INCREASED ESCHERICHIA COLI TOLERANCE TO LOW TEMPERATURE BY EXPRESSION OF GLUTATHIONE-S-TRANSFERASE GENE FROM THE CHLAMYDOMONAS SP. ICE-L

Glutathione-S-transferase (GST, EC2.5.1.18) is the key antioxidant enzyme that catalyzes the conjugation of glutathione to several electrophilic substrates in living beings. Chlamydomonas sp. ICE-L is a rare ice algae living in the Antarctica where cold conditions and strong ultraviolet radiation are present all year round. In order to find out the role that GST in Chlamydomonas sp. ICE-L plays in acclimatizing to freezing polar environment, the expression of GST gene in Chlamydomonas sp. ICE-L was analyzed using real-time PCR under different temperatures. To evaluate the amount of template RNA in each real-time PCR reaction, gene fragments of β-actin was also amplified. The results showed that the Chlamydomonas sp. ICE-L GST gene could be expressed under all experimental temperatures. In the control group, as the temperature was 8℃, the accumulation of GST mRNA was maintained at identical level during 72h. At 0℃, GST mRNA accumulation decreased in the first 6h, which was followed by an increase and peaked at 36h (PGST mRNA accumulation recovered to the level of control. At 14℃, the accumulation of GST mRNA was lower than the control group and slowly decreased during the entire experimental period and reached a quarter of the level in the control group at 72h. In addition, the pET-28a(+)-GST prokaryocyte expression vector was constructed and then transformed into E. coli BL21(DE3) to express the GST protein. The optimal expression conditions of pET-28a(+)-GST in E. coli BL21 included by 0.2 mmol/L concentration of IPTG, 37℃ and induction for 4h, and the product was mainly in the form of inclusion body. Using the HisTrap HP Columns, the expression product was separated and purified. Then the product was analyzed and confirmed by SDS-PAGE and Western blot, both results showed that the expression products with the molecular weight of 26 kD in the E. coli BL21(DE3) was the GST protein encoded by the GST gene from Chlamydomonas sp. ICE-L. At last, the E. coli BL21 containing the recombination plasmid (pET-28a(+)-GST) was treated with low temperature before growing on the agarose plate. As the recombination plasmid expressed mainly in the form of non-active aggregated monomers in E. coli BL21 induced with IPTG, we slowed the revolving speed and extended the induction time to express the GST protein with enzyme activation. The results of recombination plasmid treated with low temperature showed that the survival level of E. coli BL21 containing recombination plasmid (pET-28a(+)-GST) was higher than that of E. coli BL21 without this recombination plasmid in the first 4 days and reached the normal level at the 5th and 6th days. Our results revealed that Chlamydomonas sp. ICE-L GST expressed in E. coli BL21 could improve the tolerance of E. coli to cold conditions, suggesting that GST may play an important role in the defense against freeze in the Chlamydomonas sp. ICE-L in Antarctica. These results provided valuable information on further investigation of the molecular mechanism of Chlamydomonas sp. ICE-L GST gene.