类受体蛋白激酶在铝诱导花生程序性细胞死亡中的调控机制

Aluminum (Al) toxicity is an important factor that affect the yield of peanut (Arachis hypogaea) in red-yellow soil of south China. Revealing the signal transduction process of Al-induced programmed cell death (PCD) in peanut is helpful to finally elucidate the mechanism of Al stress. Receptor-like protein kinases (RLKs), as important factors in signal transduction, play critical roles in plant growth and development as well as in the response to many environment stresses. Based on the analysis of peanut root tip transcriptome data which acquired in our previous study, this project is going to study the role of RLK in Al-induced PCD process in peanut and carry out the following researches. 1, Clone the full-length cDNA sequence of Al-stress Associated RLK genes by RACE, and verify the RLKs biochemical function by in vitro phosphorylation experiment. 2, Combining the results of different experiments, including histochemical staining, qRT-PCR and western blotting, systematically analyze the effects of overexpression of RLKs on Al-induced PCD in transgenic plants to investigate the roles of RLK in regulation of Al-induced PCD. 3, Perform inhibitor experiments with different signal pathway inhibitors, and perform a yeast two-hybrid screen as well as protoplast transient transformation to reveal the interaction partners and subcellular localization of important RLK to study the roles of RLK in Al-induced PCD signal pathway. This work will reveal the regulation mechanism of Receptor-like protein Kinases in Al-induced PCD in Arachis hypogaea, and provide the theoretical basis for clarifying the signal mechanism of Al-induced PCD and improving the varieties of peanut.

铝毒是影响南方红黄壤地区花生产量的重要原因，对铝诱导PCD信号机制的研究有助于阐明铝胁迫机理。类受体蛋白激酶（RLK）在植物生长发育和响应环境胁迫的信号传导途径中发挥重要作用，本项目在对前期铝胁迫花生根尖转录组数据分析的基础上，关注RLK在铝诱导PCD中的作用，拟开展以下研究：①利用RACE技术获取响应铝胁迫的花生RLK基因全长序列，并用体外磷酸化技术验证其生化功能；②运用过表达技术，结合组织化学染色、qRT-PCR以及western blotting技术研究相关RLK在铝诱导PCD中的调控功能；③利用不同信号通路抑制剂开展抑制剂实验，并分别通过原生质体瞬时转化技术与酵母双杂交文库筛选技术分析重要RLK的亚细胞定位与互作组分，分析它在铝诱导PCD信号通路中的作用。以揭示类受体蛋白激酶在花生铝诱导PCD中的调控机制，为阐明铝诱导PCD的信号机制、改良花生品种提供理论依据。

本课题通过分析前期开展铝胁迫花生根尖转录组测序结果，结合已公开的栽培种花生基因组数据，寻找参与了花生铝胁迫诱导PCD过程的类受体蛋白激酶基因，对重要候选基因的功能和作用方式进行了初步研究。共鉴定出90个AhRLKs为Al响应基因，挑选了在响应铝处理过程中表达变化最强烈的RLKs基因作为候选基因，共克隆了12个AhRLKs，通过体外磷酸化技术验证了其中的两个基因产物的自磷酸化活性。结合表达分析、蛋白质生物化学分析和生物信息学分析，选择AhLRK489、AhLECRKS9和AhBAK1-like继续开展生物学功能研究。AhLRK489编码LRR型双底物特异性类受体蛋白激酶，过表达该基因使得拟南芥植株抽薹提前，衰老提前，但是不改变成熟期植株的株高；在铝胁迫条件下，AhLRK489过表达转基因植株的莲座生长受到更大的抑制，表明该基因在调控植物发育进程和铝胁迫响应方面具有重要作用。亚细胞定位分析发现AhLRK489为质膜蛋白，此外，AhLecRKS9和AhBAK1-lik也定位于质膜，而AhLr10定位于质膜和细胞质。另一方面，对AhLECRKS9和AhBAK1-like过表达转基因植株的初步观察试验发现过表达AhLECRKS9的拟南芥具有提前抽苔，莲座增大的表型，过表达AhBAK1-like的拟南芥则暂未发现明显表型变化，相关的观察和AhLRK489互作组分的筛选工作正在进行。综合上述结果，本研究为探讨AhLRK489调控花生铝诱导PCD的作用机制奠定了坚实的工作基础，对于认识RLK参与花生铝胁迫响应过程提供了重要的启示性线索。项目资助发表SCI(E)论文3篇，中文核心论文3篇，待发表论文2篇。培养硕士生3名，3名均在读。项目投入经费23万元，支出15.5202万元，各项支出与预算相符。剩余经费7.4798万元，剩余经费计划用于本项目研究后续支出。