Function Research Of Anti-freeze Genes From Chlamydomonas Sp. ICE-L

Antarctica is characterized by extreme cold, high salt and intense radiation, the specific geographic environment and climatic characteristics made it an unique cryogenic ecosystems, there are abundant of rich and unique biological resources. Antarctic ice alga Chlamydomonas sp. ICE-L was isolated from the floating ice of Antarctica. For its tolerance ability under freezing temperature, it is a good experimental material on studying the resistance biology. In this paper, we conducted a transcriptionalanalysis of 16 genes through quantitative real-time PCR. And heterogeneous expression of CiIBP （11213）, cloning this gene will be helpful in its functional study.（1） Based on the differences in high-throughput sequencing of gene expression analysis, the research team screened multiple genes, such as ice-binding proteins gene, DEAD-box RNA helicase gene and betaine lipid synthase gene. These genes may be associated with cold-adapted mechanism. In this paper, we analyzed the functional domains of these genes and the basic characteristics of their encoding proteins, compared with homologous protein from other species by constructingphylogenetic trees. The full-length of Chlamydomonas sp. ICE-L IBP gene contains 750bps encoding 248 amino acids with a DUF3494 superfamily conserved domain. The identity to it homologous genes in C. raudensis, Fragilariopsis cylindrus and Navicula glaciei are 29.34%、22.92%、20.48% respectively. The full-length of Chlamydomonas sp. ICE-L RNA helicase gene contained 2604bps encoding 868 amino acids. It contains 9 conservative motifs.Homogeneous analysis demonstrated that the gene was homologous to other RNA helicase with identity of 42.87%  48.13%、42.74%、49.49%in C. reinhardtii, Volvox carterif. Nagariensis, Coccomyxa subellipsoidea C-169 and Auxenochlorella protothecoides respectively. The full-length of Chlamydomonas sp. ICE-L Betaine lipid synthase gene contained 2049bps encoding 683 amino acids. It contains AdoMet_MTase domain and DUF3419 superfamily conserved domain. Homogeneous analysis demonstrated that the gene was homologous to other Betaine lipid synthase with identity of 65.53%、 65.30% in C. reinhardtii and V. carteri f. nagariensis respectively.（2） We conducted a transcriptional analysis of 16 genes under freezing stress conditions through batch real-time PCR analysis. The expression of 16 genes were up-regulated in the-20℃ freezing treatment period at freezing conditions compared to 7℃.And 2 genes ICE-L stearoyl-CoA desaturase （delta-9 desaturase） gene （CiSCD （20469）） and ICE-L calmodulin gene （CiCALM （8475）） had almost no change under freezing treatment.. the expression level of ICE-L chloride channel gene was significantly changed after freezing treatment for 72 h with a relative change of 7.8 fold. The expression level of ICE-L CALM gene was the highest after freezing treatment for 72 h with a relative change of 1.3 fold. The expression level of ICE-L IBP（11213） gene was the highest after freezing treatment for 3 h with a relative change of 3 fold. The expression level of the rest of the genes after freezing treatment with a relative change of 1.8-6.5 fold.（3） CilBP （11213） was ligated into plasmid pET-28a（+） and transformed into E. coli （BL21）, SDS-PAGE results showed that there was substantive expression of IBP in E. coli. The product existed as inclusion bodies in E. coli. The heterologous over-expression of genes encoding CilBP （11213） can enhance the freezing resistance of E. coli. And CilBP （11213） also was ligated into plasmid pChlamy₃ and transformed into C. reinhardtii, we found that CiIBP（11213）can enhance the freezing resistance of some of the recombination strains.In this paper, the functions of 16 ICE-L cold-resistant relative genes have been studied. The results provide the basis for revealing adaptive mechanism of ice algae to the extreme environments of Antarctic and the related functional gene development and utilization. And the results will provide theoretical basis to apply the functional gene of the Antarctic ice algae in improving the anti-stress ability of economic crops.