拟南芥DNA去甲基化基因RLL4的定位与功能分析

Mapping and Functional Characterization of RLL4 Locus Associated with DNA Demethylation Process in Arabidopsis thaliana

该研究以携带2×35S:LUC报告基因的转基因拟南芥Col-LUC为亲本系,将其种子进行甲基磺酸乙酯(EMS)诱变,在M2代筛选出1株低荧光的候选突变体,命名为rll4(reduced LUC luminescence 4)。遗传学分析表明,rll4突变位点包含1个核基因隐性突变。图位克隆技术定位结果显示,突变基因的位点位于4号染色体2个分子标记CL417-B10M1和CL418-B2M2之间,这2个分子标记分别位于F20D10和F20M13BAC(bacterial artificial chromosome)克隆。酶切PCR(Chop-PCR)结果显示,rll4突变体中基因组DNA的部分位点甲基化显著升高。反转录PCR(RT-PCR)结果显示,rll4突变体中ROS1(REPRESSOR OF SILENCING 1)的表达量并没有明显变化,而一些RNA介导的DNA甲基化(RdDM)过程靶位点的基因表达量有明显下降。研究表明,RLL4位点很可能参与了拟南芥DNA去甲基化过程。

In this study,we identified a low-luminescence mutant referred to as rll4(reduced LUC luminescence4)by screening a M2population derived from an ethyl methane sulfonate(EMS)-mutagenized transgenic parental line Col-LUC,which harbors a firefly luciferase reporter(LUC)gene under the control of2×35S promoter.Genetic analysis demonstrated that the rll4locus contains a single nuclear-encoded recessive mutation.By using map-based cloning strategy,we narrowed down the rll4locus between the molecular markers CL417-B10M1and CL418-B2M2,which are located at BAC(Bacterial Artificial Chromosome)clones F20D10and F20M13on chromosome4,respectively.Methylation-sensitive restriction enzyme PCR(Chop-PCR)results showed the DNA methylation levels of a few genomic loci were increased in rll4mutant.Reverse transcription-PCR(RT-PCR)results revealed that the expression levels of several endogenous targets of RNA-directed DNA methylation(RdDM)pathway were decreased in rll4mutant while the expression of ROS1(REPRESSOR OF SILENCING1)remained unchanged.Taken together,these results suggest that RLL4locus is probably involved in DNA demethylation process in A.thaliana.