揭示在埃及斑蚊的生物節律和光敏感性中Rh7所扮演的功能

Abstract

大多數生物體在其體內擁有特定的分子，能夠自主調節生理機能與行為。這種現象被稱為生物節律(Biological Rhythms)，而這些內生性的生物鐘會受到來自外界的刺激而調控其震盪週期以適應周遭的環境，這些刺激包括溫度、進食、光線等等，其中又以光線造成的影響最為顯著。在先前的研究中已發現果蠅具有兩個影響其日週期節律(Circadian Rhythms)的光感受蛋白，分別是隱花色素(Cryptochrome, CRY）和視紫質7(Rhodopsin 7, Rh7），它們各自作為光感測器並對果蠅的生物節律具有重要影響。在埃及斑蚊中，已經找出兩個CRY基因，分別是CRY1和CRY2，且埃及斑蚊擁有一種和果蠅Rh7結構相似的視蛋白稱為GPROP10，儘管其在果蠅體內被報導和日週期節律有相關，但它在蚊子體內的功能仍然是未知的，本篇論文的主要目的在於研究Rh7是否在埃及斑蚊身上也具有調節日週期節律的功能。在本研究中我使用CRISPR/Cas9技術產生的CRY1（AAEL004146）和GPROP10（AAEL005322）突變蚊進行研究，在分子層次我使用西方墨點法發現Rh7突變體的兩個日週期中心蛋白TIM(timeless)和PER(period)會有上升的趨勢，並且產生了長度較短的PER蛋白異構體，且這種較短的PER專一的表現在Rh7突變體的複眼中，這些結果顯示Rh7可能在PER/TIM兩種蛋白的轉錄機制上有一定的功能。在行為分析上，我利用果蠅活動力測試儀器(locomotor activity monitor, LAM)實驗記錄埃及斑蚊一天的活動力並進行分析，發現Rh7突變體在早晨的活動力(Morning Peak)會顯著下降，另外我也在夜晚期間使用了不同波長的LED光源對埃及斑蚊進行光照刺激，並分析其活動軌跡，發現Rh7突變體在藍光的刺激下活動力顯著低下，這些結果指出Rh7在埃及斑蚊身上的確具有調控日週期節律的功能並且可能是一種接收藍光波段的蛋白。

Most organisms possess specific molecules within their bodies that autonomously regulate physiological functions and behaviors. This phenomenon is referred to as Biological Rhythms, and these endogenous biological clocks are regulated by stimuli from the external environment to adapt their oscillation cycles, including factors such as temperature, feeding, and light, with light having the most significant impact. Previous studies have identified two light-sensitive proteins in fruit flies that influence their circadian rhythms, namely Cryptochrome (CRY) and Rhodopsin 7 (Rh7), each acting as photoreceptors and exerting significant effects on fruit fly biological rhythms. In Aedes aegypti, two CRY genes, CRY1 and CRY2, have been identified, along with a visual protein similar in structure to fruit fly Rh7, called GPROP10. While its relevance to circadian rhythms has been reported in fruit flies, its function within mosquitoes remains unknown. The primary aim of this study is to investigate whether Rh7 also plays a role in regulating circadian rhythms in Aedes aegypti mosquitoes. In this study, I use CRISPR/Cas9 generated mutant mosquitoes CRY1 (AAEL004146) and GPROP10 (AAEL005322) for research purposes. At the molecular level, using Western blot analysis, I found an upward trend in two circadian clock proteins, TIM (timeless) and PER (period), in Rh7 mutant variants, along with the production of shorter PER protein isoforms. This shorter PER isoform exhibited specific expression in the mutant's compound eyes, suggesting a potential role for Rh7 in the transcriptional mechanisms of PER/TIM proteins. In behavioral analysis, I recorded the daily activity of Aedes aegypti using a locomotor activity monitor (LAM) and found a significant decrease in morning peak activity in Rh7 mutants. Additionally, I stimulated mosquitoes with different wavelengths of LED light sources during the night and analyzed their activity patterns, and found significantly reduced activity in Rh7 mutants under blue light stimulation. These results indicate that Rh7 indeed regulates circadian rhythms in Aedes aegypti mosquitoes and may serve as a protein sensitive to blue light wavelengths.