Cloning And Functional Analysis Of CpFKBP Gene From Chimonanthus Praecox (L.) Link

FKBPs is a member of immunophilin family, which have been isolated and their genes have been cloned in many plants such as bean, corn, ricinus communis, tomato, grape etc. FKBPs has specific binding capacity with immunosuppressant drug FK506and rapamycin to create immune-suppression. At the same time, the FKBP has the peptidyl-prolyl cis-trans isomerase activity, which can catalyze the process of protein folding, assembling and transporting, it has played an essential role in plant growth and development. In this study, the FKBP gene was cloned from Chimonanthus praecox flower cDNA library. In this paper, we analysed CpFKBP gene’s sequence characters, the expression in prokaryotic system, isomerase activity and transformed tobaccos. The main experimental results are as follows:1. Cloning and molecular characters analysis of CpFKBPOn the basis of the constructed Chimonanthus praecox flower cDNA library and its ESTs analysis, a FKBP gene was obtained by sequencing the randomly selected clone. The cDNA of CpFKBP sequence had a length of482bp, containing ORF of339bp, encoding113amino acids with a molecular mass of12.08KD, the Isolectric Point I is8.48. It was named CpFKBP (GeneBank accession number:JQ337962). The analysis of sequence characters shows that the identity of the derived protein has the high homogeneous characteristics about90%with Populus trichocarpa, Medicago truncatula and Ricinus communis. It belongs to single-domain structure FKBP. The secondary structure of protein is composed of Radom coil(58.93%), extend strand (28.57%) and Alpha helix(12.5%). The protein does not have signal peptide and transmembrane. It has good hydrophobicity and contains4phosphorylation sites.2. The construction of recombinant vector pET-32a/CpFKBP, the optimization of expression system and the analysis of PPIase activity.BamHI and SacI restriction enzyme digestion sites were added at the two sides of CpFKBP, then CpFKBP was inserted into pET-32a(+) vector through the intermediate vector pMD19-T. Then the expression vector was transformed and induced to express in Escherichia coli BL21. At last, we optimized expression system by reviseing the temperature, lengthening time and finding proper consistence of IPTG. The result showed that the expression level of fusion protein could increase by low temperature16℃and the best expression time is about24h. The analysis of PPIase activity shows that fusion protein has a high PPIase activity.3. The construction of plant expression vector of CpFKBP and transformation of tobaccosAdd BamHI and SacI restriction enzyme digestion sites at the two ends of CpFKBP. And then link to cloning vector pMD19-T. Digest pMD19-T/CpFKBP by double restriction endonu clease BamHl and SacI, then recycle the target gene and link to expression vector pCAMBIA2301g. The recombinant vector was transformed into Agrobacterium tumefaciens LBA4404by using electro-poration method. Using the leaf disc transformation procedure mediated by Agrobacterium tumefaciens, we got kanamycin-resistant tobacco plantlets. Identified by GUS and PCR, we gain20CpFKBP transgenic plantlets.