CN115725571A

CN115725571A - Nucleic acid sequence of corn transformation event LG11 and detection method thereof

Info

Publication number: CN115725571A
Application number: CN202211606656.9A
Authority: CN
Inventors: 岳润清; 孟昭东; 张发军; 丁照华; 汪黎明
Original assignee: Shandong Academy of Agricultural Sciences
Current assignee: Shandong Academy of Agricultural Sciences
Priority date: 2022-12-13
Filing date: 2022-12-13
Publication date: 2023-03-03
Also published as: CN116200529B; CN116200529A

Abstract

The present invention relates to a nucleic acid sequence and detection method for corn transformation event LG11, wherein the nucleic acid molecule of corn LG11 comprises the sequence shown in SEQ ID NO. 1 or its reverse complement, or the sequence shown in SEQ ID NO. 2 or its reverse complement. The corn transformation event LG11 has characteristics of insect resistance and glufosinate herbicide resistance, is excellent in agronomic characters, and the detection method can accurately and quickly identify whether a biological sample contains the DNA molecule of the transgenic corn event LG11.

Description

Nucleic acid sequence of corn transformation event LG11 and detection method thereof

Technical Field

The invention relates to the technical field of plant biology. In particular, it relates to a nucleic acid sequence and method for detecting corn transformation event LG11, and in particular, to a transgenic corn event LG11 that is resistant to insect and glufosinate herbicide application, a nucleic acid sequence and method for detecting the presence of a particular transgenic corn event LG11 in a biological sample.

Background

Corn is the crop with the widest planting area in China, the planting area in 2015 is 5.7 hundred million acres, the yield is 2.2 hundred million tons, and the planting area and the total yield both reach 1/3 of the total planting area and the total yield of food crops in China. As the second major corn producing nation in the world, china has a long-term self-sufficiency of corn, but since 2010, china has been transformed from the net export nation of corn to the net import nation. According to the data of the customs administration, the imported corn in China keeps 259 kilotons and more than 7.3 hundred million dollars in value in 2012-2015, wherein the value reaches 521 kilotons and 17 hundred million dollars in 2012. In addition, the main importer countries of corn in China concentrate on a few countries such as the United states, brazil, argentina and the like, and once the export countries of corn encounter large natural disasters or trade adjustment policies, the risk of importer countries of corn in China is very large. In order to eliminate the hidden trouble, people need to stand at home to improve the yield, keep the basic balance of the supply and demand of the corn and firmly grasp the initiative of the corn industry in their hands. However, the unit yield of corn in China is insufficient and only corresponds to 60 percent of that in the United states. According to the data of the grain and agriculture organization of the United nations, the average yield per unit area of the corns in China is 388 kg/mu in the period between 2010 and 2014, and the average yield per unit area of the corns in the United states is 631 kg/mu in the same period. The high yield per unit of corn in the United states is closely related to the cultivation system, breeding technology and mechanization level of corn. Therefore, the cultivation of insect-resistant herbicide-tolerant transgenic corn varieties and the popularization of transgenic corn are effective means for improving the yield per unit, realizing the balance of supply and demand and ensuring the grain safety.

The whole growth period of corn is affected by various insect pests, among which, corn borer is the main pest in corn production, which can cause about 10% of yield reduction each year. The damage rate of the Asiatic corn borers to the spring corn in China is about 30 percent, the damage rate of the summer corn is 20 to 30 percent, the damage rate can reach 90 percent when the Asiatic corn borers are serious, and the yield is reduced by more than 30 percent. In recent years, with global warming, the damage of corn borers is further increased, and meanwhile, the large-area damage of armyworms is also generated. Spodoptera frugiperda is a novel agricultural pest invading China from Yunnan in the end of 2018 years, the number of host plants reaches 19, and the generation area of corn accounts for 98.1 percent of the total generation area of crops.

The Cry1Ab protein is developed into insect-resistant corn MON810 by Monsanto company for the first time, and aims to improve the resistance to insects of the ostrinia species (Ositriria species) such as Asiatic Corn Borer (ACB). The Vip3Aa is applied to the transgenic corn MIR162 by the Zhangda company for the first time, and has obvious control effect on Agrotis ypsilon, corn earworm Helicoverpa armigera, soybean gloomycota Loxagrotis albicostat and Spodoptera frugiperda. The M2cryAb-Vip3A protein is formed by combining main structural domains of Cry1Ab and Vip3Aa proteins through an artificial synthesis method. The main advantages of the M2cryAb-vip3A protein are: (1) The insecticidal spectrum is expanded, and the insecticidal composition contains functional structural domains of two proteins, and is expected to have resistance to corn borers, cotton bollworms, armyworms and spodoptera frugiperda; (2) The resistance management of target pests is facilitated, and a large number of researches show that Vip3 has high insecticidal activity to various lepidoptera pests, the insecticidal spectrum of Bt protein is expanded, meanwhile, the insecticidal mechanisms of Vip3 toxin and Cry toxin are different, no homology exists in evolution, and the probability of the pests for generating cross resistance to the two toxins is low; (3) The level of mycotoxin in corn ears and corn kernels is reduced, and the quality of the corn kernels is improved; (4) The protein sequence does not contain an allergen sequence, and the protein sequence has biological safety while maintaining high-efficiency insecticidal activity.

The weeds in the field compete with crops for water, fertilizer, light energy and growth space, and directly affect the yield and quality of the crops. Meanwhile, a plurality of weeds are also intermediate hosts of crop pathogenic bacteria and pests, and are one of important biological limiting factors for increasing the yield of crops. According to food and agricultural organization statistics of the united nations, the global food production loss caused by weeds is up to 950 hundred million dollars each year, which means that 3.8 million tons of wheat are lost, and the yield is about more than half of the global wheat yield in 2009. In an economic loss of $ 950 billion, the poverty of developing countries is suffering from $ 700 billion. Therefore, effective control of weeds in the field is one of the important measures for promoting grain yield increase. In addition, with the increase of the migration speed of rural population in China to cities, the scale and mechanization of agricultural planting are a foreseeable trend, so that the traditional artificial weeding mode becomes unrealistic. The popularization and the application of the herbicide can greatly reduce the labor for cotton field management and reduce the labor intensity. The development of new herbicide products with high efficiency, low toxicity and no residue is high in cost, long in time consumption and difficult. This problem can be overcome by cultivating biocidal herbicide-tolerant maize using transgenic technology. The problem of weeds can be effectively solved by spraying the herbicide for 1-2 times in the growth period of the corns, and the dosage and the input cost of the herbicide are reduced. Therefore, the herbicide-tolerant transgenic corn has very wide application value and market potential.

According to the invention, the insect-resistant gene expression cassette and the herbicide-tolerant expression cassette are connected in series, so that the insect-resistant gene expression cassette and the herbicide-tolerant expression cassette are efficiently expressed in the transgenic corn, have insect-resistant and herbicide-tolerant properties, and further enhance the application and economic value of the product.

It is known that expression of foreign genes in plants is influenced by their chromosomal location, possibly due to chromatin structure (e.g., heterochromatin) or the proximity of transcriptional regulatory elements (e.g., enhancers) to the integration site. For this reason, it is often necessary to screen a large number of events in order to be able to identify a commercializable event (i.e., an event in which the introduced gene of interest is optimally expressed). For example, it has been observed in plants and other organisms that the amount of expression of an introduced gene may vary greatly between events; differences in the spatial or temporal pattern of expression may also exist, such as differences in the relative expression of the transgene between different plant tissues, in that the actual expression pattern may not be consistent with the expression pattern expected from the transcriptional regulatory elements in the introduced gene construct, resulting in differences in the trait performance of the transformation event. Thus, it is often necessary to generate hundreds to thousands of different events and to screen those events for a single event with the amount and pattern of transgene expression expected for commercial purposes. Events with expected expression levels and patterns of transgenes can be used to introgress the transgenes into other genetic backgrounds by sexual outcrossing using conventional breeding methods. Progeny produced by this crossing mode retain the transgene expression characteristics of the original transformation event. The use of this strategy ensures reliable gene expression in many varieties that are well adapted to local growth conditions. Therefore, there is a need for trait identification and screening of more transformation events to obtain superior transformation events with superior overall trait performance and commercial prospects.

It would be beneficial to be able to detect the presence of particular events to determine whether progeny of a sexual cross contain a gene of interest. Furthermore, methods of detecting specific events will also help to comply with relevant regulations, such as that foods derived from recombinant crops need to be officially approved and labeled before being placed 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). These detection methods usually focus 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, this method described above cannot be used to distinguish between different events, particularly those produced with the same DNA construct. Therefore, it is now common to identify specific events of a transgene by PCR using a pair of primers spanning the junction of the inserted transgene and the flanking DNA, specifically a first primer containing the flanking sequence and a second primer containing the inserted sequence.

Disclosure of Invention

The invention aims to provide a corn transformation event with excellent insect-resistant and herbicide-resistant properties and excellent agronomic properties, a nucleic acid sequence for detecting corn LG11 and a detection method thereof. The transgenic corn event LG11 has excellent insect resistance and better tolerance to glufosinate herbicides, and the detection method can accurately and quickly identify whether a biological sample contains the DNA molecule of the specific transgenic corn event LG11.

In order to realize the purpose, the invention uses pCAMBIA3300+ m2cryAb-vip3A expression vector to transform corn HiIIB by an agrobacterium-mediated method to obtain more than 400 positive transformants, and after molecular detection, the maize inbred line lx03-2 is used as a recurrent parent for each generationBackcrossing to obtain BC ₅ F ₂ And the generation transgenic corn seeds LG 01-LG 20. Through the identification of insect-resistant and herbicide-resistant properties, the transformation event LG11 is a transformant which is excellent in herbicide resistance and insect resistance and has the best agronomic properties, and can be used for improving the insect-resistant and herbicide-resistant properties of corn.

In order to characterize the identity of LG11, the present invention provides a nucleic acid molecule comprising the sequence shown in SEQ ID NO. 1 and/or SEQ ID NO. 2, or the reverse complement thereof.

Further, the nucleic acid sequence comprises a sequence shown in SEQ ID NO. 3 and/or SEQ ID NO. 4, or a reverse complementary sequence thereof.

Still further, the nucleic acid sequence comprises the sequence shown in SEQ ID NO. 6 and/or SEQ ID NO. 7, or the reverse complement thereof.

Still further, the nucleic acid sequence comprises the sequence shown in SEQ ID NO. 5 or the reverse complement thereof.

The invention also provides a probe for detecting a maize transformation event, which is characterized by comprising a sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2 or SEQ ID NO. 3 or SEQ ID NO. 4 or SEQ ID NO. 6 or SEQ ID NO. 7 or a fragment thereof or a variant or reverse complement thereof.

The invention also provides a primer pair for detecting a maize transformation event, wherein an amplification product of the primer pair comprises a sequence shown as SEQ ID NO. 1 or SEQ ID NO. 2 or SEQ ID NO. 3 or SEQ ID NO. 4 or SEQ ID NO. 6 or SEQ ID NO. 7 or a fragment thereof or a variant or reverse complement thereof.

In some embodiments, the primer pair is a sequence shown as SEQ ID NO. 8 and SEQ ID NO. 9; or the sequences shown in SEQ ID NO 10 and SEQ ID NO 11.

The invention also provides a kit or microarray for detecting a maize transformation event, characterized in that it comprises the above-described probe and/or primer pair.

The invention also provides a method for detecting corn transformation events, which is characterized by detecting whether the transformation events exist in a sample to be detected by using the probe or the primer pair or the probe and primer pair or the kit or microarray.

The invention also provides a method for breeding corn, which is characterized by comprising the following steps:

1) Obtaining corn comprising the nucleic acid molecule;

2) Subjecting the corn obtained in step 1) to pollen culture, unfertilized embryo culture, doubling culture, cell culture, tissue culture, selfing or crossing or a combination thereof to obtain a corn plant, seed, plant cell, progeny plant or plant part; and optionally also (c) a second set of one or more of,

3) Identifying the progeny plants obtained in step 2) for insect resistance and/or herbicide resistance and detecting the presence or absence of said transformation event therein using the method described above.

Furthermore, the invention also provides products made of the corn plants, seeds, plant cells, progeny plants or plant parts obtained by the method, including food, feed or industrial raw materials.

The SEQ ID No. 1 is a 22 nucleotide sequence of transgenic maize event LG11 located near the insertion junction at the 5 'end of the insertion sequence, the SEQ ID No. 1 spans the left flanking genomic DNA sequence of the maize insertion site and the DNA sequence of the 5' end of the left border of the insertion sequence, the inclusion of SEQ ID No. 1 or its reverse complement identifies the presence of transgenic maize event LG11. The SEQ ID No. 2 is a 22 nucleotide sequence of transgenic maize event LG11 located near the insertion junction at the 3 'end of the insertion, the SEQ ID No. 2 spans the DNA sequence of the right border 3' end of the insertion and the right flanking genomic DNA sequence of the maize insertion site, the inclusion of SEQ ID No. 2 or its reverse complement can be identified as the presence of transgenic maize event LG11.

In the present invention, the nucleic acid sequence may be at least 11 or more consecutive polynucleotides of any portion of the transgene insert sequence in said SEQ ID NO:3 or its reverse complement (first nucleic acid sequence) or at least 11 or more consecutive polynucleotides of any portion of the 5' left flank maize genomic DNA region in said SEQ ID NO:3 or its reverse complement (second nucleic acid sequence). The nucleic acid sequence may further be homologous or reverse complementary to a portion of said SEQ ID NO. 3 comprising the entire said SEQ ID NO. 1 or SEQ ID NO. 6. 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 LG11 or its progeny can be diagnosed when the amplification product produced in the DNA amplification method using the DNA primer pair is an amplification product comprising SEQ ID NO. 1 or SEQ ID NO. 3 or SEQ ID NO. 6 or the reverse complement thereof.

The SEQ ID NO. 3 is a 517 nucleotide sequence of transgenic maize event LG11 located near the insertion junction at the 5 'end of the insertion sequence, the SEQ ID NO. 3 consists of the 280 nucleotide maize left flank genomic DNA sequence (nucleotides 1-280 of SEQ ID NO. 3) and the 237 nucleotide 5' end DNA sequence of the first expression cassette of the glufosinate-resistant gene (nucleotides 281-517 of SEQ ID NO. 3), the inclusion of the SEQ ID NO. 3 or its reverse complement identifies the presence of transgenic maize event LG11.

The nucleic acid sequence may be at least 11 or more contiguous polynucleotides of any portion of the transgene insert sequence in SEQ ID No. 4 or its reverse complement (third nucleic acid sequence) or at least 11 or more contiguous polynucleotides of any portion of the 3' right flank corn genomic DNA region in SEQ ID No. 4 or its reverse complement (fourth nucleic acid sequence). The nucleic acid sequence may further be homologous or reverse complementary to a portion of the SEQ ID NO. 4 comprising the entire SEQ ID NO. 2 or SEQ ID NO. 7. When the third nucleic acid sequence and the fourth nucleic acid sequence are used together, these nucleic acid sequences comprise a set of DNA primers in a DNA amplification method that produces an amplification product. The presence of transgenic maize event LG11 or progeny thereof can be diagnosed when the amplification product produced in a DNA amplification method using a DNA primer pair is an amplification product comprising SEQ ID No. 2 or SEQ ID No. 4 or SEQ ID No. 7 or the reverse complement thereof.

The SEQ ID No. 4 is a 1631 nucleotide sequence located near the insertion junction at the 3 'end of the insert in transgenic corn event LG11, the SEQ ID No. 4 consists of the DNA sequence at the 3' end of the second expression cassette of the insect-resistant gene of 575 nucleotides (nucleotides 1-575 of SEQ ID No. 4), the pCAMBIA3300+ m2cryAb-vip3A construct right border DNA sequence of 736 nucleotides (nucleotides 576-1311 of SEQ ID No. 4) and the corn integration site right flank genomic DNA sequence of 320 nucleotides (nucleotides 1312-1631 of SEQ ID No. 4), the inclusion of SEQ ID No. 4 or its reverse complement could be identified as the presence of transgenic corn event LG11.

5 is a 7744 nucleotide long sequence characteristic of transgenic maize event LG11, which specifically comprises the genome and genetic elements shown in table 1. The presence of transgenic maize event LG11 can be identified by inclusion of SEQ ID No. 5 or the reverse complement thereof.

Table 1 genomic and genetic elements contained in SEQ ID no

1: the unit bp.

It is well known to those skilled in the art that the first and second nucleic acid sequences or the third and fourth nucleic acid sequences need not consist of only DNA, but may also 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. In addition, the probe or primer of the invention should be at least about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 contiguous nucleotides in length, which can be selected from the nucleotides set forth in SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10 or SEQ ID NO 11. When selected from the group consisting of the nucleotides set forth in SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, and SEQ ID NO. 7, the primer may be a contiguous nucleotide of at least about 21 to about 50 or more in length.

The present invention also provides a method of protecting corn plants from herbicide-induced damage, comprising applying to a field in which at least one transgenic corn plant comprising in its genome, in order, SEQ ID No. 1, SEQ ID No. 5, nucleic acid sequences from positions 281 to 7424, and SEQ ID No. 2, or comprising in its genome SEQ ID No. 5, is grown, an effective amount of a glufosinate herbicide; the transgenic corn plants have tolerance to glufosinate herbicides.

The invention also provides a method for protecting corn plants from insect attack, which is characterized by comprising the steps of providing at least one transgenic corn plant cell in the diet of target insects, wherein the transgenic corn plant cell comprises the nucleotide sequence of SEQ ID NO. 1, the nucleotide sequence of SEQ ID NO. 5 from 281 to 7424 and the nucleotide sequence of SEQ ID NO. 2 in sequence in the genome of the transgenic corn plant cell, or comprises the nucleotide sequence of SEQ ID NO. 5 in the genome of the transgenic corn plant cell; target insects that feed on the transgenic corn plant cell are inhibited from further feeding on the corn plant.

In the nucleic acid sequences for detecting corn plants and the detection methods thereof of the present invention, the following definitions and methods may better define the invention and guide those of ordinary skill in the art in the practice of the invention, unless otherwise indicated, the terms are understood according to their conventional usage by those of ordinary skill in the art.

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

The term "comprising" means "including but not limited to".

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 granules), and plant cells intact in plants or plant parts such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, stalks, roots, root tips, anthers, and the like. It is to be understood that parts of transgenic plants within the scope of the present invention, which are derived from transgenic plants or progeny thereof which have been previously transformed with a DNA molecule of the invention and thus consist at least in part of transgenic cells, include, but are not limited to, plant cells, protoplasts, tissue, callus, embryos, and flowers, stems, fruits, leaves, and roots.

The term "gene" refers to a nucleic acid fragment that expresses a particular protein, including regulatory sequences preceding the coding sequence (5 'non-coding sequences) and regulatory sequences following the coding sequence (3' non-coding sequences). "native gene" refers to a gene that is naturally found to have its own regulatory sequences. "chimeric gene" refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found in nature. "endogenous gene" refers to a native gene that is located in its natural location in the genome of an organism. "foreign gene" is a foreign gene that exists in the genome of an organism and does not originally exist, and also refers to a gene that has been introduced into a recipient cell through a transgenic step. The foreign gene may comprise a native gene or a 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 at which the recombinant DNA has been inserted may be referred to as an "insertion site" or "target site".

"flanking DNA" may comprise the genome as it occurs naturally in an organism such as a plant or foreign (heterologous) DNA introduced by the transformation process, such as a fragment associated with the transformation event. Thus, flanking DNA may include a combination of native and foreign DNA. In the present invention, a "flanking region" or "flanking sequence" or "genomic boundary region" or "genomic boundary sequence" refers to a sequence of at least 3, 5, 10, 11, 15, 20, 50, 100, 200, 300, 400, 1000, 1500, 2000, 2500, or 5000 base pairs or more in length, which is located directly upstream or downstream of and adjacent to the originally exogenously inserted DNA molecule. When the flanking region is located upstream, it may also be referred to as "left border flanking" or "5 'genomic flanking region" or "genomic 5' flanking sequence" or the like. When the flanking region is located downstream, it may also be referred to as "right border flanking" or "3 'genomic flanking region" or "genomic 3' flanking sequence" or the like.

Transformation procedures that result in random integration of the exogenous DNA will result in transformation events that contain different flanking regions that are specific for each transformation event. When the recombinant DNA is introduced into a plant by conventional crossing, its flanking regions are not usually altered. Transformation events will also contain unique junctions between the heterologous insert DNA and segments of genomic DNA or between two segments of heterologous DNA. "junction" is the point at which two specific DNA fragments are joined. For example, the junction is present where the insert DNA joins the flanking DNA. A junction point is also present in a transformed organism where two DNA segments are joined together in a manner modified from that found in the native organism. "junction DNA" refers to DNA comprising a junction site.

The present invention provides a transgenic corn event, designated LG11, and progeny thereof, wherein transgenic corn event LG11 is corn LG11 comprising plants and seeds of transgenic corn event LG11 and plant cells or regenerable portions thereof, and plant parts of transgenic corn event LG11 including, but not limited to, cells, pollen, ovules, flowers, buds, roots, stems, leaves, and products from corn LG11, such as cottonseed, cottonseed oil, cotton coats, quilts, cotton battings, cotton cloths, and biomass left in the field of corn crops.

Transgenic corn event LG11 of the invention comprises a DNA construct that, when expressed in a plant cell, confers resistance to a pest and/or glufosinate herbicide on transgenic corn event LG11. The DNA construct comprises an expression cassette comprising a suitable promoter for expression in plants operably linked to an M2cryAb-VIP3A gene having insect resistance and a suitable polyadenylation signal sequence, the nucleic acid sequence of the M2cryAb-VIP3A protein being capable of increasing insect resistance in maize. The DNA construct comprises a further expression cassette comprising a suitable promoter for expression in plants operably linked to the bar gene encoding Phosphinothricin Acetyltransferase (PAT) protein having a nucleic acid sequence which is tolerant to glufosinate herbicides and a suitable polyadenylation signal sequence. Further, the promoter may be a suitable promoter isolated from plants, including constitutive, inducible, and/or tissue-specific promoters, including, but not limited to, cauliflower mosaic virus (CaMV) 35S promoter, figwort Mosaic Virus (FMV) 35S promoter, ubiquitin protein (Ubiquitin) promoter, actin (Actin) promoter, agrobacterium tumefaciens (Agrobacterium tumefaciens) nopaline synthase (NOS) promoter, octopine synthase (OCS) promoter, cestrum (Cestrum) yellow leaf curly virus promoter, potato tuber storage protein (Patatin) promoter, ribulose-1, 5-bisphosphate carboxylase/oxygenase (RuBisCO) promoter, glutathione thiotransferase (GST) promoter, E9 promoter, GOS promoter, alcA/alcR promoter, agrobacterium rhizogenes RolD promoter, and Arabidopsis thaliana (Arabidopsis) Suc2 promoter. The polyadenylation signal sequence may be a suitable polyadenylation signal sequence that functions in plants, including, but not limited to, polyadenylation signal sequence derived from the Agrobacterium tumefaciens nopaline synthase (NOS) gene, cauliflower mosaic virus (CaMV) 35S terminator, polyadenylation signal sequence derived from the protease inhibitor II (PIN II) gene, and polyadenylation signal sequence derived 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/transit peptides. The enhancer may enhance the expression level of a gene, and includes, but is not limited to, tobacco Etch Virus (TEV) translational activator, caMV35S enhancer, and FMV35S enhancer. The signal peptide/transit peptide may direct the transportation of the EPSPS protein to a particular organelle or compartment outside or within the cell, for example, targeting the chloroplast using a sequence encoding a chloroplast transit peptide, or targeting the endoplasmic reticulum using a 'KDEL' retention sequence.

The glufosinate-ammonium refers to a non-selective, broad-spectrum, high-efficiency and low-toxicity organophosphorus herbicide, and strongly inhibits the activity of Glutamine Synthetase (GS), an amino acid biosynthetic enzyme of bacteria and plants. GS plays an important role in the assimilation and regulation of ammonia metabolism in plants, is the only detoxifying enzyme in plants, and can detoxify ammonia released by nitric acid reduction, amino acid degradation, and light respiration. Treatment with a "glufosinate herbicide" refers to treatment with any herbicide formulation containing glufosinate. The choice of the rate of use of a certain glufosinate formulation to achieve an effective biological dose is not beyond the skill of the ordinary agronomic artisan. Treatment of a field comprising plant material derived from transgenic corn event LG11 with any one of the glufosinate-containing herbicide formulations will control weed growth in the field and will not affect the growth or pest resistance of plant material derived from transgenic corn event LG11.

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

The Agrobacterium-mediated transformation method is a commonly used 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. Said vector is transformed into an agrobacterium cell, which is subsequently used to infect plant tissue, said T-DNA region of the vector comprising the foreign DNA being inserted into the plant genome. After transformation, transgenic plants must be regenerated from the transformed plant tissue and progeny with exogenous DNA selected using appropriate markers.

A DNA construct is a combination of DNA molecules linked together to provide one or more expression cassettes. The DNA construct is preferably a plasmid capable of autonomous replication in bacterial cells and containing various restriction 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, which includes the genetic elements necessary to provide for transcription of messenger RNA, can be designed for expression in prokaryotic or eukaryotic cells. The expression cassette of the invention is designed to be most preferably 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 population of plants, regenerating said population of plants, and selecting for a particular plant characterized by the insertion of a particular genomic locus. The term "event" refers to both the original transformation event comprising the heterologous DNA and the progeny of the transformation event. The term "event" also refers to progeny resulting from sexual crosses between a transformation event and individuals of other varieties containing heterologous DNA, where the inserted DNA and flanking genomic DNA from the parent of the transformation event are present in the same chromosomal location in the progeny of the cross, even after repeated backcrosses with the backcross parents. The term "event" also refers to a DNA sequence from an original transformation event that comprises an inserted DNA and flanking genomic sequences immediately adjacent to the inserted DNA, which DNA sequence is expected to be transferred to progeny that result from sexual crossing of a parental line containing the inserted DNA (e.g., progeny resulting from the original transformation event and its selfing) with a parental line that does not contain 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 a form of DNA and/or protein and/or organism which is not normally found in nature and which is therefore produced by human intervention. Such manual intervention may result in recombinant DNA molecules and/or recombinant plants. Such "recombinant DNA molecules" are obtained by artificially combining two otherwise isolated sequence segments, for example, by chemical synthesis or by manipulating an isolated nucleic acid segment 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 heterologous nucleic acid, including the transgene body that was originally so altered, as well as progeny individuals generated from the original transgene body by sexual crossing or asexual reproduction. In the present invention, the term "transgene" does not include changes (chromosomal or extra-chromosomal) in the genome by conventional plant breeding methods or naturally occurring events such as random allofertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.

By "heterologous" in the context of 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 be derived from a first species and inserted into the genome of a second species. Such molecules are therefore heterologous to the host and are artificially introduced into the genome of the host cell.

Culturing transgenic corn event LG11 having insect-resistant properties and tolerance to glufosinate herbicides by: first sexually crossing a first parent corn plant consisting of a corn plant bred from transgenic corn event LG11 and its progeny obtained by transformation with an expression cassette of the present invention that is resistant to insects and glufosinate herbicide, with a second parent corn plant lacking insect-resistant properties or resistant to glufosinate herbicide, to produce a plurality of first generation progeny plants; progeny plants that are tolerant to the glufosinate herbicide are then selected, and maize plants that are tolerant to the glufosinate herbicide can be developed. These steps can further include backcrossing the insect-resistant and glufosinate-tolerant progeny plants with the second or third parent corn plant, and then selecting the progeny by application of a glufosinate herbicide or by identification of a trait-related molecular marker (such as a DNA molecule comprising a junction site identified at the 5 'end and 3' end of the insert sequence in transgenic corn event LG 11) to produce a corn plant that is insect-resistant and tolerant to the glufosinate herbicide.

It is also understood that two different transgenic plants may also be crossed to produce progeny that contain two separate, separately added exogenous genes. Selfing of the appropriate progeny can yield progeny plants that are homozygous for both added exogenous genes. Backcrossing of parental plants and outcrossing with non-transgenic plants as described above is also contemplated, as is asexual reproduction.

The term "probe" is an isolated nucleic acid molecule having a conventional detectable label or reporter molecule, e.g., a radioisotope, ligand, chemiluminescent agent or enzyme, bound thereto. Such a probe is complementary to one strand of the target nucleic acid, and in the present invention, the probe is complementary to one strand of DNA from the genome of transgenic corn event LG11, whether the genomic DNA is from transgenic corn event LG11 or seed or a plant or seed or extract derived from transgenic corn event LG11. Probes of the invention include not only deoxyribonucleic or ribonucleic acids, 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, annealing, forming a hybrid between the primer and the target DNA strand, and then extending along the target DNA strand under the action of a polymerase (e.g., a DNA polymerase). The primer pairs of the present invention are directed to their use in amplification of a target nucleic acid sequence, for example, by Polymerase Chain Reaction (PCR) or other conventional nucleic acid amplification methods. The length of the primer 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 primers hybridize specifically to the target sequence under highly stringent hybridization conditions. Although a primer that is different from and retains the ability to hybridize to a target DNA sequence can be designed by a conventional method, it is preferable that the primer of the present invention have complete DNA sequence identity with a contiguous nucleic acid of the target sequence.

As used herein, "kit" or "microarray" refers to a set of reagents or chips for the purpose of identification and/or detection of corn transformation events in a biological sample. For the purpose of quality control (e.g. purity of seed lot), detection of events in or in a material comprising or derived from plant material, such as, but not limited to, food or feed products, kits or chips may be used, and components thereof may be specifically adjusted.

Primers and probes based on the flanking genomic DNA and insertion sequences of the present invention can be determined by conventional methods, for example, by isolating the corresponding DNA molecule from plant material derived from transgenic corn event LG11 and determining the nucleic acid sequence of the DNA molecule. The DNA molecule comprises a transgene insert and a maize genomic flanking region, and fragments of the DNA molecule can be used as primers and probes.

The primers and probes of the invention hybridize to a target DNA sequence under stringent conditions. Any conventional amplification method can be used to identify the presence of DNA derived from transgenic corn event LG11 in a sample. Nucleic acid molecules or fragments thereof are capable of specifically hybridizing to other nucleic acid molecules under certain circumstances. As used herein, two nucleic acid molecules can be said to be capable of specifically hybridizing to each other if the two nucleic acid molecules are capable of forming an antiparallel double-stranded nucleic acid structure. Two nucleic acid molecules are said to be "complements" of one another if they exhibit complete complementarity. As used herein, a nucleic acid molecule is said to exhibit "perfect complementarity" when each nucleotide of the nucleic acid molecule is complementary to a corresponding nucleotide of another 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 to allow them to 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 to allow them to anneal and bind to each other under conventional "highly stringent" conditions. Deviations from perfect complementarity may be tolerated as long as such deviations do not completely prevent the formation of a double-stranded structure by the two molecules. In order for a nucleic acid molecule to be able to act as a primer or probe, it is only necessary to ensure sufficient complementarity in the sequence to allow formation of a stable double-stranded structure at the particular solvent and salt concentrations employed.

As used herein, a substantially homologous sequence is a nucleic acid molecule that specifically hybridizes under highly stringent conditions to the complementary strand of a matched nucleic acid molecule. Suitable stringency conditions for promoting DNA hybridization include, for example, treatment with 6.0 XSSC/sodium citrate (SSC) at about 45 ℃ followed by a wash with 2.0 XSSC at 50 ℃, as is well known to those skilled in the art. For example, the salt concentration in the washing step can be selected from the group consisting of about 2.0 XSSC for low stringency conditions, 50 ℃ to about 0.2 XSSC for high stringency conditions, 50 ℃. In addition, the temperature conditions in the washing step can be raised from about 22 ℃ at room temperature for low stringency conditions to about 65 ℃ for high stringency conditions. Both the temperature conditions and the salt concentration may be varied, or one may be held constant while the other is varied. Preferably, a nucleic acid molecule of the invention specifically hybridizes under moderately stringent conditions, for example 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 a fragment of any of the foregoing. More preferably, a nucleic acid molecule of the invention specifically hybridizes 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 a complementary sequence thereof, or to a fragment of any of the foregoing. In the context of the present invention, preferred marker nucleic acid molecules have SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 6 or SEQ ID NO 7 or the complement thereof or any fragment of the aforementioned 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 3, SEQ ID NO 4, SEQ ID NO 6 or SEQ ID NO 7 or the complement thereof or any fragment of any of the above. SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 6 and SEQ ID NO 7 can be used as markers in plant breeding methods to identify progeny of a genetic cross. Hybridization of the probe to the target DNA molecule can be detected by any method known to those skilled in the art, including, but not limited to, fluorescent labels, radioactive labels, antibody-based labels, and chemiluminescent labels.

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

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

As used herein, "amplified DNA" or "amplicon" refers to the nucleic acid amplification product of a target nucleic acid sequence that is part of a nucleic acid template. For example, to determine whether a corn plant was produced by sexual hybridization containing transgenic corn event LG11 of the invention, or whether a corn sample collected from a field contains transgenic corn event LG11, or whether a corn extract, e.g., cotton seed oil, contains transgenic corn event LG11, DNA extracted from a corn plant tissue sample or extract can be subjected to a nucleic acid amplification method using a primer pair to produce an amplicon diagnostic for the presence of DNA of transgenic corn event LG11. The primer pair includes a first primer derived from a flanking sequence adjacent to an insertion site of the inserted foreign DNA in the plant genome, and a second primer derived from the inserted foreign DNA. The amplicon has a length and sequence that is also diagnostic for the transgenic corn event LG11.

The amplicon can range in length from the bound 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, primer pairs may be derived from flanking genomic sequences flanking the inserted DNA to produce amplicons that include the entire inserted nucleotide 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 carried out by any of the nucleic acid amplification reaction methods known in the art, including the Polymerase Chain Reaction (PCR). Various nucleic acid amplification methods are well known to those skilled in the art. PCR amplification methods have been developed to amplify 22kb of genomic DNA and 42kb of phage DNA. These methods, as well as other DNA amplification methods known in the art, can be used in the present invention. The inserted foreign DNA sequence and flanking DNA sequences from transgenic corn event LG11 can be amplified by using the provided primer sequences to the genome of transgenic corn event LG11, followed by standard DNA sequencing of the PCR amplicons or cloned DNA.

DNA detection kits based on DNA amplification methods contain DNA primer molecules that specifically hybridize to the target DNA and amplify the diagnostic amplicon under appropriate reaction conditions. The kit may provide an agarose gel based detection method or a number of methods known in the art for detecting diagnostic amplicons. Kits containing DNA primers homologous or reverse complementary to any portion of the maize genomic region of SEQ ID NO 3 or SEQ ID NO 4 and homologous or reverse complementary to any portion of the transgene insert region of SEQ ID NO 5 are provided by the present invention. In particular, primer pairs useful in DNA amplification methods were identified as SEQ ID NOs 8 and 9, which amplify diagnostic amplicons homologous to a portion of the 5' transgene/genomic region of transgenic corn event LG11, wherein the amplicon includes SEQ ID NO 1. The primer pairs identified as useful in the DNA amplification method also include SEQ ID NO 10 and SEQ ID NO 11, which amplify a diagnostic amplicon homologous to a portion of the 3' transgene/genomic region of transgenic corn event LG11, wherein the amplicon includes SEQ ID NO 2. 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 such method is Genetic Bit Analysis, which designs a DNA oligonucleotide strand spanning an intervening DNA sequence and adjacent flanking genomic DNA sequences. The oligonucleotide strand is immobilized within the microwell of a microwell plate, and after PCR amplification of the target region (using one primer in each of the intervening sequence and adjacent flanking genomic sequence), 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 can be obtained by fluorescence or ELISA-like methods. The signal represents the presence of the inserted/flanking sequence, indicating that the amplification, hybridization and single base extension reactions were successful.

Another method is Pyrosequencing (Pyrosequencing) technology. The method designs an oligonucleotide strand that spans the junction of the inserted DNA sequence and the adjacent genomic DNA. The oligonucleotide strand is hybridized to the single-stranded PCR product of the target region (using one primer in each of the intervening and adjacent flanking genomic sequences), and then incubated with DNA polymerase, ATP, sulfurylase, luciferase, apyrase, adenosine-5' -phosphothioate, and luciferin. dNTPs were added separately and the resulting optical signals were measured. The light signal represents the presence of the inserted/flanking sequence, indicating that the amplification, hybridization, and single or multiple base extension reactions were successful.

Fluorescence polarization is also a method that can be used to detect the amplicons of the invention (Chen X, levine L, and Kwok PY. Fluorescence polarization in genetic nucleic acid analysis [ J ]. Genome Res,1999,9 (5): 492-8.). Using this method requires the design of an oligonucleotide strand that spans the intervening DNA sequence and adjacent genomic DNA binding sites. The oligonucleotide strand is hybridized to the single stranded PCR product of the target region (using one primer each within the insert sequence and adjacent flanking genomic sequence) and then incubated with DNA polymerase and a fluorescently labeled ddNTP. Single base extension will result in insertion of ddntps. This insertion can be measured for changes in its polarization using a fluorometer. The change in polarization represents the presence of the inserted/flanking sequence, indicating that the amplification, hybridization and single base extension reactions were successful.

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

Suitable techniques for detecting plant material derived from transgenic maize event LG11 can also include Southern blot hybridization, northern blot hybridization, and in situ hybridization based on the principle of hybridization. In particular, the suitable technique involves incubating the probe and sample, washing to remove unbound probe and detecting whether the probe has hybridised. The detection method depends on the type of label attached to the probe, for example, a radiolabeled probe can be detected by X-ray exposure and development, or an enzymatically labeled probe can be detected by a color change achieved by substrate conversion.

Sequences can also be detected using molecular markers (Tyagi S and Kramer F R. Molecular beacons: probes that are fluorescent upon hybridization [ J ]. Nat Biotechnol,1996,14 (3): 303-8.). A FRET oligonucleotide probe is designed that spans the inserted DNA sequence and the 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 a fluorescent moiety and a quenching moiety in close proximity. The FRET probe and PCR primers (one for each of the insert and adjacent flanking genomic sequence) 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 the loss of the secondary structure of the probe, thereby spatially separating the fluorescent moiety and the quencher moiety, resulting in a fluorescent signal. The generation of a fluorescent signal represents the presence of the inserted/flanking sequence, indicating that amplification and hybridization were successful.

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

The DNA detection kit can be developed using the compositions described herein and methods described or known in the DNA detection art. The kit is useful for identifying the presence or absence of DNA of transgenic corn event LG11 in a sample, and can also be used to cultivate corn plants containing DNA of transgenic corn event LG11. The kit may contain DNA primers or probes homologous or reverse complementary to at least a portion of

SEQ ID NO

1, 2, 3, 4, 5, 6 or 7, or other DNA primers or probes homologous 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 site of the maize genome containing the transgene insert and illustrated in figure 1 and table 1 comprises: the maize LG11 left flank genomic region located 5' to the transgene insert, a portion of the insert from the left border region (LB) of agrobacterium, and the first expression cassette is the 35S promoter of cauliflower mosaic virus (CaMV 35S promoter (enhanced)), operably linked to the glufosinate-ammonium resistance gene sequence (bar), and operably linked to the 35S terminator of cauliflower mosaic virus (CaMV poly (a)); the second expression cassette consisted of the cauliflower mosaic virus 35S promoter (CaMV 35 Spromoter), operably linked to the insect-resistant gene m2cryAb-vip3A, and operably linked to the nopaline synthase gene terminator (NOS terminator), a portion of the insert from the Agrobacterium right border Region (RB), and the maize LG11 right wing genome region (SEQ ID NO: 5) at the 3' end of the transgene insert. In the DNA amplification method, the DNA molecule that serves as a primer can be any portion derived from the transgene insertion sequence in transgenic corn event LG11, or any portion derived from the region of DNA flanking the corn genome in transgenic corn event LG11.

Transgenic corn event LG11 can be combined with other transgenic corn varieties, such as herbicide (e.g., glufosinate, glyphosate, etc.) tolerant corn, or transgenic corn varieties that carry insect-resistant genes. Various combinations of all of these different transgenic events, when bred with transgenic corn event LG11 of the invention, can provide improved hybrid transgenic corn varieties that are resistant to insects and to multiple herbicides. Compared with non-transgenic varieties and transgenic varieties with single characters, the varieties can show more excellent characteristics such as insect resistance, resistance to various herbicides and the like.

The invention provides a nucleic acid sequence for detecting corn plants and a detection method thereof, wherein transgenic corn event LG11 has the effects of improving insect resistance and tolerating glufosinate-ammonium herbicide. Maize plants of this trait express the M2cryAb-vip3A protein and the Phosphinothricin Acetyltransferase (PAT) protein, which confers insect resistance and tolerance to glufosinate to plants. Meanwhile, in the detection method, SEQ ID NO 1 or a reverse complementary sequence thereof, SEQ ID NO 2 or a reverse complementary sequence thereof, SEQ ID NO 3 or a reverse complementary sequence thereof, SEQ ID NO 4 or a reverse complementary sequence thereof, SEQ ID NO 6 or a reverse complementary sequence thereof, or SEQ ID NO 7 or a reverse complementary sequence thereof can be used as a DNA primer or a probe to generate an amplification product diagnosed as transgenic corn event LG11 or a progeny thereof, and the presence of plant material derived from transgenic corn event LG11 can be identified rapidly, accurately and stably.

The transgenic corn event LG11 has strong glufosinate tolerance and outstanding insect resistance. These characteristics allow LG11 to be used for improving glufosinate herbicide tolerance and insect resistance of corn, so that a new corn variety with insect resistance and herbicide tolerance is bred.

The technical solution of the present invention is further described in detail by the accompanying drawings and embodiments.

Drawings

FIG. 1 is a schematic diagram of the structure of the binding site of a transgene insert sequence to the maize genome.

FIG. 2 physical map of recombinant expression vector pCAMBIA3300+ m2cryAb-vip 3A. The English and abbreviated meanings of each element are listed as follows:

FIG. 3LG11 event vs. control lx03-2 plants for glufosinate herbicide resistance. A: lx03-2 at 0 times dose; b: lx03-2 under 1 time of dose; c: LG11 at 0-fold dose; d: LG11 at 1-fold dose; e: LG11 at 2-fold dose; f: LG11 at 4-fold dose.

FIG. 4 insect resistance performance of LG11 event versus control lx 03-2. A: a non-transgenic control lx03-2 maize plant; b: LG11 transformed event maize plants.

FIG. 5LG11 transformation event specific PCR validation results. M: marker, marking the size aside (unit: bp); n: blank control; CK: negative control (lx 03-2, lx08-8 and Ludan 9088 mix) genomic DNA;1-3: (ii) different generation transformation event LG11 genomic DNA;4: transgenic maize new hybrid LG 11-Lu single 9088 genomic DNA. A: the expected size of the left border PCR fragment is 286bp; b: the right border PCR fragment was expected to be 1447bp in size.

FIG. 6Southern hybridization cleavage and probe location.

FIG. 7 Southern blot hybridization of inserted copy number of m2cryAb-vip3A gene of interest LG11

A: hindIII enzyme digestion hybridization pattern; b: bamHI enzyme digestion hybridization pattern; c: the probe and Hind III enzyme cutting position are shown schematically; d: probe and BamHI cleavage site schematic. The lower line segment indicates the probe position, and the arrow on the right side of the picture indicates the external band.

M: DNA Marker, the size of the strip is marked aside, unit bp;

CK: negative control lx03-2;

1: a positive control plasmid;

2-4; the transformant LG11 of a different generation.

FIG. 8 Southern blot hybridization of the bar insert copy number of the LG11 target gene

M: DNA Marker, marking the size of the strip by side, and the unit bp;

CK: negative control lx03-2;

1: a positive control plasmid;

2-4; the transformant LG11 of a different generation.

Detailed Description

The transformation event LG11 related to the application refers to a maize plant which is obtained by crossing a maize inbred line lx03-2 after genetic transformation by taking maize HiIIB as a receptor and inserting an exogenous gene insert (T-DNA insert) between specific genome sequences. In a specific example, the expression vector used for the transgene has the physical map shown in FIG. 2, and the resulting T-DNA insert has the sequence shown by nucleotides 281 to 7424 of SEQ ID NO. 5. Transformation event LG11 can refer to this transgenic process, can also refer to a T-DNA insert within the genome resulting from this process, or a combination of a T-DNA insert and flanking sequences, or can refer to a maize plant resulting from this transgenic process. In a specific example, the event is also applicable to plants obtained by transforming other recipient varieties with the same expression vector and inserting the T-DNA insert into the same genomic position. Transformation event LG11 can also refer to a progeny plant derived from the above plants by vegetative propagation, sexual propagation, doubling or doubling, or a combination thereof.

Example 1 acquisition of transformation events and characterization

The M2cryAb-Vip3A protein is formed by combining main structural domains of Cry1Ab and Vip3Aa proteins through an artificial synthesis method, and the Cry1Ab and the Vip3Aa have obvious control effect on pests such as corn borers, spodoptera frugiperda and the like; the bar gene codes phosphinothricin acetyl transferase, can improve the tolerance of plant to glufosinate herbicide. The invention uses pCAMBIA3300+ m2cryAb-vip3A expression vector (the physical map of the vector is shown in figure 2, and the vector comprises m2cryAb-vip3A gene expression box and bar gene expression box), transforms receptor HiIIB by agrobacterium-mediated method, obtains more than 400 positive transformants, and carries out backcross by taking maize inbred line lx03-2 as recurrent parent in each generation after molecular detection to obtain BC ₅ F ₂ Transgenic corn seeds LG 01-LG 20 are generated, and herbicide tolerance, insect resistance and related agronomic traits of the transformed seedlings are screened and identified.

1. Screening of transformants having excellent insect-resistant and herbicide-resistant Properties

(1) Herbicide resistance screening

And (3) selecting a transformant with better herbicide tolerance by using lx03-2 as a reference and spraying glufosinate-ammonium with the amount of 1 time of the recommended field concentration in the field. The results show that only 9 transformation events were significantly more tolerant to glufosinate herbicide than the control (table 2).

TABLE 2 herbicide tolerance Performance

Values are from the mean ± standard deviation of 3 biological replicates. Statistical analysis multiple comparisons (α = 0.05) were performed using LSD, with different letters indicating significance of differences in the data from the same column at the same herbicide concentration.

(2) Resistance to insects

Transformants with better insect resistance were selected from the 9 transformation events by means of leaf chamber bioassay using lx03-2 as reference. In vitro corn leaves are used for feeding the young hatched larvae of Asiatic corn borers and Spodoptera frugiperda, and the insect resistance of the material is evaluated. The 9 transformant leaves caused significantly higher mortality rates of the ostrinia nubilalis and spodoptera littoralis than the control (table 3), wherein the resistance levels of LG04, LG06, LG09 and LG11 to the ostrinia nubilalis and spodoptera littoralis are high, and the rest are resistant or resistant.

TABLE 3 indoor bioassay

Values are expressed as mean ± standard deviation of 4 biological replicates and significance of differences from the same column data was analyzed using the LSD method (α = 0.05).

(3) Agronomic trait survey

While the resistance traits of several transformants are identified, the agronomic traits (such as plant height, leaf size, ear size and kernel weight) of the transformants are recorded in detail. Surprisingly, it was found during the data statistics that the plant heights and the grain weights of the transformants LG01, LG03, LG04, LG05, LG06, LG08, LG09, and LG14 were all significantly lower than the control lx03-2, and only the agronomic traits (plant heights and grain weights) of the transformant LG11 were not significantly different from the control (table 4).

Table 4 partial results of agronomic trait investigations

Values are from the mean ± standard deviation of 3 biological replicates. Statistical analysis multiple comparisons were performed using LSD (α = 0.05), with different letters indicating significance of differences in the same period and column data.

In summary, LG11 is a transformant that is excellent in herbicide tolerance, insect resistance, and agronomic performance.

2. Analysis of sequences flanking insertion sites of exogenous sequences on maize genome

To further clarify the insertion site of LG11 of the transformation event, the present invention analyzes the sequences flanking the insertion site of the exogenous sequence of LG11 in the maize genome.

Taking 100mg of plant leaves, quickly grinding the plant leaves by liquid nitrogen, and extracting total DNA by adopting a CTAB method. After the genomic DNA was subjected to concentration determination, the total amount of DNA was guaranteed to be > 2. Mu.g. By means of genome re-sequencing (completed by Wuhan biological sample library, inc.), each Reads has a length of 150bp to obtain at least 20Gb data, and the data quality index Q30 is ensured to be more than or equal to 80% (namely, the proportion of the basic groups with a sequencing error rate of more than 0.1% is lower than 20%). Based on the genome re-sequencing result, BWA software was used to perform a sequence homology comparison screening with the T-DNA sequence of the transgenic vector as a template and the whole sequence obtained by sequencing (BWA, http:// bio-bw. Sourceform. Net/, default). And further splicing and screening the screened sequences, and removing Reads with all sequences as carrier sequences to finally obtain a class of Reads sequences, which is characterized in that half of the class of Reads sequences is a genome sequence, and the other half of the class of Reads sequences is a carrier sequence. According to the obtained genome sequence, performing Blast sequence comparison on a maize genome website https:// www.maizegdb.org /) to obtain a specific position of the genome sequence on the genome, namely a possible insertion site.

And comparing the sequencing result with the reference genome and the exogenous T-DNA sequence respectively to obtain the insertion position information of the exogenous insert. Then, forward and reverse primers are respectively designed on the genome flanking sequence and the exogenous insertion sequence at the left and right boundaries of the insertion site, the insertion site is verified by a PCR amplification method, and a PCR product is subjected to sequencing analysis. The results showed that the exogenous fragment of transformant LG11 was inserted forward between the CHR5 maize genome: 177554037-177554053 bp.

Subsequently, primer design was performed on the genomic and T-DNA regions at the left and right borders of the insertion site using Primeblast software (https:// Blast. NCBI. Nlm. Nih. Gov/Blast) of NCBI website, and the amplification product fused a portion of the maize genomic sequence and a portion of the T-DNA sequence.

And carrying out PCR amplification by using the genome DNA of the transgenic corn strain as a template. The PCR reaction was carried out in a 20. Mu.L system. The amplification cycle program was: pre-denaturation at 94 ℃ for 3min; denaturation at 94 ℃ for 30s, annealing for 30s, extension at 72 ℃ for a certain time (set according to the size of the product fragment), 35 cycles; extension at 72 ℃ for 5min.

The LG11 transformation event was PCR amplified using the genome upstream primer (SEQ ID NO: 8) and the vector left border primer (SEQ ID NO: 9) and the vector right border primer (SEQ ID NO: 10) and the genome downstream primer (SEQ ID NO: 11) based on the results of flanking sequence and insertion position to verify the foreign fragment insertion position. The results are shown in FIG. 5. The results prove that the LG11 exogenous fragment is stably inserted into the position of chr5 of the corn genome of 177554037bp, and an insertion sequence and upstream and downstream genome flanking sequence fragments are obtained through overlapped PCR amplification and sequencing analysis, and the sequence assembly is shown in figure 6. The sequence analysis result shows that the size of the inserted sequence is 7144bp.

By analyzing the boundary sequences of the left flank and the right flank, the insertion of the exogenous sequence causes 15bp sequence mutation of a corn genome, and the left boundary sequence of the vector is deleted with 388bp (comprising a partial sequence of a herbicide-resistant gene bar and a whole terminator sequence) and the right boundary sequence is deleted with 22bp.

3. Systematic identification of LG11 herbicide tolerance and insect resistance

The invention systematically identifies the herbicide tolerance and insect resistance of the transformant LG11 in 2022 summer by sowing the corn transformant LG11 and the seeds of the reference lx03-2 in pilot-plant and industrialized bases of Longshan transgenic corn in Longshan office of Shandong province, jinan, qidun and Shanghai, spraying glufosinate ammonium with different concentrations in the fields and artificially inoculating insects.

(1) Herbicide tolerance identification

The glufosinate-ammonium spraying time was 18 days after sowing, and the number of plants (plants containing no phytotoxicity) and plant height of each phytotoxicity grade were investigated at 1 week, 2 weeks and 4 weeks after spraying, respectively, and the results are shown in table 6 and fig. 3. Compared with lx03-2, all plants have 4-5 grade phytotoxicity 1 week after glufosinate-ammonium spraying, most of the plants die, the seedling rate is 0.00%, and the damage rate reaches 100.00%; LG11 can 100.00 percent of seedlings under different dosages, but a certain phytotoxicity is generated, the phytotoxicity rate reaches 4.57-4.69 percent, and the phytotoxicity symptom disappears after 2 weeks; the plant height performance of the transformant is further investigated, and the plant heights of the transformant under different dosages have no significant difference.

The field herbicide tolerance identification result shows that the transformant LG11 can tolerate glufosinate ammonium in the dose 4 times of the field recommended dose, and the agronomic characters such as plant height and the like are not obviously different from the control. In general, deletion of a partial sequence of a gene and a terminator affects gene expression, resulting in loss of gene function, but it is surprising from the identification result that the herbicide tolerance phenotype of the LG11 transformant is not affected and is significantly stronger than that of a control.

TABLE 6 tolerance of LG11 to glufosinate herbicides

Values are expressed as mean ± standard deviation of 3 biological replicates. 0 ×,1 ×,2 ×, 4 ×, respectively, represent multiples of the amount in the recommended dosage of glufosinate-ammonium spray. The same column data were compared using LSD analysis for significance analysis (α = 0.05). "-" indicates not investigated.

(2) Identification of insect resistance

Referring to part 1 of transgenic plants and products thereof, namely, environmental safety detection of insect-resistant corn, which is published by No. 953 of agricultural Ministry of rural areas-10-2007: pest resistance was performed. The inoculation method and the investigation method are carried out according to the technical specification for identifying the disease and insect resistance of the corn in NY/T1248.5. And (3) artificially inoculating insects in the heart and leaf stage of the corn respectively, and investigating insect pest conditions after inoculating the insects for 2-3 weeks.

Results of field resistance identification to corn borer (table 7 and fig. 4):

according to the heart-leaf stage survey result, compared with lx03-2, the leaf feeding grade is 7.73 +/-0.31, the insect pest grade is 7, and the resistance grade is a feeling, the material is a local insect-susceptible material, and the quality of the artificial inoculation can meet the resistance identification requirement; meanwhile, the leaf feeding grade of LG11 was 1.33 + -0.24, which is significantly lower than the control. The pest grade is 1, which indicates that the resistance grade is high resistance.

The ear period investigation result shows that the damage grade of the female ears in comparison with lx03-2 is 6.50 +/-0.71, the resistance grade is a feeling, the material is a local pest-susceptible material, and the quality of artificial inoculation can meet the resistance identification requirement; the damage grade of the female ear of LG11 is 1.40 +/-0.06, which is obviously lower than that of a control, and the resistance grade is high resistance.

TABLE 7 identification of the field resistance of LG11 to corn borer

Values are expressed as mean ± standard deviation of 3 biological replicates, and significance of differences from the same column of data was analyzed using the t-test method (α = 0.05). Inoculating 25 strains of the transformants at the heart-leaf stage, and contrasting 15 strains; the silking period transformants were inoculated with 68 strains of insects, and the control was 15 strains.

Results of field resistance identification for spodoptera frugiperda (table 8 and fig. 4):

according to the heart-leaf stage survey result, the comparison lx03-2 shows that the leaf feeding grade is 7.83 +/-0.24, the insect pest grade is 7, and the resistance grade is a feeling, so that the material is a local pest-susceptible material, and the quality of artificial inoculation can meet the resistance identification requirement; while LG11 had a leaf feeding rating of 1.09 ± 0.09, significantly lower than the control. The transformant is rated at 1 for insect pest and rated at high resistance.

The ear period investigation result shows that the damage grade of the female ears of the control lx03-2 is 6.20 +/-0.57, the resistance grade is the feeling, the material is a local pest-sensitive material, and the quality of the artificial inoculation can meet the resistance identification requirement; while LG11 has a leaf feeding rating of 1.41 ± 0.41, significantly lower than the control, and a resistance rating of high resistance.

TABLE 8 Spodoptera frugiperda field bioassay

Values are expressed as mean ± standard deviation of 3-4 biological replicates and significance of differences from the same column data was analyzed using the t-test method (α = 0.05). Inoculating 55 strains of the transformant in the heart-leaf period and controlling 17 strains; the transformants were inoculated with 22 strains of insects at the silking period, and 10 strains of insects were used as controls.

The result of field insect resistance identification shows that the transformant LD11 can achieve high resistance level to both ostrinia nubilalis and spodoptera littoralis, and the agronomic characters such as plant height and the like have no obvious difference from a control. Therefore, the LG11 transformant can be used for improving the glufosinate herbicide tolerance and insect-resistant characters of the corn, so that a new corn variety with insect resistance and herbicide tolerance is bred.

Example 2 transformation event LG11 exogenous sequence copy number analysis

The copy number of the exogenous sequence is determined by a Southern blot hybridization method. In the Southern hybridization detection, two restriction enzymes on a T-DNA region and not in a hybridization region are selected to digest genomic DNA, each insert copy in the genome is hybridized to display a single and specific band, and after the genomic DNA is digested by the restriction enzymes, a region to be detected is selected as a probe to carry out a Southern imprinting hybridization experiment.

Southern hybridization was performed by selecting genomic DNAs of positive control plasmid, control lx03-2 and LG11 transformant digested with restriction enzymes Hind III and BamHI, and selecting sequence fragments of m2cryAb-vip3A and bar of the target genes as probes, and the probe and cleavage site are schematically shown in FIG. 6. The specific sequences of the probe primers are shown in Table 9.

TABLE 9 probes used in Southern hybridization experiments

1: the unit bp.

The inserted copy number hybridization detection of the target gene m2cryAb-vip3A selects restriction enzymes Hind III and BamHI to digest positive control plasmids, negative control lx03-2 genomic DNA and LG11 transformant genomic DNA. After running gel and transferring membrane, the membrane was labeled with m2cryAb-vip3A gene probe, and the hybridization results are shown in FIG. 7A and FIG. 7B. The positions of the probes for the foreign gene m2cryAb-vip3A and the restriction sites of the restriction enzymes are shown in FIGS. 7C and 7D. From the hybridization results, the m2cryAb-vip3A gene of LG11 was inserted as a single copy into the maize genome.

The insertion copy number hybridization detection of the target gene bar selects restriction enzymes Hind III and BamHI to cut positive control plasmids, negative control lx03-2 genome DNA and LG11 transformant genome DNA. After running the gel and transferring the membrane, the membrane was labeled with a bar gene probe, and the hybridization results are shown in FIGS. 8A and 8B. The probe position of the target gene bar and the restriction enzyme cutting site of the restriction enzyme are shown in FIG. 8C and FIG. 8D. From the hybridization results, the bar gene of LG11 was inserted as a single copy into the maize genome.

Example 3 method for producing insect-resistant herbicide tolerant corn Using transformation event LG11

LG11 stable inbred lines can be used to assemble hybrid combinations. The insect-resistant herbicide-tolerant transgenic corn LG 11-Ludan 9088 is formed by hybridizing lx08-8 serving as a female parent and a transformation event LG11 serving as a male parent, and the insect-resistant and glufosinate-resistant traits of the transgenic corn are increased on the basis of keeping the original excellent traits of the Ludan 9088 (Table 10).

The method provided by the invention can be used for detecting the LG11 in the processes of parent improvement and hybridization combination and matching so as to provide a molecular auxiliary means for variety breeding, and can also be used for identifying whether the corn variety contains the LG11 transformation event. The following method is a specific example for identifying LG11.

Molecular testing was performed on the inbred line containing LG11 and the transgenic maize new hybrid LG 11-ludan 9088 to confirm that both materials contained the transformation event LG11. PCR primer pairs were designed based on gene sequence to determine the presence of transformation event LG11 by detecting the presence of two boundaries on the left and right of the transformation event.

One detection method comprises the following steps: detecting a specific boundary sequence in the LG11 maize plant of the transformation event by using a PCR method, wherein the used PCR primer pairs are respectively SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10 and SEQ ID NO. 11, the PCR reaction system comprises:

the reaction procedure is as follows:

94 ℃ for 5min; (94 ℃,30sec, 55 ℃,30sec, 72 ℃,90 sec) × 35 cycles; 72 ℃ for 5min;4 ℃ for 5min.

Taking the PCR product to detect in 1% (w/v) 1 XTAE agarose gel electrophoresis. The expected target bands, 286bp (SEQ ID NO: 6) and 1447bp (SEQ ID NO: 7), can be amplified in the LG11 transformation event, and the results are shown in FIG. 5. Moreover, the PCR method can track the existence of transformation events, thereby being applied to breeding work.

TABLE 10 insect and glufosinate tolerance identification

Values are expressed as mean ± standard deviation, and significance of differences from the same column of data was analyzed using the t-test method (α = 0.05).

Example 4 detection method of transformation event LG11

A new variety can be developed from transgenic corn event LG11 and produce agricultural or commercial products. The agricultural or commercial product is expected to contain a nucleotide sequence capable of diagnosing the presence of transgenic corn event LG11 material in the agricultural or commercial product if a sufficient amount is detected in the agricultural or commercial product. Such agricultural or commercial products include, but are not limited to, corn oil, corn flour, corn paste, starch, and other flavorings or any other food product intended for consumption by an animal as a food source, or cosmetics, industrial products, and the like. A probe or primer pair based nucleic acid detection method and/or kit can be developed to detect a transgenic corn event LG11 nucleotide sequence such as shown in SEQ ID No. 1 or SEQ ID No. 2 in a biological sample, wherein the probe sequence or primer amplification sequence is selected from the sequences shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, and SEQ ID No. 7 to diagnose the presence of transgenic corn event LG11.

In conclusion, the transgenic corn event LG11 can improve the insect resistance of plants and has higher tolerance to glufosinate herbicides, and can be used for improving other corn germplasms and creating a novel corn hybrid combination. The detection method can accurately and quickly identify whether the biological sample contains the DNA molecule of the transgenic corn event LG11.

Finally, it should be noted that the above embodiments are only intended to illustrate the technical solutions of the present invention and not to limit the same, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A nucleic acid molecule comprising any one of:

i) Comprises the sequence shown in SEQ ID NO. 1 and/or SEQ ID NO. 2, or the reverse complementary sequence thereof;

ii) comprises the sequence shown in SEQ ID NO 3 and/or SEQ ID NO 4, or the reverse complement thereof;

iii) Comprises the sequence shown in SEQ ID NO. 6 and/or SEQ ID NO. 7, or the reverse complementary sequence thereof;

iv) comprises the sequence shown in SEQ ID NO. 5, or the reverse complement thereof.

2. A probe for detecting a maize transformation event comprising the sequence shown as SEQ ID NO 1 or 2 or 3 or 4 or 6 or 7 or a fragment or variant or reverse complement thereof.

3. A primer pair for detecting a corn transformation event, wherein an amplification product of the primer pair comprises the sequence of claim 2;

optionally, the primer pair is a sequence shown as SEQ ID NO. 8 and SEQ ID NO. 9; or the sequences shown in SEQ ID NO 10 and SEQ ID NO 11.

4. A kit or microarray for detecting a corn transformation event comprising the probe of claim 2 and/or the primer pair of claim 3.

5. A method for detecting a corn transformation event comprising detecting the presence or absence of said transformation event in a test sample using any of:

i) The probe of claim 2;

ii) the primer pair of claim 3;

iii) The probe of claim 2 and the primer pair of claim 3;

iv) the kit or microarray of claim 4.

6. A method of breeding maize, comprising the steps of:

1) Obtaining maize comprising the nucleic acid molecule of claim 1;

2) Subjecting the corn obtained in step 1) to pollen culture, unfertilized embryo culture, doubling culture, cell culture, tissue culture, selfing or crossing or a combination thereof to obtain a corn plant, seed, plant cell, progeny plant or plant part; and optionally (c) a second step of,

3) Evaluating the progeny plant obtained in step 2) for a pest resistance trait and/or for herbicide resistance identification and detecting the presence or absence of the transformation event therein using the method of claim 5.

7. A product made from the corn plant, seed, plant cell, progeny plant or plant part obtained by the method of claim 6, including a food, feed or industrial material.

8. A method of protecting a corn plant from damage caused by a herbicide comprising applying to a field in which is grown at least one transgenic corn plant comprising in its genome, in order, SEQ ID No. 1, SEQ ID No. 5, nucleic acid sequences 281 to 7424, and SEQ ID No. 2, or comprising in its genome SEQ ID No. 5, an effective amount of a glufosinate herbicide; the transgenic corn plants have tolerance to glufosinate herbicides.

9. A method of protecting a corn plant from insect infestation comprising providing at least one transgenic corn plant cell in the diet of a target insect, said transgenic corn plant cell comprising in its genome, in order, SEQ ID No. 1, SEQ ID No. 5, nucleic acid sequence 281 to 7424, and SEQ ID No. 2, or said transgenic corn plant cell comprising in its genome SEQ ID No. 5; target insects that feed on the transgenic corn plant cell are inhibited from further feeding on the corn plant.