Identifying MicroRNAs And Their Target Genes Involved In Regeneration Of Secondary Vascular System In Populus T Omentosa Carr

Wood formation is a complex process composing many biological events,such as the growth and differentiation of cambium cells and the development of new formed xylem,which is completed by the coordinate of functional genes and different regulators.microRNA(miRNA)is a negative regulator of gene expression in eukaryotes and can exert post-transcriptional regulatory roles over their target genes involved in plant growth and development through m RNA degradation and translational repression.To investigate miRNAs and their target genes involved in wood formation,we used the regeneration of the secondary vascular system established in Populus tomentosa to harvest tissues generated from differentiating xylem in a time series.We analyzed the express profiles of miRNAs and predicted the novel miRNAs during the regeneration process of secondary vascular system(SVS)in P.tomentosa by small RNA high-throughput sequencing.The target genes of miRNAs predicted and verified by degradome sequencing.The sequencing data were analyzed and led to the identification of 209 known and 189 novel miRNAs from 127 novel families during 6 time points in SVS regeneration process,e.g.7 days,10 days,12 days,16 days,18 days and 21 days after girding(AG)in P.tomentosa.The miRNA reads were normalized to total reads per million(RPM),the expression profiles of 43 conserved miRNAs and 18 non-conserved miRNAs families respectively differed noticeably during SVS regeneration.miR156,miR164,miR166 and miR168 miRNA families generally had higher expression at all 6 time points,accounted for approximately 80% of all known miRNA reads.In contrast,the expression levels of 5 conserved miRNA families,i.e.miR319,miR393,miR394,miR397,miR399 and 6 non-conserved miRNA families,i.e.miR1446,miR1448,miR473,miR475,miR477 and miR481,were very low with RPM values below 10 at the 6 time points.Except the miRNAs with RPM values below 10,most known miRNAs were highly expressed in one of the three regeneration stages: vascular cambium(VC)initiation stage(7days and 10 days AG),VC formation stage(12 days and 16 days AG),and VC differentiation stage(18 days and 21 days AG).The expressions of 15 known miRNAs were further verified by qRT-PCR.In general,the expression levels of novel miRNAs were lower than that of known miRNAs during the SVS regeneration in P.tomentosa.Among 189 novel miRNAs,only 27 miRNAs from 20 novel miRNA families had RPM values above 10 in at least one library.We cloned 21 novel miRNAs with high RPM value and detected their relative expression by universal qRT-PCR.To further understand the roles of miRNAs during the SVS regeneration process,three degradome libraries were constructed from poly(A)fraction of pooled the samples from two adjacent time points,i.e.7 and 10 days AG,12 and 16 days AG,and 18 and 21 days AG respectively.We identified a total of 223 target genes for 24 known miRNAs families and 126 target genes for 45 of the 127 novel miRNA families.These target genes were diverse and included transcription factors,signal transduction factors and other proteins involved in various biological processes.we implemented gene ontology(GO)enrichment analysis to these target genes,of which,the target genes of 8 known miRNAs and 7novel miRNAs families regulated the formation and differentiation of secondary vascular tissue.They were primarily enriched in GO categories mostly involved in auxin signaling pathway,cell differentiation,meristem development and pattern specification processes.The expression levels of 42 target genes for these 15 miRNA families were also clustered based on the microarray data from the 6 time points during SVS regeneration.In this study,we identified three large miRNA gene family clusters from three novel miRNA families,i.e.pto-miR034,pto-miR017 and pto-miR046.Each cluster contained several precursor genes from one miRNA family and closely spaced to each other on the chromosomes.The miRNA clusters originated from the intron of protein-coding transcripts or from the non-coding region between two adjacent genes,and the precursors might be derived by the promoters of the protein-coding transcripts or its own promoter.In addition,the precursors of ptc-miR396 a,ptc-miR396 b and ptc-miR1450 were found to be targeted by their own mature miRNAs and the cleave sites were in the middle of miRNAs*-coding regions.Other five pre-miRNAs,ptc-miR396 a,ptc-miR396 d,pto-miR047 a,pto-miR025 a,b and pto-miR185 were seemingly cleaved by other mature miRNAs and their cleavage sites were located at 5' ends of mature miRNAs or in the loop forming region of precursors.The research of miRNAs and their target genes during SVS regeneration process in p.tomentosa provides the basis for further analysis of these miRNAs to gain insight into their regulatory roles in wood development in trees and provide the theoretical foundation for revealing the molecular mechanism of wood formation.