Study On Cellulase Of Xanthomonas Axonopodis Pv.citri

Citrus bacterial canker(CBC) disease,caused by Xanthomonas axonopodis pv.citri,is one of most serious diseases influence the production of citrus,and also serious quarantine diseases of citrus production. Cellulase is one of the important chemical factors of the plant pathogenic bacteria causing disease.It mainly assists pathogen invades the host, digests the mechanical barrier of the plant resistance away infection and degradates cell inclusions for itself using. We found cellulases in Xac as others plant pathogen by analysis of the Xac genome and proteins,so we speculated Xac cellulase play a part in invading the host and Xac cellulase is the necessary pathogenic factor. We got the information of the isoelectric point, molecular weight,signal peptide,domains and functions of Xac cellulase by analysis of bioinformatic on Xac cellulases. We choosed tipical cellulases of Xac cellulases genes, Xacce11,Xaccel3 and Xaccel5,cloned them and expressed them in vetor of pET28a and detected the activity of the three cellulases.The results are concluded as follows:1. The protein of Xac has 22 cellulases.We divided these cellulases into 11 groups by ClustalX1.83 according to there homology and detailedly analysed their structure characteristics and functional structure domain by on line prediction.The longth of the Xac cellulase is about 350aa,63.6% has signal peptide.Xac cellulase has Glycoside Hydrolase Family 5, Glycoside Hydrolase Family 8, Glycoside Hydrolase Family 9 and Glycoside Hydrolase Family 12 activity sites.54.5% of the Xac cellulase have GH5 module.It is the main module of the Xac cellulase. In addition.there are CelD_N,CBM,TAT domains and other functional modules of the SCOP,PDB protein database. The TAT domain play an important role in ransporting the Xac protein into the periplasmic space of the plant. So we speculate Xac cellulase is likely to play a role in infecting of the plant host.2. Xac cellulase has no specific cellulase by blastp with Xanthomonas cellulase, all of the Xac cellulase has homologous protein in Xanthomonas cellulase.Xac cellulase has the highest homology with Xcv cellulase. The types and numbers of the functional domains of the Xac cellulase are analogous with Xcv's. so we speculate both Xac and Xcv cellulase have the similar way on manners of action and play roles in infecting the host. We can speculate the roles and theway that Xac in infecting the host by take example by the results that the results already been studied on Xcv cellulase.3.Cellulase of the Xac,Xaccell has 199aa, molecular weight is 22KDa, isoelectric point is 7.86 and has signal peptide and GH12 module,GH12 is its particularly module.,maybe play a important role in hydrolyzing cellulose. The gene coding Xaccell is 600bp. Xaccel3 has 474aa, molecular weight is 51KDa, isoelectric point is 5.76 and has signal peptide,GH5 module and CBM2 module,CBM2 is its particularly module. The gene coding Xaccel3 is 1,425bp. Xaccel5 has 350aa, molecular weight is 38KDa, isoelectric point is 9.48 and has signal peptide and GH5 module. The gene coding Xaccel5 is 1,053bp.These three cellulases have high homology with Xcv85-10 cellulase.We have cloned these three genes by analysis and Expressed recombinant protein by IPTG induced.4. The enzyme activity has been determined by degrade natural substrates and pNP synthetic substrates. Xaccell can degrade CMC,Avicel,Barleyβ-glucan,xylan,Xaccel3 can degrade CMC,Avicel and xylan.Xaccel5 can degrade xylan and Avicel. The three enzymes can not degrade laminarin and PASC. We have gotten the way that Xaccel1,Xaccel3 and Xaccel5 degrade the substrates by can degrading the chosen five pNP synthetic substrates, Xaccell and Xaccel3 are endo-glucanases. It is breakage theβ-1,4 glycosidic bond and degtading glucose. The endo-glucanase of Xaccel5 is not confirmed,needing further studied.