WO2021026689A1 - 用于检测大豆植物dbn8007的核酸序列及其检测方法 - Google Patents

用于检测大豆植物dbn8007的核酸序列及其检测方法 Download PDF

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WO2021026689A1
WO2021026689A1 PCT/CN2019/099996 CN2019099996W WO2021026689A1 WO 2021026689 A1 WO2021026689 A1 WO 2021026689A1 CN 2019099996 W CN2019099996 W CN 2019099996W WO 2021026689 A1 WO2021026689 A1 WO 2021026689A1
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seq
nucleic acid
sequence
dna
soybean
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PCT/CN2019/099996
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French (fr)
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韩超
于彩虹
谢香庭
王登元
杨淑靖
崔广东
康越景
鲍晓明
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北京大北农生物技术有限公司
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Priority to CN201980005159.3A priority Critical patent/CN111247255B/zh
Priority to PCT/CN2019/099996 priority patent/WO2021026689A1/zh
Publication of WO2021026689A1 publication Critical patent/WO2021026689A1/zh

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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • 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
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—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
    • C12N15/8274—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 herbicide resistance
    • C12N15/8277—Phosphinotricin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits

Definitions

  • the invention relates to the field of plant molecular biology, especially the field of genetically modified crop breeding in agricultural biotechnology research. Specifically, the present invention relates to insect-resistant and glufosinate herbicide-tolerant transgenic soybean event DBN8007 and a nucleic acid sequence for detecting whether a biological sample contains a specific transgenic soybean event DBN8007 and a detection method thereof.
  • Soybean (Glycine max) is one of the five main crops in the world. Biotechnology has been applied to soybean to improve its agronomic traits and quality. Herbicide tolerance is an important agronomic trait in soybean production, especially glyphosate herbicide tolerance. For example, there have been successful soybean events GTS40-3-2, MON89788, and major soybean planting areas such as the United States have been widely used. Planted. Another important agronomic trait is insect resistance, especially resistance to lepidopteran insects. For example, the successful soybean event MON87701 is widely planted in major soybean growing areas such as Brazil. It is worth mentioning that the mechanism of action of Vip protein is different from Cry protein.
  • Soybean resistance to lepidopteran insects can be obtained by expressing the resistance genes of lepidopteran insects in soybean plants by transgenic methods.
  • the mechanism of action of the glufosinate herbicide is different from that of the glyphosate herbicide.
  • the tolerance of soybeans to glufosinate herbicides can be obtained by expressing glufosinate herbicide tolerance genes (such as PAT) in soybean plants by transgenic methods.
  • Vip3Aa gene and PAT gene exogenous functional genes
  • the expression level of introduced genes may vary greatly between events; there may also be differences in the spatial or temporal patterns of expression, such as the relative expression of transgenes between different plant tissues There are differences, and this difference is manifested in that the actual expression pattern may be inconsistent with the expected expression pattern based on the transcription regulatory elements in the introduced gene construct. Therefore, it is usually necessary to generate hundreds or thousands of different events and screen out a single event with the expected transgene expression level and expression pattern for commercialization purposes. Events with expected transgene expression levels and expression patterns can be used to introduce transgenes into other genetic backgrounds through sexual outcrossing using conventional breeding methods. The offspring produced by this hybridization method retain the transgene expression characteristics of the original transformant. Using this strategy model can ensure reliable gene expression in many varieties, and these varieties can well adapt to local growth conditions.
  • flanking DNA a pair of primers spanning the junction of the inserted transgene and flanking DNA are often used to identify specific transgene events by PCR, specifically the first primer contained in the inserted sequence and the second primer contained in the inserted sequence.
  • the purpose of the present invention is to provide a nucleic acid sequence for detecting soybean plant DBN8007 and a detection method thereof.
  • the transgenic soybean event DBN8007 has good resistance to insects and has good tolerance to glufosinate herbicides, and
  • the detection method can accurately and quickly identify whether the biological sample contains the DNA molecule of the genetically modified soybean event DBN8007.
  • the present invention provides a nucleic acid sequence having at least 11 consecutive nucleotides in positions 1-553 of SEQ ID NO: 3 or its complementary sequence and 554th of SEQ ID NO: 3 or its complementary sequence. At least 11 consecutive nucleotides in position -1407, and/or at least 11 consecutive nucleotides in position 1-348 of SEQ ID NO: 4 or its complement, and at least 11 consecutive nucleotides of SEQ ID NO: 4 or its complement. At least 11 consecutive nucleotides in positions 349-1022.
  • the nucleic acid sequence has 22-25 contiguous nucleotides in positions 1-553 of SEQ ID NO: 3 or its complementary sequence, and 22-25 in positions 554-1407 of SEQ ID NO: 3 or its complementary sequence. 25 consecutive nucleotides, and/or SEQ ID NO: 4 or its complementary sequence, 22-25 consecutive nucleotides from positions 1-348 and SEQ ID NO: 4 or its complementary sequence, positions 349-1022 22-25 consecutive nucleotides in the middle.
  • the nucleic acid sequence comprises SEQ ID NO: 1 or its complementary sequence, and/or SEQ ID NO: 2 or its complementary sequence.
  • the SEQ ID NO: 1 or its complementary sequence is a 22-nucleotide sequence near the insertion junction at the 5'end of the inserted sequence in the transgenic soybean event DBN8007, the SEQ ID NO: 1 or its The complementary sequence spanning the flanking genomic DNA sequence of the soybean insertion site and the DNA sequence at the 5'end of the insertion sequence, including the SEQ ID NO:1 or its complementary sequence, can be identified as the existence of transgenic soybean event DBN8007.
  • the SEQ ID NO: 2 or its complementary sequence is a 22-nucleotide sequence near the insertion junction at the 3'end of the insertion sequence in the transgenic soybean event DBN8007, the SEQ ID NO: 2 or its The complementary sequence spans the DNA sequence at the 3'end of the insertion sequence and the flanking genomic DNA sequence of the soybean insertion site, and includes the SEQ ID NO: 2 or its complementary sequence to be identified as the existence of the transgenic soybean event DBN8007.
  • the nucleic acid sequence comprises SEQ ID NO: 3 or its complementary sequence, and/or SEQ ID NO: 4 or its complementary sequence.
  • the nucleic acid sequence may be at least 11 or more consecutive polynucleotides (first nucleic acid sequence) of any part of the T-DNA insertion sequence in the SEQ ID NO: 3 or its complementary sequence, or It is at least 11 or more consecutive polynucleotides (second nucleic acid sequence) of any part of the 5'flanking soybean genomic DNA region in the SEQ ID NO: 3 or its complementary sequence.
  • the nucleic acid sequence may further be homologous or complementary to a part of the SEQ ID NO: 3 including the complete SEQ ID NO: 1.
  • these nucleic acid sequences can be used as a pair of DNA primers in a DNA amplification method for generating amplification products.
  • the amplified product generated in the DNA amplification method using the DNA primer pair is an amplified product including SEQ ID NO: 1, the presence of the transgenic soybean event DBN8007 or its progeny can be diagnosed.
  • the SEQ ID NO: 3 or its complementary sequence is a sequence of 1407 nucleotides in length near the insertion junction at the 5'end of the T-DNA insertion sequence in the transgenic soybean event DBN8007.
  • the SEQ ID NO: 3 or its complementary sequence consists of the 553 nucleotide 5'flanking sequence of soybean genome (SEQ ID NO: 3 nucleotides 1-553), 356 nucleotides in the DNA sequence of pDBN4006 construct (SEQ ID No: 3 nucleotides 554-909) and 498 nucleotides prAtAct2 transcription initiation sequence (SEQ ID NO: 3 nucleotides 910-1407), including the SEQ ID NO: 3 or its complementary sequence can be identified as the existence of transgenic soybean event DBN8007.
  • the nucleic acid sequence may be at least 11 or more consecutive polynucleotides (third nucleic acid sequence) of any part of the T-DNA insertion sequence in the SEQ ID NO: 4 or its complementary sequence, or the SEQ ID NO: 4 or at least 11 consecutive polynucleotides (fourth nucleic acid sequence) of any part of the 3'flanking soybean genomic DNA region in 4 or its complementary sequence.
  • the nucleic acid sequence may further be homologous to or complementary to a part of the SEQ ID NO: 4 including the complete SEQ ID NO: 2.
  • these nucleic acid sequences can be used as a pair of DNA primers in a DNA amplification method for generating amplification products.
  • the amplified product generated in the DNA amplification method using the DNA primer pair is the amplified product including SEQ ID NO: 2, the presence of the transgenic soybean event DBN8007 or its progeny can be diagnosed.
  • the SEQ ID NO: 4 or its complementary sequence is a sequence of 1022 nucleotides in length near the T-DNA insertion junction at the 3'end of the inserted sequence in the transgenic soybean event DBN8007.
  • the SEQ ID NO: 4 or its complementary sequence consists of the 145-nucleotide DNA sequence of the t35S transcription terminator (SEQ ID NO: 4 nucleotides 1-145), and the 203 nucleotides in the DNA sequence of the pDBN4006 construct (SEQ ID NO: ID NO: 4 nucleotides 146-348) and 674 nucleotides of the soybean genome 3'flanking sequence (SEQ ID NO: 4 nucleotides 349-1022), including the SEQ ID NO: 4 or its complementary sequence can be identified as the presence of the transgenic soybean event DBN8007.
  • nucleic acid sequence includes SEQ ID NO: 5 or its complementary sequence.
  • the SEQ ID NO: 5 or its complementary sequence is a 11935 nucleotide sequence characterizing the transgenic soybean event DBN8007, and the specific genome and genetic elements contained therein are shown in Table 1. The inclusion of the SEQ ID NO: 5 or its complementary sequence can be identified as the presence of the transgenic soybean event DBN8007.
  • the first, second, third, and fourth nucleic acid sequences need not be composed of only DNA, but may also include a mixture of RNA, DNA, and RNA, or DNA, RNA, or other types of A combination of nucleotides or analogs of the polymerase template.
  • the probes or primers in the present invention should be at least about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 consecutive nucleotides in length, which can be selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5.
  • the probes and primers may be contiguous with a length of at least about 21 to about 50 or more. Nucleotides.
  • the nucleic acid sequence or its complementary sequence can be used in a DNA amplification method to generate amplicons for detecting the presence of the transgenic soybean event DBN8007 or its progeny in a biological sample; the nucleic acid sequence or its complementary sequence It can be used in nucleotide detection methods to detect the presence of transgenic soybean event DBN8007 or its descendants in biological samples.
  • the present invention also provides a method for detecting the presence of DNA of the transgenic soybean event DBN8007 in a sample, including:
  • the target amplification product includes the nucleic acid sequence.
  • the target amplification product comprises SEQ ID NO: 1 or its complementary sequence, SEQ ID NO: 2 or its complementary sequence, SEQ ID NO: 6 or its complementary sequence, and/or SEQ ID NO: 7 or its complementary sequence.
  • SEQ ID NO: 1 or its complementary sequence SEQ ID NO: 1 or its complementary sequence
  • SEQ ID NO: 2 or its complementary sequence SEQ ID NO: 6 or its complementary sequence
  • SEQ ID NO: 7 SEQ ID NO: 7 or its complementary sequence.
  • the primer includes a first primer and a second primer
  • the first primer is selected from SEQ ID NO: 1, SEQ ID NO: 8 and SEQ ID NO: 10
  • the second primer is selected from SEQ ID NO : 2, SEQ ID NO: 9 and SEQ ID NO: 11.
  • the present invention also provides a method for detecting the presence of DNA of the transgenic soybean event DBN8007 in a sample, including:
  • the probe comprising the nucleic acid sequence
  • the hybridization between the sample to be detected and the probe is detected.
  • the stringent conditions can be in 6 ⁇ SSC (sodium citrate), 0.5% SDS (sodium dodecyl sulfate) solution, hybridization at 65°C, and then 2 ⁇ SSC, 0.1% SDS and 1 ⁇ SSC, 0.1% SDS each wash the membrane once.
  • 6 ⁇ SSC sodium citrate
  • SDS sodium dodecyl sulfate
  • the probe comprises SEQ ID NO: 1 or its complementary sequence, SEQ ID NO: 2 or its complementary sequence, SEQ ID NO: 6 or its complementary sequence, and/or SEQ ID NO: 7 or its complementary sequence .
  • At least one of the probes is labeled with at least one fluorescent group.
  • the present invention also provides a method for detecting the presence of DNA of the transgenic soybean event DBN8007 in a sample, including:
  • the marker nucleic acid molecule including the nucleic acid sequence
  • the hybridization between the sample to be detected and the marker nucleic acid molecule is detected, and then the marker-assisted breeding analysis is used to determine that insect resistance and/or herbicide tolerance are genetically linked with the marker nucleic acid molecule.
  • the marker nucleic acid molecule includes at least one selected from: SEQ ID NO: 1 or its complement, SEQ ID NO: 2 or its complement, and/or SEQ ID NO: 6-11 or its complement Complementary sequence.
  • the present invention also provides a DNA detection kit, comprising at least one DNA molecule containing the nucleic acid sequence, which can be used as a DNA primer specific to the transgenic soybean event DBN8007 or its progeny One or probe.
  • the DNA molecule comprises SEQ ID NO: 1 or its complementary sequence, SEQ ID NO: 2 or its complementary sequence, SEQ ID NO: 6 or its complementary sequence, and/or SEQ ID NO: 7 or its complementary sequence .
  • the present invention also provides a plant cell comprising a nucleic acid sequence encoding an insect-resistant Vip3Aa protein, a nucleic acid sequence encoding a glufosinate herbicide-tolerant PAT protein, and a nucleic acid sequence of a specific region.
  • the nucleic acid sequence of the region includes the sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 6, and/or SEQ ID NO: 7.
  • the plant cell comprises a nucleic acid sequence encoding an insect resistance Vip3Aa protein, a nucleic acid sequence encoding a glufosinate herbicide tolerance PAT protein, and a nucleic acid sequence of a specific region, and the nucleic acid sequence of the specific region includes SEQ ID NO : 3 and/or the sequence shown in SEQ ID NO: 4.
  • the plant cell includes SEQ ID NO:1, SEQ ID NO: 5, 5594-11006 nucleic acid sequence and SEQ ID NO: 2, or the sequence shown in SEQ ID NO: 5.
  • the present invention also provides a method for protecting soybean plants from insect invasion, including providing at least one transgenic soybean plant cell in the diet of the target insect, the transgenic soybean plant cell comprising SEQ in its genome. ID NO: 1 and/or SEQ ID NO: 2, the target insect ingesting the transgenic soybean plant cell is inhibited from further ingesting the transgenic soybean plant.
  • the transgenic soybean plant cell contains the sequence shown in SEQ ID NO: 3 and/or SEQ ID NO: 4 in its genome.
  • the transgenic soybean plant cell sequentially includes SEQ ID NO:1, SEQ ID NO: 5, and SEQ ID NO: 2 or SEQ ID NO: 5 in sequence.
  • the present invention also provides a method for protecting soybean plants from damage caused by herbicides or controlling weeds in the field where soybean plants are grown, including applying an effective dose of glufosinate-ammonium herbicide to at least In a field of transgenic soybean plants, the transgenic soybean plant contains the sequence shown in SEQ ID NO: 1 and/or SEQ ID NO: 2 in its genome, and the transgenic soybean plant is resistant to glufosinate herbicide Acceptability.
  • the transgenic soybean plant contains the sequence shown in SEQ ID NO: 3 and/or SEQ ID NO: 4 in its genome.
  • the transgenic soybean plant sequentially includes SEQ ID NO: 1, SEQ ID NO: 5, the 5594-11006 nucleic acid sequence and SEQ ID NO: 2, or the sequence shown in SEQ ID NO: 5 .
  • the present invention also provides a method for cultivating soybean plants resistant to insects and/or tolerant to glufosinate-ammonium herbicide, which includes:
  • Planting at least one soybean seed the genome of the soybean seed comprising a nucleic acid sequence encoding an insect-resistant Vip3Aa protein and/or a nucleic acid sequence encoding a glufosinate herbicide-tolerant PAT protein, and a nucleic acid sequence of a specific region, or
  • the genome of the soybean seed includes the nucleic acid sequence shown in SEQ ID NO: 5;
  • the nucleic acid sequence of the specific region is SEQ ID NO:1 and/or SEQ ID NO: 2; preferably, the nucleic acid sequence of the specific region is SEQ ID NO: 3 and/or SEQ ID NO: 4 The sequence shown.
  • the present invention also provides a method for producing soybean plants resistant to insects and/or tolerant to glufosinate-ammonium herbicides, which comprises combining the encoding insects contained in the first soybean plant genome
  • the nucleic acid sequence of the specific region is SEQ ID NO: 1 and/or SEQ ID NO: 2; preferably, the nucleic acid sequence of the specific region is SEQ ID NO: 3 and/ Or the sequence shown in SEQ ID NO: 4;
  • the method includes sexually crossing the transgenic soybean event DBN8007 with soybean plants lacking insect resistance and/or glufosinate-ammonium tolerance, thereby generating a large number of progeny plants, and selecting a nucleic acid sequence having the specific region
  • progeny plants that are resistant to insects and/or tolerant to glufosinate-ammonium herbicide are selected.
  • the present invention also provides an agricultural product or commodity produced from the transgenic soybean event DBN8007, the agricultural product or commodity is lecithin, fatty acid, glycerin, sterol, soybean flakes, soybean meal, soybean protein or its concentrate Food, soybean oil, soybean protein fiber, soy milk curd or tofu.
  • soybeans (Glycine max), and includes all plant varieties that can mate with soybeans, including wild soybean 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 whole plants in plants or plant parts Cells, the plant parts such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, stems, roots, root tips, anthers and the like. It should be understood that the parts of transgenic plants within the scope of the present invention include but are not limited to plant cells, protoplasts, tissues, callus, embryos and flowers, stems, fruits, leaves and roots.
  • the above plant parts are derived from previously used A transgenic plant or its progeny transformed by a DNA molecule and thus at least partially composed of transgenic cells.
  • gene refers to a nucleic acid fragment that expresses a specific protein, including a regulatory sequence before the coding sequence (5' non-coding sequence) and a regulatory sequence after the coding sequence (3' non-coding sequence).
  • Native gene refers to a gene that is naturally found to have its own regulatory sequence.
  • Chimeric gene refers to any gene that is not a natural gene, which contains regulatory and coding sequences not found in nature.
  • Endogenous gene refers to a natural gene that is located in its natural location in the genome of an organism.
  • Formeign gene refers to a foreign gene that exists in the genome of an organism and does not originally exist, and also refers to a gene that is introduced into the recipient cell through a transgene step.
  • the foreign gene may include a natural gene inserted into a non-natural organism or a chimeric gene.
  • a "transgene” is a gene that has been introduced into the genome through a transformation procedure.
  • the site in the plant genome where the recombinant DNA has been inserted can be referred to as the "insertion site” or "target site”.
  • flanking DNA may include a genome naturally present in an organism such as a plant or foreign (heterologous) DNA introduced through a transformation process, such as a fragment associated with a transformation event. Therefore, the flanking DNA may include a combination of natural and foreign DNA.
  • flanking DNA is also called “flanking region” or “flanking sequence” or “flanking genomic sequence” or “flanking genomic DNA”, which means at least 3, 5, 10, 11, 15, 20, 50, A sequence of 100, 200, 300, 400, 1000, 1500, 2000, 2500, or 5000 base pairs or longer, which is located directly upstream or downstream of the original foreign inserted DNA molecule and adjacent to the original foreign inserted DNA molecule.
  • flanking area When the flanking area is located downstream, it can also be called “3' flanking” or “left boundary flanking” or the like. When the flanking area is located upstream, it can also be referred to as “5' flanking” or “right boundary flanking” or the like.
  • a transformation procedure that causes random integration of foreign DNA will result in transformants containing different flanking regions that are specifically contained in each transformant.
  • Transformants will also contain unique junctions between segments of heterologous insert DNA and genomic DNA or between two segments of genomic DNA or between two segments of heterologous DNA.
  • Junction is the point where two specific DNA fragments join. For example, junctions exist where the insert DNA joins the flanking DNA. Junctions are also present in transformed organisms, where two DNA fragments are joined together in a manner modified from those found in natural organisms.
  • a "junction region” or “junction sequence” refers to DNA containing junctions.
  • the present invention provides a transgenic soybean event called DBN8007 and its progeny.
  • the transgenic soybean event DBN8007 is also called soybean plant DBN8007, which includes plants and seeds of the transgenic soybean event DBN8007 and plant cells or reproducible parts thereof.
  • the plant parts of the transgenic soybean event DBN8007 including but not limited to cells, pollen, ovules, flowers, buds, roots, stems, leaves, pods, and products from the soybean plant DBN8007, such as soybean cake, flour and oil, which can be specifically lecithin , Fatty acids, glycerin, sterols, edible oil, defatted soy flakes, including defatted and roasted soy flour, soy milk curd, tofu, soy protein concentrate, isolated soy protein, hydrolyzed vegetable protein, textured soy protein and Soy protein fiber.
  • the transgenic soybean event DBN8007 of the present invention contains a DNA construct, and when it is expressed in plant cells, the transgenic soybean event DBN8007 acquires resistance to insects and tolerance to glufosinate herbicides.
  • the DNA construct comprises two expression cassettes in tandem.
  • the first expression cassette comprises a suitable promoter for expression in plants and a suitable polyadenylation signal sequence, the promoter being operably connected to Vip3Aa
  • the nucleic acid sequence of the protein, the nucleic acid sequence of the Vip3Aa protein is mainly resistant to lepidopteran insects.
  • the second expression cassette contains a suitable promoter for expression in plants and a suitable polyadenylation signal sequence, the promoter operably linked to encode phosphinothricin N-acetyltransferase (phosphinothricin N -acetyltransferase (PAT) gene, the nucleic acid sequence of the PAT protein is tolerant to glufosinate-ammonium herbicide.
  • phosphinothricin N-acetyltransferase phosphinothricin N -acetyltransferase (PAT) gene
  • the promoter may be a suitable promoter isolated from a plant, including constitutive, inducible and/or tissue-specific promoters, and the suitable promoters include, but are not limited to, cauliflower mosaic virus (CaMV) 35S Promoters, Scrophularia mosaic virus (FMV) 35S promoter, Ubiquitin (Ubiquitin) promoter, Actin (Actin) promoter, Agrobacterium (tumefaciens) nopaline synthase (NOS) promoter, Octopine synthase (OCS) promoter, Cestrum yellow leaf curl virus promoter, potato tuber storage protein (Patatin) promoter, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) promoter, glutathione sulfur transferase (GST) promoter, E9 promoter, GOS promoter, alcA/alcR promoter, Agrobacterium rhizogenes RolD promoter, and Arabidops
  • the polyadenylation signal sequence may be a suitable polyadenylation signal sequence that functions in plants, and the suitable polyadenylation signal sequence includes, but is not limited to, derived from Agrobacterium tumefaciens (Agrobacterium tumefaciens)
  • the expression cassette may also include other genetic elements including, but not limited to, enhancers and signal peptides/transit peptides.
  • the enhancer can enhance the expression level of the gene, and the enhancer includes, but is not limited to, tobacco etching virus (TEV) translation activator, CaMV35S enhancer, and FMV35S enhancer.
  • the signal peptide/transit peptide can guide the Vip3Aa protein and/or PAT protein to be transported to specific organelles or compartments outside the cell or within the cell, for example, using a sequence encoding a chloroplast transit peptide to target the chloroplast, or using'KDEL' to retain sequence targets To the endoplasmic reticulum.
  • the Vip3Aa gene can be isolated from Bacillus thuringiensis (Bt for short), and the nucleotide sequence of the Vip3Aa gene can be changed by optimizing the codon or in other ways to increase the transcript in the transformed cell.
  • Bt Bacillus thuringiensis
  • the "Lepidoptera (Lepidoptera)” includes two types of insects, moths and butterflies, and is the order with the most agricultural and forestry pests, such as cutworms, cotton bollworm, Spodoptera litura, Spodoptera litura, Peach borer, etc.
  • the phosphinothricin N-acetyltransferase (PAT) gene may be an enzyme isolated from a strain of Streptomyces viridochromogenes, which catalyzes the conversion of L-phosphinothricin into its inactive form through acetylation to give plants Tolerance to glufosinate herbicide.
  • Phosphinothricin PTC, 2-amino-4-methylphosphonobutyric acid
  • PTC is the structural unit of the antibiotic 2-amino-4-methylphosphonyl-alanyl-alanine.
  • This tripeptide has anti-Gram-positive and Gram-negative bacteria and anti-fungal Botrytis cinerea (Botrytis cinerea) activity.
  • the phosphinothricin N-acetyltransferase (PAT) gene can also be used as a selectable marker gene.
  • glufosinate-ammonium is also known as glufosinate, which refers to ammonium 2-amino-4-[hydroxy(methyl)phosphono]butyrate.
  • Treatment with "glufosinate-ammonium herbicide” means using any kind of The herbicide formulation of glufosinate-ammonium was treated. In order to achieve an effective biological dose, the choice of the use rate of a certain glufosinate-ammonium formulation does not exceed the skills of ordinary agronomic technicians.
  • any herbicide formulation containing glufosinate to treat a field containing plant material derived from transgenic soybean event DBN8007 will control the growth of weeds in the field without affecting the plant material derived from transgenic soybean event DBN8007 Growth or yield.
  • the DNA construct is introduced into the plant using transformation methods, which include, but are not limited to, Agrobacterium-mediated transformation, gene gun transformation, and pollen tube passage transformation.
  • the Agrobacterium-mediated transformation method is a common method for plant transformation.
  • the foreign DNA to be introduced into the plant is cloned between the consensus sequence on the left and right borders of the vector, that is, the T-DNA region.
  • the vector is transformed into Agrobacterium cells, and subsequently, the Agrobacterium cells are used to infect plant tissues, and the T-DNA region of the vector containing foreign DNA is inserted into the plant genome.
  • the gene gun transformation method is to bombard plant cells with vectors containing foreign DNA (particle-mediated biological bombardment transformation).
  • the pollen tube channel transformation method utilizes the natural pollen tube channel (also known as pollen tube guiding tissue) formed after plant pollination to carry the foreign DNA into the embryo sac through the nucleus channel.
  • transgenic plant After transformation, a transgenic plant must be regenerated from the transformed plant tissue, and the offspring with foreign DNA must be selected using appropriate markers.
  • a DNA construct is a combination of DNA molecules connected to each other, and the combination provides one or more expression cassettes.
  • the DNA construct is preferably a plasmid capable of self-replication in bacterial cells and contains different restriction endonuclease sites.
  • the restriction endonuclease sites contained are used to introduce functional genetic elements, namely 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 the transcription of messenger RNA, and the expression cassette can be designed to be expressed in prokaryotic cells or eukaryotic cells.
  • the expression cassette of the present invention is designed to be most preferably expressed in plant cells.
  • the transgenic "event” is obtained by transforming plant cells with a heterologous DNA construct, that is, including at least one nucleic acid expression cassette containing the target gene, inserted into the plant genome by the method of transgene to produce a plant population, and regenerate the plant population , And select specific plants that have the characteristics of inserting specific genomic sites.
  • the term “event” refers to the original transformant containing heterologous DNA and the progeny of the transformant.
  • the term “event” also refers to the offspring obtained by sexual crosses between the original transformant and other species containing heterologous DNA, even after repeated backcrossing with the backcrossed parent, the inserted DNA from the original transformant parent And flanking genomic DNA also exists in the same chromosomal position in the hybrid offspring.
  • event also refers to the DNA sequence from the original transformant, the DNA sequence comprising the insert DNA and the flanking genomic sequence closely adjacent to the insert DNA, the DNA sequence is expected to be transferred to the progeny, the progeny containing the insert DNA
  • the parental line (for example, the original transformant and its self-produced progeny) is produced by sexually crossing the parental line that does not contain the inserted DNA, and the progeny receives the inserted DNA containing the target gene.
  • recombinant refers to the form of DNA and/or protein and/or organism that cannot normally be found in nature and is therefore produced through artificial intervention. Such artificial intervention can produce recombinant DNA molecules and/or recombinant plants.
  • the "recombinant DNA molecule” is obtained by artificially combining two sequence segments that are otherwise separated, for example, by chemical synthesis or manipulation of isolated nucleic acid segments by genetic engineering techniques. The techniques for performing nucleic acid manipulations are well known.
  • transgenic includes any cell, cell line, callus, tissue, plant part or plant whose genotype has been changed due to the presence of heterologous nucleic acid.
  • the original transgenic body is an individual progeny produced by sexual cross or asexual reproduction.
  • the term “transgenic” does not include genomic (chromosomal or extrachromosomal) changes through conventional plant breeding methods or naturally occurring events such as random allogeneic fertilization, non-recombinant viral infection, non-recombinant Bacterial transformation, non-recombinant transposition or spontaneous mutation.
  • heterologous means that the first molecule in nature is usually not found in combination with the second molecule.
  • the molecule can be derived from a first species and inserted into the genome of a second species. Therefore this molecule is heterologous to the host and is artificially introduced into the genome of the host cell.
  • the transgenic soybean event DBN8007 which is resistant to lepidopteran insects and tolerant to glufosinate herbicide, through the following steps: first, the first parent soybean plant and the second parent soybean plant are sexually crossed, thereby producing Diverse first generation progeny plants, the first parent soybean plant is composed of soybean plants cultivated from the transgenic soybean event DBN8007 and its progeny, the transgenic soybean event DBN8007 and its progeny are obtained by using the lepidopteran insects of the present invention
  • the second parent soybean plant lacks resistance to lepidopteran insects and/or is tolerant to glufosinate herbicide through transformation of an expression cassette that is resistant and tolerant to glufosinate-ammonium herbicide.
  • These steps may further include backcrossing lepidopteran insect-resistant and/or glufosinate-tolerant progeny plants with the second parent soybean plant or the third parent soybean plant, and then by attacking with lepidopteran insects, Glufosinate herbicide application or identification of trait-related molecular markers (such as DNA molecules containing junction sites identified at the 5'end and 3'end of the inserted sequence in transgenic soybean event DBN8007) to select progeny, This results in soybean plants that are resistant to lepidopteran insects and tolerant to glufosinate herbicides.
  • trait-related molecular markers such as DNA molecules containing junction sites identified at the 5'end and 3'end of the inserted sequence in transgenic soybean event DBN8007
  • transgenic plants can also be mated to produce offspring that contain two independent, separate added exogenous genes. Selfing of appropriate offspring can produce offspring plants that are homozygous for the two added foreign genes.
  • the backcrossing of parent plants and outcrossing with non-transgenic plants as described above can also be expected, and the same is true for asexual reproduction.
  • probe is an isolated nucleic acid molecule to which a conventional detectable label or reporter molecule, such as a radioisotope, ligand, chemiluminescent agent, or enzyme, is bound.
  • 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 soybean event DBN8007, regardless of whether the genomic DNA is derived from the transgenic soybean event DBN8007 or seeds or from a transgene Plant or seed or extract of soybean event DBN8007.
  • the probe of the present invention not only includes deoxyribonucleic acid or ribonucleic acid, but also includes polyamide and other probe materials that specifically bind to the target DNA sequence and can be used to detect the presence of the target DNA sequence.
  • primer is a segment of isolated nucleic acid molecule that binds to a complementary target DNA strand through nucleic acid hybridization, annealing, and forms a hybrid between the primer and the target DNA strand, and then acts on the polymerase (such as DNA polymerase) Down, extend along the target DNA strand.
  • the primer pair of the present invention relates to its application in target nucleic acid sequence amplification, for example, by polymerase chain reaction (PCR) or other conventional nucleic acid amplification methods.
  • the length of the probes and primers is generally 11 polynucleotides or more, preferably 18 polynucleotides or more, more preferably 24 polynucleotides or more, and most preferably 30 polynucleotides. Acid or more.
  • Such probes and primers specifically hybridize to the target sequence under highly stringent hybridization conditions.
  • probes that are different from the target DNA sequence and maintain the ability to hybridize to the target DNA sequence can be designed by conventional methods, it is preferred that the probe and primer of the present invention have a complete DNA sequence with the continuous nucleic acid of the target sequence. Identity.
  • the primers and probes based on the flanking genomic DNA and insert sequence of the present invention can be determined by conventional methods, for example, by isolating the corresponding DNA molecule from the plant material derived from transgenic soybean event DBN8007, and determining the nucleic acid sequence of the DNA molecule.
  • the DNA molecule includes a transgene insert sequence and a soybean genome flanking sequence, and fragments of the DNA molecule can be used as primers or probes.
  • nucleic acid probes and primers of the present invention hybridize with target DNA sequences under stringent conditions. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of DNA from the transgenic soybean event DBN8007 in the sample. Nucleic acid molecules or fragments thereof can specifically hybridize with other nucleic acid molecules under certain circumstances. As used in the present invention, if two nucleic acid molecules can form an anti-parallel double-stranded nucleic acid structure, it can be said that the two nucleic acid molecules can specifically hybridize with each other. If two nucleic acid molecules show 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 the corresponding nucleotide of another nucleic acid molecule, it is said that the two nucleic acid molecules show "complete complementarity". If two nucleic acid molecules can hybridize to each other with sufficient stability so that they anneal and bind to each other under at least conventional "low stringency” conditions, the two nucleic acid molecules are said to be “minimally complementary”. Similarly, if two nucleic acid molecules can hybridize to each other with sufficient stability so that they anneal and bind to each other under conventional "highly stringent” conditions, the two nucleic acid molecules are said to have "complementarity".
  • Deviation from complete complementarity is permissible, as long as the deviation does not completely prevent the two molecules from forming a double-stranded structure.
  • a nucleic acid molecule In order for a nucleic acid molecule to be used as a primer or probe, it is only necessary to ensure that it has sufficient complementarity in sequence so that a stable double-stranded structure can be formed under the specific solvent and salt concentration used.
  • a substantially homologous sequence is a nucleic acid molecule that can specifically hybridize with the complementary strand of another matched nucleic acid molecule under highly stringent conditions.
  • Suitable stringent conditions to promote DNA hybridization for example, treatment with 6.0 ⁇ sodium chloride/sodium citrate (SSC) at approximately 45° C., and then washing with 2.0 ⁇ SSC at 50° C.
  • SSC sodium chloride/sodium citrate
  • the salt concentration in the washing step can be selected from about 2.0 ⁇ SSC, 50°C under low stringency conditions to about 0.2 ⁇ SSC, 50°C under high stringency conditions.
  • a nucleic acid molecule of the present invention can be under moderately stringent conditions, such as at about 2.0 ⁇ 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.
  • SEQ ID NO: 5 SEQ ID NO: 6 and SEQ ID NO: 7 or their complementary sequences, or any fragments of the foregoing sequences specifically hybridize.
  • a nucleic acid molecule of the present invention is compatible with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: under highly stringent conditions. 6 and SEQ ID NO: One or more nucleic acid molecules or their complementary sequences, or any fragment of the above sequence, specifically hybridize.
  • preferred marker nucleic acid molecules have SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 6, or SEQ ID NO: 7 or their complementary sequence, or any fragment of the foregoing sequence.
  • Another preferred marker nucleic acid molecule of the present invention is compatible with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 6 or SEQ ID NO: 7 or its complementary sequence, or any fragment of the above sequence has 80% to 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 the offspring of genetic crosses.
  • the hybridization between the probe and the target DNA molecule can be detected by any method well known to those skilled in the art, and these methods include, but are not limited to, fluorescent labeling, radioactive labeling, antibody labeling and chemiluminescent labeling.
  • “stringent conditions” refer to conditions that allow only the primers to hybridize to the target nucleic acid sequence in the DNA thermal amplification reaction, and have The primer of the wild-type sequence (or its complementary sequence) corresponding to the target nucleic acid sequence can bind to the target nucleic acid sequence, and preferably produces a unique amplification product, which is an amplicon.
  • target sequence means that the probe or primer only hybridizes to the target sequence in the sample containing the target sequence under stringent hybridization conditions.
  • amplicon refers to a nucleic acid amplification product of a target nucleic acid sequence that is part of a nucleic acid template.
  • amplicon refers to a nucleic acid amplification product of a target nucleic acid sequence that is part of a nucleic acid template.
  • DNA extracted from soybean plant tissue samples or extracts can be used to generate a diagnostic amplicon for the presence of DNA of transgenic soybean event DBN8007 by a nucleic acid amplification method using primer pairs.
  • the primer pair includes a first primer derived from a flanking sequence adjacent to the insertion site of the inserted foreign DNA in the plant genome, and a second primer derived from the inserted foreign DNA.
  • the amplicon has a certain length and sequence, which is also diagnostic for the transgenic soybean event DBN8007.
  • the length of the amplicon can be the combined length of the primer pair plus one nucleotide base pair, preferably plus about 50 nucleotide base pairs, more preferably plus about 250 nucleotide base pairs, Most preferably, about 450 nucleotide base pairs or more are added.
  • the primer pair may be derived from the flanking genomic sequence on both sides of the inserted DNA to generate an amplicon that includes the entire inserted nucleotide sequence.
  • One of the primer pairs derived from the plant genome sequence may be located at a certain distance from the inserted DNA sequence, and the distance may 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 achieved by any nucleic acid amplification reaction method 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 genomic DNA up to 22kb and phage DNA up to 42kb. These methods and other DNA amplification methods in the art can be used in the present invention.
  • the inserted foreign DNA sequence and the flanking DNA sequence from the transgenic soybean event DBN8007 can be amplified by using the provided primer sequences to amplify the genome of the transgenic soybean event DBN8007. After amplification, the PCR amplicon or cloned DNA can be standardized. DNA sequencing.
  • DNA detection kits based on DNA amplification methods contain DNA molecules used as primers, which specifically hybridize to target DNA under appropriate reaction conditions and amplify diagnostic amplicons.
  • the kit can provide an agarose gel-based detection method or many methods known in the art for detecting diagnostic amplicons.
  • a kit containing DNA primers homologous or complementary to any part of the soybean genome of SEQ ID NO: 3 or SEQ ID NO: 4, and homologous or complementary to any part of the transgene insertion region of SEQ ID NO: 5 is Provided by the present invention.
  • the primer pairs that are useful in DNA amplification methods are SEQ ID NO: 8 and SEQ ID NO: 9, which amplify a diagnostic amplification that is homologous to a part of the 5'transgene/genomic region of the transgenic soybean event DBN8007
  • the amplicon includes SEQ ID NO:1.
  • Other DNA molecules used as DNA primers can be selected from SEQ ID NO: 5.
  • the amplicons produced by these methods can be detected by a variety of techniques.
  • One of the methods is Genetic Bit Analysis, which designs a DNA oligonucleotide chain that spans the inserted DNA sequence and the adjacent flanking genomic DNA sequence. Fix the oligonucleotide chain in the microwells of a microwell plate, and after PCR amplification of the target region (use one primer in the insert sequence and the adjacent flanking genome sequence), the single-stranded PCR product It can hybridize with a fixed oligonucleotide chain and serve as a template for a single-base extension reaction that uses 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 the insert/flanking sequence, which indicates that the amplification, hybridization, and single base extension reactions were successful.
  • Another method is Pyrosequencing. This method designs an oligonucleotide chain that spans the inserted DNA sequence and the adjacent genomic DNA binding site. Hybridize the oligonucleotide chain with the single-stranded PCR product of the target region (using one primer in the insert sequence and the adjacent flanking genome sequence), and then hybridize with DNA polymerase, ATP, sulfurylase, and luciferin The enzymes, adenosine triphosphate bisphosphatase, adenosine-5'-phosphosulfate, and luciferin are incubated together. Add dNTPs separately, and measure the generated optical signal. The light signal represents the presence of the insertion/flanking sequence, which indicates that the amplification, hybridization, and single- or multi-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 amplicon of the present invention.
  • Using this method requires designing an oligonucleotide chain that spans the inserted DNA sequence and the adjacent genomic DNA binding site. Hybridize the oligonucleotide chain with the single-stranded PCR product of the target region (using one primer in the insert sequence and the adjacent flanking genome sequence), and then perform the hybridization with DNA polymerase and a fluorescently labeled ddNTP Incubate. Single base extension will result in the insertion of ddNTP. This insertion can use a fluorometer to measure its polarization change. The change in polarization represents the presence of insert/flanking sequences, which indicates that the amplification, hybridization, and single base extension reactions are successful.
  • Taqman is described as a method for detecting and quantifying the presence of DNA sequences. The method is described in detail in the instructions provided by the manufacturer. A brief description is as follows, design a FRET oligonucleotide probe that spans the inserted DNA sequence and the adjacent genome flanking binding site.
  • the FRET probe and PCR primers are cyclically reacted in the presence of thermostable polymerase and dNTPs.
  • the hybridization of the FRET probe leads to the splitting of the fluorescent part and the quenched part and the release of the fluorescent part on the FRET probe.
  • the generation of the fluorescent signal represents the presence of the insert/flanking sequence, which indicates that the amplification and hybridization are successful.
  • suitable techniques for detecting plant materials derived from the transgenic soybean event DBN8007 may also include Southern blot, Northern blot, and in situ hybridization.
  • the suitable technique includes incubating the probe and the sample, 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, radioactively labeled probes can be detected by X-ray film exposure and development, or enzyme-labeled probes can be detected by substrate conversion to achieve color change.
  • Tyangi et al. (Nature Biotech. 14:303-308, 1996) introduced the application of molecular markers in sequence detection.
  • a brief description is as follows, design a FRET oligonucleotide probe that spans the inserted DNA sequence and the adjacent genome flanking binding site.
  • the unique structure of the FRET probe causes it to contain a secondary structure, which can maintain the fluorescent part and the quenching part in a close range.
  • the FRET probe and PCR primers (one primer in the insert sequence and one in the adjacent flanking genome sequence) are cyclically reacted in the presence of thermostable polymerase and dNTPs.
  • the hybridization of the FRET probe and the target sequence results in the loss of the secondary structure of the probe, which results in the spatial separation of the fluorescent part and the quenched part to generate a fluorescent signal.
  • the generation of the fluorescent signal represents the presence of the insert/flanking sequence, which indicates that the amplification and hybridization are successful.
  • Optical dyes are used to detect and measure specific DNA molecules.
  • a nanotube device including an electronic sensor for detecting DNA molecules or nanobeads that bind to specific DNA molecules and thus can be detected is useful for detecting the DNA molecules of the present invention.
  • the composition of the present invention and the methods described or known in the field of DNA detection can be used to develop DNA detection kits.
  • the kit is useful for identifying whether there is DNA of transgenic soybean event DBN8007 in a sample, and can also be used to cultivate soybean plants containing the DNA of transgenic soybean event DBN8007.
  • the kit may contain DNA primers or probes which are homologous to or complementary to at least a part of SEQ ID NO: 1, 2, 3, 4 or 5, or contain other DNA primers or probes which are homologous to or
  • the DNA contained in the genetic element of the transgene that is complementary to the DNA can be used in DNA amplification reactions or as probes in DNA hybridization methods.
  • the DNA structure of the junction between the transgene insert sequence and the soybean genome contained in the soybean genome and illustrated in Figure 1 and Table 1 includes: the soybean plant DBN8007 flanking genomic region located at the 5'end of the transgene insert sequence, from the right side of Agrobacterium A part of the border region (RB) is inserted into the sequence.
  • the first expression cassette is from the Arabidopsis ACTIN2 promoter (prAtAct2), operably linked to the insect-resistant mVip3Aa gene of Bacillus thuringiensis, and operably linked to The nopaline synthase gene is composed of the transcription terminator (tNos).
  • the second expression cassette consists of a cauliflower mosaic virus 35S promoter (pr35S) that is operably linked to the glufosinate-tolerant phosphine of Streptomyces It is composed of the silk mycin N-acetyltransferase gene (cPAT) and is operably linked to the cauliflower mosaic virus 35S terminator (t35S). It is a part of the inserted sequence from the left border region (LB) of Agrobacterium , And the genomic region flanking the soybean plant DBN8007 at the 3'end of the transgene insert sequence (SEQ ID NO: 5).
  • the DNA molecule used as the primer can be any part of the transgenic insert sequence derived from the transgenic soybean event DBN8007, or any part of the soybean genome flanking DNA sequence derived from the transgenic soybean event DBN8007.
  • the transgenic soybean event DBN8007 can be combined with other genetically modified soybean varieties, such as genetically modified soybean varieties that are tolerant to herbicides (such as glyphosate, dicamba, etc.), or genetically modified soybean varieties that carry other insect-resistant genes.
  • Other genetically modified soybean varieties such as genetically modified soybean varieties that are tolerant to herbicides (such as glyphosate, dicamba, etc.), or genetically modified soybean varieties that carry other insect-resistant genes.
  • the various combinations of all these different transgenic events, bred together with the transgenic soybean event DBN8007 of the present invention can provide improved hybrid transgenic soybean varieties resistant to multiple insect pests and multiple herbicides. Compared with non-transgenic varieties and single-character genetically modified varieties, these varieties can show more excellent characteristics such as increased yield.
  • the transgenic soybean event DBN8007 of the present invention is resistant to the feeding damage of lepidopteran pests, and tolerates the phytotoxic effects of agricultural herbicides containing glufosinate-ammonium.
  • the soybean plant with dual traits expresses the Vip3Aa protein of Bacillus thuringiensis, which provides resistance to the feeding damage of lepidopteran pests (such as hawk moth) and expresses the glufosinate-resistant phosphinothricin N of Streptomyces -Acetyltransferase (PAT) protein, which confers tolerance to glufosinate-ammonium in plants.
  • PAT Streptomyces -Acetyltransferase
  • Soybeans with dual traits have the following advantages: 1) Avoid economic losses caused by lepidopteran pests (such as Spodoptera litura, Spodoptera litura, etc.), which are the main pests in soybean planting areas; 2) The ability to apply glufosinate-containing agricultural herbicides to soybean crops for broad-spectrum weed control; 3) There is no reduction in soybean yield.
  • the transgenes encoding insect resistance and glufosinate tolerance traits are linked to the same DNA segment and are present at a single locus in the genome of the transgenic soybean event DBN8007, which provides enhanced breeding efficiency and enables use Molecular markers to track transgenic inserts in breeding populations and their offspring.
  • SEQ ID NO: 1 or its complementary sequence, SEQ ID NO: 2 or its complementary sequence, SEQ ID NO: 6 or its complementary sequence, or SEQ ID NO: 7 or its complementary sequence can be used as DNA primers Or probes to generate amplified products diagnosed as transgenic soybean event DBN8007 or its progeny, and can quickly, accurately and stably identify the presence of plant material derived from transgenic soybean event DBN8007.
  • SEQ ID NO: 1 In the transgenic soybean event DBN8007, a 22-nucleotide sequence near the insertion junction at the 5'end of the insert sequence, including nucleotides 1-11 and nucleotides 12-22 The acid is located on both sides of the insertion site on the soybean genome;
  • SEQ ID NO: 2 In the transgenic soybean event DBN8007, a 22-nucleotide sequence near the insertion junction at the 3'end of the insert sequence, including nucleotides 1-11 and nucleotides 12-22 The acid is located on both sides of the insertion site on the soybean genome;
  • SEQ ID NO: 3 A sequence of 1407 nucleotides in length near the insertion junction at the 5'end of the insertion sequence in the transgenic soybean event DBN8007;
  • SEQ ID NO: 4 A sequence of 1022 nucleotides in length near the insertion junction at the 3'end of the insertion sequence in the transgenic soybean event DBN8007;
  • SEQ ID NO: 5 The entire T-DNA sequence, and the soybean genome flanking sequence at the 5'and 3'ends;
  • SEQ ID NO: 6 The sequence on SEQ ID NO: 3 spans the pDBN4006 construct DNA sequence and the prAtAct2 transcription start sequence;
  • SEQ ID NO: 7 The sequence located on SEQ ID NO: 4 spans the t35S transcription terminator sequence and the pDBN4006 construct DNA sequence;
  • SEQ ID NO: 8 Amplifies the first primer of SEQ ID NO: 3;
  • SEQ ID NO: 9 Amplifies the second primer of SEQ ID NO: 3;
  • SEQ ID NO: 10 Amplifies the first primer of SEQ ID NO: 4.
  • SEQ ID NO: 11 amplifies the second primer of SEQ ID NO: 4.
  • SEQ ID NO: 12 primers on the 5'flanking genome sequence
  • SEQ ID NO: 13 A primer on T-DNA paired with SEQ ID NO: 12;
  • SEQ ID NO: 14 The primer on the 3'flanking genome sequence, which is paired with SEQ ID NO: 12 to detect whether the transgene is homozygous or heterozygous;
  • SEQ ID NO: 15 A primer on T-DNA paired with SEQ ID NO: 14;
  • SEQ ID NO: 16 The first primer for Taqman detection of mVip3Aa gene
  • SEQ ID NO: 17 The second primer for Taqman detection of mVip3Aa gene
  • SEQ ID NO: 18 Taqman probe for detecting mVip3Aa gene
  • SEQ ID NO: 19 The first primer for Taqman detection of PAT gene
  • SEQ ID NO: 20 The second primer for Taqman detection of PAT gene
  • SEQ ID NO: 21 Probe for detecting PAT gene by Taqman
  • SEQ ID NO: 22 The first primer of soybean endogenous gene lectin
  • SEQ ID NO: 23 The second primer of soybean endogenous gene lectin
  • SEQ ID NO: 24 Probe of mVip3Aa gene in Southern hybridization detection
  • SEQ ID NO: 25 Probes for PAT gene in Southern hybridization detection
  • SEQ ID NO: 26 The primer on the T-DNA is in the same direction as SEQ ID NO: 13;
  • SEQ ID NO: 27 primer on T-DNA, opposite to SEQ ID NO: 13, used to obtain the flanking sequence;
  • SEQ ID NO: 28 primer on T-DNA, opposite to SEQ ID NO: 13, used to obtain flanking sequence;
  • SEQ ID NO: 29 The primer on the T-DNA is in the same direction as SEQ ID NO: 15;
  • SEQ ID NO: 30 primer on T-DNA, opposite to SEQ ID NO: 15, used to obtain flanking sequence;
  • SEQ ID NO: 31 The primer on the T-DNA, opposite to SEQ ID NO: 15, is used to obtain the flanking sequence.
  • Figure 1 is a schematic diagram of the present invention used to detect the nucleic acid sequence of soybean plant DBN8007 and the detection method of the transgenic insert sequence and soybean genome junction structure schematic diagram, and a schematic diagram of the relative position of the nucleic acid sequence used to detect soybean plant DBN8007 (reference for relative position diagram Wm82.a2 RefGen);
  • Figure 2 is a schematic structural diagram of the recombinant expression vector pDBN4006 used for detecting the nucleic acid sequence of soybean plant DBN8007 and its detection method according to the present invention
  • Figure 3 is a diagram showing the bioassay effect of the genetically modified soybean event DBN8007 on the cotton bollworm for detecting the nucleic acid sequence of the soybean plant DBN8007 and its detection method according to the present invention
  • Figure 4 is a diagram showing the bioassay effect of transgenic soybean event DBN8007 on Spodoptera litura which is used to detect the nucleic acid sequence of soybean plant DBN8007 and its detection method according to the present invention
  • FIG. 5 is a diagram showing the bioassay effect of the transgenic soybean event DBN8007 on the beet armyworm for detecting the nucleic acid sequence of the soybean plant DBN8007 and its detection method according to the present invention
  • Figure 6 is a diagram showing the bioassay effect of the transgenic soybean event DBN8007 on the soybean hawk moth for detecting the nucleic acid sequence of the soybean plant DBN8007 and its detection method according to the present invention
  • Fig. 7 is a field effect diagram of the cotton bollworm inoculation of the transgenic soybean event DBN8007 used for detecting the nucleic acid sequence of the soybean plant DBN8007 and the detection method thereof according to the present invention
  • Figure 8 is a field effect diagram of the transgenic soybean event DBN8007 used for detecting the nucleic acid sequence of the soybean plant DBN8007 and the detection method thereof under the natural occurrence of beet armyworm;
  • FIG. 9 is a field effect diagram of the transgenic soybean event DBN8007 used for detecting the nucleic acid sequence of the soybean plant DBN8007 and the detection method thereof under the natural occurrence of Spodoptera litura;
  • Figure 10 is a diagram showing the bioassay effect of the transgenic soybean event DBN8007 used for detecting the nucleic acid sequence of the soybean plant DBN8007 and the detection method thereof on Spodoptera frugiperda.
  • the recombinant expression vector pDBN4006 was constructed using standard gene cloning techniques (as shown in Figure 2).
  • the vector pDBN4006 contains two transgenic expression cassettes in tandem.
  • the first expression cassette is from the ACTIN2 promoter (prAtAct2) from Arabidopsis thaliana and is operably linked to the mVip3Aa gene from Bacillus thuringiensis that can provide insect resistance ( CN 103509808 B), and can be operably linked to the transcription terminator (tNos) of nopaline synthase;
  • the second expression cassette is composed of a cauliflower mosaic virus promoter (pr35S) that is operably linked to the chain
  • the fungal glufosinate-tolerant phosphinothricin N-acetyltransferase gene (cPAT) is operably linked to the cauliflower mosaic virus transcription terminator (t35S).
  • the vector pDBN4006 was transformed into Agrobacterium LBA4404 (Invitrgen, Chicago, USA; Cat. No: 18313-015) by liquid nitrogen method, and was transformed with 4-[hydroxy(methyl)phosphono]-DL-homoalanine Acid is the selection marker to screen transformed cells.
  • the conventional Agrobacterium infection method was used for transformation, and the aseptically cultured soybean cotyledon node tissue was co-cultured with the Agrobacterium described in Example 1.1 to transfer the T-DNA in the constructed recombinant expression vector pDBN4006 into Soybean genome to generate transgenic soybean event DBN8007.
  • soybean germination medium B5 salt 3.1g/L, B5 vitamin, sucrose 20g/L, agar 8g/L, pH 5.6
  • 4-6 days after germination take the aseptic soybean seedlings with enlarged cotyledon nodes, cut off the hypocotyls 3-4 mm below the cotyledon nodes, cut the cotyledons longitudinally, and remove the apical buds, lateral buds and seed roots.
  • step 1 infection step
  • Cotyledon node tissues and Agrobacterium co-culture for a period of time (3 days) (Step 2: Co-cultivation
  • the cotyledon node tissue is placed in a solid medium (MS salt 4.3g/L, B5 vitamin, sucrose 20g/L, glucose 10g/L, 2 -Morpholine ethanesulfonic acid (MES) 4g/L, zeatin 2mg/L, agar 8g/L, pH 5.6).
  • MS salt 4.3g/L
  • B5 vitamin sucrose 20g/L
  • glucose 10g/L glucose 10g/L
  • zeatin 2mg/L agar 8g/L, pH 5.6
  • the recovery medium B5 salt 3.1g/L, B5 vitamins, 2-morpholineethanesulfonic acid (MES) 1g/L, sucrose 30g/L, zeatin (ZT) 2mg/L, agar 8g/L L, cephalosporin 150mg/L, glutamic acid 100mg/L, aspartic acid 100mg/L, pH 5.6) at least one antibiotic known to inhibit the growth of Agrobacterium (cephalosporin 150-250mg/L) ), without adding a selection agent for plant transformants (step 3: recovery step).
  • B5 salt 3.1g/L, B5 vitamins, 2-morpholineethanesulfonic acid (MES) 1g/L, sucrose 30g/L, zeatin (ZT) 2mg/L, agar 8g/L L, cephalosporin 150mg/L, glutamic acid 100mg/L, aspartic acid 100mg/L, pH 5.6 at least one antibiotic known to inhibit
  • the tissue mass regenerated from the cotyledon node is cultured on a solid medium with antibiotics but no selection agent to eliminate Agrobacterium and serve as infecting cells Provide a recovery period. Then, the regenerated tissue mass of the cotyledon node is cultured on the medium containing the selection agent (4-[hydroxy(methyl)phosphono]-DL-homoalanine) and the growing transformed callus is selected (Step 4: Selection step).
  • the selection agent 4-[hydroxy(methyl)phosphono]-DL-homoalanine
  • the regenerated tissue mass of the cotyledon node is in a selective solid medium (B5 salt 3.1g/L, B5 vitamin, 2-morpholineethanesulfonic acid (MES) 1g/L , Sucrose 30g/L, 6-benzyl adenine (6-BAP) 1mg/L, agar 8g/L, cephalosporin 150mg/L, glutamic acid 100mg/L, aspartic acid 100mg/L, 4- [Hydroxy(methyl)phosphono]-DL-homoalanine 10mg/L, pH 5.6), the transformed cells can continue to grow.
  • B5 salt 3.1g/L B5 vitamin, 2-morpholineethanesulfonic acid (MES) 1g/L
  • Sucrose 30g/L sucrose 30g/L
  • 6-benzyl adenine 6-BAP
  • cephalosporin 150mg/L glutamic acid 100mg/L
  • the transformed cells regenerate into plants (step 5: regeneration step)
  • the tissue masses regenerated from the cotyledon nodes grown on the medium containing the selection agent are cultured on a solid medium (B5 differentiation medium and B5 rooting medium) to regenerate plants.
  • the screened resistant tissue pieces are transferred to the B5 differentiation medium (B5 salt 3.1g/L, B5 vitamins, 2-morpholineethanesulfonic acid (MES) 1g/L, sucrose 30g/L, zeatin (ZT) 1mg/L, agar 8g/L, cephalosporin 150mg/L, glutamic acid 50mg/L, aspartic acid 50mg/L, gibberellin 1mg/L, auxin 1mg/L, 4-[hydroxy(formaldehyde) (Base)phosphono]-DL-homoalanine 5mg/L, pH5.6), cultured and differentiated at 25°C.
  • B5 differentiation medium B5 salt 3.1g/L, B5 vitamins, 2-morpholineethanesulfonic acid (MES) 1g/L, sucrose 30g/L, zeatin (ZT) 1mg/L, agar 8g/L, cephalosporin 150mg/L, glutamic
  • the differentiated seedlings are transferred to the B5 rooting medium (B5 salt 3.1g/L, B5 vitamins, 2-morpholineethanesulfonic acid (MES) 1g/L, sucrose 30g/L, agar 8g/L, cephalosporin 150mg/L, indole-3-butyric acid (IBA) 1mg/L), in the rooting culture, cultivated at 25°C to a height of about 10cm, and then moved to the greenhouse to cultivate until it becomes firm. In the greenhouse, each day is cultured at a temperature of 26°C for 16 hours, and then at a temperature of 20°C for 8 hours.
  • B5 rooting medium B5 salt 3.1g/L, B5 vitamins, 2-morpholineethanesulfonic acid (MES) 1g/L, sucrose 30g/L, agar 8g/L, cephalosporin 150mg/L, indole-3-butyric acid (IBA) 1mg/L
  • a total of 288 independent transgenic T 0 plants were produced.
  • the above 288 independent transgenic T 0 individual plants were sent to the greenhouse for transplantation for cultivation and propagation to obtain transgenic T 1 individual plants.
  • agronomic traits of soybean plants For example, seedling vigor, growth period, plant height or lodging, etc.
  • seedling vigor For example, seedling vigor, growth period, plant height or lodging, etc.
  • the above 21 transgenic T2 generation single plants were planted in the field to identify the transgenic T2 individual plants at different stages (seedling stage-full bloom stage, initial seed stage-mature stage)
  • DBN8007 To screen whether the genetically modified soybean event DBN8007 is excellent, it has a single-copy transgene (see the second example), good insect resistance, and herbicidal glufosinate resistance. Agent tolerance and agronomic performance (see the sixth and seventh examples).
  • the second embodiment using TaqMan to detect transgenic soybean event DBN8007
  • transgenic soybean event DBN8007 About 100 mg of the leaves of transgenic soybean event DBN8007 were taken as samples, and the genomic DNA was extracted with a plant DNA extraction kit (DNeasy Plant Maxi Kit, Qiagen), and the copy number of mVip3Aa gene and PAT gene was detected by the Taqman probe fluorescence quantitative PCR method.
  • the wild-type soybean plant was used as a control, and the detection and analysis were performed according to the above method. The experiment is set to be repeated 3 times and the average value is taken.
  • Step 1 Take 100 mg of the leaves of transgenic soybean event DBN8007, grind it into a homogenate with liquid nitrogen in a mortar, and take 3 replicates for each sample;
  • Step 2 Use the plant DNA extraction kit (DNeasy Plant Maxi Kit, Qiagen) to extract the genomic DNA of the above sample, and refer to the product manual for the specific method;
  • Step 3 Use an ultra-micro spectrophotometer (NanoDrop 2000, Thermo Scientific) to measure the genomic DNA concentration of the above sample;
  • Step 4 Adjust the genomic DNA concentration of the above sample to the same concentration value, and the range of the concentration value is 80-100ng/ ⁇ L;
  • Step 5 Use Taqman probe fluorescence quantitative PCR method to identify the copy number of the sample, use the identified sample with known copy number as the standard product, and use the wild-type soybean plant sample as the control. There are 3 replicates for each sample and take the average Value; the sequence of the fluorescent quantitative PCR primer and probe are:
  • Primer 1 cgaatacagaaccctgtcggc is shown in SEQ ID NO: 16 in the sequence list;
  • Primer 2 cgtgaggaaggtctcagaaatgac as shown in SEQ ID NO: 17 in the sequence table;
  • Probe 1 cgacgatggcgtgtatatgcctcttgg is shown in SEQ ID NO: 18 in the sequence table;
  • gagggtgttgtggctggtattg is shown in SEQ ID NO: 19 in the sequence list;
  • Primer 4 tctcaactgtccaatcgtaagcg is shown in SEQ ID NO: 20 in the sequence table;
  • Probe 2 cttacgctgggccctggaaggctag is shown in SEQ ID NO: 21 in the sequence table;
  • the PCR reaction system is:
  • the 50 ⁇ primer/probe mixture contains 45 ⁇ L of each primer at 1 mM concentration, 50 ⁇ L of probe at 100 ⁇ M concentration, and 860 ⁇ L 1 ⁇ TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), and at 4°C, Store in amber test tube.
  • the PCR reaction conditions are:
  • the data was analyzed using the Fast Real-Time Fluorescent Quantitative PCR System Software (Applied Biosystems 7900HT Fast Real-Time PCR System SDS v2.3, Applied Biosystems), and the results showed that the obtained transgenic soybean event DBN8007 was a single copy.
  • the third embodiment analysis of the insertion site of the transgenic soybean event DBN8007
  • DNA extraction was performed according to the conventional CTAB (hexadecyl trimethyl ammonium bromide) method: 2g of the young leaves of transgenic soybean event DBN8007 were ground into powder in liquid nitrogen, and then 0.5 mL preheated at 65°C was added.
  • CTAB hexadecyl trimethyl ammonium bromide
  • DNA extraction CTAB buffer (20g/L CTAB, 1.4M NaCl, 100mM Tris-HCl, 20mM EDTA (ethylenediaminetetraacetic acid), adjust the pH to 8.0 with NaOH), mix well, and extract at 65°C for 90 minutes ; Add 0.5 times the volume of phenol and 0.5 times the volume of chloroform, and mix upside down; centrifuge at 12000 rpm (revolutions per minute) for 10 min; aspirate the supernatant, add 2 times the volume of absolute ethanol, gently shake the centrifuge tube, at a temperature of 4 °C Let stand for 30 min; centrifuge at 12000 rpm for another 10 min; collect DNA to the bottom of the tube; discard the supernatant and wash the precipitate with 1 mL of ethanol with a mass concentration of 70%; centrifuge at 12000 rpm for 5 min; vacuum dry or blow on a clean bench Dry; Dissolve the DNA pellet in an appropriate amount of TE buffer and store at -20°C.
  • the concentration of the extracted DNA sample is determined so that the concentration of the sample to be tested is between 80-100ng/ ⁇ L.
  • the restriction enzymes EcoR I (5' end analysis) and EcoR V (3' end analysis) were used to cut genomic DNA. Add 26.5 ⁇ L of genomic DNA, 0.5 ⁇ L of the above restriction endonuclease and 3 ⁇ L of digestion buffer to each digestion system (the restriction enzymes used are all NEB enzymes and its matching buffer or universal buffer. Called NEBCutSmart), digested for 1 hour.
  • the primer combination for isolating 5'-end genomic DNA includes SEQ ID NO: 13 and SEQ ID NO: 26 as the first primer, SEQ ID NO: 27 and SEQ ID NO: 28 as the second primer, and SEQ ID NO: 13 As a sequencing primer.
  • the primer combination for separating 3'end genomic DNA includes SEQ ID NO: 15 and SEQ ID NO: 29 as the first primer, SEQ ID NO: 30 and 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 amplified products obtained in the above PCR amplification reaction were electrophoresed on a 2.0% agarose gel to separate the PCR amplified products, and then the gel extraction kit (QIAquick Gel Extraction Kit, catalog #_28704, Qiagen Inc., Valencia, CA) separate the fragment of interest from the agarose matrix.
  • the purified PCR amplification product is then sequenced (for example, using ABI PrismTM 377, PE Biosystems, Foster City, CA) and analyzed (for example, using DNASTAR sequence analysis software, DNASTAR Inc., Madison, WI).
  • the 5'flanking sequence and the joining 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 joining sequence can be confirmed by using SEQ ID NO: 11 or SEQ ID NO: 14, in combination with SEQ ID NO: 10, SEQ ID NO: 15 or SEQ ID NO: 29.
  • the PCR reaction system and amplification conditions are shown in Table 2 and Table 3. Those skilled in the art will understand that other primer sequences can also be used to confirm flanking and joining sequences.
  • DNA sequencing of PCR amplified products provides DNA that can be used to design other DNA molecules that can be used as primers and probes to identify soybean plants or seeds derived from the transgenic soybean event DBN8007.
  • nucleotide 1-5237 of SEQ ID NO: 5 shows the soybean genome sequence flanking the right border (5' flanking sequence) of the transgenic soybean event DBN8007 insertion sequence
  • nucleotide 11210 of SEQ ID NO: 5 Position -11935 shows the soybean genome sequence flanking the left border (3' flanking sequence) of the transgenic soybean event DBN8007 insert sequence.
  • the 5'junction sequence is listed in SEQ ID NO: 1
  • the 3'junction sequence is listed in SEQ ID NO: 2.
  • the junction sequence is a relatively short polynucleotide molecule, which is a new DNA sequence, which is diagnostic for the DNA of transgenic soybean event DBN8007 when detected in a polynucleic acid detection analysis.
  • the junction sequence in SEQ ID NO: 1 and SEQ ID NO: 2 is the insertion site of the transgenic fragment in the transgenic soybean event DBN8007 and 11 polynucleotides on each side of soybean genomic DNA.
  • the longer or shorter polynucleotide junction sequence can be selected from SEQ ID NO: 3 or SEQ ID NO: 4.
  • the junction sequence (5' junction region SEQ ID NO: 1, and 3'junction region SEQ ID NO: 2) are useful as DNA probes or as DNA primer molecules in DNA detection methods.
  • the junction sequence SEQ ID NO: 6 and SEQ ID NO: 7 are also new DNA sequences in the transgenic soybean event DBN8007, which can also be used as a DNA probe or as a DNA primer molecule to detect the presence of the transgenic soybean event DBN8007 DNA.
  • the SEQ ID NO: 6 (nucleotides 794-1012 of SEQ ID NO: 3) spans the pDBN4006 construct DNA sequence and the prAtAct2 transcription initiation sequence.
  • the SEQ ID NO: 7 (SEQ ID NO: 4) Nucleotides 1-243) spanned the t35S transcription termination sequence and the pDBN4006 construct DNA sequence.
  • the amplicon is generated by using at least one primer from SEQ ID NO: 3 or SEQ ID NO: 4, which generates a diagnostic amplicon of transgenic soybean event DBN8007 when used in a PCR method.
  • a PCR amplification product is generated from the 5'end of the transgenic insert sequence, and the PCR amplification product contains the genomic DNA flanking the 5'end of the T-DNA insert in the genome of the plant material derived from the transgenic soybean event DBN8007. Part.
  • This PCR amplification product contains SEQ ID NO: 3.
  • primer 5 SEQ ID NO: 8 that hybridizes with the genomic DNA sequence flanking the 5'end of the transgene insert sequence
  • primer 6 SEQ ID NO: 8 at the transcription initiation sequence of the transgene prAtAct2 is designed. ID NO: 9).
  • a PCR amplification product is generated from the 3'end of the transgenic insert, and the PCR amplification product is a part of the genomic DNA flanking the 3'end of the T-DNA insert in the genome of the plant material derived from the transgenic soybean event DBN8007.
  • This PCR amplification product contains SEQ ID NO: 4.
  • primer 7 SEQ ID NO: 10 located in the transcription termination sequence of the transgene t35S was designed, and primer 8 (SEQ ID NO: 10) that was paired with it hybridized with the genomic DNA sequence flanking the 3'end of the transgene insert sequence. NO: 11).
  • the DNA amplification conditions described in Table 2 and Table 3 can be used in the PCR zygosity test described above to generate diagnostic amplicons of transgenic soybean event DBN8007.
  • the detection of amplicons can be performed by using Stratagene Robocycler, MJ Engine, Perkin-Elmer 9700 or Eppendorf Mastercycler Gradient thermal cycler, etc., or by methods and equipment known to those skilled in the art.
  • primers 5 and 6 when used in the PCR reaction of the genomic DNA of the transgenic soybean event DBN8007, produce a 1407bp fragment of the amplified product, when it is used in the untransformed soybean genome
  • primers 7 and 8 when used in the PCR reaction of transgenic soybean event DBN8007 genomic DNA, produced The amplified product of the 1022bp fragment, when used in the PCR reaction of untransformed soybean genomic DNA and non-DBN8007 soybean genomic DNA, no fragment was amplified.
  • PCR zygosity determination can also be used to identify whether the material derived from the transgenic soybean event DBN8007 is homozygous or heterozygous.
  • Primer 9 SEQ ID NO: 12
  • primer 10 SEQ ID NO: 13
  • primer 11 SEQ ID NO: 14
  • the DNA amplification conditions described in Tables 4 and 5 can be used in the above-mentioned zygosity test to generate diagnostic amplicons of transgenic soybean event DBN8007.
  • the biological sample containing template DNA contains DNA for diagnosing the presence of the transgenic soybean event DBN8007 in the sample.
  • the amplification reaction will generate two different DNA amplicons from a biological sample containing DNA derived from the soybean genome.
  • the DNA derived from the soybean genome will have an allele corresponding to the inserted DNA present in the transgenic soybean event DBN8007. Heterozygous.
  • These two different amplicons will correspond to the first amplicon (SEQ ID NO: 12 and SEQ ID NO: 14) derived from the wild-type soybean genomic locus and the first amplicon to diagnose the presence of the transgenic soybean event DBN8007 DNA.
  • Two amplicons SEQ ID NO: 12 and SEQ ID NO: 13).
  • primer pair of the transgenic soybean event DBN8007 is used to generate a diagnostic amplicon for the transgenic soybean event DBN8007 genomic DNA.
  • primer pairs include, but are not limited to, primers 5 and 6 (SEQ ID NO: 8 and 9), and primers 7 and 8 (SEQ ID NO: 10 and 11), which are used in the DNA amplification method.
  • a control primer 12 and 13 used to amplify endogenous soybean genes was included as an internal standard for reaction conditions.
  • the analysis of DNA extraction samples from the transgenic soybean event DBN8007 should include a positive tissue DNA extract control of the transgenic soybean event DBN8007, a negative DNA extract control derived from the non-transgenic soybean event DBN8007, and a soybean DNA extract without template. The negative control of the extract.
  • any primer pair from SEQ ID NO: 3 or its complementary sequence, or SEQ ID NO: 4 or its complementary sequence can also be used.
  • the tissue of the soybean plant DBN8007 in the transgenic event is a diagnostic amplicon containing SEQ ID NO: 1 or SEQ ID NO: 2.
  • the DNA amplification conditions described in Table 2 to Table 5 can be used to generate diagnostic amplicons of transgenic soybean event DBN8007 using appropriate primer pairs.
  • an extract of soybean plant or seed DNA that is a diagnostic amplicon for transgenic soybean event DBN8007, presumed to contain transgenic soybean event DBN8007, or a product derived from transgenic soybean event DBN8007, can be Used as a template for amplification to determine the presence of transgenic soybean event DBN8007.
  • CTAB Lysis Buffer 100mM Tris-HCl pH 8.0, 20mM EDTA pH 8.0, 1.4M NaCl, 0.2% v/v ⁇ -mercaptoethanol, 2% w/v CTAB
  • Plant tissues were incubated at a temperature of 65°C for 60 minutes. During the incubation period, the samples were mixed upside down every 10 min. After incubation, an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) was added, gently inverted and mixed for extraction, and centrifuged at 4000 rpm for 20 min.
  • the water phase was extracted with an equal volume of chloroform/isoamyl alcohol (24:1) and repeated once. Collect the aqueous phase again and add an equal volume of isopropanol. After mixing, place it at -20°C for 1 hour to precipitate the DNA, and then centrifuge at 4000 rpm for 5 minutes to obtain the DNA precipitate, and then in 1mL TE buffer (10mM Tris-HCl, 1mM EDTA , PH 8.0) resuspend the DNA pellet.
  • 1mL TE buffer (10mM Tris-HCl, 1mM EDTA , PH 8.0
  • RNA In order to degrade any RNA present, incubate DNA with 40 ⁇ L 10mg/mL RNase A at a temperature of 37°C for 30 min, centrifuge at 4000 rpm for 5 min, and at 0.1 times volume concentration of 3M sodium acetate (pH 5.2) and 2 times volume In the presence of water and ethanol, centrifuge at 12000 rpm for 10 minutes to precipitate DNA. After discarding the supernatant, wash the pellet with 70% (v/v) 1 mL ethanol, dry at room temperature, and redissolve the DNA in 1 mL TE buffer.
  • 3M sodium acetate pH 5.2
  • the genomic DNA concentration of the above samples was measured with an ultra-micro spectrophotometer (NanoDrop 2000, Thermo Scientific).
  • the DNA probe is SEQ ID NO: 24 or SEQ ID NO: 25, or is partially homologous or complementary to the foregoing sequence.
  • Use DNA Labeling and Detection Starter Kit II (Roche, Cat. No. 11585614910) for DIG labeling of probes, Southern blot hybridization, and membrane washing. For specific methods, refer to its product manual. Finally, X-ray film (Roche, Cat. No. 11666916001) was used to detect the position of probe binding.
  • Each Southern includes two control samples: (1) DNA from negative (untransformed) segregants, which are used to identify any endogenous soybean sequences that can hybridize with element-specific probes; (2) from The DNA of the negative segregant, in which Hind III-digested pDBN4006 plasmid is introduced, and the amount is equivalent to one copy number based on the length of the probe, which serves as a positive control to illustrate the sensitivity of the experiment when detecting a single gene copy in the soybean genome .
  • the hybridization data provided conclusive evidence to support the TaqMan TM PCR analysis, that the soybean plant DBN8007 contains a single copy of the mVip3Aa gene and the PAT gene.
  • Mfe I and Nco I were digested to produce single bands of about 5.7 kb and 17 kb in size, respectively;
  • PAT gene probe Mfe I and Nco I were digested to produce about 7 kb and 10 kb in size, respectively.
  • a single band indicates that one copy each of the mVip3Aa gene and the PAT gene is present in the soybean transformation event DBN8007.
  • the backbone probe no hybridization band was obtained, indicating that no pDBN4006 vector backbone sequence entered the soybean transformation event DBN8007 genome during the transformation process.
  • Vip3Aa and PAT protein in the transgenic soybean event DBN8007 can be detected by ELISA.
  • ELISA enzyme-linked immunosorbent assay
  • detection kits ENVIROLOGIX, Vip3Aa kit (AP085) and PAT kit (AP014)
  • ENVIROLOGIX enzyme-linked immunosorbent assay
  • Vip3Aa kit AP085
  • PAT kit AP014
  • the experimental results of the protein (Vip3Aa protein and PAT protein) content of the transgenic soybean event DBN8007 are shown in Table 6.
  • the average expression of Vip3Aa protein in the leaves of transgenic soybean event DBN8007 and wild-type soybean plants accounted for 15.67 and 0 in the leaf dry weight ratio ( ⁇ g/g), respectively;
  • the average expression of PAT protein in the leaves of transgenic soybean event DBN8007 and wild-type soybean plants The ratio of the amount to the dry weight of leaves ( ⁇ g/g) was 167.37 and 0, respectively.
  • Soybean transformation event DBN8007 and wild-type soybean plants (non-transgenic, NGM) 2 plants were respectively against cotton bollworm [Helicoverpa armigera, CBW], Spodoptera litura, TCW], Spodoptera exigua [Spodoptera exigua, BAW] and Bean hawk moth [Clanis bilineata, BHM] is bioassayed as follows:
  • total resistance score 100 ⁇ mortality + [100 ⁇ mortality + 90 ⁇ (initial hatching Number/total number of inoculated insects)+60 ⁇ (initial incubation-negative control number/total number of inoculated insects)+10 ⁇ (number of negative control insects/total number of inoculated insects)]+100 ⁇ (1-leaf damage rate).
  • the total number of infestations refers to the total number of infestations, that is, 10 per dish; the development progress of larvae has been reflected by the resistance total score formula; the leaf damage rate refers to the ratio of the leaf area eaten by the pest to the total leaf area.
  • 5 plants were selected from transgenic soybean event DBN8007 and wild-type soybean plants (non-transgenic, NGM) for testing, and each plant was repeated 6 times. The results are shown in Table 7-8 and Figure 3-6.
  • soybean armyworm includens, SBL], sunflower looper [Rachiplusia nu, SFL], and meadow worm [Spodoptera frugiperda, FAW] And black armyworm [Spodoptera cosmioides, BLAW] bioassay according to the following method:
  • soybean spodoptera Chrysodeixis includens, SBL], sunflower looper [Rachiplusia nu, SFL], and Li bean spodoptera [ Anticarsia gemmatalis, VBC] and Spodoptera frugiperda [Spodoptera frugiperda, FAW] were tested in the field according to the following methods:
  • bioassay cages are built in the field. Each bioassay cage is only tested for one kind of pests. The bioassay cages are not connected, and the bioassay cages are artificially planted by corn and the natural growth in the field. Grass to further increase the physical barrier.
  • the transgenic soybean event DBN8007 and wild-type soybean plants were randomly planted in each bioassay cage, with 3 replicates for each plant, and one row for each replicate (row length 3m, 30 plants/row, row spacing 50cm), conventional cultivation and management, no pesticides will be sprayed during the whole growth period.
  • leaf damage rate refers to the ratio of the leaf area eaten by the pest to the total leaf area. The results are shown in Table 13.
  • Basta herbicide (a salt water formulation of glufosinate ammonium salt with an active ingredient of 18%) was selected for spraying.
  • a random block design was used, with 3 repetitions.
  • the plot area is 15m 2 (5m ⁇ 3m), row spacing is 60cm, plant spacing is 25cm, conventional cultivation management, there is a 1m wide isolation zone between plots.
  • the transgenic soybean event DBN8007 was subjected to the following two treatments: (1) No spraying, while treatment (2) Spraying herbicides, spray an equal volume of water; (2) Press 800g ai/ha (ai/ha means " The active ingredient per hectare") is sprayed with the guarantee herbicide at the V2-V3 leaf stage (2-3 compound leaves).
  • glufosinate-ammonium herbicides such as Basta
  • contact-killing herbicides such as improper use in the field, such as excessive local accumulation of liquid medicine, phytotoxicity may appear, not the genetically modified soybean event DBN8007 tolerance problem ;
  • Different content and dosage forms of glufosinate-ammonium herbicide converted into the above-mentioned equivalent effective ingredients are applicable to the following conclusions.
  • the phytotoxicity symptoms were investigated 1 week and 2 weeks after the medication, and the yield of the plot was measured at the time of harvest; the phytotoxicity symptoms classification is shown in Table 14.
  • the damage rate of glufosinate-ammonium is determined based on the investigation results of the phytotoxicity 2 weeks after the treatment of glufosinate, and the damage rate of the herbicide (glufosinate-ammonium) is used to distinguish soybean against herbicide.
  • yield percentage (%) spray yield/no spray Yield.
  • Phytotoxicity level Description of symptoms 1 Normal growth without any symptoms of harm 2 Slight phytotoxicity, phytotoxicity is less than 10% 3 Moderate phytotoxicity, which can be recovered in the future without affecting the yield 4 The phytotoxicity is severe and difficult to recover, resulting in reduced production 5 The drug damage is serious and cannot be recovered, causing a significant reduction in production or abortion
  • the damage rate of glufosinate-ammonium herbicide is 0 under the treatment of glufosinate-ammonium herbicide (800g ai/ha); therefore, the GM soybean event DBN8007 has good glufosinate herbicide Agent tolerance.
  • the transgenic soybean event DBN8007 had no significant difference in yield under the two treatments of non-spraying and spraying 800g ai/ha glufosinate-ammonium, which further showed that the transgenic soybean event DBN8007 had good glufosinate-ammonium herbicide tolerance. It has no effect on output.
  • the agricultural product or commodity is expected to contain a nucleotide sequence capable of diagnosing the presence of the transgenic soybean event DBN8007 material in the agricultural product or commodity.
  • the agricultural products or commodities include, but are not limited to, soybean cakes, flours and oils, which can specifically be lecithin, fatty acids, glycerin, sterols, edible oils, defatted soybean flakes, including defatted and roasted soybean flour, soy milk curd, Tofu, soy protein concentrate, isolated soy protein, hydrolyzed vegetable protein, textured soy protein and soy protein fiber, and any other food that will be used as a food source for animal consumption.
  • soybean cakes, flours and oils which can specifically be lecithin, fatty acids, glycerin, sterols, edible oils, defatted soybean flakes, including defatted and roasted soybean flour, soy milk curd, Tofu, soy protein concentrate, isolated soy protein, hydrolyzed vegetable protein, textured soy protein and soy protein fiber, and any other food that will be used as a food source for animal consumption.
  • Nucleic acid detection methods and/or kits based on probes or primer pairs can be developed to detect nucleotide sequences derived from transgenic soybean event DBN8007 as shown in SEQ ID NO:1 or SEQ ID NO:2 in biological samples,
  • the probe sequence or primer sequence is selected from the sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, or a part thereof, to diagnose transgene The existence of soybean incident DBN8007.
  • the transgenic soybean event DBN8007 of the present invention has good resistance to lepidopteran insects, and at the same time has high tolerance to glufosinate herbicide, has no effect on yield, and the detection method can be accurate and rapid Identify whether the biological sample contains the DNA molecule of transgenic soybean event DBN8007.
  • the seeds corresponding to the genetically modified soybean event DBN8007 have been deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee (CGMCC for short, address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing) on February 19, 2019 in accordance with the Budapest Treaty. Institute of Microbiology, Chinese Academy of Sciences, zip code 100101), classification and designation: soybean (Glycine max), preservation status: alive, preservation number CGMCC No. 17300. The deposit will be kept in the depository for 30 years.
  • CGMCC General Microbiology Center of the China Microbial Culture Collection Management Committee

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Abstract

一种用于检测大豆植物DBN8007的核酸序列及其检测方法，所述核酸序列包括SEQ ID NO：1或其互补序列、和/或SEQ ID NO：2或其互补序列。大豆植物DBN8007对鳞翅目昆虫具有较好的抗性并对草铵膦除草剂具有较好的耐受性，对产量无影响，且检测方法可以准确快速的鉴定生物样品中是否包含转基因大豆事件DBN8007的DNA分子。

Description

用于检测大豆植物DBN8007的核酸序列及其检测方法 技术领域
本发明涉及植物分子生物学领域，特别是农业生物技术研究中的转基因农作物育种领域。具体地，本发明涉及昆虫抗性和草铵膦除草剂耐受性的转基因大豆事件DBN8007和用于检测生物样品中是否包含特定转基因大豆事件DBN8007的核酸序列及其检测方法。
背景技术
大豆(Glycine max)是世界五大主栽作物之一。生物技术已经应用于大豆以改善其农艺性状和品质。在大豆生产中除草剂耐受性是一项重要的农艺性状，特别是耐受草甘膦除草剂，如已有成功的大豆事件GTS40-3-2、MON89788，美国等大豆主要种植区域已广泛种植。另一个重要的农艺性状是昆虫抗性，特别是对鳞翅目昆虫的抗性，如已有成功的大豆事件MON87701在巴西等大豆主要种植区域广泛种植。值得一提的是，Vip蛋白与Cry蛋白的作用机制不同，其为营养期杀虫蛋白，且可以作为一种有效管理Cry蛋白抗性昆虫的手段。大豆对鳞翅目昆虫的抗性可以通过转基因的方法使鳞翅目昆虫的抗性基因在大豆植物中表达而获得。此外，草铵膦除草剂与草甘膦除草剂的作用机理不同，其为灭生性的触杀型除草剂，且可以作为一种有效管理草甘膦抗性杂草的手段。大豆对草铵膦除草剂的耐受性可以通过转基因的方法使草铵膦除草剂耐受型基因(如PAT)在大豆植物中表达而获得。
设计适用于转化大豆作物的包含外源功能基因(Vip3Aa基因和PAT基因)的表达载体且得到相应的可商业化转基因大豆事件具有重要意义。目前未有Vip蛋白在大豆植物控虫上成功应用的案例，与此同时，除草剂耐受性作为大豆生产中一项重要的农艺性状，几乎是不可或缺的，因此良好的商业化大豆转化事件要综合考虑Vip3Aa基因和PAT基因在大豆植物中的载体设计、两个表达盒的互作影响、抗虫效果、耐受除草剂效果以及对产量和其他植物生理指标的影响，使得Vip3Aa基因和PAT基因能够在大豆中适量表达并实现其相应的功能，而不影响大豆产量和其他生理指标。
已知外源基因在植物体内的表达受到它们的染色体位置的影响，可能是由于染色质结构(如异染色质)或转录调节元件(如增强子)接近整合位点。为此，通常需要筛选大量的事件才有可能鉴定出可以商业化的事件(即导入的目标基因得到最优表达的事件)。例如，在植物和其他生物体中已经观察到导入基因的表达量在事件间可能有很大差异；在表达的空间或时间模式上可能也存在差异，如在不同植物组织之间转基因的相对表达存在差异，这种差异表现在实际的表达模式可能与根据导入的基因构建体中的转录调节元件所预期的表达模式不一致。因此，通常需要产生成百上千个不同的事件并从这些事件中筛选出具有以商业化为目的所预期的转基因表达量和表达模式的单一事件。具有预期的转基因表达量和表达模式的事件可用于采用常规育种方法通过有性异型杂交将转基因渗入到其他遗传背景中。通过这种杂交方式产生的后代保持了原始转化体的转基因表达特征。应用这种策略模式可以确保在许多品种中具有可靠的基因表达，而这些品种能很好的适应当地的生长条件。
能够检测特定事件的存在以确定有性杂交的后代是否包含目的基因将是有益的。此外，检测特定事件的方法还将有助于遵守相关法规，例如来源于重组农作物的食物在投入市场前需要获得正式批准和进行标记。通过任何熟知的多核苷酸检测方法来检测转基因的存在都是可能的，例如聚合酶链式反应(PCR)或利用多核苷酸探针的DNA杂交。这些检测方法通常集中于常用的遗传元件，例如启动子、终止子、标记基因等。因此，除非与插入的转基因DNA相邻的染色体DNA(“侧翼DNA”)的序列是己知的，上述 这种方法就不能够用于区别不同的事件，特别是那些用相同的DNA构建体产生的事件。所以，目前常利用跨越了插入的转基因和侧翼DNA的接合部位的一对引物通过PCR来鉴定转基因特定事件，具体地说是包含于插入序列的第一引物和包含于插入序列的第二引物。
发明内容
本发明的目的是提供一种用于检测大豆植物DBN8007的核酸序列及其检测方法，转基因大豆事件DBN8007对昆虫具有较好的抗性并对草铵膦除草剂具有较好的耐受性，且检测方法可以准确快速的鉴定生物样品中是否包含转基因大豆事件DBN8007的DNA分子。
为实现上述目的，本发明提供了一种核酸序列，具有SEQ ID NO：3或其互补序列第1-553位中至少11个连续的核苷酸和SEQ ID NO：3或其互补序列第554-1407位中至少11个连续的核苷酸、和/或SEQ ID NO：4或其互补序列第1-348位中至少11个连续的核苷酸和SEQ ID NO：4或其互补序列第349-1022位中至少11个连续的核苷酸。
优选地，所述核酸序列具有SEQ ID NO：3或其互补序列第1-553位中22-25个连续的核苷酸和SEQ ID NO：3或其互补序列第554-1407位中22-25个连续的核苷酸、和/或SEQ ID NO：4或其互补序列第1-348位中22-25个连续的核苷酸和SEQ ID NO：4或其互补序列第349-1022位中22-25个连续的核苷酸。
优选地，所述核酸序列包含SEQ ID NO：1或其互补序列、和/或SEQ ID NO：2或其互补序列。
所述SEQ ID NO：1或其互补序列为转基因大豆事件DBN8007中在插入序列的5’末端位于插入接合部位附近的一个长度为22个核苷酸的序列，所述SEQ ID NO：1或其互补序列跨越了大豆插入位点的侧翼基因组DNA序列和插入序列的5’末端的DNA序列，包含所述SEQ ID NO：1或其互补序列即可鉴定为转基因大豆事件DBN8007的存在。所述SEQ ID NO：2或其互补序列为转基因大豆事件DBN8007中在插入序列的3’末端位于插入接合部位附近的一个长度为22个核苷酸的序列，所述SEQ ID NO：2或其互补序列跨越了插入序列的3’末端的DNA序列和大豆插入位点的侧翼基因组DNA序列，包含所述SEQ ID NO：2或其互补序列即可鉴定为转基因大豆事件DBN8007的存在。
优选地，所述核酸序列包含SEQ ID NO：3或其互补序列、和/或SEQ ID NO：4或其互补序列。
本发明中，所述核酸序列可以为所述SEQ ID NO：3或其互补序列中T-DNA插入序列的任何部分的至少11个或更多个连续多核苷酸(第一核酸序列)，或者为所述SEQ ID NO：3或其互补序列中5’侧翼大豆基因组DNA区域的任何部分的至少11个或更多个连续多核苷酸(第二核酸序列)。所述核酸序列进一步可以为同源于或互补于包含完整的所述SEQ ID NO：1的所述SEQ ID NO：3的一部分。当第一核酸序列和第二核酸序列一起使用时，这些核酸序列可作为DNA引物对用于产生扩增产物的DNA扩增方法中。使用DNA引物对在DNA扩增方法中产生的扩增产物是包括SEQ ID NO：1的扩增产物时，可以诊断转基因大豆事件DBN8007或其后代的存在。所述SEQ ID NO：3或其互补序列为转基因大豆事件DBN8007中在T-DNA插入序列的5’末端位于插入接合部位附近的一个长度为1407个核苷酸的序列，所述SEQ ID NO：3或其互补序列由553个核苷酸的大豆基因组5’侧翼序列(SEQ ID NO：3的核苷酸第1-553位)、356个pDBN4006构建体DNA序列中的核苷酸(SEQ ID NO：3的核苷酸第554-909位)和498个核苷酸的prAtAct2转录起始序列(SEQ ID NO：3的核苷酸第910-1407位)组成，包含所述SEQ ID NO：3或其互补序列即可鉴定为转基因大豆事件DBN8007的存在。
所述核酸序列可以为所述SEQ ID NO：4或其互补序列中T-DNA插入序列的任何部分的至少11个或更多个连续多核苷酸(第三核酸序列)，或者为所述SEQ ID NO：4或其互补序列中3’侧翼大豆基因组DNA区域的任何部分的至少11个或更多个连续多核苷酸(第四核酸序列)。所述核酸序列进一步可以为同源于或互补于包含完整的所述SEQ ID NO：2的所述SEQ ID NO：4的一部分。当第三核酸序列和第四核酸序列一起使用时，这些核酸序列可作为DNA引物对用于产生扩增产物的DNA扩增方法中。使用DNA引物对在DNA扩增方法中产生的扩增产物是包括SEQ ID NO：2的扩增产物时，可以诊断转基因大豆事件DBN8007或其后代的存在。所述SEQ ID NO：4或其互补序列为转基因大豆事件DBN8007中在插入序列的3’末端位于T-DNA插入接合部位附近的一个长度为1022个核苷酸的序列，所述SEQ ID NO：4或其互补序列由145个核苷酸的t35S转录终止子的DNA序列(SEQ ID NO：4的核苷酸第1-145位)、203个pDBN4006构建体DNA序列中的核苷酸(SEQ ID NO：4的核苷酸第146-348位)和674个核苷酸的大豆基因组3’侧翼序列(SEQ ID NO：4的核苷酸第349-1022位)组成，包含所述SEQ ID NO：4或其互补序列即可鉴定为转基因大豆事件DBN8007的存在。
进一步地，所述核酸序列包含SEQ ID NO：5或其互补序列。
所述SEQ ID NO：5或其互补序列为表征转基因大豆事件DBN8007的长度为11935个核苷酸的序列，其具体包含的基因组和遗传元件如表1所示。包含所述SEQ ID NO：5或其互补序列即可鉴定为转基因大豆事件DBN8007的存在。
表1、SEQ ID NO：5包含的基因组及遗传元件
Figure PCTCN2019099996-appb-000001
本领域技术人员熟知的，第一、第二、第三和第四核酸序列不必仅仅由DNA组成，也可包括RNA、DNA和RNA的混合物，或者DNA、RNA或其它不作为一种或多种聚合酶模板的核苷酸或其类似物的组合。此外，本发明中所述探针或引物应该是至少大约11、12、13、14、15、16、17、18、19、20、21或22个连续核苷酸的长度，其可以选自SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4和SEQ ID NO：5中所述的核苷酸。当选自SEQ ID NO：3、SEQ ID NO：4和SEQ ID NO：5所示的核苷酸时，所述探针和引物可以为长度是至少大约21个到大约50个或更多的连续核苷酸。
所述核酸序列或其互补序列可用于DNA扩增法中以产生扩增子，所述扩增子用于检测生物样品中转基因大豆事件DBN8007或其后代的存在；所述核酸序列或其互补序列可用于核苷酸检测法中，以检测生物样品中转基因大豆事件DBN8007或其后代的存在。
为实现上述目的，本发明还提供了一种检测样品中转基因大豆事件DBN8007的DNA存在的方法，包括：
使待检测样品与用于扩增目标扩增产物的至少两种引物在核酸扩增反应中接触；
进行核酸扩增反应；和
检测所述目标扩增产物的存在；
所述目标扩增产物包含所述核酸序列。
优选地，所述目标扩增产物包含SEQ ID NO：1或其互补序列、SEQ ID NO：2或其互补序列、SEQ ID NO：6或其互补序列、和/或SEQ ID NO：7或其互补序列。
具体地，所述引物包括第一引物和第二引物，所述第一引物选自SEQ ID NO：1、SEQ ID NO：8和SEQ ID NO：10；所述第二引物选自SEQ ID NO：2、SEQ ID NO：9和SEQ ID NO：11。
为实现上述目的，本发明还提供了一种检测样品中转基因大豆事件DBN8007的DNA存在的方法，包括：
使待检测样品与探针接触，所述探针包含所述核酸序列；
使所述待检测样品和所述探针在严格杂交条件下杂交；和
检测所述待检测样品和所述探针的杂交情况。
所述严格条件可为在6×SSC(柠檬酸钠)、0.5％SDS(十二烷基硫酸钠)溶液中，在65℃下杂交，然后用2×SSC、0.1％SDS和1×SSC、0.1％SDS各洗膜1次。
优选地，所述探针包含SEQ ID NO：1或其互补序列、SEQ ID NO：2或其互补序列、SEQ ID NO：6或其互补序列、和/或SEQ ID NO：7或其互补序列。
可选地，至少一个所述探针用至少一种荧光基团标记。
为实现上述目的，本发明还提供了一种检测样品中转基因大豆事件DBN8007的DNA存在的方法，包括：
使待检测样品与标记物核酸分子接触，所述标记物核酸分子包括所述核酸序列；
使所述待检测样品和所述标记物核酸分子在严格杂交条件下杂交；
检测所述待检测样品和所述标记物核酸分子的杂交情况，进而通过标记物辅助育种分析以确定昆虫抗性和/或除草剂耐受性与标记物核酸分子在遗传学上是连锁的。
优选地，所述标记物核酸分子包括选自以下的至少一种：SEQ ID NO：1或其互补序列、SEQ ID NO：2或其互补序列、和/或SEQ ID NO：6-11或其互补序列。
为实现上述目的，本发明还提供了一种DNA检测试剂盒，包括至少一个DNA分子，所述DNA分子包含所述核酸序列，其可以作为对于转基因大豆事件DBN8007或其后代具有特异性的DNA引物之一或探针。
优选地，所述DNA分子包含SEQ ID NO：1或其互补序列、SEQ ID NO：2或其互补序列、SEQ ID NO：6或其互补序列、和/或SEQ ID NO：7或其互补序列。
为实现上述目的，本发明还提供了一种植物细胞，包含编码昆虫抗性Vip3Aa蛋白的核酸序列、编码草铵膦除草剂耐受性PAT蛋白的核酸序列和特定区域的核酸序列，所述特定区域的核酸序列包括SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：6和/或SEQ ID NO：7所示的序列。
优选地，所述植物细胞包含编码昆虫抗性Vip3Aa蛋白的核酸序列、编码草铵膦除草剂耐受性PAT蛋白的核酸序列和特定区域的核酸序列，所述特定区域的核酸序列包括SEQ ID NO：3和/或SEQ ID NO：4所示的序列。
优选地，所述植物细胞依次包含SEQ ID NO：1、SEQ ID NO：5第5594-11006位核酸序列和SEQ ID NO：2，或者包含SEQ ID NO：5所示的序列。
为实现上述目的，本发明还提供了一种保护大豆植物免于昆虫侵袭的方法，包括在靶昆虫的膳食中提供至少一种转基因大豆植物细胞，所述转基因大豆植物细胞在其基因组中包含SEQ ID NO：1和/或SEQ ID NO：2所示的序列，摄食所述转基因大豆植物细胞的靶昆虫被抑制进一步摄食所述转基因大豆植物。
优选地，所述转基因大豆植物细胞在其基因组中包含SEQ ID NO：3和/或SEQ ID NO：4所示的序列。
优选地，所述转基因大豆植物细胞在其基因组中依次包含SEQ ID NO：1、SEQ ID NO：5第5594-11006位核酸序列和SEQ ID NO：2，或者包含SEQ ID NO：5。
为实现上述目的，本发明还提供了一种保护大豆植物免受由除草剂引起的损伤或控制种植大豆植物的大田中杂草的方法，包括将含有有效剂量草铵膦除草剂施加到种植至少一种转基因大豆植物的大田中，所述转基因大豆植物在其基因组中包含SEQ ID NO：1和/或SEQ ID NO：2所示的序列，所述转基因大豆植物对草铵膦除草剂具有耐受性。
优选地，所述转基因大豆植物在其基因组中包含SEQ ID NO：3和/或SEQ ID NO：4所示的序列。
优选地，所述转基因大豆植物在其基因组中依次包含SEQ ID NO：1、SEQ ID NO：5第5594-11006位核酸序列和SEQ ID NO：2，或者包含SEQ ID NO：5所示的序列。
为实现上述目的，本发明还提供了一种培养对昆虫具有抗性和/或耐受草铵膦除草剂的大豆植物的方法，包括：
种植至少一粒大豆种子，所述大豆种子的基因组中包含编码昆虫抗性Vip3Aa蛋白的核酸序列和/或编码草铵膦除草剂耐受性PAT蛋白的核酸序列、和特定区域的核酸序列，或者所述大豆种子的基因组中包含SEQ ID NO：5所示的核酸序列；
使所述大豆种子长成大豆植株；
用靶昆虫侵袭所述大豆植株和/或用有效剂量草铵膦除草剂喷洒所述大豆植株，收获与其他不具有特定区域的核酸序列的植株相比具有减弱的植物损伤的植株；
所述特定区域的核酸序列为SEQ ID NO：1和/或SEQ ID NO：2所示的序列；优选地，所述特定区域的核酸序列为SEQ ID NO：3和/或SEQ ID NO：4所示的序列。
为实现上述目的，本发明还提供了一种产生对昆虫具有抗性和/或对草铵膦除草剂具有耐受性的大豆植株的方法，包括将第一大豆植物基因组中包含的编码昆虫抗性Vip3Aa蛋白的核酸序列和/或编码草铵膦耐受性PAT蛋白的核酸序列、和特定区域的核酸序列，或者将所述第一大豆植物基因组中包含的SEQ ID NO：5所示的核酸序列，引入第二大豆植物，从而产生大量子代植株；选择具有所述特定区域的核酸序列的所述子代植株，且所述子代植株对昆虫具有抗性和/或对草铵膦除草剂具有耐受性；所述特定区域的核酸序列为SEQ ID NO：1和/或SEQ ID NO：2所示的序列；优选地，所述特定区域的核酸序列为SEQ ID NO：3和/或SEQ ID NO：4所示的序列；
优选地，所述方法包括将转基因大豆事件DBN8007与缺少昆虫抗性和/或草铵膦耐受性的大豆植株进行有性杂交，从而产生大量子代植株，选择具有所述特定区域的核酸序列的所述子代植株；
用靶昆虫侵袭和/或用草铵膦处理所述子代植株；
选择对昆虫具有抗性和/或对草铵膦除草剂具有耐受性的所述子代植株。
为实现上述目的，本发明还提供了一种产生自转基因大豆事件DBN8007的农产品或商品，所述农产品或商品为卵磷脂、脂肪酸、甘油、固醇、大豆片、大豆粉、大豆蛋白或其浓缩物、大豆油、大豆蛋白纤维、豆浆凝块或豆腐。
在本发明用于检测大豆植物的核酸序列及其检测方法中，以下定义和方法可以更好地定义本发明和指导本领域的普通技术人员实施本发明，除非另作说明，根据本领域普通技术人员的常规的用法来理解术语。
所述“大豆”是指黄豆(Glycine max)，并且包括可以与大豆交配的所有植物品种，包括野生大豆种。
所述“包含”、“包括”或“含有”是指“包括但不限于”。
术语“植物”包括整株植物、植物细胞、植物器官、植物原生质体、植物可以从中再生的植物细胞组织培养物、植物愈伤组织、植物丛(plant clumps)和植物或植物部分中完整的植物细胞，所述植物部分例如胚、花粉、胚珠、种子、叶、花、枝、果实、茎秆、 根、根尖、花药等。应理解为本发明范围内的转基因植物的部分包括但不限于植物细胞、原生质体、组织、愈伤组织、胚以及花、茎、果实、叶和根，以上植物部分源自事先用本发明的DNA分子转化的并因此至少部分地由转基因细胞组成的转基因植物或其子代。
术语“基因”是指表达特定蛋白的核酸片段，包括编码序列前的调节序列(5’非编码序列)和编码序列后的调节序列(3’非编码序列)。“天然基因”是指天然发现具有其自身调节序列的基因。“嵌合基因”是指不是天然基因的任何基因，其包含非天然发现的调节和编码序列。“内源基因”是指天然基因，所述天然基因位于生物体基因组中它的天然位置。“外源基因”是现存在于生物的基因组中且原来不存在的外来基因，也指经转基因步骤导入受体细胞的基因。外源基因可以包含插入非天然生物体的天然基因或嵌合基因。“转基因”是通过转化程序已经被引入基因组的基因。植物基因组中重组DNA已被插入的位点可以称为“插入位点”或“靶位点”。
“侧翼DNA”可以包含天然存在于例如植物的生物体中的基因组或通过转化过程引入的外源(异源)DNA，例如与转化事件相关的片段。因此，侧翼DNA可以包括天然和外源DNA的组合。在本发明中，“侧翼DNA”亦称“侧翼区”或“侧翼序列”或“侧翼基因组序列”或“侧翼基因组DNA”，是指至少3、5、10、11、15、20、50、100、200、300、400、1000、1500、2000、2500或5000碱基对或更长的序列，其位于最初外源插入DNA分子的直接上游或下游并且与最初外源插入DNA分子相邻。当该侧翼区位于下游时，其也可以称为“3’侧翼”或“左边界侧翼”等。当该侧翼区位于上游时，其也可以称为“5’侧翼”或“右边界侧翼”等。
引起外源DNA的随机整合的转化程序会导致含有不同侧翼区的转化体，所述不同侧翼区是每个转化体所特异性含有的。当重组DNA通过传统杂交被引入植物时，其侧翼区通常不会改变。转化体也会含有异源插入物DNA和基因组DNA的段之间或两段基因组DNA之间或两段异源DNA之间的独特的接合。“接合”是两个具体的DNA片段连接的点。例如，接合存在于插入物DNA连接侧翼DNA的位置。接合点还存在于转化的生物体中，其中两个DNA片段以修饰自天然生物体中发现的方式的连接在一起。“接合区域”或“接合序列”是指包含接合点的DNA。
本发明提供了称为DBN8007的转基因大豆事件及其后代，所述转基因大豆事件DBN8007亦称为大豆植物DBN8007，其包括转基因大豆事件DBN8007的植物和种子及其植物细胞或其可再生部分，所述转基因大豆事件DBN8007的植物部分，包括但不限于细胞、花粉、胚珠、花、芽、根、茎、叶、荚和来自大豆植物DBN8007的产物，例如大豆饼、粉和油，具体可以为卵磷脂、脂肪酸、甘油、固醇、食用油、脱脂大豆片、包括脱脂的和烘烤的大豆粉、豆浆凝块、豆腐、大豆蛋白浓缩物、分离的大豆蛋白、水解植物蛋白、组织化大豆蛋白和大豆蛋白纤维。
本发明转基因大豆事件DBN8007包含了一个DNA构建体，当其在植物细胞内表达时，所述转基因大豆事件DBN8007获得对昆虫的抗性和对草铵膦除草剂的耐受性。所述DNA构建体包含两个串联的表达盒，第一个表达盒包含用于在植物中表达的适合的启动子和适合的多聚腺苷酸化信号序列，所述启动子可操作地连接Vip3Aa蛋白的核酸序列，所述Vip3Aa蛋白的核酸序列主要对鳞翅目昆虫具有抗性。第二个表达盒包含用于在植物中表达的适合的启动子和适合的多聚腺苷酸化信号序列，所述启动子可操作地连接编码膦丝菌素N-乙酰基转移酶(phosphinothricin N-acetyltransferase，PAT)的基因，所述PAT蛋白的核酸序列对草铵膦除草剂具有耐受性。进一步地，所述启动子可以为从植物分离的适合启动子，包括组成型、诱导型和/或组织特异性启动子，所述适合启动子包括但不限于，花椰菜花叶病毒(CaMV)35S启动子、玄参花叶病毒(FMV)35S启动子、泛素蛋白(Ubiquitin)启动子、肌动蛋白(Actin)启动子、土壤农杆菌(Agrobacterium tumefaciens)胭脂碱合成酶(NOS)启动子、章鱼碱合成酶(OCS)启动子、夜香树属(Cestrum)黄 叶卷曲病毒启动子、马铃薯块茎储藏蛋白(Patatin)启动子、核酮糖-1，5-二磷酸羧化酶/加氧酶(RuBisCO)启动子、谷胱甘肽硫转移酶(GST)启动子、E9启动子、GOS启动子、alcA/alcR启动子、毛根农杆菌(Agrobacterium rhizogenes)RolD启动子和拟南芥属(Arabidopsis thaliana)Suc2启动子。所述多聚腺苷酸化信号序列可以为在植物中起作用的适合多聚腺苷酸化信号序列，所述适合多聚腺苷酸化信号序列包括但不限于，来源于土壤农杆菌(Agrobacterium tumefaciens)胭脂碱合成酶(NOS)基因的多聚腺苷酸化信号序列、来源于花椰菜花叶病毒(CaMV)35S终止子、来源于蛋白酶抑制剂||(PIN||)基因的多聚腺苷酸化信号序列和来源于α-微管蛋白(α-tubulin)基因的多聚腺苷酸化信号序列。
此外，所述表达盒还可以包括其他的遗传元件，所述遗传元件包括但不限于，增强子和信号肽/转运肽。所述增强子可以加强基因的表达水平，所述增强子包括但不限于，烟草蚀刻病毒(TEV)翻译激活因子、CaMV35S增强子和FMV35S增强子。所述信号肽/转运肽可以引导Vip3Aa蛋白和/或PAT蛋白转运到细胞外或者细胞内特定的细胞器或区室，例如，利用编码叶绿体转运肽序列靶向叶绿体，或者利用‘KDEL’保留序列靶向内质网。
所述Vip3Aa基因可以是从苏云金芽胞杆菌(Bacillus thuringiensis，简称Bt)中分离得到的，且可以通过优化密码子或者以其它方式改变Vip3Aa基因的核苷酸序列，以达到增加转化细胞中转录物的稳定性和可利用性的目的。
所述“鳞翅目(Lepidoptera)”，包括蛾、蝶两类昆虫，是农林害虫最多的一个目，如小地老虎、棉铃虫、斜纹夜蛾、二点委夜蛾、桃蛀螟等。
所述膦丝菌素N-乙酰基转移酶(PAT)基因可以是从链霉菌(Streptomyces viridochromogenes)菌株分离的酶，通过乙酰化催化L-膦丝菌素转化为其无活性形式，以赋予植物对草铵膦除草剂的耐受性。Phosphinothricin(PTC，2-氨基-4-甲膦酰丁酸)是谷氨酰胺合成酶的抑制剂。PTC是抗生素2-氨基-4-甲膦酰-丙氨酰-丙氨酸的结构单位，此三肽(PTT)具有抗革兰氏阳性和革兰氏阴性细菌以及抗真菌灰葡萄孢(Botrytis cinerea)的活性。膦丝菌素N-乙酰基转移酶(PAT)基因也可以作为选择性标记基因。
所述“草铵膦”又名草丁膦，是指2-氨基-4-[羟基(甲基)膦酰基]丁酸铵，用“草铵膦除草剂”处理是指使用任何一种含有草铵膦的除草剂制剂进行处理。为了达到有效生物学剂量而对某种草铵膦制剂使用率的选择不超过普通农艺技术人员的技能。使用任何一种含有草铵膦的除草剂制剂处理包含了来源于转基因大豆事件DBN8007的植物材料的田地，将控制所述田地中的杂草生长，并且不影响来源于转基因大豆事件DBN8007的植物材料的生长或产量。
所述DNA构建体采用转化方法被引入到植物中，所述转化方法包括但不限于，农杆菌(Agrobacterium)介导转化法、基因枪转化法和花粉管通道转化法。
所述农杆菌介导转化法是植物转化的常用方法。将要引入到植物中的外源DNA克隆到载体的左和右边界共有序列之间，即T-DNA区。所述载体被转化到农杆菌细胞中，随后，所述农杆菌细胞用于感染植物组织，包含外源DNA的载体的所述T-DNA区被插入到植物基因组中。
所述基因枪转化法即为用包含外源DNA的载体轰击植物细胞(粒子介导的生物弹击转化)。
所述花粉管通道转化法是利用植物授粉后所形成的天然的花粉管通道(又名花粉管引导组织)，经珠心通道，将外源DNA携带入胚囊。
转化后，必须从转化的植物组织再生转基因植物，并且利用适合的标记选择具有外源DNA的后代。
DNA构建体是DNA分子互相连接起来的组合，该组合提供了一个或多个表达盒。DNA构建体优选地是能够在细菌细胞内自我复制，而且含有不同的限制性内切酶位点的 质粒，所含的限制性内切酶位点用于导入提供功能性基因元件，即启动子、内含子、前导序列、编码序列、3’终止子区域和其他序列的DNA分子。DNA构建体中所含有的表达盒包括提供信使RNA的转录所必需的基因元件，所述表达盒可以设计为在原核细胞或真核细胞中表达。本发明的表达盒被设计为最优选地在植物细胞内表达。
转基因“事件”是通过用异源DNA构建体转化植物细胞而得到的，即包括至少一个含有目标基因的核酸表达盒，通过转基因的方法插入到植物基因组中以产生植物群体，再生所述植物群体，和选择具有插入特定基因组位点特征的特定植株。术语“事件”是指含有异源DNA的原始转化体和该转化体的后代。术语“事件”还指原始转化体和含有异源DNA的其它品种个体之间进行有性杂交而得到的后代，即使在与回交亲本进行反复回交后，来自于原始转化体亲本的插入DNA和侧翼基因组DNA也存在于杂交后代中的同一染色体位置。术语“事件”还指来自原始转化体的DNA序列，该DNA序列包含插入DNA和与插入DNA紧密相邻的侧翼基因组序列，该DNA序列被预期转移到子代中，该子代由含有插入DNA的亲本系(例如原始转化体和其自交产生的子代)与不含有插入DNA的亲本系进行有性杂交而产生，且该子代接受了包含目标基因的插入DNA。
本发明中“重组”是指通常不能在自然界中发现并且因此通过人工干预产生的DNA和/或蛋白和/或生物体的形式。这种人工干预可产生重组DNA分子和/或重组植物。所述“重组DNA分子”是通过人工组合两种在其它情况下是分离的序列区段而获得的，例如通过化学合成或通过遗传工程技术操作分离的核酸区段。进行核酸操作的技术是众所周知的。
术语“转基因”包括任何细胞、细胞系、愈伤组织、组织、植物部分或植物，以上的基因型由于异源核酸的存在而改变，所述“转基因”包括最初被这样改变的转基因体以及由最初的转基因体通过有性杂交或无性繁殖生成的子代个体。在本发明中，术语“转基因”不包括通过常规植物育种方法或天然发生事件的基因组的(染色体的或染色体外的)改变，所述天然发生事件例如随机异体受精、非重组病毒感染、非重组细菌转化、非重组转座或自发突变。
本发明中“异源的”是指自然界中第一分子通常不被发现与第二分子组合。例如，分子可以源自第一物种并插入到第二物种的基因组中。因此这种分子对于宿主是异源的并被人工引入宿主细胞的基因组中。
培养对鳞翅目昆虫具有抗性且对草铵膦除草剂具有耐受性的转基因大豆事件DBN8007，通过以下步骤：首先使第一亲本大豆植物与第二亲本大豆植物有性杂交，从而产生了多样的第一代子代植株，所述第一亲本大豆植物由培育自转基因大豆事件DBN8007及其后代的大豆植物组成，该转基因大豆事件DBN8007及其后代是通过利用本发明的对鳞翅目昆虫具有抗性且对草铵膦除草剂具有耐受性的表达盒进行转化而得到的，第二亲本大豆植物缺乏对鳞翅目昆虫的抗性和/或对草铵膦除草剂具有耐受性；然后选择对鳞翅目昆虫的侵袭具有抗性和/或对草铵膦除草剂具有耐受性的子代植株，可以培育出对鳞翅目昆虫具有抗性且对草铵膦除草剂具有耐受性的大豆植物。这些步骤可以进一步包括使鳞翅目昆虫抗性和/或草铵膦耐受性的子代植株与第二亲本大豆植物或第三亲本大豆植物进行回交，然后通过用鳞翅目昆虫侵袭、草铵膦除草剂施加或通过与性状相关的分子标记物(如包含转基因大豆事件DBN8007中插入序列的5’端和3’端鉴定出的接合位点的DNA分子)的鉴定来选择子代，从而产生对鳞翅目昆虫具有抗性且对草铵膦除草剂具有耐受性的大豆植物。
还应理解的是，两种不同的转基因植物也可以交配以产生含有两个独立的、分离式添加的外源基因的后代。适当后代的自交可以得到对两个添加的外源基因来说都是纯合子的后代植株。如前所述的对亲本植株的回交和与非转基因植物的异型杂交也是可以预期的，无性繁殖也是同样的。
术语“探针”是一段分离的核酸分子，其上面结合有常规的可检测标记或报告分子，例如，放射性同位素、配体、化学发光剂或酶类。这种探针与目标核酸的一条链是互补的，在本发明中，探针与来自转基因大豆事件DBN8007基因组的一条DNA链互补，不论该基因组DNA是来自转基因大豆事件DBN8007或种子还是来源于转基因大豆事件DBN8007的植物或种子或提取物。本发明的探针不仅包括脱氧核糖核酸或核糖核酸，还包括特异性地与目标DNA序列结合并可用于检测该目标DNA序列的存在的聚酰胺及其他探针材料。
术语“引物”是一段分离的核酸分子，其通过核酸杂交，退火结合到互补的目标DNA链上，在引物和目标DNA链之间形成杂合体，然后在聚合酶(例如DNA聚合酶)的作用下，沿目标DNA链延伸。本发明的引物对涉及其在目标核酸序列扩增中的应用，例如，通过聚合酶链式反应(PCR)或其他常规的核酸扩增方法。
探针和引物的长度一般是11个多核苷酸或更多，优选的是18个多核苷酸或更多，更优选的是24个多核苷酸或更多，最优选的是30个多核苷酸或更多。这种探针和引物在高度严格杂交条件下与目标序列特异性地杂交。尽管不同于目标DNA序列且对目标DNA序列保持杂交能力的探针是可以通过常规方法设计出来的，但是，优选的，本发明中的探针和引物与目标序列的连续核酸具有完全的DNA序列同一性。
基于本发明的侧翼基因组DNA和插入序列的引物和探针可以通过常规方法确定，例如，通过从来源于转基因大豆事件DBN8007的植物材料中分离相应的DNA分子，并确定该DNA分子的核酸序列。所述DNA分子包含转基因插入序列和大豆基因组侧翼序列，所述DNA分子的片段可以用作引物或探针。
本发明的核酸探针和引物在严格条件下与目标DNA序列杂交。任何常规的核酸杂交或扩增方法都可以用于鉴定样品中来源于转基因大豆事件DBN8007的DNA的存在。核酸分子或其片段在一定情况下能够与其他核酸分子进行特异性杂交。如本发明使用的，如果两个核酸分子能形成反平行的双链核酸结构，就可以说这两个核酸分子彼此间能够进行特异性杂交。如果两个核酸分子显示出完全的互补性，则称其中一个核酸分子是另一个核酸分子的“互补物”。如本发明使用的，当一个核酸分子的每一个核苷酸都与另一个核酸分子的对应核苷酸互补时，则称这两个核酸分子显示出“完全互补性”。如果两个核酸分子能够以足够的稳定性相互杂交从而使它们在至少常规的“低度严格”条件下退火且彼此结合，则称这两个核酸分子为“最低程度互补”。类似地，如果两个核酸分子能够以足够的稳定性相互杂交从而使它们在常规的“高度严格”条件下退火且彼此结合，则称这两个核酸分子具有“互补性”。从完全互补性中偏离是可以允许的，只要这种偏离不完全阻止两个分子形成双链结构。为了使一个核酸分子能够作为引物或探针，仅需保证其在序列上具有充分的互补性，以使得在所采用的特定溶剂和盐浓度下能形成稳定的双链结构。
如本发明使用的，基本同源的序列是一段核酸分子，该核酸分子在高度严格条件下能够和相匹配的另一段核酸分子的互补链发生特异性杂交。促进DNA杂交的适合的严格条件，例如，大约在45℃条件下用6.0×氯化钠/柠檬酸钠(SSC)处理，然后在50℃条件下用2.0×SSC洗涤，这些条件对本领域技术人员是公知的。例如，在洗涤步骤中的盐浓度可以选自低度严格条件的约2.0×SSC、50℃到高度严格条件的约0.2×SSC、50℃。此外，洗涤步骤中的温度条件可以从低度严格条件的室温约22℃，升高到高度严格条件的约65℃。温度条件和盐浓度可以都发生改变，也可以其中一个保持不变而另一个变量发生改变。优选地，本发明的一个核酸分子可以在中度严格条件下，例如在约2.0×SSC和约65℃下与SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6和SEQ ID NO：7中一个或多个核酸分子或其互补序列，或者上述序列的任一片段发生特异性杂交。更优选地，本发明的一个核酸分子在高度严格条件下与SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6和SEQ ID NO：7中一个或多个核酸分子或其互补序列，或者上述序列的任一片段发生特异性杂 交。本发明中，优选的标记物核酸分子具有SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：6或SEQ ID NO：7或其互补序列，或者上述序列的任一片段。本发明另一优选的标记物核酸分子与SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：6或SEQ ID NO：7或其互补序列，或者上述序列的任一片段具有80％到100％或90％到100％的序列同一性。SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：6和SEQ ID NO：7可以用作植物育种方法中的标记物以鉴定遗传杂交的后代。探针与目标DNA分子的杂交可以通过任何一种为本领域技术人员所熟知的方法进行检测，这些方法包括但不限于，荧光标记、放射性标记、抗体类标记和化学发光标记。
关于使用特定的扩增引物对目标核酸序列进行的扩增(例如，通过PCR)，“严格条件”指的是在DNA热扩增反应中仅允许引物对目标核酸序列发生杂交的条件，具有与目标核酸序列相应的野生型序列(或其互补序列)的引物，能够与所述目标核酸序列结合，并且优选产生唯一的扩增产物，扩增产物即扩增子。
术语“特异性结合(目标序列)”是指在严格杂交条件下探针或引物仅与包含目标序列的样品中的目标序列发生杂交。
如本发明使用的，“扩增子”是指作为核酸模板一部分的目标核酸序列的核酸扩增产物。例如，为了确定大豆植物是否由含有本发明转基因大豆事件DBN8007通过有性杂交方式产生，或采集自田地的大豆样品是否包含转基因大豆事件DBN8007，或大豆提取物，例如粗粉、面或油是否包含转基因大豆事件DBN8007，从大豆植物组织样品或提取物提取的DNA可以通过使用引物对的核酸扩增方法以产生对于转基因大豆事件DBN8007的DNA的存在是诊断性的扩增子。所述引物对包括一个来源于植物基因组中与插入的外源DNA插入位点相邻的侧翼序列的第一引物，和来源于插入的外源DNA的第二引物。扩增子具有一定长度和序列，所述序列对所述转基因大豆事件DBN8007也是诊断性的。扩增子的长度范围可以是引物对的结合长度加上一个核苷酸碱基对，优选加上约50个核苷酸碱基对，更优选加上约250个核苷酸碱基对，最优选加上约450个核苷酸碱基对或更多。
可选的，引物对可以来源于插入DNA两侧的侧翼基因组序列，以产生包括整个插入核苷酸序列的扩增子。来源于植物基因组序列的引物对中的一个可以位于距插入DNA序列一定距离处，该距离的范围可以为一个核苷酸碱基对到约两万个核苷酸碱基对。术语“扩增子”的使用特别排除了在DNA热扩增反应中形成的引物二聚体。
核酸扩增反应可以通过本领域已知的任何一种核酸扩增反应方法实现，包括聚合酶链式反应(PCR)。各种核酸扩增方法已是本领域技术人员所熟知的。PCR扩增方法已经发展到可扩增多达22kb的基因组DNA和多达42kb的噬菌体DNA。这些方法以及本领域的其他DNA扩增方法可以用于本发明。插入的外源DNA序列和来自转基因大豆事件DBN8007的侧翼DNA序列可以通过利用所提供的引物序列对转基因大豆事件DBN8007的基因组进行扩增，扩增后对PCR扩增子或克隆的DNA进行标准的DNA测序。
基于DNA扩增方法的DNA检测试剂盒含有用作引物的DNA分子，它们在适当的反应条件下特异性杂交到目标DNA上并扩增诊断性扩增子。试剂盒可提供基于琼脂糖凝胶的检测方法或者现有技术已知的检测诊断性扩增子的许多方法。含有与SEQ ID NO：3或SEQ ID NO：4的大豆基因组的任何部分同源或互补的、以及与SEQ ID NO：5的转基因插入区的任何部分同源或互补的DNA引物的试剂盒是本发明所提供的。特别地鉴别在DNA扩增方法中有用的引物对是SEQ ID NO：8和SEQ ID NO：9，其扩增与转基因大豆事件DBN8007的5’转基因/基因组区的一部分同源的诊断性扩增子，其中扩增子包括SEQ ID NO：1。用作DNA引物的其它DNA分子可选自SEQ ID NO：5。
这些方法所产生的扩增子可以通过多种技术进行检测。其中一个方法是遗传点分析(Genetic Bit Analysis)，该方法设计了一个跨越插入DNA序列和相邻的侧翼基因组DNA 序列的DNA寡核苷酸链。将该寡核苷酸链固定在一个微孔板的微孔内，在对目标区域进行PCR扩增后(在插入序列内和相邻的侧翼基因组序列中各使用一个引物)，单链PCR产物可与固定的寡核苷酸链进行杂交，并且作为单碱基延伸反应的模板，该延伸反应使用了DNA聚合酶和为下一个预期的碱基特定标记的ddNTPs。可以通过荧光或ELISA类方法得到结果。信号代表了插入/侧翼序列的存在，其说明扩增、杂交和单碱基延伸反应是成功的。
另一种方法是焦磷酸测序技术(Pyrosequencing)。该方法设计了一个跨越插入DNA序列和相邻的基因组DNA结合部位的寡核苷酸链。将该寡核苷酸链和目标区域的单链PCR产物(在插入序列内和相邻的侧翼基因组序列中各使用一个引物)进行杂交，然后和DNA聚合酶、ATP、硫酰基酶、荧光素酶、三磷酸腺苷双磷酸酶、腺苷-5’-磷硫酸盐和萤光素一起进行温育。分别加入dNTPs，测量产生的光信号。光信号代表了插入/侧翼序列的存在，其说明扩增、杂交、和单碱基或多碱基延伸反应是成功的。
Chen等(基因组研究(Genome Res.)9：492-498，1999)描述的荧光偏振现象也是可以用于检测本发明扩增子的一种方法。使用这种方法需要设计一个跨越插入DNA序列和相邻的基因组DNA结合部位的寡核苷酸链。将该寡核苷酸链和目标区域的单链PCR产物(在插入序列内和相邻的侧翼基因组序列中各使用一个引物)进行杂交，然后和DNA聚合酶以及一种荧光标记的ddNTP一起进行温育。单碱基延伸会导致插入ddNTP。这种插入可以利用荧光仪测量其偏振的改变。偏振的改变代表了插入/侧翼序列的存在，其说明扩增、杂交和单碱基延伸反应是成功的。
Taqman被描述为一种检测和定量分析DNA序列存在的方法，该方法在制造商所提供的使用说明中有详细介绍。现简要说明如下，设计一个跨越插入DNA序列和相邻的基因组侧翼结合部位的FRET寡核苷酸探针。该FRET探针和PCR引物(在插入序列内和相邻的侧翼基因组序列中各使用一个引物)在热稳定聚合酶和dNTPs存在下进行循环反应。FRET探针的杂交导致FRET探针上荧光部分和淬灭部分的分裂以及荧光部分的释放。荧光信号的产生代表了插入/侧翼序列的存在，其说明扩增和杂交是成功的。
基于杂交原理，用于检测来源于转基因大豆事件DBN8007的植物材料的适合技术还可以包括Southern印迹杂交(Southern blot)、Northern印迹杂交(Northern blot)和原位杂交(in situ hybridization)。特别地，所述适合技术包括温育探针和样品，洗涤以移除未结合的探针和检测探针是否已经杂交。所述的检测方法取决于探针所附标记的类型，例如，通过X光片曝光和显影可以检测放射性标记的探针，或通过底物转化实现颜色变化可以检测酶标记的探针。
Tyangi等(自然生物技术(Nature Biotech.)14：303-308，1996)介绍了分子标记在序列检测中的应用。简要说明如下，设计一个跨越插入DNA序列和相邻的基因组侧翼结合部位的FRET寡核苷酸探针。该FRET探针的独特结构导致其含有二级结构，该二级结构能够在近距离内保持荧光部分和淬灭部分。该FRET探针和PCR引物(在插入序列内和相邻的侧翼基因组序列中各使用一个引物)在热稳定聚合酶和dNTPs存在下进行循环反应。经过成功的PCR扩增，FRET探针和目标序列的杂交导致探针二级结构的丧失，从而使荧光部分和淬灭部分在空间上发生分离，产生荧光信号。荧光信号的产生代表了插入/侧翼序列的存在，其说明扩增和杂交是成功的。
其他描述的方法，例如微流体(microfluidics)提供了分离和扩增DNA样品的方法和设备。光染料用于检测和测定特定的DNA分子。包含用于检测DNA分子的电子传感器或结合特定DNA分子的纳珠并因而可被检测的纳试管(nanotube)设备对于检测本发明的DNA分子是有用的。
可以使用本发明所述的组合物和DNA检测领域描述的或已知的方法来开发DNA检测试剂盒。所述试剂盒有利于鉴定样品中是否存在转基因大豆事件DBN8007的DNA，还可以用于培育含有转基因大豆事件DBN8007的DNA的大豆植物。所述试剂盒可以含 有DNA引物或探针，其同源于或互补于SEQ ID NO：1、2、3、4或5的至少一部分，或含有其它DNA引物或探针，其同源于或互补于DNA的转基因遗传元件中所含的DNA，这些DNA序列可以用于DNA扩增反应，或作为DNA杂交方法中的探针。在大豆基因组中含有的以及在图1和表1中说明的转基因插入序列与大豆基因组结合部位的DNA结构包含：位于转基因插入序列5’末端的大豆植物DBN8007侧翼基因组区域，来自农杆菌的右侧边界区域(RB)的一部分插入序列，第一个表达盒由拟南芥的ACTIN2启动子(prAtAct2)，可操作地连接到苏云金芽孢杆菌的昆虫抗性的mVip3Aa基因上，并可操作地连接到胭脂碱合酶基因的转录终止子(tNos)上而组成，第二个表达盒由含有花椰菜花叶病毒35S启动子(pr35S)，可操作地连接到链霉菌的草铵膦耐受性的膦丝菌素N-乙酰基转移酶基因(cPAT)上，并可操作地连接到花椰菜花叶病毒35S终止子(t35S)上而组成，来自农杆菌的左侧边界区域(LB)的一部分插入序列，以及位于转基因插入序列3’末端的大豆植物DBN8007侧翼基因组区域(SEQ ID NO：5)。在DNA扩增方法中，作为引物的DNA分子可以是来源于转基因大豆事件DBN8007中转基因插入序列的任何部分，也可以是来源于转基因大豆事件DBN8007的大豆基因组侧翼DNA序列的任何部分。
转基因大豆事件DBN8007可以与其他转基因大豆品种组合，例如除草剂(如草甘膦、麦草畏等)耐受性的转基因大豆品种，或携带其他抗虫基因的转基因大豆品种。所有这些不同转基因事件的各种组合，与本发明的转基因大豆事件DBN8007一起育种，可以提供抗多种虫害并抗多种除草剂的改良杂种转基因大豆品种。这些品种相比于非转基因品种和单性状的转基因品种可以表现出产量提升等更优异的特征。
本发明转基因大豆事件DBN8007是对鳞翅目害虫的摄食损伤有抗性的，并且耐受含草铵膦的农业除草剂的植物毒性作用。该双重性状的大豆植株表达苏云金芽孢杆菌的Vip3Aa蛋白，其提供了对鳞翅目害虫(如豆天蛾)摄食损伤的抗性，并表达链霉菌的草铵膦抗性的膦丝菌素N-乙酰基转移酶(PAT)蛋白，其赋予植物对草铵膦的耐受性。双重性状大豆具有如下优点：1)免受由于鳞翅目害虫(如豆天蛾、斜纹夜蛾等)造成的经济损失，豆天蛾、斜纹夜蛾等是大豆种植区的主要害虫；2)施加含草铵膦的农业除草剂给大豆作物用于广谱杂草控制的能力；3)大豆产量没有降低。此外，编码昆虫抗性和草铵膦耐受性性状的转基因连锁在同一DNA区段上，并且存在于转基因大豆事件DBN8007基因组的单一基因座上，这一点提供了增强的育种效率并使得能够用分子标记来追踪繁殖群体及其子代中的转基因插入片段。同时本发明检测方法中SEQ ID NO：1或其互补序列、SEQ ID NO：2或其互补序列、SEQ ID NO：6或其互补序列、或者SEQ ID NO：7或其互补序列可以作为DNA引物或探针以产生诊断为转基因大豆事件DBN8007或其后代的扩增产物，且可以快速、准确、稳定的鉴定出来源于转基因大豆事件DBN8007的植物材料的存在。
序列简述
SEQ ID NO：1 转基因大豆事件DBN8007中在插入序列5’末端位于插入接合部位附近的一个长度为22个核苷酸的序列，其中第1-11位核苷酸和第12-22位核苷酸分别位于大豆基因组上插入位点的两侧；
SEQ ID NO：2 转基因大豆事件DBN8007中在插入序列3’末端位于插入接合部位附近的一个长度为22个核苷酸的序列，其中第1-11位核苷酸和第12-22位核苷酸分别位于大豆基因组上插入位点的两侧；
SEQ ID NO：3 转基因大豆事件DBN8007中在插入序列的5’末端位于插入接合部位附近的一个长度为1407个核苷酸的序列；
SEQ ID NO：4 转基因大豆事件DBN8007中在插入序列的3’末端位于插入接合部位附近的一个长度为1022个核苷酸的序列；
SEQ ID NO：5 整个T-DNA序列、5’和3’末端的大豆基因组侧翼序列；
SEQ ID NO：6 位于SEQ ID NO：3上的序列，跨越了pDBN4006构建体DNA序列和prAtAct2转录起始序列；
SEQ ID NO：7 位于SEQ ID NO：4上的序列，跨越了t35S转录终止子序列和pDBN4006构建体DNA序列；
SEQ ID NO：8 扩增SEQ ID NO：3的第一引物；
SEQ ID NO：9 扩增SEQ ID NO：3的第二引物；
SEQ ID NO：10 扩增SEQ ID NO：4的第一引物；
SEQ ID NO：11 扩增SEQ ID NO：4的第二引物；
SEQ ID NO：12 5’侧翼基因组序列上的引物；
SEQ ID NO：13 与SEQ ID NO：12配对的位于T-DNA上的引物；
SEQ ID NO：14 3’侧翼基因组序列上的引物，其与SEQ ID NO：12配对可以检测转基因是纯合子或是杂合子；
SEQ ID NO：15 与SEQ ID NO：14配对的位于T-DNA上的引物；
SEQ ID NO：16 Taqman检测mVip3Aa基因的第一引物；
SEQ ID NO：17 Taqman检测mVip3Aa基因的第二引物；
SEQ ID NO：18 Taqman检测mVip3Aa基因的探针；
SEQ ID NO：19 Taqman检测PAT基因的第一引物；
SEQ ID NO：20 Taqman检测PAT基因的第二引物；
SEQ ID NO：21 Taqman检测PAT基因的探针；
SEQ ID NO：22 大豆内源基因lectin的第一引物；
SEQ ID NO：23 大豆内源基因lectin的第二引物；
SEQ ID NO：24 Southern杂交检测中mVip3Aa基因的探针；
SEQ ID NO：25 Southern杂交检测中PAT基因的探针；
SEQ ID NO：26 位于T-DNA上的引物，与SEQ ID NO：13方向一致；
SEQ ID NO：27 位于T-DNA上的引物，与SEQ ID NO：13方向相反，用作获得侧翼序列；
SEQ ID NO：28 位于T-DNA上的引物，与SEQ ID NO：13方向相反，用作获得侧翼序列；
SEQ ID NO：29 位于T-DNA上的引物，与SEQ ID NO：15方向一致；
SEQ ID NO：30 位于T-DNA上的引物，与SEQ ID NO：15方向相反，用作获得侧翼序列；
SEQ ID NO：31 位于T-DNA上的引物，与SEQ ID NO：15方向相反，用作获得侧翼序列。
下面通过附图和实施例，对本发明的技术方案做进一步的详细描述。
附图说明
图1为本发明用于检测大豆植物DBN8007的核酸序列及其检测方法的转基因插入序列与大豆基因组接合部位的结构示意图，以及用于检测大豆植物DBN8007的核酸序列相对位置的示意图(相对位置示意图参考Wm82.a2 RefGen)；
图2为本发明用于检测大豆植物DBN8007的核酸序列及其检测方法的重组表达载体pDBN4006的结构示意图；
图3为本发明用于检测大豆植物DBN8007的核酸序列及其检测方法的转基因大豆事件DBN8007对棉铃虫生物测定效果图；
图4为本发明用于检测大豆植物DBN8007的核酸序列及其检测方法的转基因大豆事件DBN8007对斜纹夜蛾生物测定效果图；
图5为本发明用于检测大豆植物DBN8007的核酸序列及其检测方法的转基因大豆事件DBN8007对甜菜夜蛾生物测定效果图；
图6为本发明用于检测大豆植物DBN8007的核酸序列及其检测方法的转基因大豆事件DBN8007对豆天蛾生物测定效果图；
图7为本发明用于检测大豆植物DBN8007的核酸序列及其检测方法的转基因大豆事件DBN8007接种棉铃虫的田间效果图；
图8为本发明用于检测大豆植物DBN8007的核酸序列及其检测方法的转基因大豆事件DBN8007在甜菜夜蛾自然发生条件下的田间效果图；
图9为本发明用于检测大豆植物DBN8007的核酸序列及其检测方法的转基因大豆事件DBN8007在斜纹夜蛾自然发生条件下的田间效果图；
图10为本发明用于检测大豆植物DBN8007的核酸序列及其检测方法的转基因大豆事件DBN8007对草地贪夜蛾生物测定效果图。
具体实施方式
下面通过具体实施例进一步说明本发明用于检测大豆植物DBN8007的核酸序列及其检测方法的技术方案。
第一实施例、克隆与转化
1.1、载体克隆
使用标准基因克隆技术构建重组表达载体pDBN4006(如图2所示)。所述载体pDBN4006包含两个串联的转基因表达盒，第一个表达盒由来自拟南芥的ACTIN2启动子(prAtAct2)，可操作地连接到来自苏云金芽孢杆菌的可提供昆虫抗性的mVip3Aa基因(CN 103509808 B)上，并可操作地连接到胭脂碱合酶的转录终止子(tNos)上而组成；第二个表达盒由含有花椰菜花叶病毒启动子(pr35S)，可操作地连接到链霉菌的草铵膦耐受性的膦丝菌素N-乙酰基转移酶基因(cPAT)上，并可操作地连接到花椰菜花叶病毒的转录终止子(t35S)上而组成。
将所述载体pDBN4006用液氮法转化到农杆菌LBA4404(Invitrgen，Chicago，USA；Cat.No：18313-015)中，并且以4-[羟基(甲基)膦酰基]-DL-高丙氨酸为选择标记对转化细胞进行筛选。
1.2、植物转化
采用常规的农杆菌侵染法进行转化，将无菌培养的大豆子叶节组织与本实施例1.1中所述的农杆菌共培养，以将构建的重组表达载体pDBN4006中的T-DNA转入到大豆染色体组中，以产生转基因大豆事件DBN8007。
对于农杆菌介导的大豆转化，简要地，将成熟的大豆种子在大豆萌发培养基(B5盐3.1g/L，B5维他命，蔗糖20g/L，琼脂8g/L，pH5.6)中进行萌发，将种子接种于萌发培养基上，按以下条件培养：温度25±1℃；光周期(光/暗)为16/8h。萌发4-6天后取鲜绿的子叶节处膨大的大豆无菌苗，在子叶节下3-4毫米处切去下胚轴，纵向切开子叶，去顶芽、侧芽和种子根。用解剖刀的刀背在子叶节处进行创伤，用农杆菌悬浮液接触创伤过的子叶节组织，其中农杆菌能够将mVip3Aa基因的核苷酸序列和PAT基因的核苷酸序列传递至创伤过的子叶节组织(步骤1：侵染步骤)。在此步骤中，子叶节组织优选地浸入农杆菌悬浮液(OD660＝0.5-0.8，侵染培养基(MS盐2.15g/L、B5维他命、蔗糖20g/L、葡萄糖10g/L、乙酰丁香酮(AS)40mg/L、2-吗啉乙磺酸(MES)4g/L、玉米素(ZT)2mg/L，pH5.3)中以启动侵染。子叶节组织与农杆菌共培养一段时期(3天)(步骤2：共培养步骤)。优选地，子叶节组织在侵染步骤后在固体培养基(MS盐4.3g/L、B5维他命、蔗糖20g/L、葡萄糖10g/L、2-吗啉乙磺酸(MES)4g/L、玉米素2mg/L、琼脂8g/L，pH5.6)上培养。在此共培养阶段后，有一个选择性的“恢复”步骤。在“恢复”步骤中，恢复培养基(B5盐3.1g/L、B5维他命、2-吗啉乙磺酸(MES)1g/L、蔗糖30g/L、玉米素(ZT)2mg/L、琼脂8g/L，头孢霉素150mg/L，谷氨酸100mg/L，天 冬氨酸100mg/L，pH5.6)中至少存在一种己知抑制农杆菌生长的抗生素(头孢霉素150-250mg/L)，不添加植物转化体的选择剂(步骤3：恢复步骤)。优选地，子叶节再生的组织块在有抗生素但没有选择剂的固体培养基上培养，以消除农杆菌并为侵染细胞提供恢复期。接着，子叶节再生的组织块在含选择剂(4-[羟基(甲基)膦酰基]-DL-高丙氨酸)的培养基上培养并选择生长着的转化愈伤组织(步骤4：选择步骤)。优选地，子叶节再生的组织块在有选择剂的筛选固体培养基(B5盐3.1g/L、B5维他命、2-吗啉乙磺酸(MES)1g/L、蔗糖30g/L、6-苄基腺嘌呤(6-BAP)1mg/L、琼脂8g/L，头孢霉素150mg/L，谷氨酸100mg/L，天冬氨酸100mg/L，4-[羟基(甲基)膦酰基]-DL-高丙氨酸10mg/L，pH5.6)上培养，导致转化的细胞可以继续生长。然后，转化的细胞再生成植物(步骤5：再生步骤)，优选地，在含选择剂的培养基上生长的子叶节再生的组织块在固体培养基(B5分化培养基和B5生根培养基)上培养以再生植物。
筛选得到的抗性组织块转移到所述B5分化培养基(B5盐3.1g/L、B5维他命、2-吗啉乙磺酸(MES)1g/L、蔗糖30g/L、玉米素(ZT)1mg/L、琼脂8g/L、头孢霉素150mg/L、谷氨酸50mg/L、天冬氨酸50mg/L、赤霉素1mg/L、生长素1mg/L、4-[羟基(甲基)膦酰基]-DL-高丙氨酸5mg/L，pH5.6)上，25℃下培养分化。分化出来的小苗转移到所述B5生根培养基(B5盐3.1g/L、B5维他命、2-吗啉乙磺酸(MES)1g/L、蔗糖30g/L、琼脂8g/L、头孢霉素150mg/L、吲哚-3-丁酸(IBA)1mg/L)，在生根培养上，25℃下培养至约10em高，移至温室培养至结实。在温室中，每天于温度26℃下培养16小时，再于温度20℃下培养8小时。
1.3、转基因事件的鉴定和筛选
一共产生了288个独立转基因T 0植株。为了筛选表现最优异的转基因事件，将上述288个独立转基因T 0单株，送入温室移栽进行培养、繁殖获得转基因T 1单株。
由于以成熟大豆种子且以草铵膦为筛选剂进行的大豆遗传转化过程容易产生假阳性转基因事件，因此在T 1代通过喷施草铵膦的方式对转基因事件进行阳性鉴定，共获得154个阳性转基因单株；通过TaqMan TM分析检测上述154个转基因大豆植株是否存在单拷贝的mVip3Aa和PAT基因，且不含载体骨架序列，共获得90个转基因单株；通过转基因插入位点分析，共筛选到24个T-DNA两侧序列完整、T-DNA没有插入到大豆基因组的重要基因中、基因插入没有产生较大的开放阅读框(ORF)的转基因单株；通过对主要靶标昆虫(如棉铃虫、斜纹夜蛾、甜菜夜蛾)的抗性评价和比较，共筛选到21个昆虫抗性良好的转基因单株；由于遗传转化、基因插入等均可能对大豆植株造成农艺性状上的影响(例如苗势、生育期、株高或倒伏等)，因此将上述21个转基因T2代单株种于田间以鉴定转基因T2单株在不同时期(苗期-盛花期、始粒期-成熟期)的农艺性状表现；再通过自交和回交选育的方式，在不同世代、不同地理环境和/或不同背景材料的情况下，通过对转基因大豆植株的农艺性状、分子生物学、靶标昆虫抗性、草铵膦耐受性等是否可稳定遗传进行筛选，选定了转基因大豆事件DBN8007是优异的，其具有单拷贝转基因(参见第二实施例)、良好的昆虫抗性、草铵膦除草剂耐受性和农艺性状表现(参见第六实施例和第七实施例)。
第二实施例、用TaqMan进行转基因大豆事件DBN8007检测
取转基因大豆事件DBN8007的叶片约100mg作为样品，用植物DNA提取试剂盒(DNeasy Plant Maxi Kit，Qiagen)提取其基因组DNA，通过Taqman探针荧光定量PCR方法检测mVip3Aa基因和PAT基因的拷贝数。同时以野生型大豆植株作为对照，按照上述方法进行检测分析。实验设3次重复，取平均值。
具体方法如下：
步骤1、取转基因大豆事件DBN8007的叶片100mg，在研钵中用液氮研成匀浆，每个样品取3个重复；
步骤2、使用植物DNA提取试剂盒(DNeasy Plant Maxi Kit，Qiagen)提取上述样品的基因组DNA，具体方法参考其产品说明书；
步骤3、用超微量分光光度计(NanoDrop 2000，Thermo Scientific)测定上述样品的基因组DNA浓度；
步骤4、调整上述样品的基因组DNA浓度至同一浓度值，所述浓度值的范围为80-100ng/μL；
步骤5、采用Taqman探针荧光定量PCR方法鉴定样品的拷贝数，以经过鉴定已知拷贝数的样品作为标准品，以野生型大豆植株的样品作为对照，每个样品3个重复，取其平均值；荧光定量PCR引物和探针序列分别是：
以下引物和探针用来检测mVip3Aa基因序列：
引物1：cgaatacagaaccctgtcggc如序列表中SEQ ID NO：16所示；
引物2：cgtgaggaaggtctcagaaatgac如序列表中SEQ ID NO：17所示；
探针1：cgacgatggcgtgtatatgcctcttgg如序列表中SEQ ID NO：18所示；
以下引物和探针用来检测PAT基因序列：
引物3：gagggtgttgtggctggtattg如序列表中SEQ ID NO：19所示；
引物4：tctcaactgtccaatcgtaagcg如序列表中SEQ ID NO：20所示；
探针2：cttacgctgggccctggaaggctag如序列表中SEQ ID NO：21所示；
PCR反应体系为：
Figure PCTCN2019099996-appb-000002
所述50×引物/探针混合物包含1mM浓度的每种引物各45μL，100μM浓度的探针50μL和860μL 1×TE缓冲液(10mM Tris-HCl、1mM EDTA，pH 8.0)，并且在4℃，贮藏在琥珀试管中。
PCR反应条件为：
Figure PCTCN2019099996-appb-000003
利用快速实时荧光定量PCR系统软件(Applied Biosystems 7900HT Fast Real-Time PCR System SDS v2.3，Applied Biosystems)分析数据，结果表明获得的转基因大豆事件DBN8007为单拷贝。
第三实施例、分析转基因大豆事件DBN8007的插入位点
3.1、基因组DNA提取
DNA提取按照常规采用的CTAB(十六烷基三甲基溴化铵)法：取2g转基因大豆事件DBN8007的幼嫩叶片在液氮中研磨成粉后，加入0.5mL于温度65℃预热的DNA提取CTAB缓冲液(20g/L CTAB、1.4M NaCl、100mM Tris-HCl、20mM EDTA(乙二胺四乙酸)，用NaOH调pH至8.0)，充分混匀后，于温度65℃抽提90min；加入0.5倍体积苯酚和0.5倍体积氯仿，颠倒混匀；12000rpm(每分钟转数)转速下离心10min；吸取上清液，加入2倍体积无水乙醇，轻柔晃动离心管，于温度4℃静置30min；12000rpm转速下再离心10min；收集DNA到管底；弃上清液，用1mL质量浓度为70％的乙醇，洗涤沉淀；12000rpm转速下离心5min；真空抽干或在超净台吹干；DNA沉淀溶解于适量的TE缓冲液中，保存在温度-20℃条件下。
3.2、侧翼DNA序列的分析
对上述提取的DNA样品进行浓度测定，使待测样品的浓度位于80-100ng/μL之间。用限制性内切酶EcoR I(5’端分析)和EcoR V(3’端分析)分别酶切基因组DNA。每个酶切体系中加入26.5μL基因组DNA，0.5μL上述限制性内切酶以及3μL酶切缓冲液(采用的限制性酶均是NEB公司的酶及其配套的缓冲液或通用缓冲液，现称NEBCutSmart)，酶切1h。待酶切结束后，向酶切体系中加入70μL无水乙醇，冰浴30min，转速12000rpm离心7min，弃上清，吹干，之后加入8.5μL双蒸水、1μL 10×T 4-DNA连接酶缓冲液(NEB T4 DNA Ligase Reaction Buffer，其具体配方可访问NEB网站或参考https：//www.neb.com/products/restriction-endonucleases、https：//www.neb.com/products/b0202-t4-dna-ligase-reaction-buffer)以及0.5μL T 4-DNA连接酶在温度4℃连接过夜。用一系列嵌套引物进行PCR扩增分离5’端和3’端基因组DNA。具体的，分离5’端基因组DNA的引物组合包括SEQ ID NO：13和SEQ ID NO：26作为第一引物，SEQ ID NO：27和SEQ ID NO：28作为第二引物，SEQ ID NO：13作为测序引物。分离3’端基因组DNA引物组合包括SEQ ID NO：15和SEQ ID NO：29作为第一引物，SEQ ID NO：30和SEQ ID NO：31作为第二引物，SEQ ID NO：15作为测序引物，PCR反应条件如表3所示。
上述PCR扩增反应所获得的扩增产物在质量分数为2.0％琼脂糖凝胶上电泳以分离PCR扩增产物，随后使用胶回收试剂盒(QIAquick Gel Extraction Kit，目录#_28704，Qiagen Inc.，Valencia，CA)从琼脂糖基质分离目的片段。然后对纯化的PCR扩增产物测序(例如，使用ABI PrismTM 377，PE Biosystems，Foster City，CA)并分析(例如，使用DNASTAR序列分析软件，DNASTAR Inc.，Madison，WI)。
使用标准PCR方法确认5’和3’侧翼序列和接合序列。5’侧翼序列和接合序列可使用SEQ ID NO：8或SEQ ID NO：12，组合SEQ ID NO：9、SEQ ID NO：13或SEQ ID NO：26来确认。3’侧翼序列和接合序列可使用SEQ ID NO：11或SEQ ID NO：14，组合SEQ ID NO：10、SEQ ID NO：15或SEQ ID NO：29来确认。PCR反应体系和扩增条件如表2和表3所示。本领域技术人员将理解，其它引物序列也可用于确认侧翼序列和接合序列。
PCR扩增产物的DNA测序提供了可以用于设计其他DNA分子的DNA，所述其他DNA分子作为引物和探针可用于鉴定来源于转基因大豆事件DBN8007的大豆植物或种子。
发现在SEQ ID NO：5的核苷酸1-5237位显示的为大豆基因组序列在转基因大豆事件DBN8007插入序列的右边界侧翼(5’侧翼序列)，在SEQ ID NO：5的核苷酸11210-11935位显示的为大豆基因组序列在转基因大豆事件DBN8007插入序列的左边界侧翼(3’侧翼序列)。5’接合序列在SEQ ID NO：1中列出，3’接合序列在SEQ ID NO：2中列出。
3.3、PCR接合性测定
接合序列是相对短的多核苷酸分子，其是新的DNA序列，当在多核酸检测分析中检测到时对于转基因大豆事件DBN8007的DNA是诊断性的。SEQ ID NO：1和SEQ ID NO：2中的接合序列为转基因大豆事件DBN8007中转基因片段的插入位点和大豆基因组DNA的每一侧的11个多核苷酸。更长或更短的多核苷酸接合序列可以从SEQ ID NO：3或SEQ ID NO：4中选择。接合序列(5’连接区域SEQ ID NO：1，和3’连接区域SEQ ID NO：2)作为DNA探针或作为DNA引物分子在DNA检测方法中是有用的。接合序列SEQ ID NO：6和SEQ ID NO：7也是转基因大豆事件DBN8007中新的DNA序列，其也可以作为DNA探针或作为DNA引物分子检测转基因大豆事件DBN8007 DNA的存在。所述SEQ ID NO：6(SEQ ID NO：3的核苷酸794-1012位)跨越了pDBN4006构建体DNA序列和prAtAct2转录起始序列，所述SEQ ID NO：7(SEQ ID NO：4的核苷酸1-243位)跨越了t35S转录终止序列和pDBN4006构建体DNA序列。
此外，通过使用来自SEQ ID NO：3或SEQ ID NO：4的至少一个引物来产生扩增子，所述引物用于PCR方法中时产生转基因大豆事件DBN8007的诊断性扩增子。
具体地，从转基因插入序列的5’末端产生PCR扩增产物，该PCR扩增产物包含来源于转基因大豆事件DBN8007的植物材料的基因组中侧翼于T-DNA插入序列的5’末端的基因组DNA的一部分。这个PCR扩增产物包含SEQ ID NO：3。为了进行PCR扩增，设计与侧翼于转基因插入序列的5’末端的基因组DNA序列杂交的引物5(SEQ ID NO：8)，和与之配对的位于转基因prAtAct2转录起始序列的引物6(SEQ ID NO：9)。
从转基因插入序列的3’末端产生PCR扩增产物，该PCR扩增产物为包含来源于转基因大豆事件DBN8007的植物材料的基因组中侧翼于T-DNA插入序列的3’末端的基因组DNA的一部分。这个PCR扩增产物包含SEQ ID NO：4。为了进行PCR扩增，设计位于转基因t35S转录终止序列的引物7(SEQ ID NO：10)，和与之配对的与侧翼于转基因插入序列的3’末端的基因组DNA序列杂交的引物8(SEQ ID NO：11)。
表2和表3中说明的DNA扩增条件可以用于上述PCR接合性试验以产生转基因大豆事件DBN8007的诊断性扩增子。扩增子的检测可以通过使用Stratagene Robocycler、MJ Engine、Perkin-Elmer 9700或Eppendorf Mastercycler Gradient热循环仪等进行，或通过本领域技术人员已知的方法和设备进行。
表2、用于转基因大豆事件DBN8007的5’末端转基因插入物/基因组接合区域鉴定的PCR步骤和反应混合物条件
Figure PCTCN2019099996-appb-000004
表3、热循环仪扩增条件
Figure PCTCN2019099996-appb-000005
轻轻地混合，如果热循环仪上没有保温帽，可以在每个反应液上方添加1-2滴矿物油。使用表3中的循环参数在Stratagene Robocycler(Stratagene，La Jolla，CA)、MJ Engine(MJ R-Biorad，Hercules，CA)、Perkin-Elmer 9700(Perkin Elmer，Boston，MA)或Eppendorf Mastercycler Gradient(Eppendorf，Hamburg，Germany)热循环仪上进行PCR反应。MJ Engine或Eppendorf Mastercycler Gradient热循环仪应当在计算的模式下运行。Perkin-Elmer 9700热循环仪运行时要将变温速度(ramp speed)设定为最大值。
实验结果表明：引物5和6(SEQ ID NO：8和9)，当其用在转基因大豆事件DBN8007基因组DNA的PCR反应中时，产生1407bp片段的扩增产物，当其用在未转化大豆基因组DNA和非DBN8007大豆基因组DNA的PCR反应中时，没有片段被扩增；引物7和8(SEQ ID NO：10和11)，当其用在转基因大豆事件DBN8007基因组DNA的PCR反应中时，产生1022bp片段的扩增产物，当其用在未转化大豆基因组DNA和非DBN8007大豆基因组DNA的PCR反应中时，没有片段被扩增。
PCR接合性测定还可用于鉴定来源于转基因大豆事件DBN8007的材料是纯合子或是杂合子。将引物9(SEQ ID NO：12)、引物10(SEQ ID NO：13)和引物11(SEQ ID NO：14)用于扩增反应以产生转基因大豆事件DBN8007的诊断性扩增子。表4和表5中说明的DNA扩增条件可以用于上述接合性试验以产生转基因大豆事件DBN8007的诊断性扩增子。
表4、接合性测定反应液
Figure PCTCN2019099996-appb-000006
表5、接合性测定的热循环仪扩增条件
Figure PCTCN2019099996-appb-000007
使用表5中的循环参数在Stratagene Robocycler(Stratagene，La Jolla，CA)、MJ Engine(MJ R-Biorad，Hercules，CA)、Perkin-Elmer 9700(Perkin Elmer，Boston，MA)或Eppendorf Mastercycler Gradient(Eppendorf，Hamburg，Germany)热循环仪上进行PCR反应。MJ Engine或Eppendorf Mastercycler Gradient热循环仪应当在计算的模式下运行。Perkin-Elmer 9700热循环仪运行时要将变温速度(ramp speed)设定为最大值。
在所述扩增反应中，含有模板DNA的生物样品含有诊断该样品中转基因大豆事件DBN8007的存在情况的DNA。或者扩增反应将由含有来源于大豆基因组的DNA的生物样品产生两个不同的DNA扩增子，所述来源于大豆基因组的DNA相对于转基因大豆事件DBN8007中存在的插入DNA对应的等位基因是杂合的。这两个不同的扩增子将对应于来源于野生型大豆基因组基因座的第一扩增子(SEQ ID NO：12和SEQ ID NO：14)和诊断转基因大豆事件DBN8007 DNA的存在情况的第二扩增子(SEQ ID NO：12和SEQ ID NO：13)。仅产生对应于针对杂合基因组描述的第二扩增子的单个扩增子的大豆DNA样品，可诊断确定该样品中转基因大豆事件DBN8007的存在，且该样品由相对于转基因大豆植物DBN8007中存在的插入DNA对应的等位基因为纯合的大豆种子所产生。
需要说明的是，转基因大豆事件DBN8007的引物对被用于产生对转基因大豆事件DBN8007基因组DNA为诊断性的扩增子。这些引物对包括但不限于，引物5和6(SEQ ID NO：8和9)，和引物7和8(SEQ ID NO：10和11)，用于所述的DNA扩增方法中。另外，用于扩增大豆内源基因的一个对照引物12和13(SEQ ID NO：22和23)被包括在内，其作为反应条件的一个内在标准。对转基因大豆事件DBN8007 DNA抽提样品的分析应该包括一个转基因大豆事件DBN8007的阳性组织DNA抽提物对照，一个来源于非转基因大豆事件DBN8007的阴性DNA抽提物对照和一个不含有模板大豆DNA抽提物的阴性对照。除了这些引物对之外，还可以使用来自SEQ ID NO：3或其互补序列、或者SEQ ID NO：4或其互补序列的任何引物对，当它们被用于DNA扩增反应时分别产生对于来源于转基因事件大豆植物DBN8007的组织为诊断性的包含SEQ ID NO：1或SEQ ID NO：2的扩增子。表2-表5中说明的DNA扩增条件可以用于使用合适的引物对以产生转基因大豆事件DBN8007的诊断性扩增子。当在DNA扩增方法中测试时产生对转基因大豆事件DBN8007为诊断性扩增子的、推定含有转基因大豆事件DBN8007的大豆植物或种子DNA的提取物，或来源于转基因大豆事件DBN8007的产物，可以被用作扩增的模板，来确定是否存在转基因大豆事件DBN8007。
第四实施例、利用Southern印迹杂交检测转基因大豆事件DBN8007
4.1、用于Southern印迹杂交的DNA提取
利用研钵和研杵，在液氮中研磨大约5-10g植物组织。在20mL CTAB裂解缓冲液(100mM Tris-HCl pH 8.0、20mM EDTA pH 8.0、1.4M NaCl、0.2％v/vβ-疏基乙醇、2％w/v CTAB)中重悬浮约4-5g研磨过的植物组织，在温度65℃温育60min。在温育期间，每10min将样品颠倒混匀一次。温育后，加入等体积的苯酚/氯仿/异戊醇(25∶24∶1)，轻轻颠倒混匀进行抽提，以转速4000rpm离心20min。取水相用等体积氯仿/异戊醇(24∶1)重复抽提一次。再次收集水相后加入等体积异丙醇，混匀后在温度-20℃放置1h以沉淀 DNA，再以转速4000rpm离心5min得到DNA沉淀，然后在1mL TE缓冲液(10mM Tris-HCl、1mM EDTA，pH 8.0)中重悬浮DNA沉淀。为了降解任何存在的RNA，在温度37℃下，将DNA和40μL 10mg/mL RNase A温育30min，以4000rpm离心5min，并且在0.1倍体积浓度为3M醋酸钠(pH 5.2)和2倍体积无水乙醇存在的情况下，以转速12000rpm离心10min来沉淀DNA。弃掉上清液后，用70％(v/v)的1mL乙醇洗涤沉淀，室温干燥后在1mL TE缓冲液中将DNA重新溶解。
4.2、限制酶消化
用超微量分光光度计(NanoDrop 2000，Thermo Scientific)测定上述样品的基因组DNA浓度。
在100μL反应体系中，每次消化5μg DNA，用限制性内切酶Mfe I和Nco I分别消化基因组DNA，以T-DNA上mVip3Aa基因和PAT基因的部分序列作为探针。对于每种酶，在适当的温度下过夜温育消化物。利用真空离心蒸发浓缩器(speed vacuum，Thermo Scientific)旋转样品以减少体积至20μL。
4.3、凝胶电泳
向来源于本实施例4.2中的每个样品添加溴酚蓝加样染料，并且将每个样品加样到含有溴化乙锭的0.7％琼脂糖凝胶上，在TAE电泳缓冲液(40mMTris-醋酸、2mMEDTA，pH8.5)中电泳分离，在电压20V下电泳凝胶过夜。
电泳结束后，用0.25M HCl处理凝胶10min以使DNA脱嘌呤，然后分别用变性液(1.5M NaCl、0.5M NaOH)和中和液(1.5M NaCl、0.5M Tris-HCl，pH 7.2)处理凝胶各30min。在瓷盘中倒入5×SSC(3MNaCl、0.3M柠檬酸钠，pH 7.0)，搭上一块玻璃板，然后依次放浸湿的滤纸桥、凝胶、带正电的尼龙膜(Roche，Cat.No.11417240001)、三张滤纸、纸塔、重物。在室温下转膜过夜后，在去离子水中漂洗尼龙膜2次，通过紫外交联仪(UVP，UV Crosslinker CL-1000)将DNA固定在膜上。
4.4、杂交
用PCR扩增适合的DNA序列用于探针制备。所述DNA探针为SEQ ID NO：24或SEQ ID NO：25，或者与上述序列部分同源或互补。用DNA Labeling and Detection Starter Kit II试剂盒(Roche，Cat.No.11585614910)进行探针的DIG标记、Southern印迹杂交、洗膜等操作，具体方法参考其产品说明书。最后用X光片(Roche，Cat.No.11666916001)检测探针结合的位置。
每个Southern上包括两种对照样品：(1)来自阴性(未转化的)的分离子的DNA，其用于鉴定任何可与元件-特异性探针杂交的内源大豆序列；(2)来自阴性分离子的DNA，其中引入了Hind III-消化的pDBN4006质粒，其量基于探针长度等价于一个拷贝数，其作为阳性对照以说明在检测大豆基因组内的单个基因拷贝时该实验的灵敏度。
杂交数据提供了确证的证据支持TaqMan TM PCR分析，即大豆植物DBN8007含有mVip3Aa基因和PAT基因的单拷贝。利用该mVip3Aa基因探针，Mfe I和Nco I酶解后分别产生大小约5.7kb和17kb的单一条带；利用该PAT基因探针，Mfe I和Nco I酶解分别产生大小约7kb和10kb的单一条带，这表明mVip3Aa基因和PAT基因各一个拷贝存在于大豆转化事件DBN8007中。另外，对于骨架探针，未得到杂交条带，说明在转化过程中未有任何pDBN4006载体骨架序列进入大豆转化事件DBN8007基因组中。
第五实施例、通过ELISA检测转基因大豆事件DBN8007的蛋白质表达量
Vip3Aa和PAT蛋白质在转基因大豆事件DBN8007中的表达范围，可通过ELISA进行检测。
称取2mg经冷冻干燥处理后的转基因大豆事件DBN8007的叶片作为样品，液氮研磨后，加入1mL萃取缓冲液(8g/L NaCl、0.27g/L KH 2PO 4、1.42g/L Na 2HPO 4、0.2g/L KCl、5.5mL/L Tween-20，pH7.4)，混匀，温度4℃下静置30min，12000rpm的转速下 离心10min，取上清液用上述萃取缓冲液稀释至适当倍数，取80μL稀释后的上清液用于ELISA检测。
用ELISA(酶联免疫吸附测定法)检测试剂盒(ENVIROLOGIX公司，Vip3Aa试剂盒(AP085)和PAT试剂盒(AP014))对样品中蛋白质(Vip3Aa蛋白和PAT蛋白)量占叶片干重的比例进行检测分析，具体方法参考其产品说明书。同时以野生型大豆植株叶片(非转基因，NGM)作为对照，按照上述方法进行检测分析，每株重复6次。
转基因大豆事件DBN8007的蛋白质(Vip3Aa蛋白和PAT蛋白)含量的实验结果如表6所示。测得转基因大豆事件DBN8007和野生型大豆植株叶片中Vip3Aa蛋白平均表达量占叶片干重的比例(μg/g)分别为15.67和0；转基因大豆事件DBN8007和野生型大豆植株叶片中PAT蛋白平均表达量占叶片干重的比例(μg/g)分别为167.37和0。
表6、转基因大豆事件DBN8007的蛋白表达量(μg/g)测定平均结果
Figure PCTCN2019099996-appb-000008
第六实施例、事件的昆虫抗性检测
6.1、大豆植物DBN8007在中国境内的生物测定
将大豆转化事件DBN8007和野生型大豆植株(非转基因，NGM)2种植株分别对棉铃虫[Helicoverpa armigera，CBW]、斜纹夜蛾[Spodoptera litura，TCW]、甜菜夜蛾[Spodoptera exigua，BAW]和豆天蛾[Clanis bilineata，BHM]按照如下方法进行生物测定：
分别取大豆转化事件DBN8007和野生型大豆植株(非转基因，NGM)2种植株的V3期倒二叶，用无菌水冲洗干净并用纱布将叶片上的水吸干，然后去除叶脉，同时剪成约2.5cm×3cm的形状，取1-3片(根据昆虫食量确定叶片数量)剪后的叶片放入圆形塑料培养皿底部的滤纸上，所述滤纸用蒸馏水润湿，每个培养皿中放10头人工饲养的初孵幼虫，虫试培养皿加盖后，在温度26-28℃、相对湿度70％-80％、光周期(光/暗)16：8的条件下放置3天后统计结果。统计幼虫发育进度、试虫死亡率和叶片损伤率三项指标，获得抗性总分(满分300分)：抗性总分＝100×死亡率+[100×死亡率+90×(初孵虫数/接虫总数)+60×(初孵-阴性对照虫数/接虫总数)+10×(阴性对照虫数/接虫总数)]+100×(1-叶片损伤率)。其中，接虫总数是指接虫的总数量，即每皿10头；幼虫发育进度已通过抗性总分公式体现；叶片损伤率是指被害虫取食的叶片面积占叶片总面积的比例。针对每一种害虫，从转基因大豆事件DBN8007和野生型大豆植株(非转基因，NGM)分别选5株进行测试，每株重复6次。结果如表7-8和图3-6所示。
表7、转基因大豆事件DBN8007在中国境内的抗虫生物测定结果-死亡率(％)
Figure PCTCN2019099996-appb-000009
表8、转基因大豆事件DBN8007的抗虫生物测定结果-抗性总分(分)
Figure PCTCN2019099996-appb-000010
结果表明：转基因大豆事件DBN8007对上述害虫的试虫死亡率和抗性总分均显著高于NGM，说明转基因大豆事件DBN8007对棉铃虫、斜纹夜蛾、甜菜夜蛾和豆天蛾均具有较好的抗性。
6.2、转基因大豆事件DBN8007在中国境内的田间测试
将转基因大豆事件DBN8007和野生型大豆植株(非转基因，NGM)种植于田间：随机区组设计，3次重复，小区面积为30m 2(5m×6m)，行距60cm，株距10cm，常规栽培管理，全生育期不喷施杀虫剂。
(1)棉铃虫
仅在棉铃虫自然发生较为严重的地区进行自然感虫(自然虫害发生条件：为害盛期在6-7月，发育最适温度在20-30℃)。在大豆植株生长至V3期(三片复叶)，开始跟踪调查NGM叶片被棉铃虫幼虫取食的情况；当NGM的倒二叶和倒三叶不再被取食时，逐株调查棉铃虫对大豆植株的为害面积率(为害面积率＝所有单株叶片为害面积的总和/总植株叶片面积×100％)。转基因大豆事件DBN8007对棉铃虫的抗性结果如表9所示。
表9、转基因大豆事件DBN8007自然感虫条件下对棉铃虫的抗性结果
Figure PCTCN2019099996-appb-000011
结果表明：在棉铃虫自然发生条件下，与NGM相比，棉铃虫对转基因大豆事件DBN8007的为害面积率显著降低，由此说明转基因大豆事件DBN8007对棉铃虫具有较好的抗性，转基因大豆事件DBN8007在棉铃虫自然发生条件下的田间效果如图7所示。
(2)甜菜夜蛾
仅在甜菜夜蛾自然发生较为严重的地区进行自然感虫(自然虫害发生条件：为害盛期在6-7月，发育最适温度在20-30℃)。在大豆植株生长至V3期，开始跟踪调查NGM叶片被甜菜夜蛾幼虫取食的情况；当NGM的倒二叶和倒三叶不再被取食时，逐株调查甜菜夜蛾对大豆植株的为害面积率(为害面积率＝所有单株叶片为害面积的总和/总植株叶片面积×100％)。转基因大豆事件DBN8007对甜菜夜蛾的抗性结果如表10所示。表10、转基因大豆事件DBN8007自然感虫条件下对甜菜夜蛾的抗性结果
Figure PCTCN2019099996-appb-000012
结果表明：在甜菜夜蛾自然发生条件下，与NGM相比，甜菜夜蛾对转基因大豆事件DBN8007的为害面积率显著降低，由此说明转基因大豆事件DBN8007对甜菜夜蛾具有较好的抗性，转基因大豆事件DBN8007在甜菜夜蛾自然发生条件下的田间效果如图8所示。
(3)斜纹夜蛾
在大豆植株V3期进行人工接虫，接虫2次，每小区选择中心区域附近100株进行接虫，在每株大豆植株的倒二叶上接人工饲养的初孵幼虫约10头，3天后重复等量接虫一次。在接虫5-21天后，逐株调查叶片取食面积。通常接虫后14天开始调查，若NGM叶片的为害面积率(为害面积率＝所有单株叶片为害面积的总和/总植株叶片面积×100％)达到15％，则视为有效，若没有达到可适当推迟调查，但接虫后21天为害面积率仍未达15％，则本次接虫视为无效。计算各小区大豆植株V3期斜纹夜蛾对大豆叶片为害面积率平均值，转基因大豆事件DBN8007对斜纹夜蛾的抗性结果如表11所示。
表11、转基因大豆事件DBN8007人工接虫条件下对斜纹夜蛾的抗性结果
Figure PCTCN2019099996-appb-000013
结果表明：在人工接虫条件下，转基因大豆转化事件DBN8007的为害面积率显著低于NGM，由此说明转基因大豆事件DBN8007对斜纹夜蛾具有较好的抗性，转基因大豆事件DBN8007接种斜纹夜蛾的田间效果如图9所示。
6.3、大豆植物DBN8007在阿根延的生物测定
将转基因大豆事件DBN8007和野生型大豆植株(非转基因，NGM)2种植株分别对大豆夜蛾[Chrysodeixis includens，SBL]、向日葵尺蠖[Rachiplusia nu，SFL]、草地贪夜蛾[Spodoptera frugiperda，FAW]和黑黏虫[Spodoptera cosmioides，BLAW]按照如下方法进行生物测定：
分别取转基因大豆事件DBN8007和野生型大豆植株(非转基因，NGM)2种植株的V3期倒二叶，用无菌水冲洗干净并用纱布将叶片上的水吸干，然后去除叶脉，同时剪成直径约1cm的圆形，取1-3片(根据昆虫食量确定叶片数量)剪后的圆形叶片放入生测板(如图10所示)孔内的滤纸上，所述滤纸用蒸馏水润湿，每个孔内放1头初孵幼虫，盖上生测板盖，在温度26-28℃、相对湿度70％-80％、光周期(光/暗)16∶8的条件下放置5天后统计结果。统计试虫死亡率和叶片损伤率(叶片损伤率是指被害虫取食的叶片面积占叶片总面积的比例)。针对每一种害虫，从转基因大豆事件DBN8007和野生型大豆植株(非转基因，NGM)分别选长势相当的6株进行测试，每株重复32个生测孔。结果如表12和图10(草地贪夜蛾)所示。
表12、转基因大豆事件DBN8007在阿根延的生物测定结果
Figure PCTCN2019099996-appb-000014
结果表明：转基因大豆事件DBN8007对上述害虫的试虫死亡率均显著高于NGM，叶片损伤率均低于NGM，说明转基因大豆事件DBN8007对大豆夜蛾、向日葵尺蠖、草地贪夜蛾和黑黏虫(南美洲典型的大豆害虫)均具有较好的抗性。
6.4、转基因大豆事件DBN8007在阿根延的田间测试
将转基因大豆事件DBN8007和野生型大豆植株(非转基因，NGM)2种植株种植于田间，并分别对大豆夜蛾[Chrysodeixis includens，SBL]、向日葵尺蠖[Rachiplusia nu，SFL]、黎豆夜蛾[Anticarsia gemmatalis，VBC]和草地贪夜蛾[Spodoptera frugiperda，FAW]按照如下方法进行田间活体测试：
田间搭建大型生测笼(网状透气型)，每个生测笼仅进行一种害虫测试，各生测笼间不相通，且各生测笼间通过人工种植的玉米及田间自然生长的杂草来进一步增加物理阻隔。将转基因大豆事件DBN8007和野生型大豆植株(非转基因，NGM)随机种植于每个生测笼内，每种植株设3个重复，每个重复种植一行(行长3m，30株/行，行距50cm)，常规栽培管理，全生育期不喷施杀虫剂。待植株生长至V5(五片复叶)左右，释放适量上述害虫成虫入笼，10天后调查叶片损伤率(叶片损伤率是指被害虫取食的叶片面积占叶片总面积的比例)。结果如表13所示。
表13、转基因大豆事件DBN8007在阿根廷人工接虫条件下对害虫的抗性结果
Figure PCTCN2019099996-appb-000015
结果表明：在人工接虫条件下，转基因大豆事件DBN8007的叶片损伤率均低于NGM，说明转基因大豆事件DBN8007对大豆夜蛾、向日葵尺蠖、黎豆夜蛾和草地贪夜蛾(南美洲典型的大豆害虫)均具有较好的抗性。
第七实施例、事件的除草剂耐受性检测
本试验选用保试达(Basta)除草剂(有效成分为18％的草铵膦铵盐水剂)进行喷施。采用随机区组设计，3次重复。小区面积为15m 2(5m×3m)，行距60cm，株距25cm，常规栽培管理，小区之间有1m的宽隔离带。将转基因大豆事件DBN8007进行如下2种处理：(1)不喷施，在处理(2)喷洒除草剂的同时，喷洒等体积的清水；(2)按800g a.i./ha(a.i./ha是指“活性成分每公顷”)剂量在V2-V3叶期(2-3片复叶)喷洒保试达除草剂。需要说明的是，草铵膦除草剂(如Basta)为触杀型除草剂，如田间使用操作不当，如局部积累药液过多，可出现药害状，并非转基因大豆事件DBN8007耐受性存在问题；不同含量和剂型的草铵膦除草剂换算成上述等量有效成分草铵膦均适用于以下结论。
分别在用药后1周和2周调查药害症状，并在收获时测定小区的产量；药害症状分级如表14所示。用除草剂受害率作为评价转化事件的除草剂耐受性的指标，具体地，除草剂受害率(％)＝∑(同级受害株数×级别数)/(总株数×最高级别)；其中除草剂受害率是指草铵膦受害率，草铵膦受害率是根据草铵膦处理后2周的药害调查结果而确定的，并由除草剂(草铵膦)受害率判别大豆对除草剂的耐受水平。每个小区的大豆产量是称量各小区中间3行的大豆粒总产量(重量)，不同处理间的产量差异以产量百分率的形式进行度量，产量百分率(％)＝喷施产量/不喷施产量。转基因大豆事件DBN8007对除草剂耐受性的结果和大豆产量结果如表15所示。
表14、草铵膦除草剂对大豆药害程度的分级标准
药害级别 症状描述
1 生长正常，无任何受害症状
2 轻微药害，药害少于10％
3 中等药害，以后能恢复，不影响产量
4 药害较重，难以恢复，造成减产
5 药害严重，不能恢复，造成明显减产或绝产
表15、转基因大豆事件DBN8007对草铵膦除草剂耐受性的结果和大豆产量结果
Figure PCTCN2019099996-appb-000016
结果说明，在草铵膦除草剂受害率方面：转基因大豆事件DBN8007在草铵膦除草剂(800g a.i./ha)处理下受害率为0；由此，转基因大豆事件DBN8007具有良好的草铵膦除草剂耐受性。
在产量方面：转基因大豆事件DBN8007在不喷施和喷施800g a.i./ha草铵膦2种处理下产量没有明显差异，由此，进一步表明转基因大豆事件DBN8007具有良好的草铵膦除草剂耐受性，且对产量无影响。
第八实施例
可由转基因大豆事件DBN8007生产诸如农产品或商品。如果在所述农产品或商品中检测到足够的表达量，所述农产品或商品预期含有能够诊断转基因大豆事件DBN8007材料在所述农产品或商品中存在的核苷酸序列。所述农产品或商品包括但不限于大豆饼、粉和油，具体可以为卵磷脂、脂肪酸、甘油、固醇、食用油、脱脂大豆片、包括脱脂的和烘烤的大豆粉、豆浆凝块、豆腐、大豆蛋白浓缩物、分离的大豆蛋白、水解植物蛋白、组织化大豆蛋白和大豆蛋白纤维、以及将要作为食物源供动物消费的任何其它食品等。基于探针或引物对的核酸检测方法和/或试剂盒可以被开发以检测生物样品中诸如SEQ ID NO：1或SEQ ID NO：2所示的来源于转基因大豆事件DBN8007的核苷酸序列，其中探针序列或引物序列选自如SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4和SEQ ID NO：5中所示的序列或其部分，以诊断转基因大豆事件DBN8007的存在。
综上所述，本发明转基因大豆事件DBN8007对鳞翅目昆虫具有较好的抗性，同时对草铵膦除草剂具有较高的耐受性，对产量无影响，且检测方法可以准确快速的鉴定生物样品中是否包含转基因大豆事件DBN8007的DNA分子。
对应于转基因大豆事件DBN8007的种子已根据布达佩斯条约于2019年2月19日保藏在中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC，地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，邮编100101)，分类命名：大豆(Glycine max)，保藏状态：存活，保藏编号为CGMCC No.17300。保藏物将在保藏处保藏30年。
最后所应说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围。

Claims (13)

  1. 一种核酸序列，其特征在于，具有SEQ ID NO：3或其互补序列第1-553位中至少11个连续的核苷酸和SEQ ID NO：3或其互补序列第554-1407位中至少11个连续的核苷酸、和/或SEQ ID NO：4或其互补序列第1-348位中至少11个连续的核苷酸和SEQ ID NO：4或其互补序列第349-1022位中至少11个连续的核苷酸；
    优选地，所述核酸序列具有SEQ ID NO：3或其互补序列第1-553位中22-25个连续的核苷酸和SEQ ID NO：3或其互补序列第554-1407位中22-25个连续的核苷酸、和/或SEQ ID NO：4或其互补序列第1-348位中22-25个连续的核苷酸和SEQ ID NO：4或其互补序列第349-1022位中22-25个连续的核苷酸；
    优选地，所述核酸序列包含SEQ ID NO：1或其互补序列、和/或SEQ ID NO：2或其互补序列；
    优选地，所述核酸序列包含SEQ ID NO：3或其互补序列、和/或SEQ ID NO：4或其互补序列。
  2. 根据权利要求1所述的核酸序列，其特征在于，所述核酸序列包含SEQ ID NO：5或其互补序列。
  3. 一种检测样品中转基因大豆事件DBN8007的DNA存在的方法，其特征在于，包括：
    使待检测样品与用于扩增目标扩增产物的至少两种引物在核酸扩增反应中接触；
    进行核酸扩增反应；和
    检测所述目标扩增产物的存在；
    所述目标扩增产物包含权利要求1或2所述核酸序列；优选地，所述目标扩增产物包含SEQ ID NO：1或其互补序列、SEQ ID NO：2或其互补序列、SEQ ID NO：6或其互补序列、和/或SEQ ID NO：7或其互补序列。
  4. 根据权利要求3所述检测样品中转基因大豆事件DBN8007的DNA存在的方法，其特征在于，所述引物包括第一引物和第二引物，所述第一引物选自SEQ ID NO：1、SEQ ID NO：8和SEQ ID NO：10；所述第二引物选自SEQ ID NO：2、SEQ ID NO：9和SEQ ID NO：11。
  5. 一种检测样品中转基因大豆事件DBN8007的DNA存在的方法，其特征在于，包括：
    使待检测样品与探针接触，所述探针包含权利要求1所述核酸序列；优选地，所述探针包含SEQ ID NO：1或其互补序列、SEQ ID NO：2或其互补序列、SEQ ID NO：6或其互补序列、和/或SEQ ID NO：7或其互补序列；
    使所述待检测样品和所述探针在严格杂交条件下杂交；和
    检测所述待检测样品和所述探针的杂交情况。
  6. 根据权利要求5所述检测样品中转基因大豆事件DBN8007的DNA存在的方法，其特征在于，至少一个所述探针用至少一种荧光基团标记。
  7. 一种检测样品中转基因大豆事件DBN8007的DNA存在的方法，其特征在于，包括：
    使待检测样品与标记物核酸分子接触，所述标记物核酸分子包括权利要求1所述核酸序列；优选地，所述标记物核酸分子包括选自以下的至少一种：SEQ ID NO：1或其互补序列、SEQ ID NO：2或其互补序列、和/或SEQ ID NO：6-11或其互补序列；
    使所述待检测样品和所述标记物核酸分子在严格杂交条件下杂交；
    检测所述待检测样品和所述标记物核酸分子的杂交情况，进而通过标记物辅助育种分析以确定昆虫抗性和/或除草剂耐受性与标记物核酸分子在遗传学上是连锁的。
  8. 一种DNA检测试剂盒，其特征在于，包括至少一个DNA分子，所述DNA分子包含权利要求1所述核酸序列，其可以作为对于转基因大豆事件DBN8007或其后代具有特异性的DNA引物之一或探针；优选地，所述DNA分子包含SEQ ID NO：1或其互补序列、SEQ ID NO：2或其互补序列、SEQ ID NO：6或其互补序列、和/或SEQ ID NO：7或其互补序列。
  9. 一种保护大豆植物免于昆虫侵袭的方法，其特征在于，包括在靶昆虫的膳食中提供至少一种转基因大豆植物细胞，所述转基因大豆植物细胞在其基因组中包含SEQ ID NO：1和/或SEQ ID NO：2所示的序列，摄食所述转基因大豆植物细胞的靶昆虫被抑制进一步摄食所述转基因大豆植物；
    优选地，所述转基因大豆植物细胞在其基因组中包含SEQ ID NO：3和/或SEQ ID NO：4所示的序列；
    优选地，所述转基因大豆植物细胞在其基因组中依次包含SEQ ID NO：1、SEQ ID NO：5第5594-11006位核酸序列和SEQ ID NO：2，或者包含SEQ ID NO：5所示的序列。
  10. 一种保护大豆植物免受由除草剂引起的损伤或控制种植大豆植物的大田中杂草的方法，其特征在于，包括将含有有效剂量草铵膦除草剂施加到种植至少一种转基因大豆植物的大田中，所述转基因大豆植物在其基因组中包含SEQ ID NO：1和/或SEQ ID NO：2所示的序列，所述转基因大豆植物对草铵膦除草剂具有耐受性；
    优选地，所述转基因大豆植物在其基因组中包含SEQ ID NO：3和/或SEQ ID NO：4所示的序列；
    优选地，所述转基因大豆植物在其基因组中依次包含SEQ ID NO：1、SEQ ID NO：5第5594-11006位核酸序列和SEQ ID NO：2，或者包含SEQ ID NO：5所示的序列。
  11. 一种培养对昆虫具有抗性和/或耐受草铵膦除草剂的大豆植物的方法，其特征在于，包括：
    种植至少一粒大豆种子，所述大豆种子的基因组中包含编码昆虫抗性Vip3Aa蛋白的核酸序列和/或编码草铵膦除草剂耐受性PAT蛋白的核酸序列、和特定区域的核酸序列，或者所述大豆种子的基因组中包含SEQ ID NO：5所示的核酸序列；
    使所述大豆种子长成大豆植株；
    用靶昆虫侵袭所述大豆植株和/或用有效剂量草铵膦除草剂喷洒所述大豆植株，收获与其他不具有特定区域的核酸序列的植株相比具有减弱的植物损伤的植株；
    所述特定区域的核酸序列为SEQ ID NO：1和/或SEQ ID NO：2所示的序列；优选地，所述特定区域的核酸序列为SEQ ID NO：3和/或SEQ ID NO：4所示的序列。
  12. 一种产生对昆虫具有抗性和/或对草铵膦除草剂具有耐受性的大豆植株的方法，其特征在于，包括将第一大豆植物基因组中包含的编码昆虫抗性Vip3Aa蛋白的核酸序列和/或编码草铵膦耐受性PAT蛋白的核酸序列、和特定区域的核酸序列，或者将所述第一大豆植物基因组中包含的SEQ ID NO：5所示的核酸序列，引入第二大豆植物，从而产生大量子代植株；选择具有所述特定区域的核酸序列的所述子代植株，且所述子代植株对昆虫具有抗性和/或对草铵膦除草剂具有耐受性；所述特定区域的核酸序列为SEQ ID NO：1和/或SEQ ID NO：2所示的序列；优选地，所述特定区域的核酸序列为SEQ ID NO：3和/或SEQ ID NO：4所示的序列；
    优选地，所述方法包括将转基因大豆事件DBN8007与缺少昆虫抗性和/或草铵膦耐受性的大豆植株进行有性杂交，从而产生大量子代植株，选择具有所述特定区域的核酸序列的所述子代植株；
    用靶昆虫侵袭和/或用草铵膦处理所述子代植株；
    选择对昆虫具有抗性和/或对草铵膦除草剂具有耐受性的所述子代植株。
  13. 一种产生自转基因大豆事件DBN8007的农产品或商品，其特征在于，所述农产品或商品为卵磷脂、脂肪酸、甘油、固醇、大豆片、大豆粉、大豆蛋白或其浓缩物、大豆油、大豆蛋白纤维、豆浆凝块或豆腐。
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