大豆斑疹病菌tal基因Tn5插入突变体的构建
Construction of Tn5-inserted mutants in tal genes of Xanthomonas axonopodis pv.glycines

地毯草黄单胞菌大豆致病变种(Xanthomonas axonopodis pv.glycines,Xag)引起大豆斑疹病,是大豆上重要的细菌病害之一。大豆斑疹病菌通过Ⅲ型分泌系统(type-III secretion system,T3SS)将包括转录类似激活因子(transcription activator-like(tal)effectors,TALEs)在内的效应蛋白(T3SS-secreted effectors,T3SEs)注入寄主植物细胞中,从而使寄主植物产生抗病性或感病性反应。为了研究不同tal基因在Xag致病性中的作用,选择ATCC43911菌株为研究对象,根据tal基因的高度保守性,构建了可用于同源交换的含转座子Tn5的pB-C8B::Tn5质粒。通过电转化法转入受体菌细胞中,再进行Km抗性筛选,获得了tal基因插入突变体。Southern杂交结果显示,ATCC43911菌株含有6个tal基因,在Tn5转座子插入后,tal基因的位置发生了变化,杂交信号条带数目增加,并且存在Tn5的单拷贝和双拷贝插入2种形式。这表明Tn5转座子成功插入了tal基因。Tn5插入突变体的构建为进一步分离tal基因以及鉴定其在致病性中的作用奠定了工作基础。
Xanthomonas axonopodis pv.glycines (Xag)causes bacterial pustule on soybean,which is one of the most serious bacterial diseases in soybean growing area.Xag secrets large amount of effectors,including transcription activator-like(tal)effectors(TALEs),into plants via a type-III secretion system(T3SS)which is highly conserved in Gram-negative plant pathogenic bacteria.The host plant interfered by T3SS-secreted effectors(T3SEs)may be resistant or susceptible to Xag infection.In order to study the contribution of different tal genes to the bacterial pathogenicity in soybean,Xag strain ATCC43911 was used as research object in the experiment.Based on highly conserved tal genes in sequence,pB-C8B::Tn5,a plasmid with homolog-interchangeable fragments of a tal gene flanking EZ-Tn5 tansposon,was constructed.Then,the plasmid was electroporated into ATCC43911 cells and single kanamycin-resistant colonies were selected,putatively referring to Tn5-inserted tal mutants.Southern blot results demonstrated that the wild-type ATCC43911 contained 6 tal genes.The location of tal genes changed and more tal bands were showed in Tn5-inserted mutants because one or two Tn5 transposons were inserted in a tal gene.These results suggested that the Tn5 transposon was inserted into the tal genes successfully.Construction of Tn5-inserted mutants of Xag in tal genes will provide fundamental basis to understand roles of these tal genes in bacterial pathogenicity in soybean.