WO2016173361A1

WO2016173361A1 - Maize plant dbn9936 and method for use in detecting nucleic acid sequence thereof

Info

Publication number: WO2016173361A1
Application number: PCT/CN2016/077866
Authority: WO
Inventors: 丁德荣; 康越景; 张云珠; 刘海利; 庞洁; 王利君; 贾志伟; 黄金存; 郭函子; 王磊; 傅学乾; 周毅; 李风; 鲍晓明; 吕玉平; 张世平
Original assignee: 北京大北农科技集团股份有限公司; 北京大北农生物技术有限公司
Priority date: 2015-04-30
Filing date: 2016-03-30
Publication date: 2016-11-03
Also published as: UA127944C2; RU2017137790A3; RU2707527C2; CN104830847A; PH12017501967A1; CN104830847B; BR112017023304A2; RU2017137790A; AR105554A1

Abstract

Provided are a nucleic acid sequence used for detecting in a biological sample the presence of maize event DBN9936 and a detection method therefor.

Description

Maize plant DBN9936 and nucleic acid sequence and method for detecting same

Technical field

The invention relates to the field of plant molecular biology, in particular to the field of transgenic crop breeding in agricultural biotechnology research. In particular, the present invention relates to insect resistance and glyphosate herbicide tolerance transgenic maize event DBN9936 and nucleic acid sequences for detecting whether a transgenic maize event DBN9936 is included in a biological sample and a method of detecting the same.

Background technique

Maize (Zea mays L.) is a major food crop in many parts of the world. Biotechnology has been applied to corn to improve its agronomic traits and qualities. Insect resistance is an important agronomic trait in corn production, especially against lepidopteran insects such as corn borer, cotton bollworm, and armyworm. The resistance of maize to lepidopteran insects can be obtained by transgenic methods in which the resistance genes of lepidopteran insects are expressed in corn plants. Another important agronomic trait is herbicide tolerance, particularly tolerant to glyphosate herbicides. The tolerance of maize to glyphosate herbicides can be obtained by transgenic methods to express glyphosate herbicide tolerance genes (such as EPSPS) in maize plants.

It is known that the expression of foreign genes in plants is influenced by their chromosomal location, possibly due to the proximity of the chromatin structure (such as heterochromatin) or transcriptional regulatory elements (such as enhancers) to the integration site. To this end, it is often necessary to screen a large number of events to identify events that can be commercialized (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 the introduced gene may vary greatly from event to event; there may also be differences in spatial or temporal patterns of expression, such as relative expression of transgenes between different plant tissues. There is a difference in that the actual expression pattern may be inconsistent with the expression pattern expected from the transcriptional regulatory elements in the introduced gene construct. Therefore, it is often necessary to generate hundreds and thousands of different events and to screen out from these events a single event with the expected expression and expression pattern of the transgene for commercial purposes. Events with expected transgene expression levels and expression patterns can be used to infiltrate transgenes into other genetic backgrounds by sexual heterotypic hybridization using conventional breeding methods. The progeny produced by this hybridization maintain the transgene expression characteristics of the original transformants. The application of this strategy model ensures reliable gene expression in many varieties, and these varieties are well adapted to local growth conditions.

Ability to detect the presence of a specific event to determine whether a progeny of sexual crosses contains a gene of interest It will be beneficial. In addition, methods for detecting specific events will also help to comply with regulations, such as foods derived from recombinant crops that require formal approval and labeling prior to being placed on the market. It is possible to detect the presence of a transgene by any well-known polynucleotide detection method, such as polymerase chain reaction (PCR) or DNA hybridization using a polynucleotide probe. These assays typically focus on commonly used genetic elements such as promoters, terminators, marker genes, and the like. Therefore, unless the sequence of the chromosomal DNA ("flanking DNA") adjacent to the inserted transgenic DNA is known, the above method cannot be used to distinguish different events, especially those produced using the same DNA construct. event. Therefore, transgene-specific events are currently identified by PCR using a pair of primers spanning the junction of the inserted transgene and flanking DNA, specifically the first primer contained in the flanking sequence and the second primer comprising the inserted sequence.

Summary of the invention

The object of the present invention is to provide corn plant DBN9936 and nucleic acid sequence and method for detecting the same, and the transgenic corn event DBN9936 has better resistance to insects and has good tolerance to glyphosate herbicide, and the detection method It is possible to accurately and quickly identify whether the biological sample contains the DNA molecule of the corn event DBN9936.

To achieve the above object, the present invention provides a nucleic acid sequence comprising at least 11 contiguous nucleotides of SEQ ID NO: 3 or its complement, and/or at least 11 of SEQ ID NO: 4 or its complement Continuous 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 comprises SEQ ID NO: 3 or its complement, and/or SEQ ID NO: 4 or its complement.

Further, the nucleic acid sequence comprises SEQ ID NO: 5 or its complement.

The SEQ ID NO: 1 or its complement is a 22 nucleotide sequence in the transgenic maize event DBN9936 located near the insertion junction at the 5' end of the inserted sequence, or SEQ ID NO: 1 The complementary sequence spans the flanking genomic DNA sequence of the maize insertion site and the DNA sequence at the 5' end of the inserted sequence, and the SEQ ID NO: 1 or its complement can be identified as the presence of the transgenic maize event DBN9936. The SEQ ID NO: 2 or its complement is a 22 nucleotide sequence in the transgenic maize event DBN9936 located near the insertion junction at the 3' end of the inserted sequence, the SEQ ID NO: 2 or its complementary sequence spans the DNA sequence at the 3' end of the inserted sequence and the flanking genomic DNA sequence of the maize insertion site, and the SEQ ID NO: 2 or its complement can be identified as the transgenic maize event DBN9936 presence.

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 of SEQ ID NO: 3 or its complement, or At least 11 or more contiguous polynucleotides (second nucleic acid sequence) of any portion of the 5' flanking maize genomic DNA region of SEQ ID NO: 3 or its complement. The nucleic acid sequence may further be homologous or complementary to a portion of the SEQ ID NO: 3 comprising the entire SEQ ID NO: 1. When the first nucleic acid sequence is used together with the second nucleic acid sequence, these nucleic acid sequences can be used as a DNA primer pair in a DNA amplification method for producing an amplification product. When the amplification product produced in the DNA amplification method using the DNA primer pair is an amplification product comprising SEQ ID NO: 1, the presence of the transgenic maize event DBN9936 or its progeny can be diagnosed. As is well known to those skilled in the art, the first and second nucleic acid sequences need not be composed solely of DNA, but may also include RNA, a mixture of DNA and RNA, or DNA, RNA or other nucleosides that are not used as one or more polymerase templates. A combination of an acid or an analog thereof. Furthermore, the probe or primer of the present invention should be at least about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 contiguous nucleotides, which may be selected from Nucleotides as set forth 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 set forth in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, the probe and primer may be continuous from at least about 21 to about 50 or more in length. Nucleotide. The SEQ ID NO: 3 or its complement is a sequence of 1001 nucleotides in length in the transgenic maize event DBN9936 located near the insertion junction at the 5' end of the inserted sequence, or SEQ ID NO: 3 The complementary sequence consists of a 832 nucleotide maize flanking genomic DNA sequence (nucleotides 1-832 of SEQ ID NO: 3), and a nucleotide of 77 DBN10124 construct DNA sequences (nucleotides of SEQ ID NO: 3) Acid 833-909) and 92 nucleotides of the tNos (nopaline synthase) transcription terminator 3' end DNA sequence (nucleotides 910-1001 of SEQ ID NO: 3), comprising the SEQ ID NO :3 or its complement can be identified as the presence of the transgenic maize event DBN9936.

The nucleic acid sequence may be at least 11 or more contiguous polynucleotides (third nucleic acid sequence) of any portion of the transgene insertion sequence of SEQ ID NO: 4 or its complement, or the SEQ ID NO At least 11 or more contiguous polynucleotides (fourth nucleic acid sequence) of any portion of the 3' flanking maize genomic DNA region in 4 or its complement. Said The nucleic acid sequence may further be homologous or complementary to a portion of the SEQ ID NO: 4 comprising the entire SEQ ID NO: 2. When the third nucleic acid sequence is used together with the fourth nucleic acid sequence, these nucleic acid sequences can be used as a DNA primer pair in a DNA amplification method for producing an amplification product. When the amplification product produced in the DNA amplification method using the DNA primer pair is an amplification product comprising SEQ ID NO: 2, the presence of the transgenic maize event DBN9936 or its progeny can be diagnosed. The SEQ ID NO: 4 or its complement is a sequence of 1204 nucleotides in length in the transgenic maize event DBN9936 located near the insertion junction at the 3' end of the inserted sequence, or SEQ ID NO: 4 The complementary sequence consists of a 38 nucleotide t35S transcription terminator sequence (nucleotides 1-38 of SEQ ID NO: 4) and 152 DBN10124 construct DNA sequences (nucleotides of SEQ ID NO: 4) Acid 39-190) and 1014 nucleotides of the maize integration site flanking genomic DNA sequence (nucleotides 191-1204 of SEQ ID NO: 4), comprising the SEQ ID NO: 4 or its complementary sequence Identification of the presence of the transgenic maize event DBN9936.

The SEQ ID NO: 5 or its complement is a sequence that characterizes the transgenic maize event DBN9936, which is 9215 nucleotides in length, and the genomic and genetic elements specifically included are shown in Table 1. The presence of the SEQ ID NO: 5 or its complement can be identified as the presence of the transgenic maize event DBN9936.

Table 1 genomic and genetic elements encompassed by SEQ ID NO: 5

The nucleic acid sequence or the complement thereof can be used in a DNA amplification method to produce an amplicon, Detection of the amplicon to diagnose the presence of the transgenic maize event DBN9936 or its progeny in a biological sample; the nucleic acid sequence or its complement may be used in a nucleotide assay to detect the transgenic maize event DBN9936 or its progeny in a biological sample presence.

To achieve the above object, the present invention also provides a method for detecting the presence of DNA of a transgenic maize event DBN9936 in a sample, comprising:

Contacting the sample with at least two primers in a nucleic acid amplification reaction;

Performing a nucleic acid amplification reaction;

Detecting the presence of an amplification product, the presence of the amplification product indicating the presence of DNA of the transgenic maize event DBN9936 in the sample;

The amplification product comprises at least 11 contiguous nucleotides of SEQ ID NO: 3 or its complement, or at least 11 contiguous nucleotides of SEQ ID NO: 4 or its complement;

Wherein the transgenic maize event DBN9936 comprises in its genome a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6. And at least one nucleic acid sequence of the sequence of SEQ ID NO: 7.

Preferably, the amplification product comprises contiguous nucleotides 1 to 11 or 12 to 22 in SEQ ID NO: 1 or its complement, or 1 to 11 in SEQ ID NO: 2 or its complement Bit or contiguous nucleotides from positions 12-22.

Preferably, the amplification product comprises SEQ ID NO: 1 or its complement, SEQ ID NO: 2 or its complement, SEQ ID NO: 6 or its complement, or SEQ ID NO: 7 or its complement;

In the above method, the primer comprises at least one of at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 consecutive nuclei selected from the nucleic acid sequences described above. Glycosylate.

Alternatively, the primer comprises a first primer selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 10, and a second primer selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO: 11.

To achieve the above object, the present invention also provides another method for detecting the presence of DNA of the transgenic maize event DBN9936 in a sample, comprising:

Contacting the sample with a probe comprising at least 11 contiguous nucleotides of SEQ ID NO: 3 or its complement, or at least 11 contiguous nucleotides of SEQ ID NO: 4 or its complement ;

Hybridizing the sample and the probe under stringent hybridization conditions;

Detecting the hybridization of the sample and the probe, the hybridization of the sample and the probe indicates the sample There is DNA of the transgenic maize event DBN9936;

The stringent conditions may be hybridization in a solution of 6 x SSC (sodium citrate), 0.5% SDS (sodium dodecyl sulfate) at 65 ° C, followed by 2 x SSC, 0.1% SDS and 1 x SSC, The membrane was washed once for each 0.1% SDS.

Preferably, the probe comprises contiguous nucleotides 1 to 11 or 12 to 22 of SEQ ID NO: 1 or its complement, or positions 1 to 11 of SEQ ID NO: 2 or its complement Or consecutive nucleotides 12-22.

Preferably, the probe comprises SEQ ID NO: 1 or its complement, SEQ ID NO: 2 or its complement, SEQ ID NO: 6 or its complement, or SEQ ID NO: 7 or its complement.

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

Passing the sample with a label nucleic acid molecule comprising at least 11 contiguous nucleotides of SEQ ID NO: 3 or its complement, or at least 11 contiguous of SEQ ID NO: 4 or its complement Nucleotide

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

Detecting hybridization of the sample and the marker nucleic acid molecule, the hybridization of the sample and the marker nucleic acid molecule indicates the presence of DNA of the transgenic maize event DBN9936 in the sample;

Preferably, the marker nucleic acid molecule comprises SEQ ID NO: 1 or its complementary sequence at positions 1-11 or 12-22, or SEQ ID NO: 2 or its complement 1 - 11th or 12th to 22nd consecutive nucleotides.

Preferably, the marker nucleic acid molecule comprises SEQ ID NO: 1 or its complement, SEQ ID NO: 2 or its complement, SEQ ID NO: 6 or its complement, or SEQ ID NO: 7 or its complement .

Preferably, the method further comprises: identifying the insect by marker-assisted breeding analysis Resistance and/or herbicide tolerance is genetically linked to the marker nucleic acid molecule.

To achieve the above object, the present invention also provides a DNA detection kit comprising at least one DNA molecule comprising at least 11 contiguous nucleotides of the homologous sequence of SEQ ID NO: 3 or a complement thereof Or at least 11 contiguous nucleotides of the homologous sequence of SEQ ID NO: 4 or its complement, which can serve as a DNA primer or probe specific for the transgenic maize event DBN9936 or a progeny thereof, wherein The transgenic maize event DBN9936 comprises in its genome a 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 of the sequence of SEQ ID NO: 7.

Preferably, the DNA molecule comprises contiguous nucleotides 1 to 11 or 12 to 22 in SEQ ID NO: 1 or its complement, or 1 to 11 in SEQ ID NO: 2 or its complement Or consecutive nucleotides 12-22.

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

To achieve the above object, the present invention also provides a plant cell or part comprising a nucleic acid sequence encoding an insect resistance Cry1Ab protein, a nucleic acid sequence encoding a glyphosate herbicide-tolerant EPSPS protein, and a nucleic acid sequence of a specific region, The nucleic acid sequence of the particular region comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 6, or SEQ ID NO: 7, from which the whole plant cannot be regenerated.

Preferably, the corn plant cell or part comprises a nucleic acid sequence encoding an insect resistant Cry1Ab protein, a nucleic acid sequence encoding a glyphosate herbicide-tolerant EPSPS protein, and a nucleic acid sequence of a specific region, the nucleic acid sequence of the specific region comprising SEQ ID NO: 3 or SEQ ID NO: 4.

Preferably, the maize plant cell or part comprises the nucleic acid sequence set forth in SEQ ID NO:5.

To achieve the above object, the present invention also provides a method of protecting a corn plant from insect attack comprising providing at least one transgenic maize plant cell or part in a diet of a target insect, the transgenic maize plant cell or part thereof The genome comprises a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7. At least one nucleic acid sequence, the target insect ingesting the transgenic maize plant cell or part is inhibited from further ingesting the corn plant.

To achieve the above object, the present invention also provides a method of protecting a corn plant from damage caused by a herbicide, comprising applying an effective amount of a glyphosate herbicide to a field planted with at least one transgenic corn plant, The transgenic maize plant comprises in its genome a cell 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 Figure 7, said transgenic corn plant having tolerance to a glyphosate herbicide.

To achieve the above object, the present invention also provides a method for controlling weeds in a field of planting corn plants, comprising applying an effective amount of a glyphosate herbicide to a field planted with at least one transgenic corn plant, said transgene The maize plant comprises in its genome a cell 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 of the sequence shown, the transgenic maize plant having tolerance to a glyphosate herbicide.

To achieve the above object, the present invention also provides a method of cultivating a corn plant resistant to insects, comprising:

Planting at least one corn seed, the genome of the corn seed comprising a nucleic acid sequence encoding an insect resistance Cry1Ab protein and a nucleic acid sequence of a specific region;

Growing the corn seed into a corn plant;

Infesting the maize plant with a target insect, harvesting plants having reduced plant damage compared to other plants having no nucleic acid sequence of a particular region;

The nucleic acid sequence of the specific region is selected from at least one of the sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 7. A nucleic acid sequence.

Preferably, the method comprises:

Planting at least one corn seed, the genome of the corn seed comprising the nucleic acid sequence set forth in SEQ ID NO: 5;

Growing the corn seed into a corn plant;

The maize plants were challenged with target insects, and plants having reduced plant damage compared to other plants not having SEQ ID NO: 5 were harvested.

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

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

Growing the corn seed into a corn plant;

Spraying the corn plant with an effective amount of a glyphosate herbicide to harvest plants having reduced plant damage compared to other plants having no nucleic acid sequence of a particular region;

Preferably, the method comprises:

Growing the corn seed into a corn plant;

The corn plants were sprayed with an effective dose of glyphosate herbicide to harvest plants having reduced plant damage compared to other plants not having SEQ ID NO: 5.

To achieve the above object, the present invention also provides a method for cultivating an insect-resistant and glyphosate-tolerant herbicide-containing corn plant, comprising:

Planting at least one corn seed, the genome of the corn seed comprising a nucleic acid sequence encoding an insect resistance Cry1Ab protein, a nucleic acid sequence encoding a glyphosate herbicide-tolerant EPSPS protein, and a nucleic acid sequence of a specific region;

Growing the corn seed into a corn plant;

Spraying the corn plant with an effective amount of a glyphosate herbicide to harvest plants having reduced plant damage compared to other plants having no nucleic acid sequence of a particular region, the plant having damage to the insect damage to the insect Also resistant;

Preferably, the method comprises:

Growing the corn seed into a corn plant;

The corn plants are sprayed with an effective amount of a glyphosate herbicide to harvest plants having reduced plant damage compared to other plants not having SEQ ID NO: 5, the feeding damage of the plants with reduced plant damage to the insects Also resistant.

In order to achieve the above object, the present invention also provides a corn which is resistant to insects. A method of planting, comprising introducing into a genome of the corn plant a nucleic acid sequence encoding an insect resistance Cry1Ab protein and a nucleic acid sequence of a specific region, wherein the nucleic acid sequence of the specific region is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2. At least one nucleic acid sequence of the sequences of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 7.

Preferably, the method comprises introducing into the genome of the maize plant the nucleic acid sequence set forth in SEQ ID NO: 5.

Specifically, the method of producing a corn plant resistant to insects comprises:

The insect-resistant transgenic maize event DBN9936 first parental maize plant is sexually crossed with the second parental maize plant lacking insect resistance, thereby producing a large number of progeny plants;

Invading the progeny plant with a target insect;

Selecting said progeny plants having reduced plant damage compared to other nucleic acid sequences that do not have a particular region or plants of SEQ ID NO: 5;

To achieve the above object, the present invention also provides a method of producing a maize plant resistant to a glyphosate herbicide comprising introducing a glyphosate-tolerant EPSPS protein into the genome of the corn plant. a nucleic acid sequence and a nucleic acid sequence of a specific region, wherein 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: 6, and SEQ ID NO: At least one nucleic acid sequence in the sequence shown in 7.

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

The first parental maize plant of the transgenic maize event DBN9936, which is tolerant to the glyphosate herbicide, is sexually crossed with the second parental maize plant lacking glyphosate tolerance, thereby producing a large number of progeny plants;

Treating the progeny plants with a glyphosate herbicide;

Selecting the progeny plants that are tolerant to glyphosate;

To achieve the above object, the present invention also provides a method of producing a corn plant resistant to insects and tolerant to glyphosate herbicide application, comprising:

The glyphosate-tolerant and insect-resistant transgenic maize event DBN9936 first parent corn plant is sexually crossed with a second parental maize plant lacking glyphosate tolerance and/or insect resistance, thereby producing a large number of progeny plants ;

Treating the progeny plants with glyphosate;

The progeny plants that are tolerant to glyphosate are selected, and the progeny plants that are tolerant to glyphosate are also resistant to insect feeding damage;

To achieve the above object, the present invention also provides a composition comprising the polynucleotide of SEQ ID NO: 1 or SEQ ID NO: 2, which is corn flour, cornmeal, corn oil, corn silk or corn starch.

To achieve the above object, the present invention also provides an agricultural product or commodity comprising the polynucleotide of SEQ ID NO: 1 or SEQ ID NO: 2, which is corn flour, corn flour, corn oil, corn starch, Corn gluten, tortillas, cosmetics or fillers.

In the nucleic acid sequences of the present invention for detecting corn plants and methods for detecting the same, the following definitions and methods can better define the present invention and guide those skilled in the art to practice the present invention, unless otherwise stated, according to the ordinary skill in the art. The general usage of personnel to understand terms.

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

The terms "including" and "including" mean "including but not limited to".

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

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

"Flanking DNA" may comprise a genome naturally present in an organism such as a plant or an 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 natural and exogenous DNA. In the present invention, "flanking sequence", "flanking genomic sequence" or "flanking genomic DNA" or "genome flanking sequence" or "DNA sequence of flanking genome" means at least 3, 5, 10, 11, 15, 20, a sequence of 50, 100, 200, 300, 400, 1000, 1500, 2000, 2500 or 5000 base pairs or longer located directly upstream or downstream of the original exogenously inserted DNA molecule and interposed with the original exogenously inserted DNA molecule adjacent. When the flanking region is located downstream, it may also be referred to as "left border flanking" or "3' flanking" or "3' genomic border region" or "genome 3' border sequence" and the like. When the flanking region is located upstream, it may also be referred to as "right border flanking" or "5' flanking" or "5' genomic border region" or "genome 5' border sequence" and the like.

A transformation program that causes random integration of foreign DNA results in transformants containing different flanking regions that are specifically contained by each transformant. When recombinant DNA is introduced into a plant by conventional hybridization, its flanking regions generally do not change. Transformants also contain unique junctions between heterologous insert DNA and segments of genomic DNA or between two segments of genomic DNA or between two heterologous DNAs. "Joining" is the point at which two specific DNA fragments are joined. For example, the junction is present at a position where the insert DNA joins the flanking DNA. The junction is also present in the transformed organism, where the two DNA fragments are joined together in a manner modified from that found in the native organism. "Joining DNA" and "joining region" refer to DNA containing a junction.

The present invention provides a transgenic maize event called DBN9936 and its progeny, which is a maize plant DBN9936 comprising plants and seeds of the transgenic maize event DBN9936 and plant cells thereof or a regenerable portion thereof, said transgene The plant part of the maize event DBN9936, including but not limited to cells, pollen, ovules, flowers, buds, Roots, stems, silks, inflorescences, ear ears, leaves and products from corn plant DBN9936, such as corn flour, cornmeal, corn oil, corn syrup, corn silk, corn starch, and biomass left in the corn crop field.

The transgenic maize event DBN9936 of the present invention comprises a DBN10124 DNA construct which, when expressed in plant cells, acquires resistance to insects and tolerance to glyphosate herbicides. The DNA construct comprises two tandem expression cassettes, the first expression cassette comprising a suitable promoter for expression in a plant and a suitable polyadenylation signal sequence operably linked to a Cry1Ab The nucleic acid sequence of the protein, the nucleic acid sequence of the Cry1Ab protein is primarily resistant to lepidopteran insects. A second expression cassette comprises a suitable promoter for expression in a plant and a suitable polyadenylation signal sequence operably linked to encode a 5-enol-pyruvylshikimate-3-phosphate The gene of the synthase (EPSPS), the nucleic acid sequence of which is resistant to glyphosate herbicides. Further, the promoter may be a suitable promoter isolated from a plant, including constitutive, inducible and/or tissue-specific promoters including, but not limited to, cauliflower mosaic virus (CaMV) 35S Promoter, Scrophularia mosaic virus (FMV) 35S promoter, Ubiquitin promoter, actin promoter, Agrobacterium tumefaciens nopaline synthase (NOS) promoter, Octopus synthase (OCS) promoter, Cestrum yellow leaf curling virus promoter, potato tuber storage protein (Patatin) promoter, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) promoter, glutathione S-transferase (GST) promoter, E9 promoter, GOS promoter, alcA/alcR promoter, Agrobacterium rhizogenes RolD promoter and Arabidopsis Thaliana) Suc2 promoter. The polyadenylation signal sequence may be a suitable polyadenylation signal sequence that functions in plants, including, but not limited to, from Agrobacterium tumefaciens. A polyadenylation signal sequence of the nopaline synthase (NOS) gene, a polyadenylation signal sequence derived from the cauliflower mosaic virus (CaMV) 35S terminator, a protease inhibitor II (PINII) gene, and a source A polyadenylation signal sequence of the α-tubulin gene.

Furthermore, the expression cassette may also include other genetic elements including, but not limited to, enhancers and signal peptides/transport peptides. The enhancer can enhance the expression level of a gene including, but not limited to, Tobacco Etch Virus (TEV) Translational Activating Factor, CaMV35S Enhancer, and FMV35S Enhancer. The signal peptide/transport peptide can be guided The Cry1Ab protein and/or EPSPS protein is transported to extracellular or specific organelles or compartments within the cell, for example, to target chloroplasts using a chloroplast transit peptide sequence, or to the endoplasmic reticulum using a 'KDEL' retention sequence.

The Cry1Ab gene may be isolated from Bacillus thuringiensis (Bt), and the nucleotide sequence of the Cry1Ab gene may be altered by optimizing codons or otherwise to increase the transcripts in the transformed cells. The purpose of stability and availability.

The "Lepidoptera", including moths and butterflies, is one of the most pests of agricultural and forestry pests, such as corn borer, cotton bollworm, oriental armyworm, two-pointed moth, and peach aphid.

The 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) gene may be isolated from Agrobacterium tumefaciens CP4 strain and may be altered by optimizing codons or otherwise Polynucleotides of the EPSPS gene for the purpose of increasing the stability and availability of transcripts in transformed cells. The 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) gene can also be used as a selectable marker gene.

The "glyphosate" refers to N-phosphonomethylglycine and its salts, and treatment with "glyphosate herbicide" means treatment with any herbicide preparation containing glyphosate. The choice of the use rate of a certain glyphosate formulation in order to achieve an effective biological dose does not exceed the skill of a general agronomic technician. Treatment of plants containing plant material derived from the transgenic maize event DBN9936 using any of the glyphosate-containing herbicide formulations will control weed growth in the field and will not affect plant material derived from the transgenic maize event DBN9936 Growth or yield.

The DNA construct is introduced into a plant using a transformation method including, but not limited to, Agrobacterium-mediated transformation, gene gun transformation, and pollen tube pathway 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 to obtain a T-DNA sequence. The vector is transformed into Agrobacterium cells, and subsequently, the Agrobacterium cells are used to infect plant tissues, and the T-DNA sequence of the vector containing the foreign DNA is inserted into the plant genome.

The gene gun transformation method is to bombard plant cells (particle-mediated biological bombardment transformation) with a vector containing foreign DNA.

The pollen tube channel transformation method utilizes a natural pollen tube formed by pollination of plants. The road (also known as the pollen tube guides the tissue) carries the foreign DNA into the embryo sac via the nucellus channel.

After transformation, the transgenic plants must be regenerated from the transformed plant tissue and the progeny with the exogenous DNA selected using appropriate markers.

A DNA construct is a combination of DNA molecules interconnected that provides one or more expression cassettes. The DNA construct is preferably a plasmid capable of self-replication in bacterial cells and containing different restriction enzyme sites, and the restriction enzyme sites contained therein are introduced for providing functional gene elements, ie, promoters. , introns, leader sequences, coding sequences, 3' terminator regions, and other sequences of DNA molecules. The expression cassette contained in the DNA construct includes genetic elements necessary for providing transcription of messenger RNA, which can be designed to be expressed in prokaryotic or eukaryotic cells. The expression cassettes of the invention are designed to be most preferably expressed in plant cells.

A transgenic "event" is obtained by transforming a plant cell with a heterologous DNA construct, that is, comprising at least one nucleic acid expression cassette containing the gene of interest, inserted into the plant genome by a transgenic method to produce a plant population, and regenerating the plant population And selecting a particular plant with the characteristics of insertion into a particular genomic locus. The term "event" includes the original transformant of a heterologous DNA and the progeny of the transformant. The term "event" also includes progeny obtained by sexual crossing between a transformant and another variety of individuals containing heterologous DNA, even after repeated backcrossing with the backcross parent, insert DNA and flanking from the transformant parent. Genomic DNA is also present at the same chromosomal location in the progeny of the cross. The term "event" also refers to a DNA sequence from an original transformant comprising an insert DNA and a flanking genomic sequence immediately adjacent to the inserted DNA, the DNA sequence being expected to be transferred into a progeny containing the insert DNA The parental line (eg, the original transformant and its progeny produced by selfing) is produced by sexual crossing with a parental line that does not contain the inserted DNA, and the progeny receives the inserted DNA comprising the gene of interest.

"Recombinant" in the context of the invention refers to the form of DNA and/or proteins and/or organisms that are normally not found in nature and are thus produced by human intervention. Such manual intervention can produce recombinant DNA molecules and/or recombinant plants. The "recombinant DNA molecule" is obtained by artificially combining two sequence segments which are otherwise isolated, for example by chemical synthesis or by manipulation of isolated nucleic acid segments by genetic engineering techniques. Techniques for performing nucleic acid manipulation are well known.

The term "transgene" includes any cell, cell line, callus, tissue, plant part or plant, and the above genotypes are altered by the presence of a heterologous nucleic acid, including the transgene that was originally altered as such and by The original transgenic body is a progeny individual that is produced by sexual or asexual reproduction. In the present invention, the term "transgenic" is not included A plant breeding method or a genomic (chromosomal or extrachromosomal) alteration of a naturally occurring event, such as random allogeneic fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition or spontaneous mutation.

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

The transgenic maize event DBN9936, which is resistant to lepidopteran insects and resistant to glyphosate herbicides, is cultured by first sexually crossing the first parent corn plant with the second parent corn plant, thereby producing a diverse first generation progeny plant consisting of a maize plant grown from the transgenic maize event DBN9936 and its progeny, the transgenic maize event DBN9936 and its progeny being by using the lepidopteran insect of the invention A control plant that is resistant and resistant to glyphosate herbicides, the second parent corn plant lacks resistance to lepidopteran insects and/or is resistant to glyphosate herbicides And then selecting progeny plants that are resistant to the invasion of lepidopteran insects and/or resistant to glyphosate herbicides, can be developed to be resistant to lepidopteran insects and have a glyphosate herbicide Tolerant corn plants. These steps may further comprise backcrossing the lepidopteran resistant and/or glyphosate-tolerant progeny plants with the second parental maize plant or the third parental maize plant, and then by attacking with lepidopteran insects, The progeny are selected by glyphosate herbicide application or by identification of a molecular marker associated with the trait (eg, a DNA molecule comprising a junction site identified at the 5' and 3' ends of the inserted sequence in the transgenic maize event DBN9936), Thereby producing corn plants that are resistant to lepidopteran insects and tolerant to glyphosate herbicides.

It will also be appreciated that two different transgenic plants can also be crossed to produce progeny containing two separate, separately added exogenous genes. Selfing of appropriate offspring can result in progeny plants that are homozygous for both added exogenous genes. Backcrossing of parental plants and outcrossing with non-transgenic plants as previously described are also contemplated, as are asexual reproduction.

The term "probe" is an isolated nucleic acid molecule to which is incorporated a conventional detectable label or reporter molecule, for example, a radioisotope, a ligand, a chemiluminescent agent, or an enzyme. This probe is complementary to a strand of the target nucleic acid. In the present invention, the probe is complementary to a DNA strand from the genome of the transgenic maize event DBN9936, whether the genomic DNA is derived from the transgenic maize event DBN9936 or from a transgene. Plant or seed or extract of corn event DBN9936. The probe of the present invention includes not only deoxyribonucleic acid Or ribonucleic acid, further comprising a polyamide and other probe material that specifically binds to the 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 hybridizes to a complementary target DNA strand by nucleic acid hybridization, forms a hybrid between the primer and the target DNA strand, and then acts as a polymerase (eg, DNA polymerase). Next, extending along the target DNA strand. Primer pairs of the invention are directed to their use in amplification of a target nucleic acid sequence, for example, by polymerase chain reaction (PCR) or other conventional nucleic acid amplification methods.

The length of the probe and primer is generally 11 polynucleotides or more, preferably 18 polynucleotides or more, more preferably 24 polynucleotides or more, and most preferably 30 polynucleosides. Sour or more. Such probes and primers specifically hybridize to the target sequence under highly stringent hybridization conditions. Although a probe different from the target DNA sequence and capable of maintaining hybridization ability to the target DNA sequence can be designed by a conventional method, preferably, the probe and the primer of the present invention have a complete DNA sequence with the contiguous nucleic acid of the target sequence. Identity.

The primers and probes based on the flanking genomic DNA and the inserted sequences of the present invention can be determined by a conventional method, for example, by isolating a corresponding DNA molecule from a plant material derived from the transgenic maize event DBN9936, and determining the nucleic acid sequence of the DNA molecule. The DNA molecule comprises a transgene insert and a maize genome flanking sequence, and a fragment of the DNA molecule can be used as a primer or probe.

The nucleic acid probes and primers of the invention hybridize to the target DNA sequence under stringent conditions. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of DNA derived from the transgenic maize event DBN9936 in the sample. A nucleic acid molecule or fragment thereof is capable of specifically hybridizing to other nucleic acid molecules under certain circumstances. As used in the present invention, if two nucleic acid molecules are capable of forming an anti-parallel double-stranded nucleic acid structure, it can be said that the two nucleic acid molecules are capable of specifically hybridizing to each other. If two nucleic acid molecules exhibit complete complementarity, one of the nucleic acid molecules is said to be the "complement" of the other nucleic acid molecule. As used herein, when each nucleotide of one nucleic acid molecule is complementary to a corresponding nucleotide of another nucleic acid molecule, the two nucleic acid molecules are said to exhibit "complete complementarity." Two nucleic acid molecules are said to be "minimally complementary" if they are capable of hybridizing to one another with sufficient stability such that they anneal under at least conventional "low stringency" conditions and bind to each other. Similarly, two nucleic acid molecules are said to be "complementary" if they are capable of hybridizing to one another with sufficient stability such that they anneal under conventional "highly stringent" conditions and bind to each other. Deviation from complete complementarity is permissible as long as this deviation does not completely prevent the two molecules from forming a double Chain structure. In order for a nucleic acid molecule to function as a primer or probe, it is only necessary to ensure that it is sufficiently complementary in sequence to allow for the formation of a stable double-stranded structure at 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 complementary strand of another nucleic acid molecule that is matched under highly stringent conditions. Suitable stringent conditions for promoting DNA hybridization, for example, treatment with 6.0 x sodium chloride / sodium citrate (SSC) at about 45 ° C, followed by washing with 2.0 x SSC at 50 ° C, these conditions are known to those skilled in the art. It is well known. For example, the salt concentration in the washing step can be selected from about 2.0 x SSC under low stringency conditions, 50 ° C to about 0.2 x SSC, 50 ° C under highly stringent conditions. Further, the temperature conditions in the washing step can be raised from a low temperature strict room temperature of about 22 ° C to about 65 ° C under highly stringent conditions. Both the temperature conditions and the salt concentration can be changed, or one of them remains unchanged while the other variable changes. Preferably, a nucleic acid molecule of the invention can be under moderate stringency conditions, for example at about 2.0 x SSC and about 65 ° C with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4. One or more nucleic acid molecules of SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7 or a complement thereof, or any fragment of the above sequence that specifically hybridizes. More preferably, a nucleic acid molecule of the invention is under highly stringent conditions 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 and one or more of the nucleic acid molecules of SEQ ID NO: 7 or a complement thereof, or any of the above sequences, specifically hybridize. In the present invention, a 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 complement, or any fragment of the above sequence. Another preferred marker nucleic acid molecule of the invention has 80% to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 6 or SEQ ID NO: 7 or a complement thereof, or any fragment of the above sequence 100% 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. 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 labeling, and chemiluminescent labeling.

Regarding amplification of a target nucleic acid sequence using a specific amplification primer (for example, by PCR), "stringent conditions" refer to conditions in which only primers are allowed to hybridize to a target nucleic acid sequence in a DNA thermal amplification reaction, and a primer for a corresponding wild-type sequence (or its complement) of a target nucleic acid sequence capable of binding to the target nucleic acid sequence, and preferably producing a unique The amplification product, the amplification product is an amplicon.

The term "specific binding (target sequence)" means that the probe or primer hybridizes only to the target sequence in the sample containing the target sequence under stringent hybridization conditions.

As used herein, "amplified DNA" or "amplicon" refers to a nucleic acid amplification product of a target nucleic acid sequence that is part of a nucleic acid template. For example, to determine whether a corn plant is produced by sexual hybridization with the transgenic maize event DBN9936 of the invention, or whether the corn sample collected from the field contains the transgenic maize event DBN9936, or a corn extract, such as a meal, flour or oil, Transgenic maize event DBN9936, DNA extracted from corn plant tissue samples or extracts can be diagnostic amplicon by the nucleic acid amplification method using primer pairs to generate the presence of DNA for the transgenic maize event DBN9936. The primer pair includes a first primer derived from a flanking sequence adjacent to the inserted foreign DNA insertion site 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 DBN9936. The length of the amplicon may be the binding length of the primer pair plus one nucleotide base pair, preferably plus about fifty nucleotide base pairs, more preferably about two hundred and fifty nucleotides. Base pairs, most preferably plus about four hundred and fifty nucleotide base pairs or more.

Alternatively, the primer pair can be derived from a flanking genomic sequence inserted on both sides of the DNA to produce an amplicon comprising the entire inserted nucleotide sequence. One of the primer pairs derived from the plant genome sequence can be located at a distance from the inserted DNA sequence, which can range from one nucleotide base pair to about 20,000 nucleotide base pairs. The use of the term "amplicon" specifically excludes primer dimers formed in DNA thermal amplification reactions.

The nucleic acid amplification reaction can be carried out by any of the nucleic acid amplification reaction methods 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 22 kb of genomic DNA and 42 kb of phage DNA. These methods, as well as other DNA amplification methods 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 DBN9936 can be amplified by using the provided primer sequences for the genome of the transgenic maize event DBN9936, and the PCR amplicon or cloned DNA is subjected to standardization after amplification. DNA sequencing.

A DNA detection kit based on a DNA amplification method contains DNA primer molecules which specifically hybridize to a target DNA under appropriate reaction conditions and amplify a diagnostic amplicon. The kit can provide an agarose gel-based detection method or a detection diagnostic expansion known in the prior art. Many ways to increase the number of children. Kit comprising a DNA primer homologous or complementary to any portion of the maize genomic region of SEQ ID NO: 3 or SEQ ID NO: 4, and homologous or complementary to any portion of the transgene insertion region of SEQ ID NO: 5 It is provided by the present invention. A primer pair that is particularly useful in identifying DNA amplification methods is SEQ ID NO: 8 and SEQ ID NO: 9, which amplify a diagnostic amplification homologous to a portion of the 5' transgene/genomic region of the transgenic maize event DBN9936 , wherein the amplicon comprises SEQ ID NO: 1. Other DNA molecules used as DNA primers may be selected from SEQ ID NO:5.

The amplicons produced by these methods can be detected by a variety of techniques. One such method is Genetic Bit Analysis, which designs a DNA oligonucleotide strand spanning the inserted DNA sequence and adjacent flanking genomic DNA sequences. The oligonucleotide strand is immobilized in the microwell of a microplate, and after PCR amplification of the target region (one primer is used in each of the inserted sequences and adjacent flanking genomic sequences), the single-stranded PCR product Hybridization can be performed with a fixed oligonucleotide strand and as a template for a single base extension reaction using a DNA polymerase and ddNTPs specifically labeled for the next expected base. The results can be obtained by fluorescence or ELISA methods. The signal represents the presence of an insert/flanking sequence indicating that amplification, hybridization and single base extension reactions were successful.

Another method is the pyrosequencing technique. This method designs an oligonucleotide chain spanning the inserted DNA sequence and the adjacent genomic DNA binding site. Hybridization of the oligonucleotide strand and the single-stranded PCR product of the target region (using one primer in each of the inserted sequences and adjacent flanking genomic sequences), followed by DNA polymerase, ATP, sulfurylase, fluorescein The enzyme, apyrase, adenosine-5'-phosphorus sulphate and luciferin are incubated together. The dNTPs were separately added and the generated optical signal was measured. The light signal represents the presence of an insertion/flanking sequence indicating that amplification, hybridization, and single base or multiple base extension reactions are successful.

The fluorescence polarization phenomenon described by Chen et al. (Genome Res., 1999, 9: 492-498) is also a method that can be used to detect the amplicons of the present invention. Using this approach requires designing an oligonucleotide strand spanning the inserted DNA sequence and the adjacent genomic DNA binding site. Hybridization of the oligonucleotide strand and the single-stranded PCR product of the target region (using one primer in each of the inserted sequences and adjacent flanking genomic sequences), followed by DNA polymerase and a fluorescently labeled ddNTP Incubation. Single base extensions result in the insertion of ddNTPs. This insertion can be used to measure changes in its polarization using a fluorometer. The change in polarization represents the presence of an insert/flanking sequence indicating that amplification, hybridization and single base extension reactions were successful.

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

Suitable techniques for detecting plant material derived from the transgenic maize event DBN9936 can also include Southern blot, Northern blot, and In situ hybridization based on the principle of hybridization. In particular, such suitable techniques include incubating probes and samples, washing to remove unbound probes and detecting whether the probes have hybridized. The detection method depends on the type of label attached to the probe, for example, the radiolabeled probe can be detected by X-ray exposure and development, or the enzyme-labeled probe can be detected by color change by substrate conversion.

Tyangi et al. (Nat. Biotech., 1996, 14: 303-308) describe the use of molecular markers in sequence detection. Briefly described below, a FRET oligonucleotide probe spanning the inserted DNA sequence and the adjacent genomic flanking binding site was designed. The unique structure of the FRET probe results in a secondary structure that is capable of maintaining the fluorescent moiety and the quenching moiety at close distances. The FRET probe and PCR primers (using one primer in each of the inserted sequences and adjacent flanking genomic sequences) are subjected to a circular reaction in the presence of a thermostable polymerase and dNTPs. Upon successful PCR amplification, hybridization of the FRET probe to the target sequence results in loss of the secondary structure of the probe, thereby spatially separating the fluorescent moiety from the quenching moiety, producing a fluorescent signal. The generation of the fluorescent signal represents the presence of an insert/flanking sequence indicating that amplification and hybridization were successful.

Other described methods, such as microfluidics, provide methods and apparatus for isolating and amplifying DNA samples. Light dyes are used to detect and measure specific DNA molecules. A nanotube device comprising an electronic sensor for detecting a DNA molecule or a nanobead that binds to a specific DNA molecule and thus detectable is useful for detecting the DNA molecule of the present invention.

DNA detection kits can be developed using the compositions described herein and methods described or known in the art of DNA detection. The kit facilitates the identification of the presence of a transgene in a sample The DNA of the maize event DBN9936 can also be used to grow corn plants containing the DNA of the transgenic maize event DBN9936. The kit may contain a DNA primer or probe that is homologous or complementary to at least a portion of SEQ ID NO: 1, 2, 3, 4, or 5, or contains other DNA primers or probes that are homologous to or DNA complementary to DNA-transgenic genetic elements that can be used in DNA amplification reactions or as probes in DNA hybridization methods. The DNA structure contained in the maize genome and the transgenic insert sequence described in Figure 1 and Table 1 and the maize genome binding site comprises: the maize DBN9936 flanking genomic region at the 5' end of the transgene insert, from the left border of Agrobacterium A portion of the region (LB) is inserted into the sequence, and the first expression cassette is operably linked to the maize heat shock 70 kDa intron (iZmHSP70) by a tandem repeat of the cauliflower mosaic virus 35S promoter (pr35S) containing an enhancer region. Alternatively, operably linked to an insect-resistant Cry1Ab protein (cCry1Ab) of Bacillus thuringiensis, and operably linked to a transcriptional terminator (tNos) of nopaline synthase, the second expression cassette consisting of rice The actin 1 promoter (prOsAct1), operably linked to the Arabidopsis EPSPS chloroplast transit peptide (spAtCTP2), is operably linked to the glyphosate-tolerant 5-enol of the Agrobacterium CP4 strain - Pyruvate oxalic acid-3-phosphate synthase (cEPSPS), operably linked to the cauliflower mosaic virus 35S terminator (t35S), from the right border region (RB) of Agrobacterium Sequence is partially inserted, and transgene insert sequence positioned 3 'flanking genomic maize plants DBN9936 end region (SEQ ID NO: 5). In the DNA amplification method, the DNA molecule as a primer may be any part derived from the transgene insertion sequence in the transgenic maize event DBN9936, or may be any part of the DNA sequence derived from the flanking maize genome in the transgenic maize event DBN9936.

The transgenic maize event DBN9936 can be combined with other transgenic maize varieties, such as herbicides (such as glufosinate, dicamba, etc.) tolerant maize, or transgenic maize varieties carrying other insect resistance genes. Various combinations of all of these different transgenic events, bred together with the transgenic maize event DBN9936 of the present invention, can provide improved hybrid transgenic maize varieties that are resistant to a variety of pests and resistant to multiple herbicides. These varieties can exhibit more excellent characteristics such as yield increase than non-transgenic varieties and single-trait transgenic varieties.

The transgenic maize event DBN9936 is resistant to feeding damage by lepidopteran pests and tolerant to the phytotoxic effects of glyphosate-containing agricultural herbicides. The dual-characterized maize plant expresses a Cry1Ab protein of Bacillus thuringiensis which provides resistance to feeding damage to lepidopteran pests such as Asian corn borer and expresses glyphosate resistance of Agrobacterium strain CP4 5- An enol-pyruvylshikimate-3-phosphate synthase (EPSPS) protein that confers tolerance to glyphosate in plants. The dual-trait corn has the following advantages: 1) It is protected from economic losses caused by lepidopteran pests such as Asian corn borer, oriental armyworm and peach aphid. Asian corn borer, oriental armyworm and peach aphid are corn. The main pests in the growing area; 2) the ability to apply glyphosate-containing agricultural herbicides to the corn crop for broad-spectrum weed control; 3) the corn yield was not reduced. In addition, genes encoding insect resistance and glyphosate tolerance traits are linked to the same DNA segment and are present at a single locus in the genome of the transgenic maize event DBN9936, which provides enhanced breeding efficiency and enables Molecular markers to track transgenic inserts in the breeding population and its progeny. In the detection method of the present invention, SEQ ID NO: 1 or its complement, SEQ ID NO: 2 or its complement, SEQ ID NO: 6 or its complement, or SEQ ID NO: 7 or its complement may be used as DNA primers. Or a probe to generate an amplification product diagnosed as transgenic maize event DBN9936 or a progeny thereof, and the presence of plant material derived from the transgenic maize event DBN9936 can be identified quickly, accurately, and stably.

Sequence description

SEQ ID NO: 1 A sequence of 22 nucleotides in length in the transgenic maize event DBN9936 located near the insertion junction at the 5' end of the inserted sequence, wherein nucleotides 1-11 and 12-22 Glycosylates are located on both sides of the insertion site on the maize genome;

SEQ ID NO: 2 A sequence of 22 nucleotides in length in the transgenic maize event DBN9936 located near the insertion junction at the 3' end of the inserted sequence, wherein nucleotides 1-11 and 12-22 Glycosylates are located on both sides of the insertion site on the maize genome;

SEQ ID NO: 3 A sequence of 1001 nucleotides in length in the transgenic maize event DBN9936 located at the 5' end of the inserted sequence near the insertion junction;

SEQ ID NO: 4 A sequence of 1204 nucleotides in length in the transgenic maize event DBN9936 located near the insertion junction at the 3' end of the inserted sequence;

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

SEQ ID NO: 6 is a sequence located within SEQ ID NO: 3 spanning the nucleotide sequence of the DBN10124 construct DNA sequence and the tNos transcription termination sequence;

SEQ ID NO:7 is a sequence located within SEQ ID NO: 4 spanning the nucleotide sequence of the t35S transcription termination sequence and the DBN10124 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 Amplification of the second primer of SEQ ID NO: 4;

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

SEQ ID NO: 13 is a primer paired with SEQ ID NO: 12 on the T-DNA;

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

SEQ ID NO: 15 is a primer paired with SEQ ID NO: 14 on the T-DNA;

SEQ ID NO: 16 Taqman detects primer 1 of Cry1Ab;

SEQ ID NO: 17 Taqman detects primer 2 of Cry1Ab;

SEQ ID NO: 18 Taqman detects the probe 1 of the Cry1Ab;

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

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

SEQ ID NO: 21 Taqman probe 2 for detecting EPSPS;

SEQ ID NO: 22 First primer for maize endogenous gene ubiquitin protein;

SEQ ID NO: 23 second primer for maize endogenous gene ubiquitin protein;

SEQ ID NO: 24 probe for Cry1Ab in Southern blot hybridization assay;

SEQ ID NO: 25 probe for EPSPS in Southern blot hybridization assay;

SEQ ID NO:26 Primer on T-DNA, in the same orientation as SEQ ID NO:13;

SEQ ID NO:27 Primer on T-DNA, opposite to SEQ ID NO: 13 for use as a flanking sequence;

SEQ ID NO:28 Primer on T-DNA, opposite to SEQ ID NO: 13 for use as a flanking sequence;

SEQ ID NO:29 Primer on T-DNA, aligned with SEQ ID NO:15;

SEQ ID NO:30 Primer on T-DNA, opposite to SEQ ID NO: 15 for use as a flanking sequence;

SEQ ID NO: 31 Primer on T-DNA, opposite to SEQ ID NO: 15, used to obtain flanking sequences.

The technical solution of the present invention will be further described in detail below through the accompanying drawings and embodiments.

DRAWINGS

Figure 1 is a schematic diagram showing the structure of the maize gene DBN9936 transgene insert and the maize genome junction site, and the nucleic acid sequence used in the method for detecting the maize event DBN9936. Schematic diagram of relative position;

Figure 2 is a schematic diagram showing the structure of a recombinant expression vector DBN10124 for detecting the maize event DBN9936;

Figure 3 is a comparison of field effects of maize event DBN9936 and wild-type maize plants (non-transgenic, NGM) inoculated with Asian corn borer during heart and silk stage;

Figure 4 is a comparison of the field effects of maize event DBN9936 and wild-type maize plants (non-transgenic, NGM) inoculated with oriental armyworms;

Figure 5 is a comparison of field effects of maize event DBN9936 and wild-type maize plants (non-transgenic, NGM) inoculated with cotton bollworm;

Figure 6 is a comparison of field effects of maize event DBN9936 and wild-type maize plants (non-transgenic, NGM) under natural conditions of Myzus persicae;

Figure 7 is a comparison of field effects of maize event DBN9936 and wild-type maize plants (non-transgenic, NGM) under natural conditions of beet armyworm.

detailed description

Certain preferred embodiments of the invention are further illustrated by the following examples.

The first embodiment, the acquisition of the genetically modified corn event DBN9936

1.1, vector cloning

The recombinant expression vector DBN10124 (shown in Figure 2) was constructed using standard gene cloning techniques. The vector DBN10124 comprises two tandem transgenic expression cassettes, the first expression cassette being operably linked to the corn heat shock 70 kDa protein by a tandem repeat of the cauliflower mosaic virus 35S promoter (pr35S) containing an enhancer region On the (iZmHSP70), operably linked to the insect-resistant Cry1Ab protein (cCry1Ab) of Bacillus thuringiensis, and operably linked to the transcriptional terminator (tNos) of nopaline synthase; The expression cassette is operably linked to the Arabidopsis EPSPS chloroplast transit peptide (spAtCTP2) by the rice actin 1 promoter (prOsAct1), operably linked to the glyphosate tolerance of the Agrobacterium CP4 strain 5 - enol-pyruvylshikimate-3-phosphate synthase (cEPSPS), and is operably linked to the cauliflower mosaic virus 35S terminator (t35S).

The vector DBN10124 was transformed into Agrobacterium LBA4404 (Invitrgen, Chicago, USA; Cat. No: 18313-015) by liquid nitrogen method, and 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) Transformed cells are screened for selection markers.

1.2, plant transformation

Transformation was carried out by a conventional Agrobacterium infection method, and the sterile cultured maize immature embryos were co-cultured with the Agrobacterium described in the present Example 1.1 to transfer the T-DNA in the constructed recombinant expression vector DBN10124 to the corn. In the genome, to generate the transgenic maize event DBN9936.

For Agrobacterium-mediated transformation of maize, briefly, immature immature embryos are isolated from maize, and the immature embryos are contacted with Agrobacterium suspension, wherein Agrobacterium can express the nucleotide sequence of Cry1Ab gene and the nucleotide of EPSPS gene The sequence is passed to at least one cell of one of the young embryos (step 1: infection step), in which the immature embryo is preferably immersed in an Agrobacterium suspension (OD ₆₆₀ = 0.4-0.6, infecting medium (MS salt 4.3) g/L, MS vitamin, casein 300mg/L, sucrose 68.5g/L, glucose 36g/L, acetosyringone (AS) 40mg/L, 2,4-dichlorophenoxyacetic acid (2,4-D) Inoculation was initiated with 1 mg/L, pH 5.3)). The immature embryo is co-cultured with Agrobacterium for a period of time (3 days) (step 2: co-cultivation step). Preferably, the immature embryo is in solid medium after the infection step (MS salt 4.3 g/L, MS vitamin, casein 300 mg/L, sucrose 20 g/L, glucose 10 g/L, acetosyringone (AS) 100 mg/L) 2,4-Dichlorophenoxyacetic acid (2,4-D) 1 mg/L, agar 8 g/L, pH 5.8). After this co-cultivation phase, there can be an optional "recovery" step. In the "recovery" step, the medium was restored (MS salt 4.3 g / L, MS vitamin, casein 300 mg / L, sucrose 30 g / L, 2,4-dichlorophenoxyacetic acid (2,4-D) 1 mg / At least one antibiotic (cephalosporin) known to inhibit the growth of Agrobacterium is present in L, plant gel 3 g/L, pH 5.8), and no selection agent for plant transformants is added (step 3: recovery step). Preferably, the immature embryos are cultured on a solid medium with antibiotics but no selection agent to eliminate Agrobacterium and provide a recovery period for the infected cells. Next, the inoculated immature embryos are cultured on a medium containing a selective agent (N-(phosphocarboxymethyl)glycine) and the grown transformed callus is selected (step 4: selection step). Preferably, the immature embryo is screened in a solid medium with a selective agent (MS salt 4.3 g/L, MS vitamin, casein 300 mg/L, sucrose 30 g/L, N-(phosphocarboxymethyl)glycine 0.25 mol/L, 2,4-Dichlorophenoxyacetic acid (2,4-D) 1 mg/L, plant gel 3 g/L, pH 5.8) was cultured, resulting in selective growth of transformed cells. Then, the callus regenerates the plant (step 5: regeneration step), preferably, the callus grown on the medium containing the selection agent is cultured on a solid medium (MS differentiation medium and MS rooting medium) Recycled plants.

The selected resistant callus was transferred to the MS differentiation medium (MS salt 4.3 g/L, MS vitamin, casein 300 mg/L, sucrose 30 g/L, 6-benzyl adenine 2 mg/L, N- (phosphonocarboxymethyl)glycine 0.125 mol/L, plant gel 3 g/L, pH 5.8), cultured and differentiated at 25 °C. The differentiated seedlings were transferred to the MS rooting medium (MS salt 2.15 g/L, MS vitamin, casein 300 mg / L, sucrose 30 g / L, indole-3-acetic acid 1 mg / L, agar 8 g / L, pH 5.8), cultured at 25 ° C to about 10 cm high, moved to the greenhouse to grow to firm. In the greenhouse, the cells were cultured at 28 ° C for 16 hours and then at 20 ° C for 8 hours.

1.3. Identification and screening of transgenic events

A total of 770 independent transgenic T ₀ plants were produced.

By TaqMan ^TM analysis (see the second embodiment) Transgenic Maize Plant Regeneration detecting the presence or absence of Cry1Ab and EPSPS genes, the copy number and characterization of insect resistance and glyphosate herbicide tolerant lines. According to the copy number of the gene of interest, good insect resistance, glyphosate herbicide tolerance, and agronomic traits (see the fifth and sixth examples), the event DBN9936 was selected to be excellent by screening. It has a single copy transgene, good insect resistance, glyphosate herbicide tolerance, and agronomic trait performance (participating in the fifth and sixth embodiments).

Second embodiment, detection of transgenic corn event DBN9936 with TaqMan

Approximately 100 mg of the leaves of the transgenic maize event DBN9936 were taken as samples, and the genomic DNA was extracted using a plant DNA extraction kit (DNeasy Plant Maxi Kit, Qiagen), and the copy number of Cry1Ab and EPSPS was detected by Taqman probe fluorescent quantitative PCR. At the same time, the wild type corn plants were used as a control, and the detection and analysis were carried out according to the above method. The experiment was set to repeat 3 times and averaged.

The specific method is as follows:

Step 11. Take 100 mg of the leaves of the transgenic corn event DBN9936, and homogenize it with liquid nitrogen in a mortar, and take 3 replicates for each sample;

Step 12. Extract the genomic DNA of the above sample using Qiagen's DNeasy Plant Mini Kit, and refer to the product manual for the specific method;

Step 13. Determine the genomic DNA concentration of the above sample by using an ultra-micro spectrophotometer (NanoDrop 2000, Thermo Scientific);

Step 14, adjusting the genomic DNA concentration of the above sample to the same concentration value, the concentration value ranges from 80 to 100 ng / μl, the specific method is well known to those skilled in the art, or can refer to the product specification thereof;

Step 15. The Taqman probe real-time PCR method is used to identify the copy number of the sample, and the sample with the known copy number is used as a standard, and the sample of the wild type corn plant is used as a control, and each sample has 3 replicates, and the average is taken. Value; the fluorescent PCR primers and probe sequences are:

The following primers and probes were used to detect the Cry1Ab gene sequence:

Primer 1: CGAACTACGACTCCCGCAC is shown in SEQ ID NO: 16 in the Sequence Listing;

Primer 2: GTAGATTTCGCGGGTCAGTTG is shown in SEQ ID NO: 17 in the Sequence Listing;

Probe 1: CTACCCGATCCGCACCGTGTCC is shown in SEQ ID NO: 18 in the Sequence Listing;

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

Primer 3: CTGGAAGGCGAGGACGTCATCAATA is shown in SEQ ID NO: 19 in the Sequence Listing;

Primer 4: TGGCGGCATTGCCGAAATCGAG is shown in SEQ ID NO: 20 in the Sequence Listing;

Probe 2: ATGCAGGCGATGGGCGCCCGCATCCGTA as shown in SEQ ID NO: 21 in the Sequence Listing;

The PCR reaction system is:

The 50× primer/probe mixture 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 (10 mM Tris-HCl, pH 8.0; 1 mM EDTA, pH 8.0), and Store at 4 ° C in amber tubes.

The PCR reaction conditions are:

The data was analyzed using the Rapid Real-Time PCR System software (Applied Biosystems 7900HT Fast Real-Time PCR System SDS v2.3, Applied Biosystems), demonstrating that a single copy of the transgenic maize event DBN9936 was obtained.

Third embodiment, detection of genetically modified corn event DBN9936

3.1, genomic DNA extraction

DNA extraction according to the conventionally used CTAB (cetyltrimethylammonium bromide) method: 2 g of young transgenic corn event DBN9936 leaves were ground into powder in liquid nitrogen, and 0.5 mL was added at a temperature of 65 ° C. Hot DNA extraction CTAB buffer (20g/L CTAB, 1.4M NaCl, 100mM Tris-HCl, 20mM EDTA (ethylenediaminetetraacetic acid), adjusted to pH 8.0 with NaOH), fully mixed, and pumped at a temperature of 65 ° C Add 90 times; add 0.5 times volume of phenol, 0.5 volume of chloroform, mix by inversion; centrifuge at 102000 rpm (revolutions per minute) for 10 min; pipette the supernatant, add 2 volumes of absolute ethanol, gently shake the centrifuge tube, at temperature After standing at 4 ° C for 30 min; centrifuge again for 10 min at 12000 rpm; collect DNA to the bottom of the tube; discard the supernatant, wash the pellet with 1 mL of 70% ethanol, centrifuge for 5 min at 12000 rpm, vacuum or dry. The table was blown dry; the DNA pellet was dissolved in an appropriate amount of TE buffer and stored at a temperature of -20 °C.

3.2. Analysis of flanking DNA sequences

The above-prepared DNA sample was subjected to concentration measurement (same as step 13 in the second embodiment) so that the concentration of the sample to be tested was between 80 and 100 ng/μL. Genomic DNA was digested with the selected restriction enzymes Sac I, Kpn I, Xma I, Nhe I (5' end analysis) and Spe I, Pst I, BssH II (3' end analysis), respectively. 26.5 μL of genomic DNA, 0.5 μL of the selected restriction enzyme and 3 μL of restriction enzyme buffer were added to each digestion system (the restriction enzymes used were NEB enzymes and their buffers or universal buffers). Liquid (now known as NEBCutSmart), so 3μL of enzyme digestion buffer can be specifically 3μL NEBCutSmart buffer), and digested for 1 hour. After the end of the enzyme digestion, 70 μL of absolute ethanol was added to the digestion system, and the mixture was centrifuged for 30 min at 302000 rpm for 7 min. The supernatant was discarded and dried. Then, 8.5 μL of double distilled water and 1 μL of 10×T4-DNA ligase buffer were added. (NEB T4 DNA Ligase Reaction Buffer, its specific recipe can be found on the NEB website or refer to https://www.neb.com/products/restriction-endonucleases, https://www.neb.com/products/b0202-t4-dna -ligase-reaction-buffer) and 0.5 μL of T4-DNA ligase were ligated overnight at 4 °C. The 5' and 3' transgene/genomic DNA were isolated by PCR amplification using a series of nested primers. Specifically, the isolated 5' transgene/genomic DNA primer combination comprises SEQ ID NO: 13, SEQ ID NO: 26 as a first primer, SEQ ID NO: 27, SEQ ID NO: 28 as a second primer, SEQ ID NO: 13. As a sequencing primer. The isolated 3' transgene/genomic DNA primer combination comprises SEQ ID NO: 15, SEQ ID NO: 29 as the first primer, SEQ ID NO: 30, SEQ ID NO: 31 as the second primer, SEQ ID NO: 15 was used as a sequencing primer, and the PCR reaction conditions are shown in Table 3.

The obtained amplicons were electrophoresed on a 2.0% agarose gel to separate the PCR reactions, followed by separation from the agarose matrix using a QIAquick Gel Extraction Kit (QIAquick Gel Extraction Kit, catalog #_28704, Qiagen Inc., Valencia, CA). Target segment. The purified PCR product is then sequenced (eg, using ABI PrismTM 377, PE Biosystems, Foster City, CA) and analyzed (eg, using DNASTAR sequence analysis software, DNASTAR Inc., Madison, WI).

The 5' and 3' flanking sequences and junction sequences were confirmed using standard PCR methods. The 5' flanking sequence and the contact sequence can be confirmed 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 sequence and the contact sequence can be confirmed 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 Tables 2 and 3. Those skilled in the art will appreciate that other primer sequences can also be used to confirm flanking sequences and junction sequences.

DNA sequencing of PCR products provides DNA that can be used to design other DNA molecules for use as primers and probes for the identification of maize plants or seeds derived from the transgenic maize event DBN9936.

It was found that the position of the maize genome in position 1-832 of SEQ ID NO: 5 is flanking the right border of the transgenic maize event DBN9936 insert (5' flanking sequence), at nucleotide 8202 of SEQ ID NO: 5. The -9215 position shows the maize genome sequence flanking the left border of the transgenic maize event DBN9936 insertion sequence (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

The ligation sequence is a relatively short polynucleotide molecule that is a new DNA sequence that is diagnostic for the DNA of the transgenic maize event DBN9936 when detected in a polynucleic acid detection assay. The junction sequence in SEQ ID NO: 1 and SEQ ID NO: 2 is the insertion site of the transgene fragment in the transgenic maize event DBN9936 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. The junction sequence (5' junction region SEQ ID NO: 1, and 3' junction region SEQ ID NO: 2) is useful as a DNA probe or as a DNA primer molecule in a DNA detection method. The ligating sequences SEQ ID NO: 6 and SEQ ID NO: 7 are also novel DNA sequences in the transgenic maize event DBN9936, which can also serve as DNA probes or as DNA primers. Molecular detection of the presence of the transgenic maize event DBN9936 DNA. The SEQ ID NO: 6 (positions 833-1001 of nucleotides of SEQ ID NO: 3) spans the DBN10124 construct DNA sequence and the tNos transcription termination sequence, the SEQ ID NO: 7 (nucleus of SEQ ID NO: 4) The nucleotide sequence 1-190) spans the t35S transcription termination sequence and the DBN10124 construct DNA sequence.

Furthermore, an amplicon is produced by using at least one primer from SEQ ID NO: 3 or SEQ ID NO: 4, which is used in a PCR method to generate a diagnostic amplicon of the transgenic maize event DBN9936.

Specifically, a PCR product was generated from the 5' end of the transgene insert, which is part of the genomic DNA flanked by the 5' end of the T-DNA insert in the genome of the plant material derived from the transgenic maize event DBN9936. This PCR product comprises SEQ ID NO:3. For PCR amplification, primer 5 (SEQ ID NO: 8), which hybridizes to the genomic DNA sequence flanking the 5' end of the transgene insert, and primer 6 (SEQ ID) located in the transgene t35S transcription termination sequence, were designed. NO: 9).

A PCR product was generated from the 3' end of the transgene insert, which contained a portion of the genomic DNA flanking the 3' end of the T-DNA insert in the genome of the plant material derived from the transgenic maize event DBN9936. This PCR product comprises SEQ ID NO:4. For PCR amplification, primer 8 (SEQ ID NO: 11), which hybridizes to the genomic DNA sequence flanking the 3' end of the transgene insert, and the paired tNos transcription termination sequence at the 3' end of the insert were designed. Primer 7 (SEQ ID NO: 10).

The DNA amplification conditions illustrated in Tables 2 and 3 can be used in the PCR zygosity assay described above to generate a diagnostic amplicon of the transgenic maize event DBN9936. Detection of the amplicon can be carried out by using Stratagene Robocycler, MJ Engine, Perkin-Elmer 9700 or Eppendorf Mastercycler Gradien thermal cycler, or the like, or by methods and equipment known to those skilled in the art.

Table 2 PCR procedure and reaction mixture conditions for 5' transgenic insert/genomic junction region identification of transgenic maize event DBN9936

Table 3 Thermal cycler conditions

Mix gently. If there is no cap on the thermocycler, add 1-2 drops of mineral oil above each reaction. Using the above cycling parameters (Table 3) in Stratagene Robocycler (Stratagene, La Jolla, CA), MJ Engine (MJ R-Biorad, Hercules, CA), Perkin-Elmer 9700 (Perkin Elmer, Boston, MA) or Eppendorf Mastercycler Gradient ( Eppendorf, Hamburg, Germany) on thermal cycler Perform PCR. The MJ Engine or Eppendorf Mastercycler Gradient thermal cycler should operate in the calculated mode. The Perkin-Elmer 9700 Thermal Cycler is programmed to set the ramp speed to its maximum value.

The results of the experiments indicated that primers 5 and 6 (SEQ ID NOS: 8 and 9), when used in the PCR reaction of the transgenic maize event DBN9936 genomic DNA, generated an amplification product of a 1001 bp fragment when used in the untransformed maize genome. When PCR was used in PCR reactions with non-DBN9936 maize genomic DNA, no fragments were amplified; primers 7 and 8 (SEQ ID NOS: 10 and 11) were generated when used in the PCR reaction of the transgenic maize event DBN9936 genomic DNA. The amplified product of the 1204 bp fragment, when used in the PCR reaction of untransformed maize genomic DNA and non-DBN9936 maize genomic DNA, no fragments were amplified.

PCR zygosity assays can also be used to identify homozygous or heterozygous materials derived from the transgenic maize event DBN9936. Primer 9 (SEQ ID NO: 12), primer 10 (SEQ ID NO: 13) and primer 11 (SEQ ID NO: 14) were used in the amplification reaction to generate a diagnostic amplicon of the transgenic maize event DBN9936. The DNA amplification conditions illustrated in Tables 4 and 5 can be used in the above zygosity assay to generate a diagnostic amplicon of the transgenic maize event DBN9936.

Table 4 zygosity determination reaction solution

Table 5 Thermal cycler conditions for zygosity measurements

Using the above cycling parameters (Table 5) in Stratagene Robocycler (Stratagene, La Jolla, CA), MJ Engine (MJ R-Biorad, Hercules, CA), Perkin-Elmer 9700 (Perkin Elmer, Boston, MA) or Eppendorf Mastercycler Gradient ( PCR was performed on a thermocycler on Eppendorf, Hamburg, Germany. The MJ Engine or Eppendorf Mastercycler Gradient thermal cycler should operate in the calculated mode. The Perkin-Elmer 9700 Thermal Cycler is programmed to set the ramp speed to its maximum value.

In the amplification reaction, the biological sample containing the template DNA contains DNA for diagnosing the presence of the transgenic maize event DBN9936 in the sample. Or the reaction will produce two different DNA amplicons from a biological sample containing DNA derived from the maize genome, which is heterozygous for the allele corresponding to the inserted DNA present in the transgenic maize event DBN9936. of. These two different amplicons will correspond to the first amplicon derived from the wild-type maize genomic locus and the second amplicon that diagnoses the presence of the transgenic maize event DBN9936 DNA. A maize DNA sample that produces only a single amplicon corresponding to a second amplicon described for the hybrid genome can be diagnostically determined to exist for the transgenic maize event DBN9936 in the sample, and the sample is present in relation to the transgenic maize plant DBN9936 The allele corresponding to the inserted DNA is produced by homozygous corn seed.

It should be noted that the primer pair of the transgenic maize event DBN9936 was used to generate a diagnostic amplicon for the transgenic maize event DBN9936 genomic DNA. These primer pairs include, but are not limited to, primers 5 and 6 (SEQ ID NOS: 8 and 9), and primers 7 and 8 (SEQ ID NOS: 10 and 11), which are used in the DNA amplification method described. In addition, for the amplification of maize endogenous genes A control primer 12 and 13 (SEQ ID NOS: 22 and 23) was included as an intrinsic standard for the reaction conditions. Analysis of the transgenic maize event DBN9936 DNA extraction sample should include a positive tissue DNA extract control of the transgenic maize event DBN9936, a negative DNA extract control derived from the non-transgenic maize event DBN9936 and a DNA extraction without template DNA. Negative control of the substance. In addition to these primer pairs, any primer pair from SEQ ID NO: 3 or SEQ ID NO: 4, or its complement, can be used, which, when used in DNA amplification reactions, produces maize for transgenic events, respectively. The tissue of plant DBN9936 is a diagnostic amplicon comprising SEQ ID NO: 1 or SEQ ID NO: 2. The DNA amplification conditions illustrated in Tables 2 through 5 can be used to generate a diagnostic amplicon of the transgenic maize event DBN9936 using a suitable primer pair. An extract derived from a maize plant or seed DNA containing the transgenic maize event DBN9936, or a product derived from the transgenic maize event DBN9936, when tested in a DNA amplification method, yielding a transgenic maize event DBN9936 as a diagnostic amplicon Can be used as a template for amplification to determine the presence of the transgenic maize event DBN9936.

Fourth Example, Detection of Transgenic Corn Event DBN9936 by Southern Blot Hybridization

4.1, DNA extraction for Southern blot hybridization

Southern blot analysis was performed using T4, T5 generation homozygous transformation events. Approximately 5 to 10 g of plant tissue is ground in liquid nitrogen using a mortar and pestle. Resuspend plant tissue in 12.5 mL of Extraction Buffer A (0.2 M Tris pH 8.0, 50 mM EDTA, 0.25 M NaCl, 0.1% v/v β-mercaptoethanol, 2.5% w/v polyethylene-pyrrolidone), centrifuge at 4000 rpm 10 minutes (2755g). After discarding the supernatant, 2.5 mL of extraction buffer B (0.2 M Tris pH 8.0, 50 mM EDTA, 0.5 M NaCl, 1% v/v β-mercaptoethanol, 2.5% w/v polyethylene-pyrrolidone, 3%) The pellet was resuspended in sarcosyl, 20% ethanol) and incubated for 30 minutes at 37 °C. The samples were mixed once with a sterile loop during incubation. After the incubation, an equal volume of chloroform/isoamyl alcohol (24:1) was added, gently mixed by inversion, and centrifuged at 4000 rpm for 20 minutes. The aqueous layer was collected, and centrifuged at 4000 rpm for 5 minutes after the addition of 0.54 volumes of isopropyl alcohol to precipitate DNA. The supernatant was discarded and the DNA pellet was resuspended in 500 μL of TE. To degrade any RNA present, the DNA was incubated with 1 μL of 30 mg/mL RNAase A for 30 minutes at 37 ° C, centrifuged at 4000 rpm for 5 minutes, and in the presence of 0.5 volume of 7.5 M ammonium acetate and 0.54 volume of isopropanol, The DNA was precipitated by centrifugation at 14,000 rpm for 10 minutes. Discard After the supernatant, the precipitate was washed with 500 μL of 70% ethanol, and dried and resuspended in 100 μL of TE buffer.

4.2, restriction enzyme digestion

Quantitative detection of DNA concentration using a spectrophotometer or fluorometer (using 1×TNE buffer (TNE buffer: 0.01 M Tris, 0.1 M NaCl, 0.001 M EDTA, pH 7.4) and Hoechst dye (Hoechst 33258, Hoechst AG)) .

5 μg of DNA was digested each time in a 100 μL reaction system. Genomic DNA was digested with restriction endonucleases EcoR V and Hind III, respectively, using a partial sequence of Cry1Ab and EPSPS on T-DNA as a probe. For each enzyme, the digest was incubated overnight at the appropriate temperature. The sample was spun using a vacuum centrifugal evaporation concentrator (speedVac, Thermo Scientific) to reduce the volume to 30 μL.

4.3, gel electrophoresis

A bromophenol blue loading dye was added to each sample derived from this Example 4.2, and each sample was applied to a 0.7% agarose gel containing ethidium bromide in TBE running buffer (89 mM Tris). Electrophoresis was carried out in -boric acid, 2 mM EDTA, pH 8.3), and the gel was electrophoresed overnight at 20 volts.

The gel was washed in 0.25 M HCl for 15 minutes to dissociate the DNA and then washed with water. Southern blot hybridization was set as follows: 20 sheets of thick blotted paper were placed in the pan on which 4 thin dry blotting papers were placed. One thin blotting paper was pre-wetted in 0.4 M NaOH and placed on the stack, followed by a nylon membrane N+ transfer film (Hybond-N+, Amersham Pharmacia Biotech, #RPN303B) pre-wetted in 0.4 M NaOH. . The gel is placed on top to ensure that there are no air bubbles between the gel and the membrane. Three additional pre-soaked blotting papers were placed on top of the gel and the buffer pan was filled with 0.4 M NaOH. The gel was transferred to the membrane using a wick pre-soaked in 0.4 M NaOH to connect the gel stack to the buffer pan. DNA transfer was carried out for about 4 hours at room temperature. After transfer, the transfer membrane was rinsed in 2 x SSC for 10 seconds, and the DNA was bound to the membrane by UV crosslinking.

4.4, hybridization

Suitable DNA sequences were amplified by PCR for probe preparation. The DNA probe is SEQ ID NO: 24 and SEQ ID NO: 25, or is homologous or complementary to the above sequence. 25 ng of probe DNA was boiled in 45 μL of TE buffer 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 ³² P-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 transfer membrane was pre-hybridized by wetting the transfer membrane with 20 mL of pre-warmed Church pre-hybridization solution (500 mM Na ₃ P0 ₄ , 1 mM EDTA, 7% SDS, 1% BSA) for 30 minutes at 65 °C. The labeled probe was boiled for 5 minutes and placed on ice for 10 minutes. An appropriate amount of probe (1 million counts per 1 mL of pre-hybrid solution) was added to the pre-hybridization solution, and hybridization was carried out overnight at 65 °C. On the next day, the hybridization solution was discarded, rinsed with 20 mL of Church Wash Solution 1 (40 mM Na ₃ P0 ₄ , 1 mM EDTA, 5% SDS, 0.5% BSA), and then washed at 150 ° C in 150 mL of Church Wash Solution 1. 20 minutes. This procedure was repeated twice with Church Wash Solution 2 (40 mM Na ₃ P0 ₄ , 1 mM EDTA, 1% SDS). The film was exposed to a phosphor screen or X-ray film (4X light film: Model XBT, Carestream Health) to detect the position of probe binding.

Three control samples were included on each Southern: (1) DNA from a negative (untransformed) segregant used to identify any endogenous maize sequence that can hybridize to a component-specific probe; (2) from DNA of the negative segregant into which Hind III-digested DBN10124 was introduced, the amount of which is equivalent to a copy number based on the length of the probe to account for the sensitivity of the assay when detecting a single copy of the gene within the maize genome; and (3) A Hind III-digested DBN10124 plasmid equivalent to one copy number based on the length of the probe was used as a positive control for hybridization and used to demonstrate the sensitivity of the experiment.

Confirmatory hybridization data provide evidence to support TaqMan ^TM PCR analysis of that corn plants contain a single copy DBN9936 Cry1Ab and EPSPS genes. Using the Cry1Ab probe, EcoR V and Hind III were digested to generate a single band of approximately 10 kb and 9 kb, respectively; using the EPSPS probe, EcoR V and Hind III were digested to generate a single band of approximately 8 kb and 15 kb, respectively. This indicates that one copy of each of Cry1Ab and EPSPS is present in the maize transformation event DBN9936.

Fifth embodiment, insect resistance detection of corn event DBN9936

5.1, bioassay

The transgenic maize event DBN9936 and the wild-type maize plant (non-transgenic, NGM) 2 plants were respectively treated with Ostrinia furnacalis (ACB), Conogethes punctiferalis (YPM), and Athetis lepigone (Athetis lepigone, LPG), Sesamia inferens (PSB), Mythimna seperata (OAW), Spodoptera litura (TCW), Chilo suppressalis (SSB), Helicoverpa armigera (CBW) and Spodoptera exigua (BAW) were bioassay as follows:

Fresh leaves (V3-V4 period) of transgenic maize event DBN9936 and wild-type maize plants (non-transgenic, NGM) were planted separately, rinsed with sterile water and the water on the leaves was blotted with gauze, then the corn leaves were removed. Remove the veins and cut into strips of about 1cm × 3cm, take 1-3 pieces (determine the number of leaves according to the amount of insects). The cut strips are placed on the filter paper at the bottom of the round plastic dish. Wet with distilled water, put 10 captive larvae in each dish, and cover at a temperature of 26-28 ° C, relative humidity 70%-80%, photoperiod (light / dark) 16: The results were statistically placed after 3 days under conditions of 8. Asian corn borer statistical mortality, corrected for mortality by correcting mortality, corrected mortality (%) = (1 - survival / number of insects - wild type control mortality) / (1 - wild type control mortality) ×100%. Other insects count larval development progress, mortality and leaf damage rate, get the total resistance score (out of 300 points): total resistance score = 100 × corrected mortality + [100 × mortality + 90 × (initial hatching) Number of insects / number of insects + 60 × (number of initial hatching - negative control insects / number of insects) + 10 × (number of negative control insects / number of insects) + 100 × (1 - blade damage rate). Among them, the number of insects refers to the number of insects, that is, 10 per dish; the development progress of larvae has been reflected by the formula of total resistance; the rate of damage of leaves refers to the proportion of the feeding area of the pests to the total area of the leaves. Five strains were selected from the transgenic maize event DBN9936 and the wild-type maize plants (non-transgenic, NGM), and each plant was repeated 6 times. The results are shown in Tables 6 and 7.

Table 6 Anti-insect bioassay results of transgenic corn event DBN9936 - mortality (%)

Table 7 Anti-insect bioassay results of the transgenic corn event DBN9936 - total score of resistance

The results showed that the transgenic maize event DBN9936 had good resistance to Asian corn borer, peach aphid, B. californica, giant salamander, oriental armyworm, Spodoptera litura, stem borer, cotton bollworm and beet armyworm. And the test insect mortality and resistance total score of the transgenic corn event DBN9936 was significantly higher than that of the wild type corn plant.

5.2, field effect

Seeds of transgenic maize event DBN9936 and wild-type maize plants (non-transgenic, NGM) 2 plants were set to 2 treatments, each treatment was designed according to random blocks, 3 replicates, and the plot area was 30 m ² (5 m × 6 m) , line spacing 60cm, plant spacing 25cm, conventional cultivation management, do not spray insecticides during the whole growth period. There are 2m intervals between different insect insect test plots to avoid the spread of insects between different communities.

(1) Asian corn borer

In the corn leaf stage (small trumpet stage, corn plant development to the 6-8 leaf stage) and artificial silkworms in the silking stage, each insect was taken twice. There are no less than 40 artificial insects in each plot. About 60 heads of newly hatched larvae are planted in each corn leaf/filament. After 3 days of inoculation, the second time, the number of insects is the same as the first time. After 14-21 days of inoculation, the corn damage was investigated on a plant-by-plant basis. Usually, the investigation starts 14 days after the insects are received. If the negative control material (NGM) has a sense of sensation or high sensation (see Table 8 and Table 9), it is considered effective. If it is not reached, the investigation can be postponed appropriately, but after the worm is received. If the corresponding level has not been reached in 21 days, the insect is considered invalid. After the inoculation of the heart leaf, the upper part of the maize plant was investigated by the Asian corn borer. The degree of damage to the ear and the damage of the plant were investigated after the inoculation. 15-20 strains/row were randomly selected for each treatment.

Heart leaf stage: The leaf size of Asian corn borer was recorded as described in Table 8. Calculate the average value of Asian corn borer damage to each treated leaf (leaf level): average leaf level = ∑ (food leaf level × number of plants at this level) / total number of plants investigated. The level of resistance to each Asian corn borer was divided according to the average of the leaf level, as shown in Table 9. Genetically modified corn event The results of resistance of DBN9936 heart leaf stage to Asian corn borer are shown in Table 12.

Spinning stage: According to the damage of the ear, the number of pupils, the length of the pupil tunnel (cm), and the age of the larvae and the number of surviving larvae, calculate the average value of the resistance level of the Asian corn borer to the ear at the ear of each plot. The criteria are shown in Table 10, and then the level of resistance to corn borer in the corn ear is determined according to the criteria in Table 11. The results of the resistance of the transgenic maize event DBN9936 to the Asian corn borer at the silking stage are shown in Table 13.

Table 8 Classification criteria for Asian corn borer damage to corn heart

食叶级别Leaf level	症状描述Symptom description
11	仅个别叶片上有1-2个孔径≤1mm虫孔Only 1-2 holes with a pore size ≤ 1mm on individual leaves
22	仅个别叶片上有3-6个孔径≤1mm虫孔Only 3-6 wormholes with a hole diameter ≤ 1mm on individual blades
33	少数叶片有7个以上孔径≤1mm虫孔A few leaves have more than 7 pores ≤ 1mm
44	个别叶片上有1-2个孔径≤2mm虫孔1-2 holes with ≤ 2mm aperture on individual blades
55	少数叶片上有3-6个孔径≤2mm虫孔A few leaves have 3-6 wormholes with a pore size ≤ 2mm
66	部分叶片有7个以上孔径≤2mm虫孔Some blades have more than 7 wormholes with a pore size ≤ 2mm
77	少数叶片上有1-2个孔径大于2mm的虫孔There are 1-2 wormholes with a hole diameter greater than 2mm on a few blades
88	部分叶片上有3-6个孔径大于2mm的虫孔There are 3-6 wormholes with a diameter greater than 2mm on some of the blades.
99	大部分叶片上有7个以上孔径大于2mm的虫孔There are more than 7 wormholes with a diameter greater than 2mm on most of the blades.

Table 9 Evaluation criteria for maize resistance to Asian corn borer

心叶期食叶级别平均值Mean leaf level	抗性水平Resistance level
1.0-2.91.0-2.9	高抗(HR)High resistance (HR)
3.0-4.93.0-4.9	抗(R)Anti-(R)
5.0-6.95.0-6.9	中抗(MR)Medium resistance (MR)
7.0-8.97.0-8.9	感(S)Sense (S)
9.09.0	高感(HS)High sense (HS)

Table 10 Classification criteria for the degree of damage caused by Asian corn borer at the ear of corn

Table 11 Evaluation criteria for resistance to Asian corn borer at the ear of corn

雌穗被害级别平均值Average value of the level of damage to the ear	抗性水平Resistance level
1.0-2.01.0-2.0	高抗(HR)High resistance (HR)
2.1-3.02.1-3.0	抗(R)Anti-(R)
3.1-5.03.1-5.0	中抗(MR)Medium resistance (MR)
5.1-7.05.1-7.0	感(S)Sense (S)
≥7.1≥7.1	高感(HS)High sense (HS)

Table 12 Resistance Results of Asian Corn Borer in DBN9936 Heart Stage of Transgenic Corn Event

Table 13 Resistance results of Asian corn borer in DBN9936 silking stage of transgenic corn event

The results showed that the transgenic maize event DBN9936 had a good resistance level to Asian corn borer in both heart and silk stage. At the heart leaf stage, the average leaf level of DBN9936 in the transgenic corn event was significantly lower than that of wild type corn. Plant. During the silking stage, the ear damage rate, larval survival number, tunnel length and ear damage level of DBN9936 were significantly lower than those of wild-type maize plants. The field effect of transgenic maize event DBN9936 inoculated with Asian corn borer in the heart and silk stage is shown in Figure 3.

(2) Oriental armyworm

The experimental design and test methods are essentially consistent with the evaluation of Asian corn borer resistance as described above. The difference is that the artificial only in the heart stage of the corn (the development of the corn plant to the 4-6 leaf stage) Inoculation, inoculation 2 times, in each corn heart leaf, about 20 second-instar larvae were artificially reared. After 3 days of inoculation, the second time the insects were caught, the number of insects was the same as the first time. After 14 days of inoculation, the corn leaves were investigated for damage to oriental armyworms. According to the degree of damage of the eastern leaves of the corns, the average value of the oriental armyworms in each plot on the damage level of the corn leaves (food leaf grade) is calculated. The judgment criteria are as shown in Table 14, and then the corn is determined according to the criteria in Table 15. The level of resistance of the armyworm. The results of the resistance of the transgenic maize event DBN9936 to the oriental armyworm were shown in Table 16.

Table 14 Grading criteria for the degree of damage of maize leaves to oriental armyworms

食叶级别Leaf level	症状描述Symptom description
11	叶片无被害，或仅叶片上有针刺状(≤1mm)虫孔The leaves are not damaged, or only the needles have a needle-like (≤1mm) wormhole
22	仅个别叶片上有少量弹孔大小(≤5mm)虫孔Only a small number of bullet holes (≤5mm) on individual leaves
33	少数叶片有弹孔大小(≤5mm)虫孔A few blades have bullet holes (≤5mm) wormholes
44	个别叶片上缺刻(≤10mm)Defects on individual blades (≤10mm)
55	少数叶片上有缺刻(≤10mm)A few leaves have nicks (≤10mm)
66	部分叶片上有缺刻(≤10mm)Some blades have nicks (≤10mm)
77	个别叶片部分被取食，少数叶片上有大片缺刻(≤10mm)Individual blade parts are fed, and a few leaves have large nicks (≤10mm)
88	少数叶片被取食，部分叶片上有大片缺刻(≤10mm)A few leaves are fed, and some leaves have large nicks (≤10mm)
99	大部分叶片被取食Most of the leaves are fed

Table 15 Evaluation criteria for resistance of maize to oriental armyworm

心叶期食叶级别平均值Mean leaf level	抗性水平Resistance level
1.0-2.01.0-2.0	高抗(HR)High resistance (HR)
2.1-4.02.1-4.0	抗(R)Anti-(R)
4.1-6.04.1-6.0	中抗(MR)Medium resistance (MR)
6.1-8.06.1-8.0	感(S)Sense (S)
8.1-9.08.1-9.0	高感(HS)High sense (HS)

Table 16 Results of resistance to oriental armyworm in DBN9936 heart stage of transgenic maize event

The results showed that the transgenic maize event DBN9936 had a good resistance level to oriental armyworm, and the ratio of nick and leaf level of transgenic corn event DBN9936 was significantly lower than that of wild. The field effect of maize plant, transgenic corn event DBN9936 inoculated with oriental armyworm is shown in Figure 4.

(3) Helicoverpa armigera

The experimental design and test methods are essentially consistent with the evaluation of Asian corn borer resistance as described above. The difference is that only in the silking stage of the corn, artificial insects, insects 2 times, in each of the corn filaments, about 20 artificially reared larvae, 3 days after the insects, the second insect, insects The number is the same as the first time. After 14-21 days of inoculation, the rate of damage to the ear, the number of larvae per ear, and the length of the ear were investigated on a plant-by-plant basis. Usually, the investigation starts 14 days after the insects are received. If the negative control material (NGM) has a sense of sensation or high sensation (see Table 17 and Table 18), it is considered to be effective. If it is not reached, the investigation can be postponed appropriately, but after the worm is received. If the corresponding level has not been reached in 21 days, the insect is considered invalid. According to the damage rate of the ear, the number of surviving larvae, and the length of the ear damage (cm), the average value of the damage level of the cotton bollworm to the ear in the corn ear of each plot was calculated. The judgment criteria are shown in Table 17, and then judged according to the criteria of Table 18. The level of resistance to cotton bollworm at the ear of corn. The results of resistance to cotton bollworm in the silking stage of the transgenic maize event DBN9936 are shown in Table 19.

Table 17 Classification criteria for the degree of damage of cotton ears to cotton bollworm

雌穗被害级别Ear damage level	症状描述Symptom description
00	雌穗没有受害Ears are not victimized
11	仅花丝被害 Only filigree
22	穗顶被害1cmSpike top damage 1cm
3+3+	穗顶下被害每增加1cm，相应的被害级别增加1级For each increase of 1cm in the top of the ear, the corresponding damage level is increased by 1 level.
…N...N

Table 18 Evaluation criteria for resistance of cotton ears to cotton bollworm

雌穗被害级别平均值Average value of the level of damage to the ear	抗性水平Resistance level
0-1.00-1.0	高抗(HR)High resistance (HR)
1.1-3.01.1-3.0	抗(R)Anti-(R)
3.1-5.03.1-5.0	中抗(MR)Medium resistance (MR)
5.1-7.05.1-7.0	感(S)Sense (S)
≥7.1≥7.1	高感(HS)High sense (HS)

Table 19 Resistance to cotton bollworm in the silking stage of transgenic maize event DBN9936

The results showed that the transgenic maize event DBN9936 had a good resistance level to cotton bollworm, and the ear damage rate, larval survival number, ear damage length and ear damage level of the transgenic corn event DBN9936 were significantly lower than that of wild type maize plants. The field effect of inoculation of cotton bollworm by DBN9936 in the transgenic maize event is shown in Figure 5.

(4) Taoyuan

The experimental design and test methods are essentially consistent with the evaluation of Asian corn borer resistance as described above. The difference is that corn is only naturally susceptible to insects (natural pest occurrence conditions) in areas where the natural occurrence of peach aphid is more serious. After 14-21 days of initial pest occurrence, and NGM was mostly 4-5-year-old larvae hazard, the damage rate of peach aphid to maize plants was investigated on a plant-by-plant basis (the proportion of corn plants fed by pests to the total plants investigated). The results of the resistance of the transgenic maize event DBN9936 to Myzus persicae are shown in Table 20.

Table 20 Resistance to peach aphid under the condition of DBN9936 natural susceptibility in transgenic corn event

The results showed that under the natural conditions of the peach aphid, compared with the wild-type maize plants, the damage rate of the peach aphid to the transgenic corn event DBN9936 was significantly reduced, which indicated that the transgenic maize event DBN9936 had a good resistance to the peach aphid. The field effect of the transgenic maize event DBN9936 under natural conditions of Myzus persicae is shown in Figure 6.

(5) Beet armyworm

The experimental design and test methods were essentially consistent with the evaluation of the resistance of the peach aphid as described above. The difference is that after 10-15 days of initial pest occurrence, and NGM is mostly 4-6 instar larvae, the damage rate of beet armyworm to corn plants is investigated on a plant-by-plant basis. The results of resistance of the transgenic maize event DBN9936 to Spodoptera exigua are shown in Table 21.

Table 21 Resistance results of beet armyworm under DBN9936 natural susceptibility to GM maize event

The results showed that under the natural conditions of beet armyworm, compared with wild-type maize plants, the damage rate of beet armyworm to DBN9936 was significantly lower, indicating that the transgenic maize event DBN9936 had better resistance to beet armyworm. The field effect of the transgenic maize event DBN9936 under naturally occurring conditions of Spodoptera exigua is shown in Figure 7.

It is particularly worth mentioning that, according to the contents described in Chinese Patent Application Nos. 201210509817.2, 201210511214.6, 201310576970.1, 201310578129.6 and 201310681139.2, and the field efficacy of the transgenic corn event DBN9936 for insects and their bioassay results, the GM corn of the present application is indicated. Event DBN9936 implements methods and/or uses for controlling pests, specifically for the genus Diptera, Myzus persicae, Spodoptera litura, Euphorbia and Oriental armyworm; that is, any transgenic maize plant expressing the Cry1Ab protein can be controlled. Methods and/or uses of the two-pointed moth, the peach aphid, the Spodoptera litura, the giant salamander, and/or the oriental armyworm pest.

Sixth Example, Herbicide Tolerance Test of Corn Event DBN9936

This test was carried out by using Roundup herbicide (41% glyphosate isopropyl ammonium salt solution, Monsanto Company). A random block design was used with 3 repetitions. The area of the plot is 15m ² (5m×3m), the row spacing is 60cm, the plant spacing is 25cm, and the cultivation management is routine. There is a 1m wide isolation zone between the cells. The transgenic corn event DBN9936 was treated as follows: 1) no spraying; 2) spraying the Roundup herbicide at the V3 leaf stage at a dose of 1680 g ae/ha (ae/ha means "active ingredient equivalent acid per hectare") Then, the Roundup herbicide was sprayed again at the same dose in the V8 period. Parallel control experiments were performed using wild-type maize plants (non-transgenic, NGM). It should be noted that the glyphosate herbicides of different contents and dosage forms are converted into the equivalent glyphosate acid and are suitable for the following conclusions.

The symptoms of the phytotoxicity were investigated 1 week and 2 weeks after the administration, and the yield of the plot was measured at the time of harvest. The classification of symptoms of phytotoxicity is shown in Table 22. The herbicide victimization rate was used as an evaluation index to evaluate the tolerance of the herbicide tolerance in the conversion event. Specifically, the herbicide victimization rate (%) = ∑ (number of affected plants × level number) / (total number of plants × highest level); The herbicide victimization rate refers to the gargalin damage rate, and the glyphosate damage rate is determined based on the results of the phytotoxicity investigation 2 weeks after glyphosate treatment. The corn yield per plot is the total yield (weight) of corn kernels in the middle 3 rows of each plot. The yield difference between different treatments is measured as the percentage of yield. The percentage of yield (%) = spray yield / no spray Yield. The results of the tolerant tolerance of the transgenic maize event DBN9936 and corn yield results are shown in Table 23.

Table 22 Grading criteria for the degree of phytotoxicity of glyphosate herbicides on corn

药害级别Phytotoxicity level	症状描述Symptom description
11	生长正常，无任何受害症状Normal growth, no symptoms of any damage
22	轻微药害，药害少于10％Minor phytotoxicity, less than 10% phytotoxicity
33	中等药害，以后能恢复，不影响产量Moderate phytotoxicity, can recover later, does not affect production
44	药害较重，难以恢复，造成减产The phytotoxicity is heavier and difficult to recover, resulting in reduced production.
55	药害严重，不能恢复，造成明显减产或绝产Serious phytotoxicity, can not be restored, resulting in significant reduction or production

Table 23 Results of tolerance to glyphosate herbicides by DBN9936 in transgenic maize events and maize yield results

The results showed that in terms of herbicide (glyphosate) damage rate: 1) The genetically modified corn event DBN9936 was basically zero under the treatment of glyphosate herbicide (1680 g ae/ha), thus the transgenic corn event DBN9936 had good Glyphosate herbicide tolerance.

In terms of yield: there was no significant difference in the yield of DBN9936 in the transgenic corn event without spraying and spraying 1680g ae/ha glyphosate. After spraying glyphosate herbicide, the yield of transgenic corn event DBN9936 was slightly lower. This, in turn, further indicates that the transgenic maize event DBN9936 has good glyphosate herbicide tolerance.

Seventh embodiment

Such as agricultural products or commodities can be produced by the genetically modified corn event DBN9936. 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 transgenic maize event DBN9936 material present in the agricultural product or commodity. The agricultural products or commodities include, but are not limited to, corn oil, corn meal, cornmeal, jade Rice gluten, tortillas, corn starch, and any other food to be consumed as a food source for animals, or otherwise used as an ingredient in a bulking or cosmetic composition for cosmetic use and the like. A nucleic acid detection method and/or kit based on a probe or primer pair can be developed to detect a transgenic maize event DBN9936 nucleotide sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2 in a biological sample, wherein the probe The sequence or primer sequence is selected from the sequences set forth 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 the transgenic maize event DBN9936.

In summary, the transgenic maize event DBN9936 has good resistance to lepidopteran insects, and has high tolerance to glyphosate herbicides, has no effect on yield, and the detection method can be accurate and rapid. Identify whether the biological sample contains the DNA molecule of the transgenic maize event DBN9936.

The seed corresponding to the transgenic corn event DBN9936 was deposited on December 24, 2014 at the General Microbiology Center of the China Microbial Culture Collection Management Committee (CGMCC, Address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China) Institute, Zip Code 100101), classification: Maize (Zea mays), the deposit number is CGMCC No.10219. The deposit will be kept at the depository for 30 years.

It should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention, and are not intended to be limiting, although the present invention will be described in detail with reference to the preferred embodiments. Modifications or equivalents may be made without departing from the spirit and scope of the invention.

Claims

A nucleic acid sequence comprising at least 11 contiguous nucleotides of SEQ ID NO: 3 or its complement, and/or at least 11 contiguous nucleotides of SEQ ID NO: 4 or its complement .
The nucleic acid sequence according to claim 1, wherein the nucleic acid sequence comprises SEQ ID NO: 1 or its complement, and/or SEQ ID NO: 2 or its complement.
The nucleic acid sequence according to claim 2, wherein the nucleic acid sequence comprises SEQ ID NO: 3 or its complement, and/or SEQ ID NO: 4 or its complement.
The nucleic acid sequence according to claim 3, wherein the nucleic acid sequence comprises SEQ ID NO: 5 or a complement thereof.
A method for detecting the presence of DNA in a transgenic maize event DBN9936 in a sample, comprising:

Contacting the sample with at least two primers in a nucleic acid amplification reaction;

Performing a nucleic acid amplification reaction;

Detecting the presence of an amplification product, the presence of the amplification product indicating the presence of DNA of the transgenic maize event DBN9936 in the sample;

The amplification product comprises at least 11 contiguous nucleotides of SEQ ID NO: 3 or its complement, or at least 11 contiguous nucleotides of SEQ ID NO: 4 or its complement;

Preferably, the amplification product comprises contiguous nucleotides 1 to 11 or 12 to 22 in SEQ ID NO: 1 or its complement, or 1 to 11 in SEQ ID NO: 2 or its complement Bit or contiguous nucleotides from positions 12-22;

Preferably, the amplification product comprises SEQ ID NO: 1 or its complement, SEQ ID NO: 2 or its complement, SEQ ID NO: 6 or its complement, or SEQ ID NO: 7 or its complement.
The method according to claim 5, wherein said primer comprises at least one of at least 11, 12, 13, 14, 15, 16, 17 selected from the nucleic acid sequences of claims 1-4. , 18, 19, 20, 21 or 22 contiguous nucleotides; or, the primer comprises a first primer and a second primer, wherein the first primer is selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 10, second Primers are selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO: 11.
A method for detecting the presence of DNA in a transgenic maize event DBN9936 in a sample, comprising:

Contacting the sample with a probe comprising at least 11 contiguous nucleotides of SEQ ID NO: 3 or its complement, or at least 11 contiguous nucleotides of SEQ ID NO: 4 or its complement ;

Hybridizing the sample and the probe under stringent hybridization conditions;

Detecting the hybridization of the sample and the probe, the hybridization of the sample and the probe indicates the presence of DNA of the transgenic maize event DBN9936 in the sample;

Preferably, the probe comprises contiguous nucleotides 1 to 11 or 12 to 22 of SEQ ID NO: 1 or its complement, or positions 1 to 11 of SEQ ID NO: 2 or its complement Or contiguous nucleotides 12-22;

Preferably, the probe comprises SEQ ID NO: 1 or its complement, SEQ ID NO: 2 or its complement, SEQ ID NO: 6 or its complement, or SEQ ID NO: 7 or its complement.
The method of claim 7 wherein at least one of said probes is labeled with at least one fluorophore.
A method for detecting the presence of DNA in a transgenic maize event DBN9936 in a sample, comprising:

Passing the sample with a label nucleic acid molecule comprising at least 11 contiguous nucleotides of SEQ ID NO: 3 or its complement, or at least 11 contiguous of SEQ ID NO: 4 or its complement Nucleotide

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

Detecting hybridization of the sample and the marker nucleic acid molecule, the hybridization of the sample and the marker nucleic acid molecule indicates the presence of DNA of the transgenic maize event DBN9936 in the sample;

Preferably, the marker nucleic acid molecule comprises SEQ ID NO: 1 or its complementary sequence at positions 1-11 or 12-22, or SEQ ID NO: 2 or its complement 1 - 11th or 12th to 22th consecutive nucleotides;

Preferably, the marker nucleic acid molecule comprises SEQ ID NO: 1 or its complement, SEQ ID NO: 2 or its complement, SEQ ID NO: 6 or its complement, or SEQ ID NO: 7 or its complement ;

Preferably, the method further comprises: aiding breeding analysis by a marker to determine that insect resistance and/or herbicide tolerance is genetically linked to the marker nucleic acid molecule.
A DNA detection kit comprising at least one DNA molecule comprising at least 11 of the homologous sequences of SEQ ID NO: 3 or a complement thereof a contiguous nucleotide, or at least 11 contiguous nucleotides of the homologous sequence of SEQ ID NO: 4 or its complement, which can serve as a DNA primer specific for the transgenic maize event DBN9936 or its progeny Or probe;

Preferably, the DNA molecule comprises contiguous nucleotides 1 to 11 or 12 to 22 in SEQ ID NO: 1 or its complement, or 1 to 11 in SEQ ID NO: 2 or its complement Or contiguous nucleotides 12-22;

Preferably, the DNA molecule comprises the homologous sequence of SEQ ID NO: 1 or its complement, the homologous sequence of SEQ ID NO: 2 or its complement, the homologous sequence of SEQ ID NO: 6, or its complement, Or the homologous sequence of SEQ ID NO: 7 or its complement.
A plant cell or part comprising a nucleic acid sequence encoding an insect resistance Cry1Ab protein, a nucleic acid sequence encoding a glyphosate herbicide-tolerant EPSPS protein, and a nucleic acid sequence of a specific region, the nucleic acid sequence of the specific region Including SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 6 or SEQ ID NO: 7;

Preferably, the corn plant cell or part comprises a nucleic acid sequence encoding an insect resistant Cry1Ab protein, a nucleic acid sequence encoding a glyphosate herbicide-tolerant EPSPS protein, and a nucleic acid sequence of a specific region, the nucleic acid sequence of the specific region comprising SEQ ID NO: 3 or SEQ ID NO: 4;

Preferably, the maize plant cell or part comprises the nucleic acid sequence set forth in SEQ ID NO:5.
A method of protecting a corn plant from insect infestation, the method comprising providing at least one transgenic maize plant cell or part of claim 11 in a diet of a target insect, ingesting the transgenic corn plant The cell or part of the target insect is inhibited from further feeding the corn plant.
A method of protecting a corn plant from damage caused by a herbicide and/or controlling weeds in a field planting a corn plant, characterized in that the method comprises applying an effective amount of a glyphosate herbicide to at least one planting In the field of the transgenic maize plant, the transgenic maize plant comprises in its genome a cell 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: At least one nucleic acid sequence of the sequences set forth in SEQ ID NO: 6 and SEQ ID NO: 7, the transgenic maize plant having tolerance to a glyphosate herbicide.
A method of cultivating a corn plant resistant to insects, comprising:

Planting at least one corn seed comprising a coding insect resistance in the genome of the corn seed a nucleic acid sequence of a Cry1Ab protein and a nucleic acid sequence of a specific region;

Growing the corn seed into a corn plant;

Infesting the maize plant with a target insect, harvesting plants having reduced plant damage compared to other plants having no nucleic acid sequence of a particular region;

The nucleic acid sequence of the specific region is selected from at least one of the sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 7. Nucleic acid sequence;

Preferably, the method comprises:

Planting at least one corn seed, the genome of the corn seed comprising the nucleic acid sequence set forth in SEQ ID NO: 5;

Growing the corn seed into a corn plant;

The maize plants were challenged with target insects, and plants having reduced plant damage compared to other plants not having SEQ ID NO: 5 were harvested.
A method of cultivating a corn plant that is tolerant to a glyphosate herbicide, comprising:

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

Growing the corn seed into a corn plant;

Spraying the corn plant with an effective amount of a glyphosate herbicide to harvest plants having reduced plant damage compared to other plants having no nucleic acid sequence of a particular region;

The nucleic acid sequence of the specific region is selected from at least one of the sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 7. Nucleic acid sequence;

Preferably, the method comprises:

Planting at least one corn seed, the genome of the corn seed comprising the nucleic acid sequence set forth in SEQ ID NO: 5;

Growing the corn seed into a corn plant;

The corn plants were sprayed with an effective dose of glyphosate herbicide to harvest plants having reduced plant damage compared to other plants not having SEQ ID NO: 5.
A method for cultivating an insect-resistant and glyphosate-tolerant herb-tolerant corn plant, comprising:

Planting at least one corn seed comprising a coding insect resistance in the genome of the corn seed a nucleic acid sequence of a Cry1Ab protein, a nucleic acid sequence encoding a glyphosate herbicide-tolerant EPSPS protein, and a nucleic acid sequence of a specific region;

Growing the corn seed into a corn plant;

Spraying the corn plant with an effective amount of a glyphosate herbicide to harvest plants having reduced plant damage compared to other plants having no nucleic acid sequence of a particular region, the plant having damage to the insect damage to the insect Also resistant;

The nucleic acid sequence of the specific region is selected from at least one of the sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 7. Nucleic acid sequence;

Preferably, the method comprises:

Planting at least one corn seed, the genome of the corn seed comprising the nucleic acid sequence set forth in SEQ ID NO: 5;

Growing the corn seed into a corn plant;

The corn plants are sprayed with an effective amount of a glyphosate herbicide to harvest plants having reduced plant damage compared to other plants not having SEQ ID NO: 5, the feeding damage of the plants with reduced plant damage to the insects Also resistant.
A method of producing a maize plant resistant to insects, the method comprising introducing into a genome of the corn plant a nucleic acid sequence encoding an insect resistance Cry1Ab protein and a nucleic acid sequence of a specific region, the specific The nucleic acid sequence of the region is selected from at least one of the sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 7. ;

Preferably, the method comprises introducing into the genome of the maize plant the nucleic acid sequence set forth in SEQ ID NO: 5;

Preferably, the method comprises:

The insect-resistant transgenic maize event DBN9936 first parental maize plant is sexually crossed with the second parental maize plant lacking insect resistance, thereby producing a large number of progeny plants;

Invading the progeny plant with a target insect;

Selecting said progeny plants having reduced plant damage compared to other nucleic acid sequences that do not have a particular region or plants of SEQ ID NO: 5;

Wherein the transgenic maize event DBN9936 comprises in its genome a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6. And at least one nucleic acid sequence of the sequence of SEQ ID NO: 7.
Method for producing a corn plant that is tolerant to glyphosate herbicides, characterized In the method, the method comprises introducing into the genome of the corn plant a nucleic acid sequence encoding a glyphosate-tolerant EPSPS protein and a nucleic acid sequence of a specific region, wherein the nucleic acid sequence of the specific region is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, at least one nucleic acid sequence of the sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 7;

Preferably, the method comprises introducing into the genome of the maize plant the nucleic acid sequence set forth in SEQ ID NO: 5;

Preferably, the method comprises:

The first parental maize plant of the transgenic maize event DBN9936, which is tolerant to the glyphosate herbicide, is sexually crossed with the second parental maize plant lacking glyphosate tolerance, thereby producing a large number of progeny plants;

Treating the progeny plants with a glyphosate herbicide;

Selecting the progeny plants that are tolerant to glyphosate;

Wherein the transgenic maize event DBN9936 comprises in its genome a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6. And at least one nucleic acid sequence of the sequence of SEQ ID NO: 7.
A method of producing a corn plant that is resistant to insects and resistant to glyphosate herbicide application, comprising:

The glyphosate-tolerant and insect-resistant transgenic maize event DBN9936 first parent corn plant is sexually crossed with a second parental maize plant lacking glyphosate tolerance and/or insect resistance, thereby producing a large number of progeny plants ;

Treating the progeny plants with glyphosate;

The progeny plants that are tolerant to glyphosate are selected, and the progeny plants that are tolerant to glyphosate are also resistant to insect feeding damage;

Wherein the transgenic maize event DBN9936 comprises in its genome a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6. And at least one nucleic acid sequence of the sequence of SEQ ID NO: 7.
A composition comprising a polynucleotide of SEQ ID NO: 1 or SEQ ID NO: 2, characterized in that the composition is corn flour, corn flour, corn oil, corn silk or corn starch.
An agricultural product or commodity comprising the polynucleotide of SEQ ID NO: 1 or SEQ ID NO: 2, characterized in that the agricultural product or commodity is corn flour, cornmeal, corn oil, corn starch, corn gluten, tortilla, Cosmetics or fillers.