Transcriptome-wide identification and characterization of miRNAs from Pinus densata

Li-Chuan Wan,Shanfa Lu,Zongbo Qiu,Liang Zhang,Qing-Yin Zeng,Jinxing Lin,Yuanyuan Zhao,Haiyan Zhang

doi:10.1186/1471-2164-13-132

Abstract

BackgroundMicroRNAs (miRNAs) play key roles in diverse developmental processes, nutrient homeostasis and responses to biotic and abiotic stresses. The biogenesis and regulatory functions of miRNAs have been intensively studied in model angiosperms, such as Arabidopsis thaliana, Oryza sativa and Populus trichocarpa. However, global identification of Pinus densata miRNAs has not been reported in previous research.ResultsHere, we report the identification of 34 conserved miRNAs belonging to 25 miRNA families from a P. densata mRNA transcriptome database using local BLAST and MIREAP programs. The primary and/or precursor sequences of 29 miRNAs were further confirmed by RT-PCR amplification and subsequent sequencing. The average value of the minimal folding free energy indexes of the 34 miRNA precursors was 0.92. Nineteen (58%) mature miRNAs began with a 5' terminal uridine residue. Analysis of miRNA precursors showed that 19 mature miRNAs were novel members of 14 conserved miRNA families, of which 17 miRNAs were further validated by subcloning and sequencing. Using real-time quantitative RT-PCR, we found that the expression levels of 7 miRNAs were more than 2-fold higher in needles than in stems. In addition, 72 P. densata mRNAs were predicted to be targets of 25 miRNA families. Four target genes, including a nodal modulator 1-like protein gene, two GRAS family transcription factor protein genes and one histone deacetylase gene, were experimentally verified to be the targets of 3 P. densata miRNAs, pde-miR162a, pde-miR171a and pde-miR482a, respectively.ConclusionsThis study led to the discovery of 34 conserved miRNAs comprising 25 miRNA families from Pinus densata. These results lay a solid foundation for further studying the regulative roles of miRNAs in the development, growth and responses to environmental stresses in P. densata.

Highlights

MicroRNAs play key roles in diverse developmental processes, nutrient homeostasis and responses to biotic and abiotic stresses
The hairpin structures of P. densata miRNA precursors predicted by MFOLD are shown in Additional file 3 and Additional file 4
Through the analysis of the 34 P. densata miRNA precursors, we identified 19 novel mature miRNAs belonging to 14 conserved miRNA families

Summary

Introduction

MicroRNAs (miRNAs) play key roles in diverse developmental processes, nutrient homeostasis and responses to biotic and abiotic stresses. MicroRNAs (miRNAs), generally 21-24 nt in length, spatiotemporally regulate gene expression at transcriptional and/or posttranscriptional level in most eukaryotes [1] They play important roles in plant development, nutrient homeostasis, responses to biotic and abiotic stresses and antibacterial reactions [2,3,4,5]. In silico prediction of miRNAs includes searching genomic or EST databases for orthologous sequences of known miRNAs and analyzing their pre-miRNA hairpin structures [12]. The limitation of this approach is that only highly conserved miRNAs can be identified. For plant species with complete genome information, conserved and novel miRNAs can be conveniently identified based on their alignments to the genome and known miRNAs in the miRBase and analysis of their pre-miRNA stem-loop structures

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Discussion

Conclusion