Identification And Characterization Of A MicroRNA319 Gene And Its Putative Target Gene PvPCF5 In Switchgrass

Switchgrass is a kind of perennial herbaceous plant originating in North America, which belongs to Gramineae Panicum. Recently, switchgrass has received wide attention because of its excellent characteristics, including high yield, Good ecological benefits, strong adaptability and high lignin content. It has been proved that miRNA and its target genes played a crucial role in plant growth and development, nutrient balance and a variety of biotic and abiotic stresses. However, miRNAs were rarely reported in switchgrass as a model energy plant. In this study, we cloned and identified a gene named pvi-miR319a according to the stem-loop structure of the rice Osa-miR319a in switchgrass. A total of 17 transgenic rice plants with over expression of Pvi-miR319a were obtained and identified, and most of them exhibited a wider and darker leaf phenotype and delayed development compared with WT controls. Further, a gene PvPCF5 targeted by pvi-miR319a was identified in switchgrass, which encoded 444 amino acids.Based on the phylogenetic trees and multiple sequence alignment analysis, it is indicated that PvPCF5 is a member of TCP transcription factor family and found that a conserved TCP domain in the N-terminal and a complementary sequence of mature Pvi-miR319a in the C-terminal. Then, we found the largest expression of PvPCF5 is in mature panicle, while the expression levels of Pvi-miR319a is highest in young panicle by spatial expression analysis. Further tobacco transient expression experiment showed PvPCFS could be negatively regulated by Pvi-miR319a, which suggested that PvPCF5 is a target of Pvi-miR319a in switchgrass. In addition, we also validate the function of target gene by subcellular localization and transcriptional activation experiment, which suggested that PvPCF5 encoded a nuclear localization protein with transcriptional activation activity and the major transcriptional activation region is TCP domain. Thus we suspected that PvPCFS is a member of TCP transcription factor family. In conclusion, it is suggested that pvi-miR319a and its target PvPCF5 is likely to be an important regulatory factors, which will play a greater potential in genetic improvement in swhchsrass.