轉醛醇縮酶(Transaldolase, Tal)是五碳糖磷酸化路徑中非氧化途徑的速率決定酵素，在此以果蠅常用的基因表現系統( Gal4/ UAS)進行研究，我們先前的研究顯示出在特定組織降低Tal表現會產生眼睛後方凸眼及翅膀皺縮等性狀，且在另一個基因Atg14也產生類似的性狀，然而這些性狀卻是VDRC RNAi中心所提供的特定基因KK品系所特有，在VDRC所提供的GD品系卻未觀察到類似的性狀產生。近期有篇研究指出VDRC的部分KK品系可能含有兩個的干擾RNA (shRNA)標註位點，分別是VDRC標註的位點 (annotated insertion)及未被VDRC報導的標註位點 (non-annotated insertion)，且在該篇研究也顯示標註的插入位點和特異性的表達Gal4，如elavc155-Gal4及actin-Gal4雜交而產生之某些特殊的性狀很有關係，而這些結果可能是由Gal4介導錯誤表現tiptop基因而造成的。我們發現無論是Tal或Atg14的KK品系皆插入在兩個干擾RNA插入位點，且在Tal或Atg14的KK品系中降低tiptop，便可完全治癒眼睛後方凸眼的性狀，另外，過量表現tiptop也會產生類似粗眼及翅膀皺縮的性狀，為了更進一步了解插入位置對於Tal和Atg14所生成的性狀影響，我們利用遺傳方法產生重組品系移除標註的插入位點之干擾RNA，僅剩單一干擾RNA位於非標註位點，在這些重組品系中仍保有凸眼及翅膀皺縮的性狀，但性狀明顯較弱於正常的KK品系所造成的，並且我們觀察到過量表現tiptop所產生的粗眼性狀無法被改正由那些可反轉Tal和Atg14品系所造成的凸眼所恢復成正常眼睛，綜合這些實驗成果，我們證明Tal和Atg14的KK品系所造成的凸眼和皺翅性狀並非單單由插入位點造成之tiptop基因過表量表現而導致，總而言之，tiptop之過量錯誤之表現和其他未知因子可成Tal和Atg14之KK品系表現出凸眼皺翅之性狀。

Transaldolase (Tal) is a rate-limiting enzyme in the non-oxidative branch of pentose phosphate pathway. Our previous study indicates that knockdown of Tal by tissue-specific Gal4 results in posterior bulgy eye and shrunk wing in Drosophila. Also, we found that knockdown of Atg14 produces similar phenotypes. However, these phenotypes are specific to Tal and Atg14 knockdown lines from VDRC RNAi KK line collection, but not by the VDRC RNAi GD line collection. A recent report indicated that some of VDRC RNAi KK collection contained two copies of the short hairpin RNA (shRNA) target site: the annotated insertion reported by the VDRC and a previously non-annotated insertion. This suggests that the insertion locus may affect some gene expression for the special phenotypes. In addition, annotated insertion is a major contributor to some special phenotypes when crossed with tissue-specific Gal4, such as elavc155-Gal4 and actin-Gal4, which may result from Gal4-mediated toxicity through misexpression of tiptop (tio). We discover that both the UAS-TalRNAi KK line and the UAS-Atg14RNAi KK line contained two copies shRNA insertion sites. To our surprise, knockdown of tiptop can fully rescue the posterior bulgy eye and overexpression of tiptop also produces similar rough eye and shrunk wing. Furthermore, we use a genetic approach to establish recombinant lines to remove the addition shRNA insertion site in the KK lines, which leaves only one copy of the shRNA in non-annotated insertion site, of the Tal and Atg14 VDRC RNAi KK lines. Despite the phenotypes are weaker than normal KK lines, recombinant lines still conserve the phenotypes of bulgy eye and shrunk wing. However, the phenotypes by tiptop overexpression cannot be rescued by the same genes which previously had rescued the phenotypes by the RNAi knockdown of Tal and Atg14 by KK lines. These data suggest that the phenotypes are not affected only by the insertion site. In summary, expression of tiptop plays a role in the specific phenotypes by the RNAi knockdown of Tal and Atg14 in the VDRC KK lines. The phenotypes of Tal and Atg14 knockdown in the KK lines may derive from tiptop overexpression and other unknown factors.

Abstract 1
中文摘要 3
Introduction 7
Materials and Methods 12
Results 16
Discussion 23
Figures 27
Tables 49
Appendix 50
References 51

 

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