Project description:New Generation of Artificial MicroRNA and Synthetic Trans-Acting Small Interfering RNA Vectors for Efficient Gene Silencing in Arabidopsis
Project description:Cytosine DNA methylation (mC) is a genome modification that can regulate the expression of coding and non-coding genetic elements. However, little is known about the involvement of mC in response to environmental cues. We performed whole genome bisulfite sequencing to assess the spatio-temporal dynamics of mC in Arabidopsis grown under phosphate starvation.
Project description:We obtained an Arabidopsis mutant from the Arabidopsis Biological Resource Center stock collection and verified that it was homozygous for a T-DNA insertion in the first exon of ORRM1 (SALK_072648, designated here as orrm1). The homozygous mutant did not show any phenotypic defect when grown under growth room conditions. We examined the organelle transcriptome of the mutant for editing defects because other proteins carrying RIP domains have been shown to be editing factors. We analyzed the plastid RNA editing extent with a new methodology based on RNA-seq. Briefly, total RNA is isolated from leaves and RT-PCR products corresponding to known organelle genes are obtained by using gene-specific primers. The products are mixed in equimolar ratio, sheared, and used as templates to produce an Illumina TruSeq library. This RNA-seq analysis demonstrated that ORRM1 is a plastid editing factor; 12 among 34 plastid sites exhibit a severe reduction of editing extent in the mutant relative to the wild-type