Transgenic zea mays l. event ka-1g2ø15-1 and detection method therefor

Abstract

The present invention relates to a transgenic Zea mays L. event KA-1G2Ø15-1 and a related nucleic acid sequence, a detection method, a Zea mays L. plant, and a cultivation method. The Zea mays L. plant has herbicide tolerance.

Description

Genetically modified maize incident KA-1GKA-1G2Ø15-1 and its detection method Technical Field

[0001] This invention relates to the KA-1G2 transgenic maize incident. 15-1 also relates to a method for detecting KA-1G2 in maize plants. Nucleic acid sequence of 15-1 and its detection method. Background Technology

[0002] Maize (Zea mays L.) is an important food and feed crop, and the world's highest-yielding crop. It is rich in protein, fat, vitamins, trace elements, and fiber, and has enormous potential for developing highly nutritious and biologically functional foods. However, due to its complex genetic makeup and abundant variations, conventional breeding methods suffer from drawbacks such as excessively long cycles, high coefficients of variation, and negative impacts on offspring growth and development. Modern bio-breeding techniques have not only overcome these shortcomings but also improved breeding speed and quality. Currently, biotechnology is widely used in maize to improve its agronomic traits and quality.

[0003] Herbicide tolerance is an important agronomic trait in maize production, especially tolerance to glufosinate and PPO inhibitor herbicides. Maize 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 maize 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 maize event KA-1G2. 15-1 and KA-1G2 for detecting genetically modified maize events Nucleic acid sequence and detection method of 15-1.

[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] or

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

[0010] In one specific embodiment, the nucleic acid sequence is derived from the transgenic maize event KA-1G2. 15-1.

[0011] In another specific embodiment, the nucleic acid sequence is used to diagnose maize event KA-1G2. The amplicon present in 15-1.

[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 OsAct1 with nucleic acid sequence as shown in SEQ ID NO:31, a chloroplast localizing peptide CTP-MDH with nucleic acid sequence as shown in SEQ ID NO:32, a chloroplast localizing peptide CTP-OsPPO2 with nucleic acid sequence as shown in SEQ ID NO:33, a gene coding region of OsPPO2-k1 with nucleic acid sequence as shown in SEQ ID NO:34, and a terminator T-NOS for terminating gene expression with nucleic acid sequence as shown in SEQ ID NO:35;

[0014] b) The second expression cassette contains, in an operative linker, a promoter P-E35S with a nucleic acid sequence as shown in SEQ ID NO:36, a pat gene coding region with a nucleic acid sequence as shown in SEQ ID NO:37, and a terminator T-35S with a nucleic acid sequence as shown in SEQ ID NO:38 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 transgenic maize event KA-1G2. 15-1 A DNA probe that exists, 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, wherein the DNA primer pair is associated with the transgenic maize event KA-1G2. Samples 15-1 were used together in an amplification reaction to generate KA-1G2 for diagnosing transgenic maize events in the samples. The amplicon present in 15-1.

[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, SEQ ID NO:15 and SEQ ID NO:16, or SEQ ID NO:39 and SEQ ID NO:40.

[0020] The present invention also provides a method for detecting transgenic maize event KA-1G2 in samples. The kit of 15-1 contains at least one of the DNA primer pairs described above.

[0021] This invention also provides a method for detecting transgenic maize event KA-1G2 in samples. The method for the presence of DNA in 15-1 includes:

[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 was derived from the transgenic maize event KA-1G2. 15-1;

[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 detection method described above can diagnose the KA-1G2 transgenic maize event in the sample. DNA of 15-1 is present.

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

[0032] The present invention also provides a DNA detection kit comprising at least one DNA molecule, said DNA molecule comprising a continuous sequence or a complementary sequence thereof from 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 or SEQ ID NO:8, which can be used as a detection kit for transgenic maize event KA-1G2. 15-1 or its descendants have one of the specific DNA primers or probes.

[0033] In one specific embodiment, when the DNA molecule is used as a probe, it further includes the consecutive sequences or complementary sequences of SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:42;

[0034] In another specific embodiment, the probe is SEQ ID NO:41 or its complementary sequence.

[0035] The present invention also provides a method for protecting maize 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 maize plant is planted, wherein the transgenic maize plant contains, in sequence, the nucleic acid sequences of SEQ ID NO:1, SEQ ID NO:10 positions 995-5952 and SEQ ID NO:2, or SEQ ID NO:3, SEQ ID NO:10 positions 995-5952 and SEQ ID NO:4, or SEQ ID NO:5, SEQ ID NO:10 positions 995-5952 and SEQ ID NO:6, or the genome of the transgenic maize plant contains SEQ ID NO:10; wherein the transgenic maize plant is tolerant to the glutamine synthase inhibitor and / or protoporphyrinogen oxidase inhibitor herbicide.

[0036] The present invention also provides a method for controlling weeds in a field planted with maize, comprising applying a herbicide containing an effective dose of a glutamine synthase inhibitor and / or a protoporphyrinogen oxidase inhibitor to a field planted with at least one transgenic maize plant, wherein the transgenic maize plant contains, in sequence, the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:10 (positions 995-5952) and SEQ ID NO:2, or SEQ ID NO:3, SEQ ID NO:10 (positions 995-5952) and SEQ ID NO:4, or SEQ ID NO:5, SEQ ID NO:10 (positions 995-5952) and SEQ ID NO:6, or the genome of the transgenic maize plant contains SEQ ID NO:10; the transgenic maize plant is tolerant to the glutamine synthase inhibitor and / or protoporphyrinogen oxidase inhibitor herbicide.

[0037] The present invention also provides a method for culturing maize plants tolerant to glutamine synthase inhibitor herbicides and / or protoporphyrinogen oxidase inhibitor herbicides, comprising: planting at least one maize seed, wherein the genome of the maize 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 positions 995-5952 and SEQ ID NO:2, or SEQ ID NO:3, the nucleic acid sequence of SEQ ID NO:10 positions 995-5952 and SEQ ID NO:4, or SEQ ID NO:5, the nucleic acid sequence of SEQ ID NO:10 positions 995-5952 and SEQ ID NO:6, or the specific region of nucleic acid sequence comprises SEQ ID NO:10;

[0038] The corn seeds are allowed to grow into corn plants;

[0039] The corn 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.

[0040] This invention also provides a method for producing maize plants resistant to glutamine synthase inhibitor herbicides and / or protoporphyrinogen oxidase inhibitor herbicides, comprising: hybridizing a maize plant whose genome sequentially contains the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:10 positions 995-5952 and SEQ ID NO:2, or SEQ ID NO:3, SEQ ID NO:10 positions 995-5952 and SEQ ID NO:4, or SEQ ID NO:5, SEQ ID NO:10 positions 995-5952 and SEQ ID NO:6 with another maize 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 995-5952 and SEQ ID NO:2, or SEQ ID NO:3, SEQ ID NO:10 positions 995-5952 and SEQ ID NO:4, or SEQ ID NO:6; The nucleic acid sequence of SEQ ID NO:5, SEQ ID NO:10 from position 995 to 5952 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.

[0041] In one specific embodiment, the method includes: ka-1G2 transgenic maize event that is tolerant to glutamine synthase inhibitor herbicides and / or protoporphyrinogen oxidase inhibitor herbicides. 15-1 The first parent maize plant was sexually crossed with the second parent maize plant, which lacked tolerance to herbicides such as glutamine synthase inhibitors and / or protoporphyrinogen oxidase inhibitors, to produce a large number of offspring plants.

[0042] The progeny plants were treated with glutamine synthase inhibitor herbicides and / or protoporphyrinogen oxidase inhibitor herbicides;

[0043] The progeny plants were selected from those resistant to herbicides of glutamine synthase inhibitors and / or protoporphyrinogen oxidase inhibitors.

[0044] The present invention also provides a method for improving the tolerance of maize plants, the method comprising:

[0045] a) Construct the DNA construct as described above;

[0046] b) Insert the DNA construct into the genome of maize cells;

[0047] c) regenerate the corn cells into a corn plant; and

[0048] d) Select a maize plant containing the DNA construct.

[0049] In one specific embodiment, improving maize plant tolerance includes its resistance to an effective amount of at least one herbicide, preferably a glutamine synthase inhibitor herbicide and / or a protoporphyrinogen oxidase inhibitor herbicide.

[0050] This invention also provides a method derived from the transgenic maize event KA-1G2. The composition of 15-1, wherein the composition is corn flour, cornmeal, corn oil, corn shreds or corn starch.

[0051] This invention also provides a method derived from the transgenic maize event KA-1G2. 15-1 refers to agricultural products or commodities, wherein the agricultural products or commodities are corn flour, cornmeal, corn oil, corn starch, corn gluten, corn cakes, cosmetics or fillers.

[0052] The present invention also provides a plant cell, plant part, plant or seed comprising the aforementioned nucleic acid sequence.

[0053] The present invention also provides an inanimate plant material comprising the aforementioned nucleic acid sequence.

[0054] Some of the terms used in this specification are defined as follows.

[0055] The term "maize" as used in this invention refers to maize (Zea mays) and includes all plant species that can interbreed with maize, including wild maize species.

[0056] In this invention, "comprising" means "including but not limited to".

[0057] 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, succinate, and dipropylphosphonic acid; 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.

[0058] 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.

[0059] PPO-inhibiting herbicides and / or other herbicidal compounds having carboxyl groups, as described herein, may 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.

[0060] 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.

[0061] 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).

[0062] 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).

[0063] 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).

[0064] Thiadiazole herbicides include, but are not limited to, methyl methacrylate (CAS NO: 117337-19-6), methacrylate (CAS NO: 149253-65-6), and thiamethoxam (CAS NO: 123249-43-4).

[0065] Oxadiazole herbicides include, but are not limited to, propyzinoxadiazon (CAS NO: 39807-15-3) and oxadiazon (CAS NO: 19666-30-9).

[0066] 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).

[0067] Oxazolidinone herbicides include, but are not limited to, cyclooxadiazon (CAS NO: 110956-75-7).

[0068] 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: 948893-00-3), 3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]ethyl acetate (CAS: 353292-31-6, S-3100), 1,5-dimethyl-6-thio-3-(2,2,7-trifluoro-3-oxo-4-(propyl-2- 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-pyrimidine-2,4-dione (CAS: 1258836-72-4), 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-pyrimidine-2,4-dione (CAS: 1304113-05-0), 3-[7-chloro-5-fluoro-2-(trifluoromethyl)-1H-benzimidazol-4-yl]-1-methyl-6-(trifluoromethyl)-1H-pyrimidin-2,4-dione (CAS: 212754-02-4), phenylpyridine derivatives disclosed in WO2016 / 120116, and benzoxazinone derivatives disclosed in EP09163242.2.

[0069] 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.

[0070] 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."

[0071] 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.

[0072] 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).

[0073] 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-crossed offspring) and a parental line not containing the inserted DNA.

[0074] 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.

[0075] 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.

[0076] 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 those found in natural organisms. "Junction DNA" or "junction sequence" refers to DNA containing the junction site.

[0077] A “joining sequence” spans the point where the inserted genomic DNA joins with DNA from the maize natural 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 maize event 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.

[0078] 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 bound to a strand from the transgenic maize event KA-1G2. One strand of the 15-1 genome is complementary, regardless of whether the genomic DNA originated from the transgenic maize event KA-1G2. 15-1 or the seed may have originated from the genetically modified corn incident KA-1G2 15-1 plants, seeds, or extracts. 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.

[0079] 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.

[0080] 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.

[0081] 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 as to form a stable double-stranded structure under the specific solvent and salt concentration used.

[0082] 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 maize plant is infected with the transgenic maize KA-1G2 of this invention... 15-1 Whether maize samples produced through sexual hybridization or collected from the field contain genetically modified maize (Event KA-1G2) 15-1, or whether corn extracts, such as coarse flour, powder, or oil, contain genetically modified corn (Event KA-1G2) 15-1, DNA extracted from maize plant tissue samples or extracts can be used to generate nucleic acid amplification methods for transgenic maize event KA-1G2 using primer pairs. The presence of DNA from 15-1 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 corresponding to the transgenic maize event KA-1G2. 15-1 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] Vector transformation methods include Agrobacterium-mediated transformation, electroporation, microparticle bombardment, and polyethylene glycol-medium absorption to introduce recombinant plasmids into plants.

[0092] In this invention, plant transformation receptors include plant cells (including suspension cultured cells), protoplasts, callus tissue, hypocotyls, seeds, cotyledons, buds, and mature plants.

[0093] 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.

[0094] 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 maize varieties or lines derived from the proprietary transgenic maize of this invention.

[0095] Sequence Summary

[0096] SEQ ID NO:1 Genetically Modified Corn Incident KA-1G2 The insertion site of the 5' transgenic fragment in 15-1 and 10 nucleotides on each side of the maize genomic DNA;

[0097] SEQ ID NO:2 Genetically Modified Corn Incident KA-1G2 The insertion site of the 3' transgenic fragment in 15-1 and 10 nucleotides on each side of the maize genomic DNA;

[0098] SEQ ID NO:3 Genetically Modified Corn Incident KA-1G2 The insertion site of the 5' transgenic fragment in 15-1 and 20 nucleotides on each side of the maize genomic DNA;

[0099] SEQ ID NO:4 Genetically Modified Corn Incident KA-1G2 The insertion site of the 3' transgenic fragment in 15-1 and 20 nucleotides on each side of the maize genomic DNA;

[0100] SEQ ID NO:5 Genetically Modified Corn Incident KA-1G2 The insertion site of the 5' transgenic fragment in 15-1 and 50 nucleotides on each side of the maize genomic DNA;

[0101] SEQ ID NO:6 Genetically Modified Corn Incident KA-1G2 The insertion site of the 3' transgenic fragment in 15-1 and 50 nucleotides on each side of the maize genomic DNA;

[0102] SEQ ID NO:7 is the genetically modified corn incident KA-1G2 15-1 is a 987-nucleotide sequence located near the insertion junction at the 5' end of the inserted sequence;

[0103] SEQ ID NO:8 is the genetically modified corn incident KA-1G2 15-1 is a 915-nucleotide sequence located near the insertion junction at the 3' end of the inserted sequence;

[0104] SEQ ID NO:9 Transgenic maize event KA-1G2 derived from the T-DNA region of vector pKG002015. 15-1 Insertion sequence;

[0105] SEQ ID NO:10 The entire T-DNA sequence, and the flanking maize genome sequences at 5' and 3';

[0106] 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;

[0107] 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;

[0108] Specific PCR primers for the flanking genome sequence of SEQ ID NO:135';

[0109] Specific PCR primers located on T-DNA, paired with SEQ ID NO:14 and SEQ ID NO:13;

[0110] Specific PCR primers on the flanking genome sequence of SEQ ID NO:153';

[0111] Specific PCR primers located on T-DNA, paired with SEQ ID NO:16 and SEQ ID NO:15;

[0112] SEQ ID NO:17 Primer 1 for RT-qPCR detection of OsPPO2-k1 copy number;

[0113] SEQ ID NO:18 Primer 2 for RT-qPCR detection of OsPPO2-k1 copy number;

[0114] SEQ ID NO:19 Primer 1 for RT-qPCR detection of pat copy number;

[0115] SEQ ID NO:20 Primer 2 for RT-qPCR detection of pat copy number;

[0116] SEQ ID NO:21 Primer 1 for RT-qPCR detection of HMGA standards;

[0117] SEQ ID NO:22 Primer 2 for RT-qPCR detection of HMGA standard;

[0118] SEQ ID NO:23 Probe primer 1 for Southern hybridization detection of OsPPO2-k1;

[0119] SEQ ID NO:24 Probe primer 2 for Southern hybridization detection of OsPPO2-k1;

[0120] SEQ ID NO:25 Probe primer 1 for pat in Southern hybridization detection;

[0121] SEQ ID NO:26 Probe primer 2 for pat in Southern hybridization detection;

[0122] SEQ ID NO:27 Primer 1 for PCR detection of OsPPO2-k1;

[0123] SEQ ID NO:28 Primer 2 for PCR detection of OsPPO2-k1;

[0124] SEQ ID NO:29 Primer 1 for PCR detection of pat;

[0125] SEQ ID NO:30 Primer 2 for PCR detection of pat;

[0126] SEQ ID NO:31 is the nucleotide sequence of the promoter OsAct1;

[0127] SEQ ID NO:32 is the nucleotide sequence of the chloroplast localization peptide CTP-MDH;

[0128] SEQ ID NO:33 is the nucleotide sequence of the chloroplast localization peptide CTP-OsPPO2;

[0129] SEQ ID NO:34 is the nucleotide sequence of the coding region of the OsPPO2-k1 gene;

[0130] SEQ ID NO:35 is the nucleotide sequence of the terminator T-NOS;

[0131] SEQ ID NO:36 is the nucleotide sequence of promoter P-E35S;

[0132] SEQ ID NO:37 is the nucleotide sequence of the coding region of the pat gene;

[0133] SEQ ID NO:38 is the nucleotide sequence of the terminator T-35S;

[0134] Specific RT-qPCR primers on the flanking genome sequence of SEQ ID NO:395';

[0135] Specific RT-qPCR primers located on T-DNA, paired with SEQ ID NO:40 and SEQ ID NO:39;

[0136] SEQ ID NO:41 TaqMan probe for RB-end specific RT-qPCR;

[0137] The sequence SEQ ID NO:42, located inside SEQ ID NO:7, is the amplicon of primers SEQ ID NO:39 and SEQ ID NO:40;

[0138] Primer 1 for specific RT-qPCR detection of the zSSIIb gene (SEQ ID NO:43);

[0139] Primer 2 for specific RT-qPCR detection of the zSSIIb gene (SEQ ID NO:44);

[0140] SEQ ID NO:45 TaqMan probe for specific RT-qPCR detection of the zSSIIb gene. Attached Figure Description

[0141] Figure 1 is a schematic diagram of the pKG002015 carrier.

[0142] Figure 2KA-1G2 Schematic diagram of chromosome location for the 15-1 inserted sequence.

[0143] Figure 3 shows the detection of KA-1G2 in maize plants. A schematic diagram of the binding site between the transgenic insertion sequence of 15-1 and the maize genome and its detection method.

[0144] Figure 4KA-1G2 Results of in vitro gene amplification after transformation in 15-1. M: Marker; 1-4: KA-1G2 Results of OsPPO2-k1 and pat gene amplification in plant 15-1; P: plasmid pKG002015 results of OsPPO2-k1 and pat gene amplification; N: results of OsPPO2-k1 and pat gene amplification in non-transgenic maize receptor.

[0145] Figure 5KA-1G2 15-1 Specific PCR amplification results. M: Marker; T1-T4: Amplification results of LB and RB end specific PCR of T1-T4 generation single plants; P: Amplification results of plasmid pKG002015 specific PCR; N: Amplification results of non-transgenic maize receptor specific PCR.

[0146] Figure 6 KA-1G2 Field effect of 15-1 transgenic maize treated with 90g ai / ha compound A. CK is the non-transgenic maize recipient.

[0147] Figure 7KA-1G2 Field effect of 15-1 genetically modified maize sprayed with 1200g ai / ha glufosinate herbicide. CK is a non-genetically modified maize recipient. Detailed Implementation

[0148] 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, this document is to be considered illustrative rather than restrictive in all aspects.

[0149] Example 1: Vector Cloning and Transformation

[0150] 1.1 Vector Cloning

[0151] The recombinant expression vector pKG002015 (as shown in Figure 1) was constructed using standard gene cloning techniques. The pKG002015 vector, with a size of 11426 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 compound A resistance gene OsPPO2-k1 and the expression cassette for the glufosinate-ammonium herbicide resistance gene pat. The first expression cassette consists of the rice actin1 promoter OsAct1, operably linked to the chloroplast-localizing peptides CTP-MDH and CTP-OsPPO2, operably linked to the compound A resistance gene OsPPO2-k1, and operably linked to the NOS terminator (T-NOS); the second expression cassette consists of the cauliflower mosaic virus 35S promoter (P-E35S), operably linked to the glufosinate-resistant herbicide gene pat, and operably linked to the cauliflower mosaic virus 35S terminator (T-35S).

[0152] 1.2 Plant transformation and screening of transgenic plants

[0153] KA-1G2 Herbicide-tolerant maize (15-1) was obtained by introducing the compound A tolerance gene OsPPO2-k1 and the glufosinate-tolerant herbicide gene pat into a maize recipient via Agrobacterium-mediated embryo transformation. A total of 613 independent transgenic T0 plants were generated. The presence of the herbicide-tolerant genes OsPPO2-k1 and pat in the transgenic maize plants was detected by PCR and Southern hybridization analysis, and the copy number of the transgenes in the compound A and glufosinate-tolerant lines was characterized. Through screening, the maize event KA-1G2 was selected. 15-1 is superior, possessing a single-copy transgenic structure, good compound A and tolerance to glufosinate and succinate herbicides, as well as agronomic traits.

[0154] Example 2: The KA-1G2 transgenic maize incident 15-1 Insertion Sequence Analysis

[0155] Genetically modified corn KA-1G2 15-1 Flanking sequence analysis and specific PCR detection confirmed that the inserted sequence had been integrated into the maize chromosome. Using the sequenced non-transgenic recipient maize genome as a reference, KA-1G2... The 15-1 T-DNA insertion site was located on chromosome 9 at chr9:44,198,665-44,198,695. The sequencing results of the corresponding site in non-transgenic maize were used as a control for comparison analysis. During the integration of T-DNA into the genome, 30 bp of genomic sequence was deleted (as shown in Figure 2).

[0156] To clarify KA-1G2 Based on the insertion site and sequence of 15-1, primers were designed on T-DNA for KA-1G2. Genome walking was performed at 15-1, yielding a 5' flanking sequence of 931 bp and a 3' flanking sequence of 805 bp. Molecular characterization analysis indicated that KA-1G2 The 15-1 insert sequence originated from the T-DNA region of the pKG002015 vector, measuring 5125 bp (SEQ ID NO: 9). Sequencing results showed that the insert sequence was completely identical to the T-DNA sequence derived from the pKG002015 vector. The insert sequence included a T-DNA fragment between part of the RB and part of the LB, the structure of which is shown in Figure 3. The genetic elements contained in the insert sequence are shown in Table 1.

[0157] Table 1.KA-1G2 Genetic elements in the 15-1 insertion sequence

[0158] Example 3: RT-qPCR analysis of transgenic maize event KA-1G2 15-1 test

[0159] Genetically modified corn KA-1G2 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 OsPPO2-k1 and pat were quantified. A standard curve was first constructed using HMGA 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 maize is diploid, the exogenous gene / internal reference gene * 2 was used to obtain the copy number of the exogenous gene integrated into the maize genome. The primers for the target gene RT-qPCR amplification are shown in Table 2.

[0160] Table 2. Primers for RT-qPCR amplification of target genes

[0161] Using the QuantStudio3 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 KA-1G2... The OsPPO2-k1 and pat genes in 15-1 are single-copy insertions.

[0162] Example 4: The Transgenic Maize Incident KA-1G2 Southern Detection of 15-1 Exogenous Genes

[0163] The restriction enzyme sites selected for Southern blotting were determined by analyzing the sequence of the inserted elements on the vector. Using pKG002015 plasmid as a template, digoxigenin-labeled specific probes were prepared according to the instructions of the Roche DIG probe labeling kit (Cat No: 11745832910) using specific primers for the OsPPO2-k1 and pat genes, respectively. These probes were then hybridized with the digested maize genomic DNA for detection. Primer sequences are shown in Table 3.

[0164] Table 3. Primer information for Southern probe PCR

[0165] Southern blotting results for exogenous genes showed that both OsPPO2-k1 and pat genes yielded the expected results, with only one copy inserted into the maize genome and no other unexpected fragments inserted.

[0166] Example 5: The Transgenic Maize Incident KA-1G2 15-1 Methods for detecting exogenous genes and transformation-specific detection

[0167] 5.1 PCR detection of exogenous genes

[0168] DNA was extracted and purified according to the People's Republic of China agricultural industry standard NY / T674. The DNA was extracted from transgenic maize KA-1G2. Using genomic DNA from plant leaves of plant 15-1 as a template, the exogenous genes OsPPO2-k1 and pat were amplified to determine whether the exogenous genes had been integrated into the maize genome. The amplification primers are shown in Table 4.

[0169] Table 4. PCR primer information for detecting exogenous genes in transgenic maize

[0170] Figure 4 shows the results: the target gene amplification results indicate that the vector pKG002015 and the transformant KA-1G2 In both 15-1 samples, clear bands of OsPPO2-k1 (approximately 855 bp) and pat (395 bp) were amplified. The recipient (control) did not show any amplified exogenous gene band, confirming that KA-1G2... The 15-1 transgenic maize incident contained the exogenous genes OsPPO2-k1 and pat.

[0171] 5.2 Genetically Modified Maize Incident KA-1G2 15-1 Specificity Detection

[0172] The conjugation sequence is a relatively short polynucleotide molecule that is a novel DNA sequence that, when detected in nucleic acid detection analysis, is relevant to the transgenic maize event KA-1G2. DNA from 15-1 is diagnostic. SEQ ID NO:1 conjugate sequence is for transgenic maize event KA-1G2. The T-DNARB region insertion site of 15-1 and 10 bp on each side of the maize genomic DNA form the conjugate sequence SEQ ID NO:2, which is the transgenic maize event KA-1G2. The 15-1 T-DNA LB region insertion site consists of 10 bp on each side of the maize genomic DNA. 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, SEQ ID NO:11-12, and SEQ ID NO:42 are also from the transgenic maize event KA-1G2. The novel DNA sequence in 15-1 can also be used as a DNA probe or as a DNA primer molecule to detect the transgenic maize event KA-1G2. The presence of 15-1 DNA.

[0173] Specifically, the transgenic maize event KA-1G2 was 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 of 15-1 (Table 5).

[0174] Table 5. Primer information for transgenic maize-specific PCR detection

[0175] Figure 5 shows the results: KA-1G2 The T1-T4 generations of 15-1 amplified specific PCR fragments of the exogenous insert 5'flanking and 3'flanking, with sizes of 216 bp and 251 bp, respectively. Transformation into the recipient and vector did not yield any bands.

[0176] Primers for SEQ ID NO:39 and 40 were designed on the sequence of SEQ ID NO:7, and KA-1G2 was shown in SEQ ID NO:41. A TaqMan probe with a transformant-specific sequence (15-1) was used, with a fluorescent reporter group (FAM, HEX, etc.) labeled at the 5' end and a corresponding quencher group (TAMRA, BHQ1, etc.) labeled at the 3' end. Using zSSIIb as an internal reference gene, the transgenic maize event KA-1G2 was detected and quantified by RT-qPCR. Diagnostic amplicon SEQ ID NO:42 of 15-1 (Table 6).

[0177] Table 6. Primer and probe information for transgenic maize-specific RT-qPCR detection

[0178] The results showed that KA-1G2 Diagnostic amplicones were detected in all T1-T4 generations of 15-1 maize, but not in the transformed recipient or vector. The primers and probes described above can be used for the specific detection of the transgenic maize event KA-1G2. 15-1.

[0179] Example 6, KA-1G2 15-1 Detection of OsPPO2-k1 and PAT protein expression in maize transformants

[0180] KA-1G2 15-1 maize transformants expressed OsPPO2-k1 and PAT proteins. The expression of these new proteins in various organs and tissues was analyzed using ELISA (enzyme-linked immunosorbent assay). KA-1G2 was used. Materials from maize transformants 15-1, including V5 (leaves, roots), VT (leaves), R1 (leaves, stalks, pollen), and R6 (leaves, kernels), were collected, sealed, and frozen at -80°C. Once all samples were collected, they were analyzed on the same ELISA plate as standard proteins. Non-transgenic maize receptors were used as controls. ELISA results showed that KA-1G2... In maize transformants 15-1, the expression of OsPPO2-k1 and PAT proteins was good. Specifically, OsPPO2-k1 protein was expressed in KA-1G2... The highest content of PAT protein was found in the leaves of 15-1 transgenic maize at the VT stage (2.2 μg / gfwt, which is the content of the target protein per gram of fresh tissue). PAT protein was found in KA-1G2. The highest content (0.09 μg / g fwt) was found in the leaves of 15-1 transgenic maize at the VT stage, while the target protein was not found in any tissues of non-transgenic plants at different stages.

[0181] Example 7: The KA-1G2 transgenic maize incident 15-1 Assessment of tolerance to target herbicides

[0182] KA-1G2 The tolerance of the 15-1 transformant to the target herbicide was assessed under field conditions. The experimental design followed the guidelines of the Ministry of Agriculture Announcement No. 953-11.1-2007, employing a randomized block design with three replicates. Each plot was separated by a 1-meter-wide buffer zone, and the plot area was 24 m². 2 The corn was directly sown in the experiment, with a row spacing of 60 cm and a plant spacing of 25 cm. After emergence, field management was carried out according to local conventional cultivation methods. Foliar spraying with KA-1G2 was applied to the tested corn at the 4-5 leaf stage. 15-1 and the control receptor were treated with 22.5, 45, and 90 g ai / ha of compound A, 600 and 1200 g ai / ha of glufosinate, and 300 and 600 g ai / ha of succinate, respectively.

[0183] Seedling rate, plant height (5 plants randomly selected), and phytotoxicity symptoms (5 plants randomly selected) were investigated and recorded 1, 2, and 4 weeks after pesticide application. Analysis of variance was used to compare the results of different treatments of transgenic maize KA-1G2. Differences in seedling rate, damage rate and plant height between 15-1 and its corresponding non-transgenic recipient maize, and grading of herbicide damage symptoms shall be carried out in accordance with GB / T 17980.42.

[0184] The survey results showed that all non-GMO control corn plants were killed after being sprayed with 22.5, 45, and 90 g ai / ha compound A, respectively, resulting in a seedling survival rate of 0% and a damage rate of 100%. The genetically modified corn KA-1G2... In treatment areas sprayed with 22.5, 45, and 90 g ai / ha of compound A, the seedling survival rate was 100%, with no phytotoxicity and a damage rate of 0%. There were no significant differences in seedling survival rate and plant height compared to the untreated treatment (see Figure 6). Meanwhile, testing revealed that at the registered dosage, KA-1G2... 15-1 The transformant's performance against other PPO inhibitor herbicides such as flufenoxuron, ethoxyflufenoxuron, ethoxyflufenoxuron, quizalofop-p-ethyl, trifluralin, cyclopyrimethanil, mesotrione, pyrimisulfuron, propyzoxystrobin, cyprodinil, methyl methazine, pyrazosulfuron, oxadiazon, propyzoxystrobin, and penoxadiazon. Flupyrimethanil, trifluralin, cyclopyranil, and eppyrifenacil also exhibited high tolerability, excellent safety, and selectivity.

[0185] Non-GMO control maize, after being sprayed with 600 and 1200 g ai / ha glufosinate respectively, all died, with a seedling survival rate of 0% and a damage rate of 100%. GMO maize KA-1G2... The seedling rate in the treatment areas sprayed with 600 and 1200 g ai / ha glufosinate was 100%, with no herbicide damage and a damage rate of 0. The seedling rate and plant height were not significantly different from those in the treatment areas without herbicide spraying (see Figure 7).

[0186] In addition, all non-GMO control corn plants died after being sprayed with 300 and 600 g ai / ha glufosinate, respectively, resulting in a 0% seedling survival rate and a 100% damage rate. (GMO corn KA-1G2) The seedling rate of the treatment areas sprayed with 300 and 600 g ai / ha glufosinate-ammonium in 15-1 was 100%, with no phytotoxicity and a damage rate of 0. There was no significant difference in seedling rate and plant height compared with the treatment area without herbicide spraying.

[0187] Example 8: The Transgenic Maize Incident KA-1G2 Observation and analysis of the main agronomic traits of 15-1 and its receptor

[0188] Regarding the KA-1G2 genetically modified corn incident The growth period, plant height, ear height, anther color, silk color, plant type, kernel color, and cob color of transgenic maize 15-1 and the recipient (control) variety were investigated. The results showed that the transgenic maize and the recipient variety were basically the same in terms of agronomic traits, and no obvious changes in traits were found.

[0189] In summary, this invention relates to the transgenic maize event KA-1G2. 15-1 exhibits high tolerance to glufosinate and compound A, has no effect on agronomic traits such as yield, and the detection method can accurately and rapidly identify whether biological samples contain genetically modified maize (event KA-1G2). 15-1 DNA molecules.

[0190] Corresponding to the KA-1G2 genetically modified maize event Seed KG2015-1 of species 15-1 was deposited on February 19, 2024, at the China Center for Type Culture Collection (CCTCC, address: No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China), classified as: *Zea mays* L., KG2015-1, with accession number CCTCC NO: P202403. The deposit will be held at the collection for 30 years.

[0191] 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 transgenic corn event The transgenic maize event It is deposited in seed form at the China Center for Type Culture Collection, accession number CCTCC NO: P202403; More preferably, the nucleic acid sequence is diagnostic for the corn event Existing amplicon.

2. A DNA construct comprising two expression cassettes, wherein, a) The first expression cassette contains, in an operable link, a promoter OsAct1 with nucleic acid sequence as shown in SEQ ID NO:31, a chloroplast localizing peptide CTP-MDH with nucleic acid sequence as shown in SEQ ID NO:32, a chloroplast localizing peptide CTP-OsPPO2 with nucleic acid sequence as shown in SEQ ID NO:33, a gene coding region of OsPPO2-k1 with nucleic acid sequence as shown in SEQ ID NO:34, and a terminator T-NOS for terminating gene expression with nucleic acid sequence as shown in SEQ ID NO:35; b) The second expression cassette contains, in an operable link, a promoter P-E35S with a nucleic acid sequence as shown in SEQ ID NO:36, a pat gene coding region with a nucleic acid sequence as shown in SEQ ID NO:37, and a terminator T-35S with a nucleic acid sequence as shown in SEQ ID NO:38 to terminate the expression of the gene. Preferably, the nucleic acid sequence of the DNA construct includes SEQ ID NO:

9.

3. A diagnostic transgenic corn event DNA probes exist which are of sufficient length to bind the nucleic acid sequence of claim 1 which hybridize to the nucleic acid sequence of claim 1 under stringent hybridization conditions and do 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 transgenic maize events. The samples were used together in an amplification reaction to generate a diagnostic result for transgenic maize events in the samples. Existing amplicon; 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, SEQ ID NO:15 and SEQ ID NO:16, or SEQ ID NO:39 and SEQ ID NO:

40.

5. A method for detecting genetically modified corn in samples. The kit contains at least one DNA primer pair as described in claim 4.

6. A method of detecting the presence of DNA of a transgenic corn event comprising: 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 is derived from a transgenic corn event 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; wherein said detecting can diagnose a transgenic maize event in the sample The presence of DNA; Preferably, the transgenic maize event It is deposited in seed form at the China Center for Type Culture Collection, accession number CCTCC NO: P202403.

7. A DNA detection kit comprising at least one DNA molecule, said DNA molecule comprising a sequential sequence or complementary sequence of 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 or SEQ ID NO:8, which can be used as a diagnostic tool for transgenic maize events. Or its descendants possess one of the specific DNA primers or probes; Preferably, the transgenic maize event It is deposited in seed form at the China Center for Type Culture Collection, accession number CCTCC NO: P202403; More preferably, when the DNA molecule is used as a probe, it further includes the consecutive sequences or complementary sequences of SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:42; More preferably, the probe is SEQ ID NO:41 or its complementary sequence.

8. A method of protecting a maize plant from damage caused by a herbicide, characterized in that, This 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 maize plant is planted, wherein the transgenic maize plant contains, in sequence, the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:10 positions 995-5952 and SEQ ID NO:2, or SEQ ID NO:3, SEQ ID NO:10 positions 995-5952 and SEQ ID NO:4, or SEQ ID NO:5, SEQ ID NO:10 positions 995-5952 and SEQ ID NO:6, or the genome of the transgenic maize plant contains SEQ ID NO:10; the transgenic maize plant is tolerant to the glutamine synthase inhibitor and / or protoporphyrinogen oxidase inhibitor herbicide.

9. A method of controlling weeds in a field planted with corn plants, characterized by, This 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 maize plant is planted, wherein the transgenic maize plant contains, in sequence, the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:10 positions 995-5952 and SEQ ID NO:2, or SEQ ID NO:3, SEQ ID NO:10 positions 995-5952 and SEQ ID NO:4, or SEQ ID NO:5, SEQ ID NO:10 positions 995-5952 and SEQ ID NO:6, or the genome of the transgenic maize plant contains SEQ ID NO:10; the transgenic maize plant is tolerant to the glutamine synthase inhibitor and / or protoporphyrinogen oxidase inhibitor herbicide.

10. A method of growing a corn plant that is tolerant to a glutamine synthetase inhibitor herbicide and / or a protoporphyrinogen oxidase inhibitor herbicide, characterized in that, include: Plant at least one corn seed, wherein the genome of the corn 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 positions 995-5952 and SEQ ID NO:2, or SEQ ID NO:3, the nucleic acid sequence of SEQ ID NO:10 positions 995-5952 and SEQ ID NO:4, or SEQ ID NO:5, the nucleic acid sequence of SEQ ID NO:10 positions 995-5952 and SEQ ID NO:6, or the specific region of nucleic acid sequence comprises SEQ ID NO:10; The corn seeds are allowed to grow into corn plants; The corn 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.

11. A method of producing a maize plant that is tolerant to a glutamine synthetase inhibitor herbicide and / or a protoporphyrinogen oxidase inhibitor herbicide, characterized in that, This includes hybridizing a maize plant whose genome sequentially contains the nucleic acid sequences of SEQ ID NO:1, SEQ ID NO:10 positions 995-5952, and SEQ ID NO:2; or SEQ ID NO:3, SEQ ID NO:10 positions 995-5952, and SEQ ID NO:4; or SEQ ID NO:5, SEQ ID NO:10 positions 995-5952, and SEQ ID NO:6, with another maize 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 sequences of SEQ ID NO:1, SEQ ID NO:10 positions 995-5952, and SEQ ID NO:2; or SEQ ID NO:3, SEQ ID NO:10 positions 995-5952, and SEQ ID NO:4; or SEQ ID NO:5, SEQ ID NO:10 positions 995-5952, and SEQ ID NO:6, or the specific region of nucleic acid sequence contains SEQ ID NO:

6. NO:10, and the progeny plants have tolerance to glutamine synthase inhibitor herbicides and / or protoporphyrinogen oxidase inhibitor herbicides; Preferably, the method comprises: a transgenic maize event having tolerance to glutamine synthetase inhibitor herbicides and / or protoporphyrinogen oxidase inhibitor herbicides The first parent maize plant is sexually crossed with the second parent maize plant, which lacks 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; Preferably, the transgenic maize event It is deposited in seed form at the China Center for Type Culture Collection, accession number CCTCC NO: P202403.

12. A method for improving the tolerance of maize plants, the method comprising: a) Construct the DNA construct as described in claim 2; b) Insert the DNA construct into the genome of maize cells; c) regenerate the corn cells into a corn plant; and d) Select maize plants containing the DNA construct; Preferably, improving maize plant tolerance includes its resistance to an effective amount of at least one herbicide, said herbicide preferably a glutamine synthase inhibitor and / or a protoporphyrinogen oxidase inhibitor.

13. A composition produced from a transgenic maize event , characterized in that, The composition is corn flour, corn meal, corn oil, corn silk or corn starch; preferably the transgenic corn event Deposited with the China Center for Type Culture Collection in the form of seeds under the accession number CCTCC NO: P202403.

14. An event arising from genetically modified corn Agricultural products or commodities, characterized in that, The agricultural product or commodity is corn flour, corn meal, corn oil, corn starch, corn gluten, tortillas, cosmetics, or fillers; preferably, the transgenic corn event Deposited with the China Center for Type Culture Collection in the form of seeds under accession number CCTCC NO: P202403.

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

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