十字花科黑腐病菌III型效应物基因avrACXcc8004推测的启动子区

Putative promoter region of type III effector gene avrACXcc8004 in Xanthomonas campestris pv. campestris

摘要:

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－10box，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avrACXcc8004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avrACXcc8004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8bp处，而－10区与－10 box重叠;avrACXcc8004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avrACXcc8004的转录活性有较大的影响，－ 10 box突变前后avrACXcc8004均受HrpG 和HrpX 正向调控。

Abstract:

Abstract:［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 － 10 region．The whole motif of PIP-box，－ 35 region，and － 10 box was then counted as: TTCAC-N15 -TTCGC-N8 -TTGATGN18 -TACGTT． The GUS assay results demonstrate that the site-mutagenesis of － 10 box caused a higher expression of avrACXcc8004．The GUS activities in the mutant strains ΔhrpX and ΔhrpG 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 avrACXcc8004，despite of － 10 box site-mutagenesis.