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Functional Identification and Transcriptome Analysis of Cold Shock Protein in Deinococcus Radiodurans

Author: LiXinNa
Tutor: ZhangWei
School: Chinese Academy of Agricultural Sciences
Course: Microbiology
Keywords: Resistant to radiation anomalies radiodurans R1 Cold stress Cold shock proteins Transcriptome Regulatory mechanism
CLC: Q78
Type: Master's thesis
Year: 2011
Downloads: 58
Quote: 0
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Abstract

Cold shock proteins (cold shock protein, Csp) a large number of synthetic, usually in cold environments commonly found in animals, plants and eubacteria. The type of protein that contains a specific combination of RNA and ssDNA cold shock domain RNA chaperone or transcriptional enhancer function, plays an important role in the regulation of cold stress in biological adaptation reaction. Resistant to radiation the exception cocci (Deinococcus radiodurans.) R1 contain a CSP-like gene, the mutation causes the cells to ionizing radiation sensitive, but the gene having a cold shock protein function is unclear. Csp-like genes function and its regulation this paper, through gene mutation, Biolog technology and transcriptome analysis, in-depth study research progress as follows: csp-like gene encodes a 10 kDa protein, containing 86 amino acids. residues. Sequence analysis showed it with Bacillus subtilis CspB consistency was 50%, containing a cold shock domain of the specific binding of RNA and ssDNA. Real-time quantitative PCR analysis showed that csp-like genes 15 ° C 2 h of cold shock increased 1.87-fold expression induced by cold shock characteristics. △ _csp mutant in optimum temperature when compared with the wild-type R1 strains grow slowly to 15 ° C low temperature growth arrest. The results show that the gene product is a typical cold shock proteins. Biolog analysis found that, △ _csp mutant significant change in the carbon utilization levels than the wild-type strain R1 csp missing cause cells to glucose and other carbon utilization decreased significantly, three kinds of carbon utilization improve. The gel retardation experimental results show that, D. radiodurans Csp binding thymine-rich ssDNA, binding sequence or the reverse complementary sequence of the Y-box (ATTGG), combining the estimated sequence analysis, Csp may be provided through a combination of its promoter upstream Y -box reverse complementary sequence to enhance their own expression. To further study the role of csp gene regulation of cellular metabolism, the paper carried the wild-type R1 strain under normal growth conditions and △ _csp Transcriptome analysis of the mutant. The results show that, the csp mutation resulting in 466 genes were up-regulated and 322 genes were down-regulated expression of different gene products and cell wall / membrane / cell envelope biosynthesis, Translation / ribosomal structure and biosynthesis, replication recombination and repair, signal transduction mechanism, the energy generating and conversion, amino acid transport and metabolism, transport and metabolism of lipids and secondary metabolites of the synthetic transport and catabolism and so on. Real-time quantitative validation of the results. Which carotenoid synthesis gene the overall enhanced expression, the of synthetic key gene crtI raised 2.8 times. Determination of cell morphology and pigment content found that the csp mutant cultures colony color deepened, the intracellular and extracellular carotenoid content increased significantly, suggesting that the csp genes involved in the regulation of cytochrome synthesis. responses the ddrB, ddrI, recA, pprA and irrE DNA damage repair proteins majority induced expression of oxidation resistance protein genes were downregulated speculated csp cause oxidation resistance protein decline is caused by a mutant strain sensitive mainly due to UV radiation. Peptidoglycan synthesis gene and cell envelope of the S-layer protein gene upregulation, the cell ultrastructure electronic microscope substances increased the outermost layer of the cell wall of the mutant was bubbly overflow or even fall off; ABC transporter-related gene expression significantly change, developing mass spectrometry showed that the mutant cells polypeptide or lipid-peptide material distribution change that csp impact the normal cell envelope morphogenesis and material transport. This study showed that resistant to radiation abnormal cocci cold shock protein Csp through the direct or indirect role, affecting a variety of physiological stress response of the cells, including the antioxidant protection of cells, DNA damage repair and pigment synthesis process to further reveal the resistance of radiation anomalies cocci extreme environmental adaptation mechanism laid a theoretical foundation.

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