Functional Characterization Of Anthocyanin Synthase CpANS1 And Transcription Factor CpMYB2 In Chimonanthus Praecoxtepals

Wintersweet(Chimonanthus praecox L.),with an over-one-thousand-years long history in cultivation,is still a popular ornamental woody plant in China.The tepals of wintersweet flower are waxy in nature and the overall color of the flower is determined by the yellow middle tepals,and other colors have been unreported yet.At present,the study of color formation of wintersweet mainly focused on the identification and isolation of the pigments.Although the pigment compounds were different among varieties,the flavonoids were the predominant compounds in wintersweet tepals.As an important landscape plant,the flower color of wintersweet is an important ormamental trait.Therefore,it is of great significance to study the molecular mechanism of color formation biosynthesis in wintersweet.This study has laid a molecular foundation for the biosynthesis of pigments in the wintersweet and provided a theoretical basis for revealing the regulatory network of color formation.In this study,the wintersweet varieties with different inner tepals were taken as the research materials.Various omics tools were used to detect the genes related color formation.At the same time,the function and regulation mechanism of CpANS1 and transcription factors CpMYB2,which were both important to anthocyanin biosynthesis,were investigated.This study mainly includes three parts:the first part was the screen out the candidate genes responsible for different color biosynthesis,the second part was the isolation and functional analysis of CpANS1,the third part was the isolation and functional analysis of CpMYB2.The main results are as follows:1.The identification of genes related to flavonoids synthesis in wintersweet.By constructing transcriptome and proteome databases of H29 and H64,134 unigene and 30 protein sequences related in flavonoids synthesis were isolated.XAS functions as an enzymatic switch-on-off for anthocyanin biosynthesis.The expression pattern of FLS was positively correlated with that of which means that the biosynthesis of anthocyanins and flavonols does not have a competition mechanism for common substrates.2.The functional analysis of CpANS1.The expression level of CpANS1 gene was analyzed by real-time PCR,the result showed that CpANS1 was barely expressed in yellow tepals,and this gene responded to cold stress.We over-expression introduced CpANS1 into Arabidopsis thaliana tds4-2 mutant.CpANS1 can functionally complemented the tds4-2 mutant plants,which has recovered PAs and anthocyain accumulation in seed coats.And restoring the synthesis of anthocyanin in the vegetative tissue,just like young leaf epidermis,epicotyl,stem and siliques.So that has proved the CpANS1 had the ability to catalyze the substrate leucocyanidin to generate anthocyanin.3.The functional analysis of CpMYB2The CpMYB2 gene clustered into the 6th subgroup of Arabidopsis MYB family,of which MYBs has been experimentally proved to be related to anthocyanin synthesis.By gene expression analysis,we found the expression pattern of CpMYB2 was consistent with that of CpANS1.Double luciferase assay showed that CpMYB2 gene eould slightly induce the promoter activity of CpANS1.CpMYB2 protein possessed transcriptional activity,and transcriptional activation domain is located at the C-terminal.Yeast two-hybrid assay showed that CpMYB2 could interact with two types of bHLH:CpbHLH1 and CpbHLH2,which related to antocyanin synthesis.GUS enzyme activity assay showed that co-expression of bHLH2 protein with CpMYB2,the promoter activity of CpANS1 was increased.After capillary electrophoresis sequencing,we found that CpMYB2a/b/c/d/e/f total 6 alleles present in 87 wintersweet varieties.These alleles(CpMYB2 are due to the different number of 3-base repeats(GGA)sites in CpMYB2 C-terminal.Whlie the allele CpMYB2f are due to the 4 bp AAAG deletion mutation in CpMYB2 C-terminal.And only homozygous CpMYB2ff can be deceted in concolor wintersweet varieties.