巴西橡胶树乙烯信号途径关键转录因子的功能鉴定

Simulation of hevea latex drainage by ethylene is the most important technical revolution for rubber production in 20th century. However, no key ethylene-related genes of Hevea brasiliensis have been cloned and characterized,which strongly hampered the advance of further reseach of molecular mechanism for latex synthesis and drainage. In the previous work, we first cloned the full-length cDNAs of these transcriptional factror and many ethylene-related regulators. In this work,(1) a complementation and autotranscription test would be done through transformation of ethylene-related genes into Arabidopsis mutants;(2) Screening the ehtylene-induced genes by DEG analysis and cloning promoter of the key ethylene-related genes by genomic walking;(3) Verification of the interaction between the ethylene-related tansscription factor and promoter of ethlene-induced genes; (4) Identification of the inteaction between ethylene-related genes and other regulators,such as JAZ, HDA6 and EBF1, through Yeast two hybrid, Pull-down assay, and coimmunoprecipitation assay, so as to reveal the regulation pathway of ethylene signalling.Implement of the project is of great significance for characterization of ethylene-related key genes in Hevea brasiliensis and clarification of the molecular mechanism of latex drainage during ethylene stimulation.

利用乙烯刺激橡胶排胶是20世纪橡胶生产最重大的一次技术变革，但橡胶乙烯相关关键基因都未被克隆与鉴定，成为橡胶产排胶分子机理研究的瓶颈。在前期工作中，我们首次完成橡胶转录组测序和乙烯应答基因数字表达谱分析，在此基础上成功克隆了橡胶乙烯信号通路关键转录因子HbEIN3、HbEIL1和若干个关键调控因子的全长cDNA序列。本项目拟通过转化拟南芥突变体和自转录活性鉴定来验证其转录因子的功能；制备橡胶HbEIN3和HbERF1的蛋白抗体，通过染色质免疫共沉淀筛选其调控下游基因的启动子；还将利用基因组行走技术克隆乙烯诱导的与橡胶产排胶相关的基因及启动子，通过酵母单杂和EMSA技术验证乙烯关键转录因子与哪些产排胶相关基因的启动子结合并调控其转录;最后利用酵母双杂、Pull-down和免疫共沉淀技术验证乙烯转录因子与调控因子的互作，揭示其调控途径。本项目的实施对阐明乙烯在橡胶产排胶中的调控机理有重要意义。

乙烯是调控橡胶产排胶的重要信号分子，本项目克隆了4个橡胶关键转录因子， 制备了转录因子HbEIN3和 HbERF1的抗体，完成West-blot鉴定。鉴定HbEIN3， HbEIL1， HbERF1和HbERF2基因的自转录活性分析和亚细胞定位。完成橡胶树数字表达谱分析和全基因组水平筛选乙烯诱导基因，克隆乙烯诱导的橡胶产排胶相关基因的编码区和启动子区。通过HbEIN3和 HbEIL1蛋白与多个顺式作用元件的互作筛选，确定HbEIN3和 HbEIL1蛋白能与GCC-box发生互作。随后进行全基因组的启动子的生物信息学分析，预测HbEIN3和 HbEIL1蛋白调控的橡胶产排胶相关靶基因，并完成HbEIN3 与HbEIL1转录因子和靶基因的验证。项目执行过程中还开发了一个巴西橡胶树基因组信息网（www.h-brasiliensis.com），为在线搜索橡胶树基因组信息提供操作平台。本项目发表学术论文10篇，其中 SCI论文6篇。培养博士研究生2名，硕士研究生2人，参加国际学术会议2次。