Cloning And Expression Analysis Of Glutathione Reductase Gene In Chlamydomonas Sp. Ice-l From Antarctica

The full-length glutathione reductase gene of Antarctica microalgae (ICE-LGR) was cloned, and its recombinant of prokaryotic expression vector was constructed in this paper. Expression level of ICE-L GR under low salinity, high salinity and Cadmium chloride was analyzed. Partial cDNA sequence ofβ-actin gene (ICE-L actin) and Glyceraldehyde -3 - phosphate dehydrogenase gene (ICE-L GAPDH) were cloned as well.RNA was extracted and separated from Chlamydomonas sp. ICE-L at first.And then, primers for PCR were designed according to two conserved regions of glutathione reductase reported in many other species. ICE-L GR (GenBank accession number: GU395492) was successfully cloned by Rapid Amplification of cDNA Ends (RACE) technique, and characterized by bioinformatics analysis. The full-length of ICE-L GR cDNA contains 1913 bp nucleotides with a 90bp 5 'untranslated region (UTR), 365bp of 3' UTR and an open reading frame (ORF) of 1458 bp, comprising 485 amino acid residues with a theoretical molecular weight(MW) of 52.155 kDa and an estimated isoelectric point(IP) of 5.56.The deduced amino acid sequence shows high identities with GR from Chlamydomonas reinhardtii, Ostreococcus lucimarinus, Thalassiosira pseudonana, Ulva fasciata, Arabidopsis thaliana, Brassica juncea, Glycine max and Oryza sativa.A recombinant of prokaryotic expression vector pET-GR was constructed and transformed to E.coli. BL21 to express ICE-GR protein. SDS-PAGE sesults indicated that the molecular weight of the target protein was similar to theoretic molecular weight. The optimum expression condition was 1.0mmol/L concentration of IPTG, 28℃induced temperature, and 3 hours induced time. ICE-L GR protein was expressed as inclusion body.The expression patterns of ICE-L GR under the challenge of low salinity, high salinity and Cadmium chloride stress were examined by Real-time PCR analysis.The expression reached a higher level after 24h in the salinity of 11, 22, 66; In the salinity of 99 group, expression level reached the maximum at 12h. Low concentration of heavy metal CdCl2 upregulated GR expression. In 20 and 40umol/L groups, GR gene expressed most abundantly at 12h, but expression reached the peak at 18h in 60 and 80 umol/L group. Results obtained from this study provide valuable information on further investigating the molecular mechanism of ICE-L GR, and make biological universality and diversity of glutathione reductase gene more rich. There is also an important theoretical significance to comprehensively understand the structure, function and features of Antarctic glutathione- related enzyme genes. And it is helpful to clarify effectively adaptative mechanism of Antarctic microbes various stress. Furthermore, the study can offer new ideas and new gene for genetic engineering breeding on stress-tolerance species.