山葡萄特异CBL-CIPK互作子介导的低温胁迫应答机理研究

Calcineurin B-like proteins (CBLs) represent a family of newly emerging plant-specific Ca2+ sensors, which could selectively interplay with their respective kinase effectors (CBL-interacting protein kinases, CIPKs), thus playing critical roles in response to signaling transduction of various abiotic stresses. Chilling/Freezing damage is a key environmental limitation factor that adversely affects grapevine cultivation and production in cold regions. The exact regulatory mechanism of the CBL-CIPK complexes in grapevine in response to cold stress remains to be elucidated. Based on the cloning and expression characteristics of CIPKs from our previous RNA-seq data, the specific CBL-CIPK signaling components that involved in cold stress will be identified and functional characterized in this project. We will further characterize the roles of the key candidate CIPKs in stable integrated transgenic V. vinifera plants by either overexpressing or inhibiting the target gene, thereby exploring their connection with cold hardiness. Combined with cDNA-AFLP, CoIP and omics analysis, a regulatory signaling network model of specific CBL-CIPK components in cold stress will be constructed in V. amurensis. The implementation of this project will not only help in understanding in-depth mechanistic interplay of CBL/CIPK mediated signaling pathway associated with cold tolerance in grapevine, but also possess a realistic meaning in resolving the existing chilling/freezing damage problem that limits the development of Ningxia grapevine industry in China.

类钙调磷酸酶B亚基蛋白（CBLs）作为近年来新发现的一类植物Ca2+传感器蛋白，能与其互作的激酶CIPKs选择性特异结合，在调控植物响应非生物胁迫信号转导中发挥重要的作用。低温冷（冻）害是严重危害寒地葡萄栽培生产的主要逆境限制因子，有关葡萄CBL-CIPK参与低温胁迫调控的确切机制尚未阐明。本项目在前期对高抗寒山葡萄转录组测序获得CIPKs基因克隆、表达特性鉴定的基础上，拟挖掘参与低温胁迫的CBL-CIPK特异组合；明确其参与低温胁迫的功能特性，构建重要候选CIPK过量与RNAi表达载体，研究其在欧洲葡萄中过表达/被抑制与抗寒性的关系；结合cDNA-AFLP、CoIP与组学分析，初步构建特异CBL-CIPK调控的低温胁迫应答网络。课题的实施不仅有助于深层次理解山葡萄CBL/CIPK信号转导系统在抗寒代谢中的作用，而且对于通过分子育种解决制约我国宁夏葡萄产业存在的冷（冻）害问题具有现实意义。

本项目从中国野生山葡萄‘左山-1’中克隆了8个类钙调素磷酸酶B亚基蛋白基因（VaCBLs）与19个类钙调素磷酸酶B亚基蛋白互作蛋白基因（VaCIPKs）。酵母Y2H系统分析表明，山葡萄8个VaCBLs 与19个VaCIPKs 在酵母中的互作特异性及强度不同，其中VaCIPK18 可与所有的8条VaCBLs 强烈互作。分别对山葡萄VaCBL01、VaCIPK02与VaCIPK18的功能进行了解析。VaCBL01基因受低温与ABA诱导，在酵母中可与12个VaCIPKs强烈互作,与5个VaCIPKs互作强度较弱，但与 2个VaCIPKs没有互作。对山葡萄VaCIPK02基因功能研究表明，VaCIPK2在盐和干旱胁迫下具有重要作用。VaCIPK02的C末端具有转录活性，定位于细胞核与质。VaCIPK02与4种Ca2+传感蛋白VaCBLs强烈互作。基于双杂筛选,分离了与VaCIPK02互作的候选蛋白基因12个，在体内验证了VaCIPK02相互作用的ABA受体PYL9。VaCIPK02在拟南芥中组成型表达对盐胁迫更加敏感，但显著增强干旱耐受性，这一过程伴随着ABA的超敏反应、气孔关闭，并提高了节水能力。此外，VaCIPK02的过表达通过调节ABA响应基因的表达来清除和调节ROS，从而降低了ABA在细胞水平诱导的ROS活性。对山葡萄VaCIPK18的功能分析，表明其受低温、ABA、干旱与盐的诱导，定位于细胞核与质中。VaCIPK18蛋白以及其激酶与NAF结构域均无自激活活性。酵母与双分子荧光互补分析表明，VaCIPK18 可形成二聚体；位于该蛋白C端NAF结构域对于VaCIPK18与所有8个VaCBLs在酵母中的互作是必需的。在拟南芥中进行了抗寒功能分析，表明山葡萄VaCIPK18参与低温胁迫正调控。获得了VaCIPK18转化葡萄的过量与干扰表达植株。筛选了VaCIPK18的候选互作蛋白，并验证了其与VaMYB4a互作关系。项目的开展为揭示葡萄属植物CBLs 与CIPKs的功能奠定了基础，为利用基因工程开展抗寒分子育种提供了重要的基因资源。共发表论文3篇，获得个人奖项和荣誉称号2项，培养硕士研究生4人。