DNA methylation is a relatively stable epigenetic modification,and it plays critical roles in plant growth and development as well as stress resistance.Currently,much is understood about DNA methylation processes,but the understanding of DNA demethylation processes remains as yet incomplete.To better unravel the molecular mechanisms of DNA demethylation,this study performed mutant screening and consequently isolated a low luminescence mutant,referred to as rll2 (for reduced LUC luminescence 2),from an ethyl methanesulfonate (EMS)-mutagenized M2 population derived from a Col-LUC transgenic line that carries a 2 × 35S:LUC (LUC is the abbreviation of luciferase) reporter and emits high LUC luminescence in regular growth conditions.The wild-type gene defined by the mutation was hence termed as RLL2.Compared to the Col-LUC,the rll2 mutant emitted quite low luminescence,and exhibited obvious dwarfism.Genetic analysis revealed that the rll2 mutant carried a single nuclear-encoded recessive mutation that proved to be responsible for the low LUC phenotype.The RLL2 gene was mapped on chromosome 5 and then narrowed down the rll2 mutation to a small region between the markers CL506-B 14M 1 and CL507-B6M 1 which were located at BAC(bacterial artificial chromosome) clones F5O24 and T6G21,respectively.Chop-PCR results demonstrated that several genomic loci were hypermethylated in the rll2 mutant,and reverse transcription PCR (RT-PCR) data indicated the expression levels of a few endogenous genes targeted by RNA-directed DNA methylation (RdDM) pathway were decreased to varying extents in such a mutant.In summary,in this research a low luminescence mutant rll2 was obtained,and the RLL2 gene was mapped on chromosome 5 by map-based cloning strategy.This study revealed that RLL2 is presumably involved in the DNA demethylation processes,which is going to further contribute to our understanding of the molecular mechanisms of such DNA demethylation processes.%DNA甲基化是一种比较稳定的表观遗传修饰,已有的研究表明,DNA甲基化修饰对植物的生长、发育和逆境抗性等有至关重要的作用.目前对DNA甲基化产生过程的研究已经比较深入,但是对于DNA去甲基化机理的研究仍比较浅显.为了完善植物DNA去甲基化的分子机制,本研究以包含2×35S:LUC(luciferase)报告基因的拟南芥(Arabidopsis thaliana)转基因系Col-LUC为遗传筛选的起始亲本材料,将此亲本系进行甲基磺酸乙酯(ethyl methanesulfonate,EMS)诱变,从自交的M2代群体中筛选到了一株低荧光的突变体,命名为rll2 (reduced LUC luminescence 2),此突变对应的野生型基因因此被命名为RLL2.与高荧光的Col-LUC亲本系相比,rll2突变体荧光强度明显降低.在生长发育方面,rll2突变体表现出明显的发育缺陷,如植株变矮、生长势明显比Col-LUC差.遗传学分析表明,rll2突变体携带一个细胞核单基因隐性突变.图位克隆结果表明,候选基因RLL2定位于5号染色体CL506-B 14M1和CL507-B6M1两个分子标记之间,这两个分子标记分别位于F5O24和T6G21两个细菌人工染色体(bacterial artificialchromosome,BAC)克隆上.酶切-PCR(Chop-PCR)结果表明,rll2突变体中染色体上一些位点的DNA甲基化水平明显升高.反转录PCR (reverse transcription PCR,RT-PCR)结果显示,rll2突变体中,RNA介导的DNA甲基化(RNA-directed DNA methylation,RdDM)途径的几个内源靶基因的表达有不同程度的降低.综上所述,本研究筛选到一株低荧光候选突变体rll2,并初步定位了RLL2基因;实验结果表明,其很可能参与了DNA去甲基化过程.这一研究为深入探索DNA去甲基化的分子机理提供了理论依据.
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