蛋白激酶EnBIN2-like调控西藏野生垂穗披碱草低温应答的分子机制

Elymus nutans Griseb., an important alpine perennial forage, is often used for ecological construction and establishment of cultivated pastures in northern regions. The wild populations distributed in the Qinghai-Tibetan Plateau with a superior cold-tolerance acts as an ideal forage to study the adpative mechanism of cold stress. Brassinosteroids, the sixth type of plant hormone, has been found to be a ubiquitous modulator in multiple plant developmental processes. Moreover, it is involved in the regulation of various abiotic stresses, including cold stress. Our recent study showed that brassinosteroid-mediated signaling pathway plays a positive role in regulation of cold tolerance in E. nutans. However, the underlying molecular mechanisms in the regulation of cold tolerance in E. nutans have not been elucidated. . To get a deeper insight into its molecular mechanisms in mediating cold tolerance in E. nutans, a key gene in brassinosteroid-mediated signaling pathway, BIN2-like has been cloned. The over-expression, RNA interference (RNAi), and subcellular localization analysis are to be conducted. These results will clarify the roles of EnBIN2-like in brassinosteroid signaling pathway in response to cold stress. Furthermore, pull-down combined with mass spectrometry, co-immunoprecipitation (Co-IP) assay, as well as biomolecular fluorescence complementation (BiFC) assay will help us detect target protein interacted with EnBIN2-like in cold response, and further increase our ability to understand the mechanism of brassinosteroids mediating cold stress response. The study will build a foundation for improving E. nutans production under cold stress, and further provide important clues for breeding and genetic improvement of E. nutans and other gramineous forage grass.

垂穗披碱草是青藏高原地区的重要牧草，因其极强的抗性，已被广泛应用于该地区生态建设和人工草地建植。广布于西藏的野生居群是研究低温应答机制的理想材料。油菜素内酯(BRs)不仅能够调节植物的生长发育，还参与调控多种非生物逆境的应答。申请人前期RNA-Seq研究初步发现，BRs信号在垂穗披碱草低温应答中有积极的调控作用，外源BRs能够提高其低温抗性。然而，目前对于BRs调控垂穗披碱草低温应答的机制尚不明晰。我们筛选并克隆了1个BRs信号中的关键基因BIN2-like，本项目拟以西藏野生垂穗披碱草为研究对象，在基因功能与Pull down、免疫共沉淀和双分子荧光互补等方面开展研究，阐明EnBIN2-like调控垂穗披碱草低温胁迫应答机制，揭示EnBIN2-like低温应答的互作蛋白，旨在为BRs调控植物的低温应答提供新理论，为垂穗披碱草及其他禾本科牧草抗逆栽培管理及品种选育提供理论依据。

低温灾害是制约西藏草牧业发展的重要障碍。本项目以广布于青藏高原的野生垂穗披碱草为研究材料，基于RNA-Seq分析，筛选并成功克隆了EnBIN2-like基因。采用酶联免疫检测和荧光定量方法，阐明了西藏野生垂穗披碱草内源油菜素内酯对低温胁迫的响应；通过外源添加油菜素内酯结合拟南芥bak1突变体表型验证，揭示了油菜素内酯通过CBF信号途径调控西藏野生垂穗披碱草的低温适应机制；采用在水稻中异源过表达和vigs沉默技术，明确了EnBIN2-like基因负向调控植物的抗寒性；通过酵母双杂交、萤火素酶互补及免疫共沉淀技术，解析了EnBIN2-like与EnCBL10蛋白互作，阐明了EnBIN2-like介导的低温信号应答通路；采用大麦条纹花叶病毒诱导EnCBL10基因沉默和在烟草中异源过表达该基因，明确了EnCBL10基因正向调控植物的抗寒性。该研究为高寒牧草抗寒机理研究提供了理论基础，为垂穗披碱草及其近缘物种麦类作物育种提供了优良基因资源。