Use and utilizing method of genes dtrA and dtr B of two-component system

A two-component, gene technology, applied in the field of genetic engineering

Active Publication Date: 2014-04-16
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no report on the effects of dtrA (dr a0009, Gene ID: 1798204) and dtrB (dr a0010, Gene ID: 1798154) genes in Deinococcus radiodurans R1 (Deinococcus radiodurans R1) on heat shock response and UV irradiation

Method used

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  • Use and utilizing method of genes dtrA and dtr B of two-component system
  • Use and utilizing method of genes dtrA and dtr B of two-component system
  • Use and utilizing method of genes dtrA and dtr B of two-component system

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Experimental program
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Effect test

Embodiment 1

[0049] The cloning of embodiment 1dtrA and dtrB nucleotide sequence SEQ ID NO:1

[0050] According to the published genome sequence of Deinococcus radioduransR1 strain, specific primers for PCR amplification of dtrA and dtrB genes were designed. The complete dtrA and dtrB nucleotide sequences were amplified from Deinococcus radiodurans genomic DNA:

[0051]dtrA-Up 5'CGCCACGAACACGGTCAG 3'

[0052] dtrA-Down 5’CACTCAGCGGCACGAAGG 3’

[0053] dtrB-up: 5'CACCGGCCTGAACTCCAT 3'

[0054] dtrB-down: 5'TCGCACCGTTCCTACACC 3'

[0055] PCR reaction conditions were 95°C, 5min, [95°C 30sec, 57°C 30sec, 72°C 90sec] 30 cycles, 72°C extension for 10min, PCR products were purified by column, dtrA was cloned on pMD19T vector, dtrB was cloned on pGEM-T Easy carrier.

Embodiment 2

[0056] Example 2 Construction of ΔdtrA and ΔdtrB mutant strains

[0057] The dtrA gene contains a SacII restriction site, and the dtrB gene contains an EcoRV restriction site. Use SacII enzyme and EcoRV enzyme to cut the dtrA gene and dtrB gene connected to the pMD19T vector and pGEM-T Easy vector from the middle, Insert the Kana resistance cassette with the same restriction site at both ends, and verify the correct cloned gene by sequencing; amplify the target fragment inserted into the Kana resistance cassette by PCR; pass the DNA fragment inserted into the Kana resistance cassette through The method of homologous recombination was directly transformed into Deinococcus radiodurans R1, its genome was extracted, and the target fragment was amplified with verification primers and electrophoresed. For the verification map, see figure 1 and figure 2 .

[0058] The specific conversion method is as follows:

[0059] 1) Pick a single colony of Deinococcus radiodurans R1 in TGY l...

Embodiment 3

[0066] Example 3 Heat Shock Response Experiment of Wild Type R1 and Mutants ΔdtrA and ΔdtrB

[0067] 1. Experimental method

[0068] After wild-type R1 and mutant strains ΔdtrA and ΔdtrB were activated, they were inoculated in TGY liquid medium (R1) without any antibiotics and TGY liquid medium containing 8 μg / mL kanamycin ( ΔdtrA and ΔdtrB), cultured with shaking at 30°C for 10 h, and cultured to the logarithmic phase (OD 600 ≈0.8), for heat shock treatment.

[0069] 1) Take 20mL of wild-type R1 and mutant strains ΔdtrA and ΔdtrB, centrifuge at 6000×g for 10min, collect the bacteria, wash twice with 10mM pH 7.0 phosphate buffer, resuspend in 20mL phosphate buffer culture solution at 30°C for 3 hours, take 1 mL each in a 1.5 mL EP tube, and dilute with sterile deionized water (10 -1 to 10 -5 ), take 10 μL and dilute to 10 -1 to 10 -5 Wild-type R1 and mutant strains ΔdtrA and ΔdtrB were added dropwise on TGY solid medium without any antibiotics, and the growth of wild-typ...

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Abstract

The invention provides use of genes dtrA and dtr B of a two-component system for improving the heat sensitivity of strains and UV radiation resistance.. The invention further provides a utilizing method of the genes dtrA and dtr B of the two-component system, and the utilizing method comprises the following steps of: cloning genes dtrA and dtr B of the two-component system, wherein sequences of the genes of the two-component system are shown as SEQ ID NO:1 and SEQ ID NO:2; and further constructing deltadtrA and deltadtrB mutant strains with heat sensitivity and UV (Ultra Violet) radiation resistance. The function of the genes dtrA and dtr B of the two-component system for improving the heat sensitivity of strains and UV radiation resistance can be utilized in fermenting industry and cosmetic industry for production of UV radiation resistance products, and the genes dtrA and dtr B of the two-component system are good in application prospect.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to the use and utilization method of a pair of two-component system genes dtrA and dtrB in Deinococcus radiodurans R1 for improving strain heat sensitivity and UV radiation resistance. Background technique [0002] Deinococcus radiodurans R1 (Deinococcus radiodurans R1) has super strong resistance to ionizing radiation, ultraviolet rays, DNA damage agents and drying, and has attracted the attention and attention of scientists from all over the world. For example, Song Daojun and Yu Zengliang's "New Progress in the Research on the Radiation Resistance Mechanism of Deinococcus radiodurans" reported the physiological, biochemical and genetic characteristics of Deinococcus radiodurans, the special cell membrane structure, the DNA damage caused by various mutagenic factors and its relationship with other countries. Research on efficient repair mechanism; Genome analysis shows that ther...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/90C12N1/20C12R1/01
Inventor 陈明郝艳华张维王文涛赵鹏林敏
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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