Identification of abscisic acid-associated known and novel miRNAs and their target genes in Arabidopsis thaliana

Using RNA-Seq, we identified miRNAs in the WT Col-0 strain of Arabidopsis and its abi1td, mkkk17 and mkkk18 mutant lines that are differentially expressed in response to ABA treatment. Both known and novel miRNAs were characterized and the expression profiles of a subset were validated by qRT-PCR; we also verified the potential target genes of selected novel miRNAs by 5′ RLM-RACE. GO annotation revealed that the top-ranked miRNA target genes were involved in diverse biological processes, including regulation of transcription, response to stress, biosynthetic processes, and signaling pathways. Seeds of WT Arabidopsis thaliana, Columbia ecotype (Col-0), as well as abi1td, mapkkk17td, and mapkkk18td mutants were used in this study. After two days of stratification at 4°C, the plants were grown in Half Murashige and Skoog Basal Medium supplemented with sucrose. Seedlings were grown weeks for two weeks, then treated with ABA (±100 µM) for 4 hours. The harvested plant materials were freezed in liquid nitrogen and stored in -80°C freezer until further use. For each condition (mock and ABA treated), three independent biological replicates were used and small RNA was extracted using Tri-reagent according to the manufacturer’s protocol (Direct-zol RNA Miniprep; ZYMO Research Corporation). And samples were used for miRNA sequencing.