十字花科黑腐病菌Ⅲ型效应物基因avrAC_(Xcc8004)推测的启动子区  

Putative promoter region of type Ⅲ effector gene avrAC_(Xcc8004) in Xanthomonas campestris pv. campestris

作　　者：蒋国凤[1,2] 吴秋菊[3] 梁晓夏[2,3] 杨丽超[3] 阳丽艳 王凛[3] 吴小建[3] 姜伯乐[2,3] 

机构地区：[1]广西大学林学院,广西南宁530004 [2]亚热带农业生物资源保护与利用国家重点实验室,广西南宁530004 [3]广西大学生命科学与技术学院,广西南宁530004

出　　处：《微生物学报》2014年第2期159-166,共8页Acta Microbiologica Sinica

基　　金：广西自然科学基金项目(2010GXNSFB013020)~~

摘　　要：【目的】十字花科黑腐病菌(Xanthomonas campestris pv.campestris,Xcc),能侵染所有十字花科植物,引起黑腐病。Xcc通过III型分泌系统(Type III Secretion System,T3SS)将III型效应物(T3SS effector,T3SE)蛋白直接转运到植物细胞内,T3SEs对于病原菌致病性至关重要。许多已鉴定的T3SEs基因的启动子区都存在植物诱导启动子盒(Plant-inducible promoter,PIP-box)和-10 box,但PIP-box及-10 box与经典的启动子-10区及-35区之间的关系如何未见报道,-10 box的序列保守性如何也未见报道。本研究旨在对T3SE基因avrAC Xcc8004推测的启动子区进行研究。【方法】首先,通过5'RACE确定其转录起始位点,接着用Fusion PCR对-10 box TACGTT序列中倒数第二个碱基T进行点突变为A/C/G,即:TACGAT、TACGCT和TACGGT,构建GUS融合报告菌株,定量测定GUS酶活。【结果】5'RACE结果显示avrAC Xcc8004的转录起始位点为A,对比分析得到启动子的-35区位于PIP-box之后8 bp处,而-10区与-10 box重叠;avrAC Xcc8004启动子区的PIP-box和-35区、-10 box的整个模体为:TTCAC-N15-TTCGC-N8-TTGATG-N18-TACGTT。最后,GUS定量测定结果表明,突变为C时(TACGCT)的菌株的GUS酶活最高,突变为G时(TACGGT)的酶活增高最少。在ΔhrpX和ΔhrpG中的GUS酶活均比在Xcc 8004中有显著的降低。【结论】Xcc的T3SE基因PIP-box与-35区前后相衔,-10 box即-10区,-10 box对于avrAC Xcc8004的转录活性有较大的影响,-10 box突变前后avrAC Xcc8004均受HrpG和HrpX正向调控。[ Objective] Xanthomonas campestris pv. campestris （Xcc）is the cause agent of black rot of crucifers. Xcc uses type III secretion system（T3SS） to deliver T3SS effectors（T3SEs） directly into host cells, where they play important roles in pathogenesis. Many identified T3SEs genes contain plant-inducible promoter（PIP） box and - 10 box in their promoter regions. However, the relation among PIP-box, - 10 box and - 10 region, -35 region of the classic promoter is unclear, and the conservative characteristic of - 10 box sequence is hardly reported. The aim of this study was to analyze the putative promoter region of T3SE gene avrACXcc8004. [ Methods ] Through 5＇ RACE, the transcriptional start site of avrACXcc8004 was identified. Fusion PCR was introduced to generate the site-mutagenesis of - 10 box for constructing the GUS fusion report strains. [ Results] The 5＇ RACE results indicate that the transcription start site was A. After analysis, we found that - 35 region was located 8 bp downstream of PIP-box, and - 10 box was exactly overlapped with - l0 region. The whole motif of PIP-box, -35 region, and -10 box was then counted as： TTCAC-Ns-TTCGC-Ns-TTGATG- Nn-TACGTT. The GUS assay results demonstrate that the site-mutagenesis of - 10 box caused a higher expression ofavrACXcc8004. The GUS activities in the mutant strains AhrpX and AhrpG were significantly lower than that in the wild type Xcc strains. [ Conclusion] PIP-box is tandem with -35 region, -10 box is just the same as -10 region, -10 box is important for the transcription of avrACXcc8004 and HrpG and HrpX activate the expression of avrACxoosoo4 , despite of - 10 box site-mutagenesis.

关 键 词：十字花科黑腐病菌 Ⅲ型效应物 HrpX avrACXcc8004 PIP-box -10 BOX 

分 类 号：S432.4[农业科学—植物病理学]