WO2016173362A1 - Plant de maïs dbn9978 et procédé d'utilisation dans la détection de la séquence d'acide nucléique associée - Google Patents

Plant de maïs dbn9978 et procédé d'utilisation dans la détection de la séquence d'acide nucléique associée Download PDF

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WO2016173362A1
WO2016173362A1 PCT/CN2016/077867 CN2016077867W WO2016173362A1 WO 2016173362 A1 WO2016173362 A1 WO 2016173362A1 CN 2016077867 W CN2016077867 W CN 2016077867W WO 2016173362 A1 WO2016173362 A1 WO 2016173362A1
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seq
nucleic acid
acid sequence
corn
plant
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PCT/CN2016/077867
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Chinese (zh)
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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
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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
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Publication of WO2016173362A1 publication Critical patent/WO2016173362A1/fr

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    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
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    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits

Definitions

  • the invention relates to the field of plant molecular biology, in particular to the field of transgenic crop breeding in agricultural biotechnology research.
  • the present invention relates to insect resistance and glyphosate herbicide tolerance transgenic maize event DBN9978 and nucleic acid sequences for detecting whether a transgenic maize event DBN9978 is included in a biological sample and a method of detecting the same.
  • 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.
  • EPSPS glyphosate herbicide tolerance genes
  • 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.
  • the object of the present invention is to provide a corn plant DBN9978 and a nucleic acid sequence and method for detecting the same, and the transgenic corn event DBN9978 has good 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 DBN9978.
  • 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.
  • the nucleic acid sequence comprises SEQ ID NO: 1 or its complement, and/or SEQ ID NO: 2 or its complement.
  • nucleic acid sequence comprises SEQ ID NO: 3 or its complement, and/or SEQ ID NO: 4 or its complement.
  • 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 DBN9978 located at the 5' end of the inserted sequence near the insertion junction, SEQ ID NO: 1 or 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 DBN9978.
  • the SEQ ID NO: 2 or its complement is a 22 nucleotide sequence in the transgenic maize event DBN9978 located at the 3' end of the inserted sequence near the insertion junction, 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 DBN9978 presence.
  • 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.
  • the first nucleic acid sequence 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.
  • 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 DBN9978 or its progeny can be diagnosed.
  • 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.
  • 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.
  • 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 1557 nucleotides in length in the transgenic maize event DBN9978 located at the 5' end of the inserted sequence near the insertion junction, SEQ ID NO: 3 or The complementary sequence consists of a 1374 nucleotide maize flanking genomic DNA sequence (nucleotides 1-1374 of SEQ ID NO: 3) and 84 nucleotides of the DBN10124 construct DNA sequence (nucleotides of SEQ ID NO: 3) The glycerol 1375-1458) and the 99-nucleotide tNos (nopaline synthase) transcription terminator 3' end DNA sequence (nucleotides 1459-1557 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 DBN9978.
  • 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.
  • these nucleic acid sequences can be used as a DNA primer pair in a DNA amplification method for producing an amplification product.
  • the amplification product produced in the DNA amplification method using the DNA primer pair is the amplification product comprising SEQ ID NO: 2, the presence of the transgenic maize event DBN9978 or its progeny can be diagnosed.
  • the SEQ ID NO: 4 or its complement is a 672 nucleotide sequence in the transgenic maize event DBN9978 located at the 3' end of the inserted sequence near the insertion junction, SEQ ID NO: 4 or
  • the complementary sequence consists of a 51 nucleotide t35S transcription terminator sequence (nucleotides 1-51 of SEQ ID NO: 4), and a nucleotide of 141 DBN10124 construct DNA sequences (nucleotides of SEQ ID NO: 4) Acid 52-192) and a 480 nucleotide maize integration site flanking genomic DNA sequence (nucleotides 193-672 of SEQ ID NO: 4) comprising the SEQ ID NO: 4 or its complement Identification of the presence of the transgenic maize event DBN9978.
  • the SEQ ID NO: 5 or its complement is a sequence of 9219 nucleotides in length that characterizes the transgenic maize event DBN9978, 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 DBN9978.
  • 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 DBN9978 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 DBN9978 or its progeny in a biological sample presence.
  • the present invention also provides a method for detecting the presence of DNA in a transgenic maize event DBN9978 in a sample, comprising:
  • 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;
  • transgenic maize event DBN9978 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.
  • 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.
  • 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 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.
  • 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.
  • the present invention also provides another method for detecting the presence of DNA in the transgenic maize event DBN9978 in a sample, comprising:
  • transgenic maize event DBN9978 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.
  • 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.
  • 6 x SSC sodium citrate
  • SDS sodium dodecyl sulfate
  • 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.
  • 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.
  • At least one of the probes is labeled with at least one fluorophore.
  • the present invention also provides another method for detecting the presence of DNA in the transgenic maize event DBN9978 in a sample, comprising:
  • the hybridization of the sample and the marker nucleic acid molecule indicates the presence of DNA of the transgenic maize event DBN9978 in the sample;
  • transgenic maize event DBN9978 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.
  • 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.
  • 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 .
  • 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.
  • 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 DBN9978 or a progeny thereof, wherein
  • the transgenic maize event DBN9978 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the maize plant cell or part comprises the nucleic acid sequence set forth in SEQ ID NO:5.
  • 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.
  • 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.
  • 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.
  • the present invention also provides a method of cultivating a corn plant resistant to insects, comprising:
  • 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.
  • 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;
  • 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.
  • 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;
  • 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.
  • 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;
  • 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.
  • 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;
  • 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.
  • 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;
  • 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.
  • 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.
  • the method comprises introducing into the genome of the maize plant the nucleic acid sequence set forth in SEQ ID NO: 5.
  • the method of producing a corn plant resistant to insects comprises:
  • the insect-resistant transgenic maize event DBN9978 first parental maize plant is sexually crossed with the second parental maize plant lacking insect resistance, thereby producing a large number of progeny plants;
  • transgenic maize event DBN9978 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.
  • 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.
  • the method comprises introducing into the genome of the maize plant the nucleic acid sequence set forth in SEQ ID NO: 5.
  • 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 DBN9978 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;
  • transgenic maize event DBN9978 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.
  • 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 DBN9978 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 ;
  • 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;
  • transgenic maize event DBN9978 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.
  • 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.
  • 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.
  • 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.
  • corn refers to Zea mays and includes all plant species that can be mated with corn, including wild corn species.
  • 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.
  • 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.
  • gene refers to a nucleic acid fragment that expresses a particular protein, including the modulation before the coding sequence.
  • sequence 5' non-coding sequence
  • 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.
  • flanking DNA can include a combination of natural and exogenous DNA.
  • flanking sequence 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.
  • flanking region 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.
  • 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 known as DBN9978 and its progeny, which is a maize plant DBN9978 comprising plants and seeds of the transgenic maize event DBN9978 and plant cells thereof or a regenerable portion thereof, said transgene
  • the plant part of the maize event DBN9978 including but not limited to cells, pollen, ovules, flowers, buds, Roots, stems, silks, inflorescences, ear ears, leaves and products from corn plant DBN9978, 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 DBN9978 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
  • EPSPS synthase
  • 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.
  • CaMV cauliflower mosaic virus
  • 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
  • NOS nopaline synthase
  • CaMV cauliflower mosaic virus
  • PINII protease inhibitor II
  • 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.
  • TSV Tobacco Etch Virus
  • CaMV35S Enhancer CaMV35S Enhancer
  • FMV35S Enhancer 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.
  • Bt Bacillus thuringiensis
  • 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.
  • glyphosate refers to N-phosphonomethylglycine and its salts
  • 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 DBN9978 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 DBN9978 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
  • the transgenic plants 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
  • the parental line 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.
  • transgenic 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.
  • 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.
  • a molecule can be derived from a first species and inserted into the genome of a second species.
  • the molecule is heterologous to the host and is artificially introduced into the genome of the host cell.
  • the transgenic maize event DBN9978 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 plurality of first generation progeny plants consisting of maize plants grown from the transgenic maize event DBN9978 and its progeny, the transgenic maize event DBN9978 and its progeny being by using the lepidopteran insect of the present 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
  • 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.
  • 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 DBN9978), Thereby producing corn plants that are resistant to lepidopteran insects and tolerant to glyphosate herbicides.
  • 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.
  • 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.
  • the probe is complementary to a DNA strand from the genome of the transgenic maize event DBN9978, whether the genomic DNA is derived from the transgenic maize event DBN9978 or from a seed or from a transgene. Plant or seed or extract of corn event DBN9978.
  • 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.
  • 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.
  • 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.
  • Primers and probes based on the flanking genomic DNA and insert sequences of the present invention can be determined by conventional methods, for example, by isolating a corresponding DNA molecule from a plant material derived from the transgenic maize event DBN9978, 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 DBN9978 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.
  • nucleic acid molecules 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.
  • 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.
  • a nucleic acid molecule 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.
  • 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.
  • 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.
  • 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.
  • SEQ ID NO: 1 SEQ ID NO: 2
  • SEQ ID NO: 3 SEQ ID NO: 4.
  • 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.
  • 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.
  • 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.
  • 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.
  • amplified DNA refers to a nucleic acid amplification product of a target nucleic acid sequence that is part of a nucleic acid template.
  • a corn plant is produced by a sexual hybridization method comprising the transgenic maize event DBN9978 of the invention, or whether the corn sample collected from the field comprises a transgenic maize event DBN9978, or a corn extract, such as a meal, powder or oil, comprises Transgenic maize event DBN9978
  • 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 DBN9978.
  • 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 DBN9978.
  • 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.
  • 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).
  • PCR polymerase chain reaction
  • 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 DBN9978 can be amplified by using the provided primer sequences for the genome of the transgenic maize event DBN9978, 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.
  • the primer pairs that are particularly useful in identifying DNA amplification methods are 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 DBN9978. , 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. 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 DBN9978 may also include Southern blot, Northern blot, and In situ hybridization based on the principle of hybridization.
  • 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.
  • 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.
  • 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 DBN9978 can also be used to grow corn plants containing the DNA of the transgenic maize event DBN9978.
  • 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 DBN9978 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.
  • iZmHSP70 maize heat shock 70 kDa intron
  • pr35S cauliflower mosaic virus 35S promoter
  • cCry1Ab insect-resistant Cry1Ab protein
  • tNos transcriptional terminator
  • tNos transcriptional terminator
  • the actin 1 promoter (prOsAct1) operably linked to the Arabidopsis EPSPS chloroplast transit peptide (spAtCTP2)
  • cEPSPS glyphosate-tolerant 5-enol of the Agrobacterium CP4 strain - Pyruvate oxalic acid-3-phosphate synthase
  • t35S cauliflower mosaic virus 35S terminator
  • RB right border region
  • the DNA molecule as a primer may be any part derived from the transgene insertion sequence in the transgenic maize event DBN9978, or may be any part of the DNA sequence derived from the flanking maize genome in the transgenic maize event DBN9978.
  • the genetically modified maize event DBN9978 can be combined with other genetically modified maize varieties, such as corns that are tolerant to herbicides (such as glufosinate, dicamba, etc.) or transgenic maize varieties that carry other insect-resistant genes.
  • herbicides such as glufosinate, dicamba, etc.
  • transgenic maize varieties that carry other insect-resistant genes.
  • Various combinations of all of these different transgenic events, bred together with the transgenic maize event DBN9978 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 DBN9978 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.
  • EPSPS enol-pyruvylshikimate-3-phosphate synthase
  • 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.
  • 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 DBN9978, which provides enhanced breeding efficiency and enables Molecular markers to track transgenic inserts in the breeding population and its progeny.
  • 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.
  • a probe to generate an amplification product diagnosed as a transgenic maize event DBN9978 or a progeny thereof, and the presence of plant material derived from the transgenic maize event DBN9978 can be identified quickly, accurately, and stably.
  • SEQ ID NO: 1 A sequence of 22 nucleotides in length near the insertion junction at the 5' end of the inserted sequence in the transgenic maize event DBN9978, with 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 near the insertion junction at the 3' end of the inserted sequence in the transgenic maize event DBN9978, with 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 1557 nucleotides in length in the transgenic maize event DBN9978 located at the 5' end of the inserted sequence near the insertion junction;
  • SEQ ID NO: 4 A sequence of 672 nucleotides in length in the transgenic maize event DBN9978 at the 3' end of the inserted sequence near the insertion junction;
  • 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.
  • Figure 1 is a schematic diagram showing the structure of the maize gene DBN9978 transgenic insert and the maize genome junction site, and the nucleic acid sequence used in the method for detecting the maize event DBN9978. Schematic diagram of relative position;
  • FIG. 2 is a schematic view showing the structure of a recombinant expression vector DBN10124 for detecting a corn event DBN9978;
  • Figure 3 is a comparison of field effects of maize event DBN9978 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 DBN9978 and wild-type maize plants (non-transgenic, NGM) inoculated with oriental armyworms;
  • Figure 5 is a comparison of field effects of maize event DBN9978 and wild-type maize plants (non-transgenic, NGM) inoculated with cotton bollworm;
  • Figure 6 is a comparison of field effects of maize event DBN9978 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 DBN9978 and wild-type maize plants (non-transgenic, NGM) under natural conditions of beet armyworm.
  • the first embodiment the acquisition of the genetically modified corn event DBN9978
  • 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
  • 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.
  • EPSPS 5-enol-pyruvylshikimate-3-phosphate synthase
  • 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 DBN9978.
  • 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 immature embryo is co-cultured with Agrobacterium for a period of time (3 days) (step 2: co-cultivation step).
  • 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).
  • 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.
  • 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).
  • 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.
  • 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).
  • 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.
  • 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.
  • the second embodiment using TaqMan for transgenic corn event DBN9978 detection
  • Step 11 Take 100 mg of the leaves of the transgenic corn event DBN9978, 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 / ⁇ l, 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; fluorescent quantitative PCR primers and probe sequences The columns are:
  • Primer 2 GTAGATTTCGCGGGTCAGTTG is shown in SEQ ID NO: 17 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 ⁇ L of each primer at a concentration of 1 mM, 50 ⁇ L of a probe at a concentration of 100 ⁇ M, and 860 ⁇ L 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:
  • 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 ⁇ L of genomic DNA, 0.5 ⁇ L of the selected restriction enzyme and 3 ⁇ L of restriction enzyme buffer were added to each digestion system (the restriction enzymes used were NEB enzymes and their buffers or universal buffers).
  • NEB T4DNA Ligase Reaction Buffer the specific formula 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 ⁇ L 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.
  • 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.
  • 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, and SEQ ID NO: 15 as the sequencing primer
  • the PCR reaction conditions are shown in Table 3.
  • the obtained amplicons were electrophoresed on a 2.0% agarose gel to separate the PCR reactions, 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 the PCR product provides DNA that can be used to design other DNA molecules that serve as primers and probes for the identification of corn plants or seeds derived from the transgenic maize event DBN9978.
  • the maize genomic sequence shown at positions 1-1374 of nucleotides of SEQ ID NO: 5 is flanking the right border of the transgenic maize event DBN9978 insertion sequence (5' flanking sequence), at nucleotide 8740 of SEQ ID NO: 5.
  • the -9219 position shows the maize genome sequence flanking the left border of the transgenic maize event DBN9978 insertion sequence (3' flanking sequence).
  • the 5' junction sequence is set forth in SEQ ID NO: 1
  • the 3' junction sequence is set forth in SEQ ID NO: 2.
  • 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 DBN9978 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 DBN9978 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.
  • a junction sequence (5' junction region SEQ ID NO: 1, and 3' junction region SEQ ID NO: 2) as a DNA probe or as a DNA primer molecule in a DNA detection method is useful.
  • the ligating sequences SEQ ID NO: 6 and SEQ ID NO: 7 are also new DNA sequences in the transgenic maize event DBN9978, which can also be used as DNA probes or as DNA primer molecules to detect the presence of transgenic maize event DBN9978 DNA.
  • the SEQ ID NO: 6 (positions 1375-1557 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 1-192) spans the t35S transcription termination sequence and the DBN10124 construct DNA sequence.
  • 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 DBN9978.
  • 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 DBN9978.
  • This PCR product comprises SEQ ID NO:3.
  • primer 5 SEQ ID NO: 8
  • primer 6 SEQ ID located in the transgene t35S transcription termination sequence
  • 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 DBN9978.
  • This PCR product comprises SEQ ID NO:4.
  • primer 8 SEQ ID NO: 11
  • 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 DBN9978. 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.
  • thermocycler Mix gently. If there is no cap on the thermocycler, add 1-2 drops of mineral oil above each reaction.
  • 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.
  • primers 5 and 6 when used in the PCR reaction of the transgenic maize event DBN9978 genomic DNA, generated an amplification product of the 1557 bp fragment when used in the untransformed maize genome.
  • primers 7 and 8 SEQ ID NOS: 10 and 11 were generated when used in the PCR reaction of the transgenic maize event DBN9978 genomic DNA.
  • the amplified product of the 672 bp fragment when used in the PCR reaction of untransformed maize genomic DNA and non-DBN9978 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 DBN9978.
  • Primer 9 SEQ ID NO: 12
  • primer 10 SEQ ID NO: 13
  • primer 11 SEQ ID NO: 14
  • 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 DBN9978.
  • the biological sample containing the template DNA contains DNA for diagnosing the presence of the transgenic maize event DBN9978 in the sample.
  • 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 DBN9978. of. These two different amplicons will correspond to the first amplicon derived from the wild-type maize genomic locus and the second amplicon from the presence of the diagnostic transgenic maize event DBN9978 DNA.
  • a maize DNA sample that produces only a single amplicon corresponding to the second amplicon described for the hybrid genome can be diagnostically determined to determine the presence of the transgenic maize event DBN9978 in the sample, and the sample is present in relation to the transgenic maize plant DBN9978
  • the allele corresponding to the inserted DNA is produced by homozygous corn seed.
  • primer pair of the transgenic maize event DBN9978 was used to generate a diagnostic amplicon for the transgenic maize event DBN9978 genomic DNA.
  • 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 NO: 10). And 11) are used in the DNA amplification method described.
  • one of the control primers 12 and 13 (SEQ ID NOS: 22 and 23) for amplifying the maize endogenous gene was included as an intrinsic standard for the reaction conditions.
  • Analysis of the DNA sample of the transgenic maize event DBN9978 DNA should include a positive tissue DNA extract control of the transgenic maize event DBN9978, a negative DNA extract control derived from the non-transgenic maize event DBN9978, and a DNA extraction without template DNA. Negative control of the substance.
  • 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 DBN9978 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 DBN9978 using a suitable primer pair.
  • An extract derived from a maize plant or seed DNA containing the transgenic maize event DBN9978, or a product derived from the transgenic maize event DBN9978, which was generated as a diagnostic amplicon for the transgenic maize event DBN9978 when tested in a DNA amplification method. Can be used as a template for amplification to determine the presence of the transgenic maize event DBN9978.
  • 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 ⁇ -mercaptoethanol, 2.5% w/v polyethylene-pyrrolidone), centrifuge at 4000 rpm 10 minutes (2755g).
  • Extraction Buffer A 0.2 M Tris pH 8.0, 50 mM EDTA, 0.25 M NaCl, 0.1% v/v ⁇ -mercaptoethanol, 2.5% w/v polyethylene-pyrrolidone
  • extraction buffer B 0.2 M Tris pH 8.0, 50 mM EDTA, 0.5 M NaCl, 1% v/v ⁇ -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 ⁇ L of TE.
  • the DNA was incubated with 1 ⁇ L of 30 mg/mL RNAase A for 30 minutes at 37 ° C, centrifuged at 4000 rpm for 5 minutes, and at 0.5 volume of 7.5 M ammonium acetate and 0.54 body.
  • the DNA was precipitated by centrifugation at 14,000 rpm for 10 minutes. After discarding the supernatant, the precipitate was washed with 500 ⁇ L of 70% ethanol, and dried and resuspended in 100 ⁇ L of TE buffer.
  • 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)) .
  • TNE buffer 0.01 M Tris, 0.1 M NaCl, 0.001 M EDTA, pH 7.4
  • Hoechst dye Hoechst 33258, Hoechst AG
  • Genomic DNA was digested each time in a 100 ⁇ L 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 ⁇ L.
  • a vacuum centrifugal evaporation concentrator speedVac, Thermo Scientific
  • 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.
  • 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.
  • 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 ⁇ L 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 ⁇ l 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.
  • 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.
  • Church Wash Solution 1 40 mM Na 3 P0 4 , 1 mM EDTA, 5% SDS, 0.5% BSA
  • 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 DBN9978 Cry1Ab and EPSPS genes.
  • Cry1Ab probe EcoR V and Hind III were digested to generate a single band of approximately 11 kb and 12 kb, respectively; using the EPSPS probe, EcoR V and Hind III were digested to generate a single band of approximately 10 kb and 4 kb, respectively. This indicates that one copy of each of Cry1Ab and EPSPS is present in the maize transformation event DBN9978.
  • LPG Sesamia inferens
  • PSB Mythimna seperata
  • SAB Spodoptera litura
  • SSB Chilo suppressalis
  • CBW Helicoverpa armigera
  • BAW was bioassay as follows:
  • 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 DBN9978 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.
  • transgenic maize event DBN9978 had good resistance to Asian corn borer, peach aphid, larvae, giant salamander, oriental armyworm, Spodoptera litura, stem borer, cotton bollworm and beet armyworm.
  • the test insect mortality and resistance scores of the transgenic maize event DBN9978 were significantly higher than those of the wild-type maize plants.
  • seeds of transgenic maize event DBN9978 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.
  • 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.
  • NMM negative control material
  • the insect is considered invalid.
  • 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.
  • 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 DBN9978 to the Asian corn borer at the silking stage are shown in Table 13.
  • Leaf level Symptom description 1 Only 1-2 holes with a pore size ⁇ 1mm on individual leaves 2 Only 3-6 wormholes with a hole diameter ⁇ 1mm on individual blades 3 A few leaves have more than 7 pores ⁇ 1mm 4 1-2 holes with ⁇ 2mm aperture on individual blades 5 A few leaves have 3-6 wormholes with a pore size ⁇ 2mm 6 Some blades have more than 7 wormholes with a pore size ⁇ 2mm 7 There are 1-2 wormholes with a hole diameter greater than 2mm on a few blades 8 There are 3-6 wormholes with a diameter greater than 2mm on some of the blades. 9 There are more than 7 wormholes with a diameter greater than 2mm on most of the blades.
  • transgenic maize event DBN9978 had a good resistance level to Asian corn borer in both heart leaf stage and silking stage.
  • the average leaf level of DBN9978 in transgenic corn event was significantly lower than that of wild type corn. Plant.
  • the ear damage rate, larval survival number, tunnel length and ear damage level of the transgenic corn event DBN9978 were significantly lower than those of the wild type maize plants.
  • the field effect of transgenic maize event DBN9978 inoculated with Asian corn borer in the heart and silk stage is shown in Figure 3.
  • the experimental design and test method basically evaluate the resistance of Asian corn borer as described above. To. The difference is that only the corn leaf stage (the development of the corn plant to the 4-6 leaf stage) is artificially inoculated, and the insects are harvested twice, and about 20 second-instar larvae are artificially reared in each corn heart. 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 DBN9978 to the oriental armyworm in the heart stage are shown in Table 16.
  • Leaf level Symptom description 1 The leaves are not damaged, or only the needles have a needle-like ( ⁇ 1mm) wormhole 2 Only a small number of bullet holes ( ⁇ 5mm) on individual leaves 3 A few blades have bullet holes ( ⁇ 5mm) wormholes 4 Defects on individual blades ( ⁇ 10mm) 5 A few leaves have nicks ( ⁇ 10mm) 6 Some blades have nicks ( ⁇ 10mm) 7 Individual blade parts are fed, and a few leaves have large nicks ( ⁇ 10mm) 8 A few leaves are fed, and some leaves have large nicks ( ⁇ 10mm) 9 Most of the leaves are fed
  • 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.
  • NNM negative control material
  • transgenic maize event DBN9978 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 DBN9978 were significantly lower than that of wild type maize plants.
  • the field effect of inoculation of cotton bollworm in the transgenic corn event DBN9978 is shown in Figure 5.
  • 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 DBN9978 to Myzus persicae are shown in Table 20.
  • Event DBN9978 implements the method and/or use of controlling pests, specifically the larvae, the peach aphid, the Spodoptera litura, the giant salamander and the oriental armyworm; that is, any transgenic corn 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.
  • 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 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 herbicide tolerance and maize yield results for the transgenic maize event DBN9978 are shown in Table 23.
  • Production of commodities such as agricultural products or commodities can be produced by the genetically modified corn event DBN9978. If sufficient expression levels are detected in the agricultural product or commodity, the agricultural product or commodity is expected to contain nucleotides capable of diagnosing the transgenic maize event DBN9978 material present in the agricultural product or commodity sequence.
  • the agricultural product or commodity includes, but is not limited to, corn oil, corn meal, cornmeal, corn gluten, tortilla, corn starch, and any other food product to be consumed as a food source for the animal, or otherwise as a swelling or cosmetic composition.
  • the ingredients are used for cosmetic purposes 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 DBN9978 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 DBN9978.
  • the transgenic maize event DBN9978 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 DBN9978.
  • the seed corresponding to the transgenic corn event DBN9978 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: corn (Zea mays), deposit number is CGMCC No. 10292. The deposit will be kept at the depository for 30 years.
  • CGMCC China Microbial Culture Collection Management Committee

Abstract

L'invention concerne un événement de maïs transgénique DBN9978. Un plant de maïs comprenant l'événement est résistant à des insectes Lépidoptères et est tolérant à un herbicide au glyphosate. L'invention concerne également une séquence d'acide nucléique utilisée pour détecter dans un échantillon biologique la présence d'un événement de maïs DBN9978 ainsi que son procédé de détection.
PCT/CN2016/077867 2015-04-30 2016-03-30 Plant de maïs dbn9978 et procédé d'utilisation dans la détection de la séquence d'acide nucléique associée WO2016173362A1 (fr)

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CN109971880B (zh) * 2019-04-09 2022-11-04 北京大北农生物技术有限公司 用于检测玉米植物dbn9508的核酸序列及其检测方法
CN112852991B (zh) * 2021-01-27 2023-08-04 隆平生物技术(海南)有限公司 转基因玉米事件lp007-7及其检测方法
CN112852801B (zh) * 2021-01-27 2023-08-08 隆平生物技术(海南)有限公司 转基因玉米事件lp007-1及其检测方法
CN113151533B (zh) * 2021-01-27 2023-08-08 隆平生物技术(海南)有限公司 转基因玉米事件lp007-6及其检测方法
CN113151534B (zh) * 2021-01-27 2023-06-20 隆平生物技术(海南)有限公司 转基因玉米事件lp007-5及其检测方法
CN112831585B (zh) * 2021-01-27 2023-06-16 隆平生物技术(海南)有限公司 转基因玉米事件lp007-4及其检测方法
CN113980958B (zh) * 2021-10-12 2023-08-11 隆平生物技术(海南)有限公司 转基因玉米事件lp007-8及其检测方法
CN116144672B (zh) * 2022-09-23 2023-11-07 隆平生物技术(海南)有限公司 转基因玉米事件lp026-1及其检测方法
CN116144818B (zh) * 2022-09-23 2023-11-07 隆平生物技术(海南)有限公司 转基因玉米事件lp026-2及其检测方法

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