WO2023221554A1

WO2023221554A1 - Herbicide-resistant transgenic corn event ncx-1, nucleic acid sequence and detection method therefor

Info

Publication number: WO2023221554A1
Application number: PCT/CN2023/074172
Authority: WO
Inventors: 沈志成; 于小星; 郑挺; 林朝阳; 王鹏飞; 许超
Original assignee: 杭州瑞丰生物科技有限公司
Priority date: 2022-05-17
Filing date: 2023-02-02
Publication date: 2023-11-23
Also published as: CN116103423A

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

Herbicide-resistant transgenic corn event nCX-1, nucleic acid sequence and detection method

(1) Technical field

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.

(2) Background technology

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. 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.

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. 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.

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.

(3) Contents of the invention

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.

In order to achieve the above objects, the technical solutions adopted by the present invention are as follows:

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.

Preferably, the nucleic acid sequence of the transgenic maize event nCX-1 is shown in SEQ ID NO.7.

Preferably, 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.

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.

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.

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.

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.

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, 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.

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.

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.

Furthermore, the 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.

Table 1. Genome and genetic elements contained in SEQ ID NO.7

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.

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.

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.

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.

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.

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.

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.

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.

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. .

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.

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.

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).

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.

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.

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.

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.

The DNA construct is introduced into plants using transformation methods, including Agrobacterium-mediated transformation, gene gun transformation and pollen tube channel transformation.

Description of the nucleotide sequence of the present invention:

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. 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.

Compared with the existing technology, the beneficial effects of the present invention are mainly reflected in:

(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) 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) 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) 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) Agricultural products or commodities containing the transgenic corn event nCX-1 obtained by the method of the present invention.

(4) Description of drawings:

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.

(5) Specific implementation methods

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.

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.

"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.

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. 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.

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.

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.

The technical solutions of the present invention will be further described below through specific examples.

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). 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.

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. 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.

Example 3: Screening of plants nCX-1 containing transgenic maize event nCX-1

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).

Table 2 Tolerance of T ₀ generation events to the compound herbicides glyphosate and metasulfuron-methyl

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.

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:

Table 3 Quantitative PCR primers

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.

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).

Table 4 T ₁ generation event tolerance to glyphosate and pensulfuron-methyl herbicides

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.

CP4 EPSPS kit operation steps:

(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) 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) 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) 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) 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) 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) 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) 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) 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) 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 kit operation steps:

(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) 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) 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) Preparation of washing solution: Dilute the concentrated washing solution in the kit with double-distilled water at a volume ratio of 1:19.

(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: Take 1μL Streptavidin-HRP and dilute it with 10mL sample diluent, that is, 1:10000 dilution.

(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) 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) 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) 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) 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) 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) 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) 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) 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) 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.

Table 5 Expression amounts of foreign proteins in different events

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.

Example 4: Detection of maize transgenic event nCX-1

1. Extraction of corn genome

The CTAB (cetyltrimethylammonium bromide) method was used to extract the genomic DNA of event nCX-1 screened in Example 3.

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;

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;

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.

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;

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. Analysis of flanking DNA sequences

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.

Table 6 hiTAIL-PCR primer sequences

Table 7 hiTAIL-PCR reaction system

Table 8 hiTAIL-PCR reaction conditions

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 right wing region

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 left wing region

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 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 (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.

Example 5: Specific detection of transgenic maize event nCX-1

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.

Table 9 Primer information

Table 10 PCR reaction system

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.

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.

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.

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).

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.

Example 6: nCX-1 Cisosulfuron 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 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.

Herbicide damage rate calculation formula:

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.

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 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).

Table 11 nCX-1 Tolerance Questionnaire to Risulfuron-methyl

Example 7: nCX-1 Glyphosate 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. 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.

Herbicide damage rate calculation formula:

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.

Glyphosate field test results show that transgenic corn nCX-1 is highly tolerant to glyphosate (Table 12 and Figure 8).

Table 12 nCX-1 tolerance questionnaire to glyphosate

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. 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.

Herbicide damage rate calculation formula:

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).

Table 13 nCX-1 tolerance survey to glyphosate and dimethyltetrachloride compound herbicides

Example 9: Tolerance test of nCX-1 glyphosate and 2,4-D isooctyl compound herbicide (2,4-D)

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.

Herbicide damage rate calculation formula:

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.

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).

Table 14 nCX-1 tolerance questionnaire to glyphosate and 2,4-D compound herbicides

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. 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.

Herbicide damage rate calculation formula:

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).

Table 15 nCX-1 tolerance survey to glyphosate and nicosulfuron compound herbicides

Claims

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.
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.
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.
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.
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.
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.
The nucleic acid sequence of claim 4, wherein the nucleic acid sequence includes SEQ ID NO. 7 or its complementary sequence.
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.
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.
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.
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.
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.
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.