Cloning Of GCL Gene And Expression Analysis Of GCL And GST Gene In Chlamydomonas SP. ICE-L From Antarctica

Glutathione (GSH) plays an important role in antioxidation and anti-aging in organisms and was widely used in the area of biochemical drugs, nutritional foods, and food additives. GSH is a tripeptide thiol composing of Glutamic acid, Cysteine and Glycine, and its synthesis is catalyzed by γ-glutamate cysteine ligase (GCL EC:6.3.2.2) and glutathione synthetase (GS EC:6.3.2.3) in the presence of ATP. In this study, GCL was cloned from Chlamydomonas sp. ICE-L by RT-PCR and RACE-PCR methods, and denoted it as ICE-LGCL. Expression levels of ICE-LGCL under different temperatures, salinities condition and ICE-LGST under different temperatures were analyzed by real-time PCR analysis. As well, the ICE-LGCL and ICE-LGST genes were successfully expressed in the Kcoli BL21with prokaryotic expression vectors of pET-28a(+).The ICE-LGCL full-length cDNA sequence was2199bp, including a5’-terminal untranslated region (UTR) of36bp, a3’-terminal UTR of711bp with a poly(A) tail, and an open reading frame (ORF) of1452bp encoding a polypeptide of453amino acids. According to the deduced amino acid sequence, the molecular weight of ICE-LGCL was55.17kDa and with an estimated isoeletric point (pi) of5.97. The deduced amino acid sequence was submitted to a BLASTP search at the NCBI, result showed that ICE-LGCL was shared51%-70%amino acids sequence identity with those of other plants, in which the highest with Chlamydomonas reinhardtii, followed by Oryza sativa, Chorispora bungeana, Zinnia elegans, Arabidopsis thaliana and Lotus japonicus. Subcellular localization prediction showed that the ICE-LGCL may exist in chloroplast of Chlamydomonas sp.ICE-L. There are no signal peptide, transmembrane region or N-Glycosylation sites, but with27phosphorylation sites including15Serine phosphorylation sites,15tyrosine phosphorylation sites and7threonine phosphorylation sites and a structure domain of the GCL family.The expression patterns under the temperature and salinity stresses were examined by Real-time PCR analysis. Results showed that the ICE-LGCL and ICE-LGST can express at different temperatures and salinities, and the expression in control group kept at a proportionate level. As to temperature, the ICE-LGCL mRNA accumulation increased in the first24h, and come to twice as much as control group at24h under0℃. However, the accumulation of ICE-LGCL mRNA decreased in the first6h and then come to a lower level about half as much as control under14℃. Under0℃, the ICE-LGST mRNA accumulation increased in the first12h and come to the highest level at12h about two times of the control, and come back to the normal level in the following time. Under14℃, the ICE-LGST mRNA accumulation increased from24h and reached the highest level at36h. As to salinity(control is33%。), results showed that low salinities stimulated the ICE-LGCL expression and the accumulation of ICE-LGCL mRNA was less than control after treated with high salinities. In low salinity groups (11and22), the expression of ICE-LGCL kept a successive increase in the first48h and both reached the highest mRNA accumulation about more than two times as much as control. In high salinity groups (66and99), the expression level of ICE-LGCL decreased in the first6h and came to a stable level about one third of the control in the following experiment period.At the same time, the ICE-LGCL and GST were expressed in the BL21using the prokaryotic expression vectors of pET-28a and analyzed by western blot analysis. The recommendation expression plasmids were respectively named as pET-GCL and pET-GST. The optimum expression conditions of pET-GCL in E.coli BL21are0.6mmol·L-1concentration of IPTG,37℃and induced4h, and the pET-GST highly expressed at the temperature of37℃, induced time4h under0.2mmol·L-1concentration of IPTG, and both of the products were mainly in the form of inclusion body. The expression products were analyzed by SDS-PAGE and western blot, which results certified that the expression protein were respectively ICE-LGCL and ICE-LGST. And the expression of pET-GST can increase the survival of E.coli BL21under low temperature.