草原毒草黄花棘豆抗旱转录因子OoABF2的调控机理研究

Oxytropis ochrocephala is one of the most hazardous poisonous grass species in the grassland of Western China. This species causes great economic losses and poses a huge threat to the grassland ecosystem. O. ochrocephala is widely distributed in dry areas due to its strong stress resistance. Therefore, studying the mechanisms of stress tolerance in this species will be valuable for understanding the quick colonization of O.ochrocephala. Our previous studies on O.ochrocephala showed that OoABF2 transcription factor contributes to O. ochrocephala resistance against drought. However, the regulation mechanism of OoABF2 is still unclear. Based on our previous studies, we will further study the regulation mechanism of OoABF2 transcription factor. Our strategies are: 1) identifying the interacting and regulatory proteins of OoABF2 by yeast two-hybrid, bimolecular fluorescence complementation, and in vitro phosphorylation assays; 2) using ChIP-seq, yeast one-hybrid, dual-luciferase reporter assay system, qRT-PCR and transgenic Arabidopsis thaliana, to identify and analyze the target genes of OoABF2, and finally reveal to its regulatory mechanism. Results from this research will provide a new theoretical basis for understanding the quick colonziation of O.ochrocephala.

黄花棘豆作为我国西部草原地区的主要毒害草之一，给畜牧业和生态环境造成巨大损失和破坏。黄花棘豆优势分布区多为干旱少雨的地区，其迅速蔓延与自身极强的抗逆性密切相关。因此，对其抗逆机理研究成为解析黄花棘豆蔓延机制的重要突破点，但目前尚未有相关机理研究。本课题组前期研究显示，OoABF2转录因子参与黄花棘豆对干旱胁迫的应答反应，但其上游互作蛋白和下游调控靶标均不清楚。因此，本项目拟在课题组前期研究的基础上，对OoABF2的调控机理展开深入研究，采用酵母双杂交、双分子荧光互补和体外蛋白磷酸化等试验寻找其上游互作与调控蛋白，同时综合利用ChIP-seq、酵母单杂交、双荧光素酶报告基因、qRT-PCR和拟南芥转基因等技术探明其下游调控靶基因，进而从转录调控层面来探究黄花棘豆抗旱性的分子机制，以期为解析其蔓延的内在机制提供新的依据。

黄花棘豆作为我国西部草原地区的主要毒害草之一，给畜牧业和生态环境造成巨大损失和破坏。黄花棘豆优势分布区多为干旱少雨的地区，其迅速蔓延与自身极强的抗逆性密切相关。因此，对其抗逆机理研究成为解析黄花棘豆蔓延机制的重要突破点。本项目在课题组前期研究的基础上，对OoABF2的调控机理展开深入研究。利用同源克隆方法获得了黄花棘豆中的全部SnRK2s基因（OoSnRK2.1-OoSnRK2.8）。随后利用酵母双杂交技术、双分子荧光互补试验和pull-down方法证明OoABF2转录因子的上游调控基因是OoSnRK2.1和OoSnRK2.6两个蛋白激酶。同时利用CHIP-seq技术筛选到7个可能的OoABF2下游调控靶标基因。再利用酵母单杂交技术和ESMA实验验证OoLEA3蛋白是OoABF2的直接下游靶基因。综合所有实验结果，本项目部分解析了黄花棘豆中ABA信号通路，即：OoSnRK2.1/OoSnRK2.6蛋白激酶调控转录因子OoABF2，OoABF2再通过调控抗逆蛋白OoLEA3来响应ABA信号通路，从而提高黄花棘豆的抗旱性。本项目从转录调控层面探究了黄花棘豆抗旱性的分子机制，为解析其蔓延的内在机制提供了新的依据。