WO2020207125A1 - 用于检测玉米植物dbn9501的核酸序列及其检测方法 - Google Patents
用于检测玉米植物dbn9501的核酸序列及其检测方法 Download PDFInfo
- Publication number
- WO2020207125A1 WO2020207125A1 PCT/CN2020/076208 CN2020076208W WO2020207125A1 WO 2020207125 A1 WO2020207125 A1 WO 2020207125A1 CN 2020076208 W CN2020076208 W CN 2020076208W WO 2020207125 A1 WO2020207125 A1 WO 2020207125A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- seq
- nucleic acid
- sequence
- dna
- corn
- Prior art date
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
- A01H5/10—Seeds
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
- A01H6/46—Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N57/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds
- A01N57/18—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-carbon bonds
- A01N57/20—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-carbon bonds containing acyclic or cycloaliphatic radicals
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
Definitions
- the invention relates to the field of plant molecular biology, especially the field of genetically modified crop breeding in agricultural biotechnology research. Specifically, the present invention relates to insect resistance and glufosinate herbicide tolerance transgenic corn event DBN9501, and a nucleic acid sequence for detecting whether a biological sample contains a specific transgenic corn event DBN9501 and a detection method thereof.
- Corn (Zea mays L.) is the main food crop in many parts of the world. Biotechnology has been applied to corn to improve its agronomic traits and quality. Insect resistance is an important agronomic trait in corn production, especially resistance to lepidopteran insects, such as corn borer, cotton bollworm, cutworm, etc. The resistance of corn to lepidopteran insects can be obtained by expressing the resistance genes of lepidopteran insects in corn plants by transgenic methods. Another important agronomic trait is herbicide tolerance. For example, there have been successful corn transformation events NK603, GA21, etc., and corn has been widely planted in major corn growing areas such as the United States.
- glufosinate herbicides is different from that of glyphosate herbicides. It is a biocidal contact herbicide and can be used as a means to effectively manage glyphosate-resistant weeds.
- the tolerance of corn to glufosinate-ammonium herbicide can be obtained by expressing glufosinate-ammonium herbicide tolerance gene (such as pat) in corn plants by transgenic method.
- the expression level of introduced genes may vary greatly between events; there may also be differences in the spatial or temporal patterns of expression, such as the relative expression of transgenes between different plant tissues There are differences, and this difference is manifested in that the actual expression pattern may be inconsistent with the expected expression pattern based on the transcription regulatory elements in the introduced gene construct. Therefore, it is usually necessary to generate hundreds or thousands of different events and screen out a single event with the expected transgene expression level and expression pattern for commercialization purposes. Events with expected transgene expression levels and expression patterns can be used to introduce transgenes into other genetic backgrounds through sexual outcrossing using conventional breeding methods. The offspring produced by this hybridization method retain the transgene expression characteristics of the original transformant. Using this strategy model can ensure reliable gene expression in many varieties, and these varieties can well adapt to local growth conditions.
- flanking DNA a pair of primers spanning the junction of the inserted transgene and flanking DNA are often used to identify specific transgene events by PCR, specifically the first primer contained in the inserted sequence and the second primer contained in the inserted sequence.
- the purpose of the present invention is to provide a nucleic acid sequence for detecting the corn plant DBN9501 and its detection method.
- the transgenic corn event DBN9501 has good resistance to insects and has good tolerance to glufosinate herbicides, and The detection method can accurately and quickly identify whether the biological sample contains the DNA molecule of the transgenic corn event DBN9501.
- the present invention provides a nucleic acid sequence having at least 11 consecutive nucleotides in positions 1-384 of SEQ ID NO: 3 or its complementary sequence, and the first nucleotide sequence of SEQ ID NO: 3 or its complementary sequence. At least 11 consecutive nucleotides in positions 385-768; and/or at least 11 consecutive nucleotides in positions 1-564 of SEQ ID NO: 4 or its complement, and SEQ ID NO: 4 or its complement At least 11 consecutive nucleotides in positions 565-1339 of the sequence.
- the nucleic acid sequence has 22-25 consecutive nucleotides in positions 1-384 of SEQ ID NO: 3 or its complementary sequence, and 22-25 consecutive nucleotides in positions 385-768 of SEQ ID NO: 3 or its complementary sequence. -25 consecutive nucleotides; and/or SEQ ID NO: 4 or its complementary sequence at positions 1-564 22-25 consecutive nucleotides, and SEQ ID NO: 4 or its complementary sequence at positions 565- 22-25 consecutive nucleotides in position 1339.
- the nucleic acid sequence comprises SEQ ID NO: 1 or its complementary sequence, and/or SEQ ID NO: 2 or its complementary sequence.
- the SEQ ID NO:1 or its complementary sequence is a 22-nucleotide sequence near the insertion junction at the 5'end of the inserted sequence in the transgenic corn event DBN9501, the SEQ ID NO:1 or its The complementary sequence spans the flanking genomic DNA sequence of the corn insertion site and the DNA sequence at the 5'end of the insertion sequence.
- the DNA sequence containing the SEQ ID NO:1 or its complementary sequence can be identified as the existence of the transgenic corn event DBN9501.
- the SEQ ID NO: 2 or its complementary sequence is a 22-nucleotide sequence near the insertion junction at the 3'end of the inserted sequence in the transgenic corn event DBN9501, and the SEQ ID NO: 2 or its complement
- the complementary sequence spans the DNA sequence at the 3'end of the insert sequence and the flanking genomic DNA sequence of the corn insertion site, and includes the SEQ ID NO: 2 or its complementary sequence to be identified as the existence of the transgenic corn event DBN9501.
- the nucleic acid sequence comprises SEQ ID NO: 3 or its complementary sequence, and/or SEQ ID NO: 4 or its complementary sequence.
- the nucleic acid sequence may be at least 11 or more consecutive polynucleotides (first nucleic acid sequence) of any part of the T-DNA insertion sequence in the SEQ ID NO: 3 or its complementary sequence, or It is at least 11 or more continuous polynucleotides (second nucleic acid sequence) of any part of the 5'flanking maize genomic DNA region in the SEQ ID NO: 3 or its complementary sequence.
- the nucleic acid sequence may further be homologous to or complementary to a part of the SEQ ID NO: 3 including the complete SEQ ID NO: 1.
- these nucleic acid sequences can be used as a pair of DNA primers in a DNA amplification method for generating amplification products.
- the amplified product generated in the DNA amplification method using the DNA primer pair is the amplified product including SEQ ID NO: 1, the presence of the transgenic corn event DBN9501 or its progeny can be diagnosed.
- the SEQ ID NO: 3 or its complementary sequence is a sequence of 768 nucleotides in length near the insertion junction at the 5'end of the T-DNA insertion sequence in the transgenic corn event DBN9501.
- the SEQ ID NO: 3 or its complementary sequence consists of a 384-nucleotide maize genome 5'flanking sequence (SEQ ID NO: 3 nucleotides 1-384), a 168-nucleotide DBN10707 construct DNA sequence (SEQ ID NO : 3 nucleotide 385-552) and 216 nucleotide tNos (nopaline synthase) transcription terminator DNA sequence (SEQ ID NO: 3 nucleotide 553-768), The inclusion of the SEQ ID NO: 3 or its complementary sequence can be identified as the existence of the transgenic corn event DBN9501.
- the nucleic acid sequence may be at least 11 or more consecutive polynucleotides (the third nucleic acid sequence) of any part of the T-DNA insertion sequence in the SEQ ID NO: 4 or its complementary sequence, or the SEQ ID NO: 4 or its complementary sequence. ID NO: 4 or at least 11 or more consecutive polynucleotides (fourth nucleic acid sequence) in any part of the 3'flanking maize genomic DNA region in 4 or its complementary sequence.
- the nucleic acid sequence may further be homologous to or complementary to a part of the SEQ ID NO: 4 including the complete SEQ ID NO: 2.
- these nucleic acid sequences can be used as a pair of DNA primers in a DNA amplification method for generating amplification products.
- the amplified product generated in the DNA amplification method using the DNA primer pair is an amplified product including SEQ ID NO: 2, the presence of the transgenic corn event DBN9501 or its progeny can be diagnosed.
- the SEQ ID NO: 4 or its complementary sequence is a sequence of 1,339 nucleotides in length near the T-DNA insertion junction at the 3'end of the inserted sequence in the transgenic corn event DBN9501.
- the SEQ ID NO: 4 or its complementary sequence consists of the 271-nucleotide pr35S transcription initiation sequence (SEQ ID NO: 4 nucleotide Nos. 1-271) and the 293-nucleotide DBN10707 construct DNA sequence (SEQ ID NO: 4 nucleotides 272-564) and 775 nucleotides of the maize genome 3'flanking sequence (SEQ ID NO: 4 nucleotides 565-1339), including the SEQ ID NO: 4 Or its complementary sequence can be identified as the existence of transgenic corn event DBN9501.
- nucleic acid sequence includes SEQ ID NO: 5 or its complementary sequence.
- the SEQ ID NO: 5 or its complementary sequence is a sequence characterizing the transgenic corn event DBN9501 with a length of 8559 nucleotides, and the specific genome and genetic elements contained therein are shown in Table 1. Including the SEQ ID NO: 5 or its complementary sequence can be identified as the existence of the transgenic corn event DBN9501.
- the first, second, third, and fourth nucleic acid sequences need not be composed of only DNA, but may also include a mixture of RNA, DNA, and RNA, or DNA, RNA, or other types of A combination of nucleotides or analogs of the polymerase template.
- the probes or primers in the present invention should be at least about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 consecutive nucleotides in length, which can be selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5.
- the probes and primers may be continuous with a length of at least about 21 to about 50 or more Nucleotides.
- the nucleic acid sequence or its complementary sequence can be used in a DNA amplification method to generate amplicons for detecting the presence of transgenic corn event DBN9501 or its progeny in a biological sample; the nucleic acid sequence or its complementary sequence It can be used in nucleotide detection methods to detect the presence of transgenic corn event DBN9501 or its progeny in biological samples.
- the present invention also provides a method for detecting the presence of DNA of transgenic corn event DBN9501 in a sample, which includes:
- the target amplification product includes the nucleic acid sequence.
- the target amplification product comprises SEQ ID NO: 1 or its complementary sequence, SEQ ID NO: 2 or its complementary sequence, SEQ ID NO: 6 or its complementary sequence, and/or SEQ ID NO: 7 or its complementary sequence Complementary sequence.
- the primer includes a first primer and a second primer
- the first primer is selected from SEQ ID NO: 1, SEQ ID NO: 8 and SEQ ID NO: 10
- the second primer is selected from SEQ ID NO :2, SEQ ID NO: 9 and SEQ ID NO: 11.
- the present invention also provides a method for detecting the presence of DNA of transgenic corn event DBN9501 in a sample, which includes:
- the probe comprising the nucleic acid sequence
- the hybridization between the sample to be detected and the probe is detected.
- the stringent conditions can be in 6 ⁇ SSC (sodium citrate), 0.5% SDS (sodium dodecyl sulfate) solution, hybridization at 65°C, and then 2 ⁇ SSC, 0.1% SDS and 1 ⁇ SSC, 0.1% SDS each wash the membrane once.
- 6 ⁇ SSC sodium citrate
- SDS sodium dodecyl sulfate
- the probe comprises SEQ ID NO: 1 or its complementary sequence, SEQ ID NO: 2 or its complementary sequence, SEQ ID NO: 6 or its complementary sequence, and/or SEQ ID NO: 7 or its complementary sequence .
- At least one of the probes is labeled with at least one fluorescent group.
- the present invention also provides a method for detecting the presence of DNA of transgenic corn event DBN9501 in a sample, which includes:
- the marker nucleic acid molecule including the nucleic acid sequence
- the hybridization between the sample to be detected and the marker nucleic acid molecule is detected, and then the marker-assisted breeding analysis is used to determine that insect resistance and/or herbicide tolerance are genetically linked with the marker nucleic acid molecule.
- the marker nucleic acid molecule includes at least one selected from: SEQ ID NO: 1 or its complementary sequence, SEQ ID NO: 2 or its complementary sequence, and/or SEQ ID NO: 6-11 or its complementary sequence. Complementary sequence.
- the present invention also provides a DNA detection kit, comprising at least one DNA molecule containing the nucleic acid sequence, which can be used as a DNA primer specific to the transgenic corn event DBN9501 or its progeny One or probe.
- the DNA molecule comprises SEQ ID NO: 1 or its complementary sequence, SEQ ID NO: 2 or its complementary sequence, SEQ ID NO: 6 or its complementary sequence, and/or SEQ ID NO: 7 or its complementary sequence .
- the present invention also provides a plant cell comprising a nucleic acid sequence encoding an insect-resistant Vip3Aa protein, a nucleic acid sequence encoding a glufosinate herbicide-tolerant PAT protein, and a nucleic acid sequence of a specific region.
- the nucleic acid sequence of the region includes the sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 6 and/or SEQ ID NO: 7.
- the plant cell comprises a nucleic acid sequence encoding an insect resistance Vip3Aa protein, a nucleic acid sequence encoding a glufosinate herbicide tolerance PAT protein, and a nucleic acid sequence of a specific region, and the nucleic acid sequence of the specific region includes SEQ ID NO :3 and/or SEQ ID NO: 4.
- the plant cell comprises SEQ ID NO: 1, SEQ ID NO: 5, 553-7491 nucleic acid sequence and SEQ ID NO: 2, or comprises the sequence shown in SEQ ID NO: 5.
- the present invention also provides a method for protecting corn plants from insect invasion, including providing at least one transgenic corn plant cell in the diet of the target insect, the transgenic corn plant cell comprising SEQ in its genome ID NO: 1 and/or SEQ ID NO: 2, the target insect that ingests the transgenic corn plant cell is inhibited from further ingesting the transgenic corn plant.
- the transgenic corn plant cell contains the sequence shown in SEQ ID NO: 3 and/or SEQ ID NO: 4 in its genome.
- the transgenic corn plant cell sequentially includes SEQ ID NO:1, SEQ ID NO: 5, and SEQ ID NO: 2, or SEQ ID NO: 5 in sequence.
- the present invention also provides a method for protecting corn plants from damage caused by herbicides or controlling weeds in the field where corn plants are grown, which includes applying an effective dose of glufosinate-ammonium herbicide to plant at least In a field of transgenic corn plants, the transgenic corn plant includes the sequence shown in SEQ ID NO:1 and/or SEQ ID NO:2 in its genome, and the transgenic corn plant is resistant to glufosinate herbicide Acceptability.
- the transgenic corn plant contains the sequence shown in SEQ ID NO: 3 and/or SEQ ID NO: 4 in its genome.
- the transgenic corn plant sequentially includes SEQ ID NO: 1, SEQ ID NO: 5 553-7491 nucleic acid sequence and SEQ ID NO: 2, or includes the sequence shown in SEQ ID NO: 5 .
- the present invention also provides a method for cultivating corn plants resistant to insects and/or tolerant to glufosinate-ammonium herbicide, including:
- the genome of the corn seed contains a nucleic acid sequence encoding an insect-resistant Vip3Aa protein and/or a nucleic acid sequence encoding a glufosinate herbicide-tolerant PAT protein, and a nucleic acid sequence of a specific region, or
- the genome of the corn seed includes the nucleic acid sequence shown in SEQ ID NO: 5;
- the nucleic acid sequence of the specific region is SEQ ID NO:1 and/or SEQ ID NO: 2; preferably, the nucleic acid sequence of the specific region is SEQ ID NO: 3 and/or SEQ ID NO: 4 The sequence shown.
- the present invention also provides a method for producing a corn plant resistant to insects and/or tolerant to glufosinate-ammonium herbicide, which comprises combining the encoding insects contained in the genome of the first corn plant
- the nucleic acid sequence of the specific region is SEQ ID NO: 1 and/or SEQ ID NO: 2; preferably, the nucleic acid sequence of the specific region is SEQ ID NO: 3 and/ Or the sequence shown in SEQ ID NO
- the method includes sexually crossing transgenic corn event DBN9501 with corn plants lacking insect resistance and/or glufosinate-ammonium tolerance, thereby generating a large number of progeny plants, and selecting nucleic acid sequences with the specific region
- the progeny plant; the nucleic acid sequence of the specific region includes the sequence shown in SEQ ID NO:1 and/or SEQ ID NO: 2; preferably, the nucleic acid sequence of the specific region includes SEQ ID NO: 3 and / Or the sequence shown in SEQ ID NO: 4.
- the present invention also provides an agricultural product or commodity produced from the transgenic corn event DBN9501.
- the agricultural product or commodity is corn meal, corn flour, corn oil, corn silk, corn starch, corn gluten, corn tortillas , Cosmetics or fillers.
- corn refers to Zea mays, and includes all plant varieties that can mate with corn, including wild corn species.
- plant includes whole plants, plant cells, plant organs, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant callus, plant clumps, and whole plants in plants or plant parts Cells, the plant parts such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, stalks, roots, root tips, anthers and the like. It should be understood that the parts of transgenic plants within the scope of the present invention include but are not limited to plant cells, protoplasts, tissues, callus, embryos, and flowers, stems, fruits, leaves and roots. A transgenic plant or its progeny transformed by a DNA molecule and thus at least partially composed of transgenic cells.
- gene refers to a nucleic acid fragment that expresses a specific protein, including a regulatory sequence before the coding sequence (5' non-coding sequence) and a regulatory sequence after the coding sequence (3' non-coding sequence).
- Native gene refers to a gene that is naturally found to have its own regulatory sequence.
- Chimeric gene refers to any gene that is not a natural gene, which contains regulatory and coding sequences not found in nature.
- Endogenous gene refers to a natural gene that is located in its natural location in the genome of an organism.
- Formeign gene refers to a foreign gene that exists in the genome of an organism and does not originally exist, and also refers to a gene that is introduced into the recipient cell through a transgene step.
- the foreign gene may include a natural gene inserted into a non-natural organism or a chimeric gene.
- a "transgene” is a gene that has been introduced into the genome through a transformation procedure.
- the site in the plant genome where the recombinant DNA has been inserted can be referred to as the "insertion site” or "target site”.
- flanking DNA may include a genome naturally present in an organism such as a plant or foreign (heterologous) DNA introduced through a transformation process, such as a fragment associated with a transformation event. Therefore, the flanking DNA may include a combination of natural and foreign DNA.
- flanking DNA is also called “flanking region” or “flanking sequence” or “flanking genomic sequence” or “flanking genomic DNA”, which means at least 3, 5, 10, 11, 15, 20, 50, A sequence of 100, 200, 300, 400, 1000, 1500, 2000, 2500, or 5000 base pairs or longer, which is located directly upstream or downstream of the original foreign inserted DNA molecule and adjacent to the original foreign inserted DNA molecule.
- flanking area When the flanking area is located downstream, it can also be referred to as "3' flanking” or “right boundary flanking” or the like. When the flanking area is located upstream, it can also be referred to as “5' flanking” or “left boundary flanking” or the like.
- a transformation procedure that causes random integration of foreign DNA will result in transformants containing different flanking regions that are specifically contained in each transformant.
- Transformants will also contain unique junctions between segments of heterologous insert DNA and genomic DNA or between two segments of genomic DNA or between two segments of heterologous DNA.
- Junction is the point where two specific DNA fragments join. For example, junctions exist where the insert DNA joins the flanking DNA. Junctions are also present in transformed organisms, where two DNA fragments are joined together in a manner modified from those found in natural organisms.
- a "junction region” or “junction sequence” refers to DNA containing junctions.
- the present invention provides a transgenic corn event called DBN9501 and its progeny.
- the transgenic corn event DBN9501 is also called a corn plant DBN9501, which includes plants and seeds of the transgenic corn event DBN9501 and plant cells or reproducible parts thereof.
- the transgenic corn event DBN9501 of the present invention contains a DNA construct, and when it is expressed in plant cells, the transgenic corn event DBN9501 acquires resistance to insects and tolerance to glufosinate herbicides.
- the DNA construct comprises two expression cassettes in tandem, the first expression cassette comprises a suitable promoter for expression in plants and a suitable polyadenylation signal sequence, the promoter is operably connected to Vip3Aa19
- the nucleic acid sequence of the protein, the nucleic acid sequence of the Vip3Aa19 protein is mainly resistant to lepidopteran insects.
- the second expression cassette contains a suitable promoter for expression in plants and a suitable polyadenylation signal sequence, the promoter operably linked to encode phosphinothricin N-acetyltransferase (phosphinothricin N -acetyltransferase (PAT) gene, the nucleic acid sequence of the PAT protein is tolerant to glufosinate-ammonium herbicide.
- phosphinothricin N-acetyltransferase phosphinothricin N -acetyltransferase (PAT) gene
- the promoter may be a suitable promoter isolated from a plant, including constitutive, inducible and/or tissue-specific promoters, and the suitable promoters include, but are not limited to, cauliflower mosaic virus (CaMV) 35S Promoters, Scrophularia mosaic virus (FMV) 35S promoter, Ubiquitin (Ubiquitin) promoter, Actin (Actin) promoter, Agrobacterium (tumefaciens) nopaline synthase (NOS) promoter, Octopine synthase (OCS) promoter, Cestrum yellow leaf curl virus promoter, potato tuber storage protein (Patatin) promoter, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) promoter, glutathione sulfur transferase (GST) promoter, E9 promoter, GOS promoter, alcA/alcR promoter, Agrobacterium rhizogenes (Agrobacterium rhizogenes
- the polyadenylation signal sequence may be a suitable polyadenylation signal sequence that functions in plants, and the suitable polyadenylation signal sequence includes, but is not limited to, derived from Agrobacterium tumefaciens (Agrobacterium tumefaciens)
- the polyadenylation signal sequence of nopaline synthase (NOS) gene, the 35S terminator of cauliflower mosaic virus (CaMV), the polyadenylation signal sequence of protease inhibitor II (PINII) gene and its source The polyadenylation signal sequence of the ⁇ -tubulin gene.
- the expression cassette may also include other genetic elements including, but not limited to, enhancers and signal peptides/transit peptides.
- the enhancer can enhance the expression level of the gene, and the enhancer includes, but is not limited to, tobacco etching virus (TEV) translation activator, CaMV35S enhancer, and FMV35S enhancer.
- the signal peptide/transit peptide can guide the Vip3Aa19 protein and/or PAT protein to be transported to specific organelles or compartments outside the cell or within the cell, for example, using a sequence encoding a chloroplast transit peptide to target the chloroplast, or using'KDEL' to retain sequence targets To the endoplasmic reticulum.
- the Vip3Aa19 gene can be isolated from Bacillus thuringiensis (Bt for short), and the nucleotide sequence of the Vip3Aa19 gene can be changed by optimizing the codon or in other ways to increase the transcript in the transformed cell.
- Bt Bacillus thuringiensis
- the "Lepidoptera (Lepidoptera)” includes two types of insects, moths and butterflies, and is the order with the most agricultural and forestry pests, such as cutworms, cotton bollworm, Spodoptera litura, Spodoptera litura, Peach borer, etc.
- the phosphinothricin N-acetyltransferase (PAT) gene may be an enzyme isolated from a strain of Streptomyces viridochromogenes, which catalyzes the conversion of L-phosphinothricin into its inactive form through acetylation to give plants Tolerance to glufosinate herbicide.
- Phosphinothricin PTC, 2-amino-4-methylphosphonobutyric acid
- PTC is the structural unit of the antibiotic 2-amino-4-methylphosphonyl-alanyl-alanine.
- This tripeptide has anti-Gram-positive and Gram-negative bacteria and anti-fungal Botrytis cinerea (Botrytis cinerea) activity.
- the phosphinothricin N-acetyltransferase (PAT) gene can also be used as a selectable marker gene.
- glufosinate-ammonium is also known as glufosinate, which refers to ammonium 2-amino-4-[hydroxy(methyl)phosphono]butyrate.
- Treatment with "glufosinate-ammonium herbicide” means using any kind of The herbicide formulation of glufosinate-ammonium was treated. In order to achieve an effective biological dose, the choice of the use rate of a certain glufosinate-ammonium formulation does not exceed the skills of ordinary agronomic technicians.
- any herbicide formulation containing glufosinate to treat a field containing plant material derived from transgenic corn event DBN9501 will control the growth of weeds in the field and will not affect the plant material derived from transgenic corn event DBN9501 Growth or yield.
- the DNA construct is introduced into the plant using transformation methods, which include, but are not limited to, Agrobacterium-mediated transformation, gene gun transformation, and pollen tube passage transformation.
- the Agrobacterium-mediated transformation method is a common method for plant transformation.
- the foreign DNA to be introduced into the plant is cloned between the consensus sequence on the left and right borders of the vector, that is, the T-DNA region.
- the vector is transformed into Agrobacterium cells, and subsequently, the Agrobacterium cells are used to infect plant tissues, and the T-DNA region of the vector containing foreign DNA is inserted into the plant genome.
- the gene gun transformation method is to bombard plant cells with vectors containing foreign DNA (particle-mediated biological bombardment transformation).
- the pollen tube channel transformation method utilizes the natural pollen tube channel (also known as pollen tube guiding tissue) formed after plant pollination to carry the foreign DNA into the embryo sac through the nucleus channel.
- transgenic plant After transformation, a transgenic plant must be regenerated from the transformed plant tissue, and the offspring with foreign DNA must be selected using appropriate markers.
- a DNA construct is a combination of DNA molecules connected to each other, and the combination provides one or more expression cassettes.
- the DNA construct is preferably a plasmid capable of self-replication in bacterial cells and contains different restriction endonuclease sites.
- the restriction endonuclease sites contained are used to introduce functional genetic elements, namely promoters. , Introns, leader sequences, coding sequences, 3'terminator regions and other sequences of DNA molecules.
- the expression cassette contained in the DNA construct includes genetic elements necessary for the transcription of messenger RNA, and the expression cassette can be designed to be expressed in prokaryotic cells or eukaryotic cells.
- the expression cassette of the present invention is designed to be most preferably expressed in plant cells.
- the transgenic "event” is obtained by transforming plant cells with a heterologous DNA construct, that is, including at least one nucleic acid expression cassette containing the target gene, inserted into the plant genome by the method of transgene to produce a plant population, and regenerate the plant population , And select specific plants that have the characteristics of inserting specific genomic sites.
- the term “event” refers to the original transformant containing heterologous DNA and the progeny of the transformant.
- the term “event” also refers to the offspring obtained by sexual crosses between the original transformant and other species containing heterologous DNA, even after repeated backcrossing with the backcrossed parent, the inserted DNA from the original transformant parent And flanking genomic DNA also exists in the same chromosomal position in the hybrid offspring.
- event also refers to the DNA sequence from the original transformant, the DNA sequence comprising the insert DNA and the flanking genomic sequence closely adjacent to the insert DNA, the DNA sequence is expected to be transferred to the progeny, the progeny containing the insert DNA
- the parental line of (for example, the original transformant and its self-produced progeny) is produced by sexually crossing the parental line that does not contain the inserted DNA, and the progeny receives the inserted DNA containing the target gene.
- recombinant refers to the form of DNA and/or protein and/or organism that cannot normally be found in nature and is therefore produced through artificial intervention. Such artificial intervention can produce recombinant DNA molecules and/or recombinant plants.
- the "recombinant DNA molecule” is obtained by artificially combining two sequence segments that are otherwise separated, for example, by chemical synthesis or manipulation of isolated nucleic acid segments by genetic engineering techniques. The techniques for performing nucleic acid manipulations are well known.
- transgenic includes any cell, cell line, callus, tissue, plant part or plant whose genotype has been changed due to the presence of heterologous nucleic acid.
- the original transgenic body is an individual progeny produced by sexual cross or asexual reproduction.
- the term “transgenic” does not include genomic (chromosomal or extrachromosomal) changes through conventional plant breeding methods or naturally occurring events such as random allogeneic fertilization, non-recombinant viral infection, non-recombinant Bacterial transformation, non-recombinant transposition or spontaneous mutation.
- heterologous means that the first molecule in nature is usually not found in combination with the second molecule.
- the molecule can be derived from a first species and inserted into the genome of a second species. Therefore this molecule is heterologous to the host and is artificially introduced into the genome of the host cell.
- the transgenic corn event DBN9501 which is resistant to lepidopteran insects and tolerant to glufosinate herbicide, through the following steps: first, the first parent corn plant is sexually crossed with the second parent corn plant, thereby producing Diverse first generation progeny plants, the first parent corn plant is composed of corn plants cultivated from transgenic corn event DBN9501 and its progeny, the transgenic corn event DBN9501 and its progeny are obtained by using the lepidopteran insects of the present invention
- the second parent maize plant lacks resistance to lepidopteran insects and/or is tolerant to glufosinate herbicide by transforming an expression cassette that is resistant and tolerant to glufosinate-ammonium herbicide.
- These steps may further include backcrossing lepidopteran insect-resistant and/or glufosinate-tolerant progeny plants with the second parent corn plant or the third parent corn plant, and then by attacking with lepidopteran insects, Glufosinate herbicide application or identification of trait-related molecular markers (such as DNA molecules containing the junction sites identified at the 5'end and 3'end of the inserted sequence in transgenic corn event DBN9501) to select progeny, This results in corn plants that are resistant to lepidopteran insects and tolerant to glufosinate herbicides.
- trait-related molecular markers such as DNA molecules containing the junction sites identified at the 5'end and 3'end of the inserted sequence in transgenic corn event DBN9501
- transgenic plants can also be mated to produce offspring that contain two independent, separate added exogenous genes. Selfing of appropriate offspring can produce offspring plants that are homozygous for the two added foreign genes.
- the backcrossing of parent plants and outcrossing with non-transgenic plants as described above can also be expected, and the same is true for asexual reproduction.
- probe is an isolated nucleic acid molecule to which a conventional detectable label or reporter molecule, such as a radioisotope, ligand, chemiluminescent agent, or enzyme, is bound.
- This probe is complementary to a strand of the target nucleic acid.
- the probe is complementary to a DNA strand from the genome of the transgenic corn event DBN9501, regardless of whether the genomic DNA is from the transgenic corn event DBN9501 or seeds or from a transgene Plant or seed or extract of corn event DBN9501.
- the probe of the present invention not only includes deoxyribonucleic acid or ribonucleic acid, but also includes polyamide and other probe materials that specifically bind to the target DNA sequence and can be used to detect the presence of the target DNA sequence.
- primer is an isolated nucleic acid molecule that binds to a complementary target DNA strand through nucleic acid hybridization and annealing, forms a hybrid between the primer and the target DNA strand, and then acts on the polymerase (such as DNA polymerase) Down, extend along the target DNA strand.
- the primer pair of the present invention relates to its application in target nucleic acid sequence amplification, for example, by polymerase chain reaction (PCR) or other conventional nucleic acid amplification methods.
- the length of the probes and primers is generally 11 polynucleotides or more, preferably 18 polynucleotides or more, more preferably 24 polynucleotides or more, and most preferably 30 polynucleotides. Acid or more.
- Such probes and primers specifically hybridize to the target sequence under highly stringent hybridization conditions.
- probes that are different from the target DNA sequence and maintain the ability to hybridize to the target DNA sequence can be designed by conventional methods, it is preferred that the probe and primer of the present invention have a complete DNA sequence with the continuous nucleic acid of the target sequence. Identity.
- the primers and probes based on the flanking genomic DNA and insert sequence of the present invention can be determined by conventional methods, for example, by isolating the corresponding DNA molecule from the plant material derived from transgenic corn event DBN9501 and determining the nucleic acid sequence of the DNA molecule.
- the DNA molecule contains a transgene insert sequence and a maize genome flanking sequence, and fragments of the DNA molecule can be used as primers or probes.
- nucleic acid probes and primers of the present invention hybridize with target DNA sequences under stringent conditions. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of DNA from transgenic corn event DBN9501 in the sample. Nucleic acid molecules or fragments thereof can specifically hybridize with other nucleic acid molecules under certain circumstances. As used in the present invention, if two nucleic acid molecules can form an anti-parallel double-stranded nucleic acid structure, it can be said that the two nucleic acid molecules can specifically hybridize with each other. If two nucleic acid molecules show complete complementarity, one of the nucleic acid molecules is said to be the "complement" of the other nucleic acid molecule.
- nucleic acid molecules when each nucleotide of one nucleic acid molecule is complementary to the corresponding nucleotide of another nucleic acid molecule, it is said that the two nucleic acid molecules show "complete complementarity". If two nucleic acid molecules can hybridize to each other with sufficient stability so that they anneal and bind to each other under at least conventional "low stringency” conditions, the two nucleic acid molecules are said to be “minimally complementary”. Similarly, if two nucleic acid molecules can hybridize to each other with sufficient stability so that they anneal and bind to each other under conventional "highly stringent” conditions, the two nucleic acid molecules are said to have "complementarity".
- Deviation from complete complementarity is permissible, as long as the deviation does not completely prevent the two molecules from forming a double-stranded structure.
- a nucleic acid molecule In order for a nucleic acid molecule to be used as a primer or probe, it is only necessary to ensure that it has sufficient complementarity in sequence, so that a stable double-stranded structure can be formed under the specific solvent and salt concentration used.
- a substantially homologous sequence is a nucleic acid molecule that can specifically hybridize with the complementary strand of another matched nucleic acid molecule under highly stringent conditions.
- Suitable stringent conditions to promote DNA hybridization for example, treatment with 6.0 ⁇ sodium chloride/sodium citrate (SSC) at approximately 45° C., and then washing with 2.0 ⁇ SSC at 50° C.
- SSC sodium chloride/sodium citrate
- the salt concentration in the washing step can be selected from about 2.0 ⁇ SSC, 50°C under low stringency conditions to about 0.2 ⁇ SSC, 50°C under high stringency conditions.
- the temperature conditions in the washing step can be raised from room temperature of about 22°C under low stringency conditions to approximately 65°C under high stringency conditions.
- the temperature conditions and the salt concentration can both change, or one of them can remain unchanged while the other variable changes.
- a nucleic acid molecule of the present invention can be compared with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: under moderately stringent conditions, for example, at about 2.0 ⁇ SSC and about 65°C. 4.
- One or more of the nucleic acid molecules in SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7 or their complementary sequences, or any fragment of the foregoing sequence specifically hybridizes.
- a nucleic acid molecule of the present invention is compatible with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: under highly stringent conditions.
- One or more nucleic acid molecules or their complementary sequences in 6 and SEQ ID NO: 7, or any fragment of the above sequence specifically hybridizes.
- a preferred marker nucleic acid molecule has SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 6, or SEQ ID NO: 7 or its complementary sequence, or any fragment of the foregoing sequence.
- Another preferred marker nucleic acid molecule of the present invention is compatible with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 6 or SEQ ID NO: 7 or its complementary sequence, or any fragment of the above sequence has 80% to 100% or 90% to 100% sequence identity.
- SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 6, and SEQ ID NO: 7 can be used as markers in plant breeding methods to identify the offspring of genetic crosses.
- the hybridization between the probe and the target DNA molecule can be detected by any method well known to those skilled in the art, and these methods include, but are not limited to, fluorescent labeling, radioactive labeling, antibody labeling and chemiluminescent labeling.
- “stringent conditions” refer to conditions that allow only the primers to hybridize to the target nucleic acid sequence in the DNA thermal amplification reaction, and have The primer of the wild-type sequence (or its complementary sequence) corresponding to the target nucleic acid sequence can bind to the target nucleic acid sequence, and preferably produces a unique amplification product, which is an amplicon.
- target sequence means that the probe or primer only hybridizes to the target sequence in the sample containing the target sequence under stringent hybridization conditions.
- amplicon refers to a nucleic acid amplification product of a target nucleic acid sequence that is part of a nucleic acid template.
- amplicon refers to a nucleic acid amplification product of a target nucleic acid sequence that is part of a nucleic acid template.
- DNA extracted from corn plant tissue samples or extracts can be used to generate a diagnostic amplicon for the presence of DNA of transgenic corn event DBN9501 through a nucleic acid amplification method using primer pairs.
- the primer pair includes a first primer derived from a flanking sequence adjacent to the insertion site of the inserted foreign DNA in the plant genome, and a second primer derived from the inserted foreign DNA.
- the amplicon has a certain length and sequence, which is also diagnostic for the transgenic corn event DBN9501.
- the length of the amplicon can be the combined length of the primer pair plus one nucleotide base pair, preferably plus about 50 nucleotide base pairs, more preferably plus about 250 nucleotide base pairs, Most preferably, about 450 nucleotide base pairs or more are added.
- the primer pair may be derived from the flanking genomic sequence on both sides of the inserted DNA to generate an amplicon that includes the entire inserted nucleotide sequence.
- One of the primer pairs derived from the plant genome sequence may be located at a certain distance from the inserted DNA sequence, and the distance may range from one nucleotide base pair to about 20,000 nucleotide base pairs.
- the use of the term "amplicon" specifically excludes primer dimers formed in DNA thermal amplification reactions.
- the nucleic acid amplification reaction can be achieved by any nucleic acid amplification reaction method known in the art, including polymerase chain reaction (PCR).
- PCR polymerase chain reaction
- Various nucleic acid amplification methods are well known to those skilled in the art.
- PCR amplification methods have been developed to amplify genomic DNA up to 22kb and phage DNA up to 42kb. These methods and other DNA amplification methods in the art can be used in the present invention.
- the inserted foreign DNA sequence and the flanking DNA sequence from the transgenic corn event DBN9501 can be amplified by using the provided primer sequences to amplify the genome of the transgenic corn event DBN9501. After amplification, the PCR amplicon or cloned DNA can be standardized. DNA sequencing.
- DNA detection kits based on DNA amplification methods contain DNA molecules used as primers, which specifically hybridize to target DNA under appropriate reaction conditions and amplify diagnostic amplicons.
- the kit can provide detection methods based on agarose gel or many methods known in the art to detect diagnostic amplicons.
- a kit containing DNA primers homologous or complementary to any part of the maize genome of SEQ ID NO: 3 or SEQ ID NO: 4, and homologous or complementary to any part of the transgene insertion region of SEQ ID NO: 5 is Provided by the present invention.
- the primer pairs that are useful in DNA amplification methods are SEQ ID NO: 8 and SEQ ID NO: 9, which amplify a diagnostic amplification that is homologous to a part of the 5'transgene/genomic region of the transgenic corn event DBN9501 Where the amplicon includes SEQ ID NO:1.
- Other DNA molecules used as DNA primers can be selected from SEQ ID NO: 5.
- the amplicons produced by these methods can be detected by a variety of techniques.
- One of the methods is Genetic Bit Analysis, which designs a DNA oligonucleotide chain that spans the inserted DNA sequence and the adjacent flanking genomic DNA sequence. Fix the oligonucleotide chain in the microwells of a microwell plate, and after PCR amplification of the target region (use one primer in the insert sequence and the adjacent flanking genome sequence), the single-stranded PCR product It can hybridize with a fixed oligonucleotide chain and serve as a template for a single-base extension reaction that uses DNA polymerase and ddNTPs specifically labeled for the next expected base. The results can be obtained by fluorescence or ELISA methods. The signal represents the presence of the insert/flanking sequence, which indicates that the amplification, hybridization, and single base extension reactions were successful.
- Another method is Pyrosequencing. This method designs an oligonucleotide chain that spans the inserted DNA sequence and the adjacent genomic DNA binding site. Hybridize the oligonucleotide chain with the single-stranded PCR product of the target region (using one primer in the insert sequence and the adjacent flanking genome sequence), and then hybridize with DNA polymerase, ATP, sulfurylase, and luciferin The enzymes, adenosine triphosphate bisphosphatase, adenosine-5'-phosphosulfate, and luciferin are incubated together. Add dNTPs separately, and measure the generated optical signal. The light signal represents the presence of the insertion/flanking sequence, which indicates that the amplification, hybridization, and single- or multi-base extension reactions are successful.
- the fluorescence polarization phenomenon described by Chen et al. is also a method that can be used to detect the amplicon of the present invention.
- Using this method requires designing an oligonucleotide chain that spans the inserted DNA sequence and the adjacent genomic DNA binding site. Hybridize the oligonucleotide chain with the single-stranded PCR product of the target region (using one primer in the insert sequence and the adjacent flanking genome sequence), and then perform the hybridization with DNA polymerase and a fluorescently labeled ddNTP Incubate. Single base extension will result in the insertion of ddNTP. This insertion can use a fluorometer to measure its polarization change. The change in polarization represents the presence of insert/flanking sequences, which indicates that the amplification, hybridization, and single base extension reactions are successful.
- Taqman is described as a method for detecting and quantifying the presence of DNA sequences. The method is described in detail in the instructions provided by the manufacturer. A brief description is as follows, design a FRET oligonucleotide probe that spans the inserted DNA sequence and the adjacent genome flanking binding site.
- the FRET probe and PCR primers are cyclically reacted in the presence of thermostable polymerase and dNTPs.
- the hybridization of the FRET probe leads to the splitting of the fluorescent part and the quenched part and the release of the fluorescent part on the FRET probe.
- the generation of the fluorescent signal represents the presence of the insert/flanking sequence, which indicates that the amplification and hybridization are successful.
- suitable techniques for detecting plant materials derived from transgenic corn event DBN9501 may also include Southern blot, Northern blot, and in situ hybridization.
- the suitable technique includes incubating the probe and the sample, washing to remove unbound probes and detecting whether the probes have hybridized.
- the detection method depends on the type of label attached to the probe. For example, radioactively labeled probes can be detected by X-ray film exposure and development, or enzyme-labeled probes can be detected by substrate conversion to achieve color change.
- Tyangi et al. (Nature Biotech. 14:303-308, 1996) introduced the application of molecular markers in sequence detection.
- a brief description is as follows, design a FRET oligonucleotide probe that spans the inserted DNA sequence and the adjacent genome flanking binding site.
- the unique structure of the FRET probe causes it to contain a secondary structure, which can maintain the fluorescent part and the quenching part in a close range.
- the FRET probe and PCR primers (one primer in the insert sequence and one in the adjacent flanking genome sequence) are cyclically reacted in the presence of thermostable polymerase and dNTPs.
- the hybridization of the FRET probe and the target sequence results in the loss of the secondary structure of the probe, which results in the spatial separation of the fluorescent part and the quenched part to generate a fluorescent signal.
- the generation of the fluorescent signal represents the presence of the insert/flanking sequence, which indicates that the amplification and hybridization are successful.
- Optical dyes are used to detect and measure specific DNA molecules.
- a nanotube device including an electronic sensor for detecting DNA molecules or nanobeads that bind to specific DNA molecules and thus can be detected is useful for detecting the DNA molecules of the present invention.
- the composition of the present invention and the methods described or known in the field of DNA detection can be used to develop DNA detection kits.
- the kit is useful for identifying whether there is DNA of transgenic corn event DBN9501 in a sample, and can also be used to cultivate corn plants containing the DNA of transgenic corn event DBN9501.
- the kit may contain DNA primers or probes which are homologous to or complementary to at least a part of SEQ ID NO: 1, 2, 3, 4 or 5, or contain other DNA primers or probes which are homologous to or
- the DNA contained in the genetic element of the transgene that is complementary to the DNA can be used in DNA amplification reactions or as probes in DNA hybridization methods.
- the DNA structure of the binding site of the transgenic insert sequence and the maize genome contained in the maize genome and illustrated in Figure 1 and Table 1 includes: the maize plant DBN9501 flanking genomic region located at the 5'end of the transgenic insert sequence, from the left side of Agrobacterium A part of the border region (LB) is inserted into the sequence.
- the first expression cassette consists of the maize ubiquitin gene 1 promoter (prZmUbi1), which is operably linked to the glufosinate-tolerant phosphinothricin N-acetyl of Streptomyces Base transferase (cPAT) and operably linked to the nopaline synthase transcription terminator (tNos).
- the second expression cassette is composed of the cauliflower mosaic virus 35S promoter containing the tandem repeat of the enhancer region (pr35S), operably linked to the maize heat shock 70kDa protein intron (iZmHSP70), operably linked to the insect resistant Vip3Aa19 protein (cVip3Aa19) of Bacillus thuringiensis, and operably linked to nopaline
- the synthase transcription terminator (tNos) is composed of a part of the insert sequence from the right border region (RB) of Agrobacterium, and the genomic region flanking the corn plant DBN9501 at the 3'end of the transgene insert sequence (SEQ ID NO: 5). ).
- the DNA molecule used as the primer can be any part of the transgenic insert sequence derived from the transgenic corn event DBN9501, or any part of the flanking DNA sequence of the maize genome derived from the transgenic corn event DBN9501.
- the transgenic maize event DBN9501 can be combined with other transgenic maize varieties, such as herbicide (such as glyphosate, dicamba, etc.) tolerant transgenic maize varieties, or transgenic maize varieties carrying other insect resistance genes.
- herbicide such as glyphosate, dicamba, etc.
- transgenic maize varieties carrying other insect resistance genes can provide improved hybrid transgenic corn varieties resistant to multiple pests and multiple herbicides. Compared with non-transgenic varieties and single-character genetically modified varieties, these varieties can show more excellent characteristics such as increased yield.
- the transgenic corn event DBN9501 of the present invention is resistant to the feeding damage of lepidopteran pests, and tolerates the phytotoxic effects of agricultural herbicides containing glufosinate.
- the dual-character corn plant expresses the Vip3Aa19 protein of Bacillus thuringiensis, which provides resistance to the feeding damage of lepidopteran pests (such as cutworms), and expresses the glufosinate-resistant phosphinothricin N of Streptomyces -Acetyltransferase (PAT) protein, which confers tolerance to glufosinate-ammonium in plants.
- PAT Streptomyces -Acetyltransferase
- Dual trait corn has the following advantages: 1) It is protected from economic losses caused by lepidopteran pests (such as cutworms, cotton bollworms, etc.). Cutworms and cotton bollworms are the main pests in corn growing areas; The agricultural herbicide of glufosinate-ammonium gives corn crops the ability to be used for broad-spectrum weed control; 3) corn yield is not reduced.
- the transgenes encoding insect resistance and glufosinate tolerance traits are linked to the same DNA segment and are present on a single locus in the genome of transgenic maize event DBN9501, which provides enhanced breeding efficiency and enables use Molecular markers to track transgenic inserts in breeding populations and their offspring.
- SEQ ID NO: 1 or its complementary sequence, SEQ ID NO: 2 or its complementary sequence, SEQ ID NO: 6 or its complementary sequence, or SEQ ID NO: 7 or its complementary sequence can be used as DNA primers Or probes to generate amplified products diagnosed as transgenic maize event DBN9501 or its progeny, and can quickly, accurately and stably identify the presence of plant material derived from transgenic maize event DBN9501.
- SEQ ID NO: 1 A sequence of 22 nucleotides in length near the insertion junction at the 5'end of the insert sequence in the transgenic corn event DBN9501, in which nucleotides 1-11 and nucleotides 12-22 are nucleosides The acid is located on both sides of the insertion site on the maize genome;
- SEQ ID NO: 2 In the transgenic corn event DBN9501, a 22-nucleotide sequence near the insertion junction at the 3'end of the insert sequence, in which nucleotides 1-11 and nucleotides 12-22 are nucleosides The acid is located on both sides of the insertion site on the maize genome;
- SEQ ID NO: 3 A sequence of 768 nucleotides in length near the insertion junction at the 5'end of the insertion sequence in the transgenic corn event DBN9501;
- SEQ ID NO: 4 A sequence of 1339 nucleotides in length near the insertion junction at the 3'end of the insertion sequence in the transgenic corn event DBN9501;
- SEQ ID NO: 5 The entire T-DNA sequence, the flanking sequence of the maize genome at the 5'and 3'ends;
- SEQ ID NO: 6 The sequence on SEQ ID NO: 3, which straddles the left border region (LB) and the tNos transcription termination sequence;
- SEQ ID NO: 7 The sequence on SEQ ID NO: 4, spanning the pr35S transcription start sequence and the right border region (RB);
- SEQ ID NO: 8 amplifies the first primer of SEQ ID NO: 3;
- SEQ ID NO: 9 amplifies the second primer of SEQ ID NO: 3;
- SEQ ID NO: 10 Amplifies the first primer of SEQ ID NO: 4.
- SEQ ID NO: 11 amplifies the second primer of SEQ ID NO: 4.
- SEQ ID NO: 12 Primer on the 5'flanking genome sequence
- SEQ ID NO: 13 A primer on T-DNA paired with SEQ ID NO: 12;
- the primer on the 3'flanking genomic sequence of SEQ ID NO:14, paired with SEQ ID NO: 12 can detect whether the transgene is homozygous or heterozygous;
- SEQ ID NO: 16 The first primer for Taqman to detect Vip3Aa19 gene
- SEQ ID NO: 17 The second primer for Taqman to detect Vip3Aa19 gene
- SEQ ID NO: 18 Taqman probe for detecting Vip3Aa19 gene
- SEQ ID NO: 19 The first primer for Taqman detection of pat gene
- SEQ ID NO: 20 The second primer for Taqman to detect pat gene
- SEQ ID NO: 21 Taqman probe for detecting pat gene
- SEQ ID NO: 22 The first primer of corn endogenous gene SSIIb;
- SEQ ID NO: 23 The second primer of corn endogenous gene SSIIb;
- SEQ ID NO: 24 Probe for Vip3Aa19 gene in Southern hybridization detection
- SEQ ID NO: 25 Probes for pat gene in Southern hybridization detection
- SEQ ID NO: 26 The primer on T-DNA is in the same direction as SEQ ID NO: 13;
- SEQ ID NO: 27 primer on T-DNA, opposite to SEQ ID NO: 13, used to obtain the flanking sequence;
- SEQ ID NO: 28 primer on T-DNA, opposite to SEQ ID NO: 13, used to obtain the flanking sequence;
- SEQ ID NO: 29 The primer on the T-DNA is in the same direction as SEQ ID NO: 15;
- SEQ ID NO: 30 primer on T-DNA, opposite to SEQ ID NO: 15, used to obtain flanking sequence;
- SEQ ID NO: 31 primer on T-DNA, opposite to SEQ ID NO: 15, used to obtain flanking sequence.
- Figure 1 is a schematic diagram of the present invention for detecting the nucleic acid sequence of the corn plant DBN9501 and the detection method of the transgenic insert sequence and the maize genome junction structure diagram, and a schematic diagram of the relative position of the nucleic acid sequence for detecting the corn plant DBN9501 (reference for the relative position diagram B73 RefGen v3);
- FIG. 2 is a schematic diagram of the structure of the recombinant expression vector DBN10707 used for detecting the nucleic acid sequence of the corn plant DBN9501 and its detection method according to the present invention
- Figure 3 is a field effect diagram of the transgenic corn event DBN9501 used to detect the nucleic acid sequence of the corn plant DBN9501 and its detection method according to the present invention under natural occurrence conditions of the cutworm;
- Fig. 4 is a field effect diagram of the cotton bollworm inoculation of the transgenic corn event DBN9501 used for detecting the nucleic acid sequence of the corn plant DBN9501 and its detection method according to the present invention
- FIG. 5 is a field effect diagram of the transgenic corn event DBN9501 used for detecting the nucleic acid sequence of the corn plant DBN9501 and the detection method thereof under the natural occurrence of Spodoptera litura;
- Fig. 6 is a field effect diagram of the transgenic corn event DBN9501 used for detecting the nucleic acid sequence of the corn plant DBN9501 and the detection method thereof under the natural occurrence of beet armyworm.
- the recombinant expression vector DBN10707 was constructed using standard gene cloning techniques (as shown in Figure 2).
- the vector DBN10707 contains two transgenic expression cassettes in tandem.
- the first expression cassette consists of the cauliflower mosaic virus 35S promoter (pr35S) containing the tandem repeat of the enhancer region, which is operably linked to the maize heat shock 70kDa protein.
- the second one is composed of iZmHSP70, which is operably connected to the insect-resistant Vip3Aa19 protein (cVip3Aa19) of Bacillus thuringiensis, and is operably connected to the nopaline synthase transcription terminator (tNos);
- the second The expression cassette contains the maize ubiquitin gene 1 promoter (prZmUbi1), which is operably linked to the glufosinate-ammonium-tolerant phosphinothricin N-acetyltransferase (cPAT) of Streptomyces, and is operably linked To the nopaline synthase transcription terminator (tNos).
- the vector DBN10707 was transformed into Agrobacterium LBA4404 (Invitrgen, Chicago, USA; Cat. No: 18313-015) by liquid nitrogen method, and used as 4-[hydroxy(methyl)phosphono]-DL-homoalanine Acid is the selection marker to screen transformed cells.
- the conventional Agrobacterium infection method was used for transformation, and the aseptically cultivated maize immature embryos were co-cultured with the Agrobacterium described in Example 1.1 to transfer the T-DNA in the constructed recombinant expression vector DBN10707 to maize Chromosome to generate transgenic corn event DBN9501.
- immature immature embryos are separated from maize, and the immature embryos are contacted with Agrobacterium suspension, where Agrobacterium can combine the nucleotide sequence of the Vip3Aa19 gene and the nucleotide sequence of the pat gene.
- the sequence is transferred to at least one cell of one of the immature embryos (step 1: infection step).
- the immature embryos are co-cultured with Agrobacterium for a period of time (3 days) (step 2: co-cultivation step).
- the immature embryos are placed on a solid medium (MS salt 4.3g/L, MS vitamins, casein 300mg/L, sucrose 20g/L, glucose 10g/L, AS 100mg/L, 2,4- D 1mg/L, agar 8g/L, pH 5.8).
- a solid medium MS salt 4.3g/L, MS vitamins, casein 300mg/L, sucrose 20g/L, glucose 10g/L, AS 100mg/L, 2,4- D 1mg/L, agar 8g/L, pH 5.8.
- the recovery medium (MS salt 4.3g/L, MS vitamins, casein 300mg/L, sucrose 30g/L, 2,4-D 1mg/L, cephalosporin 250mg/L, plant coagulation
- immature embryos are cultured on a solid medium with antibiotics but no selective agent to eliminate Agrobacterium and provide a recovery period for infected cells.
- the inoculated immature embryos are cultured on a medium containing a selection agent (4-[hydroxy(methyl)phosphono]-DL-homoalanine) and the growing transformed callus is selected (Step 4: Selection Step ).
- immature embryos are selected in solid medium (MS salt 4.3g/L, MS vitamins, casein 300mg/L, sucrose 30g/L, cephalosporin 250mg/L, 4-[hydroxy(methyl) ) Phosphono]-DL-homoalanine 10mg/L, 2,4-D 1mg/L, plant gel 3g/L, pH 5.8) cultured on it, resulting in selective growth of transformed cells.
- the callus regenerates plants (step 5: regeneration step), preferably, the callus grown on the medium containing the selection agent is cultured on a solid medium (MS differentiation medium and MS rooting medium) to Regenerate plants.
- the screened resistant callus was transferred to the MS differentiation medium (MS salt 4.3g/L, MS vitamins, casein 300mg/L, sucrose 30g/L, 6-benzyl adenine 2mg/L, cephalosporin Cultivation and differentiation were carried out at 250 mg/L 4-[hydroxy(methyl)phosphono]-DL-homoalanine 5 mg/L, plant gel 3 g/L, pH 5.8) at 25°C.
- MS differentiation medium MS salt 4.3g/L
- MS vitamins, casein 300mg/L, sucrose 30g/L, 6-benzyl adenine 2mg/L, cephalosporin Cultivation and differentiation were carried out at 250 mg/L 4-[hydroxy(methyl)phosphono]-DL-homoalanine 5 mg/L, plant gel 3 g/L, pH 5.8) at 25°C.
- the differentiated seedlings are transferred to the MS rooting medium (MS salt 2.15g/L, MS vitamins, casein 300mg/L, sucrose 30g/L, cephalosporin 250mg/L, indole-3-acetic acid 1mg/L , Plant gel 3g/L, pH 5.8), cultivate it at a temperature of 25°C to a height of about 10cm, move it to the greenhouse and cultivate until it becomes firm. In the greenhouse, culture at a temperature of 28°C for 16 hours a day, and then at a temperature of 20°C for 8 hours.
- MS rooting medium MS salt 2.15g/L, MS vitamins, casein 300mg/L, sucrose 30g/L, cephalosporin 250mg/L, indole-3-acetic acid 1mg/L , Plant gel 3g/L, pH 5.8
- Transgenic T 0 may affect the agronomic traits of maize plants (such as dwarf and thick, clumping leaves, mosaic, opposing leaves, abnormal spinning and loose powder, or poor fruit setting, etc.)
- the above 200 independent Transgenic T 0 individual plants are sent to the greenhouse for transplantation and culture to identify the agronomic performance of transgenic T 0 individual plants at different stages (seedling stage-jointing stage, jointing stage-powdering stage and filling stage-maturity stage).
- seedling stage-jointing stage, jointing stage-powdering stage and filling stage-maturity stage 136 transgenic T 0 plants with normal agronomic traits.
- TaqMan TM analysis was used to detect the presence of single copies of Vip3Aa19 and pat genes in the above-mentioned 136 transgenic maize plants without vector backbone sequences, and a total of 83 transgenic T 0 plants were obtained; through transgene insertion site analysis, a total of 28 were screened.
- the T-DNA flanking sequence is complete, the T-DNA is not inserted into the important genes of the maize genome, and the gene insertion does not produce a new open reading frame (ORF) transgenic T 0 single plant; through the main target insects (such as cutworms) , Helicoverpa armigera, Spodoptera litura or Spodoptera exigua) resistance evaluation and comparison, a total of 13 transgenic T 0 plants with good insect resistance were screened; through the evaluation and comparison of tolerance to glufosinate-ammonium herbicide, A total of 12 transgenic T 0 plants with good tolerance to glufosinate-ammonium herbicide were screened; under different generations, different
- the selected transgenic corn event DBN9501 is excellent, which has a single copy of the transgene (see the second example), good insect resistance, Glufosinate herbicide tolerance and agronomic performance (see the fifth and sixth examples).
- the second embodiment using TaqMan to detect transgenic corn event DBN9501
- transgenic corn event DBN9501 About 100 mg of the leaves of transgenic corn event DBN9501 were taken as samples, and their genomic DNA was extracted with a plant DNA extraction kit (DNeasy Plant Maxi Kit, Qiagen), and the copy number of Vip3Aa19 gene and pat gene was detected by Taqman probe fluorescence quantitative PCR method. At the same time, wild-type corn plants were used as controls, and the detection and analysis were performed according to the above methods. The experiment is set to be repeated 3 times and the average value is taken.
- DNeasy Plant Maxi Kit, Qiagen DNeasy Plant Maxi Kit, Qiagen
- Step 1 Take 100 mg of the leaves of transgenic corn event DBN9501 (after pollination), grind it into a homogenate with liquid nitrogen in a mortar, and take 3 replicates for each sample;
- Step 2 Use the plant DNA extraction kit (DNeasy Plant Maxi Kit, Qiagen) to extract the genomic DNA of the above sample, and refer to the product manual for the specific method;
- Step 3 Use an ultra-micro spectrophotometer (NanoDrop 2000, Thermo Scientific) to measure the genomic DNA concentration of the above sample;
- Step 4 Adjust the genomic DNA concentration of the above sample to the same concentration value, and the range of the concentration value is 80-100ng/ ⁇ L;
- Step 5 Use Taqman probe fluorescence quantitative PCR method to identify the copy number of the sample, use the identified sample with known copy number as the standard product, and use the wild-type corn plant sample as the control. There are 3 replicates for each sample and take the average Value; the sequence of the fluorescent quantitative PCR primer and probe are:
- Primer 1 cgaatacagaaccctgtcggc is shown in SEQ ID NO: 16 in the sequence list;
- Primer 2 cgtgaggaaggtctcagaaatgac is shown in SEQ ID NO: 17 in the sequence table;
- Probe 1 cgacgatggcgtgtatatgcctcttgg is shown in SEQ ID NO: 18 in the sequence table;
- gagggtgttgtggctggtattg is shown in SEQ ID NO: 19 in the sequence list;
- Primer 4 tctcaactgtccaatcgtaagcg is shown in SEQ ID NO: 20 in the sequence table;
- Probe 2 cttacgctgggccctggaaggctag is shown in SEQ ID NO: 21 in the sequence table;
- the PCR reaction system is:
- the 50 ⁇ primer/probe mixture contains 45 ⁇ L of each primer at a concentration of 1 mM, 50 ⁇ L of probe at a concentration of 100 ⁇ M, and 860 ⁇ L 1 ⁇ TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), and the temperature is at 4°C. , Stored in amber test tube.
- the PCR reaction conditions are:
- the data was analyzed using the Fast Real-Time Fluorescent Quantitative PCR System Software (Applied Biosystems 7900HT Fast Real-Time PCR System SDS v2.3, Applied Biosystems), and the results showed that the obtained transgenic corn event DBN9501 was a single copy.
- the third embodiment analysis of the insertion site of the transgenic corn event DBN9501
- DNA extraction was carried out according to the conventional CTAB (hexadecyl trimethyl ammonium bromide) method: 2g of the young leaves of transgenic corn DBN9501 were ground into powder in liquid nitrogen, and then 0.5 mL preheated at 65°C 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 90 minutes ; Add 0.5 times the volume of phenol and 0.5 times the volume of chloroform, and mix upside down; centrifuge at 12000 rpm (revolutions per minute) for 10 min; aspirate the supernatant, add 2 times the volume of absolute ethanol, gently shake the centrifuge tube, at a temperature of 4 °C Let stand for 30 min; centrifuge at 12000 rpm for another 10 min; collect DNA to the bottom of the tube; discard the supernatant and wash
- the concentration of the extracted DNA sample is determined so that the concentration of the sample to be tested is between 80-100ng/ ⁇ L.
- the restriction enzymes Kpn I (5' end analysis) and Spe I (3' end analysis) were used to cut genomic DNA. Add 26.5 ⁇ L of genomic DNA, 0.5 ⁇ L of the above restriction endonuclease and 3 ⁇ L of digestion buffer to each digestion system (the restriction enzymes used are all NEB enzymes and its matching buffer or universal buffer. Called NEBCutSmart), digested for 1 hour.
- the primer combination for isolating 5'-end genomic DNA includes SEQ ID NO: 13 and SEQ ID NO: 26 as the first primer, SEQ ID NO: 27 and SEQ ID NO: 28 as the second primer, and SEQ ID NO: 13 As a sequencing primer.
- the primer combination for isolating 3'end genomic DNA includes SEQ ID NO: 15 and SEQ ID NO: 29 as the first primer, SEQ ID NO: 30 and SEQ ID NO: 31 as the second primer, and SEQ ID NO: 15 as the sequencing primer,
- the PCR reaction conditions are shown in Table 3.
- the amplified products obtained by the above PCR amplification reaction were electrophoresed on a 2.0% agarose gel to separate the PCR amplified products, and then the gel recovery kit (QIAquick Gel Extraction Kit, catalog #_28704, Qiagen Inc., Valencia, CA) separate the target fragment from the agarose matrix. Then the purified PCR amplification product is sequenced (for example, using ABI PrismTM 377, PE Biosystems, Foster City, CA) and analyzed (for example, using DNASTAR sequence analysis software, DNASTAR Inc., Madison, WI).
- the 5'flanking sequence and the joining sequence can be confirmed using SEQ ID NO: 8 or SEQ ID NO: 12, in combination with SEQ ID NO: 9, SEQ ID NO: 13 or SEQ ID NO: 26.
- the 3'flanking sequence and the joining sequence can be confirmed using SEQ ID NO: 11 or SEQ ID NO: 14, combined with SEQ ID NO: 10, SEQ ID NO: 15 or SEQ ID NO: 29.
- the PCR reaction system and amplification conditions are shown in Table 2 and Table 3. Those skilled in the art will understand that other primer sequences can also be used to confirm flanking and junction sequences.
- DNA sequencing of PCR amplified products provides DNA that can be used to design other DNA molecules that can be used as primers and probes to identify corn plants or seeds derived from transgenic corn event DBN9501.
- nucleotides 1-384 of SEQ ID NO: 5 show that the maize genome sequence is flanking the left border (5' flanking sequence) of the transgenic maize event DBN9501 insert sequence, and the nucleotides in SEQ ID NO: 5 Positions 7785-8559 show the maize genome sequence flanking the right boundary (3' flanking sequence) of the transgenic maize event DBN9501 insert sequence.
- the 5'junction sequence is listed in SEQ ID NO: 1
- 3'junction sequence is listed in SEQ ID NO: 2.
- the junction sequence is a relatively short polynucleotide molecule, which is a new DNA sequence that is diagnostic for the DNA of transgenic corn event DBN9501 when detected in a polynucleic acid detection analysis.
- the junction sequences in SEQ ID NO: 1 and SEQ ID NO: 2 are the insertion site of the transgenic fragment in the transgenic corn event DBN9501 and 11 polynucleotides on each side of the corn genomic DNA.
- the longer or shorter polynucleotide junction sequence can be selected from SEQ ID NO: 3 or SEQ ID NO: 4.
- the junction sequences (5' junction region SEQ ID NO: 1 and 3'junction region SEQ ID NO: 2) are useful as DNA probes or as DNA primer molecules in DNA detection methods.
- the junction sequence SEQ ID NO: 6 and SEQ ID NO: 7 are also new DNA sequences in the transgenic corn event DBN9501, and they can also be used as DNA probes or as DNA primer molecules to detect the presence of the transgenic corn event DBN9501 DNA.
- the SEQ ID NO: 6 (nucleotide 385-574 of SEQ ID NO: 3) spans the DBN10707 construct DNA sequence and the tNos transcription termination sequence
- the SEQ ID NO: 7 (SEQ ID NO: 4 nuclear The nucleotides 252-451) span the pr35S transcription initiation sequence and the DNA sequence of the DBN10707 construct.
- the amplicon is generated by using at least one primer from SEQ ID NO: 3 or SEQ ID NO: 4, which generates a diagnostic amplicon of transgenic corn event DBN9501 when used in a PCR method.
- a PCR amplification product is generated from the 5'end of the transgenic insert sequence, and the PCR amplification product is a product containing genomic DNA flanking the 5'end of the T-DNA insert in the genome of the plant material derived from the transgenic corn event DBN9501 Part.
- This PCR amplification product contains SEQ ID NO: 3.
- design primer 5 SEQ ID NO: 8 that hybridizes with the genomic DNA sequence flanking the 5'end of the transgene insert sequence, and the primer paired with the tNos transcription termination sequence in the T-DNA insert sequence 6 (SEQ ID NO: 9).
- a PCR amplification product is generated from the 3'end of the transgene insert sequence, and the PCR amplification product contains a part of the genomic DNA flanking the 3'end of the T-DNA insert sequence in the genome of the plant material derived from transgenic corn event DBN9501.
- This PCR amplification product contains SEQ ID NO: 4.
- design primer 7 SEQ ID NO: 10
- the primer 8 SEQ ID NO: 11).
- the DNA amplification conditions described in Table 2 and Table 3 can be used in the PCR zygosity test described above to generate the diagnostic amplicon of transgenic corn event DBN9501.
- the detection of amplicons can be performed by using Stratagene Robocycler, MJ Engine, Perkin-Elmer 9700 or Eppendorf Mastercycler Gradient thermal cycler, etc., or by methods and equipment known to those skilled in the art.
- primers 5 and 6 when used in the PCR reaction of transgenic maize event DBN9501 genomic DNA, produce 768bp fragments of amplified products, when they are used in the untransformed maize genome
- primers 7 and 8 when used in the PCR reaction of transgenic corn event DBN9501 genomic DNA, produced The amplified product of the 1339bp fragment, when it was used in the PCR reaction of untransformed corn genomic DNA and non-DBN9501 corn genomic DNA, no fragment was amplified.
- the PCR zygosity assay can also be used to identify whether the material derived from the transgenic corn event DBN9501 is homozygous or heterozygous.
- Primer 9 SEQ ID NO: 12
- primer 10 SEQ ID NO: 13
- primer 11 SEQ ID NO: 14
- the DNA amplification conditions described in Tables 4 and 5 can be used in the above-mentioned zygosity test to generate diagnostic amplicons of transgenic corn event DBN9501.
- the biological sample containing template DNA contains DNA for diagnosing the presence of the transgenic corn event DBN9501 in the sample.
- the amplification reaction will produce two different DNA amplicons from a biological sample containing DNA derived from the maize genome, the alleles corresponding to the inserted DNA present in the transgenic maize event DBN9501 Heterozygous. These two different amplicons will correspond to the first amplicon (SEQ ID NO: 12 and SEQ ID NO: 14) derived from the wild-type corn genomic locus and the first amplicon to diagnose the presence of the transgenic corn event DBN9501 DNA. Two amplicons (SEQ ID NO: 12 and SEQ ID NO: 13).
- primer pair of transgenic corn event DBN9501 is used to generate amplicons that are diagnostic for the genomic DNA of transgenic corn event DBN9501.
- primer pairs include, but are not limited to, primers 5 and 6 (SEQ ID NO: 8 and 9), and primers 7 and 8 (SEQ ID NO: 10 and 11), which are used in the DNA amplification method.
- a control primer 12 and 13 used to amplify endogenous maize genes was included as an internal standard for the reaction conditions.
- the analysis of DNA extraction samples from transgenic maize event DBN9501 should include a positive tissue DNA extract control of transgenic maize event DBN9501, a negative DNA extract control derived from non-transgenic maize event DBN9501, and a maize DNA extract without template. The negative control of the extract.
- any primer pair derived from SEQ ID NO: 3 or its complementary sequence, or SEQ ID NO: 4 or its complementary sequence can also be used.
- the tissue of the corn plant DBN9501 at the transgenic event is a diagnostic amplicon comprising SEQ ID NO: 1 or SEQ ID NO: 2.
- the DNA amplification conditions described in Table 2 to Table 5 can be used to generate diagnostic amplicons of transgenic corn event DBN9501 using appropriate primer pairs.
- an extract of maize plant or seed DNA that is a diagnostic amplicon for transgenic maize event DBN9501, presumed to contain transgenic maize event DBN9501, or a product derived from transgenic maize event DBN9501, can be Used as a template for amplification to determine the presence of transgenic corn event DBN9501.
- the water phase was extracted with an equal volume of chloroform/isoamyl alcohol (24:1) and repeated once. Collect the aqueous phase again and add an equal volume of isopropanol. After mixing, place it at -20°C for 1 hour to precipitate the DNA, and then centrifuge at 4000 rpm for 5 minutes to obtain the DNA precipitate, and then in 1mL TE buffer (10mM Tris-HCl, 1mM EDTA , PH 8.0) resuspend the DNA pellet.
- 1mL TE buffer (10mM Tris-HCl, 1mM EDTA , PH 8.0
- RNA was precipitated by centrifugation at 12000 rpm for 10 min. After discarding the supernatant, wash the pellet with 70% (v/v) 1 mL ethanol, dry at room temperature, and redissolve the DNA in 1 mL TE buffer.
- the genomic DNA concentration of the above samples was measured with an ultra-micro spectrophotometer (NanoDrop 2000, Thermo Scientific).
- bromophenol blue loading buffer to each sample derived from this example 4.2, and load each sample on a 0.7% TAE agarose gel containing ethidium bromide, and then add bromophenol blue loading buffer to the TAE running buffer ( Electrophoresis separation in 40mM Tris-acetic acid, 2mM EDTA, pH 8.5), and electrophoresis the gel overnight at a voltage of 20V.
- the DNA probe is SEQ ID NO: 24 or SEQ ID NO: 25, or is partially homologous or complementary to the above sequence.
- Use DNA Labeling and Detection Starter Kit II (Roche, Cat. No. 11585614910) for DIG labeling of probes, Southern blot hybridization, and membrane washing. For specific methods, refer to its product manual.
- X-ray film (Roche, Cat. No. 11666916001) was used to detect the position of probe binding.
- Each Southern includes two control samples: (1) DNA from negative (untransformed) segregants, which are used to identify any endogenous corn sequences that can hybridize with element-specific probes; (2) from negative Ionized DNA, into which Nco I-digested DBN10707 plasmid is introduced, the amount is equivalent to one copy number based on the length of the probe, which serves as a positive control for hybridization and is used to illustrate the sensitivity of the experiment.
- the hybridization data provided conclusive evidence to support TaqMan TM PCR analysis, that is, the corn plant DBN9501 contains a single copy of the Vip3Aa19 gene and the pat gene.
- Nco I and Ned I enzymatic digestion produced single bands of approximately 11 kb and 9 kb, respectively;
- the pat gene probe Nco I and Ned I enzymatic digestion produced approximately 8.5 kb and 1.3 kb, respectively
- a single band indicates that one copy of the Vip3Aa19 gene and one copy of the pat gene exist in the corn plant DBN9501.
- the backbone probe no hybridization band was obtained, indicating that no DBN10707 vector backbone sequence entered the maize plant DBN9501 genome during the transformation process.
- transgenic maize event DBN9501 and wild-type maize plants (non-transgenic, NGM) 2 plants were tested against Agrotis ypsilon Rottemberg (BCW), Spodoptera litura (TCW), Spodoptera exigua (BAW). ), Sesamia inferens (PSB), Sorghum striped borer (Chilo sacchariphagus, SGB) and Millet ash borer (Chilo infuscatellus, MSB) were bioassayed according to the following methods:
- total resistance 100 ⁇ mortality + [100 ⁇ mortality + 90 ⁇ (number of newly hatched insects/ Insect number)+60 ⁇ (Incubation-negative control number/Insect number)+10 ⁇ (Negative control number/Insect number)]+100 ⁇ (1-leaf damage rate).
- the number of catching insects refers to the number of catching insects, that is, 5 or 10 per dish (depending on the feeding amount of the pests); the development progress of larvae has been reflected by the resistance total score formula; the leaf damage rate refers to the number of pests The ratio of the leaf area that eats to the total leaf area. From the transgenic corn event DBN9501 and wild-type corn plants (non-transgenic, NGM), 5 plants were selected for testing, and each plant was repeated 6 times. The results are shown in Table 6 and Table 7.
- transgenic maize event DBN9501 had good resistance to cutworms, Spodoptera litura, beet armyworm, Chinese rice borer, sorghum stripe borer and millet gray borer, and the transgenic maize event DBN9501 had better resistance to test insect mortality and resistance.
- the total scores of sex were significantly higher than NGM.
- the damage rate the number of corn plants eaten by the pests/the total number of plants ⁇ 100%).
- the resistance results of transgenic corn event DBN9501 to cutworms are shown in Table 8.
- Ear damage level Description of symptoms 0 No ear damage 1 Only the filaments are killed 2 Ear damage 1cm 3+ For every increase of 1cm of damage under the ear top, the corresponding damage level increases by 1 level ...N To
- the resistance results of transgenic corn event DBN9501 to Spodoptera exigua are shown in Table 13.
- transgenic maize event DBN9501 realizes the method and/or use of controlling pests, specifically the rice borer, Spodoptera litura, S. chinensis, sorghum stripe borer and peach borer; that is, any transgenic corn plant expressing Vip3Aa19 protein can be controlled Methods and/or uses of P. chinensis, Spodoptera litura, P. sinensis, P. sorghum and/or Peach borer pests.
- Basta herbicide (a salt water formulation of glufosinate ammonium salt with an active ingredient of 18%) was selected for spraying.
- a random block design was used, with 3 repetitions.
- the plot area is 15m 2 (5m ⁇ 3m), row spacing is 60cm, plant spacing is 25cm, conventional cultivation management, there is a 1m wide isolation zone between plots.
- the transgenic corn event DBN9501 was subjected to the following two treatments: (1) No spraying, while the treatment (2) Spraying herbicides, spray an equal volume of water; (2) Press 800g ai/ha (ai/ha means " Active ingredient per hectare") The dose is sprayed with the guarantee herbicide during the V2-V3 leaf stage.
- glufosinate-ammonium herbicides such as Basta
- Basta contact herbicides. If they are used improperly in the field, such as excessive local accumulation of liquid medicine, they may cause phytotoxicity. It is not that there is a problem with the tolerance of the transgenic corn event DBN9501 ; Different content and dosage forms of glufosinate-ammonium herbicide converted into the above-mentioned equivalent effective ingredients are applicable to the following conclusions.
- the phytotoxicity symptoms were investigated 1 week and 2 weeks after the medication, and the yield of the plot was measured at the time of harvest; the phytotoxicity symptoms classification is shown in Table 14.
- the damage rate of the herbicide refers to the damage rate of glufosinate-ammonium.
- the damage rate of glufosinate-ammonium is determined based on the investigation results of the phytotoxicity 2 weeks after the treatment of glufosinate-ammonium. The tolerance level.
- the corn yield of each plot is the total yield (weight) of corn kernels in the middle 3 rows of each plot.
- yield percentage (%) spray yield/no spray Yield.
- Phytotoxicity level Description of symptoms 1 Normal growth without any symptoms of harm 2 Slight phytotoxicity, phytotoxicity is less than 10% 3 Moderate phytotoxicity, which can be recovered in the future without affecting the yield 4 The phytotoxicity is severe and difficult to recover, resulting in reduced production 5 The drug damage is serious and cannot be recovered, causing a significant reduction in production or abortion
- the damage rate of the transgenic corn event DBN9501 is 0 under the treatment of glufosinate-ammonium herbicide (800g ai/ha); therefore, the transgenic corn event DBN9501 has good glufosinate herbicide Agent tolerance.
- the agricultural product or commodity is expected to contain a nucleotide sequence capable of diagnosing the presence of the transgenic corn event DBN9501 material in the agricultural product or commodity.
- the agricultural products or commodities include, but are not limited to, corn oil, corn meal, corn flour, corn gluten, corn tortillas, corn starch, and any other food that will be used as a food source for animal consumption, or otherwise used as a bulking agent or cosmetic composition
- the ingredients are used for cosmetic purposes.
- Nucleic acid detection methods and/or kits based on probes or primer pairs can be developed to detect nucleotide sequences derived from transgenic corn event DBN9501, such as SEQ ID NO: 1 or SEQ ID NO: 2, in biological samples,
- the probe sequence or primer sequence is selected from the sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, or a part thereof, to diagnose transgene The existence of the corn event DBN9501.
- the transgenic corn event DBN9501 of the present invention has good resistance to lepidopteran insects, and at the same time has high tolerance to glufosinate herbicide, has no effect on yield, and the detection method can be accurate and rapid Identify whether the biological sample contains the DNA molecule of transgenic corn event DBN9501.
- the seeds corresponding to the genetically modified corn event DBN9501 have been deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee (CGMCC for short) according to the Budapest Treaty on January 23, 2019. Address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing. Institute of Microbiology, Chinese Academy of Sciences, 100101), classified and named: Zea mays, and the preservation number is CGMCC No. 17099. The deposit will be kept in the depository for 30 years.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Environmental Sciences (AREA)
- Botany (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Developmental Biology & Embryology (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physiology (AREA)
- Analytical Chemistry (AREA)
- Plant Pathology (AREA)
- Agronomy & Crop Science (AREA)
- Pest Control & Pesticides (AREA)
- Dentistry (AREA)
- Mycology (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Cell Biology (AREA)
- Immunology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
Description
雌穗被害级别 | 症状描述 |
0 | 雌穗没有受害 |
1 | 仅花丝被害 |
2 | 穗顶被害1cm |
3+ | 穗顶下被害每增加1cm,相应的被害级别增加1级 |
…N |
雌穗被害级别平均值 | 抗性水平 |
0-1.0 | 高抗(HR) |
1.1-3.0 | 抗(R) |
3.1-5.0 | 中抗(MR) |
5.1-7.0 | 感(S) |
≥7.1 | 高感(HS) |
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 |
Claims (13)
- 一种核酸序列,其特征在于,具有SEQ ID NO:3或其互补序列第1-384位中至少11个连续的核苷酸和SEQ ID NO:3或其互补序列第385-768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1-564位中至少11个连续的核苷酸和SEQ ID NO:4或其互补序列第565-1339位中至少11个连续的核苷酸;优选地,所述核酸序列具有SEQ ID NO:3或其互补序列第1-384位中22-25个连续的核苷酸和SEQ ID NO:3或其互补序列第385-768位中22-25个连续的核苷酸、和/或SEQ ID NO:4或其互补序列第1-564位中22-25个连续的核苷酸和SEQ ID NO:4或其互补序列第565-1339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。
- 根据权利要求1所述的核酸序列,其特征在于,所述核酸序列包含SEQ ID NO:5或其互补序列。
- 一种检测样品中转基因玉米事件DBN9501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。
- 根据权利要求3所述检测样品中转基因玉米事件DBN9501的DNA存在的方法,其特征在于,所述引物包括第一引物和第二引物,所述第一引物选自SEQ ID NO:1、SEQ ID NO:8和SEQ ID NO:10;所述第二引物选自SEQ ID NO:2、SEQ ID NO:9和SEQ ID NO:11。
- 一种检测样品中转基因玉米事件DBN9501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。
- 根据权利要求5所述检测样品中转基因玉米事件DBN9501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。
- 一种检测样品中转基因玉米事件DBN9501的DNA存在的方法,其特征在于,包括:使待检测样品与标记物核酸分子接触,所述标记物核酸分子包括权利要求1所述核酸序列;优选地,所述标记物核酸分子包括选自以下的至少一种:SEQ ID NO:1或其互补序列、SEQ ID NO:2或其互补序列、和/或SEQ ID NO:6-11或其互补序列;使所述待检测样品和所述标记物核酸分子在严格杂交条件下杂交;检测所述待检测样品和所述标记物核酸分子的杂交情况,进而通过标记物辅助育种分析以确定昆虫抗性和/或除草剂耐受性与标记物核酸分子在遗传学上是连锁的。
- 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玉米事件DBN9501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。
- 一种保护玉米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玉米植物细胞,所述转基因玉米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,摄食所述转基因玉米植物细胞的靶昆虫被抑制进一步摄食所述转基因玉米植物;优选地,所述转基因玉米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,所述转基因玉米植物细胞在其基因组中依次包含SEQ ID NO:1、SEQ ID NO:5第553-7491位核酸序列和SEQ ID NO:2,或者包含SEQ ID NO:5所示的序列。
- 一种保护玉米植物免受由除草剂引起的损伤或控制种植玉米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玉米植物的大田中,所述转基因玉米植物 在其基因组中包含SEQ ID NO:1和/或SEQ ID NO:2所示的序列,所述转基因玉米植物对草铵膦除草剂具有耐受性;优选地,所述转基因玉米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,所述转基因玉米植物在其基因组中依次包含SEQ ID NO:1、SEQ ID NO:5第553-7491位核酸序列和SEQ ID NO:2,或者包含SEQ ID NO:5所示的序列。
- 一种培养对昆虫具有抗性和/或耐受草铵膦除草剂的玉米植物的方法,其特征在于,包括:种植至少一粒玉米种子,所述玉米种子的基因组中包含编码昆虫抗性Vip3Aa蛋白的核酸序列和/或编码草铵膦除草剂耐受性PAT蛋白的核酸序列、和特定区域的核酸序列,或者所述玉米种子的基因组中包含SEQ ID NO:5所示的核酸序列;使所述玉米种子长成玉米植株;用靶昆虫侵袭所述玉米植株和/或用有效剂量草铵膦除草剂喷洒所述玉米植株,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。
- 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玉米植株的方法,其特征在于,包括将第一玉米植物基因组中包含的编码昆虫抗性Vip3Aa蛋白的核酸序列和/或编码草铵膦耐受性PAT蛋白的核酸序列、和特定区域的核酸序列,或者将所述第一玉米植物基因组中包含的SEQ ID NO:5所示的核酸序列,引入第二玉米植物,从而产生大量子代植株;选择具有所述特定区域的核酸序列的所述子代植株,且所述子代植株对昆虫具有抗性和/或对草铵膦除草剂具有耐受性;所述特定区域的核酸序列为SEQ ID NO:1和/或SEQ ID NO:2所示的序列;优选地,所述特定区域的核酸序列为SEQ ID NO:3和/或SEQ ID NO:4所示的序列;优选地,所述方法包括将转基因玉米事件DBN9501与缺少昆虫抗性和/或草铵膦耐受性的玉米植株进行有性杂交,从而产生大量子代植株,选择具有所述特定区域的核酸序列的所述子代植株;用靶昆虫侵袭和/或用草铵膦处理所述子代植株;选择对昆虫具有抗性和/或对草铵膦除草剂具有耐受性的所述子代植株。
- 一种产生自转基因玉米事件DBN9501的农产品或商品,其特征在于,所述农产品或商品为玉米粗粉、玉米面、玉米油、玉米穗丝、玉米淀粉、玉米面筋、玉米饼、化妆品或填充剂。
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
MX2021010933A MX2021010933A (es) | 2019-04-09 | 2020-02-21 | Secuencia de acido nucleico para detectar la planta de maiz dbn9501 y metodo de deteccion para la misma. |
BR112021000302-9A BR112021000302A2 (pt) | 2019-04-09 | 2020-02-21 | Sequência de ácidos nucleicos, método para detectar a presença do dna do evento de milho transgênico dbn9501 em uma amostra, kit de detecção de dna, métodos para proteger uma planta de milho, método para criar uma planta de milho resistente a insetos e/ou tolerante ao herbicida glufosinato e produto ou commodity agrícola derivado do evento de milho transgênico dbn9501 |
ZA2021/04864A ZA202104864B (en) | 2019-04-09 | 2021-07-12 | Nucleic acid sequence for detecting maize plant dbn9501 and detection method therefor |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910280088.XA CN109868273B (zh) | 2019-04-09 | 2019-04-09 | 用于检测玉米植物dbn9501的核酸序列及其检测方法 |
CN201910280088.X | 2019-04-09 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2020207125A1 true WO2020207125A1 (zh) | 2020-10-15 |
Family
ID=66922273
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CN2020/076208 WO2020207125A1 (zh) | 2019-04-09 | 2020-02-21 | 用于检测玉米植物dbn9501的核酸序列及其检测方法 |
Country Status (6)
Country | Link |
---|---|
CN (1) | CN109868273B (zh) |
AR (1) | AR118492A1 (zh) |
BR (1) | BR112021000302A2 (zh) |
MX (1) | MX2021010933A (zh) |
WO (1) | WO2020207125A1 (zh) |
ZA (1) | ZA202104864B (zh) |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112877454A (zh) * | 2021-01-27 | 2021-06-01 | 隆平生物技术(海南)有限公司 | 转基因玉米事件lp007-3及其检测方法 |
CN116144672A (zh) * | 2022-09-23 | 2023-05-23 | 隆平生物技术(海南)有限公司 | 转基因玉米事件lp026-1及其检测方法 |
CN112852801B (zh) * | 2021-01-27 | 2023-08-08 | 隆平生物技术(海南)有限公司 | 转基因玉米事件lp007-1及其检测方法 |
CN116676304A (zh) * | 2023-07-20 | 2023-09-01 | 隆平生物技术(海南)有限公司 | 转基因玉米事件lp016-1及其检测方法 |
CN116694627A (zh) * | 2023-07-26 | 2023-09-05 | 隆平生物技术(海南)有限公司 | 转基因玉米事件lp035-1及其检测方法 |
CN116694629A (zh) * | 2023-07-31 | 2023-09-05 | 隆平生物技术(海南)有限公司 | 转基因玉米事件lp038-1及其检测方法 |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109868273B (zh) * | 2019-04-09 | 2023-02-03 | 北京大北农生物技术有限公司 | 用于检测玉米植物dbn9501的核酸序列及其检测方法 |
CN113151533B (zh) * | 2021-01-27 | 2023-08-08 | 隆平生物技术(海南)有限公司 | 转基因玉米事件lp007-6及其检测方法 |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101402996A (zh) * | 2008-11-06 | 2009-04-08 | 东北农业大学 | 粮食作物深加工制品高通量五重巢式pcr转基因检测方法 |
CN102762725A (zh) * | 2009-12-23 | 2012-10-31 | 拜尔知识产权有限公司 | 耐受hppd抑制剂型除草剂的植物 |
CN103039494A (zh) * | 2012-12-05 | 2013-04-17 | 北京大北农科技集团股份有限公司 | 控制害虫的方法 |
CN103140585A (zh) * | 2010-07-07 | 2013-06-05 | 先正达参股股份有限公司 | 鞘翅目害虫的控制 |
CN103719136A (zh) * | 2013-11-15 | 2014-04-16 | 北京大北农科技集团股份有限公司 | 控制害虫的方法 |
CN109868273A (zh) * | 2019-04-09 | 2019-06-11 | 北京大北农生物技术有限公司 | 用于检测玉米植物dbn9501的核酸序列及其检测方法 |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103555716B (zh) * | 2013-11-06 | 2016-01-20 | 北京大北农科技集团股份有限公司 | 增强表达的内含子序列及其用途 |
CN104878092B (zh) * | 2015-04-30 | 2018-10-26 | 北京大北农科技集团股份有限公司 | 用于检测玉米植物dbn9953的核酸序列及其检测方法 |
CN104878096B (zh) * | 2015-04-30 | 2019-01-15 | 北京大北农科技集团股份有限公司 | 用于检测除草剂耐受性玉米植物dbn9868的核酸序列及其检测方法 |
-
2019
- 2019-04-09 CN CN201910280088.XA patent/CN109868273B/zh active Active
-
2020
- 2020-02-21 MX MX2021010933A patent/MX2021010933A/es unknown
- 2020-02-21 WO PCT/CN2020/076208 patent/WO2020207125A1/zh active Application Filing
- 2020-02-21 BR BR112021000302-9A patent/BR112021000302A2/pt unknown
- 2020-03-26 AR ARP200100835A patent/AR118492A1/es unknown
-
2021
- 2021-07-12 ZA ZA2021/04864A patent/ZA202104864B/en unknown
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101402996A (zh) * | 2008-11-06 | 2009-04-08 | 东北农业大学 | 粮食作物深加工制品高通量五重巢式pcr转基因检测方法 |
CN102762725A (zh) * | 2009-12-23 | 2012-10-31 | 拜尔知识产权有限公司 | 耐受hppd抑制剂型除草剂的植物 |
CN103140585A (zh) * | 2010-07-07 | 2013-06-05 | 先正达参股股份有限公司 | 鞘翅目害虫的控制 |
CN103039494A (zh) * | 2012-12-05 | 2013-04-17 | 北京大北农科技集团股份有限公司 | 控制害虫的方法 |
CN103719136A (zh) * | 2013-11-15 | 2014-04-16 | 北京大北农科技集团股份有限公司 | 控制害虫的方法 |
CN109868273A (zh) * | 2019-04-09 | 2019-06-11 | 北京大北农生物技术有限公司 | 用于检测玉米植物dbn9501的核酸序列及其检测方法 |
Non-Patent Citations (2)
Title |
---|
WANG, CUIYUN ET AL.: "Analysis of the Integration Site of Exogenous Gene in Transgenic Maize", BIOTECHNOLOGY BULLETIN, vol. 35, no. 3, 31 March 2019 (2019-03-31), XP055741490, ISSN: 1002-5464 * |
ZHANG, XIAOJING: "Molecular Characterization and Inheritance Stability Analysis of An Insect-resistant and Herbicide-tolerant Transgenic Maize", CHINESE MASTER’S THESES FULL-TEXT DATABASE, AGRICULTURAL SCIENCE AND TECHNOLOGY, no. 1,, 15 January 2019 (2019-01-15), pages 1 - 66, XP055741499, ISSN: 1674-0246 * |
Cited By (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112877454A (zh) * | 2021-01-27 | 2021-06-01 | 隆平生物技术(海南)有限公司 | 转基因玉米事件lp007-3及其检测方法 |
CN112852801B (zh) * | 2021-01-27 | 2023-08-08 | 隆平生物技术(海南)有限公司 | 转基因玉米事件lp007-1及其检测方法 |
CN112877454B (zh) * | 2021-01-27 | 2023-08-08 | 隆平生物技术(海南)有限公司 | 转基因玉米事件lp007-3及其检测方法 |
CN116144672A (zh) * | 2022-09-23 | 2023-05-23 | 隆平生物技术(海南)有限公司 | 转基因玉米事件lp026-1及其检测方法 |
CN116144672B (zh) * | 2022-09-23 | 2023-11-07 | 隆平生物技术(海南)有限公司 | 转基因玉米事件lp026-1及其检测方法 |
CN116676304A (zh) * | 2023-07-20 | 2023-09-01 | 隆平生物技术(海南)有限公司 | 转基因玉米事件lp016-1及其检测方法 |
CN116676304B (zh) * | 2023-07-20 | 2023-09-26 | 隆平生物技术(海南)有限公司 | 转基因玉米事件lp016-1及其检测方法 |
CN116694627A (zh) * | 2023-07-26 | 2023-09-05 | 隆平生物技术(海南)有限公司 | 转基因玉米事件lp035-1及其检测方法 |
CN116694627B (zh) * | 2023-07-26 | 2023-09-29 | 隆平生物技术(海南)有限公司 | 转基因玉米事件lp035-1及其检测方法 |
CN116694629A (zh) * | 2023-07-31 | 2023-09-05 | 隆平生物技术(海南)有限公司 | 转基因玉米事件lp038-1及其检测方法 |
CN116694629B (zh) * | 2023-07-31 | 2023-09-29 | 隆平生物技术(海南)有限公司 | 转基因玉米事件lp038-1及其检测方法 |
Also Published As
Publication number | Publication date |
---|---|
BR112021000302A2 (pt) | 2021-04-06 |
CN109868273A (zh) | 2019-06-11 |
MX2021010933A (es) | 2021-10-13 |
ZA202104864B (en) | 2022-05-25 |
AR118492A1 (es) | 2021-10-20 |
CN109868273B (zh) | 2023-02-03 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO2020207125A1 (zh) | 用于检测玉米植物dbn9501的核酸序列及其检测方法 | |
CN112852801B (zh) | 转基因玉米事件lp007-1及其检测方法 | |
WO2016173361A1 (zh) | 玉米植物dbn9936及用于检测其的核酸序列和方法 | |
WO2024060732A1 (zh) | 转基因玉米事件lp026-2及其检测方法 | |
WO2016173362A1 (zh) | 玉米植物dbn9978及用于检测其的核酸序列和方法 | |
CN112852991B (zh) | 转基因玉米事件lp007-7及其检测方法 | |
WO2021026689A1 (zh) | 用于检测大豆植物dbn8007的核酸序列及其检测方法 | |
CN116144817B (zh) | 转基因玉米事件lp026-4及其检测方法 | |
CN109971880B (zh) | 用于检测玉米植物dbn9508的核酸序列及其检测方法 | |
WO2021026688A1 (zh) | 用于检测大豆植物dbn8002的核酸序列及其检测方法 | |
CN113151533B (zh) | 转基因玉米事件lp007-6及其检测方法 | |
CN112831585A (zh) | 转基因玉米事件lp007-4及其检测方法 | |
CN113151534B (zh) | 转基因玉米事件lp007-5及其检测方法 | |
WO2023155193A1 (zh) | 用于检测大豆植物dbn8205的核酸序列及其检测方法 | |
CN116144671A (zh) | 转基因玉米事件lp026-3及其检测方法 | |
CN116144672B (zh) | 转基因玉米事件lp026-1及其检测方法 | |
WO2016173360A1 (zh) | 玉米植物dbn9927及用于检测其的核酸序列和方法 | |
CN113278721A (zh) | 转基因玉米事件lw2-2及其检测方法 | |
CN116640761B (zh) | 转基因玉米事件lp018-1及其检测方法 | |
CN116694626B (zh) | 转基因玉米事件lp035-2及其检测方法 | |
CN116694627B (zh) | 转基因玉米事件lp035-1及其检测方法 | |
CN116694629B (zh) | 转基因玉米事件lp038-1及其检测方法 | |
CN113980958B (zh) | 转基因玉米事件lp007-8及其检测方法 | |
CN116219063A (zh) | 用于检测玉米植物dbn9235的核酸序列及其检测方法 | |
CN116732215A (zh) | 用于检测玉米植物dbn9229的核酸序列及其检测方法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 20786977 Country of ref document: EP Kind code of ref document: A1 |
|
REG | Reference to national code |
Ref country code: BR Ref legal event code: B01A Ref document number: 112021000302 Country of ref document: BR |
|
ENP | Entry into the national phase |
Ref document number: 112021000302 Country of ref document: BR Kind code of ref document: A2 Effective date: 20210108 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
32PN | Ep: public notification in the ep bulletin as address of the adressee cannot be established |
Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205 DATED 04.03.2022) |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 20786977 Country of ref document: EP Kind code of ref document: A1 |