利用電泳分析程式預測之阿拉伯芥中可能形成 R-loop結構之基因

Abstract

R-loop為RNA與互補DNA雜合後使另一股DNA分離而單獨存在所形成的三股核酸結構。R-loop的結構包含由轉錄後的RNA與模板DNA (template strand DNA ) 重新配對，及單股的非模板DNA (non-template strand DNA)。植物中第一個R-loop有關之研究，是COOLAIR基因形成R-loop後，失去抑制FLC的作用，而共同調控植物開花。為了深入了解 R-loop是否在植物扮演其他角色，本實驗室謝閔翔與生物產業機電工程系陳倩瑜老師實驗室合作，使用程式語言「C++」來設計出可以搜尋出可能形成R-loop的基因的程式，篩選出了在53個在阿拉伯芥中可能形成R-loop的基因。謝 (2015)已研究LBD18在in vitro及in vivo形成R-loop。本實驗挑選13個基因以胞外轉錄及三種gel mobility shift assay進行研究。13個基因中，部分出現in vitro R-loop形成之特徵性色帶模糊。但同一種基因中，三種gel mobility shift assay得到的實驗結果不盡相同，且LBD18在三種方法中都沒有呈現色帶模糊。另外，本研究發現之前使用urea於染色體DNA抽取可能有缺點，因此使用改良的抽取方法，唯此方法未能再現LBD18在native bisulfite sequencing assay之結果。

R-loop is a triple helix structure consisting of a DNA/RNA hybrid plus a single strand DNA. R-loops are formed when newly synthesized RNA threads back to anneal with the template strand DNA, leaving the non-template strand DNA remaining as a single strand. In Arabidopsis, the expression level of COOLAIR, the first gene discovered that can form R-loops in plants, coordinates flowering with FLC. To explore if R-loop formation is widespread in plants, Min-Hsiang Hsieh designed an R-loop searching program written using C++ together with Dr. Chien-Yu Chen (Department of Bio-industrial Mechatronics Engineering of National Taiwan University) .Fifty-three genes were predicted to be able to form R-loops by the program. Hsieh (2015) found that LOB domain-containing protein 18 (LBD18) form DNA/RNA hybrids in vitro and in vivo. In this study, 13 genes were tested by in vitro transcription and three kinds of gel mobility shift assay. Part of the 13 genes had the distinctive smear of DNA/RNA hybrid in vitro, but different protocol caused different consequences in some genes. In this study, LBD18 did not have smear pattern by three kinds of gel mobility shift assay. Moreover, there was shortcoming of using urea to extract genomic DNA by Hsieh. (2015). Modified genomic DNA extraction protocol did not repeat the native bisulfite sequencing result of LBD18.