探討東方果實蠅序列突變與其抗藥性的相關性

Abstract

東方果實蠅（Bactrocra dorsalis）是危害世界各地經濟作物的主要害蟲之一，其抗藥性是一項亟待解決的問題。目前被發表出來、和抗藥性相關的基因如AChE、GSTs、COEs 和P450s 都和基因突變有關。為了調查基因突變和抗藥性的相關性，本研究設計了一項方法，企圖找出與抗藥性相關的點突變，並對這些點突變做定性分析。 本研究所用到的樣本包含一個感性品系、三個抗性品系（分別抗福木松、芬殺 松和納乃得）和三個復性品系（福木松、芬殺松和納乃得）。樣本經過定序以後，再透過VelVet 和Oases 軟體做序列組裝。利用blastx 將組裝好的isotigs 和黃果蠅（Drosophila melanogaster）蛋白質序列做序列比對，找出每個isotig 所比對到的黃果蠅蛋白質序列，作為其同源基因（homologous gene）。接著對於每個殺蟲劑實驗組，透過多重序列比對（multiple sequence alignment）比較同源基因中的感性、抗性和復性isotigs。結果顯示，總共有26 個感性isotigs，在三種殺蟲劑實驗組中都可以找到發生在同一位置的點突變，這些點突變很有可能和抗藥性有關。針對這26 個感性isotigs 做四種特性分析：突變頻率、突變位置是否在蛋白質表面、基因表現量和跨物種保留性，以探討突變和抗藥性的相關性。突變頻率分析顯示了這些突變確實存在，例如抗性isotig 具有較高的抗性品系read 回貼數、但是感性isotig 和復性isotig 則具有較低的抗性品系read 回貼數。在蛋白質表面發生的突變很可能和殺蟲劑分子結合有關。如果具有點突變的isotigs 在基因表現上產生調控（正調控或負調控），它們很可能和殺蟲劑代謝有關。此外，本研究也利用了黃果蠅的AChE、GSTs、COEs 和P450s 相關基因來驗證點突變，結果顯示東方果實蠅的基因在這幾個分類，皆有為數不少的基因具有點突變。最後，透過資料庫的建立，提供友善的使用介面方便存取本篇研究的結果，將有助於後續應用。

Bactrocera dorsalis is one of the serious agricultural pests that result in the economic injury worldwide. The insecticide resistance of B. dorsalis is an important issue to be addressed. Several known insecticide related genes, such as AChE, GSTs, COEs, and P450s, were reported to be involved in gene mutations. To investigate the sequence variations related to insecticide resistance, an approach is proposed in this thesis for mutation discovery and characterization. The samples for this study include a susceptible line, three resistant lines of formothion, fenthion and methomyl, and three recovered lines of formothion, fenthion and methomyl, respectively. The samples were sequenced and assembled into isotigs by using Velvet followed by Oases. Blastx was performed against proteins of Drosophila melanogaster to search for homologou genes for each isotig. For each insecticide group, isotigs of the susceptible line, the resistant line and the recovered line with the same homologou gene were compared by invoking multiple sequence alignment. In the results, there were 26 susceptible isotigs of which the mutations were commonly observed in three insecticide groups and were highly considered related to insecticide resistance. Four characterizations were applied to these isotigs: mutation frequency, protein surface, gene expression and evolutionary conservation, to investigate how these mutations might be related to insecticide resistance. Mutation frequency analysis supports the validity of most of the mutations, i.e. high read counts in the resistant line but low in both of the susceptible and the recovered line. Some mutations were predicted to be on the protein surface and might be involved in insecticide binding. In addition, AChE, GSTs, COEs and P450s related genes of D. melanogaster were examined to see if they contain point mutations discovered in this study, and the results showed that there are several B. dorsalis genes in each category found to have point mutations. In the end, a database was established with a friendly user interface for conveniently accessing data generated in this study for future applications.