Zn/Cd超积累植物东南景天SaREFl耐镉功能的探索研究

With the acceleration of the social industrialization,soil pollution by heavy metals are becoming more and more serious. The remediation of metal-contaminated soils is of great importance to our social and economical sustainable development. Using plants to remove heavy metals from contaminated soils has spurred great researching interest and becomes a developing tendency. However,there are only a few metal-related genes identified and characterized.The related mechanisms and the applications in culturing new heavy-metal tolerant plants still need to be studied. Hence, based on REFl, an highly heavy-metal tolerant gene obtained formerly from sedum alfredii, this project will focus on the study of REFl function characterization and cadium-tolerant mechanisms. Firstly, we will study the transcript length, the copy numbers, the expression profile, the phylogenetic analysis and the functional domains by the molecular hybrization and biological softwares. Then the expression profile of REFl response to cadmium will be researched by the means of quantitive PCR. The indispensable functional sites will be identified by the recombinant expression.Confocal microscopy will suggest the exact subcellular location of this gene. More importantly, we will transform the Sedum alfredii plants with RNA inference vector containing REFl and study the physiological changes of REFl-knockout mutants under cadium stress. Meanwhile, we will transform the Arabidopsis and polars with the overexpressing vector containing REFl and compare the physiological changes of REFl-overexpressing plants under cadium stress. These studies will further verify the cadium-tolerant function of REFl. Through these above experiments, the cadium-tolerant mechanisms of REFl will be deeply studied and verified. More significantly, these results will provide valuable data for the application of REFl in producing new heavy-metal tolerant breed.

随着工业化进程的加快，我国土壤重金属污染日趋严重，造成巨大损失，威胁人类健康。污染土地的修复对我国社会、经济的可持续发展具有重要意义。采用植物修复是重金属污染治理的发展趋势。但目前已知的重金属抗性基因较少，相关基因的抗性机制及在重金属高抗林木新品种培育中的应用还有待研究。本项目以前期得到的东南景天高抗镉基因REFl为研究对象，在研究其转录本、拷贝数、组织表达模式、进化关系、结构域等的基础上，借助实时定量技术研究其对镉胁迫的应答模式；运用原核表达系统获得定点缺失的重组蛋白，研究其镉功能的变化，寻找关键的抗镉依赖作用位点；构建RNA干涉载体进行东南景天转基因，构建超表达载体进行拟南芥和杨树转基因，通过研究转基因植株对镉胁迫的响应变化进一步验证该基因的抗镉功能，并为该基因的应用提供前期基础。通过上述研究，深入地研究REFl的抗镉机制，明确其抗镉功能，为该基因在培育林木高抗品种的应用提供理论依据。

镉是一种主要的重金属污染物，由于其易富集、难降解以及难清除而对环境和生物有着潜在且持续的危害作用。超积累型东南景天是在浙江衢州废弃的铅锌矿区中发现的，对其重金属抗性及超积累特性的分子机制研究有着重要的意义。. 前期工作中将东南景天cDNA文库进行酵母转化，进行耐镉基因的初步筛选，从中选取一耐镉基因，扩增其全长cDNA序列并进行同源比对、进化树构建等生物信息学分析，发现其与其他物种的与橡胶延伸因子(rubber elongation factor, REF)同源性更高，因此我们将其命名为SaREFl。构建酵母表达载体，将该基因在镉敏感型酵母菌株(ycf1)中进行异源表达，结果显示该基因的表达可以在一定程度上恢复镉敏感型酵母在含镉培养基上的生长，转化空载体的酵母在含镉培养基中的生长则受到抑制。构建植物表达载体将该基因在拟南芥中进行超表达，将拟南芥幼苗在含镉培养基培养后测量根长及生物量，发现转基因拟南芥幼苗长势优于野生型；将拟南芥成苗进行镉胁迫处理，生理指标显示转基因拟南芥的耐镉能力优于野生型，并累积更多的镉离子。. 借助实时定量技术研究SaREFl对其他重金属的响应模式，结果发现东南景天SaREFl基因对铅锌铜的应答水平不及镉且在根中响应比茎和叶中敏感。酵母双杂的结果显示SaREFl基因可能与细胞中负责囊泡运输的PRA1蛋白存在互作的可能性，而亚细胞定位显示SaREFl主要在胞质中表达，因此推测SaREFl基因可能通过减轻镉离子的胞质毒性发挥耐镉功能。. REF基因在前期研究中证实其与橡胶粒子周围的脂膜相关，是橡胶分子延伸的关键组分，该基因或者类似基因与胁迫相关的功能鲜有报道。通过对东南景天SaREFl的研究丰富了REF基因及其类似物的功能解读，并为该基因在耐抗镉植物中的应用提供理论支持和指导。