CN116200529A - Nucleic acid sequence of corn transformation event LG11 and detection method thereof - Google Patents

Abstract

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

Description

Nucleic acid sequence and detection method of maize transformation event LG11

technical field

The invention relates to the field of plant biotechnology. Specifically, it relates to a nucleic acid sequence and detection method of a maize transformation event LG11, especially a transgenic maize event LG11 that is insect-resistant and tolerant to the application of glufosinate-ammonium herbicides and is used to detect whether a specific transgene is contained in a biological sample Nucleic acid sequence of maize event LG11 and its detection method.

Background technique

The whole growth period of corn will be affected by various insect pests, among which corn borer is the main pest in corn production, which can cause about 10% yield reduction every year. In recent years, with global warming, the harm of corn borer has been further aggravated, and at the same time, large-scale damage by armyworms has also occurred from time to time. Spodoptera frugiperda is a new type of agricultural pest that invaded my country from Yunnan at the end of 2018. The number of host plants has reached 19, and the occurrence area of corn accounts for 98.1% of the total crop area.

The Cry1Ab protein was first developed by Monsanto into insect-resistant maize MON810 to increase resistance to pests of the Ositrinia species such as the Asian Corn Borer (ACB). Vip3Aa was first applied by Syngenta to the transgenic maize MIR162, and it has a significant control effect on Agrotis ypsilon, Helicoverpa armigera, Loxagrotis albicosta and Spodoptera frugiperda. M2cryAb-vip3A protein is synthesized by combining the main domains of Cry1Ab and Vip3Aa proteins. The main advantages of the M2cryAb-vip3A protein are: (1) expanding the insect resistance spectrum, and containing the functional domains of the two proteins at the same time, it is expected to be resistant to corn borer, cotton bollworm, armyworm, and fall armyworm; (2) It is beneficial to the management of target pest resistance. A large number of studies have shown that Vip3 has high insecticidal activity against a variety of Lepidoptera pests, expanding the insecticidal spectrum of Bt protein. At the same time, there are differences in the insecticidal mechanism of Vip3 toxin and Cry toxin. There is no homology in evolution, and the chance of pests developing cross-resistance to these two toxins is very small; (3) reduce the level of mycotoxins in corn ears and grains, and improve the quality of corn grains; (4) the protein sequence does not contain Allergen sequence, while maintaining high-efficiency insecticidal activity, has biosafety.

Field weeds compete with crops for water, fertilizer, light energy and growth space, directly affecting crop yield and quality. At the same time, many weeds are intermediate hosts of crop pathogenic bacteria and pests, and are one of the important biological limiting factors for crop production. Therefore, effective control of field weeds is one of the important measures to promote grain production. In addition, the scale and mechanization of agricultural planting is a foreseeable trend, which makes the traditional manual weeding method unrealistic. The promotion and use of herbicides can greatly reduce the labor and labor intensity of cotton field management. Developing new herbicide products with high efficiency, low toxicity and no residue is expensive, time-consuming and difficult. Breeding maize resistant to herbicides through transgenic technology can overcome this problem. Spraying 1-2 times during the corn growth period can effectively solve the weed problem, reducing the amount of herbicides and input costs. Therefore, herbicide-tolerant transgenic corn has very broad application value and market potential.

The invention connects the insect-resistant gene expression box and the herbicide-resistant expression box in series, so that it can be expressed efficiently in the transgenic corn, and has both insect-resistant and herbicide-resistant traits, and further enhances the application and economic value of the product.

The expression of exogenous genes in plants is known to be influenced by their chromosomal location, possibly due to the proximity of chromatin structure (such as heterochromatin) or transcriptional regulatory elements (such as enhancers) to the integration site. For this reason, it is usually necessary to screen a large number of events before it is possible to identify commercially viable events (ie, events in which the introduced target gene is optimally expressed). For example, it has been observed in plants and other organisms that the amount of expression of an introduced gene can vary considerably between events; there may also be differences in the spatial or temporal pattern of expression, such as the relative expression of the transgene between different plant tissues There are differences in which the actual expression pattern may not be consistent with the expression pattern expected based on the transcriptional regulatory elements in the introduced gene construct, resulting in differences in the trait expression of the transformation event. Therefore, it is often necessary to generate hundreds to thousands of different events and to screen these events for a single event with the expected amount and pattern of transgene expression for commercialization purposes. Events with the expected amount and pattern of transgene expression can be used to introgress the transgene into other genetic backgrounds by sexual outcrossing using conventional breeding methods. The progeny produced by this crossing maintain the transgene expression characteristics of the original transformation event. Applying this pattern of strategies can ensure reliable gene expression in many varieties that are well adapted to local growing conditions. Therefore, more transformation events need to be characterized and screened to obtain excellent transformation events with excellent comprehensive traits and commercial prospects.

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 the gene of interest. In addition, methods to detect specific events will help to comply with regulations, such as the need for formal approval and labeling of food derived from recombinant crops before being placed on the market. Detection of the presence of the transgene is possible by any of the well-known polynucleotide detection methods, such as polymerase chain reaction (PCR). These assays usually focus on commonly used genetic elements such as promoters, terminators, marker genes, etc. Therefore, unless the sequence of the chromosomal DNA adjacent to the inserted transgenic DNA ("flanking DNA") is known, this approach cannot be used to distinguish between different events, especially those produced with the same DNA construct. event. Therefore, transgene-specific events are now commonly identified by PCR using a pair of primers spanning the junction of the inserted transgene and flanking DNA, specifically a first primer containing the flanking sequence and a second primer containing the inserted sequence.

Contents of the invention

The purpose of the present invention is to provide a maize transformation event with excellent insect resistance and herbicide resistance and agronomic performance, as well as a nucleic acid sequence for detecting maize LG11 and a detection method thereof. The transgenic corn event LG11 has excellent insect resistance and good tolerance to glufosinate-ammonium herbicide, and the detection method can accurately and quickly identify whether the DNA molecule of the specific transgenic corn event LG11 is contained in the biological sample.

In order to achieve the above purpose, the present invention uses the pCAMBIA3300+m2cryAb-vip3A expression vector to transform maize HiIIIB through the method mediated by Agrobacterium, and obtains more than 400 positive transformants. After molecular detection, maize inbred line Chang 7-2 was backcrossed as a recurrent parent to obtain BC ₅ F _2nd generation transgenic maize seeds LG01-LG20. Through the identification of insect resistance and herbicide resistance traits, it was found that the transformation event LG11 is a transformant with herbicide resistance, excellent insect resistance and the best agronomic traits, which can be used to improve the insect resistance and herbicide tolerance traits of maize.

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 its reverse complementary sequence.

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

Further, the nucleic acid sequence comprises the sequence shown in SEQ ID NO:6 and/or SEQ ID NO:7, or its reverse complementary sequence.

Furthermore, the nucleic acid sequence comprises the sequence shown in SEQ ID NO: 5 or its reverse complementary sequence.

The present invention also provides a probe for detecting maize transformation events, characterized in that it comprises 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 The sequence shown in SEQ ID NO: 7 or a fragment thereof or a variant thereof or a reverse complement thereof.

The present invention also provides a pair of primers for detecting maize transformation events, characterized in that, the amplified product of the pair of primers comprises SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or the sequence shown in SEQ ID NO: 6 or SEQ ID NO: 7 or a fragment or variant or reverse complementary sequence thereof.

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

The present invention also provides a kit or microarray for detecting maize transformation events, which is characterized by comprising the above-mentioned probe and/or primer pair.

The present invention also provides a method for detecting maize transformation events, which is characterized in that it includes using the above-mentioned probe or the above-mentioned primer pair or the above-mentioned probe and primer pair or the above-mentioned kit or microarray to detect whether The transformation event is present.

The present invention also provides a method for breeding corn, characterized in that the method comprises the following steps:

1) obtaining corn comprising the above-mentioned nucleic acid molecules;

2) Obtain corn plants, seeds, plant cells, progeny plants or plant parts from the corn obtained in step 1) through pollen culture, unfertilized embryo culture, double culture, cell culture, tissue culture, selfing or hybridization or a combination of the above ; and optionally,

3) Identify insect resistance traits and/or herbicide resistance on the progeny plants obtained in step 2), and use the above-mentioned method to detect whether the transformation event exists.

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

The SEQ ID NO: 1 is a sequence of 22 nucleotides in length near the insertion junction at the 5' end of the insertion sequence in the transgenic maize event LG11, and the SEQ ID NO: 1 spans the maize insertion site The genomic DNA sequence of the left flank and the DNA sequence of the 5' end of the left border of the inserted sequence, including the SEQ ID NO: 1 or its reverse complement sequence, can be identified as the presence of the transgenic maize event LG11. The SEQ ID NO: 2 is a sequence of 22 nucleotides in length near the insertion junction at the 3' end of the insertion sequence in the transgenic maize event LG11, and the SEQ ID NO: 2 spans the right border of the insertion sequence 3 The DNA sequence at the 'end and the right flank genomic DNA sequence of the maize insertion site, comprising said SEQ ID NO: 2 or its reverse complement sequence, can be identified as the presence of the transgenic maize event LG11.

In the present invention, the nucleic acid sequence may be at least 11 or more continuous polynucleotides (first nucleic acid sequence) of any part of the transgene insertion sequence in the SEQ ID NO: 3 or its reverse complementary sequence, or It is at least 11 or more contiguous polynucleotides (second nucleic acid sequence) of any part of the 5' left flank maize genomic DNA region in said SEQ ID NO: 3 or its reverse complementary sequence. Said nucleic acid sequence may further be homologous or reverse complementary to a portion of said SEQ ID NO:3 comprising the entirety of said SEQ ID NO:1 or SEQ ID NO:6. When a first nucleic acid sequence and a 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 amplified product. A transgenic maize event 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 or SEQ ID NO: 3 or SEQ ID NO: 6 or its reverse complement The presence of LG11 or its descendants.

The SEQ ID NO: 3 is a sequence of 517 nucleotides in length near the insertion junction at the 5' end of the insertion sequence in the transgenic maize event LG11, and the SEQ ID NO: 3 consists of 280 nucleotides The 5' terminal DNA sequence (SEQ ID NO: nucleotide 1-280 of the maize left flank genomic DNA sequence (SEQ ID NO:3 nucleotide 1-280) and the first expression cassette of the glufosinate-ammonium-resistant gene of 237 nucleotides 3 nucleotides 281-517), comprising said SEQ ID NO: 3 or its reverse complementary sequence can be identified as the presence of transgenic maize event LG11.

The nucleic acid sequence can be at least 11 or more continuous polynucleotides (the third nucleic acid sequence) of any part of the transgene insertion sequence in the SEQ ID NO: 4 or its reverse complementary sequence, or the SEQ ID NO: At least 11 or more contiguous polynucleotides (fourth nucleic acid sequence) of any part of the 3' right flank maize genomic DNA region in ID NO: 4 or its reverse complement. Said nucleic acid sequence may further be homologous or reverse complementary to a portion of said SEQ ID NO:4 comprising the entirety of said SEQ ID NO:2 or SEQ ID NO:7. When a third nucleic acid sequence and a 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 amplified product. Transgenic maize can be diagnosed when the amplification product produced in the DNA amplification method using the DNA primer pair is an amplification product comprising SEQ ID NO: 2 or SEQ ID NO: 4 or SEQ ID NO: 7 or its reverse complement The existence of event LG11 or its descendants.

The SEQ ID NO: 4 is a sequence of 1631 nucleotides in length near the insertion junction at the 3' end of the insertion sequence in the transgenic maize event LG11, and the SEQ ID NO: 4 consists of 575 nucleotides The 3' end DNA sequence (nucleotide 1-575 of the nucleotide 1-575 of SEQ ID NO:4), the pCAMBIA3300+m2cryAb-vip3A construct right border DNA sequence of 736 nucleotides of the second expression cassette of the anti-insect gene (SEQ ID NO: 4 nucleotides 576-1311) and the maize integration site right flank genomic DNA sequence (SEQ ID NO: 4 nucleotides 1312-1631) of 320 nucleotides, comprising said SEQ ID NO: 4 or its reverse complementary sequence can be identified as the presence of transgenic maize event LG11.

The SEQ ID NO:5 is a sequence of 7744 nucleotides in length characterizing the transgenic maize event LG11, and its specific genome and genetic elements are shown in Table 1. The presence of the transgenic maize event LG11 can be identified by the inclusion of said SEQ ID NO: 5 or its reverse complement.

Table 1 The genome and genetic elements contained in SEQ ID NO: 5

1: The unit is bp.

As is well known to those skilled in the art, the first and second nucleic acid sequences or the third and fourth nucleic acid sequences do not have to be composed only of DNA, but may also include RNA, a mixture of DNA and RNA, or DNA, RNA or other sequences that do not serve as one or Combinations of nucleotides or analogs thereof for multiple polymerase templates. In addition, the probes or primers of the present invention should be at least about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 consecutive nucleotides in length, which can be selected from 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 , a nucleotide set forth in SEQ ID NO: 10 or SEQ ID NO: 11. When selected from the nucleotides shown in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, the primer can be at least about 21 to 21 in length. About 50 or more contiguous nucleotides.

The present invention also provides a method of protecting corn plants from damage caused by herbicides, comprising applying an effective dose of glufosinate-ammonium herbicide to a field in which at least one transgenic corn plant is planted, said The transgenic corn plant comprises SEQ ID NO:1, the 281-7424 nucleotide sequence of SEQ ID NO:5 and SEQ ID NO:2 in its genome, or the genome of the transgenic corn plant comprises SEQ ID NO:5; The transgenic corn plants are tolerant to glufosinate-ammonium herbicide.

The present invention also provides a method of protecting maize plants from insect attack, characterized in that it comprises providing in the diet of the target insect at least one transgenic maize plant cell, said transgenic maize plant cell comprising sequentially in its genome SEQ ID NO:1, the 281-7424th nucleic acid sequence of SEQ ID NO:5 and SEQ ID NO:2, or the genome of the transgenic maize plant cell comprises SEQ ID NO:5; the target of ingesting the transgenic maize plant cell Insects are inhibited from further feeding on the corn plants.

In the nucleic acid sequence and its detection method for detecting corn plants in the present invention, the following definitions and methods can better define the present invention and guide those of ordinary skill in the art to implement the present invention, unless otherwise specified, according to the ordinary skills in the art common usage by people to understand the term.

The term "maize" refers to Zea mays and includes all plant species that can be crossed with maize, including wild maize species.

The "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 callus, plant clumps and whole plants in plants or plant parts Cells, said plant parts such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, stalks, roots, root tips, anthers, and the like. Parts of transgenic plants that are understood to be within the scope of the present invention include, but are not limited to, plant cells, protoplasts, tissues, callus, embryos, as well as flowers, stems, fruits, leaves and roots, the above plant parts being derived from the A transgenic plant transformed with a DNA molecule and thus consisting at least in part of a transgenic cell, or its progeny.

The term "gene" refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences before the coding sequence (5' non-coding sequence) and regulatory sequences after the coding sequence (3' non-coding sequence). "Native gene" refers to a gene that is found in nature with its own regulatory sequences. "Chimeric gene" refers to any gene that is not a native gene, comprising regulatory and coding sequences not found in nature. "Endogenous gene" refers to a native gene in its natural location in the genome of an organism. "Exogenous gene" is a foreign gene that exists in the genome of an organism and does not exist before, and also refers to a gene that is introduced into a recipient cell through a transgenic procedure. Foreign genes may comprise native or chimeric genes 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 the "insertion site" or "target site".

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

Transformation procedures that result in random integration of foreign DNA will result in transformation events that contain different flanking regions that each transformation event specifically contains. When recombinant DNA is introduced into plants by conventional crossing, its flanking regions are usually not altered. Transformation events will also contain unique junctions between the heterologous insert DNA and segments of genomic DNA or between two segments of genomic DNA or between two segments of heterologous DNA. A "junction" is the point at which two specific DNA segments join. For example, junctions exist where insert DNA joins flanking DNA. Junctions also exist in transformed organisms where two segments of DNA join together in a manner modified from that found in natural organisms. "Junction DNA" refers to DNA comprising a junction.

The present invention provides a transgenic maize event designated as LG11 and progeny thereof, said transgenic maize event LG11 being maize LG11, including plants and seeds of transgenic maize event LG11 and plant cells or regenerable parts thereof, said transgenic maize event LG11 Plant parts of 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 wool, cotton cloth, and leftover corn Biomass in crop fields.

The transgenic corn event LG11 of the present invention comprises a DNA construct that, when expressed in plant cells, acquires insect resistance and/or tolerance to glufosinate-ammonium herbicides. The DNA construct comprises an expression cassette comprising a suitable promoter for expression in plants and a suitable polyadenylation signal sequence, the promoter is operably linked to the m2cryAb- The vip3A gene, the nucleic acid sequence of the M2CryAb-VIP3A protein can improve corn insect resistance. The DNA construct comprises a further expression cassette comprising a suitable promoter for expression in plants and a suitable polyadenylation signal sequence, said promoter being operably linked to a protein encoding phosphinothricin acetyl Gene bar of transferase (PAT), the nucleic acid sequence of the PAT protein has tolerance to glufosinate-ammonium herbicide. Further, the promoter can be a suitable promoter isolated from a plant, including a constitutive, inducible and/or tissue-specific promoter, and the suitable promoter includes, but is not limited to, cauliflower mosaic virus (CaMV) 35S Promoter, Scrophulariaceae mosaic virus (FMV) 35S promoter, Ubiquitin promoter, Actin promoter, Agrobacterium tumefaciens nopaline synthase (NOS) promoter, Octopine synthase (OCS) promoter, Cestrum yellow leaf curl virus promoter, potato tuber storage protein (Patatin) promoter, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) promoter, glutathione sulfur transferase (GST) promoter, E9 promoter, GOS promoter, alcA/alcR promoter, Agrobacterium rhizogenes (Agrobacterium rhizogenes) RolD promoter and Arabidopsis (Arabidopsis thaliana) Suc2 promoter. The polyadenylation signal sequence may be a suitable polyadenylation signal sequence that functions in plants, and the suitable polyadenylation signal sequence includes, but is not limited to, derived from Agrobacterium tumefaciens The polyadenylation signal sequence of the nopaline synthase (NOS) gene, the 35S terminator from the cauliflower mosaic virus (CaMV), the polyadenylation signal sequence and the source of the protease inhibitor Ⅱ (PINⅡ) gene The polyadenylation signal sequence of the α-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 can enhance the expression level of the gene, and the enhancer includes, but is not limited to, tobacco etch virus (TEV) translation activator, CaMV35S enhancer and FMV35S enhancer. The signal peptide/transit peptide can guide the EPSPS protein to be transported to a specific organelle or compartment outside the cell or within the cell, for example, using the sequence encoding the chloroplast transit peptide to target the chloroplast, or using the 'KDEL' retention sequence to target the endoplasmic reticulum.

The "glufosinate-ammonium" refers to a non-selective, broad-spectrum, high-efficiency, low-toxicity organophosphate herbicide, which strongly inhibits the activity of the amino acid biosynthetic enzyme-glutamine synthetase (Glutamine Synthetase, GS) of bacteria and plants . GS plays an important role in plant ammonia assimilation and regulation of ammonia metabolism. It is the only detoxification enzyme in plants, which can relieve the toxicity of ammonia released from nitrate reduction, amino acid degradation and photorespiration. Treatment with "glufosinate-ammonium herbicide" means treatment with any herbicide formulation containing glufosinate-ammonium. The selection of the application rate of a glufosinate-ammonium formulation to achieve a biologically effective dose is within the skill of the average agronomist. Treatment of a field containing plant material derived from transgenic maize event LG11 with any of the herbicide formulations containing glufosinate will control weed growth in said field without affecting plant material derived from transgenic maize event LG11 growth or insect resistance.

The DNA construct is introduced into plants using transformation methods including, but not limited to, Agrobacterium-mediated transformation, biolistic transformation, and pollen tube passage 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, ie the T-DNA region. The vector is transformed into Agrobacterium cells, which are then used to infect plant tissues, and the T-DNA region of the vector containing foreign DNA is inserted into the plant genome. Following transformation, transgenic plants must be regenerated from the transformed plant tissue, and appropriate markers used to select for progeny bearing the foreign DNA.

A DNA construct is an interconnected assembly of DNA molecules that provides one or more expression cassettes. The DNA construct is preferably a plasmid capable of self-replicating in bacterial cells and containing various restriction endonuclease sites for introduction to provide a functional genetic element, i.e. a promoter , introns, leader sequences, coding sequences, 3' terminator regions and other sequences of DNA molecules. The expression cassette contained in the DNA construct includes the genetic elements necessary to provide transcription of the messenger RNA and can be designed for expression in prokaryotic or eukaryotic cells. The expression cassettes of the invention are designed for expression most preferably 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, inserted transgenically into the plant genome to produce a plant population, which is regenerated , and select specific plants with features inserted into specific genomic loci. The term "event" refers to the original transformation event comprising heterologous DNA and the progeny of that transformation event. The term "event" also refers to the progeny of a sexual cross between a transformation event and an individual of another breed that contains heterologous DNA, even after repeated backcrosses with the backcross parent, the inserted DNA and flanking Genomic DNA is also present at the same chromosomal location in hybrid offspring. The term "event" also refers to a DNA sequence from an original transformation event comprising an insert DNA and flanking genomic sequences in close proximity to the insert DNA that is expected to be transferred to progeny derived from The parental line (eg, the original transformation event and its progeny resulting from selfing) is sexually crossed with a parental line that does not contain the inserted DNA, and the progeny receive the inserted DNA containing the gene of interest.

"Recombinant" in the present invention refers to a form of DNA and/or protein and/or organism that is not normally found in nature and thus produced by human intervention. Such human intervention can produce recombinant DNA molecules and/or recombinant plants. Said "recombinant DNA molecule" is obtained by the artificial combination of two otherwise separate sequence segments, such as by chemical synthesis or by manipulation of separate nucleic acid segments by genetic engineering techniques. The techniques for performing nucleic acid manipulations are well known.

The term "transgenic" includes any cell, cell line, callus, tissue, plant part or plant, the genotype of which is altered by the presence of heterologous nucleic acid, said "transgenic" including transgenics originally so altered as well as those derived from The offspring individuals produced by the original transgenic body through sexual crossing or asexual reproduction. In the present invention, the term "transgenic" does not include alterations of the genome (chromosomal or extrachromosomal) by conventional plant breeding methods or naturally occurring events such as random heterozygous fertilization, non-recombinant viral infection, non-recombinant Bacterial transformation, non-recombinant transposition, or spontaneous mutation.

"Heterologous" in the context of the present invention means that a first molecule is not normally found in combination with a second molecule in nature. For example, a molecule can be derived from a first species and inserted into the genome of a second species. This molecule is thus heterologous to the host and is artificially introduced into the genome of the host cell.

Transgenic maize event LG11 with insect resistance properties and tolerance to the glufosinate herbicide was developed by first sexually crossing a first parental maize plant with a second parental maize plant, resulting in a diverse first Progeny plants, the first parent corn plant consisting of corn plants bred from the transgenic corn event LG11 and its progeny by utilizing the insect-resistant and glufosinate-ammonium herbicide of the present invention Transformed with a tolerant expression cassette, the second parent maize plant lacks the insect resistance trait or is tolerant to glufosinate-ammonium herbicide; progeny plants are then selected for glufosinate-ammonium-tolerant herbicide , corn plants can be bred to be tolerant to glufosinate-ammonium herbicide. These steps may further comprise backcrossing the insect-resistant and glufosinate-tolerant progeny plants to a second parent corn plant or a third parent corn plant, either by application of the herbicide glufosinate or by a molecule associated with the trait Identification of markers (such as DNA molecules containing the junction sites identified at the 5' and 3' ends of the insert in transgenic maize event LG11) to select progeny for insect resistance and glufosinate herbicide Tolerant corn plants.

It should also be understood that two different transgenic plants can also be crossed to produce progeny that contain two independent, segregated additions of the exogenous gene. Selfing of suitable progeny can result in progeny plants that are homozygous for both added exogenous genes. Backcrossing to parental plants and outcrossing to non-transgenic plants are also contemplated as previously described, as is vegetative propagation.

The term "probe" is an isolated nucleic acid molecule to which is bound a conventional detectable label or reporter molecule, eg, a radioisotope, ligand, chemiluminescent agent or enzyme. Such probes are 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 the transgenic maize event LG11, whether the genomic DNA is from the transgenic maize event LG11 or the seed or is derived from the transgene Plants or seeds or extracts of maize event LG11. The 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, by nucleic acid hybridization, anneals to a complementary target DNA strand, forms a hybrid between the primer and target DNA strand, and then reacts with a polymerase (eg, DNA polymerase) Bottom, stretches along the target DNA strand. The primer pairs of the present invention relate to their use in the amplification of target nucleic acid sequences, for example, by polymerase chain reaction (PCR) or other conventional nucleic acid amplification methods. Primers are generally 11 polynucleotides or more in length, preferably 18 polynucleotides or more, more preferably 24 polynucleotides or more, most preferably 30 polynucleotides or more many. Such primers hybridize specifically to the target sequence under highly stringent hybridization conditions. Although primers that are different from the target DNA sequence and maintain hybridization ability to the target DNA sequence can be designed by conventional methods, preferably, the primers in the present invention have complete DNA sequence identity with the continuous nucleic acid of the target sequence.

As used herein, "kit" or "microarray" refers to a reagent set or chip used for the purpose of identification and/or detection of maize transformation events in a biological sample. For the purpose of quality control (such as the purity of a seed batch), the detection of events in or comprising or derived from plant material, such as but not limited to food or feed products, the detection of events in plant material, and its The components can be specifically adjusted.

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

Primers and probes of the invention hybridize to target DNA sequences under stringent conditions. Any conventional amplification method can be used to identify the presence of DNA derived from transgenic maize 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 are 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. A nucleic acid molecule is said to be the "complement" of another nucleic acid molecule if two nucleic acid molecules exhibit perfect complementarity. As used herein, two nucleic acid molecules are said to exhibit "perfect complementarity" when every nucleotide of one nucleic acid molecule is complementary to the 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 be "complementary" if they are capable of hybridizing to each other with sufficient stability such that they anneal and bind to each other under conventional "high stringency" conditions. Deviations from perfect complementarity are permissible as long as the deviation does not completely prevent the two molecules from forming a double-stranded structure. In order for a nucleic acid molecule to serve as a primer or probe, it only needs to be sufficiently complementary in sequence to form a stable double-stranded structure under 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 to a matching complementary strand of another nucleic acid molecule under highly stringent conditions. Suitable stringent conditions to promote DNA hybridization, for example, treatment with 6.0× sodium chloride/sodium citrate (SSC) at about 45° C., followed by washing with 2.0× SSC at 50° C., are known to those skilled in the art. is well known. For example, the salt concentration in the washing step can be selected from about 2.0×SSC, 50°C for low stringency conditions to about 0.2×SSC, 50°C for high stringency conditions. In addition, the temperature conditions in the washing step can be increased from about 22°C at room temperature for low stringency conditions to about 65°C for high stringency conditions. Both the temperature condition and the salt concentration can be changed, or one can be kept constant while the other variable is changed. Preferably, a nucleic acid molecule of the present invention can be combined with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO: 4. One or more nucleic acid molecules in SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7 or their complementary sequences, or any fragment of the above sequences specifically hybridize. More preferably, a nucleic acid molecule of the present invention is combined with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 under highly stringent conditions Specific hybridization occurs with one or more nucleic acid molecules in SEQ ID NO: 7 or its complementary sequence, or any fragment of the above sequence. In the present invention, preferred marker nucleic acid molecule has 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 its complementary sequence, or Any fragment of the above sequence.

Another preferred marker nucleic acid molecule of the present invention and 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 its complementary sequence , or any fragment of the above sequence having 80% to 100% or 90% to 100% sequence identity. 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 the progeny of genetic crosses . The hybridization of the probe to the target DNA molecule can be detected by any method known to those skilled in the art, including but not limited to fluorescent labeling, radioactive labeling, antibody-based labeling and chemiluminescent labeling.

With respect to the amplification of a target nucleic acid sequence using specific amplification primers (for example, by PCR), "stringent conditions" refer to conditions that allow only the primers to hybridize to the target nucleic acid sequence in a DNA thermal amplification reaction, with the same The primer corresponding to the wild-type sequence (or its complementary sequence) of the target nucleic acid sequence is capable of binding to the target nucleic acid sequence, and preferably produces a unique amplification product, the amplification product being an amplicon.

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

As used herein, "amplified DNA" or "amplicon" refers to the product of nucleic acid amplification of a target nucleic acid sequence that is part of a nucleic acid template. For example, in order to determine whether corn plants were sexually crossed containing the transgenic corn event LG11 of the present invention, or whether a corn sample collected from a field contained the transgenic corn event LG11, or whether corn extracts, such as cotton wool, cottonseed oil, contained the transgenic corn Event LG11, DNA extracted from maize plant tissue samples or extracts can be subjected to nucleic acid amplification methods using primer pairs to generate amplicons that are diagnostic for the presence of DNA from transgenic maize event LG11. The pair of primers includes a first primer derived from a flanking sequence adjacent to the 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 maize event LG11.

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 and fifty nucleotides base pairs, most preferably plus about four hundred and fifty nucleotide base pairs or more.

Alternatively, primer pairs can be derived from flanking genomic sequences flanking the insert DNA to generate amplicons that include the entire insert nucleotide sequence. One of the primer pairs derived from the plant genomic sequence can be located at a distance from the insert DNA sequence, which distance can 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 DNA thermal amplification reactions.

Nucleic acid amplification reaction can be achieved by any nucleic acid amplification reaction method known in the art, including 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. Inserted exogenous DNA sequences and flanking DNA sequences from transgenic maize event LG11 can be amplified from the genome of transgenic maize event LG11 using the primer sequences provided, followed by standard analysis of PCR amplicons or cloned DNA. DNA sequencing.

DNA detection kits based on DNA amplification methods contain DNA primer molecules that, under appropriate reaction conditions, specifically hybridize to target DNA and amplify diagnostic amplicons. Kits may provide agarose gel-based detection methods or a number of methods known in the art to detect diagnostic amplicons. DNA primers that are homologous or reverse complementary to any part of the maize genomic region of SEQ ID NO:3 or SEQ ID NO:4 and that are homologous or reverse complementary to any part of the transgene insertion region of SEQ ID NO:5 The test kit is provided by the present invention. In particular, a primer pair identified as useful in DNA amplification methods was SEQ ID NO: 8 and SEQ ID NO: 9, which amplified a diagnostic amplicon homologous to a portion of the 5' transgene/genomic region of transgenic maize event LG11. An amplicon, wherein the amplicon comprises SEQ ID NO:1. Primer pairs identified as useful in DNA amplification methods 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 maize event LG11 , wherein the amplicon comprises SEQ ID NO:2. Other DNA molecules used as DNA primers may be selected from SEQ ID NO:5.

Amplicons generated by these methods can be detected by a variety of techniques. One such method is GeneticBit Analysis, which designs a DNA oligonucleotide strand spanning the insert DNA sequence and the adjacent flanking genomic DNA sequence. The oligonucleotide strands are immobilized in the microwells of a microplate, and after PCR amplification of the region of interest (using one primer each within the insert sequence and adjacent flanking genomic sequences), the single-stranded PCR product Can hybridize to immobilized oligonucleotide strands and serve as templates for single-base extension reactions using a DNA polymerase and ddNTPs specifically labeled for the next expected base. Results can be obtained by fluorescent or ELISA-like methods. The signal represents the presence of the insertion/flanking sequence, which indicates that the amplification, hybridization and single base extension reactions were successful.

Another method is Pyrosequencing (pyrosequencing) technology. This method designs an oligonucleotide strand that spans the insert DNA sequence and the adjacent genomic DNA binding site. This oligonucleotide strand is hybridized to a single-stranded PCR product of the region of interest (using one primer each within the insert and adjacent flanking genomic sequences), followed by DNA polymerase, ATP, sulfurylase, luciferin The enzyme, apyrase, adenosine-5'-phosphosulfate and luciferin are incubated together. Add dNTPs respectively, and measure the light signal generated. The light signal represents the presence of the insertion/flanking sequence, which indicates that the amplification, hybridization, and single-base or multi-base extension reactions were successful.

Fluorescence polarization is also a method that can be used to detect amplicons of the present invention (Chen X, Levine L, and Kwok PY. Fluorescence polarization in homogeneous nucleic acid analysis [J]. Genome Res, 1999, 9 (5): 492 -8.). Using this method requires the design of an oligonucleotide strand that spans the insert DNA sequence and the adjacent genomic DNA binding site. The oligonucleotide strand is hybridized to a single-stranded PCR product of the region of interest (using one primer each within the insert and adjacent flanking genomic sequences) with DNA polymerase and a fluorescently labeled ddNTP Incubation. Single base extensions result in the insertion of ddNTPs. This insertion can be measured as a change in polarization using a fluorometer. A change in polarization represents the presence of insertion/flanking sequences, which indicates that the amplification, hybridization and single base extension reactions were successful.

Taqman is described as a method for the detection and quantification of the presence of DNA sequences, which is described in detail in the instructions for use provided by the manufacturer. As a brief example, design a FRET oligonucleotide probe spanning the insertion DNA sequence and the adjacent genomic flanking junction site as follows. The FRET probe and PCR primers (one each within the insert 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 and quencher moieties on the FRET probe and release of the fluorescent moiety. The generation of a fluorescent signal represents the presence of the insertion/flanking sequence, which indicates that the amplification and hybridization were successful.

Suitable techniques for detecting plant material derived from transgenic maize event LG11 may also include Southern blot hybridization, Northern blot hybridization, and in situ hybridization based on hybridization principles. In particular, such suitable techniques include 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 film exposure and visualization, or enzyme-labeled probes can be detected by substrate conversion to achieve a color change.

Molecular markers can also be used to detect sequences (Tyagi S and Kramer F R. Molecular beacons: probes that fluoresce upon hybridization [J]. Nat Biotechnol, 1996, 14(3): 303-8.). Design a FRET oligonucleotide probe that spans the insertion DNA sequence and the adjacent genomic flanking junction site. The unique structure of this FRET probe results in it containing a secondary structure that is capable of maintaining a fluorescent moiety and a quencher moiety in close proximity. The FRET probe and PCR primers (one each within the insert and adjacent flanking genomic sequences) are cycled in the presence of a thermostable polymerase and dNTPs. After successful PCR amplification, the hybridization of the FRET probe to the target sequence leads to the loss of the secondary structure of the probe, thereby spatially separating the fluorescent part and the quencher part, resulting in a fluorescent signal. The generation of a fluorescent signal represents the presence of the insertion/flanking sequence, which indicates that the amplification and hybridization were successful.

Other described methods such as microfluidics provide methods and devices for isolating and amplifying DNA samples. Optical dyes are used to detect and measure specific DNA molecules. Nanotube devices comprising electronic sensors for the detection of DNA molecules or nanobeads which bind specific DNA molecules and thus can be detected are useful for the detection of the DNA molecules of the present invention.

DNA detection kits can be developed using the compositions described herein and methods described or known in the field of DNA detection. The kit facilitates the identification of the DNA of the transgenic maize event LG11 in a sample, and can also be used to breed maize plants containing the DNA of the transgenic maize 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 , which are homologous or complementary to the DNA contained in the transgenic genetic element of the DNA, these DNA sequences can be used in DNA amplification reactions, or as probes in DNA hybridization methods. The DNA structure of the junction of the transgene insert sequence and the maize genome contained in the maize genome and illustrated in Figure 1 and Table 1 contains: the maize LG11 left flank genomic region located at the 5' end of the transgene insert sequence, from the left of Agrobacterium Part of the insert sequence in the lateral border region (LB), the first expression cassette is the 35S promoter (CaMV35S promoter (enhanced)) of cauliflower mosaic virus, operably linked to the glufosinate-ammonium resistance gene sequence (bar), and operably connected to the 35S terminator (CaMV poly(A)) of cauliflower mosaic virus; the second expression cassette consists of the 35S promoter (CaMV 35Spromoter) of cauliflower mosaic Insect gene m2cryAb-vip3A, and operably linked to the nopaline synthase gene terminator (NOSterminator), a part of the insertion sequence from the right border region (RB) of Agrobacterium, and the insertion sequence 3' of the transgene Maize LG11 right flank genomic region at the end (SEQ ID NO:5). In the DNA amplification method, the DNA molecules used as primers can be any part of the transgene insertion sequence in the transgenic maize event LG11, or any part of the DNA region of the flanking maize genome in the transgenic maize event LG11.

The transgenic maize event LG11 can be combined with other transgenic maize varieties, such as herbicide (such as glufosinate, glyphosate, etc.) tolerant maize, or transgenic maize varieties carrying insect resistance genes. Various combinations of all of these different transgenic events, bred together with the transgenic maize event LG11 of the present invention, can provide improved hybrid transgenic maize varieties that are insect resistant and resistant to multiple herbicides. Compared with non-transgenic varieties and single-trait transgenic varieties, these varieties can show more excellent characteristics such as insect resistance and multi-herbicide resistance.

The invention provides a nucleic acid sequence for detecting corn plants and a detection method thereof. The transgenic corn event LG11 has the effect of improving insect resistance traits and tolerance to glufosinate-ammonium herbicides. 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 the plant. Meanwhile, in the detection method of the present invention, SEQ ID NO: 1 or its reverse complementary sequence, SEQ ID NO: 2 or its reverse complementary sequence, SEQ ID NO: 3 or its reverse complementary sequence, SEQ ID NO: 4 or its reverse Complementary sequences, SEQ ID NO: 6 or its reverse complement, or SEQ ID NO: 7 or its reverse complement can be used as DNA primers or probes to generate amplification products diagnostic of transgenic maize event LG11 or progeny thereof, And the existence of the plant material derived from the transgenic maize event LG11 can be quickly, accurately and stably identified.

The transgenic maize event LG11 had strong glufosinate-ammonium tolerance and outstanding insect resistance. These characteristics make LG11 a transformant that can be used to improve the glufosinate-ammonium herbicide tolerance and insect resistance traits of corn, so as to breed new corn varieties that are resistant to insects and herbicides.

The technical solutions of the present invention will be described in further detail below with reference to the accompanying drawings and embodiments.

Description of drawings

Figure 1 Schematic diagram of the structure of the binding site between the transgene insertion sequence and the maize genome.

Fig. 2 The physical map of the recombinant expression vector pCAMBIA3300+m2cryAb-vip3A. The English and abbreviation meanings of each component are listed as follows:

LB T-DNA left border sequence of Agrobacterium.

CaMV poly(A) 35S terminator of cauliflower mosaic virus (CaMV).

bar encodes PAT protein, which relieves the toxicity of glufosinate-ammonium.

CaMV 35S promoter (enhanced) The 35S promoter of cauliflower mosaic virus, which can initiate the transcription of the target gene NOS terminator The terminator of the nopaline synthase gene.

m2cryAb-vip3A M2cryAb-vip3A protein coding gene.

CaMV 35S promoter The 35S promoter of cauliflower mosaic virus (CaMV).

RB T-DNA right border sequence of Agrobacterium.

Plasmid stabilization site for pVS1 staA pVS1 plasmid.

pVS1 RepA Replication origin site of pVS1 plasmid.

bom The bom site of the pBR322 plasmid.

Origin of replication of the ori pBR322 plasmid.

kanR encodes an aminoglycoside phosphotransferase protein that confers kanamycin resistance to bacteria.

Fig. 3 The situation of resistance to glufosinate-ammonium herbicide of LG11 event and control Chang 7-2 plants. A: 0 times the dose of Xiachang 7-2; B: 1 times the dose of Xiachang 7-2; C: 0 times the dose of LG11; D: 1 times the dose of LG11; E: 2 times the dose of LG11; F: 4 times the dose Lower dosage of LG11.

Fig. 4 Insect resistance performance of LG11 event and control Chang 7-2. A: Non-transgenic control Chang7-2 maize plant; B: LG11 transformation event maize plant.

Figure 5 The results of LG11 transformation event-specific PCR verification. M: Marker, size marked next to it (unit: bp); N: Blank control; CK: Negative control (Chang 7-2 and Zhengdan 958 mixture) genomic DNA; 1-3: LG11 genomic DNA of transformation events of different generations; 4 : Genomic DNA of a new transgenic maize hybrid LG11-Zhengdan 958. A: The expected size of the left border PCR fragment is 286bp; B: The expected size of the right border PCR fragment is 1447bp.

Fig. 6 Southern hybridization digestion and probe position.

Figure 7 Southern blot hybridization of the target gene m2cryAb-vip3A insertion copy number in LG11

A: HindⅢ enzyme digestion hybridization map; B: BamHI enzyme digestion hybridization map; C: probe and HindⅢ enzyme digestion position schematic diagram; D: probe and BamHI enzyme digestion position schematic diagram. The line segment below marks the position of the probe, and the arrow on the right side of the picture marks the exogenous band.

M: DNA Marker, the band size is marked next to it, in bp;

CK: negative control Chang 7-2;

1: positive control plasmid;

2-4; transformant LG11 of different generations.

Figure 8 Southern blot hybridization of target gene bar insertion copy number of LG11

A: HindⅢ enzyme digestion hybridization map; B: BamHI enzyme digestion hybridization map; C: probe and HindⅢ enzyme digestion position schematic diagram; D: probe and BamHI enzyme digestion position schematic diagram. The line segment below marks the position of the probe, and the arrow on the right side of the picture marks the exogenous band.

M: DNAMarker, the size of the band is marked next to it, and the unit is bp;

CK: negative control Chang 7-2;

1: positive control plasmid;

2-4; transformant LG11 of different generations.

Detailed ways

The transformation event LG11 involved in this application refers to the insertion of foreign gene inserts between specific genome sequences after genetic transformation with the recurrent parent maize inbred line Chang 7-2, backcrossing, and selfing after genetic transformation (T-DNA insert) of maize plants. In a specific embodiment, the expression vector used for the transgene has the physical map shown in Figure 2, and the obtained T-DNA insert has the sequence shown in nucleotides 281-7424 of SEQ ID NO:5. Transformation event LG11 may refer to this transgenic process, or to the T-DNA insert in the genome obtained by this process, or the combination of T-DNA insert and flanking sequence, or may refer to this transgenic process. corn plant. In a specific example, this event is also applicable to plants obtained by transformation of other recipient species with the same expression vector, thereby inserting the T-DNA insert at the same genomic location. The transformation event LG11 may also refer to the progeny plants obtained by asexual reproduction, sexual reproduction, reduction or doubling reproduction or a combination of the above-mentioned plants.

The acquisition of embodiment 1 transformation event and character identification

M2cryAb-vip3A protein is artificially synthesized by combining the main domains of Cry1Ab and Vip3Aa proteins. Cry1Ab and Vip3Aa have significant control effects on corn borer, Spodoptera frugiperda and other pests; bar gene encodes phosphinothricin Acetyltransferase can improve plant tolerance to glufosinate-ammonium herbicide. The present invention uses the pCAMBIA3300+m2cryAb-vip3A expression vector (see Figure 2 for the physical map of the carrier, including the m2cryAb-vip3A gene expression cassette and the bar gene expression cassette) to transform the receptor HiIIB through an Agrobacterium-mediated method, and obtain more than 400 positive Transformants, after molecular detection, were backcrossed with the maize inbred line Chang7-2 as the recurrent parent in each generation to obtain BC ₅ F _2nd generation transgenic maize seeds LG01-LG20, and the herbicide tolerance and resistance of these transformed seedlings were tested. Pests and related agronomic traits were screened and identified.

1. Screening transformants with excellent insect resistance and herbicide resistance

(1) Herbicide resistance screening

Taking the recurrent parent Chang 7-2 as a reference, the transformants with better herbicide tolerance were screened by spraying glufosinate-ammonium at 1 times the recommended concentration in the field. The results showed that only 9 transformation events were significantly more tolerant to glufosinate-ammonium herbicide than the control (Table 2).

Table 2 Herbicide tolerance performance

Values are mean ± SD of 3 biological replicates. Statistical analysis uses LSD for multiple comparisons (α=0.05), and different letters indicate the significance of the difference between the data in the same column under the same herbicide concentration.

(2) Insect resistance

Using the recurrent parent Chang 7-2 as a reference, the transformants with better insect resistance were screened from the above 9 transformation events by leaf bioassay. Insect resistance of materials was evaluated by feeding detached maize leaves to newly hatched larvae of Ostrinia corn borer and Spodoptera frugiperda. The mortality of corn borer and fall armyworm caused by 9 transformant leaves was significantly higher than that of the control (Table 3), wherein LG04, LG06, LG09, and LG11 were highly resistant to corn borer and fall armyworm. The rest are neutral or resistant.

Table 3 Indoor biometrics

Values are expressed as the mean ± standard deviation of 4 biological repeats, and the significance of the difference between the data in the same column was analyzed by the LSD method (α = 0.05).

(3) Investigation of agronomic traits

While identifying the resistance traits of several transformants, their agronomic traits (such as plant height, leaf size, ear size and grain weight, etc.) were also recorded in detail. During data statistics, it was unexpectedly found that the plant height and 100-grain weight of the transformants LG01, LG03, LG04, LG05, LG06, LG08, LG09 and LG14 were significantly lower than that of the control Chang 7-2, and only the agronomic traits of the transformant LG11 (Plant height and 100-kernel weight) had no significant difference with the control (Table 4).

Table 4 Part of the survey results of agronomic traits

Values are mean ± SD of 3 biological replicates. Statistical analysis uses LSD for multiple comparisons (α=0.05), and different letters indicate significant differences between data in the same column in the same period.

Taken together, LG11 is a transformant with excellent herbicide resistance, insect resistance and the best agronomic traits.

2. Analysis of the flanking sequence of the insertion site of the exogenous sequence in the maize genome

In order to further clarify the insertion site of the transformation event LG11, the present invention analyzed the flanking sequence of the insertion site of the LG11 exogenous sequence on the maize genome.

Take 100 mg of plant leaves, grind them quickly with liquid nitrogen, and extract total DNA by CTAB method. After measuring the concentration of genomic DNA, ensure that the total amount of DNA is >2 μg. Using genome resequencing (completed by Wuhan Biobank Co., Ltd.), the length of each Reads is 150bp, and at least 20Gb of data are obtained, and the data quality index Q30≥80% (ie: the proportion of bases with a sequencing error rate greater than 0.1%) ratio below 20%). According to the results of genome resequencing, use the BWA software to use the T-DNA sequence of the transgenic carrier as a template to compare and screen the sequence homology with all the sequences obtained by sequencing (BWA, http://bio-bwa.sourceforge.net/, the default setting ). The screened sequences were further assembled and screened, and the Reads whose sequences were all carrier sequences were removed, and finally a type of Reads sequence was obtained, which was characterized in that half of it was genome sequence and the other half was carrier sequence. According to the obtained genome sequence, perform Blast sequence alignment on the maize genome website (https://www.maizegdb.org/) to obtain the specific position of the genome sequence on the genome, which is the possible insertion site.

The sequencing results were compared with the reference genome and the exogenous T-DNA sequence to obtain the insertion position information of the exogenous insert. Subsequently, forward and reverse primers were designed on the genomic flanking sequence and the foreign insertion sequence at the left and right borders of the insertion site, the insertion site was verified by PCR amplification, and the PCR products were sequenced and analyzed . The results showed that the exogenous fragment of the transformant LG11 was inserted into the maize genome between chr5: 177554037-177554053bp forward.

Then, use the Primerblast software on the NCBI website (https://blast.ncbi.nlm.nih.gov/Blast) to design primers in the genome and T-DNA regions at the left and right borders of the insertion site, and a part of the amplified product is fused Maize genome sequence and a part of T-DNA sequence.

The genomic DNA of the transgenic maize line was used as a template for PCR amplification. The PCR reaction was carried out in a 20 μL system. The amplification cycle program was: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing for 30 s, extension at 72°C for a certain time (set according to the size of the product fragment), 35 cycles; extension at 72°C for 5 min.

According to the results of the flanking sequence and insertion position, the genome upstream primer (SEQ ID NO: 8) and the vector left border primer (SEQ ID NO: 9) were used to amplify and the vector right border primer (SEQ ID NO: 10) and the genome downstream primer ( SEQ ID NO: 11) PCR amplification was performed on the LG11 transformation event to verify the insertion position of the exogenous fragment. The results are shown in Figure 5. The results proved that the exogenous LG11 fragment was stably inserted into the maize genome chr 5:177554037bp position, and further by overlapping PCR amplification and sequencing analysis, the inserted sequence and upstream and downstream genome flanking sequence fragments were obtained. The sequence assembly is shown in Figure 6. Sequence analysis results showed that the size of the inserted sequence was 7144bp.

By analyzing the border sequences on the left and right flanks, it can be seen that the insertion of foreign sequences caused a sequence mutation of 15 bp in the maize genome, and at the same time, the left border sequence of the vector was missing 388 bp (including part of the sequence of the herbicide tolerance gene bar and the entire terminator sequence), and the right The border sequence is missing 22bp.

3. Systematic identification of herbicide tolerance and insect resistance of LG11

In the summer of 2022, the seeds of corn transformant LG11 and contrast Chang 7-2 were sown in Huanghuaihai Transgenic Corn Pilot Test and Industrialization Base, Dangjia Village, Longshan Office, Zhangqiu District, Jinan, Shandong Province, and sprayed with different concentrations and doses in the field. The herbicide tolerance and insect resistance traits of the transformant LG11 were systematically identified by means of glufosinate-ammonium and artificial inoculation.

(1) Herbicide tolerance identification

The spraying time of glufosinate-ammonium was 18 days after sowing, and the number of plants (including plants without phytotoxicity) and plant height of each level of phytotoxicity were investigated at 1 week, 2 weeks and 4 weeks after spraying respectively. The results are shown in Table 6 and Fig. 3. In the control Chang 7-2, one week after spraying glufosinate-ammonium, all the plants had phytotoxicity of grade 4 to 5, most of the plants died, the seedling rate was 0.00%, and the damage rate was 100.00%. 100.00% of the seedlings were established, but there was some phytotoxicity, the phytotoxicity rate was 4.57%-4.69%, and the symptoms of phytotoxicity disappeared after 2 weeks; further investigation of the plant height showed that there was no significant difference in the plant height of the transformants under different doses.

The field herbicide tolerance identification results showed that the transformant LG11 could tolerate glufosinate-ammonium at 4 times the recommended dose in the field, and the agronomic traits such as plant height were not significantly different from those of the control. Normally, partial gene sequence and terminator deletion will affect gene expression and lead to loss of gene function, but surprisingly, the herbicide tolerance phenotype of LG11 transformants is not affected from the identification results, which is significantly stronger than control.

Table 6LG11 tolerance to glufosinate-ammonium herbicide

Values are expressed as mean ± standard deviation of 3 biological replicates. 0×, 1×, 2×, 4× represent the multiples of the recommended dose of glufosinate-ammonium spraying respectively. The significant difference analysis of data in the same column was compared using LSD analysis (α=0.05). "-" indicates not surveyed.

(2) Identification of insect resistance

Refer to the "Announcement No. 953 of the Ministry of Agriculture and Rural Affairs - 10-2007 Environmental Safety Testing of Transgenic Plants and Their Products, Insect-resistant Corn Part 1: Insect Resistance". The inoculation method and investigation method were carried out in accordance with the "NY/T 1248.5 Technical Specification for Identification of Corn Disease and Pest Resistance". Artificial inoculation was carried out at the heart leaf stage of corn respectively, and the pest damage was investigated 2-3 weeks after inoculation.

Field resistance identification results to corn borer (Table 7 and Figure 4):

The results of the survey at the heart-leaf stage found that the leaf-eating level of Chang 7-2 was 7.73±0.31, the pest level was 7, and the resistance level was sensitive, indicating that the material is a local insect-susceptible material, and the quality of this artificial inoculation can meet the requirements. Requirements for resistance identification; meanwhile, the leaf-eating level of LG11 was 1.33±0.24, which was significantly lower than that of the control. The pest level is 1, indicating that its resistance level is high resistance.

The results of the earing stage survey found that the damage level of the female ears of Chang 7-2 was 6.50±0.71, and the resistance level was sensitive, indicating that the material is a local susceptible material. This time it shows that the quality of artificial inoculation can meet the requirements of resistance identification ; The ear damage level of LG11 was 1.40±0.06, which was significantly lower than that of the control, and the resistance level was high resistance.

Table 7 Identification of field resistance of LG11 to corn borer

Values are expressed as the mean ± standard deviation of three biological repeats, and the significance of the difference between the data in the same column was analyzed by t-test method (α = 0.05). There were 25 transformants at the heart-leaf stage and 15 control strains; 68 transformants at the silk-spinning stage and 15 control strains.

Field resistance identification results to Spodoptera frugiperda (Table 8 and Figure 4):

The results of the survey at the heart-leaf stage found that the leaf-eating level of the control Chang 7-2 was 7.83±0.24, the pest level was 7, and the resistance level was sensitive, indicating that the material is a local insect-susceptible material, indicating that the quality of artificial inoculation can meet the requirements of anti-infestation. Sex identification requirements; while the leaf-eating level of LG11 was 1.09±0.09, which was significantly lower than that of the control. The pest level of the transformant is 1, and the resistance level is high resistance.

The results of the investigation at the earing stage found that the damage level of the female ears of Chang 7-2 was 6.20±0.57, and the resistance level was sensitive, indicating that the material was a local susceptible material, indicating that the quality of artificial inoculation could meet the requirements of resistance identification; The leaf-eating level of LG11 was 1.41±0.41, which was significantly lower than that of the control, and the resistance level was high resistance.

Table 8 Field bioassay of Spodoptera frugiperda

Values are expressed as the mean ± standard deviation of 3 to 4 biological repetitions, and the significance of the difference between the data in the same column is analyzed by t-test method (α = 0.05). There were 55 transformants at the heart-leaf stage and 17 control strains; 22 transformants at the silk-spinning stage and 10 control strains.

The results of field insect resistance identification showed that the transformant LD11 could achieve high levels of resistance to corn borer and fall armyworm, and the agronomic traits such as plant height were not significantly different from those of the control. Therefore, the LG11 transformant can be used to improve the glufosinate-ammonium herbicide tolerance and insect resistance traits of corn, so as to breed new corn varieties resistant to insects and herbicides.

Example 2 Analysis of the copy number of the exogenous sequence of the transformation event LG11

The copy number of exogenous sequence was determined by Southern blot hybridization method. In the Southern hybridization detection, two restriction endonucleases on the T-DNA region and not in the hybridization region are selected to digest the genomic DNA, and each inserted copy in the genome will be displayed as a single and specific band after hybridization. After restriction endonuclease digestion, the region to be tested was selected as a probe for Southern blot hybridization experiments.

Southern hybridization selects restriction endonucleases HindⅢ and BamHI to digest the positive control plasmid, control Chang 7-2 and LG11 transformant genomic DNA, and selects the sequence fragments of the target gene m2cryAb-vip3A and bar as probes, probes and enzyme cutting positions The schematic diagram is shown in Figure 6. The specific sequences of the probe primers are shown in Table 9.

Table 9 Probes used in Southern hybridization experiments

1: The unit is bp.

Insertion copy number hybridization detection of the target gene m2cryAb-vip3A Selection of restriction endonucleases HindⅢ and BamHI digestion positive control plasmid, negative control Chang 7-2 genomic DNA and LG11 transformant genomic DNA. After running the gel and transferring to the membrane, it was labeled with the m2cryAb-vip3A gene probe, and the hybridization results are shown in Figure 7A and Figure 7B. The position of the probe of the exogenous gene m2cryAb-vip3A and the cutting site of the restriction endonuclease are shown in Figure 7C and Figure 7D. From the hybridization results, the m2cryAb-vip3A gene of LG11 was inserted into the maize genome in a single copy.

For the hybridization detection of the insertion copy number of the target gene bar, restriction endonucleases HindⅢ and BamHI were selected to digest the positive control plasmid, the negative control Chang 7-2 genomic DNA and the LG11 transformant genomic DNA. After the gel was transferred to the membrane, it was labeled with the bar gene probe, and the hybridization results are shown in Figure 8A and Figure 8B. The position of the probe of the target gene bar and the cutting site of the restriction endonuclease are shown in Figure 8C and Figure 8D. According to the hybridization results, the bar gene of LG11 was inserted into the maize genome in a single copy.

Example 3 The method for producing insect-resistant and herbicide-resistant corn using the transformation event LG11

Through the preliminary experiments, it was found that the resistance and other agronomic traits of the transformation event LG11 are very good, so this event can be used to quickly transform and improve the backbone parents belonging to the same group in production, so as to improve the insect resistance and herbicide tolerance of these backbone parents . In addition, the transformation event LG11 can be directly used to assemble hybrid groups. The insect-resistant and herbicide-resistant transgenic corn LG11-Zhengdan 958 was directly assembled from Zheng 58 as the female parent and the transformation event LG11 as the male parent; LG11-Zheng Shan 958 added insect resistance and glufosinate-resistant traits on the basis of retaining the original good traits (Table 10).

The method provided by the invention can not only detect LG11 in the process of parental improvement and hybridization combination, so as to provide molecular auxiliary means for the selection of varieties, but also can be used to identify whether LG11 transformation events are contained in maize varieties. The following method is a specific example of identifying LG11.

Molecular detection was carried out on the transformation event LG11 and the new transgenic maize hybrid LG11-Zhengdan 958 to confirm that the material contained the transformation event LG11. A pair of PCR primers was designed according to the gene sequence, and the existence of the transformation event LG11 was determined by detecting the presence of two borders on the left and right of the transformation event.

One of the detection methods is: using the PCR method to detect the specific border sequence in the transformation event LG11 corn plant, the PCR primers used are respectively SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: IDNO:11, PCR reaction system:

The reaction procedure is:

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

The PCR products were detected by electrophoresis in 1% (w/v) 1×TAE agarose gel. In the LG11 transformation event, the expected target bands can be amplified, with sizes of 286bp (SEQ ID NO:6) and 1447bp (SEQ ID NO:7), respectively, and the results are shown in FIG. 5 . Moreover, the PCR method can track the existence of transformation events, so that it can be applied to breeding work.

Table 10 Insect resistance (corn borer and fall armyworm) and herbicide tolerance identification

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

Example 4 Detection method of transformation event LG11

New varieties can be bred from the transgenic corn event LG11 to produce agricultural products or commodities. If a sufficient amount is detected in said produce or commodity, said produce or commodity is expected to contain a nucleotide sequence capable of diagnosing the presence of transgenic maize Event LG11 material in said produce or commodity. Such agricultural products or commodities include, but are not limited to, corn oil, cornmeal, cornmeal, cornstarch, starches, and other condiments or any other foodstuffs used as food sources for animal consumption, or cosmetics, industrial supplies, and the like. Nucleic acid detection methods and/or kits based on probes or primer pairs can be developed to detect the transgenic maize event LG11 nucleotide sequence such as shown in SEQ ID NO:1 or SEQ ID NO:2 in biological samples, wherein the probe The sequence or primer amplified sequence is selected from the group consisting of as 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 Sequences shown to diagnose the presence of transgenic maize event LG11.

In summary, the transgenic corn event LG11 of the present invention can improve plant insect resistance and have higher tolerance to glufosinate-ammonium herbicide, and it can be used to improve other corn germplasm and create new corn hybrid combinations . The detection method can accurately and quickly identify whether the DNA molecule of the transgenic corn event LG11 is contained in the biological sample.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention without limitation. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be The scheme shall be modified or equivalently replaced without departing from the spirit and scope of the technical scheme of the present invention.

Claims (9)

1. A nucleic acid molecule comprising any one of the following:

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

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

iii) Comprising the sequence shown in SEQ ID NO. 6 and/or SEQ ID NO. 7, or the reverse complement 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 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 or variant or reverse complement thereof.

3. A primer pair for detecting a maize transformation event, wherein the 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 SEQ ID NO. 10 and SEQ ID NO. 11.

4. Kit or microarray for detecting maize transformation events, characterized in that it comprises a probe according to claim 2 and/or a primer pair according to claim 3.

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

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, the method comprising the steps of:

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

2) Obtaining a maize plant, seed, plant cell, progeny plant or plant part from the maize obtained in step 1) by pollen culture, unfertilized embryo culture, doubling culture, cell culture, tissue culture, selfing or crossing or a combination thereof; optionally, the composition may be in the form of a gel,

3) Assessing the pest resistance trait and/or identifying the herbicide resistance of the progeny plant obtained in step 2) and detecting the presence or absence of said transformation event using the method of claim 5.

7. A product made from the maize plant, seed, plant cell, progeny plant or plant part obtained by the method of claim 6, comprising food, feed or industrial feedstock.

8. A method of protecting a maize plant from injury caused by a herbicide comprising applying to a field where at least one transgenic maize plant comprising in its genome the nucleic acid sequence of SEQ ID No. 1, SEQ ID No. 5 at positions 281-7424 and SEQ ID No. 2 in that order or comprising in its genome SEQ ID No. 5 an effective dose of a glufosinate herbicide; the transgenic corn plants have tolerance to glufosinate herbicide.

9. A method of protecting a maize plant from insect infestation comprising providing at least one transgenic maize plant cell comprising in its genome the nucleic acid sequence of SEQ ID No. 1, SEQ ID No. 5 at positions 281-7424 and SEQ ID No. 2 in that order, or comprising in its genome SEQ ID No. 5; target insects that ingest the cells of the transgenic corn plant are inhibited from further ingestion of the corn plant.

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