杨树CCH铜伴侣蛋白家族参与铜胁迫相关代谢途径解析

项目名称： 杨树CCH铜伴侣蛋白家族参与铜胁迫相关代谢途径解析

项目编号： No.31470664

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 农业科学

项目作者： 许志茹

作者单位： 东北林业大学

项目金额： 87万元

中文摘要： 植物Atx1-like铜伴侣蛋白包括Atx1-type和CCH-type两类。CCH-type铜伴侣具有植物特异的羧基末端伸长区；目前，此类铜伴侣的研究相对滞后，蛋白及其羧基末端的主要功能、表达调控方式、参与的代谢途径等均未得到揭示。本研究结合生物信息学分析，在杨树全基因组中鉴定了11个CCH-type铜伴侣蛋白。在此基础上，将研究杨树CCH铜伴侣家族基因应答不同生物胁迫的表达特性及亚细胞定位。构建CCH家族基因及C端结构域突变基因的过量表达和RNAi载体后遗传转化小黑杨和拟南芥突变体；结合表达谱深度挖掘CCH铜伴侣家族成员的功能及参与的代谢途径。分离CCH家族基因的启动子并确定响应CuSO4、H2O2处理的顺式作用元件。筛选CCH家族基因的调控因子；分离CCH蛋白家族成员的互作蛋白并确定C端结构域在互作中的作用。这些工作将为阐释植物CCH-type铜伴侣的功能特异性和多样性提供依据。

中文关键词： 小黑杨；CCH铜伴侣蛋白；功能鉴定；表达谱；启动子分析

英文摘要： Nowadays, Atx1-like copper chaperones in yeast and Atox1 copper chaperones, the orthologues in animals, have been studied extensively. There are two types of Atx1-like copper chaperones in plants, such as Atx1-type and CCH-type. CCH-type copper chaperone has the plant exclusive C-terminal extension. At present, the study on this kind of copper chaperone was lagged relatively, and the main function of CCH protein and its C-domain, the expression regulation pattern, the metabolic pathways which CCH participated, and so on, has not been revealed yet. Populus simonii × P. nigra would be used as material in this study. Using bioinformatics method, 11 CCH-type copper chaperones genes have been identified in poplar total genome. Based on this, the expression feature responded to different biotic stress and the subcellular localization of CCH copper chaperones family genes would be studied. The over-expression vectors and RNAi vectors of CCH family genes and the genes with mutant C-terminal would be constructed by Gateway technique. After transferred into Arabidopsis mutant and Populus simonii × P. nigra, the transcriptome of these transgenic and wild plants could be built using the high throughput sequencing method, and the function of CCH-type copper chaperones family members and the involved metabolic pathways will be excavated deeply by comparing the differentially expressed genes between overexpression transgenic plants and the inhibited expression ones. The promoter sequences of CCH family genes would be cloned and the cis-acting elements responding to CuSO4 and H2O2 would be ascertained by deletion analysis. Using yeast one-hybrid system, CHIP and electrophoretic mobility shift assay (EMSA) technology, we could screen the regulatory factors interacting with the cis-acting elements of CCH family genes in poplar. The interacting protein of CCH copper chaperones family members would be filtered and the role of the C-terminal in these interactions could be confirmed. This study will provide important basis data for further clarifying the function specificity and diversity of CCH copper chaperones in plant.

英文关键词： Populus simonii × P. nigra;CCH copper chaperone protein;functional identification;expression profile;promoter analysis