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| Identification and Functional Analysis of a Candidate Mutant rll2 Associated with DNA Demethylation Processes in Arabidopsis thaliana |
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Abstract Abstract 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-B14M1 and CL507-B6M1 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.
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Received: 27 February 2017
Published: 20 July 2017
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