CN106119245B - Nucleic acid sequence and detection method for detecting herbicide-tolerant soybean plant DBN9001 - Google Patents

Abstract

The present invention relates to a kind of for detecting the nucleic acid sequence and its detection method of herbicide-tolerant soybean plant DBN9001, and the nucleic acid sequence of the bean plant includes SEQ ID NO:1 or its complementary series or SEQ ID NO:2 or its complementary series.Transgenic soybean event DBN9001 of the present invention has preferable tolerance to glyphosate herbicidal and glufosinate-ammonium herbicide, on yield without influence, and detection method can quickly and accurately identify in biological sample whether include transgenic soybean event DBN9001 DNA molecular.

Description

Nucleic acid sequence and detection method for herbicide-tolerant soybean plant DBN9001 test method

technical field

The present invention relates to a nucleic acid sequence for detecting herbicide-tolerant soybean plant DBN9001 and a detection method thereof, in particular to a soybean plant DBN9001 tolerant to glyphosate and glufosinate-ammonium and detecting whether specific Methods of DNA Molecules for Transgenic Soybean Event DBN9001.

Background technique

N-phosphonomethylglycine, also known as glyphosate, is a systemic, chronic broad-spectrum herbicide. Glyphosate is a competitive inhibitor of phosphoenolpyruvate (PEP), the synthetic substrate of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), and can inhibit the synthesis of PEP and 3-phosphoshikimate. The conversion of the two substrates to 5-enolpyruvylshikimic acid-3-phosphoshikimic acid under the catalysis of EPSPS blocks the synthetic pathway of shikimic acid, the precursor of aromatic amino acid synthesis, and interferes with the synthesis of proteins leading to plant and bacteria die.

Glyphosate tolerance can be achieved by expressing modified EPSPS. The modified EPSPSs have lower affinity to glyphosate, so EPSPSs maintain their catalytic activity in the presence of glyphosate, that is, glyphosate tolerance is obtained.

Soybean (Glycine max) is one of the five major crops in the world. Herbicide tolerance is an important agronomic trait in soybean production, especially tolerance to glyphosate herbicide. Soybean tolerance to glyphosate herbicides can be obtained by expressing glyphosate herbicide tolerance genes (EPSPS, CP4) in soybean plants through transgenic methods, such as soybean event GTS40-3-2, soybean event MON89788 and others.

The widespread adoption of glyphosate-tolerant farming systems and the increasing use of glyphosate have led to the prevalence of glyphosate-resistant weeds in recent years. In areas where growers are faced with glyphosate-resistant weeds or are transitioning to more difficult-to-control weed species, growers can compensate for glyphosate's weakness by mixing or alternating applications with other herbicides capable of controlling missed weeds .

Glufosinate-ammonium is a non-systemic and non-selective herbicide among the phosphinothricin herbicides. It is mainly used for the post-emergence control of annual or perennial broadleaf weeds by L-phosphinothricin (the active ingredient in glufosinate-ammonium) on glutamine synthase (an enzyme necessary for the detoxification of ammonia in plants) ) to control weeds by irreversible inhibition. Unlike glyphosate that kills roots, glufosinate kills leaves first, and can conduct transpiration in plant xylem through plant transpiration, and its quick-acting effect is between paraquat and glyphosate.

Phosphinothricin N-acetyltransferase (PAT) isolated from Streptomyces catalyzes the conversion of L-phosphinothricin to its inactive form by acetylation. Genes expressing plant-optimized versions of PAT have been used in soybeans to confer tolerance to the herbicide glufosinate in soybeans, eg soybean event A5547-127. Therefore, the use of glufosinate-ammonium herbicides in combination with glufosinate-ammonium tolerance traits can be an effective non-selective means of managing glyphosate-resistant weeds.

In the future, with the promotion and large-scale planting of transgenic insect-resistant soybeans, a small number of surviving insects/pests may develop resistance after several generations of reproduction. Genetically modified herbicide-tolerant soybeans, as non-insect-resistant genetically modified soybeans, are planted together with genetically modified insect-resistant soybeans in a certain proportion, which can delay insects/pests from developing resistance.

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 correspond to that expected based on the transcriptional regulatory elements in the introduced gene construct. 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 method maintained the transgene expression characteristics of the original transformant. Applying this pattern of strategies can ensure reliable gene expression in many varieties that are well adapted to local growing conditions.

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 would 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) or DNA hybridization using polynucleotide probes. 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 object of the present invention is to provide a nucleic acid sequence and detection method for detecting herbicide-tolerant soybean plant DBN9001, and the transgenic soybean event DBN9001 has good tolerance to glyphosate herbicide and glufosinate-ammonium herbicide , and the detection method can accurately and quickly identify whether the biological sample contains the DNA molecule of the specific transgenic soybean event DBN9001.

To achieve the above object, the present invention provides a nucleic acid molecule having the following nucleic acid sequence, said nucleic acid sequence comprising at least 11 consecutive nucleotides in SEQ ID NO: 3 or its complementary sequence, and/or SEQ ID NO: 4 or at least 11 consecutive nucleotides in its complementary sequence.

Preferably, the nucleic acid sequence comprises SEQ ID NO: 1 or its complement, and/or SEQ ID NO: 2 or its complement.

Further, the nucleic acid sequence includes SEQ ID NO: 3 or its complementary sequence, and/or SEQ ID NO: 4 or its complementary sequence.

Furthermore, the nucleic acid sequence includes SEQ ID NO: 5 or its complementary sequence.

Said SEQ ID NO: 1 or its complementary sequence is a sequence of 22 nucleotides in length near the insertion junction at the 5' end of the inserted sequence in the transgenic soybean event DBN9001, said SEQ ID NO: 1 or its complementary sequence The complementary sequence spans the flanking genomic DNA sequence of the soybean insertion site and the DNA sequence at the 5' end of the insertion sequence, including said SEQ ID NO: 1 or its complementary sequence can be identified as the presence of the transgenic soybean event DBN9001. Said SEQ ID NO: 2 or its complementary sequence is a sequence of 22 nucleotides in length near the insertion junction at the 3' end of the inserted sequence in the transgenic soybean event DBN9001, said SEQ ID NO: 2 or its complementary sequence The complementary sequence spans the DNA sequence at the 3' end of the insertion sequence and the flanking genomic DNA sequence of the soybean insertion site, including said SEQ ID NO: 2 or its complementary sequence can be identified as the presence of the transgenic soybean event DBN9001.

In the present invention, the nucleic acid sequence may be at least 11 or more contiguous polynucleotides (first nucleic acid sequence) of any part of the transgene insertion sequence in the SEQ ID NO: 3 or its complementary sequence, or the At least 11 or more contiguous polynucleotides (second nucleic acid sequence) of any part of the 5' flanking soybean genomic DNA region in said SEQ ID NO: 3 or its complementary sequence. Said nucleic acid sequence may further be homologous or complementary to a portion of said SEQ ID NO:3 comprising the entirety of said SEQ ID NO:1. When a first nucleic acid sequence and a second nucleic acid sequence are used together, these nucleic acid sequences can be used as a DNA primer pair in a DNA amplification method to generate an amplification product. The presence of transgenic soybean event DBN9001 or progeny thereof 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. As is well known to those skilled in the art, the first and second nucleic acid sequences need not consist solely of DNA, but may also include RNA, a mixture of DNA and RNA, or DNA, RNA or other nucleosides that do not serve as templates for one or more polymerases Combinations of acids or their analogs. 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 Nucleotides described in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5. When selected from the nucleotides shown in SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, the probes and primers may be at least about 21 to about 50 or more contiguous Nucleotides. Said SEQ ID NO: 3 or its complementary sequence is a sequence of 780 nucleotides in length near the insertion junction at the 5' end of the inserted sequence in the transgenic soybean event DBN9001, said SEQ ID NO: 3 or its complementary sequence Complementary sequence consists of soybean flank genomic DNA sequence (nucleotide 1-476 of SEQ ID NO:3) of 476 nucleotides, junction region sequence (nucleotide 477-476 of SEQ ID NO:3) of 39 nucleotides 515) and the 3' end DNA sequence (nucleotides 516-780 of SEQ ID NO:3) of the prGm17gTsf1 promoter sequence of 265 nucleotides, comprising said SEQ ID NO:3 or its complementary sequence can be identified for the presence of GM soybean event DBN9001.

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 complementary sequence, or the SEQ ID NO :4 or at least 11 or more contiguous polynucleotides (the fourth nucleic acid sequence) of any part of the 3' flanking soybean genomic DNA region in 4 or its complementary sequence. Said nucleic acid sequence may further be homologous or complementary to a portion of said SEQ ID NO:4 comprising the entirety of said SEQ ID NO:2. When the third nucleic acid sequence and the fourth nucleic acid sequence are used together, these nucleic acid sequences can be used as a DNA primer pair in a DNA amplification method to generate an amplification product. The presence of transgenic soybean event DBN9001 or progeny thereof can be diagnosed when the amplification product generated in the DNA amplification method using the DNA primer pair is an amplification product comprising SEQ ID NO:2. Said SEQ ID NO: 4 or its complementary sequence is a sequence of 877 nucleotides in length near the insertion junction at the 3' end of the inserted sequence in the transgenic soybean event DBN9001, said SEQ ID NO: 4 or its complementary sequence The complementary sequence consists of the phosphinothricin N-acetyltransferase cPAT sequence (nucleotide 1-183 of SEQ ID NO:4) of 183 nucleotides, the carrier spacer sequence of 21 nucleotides (SEQ ID NO: nucleotides 184-204 of 4), t35S terminator sequence of 195 nucleotides (nucleotides 205-399 of SEQ ID NO:4), nucleotides in 10 pDBN4003 construct DNA sequences (SEQ ID NO: 4 nucleotides 400-409) and the soybean integration site flanking genomic DNA sequence (SEQ ID NO: 4 410-879) of 468 nucleotides, comprising said SEQ ID NO: 4 or its complementary The sequence can be identified as the presence of transgenic soybean event DBN9001.

The SEQ ID NO:5 or its complementary sequence is a sequence of 6644 nucleotides in length characterizing the transgenic soybean event DBN9001, and its specific genome and genetic elements are shown in Table 1. The presence of the transgenic soybean event DBN9001 can be identified by including said SEQ ID NO: 5 or its complementary sequence.

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

The nucleic acid sequence or its complement can be used in a DNA amplification method to produce amplicons, the detection of which is diagnostic of the presence of transgenic soybean event DBN9001 or its progeny in a biological sample; the nucleic acid sequence or its complement Can be used in nucleotide assays to detect the presence of transgenic soybean event DBN9001 or its progeny in biological samples.

To achieve the above object, the present invention also provides a method for detecting the presence of DNA of the transgenic soybean event DBN9001 in a sample, comprising:

contacting the sample to be detected with at least two primers for amplifying the target amplification product in a nucleic acid amplification reaction;

performing a nucleic acid amplification reaction; and

detecting the presence of said target amplification product;

The target amplification product includes at least 11 consecutive nucleotides in SEQ ID NO: 3 or its complementary sequence, and/or at least 11 consecutive nucleotides in SEQ ID NO: 4 or its complementary sequence.

Further, the target amplification product includes the 1st-11th or 12th-22nd consecutive nucleotides in SEQ ID NO: 1 or its complementary sequence, and/or the 1st nucleotide in SEQ ID NO: 2 or its complementary sequence 1-11 or 12-22 consecutive nucleotides.

Further, the target amplification product includes at least one selected from the following: SEQ ID NO: 1 or its complementary sequence, SEQ ID NO: 2 or its complementary sequence, SEQ ID NO: 6 or its complementary sequence, and SEQ ID NO: 7 or its complement.

In the above technical solution, at least one of the primers includes the nucleic acid sequence or a fragment thereof or a sequence complementary thereto.

Specifically, the primers include a first primer and a second primer, the first primer is selected from SEQ ID NO:8 and SEQ ID NO:10; the second primer is selected from SEQ ID NO:9 and SEQ ID NO :11.

To achieve the above object, the present invention also provides a method for detecting the presence of DNA of the transgenic soybean event DBN9001 in a sample, comprising:

The sample to be detected is contacted with a probe comprising at least 11 consecutive nucleotides of SEQ ID NO: 3 or its complement, and/or at least 11 consecutive nucleotides of SEQ ID NO: 4 or its complement of nucleotides;

Hybridizing the sample to be detected and the probe under stringent hybridization conditions; and

Detecting the hybridization between the sample to be detected and the probe.

The stringent conditions can be hybridized at 65° C. in 6×SSC (sodium citrate), 0.5% SDS (sodium dodecyl sulfate) solution, and then mixed with 2×SSC, 0.1% SDS and 1×SSC, Wash each membrane once with 0.1% SDS.

Further, the probe includes SEQ ID NO: 1 or its complementary sequence 1-11 or 12-22 consecutive nucleotides, and/or SEQ ID NO: 2 or its complementary sequence 1-1 The 11th or 12th-22nd consecutive nucleotides.

Further, the probe comprises at least one selected from the following: SEQ ID NO: 1 or its complementary sequence, SEQ ID NO: 2 or its complementary sequence, SEQ ID NO: 6 or its complementary sequence, and SEQ ID NO :7 or its complement.

Optionally, at least one of said probes is labeled with at least one fluorophore.

To achieve the above object, the present invention also provides a method for detecting the presence of DNA of the transgenic soybean event DBN9001 in a sample, comprising:

The sample to be detected is contacted with a marker nucleic acid molecule comprising at least 11 consecutive nucleotides of SEQ ID NO: 3 or its complement, and/or SEQ ID NO: 4 or its complement At least 11 consecutive nucleotides;

Hybridizing the sample to be detected and the marker nucleic acid molecule under stringent hybridization conditions; and

Detecting the hybridization between the sample to be detected and the marker nucleic acid molecule, and then analyzing the marker-assisted breeding to determine the genetic relationship between glyphosate tolerance and/or glufosinate tolerance and the marker nucleic acid molecule are chained.

Further, the marker nucleic acid molecule includes the 1-11 or 12-22 consecutive nucleotides in SEQ ID NO: 1 or its complementary sequence, and/or the nucleotides in SEQ ID NO: 2 or its complementary sequence 1-11 or 12-22 consecutive nucleotides.

Further, the marker nucleic acid molecule comprises at least one selected from the following: SEQ ID NO: 1 or its complement, SEQ ID NO: 2 or its complement, SEQ ID NO: 6 or its complement, and SEQ ID NO: 7 or its complement.

To achieve the above object, the present invention also provides a DNA detection kit, comprising at least one DNA molecule, said DNA molecule comprising at least 11 consecutive nucleotides in the homologous sequence of SEQ ID NO:3 or its complementary sequence , and/or at least 11 consecutive nucleotides in the homologous sequence of SEQ ID NO:4 or its complementary sequence, which can be used as a specific DNA primer or probe for the transgenic soybean event DBN9001 or its progeny.

Further, the DNA molecule includes SEQ ID NO: 1 or its complementary sequence 1-11 or 12-22 consecutive nucleotides, and/or SEQ ID NO: 2 or its complementary sequence 1- The 11th or 12th-22nd consecutive nucleotides.

Furthermore, the DNA molecule includes at least one selected from the following: a homologous sequence of SEQ ID NO: 1 or its complement, a homolog of SEQ ID NO: 2 or its complement, SEQ ID NO: 6 The homologous sequence of or its complement, and the homologous sequence of SEQ ID NO: 7 or its complement.

To achieve the above object, the present invention also provides a plant cell or part, comprising a nucleic acid sequence encoding a glyphosate-tolerant EPSPS protein, a nucleic acid sequence encoding a glufosinate-ammonium-tolerant PAT protein, and a nucleic acid sequence in a specific region, The nucleic acid sequence of the specific region includes at least one selected from the following: the sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:6 and SEQ ID NO:7.

In order to achieve the above object, the present invention also provides a method for producing soybean plants tolerant to glyphosate herbicides and/or glufosinate-ammonium herbicides, comprising introducing into the genome of the soybean plants encoding glyphosate The nucleic acid sequence of the phosphine-tolerant EPSPS protein and/or the nucleic acid sequence encoding the glufosinate-ammonium-tolerant PAT protein, and the nucleic acid sequence of the specific region, the nucleic acid sequence of the specific region is selected from SEQ ID NO:1, SEQ ID NO :2, at least one nucleic acid sequence in the sequences shown in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7.

Specifically, the method for producing soybean plants tolerant to glyphosate herbicides and/or glufosinate-ammonium herbicides comprises:

Transgenic soybean event DBN9001 first parent soybean plants tolerant to glyphosate and/or glufosinate herbicides were compared with second parent soybean plants lacking glyphosate and/or glufosinate tolerance Sexual hybridization, resulting in a large number of progeny plants;

Treating said progeny plants with glyphosate herbicide and/or glufosinate-ammonium herbicide; and

The progeny plants are selected for tolerance to glyphosate and/or glufosinate.

To achieve the above object, the present invention also provides a method of cultivating soybean plants tolerant to glyphosate herbicides and/or glufosinate-ammonium herbicides, comprising:

Planting at least one soybean seed, the genome of the soybean seed includes a nucleic acid sequence encoding a glyphosate-tolerant EPSPS protein and/or a nucleic acid sequence encoding a glufosinate-ammonium-tolerant PAT protein, and a nucleic acid sequence in a specific region;

growing the soybean seeds into soybean plants; and

Spraying the soybean plants with an effective dose of glyphosate herbicide and/or glufosinate-ammonium herbicide, and harvesting plants with weakened plant damage compared with other plants that do not have the nucleic acid sequence of the specific region;

The nucleic acid sequence of the specific region 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 At least one nucleic acid sequence in the sequence shown.

To achieve the above object, the present invention also provides a method for protecting plants from damage caused by herbicides, comprising applying herbicides containing effective doses of glyphosate and/or glufosinate-ammonium to planting at least one transgenic soybean In a field of plants, the transgenic soybean plant comprises in its genome a gene 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 at least one nucleic acid sequence in the sequence shown in SEQ ID NO: 7, the transgenic soybean plant has tolerance to glyphosate herbicide and/or glufosinate-ammonium herbicide.

To achieve the above object, the present invention also provides a method of controlling field weeds, comprising applying herbicides containing effective doses of glyphosate and/or glufosinate-ammonium to a field where at least one transgenic soybean plant is planted, so that The transgenic soybean plant comprises in its genome a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO At least one nucleic acid sequence in the sequence shown in 7, the transgenic soybean plant has tolerance to glyphosate herbicide and/or glufosinate-ammonium herbicide.

To achieve the above object, the present invention also provides a method for controlling glyphosate-resistant weeds in the field of glyphosate-tolerant plants, comprising applying a herbicide containing an effective dose of glufosinate to planting at least one In a field of a glyphosate-tolerant transgenic soybean plant comprising in its genome a gene 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 at least one nucleic acid sequence in the sequences shown in SEQ ID NO: 7, the glyphosate-tolerant transgenic soybean plant has paraglutamic acid ammonium at the same time Phosphate herbicide tolerance.

To achieve the above object, the present invention also provides a method for delaying insect resistance, comprising planting at least one transgenic soybean plant tolerant to glyphosate and/or glufosinate in a field where insect-resistant soybean plants are grown , the transgenic soybean plant with tolerance to glyphosate and/or glufosinate contains in its genome a group selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, At least one nucleic acid sequence in the sequences shown in SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7.

To achieve the above object, the present invention also provides an agricultural product or commodity comprising a polynucleotide of SEQ ID NO: 1 or SEQ ID NO: 2, wherein the agricultural product or commodity is lecithin, fatty acid, glycerin, sterol, soybean flakes, soy flour, soy protein or concentrates thereof, soy oil, soy protein fiber, soy milk curd or tofu.

In the nucleic acid sequence of the present invention for detecting herbicide-tolerant soybean plant DBN9001 and its detection method, 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 , the terms are understood according to conventional usage by those of ordinary skill in the art.

The "soybean" refers to soybean (Glycine max), and includes all plant varieties that can be crossed with soybean, including wild soybean species.

The terms "comprising", "including" mean "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 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 through 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 of sequence that is located immediately upstream or downstream of and adjacent to the original exogenously inserted DNA molecule. When the flanking region is located downstream, it may also be referred to as "left border flank" or "3' flank" or "3' genomic border region" or "genomic 3' border sequence" and the like. When the flanking region is located upstream, it may also be referred to as "right border flank" or "5' flank" or "5' genomic border region" or "genomic 5' border sequence" and the like.

Transformation procedures that result in random integration of exogenous DNA will result in transformants containing different flanking regions that are specific to each transformant. When recombinant DNA is introduced into plants by conventional crossing, its flanking regions are usually not altered. Transformants will also contain unique junctions between the heterologous insert DNA and the segment 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" and "junction region" refer to DNA including junction points.

The present invention provides a transgenic soybean event designated DBN9001, which is soybean plant DBN9001, including plants and seeds of transgenic soybean event DBN9001 and plant cells or regenerable parts thereof, and progeny thereof, said transgenic soybean event DBN9001 Plant parts of soybean event DBN9001, including but not limited to cells, pollen, ovules, flowers, buds, roots, stems, leaves, pods and products from soybean plant DBN9001, such as soybean cake, meal and oil, specifically lecithin, Fatty acids, glycerin, sterols, edible oils, defatted soy flakes, soy flour including defatted and roasted, soy milk curd, tofu, soy protein concentrate, isolated soy protein, hydrolyzed vegetable protein, textured soy protein and soy protein fibers.

The transgenic soybean event DBN9001 of the present invention comprises a DNA construct that, when expressed in plant cells, acquires tolerance to glyphosate herbicides and glufosinate-ammonium herbicides. The DNA construct comprises two expression cassettes in tandem, the first expression cassette comprising a suitable promoter for expression in plants and a suitable polyadenylation signal sequence, said promoter being operably linked to the Gene for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), which confers tolerance to the glyphosate herbicide. The second expression cassette comprises a suitable promoter for expression in plants operably linked to an enzyme encoding phosphinothricin N-acetyltransferase (PAT) and a suitable polyadenylation signal sequence The gene, the nucleic acid sequence of the PAT protein has tolerance to glufosinate-ammonium herbicide. Further, the promoter may 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, Tsf1 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 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 Polyadenylation signal sequence of nopaline synthase (NOS) gene, derived from cauliflower mosaic virus (CaMV) 35S terminator, derived from pea ribulose-1,5-bisphosphate carboxylase/oxygenase E9 terminator, polyadenylation signal sequence derived from protease inhibitor II (PINII) gene and polyadenylation signal sequence derived from 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 and/or PAT 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' retained sequence target To the endoplasmic reticulum.

The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene can be isolated from the Agrobacterium tumefaciens sp. CP4 strain, and can be changed by optimizing codons or in other ways Polynucleotides encoding EPSPS for the purpose of increasing the stability and availability of transcripts in transformed cells. The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene can also serve as a selectable marker gene.

The "glyphosate" refers to N-phosphonomethylglycine and its salts, and the treatment with "glyphosate herbicide" refers to the use of any herbicide formulation containing glyphosate. The selection of the application rate of a glyphosate formulation to achieve a biologically effective dose is within the skill of the ordinary agronomist. Treatment of a field containing plant material derived from herbicide-tolerant soybean plant DBN9001 with any of the herbicide formulations containing glyphosate will control the growth of weeds in said field without affecting the Growth or yield of plant material from receptive soybean plant DBN9001.

The phosphinothricin N-acetyltransferase (PAT) gene isolated from Streptomyces viridochromogenes catalyzes the conversion of L-phosphinothricin to its inactive form through acetylation to endow plants with Tolerance of ammonium phosphonate herbicides. Phosphinothricin (PTC, 2-amino-4-methylphosphonobutyric acid) is an inhibitor of glutamine synthetase. PTC is the structural unit of the antibiotic 2-amino-4-methylphosphonyl-alanyl-alanine. This tripeptide (PTT) has anti-gram-positive and gram-negative bacteria and anti-fungal Botrytis cinerea (Botrytis cinerea). cinerea) activity. The phosphinothricin N-acetyltransferase (PAT) gene can also serve as a selectable marker gene.

The "glufosinate-ammonium", also known as glufosinate, refers to 2-amino-4-[hydroxyl (methyl) phosphono] ammonium butyrate, and the treatment with "glufosinate-ammonium herbicide" refers to the use of any one containing Glufosinate-ammonium herbicide formulations. 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 herbicide-tolerant soybean plant DBN9001 with any of the herbicide formulations containing glufosinate will control the growth of weeds in said field without affecting the Growth or yield of plant material from receptive soybean plant DBN9001.

The DNA construct is introduced into the plant using a transformation method 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.

The gene bombardment transformation method is bombarding plant cells with a vector containing foreign DNA (particle-mediated biolistic transformation).

The pollen tube channel transformation method utilizes the natural pollen tube channel (also known as pollen tube guiding tissue) formed after plant pollination to carry exogenous DNA into the embryo sac through the nucellus channel.

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 a nucleic acid expression cassette containing the gene of interest, inserted transgenically into the plant genome to produce a plant population, regenerating said plant population , and select specific plants with features inserted into specific genomic loci. The term "event" includes the original transformant of heterologous DNA and the progeny of that transformant. The term "event" also includes the progeny of a sexual cross between a transformant and an individual of another breed containing heterologous DNA, even after repeated backcrosses with the backcross parent, the insert 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 transformant comprising the insert DNA and flanking genomic sequences in close proximity to the insert DNA, which DNA sequence is expected to be transferred to progeny derived from cells containing the insert DNA The parental line (such as the original transformant and its progeny produced by 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 soybean event DBN9001 tolerant to glyphosate herbicides and glufosinate-ammonium herbicides was developed by first sexually crossing a first parent soybean plant with a second parent soybean plant, resulting in a diverse second A generation of progeny plants, the first parent soybean plant consisting of soybean plants grown from transgenic soybean event DBN9001 and its progeny obtained by using the glyphosate herbicides and grasses of the present invention Transformed with an expression cassette for tolerance to glyphosate herbicides, the second parent soybean plant lacks tolerance to glyphosate herbicides and/or glufosinate-ammonium herbicides; then selects for glyphosate herbicides And/or progeny plants tolerant to the application of glufosinate-ammonium herbicide can breed soybean plants tolerant to glyphosate-ammonium herbicide and glufosinate-ammonium herbicide. These steps may further comprise backcrossing progeny plants tolerant to the application of glyphosate herbicides and/or glufosinate-ammonium herbicides to the second parent soybean plant or the third parent soybean plant, followed by the application of grass Glyphosate herbicides, glufosinate-ammonium herbicides, or through the identification of molecular markers associated with traits (such as DNA molecules containing the junction sites identified at the 5' and 3' ends of the inserted sequence in transgenic soybean event DBN9001) Progeny are selected to produce soybean plants tolerant to glyphosate and glufosinate herbicides.

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.

Bt gene-transformed soybeans can kill insects/pests such as Lepidoptera, but there are also a small number of surviving insects/pests. After several generations of reproduction, resistant insects/pests against Bt protein may be produced. In order to solve the problem of insect/pest resistance, the US Environmental Protection Agency has given the following guidance for the use of genetically modified crops to provide a certain percentage of refuge soybeans (which can be non-insect-resistant genetically modified soybeans (such as herbicide-tolerant genetically modified soybeans) , or GM soybeans resistant to non-target pests, or non-GM soybeans). When the vast majority of insects/pests were killed on the corresponding GM insect-resistant soybeans, some insects/pests did not die on the shelter soybeans , to ensure that the non-resistant insects/pests dominate the population. In this way, even if there are a small number of surviving resistant insects/pests, the resistance genes are significantly diluted after mating with the dominant non-resistant insects/pests.

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 soybean event DBN9001, whether the genomic DNA is from the transgenic soybean event DBN9001 or the seed or derived from the transgene Plant or seed or extract of soybean event DBN9001. 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.

Probes and primers are generally 11 polynucleotides or more in length, preferably 18 polynucleotides or more, more preferably 24 polynucleotides or more, most preferably 30 polynucleotides in length sour or more. Such probes and primers hybridize specifically to the target sequence under highly stringent hybridization conditions. Although probes that are different from the target DNA sequence and maintain the ability to hybridize to the target DNA sequence can be designed by conventional methods, preferably, the probes and primers in the present invention have complete DNA sequences with the continuous nucleic acid of the target sequence identity.

The primers and probes based on the flanking genomic DNA and the insert sequence of the present invention can be determined by conventional methods, for example, by isolating the corresponding DNA molecule from the plant material derived from the transgenic soybean event DBN9001, and determining the nucleic acid sequence of the DNA molecule. The DNA molecule contains transgene insertion sequence and soybean genome flanking sequence, and the fragments of the DNA molecule can be used as primers or probes.

The nucleic acid probes and primers of the invention hybridize to target DNA sequences under stringent conditions. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of DNA derived from transgenic soybean event DBN9001 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, the preferred marker nucleic acid molecule has SEQ ID NO: 1, SEQ ID NO: 2, 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 has 80% to 100% to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 6 or SEQ ID NO: 7 or its complementary sequence, or any fragment of the above sequence % or 90% to 100% sequence identity. SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:6 and SEQ ID NO:7 can be used as markers in plant breeding methods to identify 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 probe or 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, to determine whether soybean plants have been sexually crossed from soybeans containing the transgenic soybean event DBN9001 of the present invention, or whether soybean samples collected from the field contain transgenic soybean event DBN9001, or whether soybean extracts, such as meal, meal or oil, contain Transgenic soybean event DBN9001, DNA extracted from soybean 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 soybean event DBN9001. 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 was of a length and sequence that was also diagnostic for the transgenic soybean event DBN9001. 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 up to 22 kb of genomic DNA and up to 42 kb of phage DNA. These methods, as well as other DNA amplification methods known in the art, can be used in the present invention. The inserted exogenous DNA sequence and the flanking DNA sequence from the transgenic soybean event DBN9001 can be amplified from the genome of the transgenic soybean event DBN9001 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. Kit containing DNA primers homologous or complementary to any part of the soybean genomic region of SEQ ID NO:3 or SEQ ID NO:4, and to any part of the transgene insertion region of SEQ ID NO:5 provided by the present invention. A primer pair specifically identified as useful in DNA amplification methods is SEQ ID NO: 8 and SEQ ID NO: 9, which amplifies diagnostic amplification of homology to a portion of the 5' transgene/genomic region of transgenic soybean event DBN9001 sub, wherein the amplicon comprises SEQ ID NO: 1. 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 Genetic Bit 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.

The fluorescence polarization phenomenon described by Chen et al. (Genome Res. 9:492-498, 1999) is also a method that can be used to detect amplicons of the invention. 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 sequence) 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 herbicide tolerant transgenic soybean event DBN9001 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.

Tyangi et al. (Nat. Biotech. 14:303-308, 1996) describe the use of molecular markers in sequence detection. Briefly described below, a FRET oligonucleotide probe is designed to span 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 presence of DNA from transgenic soybean event DBN9001 in a sample, and can also be used to breed soybean plants containing DNA from transgenic soybean event DBN9001. The kit may contain DNA primers or probes homologous to or complementary to at least a portion of SEQ ID NO: 1, 2, 3, 4 or 5, or other DNA primers or probes homologous to or complementary to at least a portion of SEQ ID NO: 1, 2, 3, 4 or 5 The DNA contained in the transgenic genetic element is complementary to the DNA, which 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 soybean genome contained in the soybean genome and illustrated in Figure 1 and Table 1 comprises: the soybean plant DBN9001 flanking genomic region located at the 5' end of the transgene insert sequence, the first expression cassette consisting of Soybean Tsf1 gene (encoding elongation factor EF-1α) promoter (prGm17gTsf1), operably linked to the coding sequence of Arabidopsis EPSPS chloroplast transit peptide (spAtCTP2), operably linked to the glyphosate-resistant Agrobacterium CP4 strain Receptive 5-enol-pyruvylshikimate-3-phosphate synthase (cEPSPS), operably linked to the 3' untranslated sequence (tPse9 ), a second expression cassette consisting of a tandem repeat of the cauliflower mosaic virus 35S promoter (pr35S) containing an enhancer region, operably linked to the glufosinate-resistant phosphinothricin N of Streptomyces - acetyltransferase (cPAT), and is operably linked to the cauliflower mosaic virus 35S terminator (t35S), part of the insert sequence from the left border region (LB) of Agrobacterium, and the transgene insert The soybean plant DBN9001 flanking genomic region at the 3' end of the sequence (SEQ ID NO:5). In the DNA amplification method, the DNA molecules used as primers can be any part of the transgene insert sequence in soybean plant DBN9001, or any part of the DNA region flanking the soybean genome in the transgenic soybean event DBN9001.

GM soybean event DBN9001 can be combined with other GM soybean varieties, such as soybeans with herbicide tolerance (such as 2,4-D, dicamba, etc.), or GM soybean varieties carrying other insect resistance genes (such as Cry1Ac, Cry2Ab, etc.) . Various combinations of all these different transgenic events, bred together with the transgenic soybean event DBN9001 of the present invention, can provide improved hybrid transgenic soybean varieties resistant to various insect pests and tolerant to various herbicides. Compared with non-transgenic varieties and single-trait transgenic varieties, these varieties can show more excellent characteristics such as yield improvement.

The invention provides a nucleic acid sequence and detection method for detecting herbicide-tolerant soybean plant DBN9001. The transgenic soybean event DBN9001 is tolerant to the phytotoxic effect of agricultural herbicides containing glyphosate and/or glufosinate-ammonium. The dual-trait soybean plants express the glyphosate-resistant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) protein of Agrobacterium strain CP4, which confers tolerance to glyphosate in plants, And express the Streptomyces glufosinate-resistant phosphinothricin N-acetyltransferase (PAT) protein, which confers plant tolerance to glufosinate. Dual-trait soybeans have the following advantages: 1) the ability to apply glyphosate-containing agricultural herbicides to soybean crops for broad-spectrum weed control; 2) use of glufosinate-ammonium herbicides in combination with glufosinate-ammonium Glyphosate herbicide mixed or used alternately) can be used as a non-selective means to effectively manage glyphosate-resistant weeds; 3) herbicide-tolerant transgenic soybeans are non-insect-resistant transgenic soybeans, and transgenic insect-resistant soybeans Planting together in a certain proportion can delay the resistance of insects/pests; 4) The yield of soybeans is not reduced. In addition, genes encoding glyphosate tolerance and glufosinate-ammonium tolerance traits are linked on the same DNA segment and present at a single locus in the genome of transgenic soybean event DBN9001, which provides enhanced breeding efficiency and Enables molecular markers to track transgene insertions in breeding populations and their progeny. Meanwhile, in the detection method of the present invention, SEQ ID NO: 1 or its complementary sequence, SEQ ID NO: 2 or its complementary sequence, SEQ ID NO: 6 or its complementary sequence, or SEQ ID NO: 7 or its complementary sequence can be used as DNA primer or probe The aim is to produce an amplification product that is diagnosed as the transgenic soybean event DBN9001 or its progeny, and can quickly, accurately and stably identify the existence of the plant material derived from the transgenic soybean event DBN9001.

sequence description

SEQ ID NO: 1 A sequence of 22 nucleotides in length near the insertion junction at the 5' end of the insertion sequence in transgenic soybean event DBN9001, wherein the 1st-11th nucleotides and the 12th-22nd core The nucleotides are respectively located on both sides of the insertion site on the soybean genome;

SEQ ID NO:2 A sequence of 22 nucleotides in length near the insertion junction at the 3' end of the insertion sequence in transgenic soybean event DBN9001, wherein the 1st-11th nucleotides and the 12th-22nd core The nucleotides are respectively located on both sides of the insertion site on the soybean genome;

SEQ ID NO:3 A sequence of 780 nucleotides in length near the insertion junction at the 5' end of the insertion sequence in transgenic soybean event DBN9001;

SEQ ID NO: 4 A sequence of 877 nucleotides in length near the insertion junction at the 3' end of the insertion sequence in transgenic soybean event DBN9001;

SEQ ID NO:5 whole T-DNA sequence, 5' and 3' flanking soybean genome sequence;

SEQ ID NO:6 is located at the sequence within SEQ ID NO:3, spanning the nucleotide sequence of the junction region sequence between the pDBN4003 construct and the genome and the prGm17gTsf1 promoter;

SEQ ID NO:7 is located in the sequence within SEQ ID NO:4, spanning the nucleotide sequence in the phosphinothricin N-acetyltransferase cPAT, the t35S transcription terminator sequence and the pDBN4003 construct DNA sequence;

SEQ ID NO:8 amplifies the first primer of SEQ ID NO:3;

SEQ ID NO:9 amplifies the second primer of SEQ ID NO:3;

SEQ ID NO:10 amplifies the first primer of SEQ ID NO:4;

SEQ ID NO:11 amplifies the second primer of SEQ ID NO:4;

Primer on SEQ ID NO:12 5' flanking genomic sequence;

The primer located on T-DNA paired with SEQ ID NO:13 and SEQ ID NO:12;

The primer on the genomic sequence of the flanking genome of SEQ ID NO:14 3', which can be paired with SEQ ID NO:12 to detect whether the transgene is homozygous or heterozygous;

The primer located on T-DNA paired with SEQ ID NO:15 and SEQ ID NO:14;

SEQ ID NO:16 Taqman detects primer 1 for EPSPS;

SEQ ID NO:17 Taqman detects the primer 2 of EPSPS;

SEQ ID NO:18 Taqman probe 1 for detecting EPSPS;

SEQ ID NO:19 Taqman detects primer 3 of PAT;

SEQ ID NO:20 Taqman detects primer 4 of PAT;

SEQ ID NO:21 Taqman detects the probe 2 of PAT;

SEQ ID NO:22 The first primer of soybean endogenous gene ubiquitin protein (Ubiquitin);

SEQ ID NO:23 The second primer of soybean endogenous gene ubiquitin protein (Ubiquitin);

A probe for EPSPS in SEQ ID NO:24 Southern blot hybridization detection;

The probe of PAT in SEQ ID NO:25 Southern blotting hybridization detection;

The primer of SEQ ID NO:26 located on the T-DNA is consistent with the direction of SEQ ID NO:13;

The primer of SEQ ID NO:27 located on the T-DNA is opposite to the direction of SEQ ID NO:13, and is used to obtain flanking sequences;

The primer of SEQ ID NO:28 located on the T-DNA is opposite to the direction of SEQ ID NO:13, and is used to obtain flanking sequences;

SEQ ID NO:29 is a primer located on the T-DNA, which is in the same direction as SEQ ID NO:15;

The primer of SEQ ID NO:30 located on the T-DNA is opposite to the direction of SEQ ID NO:15, and is used to obtain flanking sequences;

The primer of SEQ ID NO:31 located on the T-DNA, in the opposite direction to that of SEQ ID NO:15, was used to obtain flanking sequences.

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

Fig. 1 is a schematic diagram of the structure of the transgene insertion sequence and soybean genome junction for detecting the nucleic acid sequence of the herbicide-tolerant soybean plant DBN9001 and the detection method of the present invention;

Fig. 2 is a schematic structural view of the recombinant expression vector pDBN4003 used for detecting the nucleic acid sequence of the herbicide-tolerant soybean plant DBN9001 and the detection method of the present invention.

Detailed ways

The technical scheme for detecting the nucleic acid sequence of the herbicide-tolerant soybean plant DBN9001 and the detection method thereof of the present invention is further illustrated by specific examples below.

The first embodiment, cloning and transformation

1.1. Vector cloning

The recombinant expression vector pDBN4003 (as shown in Figure 2) was constructed using standard gene cloning techniques. The vector pDBN4003 contains two tandem transgene expression cassettes, the first expression cassette is operably linked to the coding sequence of the Arabidopsis EPSPS chloroplast transit peptide by the soybean Tsf1 gene (encoding elongation factor EF-1α) promoter (prGm17gTsf1) (spAtCTP2), operably linked to the glyphosate-tolerant 5-enol-pyruvylshikimate-3-phosphate synthase (cEPSPS) of Agrobacterium sp. CP4 strain, operably linked to sugar-1,5-bisphosphate carboxylase on the 3' untranslated sequence (tPse9); the second expression cassette consists of a tandem repeat of the cauliflower mosaic virus 35S promoter (pr35S) containing an enhancer region, which can The composition is operatively linked to the glufosinate-tolerant phosphinothricin N-acetyltransferase (cPAT) of Streptomyces and is operably linked to the cauliflower mosaic virus 35S terminator (t35S).

The vector pDBN4003 was transformed into Agrobacterium LBA4404 (Invitrgen, Chicago, USA; Cat.No: 18313-015) with liquid nitrogen method, and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) Transformed cells are screened for selectable markers.

1.2. Plant transformation

The conventional Agrobacterium infection method was used for transformation, and the aseptically cultured soybean cotyledon node tissue was co-cultured with the Agrobacterium described in Example 1.1 to transfer the T-DNA in the constructed recombinant expression vector pDBN4003 into soybean genome to generate the transgenic soybean event DBN9001.

For Agrobacterium-mediated soybean transformation, briefly, mature soybean seeds were germinated in soybean germination medium (B5 salt 3.1 g/L, B5 vitamin, sucrose 20 g/L, agar 8 g/L, pH 5.6) , inoculate the seeds on the germination medium, and cultivate according to the following conditions: temperature 25±1°C; photoperiod (light/dark) 16/8h. After 4-6 days of germination, get the expanded aseptic seedling of soybean at the bright green cotyledon node, cut off the hypocotyl at 3-4 mm below the cotyledon node, cut the cotyledon longitudinally, and remove the terminal bud, lateral bud and seed root. Wound the cotyledon node with the back of a scalpel, and contact the wounded cotyledon node tissue with the Agrobacterium suspension, wherein the Agrobacterium can transfer the nucleotide sequence of the EPSPS gene and the nucleotide sequence of the PAT gene to the wounded Cotyledon node tissue (step 1: infection step). In this step, the cotyledon node tissue is preferably immersed in the Agrobacterium suspension (OD ₆₆₀ =0.5-0.8, infection medium (MS salt 2.15g/L, B5 vitamin, sucrose 20g/L, glucose 10g/L, acetosyring Ketone (AS) 40mg/L, 2-morpholineethanesulfonic acid (MES) 4g/L, zeatin (ZT) 2mg/L, pH5.3) to initiate infection. Cotyledon node tissue and Agrobacterium co-culture for a period Period (3 days) (step 2: co-cultivation step). Preferably, the cotyledon node tissue is in solid medium (MS salt 4.3g/L, B5 vitamin, sucrose 20g/L, glucose 10g/L, 2-morpholineethanesulfonic acid (MES) 4g/L, zeatin 2mg/L, agar 8g/L, pH 5.6). After this co-cultivation phase, there is an optional "recovery" step. In In the "recovery" step, recovery medium (B5 salt 3.1g/L, B5 vitamin, 2-morpholineethanesulfonic acid (MES) 1g/L, sucrose 30g/L, zeatin (ZT) 2mg/L, agar 8g /L, cephalosporin 150mg/L, glutamic acid 100mg/L, aspartic acid 100mg/L, pH5.6) there is at least one antibiotic known to inhibit the growth of Agrobacterium (cephalosporin 150-250mg/L L) without addition of a selection agent for plant transformants (step 3: recovery step). Preferably, tissue pieces regenerated from the cotyledonary nodes are cultured on solid media with antibiotics but no selection agent to eliminate Agrobacterium and provide protection for infection The cells provide a recovery period. Next, the tissue pieces regenerated from the cotyledonary nodes are cultured on a medium containing a selection agent (glyphosate) and selected for growing transformed calli (step 4: selection step). Preferably, the cotyledonal nodes are regenerated The tissue blocks were screened in solid media with selective agents (B5 salt 3.1g/L, B5 vitamins, 2-morpholineethanesulfonic acid (MES) 1g/L, sucrose 30g/L, 6-benzyl adenine (6 -BAP) 1mg/L, agar 8g/L, cephalosporin 150mg/L, glutamic acid 100mg/L, aspartic acid 100mg/L, glyphosate isopropylamine salt 10mg/L, pH5.6) , resulting in transformed cells that can continue to grow. Then, transformed cells regenerate into plants (step 5: regeneration step), preferably, tissue pieces regenerated from cotyledonary nodes grown on medium containing the selection agent are grown on solid medium (B5 Differentiation medium and B5 rooting medium) to regenerate plants.

The resistant tissue blocks obtained by screening were transferred to the B5 differentiation medium (B5 salt 3.1g/L, B5 vitamins, 2-morpholineethanesulfonic acid (MES) 1g/L, sucrose 30g/L, zeatin (ZT) 1mg/L, agar 8g/L, cephalosporin 150mg/L, glutamic acid 50mg/L, aspartic acid 50mg/L, gibberellin 1mg/L, auxin 1mg/L, glyphosate isopropylamine salt 10mg/L, pH 5.6), cultured and differentiated at 25°C. Differentiated seedlings were transferred to the B5 rooting medium (B5 salt 3.1g/L, B5 vitamins, 2-morpholineethanesulfonic acid (MES) 1g/L, sucrose 30g/L, agar 8g/L, cephalosporin 150mg/L, indole-3-butyric acid (IBA) 1mg/L), on the rooting culture, culture at 25°C to a height of about 10cm, and move to the greenhouse for cultivation until fruiting. In the greenhouse, culture was carried out at 26°C for 16 hours and at 20°C for 8 hours every day.

1.3. Identification and screening of transgenic events

A total of 288 independent transgenic _T0 plants were generated.

Regenerated transgenic soybean plants were tested for the presence of EPSPS and PAT genes by TaqMan ^™ analysis (see second example), and the copy number of glyphosate- and glufosinate-tolerant lines was characterized. Through screening, event DBN9001 was selected to be superior with a single copy transgene, good glyphosate herbicide tolerance, glufosinate herbicide tolerance and performance of agronomic traits (see sixth example).

The second embodiment, detection of transgenic soybean event DBN9001 with TaqMan

About 100 mg of the leaves of the transgenic soybean event DBN9001 were taken as a sample, and the genomic DNA was extracted with a plant DNA extraction kit (DNeasy Plant Maxi Kit, Qiagen), and the copy numbers of the EPSPS gene and the PAT gene were detected by fluorescent quantitative PCR with Taqman probes. At the same time, the wild-type soybean plants were used as a control, and the detection and analysis were carried out according to the above method. The experiment was repeated 3 times, and the average value was taken.

The specific method is as follows:

Step 11, take 100 mg of leaves of the transgenic soybean event DBN9001, grind it into a homogenate with liquid nitrogen in a mortar, and take 3 replicates for each sample;

Step 12, using the DNeasy Plant Mini Kit of Qiagen to extract the genomic DNA of the above sample, the specific method refers to its product manual;

Step 13, measure the genomic DNA concentration of above-mentioned sample with NanoDrop 2000 (Thermo Scientific);

Step 14, adjusting the genomic DNA concentration of the above samples to the same concentration value, the concentration value ranges from 80-100ng/μl;

Step 15, using the Taqman probe fluorescent quantitative PCR method to identify the copy number of the sample, using the sample with known copy number as the standard, and using the sample of the wild-type soybean plant as the control, each sample is repeated 3 times, and the average Value; Fluorescence quantitative PCR primer and probe sequences are respectively:

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

Primer 1: TTGGTGCTAACCTTACCGTTGAG as shown in SEQ ID NO: 16 in the sequence listing;

Primer 2: GCTTACCACGACCTTCAAGACG as shown in SEQ ID NO: 17 in the sequence listing;

Probe 1: CTGATGCTGACGGTGTGCGTACCATC as shown in SEQ ID NO: 18 in the sequence listing;

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

Primer 3: CAGTTGAGATTAGGCCAGCTACAG as shown in SEQ ID NO: 19 in the sequence listing;

Primer 4: TTCACTGTAGACGTCTCAATGTAATGG as shown in SEQ ID NO: 20 in the sequence listing;

Probe 2: CAGCTGATATGGCCGCGGTTTGTG as shown in SEQ ID NO: 21 in the sequence listing;

The PCR reaction system is:

The 50X primer/probe mix contained 45 μL of each primer at a concentration of 1 mM, 50 μL of a probe at a concentration of 100 μM, and 860 μL of 1X TE buffer, and was stored at 4°C in amber tubes.

The PCR reaction conditions are:

The data was analyzed using SDS2.3 software (Applied Biosystems), and a single copy of the transgenic soybean event DBN9001 was obtained.

The third embodiment, detection of genetically modified soybean event DBN9001

3.1. Genomic DNA extraction

The DNA was extracted according to the conventional CTAB (cetyltrimethylammonium bromide) method: take 2 grams of tender leaves of the transgenic soybean event DBN9001 and grind them into powder in liquid nitrogen, add 0.5mL and pre-cook at a temperature of 65°C. Hot DNA extraction CTAB buffer (20g/L CTAB, 1.4M NaCl, 100mM Tris-HCl, 20mM EDTA (ethylenediaminetetraacetic acid), adjust the pH to 8.0 with NaOH), mix thoroughly, and pump at a temperature of 65°C Extract for 90 minutes; add 0.5 times the volume of phenol and 0.5 times the volume of chloroform, mix upside down; centrifuge at 12,000 rpm (revolutions per minute) for 10 minutes; absorb the supernatant, add 2 times the volume of absolute ethanol, and gently shake the centrifuge tube. Let stand at 4°C for 30 minutes; centrifuge at 12,000 rpm for 10 minutes; collect DNA to the bottom of the tube; discard the supernatant and wash the precipitate with 1 mL of 70% ethanol; centrifuge at 12,000 rpm for 5 minutes; vacuum Dry or blow-dry on an ultra-clean bench; the DNA precipitate is dissolved in an appropriate amount of TE buffer (10mM Tris-HCl, 1mM EDTA, pH 8.0), and stored at -20°C.

3.2. Analysis of flanking DNA sequences

Concentration determination is performed on the extracted DNA sample, so that the concentration of the sample to be tested is between 80-100 ng/μL. Genomic DNA was digested with the selected restriction enzymes PsiI, DraI and TaqI (5' end analysis) and AflII, TaqI and PsiI (3' end analysis). Add 26.5 μL of genomic DNA, 0.5 μL of the restriction endonuclease selected above, and 3 μL of digestion buffer to each enzyme digestion system, and digest for 1 hour. After the enzyme digestion, add 70 μL of absolute ethanol to the enzyme digestion system, ice bath for 30 minutes, centrifuge at 12,000 rpm for 7 minutes, discard the supernatant, blow dry, then add 8.5 μL double distilled water (dd H ₂ O), 1 μL 10XT ₄ -DNA Ligase Buffer (NEB T4DNA Ligase Reaction Buffer, its specific formula can visit NEB website or refer to https://www.neb.com/products/restriction-endonucleases, https://www.neb.com/ products/b0202-t4-dna-ligase-reaction-buffer) and 0.5 μL T ₄ -DNA ligase were ligated overnight at 4°C. PCR amplification using a series of nested primers isolates 5' and 3' transgene/genomic DNA. Specifically, the primer combination for isolating the 5' transgene/genomic DNA includes SEQ ID NO:13, SEQ ID NO:26 as the first primer, SEQ ID NO:27, SEQ ID NO:28 as the second primer, and SEQ ID NO:13 as a sequencing primer. Isolation of 3' transgene/genomic DNA primer combination including SEQ ID NO:15, SEQ ID NO:29 as first primer, SEQ ID NO:30, SEQ ID NO:31 as second primer, SEQ ID NO:15 as sequencing primer, The PCR reaction conditions are shown in Table 3.

The obtained amplicons were electrophoresed on a 2.0% agarose gel to separate the PCR reactions and subsequently separated from the agarose matrix using a Gel Extraction Kit (QIAquick Gel Extraction Kit, catalog #_28704, Qiagen Inc., Valencia, CA). target fragment. Purified PCR products are then sequenced (eg, ABI Prism™ 377, PE Biosystems, Foster City, CA) and analyzed (eg, DNASTAR Sequence Analysis Software, DNASTAR Inc., Madison, WI).

The 5' and 3' flanking and junction sequences were confirmed using standard PCR methods. The 5' flanking and junction sequences can be identified using SEQ ID NO:8 or SEQ ID NO:12 in combination with SEQ ID NO:9, SEQ ID NO:13 or SEQ ID NO:26. The 3' flanking and junction sequences can be identified using SEQ ID NO:11 or SEQ ID NO:14, in combination with SEQ ID NO:10, SEQ ID NO:15 or SEQ ID NO:29. The PCR reaction system and amplification conditions are shown in Table 2 and Table 3. Those skilled in the art will understand that other primer sequences can also be used to confirm flanking and junction sequences.

DNA sequencing of the PCR products provided DNA that could be used to design additional DNA molecules as primers and probes for the identification of soybean plants or seeds derived from transgenic soybean event DBN9001.

It was found that the soybean genome sequence shown at nucleotides 1-859 of SEQ ID NO: 5 is flanking the right border (5' flanking sequence) of the inserted sequence of the transgenic soybean event DBN9001, at nucleotide 5564 of SEQ ID NO: 5 Position -6644 shows the soybean genome sequence flanking the left border of the transgenic soybean event DBN9001 insert (3' flanking sequence). The 5' junction sequence is set forth in SEQ ID NO:1 and the 3' junction sequence is set forth in SEQ ID NO:2.

3.3. PCR zygosity determination

Junction sequences are relatively short polynucleotide molecules that are novel DNA sequences that are diagnostic for the DNA of transgenic soybean Event DBN9001 when detected in a polynucleotide detection assay. The junction sequence in SEQ ID NO: 1 and SEQ ID NO: 2 is the insertion site of the transgene fragment in the transgenic soybean event DBN9001 and 11 polynucleotides on each side of the soybean genomic DNA. Longer or shorter polynucleotide junction sequences can be selected from SEQ ID NO:3 or SEQ ID NO:4. Junction sequences (5' junction region SEQ ID NO: 1, and 3' junction region SEQ ID NO: 2) are useful in DNA detection methods as DNA probes or as DNA primer molecules. The junction sequences SEQ ID NO:6 and SEQ ID NO:7 are also novel DNA sequences in the transgenic soybean event DBN9001, which can also be used as DNA probes or as DNA primer molecules to detect the presence of transgenic soybean event DBN9001 DNA. Said SEQ ID NO:6 (the 477-780th nucleotide of SEQ ID NO:3) spans the nucleotide sequence of the junction region between the pDBN4003 construct and the genome and the prGm17gTsf1 promoter, said SEQ ID NO:7 (nucleotides 147-409 of SEQ ID NO:4) spanned the phosphinothricin N-acetyltransferase cPAT, the t35S transcription terminator sequence, and the pDBN4003 construct DNA sequence.

In addition, an amplicon is generated by using at least one primer from SEQ ID NO:3 or SEQ ID NO:4, which when used in a PCR method generates a diagnostic amplicon of transgenic soybean event DBN9001.

Specifically, a PCR product was generated from the 5' end of the transgene insert that was a portion of the genomic DNA flanking the 5' end of the T-DNA insert in the genome comprising plant material derived from transgenic soybean event DBN9001. This PCR product comprises SEQ ID NO:3. For PCR amplification, primer 5 (SEQ ID NO: 8) was designed to hybridize to the genomic DNA sequence flanking the 5' end of the transgene insert sequence, and its paired primer 6 (SEQ ID NO: 8) located at the transgene prGm17gTsf1 promoter sequence NO:9).

A PCR product comprising a portion of the genomic DNA flanking the 3' end of the T-DNA insert in the genome of plant material derived from transgenic soybean Event DBN9001 was generated from the 3' end of the transgene insert. This PCR product comprises SEQ ID NO:4. For PCR amplification, primer 8 (SEQ ID NO: 11) was designed to hybridize to the genomic DNA sequence flanking the 3' end of the transgene insert, and paired with phosphinothricin at the 3' end of the insert Primer 7 for N-acetyltransferase cPAT sequence (SEQ ID NO: 10).

The DNA amplification conditions described in Tables 2 and 3 can be used in the PCR zygosity assay described above to generate diagnostic amplicons for transgenic soybean event DBN9001. The detection of the amplicon can be performed by using Stratagene Robocycler, MJEngine, Perkin-Elmer 9700 or Eppendorf Mastercycler Gradien thermal cycler, etc., or by methods and equipment known to those skilled in the art.

Table 2. PCR steps and reaction mixture conditions for identification of the 5' transgene insert/genome junction region of transgenic soybean event DBN9001

Table 3. Perkin-Elmer9700 thermal cycler conditions

Mix gently and add 1-2 drops of mineral oil on top of each reaction if there is no thermal cap on the thermal cycler. Using the above cycling parameters (Table 3) in Stratagene Robocycler (Stratagene, La Jolla, CA), MJEngine (MJ R-Biorad, Hercules, CA), Perkin-Elmer 9700 (Perkin Elmer, Boston, MA) or Eppendorf Mastercycler Gradient (Eppendorf , Hamburg, Germany) thermal cycler for PCR. The MJ Engine or Eppendorf Mastercycler Gradient thermal cycler should be run in calculation mode. The Perkin-Elmer 9700 Thermal Cycler was run with the ramp speed set to the maximum value.

The experimental results show that: primers 5 and 6 (SEQ ID NO: 8 and 9), when used in the PCR reaction of the transgenic soybean event DBN9001 genomic DNA, produce an amplification product of a 780bp fragment, when used in the non-transformed soybean genome DNA and non-DBN9001 soybean genomic DNA in the PCR reaction, no fragment was amplified; primers 7 and 8 (SEQ ID NO: 10 and 11), when used in the PCR reaction of transgenic soybean event DBN9001 genomic DNA, produced The amplified product of the 877 bp fragment, when it was used in a PCR reaction of non-transformed soybean genomic DNA and non-DBN9001 soybean genomic DNA, no fragment was amplified.

PCR zygosity assays can also be used to identify whether material derived from transgenic soybean event DBN9001 is homozygous or heterozygous. Primer 9 (SEQ ID NO: 12), Primer 10 (SEQ ID NO: 13) and Primer 11 (SEQ ID NO: 14) were used in the amplification reaction to generate a diagnostic amplicon of transgenic soybean event DBN9001. The DNA amplification conditions described in Tables 4 and 5 can be used in the zygosity assay described above to generate diagnostic amplicons for transgenic soybean event DBN9001.

Table 4. Reaction solution for zygosity determination

Table 5. Zygosity determination Perkin-Elmer9700 thermal cycler conditions

Using the above cycle parameters (Table 5) in Stratagene Robocycler (Stratagene, La Jolla, CA), MJ Engine (MJ R-Biorad, Hercules, CA), Perkin-Elmer 9700 (Perkin Elmer, Boston, MA) or Eppendorf Mastercycler Gradient ( Eppendorf, Hamburg, Germany) thermal cycler for PCR. The MJ Engine or Eppendorf Mastercycler Gradient thermal cycler should be run in calculation mode. The Perkin-Elmer 9700 Thermal Cycler was run with the ramp speed set to the maximum value.

In the amplification reaction, the biological sample containing template DNA contains DNA diagnostic for the presence of transgenic soybean event DBN9001 in the sample. Or the reaction will produce two different DNA amplicons from a biological sample containing DNA derived from the soybean genome that is heterozygous for the allele corresponding to the inserted DNA present in transgenic soybean event DBN9001 of. These two distinct amplicons will correspond to the first amplicon derived from the wild-type soybean genomic locus and the second amplicon diagnostic for the presence of transgenic soybean event DBN9001 DNA. Generating only a soybean DNA sample corresponding to a single amplicon of the second amplicon described for the heterozygous genome, the presence of transgenic soybean event DBN9001 in the sample can be diagnostically determined, and the sample is generated relative to the presence in the transgenic soybean plant DBN9001 The allele corresponding to the inserted DNA is produced in soybean seeds that are homozygous.

It should be noted that the primer pair for GM soybean event DBN9001 was used to generate amplicons that were diagnostic for GM soybean event DBN9001 genomic DNA. These primer pairs include, but are not limited to, primers 5 and 6 (SEQ ID NO: 8 and 9), and primers 7 and 8 (SEQ ID NO: 10 and 11), used in the DNA amplification method. Additionally, a control primer 12 and 13 (SEQ ID NO: 22 and 23) for amplifying endogenous soybean genes was included as an internal standard for reaction conditions. Analysis of DNA extract samples from GM soybean event DBN9001 should include a positive tissue DNA extract control from GM soybean event DBN9001, a negative DNA extract control from non-GM soybean event DBN9001, and a soybean DNA extract containing no template. Extract negative control. In addition to these primer pairs, any primer pair from SEQ ID NO: 3 or SEQ ID NO: 4, or the complement thereof, which when used in a DNA amplification reaction respectively produces Tissues from plant DBN9001 are diagnostic comprising the amplicon of SEQ ID NO:1 or SEQ ID NO:2. The DNA amplification conditions described in Tables 2-5 can be used to generate diagnostic amplicons for transgenic soybean event DBN9001 using appropriate primer pairs. An extract that putatively contains DNA from a soybean plant or seed comprising transgenic soybean event DBN9001, or a product derived from transgenic soybean event DBN9001, that produces an amplicon that is diagnostic for transgenic soybean event DBN9001 when tested in a DNA amplification method , can be used as a template for amplification to determine the presence or absence of transgenic soybean event DBN9001.

The fourth embodiment, detection of transgenic soybean event DBN9001 by Southern blot hybridization

4.1. DNA extraction for Southern blot hybridization

Southern blot analysis was performed using homozygous transformation events in the T4, T5 and T6 generations. Plant tissue was ground in liquid nitrogen using a mortar and pestle. Resuspend 4-5 g of ground lysate in 20 mL of CTAB lysis buffer (100 mM Tris pH 8.0, 20 mM EDTA pH 8.0, 1.4 M NaCl, 0.2% v/v β-mercaptoethanol, 2% w/v polyvinyl-pyrrolidone) plant tissue and incubated at a temperature of 65°C for 60 minutes. During the incubation period, the samples were mixed by inversion every 10 minutes. After incubation, an equal volume of chloroform/isoamyl alcohol (24:1) was added, mixed gently by inversion, and centrifuged at 4000 rpm for 20 minutes. Collect the aqueous phase, add an equal volume of isopropanol and place it at -20°C for 1 hour to precipitate DNA, then centrifuge at 4000 rpm for 5 minutes to obtain a DNA pellet, and then resuspend the DNA pellet in 1 mL of TE buffer. To degrade any RNA present, incubate the DNA with 5 μL RNAase A at a concentration of 30 mg/mL for 30 minutes at 37°C, centrifuge at 4000 rpm for 5 minutes, and then dissolve in 0.1 volume of 3M sodium acetate and 2 volumes of absolute ethanol If present, centrifuge at 14,000 rpm for 10 minutes to pellet the DNA. After discarding the supernatant, the pellet was washed with 1 mL of 70% (v/v) ethanol, dried and redissolved in 1 mL of TE buffer. The genomic DNA concentration of the above samples was measured with an ultramicro spectrophotometer (NanoDrop 2000, Thermo Scientific).

4.2. Restriction enzyme digestion

In a 100 μL reaction system, digest 5 μg of DNA each time. Genomic DNA was digested with restriction endonucleases EcoRI and EcoRV, and partial sequences of EPSPS and PAT on T-DNA were used as probes. For each enzyme, digests were incubated overnight at the appropriate temperature. Samples were spun down using a speed vacuum, Thermo Scientific to reduce the volume to 20 μL.

4.3. Gel electrophoresis

Bromophenol blue loading dye was added to each sample derived from Example 4.2, and each sample was loaded onto a 0.7% agarose gel containing ethidium bromide in TAE electrophoresis buffer (40 mM Tris -Acetic acid, 2mM EDTA, pH 8.0), run the gel overnight at 20 volts.

The gel was treated with denaturing solution (1.5M NaCl, 0.5M NaOH) and neutralizing solution (1.5M NaCl, 0.5M Tris, pH7.2) for 30 minutes each. Pour 5×SSC into a porcelain dish, put a glass plate on it, and then put the soaked filter paper bridge, gel, positively charged nylon membrane (Roche, Cat.No.11417240001), three layers of filter paper, and paper tower , heavy objects. After transferring the membrane overnight at room temperature, the nylon membrane was rinsed in 2×SSC for 10 seconds, and the DNA was immobilized on the membrane by UV crosslinking.

4.4 Hybridization

Appropriate DNA sequences were amplified by PCR for probe preparation. The DNA probes are SEQ ID NO: 24 and SEQ ID NO: 25, or are partially homologous or complementary to the above sequences. DIG labeling of probes, Southern blot hybridization, and signal detection were performed using DNA High Prime DNA Labeling and Detection Starter Kit II (DIG HighPrime DNA Labeling and Detection Starter Kit II kit, Roche, Cat. No. 11585614910). For the method, refer to its product manual.

Two control samples were included on each Southern: (1) DNA from negative (untransformed) segregants, which was used to identify any endogenous soybean sequences that could hybridize with element-specific probes; (2) based on The probe length was equivalent to one copy number of Hind III-digested pDBN4003 plasmid, which served as a positive control for hybridization and was used to illustrate the sensitivity of the experiment.

Hybridization data provided corroborating evidence in support of TaqMan ^™ PCR analysis that soybean plant DBN9001 contained single copies of the EPSPS and PAT genes. Using this EPSPS probe, EcoR I and EcoR V enzymatic digestions produced single bands of about 8 kb and 13 kb in size respectively; using this PAT probe, EcoR I and EcoR V enzymatic digestions produced single bands of about 7.5 kb and 2.5 kb in size, respectively bring. This indicates that one copy each of EPSPS and PAT is present in soybean transformation event DBN9001.

The fifth embodiment, detection of transgenic soybean event DBN9001 protein by ELISA

The expression range of EPSPS and PAT proteins in the transgenic soybean event DBN9001 can be detected by ELISA.

Take 2mg of fresh leaves of transgenic soybean event DBN9001 as a sample, grind with liquid nitrogen and add 1mL of the extraction buffer (8g/L NaCl, 0.27g/L KH ₂ PO ₄ , 1.42g/L Na ₂ HPO ₄ , 0.2g/L L KCl, 5.5ml/L Tween-20, pH7.4), centrifuge at 4000rpm for 10 minutes, take the supernatant and dilute 40 times with the extraction buffer, take 80 μl of the diluted supernatant for ELISA detection .

ELISA (enzyme-linked immunosorbent assay) kit (ENVIRLOGIX company, EPSPS kit and PAT kit) was used to detect and analyze the ratio of the amount of protein (EPSPS protein and PAT protein) in the sample to the fresh weight of the leaves. For specific methods, refer to its Product Manual. At the same time, wild-type soybean plant leaves (non-transgenic, NGM) were used as a control, and detection and analysis were carried out according to the above method, and each plant was repeated 6 times.

The experimental results of the protein (EPSPS protein and PAT protein) content of the transgenic soybean event DBN9001 are shown in Table 6. The percentages (μg/g) of the average expression of EPSPS protein in the fresh leaves of transgenic soybean event DBN9001 and wild-type soybean plants were measured to be 170.1 and 0, respectively; the fresh leaves of transgenic soybean event DBN9001 and wild-type soybean plants The ratio of the average expression of PAT protein to the fresh weight of leaves (μg/g) was 260.2 and 0 respectively.

Table 6. Average results of protein expression (μg/g) determination of transgenic soybean event DBN9001

Sixth embodiment, detection of herbicide tolerance of transgenic soybean event DBN9001

In this experiment, Roundup herbicide (41% glyphosate isopropyl ammonium salt solution) and Basta herbicide (18% glufosinate-ammonium active ingredient) were selected for spraying. A random block design was adopted with 3 repetitions. The area of the plot is 18m ² (5m×3.6m), the row spacing is 60cm, the plant spacing is 8cm, conventional cultivation and management, and there is a 1m wide isolation zone between the plots. The transgenic soybean event DBN9001 was subjected to the following three treatments: 1) No spraying, artificial grass control; 2) Spraying at the V3 leaf stage at a dose of 1680g ae/ha (ae/ha refers to "active ingredient equivalent acid per hectare") Roundup herbicide, and then spray Roundup herbicide again at the same dosage at the R2 stage (full flowering stage); 3) Spray the protective herbicide at the V3 leaf stage by 800g ai/ha (ai/ha refers to "active ingredient per hectare") dosage 4) Spray 800g ai/ha herbicide at V3 leaf stage, then spray 1680g ae/ha at R2 stage Roundup herbicide. Parallel control experiments were performed with wild-type soybean plants (non-transgenic, NGM). It should be noted that the conversion of glyphosate herbicides with different contents and formulations into the equivalent amount of glyphosate acid, and the conversion of different concentrations of glufosinate-ammonium solutions into the above-mentioned equivalent active ingredient glufosinate-ammonium are applicable to the following conclusions.

The symptoms of phytotoxicity were investigated 1 week and 2 weeks after the application, and the soybean yield of the plot was measured at the time of harvest. The grading of phytotoxicity symptoms is shown in Table 7. The herbicide damage rate is used as an index to evaluate the herbicide tolerance of the transformation event, specifically, the herbicide damage rate (%)=∑(the number of injured plants of the same level×the number of levels)/(the total number of plants×the highest level); Herbicide damage rate includes glyphosate damage rate and glufosinate-ammonium damage rate, herbicide damage rate is determined according to the phytotoxicity investigation results 2 weeks after glyphosate or glufosinate-ammonium treatment. The soybean yield of each plot is the total yield (weight) of soybean grains in the middle 3 rows of each plot, and the yield difference between different treatments is measured in the form of yield percentage, yield percentage (%)=sprayed yield/no spraying Yield. The results of herbicide tolerance and soybean yield of transgenic soybean event DBN9001 are shown in Table 8.

Table 7. Grading standards for the degree of herbicide damage to soybeans

Phytotoxicity level symptom description 1 Normal growth without any symptoms of injury 2 Slight phytotoxicity, phytotoxicity less than 10% 3 Moderate phytotoxicity, can recover later, does not affect yield 4 The drug damage is heavy, it is difficult to recover, resulting in reduced production 5 The phytotoxicity is serious and cannot be recovered, resulting in obvious production reduction or cessation of production

Table 8. Results of herbicide tolerance and yield test results of transgenic soybean event DBN9001

The results show that in terms of herbicide (glyphosate and glufosinate) damage rate: 1) the damage rate of the genetically modified soybean event DBN9001 is basically 0 under the treatment of glyphosate herbicide (1680g a.e./ha); 2) the genetically modified soybean event The damage rate of DBN9001 under the treatment of glufosinate-ammonium herbicide (800g a.i./ha) was basically 0; The damage rate under the ha) treatment was basically 0; thus, the transgenic soybean event DBN9001 has good herbicide (glyphosate and glufosinate) tolerance.

In terms of yield: GM soybean event DBN9001 was sprayed with glyphosate (1680g a.e./ha), glufosinate (800g a.i./ha) and glufosinate (800g a.i./ha) + glyphosate The yield under the three treatments of herbicide (1680g a.e./ha) was slightly increased compared with the treatment without spraying, thus further indicating that the transgenic soybean event DBN9001 has good tolerance to herbicides (glyphosate and glufosinate) .

Seventh embodiment

Agricultural products or commodities such as can be produced from transgenic soybean event DBN9001. If a sufficient expression level is detected in the agricultural product or commodity, the agricultural product or commodity is expected to contain a nucleotide sequence capable of diagnosing the presence of the transgenic soybean event DBN9001 material in the agricultural product or commodity. Such foods or commodities include, but are not limited to, products from the soybean plant DBN9001, such as soybean cakes, flours, and oils, specifically lecithin, fatty acids, glycerin, sterols, edible oils, defatted soybean flakes, including defatted and roasted Soy flour, soy milk curd, tofu, soy protein concentrate, isolated soy protein, hydrolyzed vegetable protein, textured soy protein, and soy protein fiber. Nucleic acid detection methods and/or kits based on probes or primer pairs can be developed to detect the transgenic soybean event DBN9001 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 sequence is selected from the sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5 or a fragment thereof or a sequence complementary thereto, To diagnose the presence of GM soybean event DBN9001.

In summary, the transgenic soybean event DBN9001 of the present invention has good tolerance to glyphosate herbicides and glufosinate-ammonium herbicides, has no effect on yield, and the detection method can accurately and quickly identify whether the biological sample contains transgene DNA molecule of soybean event DBN9001.

The seeds corresponding to the genetically modified soybean event DBN9001 were preserved on November 27, 2015 in the General Microbiology Center of the China Committee for Microorganism Culture Collection (CGMCC for short, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Microbiology, Chinese Academy of Sciences Institute, Zip code 100101), classification name: soybean (Glycine max), deposit number is CGMCCNo.11043. The deposit will be kept in the depository for 30 years.

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 (23)

1. a kind of nucleic acid molecules with following nucleic acid sequence, which is characterized in that the nucleic acid sequence include SEQ ID NO:1 or Its complementary series, and/or SEQ ID NO:2 or its complementary series, the nucleic acid molecules are originated from transgenic soybean event DBN9001, the transgenic soybean event DBN9001 are preserved in the form of seed and with deposit number CGMCC No.11043 China Committee for Culture Collection of Microorganisms's common micro-organisms center.

2. nucleic acid molecules according to claim 1, which is characterized in that the nucleic acid sequence include SEQ ID NO:3 or its Complementary series, and/or SEQ ID NO:4 or its complementary series.

3. nucleic acid molecules according to claim 2, which is characterized in that the nucleic acid sequence include SEQ ID NO:5 or its Complementary series.

4. a kind of method existing for DNA of test sample transgenic soybean event DBN9001 characterized by comprising

Contact sample to be tested in nucleic acid amplification reaction at least two primers for expanding target amplification product；

Carry out nucleic acid amplification reaction；With

Detect the presence of the target amplification product；

The target amplification product includes SEQ ID NO:1 or its complementary series, and/or SEQ ID NO:2 or its complementary series, The target amplification product is originated from transgenic soybean event DBN9001, and the transgenic soybean event DBN9001 is with the shape of seed Formula and China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved in deposit number CGMCC No.11043.

5. method existing for the DNA of test sample transgenic soybean event DBN9001 according to claim 4, feature It is, the target amplification product further includes at least one selected from the following: SEQ ID NO:6 or its complementary series and SEQ ID NO:7 or its complementary series.

6. method existing for the DNA of test sample transgenic soybean event DBN9001 according to claim 4 or 5, special Sign is that described two primers include SEQ ID NO:8 and SEQ ID NO:9 or SEQ ID NO:10 and SEQ ID NO: 11。

7. a kind of method existing for DNA of test sample transgenic soybean event DBN9001 characterized by comprising

Contact sample to be tested with probe, the probe includes SEQ ID NO:1 or its complementary series or SEQ ID NO: 2 or its complementary series, the source probe transgenic soybean event DBN9001, the transgenic soybean event DBN9001 is to plant Son form and China Committee for Culture Collection of Microorganisms's commonly micro- life is preserved in deposit number CGMCC No.11043 Object center；

Hybridize the sample to be tested and the probe under stringent hybridization conditions；With

Detect the hybridisation events of the sample to be tested and the probe.

8. method existing for the DNA of test sample transgenic soybean event DBN9001 according to claim 7, feature It is, the probe also includes at least one selected from the following: SEQ ID NO:6 or its complementary series and SEQ ID NO:7 Or its complementary series.

9. special according to method existing for the DNA of the test sample transgenic soybean event DBN9001 of claim 7 or 8 Sign is that at least one described probe is marked at least one fluorophor.

10. a kind of method existing for DNA of test sample transgenic soybean event DBN9001 characterized by comprising

Contact sample to be tested with marker nucleic acid molecules, the marker nucleic acid molecules include SEQ ID NO:1 or it is mutual Complementary series or SEQ ID NO:2 or its complementary series, the marker nucleic acid molecules are originated from transgenic soybean event DBN9001, the transgenic soybean event DBN9001 are preserved in the form of seed and with deposit number CGMCC No.11043 China Committee for Culture Collection of Microorganisms's common micro-organisms center；

Hybridize the sample to be tested and the marker nucleic acid molecules under stringent hybridization conditions；With

The hybridisation events of the sample to be tested and the marker nucleic acid molecules are detected, and then pass through marker assistant breeding point Analysis is to determine that glyphosate tolerant and/or glufosinate tolerant and marker nucleic acid molecules are chain on science of heredity.

11. method existing for the DNA of test sample transgenic soybean event DBN9001 according to claim 10, special Sign is that the marker nucleic acid molecules further include at least one selected from the following: SEQ ID NO:6 or its complementary series and SEQ ID NO:7 or its complementary series.

12. a kind of DNA detection kit, which is characterized in that including at least one DNA molecular, the DNA molecular includes SEQ ID NO:1 or its complementary series or SEQ ID NO:2 or its complementary series, can be used as transgenic soybean event DBN9001 or its offspring have one of DNA primer of specificity or probe；The DNA molecular is originated from transgenic soybean event DBN9001, the transgenic soybean event DBN9001 are preserved in the form of seed and with deposit number CGMCC No.11043 China Committee for Culture Collection of Microorganisms's common micro-organisms center.

13. DNA detection kit according to claim 12, which is characterized in that further include when the DNA molecular is as probe At least one selected from the following: SEQ ID NO:6 or its complementary series and SEQ ID NO:7 or its complementary series.

14. a kind of method for generating the soybean plant strain for having tolerance to glyphosate herbicidal and/or glufosinate-ammonium herbicide, special Sign is, including will successively include SEQ ID NO:1,899-5553 nucleic acid sequences of SEQ ID NO:5 and SEQ in genome The soybean plant strain of ID NO:2 hybridizes with another soybean plant strain, to generate a large amount of progeny plants；Selection genome successively wraps ID containing SEQ NO:1,899-5553 nucleic acid sequences of SEQ ID NO:5 and SEQ ID NO:2 or genome include SEQ The progeny plant of ID NO:5, and the progeny plant has glyphosate and/or glufosinate tolerant.

15. 4 soybean generated to glyphosate herbicidal and/or glufosinate-ammonium herbicide with tolerance according to claim 1 The method of plant characterized by comprising

To there is the first parent of transgenic soybean event DBN9001 of tolerance to glyphosate herbicidal and/or glufosinate-ammonium herbicide This soybean plant strain and the second parental soybean plant sexual hybridization for lacking glyphosate and/or glufosinate tolerant, to generate big Measure progeny plant；

The progeny plant described in glyphosate herbicidal and/or glufosinate-ammonium herbicide treatment；With

The progeny plant of selection tolerance glyphosate and/or glufosinate-ammonium herbicide；

The transgenic soybean event DBN9001 is preserved in China in the form of seed and with deposit number CGMCC No.11043 Microbiological Culture Collection administration committee common micro-organisms center.

16. a kind of method that culture has the bean plant of tolerance to glyphosate herbicidal and/or glufosinate-ammonium herbicide, special Sign is, comprising:

An at least soya seeds are planted, include the nucleic acid sequence of specific region, the spy in the genome of the soya seeds The nucleic acid sequence for determining region successively includes SEQ ID NO:1,899-5553 nucleic acid sequences of SEQ ID NO:5 and SEQ ID The nucleic acid sequence of NO:2 or the specific region includes SEQ ID NO:5；

The soya seeds are made to grow up to soybean plant strain；With

The soybean plant strain described in effective dose glyphosate herbicidal and/or glufosinate-ammonium herbicide spray, harvest do not have with other The plant of the nucleic acid sequence of specific region compares the plant with the plant injury weakened.

17. a kind of method for protecting the plants from the damage as caused by herbicide, which is characterized in that including effective dose will be contained Glyphosate and/or the herbicide of glufosinate-ammonium are applied to the big Tanaka for planting at least one transgenic soy bean plant, the transgenosis Bean plant successively includes SEQ ID NO:1,899-5553 nucleic acid sequences of SEQ ID NO:5 and SEQ in its genome It include SEQ ID NO:5 in the genome of ID NO:2 or the transgenic soy bean plant；The transgenic soy bean plant tool There is the tolerance to glyphosate herbicidal and/or glufosinate-ammonium herbicide.

18. a kind of method for controlling weeds in field, which is characterized in that including effective dose glyphosate and/or glufosinate-ammonium will be contained Herbicide be applied to the big Tanaka for planting at least one transgenic soy bean plant, the transgenic soy bean plant is in its genome In successively include SEQ ID NO:1,899-5553 nucleic acid sequences of SEQ ID NO:5 and SEQ ID NO:2 or described turn It include SEQ ID NO:5 in the genome of transgenic soybean plant；The transgenic soy bean plant have to glyphosate herbicidal and/ Or the tolerance of glufosinate-ammonium herbicide.

19. a kind of method for the crop field glyphosate resistance weeds for controlling glyphosate-tolerant plant, which is characterized in that including inciting somebody to action Herbicide containing effective dose glufosinate-ammonium is applied to the big of the transgenic soy bean plant for planting at least one glyphosate tolerant Tanaka, the transgenic soy bean plant of the glyphosate tolerant successively include SEQ ID NO:1, SEQ ID in its genome The transgenic soy bean plant of 899-5553 nucleic acid sequences of NO:5 and SEQ ID NO:2 or the glyphosate tolerant It include SEQ ID NO:5 in genome；The transgenic soy bean plant of the glyphosate tolerant has to glufosinate-ammonium weeding simultaneously The tolerance of agent.

20. a kind of method for delaying insect-resistant, which is characterized in that including planted in the big Tanaka for planting pest-resistant bean plant to A kind of few transgenic soy bean plant with glyphosate and/or glufosinate tolerant, it is described to have glyphosate and/or glufosinate-ammonium resistance to Transgenic soy bean plant by property successively includes 899-5553 SEQ ID NO:1, SEQ ID NO:5 cores in its genome The gene of acid sequence and SEQ ID NO:2 or the transgenic soy bean plant with glyphosate and/or glufosinate tolerant It include SEQ ID NO:5 in group.

21. a kind of agricultural product or commodity for being produced from transgenic soybean event DBN9001, which is characterized in that the agricultural product or Commodity are soybean piece, soy meal, soybean protein or its concentrate, soybean oil, soybean fiber, soya-bean milk grumeleuse；It is described to turn base Chinese microorganism strain guarantor is preserved in because of soybean event DBN9001 in the form of seed and with deposit number CGMCC No.11043 Hide administration committee's common micro-organisms center.

22. being produced from the agricultural product or commodity of transgenic soybean event DBN9001 according to claim 21, feature exists In the soya-bean milk grumeleuse is bean curd.

23. a kind of agricultural product or commodity for being produced from transgenic soybean event DBN9001, which is characterized in that the agricultural product or Commodity are lecithin, fatty acid, glycerol, sterol；The transgenic soybean event DBN9001 is compiled in the form of seed and with preservation Number CGMCC No.11043 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center.