- 學位論文
利用RNA干擾探討東方果實蠅 (Bactrocera dorsalis) 榖胱甘肽硫轉移酶在有機磷類藥劑抗性的角色
Using RNA interference to study the role of glutathione S-transferase genes in organophosphate-resistant Bactrocera dorsalis
摘要
東方果實蠅 Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) 為重要果樹害蟲。殺蟲劑如有機磷類常用來防治東方果實蠅，而在實驗室中已經得知果實蠅科會對有機磷類藥劑產生抗性，目前已確認在田間也有果實蠅科的抗性族群。在雙翅目蚊科 (Culicidae) 的研究中，指出有一群稱作榖胱甘肽硫轉移酶 (glutathione S-transferase, GST) 的蛋白質表現與對有機磷抗藥性具高度相關性。GST 的解毒機制為降低外來物質的毒性並使之易於排出生物體外，因此尋找東方果實蠅的GST 基因並進一步確認其功能對於未來的防治會相當有幫助。為找尋與有機磷類藥劑抗性相關的東方果實蠅 GST 基因，本論文使用以次世代定序 (next generation sequencing, NGS) 技術已經建立之 5 種東方果實蠅有機磷抗性品系和 2 個感性品系的轉錄體 (transcriptome) 資料庫，篩選出 22 個 GST 基因共 108 條轉錄子 (transcripts)，使用定量即時聚合酶鏈鎖反應 (qPCR) 挑選出在特定抗性品系與 NGS 資料中RPKM (Reads per kilobase of transcript per million) 比值相關的基因，發現在芬殺松抗性品系中，BdGSTD2、BdGSTD5 和 BdGSTE9 這 3 個基因的表現量與 NGS 資料中 RPKM 的比值相近。接下來同樣利用 qrt-PCR 檢視這 3 個 GST 基因在芬殺松抗性品系不同性別的頭、胸、腹部及消化道，和第二、三齡幼蟲及蛹期的表現量，發現雖然基因在不同部位及齡期的表現量不盡相同，但都大幅高於感性品系。確認此 3 個 GST 基因會在芬殺松抗性品系的表現量之後，使用微針注射法進行 RNA 干擾，將上述 3 種 GST 基因及當對照用的增強性綠色螢光蛋白 (Enhanced Green Fluorescent Protein, EGFP) 的雙股 RNA (dsRNA) 注射羽化當日的芬殺松抗性品系成蟲中，蒐集注射之後不同日齡的成蟲以 qrt-PCR 確認注射後基因靜默的效果以及隨時間的影響，發現 BdGSTE9 在注射後第 1 日表現下降，但隨後回升；而 BdGSTD2 及 BdGSTD5則分別在第 1 日及第 3 日下降。最後使用局部滴定法 (topical application) 施用芬殺松，選擇診斷劑量分別測試 3 個 GST 基因分別下降時的日齡，並以 EGFP 作為對照組。結果 BdGSTE9 的死亡率與對照組無差異；而 BdGSTD2 和 BdGSTD5 的死亡率大於對照組，證實了後兩者與有機磷藥劑抗性具相關性。本試驗所得到的 BdGSTE9 轉錄子短於 NCBI 上的全長，可能是造成 RNAi 和生物檢定實驗與生物體內表現量實驗不符的原因，未來會藉快速擴增 cDNA 末端技術增幅轉錄子並重新設計 RNAi 試驗。而 BdGSTD2 和 BdGSTD5 未來可以在芬殺松抗性品系上測試其他有機磷劑和不同作用機制的交互抗性，也可表現出這兩個基因的蛋白質以動力酵素學測試對於有機磷劑的分解速率。
並列摘要
The oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), is a notorious pest of fruit plants. The application of chemical agents such as organophosphates (OPs) is the most common method used to control B. dorsalis. However, in Tephritids, resistance to such chemical agents was first discovered in the laboratory, and to date it has also been confirmed that there is resistance to OPs in field populations. Research indicates that the enzymes known as glutathione S-transferases (GSTs) play an important role in OP resistance of B. dorsalis as well as in a number of other Dipteran species. In GSTs, the mechanism of insecticide resistance involves the metabolism of allellochemicals that make insecticides less toxic and easier to excrete from the body. Hence, for the future of pest control, it would be helpful to identify and further characterize these GST genes in B. dorsalis. In order to find GST genes related to OP resistance, we utilized a transcriptome dataset from five different resistance strains and two susceptible strains of B. dorsalis using the Next Generation Sequencing (NGS) technique. From this dataset, screening was used to identify 22 GST genes from 108 transcripts. We next used qrt-PCR to confirm the expression level of candidate genes identified and confirmed that expression levels of BdGSTD2, BdGSTD5 and BdGSTE9 in fenthion-resistance strain matched RPKM (Reads per kilobase of transcript per million) ratios from the NGS dataset. Of the three genes that met our expectations in terms of expression levels, we tested expression levels of heads, thoraces, abdomens, and digestive tracts from flies of both genders and second, third stage fly larvae and pupae. Although expression levels of these three genes differed, they were all higher than the susceptible samples. After confirming that these three genes could be expressed in fenthion-resistant flies, we used micro-injections to perform RNAi assays by injecting dsRNA of these three genes and an enhanced green fluorescent protein (EGFP) gene used as reference into fenthion-resistance strains, and used qrt-PCR to test expression levels on different days after injections. We found that in BdGSTE9, expressions declined after 1 day but then increased; in BdGSTD2 and BdGSTD5, expressions declined after 1 day and 3 days, respectively. Finally, we used topical application of diagnostic dosages on flies that had been given injections and showed declining expressions levels. EGFP was used as a reference group. In BdGSTE9, mortality was not significantly different from the reference group, while in BdGSTD2 and BdGSTD5, mortalities were greater than the reference group, which suggests that the latter two genes are associated with OP resistance. The transcript of BdGSTE9 we used was shorter than the sequence from the NCBI database, which may be the reason that the results of RNAi, topical application, and expression levels of BdGSTE9 did not match. In the future we will expand the sequence of this BdGSTE9 transcript by RACE (rapid amplification of cDNA ends), and redesign our RNAi experiments. In terms of BdGSTD2 and BdGSTD5, we plan to test their contribution to cross-resistance of other organophosphates and pesticides using different mode of actions, and we also hope to express proteins of these two genes in order to test their rates of metabolite organophosphates by enzyme kinetics.
參考文獻
延伸閱讀
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