- 學位論文
東方果實蠅與有機磷抗藥性相關之羧酸酯酶研究
A study of carboxylesterases associated with organophosphate resistance in Bactrocera dorsalis
摘要
羧酸酯酶 (carboxylesterase, CBE) 具有水解羧酸酯及隔離殺蟲劑等外來物質之生理功能，為昆蟲外來物防禦系統的重要組成，並於東方果實蠅 (Bactrocera dorsalis) 與其他昆蟲中參與對有機磷類 (organophosphate) 殺蟲劑之抗藥性機制。先前研究指出東方果實蠅乃力松抗性品系與其他有機磷抗性以及感性品系相比，在酯酶活性圖譜上有一特定高強度之羧酸酯酶條帶 BdE5，且可被氧化巴拉松與乃力松等有機磷劑抑制，其在族群中出現頻度亦證實與乃力松抗藥性有關。在本研究中，體內抑制實驗發現診斷劑量之乃力松對於感性品系酯酶之總體抑制程度影響較抗性品系大，透過 NanoLC-nanoESi-MS/MS 針對 BdE5 進行蛋白質鑑定並將其註解為 esterase FE4-like (XP_011200445.1)，利用快速擴增 cDNA 末端法 (rapid amplification of cDNA ends) 取得其 2,012 bp 之 cDNA 全長序列，並包含長度為1,770 bp 且可轉譯 590 個胺基酸殘基之開放閱讀框架 (open reading frame)。親緣分析亦指出其屬於分泌性 β 酯酶 (secreted β esterases)(E 支群) 並與黑腹果蠅 (Drosophila melanogaster) 之羧酸酯酶 CG6414 (NP_570089) 具有親緣關係。另一方面，由於昆蟲羧酸酯酶基因屬於羧酸/膽鹼酯酶 (carboxyl/cholinesterase, CCE) 多基因家族，許多與有機磷抗性相關之羧酸酯酶仍尚未被發現與個別探討。為發掘更多有機磷抗藥性相關之羧酸酯酶，本研究透過 RNA 測序 (RNA sequencing, RNA-seq) 取得東方果實蠅轉錄體資訊進行分析，以 5 個有機磷抗性品系對感性品系間轉錄本 (transcript) 之 RPKM (reads per kilobase per million mapped reads) 比值篩選，由 33 條酯酶基因中選取 6 條比值大於 2 之候選基因。以即時定量聚合酶連鎖反應 (RT-qPCR) 測定候選基因於乃力松、三氯松及馬拉松抗性品系比對感性品系計算所得之基因相對表現量，結果顯示其中 3 條在 3 個抗性品系中表現量皆顯著高於感性品系 (或高於 2 倍)，可能與有機磷抗藥性相關。BdE5 基因亦為其中之一，與先前其在酯酶圖譜及活性實驗之發現相符。而相對表現量最高之基因被註解為 carboxylesterase B2 (AGU42836.1)(為 alpha-Esterase-7 (αE7) 之直系同源基因)，最終亦成功於秋行軍蟲 (Spodoptera frugiperda) Sf21 細胞中，以昆蟲桿狀病毒 (baculovirus) 表現系統表現出具有活性之 αE7 重組蛋白。本研究提供對東方果實蠅有機磷抗藥性相關之羧酸酯酶更深入之認識，並生產東方果實蠅 αE7 重組蛋白供後續抗藥性機制研究或相關技術開發之應用。
並列摘要
Physiological functions of carboxylesterases (CBEs) include hydrolysis of carboxylic esters and sequestration of xenobiotics such as insecticides, making CBEs the most important component of the xenobiotic defense system in insects. CBEs are also involved in the mechanism of organophosphorus insecticide resistance in many insects such as Bactrocera dorsalis. Previous studies have compared organophosphate (OP)-resistant and -susceptible lines of Bactrocera dorsalis and discovered a CBE BdE5 in the naled-resistant line with remarkable quantitative elevation in isoenzyme patterns of esterase activity, which was also inhibited by OPs such as paraoxon and naled. Correlation was found between naled resistance and flies with a higher percentage of intensive BdE5 bands. The current study used in vivo inhibition assays to show that, under diagnostic doses of naled, overall extent of inhibition on CBEs was much greater in the susceptible line than in the naled-resistant line. Through NanoLC-nanoESi-MS/MS analysis, BdE5 was identified and annotated as an esterase FE4-like (XP_011200445.1). Rapid amplification of cDNA ends was used to obtain the full-length 2,012-bp BdE5 cDNA, which contained an open reading frame of 1,770 bp and encoded a putative protein of 590 amino acid residues. Phylogenetic analysis revealed that BdE5 is a secreted β esterases (E clade) closely related to CG6414 (NP_570089), a CBE in Drosophila melanogaster. As insect CBE genes belong to a large multigene carboxyl/cholinesterases (CCE) family, many CBEs related to OP resistance have not yet been found and their specific roles have yet to be studied. To discover more OP resistance-related CBEs, in this study, we used RNA-seq analysis to obtain transcript RPKM ratios of five OP-resistant lines to one susceptible line, screening out CCE genes. Six genes had ratios of more than two and were chosen as candidate genes from a total of 33 genes. RT-qPCR was used to check the relative expression levels of candidate genes in the naled-, trichlorfon-, malathion-resistant lines compared to the susceptible line, and three of these candidate genes were thought to be related to OP resistance due to their significantly higher (or more than two-fold) expression levels in the three resistant lines. BdE5 was one of these genes and its transcript level result was consistent with previous findings of esterase patterns and activity assays. The gene with the highest relative expression level was annotated as carboxylesterase B2 (AGU42836.1)(orthologous gene of alpha-Esterase-7, αE7), and was successfully used to express recombinant αE7 with CBE catalytic activity in Spodoptera frugiperda Sf21 cells using the baculovirus expression system. The results in this study provide further understanding on how CBEs relate to OP resistance, and the B. dorsalis recombinant αE7 provides a basis for future studies of resistance mechanisms or development of related technical applications.