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采用Genome walking方法从辣椒基因组DNA中分离获得辣椒CaWRKY5基因上游的启动子序列,命名为CaWRKY5p.为了分析CaWRKY5p对病原菌和机械损伤的应答以及可能的调控区域,将该基因5’端顺序4个不同缺失片段分别与GUS报告基因相融合构成表达载体,在农杆菌介导的烟草叶片瞬间表达系统中表达.结果表明:启动子TATA框为起始密码子ATG上游-140 bp至-136 bp之间的TATATAA,同时含有多种与逆境胁迫相关的顺式作用元件,如W-box、S-box、MeJA应答元件和ABRE类似元件等;CaWRKYK5基因上游-936 bp启动子可以应答青枯病菌、抗病信号分子以及机械损伤;CaWRKY5p应答青枯病菌和茉莉酸甲酯(MeJA)的核心元件可能位于启动子-886 bp至-489 bp之间,应答乙烯(ET)核心调控区域位于-936 bp和-886 bp之间,而应答机械损伤的核心元件位于启动子-336 bp以及ATG之间.
The CaWRKY5primer promoter sequence was isolated from the capsicum genomic DNA by Genome walking method and named CaWRKY5p.To analyze the CaWRKY5p response to pathogenic bacteria and mechanical damage and the possible regulatory regions, The deletion fragment was fused with the GUS reporter gene to construct an expression vector and expressed in Agrobacterium tumefaciens-mediated tobacco leaf transient expression system.The results showed that the promoter TATA box was between -140 bp and -136 bp upstream of the start codon ATG Of TATATAA, and contain a variety of cis-acting elements related to stress such as W-box, S-box, MeJA response element and ABRE similar elements. The -936 bp upstream of CaWRKYK5 gene can response to bacterial wilt, The molecular mechanism of CaWRKY5p response to bacterial wilt and methyl jasmonate (MeJA) may lie between -886 bp and -489 bp in the promoter and -936 bp in the core regulatory region of ethylene (ET) -886 bp, whereas the core element that responds to mechanical damage is located between the -336 bp promoter and ATG.