摘要：胰島素是生物體內用來反應環境壓力的內分泌物，目前已知其可影響生物對溫度、饑餓以及氧化劑的耐受性。在線蟲、果蠅及老鼠的老化研究中更指出胰島素扮演重要的角色，而且其訊息的傳遞路徑保守性很高。有趣的是，胰島素被發現可以影響果蠅時鐘基因period 的表現，但其作用的機制與途徑仍不清楚。為了瞭解內分泌如何調控生物時鐘的表現，以及其與老化的關係，我們選擇德國蜚蠊作為實驗材料進行研究，探討環境壓力如何影響胰島素之量的變化，進而反應在日週律動的表現上。首先將選殖德國蜚蠊的胰島素受器(Insulin Receptor) 基因以得到其cDNA 序列資料，接著以此資料可以合成雙股RNA，然後將其注射至德國蜚蠊的體內，以RNA干擾(RNA interference) 的分子技術讓胰島素受器基因無法表現，最後記錄這些德國蜚蠊在日週律動表現(包括活動行為及時鐘基因period) 、壽命，以及其對環境壓力(包括溫度、饑餓與氧化劑) 的耐受性等指標的變化。此外，青春激素(Juvenile Hormone) 是昆蟲獨有的內分泌激素，它在表現量的變化會造成昆蟲在生殖、代謝以及對環境壓力的抗性的改變。由於青春激素量的變化受胰島素所調控，因此胰島素是否經由青春激素的途徑改變德國蜚蠊日週律動的表現則是本計畫將探討的另一個重點。為了確認青春激素與德國蜚蠊日週律動表現的關係，我們將針對3-Hydroxy-3-methylglutaryl CoA reductase 進行分析，其與青春激素的生合成有關且該基因在德國蜚蠊已經被選殖出來，利用RNA干擾的方法讓其無法表現，看德國蜚蠊在日週律動的表現是否改變；另一方面，本實驗室之前的研究發現德國蜚蠊雌、雄蟲在活動行為之規律性的表現不同，推測與雌蟲周期性的生殖行為有關，因此本計劃也將以RNA干擾的方法探討青春激素如何影響德國蜚蠊雌蟲的生殖行為，以釐清活動行為日週律動與雌蟲生殖行為的關係。
Abstract: Insulin is the endocrine factor which responds directly to environmental stress such as temperature, starvation and oxidative stress. Based on the researches ofnematodes, flies and mouse, insulin plays an important role on aging, and its signaling pathways are conserved. Interestingly, insulin was found to affect the expression of clock gene period in Drosophila, but the possible underlying mechanisms and pathways remains unclear. In order to understand the endocrine control of circadian clock as well as its relationships with aging, we have chosen the German cockroach (Blattella germanica) as the experimental material to conduct studies on the impact of environmental stress on the level of insulin, thereby reflected in the expression of
circadian rhythms. Firstly, insulin receptor gene will be cloned in order to know the target sites of insulin following by sequence analysis. By using RNA interference (RNAi) technique, double-strand RNA can be synthesized based on the sequence of cDNA, and then injected into the German cockroach to disrupt the expression of insulin receptor gene. Some indicators including expression of circadian rhythm (locomotion and period gene), lifespan and resistance against environmental stress (temperature, starvation and oxidative agents) will be monitored to understand the
functions of insulin. In addition, the Juvenile Hormone (JH) is a multifunctional hormone in insect, and changes in JH level will affect the condition of reproduction, metabolism, as well as the ability to resist environmental stress. Since JH fluctuation is regulated by insulin, we are going to explore the possibility of insulin altering the expression of circadian rhythm through JH signaling pathway. The relationship between JH and expression of circadian rhythm will be determined by analysis of 3-Hydroxy-3-methylglutaryl CoA reductase because of its involvement in the biosynthesis of JH. This enzyme is already cloned in the German cockroach so that the method of RNA interference can be used to analyze the effects of JH on the expression of circadian rhythm. Finally, our previous study has found that sexual differential expression of locomotor circadian rhythm in the German cockroach was related to reproductive cycle of female adults. Hence, the RNA interference ofn JH synthesis will reveal the effects of JH on reproductive behavior, and clarify its relationships with the expression of locomotor circadian rhythm.