The isolation and identification of salt-responsive novel microRNAs from Salicornia brachiata, an extreme halophyte

Abstract

The small, non-coding RNA species (20–24 nucleotides), including microRNAs (miRNAs) and small interfering RNAs, are key regulators of gene expression in eukaryotic cells. Mostly, miRNAs have been identified from glycophytes under different abiotic stresses. The isolation and identification of miRNAs from halophytes are challenging due to the presence of high salt concentrations. In this study, small RNA cDNA libraries were constructed using an improved method to identify salt-responsive novel miRNAs from Salicornia brachiata. A total of 159 sequences were cloned and analysed. There was no adapter contamination and low-abundance small RNAs were amplified efficiently. Twelve putative miRNAs/pre-miRNAs and a small RNA were identified and confirmed by northern blot. Among the putative miRNAs, nine are novel and three belong to the conserved miRNA families (MIR169g, MIR1433, MIR138b). Northern hybridisation and real-time PCR showed that nine miRNAs (seven novel and two conserved) and one small RNA were highly expressed at 2.0 M NaCl, two novel miRNAs at 1.5 M NaCl, and one conserved miRNA at 1.0 M NaCl. Furthermore, 67 putative target genes of the isolated miRNA were predicted on the basis of sequence homology, of which more than 50 % are associated with abiotic stresses. The target genes of miRNA 169g (Sb-miRNA10), and Sb-miRNA7 (NF-YA transcription factor and cytochrome P450-like TATA box binding protein respectively) were analysed by real-time PCR. The study of miRNAs from an extreme halophyte broadens our understanding of the important role played by miRNAs in plant abiotic stress.