杨树CCH铜伴侣蛋白家族参与铜胁迫相关代谢途径解析

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.

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。

植物CCH铜伴侣蛋白具有植物特异的羧基末端伸长区；目前，此类铜伴侣蛋白的研究相对滞后且集中于模式植物拟南芥，CCH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。本研究以拟南芥CCH蛋白（NP_564870.1）为探针，在毛果杨数据库中鉴定了21个PtCCHs铜伴侣蛋白家族成员；这些蛋白的N端均具有重金属结合基序MXCXXC，并能够形成典型的βαββαβ二级结构。利用生物信息学软件分析了PtCCHs家族成员的氨基酸数目、分子量、等电点、染色体定位、内含子数量等信息。利用PopGenIE中的表达数据鉴定了毛果杨PtCCHs基因的组织表达特异性，同时通过qRT-PCR检测了小黑杨PnCCHs基因的组织特异性表达模式。利用含有高浓度和缺少铜离子的霍格兰德营养液处理小黑杨后，PnCCHs基因应答铜处理在幼叶、成熟叶片、茎及根中的表达具有特异性。同时检测了处理后小黑杨扦插苗的叶面积、叶绿素含量、光合速率、鲜重、干重、相对含水率、蒸腾速率、叶片胞间CO2浓度、SOD和POD酶活等生理生化指标。成功克隆了14个小黑杨PnCCHs基因的全长cDNA序列，核苷酸序列已经登录到NCBI数据库。构建了14个PnCCHs基因的酵母表达载体并转化酵母sod1△和atx1△突变体细胞。转化后的酵母sod1∆突变体细胞都可以在缺少赖氨酸的培养基上生长，证明14个小黑杨PnCCHs蛋白都具有抗氧化功能。转化PnCCH5、6、7、8、10、11或PnCCH20基因的酵母atx1Δ突变体细胞能够在含有菲啰嗪的培养基上生长，证明这7个PnCCHs蛋白具有铜伴侣功能，可以恢复酵母突变体细胞的高亲和铁吸收能力。成功构建了PnCCH5、6、7、8、10、11、15、17、19和PnCCH20基因的过量表达载体。遗传转化拟南芥野生型、atx1和cch突变体植株后获得了T2代植株。遗传转化小黑杨后已经获得了PnCCH10的小黑杨转基因株系，同时获得了PnCCH5、6、7、15、17和PnCCH19转基因抗性芽，正在进行分子生物学鉴定。成功构建了小黑杨PnCCH5、6、7、10、15、17和PnCCH19基因的亚细胞定位表达载体，并成功转化了农杆菌细胞，正在进行这些PnCCHs蛋白的亚细胞定位分析。上述研究工作将为鉴定和阐释杨树CCH铜伴侣蛋白的功能特异性和多样性提供依据。