利用桥梁载体策略构建水稻条斑病菌T3SS基因诱导表达检测体系

Establishment of the inducing expression detection system of Xanthomonas oryzae pv. oryzicola T3SS genes using bridge vector strategy

水稻条斑病菌(Xanthomonas oryzae pv.oryzicola,Xoc)侵染水稻,引起细菌性条斑病(bacterial leaf streak,BLS)。Xoc主要依靠hrp基因簇(T3SS基因)编码的III型分泌系统(type III secretion system,T3SS)将效应蛋白注入水稻细胞中,激发寄主水稻的感(抗)病性。为了准确在Xoc中进行T3SS基因表达调控的分析,本研究设计出桥梁载体策略,将目标基因的启动子或者功能片段构建在高拷贝的桥梁载体,获得融合表达元件,通过亚克隆的方式将融合元件转入骨架载体上,获得用于转录表达和蛋白表达分析的载体。为了验证该策略的可行性,选取hrpG、hrpX和hrcC基因构建了相应的启动子探针载体和蛋白表达载体,将这些载体导入毒性调控子基因trh、lrpX和zur的突变体中,GUS活性测定、荧光定量PCR以及蛋白免疫杂交结果显示:在trh、lrpX和zur的突变体中,hrpG、hrpX和hrcC的转录表达水平、mRNA水平和蛋白表达水平呈现一致。这表明这套桥梁载体策略能够有效应用于T3SS基因转录表达和蛋白表达的分析。综上所述,运用桥梁载体能够克服低拷贝载体DNA遗传操作效率低的缺陷,这一策略也适用于毒性相关基因调控机理的研究,这将为Xoc-水稻互作研究提供高效的工作系统。

Xanthomonas oryzae pv.oryzicola(Xoc)causes bacterial leaf streak(BLS)in host plant rice.Xoc is dependent on the type III secretion system(T3 SS)encoded by hrp cluster(T3 SS genes)for the secretion of the type III effector proteins into rice cells leading to disease resistance or susceptibility in rice.To analyze the regulating expression of T3 SS gene in Xoc,in this study we designed a bridge vector strategy in which a promo-ter segment or complementary fragment of target gene was constructed on a high-copy bridge vector to obtain a fusion element which then was subcloned into a backbone vector to obtain a vector for transcriptional expression or protein expression analysis.In order to verify the feasibility of this strategy,the promoter-probe-vectors and protein expression vectors of selected hrpG,hrpX and hrcC genes were constructed,and these vectors were introduced into the Xoc mutants of the virulence regulator genes trh,lrpX and zur.The results of the GUS activities,quantitative real-time PCR and protein immunohybridization showed that the transcriptional expression le-vels,mRNA levels and protein expression levels of hrpG,hrpX and hrcC genes were consistent in the mutants of trh,lrpX and zur.This indicates that this bridge vector strategy can be effectively used to analyze the T3 SS gene transcriptional expression and protein expression.In conclusion,the application of bridge vector can overcome the shortcomings of low-copy vector DNA genetic manipulation efficiency.This strategy is also applicable to study the regulating expression mechanism of virulence-associated genes,which will provide an efficient working system for Xoc-rice interaction research.