Clone,Expression Patterns Of Key Structural Genes In Anthcyanin Synthesis Pathway Of Nelumbo Nucifera

Nelumbo nucifera is an aquatic plant,belonging to Nelumbo of Nelumbonaceae,also known as one of the top-ten traditional famous flowers in China,which is of high ornamental value because of its grateful gestures and wonderful flower colors.Furthermore,lotus is popular for its medicinal value of roots,leaves and petioles and edibleness of rhizomes.Among different flower color cultivars of lotus,the pink flower color cultivars account for a large part,generating drab colors of this species.So far,molecular breeding of flower color for lotus is difficult to have further development because there are few researches doing for flower color formation and its regulatory mechanism is still unclear.In this study,the petals from red,pink,white and yellow flower color cultivars,respectively,are objects.At the first of all,the apparent traits and total pigment content of petals were determined.And then,we clone 7 key structure genes and analyze their expression level in petals of different flower development stages(bud stage,initial stage,full-opening stage and wilting stage)from different cultivars,and tissues(petal,carpel,stamen,petiole and leaf)with the same variety to illuminate regulatory mechanism in anthocyanin synthesis pathway,which would provide important theoretical basic for molecular breeding of flower color improvement and innovation.1 Apparent traits analysis and pigments content determination in petals of different flower development stages from four cultivarsAccording to the results,total anthocyanin content changed with the turn of apparent traits in petals of red and pink cultivars while petals of white cultivars had hardly any anthocyanin,which implied content of anthocyanin plays an important role in flower color formation of red and pink lotus cultivars.Furthermore,we supposed that the accumulation of total flavoniod relate to yellow color formation because there was hardly any anthocyanin but the highest total flavoniod content of yellow cultivar in four cultivars.2 Clone and sequence analysis of key structural genes in anthocyanin biosynthetic pathway7 key structural genes in anthocyanin biosynthetic pathway were isolated from lotus by gene clone.NnCHS,NnCHI,NnF3H,NnF3’H,NnF3’5’H,NnDFR,NnANS were recognized respectively through sequence alignment.The open reading frame(ORF)of NnCHS is 1170bp,coding 389 amino acids.The ORF of NnCHI is 630 bp,coding 209 amino acids.The ORF of NnF3H,NnF3’H,NnF3’S5’H are 996 bp,1542 bp and 1524 bp,respectively,coding 331,513 and 507 amino acids apart.The ORF of NnDFR and NnANS are 1029 bp and 1080 bp,coding 342 and 389 amino acids separately.Homology analysis showed that CHS,CHI,F3’H,F3’5’H and ANS shared high homology with other species while F3H and DFR had low homology with other species.Phylogenetic analysis based on amino acid sequence indicated F3H and ANS have distant relationship with other species.Furthermore,bioinformatics was utilized to analyze protein physicochemical property,domain and secondary structural.The results show that only F3,H is hydrophilic and CHS,CHI,F3H,DFR,ANS are acidic.CHS,CHI,F3,H and DFR belong to stable proteins.Secondary structural of 7 proteins consist of alpha helix,extended strand,beta turn and random coil.Moreover,7 proteins in this study have relevant structural domain.3 Expression patterns of 7 key structural genes in anthocyanin synthesis pathwayThe most suitable reference genes in gene expression research of different flower development stages from four color cultivars of lotus are ACT and EFla through stability evaluation of 8 housekeeping genesby qPCR.Based on the selection of suitable reference genes,we detected the expression level of 7 structural genes in anthocyanin synthetic pathway of lotus.Results show that Expression level of the genes all gradually increased with the development periods reaching peak in the full-opening stage and then decreased in petals of red cultivar.Whilein pink cultivar,there is little expression of F3 ’H during flower development stages and expression level of other genes existed and gradually decreased.In the petals of white cultivar,it hardly detected the expression of DFR and ANS,while in yellow cultivar DFR had hardly any transcription but CHI expressed exceptionally high level in bud stage.Besides,expression levels of 7 structural genes in petals were higher than other tissues.In conclusion,the transcription level of CHI,F3’H,DFR,ANS played an important role in anthocyanin synthesis and accumulation which might lead to different flower colors of lotus.