巴西橡胶树HbJAZ1互作蛋白的筛选与功能鉴定研究

Latex drainage after tapping is a woundng response of rubber trees (Hevea brasiliensis).Jasmonic acid (JA) is one of most important signal of plant wounding response.Tapping and treatment with exogenous JA induces laticifer differentiation and latex synthesis of rubber tree, JA is therefor a key signal regulator for laticifer differentiation and latex synthesis.JAZ is a negative factor of JA signalling pathway, which plays a key role in this pathway by repressing the JA-related transcriptional factors. JAZ protein was degraduated after JA binding to its receptor and related transcriptional factors were released . Identification of JAZ target proteins is a shortcut to reveal the mechanism of laticifer differentiation and rubber production of rubber tree. In the previous study, we constructed the bait vector of HbJAZ1 and screened more than 10 proteins interacted with HbJAZ1 through yeast two-hybrid method, which including 2 transcriptional factors. Furthermore, full-length cDNA sequences of seven interacted genes were cloned. In this work, we intend to verify the interaction between HbJAZ1 and the target gene using bimolecular flurescent complementation (BiFC). Additionally, we plan to investigate whether the HbJAZ1 related transcription factor could specifically bind the promoter of latex synthesis genes and activate their transcription.Finally, we are investigating the gene expression and doing the plant transformation to study their physiological function. This work will lay a theory foundation for basic research of rubber tree and technical improvement of rubber production.

排胶是巴西橡胶树的一种创伤反应,茉莉酸是植物创伤反应关键信号分子。割胶和茉莉酸处理促进橡胶乳管分化和胶乳合成，茉莉酸是调控乳管分化和产胶的关键激素。JAZ蛋白遏制茉莉酸下游转录因子的转录活性，茉莉酸与受体结合后促进JAZ蛋白降解而激活其下游基因的转录。鉴定橡胶JAZ蛋白（HbJAZ1）的目标基因是研究橡胶乳管分化和产胶机理的关键途径。我们构建了橡胶HbJAZ1基因的诱饵载体，通过酵母双杂在橡胶cDNA文库中筛选到10多个HbJAZ1的互作蛋白（其中包括两个转录因子），并克隆了其中7个基因的全长cDNA序列。本项目拟利用双分子荧光互补的方法验证HbJAZ1与目标蛋白之间的互作，并通过酵母单杂的方法探讨与HbJAZ1互作的转录因子是否能够与胶乳合成相关基因的启动子发生互作，最后通过分析基因差异表达和植物转化来验证其基因功能，为橡胶产胶机理研究和生产技术的改进提供了重要理论依据。

茉莉酸是调控橡胶创伤反应和产排胶的重要信号分子，本项目克隆7个橡胶茉莉酸信号抑制因子JAZ蛋白，通过酵母双杂技术筛选HbJAZ1基因互作的转录因子，包括3个与MYC转录因子和一个锌指蛋白，通过酵母双杂交和双分子荧光互补技术验证JAZ与HbMYC3基因的互作。利用荧光定量RT-PCR研究了HbJAZs基因和HbMYC2， HbMYC3和HbMYC4基因的差异表达情况，也完成了HbMYC2的遗传转化。首次系统地鉴定了橡胶茉莉酸信号传递通路中的关键基因。此外，还通过酵母单杂交技术筛选到HbMYCs互作的顺式作用元件G-box,并通过数字表达谱和生物信息学技术筛选HbMYCs的靶基因。 HbMYCs潜在的靶基因包括一系列产排胶相关基因，包括已知的橡胶排胶相关的水通道蛋白基因，该发现为揭示茉莉酸信号如何调控橡胶产排胶的分子机理打下坚实的基础。.本项目已发表SCI论文3篇，还有两篇提交的论文在审稿中，另外还发表中文期刊论文4篇，培养研究生5名，本科生2人。参加国际学术会议一次。建立橡胶基因组信息及顺式作用元件搜索网站一个：www.H-brasiliensis.com。