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標題: 東方果實蠅transformer基因功能研究
Investigation of transformer function in Bactrocera dorsalis
作者: 李嘉欣
Li, Chia-Hsin
關鍵字: 東方果實蠅(Bactrocera dorsalis)
Bactrocera dorsalis
transformer (tra)
transformer-2 (tra-2)
doublesex (dsx)
yolk protein 1 (yp 1)
transformer (tra)
transformer-2 (tra-2)
doublesex (dsx)
yolk protein 1 (yp 1)
出版社: 昆蟲學系所
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摘要: 東方果實蠅是臺灣果樹最重要的害蟲之一,其危害主因是雌成蟲在果實產卵,導致果實損壞及幼蟲蛀食果肉;若能減低雌成蟲數量,可直接降低經濟損失。因此全面性了解東方果實蠅的性別決定機制將有助於新的防治策略擬定,以降低田間雌成蟲的數量。文獻中指出transformer (tra) 是果實蠅雌性別決定的開關,控制tra的表現即可影響其性別表現。因此本研究首先萃取東方果實蠅不同時期的RNA,以RT-PCR的技術偵測tra、transformer-2 (tra-2)、doublesex (dsx) 及卵黃蛋白基因 (yolk protein 1, yp 1) 等性別決定基因的表現,並探討基因與個體發展的相互關係。結果顯示卵期的traf相對表現量高,證實母體會給予胚胎traf mRNA幫助胚胎的性別決定基因進行RNA選擇性接合;幼蟲、蛹期處於性別特徵不明顯的階段,各性別相關基因的表現量皆不高。羽化之後,雌成蟲Bdtraf、Bddsxf及Bdyp 1的表現量與雄成蟲Bddsxm的表現量皆隨日齡漸增,應與成蟲需啟動下游大量性別專一性基因所致。由於Bdtra-2為雌雄皆有的輔助性基因,觀察到其於幼蟲、蛹、成蟲時期皆呈現穩定表現。其次,利用Bdtra siRNA餵食剛孵化幼蟲及Bdtra dsRNA注射0日齡蛹的RNA干擾 (RNAi) 實驗進一步探討Bdtra的生理功能。利用Bdtra siRNA餵食幼蟲的實驗中,30隻幼蟲有23隻順利羽化為成蟲,雌雄成蟲的外觀皆正常,且雄成蟲子代雌雄比例接近1:1,並未發現中間性或偽雄性個體。蛹注射實驗結果則觀察到在68個經Bdtra dsRNA注射的蛹中,16隻雄成蟲及19隻雌成蟲成功羽化。存活雌成蟲中有8隻產卵器產生變形,其中的6隻雌成蟲沒有採集到卵;而雄成蟲大部分皆具有正常的外生殖器,但部分個體無子代產生。解剖觀察經Bdtra RNAi處理雌雄成蟲,精巢或卵巢外形與對照組個體無明顯差異。
The oriental fruit fly, Bactrocera dorsalis (Hendel), invades more than 150 fruit crops; it causes serious damage to fruits and leads to huge economic loss in Taiwan. Owing to the number of female flies is directly correlated to crops; a comprehensive understanding of sexual determination will be useful to find a new way to reducing female flies in the field. In the tephritid flies, transformer (tra) is a switch in female sex-determination. To learn more matter of this system, the expression patterns of sex-related genes including tra, transformer-2 (tra-2), doublesex (dsx) and yolk protein gene (Bdyp1) in Bactrocera dorsalis were examined in this study. RNA preparations from the different developmental stages of the oriental fruit flies were extracted to detect the expressions of these genes by Reverse transcription polymerase chain reaction (RT-PCR). High levels of Bdtraf observed in both unfertilized and fertilized eggs and consist with the previous observation from Ceratitis capitata that the female will deposit traf mRNA into eggs for its auto-regulatory function in embryonic stage. In the larval and pupal stage, many gender characters are still indefinite, expression levels of these sex-related genes are relatively low compared to those of adults. After eclosion, the expressions of Bdtraf, Bddsxf and Bdyp1 in female and the expression of Bddsxm in male are increasing with age. The phenomena indicate these products are functional and critical in sex differential pathways. TRA-2 is a protein present in both male and female, its expression level is constant in all stages as expected. Two RNA interference (RNAi) experiments were performed for Bdtra functional analyses. In the feeding experiment, 13 male and 10 female flies enclosed successful after the Bdtra siRNA treatment. However, all flies are normal in appearance, and the sex ratio of the F1 offspring of treated males close to 1:1 without expected intersex or pseudomales detected. In addition, Bdtra doubles-strand RNA covering exon 2 to exon 4 was prepared, and injected into 68 early pupae. Total 16 male and 19 female flies were successful eclosion. Eight RNAi treated female had deformed ovipositor; six of these female were unable to lay eggs. Most of male adults possessed normal external genitalia, but some individuals did not have offspring. No morphological changes were detected on ovaries or spermaries of Bdtra dsRNA treated flies.
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