SaREFl基因参与超积累型东南景天镉抗性的分子机理研究

With the aggravation of heavy-metal contaminated soil, remediation of polluted soil has become a serious concern needed to be solved urgently. Characterization of genes related to heavy-metal resistance is vital for the culture of genetically engineering breeds with novel traits of high tolerance. In our previous study, a gene containing the REF domain in Sedum alfredii Hance (HE) designated as SaREFl was detected to be endowed with novel functions of cadmium tolerance, however, neither the precise mechanism underlying the functional divergence nor the regulatory network are completely understood. Herein, we first perform the cloning of promoter region and validate key cis-elements associated with cadmium tolerance through promoter deletion. Constructs enclosing key elements would be applied in the yeast one-hybrid system to screen for potential regulatory genes aimed to analyze the upstream regulatory pathways. Meanwhile, pull down and other protein-interacting techniques would be performed to characterize the candidate proteins functionally related to SaREFl. To investigate the roles of SaREFl in more details, omics sequencing would be conducted to identify differentially expressed genes among the transformed SaREFl overexpressing or interfering Sedum alfredii Hance plants and wild types. Combined with the above findings, we expect to elucidate the precise mechanism underlying the cadmium tolerance of SaREFl and explore whether a novel regulatory pathway responding to heavy-metal stress exists. More importantly, our work also provides theoretical assistances for development of plants with high resistance to heavy metals which would be applied in decontamination of heavy-metal polluted soils.

我国土壤重金属污染日益加剧，研究重金属抗性基因的功能机制，用于培育林木高抗新种质，对于提高污染土壤的植物修复效率具有至关重要的意义。前期研究发现超积累型东南景天SaREFl具有镉抗性功能，作为非传统的重金属抗性基因，如何在镉胁迫中发挥抗性功能仍有待研究。本项目拟通过启动子系列缺失突变及酵母单杂交，筛选调控SaREFl基因的上游因子；借助蛋白互作技术，鉴定与SaREFl存在直接相互作用的蛋白；比较SaREFl超表达和干涉转基因东南景天镉抗性差异，采用组学测序技术筛选差异基因及蛋白，与转录调控、蛋白互作等相结合，解析其调控网络及功能通路。通过本项目明确SaREFl基因的调控因子及互作蛋白，探究其通过参与或影响何种生理进程行使镉抗性功能，解析SaREFl基因参与超积累型东南景天镉抗性的分子机理，为培育林木镉高抗新种质提供理论基础和科学依据。

我国土壤重金属污染日益加剧，研究重金属抗性基因的功能机制，用于培育林木高抗新种质，对于提高污染土壤的植物修复效率具有至关重要的意义。近年来超积累植物耐受和积累镉的机制取得较大进展，但其调控机制仍不清楚。前期研究发现超积累型东南景天橡胶延伸因子类似基因SaREFl具有镉抗性功能。本研究对SaREFl进行了东南景天过表达研究，发现其可提高转基因植株镉抗性。通过酵母单杂交技术从东南景天cDNA文库中筛选了SaREFl基因两个潜在调控因子；同时，借助蛋白互作技术验证了BPL6和CSN5均与SaREFl存在互作关系，并对BPL6和CSN5进行了初步镉抗性功能研究。此外，我们建立了镉敏感酵母的筛选体系，借助cDNA文库的表达筛选鉴定48个提高Cd抗性的基因，并对SaCTP2和SaDRT100在酵母和拟南芥中的作用机理以及在东南景天中的表达模式进行了深入研究，同时鉴定了SaABCC在促进Cd的吸收转运中的作用。本研究初步探索了SaREFl等镉关键基因参与东南景天镉抗性的分子机制，发掘与其相关联的上下游关键基因，提示超积累型东南景天的镉抗性是极为复杂的生理进程，与抗氧化、抗性基因调控及信号传递有着密切的关联。该研究为镉抗性功能基因应用于植物修复及林木高抗新种质培育提供重要资源和理论基础。