Cloning And Functional Analysis Of Cyclophilins A(CyPA) Gene Form Chlorella

Cyclophilin A (CyPA) is a kind of proteins which have an activity of peptidyl-prolyl cis-trans isomerase (PPIase) with aconservative structure. It catalyses the cis-trans folding of Xaa-Pro peptide bonds and accelerates conformation changes of protein that rate-limiting step is peptide bond folding of proline, and also help protein keeping proper three dimensional structure. CyPA widely distributes in different tissues of prokaryotic and eukaryocyte with a vital important function. Therefore, the present study used chlorella as materials, which obtained from saline soil in Northeast of China, constructed cDNA libraries and then transformed into saccharomyces cerevisiae, and found several genes related to salt-stress using NaCl as screening concentration, and then researched their related biological function.Among45cloned genes from resistance selection,4genes were finally identified. Based on relevant literature, we selected CyPA gene as materials for further research, and confirmed that its protein has the conservative and typical feature structure. As CyPA distributed abroad in cell, this study researched its cellular localization in arabidopsis protoplast. The results showed that CyPA was found in chloroplast.In order to investigate the relationship between CyPA and abiotic stress, we firstly identified CyPA gene expression of chlorella under NaCl, NaHCO3and Sorbital stresses using Northern blotting method, which suggested that its expression was up regulation caused by those stresses. In addition, we got yeast with over-expressed CyPA gene by chemical transformation, and then transformed rice and Arabidopsis for discovering the ability of plant response to abiotic stress. The above results revealed that transgenic yeast with over-expressed CyPA gene could improve response to salt stress (NaCl), alkaline salt stress (NaHCO3), oxidative stress (H2O2) and drought stress (Sorbital); transgenic Arabidopsis with over-expressed CyPA gene could highly response to salt stress (NaCl), alkaline salt stress (NaHCO3); and transgenic rice with over-expressed CyPA gene had a increasing ability to salt stress (NaCl), alkaline salt stress (NaHC03) and Me-JA stress (jasmonic acid methyl ester).With a view to further explore the peptidyl-prolyl cis-trans isomerase (PPIase) activity of CyPA in chlorella, we optimized prokaryotic expression conditions and obtained lager amounts of purified CyPA which possessed PPIase activity that was determined by the method of two-step reaction coupled to chymotrypsin.