Glutathione S-Transferase 調控德國蜚蠊對百滅寧抗性之日週律動表現

Abstract

在過去研究中，我們已知動物對於外來有害的物質之感性程度會表現出日週的節律現象，但在昆蟲的研究上，對於分子層次調控此節律現象的報告相當稀少，在此篇研究中，我們針對德國蜚蠊 (Blattella germanica) 對於百滅寧 (permethrin)之抗性節律以及 delta 類群的 glutathione S-transferase (BgGSTD1) 之分子調控進行探討。將雄性的德國蜚蠊，在不同時間點暴露於相同濃度劑量的百滅寧，結果顯示在光暗週期的環境下，德國蜚蠊在暗期對於百滅寧有較高的抗性，同樣的在全暗的環境下，德國蜚蠊在相時夜晚 (subjective night) 也表現出較高的抗性，證實了德國蜚蠊對於百滅寧的抗性是受到約日時鐘 (circadian clock) 調控。在分子調控的探討中，德國蜚蠊脂肪體中的 BgGSTD1 基因在亮期的初期，呈現表現量的高峰，且在光暗週期或是全暗的環境下，BgGSTD1 表現量皆呈現一日的節律現象，意味著 BgGSTD1 所調控的解毒反應呈現著日週律動，然而在 cytosolic GSTs (cGSTs) 酵素活性的實驗結果中，並沒有辦法觀察到與 BgGSTD1 基因表現相同的節律現象。接著利用核醣核酸干擾技術，對於 BgGSTD1 參與百滅寧的日週節律抗性上的功能性探討。注射 BgGSTD1 雙股的核糖核酸三天後，在 BgGSTD1 的基因表現以及 cGSTs 的酵素活性上皆顯著的降低，且在核糖核酸干擾造成 BgGSTD1 靜默效應下，德國蜚蠊在暗期對於百滅寧的抗性明顯降低，但是亮期沒有顯著的差異。從以上的結果中，我們證實 BgGSTD1 在德國蜚蠊對於百滅寧的日週律動抗性上，扮演著參與調控的角色。

Even though numerous studies have confirmed the daily variation of susceptibility to xenobiotic compounds in animals, there are a few reports studying molecular mechanisms underlying the daily rhythm of insecticide resistance in insects. In the present study, we investigated the daily susceptibility rhythm to permethrin and the expression level of the delta class glutathione S-transferase (BgGSTD1) gene in Blattella germanica. Male cockroaches were exposed to the same concentration of permethrin at different time points in a light-dark cycle, and results showed that the highest resistance occurred at night. Furthermore, the circadian rhythm of permethrin resistance can be demonstrated by the highest resistance at subjective night under constant darkness. The mRNA level of the BgGSTD1 gene in the fat body of B. germanica peaked at the early day time under light-dark and constant dark conditions, whereas the enzyme activity of cytosolic GSTs wasn’t reflected the rhythmic pattern as well as BgGSTD1 expression. RNA interference (RNAi) was employed to study the function of BgGSTD1 in the circadian rhythm of permethrin resistance in B. germanica. Both BgGSTD1 mRNA level and cytosolic GSTs activity were significantly decreased by dsGSTD1 injection. In addition, the survival rate of B. germanica with silenced BgGSTD1 was significantly decreased at night but not in the day time when the cockroaches were exposed to permethrin. These results have demonstrated that BgGSTD1 plays an important role in circadian regulation of permethrin resistance in B. germanica.