南极衣藻谷胱甘肽-S-转移酶基因的表达及其对大肠杆菌低温耐受性的增强作用

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

谷胱甘肽-S-转移酶(Glutathione-S-transferase,GST,EC2.5.1.18)是生物体内一种重要的抗氧化酶,为阐明GST在南极衣藻(Chlamydomonas sp.ICE-L)中的具体地位,采用实时荧光定量PCR对不同温度下南极衣藻的GST基因的表达进行了分析;并构建了原核表达载体pET28a(+)-GST,转化至大肠杆菌BL21(DE3)中进行诱导表达,通过平板培养法探讨了重组菌E.coli BL21(pET28a(+)-GST)对低温胁迫的耐受性。结果显示,GST在0℃时表达量最高,最高可达对照组的两倍多;pET28a(+)-GST重组表达载体在E.coli BL21中实现了高效表达,且主要以包涵体形式存在,经HisTrap HP柱分离纯化获得高纯度的GST融合蛋白,并通过SDS-PAGE及Western blot分析得以验证;对低温胁迫实验发现南极衣藻GST蛋白的表达可以提高重组菌E.coli BL21对低温的耐受性,说明GST基因对南极衣藻适应南极低温环境具有重要作用。

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（P0.01） about more than twice of that in the control group,in the rest time the GST 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.