轉位子mini Tag與miRNA形成之研究

Abstract

近來研究報告證實miRNA與轉位子 ( transposable element，TE ) 間有著密切的關聯性，且發現有些特定的轉位子序列經演化後，可形成類似miRNA的hairpin結構。本實驗利用阿拉伯芥Landsberg品種的內生轉位子Tag1 ( 3.3 kb ) 探討轉位子與 miRNA 之關係。轉位子Tag1前後兩端含有22 bp可完全互補的反向重複序列 ( terminal inverted repeat，TIR )，欲了解此轉位子序列在RNA層級是否會形成hairpin，繼而以miRNA形式調控基因表現，本研究以PCR擴增Tag1 5端262 bp與 3端270 bp 構築成532 bp的mini Tag。另使用PCR合成不含3’端TIR的510，以破壞hairpin結構的形成。將以上片段分別插入報導基因luc 的5’ UTR及3’ UTR，利用農桿菌轉殖至菸草觀察基因表現變化。冷光酶反應in vivo檢驗結果顯示，菸草轉殖之癒合組織中所有構築皆具有冷光酶活性。推測轉位子mini Tag在細胞中可能不會形成有效的hairpin二級結構或是有二級結構形成但未成功誘發RNA干擾途徑；但關於luc基因表現量是否有尚未觀察到的細微變化以及細胞中是否形成預期中的hairpin結構，還須以其他實驗進行驗證。本實驗模擬轉位子可能扮演miRNA之角色，更進一步探討轉位子與miRNA之間的關係，並提供未來調控基因表現之新工具。

It has been proved that there is a close association between miRNA and transposable element (TE). Some TE may form a secondary structure which is similar to the hairpin structure of miRNA. In this study, we use an intrinsic TE, Tag1, from Arabidopsis thaliana Landsberg erecta ecotype. Tag1 is 3.3 kb in length and has 22-bp terminal inverted repeat ( TIR ) which can perfectly match. We questioned that whether these sequences can form a hairpin structure at RNA level, and then control the target gene expression by miRNA formation. PCR technique was employed to amplify the 5’ and 3’ subterminal fragments ( 270 and 262 bp ) of Tag1 sequence to yield the mini Tag (532 bp). To analyze the expression, the mini Tag was inserted into the 5’ UTR and 3’ UTR of a reporter gene luc, and was transformed into Nicotiana tabacum by Agrobacterium tumefaciens. The results of in vivo luciferase assay show that all the transformants have the luciferase activity. This outcome indicates that the mini Tag element may not form an effective hairpin structure or the formation of secondary structure occurs but it cannot induce to RNA interference pathway. Finally, we discuss the correlation between the TE and miRNA, and introduce a new tool of gene regulation.