非ABA依赖型SnRK2激酶调控马铃薯响应干旱胁迫的机制解析

Sucrose Non-Fermenting-1 Related Kinases 2（SnRK2）is a family of highly conserved protein kinases in plants. They play important roles in the development of abscisic acid-dependent plants, and they are involved in the stress responses of plants to osmotic stress caused by drought or saline. To reveal the functional importance of SnRK2s in potato , we propose to employ the methods of TALEN (transcription activator-like effector nucleases) and combined with the pollen protoplast transformation technique to construct the SnRK2s knock-out potato plant. The physiological and biochemical characteristics of knock-out potato will be evaluated to reveal the regulatory function of SnRK2s. To study the activities of the SnRK2s in response to ABA, the His-tagged SnRK2s protein will be purified and the In-Gel Kinase assay will be conducted to analyze the in vitro activities of SnRK2s. To further characterize the function of non-ABA dependent SnRk2 in response to the abiotic stresses such as drought, the green fluorescence protein (GFP) labeling, real-time quantitative PCR will be employed to analyze the subcellular distribution and the stress induced tissue specific transcriptional expression. Final, the RNA-Seq will be conducted to analyze the regulatory roles of non-ABA dependent SnRk2. The results of this study should reveal the functional importance of non-ABA dependent SnRK2 in potato and would make it possible to explore the potentiality of improving the stress tolerance of potato through regulating the expression and activities of SnRK2s.

蔗糖非酵解相关蛋白激酶家族2 (Sucrose Non-Fermenting Related Kinases，SnRK2) 是一类植物中高度保守的蛋白激酶，也是植物响应干旱盐碱等导致的渗透性胁迫的主要调控原件，但关于其在重要作物中的抗逆机理研究还很薄弱。本项目以马铃薯SnRK2类激酶为研究对象，利用TALEN (transcription activator-like effector nucleases) 基因组定向修饰与花粉原生质体转化相结合的技术手段构建SnRK2s敲除植株；利用In-Gel Kinase分析SnRK2s的体外激酶活性并确定非ABA依赖型SnRK2；利用荧光蛋白标记，实时定量RT-PCR，RNA-Seq等技术手段分析干旱胁迫下非ABA依赖型SnRK2激酶的组织特异性表达与遗传调控途径。研究结果将为解析马铃薯干旱胁迫响应代谢网络以及马铃薯抗逆育种提供理论依据与技术基础。

蔗糖非酵解相关蛋白激酶家族2 (Sucrose Non-Fermenting Related Kinases，SnRK2) 是一类植物中高度保守的蛋白激酶，也是植物响应干旱盐碱等导致的渗透性胁迫的主要调控原件。经过4年的研究，本项目对马铃薯中SnRK相关基因进行功能研究，取得了以下成果：（1）完成了马铃薯基因组中StSnRK2基因家族成员的筛选、克隆、组织特异性表达及亚细胞定位分析；并对其进行非生物胁迫处理下，StSnRK2基因家族的表达及生理生化指标的关联性分析；转StSnRK2的烟草中，抗逆相关基因的表达特性分析。（2）完成了CRISPR/Cas9、RNAi以及过表达载体的构建，原核表达载体的构建及蛋白纯化，抗体的制备及纯化。（3）完成了过表达转基因植株的筛选、鉴定，及胁迫处理下表型、生理生化指标、基因表达及转录组分析。我们的研究结果表明：马铃薯StSnRK2基因家族成员参与马铃薯响应逆境胁迫，虽然在非生物胁迫下，该家族成员基因表达量均有不同程度的升高，但是SnRK2.1/7基因的过量表达，反向抑制了其上下游基因的表达，从而抑制其响应胁迫的信号通路的表达，最终导致其响应胁迫的能力降低，严重影响植株的生长发育。后续我们对SnRK2.1/7基因RNAi植株的表型、生理生化及基因表达分析，将为我们进一步揭示马铃薯SnRK2家族成员的功能提供依据。