| Mango bacterial leaf spot caused by Xanthomonas critis pv.mangiferaeindicae(Xcm)is a destructive leaf,stem,and fruit diseases in many production areas,and is one of the most important diseases of mango in the world.Cell wall degrading enzymes secreted by the pathogen are important pathogenic factors destroying host tissue epidermal cells and invading into plant tissues causing further cell destruction.A variety of degrading enzymes of the cell wall were identified in previous Xcm study during infecting mango or natural fermentation.In order to t’urther clarify the key enzymes,tour genes,cel,xyl1,xyl2 and xynA of Xcm were cloned and sequenced,and its bioinformatics prediction were carried out by using bioinformatics software.The qRT-PCR was used to analyzing the expression level of those four genes before and after invading leaves tissue.And then,the genes expression of xyl1,xyl2 and xynA were further analylied.The results were as follows:1.The genes of cel,xyl1,xyl2 and xynA on Xcm were cloned and sequenced by homologous cloning method,and the sequence characteristics and functional structures of encoded proteins were analyzed using bioinformatics software.By phylogenetic trees analyses,the results showed that the four genes of Xcm were highly conserved among different species,with X.citri pv.pufnicae str.LMG 859(CCF67690.1),X.campestris pv.centellae(OOW50899.1),X.axonopodis pv.melhusii(OOW66465.1)and X.cilri pv.glycines(OEY88849.1),which have a closest relationship.2.In order to analyze the expression levels furtherly of cel9 xyl1,xyl2 and xynA genes in the process of diseases infecting,the five housekeeping genes of 16s rRNA,gyrB,GAPHD,danK and rpoD were selected as candidate reference genes from Xcm genome.A comprehensive evaluation of the stability of the expression of these five reference genes before and after invasion of the leaves tissue of the mango by the method of qRT-PCR.The results showed that the internal reference genes of gyrB,GAPHD and rpoD were the most compatible combination genes before disease invasion in the samples on leaf surface.And the gyrB and GAPHD were the most compatible combination internal reference genes after disease invasion inside the leaves tissue.3.The expression levels of the four genes cel,xyl1,xyl2 and xynA were analyzed by qRT-PCR before and after Xcm invasion into the leaves of the mango and analyzing in term of selected internal reference gene combinations.The result showed these four genes were continuously and efficiently expressing before Xcm invading the leaves tissue of the mango.The expression trend of cel gene was gradually increasing,up to the highest at 72 h;the expression level of xyl1 gene was gradually increased to the highest expression level at 12 h,and followed by a small peak at 48 h,then decreased gradually;the expression level of xyl2 gene gradually increased to the maximum at 48 h,and then decreased;The expression level of xynA gene gradually increased to 12 h and then decreased slightly,and then increased to the maximum expression level at 48 h and then decreased.After Xcm invaded the leaves tissue of mango,the four genes were highly expressed in this process,and the expressions were the highest at 6 h,3 h,24 h and 3 h.It is speculated that these four genes play different roles in different stage in the process of disease invasion.4.The prokaryotic expression vector pET-32a was used to connect the three genes of xyl1,xyl2 and xynA with cutting off their signal peptide,respectively.The recombinant expression plasmids were successfully transferred into E.coli BL21(DE3),and protein expressions were carried out by IPTG induction.The results showed that the induced proteins were mainly in the main precipitates by testing of SDS-PAGE,which were 70 kD,70 kD and 45 kD,respectively,indicating that three proteins were all inclusion bodies and had not enzymatic activity.The optimal protein induction conditions were screened out with the different factors of medium,temperature,IPTG induction concentration and sampling time. |
PDF Full Text Request