蜈蚣草不同ACR3亚砷酸逆转运蛋白的功能研究和比较

Arsenic (As) hyperaccumulating fern Pteris vittata is an ideal plant to remediate As-contaminated soils. However, the molecular mechanism of As hyperaccumulation in this plant has not been fully elucidated yet. ACR3 (Arsenic Compounds Resistance 3) arsenite (AsIII) antiporter plays a key role in As detoxification in organisms, as well as in As detoxification and As hyperaccumulation in P. vittata. So far, there are few reports of P. vittata ACR3s and the functional differences among ACR3s remain unclear. In this proposal, four ACR3 genes were cloned from P. vittata and their functions and differences were investigated in yeast, Arabidopsis and P. vittata. We will transform the ACR3s into yeast, test As tolerance of and determine As accumulation in yeast transformants to investigate yeast functions of the ACR3s. Besides, we will also introduce different ACR3 genes into Arabidopsis and analyze As uptake, As efflux, As translocation, As detoxification and As accumulation in the transgenic plants. To further elucidate the functions and the differences of the ACR3s in plants, subcellular localization of the ACR3s in Arabidopsis will also be investigated using ACR3-GFP fusion proteins. At last, we will also study the transcriptional expression pattern and the subcellular localization of different ACR3s in P. vittata by real-time qRT-PCR and western blots. Based on studies and comparisons of these ACR3s in yeast, Arabidopsis and P. vittata systems, we aim to reveal the different roles of different ACR3s in arsenic hyperaccumulation of P. vittata, which is believed to improve the efficiency of phytoremediation of arsenic contaminated soils by P. vittata.

蜈蚣草是一种理想的砷污染修复植物，但其砷富集的分子机制仍不完全清楚。ACR3亚砷酸逆转运蛋白是生物砷解毒的关键转运蛋白，也是蜈蚣草砷解毒和砷富集的关键蛋白。目前，关于蜈蚣草ACR3的研究十分匮乏，且不同ACR3的功能差异仍不清楚。基于此，本项目以ACR3功能研究和比较为切入点，以蜈蚣草中4个不同ACR3为研究对象，在酵母、拟南芥和蜈蚣草三种系统中开展相关研究。通过分析ACR3对酵母砷抗性和砷积累的影响，阐明不同ACR3的酵母功能；通过分析ACR3在拟南芥砷吸收、外排、转运、解毒以及砷积累中的作用，结合ACR3-GFP融合蛋白在拟南芥中的亚细胞定位，阐明不同ACR3的植物功能；最后，通过qRT-PCR、膜组分分离和蛋白免疫印迹分析ACR3在蜈蚣草中的表达模式和亚细胞定位，并结合酵母实验和拟南芥实验，系统解析不同ACR3在蜈蚣草砷富集过程中的作用，为科学利用蜈蚣草开展砷污染修复提供理论支撑。

随着我国工业化和城市化进程的加快，土壤砷污染加剧。砷的致癌性、砷污染土壤的广泛性以及砷被农作物吸收进入食物链导致的食品安全问题引起了国内外学者的广泛关注。蕨类植物蜈蚣草（Pteris vittata）是世界上第一种被发现的砷超富集植物，是一种理想的修复土壤砷污染的植物，也是研究植物砷吸收、转运、积累和解毒机制的理想植物材料。.ACR3基因是参与蜈蚣草砷解毒、转运和积累的关键基因，本项目从蜈蚣草中克隆得到了4个不同的ACR3基因：PvACR3、PvACR3;1、 PvACR3;2和PvACR3;3。本项目分析了不同蜈蚣草ACR3基因的酵母功能，发现4个ACR3基因均编码有功能的亚砷酸（AsIII）逆转运蛋白，但其转运AsIII的效率存在差异。在此基础上，项目首先重点分析了蜈蚣草PvACR3;1的植物功能，发现PvACR3;1定位于植物液泡膜上，其在模式植物拟南芥和烟草中异源表达可将砷转运到根部液泡中进行区隔化，有效将砷滞留在根部，降低了砷向地上部的运移。基于此，项目利用PvACR3;1培育低砷水稻，发现在水稻中表达PvACR3;1基因能显著降低水稻糙米中的总砷，尤其是无机砷含量。最后，项目分析蜈蚣草PvACR3;2和PvACR3;3的植物功能，比较了不同ACR3的异同。.综上所述，项目分析了蜈蚣草不同ACR3的植物功能，探究了ACR3在蜈蚣草砷富集过程中的作用，并利用不同ACR3调控植物砷吸收，对培育砷积累修复植物或阻控砷在粮食作物中的积累、保障食品安全具有重要意义。