Characteristic of Glutamate Cysteine Ligase Gene and its response to the salinity and temperature stress in Chlamydomonas sp. ICE-L from Antarctica

Abstract

Chlamydomonas sp. ICE-L, an Antarctic ice alga, has high tolerance ability to freezing and salinity. Glutathione (GSH) is animportant small antioxidative molecule in the growth and stress responses of plants including algae. We cloned a full-length cDNAencoding glutamate cysteine ligase (ICE-LGCL), the key enzyme of GSH synthesis, from Chlamydomonas sp. ICE-L by RT-PCR andrapid amplification of cDNA ends technique (RACE). The cDNA has 2199 bp nucleotides with an open reading frame (ORF) of 1452 bpencoding a polypeptide of 453 amino acids. BLASTP algorithm results showed that ICE-LGCL shared 51%–70% amino acid sequenceidentity with the reported GCLs and shared the highest identity with Chlamydomonas reinhardtii. We also successfully made ICE-LGCLprotein express in E. coli BL21. The optimum expression conditions are the induced reagent IPTG of 0.2 mmol/L, temperature of 37°C, and the induced time of 4 h. The expression patterns of ICE-LGCL in mRNA by real-time PCR analysis showed that ICE-LGCLexpressed under the different temperature and salinity challenges. Low temperature and low salinities stimulated the accumulation ofICE-LGCL mRNA in ICE-L cells. These results indicate that GCL might play an important role in Antarctic Chlamydomonas sp. ICE-L.