Abstract
BackgroundMicroRNAs (miRNAs) constitute a family of small RNA (sRNA) population that regulates the gene expression and plays an important role in plant development, metabolism, signal transduction and stress response. Extensive studies on miRNAs have been performed in different plants such as Arabidopsis thaliana, Oryza sativa etc. and volume of the miRNA database, mirBASE, has been increasing on day to day basis. Stevia rebaudiana Bertoni is an important perennial herb which accumulates high concentrations of diterpene steviol glycosides which contributes to its high indexed sweetening property with no calorific value. Several studies have been carried out for understanding molecular mechanism involved in biosynthesis of these glycosides, however, information about miRNAs has been lacking in S. rebaudiana. Deep sequencing of small RNAs combined with transcriptomic data is a powerful tool for identifying conserved and novel miRNAs irrespective of availability of genome sequence data.ResultsTo identify miRNAs in S. rebaudiana, sRNA library was constructed and sequenced using Illumina genome analyzer II. A total of 30,472,534 reads representing 2,509,190 distinct sequences were obtained from sRNA library. Based on sequence similarity, we identified 100 miRNAs belonging to 34 highly conserved families. Also, we identified 12 novel miRNAs whose precursors were potentially generated from stevia EST and nucleotide sequences. All novel sequences have not been earlier described in other plant species. Putative target genes were predicted for most conserved and novel miRNAs. The predicted targets are mainly mRNA encoding enzymes regulating essential plant metabolic and signaling pathways.ConclusionsThis study led to the identification of 34 highly conserved miRNA families and 12 novel potential miRNAs indicating that specific miRNAs exist in stevia species. Our results provided information on stevia miRNAs and their targets building a foundation for future studies to understand their roles in key stevia traits.
Highlights
MicroRNAs constitute a family of small RNA population that regulates the gene expression and plays an important role in plant development, metabolism, signal transduction and stress response
MicroRNAs constitute a class of single stranded 19–24 nucleotide long non-coding RNAs that mediate endogenous gene silencing by binding to their target genes. miRNAs binds either to the open reading frame (ORF) or untranslated regions (UTRs) of target mRNAs and silencing occurs either by target cleavage or by inhibiting mRNA translation [1,2] through perfect or partial complementarity respectively [3]. miRNA feature to regulate gene expression is exemplified by their indispensable roles in critical biological and cellular events in plants including lateral root formation, leaf morphology
Deep sequencing of small RNA (sRNA) Using 36 cycled single end sequencing by genome analyzer II [32], a total of 30,472,534 sequences were obtained from smRNAome of S. rebaudiana
Summary
MicroRNAs (miRNAs) constitute a family of small RNA (sRNA) population that regulates the gene expression and plays an important role in plant development, metabolism, signal transduction and stress response. MiRNA biogenesis involves a meshwork of various enzymes These are produced from their own transcriptional units [11] generated by stepwise processing of RNA polymerase II dependent primary miRNA transcripts (primiRNAs) which are subsequently capped, spliced and polyadenylated [5]. RNA binding protein DWADDLE (DDL) stabilizes pri-miRNAs for their conversion into processing centers called nuclear dicing bodies (D bodies) to stem loop precursor miRNAs (premiRNAs). This conversion further requires concerted assistance of Serrate (C2-H2 zinc finger protein), HYPONASTIC LEAVES1 (HYL-1, a double stranded-RNA binding protein), nuclear cap binding complex (CBC) and Dicer-like 1 (DCL-1, RNase-III class of enzymes). The cleaved halves of target mRNA are degraded by exoribonucleases 4 (XRN-4) and secondary siRNAs [12,13]
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