Characterization and primary functional analysis of Pinus densata miR171

The miR171 is a conserved microRNA (miRNA) family and has been shown to participate in plant growth and development. However, the precise function of miR171 in Pinus densata remains largely unclear. Mature miR171 sequence comparison reveals high similarity between Arabidopsis thaliana and P. densata and the pre-miR171 could fold into a characteristic stem-loop hairpin structure. Genes encoding GRAS (GAI-RGA-SCR) family transcription factors and actin binding protein were identified as targets of pde-miR171 using a modified RNA ligase mediated 5’ rapid amplification of cDNA ends (RLM-RACE). Furthermore, the interaction between pde-miR171 and Arabidopsis SCL6 (SCARECROW-LIKE6) was further validated through transient co-expression of both genes in Nicotiana benthamiana leaves. Next, results of real-time quantitative PCR demonstrated that the expression of pde-miR171 was significantly up-regulated in miR171 -overexpressing plants than in wild-type plants, which was inversely correlated with the expression of Arabidopsis SCL6 genes. In addition, overexpression of pde-miR171 in Arabidopsis induced larger leaves and earlier flowering under long-day conditions compared with the wild type. The findings presented here suggest that miR171 derived from a P. densata precursor together with its target gene SCL6 may play important roles in the regulation of primary root growth, leaf shape, and flowering time in plants.