Effects Of MYB Transcription Factors On Biosynthesis Of Chlorogenic Acid In Lonicera Japonica Via Multi-Omics Analysis

Objective: We performed full-length transcriptome sequencing,RNA-seq and targeted metabolic profiling at five different flowering stages using Lonicera japonica cultivar“Huajin 6” to explore the role of MYB transcription factors in the regulation of chlorogenic acid biosynthesis,and plant growth and development.Methods:The full-length transcriptome of “Huajin 6” was determined using Pac Bio Sequel system,and used as reference for the subsequent RNA-seq data analysis.Transcriptome in flower buds at five flowering stages of “Huajin 6” was measured using Illumina Hiseq system.Sequences were analyzed using a serial of bioinformatics tools,and transcription difference of thousands of transcripts were revealed and categorized into different groups according to their functional annotation such as MYB transcription factors.LC-MS/MS analysis were used to determine metabolite contents in flower buds.Overexpression vectors of several MYBs were constructed using gateway cloning technology.Results: 1.The full-length transcriptome of Lonicera japonica were obtained and annotated using seven databases including NCBI non-redundant protein sequences(Nr),NCBI nucleotide sequences(Nt),Protein family(Pfam),Clusters of Orthologous Groups of proteins/eu Karyotic Ortholog Groups(COG/KOG),Swiss-Prot,Kyoto Encyclopedia of Genes and Genomes(KEGG),Gene Ontology(GO).In total,97588 genes were identified,each of which may have different numbers of transcripts.Unigene sequence analysis revealed coding sequences of proteins,long non-coding RNA(lnc RNA)and simple sequence repeats(SSR)markers.Transcription factors were predicted using iTAK software and categorized into different groups.2.Based on the full-length transcriptome of “Huajin 6”,we investigated transcriptome profiles in flowers of five different stages and identified thousands of genes differentially expressed.Genes with various transcript abundance at stage 2 to stage 5 compared to stage1 were categorized into different biological processes,cellular compartments and molecular functions.In order to understand the regulation mechanism of chlorogenic acid biosynthesis in Lonicera japonica flowers,we isolated homologous genes in chlorogenic acid biosynthetic pathway,some of which had differential expression at different stages.3.MYB transcription factors plays important roles in the regulation of biosynthesis of secondary metabolites such as anthocyanin and flavonoids.We isolated all MYB and MYB-related transcript factors and analyzed their transcript abundance at five different stages.Correlation analysis revealed several MYB transcription factors associated with the expression of HQT genes which catalyze the last step of chlorogenic acid biosynthesis.4.The allocation of primary metabolites is important for the accumulation of secondary metabolites.Thus,we analyzed the accumulation of primary metabolites in flower buds at different stages.The fluctuation of primary metabolite contents suggested different accumulation of chlorogenic acid at different stages.Conclusion: We conducted the full-length transcriptome and RNA-seq analysis of flower buds at five different stages,and isolated MYB transcription factors and homologous genes in chlorogenic acid biosynthesis pathway.The relationship between MYB transcription factors and homologous genes remains to be further analyzed.We also analyzed the accumulation of primary metabolites in flower buds of different stages.All of my work provide the platform to analyze the gene function in Lonicera japonica andelucidate the regulation mechanism of chlorogenic acid biosynthesis,which is helpful to improve the quality of Lonicera japonica.