Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/89071
標題: Structural and functional analyses of vestigial and scalloped genes in Bactrocera dorsalis Hendel
東方果實蠅vestigial與scalloped基因之結構及功能分析
作者: 李佳容
Chia-Jung Lee
關鍵字: 東方果實蠅
翅發育
遺跡基因
扇貝基因
Bactrocera dorsalis
vestigial
scalloped
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摘要: 東方果實蠅 (Bactrocera dorsalis Hendel)是台灣果樹的主要害蟲之一,其危害嚴重影響水果的品質與產量。由於此害蟲的飛行能力強,使得危害面積易於擴大,也增加防治上的困難度;因此,深入瞭解東方果實蠅翅的發育與調節機制,設法降低其移動能力,將有助於未來對此蟲的防治。有文獻指出扇貝基因 (scalloped, sd)及遺跡基因 (vestigial, vg)與昆蟲翅的發育有關,因此本研究針對這兩個基因的結構、功能與表現進行研究,以證實它們對東方果實蠅的翅發育調控。首先根據茄二十八星瓢蟲(Henosepilachna vigintioctopunctata)及異色瓢蟲(Harmonia axyridis)的VG與SD已知蛋白質序列設計簡併式引子(degenerate primers);接著完成反轉錄聚合酶連鎖反應 (reverse transcription-PCR)來選殖東方果實蠅之sd與vg部份cDNA序列;最後再利用快速放大cDNA末端技術 (rapid amplification of cDNA ends)技術決定基因全長。經過與黑腹果蠅 (Drosophila melanogaster)之相應cDNA序列比對後,發現東方果實蠅這兩個調節因子與黑腹果蠅的SD及VG具有相似的蛋白質功能區,從基因結構顯示這兩個基因在東方果實蠅與黑腹果蠅間具有同源性。此外,利用即時定量聚合酶連鎖反應 (Real-time Quantitative PCR)探討從卵至成蟲共十個發育期間vg與sd之mRNA相對表現量,結果指出這兩個基因皆在五日齡蛹期表現達到最高峰,這也間接反映出它們的功能與翅的發育相關。最後利用RNA干擾 (RNA interference)探討東方果實蠅VG與SD之生理功能。先分別製作vg及sd雙股RNA (double-strand RNA),再分別或同時注射入老熟幼蟲與一日齡蛹的腹部末端。經RNAi處理後,可觀察到成蟲翅之形態變異及對vg及sd mRNA的專一性抑制。綜合上述實驗結果,可以確認此二蛋白質的確參與東方果實蠅翅的發育過程,並且其正常的表現對東方果實蠅的飛行功能極為重要。此基礎研究成果未來可繼續延伸,並配合轉殖技術限制東方果實蠅的飛行能力,具有應用於生物防治的潛力。
Oriental fruit fly (Bactrocera dorsalis Hendel), a major insect pest causing severe destruction of fruit production in Taiwan, is difficult to manage partly due to its flying ability. Knowledge of wing development will be valuable and have the potential to limit the movement and distribution of this pest in the field. Two genes, vestigial (vg) and scalloped (sd), have been reported to play important roles in wing formation of Drosophila melanogaster and other insects, therefore their orthologue in B. dorsalis were chosen as targets in this study. Based on gene homology among insect species, degenerated primers were designed to obtain cDNA of Bdsd and Bdvg by RT-PCR. After sequence analyses, putative SD interaction domain and partial N-terminus transactivation domain were found in Bdvg. In addition, TEA domain and VG interaction domain were also identified and characterized in Bdsd. With quantitative real time PCR, the gene expression patterns of ten different developmental stages indicated that Bdvg expression in pupae gave the highest expression level; and similar trend was also detected in Bdsd. For functional assay, RNA interference (RNAi) resulted in wing deficiency of some Bdvg or Bdsd dsRNA treated individuals. These results suggest that both Bdvg and Bdsd genes are related to the wing development of oriental fruit fly, and interference of Bdvg and Bdsd can lead to the limitation of fly. The ability to reduce the distribution of the pest has its potential as a useful candidate in future management.
URI: http://hdl.handle.net/11455/89071
其他識別: U0005-2008201513215500
文章公開時間: 2018-08-26
Appears in Collections:昆蟲學系

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