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F20M13 BAC(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,RL L4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 lumines-cence 4) by screening a M2 population derived from an ethyl methane sulfonate (EMS)-mutagenized trans-genic parental line Col-LUC ,which harbors a firefly luciferase reporter (LUC) gene under the control of 2 × 35S promoter.Genetic analysis demonstrated that the rll4 locus contains a single nuclear-encoded reces-sive mutation .By using map-based cloning strategy ,we narrowed down the rll4 locus between the molec-ular markers CL417-B10M1 and CL418-B2M2 ,which are located at BAC (Bacterial Artificial Chromo-some) clones F20D10 and F20M13 on chromosome 4 ,respectively .Methylation-sensitive restriction en-zyme PCR (Chop-PCR) results showed the DNA methylation levels of a few genomic loci were increased in rll4 mutant .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 rll4 mutant while the expression of ROS1 (REPRESSOR OF SILENCING 1) remained unchanged .Taken together , these results suggest that RL L4 locus is probably involved in DNA demethylation process in A .thaliana .
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