Studies On The Antifreeze Mechanism Of Chlorella Vulgaris Strain NJ-7

Chlorella vulgaris is a unicellular green alga of the genus Chlorella, Oocystaceae, Chlorococcales, Chlorophyceae, Chlorophyta. C. vulgaris NJ-7 was found on the surface of rocks nearby the Zhongshan Station (China) of Antarctica, isolated and purified by our laboratory. Unlike most other C. vulgaris strains, it grows normally at 4℃and above 20℃. To explore the molecular mechanism of its adaption to low temperature, I analyzed the differential expression of genes in C. vulgaris NJ-7 at the two temperatures. The complete cDNA and genomic DNA sequences were obtained for several genes up-regulated at the low temperature. The expression mode of these genes was compared in C. vulgaris NJ-7 and a temperate strain C. vulgaris UTEX259. Moreover, two antifreeze protein genes of C. vulgaris NJ-7 were expressed in Chlamydomonas reinhardtii.The main researches and results are described in three parts:(1) By using Suppression Subtractive Hybridization (SSH) technology, two subtractive cDNA libraries were constructed with C. vulgaris NJ-7 growing at 4℃and 20℃. After two rounds of dot blot hybridizations, 20 cDNA fragments up-regulated at 4℃were identified from 400 clones. Of these fragments, six were homologous to certain functional genes, including genes encoding two antifreeze proteins in C. vulgaris C-27 (hiC6, hiC12), thiazole biosynthetic enzyme (thi), hot shock protein (hsp70), ribosomal proteins L10A and L18. The other 14 cDNA fragments showed no significant similarity to any gene in GenBank and were presumptively novel genes. Northern blot hybridizations confirmed the differential expression of these cDNA fragments at the two temperatures. In addition, 19 cDNA were found to be down-regulated at 4℃.(2) Using Rapid Amplification of cDNA End (RACE) technology, complete cDNA sequences of 5 genes similar to known genes were obtained. By screening a genomic library, the complete genomic DNA sequences of these genes were also obtained. Analysis showed that these genes contained no TATA and CAAT boxes at 5'-UTR usually found in eukaryotic genes. However, all the genes contain the typical algal tailing signal sequence at 3'-UTR. Comparison of the expression of these genes in the two strains using Northern blot hybridization revealed their remarkable difference in response to low temperature. Genes were up-regulated by low temperature quickly in temperate strain, but much more slowly in the Antarctic strain.(3) Four plasmids were constructed in which antifreeze protein genes NJ7hiC6 and NJ7hiC12 were positioned downstream of rbcL or atpA promoters of Chlanmydomonas reinhardtii. Using the glass bead method, these plasmids were transferred into C. reinhardtii cc-849. Western blot analyses confirmed the high efficient expression of antifreeze proteins in C. reinhardtii cc-849.The main conclusions of this study are:(1) At 4℃and 20℃, antifreeze protein genes NJ7hiC6 and NJ7hiC12 and other genes showed significant difference in expression in C. vulgaris NJ-7.(2) Transcriptional regulation sequences for genes in C. vulgaris NJ-7 were different from those in most other eukaryotes. The response of gene regulation to low temperature in the Antarctic strain was greatly reduced compared to that of the temperate strain.(3) C. reinhardtii cc-849 could be used as a novel type of bioreactor to produce exogenous antifreeze proteins for industry purpose.