CN116144672B

CN116144672B - Transgenic corn event LP026-1 and detection method thereof

Info

Publication number: CN116144672B
Application number: CN202211161751.2A
Authority: CN
Inventors: 贾志伟; 吕玉平; 李晓娇; 王强; 李树秀; 孙宇; 王建海; 赵丽媛; 石小堰; 李涛
Original assignee: Longping Biotechnology Hainan Co ltd
Current assignee: Longping Biotechnology Hainan Co ltd
Priority date: 2022-09-23
Filing date: 2022-09-23
Publication date: 2023-11-07
Anticipated expiration: 2042-09-23
Also published as: CN116144672A

Abstract

The present invention provides a nucleic acid sequence comprising one or more selected from the group consisting of the sequences SEQ ID NO. 1-7 and the complements thereof, said nucleic acid sequence being derived from a plant, seed or cell comprising transgenic maize event LP026-1, a representative sample of seed comprising said event having been deposited under accession number CCTCC NO. P202206. The transgenic corn event LP026-1 of the present invention is not only well resistant to ingestion by lepidopteran pests, but also is tolerant to glyphosate-containing agricultural herbicides. The maize plant with the double characters has the following advantages: the economic loss caused by lepidopteran pests is avoided; corn crops that can tolerate the common commercial herbicide glyphosate; the corn yield is not reduced; enhancing breeding efficiency, enabling the use of molecular markers to track transgene inserts in the breeding populations and their offspring. Meanwhile, the detection method provided by the invention can rapidly, accurately and stably identify the existence of the plant material derived from the transgenic corn event LP 026-1.

Description

Transgenic corn event LP026-1 and detection method thereof

Technical Field

The invention belongs to the technical field of molecular biology, relates to a detection method of transgenic plants and products thereof, and in particular relates to a transgenic corn event LP026-1 which is resistant to insects and resistant to glyphosate herbicide application, and a nucleic acid sequence and a method for detecting the transgenic corn LP 026-1.

Background

Corn (Zea mays l.) is the predominant food crop in many parts of the world. Biotechnology has been applied to corn to improve its agronomic traits and quality. Insect resistance is an important agronomic trait in corn production, particularly resistance to lepidopteran insects (e.g., corn borer, cotton bollworm, spodoptera frugiperda, armyworm, etc.). Resistance of maize to lepidopteran insects can be obtained by transgenic expression of a lepidopteran insect resistance gene in a maize plant. Another important agronomic trait is herbicide tolerance, particularly glyphosate tolerance. Tolerance to glyphosate herbicide in corn can be achieved by transgenic approaches to express glyphosate herbicide tolerance genes (e.g., epsps) in corn plants.

Expression of exogenous genes in plants is known to be affected by their insertion into maize genome, 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 for 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 have excellent lepidopteran pest (e.g., asian corn borer, spodoptera frugiperda, oriental armyworm, spodoptera frugiperda, cotton bollworm, cutworm, and Pink borer, etc.) and glyphosate herbicide resistance without affecting corn yield, and conventional breeding methods can be used to backcross transgenic traits 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 lepidoptera pests (such as Asian corn borer, spodoptera frugiperda, oriental armyworm, spodoptera frugiperda, cotton bollworm, cutworm, carpoth borer and the like) and glyphosate herbicide resistance, prevents the varieties from being damaged by main lepidoptera pests, 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 polynucleotide detection method, such as Polymerase Chain Reaction (PCR) or DNA hybridization using polynucleotide 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 T-DNA 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 event LP026-1, a nucleic acid sequence for detecting corn plant LP026-1 event and a detection method thereof, which can accurately and rapidly identify whether a biological sample contains a DNA molecule of a specific transgenic corn event LP 026-1.

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 LP026-1, and a representative sample of seed (taxonomically named Zea mays L) comprising the event has been deposited at the China center for type culture collection (CCTCC, address: wuhan, hubei, wuhan, university, eight 299, and university, wuhan, university, center of post code 430072) at a accession number of CCTCC NO: P202206 on 4 months 15 of 2022. In some embodiments, the nucleic acid sequence is an amplicon diagnostic for the presence of corn event LP 026-1.

In some embodiments of the invention, the invention provides a nucleic acid sequence comprising at least 11 consecutive nucleotides of SEQ ID NO. 3 or a complement thereof and/or at least 11 consecutive nucleotides of SEQ ID NO. 4 or a complement thereof. 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 LP026-1, 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 LP026-1 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 22 nucleotides, which is positioned near the insertion junction at the 3 '-end of the insertion sequence in the transgenic corn event LP026-1, 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 LP026-1 can be identified by the SEQ ID NO. 2 or the complementary sequence thereof.

The nucleic acid sequences provided herein can be at least 11 or more contiguous polynucleotides (first nucleic acid sequences) of any portion of the transgene insert sequence in SEQ ID No. 3 or its complement, or at least 11 or more contiguous polynucleotides (second nucleic acid sequences) of any portion of the 5' flanking maize genomic DNA region in SEQ ID No. 3 or its complement. 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 pair of DNA primers in a DNA amplification method that produces an amplification product. The presence of transgenic maize event LP026-1 or its progeny can be diagnosed when the amplification product generated 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 nucleotides 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 nucleotides 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 nucleotides set forth in SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5, the probes and primers may be about 17 to 50 or more consecutive nucleotides in length. The sequence of SEQ ID NO. 3 or its complement is a 1359 nucleotide sequence near the 5 'end of the insertion junction in transgenic maize event LP026-1, the SEQ ID NO. 3 or its complement consists of a 890 nucleotide maize flanking genomic DNA sequence (nucleotides 1-890 of SEQ ID NO. 3), a 352 nucleotide pLP026 construct DNA sequence (nucleotides 891-1242 of SEQ ID NO. 3) and a 117 nucleotide 3' end DNA sequence of the Nos terminator (nucleotides 1243-1359 of SEQ ID NO. 3), comprising the SEQ ID NO. 3 or its complement can be identified as the presence of transgenic maize event LP 026-1.

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 nucleotides (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, the method of amplifying DNA to produce an amplified product includes a pair of DNA primers. The presence of transgenic maize event LP026-1 or its progeny can be diagnosed when the amplification product generated in the DNA amplification method using the DNA primer pair is an amplification product comprising SEQ ID NO. 2. The sequence of SEQ ID NO. 4 or its complement is 584 nucleotides in length near the 3' -end of the insertion junction in the transgenic maize event LP026-1, the SEQ ID NO. 4 or its complement consists of 53 nucleotide tNos (nopaline synthase) transcription terminator sequence (nucleotides 1-53 of SEQ ID NO. 4), 199 nucleotide pLP026 construct DNA sequence (nucleotides 54-252 of SEQ ID NO. 4) and 332 nucleotide maize integration site flanking genomic DNA sequence (nucleotides 253-584 of SEQ ID NO. 4), comprising the SEQ ID NO. 4 or its complement can be identified as the presence of the transgenic maize event LP 026-1.

The SEQ ID NO. 5 or its complement is a sequence of 17345 nucleotides in length characterizing transgenic maize event LP026-1, which specifically contains the genome and genetic elements as shown in Table 1. The presence of transgenic maize event LP026-1 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 may be used in a DNA amplification method to produce an amplification product, the presence of transgenic corn event LP026-1 or its progeny in a biological sample being diagnosed by detection of the amplification product; the nucleic acid sequence or the complement thereof may be used in a nucleotide assay to detect the presence of transgenic maize event LP026-1 or a progeny thereof in a biological sample.

The present invention provides a DNA primer pair comprising a first primer and a second primer, wherein each of the first primer and the second primer comprises a fragment of SEQ ID No. 5 or a complement thereof and when used in an amplification reaction with DNA comprising corn event LP026-1, produces an amplification product of detecting corn event LP026-1 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. 10; 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.

Further, the amplification product comprises consecutive nucleotides 1 to 11 or 12 to 22 in SEQ ID NO. 1 or its complement, or consecutive nucleotides 1 to 11 or 12 to 22 in SEQ ID NO. 2 or its complement.

Still further, 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. 10 or SEQ ID NO. 15, and the second primer is selected from SEQ ID NO. 11 or SEQ ID NO. 14.

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 fragment of SEQ ID NO. 5 or a complement thereof, which hybridizes under stringent hybridization conditions with 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 with 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 one embodiment, 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 for detecting the presence of DNA comprising transgenic maize event LP026-1 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 transgenic maize event LP026-1 in the test sample.

The invention also provides a method of detecting the presence of DNA comprising transgenic maize event LP026-1 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.

Wherein hybridization of the sample to be tested and the marker nucleic acid molecule is detected, and further by marker assisted breeding analysis to determine that insect resistance and/or herbicide tolerance is genetically linked to the marker nucleic acid molecule.

The invention also provides a DNA detection kit, comprising: a DNA primer pair that produces an amplicon diagnostic for transgenic maize event LP026-1, a probe specific for SEQ ID NOs 1-7 or a marker nucleic acid molecule specific for SEQ ID NOs 1-7. Specifically, the detection kit comprises the probe, the primer pair or the marker nucleic acid molecule.

In some embodiments, the invention provides a DNA detection kit comprising 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 be used as a DNA primer or probe specific for transgenic maize event LP026-1 or a progeny thereof.

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 comprises a homologous sequence of SEQ ID NO. 1 or a complement thereof, a homologous sequence of SEQ ID NO. 2 or a complement thereof, a homologous sequence of SEQ ID NO. 6 or a complement thereof, or a homologous sequence of SEQ ID NO. 7 or a complement thereof. To achieve the above object, the present invention also provides a plant cell comprising nucleic acid sequences encoding insect-resistant Cry1Ab, cry2Ab and Cry1Fa proteins, a nucleic acid sequence encoding a glyphosate herbicide tolerance EPSPS protein and a nucleic acid sequence of a specific region comprising the sequence set forth in 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

The invention also provides a method of protecting a maize plant from insect infestation comprising providing at least one transgenic maize plant cell comprising transgenic maize event LP026-1 in the diet of a target insect; target insects that ingest the transgenic corn plant cells are inhibited from further ingest the corn plant.

The invention also provides a method of protecting a maize plant from injury caused by a herbicide, planting at least one transgenic maize plant comprising transgenic maize event LP026-1. In some embodiments, the method comprises applying an effective dose of a glyphosate herbicide to a field in which at least one transgenic corn plant comprising transgenic corn event LP026-1 is grown.

The invention also provides a method of controlling weeds in a field in which corn plants are planted, comprising applying to the field in which at least one transgenic corn plant comprising transgenic corn event LP026-1 is planted an effective dose of a glyphosate herbicide.

The present invention also provides a method of growing a maize plant that is resistant to insects comprising: planting at least one corn seed comprising transgenic corn event LP 026-1;

growing the corn seed into a corn plant;

attack the maize plant with the target insect and/or spray the maize plant with an effective dose of glyphosate herbicide, harvesting plants having reduced plant damage as compared to other plants not comprising the transgenic maize event LP026-1.

In some embodiments, the invention provides a method of culturing a corn plant that is resistant to insects and tolerant to a glyphosate herbicide comprising:

planting at least one corn seed comprising transgenic corn event LP 026-1;

growing the corn seed into a corn plant;

spraying the maize plants with an effective dose of a glyphosate herbicide, and harvesting plants having reduced plant damage as compared to other plants not having the transgenic maize event LP026-1, the plants having reduced plant damage also being resistant to feeding damage by insects.

In some embodiments, the invention also provides a method of producing a maize plant that is resistant to insects, comprising introducing into the genome of said maize plant a transgenic maize event LP026-1, selecting a maize plant that has reduced plant damage to insect ingestion. In some embodiments, the method comprises: sexual crossing a transgenic corn event LP026-1 first parent corn plant having resistance to an insect with a second parent corn plant lacking insect resistance, thereby producing a plurality of progeny plants; attack the progeny plant with a target insect; selecting said progeny plants having reduced plant damage compared to other plants not having transgenic maize event LP 026-1.

In some embodiments, the invention also provides a method of producing a maize plant that is tolerant to a glyphosate herbicide, comprising introducing into the genome of the maize plant transgenic maize event LP026-1, selecting a maize plant that is tolerant to glyphosate. In some embodiments, the method comprises: sexual crossing a transgenic corn event LP026-1 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 corn plant that is resistant to insects and tolerant to glyphosate herbicide application, comprising: transgenic maize event LP026-1 was introduced into the genome of the maize plant and a maize plant was selected that was glyphosate tolerant and insect resistant. In some embodiments, the methods comprise sexually crossing a transgenic corn event LP026-1 of glyphosate tolerance and insect resistance with a second parent corn plant lacking glyphosate tolerance and/or insect resistance, thereby producing a plurality of progeny plants; treating said progeny plants with glyphosate; the progeny plants that are tolerant to glyphosate are also selected to be resistant to insect feeding damage.

The invention also provides a composition of matter resulting from transgenic corn event LP026-1, said composition being corn meal, corn flour, corn oil, corn silk or corn starch. In some embodiments, the composition may be an agricultural product or commodity such as corn meal, corn flour, corn oil, corn starch, corn gluten, tortilla, cosmetics, or bulking agent. If sufficient expression is detected in the composition, the composition is expected to contain a nucleic acid sequence capable of diagnosing the presence of transgenic maize event LP026-1 material in the composition. In particular, the compositions include, but are not limited to, corn oil, corn meal, corn flour, corn gluten, tortilla, corn starch, any other food product 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.

The probe or primer pair-based detection methods and/or kits of the invention can be employed to detect a transgenic maize event LP026-1 nucleic acid sequence, such as that 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 group consisting of the sequences shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, and SEQ ID NO. 5, to diagnose the presence of transgenic maize event LP 026-1.

In conclusion, the transgenic corn event LP026-1 has the dual characteristics of insect resistance and herbicide resistance, and has the following advantages: 1) Avoiding economic losses caused by lepidoptera pests (such as Asiatic corn borer, oriental armyworm, spodoptera frugiperda, cotton bollworm, cutworm, and carpopodium borer, etc.), which are the main pests in corn planting areas; 2) The ability to apply glyphosate-containing agricultural herbicides to corn crops for broad spectrum weed control; 3) The corn yield was not reduced. Specifically, the event LP026-1 of the invention has high resistance to target pests, can lead the death rate of the pests to be up to 100 percent, and protects plants to lead the pest rate to be as low as 0 percent; the herbicide composition has high tolerance to glyphosate herbicide, and can protect plants to ensure that the damage rate is as low as 0 percent; and the agronomic characters of the plants containing the event are excellent, and the yield percentage can reach as high as 100 percent. Furthermore, genes encoding insect resistance and glyphosate tolerance traits are linked on the same DNA segment and are present at a single locus in the transgenic maize event LP026-1 genome, which increases breeding efficiency and enables molecular markers to be used to track transgene inserts in the breeding populations and their progeny. Meanwhile, the primer or probe sequence provided in the detection method can generate an amplification product identified as the transgenic corn event LP026-1 or a progeny thereof, and can rapidly, accurately and stably identify the existence of plant material derived from the transgenic corn event LP 026-1.

Terminology

The following definitions and methods may better define the present invention and instruct those of ordinary skill in the art to practice the present invention, and unless otherwise indicated, terms are understood according to their conventional usage by those 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 invention 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 invention 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 event, designated as LP026-1, and progeny thereof, which transgenic corn event LP026-1 is corn plant LP026-1, which includes plants and seeds of transgenic corn event LP026-1 and plant cells thereof or regenerable parts thereof, plant parts of transgenic corn event LP026-1 including, but not limited to, cells, pollen, ovules, flowers, shoots, roots, stems, silks, inflorescences, ears, leaves and products from corn plant LP026-1, such as corn meal, corn oil, corn steep liquor, corn cobs, corn starch and biomass left in the corn crop field.

The transgenic maize event LP026-1 of the present invention comprises a DNA construct that, when expressed in a plant cell, confers resistance to insects and tolerance to glyphosate herbicide to the transgenic maize event LP 026-1.

In some embodiments of the invention, the DNA construct comprises four expression cassettes in tandem, the first expression cassette comprising a suitable promoter for expression in a plant operably linked to a nucleic acid sequence of a bacillus thuringiensis insect-resistant Cry2Ab protein (Cry 2 Ab), said Cry2Ab protein having lepidopteran insect resistance, and a suitable polyadenylation signal sequence; the second expression cassette consists of a nucleic acid sequence comprising a suitable promoter for expression in plants operably linked to an insect-resistant Cry1Fa protein of bacillus thuringiensis (Cry 1 Fa), said Cry1Fa having lepidopteran insect resistance, and a suitable polyadenylation signal sequence; the third expression cassette comprises a suitable promoter for expression in plants operably linked to a nucleic acid sequence of a Cry1Ab protein that is predominantly resistant to lepidopteran insects and a suitable polyadenylation signal sequence. The fourth expression cassette comprises a suitable promoter for expression in plants operably linked to a gene encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and a suitable polyadenylation signal sequence, the nucleic acid sequence of which EPSPS protein is tolerant to glyphosate herbicide. 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 Ubiquitin protein (Ubiquitin) promoter, the Actin (action) promoter, the agrobacterium (Agrobacterium tumefaciens) nopaline synthase (NOS) promoter, the octopine synthase (OCS) promoter, the night yellow leaf curly virus (cestron) promoter, the tuber storage protein (Patatin) promoter, the ribulose-1, 5-bisphosphate carboxylase/oxygenase (rusco) promoter, the Glutathione S Transferase (GST) promoter, the E9 promoter, the GOS promoter, the alcA/alcR promoter, the agrobacterium (Agrobacterium rhizogenes) roller promoter, and the arabidopsis (Arabidopsis thaliana) 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 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 peptide/transit peptide nucleic acid coding sequences. 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 can direct the transport of the Cry1Ab protein and/or EPSPS protein to a particular organelle or compartment 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 Cry1Ab, cry2Ab, and Cry1Fa genes may be isolated from bacillus thuringiensis (Bacillus thuringiensis, bt for short), and the nucleic acid sequences of the Cry1Ab, cry2Ab, and Cry1Fa genes may be optimized or otherwise altered to increase the stability and availability of transcripts in the transformed cells.

In some embodiments of the invention, a maize cell, seed or plant comprising transgenic maize event LP026-1 comprises in its genome the nucleic acid sequence of SEQ ID NO. 1, SEQ ID NO. 5 at positions 891-17013 and SEQ ID NO. 2, or SEQ ID NO. 5.

The Lepidoptera, the school name Lepidotera, including moths and butterflies, is one of the most agricultural and forestry pests, such as corn borers, cotton bollworms, oriental armyworms, spodoptera frugiperda, athetis lepigone, and carpopodium borers.

The 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) gene may be isolated from agrobacterium tumefaciens (Agrobacterium tumefaciens sp.) CP4 strain and the polynucleotide encoding the EPSPS gene may be modified by optimizing codons or otherwise achieving the goal of increasing the stability and availability of transcripts in transformed cells. The 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) gene can 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 transgenic corn event LP026-1 with any herbicide formulation containing glyphosate will control weed growth in the field and will not affect the growth or yield of plant material derived from transgenic corn event LP 026-1.

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 in particular 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 most specifically expressed in plant cells.

A transgenic "event" is obtained by transforming a plant cell with a heterologous DNA construct, i.e., comprising at least one 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 LP026-1 resistant to lepidopteran insects and resistant to glyphosate herbicide is grown by the steps of: first sexually crossing a first parent corn plant consisting of a corn plant grown from transgenic corn event LP026-1 and progeny thereof obtained by transformation with an expression cassette of the invention that is lepidopteran insect resistant and tolerant to a glyphosate herbicide, with a second parent corn plant lacking lepidopteran insect resistance and/or tolerant to a glyphosate herbicide, thereby producing a plurality of first generation progeny plants; progeny plants that are resistant to attack by lepidopteran insects and/or tolerant to glyphosate herbicide are then selected, and maize plants that are resistant to lepidopteran insects and tolerant to glyphosate herbicide can be grown. These steps may further include backcrossing the lepidopteran insect-resistant and/or glyphosate-tolerant progeny plant with the second parent corn plant or the third parent corn plant, and selecting the progeny by infestation with the lepidopteran insect, application of a glyphosate 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 LP 026-1), thereby producing a lepidopteran insect-resistant and glyphosate herbicide-tolerant corn plant.

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, can be 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 maize event LP026-1, whether the genomic DNA is from transgenic maize event LP026-1 or seed or plant or seed or extract derived from transgenic maize event LP 026-1. 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. The DNA molecules comprising the full length or fragments of SEQ ID NOS: 1-7 can be used as primers and probes for detecting the maize event LP026-1 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 the flanking genomic DNA and insert sequences of the invention may be determined by conventional methods, for example, by isolating the corresponding DNA molecule from plant material derived from transgenic maize event LP026-1 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 derived from transgenic maize event LP026-1 in a sample. 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. In particular, a nucleic acid molecule of the invention may 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 specifically, 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 a sequence complementary thereto, or a fragment of any 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," "amplification product," or "amplicon" refers to a nucleic acid amplification product of a target nucleic acid sequence that is part of a nucleic acid template. For example, to determine whether a maize plant is produced by sexual hybridization with a maize event LP026-1 containing the invention, or whether a maize sample collected from a field contains a transgenic maize event LP026-1, or whether a maize extract, such as meal, flour or oil, contains a transgenic maize event LP026-1, DNA extracted from a maize 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 maize event LP 026-1. 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 maize event LP 026-1. 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 22kb genomic DNA and 42kb phage DNA. 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 maize event LP026-1 can be amplified by using the provided primer sequences to amplify the genome of transgenic maize event LP026-1, after which standard DNA sequencing can be performed on the PCR amplicon or cloned DNA.

DNA detection kits based on DNA amplification methods may contain DNA primer molecules that specifically hybridize to target DNA and amplify diagnostic amplicons under appropriate reaction conditions. 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 LP026-1, 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 Genetic Bit 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 ddNTPs. Single base extension results in insertion of ddNTPs. 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 transgenic maize event LP026-1 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 from transgenic corn event LP026-1 in a sample, and can also be used to cultivate corn plants containing DNA from transgenic corn event LP 026-1. 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 LP026-1 flanking genomic region at the 5' end of the transgene insert, a portion of the insert from the right border Region (RB) of agrobacterium, a first expression cassette consisting of a figwort mosaic virus 35s promoter (prFMV), operably linked to the maize heat shock protein gene HSP70 protein intron (iZmHSP 70), operably linked to maize chloroplast transit peptide 2 (spzmcp 2), operably linked to the insect-resistant Cry2Ab protein of bacillus thuringiensis (Cry 2 Ab), operably linked to the nopaline synthase transcription terminator (tNos); the second expression cassette consisted of the maize ubiquitin gene promoter Ubi (przmbi) containing tandem repeats of the enhancer region, operably linked to the insect resistance gene Cry1Fa of bacillus thuringiensis (Cry 1 Fa), operably linked to the terminator ORF25poly a from agrobacterium tumefaciens pTi 15955; the third expression cassette consisted of a cauliflower mosaic virus 35S promoter (pr 35S), operably linked to the 5' untranslated leader sequence of the wheat chloroplast a/b binding protein (lWtCab), operably linked to the rice actin gene 1 intron (iOsAct 1), operably linked to the insect-resistant Cry1Ab protein of bacillus thuringiensis (Cry 1 Ab), and operably linked to the terminator of the benzenesulfonamide induction gene 2 (tIn 2); the fourth expression cassette consisted of a rice actin 1 promoter (prOsAct 1), operably linked to an Arabidopsis chloroplast transit peptide (spatCTP 2), operably linked to a glyphosate tolerant 5-enol-pyruvylshikimate-3-phosphate synthase (cEPSPS) of the Agrobacterium CP4 strain, and operably linked to a nopaline synthase transcription terminator (tNos). A part of the insert from the left border region (LB) of Agrobacterium, and the region flanking the maize plant LP026-1 genome at the 3' -end of the transgenic insert (SEQ ID NO: 5). In the DNA amplification method, the DNA molecule used as a primer may be any part derived from the transgene insert sequence in transgenic maize event LP026-1, or any part derived from the DNA region of the flanking maize genome in transgenic maize event LP 026-1.

Transgenic corn event LP026-1 can be combined with other transgenic corn varieties, such as herbicide (e.g., glufosinate, dicamba, etc.) tolerant corn, or transgenic corn varieties carrying other insect-resistant genes (e.g., chafer, grub, diabrotica, etc.). Various combinations of all of these different transgenic events, when bred with transgenic maize event LP026-1 of the present invention, can provide improved hybrid transgenic maize varieties that are resistant to multiple 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 transgenic corn event LP026-1, which is resistant to feeding injury by lepidopteran pests and is tolerant to the phytotoxic effects of glyphosate-containing agricultural herbicides, for detecting nucleic acid sequences of corn plants comprising the event, and methods of detecting the same. The dual trait maize plants express the Cry1Ab, cry2Ab, and Cry1Fa proteins of bacillus thuringiensis, which provide resistance to ingestion damage by lepidopteran pests (e.g., asian corn borers, spodoptera frugiperda); and which expresses a glyphosate resistant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) protein of agrobacterium strain CP4, which confers on plants tolerance to glyphosate. The dual-trait corn has the following advantages: 1) Avoiding economic losses due to lepidopteran pests (such as Asiatic corn borer, oriental mythidae, spodoptera frugiperda, cotton bollworm, cutworm, and carpopodium borer); 2) The ability to apply glyphosate-containing agricultural herbicides to corn crops for broad spectrum weed control; 3) The corn yield is not reduced. Specifically, the event LP026-1 of the invention has high resistance to target pests, can lead the death rate of the pests to be up to 100 percent, and protects plants to lead the pest rate to be as low as 0 percent; the herbicide composition has high tolerance to glyphosate herbicide, and can protect plants to ensure that the damage rate is as low as 0 percent; and the agronomic characters of the plants containing the event are excellent, and the yield percentage can reach as high as 100 percent. Furthermore, genes encoding insect resistance and glyphosate tolerance traits are linked on the same DNA segment and are present at a single locus in the transgenic maize event LP026-1 genome, which provides enhanced breeding efficiency and enables molecular markers to be used to track transgenic 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 LP026-1 or the progeny thereof, and can rapidly, accurately and stably identify the existence of plant materials derived from the transgenic corn event LP 026-1.

Drawings

FIG. 1 is a schematic structural diagram of a transgene insert sequence and a maize genomic binding site for detecting a nucleic acid sequence of maize plant LP026-1 and a method of detecting the same according to the present invention;

FIG. 2 is a schematic diagram showing the structure of a recombinant expression vector pLP026 for detecting the nucleic acid sequence of maize plant LP026-1 and a detection method thereof;

FIG. 3 is an in vitro resistance effect of transgenic corn comprising transgenic corn event LP026-1 of the present invention against lepidopteran pests;

FIG. 4 is a graph of the artificial insemination effect of transgenic corn comprising transgenic corn event LP026-1 of the present invention in corn borer fields;

FIG. 5 is a graph of the artificial insemination effect of transgenic corn comprising transgenic corn event LP026-1 of the present invention in a cotton bollworm field;

FIG. 6 is a graph of the field effect of transgenic corn comprising transgenic corn event LP026-1 of the present invention under conditions in which borer on peach occurs naturally;

FIG. 7 is a graph of the field effect of transgenic corn comprising transgenic corn event LP026-1 of the present invention under spodoptera exigua naturally occurring conditions;

FIG. 8 is a graph of the field effect of transgenic corn comprising transgenic corn event LP026-1 of the present invention under spodoptera frugiperda naturally occurring conditions.

FIG. 9 is a plot of the field effect of the proposed spray concentration of transgenic corn of the invention comprising transgenic corn event LP026-1 in a field sprayed with 4-fold doses of glyphosate herbicide.

Detailed Description

The present invention will be described in further detail by way of examples. The features and advantages of the present invention will become more apparent from the exemplary description.

The word "exemplary" is used herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

In addition, the technical features described below in the different embodiments of the present invention may be combined with each other as long as they do not collide with each other.

The following is a detailed description of the nucleic acid sequence and detection method of the present invention for detecting LP026-1 in maize plants.

EXAMPLE 1 cloning and transformation

1.1 vector cloning

The recombinant expression vector pLP026 (shown in FIG. 2) was constructed using standard gene cloning techniques. The vector pLP026 comprises 4 transgene expression cassettes in tandem, the first expression cassette consisting of a figwort mosaic virus 35s promoter (prFMV), operably linked to the maize heat shock protein gene HSP70 protein intron (izmsp 70), operably linked to maize chloroplast transit peptide 2 (spzmcp 2), operably linked to the bacillus thuringiensis insect resistant Cry2Ab protein (Cry 2 Ab), and operably linked to the nopaline synthase transcription terminator (tNos); the second expression cassette consisted of the maize ubiquitin gene promoter Ubi (przmbi) containing tandem repeats of the enhancer region, operably linked to the insect resistance gene Cry1Fa of bacillus thuringiensis (Cry 1 Fa), operably linked to the terminator ORF25poly a from agrobacterium tumefaciens pTi 15955; the third expression cassette consisted of a cauliflower mosaic virus 35S promoter (pr 35S), operably linked to the 5' untranslated leader sequence of the wheat chloroplast a/b binding protein (lWtCab), operably linked to the rice actin gene 1 intron (iOsAct 1), operably linked to the insect-resistant Cry1Ab protein of bacillus thuringiensis (Cry 1 Ab), and operably linked to the terminator of the benzenesulfonamide induction gene 2 (tIn 2); the fourth expression cassette consisted of the rice actin 1 promoter (prOsAct 1), operably linked to the Arabidopsis 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 nopaline synthase transcription terminator (tNos). The vector pLP026 was transformed into Agrobacterium LBA4404 (Invitrogen, chicago, USA; cat. No. 18313-015) by liquid nitrogen method, and the transformed cells were screened using 5-enol-pyruvylshikimate-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 pLP026 into the maize genome to generate transgenic maize events.

For agrobacterium-mediated maize transformation, briefly, immature chick embryos are isolated from maize, and the chick embryos are contacted with an agrobacterium suspension, wherein the agrobacterium is capable of delivering the nucleic acid sequences of cry1Ab, cry2Ab, cry1Fa genes and the nucleic acid sequences of epsps genes to at least one cell of one of the chick embryos (step 1: an infestation step) in which the young embryos are immersed in a suspension of agrobacteria (OD 660 = 0.4-0.6), in a infestation 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, in a period of time (3 days) after the infestation step (step 2: co-cultivation step) in particular, the young embryos are cultivated after the infestation step on a 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) in a period of co-cultivation in which the young embryos can be recovered in a selective recovery medium (MS salt 4.3 g/3 mg, 2, 35 g/L) of 2, 4-dichlorophenoxyacetic acid, pH 5.8) at least one antibiotic known to inhibit agrobacterium growth (e.g., cephalosporin), without the addition of a selection agent for plant transformants (step 3: and (5) a recovery step). Specifically, 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 chick embryos are then cultured on a medium containing a selection agent (N- (phosphonomethyl) glycine) and the growing transformed calli are selected (step 4: selection step). Specifically, the young embryos are cultured on a selective solid medium (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) with a selective agent, resulting in selective growth of the transformed cells. Then, the callus is regenerated into plants (step 5: regeneration step), specifically, the callus grown on the medium containing the selection agent is cultured on solid medium (MS differentiation medium and MS rooting medium) to regenerate the plants.

The selected resistant calli were transferred to 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 seedlings were transferred to 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 at 25℃to a height of about 10cm, 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 1500 independent transgenic T0 individuals were generated. Molecular detection (including target gene copy number, insertion position and the like), target character (insect resistance and herbicide resistance) and agronomic character evaluation are carried out on all T0 plants, and LP026-1 is obtained after abnormal transformant plants are removed.

Example 2 detection of transgenic maize event LP026-1 with TaqMan

About 100mg of leaf of transgenic maize event LP026-1 was taken as a sample, its genomic DNA was extracted with Qiagen DNeasy Plant Maxi Kit and the copy numbers of cry1Ab, cry2Ab, cry1Fa and epsps 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 LP026-1, 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 cry1Ab gene sequence:

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

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

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

the following primers and probes were used to detect cry2Ab gene sequence:

Primer 3: GGACAGAGGCACCGCATT is shown as SEQ ID NO. 19 in the sequence table;

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

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

the following primers and probes were used to detect cry1Fa gene sequence:

primer 5: GCTATGTCCAGTCCCCAACCT is shown as SEQ ID NO. 22 in the sequence table;

primer 6: CAAGCTGCTAACCTGCACTTGT is shown as SEQ ID NO. 23 in the sequence table;

probe 3: CCCAAACGACACAGCGTCGCG is shown as SEQ ID NO. 24 in the sequence table;

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

primer 7: GCAAATCCTCTGGCCTTTCC is shown as SEQ ID NO. 25 in the sequence table;

primer 8: TGAAGGACCGGTGGGAGAT is shown as SEQ ID NO. 26 in the sequence table;

probe 4: CGTCCGCATTCCCGGCGA is shown as SEQ ID NO 27 in the sequence table;

the PCR reaction system is that

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 LP026-1.

Example 3 transgenic maize event LP026-1 detection

3.1 genomic DNA extraction

DNA extraction according to the conventionally employed CTAB (cetyltrimethylammonium bromide) method: 2 g of tender transgenic corn event LP026-1 leaves are ground into powder in liquid nitrogen, 0.5mL of DNA preheated at 65 ℃ is added to extract CTAB Buffer [20g/L CTAB,1.4M NaCl,100mM Tris-HCl,20mM EDTA (ethylenediamine tetraacetic acid) ], naOH is used for regulating the pH to 8.0, and the mixture is fully and uniformly mixed and extracted for 90min at 65 ℃; adding 0.5 volume of phenol and 0.5 volume of chloroform, and mixing the mixture upside down; centrifuging at 12000rpm for 10min; sucking the supernatant, adding 1-time volume of isopropanol, gently shaking the centrifuge tube, and standing at-20deg.C for 30min; further centrifuging at 12000rpm for 10min; collecting DNA to the bottom of the tube; discarding the supernatant, washing the precipitate with 0.5mL of 70% ethanol by volume; centrifuging at 12000rpm for 5min; vacuum pumping or blow-drying in an ultra clean bench; the DNA precipitate 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, pstI, bssHII (5 'end assay) and SacI, kpnI, xmaI, nheI (3' end assay), respectively. 26.5. Mu.L of genomic DNA, 0.5. Mu.L of the above-selected restriction enzyme and 3. Mu.L of the cleavage buffer were added to each cleavage system, and the cleavage was performed at an appropriate temperature for 1 hour. After the enzyme digestion is finished, 70 mu L of absolute ethyl alcohol is added into an enzyme digestion system, ice bath is carried out for 30min, centrifugal separation is carried out for 7min at the rotating speed of 12000rpm, supernatant is discarded, drying is carried out, and then 8.5 mu L of double distilled water (ddH) is added ₂ O), 1. Mu.L of 10 XT4 Buffer and 0.5. Mu. L T4 ligase were ligated overnight at 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. 34 as a first primer, SEQ ID NO. 35, SEQ ID NO. 36 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. 37 as the first primer, SEQ ID NO. 38, SEQ ID NO. 39 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 QIAquick Gel extraction kit (catalogue # 28704, qiagen Inc., valencia, CA). The purified PCR product is then sequenced (e.g., abiprism 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. 34. 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. 37. The PCR reaction system and amplification conditions are shown in Table 3. 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 product 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 LP 026-1.

Nucleotide 1-890 of SEQ ID No. 5 was found to flank the maize genomic sequence at the right border (5 'flanking sequence) of the insert sequence of transgenic maize event LP026-1, and nucleotide 17014-17345 of SEQ ID No. 5 was found to flank the maize genomic sequence at the left border (3' flanking sequence) of the insert sequence of transgenic maize event LP 026-1. 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 the DNA of transgenic maize event LP026-1 when detected in a polynucleic acid detection assay. The binding sequence of SEQ ID NO. 1 is composed of 11bp on one side of the T-DNA RB region insertion site and 11bp on one side of the corn genome DNA insertion site of the transgenic corn event LP026-1, and the binding sequence of SEQ ID NO. 2 is composed of 11bp on the other side of the T-DNA LB region insertion site and the corn genome DNA insertion site of the transgenic corn event LP 026-1. 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 LP026-1, which can also be used as DNA probes or as DNA primer molecules to detect the presence of transgenic maize event LP026-1 DNA. The sequence of SEQ ID NO. 6 (nucleotides 891-1359 of SEQ ID NO. 3) spans the LP026 construct DNA sequence and the tNos transcription termination sequence, and the sequence of SEQ ID NO. 7 (nucleotides 1-252 of SEQ ID NO. 4) spans the tNos transcription termination sequence and the LP026 construct DNA sequence.

Furthermore, the amplicon is generated by using primers from at least one of SEQ ID NO. 3 or SEQ ID NO. 4, which primers when used in a PCR method generate a diagnostic amplicon for transgenic maize event LP 026-1.

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 LP 026-1. This PCR product contains SEQ ID NO 3. For PCR amplification, primers 11 (SEQ ID NO: 8) hybridizing to the genomic DNA sequence flanking the 5' -end of the transgene insert and primers 12 (SEQ ID NO: 9) located in the transcription termination sequence of the transgene tNos were designed to pair with them.

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 plant material derived from transgenic maize event LP 026-1. This PCR product contains SEQ ID NO. 4. For PCR amplification, primers 14 (SEQ ID NO: 11) hybridizing to the genomic DNA sequences flanking the 3 '-end of the transgene insert and primers 13 (SEQ ID NO: 10) of the tNos transcription termination sequence at the 3' -end of the insert were designed to pair with.

The DNA amplification conditions described in tables 3 and 4 can be used in the PCR zygosity assay described above to generate the diagnostic amplicon of transgenic maize event LP 026-1. Detection of the amplicon may be performed by using a Stratagene Robocycle, MJ Engine, perkin-Elmer9700 or Eppendorf Mastercycler Gradien thermocycler, or the like, 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 of transgenic maize event LP026-1

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 thermal cycler of Stratagene Robocycler (Stratagene, la Jolla, calif.), MJ Engine (MJ R-Biorad, hercules, calif.), perkin-Elmer9700 (Perkin Elmer, boston, mass.) or Eppendorf Mastercycler Gradient (Eppendorf, hamburg, germany) using the cycling parameters of 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 11 and 12 (SEQ ID NOS: 8 and 9), which when used in a PCR reaction of transgenic maize event LP026-1 genomic DNA, produced an amplification product of a 1359bp fragment, NO fragment being amplified when used in a PCR reaction of untransformed maize genomic DNA and non-LP 026-1 maize genomic DNA; primers 13 and 14 (SEQ ID NOS: 10 and 11), when used in the PCR reaction of transgenic maize event LP026-1 genomic DNA, produced an amplification product of 584bp fragments, and when used in the PCR reaction of untransformed maize genomic DNA and non-LP 026-1 maize genomic DNA, NO fragments were amplified.

The PCR zygosity assay can also be used to identify whether the material derived from transgenic maize event LP026-1 is homozygous or heterozygous. Primer 15 (SEQ ID NO: 12), primer 16 (SEQ ID NO: 13) and primer 17 (SEQ ID NO: 14), or primer 16 (SEQ ID NO: 13), primer 17 (SEQ ID NO: 14) and primer 18 (SEQ ID NO: 15) are used in an amplification reaction to generate a diagnostic amplicon for transgenic maize event LP 026-1. The DNA amplification conditions described in tables 5 and 6 can be used in the zygosity assay described above to generate a diagnostic amplicon of transgenic maize event LP 026-1.

TABLE 5 reaction solution for measuring the bondability

/>

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

PCR was performed on a thermal cycler of Stratagene Robocycler (Stratagene, la Jolla, calif.), MJ Engine (MJ R-Biorad, hercules, calif.), perkin-Elmer9700 (Perkin Elmer, boston, mass.) or Eppendorf Mastercycler Gradient (Eppendorf, hamburg, germany) using the cycling parameters of Table 6. 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.

In the amplification reaction, the biological sample containing the template DNA contains DNA diagnostic for the presence of transgenic maize event LP026-1 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 allele corresponding to the insert DNA present in transgenic corn event LP026-1. These two different amplicons would correspond to a first amplicon derived from the wild-type maize genomic locus and a second amplicon that diagnoses the presence of transgenic maize event LP026-1 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 LP026-1 can be diagnostically determined in the sample, and the sample is generated from a corn seed homozygous for the allele corresponding to the insert DNA present in transgenic corn plant LP026-1. It should be noted that the primer pair for transgenic maize event LP026-1 was used to generate an amplicon diagnostic for transgenic maize event LP026-1 genomic DNA. These primer pairs include, but are not limited to, primers 11 and 12 (SEQ ID NOS: 8 and 9), and primers 13 and 14 (SEQ ID NOS: 10 and 11) for use in the DNA amplification method. In addition, a control primer 9 and 10 (SEQ ID NO:28 and SEQ ID NO: 29) for amplifying the maize endogenous gene was included as an intrinsic criterion for the reaction conditions. Analysis of the DNA extract sample of transgenic corn event LP026-1 should include a positive tissue DNA extract control of transgenic corn event LP026-1, a negative DNA extract control derived from non-transgenic corn event LP026-1 and a negative control not containing the 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, respectively, that is diagnostic for tissue derived from transgenic event maize plant LP026-1, may be used. The DNA amplification conditions described in tables 2-5 can be used to generate diagnostic amplicons of transgenic maize event LP026-1 using appropriate primer pairs. Extracts that are presumed to contain corn plant or seed DNA comprising transgenic corn event LP026-1, or products derived from transgenic corn event LP026-1, that when tested in a DNA amplification method produce an amplicon diagnostic for transgenic corn event LP026-1, can be used as templates for amplification to determine the presence or absence of transgenic corn event LP026-1.

Example 4 detection of transgenic maize event LP026-1 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. Mu.L TE. To degrade any RNA present, the DNA was incubated with 1. Mu.L of 30mg/mL RNAaseA 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 detected using a spectrophotometer or fluorometer (using 1 xTAE and GelRED dyes). In a 100. Mu.L reaction system, 5. Mu.g of DNA was digested each time. Respectively digesting the genome DNA by using restriction enzymes BamHI and HindIII, and taking partial sequences of Cry2Ab and EPSPS on the T-DNA as probes; genomic DNA was digested with restriction enzymes AvrII and HindIII, respectively, and partial sequences of Cry1Ab and Cry1Fa 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. In 0.4M NaOH, 1 sheet of Bao Yinji paper was pre-moistened 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. 30,SEQ ID NO:31,SEQ ID NO:32 and SEQ ID NO. 33, 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 after rinsing with 20mL Church rinse solution 1 (40mM Na3P04,1mM EDTA,5%SDS,0.5%BSA), the membrane was washed in 150mL Church rinse solution 1 at 65℃for 20 minutes. Washing with Church rinse solution 2 (40 mM Na ₃ P0 ₄ ，1mM EDTA,1% sds) the procedure 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; (2) DNA from positive segregants, into which HindIII digested pLP026 was introduced in an amount equivalent to one copy number based on probe length, to demonstrate the sensitivity of the experiment when detecting single gene copies within the maize genome.

Hybridization data provided corroborated evidence to support TaqMan ^TM PCR analysis, i.e., maize plant LP026-1 contained a single copy of the Cry2Ab, cry1Fa, cry1Ab, and EPSPS genes. With this Cry2Ab probe, bamHI and HindIII enzymatic hydrolysis produced single bands of about 7.8kb and 10.4kb in size, respectively; using the Cry1Fa probe, avrII and HindIII enzymatic hydrolysis produced single bands of about 13.6kb and 20.8kb, respectively; using the Cry1Ab probe, avrII and HindIII enzymatic hydrolysis produced single bands of about 18.1kb and 20.8kb in size, respectively; using this EPSPS probe, bamHI and HindIII enzymatic hydrolysis produced single bands of approximately 5.1kb and 20.8kb, respectively. This indicates that Cry1Ab, cry2Ab, cry1Fa, and one copy of EPSPS each are present in maize transformation event LP 026-1.

Example 5 insect resistance detection

5.1 bioassay of maize plant LP026-1

Transgenic maize event LP026-1 and wild type maize plants (non-transgenic, transformed recipient control (CK-)) 2 plants were bioassay of Asian corn borer (Ostrinia furnacalis), spodoptera frugiperda (Spodoptera frugiperda), spodoptera frugiperda (Conogethes punctiferalis), athetis lepigone (Athetis lepigone), oriental armyworm (Mythimna seperata), gekko Swinhonis (Agrotis ypsilon), cotton bollworm (Helicoverpa armigera), and beet armyworm (Spodoptera exigua), respectively, as follows:

fresh leaves (V3-V4 period) of 2 plants of transgenic corn event LP026-1 and wild type corn plants (non-transgenic, transformed acceptor control (CK-)) were taken respectively, washed clean with sterile water and blotted dry with absorbent paper, then corn leaves were vein removed while being cut into strips of about 1cm by 3cm in size, 1-3 (leaf number was determined according to insect diet) pieces of cut strips were placed on filter paper at the bottom of a circular plastic petri dish, the filter paper was moistened with distilled water, 10 artificially fed initially hatched larvae were inoculated into each petri dish, and after the petri dish was capped with an insect test dish, the temperature was 26-28 ℃, relative humidity was 70% -80%, photoperiod (light/dark) was 16: statistics were carried out after 5 days of standing under 8 conditions. Mortality was counted and the results were identified by correcting the level of resistance by mortality (corrected mortality (%) = (1-survival/number of grafts-wild type control mortality)/(1-wild type control mortality) ×100%), and are shown in table 7 and fig. 3.

Table 7, in vitro anti-insect bioassay results for transgenic maize event LP 026-1-mortality (%)

5.2 determination of the field pest control Effect of transgenic maize event LP026-1

(1) Corn borer

The living body insect-grafting method is adopted to identify the resistance of transgenic corn event LP026-1 to Asiatic corn borer which is a main target pest in the field. The insects are grafted in the 4-6 leaf period and the silk-laying period (3-5 cm of female silk) of corn, the insects are grafted for 2 times in each period, 50 heads are grafted each time, and the interval between the two times of grafting is one week. After 14d of the leaf-picking period, the feeding condition of upper leaves in corn plants by Asian corn borers is investigated by each plant, and the leaf feeding grade of the Asian corn borers is recorded. After the silk-laying period is inoculated, the damage degree of female ears and the damage condition of plants are investigated before harvesting, wherein the damage degree comprises the damage length of the female ears, the number of boreholes, the tunnel length of the boreholes, the age of surviving larvae and the survival number of the surviving larvae. The resistance of transgenic corn event LP026-1 to Asian corn borers was evaluated using "grading criteria for the extent of corn cob leaf damage by Asian corn borers" as an indicator, and the results are shown in FIG. 4 and Table 12. The cutting investigation was carried out on the ears during the silking period, and the statistical results are shown in Table 13. The results indicate that the leaf-level average for transgenic maize event LP026-1 is significantly lower than the transformed recipient control (CK-); during the laying period, the female ear damage rate, larval survival, tunnel length, and female ear damage level of transgenic maize event LP026-1 were significantly lower than the transformation recipient control (CK-). Overall, transgenic corn event LP026-1 had good resistance to asian corn borers during both the cotyledonary and silking phases.

Table 8, grading criteria for extent of corn borer damage to corn cob

Leaf grade	Description of symptoms
		1	The blade is harmless, or only has needle-like insect holes (less than or equal to 1 mm)
2	Only a small number of wormholes with the size of the bullet holes (less than or equal to 5 mm) are formed on the individual blades
		3	Few leaves have wormholes with the size of bullet holes (less than or equal to 5 mm)
4	The individual blade is notched (less than or equal to 10 mm)
		5	Few leaves are provided with nicks (less than or equal to 10 mm)
6	Part of the blades are provided with notches (less than or equal to 10 mm)
		7	Individual onesThe leaf part is eaten, and a few leaves are provided with large-piece nicks (less than or equal to 10 mm)
8	A few leaves are eaten, and a part of the leaves are provided with large-piece nicks (less than or equal to 10 mm)
		9	Most of the leaves are fed

Table 9 evaluation criteria for resistance of corn to Asian corn borer

Mean value of leaf grade in heart leaf stage	Resistance level
		1.0-2.9	High Resistance (HR)
3.1-4.9	Anti (R)
		5.0-6.9	Middle Resistance (MR)
7.0-8.9	Sense (S)
		9.0	High Sense (HS)

TABLE 10 grading Standard for the extent to which the ear period is affected by Asian corn borer

Table 11 evaluation criteria for resistance of ear stage to Asian corn borer

Average value of the damage level of female spike	Type of resistance
		1-2.0	High Resistance (HR)
2.1-3.0	Anti (R)
		3.1-5.0	Middle Resistance (MR)
5.1-7.0	Sense (S)
		≥7.1	High Sense (HS)

Table 12 results of resistance of transgenic corn event LP026-1 to Asian corn borer at the heart leaf stage

Project/plant	LP026-1	CK-
			Average value of leaf grade	1.15	9.1
Resistance level	Gao Kang	High sense

TABLE 13 results of resistance of transgenic maize event LP026-1 to Asian corn borer during the laying period

Project/plant	LP026-1	CK-
			Female ear pest rate (%)	0	100
Number of larvae surviving	0	16
			Tunnel length (cm)	0	1.9
Grade of female ear damage	0	6.7
			Resistance level	Gao Kang	Feel of the sense

(2) Oriental armyworm

Artificial inoculation is carried out in the corn cob leaf stage (4-6 leaf stage) for 2 times, and 20 heads of artificially fed two-year Oriental armyworm are inoculated in each corn cob leaf. After 3 days of insect inoculation, the second insect inoculation is carried out, and the insect inoculation quantity is the same as that of the first insect inoculation. After 14 days of inoculation, the extent to which corn leaves are damaged by Oriental armyworms was investigated. According to the damage degree of the corn leaf to the Oriental myxoworm, the average value of the damage level (leaf feeding level) of the Oriental myxoworm to the corn leaf in each cell is calculated, the judgment standard is shown in the table 14, and then the resistance level of the corn to the Oriental myxoworm is judged according to the standard of the table 15. The results of resistance to Oriental myxoplasma in the cardiac stage of transgenic maize event LP026-1 are shown in Table 16. The results indicate that transgenic maize event LP026-1 has a better level of resistance to Oriental myxoma, and that the proportion of imprinting and leaf feeding of transgenic maize event LP026-1 are significantly lower than the transformation recipient control (CK-).

TABLE 14 grading Standard for the extent to which corn leaf is damaged by Oriental Clay

Grade of female ear damage	Description of symptoms
		1	The blade is harmless, or only has needle-like insect holes (less than or equal to 1 mm)
2	Only a small number of wormholes with the size of the bullet holes (less than or equal to 5 mm) are formed on the individual blades
		3	The small number of blades are provided with wormholes with the sizes of bullet holes (less than or equal to 5 mm)
4	The nicks on the individual leaves are less than or equal to 10mm
		5	Few leaves are provided with nicks (less than or equal to 10 mm)
6	Part of the blades are provided with notches (less than or equal to 10 mm)
		7	Individual leaf portions are fed, and few leaves are provided with large cuts (less than or equal to 10 mm)
8	A few leaves are eaten, and a part of the leaves are provided with large-piece nicks (less than or equal to 10 mm)
		9	Most of the leaves are fed

TABLE 15 evaluation criteria for resistance of corn to Oriental armyworm

Average value of the damage level of female spike	Type of resistance
		1.0-2.0	High Resistance (HR)
2.1-4.0	Anti (R)
		4.1-6.0	Middle Resistance (MR)
6.1-8.0	Sense (S)
		8.1-9.0	High Sense (HS)

TABLE 16 results of resistance of transgenic maize event LP026-1 to Oriental myxoplasma in the cardiac stage

Project/plant	LP026-1	CK-
			Average value of leaf grade	1.12	7.6
Resistance level	Gao Kang	Feel of the sense

(3) Bollworm (Bowls)

And (3) artificially inoculating the transgenic corn event LP026-1 for 2 times in the corn silk period, inoculating 20 newly hatched larvae artificially fed in each corn silk, and inoculating the first time 3 days later, wherein the number of the second time of insect inoculation is the same as that of the first time. After 14-21 days of insect inoculation, the pest rate of female ears, the number of surviving larvae of each female ear and the pest length of the female ears are investigated plant by plant. Investigation was usually started 14 days after insect inoculation, and if the harmful level of the negative control material (CK-) reached the sense or high sense, then the investigation was considered to be effective, and if the investigation was not properly postponed, but the corresponding level was not reached yet 21 days after insect inoculation, then the insect inoculation was considered to be ineffective. And calculating the average value of the damage level of the cotton bollworms in the corn ear period of each cell to the female spike according to the damage rate of the female spike, the number of surviving larvae and the damage length (cm) of the female spike, wherein the judging standard is shown in a table 17, and then judging the resistance level of the cotton bollworms in the corn ear period according to the standard of a table 18. The results of resistance to cotton bollworms during the silking period of transgenic maize event LP026-1 are shown in FIG. 5 and Table 19. The results indicate that transgenic corn event LP026-1 has a higher level of resistance to cotton bollworms, and that the female ear pest rate, larval survival, female ear pest length, and female ear pest level of transgenic corn event LP026-1 are significantly lower than the transformation recipient control (CK-).

TABLE 17 grading Standard for the extent to which corn females are damaged by Helicoverpa armigera

Grade of female ear damage	Description of symptoms
		0	The female ear is not damaged
1	Only the filament is damaged
		2	The head of the ear is damaged by 1cm
3+	Every 1cm of pest under the top of the ear, the corresponding pest level is increased by 1 level
		…N

TABLE 18 evaluation criteria for resistance of maize female ear to Heliothis armigera

Average value of the damage level of female spike	Type of resistance
		0-1.0	High Resistance (HR)
1.1-3.0	Anti (R)
		3.1-5.0	Middle Resistance (MR)
5.1-7.0	Sense (S)
		≥7.1	High Sense (HS)

TABLE 19 results of resistance to cotton bollworms during the silking period of transgenic maize event LP026-1

(4) Peach borer

And 2021, 7 months, and performing a field natural pest-sensing test of the carpopodium punctosum at a transgenic corn planting base in an Anoectochilus formosanus Xu Changshi construction and installation area in Henan province. And (3) after 14-21 days of the primary insect damage, and when most of control (CK-) plants are damaged by 4-5-year-old larvae, the pest rate of the carpopodium borer to the corn plants is investigated plant by plant. The results of resistance of transgenic maize event LP026-1 to carpenter's borer are shown in FIG. 6 and Table 20. The results show that under the condition that the carpopodium borer naturally occurs, compared with the control (CK-) the damage rate of the carpopodium borer to the transgenic corn event LP026-1 is obviously reduced, thereby indicating that the transgenic corn event LP026-1 has higher resistance to the carpopodium borer.

Table 20, resistance results of transgenic maize event LP026-1 to carpopodium borer under natural pest-sensing conditions

Project/plant	LP026-1	CK-
			Rate of damage (%)	0	71

(5) Beet armyworm

The field natural pest-sensing test of asparagus caterpillar was performed in 2021 at 3 months on a transgenic corn planting base in the cliff region of three-city, south-hainan province. After 10-15 days of initial pest occurrence, and when most of control (CK-) plants are damaged by 4-6-year-old larvae, the pest rate of asparagus caterpillar to corn plants is investigated plant by plant. The results of resistance of transgenic maize event LP026-1 to asparagus caterpillar are shown in FIG. 7 and Table 21. The results show that the pest rate of the spodoptera exigua on the transgenic corn event LP026-1 is significantly reduced compared with the control (CK-) under the naturally occurring condition of the spodoptera exigua, thereby indicating that the transgenic corn event LP026-1 has higher resistance to the spodoptera exigua.

Table 21, results of resistance to spodoptera exigua under the Natural pest-sensing conditions of transgenic corn event LP026-1

Project/plant	LP026-1	CK-
			Rate of damage (%)	0	93

(6) Spodoptera frugiperda (L.) kurz

The field natural pest-sensing test of spodoptera frugiperda was performed at 2021 at 3 months on a transgenic corn planting base in the cliff region of three-city, south-hainan province. After 10-15 days of initial pest occurrence, and when the control (CK-) is mostly the damage of 4-6-year-old larvae, the pest rate of spodoptera frugiperda to corn plants is investigated plant by plant. The results of resistance of transgenic corn event LP026-1 to Spodoptera frugiperda are shown in Table 22 and the field resistance effect is shown in FIG. 8. The results show that the spodoptera littoralis has a significantly reduced rate of damage to the transgenic corn event LP026-1 under naturally occurring conditions of spodoptera littoralis as compared to the control (CK-) thereby demonstrating that the transgenic corn event LP026-1 has a higher resistance to spodoptera littoralis.

Table 22, results of resistance of transgenic corn event LP026-1 to Spodoptera frugiperda under natural insect-sensing conditions

Project/plant	LP026-1	CK-
			Rate of damage (%)	0	100

Example 6 herbicide tolerance detection of maize transformation event

The test selects the pesticide (41% glyphosate isopropyl ammonium salt aqua) for spraying. 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. Separate entry of transgenic maize event LP026-1The following 2 treatments were performed: 1) Spraying is not performed; 2) The pesticide was sprayed at 1680g a.e./ha dose during the V3 leaf stage and then again at the same dose during the V8 stage. It should be noted that the conversion of glyphosate herbicide of different content and dosage forms to equivalent amounts of glyphosate acid is applicable to the following conclusion. The phytotoxicity symptoms were investigated at 1 and 2 weeks after dosing, respectively, and the yield of the cells was determined at harvest. The phytotoxicity symptoms were ranked as shown in table 23. The herbicide damage rate is used as an evaluation index to evaluate an index of herbicide tolerance of a transformation event, specifically, the herbicide damage rate (%) Σ (peer damage number×number of ranks)/(total number×highest rank); the herbicide damage rate refers to the glyphosate damage rate, and the glyphosate damage rate is determined according to the phytotoxicity investigation result of 2 weeks after the glyphosate treatment. 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 = sprayed yield/non-sprayed yield). The results of transgenic corn event LP026-1 for herbicide tolerance and corn yield are shown in FIG. 9 and Table 24.

Table 23, grading Standard of extent of phytotoxicity of glyphosate herbicide to corn

Table 24, results of transgenic corn event LP026-1 for tolerance to glyphosate herbicide and corn yield results

Project/plant	LP026-1	CK-
			Glyphosate rate (%) (spraying clear water)	0	0
Glyphosate rate (%) (farmer reaches 800 ml/mu, recommended concentration 150-250 ml/mu)	0	100
			Yield percentage (farm 800 ml/mu, recommended concentration of 150-250 ml/mu)	100	0

The results show that in terms of herbicide (glyphosate) damage: 1) The transgenic corn event LP026-1 had a victim rate of substantially 0 under glyphosate herbicide (800 ml/mu) treatment, and thus the transgenic corn event LP026-1 had good glyphosate herbicide tolerance.

In terms of yield: the yield of the transgenic corn event LP026-1 is not obviously different under the condition that 800 ml/mu of glyphosate is not sprayed and subjected to 2 treatments, and the yield of the transgenic corn event LP026-1 is not basically reduced after the glyphosate herbicide is sprayed, so that the transgenic corn event LP026-1 is further indicated to have good glyphosate herbicide tolerance.

In conclusion, through TaqMan ^TM Analysis (see example 2) the regenerated transgenic maize plants were tested for the presence of cry1Ab, cry2Ab, cry1Fa and epsps genes and characterized for insect resistance and copy number of glyphosate herbicide tolerant lines. Based on copy number of the gene of interest, good insect resistance, glyphosate herbicide tolerance and agronomic performance (see example 5 and example 6), events were selected by screening LP026-1 is excellent with single copy transgenes, good insect resistance, glyphosate herbicide tolerance, and agronomic performance (examples 5 and 6).

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.

Claims

1. A nucleic acid molecule comprising one selected from the group consisting of the sequences SEQ ID NOs 1-5 and complements thereof, which is derived from a transgenic plant, seed or cell comprising the transgenic maize event LP026-1, the maize seed comprising said event having been deposited under accession number cctccc No. P202206.

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 comprising transgenic corn event LP026-1, an amplicon is generated for the transgenic corn event LP026-1 in a test sample, corn seed comprising said event has been deposited with accession number cctccc NO: P202206, said first primer being selected from the group consisting of SEQ ID NO:8 or SEQ ID NO:12, said second primer being selected from the group consisting of SEQ ID NO:9 or SEQ ID NO:13; or the first primer is selected from SEQ ID NO. 10 and the second primer is selected from SEQ ID NO. 11.

3. A method of detecting the presence of DNA from transgenic maize event LP026-1 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 sequence SEQ ID NO. 3 or SEQ ID NO. 4 or a complementary sequence thereof, namely the DNA containing the transgenic corn event LP026-1 in the detection sample, and the corn seeds containing the event are preserved with a preservation number CCTCC NO. P202206.

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

5. A method of protecting a maize plant from insect infestation comprising providing at least one transgenic maize plant cell comprising a nucleic acid molecule of transgenic maize event LP026-1 in the diet of a target insect; target insects feeding on cells of the transgenic maize plant are inhibited from further feeding on the maize plant, the nucleic acid molecule comprising the sequence SEQ ID NO. 5 or a complement thereof.

6. A method of protecting a corn plant from injury caused by a herbicide, comprising planting at least one transgenic corn plant comprising a nucleic acid molecule of transgenic corn event LP026-1 comprising the sequence of SEQ ID No. 5 or a complement thereof, and applying an effective dose of a glyphosate herbicide.

7. A method of controlling weeds in a field in which corn plants are grown, comprising applying an effective dose of a glyphosate herbicide to the field in which at least one transgenic corn plant is grown, said transgenic corn plant comprising a nucleic acid molecule of transgenic corn event LP026-1 comprising the sequence of SEQ ID No. 5 or a complement thereof.

8. A method of growing a corn plant that is resistant to insects and/or tolerant to glyphosate herbicide, comprising: planting at least one corn seed comprising a nucleic acid molecule of transgenic corn event LP 026-1;

growing the corn seed into a corn plant;

attack the maize plant with a target insect and/or spray the maize plant with an effective dose of a glyphosate herbicide, harvesting plants having reduced plant damage as compared to other plants not having the transgenic maize event LP026-1, the nucleic acid molecule comprising the sequence SEQ ID NO 5 or a complement thereof.