α-葡糖苷酶在烟粉虱传播番茄褪绿病毒过程中的作用机制研究

Tomato chlorosis virus (ToCV) has spread in China in recent years and causes serious losses to tomatoes and other vegetables. Previous studies found that the rapid spread of ToCV to the whole country in recent years is associated with the widespread distribution of Bemisia tabaci Q. However, little is known about the key factor that affects the process of virus acquisition, retention and transmission by B. tabaci Q. Combined with the previous transcriptome, genomic sequencing and qPCR results, we found that with the increase of ToCV content in B. tabaci, the expression of α-glucosidase gene increased significantly. This study intends to determine the effect of α-glucosidase on virus acquisition, retention and transmission by B. tabaci Q, using the techniques and methods of insect physiological biochemistry and molecular biology. After that, α-glucosidase activity and gene expression will be inhibited by enzyme inhibitor and RNAi treatment, then the inhibition effects on virus acquisition, retention and transmission by B. tabaci Q will be determined, and the inhibition effects on growth and development and glucose metabolism of B. tabaci Q will also be determined. Our expected results will help us to deeply understand the transmission process of semi-persistent virus, to determine the mechanism of α-glucosidase in ToCV transmission process by B. tabaci, and to provide new ideas for control ToCV effectively.

番茄褪绿病毒（ToCV）是近年来在我国大面积发生的给番茄等蔬菜生产造成严重损失的病毒，前期研究发现Q烟粉虱的广泛分布是造成ToCV在最近几年迅速蔓延至全国的重要原因，然而关于影响Q烟粉虱获毒、持毒和传毒过程的关键因子仍缺乏研究，结合前期的转录组、基因组测序和qPCR验证结果，我们发现随着烟粉虱体内ToCV含量升高，α-葡糖苷酶基因表达量显著上升。本研究拟采用昆虫生理生化和分子生物学的技术与方法，重点研究α-葡糖苷酶在烟粉虱获毒、持毒、传毒过程中的作用机制，并通过酶抑制剂处理和RNAi的方法抑制α-葡糖苷酶的活性和基因表达情况，研究其活性降低后对烟粉虱获毒、持毒、传毒的影响及对烟粉虱生长发育和糖代谢的影响。预期研究结果将丰富我们对半持久病毒传播过程的认识，明确α-葡糖苷酶在烟粉虱传播ToCV过程中的作用机制，为有效控制ToCV提供新思路。

研究了健康与带毒烟粉虱体内α-葡萄糖苷酶表达量和酶活；烟粉虱体内ToCV病毒量与α-葡萄糖苷酶表达量的关系；饲喂Bt11975 dsRNA后对烟粉虱获取和传播ToCV的影响；α-葡萄糖苷酶抑制剂处理后对烟粉虱获取和传播ToCV的影响。研究表明α-葡萄糖苷酶在烟粉虱传播ToCV过程中发挥了重要作用。在不同的获毒时间内，烟粉虱体内α-葡萄糖苷酶基因Bt11975相对表达量和酶活显著高于健康烟粉虱。获毒24-72 h的烟粉虱体内Bt11975相对表达量为健康烟粉虱的2倍以上，获毒24 h的烟粉虱体内α-葡萄糖苷酶酶活增加了0.70倍；同时Bt11975在带毒烟粉虱的唾液腺和前肠中高表达。与烟粉虱饲喂GFP dsRNA对比，烟粉虱在喂食Bt11975 dsRNA后，随着时间的延长，烟粉虱体内Bt11975的相对分子表达量显著降低，同时烟粉虱在获毒48 h和72 h时，烟粉虱获毒率和体内病毒量也逐渐降低。通过25头和50头烟粉虱进行传毒时，有效控制了被番茄褪绿病毒侵染的番茄植株的数量，且番茄植株感染的病毒量明显减少。与烟粉虱仅饲喂蔗糖液对比，饲喂不同浓度酶抑制剂48 h后发现，酶抑制剂浓度升高，烟粉虱死亡数量增多，同时饲喂后的烟粉虱体内α-葡萄糖苷酶的酶活性下降，烟粉虱获毒能力和传毒能力均显著降低。研究结果表明，α-葡萄糖苷酶在烟粉虱传播ToCV过程中发挥了重要作用，针对该基因设计的dsRNAs可抑制Bt11975的表达量，酶抑制剂可抑制α-葡萄糖苷酶酶活，从而导致烟粉虱死亡，降低其对番茄褪绿病毒病的传播。研究结果可以应用于农业生产中对烟粉虱的防治，及降低番茄褪绿病毒病的发生。