WO2023155193A1 - 用于检测大豆植物dbn8205的核酸序列及其检测方法 - Google Patents

用于检测大豆植物dbn8205的核酸序列及其检测方法 Download PDF

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WO2023155193A1
WO2023155193A1 PCT/CN2022/077093 CN2022077093W WO2023155193A1 WO 2023155193 A1 WO2023155193 A1 WO 2023155193A1 CN 2022077093 W CN2022077093 W CN 2022077093W WO 2023155193 A1 WO2023155193 A1 WO 2023155193A1
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seq
nucleic acid
dbn8205
soybean
sequence
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PCT/CN2022/077093
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English (en)
French (fr)
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于彩虹
谢香庭
狄少康
韩超
李运亭
张林林
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 CN202280000580.7A priority Critical patent/CN114787389A/zh
Priority to PCT/CN2022/077093 priority patent/WO2023155193A1/zh
Priority to BR112023005093A priority patent/BR112023005093A2/pt
Priority to ARP230100274A priority patent/AR128454A1/es
Publication of WO2023155193A1 publication Critical patent/WO2023155193A1/zh

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Definitions

  • the invention relates to the field of plant molecular biology, in particular to the field of transgenic crop breeding in agricultural biotechnology research. Specifically, the present invention relates to an insect-resistant and glufosinate-tolerant transgenic soybean event DBN8205, a nucleic acid sequence for detecting whether a specific transgenic soybean event DBN8205 is contained in a biological sample, and a detection method thereof.
  • Soybean (Glycine max) is one of the five major crops in the world. Biotechnology methods have been applied to this crop to produce soybean varieties with desirable traits. Two of the most important agronomic traits in soybean production are insect resistance and herbicide tolerance. Insect resistance in soybeans can be acquired by transgenic approaches to express insect resistance genes in soybean plants, transgenic soybeans that rely on the expression of a single insect resistance protein against insect infestation are at risk of limited durability because Insects will evolve resistance to insecticidal proteins expressed in GM soybeans under continuous selection pressure. Once such resistance occurs and cannot be effectively controlled, it will undoubtedly limit the commercial value of GM soybean varieties containing insecticidal proteins .
  • Phosphinothricin N-acetyltransferase isolated from Streptomyces catalyzes the conversion of L-phosphinothricin to its inactive form by acetylation.
  • PAT Phosphinothricin N-acetyltransferase isolated from Streptomyces catalyzes the conversion of L-phosphinothricin to its inactive form by acetylation.
  • Genes expressing plant-optimized versions of PAT have been used in soybeans to confer tolerance to the herbicide glufosinate in soybeans, eg soybean event A5547-127.
  • a good commercial soybean transformation event should comprehensively consider the vector design of the cCry2Ab gene, cCry1Ac gene and cPAT gene in soybean plants, the interaction of the three expression cassettes, insect resistance, herbicide tolerance and The impact on yield and other plant physiological indicators enables cCry2Ab gene, cCry1Ac gene and cPAT gene to be expressed in soybeans in an appropriate amount and realize their corresponding functions without affecting soybean yield and other physiological indicators.
  • exogenous genes in plants is known to be influenced by their chromosomal location, possibly due to the proximity of chromatin structure (such as heterochromatin) or transcriptional regulatory elements (such as enhancers) to the integration site. For this reason, it is usually necessary to screen a large number of events before it is possible to identify commercially viable events (ie, events in which the introduced target gene is optimally expressed). For example, it has been observed in plants and other organisms that the amount of expression of an introduced gene can vary considerably between events; there may also be differences in the spatial or temporal pattern of expression, such as the relative expression of the transgene between different plant tissues There are differences in which the actual expression pattern may not correspond to that expected based on the transcriptional regulatory elements in the introduced gene construct.
  • Events with the expected amount and pattern of transgene expression can be used to introgress the transgene into other genetic backgrounds by sexual outcrossing using conventional breeding methods.
  • the progeny produced by this crossing method maintained the transgene expression characteristics of the original transformant. Applying this pattern of strategies can ensure reliable gene expression in many varieties that are well adapted to local growing conditions.
  • Detection of the presence of the transgene is possible by any of the well-known polynucleotide detection methods, such as polymerase chain reaction (PCR) or DNA hybridization using polynucleotide probes. These assays usually focus on commonly used genetic elements such as promoters, terminators, marker genes, etc.
  • transgenic DNA the sequence of the chromosomal DNA adjacent to the inserted transgenic DNA
  • this approach cannot be used to distinguish between different events, especially those produced with the same DNA construct. event. Therefore, transgene-specific events are now commonly identified by PCR using a pair of primers spanning the junction of the inserted transgene and flanking DNA, specifically a first primer contained within the inserted sequence and a second primer contained within the inserted sequence.
  • the object of the present invention is to provide a nucleic acid sequence and detection method for detecting soybean plant DBN8205, the transgenic soybean event DBN8205 has better resistance to insects and better tolerance to glufosinate-ammonium herbicide, and The detection method can accurately and quickly identify whether the DNA molecule of the transgenic soybean event DBN8205 is contained in the biological sample.
  • the present invention provides a nucleic acid molecule having the following nucleic acid sequence, said nucleic acid sequence comprising SEQ ID NO: 3 or its complementary sequence at least 11 consecutive nucleotides and SEQ ID NO: 1-462 NO: 3 or its complementary sequence at least 11 consecutive nucleotides at positions 463-634, and/or SEQ ID NO: 4 or its complementary sequence at least 11 consecutive nucleotides at positions 1-225 and SEQ ID NO: 4 or its complementary sequence at least 11 consecutive nucleotides ID NO: At least 11 consecutive nucleotides in positions 226-642 of 4 or its complementary sequence.
  • the nucleic acid sequence comprises 22-25 consecutive nucleotides in SEQ ID NO: 3 or its complementary sequence 1-462 and 22-22 in SEQ ID NO: 3 or its complementary sequence 463-634 25 consecutive nucleotides, and/or 22-25 consecutive nucleotides in SEQ ID NO: 4 or its complementary sequence 1-225 and SEQ ID NO: 4 or its complementary sequence 226-642 22-25 consecutive nucleotides.
  • the nucleic acid sequence comprises SEQ ID NO: 1 or its complement, and/or SEQ ID NO: 2 or its complement.
  • Said SEQ ID NO: 1 or its complementary sequence is a sequence of 22 nucleotides in length near the insertion junction at the 5' end of the inserted sequence in the transgenic soybean event DBN8205, said SEQ ID NO: 1 or its complementary sequence Complementary sequences spanning the flanking genomic DNA sequence of the soybean insertion site and the DNA sequence at the 5' end of the insertion sequence, comprising said SEQ ID NO: 1 or its complementary sequence can be identified as the presence of transgenic soybean event DBN8205.
  • Said SEQ ID NO: 2 or its complementary sequence is a sequence of 22 nucleotides in length near the insertion junction at the 3' end of the inserted sequence in the transgenic soybean event DBN8205, said SEQ ID NO: 2 or its complementary sequence
  • the complementary sequence spans the DNA sequence at the 3' end of the insertion sequence and the flanking genomic DNA sequence of the soybean insertion site, including said SEQ ID NO: 2 or its complementary sequence can be identified as the presence of the transgenic soybean event DBN8205.
  • the nucleic acid sequence comprises SEQ ID NO: 3 or its complement, and/or SEQ ID NO: 4 or its complement.
  • the nucleic acid sequence comprises at least 11 or more continuous polynucleotides (first nucleic acid sequence) of any part of the T-DNA insertion sequence in the SEQ ID NO: 3 or its complementary sequence, and the At least 11 or more continuous polynucleotides (second nucleic acid sequences) of any part of the 5' flanking soybean genomic DNA region in said SEQ ID NO: 3 or its complementary sequence.
  • Said nucleic acid sequence may further be homologous or complementary to a part of said SEQ ID NO:3 comprising the entirety of said SEQ ID NO:1.
  • nucleic acid sequences can be used as a DNA primer pair in a DNA amplification method to generate an amplification product.
  • the presence of transgenic soybean event DBN8205 or progeny thereof can be diagnosed when the amplification product generated in the DNA amplification method using the DNA primer pair is an amplification product comprising SEQ ID NO: 1.
  • the SEQ ID NO: 3 or its complementary sequence is a sequence of 634 nucleotides in length near the insertion junction at the 5' end of the T-DNA insertion sequence in the transgenic soybean event DBN8205, and the SEQ ID NO: 3 or its complementary sequence consists of 462 nucleotides of the soybean genome 5' flanking sequence (SEQ ID NO: 3 nucleotides 1-462) and 172 nucleotides in the pDBN4031 construct DNA sequence (SEQ ID NO: 463-634 nucleotides of 3), comprising said SEQ ID NO: 3 or its complementary sequence can be identified as the presence of transgenic soybean event DBN8205.
  • nucleic acid sequence comprises at least 11 or more contiguous polynucleotides (the third nucleic acid sequence) of any part of the T-DNA insertion sequence in said SEQ ID NO: 4 or its complementary sequence, and said SEQ ID NO : at least 11 or more contiguous polynucleotides (fourth nucleic acid sequence) of any part of the 3' flanking soybean genomic DNA region in 4 or its complement.
  • Said nucleic acid sequence may further be homologous or complementary to a part of said SEQ ID NO:4 comprising the entirety of said SEQ ID NO:2.
  • these nucleic acid sequences can be used as a DNA primer pair in a DNA amplification method to generate an amplification product.
  • the presence of transgenic soybean event DBN8205 or progeny thereof can be diagnosed when the amplification product generated in the DNA amplification method using the DNA primer pair is an amplification product comprising SEQ ID NO: 2.
  • Said SEQ ID NO: 4 or its complementary sequence is a sequence of 642 nucleotides in length near the T-DNA insertion junction site at the 3' end of the inserted sequence in the transgenic soybean event DBN8205, said SEQ ID NO: 4 or its complementary sequence consists of 21 nucleotides of the nucleotide sequence (SEQ ID NO: nucleotide 1-21 of 4), 204 nucleotides in the pDBN4031 construct DNA sequence (SEQ ID NO: 4 nucleotides 22-225) and the soybean genome 3' flanking sequence (SEQ ID NO: 4 nucleotides 226-642) of 417 nucleotides, comprising the SEQ ID NO: 4 or its complementary sequence can be identified as the existence of the transgenic soybean event DBN8205.
  • nucleic acid sequence comprises SEQ ID NO: 5 or its complementary sequence.
  • the SEQ ID NO: 5 or its complementary sequence is a sequence of 12813 nucleotides in length that characterizes the transgenic soybean event DBN8205, and its specific genome and genetic elements are shown in Table 1.
  • the presence of the transgenic soybean event DBN8205 can be identified as comprising said SEQ ID NO: 5 or its complementary sequence.
  • the first, second, third and fourth nucleic acid sequences need not consist solely of DNA, but may also include RNA, a mixture of DNA and RNA, or DNA, RNA or other sequences that do not serve as one or more Combinations of nucleotides or analogs thereof for the polymerase template.
  • the probes or primers of the present invention should be at least about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 consecutive nucleotides in length, which can be selected from Nucleotides described in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.
  • the probes and primers can be at least about 21 to about 50 or more contiguous Nucleotides.
  • the nucleic acid sequence or its complement can be used in DNA amplification methods to generate amplicons for detecting the presence of transgenic soybean event DBN8205 or its progeny in a biological sample; the nucleic acid sequence or its complement Can be used in nucleotide assays to detect the presence of transgenic soybean event DBN8205 or its progeny in biological samples.
  • the present invention also provides a method for detecting the presence of DNA of the transgenic soybean event DBN8205 in a sample, comprising:
  • the target amplification product comprises 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 complementary sequence.
  • the two primers include complementary sequences of SEQ ID NO: 8 and SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, or SEQ ID NO: 1 and SEQ ID NO: 2.
  • the present invention also provides a method for detecting the presence of DNA of the transgenic soybean event DBN8205 in a sample, comprising:
  • the stringent conditions can be hybridized at 65° C. in 6 ⁇ SSC (sodium citrate), 0.5% SDS (sodium dodecyl sulfate) solution, and then mixed with 2 ⁇ SSC, 0.1% SDS and 1 ⁇ SSC, Wash each membrane once with 0.1% SDS.
  • 6 ⁇ SSC sodium citrate
  • SDS sodium dodecyl sulfate
  • the probe comprises SEQ ID NO: 1 or its complement, SEQ ID NO: 2 or its complement, SEQ ID NO: 6 or its complement, and/or SEQ ID NO: 7 or its complement .
  • At least one of said probes is labeled with at least one fluorophore.
  • the present invention also provides a method for detecting the presence of DNA of the transgenic soybean event DBN8205 in a sample, comprising:
  • the marker nucleic acid molecule comprises at least one selected from the group consisting of SEQ ID NO: 1 or its complement, SEQ ID NO: 2 or its complement, and SEQ ID NOs: 6-11 or its complement .
  • the present invention also provides a DNA detection kit, comprising at least one DNA molecule comprising the nucleic acid sequence, which can be used as a DNA primer specific for the transgenic soybean event DBN8205 or its progeny One or probe.
  • the DNA molecule comprises SEQ ID NO: 1 or its complement, SEQ ID NO: 2 or its complement, SEQ ID NO: 6 or its complement, and/or SEQ ID NO: 7 or its complement .
  • the present invention also provides a plant cell or part, comprising a nucleic acid sequence encoding an insect-resistant Cry2Ab protein, a nucleic acid sequence encoding an insect-resistant Cry1Ac protein, and a nucleic acid sequence encoding a glufosinate-resistant PAT protein
  • the nucleic acid sequence of described specific region comprises the sequence shown in SEQ ID NO:1 and/or SEQ ID NO:2;
  • the nucleic acid sequence of described specific region comprises SEQ ID NO:3 and /or the sequence shown in SEQ ID NO:4.
  • the plant cell or part comprises SEQ ID NO: 1, the nucleotide sequence at positions 866-12192 of SEQ ID NO: 5 and SEQ ID NO: 2 in sequence, or comprises the sequence shown in SEQ ID NO: 5.
  • said plant cell or part comprises transgenic soybean event DBN8205;
  • said plant cells or parts further comprise at least one other transgenic soybean event than transgenic soybean event DBN8205; preferably said other transgenic soybean event is transgenic soybean event DBN9004 and/or transgenic soybean event DBN8002.
  • the present invention also provides a method for protecting soybean plants from insect attack, comprising providing in the diet of target insects at least one transgenic soybean plant cell, said transgenic soybean plant cell comprising in its genome SEQ ID NO: 1 and/or the sequence shown in SEQ ID NO: 2, the target insects that ingest the transgenic soybean plant cells are inhibited from further ingesting the transgenic soybean plants.
  • the transgenic soybean plant cell comprises the sequence shown in SEQ ID NO: 3 and/or SEQ ID NO: 4 in its genome.
  • the transgenic soybean plant cell sequentially comprises SEQ ID NO: 1, the 866-12192 nucleotide sequence of SEQ ID NO: 5 and SEQ ID NO: 2 in its genome, or comprises SEQ ID NO: 5.
  • the present invention also provides a method for protecting soybean plants from damage caused by herbicides or controlling the weeds in the field of planting soybean plants, comprising applying an effective dose of glufosinate-ammonium herbicide to the planting at least
  • described transgenic soybean plant comprises the sequence shown in SEQ ID NO:1 and/or SEQ ID NO:2 in its genome, and described transgenic soybean plant has resistance to glufosinate-ammonium herbicide Receptivity.
  • the transgenic soybean plant comprises the sequence shown in SEQ ID NO: 3 and/or SEQ ID NO: 4 in its genome.
  • the transgenic soybean plant sequentially comprises SEQ ID NO: 1, the 866-12192 nucleotide sequence of SEQ ID NO: 5 and SEQ ID NO: 2 in its genome, or comprises the sequence shown in SEQ ID NO: 5 .
  • the present invention also provides a method of cultivating insect-resistant and/or glufosinate-tolerant soybean plants, comprising:
  • Plant at least one soybean seed, the genome of the soybean seed comprises a nucleic acid sequence encoding an insect-resistant Cry2Ab protein and/or a nucleic acid sequence encoding an insect-resistant Cry1Ac protein and/or an encoding glufosinate herbicide tolerance PAT protein
  • the nucleic acid sequence of the soybean seed, and the nucleic acid sequence of a specific region, or the genome of the soybean seed comprises 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 the sequence shown in SEQ ID NO:2; preferably, the nucleic acid sequence of the specific region is SEQ ID NO:3 and/or SEQ ID NO:4 sequence shown.
  • the present invention also provides a method for producing a soybean plant resistant to insects and/or glufosinate-ammonium herbicide tolerance, comprising encoding the insect-resistant insect resistance gene contained in the genome of the first soybean plant
  • the nucleotide sequence of Cry2Ab protein and/or the nucleotide sequence of coding insect resistance Cry1Ac protein and/or the nucleotide sequence of coding glufosinate-ammonium tolerance PAT protein, and the nucleotide sequence of specific region, or the first soybean plant genome The nucleic acid sequence shown in SEQ ID NO: 5 contained in is introduced into the second soybean plant, thereby producing a large number of progeny plants; the progeny plants having the nucleic acid sequence of the specific region are selected, and the progeny plants are to Insects have resistance and/or have tolerance to glufosinate-ammonium herbicide;
  • the nucleic acid sequence of described specific region is the sequence shown in SEQ ID NO:1 and/or SEQ
  • the method comprises sexually crossing a first soybean plant comprising the transgenic soybean event DBN8205 with a second soybean plant to generate a large number of progeny plants, and selecting the progeny plants having the nucleic acid sequence of the specific region ;
  • Such progeny plants are selected for resistance to the target insect and/or tolerance to the glufosinate-ammonium herbicide.
  • the present invention also provides an agricultural product or commodity produced from a soybean plant comprising the transgenic soybean event DBN8205, said agricultural product or commodity is lecithin, fatty acid, glycerin, sterol, soybean flakes, soybean flour, soybean protein or its concentrate, soybean oil, soy protein fiber, soy milk curd or tofu.
  • the present invention also provides an agricultural product or commodity produced from a soybean plant comprising the transgenic soybean event DBN8205, said soybean plant further comprising at least one other transgenic soybean event different from the transgenic soybean event DBN8205;
  • said other transgenic soybean event is transgenic soybean event DBN9004 and/or genetically modified soybean event DBN8002.
  • the present invention also provides a method for expanding the plant insect resistance spectrum and/or the range of herbicide tolerance, by combining the transgenic soybean event DBN8205 with at least one plant that is different from the transgenic soybean event DBN8205 Expressed together with other transgenic soybean events;
  • said other transgenic soybean event is transgenic soybean event DBN9004 and/or genetically modified soybean event DBN8002.
  • the DBN9004 provided by the present invention is the transgenic soybean event disclosed in the patent CN106086011A, and the transgenic soybean event DBN9004 is stored in the General Microorganism Center of China Microbiological Culture Collection Management Committee in the form of seeds and with the preservation number CGMCC No.11171.
  • the DBN8002 provided by the present invention is the transgenic soybean event disclosed in the patent CN111406117A, and the transgenic soybean event DBN8002 is stored in the General Microorganism Center of China Microbiological Culture Collection Management Committee in the form of seeds and with the preservation number CGMCC No.17299.
  • nucleic acid sequence and its detection method for detecting soybean plants in the present invention the following definitions and methods can better define the present invention and guide those of ordinary skill in the art to implement the present invention, unless otherwise specified, according to the ordinary skills in the art common usage by people to understand the term.
  • soybean refers to soybean (Glycine max), and includes all plant varieties that can be crossed with soybean, 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 calli, plant clumps and whole plants in plants or plant parts Cells, said plant parts such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, stalks, roots, root tips, anthers, and the like.
  • transgenic plants that are understood to be within the scope of the present invention include, but are not limited to, plant cells, protoplasts, tissues, callus, embryos, as well as flowers, stems, fruits, leaves and roots, the above plant parts being derived from the A transgenic plant transformed with a DNA molecule and thus consisting at least in part of a transgenic cell, or its progeny.
  • gene refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences before the coding sequence (5' non-coding sequence) and regulatory sequences after the coding sequence (3' non-coding sequence).
  • Native gene refers to a gene that is found in nature with its own regulatory sequences.
  • Chimeric gene refers to any gene that is not a native gene, comprising regulatory and coding sequences not found in nature.
  • Endogenous gene refers to a native gene in its natural location in the genome of an organism.
  • Exogenous gene is a foreign gene that exists in the genome of an organism and does not exist before, and also refers to a gene that is introduced into a recipient cell through a transgenic procedure.
  • Foreign genes may comprise native or chimeric genes inserted into a non-native organism.
  • a "transgene” is a gene that has been introduced into the genome by a transformation procedure.
  • the site in the plant genome where the recombinant DNA has been inserted may be referred to as the "insertion site” or "target site”.
  • flanking DNA may comprise the genome naturally present in an organism such as a plant or exogenous (heterologous) DNA introduced by a transformation process, such as a fragment associated with a transformation event.
  • flanking DNA can include a combination of native and foreign DNA.
  • flanking DNA is also called “flank region” or “flank sequence” or “flank genomic sequence” or “flank 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 that is immediately upstream or downstream of and adjacent to the original exogenously inserted DNA molecule.
  • flanking region When this flanking region is located downstream, it may also be referred to as a "3' flank” or a “left border flank” or the like. When this flanking region is located upstream, it may also be referred to as the "5' flank” or the “right border flank” or the like.
  • Transformants will also contain a unique junction between the heterologous insert DNA and the segment of genomic DNA or between two segments of genomic DNA or between two segments of heterologous DNA.
  • a "junction” is the point at which two specific DNA segments join. For example, junctions exist where insert DNA joins flanking DNA. Junctions also exist in transformed organisms where two segments of DNA join together in a manner modified from that found in natural organisms. "Junction region” or “junction sequence” refers to the DNA comprising the junction.
  • the present invention provides a transgenic soybean event designated DBN8205, also known as soybean plant DBN8205, including plants and seeds of transgenic soybean event DBN8205 and plant cells or regenerable parts thereof, and progeny thereof, Plant parts of transgenic soybean Event DBN8205, including but not limited to cells, pollen, ovules, flowers, buds, roots, stems, leaves, pods and products from soybean plant DBN8205, such as soybean cake, meal and oil, which may specifically be lecithin , fatty acids, glycerin, sterols, edible oils, defatted soy flakes, including defatted and roasted soy flour, soymilk curd, tofu, soy protein concentrate, isolated soy protein, hydrolyzed vegetable protein, textured soy protein and Soy protein fiber.
  • DBN8205 also known as soybean plant DBN8205
  • Plant parts of transgenic soybean Event DBN8205 including but not limited to cells, pollen, ovules, flowers, buds, roots, stems, leaves,
  • the transgenic soybean event DBN8205 of the present invention comprises a DNA construct that, when expressed in plant cells, acquires insect resistance and tolerance to glufosinate herbicide.
  • the DNA construct comprises three expression cassettes in tandem, the first expression cassette comprising a suitable promoter for expression in plants, a nucleic acid sequence encoding a signal peptide/transit peptide, a nucleic acid sequence encoding a Cry2Ab protein and a suitable polyadenylation signal sequence, the Cry2Ab protein is mainly resistant to Lepidoptera insects.
  • the second expression cassette comprises a suitable promoter for expression in plants, a nucleic acid sequence encoding a signal peptide/transit peptide, a nucleic acid sequence encoding a Cry1Ac protein which is also a polyadenylation signal sequence and a suitable polyadenylation signal sequence Mainly resistant to Lepidoptera insects.
  • the third expression cassette comprises a suitable promoter for expression in plants, a nucleic acid sequence encoding phosphinothricin N-acetyltransferase (phosphinothricin N-acetyltransferase, PAT) and a suitable polyadenylation signal sequence , the PAT protein has tolerance to glufosinate-ammonium herbicide.
  • the promoter may be a suitable promoter isolated from a plant, including a constitutive, inducible and/or tissue-specific promoter, and the suitable promoter includes, but is not limited to, cauliflower mosaic virus (CaMV) 35S Promoter, Scrophulariaceae mosaic virus (FMV) 35S promoter, Ubiquitin promoter, Actin promoter, Agrobacterium tumefaciens nopaline synthase (NOS) promoter, Octopine synthase (OCS) promoter, Cestrum yellow leaf curl virus promoter, potato tuber storage protein (Patatin) promoter, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) promoter, glutathione sulfur transferase (GST) promoter, E9 promoter, GOS promoter, alcA/alcR promoter, Agrobacterium rhizogenes RolD promoter and Arabidopsis (Arabidopsis thaliana
  • the signal peptide/transit peptide can guide the transport of Cry2Ab protein and/or Cry1Ac protein to a specific organelle or compartment in the cell, for example, using the sequence encoding the chloroplast transit peptide to target the chloroplast, or using the 'KDEL' retention sequence to target the endoplasm net.
  • 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
  • NOS nopaline synthase
  • CaMV cauliflower mosaic virus
  • PIN II protease inhibitor II
  • the expression cassette may also include other genetic elements, including but not limited to enhancers.
  • the enhancer can enhance the expression level of the gene, and the enhancer includes but not limited to, tobacco etch virus (TEV) translation activator, CaMV35S enhancer and FMV35S enhancer.
  • TMV tobacco etch virus
  • Cry2Ab insecticidal protein and Cry1Ac insecticidal protein are two kinds of insecticidal proteins, which are insoluble parasporal crystal proteins produced by Bacillus thuringiensis (Bt for short).
  • Cry2Ab protein or Cry1Ac protein is ingested by insects into the midgut, the protoxin of the toxic protein is dissolved in the alkaline pH environment of the insect midgut, and the N- and C-terminals of the protein are digested by alkaline protease to convert the protoxin into active fragments , the active fragment binds to the receptor on the upper surface of the insect midgut epithelial cell membrane, inserts into the intestinal membrane, causes the cell membrane to perforate the lesion, destroys the osmotic pressure change and pH balance inside and outside the cell membrane, disturbs the digestive process of the insect, and eventually leads to its death.
  • the "Lepidoptera” includes two types of insects, moths and butterflies, and is an order with the largest number of agricultural and forestry pests, such as cutworms, cotton bollworms, Spodoptera litura, Spodoptera spp., Peach borer and the like.
  • 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 by acetylation, to endow plants with Tolerance to glufosinate-ammonium 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 serve as a selectable marker gene.
  • glufosinate-ammonium also known as glufosinate, refers to 2-amino-4-[hydroxyl (methyl) phosphono] ammonium butyrate
  • the treatment with "glufosinate-ammonium herbicide” refers to the use of any one containing Glufosinate-ammonium herbicide formulations.
  • the selection of the application rate of a glufosinate-ammonium formulation to achieve a biologically effective dose is within the skill of the average agronomist.
  • Treatment of a field containing plant material derived from GM soybean event DBN8205 with any of the herbicide formulations containing glufosinate will control weed growth in said field without affecting the plant material derived from GM soybean event DBN8205 growth or yield.
  • the DNA construct is introduced into plants using transformation methods including, but not limited to, Agrobacterium-mediated transformation, biolistic transformation, and pollen tube passage transformation.
  • the Agrobacterium-mediated transformation method is a common method for plant transformation.
  • the foreign DNA to be introduced into the plant is cloned between the left and right border consensus sequences of the vector, ie the T-DNA region.
  • the vector is transformed into Agrobacterium cells, which are then used to infect plant tissues, and the T-DNA region of the vector containing foreign DNA is inserted into the plant genome.
  • the gene bombardment transformation method is bombarding plant cells with a vector containing foreign DNA (particle-mediated biolistic transformation).
  • the pollen tube channel transformation method utilizes the natural pollen tube channel (also known as pollen tube guiding tissue) formed after plant pollination to carry exogenous DNA into the embryo sac through the nucellus channel.
  • transgenic plants Following transformation, transgenic plants must be regenerated from the transformed plant tissue, and appropriate markers used to select for progeny bearing the foreign DNA.
  • a DNA construct is an interconnected assembly of DNA molecules that provides one or more expression cassettes.
  • the DNA construct is preferably a plasmid capable of self-replicating in bacterial cells and containing various restriction endonuclease sites for introduction to provide a functional genetic element, i.e. a promoter , introns, leader sequences, coding sequences, 3' terminator regions and other sequences of DNA molecules.
  • the expression cassette contained in the DNA construct includes the genetic elements necessary to provide transcription of the messenger RNA and can be designed for expression in prokaryotic or eukaryotic cells.
  • the expression cassettes of the invention are designed for expression most preferably in plant cells.
  • a transgenic "event” is obtained by transforming a plant cell with a heterologous DNA construct, i.e., comprising at least one nucleic acid expression cassette containing a gene of interest, inserted transgenically into the plant genome to produce a plant population, which is regenerated , and select specific plants with features inserted into specific genomic loci.
  • a heterologous DNA construct i.e., comprising at least one nucleic acid expression cassette containing a gene of interest, inserted transgenically into the plant genome to produce a plant population, which is regenerated , and select specific plants with features inserted into specific genomic loci.
  • the term “event” refers to the original transformant containing heterologous DNA and the progeny of that transformant.
  • event also refers to the progeny of a sexual cross between an original transformant and an individual of another breed that contains heterologous DNA, even if, after repeated backcrosses with the backcross parent, the inserted DNA from the original transformant parent and flanking genomic DNA are also present at the same chromosomal location in the hybrid offspring.
  • event also refers to a DNA sequence from an original transformant comprising the insert DNA and flanking genomic sequences in close proximity to the insert DNA, which DNA sequence is expected to be transferred to progeny derived from cells containing the insert DNA
  • the parental line (such as the original transformant and its progeny produced by selfing) is sexually crossed with a parental line that does not contain the inserted DNA, and the progeny receive the inserted DNA containing the gene of interest.
  • Recombinant in the present invention refers to a form of DNA and/or protein and/or organism that is not normally found in nature and thus produced by human intervention. Such human intervention can produce recombinant DNA molecules and/or recombinant plants. Said "recombinant DNA molecule” is obtained by the artificial combination of two otherwise separate sequence segments, such as by chemical synthesis or by manipulation of separate nucleic acid segments by genetic engineering techniques. The techniques for performing nucleic acid manipulations are well known.
  • transgenic includes any cell, cell line, callus, tissue, plant part or plant, the genotype of which is altered by the presence of heterologous nucleic acid, said “transgenic” including transgenics originally so altered as well as those derived from The offspring individuals produced by the original transgenic body through sexual crossing or asexual reproduction.
  • the term “transgenic” does not include alterations of the genome (chromosomal or extrachromosomal) by conventional plant breeding methods or naturally occurring events such as random heterozygous fertilization, non-recombinant viral infection, non-recombinant Bacterial transformation, non-recombinant transposition, or spontaneous mutation.
  • Heterologous in the context of the present invention means that a first molecule is not normally found in combination with a second molecule in nature.
  • a molecule can be derived from a first species and inserted into the genome of a second species. This molecule is thus heterologous to the host and is artificially introduced into the genome of the host cell.
  • the second parent soybean plant lacks resistance to Lepidoptera insects and/or has tolerance to glufosinate-ammonium herbicide and then selecting progeny plants that are resistant to lepidopteran attack and/or glufosinate-ammonium herbicide tolerance can produce lepidopteran-resistant and glufosinate-ammonium herbicides agent-tolerant soybean plants.
  • These steps may further comprise backcrossing lepidopteran resistant and/or glufosinate-tolerant progeny plants to a second parent soybean plant or a third parent soybean plant, followed by infestation with lepidopteran insects, glufosinate herbicide application or selection of progeny by identification of trait-associated molecular markers such as DNA molecules containing junction sites identified at the 5' and 3' ends of the inserted sequence in transgenic soybean event DBN8205, This results in soybean plants that are resistant to Lepidoptera insects and tolerant to the glufosinate herbicide.
  • transgenic plants can also be mated to produce offspring containing two independent, segregated additions of the exogenous gene. Selfing of suitable progeny can result in progeny plants that are homozygous for both added exogenous genes. Backcrossing to parental plants and outcrossing to non-transgenic plants are also contemplated as previously described, as is vegetative propagation.
  • probe is an isolated nucleic acid molecule to which is bound a conventional detectable label or reporter molecule, eg, a radioisotope, ligand, chemiluminescent agent or enzyme.
  • This probe is complementary to one strand of the target nucleic acid, and in the present invention, the probe is complementary to one strand of DNA from the genome of the transgenic soybean event DBN8205, whether the genomic DNA is from the transgenic soybean event DBN8205 or the seed or is derived from the transgene Plant or seed or extract of soybean event DBN8205.
  • the probes of the present invention include not only deoxyribonucleic acid or ribonucleic acid, but also polyamides and other probe materials that specifically bind to a target DNA sequence and can be used to detect the presence of the target DNA sequence.
  • primer is an isolated nucleic acid molecule that, by nucleic acid hybridization, anneals to a complementary target DNA strand, forms a hybrid between the primer and target DNA strand, and then reacts with a polymerase (eg, DNA polymerase) Bottom, stretches along the target DNA strand.
  • a polymerase eg, DNA polymerase
  • the primer pairs of the present invention relate to their use in the amplification of target nucleic acid sequences, for example, by polymerase chain reaction (PCR) or other conventional nucleic acid amplification methods.
  • Probes and primers are generally 11 polynucleotides or more in length, preferably 18 polynucleotides or more, more preferably 24 polynucleotides or more, most preferably 30 polynucleotides in length sour or more. Such probes and primers hybridize specifically to the target sequence under highly stringent hybridization conditions. Although probes that are different from the target DNA sequence and maintain the ability to hybridize to the target DNA sequence can be designed by conventional methods, preferably, the probes and primers in the present invention have complete DNA sequences with the continuous nucleic acid of the target sequence identity.
  • the primers and probes based on the flanking genomic DNA and the insert sequence of the present invention can be determined by conventional methods, for example, by isolating the corresponding DNA molecule from the plant material derived from the transgenic soybean event DBN8205, and determining the nucleic acid sequence of the DNA molecule.
  • the DNA molecule contains transgene insertion sequence and soybean genome flanking sequence, and the fragments of the DNA molecule can be used as primers or probes.
  • nucleic acid probes and primers of the invention hybridize to target DNA sequences under stringent conditions. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of DNA derived from transgenic soybean Event DBN8205 in a sample.
  • Nucleic acid molecules or fragments thereof are capable of specifically hybridizing to other nucleic acid molecules under certain circumstances. As used herein, two nucleic acid molecules are said to be capable of specifically hybridizing to each other if the two nucleic acid molecules are capable of forming an antiparallel double-stranded nucleic acid structure.
  • a nucleic acid molecule is said to be the "complement" of another nucleic acid molecule if two nucleic acid molecules exhibit perfect complementarity.
  • nucleic acid molecules are said to exhibit "perfect complementarity" when every nucleotide of one nucleic acid molecule is complementary to the corresponding nucleotide of the other nucleic acid molecule.
  • Two nucleic acid molecules are said to be “minimally complementary” if they are capable of hybridizing to each other with sufficient stability such that they anneal and bind to each other under at least conventional "low stringency” conditions.
  • two nucleic acid molecules are said to be “complementary” if they are capable of hybridizing to each other with sufficient stability such that they anneal and bind to each other under conventional "high stringency” conditions.
  • Deviations from perfect complementarity are permissible as long as the deviation does not completely prevent the two molecules from forming a double-stranded structure.
  • a nucleic acid molecule In order for a nucleic acid molecule to serve as a primer or probe, it only needs to be sufficiently complementary in sequence to form a stable double-stranded structure under the particular solvent and salt concentration employed.
  • a substantially homologous sequence is a nucleic acid molecule that is capable of specifically hybridizing to a matching complementary strand of another nucleic acid molecule under highly stringent conditions.
  • Suitable stringent conditions to promote DNA hybridization for example, treatment with 6.0 ⁇ sodium chloride/sodium citrate (SSC) at about 45° C., followed by washing with 2.0 ⁇ SSC at 50° C., are known to those skilled in the art. is well known.
  • the salt concentration in the washing step can be selected from about 2.0 ⁇ SSC, 50°C for low stringency conditions to about 0.2 ⁇ SSC, 50°C for high stringency conditions.
  • the temperature conditions in the washing step can be increased from about 22°C at room temperature for low stringency conditions to about 65°C for high stringency conditions.
  • Both the temperature condition and the salt concentration can be changed, or one can be kept constant while the other variable is changed.
  • a nucleic acid molecule of the present invention can be mixed with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4.
  • a nucleic acid molecule of the present invention is combined under highly stringent conditions with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: One or more nucleic acid molecules or their complementary sequences in 6 and SEQ ID NO: 7, or any fragment of the above-mentioned sequences specifically hybridizes.
  • the preferred marker nucleic acid molecule has SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 6 or SEQ ID NO: 7 or its complementary sequence, or any fragment of the above sequence.
  • Another preferred marker nucleic acid molecule of the present invention has 80% to 100% 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 progeny of genetic crosses.
  • the hybridization of the probe to the target DNA molecule can be detected by any method known to those skilled in the art, including but not limited to fluorescent labeling, radioactive labeling, antibody-based labeling and chemiluminescent labeling.
  • stringent conditions refer to conditions that allow only the primers to hybridize to the target nucleic acid sequence in a DNA thermal amplification reaction, with the same
  • the primer corresponding to the wild-type sequence (or its complementary sequence) of the target nucleic acid sequence is capable of binding to the target nucleic acid sequence, and preferably produces a unique amplification product, the amplification product being an amplicon.
  • the term "specifically binds (to a target sequence)" means that under stringent hybridization conditions a probe or primer hybridizes only to a target sequence in a sample containing the target sequence.
  • amplicon refers to the product of nucleic acid amplification of a target nucleic acid sequence that is part of a nucleic acid template. For example, to determine whether soybean plants have been sexually crossed from soybeans containing the transgenic soybean event DBN8205 of the present invention, or whether soybean samples collected from a field contain transgenic soybean event DBN8205, or whether soybean extracts, such as meal, flour or oil, contain Transgenic soybean event DBN8205, DNA extracted from soybean plant tissue samples or extracts can be subjected to nucleic acid amplification methods using primer pairs to generate amplicons that are diagnostic for the presence of DNA from transgenic soybean event DBN8205.
  • the pair of primers includes a first primer derived from a flanking sequence adjacent to the insertion site of the inserted foreign DNA in the plant genome, and a second primer derived from the inserted foreign DNA.
  • the amplicon was of a length and sequence that was also diagnostic for the transgenic soybean event DBN8205.
  • the length of the amplicon may range from 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 plus about 450 nucleotide base pairs or more.
  • primer pairs can be derived from flanking genomic sequences flanking the insert DNA to generate amplicons that include the entire insert nucleotide sequence.
  • One of the primer pairs derived from the plant genomic sequence can be located at a distance from the insert DNA sequence, which distance can range from one nucleotide base pair to about twenty thousand nucleotide base pairs.
  • the use of the term "amplicon" specifically excludes primer-dimers formed in DNA thermal amplification reactions.
  • Nucleic acid amplification reactions 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 up to 22 kb of genomic DNA and up to 42 kb of phage DNA. These methods, as well as other DNA amplification methods known in the art, can be used in the present invention.
  • the inserted exogenous DNA sequence and the flanking DNA sequence from the transgenic soybean event DBN8205 can be amplified by using the provided primer sequences to the genome of the transgenic soybean event DBN8205, after amplification, the PCR amplicon or cloned DNA is subjected to standard analysis. DNA sequencing.
  • DNA detection kits based on DNA amplification methods contain DNA molecules used as primers that, under appropriate reaction conditions, specifically hybridize to target DNA and amplify diagnostic amplicons.
  • Kits may provide agarose gel-based detection methods or a number of methods known in the art to detect diagnostic amplicons.
  • a kit containing DNA primers that are homologous or complementary to any part of the soybean genome of SEQ ID NO: 3 or SEQ ID NO: 4, and to any part of the transgene insertion region of SEQ ID NO: 5 is provided by the present invention.
  • Primer pairs specifically identified as useful in DNA amplification methods are SEQ ID NO: 8 and SEQ ID NO: 9, which amplify diagnostic amplification of a portion of the 5' transgene/genomic region homologous to transgenic soybean event DBN8205
  • the amplicon, wherein the amplicon comprises SEQ ID NO: 1.
  • Other DNA molecules used as DNA primers may be selected from SEQ ID NO:5.
  • Amplicons generated by these methods can be detected by a variety of techniques.
  • One such method is Genetic Bit Analysis, which designs a DNA oligonucleotide strand that spans the insert DNA sequence and the adjacent flanking genomic DNA sequence.
  • the oligonucleotide strands are immobilized in the microwells of a microplate, and after PCR amplification of the region of interest (using one primer each within the insert sequence and adjacent flanking genomic sequences), the single-stranded PCR product Can hybridize to immobilized oligonucleotide strands and serve as templates for single-base extension reactions using a DNA polymerase and ddNTPs specifically labeled for the next expected base.
  • Results can be obtained by fluorescent or ELISA-like methods.
  • the signal represents the presence of the insertion/flanking sequence, which indicates that the amplification, hybridization and single base extension reactions were successful.
  • Another method is pyrosequencing technology (Pyrosequencing).
  • This method designs an oligonucleotide strand that spans the insert DNA sequence and the adjacent genomic DNA binding site.
  • This oligonucleotide strand is hybridized to a single-stranded PCR product of the region of interest (using one primer each within the insert and adjacent flanking genomic sequences), followed by DNA polymerase, ATP, sulfurylase, luciferin
  • the enzyme, apyrase, adenosine-5'-phosphosulfate and luciferin are incubated together. Add dNTPs respectively, and measure the light signal generated.
  • the light signal represents the presence of the insertion/flanking sequence, which indicates that the amplification, hybridization, and single-base or multi-base extension reactions were successful.
  • the fluorescence polarization phenomenon described by Chen et al. is also a method that can be used to detect amplicons of the invention.
  • Using this method requires the design of an oligonucleotide strand that spans the insert DNA sequence and the adjacent genomic DNA binding site.
  • the oligonucleotide strand is hybridized to a single-stranded PCR product of the region of interest (using one primer each within the insert and adjacent flanking genomic sequences) with DNA polymerase and a fluorescently labeled ddNTP Incubation.
  • Single base extensions result in the insertion of ddNTPs.
  • This insertion can be measured as a change in polarization using a fluorometer.
  • a change in polarization represents the presence of insertion/flanking sequences, which indicates that the amplification, hybridization and single base extension reactions were successful.
  • Taqman is described as a method for the detection and quantification of the presence of DNA sequences, which is described in detail in the instructions for use provided by the manufacturer.
  • a FRET oligonucleotide probe is designed to span the insertion DNA sequence and the adjacent genomic flanking junction site.
  • the FRET probe and PCR primers are cycled in the presence of a thermostable polymerase and dNTPs.
  • Hybridization of the FRET probe results in cleavage of the fluorescent and quencher moieties on the FRET probe and release of the fluorescent moiety.
  • the generation of a fluorescent signal represents the presence of the insertion/flanking sequence, which indicates that the amplification and hybridization were successful.
  • suitable techniques for detecting plant material derived from transgenic soybean event DBN8205 may also include Southern blot, Northern blot, and in situ hybridization.
  • suitable techniques include incubating the probe and sample, washing to remove unbound probe and detecting whether the probe has hybridized.
  • the detection method depends on the type of label attached to the probe, for example, radiolabeled probes can be detected by X-ray film exposure and visualization, or enzyme-labeled probes can be detected by substrate conversion to achieve a color change.
  • Tyangi et al. (Nature Biotech. 14:303-308, 1996) describe the use of molecular markers in sequence detection. Briefly described below, a FRET oligonucleotide probe is designed to span the insertion DNA sequence and the adjacent genomic flanking junction site. The unique structure of this FRET probe results in it containing a secondary structure that is capable of maintaining a fluorescent moiety and a quencher moiety in close proximity.
  • the FRET probe and PCR primers are cycled in the presence of a thermostable polymerase and dNTPs.
  • the hybridization of the FRET probe to the target sequence leads to the loss of the secondary structure of the probe, thereby spatially separating the fluorescent part and the quencher part, resulting in a fluorescent signal.
  • the generation of a fluorescent signal represents the presence of the insertion/flanking sequence, which indicates that the amplification and hybridization were successful.
  • microfluidics provide methods and devices for isolating and amplifying DNA samples.
  • Optical dyes are used to detect and measure specific DNA molecules.
  • Nanotube devices comprising electronic sensors for the detection of DNA molecules or nanobeads which bind specific DNA molecules and thus can be detected are useful for the detection of the DNA molecules of the present invention.
  • DNA detection kits can be developed using the compositions described herein and methods described or known in the field of DNA detection.
  • the kit facilitates the identification of the presence of DNA from transgenic soybean event DBN8205 in a sample and can also be used to breed soybean plants containing DNA from transgenic soybean event DBN8205.
  • the kit may contain DNA primers or probes homologous to or complementary to at least a portion of SEQ ID NO: 1, 2, 3, 4 or 5, or other DNA primers or probes homologous to or complementary to at least a portion of SEQ ID NO: 1, 2, 3, 4 or 5
  • the DNA contained in the transgenic genetic element is complementary to the DNA, which DNA sequences can be used in DNA amplification reactions, or as probes in DNA hybridization methods.
  • the DNA structure of the junction of the transgene insert sequence and the soybean genome contained in the soybean genome and illustrated in Figure 1 and Table 1 contains: the soybean plant DBN8205 flanking genomic region located at the 5' end of the transgene insert sequence, from the right side of the Agrobacterium A portion of the insert sequence in the border region (RB), the first expression cassette from the Arabidopsis ACT2 promoter (prAtAct2-01), is operably linked to the Arabidopsis chloroplast transit peptide gene (spAtCTP2) and is operably Linked to the insect-resistant cCry2Ab gene of Bacillus thuringiensis and operably linked to the terminator (tPsE9) of the pea RbcS gene; the second expression cassette consists of Arabidopsis ribulose 1,5-di Phosphate carboxylase small subunit gene promoter (prAtRbcS4), operably linked to Arabidopsis ribulose 1,5-bisphosphate carboxylase small subunit gene chlor
  • the DNA molecule used as the primer can be any part of the transgene insert sequence derived from the transgenic soybean event DBN8205, or any part of the flanking DNA sequence of the soybean genome derived from the transgenic soybean event DBN8205.
  • GM soybean event DBN8205 can be combined with other GM soybean varieties, such as GM soybean varieties tolerant to herbicides (such as glyphosate, dicamba, etc.), or GM soybean varieties carrying other insect resistance genes.
  • GM soybean varieties tolerant to herbicides such as glyphosate, dicamba, etc.
  • GM soybean varieties carrying other insect resistance genes Various combinations of all these different transgenic events, bred together with the transgenic soybean event DBN8205 of the present invention, can provide improved hybrid transgenic soybean varieties resistant to various insect pests and various herbicides. These varieties can exhibit superior characteristics compared with non-transgenic varieties and single-trait transgenic varieties.
  • the present invention provides the stacked transgenic soybean event DBN8205 x DBN8002 x DBN9004 obtained by crossing DBN8205, DBN8002 and DBN9004.
  • the stacked transgenic soybean event DBN8205 x DBN8002 x DBN9004 contains the cCry2Ab gene, cCry1Ac gene, cPAT gene, cVip3Aa gene and cEPSPS gene inserted into specific sites in the soybean cell genome, which effectively control South America (A Genyan and Brazil) and major soybean lepidopteran pests in China, and the cPAT gene and cEPSPS gene in the superimposed transgenic soybean event can endow soybean plants with tolerance to glufosinate herbicide and glyphosate herbicide, and No effect on yield.
  • Said “stacking” is the combination of at least two transgenic events with the desired trait into the same plant. Transgenic events are stacked by crossing between parents of transgenic events with desired traits and then identifying offspring with all of these desired traits. Stacking of transgenic events can be used to combine two or more different traits, including, for example, two or more different insect resistance traits, two or more herbicide resistance traits, and/or insect resistance traits and Herbicide resistance traits.
  • the superimposed transgenic soybean event DBN8205 x DBN8002 x DBN9004 described in the present invention is also called soybean plant DBN8205 x DBN8002 x DBN9004, which includes the superimposed transgenic soybean event DBN8205 x DBN8002 x DBN9004 plants and seeds and plant cells or their regenerable parts.
  • Plant parts of the stacked transgenic soybean event DBN8205 x DBN8002 x DBN9004 including but not limited to cells, pollen, ovules, flowers, buds, roots, stems, leaves, pods and products from the stacked transgenic soybean event DBN8205 x DBN8002 x DBN9004, e.g.
  • the transgenic soybean event DBN8205 of the present invention is resistant to feeding damage by Lepidoptera pests and is resistant to the phytotoxic effects of agricultural herbicides containing glufosinate-ammonium.
  • This dual-trait soybean plant expresses the Cry2Ab and Cry1Ac proteins of Bacillus thuringiensis, which confer resistance to feeding damage from lepidopteran pests, and the glufosinate-resistant phosphinothricin N-acetyl of Streptomyces Transferase (PAT) protein, which confers tolerance to glufosinate in plants.
  • PAT Streptomyces Transferase
  • the dual character soybean has the following advantages: 1) from the economic losses caused by Lepidoptera pests (such as cotton bollworm, Spodoptera litura, Spodoptera litura and cutworm, etc.), cotton bollworm, Spodoptera litura, Spodoptera litura and Spodoptera litura and Cutworms etc. are the main pests in soybean planting areas; 2) the ability to apply agricultural herbicides containing glufosinate-ammonium to soybean crops for broad-spectrum weed control; 3) soybean yield is not reduced.
  • Lepidoptera pests such as cotton bollworm, Spodoptera litura, Spodoptera litura and cutworm, etc.
  • soybean yield is not reduced.
  • transgenes encoding insect resistance and glufosinate-ammonium tolerance traits are linked on the same DNA segment and are present at a single locus in the genome of transgenic soybean event DBN8205, which provides enhanced breeding efficiency and enables the use of Molecular markers to track transgene insertions in breeding populations and their progeny.
  • 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 amplification products that are diagnosed as transgenic soybean event DBN8205 or its progeny, and can quickly, accurately and stably identify the presence of plant materials derived from transgenic soybean event DBN8205.
  • SEQ ID NO: 1 A sequence of 22 nucleotides in length near the insertion junction at the 5' end of the insertion sequence in the transgenic soybean event DBN8205, wherein the 1st-11th nucleotides and the 12th-22nd nucleotides Acids are respectively located on both sides of the insertion site on the soybean genome;
  • SEQ ID NO: 2 A sequence of 22 nucleotides in length near the insertion junction at the 3' end of the insertion sequence in the transgenic soybean event DBN8205, wherein the 1st-11th nucleotides and the 12th-22nd nucleotides Acids are respectively located on both sides of the insertion site on the soybean genome;
  • SEQ ID NO: 3 A sequence of 634 nucleotides in length near the insertion junction at the 5' end of the insertion sequence in transgenic soybean event DBN8205;
  • SEQ ID NO: 4 A sequence of 642 nucleotides in length near the insertion junction at the 3' end of the insertion sequence in transgenic soybean event DBN8205;
  • SEQ ID NO: 5 The entire T-DNA sequence, soybean genome flanking sequences at the 5' and 3' ends;
  • SEQ ID NO: 6 spans the pDBN4031 construct DNA sequence and the prAtAct2-01 transcription initiation sequence
  • SEQ ID NO: 7 spans the t35S transcription terminator sequence and the pDBN4031 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: 13 The primer located on the T-DNA paired with SEQ ID NO: 12;
  • SEQ ID NO: 15 The primer located on the T-DNA paired with SEQ ID NO: 14;
  • SEQ ID NO: 16 Taqman detects the first primer of cCry2Ab gene
  • SEQ ID NO: 17 Taqman detects the second primer of cCry2Ab gene
  • SEQ ID NO: 18 Taqman probe for detecting cCry2Ab gene
  • SEQ ID NO: 19 Taqman detects the first primer of cCry1Ac gene
  • SEQ ID NO: 20 Taqman detects the second primer of cCry1Ac gene
  • SEQ ID NO: 21 Taqman probe for detecting cCry1Ac gene
  • SEQ ID NO: 22 Taqman detects the first primer of cPAT gene
  • SEQ ID NO: 23 Taqman detects the second primer of cPAT gene
  • SEQ ID NO: 24 Taqman probe for detecting cPAT gene
  • SEQ ID NO: 25 The first primer of soybean endogenous gene lectin
  • SEQ ID NO: 26 The second primer of soybean endogenous gene lectin
  • SEQ ID NO: 27 The probe of cCry2Ab gene in Southern hybridization detection
  • SEQ ID NO: 28 Probe for cCry1Ac gene in Southern hybridization detection
  • SEQ ID NO: 29 The probe of cPAT gene in Southern hybridization detection
  • SEQ ID NO: 30 is a primer located on the T-DNA, which is in the same direction as SEQ ID NO: 13;
  • SEQ ID NO: 31 is a primer located on the T-DNA, which is in the same direction as SEQ ID NO: 15;
  • SEQ ID NO: 32 is a primer located on the T-DNA, opposite to that of SEQ ID NO: 13;
  • SEQ ID NO: 33 is a primer located on the T-DNA, opposite to that of SEQ ID NO: 13;
  • SEQ ID NO: 34 is a primer located on the T-DNA, opposite to that of SEQ ID NO: 15;
  • SEQ ID NO: 35 is a primer located on the T-DNA, opposite to that of SEQ ID NO: 15;
  • SEQ ID NO: 36 The nucleotide sequence of prAtAct2-02 on the recombinant expression vector pDBN4032;
  • SEQ ID NO: 37 The nucleotide sequence of tOsMth on the recombinant expression vector pDBN4032;
  • SEQ ID NO: 38 The nucleotide sequence of tMtPt1 on the recombinant expression vector pDBN4032.
  • Fig. 1 is a schematic diagram of the structure of the transgene insertion sequence and soybean genome junction site used to detect the nucleic acid sequence of soybean plant DBN8205 and its detection method according to the present invention, and a schematic diagram of the relative position of the nucleic acid sequence used to detect soybean plant DBN8205 (the relative position diagram refers to Wm82.a4 RefGen);
  • Fig. 2 is a structural representation of the recombinant expression vector pDBN4031 used to detect the nucleic acid sequence of soybean plant DBN8205 and its detection method in the present invention
  • Fig. 3 is a schematic structural diagram of the recombinant expression vector pDBN4032 of the present invention.
  • Figure 4 is a field effect diagram of the transgenic soybean event DBN8205 under the natural occurrence condition of cotton bollworm;
  • Figure 5 is a field effect diagram of the transgenic soybean event DBN8205 under natural occurrence conditions of beet armyworm
  • Figure 6 is a field effect diagram of the transgenic soybean event DBN8205 under the condition of natural occurrence of silver-leaved armyworm;
  • Fig. 7 is a field effect diagram of the transgenic soybean event DBN8205 under the condition of soybean borer natural occurrence in area 1.
  • the recombinant expression vector pDBN4031 (as shown in Figure 2) was constructed using standard gene cloning techniques.
  • the vector pDBN4031 contains three transgenic expression cassettes in series, the first expression cassette is operably connected to the Arabidopsis thaliana chloroplast transit peptide gene (spAtCTP2) from the ACT2 promoter (prAtAct2-01) of Arabidopsis, and operably linked to the insect resistance cCry2Ab gene of Bacillus thuringiensis and operably linked to the terminator (tPsE9) of the pea RbcS gene;
  • the second expression cassette consists of Arabidopsis ribulose 1, 5-bisphosphate carboxylase small subunit gene promoter (prAtRbcS4), operably linked to Arabidopsis ribulose 1,5-bisphosphate carboxylase small subunit gene chloroplast transit peptide gene (spAtRbcS4), And operably connected to the insect resistance cC
  • the recombinant expression vector pDBN4032 (as shown in Figure 3) was constructed using standard gene cloning techniques.
  • the vector pDBN4032 contains three tandem transgene expression cassettes, the first expression cassette consists of the ACT2 promoter (prAtAct2-02) (SEQ ID NO: 36) of Arabidopsis thaliana and is operably linked to the Arabidopsis chloroplast transit peptide Gene (spAtCTP2), and operably linked to the insect resistance cCry2Ab gene of Bacillus thuringiensis, and operably linked to the metallothionein-like protein gene transcription terminator (tOsMth) (SEQ ID NO: 37)
  • the second expression cassette consists of the Arabidopsis ribulose 1,5-bisphosphate carboxylase small subunit gene promoter (prAtRbcS4), which is operably linked to the Arabidopsis ribulose 1,5- The bisphosphate carboxylase small subunit gene is on
  • the vectors pDBN4031 and pDBN4032 were respectively transformed into Agrobacterium LBA4404 (Invitrgen, Chicago, USA; Cat.No: 18313-015) by liquid nitrogen method, and 4-[hydroxyl (methyl)phosphono]-DL- Homoalanine was used as a selectable marker to screen transformed cells.
  • the conventional Agrobacterium infection method is used for transformation, and the aseptically cultured soybean cotyledon node tissue is co-cultured with the Agrobacterium containing the vector pDBN4031 in Example 1.1, so that the T-DNA in the constructed recombinant expression vector pDBN4031 Transformation into the soybean genome to generate transgenic soybean events containing the recombinant expression vector pDBN4031.
  • the aseptically cultivated soybean cotyledon node tissue was co-cultured with the Agrobacterium containing the vector pDBN4032 in Example 1.1, so as to transfer the T-DNA in the constructed recombinant expression vector pDBN4032 into the soybean genome , to generate transgenic soybean events containing the recombinant expression vector pDBN4032.
  • soybean germination medium B5 salt 3.1 g/L, B5 vitamin, sucrose 20 g/L, agar 8 g/L, pH 5 .6
  • soybean germination medium B5 salt 3.1 g/L, B5 vitamin, sucrose 20 g/L, agar 8 g/L, pH 5 .6
  • inoculate the seeds on the germination medium and cultivate according to the following conditions: temperature 25 ⁇ 1°C; photoperiod (light/dark) 16/8h.
  • 4-6 days of germination get the aseptic soybean seedlings enlarged at the bright green cotyledon nodes, cut off the hypocotyl at 3-4mm below the cotyledon nodes, cut the cotyledons longitudinally, and remove terminal buds, lateral buds and seed roots.
  • Cotyledon node tissue and Agrobacterium co-culture for a period period (3 days) (step 2: co-cultivation step).
  • the cotyledon node tissue is in solid medium (MS salt 4.3g/L, B5 vitamin, sucrose 20g/L, glucose 10g/L, MES 4g/L, ZT 2mg/L, agar 8g/L, pH 5.6).
  • solid medium MS salt 4.3g/L, B5 vitamin, sucrose 20g/L, glucose 10g/L, MES 4g/L, ZT 2mg/L, agar 8g/L, pH 5.6.
  • the recovery medium B5 salt 3.1g/L, B5 vitamin, MES 1g/L, sucrose 30g/L, ZT 2mg/L, agar 8g/L, cephalosporin 150mg/L, glutamic acid 100mg/L, aspartic acid 100mg/L L, pH 5.6
  • antibiotic cephalosporin 150-250 mg/L
  • the cotyledons The tissue pieces regenerated from the cotyledon nodes were cultured on solid medium with antibiotics but no selection agent to eliminate Agrobacterium and provide a recovery period for infected cells. Next, the tissue pieces regenerated from the cotyledon nodes were cultured on solid medium containing the selection agent (4-[hydroxy( (methyl) phosphono]-DL-homoalanine) culture medium and select the growing transformed callus (step 4: selection step).
  • the selection agent 4-[hydroxy( (methyl) phosphono]-DL-homoalanine
  • the tissue pieces regenerated from the cotyledon nodes are in the presence of the selection agent Screen solid medium (B5 salt 3.1g/L, B5 vitamin, MES 1g/L, sucrose 30g/L, 6-benzyl adenine (6-BAP) 1mg/L, agar 8g/L, cephalosporin 150mg/L L, glutamic acid 100mg/L, aspartic acid 100mg/L, 4-[hydroxyl (methyl) phosphono]-DL-homoalanine 10mg/L, pH5.6) cultured, resulting in transformed cells Can continue to grow.
  • the transformed cell regenerates plant (step 5: regeneration step), preferably, the tissue block that the cotyledon node growth on the substratum that contains selection agent grows in solid medium (B5 differentiation medium and B5 rooting medium) to regenerate plants.
  • the resistant tissue blocks obtained by screening were transferred to the B5 differentiation medium (B5 salt 3.1g/L, B5 vitamin, MES 1g/L, sucrose 30g/L, 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(methyl)phosphono]-DL-homoalanine 5mg /L, pH 5.6), cultured and differentiated at 25°C.
  • B5 differentiation medium B5 salt 3.1g/L, B5 vitamin, MES 1g/L, sucrose 30g/L, 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(methyl)phospho
  • B5 rooting medium B5 salt 3.1g/L, B5 vitamins, MES 1g/L, sucrose 30g/L, agar 8g/L, cephalosporin 150mg/L, indole-3- Butyric acid (IBA) 1mg/L
  • IBA indole-3- Butyric acid
  • a total of 1037 independent transgenic T 0 plants were generated from the vector pDBN4031.
  • the above 1037 independent transgenic T 0 individual plants were sent to the greenhouse for transplanting for cultivation and propagation to obtain transgenic T 1 individual plants.
  • transgenic events Since the soybean genetic transformation process with mature soybean seeds and glufosinate-ammonium as a screening agent is prone to false positive transgenic events, the positive identification of transgenic events was carried out by spraying glufosinate-ammonium in the T1 generation, and a total of 137 transgenic events were obtained.
  • transgenic soybean events DBN8205, pDBN4031-1 and pDBN4031-2 screened by the vector pDBN4031 With the transgenic soybean events pDBN4032-1, pDBN4032-2 and pDBN4032-3 screened by the vector pDBN4032 on the main target insects (Spodoptera litura and Beet Spodoptera) resistance evaluation (see the sixth example), it shows that the design of carrier pDBN4031 is relatively excellent, it is an excellent carrier obtained by fully considering and analyzing the combination and interaction of regulatory elements, and it also shows that the transgenic soybean event DBN8205 Optimal resistance to primary target insects (Spodoptera litura and Spodoptera litura).
  • Step 1 Take 100 mg of leaves of the transgenic soybean event DBN8205, 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 its product manual for specific methods;
  • Step 3 measure the genomic DNA concentration of above-mentioned sample with ultra-micro spectrophotometer (NanoDrop 2000, Thermo Scientific);
  • Step 4 adjusting the genomic DNA concentration of the above sample to the same concentration value, the range of the concentration value is 80-100ng/ ⁇ L;
  • Step 5 using the Taqman probe fluorescent quantitative PCR method to identify the copy number of the sample, using the sample with known copy number as the standard, and using the sample of the wild-type soybean plant as the control, each sample was repeated 3 times, and the average Value; Fluorescence quantitative PCR primer and probe sequences are respectively:
  • Primer 1 gtccacgagaatggatcaatga as shown in SEQ ID NO: 16 in the sequence listing;
  • Primer 2 gtgtggcgtgaataggtgaaatag as shown in SEQ ID NO: 17 in the sequence listing;
  • Probe 1 ctggctcccaacgactataccgggttt as shown in SEQ ID NO: 18 in the sequence listing;
  • Primer 3 gacacagtttctgctcagcgag as shown in SEQ ID NO: 19 in the sequence listing;
  • Primer 4 cccagatgatgtcaactagtccg as shown in SEQ ID NO: 20 in the sequence listing;
  • Probe 2 cgtgccaggtgctgggttcgttc as shown in SEQ ID NO: 21 in the sequence listing;
  • Primer 6 tctcaactgtccaatcgtaagcg as shown in SEQ ID NO: 23 in the sequence listing;
  • Probe 3 ccttacgctgggccctggaaggctag as shown in SEQ ID NO: 24 in the sequence listing;
  • the PCR reaction system is:
  • the 50 ⁇ primer/probe mixture contained 45 ⁇ L of each primer at a concentration of 1 mM, 50 ⁇ L of probes at a concentration of 100 ⁇ M and 860 ⁇ L of 1 ⁇ TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), and at 4 ° C, Stored in amber test tubes.
  • the PCR reaction conditions are:
  • the data was analyzed using 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 DBN8205 was a single copy.
  • the third embodiment analysis of the insertion site of the transgenic soybean event DBN8205
  • the DNA was extracted according to the conventional CTAB (cetyltrimethylammonium bromide) method: take 2g of the young leaves of the transgenic soybean event DBN8205 and grind them into powder in liquid nitrogen, add 0.5mL of DNA extraction CTAB buffer (20g/L CTAB, 1.4M NaCl, 100mM Tris-HCl, 20mM EDTA (ethylenediaminetetraacetic acid), adjust the pH to 8.0 with NaOH), mix thoroughly, and extract at 65°C for 90min ; Add 0.5 times the volume of phenol and 0.5 times the volume of chloroform, mix upside down; centrifuge at 12,000 rpm (revolutions per minute) for 10 minutes; absorb the supernatant, add 2 times the volume of absolute ethanol, shake the centrifuge tube gently, and store at 4°C Let stand for 30min; centrifuge at 12000rpm for 10min; collect DNA to the bottom of the tube; discard the supernatant and wash the precipitate with 1mL of 70% ethanol; centrifuge at 12
  • Concentration determination is performed on the extracted DNA sample, so that the concentration of the sample to be tested is between 80-100 ng/ ⁇ L.
  • Genomic DNA was digested with restriction endonucleases EcoR I (5' end analysis) and EcoR V (3' end analysis). Add 26.5 ⁇ L of genomic DNA, 0.5 ⁇ L of the aforementioned restriction enzymes, and 3 ⁇ L of restriction enzyme digestion buffer to each enzyme digestion system (the restriction enzymes used are all enzymes from NEB Company and their supporting buffers or universal buffers, now called NEBCutSmart), digested for 1h.
  • the primer combination for isolating 5' end genomic DNA includes SEQ ID NO: 13 and SEQ ID NO: 30 as the first primer, SEQ ID NO: 32 and SEQ ID NO: 33 as the second primer, and SEQ ID NO: 13 as a sequencing primer.
  • the combination of primers for isolating 3' end genomic DNA includes SEQ ID NO: 15 and SEQ ID NO: 31 as the first primer, SEQ ID NO: 34 and SEQ ID NO: 35 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 from 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) to isolate the fragment of interest from an agarose matrix.
  • Purified PCR amplification products were then sequenced (e.g., using ABI PrismTM 377, PE Biosystems, Foster City, CA) and analyzed (e.g., using DNASTAR sequence analysis software, DNASTAR Inc., Madison, WI).
  • flanking and junction sequences were confirmed using standard PCR methods.
  • the 5' flanking and junction sequences can be identified using SEQ ID NO:8 or SEQ ID NO:12 in combination with SEQ ID NO:9, SEQ ID NO:13 or SEQ ID NO:30.
  • the 3' flanking and junction sequences can be identified using SEQ ID NO: 11 or SEQ ID NO: 14 in combination with SEQ ID NO: 10, SEQ ID NO: 15 or SEQ ID NO: 31.
  • the PCR reaction system and amplification conditions are shown in Table 2 and Table 3. Those skilled in the art will appreciate that other primer sequences can also be used to confirm flanking and junction sequences.
  • DNA sequencing of the PCR amplification products provided DNA that could be used to design additional DNA molecules that could be used as primers and probes to identify soybean plants or seeds derived from transgenic soybean event DBN8205.
  • soybean genome sequence shown at nucleotides 1-481 of SEQ ID NO: 5 is flanking the right border of the transgenic soybean event DBN8205 insertion sequence (5' flanking sequence), at nucleotide 12397 of SEQ ID NO: 5 Position -12813 shows the soybean genomic sequence flanking the left border of the transgenic soybean event DBN8205 insert (3' flanking sequence).
  • the 5' junction sequence is set forth in SEQ ID NO:1 and the 3' junction sequence is set forth in SEQ ID NO:2.
  • junction sequences are relatively short polynucleotide molecules that are novel DNA sequences that are diagnostic for the DNA of transgenic soybean Event DBN8205 when detected in a polynucleotide detection assay.
  • the junction sequence in SEQ ID NO: 1 and SEQ ID NO: 2 is the insertion site of the transgene fragment in the transgenic soybean event DBN8205 and 11 polynucleotides on each side of the soybean genomic DNA.
  • Longer or shorter polynucleotide junction sequences can be selected from SEQ ID NO:3 or SEQ ID NO:4.
  • Junction sequences (5' junction region SEQ ID NO: 1, and 3' junction region SEQ ID NO: 2) are useful in DNA detection methods as DNA probes or as DNA primer molecules.
  • SEQ ID NO: 6 and SEQ ID NO: 7 are also new DNA sequences in transgenic soybean event DBN8205, which can also be used as DNA probes or as DNA primer molecules to detect the presence of transgenic soybean event DBN8205 DNA.
  • Said SEQ ID NO: 6 spans the pDBN4031 construct DNA sequence and the prAtAct2-01 transcription initiation sequence
  • said SEQ ID NO: 7 spans the t35S transcription termination sequence and the pDBN4031 construct DNA sequence.
  • an amplicon is generated by using at least one primer from SEQ ID NO: 3 or SEQ ID NO: 4, which when used in a PCR method generates a diagnostic amplicon for transgenic soybean event DBN8205.
  • a PCR amplification product comprising genomic DNA flanking the 5' end of the T-DNA insert in the genome of plant material derived from transgenic soybean event DBN8205 was generated from the 5' end of the transgene insert. part.
  • This PCR amplification product comprises SEQ ID NO:3.
  • primer 7 SEQ ID NO: 8 was designed to hybridize to the genomic DNA sequence flanking the 5' end of the transgene insert, and paired with the prAtAct2-01 transcriptional start located in the T-DNA insert.
  • Primer 8 (SEQ ID NO: 9) of the original sequence.
  • a PCR amplification product was generated from the 3' end of the transgene insert that was a portion of the genomic DNA flanking the 3' end of the T-DNA insert in the genome of plant material derived from transgenic soybean event DBN8205.
  • This PCR amplification product comprises SEQ ID NO:4.
  • primer 9 located at the t35S transcription termination sequence in the T-DNA insert was designed, and its paired primer hybridized to the genomic DNA sequence flanking the 3' end of the transgene insert.
  • Primer 10 (SEQ ID NO: 11).
  • the DNA amplification conditions described in Tables 2 and 3 can be used in the PCR zygosity assay described above to generate diagnostic amplicons for transgenic soybean event DBN8205.
  • the detection of the amplicon can be carried out by using Stratagene Robocycler, MJ Engine, Perkin-Elmer 9700 or Eppendorf Mastercycler Gradient thermocycler, etc., or by methods and equipment known to those skilled in the art.
  • primers 7 and 8 when used in the PCR reaction of the transgenic soybean event DBN8205 genomic DNA, produce an amplification product of a 634bp fragment, when used in the non-transformed soybean genome No fragment was amplified when used in a PCR reaction of DNA and non-DBN8205 soybean genomic DNA
  • primers 9 and 10 when used in a PCR reaction of transgenic soybean event DBN8205 genomic DNA, produced The amplified product of the 642 bp fragment, when it was used in a PCR reaction of non-transformed soybean genomic DNA and non-DBN8205 soybean genomic DNA, no fragment was amplified.
  • PCR zygosity assays were also used to identify whether material derived from transgenic soybean event DBN8205 was homozygous or heterozygous.
  • Primer 11 SEQ ID NO: 12
  • Primer 12 SEQ ID NO: 13
  • Primer 13 SEQ ID NO: 14
  • the DNA amplification conditions described in Tables 4 and 5 can be used in the zygosity assay described above to generate diagnostic amplicons for transgenic soybean event DBN8205.
  • cycle parameters in Table 5 on Stratagene Robocycler (Stratagene, La Jolla, CA), MJ Engine (MJ R-Biorad, Hercules, CA), Perkin-Elmer 9700 (Perkin Elmer, Boston, MA) or Eppendorf Mastercycler Gradient (Eppendorf , Hamburg, Germany) thermal cycler for PCR reactions.
  • the MJ Engine or Eppendorf Mastercycler Gradient thermal cycler should be run in calculation mode.
  • the Perkin-Elmer 9700 Thermal Cycler was run with the ramp speed set to the maximum value.
  • the biological sample containing template DNA contains DNA diagnostic for the presence of transgenic soybean event DBN8205 in the sample.
  • the amplification reaction will produce two different DNA amplicons from a biological sample containing DNA derived from the soybean genome that corresponds to the allele corresponding to the inserted DNA present in transgenic soybean event DBN8205 is heterozygous. These two distinct 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 second to diagnose the presence of GM soybean event DBN8205 DNA. Amplicons (SEQ ID NO: 12 and SEQ ID NO: 13).
  • the presence of transgenic soybean event DBN8205 in the sample can be diagnostically determined, and the sample is generated relative to the presence in the transgenic soybean plant DBN8205 The allele corresponding to the inserted DNA is produced in soybean seeds that are homozygous.
  • primer pair for GM soybean event DBN8205 was used to generate amplicons that were diagnostic for GM soybean event DBN8205 genomic DNA. These primer pairs include, but are not limited to, primers 7 and 8 (SEQ ID NO: 8 and 9), and primers 9 and 10 (SEQ ID NO: 10 and 11), used in the DNA amplification method described. Additionally, a control primer 14 and 15 (SEQ ID NO: 25 and 26) for amplifying soybean endogenous genes was included as an internal standard for reaction conditions. Analysis of DNA extract samples from GM soybean event DBN8205 should include a positive tissue DNA extract control from GM soybean event DBN8205, a negative DNA extract control from non-GM soybean event DBN8205, and a soybean DNA extract containing no template.
  • any primer pair from SEQ ID NO: 3 or its complement, or SEQ ID NO: 4 or its complement which when used in a DNA amplification reaction yields a Tissues from the transgenic event soybean plant DBN8205 diagnostically contained the amplicon of SEQ ID NO: 1 or SEQ ID NO: 2.
  • the DNA amplification conditions described in Tables 2-5 can be used to generate diagnostic amplicons for transgenic soybean event DBN8205 using appropriate primer pairs.
  • Extracts of DNA from soybean plants or seeds putatively containing transgenic soybean event DBN8205 that yield diagnostic amplicons for transgenic soybean event DBN8205, or products derived from transgenic soybean event DBN8205, when tested in a DNA amplification method may Used as a template for amplification to determine the presence of GM soybean event DBN8205.
  • CTAB lysis buffer 100 mM Tris-HCl pH 8.0, 20 mM EDTA pH 8.0, 1.4 M NaCl, 0.2% v/v ⁇ -mercaptoethanol, 2% w/v CTAB
  • Tissues were incubated at 65°C for 60min. During the incubation period, the samples were mixed by inversion 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 aqueous phase was repeatedly extracted once with an equal volume of chloroform/isoamyl alcohol (24:1). After collecting the aqueous phase again, add an equal volume of isopropanol, mix well and place at -20°C for 1 hour to precipitate DNA, then centrifuge at 4000rpm for 5 minutes to obtain the DNA precipitate, and then in 1mL TE buffer (10mM Tris-HCl, 1mM EDTA , pH 8.0) to resuspend the DNA pellet.
  • 1mL TE buffer (10mM Tris-HCl, 1mM EDTA , pH 8.0
  • DNA was incubated with 40 ⁇ L of 10 mg/mL RNase A for 30 min at 37°C, centrifuged at 4000 rpm for 5 min, and 3 M sodium acetate (pH 5.2) at a concentration of 0.1 volume and 2 volumes of no In the presence of water and ethanol, DNA was precipitated by centrifugation at 12000 rpm for 10 min. After discarding the supernatant, wash the pellet with 1 mL of 70% (v/v) ethanol, dry at room temperature and redissolve the DNA in 1 mL of TE buffer.
  • the genomic DNA concentration of the above samples was determined with an ultramicro spectrophotometer (NanoDrop 2000, Thermo Scientific).
  • digest 5 ⁇ g of DNA each time digest genomic DNA with restriction endonucleases Mfe I, Spe I, Hind III and Sph I respectively, and use partial sequences of cCry2Ab gene, cCry1Ac gene and cPAT gene on T-DNA as a probe.
  • digests were incubated overnight at the appropriate temperature. The sample was spun down using a speed vacuum, Thermo Scientific to reduce the volume to 20 ⁇ L.
  • DNA sequences were amplified by PCR for probe preparation.
  • the DNA probe is SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 29, or is partially homologous or complementary to the above sequence.
  • Use the DNA Labeling and Detection Starter Kit II kit (Roche, Cat. No. 11585614910) for DIG labeling of probes, Southern blot hybridization, membrane washing and other operations. For specific methods, refer to its product manual.
  • X-ray film (Roche, Cat. No. 11666916001) was used to detect the binding position of the probe.
  • Hybridization data provided corroborating evidence in support of TaqMan TM PCR analysis that soybean plant DBN8205 contained a single copy of the cCry2Ab gene, cCry1Ac gene and cPAT gene.
  • the cCry2Ab gene probe the enzymatic digestion of Mfe I and Spe I produced single bands of about 8.0 kb and 6.0 kb in size; A single band of kb; using the cPAT gene probe, Mfe I and Spe I produced single bands of about 9.5 kb and 13 kb in size after enzymatic digestion, which indicated that one copy of cCry2Ab gene, cCry1Ac gene and cPAT gene existed in soybean Plant DBN8205.
  • the backbone probe no hybridization band was obtained, indicating that no backbone sequence of the pDBN4031 vector entered the soybean plant DBN8205 genome during the transformation process.
  • the fifth embodiment detecting the protein expression level of the transgenic soybean event DBN8205 by ELISA
  • the expression range of Cry1Ac protein in the transgenic soybean event DBN8205 can be detected by ELISA.
  • V5 leaves, R2 stems and flowers, and R6 roots, stems and seeds of the transgenic soybean event DBN8205 were extracted, and the different soybean tissues of the above-mentioned different growth stages were freeze-dried as samples, and 20 mg were weighed for liquid nitrogen grinding.
  • 1mL extraction buffer (8g/L NaCl, 0.27g/L KH 2 PO 4 , 1.42g/LNa 2 HPO 4 , 0.2g/L KCl, 5.5mL/L Tween-20, pH7.4), mix Evenly, let stand at 4°C for 30min, centrifuge at 12000rpm for 10min, take the supernatant and dilute to an appropriate multiple with the above extraction buffer, take 80 ⁇ L of the diluted supernatant for ELISA detection.
  • 1mL extraction buffer 8g/L NaCl, 0.27g/L KH 2 PO 4 , 1.42g/LNa 2 HPO 4 , 0.2g/L KCl, 5.5mL/L Tween-20, pH7.4
  • ELISA enzyme-linked immunosorbent assay
  • the experimental results of Cry1Ac protein content in transgenic soybean event DBN8205 are shown in Table 6.
  • the ratio of the average expression of Cry1Ac protein in different tissues at different growth stages in the transgenic soybean event DBN8205 to the dry weight of the corresponding tissues was measured to be 1.11 to 342.05 ( ⁇ g/g).
  • the ratio of stem dry weight in R2 stage was 5.3 ⁇ g/g
  • the average expression of Cry1Ac protein in soybean R2 flower dry weight was 12.45 ⁇ g/g
  • the average expression of Cry1Ac protein in soybean R6 root The proportion of Cry1Ac protein expression in R6 root dry weight was 1.11 ⁇ g/g, and the average expression level of Cry1Ac protein in soybean R6 seed dry weight was 8.44 ⁇ g/g.
  • Table 6 fully demonstrates that the Cry1Ac protein in the transgenic soybean event DBN8205 is expressed in different soybean tissues at different growth stages of soybean, especially in leaves, flowers and seeds. resistance, and at the same time indicate that the design of the vector pDBN4031 is excellent.
  • the second leaves of the V3 stage of the above-mentioned transgenic soybean plants and wild-type soybean plants were taken respectively, rinsed with sterile water, and the water on the leaves was blotted dry with gauze, then the veins were removed, and cut into The shape of about 2.5cm ⁇ 3cm, take 1-3 pieces (determine the number of leaves according to the food intake of insects) and put the cut leaves on the filter paper at the bottom of the circular plastic petri dish.
  • the filter paper is moistened with distilled water.
  • total resistance score 100 ⁇ mortality+[100 ⁇ mortality+90 ⁇ (newly hatched number/total number of inoculated worms)+60 ⁇ (number of larvae larger than the size of newly hatched larvae to smaller than the size of negative control/total number of inoculated worms)+10 ⁇ (number of negative control worms/total number of inoculated worms)]+100 ⁇ (1-leaf damage rate ).
  • the total number of inoculated insects refers to the total number of inoculated insects, that is, 10 per dish; the development progress of larvae has been reflected by the total resistance score formula; the leaf damage rate refers to the ratio of the leaf area eaten by pests to the total leaf area.
  • 5 plants were selected from transgenic soybean plants DBN8205, pDBN4031-1, pDBN4031-2, pDBN4032-1, pDBN4032-2, pDBN4032-3 and wild-type soybean plants (non-transgenic, NGM) for testing. Strains were repeated 3 times. The results are shown in Table 7.
  • transgenic soybean events (DBN8205, pDBN4031-1, pDBN4031-2) screened by vector pDBN4031 and transgenic soybean events (pDBN4032-1, pDBN4032-2, pDBN4032-2) screened by vector pDBN4032 3)
  • the resistance to beet armyworm and Spodoptera litura was significantly better than NGM
  • the transgenic soybean events (DBN8205, pDBN4031-1, pDBN4031-2) screened by vector pDBN4031 were resistant to beet armyworm and Spodoptera litura
  • the resistance of the vector pDBN4031 was better than that of the transgenic soybean events (pDBN4032-1, pDBN4032-2, pDBN4032-3) screened by the vector pDBN4032, indicating that the design of the vector pDBN4031 is excellent, which fully considers and analyzes the combination and interaction of regulatory elements.
  • the seventh embodiment detection of DBN8205 event resistance to insects
  • the transgenic soybean event DBN8205 and wild-type soybean plants were planted against cotton bollworm [Helicoverpa armigera, CBW], small cutworm [Agrotis ypsilon, BCW], bean hornworm [Clanis bilineata, BHM] and The bioassay of the fall armyworm [Spodoptera frugiperda, FAW] was performed as follows:
  • the second leaves of the V3 stage of the transgenic soybean event DBN8205 and the wild-type soybean plant (non-transgenic, NGM) were taken respectively, washed with sterile water, and the water on the leaves was blotted dry with gauze, then the veins were removed, and cut into The shape of about 2.5cm ⁇ 3cm, take 1-3 pieces (determine the number of leaves according to the food intake of insects) and put the cut leaves on the filter paper at the bottom of the circular plastic petri dish.
  • the filter paper is moistened with distilled water.
  • total resistance score 100 ⁇ mortality+[100 ⁇ mortality+90 ⁇ (newly hatched number/total number of inoculated worms)+60 ⁇ (number of larvae larger than the size of newly hatched larvae to smaller than the size of negative control/total number of inoculated worms)+10 ⁇ (number of negative control worms/total number of inoculated worms)]+100 ⁇ (1-leaf damage rate ).
  • the total number of inoculated insects refers to the total number of inoculated insects, that is, 10 per dish; the development progress of larvae has been reflected by the total resistance score formula; the leaf damage rate refers to the ratio of the leaf area eaten by pests to the total leaf area.
  • 5 plants were selected from the transgenic soybean event DBN8205 and wild-type soybean plants (non-transgenic, NGM) for testing, and each plant was repeated 6 times. The experimental results are shown in Table 8.
  • Transgenic soybean event DBN8205 and wild-type soybean plants were planted in the field: random block design, 3 repetitions, plot area of 30m 2 (5m ⁇ 6m), row spacing 60cm, row spacing 10cm, conventional cultivation management, Target pest insecticides are not sprayed during the whole growth period.
  • Natural pest detection is only carried out in areas where the natural occurrence of silver-leaved armyworm is relatively serious (conditions for natural pest occurrence: the peak period of damage is from June to September, and the optimum temperature for development is 20-30°C).
  • Table 11 shows the resistance results of the transgenic soybean event DBN8205 to silver-leaved armyworm.
  • FIG. 6 is a comparative effect diagram of the transgenic soybean event DBN8205 and NGM under the natural occurrence conditions of Autographa.
  • the transgenic soybean event DBN8205 and wild-type soybean plants were planted respectively against Rachiplusia nu, SFL, Anticarsia gemmatalis, VBC, and Chrisiodexys includens, respectively.
  • SBL South American cotton bollworm [Helicoverpa gelotopoeon, SABW], tobacco leaf moth [Chloridea virescens, TBW], grassland armyworm [Spodoptera frugiperda, FAW], carbon black armyworm [Spodoptera cosmioides, BLAW], Helicoptera [Helicoverpa zea, SPW], southern spodoptera [Spodoptera eridania, SAW] and Albula litura [Spodoptera albula, GSAW] were bioassayed as follows:
  • the second leaves of the V3 stage of the transgenic soybean event DBN8205 and the wild-type soybean plant (non-transgenic, NGM) were taken respectively, washed with sterile water, and the water on the leaves was blotted dry with gauze, then the veins were removed, and cut into A circle with a diameter of about 1.6 cm, take 1-3 pieces (the number of leaves is determined according to the food intake of insects), and put the cut round leaves on a round plastic petri dish with 2 mL of agar, and put 1 primordial on each petri dish.
  • leaf damage rate refers to the ratio of the area of leaves eaten by pests to the total area of leaves.
  • transgenic soybean event DBN8205 and wild-type soybean plants were tested against corn stem borer [Elasmopalpus lignosellus, LSCB] according to the following methods to determine the pest mortality, plant damage rate and plant mortality.
  • the method for measuring the mortality of test insects the three-day-germinated transgenic soybean event DBN8205 seedlings and wild-type soybean plant seedlings (non-transgenic, NGM) (32 each) cultivated under greenhouse conditions were rooted out, and then placed in separate in a small plastic box.
  • the bottom of the chamber plastic box contains 2% agar to maintain the normal development of the plants.
  • the method for measuring the plant damage rate and plant mortality Take 32 plants of transgenic soybean event DBN8205 plants and wild-type soybean plants (non-transgenic, NGM) that have been cultivated for 7 days under greenhouse conditions, and place them in the flowerpots where the above soybean plants are planted. Place PVC pipes around to increase physical barrier to avoid migration of insects. Two 12-hour-incubated corn stem borer larvae were then placed at the base of the stem of each plant. After 15 days of inoculation, the plant damage rate (plant damage rate refers to the ratio of the total number of surviving plants damaged by pests to the total number of all tested plants) and plant mortality (plant mortality refers to the ratio of the total number of plants damaged to death by pests to all ratio of the total number of test plants). Each plant was repeated 6 times. The experimental results are shown in Table 14.
  • Basta herbicide (glufosinate-ammonium ammonium saline solution with an active ingredient of 18%) was selected for spraying.
  • a random block design was adopted with 3 repetitions.
  • the area of the plot is 15m 2 (5m ⁇ 3m), the row spacing is 60cm, the plant spacing is 10cm, conventional cultivation management, and there is a 1m wide isolation zone between the plots.
  • the transgenic soybean event DBN8205 was subjected to the following two treatments: (1) without spraying herbicides, artificial weed control to remove the influence of weeds on soybean growth; (2) according to 800g ai/ha (ai/ha refers to "active Components per hectare") Dosage spraying Baoshida herbicide in V2 ⁇ V3 period.
  • the symptoms of phytotoxicity were investigated 1 week and 2 weeks after the application, and the yield of the plot was measured at the time of harvest; the grading of phytotoxicity symptoms is shown in Table 15.
  • the injury rate of glufosinate refers to the injury rate of glufosinate-ammonium.
  • the injury rate of glufosinate-ammonium is determined according to the results of the phytotoxicity investigation 2 weeks after the treatment of glufosinate-ammonium. tolerance level.
  • yield percentage sprayed yield/no spray Construction volume.
  • Phytotoxicity level symptom description 1 Normal growth without any symptoms of injury 2 Slight phytotoxicity, phytotoxicity less than 10% 3 Moderate phytotoxicity, can recover later, does not affect yield 4 The drug damage is heavy, it is difficult to recover, resulting in reduced production 5 The phytotoxicity is serious and cannot be recovered, resulting in obvious production reduction or cessation of production
  • the damage rate of transgenic soybean event DBN8205 is 0 under the treatment of glufosinate-ammonium herbicide (800g a.i./ha); thus, the transgenic soybean event DBN8205 has good glufosinate-ammonium herbicide drug tolerance.
  • the yield under spraying 800g a.i./ha glufosinate-ammonium treatment was not significantly different from that without spraying treatment, thus further indicating that the transgenic soybean event DBN8205 has good tolerance to glufosinate-ammonium herbicide , and has no effect on yield.
  • the transgenic soybean event DBN8205 whose transformation background was the soybean Jack plant was transferred into the soybean plants whose parental background was Heihe 43 and the parental background were Zhonghuang 35 respectively by backcrossing.
  • the transgenic soybean event DBN8205 with the transformation background of Heihe 43 and the transgenic soybean event DBN8205 with the transformation background of Zhonghuang 35 were respectively obtained by selfing, and the integrity of the transgenic soybean event DBN8205 was detected by PCR in each generation (see the third example).
  • the soybean transformation event DBN8205 whose transformation backgrounds were Jack, Heihe 43 and Zhonghuang 35 were compared with wild-type soybean Jack plants, wild-type soybean Heihe 43 plants and wild-type soybean Zhonghuang 35 plants (non-transgenic, NGM) respectively.
  • Cotton bollworm [Helicoverpa armigera, CBW] was subjected to bioassay according to the bioassay method in Example 7.1, and each plant was repeated 6 times. The experimental results are shown in Table 17.
  • the genetically modified soybean event DBN9004 (CN106086011A) (male parent) was crossed with the genetically modified soybean event DBN8002 (female parent) to obtain heterozygous plants superimposed with the genetically modified soybean event DBN8002 x DBN9004, and then after two generations of selfing, the target gene copy was detected by TaqMan number (referring to the second embodiment) and the homozygous (referring to the third embodiment) of PCR zygosity detection site, obtain superimposed transgenic soybean event DBN8002 x DBN9004 homozygous plant, use it as male parent and transgenic soybean event DBN8205 ( female parent) to obtain superimposed transgenic soybean event DBN8205 x DBN8002 x DBN9004.
  • the superimposed transgenic soybean event DBN8205 x DBN8002 x DBN9004 and wild-type soybean plants (non-transgenic, NGM) 2 plants were respectively effective against cotton bollworm [Helicoverpa armigera, CBW], beet armyworm [Spodoptera exigua, BAW] and frugiperda [ Spodoptera frugiperda, FAW] carry out bioassay according to the method of the seventh embodiment 7.1.
  • the experimental results are shown in Table 18.
  • the superimposed transgenic soybean event DBN8205 x DBN8002 x DBN9004 and wild-type soybean plants (non-transgenic, NGM) 2 plants were respectively effective against Rachiplusia nu, SFL, Anticarsia gemmatalis, VBC, soybean Moth [Chrisiodexys includesens, SBL], South American cotton bollworm [Helicoverpa gelotopoeon, SABW], Spodoptera frugiperda, FAW], Spodoptera cosmioides, BLAW and Helicoverpa zea, SPW Bioassays were performed according to the method according to the seventh example 7.3(1). The experimental results are shown in Table 19.
  • the superimposed transgenic soybean event DBN8205 x DBN8002 x DBN9004 were subjected to the following two treatments: 1) no herbicide spraying, artificial weed control to remove the impact of weeds on soybean growth; 2) according to 1680g ae/ha (ae/ha Refers to the "active ingredient equivalent acid per hectare") dose spraying Roundup herbicide at the V3 leaf stage, and then spraying Roundup herbicide again at the same dosage at the R2 stage (full flowering stage); 3) by 800g ai/ha (ai/ ha refers to "active ingredient per hectare") spraying Baoshida herbicide at the V3 leaf stage, and then spraying Baoshida herbicide again at the V6 stage at the same dose; 4) Spraying 800g ai/ha at the V3 leaf stage Baoshida herbicide, and then spray Roundup herbicide at 1680g ae/ha dose in R2 period.
  • the symptoms of phytotoxicity were investigated 1 week and 2 weeks after the application, and the soybean yield of the plot was measured at the time of harvest.
  • Herbicide damage rate includes glyphosate damage rate and glufosinate-ammonium damage rate, herbicide damage rate is determined according to the phytotoxicity investigation results 2 weeks after glyphosate or glufosinate-ammonium treatment.
  • yield percentage sprayed yield/no spraying Yield.
  • Agricultural products or commodities such as soybean plants can be produced from soybean plants comprising transgenic soybean event DBN8205 or from soybean plants comprising transgenic soybean event DBN8205 and at least one other transgenic soybean event different from transgenic soybean event DBN8205. If a sufficient expression level is detected in the agricultural product or commodity, the agricultural product or commodity is expected to contain a nucleotide sequence capable of diagnosing the presence of the transgenic soybean event DBN8205 material in the agricultural product or commodity.
  • the agricultural products or commodities include, but are not limited to, soybean cakes, flours, and oils, specifically lecithin, fatty acids, glycerin, sterols, edible oils, defatted soybean flakes, including defatted and roasted soybean flour, soybean milk curd, Tofu, soy protein concentrate, isolated soy protein, hydrolyzed vegetable protein, textured soy protein and soy protein fiber, and any other food product to be consumed by animals as a food source, etc.
  • Nucleic acid detection methods and/or kits based on probe or primer pairs can be developed to detect the nucleotide sequence derived from transgenic soybean event DBN8205 such as shown in SEQ ID NO: 1 or SEQ ID NO: 2 in biological samples, wherein the probe sequence or primer sequence is selected from sequences or parts thereof shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 to diagnose transgenes Existence of soybean event DBN8205.
  • the transgenic soybean event DBN8205 of the present invention has good resistance to Lepidoptera insects and high tolerance to glufosinate-ammonium herbicide without affecting other agronomic traits and yield of the plant itself.
  • the detection method can accurately and quickly identify whether the DNA molecule of the transgenic soybean event DBN8205 is contained in the biological sample.
  • the seeds corresponding to the genetically modified soybean event DBN8205 have been deposited in the General Microorganism Center of China Committee for Microorganism Culture Collection (CGMCC for short) in accordance with the Budapest Treaty on December 27, 2021, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Zip code 100101), classification name: soybean (Glycine max), preservation state: living, preservation number is CGMCC No.45071. The deposit will be kept in the depository for 30 years.
  • CGMCC General Microorganism Center of China Committee for Microorganism Culture Collection

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Abstract

提供了一种用于检测大豆植物DBN8205的核酸序列及其检测方法，该核酸序列包括SEQ ID NO：1或其互补序列、和/或SEQ ID NO：2或其互补序列。该大豆植物DBN8205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，且对产量无影响。该检测方法可以准确快速地鉴定生物样品中是否包含转基因大豆事件DBN8205的DNA分子。

Description

用于检测大豆植物DBN8205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DBN8205和用于检测生物样品中是否包含特定转基因大豆事件DBN8205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。已将生物技术方法应用于这种作物以产生具有所需性状的大豆品种。在大豆生产中最重要的两个农艺性状为昆虫抗性和除草剂耐受性。大豆对昆虫的抗性可以通过转基因的方法使昆虫抗性基因在大豆植物中表达而获得，依赖于针对昆虫侵染的单一抗虫蛋白表达的转基因大豆处于有限耐久性的风险中，这是因为昆虫在持续的选择压力下将进化出针对转基因大豆中表达的杀虫蛋白的抗性，这样的抗性一旦产生并且不能被有效控制的话，无疑将限制含有杀虫蛋白的转基因大豆品种的商业价值。因此将两种或两种以上杀虫蛋白组合使用可以作为一种延缓昆虫抗性的方法，同时可以拓宽抗虫谱。从链霉菌分离的膦丝菌素N-乙酰基转移酶(PAT)通过乙酰化催化L-膦丝菌素转化为其无活性形式。表达PAT的植物优化形式的基因已经在大豆中使用以赋予大豆对草铵膦除草剂的耐受性，例如大豆事件A5547-127。
设计适用于转化大豆作物的包含外源功能基因(cCry2Ab基因、cCry1Ac基因和cPAT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。除了功能基因(cCry2Ab基因、cCry1Ac基因和cPAT基因)本身，调控元件的选择对于获得良好的转化事件是至关重要且技术效果是难以预料的。例如，商业化抗草甘膦大豆转化事件GTS 40-3-2(US5633435)和MON89788(CN101252831B)，两者都转入了CP4-EPSPS基因(氨基酸序列相同)，且均采用单表达盒的分子设计，但由于选择了不同的调控元件，两个转化事件的EPSPS蛋白表达水平存在明显差异，产量水平也不尽相同。因此，在设计表达载体时，需要充分考量和分析调控元件的组合和互作以及在T-DNA上的排布设计。与此同时，良好的商业化大豆转化事件还要综合考虑cCry2Ab基因、cCry1Ac基因和cPAT基因在大豆植物中的载体设计、三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cCry2Ab基因、cCry1Ac基因和cPAT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DBN8205的核酸序列及其检测方法，转基因大豆事件DBN8205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DBN8205的DNA分子。
为实现上述目的，本发明提供了一种具有以下核酸序列的核酸分子，所述核酸序列包含SEQ ID NO：3或其互补序列第1-462位中至少11个连续的核苷酸和SEQ ID NO：3或其 互补序列第463-634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1-225位中至少11个连续的核苷酸和SEQ ID NO：4或其互补序列第226-642位中至少11个连续的核苷酸。
优选地，所述核酸序列包含SEQ ID NO：3或其互补序列第1-462位中22-25个连续的核苷酸和SEQ ID NO：3或其互补序列第463-634位中22-25个连续的核苷酸、和/或SEQ ID NO：4或其互补序列第1-225位中22-25个连续的核苷酸和SEQ ID NO：4或其互补序列第226-642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DBN8205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DBN8205的存在。所述SEQ ID NO：2或其互补序列为转基因大豆事件DBN8205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DBN8205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，所述核酸序列包含所述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)，和所述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DBN8205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DBN8205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634个核苷酸的序列，所述SEQ ID NO：3或其互补序列由462个核苷酸的大豆基因组5’侧翼序列(SEQ ID NO：3的核苷酸第1-462位)和172个pDBN4031构建体DNA序列中的核苷酸(SEQ ID NO：3的核苷酸第463-634位)，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DBN8205的存在。
所述核酸序列包含所述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)，和所述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DBN8205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DBN8205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642个核苷酸的序列，所述SEQ ID NO：4或其互补序列由21个核苷酸的t35S转录终止子的DNA序列(SEQ ID NO：4的核苷酸第1-21位)、204个pDBN4031构建体DNA序列中的核苷酸(SEQ ID NO：4的核苷酸第22-225位)和417个核苷酸的大豆基因组3’侧翼序列(SEQ ID NO：4的核苷酸第226-642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DBN8205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DBN8205的长度为12813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DBN8205的存在。
表1、SEQ ID NO：5包含的基因组及遗传元件
Figure PCTCN2022077093-appb-000001
Figure PCTCN2022077093-appb-000002
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DBN8205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DBN8205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DBN8205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，所述两种引物包括SEQ ID NO：8和SEQ ID NO：9、SEQ ID NO：10和SEQ ID NO：11、或者SEQ ID NO：1和SEQ ID NO：2的互补序列。
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DBN8205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DBN8205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DBN8205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或部分，包含编码昆虫抗性Cry2Ab蛋白的核酸序列、编码昆虫抗性Cry1Ac蛋白的核酸序列、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，所述特定区域的核酸序列包含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包含SEQ ID NO：3和/或SEQ ID NO：4所示的序列。
优选地，所述植物细胞或部分依次包含SEQ ID NO：1、SEQ ID NO：5第866-12192位核酸序列和SEQ ID NO：2，或者包含SEQ ID NO：5所示的序列。
优选地，所述植物细胞或部分包含转基因大豆事件DBN8205；
可选地，所述植物细胞或部分还包含至少一种不同于转基因大豆事件DBN8205的其他转基因大豆事件；优选地，所述其他转基因大豆事件为转基因大豆事件DBN9004和/或转基因大豆事件DBN8002。
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866-12192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866-12192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Cry2Ab蛋白的核酸序列和/或编码昆虫抗性Cry1Ac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Cry2Ab蛋白的核酸序列和/或编码昆虫抗性Cry1Ac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，所述方法包括将包含转基因大豆事件DBN8205的第一大豆植物与第二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；
选择对靶昆虫具有抗性和/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包含转基因大豆事件DBN8205大豆植物的农产品或商品，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。
为实现上述目的，本发明还提供了一种产生自包含转基因大豆事件DBN8205大豆植物的农产品或商品，所述大豆植物还包含至少一种不同于转基因大豆事件DBN8205的其他转基因大豆事件；
优选地，所述其他转基因大豆事件为转基因大豆事件DBN9004和/或转基因大豆事件DBN8002。
为实现上述目的，本发明还提供了一种扩展植物抗虫谱和/或所耐受除草剂范围的方法，将所述转基因大豆事件DBN8205在植物中与至少一种不同于转基因大豆事件DBN8205的其他转基因大豆事件一起表达；
优选地，所述其他转基因大豆事件为转基因大豆事件DBN9004和/或转基因大豆事件DBN8002。
本发明提供的DBN9004为专利CN106086011A中公开的转基因大豆事件，所述转基因大豆事件DBN9004以种子的形式且以保藏编号CGMCC No.11171保藏于中国微生物菌种保藏管理委员会普通微生物中心。
本发明提供的DBN8002为专利CN111406117A中公开的转基因大豆事件，所述转基因大豆事件DBN8002以种子的形式且以保藏编号CGMCC No.17299保藏于中国微生物菌种保藏管理委员会普通微生物中心。
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。
术语“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DBN8205的转基因大豆事件及其后代，所述转基因大豆事件DBN8205亦称为大豆植物DBN8205，其包括转基因大豆事件DBN8205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DBN8205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DBN8205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DBN8205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DBN8205获得对昆虫的抗性和对草铵膦除草剂的耐受性。所述DNA构建体包含三个串联的表达盒，第一个表达盒包含用于在植物中表达的适合的启动子，编码信号肽/转运肽的核酸序列，编码Cry2Ab蛋白的核酸序列和适合的多聚腺苷酸化信号序列，所述Cry2Ab蛋白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，编码信号肽/转运肽的核酸序列，编码Cry1Ac蛋白的核酸序列和适合的多聚腺苷酸化信号序列，所述Cry1Ac蛋白亦主要对鳞翅目昆虫具有抗性。第三个表达盒包含用于在植物中表达的适合的启动子，编码膦丝菌素N-乙酰基转移酶(phosphinothricin N-acetyltransferase，PAT)的核酸序列和适合的多聚腺苷酸化信号序列，所述PAT蛋白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。所述信号肽/转运肽可以引导Cry2Ab蛋白和/或Cry1Ac蛋白转运到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II(PIN II)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。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。
Cry2Ab杀虫蛋白和Cry1Ac杀虫蛋白是众多杀虫蛋白中的两种，其是由苏云金芽孢杆菌(Bacillus thuringiensis，简称Bt)产生的不溶性伴孢结晶蛋白。Cry2Ab蛋白或Cry1Ac蛋白被昆虫摄入进入中肠，毒蛋白原毒素被溶解在昆虫中肠的碱性pH环境下，蛋白N-和C-末端被碱性蛋白酶消化，将原毒素转变成活性片段，活性片段和昆虫中肠上皮细胞膜上表面上受体结合，插入肠膜，导致细胞膜出现穿孔病灶，破坏细胞膜内外的渗透压变化及pH平衡等，扰乱昆虫的消化过程，最终导致其死亡。
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DBN8205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DBN8205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。
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转基因大豆事件DBN8205的方法，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DBN8205及其后代的大豆植物组成，该转基因大豆事件DBN8205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，可以产生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DBN8205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DBN8205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DBN8205或种子还是来源于转基因大豆事件DBN8205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DBN8205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DBN8205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DBN8205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DBN8205，或大豆提取物，例如粗粉、面或油是否包含转基因大豆事件DBN8205，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DBN8205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DBN8205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DBN8205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DBN8205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DBN8205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DBN8205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DBN8205的DNA，还可以用于培育含有转基因大豆事件DBN8205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DBN8205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ACT2启动子(prAtAct2-01)，可操作地连接到拟南芥叶绿体转运肽基因(spAtCTP2)上，并可操作地连接到苏云金芽孢杆菌的昆虫抗性的cCry2Ab基因上，并可操作地连接到豌豆RbcS基因的终止子(tPsE9)上而组成；第二个表达盒由拟南芥核酮糖1，5-二磷酸羧化酶小亚基基因启动子(prAtRbcS4)，可操作地连接到拟南芥核酮糖1，5-二磷酸羧化酶小亚基基因叶绿体转运肽基因(spAtRbcS4)上，并可操作地连接到苏云金芽孢杆菌的昆虫抗性的cCry1Ac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；第三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DBN8205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DBN8205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DBN8205的大豆基因组侧翼DNA序列的任何部分。
转基因大豆事件DBN8205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DBN8205一起育种，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更优异的特征。
例如，本发明提供了叠加转基因大豆事件DBN8205 x DBN8002 x DBN9004，其是通过DBN8205、DBN8002和DBN9004杂交获得。所述叠加转基因大豆事件DBN8205 x DBN8002 x DBN9004包含插入大豆细胞基因组内特定位点中的cCry2Ab基因、cCry1Ac基因、cPAT基因、cVip3Aa基因和cEPSPS基因，其以三种抗虫机制有效控制南美洲(阿根延和巴西)和中国境内的大豆主要鳞翅目害虫，且叠加转基因大豆事件中的cPAT基因和cEPSPS基因可以赋予大豆植物对草铵磷除草剂和草甘膦除草剂的耐受性，并对产量无影响。
所述“叠加”是将具有所需性状的至少两个转基因事件组合入同一植物。通过在具有所需性状的转基因事件的亲本之间进行杂交，然后鉴定具有所有这些所需性状的后代来将 转基因事件叠加。可使用转基因事件叠加以组合两种或更多种不同性状，包括例如两种或更多种不同昆虫抗性性状、两种或更多种除草剂抗性性状、和/或昆虫抗性性状和除草剂抗性性状。
本发明所述叠加转基因大豆事件DBN8205 x DBN8002 x DBN9004亦称为大豆植物DBN8205 x DBN8002 x DBN9004，其包括叠加转基因大豆事件DBN8205 x DBN8002 x DBN9004的植物和种子及其植物细胞或其可再生部分，所述叠加转基因大豆事件DBN8205 x DBN8002 x DBN9004的植物部分，包括但不限于细胞、花粉、胚珠、花、芽、根、茎、叶、荚和来自叠加转基因大豆事件DBN8205 x DBN8002 x DBN9004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DBN8205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Cry2Ab蛋白和Cry1Ac蛋白，其提供了对鳞翅目害虫摄食损伤的抗性，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(如棉铃虫、斜纹夜蛾、甜菜夜蛾和小地老虎等)造成的经济损失，棉铃虫、斜纹夜蛾、甜菜夜蛾和小地老虎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DBN8205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DBN8205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DBN8205的植物材料的存在。
序列简述
SEQ ID NO：1 转基因大豆事件DBN8205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DBN8205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DBN8205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634个核苷酸的序列；
SEQ ID NO：4 转基因大豆事件DBN8205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642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 跨越了pDBN4031构建体DNA序列和prAtAct2-01转录起始序列；
SEQ ID NO：7 跨越了t35S转录终止子序列和pDBN4031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cCry2Ab基因的第一引物；
SEQ ID NO：17 Taqman检测cCry2Ab基因的第二引物；
SEQ ID NO：18 Taqman检测cCry2Ab基因的探针；
SEQ ID NO：19 Taqman检测cCry1Ac基因的第一引物；
SEQ ID NO：20 Taqman检测cCry1Ac基因的第二引物；
SEQ ID NO：21 Taqman检测cCry1Ac基因的探针；
SEQ ID NO：22 Taqman检测cPAT基因的第一引物；
SEQ ID NO：23 Taqman检测cPAT基因的第二引物；
SEQ ID NO：24 Taqman检测cPAT基因的探针；
SEQ ID NO：25 大豆内源基因lectin的第一引物；
SEQ ID NO：26 大豆内源基因lectin的第二引物；
SEQ ID NO：27 Southern杂交检测中cCry2Ab基因的探针；
SEQ ID NO：28 Southern杂交检测中cCry1Ac基因的探针；
SEQ ID NO：29 Southern杂交检测中cPAT基因的探针；
SEQ ID NO：30 位于T-DNA上的引物，与SEQ ID NO：13方向一致；
SEQ ID NO：31 位于T-DNA上的引物，与SEQ ID NO：15方向一致；
SEQ ID NO：32 位于T-DNA上的引物，与SEQ ID NO：13方向相反；
SEQ ID NO：33 位于T-DNA上的引物，与SEQ ID NO：13方向相反；
SEQ ID NO：34 位于T-DNA上的引物，与SEQ ID NO：15方向相反；
SEQ ID NO：35 位于T-DNA上的引物，与SEQ ID NO：15方向相反；
SEQ ID NO：36 重组表达载体pDBN4032上prAtAct2-02的核苷酸序列；
SEQ ID NO：37 重组表达载体pDBN4032上tOsMth的核苷酸序列；
SEQ ID NO：38 重组表达载体pDBN4032上tMtPt1的核苷酸序列。
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DBN8205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DBN8205的核酸序列相对位置的示意图(相对位置示意图参考Wm82.a4 RefGen)；
图2为本发明用于检测大豆植物DBN8205的核酸序列及其检测方法的重组表达载体pDBN4031的结构示意图；
图3为本发明重组表达载体pDBN4032的结构示意图；
图4为转基因大豆事件DBN8205在棉铃虫自然发生条件下的田间效果图；
图5为转基因大豆事件DBN8205在甜菜夜蛾自然发生条件下的田间效果图；
图6为转基因大豆事件DBN8205在银纹夜蛾自然发生条件下的田间效果图；
图7为转基因大豆事件DBN8205在大豆食心虫自然发生条件下在地区一的田间效果图。
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DBN8205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pDBN4031(如图2所示)。所述载体 pDBN4031包含三个串联的转基因表达盒，第一个表达盒由拟南芥的ACT2启动子(prAtAct2-01)，可操作地连接到拟南芥叶绿体转运肽基因(spAtCTP2)上，并可操作地连接到苏云金芽孢杆菌的昆虫抗性的cCry2Ab基因上，并可操作地连接到豌豆RbcS基因的终止子(tPsE9)上而组成；第二个表达盒由拟南芥核酮糖1，5-二磷酸羧化酶小亚基基因启动子(prAtRbcS4)，可操作地连接到拟南芥核酮糖1，5-二磷酸羧化酶小亚基基因叶绿体转运肽基因(spAtRbcS4)上，并可操作地连接到苏云金芽孢杆菌的昆虫抗性的cCry1Ac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，第三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。
使用标准基因克隆技术构建重组表达载体pDBN4032(如图3所示)。所述载体pDBN4032包含三个串联的转基因表达盒，第一个表达盒由拟南芥的ACT2启动子(prAtAct2-02)(SEQ ID NO：36)，可操作地连接到拟南芥叶绿体转运肽基因(spAtCTP2)上，并可操作地连接到苏云金芽孢杆菌的昆虫抗性的cCry2Ab基因上，并可操作地连接到金属硫蛋白样蛋白基因转录终止子(tOsMth)上(SEQ ID NO：37)上而组成；第二个表达盒由拟南芥核酮糖1，5-二磷酸羧化酶小亚基基因启动子(prAtRbcS4)，可操作地连接到拟南芥核酮糖1，5-二磷酸羧化酶小亚基基因叶绿体转运肽基因(spAtRbcS4)上，并可操作地连接到苏云金芽孢杆菌的昆虫抗性的cCry1Ac基因上，并可操作地连接到蒺藜苜蓿磷酸盐转运蛋白1基因的转录终止子(tMtPt1)(SEQ ID NO：38)上而组成；第三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。
将所述载体pDBN4031和pDBN4032分别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中含有所述载体pDBN4031的农杆菌共培养，以将构建的重组表达载体pDBN4031中的T-DNA转入到大豆染色体组中，以产生含有重组表达载体pDBN4031的转基因大豆事件。
按照上述方法，将无菌培养的大豆子叶节组织与本实施例1.1中含有所述载体pDBN4032的农杆菌共培养，以将构建的重组表达载体pDBN4032中的T-DNA转入到大 豆染色体组中，以产生含有重组表达载体pDBN4032的转基因大豆事件。
对于农杆菌介导的大豆转化，简要地，将成熟的大豆种子(大豆品种为Jack)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3-4mm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pDBN4031中cCry2Ab基因的核苷酸序列、cCry1Ac基因的核苷酸序列和cPAT基因的核苷酸序列(或pDBN4032中cCry2Ab基因的核苷酸序列、cCry1Ac基因的核苷酸序列和cPAT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(OD 660＝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、MES 4g/L、ZT 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、MES 1g/L、蔗糖30g/L、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、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、MES 1g/L、蔗糖30g/L、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，温度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、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，温度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16h，再于温度20℃下培养8h。
1.3、转基因事件的鉴定和筛选
载体pDBN4031一共产生了1037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1037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137个阳性转基因单株；通过TaqMan TM分析检测上述137个转基因大豆植株是否存在单拷贝的cCry2Ab基因、cCry1Ac基因和cPAT基因，且不含载体骨架序列，共获得84个转基因单株；通过转基因插入位点分析，共筛选到30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(棉铃虫、斜纹夜蛾、甜菜夜蛾、小地老虎)的抗性评价和比较，共筛选到25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25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3个优异的转基因大豆事件DBN8205、pDBN4031-1和pDBN4031-2，其中，转基因大豆事件DBN8205的性状最优(参见第六实施例至第九实施例)。
按照上述载体pDBN4031筛选优异转基因大豆事件DBN8205、pDBN4031-1和pDBN4031-2的方法，将构建的重组表达载体载体pDBN4032筛选出3个优异的转基因大豆事件pDBN4032-1、pDBN4032-2和pDBN4032-3，其均具有单拷贝转基因。
通过比较由载体pDBN4031筛选得到的转基因大豆事件DBN8205、pDBN4031-1和pDBN4031-2与由载体pDBN4032筛选得到的转基因大豆事件pDBN4032-1、pDBN4032-2和pDBN4032-3对主要靶标昆虫(斜纹夜蛾和甜菜夜蛾)的抗性评价(参见第六实施例)，说明载体pDBN4031的设计是较优异的，其是充分考量和分析调控元件的组合和互作获得的优异载体，同时说明转基因大豆事件DBN8205对主要靶标昆虫(斜纹夜蛾和甜菜夜蛾) 的抗性最优。
第二实施例、用TaqMan进行转基因大豆事件DBN8205检测
取转基因大豆事件DBN8205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cCry2Ab基因、cCry1Ac基因和cPAT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DBN8205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cCry2Ab基因序列：
引物1：gtccacgagaatggatcaatga如序列表中SEQ ID NO：16所示；
引物2：gtgtggcgtgaataggtgaaatag如序列表中SEQ ID NO：17所示；
探针1：ctggctcccaacgactataccgggttt如序列表中SEQ ID NO：18所示；
以下引物和探针用来检测cCry1Ac基因序列：
引物3：gacacagtttctgctcagcgag如序列表中SEQ ID NO：19所示；
引物4：cccagatgatgtcaactagtccg如序列表中SEQ ID NO：20所示；
探针2：cgtgccaggtgctgggttcgttc如序列表中SEQ ID NO：21所示；
以下引物和探针用来检测cPAT基因序列：
引物5：gagggtgttgtggctggtattg如序列表中SEQ ID NO：22所示；
引物6：tctcaactgtccaatcgtaagcg如序列表中SEQ ID NO：23所示；
探针3：cttacgctgggccctggaaggctag如序列表中SEQ ID NO：24所示；
PCR反应体系为：
Figure PCTCN2022077093-appb-000003
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 PCTCN2022077093-appb-000004
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DBN8205为单拷贝。
第三实施例、分析转基因大豆事件DBN8205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DBN8205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，12000rpm转速下离心7min，弃上清，吹干，之后加入8.5μL双蒸水(dd H 2O)、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：30作为第一引物，SEQ ID NO：32和SEQ ID NO：33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：31作为第一引物，SEQ ID NO：34和SEQ ID NO：35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：30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：31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DBN8205的大豆植物或种子。
发现在SEQ ID NO：5的核苷酸1-481位显示的为大豆基因组序列在转基因大豆事件DBN8205插入序列的右边界侧翼(5’侧翼序列)，在SEQ ID NO：5的核苷酸12397-12813位显示的为大豆基因组序列在转基因大豆事件DBN8205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DBN8205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DBN8205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DBN8205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DBN8205 DNA的存在。所述SEQ ID NO：6跨越了pDBN4031构建体DNA序列和prAtAct2-01转录起始序列，所述SEQ ID NO：7跨越了t35S转录终止序列和pDBN4031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DBN8205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DBN8205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7(SEQ ID NO：8)，和与之配对的位于T-DNA插入序列中prAtAct2-01转录起始序列的引物8(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DBN8205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T-DNA插入序列中t35S转录终止序列的引物9(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10(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DBN8205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DBN8205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 PCTCN2022077093-appb-000005
Figure PCTCN2022077093-appb-000006
表3、热循环仪扩增条件
Figure PCTCN2022077093-appb-000007
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7和8(SEQ ID NO：8和9)，当其用在转基因大豆事件DBN8205基因组DNA的PCR反应中时，产生634bp片段的扩增产物，当其用在未转化大豆基因组DNA和非DBN8205大豆基因组DNA的PCR反应中时，没有片段被扩增；引物9和10(SEQ ID NO：10和11)，当其用在转基因大豆事件DBN8205基因组DNA的PCR反应中时，产生642bp片段的扩增产物，当其用在未转化大豆基因组DNA和非DBN8205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DBN8205的材料是纯合子或是杂合子。将引物11(SEQ ID NO：12)、引物12(SEQ ID NO：13)和引物13(SEQ ID NO：14)用于扩增反应以产生转基因大豆事件DBN8205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DBN8205的诊断性扩增子。
表4、接合性测定反应液
Figure PCTCN2022077093-appb-000008
Figure PCTCN2022077093-appb-000009
表5、接合性测定的热循环仪扩增条件
Figure PCTCN2022077093-appb-000010
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DBN8205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DBN8205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DBN8205DNA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DBN8205的存在，且该样品由相对于转基因大豆植物DBN8205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DBN8205的引物对被用于产生对转基因大豆事件DBN8205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7和8(SEQ ID NO：8和9)，和引物9和10(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14和15(SEQ ID NO：25和26)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DBN8205 DNA抽提样品的分析应该包括一个转基因大豆事件DBN8205的阳性组织DNA抽提物对照，一个来源于非转基因大 豆事件DBN8205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DBN8205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DBN8205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DBN8205为诊断性扩增子的、推定含有转基因大豆事件DBN8205的大豆植物或种子DNA的提取物，或来源于转基因大豆事件DBN8205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DBN8205。
第四实施例、利用Southern印迹杂交检测转基因大豆事件DBN8205
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4-5g研磨后的植物组织，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、Spe I、Hind III和Sph I分别消化基因组DNA，以T-DNA上cCry2Ab基因、cCry1Ac基因和cPAT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(3M NaCl、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：27、SEQ ID NO：28或SEQ ID NO：29，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pDBN4031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DBN8205含有cCry2Ab基因、cCry1Ac基因和cPAT基因的单拷贝。利用该cCry2Ab基因探针，Mfe I和Spe I酶解后分别产生大小约8.0kb和6.0kb的单一条带；利用该cCry1Ac基因探针，Hind III和Sph I酶解分别产生大小约13kb和7.5kb的单一条带；利用该cPAT基因探针，Mfe I和Spe I酶解后分别产生大小约9.5kb和13kb的单一条带，这表明cCry2Ab基因、cCry1Ac基因和cPAT基因各一个拷贝存在于大豆植物DBN8205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pDBN4031载体骨架序列进入大豆植物DBN8205基因组中。
第五实施例、通过ELISA检测转基因大豆事件DBN8205的蛋白质表达量
Cry1Ac蛋白质在转基因大豆事件DBN8205中的表达范围，可通过ELISA进行检测。
提取转基因大豆事件DBN8205的V5期叶片，R2期茎和花，R6期根、茎和种子，将 上述不同生长阶段的不同大豆组织经冷冻干燥处理后作为样品，分别称取20mg进行液氮研磨，然后分别加入1mL萃取缓冲液(8g/L NaCl、0.27g/L KH 2PO 4、1.42g/LNa 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，Cry1Ac试剂盒(AP003))对样品中蛋白质(Cry1Ac蛋白)含量占不同大豆组织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相应组织(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DBN8205中Cry1Ac蛋白含量的实验结果如表6所示。测得转基因大豆事件DBN8205中Cry1Ac蛋白在不同生长阶段的不同组织中的蛋白平均表达量占相应组织干重的比例为1.11至342.05(μg/g)。
表6、转基因大豆事件DBN8205中Cry1Ac蛋白表达量(μg/g)测定结果
Figure PCTCN2022077093-appb-000011
表6的结果表明，转基因大豆事件DBN8205中Cry1Ac蛋白在大豆V5期叶片中的平均表达量占V5期叶片干重的比例为342.05μg/g，Cry1Ac蛋白在大豆R2期茎中的平均表达量占R2期茎干重的比例为5.3μg/g，Cry1Ac蛋白在大豆R2期花中的平均表达量占R2期花干重的比例为12.45μg/g，Cry1Ac蛋白在大豆R6期根中的平均表达量占R6期根干重的比例为1.11μg/g，Cry1Ac蛋白在大豆R6期种子中的平均表达量占R6期种子干重的比例为8.44μg/g。表6充分说明转基因大豆事件DBN8205中Cry1Ac蛋白在大豆不同生长阶段的不同大豆组织中均有表达，尤其在叶片、花和种子中的表达量较高，可以对鳞翅目昆虫表现出较好的抗性，同时表明载体pDBN4031的设计是优异的。
第六实施例、载体pDBN4031和pDBN4032的抗虫水平评估
将由载体pDBN4031筛选得到的3个优异的转基因大豆植株DBN8205、pDBN4031-1和pDBN4031-2和由载体pDBN4032筛选得到的3个优异的转基因大豆植株pDBN4032-1、pDBN4032-2和pDBN4032-3以及野生型大豆植株(非转基因，NGM)7种植株分别对中国境内的主要害虫(甜菜夜蛾[Spodoptera exigua，BAW]和斜纹夜蛾[Spodoptera litura，TCW])按照如下方法进行生物测定：
分别取上述转基因大豆植株和野生型大豆植株(非转基因，NGM)7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×(大于初孵幼虫大小至小于阴性对照大小虫数/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，从转基因大豆植株DBN8205、pDBN4031-1、pDBN4031-2、pDBN4032-1、pDBN4032-2、pDBN4032-3以及野生型大豆植株(非转基因，NGM)分别选5株进行测试，每株重复3次。结果如表7所示。
表7、转基因大豆植株DBN8205、pDBN4031-1、pDBN4031-2、pDBN4032-1、pDBN4032-2、pDBN4032-3的抗虫生物测定结果-死亡率(％)及抗性总分(分)
Figure PCTCN2022077093-appb-000012
表7的结果表明，(1)由载体pDBN4031筛选得到的转基因大豆事件(DBN8205、 pDBN4031-1、pDBN4031-2)和由载体pDBN4032筛选得到的转基因大豆事件(pDBN4032-1、pDBN4032-2、pDBN4032-3)对甜菜夜蛾和斜纹夜蛾的抗性均显著优于NGM；(2)由载体pDBN4031筛选得到的转基因大豆事件(DBN8205、pDBN4031-1、pDBN4031-2)对甜菜夜蛾和斜纹夜蛾的抗性优于由载体pDBN4032筛选得到的转基因大豆事件(pDBN4032-1、pDBN4032-2、pDBN4032-3)，表明载体pDBN4031的设计是较优异的，其是充分考量和分析调控元件的组合和互作获得的优异载体；(3)转基因大豆事件DBN8205对甜菜夜蛾和斜纹夜蛾的抗性最优。
第七实施例、DBN8205事件对昆虫的抗性检测
为了进一步验证DBN8205事件对昆虫的抗性效果，选取中国境内和南美洲(阿根延和巴西)主要害虫进行生物测定实验和田间效力实验。
7.1、大豆植物DBN8205对中国境内主要害虫的生物测定
将转基因大豆事件DBN8205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]、小地老虎[Agrotis ypsilon，BCW]、豆天蛾[Clanis bilineata，BHM]和草地贪夜蛾[Spodoptera frugiperda，FAW]按照如下方法进行生物测定：
分别取转基因大豆事件DBN8205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×(大于初孵幼虫大小至小于阴性对照大小虫数/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DBN8205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。实验结果如表8所示。
表8、转基因大豆事件DBN8205对中国境内主要害虫的抗虫生物测定结果-死亡率(％)及抗性总分(分)
Figure PCTCN2022077093-appb-000013
Figure PCTCN2022077093-appb-000014
结果表明：转基因大豆事件DBN8205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DBN8205对棉铃虫、小地老虎、豆天蛾和草地贪夜蛾均具有较好的抗性。
7.2、转基因大豆事件DBN8205在中国境内的田间测试
将转基因大豆事件DBN8205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，全生育期不喷施靶标害虫杀虫剂。
(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V2期，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DBN8205对棉铃虫的抗性结果如表9所示。
表9、转基因大豆事件DBN8205自然感虫条件下对棉铃虫的抗性结果
Figure PCTCN2022077093-appb-000015
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DBN8205的为害面积率为0，说明棉铃虫对转基因大豆事件DBN8205的叶片基本没有造成损伤，同时说明转基因大豆事件DBN8205对棉铃虫具有较好的抗性，转基因大豆事件DBN8205在棉铃虫自然发生条件下的田间效果如图4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V2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DBN8205对甜菜夜蛾的抗性结果如表10所示。
表10、转基因大豆事件DBN8205自然感虫条件下对甜菜夜蛾的抗性结果
Figure PCTCN2022077093-appb-000016
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DBN8205的为害面积率显著降低，由此说明转基因大豆事件DBN8205对甜菜夜蛾具有较好的抗性，转基因大豆事件DBN8205在甜菜夜蛾自然发生条件下的田间效果如图5所示。
(3)银纹夜蛾
仅在银纹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6-9月，发育最适温度在20-30℃)。在大豆植株生长至V2期，开始跟踪调查NGM叶片被银纹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，逐株调查银纹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DBN8205对银纹夜蛾的抗性结果如表11所示。
表11、转基因大豆事件DBN8205自然感虫条件下对银纹夜蛾的抗性结果
Figure PCTCN2022077093-appb-000017
结果表明：在银纹夜蛾自然发生条件下，与NGM相比，银纹夜蛾对转基因大豆事件DBN8205的为害面积率显著降低，由此说明转基因大豆事件DBN8205对银纹夜蛾具有较好的抗性，图6为转基因大豆事件DBN8205和NGM在银纹夜蛾自然发生条件下的对比效果图。
(4)大豆食心虫
仅在中国境内大豆食心虫自然发生较为严重的3个地区进行自然感虫(自然虫害发生条件：为害盛期在8-9月，发育最适温度在20-25℃)。在大豆植株生长至R6期，开始跟踪调查NGM豆荚被大豆食心虫幼虫取食的情况；当植株完全成熟时，逐株调查大豆豆荚的被食粒率(被食粒率＝所有单株豆荚被虫食破损的籽粒数目的总和/总植株豆荚籽粒数目×100％)。转基因大豆事件DBN8205对大豆食心虫的抗性结果如表12所示。
表12、转基因大豆事件DBN8205自然感虫条件下对大豆食心虫的抗性结果
Figure PCTCN2022077093-appb-000018
Figure PCTCN2022077093-appb-000019
结果表明：在大豆食心虫自然发生条件下，与NGM相比，大豆食心虫对转基因大豆事件DBN8205的豆荚籽粒被食粒率均为0，说明大豆食心虫对转基因大豆事件DBN8205的豆荚基本没有造成损伤，同时说明转基因大豆事件DBN8205对大豆食心虫具有较好的抗性。转基因大豆事件DBN8205在大豆食心虫自然发生条件下在地区一的田间效果如图7所示。
7.3、大豆植物DBN8205对南美洲(阿根延和巴西)主要害虫的生物测定
(1)大豆植物DBN8205对南美洲(阿根延和巴西)主要食叶类害虫的生物测定
将转基因大豆事件DBN8205和野生型大豆植株(非转基因，NGM)2种植株分别对薄荷灰夜蛾[Rachiplusia nu，SFL]、黎豆夜蛾[Anticarsia gemmatalis，VBC]、大豆夜蛾[Chrisiodexys includens，SBL]、南美棉铃虫[Helicoverpa gelotopoeon，SABW]、烟芽叶蛾[Chloridea virescens，TBW]、草地贪夜蛾[Spodoptera frugiperda，FAW]、炭黑夜蛾[Spodoptera cosmioides，BLAW]、谷实夜蛾[Helicoverpa zea，SPW]、南部灰翅夜蛾[Spodoptera eridania，SAW]和阿尔布拉斜纹夜蛾[Spodoptera albula，GSAW]按照如下方法进行生物测定：
分别取转基因大豆事件DBN8205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1.6cm的圆形，取1-3片(根据昆虫食量确定叶片数量)剪后的圆形叶片放在已加入2mL琼脂的圆形塑料培养皿上，每个培养皿上放1头初孵幼虫，盖上培养皿盖，在温度26-28℃、相对湿度60％-80％、光周期(光/暗)14∶10的条件下放置3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DBN8205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个培养皿。实验结果如表13所示。
表13、转基因大豆事件DBN8205对南美洲(阿根延和巴西)主要害虫的生物测定
Figure PCTCN2022077093-appb-000020
Figure PCTCN2022077093-appb-000021
结果表明：转基因大豆事件DBN8205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DBN8205对薄荷灰夜蛾、黎豆夜蛾、大豆夜蛾、南美棉铃虫、烟芽叶蛾、草地贪夜蛾、炭黑夜蛾、谷实夜蛾、南部灰翅夜蛾和阿尔布拉斜纹夜蛾均具有较好的抗性。
(2)大豆植物DBN8205对南美洲(阿根延和巴西)大豆蛀茎类害虫的抗性效果
将转基因大豆事件DBN8205和野生型大豆植株(非转基因，NGM)2种植株幼苗对玉米蛀茎虫[Elasmopalpus lignosellus，LSCB]按照如下方法测定害虫死亡率、植物受损率和植株死亡率。
测定试虫死亡率的方法：将在温室条件下培养的萌发三天的转基因大豆事件DBN8205幼苗和野生型大豆植株幼苗(非转基因，NGM)(各32株)连根取出，然后分别放置在单独的小室塑料盒中。在小室塑料盒底部含有2％的琼脂，以保持植株正常发育。然后在每株植物上放入1头孵化12小时后的玉米蛀茎虫幼虫，用塑料盖封闭小室塑料盒，在温度23-27℃，相对湿度60％-80％、光周期(光/暗)14∶10的条件下放置5天后统计试虫死亡率。每株重复6次。
测定植株受损率和植株死亡率的方法：取在温室条件下培养出苗7天的转基因大豆事件DBN8205植株和野生型大豆植株(非转基因，NGM)各32株，在种植上述大豆植株的 花盆周围放置PVC管增加物理阻隔，以避免昆虫的迁移。然后在每株植物的茎基部放入2头孵化12小时的玉米蛀茎虫幼虫。接虫15天后统计植株受损率(植物受损率是指被害虫损害的存活植株总数占所有测试植株总数的比例)和植株死亡率(植株死亡率是指被害虫损害致死的植株总数占所有测试植株总数的比例)。每株重复6次。实验结果如表14所示。
表14、转基因大豆事件DBN8205对玉米蛀茎虫的抗性效果
Figure PCTCN2022077093-appb-000022
结果表明：转基因大豆事件DBN8205对玉米蛀茎虫的试虫死亡率显著高于NGM，植株受损率和植株死亡率均显著低于NGM，说明转基因大豆事件DBN8205对玉米蛀茎虫具有较好的抗性。
第八实施例、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10cm，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DBN8205进行如下2种处理：(1)不喷施除草剂，人工控草，以去除杂草对大豆生长的影响；(2)按800g a.i./ha(a.i./ha是指“活性成分每公顷”)剂量在V2～V3期喷洒保试达除草剂。用野生型大豆植株(非转基因，NGM)做平行对照实验。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DBN8205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15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行植株的大豆粒总产量(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DBN8205对除草剂耐受性的结果和大豆产量结果如表16所示。
表15、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16、转基因大豆事件DBN8205对草铵膦除草剂耐受性的结果和大豆产量结果
Figure PCTCN2022077093-appb-000023
结果说明，在草铵膦除草剂受害率方面：转基因大豆事件DBN8205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DBN8205具有良好的草铵膦除草剂耐受性。
在产量方面：在喷施800g a.i./ha草铵膦处理下的产量与不喷施处理相比，产量无明显差异，由此，进一步表明转基因大豆事件DBN8205具有良好的草铵膦除草剂耐受性，且对产量无影响。
第九实施例、不同背景下转基因大豆事件DBN8205的抗虫效果
将第一实施例中转化背景为大豆Jack植株的转基因大豆事件DBN8205通过回交的方式分别转入亲本背景为黑河43和亲本背景为中黄35的大豆植株中，通过5代回交及3代自交分别获得转化背景为黑河43的转基因大豆事件DBN8205和转化背景为中黄35的转基因大豆事件DBN8205，每代通过PCR检测转基因大豆事件DBN8205的完整性(参见第三实施例)。
将转化背景分别为Jack、黑河43和中黄35的大豆转化事件DBN8205与野生型大豆Jack植株、野生型大豆黑河43植株和野生型大豆中黄35植株(非转基因，NGM)6种植株分别对棉铃虫[Helicoverpa armigera，CBW]按照第七实施例7.1中的生物测定方法进行生物测定，每株重复6次。实验结果如表17所示。
表17、转基因大豆事件DBN8205在不同转化背景下的抗虫生物测定结果-死亡率(％) 及抗性总分(分)
Figure PCTCN2022077093-appb-000024
表17的结果表明：转基因大豆事件DBN8205在不同的转化背景下对棉铃虫均具有较好的抗性，表明转基因大豆事件DBN8205在不同转化背景下对靶标害虫的抗性效果稳定。
第十实施例、叠加转基因大豆事件DBN8205 x DBN8002 x DBN9004对昆虫的抗性检测
1、获得叠加转基因大豆事件DBN8205 x DBN8002 x DBN9004
将转基因大豆事件DBN9004(CN106086011A)(父本)与转基因大豆事件DBN8002(母本)杂交，获得叠加转基因大豆事件DBN8002 x DBN9004的杂合植株，然后经过两代自交，并通过TaqMan检测目标基因拷贝数(参考第二实施例)和PCR接合性检测位点的纯杂合(参考第三实施例)，获得叠加转基因大豆事件DBN8002 x DBN9004纯合植株，将其作为父本与转基因大豆事件DBN8205(母本)杂交，获得叠加转基因大豆事件DBN8205 x DBN8002 x DBN9004。
2、叠加转基因大豆事件DBN8205 x DBN8002 x DBN9004对中国境内主要害虫的生物测定
将叠加转基因大豆事件DBN8205 x DBN8002 x DBN9004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]、甜菜夜蛾[Spodoptera exigua，BAW]和草地贪夜蛾[Spodoptera frugiperda，FAW]按照第七实施例7.1的方法进行生物测定。实验结果如表18所示。
表18、叠加转基因大豆事件DBN8205 x DBN8002 x DBN9004对中国境内主要害虫的生物测定结果-死亡率(％)及抗性总分(分)
Figure PCTCN2022077093-appb-000025
Figure PCTCN2022077093-appb-000026
结果表明：叠加转基因大豆事件DBN8205 x DBN8002 x DBN9004对上述害虫的试虫死亡率和抗性总分均显著高于NGM，说明叠加转基因大豆事件DBN8205 x DBN8002 x DBN9004对棉铃虫、甜菜夜蛾和草地贪夜蛾均具有较好的抗性。
3、叠加转基因大豆事件DBN8205 x DBN8002 x DBN9004对南美洲(阿根延和巴西)主要害虫的生物测定
将叠加转基因大豆事件DBN8205 x DBN8002 x DBN9004和野生型大豆植株(非转基因，NGM)2种植株分别对薄荷灰夜蛾[Rachiplusia nu，SFL]、黎豆夜蛾[Anticarsia gemmatalis，VBC]、大豆夜蛾[Chrisiodexys includens，SBL]、南美棉铃虫[Helicoverpa gelotopoeon，SABW]、草地贪夜蛾[Spodoptera frugiperda，FAW]、炭黑夜蛾[Spodoptera cosmioides，BLAW]和谷实夜蛾[Helicoverpa zea，SPW]按照按照第七实施例7.3(1)的方法进行生物测定。实验结果如表19所示。
表19、叠加转基因大豆事件DBN8205 x DBN8002 x DBN9004对南美洲(阿根延和巴西)主要害虫的生物测定
Figure PCTCN2022077093-appb-000027
Figure PCTCN2022077093-appb-000028
结果表明：叠加转基因大豆事件DBN8205 x DBN8002 x DBN9004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，说明叠加转基因大豆事件DBN8205 x DBN8002 x DBN9004对薄荷灰夜蛾、黎豆夜蛾、大豆夜蛾、南美棉铃虫、草地贪夜蛾、炭黑夜蛾和谷实夜蛾均具有较好的抗性。
第十实施例、叠加转基因大豆事件DBN8205 x DBN8002 x DBN9004的除草剂耐受性检测
本试验选用农达除草剂(41％草甘膦异丙铵盐水剂)和保试达(Basta)除草剂(有效成分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。将叠加转基因大豆事件DBN8205 x DBN8002 x DBN9004分别进行如下2种处理：1)不喷施除草剂，人工控草，以去除杂草对大豆生长的影响；2)按1680g a.e./ha(a.e./ha是指“活性成分当量酸每公顷”)剂量在V3叶期喷洒农达除草剂，然后在R2期(盛花期)按相同剂量再次喷洒农达除草剂；3)按800g a.i./ha(a.i./ha是指“活性成分每公顷”)剂量在V3叶期喷洒保试达除草剂，然后在V6期按相同剂量再次喷洒保试达除草剂；4)按800g a.i./ha剂量在V3叶期喷洒保试达除草剂，然后在R2期按1680g a.e./ha剂量喷洒农达除草剂。用野生型大豆植株(非转基因，NGM)做平行对照实验。需要说明的是，不同含量和剂型的草甘膦除草剂换算成等量草甘膦酸的形式，以及不同浓度的草铵膦溶液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大豆产量。药害症状分级参见第七实施例表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)；其中除草剂受害率包括草甘膦受害率和草铵膦受害率，除草剂受害率是根据草甘膦或草铵膦处理后2周的药害调查结果而确定的。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。叠加转基因大豆事件DBN8205 x DBN8002 x DBN9004对除草剂耐受性的结果和大豆产量结果如表20所示。
表20、叠加转基因大豆事件DBN8205 x DBN8002 x DBN9004对除草剂耐受性的结果和产量测试结果
Figure PCTCN2022077093-appb-000029
Figure PCTCN2022077093-appb-000030
结果说明，在除草剂(草甘膦和草铵膦)受害率方面：1)叠加转基因大豆事件DBN8205 x DBN8002 x DBN9004在草甘膦除草剂(1680g a.e./ha)处理下受害率基本为0；2)叠加转基因大豆事件DBN8205 x DBN8002 x DBN9004在草铵膦除草剂(800g a.i./ha)处理下受害率也基本为0；3)叠加转基因大豆事件DBN8205 x DBN8002 x DBN9004在草铵膦除草剂(800g a.i./ha)和草甘膦除草剂(1680g a.e./ha)处理下受害率也基本为0；由此，叠加转基因大豆事件DBN8205 x DBN8002 x DBN9004具有良好的除草剂(草甘膦和草铵膦)耐受性。
在产量方面：叠加转基因大豆事件DBN8205 x DBN8002 x DBN9004在喷施草甘膦除草剂(1680g a.e./ha)、草铵膦除草剂(800g a.i./ha)和草铵膦除草剂(800g a.i./ha)+草甘膦除草剂(1680g a.e./ha)3种处理下的产量与不喷施处理相比无明显差异，由此，进一步表明叠加转基因大豆事件DBN8205 x DBN8002 x DBN9004具有良好的除草剂(草甘膦和草铵膦)耐受性。
第十一实施例
可由包含转基因大豆事件DBN8205的大豆植物或包含转基因大豆事件DBN8205和至少一种不同于转基因大豆事件DBN8205的其他转基因大豆事件的大豆植物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DBN8205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DBN8205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DBN8205的存在。
综上所述，本发明转基因大豆事件DBN8205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，且不影响植物本身其他的农艺性状和产量。此外，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DBN8205的DNA分子。
对应于转基因大豆事件DBN8205的种子已根据布达佩斯条约于2021年12月27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.45071。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。

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  1. 一种具有以下核酸序列的核酸分子，其特征在于，所述核酸序列包含SEQ ID NO：3或其互补序列第1-462位中至少11个连续的核苷酸和SEQ ID NO：3或其互补序列第463-634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1-225位中至少11个连续的核苷酸和SEQ ID NO：4或其互补序列第226-642位中至少11个连续的核苷酸；
    优选地，所述核酸序列包含SEQ ID NO：3或其互补序列第1-462位中22-25个连续的核苷酸和SEQ ID NO：3或其互补序列第463-634位中22-25个连续的核苷酸、和/或SEQ ID NO：4或其互补序列第1-225位中22-25个连续的核苷酸和SEQ ID NO：4或其互补序列第226-642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所述的核酸分子，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DBN8205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DBN8205的DNA存在的方法，其特征在于，所述两种引物包括SEQ ID NO：8和SEQ ID NO：9、SEQ ID NO：10和SEQ ID NO：11、或者SEQ ID NO：1和SEQ ID NO：2的互补序列。
  5. 一种检测样品中转基因大豆事件DBN8205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DBN8205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DBN8205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DBN8205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. 一种植物细胞或部分，其特征在于，包含编码昆虫抗性Cry2Ab蛋白的核酸序列、编码昆虫抗性Cry1Ac蛋白的核酸序列、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，所述特定区域的核酸序列包含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包含SEQ ID NO：3和/或SEQ ID NO：4所示的序列；
    优选地，所述植物细胞或部分包含转基因大豆事件DBN8205；
    可选地，所述植物细胞或部分还包含至少一种不同于转基因大豆事件DBN8205的其他转基因大豆事件；优选地，所述其他转基因大豆事件为转基因大豆事件DBN9004和/或转基因大豆事件DBN8002。
  10. 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866-12192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866-12192位核酸序列和SEQ ID NO：2，或者包含SEQ ID NO：5所示的序列。
  12. 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Cry2Ab蛋白的核酸序列和/或编码昆虫抗性Cry1Ac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包含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包含SEQ ID NO：3和/或SEQ ID NO：4所示的序列。
  13. 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Cry2Ab蛋白的核酸序列和/或编码昆虫抗性Cry1Ac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包含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包含SEQ ID NO：3和/或SEQ ID NO：4所示的序列；
    优选地，所述方法包括将包含转基因大豆事件DBN8205的第一大豆植物与第二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；
    选择对靶昆虫具有抗性和/或对草铵膦除草剂具有耐受性的所述子代植株。
  14. 一种产生自包含转基因大豆事件DBN8205大豆植物的农产品或商品，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。
  15. 根据权利要求14所述的产生自包含转基因大豆事件DBN8205大豆植物的农产品或商品，其特征在于，所述大豆植物还包含至少一种不同于转基因大豆事件DBN8205的其他转基因大豆事件；
    优选地，所述其他转基因大豆事件为转基因大豆事件DBN9004和/或转基因大豆事件DBN8002。
  16. 一种扩展植物抗虫谱和/或所耐受除草剂范围的方法，其特征在于，将所述转基因大豆事件DBN8205在植物中与至少一种不同于转基因大豆事件DBN8205的其他转基因大豆事件一起表达；
    优选地，所述其他转基因大豆事件为转基因大豆事件DBN9004和/或转基因大豆事件DBN8002。
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