Cloning And Functional Characterization Of Cyclophilins Gene From Chimonanthus Praecox (L.) Link

Cyclophilins(CyPs) is one of immunophilin family, which was cloned an indetificated in many plants such as tomato,corn.rape,cabbage,rice,arabidopsis and beans. CyPs is the intracellular target of immunosuppressant drug cyclosporin A (CsA). Binding of CsA to CyPs leads to a complexes which inhibits communication in T cell and the complexes play a role of immunosuppression. CyPs has peptidyl-prolyl cis-trans isomerase activity, which can catalyse the process of protein especial proline folding assembling and transporting. CyPs take part in process of photoprotection of chloroplast and regulation of oxidation-reduction. CyPs also plays important role in stress response and antipathogen immunity.In this study.the CyPs gene was cloned from Chimonanthus praecox flower cDNA library.The analysis of sequence characters and the expression in prokaryotic system were carried out. The analysis of transformed tobaccos were also carried out. The main findings are as follows:1. Cloning and molecular characters analysis of CpCYPA CyPs gene was obtained by sequencing the randomly selected clone, on the basis of Chimonanthus praecox flower cDNA library construction and its ESTs analysis. The cDNA of CpCYP (GeneBank accession number:DW223084) sequence had a length of 927bp, containing ORF of 495bp, encoding 90 amino acids with a molecular mass of 18.26KD. This sequence exhibited homology to beans and graps etc. The identity of the derived protein has the high homogeneous characteristics of 90%. The protein has single domain and 9 PPIase active sites. The secondary structure of protein composed of Alpha helix(23.97%), Radom coil,(43.90%),extend strand (27.44%). The protein do not have signal peptide and transmembrane.The protein contained 5 phosphorylation sites and has good hydrophiliciry.2.The construction of recombinant vector of pET32a/CpCYP and the optimization of expression system. The CpCYP was amplified by PCR using the primers with BamHâ… and Hindâ…¢digestion sites.PMD19-T was used the intermediate vector, while the CpCYP was inserted into PET-32(a) vector. Then the expression vector was transformed and expressed Escherichia coli BL21. The expression protein is a fusion protein. We got the recombinant protein finally.We optimized expression system by adjustment of inducing temperature, conditions of IPTG and inducible time.The result showed that the expression level of fusion protein can increase by raising the temperature and time. The expression level of fusion protein do not increase after 4h.2. The genetic transformation of tobaccosThe CpCYP was amplified by PCR using the primers with Xbaâ… and Sacâ… . Recycle target gene and then link to cloning vector pMD-19T. Digest pMD-19TM/CpCYP by double restriction endonuclease Xbaâ… and Sacâ… ,then recycle the target gene and link to expression vector pCAMBIA2301.,The recombinant vector was transformed Agrobacterium tumefaciens LBA4404 by using freeze-thaw method. Using the leaf disc transformation procedure mediated by Agrobacterium tumefaciens,we got kanamycin-resistant tobacco plantlets, in which 17 CpCYP transgenic plantlets were identified by GUS,PCR and RT-PCR.