The Cdna Cloning Of Cyclophilin And Calmodulin Genes From Camellia Oleifera And Cyclophilin Gene Expression In Prokaryote

Tea-oil tree(Camellia oleifera) is one of the most important ligneous edible oil trees in China. It has large cultivated area, adsperse distribution and strong adaptivity.90% of tea-oil is unsaturated fatty acid, such as oleic acid, linolaic acid, linolenic acid and so on. So it is a kind of excellent vegetable oil. Its byproduct also has important use. Cyclophilins (CyPs) are supperfamily members and ubiquitously distributed in endoplasmmic reticulum, Gologi appparatus, mitochondion, chloroplast and cytosol, et al. they include CyPA, CyPB, CyPC and CyPD, etc. CyPs are the major high-affinity binding protein in vertebrates for the immunosuppressive drug cyclosporin A (CSA). They exhibit a peptidyl-prolyl cis-trans isomerase activity (PPIase or rotamase), which accelerates protein folding by catalyzing the cis-trans isomerization of proline imidic peptide bonds in oligopeptides. They also work as the molecular chaperones and are involved in biological processes including the cellar signaling, spliceosome assemble, cellar multiplication, cell development, apoptosis and intercellular communication, etc. Plant CyPs also play important roles in biology, adverse stresses and immunosuppression. Based on the constructed cDNA library and EST library by the Key Lab of Non-wood Forest Product of State Forestry Administration, Central South University of Forestry and Technology, the Cyclopholin and Calmodulin genes were isolated, cloned. And the cyclophilin protein was also expressed by constructing prokaryotic vector. For this paper, major research conclusion is as follow.1. The C. oleifera CyP gene was firstly and successfully isolated and cloned. And its size of full-length cDNA is 975 bp containing an open reading frame (ORF) of 621bp with 5'and 3'untranslated regions (UTRs) and a long Poly (A) tail. It encodes a 22.24-kDa protein of 207 amino acid residues with a 26-amino-acid sequence of signal peptides,14-amino acid residues in coiled coil domain,2 N-glycosylation sites and other structural domains. The whole protein may be globular. The deduced amino acid of C. oleifera cyclophilin gene contains a unique 7 amino acid residues (KSGKPLH) of the plants relative to the amino acid sequences of mammalian and fungal CyPs. It displays high homology and similarity with those of other organisms, with overall identities of 80-94% to the homologous cyclophilins from prokaryotics and eukaryotic organisms. The C. oleifera CyP might be involved in the cellular processes, such as signal transduction, stress responsiveness, and immunosuppressive activity.2. The CaM1 and CaM2 genes of C. oleifera were firstly and successfully seperated and identified. They are 953bp and 1024bp in the full-length cDNA, respectively. They contain ORFs of 450bp, which encode the same protein of 150 amino acids. The characteristics of C. oleifera CaM protein is corresponded with the hypothesis theory Multigenes stand for the same protein". The two CaM sequences display highly homologous and conservative comparing with those of other higher plants, and the identifaction of above 90 percent. The C. oleifera CaM protein is comprised of 19 amino acids, and its theoretical isoelectric point is 4.10, which belongs to a hydrophilic protein. Its secondary structure contain four EF-hand domains, Ca2+ ions binding sites, Cys-27 residue, death effector daim, dockenn typeⅠrepeat and a domain of unknown function, but no signal peptides and transmembrane regions. The C. oleifera CaM might be involved in cellular processes that are critical for growth and development, regulation of many Ca2+ -dependent processes, etc.3. The C. oleifera gene was expressed visa constructing prokaryotic expressed vector, and then was induced to obtain the C. oleifera protein. According to the experimential resut, the induced protein, whose molecular weight is 22.24 kDa, and belongs to CyB. The protein can be as the further fundamental basis of studying Rotamase activity assay, IC50 determination and the Kd of CsA binding, etc. The paper makes use of gene engineering approach to obtain CoCyP protein, which could be as the basis of exploiting the relationship between structure and function.