蜈蚣草砷液泡区隔化基因的挖掘和功能分析及其应用研究

Arsenic contamination in the environment has become more serious in our country. It is more urgent to research and develop phytoremediation technology using a hyperaccumulator Pteris vittata, which provides a cost-effective and environmentally friendly option for land remediation. Previous studies have suggested that arsenic vacuole compartmentalization may play an important role in the As-hyperaccumulation in P. vittata, but the arsenic transport mechanisms of vacuole are largely unknown, and the identification of related genes are limited so far. This plan of the project is to identify more vacuole membrane protein genes involving in arsenic vacuole compartmentalization, by using the "1 KP program" which contains the transcriptome sequences of P. vittata, together with the early stage of the analysis of the vacuole membrane proteomics data of P. vittata in our lab. Then we tried to clone and characterization the function of the key target genes by bioinformatics analysis, subcellular localization, gene expression pattern analysis, yeast mutants and heterologous expression in Arabidopsis and so on. Furthermore, the Miscanthus with the features of wide adaptability and strong stress resistance is as genetically modified receptor for the identified and functional genes, to build engineering plant, for detecting arsenic enrichment ability and studying its application prospect. Our research is not only of great significance for understanding the arsenic hyperaccumulation mechanism P. vittata, but for breeding new engineering plant which can be used in phytoremediation, and provides the basis of the theory and application for the concrete implementation of phytoremediation technology.

我国砷污染严重，以砷超富集植物蜈蚣草为材料研发砷污染的植物修复技术迫在眉睫。砷液泡区隔化是蜈蚣草超富集的关键特性，研究蜈蚣草液泡膜砷转运和区隔化基因是该技术的重要环节，但目前为止，鉴定的相关基因有限，分子机制尚不清楚。本项目拟利用"1KP计划"近日公布的蜈蚣草"转录组序列"，分析前期获得的蜈蚣草液泡膜蛋白质组学数据，鉴定液泡膜砷区隔化相关的蛋白，特别是蜈蚣草特有的蛋白，进而克隆得到目标蛋白基因；根据关键基因的特性，通过生物信息学分析、亚细胞定位、基因表达模式分析、酵母突变体和拟南芥异源表达等多种方法研究其功能；利用鉴定的功能基因，以适生性广、抗逆性强的芒草作为"载体"构建工程植株，对其进行砷富集能力检测以探讨其应用前景。研究结果不仅对深入了解蜈蚣草液泡区隔化的砷超富集机制具有重要意义，还可为培育用于植物修复新型工程植株挖掘分子元件，而且将为植物修复技术的具体实施提供理论和应用依据。

全球特别是我国砷污染严重，以砷超富集植物蜈蚣草为材料研发砷污染的植物修复技术迫在眉睫，蜈蚣草具有液泡区隔化的砷超富集特性，研究蜈蚣草砷转运和区隔化液泡膜蛋白是研发该技术的关键环节。本项目利用“1KP计划”公布的蜈蚣草“转录组序列”，分析了前期获得的蜈蚣草液泡膜蛋白质组学数据，鉴定了多个液泡膜砷区隔化相关的蛋白，进而针对目标蛋白基因进行了克隆；对其通过亚细胞定位验证、基因表达模式分析、酵母突变体和拟南芥异源表达等多种方法研究其功能。建立了适生性广、抗逆性强的芒草的再生体系和遗传转化体系，利用该功能基因，以芒草为“载体”构建工程植株，并对其砷抗性和砷富集能力进行了验证。本项目研究结果不仅对深入了解蜈蚣草液泡区隔化的砷超富集机制具有重要意义，还为培育用于植物修复新型工程植株挖掘了分子元件，可为植物修复技术的具体实施提供理论和应用依据。