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Method for detecting activity of chloroplast promoter on basis of tetracycline regulation system

A technique for regulating systems and promoters, applied in the field of plant plastid genetic engineering research, can solve the problems of few reports on tetracycline-induced expression systems

Active Publication Date: 2016-07-06
YUNNAN AGRICULTURAL UNIVERSITY
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Problems solved by technology

[0005] At present, the Tet regulatory system has been widely used in medical treatment, and it is the most widely used induction system at present. It is used in combination with the Cre / loxP recombinase system to control the expression of the Cre recombinase gene, but it has not been successful in many plants so far. , there are very few reports on the use of tetracycline-induced expression systems in plastid genetic engineering

Method used

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  • Method for detecting activity of chloroplast promoter on basis of tetracycline regulation system
  • Method for detecting activity of chloroplast promoter on basis of tetracycline regulation system
  • Method for detecting activity of chloroplast promoter on basis of tetracycline regulation system

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Embodiment 1

[0026] 1. Prepare materials

[0027] 1.1 Instrument

[0028] PCR instrument, ultra-low temperature refrigerator, centrifuge, ultra-clean operating table, electrophoresis instrument, ultraviolet gel imaging system, ultra-pure water system.

[0029] 1.2 Experimental reagents

[0030] Competent cells, pEASY-Blunt cloning vector, and DNA polymerase were purchased from Beijing Quanshijin Biological Company; T4 ligase and various restriction endonucleases were purchased from Takara Biological Company; plasmid recovery kits and gel recovery kits were purchased from Yu Zhuangmeng International.

[0031] 1.3 Primer design

[0032] Primer sequence SEQID used in the present invention

[0033] NO:1(5'-CAAGCTTGCTCCCCCGCCGTCGTTCAAT-3'),

[0034] NO:2(5'-GTCTAGAGCTAGCCTGTCCACCAGTCAT-3'),

[0035] NO:3(5'-CGTCGACATGTCTAGATTAGATAAAAGTA-3'),

[0036] NO:4(5'-CGGTACCTTTAAGACCCACTTTTCACATTTA-3'),

[0037] NO:5 (5'-CAAGCTTCATGAATAAATGCAAGAAAAT-3') was synthesized by Beijing Huada Gene Compan...

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Abstract

The invention relates to a method for detecting activity of a chloroplast promoter on the basis of a tetracycline regulation system, belonging to the technical field of plant plastogene engineering research. The method comprises the following steps: on the basis of the tetracycline gene and tetracycline specific recognition sequence core control region, biosynthesizing a prrnO1O2 promoter with the tetracycline core control region, verifying the activity of the promoter in prokaryotic cells, and carrying out GFP (green fluorescent protein) gene expression under the driving action of the promoter; establishing a tetracycline-induced expression vector, and screening out the tetracycline suitable for bacterium growth, of which the maximum concentration is 5 mu g / ml; and finally, establishing the GFP expression vector under the tetracycline regulation system, wherein the function of the prrnO1O2 promoter is inhibited when the tetracycline is not added, and the GFP gene expresses green fluorescent proteins after the tetracycline is added. The tetracycline regulation system is utilized to control the activity of the chloroplast promoter, thereby avoiding the regulation interference of the nuclear genome on the plastome, and providing an effective method and way for further plastogene engineering breeding.

Description

Technical field: [0001] The invention relates to a method for detecting chloroplast promoter activity based on a tetracycline regulation system, which belongs to the technical field of plant plastid genetic engineering research. Background technique: [0002] Chloroplast genetic engineering is accompanied by the continuous deepening of chloroplast genome research. At present, chloroplast genome transformation technology has been obtained in higher plants such as tobacco, tomato, potato, Arabidopsis, soybean, carrot, cauliflower, rape, rice and cotton. success. [0003] Compared with nuclear transgenic technology, chloroplast gene transformation has obvious advantages. (1) Through the homologous recombination of DNA fragments, the insertion site of the foreign gene can be precisely controlled; (2), the safety and stability are good. The chloroplast genes of most plants are inherited from the maternal line, which greatly reduces the ecological risk caused by gene diffusion; ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/70
CPCC12Q1/6897
Inventor 许俊强张应华曹绍玉毕保良董玉梅于晓俊
Owner YUNNAN AGRICULTURAL UNIVERSITY
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