CN110881367A

CN110881367A - Corn event Ttrans-4 and methods of use thereof

Info

Publication number: CN110881367A
Application number: CN201910374145.0A
Authority: CN
Inventors: 余桂容; 杜文平; 宋军; 徐利远; 陈谦; 邓自圆; 张敏; 林杨
Original assignee: SAAS BIOTECHNOLOGY AND NUCLEAR TECHNOLOGY RESEARCH INSTITUTE
Current assignee: SAAS BIOTECHNOLOGY AND NUCLEAR TECHNOLOGY RESEARCH INSTITUTE
Priority date: 2019-01-02
Filing date: 2019-04-26
Publication date: 2020-03-17
Also published as: CN110195122A; CN110184276A

Abstract

The present invention relates to a corn event T-anti-4 and methods of use thereof, the nucleic acid sequence of the corn plant comprising SEQ ID NO: 1 or a complementary sequence thereof, or SEQ ID NO: 2 or the complement thereof. Plants produced by the maize event T-resistant-4 of the present invention have better tolerance to glyphosate herbicides.

Description

Corn event Ttrans-4 and methods of use thereof

Technical Field

The present invention relates to a novel corn transformation event tara-4 which exhibits enhanced tolerance to glyphosate herbicides. The invention also relates to the application method of the corn plant parts and seeds related to the Ttrans-4 and the products thereof.

Background

The weeds in the field compete with crops for water, fertilizer, light 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 million, poverty developing countries bear approximately $ 700 million (FAO. the lurking company of waters [ J/OL ] (http:// www.fao.org/news/store/en/item/29402/icode /), 2009-08-11.). Therefore, effective control of weeds in the field is one of the important measures for promoting the yield increase of grains. In China, more than 40 types of weeds which harm corn are available, and more than 10 types of weeds which harm corn are available. The weeds can reduce the yield of the corn by 10-20% in general years, and the yield is higher by 30-50% in serious cases. In addition, with the increase of the migration speed of rural population in China to cities, the scale and mechanization of corn planting are a foreseeable trend, so that the traditional artificial weeding mode becomes unrealistic. At present, the selective herbicide widely applied in the market has large application amount and long residual period, and is easy to influence the normal growth of the next-stubble crops. The biocidal herbicide such as glyphosate has the characteristics of high efficiency, low toxicity, easy degradation, no residue and the like. However, they are not selective in weed control and cannot be used directly in the growing period of crops. The difficult problem can be overcome by cultivating the corn which is resistant to the biocidal herbicide by a 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.

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 may also exist in the spatial or temporal pattern of expression, 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. 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 pattern retain the transgene expression characteristics of the original transformant. The use of this strategy ensures reliable gene expression in many varieties that are well adapted to local growth conditions.

Disclosure of Invention

The invention aims to provide a corn transformation event Ttrans-4 and a using method thereof, wherein the transgenic corn event Ttrans-4 has better tolerance to glyphosate herbicide, and other corn plants with tolerance to the glyphosate herbicide can be cultivated by utilizing the transformation event.

To achieve the above objects, the present invention provides a method for protecting corn plants from herbicide-induced damage comprising applying to a field planted with at least one transgenic corn plant comprising in its genome, in order, SEQ ID NO: 1. SEQ ID NO: 5, 399-: 2, or the genome of said transgenic maize plant comprises SEQ ID NO: 5; the transgenic corn plants have tolerance to glyphosate herbicides.

To achieve the above objects, the present invention also provides a method of controlling weeds in a field planted with corn plants, comprising applying to the field planted with at least one transgenic corn plant comprising in sequence in its genome SEQ ID NO: 1. SEQ ID NO: 5, 399-: 2, or the genome of said transgenic maize plant comprises SEQ ID NO: 5; the transgenic corn plants have tolerance to glyphosate herbicides.

To achieve the above objects, the present invention also provides a method of growing a corn plant tolerant to glyphosate herbicide comprising:

planting at least one corn seed, the genome of the corn seed comprising a nucleic acid sequence of a particular region, the nucleic acid sequence of the particular region comprising, in order, the nucleic acid sequence of SEQ ID NO: 1. SEQ ID NO: 5, sequence 399-: 2, or the nucleic acid sequence of said specific region comprises SEQ ID NO: 5;

growing the corn seed into a corn plant;

spraying said corn plants with an effective amount of glyphosate herbicide to harvest plants having reduced plant damage compared to other plants not having the nucleic acid sequence of said specific region.

To achieve the above objects, the present invention also provides a method of producing a corn plant tolerant to glyphosate herbicide comprising introducing into the genome of said corn plant the nucleotide sequence of SEQ ID NO: 5, 399-4244 and such that the genome of said maize plant comprises in sequence the nucleic acid sequence of SEQ ID NO: 1. SEQ ID NO: 5, 399-: 2 or such that the genome of said maize plant comprises SEQ ID NO: and 5, selecting the corn plants tolerant to the glyphosate.

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

sexually crossing a transgenic corn event T-resistant-4 first parental corn plant having tolerance to glyphosate herbicide with a second parental corn plant lacking glyphosate tolerance, thereby producing a plurality of progeny plants;

treating said progeny plants with glyphosate herbicide;

selecting said progeny plants that are tolerant to glyphosate.

To achieve the above objects, the present invention also provides a composition for producing autoradiogenic corn event Tanti-4, wherein the composition is corn flour, corn oil, corn silk or corn starch.

To achieve the above objects, the present invention also provides an agricultural or commercial product produced by transgenic corn event tadant-4, which is corn flour, corn oil, corn starch, corn gluten, corn cake, cosmetics or bulking agent.

In using the present invention for methods of creating and using transformants, 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 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 foreign DNA will result in transformants that contain different flanking regions that are specifically contained by each transformant. When the recombinant DNA is introduced into a plant by conventional crossing, its flanking regions are not usually altered. Transformants 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 fragments are joined together in a manner that is modified in the way found in the native organism. "junction DNA" refers to DNA comprising a junction site.

The present invention provides a transgenic corn event and its progeny, said transgenic corn event tadista-4 being a corn plant tadista-4 comprising plants and seeds and plant cells thereof or regenerable parts thereof of transgenic corn event tadista-4, said plant parts of transgenic corn event tadista-4 including but not limited to cells, pollen, ovule, flowers, shoots, roots, stems, silks, inflorescences, ears, leaves, and products from corn plant tadista-4, such as corn flour, corn oil, corn steep liquor, corn silks, corn starch, and biomass left in a field of corn crops.

The present invention relates to a transgenic maize event T-anti-4 comprising a DNA construct which when expressed in a plant cell, the transgenic maize event T-anti-4 gains tolerance to glyphosate herbicide, the DNA construct comprising an expression cassette comprising a suitable promoter for expression in a plant operably linked to a gene encoding a 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) and a suitable polyadenylation signal sequence, the nucleic acid sequence of the EPSPS protein being tolerant to glyphosate herbicide, further the promoter may be a suitable promoter isolated from a plant, including constitutive, inducible and/or tissue specific promoters, including but not limited to, the cauliflower mosaic virus (CaMV)35S promoter, the Figwort Mosaic Virus (FMV)35S promoter, the Ubiquitin protein (uquitin) promoter, the Actin (Actin) promoter, the Agrobacterium (Agrobacterium) promoter, the Agrobacterium (agroteramefaciens) promoter, the synthase (agufactein) promoter, the octopine synthase (aghui) promoter, the promoter derived from Agrobacterium tumefaciens (agtulicphosphate synthase) promoter, the promoter derived from Agrobacterium tumefaciens (agjuvenin) promoter, the promoter derived from the promoter, the promoter suitable promoter for the promoter, the promoter for the promoter of the Agrobacterium tumefaciens protein (agtulicutebaccific protein (aguacilobulins) isolated from the promoter, the promoter of the Agrobacterium tumefaciens, the promoter of the Agrobacterium, the promoter of the.

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, including, but 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 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) gene may be isolated from Agrobacterium tumefaciens (Agrobacterium tumefaciens sp) CP4 strain or Pseudomonas fluorescens (Pseudomonas fluorescens) G2 strain, and the polynucleotide encoding the EPSPS gene may be altered by codon optimization or in other ways to increase the stability and availability of the transcript in the transformed cell. The 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) gene can also be used as a selectable marker gene.

By "glyphosate" is meant N-phosphonomethylglycine and its salts and by "treatment with glyphosate herbicide" is meant treatment with any herbicide formulation containing glyphosate. The rate of use of a certain glyphosate formulation to achieve an effective biological dose is not chosen beyond the skill of the ordinary agronomic artisan. Treatment of a field containing plant material derived from transgenic corn event tdain-4 with any one of the herbicide formulations containing glyphosate will control weed growth in the field and not affect the growth or yield of plant material derived from transgenic corn event tdain-4.

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.

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

The pollen tube channel transformation method is characterized in that a natural pollen tube channel (also called a pollen tube guide tissue) formed after plant pollination is utilized, and exogenous DNA is carried into an embryo sac through a nucellar channel.

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 different restriction enzyme sites for the introduction of DNA molecules providing functional genetic elements, i.e. promoters, introns, leader sequences, coding sequences, 3' terminator regions and other sequences. The expression cassette contained in the DNA construct, 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 transformant comprising the heterologous DNA and progeny of the transformant. The term "event" also refers to progeny resulting from sexual crosses between a transformant and an individual of another variety containing heterologous DNA, even after repeated backcrossing with the backcross parent, where the inserted DNA and flanking genomic DNA from the transformant parent are present at the same chromosomal location in the progeny of the cross. The term "event" also refers to a DNA sequence from the original transformant that comprises the inserted DNA and flanking genomic sequences immediately adjacent to the inserted DNA, which DNA sequence is expected to be transferred into progeny; the progeny is produced by sexual crossing of a parental line containing the inserted DNA (e.g., the original transformant and progeny resulting from selfing thereof) with a parental line that does not contain the inserted DNA, and the progeny receives 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 that is not normally found in nature and 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 a transgenic corn event T anti-4 that is tolerant to glyphosate herbicide by: first sexually crossing a first parent corn plant consisting of a corn plant bred from a transgenic corn event T-resistant-4 and its progeny, which is obtained by transformation using the expression cassette of the present invention that is tolerant to glyphosate herbicide, with a second parent corn plant lacking tolerance to glyphosate herbicide, to produce a variety of first generation progeny plants; and then selecting the progeny plants with tolerance to the glyphosate herbicide to breed the corn plants with tolerance to the glyphosate herbicide. These steps can further include backcrossing the glyphosate tolerant progeny plant with a second or third parent corn plant, and then selecting the progeny by application with the glyphosate herbicide or by identification of a trait-related molecular marker, such as a DNA molecule comprising a splice site identified by the 5 'and 3' ends of the insert sequence in transgenic corn event T-anti-4, to produce a corn plant that is tolerant to the glyphosate herbicide.

It is also understood that two different transgenic plants may also be crossed to produce progeny containing two separate, separately added exogenous genes. Selfing of 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 propagation.

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 probes are complementary to one strand of the target nucleic acid, and in the present invention, the probes are complementary to one strand of DNA from the transgenic corn event T-anti-4 genome, whether the genomic DNA is from the transgenic corn event T-anti-4 or seed or a plant or seed or extract derived from the transgenic corn event T-anti-4. 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 probes and primers is generally 11 polynucleotides or more, preferably 18 polynucleotides or more, more preferably 24 polynucleotides or more, and most preferably 30 polynucleotides or more. Such probes and primers hybridize specifically to the target sequence under highly stringent hybridization conditions. Although probes that are different from and maintain the ability to hybridize to the target DNA sequence can be designed by conventional methods, it is preferred that the probes and primers of the present invention have complete DNA sequence identity to the contiguous nucleic acid of the target sequence.

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 maize event T anti-4 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 or probes.

The nucleic acid probes and primers of the invention hybridize to a target DNA sequence under stringent conditions. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of DNA derived from transgenic corn event T anti-4 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 they 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 to allow a nucleic acid molecule to act as a primer or probe, it is only necessary to ensure sufficient complementarity in sequence to allow the formation of a stable double-stranded structure in the particular solvent and salt concentrations employed.

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 a 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), a 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 P Y. 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 T anti-4 can also include Southern blot hybridization, Northern blot hybridization and in situ hybridization based on hybridization principles. In particular, the suitable techniques include 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

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 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 FRET probes to target sequences by successful PCR amplificationResulting in a loss of 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 of transgenic corn event T anti-4 DNA in a sample and can also be used to cultivate corn plants containing transgenic corn event T anti-4 DNA. The kit may contain DNA primers or probes homologous or complementary to SEQ ID NO: 1. 2, 3, 4 or 5, 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 T anti-4 left flanking genomic region located 5' to the transgene insert, a portion of the insert from the Agrobacterium left border region (LB), the first expression cassette consists of the maize ubiquitin gene promoter (ubiquitin promoter), operably linked to a pea rbcS chloroplast transit peptide Sequence (SP), operably linked to a glyphosate tolerant 5-enol-pyruvylshikimate-3-phosphate synthase gene (2mG2-epsps) of the fluorescent Pseudomonas G2 strain, and operably linked to a nopaline synthase gene terminator (nonsteroider), a part of an insertion sequence from the right border Region (RB) of Agrobacterium, and a maize plant T anti-4 right flank genomic region located at the 3' end of the transgene insert (SEQ ID NO: 5). In the DNA amplification method, the DNA molecule used as a primer can be any part of the DNA sequence derived from the transgene insert in transgenic corn event T anti-4, or any part of the DNA region flanking the corn genome in transgenic corn event T anti-4.

Transgenic corn event T-anti-4 can be combined with other transgenic corn varieties, such as herbicide (e.g., glufosinate, dicamba, etc.) tolerant corn, or transgenic corn varieties carrying insect-resistant genes. All of these various combinations of different transgenic events, when bred with transgenic corn event T-resistant-4 of the present invention, can provide improved hybrid transgenic corn varieties that are resistant to insects and to multiple herbicides. These varieties can exhibit more excellent characteristics such as an increase in yield as compared with non-transgenic varieties and single-trait transgenic varieties.

The invention provides a transgenic corn event and a method of using the same, wherein the transgenic corn event Ttrans-4 is tolerant to the phytotoxic effect of an agricultural herbicide containing glyphosate. The maize plants of this trait express the glyphosate resistant 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) protein of pseudomonas fluorescens strain G2, which confers tolerance to glyphosate to plants. Meanwhile, in the detection method of the invention, SEQ ID NO: 1 or its complementary sequence, SEQ ID NO: 2 or the complement thereof, SEQ ID NO: 6 or a complementary sequence thereof, or SEQ ID NO: 7 or its complement can be used as DNA primers or probes to produce amplification products that are diagnostic for transgenic corn event T anti-4 or its progeny, and can quickly, accurately, and stably identify the presence of plant material derived from transgenic corn event T anti-4.

Brief description of the sequence:

SEQ ID NO: 1 the 5' transgene insertion site left flank corn genomic DNA and 11 nucleotide sequences of the left border of the transgene fragment in transgenic corn event T anti-4;

SEQ ID NO: 2 each of 11 nucleotide sequences of the right border and right flank corn genomic DNA of the 3' transgene insertion site transgene segment in transgenic corn event T anti-4;

SEQ ID NO: 3 transgenic maize event T anti-4 a sequence of 448 nucleotides in length located near the insertion junction at the 5' end of the insertion sequence;

SEQ ID NO: 4 transgenic maize event T anti-4 a sequence of 611 nucleotides in length located near the insertion junction at the 3' end of the insertion sequence;

SEQ ID NO: 55 'left flank maize genomic sequence, entire T-DNA sequence and 3' right flank maize genomic sequence;

SEQ ID NO: 6 is located in SEQ ID NO: 3, spanning the 5' left flank maize genomic sequence, the 2mG2-epsps-pC3301 construct left border DNA sequence, and the ubiquitin promoter sequence;

SEQ ID NO: 7 is located in SEQ ID NO: 4, spanning the nos transcription termination sequence, the 2mG2-epsps-pC3301 construct right border DNA sequence and the 3' right flank maize genomic sequence;

SEQ ID NO: 8 amplifying SEQ ID NO: 6;

SEQ ID NO: 9 amplification of SEQ ID NO: 6, a second primer;

SEQ ID NO: 10 amplifying SEQ ID NO: 7;

SEQ ID NO: 11 amplifying SEQ ID NO: 7, a second primer;

SEQ ID NO: 12 PCR detection of the first primer of 2mG 2-epsps;

SEQ ID NO: 13 PCR detection of a second primer of 2mG 2-epsps;

SEQ ID NO: 14 obtaining primers for the right border flanking sequence;

SEQ ID NO: 15 obtaining primers of the right border flanking sequence;

SEQ ID NO: 16 obtaining primers for the right border flanking sequence;

SEQ ID NO: 17 obtaining primers of the right border flanking sequence;

SEQ ID NO: 18 probes in Southern hybridization assays;

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 the transgene insert to the maize genome.

FIG. 2 physical map of recombinant expression vector 2mG2-epsps-pC 3301. The English and abbreviated meanings of each element are listed as follows:

T-DNA left border sequence of T-Border (left) Agrobacterium C58.

A promoter of ubiquitin promoter maize ubiquitin gene.

rbcS chloroplast transit peptide in SP pea.

2mG2-epsps was derived from the glyphosate tolerance gene of strain G2.

The terminator of the nos terminator nopaline synthase gene.

T-DNA right border sequence of T-Border (right) Agrobacterium C58.

Plasmid stabilization site of PVS1 sta pVS1 plasmid.

The replication initiation site of the PVS1 rep pVS1 plasmid.

The bom site of the PBR322 bom pBR322 plasmid.

Origin of replication of the PBR322 ori pBR322 plasmid.

kanamycins (R) encode aminoglycoside phosphotransferase proteins that confer kanamycin resistance to bacteria.

FIG. 3 Southern blot hybridization of insertion copy number of anti-4 foreign gene. A: carrying out SacI enzyme digestion; b: BstEII enzyme digestion; c: the restriction enzyme site of the T-DNA region and the position of the probe are shown schematically, the number indicates the size of the band after restriction enzyme, the unit is kb, and the position of the used probe is marked on the lower part. Lane 1: DNA Marker III, DIG-labeled (Roche), the size of the band is marked aside, unit kb; lane 2: blank; lane 3: 2mG2-epsps-pC3301 plasmid and recipient 18599 genomic DNA mixture; lane 4: receptor 18599 genomic DNA; lane 5: Tanti-4T₆A surrogate genomic DNA; lane 6: Tanti-4T₇The generation of genomic DNA.

Figure 4 field performance of transformation event T anti-4 after 4 weeks of glyphosate spray. A: the receptor 18599 did not spray glyphosate; b: spraying glyphosate on a receptor 18599; c: the transformation event T anti-4 is not sprayed with glyphosate; d: and (4) spraying glyphosate on a transformation event T-resistance-4.

FIG. 5 results of detection of transformation event Tanti-4. A: detecting the left boundary, wherein the expected size of a target band is 353 bp; b: detecting the right boundary, wherein the expected size of a target band is 414 bp; m: the molecular weight standard is 2kb, 1kb, 750bp, 500bp, 250bp and 100bp from top to bottom; 1: water; 2: corn material 0151; 3: corn material sheet 30; 4: corn material T-resistant-4-0151; 5: corn material 16-1.

Detailed Description

The transformation event Tanti-4 referred to in this application refers to a maize plant obtained by genetic transformation using maize inbred line 18599 as recipient to insert foreign gene inserts (T-DNA inserts) between specific genomic sequences. In a specific example, the expression vector used for the transgene has the physical map shown in FIG. 1, and the resulting T-DNA insert has the nucleotide sequence shown in SEQ ID NO: 5, the 293-position and 4277 nucleotide sequence. Transformation event T anti-4 may refer to this transgenic process, may also refer to the T-DNA insert within the genome resulting from this process, or the combination of the T-DNA insert and flanking sequences, or may refer to the 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 Tanti-4 may also refer to progeny plants resulting from vegetative propagation, sexual propagation, doubling or doubling of the above plants or a combination thereof.

EXAMPLE 1 construction of transformation vectors

(1) According to the requirement of expressing the gene function in the plant, a chloroplast leader SP sequence is added at the 5' end of the coding region of the 2mG2-EPSPS gene to ensure that the translated EPSPS protein is transported to chloroplast in the synthesis site of aromatic amino acid. (2) Synthesizing a 2mG2-epsps sequence, and respectively introducing BamHI and SacI enzyme cutting sites at two ends of the sequence; the SP sequence is cloned, and PstI and BamHI enzyme cutting sites are respectively introduced at two ends of the sequence.

(3) The 2mG2-epsps sequence and the PUC19 vector were treated with BamHI + SacI and ligated to obtain 2mG2-epsps-PUC19 vector. The SP sequence was treated with PstI + BamHI and ligated with the 2mG2-epsps-PUC19 vector to obtain SP-2mG2-epsps-PUC19 vector.

(4) The HindIII + PstI enzyme digestion PAH17 vector obtains a ubiquitin promoter, and the ubitin promoter is connected into a SP-2mG2-epsps-PUC19 vector treated by HindIII + PstI to obtain a Pubi-SP-2mG2-epsps-PUC19 vector.

(5) And (3) processing the pCABIA3301 vector and DNA fragments with the left end of AseI + HindIII and the right end of SacI + BstEII by AseI + BstEII, and connecting to obtain the modified pCAMBIA3301 vector.

(6) HindIII + SacI is used for enzyme digestion of Pubi-SP-2mG2-epsps-PUC19 vector and modified pCAMBIA3301 vector, Pubi-SP-am79epsps is connected to pCAMBIA3301 vector, and the vector named as 2mG2-epsps-pC3301 is obtained.

The obtained vector has a size of 10.261kb, and its physical map is shown in FIG. 1.

Example 2 genetic transformation of maize

The method used for transforming the corn is an agrobacterium-mediated method, and the operation procedure is as follows:

(1) selecting corn ears pollinated for 10-13 days, and stripping young embryos (1.5-2.0mm) with proper size from the corn ears;

(2) using an inoculating loop to pick agrobacterium (containing an expression vector) from a YEP plate growing for three days at 19 ℃, suspending the agrobacterium in an infection culture solution at room temperature and 75rpm for 2-4h until OD550 is 0.3-0.5, and infecting the stripped immature embryos;

(3) transferring the infected young embryo to a co-culture medium, and culturing for 3 days at 20 ℃ in the dark;

(4) transferring the young embryo into a recovery culture medium after 3 days, and culturing in the dark at 28 ℃ for 7 days;

(5) transferring the young embryo to a screening culture medium after recovery culture, and performing conversion once every two weeks to select a callus which grows rapidly;

(6) the selected callus germinates under light, and a transgenic plant is determined by molecular detection after the callus grows into a regeneration plant;

example 3 selection of transformants

(I) And performing target gene molecule detection on 810T 0 transformed seedlings obtained by transformation. Designing PCR primer pairs according to gene sequences, wherein the primer sequences are respectively SEQ ID NO: 12 and SEQ ID NO: 13. extracting genome DNA from the leaves of the transformed seedlings, and amplifying according to the following PCR parameters:

reaction system:

reaction procedure:

the size of the target gene amplified fragment of the positive transformant is consistent with that of the amplified fragment of the positive plasmid control PCR, and the target gene amplified fragment is 499bp, and 13 positive single-plant T1 seeds are harvested.

(2) The 6 strains with the serial numbers of T-resistant-1, T-resistant-2, T-resistant-3, T-resistant-4, T-resistant-5 and T-resistant-6 are obtained by screening T1-T3 generation plants in a greenhouse through herbicide screening, segregation ratio inspection and the like.

(3) For T in greenhouses₄-T₆The generation plants are further investigated and screened for agronomic characters to obtain a transformant T-resistant-4.

In terms of yield traits, Titan-4 has a cone-shaped ear, a hard grain type, a yellow grain color and a white axis, all of which are consistent with 18599. Tanti-4 and 18599 were not significantly different (p > 0.05) in ear length, spiky, ear thickness, ear row number, row grain number, hundred grain weight, and yield (Table 2).

TABLE 2 investigation results of T anti-4 yield and seed test traits

The data in the same column are tested for significance of difference by using the LSD method (α ═ 0.05).

Example 4 flanking sequences of transformation event T anti-4 exogenous sequence and maize genomic insertion site

Primers (SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17) for flanking sequence separation were designed, the right flank sequence (position 4278 and 4601 of SEQ ID NO: 5) of the transformant T-antibody 4 was obtained by a Tail-PCR amplification and sequencing method, and the left flank sequence (position 1 to 292 of SEQ ID NO: 5) was obtained by PCR amplification sequencing based on the genomic sequence (primer sequences SEQ ID NO: 8 and SEQ ID NO: 9). The flanking sequences were analyzed for homology alignment with the maize genomic sequence in the plantagdb database (http:// www.plantgdb.org/ZmGDB/cgi-bin/blastgdb. p1) using the BLASTN tool, with B73 RefGen _ V2 as the reference sequence. Analysis shows that the transformant T anti-4 is inserted into the corn genome Chr 01: 227594470 position.

The operation steps of Tail-PCR are as follows:

1) and extracting corn genome DNA.

2) Plant genome DNA is used as a template of a first round of PCR reaction, and the reaction system is as follows:

the reaction procedure is as follows: 94 ℃ for 5 min; (94 ℃, 30 sec; 62 ℃, 2 min; 72 ℃, 2.5 min). times.5 cycles; 94 ℃, 30 sec; at 25 ℃ for 3 min; 72 deg.C (32% ramp), 3 min; (94 ℃, 30 sec; 62 ℃, 1 min; 72 ℃, 2.5 min; 94 ℃, 30 sec; 62 ℃, 1 min; 72 ℃, 2.5 min; 94 ℃, 30 sec; 45 ℃, 1 min; 72 ℃, 2.5 min). times.15 cycles; 72 ℃ for 7 min; 20 ℃ for 10 min.

3) And (3) carrying out second round PCR amplification by taking the corresponding PCR product (mother liquor) in the step 2) as a template. The reaction system is as follows:

the reaction procedure is as follows: 94 ℃ for 5 min; (94 ℃, 30 sec; 65 ℃, 1 min; 72 ℃, 2.5 min; 94 ℃, 30 sec; 65 ℃, 1 min; 72 ℃, 2.5 min; 94 ℃, 30 sec; 45 ℃, 1 min; 72 ℃, 2.5 min). times.20 cyclos; 72 ℃ for 7 min; 20 ℃ for 10 min.

4) And (3) performing third PCR amplification by using the corresponding PCR product (diluted by 30 times) in the step 3) as a template. The reaction system is as follows:

the reaction procedure is as follows: 94 ℃ for 5 min; (94 ℃, 30 sec; 65 ℃, 1 min; 72 ℃, 2.5 min; 94 ℃, 30 sec; 65 ℃, 1 min; 72 ℃, 2.5 min; 94 ℃, 30 sec; 45 ℃, 1 min; 72 ℃, 2.5 min). times.20 cycles; 72 ℃ for 7 min; 20 ℃ for 10 min.

5) Taking the product of the third round of PCR in the step 4) to carry out electrophoresis detection in 1% (w/v)1 XTAE agarose gel, and recovering the DNA fragment with the size meeting the purpose.

6) The recovered fragments were ligated to T-vector and ligated overnight at 16 ℃.

7) Conversion of the ligation product of 6).

8) Amplifying the transformation product in the step 7), and picking positive clone and shaking the bacterium to extract plasmid.

9) Sending the plasmid for sequencing.

The full-length insert of anti-4 was further amplified and sequenced by overlapping PCR steps. The actual insertion sequence of the transformant T-anti-4 and the genome sequences of the left and right flanking corns are shown as SEQ ID NO: 5, respectively.

TABLE 3 primer information for flanking sequence isolation

N is A/T/C/G; v is G/C/A; 1: the unit bp.

Example 5 copy number detection of transformation event Tanti-4

The copy number of the inserted foreign gene is determined by a Southern blot hybridization method. The insertion copy number hybridization detection of 2mG2-epsps gene fragment selects SacI restriction enzyme and BstEII restriction enzyme to respectively cut the mixture of positive control 2mG2-epsps-pC3301 plasmid and wild type 18599 genomic DNA, negative control wild type 18599 genomic DNA and T₆、T₇Generation T anti-4 transformant genomic DNA. After running the gel and transferring the membrane, the membrane is labeled with a 2mG2-epsps gene probe. The hybridization results are shown in FIG. 4A, B. The probe position of the target gene 2mG2-epsps and the restriction sites of the restriction enzymes SacI and BstEII are shown in figure 4C.

The restriction enzyme cutting site of SacI in the T-DNA region is only one, and is positioned at the right side of 2mG2-epsps gene probe, the marker band obtained after the restriction enzyme cutting T anti-4 genome DNA is hybridized with specific probe should include 3.6kb of T-DNA sequence and its left genome sequence with unknown size, the length of the whole fragment is greater than 3.6kb, a single hybridization band is marked in the experiment, the size is about 15kb (fig. 4A lanes 5 and 6), and the expected size is met.

BstEII has two enzyme cutting sites in a T-DNA region, both of which are positioned at the right side of a 2mG2-epsps gene probe, and a marker band obtained after the genomic DNA of the T-anti-4 transformant subjected to enzyme cutting is hybridized with a specific probe should comprise a T-DNA sequence of 2.6kb and a sequence with unknown size on the left genome, the length of the whole fragment is more than 2.6kb, a single hybridization band is marked in an experiment, and the size is about 2.7kb (

lanes

5 and 6 of figure 4B), which is in line with the expectation.

Meanwhile, neither the blank control nor the negative control receptor 18599 was marked with restriction enzymes SacI and BstEII, and no band was observed (lanes 2, 4); the positive control 2mG2-epsps-pC3301 plasmid and the receptor 18599 genomic DNA mixture were labeled with SacI and BstEII restriction enzymes and the hybridized band matched to plasmid size 10.3kb or 10.1 kb (lane 3), indicating the specificity of the hybridized probe.

The above experimental results show that the T-DNA region of T anti-4 contains a single copy of 2mG2-epsps gene fragment, and that the T-DNA region is inserted in a single site into the maize genome. Exogenous inserts from generations to generations can be stably inherited by sexual reproduction.

Example 6 tolerance of transformation event T anti-4 to target herbicides

Under the natural environment of the field, the tolerance of the T-resistant-4 transformant to the herbicide glyphosate is measured by a method of artificially spraying the herbicide, so as to determine the effectiveness of the target character of herbicide tolerance of the transformant. The herbicide is a pesticide, is produced by the company of Tondong, Meng, and has an active ingredient glyphosate isopropylamine salt content of 41 percent and a water aqua formulation. The recommended dosage in the field is 150-250 mL/mu, 200 mL/mu (the content of active ingredients is 82 g/mu) is taken, and 30L/mu of water is added for spraying.

Experimental treatment 4 treatments were set up:

(1) the transgenic corn is not sprayed with herbicide;

(2) spraying a target herbicide on the transgenic corn;

(3) the corresponding non-transgenic corn is not sprayed with herbicide;

(4) spraying a target herbicide on the corresponding non-transgenic corn;

the herbicide spraying dosage is 1 time (1 x, the content of active ingredients is 82 g/mu), 2 times (2 x, the content of active ingredients is 164 g/mu) and 4 times (4 x, the content of active ingredients is 328 g/mu) of the spraying clear water (0 x) and the recommended dosage in the field respectively.

And (3) character investigation: the seedling rate, plant height (the highest 5 plants were selected), and phytotoxicity symptoms (the lowest 5 plants were selected) were investigated and recorded at 1 week, 2 weeks, and 4 weeks after the application, respectively.

(1) If the phytotoxicity can be counted or measured, it is expressed in absolute numbers, such as the number of plants (number of sprayed plants, number of dead plants) or the height of the plants (the highest 5 plants are selected).

(2) After 1 week of application, each treatment was rated according to Table 4.

TABLE 4 grading table of phytotoxicity symptoms of herbicides

The herbicide damage rate was calculated by formula (1).

In the formula:

x-damage in percent (%);

n-number of victim strains at the same level;

s-number of levels;

t-total number of plants;

m-highest level.

After spraying 0-fold glyphosate solution for 1 week, both transformants and non-transgenic recipient controls were able to grow and survive, but weeds were bushy and corn was poorly grown (fig. 4A, C). After 1 week of spraying glyphosate solution with the medium amount of 1 time of the recommended field dosage, the non-transgenic receptor control corn and weeds are completely dried and dead (figure 4B), the plants of the transformant can normally grow, and the corn grows better than the corn without spraying the herbicide due to the complete killing of the weeds (figure 4D). With the lapse of time, the transformant plants sprayed with different doses of glyphosate still grew normally after 4 weeks of spraying the glyphosate solution, and the appearance was the same under different doses.

The phytotoxicity test results are shown in table 5, and the plants died without forming live seedlings when the control receptor 18599 was sprayed with 1-fold the dose of herbicide. The transformation event Ttrans-4 is not completely survived after being investigated for 1 week after being sprayed, the damage rate is 0 percent, and the plant height has no significant difference between clear water treatment and herbicide treatment; no phytotoxicity or plant death occurs in 2 weeks and 4 weeks after the pesticide application, and the plant growth is not inhibited.

TABLE 5 tolerance Performance of T anti-4 to Glyphosate

The values are expressed as mean ± standard deviation of 3 biological replicates, and the data were tested for significance by LSD between different spray doses in the same column (α ═ 0.05).

These results indicate that the transformation event, Tanti-4, has a strong tolerance to glyphosate. When the recommended dosage is treated by 4 times, the phytotoxicity rate is still 0, and the tolerance level is excellent. Thus, transformation event tadiax 4 can protect corn from damage caused by herbicides. Weeds in corn fields can be controlled by planting transformation event tad-4 and spraying the appropriate dose of herbicide.

Example 7 method for generating maize plants tolerant to glyphosate herbicide Using transformation event Tanti-4

Early tests show that the resistance of the T-resistant-4 and other agronomic characters are excellent, so that the event is used for transforming and improving backbone parents 0151, 08-641, G533 and the like which are very wide in production in southwest areas so as to improve the herbicide resistance of the backbone parents. In 2010, the first filial generation is made, then backcrossed with the receptor parent for 3 generations, and then selfed for more than 4 generations, so that the material is basically stable. Through two-field shuttling breeding in Sichuan province and Hainan province, two generations of seeds are planted every year. And (3) selecting the herbicide-resistant glyphosate with a target character at high concentration (4 times of the recommended dosage of the glyphosate) in each generation, and selecting excellent single plants and ear rows for seed reservation by combining other agronomic characters until the characters are stably purified.

In addition, T-anti-4 stable inbred lines can also be combined into hybrid combinations. The new transgenic corn combination 16-1 is formed by selecting and matching a backbone inbred line P9325 as a female parent and a backbone parent 0151 as a background and a parent containing a transformation event T anti-4 as a male parent in 2015; the new corn combination 16-1 is participated in the 2016 new corn combination ratio test, the average yield is 570.68 kg/mu, and the yield is increased by 9.2 percent compared with the control yield of 30 percent.

The method provided by the invention can be used for detecting the T-resistance-4 in the processes of parent improvement and hybridization combination and assembly to provide a molecular auxiliary means for variety breeding, and can also be used for identifying whether the corn variety contains the T-resistance-4 transformation event. The following method is a specific example for identifying T anti-4.

Molecular assays were performed on inbred T anti-4-0151 and new transgenic maize combination 16-1 obtained by parental 0151 modification to confirm that transformation event T anti-4 was included in both materials. PCR primer pairs were designed based on gene sequence to determine the presence of the transformation event T anti-4 by detecting the presence of two boundaries on the left and right of the transformation event. Wherein the primer sequences for detecting the left boundary are respectively SEQ ID NO: 8 and SEQ ID NO: 9, the primer sequences detected by the left border are respectively SEQ ID NO: 10 and SEQ ID NO: 11. extracting genome DNA from leaves of a corn plant, and amplifying according to the following PCR parameters:

and (3) PCR reaction system:

the reaction procedure is as follows:

94 ℃ for 5 min; (94 ℃, 30 sec; 55 ℃, 30 sec; 72 ℃, 1.0 min). times.35 cycles; 72 ℃ for 7 min; 4 ℃ for 5 min.

Taking the PCR product to detect in 1% (w/v)1 XTAE agarose gel electrophoresis.

The results are shown in FIG. 5. Samples of T anti-4-0151 and 16-1 were able to amplify the corresponding target bands, whereas maize samples with the same background but without the T anti-4 event were unable to amplify the bands, demonstrating that the T anti-4 event was successfully transferred into parental 0151 and that the hybrid combination 16-1 formulated therewith also included the T anti-4 event.

The herbicide tolerance of Tanti-4-0151 and 16-1 was identified by reference to example 7, and the results are shown in Table 6. The herbicide did not cause damage to the Ttrans-4-0151 and 16-1 materials, but caused death of the control material 0151 and Tanbo 30 without Ttrans-4, indicating that the use of the transformation event Ttrans-4 can produce maize plants that are tolerant to glyphosate herbicide.

TABLE 6 tolerance Performance of T anti-4-0151 and 16-1 to glyphosate

The values are expressed as mean ± standard deviation of 3 biological replicates and the data in the same column are tested for significance by LSD (α ═ 0.05).

EXAMPLE 8 detection of transformation event Tanti-4

Such as agricultural or commodity products can be produced from transgenic corn event tad-4. If a sufficient amount of expression is detected in the agricultural or commodity product, the agricultural or commodity product is expected to contain a nucleotide sequence capable of diagnosing the presence of transgenic corn event T anti-4 material in the agricultural or commodity product. Such agricultural or commercial products include, but are not limited to, corn oil, corn meal, corn flour, corn gluten, corn tortillas, corn starch, and any other food product intended for consumption by an animal as a food source, or otherwise for cosmetic use as an ingredient in a bulking or cosmetic composition, and the like. Nucleic acid detection methods and/or kits based on probe or primer pairs can be developed to detect nucleic acid sequences such as SEQ ID NOs: 1 or SEQ ID NO: 2, wherein the probe sequence or primer sequence is selected from the group consisting of SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6 and SEQ ID NO: 7 to diagnose the presence of transgenic maize event T anti-4.

In conclusion, the transgenic corn event Tanti-4 has higher tolerance to glyphosate herbicide, and the application of the Tanti-4 can quickly cultivate and produce plants or products with herbicide tolerance.

Finally, it should be noted that the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, 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 may be made on 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 method of protecting corn plants from herbicide-induced damage comprising applying an effective dose of a glyphosate herbicide 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, 399-: 2, or the genome of said transgenic maize plant comprises SEQ ID NO: 5; the transgenic corn plants have tolerance to glyphosate herbicides.

2. A method of controlling weeds in a field planted with corn plants, comprising applying to the field planted with at least one transgenic corn plant comprising in its genome, in order, SEQ ID NO: 1. SEQ ID NO: 5, 399-: 2, or the genome of said transgenic maize plant comprises SEQ ID NO: 5; the transgenic corn plants have tolerance to glyphosate herbicides.

3. A method of growing a corn plant tolerant to glyphosate herbicide comprising:

planting at least one corn seed, the genome of the corn seed comprising a nucleic acid sequence of a particular region, the nucleic acid sequence of the particular region comprising, in order, the nucleic acid sequence of SEQ ID NO: 1. SEQ ID NO: 5, 399-: 2, or the nucleic acid sequence of said specific region comprises SEQ ID NO: 5;

growing the corn seed into a corn plant;

4. A method of producing a corn plant tolerant to glyphosate herbicide comprising introducing into the genome of said corn plant the amino acid sequence of SEQ ID NO: 5, 399-4244 and such that the genome of said maize plant comprises in sequence the nucleic acid sequence of SEQ ID NO: 1. SEQ ID NO: 5, 399-: 2 or such that the genome of said maize plant comprises SEQ ID NO: and 5, selecting the corn plants tolerant to the glyphosate.

5. The method of producing a corn plant tolerant to glyphosate herbicide as claimed in claim 7, comprising:

treating said progeny plants with glyphosate herbicide;

selecting said progeny plants that are tolerant to glyphosate.

6. A composition for producing autoregfed corn event tadant-4, wherein said composition is corn flour, corn oil, corn cobs, or corn starch.

7. An agricultural or commercial product produced by transgenic corn event tadant-4, wherein the agricultural or commercial product is corn flour, corn oil, corn starch, corn gluten, corn tortilla, cosmetics, or bulking agents.