鼠耳芥ARGONAUTE2/5基因在植物锌/镉耐受性和超富集中的作用机制

Arabidopsis halleri, a close species of Arabidopsis thaliana, is a typical hyperaccumulator with strong tolerance and accumulation of Zn/Cd. ARGONAUTE2/5 function as a component of RNA-induced silencing complex (RISC) in A. halleri. Our transcriptome results revealed that AGO2/5 display significantly higher transcript abundance in A. halleri than their homologs in A. thaliana. More importantly, the A. thaliana cells overexpressing ARGONAUTE 2（AGO2）and AGO5 of A. halleri displayed significantly enhanced Zn/Cd tolerance and accumulation. The role and regulatory mechanisms of AGO-miRNA modules in Zn/Cd resistance and hyperaccumulation are currently unclear. Based on this, this study aims to demonstrate function and regulation of AGO2/5 in Zn/Cd tolerance and hyperaccumulation. 1) Gene expression patterns and subcellular localization firstly will be investigated. Phenotypic analysis will be performed using AGO2/5 RNAi and overexpressing lines to validate their roles in Zn/Cd tolerance and accumulation. 2) miRNA and transcript profiling using AGO2/5 RNAi and overexpressing lines as well as pull-down of AGO2/5-miRNA complex will reveal AGO2/5-mediated miRNA pathways that participate in regulatory network of Zn/Cd tolerance and accumulation in A. halleri. The results will benefit for dissecting regulatory mechanisms of heavy metal adaptions in plants and provide basic information for improving the performance of plants in heavy meal excessive environment.

拟南芥近缘种鼠耳芥是典型的超富集植物，具有极强的锌/镉耐受性和累积能力。鼠耳芥ARGONAUTE2/5（AGO2/5）蛋白是RNA沉默复合体的重要成员。转录组分析显示鼠耳芥AGO2/5表达水平分别远高于拟南芥，在拟南芥中过表达AGO2/5显著提高了其细胞的锌/镉耐受和累积能力。目前有关AGO蛋白-miRNA模块在植物锌/镉耐受性和超富集中的作用及调控机制不清楚。基于此，本研究拟在鼠耳芥中研究AGO2/5在植物锌/镉耐受性和超富集中的作用机制，包括1）分析AGO2/5组织表达模式及亚细胞定位，利用RNAi和过表达系确定AGO2/5对锌/镉耐受和累积的作用；2）利用RNAi和过表达系开展小RNA表达谱和转录组分析，并结合免疫沉淀结果，分析AGO2/5及其介导的miRNA参与调控锌/镉耐受和累积的作用网络。上述结果将促进植物重金属适应性调控机制的解析，为改良植物重金属环境适应能力提供理论依据。

与其近缘种拟南芥相比，鼠耳芥具有很强的Zn/Cd耐受和富集能力，因此成为研究植物重金属耐受和累积调控机制的良好材料。本研究首先对Zn/Cd胁迫下鼠耳芥和拟南芥植株做了比较转录组分析，发现AGONAUTE2 (AGO2)和AGO5两个同源基因在鼠耳芥中的表达水平均显著高于拟南芥。qPCR实验进一步确认了AGO2/5基因在两个物种间的表达水平差异。克隆得到AGO2/5基因的编码区全长序列，发现其编码的蛋白均具有完整的PAZ，MID和PiWi结构域，属于典型的AGO蛋白家族成员。qPCR和启动子报告株系均表明AGO2在地上部分高表达，而AGO5特异性地高表达在根组织中。亚细胞定位实验表明AGO2/5蛋白在细胞核和细胞质均有分布。过表达转基因拟南芥的表型分析发现，AGO2能够显著大幅减缓Zn/Cd胁迫下地上部分生长抑制状况，增强了植株耐受性。原生质体和重金属含量测定实验表明AGO2增强了细胞对Zn/Cd的耐受性，而并未改变植株内的Zn/Cd含量。AGO5过表达导致转基因植株地上部分重金属耐受性明显减弱，却增强了根组织的Zn/Cd耐受性。进一步的实验结果表明，AGO5促进了植株体内Zn/Cd离子由根向地上部分的转运，而并未改变植物细胞对重金属的耐受性。用Pull-down结合小RNA测序的方法，鉴定16个和13个分别能够与AGO2和AGO5直接结合的小RNA，并通过AGO2/5转基因植株的比较转录组测序构建了AGO2/5对下游基因表达的调控网络。本研究的结果明确了AGO2/5蛋白在耐受和累积Zn/Cd过程中的作用，初步搞清了AGO蛋白通过介导miRNA参与植物重金属适应性的作用方式，为阐明植物重金属抗性和累积调控机制提供了证据，也为植物重金属环境适应性遗传改良提供了候选基因资源。