调控拟南芥camalexin合成途径上多基因协同转录的转录因子基因克隆及MAPK磷酸化对其功能调控的研究

项目名称： 调控拟南芥camalexin合成途径上多基因协同转录的转录因子基因克隆及MAPK磷酸化对其功能调控的研究

项目编号： No.30870220

项目类型： 面上项目

立项/批准年度： 2009

项目学科： 轻工业、手工业

项目作者： 任东涛

作者单位： 中国农业大学

项目金额： 36万元

中文摘要： Camalexin是拟南芥产生的主要植保素，对拟南芥抵抗多种病原菌侵染具重要作用。研究表明合成途径上多基因的协同激活能导致Camalexin的合成。我们的结果表明，MKKs-MPK3/MPK6的激活，诱导了Trp及Camalexin合成途径上多个关键基因的协同、大量转录，导致Camalexin大量合成。利用其中的PAD3等的启动子多个删除片段驱动GUS基因表达、凝胶阻滞及亲和纯化的方法证明，参加整个合成途径基因的转录因子应该包括MYB及WRKY等；利用点突变等方法证明了MYBa的T4和T244位点为激活的MPK3/MPK6磷酸化位点；突变基因转基因植株分析及基因敲除分析发现，仅敲除该基因和表达MYBa模拟磷酸化状态蛋白不能调控Camalexin合成，应该存在其他转录因子共同调控。利用蛋白质组学和代谢组学方法鉴定出，GSTF6参与Camalexin中噻唑环合成的调控，该调控通过使GSH连接至吲哚乙腈(IAN)上发生的，揭示了噻唑环的原始供体可能是GSH，揭示了GSTF6是Camalexin合成途径的新组分。

中文关键词： 拟南芥；Camalexin；MAPK级联系统；基因协同调控；磷酸化

英文摘要： Camalexin (3-thiazol-2'-yl-indole) is the main phytoalexin in Arabidopsis. Camalexin is synthesized from tryptophan (Trp) via IAOx, a key branch point between primary and secondary metabolism in Arabidopsis. Overexpression of any of genes in Trp and camalexin biosynthesis pathways in Arabidopsis plants alone dose not increase camalexin accumulation. These results imply that coordinate activation of multiple genes in the camalexin biosynthesis pathway, even those in Trp biosynthesis pathway, is needed for the camalexin accumulation. Our recent results have showed that activation of MPK3/MPK6 induce coordinate activation of multiple genes in the Trp and camalexin biosynthesis pathways,and lead accumulation of camalexin in transgenic plants. MPK3/MPK6 may activate transcription factor(s),including MYBs and WRKYs through phosphorylation and regulate multiple genes expression. By using truncated DNA fragments from PAD3 and other gene's promoter,MYB and WRKY transcription factors were identified. Assay on the phosphorylation sites have show that,Thr4 and Thr244 are the two phosphorylation sites on MYBa. Camalexin production in the gene's knockout mutant or phosphomimic form overexpressing plants do not show significant changes. These results suggest that the MYBa and other gene may functional together to regulate camalexin biosynthetic pathway. In addition, we found that GSTF6 participtates camalexin biosynthesis through catalyzing the conjugation of GSH with IAN. The thiazole ring of camalexin molecular is originated from GSH. The GSTF6 is a newly identified component in camalexin biosynthetic pathway.

英文关键词： Camalexin;MAPK cascade;Coordinate regulation of genes expression by transcription factor;Phosphorylation