Standard plasmid molecular used for detecting transgenic soybean, corn and cotton and construction of the standard plasmid molecular

A technology of transgenic soybean and transgenic corn, 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 standard samples of transgenic crops, and achieve the effects of high sensitivity, high specificity and repeatability

Active Publication Date: 2012-07-11
FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Genomic DNA extracted from positive standards (CRMs) is usually used as a positive control, but due to the biosafety of genetically modified crops and the limitations of existing techn...

Method used

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  • Standard plasmid molecular used for detecting transgenic soybean, corn and cotton and construction of the standard plasmid molecular
  • Standard plasmid molecular used for detecting transgenic soybean, corn and cotton and construction of the standard plasmid molecular
  • Standard plasmid molecular used for detecting transgenic soybean, corn and cotton and construction of the standard plasmid molecular

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Construction of standard plasmid molecules

[0029] 1. Experimental materials

[0030] Transgenic maize lines Bt176 and Mon810; non-transgenic maize varieties.

[0031] 2. Experimental reagents

[0032] pMD TM 19-T Simple Vector was purchased from Dalian Bao Bioengineering Company; dNTPs, Taq DNA polymerase, DNA marker I, II and marker III were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; restriction endonucleases 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 subpackages or domestic analytical grades.

[0033] 3. Experimental equipment

[0034]TC-512 PCR Amplifier (TECHNE, UK); Geliance 200 DNA Electrophoresis Gel Imager (PerkinElmer, USA); Nano-Drop ND-1000 Nucleic Acid Protein Analyzer (Nano Drop, USA); centrifuge; constant tem...

Embodiment 2

[0066] Example 2: Application of constructed standard plasmid molecules in actual detection

[0067] 1. Enzymes and Reagents

[0068] Real-time PCR detection kit (Premix Ex Taq TM (Perfect Real Time)) were purchased from Bao Bioengineering (Dalian) Co., Ltd.; primers and TaqMan probes were synthesized by Shanghai Jikang Biotechnology Co., Ltd.; other biochemical reagents were imported subpackage or domestic analytical grade.

[0069] 2. Experimental equipment

[0070] Real-time fluorescence quantitative PCR instrument 7500 (ABI company).

[0071] 3. Experimental method and process

[0072] 1. Design and synthesis of fluorescent quantitative PCR primers and probes

[0073] Primers and probes for quantitative PCR were designed using the software Primer Premier 5.0 according to the transformation event sequences at the 3' ends of Hmg and Bt176 and Mon810 strains in standard plasmid molecules, as shown in Table 4. The 5' end of the TaqMan probe is labeled with the reporter fl...

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Abstract

The invention discloses a necessary standard plasmid molecular and a construction method of the standard plasmid molecular in the field of transgenic crop detection. The construction method comprises the following steps: modifying an octo-gene plasmid molecular pTLE8 (Patent Application No.201010286884.3), adding a fusion fragment of 3'-transformation event specific sequences of transgenic corn Bt176 strain and Mon810 strain and corn internal reference gene Hmg specific sequence, and modifying into a deca-gene plasmid molecular pTLH10 (Lec1, 35S, NOS, PAT, Sad1, Cry1, Ab/c, Bt176, Mon810 and Hmg). The standard plasmid molecular constructed by the invention can completely replace a positive standard sample, and is suitable for screening transgenic soybean GTS40-3-2 strain, transgenic corn Bt176 and Mon810 strains and transgenic Bt cottons, and qualitative and quantitative PCR analysis and detection of gene specificity and strain specificity.

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 detection of transgenic soybean, corn and cotton and a construction method thereof. Background technique [0002] Since the commercialization of transgenic plants, the global GM planting area has continued to expand. From 1996 to 2009, the global GM crop planting area has reached 690 million hectares, and the GM crop planting area has increased by 67 times in the past 10 years. The GM crops grown are mainly GM soybean, GM corn and GM cotton. The GM soybean planted the largest area with 58.6 million hectares, accounting for 57% of the global GM crop area, followed by GM corn (25.2 million hectares, or 25%) and GM cotton (13.4 million hectares, or 13%). With the widespread cultivation of genetically modified crops, their safety issues have attracted widespread attention from the global public. In many countries, nota...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/63
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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