滞育烟蚜茧蜂的海藻糖积累机制及滞育关联蛋白功能研究

The aphid parasitoid Aphidius gifuensis offer tremendous potential as biological control agents, and it is capable of entering a factultative, final-instar larval diapause that is programmed by low temperature and short-day daylength. The attribute of diapause could improve shelf-life and increase fecundity, survival period and stress resistance of the parasitoid that terminated diapause. This feature is most attractive for mass culture and application of this aphid parasitoid. We previously observed that the aphid parasitoid can dramatically accumulate trehalose, and we also classified the diapause-associated proteins and determined their expression features. In this program, techniques of insect biochemistry, transcriptomics and RNAi are exploited to detect transformation between sugars and polyols, association between trahalose accumulation and diapause depth, and to disentangle mechanism of trehalose accumulation during diapause of the aphid parasitoid. As well as the class groups of diapause-associated proteins and their functions will be analysed. Studies are focused on the diapause-associated proteins that involved in glucose and lipid metabolism. Differential gene expression of trehalase, calcium calmodulin-dependent protein kinases and insulin-like growth factor binding proteins will be discovered, and pathways related to hormonal regulation of diapause, such as the insulin signalling pathway, will also be studied. Our program could provide references for fundamental research of diapause in parasitoids, and could also suggest techniques that may be exploited to increase efficency of diapause induction and shelf-life of biological control agents.

烟蚜茧蜂是优良的寄生性天敌昆虫，低温短日照能诱导其进入滞育态，可显著延长产品贮存期，滞育解除后雌蜂的产卵量、存活期、抗逆性均有提高，这一特性对扩繁及应用具重要意义。前期研究发现，该虫滞育诱导过程存在显著的海藻糖积累现象，并伴有滞育关联蛋白表达量发生变化。本项目拟在已有研究的基础上，利用昆虫生物化学、转录组学、RNA干扰等技术，分析烟蚜茧蜂滞育过程海藻糖积累与滞育深度的关联性，监测糖醇转化的过程，解析烟蚜茧蜂滞育的海藻糖积累机制，以与糖醇代谢相关的滞育关联蛋白为重点，系统研究海藻糖酶、钙调素依赖性蛋白激酶、胰岛素样生长因子结合蛋白等在滞育进程中的差异表达规律及其功能，探讨胰岛素信号通路调节糖醇代谢的机理，为深入寄生蜂滞育基础理论研究提供参考依据，也为提高寄生蜂滞育诱导效率、延长天敌产品货架期提供技术支撑。

烟蚜茧蜂是优良的寄生性天敌昆虫，滞育能延长其产品贮存期，滞育解除后雌蜂的产卵量和抗逆性均有提高，对扩繁及应用具重要意义。针对烟蚜茧蜂滞育期存在海藻糖积累，并伴有滞育关联蛋白表达量波动的现象，利用昆虫生物化学、转录组学、RNA干扰等技术深入研究，一是检测了烟蚜茧蜂在发育期、滞育期、滞育解除后，各时段体内生化指标和糖代谢关键酶的变化规律，证实滞育前糖原和海藻糖含量均处于高水平，进入滞育期糖原逐渐转化为海藻糖，滞育阶段海藻糖含量是非滞育个体的1.87倍。二是分析滞育关联蛋白的类群、表达特点及生物学功能，确定上调的滞育关联蛋白86个，下调滞育关联蛋白98个，主要集中代谢过程、次生物代谢生物合成、糖酵解、三羧酸循环、丙酮酸代谢等过程。三是克隆了4个关键的滞育关联基因，肌钙蛋白C基因全长703 bp，能增强肌肉细胞骨架作用，提高滞育期间的耐寒性；海藻糖合成酶基因AgTPS基因全长2971bp，可催化生成海藻糖-6-磷酸，参与催化海藻糖-6-磷酸去磷酸化生成海藻糖；糖原合成酶基因AgGYS基因全长2547bp，为有糖原磷酸化活性的糖基转移酶结构域；GYS基因全长2529bp，可催化UDP-葡萄糖的糖基转移到糖原分子的分支末段。四是确定了滞育海藻糖积累的的分子机理，在滞育准备前期烟蚜茧蜂会大量积累糖原的同时，降解部分糖原用于合成甘油及山梨酸等抗寒物质；在4龄末期，AgTPS基因表达量急剧上升，AgGP基因表达量维持不变，AgGYS基因表达量有所下降但仍高于同龄期非滞育个体；当进入滞育阶段，促进体内大量合成海藻糖，导致海藻糖积累量的迅速上升；进入滞育末期，烟蚜茧蜂4个滞育基因表达量均显著下降，代谢相关酶的活性和基因表达量的降低，不再进行糖原或海藻糖的大量合成。上述研究结论清晰地揭示了烟蚜茧蜂滞育过程海藻糖积累与滞育深度的关联性，解析了烟蚜茧蜂滞育的海藻糖积累机制，了解了部分滞育关联蛋白的功能，丰富了寄生蜂滞育基础理论，也为提高寄生蜂滞育诱导效率、延长天敌产品货架期提供技术支撑。