小球藻亲环蛋白A的酶活性及过表达拟南芥抗盐性分析

在前期研究中分离到噬碳酸盐小球藻（Chlorella sp.X1）的亲环蛋白基因CsCyp1A（登录号：KY207381），编码CsCyp1A蛋白是否具有肽酰脯氨酰顺反异构酶（PPIase）活性功能是进一步研究它参与小球藻耐盐生物学过程的基础。通过His标签的pQE-30-CsCyp1A原核蛋白表达载体，经IPTG诱导它的E.coli M15菌株表达融合蛋白，Nitra-柱纯化后获得纯化的30 kDa左右的His-CsCyp1A融合蛋白质，Western杂交检测到HisCsCyp1A蛋白的杂交信号。酶活性分析显示，HisCsCyp1A融合蛋白中生色基团的产生速度明显快于对照，表明CsCyp1A蛋白可以催化底物N-succinyl-Xaa-Pro-Phe-p-nitroanilide中Xaa-Pro肽键的顺反折叠，说明纯化的CsCyp1A蛋白具有PPIase活性。典型的亲环蛋白家族成员具有肽酰脯氨酰顺反异构酶（PPIase）活性，参与折叠和转运、信号转导、免疫调节、细胞凋亡等生物学过程。真空渗入法侵染转化的拟南芥在35S启动子驱动下超表达CsCyp1A基因，耐盐性研究表明它提高了过表达株系对NaCl胁迫的耐性，揭示小球藻CsCyp1A基因的抗盐性功能，它将成为抗逆育种的基因资源。本研究也为探索小球藻亲环蛋白A的抗盐碱胁迫生物学作用和分子育种奠定了基础。

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

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.