Isolation And Screening Of Cold-Induced Promoter By Promoter Probe Vector From Arthrobacter Sp.A3

Transcription is one of the most important biological processes of organisms. Promoter, the core component of gene transcription and regulation, can directly affect the formation of biological characteristic and the biological adaptability to the external environment. Arthrobacter sp.A3 is isolated from the Number 1 Glacier permafrost, which is a strain of high-G+C Gram-positive psychrotrophic bacterium. In order to study and explain the resistant mechanisms of Arthrobacter sp.A3, we have carried out the research on screening cold-induced promoter in Arthrobacter sp.A3. Promoter probe vector is constructed in this study to screen cold-induced promoters. As a result, nine cold-induced promoters are discovered and their intensities are measured. All these results establish the foundation for subsequent functional study. The major findings are as follows:(1) In the present study we have moved promoter fragments from pART2 plasmid and inserted Amp-gfp fusion gene with three different open reading frames as a reporter gene to construct a promoter-probe plasmid pART2-SP-Amp-gfp which can replicate normally in both Arthrobacter sp. A3 and Escherichia coli.(2) We have extracted the genomic DNA from Arthrobacter sp.A3 using CTAB method. After partial digestion with Sau3AI, the genomic DNA fragments are connected with the probe plasmid pART2-SP-Amp-gfp which is digested by BamHI and dephosphorylated in advance to compose the promoter probe plasmid library. The plasmid library is electrically transformed into Arthrobacter sp.A3, and a total of about 900 000 transformed colonies is acquired.(3) Construction of a recombinant plasmid pET-28-gfp.We have induced and expressed GFP-His6 fusion protein and obtained recombinant protein by affinity chromatography. The purified fusion protein GFP-His6 is used as antigen to immunise rabbit to produce polyclonal antibody. The sensitivity and specificity of the antibody are detected using enzyme-linked immunosorbent assay (ELISA) and Western blot. The results show that, the titer of GFP polyclonal antibody are more than 1:100,000. The GFP polyclonal antibody fragment is purified by hybridizing the GFP-Sepharose CL-6B with the antisera of GFP.(4) Nine cold-induced highly-expressed under low temperature promoters are preliminary screened with whole-wavelength microplate reader. The expression levels of GFP in Arthrobacter sp. A3 under low temperature are detected with Western Blot to verify the strengths of the nine cold-induced promoters.The size of cold-induced promoters is about 200bp-400bp, which is validated by PCR amplification. In summary, our study has constructed pART2-SP-Amp-gfp promoter probe plasmid and acquired the screened strain library of the promoter of Arthrobacter. We have expressed in vitro and purified GFP fusion protein, acquired the polyclonal antibody for it, and screened and verified nine cold inducible promoters with whole-wavelength microplate reader and GFP antibody.The size of cold-induced promote is about 200bp-400bp. These results lay a substantial foundation for further study of the regulating mechanism of gene expression, and are valuable to better interpret the resistant mechanism of microbial survival in frozen soil at low temperature. In a word, the studies contain theoretical significance and application prospects.