Two-gene standard plasmid molecule used for detecting genetically modified soybeans and building method thereof

A technology of transgenic soybeans and standard plasmids, which is applied in the field of plasmid molecules in the field of molecular biology technology, can solve problems such as the lack of positive standard products, and achieve the effect of good specificity and high sensitivity

Inactive Publication Date: 2012-04-11
FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] An object of the present invention is to provide a standard molecular control for the detection of genetically modified soybeans and its construction met

Method used

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  • Two-gene standard plasmid molecule used for detecting genetically modified soybeans and building method thereof
  • Two-gene standard plasmid molecule used for detecting genetically modified soybeans and building method thereof
  • Two-gene standard plasmid molecule used for detecting genetically modified soybeans and building method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] 1. Experimental materials

[0025] GM soybean line GTS40-3-2; non-GM soybean variety.

[0026] 2. Experimental reagents

[0027] pMDTM19-T Simple Vector was purchased from Dalian Bao Biological Engineering Company; dNTPs, Taq DNA polymerase, DNA marker Ⅰ, Ⅱ and marker Ⅲ were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; restriction enzymes Not Ⅰ, Sal Ⅰ, EcoR Ⅰ and Xba Ⅰ were purchased from NEB Beijing Co., Ltd.; plasmid genomic DNA was extracted and purified using a plasmid mini-extraction kit developed by Beijing Tiangen Biochemical Technology Co., Ltd.; other biochemical reagents were imported or domestically produced analytically pure.

[0028] 3. Experimental equipment

[0029] TC-512 PCR amplification instrument (Techne, UK); Geliance 200 DNA electrophoresis gel imager (PerkinElmer, USA); Nano-Drop ND-1000 nucleic acid protein analyzer (Nano Drop, USA); centrifuge; constant temperature water bath ; Incubator; Balance etc.

[0030] 4. Test method an...

Embodiment 2

[0088] 1. Enzymes and Reagents

[0089] Real-time fluorescent quantitative PCR detection kit (Premix Ex Taq TM (Perfect Real Time)) were purchased from Treasure Bioengineering (Dalian) Co., Ltd.; primers and TaqMan probes were synthesized by Treasure Bioengineering (Dalian) Co., Ltd.; other biochemical reagents were imported or domestic analytically pure.

[0090] 2. Experimental equipment

[0091] Real-time fluorescent quantitative PCR instrument 7500 (ABI company).

[0092] 3. Experimental method and process

[0093] 1. Design and synthesis of fluorescent quantitative PCR primers and probes

[0094] According to the sequences of the Lectin 1 and EPSPS genes in the standard plasmid molecules, quantitative PCR primers and probes were designed using the software Primer Premier 5.0, as shown in Table 6. The 5' ends of the TaqMan probes were labeled with reporter fluorescent dyes FAM and TET, and the 3' ends were labeled with quencher non-fluorescent dyes BHQ and Eclipse. Pr...

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Abstract

The invention discloses an essential standard plasmid molecule used for detecting genetically modified crops and a building method thereof. The standard plasmid molecule contains specific fragments of a soybean endogenous gene Lectin and specific fragments of an exogenous gene EPSPS in the genetically modified soybean strain GTS40-3-2. The standard plasmid molecule and the building method have the following beneficial effects: the two target gene fragments are obtained by analyzing sequences, designing primers and amplifying the genes; the standard artificial recombinant plasmid molecule pTLE2 is obtained through such gene cloning technologies as fusion PCR (polymerase chain reaction), link, transform and the like; the standard plasmid molecule built in the invention can absolutely replace the positive controls and is suitable for qualitative and quantitative PCR analysis and detection of the specificity of the structural genes in the genetically modified soybean strain GTS40-3-2; and new exogenous gene fragments can be added on the basis of the original standard molecule if new genetically modified materials occur, thus meeting the increasing detection requirements of the genetically modified crop materials.

Description

technical field [0001] The invention relates to a plasmid molecule in the technical field of molecular biology, in particular to a standard plasmid molecule for transgenic soybean detection and a construction method thereof. Background technique [0002] Since 1996, the global planting area of ​​genetically modified crops has grown at an average annual rate of more than 10%. In 2009, the global planting area of ​​genetically modified crops approved reached 134 million hectares, of which the planting area of ​​genetically modified soybeans reached 69 million hectares, accounting for 77% of the total planting area of ​​soybeans. %. With the widespread cultivation of genetically modified crops, their safety issues have aroused the general attention of the global public. The European Union signed the first bill in the world in 1998, requiring labeling of genetically modified products; in 1999, it required that non-genetically modified products exported to the EU must not contai...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/63C12N15/66
Inventor 王建华王秀敏滕达杨雅麟
Owner FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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