杨树ATX1-type铜伴侣蛋白基因功能及调控机制研究

Nowadays, ATX-like copper chaperones in yeast and Atox1 copper chaperones, the orthologues in animals, have studied intensively. In plants, however, the research on ATX1-type copper chaperones is relatively backward, and there are still many problems remaining to be investigated, such as the function of the protein in plants, the molecular mechanisms of gene regulation pattern, the metabolic pathways that the proteins involved in, and whether these proteins act as rugulation factors, etc. Based on the known research results and bioinformatics analysis, we identified 2 ATX1-type copper chaperones genes in poplar total genome. The promoter sequences of these two genes would be cloned and the cis-acting elements responding to various biotic factors, for example, JA, NaCl, CuSO4 and H2O2, could be ascertained by deletion analysis. Using yeast one-hybrid system and electrophoretic mobility shift assay (EMSA) technology, we could filter the protein factors interacting with the cis-acting elements of ATX1-type genes in poplar. The interaction between ATX1-type genes and the promoter elements of the cell cycle proteins would be analyzed further then. In that case, we would confirm whether the ATX1-type proteins have the function of the regulation factors. The over-expression vectors and RNAi vectors of ATX1-type genes would be constructed by Gateway technique. The transgenic populas plants will be obtained and the phenotype of the plants will be identified. The transcriptoms of these transgenic and wild plants are built using the high throughput sequencing method. The target genes of ATX1-type copper chaperones will be determined by comparing the differentially expressed genes between overexpression and inhibit expression of populas transgenic plants. These results will provide important study data for further clarifying the function of the ATX1-like copper chaperones.

酵母铜伴侣蛋白ATX-like及动物中的直系同源物Atox1研究已较为深入，植物直系同源物ATX1-type铜伴侣蛋白的研究则相对滞后，包括调控方式、参与的代谢途径及是否具有转录因子功能等均未得到揭示。本研究以杨树为材料，结合生物信息学分析，在杨树全基因组中鉴定了2个ATX1-type铜伴侣蛋白基因。在此基础上，将分离其启动子并通过缺失分析试验确定响应JA、NaCl、CuSO4、H2O2处理的顺式作用元件。利用酵母单杂交和凝胶阻滞技术筛选ATX1-type铜伴侣蛋白基因的调控因子，同时鉴定该蛋白是否与细胞周期蛋白基因启动子相互作用，以此验证其转录因子功能。构建ATX1-type基因的过量表达和RNAi载体，转化小黑杨后测定表达谱。通过比较转基因植株表达谱确定受ATX1-type铜伴侣蛋白调控的下游靶基因群。这些研究工作将为阐释植物ATX1-like基因的功能提供依据。

酵母铜伴侣蛋白ATX1及动物中同源物Atox1的研究已较为深入，木本植物ATX1-type铜伴侣蛋白的研究相对滞后。本项目克隆了小黑杨PnATXs基因并对其核苷酸序列进行了详细的生物信息学分析。qRT-PCR检测结果显示，PnATX1基因在茎和根中的表达量较高，PnATX2基因在叶中的表达量较高。缺铜、高铜、锌离子、铁离子、茉莉酸和水杨酸处理后小黑杨PnATXs基因的表达模式存在差异。细胞定位结果显示，小黑杨PnATX1蛋白定位于细胞核和细胞质，PnATX2蛋白定位于细胞质。小黑杨PnATXs基因的表达载体转化酵母sod1△和atx1△细胞后，成功鉴定了PnATXs蛋白具有抗氧化活性和铜伴侣功能。表达PnATXs的拟南芥转基因植株在受到铜离子胁迫时受伤害程度明显降低，生物量增长明显增加。利用植物过量表达载体pROKⅡ-PnATX1和pROKⅡ-PnATX2，通过农杆菌介导遗传转化小黑杨后获得了多个转基因株系，并进行了分子生物学鉴定。成功克隆了PnATX1和PnATX2基因的启动子序列，并利用生物信息学手段分析了启动子中的顺式作用元件组成。组织化学染色结果表明，PnATXs启动子能够驱动报告基因GUS表达，具有生物学活性。克隆的拟南芥CyclinB1;1和CyclinD2;1启动子序列也具有生物学活性，可以用于与小黑杨PnATXs蛋白的体外互作分析。构建了小黑杨PnATXs基因的原核表达载体并获得了转化成功的大肠杆菌BL21菌株。此外，在毛果杨基因组中鉴定了11个新型重金属结合蛋白，家族成员都含有2个重金属结合基序MXCXXC和2个βαββαβ结构。qRT-PCR检测结果表明，小黑杨新型重金属结合蛋白家族成员在铜胁迫条件下的表达模式存在特异性。这些研究工作为阐释木本植物ATX1-like基因的功能多样性及在维持细胞铜稳态过程中的作用提供了试验依据。