WO2023221554A1 - Herbicide-resistant transgenic corn event ncx-1, nucleic acid sequence and detection method therefor - Google Patents
Herbicide-resistant transgenic corn event ncx-1, nucleic acid sequence and detection method therefor Download PDFInfo
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Classifications
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- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
-
- 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 present invention relates to a herbicide-resistant transgenic corn event and its detection. Specifically, it relates to a herbicide-resistant transgenic corn event nCX-1 and a nucleic acid sequence for detecting whether a biological sample contains a specific transgenic corn event nCX-1. its detection method.
- Corn is one of the most widely grown food crops in the world. It has multiple uses such as food, feed, and energy. It is not only the main food crop, but also the main raw material for animal husbandry and industrial production. The yield and quality of corn play an important role in agricultural production and global economic development. However, during corn planting, weeds compete with corn for nutrients and growth space, affecting corn growth and reducing corn yield. Weed control is an important step in corn production. Using herbicides to control weeds can reduce the impact of weeds on corn growth, stabilize and increase corn yields. By introducing herbicide-resistant genes into corn through genetic engineering methods, obtaining herbicide-resistant transgenic corn can improve the efficiency of corn weed control, reduce production costs, and bring huge economic benefits to agricultural production, thereby achieving positive social and ecological benefits. .
- Crops can gain herbicide tolerance through genetic improvement techniques.
- the cp4 epsps gene derived from Agrobacterium tumefaciens sp strain CP4 strain CP4 is introduced into crops through Agrobacterium-mediated transformation, and crops expressing 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) are obtained.
- EPSPS 5-enolpyruvylshikimate-3-phosphate synthase
- Tolerance to the herbicide glyphosate However, long-term use of a single herbicide often results in the emergence of a large number of resistant weeds. Glyphosate-resistant crops have been promoted in the Americas for more than two decades, and a large number of glyphosate-resistant weeds have been produced. It is difficult to effectively control weeds in the field using glyphosate alone. Breeding genetically modified crops that are tolerant to two or more herbicides can provide diversified options for weed control and can
- Cytochrome P450 is a large gene family. Research has found that some P450 genes can degrade herbicides.
- a P450 gene N-Z1 in bermudagrass (US Patent: US9657303, Canadian Patent: CA2818581C and Brazilian Patent: BR112013012678B1) encodes a protein that can confer tolerance to a variety of herbicides in crops.
- the invention provides a transgenic maize event nCX-1, which simultaneously expresses two proteins, N-Z1 and CP4 EPSPS, and is resistant to pyrosulfuron-methyl, nicosulfuron-methyl, dimethyltetrachloride, 2,4-D and grass. Glyphosate is highly tolerant, genetically stable, and has no adverse effects on agronomic traits.
- a transformation event is a molecular structure composed of exogenous DNA sequences in the upstream and downstream flanking regions of the genome insertion site and exogenous genes.
- exogenous DNA sequences can be randomly inserted into any site on any chromosome of the plant genome, each event obtained is unique.
- the expression of exogenous DNA in plants is affected by the chromosomal position where the exogenous DNA is inserted.
- the position of exogenous DNA on the chromosome is different, and there will be huge differences in the expression level, expression space and time pattern of exogenous DNA, and thus the impact on the agronomic traits of plants will also be different.
- Different transformation events obtained by transformation of the same exogenous gene often have huge differences in traits.
- transformation events whose expression level, expression pattern and functional traits of the target gene can meet the needs of production and application.
- the ideal transformation event obtained through screening can be transgeneed into other genetic backgrounds using conventional breeding methods of sexual hybridization, and the offspring produced through hybridization can maintain the transgenic characteristics of the original event.
- the invention provides a transgenic corn event with single copy insertion of exogenous genes, good inheritance stability, high resistance to pyrosulfuron-methyl, nicosulfuron-methyl, dimethyltetrachloride, 2,4-D and glyphosate and other excellent traits.
- the present invention can accurately and quickly identify whether the biological sample contains the transgenic corn event nCX-1. DNA molecules of nCX-1.
- the present invention provides a herbicide-resistant transgenic maize event nCX-1.
- the transgenic maize event nCX-1 is an exogenous DNA molecule (i.e., T-DNA) inserted into chromosome 7 of the maize genome SEQ ID NO.
- T-DNA exogenous DNA molecule
- the DNA molecule obtained between the 3' end of 22 and the 5' end of SEQ ID NO.
- the corn genome nucleic acid sequence is derived from the database Maize Genetics and Genomics Database (Zm-B73-REFERENCE-NAM-5.0);
- the Exogenous DNA molecules include N-Z1 gene expression cassette and cp4 epsps gene expression cassette;
- the N-Z1 gene expression cassette includes: Actin promoter used for N-Z1 gene initiation, N-Z1 gene coding frame, -CaMV35S terminator that terminates the Z1 gene;
- the cp4 epsps gene expression cassette includes: the ZmUbi promoter derived from the maize polyubiquitin-1 gene used to initiate the cp4 epsps gene, the cp4 epsps gene coding frame, and the CaMV35S that terminates the cp4 epsps gene terminator.
- nucleic acid sequence of the transgenic maize event nCX-1 is shown in SEQ ID NO.7.
- the herbicide includes one or more of glyphosate, penosulfuron-methyl, nicosulfuron-methyl, dimethyltetrachloride or 2,4-D.
- the transgenic maize event nCX-1 of the present invention is deposited in the China Typical Culture Collection Center in the form of maize (Zea mays L.) nCX-1 seeds, with the preservation number: CCTCC NO.P202213, and the preservation date is April 20, 2022. Address: Wuhan University, Wuhan, China, Postal Code 430072.
- the genome database referenced by the transgenic maize event nCX-1 described in the present invention comes from Maize Genetics and Genomics Database (Zm-B73-REFERENCE-NAM-5.0).
- Maize Genetics and Genomics Database Zm-B73-REFERENCE-NAM-5.0.
- researchers in this field know that there are a large number of active transposon sequences in the maize genome, and there may be sequence position shifts in the maize genome under different genetic backgrounds.
- researchers in the field can obtain the progeny of the present invention through hybridization and other methods.
- the corn event in which the flanking sequences of the exogenous T-DNA in the genome of any event are SEQ ID NO. 22 and SEQ ID NO. 23 should be regarded as the present invention. content of the invention.
- the present invention provides a nucleic acid sequence for detecting the transgenic maize event nCX-1, the nucleic acid sequence Includes SEQ ID NO. 1 or its complement and/or SEQ ID NO. 2 or its complement.
- the SEQ ID NO.1 or its complementary sequence is a 26-nucleotide sequence of transgenic maize event nCX-1 located at the insertion junction at the 5' end of the inserted sequence, which in turn includes 13 nuclei from maize genes. and 13 nucleotides from the inserted T-DNA. Therefore, the SEQ ID NO.1 or its complementary sequence spans the flanking genomic DNA sequence of the insertion site of the foreign DNA molecule in the transgenic maize event nCX-1 And the 5' end sequence of the exogenous DNA molecule, including the SEQ ID NO. 1 or its complementary sequence, can be identified as the presence of the transgenic maize event nCX-1.
- the SEQ ID NO.2 or its complementary sequence is a 26-nucleotide sequence of transgenic maize event nCX-1 located at the insertion junction at the 3' end of the insertion sequence, which in turn includes 13 T-s from the insertion. DNA nucleotides and 13 nucleotides from the maize gene. Therefore, the SEQ ID NO.2 or its complementary sequence spans the exogenous DNA molecule at the insertion site of the exogenous DNA molecule in the transgenic maize event nCX-1.
- the 3' end sequence and flanking genomic DNA sequence, including the SEQ ID NO. 2 or its complementary sequence, can be identified as the presence of transgenic maize event nCX-1.
- nucleic acid sequence provided by the invention also includes SEQ ID NO. 3 or its complementary sequence, and/or SEQ ID NO. 4 or its complementary sequence.
- the SEQ ID NO.3 or its complementary sequence is a 362-nucleotide sequence of transgenic maize event nCX-1 located at the insertion junction region at the 5' end of the inserted sequence, in which base 1 of SEQ ID NO.3 -225bp is the flanking maize genomic DNA sequence inserted near the binding site, and base 226-362bp is the 5' end sequence of the nucleotides of the T-DNA inserted near the binding site, including the SEQ ID NO.3 or its complementary sequence This can identify the presence of transgenic maize event nCX-1.
- the SEQ ID NO.4 or its complementary sequence is a 494bp nucleotide sequence in the insertion junction region at the 3' end of the inserted sequence of the transgenic maize event nCX-1
- the SEQ ID NO.4 No. 1- 48bp is the 3' end sequence of the nucleotide of the T-DNA inserted near the binding site
- 49-494bp is the flanking maize genomic DNA sequence inserted near the binding site, which can be identified if it contains the SEQ ID NO.4 or its complementary sequence for the presence of transgenic maize event nCX-1.
- nucleic acid sequence provided by the invention also includes SEQ ID NO. 5 or its complementary sequence, and/or SEQ ID NO. 6 or its complementary sequence.
- the SEQ ID NO.5 or its complementary sequence is a 957-nucleotide sequence of transgenic maize event nCX-1 located at the 5' end of the inserted sequence in the insertion junction region, in which base 1 of SEQ ID NO.5 -729bp is the flanking maize genomic DNA sequence inserted near the binding site, and bases 730-957bp is the 5' end sequence of the nucleotides of the T-DNA inserted near the binding site, including the SEQ ID NO.5 or its complementary sequence This can identify the presence of transgenic maize event nCX-1.
- the SEQ ID NO.6 or its complementary sequence is a 946bp nucleotide sequence in the insertion junction region at the 3' end of the inserted sequence of the transgenic maize event nCX-1, and the SEQ ID NO.6 No. 1- 265bp is the 3' end sequence of the nucleotide of the T-DNA inserted near the binding site, and 266-946bp is the flanking maize genomic DNA sequence inserted near the binding site. It can be identified if it contains the SEQ ID NO.6 or its complementary sequence. for the presence of transgenic maize event nCX-1.
- nucleic acid sequence provided by the invention includes SEQ ID NO. 7 or its complementary sequence.
- the SEQ ID NO.7 or its complementary sequence is a sequence of 8695 nucleotides unique to the transgenic maize event nCX-1, which includes the entire T-DNA sequence and the flanking maize genes at its 5' and 3' ends. type sequence. The specific genome and genetic elements included are shown in Table 1. The presence of the transgenic maize event nCX-1 can be identified by including the SEQ ID NO.7 or its complementary sequence.
- the present invention provides a continuous nucleotide sequence unique to the transgenic maize event nCX-1.
- the continuous nucleotide sequence can be used to characterize the transgenic maize event nCX-1, and thus can be used to detect whether the transgenic maize event nCX-1 exists in a sample. Specifically, the presence of at least 13 consecutive nucleotides in the nucleic acid molecules represented by one or more of SEQ ID NO. 1-7 in the sample indicates the presence of transgenic corn event nCX-1 in the sample.
- SEQ ID NO.1 or its complement and/or SEQ ID NO.2 or its complement in a sample indicates the presence of transgenic maize event nCX-1 in the sample.
- SEQ ID NO.3 or its complementary sequence and/or SEQ ID NO.4 or its complementary sequence in the sample indicates the presence of transgenic maize event nCX-1 in the sample.
- SEQ ID NO.5 or its complementary sequence and/or SEQ ID NO.6 or its complementary sequence in the sample indicates the presence of transgenic maize event nCX-1 in the sample.
- SEQ ID NO.7 or its complementary sequence in the sample indicates the presence of transgenic maize event nCX-1 in the sample.
- the presence of SEQ ID NO.1 or its complementary sequence and SEQ ID NO.4 or its complementary sequence in the sample can also indicate the presence of transgenic maize event nCX-1 in the sample
- the presence of SEQ ID NO.1 or its complementary sequence and SEQ ID NO.6 or its complementary sequence in the sample can also indicate the presence of transgenic corn event nCX-1 in the sample, or, the presence of SEQ ID NO.2 or its complementary sequence in the sample
- the presence of its complementary sequence and SEQ ID NO.3 or its complementary sequence can also indicate the presence of transgenic maize event nCX-1 in the sample; alternatively, the presence of SEQ ID NO.2 or its complementary sequence and SEQ ID NO.5 or its complementary sequence in the sample
- the presence of complementary sequences can also indicate the presence of transgenic maize event nCX-1 in the sample; alternatively, the presence of SEQ ID NO.3 or its complement and SEQ ID NO.6 or its complement in the sample
- the present invention provides a method for detecting the presence of DNA molecules of transgenic maize event nCX-1 in a sample, including: (1) adding the sample to be detected with a first primer and a second primer in a nucleic acid amplification reaction solution Contact; the first primer is one of SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12 or SEQ ID NO.14; the second primer is SEQ ID NO.9, SEQ ID One of NO.11, SEQ ID NO.13 or SEQ ID NO.15; (2) perform a nucleic acid amplification reaction; (3) detect the presence of amplification products; the amplification products include SEQ ID NO.1 or its complement, SEQ ID NO.2 or its complement.
- the amplification product includes at least 13 consecutive nucleotides in SEQ ID NO.3 or its complementary sequence, and/or at least 13 consecutive nucleotides in SEQ ID NO.4 or its complementary sequence.
- the amplification product includes at least 13 consecutive nucleotides in SEQ ID NO.5 or its complementary sequence, and/or at least 13 consecutive nucleotides in SEQ ID NO.6 or its complementary sequence. .
- the amplification product includes SEQ ID NO.1 or its complementary sequence, SEQ ID NO.2 or its complementary sequence, SEQ ID NO.3 or its complementary sequence, SEQ ID NO.4 or its complementary sequence, SEQ ID NO. .5 or its complementary sequence, SEQ ID NO.6 or its complementary sequence, and/or at least 13 consecutive nucleotides in SEQ ID NO.7 or its complementary sequence, can be identified as the transgenic maize event nCX-1 exist.
- the primer includes at least one of the nucleotide sequences.
- the first primers are respectively composed of SEQ ID NO.3 (primer name: RB-F1, corresponding number: SEQ ID NO.8), SEQ ID NO.4 (primer name: LB-F1, corresponding number: SEQ ID NO.10), SEQ ID NO.5 (primer name: RB-F2, corresponding number: SEQ ID NO.12) and SEQ ID NO.6 (primer name: LB-F2, corresponding number: SEQ ID NO.14 ) designed;
- the second primers are respectively composed of SEQ ID NO.3 (Primer name: RB-R1, corresponding number: SEQ ID NO.9), SEQ ID NO.4 (Primer name: LB-R1, corresponding number: SEQ ID NO.11), SEQ ID NO.5 ( Primer name: RB-R2, corresponding number: SEQ ID NO.13) and SEQ ID NO.6 (primer name: LB-R2, corresponding number:
- the present invention also provides a method for cultivating herbicide-resistant corn plants containing the transgenic corn event nCX-1.
- the method includes: planting corn seeds containing a specific region of nucleic acid sequence, so that the corn Grow into corn plants, spray the corn plants with herbicides, and harvest plants with significantly improved herbicide tolerance compared with other corn plants that do not contain the nucleic acid sequence of the specific region; the nucleic acid sequence of the specific region is from transgenic corn Event nCX-1, the nucleic acid sequence of the specific region includes SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or SEQ One of the nucleotide sequences shown in ID NO.7 or its complementary sequence; the herbicide is glyphosate, pyrosulfuron-methyl, nicosulfuron-methyl, dimethyltetrachloride or 2,4-D isooctyl One or more of the esters (2
- the present invention also provides a method for obtaining herbicide-resistant corn plants containing the transgenic corn event nCX-1.
- the method includes combining a corn plant containing a specific region nucleic acid sequence with another corn plant.
- the plants are hybridized to produce progeny plants; the tolerance to herbicides is significantly improved compared with other plants that do not contain the nucleic acid sequence of the specific region; the nucleic acid sequence of the specific region is from the transgenic maize event nCX-1, so
- the nucleic acid sequence of the specific region includes SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO.7.
- the herbicide is one or more of glyphosate, pyrosulfuron-methyl, nicosulfuron-methyl, dimethyltetrachloride, and 2,4-D.
- the present invention provides a method for controlling weeds in transgenic corn fields containing the transgenic corn event nCX-1.
- the method includes spraying herbicides into fields where transgenic corns are planted, and the weeds in the corn fields are covered with Kill;
- the transgenic corn genome contains a specific region nucleic acid sequence from the transgenic corn event nCX-1, and the specific region nucleic acid sequence includes SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID One of the nucleotide sequences shown in NO.4, SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO.7 or its complementary sequence;
- the herbicides are glyphosate, penisulfuron-methyl, One or more of nicosulfuron, dimethyltetrachloride, and 2,4-D.
- the present invention also provides an agricultural product or commodity produced from the transgenic corn event nCX-1.
- the agricultural product or commodity includes corn flour, corn flour, corn oil, corn starch, corn gluten, corn tortillas, and corn-containing ingredients. Cosmetics, or excipients containing corn ingredients.
- the transgenic maize event nCX-1 of the present invention includes a DNA construct. When expressed in plant cells, the transgenic maize event nCX-1 obtains p-cisulfuron-methyl, nicosulfuron-methyl, dimethyltetrachloride, 2 , 4-D and glyphosate herbicide tolerance.
- the T-DNA construct contains two expression cassettes in series, the first expression cassette containing a suitable promoter for expressing N-Z1 protein in plants and a suitable terminator, the promoter operably linked to N -The nucleotide sequence of the Z1 protein, the N-Z1 protein is tolerant to azosulfuron-methyl, nicosulfuron-methyl, dimethyltetrachloride, and 2,4-D herbicides, and the promoter includes actin Protein (Actin) promoter, the terminator includes the cauliflower mosaic virus (CaMV) 35S terminator.
- Actin actin Protein
- the second expression cassette contains a suitable promoter for expression of the EPSPS protein in plants, operably linked to a promoter encoding 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) gene, the EPSPS protein is tolerant to glyphosate herbicide, the promoter includes the maize polyubiquitin-1 gene promoter (ZmUbi), and the terminator includes the CaMV 35S terminator.
- EPSPS 5-enol-pyruvylshikimate-3-phosphate synthase
- the DNA construct is introduced into plants using transformation methods, including Agrobacterium-mediated transformation, gene gun transformation and pollen tube channel transformation.
- SEQ ID NO.1 represents 13 nucleotides on each side of the flanking maize genome and 5'-end transgene insertion site in the transgenic maize event nCX-1;
- SEQ ID NO.2 represents the 3’-end transgene insertion site and 13 nucleotides on each side of the flanking maize genome in the transgenic maize event nCX-1;
- SEQ ID NO.3 represents the 362 nucleotides near the junction region of the 5'-end transgene insertion site in the transgenic maize event nCX-1 sequence;
- SEQ ID NO.4 represents the 494 nucleotide sequence near the junction region of the 3'-end transgene insertion site in the transgenic maize event nCX-1;
- SEQ ID NO.5 represents the 957 nucleotide sequence near the junction region of the 5'-end transgene insertion site in the transgenic maize event nCX-1;
- SEQ ID NO.6 represents the 946 nucleotide sequence near the junction region of the 3'-end transgene insertion site in the transgenic maize event nCX-1;
- SEQ ID NO.7 represents the transgenic maize event nCX-1 insertion T-DNA sequence and the maize genome sequence at its 5’ end and 3’ end;
- SEQ ID NO.8 represents the first primer (RB-F1) for detecting SEQ ID NO.3;
- SEQ ID NO.9 represents the second primer (RB-R1) for detecting SEQ ID NO.3;
- SEQ ID NO.10 represents the first primer (LB-F1) for detecting SEQ ID NO.4;
- SEQ ID NO.11 represents the second primer (LB-R1) for detecting SEQ ID NO.4;
- SEQ ID NO.12 represents the first primer (RB-F2) for detecting SEQ ID NO.5;
- SEQ ID NO.13 represents the second primer (RB-R2) for detecting SEQ ID NO.5;
- SEQ ID NO.14 represents the first primer (LB-F2) for detecting SEQ ID NO.6;
- SEQ ID NO.15 represents the second primer (LB-R2) for detecting SEQ ID NO.6.
- the transgenic corn event nCX-1 of the present invention expresses two proteins, N-Z1 and CP4 EPSPS, and is highly resistant to azosulfuron-methyl, nicosulfuron-methyl, dimethyltetrachloride, 2,4-D and glyphosate herbicides.
- one or more herbicides such as glyphosate, pyrosulfuron-methyl, nicosulfuron-methyl, dimethyltetrachloride, and 2,4-D can be used for transgenic screening and planting of transgenic corn.
- one or more herbicides such as glyphosate, penosulfuron-methyl, nicosulfuron-methyl, dimethyltetrachloride, and 2,4-D can be used for weed control.
- the present invention provides a herbicide-resistant transgenic corn event nCX-1, which inserts a single copy of the target gene N-Z1 and cp4epsps into a specific site of the corn genome, so that the transgenic corn event nCX-1 contains Different generations and corn materials containing the transgenic corn event nCX-1 have stable integration, stable expression, and stable resistance to herbicides.
- the transgenic corn event nCX-1 is deposited in the China Typical Culture Collection Center in the form of corn seeds, with the preservation number: CCTCC NO.P202213. D and glyphosate tolerance.
- the nucleotide sequence for specifically detecting the transgenic maize event nCX-1 provided by the invention is SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO. 5.
- SEQ ID NO.6 or SEQ ID NO.7 can specifically detect the transgenic corn event nCX-1.
- the transgenic corn event nCX-1 of the present invention can use one or more of glyphosate, pyrosulfuron-methyl, nicosulfuron-methyl, dimethyltetrachloride, and 2,4-D during the cross-breeding process.
- Herbicides are used for transgenic screening.
- one or more herbicides such as glyphosate, pyrosulfuron-methyl, nicosulfuron-methyl, dimethyltetrachloride, and 2,4-D can be used for weed control. , effectively improve weed control efficiency, reduce the risk of herbicide-resistant weeds, and reduce weed control costs.
- Figure 1 shows the transformation vector map
- Figure 2 shows the electrophoresis diagram of hiTAIL-PCR amplification product.
- Figure 3 is a schematic diagram of the exogenous inserted gene and the maize genome structure.
- Figure 4 shows the electrophoresis chart of maize genome-specific detection.
- M Marker; 1: Blank control (no genome added); 2: nCX-1; 3: Conventional corn Zhengdan 958; 4: Transgenic corn mixed sample (Ruifeng 125, Zhejiang University Ruifeng 8); 5: Conventional soybean day Long No. 1; 6: Genetically modified soybean mixed sample (Zhonghuang 6106, SHZD3201); 7: Conventional rice Xiushui 134; 8: Genetically modified insect-resistant cotton (GHB614, COT102).
- Figure 5 shows the electrophoresis pattern of nCX-1 5’ end specific detection.
- M Marker; 1: nCX-1; 2: Conventional corn Zhengdan 958; 3: Genetically modified corn mixed sample (Ruifeng 125, Zhejiang University Ruifeng 8); 4: Conventional soybean Tianlong No. 1; 5: Genetically modified soybean mixed sample (Zhonghuang 6106, SHZD3201); 6: conventional rice Xiushui 134; 7: transgenic insect-resistant cotton (GHB614, COT102).
- Figure 6 shows the electrophoresis pattern of nCX-1 3’ end specific detection.
- 1 Conventional corn Zhengdan 958; 2: Conventional soybean Tianlong No. 1; 3: Conventional rice Xiushui 134; 4: nCX-1; M: Marker; 5: Genetically modified corn mixed sample (Ruifeng 125, Zhejiang University Ruifeng 8) ; 6: Genetically modified soybean mixed sample (Zhonghuang 6106, SHZD3201); 7: Genetically modified insect-resistant cotton (GHB614, COT102).
- Figure 7 is a picture of the tolerance of nCX-1 and control to glyphosate.
- Figure 8 is a picture of the tolerance of nCX-1 and control to rimisulfuron.
- Figure 9 is a picture of the tolerance of nCX-1 and the control to glyphosate + dimethyltetrachloride.
- Figure 10 is a picture of the tolerance of nCX-1 and control to glyphosate + 2,4-D isooctyl ester (2,4-D).
- Figure 11 is a picture of the tolerance of nCX-1 and control to glyphosate + nicosulfuron.
- Corn refers to maize (Zea mays L.), including all plant varieties that mate with corn, including wild corn species.
- the “including” is synonymous with “including” and “containing”, and means “including but not limited to”.
- the plant includes whole plants, plant cells, plant organs, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant callus, plant clumps and intact plant cells in plants or plant parts, said plants Parts include embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, stems, roots, root tips, anthers, etc.
- Said transgenic plant is derived from a transgenic plant transformed with the DNA molecule of the invention and thus consisting at least partly of transgenic cells or its progeny.
- the transgenic "event” is obtained by transforming plant cells with a construct of exogenous DNA (for example, including at least one nucleic acid expression cassette containing the target gene), which is inserted into the plant genome by a transgenic method to generate a plant population and regenerate The plant population, and selection of specific plants characterized by insertion of specific genomic loci.
- the term "event” refers to the original event that includes exogenous DNA and/or the descendants of that event.
- the term “event” also refers to the offspring resulting from a sexual cross between an event and individuals of other breeds containing foreign DNA. Even after repeated backcrossing to the backcross parent, the inserted DNA and flanking genomic DNA from the event parent remain unchanged. The same chromosomal location present in the hybrid offspring.
- event also refers to a DNA sequence from an original event that contains the inserted DNA and flanking genomic sequences in close proximity to the inserted DNA, which DNA sequence is expected to be transferred to progeny consisting of the progeny containing the inserted DNA.
- a parent line eg, the original event and its self-produced progeny
- a parent line is produced by sexual crossing with a parent line that does not contain the inserted DNA, and the progeny receives the inserted DNA containing the gene of interest.
- transgene includes any cell, cell line, callus, tissue, plant part or plant whose genotype is altered by the presence of exogenous nucleic acid, and the term “transgene” includes a transgene originally so altered and Offspring individuals produced from the original transgenic body through sexual crossing or asexual reproduction.
- the term “transgene” does not include changes in the genome (chromosomal or extrachromosomal) by conventional plant breeding methods or naturally occurring events, such as those that occur with Allogeneic fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition or spontaneous mutation.
- the "transgenic corn event nCX-1" is a DNA molecule obtained by inserting an exogenous DNA molecule between chr7:176570691-176570736bp of the corn genome, and also includes plant nCX-1, seeds and plants containing the transgenic corn event nCX-1 Cells or renewable parts thereof, including but not limited to cells, pollen, ovules, flowers, shoots, roots, stems, silks, catkins, ears, leaves and products from the corn plant nCX-1, such as Zea mays Meal, cornmeal, corn steep liquor, corn silk, cornstarch and biomass left in the corn crop field.
- the "primer” is an isolated nucleic acid molecule that anneals and binds to a complementary target DNA strand through nucleic acid hybridization, forming a hybrid between the primer and the target DNA strand, and then reacts with a polymerase (such as DNA polymerase) Under the influence, it extends along the target DNA strand.
- the primer pairs of the present invention relate to their use in amplification of target nucleic acid sequences, for example, by polymerase chain reaction (PCR) or other conventional nucleic acid amplification methods.
- Example 1 Obtaining plasmid vectors containing foreign genes
- the vector map of the present invention for maize transformation is shown in Figure 1.
- the transformation plasmid vector uses pCambia1300 (GenBank: AF234296.1) as the plant transformation vector framework, and in its multiple cloning site region is added an expression box containing the complete expression CP4 EPSPS protein and
- the T-DNA that expresses the N-Z1 protein expression cassette specifically consists of the following parts. It expresses the CP4 EPSPS protein expression cassette: CP4EPSPS.
- the promoter driving CP4 EPSPS is derived from the maize polyubiquitin-1 gene promoter (pZmUbi-1).
- the promoter is the 35S gene terminator of CaMV; the N-Z1 protein expression cassette is: N-Z1, the promoter driving N-Z1 is the Actin promoter, the terminator is the 35S gene terminator of CaMV, and the T-DNA sequence is SEQ ID Nucleotides 730-8014 in NO.7.
- the obtained transformation plasmid was introduced into Agrobacterium LBA4404 using electroporation method (2500V) to obtain Agrobacterium containing the transformation vector.
- Example 2 Obtaining plant nCX-1 containing transgenic maize event nCX-1
- Agrobacterium-mediated genetic transformation of corn was carried out, specifically according to the method and culture medium formula reported by Frame et al. (Plant Physiol, 2002, 129:13-22). Glyphosate was used as the screening reagent. The steps were as follows: Take pollination After 8 to 10 days of corn ears, immature embryos with a size of 1.0-1.5mm are collected. Mix the Agrobacterium containing the transformation vector into the infection medium, and adjust the bacterial concentration OD660 to 0.5-0.6. Put the collected immature embryos into the infection solution containing Agrobacterium and let it stand at room temperature for 5 minutes.
- the cultured immature embryos were transferred to callus induction medium containing a final concentration of 200 mg/L timentin antibiotic (GlaxoSmithKline, USA), and cultured in the dark at 28°C for 10-14 days to kill Agrobacterium. All calli after induction culture were transferred to the selection medium containing a final concentration of 2mM glyphosate and cultured in the dark at 28°C for 2-3 weeks. After induction culture, all calli were transferred to fresh selection medium containing 2mM glyphosate and cultured in the dark at 28°C for 2-3 weeks.
- T 0 generation events obtained in Example 2 were transplanted into the greenhouse after hardening, and 801 seedlings survived transplantation in the greenhouse.
- spray glyphosate and penisulfuron-methyl compound herbicides (the effective dose of glyphosate is 60g/acre; the effective dose of penisulfuron-methyl is 0.8g/acre) ), there were 110 incidents without phytotoxicity, 640 incidents with phytotoxicity, and 100 incidents of death (Table 2).
- Quantitative PCR testing was performed on events without phytotoxicity, and the content of foreign genes in 110 surviving events was measured to evaluate the insertion copy number of T-DNA, and events with two or more copies were discarded. Take the plants from the above events and extract the plant genome using CTAB method. The copy number of the cp4 epsps gene was detected by the SYBR Green fluorescence quantitative PCR method to determine the copy number of the foreign gene. Select zSSIIb in the corn genome as the internal reference gene, randomly select a corn event as the benchmark, and calculate the relative content of the target gene at the initial stage of the reaction.
- the SYBR Green fluorescence quantitative PCR kit (BIO RAD) was used to perform the reaction in the Bio-Rad Rad CFX96 TM Real-Time PCR instrument, and the results were analyzed using the Ct value comparison method.
- the system and procedures refer to the instructions of the SYBR Green fluorescence quantitative PCR kit.
- the primer sequences are as follows:
- nCX-1 to nCX-7 The expression levels of 7 events (denoted as nCX-1 to nCX-7) with good herbicide tolerance were measured.
- the CP4 EPSPS enzyme-linked immunoassay quantitative detection kit used in this study was purchased from Shanghai Youlong Biotechnology Co., Ltd., and the N-Z1 kit was purchased from Zhongding Biotech’s protein-specific ELISA detection kit.
- a standard curve can be drawn based on the OD 450 value of the standard sample. In order to eliminate systematic errors between each measurement, a standard curve is produced simultaneously for each sample measurement.
- washing solution Dilute the concentrated washing solution in the kit with double-distilled water at a volume ratio of 1:19.
- Biotinylated antibody Add 20 ⁇ L of double-distilled water to the biotinylated antibody. After it is fully dissolved, take 1 ⁇ L of biotinylated antibody and dilute it with 10 mL of sample diluent, that is, 1:10000 dilution.
- Streptavidin-HRP Take 1 ⁇ L Streptavidin-HRP and dilute it with 10mL sample diluent, that is, 1:10000 dilution.
- TMB substrate solution
- a standard curve can be drawn based on the OD 450 value of the standard sample. In order to eliminate the systematic error between each measurement, a standard curve is produced simultaneously for each sample measurement.
- CTAB cetyltrimethylammonium bromide
- CTAB buffer (20 g/L CTAB, 1.4 M NaCl, 100 mM Tris-HCl) preheated in a 65°C water bath. , 20mM EDTA, pH 8.0), mix thoroughly, and then bathe in a 65°C water bath for 60 minutes;
- TE buffer 10mM Tris-HCl, 1mM EDTA, pH 8.0
- Measure the DNA concentration with Nanodrop and store it for later use.
- the third round of PCR amplification products were recovered using Axygen's PCR product recovery kit (Figure 2), connected to the PMD19-T cloning vector (TaKaRa, Code: D102A), transformed into E. coli, and the obtained positive clones were Kang Biotechnology has Co., Ltd. for sequencing.
- the obtained sequence information was compared and analyzed with the maize online database (http://www.maizegdb.org) to retrieve similar maize genome sequences.
- the fragment identified as containing the 5' flanking region obtained by the hiTAIL-PCR method was sequenced.
- the sequencing result was SEQ ID NO.5.
- the 1-729 bp sequence corresponds to the maize genomic DNA, and the 730-957 bp sequence corresponds to the foreign genome DNA. Source DNA.
- the fragment identified as containing the 3' flanking region was sequenced, and the sequencing result was SEQ ID NO. 6. 1-265 bp were the nucleotide sequence of the inserted gene, and 266-946 bp were the nucleotide sequence of the maize flanking genome.
- nCX-1 integrates into genome sequence information
- the above sequenced, aligned and verified upstream and downstream flanking sequences of the insertion site and the herbicide-resistant gene expression cassette sequence are spliced to form this version.
- the nucleotide sequence of the transgenic maize event nCX-1 according to the invention is SEQ ID NO. 7, and the gene structure diagram is shown in Figure 3.
- the corresponding transgenic maize event nCX-1 is deposited in the China Type Culture Collection Center in the form of maize (Zea mays L.) nCX-1 seeds, with the collection number: CCTCC NO.P202213.
- the 5' and 3' flanking sequences of the transgenic corn event nCX-1 are shown in nucleotides 1-729 and 8015-8695 of SEQ ID NO.7 respectively.
- the PCR reaction program was: denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, a total of 35 cycles, and a final extension at 72°C for 7 min.
- the internal reference primers zSSIIb-1F (SEQ ID NO.16) and zSSIIb-1R (SEQ ID NO.17) were used to test the quality of the above genome, and the genome without addition was used as a blank control.
- the primer pair RB-F1 (SEQ ID NO.8) and RB-R1 (SEQ ID NO.9) were used to target nCX-1, conventional corn (Zhengdan 958), and transgenic corn mixed sample (Ruifeng 125, Zhejiang University Ruifeng 8), conventional soybeans (Tianlong 1), genetically modified soybean mixed samples (Zhonghuang 6106, SHZD3201), conventional rice Xiushui 134 and genetically modified insect-resistant cotton (GHB614 and COT102) were used for nucleic acid specific detection, and the amplification products
- the electrophoresis results showed that only the nCX-1 sample could detect a band of approximately 360 bp, and the band size was consistent with expectations. Other samples that did not contain the nCX-1 genome could not detect specific bands.
- the primer pair provided by the invention The presence of nCX-1 can be specifically detected ( Figure 5).
- the primer pair LB-F1 (SEQ ID NO. 10) and LB-R1 (SEQ ID NO. 11) were used to target nCX-1, conventional corn (Zhengdan 958), and transgenic corn mixed sample (Ruifeng 125, Zhejiang University Ruifeng 8), conventional soybean Tianlong No. 1, genetically modified soybean mixed sample (Zhonghuang 6106, SHZD3201), conventional rice (Xiushui 134) and genetically modified insect-resistant cotton (GHB614 and COT102) PCR amplification was performed, and the electrophoresis results of the amplification products showed that only the nCX-1 sample could detect a band of about 500 bp, and the band size was consistent with expectations (Figure 6).
- the primer pair provided by the present invention can specifically detect the presence of nCX-1. Therefore, the test primer pair provided by the present invention can specifically detect the presence of nCX-1-containing samples.
- the seeds of the transgenic corn event nCX-1 in Example 4 and the common corn Zhengdan 958 (as a control) were used in a random block design, repeated three times, with a total of 24 plots, each plot area 4m ⁇ 6m, and sown with double seeds.
- the spacing between plants is 25cm
- the spacing between rows is 50cm
- intervals of 1m are set between plots.
- Spray azosulfuron-methyl at the 3-5 leaf stage, and the treatments are as follows: 1) No spraying; 2) Spray azosulfuron-methyl at an effective dose of 1 g/mu; 3) Spray an effective dose of 2 g/mu of imisulfuron-methyl; 4) Spray an effective dose of 4 g/mu of imisulfuron-methyl.
- the phytotoxicity symptoms are graded according to GB/T 17980.42 -2000 execution.
- X damage rate, unit is percentage (%); N: number of damaged plants at a certain level; S: level value; Z: total number of plants; M is the highest level.
- the variance analysis method was used to compare the differences in seedling emergence rate, seedling establishment rate and damage rate between transgenic herbicide-resistant corn and non-transgenic corn under different treatments. Determine the herbicide tolerance level of transgenic herbicide-resistant corn.
- the seeds of the transgenic corn event nCX-1 in Example 4 and the common corn Zhengdan 958 (as a control) were used in a random block design, repeated three times, with a total of 24 plots, each plot area 4m ⁇ 6m, and sown with double seeds.
- the spacing between plants is 25cm, the spacing between rows is 50cm, and there are 1m intervals between plots.
- the treatments are as follows: 1) No spraying; 2) Spray a medium dose of glyphosate, with an effective dose of 60 g/mu.
- X damage rate, unit is percentage (%); N: number of damaged plants at a certain level; S: level value; Z: total number of plants; high-level.
- the variance analysis method was used to compare the differences in seedling emergence rate, seedling establishment rate and damage rate between transgenic herbicide-resistant corn and non-transgenic corn under different treatments. Determine the herbicide tolerance level of transgenic herbicide-resistant corn.
- Example 8 nCX-1 glyphosate and dimethyltetrachloride compound herbicide tolerance test
- the seeds of the transgenic corn event nCX-1 in Example 4 and the common corn Zhengdan 958 (as a control) were used in a random block design, repeated three times, with a total of 24 plots, each plot area 4m ⁇ 6m, and sown with double seeds.
- the spacing between plants is 25cm, the spacing between rows is 50cm, and there are 1m intervals between plots.
- the treatments are as follows: 1) No spraying; 2) Spray an effective dose of 60g/ Glyphosate per mu + effective dose of 50 g/mu of methylene chloride; 3) Spray effective dose of 120 g/mu of glyphosate + effective dose of 100 g/mu of methylene chloride; 4) Spray effective dose of 240 g/mu Mu glyphosate + effective dose 200 grams/mu dimethyltetrachloride. Observe the seedling establishment rate, plant height (select the 5 highest plants), and phytotoxicity symptoms (select the 5 plants with the mildest phytotoxicity symptoms) 1 week, 2 weeks, and 4 weeks after treatment. The phytotoxicity symptoms are graded according to GB/T 17980.42 -2000 execution.
- X damage rate, unit is percentage (%); N: number of damaged plants at a certain level; S: level value; Z: total number of plants; M is the highest level.
- the variance analysis method was used to compare the differences in seedling emergence rate, seedling establishment rate and damage rate between transgenic herbicide-resistant corn and non-transgenic corn under different treatments. Determine the herbicide tolerance level of transgenic herbicide-resistant corn.
- the seeds of the transgenic corn event nCX-1 in Example 4 and the common corn Zhengdan 958 (as a control) were used in a random block design, repeated three times, with a total of 24 plots, each plot area 4m ⁇ 6m, and sown with double seeds.
- the spacing between plants is 25cm, the spacing between rows is 50cm, and there are 1m intervals between plots.
- the treatments are as follows: 1) No spraying; 2) Spray an effective dose of 60 grams.
- X damage rate, unit is percentage (%); N: number of damaged plants at a certain level; S: level value; Z: total number of plants; M is the highest level.
- the variance analysis method was used to compare the differences in seedling emergence rate, seedling establishment rate and damage rate between transgenic herbicide-resistant corn and non-transgenic corn under different treatments. Determine the herbicide tolerance level of transgenic herbicide-resistant corn. Common corn (Zhengdan 958) was used as a control.
- Example 10 nCX-1 glyphosate and nicosulfuron compound herbicide tolerance test
- the seeds of the transgenic corn event nCX-1 in Example 4 and the common corn Zhengdan 958 (as a control) were used in a random block design, repeated three times, with a total of 24 plots, each plot area 4m ⁇ 6m, and sown with double seeds.
- the spacing between plants is 25cm, the spacing between rows is 50cm, and there are 1m intervals between plots.
- the treatments are as follows: 1) No spraying; 2) Spray Apply an effective dose of 60 g/mu glyphosate + an effective dose of 1.6 g/mu nicosulfuron; 3) Spray an effective dose of 120 g/mu glyphosate + an effective dose of 3.2 g/mu nicosulfuron; 4) Spray Apply an effective dose of 240 g/mu of glyphosate + an effective dose of 6.4 g/mu of nicosulfuron.
- 1 week, 2 weeks, and 4 weeks after treatment the seedling establishment rate, plant height (select the 5 plants with the highest symptoms), and phytotoxicity symptoms (select the 5 plants with the mildest phytotoxicity symptoms) were investigated.
- the phytotoxicity symptoms were graded according to GB/T 17980.42. -2000 execution.
- X damage rate, unit is percentage (%); N: number of damaged plants at a certain level; S: level value; Z: total number of plants; M is the highest level.
- the variance analysis method was used to compare the differences in seedling emergence rate, seedling establishment rate and damage rate between transgenic herbicide-resistant corn and non-transgenic corn under different treatments. Determine the herbicide tolerance level of transgenic herbicide-resistant corn. Common corn (Zhengdan 958) was used as a control.
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Abstract
Disclosed in the present invention are an herbicide-resistant transgenic corn event nCX-1, a nucleic acid sequence and a detection method therefor. The nucleic acid molecule of the transgenic corn event nCX-1 is a DNA molecule obtained by inserting exogenous DNA molecules N-Z1 and cp4 epsps into a corn genome seven chromosome; the nucleic acid sequence of the specific detection transgenic corn event nCX-1 comprises SEQ ID NO. 1 or a complementary sequence thereof, and/or SEQ ID NO. 2 or a complementary sequence thereof. Also disclosed in the present invention are a method for cultivating and obtaining a transformed plant of a transgenic corn event nCX-1, a method for controlling field weeds, and produced agricultural products or commodities. The transformed plant of the transgenic corn event nCX-1 has a good tolerance to herbicides flazasulfuron, nicosulfuron, dimethyltetrachloro, 2,4-D, and glyphosate.
Description
本发明涉及一种抗除草剂转基因玉米事件及检测,具体的说,涉及一种耐除草剂转基因玉米事件nCX-1和用于检测生物样品中是否包含特定转基因玉米事件nCX-1的核酸序列及其检测方法。The present invention relates to a herbicide-resistant transgenic corn event and its detection. Specifically, it relates to a herbicide-resistant transgenic corn event nCX-1 and a nucleic acid sequence for detecting whether a biological sample contains a specific transgenic corn event nCX-1. its detection method.
玉米是世界上种植最广泛的粮食作物之一,具有粮食、饲料、能源等多种用途,不仅是主要的粮食作物,也是畜牧业和工业生产的主要原料。玉米的产量和品质对农业生产和全球经济发展起着重要的作用。但是在玉米种植过程中,杂草与玉米竞争营养和生长空间,影响玉米生长导致玉米产量降低,杂草防治是玉米生产中的重要环节。利用除草剂控制杂草可以减少杂草对玉米生长的影响,稳定和提高玉米产量。通过基因工程方法将耐除草剂基因导入到玉米中,获得耐除草剂转基因玉米可以提高玉米杂草防治效率,降低生产成本,为农业生产带来巨大的经济效益,从而实现积极的社会和生态效益。Corn is one of the most widely grown food crops in the world. It has multiple uses such as food, feed, and energy. It is not only the main food crop, but also the main raw material for animal husbandry and industrial production. The yield and quality of corn play an important role in agricultural production and global economic development. However, during corn planting, weeds compete with corn for nutrients and growth space, affecting corn growth and reducing corn yield. Weed control is an important step in corn production. Using herbicides to control weeds can reduce the impact of weeds on corn growth, stabilize and increase corn yields. By introducing herbicide-resistant genes into corn through genetic engineering methods, obtaining herbicide-resistant transgenic corn can improve the efficiency of corn weed control, reduce production costs, and bring huge economic benefits to agricultural production, thereby achieving positive social and ecological benefits. .
农作物可以通过遗传改良技术获得耐除草剂性状。例如,通过农杆菌介导转化法将来源于农杆菌(Agrobacterium tumefaciens sp strain CP4)CP4菌株的cp4 epsps基因导入农作物,表达5-烯醇丙酮莽草酸-3-磷酸合成酶(EPSPS)的农作物获得了对除草剂草甘膦的耐受性。但长时间使用单一除草剂往往造成抗性杂草的大量产生。美洲推广耐草甘膦作物超过二十年,已经产出大量草甘膦抗性杂草,单独使用草甘膦难以实现田间杂草的有效防治。培育耐受两种或两种以上除草剂的转基因农作物可以为杂草控制提供多样化的选择,也能够有效延缓抗性杂草的产生。Crops can gain herbicide tolerance through genetic improvement techniques. For example, the cp4 epsps gene derived from Agrobacterium tumefaciens sp strain CP4 strain CP4 is introduced into crops through Agrobacterium-mediated transformation, and crops expressing 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) are obtained. Tolerance to the herbicide glyphosate. However, long-term use of a single herbicide often results in the emergence of a large number of resistant weeds. Glyphosate-resistant crops have been promoted in the Americas for more than two decades, and a large number of glyphosate-resistant weeds have been produced. It is difficult to effectively control weeds in the field using glyphosate alone. Breeding genetically modified crops that are tolerant to two or more herbicides can provide diversified options for weed control and can also effectively delay the emergence of resistant weeds.
细胞色素P450是一个庞大的基因家族。研究发现,部分P450基因可以降解除草剂。狗牙根中的一种P450基因N-Z1(美国专利:US9657303、加拿大专利:CA2818581C和巴西专利:BR112013012678B1)编码的蛋白质能够赋予农作物对多种除草剂的耐受性。本发明提供一种转基因玉米事件nCX-1,该事件同时表达了N-Z1和CP4 EPSPS两种蛋白,对啶嘧磺隆、烟嘧磺隆、二甲四氯、2,4-D和草甘膦具有高耐受力、稳定遗传、而且对农艺性状没有不良影响。Cytochrome P450 is a large gene family. Research has found that some P450 genes can degrade herbicides. A P450 gene N-Z1 in bermudagrass (US Patent: US9657303, Canadian Patent: CA2818581C and Brazilian Patent: BR112013012678B1) encodes a protein that can confer tolerance to a variety of herbicides in crops. The invention provides a transgenic maize event nCX-1, which simultaneously expresses two proteins, N-Z1 and CP4 EPSPS, and is resistant to pyrosulfuron-methyl, nicosulfuron-methyl, dimethyltetrachloride, 2,4-D and grass. Glyphosate is highly tolerant, genetically stable, and has no adverse effects on agronomic traits.
转化事件(event)是由外源DNA序列在基因组插入位点的上下游侧翼区和外源基因构成的分子结构。在遗传转化时,由于外源DNA序列可以随机插入到植物基因组的任何一条染色体上的任何位点,因此获得的每个事件都是独特的。外源DNA在植物中的表达受到外源DNA所插入的染色体位置的影响。外源DNA在染色体上的位置不同,外源DNA的表达水平、表达空间和时间模式上会存在巨大差异,从而对植物的农艺性状的影响也各不相同。相同外源基因转化得到的不同转化事件间往往性状差异巨大,因此,通常需要筛选大量转化事件,才能够筛选出目标基因表达水平、表达模式和功能性状都能够满足生产应用需要的转化事件。筛选得到的理想转化事件可以采用有性杂交的常规育种方法将转基因渗入到其他遗传背景中,通过杂交方式产生的后代可以保持原始事件的转基因特征。A transformation event is a molecular structure composed of exogenous DNA sequences in the upstream and downstream flanking regions of the genome insertion site and exogenous genes. During genetic transformation, since exogenous DNA sequences can be randomly inserted into any site on any chromosome of the plant genome, each event obtained is unique. The expression of exogenous DNA in plants is affected by the chromosomal position where the exogenous DNA is inserted. The position of exogenous DNA on the chromosome is different, and there will be huge differences in the expression level, expression space and time pattern of exogenous DNA, and thus the impact on the agronomic traits of plants will also be different. Different transformation events obtained by transformation of the same exogenous gene often have huge differences in traits. Therefore, it is usually necessary to screen a large number of transformation events to select transformation events whose expression level, expression pattern and functional traits of the target gene can meet the needs of production and application. The ideal transformation event obtained through screening can be transgeneed into other genetic backgrounds using conventional breeding methods of sexual hybridization, and the offspring produced through hybridization can maintain the transgenic characteristics of the original event.
鉴定外源基因在基因组中的整合位点对转基因作物的杂交育种、生产应用、商业化注册和法律法规监管上具有重要的价值。提供转化事件外源基因整合位点信息,才能够通过现有的多核苷酸的检测方法来检测植物中转化事件的存在。常规的多核苷酸和蛋白检测的方法可以检测是否为转基因,但不能有效区别不同的转化事件,特别是使用相同基因或相同转化载体产生的转化事件。因此,只有针对插入基因和侧翼序列的检测才能够准确判断是否存在目标转基因事件。Identification of the integration site of foreign genes in the genome is of great value to the cross-breeding, production application, commercial registration and legal and regulatory supervision of transgenic crops. Only by providing information on the exogenous gene integration site of the transformation event can the presence of transformation events in plants be detected using existing polynucleotide detection methods. Conventional polynucleotide and protein detection methods can detect whether a transgene is present, but cannot effectively distinguish between different transformation events, especially transformation events produced using the same gene or the same transformation vector. Therefore, only detection of the inserted gene and flanking sequences can accurately determine whether the target transgenic event is present.
(三)发明内容(3) Contents of the invention
本发明提供一种外源基因单拷贝插入、遗产稳定性良好、高抗啶嘧磺隆、烟嘧磺隆、二甲四氯、2,4-D和草甘膦等优秀性状的转基因玉米事件nCX-1及用于检测所述转基因玉米事件nCX-1的核酸分子及其特异性检测方法,本发明能够准确快速地鉴定生物样品中是否包含转基因玉米事
件nCX-1的DNA分子。The invention provides a transgenic corn event with single copy insertion of exogenous genes, good inheritance stability, high resistance to pyrosulfuron-methyl, nicosulfuron-methyl, dimethyltetrachloride, 2,4-D and glyphosate and other excellent traits. nCX-1 and the nucleic acid molecules used to detect the transgenic corn event nCX-1 and its specific detection method. The present invention can accurately and quickly identify whether the biological sample contains the transgenic corn event nCX-1. DNA molecules of nCX-1.
为了实现上述目的,本发明采用的技术方案如下:In order to achieve the above objects, the technical solutions adopted by the present invention are as follows:
第一方面,本发明提供一种抗除草剂转基因玉米事件nCX-1,所述转基因玉米事件nCX-1是将外源DNA分子(即T-DNA)插入玉米基因组7号染色体上SEQ ID NO.22的3’端和SEQ ID NO.23的5’端之间获得的DNA分子;所述玉米基因组核酸序列来源于数据库Maize Genetics and Genomics Database(Zm-B73-REFERENCE-NAM-5.0);所述外源DNA分子包括N-Z1基因表达盒和cp4 epsps基因表达盒;所述N-Z1基因表达盒包括:用作N-Z1基因启动的Actin启动子、N-Z1基因编码框、用作N-Z1基因终止的CaMV35S终止子;所述cp4 epsps基因表达盒包括:用作cp4 epsps基因启动的源自玉米polyubiquitin-1基因的ZmUbi启动子、cp4 epsps基因编码框、作为cp4 epsps基因终止的CaMV35S终止子。
In a first aspect, the present invention provides a herbicide-resistant transgenic maize event nCX-1. The transgenic maize event nCX-1 is an exogenous DNA molecule (i.e., T-DNA) inserted into chromosome 7 of the maize genome SEQ ID NO. The DNA molecule obtained between the 3' end of 22 and the 5' end of SEQ ID NO. 23; the corn genome nucleic acid sequence is derived from the database Maize Genetics and Genomics Database (Zm-B73-REFERENCE-NAM-5.0); the Exogenous DNA molecules include N-Z1 gene expression cassette and cp4 epsps gene expression cassette; the N-Z1 gene expression cassette includes: Actin promoter used for N-Z1 gene initiation, N-Z1 gene coding frame, -CaMV35S terminator that terminates the Z1 gene; the cp4 epsps gene expression cassette includes: the ZmUbi promoter derived from the maize polyubiquitin-1 gene used to initiate the cp4 epsps gene, the cp4 epsps gene coding frame, and the CaMV35S that terminates the cp4 epsps gene terminator.
In a first aspect, the present invention provides a herbicide-resistant transgenic maize event nCX-1. The transgenic maize event nCX-1 is an exogenous DNA molecule (i.e., T-DNA) inserted into chromosome 7 of the maize genome SEQ ID NO. The DNA molecule obtained between the 3' end of 22 and the 5' end of SEQ ID NO. 23; the corn genome nucleic acid sequence is derived from the database Maize Genetics and Genomics Database (Zm-B73-REFERENCE-NAM-5.0); the Exogenous DNA molecules include N-Z1 gene expression cassette and cp4 epsps gene expression cassette; the N-Z1 gene expression cassette includes: Actin promoter used for N-Z1 gene initiation, N-Z1 gene coding frame, -CaMV35S terminator that terminates the Z1 gene; the cp4 epsps gene expression cassette includes: the ZmUbi promoter derived from the maize polyubiquitin-1 gene used to initiate the cp4 epsps gene, the cp4 epsps gene coding frame, and the CaMV35S that terminates the cp4 epsps gene terminator.
优选的,所述转基因玉米事件nCX-1核酸序列如SEQ ID NO.7所示。Preferably, the nucleic acid sequence of the transgenic maize event nCX-1 is shown in SEQ ID NO.7.
优选的,所述除草剂包括草甘膦、啶嘧磺隆、烟嘧磺隆、二甲四氯或2,4-D中一种或者多种。Preferably, the herbicide includes one or more of glyphosate, penosulfuron-methyl, nicosulfuron-methyl, dimethyltetrachloride or 2,4-D.
本发明所述转基因玉米事件nCX-1以玉米(Zea mays L.)nCX-1种子的形式保藏于中国典型培养物保藏中心,保藏编号:CCTCC NO.P202213,保藏日期2022年4月20日,地址:中国武汉,武汉大学,邮编430072。The transgenic maize event nCX-1 of the present invention is deposited in the China Typical Culture Collection Center in the form of maize (Zea mays L.) nCX-1 seeds, with the preservation number: CCTCC NO.P202213, and the preservation date is April 20, 2022. Address: Wuhan University, Wuhan, China, Postal Code 430072.
特别需要指出的是,本发明所述的转基因玉米事件nCX-1参考的基因组数据库来源于Maize Genetics and Genomics Database(Zm-B73-REFERENCE-NAM-5.0)。本领域的研究人员都知道,玉米基因组中存在大量活跃的转座子序列,不同遗传背景下的玉米基因组中可能存在序列位置的偏移。本领域的研究人员可以通过杂交等方式获得的本发明的后代,任意事件的基因组中外源T-DNA的旁侧序列为SEQ ID NO.22和SEQ ID NO.23的玉米事件都应视为本发明的内容。It should be pointed out in particular that the genome database referenced by the transgenic maize event nCX-1 described in the present invention comes from Maize Genetics and Genomics Database (Zm-B73-REFERENCE-NAM-5.0). Researchers in this field know that there are a large number of active transposon sequences in the maize genome, and there may be sequence position shifts in the maize genome under different genetic backgrounds. Researchers in the field can obtain the progeny of the present invention through hybridization and other methods. The corn event in which the flanking sequences of the exogenous T-DNA in the genome of any event are SEQ ID NO. 22 and SEQ ID NO. 23 should be regarded as the present invention. content of the invention.
第二方面,本发明提供一种用于检测所述转基因玉米事件nCX-1的核酸序列,所述核酸序列
包括SEQ ID NO.1或其互补序列、和/或SEQ ID NO.2或其互补序列。In a second aspect, the present invention provides a nucleic acid sequence for detecting the transgenic maize event nCX-1, the nucleic acid sequence Includes SEQ ID NO. 1 or its complement and/or SEQ ID NO. 2 or its complement.
所述SEQ ID NO.1或其互补序列为转基因玉米事件nCX-1在插入序列的5’末端位于插入接合部位的一个长度为26个核苷酸序列,其中依次包括13个来自玉米基因的核苷酸和13个来自于插入的T-DNA的核苷酸,因此,所述SEQ ID NO.1或其互补序列跨越了转基因玉米事件nCX-1中外源DNA分子插入位点的侧翼基因组DNA序列和外源DNA分子的5’末端序列,包含所述SEQ ID NO.1或其互补序列即可鉴定为转基因玉米事件nCX-1的存在。The SEQ ID NO.1 or its complementary sequence is a 26-nucleotide sequence of transgenic maize event nCX-1 located at the insertion junction at the 5' end of the inserted sequence, which in turn includes 13 nuclei from maize genes. and 13 nucleotides from the inserted T-DNA. Therefore, the SEQ ID NO.1 or its complementary sequence spans the flanking genomic DNA sequence of the insertion site of the foreign DNA molecule in the transgenic maize event nCX-1 And the 5' end sequence of the exogenous DNA molecule, including the SEQ ID NO. 1 or its complementary sequence, can be identified as the presence of the transgenic maize event nCX-1.
所述SEQ ID NO.2或其互补序列为转基因玉米事件nCX-1在插入序列的3’末端位于插入接合部位的一个长度为26个核苷酸序列,其中依次包括13个来自插入的T-DNA的核苷酸和13个来自玉米基因的核苷酸,因此,所述SEQ ID NO.2或其互补序列跨越了转基因玉米事件nCX-1中外源DNA分子插入位点的外源DNA分子的3’末端序列和侧翼基因组DNA序列,包含所述SEQ ID NO.2或其互补序列即可鉴定为转基因玉米事件nCX-1的存在。The SEQ ID NO.2 or its complementary sequence is a 26-nucleotide sequence of transgenic maize event nCX-1 located at the insertion junction at the 3' end of the insertion sequence, which in turn includes 13 T-s from the insertion. DNA nucleotides and 13 nucleotides from the maize gene. Therefore, the SEQ ID NO.2 or its complementary sequence spans the exogenous DNA molecule at the insertion site of the exogenous DNA molecule in the transgenic maize event nCX-1. The 3' end sequence and flanking genomic DNA sequence, including the SEQ ID NO. 2 or its complementary sequence, can be identified as the presence of transgenic maize event nCX-1.
进一步地,本发明提供的核酸序列还包括SEQ ID NO.3或其互补序列、和/或SEQ ID NO.4或其互补序列。Further, the nucleic acid sequence provided by the invention also includes SEQ ID NO. 3 or its complementary sequence, and/or SEQ ID NO. 4 or its complementary sequence.
所述SEQ ID NO.3或其互补序列为转基因玉米事件nCX-1在插入序列的5’末端位于插入接合区的一个长度为362个核苷酸序列,其中SEQ ID NO.3的碱基1-225bp为插入结合部位附近的侧翼玉米基因组DNA序列,碱基226-362bp为插入结合部位附近的T-DNA的核苷酸的5’末端序列,包含所述SEQ ID NO.3或其互补序列即可鉴定为转基因玉米事件nCX-1的存在。The SEQ ID NO.3 or its complementary sequence is a 362-nucleotide sequence of transgenic maize event nCX-1 located at the insertion junction region at the 5' end of the inserted sequence, in which base 1 of SEQ ID NO.3 -225bp is the flanking maize genomic DNA sequence inserted near the binding site, and base 226-362bp is the 5' end sequence of the nucleotides of the T-DNA inserted near the binding site, including the SEQ ID NO.3 or its complementary sequence This can identify the presence of transgenic maize event nCX-1.
所述SEQ ID NO.4或其互补序列为转基因玉米事件nCX-1在插入序列的3’末端位于插入接合区的一个长度为494bp的核苷酸序列,所述SEQ ID NO.4第1-48bp为插入结合部位附近的T-DNA的核苷酸的3’末端序列,49-494bp为插入结合部位附近的侧翼玉米基因组DNA序列,包含所述SEQ ID NO.4或其互补序列即可鉴定为转基因玉米事件nCX-1的存在。The SEQ ID NO.4 or its complementary sequence is a 494bp nucleotide sequence in the insertion junction region at the 3' end of the inserted sequence of the transgenic maize event nCX-1, and the SEQ ID NO.4 No. 1- 48bp is the 3' end sequence of the nucleotide of the T-DNA inserted near the binding site, 49-494bp is the flanking maize genomic DNA sequence inserted near the binding site, which can be identified if it contains the SEQ ID NO.4 or its complementary sequence for the presence of transgenic maize event nCX-1.
进一步地,本发明提供的核酸序列还包括SEQ ID NO.5或其互补序列、和/或SEQ ID NO.6或其互补序列。Further, the nucleic acid sequence provided by the invention also includes SEQ ID NO. 5 or its complementary sequence, and/or SEQ ID NO. 6 or its complementary sequence.
所述SEQ ID NO.5或其互补序列为转基因玉米事件nCX-1在插入序列的5’末端位于插入接合区的一个长度为957个核苷酸序列,其中SEQ ID NO.5的碱基1-729bp为插入结合部位附近的侧翼玉米基因组DNA序列,碱基730-957bp为插入结合部位附近的T-DNA的核苷酸的5’末端序列,包含所述SEQ ID NO.5或其互补序列即可鉴定为转基因玉米事件nCX-1的存在。The SEQ ID NO.5 or its complementary sequence is a 957-nucleotide sequence of transgenic maize event nCX-1 located at the 5' end of the inserted sequence in the insertion junction region, in which base 1 of SEQ ID NO.5 -729bp is the flanking maize genomic DNA sequence inserted near the binding site, and bases 730-957bp is the 5' end sequence of the nucleotides of the T-DNA inserted near the binding site, including the SEQ ID NO.5 or its complementary sequence This can identify the presence of transgenic maize event nCX-1.
所述SEQ ID NO.6或其互补序列为转基因玉米事件nCX-1在插入序列的3’末端位于插入接合区的一个长度为946bp的核苷酸序列,所述SEQ ID NO.6第1-265bp为插入结合部位附近的T-DNA的核苷酸的3’末端序列,266-946bp为插入结合部位附近的侧翼玉米基因组DNA序列,包含所述SEQ ID NO.6或其互补序列即可鉴定为转基因玉米事件nCX-1的存在。The SEQ ID NO.6 or its complementary sequence is a 946bp nucleotide sequence in the insertion junction region at the 3' end of the inserted sequence of the transgenic maize event nCX-1, and the SEQ ID NO.6 No. 1- 265bp is the 3' end sequence of the nucleotide of the T-DNA inserted near the binding site, and 266-946bp is the flanking maize genomic DNA sequence inserted near the binding site. It can be identified if it contains the SEQ ID NO.6 or its complementary sequence. for the presence of transgenic maize event nCX-1.
更进一步地,本发明提供的核酸序列包括SEQ ID NO.7或其互补序列。Furthermore, the nucleic acid sequence provided by the invention includes SEQ ID NO. 7 or its complementary sequence.
所述SEQ ID NO.7或其互补序列是转基因玉米事件nCX-1特有的长度为8695个核苷酸的序列,其包括了整个T-DNA序列及其5’和3’末端的侧翼玉米基因型序列。具体包含的基因组和遗传元件如表1所示。包含所述SEQ ID NO.7或其互补序列即可鉴定为转基因玉米事件nCX-1的存在。The SEQ ID NO.7 or its complementary sequence is a sequence of 8695 nucleotides unique to the transgenic maize event nCX-1, which includes the entire T-DNA sequence and the flanking maize genes at its 5' and 3' ends. type sequence. The specific genome and genetic elements included are shown in Table 1. The presence of the transgenic maize event nCX-1 can be identified by including the SEQ ID NO.7 or its complementary sequence.
表1.SEQ ID NO.7包含的基因组和遗传元件
Table 1. Genome and genetic elements contained in SEQ ID NO.7
Table 1. Genome and genetic elements contained in SEQ ID NO.7
本发明提供了转基因玉米事件nCX-1所特有的连续核苷酸序列,该连续核苷酸序列可用于表征转基因玉米事件nCX-1,从而可用于检测样品中是否存在转基因玉米事件nCX-1。具体而言,样品中SEQ ID NO.1-7中的一者或者多者所示的核酸分子中至少13个连续的核苷酸的存在表明该样品中存在转基因玉米事件nCX-1。The present invention provides a continuous nucleotide sequence unique to the transgenic maize event nCX-1. The continuous nucleotide sequence can be used to characterize the transgenic maize event nCX-1, and thus can be used to detect whether the transgenic maize event nCX-1 exists in a sample. Specifically, the presence of at least 13 consecutive nucleotides in the nucleic acid molecules represented by one or more of SEQ ID NO. 1-7 in the sample indicates the presence of transgenic corn event nCX-1 in the sample.
样品中SEQ ID NO.1或其互补序列和/或SEQ ID NO.2或其互补序列的存在表明该样品中存在转基因玉米事件nCX-1。The presence of SEQ ID NO.1 or its complement and/or SEQ ID NO.2 or its complement in a sample indicates the presence of transgenic maize event nCX-1 in the sample.
进一步地,样品中SEQ ID NO.3或其互补序列和/或SEQ ID NO.4或其互补序列的存在表明该样品中存在转基因玉米事件nCX-1。Further, the presence of SEQ ID NO.3 or its complementary sequence and/or SEQ ID NO.4 or its complementary sequence in the sample indicates the presence of transgenic maize event nCX-1 in the sample.
进一步地,样品中SEQ ID NO.5或其互补序列和/或SEQ ID NO.6或其互补序列的存在表明该样品中存在转基因玉米事件nCX-1。Further, the presence of SEQ ID NO.5 or its complementary sequence and/or SEQ ID NO.6 or its complementary sequence in the sample indicates the presence of transgenic maize event nCX-1 in the sample.
进一步地,样品中SEQ ID NO.7或其互补序列的存在表明该样品中存在转基因玉米事件nCX-1。Further, the presence of SEQ ID NO.7 or its complementary sequence in the sample indicates the presence of transgenic maize event nCX-1 in the sample.
本领域技术人员容易理解的是,与上述方案类似,样品中SEQ ID NO.1或其互补序列和SEQ ID NO.4或其互补序列的存在也可以表明该样品中存在转基因玉米事件nCX-1,或者,样品中SEQ ID NO.1或其互补序列和SEQ ID NO.6或其互补序列的存在也可以表明该样品中存在转基因玉米事件nCX-1,或者,样品中SEQ ID NO.2或其互补序列和SEQ ID NO.3或其互补序列的存在也可以表明该样品中存在转基因玉米事件nCX-1;或者,样品中SEQ ID NO.2或其互补序列和SEQ ID NO.5或其互补序列的存在也可以表明该样品中存在转基因玉米事件nCX-1;或者,样品中SEQ ID NO.3或其互补序列和SEQ ID NO.6或其互补序列的存在也可以表明该样品中存在转基因玉米事件nCX-1;或者,样品中SEQ ID NO.4或其互补序列和SEQ ID NO.5或其互补序列的存在也可以表明该样品中存在转基因玉米事件nCX-1。It is easy for those skilled in the art to understand that, similar to the above scheme, the presence of SEQ ID NO.1 or its complementary sequence and SEQ ID NO.4 or its complementary sequence in the sample can also indicate the presence of transgenic maize event nCX-1 in the sample , alternatively, the presence of SEQ ID NO.1 or its complementary sequence and SEQ ID NO.6 or its complementary sequence in the sample can also indicate the presence of transgenic corn event nCX-1 in the sample, or, the presence of SEQ ID NO.2 or its complementary sequence in the sample The presence of its complementary sequence and SEQ ID NO.3 or its complementary sequence can also indicate the presence of transgenic maize event nCX-1 in the sample; alternatively, the presence of SEQ ID NO.2 or its complementary sequence and SEQ ID NO.5 or its complementary sequence in the sample The presence of complementary sequences can also indicate the presence of transgenic maize event nCX-1 in the sample; alternatively, the presence of SEQ ID NO.3 or its complement and SEQ ID NO.6 or its complement in the sample can also indicate the presence of Transgenic maize event nCX-1; alternatively, the presence of SEQ ID NO.4 or its complement and SEQ ID NO.5 or its complement in a sample may also indicate the presence of transgenic maize event nCX-1 in the sample.
第三方面,本发明提供了一种检测样品中转基因玉米事件nCX-1的DNA分子存在的方法,包括:(1)将待检测样品与第一引物和第二引物在核酸扩增反应液中接触;所述第一引物为SEQ ID NO.8、SEQ ID NO.10、SEQ ID NO.12或SEQ ID NO.14中的一种;所述第二引物为SEQ ID NO.9、SEQ ID NO.11、SEQ ID NO.13或SEQ ID NO.15中的一种;(2)进行核酸扩增反应;(3)检测扩增产物的存在;所述扩增产物包括SEQ ID NO.1或其互补序列、SEQ ID NO.2或其互补序列。In a third aspect, the present invention provides a method for detecting the presence of DNA molecules of transgenic maize event nCX-1 in a sample, including: (1) adding the sample to be detected with a first primer and a second primer in a nucleic acid amplification reaction solution Contact; the first primer is one of SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12 or SEQ ID NO.14; the second primer is SEQ ID NO.9, SEQ ID One of NO.11, SEQ ID NO.13 or SEQ ID NO.15; (2) perform a nucleic acid amplification reaction; (3) detect the presence of amplification products; the amplification products include SEQ ID NO.1 or its complement, SEQ ID NO.2 or its complement.
优选的,所述扩增产物包括SEQ ID NO.3或其互补序列中至少13个连续的核苷酸、和/或SEQ ID NO.4或其互补序列中至少13个连续的核苷酸。Preferably, the amplification product includes at least 13 consecutive nucleotides in SEQ ID NO.3 or its complementary sequence, and/or at least 13 consecutive nucleotides in SEQ ID NO.4 or its complementary sequence.
更优选的,所述扩增产物包括SEQ ID NO.5或其互补序列中至少13个连续的核苷酸、和/或SEQ ID NO.6或其互补序列中至少13个连续的核苷酸。More preferably, the amplification product includes at least 13 consecutive nucleotides in SEQ ID NO.5 or its complementary sequence, and/or at least 13 consecutive nucleotides in SEQ ID NO.6 or its complementary sequence. .
进一步地,扩增产物包括SEQ ID NO.1或其互补序列、SEQ ID NO.2或其互补序列、SEQ ID NO.3或其互补序列、SEQ ID NO.4或其互补序列、SEQ ID NO.5或其互补序列、SEQ ID NO.6或其互补序列、和/或SEQ ID NO.7或其互补序列中至少13个连续的核苷酸,即可鉴定为转基因玉米事件nCX-1的存在。Further, the amplification product includes SEQ ID NO.1 or its complementary sequence, SEQ ID NO.2 or its complementary sequence, SEQ ID NO.3 or its complementary sequence, SEQ ID NO.4 or its complementary sequence, SEQ ID NO. .5 or its complementary sequence, SEQ ID NO.6 or its complementary sequence, and/or at least 13 consecutive nucleotides in SEQ ID NO.7 or its complementary sequence, can be identified as the transgenic maize event nCX-1 exist.
在上述技术方案中,所述引物包括至少一种所述核苷酸序列。具体地,所述第一引物分别由SEQ ID NO.3(引物名称:RB-F1,对应编号:SEQ ID NO.8)、SEQ ID NO.4(引物名称:LB-F1,对应编号:SEQ ID NO.10)、SEQ ID NO.5(引物名称:RB-F2,对应编号:SEQ ID NO.12)和SEQ ID NO.6(引物名称:LB-F2,对应编号:SEQ ID NO.14)设计而成;所述第二引物分别由SEQ ID
NO.3(引物名称:RB-R1,对应编号:SEQ ID NO.9)、SEQ ID NO.4(引物名称:LB-R1,对应编号:SEQ ID NO.11)、SEQ ID NO.5(引物名称:RB-R2,对应编号:SEQ ID NO.13)和SEQ ID NO.6(引物名称:LB-R2,对应编号:SEQ ID NO.15)设计而成。In the above technical solution, the primer includes at least one of the nucleotide sequences. Specifically, the first primers are respectively composed of SEQ ID NO.3 (primer name: RB-F1, corresponding number: SEQ ID NO.8), SEQ ID NO.4 (primer name: LB-F1, corresponding number: SEQ ID NO.10), SEQ ID NO.5 (primer name: RB-F2, corresponding number: SEQ ID NO.12) and SEQ ID NO.6 (primer name: LB-F2, corresponding number: SEQ ID NO.14 ) designed; the second primers are respectively composed of SEQ ID NO.3 (Primer name: RB-R1, corresponding number: SEQ ID NO.9), SEQ ID NO.4 (Primer name: LB-R1, corresponding number: SEQ ID NO.11), SEQ ID NO.5 ( Primer name: RB-R2, corresponding number: SEQ ID NO.13) and SEQ ID NO.6 (primer name: LB-R2, corresponding number: SEQ ID NO.15) were designed.
第四方面,本发明还提供了一种培育耐除草剂的含所述转基因玉米事件nCX-1的玉米植物的方法,所述方法包括:种植含有特定区域核酸序列的玉米种子,使所述玉米长成玉米植株,用除草剂喷洒所述玉米植株,收获与其他不含有所述特定区域核酸序列的玉米植株相比,耐除草剂能力显著提高了的植株;所述特定区域核酸序列来自转基因玉米事件nCX-1,所述特定区域的核酸序列包含SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6或者SEQ ID NO.7所示核苷酸序列中的一种或其互补序列;所述除草剂为草甘膦、啶嘧磺隆、烟嘧磺隆、二甲四氯或2,4-滴异辛酯(2,4-D)中一种或者多种。In a fourth aspect, the present invention also provides a method for cultivating herbicide-resistant corn plants containing the transgenic corn event nCX-1. The method includes: planting corn seeds containing a specific region of nucleic acid sequence, so that the corn Grow into corn plants, spray the corn plants with herbicides, and harvest plants with significantly improved herbicide tolerance compared with other corn plants that do not contain the nucleic acid sequence of the specific region; the nucleic acid sequence of the specific region is from transgenic corn Event nCX-1, the nucleic acid sequence of the specific region includes SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or SEQ One of the nucleotide sequences shown in ID NO.7 or its complementary sequence; the herbicide is glyphosate, pyrosulfuron-methyl, nicosulfuron-methyl, dimethyltetrachloride or 2,4-D isooctyl One or more of the esters (2,4-D).
第五方面,本发明还提供了一种获得耐除草剂的含所述转基因玉米事件nCX-1的玉米植株的方法,所述方法包括将含有特定区域核酸序列的玉米植株,与另一种玉米植株杂交,从而产生子代植株;收获与其他不含有所述特定区域核酸序列的植株相比,对除草剂的耐受性显著提高;所述特定区域核酸序列来自转基因玉米事件nCX-1,所述特定区域的核酸序列包含SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6或SEQ ID NO.7所示核苷酸序列中的一种或其互补序列;所述除草剂为草甘膦、啶嘧磺隆、烟嘧磺隆、二甲四氯、2,4-D中一种或者多种。In a fifth aspect, the present invention also provides a method for obtaining herbicide-resistant corn plants containing the transgenic corn event nCX-1. The method includes combining a corn plant containing a specific region nucleic acid sequence with another corn plant. The plants are hybridized to produce progeny plants; the tolerance to herbicides is significantly improved compared with other plants that do not contain the nucleic acid sequence of the specific region; the nucleic acid sequence of the specific region is from the transgenic maize event nCX-1, so The nucleic acid sequence of the specific region includes SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO.7. One of the nucleotide sequences or the complementary sequence thereof; the herbicide is one or more of glyphosate, pyrosulfuron-methyl, nicosulfuron-methyl, dimethyltetrachloride, and 2,4-D.
第六方面,本发明提供一种控制含所述转基因玉米事件nCX-1的转基因玉米田间杂草的方法,所述方法包括将除草剂喷施到种植转基因玉米的大田中,玉米田间杂草被杀灭;所述转基因玉米基因组中包含来自转基因玉米事件nCX-1的特定区域核酸序列,所述特定区域核酸序列包含SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6或者SEQ ID NO.7所示核苷酸序列中的一种或其互补序列;所述除草剂为草甘膦、啶嘧磺隆、烟嘧磺隆、二甲四氯、2,4-D中一种或者多种。In a sixth aspect, the present invention provides a method for controlling weeds in transgenic corn fields containing the transgenic corn event nCX-1. The method includes spraying herbicides into fields where transgenic corns are planted, and the weeds in the corn fields are covered with Kill; the transgenic corn genome contains a specific region nucleic acid sequence from the transgenic corn event nCX-1, and the specific region nucleic acid sequence includes SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID One of the nucleotide sequences shown in NO.4, SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO.7 or its complementary sequence; the herbicides are glyphosate, penisulfuron-methyl, One or more of nicosulfuron, dimethyltetrachloride, and 2,4-D.
第七方面,本发明还提供一种产生自转基因玉米事件nCX-1的农产品或商品,所述农产品或商品包括玉米粉、玉米面、玉米油、玉米淀粉、玉米面筋、玉米饼、含玉米成分的化妆品、或者含玉米成分的辅料。In a seventh aspect, the present invention also provides an agricultural product or commodity produced from the transgenic corn event nCX-1. The agricultural product or commodity includes corn flour, corn flour, corn oil, corn starch, corn gluten, corn tortillas, and corn-containing ingredients. Cosmetics, or excipients containing corn ingredients.
本发明转基因玉米事件nCX-1包含了一个DNA构建体,当其在植物细胞内表达时,所述转基因玉米事件nCX-1获得对啶嘧磺隆、烟嘧磺隆、二甲四氯、2,4-D和草甘膦除草剂的耐受性。所述T-DNA构建体包含两个串联的表达盒,第一个表达盒包含用于在植物中表达N-Z1蛋白的合适启动子和合适的终止子,所述启动子可操作地连接N-Z1蛋白的核苷酸序列,所述N-Z1蛋白对啶嘧磺隆、烟嘧磺隆、二甲四氯、2,4-D除草剂具有耐受性,所述启动子包括肌动蛋白(Actin)启动子,所述终止子包括花椰菜花叶病毒(CaMV)35S终止子。第二个表达盒包含用于在植物中表达EPSPS蛋白的合适的启动子和合适的终止子,所述启动子可操作地连接编码5-烯醇-丙酮酰莽草酸-3-磷酸合酶(EPSPS)的基因,所述EPSPS蛋白对草甘膦除草剂具有耐受性,所述启动子包括玉米polyubiquitin-1基因启动子(ZmUbi),所述终止子包括CaMV 35S终止子。The transgenic maize event nCX-1 of the present invention includes a DNA construct. When expressed in plant cells, the transgenic maize event nCX-1 obtains p-cisulfuron-methyl, nicosulfuron-methyl, dimethyltetrachloride, 2 , 4-D and glyphosate herbicide tolerance. The T-DNA construct contains two expression cassettes in series, the first expression cassette containing a suitable promoter for expressing N-Z1 protein in plants and a suitable terminator, the promoter operably linked to N -The nucleotide sequence of the Z1 protein, the N-Z1 protein is tolerant to azosulfuron-methyl, nicosulfuron-methyl, dimethyltetrachloride, and 2,4-D herbicides, and the promoter includes actin Protein (Actin) promoter, the terminator includes the cauliflower mosaic virus (CaMV) 35S terminator. The second expression cassette contains a suitable promoter for expression of the EPSPS protein in plants, operably linked to a promoter encoding 5-enol-pyruvylshikimate-3-phosphate synthase ( EPSPS) gene, the EPSPS protein is tolerant to glyphosate herbicide, the promoter includes the maize polyubiquitin-1 gene promoter (ZmUbi), and the terminator includes the CaMV 35S terminator.
所述DNA构建体采用转化方法引入植物中,包括农杆菌介导转化法、基因枪转化法和花粉管通道转化法。The DNA construct is introduced into plants using transformation methods, including Agrobacterium-mediated transformation, gene gun transformation and pollen tube channel transformation.
本发明核苷酸序列的说明:Description of the nucleotide sequence of the present invention:
SEQ ID NO.1表示转基因玉米事件nCX-1中侧翼玉米基因组和5’端转基因插入位点每侧各13个核苷酸;SEQ ID NO.1 represents 13 nucleotides on each side of the flanking maize genome and 5'-end transgene insertion site in the transgenic maize event nCX-1;
SEQ ID NO.2表示转基因玉米事件nCX-1中3’端转基因插入位点和侧翼玉米基因组每侧各13个核苷酸;SEQ ID NO.2 represents the 3’-end transgene insertion site and 13 nucleotides on each side of the flanking maize genome in the transgenic maize event nCX-1;
SEQ ID NO.3表示转基因玉米事件nCX-1中5’端转基因插入位点接合区附近362个核苷酸
序列;SEQ ID NO.3 represents the 362 nucleotides near the junction region of the 5'-end transgene insertion site in the transgenic maize event nCX-1 sequence;
SEQ ID NO.4表示转基因玉米事件nCX-1中3’端转基因插入位点接合区附近494个核苷酸序列;SEQ ID NO.4 represents the 494 nucleotide sequence near the junction region of the 3'-end transgene insertion site in the transgenic maize event nCX-1;
SEQ ID NO.5表示转基因玉米事件nCX-1中5’端转基因插入位点接合区附近957个核苷酸序列;SEQ ID NO.5 represents the 957 nucleotide sequence near the junction region of the 5'-end transgene insertion site in the transgenic maize event nCX-1;
SEQ ID NO.6表示转基因玉米事件nCX-1中3’端转基因插入位点接合区附近946个核苷酸序列;SEQ ID NO.6 represents the 946 nucleotide sequence near the junction region of the 3'-end transgene insertion site in the transgenic maize event nCX-1;
SEQ ID NO.7表示转基因玉米事件nCX-1插入T-DNA序列及其5’端和3’端的玉米基因组序列;SEQ ID NO.7 represents the transgenic maize event nCX-1 insertion T-DNA sequence and the maize genome sequence at its 5’ end and 3’ end;
SEQ ID NO.8表示检测SEQ ID NO.3的第一引物(RB-F1);SEQ ID NO.8 represents the first primer (RB-F1) for detecting SEQ ID NO.3;
SEQ ID NO.9表示检测SEQ ID NO.3的第二引物(RB-R1);SEQ ID NO.9 represents the second primer (RB-R1) for detecting SEQ ID NO.3;
SEQ ID NO.10表示检测SEQ ID NO.4的第一引物(LB-F1);SEQ ID NO.10 represents the first primer (LB-F1) for detecting SEQ ID NO.4;
SEQ ID NO.11表示检测SEQ ID NO.4的第二引物(LB-R1);SEQ ID NO.11 represents the second primer (LB-R1) for detecting SEQ ID NO.4;
SEQ ID NO.12表示检测SEQ ID NO.5的第一引物(RB-F2);SEQ ID NO.12 represents the first primer (RB-F2) for detecting SEQ ID NO.5;
SEQ ID NO.13表示检测SEQ ID NO.5的第二引物(RB-R2);SEQ ID NO.13 represents the second primer (RB-R2) for detecting SEQ ID NO.5;
SEQ ID NO.14表示检测SEQ ID NO.6的第一引物(LB-F2);SEQ ID NO.14 represents the first primer (LB-F2) for detecting SEQ ID NO.6;
SEQ ID NO.15表示检测SEQ ID NO.6的第二引物(LB-R2)。SEQ ID NO.15 represents the second primer (LB-R2) for detecting SEQ ID NO.6.
本发明转基因玉米事件nCX-1表达了N-Z1和CP4 EPSPS两个蛋白,对啶嘧磺隆、烟嘧磺隆、二甲四氯、2,4-D和草甘膦除草剂具有高耐受性,在杂交育种过程中可以应用草甘膦、啶嘧磺隆、烟嘧磺隆、二甲四氯、2,4-D中一种或者多种除草剂进行转基因筛选,在转基因玉米种植过程中可以应用草甘膦、啶嘧磺隆、烟嘧磺隆、二甲四氯、2,4-D中一种或者多种除草剂进行杂草防治。The transgenic corn event nCX-1 of the present invention expresses two proteins, N-Z1 and CP4 EPSPS, and is highly resistant to azosulfuron-methyl, nicosulfuron-methyl, dimethyltetrachloride, 2,4-D and glyphosate herbicides. In the process of cross-breeding, one or more herbicides such as glyphosate, pyrosulfuron-methyl, nicosulfuron-methyl, dimethyltetrachloride, and 2,4-D can be used for transgenic screening and planting of transgenic corn. During the process, one or more herbicides such as glyphosate, penosulfuron-methyl, nicosulfuron-methyl, dimethyltetrachloride, and 2,4-D can be used for weed control.
与现有技术相比,本发明有益效果主要体现在:Compared with the existing technology, the beneficial effects of the present invention are mainly reflected in:
(1)本发明提供了一种耐除草剂的转基因玉米事件nCX-1,该事件将目的基因N-Z1和cp4epsps单拷贝插入玉米基因组的特定位点,使得含所述转基因玉米事件nCX-1的不同世代及含有该转基因玉米事件nCX-1的玉米材料中整合稳定、表达稳定、对除草剂抗性稳定。所述转基因玉米事件nCX-1以玉米种子的形式保藏于中国典型培养物保藏中心,保藏编号:CCTCC NO.P202213,对啶嘧磺隆、烟嘧磺隆、二甲四氯、2,4-D和草甘膦具有耐受性。(1) The present invention provides a herbicide-resistant transgenic corn event nCX-1, which inserts a single copy of the target gene N-Z1 and cp4epsps into a specific site of the corn genome, so that the transgenic corn event nCX-1 contains Different generations and corn materials containing the transgenic corn event nCX-1 have stable integration, stable expression, and stable resistance to herbicides. The transgenic corn event nCX-1 is deposited in the China Typical Culture Collection Center in the form of corn seeds, with the preservation number: CCTCC NO.P202213. D and glyphosate tolerance.
(2)本发明提供的特异性检测转基因玉米事件nCX-1的核苷酸序列,SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6或SEQ ID NO.7,能够特异性检测转基因玉米事件nCX-1。(2) The nucleotide sequence for specifically detecting the transgenic maize event nCX-1 provided by the invention is SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO. 5. SEQ ID NO.6 or SEQ ID NO.7 can specifically detect the transgenic corn event nCX-1.
(3)本发明用于检测转基因玉米事件nCX-1的检测方法中,针对nCX-1特有序列SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6或SEQ ID NO.7设计特异性检测引物对,应用核酸扩增方法准确、稳定鉴定出nCX-1转基因事件的存在,能够实现对nCX-1的研究、生产加工和应用进行溯源和全流程监管。(3) In the detection method of the present invention for detecting the transgenic corn event nCX-1, the nCX-1 unique sequence SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ Design specific detection primer pairs for ID NO.5, SEQ ID NO.6 or SEQ ID NO.7, and use nucleic acid amplification methods to accurately and stably identify the presence of nCX-1 transgenic events, enabling research on nCX-1. Production, processing and application are traceable and monitored throughout the entire process.
(4)本发明所述转基因玉米事件nCX-1,在杂交育种过程中可以应用草甘膦、啶嘧磺隆、烟嘧磺隆、二甲四氯、2,4-D中一种或者多种除草剂进行转基因筛选,在玉米种植过程中可以应用草甘膦、啶嘧磺隆、烟嘧磺隆、二甲四氯、2,4-D中一种或者多种除草剂进行杂草防治,有效提高杂草防治效率,降低除草剂抗性杂草产生的风险,降低杂草防治成本。(4) The transgenic corn event nCX-1 of the present invention can use one or more of glyphosate, pyrosulfuron-methyl, nicosulfuron-methyl, dimethyltetrachloride, and 2,4-D during the cross-breeding process. Herbicides are used for transgenic screening. During the corn planting process, one or more herbicides such as glyphosate, pyrosulfuron-methyl, nicosulfuron-methyl, dimethyltetrachloride, and 2,4-D can be used for weed control. , effectively improve weed control efficiency, reduce the risk of herbicide-resistant weeds, and reduce weed control costs.
(5)本发明方法获得的含所述转基因玉米事件nCX-1的农产品或商品。(5) Agricultural products or commodities containing the transgenic corn event nCX-1 obtained by the method of the present invention.
图1为转化载体图谱。Figure 1 shows the transformation vector map.
图2为hiTAIL-PCR扩增产物的电泳图。Figure 2 shows the electrophoresis diagram of hiTAIL-PCR amplification product.
图3为外源插入基因与玉米基因组结构示意图。
Figure 3 is a schematic diagram of the exogenous inserted gene and the maize genome structure.
图4为玉米基因组特异性检测电泳图。M:Marker;1:空白对照(不添加基因组);2:nCX-1;3:常规玉米郑单958;4:转基因玉米混合样(瑞丰125,浙大瑞丰8);5:常规大豆天隆1号;6:转基因大豆混合样(中黄6106,SHZD3201);7:常规水稻秀水134;8:转基因抗虫棉(GHB614,COT102)。Figure 4 shows the electrophoresis chart of maize genome-specific detection. M: Marker; 1: Blank control (no genome added); 2: nCX-1; 3: Conventional corn Zhengdan 958; 4: Transgenic corn mixed sample (Ruifeng 125, Zhejiang University Ruifeng 8); 5: Conventional soybean day Long No. 1; 6: Genetically modified soybean mixed sample (Zhonghuang 6106, SHZD3201); 7: Conventional rice Xiushui 134; 8: Genetically modified insect-resistant cotton (GHB614, COT102).
图5为nCX-1 5’端特异性检测电泳图。M:Marker;1:nCX-1;2:常规玉米郑单958;3:转基因玉米混合样(瑞丰125,浙大瑞丰8);4:常规大豆天隆1号;5:转基因大豆混合样(中黄6106,SHZD3201);6:常规水稻秀水134;7:转基因抗虫棉(GHB614,COT102)。Figure 5 shows the electrophoresis pattern of nCX-1 5’ end specific detection. M: Marker; 1: nCX-1; 2: Conventional corn Zhengdan 958; 3: Genetically modified corn mixed sample (Ruifeng 125, Zhejiang University Ruifeng 8); 4: Conventional soybean Tianlong No. 1; 5: Genetically modified soybean mixed sample (Zhonghuang 6106, SHZD3201); 6: conventional rice Xiushui 134; 7: transgenic insect-resistant cotton (GHB614, COT102).
图6为nCX-1 3’端特异性检测电泳图。1:常规玉米郑单958;2:常规大豆天隆1号;3:常规水稻秀水134;4:nCX-1;M:Marker;5:转基因玉米混合样(瑞丰125,浙大瑞丰8);6:转基因大豆混合样(中黄6106,SHZD3201);7:转基因抗虫棉(GHB614,COT102)。Figure 6 shows the electrophoresis pattern of nCX-1 3’ end specific detection. 1: Conventional corn Zhengdan 958; 2: Conventional soybean Tianlong No. 1; 3: Conventional rice Xiushui 134; 4: nCX-1; M: Marker; 5: Genetically modified corn mixed sample (Ruifeng 125, Zhejiang University Ruifeng 8) ; 6: Genetically modified soybean mixed sample (Zhonghuang 6106, SHZD3201); 7: Genetically modified insect-resistant cotton (GHB614, COT102).
图7为nCX-1和对照对草甘膦耐受性图片。Figure 7 is a picture of the tolerance of nCX-1 and control to glyphosate.
图8为nCX-1和对照对啶嘧磺隆耐受性图片。Figure 8 is a picture of the tolerance of nCX-1 and control to rimisulfuron.
图9为nCX-1和对照对草甘膦+二甲四氯耐受性图片。Figure 9 is a picture of the tolerance of nCX-1 and the control to glyphosate + dimethyltetrachloride.
图10为nCX-1和对照对草甘膦+2,4-滴异辛酯(2,4-D)耐受性图片。Figure 10 is a picture of the tolerance of nCX-1 and control to glyphosate + 2,4-D isooctyl ester (2,4-D).
图11为nCX-1和对照对草甘膦+烟嘧磺隆耐受性图片。Figure 11 is a picture of the tolerance of nCX-1 and control to glyphosate + nicosulfuron.
以下将参照附图更完整地描述本发明,其中显示了本发明部分但非全部的实施方案。实际上,可以以许多不同的形式实施本发明,而不应当理解为限制至本文所列出的实施方案。下述定义和方法将更好地定义本发明并且知道本领域的普通技术人员实施本发明。本发明所属领域的技术人员可根据本文的描述和附图中的相关信息的指导提出的本发明的许多修改和其它实施方案。因此,应当理解,本发明不限于所公开的特定实施方案,基于本发明的教导所提出的修改方案和其它实施方案也包括在所附权利要求的范围之内。The invention will be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all, embodiments of the invention are shown. Indeed, the invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. The following definitions and methods will better define the invention and enable those of ordinary skill in the art to practice the invention. Many modifications and other embodiments of the invention may be devised by those skilled in the art to which this invention pertains upon the guidance of the description herein and the associated information in the accompanying drawings. Therefore, it is to be understood that the present invention is not limited to the specific embodiments disclosed and that modifications and other embodiments based on the teachings of the present invention are included within the scope of the appended claims.
除非另做说明,本发明使用的术语应该根据相关领域的普通技术人员的常规用法来理解。Unless otherwise stated, the terms used in the present invention should be understood according to conventional usage by those of ordinary skill in the relevant art.
本发明所述“玉米”是指玉蜀黍(Zea mays L.),包括与玉米交配的所有植物品种,包括野生玉米种。"Corn" as mentioned in the present invention refers to maize (Zea mays L.), including all plant varieties that mate with corn, including wild corn species.
所述“包括”与“包含”、“含有”同义,并且指“包括但不限于”。所述植物包括整株植物、植物细胞、植物器官、植物原生质体、植物可以从中再生的植物细胞组织培养物、植物愈伤组织、植物丛和植物或植物部分中完整的植物细胞,所述植物部分例如胚、花粉、胚珠、种子、叶、花、枝、果实、茎秆、根、根尖、花药等。所述转基因植物源自本发明DNA分子转化的并因此至少部分由转基因细胞组成的转基因植物或其子代。The "including" is synonymous with "including" and "containing", and means "including but not limited to". The plant includes whole plants, plant cells, plant organs, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant callus, plant clumps and intact plant cells in plants or plant parts, said plants Parts include embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, stems, roots, root tips, anthers, etc. Said transgenic plant is derived from a transgenic plant transformed with the DNA molecule of the invention and thus consisting at least partly of transgenic cells or its progeny.
所述转基因“事件”是通过用外源DNA(例如,包括至少一个含有目标基因的核酸表达盒)构建体转化植物细胞而得到的,通过转基因的方法插入到植物基因组中以产生植物群体,再生所述植物群体,和选择具有插入特定基因组位点特征的特定植株。术语“事件”指包括外源DNA的原始事件和/或该事件的后代。术语“事件”也指事件和含有外源DNA的其它品种个体之间进行有性杂交而得到的后代,即使在重复与回交亲本回交后,来自于事件亲本的插入DNA和侧翼基因组DNA也存在于杂交后代中的同一染色体位置。术语“事件”也指来自原始事件的DNA序列,该DNA序列包含插入DNA和与插入DNA紧密相邻的侧翼基因组序列,该DNA序列被预期转移到子代中,该子代由含有插入DNA的亲本品系(例如原始事件和其自交产生的子代)与不含有插入DNA的亲本品系进行有性杂交而产生,且该子代接受了包含目标基因的插入DNA。The transgenic "event" is obtained by transforming plant cells with a construct of exogenous DNA (for example, including at least one nucleic acid expression cassette containing the target gene), which is inserted into the plant genome by a transgenic method to generate a plant population and regenerate The plant population, and selection of specific plants characterized by insertion of specific genomic loci. The term "event" refers to the original event that includes exogenous DNA and/or the descendants of that event. The term "event" also refers to the offspring resulting from a sexual cross between an event and individuals of other breeds containing foreign DNA. Even after repeated backcrossing to the backcross parent, the inserted DNA and flanking genomic DNA from the event parent remain unchanged. The same chromosomal location present in the hybrid offspring. The term "event" also refers to a DNA sequence from an original event that contains the inserted DNA and flanking genomic sequences in close proximity to the inserted DNA, which DNA sequence is expected to be transferred to progeny consisting of the progeny containing the inserted DNA. A parent line (eg, the original event and its self-produced progeny) is produced by sexual crossing with a parent line that does not contain the inserted DNA, and the progeny receives the inserted DNA containing the gene of interest.
所述“转基因”包括任何细胞、细胞系、愈伤组织、组织、植物部分或植物,以上的基因型由于外源核酸的存在而改变,所述“转基因”包括最初被这样改变的转基因体以及由最初的转基因体通过有性杂交或无性繁殖生成的子代个体。在本发明中,术语“转基因”不包括通过常规植物育种方法或天然发生事件的基因组的(染色体的或染色体外的)改变,所述天然发生事件例如随
机异体受精、非重组病毒感染、非重组细菌转化、非重组转座或自发突变。The term "transgene" includes any cell, cell line, callus, tissue, plant part or plant whose genotype is altered by the presence of exogenous nucleic acid, and the term "transgene" includes a transgene originally so altered and Offspring individuals produced from the original transgenic body through sexual crossing or asexual reproduction. In the present invention, the term "transgene" does not include changes in the genome (chromosomal or extrachromosomal) by conventional plant breeding methods or naturally occurring events, such as those that occur with Allogeneic fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition or spontaneous mutation.
所述“转基因玉米事件nCX-1”即为外源DNA分子插入玉米基因组的chr7:176570691-176570736bp之间获得的DNA分子,还包括含转基因玉米事件nCX-1的植物nCX-1、种子及植物细胞或其可再生部分,所述植物部分包括但不限于细胞、花粉、胚珠、花、芽、根、茎、穗丝、花絮、耳穗、叶和来自玉米植物nCX-1的产物,如玉米粉、玉米面、玉米浆、玉米穗丝、玉米淀粉和留在玉米作物田间的生物量。The "transgenic corn event nCX-1" is a DNA molecule obtained by inserting an exogenous DNA molecule between chr7:176570691-176570736bp of the corn genome, and also includes plant nCX-1, seeds and plants containing the transgenic corn event nCX-1 Cells or renewable parts thereof, including but not limited to cells, pollen, ovules, flowers, shoots, roots, stems, silks, catkins, ears, leaves and products from the corn plant nCX-1, such as Zea mays Meal, cornmeal, corn steep liquor, corn silk, cornstarch and biomass left in the corn crop field.
所述“引物”是一段分离的核酸分子,其通过核酸杂交,退火结合到互补的目标DNA链上,在引物和目标DNA链之间形成杂合体,然后在聚合酶(例如DNA聚合酶)的作用下,沿着目标DNA链延伸。本发明的引物对涉及其在目标核酸序列扩增中的应用,例如,通过聚合酶链式反应(PCR)或其他常规的核酸扩增方法。The "primer" is an isolated nucleic acid molecule that anneals and binds to a complementary target DNA strand through nucleic acid hybridization, forming a hybrid between the primer and the target DNA strand, and then reacts with a polymerase (such as DNA polymerase) Under the influence, it extends along the target DNA strand. The primer pairs of the present invention relate to their use in amplification of target nucleic acid sequences, for example, by polymerase chain reaction (PCR) or other conventional nucleic acid amplification methods.
以下通过具体实施例进一步说明本发明的技术方案。The technical solutions of the present invention will be further described below through specific examples.
实施例1:含有外源基因的质粒载体的获得Example 1: Obtaining plasmid vectors containing foreign genes
本发明用于玉米转化的载体图谱如图1所示,转化质粒载体以pCambia1300(GenBank:AF234296.1)为植物转化载体框架,在其多克隆位点区域加入包含完整表达CP4 EPSPS蛋白表达框和表达N-Z1蛋白表达框的T-DNA,具体由如下部分组成,表达CP4 EPSPS蛋白表达框:CP4EPSPS,驱动CP4 EPSPS的启动子为来源于玉米polyubiquitin-1基因启动子(pZmUbi-1),终止子是CaMV的35S基因终止子;表达N-Z1蛋白表达框:N-Z1,驱动N-Z1的启动子为Actin启动子,终止子为CaMV的35S基因终止子,T-DNA序列为SEQ ID NO.7中730-8014位核苷酸。The vector map of the present invention for maize transformation is shown in Figure 1. The transformation plasmid vector uses pCambia1300 (GenBank: AF234296.1) as the plant transformation vector framework, and in its multiple cloning site region is added an expression box containing the complete expression CP4 EPSPS protein and The T-DNA that expresses the N-Z1 protein expression cassette specifically consists of the following parts. It expresses the CP4 EPSPS protein expression cassette: CP4EPSPS. The promoter driving CP4 EPSPS is derived from the maize polyubiquitin-1 gene promoter (pZmUbi-1). Termination The promoter is the 35S gene terminator of CaMV; the N-Z1 protein expression cassette is: N-Z1, the promoter driving N-Z1 is the Actin promoter, the terminator is the 35S gene terminator of CaMV, and the T-DNA sequence is SEQ ID Nucleotides 730-8014 in NO.7.
利用电击法(2500V)将获得的转化质粒导入农杆菌LBA4404中,获得含有转化载体的农杆菌。The obtained transformation plasmid was introduced into Agrobacterium LBA4404 using electroporation method (2500V) to obtain Agrobacterium containing the transformation vector.
实施例2:含转基因玉米事件nCX-1的植物nCX-1获得Example 2: Obtaining plant nCX-1 containing transgenic maize event nCX-1
采用农杆菌介导进行玉米遗传转化,具体是根据Frame等(Plant Physiol,2002,129:13-22)报道的方法和培养基配方进行转化,使用草甘膦为筛选试剂,步骤如下:取授粉后8~10天的玉米穗,收集大小为1.0-1.5mm未成熟胚。将含有转化载体的农杆菌混匀在侵染培养基中,调整菌液浓度OD660为0.5-0.6。将收集的未成熟胚放入含有农杆菌的侵染液中,室温静置5min,将胚倒在共培养基中,吸干液体,将胚平面朝下放置在共培养基上,22℃培养3-5天。将培养后的未成熟胚转移到含有终浓度200mg/L特美汀抗生素(葛兰素史克,美国)的愈伤组织诱导培养基上,28℃暗培养10-14天杀灭农杆菌。诱导培养后的所有愈伤组织转到含有终浓度2mM草甘膦的筛选培养基上,28℃暗培养2-3周。诱导培养后将所有的愈伤组织转到新鲜的含有2mM草甘膦的筛选培养基上,28℃暗培养2-3周。将成活的胚性组织转到再生培养基上,28℃暗培养10-14天后转移到新鲜的再生培养基上,26℃光照培养10-14天。挑取发育完全的植株到生根培养基上,26℃光照培养直到根发育完全,将生根后的再生苗移植到温室中生长繁种,一共产生850个独立转基因单株,作为T0代事件,用于筛选分析。Agrobacterium-mediated genetic transformation of corn was carried out, specifically according to the method and culture medium formula reported by Frame et al. (Plant Physiol, 2002, 129:13-22). Glyphosate was used as the screening reagent. The steps were as follows: Take pollination After 8 to 10 days of corn ears, immature embryos with a size of 1.0-1.5mm are collected. Mix the Agrobacterium containing the transformation vector into the infection medium, and adjust the bacterial concentration OD660 to 0.5-0.6. Put the collected immature embryos into the infection solution containing Agrobacterium and let it stand at room temperature for 5 minutes. Pour the embryos into the co-culture medium, suck up the liquid, place the embryos flat side down on the co-culture medium, and culture at 22°C. 3-5 days. The cultured immature embryos were transferred to callus induction medium containing a final concentration of 200 mg/L timentin antibiotic (GlaxoSmithKline, USA), and cultured in the dark at 28°C for 10-14 days to kill Agrobacterium. All calli after induction culture were transferred to the selection medium containing a final concentration of 2mM glyphosate and cultured in the dark at 28°C for 2-3 weeks. After induction culture, all calli were transferred to fresh selection medium containing 2mM glyphosate and cultured in the dark at 28°C for 2-3 weeks. Transfer the viable embryonic tissue to the regeneration medium, culture it in the dark at 28°C for 10-14 days, then transfer it to fresh regeneration medium, and culture it in the light at 26°C for 10-14 days. Pick fully developed plants onto the rooting medium and culture them under light at 26°C until the roots are fully developed. The regenerated seedlings after rooting are transplanted to the greenhouse for growth and propagation. A total of 850 independent transgenic plants are produced, which are regarded as the T 0 generation event. For screening analysis.
实施例3:含转基因玉米事件nCX-1的植物nCX-1的筛选Example 3: Screening of plants nCX-1 containing transgenic maize event nCX-1
实施例2获得的850个T0代事件经炼苗后移栽至温室内,在温室中移栽成活801棵幼苗。待T0代转基因玉米长至4-5叶期时,喷施草甘膦和啶嘧磺隆复合除草剂(草甘膦有效剂量为60g/亩;啶嘧磺隆有效剂量为0.8g/亩),没有药害的事件为110个,有药害的事件为640个,死亡的事件为100个(表2)。The 850 T 0 generation events obtained in Example 2 were transplanted into the greenhouse after hardening, and 801 seedlings survived transplantation in the greenhouse. When the T 0 generation transgenic corn reaches the 4-5 leaf stage, spray glyphosate and penisulfuron-methyl compound herbicides (the effective dose of glyphosate is 60g/acre; the effective dose of penisulfuron-methyl is 0.8g/acre) ), there were 110 incidents without phytotoxicity, 640 incidents with phytotoxicity, and 100 incidents of death (Table 2).
表2 T0代事件对对草甘膦和啶嘧磺隆复合除草剂的耐受性
Table 2 Tolerance of T 0 generation events to the compound herbicides glyphosate and metasulfuron-methyl
Table 2 Tolerance of T 0 generation events to the compound herbicides glyphosate and metasulfuron-methyl
对没有药害的事件进行定量PCR检测,测定外源基因在110个存活的事件中的含量,从而评估T-DNA的插入拷贝数,放弃带有两个或者两个以上拷贝的事件。取上述事件的植株,用CTAB法提取植物基因组。通过SYBR Green荧光定量PCR方法检测cp4 epsps基因的拷贝数以确定外源基因的拷贝数。选取玉米基因组中的zSSIIb作为内参基因,随机选取一个玉米事件作为基准,计算目的基因在反应初始的相对含量。Quantitative PCR testing was performed on events without phytotoxicity, and the content of foreign genes in 110 surviving events was measured to evaluate the insertion copy number of T-DNA, and events with two or more copies were discarded. Take the plants from the above events and extract the plant genome using CTAB method. The copy number of the cp4 epsps gene was detected by the SYBR Green fluorescence quantitative PCR method to determine the copy number of the foreign gene. Select zSSIIb in the corn genome as the internal reference gene, randomly select a corn event as the benchmark, and calculate the relative content of the target gene at the initial stage of the reaction.
本实施例使用SYBR Green荧光定量PCR试剂盒(BIO RAD),在Bio-Rad Rad CFX96TMReal-Time PCR仪中进行反应,利用Ct值比较法分析结果。体系及程序参照SYBR Green荧光定量PCR试剂盒的说明书,引物序列如下:In this example, the SYBR Green fluorescence quantitative PCR kit (BIO RAD) was used to perform the reaction in the Bio-Rad Rad CFX96 TM Real-Time PCR instrument, and the results were analyzed using the Ct value comparison method. The system and procedures refer to the instructions of the SYBR Green fluorescence quantitative PCR kit. The primer sequences are as follows:
表3定量PCR引物
Table 3 Quantitative PCR primers
Table 3 Quantitative PCR primers
通过分析cp4 epsps基因拷贝数的实验结果,进而证实外源基因己整合到所检测的玉米植株的染色体组中,其中有36个单拷贝的转基因玉米植株。By analyzing the experimental results of the cp4 epsps gene copy number, it was confirmed that the foreign gene has been integrated into the chromosomes of the tested corn plants, including 36 single-copy transgenic corn plants.
对36个选定单拷贝事件的后代喷施草甘膦和啶嘧磺隆复合除草剂(草甘膦有效剂量为120g/亩;啶嘧磺隆有效剂量为1g/亩),结果显示,对更高浓度复合除草剂具有耐受性的事件有7个(表4)。The progeny of 36 selected single-copy events were sprayed with a compound herbicide of glyphosate and azosulfuron-methyl (the effective dose of glyphosate is 120g/mu; the effective dose of abisulfuron-methyl is 1g/mu). The results show that the effect on There were 7 events with tolerance to higher concentrations of compound herbicides (Table 4).
表4 T1代事件对对草甘膦和啶嘧磺隆复合除草剂的耐受性
Table 4 T 1 generation event tolerance to glyphosate and pensulfuron-methyl herbicides
Table 4 T 1 generation event tolerance to glyphosate and pensulfuron-methyl herbicides
对除草剂耐受性良好的7个事件(记为nCX-1~nCX-7)进行表达量测定。The expression levels of 7 events (denoted as nCX-1 to nCX-7) with good herbicide tolerance were measured.
本研究使用的CP4 EPSPS酶联免疫定量检测试剂盒购自上海佑隆生物科技有限公司,N-Z1试剂盒购自钟鼎生物的蛋白质特制ELISA检测试剂盒。The CP4 EPSPS enzyme-linked immunoassay quantitative detection kit used in this study was purchased from Shanghai Youlong Biotechnology Co., Ltd., and the N-Z1 kit was purchased from Zhongding Biotech’s protein-specific ELISA detection kit.
CP4 EPSPS试剂盒操作步骤:CP4 EPSPS kit operation steps:
(1)将试剂盒从冷藏环境中取出,置于室温(20-25℃)平衡30min以上,使所有试剂和所需板条温度回升至室温,每种液体试剂使用前均摇匀。(1) Take the kit out of the refrigerated environment and place it at room temperature (20-25°C) for more than 30 minutes to allow the temperatures of all reagents and required strips to return to room temperature. Shake each liquid reagent evenly before use.
(2)取40-50mg玉米组织,放入2mL离心管中,加入钢珠,液氮冷冻,研磨仪打碎。加入600μL样品提取液,振荡混匀5min,室温静置5min,12000rpm,离心10min。根据需要用样品提取液稀释样品后用于测定,将OD450值控制在可测量范围内。(2) Take 40-50mg of corn tissue, put it into a 2mL centrifuge tube, add steel beads, freeze it in liquid nitrogen, and grind it with a grinder. Add 600 μL of sample extraction solution, shake and mix for 5 minutes, let stand at room temperature for 5 minutes, and centrifuge at 12,000 rpm for 10 minutes. Dilute the sample with sample extraction solution as needed and use it for measurement to control the OD 450 value within the measurable range.
(3)将标准品浓度稀释为48ppb,24ppb,12ppb,6ppb和3ppb。将20×浓缩洗涤液用去离子水稀释成1×洗涤工作液。用酶稀释液将11×浓缩酶标液按1:10体积比进行稀释。(3) Dilute the standard concentration to 48ppb, 24ppb, 12ppb, 6ppb and 3ppb. Dilute the 20× concentrated washing solution with deionized water into 1× working washing solution. Dilute the 11× concentrated enzyme standard solution with enzyme diluent at a volume ratio of 1:10.
(4)向酶联板的每个微孔中加入样品提取液(空白对照)/标准品/样品100μL,轻轻振荡混匀,用Parafilm膜把酶联板封住,室温下在水平摇床上避光环境震荡45min。(4) Add 100 μL of sample extraction solution (blank control)/standard/sample to each microwell of the enzyme-linked plate, shake gently to mix, seal the enzyme-linked plate with Parafilm membrane, and place on a horizontal shaker at room temperature. Shake in dark environment for 45 minutes.
(5)反应完成后,将板内的液体倒出,每孔加入250μL的洗涤工作液充分洗涤4-5次,将板在吸水纸上拍干。(5) After the reaction is completed, pour out the liquid in the plate, add 250 μL of washing working solution to each well and wash thoroughly 4-5 times, and pat the plate dry on absorbent paper.
(6)每孔加入100μL酶标工作液,轻轻震荡混匀,用Parafilm膜把酶联板封住,室温下在
水平摇床上避光环境震荡30min。重复步骤5。(6) Add 100 μL of enzyme-labeled working solution to each well, shake gently to mix, seal the enzyme-linked plate with Parafilm membrane, and store at room temperature. Shake on a horizontal shaker in a dark environment for 30 minutes. Repeat step 5.
(7)每孔加入100μL显色剂,轻轻震荡混匀,用Parafilm膜把酶联板封住,室温下在水平摇床上避光环境震荡15min。(7) Add 100 μL of chromogenic reagent to each well, shake gently to mix, seal the enzyme-linked plate with Parafilm membrane, and shake for 15 minutes on a horizontal shaker in a dark environment at room temperature.
(8)每孔加入100μL终止液,轻轻震荡混匀,5min内在酶标仪中测定OD450。(8) Add 100 μL of stop solution to each well, shake gently to mix, and measure OD 450 in a microplate reader within 5 minutes.
(9)根据标准样品的OD450值可以绘制出一条标准曲线图,为了排除每次测定之间的系统误差,每次测定样品都同时制作标准曲线。其中一次测定的标准曲线公式是:y=0.0334x+0.0089(R2=0.9976)。(9) A standard curve can be drawn based on the OD 450 value of the standard sample. In order to eliminate systematic errors between each measurement, a standard curve is produced simultaneously for each sample measurement. The standard curve formula for one of the measurements is: y=0.0334x+0.0089 (R 2 =0.9976).
(10)将样本的OD450值代入标准曲线中,从标准曲线上读出样本所对应的浓度,即可算出CP4 EPSPS蛋白含量(μg/g)=样本浓度(ppb)*稀释倍数*样品提取液体积(μL)/叶片重量(mg)/1000。(10) Substitute the OD 450 value of the sample into the standard curve and read the concentration corresponding to the sample from the standard curve to calculate the CP4 EPSPS protein content (μg/g) = sample concentration (ppb) * dilution factor * sample extraction Liquid volume (μL)/leaf weight (mg)/1000.
N-Z1试剂盒操作步骤:N-Z1 kit operation steps:
(1)将试剂盒提前从冷藏环境中取出,置于室温(20-25℃)平衡20min以上,使所有试剂和所需板条温度回升至室温,每种液体试剂使用前均摇匀。(1) Take the kit out of the refrigerated environment in advance and place it at room temperature (20-25°C) to equilibrate for more than 20 minutes to bring the temperature of all reagents and required strips back to room temperature. Shake each liquid reagent evenly before use.
(2)取30mg左右玉米组织(种子先敲碎),放入2mL离心管中,加入钢珠,液氮冷冻,研磨仪打碎。加入1000μL PBS缓冲液,4℃震荡孵育1h后12000rpm,离心10min,取上清,将上清稀释一定倍数后再进行检测,将OD450值控制在可测量范围内。(2) Take about 30 mg of corn tissue (seeds are crushed first), put it into a 2mL centrifuge tube, add steel beads, freeze it in liquid nitrogen, and crush it with a grinder. Add 1000 μL PBS buffer, incubate with shaking at 4°C for 1 hour and then centrifuge at 12000 rpm for 10 min. Take the supernatant and dilute the supernatant to a certain multiple before testing to control the OD 450 value within the measurable range.
(3)取1mL双蒸水加入标准蛋白中,旋紧管盖,上下颠倒数次,待其充分溶解后,轻轻混匀(浓度200ng/ml)。然后用PBS缓冲液进行倍比稀释(注:不要直接在反应孔中进行倍比稀释,在EP管中进行)。建议配制成以下浓度200、100、50、25、12.5、6.25、3.125、1.5625、0.78125、0ng/mL,PBS缓冲液直接作为空白孔0ng/mL。(3) Add 1 mL of double-distilled water to the standard protein, tighten the cap of the tube, invert it several times, wait until it is fully dissolved, and mix gently (concentration 200ng/ml). Then use PBS buffer for doubling dilution (note: do not do doubling dilution directly in the reaction well, do it in an EP tube). It is recommended to prepare the following concentrations: 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, 0ng/mL, and the PBS buffer is directly used as the blank well at 0ng/mL.
(4)洗涤液的配制:将试剂盒中的浓缩洗涤液用双蒸水按1:19体积比稀释。(4) Preparation of washing solution: Dilute the concentrated washing solution in the kit with double-distilled water at a volume ratio of 1:19.
(5)生物素化抗体:取20μL双蒸水加入生物素化抗体中,待其充分溶解后,取1μL生物素化抗体用10mL样品稀释液稀释,即1:10000稀释。(5) Biotinylated antibody: Add 20 μL of double-distilled water to the biotinylated antibody. After it is fully dissolved, take 1 μL of biotinylated antibody and dilute it with 10 mL of sample diluent, that is, 1:10000 dilution.
(6)Streptavidin-HRP:取1μL Streptavidin-HRP用10mL样品稀释液稀释,即1:10000稀释。(6) Streptavidin-HRP: Take 1μL Streptavidin-HRP and dilute it with 10mL sample diluent, that is, 1:10000 dilution.
(7)向酶联板的每个微孔中加入PBS缓冲液(空白对照)/标准品/样品100μL,用Parafilm膜把酶联板封住,37℃孵育60min。(7) Add 100 μL of PBS buffer (blank control)/standard/sample to each microwell of the enzyme-linked plate, seal the enzyme-linked plate with Parafilm membrane, and incubate at 37°C for 60 minutes.
(8)反应完成后,将板内的液体倒出,甩干,每孔加入300μL的洗涤工作液充分洗涤3次,将孔内的液体在吸水纸上拍干。(8) After the reaction is completed, pour out the liquid in the plate, spin it dry, add 300 μL of washing working solution to each well and wash thoroughly three times, and pat the liquid in the well dry on absorbent paper.
(9)每孔加入100μL稀释好的生物素化抗体,用Parafilm膜把酶联板封住,37℃孵育60min。(9) Add 100 μL of diluted biotinylated antibody to each well, seal the enzyme-linked plate with Parafilm membrane, and incubate at 37°C for 60 minutes.
(10)反应完成后,将板内的液体倒出,甩干,每孔加入300μL的洗涤工作液充分洗涤3次,将孔内的液体在吸水纸上拍干。(10) After the reaction is completed, pour out the liquid in the plate and spin dry. Add 300 μL of washing working solution to each well and wash thoroughly three times. Pat the liquid in the well dry on absorbent paper.
(11)每孔加入100μL稀释好的Streptavidin-HRP,用Parafilm膜把酶联板封住,37℃孵育60min。(11) Add 100 μL of diluted Streptavidin-HRP to each well, seal the enzyme-linked plate with Parafilm membrane, and incubate at 37°C for 60 minutes.
(12)反应完成后,将板内的液体倒出,甩干,每孔加入300μL的洗涤工作液充分洗涤4次,将孔内的液体在吸水纸上拍干。(12) After the reaction is completed, pour out the liquid in the plate, spin dry, add 300 μL of washing working solution to each well and wash thoroughly 4 times, and pat the liquid in the well dry on absorbent paper.
(13)每孔加入100μL底物溶液(TMB),用Parafilm膜把酶联板封住,37℃孵育15min左右(根据实际显色情况酌情缩短,但不可超过15min。当标准孔出现明显梯度时,即可终止)。(13) Add 100 μL substrate solution (TMB) to each well, seal the enzyme-linked plate with Parafilm membrane, and incubate at 37°C for about 15 minutes (shorten it as appropriate according to the actual color development, but not exceed 15 minutes. When there is an obvious gradient in the standard wells , can be terminated).
(14)每孔加入100μL终止液,终止反应,此时蓝色立转黄色。终止液的加入顺序应尽量与底物溶液的加入顺序相同。立即用酶标仪在450nm波长测量各孔的光密度(OD值)。应提前打开酶标仪电源,预热仪器,设置好检测程序。.(14) Add 100 μL stop solution to each well to stop the reaction. At this time, the blue color immediately turns to yellow. The order in which the stop solution is added should be as consistent as possible in the order in which the substrate solution is added. Immediately measure the optical density (OD value) of each well with a microplate reader at a wavelength of 450 nm. The power of the microplate reader should be turned on in advance, the instrument should be preheated, and the detection program should be set. .
(15)根据标准样品的OD450值可以绘制出一条标准曲线图,为了排除每次测定之间的系统误差,每次测定样品都同时制作标准曲线。其中一次测定的标准曲线公式是:y=0.5173x+0.6229(R2=0.9975)。
(15) A standard curve can be drawn based on the OD 450 value of the standard sample. In order to eliminate the systematic error between each measurement, a standard curve is produced simultaneously for each sample measurement. The standard curve formula for one of the measurements is: y=0.5173x+0.6229 (R 2 =0.9975).
(16)将样本的OD450值代入标准曲线中,从标准曲线上读出样本所对应的浓度,即可算出N-Z1蛋白含量(μg/g)=样本浓度(ppb)*稀释倍数*样品提取液体积(μL)/叶片重量(mg)/1000。(16) Substitute the OD 450 value of the sample into the standard curve, read the concentration corresponding to the sample from the standard curve, and calculate the N-Z1 protein content (μg/g) = sample concentration (ppb) * dilution factor * sample Extract volume (μL)/leaf weight (mg)/1000.
表5不同事件的外源蛋白表达量
Table 5 Expression amounts of foreign proteins in different events
Table 5 Expression amounts of foreign proteins in different events
选取CP4 EPSPS和N-Z1蛋白表达量适中的事件,并结合田间农艺性状表现,通过筛选,最终选定出更为优异的事件nCX-1,该事件具有外源基因单拷贝插入位点、啶嘧磺隆和草甘膦耐受性强、外源基因及性状稳定遗传和农艺性状表现突出的特点。Events with moderate CP4 EPSPS and N-Z1 protein expression were selected, combined with field agronomic performance, and through screening, the more excellent event nCX-1 was finally selected. This event has a single copy insertion site of foreign genes, It has the characteristics of strong tolerance to rimsulfuron-methyl and glyphosate, stable inheritance of foreign genes and traits, and outstanding agronomic traits.
实施例4:玉米转基因事件nCX-1检测Example 4: Detection of maize transgenic event nCX-1
1、玉米基因组的提取1. Extraction of corn genome
采用CTAB(十六烷基三甲基溴化铵)法提取实施例3筛选的事件nCX-1基因组DNA。The CTAB (cetyltrimethylammonium bromide) method was used to extract the genomic DNA of event nCX-1 screened in Example 3.
取1000mg克幼嫩的事件nCX-1的叶片在液氮中研磨成粉后,加入0.8mL于65℃水浴锅中预热的CTAB缓冲液(20g/L CTAB,1.4M NaCl,100mM Tris-HCl,20mM EDTA,pH 8.0),充分混匀后,于65℃水浴锅中水浴60min;Take 1000 mg of young incident nCX-1 leaves and grind them into powder in liquid nitrogen, then add 0.8 mL of CTAB buffer (20 g/L CTAB, 1.4 M NaCl, 100 mM Tris-HCl) preheated in a 65°C water bath. , 20mM EDTA, pH 8.0), mix thoroughly, and then bathe in a 65°C water bath for 60 minutes;
加入等体积的三氯甲烷,颠倒混匀,12000rpm(每分钟转数)转速下离心10min,吸取上清液至新的离心管中;Add an equal volume of chloroform, mix by inverting, centrifuge at 12,000 rpm (revolutions per minute) for 10 minutes, and pipet the supernatant into a new centrifuge tube;
加入0.7倍体积的异丙醇,轻柔晃动离心管,12000rpm转速下离心1min,收集DNA到管底;弃上清液,Add 0.7 times the volume of isopropyl alcohol, shake the centrifuge tube gently, and centrifuge at 12,000 rpm for 1 minute to collect DNA to the bottom of the tube; discard the supernatant.
加入1mL质量浓度为75%的乙醇,洗涤沉淀,12000rpm转速下离心1min,重复洗涤一次,在超净台吹干;Add 1 mL of ethanol with a mass concentration of 75%, wash the precipitate, centrifuge at 12,000 rpm for 1 min, repeat washing once, and blow dry on a clean bench;
DNA沉淀溶解于适量的TE缓冲液(10mM Tris-HCl,1mM EDTA,pH 8.0)中,Nanodrop测定DNA的浓度,保存备用。Dissolve the DNA precipitate in an appropriate amount of TE buffer (10mM Tris-HCl, 1mM EDTA, pH 8.0), measure the DNA concentration with Nanodrop, and store it for later use.
2、侧翼DNA序列的分析2. Analysis of flanking DNA sequences
使用Liu等报道的hiTAIL-PCR(High-efficiency thermal asymmetric interlaced PCR)方法(Liu,Yao Guang,and Yuanling Chen.2007.High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences.Biotechniques,43:649-650.)对实施例3筛选的优良事件nCX-1外源转基因DNA插入点两侧的区域序列进行测定。该方法通过3个嵌套的特异性引物分别和简并引物组合进行连续的PCR扩增,利用不同退火温度选择性地扩增目标片段。引物序列见表6,PCR反应体系见表7,PCR反应条件见表8。Use the hiTAIL-PCR (High-efficiency thermal asymmetric interlaced PCR) method reported by Liu et al. (Liu, Yao Guang, and Yuanling Chen. 2007. High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences. Biotechniques, 43:649- 650.) Determine the sequence of the regions on both sides of the insertion point of the exogenous transgenic DNA of nCX-1, the excellent event screened in Example 3. This method performs continuous PCR amplification by combining three nested specific primers with degenerate primers, and uses different annealing temperatures to selectively amplify target fragments. The primer sequences are shown in Table 6, the PCR reaction system is shown in Table 7, and the PCR reaction conditions are shown in Table 8.
表6 hiTAIL-PCR引物序列
Table 6 hiTAIL-PCR primer sequences
Table 6 hiTAIL-PCR primer sequences
表7 hiTAIL-PCR反应体系
Table 7 hiTAIL-PCR reaction system
Table 7 hiTAIL-PCR reaction system
表8 hiTAIL-PCR反应条件
Table 8 hiTAIL-PCR reaction conditions
Table 8 hiTAIL-PCR reaction conditions
采用Axygen公司的PCR产物回收试剂盒回收第3轮PCR扩增产物(图2),连接到PMD19-T克隆载体上(TaKaRa,Code:D102A),转化大肠杆菌,将得到的阳性克隆经杭州有康生物科技有
限公司进行测序。获得的序列信息与玉米网上数据库(http://www.maizegdb.org)进行比对分析,检索相似的玉米基因组序列。The third round of PCR amplification products were recovered using Axygen's PCR product recovery kit (Figure 2), connected to the PMD19-T cloning vector (TaKaRa, Code: D102A), transformed into E. coli, and the obtained positive clones were Kang Biotechnology has Co., Ltd. for sequencing. The obtained sequence information was compared and analyzed with the maize online database (http://www.maizegdb.org) to retrieve similar maize genome sequences.
3、T-DNA右侧翼区3. T-DNA right wing region
对通过hiTAIL-PCR方法获得的鉴定为包含5’侧翼区的片段进行测序,测序结果为SEQ ID NO.5,第1-729bp的序列对应于玉米基因组DNA,第730-957bp的序列对应于外源DNA。The fragment identified as containing the 5' flanking region obtained by the hiTAIL-PCR method was sequenced. The sequencing result was SEQ ID NO.5. The 1-729 bp sequence corresponds to the maize genomic DNA, and the 730-957 bp sequence corresponds to the foreign genome DNA. Source DNA.
4、T-DNA左侧翼区4. T-DNA left wing region
鉴定为包含3’侧翼区的片段进行测序,测序结果为SEQ ID NO.6,第1-265bp为插入基因核苷酸序列,第266-946bp为玉米侧翼基因组核苷酸序列。The fragment identified as containing the 3' flanking region was sequenced, and the sequencing result was SEQ ID NO. 6. 1-265 bp were the nucleotide sequence of the inserted gene, and 266-946 bp were the nucleotide sequence of the maize flanking genome.
4、nCX-1整合入基因组序列信息4. nCX-1 integrates into genome sequence information
上述经测序比对和验证过的插入位点上下游旁侧序列和抗除草剂基因表达框序列(含完整表达CP4 EPSPS蛋白表达框和表达N-Z1蛋白表达框的T-DNA)拼接形成本发明所述的转基因玉米事件nCX-1,核苷酸序列为SEQ ID NO.7,基因结构图如图3所示。对应转基因玉米事件nCX-1以玉米(Zea mays L.)nCX-1种子的形式保藏于中国典型培养物保藏中心,保藏编号:CCTCC NO.P202213。
The above sequenced, aligned and verified upstream and downstream flanking sequences of the insertion site and the herbicide-resistant gene expression cassette sequence (including T-DNA for the complete expression of the CP4 EPSPS protein expression cassette and the expression of the N-Z1 protein expression cassette) are spliced to form this version. The nucleotide sequence of the transgenic maize event nCX-1 according to the invention is SEQ ID NO. 7, and the gene structure diagram is shown in Figure 3. The corresponding transgenic maize event nCX-1 is deposited in the China Type Culture Collection Center in the form of maize (Zea mays L.) nCX-1 seeds, with the collection number: CCTCC NO.P202213.
The above sequenced, aligned and verified upstream and downstream flanking sequences of the insertion site and the herbicide-resistant gene expression cassette sequence (including T-DNA for the complete expression of the CP4 EPSPS protein expression cassette and the expression of the N-Z1 protein expression cassette) are spliced to form this version. The nucleotide sequence of the transgenic maize event nCX-1 according to the invention is SEQ ID NO. 7, and the gene structure diagram is shown in Figure 3. The corresponding transgenic maize event nCX-1 is deposited in the China Type Culture Collection Center in the form of maize (Zea mays L.) nCX-1 seeds, with the collection number: CCTCC NO.P202213.
实施例5:转基因玉米事件nCX-1特异性检测Example 5: Specific detection of transgenic maize event nCX-1
转基因玉米事件nCX-1的5’及3’侧翼序列分别如SEQ ID NO.7的第1-729位核苷酸及第8015-8695位核苷酸所示,针对转基因玉米事件nCX-1的5’端及3’端插入位点序列分别设计引物,进行PCR反应,引物信息见表9,反应体系下表10所示。The 5' and 3' flanking sequences of the transgenic corn event nCX-1 are shown in nucleotides 1-729 and 8015-8695 of SEQ ID NO.7 respectively. For the transgenic corn event nCX-1 Design primers for the 5' end and 3' end insertion site sequences respectively, and perform PCR reactions. The primer information is shown in Table 9, and the reaction system is shown in Table 10 below.
表9引物信息
Table 9 Primer information
Table 9 Primer information
表10 PCR反应体系
Table 10 PCR reaction system
Table 10 PCR reaction system
PCR反应程序为:94℃变性5min,94℃变性30s,58℃退火30s,72℃延伸30s,共进行35个循环,最后72℃延伸7min。The PCR reaction program was: denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, a total of 35 cycles, and a final extension at 72°C for 7 min.
选取转基因玉米事件nCX-1种子、常规玉米(郑单958)、转基因玉米混合样(分别取等量的瑞丰125和浙大瑞丰8各自提取DNA后混合)、常规大豆(天隆1号)、转基因大豆混合样(分别取等量的中黄6106和SHZD3201各自提取DNA后混合)、常规水稻(秀水134)、转基因抗虫棉(分别取等量GHB614和COT102各自提取DNA后混合)进行核酸特异性检测。首先采用内参照引物zSSIIb-1F(SEQ ID NO.16)和zSSIIb-1R(SEQ ID NO.17)测试上述基因组的质量,以不添加基因组为空白对照。Select genetically modified corn event nCX-1 seeds, conventional corn (Zhengdan 958), genetically modified corn mixed samples (take equal amounts of Ruifeng 125 and Zhejiang University Ruifeng 8 to extract DNA and mix them), conventional soybeans (Tianlong 1) , mixed samples of genetically modified soybeans (take equal amounts of Zhonghuang 6106 and SHZD3201 to extract DNA and mix them), conventional rice (Xiushui 134), and genetically modified insect-resistant cotton (take equal amounts of GHB614 and COT102 to extract DNA and mix them) for nucleic acid testing. Specific testing. First, the internal reference primers zSSIIb-1F (SEQ ID NO.16) and zSSIIb-1R (SEQ ID NO.17) were used to test the quality of the above genome, and the genome without addition was used as a blank control.
扩增产物琼脂糖电泳结果显示,含玉米基因组的样品(常规玉米、nCX-1、瑞丰125和浙大瑞丰8混合样)能够检测到大约150bp左右的条带(图4),与预期条带大小一致,不含玉米基因组的样品检测不到条带,说明用来测试的玉米基因组质量符合要求。The agarose electrophoresis results of the amplification products showed that samples containing corn genome (conventional corn, nCX-1, Ruifeng 125 and Zhejiang University Ruifeng 8 mixed samples) can detect a band of about 150 bp (Figure 4), which is consistent with the expected band. The band sizes are consistent, and no bands are detected in samples that do not contain corn genome, indicating that the quality of the corn genome used for testing meets the requirements.
同样方法,应用引物对RB-F1(SEQ ID NO.8)和RB-R1(SEQ ID NO.9)分别对nCX-1、常规玉米(郑单958)、转基因玉米混合样(瑞丰125,浙大瑞丰8)、常规大豆(天隆1号)、转基因大豆混合样(中黄6106,SHZD3201)、常规水稻秀水134和转基因抗虫棉(GHB614和COT102)进行核酸特异性检测,扩增产物电泳结果显示,仅有nCX-1样品能够检测到约360bp大小的条带,条带大小与预期一致,不含nCX-1基因组的其他样品检测不到特异性条带,本发明提供的引物对能够特异性检测出nCX-1的存在(图5)。In the same method, the primer pair RB-F1 (SEQ ID NO.8) and RB-R1 (SEQ ID NO.9) were used to target nCX-1, conventional corn (Zhengdan 958), and transgenic corn mixed sample (Ruifeng 125, Zhejiang University Ruifeng 8), conventional soybeans (Tianlong 1), genetically modified soybean mixed samples (Zhonghuang 6106, SHZD3201), conventional rice Xiushui 134 and genetically modified insect-resistant cotton (GHB614 and COT102) were used for nucleic acid specific detection, and the amplification products The electrophoresis results showed that only the nCX-1 sample could detect a band of approximately 360 bp, and the band size was consistent with expectations. Other samples that did not contain the nCX-1 genome could not detect specific bands. The primer pair provided by the invention The presence of nCX-1 can be specifically detected (Figure 5).
同样方法,应用引物对LB-F1(SEQ ID NO.10)和LB-R1(SEQ ID NO.11)分别对nCX-1、常规玉米(郑单958)、转基因玉米混合样(瑞丰125,浙大瑞丰8)、常规大豆天隆1号、转基因大豆混合样(中黄6106,SHZD3201)、常规水稻(秀水134)和转基因抗虫棉(GHB614和COT102)
进行PCR扩增,扩增产物电泳结果显示,仅有nCX-1样品能够检测到约500bp大小的条带,条带大小与预期一致(图6),不含nCX-1基因组的其他样品没有条带检出,本发明提供的引物对能够特异性检测出nCX-1的存在。因此,本发明提供的测试引物对能够特异性检测到含有nCX-1样品的存在。In the same method, the primer pair LB-F1 (SEQ ID NO. 10) and LB-R1 (SEQ ID NO. 11) were used to target nCX-1, conventional corn (Zhengdan 958), and transgenic corn mixed sample (Ruifeng 125, Zhejiang University Ruifeng 8), conventional soybean Tianlong No. 1, genetically modified soybean mixed sample (Zhonghuang 6106, SHZD3201), conventional rice (Xiushui 134) and genetically modified insect-resistant cotton (GHB614 and COT102) PCR amplification was performed, and the electrophoresis results of the amplification products showed that only the nCX-1 sample could detect a band of about 500 bp, and the band size was consistent with expectations (Figure 6). Other samples that did not contain the nCX-1 genome had no bands. Band detection, the primer pair provided by the present invention can specifically detect the presence of nCX-1. Therefore, the test primer pair provided by the present invention can specifically detect the presence of nCX-1-containing samples.
实施例6:nCX-1啶嘧磺隆耐受性测试Example 6: nCX-1 Cisosulfuron Tolerance Test
将实施例4转基因玉米事件nCX-1的种子和普通玉米郑单958(作为对照),采用随机区组设计,3次重复,共24个小区,每个小区面积4m×6m,双粒播种,株距25cm,行距50cm,小区间设1m间隔,在3-5叶期喷施啶嘧磺隆,处理如下:1)不喷施;2)喷施有效剂量1克/亩的啶嘧磺隆;3)喷施有效剂量2克/亩的啶嘧磺隆;4)喷施有效剂量4克/亩的啶嘧磺隆。在用药后1周、2周、4周调查成苗率,植株高度(选取最高的5株)、药害症状(选取药害症状最轻的5株),药害症状分级按GB/T 17980.42-2000执行。The seeds of the transgenic corn event nCX-1 in Example 4 and the common corn Zhengdan 958 (as a control) were used in a random block design, repeated three times, with a total of 24 plots, each plot area 4m×6m, and sown with double seeds. The spacing between plants is 25cm, the spacing between rows is 50cm, and intervals of 1m are set between plots. Spray azosulfuron-methyl at the 3-5 leaf stage, and the treatments are as follows: 1) No spraying; 2) Spray azosulfuron-methyl at an effective dose of 1 g/mu; 3) Spray an effective dose of 2 g/mu of imisulfuron-methyl; 4) Spray an effective dose of 4 g/mu of imisulfuron-methyl. Observe the seedling establishment rate, plant height (select the 5 highest plants), and phytotoxicity symptoms (select the 5 plants with the mildest phytotoxicity symptoms) 1 week, 2 weeks, and 4 weeks after treatment. The phytotoxicity symptoms are graded according to GB/T 17980.42 -2000 execution.
除草剂受害率计算公式:
Herbicide damage rate calculation formula:
Herbicide damage rate calculation formula:
式中,X:受害率,单位为百分率(%);N:某级受害株数;S:级别值;Z:总株数;M最高级别。In the formula, X: damage rate, unit is percentage (%); N: number of damaged plants at a certain level; S: level value; Z: total number of plants; M is the highest level.
用方差分析方法比较不同处理的转基因抗除草剂玉米、非转基因玉米在出苗率、成苗率和受害率方面的差异。判别转基因抗除草剂玉米对除草剂的耐受水平。The variance analysis method was used to compare the differences in seedling emergence rate, seedling establishment rate and damage rate between transgenic herbicide-resistant corn and non-transgenic corn under different treatments. Determine the herbicide tolerance level of transgenic herbicide-resistant corn.
啶嘧磺隆田间测试结果,转基因玉米nCX-1对啶嘧磺隆具有高度耐受性(表11和图7)。The results of the field test of penisulfuron-methyl showed that the transgenic maize nCX-1 was highly tolerant to penisulfuron-methyl (Table 11 and Figure 7).
表11 nCX-1对啶嘧磺隆耐受性调查表
Table 11 nCX-1 Tolerance Questionnaire to Risulfuron-methyl
Table 11 nCX-1 Tolerance Questionnaire to Risulfuron-methyl
实施例7:nCX-1草甘膦耐受性测试Example 7: nCX-1 Glyphosate Tolerance Test
将实施例4转基因玉米事件nCX-1的种子和普通玉米郑单958(作为对照),采用随机区组设计,3次重复,共24个小区,每个小区面积4m×6m,双粒播种,株距25cm,行距50cm,小区间设1m间隔,在3-5叶期喷施草甘膦,处理如下:1)不喷施;2)喷施中剂量草甘膦,有效剂量为60克/亩;3)2倍中剂量草甘膦,有效剂量为120克/亩;4)4倍中剂量草甘膦,有效剂量为240克/亩。在用药后1周、2周、4周调查成苗率,植株高度(选取最高的5株)、药害症状(选取药害症状最轻的5株),药害症状分级按GB/T 17980.42-2000执行。The seeds of the transgenic corn event nCX-1 in Example 4 and the common corn Zhengdan 958 (as a control) were used in a random block design, repeated three times, with a total of 24 plots, each plot area 4m×6m, and sown with double seeds. The spacing between plants is 25cm, the spacing between rows is 50cm, and there are 1m intervals between plots. Spray glyphosate at the 3-5 leaf stage. The treatments are as follows: 1) No spraying; 2) Spray a medium dose of glyphosate, with an effective dose of 60 g/mu. ; 3) 2 times the medium dose glyphosate, the effective dose is 120 g/mu; 4) 4 times the medium dose glyphosate, the effective dose is 240 g/mu. Observe the seedling establishment rate, plant height (select the 5 highest plants), and phytotoxicity symptoms (select the 5 plants with the mildest phytotoxicity symptoms) 1 week, 2 weeks, and 4 weeks after treatment. The phytotoxicity symptoms are graded according to GB/T 17980.42 -2000 execution.
除草剂受害率计算公式:
Herbicide damage rate calculation formula:
Herbicide damage rate calculation formula:
式中,X:受害率,单位为百分率(%);N:某级受害株数;S:级别值;Z:总株数;M最
高级别。In the formula, X: damage rate, unit is percentage (%); N: number of damaged plants at a certain level; S: level value; Z: total number of plants; high-level.
用方差分析方法比较不同处理的转基因抗除草剂玉米、非转基因玉米在出苗率、成苗率和受害率方面的差异。判别转基因抗除草剂玉米对除草剂的耐受水平。The variance analysis method was used to compare the differences in seedling emergence rate, seedling establishment rate and damage rate between transgenic herbicide-resistant corn and non-transgenic corn under different treatments. Determine the herbicide tolerance level of transgenic herbicide-resistant corn.
草甘膦田间测试结果,转基因玉米nCX-1对草甘膦具有高度耐受性(表12和图8)。Glyphosate field test results show that transgenic corn nCX-1 is highly tolerant to glyphosate (Table 12 and Figure 8).
表12 nCX-1对草甘膦耐受性调查表
Table 12 nCX-1 tolerance questionnaire to glyphosate
Table 12 nCX-1 tolerance questionnaire to glyphosate
实施例8:nCX-1草甘膦和二甲四氯复合除草剂耐受性测试Example 8: nCX-1 glyphosate and dimethyltetrachloride compound herbicide tolerance test
将实施例4转基因玉米事件nCX-1的种子和普通玉米郑单958(作为对照),采用随机区组设计,3次重复,共24个小区,每个小区面积4m×6m,双粒播种,株距25cm,行距50cm,小区间设1m间隔,在3-5叶期喷施草甘膦和二甲四氯复合除草剂,处理如下:1)不喷施;2)喷施有效剂量60克/亩草甘膦+有效剂量50克/亩二甲四氯;3)喷施有效剂量120克/亩草甘膦+有效剂量100克/亩二甲四氯;4)喷施有效剂量240克/亩草甘膦+有效剂量200克/亩二甲四氯。在用药后1周、2周、4周调查成苗率,植株高度(选取最高的5株)、药害症状(选取药害症状最轻的5株),药害症状分级按GB/T 17980.42-2000执行。The seeds of the transgenic corn event nCX-1 in Example 4 and the common corn Zhengdan 958 (as a control) were used in a random block design, repeated three times, with a total of 24 plots, each plot area 4m×6m, and sown with double seeds. The spacing between plants is 25cm, the spacing between rows is 50cm, and there are 1m intervals between plots. Spray glyphosate and dimethyltetrachloride compound herbicide at the 3-5 leaf stage. The treatments are as follows: 1) No spraying; 2) Spray an effective dose of 60g/ Glyphosate per mu + effective dose of 50 g/mu of methylene chloride; 3) Spray effective dose of 120 g/mu of glyphosate + effective dose of 100 g/mu of methylene chloride; 4) Spray effective dose of 240 g/mu Mu glyphosate + effective dose 200 grams/mu dimethyltetrachloride. Observe the seedling establishment rate, plant height (select the 5 highest plants), and phytotoxicity symptoms (select the 5 plants with the mildest phytotoxicity symptoms) 1 week, 2 weeks, and 4 weeks after treatment. The phytotoxicity symptoms are graded according to GB/T 17980.42 -2000 execution.
除草剂受害率计算公式:
Herbicide damage rate calculation formula:
Herbicide damage rate calculation formula:
式中,X:受害率,单位为百分率(%);N:某级受害株数;S:级别值;Z:总株数;M最高级别。In the formula, X: damage rate, unit is percentage (%); N: number of damaged plants at a certain level; S: level value; Z: total number of plants; M is the highest level.
用方差分析方法比较不同处理的转基因抗除草剂玉米、非转基因玉米在出苗率、成苗率和受害率方面的差异。判别转基因抗除草剂玉米对除草剂的耐受水平。The variance analysis method was used to compare the differences in seedling emergence rate, seedling establishment rate and damage rate between transgenic herbicide-resistant corn and non-transgenic corn under different treatments. Determine the herbicide tolerance level of transgenic herbicide-resistant corn.
草甘膦和二甲四氯复合除草剂田间测试结果,转基因玉米nCX-1对草甘膦和二甲四氯复合除草剂具有高度耐受性(表13和图9)。Field test results of glyphosate and dimethyltetrachloride compound herbicides showed that transgenic corn nCX-1 is highly tolerant to glyphosate and dimethyltetrachloride compound herbicides (Table 13 and Figure 9).
表13 nCX-1对草甘膦和二甲四氯复合除草剂耐受性调查表
Table 13 nCX-1 tolerance survey to glyphosate and dimethyltetrachloride compound herbicides
Table 13 nCX-1 tolerance survey to glyphosate and dimethyltetrachloride compound herbicides
实施例9:nCX-1草甘膦和2,4-滴异辛酯(2,4-D)复合除草剂耐受性测试Example 9: Tolerance test of nCX-1 glyphosate and 2,4-D isooctyl compound herbicide (2,4-D)
将实施例4转基因玉米事件nCX-1的种子和普通玉米郑单958(作为对照),采用随机区组设计,3次重复,共24个小区,每个小区面积4m×6m,双粒播种,株距25cm,行距50cm,小区间设1m间隔,在3-5叶期喷施草甘膦和2,4-D复合除草剂,处理如下:1)不喷施;2)喷施有效剂量60克/亩草甘膦+有效剂量12克/亩2,4-D;3)喷施有效剂量120克/亩草甘膦+有效剂量24克/亩2,4-D;4)喷施有效剂量240克/亩草甘膦+有效剂量48克/亩2,4-D。在用药后1周、2周、4周调查成苗率,植株高度(选取最高的5株)、药害症状(选取药害症状最轻的5株),药害症状分级按GB/T 17980.42-2000执行。The seeds of the transgenic corn event nCX-1 in Example 4 and the common corn Zhengdan 958 (as a control) were used in a random block design, repeated three times, with a total of 24 plots, each plot area 4m×6m, and sown with double seeds. The spacing between plants is 25cm, the spacing between rows is 50cm, and there are 1m intervals between plots. Spray glyphosate and 2,4-D compound herbicide at the 3-5 leaf stage. The treatments are as follows: 1) No spraying; 2) Spray an effective dose of 60 grams. /mu glyphosate + effective dose 12g/mu 2,4-D; 3) Spray effective dose 120g / mu glyphosate + effective dose 24g / mu 2,4-D; 4) Spray effective dose 240 g/mu glyphosate + effective dose 48 g/mu 2,4-D. Observe the seedling establishment rate, plant height (select the 5 highest plants), and phytotoxicity symptoms (select the 5 plants with the mildest phytotoxicity symptoms) 1 week, 2 weeks, and 4 weeks after treatment. The phytotoxicity symptoms are graded according to GB/T 17980.42 -2000 execution.
除草剂受害率计算公式:
Herbicide damage rate calculation formula:
Herbicide damage rate calculation formula:
式中,X:受害率,单位为百分率(%);N:某级受害株数;S:级别值;Z:总株数;M最高级别。In the formula, X: damage rate, unit is percentage (%); N: number of damaged plants at a certain level; S: level value; Z: total number of plants; M is the highest level.
用方差分析方法比较不同处理的转基因抗除草剂玉米、非转基因玉米在出苗率、成苗率和受害率方面的差异。判别转基因抗除草剂玉米对除草剂的耐受水平。以普通玉米(郑单958)为对照。The variance analysis method was used to compare the differences in seedling emergence rate, seedling establishment rate and damage rate between transgenic herbicide-resistant corn and non-transgenic corn under different treatments. Determine the herbicide tolerance level of transgenic herbicide-resistant corn. Common corn (Zhengdan 958) was used as a control.
草甘膦和2,4-D复合除草剂田间测试结果,转基因玉米nCX-1对草甘膦和2,4-D复合除草剂具有高度耐受性(表14和图10)。Field test results of glyphosate and 2,4-D compound herbicides showed that transgenic corn nCX-1 is highly tolerant to glyphosate and 2,4-D compound herbicides (Table 14 and Figure 10).
表14 nCX-1对草甘膦和2,4-D复合除草剂耐受性调查表
Table 14 nCX-1 tolerance questionnaire to glyphosate and 2,4-D compound herbicides
Table 14 nCX-1 tolerance questionnaire to glyphosate and 2,4-D compound herbicides
实施例10:nCX-1草甘膦和烟嘧磺隆复合除草剂耐受性测试Example 10: nCX-1 glyphosate and nicosulfuron compound herbicide tolerance test
将实施例4转基因玉米事件nCX-1的种子和普通玉米郑单958(作为对照),采用随机区组设计,3次重复,共24个小区,每个小区面积4m×6m,双粒播种,株距25cm,行距50cm,小区间设1m间隔,在3-5叶期喷施草甘膦和烟嘧磺隆复合除草剂,处理如下:1)不喷施;2)喷
施有效剂量60克/亩草甘膦+有效剂量1.6克/亩烟嘧磺隆;3)喷施有效剂量120克/亩草甘膦+有效剂量3.2克/亩烟嘧磺隆;4)喷施有效剂量240克/亩草甘膦+有效剂量6.4克/亩烟嘧磺隆。在用药后1周、2周、4周调查成苗率,植株高度(选取最高的5株)、药害症状(选取药害症状最轻的5株),药害症状分级按GB/T 17980.42-2000执行。The seeds of the transgenic corn event nCX-1 in Example 4 and the common corn Zhengdan 958 (as a control) were used in a random block design, repeated three times, with a total of 24 plots, each plot area 4m×6m, and sown with double seeds. The spacing between plants is 25cm, the spacing between rows is 50cm, and there are 1m intervals between plots. Spray glyphosate and nicosulfuron compound herbicides at the 3-5 leaf stage. The treatments are as follows: 1) No spraying; 2) Spray Apply an effective dose of 60 g/mu glyphosate + an effective dose of 1.6 g/mu nicosulfuron; 3) Spray an effective dose of 120 g/mu glyphosate + an effective dose of 3.2 g/mu nicosulfuron; 4) Spray Apply an effective dose of 240 g/mu of glyphosate + an effective dose of 6.4 g/mu of nicosulfuron. 1 week, 2 weeks, and 4 weeks after treatment, the seedling establishment rate, plant height (select the 5 plants with the highest symptoms), and phytotoxicity symptoms (select the 5 plants with the mildest phytotoxicity symptoms) were investigated. The phytotoxicity symptoms were graded according to GB/T 17980.42. -2000 execution.
除草剂受害率计算公式:
Herbicide damage rate calculation formula:
Herbicide damage rate calculation formula:
式中,X:受害率,单位为百分率(%);N:某级受害株数;S:级别值;Z:总株数;M最高级别。In the formula, X: damage rate, unit is percentage (%); N: number of damaged plants at a certain level; S: level value; Z: total number of plants; M is the highest level.
用方差分析方法比较不同处理的转基因抗除草剂玉米、非转基因玉米在出苗率、成苗率和受害率方面的差异。判别转基因抗除草剂玉米对除草剂的耐受水平。以普通玉米(郑单958)为对照。The variance analysis method was used to compare the differences in seedling emergence rate, seedling establishment rate and damage rate between transgenic herbicide-resistant corn and non-transgenic corn under different treatments. Determine the herbicide tolerance level of transgenic herbicide-resistant corn. Common corn (Zhengdan 958) was used as a control.
草甘膦和烟嘧磺隆复合除草剂田间测试结果,转基因玉米nCX-1对草甘膦和烟嘧磺隆复合除草剂具有高度耐受性(表15和图11)。Field test results of glyphosate and nicosulfuron-methyl herbicides showed that transgenic corn nCX-1 is highly tolerant to glyphosate and nicosulfuron-methyl herbicides (Table 15 and Figure 11).
表15 nCX-1对草甘膦和烟嘧磺隆复合除草剂耐受性调查表
Table 15 nCX-1 tolerance survey to glyphosate and nicosulfuron compound herbicides
Table 15 nCX-1 tolerance survey to glyphosate and nicosulfuron compound herbicides
Claims (13)
- 一种抗除草剂转基因玉米事件nCX-1,其特征在于,所述转基因玉米事件nCX-1是将外源DNA分子插入玉米基因组7号染色体上SEQ ID NO.22所示的3’端和SEQ ID NO.23所示的5’端之间获得的DNA分子;所述外源DNA分子包括N-Z1基因表达盒和cp4 epsps基因表达盒。A herbicide-resistant transgenic corn event nCX-1, characterized in that the transgenic corn event nCX-1 inserts an exogenous DNA molecule into the 3' end shown in SEQ ID NO. 22 and SEQ The DNA molecule obtained between the 5' ends shown in ID NO. 23; the exogenous DNA molecule includes the N-Z1 gene expression cassette and the cp4 epsps gene expression cassette.
- 如权利要求1所述的抗除草剂转基因玉米事件nCX-1,其特征在于,所述N-Z1基因表达盒包括:用作N-Z1基因启动的Actin启动子、N-Z1基因编码框、用作N-Z1基因终止的CaMV35S终止子;所述cp4 epsps基因表达盒包括:用作cp4 epsps基因启动的ZmUbi启动子、cp4 epsps基因编码框、作为cp4 epsps基因终止的CaMV35S终止子。The herbicide-resistant transgenic maize event nCX-1 according to claim 1, characterized in that the N-Z1 gene expression cassette includes: an Actin promoter used for starting the N-Z1 gene, an N-Z1 gene coding frame, CaMV35S terminator used as N-Z1 gene termination; the cp4 epsps gene expression cassette includes: ZmUbi promoter used as cp4 epsps gene initiation, cp4 epsps gene coding frame, and CaMV35S terminator used as cp4 epsps gene termination.
- 如权利要求1所述的抗除草剂转基因玉米事件nCX-1,其特征在于,所述转基因玉米事件nCX-1核酸序列如SEQ ID NO.7所示。The herbicide-resistant transgenic corn event nCX-1 as claimed in claim 1, characterized in that the nucleic acid sequence of the transgenic corn event nCX-1 is as shown in SEQ ID NO.7.
- 一种用于检测权利要求1所述转基因玉米事件nCX-1的核酸序列,其特征在于,所述核酸序列包括SEQ ID NO.1或其互补序列、和/或SEQ ID NO.2或其互补序列。A nucleic acid sequence for detecting the transgenic maize event nCX-1 described in claim 1, characterized in that the nucleic acid sequence includes SEQ ID NO.1 or its complement, and/or SEQ ID NO.2 or its complement sequence.
- 如权利要求4所述的核酸序列,其特征在于,所述核酸序列包括SEQ ID NO.3或其互补序列、和/或SEQ ID NO.4或其互补序列。The nucleic acid sequence of claim 4, wherein the nucleic acid sequence includes SEQ ID NO.3 or its complementary sequence, and/or SEQ ID NO.4 or its complementary sequence.
- 如权利要求4所述的核酸序列,其特征在于所述核酸序列包括SEQ ID NO.5或其互补序列、和/或SEQ ID NO.6或其互补序列。The nucleic acid sequence of claim 4, characterized in that the nucleic acid sequence includes SEQ ID NO.5 or its complementary sequence, and/or SEQ ID NO.6 or its complementary sequence.
- 如权利要求4所述的核酸序列,其特征在于所述核酸序列包括SEQ ID NO.7或其互补序列。The nucleic acid sequence of claim 4, wherein the nucleic acid sequence includes SEQ ID NO. 7 or its complementary sequence.
- 一种检测权利要求1所述转基因玉米事件nCX-1的方法,其特征在于,所述方法包括:(1)将待检测样品与第一引物和第二引物在核酸扩增反应液中接触;所述第一引物为SEQ ID NO.8、SEQ ID NO.10、SEQ ID NO.12或SEQ ID NO.14中的一种;所述第二引物为SEQ ID NO.9、SEQ ID NO.11、SEQ ID NO.13或SEQ ID NO.15中的一种;(2)进行核酸扩增反应;(3)检测扩增产物的存在;所述扩增产物包括SEQ ID NO.1或其互补序列、或者SEQ ID NO.2或其互补序列。A method for detecting the transgenic maize event nCX-1 according to claim 1, characterized in that the method includes: (1) contacting the sample to be detected with the first primer and the second primer in the nucleic acid amplification reaction solution; The first primer is one of SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12 or SEQ ID NO.14; the second primer is SEQ ID NO.9, SEQ ID NO. 11. One of SEQ ID NO.13 or SEQ ID NO.15; (2) perform a nucleic acid amplification reaction; (3) detect the presence of an amplification product; the amplification product includes SEQ ID NO.1 or its The complementary sequence, or SEQ ID NO.2 or its complementary sequence.
- 如权利要求8所述的方法,其特征在于,所述扩增产物包括SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7所示核苷酸序列或其互补序列中至少13个连续的核苷酸。The method of claim 8, wherein the amplification product includes SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7. At least 13 consecutive nucleotides in the nucleotide sequence or its complement.
- 一种培育耐除草剂的含权利要求1所述转基因玉米事件nCX-1的玉米植物的方法,其特征在于,所述方法包括:种植含有特定区域核酸序列的玉米种子,使所述玉米长成玉米植株,用除草剂喷洒所述玉米植株,收获与其他不含有所述特定区域核酸序列的玉米植株相比,耐除草剂能力显著提高了的植株;所述特定区域核酸序列来自转基因玉米事件nCX-1,所述特定区域核酸序列包含SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6或者SEQ ID NO.7所示核苷酸序列中的一种或其互补序列;所述除草剂为草甘膦、啶嘧磺隆、烟嘧磺隆、二甲四氯、2,4-D中一种或者多种。A method for cultivating herbicide-resistant corn plants containing the transgenic corn event nCX-1 according to claim 1, characterized in that the method includes: planting corn seeds containing a specific region of nucleic acid sequence, so that the corn grows into Corn plants are sprayed with herbicides to harvest plants with significantly improved herbicide tolerance compared to other corn plants that do not contain the nucleic acid sequence of the specific region; the nucleic acid sequence of the specific region is from the transgenic corn event nCX -1, the specific region nucleic acid sequence includes SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO. One of the nucleotide sequences shown in 7 or its complementary sequence; the herbicide is one or more of glyphosate, pyrosulfuron-methyl, nicosulfuron-methyl, dimethyltetrachloride, and 2,4-D. kind.
- 一种获得耐除草剂的含权利要求1所述转基因玉米事件nCX-1的玉米植株的方法,其特征在于,将含有特定区域核酸序列的玉米植株,与另一种玉米植株杂交,从而产生子代植株;收获与其他不含有所述特定区域核酸序列的植株相比,对除草剂的耐受性显著提高;所述特定区域核酸序列来自转基因玉米事件nCX-1,所述特定区域的核酸序列包含SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6或SEQ ID NO.7所示核苷酸序列中的一种或其互补序列; 所述除草剂为草甘膦、啶嘧磺隆、烟嘧磺隆、二甲四氯、2,4-D中一种或者多种。A method for obtaining herbicide-resistant corn plants containing transgenic corn event nCX-1 according to claim 1, characterized in that a corn plant containing a specific region nucleic acid sequence is hybridized with another corn plant to produce a progeny Generation of plants; Harvest has significantly improved tolerance to herbicides compared with other plants that do not contain the nucleic acid sequence of the specific region; the nucleic acid sequence of the specific region is from the transgenic maize event nCX-1, and the nucleic acid sequence of the specific region Comprising the nucleotide sequence shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO.7 one or its complementary sequence; The herbicide is one or more of glyphosate, penosulfuron-methyl, nicosulfuron-methyl, dimethyltetrachloride, and 2,4-D.
- 一种控制含权利要求1所述转基因玉米事件nCX-1的转基因玉米田间杂草的方法,其特征在于,所述方法包括将除草剂喷施到种植转基因玉米的大田中,玉米田间杂草被杀灭;所述转基因玉米基因组中包含来自转基因玉米事件nCX-1的特定区域核酸序列,所述特定区域核酸序列包含SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6或者SEQ ID NO.7所示核苷酸序列中的一种或其互补序列;所述除草剂为草甘膦、啶嘧磺隆、烟嘧磺隆、二甲四氯、2,4-D中一种或者多种。A method for controlling weeds in transgenic corn fields containing transgenic corn event nCX-1 according to claim 1, characterized in that the method includes spraying herbicides into fields where transgenic corns are planted, and the weeds in the corn fields are covered with Kill; the transgenic corn genome contains a specific region nucleic acid sequence from the transgenic corn event nCX-1, and the specific region nucleic acid sequence includes SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID One of the nucleotide sequences shown in NO.4, SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO.7 or its complementary sequence; the herbicides are glyphosate, penisulfuron-methyl, One or more of nicosulfuron, dimethyltetrachloride, and 2,4-D.
- 一种产生自权利要求1所述转基因玉米事件nCX-1的农产品或商品,其特征在于,所述农产品或商品包括玉米粉、玉米面、玉米油、玉米淀粉、玉米面筋、玉米饼、含玉米成分的化妆品或者含玉米成分的辅料。 An agricultural product or commodity produced from the transgenic corn event nCX-1 of claim 1, characterized in that the agricultural product or commodity includes corn flour, corn flour, corn oil, corn starch, corn gluten, corn tortillas, corn-containing ingredients Cosmetics or excipients containing corn ingredients.
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