海藻糖TPS/TPP合成途径调控褐飞虱蜕皮及几丁质代谢的分子机制

Trehalose content changed in insect blood would affect insect’s development, ecdysis, metamorphosis and other physiological activities. So the study of trehalose synthesis pathway has important theory significance and application value in the role of regulation on trehalose supply ability and regulation on chitin degradation during the insect molting process. The project intends to important pests of Nilaparvate lugens as the research object, and first to identifying trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) genes’ function in trehalose synthesis. Second, this project based on previous study use RNAi technology combined with DGE (digital gene expression profiling) and iTRAQ (isobaric tags for relative and absolute quantitation) quantitative analysis to choose differentially expressed genes, including transcription factors and other functional genes, by TPS and TPP regulating in chitin metabolism pathway of N. lugens. Then RNAi technology is used and excess glucose or trehalose injection to restrain or enhance the expression of TPS and TPP genes. In these experiments, differential genes’ expression on mRNA and protein level of chitin synthase and chitinase should be detected, and the enzyme activity of trehalase, trehalose-6-phosphate synthase, chitinase and chitin synthase and the content of chitin, trehalose and glucose also should be detected. All of these record including malformation and mortality of N. lugens and identify structural change of chitin will be recorded. And it is very important to value the safety of low expression of TPS and TPP genes to non-target insects, explain regulation of TPS and TPP genes to chitin metabolism. These results will further improve the molecular mechanism of insect growth regulation to energy metabolism and chitin metabolism, which all would establish the theoretical foundation about controlling pest by regulating trehalose’ supplying.

海藻糖供给直接关系到昆虫发育、蜕皮和变态等生理活动。因此，研究海藻糖合成途径对昆虫海藻糖供给的调节能力及蜕皮和几丁质代谢的调控功能具有重要意义。本项目拟以重要害虫褐飞虱作为研究对象，首先鉴定TPS/TPP在海藻糖合成中的具体功能；其次，采用RNAi结合数字表达谱和蛋白定量分析，筛选鉴定TPS/TPP调控几丁质代谢通路中包括转录因子在内的关键基因等差异表达情况；再次通过抑制或提高TPS/TPP的表达，检测几丁质代谢通路中差异基因、几丁质合成酶及几丁质酶等在mRNA和蛋白水平上的表达情况，测定海藻糖合成酶、几丁质代谢相关酶类的酶活变化及几丁质、海藻糖和葡萄糖等含量的变化，统计褐飞虱的畸形率、死亡率并检测几丁质结构变化。同时评价TPS/TPP低表达对其它非靶标昆虫的安全性，阐明TPS/TPP对几丁质代谢的调控作用，进一步完善昆虫发育调控的分子机制，为分析和评估调节海藻糖供给控制害虫奠定理论基础。

海藻糖供给直接关系到昆虫发育、蜕皮和变态等生理活动，其中海藻糖合成途径对昆虫海藻糖供给的调节能力及蜕皮和几丁质代谢的调控功能具有重要作用。首先，本研究以褐飞虱3个海藻糖合成酶基因（TPS）为主要研究对象。采用注射法RNAi技术抑制TPS基因的表达，直接导致昆虫的几丁质合成障碍，包括蜕皮障碍和翅膀畸形等。其次，通过制备褐飞虱3个TPS基因上调和下调样品，采用数字表达谱（DGE）及同位素标记相对和绝对定量（iTRAQ）技术进行测序和分析，研究结果发现不同的TPS基因调控糖代谢、氨基酸代谢、脂质代谢、核酸代谢、激素代谢、维生素代谢、能量代谢等通路存在差异，但是它们都能够调控下游的几丁质合成与几丁质降解通路，具体表现在不同的TPS低表达后，几丁质合成通路相关基因和几丁质酶等表达出现显著或者极显著下降。再次，通过注射过量的海藻糖、过量的葡萄糖、注射不同的dsTPSs及注射不同浓度的海藻糖酶抑制剂等，来上调和下调褐飞虱体内TPS基因的表达，检测几丁质合成通路、几丁质降解途径、海藻糖代谢和糖原代谢等途径相关基因的表达结果发现：TPS主要通过影响海藻糖含量的平衡从而调控表皮和翅基等组织几丁质代谢途径基因表达变化，导致几丁质含量降低，无法正常完成蜕皮和翅膀发育等过程，在褐飞虱和赤拟谷盗中甚至出现高达30%的死亡率，表明TPS基因具有作为害虫控制靶标的潜力。最后，本研究选取一种天敌昆虫异色瓢虫来作为非靶标昆虫安全性评价的对象，以dsGFP注射为对照组，注射dsTPS为处理组，异色瓢虫在24 h取食处理的褐飞虱数量无显著差异；但是除异色瓢虫TRE1-1外，其他都显著增加，而三种糖含量和两种海藻糖酶活性变化均无明显差异。这些结果表明TPS具备作为害虫控制的靶标基因，对非靶标昆虫来说相对安全。该项目不仅从理论上阐明了TPS作为潜在害虫靶标的控害分子机理，而且还研究评估了TPS基因在害虫控制中对非靶标昆虫的安全性。