大豆斑疹病菌内切甘露聚糖酶ManA的功能研究

Study on the function of mannan endo-1,4-β-mannosidase(ManA)in Xanthomonas axonopodis pv.glycines

为探析大豆斑疹病菌(Xanthomonas axonopodis pv.glycines,Xag)基因组中唯一注释编码内切甘露聚糖酶(mannan endo-1,4-β-mannosidase,ManA)基因是否参与Xag的内切甘露聚糖酶活性及其他功能,采用同源重组策略创制了Xag的manA基因缺失突变株(ΔmanA),并对该突变株进行功能分析.结果表明:相对于野生型,该突变株的胞外纤维素酶活性和内切甘露聚糖酶活性均显著增强,而且能形成明显的胶状物;功能互补使ΔmanA的上述3种表型均恢复至野生型水平;manA基因突变不影响菌株的生物膜形成、胞外多糖合成及蛋白酶和淀粉酶活性;负责胞外纤维素酶合成的egl2和engXCA基因也共同控制Xag的内切甘露聚糖酶活性;荧光定量PCR结果显示,ManA涉及负调控egl2和engXCA基因的表达.总之,manA基因突变导致egl2和engXCA基因的表达水平上调,从而增强菌株的内切甘露聚糖酶活性.研究为深度解析植物病原黄单胞菌Ⅱ型分泌系统胞外酶类的生物学特性提供了科学线索.

Despite there was only one gene encoded mannan endo-1,4-β-mannosidase(ManA)in genome of Xanthomonas axonopodis pv.glycines(Xag),however,it kept unknown whether ManA had been involved in endo-mannosidase activity and the other functions of Xag.In order to elucidate ManA biological functions,the manA deletion mutant(ΔmanA)was generated by allelic exchange.Compared with the wild-type,the resulting manA mutant exhibited significantly enhanced on the activity of both cellulase and endo-mannosidase,and developed obvious"gum-like"substances.The above properties inΔmanA were completely restored by the wild-type manA.However,mutation in manA did not affect biofilm formation,extracellular polysaccharide synthesis,and the activities of both protease and amylase.Interestingly,egl2 and engXCA,which were responsible for extracellular cellulase synthesis,also determined endo-mannosidase activity of Xag.qRT-PCRs confirm that ManA negatively regulated egl2 and engXCA at the mRNA level.It was concluded that the resulting manA mutant resulted in up-regulated expression levels of egl2 and engXCA,and thereby enhanced the endo-mannosidase activity of Xag.The conclusions could provide scientific clues for deeper analysis of biological characteristics of extracellular enzymes secreted by typeⅡsecretory system in plant pathogen Xanthomonas.