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Standard molecule for specifically detecting transgenic rice strain Kefeng No.6 and application thereof

A technology of encoding genes and sequences, which is applied in the field of specific detection of standard molecules of the transgenic rice line Kefeng 6, which achieves the effects of wide linear range, strong stability and good uniformity

Active Publication Date: 2013-06-19
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no relevant report on the plasmid standard molecule for specific detection of the transgenic rice line Kefeng 6

Method used

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  • Standard molecule for specifically detecting transgenic rice strain Kefeng No.6 and application thereof
  • Standard molecule for specifically detecting transgenic rice strain Kefeng No.6 and application thereof
  • Standard molecule for specifically detecting transgenic rice strain Kefeng No.6 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1, the construction of standard molecule pEASY-KF6 and the synthesis of specific primers and probes

[0040] 1. Construction of the standard molecule pEASY-KF6

[0041] The DNA fragment shown in Sequence 1 in the synthetic sequence listing, from the 5' end to the 3' end is as follows: GCG-Hind III-cowpea trypsin inhibitor coding gene (CpTI gene) fragment (position 10-101)-BamH I -Right border sequence (right border sequence RB) fragment of Ti plasmid T-DNA (108-184) -BamH I-rice endogenous gene GOS fragment (191-258) -Hind III-Bacillus thuringiensis insecticide Crystal protein coding gene (Bt gene) fragment (position 265-336)-XbaⅠ-GCG.

[0042] The DNA fragment shown in sequence 1 in the sequence list synthesized above was connected to the pEASY-T3 vector in the form of T-A cloning, and the ligated product was transformed into Escherichia coli, and the recombinant bacterial suspension was detected by PCR using primers KF-F and KF-R . At the same time, the ...

Embodiment 2

[0051] The specific detection of embodiment 2, specific primer and probe

[0052] The transgenic rice line Kefeng 6 (GMO+), the non-transgenic rice line Minghui 86 (GMO-), the transgenic tomato line Huafan 1, the transgenic rice line Bt63, and the transgenic cotton (Bt insect-resistant gene) line 41 and the total DNA of the phytase gene-transformed corn line BVLA430101 were used as templates, and the primers and probes in Table 1 of Example 1 were used to perform real-time fluorescent PCR reactions on the corresponding genes to detect the specificity of the primers and probes. The experiment was repeated three times.

[0053] Real-time fluorescent PCR reaction system: Mix 12.5 μl AB TaqMan Gene Expression Master Mix, 5 μl DNA template, 5 μl sterilized ultrapure water, 1 μl forward primer, 1 μl reverse primer and 0.5 μl specific probe to obtain a 25 μl reaction system; In the reaction system, at the beginning of the reaction, the concentrations of the forward and reverse prime...

Embodiment 3

[0057] Embodiment 3, the sensitivity detection of standard system and the linear range detection of standard curve

[0058] Adjust the concentration of the standard molecule pEASY-KF6 constructed in Example 1 to 3×10 9 copy / μl, and then diluted 10 times according to the concentration gradient, the dilution was from 3×10 6 copies / μl to 3×10 -1 A total of 8 gradients per copy / μl, using the primers and probes in Table 1 of Example 1 to carry out real-time fluorescent PCR reactions on the corresponding genes, thereby determining the sensitivity and standard curve of the standard system composed of standard molecules and specific primers and probes the linear range. The experiment was repeated 3 times, and the results were averaged.

[0059] Real-time fluorescent PCR reaction system: Mix 12.5 μl AB TaqMan Gene Expression Master Mix, 5 μl DNA template, 5 μl sterilized ultrapure water, 1 μl forward primer, 1 μl reverse primer and 0.5 μl specific probe to obtain a 25 μl reaction sy...

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Abstract

The invention discloses a standard molecular for specifically detecting a transgenic rice strain Kefeng No.6 and an application thereof. The standard molecule disclosed by the invention is an annular carrier which comprises a cowpea trypsin inhibitor coding gene segment shown by the 10th-101st sites of a sequence 1, a bacillus thuringiensis insecticidal crystal protein coding gene segment shown by the 256th-336th sites of the sequence 1, a rice endogenous gene GOS segment shown by the 191th-258th sites of the sequence 1 and a border sequence segment of Ti plasmid T-DNA shown by the 108th-184th sites of the sequence 1. Experiments prove that the standard molecule disclosed by the invention has high sensitivity, wide linear range, good uniformity and strong stability, and is of important significance to the qualitative and quantitative detection of the transgenic rice strain Kefeng No.6; and the lowest detection line of the standard molecule can reach 2 copies.

Description

technical field [0001] The invention relates to a standard molecule for specifically detecting transgenic rice line Kefeng No. 6 and its application. Background technique [0002] Rice is one of the most important food crops in my country and even in the world. It is the crop with the largest population and the longest history in the world. With the development of biotechnology, transgenic technology is being widely used to improve the quality of crops. Several transgenic rice strains have been released in the environment and applied for commercial planting. Kefeng 6 is a transgenic rice line with a bivalent insect-resistant gene, and the foreign gene introduced is Bt / CpTI. [0003] In the detection of genetically modified products, standard substances must be used as positive controls or standard curves must be used to accurately detect genetically modified products. The standard plasmid molecule is a recombinant plasmid molecule developed in recent years that can replace...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12Q1/68
Inventor 黄新任鹤
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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