CN113278721B

CN113278721B - Transgenic corn event LW2-2 and detection method thereof

Info

Publication number: CN113278721B
Application number: CN202110461014.3A
Authority: CN
Inventors: 李晓娇; 贾志伟; 王强; 吕玉平; 刘枫; 赵晓娜; 孙宇; 董雷; 李胜兵; 赵丽媛; 李涛; 张原�; 卢娟; 韩雨颖; 易金麒
Original assignee: Longping Biotechnology Hainan Co ltd
Current assignee: Longping Biotechnology Hainan Co ltd
Priority date: 2021-04-27
Filing date: 2021-04-27
Publication date: 2023-08-15
Anticipated expiration: 2041-04-27
Also published as: CN113278721A

Abstract

The application discloses a nucleic acid sequence, which comprises one or more selected from the sequences SEQ ID NO. 1-7 and the complementary sequences thereof, wherein the nucleic acid sequence is derived from plants, seeds or cells comprising corn event LW2-2, and a representative sample of the seeds comprising the event has been preserved with the preservation number CCTCC NO. P202022; the application also discloses a detection method for detecting the LW2-2 event. The transgenic corn event LW2-2 has better tolerance to glyphosate herbicide and glufosinate herbicide, and the detection method can accurately and rapidly identify whether plant materials comprising the transgenic corn event LW2-2 exist in a biological sample.

Description

Transgenic corn event LW2-2 and detection method thereof

Technical Field

The application relates to the technical field of molecular biology, relates to a detection method of transgenic plants and products thereof, in particular to a transgenic corn event LW2-2 which is tolerant to glyphosate herbicide application, and a nucleic acid sequence and a method for detecting the transgenic corn LW 2-2.

Background

N-phosphonomethylglycine, also known as glyphosate, is a systemic conduction type chronic broad spectrum biocidal herbicide. Glyphosate is a competitive inhibitor of phosphoenolpyruvic acid (PEP) which is a 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 aromatic amino acid synthesis precursor-shikimate, and causing the death of plants and bacteria due to interference of protein synthesis.

Glyphosate tolerance can be achieved by expression of modified EPSPS. The modified EPSPS have a lower affinity for glyphosate and thus in the presence of glyphosate, EPSPS retain their catalytic activity, i.e., glyphosate tolerance is achieved.

Corn (Zea mays l.) is the predominant food crop in many parts of the world. Herbicide tolerance is an important agronomic trait in corn production, particularly tolerance to glyphosate herbicides. Tolerance of corn to glyphosate herbicide can be obtained by transgenic approaches to express the glyphosate herbicide tolerance gene (EPSPS, CP 4) in corn plants, such as corn event NK603, corn event MON88017, and the like.

The widespread adoption of glyphosate-tolerant farming systems and the increasing use of glyphosate have led to the recent popularity of glyphosate-resistant weeds. In areas where growers are facing glyphosate resistant weeds or are transitioning to more difficult weed species to control, growers can compensate for the vulnerability of glyphosate by mixing or alternating with other herbicides that are capable of controlling the missing weeds.

Glufosinate is a non-systematic, non-selective herbicide among phosphinothricin herbicides. The method is mainly used for controlling annual or perennial broadleaf weeds after soil emergence, and is used for controlling the weeds through irreversible inhibition of glutamine synthase (an enzyme necessary for detoxification of ammonia in plants) by L-phosphinothricin (an active ingredient in glufosinate). Unlike glyphosate root killing, glufosinate leaf killing is performed first, and can be conducted on plant xylem through plant transpiration, and the quick acting property of glufosinate leaf killing is between paraquat and glyphosate.

The enzyme phosphinothricin N-acetyltransferase (PAT) isolated from Streptomyces catalyzes the conversion of L-phosphinothricin to its inactive form by acetylation. Plant-optimized forms of genes expressing PAT have been used in soybeans to confer tolerance to glufosinate herbicides to soybeans, such as soybean event a5547-127. The use of glufosinate herbicide in combination with glufosinate tolerance traits can therefore be a non-selective means of effectively managing glyphosate resistant weeds.

Meanwhile, with large-area planting of transgenic insect-resistant corn, a small number of surviving insects/pests may develop resistance after several generations of propagation. The herbicide-resistant transgenic corn is used as non-insect-resistant transgenic corn, and is planted together with the transgenic insect-resistant corn in a certain proportion, so that the generation of drug resistance of insects/pests can be delayed.

Expression of exogenous genes in plants is known to be affected by their chromosomal location, possibly due to the proximity of chromatin structures (e.g., heterochromatin) or transcriptional regulatory elements (e.g., enhancers) to the integration site. For this reason, it is often necessary to screen a large number of events to make it possible to identify events that can be commercialized (i.e., events in which the introduced target gene is optimally expressed). For example, it has been observed in plants and other organisms that the expression level of the introduced gene may vary greatly between events; there may also be differences in the spatial or temporal pattern of expression, such as differences in the relative expression of transgenes between different plant tissues, which differences may be manifested in actual expression patterns that are inconsistent with the expression patterns expected from the transcriptional regulatory elements in the introduced gene construct. Thus, it is often desirable to generate hundreds or thousands of different events and screen those events for a single event having transgene expression levels and patterns that are expected for commercialization purposes. Such transformation events are dual resistant to glyphosate herbicide and glufosinate herbicide and do not affect corn yield, and conventional breeding methods can be used to backcross transgenes into other genetic backgrounds by crossing. The progeny produced by this crossing maintains the transgene expression characteristics and trait performance of the original transformant. The application of the strategy mode can ensure reliable gene expression in a plurality of varieties, has stable glyphosate and glufosinate herbicide resistance, has broad-spectrum weed control capability, and can be well adapted to the growth conditions of places.

It would be beneficial to be able to detect the presence of a particular event to determine whether the progeny of a sexual cross contain a gene of interest. In addition, methods of detecting specific events will also help to comply with relevant regulations, such as the need for formal approval and marking of foods derived from recombinant crops prior to their being put on the market. It is possible to detect the presence of the transgene by any well known method of nucleic acid detection, such as Polymerase Chain Reaction (PCR) or DNA hybridization using nucleic acid probes. These detection methods are generally focused on commonly used genetic elements such as promoters, terminators, marker genes, and the like. Thus, unless the sequence of chromosomal DNA adjacent to the inserted transgenic DNA ("flanking DNA") is known, such a method as described above cannot be used to distinguish between different events, particularly those generated with the same DNA construct. Therefore, it is common today to identify a transgene specific event by PCR using a pair of primers spanning the junction of the inserted transgene and flanking DNA, specifically a first primer comprising the flanking sequence and a second primer comprising the inserted sequence.

Disclosure of Invention

The invention aims to provide a transgenic corn plant LW2-2 with better tolerance to glyphosate herbicide and glufosinate herbicide, a nucleic acid sequence for detecting herbicide-tolerant corn plant LW2-2 and a detection method thereof, wherein the transgenic corn event LW2-2 has better tolerance to glyphosate herbicide and glufosinate herbicide, and the detection method can accurately and quickly identify whether a biological sample contains DNA molecules of specific transgenic corn event LW 2-2.

To achieve the above object, the present invention provides a nucleic acid sequence comprising one or more selected from the group consisting of sequences SEQ ID NO 1-7 (i.e., 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) and the complements thereof. In some embodiments, the nucleic acid sequence is derived from a plant, seed, or cell comprising corn event LW2-2, and a representative sample of seed comprising the event has been deposited at China center for type culture collection (CCTCC, address: wuchan district, wuhan, hubei province, eight-way 299, university of Wuhan, inc., post code 430072) with a deposit number of CCTCC NO: P202022. In some facts, the nucleic acid sequence is an amplicon diagnostic for the presence of corn event LW 2-2.

In some embodiments, the nucleic acid sequence comprises at least 11 consecutive nucleotides of SEQ ID NO. 3 or its complement, and/or at least 11 consecutive nucleotides of SEQ ID NO. 4 or its complement. In some embodiments, the nucleic acid sequence comprises SEQ ID NO. 1 or a complement thereof, and/or SEQ ID NO. 2 or a complement thereof. In some embodiments, the nucleic acid sequence comprises SEQ ID NO. 3 or a complement thereof, and/or SEQ ID NO. 4 or a complement thereof. In some embodiments, the nucleic acid sequence comprises SEQ ID NO. 5 or a complement thereof.

The SEQ ID NO. 1 or the complementary sequence thereof is a sequence with the length of 22 nucleotides, which is positioned near the insertion junction at the 5 '-end of the insertion sequence in the transgenic corn event LW2-2, the SEQ ID NO. 1 or the complementary sequence thereof spans the flanking genomic DNA sequence of the corn insertion site and the DNA sequence at the 5' -end of the insertion sequence, and the existence of the transgenic corn event LW2-2 can be identified by the SEQ ID NO. 1 or the complementary sequence thereof. The SEQ ID NO. 2 or the complementary sequence thereof is a sequence with the length of 26 nucleotides, which is positioned near the insertion junction at the 3 '-end of the insertion sequence in the transgenic corn event LW2-2, the SEQ ID NO. 2 or the complementary sequence thereof spans the DNA sequence at the 3' -end of the insertion sequence and the flanking genomic DNA sequence of the corn insertion site, and the existence of the transgenic corn event LW2-2 can be identified by the SEQ ID NO. 2 or the complementary sequence thereof.

In the present invention, the nucleic acid sequence may be at least 11 or more contiguous polynucleotides of any portion of the transgene insert sequence in SEQ ID NO. 3 or its complement (first nucleic acid sequence), or at least 11 or more contiguous polynucleotides of any portion of the 5' flanking maize genomic DNA region in SEQ ID NO. 3 or its complement (second nucleic acid sequence). The nucleic acid sequence may further be homologous or complementary to a portion of the SEQ ID NO. 3 comprising 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 comprise a set of DNA primers in a DNA amplification method that produces an amplification product. The presence of transgenic maize event LW2-2 or its progeny can be diagnosed when the amplification product produced in the DNA amplification method using the DNA primer pair is an amplification product comprising SEQ ID NO. 1. It is well known to those skilled in the art that the first and second nucleic acid sequences need not consist of only DNA, but may include RNA, a mixture of DNA and RNA, or a combination of DNA, RNA, or other nucleic acids or analogs thereof that do not serve as templates for one or more polymerases. Furthermore, the probes or primers described in 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 may be selected from the nucleic acids set forth in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5. When selected from the group consisting of the nucleic acids set forth in SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5, the probes and primers may be at least about 17 to about 50 or more contiguous nucleotides in length. The sequence of SEQ ID NO. 3 or its complement is 1630 nucleotides in length near the 5 'end of the insertion junction in transgenic maize event LW2-2, the SEQ ID NO. 3 or its complement consists of 1070 nucleotides of maize flanking genomic DNA sequence (nucleotides 1-1070 of SEQ ID NO. 3), 366 nucleotides of pLW2 construct DNA sequence (nucleotides 1071-1436 of SEQ ID NO. 3) and 194 nucleotides of the 3' end DNA sequence of the CaMv35S terminator sequence (nucleotides 1437-1630 of SEQ ID NO. 3) comprising the SEQ ID NO. 3 or its complement can be identified as the presence of transgenic maize event LW 2-2.

The nucleic acid sequence may be at least 11 or more contiguous polynucleotides (third nucleic acid sequence) of any portion of the transgene insert sequence in the SEQ ID NO. 4 or its complement, or at least 11 or more contiguous polynucleotides (fourth nucleic acid sequence) of any portion of the 3' flanking maize genomic DNA region in the SEQ ID NO. 4 or its complement. The nucleic acid sequence may further be homologous or complementary to a portion of the SEQ ID NO. 4 comprising 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 comprise a set of DNA primers in a DNA amplification method that produces an amplification product. The presence of transgenic maize event LW2-2 or its progeny can be diagnosed when the amplification product produced in the DNA amplification method using the DNA primer pair is an amplification product comprising SEQ ID NO. 2. The SEQ ID NO. 4 or its complement is a 1515 nucleotide long sequence near the insertion junction at the 3' end of the insertion sequence in transgenic maize event LW2-2, consisting of a 195 nucleotide t35S terminator sequence (nucleotides 1-195 of SEQ ID NO. 4), a 152 nucleotide pLW2 construct DNA sequence (nucleotides 196-347 of SEQ ID NO. 4) and 1132 nucleotide maize integration site flanking genomic DNA sequence (348-1479 of SEQ ID NO. 4), comprising the SEQ ID NO. 4 or its complement, can be identified as the presence of transgenic maize event LW 2-2.

The SEQ ID NO. 5 or its complement is a 7280 nucleotide sequence characterizing transgenic maize event LW2-2, which specifically comprises the genome and genetic elements as shown in Table 1. The presence of transgenic maize event LW2-2 can be identified by inclusion of the SEQ ID NO 5 or its complement.

Table 1, genome and genetic element contained in SEQ ID NO. 5

The nucleic acid sequence or the complement thereof can be used in a DNA amplification method to produce an amplicon, the detection of which diagnoses the presence of transgenic corn event LW2-2 or a progeny thereof in a biological sample; the nucleic acid sequence or its complement can be used in a nucleic acid detection method to detect the presence of transgenic corn event LW2-2 or its progeny in a biological sample.

The present invention provides a DNA primer pair comprising a first primer and a second primer, each comprising a partial sequence of SEQ ID No. 5 or a complement thereof, and when used in an amplification reaction with DNA comprising corn event LW2-2, produces an amplicon that detects corn event LW2-2 in a sample.

In some embodiments, the first primer is selected from the group consisting of SEQ ID NO. 1 or a complement thereof, SEQ ID NO. 8 or SEQ ID NO. 12; the second primer is selected from SEQ ID NO. 2 or a complementary sequence thereof, SEQ ID NO. 11 or SEQ ID NO. 14.

In some embodiments of the invention, the amplification product comprises at least 11 consecutive nucleotides of SEQ ID NO. 3 or its complement, or at least 11 consecutive nucleotides of SEQ ID NO. 4 or its complement.

In some embodiments, the amplification product comprises consecutive nucleotides 1-11 or 12-22 of SEQ ID NO. 1 or its complement, or consecutive nucleotides 1-11 or 12-22 of SEQ ID NO. 2 or its complement.

In some embodiments, the amplification product comprises SEQ ID NO. 1 or its complement, SEQ ID NO. 2 or its complement, SEQ ID NO. 6 or its complement, or SEQ ID NO. 7 or its complement.

In the above technical scheme, the primer comprises at least one of the nucleic acid sequences. Specifically, the primer comprises a first primer and a second primer, wherein the first primer is selected from SEQ ID NO. 1 or a complementary sequence thereof, SEQ ID NO. 8 or SEQ ID NO. 12, and the second primer is selected from SEQ ID NO. 9 or SEQ ID NO. 13; or the first primer is selected from SEQ ID NO. 2 or a complementary sequence thereof, SEQ ID NO. 11 or SEQ ID NO. 14, and the second primer is selected from SEQ ID NO. 10 or SEQ ID NO. 15.

The present invention also provides a DNA probe comprising a fragment of SEQ ID NO. 5 or a complementary sequence thereof, which hybridizes under stringent hybridization conditions to a DNA molecule comprising a nucleic acid sequence selected from SEQ ID NO. 1-7 or a complementary sequence thereof and does not hybridize under stringent hybridization conditions to a DNA molecule not comprising a nucleic acid sequence selected from SEQ ID NO. 1-7 or a complementary sequence thereof.

In some embodiments, the DNA probe comprises a sequence selected from the group consisting of SEQ ID NO. 1 or its complement, SEQ ID NO. 2 or its complement, SEQ ID NO. 6 or its complement, and SEQ ID NO. 7 or its complement.

In some embodiments, the DNA probe is labeled with a fluorescent group.

In some embodiments, the probe comprises at least 11 consecutive nucleotides of SEQ ID NO. 3 or its complement, or at least 11 consecutive nucleotides of SEQ ID NO. 4 or its complement; further, the probe comprises continuous nucleotides at positions 1-11 or 12-22 in SEQ ID NO. 1 or the complementary sequence thereof, or continuous nucleotides at positions 1-11 or 12-22 in SEQ ID NO. 2 or the complementary sequence thereof.

The present invention also provides a marker nucleic acid molecule comprising a partial sequence of SEQ ID NO. 5 or a complement thereof, which marker nucleic acid molecule hybridizes under stringent hybridization conditions to a DNA molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO. 1-7 or a complement thereof and does not hybridize under stringent hybridization conditions to a DNA molecule not comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO. 1-7 or a complement thereof.

In some embodiments, the marker nucleic acid molecule comprises a sequence selected from the group consisting of SEQ ID NO. 1 or its complement, SEQ ID NO. 2 or its complement, SEQ ID NO. 6 or its complement, and SEQ ID NO. 7 or its complement.

In some embodiments, the marker nucleic acid molecule comprises at least 11 consecutive nucleotides of SEQ ID NO. 3 or its complement, or at least 11 consecutive nucleotides of SEQ ID NO. 4 or its complement;

in some embodiments, the marker nucleic acid molecule comprises consecutive nucleotides 1-11 or 12-22 of SEQ ID NO. 1 or its complement, or consecutive nucleotides 1-11 or 12-22 of SEQ ID NO. 2 or its complement.

Further, the present invention provides a method of detecting the presence of DNA comprising transgenic corn event LW2-2 in a sample comprising:

(1) Contacting a sample to be detected with the pair of DNA primers in a nucleic acid amplification reaction;

(2) Performing a nucleic acid amplification reaction;

(3) Detecting the presence of an amplification product;

the amplification product comprises a nucleic acid sequence selected from the group consisting of the sequences SEQ ID NOs 1-7 and their complements, i.e., is indicative of the presence of DNA comprising the transgenic corn event LW2-2 in the test sample.

The invention also provides a method of detecting the presence of DNA comprising transgenic maize event LW2-2 in a sample comprising:

(1) Contacting a sample to be detected with said DNA probe, and/or said marker nucleic acid molecule;

(2) Hybridizing the sample to be detected with the probe and/or the marker nucleic acid molecule under stringent hybridization conditions;

(3) Detecting hybridization of the sample to be detected with the probe and/or the marker nucleic acid molecule.

The stringent conditions may be hybridization in 6 XSSC (sodium citrate), 0.5% SDS (sodium dodecyl sulfate) solution at 65℃and then washing the membrane 1 time with 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS, respectively.

In some embodiments of the invention, the marker nucleic acid molecule comprises at least 11 consecutive nucleotides of SEQ ID NO. 3 or its complement, or at least 11 consecutive nucleotides of SEQ ID NO. 4 or its complement;

detecting hybridization of the sample to be detected and the marker nucleic acid molecule, and further determining that glyphosate tolerance and/or glufosinate tolerance are genetically linked to the marker nucleic acid molecule by marker-assisted breeding analysis.

Further, the marker nucleic acid molecule comprises the 1 st to 11 th or 12 th to 22 th continuous nucleotide in SEQ ID NO. 1 or the complementary sequence thereof, or the 1 st to 11 th or 12 th to 22 th continuous nucleotide in SEQ ID NO. 2 or the complementary sequence thereof.

Still further, the marker nucleic acid molecule has SEQ ID NO. 1 or a complement thereof, SEQ ID NO. 2 or a complement thereof, SEQ ID NO. 6 or a complement thereof, or SEQ ID NO. 7 or a complement thereof.

In some embodiments, the invention also provides a DNA detection kit comprising: a probe specific for SEQ ID NO. 5, a DNA primer pair that produces an amplicon diagnostic for transgenic maize event LW2-2, or a marker nucleic acid molecule specific for SEQ ID NO. 5. In some embodiments, the kit comprises a primer pair, probe or marker nucleic acid molecule of the invention. In some embodiments, the kit comprises at least one DNA molecule comprising at least 11 contiguous nucleotides of the homologous sequence of SEQ ID NO. 3 or the complement thereof, or at least 11 contiguous nucleotides of the homologous sequence of SEQ ID NO. 4 or the complement thereof, which can serve as a DNA primer or probe specific for transgenic maize event LW2-2 or its progeny.

Further, the DNA molecule comprises the continuous nucleotides at positions 1-11 or 12-22 in SEQ ID NO. 1 or the complementary sequence thereof, or the continuous nucleotides at positions 1-11 or 12-22 in SEQ ID NO. 2 or the complementary sequence thereof.

Still further, the DNA molecule has a sequence homologous to SEQ ID NO. 1 or a sequence complementary thereto, a sequence homologous to SEQ ID NO. 2 or a sequence complementary thereto, a sequence homologous to SEQ ID NO. 6 or a sequence complementary thereto, or a sequence homologous to SEQ ID NO. 7 or a sequence complementary thereto. To achieve the above object, the present invention also provides a plant cell comprising a nucleic acid sequence encoding a glufosinate herbicide tolerant PAT protein, a nucleic acid sequence encoding a glyphosate herbicide tolerant EPSPS protein, and a nucleic acid sequence of a specific region, wherein the nucleic acid sequence of the specific region comprises a sequence shown as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 6, or SEQ ID No. 7.

The sequences provided by the present invention include the sequences listed in table 2 below:

TABLE 2 related sequences of the invention

In some embodiments, the invention also provides a plant cell comprising a nucleic acid sequence encoding a glyphosate tolerant EPSPS protein, a nucleic acid sequence encoding a glufosinate tolerant PAT protein, and a specific region of a nucleic acid sequence comprising a sequence set forth in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 6, or SEQ ID No. 7.

In some embodiments, the invention also provides a method of producing a maize plant that is tolerant to glyphosate herbicide, comprising introducing into the genome of the maize plant transgenic maize event LW2-2, and selecting a maize plant that is tolerant to glyphosate. In some embodiments of the invention, the method comprises introducing into the genome of the maize plant a nucleic acid sequence encoding a glyphosate tolerant EPSPS protein and a specific region of a nucleic acid sequence selected from at least one of the sequences set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO: 7.

In particular, the method of producing a corn plant tolerant to glyphosate herbicide comprises:

Sexual crossing a transgenic corn event LW2-2 first parent corn plant having tolerance to a glyphosate herbicide with a second parent corn plant lacking glyphosate tolerance, thereby producing a plurality of progeny plants;

treating said progeny plants with a glyphosate herbicide;

selecting said progeny plants that are tolerant to glyphosate.

In some embodiments, the invention also provides a method of producing a maize plant that is tolerant to glufosinate herbicide, comprising: transgenic maize event LW2-2 was introduced into the genome of the maize plants and glufosinate tolerant maize plants were selected. In some embodiments, the method comprises introducing into the genome of the maize plant a nucleic acid sequence encoding a glufosinate-tolerant PAT protein and a specific region of a nucleic acid sequence selected from at least one of the sequences set forth in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, and SEQ ID No. 7.

In particular, the method of producing a maize plant that is tolerant to glufosinate herbicide comprises:

sexual crossing a transgenic corn event LW2-2 first parent corn plant having tolerance to glufosinate herbicide with a second parent corn plant lacking glufosinate tolerance, thereby producing a plurality of progeny plants;

Treating said progeny plants with a glufosinate herbicide;

selecting said progeny plants that are tolerant to glyphosate.

In some embodiments, the invention also provides a method of producing a corn plant tolerant to glyphosate herbicide and glufosinate herbicide comprising: transgenic maize event LW2-2 was introduced into the genome of the maize plants and maize plants tolerant to glyphosate and glufosinate were selected. In some embodiments, the method comprises introducing into the genome of the maize plant a nucleic acid sequence encoding a glyphosate tolerant EPSPS protein, a nucleic acid sequence encoding a glufosinate tolerant PAT protein, and a nucleic acid sequence of a particular region selected from at least one of the sequences set forth in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, and SEQ ID No. 7.

In particular, the method of producing a corn plant tolerant to glyphosate herbicide and glufosinate herbicide comprises:

sexual crossing a transgenic corn event LW2-2 first parent corn plant having tolerance to glyphosate herbicide and glufosinate herbicide with a second parent corn plant lacking glyphosate and/or glufosinate tolerance, thereby producing a plurality of progeny plants;

Treating the progeny plants with a glyphosate herbicide and a glufosinate herbicide;

selecting said progeny plants that are tolerant to glyphosate and glufosinate.

In some embodiments, the invention also provides a method of culturing a corn plant tolerant to a glyphosate herbicide comprising:

planting at least one corn seed comprising transgenic corn event LW 2-2;

growing the corn seed into a corn plant;

spraying the corn plants with an effective dose of glyphosate herbicide, and harvesting plants having reduced plant damage as compared to other plants not having the transgenic corn event LW 2-2.

In some embodiments, the invention provides a method of culturing a corn plant tolerant to a glyphosate herbicide comprising:

planting at least one corn seed comprising in its genome a nucleic acid sequence encoding a glyphosate tolerant EPSPS and a nucleic acid sequence of a specified region;

growing the corn seed into a corn plant;

spraying the maize plant with an effective dose of a glyphosate herbicide to harvest plants having reduced plant damage as compared to other plants not having the nucleic acid sequence of the specified region;

The nucleic acid sequence of 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, SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7.

In some embodiments, the present invention also provides a method of culturing a maize plant that is tolerant to glufosinate herbicide, comprising:

planting at least one corn seed comprising transgenic corn event LW 2-2;

growing the corn seed into a corn plant;

spraying the maize plants with an effective dose of glufosinate herbicide, and harvesting plants having reduced plant damage compared to other plants not having the transgenic maize event LW 2-2.

In some embodiments of the invention, there is provided a method of culturing a maize plant that is tolerant to glufosinate herbicide comprising:

planting at least one corn seed comprising in its genome a nucleic acid sequence encoding a glufosinate-tolerant PAT protein and a nucleic acid sequence of a specific region;

growing the corn seed into a corn plant;

spraying the maize plant with an effective dose of glufosinate herbicide to harvest plants having reduced plant damage compared to other plants not having the nucleic acid sequence of the specific region;

In some embodiments, the present invention provides a method of growing a corn plant tolerant to a glufosinate herbicide and a glufosinate herbicide comprising:

planting at least one corn seed comprising transgenic corn event LW 2-2;

growing the corn seed into a corn plant;

spraying the corn plants with an effective dose of glufosinate herbicide and glufosinate herbicide to harvest plants having reduced plant damage compared to other plants not having the transgenic corn event LW 2-2.

In some embodiments, the invention also provides a method of culturing a corn plant tolerant to glyphosate herbicide and glufosinate herbicide comprising:

planting at least one corn seed comprising in its genome a nucleic acid sequence encoding a glyphosate tolerant EPSPS protein, a nucleic acid sequence encoding a glufosinate tolerant PAT protein, and a nucleic acid sequence of a specific region;

growing the corn seed into a corn plant;

Spraying the maize plants with an effective dose of a glyphosate herbicide and a glufosinate herbicide to harvest plants having reduced plant damage compared to other plants not having the nucleic acid sequence of the particular region;

In some embodiments, the present invention also provides a method of protecting a corn plant from herbicide-induced damage comprising growing at least one transgenic corn plant comprising transgenic corn event LW2-2, in particular comprising applying an effective dose of a herbicide comprising glyphosate and/or glufosinate to a field in which at least one transgenic corn plant comprising transgenic corn event LW2-2, in some embodiments, said transgenic corn plant comprising 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, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7 has tolerance to a glyphosate herbicide and/or glufosinate herbicide.

In some embodiments, the present invention also provides a method of controlling weeds in a field comprising applying an effective dose of glyphosate and/or glufosinate herbicide to a field in which at least one transgenic corn plant comprising transgenic corn event LW2-2 is grown. In some embodiments, the transgenic corn plant comprises in its genome at least one nucleic acid sequence selected from the group consisting of the sequences set forth in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, and SEQ ID NO. 7, said transgenic corn plant having tolerance to glyphosate herbicide and/or glufosinate herbicide.

In some embodiments, the present invention also provides a method of controlling glyphosate resistant weeds in a field of glyphosate tolerant plants, comprising applying an effective dose of a herbicide comprising glufosinate to the field in which at least one glyphosate tolerant transgenic corn plant comprising transgenic corn event LW2-2 is grown. In particular, in some embodiments, the glyphosate tolerant transgenic corn plant comprises in its genome at least one nucleic acid sequence selected from the group consisting of the sequences set forth in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, and SEQ ID NO. 7, which is also tolerant to a glufosinate herbicide.

In some embodiments, the invention also provides a processed product resulting from transgenic corn event LW2-2, characterized in that the processed product is corn flour, corn oil, corn starch, corn gluten, tortilla, a cosmetic, or a bulking agent.

In summary, the transgenic corn event LW2-2 of the present invention has dual herbicide resistance, and has the following advantages: 1) The ability to apply glyphosate-containing agricultural herbicides to corn crops for broad spectrum weed control; 2) The ability to apply glufosinate-containing agricultural herbicides to corn crops for broad spectrum weed control; 3) The corn yield is not reduced. Specifically, the event LW2-2 of the invention has high tolerance to glyphosate and glufosinate herbicides, and can protect plants to reduce the rate of damage to 0%; and the agronomic characters of the plants containing the event are excellent, and the yield percentage can reach as high as 100 percent. Furthermore, the genes encoding the two herbicide tolerance traits are linked on the same DNA segment and are present at a single locus in the transgenic maize event LW2-2 genome, which provides enhanced breeding efficiency and enables the use of molecular markers to track transgene inserts in the breeding populations and their progeny. Meanwhile, the primer or probe sequence provided in the detection method can generate amplification products diagnosed as transgenic corn event LW2-2 or the progeny thereof, and can rapidly, accurately and stably identify the existence of plant materials derived from the transgenic corn event LW 2-2.

Drawings

The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application and do not constitute a limitation on the application. In the drawings:

FIG. 1 is a schematic representation of the structure of the binding site between the transgene insert sequence and the maize genome of the nucleic acid sequence and the detection method thereof for detecting herbicide tolerant maize plants LW2-2 of the present application;

FIG. 2 is a schematic diagram of the structure of a recombinant expression vector pLW2 for detecting herbicide tolerant corn plants LW2-2 and a detection method thereof according to the present application.

FIG. 3 is a graph of the field effect of a recommended spray concentration of glyphosate herbicide pesticide of a transgenic corn of the present application comprising transgenic corn event LW2-2 in a field sprayed at 4-fold dosage.

FIG. 4 is a graph showing the recommended spray concentration of glufosinate herbicide for field effectiveness of transgenic corn comprising transgenic corn event LW2-2 of the present application in a field sprayed with 2-fold doses.

Detailed Description

The following description of the embodiments of the present application will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present application, but not all embodiments. The following description of at least one exemplary embodiment is merely exemplary in nature and is in no way intended to limit the application, its application, or uses. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present application. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, components, compositions, and/or combinations thereof.

In the context of the present application for detecting a nucleic acid sequence of a herbicide-resistant corn plant and methods of detecting the same, the following definitions and methods may better define the application and direct one of ordinary skill in the art to practice the application, unless otherwise indicated, terms are to be understood according to the routine usage of one of ordinary skill in the art.

The term "maize" refers to maize (Zea mays) and includes all plant varieties that can be mated to maize, including wild maize varieties.

The term "comprising" means "including but not limited to". The "processed product" refers to a product obtained by processing a raw material such as a plant or a seed, for example, a composition.

The term "plant" includes whole plants, plant cells, plant organs, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps (plant cones), and intact plant cells in plants or plant parts, such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, stems, roots, root tips, anthers, and the like. It is to be understood that parts of transgenic plants within the scope of the present application include, but are not limited to, plant cells, protoplasts, tissues, calli, embryos and flowers, stems, fruits, leaves and roots, which are derived from transgenic plants or their progeny which have been previously transformed with the DNA molecules of the present application and thus at least partially consist of the transgenic cells.

The term "gene" refers to a nucleic acid fragment that expresses a particular protein, including regulatory sequences preceding (5 'non-coding sequences) and regulatory sequences following (3' non-coding sequences) the coding sequences. "native gene" refers to a gene that is found naturally to have its own regulatory sequences. By "chimeric gene" is meant any gene that is not a native gene, comprising regulatory and coding sequences found in a non-native manner. "endogenous gene" refers to a native gene that is located in its natural location in the genome of an organism. "exogenous gene" is a foreign gene that is present in the genome of an organism and that is not originally present, and also refers to a gene that has been introduced into a recipient cell by a transgenic procedure. The exogenous gene may comprise a native gene or chimeric gene inserted into a non-native organism. A "transgene" is a gene that has been introduced into the genome by a transformation procedure. The site in the plant genome where the recombinant DNA has been inserted may be referred to as an "insertion site" or "target site".

"flanking DNA" may comprise genomic or foreign (heterologous) DNA introduced by a transformation process, such as fragments associated with a transformation event, naturally occurring in an organism such as a plant. Thus, flanking DNA may include a combination of native and foreign DNA. In the present invention, a "flanking region" or "flanking sequence" or "genomic border region" or "genomic border 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 more that is immediately upstream or downstream of and adjacent to the initial exogenous inserted DNA molecule. When this flanking region is located downstream, it may also be referred to as a "left border flanking" or a "3 'genomic border region" or a "genomic 3' border sequence", etc. When this flanking region is located upstream, it may also be referred to as a "right-hand border flanking" or a "5 'genomic border region" or a "genomic 5' border sequence", etc.

Transformation procedures that cause random integration of the foreign DNA will result in transformants that contain different flanking regions that each transformant specifically contains. When recombinant DNA is introduced into plants by conventional hybridization, its flanking regions are generally not altered. Transformants will also contain unique junctions between the heterologous insert DNA and segments of genomic DNA or between two segments of heterologous DNA. "ligation" is the point at which two specific DNA fragments are ligated. For example, the junction exists where the insert DNA joins the flanking DNA. The junction point is also present in transformed organisms, where the two DNA fragments are joined together in a manner that modifies what is found in the native organism. "adapter DNA" refers to DNA that contains an adapter.

The present invention provides transgenic corn events, termed LW2-2, and progeny thereof, wherein the transgenic corn event LW2-2 is corn plant LW2-2, which includes plants and seeds of the transgenic corn event LW2-2 and plant cells thereof or regenerable portions thereof, and plant portions of the transgenic corn event LW2-2, including but not limited to cells, pollen, ovules, flowers, shoots, roots, stems, silks, inflorescences, ears, leaves and products from the corn plant LW2-2, such as corn meal, corn oil, corn steep liquor, corn cobs, corn starch and biomass left in the corn crop field.

The transgenic corn event LW2-2 of the invention comprises a DNA construct that, when expressed in a plant cell, confers tolerance to glyphosate herbicide and glufosinate herbicide on said transgenic corn event LW 2-2. The DNA construct comprises two expression cassettes in tandem, the first comprising a suitable promoter for expression in a plant operably linked to a gene encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) that is tolerant to glyphosate herbicide and a suitable polyadenylation signal sequence. The second expression cassette comprises a suitable promoter for expression in plants operably linked to a gene encoding phosphinothricin N-acetyl transferase (PAT), the nucleic acid sequence of which is tolerant to glufosinate herbicide, and a suitable polyadenylation signal sequence. Further, the promoter may be a suitable promoter isolated from plants, including constitutive, inducible and/or tissue-specific promoters, including, but not limited to, the cauliflower mosaic virus (CaMV) 35S promoter, the Figwort Mosaic Virus (FMV) 35S promoter, the Tsf1 promoter, ubiquitin protein (Ubiquitin) promoter, actin (action) promoter, agrobacterium (Agrobacterium tumefaciens) nopaline synthase (NOS) promoter, octopine synthase (OCS) promoter, the night tree (cestrom) yellow leaf curlvirus promoter, tuber storage protein (Patatin) promoter, ribulose-1, 5-bisphosphate carboxylase/oxygenase (rusco) promoter, glutathione transferase (GST) promoter, E9 promoter, GOS promoter, alcA/alcR promoter, agrobacterium rhizogenes (Agrobacterium rhizogenes) rod promoter and Arabidopsis thaliana (sub 2) promoter. The polyadenylation signal sequence may be a suitable polyadenylation signal sequence for functioning in plants, including, but not limited to, polyadenylation signal sequences derived from the Agrobacterium tumefaciens (Agrobacterium tumefaciens) nopaline synthase (NOS) gene, from the cauliflower mosaic virus (CaMV) 35S terminator, from the pea ribulose-1, 5-bisphosphate carboxylase/oxygenase E9 terminator, from the protease inhibitor II (PIN II) gene, and from the alpha-tubulin (alpha-tubulin) gene.

In addition, the expression cassette may also include other genetic elements including, but not limited to, enhancers and signal peptides/transit peptides. The enhancer may enhance the expression level of a gene, including, but not limited to, tobacco Etch Virus (TEV) translational activator, caMV35S enhancer, and FMV35S enhancer. The signal peptide/transit peptide may direct the transit of EPSPS proteins and/or PAT proteins to specific organelles or compartments outside or inside the cell, for example, targeting to the chloroplast using a sequence encoding a chloroplast transit peptide, or targeting to the endoplasmic reticulum using a 'KDEL' retention sequence.

The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene may be isolated from Agrobacterium tumefaciens (Agrobacterium tumefaciens sp.) CP4 strain and may be modified by optimizing codons or otherwise altering the polynucleotide encoding the EPSPS for the purpose of increasing the stability and availability of transcripts in the transformed cells. The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene may also be used as a selectable marker gene.

The term "glyphosate" refers to N-phosphonomethylglycine and its salts, and treatment with a "glyphosate herbicide" refers to treatment with any herbicide formulation containing glyphosate. The rate of use of a glyphosate formulation is selected to achieve an effective biological dosage that does not exceed the skill of an ordinarily skilled artisan. Treatment of a field containing plant material derived from herbicide-resistant corn plants LW2-2 with any herbicide formulation that contains glyphosate will control weed growth in the field and will not affect the growth or yield of plant material derived from herbicide-tolerant corn plants LW 2-2.

The enzyme phosphinothricin N-acetyltransferase (PAT) gene isolated from Streptomyces (Streptomyces viridochromogenes) catalyzes the conversion of L-phosphinothricin to its inactive form by acetylation to confer tolerance to glufosinate herbicide to plants. Phosphinothrin (PTC, 2-amino-4-methylphosphonobutyric acid) is an inhibitor of glutamine synthetase. PTC is the structural unit of the antibiotic 2-amino-4-methylphosphono-alanyl-alanine, and this tripeptide (PTT) has activity against gram-positive and gram-negative bacteria and against the fungus Botrytis cinerea. The phosphinothricin N-acetyl transferase (PAT) gene may also be used as a selectable marker gene.

The term "glufosinate" is also known as glufosinate, and refers to 2-amino-4- [ hydroxy (methyl) phosphono ] butanoic acid ammonium, and treatment with a "glufosinate herbicide" refers to treatment with any herbicide formulation containing glufosinate. The choice of the rate of use of a certain glufosinate formulation in order to achieve an effective biological dose is not beyond the skills of the average agronomic technician. Treatment of a field containing plant material derived from herbicide tolerant corn plant LW2-2 with any herbicide formulation containing glufosinate will control weed growth in the field and will not affect the growth or yield of plant material derived from herbicide tolerant corn plant LW 2-2.

The DNA construct is introduced into a plant using transformation methods including, but not limited to, agrobacterium (Agrobacterium) -mediated transformation, gene gun transformation, and pollen tube channel transformation.

The agrobacterium-mediated transformation method is a common method for plant transformation. The foreign DNA to be introduced into the plant is cloned between the left and right border consensus sequences of the vector, i.e., the T-DNA region. The vector is transformed into an agrobacterium cell, which is subsequently used to infect plant tissue, and the T-DNA region of the vector comprising exogenous DNA is inserted into the plant genome.

The gene gun transformation method is to bombard plant cells (particle-mediated biolistic transformation) with a vector containing exogenous DNA.

The pollen tube channel transformation method utilizes a natural pollen tube channel (also called pollen tube guiding tissue) formed after plant pollination to carry exogenous DNA into embryo sacs through a bead core channel.

After transformation, the transgenic plants must be regenerated from the transformed plant tissue and offspring with the exogenous DNA selected using appropriate markers.

A DNA construct is a combination of DNA molecules that are linked to one another to provide one or more expression cassettes. The DNA construct is preferably a plasmid capable of self replication in bacterial cells and containing various restriction enzyme sites for the introduction of DNA molecules providing functional genetic elements, i.e. promoters, introns, leader sequences, coding sequences, 3' terminator regions and other sequences. The expression cassette contained in the DNA construct includes the genetic elements necessary to provide for transcription of messenger RNA, and can be designed for expression in prokaryotic or eukaryotic cells. The expression cassette of the invention is designed to be expressed most preferably in plant cells.

A transgenic "event" is obtained by transforming plant cells with a heterologous DNA construct, i.e., comprising a nucleic acid expression cassette containing a gene of interest, inserting into the plant genome by transgenic means to produce a plant population, regenerating the plant population, and selecting a particular plant having the characteristics of being inserted into a particular genomic locus. The term "event" refers to both the original transformant comprising the heterologous DNA and the progeny of the transformant. The term "event" also refers to the progeny of a sexual cross between a transformant and other species of individuals containing heterologous DNA, even after repeated backcrosses with a backcross parent, the inserted DNA and flanking genomic DNA from the transformant parent are present at the same chromosomal location in the hybrid progeny. The term "event" also refers to a DNA sequence from an original transformant that comprises an inserted DNA and flanking genomic sequences immediately adjacent to the inserted DNA, which DNA sequence is expected to be transferred into progeny resulting from sexual crossing of a parental line containing the inserted DNA (e.g., the original transformant and progeny resulting from its selfing) with a parental line not containing the inserted DNA, and which progeny received the inserted DNA comprising the gene of interest.

"recombinant" in the context of the present invention refers to forms of DNA and/or proteins and/or organisms that are not normally found in nature and are therefore produced by manual intervention. Such manual intervention may result in recombinant DNA molecules and/or recombinant plants. The "recombinant DNA molecule" is obtained by artificially combining two otherwise isolated sequence segments, for example by chemical synthesis or by manipulation of isolated nucleic acid segments by genetic engineering techniques. Techniques for performing nucleic acid manipulations are well known.

The term "transgene" includes any cell, cell line, callus, tissue, plant part or plant, the genotype of which is altered by the presence of a heterologous nucleic acid, and includes the transgene originally so altered as well as progeny individuals produced from the original transgene by sexual crosses or asexual propagation. In the present invention, the term "transgene" does not include genomic (chromosomal or extrachromosomal) alterations by conventional plant breeding methods or naturally occurring events such as random allofertilisation, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition or spontaneous mutation.

By "heterologous" in the present invention is meant that the first molecule is not normally found in combination with the second molecule in nature. For example, a molecule may originate from a first species and be inserted into the genome of a second species. Such molecules are thus heterologous to the host and are artificially introduced into the genome of the host cell.

Transgenic corn event LW2-2 tolerant to glyphosate and glufosinate herbicides is cultivated by: first sexually crossing a first parent corn plant consisting of a corn plant grown from transgenic corn event LW2-2 and progeny thereof obtained by transformation with an expression cassette of the invention that is tolerant to glyphosate herbicide and glufosinate herbicide, with a second parent corn plant lacking tolerance to glyphosate herbicide and/or glufosinate herbicide, thereby producing a multiplicity of first generation progeny plants; progeny plants that are tolerant to the application of the glyphosate herbicide and/or glufosinate herbicide are then selected, and corn plants that are tolerant to the glyphosate herbicide and glufosinate herbicide can be grown. These steps may further include backcrossing a progeny plant that is tolerant to the application of the glyphosate herbicide and/or glufosinate herbicide with the second parent corn plant or the third parent corn plant, and selecting the progeny plant by applying the glyphosate herbicide, the glufosinate herbicide, or by identification of a molecular marker associated with the trait (e.g., a DNA molecule comprising the junction site identified at the 5 'and 3' ends of the insertion sequence in transgenic corn event LW 2-2), thereby producing a corn plant that is tolerant to the glyphosate herbicide and glufosinate herbicide.

It will also be appreciated that two different transgenic plants can also be crossed to produce offspring containing two independent, separately added exogenous genes. Selfing of appropriate offspring can result in offspring plants that are homozygous for both added exogenous genes. Backcrossing of parent plants and outcrossing with non-transgenic plants as previously described are also contemplated, as are asexual propagation.

The term "probe" is an isolated nucleic acid molecule to which a conventional detectable label or reporter molecule, e.g., a radioisotope, ligand, chemiluminescent agent, or enzyme, is attached. Such a probe is complementary to one strand of the target nucleic acid, and in the present invention, the probe is complementary to one strand of DNA from the genome of transgenic corn event LW2-2, whether the genomic DNA is from transgenic corn event LW2-2 or seed or plant derived from transgenic corn event LW2-2 or seed or extract. Probes of the present invention include 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.

The term "primer" is an isolated nucleic acid molecule that binds to a complementary target DNA strand by nucleic acid hybridization, anneals to form 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 the invention relate to their use in the amplification of a target nucleic acid sequence, for example, by the Polymerase Chain Reaction (PCR) or other conventional nucleic acid amplification methods.

Methods of designing and using primers and probes are well known in the art. DNA molecules comprising the full or partial sequence of SEQ ID NOS: 1-7 can be used as primers and probes for detecting corn event LW2-2 and can be readily designed by one skilled in the art using the sequences provided herein.

The length of the probes and primers is generally 11 polynucleotides or more, preferably 18 polynucleotides or more, more preferably 24 polynucleotides or more, and most preferably 30 polynucleotides or more. Such probes and primers hybridize specifically to the target sequence under highly stringent hybridization conditions. Although probes other than the target DNA sequence and maintaining hybridization ability to the target DNA sequence can be designed by conventional methods, it is preferred that the probes and primers of the present invention have complete DNA sequence identity to a contiguous nucleic acid of the target sequence.

Primers and probes based on flanking genomic DNA and insert sequences of the invention can be determined by conventional methods, for example, by isolating the corresponding DNA molecule from plant material derived from transgenic maize event LW2-2 and determining the nucleic acid sequence of the DNA molecule. The DNA molecule comprises a transgene insert and maize genomic flanking regions, and fragments of the DNA molecule may be used as primers or probes.

The nucleic acid probes and primers of the invention hybridize to a target DNA sequence under stringent conditions. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of DNA in the sample derived from transgenic corn event LW 2-2. The nucleic acid molecule or fragment thereof is capable of specifically hybridizing to other nucleic acid molecules under certain conditions. As used herein, two nucleic acid molecules can be said to specifically hybridize to each other if they are capable of forming antiparallel double-stranded nucleic acid structures. Two nucleic acid molecules are said to be "complements" of one nucleic acid molecule if they exhibit complete complementarity. As used herein, a nucleic acid molecule is said to exhibit "complete complementarity" when each nucleotide of the two molecules is complementary to a corresponding nucleotide of the other nucleic acid molecule. Two nucleic acid molecules are said to be "minimally complementary" if they are capable of hybridizing to each other with sufficient stability such that they anneal and bind to each other under at least conventional "low stringency" conditions. Similarly, two nucleic acid molecules are said to have "complementarity" if they are capable of hybridizing to each other with sufficient stability such that they anneal and bind to each other under conventional "highly stringent" conditions. Deviations from complete complementarity are permissible provided that such deviations do not completely prevent the formation of double-stranded structures by the two molecules. In order to enable a nucleic acid molecule to act as a primer or probe, it is only necessary to ensure sufficient complementarity in sequence to allow the formation of a stable double-stranded structure at the particular solvent and salt concentration employed.

As used herein, a substantially homologous sequence is a nucleic acid molecule that is capable of specifically hybridizing under highly stringent conditions to the complementary strand of a matching other nucleic acid molecule. Suitable stringent conditions for promoting DNA hybridization, for example, treatment with 6.0 XSSC/sodium citrate (SSC) at about 45℃followed by washing with 2.0 XSSC at 50℃are well known to those skilled in the art. For example, the salt concentration in the washing step may be selected from about 2.0 XSSC at low stringency conditions, about 0.2 XSSC at 50℃to high stringency conditions, about 50 ℃. In addition, the temperature conditions in the washing step may be raised from about 22 ℃ at room temperature under low stringency conditions to about 65 ℃ under high stringency conditions. The temperature conditions and salt concentration may both be varied, or one may remain unchanged while the other variable is varied. Preferably, a nucleic acid molecule of the invention can specifically hybridize under moderately stringent conditions, e.g., at about 2.0 XSSC and about 65℃to one or more 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 and SEQ ID NO. 7, or to a complement thereof, or to any fragment of the foregoing. More preferably, a nucleic acid molecule of the invention hybridizes specifically under highly stringent conditions to one or more 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 and SEQ ID NO. 7 or to the complement thereof, or to any fragment of the above sequences. In the present invention, preferred marker nucleic acid molecules have SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 6 or SEQ ID NO. 7 or the complement thereof, or any fragment of the above sequences. Another preferred marker nucleic acid molecule of the invention has 80% to 100% or 90% to 100% sequence identity with SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 6 or SEQ ID NO. 7 or the complement thereof, or any fragment of the above sequences. SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 6 and SEQ ID NO. 7 can be used as markers in plant breeding methods to identify offspring of genetic crosses. Hybridization of the probe to the target DNA molecule may be detected by any method known to those skilled in the art, including, but not limited to, fluorescent labels, radiolabels, antibody-based labels, and chemiluminescent labels.

With respect to amplification (e.g., by PCR) of a target nucleic acid sequence using specific amplification primers, "stringent conditions" refer to conditions that allow hybridization of only the primer pair to the target nucleic acid sequence in a DNA thermal amplification reaction, and primers having a wild-type sequence (or its complement) corresponding to the target nucleic acid sequence are capable of binding to the target nucleic acid sequence and preferably produce a unique amplification product, i.e., an amplicon.

The term "specific binding (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.

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, to determine whether a corn plant is produced by sexual hybridization with a corn containing the transgenic corn event LW2-2 of the invention, or whether a corn sample collected from a field contains the transgenic corn event LW2-2, or whether a corn extract, such as meal, flour, or oil, contains the transgenic corn event LW2-2, DNA extracted from a corn plant tissue sample or extract can be amplified by a nucleic acid amplification method using a primer pair to produce an amplicon diagnostic for the presence of DNA of the transgenic corn event LW 2-2. The primer pair includes a first primer derived from a flanking sequence in the genome of the plant adjacent to the insertion site of the inserted foreign DNA, and a second primer derived from the inserted foreign DNA. The amplicon has a length and sequence that is also diagnostic for the transgenic corn event LW 2-2. The length of the amplicon may 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 fifty nucleotide base pairs, and most preferably plus about four hundred fifty nucleotide base pairs or more.

Alternatively, the primer pair may be derived from flanking genomic sequences flanking the inserted DNA to produce an amplicon comprising the entire inserted nucleic acid sequence. One of the primer pairs derived from the plant genomic sequence may be located at a distance from the inserted DNA sequence that may range from one nucleotide base pair to about twenty thousand nucleotide base pairs. The use of the term "amplicon" specifically excludes primer dimers formed in the DNA thermal amplification reaction.

The nucleic acid amplification reaction may be accomplished by any nucleic acid amplification reaction method known in the art, including the Polymerase Chain Reaction (PCR). Various methods of nucleic acid amplification are well known to those skilled in the art. PCR amplification methods have been developed to amplify genomic DNA up to 22kb and phage DNA up to 42 kb. These methods, as well as other DNA amplification methods in the art, may be used in the present invention. The inserted exogenous DNA sequence and flanking DNA sequences from transgenic corn event LW2-2 can be obtained by amplifying the genome of transgenic corn event LW2-2 using the provided primer sequences, and standard DNA sequencing of the PCR amplicon or cloned DNA after amplification.

DNA detection kits based on DNA amplification methods contain DNA primer molecules that hybridize specifically to the target DNA under appropriate reaction conditions and amplify the diagnostic amplicon. The kit may provide agarose gel-based detection methods or a number of methods known in the art for detecting diagnostic amplicons. Kits comprising DNA primers homologous or complementary to any portion of the maize genomic region of SEQ ID NO. 3 or SEQ ID NO. 4 and homologous or complementary to any portion of the transgene insertion region of SEQ ID NO. 5 are provided by the invention. In particular, primer pairs identified as useful in DNA amplification methods are SEQ ID NO. 8 and SEQ ID NO. 9, which amplify a diagnostic amplicon homologous to a portion of the 5' transgene/genomic region of transgenic maize event LW2-2, wherein the amplicon comprises SEQ ID NO. 1. Other DNA molecules used as DNA primers may be selected from SEQ ID NO. 5.

Amplicons produced by these methods can be detected by a variety of techniques. One of the methods is GeneticBit Analysis, which designs a DNA oligonucleotide strand that spans the insert DNA sequence and adjacent flanking genomic DNA sequences. The oligonucleotide strand is immobilized in a microwell of a microwell plate, and after PCR amplification of the target region (using one primer in each of the insert sequence and adjacent flanking genomic sequences), the single-stranded PCR product can hybridize to the immobilized oligonucleotide strand and serve as a template for a single base extension reaction using DNA polymerase and ddNTPs specifically labeled for the next desired base. The results may be obtained by fluorescence or ELISA-like methods. The signal represents the presence of an insertion/flanking sequence, which indicates that the amplification, hybridization and single base extension reactions were successful.

Another method is Pyrosequencing technology. The method contemplates an oligonucleotide strand spanning the insertion DNA sequence and adjacent genomic DNA binding sites. The oligonucleotide strand and the single stranded PCR product of the target region (using one primer in each of the insert sequence and adjacent flanking genomic sequences) are hybridized and then incubated with DNA polymerase, ATP, sulfurylase, luciferase, apyrase, adenosine-5' -phosphosulfate and luciferin. dNTPs are added separately and the resulting optical signal is measured. The optical signal represents the presence of an insertion/flanking sequence, which indicates that amplification, hybridization, and single base or multiple base extension reactions were successful.

Fluorescence polarization as described by Chen et al (Genome Res.) 9:492-498, 1999) is also one method that may be used to detect the amplicons of the present invention. The use of this method requires the design of an oligonucleotide strand spanning the insertion DNA sequence and adjacent genomic DNA binding sites. The oligonucleotide strand is hybridized to a single stranded PCR product of the target region (using one primer in each of the insert sequence and adjacent flanking genomic sequences) and then incubated with DNA polymerase and a fluorescent labeled ddNTP. Single base extension will result in insertion of ddNTP. Such an insertion can measure the change in its polarization using a fluorometer. The change in polarization represents the presence of an insertion/flanking sequence, which indicates that amplification, hybridization, and single base extension reactions were successful.

Taqman is described as a method for detecting and quantifying the presence of a DNA sequence, which is described in detail in the instructions provided by the manufacturer. Briefly, a FRET oligonucleotide probe is designed that spans the intervening DNA sequence and adjacent genomic flanking binding sites, as described below. The FRET probe and PCR primers (one primer in each of the insert sequence and adjacent flanking genomic sequences) are cycled in the presence of a thermostable polymerase and dNTPs. Hybridization of the FRET probe results in cleavage of the fluorescent moiety and the quencher moiety on the FRET probe and release of the fluorescent moiety. The generation of a fluorescent signal is representative of the presence of the insertion/flanking sequences, which indicates that amplification and hybridization were successful.

Suitable techniques for detecting plant material derived from herbicide tolerant transgenic corn event LW2-2 based on hybridization principles may also include Southern blot hybridization, northern blot hybridization, and in situ hybridization. In particular, the suitable technique includes incubating the probe and sample, washing to remove unbound probe and detecting whether the probe has hybridized. The detection method depends on the type of label attached to the probe, for example, radiolabeled probes can be detected by X-ray exposure and development, or enzymatically labeled probes can be detected by substrate conversion to effect a color change.

Tyangi et al (Nat. Biotech.) 14:303-308,1996) describe the use of molecular markers in sequence detection. Briefly described, a FRET oligonucleotide probe is designed that spans the intervening DNA sequence and adjacent genomic flanking binding sites. The unique structure of the FRET probe results in it containing a secondary structure that is capable of retaining both the fluorescent moiety and the quenching moiety in close proximity. The FRET probe and PCR primers (one primer in each of the insert sequence and adjacent flanking genomic sequences) are cycled in the presence of a thermostable polymerase and dNTPs. Upon successful PCR amplification, hybridization of the FRET probe to the target sequence results in a loss of secondary structure of the probe, thereby spatially separating the fluorescent moiety from the quenching moiety, producing a fluorescent signal. The generation of a fluorescent signal is representative of the presence of the insertion/flanking sequences, which indicates that amplification and hybridization were successful.

Other described methods, such as microfluidics (microfluidics), provide methods and apparatus for isolating and amplifying DNA samples. The photodyes are used to detect and determine specific DNA molecules. A nano tube (nano tube) device comprising an electronic sensor for detecting DNA molecules or a nano bead binding to a specific DNA molecule and thus being detectable is useful for detecting the DNA molecules of the invention.

DNA detection kits may be developed using the compositions of the present invention and methods described in or known to the DNA detection arts. The kit facilitates the identification of the presence or absence of DNA for transgenic corn event LW2-2 in a sample, and can also be used to cultivate corn plants containing DNA for transgenic corn event LW 2-2. The kit may contain DNA primers or probes homologous to or complementary to at least a portion of SEQ ID NO. 1, 2, 3, 4 or 5, or other DNA primers or probes homologous to or complementary to DNA contained in the transgenic genetic element of DNA, which DNA sequences may be used in DNA amplification reactions or as probes in DNA hybridization methods. The DNA structure of the transgene insert contained in the corn genome and the binding site to the corn genome illustrated in fig. 1 and table 1 comprises: a maize plant LW2-2 flanking genomic region at the 5' end of the transgene insert, a portion of the insert sequence from the right border Region (RB) of agrobacterium, a first expression cassette consisting of a tandem repeat of the cauliflower mosaic virus 35S promoter (p 35S) containing the enhancer region, operably linked to the coding sequence for the arabidopsis EPSPS chloroplast transit peptide (spactp 2), operably linked to the glyphosate tolerant 5-enol-pyruvylshikimate-3-phosphate synthase (cresps) of the agrobacterium CP4 strain, operably linked to the cauliflower mosaic virus transcription terminator (CaMv 35S), a second expression cassette consisting of the tandem repeat of the cauliflower mosaic virus 35S promoter (p 35S) containing the enhancer region, operably linked to the phosphinothricin N-acetyl transferase (cPAT 35S) of streptomyces and to the cauliflower mosaic virus 35S terminator (t 35S), and a portion of the insert sequence from the left border region (SEQ ID 2) of the maize plant insert region. In the DNA amplification method, the DNA molecule used as a primer may be any part derived from the transgene insert sequence in maize plant LW2-2, or may be any part derived from the DNA region of the flanking maize genome in transgenic maize event LW 2-2.

Transgenic corn event LW2-2 can be combined with other transgenic corn varieties, such as herbicide tolerant (e.g., 2,4-D, dicamba, etc.) corn, or transgenic corn varieties carrying other insect-resistant genes (e.g., cry1Ab, vip3A, etc.). Various combinations of all of these different transgenic events, when bred with the transgenic maize event LW2-2 of the present invention, can provide improved hybrid transgenic maize varieties that are resistant to multiple insect pests and tolerant to multiple herbicides. These varieties may exhibit superior characteristics such as yield enhancement compared to non-transgenic varieties and transgenic varieties of single trait.

The present invention provides a nucleic acid sequence for detecting herbicide tolerant corn plants LW2-2 and methods for detecting the same, the transgenic corn event LW2-2 being tolerant to the phytotoxic effects of glyphosate and/or glufosinate-containing agricultural herbicides. The dual trait maize plants express a glyphosate resistant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) protein of agrobacterium strain CP4, which confers tolerance to glyphosate on plants, and a phosphinothricin N-acetyl transferase (PAT) protein of streptomyces, which confers tolerance to glufosinate on plants. The dual-trait corn has the following advantages: 1) The ability to apply glyphosate-containing agricultural herbicides to corn crops for broad spectrum weed control; 2) The combined use of glufosinate herbicide (either mixed with or alternating with glyphosate herbicide) can serve as a non-selective means for effectively managing glyphosate resistant weeds; 3) The herbicide tolerance transgenic corn is used as non-insect-resistant transgenic corn, and is planted together with the transgenic insect-resistant corn in a certain proportion, so that the generation of resistance of insects/pests can be delayed; 4) The corn yield is not reduced. Furthermore, genes encoding glyphosate tolerance and glufosinate tolerance traits are linked on the same DNA segment and are present at a single locus in the genome of transgenic maize event LW2-2, which provides enhanced breeding efficiency and enables molecular markers to be used to track transgenic inserts in the breeding populations and their progeny. Meanwhile, SEQ ID NO. 1 or the complementary sequence thereof, SEQ ID NO. 2 or the complementary sequence thereof, SEQ ID NO. 6 or the complementary sequence thereof, or SEQ ID NO. 7 or the complementary sequence thereof can be used as a DNA primer or a probe to generate an amplified product diagnosed as transgenic corn event LW2-2 or the progeny thereof in the detection method of the invention, and the existence of plant materials derived from the transgenic corn event LW2-2 can be rapidly, accurately and stably identified.

Such as agricultural or commodity products can be produced from transgenic corn event LW 2-2. If sufficient expression is detected in the agricultural product or commodity, the agricultural product or commodity is expected to contain a nucleic acid sequence capable of diagnosing the presence of transgenic corn event LW2-2 material in the agricultural product or commodity. Such agricultural products or commodity products include, but are not limited to, corn oil, corn meal, corn flour, corn gluten, tortilla, corn starch, any other food product that is to be consumed by an animal as a food source, or otherwise used for cosmetic purposes, etc., as an ingredient in an expanding agent or cosmetic composition. Nucleic acid detection methods and/or kits based on probe or primer pairs can be developed to detect a transgenic corn event LW2-2 nucleic acid sequence such as shown in SEQ ID NO. 1 or SEQ ID NO. 2 in a biological sample, wherein the probe sequence or primer sequence is selected from the sequences shown as SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5 to diagnose the presence of transgenic corn event LW 2-2.

In conclusion, the transgenic corn event LW2-2 has better tolerance to glyphosate herbicide and glufosinate herbicide, has no influence on yield, and the detection method can accurately and rapidly identify whether the biological sample contains DNA molecules of the transgenic corn event LW 2-2.

Seeds corresponding to transgenic corn event LW2-2 have been deposited at China center for type culture collection (CCTCC, address: eight 299 universities of Wuhan, inc., post code 430072) at 12 months 6 of 2020), and are designated by the taxonomic designation: corn (Zea mays) with a preservation number of CCTCC NO: P202022, and the deposit will be preserved for 30 years at the point of preservation.

EXAMPLE 1 cloning and transformation

1.1 vector cloning

The recombinant expression vector pLW2 (as shown in fig. 2) was constructed using standard gene cloning techniques. The vector pLW2 comprises two transgene expression cassettes in tandem, the first expression cassette consisting of a rice actin 1 promoter (pOsAct 1), operably linked to the coding sequence of the Arabidopsis thaliana EPSPS chloroplast transit peptide (spatCTP 2), operably linked to the glyphosate tolerant 5-enol-pyruvylshikimate-3-phosphate synthase (cEPSPS) of the Agrobacterium CP4 strain, and operably linked to the transcription terminator of cauliflower mosaic virus 35S (CaMv 35S); the second expression cassette consists of a tandem repeat of the cauliflower mosaic virus 35S promoter (p 35S) containing an enhancer region operably linked to a phosphinothricin N-acetyltransferase (cPAT) resistant to the phosphinothricin of Streptomyces, and operably linked to a cauliflower mosaic virus 35S terminator (t 35S).

The vector pLW2 was transformed into Agrobacterium LBA4404 (Invitrogen, chicago, USA; cat. No. 18313-015) by liquid nitrogen method, and the transformed cells were screened using 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) as a selection marker.

1.2 plant transformation

Transformation was performed using conventional agrobacterium infection, and the aseptically cultured maize young embryos were co-cultured with agrobacterium as described in this example 1.1 to transfer the T-DNA in the constructed recombinant expression vector pLW2 into the maize chromosome set to generate transgenic maize event LW2-2.

For Agrobacterium-mediated transformation of maize, briefly, immature embryos are isolated from maize, the immature embryos are contacted with an Agrobacterium suspension, wherein the Agrobacterium is capable of transferring the nucleic acid sequence of the EPSPS gene and the nucleic acid sequence of the PAT gene to at least one cell of one of the immature embryos (step 1: an infection step), in which step the immature embryos are preferably immersed in the Agrobacterium suspension (OD ₆₆₀ =0.4-0.6), in an infected medium (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 68.5g/L, glucose 36g/L, acetosyringone (AS) 40mg/L, 2, 4-dichlorophenoxyacetic acid (2, 4-D) 1mg/L, ph=5.3) to initiate inoculation. The young embryo is co-cultured with Agrobacterium for a period of time (3 days) (step 2: co-culturing step). Preferably, the young embryos are cultured after the infection step on solid medium (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 20g/L, glucose 10g/L, acetosyringone (AS) 100mg/L, 2, 4-dichlorophenoxyacetic acid (2, 4-D) 1mg/L, agar 8g/L, pH=5.8). After this co-cultivation stage, there may be an optional "recovery" step. In the "recovery" step, recovery medium (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 30g/L, 2, 4-dichlorophenoxyacetic acid (2, 4-D) 1mg/L, plant) The organogel 3g/L, ph=5.8) had at least one antibiotic known to inhibit agrobacterium growth (cephalosporin), and no selection agent for plant transformants was added (step 3: and (5) a recovery step). Preferably, the young embryos are cultured on solid medium with antibiotics but no selection agent to eliminate agrobacterium and provide a recovery period for the infected cells. The inoculated young embryos are then cultured on a medium containing a selection agent (glyphosate) and the growing transformed calli are selected (step 4: selection step). Preferably, the young embryos are cultured on selection solid medium with selection agent (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 30g/L, N- (phosphonomethyl) glycine 0.25mol/L, 2, 4-dichlorophenoxyacetic acid (2, 4-D) 1mg/L, plant gel 3g/L, pH=5.8) resulting in selective growth of the transformed cells. Then, the callus is regenerated into a plant (step 5: regeneration step), preferably, the callus grown on the medium containing the selection agent is cultured on a solid medium (MS differentiation medium and MS rooting medium) to regenerate the plant.

The selected resistant calli were transferred to the MS differentiation medium (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 30g/L, 6-benzyl adenine 2mg/L, N- (phosphonomethyl) glycine 0.125mol/L, plant gel 3g/L, pH=5.8) and cultured at 25 ℃. The differentiated plantlets were transferred to the MS rooting medium (MS salt 2.15g/L, MS vitamin, casein 300mg/L, sucrose 30g/L, indole-3-acetic acid 1mg/L, agar 8g/L, pH=5.8), cultured to about 10cm high at 25℃and transferred to a greenhouse for cultivation until set. In the greenhouse, the cells were cultured at 28℃for 16 hours and at 20℃for 8 hours each day.

1.3 identification and screening of transgenic events

A total of 485 independent transgenic T0 plants were produced.

Example 2 detection of transgenic maize event LW2-2 Using TaqMan

About 100mg of leaf of transgenic corn event LW2-2 was taken as a sample, its genomic DNA was extracted with Qiagen's DNeasyPlantMaxi Kit, and the copy numbers of EPSPS gene and PAT gene were detected by Taqman probe fluorescent quantitative PCR method. Meanwhile, wild corn plants are used as a control, and detection and analysis are carried out according to the method. Experiments were repeated 3 times and averaged.

The specific method comprises the following steps:

step 11, taking 100mg of leaves of transgenic corn event LW2-2, grinding into homogenate in a mortar by using liquid nitrogen, and taking 3 repeats of each sample;

step 12, extracting genomic DNA of the sample by using DNeasy Plant Mini Kit of Qiagen, wherein the specific method refers to the product instruction;

step 13, determining the concentration of the genomic DNA of the sample by using NanoDrop 2000 (Thermo Scientific);

step 14, adjusting the concentration of the genomic DNA of the sample to the same concentration value, wherein the concentration value ranges from 80 ng/mu L to 100 ng/mu L;

step 15, identifying the copy number of the sample by adopting a Taqman probe fluorescent quantitative PCR method, taking the sample with the identified known copy number as a standard substance, taking the sample of a wild type corn plant as a control, repeating each sample for 3 times, and taking the average value; the fluorescent quantitative PCR primer and the probe sequences are respectively as follows:

The following primers and probes were used to detect the epps gene sequence:

primer 1: GCAAATCCTCTGGCCTTTCC is shown as SEQ ID NO. 16 in the sequence table;

primer 2: TGAAGGACCGGTGGGAGAT is shown as SEQ ID NO. 17 in the sequence table;

probe 1: CGTCCGCATTCCCGGCGA is shown as SEQ ID NO. 18 in the sequence table;

the following primers and probes were used to detect the pat gene sequence:

primer 3: CCGCGGTTTGTGATATCGTT is shown as SEQ ID NO. 19 in the sequence table;

primer 4: TCTTGCAACCTCTCTAGATCATCAA is shown as SEQ ID NO. 20 in the sequence table;

probe 2: TAGGACAGAGCCACAAACACCACAAGAGTG is shown as SEQ ID NO. 21 in the sequence table;

the PCR reaction system is as follows:

the 50 Xprimer/probe mixture contained 45. Mu.L of each primer at a concentration of 1mM, 50. Mu.L of probe at a concentration of 100. Mu.M and 860. Mu.L of 1 XTE buffer, and was stored in amber tubes at 4 ℃.

The PCR reaction conditions were:

the data were analyzed using SDS2.3 software (Applied Biosystems) to obtain a single copy of transgenic maize event LW2-2.

Example 3 transgenic maize event LW2-2 detection

3.1 genomic DNA extraction

DNA extraction according to the conventionally employed CTAB (cetyltrimethylammonium bromide) method: 2 g of young transgenic corn event LW2-2 leaves are ground into powder in liquid nitrogen, added with 0.5mL of DNA preheated at 65 ℃ to extract CTAB Buffer (20 g/L CTAB,1.4M NaCl,100mM Tris-HCl,20mM EDTA (ethylenediamine tetraacetic acid), pH is regulated by NaOH=8.0), fully and uniformly mixed, and extracted for 90 minutes at 65 ℃; adding 0.5 volume of phenol and 0.5 volume of chloroform, and mixing the mixture upside down; centrifugation is carried out at 12000rpm (revolutions per minute) for 10 minutes; absorbing the supernatant, adding 2 times of absolute ethyl alcohol, gently shaking the centrifuge tube, and standing at 4 ℃ for 30 minutes; further centrifuging at 12000rpm for 10 minutes; collecting DNA to the bottom of the tube; discarding the supernatant, washing the precipitate with 1mL of ethanol with a mass concentration of 70%; centrifuging at 12000rpm for 5 minutes; vacuum pumping or blow-drying in an ultra clean bench; the DNA pellet was dissolved in an appropriate amount of TE buffer (10 mM Tris-HCl,1mM EDTA,PH =8.0) and stored at a temperature of-20 ℃.

3.2 analysis of flanking DNA sequences

And (3) carrying out concentration measurement on the extracted DNA sample, so that the concentration of the sample to be measured is between 80 and 100 ng/. Mu.L. Genomic DNA was digested with selected restriction enzymes SpeI (NEB, # R3133L), pstI (NEB, # R0140L), ecoRI (NEB, # R3101L), and BamHI (NEB, # R3136L), xmaI (NEB, # R0180L), kpnI (NEB, # R3142L), sacII (NEB, # R0157L) for 3' end analysis, respectively. 26.5. Mu.L of genomic DNA, 0.5. Mu.L of the restriction enzyme selected above, and 3. Mu.L of the cleavage buffer were added to each cleavage system, and the cleavage was performed for 1 hour. After completion of the cleavage, 70. Mu.L of absolute ethanol was added to the cleavage system, the mixture was ice-washed for 30 minutes, centrifuged at 12000rpm for 7 minutes, the supernatant was discarded, and the dried product was blown dry, followed by addition of 8.5. Mu.L of double distilled water (ddH 2O), 1. Mu.L of 10X T4 Buffer and 0.5. Mu. L T4 ligase (NEB, # M0202L) and ligation overnight at a temperature of 4 ℃. PCR amplification was performed with a series of nested primers to isolate 5 'and 3' transgenes/genomic DNA. Specifically, the isolated 5' transgene/genomic DNA primer combination includes SEQ ID NO. 13, SEQ ID NO. 26 as a first primer, SEQ ID NO. 27, SEQ ID NO. 28 as a second primer, and SEQ ID NO. 13 as a sequencing primer. The isolated 3' transgene/genomic DNA primer combination included SEQ ID NO. 15, SEQ ID NO. 29 as the first primer, SEQ ID NO. 30, SEQ ID NO. 31 as the second primer, SEQ ID NO. 15 as the sequencing primer, and the PCR reaction conditions are shown in Table 3.

The resulting amplicons were electrophoresed on a 2.0% agarose gel to isolate the PCR reaction, followed by isolation of the fragment of interest from the agarose matrix using the QIAquickGel extraction kit (catalogue # 28704, qiagen Inc., valencia, CA). The purified PCR product is then sequenced (e.g., ABI prism (tm) 377,PE Biosystems,Foster City,CA) and analyzed (e.g., DNASTAR sequence analysis software, DNASTAR inc., madison, WI).

The 5 'and 3' flanking sequences and the junction sequences were confirmed using standard PCR methods. The 5' flanking sequences and the junction sequences can be confirmed using SEQ ID NO. 8 or SEQ ID NO. 12, in combination with SEQ ID NO. 9, SEQ ID NO. 13 or SEQ ID NO. 26. The 3' flanking sequences and the junction sequences can be confirmed using SEQ ID NO. 11 or SEQ ID NO. 14 in combination with SEQ ID NO. 10, SEQ ID NO. 15 or SEQ ID NO. 29. The PCR reaction system and the amplification conditions are shown in tables 3 and 4. Those skilled in the art will appreciate that other primer sequences may be used to confirm flanking and junction sequences.

DNA sequencing of the PCR products provides DNA that can be used to design other DNA molecules as primers and probes for identification of maize plants or seeds derived from transgenic maize event LW 2-2.

It was found that nucleotide 1-1131 of SEQ ID NO. 5 shows the maize genomic sequence flanking the right border of the insertion sequence of transgenic maize event LW2-2 (5 'flanking sequence) and nucleotide 6257-7144 of SEQ ID NO. 5 shows the maize genomic sequence flanking the left border of the insertion sequence of transgenic maize event LW2-2 (3' flanking sequence). The 5 'junction sequence is set forth in SEQ ID NO. 1 and the 3' junction sequence is set forth in SEQ ID NO. 2.

3.3 PCR zygosity assay

The junction sequence is a relatively short polynucleotide molecule that is a novel DNA sequence that is diagnostic for DNA of transgenic corn event LW2-2 when detected in a nucleic acid detection assay. The junction sequence of SEQ ID NO. 1 is composed of 11bp on one side of the T-DNARB region insertion site and the corn genome DNA insertion site of the transgenic event LW2-2, and the junction sequence of SEQ ID NO. 2 is composed of 11bp on the other side of the T-DNALB region insertion site and the corn genome DNA insertion site of the transgenic corn event LW 2-2. Longer or shorter polynucleotide binding sequences may be selected from SEQ ID NO. 3 or SEQ ID NO. 4. The junction sequences (5 'junction region SEQ ID NO:1, and 3' junction region SEQ ID NO: 2) are useful as DNA probes or as DNA primer molecules in DNA detection methods. The junction sequences SEQ ID NO. 6 and SEQ ID NO. 7 are also novel DNA sequences in transgenic maize event LW2-2, which can also be used as DNA probes or as DNA primer molecules to detect the presence of transgenic maize event LW2-2 DNA. The sequence of SEQ ID NO. 6 (nucleotide numbers 1071-1630 of SEQ ID NO. 3) spans the pLW2 construct DNA sequence and the CaMv35S transcription termination sequence, and the sequence of SEQ ID NO. 7 (nucleotide numbers 1-347 of SEQ ID NO. 4) spans the t35S transcription terminator sequence and the pLW2 construct DNA sequence.

Furthermore, the amplicon is generated by using at least one primer from SEQ ID NO. 3 or SEQ ID NO. 4, which primer when used in a PCR method generates a diagnostic amplicon for transgenic maize event LW 2-2.

Specifically, a PCR product is generated from the 5 'end of the transgenic insert that is a portion of genomic DNA flanking the 5' end of the T-DNA insert in the genome comprising plant material derived from transgenic maize event LW 2-2. This PCR product contains SEQ ID NO 3. For PCR amplification, primer 5 (SEQ ID NO: 8) hybridizing to the genomic DNA sequence flanking the 5' end of the transgene insert and primer 6 (SEQ ID NO: 9) located in the transgene CaMv35S transcription termination sequence was designed to pair with it.

A PCR product is generated from the 3 'end of the transgenic insert comprising a portion of genomic DNA flanking the 3' end of the T-DNA insert in the genome of the plant material derived from transgenic maize event LW 2-2. This PCR product contains SEQ ID NO. 4. For PCR amplification, primer 8 (SEQ ID NO: 11) hybridizing to the genomic DNA sequence flanking the 3 '-end of the transgene insert and primer 7 (SEQ ID NO: 10) of the p35S promoter sequence at the 3' -end of the insert were designed to pair with it.

The DNA amplification conditions illustrated in tables 3 and 4 can be used in the PCR zygosity assay described above to generate a diagnostic amplicon for transgenic corn event LW 2-2. Detection of the amplicon may be performed by using a Stratagene Robotcycler, MJ Engine, perkin-Elmer9700 or Eppendorf MastercyclerGradien thermal cycler, or the like, as shown in Table 4, or by methods and apparatus known to those skilled in the art.

TABLE 3 PCR step and reaction mixture conditions for identification of 5' transgenic insert/genome combination region for transgenic maize event LW2-2

Table 4, perkin-Elmer9700 thermal cycler conditions

Mix gently, if there is no thermal cap on the thermocycler, 1-2 drops of mineral oil can be added above each reaction solution. PCR was performed on a Stratagene Robotcycler (Stratagene, la Jolla, calif.), MJENG (MJ R-Biorad, hercules, calif.), perkin-Elmer9700 (Perkin Elmer, boston, mass.) or Eppendorf Mastercycler Gradient (Eppendorf, hamburg, germany) thermocycler using the above cycling parameters (Table 4). The MJ Engine or Eppendorf Mastercycler Gradient thermocycler should operate in a calculated mode. The Perkin-Elmer9700 thermocycler is operated with a ramp rate (ramp speed) set to a maximum value.

The experimental results show that: primers 5 and 6 (SEQ ID NOS: 8 and 9), which when used in a PCR reaction of transgenic maize event LW2-2 genomic DNA, produce an amplification product of 1411bp fragment, and when used in a PCR reaction of untransformed maize genomic DNA and non-LW 2-2 maize genomic DNA, NO fragment is amplified; primers 7 and 8 (SEQ ID NOS: 10 and 11), when used in a PCR reaction of transgenic maize event LW2-2 genomic DNA, produced an amplified product of the 1461bp fragment, and when used in a PCR reaction of untransformed maize genomic DNA and non-LW 2-2 maize genomic DNA, NO fragment was amplified.

The PCR zygosity assay can also be used to identify whether the material derived from transgenic maize event LW2-2 is homozygous or heterozygous. Primer 9 (SEQ ID NO: 12), primer 10 (SEQ ID NO: 13) and primer 11 (SEQ ID NO: 14) or primer 9 (SEQ ID NO: 12), primer 10 (SEQ ID NO: 13) and primer 12 (SEQ ID NO: 15) are used in an amplification reaction to generate a diagnostic amplicon for transgenic corn event LW 2-2. The DNA amplification conditions illustrated in tables 5 and 6 can be used in the zygosity assay described above to generate a diagnostic amplicon for transgenic corn event LW 2-2.

TABLE 5 reaction solution for measuring the bondability

TABLE 6 determination of the bondability Perkin-Elmer9700 thermal cycler conditions

PCR was performed on a Stratagene Robotcycler (Stratagene, la Jolla, calif.), MJ Engine (MJ R-Biorad, hercules, calif.), perkin-Elmer 9700 (Perkin Elmer, boston, mass.) or Eppendorf Mastercycler Gradient (Eppendorf, hamburg, germany) thermocycler using the above cycling parameters (Table 6). The MJ Engine or Eppendorf Mastercycler Gradient thermocycler should operate in a calculated mode. The Perkin-Elmer 9700 thermocycler is operated with a ramp rate (ramp speed) set to a maximum value.

In the amplification reaction, the biological sample containing the template DNA contains DNA diagnostic for the presence of transgenic corn event LW2-2 in the sample. Or the reaction will produce two different DNA amplicons from a biological sample containing DNA derived from the corn genome that is heterozygous for the corresponding allele of the insert DNA present in transgenic corn event LW 2-2. These two different amplicons would correspond to a first amplicon derived from the wild-type maize genomic locus and a second amplicon diagnostic for the presence of transgenic maize event LW2-2 DNA. Only a corn DNA sample corresponding to a single amplicon of the second amplicon described for the heterozygous genome is generated, the presence of transgenic corn event LW2-2 can be diagnostically determined in the sample, and the sample is generated from a corn seed homozygous for the allele corresponding to the inserted DNA present in the transgenic corn plant LW 2-2.

It should be noted that the primer pair for transgenic corn event LW2-2 was used to generate amplicons diagnostic for the genomic DNA of transgenic corn event LW2-2. These primer pairs include, but are not limited to, primers 5 and 6 (SEQ ID NOS: 8 and 9), and primers 7 and 8 (SEQ ID NOS: 10 and 11) for use in the DNA amplification method described. In addition, a control primer 13 and primer 14 (SEQ ID NOS: 22 and 23) for amplifying maize endogenous genes are included as an intrinsic criterion for the reaction conditions. The analysis of the DNA extract sample of transgenic corn event LW2-2 should include a positive tissue DNA extract control of transgenic corn event LW2-2, a negative DNA extract control derived from non-transgenic corn event LW2-2 and a negative control that does not contain a template corn DNA extract. In addition to these primer pairs, any primer pair from SEQ ID NO. 3 or SEQ ID NO. 4, or the complement thereof, which when used in a DNA amplification reaction, produces an amplicon comprising SEQ ID NO. 1 or SEQ ID NO. 2 that is diagnostic for tissue derived from transgenic event maize plants LW2-2, respectively, may be used. The DNA amplification conditions illustrated in tables 3-6 can be used to generate diagnostic amplicons of transgenic corn event LW2-2 using appropriate primer pairs. Extracts that are presumed to contain corn plant or seed DNA comprising transgenic corn event LW2-2, or products derived from transgenic corn event LW2-2, that when tested in a DNA amplification method produce amplicons diagnostic for transgenic corn event LW2-2, can be used as templates for amplification to determine the presence or absence of transgenic corn event LW2-2.

Example 4 detection of transgenic maize event LW2-2 by Southern blot hybridization

4.1 DNA extraction for Southern blot hybridization

Southern blot analysis was performed using T4, T5 generation homozygous transformation events. Approximately 5 to 10g of plant tissue was ground in liquid nitrogen using a mortar and pestle. Plant tissue was resuspended in 12.5mL extraction buffer a (0.2M Tris ph=8.0, 50mM EDTA,0.25M NaCl,0.1%v/v β -mercaptoethanol, 2.5% w/v polyvinylpyrrolidone) and centrifuged at 4000rpm for 10 min (2755 g). After discarding the supernatant, the pellet was resuspended in 2.5mL of extraction buffer B (0.2M Tris ph=8.0, 50mM EDTA,0.5M NaCl,1%v/v β -mercaptoethanol, 2.5% w/v polyvinylpyrrolidone, 3% myo-aminoacyl, 20% ethanol) and incubated for 30 min at 37 ℃. During the incubation period, the samples were mixed once with a sterile loop. After incubation, an equal volume of chloroform/isoamyl alcohol (24:1) was added, gently mixed by inversion and centrifuged at 4000rpm for 20 minutes. The aqueous layer was collected and centrifuged at 4000rpm for 5 minutes after the addition of 0.54 volume of isopropanol to precipitate the DNA. The supernatant was discarded and the DNA pellet was resuspended in 500 μlte. To degrade any RNA present, the DNA was incubated with 1. Mu.L of 30mg/ml LRNAase A for 30 min at 37℃and centrifuged at 4000rpm for 5 min, and the DNA was precipitated by centrifugation at 14000rpm for 10 min in the presence of 0.5 volumes of 7.5M ammonium acetate and 0.54 volumes of isopropanol. After discarding the supernatant, the pellet was washed with 500. Mu.L of 70% ethanol and dried and resuspended in 100. Mu.L TE.

4.2 restriction enzyme digestion

DNA concentrations were quantitatively determined using a spectrophotometer or fluorometer (using 1 XTNE and Hoechst dyes).

In a 100. Mu.L reaction system, 5. Mu.g of DNA was digested each time. Genomic DNA was digested with restriction enzymes, bamHI and HindIII, ecoRI and HindIII, respectively, and partial sequences of EPSPS, PAT on T-DNA were used as probes. For each enzyme, the digestate was incubated at the appropriate temperature overnight. The samples were spun down to a volume of 30 μl using a vacuum centrifugal evaporative concentrator (speed vacuum).

4.3 gel electrophoresis

Bromophenol blue loading dye was added to each sample from this example 4.2, and each sample was loaded onto a 0.7% agarose gel containing ethidium bromide, electrophoretically separated in TBE electrophoresis buffer, and the gel was electrophoresed overnight at 20 volts.

The gel was washed in 0.25M HCl for 15 minutes to depurinate the DNA, then washed with water. Southern blot hybridization was set as follows: in the tray 20 thick dry blotting papers were placed, and 4 thin dry blotting papers were placed thereon. 1 sheet of Bao Yinji paper was pre-moistened in 0.4M NaOH and placed on the paper stack, followed by 1 sheet of Hybond-N+ transfer film pre-moistened in 0.4M NaOH (Amersham Pharmacia Biotech, # RPN 303B). The gel is placed on top, ensuring that there are no bubbles between the gel and the membrane. 3 additional pre-soaked blotters were placed on top of the gel and the buffer tray was filled with 0.4M NaOH. The gel stack and the buffer disc were connected with a wick pre-immersed in 0.4M NaOH, and the DNA was transferred to the membrane. DNA transfer was performed at room temperature for about 4 hours. After transfer, the Hybond membranes were rinsed in 2 XSSC for 10 seconds and the DNA was bound to the membrane by UV cross-linking.

4.4 hybridization

PCR was used to amplify the appropriate DNA sequences for probe preparation. The DNA probes are SEQ ID NO. 24 and SEQ ID NO. 25, or are homologous or complementary to the sequence parts. 25ng of probe DNA was boiled in 45. Mu.L TE for 5 minutes, placed on ice for 7 minutes, and then transferred to a Rediprime II (Amersham Pharmacia Biotech, #RPN1633) tube. After adding 5. Mu.L of 32P-labeled dCTP to the Rediprime tube, the probe was incubated at 37℃for 15 minutes. The probe was purified by centrifugation through a microcentrifuge G-50 column (Amersham Pharmacia Biotech, # 27-5330-01) according to the manufacturer's instructions to remove unincorporated dNTPs. Probe activity was measured using a scintillation counter.

By prehybridization with Church prehybridization solution (500 mM Na ₃ P0 ₄ 1mM EDTA,7%SDS,1%BSA) wet the Hybond membrane for 30 minutes, prehybridized the Hybond membrane. The labeled probe was boiled for 5 minutes and placed on ice for 10 minutes. To the pre-hybridization buffer, an appropriate amount of probe (1 million counts per 1mL of pre-hybridization buffer) was added and hybridization was performed overnight at 65 ℃. The next day, hybridization buffer was discarded and solution 1 (40 mM Na was washed with 20mL of CH ₃ P0 ₄ 1mM EDTA,5%SDS,0.5%BSA) the membranes were washed in 150mL Church rinse solution 1 for 20 minutes at 65 ℃. Washing with Church rinse solution 2 (40 mM Na ₃ P0 ₄ 1mM EDTA,1% SDS) was repeated 2 times. The membrane is exposed to a phosphor screen or X-ray film to detect the location of probe binding.

Two control samples were included on each Southern: (1) DNA from negative (untransformed) isolates that are used to identify any endogenous maize sequences that can hybridize to the element-specific probe; and (2) HindIII-digested pLW2 plasmid equivalent to one copy number based on probe length, which served as a positive control for hybridization and was used to demonstrate the sensitivity of the experiment.

The hybridization data provided corroborated evidence that supports TaqMan PCR analysis, i.e., that corn plants LW2-2 contain a single copy of the EPSPS and PAT genes. Using the EPSPS probe, bamHI and HindIII enzymatic hydrolysis produced single bands of about 6.5kb and 13.8kb, respectively; using the PAT probe, ecoRI and HindIII were digested to yield single bands of about 6.7kb and 13.8kb, respectively. This indicates that one copy of each of EPSPS and PAT is present in maize transformation event LW 2-2.

Example 5 herbicide tolerance assay

The test selects pesticide (41% glyphosate isopropyl ammonium salt aqua) and pesticide (glufosinate with 18% active ingredient) to spray. A random block design was used, 3 replicates. The cell area is 15m ² (5 m is multiplied by 3 m), the row spacing is 60cm, the plant spacing is 25cm, the conventional cultivation management is carried out, and a 1m wide isolation belt is arranged between the cells. Transgenic maize event LW2-2 was subjected to the following 3 treatments, respectively: 1) Spraying is not performed; 2) Spraying the pesticide in the period of the V3 leaves according to the dosage of 800 ml/mu, and then spraying the pesticide again in the period of the V8 according to the same dosage; 3) The up-to-protection (Basta) herbicide was sprayed at a dose of 400 ml/mu at the V3 leaf stage, and then again at the same dose at the V8 stage. It should be noted that the glyphosate herbicide of different contents and dosage forms is converted into the form of equivalent glyphosate acid, and the glufosinate solution of different concentrations is converted into the equivalent effective component glufosinate, which is suitable for the following conclusion.

Phytotoxicity symptoms were investigated at 1 and 2 weeks after dosing, respectively, and the corn yield of the cells was determined at harvest. The phytotoxicity symptoms were ranked as shown in table 7. Herbicide damage rate is used as an index for evaluating herbicide tolerance of a transformation event, specifically, herbicide damage rate (%) = Σ (peer damage number×number of ranks)/(total number×highest rank); wherein the herbicide damage rate comprises a glyphosate damage rate and a glufosinate damage rate, and the herbicide damage rate is determined according to the phytotoxicity investigation result of 2 weeks after the treatment of the glyphosate or the glufosinate. The corn yield per cell is measured as the total yield (weight) of corn kernels in the middle 3 rows of each cell, and the yield difference between the different treatments is measured as a yield percentage (% yield = yield/no-spray control yield). The results of the tolerance of transgenic corn event LW2-2 to herbicide and the corn yield results are shown in Table 8.

TABLE 7 grading Standard for the extent of phytotoxicity of herbicides on corn

Grade of phytotoxicity	Description of symptoms
		1	Normal growth without any signs of injury
2	Slight phytotoxicity, less than 10%
		3	Moderate phytotoxicity, recovery after that, no influence on yield
4	Heavy phytotoxicity and difficult recovery, resulting in yield reduction
		5	Serious phytotoxicity and failure to recover, resulting in obvious yield reduction or absolute yield

TABLE 8 results of tolerance to herbicide and corn yield results for transgenic corn event LW2-2

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The results demonstrate that in terms of herbicide (glyphosate and glufosinate) damage: the damage rate of the transgenic corn event LW2-2 under the treatment of glyphosate herbicide agricultural reaches (800 ml/mu) is basically 0, and the effect of the transgenic corn event LW2-2 after 7 days of spraying glyphosate herbicide in the V3 phase is shown in figure 3; the damage rate of the transgenic corn event LW2-2 under the treatment of the glufosinate herbicide is basically 0 when the glufosinate herbicide is guaranteed (400 ml/mu), and the effect of the transgenic corn event LW2-2 after 7 days of the glufosinate herbicide is sprayed in the V3 stage is shown in FIG. 4; thus, transgenic corn event LW2-2 has good herbicide (glyphosate and glufosinate) tolerance.

In terms of yield: the yield of the transgenic corn event LW2-2 is not obviously different under the conditions of no spraying, glyphosate herbicide pesticide reaching (800 ml/mu) and glufosinate herbicide protecting reaching (400 ml/mu) 3 treatments; the yield of transgenic corn event LW2-2 was instead slightly increased after herbicide application, thereby further indicating that transgenic corn event LW2-2 had good herbicide (glyphosate and glufosinate) tolerance.

In summary, regenerated transgenic maize plants were tested for the presence of EPSPS and PAT genes by taqman analysis (example 2) and characterized for copy number of glyphosate and glufosinate tolerant lines. Through screening, event LW2-2 was selected to be excellent with single copy transgenes, good glyphosate herbicide tolerance, glufosinate herbicide tolerance, and manifestation of agronomic traits (example 5).

Finally, it should be noted that the above-mentioned embodiments are merely for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention.

Sequence listing

<110> Longping biotechnology (Hainan) Co., ltd

<120> transgenic maize event LW2-2 and methods of detecting the same

<160> 31

<170> SIPOSequenceListing 1.0

<210> 1

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 1

aacgatatgt gtaaactgaa gg 22

<210> 2

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

ctggccgtat cccggtagtg ac 22

<210> 3

<211> 1630

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

agggcccatg tatcctattc ttgtttccct tgtttccttc ctccaagaag cgagaagaat 60

tggaaatttc agtgaaaagc ctggactcca aggagatcaa caaagataag cagtgagtcc 120

aagaaaccgg cgaagatggg accaaatccc cttatacaac aacctctgag gtcacatgct 180

gatcctgaac ttgctggcaa actccaccgt acagagaaca aatatgtcgt gtgtccatta 240

gttgctcaga cgagagcagc agaatcgaga tgcttgacag ttcatgattg atcaactcat 300

gtcaatggaa agatttaaga agacttccat gatggacttg caccggcagc aaggaacatt 360

cagatccaac tgcacacaga agaatcaact caattgtgag aggataatga catctgatga 420

ctacacgtct gaagccaata tctaagtatg tgtgcaacac cattcactca aactaaagaa 480

acccattaat gccaaaaata ccaacagcat tgtgcttgca ggcctggcgg cactagaaat 540

catgtacaca gaaaagaaat ggctacttga ataggcagag aaaatataaa gtattagact 600

aaaccaaaag acttaatacc atcaattgcg ttgcctaacc tcattttccc ttgcacaagt 660

attactcctg ctagctcctg ttttgttgtg tgcctcaggc tctgtttcag tgtttgtcat 720

actaatgagc ataaaatagt ggagcaaggc agccggctgt tatcgagcac attgagggca 780

tcatccatgg aatgagtagg agctgccagc cgccgctcta ccatgtgccc atttcatcga 840

gcacaatgag ggcatcatcc atggaatgaa ttggagcggc aacagatgag gagtagaaga 900

ggaagtttgc tccatccagc ctgttgacgc taccggctag tcctctgctg cccccaggtt 960

atgtctaacg cacttgctgg atctgataca gaaaaggtga gaagcaaatc tgggagtata 1020

aatgattata aatgggtcat gggtgaaaga gagggagaac gatatgtgta aactgaaggc 1080

gggaaacgac aatctgatca agagcggaga attaagggag tcacgttatg acccccgccg 1140

atgacgcggg acaagccgtt ttacgtttgg aactgacaga accgcaacgc tgcaggaatt 1200

ggccgcaggt ggatttgtat taaactaatg actaattagt ggcactagcc tcaccgactt 1260

cgcagacgag gccgctaagt cgcagctacg ctctcaacgg cactgactag gtagtttaaa 1320

cgtgcactta attaaggtac cgggaattta aatcccggga ggtctcgcag acctagctag 1380

ttagaatccc gagacctaag tgactagggt cacgtgaccc tagtcactta aagcttaatt 1440

gacgcttaga caacttaata acacattgcg gacgttttta atgtactgaa ttaacgccga 1500

attaattcgg gggatctgga ttttagtact ggattttggt tttaggaatt agaaatttta 1560

ttgatagaag tattttacaa atacaaatac atactaaggg tttcttatat gctcaacaca 1620

tgagcgaaac 1630

<210> 4

<211> 1479

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

ctgaaatcac cagtctctct ctacaaatct atctctctct ataataatgt gtgagtagtt 60

cccagataag ggaattaggg ttcttatagg gtttcgctca tgtgttgagc atataagaaa 120

cccttagtat gtatttgtat ttgtaaaata cttctatcaa taaaatttct aattcctaaa 180

accaaaatcc agtggcctgc agggaattct taattaagtg cacgcggccg cctacttagt 240

caagagcctc gcacgcgact gtcacgcggc caggatcgcc tcgtgagcct cgcaatctgt 300

acctagttta gctagttagg acgttaacag ggacgcgcct ggccgtatcc cggtagtgac 360

ctgtcatggt acgagttctt aagggtccgt atgtgtggat tgctaaaaca cgtctgaagc 420

tgatgcgttg gacgacgcat gtgtggattg cagatgaaat cgtgtgcttc ggtaggattt 480

tgaataaatt ttgcaaaaca tgatccgtga gtgcatacct caacggctca acccatgacg 540

atacgatgca atatgtggct gagccggtgg gttcggtttc aaccagacgc aagaatttat 600

agggtctaga aagaccacat aaaagtcatg gtttgttgtt cgtcaagctc gctaactatc 660

ggaaatgaat cacattgttg cgacgagcga gatctagttt gtcgaacaga ttggcatcac 720

cacgacaatg gggcaaggat gaatgcgcta ggaaggtgcg aggttgggcg gcggcggaaa 780

cggagtagcg agcaaactgg tttaggtagt atcgagaaga ttaaggtcag agtgatagat 840

gcaaggacta ggccgggaag gatccaataa ggaggctcat gaatgggttg ttttatgtca 900

tgttccaaat atctagagct aattgttatc tggttttagc tagtttgatg tggctaacaa 960

ctaatgctta tttgagggta taactaacaa ttagttgatt cattctaaac ctcttagtta 1020

attttagccg ctaattatta gtttcagagg ttcagaacaa gactttatat tatctaatat 1080

ttctattttt ttctacgctc gacagtaaat aatcattaaa cataaatgat gtataaaatt 1140

taaggggggc atggcctact ttgccctctt tgaatttacc tctggtttca acgccattcg 1200

gttcgaccgg ctgatcgatc ggtgtgccta actggtcagt cgagaccaat accgacccgg 1260

aatgccccag agaaccggtt gcatgagtat tagaggagat taggagcata taacccctta 1320

atctcctcct atatccgtgt atctaaacaa gcctttatag attatatcga ggaatataat 1380

tttatatgca agtatgattt tttaaatggt tttagactag tgtgatatgt aactgttgga 1440

ttaccaagtc atttacataa aaatataggc tcacggccc 1479

<210> 5

<211> 7280

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

agggcccatg tatcctattc ttgtttccct tgtttccttc ctccaagaag cgagaagaat 60

tggaaatttc agtgaaaagc ctggactcca aggagatcaa caaagataag cagtgagtcc 120

aagaaaccgg cgaagatggg accaaatccc cttatacaac aacctctgag gtcacatgct 180

gatcctgaac ttgctggcaa actccaccgt acagagaaca aatatgtcgt gtgtccatta 240

gttgctcaga cgagagcagc agaatcgaga tgcttgacag ttcatgattg atcaactcat 300

gtcaatggaa agatttaaga agacttccat gatggacttg caccggcagc aaggaacatt 360

cagatccaac tgcacacaga agaatcaact caattgtgag aggataatga catctgatga 420

ctacacgtct gaagccaata tctaagtatg tgtgcaacac cattcactca aactaaagaa 480

acccattaat gccaaaaata ccaacagcat tgtgcttgca ggcctggcgg cactagaaat 540

catgtacaca gaaaagaaat ggctacttga ataggcagag aaaatataaa gtattagact 600

aaaccaaaag acttaatacc atcaattgcg ttgcctaacc tcattttccc ttgcacaagt 660

attactcctg ctagctcctg ttttgttgtg tgcctcaggc tctgtttcag tgtttgtcat 720

actaatgagc ataaaatagt ggagcaaggc agccggctgt tatcgagcac attgagggca 780

tcatccatgg aatgagtagg agctgccagc cgccgctcta ccatgtgccc atttcatcga 840

gcacaatgag ggcatcatcc atggaatgaa ttggagcggc aacagatgag gagtagaaga 900

ggaagtttgc tccatccagc ctgttgacgc taccggctag tcctctgctg cccccaggtt 960

atgtctaacg cacttgctgg atctgataca gaaaaggtga gaagcaaatc tgggagtata 1020

aatgattata aatgggtcat gggtgaaaga gagggagaac gatatgtgta aactgaaggc 1080

gggaaacgac aatctgatca agagcggaga attaagggag tcacgttatg acccccgccg 1140

atgacgcggg acaagccgtt ttacgtttgg aactgacaga accgcaacgc tgcaggaatt 1200

ggccgcaggt ggatttgtat taaactaatg actaattagt ggcactagcc tcaccgactt 1260

cgcagacgag gccgctaagt cgcagctacg ctctcaacgg cactgactag gtagtttaaa 1320

cgtgcactta attaaggtac cgggaattta aatcccggga ggtctcgcag acctagctag 1380

ttagaatccc gagacctaag tgactagggt cacgtgaccc tagtcactta aagcttaatt 1440

gacgcttaga caacttaata acacattgcg gacgttttta atgtactgaa ttaacgccga 1500

attaattcgg gggatctgga ttttagtact ggattttggt tttaggaatt agaaatttta 1560

ttgatagaag tattttacaa atacaaatac atactaaggg tttcttatat gctcaacaca 1620

tgagcgaaac cctatagaaa ccctaattcc cttatctggg aactactcac acattattat 1680

ggagaaactc gactagttca ggcagccttc gtatcggaga gttcgatctt cgcgcccagc 1740

ccggccatca ggtccatgaa ctccgggaag ctcgtggcga tcatcgtggc atcgtccacc 1800

gtgacagggt tttccgacac gaggcccatg acgaggaagc tcatggcgat gcggtgatcg 1860

agatgggtgg cgacggcggc gcccgaggcg ttgccgagcc ccttgccgtc agggcggcca 1920

cgcacgacga gcgacgtctc gccctcatcg caatccacgc cattgagctt gaggccattg 1980

gcgacggccg agaggcggtc gctttccttg acgcggagtt cttccagacc gttcatcacg 2040

gtcgcccctt ccgcgaaggc ggcggcgaca gcgagaatcg gatattcgtc gatcatcgaa 2100

ggcgcgcggt cttccggcac cgtgacgccc ttcagcgtgg aggagcgaac gcgcaggtcc 2160

gccacgtctt cgccgccggc aaggcgcggg ttgatgactt cgatgtcggc gcccatttcc 2220

tgcagcgtca ggatgaggcc ggtgcgggtg gggttcatca gcacgttgag gatggtgacg 2280

tcggagcccg gaacaagcag ggccgcaacc agcgggaagg ccgtcgagga cgggtcgccc 2340

ggcacgtcga tgacttggcc ggtgagcttg ccgcggcctt ccaggcggat ggtgcgcacg 2400

ccgtccgcat ccgtctcgac ggtaaggttg gcgccaaagc cctgcagcat cttttccgta 2460

tgatcgcgcg tcatgatcgg ctcgatgacc gtcgtgatgc cgggcgtgtt gaggccggcg 2520

agcagcacgg cggacttcac ctgtgcggag gccatcggca cgcggtaggt gatcggcgtc 2580

ggcgtcttcg gcccgcgcaa ggtaacggga agacggtcac cgtcttccga tttcacctgc 2640

acgcccattt cgcgcagcgg gttcaacacg cggcccatcg ggcgctttgt gagcgaggcg 2700

tcgccgatga aggtgctgtc gaaatcgtag accccgacga ggcccatcgt caggcggcag 2760

cccgtggcgg cattgccgaa atcgagcggc gcctcaggcg ccaggaggcc gccattgccg 2820

acgccatcga tgatccaggt gtcgccttcc ttacggatgc gggcgcccat cgcctgcatg 2880

gccttgcccg tattgatgac gtcctcgcct tccagaaggc cggtgatgcg cgtttcaccg 2940

ctcgcgagac cgccgaacat gaaggaccgg tgggagatcg acttgtcgcc gggaatgcgg 3000

acggttccgg aaaggccaga ggatttgcgg gcggttgcgg gccggctgct tgcaccgtga 3060

agcatgcacg ccgtggaaac agaagacatg accttaagag gacgaagctc agagccaatt 3120

aacgtcatcc cactcttctt caatccccac gacgacgaaa tcggataagc tcgtggatgc 3180

tgctgcgtct tcagagaaac cgataaggga gatttgcgtt gactggattt cgagagattg 3240

gagataagag atgggttctg cacaccattg cagattctgc taacttgcgc catggttgat 3300

cacttctacc tacaaaaaag ctccgcacga ggctgcattt gtcacaaatc atgaaaagaa 3360

aaactaccga tgaacaatgc tgagggattc aaattctacc cacaaaaaga agaaagaaag 3420

atctagcaca tctaagcctg acgaagcagc agaaatatat aaaaatataa accatagtgc 3480

ccttttcccc tcttcctgat cttgtttagc atggcggaaa ttttaaaccc cccatcatct 3540

cccccaacaa cggcggatcg cagatctaca tccgagagcc ccattccccg cgagatccgg 3600

gccggatcga tccacgccgg cgagagcccc agccgcgaga tcccgcccct cccgcgcacc 3660

gatctgggcg cgcacgaagc cgcctctcgc ccacccaaac taccaaggcc aaagatcgag 3720

accgagacgg aaaaaaaaaa cggagaaaga aagaggagag gggcggggtg gttaccggcg 3780

cggcggcggc ggagggggag gggggaggag ctcgtcgtcc ggcagcgagg ggggaggagg 3840

tggaggtggt ggtggtggtg gtggtagggt tggggggatg ggaggagagg ggggggtatg 3900

tatatagtgg cgatgggggg cgtttctttg gaagcggagg gagggccggc ctcgtcgctg 3960

gctcgcgatc ctcctcgcgt ttccggcccc cacgacccgg acccacctgc tgttttttct 4020

ttttcttttt tttctttctt tttttttttt ggctgcgaga cgtgcggtgc gtgcggacaa 4080

ctcacggtga tagtgggggg gtgtggagac tattgtccag ttggctggac tggggtgggt 4140

tgggttgggt tgggttgggc tgggcttgct atggatcgtg gatagcactt tgggctttag 4200

gaactttagg ggttgttttt gtaaatgttt tgagtctaag tttatctttt atttttacta 4260

gaaaaaatac ccatgcgctg caacggggga aagctatttt aatcttatta ttgttcattg 4320

tgagaattaa ttcgcctgaa tatatatttt tctcaaaaat tatgtcaaat tagcatatgg 4380

gtttttttaa agatatttct tatacaaatc cctctgtatt tacaaaagca aacgaactta 4440

aaacccgact caaatacaga tatgcatttc caaaagcgaa taaacttaaa aaccaattca 4500

tacaaaaatg acgtatcaaa gtaccgacaa aaacatcctc aatttttata atagtagaaa 4560

agagtaaatt tcactttggg ccacctttta ttaccgatat tttactttat accacctttt 4620

aactgatgtt ttcacttttg accaggtaat cttacctttg ttttattttg gactatcccg 4680

actctcttct caagcatatg aatgacctcg aggcgcgccc catggagtca aagattcaaa 4740

tagaggacct aacagaactc gccgtaaaga ctggcgaaca gttcatacag agtctcttac 4800

gactcaatga caagaagaaa atcttcgtca acatggtgga gcacgacacg cttgtctact 4860

ccaaaaatat caaagataca gtctcagaag accaaagggc aattgagact tttcaacaaa 4920

gggtaatatc cggaaacctc ctcggattcc attgcccagc tatctgtcac tttattgtga 4980

agatagtgga aaaggaaggt ggctcctaca aatgccatca ttgcgataaa ggaaaggcca 5040

tcgttgaaga tgcctctgcc gacagtggtc ccaaagatgg acccccaccc acgaggagca 5100

tcgtggaaaa agaagacgtt ccaaccacgt cttcaaagca agtggattga tgtgatatct 5160

ccactgacgt aagggatgac gcacaatccc actatccttc gcaagaccct tcctctatat 5220

aaggaagttc atttcatttg gagaggacaa tgtctccgga gaggagacca gttgagatta 5280

ggccagctac agcagctgat atggccgcgg tttgtgatat cgttaaccat tacattgaga 5340

cgtctacagt gaactttagg acagagccac aaacaccaca agagtggatt gatgatctag 5400

agaggttgca agatagatac ccttggttgg ttgctgaggt tgagggtgtt gtggctggta 5460

ttgcttacgc tgggccctgg aaggctagga acgcttacga ttggacagtt gagagtactg 5520

tttacgtgtc acataggcat caaaggttgg gcctaggatc cacattgtac acacatttgc 5580

ttaagtctat ggaggcgcaa ggttttaagt ctgtggttgc tgttataggc cttccaaacg 5640

atccatctgt taggttgcat gaggctttgg gatacacagc ccggggtaca ttgcgcgcag 5700

ctggatacaa gcatggtgga tggcatgatg ttggtttttg gcaaagggat tttgagttgc 5760

cagctcctcc aaggccagtt aggccagtta cccagatctg actgaaatca ccagtctctc 5820

tctacaaatc tatctctctc tataataatg tgtgagtagt tcccagataa gggaattagg 5880

gttcttatag ggtttcgctc atgtgttgag catataagaa acccttagta tgtatttgta 5940

tttgtaaaat acttctatca ataaaatttc taattcctaa aaccaaaatc cagtggcctg 6000

cagggaattc ttaattaagt gcacgcggcc gcctacttag tcaagagcct cgcacgcgac 6060

tgtcacgcgg ccaggatcgc ctcgtgagcc tcgcaatctg tacctagttt agctagttag 6120

gacgttaaca gggacgcgcc tggccgtatc ccggtagtga cctgtcatgg tacgagttct 6180

taagggtccg tatgtgtgga ttgctaaaac acgtctgaag ctgatgcgtt ggacgacgca 6240

tgtgtggatt gcagatgaaa tcgtgtgctt cggtaggatt ttgaataaat tttgcaaaac 6300

atgatccgtg agtgcatacc tcaacggctc aacccatgac gatacgatgc aatatgtggc 6360

tgagccggtg ggttcggttt caaccagacg caagaattta tagggtctag aaagaccaca 6420

taaaagtcat ggtttgttgt tcgtcaagct cgctaactat cggaaatgaa tcacattgtt 6480

gcgacgagcg agatctagtt tgtcgaacag attggcatca ccacgacaat ggggcaagga 6540

tgaatgcgct aggaaggtgc gaggttgggc ggcggcggaa acggagtagc gagcaaactg 6600

gtttaggtag tatcgagaag attaaggtca gagtgataga tgcaaggact aggccgggaa 6660

ggatccaata aggaggctca tgaatgggtt gttttatgtc atgttccaaa tatctagagc 6720

taattgttat ctggttttag ctagtttgat gtggctaaca actaatgctt atttgagggt 6780

ataactaaca attagttgat tcattctaaa cctcttagtt aattttagcc gctaattatt 6840

agtttcagag gttcagaaca agactttata ttatctaata tttctatttt tttctacgct 6900

cgacagtaaa taatcattaa acataaatga tgtataaaat ttaagggggg catggcctac 6960

tttgccctct ttgaatttac ctctggtttc aacgccattc ggttcgaccg gctgatcgat 7020

cggtgtgcct aactggtcag tcgagaccaa taccgacccg gaatgcccca gagaaccggt 7080

tgcatgagta ttagaggaga ttaggagcat ataacccctt aatctcctcc tatatccgtg 7140

tatctaaaca agcctttata gattatatcg aggaatataa ttttatatgc aagtatgatt 7200

ttttaaatgg ttttagacta gtgtgatatg taactgttgg attaccaagt catttacata 7260

aaaatatagg ctcacggccc 7280

<210> 6

<211> 560

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

aactgaaggc gggaaacgac aatctgatca agagcggaga attaagggag tcacgttatg 60

acccccgccg atgacgcggg acaagccgtt ttacgtttgg aactgacaga accgcaacgc 120

tgcaggaatt ggccgcaggt ggatttgtat taaactaatg actaattagt ggcactagcc 180

tcaccgactt cgcagacgag gccgctaagt cgcagctacg ctctcaacgg cactgactag 240

gtagtttaaa cgtgcactta attaaggtac cgggaattta aatcccggga ggtctcgcag 300

acctagctag ttagaatccc gagacctaag tgactagggt cacgtgaccc tagtcactta 360

aagcttaatt gacgcttaga caacttaata acacattgcg gacgttttta atgtactgaa 420

ttaacgccga attaattcgg gggatctgga ttttagtact ggattttggt tttaggaatt 480

agaaatttta ttgatagaag tattttacaa atacaaatac atactaaggg tttcttatat 540

gctcaacaca tgagcgaaac 560

<210> 7

<211> 347

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

ctgaaatcac cagtctctct ctacaaatct atctctctct ataataatgt gtgagtagtt 60

cccagataag ggaattaggg ttcttatagg gtttcgctca tgtgttgagc atataagaaa 120

cccttagtat gtatttgtat ttgtaaaata cttctatcaa taaaatttct aattcctaaa 180

accaaaatcc agtggcctgc agggaattct taattaagtg cacgcggccg cctacttagt 240

caagagcctc gcacgcgact gtcacgcggc caggatcgcc tcgtgagcct cgcaatctgt 300

acctagttta gctagttagg acgttaacag ggacgcgcct ggccgta 347

<210> 8

<211> 27

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

agggcccatg tatcctattc ttgtttc 27

<210> 9

<211> 27

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

gtttcgctca tgtgttgagc atataag 27

<210> 10

<211> 29

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

ctgaaatcac cagtctctct ctacaaatc 29

<210> 11

<211> 30

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

gggccgtgag cctatatttt tatgtaaatg 30

<210> 12

<211> 28

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

gtatgtgtgc aacaccattc actcaaac 28

<210> 13

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

agctaggtct gcgagacctc cc 22

<210> 14

<211> 30

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

agcattagtt gttagccaca tcaaactagc 30

<210> 15

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

ccaaaatcca gtggcctgca ggga 24

<210> 16

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

gcaaatcctc tggcctttcc 20

<210> 17

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

tgaaggaccg gtgggagat 19

<210> 18

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 18

cgtccgcatt cccggcga 18

<210> 19

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 19

ccgcggtttg tgatatcgtt 20

<210> 20

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 20

tcttgcaacc tctctagatc atcaa 25

<210> 21

<211> 30

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 21

taggacagag ccacaaacac cacaagagtg 30

<210> 22

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 22

agcagacggc acggcatctc tgt 23

<210> 23

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 23

cagaagtaga actaccgggc cct 23

<210> 24

<211> 655

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 24

ctacgatttc gacagcacct tcatcggcga cgcctcgctc acaaagcgcc cgatgggccg 60

cgtgttgaac ccgctgcgcg aaatgggcgt gcaggtgaaa tcggaagacg gtgaccgtct 120

tcccgttacc ttgcgcgggc cgaagacgcc gacgccgatc acctaccgcg tgccgatggc 180

ctccgcacag gtgaagtccg ccgtgctgct cgccggcctc aacacgcccg gcatcacgac 240

ggtcatcgag ccgatcatga cgcgcgatca tacggaaaag atgctgcagg gctttggcgc 300

caaccttacc gtcgagacgg atgcggacgg cgtgcgcacc atccgcctgg aaggccgcgg 360

caagctcacc ggccaagtca tcgacgtgcc gggcgacccg tcctcgacgg ccttcccgct 420

ggttgcggcc ctgcttgttc cgggctccga cgtcaccatc ctcaacgtgc tgatgaaccc 480

cacccgcacc ggcctcatcc tgacgctgca ggaaatgggc gccgacatcg aagtcatcaa 540

cccgcgcctt gccggcggcg aagacgtggc ggacctgcgc gttcgctcct ccacgctgaa 600

gggcgtcacg gtgccggaag accgcgcgcc ttcgatgatc gacgaatatc cgatt 655

<210> 25

<211> 377

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 25

agattaggcc agctacagca gctgatatgg ccgcggtttg tgatatcgtt aaccattaca 60

ttgagacgtc tacagtgaac tttaggacag agccacaaac accacaagag tggattgatg 120

atctagagag gttgcaagat agataccctt ggttggttgc tgaggttgag ggtgttgtgg 180

ctggtattgc ttacgctggg ccctggaagg ctaggaacgc ttacgattgg acagttgaga 240

gtactgttta cgtgtcacat aggcatcaaa ggttgggcct aggatccaca ttgtacacac 300

atttgcttaa gtctatggag gcgcaaggtt ttaagtctgt ggttgctgtt ataggccttc 360

caaacgatcc atctgtt 377

<210> 26

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 26

cgatacgaag gctgcctgaa 20

<210> 27

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 27

ccatcaggtc catgaactcc 20

<210> 28

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 28

gtgacagggt tttccgacac 20

<210> 29

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 29

tccaaggcca gttaggccag ttac 24

<210> 30

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 30

gggctgtgta tcccaaagcc tca 23

<210> 31

<211> 26

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 31

ggcctataac agcaaccaca gactta 26

Claims

1. A nucleic acid molecule comprising any one selected from the group consisting of sequences SEQ ID NOs 1-5 and complements thereof, said nucleic acid molecule being derived from a transgenic plant, seed or cell comprising corn event LW2-2, corn seed comprising said event having been deposited under accession number cctccc No. P202022.

2. A DNA primer pair comprising a first primer and a second primer, wherein when said first primer and said second primer are used in an amplification reaction with DNA containing corn event LW2-2, an amplicon is produced for detection of corn event LW2-2 in a sample,

the first primer is selected from SEQ ID NO. 8 or SEQ ID NO. 12; the second primer is selected from SEQ ID NO. 11 or SEQ ID NO. 14; corn seeds containing the event have been preserved with a preservation number CCTCC NO: P202022.

3. A method of detecting the presence of DNA from transgenic corn event LW2-2 in a sample, comprising:

(1) Contacting a sample to be detected with the DNA primer pair of claim 2 in a nucleic acid amplification reaction;

(2) Performing a nucleic acid amplification reaction;

(3) Detecting the presence of an amplification product;

the amplified product comprises a nucleic acid sequence with a sequence SEQ ID NO. 3-5 or a complementary sequence thereof, namely, DNA (deoxyribonucleic acid) representing that the detected sample contains transgenic corn event LW2-2, and corn seeds containing the event are preserved with a preservation number CCTCC NO. P202022.

4. A DNA detection kit comprising: the DNA primer pair of claim 2.

5. A method of protecting a corn plant from injury caused by a herbicide comprising planting at least one transgenic corn plant comprising transgenic corn event LW2-2, applying an effective dose of glyphosate and/or glufosinate herbicide; corn seeds containing the event have been preserved with a preservation number CCTCC NO: P202022.

6. A method of controlling weeds in a field comprising applying an effective dose of glyphosate and/or glufosinate herbicide to a field in which at least one transgenic corn plant comprising transgenic corn event LW2-2 has been deposited with the corn seeds of said event under deposit number cctccc No. P202022.

7. A method of growing a corn plant tolerant to glyphosate and/or glufosinate herbicide comprising: planting at least one corn seed comprising transgenic corn event LW 2-2;

growing the corn seed into a corn plant;

spraying the maize plants with an effective dose of glyphosate and/or glufosinate herbicide, harvesting plants having reduced plant damage as compared to other plants not having the transgenic maize event LW2-2, the maize seed comprising the event having been preserved at a preservation number cctccc No. P202022.