连作地黄应答自毒物质的LRR-RLKs受体激酶鉴定与功能分析

LRR-RLKs (Leucine Rich-Repeat Receptor-Like Kinases) play a critical roles in perceiving signals from environmental stress in plant. Our preliminary research found that self-allelochemicals, which derived from rhizosphere of continuous cropping R. glutinosa were sensed by LRR-LRKs receptors. The following four research will be conducted in this project on the first cropping and continuous cropping R. glutinosa sampled from the field and simulation experiments：(1) The specific receptors responding to allelopathic chemicals were determined through analyzing its spatial and temporal expression in continuous cropping R. glutinosa and various R. glutinosa that were treated with different stresses factors. (2) RACE technology were used to clone full length of LRR-RLKs cDNA. (3) RNAi and over expressed technology were used to confirm the function of LRR-RLKs from above step that used to perceive allelopathic chemicals. (4) A series of proteins that interact with LRR-RLKs receptors identified from step 2 were separated by using Co-IP and GST pull-down experiments. This study was aimed to uncover key signaling pathway that used to recognize and transmit self-allelochemicals from R. glutinosa, reveal the underlying molecular mechanism of R. glutinosa consecutive monoculture problems, and provide theoretical basis and technical support for solving the problem of continuous cropping obstacles.

植物富含亮氨酸重复类受体蛋白激酶（LRR-RLKs）是植物感知胁迫信号的重要受体之一。 本课题组前期研究发现LRR-RLKs受体应答了连作地黄根际自毒物质信号。本项目拟在大田和组培模拟条件下开展以下研究：①采用qRT-PCR/Northern Blot 技术研究LRR-LRKs在连作以及不同胁迫因子处理下地黄体内的表达模式，鉴定响应自毒物质的特异LRR-LRKs基因；②利用RACE技术克隆LRR-RLKs全长序列，并进行亚细胞定位；③利用过表达和RNAi方法确证接收自毒物质信号的LRR-RLKs受体蛋白；④利用免疫共沉淀（Co-IP）和GST pull-down 技术获取与LRR-RLKs互作的蛋白，寻找地黄应答自毒物质的信号通路。为揭示地黄连作障碍的分子机理，解决中药材生产中连作障碍难题提供理论依据。

前期研究发现地黄LRR-RLKs感知和引发了连作地黄的伤害进程。为深入揭示LRR-RLKs在地黄连作障碍形成中的角色，本项目搭建了田间、受控和模拟连作体系，精细鉴定了连作特异响应LRR-RLKs，确证了其在连作伤害分子链上的核心位置，完善了地黄连作形成分子证据链。.（1）鉴定了响应连作的特异LRR-RLKs清单。筛选、确证了地黄LRR-RLKs基因，详细分析了其在连作及不同胁迫因子下表达特征，遴选出了与连作密切相关基因。结果在地黄中鉴定到40个地黄LRR-RLKs蛋白，12个蛋白与连作形成密切关联。.（2）克隆了LRR-RLKs基因全长编码区并确认了其亚细胞位置。克隆了地黄LRR-RLKs基因全长编码区，制备了其抗体信息，并通过Western blot技术确证了其蛋白功能。结果亚细胞定位显示LRR-RLKs均位于细胞膜上，蛋白水平上确认了地黄LRR-RLKs与连作形成密切关联。.（3）利用过表达和RNAi确证了地黄LRR-RLKs基因功能。在优化地黄遗传转化体系基础上，对连作特异LRR-RLKs进行过表达和RNAi，结合其在连作下的表达特征和伤害特性，确证了LRR-RLKs在连作中角色。发现LRR-RLKs在连作中存在着2种效应，一种是LRR-RLKs丰度增加降低连作地黄死亡率，一种则相反，表明在地黄连作形成中LRR-RLKs具有正负效应。.（4）鉴定了LRR-RLKs互作蛋白。对比LRR-RLKs在原核、真核中表达效率，发现杆状病毒能有效表达地黄膜蛋白。利用GST pull-down鉴定连作关键LRR-RLKs互作蛋白，发现其参与了地黄病害响应进程。进一步通过BiFC验证LRR-RLKs互作蛋白，发现其与MinD蛋白能互作。利用BiFC分析植物保守LRR-RLKs互作对，发现RgNIK1和RgBAK1、RgSERK2能互作，RgSOBIR1与RgBAK1、RgSERK2不互作。利用分子对接发现地黄关键化感物质与RgSOBIR1感知点结合，阻滞RgSOBIR1对病原菌感知，这暗示连作中化感物质、致病菌与LRR-RLKs互作引发了连作伤害。.本项目结合多层模拟连作和多元分子生物学技术揭示了LRR-RLKs在地黄连作形成中的角色，梳理出连作形成中化感自毒物质、根际微生物和植物间互作机制，阐明了连作地黄死亡的内在机制，结果为全面解读地黄连作障碍形成机制和消减连作奠定基础。