Study On MicroRNA Sequencing And Its Function Prediction Of Regulatory Anthocyanin Synthesis In Flowering Process Of Lonicera Japonica Thunb

Lonicera japonica Thunb.is used as an important medicinal plant mainly due to its dried buds or primordial flowers.In China,L.japonica has been known for its wide range of medicinal uses since ancient times,its effects are mainly antipyretic,antibacterial and anti-inflammatory,etc.Micro RNAs(miRNAs),a class of small noncoding RNAs,play important roles in the growth and development of plants.Currently,studies have been well reported on L.japonica at the genomic,metabolomic,transcriptomic,and protein levels,however,little has been reported on posttranscriptional modifications during L.japonica development and the regulatory functions of miRNAs.In this study,miRNA sequencing was performed in five stages of L.japonica: young alabastrum stage,green alabastrum stage,whole white alabastrum stage,silvery flower stage,golden flower stage,and combined with L.japonica transcriptome and metabolome studies,miRNAs and their target genes in different flowering stages of L.japonica were analyzed and identified,and the regulatory functions of miRNAs and target genes were predicted.The results of the specific experimental analysis are as follows:(1)MiRNA and target gene identification: 425,426,487,524,489 miRNAs were identified in the young alabastrum stage,the green alabastrum stage,the whole white alabastrum stage,the silvery flower stage,and the golden flower stage,respectively,of which 44 new miRNAs were identified,and 326 miRNAs were co-identified in the five periods.The maturation lengths of all new miRNAs in L.japonica were analyzed,and the maturation sequence lengths were distributed in 21 nt,22 nt,and 24 nt.234 miRNAs with significant differences in expression between young alabastrum stage and other stages in L.japonica were predicted for target genes,and 2678 target genes were obtained,and the expression patterns of miRNAs and their target genes were correlated by combining L.japonica transcriptome data Finally,83 miRNAs and 182 target genes were obtained.(2)Analysis of the regulatory effect of miRNA on the flowering period of L.japonica: correlation analysis of miRNA and target gene expression patterns revealed that Car-miRN777 regulates the most target genes,mainly involved in RNA,protein,lipid metabolism,transport,and secondary metabolism,among which target genes ELF7 and CPK33 are involved in flowering regulation,indicating that Car-miRN777 may be involved in flowering regulation by targeting ELF7/CPK33,suggesting that Car-miRN777 may be involved in the regulation of flowering development by targeting ELF7/CPK33.Functional clustering of target genes revealed that the miR319 familyregulated target genes MYB33 and TCP could regulate hormone levels in petals and anthers,suggesting that some members of the miR319 family may influence flower development by regulating MYB33/TCP during L.japonica flowering.The expression patterns of randomly selected miRNAs and their target genes at five periods were analyzed using real-time fluorescence quantitative PCR,and the quantitative results were consistent with the analytical results.(3)Analysis and prediction of the relevance of miRNA on the regulation of secondary metabolism in L.japonica: metabolomic integration analysis of five periods of L.japonica revealed that the target gene of Hsy-miR160 I and Arogenate Dehydrataseand(ADT),were mapped to the phenylalanine synthesis pathway,catalyzing the last step of phenylalanine biosynthesis.In addition,the expression of Hsy-miR160 I and ADT gradually decreased during the flowering process of L.japonica,and the content of related metabolites also gradually decreased during the flowering process of L.japonica.It indicates that Hsy-miR160 I has the potential to control the content of downstream secondary metabolites by regulating the upstream phenylalanine flux during different flowering stages of L.japonica.The prediction analysis of transcription factors and their binding sites showed that Hbr-miR482 d could target MYB1,while MYB1 binding sites were present in the dihydroflavonol 4-reductase(DFR)promoter sequence.real-time fluorescence quantitative PCR of Hbr-miR482 d,MYB1,DFR and the determination of anthocyanin content indicated that during L.japonica flowering Hbr-miR482 d may induce anthocyanin accumulation by positively regulating the binding of transcription factors MYB1 and DFR genes.In this study,miRNAs and their target genes were identified and predicted to regulate flower development and secondary metabolism,especially anthocyanin-like synthesis,during L.japonica flowering,through miRNA sequencing and integration of L.japonica transcriptome and metabolome data,and the potential mechanism of miRNA regulation of secondary metabolite accumulation during L.japonica flowering was preliminarily revealed;this study has laid a certain foundation for further revealing the regulatory mechanism of L.japonica growth and development and improving the content of L.japonica secondary metabolites..