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Cloning and Expression Analysis of GPx, GST and SAHH Genes in Chlamydomonas Sp. ICE-L from Antarctica

Author: WangJinHui
Tutor: Ding
School: Guangdong Ocean University
Course: Aquaculture
Keywords: Chlamydomonas sp. ICE-L Glutathione reductase Glutathione S-transferase S-adenosyl-L-homocysteine hydrolase Clone Bioinformatics Prokaryotic expression
CLC: Q943.2
Type: Master's thesis
Year: 2011
Downloads: 11
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Abstract


Glutathione peroxidase and glutathione S-transferase are two important enzymes of glutathione antioxidant system, and they can clear away the H2O2 and organic peroxide produced in physiological and pathological processes. S-adenosyl-L-homocysteine hydrolase, which plays an important role in multitude cellular methylate reaction, is widely exist in living cells. cDNAs encoding glutathione peroxidase, glutathione S-transferase and S-adenosyl-L-homocysteine hydrolase were cloned from Antarctic ice algae Chlamydomonas sp. ICE-L by RT-PCR and RACE-PCR methods, and named ICE-LGPx, ICE-LGST and ICE-LSAHH, respectively. Expression levels of ICE-LGPx and ICE-LGST under different salinity and Cadmium chloride were analyzed. Recombinant of prokaryotic expression vectors of ICE-LGPx and ICE-LGST were also constructed in this paper.The ICE-LGPx full-length cDNA sequence was 1956 bp, encoded a polypeptide of 255 amino acids. The predicted molecular weight (Mw) of ICE-LGPx was 28.3 KD with an estimated pI of 9.07. Phylogenetic analysis showed that Chlamydomonas sp. ICE-LGPx was parallel evolution with Micromonas sp. RCC299 and Micromonas pusilla. A 102 bp SECIS (SElenoCysteine Insertion Sequence) element was identified in the 3’-UTR of ICE-LGPx. The 3D molecular modeling showed ICE-LGPx consisted of 7β-sheets and 5α-helices. The ICE-LGST full-length cDNA sequence was 966 bp, encoded a polypeptide of 321 amino acids with a deduced molecular mass of 24.1 KD and an estimated pI of 9.69. ICE-LGST gene sequence shared most identity with that of Chlamydomonas reinhardtii mitochondrial GST gene. The subcellular localization prediction showed the ICE-LGST protein might come from mitochondrion. The 3D molecular modeling showed ICE-LGST consisted of 4β-sheets and 9α-helices, and had two function domains: GSH binding site (G-site) and substrate binding site (H-site). ICE-LSAHH full-length cDNA sequence was 1844 bp, encoded a polypeptide of 487 amino acids. The predicted molecular weight of ICE-LSAHH was 53 KD with an estimated pI of 5.16. Sequence analysis showed that the ICE-LSAHH gene shared high sequence identity with other SAHHs. ICE-LSAHH was parallel evolution with Dunaliella salina, Chlamydomonas reinhardtii and Chlorella variabilis. The 3D molecular modeling showed the ICE-LSAHH subunit consisted of three domains which were substrate-binding domain, NAD-binding domain and C-terminal domain, and the subunit consisted of 12β-sheets and 20α-helices.The expression patterns of ICE-LGPx and ICE-LGST under the challenge of salinity and Cadmium chloride stress were examined by Real-time PCR analysis. Experimental results showed that there were gene expression on ICE-LGPx and ICE-LGST at different NaCl and CdCl2 concentration stress, with the trend of first increasing and then decreasing. The expression of ICE-LGPx reached a higher level at 24 h in the salinity of 11 and 66 while at 12 h in the salinity of 22 and 99. The highest expression of ICE-LGST appreared at 24 h in low salinity 11 and 22, but at 12 h in high salinity 99. ICE-LGST has a more expression than ICE-LGPx when treated by heavy metal CdCl2. In 10 and 20μmol·L-1 groups, ICE-LGPx gene expressed most abundantly at 48 h and 24 h, respectively, while in 30 and 40μmol·L-1 groups the highest expression were at 12 h. The highest expression of ICE-LGST was at 36 h and 48 h in 10 and 20μmol·L-1 while at 36 h and 12h in 30 and 40μmol·L-1 proups, respectively.Recombinant of prokaryotic expression vectors pET28-GPx and pET28-GST were successfully constructed using pET-28a vector, respectively.

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