《植物生理学报》 2017, 53(4): 721-728

一个参与拟南芥DNA去甲基化过程候选基因的定位与功能分析

罗俊鹏, 王敏, 腊红桂^*

南京农业大学生命科学学院, 南京210095

通信作者：腊红桂；E-mail: hongguila@njau.edu.cn

摘　要：

以转基因拟南芥(Arabidopsis thaliana) Col-LUC为亲本材料, 把它的种子进行甲基磺酸乙酯(EMS)诱变, 在M2代幼苗中筛选出一株荧光降低的候选突变体, 命名为rll1 (reduced LUC luminescence 1), 此突变基因对应的野生型基因位点因此被命名为AtRLL1位点。该突变体在生长发育方面表现出明显矮化、角果较短、叶片较小的发育缺陷表型。遗传学分析表明该突变属于细胞核单基因隐性突变。通过图位克隆技术把AtRLL1位点定位于2号染色体T3K9克隆。酶切PCR (chop-PCR)结果表明该突变体中35S启动子和基因组DNA上的多个位点均表现出甲基化升高的现象。反转录PCR (RT-PCR)结果表明rll1中一些RNA介导的DNA甲基化(RdDM)途径的内源靶基因的表达量明显下降。综上所述, 这些研究结果表明AtRLL1位点很可能参与了拟南芥DNA去甲基化过程。

关键词：拟南芥; rll1; 图位克隆; DNA去甲基化

收稿：2017-02-20   修定：2017-03-22

资助：南京农业大学中央高校基本科研业务费(KYRC201409)。

Mapping and functional analysis of a candidate gene involved in DNA demethylation process in Arabidopsis thaliana

LUO Jun-Peng, WANG Min, LA Hong-Gui^*

College of life science, Nanjing Agricultural University, Nanjing 210095, China

Corresponding author: LA Hong-Gui; E-mail: hongguila@njau.edu.cn

Abstract:

In this study, a low luminescence mutant named rll1 (reduced LUC luminescence 1) was obtained by screening a M2 population derived from an ethyl methane sulfonate (EMS) mutagenized transgenic Arabidopsis thaliana Col-LUC line. The wild-type gene locus defined by the mutation was therefore designed as AtRLL1 locus. The rll1 mutant exhibited obvious developmental defects, such as dwarfism, shorter siliques and smaller leaves when compared to Col-LUC. Genetic analysis revealed that the rll1 was a single recessive mutation on nuclear and the candidate gene locus was subsequently mapped on BAC clone T3K9 on chromosome 2 by means of map-based cloning strategy. Methylation-sensitive restriction enzyme PCR (chop-PCR) analysis demonstrates that the DNA methylation levels of 35S promoter and a few genomic loci were obviously increased in the rll1 mutant. Reverse transcription-PCR (RT-PCR) results show that the expression level of several endogenous targets of RNA-directed DNA methylation (RdDM) pathway were decreased to varying extents in rll1. Taken together, these results suggest that the AtRLL1 locus is presumably involved in DNA demethylation process in A. thaliana.

Key words: Arabidopsis thaliana; rll1; map-based cloning; DNA demethylation