Prokaryotic Expression,Activity Assay of Chlorella Cyclophilin A and Salt Tolerance Analysis of Arabidopsis Over-expressing CsCyp1A

doi:10.7525/j.issn.1673-5102.2017.05.012

Abstract

Abstract: In order to test the activity of peptidyl-prolyl cis-trans isomerase of cyclophilin encoded by CsCyp1A, which was isolated from Chlorella sp. X1 in previous research(Accession No:KY207381), the primers were designed by using pMD18-T-CsCyp1A plasmid DNA as template and the target gene fragment that included Kpn Ⅰ and Sal Ⅰ digestion sites were obtained by cloning. After enzyme digestion, ligation and sequence analysis, the His-tagged pQE-30-CsCyp1A prokaryotic expression vector was obtained. IPTG induced E.coli M15 strain which contained its plasmid to express the fusion protein. Purified soluble HisCsCypA fusion protein was obtained by purification on a Nitra-column and the relative molecular mass of CsCyp1A was about 29 kDa. HisCsCyp1A protein hybridization signal was detected by Western blot. By enzyme activity analysis, the production rate of chromogenic groups in HisCsCyp1A fusion protein was significantly faster than in control, and the CsCyp1A protein could catalyze the cis-trans folding of Xaa-Pro peptide bond in N-succinyl-Xaa-Pro-Phe-p-nitroanilide and accelerate the cleavage of blocking groups. The purified CsCyp1A protein had PPIase activity. By overexpression of CsCyp1A gene driven by 35S promoter in Arabidopsis thaliana under vacuum infiltration, the results showed that it increased the tolerance of overexpression lines to NaCl stress, and revealed the salt tolerance of Chlorella CsCyp1A gene. It will become a genetic resource for resistance breeding. The study established the foundation for exploring the biological role of chlorella cyclophilin CsCyp1A in the anti-carbonate stress of algae.

Key words: phytococcal chlorella, saline-sodic soil, cyclophilin A, peptide prolyl cis-trans isomerase, prokaryotic expression protein

CLC Number:

Q812

LIAO Xu, HE Ming-Liang, ZHANG Su-Yan, MA Hai-Yan, WANG Xu-Hui, LUO Qiu-Xiang, GUAN Qing-Jie. Prokaryotic Expression,Activity Assay of Chlorella Cyclophilin A and Salt Tolerance Analysis of Arabidopsis Over-expressing CsCyp1A[J]. Bulletin of Botanical Research, 2017, 37(5): 722-729.

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