EnCIPK31基因介导西藏野生垂穗披碱草低温应答中Ca2+信号机制研究

Elymus nutans is an important grass that has been used extensively for grassland establishment and ecological restoration in cold areas. Elymus nutans Griseb. is widely distributed in the Qinghai-Tibet Plateau areas, and some of the wild accessions show a superior cold tolerance. Thus, the wild accession is an ideal choice to study cold tolerance mechanism in Elymus nutans. In a previous study, we analyzed the transcriptomic changes in the wild accessions and selected EnCIPK31 gene under cold stress. To elucidate the Ca2+ signaling pathway and EnCIPK31 gene function of the superior low temperature tolerance in Elymus nnutans Griseb., we propose to conduct systematic study to investigate cold response in the wild accession of Tibet. We have three objects: 1) to examine physiological and biochemical responses to cold treatment. This objective is achieved by monitoring changes of signaling Ca2+ molecules and genes through blocking their synthesis or action using a pharmacological approach. 2) to identify the function of EnCIPK31gene involved in cold response. This objective is achieved by technique of CRISPR/Cas9 and over expression of EnCIPK31gene. 3) to identify the pathways of signaling Ca2+ in cold response. This objective is achieved by H2Y analyzing and GFP-Pull down analyzing. By comparing the results we have for the cold response in common Elymus nutans, we may contribute to the superior low temperature tolerance in Elymus nutans Griseb.. The knowledge developed in this study will build a foundation for improving Elymus nutans production under cold stress through breeding and other cultivation practices.

垂穗披碱草是寒冷地区草牧业发展的重要草种。广布于青藏高原的野生居群是研究其低温适应的理想选择。本项目拟开展EnCIPK31基因介导西藏野生居群低温适应中Ca2+信号分子机制研究。在前期初步分析转录组、鉴定钙信号关键作用和筛选CIPK31基因的基础上，从EnCIPK31基因调控垂穗披碱草低温适应的功能和钙信号途径入手，采用电感耦合等离子体质谱和Real-time PCR技术，通过外施CaCl2、Ca2+螯合剂和Ca2+通道抑制剂，分析内源钙离子对低温响应及基因表达模式的影响；借助过表达及CRISPR/Cas9编辑等手段，明晰EnCIPK31基因的作用；应用酵母双杂及pull down方法解析EnCIPK31基因调控低温适应的分子机制；在磷酸化水平上借助模式植物和共表达技术，验证EnCIPK31基因介导的西藏野生垂穗披碱草低温适应中Ca2+信号的分子机制，为垂穗披碱草育种栽培提供科学依据。

钙信号在西藏野生垂穗披碱草低温应答中发挥重要作用，其中EnCIPK31是钙信号通路中的重要基因，但其作用机制尚不明晰。本项目在前期 RNA-Seq 及生理生化研究、筛选了EnCIPK31 关键基因基础上，采用外源添加CaCl2与低温复合处理，揭示了外源Ca2+以浓度依赖的方式调控当雄野生垂穗披碱草的低温适应性；克隆了EnCIPK31基因的CDS全长序列，明确了该基因主要在地上部分表达，且该基因受低温调控；揭示了EnCIPK31蛋白主要定位在细胞核和细胞质；阐明了EnCIPK31基因积极地调控垂穗披碱草的低温适应性；解析了EnCIPK31的互作蛋白和下游调控信号，阐释了EnCBL2/4-EnCIPK31信号通过EnRFS依赖的棉子糖合成途径调控垂穗披碱草的低温适应性。本项目的实施可进一步丰富植物的低温适应理论，为披碱草属重要牧草的栽培育种提供科学依据。