MicroRNAs (miRNAs) are small noncoding regulatory RNAs that play key roles in the process of plant development. To date, extensive studies of miRNAs have been performed in a few model plants, but few efforts have focused on small RNAs (sRNAs) in conifers because of the lack of reference sequences for their enormous genomes. In this study, Solexa sequencing of three sRNA libraries obtained from dormant, reactivating, and active vascular cambium in Chinese fir (Cunninghamia lanceolata) using tangential cryosectioning identified 20 known miRNA families and 18 novel potential miRNAs, of which nine novel miRNA precursors were validated by RT-PCR and sequencing. More than half of these novel miRNAs displayed stage-specific expression patterns in the vascular cambium. Furthermore, analysing the 103 miRNAs and their predicted targets indicated that about 70% appeared to negatively regulate their targets, of which two target genes involved in the regulation of cambial cell division were validated via RNA ligase-mediated rapid amplification of 5'-cDNA ends (RLM 5'-RACE) and transient co-expression in Nicotiana benthamiana leaves. Interestingly, miRNA156 and miRNA172 may regulate the phase transition in vascular cambium from dormancy to active growth. These results provide new insights into the important regulatory functions of miRNAs in vascular cambium development and wood formation in conifers.
Keywords: Chinese fir (Cunninghamia lanceolata); high-throughput small RNA sequencing; microRNA; vascular cambium; wood formation..
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