桃小食心虫专性滞育和兼性滞育关键调控基因发掘与功能鉴定

Diapause is a quantitative trait in which multiple genes share complex interactions that generate the phenotype. Peach fruit moth, Carposina sasakii Matsumura is an important pest of many fruits. C. sasakii exhibits a facultative diapause that occurs on the host of Malus pumila, Crataegus pinnatifida, Prunus persica and Zizyphus jujuba, and this phenotype of facultative diapause is under photoperiodic control. However, C. sasakii exhibits an obligatory diapause on the host of Prunus armeniaca, and this phenotype is effected little by photoperiod. The obligatory diapause characteristic and the early taking place time are all adapted to short growing time and early taking place of fruit of P. armeniaca. In order to know the molecular mechanism of the diapause of C. sasakii, RNA-seq to simultaneously quantify and identify transcriptional profiles are used to explain the dramatic differences in two phenotypes. First, we get abundant gene expression data in the process of obligatory diapause and facultative diapause regulation using RNA-seq. Second, differential gene expression analysis between two diapause phenotypes and non-diapause phenotype is performed, and key genes and metabolic pathways according to obligatory diapause or facultative diapause are selected. At last, the key genes were analyzed in terms of genetic evolution, molecular characteristics, tissue expression, protein localization, RNA interference, and polypeptide activity to verify the function of their metabolic pathways in the development of diapause types. This will eventually clarify the molecular basis for the differences in different types of diapause, and provide scientific basis for interpreting the developmental mechanisms of different diapause types.

桃小食心虫(桃小)不同生物型对光周期反应不同。苹果上的桃小(苹果生物型)形成了兼性滞育，滞育受光周期调控；但杏上的桃小(杏生物型)形成了专性滞育，滞育基本不受光周期影响。滞育特性是两种生物型差别的关键，它们之间遗传分化达到了物种级。滞育是多基因参与的调控过程，预实验发现生物钟基因和激素调控相关基因在两种生物型老熟幼虫体内表达存在明显差异，其中37个候选基因的表达量差别超过4倍。为了精确筛选不同滞育类型调控的关键基因，本项目拟进一步对两种滞育形成过程的转录组进行挖掘，深度比较专性滞育、兼性滞育过程的基因表达数据，获得两种滞育类型调控差异关键基因；对关键基因进行遗传进化、分子特性、组织表达、蛋白定位、RNA干扰以及多肽活性的等方面的分析，验证其所在代谢通路在各滞育类型发育中的功能。最终将阐明桃小不同滞育类型差异调控的分子基础，为诠释不同滞育类型发育机制提供科学依据。

滞育是桃小重要的生物学特性，严格受光周期调控。首先探究了桃小滞育激素在滞育调控中的作用。在桃小生活史的每一个虫态，滞育激素受体基因（diapause hormone receptor，CsDHR）都有表达，CsDHR表达量变化与CsDH表达基本同步，进入滞育初期表达量有明显上升，在滞育过程也维持一定的表达量； CsDHR主要在滞育幼虫的脂肪体和消化道中大量表达。CsDH在桃小中的作用可能与基础生理代谢调节相关，当代谢旺盛需要大量合成新组织的时期该基因表达量高，该代谢贯穿桃小整个生命周期，表达与否并不直接决定滞育的形成。.分析了桃小整个发育阶段蜕皮激素（20E）和保幼激素（JH）的表达动态，发现了这两个激素在桃小滞育和非滞育发育调控关键期的差异。桃小滞育和非滞育发育幼虫期的20E滴度和JH滴度均是逐渐降低的过程，没有差异。但在达到老熟脱果时，滞育发育的幼虫20E滴度要显著低于非滞育发育的幼虫，说明桃小的滞育发育要求20E处于较低滴度，桃小的滞育诱导是通过降低20E的合成来抑制发育，从而进入滞育状态。桃小进入滞育后，JH滴度要低于发育期，并且在滞育解除阶段JH滴度快速升高，滞育结束后JH的合成上升对于启动发育非常重要。.对注定滞育的桃小脱果幼虫施加甲氧虫酰肼和20E，两者均能够干扰桃小的滞育发育，且干扰效果类似，但相同浓度的20E比甲氧虫酰肼的干扰作用更强。不能结滞育圆茧的比例随着甲氧虫酰肼点滴量的增加而增多，表现结非滞育长茧、畸型茧、反复结茧、分泌红色体液、失去结茧能力、虫体快速缩小以及死亡等症状，激素滴度分析表明不能结滞育圆茧虫体20E显著升高。JH类似物（烯虫酯）对长光照脱果幼虫的作用表现为不直接致死，而是影响后续蛹的羽化；短光照脱果幼虫由于接触烯虫酯，能够成功解除滞育的数量大幅度减少。激素类似物干扰桃小的滞育及其发育过程，为桃小防治新技术的开发奠定了基础。