胡杨SnRK2s及其互作蛋白基因参与ABA介导的水分胁迫响应机制研究

To reveal the stress resistance mechanism of P. euphratica is the basis for poplar genetic resources innovation utilizing important gene resources from P. euphratica. This project centers on the scientific theory that ABA signal transduction play important roles in plants abiotic stress response. In view of the defect that it is unrealistic to identify key important genes from P. euphratica through the mutant screening method, we performed a large scale of RNA-seq from a variety of stress treatments and finally isolated several water stress related SnRK2 members. We also established a yeast library using cDNA from water stress treated P.euphratica to screen SnRK2s interacting protein genes, and will use yeast-two hybrid and bimolecular fluorescence complementation (BiFC) methods to identify the interaction relationship between SnRK2s and their interacting proteins. Subcellular localization and expression patterns of SnRK2s and their interacting proteins will be analyzed by microtechnic and quantitative PCR method, respectively. Function and action mechnism of SnRK2s will be analyzed by genetic transformation in poplar. Downstream gene expression analysis will be analyzed by DNA microarray. The project will reveal the water stress response mechanism of PeSnRK2s and their interacting protein genes. The results will not only provide scientific foundation for further analysis of ABA signaling pathways in P. euphratica, but also provide theoretical basis for breeding stress resistant poplar by using SnRK2s.

揭示胡杨抗逆机制是利用胡杨优良基因资源进行杨树种质创新的基础。本项目围绕ABA信号转导是植物响应非生物逆境胁迫的重要途径这一科学理论，针对难以通过突变体筛选从胡杨中分离关键功能基因这一缺陷，利用大规模转录组测序的方法从多种胁迫处理的胡杨中筛选出水分胁迫响应相关的SnRK2类成员，建立了水分胁迫条件下的胡杨cDNA酵母文库。在此基础上，将通过酵母双杂和BiFC等手段构建胡杨SnRK2s的蛋白互作模式；通过亚细胞定位和荧光定量PCR分析SnRK2s及其互作蛋白基因的细胞定位情况和表达情况；通过杨树遗传转化分析SnRK2s功能及作用机制；通过基因芯片分析其下游调控基因。揭示胡杨SnRK2s及其互作蛋白基因参与水分胁迫响应的机制，不仅为进一步解析胡杨ABA信号途径提供科学依据，也将为探索利用SnRK2s创建杨树抗逆性优良种质资源提供相关理论依据。

ABA信号响应是植物响应非生物逆境胁迫的重要途径。通过该项目的实施，我们从胡杨中克隆获得三个参与胡杨ABA信号的SnRK2类基因，构建植物表达载体，通过农杆菌介导法实现了其中PeSnRK2.6在拟南芥、杨树中的遗传转化，得到了抗非生物胁迫逆性增强的转基因植株。同时，通过酵母杂交及双分子荧光标记技术，确定了胡杨中PePYL4－PeHAB1－PeSnRK2.6这一ABA逆境信号通路的存在，并对互作关系中的因子通过转基因的方法进行了功能研究，初步探明了胡杨SnRK2类基因参与胡杨ABA信号通路的依据。在此基础上，进一步确定了两个新的PeSnRK2.6的特异性互作蛋白基因Tify1和Tify2，这种互作关系在其他物种中未见到类似报道。通过研究这两个蛋白基因的表达模式，确定了Tify1和Tify2在干旱、高盐、寒冷等各种逆境胁迫下的诱导特性。综上，我们确定了胡杨SnRK基因参与逆境响应的分子网络，并为利用杨树自身的SnRK基因等ABA信号组分成员进行林木种质创新提供了理论依据。