ABSTRACT: 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 in conifers because of the lack of reference sequences for these enormous genomes. In this study, Solexa sequencing of three small RNA libraries obtained from dormant, reactivating, and active vascular cambium in Chinese fir samples identified 76 known miRNAs from 27 miRNA families and 18 new potential miRNAs, of which 15 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, analyzing the 103 miRNAs and their predicted targets indicated that 30% of miRNAs appeared to negatively regulate their targets, of which 4 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). 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. The aim of this study is to investigate the small RNA transcriptome in different stages of vascular cambium development in Cunninghamia lanceolata. We herein conducted our comprehensive analysis of a plant sRNAome during development of the vascular cambium in Chinese fir using cryosectioning to isolate cambial meristem, which allowed a higher cellular resolution for defining small RNA profiles and overcomed the limitations resulting from the mixed samples in many previous investigations. Total RNAs of dormant, reactivating, and active cambial meristem, respectively were isolated using the Concert Plant RNA Reagent (Invitrogen, Carlsbad, CA, USA), and were then treated with RNase-free DNase I (Promega, Madison, WI, USA). Twenty micrograms of total RNAs were used and 16 to 30-nt sRNAs were purified using Novex 15% TBE-¬Urea gel (Invitrogen). Two adaptors were sequentially ligated to the 5' and 3' ends of purified sRNAs. The ligation products were further purified from Novex 10% TBE-Urea gel. Reverse transcriptase SuperScript II (Invitrogen) and high-fidelity DNA polymerase Phusion (New England Biolabs, Ipswich, MA, USA) were used in the following RT-PCR reaction. The amplification products were cut from Novex 6% TBE-Urea gel. The purified DNA fragments were used for sequencing on an Illumina 2G Genome Analyzer at the Beijing Genomics Institute, Shenzhen, China.
