Analysis of the program-predicted R-loop forming genes in Arabidopsis by gel mobility shift assay

• Main Authors: Ya-Jhen Chen, 陳亞甄
• Other Authors: 楊健志
• Format: Others
• Language: zh-TW
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/49385097482072554527

碩士 === 國立臺灣大學 === 生化科技學系 === 104 === 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.