Authors: YUEYUE PENG, YU DING, XIAOCHEN TANG, JICHANG JIAN, JINHUI WANG, QUANFU WANG
Abstract: Chlamydomonas sp. ICE-L, an Antarctic ice alga, has high tolerance ability to freezing and salinity. Glutathione (GSH) is an important small antioxidative molecule in the growth and stress responses of plants including algae. We cloned a full-length cDNA encoding glutamate cysteine ligase (ICE-LGCL), the key enzyme of GSH synthesis, from Chlamydomonas sp. ICE-L by RT-PCR and rapid amplification of cDNA ends technique (RACE). The cDNA has 2199 bp nucleotides with an open reading frame (ORF) of 1452 bp encoding a polypeptide of 453 amino acids. BLASTP algorithm results showed that ICE-LGCL shared 51%-70% amino acid sequence identity with the reported GCLs and shared the highest identity with Chlamydomonas reinhardtii. We also successfully made ICE-LGCL protein 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-LGCL expressed under the different temperature and salinity challenges. Low temperature and low salinities stimulated the accumulation of ICE-LGCL mRNA in ICE-L cells. These results indicate that GCL might play an important role in Antarctic Chlamydomonas sp. ICE-L.
Keywords: Chlamydomonas sp. ICE-L, glutamate cysteine ligase (GCL), temperature, salinity, expression analysis, prokaryotic expression
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