微丝骨架与微丝结合蛋白调控小麦对条锈病抗性的作用机理研究

Puccinia striiformis f. sp. tritici is an obligate fungus. Cultivating resistant cultivars is the most effective measure to prevent rust disease in wheat. However, many aspects of the resistance mechanism of wheat against P. striiformis f. sp. tritici remain to be elucidated. In previous studies, we found that actin filaments are regulated by actin-binding proteins (ABPs) and the depolymerization of actin filaments resulted in a weakening of wheat resistance. The full-length of the ABP genes TaPRO and TaADF which promote the depolymerization of actin filaments have been successfully cloned. The expression of these genes is downregulated in incompatible interactions. In this project, we attempt to reveal the roles of TaPRO and other ABPs which promote the depolymerization of actin filaments in the resistance of wheat against P. striiformis f. sp. tritici by means of virus-induced gene silencing; and use qRT-PCR to analyze the expression profile; the difference in the expression of the genes will be detected by Western blot; their functional loci and spatiotemporal characteristics are elucidated by means of subcellular localization and immunocytochemical localization; the functions of these genes will be investigated in transgenetic Arabidopsis plants. This research is aimed at clarifying the resistant mechanism of wheat against the stripe rust from a new angle, generating novel ideas of controlling wheat stripe rust and wheat resistance breeding.

小麦条锈菌为专性寄生菌，选用抗病品种是防治条锈病最为有效措施，然而小麦抗条锈病的机制还有许多方面尚未搞清。我们在前期工作中，发现微丝骨架受微丝结合蛋白的调控,微丝骨架的解聚导致小麦抗病性降低；并克隆了促进微丝解聚的小麦微丝结合蛋白基因TaPRO和TaADF1，它们在非亲和互作中表达下调。本项目将以TaPRO等促进微丝解聚的微丝结合蛋白基因为切入点，通过病毒诱导的基因沉默技术揭示其在小麦抗条锈病中的作用；利用qRT-PCR进行表达谱分析；Western blot检测在蛋白水平上的表达差异；通过亚细胞定位和免疫细胞化学定位，确定其作用位点以及时空变化特征；并转化拟南芥研究这些基因的功能。本研究有望从全新的角度阐明小麦抗条锈性机制，为该病防治和小麦抗锈育种提供新思路。

小麦条锈病是世界范围内小麦的最重要病害,揭示小麦抗条锈菌侵染的分子机理对于植物抗病性研究具有重要的意义。本项目从小麦-条锈菌互作差异表达的cDNA消减文库中分离并克隆得到高度表达的微丝结合蛋白基因TaPro和TaADF4；采用qRT-PCR技术对其在小麦组织中分布的特异性，非生物因子（高温、低温、耐旱性、盐）胁迫及其在小麦与条锈菌不同组合互作过程中的表达情况进行了研究；利用大麦条纹花叶病毒（BSMV）诱导的基因沉默体系研究了小麦微丝骨架相关蛋白TaPro和TaADF4 的功能；运用酵母双杂交技术，筛选到与TaADF4互作的元件TaACT1，并通过免疫共沉淀以及免疫荧光蛋白的共定位技术验证其直接互作；利用绿色荧光蛋白GFP对其进行了的亚细胞定位。采用外源激素和LC/MS技术，获得微丝相关基因在小麦抗条锈菌侵染过程中参与的信号转导途径。通过微丝骨架解聚，证实了微丝骨架在小麦抵抗条锈菌侵染过程中的重要作用。共发表9篇学术论文，其中SCI论文7篇。通过综合研究认为，本项目研究以全新的角度阐明了小麦-条锈菌互作的抗锈性机制，推进了植物抗病性研究进程。