Research On Anthocyanin Related Genes And Its Relationship With Anthocyanin Content In Mulberry Fruits

Anthocyanins are widely distributed in nature and the main water-soluble pigments in plants.As a natural food pigments,anthocyanins are safe,non-toxic and have a certain nutritional and pharmacological effects which has great application potential in food,cosmetics and medicine.As an important cash crop,there has thousands years of mulberry planting history,which can not be the silkworm food but has important medicinal value.Mulberry fruits containing plenty of anthocyanins and is beneficial to human body.From the former studies,we had studied total flavonoids content,anthocyanins content,compounds isolation and purification.In this experiment,Chalcone isomerase(MmCHI),Anthocyanidin synthase(MmANS)and Dihydroflavonol 4-reductase(MmDFR)were cloned from mulberry and analysed the expression characteristics of the three genes and the former obtained genes Chalcone synthase(MmCHS),Flavonol synthase(MmFLS),Flavanone 3β-hydroxylase(MmF3H)and Cinnamate-4-hydroxylase(MmC4H)in different mature mulberry cultivars and different stages of mulberry fruits development.This study laid basis on mulberry color of molecular mechanism,and had some theoretical guiding significance in the later research.The main research results are as follows:1、Based on the nucleotide sequence of Chalcone isomerase(CHI),Anthocyanidin synthase(ANS)and Dihydroflavonol 4-reductase(DFR).Primers of three genes were designed.All the PCR products were ligated to pMD19-T vector and then transformed recombinant vector into E.coli Top10 strain.The clone results were checked by PCR and cultured the right strains to sent for sequencing.We obtained the correct sequence of CHI,ANS and DFR from experiment,and called them MmCHI(GeneBank:MK287781),MmANS(GeneBank:MK287780)and MmDFR(GeneBank:MK287779)respectively.The ORF sequence length of MmCHI gene was 519 bp,encoding 172 amino acids with predicted protein molecular weight was 19.7 kD and isoelectric point was 5.5.The sequence length of MmANS gene ORF region was 1 077 bp,encoding 358 amino acids with predicted protein molecular weight was 42.23 kD and isoelectric point was 5.25.The ORF sequence length of MmDFR gene was 1 011 bp,encoding 336 amino acids with predicted protein molecular weight was 39.51 kD and isoelectric point was 5.34.Amino acid sequence alignment and Phylogenetic tree were constructed of three genes from Morus multicaulis and other plant species.The results indicated that the amino acid sequences of MmCHI,MmANS and MmDFR had highly similarity with other plants,especially Morus notabilis,Rosaceous plants.2、In order to know the relationship between mulberry anthocyanin content and expression level of related genes,the fluorescence quantitative PCR and solvent extraction were used to detect 7 related genes(CHI、ANS、DFR、C4H、CHS、FLS、F3H)expression level and the anthocyanins content of seven mulberry cultivars(GHM,BYW,SZ2,HG2,DS,M4,SZ1).From results we can see related genes expression level had great relationship with anthocyanin content and had some influence in forming the mulberry fruits color in anthocyanin synthesis pathway,that means genes expression was positively correlated with the content of anthocyanin.3、In this study,qRT-PCR and solvent extraction methods were used to analyze the relationship between mulberry anthocyanin content and related genes expression level in anthocyanin synthesis pathway during two different cultivars development.The results showed that anthocyanins content were increased by fruit ripening.There are positive correlation in genes expression level and anthocyanins content in anthocyanin synthesis pathway and will make the appearance of mulberry color deeper.