Cloning, Prokaryotic Expression And Characterization Of A Chitinase Gene From Chimonanthus Praecox (L.) Link

As a important PR protein,chitinase has been a hot point of genetic engineering of plant resistance to fungal diseases all time.in this paper,the biological characteristics,classifications, biological function of plant chitinase were Summarizsed,as well as expression regulation of its gene.It emphasis on the Progress in Research and Application of plant chitinase.With the development of study,the Research and Application of plant chifinase in many fields has attracted more and more attention with a wide prospect.Chimonanthus praecox(L.) Link,a shrub originated from China,blossoms lordly in deep winter,possessing many advantages,such as resistance of coldness,drought,and secateur.Among them,chilling tolerance is its main characteristic.As long as the temperature isn't below -15℃,it can safely live through the winter.In our rearch,a chitinase gene(Cpchia) was cloned from imonanthus,praecox flower,and expressed in Escherichia coli,then we mensurated the effects of its' activity and stability at the different temperature and pH.The main results are as follows:1 Cloning of Cpchia from Ch.praecox flowerA chitinase gene,designated as Cpchia(GenBank accession number:FJ749130),was obtained by sequencing the randomly selected clones,on the basis of Chimonanthus praecox flower cDNA library construction and its ESTs analysis.This sequence exhibited homology to Persea Americana and Poa pratensis etc.The identity of the derived protein has the high homogeneous characteristics of 71%～73%.The further analysis showed that Cpchia is a chitinase gene from from Ch.Praecox.The full-length cDNA of Cpchia is 1148 bp,containing a 954 bp open reading frame(ORF) and encoding a predicted protein of 317 amino acids.2 Structural characteristics analysis and function prediction of Cpchia protein The structure characteristics of Cpchia protein were analyzed with bioinformatic method,The results showed that Cpchia protein contained a N_terminal signal region,a chitin binding domain rich in cys and gly,a P-rich hinge region and a catalytic domain,and it didn't has C_terminal region.Therefor,we preliminary estimated it is a Class Ib chitinase.Its N terminal had a 20aa signal peptide sequence,secondary structure of Cpchia protein composed of Alpha helix(65.29%), extend strand(5.36%) and loops(29.33%),meanwhile,this protein contained many phosphorylation sites,we conjectured the protein was diversified modified after translation,related to the multiple resistance of chitinase.3 The construction of prokaryotic expression vector of Cpchia and the optimization of expression systemCpchia was inserted into an prokaryotic expression vector pET28a(+) with Resection of N_terminal signal region.Then the expression vector was transformed and expressed Escherichia coli BL21.We got the recombinant protein finally.We optimized expression system by adjustment of inducing temperature,conditions of IPTG and inducible time.The SDS-PAGE electrophoresis analysis showed that the recombinant protein was only present in the inclusion body.In this experiment,it show that expression level of target protein was decreased by lowering temperature but no soluble protein produced.4 The purification,renaturation and characterization of prokaryotic expression proteinWe broke cell by lysozyme and ultrasonic disruption.The purity and recovery of inclusion body were significantly increased by this method.We used dissolution buffer contained 8M Urea dissolved the inclusion body.Then the supermatant was dialysis refolded after high speed centrifugal in 4℃.The enzymatic activation analysis show that the refolded protein had more higher activation.The gradient test of temperature showed that the chitinase activation was highest in 40℃,kept activity stable below 50℃,and closed to deactivation in 80℃.The gradient test of PH showed that the chitinase activation and stablition were highest in PH7.0.