Study On The Regulation Genes Related To Anthocyanin Synthesis In The Fruit Of Lonicera Edulis

The fruit of Lonicera edulis is rich in anthocyanins,which are important secondary metabolites in plants and have antioxidant activity.They can scavenge free radicals and anti-tumor,which is beneficial to human health.Therefore,the fruit of Lonicera edulis has both medicinal value,nutritional value and edible value.To study the molecular regulation mechanism of anthocyanin synthesis during fruit ripening,it can provide a theoretical basis for cultivating and screening Lonicera edulis varieties with high content of anthocyanins.In this study,different cultivars of Lonicera edulis were used as materials to analyze the mechanism of anthocyanin accumulation during mulberry fruit ripening from physiological,molecular and bioinformatic analysis.The following results were obtained:The content of anthocyanins in Lonicera edulis of the two varieties of "6" and "9" were detected by solvent extraction and pH differential method.The results showed a trend of increasing with the ripening of the fruit,and the anthocyanins content of the two varieties at maturity were: 253.73 mg/100 g and 75.24 mg/100 g,respectively."No.6" is higher than "No.9".Combined with the expression level of structural genes and the accumulation of anthocyanins,the enzymes of ANS and UFGT,which play a key role in the accumulation of anthocyanins.Enzyme activity detection of ANS and UFGT was performed using an ELISA kit.The results showed that ANS enzymes maintained high activity in different varieties of Lonicera edulis.The expression levels of MYB,bHLH and WD40 in different stages of fruit ripening of "No.6" and "No.9" were analyzed by qRT-PCR.The results showed that the relative expression of MYB in the "6" and "9" varieties decreased first and then increased.The overall expression trend was opposite to the accumulation trend of anthocyanins,which may inhibit the accumulation of anthocyanins.The relative expression of bHLH in the two varieties at different periods showed a pattern of decreasing first,then rising and then decreasing.The expression level of WD40 was up-regulated in the early stage to the maturity stage of the two varieties except for the“No.9” three stage,indicating that WD40 promoted the accumulation of anthocyaninsas the fruit matured.The results indicated that the high-efficiency expression of most of the regulatory genes was in the stage of coloring to maturity of the Lonicera edulis.Primers were designed based on the known gene-related gene sequences.Primers were designed based on the known gene-related gene sequences,and the complete CDS region cloning and bioinformatics analysis of the anthocyanins synthesis-related regulatory gene WD40 were performed.Physical and chemical analysis showed that the nucleic acid sequence was 1230 bp in length and encoded 409 amino acids,belonging to hydrophilic and extramembranous proteins.Sequence analysis and homology alignment confirmed that the cloned sequence was WD40.miRNA sequencing and bioinformatics analysis of fruits of "No.6" variety by high-throughput sequencing.A total of 507 miRNAs were identified,of which 16 were new and 491 were conserved,and 9 miRNAs related to anthocyanin synthesis were screened.qRT-PCR quantitatively analyzed miRNAs with high expression levels and their target genes.The study found that miR156h-3p and miR396a-3p were negatively correlated with the expression of target gene UFGT,and further clarified the key miRNAs involved in anthocyanin synthesis in Lonicera edulis.