage caused by herbicides, comprising applying a herbicide containing an effective dose of a glutamine synthase inhibitor and / or a protoporphyrinogen oxidase inhibitor to a field in which at least one transgenic soybean plant is planted, wherein the transgenic soybean plant contains, in sequence, the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:10 (positions 1049-5292) and SEQ ID NO:2, or SEQ ID NO:3, SEQ ID NO:10 (positions 1049-5292) and SEQ ID NO:4, or SEQ ID NO:5, SEQ ID NO:10 (positions 1049-5292) and SEQ ID NO:6, or the genome of the transgenic soybean plant contains SEQ ID NO:10; wherein the transgenic soybean plant is tolerant to the glutamine synthase inhibitor and / or protoporphyrinogen oxidase inhibitor herbicide.

[0033] The present invention also provides a method for controlling weeds in a field where soybean plants are grown, comprising applying a herbicide containing an effective dose of a glutamine synthase inhibitor and / or a protoporphyrinogen oxidase inhibitor to a field where at least one transgenic soybean plant is grown, wherein the transgenic soybean plant contains, in sequence, the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:10 (positions 1049-5292) and SEQ ID NO:2, or SEQ ID NO:3, SEQ ID NO:10 (positions 1049-5292) and SEQ ID NO:4, or SEQ ID NO:5, SEQ ID NO:10 (positions 1049-5292) and SEQ ID NO:6, or the genome of the transgenic soybean plant contains SEQ ID NO:10; the transgenic soybean plant is tolerant to the glutamine synthase inhibitor and / or protoporphyrinogen oxidase inhibitor herbicide.

[0034] The present invention also provides a method for culturing soybean plants tolerant to glutamine synthase inhibitor herbicides and / or protoporphyrinogen oxidase inhibitor herbicides, comprising: planting at least one soybean seed, wherein the genome of the soybean seed contains a specific region of nucleic acid sequence, wherein the specific region of nucleic acid sequence sequentially comprises SEQ ID NO:1, SEQ ID NO:10 nucleic acid sequence positions 1049-5292 and SEQ ID NO:2, or SEQ ID NO:3, SEQ ID NO:10 nucleic acid sequence positions 1049-5292 and SEQ ID NO:4, or SEQ ID NO:5, SEQ ID NO:10 nucleic acid sequence positions 1049-5292 and SEQ ID NO:6, or the specific region of nucleic acid sequence comprises SEQ ID NO:10;

[0035] The soybean seeds are then allowed to grow into soybean plants.

[0036] The soybean plants were sprayed with an effective dose of a glutamine synthase inhibitor herbicide and / or a protoporphyrinogen oxidase inhibitor herbicide, and the plants with reduced plant damage compared to other plants that do not have the nucleic acid sequence of the specific region were harvested.

[0037] This invention also provides a method for producing soybean plants resistant to glutamine synthase inhibitor herbicides and / or protoporphyrinogen oxidase inhibitor herbicides, comprising: hybridizing a soybean plant whose genome sequentially contains the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:10 positions 1049-5292 and SEQ ID NO:2, or SEQ ID NO:3, SEQ ID NO:10 positions 1049-5292 and SEQ ID NO:4, or SEQ ID NO:5, SEQ ID NO:10 positions 1049-5292 and SEQ ID NO:6 with another soybean plant to produce a large number of progeny plants; selecting progeny plants whose genome contains a specific region of nucleic acid sequence, wherein the specific region of nucleic acid sequence sequentially contains the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:10 positions 1049-5292 and SEQ ID NO:2, or SEQ ID NO:3, SEQ ID NO:10 positions 1049-5292 and SEQ ID NO:6; and... The nucleic acid sequence of SEQ ID NO:4, or SEQ ID NO:5, SEQ ID NO:10 from position 1049 to 5292 and SEQ ID NO:6, or the nucleic acid sequence of the specific region contains SEQ ID NO:10, and the progeny plants have tolerance to glutamine synthase inhibitor herbicides and / or protoporphyrinogen oxidase inhibitor herbicides.

[0038] In one specific embodiment, the method includes: [the process involves] transgenic soybeans tolerant to glutamine synthase inhibitor herbicides and / or protoporphyrinogen oxidase inhibitor herbicides. The first parent soybean plant was sexually crossed with the second parent soybean plant, which lacked tolerance to herbicides such as glutamine synthase inhibitors and / or protoporphyrinogen oxidase inhibitors, thereby producing a large number of offspring plants.

[0039] The progeny plants were treated with glutamine synthase inhibitor herbicides and / or protoporphyrinogen oxidase inhibitor herbicides;

[0040] The progeny plants were selected from those resistant to herbicides of glutamine synthase inhibitors and / or protoporphyrinogen oxidase inhibitors.

[0041] The present invention also provides a method for improving the tolerance of soybean plants, the method comprising:

[0042] a) Construct the DNA construct as described above;

[0043] b) Insert the DNA construct into the genome of soybean cells;

[0044] c) regenerate the soybean cells into a soybean plant; and

[0045] d) Select soybean plants containing the DNA construct.

[0046] In one specific embodiment, improving the tolerance of soybean plants includes their resistance to an effective amount of at least one herbicide, preferably a glutamine synthase inhibitor herbicide and / or a protoporphyrinogen oxidase inhibitor herbicide.

[0047] This invention also provides a method arising from genetically modified soybean events. Compositions thereof or their derivatives, wherein the composition is soybean oil, soybean protein, soybean flour or soybean starch.

[0048] This invention also provides a method arising from genetically modified soybean events. Agricultural products or commodities thereof, including soybean oil, soybean protein, soybean flour, soybean starch, soybean meal, soybean flakes, soybean hulls, soy milk, soybean cheese, soybean wine, animal feed containing soybeans, paper containing soybeans, cheese containing soybeans, soybean biomass, or fuel products produced using soybean plants and parts thereof.

[0049] The present invention also provides a plant cell, plant part, plant or seed comprising the aforementioned nucleic acid sequence.

[0050] The present invention also provides an inanimate plant material comprising the aforementioned nucleic acid sequence.

[0051] Some of the terms used in this specification are defined as follows.

[0052] The term "soybean" as used in this invention refers to soybean (Glycine max) and all plant varieties that can be interbred with soybean, including wild soybean species.

[0053] In this invention, "comprising" means "including but not limited to".

[0054] The term "herbicide" as used in this invention refers to an active ingredient capable of killing, controlling, or hindering plant growth. The terms "herbicide tolerance," "herbicide resistance," "herbicide tolerance," or "herbicide resistance" in this invention refer to the continued growth of a plant even after the use of herbicides that kill common or wild plants, resist plant growth, or weaken or stop the plant's growth compared to wild plants. Among these, glutamine synthetase inhibitor herbicides include, but are not limited to, glufosinate and glufosinate-ammonium; protoporphyrinogen oxidase (PPO) inhibitor herbicides can be classified into pyrimidinediones, diphenyl-ethers, phenylpyrazoles, N-phenylphthalimides, thiadiazoles, oxadiazoles, triazolinones, oxazolidinediones, and other herbicides with different chemical structures.

[0055] Generally, if the PPO-inhibiting herbicides and / or other herbicides, as described herein and usable in the context of this invention, are capable of forming geometric isomers, such as E / Z isomers, then both, pure isomers, and mixtures thereof may be used in compositions according to the invention. If the PPO-inhibiting herbicides and / or other herbicides, as described herein, have one or more chiral centers and are thus present as enantiomers or diastereomers, then both, pure enantiomers, diastereomers, and mixtures thereof may be used in compositions according to the invention. If the PPO-inhibiting herbicides and / or other herbicides, as described herein, have ionizable functional groups, then they may also be used in the form of their agriculturally acceptable salts. Typically, salts of those cations and acid addition salts of those acids are suitable, whose cations and anions do not have adverse effects on the activity of the active compound, respectively. The preferred cations are alkali metal ions, preferably lithium, sodium, and potassium ions; alkaline earth metal ions, preferably calcium and magnesium ions; and transition metal ions, preferably manganese, copper, zinc, and iron ions, further preferably ammonium and substituted ammonium ions, wherein one to four hydrogen atoms are substituted by C1-C4-alkyl, hydroxy-C1-C4-alkyl, C1-C4-alkoxy-C1-C4-alkyl, hydroxy-C1-C4-alkoxy-C1-C4-alkyl, phenyl, or benzyl, preferably ammonium, methylammonium, isopropylammonium, dimethylammonium, diisopropylammonium, trimethylammonium, heptylammonium, dodecylammonium, tetradecylammonium, tetramethylammonium, tetraethylammonium, tetrabutylammonium, 2 - Hydroxyethylammonium (olamine salt), 2-(2-hydroxyethyl-1-oxy)ethyl-1-ylammonium (diethylene glycolamine salt), di(2-hydroxyethyl-1-yl)ammonium (diethylene glycolamine salt), tri(2-hydroxyethyl)ammonium (trinitroethanolamine salt), tri(2-hydroxypropyl)ammonium, benzyltrimethylammonium, benzyltriethylammonium, N,N,N-trimethylethanolammonium (choline salt), in addition to phosphonium ions, sulfonium ions, preferably tri(C1-C4-alkyl)sulfonium such as trimethylsulfonium, and sulfonium oxide ions, preferably tri(C1-C4-alkyl)sulfonium oxide ions, and finally, salts of polyamines such as N,N-bis-(3-aminopropyl)methylamine and diethylenetriamine. The main anions that can be used for acid addition salts are chloride, bromide, fluoride, iodide, hydrogen sulfate, methyl sulfate, sulfate, dihydrogen phosphate, hydrogen phosphate, nitrate, bicarbonate, carbonate, hexafluorosilicate, hexafluorophosphate, benzoate, and anions of C1-C4-alkanoic acids, with formate, acetate, propionate, and butyrate being preferred.

[0056] PPO-inhibiting herbicides and / or other herbicidal compounds having carboxyl groups, as described herein, can be used in the form of acids, agriculturally suitable salts as mentioned above, or otherwise agriculturally acceptable derivatives, for example as amides such as mono- and di-C1-C6-alkylamides or arylamides, and as esters such as allyl esters, propargyl esters, C1-C6-alkyl esters, etc.10 -alkyl esters, alkoxyalkyl esters, tefuryl ((tetrahydrofuran-2-yl)methyl) esters, and also as thioesters, for example as C1-C 10 -Alkyl thioesters. Preferred mono- and di-C1-C6-alkylamides are methyl and dimethylamides. Preferred arylamides are, for example, N-anilide and 2-chloroanilide. Preferred alkyl esters are, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, mexyl (1-methylhexyl), meptyl (1-methylheptyl), heptyl, octyl, or isooctyl (2-ethylhexyl) esters. Preferred C1-C4-alkoxy-C1-C4-alkyl esters are straight-chain or branched C1-C4-alkoxyethyl esters, such as 2-methoxyethyl ester, 2-ethoxyethyl ester, 2-butoxyethyl ester, 2-butoxypropyl ester, or 3-butoxypropyl ester. Straight-chain or branched C1-C 10 An example of an alkyl thioester is an ethyl thioester.

[0057] In one exemplary embodiment, pyrimidinid herbicides include, but are not limited to, flufenacet (CAS NO: 134605-64-4), fensulfuron-methyl (CAS NO: 372137-35-4), bispyribac-methyl (CAS NO: 158755-95-4), tiafenacil (CAS NO: 1220411-29-9), epyrifenacil (CAS NO: 353292-31-6), 1-methyl-6-trifluoromethyl-3-(2,2,7-trifluoro-3-oxo-4-prop-2-ynyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-1H-pyrimidin-2,4-dione (CAS NO: 1220411-29-9), Epyrifenacil (CAS NO: 353292-31-6), and 1-methyl-6-trifluoromethyl-3-(2,2,7-trifluoro-3-oxo-4-prop-2-ynyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-1H-pyrimidin-2,4-dione (CAS NO: 134605-64-4), pyrimidinidyl-6-yl)-1H-pyrimidin-2,4-dione (CAS NO: 1220411-29-9). NO: 1304113-05-0), 3-[7-chloro-5-fluoro-2-(trifluoromethyl)-1H-benzimidazol-4-yl]-1-methyl-6-(trifluoromethyl)-1H-pyrimidin-2,4-dione (CAS NO: 212754-02-4), flupropacil (CAS NO: 120890-70-2), Uracil-pyridine disclosed in WO2017 / 202768 and uracil derivatives disclosed in WO2018 / 019842.

[0058] Diphenyl ether herbicides include, but are not limited to, flufenoxuron (CAS NO: 72178-02-0), ethoxyflufen (CAS NO: 42874-03-3), bensulfuron (CAS NO: 74070-46-5), quizalofop-p-ethyl (CAS NO: 77501-63-4), methoxyflufen (CAS NO: 32861-85-1), glufosinate (CAS NO: 1836-77-7), ethoxyflufen (CAS NO: 77501-90-7), trifluralin or its sodium salt (CAS NO: 50594-66-6 or 62476-59-9), methoxyflufen (CAS NO: 42576-02-3), ethoxyfen (CAS NO: 188634-90-4), chlorfluazuron ethyl (CAS NO: 131086-42-5), and fluoronitrofen (CAS NO: 131086-42-5). NO: 13738-63-1), furyloxyfen (CAS NO: 80020-41-3), nitrofluorfen (CAS NO: 42874-01-1) and halosafen (CAS NO: 77227-69-1).

[0059] Phenylepiazole herbicides include, but are not limited to, imidacloprid (CAS NO: 129630-19-9), isopyrazosulfuron (CAS NO: 174514-07-9) and ethyl 2-[1-(2,3,4-trichlorophenyl)-4-nitropyrazolyl-5-oxo]propionate (CAS: 118237-10-8).

[0060] N-phenylimide herbicides include, but are not limited to, propyzamide (CAS NO: 103361-09-7), indole-3-methyl (CAS NO: 142891-20-1), flumipropyn (CAS NO: 84478-52-4), fluazinam (CAS NO: 87546-18-7), chlorophthalim (CAS: 39985-63-2), and N-(4-chlorophenyl)-3,4,5,6-tetrahydrophenyl-o-dicarboximide (CAS: 7386-21-2).

[0061] Thiadiazole herbicides include, but are not limited to, methyl methacrylate (CAS NO: 117337-19-6), methoxyfenozide (CAS NO: 149253-65-6), and thiamethoxam (CAS NO: 123249-43-4).

[0062] Oxadiazole herbicides include, but are not limited to, propyzinoxadiazon (CAS NO: 39807-15-3) and oxadiazon (CAS NO: 19666-30-9).

[0063] Triazoline herbicides include, but are not limited to, oxadiazon (CAS NO: 128621-72-7), oxadiazon / oxadiazon (CAS NO: 128639-02-1), mesotrione (CAS NO: 122836-35-5), oxadiazon (CAS NO: 68049-83-2), and oxadiazon (CAS NO: 173980-17-1).

[0064] Oxazolidinone herbicides include, but are not limited to, cyclooxadiazon (CAS NO: 110956-75-7).

[0065] Other herbicides include, but are not limited to, bispyribac-methyl (CAS NO: 158353-15-2), flupyrazosulfuron-methyl (CAS NO: 188489-07-8), cyclopyranil (CAS NO: 1651191-47-7), flupyrazosulfuron-methyl (CAS NO: 190314-43-3), trifludimoxazin (CAS NO: 1258836-72-4), phenopylate (CAS: 40575-34-6), N-ethyl-3-(2,6-dichloro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS NO: 452098-92-9), and N-tetrahydrofurfuryl-3-(2,6-dichloro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS NO: 452098-92-9). NO: 915396-43-9), N-ethyl-3-(2-chloro-6-fluoro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS NO: 452099-05-7), N-tetrahydrofurfuryl-3-(2-chloro-6-fluoro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS NO: 452100-03-7), 3-[7-fluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl]-1,5-dimethyl-6-thio-[1,3,5]triazin-2,4-dione (CAS NO: 915396-43-9), N-ethyl ...099-05-7), 3-tetrahydrofurfuryl-3-(2-chloro-6-fluoro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS NO: 452100-03-7), 3-[7- NO: 451484-50-7), 2-(2,2,7-trifluoro-3-oxo-4-prop-2-ynyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-4,5,6,7-tetrahydro-isoindole-1,3-Diketone (CAS NO: 1300118-96-0), (E)-4-[2-chloro-5-[4-chloro-5-(difluoromethoxy)-1H-methyl-pyrazol-3-yl]-4-fluoro-phenoxy]-3-methoxy-but-2-enoic acid methyl ester (CAS NO: 1300118-96-0), (E)-4 ...[2-chloro-5-[4-chloro-5-(difluoromethoxy)-1H-methyl-pyrazol-3-yl]-4-fluoro-phenoxy]-3-methoxy-but-2-enoic acid methyl ester (CAS NO NO: 948893-00-3), ethyl acetate of 3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy] (CAS: 353292-31-6, S-3100), 1,5-dimethyl-6-thio-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS: 1258836-72-4), 1-methyl Benzoxazinone derivatives disclosed in WO2016 / 120116 and EP09163242.2.

[0066] In another exemplary embodiment, the compound represented by general formula I is selected from compound A: methyl(2R)-2-{[(E)-{2-chloro-4-fluoro-5-[3-methyl-2,6-dioxo-4-(trifluoromethyl)-3,6-dihydropyrimidin-1(2H)-yl]benzyl}amino]oxy}propionate, flusulfanilamide, oxyfluorfen, ethoxyflufenoxam, haloxyfop-methyl, quizalofop-p-ethyl, trifluralofop-p-ethyl, pyrazosulfuron, mesotrione, pyrimisulfuron-methyl, propyzoxystrobin, cyproconazole, methyl methazine, pyrazosulfuron-methyl, oxadiazon, propyzoxystrobin, cyclopentoxadiazon, pyrazosulfuron-methyl Flupyrimethanil, trifluralin, Cyclopyranil, and Epyrifenacil.

[0067] The term "gene" refers to a nucleic acid fragment that expresses a specific protein, including the regulatory sequence preceding the coding sequence (5' non-coding sequence) and the regulatory sequence following the coding sequence (3' non-coding sequence). A "natural gene" is a gene that is naturally found to have its own regulatory sequence. A "chimeric gene" is any gene that is not a natural gene but contains regulatory and coding sequences not naturally found. An "endogenous gene" is a natural gene located at its natural position in an organism's genome. A "foreign gene" is a foreign gene that is currently present in an organism's genome and was not originally present; it also refers to a gene introduced into a recipient cell through a transgenic process. Foreign genes can include natural genes inserted into non-natural organisms or chimeric genes. A "transgenic gene" is a gene that has been introduced into the genome through a transformation process. The site where recombinant DNA has been inserted into the plant genome can be called an "insertion site" or a "target site."

[0068] The terms "nucleotide sequence" or "nucleic acid sequence" are used interchangeably and refer to oligonucleotides, nucleotides, or polynucleotides and segments or portions thereof, which may be single-stranded or double-stranded, and denote sense or antisense strands. Nucleic acids include DNA, RNA, or hybrids thereof, and may have natural or synthetic origins. For example, nucleic acids may include mRNA or cDNA. Nucleic acids may include nucleic acids that have been amplified (e.g., using polymerase chain reaction). The single-letter codes for nucleotides are as described in Table 1 of Section 2422 of the U.S. Patent Examination Procedure Manual.

[0069] The term "transgenic plant" refers to a plant containing heteropolynucleotides. Preferably, the heteropolynucleotides are stably integrated into the genome, allowing the polynucleotides to be passed on to successive generations. Heteropolynucleotides may be integrated into the genome alone or as part of a recombinant expression cassette. "Transgenic" is used herein to refer to any cell, cell line, callus, tissue, plant part, or plant whose genotype has been altered due to the presence of heteronucleotides, including those originally altered transgenic organisms or cells, and those produced from hybridization or asexual reproduction of the initial transgenic organism or cell. As used herein, the term "transgenic" is not intended to include changes to the genome (chromosomal or extrachromosomal) made through conventional plant breeding methods (e.g., hybridization) or through naturally occurring events (e.g., autofertilization, random hybridization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation).

[0070] A transgenic "event" occurs when a plant population is regenerated by transforming plant cells with heterologous DNA (i.e., a nucleic acid construct containing the target transgene), resulting in the insertion of the transgene into the plant genome, and specific plants are selected based on the insertion fragment at a specific genomic location. The term "event" refers to the initial transformant containing the heterologous DNA and its offspring. The term "event" also refers to the offspring produced by outcrossing a transformant with another variety containing the genomic / transgenic DNA. Even after repeated backcrosses with a recurrent parent, the inserted transgenic DNA and flanking genomic DNA (genomic / transgenic DNA) from the transformant parent remain at the same chromosomal location in the hybrid offspring. The term "event" also refers to DNA from the initial transformant containing the inserted DNA and the flanking genomic sequence immediately adjacent to the inserted DNA, and its offspring, which will be transferred to the offspring. The offspring obtain DNA containing the target transgene through sexual crosses between a parental line containing the inserted DNA (e.g., the initial transformant and its self-pollinated offspring) and a parental line not containing the inserted DNA.

[0071] Genetically modified soybean incident The "derivative lines," namely those using genetically modified soybeans, are... Soybean varieties selected as genetic parents possess genetic traits inherited from transgenic soybeans. Herbicide resistance.

[0072] The term "inserted DNA" or "inserted sequence" refers to the heterologous DNA within the expression cassette used to transform plant material. The inserted DNA is derived from T-DNA and is contained in binary vectors used in Agrobacterium-mediated plant transformation.

[0073] The "flanking DNA" or "flanking sequence" described in this invention can comprise the genome naturally present in organisms such as plants or exogenous (heterologous) DNA introduced through a transformation process, such as fragments associated with a transformation event. Therefore, flanking DNA can comprise a combination of natural and exogenous DNA. In this invention, a "flanking region," "flanking sequence," "genome boundary region," or "genome boundary sequence" refers to a sequence of at least 3, 5, 10, 11, 15, 20, 50, 100, 200, 300, 400, 1000, 1500, 2000, 2500, or 5000 base pairs or longer, located directly upstream or downstream of and adjacent to the initially exogenous inserted DNA molecule. When the flanking region is downstream, it can also be referred to as a "left boundary flanking," "3' flanking," "3' genome boundary region," or "genome 3' boundary sequence," etc. When this flanking region is located upstream, it can also be referred to as the "right boundary flanking region", "5' flanking region", "5' genome boundary region", or "genome 5' boundary sequence", etc.

[0074] The term "junction" refers to the point where two specific DNA segments join together. For example, a junction exists where an insert DNA joins flanking DNA. Junction sites also exist in transformed organisms, where two DNA segments are joined together in a manner modified from that found in natural organisms. "Junction DNA" or "junction sequence" refers to DNA containing the junction site.

[0075] A “joining sequence” spans the point where the inserted genomic DNA joins with DNA from the soybean native genome flanking the insertion site, wherein the identification or detection of one or more joining sequences in the plant genetic material is sufficient to diagnose the event. This includes DNA sequences spanning the insertion and similarly long flanking DNA in the soybean events described herein. Specific examples of such diagnostic sequences are provided herein; however, other sequences overlapping the insertion joint or the joint between the insertion and the genomic sequence are also diagnostic and can be used according to the invention.

[0076] The term "probe" refers to a segment of isolated nucleic acid molecule to which a conventionally detectable label or reporter molecule is bound, such as a radioactive isotope, ligand, chemiluminescent agent, or enzyme. This probe is complementary to one strand of the target nucleic acid; in this invention, the probe is associated with a transgenic soybean event. One strand of the genome is complementary, regardless of whether the genome DNA originated from genetically modified soybeans. The question remains whether the seeds originated from genetically modified soybeans. The probes of this invention include not only deoxyribonucleic acid or ribonucleic acid, but also polyamides and other probe materials that specifically bind to the target DNA sequence and can be used to detect the presence of the target DNA sequence.

[0077] The term "primer" refers to a segment of isolated nucleic acid molecule that binds to a complementary target DNA strand through nucleic acid hybridization and annealing, forming a hybrid between the primer and the target DNA strand, and then extends along the target DNA strand under the action of a polymerase (e.g., DNA polymerase). The primer pairs of this invention relate to their application in the amplification of target nucleic acid sequences, for example, by polymerase chain reaction (PCR) or other conventional nucleic acid amplification methods.

[0078] The nucleic acid probes and primers of this invention hybridize with target DNA molecules under stringent conditions. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of DNA from transgenic plants in a sample. Polynucleotide molecules, also known as nucleic acid segments, or fragments thereof, can specifically hybridize with other nucleic acid molecules under certain conditions.

[0079] As used herein, two polynucleotide molecules are said to be capable of specifically hybridizing with each other if they can form antiparallel double-stranded nucleic acid structures. One nucleic acid molecule is said to be “complementary” to the other if it exhibits perfect complementarity. As used herein, a molecule is said to exhibit “perfect complementarity” when every nucleotide of one molecule is complementary to a nucleotide of the other molecule. Two molecules are said to be “minimally complementary” if they can hybridize with sufficient stability to remain bound together under at least conventional “low-tightness” conditions. Similarly, molecules are said to be “complementary” if they can hybridize with sufficient stability to remain bound together under conventional “high-tightness” conditions. Conventional tightness conditions are described in Sambrook et al., 1989, and Hames et al., Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985). Therefore, deviations from perfect complementarity are permissible, as long as such deviations do not completely preclude the ability of molecules to form double-stranded structures. In order for nucleic acid molecules to be used as primers or probes, the nucleic acid molecules only need to be sufficiently complementary in sequence so that they can form a stable double-stranded structure under the specific solvent and salt concentration used.

[0080] The term "amplifier" refers to the nucleic acid amplification product of a target nucleic acid sequence that is part of a nucleic acid template. In this invention, "amplifier" and "amplification product" are used interchangeably. For example, to determine whether a soybean plant is a genetically modified soybean containing the present invention... Whether soybeans produced through sexual hybridization or collected from fields contain genetically modified soybeans. Or whether soybean extracts, such as semolina, flour, or oil, contain genetically modified soybeans. DNA extracted from soybean plant tissue samples or extracts can be used to generate nucleic acid amplification methods for genetically modified soybean events using primer pairs. The presence of the foreign DNA is a diagnostic amplicon. The primer pair comprises a first primer derived from a flanking sequence in the plant genome adjacent to the insertion site of the foreign DNA, and a second primer derived from the inserted foreign DNA. The amplicon has a specific length and sequence, which is specific to the transgenic soybean event. It is also diagnostic. The length of the amplicon can be the binding length of the primer pair plus one nucleotide base pair, preferably plus about fifty nucleotide base pairs, more preferably plus about two hundred and fifty nucleotide base pairs, and most preferably plus about four hundred and fifty nucleotide base pairs or more.

[0081] The term "plant" is used in its broadest sense because it refers to organic matter and is intended to encompass eukaryotes belonging to the plant kingdom, including but not limited to vascular plants, vegetables, seeds, flowers, trees, herbs, shrubs, grasses, vines, ferns, mosses, fungi, and algae, as well as clones, suckers, and plant parts used for asexual reproduction (e.g., cuttings, tubes, seedlings, rhizomes, underground stems, clumps, crowns, bulbs, corms, tubers, rhizomes, plants / tissues produced in tissue culture, etc.). The term "plant" also encompasses the whole plant, the ancestor and descendants of plants and plant parts, including seeds, seedlings, stems, leaves, roots (including tubers), flowers, florets, fruits, pedicels, pedicels, stamens, anthers, stigmas, styles, ovaries, petals, sepals, carpels, root tips, root caps, root hairs, leaf hairs, seed hairs, pollen grains, microspores, cotyledons, hypocotyls, epicotyls, xylem, phloem, parenchyma, endosperm, companion cells, guard cells, and any other known organs, tissues, and cells of a plant, and tissues and organs therein that each contains the target gene / nucleic acid. The term "plant" also encompasses plant cells, suspension cultures, callus, embryos, meristematic regions, gametophytes, sporophytes, pollen, and microspores, again wherein each of the foregoing contains the target gene / nucleic acid.

[0082] The term "plant tissue" or "plant part" includes plant cells, protoplasts, plant tissue cultures, plant callus, plant masses, as well as plant embryos, pollen, ovules, seeds, leaves, stems, flowers, branches, seedlings, fruits, kernels, spikes, roots, root tips, anthers, etc.

[0083] The term “plant cell” should be understood as any cell that is derived from or found in a plant and is capable of forming, for example: undifferentiated tissues such as callus, differentiated tissues such as embryos, components of a plant, or seeds.

[0084] The genome of a plant, plant tissue, or plant cell as described in this invention refers to any genetic material within a plant, plant tissue, or plant cell, including the nucleus and plastid genome and the mitochondrial genome.

[0085] The term "expression cassette" refers to the complete element required to express a gene, which contains a nucleotide sequence that encodes a target protein and has a start codon and a stop codon.

[0086] The term “kit” refers to any article (e.g., packaging or container) that includes at least one device, and a kit may further include instructions for use, supplementary reagents and / or components or parts used in the methods or steps described herein.

[0087] In some embodiments, the kit further comprises one or more of the following: nucleic acid extraction reagents, reagents for nucleic acid amplification, positive controls, and negative controls.

[0088] The terms "DNA construct" and "recombinant vector" refer to vectors containing heterologous or recombinant nucleotide sequences. The term "vector" refers to a nucleic acid or polynucleotide fragment used to introduce or transfer one or more nucleic acids or one or more polynucleotides into target cells or tissues.

[0089] Vector transformation methods include Agrobacterium-mediated transformation, electroporation, microparticle bombardment, and polyethylene glycol-medium absorption to introduce recombinant plasmids into plants.

[0090] In this invention, plant transformation receptors include plant cells (including suspension cultured cells), protoplasts, callus tissue, hypocotyls, seeds, cotyledons, buds, and mature plants.

[0091] The scope of transgenic plants includes not only contemporary plants from which genes have been introduced, but also their clones and offspring (T1, T2, or subsequent generations). The scope of this invention also includes all mutants and variants of the aforementioned transgenic plants that exhibit characteristics of the primary transgenic plant after hybridization and fusion. The scope of this invention also includes parts of a plant, such as seeds, flowers, stems, fruits, leaves, roots, tubers, or rhizomes, derived from a plant that has been genetically modified in advance using the methods mentioned in this invention, or its offspring, and which must consist at least of a portion of genetically modified cells.

[0092] This invention relates to the identification of such flanking, junctional, and inserted sequences. The invention includes associated PCR primers and amplicones. The PCR analysis method of this invention, which analyzes amplicones across the inserted DNA and its boundaries, can be used to detect or identify commercially available transgenic soybean varieties or lines derived from the proprietary transgenic soybeans of this invention.

[0093] Sequence Summary

[0094] SEQ ID NO:1 Genetically Modified Soybean Incident The insertion site of the 5' transgenic fragment and 10 nucleotides on each side of the soybean genomic DNA;

[0095] SEQ ID NO:2 Genetically Modified Soybean Incident The insertion site of the 3' transgenic fragment and 10 nucleotides on each side of the soybean genomic DNA;

[0096] SEQ ID NO:3 Genetically Modified Soybean Incident The insertion site of the 5' transgenic fragment and 20 nucleotides on each side of the soybean genomic DNA;

[0097] SEQ ID NO:4 Genetically Modified Soybean Incident The insertion site of the 3' transgenic fragment and 20 nucleotides on each side of the soybean genomic DNA;

[0098] SEQ ID NO:5 Genetically Modified Soybean Incident The insertion site of the 5' transgenic fragment and 50 nucleotides on each side of the soybean genomic DNA;

[0099] SEQ ID NO:6 Genetically Modified Soybean Incident The insertion site of the 3' transgenic fragment and 50 nucleotides on each side of the soybean genomic DNA;

[0100] SEQ ID NO:7 Genetically Modified Soybean Incident A 1211-nucleotide sequence located near the insertion junction at the 5' end of the inserted sequence;

[0101] SEQ ID NO:8 Genetically Modified Soybean Incident A 1020-nucleotide sequence located near the insertion junction at the 3' end of the inserted sequence;

[0102] SEQ ID NO:9 Transgenic soybean event originating from the T-DNA region of vector pKG002016 Insert sequence;

[0103] SEQ ID NO:10 The entire T-DNA sequence, and the flanking soybean genome sequences at 5' and 3';

[0104] The sequence SEQ ID NO:11, located inside SEQ ID NO:7, is the amplicon of primers SEQ ID NO:13 and SEQ ID NO:14;

[0105] The sequence SEQ ID NO:12, located inside SEQ ID NO:8, is the amplicon of primers SEQ ID NO:15 and SEQ ID NO:16;

[0106] Primers on the 5' flanking genome sequence of SEQ ID NO:13;

[0107] Primers located on T-DNA that pair with SEQ ID NO:14 and SEQ ID NO:13;

[0108] Primers on the 3' flanking genome sequence of SEQ ID NO:15;

[0109] Primers located on T-DNA that pair with SEQ ID NO:16 and SEQ ID NO:15;

[0110] SEQ ID NO:17 Primer 1 for PCR detection of pat;

[0111] SEQ ID NO:18 Primer 2 for PCR detection of pat;

[0112] SEQ ID NO:19 Primer 1 for PCR detection of OsPPO2-k1;

[0113] SEQ ID NO:20 Primer 2 for PCR detection of OsPPO2-k1;

[0114] The nucleotide sequence of promoter P-CsVMV of SEQ ID NO:21;

[0115] SEQ ID NO:22 Nucleotide sequence of the coding region of the pat gene;

[0116] The nucleotide sequence of the terminator T-E9 in SEQ ID NO:23;

[0117] The nucleotide sequence of the promoter P-CLSV of SEQ ID NO:24;

[0118] SEQ ID NO:25 The nucleotide sequence of the TEV protease leader sequence L-TEV;

[0119] SEQ ID NO:26 Nucleotide sequence of the coding region of the OsPPO2-k1 gene;

[0120] The nucleotide sequence of the terminator T-NOS in SEQ ID NO:27;

[0121] SEQ ID NO:28 Primer 1 for RT-qPCR detection of pat copy number;

[0122] SEQ ID NO:29 Primer 2 for RT-qPCR detection of pat copy number;

[0123] SEQ ID NO:30 Primer 1 for RT-qPCR detection of OsPPO2-k1 copy number;

[0124] SEQ ID NO:31 Primer 2 for RT-qPCR detection of OsPPO2-k1 copy number;

[0125] SEQ ID NO:32 Primer 1 for RT-qPCR detection of LE1 standard;

[0126] SEQ ID NO:33 Primer 2 for RT-qPCR detection of LE1 standard. Attached Figure Description

[0127] Figure 1 is a schematic diagram of the pKG002016 carrier.

[0128] Figure 2 Schematic diagram of chromosome location for inserted sequence.

[0129] Figure 3 shows the detection of soybean plants. A schematic diagram of the binding site between the transgenic insertion sequence and the soybean genome, showing the nucleic acid sequence and its detection method.

[0130] Figure 4 Results of in vitro gene amplification after transformation. M: Marker; 1-3: 4: pat gene amplification results in plant plants; 5: pat gene amplification results in plasmid pKG002016; 6: pat gene amplification results in non-transgenic soybean GM11; 7: pat gene amplification results in non-transgenic soybean GM11; 8: 9: Amplification results of OsPPO2-k1 gene in plant; 10: Amplification results of OsPPO2-k1 gene in plasmid pKG002016; 11: Amplification results of OsPPO2-k1 gene in non-transgenic soybean GM11.

[0131] Figure 5 Specific PCR amplification results. M: Marker; T1: T1 generation single plant; T2: T2 generation single plant; T3: T3 generation single plant; P: plasmid pKG002016; N: non-transgenic soybean GM11.

[0132] Figure 6 Field effect of genetically modified soybeans sprayed with 3000g ai / ha glufosinate herbicide at the recommended field concentration 7 days later. CK is non-genetically modified soybean GM11.

[0133] Figure 7 Field effect of genetically modified soybeans sprayed with 600g ai / ha glufosinate-ammonium herbicide 7 days after the recommended field concentration. CK is non-genetically modified soybean GM11.

[0134] Figure 8 Field effect of genetically modified soybeans sprayed with the recommended field concentration of 60g ai / ha compound A for 7 days. CK is non-genetically modified soybean GM11. Detailed Implementation

[0135] The following embodiments are provided to provide those skilled in the art with a complete disclosure and description of how to prepare and use the invention, and these embodiments are not intended to limit the scope of the invention as viewed by the inventors, nor are they intended to represent or imply that the experiments described below are all or only the experiments performed. Those skilled in the art will understand that many variations and / or modifications can be made to the invention shown in specific aspects without departing from the spirit or scope broadly described herein. Therefore, aspects herein are to be considered illustrative rather than restrictive in all respects.

[0136] Example 1: Vector Cloning and Transformation

[0137] 1.1 Vector Cloning

[0138] The recombinant expression vector pKG002016 (as shown in Figure 1) was constructed using standard gene cloning techniques. The pKG002016 vector, with a size of 10709 bp, was artificially constructed based on pCAMBIA1300. The hygromycin resistance gene on the pCAMBIA1300 plasmid was removed by enzyme digestion, and two expression cassettes were then introduced using the Golden Gate method: the expression cassette for the glufosinate-tolerant herbicide gene pat and the expression cassette for the compound A-tolerant gene OsPPO2-k1. The first expression cassette consists of the cassava vein mosaic virus promoter P-CsVMV, operably linked to the glufosinate-resistant herbicide gene pat, and operably linked to the E9 terminator (T-E9); the second expression cassette consists of the peanut chlorotic stripe virus CLSV promoter (P-CLSV), operably linked to the TEV protease leader sequence (L-TEV) derived from tobacco etch virus, operably linked to the compound A resistance gene OsPPO2-k1, and operably linked to the Agrobacterium NOS terminator (T-NOS).

[0139] 1.2 Plant Transformation

[0140] Transformation was performed using the conventional Agrobacterium infection method, with explants being cotyledonary nodes of soybean GM11 (Tianlong 1). The Agrobacterium strain used was EHA105. Plump and healthy soybean seeds were selected, sterilized, and soaked overnight in sterile water. The roots and hypocotyl were removed using a sterile scalpel, leaving a 2-3 mm hypocotyl. The upper third of the cotyledons was removed, the seed coat was peeled off, and the hypocotyl was cut open to remove the plumule. The seeds were then infected with Agrobacterium and co-cultured in the dark for 3 days, followed by glufosinate selection to obtain resistant shoots. Different ratios of plant hormones were used to regulate the growth of the shoot clusters throughout the process. The resulting transgenic seedlings were transplanted into a greenhouse.

[0141] 1.3 Identification and Screening of Genetically Modified Events

[0142] Molecular analysis (including target gene copy number and insertion location), target trait (herbicide tolerance), and agronomic trait assessment were performed on 513 T0 plants. After removing abnormal transformants, transgenic soybean events were identified through screening. It is excellent, exhibiting single-copy transgenicity, good tolerance to glufosinate, succinate, and compound A, as well as agronomic traits.

[0143] Example 2: The Genetically Modified Soybean Incident Insertion sequence analysis

[0144] Genetically modified soybeans Flanking sequence analysis and specific PCR detection confirmed that the inserted sequence had been integrated into the chromosome. Using the sequenced soybean variety Williams82 as a reference genome, The T-DNA insertion site is located on chromosome 6 at chr6:1,658,798-1,658,822. It is a reverse insertion. During the integration of T-DNA into the genome, 25 bp of the genomic sequence is deleted, and 4 bp is inserted between the LB and the genomic sequence (as shown in Figure 2).

[0145] To clarify The insertion site and insertion sequence were determined, and primers were designed on the T-DNA to target it. Genome walking was performed, yielding a 5' flanking sequence of 976 bp and a 3' flanking sequence of 891 bp. PCR detection and sequencing results showed that the inserted sequence was completely identical to the T-DNA sequence derived from the pKG002016 vector, with an inserted sequence size of 4405 bp (SEQ ID NO: 9), including a 4 bp insertion at the LB end where it joins the genomic DNA. The inserted sequence comprises T-DNA fragments from both the RB and LB ends, and its structure is shown in Figure 3. The genetic elements contained in the inserted sequence are listed in Table 1.

[0146] Table 1. Genetic elements in the inserted sequence

[0147] Example 3: RT-qPCR for transgenic soybean events Detection

[0148] Genetically modified soybeans RT-qPCR copy number detection of exogenous gene insertion was performed in the T0 generation. Using leaf genomic DNA as a template, the exogenous genes pat and OsPPO2-k1 were quantified. A standard curve was first constructed using LE1 standards with known copy numbers. The Ct value showed a linear relationship with the logarithm of the initial template amount, with the x-axis representing the logarithm of the initial template amount and the y-axis representing the Ct value. Subsequently, the internal reference gene and exogenous gene in the transgenic sample were amplified to obtain the Ct value. The initial template amount of both was calculated using the standard curve. Since soybean is diploid, the exogenous gene / internal reference gene * 2 was used to obtain the copy number of the exogenous gene integrated into the soybean genome. The primers for the target gene RT-qPCR amplification are shown in Table 2.

[0149] Table 2. Primers for RT-qPCR amplification of target genes

[0150] Using the Roche 480II instrument, the system can automatically calculate the number of parameters for each gene in each sample based on the standard curve. The RT-qPCR results show that... The pat and OsPPO2-k1 genes are single-copy insertions.

[0151] Example 4: The Genetically Modified Soybean Incident 4.1 PCR Detection of Exogenous Genes

[0152] DNA was extracted and purified according to the People's Republic of China agricultural industry standard NY / T674. Genetically modified soybeans were used. Using plant leaf genomic DNA as a template, the exogenous genes pat and OsPPO2-k1 were amplified to determine whether the exogenous genes had been integrated into the soybean genome. The amplification primers are shown in Table 3.

[0153] Table 3. PCR primer information for detecting exogenous genes in transgenic soybeans

[0154] Figure 4 shows the results: the target gene amplification results indicate that vector pKG002016 and the transformant Both amplified clear bands of approximately 355 bp for pat and 752 bp for OsPPO2-k1. GM11 (control) did not amplify the exogenous gene band, proving that... The genetically modified soybean incident involved the exogenous genes pat and OsPPO2-k1.

[0155] 4.2 Genetically Modified Soybean Incident Specific detection

[0156] The conjugate sequence is a relatively short polynucleotide molecule that is a novel DNA sequence that, when detected in nucleic acid detection analysis, is relevant to genetically modified soybean events. The DNA is diagnostic. SEQ ID NO:1 conjugate sequence is for genetically modified soybean events. The T-DNA RB region insertion site and soybean genomic DNA are composed of 10 bp on each side of the insertion site. The conjugate sequence SEQ ID NO:2 represents a transgenic soybean event. The T-DNA LB region insertion site and soybean genomic DNA are combined, with 10 bp on each side of the insertion site. Longer or shorter polynucleotide conjugation sequences can be selected from SEQ ID NO:7 or SEQ ID NO:8. The conjugation sequences (5' conjugation region SEQ ID NO:1 and 3' conjugation region SEQ ID NO:2) are useful as DNA probes or as DNA primer molecules in DNA detection methods. Conjugation sequences SEQ ID NO:3-6 and SEQ ID NO:11-12 are also related to transgenic soybean events. The novel DNA sequence can also be used as a DNA probe or as a DNA primer molecule to detect genetically modified soybean events. The presence of DNA.

[0157] Specifically, transgenic soybean events were generated by designing primers such as SEQ ID NO:13-16 on the sequence of SEQ ID NO:7 or SEQ ID NO:8 using PCR. Diagnostic amplicon SEQ ID NO:11 and SEQ ID NO:12 (Table 4).

[0158] Table 4. PCR primer information for specific detection of transgenic soybeans

[0159] Figure 5 shows the results: The T1-T3 generations all amplified specific PCR fragments of the exogenous insert 5'flanking and 3'flanking, with sizes of 477bp and 343bp, respectively. No bands were amplified when transforming the receptor GM11 or the vector.

[0160] Example 5: The Genetically Modified Soybean Incident Detection of PAT and OsPPO2-k1 protein expression in the middle Soybean transformants were used to express PAT and OsPPO2-k1 proteins. The expression of these newly expressed proteins in various organs and tissues was analyzed using ELISA (enzyme-linked immunosorbent assay). Protein expression levels were measured in leaves, stems, and roots of soybean transformants at stage V3; leaves, stems, roots, pollen, flowers, and pods at stage R3; leaves, stems, roots, and seeds at stage R6; and leaves, stems, roots, and seeds at stage R8. Non-transgenic soybean GM11 was used as a control. (Transgenic soybean events...) The results of the detection of protein (PAT and OsPPO2-k1 protein) content are shown in Table 5.

[0161] Table 5. ELISA results of the transformant plants (ug / g fresh weight)

[0162] Note: NA indicates that the value is below the effective detection range of the standard curve and was not detected.

[0163] μg / g fwt is the content of the target protein per gram of fresh tissue.

[0164] ELISA results showed that PAT and OsPPO2-k1 proteins were present in transgenic soybeans. The target protein was detected in all tested tissue samples, but the concentration varied per gram in different tissues. Among the tested tissues, the content of PAT protein was highest in leaves at R8 stage and lowest in grains at R6 stage; the content of OsPPO2-k1 protein was highest in leaves at R8 stage and lowest in grains at R6 stage. The target protein was not present in any tissues of non-transgenic plants at different stages.

[0165] Example 6: The Genetically Modified Soybean Incident Tolerance to target herbicides

[0166] The tolerance of the transformants to the target herbicide was assessed under field conditions. The experimental design followed the guidelines in "Environmental Safety Testing of Transgenic Plants and Their Products - Herbicide-Tolerant Soybeans Part 1: Herbicide Tolerance" (Ministry of Agriculture Announcement No. 2031-1-2013). A randomized block design was used, with four replicates. Each plot had a 1-meter-wide isolation zone and an area of ​​20 m². 2 Soybeans were directly sown in the experiment, with a row spacing of 40 cm and a plant spacing of 15 cm. After emergence, field management was carried out according to local conventional cultivation methods. Foliar spraying was applied to the tested soybeans at the 3-4 compound leaf stage. The control GM11 was treated with 750, 1500, and 3000 g ai / ha of glufosinate, 300 and 600 g ai / ha of succinate, and 15, 30, and 60 g ai / ha of compound A, respectively.

[0167] After pesticide application, survival rate, soybean plant height (10 plants randomly selected), and pesticide damage symptoms (10 plants randomly selected) were investigated and recorded. Analysis of variance was used to compare the results of different treatments of transgenic soybeans. The differences in seedling rate, damage rate and plant height of the corresponding non-transgenic recipient soybean GM11 were investigated, and the grading of herbicide damage symptoms was carried out in accordance with GB / T 17980.125.

[0168] The results showed that all non-GMO control soybean GM11 plants died 7 days after application of 750g ai / ha glufosinate herbicide. (GMO soybeans...) It can tolerate 3000 g ai / ha glufosinate herbicide. After 7, 14 and 28 days of spraying 3000 g ai / ha glufosinate herbicide, there were no significant differences in seedling rate, damage rate, plant height compared with the control group without herbicide spraying and the plants treated with 750 and 1500 g ai / ha doses (Figure 6).

[0169] All non-GMO control soybean GM11 plants died 7 days after being sprayed with 300g / ha of aliphatic glufosinate-ammonium herbicide. It can tolerate 600g ai / ha glufosinate-ammonium herbicide. After 7, 14 and 28 days of spraying 600g ai / ha glufosinate-ammonium herbicide, there were no significant differences in seedling rate, damage rate, plant height compared with the control group without herbicide spraying and the plants treated with 300g ai / ha (Figure 7).

[0170] Meanwhile, all non-GMO control soybean GM11 plants died 7 days after application of 15g ai / ha compound A. (GMO soybeans) It can tolerate 60g ai / ha compound A. After 7, 14 and 28 days of spraying 60g ai / ha compound A, there were no significant differences in seedling rate, damage rate, plant height compared with the control group without herbicide spraying and plants treated with 15 and 30g ai / ha doses (Figure 8).

[0171] Furthermore, this invention has demonstrated through numerous experiments that genetically modified soybeans at the registered dosage... For other PPO inhibitor herbicides such as flufenoxuron, ethoxyflufenoxuron, ethoxyflufenoxuron, quizalofop-P-ethyl, trifluralin, cyclopyridone, mesotrione, pyrimisulfuron, propyzoxystrobin, cyprodinil, methyl methazine, pyrazosulfuron, oxadiazon, propyzoxystrobin, cyclopyridone, etc. Flupyrimethanil, trifluralin, cyclopyranil, and eppyrifenacil also exhibited high tolerability, excellent safety, and selectivity.

[0172] Example 7: The Genetically Modified Soybean Incident Observation and analysis of the main agronomic traits of its receptors

[0173] On the genetically modified soybean incident The growth period, plant height, number of pods per plant, grain weight per plant, 100-seed weight, number of branches on the main stem, and number of nodes on the main stem of the transgenic soybean and the recipient variety were investigated. The results showed that the transgenic soybean and the recipient variety were basically the same in terms of agronomic traits, and no obvious changes in traits were found.

[0174] In summary, this invention relates to the genetically modified soybean incident. It exhibits high tolerance to glufosinate, succinate, and compound A, with no impact on agronomic traits such as yield. Furthermore, the detection method can accurately and rapidly identify whether genetically modified soybeans are present in biological samples. DNA molecules.

[0175] Corresponding to the genetically modified soybean incident Representative seed samples were deposited on October 12, 2024, at the China Center for Type Culture Collection (CCTCC, address: No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China), classified as soybean (Glycine max L.), with accession number CCTCC NO: P202427. The deposited material will be held at the collection for 30 years.

[0176] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A nucleic acid sequence, characterized in that, The nucleic acid sequence includes: or (b) A nucleic acid sequence complementary to (a); Preferably, the nucleic acid sequence is derived from the transgenic soybean event KA-6G2. 16-7 or its derivatives; More preferably, the nucleic acid sequence is used to diagnose soybean event KA-6G2. Amplicon present in 16-7 or its derivatives.

2. A DNA construct comprising two expression cassettes, wherein, a) The first expression cassette contains, in an operable link, a promoter P-CsVMV with nucleic acid sequence as shown in SEQ ID NO:21, a pat gene coding region with nucleic acid sequence as shown in SEQ ID NO:22, and a terminator T-E9 with nucleic acid sequence as shown in SEQ ID NO:23 to terminate the expression of the gene. b) The second expression cassette contains, in an operable link, a promoter P-CLSV as shown in SEQ ID NO:24, a TEV protease leader sequence L-TEV as shown in SEQ ID NO:25, an OsPPO2-k1 gene coding region as shown in SEQ ID NO:26, and a terminator T-NOS as shown in SEQ ID NO:27 to terminate the expression of the gene. Preferably, the nucleic acid sequence of the DNA construct includes SEQ ID NO:

9.

3. A diagnostic method for genetically modified soybean events KA-6G2 16-7 A DNA probe present, wherein the DNA probe is of sufficient length to bind the nucleic acid sequence of claim 1, wherein the DNA probe hybridizes to the nucleic acid sequence of claim 1 under strict hybridization conditions and does not hybridize to other nucleic acid sequences.

4. A DNA primer pair comprising a first DNA primer and a second DNA primer different from the first DNA primer, said DNA primer pair being associated with the transgenic soybean event KA-6G2. Samples 16-7 were used together in an amplification reaction to generate KA-6G2, a diagnostic marker for genetically modified soybean events in the samples. 16-7 amplicons exist; Preferably, the amplicon comprises the nucleic acid sequence of claim 1; More preferably, the primer pair includes SEQ ID NO:13 and SEQ ID NO:14, or SEQ ID NO:15 and SEQ ID NO:

16.

5. A method for detecting genetically modified soybeans in samples, KA-6G2 The kit of 16-7 contains at least one DNA probe as described in claim 3 or a DNA primer pair as described in claim 4.

6. A detection method for genetically modified soybeans in samples, KA-6G2 Methods for the presence of DNA in 16-7, including: a) Contact the sample to be tested with at least one DNA primer pair as described in claim 4; b) Perform an amplification reaction sufficient to produce DNA amplicon; as well as, c) Detect the presence of the DNA amplicon in the reaction; The amplicon sequence was derived from the transgenic soybean event KA-6G2. 16-7; Preferably, the DNA amplicon comprises the nucleic acid sequence of claim 1; Or the method may include: a) Bring the sample to be tested into contact with the probe described in claim 3; b) Hybridize the sample to be tested and the probe under stringent hybridization conditions; and c) Detect the hybridization between the sample to be tested and the probe; The detection method described above can diagnose the genetically modified soybean event KA-6G2 in the sample. DNA of 16-7 is present.

7. A method for protecting soybean plants from damage caused by herbicides, characterized in that, The invention includes applying a herbicide containing an effective dose of a glutamine synthase inhibitor and / or a protoporphyrinogen oxidase inhibitor to a field where at least one transgenic soybean plant is planted, wherein the transgenic soybean plant contains, in sequence, the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:10 (positions 1049-5292) and SEQ ID NO:2, or SEQ ID NO:3, SEQ ID NO:10 (positions 1049-5292) and SEQ ID NO:4, or SEQ ID NO:5, SEQ ID NO:10 (positions 1049-5292) and SEQ ID NO:6, or the genome of the transgenic soybean plant contains SEQ ID NO:10; the transgenic soybean plant is tolerant to the glutamine synthase inhibitor and / or protoporphyrinogen oxidase inhibitor herbicide.

8. A method for controlling weeds in soybean planting fields, characterized in that, The invention includes applying a herbicide containing an effective dose of a glutamine synthase inhibitor and / or a protoporphyrinogen oxidase inhibitor to a field where at least one transgenic soybean plant is planted, wherein the transgenic soybean plant contains, in sequence, the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:10 (positions 1049-5292) and SEQ ID NO:2, or SEQ ID NO:3, SEQ ID NO:10 (positions 1049-5292) and SEQ ID NO:4, or SEQ ID NO:5, SEQ ID NO:10 (positions 1049-5292) and SEQ ID NO:6, or the genome of the transgenic soybean plant contains SEQ ID NO:10; the transgenic soybean plant is tolerant to the glutamine synthase inhibitor and / or protoporphyrinogen oxidase inhibitor herbicide.

9. A method for cultivating soybean plants tolerant to herbicides of glutamine synthase inhibitors and / or protoporphyrinogen oxidase inhibitors, characterized in that, include: Plant at least one soybean seed, wherein the genome of the soybean seed contains a specific region of nucleic acid sequence, wherein the specific region of nucleic acid sequence sequentially comprises SEQ ID NO:1, the nucleic acid sequence of SEQ ID NO:10 from position 1049 to 5292 and SEQ ID NO:2, or SEQ ID NO:3, the nucleic acid sequence of SEQ ID NO:10 from position 1049 to 5292 and SEQ ID NO:4, or SEQ ID NO:5, the nucleic acid sequence of SEQ ID NO:10 from position 1049 to 5292 and SEQ ID NO:6, or the specific region of nucleic acid sequence comprises SEQ ID NO:10; The soybean seeds are then allowed to grow into soybean plants. The soybean plants were sprayed with an effective dose of a glutamine synthase inhibitor herbicide and / or a protoporphyrinogen oxidase inhibitor herbicide, and the plants with reduced plant damage compared to other plants that do not have the nucleic acid sequence of the specific region were harvested.

10. A method for producing soybean plants resistant to glutamine synthase inhibitor herbicides and / or protoporphyrinogen oxidase inhibitor herbicides, characterized in that, This includes hybridizing soybean plants whose genomes sequentially contain the nucleic acid sequences of SEQ ID NO:1, SEQ ID NO:10 positions 1049-5292, and SEQ ID NO:2; or SEQ ID NO:3, SEQ ID NO:10 positions 1049-5292, and SEQ ID NO:4; or SEQ ID NO:5, SEQ ID NO:10 positions 1049-5292, and SEQ ID NO:6, with another soybean plant to produce a large number of progeny plants; selecting progeny plants whose genomes contain a specific region of nucleic acid sequences, wherein the specific region of nucleic acid sequences sequentially contains the nucleic acid sequences of SEQ ID NO:1, SEQ ID NO:10 positions 1049-5292, and SEQ ID NO:2; or SEQ ID NO:3, SEQ ID NO:10 positions 1049-5292, and SEQ ID NO:4; or SEQ ID NO:5, SEQ ID NO:10 positions 1049-5292, and SEQ ID NO:6, and SEQ ID NO:

6. NO:6, or the nucleic acid sequence of the specific region contains SEQ ID NO:10, and the progeny plants have tolerance to glutamine synthase inhibitor herbicides and / or protoporphyrinogen oxidase inhibitor herbicides; Preferably, the method includes: administering genetically modified soybean KA-6G2, which is tolerant to glutamine synthase inhibitor herbicides and / or protoporphyrinogen oxidase inhibitor herbicides. 16-7 The first parent soybean plant was sexually crossed with the second parent soybean plant lacking tolerance to herbicides such as glutamine synthase inhibitors and / or protoporphyrinogen oxidase inhibitors, thereby producing a large number of offspring plants; The progeny plants were treated with glutamine synthase inhibitor herbicides and / or protoporphyrinogen oxidase inhibitor herbicides; Select the progeny plants that are resistant to glutamine synthase inhibitor herbicides and / or protoporphyrinogen oxidase inhibitor herbicides; 11. A method for improving the tolerance of soybean plants, the method comprising: a) Construct the DNA construct as described in claim 2; b) Insert the DNA construct into the genome of soybean cells; c) regenerate the soybean cells into a soybean plant; and d) Select soybean plants containing the DNA construct; Preferably, improving the tolerance of soybean plants includes their resistance to an effective amount of at least one herbicide, said herbicide preferably a glutamine synthase inhibitor and / or a protoporphyrinogen oxidase inhibitor.

12. A phenomenon arising from the KA-6G2 genetically modified soybean event. A composition of 16-7 or its derivatives, characterized in that, The composition is soybean oil, soybean protein, soybean flour, or soybean starch.

13. A phenomenon arising from the KA-6G2 genetically modified soybean event. Agricultural products or commodities of varieties 16-7 or their derivatives, characterized in that, The agricultural products or commodities mentioned are soybean oil, soybean protein, soybean flour, soybean starch, soybean meal, soybean flakes, soybean hulls, soy milk, soybean cheese, soybean wine, animal feed containing soybeans, paper containing soybeans, cheese containing soybeans, soybean biomass, or fuel products produced using soybean plants and parts of soybean plants.

14. A plant cell, plant part, plant, seed or inanimate plant material comprising the nucleic acid sequence as described in claim 1.

15. The nucleic acid sequence according to claim 1, the probe according to claim 3, the primer pair according to claim 4, the kit according to claim 5, the method according to claim 6, the method according to claim 10, the composition according to claim 12, or the agricultural product or commodity according to claim 13, characterized in that, The genetically modified soybean incident KA-6G2 Representative samples from 16-7 are deposited in seed form at the China Center for Type Culture Collection, accession number CCTCC NO: P202427.

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