利用菸草原生質體細胞研究逆對稱構造對於植物基因表現之影響

Abstract

由原核生物、動物基因及菠菜葉綠體轉錄終止的研究報告中已知逆對稱序列在基因表現的調控上扮演著重要的角色。 為研究逆對稱序列在植物菸草RNA polymerase II轉錄終止的影響，本論文在實驗設計上將大腸桿菌酥胺酸衰減子以及其變異株構築到pBI221SI載體上，將構築好的載體以電擊的方法送入菸草原生質體細胞中，由GUS的表現來分析逆對稱構造對於基因表現的影響，並且探討可能的機制。 由數據的分析可知stem-loop構造可以穩定mRNA，並且提高蛋白質表現，但是stem-loop構造並不能取代poly(A)tail的功能，mRNA若不帶有poly (A) tail則蛋白質表現很差；將逆重複序列置於3'flanking區域poly (A) sites之後有提高基因表現的效果；由大腸桿菌各個不同的變異株終止轉錄的效率看來，與已知的研究報告結果相同的是stem-loop穩定度與轉錄終止的效率無關，序列上的特異性與二級結構對於轉錄終止的影響較大。

It has been known that inverted repeat sequences play important roles in regulating gene expression in prokaryotes mamalian cells and chloroplast genes. E. coil thr attenuator and its variants have been cloned into pBI221ST vector and sent into Nicotiana tabacum protoplasts to examine the effects of inverted repeat sequences on transcription termination of RNA polymerase II. The effects of attenuators can be analyzed according to GUS reporter expression. The data of this thesis show that stem-loop can stabilize mRNA and promote gene expression, but its function can not take place of that of poly (A) tail. Suprising, inverted repeat sequences after poly (A) sites can promote gene expression. Further more, stem-loop stability has no concern with ability of terminating transcription, and sequences specificity and the structure of stem-loop seem have much effect on transcription termination.