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Primers for detecting phosphomannose isomerase gene in transgenic plant product, and probe thereof

A technology of mannose phosphate, transgenic plants, applied in the direction of determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc.

Inactive Publication Date: 2014-01-01
INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no PCR detection kit for mannose phosphate isomerase gene in transgenic plants in China. The present invention can make up for the above defects, and complete the detection of mannose phosphate isomerase gene in transgenic plants by using real-time fluorescent PCR detection method

Method used

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  • Primers for detecting phosphomannose isomerase gene in transgenic plant product, and probe thereof
  • Primers for detecting phosphomannose isomerase gene in transgenic plant product, and probe thereof
  • Primers for detecting phosphomannose isomerase gene in transgenic plant product, and probe thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Detect transgenic corn 3272 according to the following method, use 59122, BT11, MON88017, MON810 transgenic corn lines as negative controls:

[0029] Including 10×PCR reaction buffer, 0.2mmol / L dNTP, 2mmol / L magnesium sulfate, 1.5U Taq enzyme, 0.4μmol / L upstream primer PMI-taqF: 5'-ccgggtgaatcagcgttta-3'; downstream primer PMI-taqR: 5 '-gccgtggcctttgacagt-3'; Taqman probe PMI-taqP:FAM-tgccgccaacgaatcaccgg-TAMARA. Wherein 10×PCR reaction buffer contains 100mmol / L tris-hydrochloric acid of pH8.8, 500mmol / L potassium chloride and 1% Triton X-100;

[0030] Follow the procedure below for testing:

[0031] (1) DNA extraction of transgenic corn samples to be tested

[0032] Using Ambio kit (GenoDNA Plant Mini Kit)

[0033] A. Full grinding

[0034] B. Add 400μg BufferAP1 and 0.5μl RNaseA to each 75mg fresh material or 15mg dry material sample at most, and mix well;

[0035] C. Incubate at 65°C for 10 minutes, and invert the centrifuge tube 2-3 times during the period;

...

Embodiment 2

[0051] Detect transgenic maize MIR604 as follows, using GTS 40-3-2, MON89788, A2704-12 transgenic soybean lines as negative controls:

[0052] Including 10×PCR reaction buffer, 0.2mmol / L dNTP, 2mmol / L magnesium sulfate, 1.5U Taq enzyme, 0.4μmol / L upstream primer PMI-taqF: 5'-ccgggtgaatcagcgttta-3'; downstream primer PMI-taqR: 5 '-gccgtggcctttgacagt-3'; Taqman-MGB probe PMI-taqP:FAM-tgccgccaacgaatcaccgg-TAMARA. Wherein 10×PCR reaction buffer contains 100mmol / L tris-hydrochloric acid of pH8.8, 500mmol / L potassium chloride and 1% Triton X-100;

[0053] Follow the procedure below for testing:

[0054] (1) DNA extraction of transgenic corn samples to be tested

[0055] Using Ambio kit (GenoDNA Plant Mini Kit)

[0056] A. Full grinding

[0057] B. Add 400μg BufferAP1 and 0.5μl RNaseA to each 75mg fresh material or 15mg dry material sample at most, and mix well;

[0058] C. Incubate at 65°C for 10 minutes, and invert the centrifuge tube 2-3 times during the period;

[0059] D. ...

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Abstract

The invention belongs to a primer and a probe of a transgenic plant product, and concretely relates to a primer for detecting a phosphomannose isomerase gene in the transgenic plant product through a real-time fluorescent PCR technology, and a probe thereof. Regents used in the invention comprise a PCR amplification reaction solution, an upstream primer PMI-taqF5'-CCGGGTGAATCAGCGTTTA-3' and a downstream primer PMI-taqR:5'-GCCGTGGCCTTTGACAGT-3', and a probe PM1-taqP:FAM-TGCCGCCAACGAATCACCGG-TAMARA. A method for detecting phosphomannose isomerase gene in transgenic plant product comprises the following steps: extracting DNA from the transgenic plant product, carrying out real-time fluorescent PCR amplification of the phosphomannose isomerase gene in the transgenic plant product, and analyzing amplification results by using fluorescent quantitative PCR instrument random software to determine the results. The method has the advantages of rapidity, strong specificity, high sensitivity and simple operation.

Description

technical field [0001] The invention belongs to the detection technology of plant source components, in particular to primers and probes for detection of phosphomannose isomerase gene of transgenic plant products by real-time fluorescent PCR technology. Background technique [0002] The mannose positive selection system belongs to one of the non-antibiotic screening systems. It uses the phosphomannose isomerase gene (PMI) of Escherichia coli as a selection gene and mannose as a selection agent to screen transgenic plants. Mannose is catalyzed and phosphorylated into mannose-6-phosphate by plant endogenous hexokinase, and the reaction consumes ATP and phosphate, which will lead to cell division and lack of energy for growth, and the excessive accumulation of mannose-6-phosphate is toxic to plant cells . Transformed cells integrated with exogenous PMI gene can convert 6-phosphate mannose isomerase into 6-phosphate fructose, so that the metabolism can continue, avoiding the la...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q2561/101C12Q2561/113C12Q2531/113
Inventor 高宏伟王述柏孙敏周茜
Owner INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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