桑树病程相关蛋白基因MuPR1-2的启动子克隆及诱导表达活性

Cloning and Induced Activity Analysis of the Promoter of Pathogenesis-related Protein Gene MuPＲ1-2 from Mulberry

研究桑树抗病关键蛋白基因的启动子及其活性,对阐明基因的功能与表达调控机制十分重要。利用Tail-PCR技术成功地从桑树叶片基因组DNA模板中扩增得到病程相关蛋白基因MuP R1-2的启动子,将之命名为pM uP R1-2。利用PlantC ARE软件分析pM uP R1-2的核苷酸序列,发现其具有多个转录起始所必需的顺式作用元件以及多种转录因子结合位点,另外还包含多种环境因子响应元件。构建由pM uP R1-2启动GUS基因表达的植物表达载体,通过农杆菌介导的烟草瞬时表达及GUS组织化学染色分析pM uP R1-2具有启动子的功能,证实烟草叶片接种Pst DC3000菌液后能驱动下游报告基因GUS表达,pM uP R1-2具有病原菌诱导表达活性。进一步构建pM uP R1-2稳定表达的转基因拟南芥植株,通过GUS组织化学染色与GUS荧光定量分析发现pM uP R1-2不仅具有病原真菌和细菌诱导表达活性,同时具有可被多种激素诱导表达的特性和组织表达特异性。

Studying the promoter of key genes related to mulberry disease resistance and its activity plays a critical role in clarifing the gene function and expressional regulatory mechanism. In present study,the promoter of pathogenesis-related protein gene MuPR1-2 was successfully cloned from genomic DNA of mulberry leaf by Tail-PCR and designated as pMuPR1-2. Sequence analysis of pMuPR1-2 was performed using software PlantC ARE and the results showed that it contains several cis-acting elements necessary for transcriptional initiation,multiple transcription factor binding sites,and a variety of environmental factor responsive elements. The plant expression vector containing GUS gene drived by pMuPR1-2 promoter was constructed. The activity of pMuPR1-2 was investigated by the method of Agrobacterium-mediated transient expression in tobacco leaves,and the result indicated that p MuP R1-2 could initiate the transcription of downstream GUS gene which was used as a reporter gene after tobacco leaves were inoculated by Pst DC3000 bacterial solu-tion and could be induced upon pathogen attack. The transgenic Arabidopsis plants stably expressing pMuPR1-2 were further constructed. GUS histochemical staining and fluorescent quantitative analysis of GUS showed that p MuP R1-2 could be induced by pathogenic fungi and bacteria,and it was expressed in special tissues after induced by some hormones.