德國蜚蠊時鐘基因靜默對時鐘基因之轉錄影響

Abstract

自從第一個時鐘基因 period (per) 在黃果蠅 (Drosophila melanogaster) 中被發現，我們知道日週律動是由基因所調控，且時鐘基因轉錄和轉譯的負回饋迴圈被視為核心時鐘的分子機制。藉研究四個德國蜚蠊 (Blattella germanica) 時鐘相關基因 per, timeless (tim)，cryptochrome1 (cry1) 和 cryptochrome2 (cry2) 之間的交互關係，進而解開德國蜚蠊生物時鐘的運作。運用 RNA interference (RNAi) 靜默時鐘基因，能簡單地探討並比較時鐘基因轉錄表現上的變化，來決定基因間的關係。除此之外，我也發現這些時鐘基因也會在觸角中表現，所以在拿頭部研究分子時鐘機制的同時，也可以拿觸角的結果來比較中央和週邊時鐘系統的差異。實驗發現注射 tim 或 cry2 的雙股核醣核酸後，cry2，per 和 tim 的相對基因轉錄表現量會被影響。有趣的是，注射cry1 的雙股核醣核酸後，cry2，per 和 tim 不管在頭部或觸角中的相對表現量，和對照組相比都呈現完全相反的表現趨勢。為了探討這四個時鐘相關基因在蛋白質層級的交互作用，我選用酵母菌雙雜合技術 yeast two-hybrid (Y2H) 來證實。雖然 cry1 可以塞入 Y2H 的質體中，其他三個時鐘基因卻因長度太長而無法成功塞入。即使沒有蛋白質交互作用的結果，從 RNAi 的實驗結果能推斷德國蜚蠊與帝王蝶 (Danaus plexippus) 的時鐘分子模式應該是不同的。在帝王蝶模式中，PER和 CRY2 會形成異二聚體並回到細胞核充當 CLK 和CYC 異二聚體的抑制者。然而在德國蜚蠊模式中，TIM 和CRY2才是轉錄抑制者它們會來調控 cry2，per 和 tim 的基因表現。此外，CRY1 的功能則是校正生物時鐘的相位，與帝王蝶模式是一樣的。

Since the first clock gene, period (per), was found in Drosophila melanogaster, the circadian rhythm is known to be regulated by genes. A negative transcription-translation feedback loop is considered to be the core of clockwork. I have obtained 4 genes per, timeless (tim), cryptochrome1 (cry1), and cryptochrome2 (cry2) which are related to clockwork in Blattella germanica and investigated their interactions to unveil the core clockwork. By using RNA interference (RNAi), we could explore and compare the variation of clock genes' expressions to determine their relationships. In addition, I discovered that clock genes also expressed in antennae. By comparing the clock genes' expression after RNAi between head and antennae, I intended to find the difference in clockwork between central and peripheral clock. I found that after injecting dsTim and dsCry2, the relative amount of cry2, per and tim were influenced. Interestingly, for dsCry1 treatment, the relative mRNA expression level of per, tim and cry2 showed totally reverse pattern compared with control group in the head and antennae. To verify the interactions among the 4 genes at protein level, yeast two-hybrid (Y2H) technique was used. Although cry1 could be inserted into yeast plasmid, the other 3 genes were too large to be inserted successfully. Even without protein interaction results, I unveiled the molecular clockwork of the German cockroach may be different to the monarch butterfly model which PER and CRY2 formed heterodimer, then entering nucleus to act as a CLK/CYC repressor. Instead, TIM and CRY2 probably function as transcriptional repressors that regulate the expression of per, tim and cry2. For CRY1's function, it served as a role to reset the clock phase as the CRY1 in the monarch butterfly model.