轉位子mini Ds插入epsps對基因表現影響之評估

Abstract

轉位子 (transposable element, TE)係可在基因組內遷移之DNA片段。近來研究指出，TE可調控其插入位置之側翼基因，扮演cis-調控因子 (cis-regulating element)而產生增強或靜默等效果。研究顯示TE調控側翼基因之能力受插入位置影響，其中以插入5端未轉譯區與第一隱子最顯著。許多研究利用高通量定序，探討TE對基因組的整體效果；較少著墨於單一TE因不同插入位置所產生影響之定量研究及有效評估。本研究以水稻為實驗平台建立四種構築，分別將mini Ds以正、反向，插入外源epsps基因之5端未轉譯區與第一隱子作為實驗組以及未插入之外源epsps基因作為對照組，二基因匣以同一段T-DNA送入，獲得轉殖株。並利用半定量及real-time PCR，評估基因表現差異。結果顯示，mini Ds插入此二區域，均傾向降低側翼基因表現。研究中亦探討構築建立之位向 (orientation)問題、TE插入之差異表現以及實驗評估成效，期能對TE與宿主之互動有更深的認識。

Transposable elements (TE) are mobile DNA fragments among genome. It has been shown that insertion of TE may regulate the flanking genes, acting as a cis-regulating element to exert enhancing or silencing effects. Such regulating capacity has been reported to influenced by the insertional site, especially significantly in 5’ UTR and the first intron. Much research has been drawn to the study of high-throughput analysis, with an eye to discuss the overall impact on genome. Relatively less, however, focuses on quantitative analysis and valid assessment of the effect of a single TE on a gene in different insertion sites. In this study, four constructs, where a mini Ds-inserted epsps gene in 5’ UTR or the first intron in either orientation and a reference gene are adjacently designed in T-DNA, were introduced into rice. The expression differences are assessed through semi-quantitative and quantitative RT-PCR. The relative expression levels of the mini Ds-inserted genes, by either orientation and by both insertion sites, were significantly lower than the reference. Based on the results, the orientations of the insertion, the influence of TE insertion on gene expression, and the assessment of the research are deliberated for deeper understanding of the interaction between the TE and the host genome.