Five-gene standard plasmid molecule for transgenic soybean detection and construction thereof
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A technology of transgenic soybean and standard plasmid, which is applied in the field of plasmid molecules in the field of molecular biology technology, can solve the problem of difficulty in obtaining positive standards of transgenic crops, and achieves the effects of high sensitivity and good specificity
Active Publication Date: 2012-04-11
FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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Problems solved by technology
However, due to the biosafety of genetically modified crops and the limitations of existing technologies, it is difficult to obtain positive standards for genetically modified crops, which has become a bottleneck in detection methods and applications
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Embodiment 1
[0034] Embodiment 1: Construction of standard plasmid molecules
[0038] 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.; other biochemical reagents were imported or domestically produced analytically pure.
[0039] 3. Experimental equipment
[0040] TC-512 PCR amplification instrument (Techne, UK); Geliance 200 DNA electrophoresis gel imager (PerkinElmer, USA); Nano-Drop ND-1000 nucleic acid protein analyzer (Nano D...
Embodiment 2
[0091] Example 2: Application of the constructed standard plasmid molecules in actual detection
[0092] 1. Enzymes and Reagents
[0093] 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.
[0097] 1. Qualitative PCR detection of standard plasmid molecule pTLE5
[0098] Qualitative PCR detection methods of soybean internal reference gene Lectin 1, transgenic soybean GTS40-3-2 and A2704-12 structural gene specificity, transgenic soybean screening sequence 35S promoter and NOS terminator were obtained by consulting relevant literature. The PCR reaction syst...
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Abstract
The invention discloses a standard plasmid molecule necessary for transgenic crop detection and a construction method thereof, and particularly discloses a standard plasmid molecule for transgenic soybean detection and a construction method thereof. The standard plasmid molecule comprises a soybean endogenous gene Lectin-specific fragment, a 35S promoter and NOS terminator-specific fragment, a transgenic soybean line A2704-12 foreign gene PAT-specific fragment, and a transgenic soybean line GTS40-3-2 foreign gene EPSPS-specific fragment. By analyzing the specific sequences of the above five genes, specific PCR (polymerase chain reaction) primers are designed, and five target gene fragments are obtained by amplification. By a fusion PCR method, the five genes are fused to form a long fragment, and the long fragment is cloned to plasmid vector pMDTM19-T Simple to obtain a recombinant plasmid molecule pTLE5. The standard plasmid molecule constructed in the invention can completely substitute for a positive standard product, and is completely suitable for screening as well as structural-gene-specific qualitative and quantitative PCR analysis and detection of transgenic soybean lines GTS40-3-2 and A2704-12. If a new transgenic material emerges, a new foreign gene fragment can be added to an original standard molecule, thus the detection requirements of increasing transgenic crop materials are met.
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...
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Patent Type & Authority Applications(China)
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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