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Standard plasmid molecule used for detecting genetically modified soybeans and cotton and building method thereof
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A technology of transgenic soybean and standard plasmid, applied in the field of molecular biology, can solve the problem of difficulty in obtaining positive standard of transgenic crops, and achieve the effect of high sensitivity and good specificity
Active Publication Date: 2012-04-11
FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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Embodiment 1
[0040] Embodiment 1: Construction of standard plasmid molecules
[0044] PPML TM 19-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.;
[0045] 3. Experimental equipment
[0046] TC-512 PCR amplification instrument (TECHNE, UK); Geliance 200 DNAelectrophoresis gel imager (PerkinElmer, USA); other biochemical reagents were imported or domestically produced analytically pure. Nano-Drop ND-1000 nucleic acid...
Embodiment 2
[0097] Example 2: Application of the constructed standard plasmid molecules in actual detection
[0098] 1. Enzymes and Reagents
[0099] 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.
[0103] 1. Design and synthesis of fluorescent quantitative PCR primers and probes
[0104] 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 dye...
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Abstract
The invention discloses a standard plasmid molecule used for detecting genetically modified soybeans and cotton and a building method thereof. The standard plasmid molecule contains eight gene specific fragments (Lec1, 35S, NOS, PAT, Sad1, Cry1Ab / c and EPSPS) of soybeans and cotton. The standard plasmid molecule and the building method have the following beneficial effects: the eight genes are fused into a large fragment through fusion PCR (polymerasechain reaction) and the large fragment is cloned on a plasmid vector to obtain a recombinant plasmid molecule pTLE8; the standard plasmid molecule built in the invention can replace the positive controls and is suitable for the screening of the genetically modified soybean strain GTS40-3-2 and Bt cotton and the qualitative and quantitative PCR analysis and detection of the gene and strain specificity; 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 belongs to the technical field of molecular biology, and in particular relates to a standard plasmid molecule for detection of transgenic soybeans and transgenic cotton and a construction method thereof. Background technique [0002] Since the first transgenic tomato "FLAVR SAVR" came out, with the continuous development of biotechnology, genetically modified plants (GMP) have been more and more used in the agricultural field. From the first successful commercialization in 1996 to 2009, herbicide-resistant products have been dominant in genetically modified crops, followed by insect-resistant genes. According to statistics, in 2009, the approved planting of transgenic crops in the world was about 134 million hectares, of which the planting area of transgenic soybeans reached 69 million hectares, accounting for 77% of the total soybean planting area. The planting area of transgenic cotton was 16.17 million hectares, accounting for 49%...
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IPC IPC(8): C12Q1/68C12N15/63C12N15/66C12N15/10
Inventor 王建华王秀敏杨雅麟滕达
Owner FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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