拟南芥microRNA395调控镉胁迫功能研究

MiRNA plays very important function in regulated abiotic stresses. Two components of sulfate assimilation, the low-affinity sulfate transporter SULTR2;1 and ATP sulfurylase, are argeted by miR395, and that the mRNAs encoding isoforms ATPS1 and ATPS4 of ATPS, and SULTR2;1, are cleaved by miR395; meanwhile, ATPS1、ATPS2、ATPS3, ATPS4 and SULTR2;1 also regulated under Cd stress. Meanwile, Overexpressionof miR395c or miR395e retarded and accelerated,respectively, the seed germination of Arabidopsis under high salt or dehydration stress conditions,which suggested miR395 can regulated other stress. We found miR395 also can regulated ATPS1, ATPS3 and ATPS4 of ATP sulfurylase, and SULTR2;1 under Cd stress in Brasscia nupas and Arabidopsis thaliana. But，the mechanism are unclearly. We will try to figure out the way of miR395 regulate Cd stress by analysis of location, overexpression, mimicry miRNA（silence the function of miR395） and target gene silence transgenic plants.By the methods of northern blot and in situ hybridization etc., the expressions of miR395 and ATPS, SULTR2;1 the characteristics of phenotype and mechanisms of physiology and biochemistry will be studied in the control and transgenic plants under heavy metal Cd stress. The study also may provide the evidences in investigation of regulatory mechanisms for plant tolerance to stresses.

miRNA在调控植物抵御非生物胁迫方面具有重要作用。相关研究发现拟南芥miR395会通过调控ATPS等硫代谢基因来响应缺硫胁迫，而miR395过表达还会影响盐胁迫和干旱下种子的萌发，这显示miR395可能对多种胁迫存在响应。课题组前期研究发现，miR395能通过调节硫代谢相关基因家族BnSultr2;1和ATPS 来参与镉胁迫响应。本研究拟构建mimicry miR395（竞争性结合并抑制miR395表达）和抗miR395-ATPS（定点突变）两个过表达载体,转化获得转基因植株，结合利用已获得的miR395过表达株系，利用northern blot、原位杂交等手段探索miR395和ATPS的时空表达模式、转基因植株的表型变化及其在重金属镉胁迫下的生理生化反应。项目研究结果可以阐明miR395和其靶基因在植物耐受重金属镉胁迫中所发挥的作用，为植物抗逆机制研究提供理论依据。

本研究构建了mimicry miR395（竞争性结合并抑制miR395表达）和过表达miR395两个过表达载体,转化获得转基因植株，利用Realtime PCR, northern blot等手段探索miR395和ATPS的时空表达模式、转基因植株的表型变化及其在重金属镉胁迫下的反应。在镉胁迫下miR395抑制了 4 个靶基因的表达， miR395参与了植物在镉胁迫下的响应机制。进一步对本课题组的红麻品种进行抗重金属胁迫方面进行研究，研究镉胁迫下红麻响应生理反应与转基因转化平台，验证红麻中miR395功能，为利用miR395调控机制应用作物选育奠定基础。选育了杂交红麻H1302，对所选育的品种进行了镉污染农田的种植实验，结果表明说明该品种具有作为镉污染农田修复材料的巨大潜力。