麦二叉蚜唾液中导致小麦迅速黄化的关键致害因子鉴定及其作用机理

Greenbug (Schizaphis graminum) feeding leads to obvious chlorosis and necrosis on wheat in a short period of time, S.graminum saliva plays important roles in the induction of chlorosis on wheat, but the virulence factors species in saliva and its mechanisms are still unknown. In this project, the full-length transcriptome of S. graminum salivary glands and proteomic profiling of watery saliva are firstly conducted to identify candidate virulence factors. Infiltration of candidate virulence factors expressed by eukaryotic expression system into wheat leaves are performed, and then the effects on the leaf symptoms, accumulation of H2O2 and the expression levels of some genes involving in the plant defense responses are detected. The virulence factors with chlorosis-induced activity are silenced using RNAi, and then the effects of gene function loss on the damage symptoms of wheat induced by aphid are detected to further confirm the roles of virulence factors. Aphid feeding induced cDNA library of wheat leaves are constructed, then Yeast Two-Hybrid System and GST-pull down are performed to discover the interacting proteins of virulence factors in wheat to reveal its mechanisms. This research will provide insight into the mechanism of greenbug-induced phytotoxic damage and aphid-host interactions, also can serve as the basis for further studies on breeding for aphid resistant varieties.

麦二叉蚜取食在短时间内即可导致小麦黄化枯萎，其唾液中存在一些致害因子在引起小麦黄枯中起着重要作用，但其种类及作用机理仍未知。本项目拟通过麦二叉蚜唾液腺全长转录组测序联合唾液蛋白质组学筛选唾液中可诱导小麦黄化的潜在致害因子；利用酵母真核表达系统体外获得潜在致害因子蛋白，渗透注射表达产物后检测对小麦叶片表型、H2O2积累及防御反应的影响；进一步通过RNAi技术沉默具有黄化诱导活性的关键致害因子，检测其功能丧失后蚜虫对小麦黄化诱导能力的变化，进而从正反两方面鉴定并验证关键致害因子种类及功能；构建麦二叉蚜取食诱导的小麦叶片cDNA文库，利用酵母双杂交系统、GST-pull down鉴定并验证致害因子在小麦内的互作蛋白，揭示其作用机理。本研究将有助于进一步阐明麦二叉蚜致害机理及蚜虫-寄主互作关系，同时为抗蚜小麦育种等新型控蚜策略提供科学依据。

麦二叉蚜为小麦主要害虫之一，其具有植物毒性，取食后可引起小麦叶片迅速黄化。蚜虫为刺吸式害虫，取食过程中可向植物体内分泌唾液，其唾液蛋白在调控蚜虫-小麦互作中具有重要作用，但麦二叉蚜致害机制及唾液蛋白在其致害性中的作用机理仍未知。本研究通过转录组测序发现麦二叉蚜取食可诱导小麦水杨酸介导的防御信号途径，同时在取食位点引用H2O2大量积累，利用NADPH氧化酶抑制剂处理小麦叶片，可显著降低蚜虫取食诱导的黄化症状、过氧化氢积累及SA途径相关基因的表达量，证实NADPH氧化酶在麦二叉蚜致害性及其诱导小麦防御反应中具有重要作用。为进一步阐明唾液蛋白在麦二叉蚜致害性中的作用，通过麦二叉蚜唾液腺转录组及唾液蛋白质组联合分析鉴定76个唾液蛋白，并选取16个功能未知唾液蛋白作为潜在致害因子；RT-qPCR结果表明16个潜在致害因子都在麦二叉蚜唾液腺特异表达；利用农杆菌介导的瞬时表达体系及细菌III型分泌系统发现一个富含半胱氨酸的唾液蛋白Sg1658过表达后可引起烟草及小麦叶片的黄化死亡，DAB染色法发现注射区域有大量的H2O2积累，验证了Sg1658作为麦二叉蚜致害因子在诱导小麦叶片黄化死亡中的重要作用。同时研究发现了一个功能未知蛋白Sg2204在麦二叉蚜取食小麦过程中高量表达，在烟草叶片中瞬时表达Sg2204可抑制BAX及INF1触发的细胞死亡和植物免疫反应，利用细菌III型分泌在小麦中瞬时表达蚜虫候选效应子Sg2204，结果表明Sg2204可抑制荧光假单胞杆菌引起的胼胝质积累及水杨酸茉莉酸介导的植物防御反应，取食表达Sg2204小麦叶片后蚜虫存活率、产仔量及韧皮部取食显著增加。相反，利用纳米载体介导的RNA干扰（RNAi）技术沉默该基因后，麦二叉蚜取食可诱导更强的植物防御反应并导致蚜虫寄主适应性显著下降，证实了Sg2204可抑制小麦防御反应，提高麦二叉蚜寄主适应性及致害性。以上研究为解析麦蚜致害机制奠定理论基础，为开发蚜虫新型防控技术及培育抗蚜小麦品种提供了新的思路。