LRR-RLKs受体蛋白激酶控制水稻花药绒毡层特征和PCD的分子机理研究

Flowering plants form male reproductive cells (microsporocytes) during sporophytic generation, which subsequently differentiate into multicellular male gametes in the gametophytic generation. The tapetum is a somatic helper tissue neighboring microsporocytes and supporting gametogenesis. The mechanism controlling the specification of the tapetal cell fate and development within the anther has long been a mystery in biology. Recent investigations revealed molecular switches and signaling pathways including Leucine-Rich Repeat Receptor-Like Kinases (LRR-RLKs) underlying the establishment of tapetal cell fate and differentiation in plants. However, the underlying mechnism of LRR-RLKs controlling tapetal cell development remains unclear. In this proposal we plan to employ two mutants of LRR-RLKs, mutiple sporocyte 1-3 (msp1-3) and abnormal tapetal degeneration1 (atd1) to investigate the downstream signalling pathways of MSP1 and ATD1 using bioehcmical and genetic approaches. The protein kinase activitiies and their crutial residues of MSP1 and ATD1 will be analyzed. Their downsteam interacting proteins will be revealed by yeast two hybrid method using the kinase domains of MSP1 and ATD1 as the bait, respectively. In addition, the msp1-3 and atd1 anthers are to be used for identifying downstream components using phosphoproteomics. The function of the confirmed downstream proteins will be analyzed by genetic approaches such as CRISPR and expression analysis. Thus this work may fill the gap between LRR-RLK sginalling and the specification of tapetal development in rice.

开花植物在孢子体发生时期形成小孢子母细胞并分化为多细胞的雄性配子；绒毡层属于体细胞组织，与小孢子母细胞、小孢子相邻，并为配子体发育提供营养和信号分子。进年来人们揭示了LRR类-受体蛋白激酶在控制绒毡层细胞特征方面具有重要作用，但其信号途径不清楚。本项目在前期工作基础上，以两个花药绒毡层发育异常的LRR受体蛋白激酶突变体msp1-3和atd1开展研究。msp1-3花药没有绒毡层、小孢子母细胞异常增多；atd1绒毡层细胞降解异常。我们拟开展MSP1以及ATD1的受体蛋白激酶活性等生化分析；并利用酵母双杂交、磷酸化蛋白质组等对MSP1以及ATD1的细胞内结构域互作蛋白、磷酸化底物进行分析，确定其下游作用蛋白。并利用CRISPR等技术研究这些下游蛋白的生物功能；从而提出这两个LRR类-受体蛋白激酶在控制绒毡层细胞发育中的信号传导途径和作用机制。

开花植物在孢子体时期的适当发育阶段可形成雄性生殖器官-雄蕊，雄蕊由花药和花丝组成。花药是形成小孢子的主要器官，在花药中央形成小孢子母细胞，通过减数分裂和有丝分裂产生雄性配子；花药发育早期，经过细胞分裂分化，多数高等植物可以在小孢子外部形成四层细胞壁结构：从外到内依次为外皮层、内皮层、中层、绒毡层。花药外壁细胞之间的有序协同发育是小孢子正常发育分化的前提。富亮氨酸（LRR）类-受体蛋白激酶能协同细胞间的信号传递。本项目在前期工作基础上，通过遗传和生化手段阐明了富亮氨酸受体激酶MSP1 (MULTIPLE SPOROCYTE1)与其配体结合OsTDL1A (TPD1-like 1A)决定早期花药细胞命运和小孢子发育。并进一步阐明了OsTDL1A-MSP1是通过调控细胞的氧化还原状态调控花药壁的细胞命运和小孢子的增殖。另外，本项目还鉴定到一个新富亮氨酸受体激酶TMS10 (Thermo-Sensitive Genic Male Sterile 10)，可以感受温度调控花药绒毡层的降解和小孢子的发育的机制。高温条件下tms10突变体表现出温敏雄性不育，低温下该突变体育性恢复正常，我们并探讨了该不育系的温度响应机制和在生产中应用的可能。此外，本研究还对一些直接参与花药绒毡层和小胞子发育的下游效应基因/蛋白进行了功能研究。例如我们分析发现，小RNA miR2275 和一个24 nt phasiRNA 及其前体PHAS在msp1和Ostpd1a的突变体中都不表达，表明它们是OsTDL1A-MSP1途径的重要效应因子。本研究还鉴定了DPW2 (Defective Pollen Wall 2)、OsGPAT3 (Glycerol-3-Phosphate Acyltransferase 3)、RMD (Rice Morphology Determinant)、OsGPAT3 (Tetraketide α-Pyrone Reductase)、DPW3 (Defective Pollen Wall 3)等参与水稻花药发育的新基因，并研究了其生物学功能。该研究提升了人们对于受体激酶在雄性生殖发育中的功能有了新的认识，相关雄性不育突变体材料在水稻杂交育种中具有一定的应用潜力。本项目执行过程中，发表标注论文12篇，累计影响因子超过85。用课题相关的材料和方法申请专利7项。

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