马铃薯甲虫结节性硬化复合物基因LdTSC1和LdTSC2基因的克隆及功能分析

【目的】通过RNAi技术明确马铃薯甲虫TOR上游的关键信号集成节点及类胰岛素信号通道下游基因结节性硬化复合物TSC1和TSC2的功能。旨在为探明马铃薯甲虫类胰岛素信号转导提供更多理论支持。【方法】在NCBI(美国国家生物技术信息中心)获取马铃薯甲虫LdTSC1/2序列,分别利用多重序列比对和系统发育分析确定该基因的完整性和系统发育关系;采用喂食幼虫dsRNA的方法,观察该基因的调低对马铃薯甲虫幼虫生长发育、糖脂代谢的影响。【结果】克隆得到马铃薯甲虫TSC1编码蛋白的氨基酸序列与鞘翅目白蜡窄吉丁直系同源蛋白的氨基酸序列的自展一致度为100%,聚为一支;TSC2编码蛋白的氨基酸序列与鞘翅目白蜡窄吉丁和赤拟谷盗的同源蛋白氨基酸序列的自展一致度为100%,聚为一支。通过分别喂食2龄幼虫LdTSC1/2的dsRNA能有效降低靶标基因的表达量,幼虫出现体重减轻,化蛹率和羽化率显著下降,葡萄糖的吸收转化效率降低,海藻糖含量升高和甘油三酯均减少。【结论】下调2龄幼虫LdTSC1/2的表达量,导致试虫出现抑制了糖脂代谢、脂肪体减少、体重减轻以及发育延迟;结果表明LdTSC1/2调控了马铃薯甲虫幼虫的糖脂代谢过程,显著影响幼虫化蛹和蜕皮过程。

Cloning and functional characterization of the tuberous sclerosis complex TSC1 and TSC2 genes in the Colorado potato beetle, Leptinotarsa decemlineata

Aim] The TSC1-TSC2 complex:a key signal-integrating node upstream of TOR and downstream of the insulin signaling pathway genes in Leptinotarsa decemlineata was identified by RNAi technology. The aim is to provide more theoretical support for probing the insuline/insuline-like signal transduction pathway of L. decemlineata.Method] The LdTSC1/2 cDNA sequences were obtained from NCBI (National Center for Biotechnology Information), and the domain structure and phylogeny of these genes were determined by multiple sequence alignment and phylogenetic analysis. The phenotype, metabolism of sugars and fatty acids of dsRNA-feeding larvae was observed, detected and analysed.Result] The L. decemlineata TSC1/2 was cloned, the LdTSC1/2 showed orthology with the Agrilus planipennis TSC1/2 and Tribolium castaneum, respectively, and the bootstrap values in plylogenetic tree were all 100. By feeding the dsRNA of the 2nd instar larva, the expression level of the target genes was effectively reduced, larvaes mass loss, pupation and emergence rate are significantly reduced, glucose absorption and conversion efficiency is reduced, and triglycerides are decreased, but trehalose content is increased.Conclusion] By feeding the dsRNA of the 2nd instar larva, the expression level of the target genes was effectively reduced, and the performance of tested insects showed inhibited in carbohydrate and lipid metabolism resulting in fat body and mass loss, and development delay. TSC1/2 regulates the carbohydrate and lipd acid metabolism in L. decemlineata larvae, with the result that leading to development delay.