Transcriptome Analysis And Identification Of MiRNAs In Lonicera Japonica By RNA-Seq Technology

The heperennial and evergreen twining vine,Lonicera japonica(L.japonica)is an important herbal medicine with great economic value.Widely cultivated in eastern Asian countries such as China,Japan and Korea.Pharmacological study has reported that L.japonica is used as a herbal medicine with anti-bacterial,anti-viral,anti-endotoxin,antiInflammatory and anti-pyretic effects.Therefore,L.japonica is often used to treat some human diseases including severe respiratory syndromes,H1N1 flu and hand-foot and mouth disease.L.japonica is also used as food,and worldwide as a healthy beverage,which recently led to the rapid increase in commercial value of Flos Lonicerae Japonicae(the L.japonica flower bud)in herbal medicine trading markets.With the development of Next-generation sequencing(NGS),various studies have reported that short sequencing reads from NGS have been successfully used for de novo genome and transcriptome assembly in organisms without genome reference.Although next-generation sequencing technology has been applied for the transcriptome studies in L.japonica.But to our knowledge,there is no report of identified microRNAs of L.japonica yet,which hindered the functional genomics and anti-disease researches of L.japonica.In this study,the transcriptome profiles of leaves and flowers in L.japonica were characterized by NGS.Over 200 million clean reads(Flower:124,617,394 clean reads,Leaf:114,787,876 clean reads)were generated using the Illumina Hiseq 2000 platform.De novo assembly of the L.japonica transcriptome was performed using the Trinity program.There were 150,523 UniGenes obtained with an average lengthof 632 bp and an N50 of 947 bp.All UniGenes were annotated with available protein and nucleotide databases.Furthermore,differentially expressed genes(DEGs)between leaves and flowers were identified.The results indicated that 35,327 UniGenes were differentially expressed between leaf and flower tissues(FDR ≤ 0.001),There were 6602 DEGs assigned with Biological Process terms: Metabolic process,Response to stimulus,Cellular process,ect.And partial DEGs were verified with qRT-PCR.KEGG pathway analysis shown that 4,564 UniGenes were assigned to the top 10 KEGG biochemical pathways and three important genes involved in the synthesis pathway of chlorogenic acid,such as SK1,C4 H and HCT were up-regulated inflowers.In addition,we screened SSRs,blasted TFs and enzyme from the unigens of L.japonica transcriptome.As miRNA regulate the gene expression at the post transcriptional level by degrading or inhibiting the translation of targeted gene mRNAs,next-generation sequencing technology has been widely applied for the sRNA research in recent years.In this study,we constructed the small RNA cDNA libraries from L.japonica leaf and flower and performed small RNA sequencing using Illumina Hi-seq 2000 platform.By taking advantage of bioinformatics approach,the conserved and novel L.japonica miRNAs were identified,respectively.A total of 2,297 million clean reads from flower and leaf tissues were obtained,which generated 146 conserved miRNAs distributed in 20 families and 110 novel miRNAs.Accordingly,72 differentially expressed mi RNAs(P≤0.001)between leaves and flowers and their potential target genes were identified and validated.The qRT-PCR validation showed that majority of the differentially expressed miRNAs showed significant tissue-specific expression in L.japonica.Furthermore,the mi RNA-mRNA and mRNA-mRNA regulatory networks were constructed using Cytoscape software.Additionally,the potential target genes of the conserved and novel miRNAs werepredicted.Annotation and network analysis of mi RNA and their putative target genes were also performed to get a better understanding of their functions in various biological processes in L.japonica.At last,RLM-5’RACE was used to test partial predicted target genes of miRNA and the broken sites of seven target genes were verified.In this study,more comprehensive transcriptome profiles and mi RNA expression profile from L.japonica were obtained using next-generation sequencing technology.Combine with the transcriptome sequence data of L.japonica,analysis of relationship between miRNAs and their target were carried out,all of these provide important reference data for functional genomics and pharmacology research in the future.