自身启动子驱动下蜈蚣草不同ACR3基因的植物功能研究

蜈蚣草是为数不多的成功应用于土壤砷污染修复的砷超富集植物，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。蜈蚣草中存在4个不同ACR3基因，是蜈蚣草区别于非超富集植物的重要分子特征，也是蜈蚣草高效富砷的关键。本项目拟以ACR3重要调控元件——自身启动子为切入点，首先通过TAIL PCR和染色体步移克隆不同ACR3自身启动子；在此基础上利用qRT-PCR和启动子融合GUS报告基因分析启动子功能和ACR3表达模式；进一步将启动子和相应ACR3分别导入模式植物烟草中表达，精准解析自身启动子驱动下ACR3在植物砷吸收、外排、转运、解毒和积累中的功能；最后在烟草中同时表达多个ACR3基因，探明不同ACR3的协同作用，系统阐明蜈蚣草砷富集分子机制，为提高蜈蚣草修复效率提供理论支撑，为培育砷积累工程植物提供重要的基因资源和调控元件。

Arsenic (As) hyperaccumulator Pteris vittata is one of the few plants that have been successfully applied in practical phytoremediation of As contaminated soil. However, the molecular mechanism of As hyperaccumulation in P. vittata is still unclear. Arsenite (AsIII) antiporter ACR3 is the key transporter for As detoxification. In the genome of P. vittata, four different ACR3 genes were found, which is different from non-hyperaccumulators. Thus, the multiple ACR3s are also supposed to play key role in As hyperaccumulation. In this proposal, considering the native promoters are important for gene expression and function, we will firstly clone the promoters of the four ACR3 genes using TAIL PCR and genome walking and conduct bioinformatic prediction. Based on the promoters, we will study the expression pattern of the ACR3 genes using qRT-PCR and GUS reporter gene driven by ACR3 promoters. Further more, we will transform different ACR3s into model plant tobacco, driven by their corresponding native promoters. We then investigate the roles of different ACR3s in As uptake, efflux, translocation, detoxification and accumulation in transgenic tobaccos. At last, we will transform multiple ACR3s into tobacco to investigate the synergistic effect of different ACR3. Based on these studies, we aim to clarify the function of different ACR3s in As transport, detoxification and hyperaccumulation in P. vittata, which is believe to provide basic knowledge for improving the efficiency of phytoremediation of arsenic contaminated soils by P. vittata and also to provide gene resources and regulatory elements for engineering As tolerance and hyperaccumulation in plants.