CN104878095B - Nucleic acid sequence and its detection method for detecting herbicide tolerant corn plant DBN9858 - Google Patents

Abstract

The present invention relates to a nucleic acid sequence for detecting herbicide-tolerant corn plant DBN9858 and a detection method thereof, wherein the nucleic acid sequence of the corn plant comprises SEQ ID NO: 1 or its complementary sequence, or SEQ ID NO: 2 or its complementary sequence. The transgenic corn event DBN9858 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 the DNA of the transgenic corn event DBN9858 molecular.

Description

Nucleic acid sequence and its detection method for the detection of herbicide tolerance maize plant DBN9858 test method

technical field

The present invention relates to a nucleic acid sequence for detecting herbicide-tolerant corn plant DBN9858 and a detection method thereof, in particular to a kind of corn plant DBN9858 tolerant to glyphosate and glufosinate and detecting whether specific Methods for DNA Molecules of Transgenic Maize Event DBN9858.

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.

Maize (Zea mays L.) is a major food crop in many parts of the world. Herbicide tolerance is an important agronomic trait in maize production, especially tolerance to glyphosate herbicide. The tolerance of maize to glyphosate herbicides can be obtained by expressing glyphosate herbicide tolerance genes (EPSPS, CP4) in maize plants by transgenic methods, such as maize event NK603, maize event MON88017 and the like.

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.

The enzyme 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 soybeans to glufosinate-ammonium herbicides, such as 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.

At the same time, with the large-scale planting of transgenic insect-resistant corn, a small number of surviving insects/pests may develop resistance after several generations of reproduction. Herbicide-resistant transgenic corn, as non-insect-resistant transgenic corn, is planted together with transgenic insect-resistant corn 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 corn plant DBN9858, and the transgenic corn event DBN9858 has better 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 maize event DBN9858.

To achieve the above object, the present invention provides a nucleic acid sequence, comprising 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 consecutive nucleotides.

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.

The 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 insertion sequence in the transgenic maize event DBN9858, the SEQ ID NO: 1 or its The complementary sequence spans the flanking genomic DNA sequence of the maize insertion site and the DNA sequence at the 5' end of the insertion sequence, and the presence of the transgenic maize event DBN9858 can be identified by including said SEQ ID NO: 1 or its complementary sequence. 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 maize event DBN9858, said SEQ ID NO: 2 or its complementary sequence Complementary sequences spanning the DNA sequence at the 3' end of the insertion sequence and the flanking genomic DNA sequence of the maize insertion site, comprising said SEQ ID NO: 2 or its complement can be identified as the presence of transgenic maize event DBN9858.

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 sequences) of any part of the 5' flanking maize 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 comprise a set of DNA primers in a DNA amplification method that produces an amplification product. The presence of transgenic maize event DBN9858 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 1301 nucleotides in length near the insertion junction at the 5' end of the inserted sequence in the transgenic maize event DBN9858, said SEQ ID NO: 3 or its complementary sequence Complementary sequence consists of the corn flank genomic DNA sequence (nucleotide 1-1067 of SEQ ID NO:3) of 1067 nucleotides, the DBN10006 construct DNA sequence of 89 nucleotides (nucleotide of 3 1068-1156) and the 5' end DNA sequence (nucleotide 1157-1301 of SEQ ID NO:3) of the pr35S promoter sequence of 145 nucleotides, comprising said SEQ ID NO:3 or its complementary sequence namely The presence of transgenic maize event DBN9858 can be identified.

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 At least 11 or more continuous polynucleotides (the fourth nucleic acid sequence) of any part of the 3' flanking maize 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 a third nucleic acid sequence and a fourth nucleic acid sequence are used together, these nucleic acid sequences comprise a set of DNA primers in a DNA amplification method that produces an amplified product. The presence of transgenic maize event DBN9858 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 1310 nucleotides in length near the insertion junction at the 3' end of the inserted sequence in the transgenic maize event DBN9858, said SEQ ID NO: 4 or its complementary sequence Complementary sequence consists of the tNos terminator sequence (nucleotide 1-164 of SEQ ID NO:4) of 164 nucleotides, the DBN10006 construct DNA sequence (nucleotide 165 of SEQ ID NO:4) of 84 nucleotides -248) and 1062 nucleotides of maize integration site flanking genomic DNA sequence (249-1310 of SEQ ID NO:4), comprising said SEQ ID NO:4 or its complementary sequence can be identified as a transgenic maize event The presence of DBN9858.

The SEQ ID NO:5 or its complementary sequence is a sequence of 6890 nucleotides in length characterizing the transgenic maize event DBN9858, and its specific genome and genetic elements are shown in Table 1. The presence of the transgenic maize event DBN9858 can be identified by the inclusion of 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 DNA amplification methods to produce amplicons, the detection of which is diagnostic of the presence of transgenic maize event DBN9858 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 maize event DBN9858 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 corn event DBN9858 in a sample, comprising:

contacting the sample to be tested with at least two primers in a nucleic acid amplification reaction;

Perform nucleic acid amplification reactions;

detecting the presence of the amplification product;

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

Further, the amplified product includes consecutive nucleotides at positions 1-11 or 12-22 in SEQ ID NO: 1 or its complementary sequence, or 1-11 in SEQ ID NO: 2 or its complementary sequence or the 12th-22nd consecutive nucleotides.

Further, the amplified product comprises 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 .

In the above technical solution, the primer includes at least one nucleic acid sequence.

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 corn event DBN9858 in a sample, comprising:

Contacting the sample to be detected with a probe comprising at least 11 consecutive nucleotides of SEQ ID NO: 3 or its complement, or at least 11 consecutive cores of SEQ ID NO: 4 or its complement nucleotide;

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

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 consecutive nucleotides at positions 1-11 or 12-22 in SEQ ID NO: 1 or its complementary sequence, or at positions 1-11 in SEQ ID NO: 2 or its complementary sequence Or the 12th-22nd consecutive nucleotides.

Furthermore, the probe has 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.

To achieve the above object, the present invention also provides a method for detecting the presence of DNA of the transgenic corn event DBN9858 in a sample, comprising:

Contacting the sample to be detected with a marker nucleic acid molecule comprising at least 11 consecutive nucleotides of SEQ ID NO: 3 or its complement, or at least 11 of SEQ ID NO: 4 or its complement consecutive nucleotides;

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

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, or the 1-1 in SEQ ID NO: 2 or its complementary sequence. The 11th or 12th-22nd consecutive nucleotides.

Further, the marker nucleic acid molecule has 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 sequence.

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 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 , 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 DNA primer or probe specific for the transgenic maize event DBN9858 or its progeny.

Further, the DNA molecule includes consecutive nucleotides at positions 1-11 or 12-22 in SEQ ID NO: 1 or its complementary sequence, or at positions 1-11 in SEQ ID NO: 2 or its complementary sequence Or the 12th-22nd consecutive nucleotides.

Further, the DNA molecule has a homologous sequence of SEQ ID NO:1 or its complement, a homologous sequence of SEQ ID NO:2 or its complement, a homologous sequence of SEQ ID NO:6 or its complement, Or a homologous sequence of SEQ ID NO: 7 or its complement.

To achieve the above object, the present invention also provides a plant cell, 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, said The nucleic acid sequence of the specific region includes the sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:6 or SEQ ID NO:7.

To achieve the above object, the present invention also provides a method for producing a corn plant tolerant to glyphosate herbicides, comprising introducing a nucleic acid encoding a glyphosate-tolerant EPSPS protein into the genome of the corn plant Sequence and the nucleic acid sequence of specific region, the nucleic acid sequence of described specific region is selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO: 6 and at least one nucleic acid sequence in the sequence shown in SEQ ID NO:7.

Specifically, the method for producing a corn plant tolerant to glyphosate herbicides comprises:

sexually crossing a transgenic corn event DBN9858 first parent corn plant tolerant to glyphosate herbicides with a second parent corn plant lacking glyphosate tolerance to produce a large number of progeny plants;

Treating the progeny plants with glyphosate herbicide;

The progeny plants are selected for tolerance to glyphosate.

To achieve the above object, the present invention also provides a method for producing a corn plant tolerant to glufosinate-ammonium herbicides, comprising introducing a nucleic acid encoding a glufosinate-ammonium-tolerant PAT protein into the genome of the corn plant Sequence and the nucleic acid sequence of specific region, the nucleic acid sequence of described specific region is selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO: 6 and at least one nucleic acid sequence in the sequence shown in SEQ ID NO:7.

Specifically, the method for producing corn plants tolerant to glufosinate-ammonium herbicides comprises:

sexually crossing a first parent corn plant of transgenic corn Event DBN9858 tolerant to glufosinate-ammonium herbicide with a second parent corn plant lacking glufosinate-ammonium tolerance to produce a large number of progeny plants;

Treat the progeny plants with glufosinate-ammonium herbicide;

The progeny plants are selected for tolerance to glyphosate.

To achieve the above object, the present invention also provides a method for producing a corn plant tolerant to glyphosate herbicides and glufosinate-ammonium herbicides, comprising introducing a glyphosate-resistant gene into the genome of the corn plant The nucleotide sequence of the tolerant EPSPS protein, the nucleotide sequence of the coding glufosinate-ammonium tolerant PAT protein and the nucleotide sequence of the specific region, the nucleotide sequence of the specific region is selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID At least one nucleic acid sequence in the sequences shown in NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7.

Specifically, the method for producing corn plants tolerant to glyphosate herbicides and glufosinate-ammonium herbicides includes:

Transgenic corn event DBN9858 first parent corn plants tolerant to glyphosate and glufosinate herbicides were sexually crossed with second parent corn plants lacking glyphosate and/or glufosinate tolerance , resulting in a large number of progeny plants;

Treating the progeny plants with glyphosate herbicide and glufosinate-ammonium herbicide;

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

To achieve the above object, the present invention also provides a method of cultivating a corn plant tolerant to glyphosate herbicides, comprising:

Planting at least one corn seed, the genome of the corn seed includes a nucleic acid sequence encoding a glyphosate-tolerant EPSPS and a nucleic acid sequence in a specific region;

growing the corn seeds into corn plants;

Spraying the corn plants with an effective dose of glyphosate herbicide, harvesting plants with reduced 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 of cultivating a corn plant tolerant to glufosinate-ammonium herbicide, comprising:

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

growing the corn seeds into corn plants;

Spraying the corn plants with an effective dose of glufosinate-ammonium herbicide, and harvesting plants with reduced 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 of cultivating a corn plant tolerant to glyphosate herbicides and glufosinate-ammonium herbicides, comprising:

Planting at least one corn seed, the genome of the corn seed includes 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;

growing the corn seeds into corn plants;

Spraying the corn plants with an effective dose of glyphosate herbicide and glufosinate-ammonium herbicide, and harvesting plants with reduced 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 to planting at least one transgenic corn In the field of plants, the transgenic maize 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 corn 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 field weeds, comprising applying herbicides containing effective doses of glyphosate and/or glufosinate-ammonium to a field where at least one transgenic corn plant is planted, so The transgenic maize 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 SEQ ID NO At least one nucleic acid sequence in the sequence shown in 7, the transgenic corn 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 maize 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 corn plant also has paraglutamic acid ammonium 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 corn plant tolerant to glyphosate and/or glufosinate in a field where insect-resistant corn plants are planted , the glyphosate-tolerant transgenic maize 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: At least one nucleic acid sequence in the sequences shown in 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 corn flour, cornmeal, corn oil, cornstarch, Corn gluten, tortillas, cosmetics or fillers.

In the nucleic acid sequence of the present invention for detecting herbicide-resistant corn plants 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, according to the present invention Conventional usage by those of ordinary skill in the art is to understand the terms.

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

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

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

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

"Flanking DNA" may comprise the genome naturally present in an organism such as a plant or exogenous (heterologous) DNA introduced by a transformation process, such as a fragment associated with a transformation event. Thus, flanking DNA can include a combination of native and foreign DNA. In the present invention, "flanking region" or "flanking sequence" or "genome border region" or "genome border sequence" means at least 3, 5, 10, 11, 15, 20, 50, 100, 200, 300, 400 , 1000, 1500, 2000, 2500, or 5000 base pairs or longer 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" refers to DNA comprising a junction.

The present invention provides a transgenic maize event designated DBN9858, which is a maize plant DBN9858, including plants and seeds of transgenic maize event DBN9858 and plant cells or regenerable parts thereof, and progeny thereof, said transgenic maize event DBN9858 Plant parts of corn event DBN9858, including but not limited to cells, pollen, ovules, flowers, buds, roots, stems, tassels, inflorescences, ears, leaves, and products from corn plant DBN9858, such as corn flour, cornmeal, corn oil , corn steep liquor, corn silk, cornstarch and biomass left in corn crop fields.

The transgenic maize event DBN9858 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 resistant corn plant DBN9858 with any of the herbicide formulations containing glyphosate will control the growth of weeds in said field without affecting tolerance to the derived herbicide Growth or yield of plant material from maize plant DBN9858.

The enzyme 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 to glufosinate-ammonium herbicide. 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 maize plant DBN9858 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 a receptive maize plant DBN9858.

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" refers to the original transformant comprising heterologous DNA and the progeny of that transformant. The term "event" also refers to 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 maize event DBN9858, tolerant to glyphosate herbicide and glufosinate herbicide, was developed by first sexually crossing a first parent maize plant with a second parent maize plant, resulting in diverse second A generation of progeny plants, the first parent corn plant consisting of corn plants bred from transgenic corn event DBN9858 and its progeny by utilizing the herbicides against glyphosate and grasses of the present invention transformed with an expression cassette for tolerance to the ammonium phosphonate herbicide, the second parent maize plant lacking tolerance to the glyphosate herbicide and/or the glufosinate-ammonium herbicide; And/or progeny plants that are tolerant to the application of glufosinate-ammonium herbicide can breed corn plants that are 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 corn plant or the third parent corn plant, followed by application of grass Glyphosate herbicides, glufosinate-ammonium herbicides, or by identification of trait-associated molecular markers such as DNA molecules containing the junction sites identified at the 5' and 3' ends of the insert sequence in transgenic maize event DBN9858 Progeny are selected to produce corn 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 corn can kill insects/pests such as Lepidoptera and Coleoptera, 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 U.S. Environmental Protection Agency has given the following guidelines for the use of genetically modified crops, and a certain proportion of shelter corn is required (the requirements for shelter corn vary from 5%, 10%, and 10% depending on the product. 20%, etc. and can be non-insect resistant GM corn (such as herbicide tolerant GM corn), or corn resistant to non-target pests, not necessarily non-GM corn). When most of the insects/pests are killed on the corresponding transgenic insect-resistant corn, some insects/pests are not dead on the shelter corn, which ensures that the insects/pest groups without resistance account for the dominant number. 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 number of 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 transgenic maize event DBN9858, whether the genomic DNA is from transgenic maize event DBN9858 or the seed or derived from the transgene Plant or seed or extract of maize event DBN9858. 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 maize event DBN9858, and determining the nucleic acid sequence of the DNA molecule. The DNA molecule contains the transgene insertion sequence and the maize genome flanking region, and the fragments of the DNA molecule can be used as primers 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 maize event DBN9858 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, in order to determine whether corn plants were produced by sexual crossing containing the transgenic corn event DBN9858 of the present invention, or whether a corn sample collected from a field contained the transgenic corn event DBN9858, or whether a corn extract, such as meal, meal or oil contained For transgenic maize event DBN9858, DNA extracted from maize plant tissue samples or extracts can be subjected to nucleic acid amplification methods using primer pairs to generate amplicons that are diagnostic for the presence of DNA from transgenic maize event DBN9858. The pair of primers includes a first primer derived from a flanking sequence adjacent to the insertion site of the inserted foreign DNA in the plant genome, and a second primer derived from the inserted foreign DNA. The amplicon has a length and sequence that is also diagnostic for the transgenic maize event DBN9858. 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 maize event DBN9858 can be amplified from the genome of the transgenic maize event DBN9858 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. A kit containing DNA primers that are homologous or complementary to any part of the maize 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 maize event DBN9858 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 GeneticBit Analysis, which designs a DNA oligonucleotide strand spanning the insert DNA sequence and the adjacent flanking genomic DNA sequence. The oligonucleotide strands are immobilized in the microwells of a microplate, and after PCR amplification of the region of interest (using one primer each within the insert sequence and adjacent flanking genomic sequences), the single-stranded PCR product Can hybridize to immobilized oligonucleotide strands and serve as templates for single-base extension reactions using a DNA polymerase and ddNTPs specifically labeled for the next expected base. Results can be obtained by fluorescent or ELISA-like methods. The signal represents the presence of the insertion/flanking sequence, which indicates that the amplification, hybridization and single base extension reactions were successful.

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

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 maize event DBN9858 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 maize event DBN9858 in a sample, and can also be used to breed maize plants containing DNA from transgenic maize event DBN9858. 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 maize genome contained in the maize genome and illustrated in Figure 1 and Table 1 contains: the flanking genomic region of maize plant DBN9858 located at the 5' end of the transgene insert sequence, from the left side of Agrobacterium Part of the insert sequence in the border region (LB), the first expression cassette consists of a tandem repeat of the cauliflower mosaic virus 35S promoter (pr35S) containing an enhancer region, operably linked to the glufosinate-resistant Streptomyces phosphinothricin N-acetyltransferase (cPAT) and is operably linked to the cauliflower mosaic virus 35S terminator (t35S), the second expression cassette consists of the rice actin 1 promoter ( prOsAct1), operably linked to the coding sequence for the Arabidopsis EPSPS chloroplast transit peptide (spAtCTP2), operably linked to the glyphosate-tolerant 5-enol-pyruvylshikimate-3 of the Agrobacterium CP4 strain - Phosphate synthase (cEPSPS), consisting of operably linked to the transcription terminator (tNos) of nopaline synthase, a part of the insert sequence from the right border region (RB) of Agrobacterium, and the insert sequence located at transgene insert 3 Maize plant DBN9858 flanking genomic region at the ' end (SEQ ID NO:5). In the DNA amplification method, the DNA molecules used as primers can be any part of the transgene insert sequence derived from the maize plant DBN9858, or any part of the DNA region flanking the maize genome in the transgenic maize event DBN9858.

GM maize event DBN9858 can be combined with other GM maize varieties, such as herbicide tolerant (such as 2,4-D, dicamba, etc.), or GM maize varieties carrying other insect resistance genes (such as Cry1Ab, Vip3A, etc.) . Various combinations of all these different transgenic events, bred together with the transgenic maize event DBN9858 of the present invention, can provide improved hybrid transgenic maize 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 for detecting herbicide-tolerant corn plant DBN9858 and a detection method thereof. The transgenic corn event DBN9858 is tolerant to the phytotoxic effect of agricultural herbicides containing glyphosate and/or glufosinate-ammonium. The dual-trait maize 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 corn has the following advantages: 1) the ability to apply glyphosate-containing agricultural herbicides to corn crops for broad-spectrum weed control; 2) the use of glufosinate-ammonium herbicides in combination with Glyphosate herbicide mixed or alternate use) can be used as a non-selective means to effectively manage glyphosate-resistant weeds; 3) herbicide-tolerant transgenic corn as non-insect-resistant transgenic corn, and transgenic insect-resistant corn and other Planting together in a certain proportion can delay the resistance of insects/pests; 4) The yield of corn 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 maize event DBN9858, 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 maize event DBN9858 or its progeny, and can quickly, accurately and stably identify the existence of the plant material derived from the transgenic maize event DBN9858.

sequence description

Insertion site of the 5' transgene fragment in SEQ ID NO: 1 transgenic maize event DBN9858 and 11 nucleotides on each side of maize genomic DNA;

Insertion site of the 3' transgene fragment in SEQ ID NO: 2 transgenic maize event DBN9858 and 11 nucleotides on each side of maize genomic DNA;

A sequence of 1301 nucleotides in length near the insertion junction at the 5' end of the insertion sequence in SEQ ID NO: 3 transgenic maize event DBN9858;

A sequence of 1310 nucleotides in length near the insertion junction at the 3' end of the insertion sequence in SEQ ID NO: 4 transgenic maize event DBN9858;

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

SEQ ID NO:6 is located at the sequence within SEQ ID NO:3, spanning the DBN10006 construct DNA sequence and the pr35S promoter sequence;

SEQ ID NO:7 is located at the sequence within SEQ ID NO:4, spanning the tNos terminator sequence and the DBN10006 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;

Primers on T-DNA paired with SEQ ID NO:13 and SEQ ID NO:12;

The primer on the genomic sequence of SEQ ID NO:14 3' flanks, and its pairing with SEQ ID NO:12 can detect whether the transgene is homozygous or heterozygous;

Primers 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 probe 2 of PAT;

SEQ ID NO:22 The first primer of endogenous gene Ubiquitin in maize;

The second primer of SEQ ID NO:23 maize endogenous gene Ubiquitin;

The probe of PAT in SEQ ID NO:24 Southern hybridization detection;

The probe of EPSPS in SEQ ID NO:25 Southern 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;

The primer of SEQ ID NO: 29 located on the T-DNA is consistent with the direction of 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 the corn genome binding site used to detect the nucleic acid sequence of the herbicide-tolerant corn plant DBN9858 and the detection method of the present invention;

Fig. 2 is a schematic structural diagram of the recombinant expression vector DBN10006 used for detecting the nucleic acid sequence of the herbicide-tolerant corn plant DBN9858 and the detection method of the present invention.

Detailed ways

The technical scheme for detecting the nucleic acid sequence of the herbicide-tolerant corn plant DBN9858 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 DBN10006 (as shown in Figure 2) was constructed using standard gene cloning techniques. The vector DBN10006 contains two tandem transgene expression cassettes, the first of which consists of a tandem repeat of the cauliflower mosaic virus 35S promoter (pr35S) containing an enhancer region, operably linked to the glufosinate-resistant gene of Streptomyces. Competent phosphinothricin N-acetyltransferase (cPAT) and operably linked to the cauliflower mosaic virus 35S terminator (t35S); the second expression cassette is initiated by rice actin 1 (prOsAct1), operably linked to the coding sequence of the Arabidopsis EPSPS chloroplast transit peptide (spAtCTP2), operably linked to the glyphosate-tolerant 5-enol-pyruvylshikimic acid of the Agrobacterium sp. CP4 strain -3-phosphate synthase (cEPSPS), which is operably linked to the transcription terminator (tNos) of nopaline synthase.

The vector DBN10006 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 a selectable marker.

1.2. Plant transformation

The conventional Agrobacterium infection method was used for transformation, and the aseptically cultured corn immature embryos were co-cultured with the Agrobacterium described in Example 1.1 to transfer the T-DNA in the constructed recombinant expression vector DBN10006 into the corn genome to generate transgenic maize event DBN9858.

For Agrobacterium-mediated transformation of maize, briefly, immature immature embryos are isolated from maize and contacted with a suspension of Agrobacterium capable of converting the nucleotide sequence of the EPSPS gene and the nucleotide sequence of the PAT gene The sequence is transferred to at least one cell of one of the immature embryos (step 1: infection step), during which the immature embryos are preferably immersed in an Agrobacterium suspension (OD660=0.4-0.6, infection medium (MS salts 4.3 g /L, MS vitamins, casein 300mg/L, sucrose 68.5g/L, glucose 36g/L, acetosyringone (AS) 40mg/L, 2,4-dichlorophenoxyacetic acid (2,4-D) 1mg /L, pH 5.3)) to initiate inoculation. The immature embryos were co-cultured with Agrobacterium for a period of time (3 days) (step 2: co-cultivation step). Preferably, immature embryos are cultured on solid medium (MS salts 4.3g/L, MS vitamins, casein 300mg/L, sucrose 20g/L, glucose 10g/L, acetosyringone (AS) 100mg/L after the infection step. , 2,4-dichlorophenoxyacetic acid (2,4-D) 1mg/L, agar 8g/L, pH 5.8). After this co-cultivation phase, there can be an optional "recovery" step. In the "recovery" step, recovery medium (MS salts 4.3g/L, MS vitamins, casein 300mg/L, sucrose 30g/L, 2,4-dichlorophenoxyacetic acid (2,4-D) 1mg/ L. At least one antibiotic (cephalosporin) known to inhibit the growth of Agrobacterium exists in the plant gel (3 g/L, pH 5.8), and no selection agent for plant transformants is added (step 3: recovery step). Preferably, immature embryos are cultured on solid medium with antibiotics but no selection agent to eliminate Agrobacterium and provide a recovery period for infected cells. Next, the inoculated immature embryos are cultured on a medium containing a selection agent (glyphosate) and the growing transformed calli are selected (step 4: selection step). Preferably, the immature embryos are cultured on a selective solid medium (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 30g/L, N-(phosphonomethyl)glycine 0.25mol/L, 2,4-dichlorophenoxyacetic acid (2,4-D) 1mg/L, phytogel 3g/L, pH 5.8), resulting in selective growth of transformed cells. Then, the callus regenerates into plants (step 5: regeneration step), preferably, the callus grown on the medium containing the selection agent is cultured on solid medium (MS differentiation medium and MS rooting medium) to regenerated plants.

The resistant callus obtained by screening was transferred to the MS differentiation medium (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 30g/L, 6-benzyl adenine 2mg/L, N- (phosphonomethyl)glycine 0.125mol/L, phytogel 3g/L, pH 5.8), cultured and differentiated at 25°C. Differentiated seedlings were transferred to the MS rooting medium (MS salt 2.15g/L, MS vitamins, casein 300mg/L, sucrose 30g/L, indole-3-acetic acid 1mg/L, agar 8g/L, pH 5.8) above, cultivate at 25°C to a height of about 10cm, and move to the greenhouse to cultivate until firm. In the greenhouse, culture was carried out at 28°C for 16 hours and at 20°C for 8 hours every day.

1.3. Identification and screening of transgenic events

A total of 332 independent transgenic T0 plants were generated.

Regenerated transgenic maize plants were tested for the presence of EPSPS and PAT genes by TaqManTM analysis (see second example), and the copy number of glyphosate and glufosinate-tolerant lines was characterized. Through screening, event DBN9858 was selected to be superior with a single copy transgene, good glyphosate herbicide tolerance, glufosinate herbicide tolerance and performance of agronomic traits (see fifth example).

The second embodiment, detection of transgenic corn event DBN9858 with TaqMan

About 100 mg of leaves of transgenic maize event DBN9858 were taken as a sample, and its genomic DNA was extracted with Qiagen's DNeasy PlantMaxi Kit, and the copy numbers of EPSPS gene and PAT gene were detected by Taqman probe fluorescence quantitative PCR method. At the same time, wild-type maize plants were used as a control, and 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 corn event DBN9858, 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 after identification as a standard, and using the sample of the wild-type corn plant as a control, each sample was 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: CTGGAAGGCGAGGACGTCATCAATA as shown in SEQ ID NO: 16 in the sequence listing;

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

Probe 1: ATGCAGGCGATGGGCGCCCGCATCCGTA 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 1×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 maize event DBN9858 was obtained.

The third embodiment, detection of transgenic corn event DBN9858

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 corn event DBN9858 and grind them into powder in liquid nitrogen, add 0.5mL and pre-cook at 65°C. Hot DNA extraction CTABBuffer (20g/L CTAB, 1.4M NaCl, 100mM Tris-HCl, 20mM EDTA (ethylenediaminetetraacetic acid), adjust the pH to 8.0 with NaOH), mix thoroughly, and extract at 65°C for 90 minutes; add 0.5 times the volume of phenol, 0.5 times the volume of chloroform, mix upside down; centrifuge at 12000rpm (revolutions per minute) for 10 minutes; absorb the supernatant, add 2 times the volume of absolute ethanol, 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 in ultra-clean bench; DNA precipitate was dissolved in 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 BamH I, Xma I, Kpn I, Sac II (5' end analysis) and Spe I, Pst I, Eco57I (3' end analysis). Add 26.5 μL of genomic DNA, 0.5 μL of the restriction enzymes selected above, and

And 3 μL enzyme digestion buffer, enzyme digestion for 1 hour. After enzyme digestion, add 70 μL of absolute ethanol to the enzyme digestion system, ice bath for 30 minutes, centrifuge at 12000 rpm for 7 minutes, discard the supernatant, blow dry, then add 8.5 μL double distilled water (dd H2O), 1 μL 10X T4Buffer And 0.5 μL T4 ligase was ligated overnight at a temperature of 4°C. PCR amplification using a series of nested primers isolates 5' and 3' transgene/genomic DNA. Specifically, the primer combination for separating 5' transgene/genome 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 the sequencing primers. Isolation 3' transgene/genomic DNA primer set includes 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 , 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 then the fragments of interest were isolated from the agarose matrix using the QIAquickGel extraction kit (catalog #_28704, Qiagen Inc., Valencia, CA). 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 maize plants or seeds derived from transgenic maize event DBN9858.

It was found that the maize genome sequence shown at nucleotides 1-1067 of SEQ ID NO:5 is flanking the right border (5' flanking sequence) of the transgenic maize event DBN9858 insert sequence, at nucleotide 5829 of SEQ ID NO:5 Position -6890 shows the maize genome sequence flanking the left border of the transgenic maize event DBN9858 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 maize event DBN9858 when detected in a polynucleotide detection assay. The junction sequences in SEQ ID NO: 1 and SEQ ID NO: 2 are the insertion site of the transgene fragment in transgenic maize event DBN9858 and 11 polynucleotides on each side of the maize 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. Junction sequences SEQ ID NO: 6 and SEQ ID NO: 7 are also novel DNA sequences in transgenic maize event DBN9858, which can also be used as DNA probes or as DNA primer molecules to detect the presence of transgenic maize event DBN9858 DNA. Said SEQ ID NO: 6 (the 1068-1301 nucleotides of SEQ ID NO: 3) spanned the DBN10006 construct DNA sequence and the pr35S promoter sequence, and said SEQ ID NO: 7 (the nucleotide of SEQ ID NO: 4) Nucleotides 1-248) span the tNos terminator sequence and the DBN10006 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 maize event DBN9858.

Specifically, a PCR product was generated from the 5' end of the transgene insert, which 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 maize event DBN9858. 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 (SEQ ID NO: 8), and its paired primer 6 (SEQ ID NO: 8) located at the transcription termination sequence of the transgene tNos 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 maize Event DBN9858 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 sequence, and paired with the pr35S promoter sequence located at the 3' end of the insert Primer 7 (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 maize event DBN9858. The detection of the amplicon can be performed by using Stratagene Robocycler, MJ Engine, Perkin-Elmer 9700 or Eppendorf Mastercycler Gradien thermal cycler as shown in Table 3, 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 maize event DBN9858

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 following 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 corn event DBN9858 genomic DNA, produce an amplification product of 1301bp fragment, when used in the non-transformed corn genome DNA and non-DBN9858 maize genomic DNA in a PCR reaction, no fragment was amplified; primers 7 and 8 (SEQ ID NO: 10 and 11), when used in a PCR reaction of transgenic maize event DBN9858 genomic DNA, produced The amplified product of the 1310 bp fragment, when it was used in a PCR reaction of non-transformed maize genomic DNA and non-DBN9858 maize genomic DNA, no fragment was amplified.

PCR zygosity assays can also be used to identify whether material derived from transgenic maize event DBN9858 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 amplification reactions to generate diagnostic amplicons for transgenic maize event DBN9858. 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 maize event DBN9858.

Table 4. Reaction solution for zygosity determination

Table 5. Zygosity determination Perkin-Elmer9700 thermal cycler conditions

Cycling was performed on a 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 maize event DBN9858 in the sample. Or the reaction will yield two different DNA amplicons from a biological sample containing DNA derived from the maize genome that is heterozygous for the allele corresponding to the inserted DNA present in transgenic maize event DBN9858 of. These two distinct amplicons would correspond to the first amplicon derived from the wild-type maize genomic locus and the second amplicon diagnostic for the presence of transgenic maize event DBN9858 DNA. Generating only a single amplicon corresponding to the second amplicon described for the heterozygous genome, a maize DNA sample in which the presence of transgenic maize event DBN9858 can be diagnostically determined is generated relative to the presence in transgenic maize plants of DBN9858 The alleles corresponding to the inserted DNA are produced in maize seeds that are homozygous.

It should be noted that the primer pair for transgenic maize event DBN9858 was used to generate amplicons that were diagnostic for the genomic DNA of transgenic maize event DBN9858. 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 maize genes was included as an internal standard for reaction conditions. Analysis of DNA extract samples from GM maize event DBN9858 should include a positive tissue DNA extract control from GM maize event DBN9858, a negative DNA extract control from non-GM maize event DBN9858 and a maize 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 their complement, which when used in a DNA amplification reaction respectively produces Tissues from plant DBN9858 were 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 maize event DBN9858 using appropriate primer pairs. An extract putatively containing DNA from a maize plant or seed comprising transgenic maize event DBN9858, or a product derived from transgenic maize event DBN9858, that produces an amplicon that is diagnostic for transgenic maize event DBN9858 when tested in a DNA amplification method , can be used as a template for amplification to determine the presence or absence of transgenic maize event DBN9858.

The fourth embodiment, detection of transgenic maize event DBN9858 by Southern blot hybridization

4.1. DNA extraction for Southern blot hybridization

Southern blot analysis was performed using homozygous transformation events in the T4 and T5 generations. Using a mortar and pestle, grind approximately 5 to 10 g of plant tissue in liquid nitrogen. Resuspend plant tissue in 12.5 mL of extraction buffer A (0.2M Tris pH 8.0, 50 mM EDTA, 0.25M NaCl, 0.1% v/v β-mercaptoethanol, 2.5% w/v polyvinyl-pyrrolidone) and centrifuge at 4000 rpm 10 minutes (2755g). After discarding the supernatant, in 2.5mL extraction buffer B (0.2M Tris pH 8.0, 50mM EDTA, 0.5M NaCl, 1% v/v β-mercaptoethanol, 2.5% w/v polyvinyl-pyrrolidone, 3% Sarkosyl, 20% ethanol) and incubated at 37°C for 30 minutes. During the incubation, the samples were mixed once with a sterile loop. 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. The aqueous layer was collected and centrifuged at 4000 rpm for 5 minutes after addition of 0.54 volumes of isopropanol to pellet the DNA. The supernatant was discarded, and the DNA pellet was resuspended in 500 μLTE. To degrade any RNA present, DNA was incubated with 1 μL of 30 mg/ml LRNAase A for 30 min at 37 °C, centrifuged at 4000 rpm for 5 min, and passed through in the presence of 0.5 volume of 7.5 M ammonium acetate and 0.54 volume of isopropanol. DNA was pelleted by centrifugation at 14000 rpm for 10 minutes. After discarding the supernatant, the pellet was washed with 500 μL of 70% ethanol, dried and resuspended in 100 μL of TE.

4.2. Restriction enzyme digestion

DNA concentration was quantified using a spectrophotometer or fluorometer (using 1X TNE and Hoechst dye).

In a 100 μL reaction system, digest 5 μg of DNA each time. Genomic DNA was digested with restriction endonucleases Sac I and Hind III respectively, 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. The sample was spun down using a speed vacuum to reduce the volume to 30 [mu]L.

4.3. Gel electrophoresis

Add bromophenol blue loading dye to each sample derived from Example 4.2, and load each sample on a 0.7% agarose gel containing ethidium bromide, and separate by electrophoresis in TBE electrophoresis buffer , run the gel overnight at 20 volts.

The gel was washed in 0.25M HCl for 15 minutes to depurinate the DNA, followed by washing with water. The Southern blot hybridization was set up as follows: 20 thick sheets of dry blotting paper were placed in a dish and 4 thin sheets of dry blotting paper were placed on top of them. A sheet of thin blotting paper was pre-wetted in 0.4M NaOH and placed on top of the stack, followed by a sheet of Hybond-N+ transfer membrane (Amersham Pharmacia Biotech, #RPN303B) pre-wetted in 0.4M NaOH. The gel is placed on top, making sure there are no air bubbles between the gel and the membrane. 3 additional pre-soaked blotting papers were placed on top of the gel and the buffer tray was filled with 0.4M NaOH. The DNA was transferred to the membrane by connecting the gel stack and the buffer disc with a wick pre-soaked in 0.4 M NaOH. Perform DNA transfer for approximately 4 hours at room temperature. After transfer, the Hybond membrane was rinsed in 2×SSC for 10 seconds, and the DNA was bound to 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. 25 ng of probe DNA was boiled in 45 μLTE for 5 minutes, placed on ice for 7 minutes, and then transferred to a Rediprime II (Amersham Pharmacia Biotech, #RPN1633) tube. After adding 5 μl of 32P-labeled dCTP to the Rediprime tube, the probe was incubated at 37° C. for 15 minutes. The probe was purified by centrifugation on a microcentrifuge G-50 column (Amersham Pharmacia Biotech, #27-5330-01 ) to remove unincorporated dNTPs according to the manufacturer's instructions. Probe activity was measured using a scintillation counter.

The Hybond membrane was prehybridized by wetting the Hybond membrane with 20 mL of pre-warmed Church prehybridization solution (500 mM Na3P04, 1 mM EDTA, 7% SDS, 1% BSA) at 65°C for 30 minutes. The labeled probes were boiled for 5 minutes and placed on ice for 10 minutes. An appropriate amount of probe (1 million counts per 1 mL of prehybridization buffer) was added to the prehybridization buffer, and hybridization was performed overnight at 65°C. The next day, the hybridization buffer was discarded, and after rinsing with 20 mL of Church Washing Solution 1 (40 mM Na3P04, 1 mM EDTA, 5% SDS, 0.5% BSA), the membrane was washed in 150 mL of Church Washing Solution 1 at 65°C for 20 minutes. This process was repeated twice with Church rinse solution 2 (40 mM Na3P04, 1 mM EDTA, 1% SDS). The membrane is exposed to a phosphor screen or X-ray film to detect where the probe binds.

Three control samples were included on each Southern: (1) DNA from negative (untransformed) segregants, which was used to identify any endogenous maize sequences that could hybridize with element-specific probes; (2) DNA from DNA from a negative segregant incorporating Hind III-digested DBN10006 in an amount equivalent to one copy number based on probe length to illustrate the sensitivity of the assay in detecting single gene copies within the maize genome; and (3 ) based on a probe length equivalent to one copy number of HindIII-digested DBN10006 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 maize plant DBN9858 contained single copies of the EPSPS and PAT genes. Using this EPSPS probe, Sac I and Hind III enzymatically digested to produce single bands of about 4kb and 11kb in size respectively; using this PAT probe, Sac I and Hind III enzymatically digested to produce single bands of about 3.5kb and 1.8kb in size respectively bring. This indicates that one copy each of EPSPS and PAT was present in maize transformation event DBN9858.

The fifth embodiment, the herbicide tolerance detection of event

In this experiment, Roundup herbicide (41% glyphosate isopropyl ammonium salt solution) and Baoshida 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 15m2 (5m×3m), the row spacing is 60cm, the plant spacing is 25cm, conventional cultivation and management, and there is a 1m wide isolation zone between the plots. The transgenic corn event DBN9858 was subjected to the following three treatments: 1) no spraying; 2) spraying Roundup herbicide at the V3 leaf stage at a dose of 1680 g a.e./ha, and then spraying Roundup herbicide again at the V8 stage at the same dose; 3) Spray Basta herbicide at V3 leaf stage according to 800g a.i./ha dose, and then spray Basta herbicide again at V8 stage with the same dosage. 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 corn yield of the plot was measured at the time of harvest. The grading of phytotoxicity symptoms is shown in Table 6. 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 corn yield of each plot is the total yield (weight) of corn kernels 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 corn yield of transgenic corn event DBN9858 are shown in Table 7.

Table 6. Grading standards for the degree of herbicide damage to corn

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 7. Results of herbicide tolerance and corn yield of transgenic corn event DBN9858

The results showed that in terms of herbicide (glyphosate and glufosinate) damage rate: 1) the damage rate of the transgenic corn event DBN9858 was basically 0 under the treatment of glyphosate herbicide (1680g a.e./ha); The damage rate under the treatment of glufosinate-ammonium herbicide (800g a.i./ha) was basically 0; thus, the transgenic corn event DBN9858 has good herbicide (glyphosate and glufosinate-ammonium) tolerance.

In terms of yield: the transgenic corn event DBN9858 had no significant difference in yield under the three treatments of no spraying, glyphosate herbicide (1680g a.e./ha) and glufosinate-ammonium herbicide (800g a.i./ha); Afterwards, the yield of the transgenic maize event DBN9858 increased slightly, thus further indicating that the transgenic maize event DBN9858 has good herbicide (glyphosate and glufosinate) tolerance.

Sixth embodiment

Agricultural products or commodities such as can be produced from transgenic maize event DBN9858. If sufficient expression levels are 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 transgenic maize event DBN9858 material in the agricultural product or commodity. Such agricultural products or commodities include, but are not limited to, corn oil, corn meal, cornmeal, corn gluten, corn tortillas, cornstarch, and any other food product that is to be consumed by an animal as a food source, or otherwise as a bulking agent or in a cosmetic composition The ingredients are used for cosmetic purposes etc. Nucleic acid detection methods and/or kits based on probes or primer pairs can be developed to detect the transgenic maize event DBN9858 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 as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5 to diagnose the presence of transgenic maize event DBN9858.

In summary, the transgenic corn event DBN9858 of the present invention has good tolerance to glyphosate herbicide and glufosinate-ammonium herbicide, has no effect on yield, and the detection method can accurately and quickly identify whether the biological sample contains transgene DNA molecule from maize event DBN9858.

The seeds corresponding to the genetically modified corn event DBN9858 were preserved on December 24, 2014 in the General Microbiology Center of 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 Research Institute, zip code 100101), classification name: corn (Zea mays), deposit number is CGMCCNo.10212. 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 (27)

1. a kind of nucleic acid sequence, which is characterized in that including SEQ ID NO:1 or its complementary series, and/or SEQ ID NO:2 or Its complementary series, the nucleic acid sequence be originated from transgenic corn events DBN9858, the transgenic corn events DBN9858 with The form of seed and to be preserved in China Committee for Culture Collection of Microorganisms with deposit number CGMCC No.10212 commonly micro- Bio-Centers.

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

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

4. a kind of method existing for DNA of detection sample transgenic corn event DBN9858, which is characterized in that including：

Detected sample is set to be contacted in nucleic acid amplification reaction at least two primers；

Carry out nucleic acid amplification reaction；

Detect the presence of amplified production；

The amplified production includes SEQ ID NO:1 or its complementary series, and/or SEQ ID NO:2 or its complementary series；

The amplified production is originated from transgenic corn events DBN9858, and the transgenic corn events DBN9858 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.10212.

5. method existing for the DNA of detection sample transgenic corn event DBN9858 according to claim 4, feature It is, the amplified production further includes SEQ ID NO:6 or its complementary series, and/or SEQ ID NO:7 or its complementary series.

6. method existing for the DNA of the detection sample transgenic corn event DBN9858 according to claim 4 or 5, special Sign is that the primer includes the first primer and the second primer, and the first primer is selected from SEQ ID NO:1 or its complementary sequence Row, SEQ ID NO:8 and SEQ ID NO:10；Second primer is selected from SEQ ID NO:2 or its complementary series, SEQ ID NO:9 and SEQ ID NO:11.

7. a kind of method existing for DNA of detection sample transgenic corn event DBN9858, which is characterized in that including：

Detected sample is set to be contacted 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 corn event DBN9858, the transgenic corn events DBN9858 is to plant Son form and China Committee for Culture Collection of Microorganisms's commonly micro- life is preserved in deposit number CGMCC No.10212 Object center；

The detected sample and the probe is set to hybridize under stringent hybridization conditions；

Detect the hybridisation events of the detected sample and the probe.

8. method existing for the DNA of detection sample transgenic corn event DBN9858 according to claim 7, feature It is, the probe also has SEQ ID NO:6 or its complementary series or SEQ ID NO:7 or its complementary series.

9. special according to method existing for the DNA of the detection sample transgenic corn event DBN9858 of claim 7 or 8 Sign is that at least one probe is marked at least one fluorophor.

10. a kind of method existing for DNA of detection sample transgenic corn event DBN9858, which is characterized in that including：

Detected sample is set to be contacted with marker nucleic acid molecules, the marker nucleic acid molecules include SEQ ID NO:1 or its mutually Complementary series or SEQ ID NO:2 or its complementary series, the marker nucleic acid molecules are originated from transgenic corn events DBN9858, the transgenic corn events DBN9858 are preserved in the form of seed and with deposit number CGMCC No.10212 China Committee for Culture Collection of Microorganisms's common micro-organisms center；

The detected sample and the marker nucleic acid molecules is set to hybridize under stringent hybridization conditions；

The hybridisation events of the detected sample 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 detection sample transgenic corn event DBN9858 according to claim 10, special Sign is that the marker nucleic acid molecules further include SEQ ID NO:6 or its complementary series or SEQ ID NO:7 or its mutually Complementary series.

12. a kind of DNA detection kits, 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, it can be used as transgenic corn events DBN9858 or its offspring have one of DNA primer of specificity or probe；The DNA molecular is originated from transgenic corn events DBN9858, the transgenic corn events DBN9858 are preserved in the form of seed and with deposit number CGMCC No.10212 China Committee for Culture Collection of Microorganisms's common micro-organisms center.

13. according to DNA detection kits described in claim 12, which is characterized in that further include when the DNA molecular is as probe SEQ ID NO:6 or its complementary series or SEQ ID NO:7 or its complementary series.

14. a kind of method generating the plant that there is tolerance to glyphosate herbicidal, which is characterized in that including to described SEQ ID NO are introduced in the genome of plant:5 1157-5744 nucleic acid sequences, and make the base of the plant Include SEQ ID NO successively because organizing:1,SEQ ID NO:5 1157-5744 nucleic acid sequences and SEQ ID NO:2, or make The genome for obtaining the plant includes SEQ ID NO:5, the plant of selection tolerance glyphosate.

15. according to the method for generating the plant that there is tolerance to glyphosate herbicidal described in claim 14, feature It is, including：

By first parental maize plants of transgenic corn events DBN9858 to glyphosate herbicidal with tolerance and lack grass Second parental maize plant sexual hybridization of sweet phosphine tolerance, to generate a large amount of progeny plants；

The progeny plant is handled with glyphosate herbicidal；

The progeny plant of selection tolerance glyphosate；

The transgenic corn events DBN9858 is preserved in China in the form of seed and with deposit number CGMCC No.10212 Microbiological Culture Collection administration committee common micro-organisms center.

16. a kind of method generating the plant that there is tolerance to glufosinate-ammonium herbicide, which is characterized in that including to described SEQ ID NO are introduced in the genome of plant:5 1157-5744 nucleic acid sequences, and make the base of the plant Include SEQ ID NO successively because organizing:1,SEQ ID NO:5 1157-5744 nucleic acid sequences and SEQ ID NO:2, or make The genome for obtaining the plant includes SEQ ID NO:5, the plant of selection tolerance glufosinate-ammonium.

17. according to the method for generating the plant that there is tolerance to glufosinate-ammonium herbicide described in claim 16, feature It is, including：

By first parental maize plants of transgenic corn events DBN9858 to glufosinate-ammonium herbicide with tolerance and lack grass Second parental maize plant sexual hybridization of ammonium phosphine tolerance, to generate a large amount of progeny plants；

The progeny plant described in glufosinate-ammonium herbicide treatment；

The progeny plant of selection tolerance glufosinate-ammonium；

The transgenic corn events DBN9858 is preserved in China in the form of seed and with deposit number CGMCC No.10212 Microbiological Culture Collection administration committee common micro-organisms center.

18. a kind of method generating the plant that there is tolerance to glyphosate herbicidal and glufosinate-ammonium herbicide, feature It is, includes introducing SEQ ID NO into the genome of the plant:5 1157-5744 nucleic acid sequences, and make The genome of the plant includes SEQ ID NO successively:1,SEQ ID NO:5 1157-5744 nucleic acid sequences and SEQ ID NO:2, or so that the genome of the plant includes SEQ ID NO:5, selection tolerance glyphosate and glufosinate-ammonium Plant.

19. according to the plant that there is tolerance to glyphosate herbicidal and glufosinate-ammonium herbicide is generated described in claim 18 Method, which is characterized in that including：

There to be the first parents of transgenic corn events DBN9858 of tolerance beautiful glyphosate herbicidal and glufosinate-ammonium herbicide Rice plant and the second parental maize plant sexual hybridization for lacking glyphosate and/or glufosinate tolerant, to generate big quantum For plant；

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

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

The transgenic corn events DBN9858 is preserved in China in the form of seed and with deposit number CGMCC No.10212 Microbiological Culture Collection administration committee common micro-organisms center.

20. a kind of method that culture has glyphosate herbicidal the corn plant of tolerance, which is characterized in that including：

An at least corn seed is planted, the nucleic acid sequence of specific region, the spy are included in the genome of the corn seed The nucleic acid sequence for determining region includes SEQ ID NO successively:1,SEQ ID NO:5 1157-5744 nucleic acid sequences and SEQ ID NO:2 or the specific region nucleic acid sequence include SEQ ID NO:5；

The corn seed is set to grow up to plant；

The plant is sprayed with effective dose glyphosate herbicidal, harvest does not have the nucleic acid of the specific region with other The plant of sequence compares the plant with the plant injury weakened.

21. a kind of method that culture has glufosinate-ammonium herbicide the corn plant of tolerance, which is characterized in that including：

An at least corn seed is planted, the nucleic acid sequence of specific region, the spy are included in the genome of the corn seed The nucleic acid sequence for determining region includes SEQ ID NO successively:1,SEQ ID NO:5 1157-5744 nucleic acid sequences and SEQ ID NO:2 or the specific region nucleic acid sequence include SEQ ID NO:5；

The corn seed is set to grow up to plant；

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

22. a kind of method that culture has glyphosate herbicidal and glufosinate-ammonium herbicide the corn plant of tolerance, feature It is, including：

An at least corn seed is planted, the nucleic acid sequence of specific region, the spy are included in the genome of the corn seed The nucleic acid sequence for determining region includes SEQ ID NO successively:1,SEQ ID NO:5 1157-5744 nucleic acid sequences and SEQ ID NO:2 or the specific region nucleic acid sequence include SEQ ID NO:5；

The corn seed is set to grow up to plant；

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

23. a kind of protecting the plants from the method damaged 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 rotaring gene corn plant, the transgenosis Corn plant includes SEQ ID NO successively in its genome:1,SEQ ID NO:5 1157-5744 nucleic acid sequence and SEQ ID NO:2 or the rotaring gene corn plant genome in include SEQ ID NO:5；The transgenic corns are planted Object has the tolerance to glyphosate herbicidal and/or glufosinate-ammonium herbicide.

24. a kind of method of control 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 rotaring gene corn plant, the rotaring gene corn plant is in its genome In successively include SEQ ID NO:1,SEQ ID NO:5 1157-5744 nucleic acid sequence and SEQ ID NO:2, Huo Zhesuo State in the genome of rotaring gene corn plant includes SEQ ID NO:5；The rotaring gene corn plant has to Gyphosate herbicice The tolerance of agent and/or glufosinate-ammonium herbicide.

25. a kind of method of the crop field glyphosate resistance weeds of control 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 rotaring gene corn plant for planting at least one glyphosate tolerant The rotaring gene corn plant of Tanaka, the glyphosate tolerant include SEQ ID NO successively in its genome:1,SEQ ID NO:5 1157-5744 nucleic acid sequence and SEQ ID NO:2 or the glyphosate tolerant rotaring gene corn plant Genome in include SEQ ID NO:5；The rotaring gene corn plant of the glyphosate tolerant has simultaneously removes glufosinate-ammonium The tolerance of careless agent.

26. a kind of method delaying insect-resistant, which is characterized in that be included in plant pest-resistant corn plant big Tanaka plant to A kind of few rotaring gene corn plant with glyphosate and/or glufosinate tolerant, the glyphosate and/or glufosinate tolerant Rotaring gene corn plant in its genome successively include SEQ ID NO:1,SEQ ID NO:5 1157-5744 cores Acid sequence and SEQ ID NO:2 or the rotaring gene corn plant of the glyphosate and/or glufosinate tolerant genome in Including SEQ ID NO:5.

27. a kind of agricultural product or commodity produced by transgenic corn events DBN9858, which is characterized in that the agricultural product or Commodity are corn flour, maize flour, corn oil, cornstarch, corn gluten, corn-dodger, cosmetics or filler；The transgenosis Corn event DBN9858 is preserved in Chinese microorganism strain preservation in the form of seed and with deposit number CGMCC No.10212 Administration committee's common micro-organisms center.