title: Transcriptome-wide identification and characterization of miRNAs from Pinus densata.
abstract: BACKGROUND
  MicroRNAs (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.
  
  
  RESULTS
  Here, 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.
  
  
  CONCLUSIONS
  This 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.
authors.affiliation: Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China.
authors.forename: Li-Chuan
authors.initials: LC
authors.lastname: Wan
authors.forename: Haiyan
authors.initials: H
authors.lastname: Zhang
authors.forename: Shanfa
authors.initials: S
authors.lastname: Lu
authors.forename: Liang
authors.initials: L
authors.lastname: Zhang
authors.forename: Zongbo
authors.initials: Z
authors.lastname: Qiu
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Zhao
authors.forename: Qing-Yin
authors.initials: QY
authors.lastname: Zeng
authors.forename: Jinxing
authors.initials: J
authors.lastname: Lin
mesh_terms: D001483:Base Sequence; D020869:Gene Expression Profiling; D018506:Gene Expression Regulation, Plant; D035683:MicroRNAs / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D028223:Pinus / Q000235:genetics*; D059367:RNA Cleavage; D012333:RNA, Messenger / Q000378:metabolism; D015203:Reproducibility of Results; D059467:Transcriptome
chemical_list: D035683:MicroRNAs; D012333:RNA, Messenger