Glyphosate-resistant transgenic corn event KJ7002 and its detection method

Abstract

The present invention discloses a nucleic acid sequence for detecting corn event KJM637002 (commercial name KJ7002), which comprises any sequence in SEQ ID NO:1, 3 and any sequence in SEQ ID NO:2, 4; or comprises SEQ ID NO:7. The nucleic acid sequence is derived from a plant, seed or cell comprising corn event KJ7002. The transgenic corn event KJ7002 of the present invention can tolerate high concentration of glyphosate herbicide, is beneficial to control field weeds, improve farming efficiency and yield, and has the advantages of high resistance to glyphosate herbicide and high yield as a shelter for insect-resistant and glyphosate-resistant transgenic corn (such as KJ1003, etc.).

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology and plant breeding, and mainly relates to transgenic plants and inspection methods thereof, and specifically relates to a transgenic corn event KJM637002 (commercial name KJ7002) that tolerates the application of glyphosate herbicides and a method for detecting whether a biological sample contains a specific transgenic corn event KJ7002 nucleic acid sequence. Background Art

[0002] Corn (Zea mays L.) is a major food crop in many parts of the world and an important feed crop. It is strategically important in the development of animal husbandry and aquaculture, and is also widely used in medicine and chemical industry. Biotechnology has been applied to improve the agronomic traits and quality of corn.

[0003] An important agronomic trait of corn is tolerance to herbicides (especially glyphosate herbicides). In corn production, field weeding consumes a lot of labor, resulting in increased planting costs. If weeding is not timely, it will also cause corn yield reduction, resulting in economic losses to corn production. N-phosphonomethylglycine, also known as glyphosate, is a systemic, chronic, broad-spectrum, lethal herbicide. Glyphosate is a competitive inhibitor of phosphoenolpyruvate (PEP), the synthetic substrate of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), and can inhibit the conversion of PEP and 3-phosphoshikimate to 5-enolpyruvylshikimate-3-phosphoshikimate under the catalysis of EPSPS, thereby blocking the synthetic pathway of shikimate, a precursor of aromatic amino acid synthesis, and interfering with protein synthesis, leading to plant and bacterial death. Glyphosate tolerance can be achieved by expressing modified EPSPS. The modified EPSPS has a lower affinity for glyphosate, so in the presence of glyphosate, EPSPS maintains their catalytic activity, and by expressing glyphosate-tolerant genes (such as EPSPS) in corn plants through transgenic methods, corn can gain tolerance to glyphosate herbicides. For example, transgenic corn events NK603, MON87427, DBN9858, nCX-1, etc.

[0004] It is known that the expression of exogenous genes in plants is affected by their chromosomal location, which may be due to the proximity of chromatin structure (such as heterochromatin) or transcriptional regulatory elements (such as enhancers) to the integration site. For this reason, it is usually necessary to screen a large number of events before it is possible to identify events that can be commercialized (i.e., events in which the introduced target gene is optimally expressed). The commercialized events screened out have excellent herbicide resistance and do not affect other agronomic traits. The transgenics can be backcrossed into other genetic backgrounds through conventional breeding methods. The offspring produced by this hybridization method maintain the transgenic expression characteristics and trait performance of the original transformant. The application of this strategic model can ensure reliable gene expression in many varieties.

[0005] Methods for detecting specific events will be useful for later hybridization and breeding, and for identifying whether the offspring contain the target gene. In addition, it will also help to comply with relevant regulations and protect products. It is possible to detect the presence of transgenics by any well-known polynucleotide detection method, such as DNA hybridization using polymerase chain reaction (PCR) or polynucleotide probes. Generally, a pair of primers that span the junction of the inserted transgenic and flanking DNA are often used to identify transgenic specific events by PCR, specifically including a first primer located in the flanking sequence and a second primer in the inserted sequence.

[0006] Long-term, large-scale planting of Bt crops may lead to Bt resistance in pests, which is a key factor affecting the sustainable application of transgenic insect-resistant corn. In order to delay the evolution of pest resistance to Bt crops, the "high dose / refuge" strategy is widely used around the world. The "high dose / refuge" strategy is to plant non-Bt crops near Bt crop areas that express Bt insecticidal proteins at high doses as a refuge for sensitive pest populations. The small number of resistant individuals that survive in the Bt crop area can freely mate with the sensitive population provided by the refuge, and the resulting resistant heterozygous offspring can still be killed by Bt crops that express Bt insecticidal proteins at high doses, thereby reducing the accumulation of insect resistance genes and delaying the development of pest resistance. Therefore, it is necessary to provide effective refuges for insects for commercially available insect-resistant and glyphosate-resistant corn such as KJ1003 and other transgenic corn events. Summary of the invention

[0007] The purpose of the present invention is to provide a transgenic corn event KJM637002 (commercial name KJ7002, hereinafter described by the commercial name) and a nucleic acid sequence and a detection method for detecting the corn event KJ7002. The corn event KJ7002 provided by the present invention can be used as a shelter to prevent insect resistance caused by the promotion of insect-resistant and glyphosate-resistant corn events, and the detection method provided by the present invention can accurately and quickly identify whether a biological sample contains a specific transgenic corn event KJ7002 DNA molecule.

[0008] To achieve the above object, the present invention provides a nucleic acid sequence comprising one of the following (1)-(2):

[0009] (1) any sequence of SEQ ID NO: 1, 3 or its complementary sequence, and any sequence of SEQ ID NO: 2, 4 or its complementary sequence;

[0010] (2) Sequence SEQ ID NO: 7 or its complementary sequence;

[0011] The nucleic acid sequence is derived from plants, seeds or cells of the transgenic corn event KJ7002. Representative samples of seeds of the corn event (Latin name: Zea mays L.) have been deposited in the China Center for Type Culture Collection (CCTCC for short, address: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University Collection Center, Postal Code 430072) with the deposit number CCTCC NO: P202409, and the deposit time is March 20, 2024.

[0012] The SEQ ID NO:1 or its complementary sequence is a 22-nucleotide sequence located near the insertion junction at the 5' end of the insertion sequence in the transgenic corn event KJ7002, and the SEQ ID NO:1 or its complementary sequence spans the flanking genomic DNA sequence of the corn insertion site and the DNA sequence at the 5' end of the insertion sequence; the SEQ ID NO:2 or its complementary sequence is a 22-nucleotide sequence located near the insertion junction at the 3' end of the insertion sequence in the transgenic corn event KJ7002, and the SEQ ID NO:2 or its complementary sequence spans the DNA sequence at the 3' end of the insertion sequence and the flanking genomic DNA sequence of the corn insertion site. The presence of the transgenic corn event KJ7002 can be identified by containing the SEQ ID NOs:1 and 2 or their complementary sequences.

[0013] In the present invention, the nucleic acid sequence may be at least 11 or more continuous polynucleotides (first nucleic acid sequence) of any part of the transgenic insertion sequence in the SEQ ID NO:3 or its complementary sequence, or at least 11 or more continuous polynucleotides (second nucleic acid sequence) of any part of the 5' flanking corn genomic DNA region in the SEQ ID NO:3 or its complementary sequence. The nucleic acid sequence may further be homologous to or complementary to a part of the SEQ ID NO:3 that includes the complete SEQ ID NO:1. When the first nucleic acid sequence and the second nucleic acid sequence are used together, these nucleic acid sequences include a DNA primer pair in a DNA amplification method for producing an amplified product. When the amplified product produced in the DNA amplification method using the DNA primer pair is an amplified product including SEQ ID NO:1, the presence of the transgenic corn event KJ7002 or its progeny can be diagnosed. It is well known to those skilled in the art that the first and second nucleic acid sequences do not have to consist solely of DNA, but may also include RNA, a mixture of DNA and RNA, or a combination of DNA, RNA or other nucleotides or their analogs that do not serve as templates for one or more polymerases. In addition, the probe or primer of the present invention should be at least about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 consecutive nucleotides in length, which can be selected from the nucleotides described in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4. When selected from the nucleotides shown in SEQ ID NO: 3 and SEQ ID NO: 4, the probe and primer can be at least about 21 to about 50 or more consecutive nucleotides in length. The SEQ ID NO: 3 or its complementary sequence is a sequence with a length of 938 nucleotides located near the insertion junction site at the 5' end of the insertion sequence in the transgenic corn event KJ7002. The SEQ ID NO: 3 or its complementary sequence consists of a 624-nucleotide 5' corn flanking genomic DNA sequence (nucleotides 1-624 of SEQ ID NO: 3) and a 314-nucleotide rice TubA promoter DNA sequence (nucleotides 625-938 of SEQ ID NO: 3). The presence of the transgenic corn event KJ7002 can be identified by containing the SEQ ID NO: 3 or its complementary sequence.

[0014] The nucleic acid sequence can be at least 11 or more continuous polynucleotides (third nucleic acid sequence) of any part of the transgenic insertion sequence in the SEQ ID NO:4 or its complementary sequence, or at least 11 or more continuous polynucleotides (fourth nucleic acid sequence) of any part of the 3' flanking corn genomic DNA region in the SEQ ID NO:4 or its complementary sequence. The nucleic acid sequence can further be homologous to or complementary to a portion of the SEQ ID NO:4 that includes the complete SEQ ID NO:2. When the third nucleic acid sequence and the fourth nucleic acid sequence are used together, these nucleic acid sequences include a DNA primer set in a DNA amplification method for producing an amplification product. When the amplification product produced in the DNA amplification method using the DNA primer pair is an amplification product including SEQ ID NO:2, the presence of the transgenic corn event KJ7002 or its progeny can be diagnosed. The SEQ ID NO:4 or its complementary sequence is a sequence with a length of 1142 nucleotides located near the insertion junction at the 3' end of the insertion sequence in the transgenic corn event KJ7002. The SEQ ID NO:4 or its complementary sequence consists of a 444-nucleotide rice TubA terminator DNA sequence (nucleotides 1-444 of SEQ ID NO:4), a 59-nucleotide vector spacer sequence (nucleotides 445-503 of SEQ ID NO:4), and a 639-nucleotide 3'-end corn integration site flanking genomic DNA sequence (nucleotides 504-1142 of SEQ ID NO:4). The presence of the transgenic corn event KJ7002 can be identified by the presence of the SEQ ID NO:4 or its complementary sequence.

[0015] The SEQ ID NO:5 or its complementary sequence is located in the internal sequence of SEQ ID NO:3, including 314 bp of the rice TubA promoter DNA sequence at the 5' end, and the SEQ ID NO:6 or its complementary sequence is located in the internal sequence of SEQ ID NO:4, including 444 bp of the rice TubA terminator DNA sequence at the 3' end and 59 bp of the vector spacer sequence, totaling 503 bp.

[0016] The SEQ ID NO: 7 or its complementary sequence is a sequence of 5046 nucleotides in length that characterizes the transgenic corn event KJ7002, comprising SEQ ID NO: 1-6, including a 5' corn genome flanking sequence, the entire transgenic expression cassette and a 3' corn genome flanking sequence, and the specific genome and genetic elements contained therein are shown in Table 1. The presence of the transgenic corn event KJ7002 can be identified by the presence of the SEQ ID NO: 7 or its complementary sequence.

[0017] Table 1 Genome and genetic elements contained in SEQ ID NO:7

[0018]

[0019]

[0020] The nucleic acid sequence or its complementary sequence can be used in a DNA amplification method to produce an amplicon, and the detection of the amplicon is used to diagnose the presence of the transgenic corn event KJ7002 or its progeny in a biological sample; the nucleic acid sequence or its complementary sequence can be used in a nucleotide detection method to detect the presence of the transgenic corn event KJ7002 or its progeny in a biological sample.

[0021] To achieve the above object, the present invention also provides a method for detecting the presence of DNA of transgenic corn event KJ7002 in a sample, comprising: contacting the sample to be detected with at least two primers in a nucleic acid amplification reaction;

[0022] performing nucleic acid amplification reactions;

[0023] detecting the presence of the amplification product;

[0024] The amplification product includes any sequence selected from SEQ ID NO: 1, 3 or its complementary sequence, and any sequence selected from SEQ ID NO: 2, 4 or its complementary sequence; or includes SEQ ID NO: 7 or its complementary sequence, indicating that the test sample contains the DNA of the transgenic corn event KJ7002.

[0025] The primers include a first primer and a second primer, wherein the first primer is selected from SEQ ID NO: 1 and its complementary sequence, SEQ ID NO: 23 or SEQ ID NO: 22, and the second primer is selected from SEQ ID NO: 2 and its complementary sequence, SEQ ID NO: 26 or SEQ ID NO: 19;

[0026] More specifically, the first primer is selected from SEQ ID NO: 1 or its complementary sequence, SEQ ID NO: 23 or SEQ ID NO: 22, and the second primer is selected from SEQ ID NO: 24 or SEQ ID NO: 21; or the first primer is selected from SEQ ID NO: 2 or its complementary sequence, SEQ ID NO: 26 or SEQ ID NO: 19, and the second primer is selected from SEQ ID NO: 25 or SEQ ID NO: 20.

[0027] To achieve the above object, the present invention also provides a method for detecting the presence of DNA of transgenic corn event KJ7002 in a sample, comprising:

[0028] Contacting the sample to be tested with a probe, wherein the probe comprises a partial sequence of SEQ ID NO: 7 and a complementary sequence thereof, wherein the probe hybridizes to a DNA molecule having a nucleic acid sequence selected from SEQ ID NO: 1-4, 7 or a complementary sequence thereof under stringent hybridization conditions, and does not hybridize to a DNA molecule not having a nucleic acid sequence selected from SEQ ID NO: 1-7 or a complementary sequence thereof under stringent conditions;

[0029] Hybridizing the sample to be detected and the probe under stringent hybridization conditions;

[0030] Detect the hybridization between the sample to be detected and the probe.

[0031] The stringent conditions may be hybridization at 65°C in a 6×SSC (sodium citrate) and 0.5% SDS (sodium dodecyl sulfate) solution, followed by washing the membrane once each with 2×SSC, 0.1% SDS and 1×SSC, 0.1% SDS.

[0032] In some embodiments, the probe comprises at least 11 consecutive nucleotides of SEQ ID NO:3 or its complementary sequence, or at least 11 consecutive nucleotides of SEQ ID NO:4 or its complementary sequence;

[0033] Furthermore, the probe includes consecutive nucleotides at positions 1-11 or positions 12-22 in SEQ ID NO: 1 or its complementary sequence, and consecutive nucleotides at positions 1-11 or positions 12-22 in SEQ ID NO: 2 or its complementary sequence.

[0034] Optionally, at least one of said probes is labeled with at least one fluorescent group.

[0035] To achieve the above object, the present invention also provides a method for detecting the presence of DNA of transgenic corn event KJ7002 in a sample, comprising:

[0036] The sample to be detected is contacted with a marker nucleic acid molecule, wherein the marker nucleic acid molecule comprises a partial sequence of SEQ ID NO: 7 and a complementary sequence thereof, and the marker nucleic acid molecule is selected from any sequence of SEQ ID NO: 1, 3 or its complementary sequence, and any sequence of SEQ ID NO: 2, 4 or its complementary sequence under stringent hybridization conditions; or hybridizes with a DNA molecule comprising a nucleic acid sequence of SEQ ID NO: 7 or its complementary sequence, and does not hybridize with a DNA molecule not containing a nucleic acid sequence selected from SEQ ID NO: 1-7 or its complementary sequence under stringent conditions.

[0037] Hybridizing the sample to be detected and the marker nucleic acid molecule under stringent hybridization conditions;

[0038] The hybridization between the sample to be tested and the marker nucleic acid molecule is detected, and then marker-assisted breeding analysis is performed to determine whether herbicide tolerance is genetically linked to the marker nucleic acid molecule.

[0039] In one embodiment, the marker nucleic acid molecule includes at least 11 consecutive nucleotides of SEQ ID NO: 3 or its complementary sequence, or at least 11 consecutive nucleotides of SEQ ID NO: 4 or its complementary sequence;

[0040] Furthermore, the marker nucleic acid molecule comprises a sequence selected from SEQ ID NO: 1 or its complementary sequence, SEQ ID NO: 2 or its complementary sequence.

[0041] To achieve the above-mentioned object, the present invention also provides a DNA detection kit, comprising at least one DNA molecule, wherein the DNA molecule comprises at least 11 consecutive nucleotides in a homologous sequence of SEQ ID NO:3 or its complementary sequence, or at least 11 consecutive nucleotides in a homologous sequence of SEQ ID NO:4 or its complementary sequence, which can be used as a DNA primer or probe specific to the transgenic corn event KJ7002 or its progeny.

[0042] Furthermore, the DNA molecule includes consecutive nucleotides at positions 1-11 or positions 12-22 in SEQ ID NO: 1 or its complementary sequence, or consecutive nucleotides at positions 1-11 or positions 12-22 in SEQ ID NO: 2 or its complementary sequence.

[0043] Furthermore, the DNA molecule includes a homologous sequence of SEQ ID NO: 1 or a complementary sequence thereof, a homologous sequence of SEQ ID NO: 2 or a complementary sequence thereof.

[0044] To achieve the above object, the present invention provides a method for protecting corn plants from damage caused by herbicides, comprising applying an effective dose of glyphosate herbicide to a field planted with at least one transgenic corn plant, wherein the transgenic corn plant comprises at least one nucleic acid sequence selected from the sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 7 in its genome, and the transgenic corn plant has tolerance to glyphosate herbicide. Specifically, the transgenic corn plant is a transgenic corn plant comprising transgenic corn event KJ7002.

[0045] To achieve the above object, the present invention also provides a method for controlling weeds in a field planted with corn plants, comprising applying an effective dose of a glyphosate herbicide to a field planted with at least one transgenic corn plant, wherein the transgenic corn plant comprises in its genome at least one nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 7, and the transgenic corn plant has tolerance to the glyphosate herbicide. Specifically, the transgenic corn plant is a transgenic corn plant comprising the transgenic corn event KJ7002.

[0046] allowing the corn seeds to grow into corn plants;

[0047] To achieve the above object, the present invention also provides a method for cultivating a corn plant tolerant to glyphosate herbicides, comprising: planting at least one corn seed, wherein the genome of the corn seed comprises a nucleic acid sequence encoding a glyphosate herbicide-tolerant CP4-EPSPS protein and a nucleic acid sequence of a specific region;

[0048] allowing the corn seeds to grow into corn plants;

[0049] Spraying the corn plants with an effective amount of glyphosate herbicide, and harvesting the plants having reduced plant damage compared to other plants not having the nucleic acid sequence of the specific region;

[0050] The nucleic acid sequence of the specific region is selected from at least one of the nucleic acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 7. Specifically, the corn seeds are corn seeds of the transgenic corn event KJ7002.

[0051] To achieve the above objectives, the present invention also provides a method for producing a corn plant tolerant to glyphosate herbicides, comprising introducing a nucleic acid sequence encoding a glyphosate-tolerant CP4-EPSPS protein and a nucleic acid sequence in a specific region into the genome of the corn plant, wherein the nucleic acid sequence in the specific region is selected from at least one of the sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:7.

[0052] Specifically, the method for producing corn plants tolerant to glyphosate herbicides comprises: sexually hybridizing a first parent corn plant of transgenic corn event KJ7002 tolerant to glyphosate herbicides with a second parent corn plant lacking glyphosate tolerance, thereby producing a large number of progeny plants;

[0053] treating the progeny plants with a glyphosate herbicide;

[0054] Selecting the progeny plants that are tolerant to glyphosate;

[0055] The transgenic corn event KJ7002 comprises in its genome at least one nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 7.

[0056] To achieve the above objectives, the present invention also provides a processed product produced from the transgenic corn event KJ7002, wherein the processed product is corn flour, corn oil, corn silk, corn starch, corn gluten, corn tortillas, cosmetics or fillers.

[0057] In the present invention for detecting nucleic acid sequences of corn plants and their detection methods, the following definitions and methods can better define the present invention and guide ordinary technicians in the field to implement the present invention. Unless otherwise specified, the terms are understood according to the conventional usage of ordinary technicians in the field.

[0058] Transformation procedures that cause random integration of foreign DNA will result in transformants containing different flanking regions that are specific to each transformant. When recombinant DNA is introduced into plants by traditional hybridization, its flanking regions are usually unchanged. Transformants will also contain unique junctions between segments of heterologous insert DNA and genomic DNA or between two segments of genomic DNA or between two segments of heterologous DNA.

[0059] The present invention provides a transgenic corn event called KJ7002 and its progeny, wherein the transgenic corn event KJ7002 is the corn plant KJ7002, which includes plants and seeds of the transgenic corn event KJ7002 and plant cells or their regenerable parts, and the plant parts of the transgenic corn event KJ7002 include but are not limited to cells, pollen, ovules, flowers, buds, roots, stems, silks, inflorescences, ears, leaves and products from the corn plant KJ7002, such as corn flour, corn meal, corn oil, corn steep liquor, corn silk, corn starch and biomass left in the corn crop field.

[0060] The transgenic corn event KJ7002 of the present invention comprises a DNA construct which, when expressed in plant cells, acquires tolerance to glyphosate herbicide.

[0061] In some embodiments of the present invention, the DNA construct comprises an expression cassette, which comprises a suitable promoter and terminator for expression in plants, and the promoter is operably linked to the cp4-epspsp gene from Agrobacterium sp. strain CP4. The CP4-EPSPS protein has a low affinity for glyphosate. When the CP4-EPSPS protein is expressed in plants, the shikimic acid synthesis pathway can be reconstructed in the plant when glyphosate is used, so that the transgenic plant continues to synthesize aromatic amino acids and resists glyphosate herbicide.

[0062] Further, the promoter may be a suitable promoter isolated from a plant, including but not limited to a rice TubA promoter. The terminator may be a suitable terminator isolated from a plant, including but not limited to a rice TubA terminator TubA Ter.

[0063] In addition, the expression cassette may also include other genetic elements, including but not limited to enhancers and signal peptide / transit peptide nucleic acid encoding sequences. The signal peptide / transit peptide includes but not limited to CTP2 (encoding chloroplast transit peptide, CP4-EPSPS protein localization is processed into mature protein in the chloroplast).

[0064] The DNA construct is introduced into the plant using a transformation method, including but not limited to Agrobacterium-mediated transformation, biolistic transformation, and pollen tube pathway transformation.

[0065] DNA construct is the combination that DNA molecule is connected to each other, and this combination provides one or more expression cassettes.DNA construct is preferably can self-replicate in bacterial cell, and contains the plasmid of different restriction endonuclease sites, and the restriction endonuclease sites contained are used to import the DNA molecule that functional gene element is provided, i.e. promoter, intron, leader sequence, encoding sequence, 3 ' terminator region and other sequences.The expression cassette contained in the DNA construct comprises the necessary gene element that provides the transcription of messenger RNA, and the expression cassette can be designed to express in prokaryotic cell or eukaryotic cell.Expression cassette of the present invention is designed to express in plant cell most preferably.

[0066] The transgenic corn event KJ7002 with tolerance to glyphosate herbicide is cultivated by the following steps: firstly, a first parent corn plant is sexually hybridized with a second parent corn plant to produce a variety of first-generation offspring plants, wherein the first parent corn plant is composed of corn plants cultivated from the transgenic corn event KJ7002 and its offspring, and the transgenic corn event KJ7002 and its offspring are obtained by transformation using the expression box with tolerance to glyphosate herbicide of the present invention, and the second parent corn plant lacks tolerance to glyphosate herbicide; and then, the offspring plants with tolerance to glyphosate herbicide are selected to cultivate corn plants with tolerance to glyphosate herbicide. These steps can further include backcrossing the glyphosate-tolerant progeny plants to the second parent corn plant or the third parent corn plant, and then selecting the progeny by applying glyphosate herbicide or by identification of molecular markers associated with the trait (such as a DNA molecule comprising the junction sites identified at the 5' and 3' ends of the insertion sequence in the transgenic corn event KJ7002), thereby producing corn plants that are tolerant to glyphosate herbicide.

[0067] It should also be understood that two different transgenic plants can also be crossed to produce offspring containing two independent, segregating added exogenous genes. Selfing of appropriate offspring can result in offspring plants that are homozygous for both added exogenous genes. Backcrossing of parental plants and outcrossing with non-transgenic plants as described above are also contemplated, as are asexual reproduction.

[0068] The term "probe" is a separate nucleic acid molecule to which a conventional detectable label or reporter molecule is bound, for example, a radioisotope, a ligand, a chemiluminescent agent or an enzyme. Such a probe is complementary to a strand of a target nucleic acid. In the present invention, the probe is complementary to a DNA strand from the genome of the transgenic corn event KJ7002, whether the genomic DNA is from the transgenic corn event KJ7002 or a seed or a plant or seed or an extract derived from the transgenic corn event KJ7002. The probe of the present invention includes not only deoxyribonucleic acid or ribonucleic acid, but also polyamides and other probe materials that specifically bind to a target DNA sequence and can be used to detect the presence of the target DNA sequence.

[0069] Primers and probes based on the flanking genomic DNA and insertion sequences of the present invention can be determined by conventional methods, for example, by isolating the corresponding DNA molecule from plant material derived from transgenic corn event KJ7002 and determining the nucleic acid sequence of the DNA molecule. The DNA molecule comprises the transgenic insertion sequence and the flanking region of the corn genome, and fragments of the DNA molecule can be used as primers or probes.

[0070] The nucleic acid probes and primers of the present invention hybridize with the target DNA sequence under stringent conditions. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of DNA derived from the transgenic corn event KJ7002 in a sample. Nucleic acid molecules or fragments thereof can be specifically hybridized with other nucleic acid molecules under certain circumstances. As used in the present invention, if two nucleic acid molecules can form an antiparallel double-stranded nucleic acid structure, it can be said that the two nucleic acid molecules can specifically hybridize with each other. If two nucleic acid molecules show complete complementarity, one of the nucleic acid molecules is said to be the "complement" of the other nucleic acid molecule. As used in the present invention, when each nucleotide of a nucleic acid molecule is complementary to the corresponding nucleotide of another nucleic acid molecule, the two nucleic acid molecules are said to show "complete complementarity". If two nucleic acid molecules can hybridize with each other with sufficient stability so that they anneal and bind to each other under at least conventional "low stringency" conditions, the two nucleic acid molecules are said to be "minimally complementary". Similarly, if two nucleic acid molecules can hybridize with each other with sufficient stability so that they anneal and bind to each other under conventional "high stringency" conditions, the two nucleic acid molecules are said to have "complementarity". Deviations from complete complementarity are permissible as long as such deviations do not completely prevent the two molecules from forming a double-stranded structure. In order for a nucleic acid molecule to serve as a primer or probe, it is only necessary to ensure that it has sufficient complementarity in sequence to form a stable double-stranded structure under the specific solvent and salt concentration used.

[0071] As used in the present invention, a substantially homologous sequence is a nucleic acid molecule that can specifically hybridize with the complementary strand of another matching nucleic acid molecule under highly stringent conditions. Suitable stringent conditions that promote DNA hybridization, for example, treatment with 6.0× sodium chloride / sodium citrate (SSC) at about 45°C, followed by washing with 2.0×SSC at 50°C, are well known to those skilled in the art. For example, the salt concentration in the washing step can be selected from about 2.0×SSC, 50°C for low stringency conditions to about 0.2×SSC, 50°C for high stringency conditions. In addition, the temperature conditions in the washing step can be increased from about 22°C at room temperature for low stringency conditions to about 65°C for high stringency conditions. Both the temperature conditions and the salt concentration can be changed, or one of them can be kept constant while the other variable is changed. Preferably, a nucleic acid molecule of the present invention can specifically hybridize with one or more nucleic acid molecules or their complementary sequences in SEQ ID NO: 1-4, 7, or any fragment of the above sequence under moderate stringency conditions, for example, at about 2.0×SSC and about 65°C. More preferably, a nucleic acid molecule of the invention specifically hybridizes with one or more nucleic acid molecules of SEQ ID NO: 1-4, 7 or their complementary sequences, or any fragments of the above sequences under highly stringent conditions. In the present invention, the preferred marker nucleic acid molecule has SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 or their complementary sequences, or any fragments of the above sequences.

[0072] SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 can be used as markers in plant breeding methods to identify offspring of genetic crosses. The hybridization of the probe to the target DNA molecule can be detected by any method known to those skilled in the art, including but not limited to fluorescent labels, radioactive labels, antibody labels and chemiluminescent labels.

[0073] With respect to amplification of a target nucleic acid sequence using specific amplification primers (e.g., by PCR), "stringent conditions" refer to conditions that only allow the primers to hybridize to the target nucleic acid sequence in a DNA thermal amplification reaction, and primers having a wild-type sequence (or its complementary sequence) corresponding to the target nucleic acid sequence are able to bind to the target nucleic acid sequence and preferably produce a unique amplification product, i.e., an amplicon.

[0074] The term "specifically binds to (target sequence)" means that under stringent hybridization conditions, the probe or primer hybridizes only to the target sequence in a sample containing the target sequence.

[0075] As used herein, "amplified DNA" or "amplicon" refers to the nucleic acid amplification product of a target nucleic acid sequence that is part of a nucleic acid template. For example, in order to determine whether a corn plant is produced by sexual hybridization containing the transgenic corn event KJ7002 of the present invention, or whether a corn sample collected from a field contains the transgenic corn event KJ7002, or whether a corn extract, such as meal, powder or oil, contains the transgenic corn event KJ7002, DNA extracted from a corn plant tissue sample or extract can be used by a nucleic acid amplification method using a primer pair to produce an amplicon that is diagnostic for the presence of DNA of the transgenic corn event KJ7002. The primer pair includes a first primer derived from a flanking sequence adjacent to the inserted exogenous DNA insertion site in the plant genome, and a second primer derived from the inserted exogenous DNA. The amplicon has a certain length and sequence, and the sequence is also diagnostic for the transgenic corn event KJ7002. The length of the amplicon can range from the combined 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.

[0076] Alternatively, the primer pairs can be derived from flanking genomic sequences on both sides of the inserted DNA to produce an amplicon comprising the entire inserted nucleotide sequence. One of the primer pairs derived from the plant genomic sequence can be located at a distance from the inserted DNA sequence, the distance ranging from one nucleotide base pair to about 20,000 nucleotide base pairs. The use of the term "amplicon" specifically excludes primer dimers formed in a DNA thermal amplification reaction.

[0077] The inserted exogenous DNA sequence and the flanking DNA sequence from the transgenic corn event KJ7002 can be amplified from the genome of the transgenic corn event KJ7002 using the provided primer sequences, and then the PCR amplicon or cloned DNA can be subjected to standard DNA sequencing.

[0078] The DNA detection kit based on the DNA amplification method contains DNA primer molecules that specifically hybridize to the target DNA under appropriate reaction conditions and amplify diagnostic amplicons. The kit can provide agarose gel-based detection methods or many methods known in the prior art for detecting diagnostic amplicons. The kit containing DNA primers homologous or complementary to any part of the corn genomic region of SEQ ID NO:3 or SEQ ID NO:4, and homologous or complementary to any part of the transgenic insertion region of SEQ ID NO:7 is provided by the present invention. The amplicons produced by these methods can be detected by a variety of techniques. One of the methods is Genetic Bit Analysis, which designs a DNA oligonucleotide chain spanning the inserted DNA sequence and the adjacent flanking genomic DNA sequence. The oligonucleotide chain is fixed in a microwell of a microwell plate, and after PCR amplification of the target region (one primer is used in the inserted sequence and in the adjacent flanking genomic sequence), the single-stranded PCR product can be hybridized with the fixed oligonucleotide chain and used as a template for a single base extension reaction, which uses a DNA polymerase and ddNTPs specifically labeled for the next expected base. The results can be obtained by fluorescence or ELISA-type methods. The signal represents the presence of the insert / flanking sequence, which indicates that the amplification, hybridization and single base extension reactions are successful.

[0079] Another method is Pyrosequencing technology. This method designs an oligonucleotide chain that spans the inserted DNA sequence and the adjacent genomic DNA binding site. The oligonucleotide chain is hybridized with the single-stranded PCR product of the target region (one primer is used in the inserted sequence and one primer is used in the adjacent flanking genomic sequence), and then incubated with DNA polymerase, ATP, sulfhydryl enzyme, luciferase, apyrase, adenosine-5'-phosphosulfate and luciferin. dNTPs are added separately and the generated light signals are measured. The light signal represents the presence of the inserted / flanking sequence, which indicates that the amplification, hybridization, and single-base or multi-base extension reaction are successful.

[0080] The fluorescence polarization phenomenon described by Chen et al. (Genome Res. 9:492-498, 1999) is also a method that can be used to detect the amplicon of the present invention. Using this method requires designing an oligonucleotide chain that spans the inserted DNA sequence and the adjacent genomic DNA binding site. The oligonucleotide chain is hybridized with the single-stranded PCR product of the target region (one primer is used in the inserted sequence and the adjacent flanking genomic sequence), and then incubated with DNA polymerase and a fluorescently labeled ddNTP. Single-base extension will result in the insertion of ddNTP. This insertion can be measured by a change in its polarization using a fluorimeter. The change in polarization represents the presence of the inserted / flanking sequence, which indicates that the amplification, hybridization and single-base extension reactions are successful.

[0081] Taqman is described as a method for detecting and quantifying the presence of a DNA sequence and is described in detail in the instructions provided by the manufacturer. As a brief example, a FRET oligonucleotide probe is designed that spans the insert DNA sequence and the adjacent genomic flanking binding site. The FRET probe and PCR primers (one primer each in the insert sequence and the adjacent flanking genomic sequence) are cycled in the presence of a thermostable polymerase and dNTPs. Hybridization of the FRET probe results in the cleavage of the fluorescent and quenching portions of the FRET probe and the release of the fluorescent portion. The generation of a fluorescent signal represents the presence of the insert / flanking sequence, which indicates that amplification and hybridization were successful.

[0082] Based on the hybridization principle, the applicable techniques for detecting the plant material derived from the transgenic corn event KJ7002 can also include Southern blot hybridization, Northern blot hybridization and in situ hybridization. Especially, the applicable techniques include incubation probe and sample, washing to remove unbound probe and detection probe whether hybridization. The detection method depends on the type of probe attached mark, for example, radiolabeled probe can be detected by X-ray film exposure and development, or enzyme-labeled probe can be detected by substrate conversion to realize color change.

[0083] Tyangi et al. (Nat. Biotech. 14:303-308, 1996) introduced the application of molecular markers in sequence detection. Briefly described below, a FRET oligonucleotide probe is designed that spans the insertion DNA sequence and the adjacent genomic flanking binding site. The unique structure of the FRET probe causes it to contain a secondary structure that can maintain the fluorescent part and the quenching part at a close distance. The FRET probe and PCR primers (one primer each is used in the insertion sequence and the adjacent flanking genomic sequence) are subjected to a cyclic reaction in the presence of a thermostable polymerase and dNTPs. After successful PCR amplification, the hybridization of the FRET probe and the target sequence results in the loss of the probe secondary structure, thereby separating the fluorescent part and the quenching part in space and generating a fluorescent signal. The generation of the fluorescent signal represents the presence of the insertion / flanking sequence, which indicates that the amplification and hybridization are successful.

[0084] Other described methods, such as microfluidics, provide methods and devices for separating and amplifying DNA samples. Optical dyes are used to detect and measure specific DNA molecules. Nanotube devices containing electronic sensors for detecting DNA molecules or nanobeads that bind specific DNA molecules and can thus be detected are useful for detecting DNA molecules of the present invention.

[0085] A DNA detection kit can be developed using the primer pairs of the present invention and methods described or known in the art of DNA detection. The kit is useful for identifying the presence of DNA of transgenic corn event KJ7002 in a sample, and can also be used to grow corn plants containing DNA of transgenic corn event KJ7002. The kit can contain DNA primers or probes that are homologous to or complementary to at least a portion of SEQ ID NO: 1, 2, 3, 4, or 7, or other DNA primers or probes that are homologous to or complementary to DNA contained in transgenic genetic elements, and these DNA sequences can be used in DNA amplification reactions, or as probes in DNA hybridization methods.

[0086] In the DNA amplification method, the DNA molecule used as a primer can be any part of the transgenic insertion sequence derived from the transgenic corn event KJ7002, or any part of the DNA region flanking the corn genome derived from the transgenic corn event KJ7002.

[0087] Transgenic corn event KJ7002 can be combined with other transgenic corn varieties, such as corn with tolerance to herbicides (such as glufosinate, dicamba, etc.), or transgenic corn varieties carrying other insect-resistant genes. Various combinations of all these different transgenic events, bred together with the transgenic corn event KJ7002 of the present invention, can provide improved hybrid transgenic corn varieties that are resistant to multiple insect pests and multiple herbicides. These varieties can show more excellent characteristics such as yield improvement compared to non-transgenic varieties and single-trait transgenic varieties.

[0088] The present invention provides a nucleic acid sequence and a detection method for detecting corn plants. The transgenic corn event KJ7002 tolerates the phytotoxic effects of agricultural herbicides containing glyphosate.

[0089] Corn plants with this trait express the glyphosate-resistant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) protein of Agrobacterium strain CP4, which confers tolerance to glyphosate. Herbicide-tolerant corn can tolerate high concentrations of glyphosate herbicides, allowing corn growers to use efficient, broad-spectrum, and inexpensive glyphosate for convenient weed management during planting, reducing labor costs.

[0090] According to international practice, the refuge accounts for 5-20% of the planting area. The glyphosate-tolerant transgenic corn event KJ7002 can be used as a refuge for insect-resistant and glyphosate-resistant transgenic corn. It has the characteristics of resistance to high concentrations of glyphosate. In years when insect pests are not serious, the refuge corn can also harvest ideal yields.

[0091] In addition, the gene encoding the glyphosate tolerance trait is present on a single locus in the genome of the transgenic corn event KJ7002, which provides enhanced breeding efficiency and enables the use of molecular markers to track transgenic inserts in breeding populations and their progeny. At the same time, the DNA primers or probes provided in the detection method of the present invention can produce amplification products that are diagnosed as transgenic corn event KJ7002 or its progeny, and can quickly, accurately and stably identify the presence of plant materials derived from transgenic corn event KJ7002. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] Figure 1 It is a schematic diagram of the structure of the transgenic insertion sequence and the binding site of the corn genome used for detecting the nucleic acid sequence of corn plant KJ7002 and the detection method thereof of the present invention;

[0093] Figure 2 It is a schematic diagram of the structure of the recombinant expression vector pk0637 used for detecting the nucleic acid sequence of corn plant KJ7002 and the detection method thereof of the present invention;

[0094] Figure 3It is the comparison result of the 3' flanking sequence of the inserted sequence in the database;

[0095] Figure 4 The 3' insertion site sequence verification result of transformant KJ7002, where M is a molecular weight marker, 1 is the amplification product using the genomic DNA of corn B104 as a template; 2 is a blank control, the amplification product using water as a template; 3 is the amplification product using pK0637 plasmid DNA as a template; 4 is the amplification product using the genomic DNA of transformant KJ7002 as a template;

[0096] Figure 5 The 3' insertion site sequence verification result of transformant KJ7002, where M is a molecular weight marker, 1 is the amplification product using the genomic DNA of corn B104 as a template; 2 is a blank control, the amplification product using water as a template; 3 is the amplification product using pK0637 plasmid DNA as a template; 4 is the amplification product using the genomic DNA of transformant KJ7002 as a template;

[0097] Figure 6 It is the electrophoresis diagram of 5' end specific PCR detection of transformant KJ7002, wherein, M: marker, 1: positive control, amplification product using pK0637 plasmid as template, 2: negative control, amplification product using genomic DNA of non-transgenic control corn (recipient B104) as template, 3: blank control, amplification product using water as template, 4: other transformant control, amplification product using genomic DNA of transformant KJM637004 plant as template, 5-7, 8-10, 11-13, 14-16: amplification products using genomic DNA of T3, T4, T5, T6 generations of corn plants of KJ7002 transformant as template, respectively;

[0098] Figure 7 The figure is the electrophoresis diagram of 3' end specific PCR detection of transformant KJ7002, wherein, M: marker, 1: positive control, amplification product using pK0637 plasmid as template, 2: negative control, amplification product using genomic DNA of non-transgenic control corn (recipient B104) as template, 3: blank control, amplification product using water as template, 4: other transformant control, amplification product using genomic DNA of transformant KJM637004 plant as template, 5-7, 8-10, 11-13, 14-16: amplification products using genomic DNA of T3, T4, T5, T6 generations of corn plants of KJ7002 transformant as template, respectively;

[0099] Figure 8Southern hybridization map of cp4-epsps gene probe, where A: hybridization map after EcoRⅠ digestion, B: hybridization map after EcoRⅤ digestion, C: schematic diagram of electrophoretic mobility deviation, 1: Marker, 2: blank, 3: CK+ (pK0637 plasmid EcoRⅠ digestion), 4: CK-negative control (receptor B104), 5: KJ7002 T4 generation, 6: KJ7002 T5 generation, 7: KJ7002 T6 generation; the left side of C is digoxigenin-labeled Marker, and the right side is 1kb Marker (Thermo);

[0100] Fig. 9 This is a field effect diagram of the transgenic corn containing the transgenic corn event KJ7002 of the present invention after spraying 1, 2, and 4 times the dose of glyphosate herbicide for two weeks; DETAILED DESCRIPTION

[0101] The present invention is further illustrated and described below in conjunction with the embodiments, but the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the present invention and the embodiments, all other inventions and embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention.

[0102] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0103] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0104] Example 1: Cloning and transformation

[0105] 1.1 Vector cloning

[0106] The recombinant expression vector pK0637 (such as Figure 2 The vector pK0637 comprises a transgenic expression cassette, which is composed of a rice TubA promoter, operably linked to a chloroplast transit peptide CTP2, localizes the CP4-EPSPS protein to the chloroplast and processes it into a mature protein, operably linked to the glyphosate-tolerant 5-enol-pyruvylshikimate-3-phosphate synthase (CP4-EPSPS) of the Agrobacterium CP4 strain, and operably linked to a rice TubA terminator. The vector pK0637 is transformed into Agrobacterium LBA4404 (Invitrgen, Chicago, USA; Cat. No: 18313-015) by liquid nitrogen method, and the transformed cells are screened using 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) as a selection marker.

[0107] 1.2 Plant transformation

[0108] The conventional Agrobacterium infection method was used for transformation. The sterile cultured maize (maize inbred line B104) immature embryos were co-cultured with Agrobacterium to transfer the T-DNA in the constructed recombinant expression vector pK0637 into the maize chromosome set to produce the transgenic maize event KJ7002. The specific operation was as follows:

[0109] 1) Prepare pK0637 Agrobacterium culture medium. Spread the culture medium stored in -80℃ refrigerator on YEB medium supplemented with antibiotics rifampicin and kanamycin, and culture at 28℃ in the dark for 48h. Before use, collect the Agrobacterium grown on the plate with an inoculation loop, suspend it with inf-AS medium, dilute the culture medium, and calculate OD 600 is 0.5.

[0110] 2) Preparation of young embryos Take corn ears about 12 days after pollination. Remove the covering leaves, treat the surface with 75% alcohol for 30 seconds, treat with 1% sodium hypochlorite for 40 minutes, and wash with sterile water three times. Peel off the young embryos at room temperature and place them in a centrifuge tube containing 2 mL of inf medium. Wash twice with new inf washing solution, and finally leave a small amount of inf in the tube just enough to immerse the young embryos.

[0111] 3) Pretreatment of young embryos: Place the centrifuge tube containing young embryos in a 45°C water bath for 5 minutes. After the water bath is completed, quickly take out the centrifuge tube and place it in an ice bath for 2 minutes.

[0112] 4) Infection of young embryos with Agrobacterium: Remove the remaining inf in the above centrifuge tube by aspirating, add 1 mL of the prepared Agrobacterium solution, invert the centrifuge tube 20 times, and let it stand for 5 minutes.

[0113] 5) After the co-culture infection is completed, the young embryos and the infection solution are transferred to the co-culture medium. The excess Agrobacterium suspension on the culture medium is sucked off with a pipette. The young embryos are placed so that the embryonic axis is in contact with the culture medium, i.e. the scutellum faces upward. The culture dish is sealed with a sealing film and cultured in the dark at 28°C for 3 days.

[0114] 6) Recovery Culture After 3 days of co-culture, the young embryos were transferred to recovery medium and cultured in the dark at 28°C for 14 days.

[0115] 7) Screening culture The immature embryos after 14 days of recovery culture were transferred to the screening medium and cultured in the dark at 28°C for 14 days. After 14 days, the embryos were subcultured once using the same medium.

[0116] 8) Differentiation culture After two rounds of screening, the vigorously growing calli were transferred to differentiation medium and cultured at 28°C under light for 14 days.

[0117] 9) After rooting and differentiation of regenerated seedlings, the differentiated seedlings of about 2 cm were transferred into a rooting pot containing rooting medium and cultured at 28°C for 14 days.

[0118] 10) Seedling Transplantation After 14 days of rooting culture, the seedlings with developed root systems were transplanted to small pots in the greenhouse and covered with a cover to keep moisture. The cover was removed after 3 days. After 2 weeks of seedling acclimatization and PCR testing, positive plants were transplanted to large pots and cultured at 26℃ for 16 hours of light until flowering and seeding.

[0119] 1.3 Identification and screening of transgenic events

[0120] By TaqMan TM The regenerated transgenic maize plants were analyzed (see Example 2) to detect the presence of the cp4-epsps gene and to characterize the copy number of the glyphosate herbicide tolerant lines. Based on the copy number of the inserted sequence, good glyphosate herbicide tolerance and agronomic performance (see Example 5), event KJ7002 was selected as superior from 375 individual transgenic events through screening, having a single copy transgene, good glyphosate herbicide tolerance and agronomic performance.

[0121] Example 2: Using TaqMan TM Detection of genetically modified corn event KJ7002

[0122] 20 mg of leaves of transgenic corn event KJ7002 were taken to extract genomic DNA. The copy number of cp4-epsps was detected by Taqman probe fluorescence quantitative PCR method, and the primer and probe sequences used are shown in Table 2. At the same time, wild-type corn plants were used as controls and the detection and analysis were performed according to the above method.

[0123] Table 2 Primer and probe sequences for detecting cp4-epsps

[0124]

[0125]

[0126] The reaction system 10ul is as follows:

[0127]

[0128] Each ml of 50× primer-probe mixture contained 15 μL of each primer at 1 mM concentration, 50 ul of probe at 100 μM concentration, and 920 ul of sterile water, and was stored at 4° C. in a brown light-proof test tube.

[0129] The amplification procedure is as follows:

[0130]

[0131] The data were analyzed using QuantStudio Design & Analysis Software (Applied Biosystems) to obtain single-copy transgenic maize events.

[0132] Example 3: Analysis of the insertion site of transformant KJ7002 and transformant-specific PCR detection

[0133] 1. Purpose of the experiment

[0134] The flanking sequences of the target gene insertion site of the transformant were amplified by Tail-PCR technology, and the transformant-specific PCR detection system of KJ7002 was established based on the flanking sequence information.

[0135] 2. Test Materials

[0136] Analysis of the insertion site of the target gene The genomic DNA of the transformant KJ002 was used as a template to perform Tail-PCR experiments to analyze the insertion site information. At the same time, specific PCR verification was performed based on the obtained flanking sequence information of the insertion site, and the recipient B104 was used as a non-transgenic control.

[0137] 3. Test methods

[0138] 1) Extraction of genome:

[0139] Extraction of DNA from plant leaves using the CTAB method

[0140] 2) Analysis method of the 3' flanking sequence of the inserted sequence:

[0141] Specific primers pk0637-R1-1, pk0637-R2-1 and pk0637-R3-1 were designed according to the literature (reference: Liu YG, High-efficiency thermalasymmetric interlaced PCR for amplification of unknown flanking sequences. Biotechniques, 2007), and Tail-PCR degenerate primers LAD1, LAD2, LAD3, LAD4 and AC1 were designed (Table 3). The transformant genomic DNA was used as a template, and the pk0637-R1-1 primer was used with LAD1, LAD2, LAD3, LAD4 respectively for the first round of Tail-PCR reaction; the first round amplification product was used as a template, and pk0637-R2-1 and AC1 were used as primers for the second round of Tail-PCR reaction; the second round of Tail-PCR reaction was carried out using the second round amplification product as a template, and pk0637-R3-1 and AC1 as primers for the third round of Tail-PCR reaction. After three rounds of amplification, the Tail PCR products were subjected to agarose gel electrophoresis, and the target band was recovered and sequenced using pk0637-R3-1 as a sequencing primer to obtain the flanking sequence of the insertion site of the target gene, and the accuracy of the insertion site was confirmed by specific PCR detection primers.

[0142] Table 3 Primer names and sequences

[0143]

[0144] Table 4 Amplification system of the first round of Tail-PCR

[0145]

[0146] Table 5 Amplification program of the first round of Tail-PCR

[0147]

[0148] Table 6 Amplification system of the second round of Tail-PCR

[0149]

[0150]

[0151] Table 7 Amplification program of the second round of Tail-PCR

[0152]

[0153] Table 8 The third round of Tail-PCR amplification system

[0154]

[0155] Table 9 The third round of Tail-PCR amplification program

[0156]

[0157]

[0158] 2) 3' flanking sequence analysis method:

[0159] According to the 3' flanking sequence information obtained by Tail PCR, primers 637-3-F (SEQ ID NO: 20) and 637-3-R (SEQ ID NO: 19) were designed on the genome and insertion sequences, respectively. Amplification was performed using KJ7002 genomic DNA as a template, and the amplified products were sequenced and analyzed to verify the accuracy of the Tail PCR results and determine the specific information of the insertion site of the target gene and the true situation of its flanking sequences.

[0160] Table 10 T-DNA 3' end insertion position specific PCR detection primers

[0161]

[0162] Table 11 T-DNA 3' end insertion position specific PCR detection amplification system

[0163] Components Initial concentration of components Dosage (μl) KOD one 2X 25 637-3-R 10μM 1.5 637-3-F 10μM 1.5 <![CDATA[ddH2O]]> 20 DNA 2 Total volume 50

[0164] Table 12 T-DNA 3' end insertion position specific PCR detection reaction program

[0165]

[0166] 4) 5' flanking sequence analysis method:

[0167] Primers 637-5-F (SEQ ID NO: 22) and 637-5-R (SEQ ID NO: 21) (Table 13) were designed upstream of the insertion site Chr6: 70988211 found at the 3' end of the target gene insertion sequence (relative to the upstream of the insertion sequence) and at the 5' end of the insertion sequence. KJ7002 genomic DNA was used as a template for amplification, and the amplified products were sequenced and compared to determine the 5' end boundary position of the target gene and its flanking sequence.

[0168] Table 13 T-DNA 5' end insertion position specific PCR detection primers

[0169]

[0170]

[0171] Table 14 T-DNA 5' end insertion position specific PCR detection amplification system

[0172] Components Initial concentration of components Dosage (μl) KOD one mix 2X 25 637-5-R 10μM 1.5 637-5-F 10μM 1.5 <![CDATA[ddH2O]]> 19 DNA 3 Total volume 50

[0173] Table 15 T-DNA 5' end insertion position specific PCR detection amplification program

[0174]

[0175] 5) Establishment of transformant-specific PCR method

[0176] According to the specific insertion sequence and flanking sequence of the obtained transformant KJ7002, 5'-end and 3'-end specific detection primers were designed for specific PCR detection to qualitatively identify the transformant KJ7002. 5'-end specific detection primers: 225-5-F (SEQ ID NO: 23) and 225-5-R (SEQ ID NO: 24), with an expected amplification length of 259 bp; 3'-end specific detection primers: 225-3-F (SEQ ID NO: 25) and 225-3-R (SEQ ID NO: 26), with an expected amplification length of 249 bp.

[0177] According to the specific insertion sequence and its flanking sequences of the obtained transformant KJ7002, the 5'-end and 3'-end specific detection primers used for specific PCR detection were designed as shown in the table.

[0178] Table 16 Specific PCR detection primers

[0179]

[0180] Specific PCR reaction

[0181] Prepare specific PCR systems with different templates (plant materials or water) and different primer pairs according to the system shown in Table 17. Place in a PCR instrument and perform the reaction according to the program in Table 18.

[0182] Table 17 Specific PCR reaction system

[0183] Components Initial concentration of components Dosage (μL) KOD one Mix 2× 10 Upstream primer 10μM 0.6 Downstream primer 10μM 0.6 Total DNA of sample 1 <![CDATA[ddH2O]]> 5.8 GC enhancer 2 Total volume 20μL

[0184] Table 18 Specific PCR program

[0185]

[0186] 4. Experimental Results

[0187] 1) Insertion site at the 3' end of the insertion sequence

[0188] The 3' end boundary of T-DNA was determined based on the Tail PCR product sequencing results: using the database B104 ISU_USDA 0.1assembly, it was found that the 468th position of the vector sequence was inserted into the 70988211th position of the Chr6 genome ( Figure 3 ).

[0189] According to the 3' flanking sequence information obtained by Tail PCR, primers 637-3-R (SEQ ID NO: 19) and 637-3-F (SEQ ID NO: 20) were designed on the genome and insert sequences, respectively. The KJ7002 genomic DNA was used as a template for amplification, and a 937 bp band was obtained ( Figure 4 ), the amplified products were sequenced and analyzed to determine that the target gene (T-DNA sequence) was reversely inserted into chromosome 6 of the genome, specifically at Chr6:70988211, and the target gene sequence connected thereto corresponded to the 468th bp of the vector sequence.

[0190] 2) Insertion site at the 5' end of the insertion sequence

[0191] Primers 637-5-F and 637-5-R were designed at the upstream of the insertion site Chr6: 70988211 found at the 3' end of the target gene insertion sequence (relative to the upstream of the insertion sequence) and at the 5' end of the insertion sequence, respectively. The KJ7002 genomic DNA was used as a template for amplification to obtain a 938 bp PCR product ( Figure 5 ), and through sequencing and alignment analysis, it was determined that the 5' end boundary position of the target gene was the TubA promoter of T-DNA, which was inserted into the 70988187bp of chromosome 6 of the maize genome.

[0192] 3) Transformant-specific PCR detection results

[0193] Three plants of each of the T3, T4, T5 and T6 generations of the transformant corn material KJ7002 that grew well after spraying glyphosate were randomly selected as test samples, pK0637 plasmid was used as a positive control, non-transgenic control corn (recipient B104) was used as a negative control, water was used as a template as a blank control, and the transformant KJM637004 was used as a control for other transformants. Specific PCR detection was performed using 5' end (SEQ ID NO: 23 and SEQ ID NO: 24) and 3' end specific detection primers (SEQ ID NO: 25 and SEQ ID NO: 26), respectively.

[0194] like Figure 6 and Figure 7As shown, in each of the three plants randomly selected from the T3-T6 generations, target fragments of 259bp and 249bp, which were consistent with the theoretical values, were amplified, and no bands were amplified in all other controls, indicating that both the 5'-end specific PCR detection and the 3'-end specific PCR detection systems of the transformant have the function of specifically detecting the transformant KJ7002. The detection results also showed that the integration of the exogenous gene of the KJ7002 transformant at chromosome 6 Chr6:70988187-70988211 can be stably inherited.

[0195] Example 4: Detection of transgenic maize event KJ7002 by Southern blot hybridization

[0196] Southern hybridization was performed using vector plasmid pk0637 as a positive control, non-transgenic maize receptor B104 as a negative control, and transformant KJ7002 plants of different generations as samples to obtain a transformant-specific molecular hybridization map. Southern hybridization was used to analyze the copy number of the exogenous insertion sequence of transgenic maize KJ7002 and the stability of the event between different generations.

[0197] 1. DNA Extraction for Southern Blot Hybridization

[0198] Take 2g of the sample leaf to be tested, add quartz sand and liquid nitrogen, grind it thoroughly, and pour the powder into a 50ml centrifuge tube;

[0199] After the liquid nitrogen in the centrifuge tube has evaporated completely, add 10 ml of DNA extraction buffer and shake vigorously at room temperature for 10-15 minutes;

[0200] Add 5 ml of phenol and shake gently for 5 to 10 minutes; then add 5 ml of chloroform and continue to shake gently for 10 to 15 minutes;

[0201] Centrifuge at 8000 rpm for 10 min at room temperature with vertical rotor and 3800 rpm for horizontal rotor.

[0202] Aspirate the supernatant, add an equal volume of isopropanol, and gently shake the centrifuge tube to fully mix the isopropanol and the supernatant. White flocculent DNA can be seen;

[0203] Pick up the DNA precipitate into a new 1.5ml centrifuge tube, add 1ml 70% ethanol, and wash the precipitate;

[0204] After the precipitate is dried, add 800ul sterile water (containing RNaseA at a final concentration of 50μg / ml) to dissolve it, and digest the RNA at 37℃ for 0.5-1hr; take 1ul from each tube and run electrophoresis to observe whether the RNase has been completely removed;

[0205] Add 10ul of proteinase K (20mg / ml) to each tube, dilute to 1x with 10x proteinase K buffer, and mix thoroughly. Incubate at 37℃ for 1 hour;

[0206] Extract once with equal volumes of phenol / chloroform and chloroform; shake gently for 10-15 minutes, centrifuge at 12000 rpm for 15 minutes;

[0207] Add 1 / 10 volume of 3M NaAC to the supernatant, mix thoroughly, then add 2.5 volumes of pre-cooled anhydrous ethanol, mix, place at -20°C for more than 30 min, centrifuge at 12,500 rpm, 4°C for 15 min, and discard the supernatant;

[0208] Wash the DNA precipitate with 70% ethanol, dry it, and dissolve it in an appropriate amount of TE or 300 μL of water;

[0209] Take a small amount of DNA, check its quality on agarose gel and calibrate the DNA amount, and store the remaining DNA samples at -20℃ for future use.

[0210] 2. Probe Design

[0211] The probe primers for target gene detection were designed based on the target gene sequence information in the pK0637 vector as shown in 19.

[0212] Table 19 Primer information used to prepare probes

[0213]

[0214] 3. Restriction Enzyme Digestion

[0215] Take 10g of DNA samples from the test plants and wild-type control plants and use 120μl system for enzyme digestion. The enzymes used include (EcoRⅠ and EcoRⅤ). Add 300μl endonuclease and digest overnight. Add 75μl endonuclease on the next day and continue digestion for 3h.

[0216] 4. Gel Electrophoresis

[0217] a. To prepare 0.7% agarose gel, EB needs to be added; to prepare large slabs of gel, use a large-pore comb;

[0218] b. After the digestion, add 500 μl ultrapure water and 600 μl isopropanol to the digestion product, precipitate at -20°C for 2 h, and centrifuge at 12000 rpm for 10 min;

[0219] c. Discard the supernatant, add 75% ethanol to wash, 6000rpm, 3min. Place on a clean bench to dry;

[0220] d. Add 50 μl ultrapure water to dissolve, add 6 μl 10*lording buffer, and place in a 56°C water bath for 3 min;

[0221] e. Load the sample, let it stand for 2 minutes, and run electrophoresis at 20 V overnight. The loading amount of the positive control is 10 ng.

[0222] 5. Hybridization

[0223] a. Transfer: Take a photo of the gel, cut off the excess gel, and cut a corner at the upper right corner; rinse with deionized water, put in the denaturing buffer, shake for 45 minutes, soak briefly in deionized water, put in the neutralization buffer, shake for 30 minutes, pour it out, replace with new neutralization buffer, and shake for 15 minutes; cut the filter paper into two pieces of 18×38cm, two pieces of the size of the gel block, and one piece of the size of the nylon membrane gel block. Wet the nylon membrane with deionized water for 5-10 minutes; find a white porcelain plate, put a glass plate on it (first wash it with detergent and dry it, then wipe it with alcohol), pour 20×SSC into the white porcelain plate, cover the two filter papers on the glass plate, wet it with 20×SSC and drive away the bubbles. Put the gel block on the filter paper at one time (inverted), cut a corner of the nylon membrane and the gel block at the same position, and two filter papers, and drive away the bubbles each time. Seal the film with a fresh-keeping bag, then put absorbent paper on it, and then put a glass plate on the absorbent paper and press the glass plate with a heavy object. Transfer the film for 1 day.

[0224] b. DNA fixation: After the transfer, write a positive line with a pencil on the side of the nylon membrane that contacts the gel block. Rinse the membrane with 10×SSC and crosslink it with a UV crosslinker for 2 minutes. Then rinse with deionized water and dry at room temperature.

[0225] c. Prehybridization: Preheat an appropriate amount of DIG Easy Hyb (10ml / 100cm 2 At the same time, place the membrane in a hybridization tube with the DNA-bound side facing upwards and add 10 ml (the area of ​​the membrane is less than 100 cm 2 )DIG Easy Hyb, prehybridization at 25°C for 30 min while rotating the hybridization tube.

[0226] d. Hybridization: Dilute the DIG-labeled probe to 25 ng / ml with DIG Easy Hyb (3.5 ml / 100 cm 2 Boil the membrane in boiling water for 5 minutes and quickly put it into an ice bath; add the diluted probe to the preheated DIG Easy Hyb and mix well while avoiding bubbles; pour out the pre-hybridization solution, add the mixed probe, and hybridize overnight at 42°C while rotating the hybridization tube.

[0227] e. Washing membrane: Pour out or recycle the hybridization solution, add 20ml 2×SSC containing 0.1% SDS, and rotate and wash in the hybridization oven at 25℃ for 5min×2; discard the waste solution, add 20ml 0.5×SSC containing 0.1% SDS, and rotate and wash in the hybridization oven at 65℃ for 15min×2; after hybridization and membrane washing, discard the waste solution, add 20ml Washing buffer, and rotate and wash in the hybridization oven at 37℃ for 5min; discard the waste solution, add 100ml Blocking solution, and rotate and incubate in the hybridization oven at 37℃ for 30min; discard the waste solution, add 20ml Antibody solution, and rotate and incubate in the hybridization oven at 37℃ for 30min; discard the waste solution, add 100ml Washing buffer, and rotate and incubate in the hybridization oven at 37℃ for 15min×2; discard the waste solution, add 10ml Detection buffer, and rotate and incubate in the hybridization oven at 37℃ for 5min; cut the hybridization bag, put the DNA side up on the hybridization bag, and add 1ml CSPD Place the membrane on the ready-to-use plate and immediately cover it with the second page of the hybridization bag without any bubbles. Incubate at room temperature for 5 minutes. Squeeze out excess liquid and any bubbles that may be present. Seal the edges with an edge sealer. Place the membrane at 37°C for 10 minutes to enhance the luminescence reaction.

[0228] f. Imaging: Place the membrane in the Tianneng luminescence imaging system and adjust the exposure time to 1-15 minutes to obtain the best Southern hybridization photos.

[0229] 6. Results and Discussion

[0230] The hybridization results of the specific probe for the cp4-epsps gene are summarized in Table 20.

[0231] (1) Hybridization results of the positive control: After the pK0637 vector plasmid was digested with EcoRI, which has only one restriction site, Southern hybridization was performed with a probe specific for the target gene cp4-epsps. Only a band of approximately 10.5 kb was detected, which was consistent with expectations (see Figure 8 A and B, Note: Figure 8 The concentration of agarose gel is 1.5%). Since the marker used is pre-labeled with digoxigenin, its migration rate is slightly slower than that of unlabeled sample DNA during electrophoresis, resulting in the indicated band size being smaller than the actual size. The specific degree of deviation is shown in Figure 8 Middle C.

[0232] (2) Analysis of hybridization results of specific probes for the cp4-epsps gene of the KJ7002 event

[0233] like Figure 8As shown in A, when EcoRI was used for digestion, the hybridization bands of KJ7002 transformants T4, T5 and T6 were all about 15 kb, which was in line with the expectation of being larger than 4.1 kb. The negative control (receptor B104) did not have any hybridization bands. Figure 8 As shown in B, when EcoRⅤ was used for digestion, the hybridization bands of KJ7002 transformant T4, T5 and T6 generations were only one hybridization band located in the middle of 2322-4361bp. The marker used in Southern hybridization was DIG marker, and the migration rate was slowed down. The difference in electrophoretic mobility was compared. Figure 8 In the middle C, the size of the 1kb marker band without DIG marker in the middle of the two bands of 2322-4361bp is 4000bp. Therefore, it is inferred that when EcoRⅤ is digested, the hybridization band size of KJ7002 T4, T5 and T6 generation materials is about 4kb, which is in line with the expectation of being greater than 3.9kb. The obtained flanking sequence was analyzed for endonuclease restriction sites, and it was found that EcoRⅤ restriction sites were indeed found on the 3' end flanking, and the distance from the insertion sequence EcoRⅤ restriction site was 3956bp, confirming this Southern hybridization band of about 4kb. The negative control (receptor B104) did not have any hybridization bands. Both enzyme digestions showed that the integration of cp4-epsps gene in the genome was single copy.

[0234] Table 20 Specific probe hybridization results of cp4-epsps gene

[0235]

[0236]

[0237] In summary, the hybridization results of the cp4-epsps probe indicated that the T-DNA region of the vector pK0637 had been integrated into the genome of event KJ7002 in the form of a single copy and could be stably inherited in different generations.

[0238] Example 5: Herbicide tolerance testing of KJ7002 event

[0239] 1. Experimental Materials and Methods

[0240] (1) Plant materials: transgenic maize event KJ7002 and non-transgenic maize recipient control B104.

[0241] (2) Herbicide: 41% Roundup produced by Monsanto (recommended dosage: 150-250 ml / mu, 200 ml / mu is 1×, 400 ml is 2×, and so on)

[0242] (3) The transformants and controls were planted in 3 replicates, with a distance of more than 1 meter between each other, a row spacing of 60 cm, and a plant spacing of 25 cm. The sowing was carried out according to the conventional sowing time, sowing method and sowing amount of summer corn. The medium dose and water control dose of 4 times the recommended dose on the pesticide label were set, and the water volume was 450L / hectare. The stem and leaf treatment was carried out after the seedlings. Glyphosate was sprayed when the corn was 3-5 leaves. A sprayer with constant pressure, step and time recording and a fan-shaped nozzle was used to spray the stems and leaves with herbicides or water. The dose of each herbicide was consistent with the water consumption of the control. Other management was carried out according to local conventional methods.

[0243] (4) Investigation and Recording

[0244] The seedling rate, plant height and drug damage symptoms were investigated and recorded 1 week, 2 weeks and 4 weeks after drug application. The drug damage symptoms included growth inhibition, chlorosis, dead spots, deformities, etc.

[0245] Seedling rate: Before spraying, investigate the number of seedlings in the plot; after spraying, investigate the number of surviving plants.

[0246] Plant height: Select the 5 tallest plants.

[0247] Symptoms of drug damage: Select 5 plants with the least drug damage.

[0248] The classification standards for pesticide injury symptoms shall be implemented in accordance with the Agricultural Office of Agriculture

[2022] No. 3, as follows:

[0249] Level 0: normal growth, no symptoms of pesticide damage, and consistent with the growth of the control sprayed with water;

[0250] Level 1: Slightly damaged by pesticide, with yellowing of the heart leaves or pesticide spots occupying less than 10% of the leaf area;

[0251] Level 2: Mild growth inhibition or chlorosis, with drug-damaged spots occupying less than 1 / 4 of the leaf area;

[0252] Level 3: Moderate damage, with a significant impact on growth and development, dwarfing of plants or leaf deformities, or damage spots occupying less than 1 / 2 of the leaf area;

[0253] Level 4: The damage caused by the drug is serious, which has a great impact on growth and development. The plant is obviously dwarfed or the leaves are seriously deformed or the drug-damaged spots occupy less than 3 / 4 of the leaf area;

[0254] Level 5: The drug damage is extremely serious, the plant dies or the drug-damaged spots occupy more than 3 / 4 of the leaf area.

[0255] After corn harvest, 10 ears were selected from the middle 2 rows of each plot for indoor testing to determine agronomic traits related to corn yield.

[0256] (3) Data processing

[0257] The victimization rate is calculated according to the formula shown:

[0258]

[0259] Note: X—damage rate, in percentage (%); N—number of damaged plants at the same level; S—level number; T—total number of plants; M—highest level. The calculation result shall be rounded to two significant figures after the decimal point.

[0260] The variance analysis method was used to compare the differences in seedling rate, damage rate and plant height between transgenic corn and non-transgenic control corn under different treatments, and to determine the tolerance level of transgenic corn to the target herbicide glyphosate.

[0261] 2. Results and Analysis

[0262] (1) Seedling rate

[0263] During the whole test period, the seedling rate of transformant KJ7002 and receptor control B104 under the condition of spraying water was 100%. As shown in Table 21, under the condition of spraying 4× glyphosate, the seedling rate of transgenic corn was 100% at 1 week, 2 weeks and 4 weeks after the drug application, which was no different from the clear water control; all the receptor controls died, and the seedling rate was 0, which was significantly lower than the clear water control.

[0264] Table 21 Seedling rate at different times of glyphosate treatment (%)

[0265]

[0266] Note: Data are expressed as mean ± standard deviation.

[0267] (2) Victim rate

[0268] Table 22: Damage rate of transformant KJ7002 after spraying 4 times glyphosate

[0269]

[0270] Note: Data are expressed as mean ± standard deviation.

[0271] As shown in Table 22 and Fig. 9 As shown in the figure, after spraying with water, there were no damaged plants in both transgenic corn and non-transgenic corn materials; the transformant KJ7002 was able to basically maintain a good growth state in 1 week, 2 weeks and 4 weeks under the treatment of spraying with 4 times the concentration, without moderate phytotoxicity symptoms such as leaf chlorosis, wilting, deformity, plant height reduction, growth slowdown or even stagnation, and the calculated damage rate was between 0.9-3.19%, all of which were minor phytotoxicity. After 4 weeks, the damage rate decreased and most of the plants returned to normal. Non-transgenic corn lost green 3 days after spraying with 1 times the concentration of glyphosate, and all plants gradually wilted, dried up and died over time, with a phytotoxicity rate of 100%. Therefore, according to GB / T19780.42, the glyphosate tolerance level of transgenic corn KJ7002 was determined to be high resistance.

[0272] (3) Output

[0273] The yield-related traits of the transformant KJ7002 after spraying with glyphosate were followed up and tested. The results are shown in Table 23, indicating that after spraying with 4 times the concentration of glyphosate, the main yield-related traits (ear length, ear diameter, number of ear rows, and 100-grain weight) of the transgenic corn KJ7002 were improved compared with the transgenic materials of the clear water control and the non-transgenic control of clear water, indicating that even when sprayed with 4 times the concentration of glyphosate, the ear and yield traits of the transgenic corn KJ7002 were slightly improved. This further indicates that the transgenic corn event KJ7002 has good tolerance to herbicides (glyphosate).

[0274] Table 23 Yield-related traits of transformant KJ7002

[0275]

[0276] Note: Data are expressed as mean ± standard deviation. The difference significance was analyzed using Turkey's model test, n = 15. The same lowercase letters indicate no significant difference in the same column of data (P < 0.05). "--" indicates that the plant died and could not be measured.

Claims

1. A nucleic acid molecule, characterized in that The sequence of the nucleic acid molecule includes the sequence SEQ ID NO:7 or its complementary sequence; The nucleic acid molecule is derived from the plant, seed or cell of the transgenic corn event KJ7002. The representative sample of the seeds of the event has been deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: P202409.

2. A DNA primer pair for detecting the presence of transgenic corn event KJ7002, the primer pair comprising a first primer and a second primer, characterized in that: The first primer is SEQ ID NO: 1, and the second primer is the complementary sequence of SEQ ID NO: 2; or the first primer is the complementary sequence of SEQ ID NO: 1, and the second primer is SEQ ID NO: 2; and a representative sample of the seeds of the event has been deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: P202409.

3. A DNA detection kit, characterized in that: Comprising the DNA primer pair described in claim 2.

4. A method for protecting corn plants from damage caused by herbicides, characterized in that At least one transgenic corn plant comprising transgenic corn event KJ7002 is planted, a representative sample of seeds of the event has been deposited in China Center for Type Culture Collection with deposit number CCTCC NO: P202409, and the herbicide is glyphosate herbicide.

5. A method for controlling weeds in a field of corn plants, characterized in that: The method comprises applying an effective amount of glyphosate herbicide to a field planted with at least one transgenic corn plant comprising transgenic corn event KJ7002, a representative sample of seeds of which has been deposited in China Center for Type Culture Collection with the deposit number CCTCC NO: P202409.

6. A method for growing corn plants tolerant to glyphosate herbicides, characterized in that: include: sexually crossing the transgenic corn event KJ7002 as a first parent corn plant with a second parent corn plant lacking tolerance to glyphosate herbicide, thereby producing a plurality of progeny plants; The corn plants were sprayed with an effective dose of glyphosate herbicide and the plants were harvested with reduced plant damage compared to other plants not having the transgenic corn event KJ7002; representative samples of seeds of the event have been deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: P202409.

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