舞毒蛾Methuselah like受体生理功能及其G蛋白偶联信号转导机制研究

Mthuselah (Mth), a G protein-coupled receptor, was originally identified in Drosophila melanogaster. Mthuselah is associated with lifespan, stress resistance and reproduction in Drosophila melanogaster. In nondrosophiline insects Mth is not present, and a number of Mthuselah-like genes (Mthl) have been identified. However, it is little known about physiological functions of these Mthl genes. Therefore, we had cloned two Mthl genes of gypsy moth. Previous studies showed effects of Mthls on growth and development of gypsy moth and regulation of down-stream P450 gene expression. These results indicated Mthl genes may be associated with regulation of physiological functions in gypsy moth. In present study, we will conduct the following projects to further explore functions of Mthl genes in gypsy moth including (1) identifying Mthl agonist genes; (2) study the effects of Mthl and its agonists on gypsy moth lifespan, reproduction and stress resistance (such as high temperature, starvation, oxidative stress); (3) characterization of G protein-coupled signal transduction of Mthl receptors in gypsy moth. These results will demonstrate physiological functions and signal transduction of Mthl genes in gypsy moth. And it will provide new pathway for understanding organism’s lifespan and formulate new pesticide by “reverse pharmacology” method based on finding selective agonist and antagonist of Mthl using high-throughout screening.

果蝇Methuselah（Mth）受体属于G蛋白偶联受体（GPCRs）家族成员，参与调节寿命、应激反应和生殖能力。在非果蝇昆虫中鉴定存在Methuselah-like（Mthl）受体基因，但对这些Mthl基因功能了解甚少。为此，我们克隆获得了2条舞毒蛾Mthl受体全长基因。前期研究发现其影响舞毒蛾生长发育，能调控下游P450基因表达，表明Mthl可能参与舞毒蛾生理功能调控。本项目拟从以下几个方面进一步探讨舞毒蛾Mthl受体基因功能：（1）鉴定Mthl受体激动剂基因；（2）Mthl受体及其激动剂对舞毒蛾寿命、生殖和抗逆性（高温、饥饿、氧化胁迫）影响；（3）Mthl受体G蛋白偶联信号转导通路分析。本项目将阐明舞毒蛾Mthl受体生理功能和信号转导通路，为揭示生物衰老机制提供新的途径，也为将来通过高通量筛选寻找选择性的Mthl受体激动剂和拮抗剂，并通过“反向药理学”的方法创制新的杀虫剂奠定基础。

Methuselah-like （Mthl）属于G蛋白偶联受体（GPCRs）家族成员，参与调节寿命、应激反应和生殖能力。我们首次以森林重大害虫舞毒蛾为对象，研究了Mthl受体及激动剂受体对舞毒蛾寿命、生殖能力和抗逆性的影响以及信号转导通路。LdMthl1基因沉默舞毒蛾幼虫相对取食量（RCR）和近似消化率（AD）均低于对照组dsGFP，而食物利用率（ECI）和食物转化率（ECD）均高于对照组dsGFP，而LdSPR基因沉默体则反之，推测LdMthl1和LdSPR基因影响营养利用。表达LdMthl1和LdSPR基因果蝇均延长了寿命；舞毒蛾LdMthl1和LdSPR基因沉默体饥饿和高温胁迫下存活率低于dsGFP。与对照dsGFP相比，LdMthl1和LdSPR沉默舞毒蛾幼虫在百草枯胁迫下存活率均低于对照。表达LdMthl1和LdSPR基因果蝇具有增加对耐高温（36℃）胁迫的应激能力，具有抗饥饿的能力。百草枯氧化胁迫下，转LdSPR基因果蝇品系死亡率低于果蝇对照品系，分别对照的54.21%和65.22%，而LdMthl1可能未参与抗氧化能力。.RNA-seq分析LdMthl1沉默体和dsGFP对照转录组文库分析发现71个差异基因，其中6个差异基因分别注释到“Phosphatidylinositol signaling system”、“Calcium signaling pathway”、“Glucagon signaling pathway”和“Ribosome biogenesisin eukaryotes”，这可能受LdMthl1基因沉默的影响，将是研究的重点。我们将LdMthl1稳定表达于HEK293T细胞，合成Dm-SP成熟肽检测细胞内钙流，结果表明瞬时加入果蝇SP成熟肽（序列为KPTKFPIPSPNPRDKWCRLNLGPAWGGRC）后，HEK293-LdMthl细胞可以被SP激活，并呈现出浓度依赖性Ca2+流，而瞬时加入美国白蛾TK成熟肽（序列为SQLGFFGMRG-NH2）和DMSO后，则没有明显Ca2+流信号产生。Mthl-HEK293细胞系中SP抑制了forskolin引发cAMP的积累，说明Mthl偶联Gai抑制了cAMP的积累。研究结果进一步丰富了Mthl在非模式昆虫中的功能，为将来以Mthl受体作为分子靶标创制新型杀虫剂提供了重要理论。