多杀菌素抗性西花蓟马与番茄斑萎病毒（TSWV）互作因子的鉴定及功能分析

Insect vector control, through the use of insecticides, plays a key role in the prevention and control of insect-borne viral diseases. In recent years, Frankliniella occidentalis (Pergande) have caused increasing serious damages in China, and the Tomato spotted wilt virus (TSWV) transmitted by F. occidentalis has resulted in great losses in some areas. Spinosyns such as spinosad, are the most effective insecticides for F. occidentalis control. However, resistance to spinosad has become an increasingly prominent problem. Whether resistance in F. occidentalis has impact on its virus transmission, to what extent of the impact and what are the molecular mechanisms for the impact are important scientific problems to be solved. So far, it is not clear the proteins interacted with TSWV in thrips and their functions need to be clarified. In the present project three aspects are to be conducted: 1) To screen the different specific binding proteins with TSWV in spinosad-susceptible and -resistant F. occidentalis using 2-D and virus overlay assay techniques. 2) To identify the proteins through protein mass spectrometry and then retrieve the transcriptomes of -susceptible and -resistant F. occidentalis to obtain the nucleotide sequences of the proteins. To clone the full-length sequences of the genes encoding the proteins by using RT-PCR and RACE. 3) To determine the functions of the genes and their differences in the two strains by using RNA interference technique and comparing the difference in virus acquisition and virus transmission efficiency between the two strains. The results will be helpful in further understanding the molecular mechanisms of the interaction between TSWV and F. occidentalis, to clarify the impact of spinosad resistance on the virus acquisition and virus transmission and the molecular mechanisms of the effect. In addition, the study will be provide theoretic bases and new thought for resistant F. occidentalis control and the IPM of TSWV.

使用杀虫剂阻断介体昆虫是防控虫传植物病毒病的主要途径。西花蓟马及其传播的番茄斑萎病毒（TSWV）病在我国的危害呈加剧趋势，西花蓟马对多杀菌素的抗性问题也日益凸显，目前蓟马体内与TSWV互作的蛋白因子及功能尚不明确，抗药性是否影响西花蓟马对TSWV的传播及其分子机制如何是亟待解决的科学问题。本项目以多杀菌素敏感和抗性西花蓟马为试虫，拟采用双向电泳和病毒覆盖技术，筛选西花蓟马体内与TSWV特异结合及具有种群差异的蛋白；通过蛋白质谱鉴定和西花蓟马转录组数据库获得匹配蛋白的核苷酸序列，用RT-PCR及RACE技术克隆不同基因的全长序列；采用RNAi技术结合西花蓟马获毒和传毒效率的比较，确定不同受体蛋白基因的功能和在两品系中的差异。研究结果有助于深入了解TSWV与西花蓟马互作的分子机制，阐明多杀菌素抗性对西花蓟马传毒的影响及分子机制，为抗性西花蓟马和TSWV的防控提供理论基础和新思路。

建立了西花蓟马酵母双杂交cDNA文库和分裂泛素酵母双杂交cDNA膜文库，番茄斑萎病毒的GN筛选到了67个西花蓟马蛋白，GC筛选到了122个西花蓟马蛋白。与GN互作的寄主因子中，选择三个筛选丰度高、酵母水平互作强以及可能参与了蓟马获毒传毒的相关基因，克隆该基因全长，构建到酵母双杂交载体pPR3-N中，与诱饵载体共转酵母和X-β-半乳糖苷酶颜色反应验证了载脂蛋白（ApoD）、表皮蛋白（Obst）和钙离子通道蛋白（Orai）与GN蛋白存在相互作用。利用荧光定量PCR技术（qPCR）分析了候选基因在西花蓟马取食TSWV各龄期以及未取食TSWV的表达量，ApoD在西花蓟马感染TSWV 1龄、2龄、蛹期以及成虫期的表达量均显著低于对应TSWV未感染的表达量，Obst在西花蓟马感染TSWV 1龄、2龄、预蛹期、蛹期以及成虫期的表达量均高于对应TSWV未感染的表达量，Orai在西花蓟马感染TSWV 1龄、2龄、预蛹期、蛹期以及成虫期的表达量均显著低于对应TSWV未感染的表达量。利用RNAi进行若虫干扰，沉默Apod基因48h后，干扰效率显著降低70%左右，沉默Obst基因72h后，干扰效率显著降低60%左右，沉默Orai基因24h后，干扰效率显著降低40%左右；进行成虫干扰，沉默Apod、Obst和Orai基因48h后，干扰效率均显著降低60%左右。进一步的干扰实验结果发现，Apod和Orai基因可能与蓟马获毒和传毒无关，Obst基因与蓟马获毒无关，但是可能在西花蓟马传毒过程中起着关键性作用，为后续研究番茄斑萎病毒与其传毒寄主西花蓟马在协同进化过程中形成了其独特的传毒机制奠定了基础。.以多杀菌素敏感和抗性近等基因系西花蓟马种群为研究对象，研究了多杀菌素抗性对蓟马获取病毒、病毒在虫体内增殖以及传播的影响，发现多杀菌素抗性显著提高了西花蓟马对病毒获取及增殖的能力，抗性西花蓟马的传毒能力高于敏感种群。发现西花蓟马携带番茄斑萎病毒后发育历期显著延长，更重要的是，延长的发育主要是成虫阶段，而成虫对病毒的扩散和传播极其重要。另外，携带番茄斑萎病毒的西花蓟马能够产生更多的子代，而大多数增加的子代是传播病毒能力更强的雄虫，导致子代的雌雄性比从1.3-7.0变为0.6-1。带毒西花蓟马的交配时间也显著延长，雌虫再次交配次数也呈降低趋势。