Screening And Functional Analysis Of The Key Structural Genes For Color Rendering Of Rhododendron Agastum

Rhododendron agastum(R.agastum)is a shrub or small tree of Rhododendron subgenus of Rhododendron in Rhododendron family,mainly distributed in eastern Guizhou.It is one of the most important constructive species and dominant species of Baili Rhododendron National Forest Park.The flowers of R.agastum are large and bright,with great ornamental value.At the same time,the R.agastum flower is pink and rich in anthocyanins,which is a natural pigment resource worth studying and has certain development and application value in food industry.Flower color is one of the important characteristics of ornamental plants,which is mainly determined by three types of pigments:carotenoids,betaine and flavonoids.Studies have shown that the main substance that determines the color of R.agastum flowers is anthocyanins,which can impart pink,red,purple,and other colors to plants and is particularly important for the fineness of tissues such as flowers and fruits,and also can greatly improve the ornamental value of flowers as well as the taste and flavor of fruits.In the biosynthesis pathway of anthocyanins,dihydroflavonol 4-reductase(DFR)is located in the middle,which can catalyze the reduction of three dihydroflavonols to corresponding leucoanthocyanidin.Due to the selective catalysis of three dihydroflavonols,DFR plays an important role in determining the type of anthocyanins in plants.Flavonoid 3-O-glucosyltransferase(3GT)is located downstream of anthocyanin pathway,which glycosylates anthocyanidins to form anthocyanins.It plays a key role in enhancing the stability,water solubility and transportability of anthocyanins,directly affecting the presentation of plant color.In this study,the kinds and content of anthocyanins from R.agastum(pink)and Rhododendron irroratum(white)flowers were measured firstly to clarify the variation rule of anthocyanins during flowering process of R.agastum.Meanwhile,on the basis of the transcriptome sequencing analysis of both flowers,two structure key genes(RaDFR1 and Ra3GT2)that regulate the color rendering of R.agastum flowers were selected through the correlation analysis between anthocyanin variation and expressions of structural gene,then their functions were identified.The research results are as follows:1.Screening of key structural genes for color presentation in R.agastumFirstly,the anthocyanins in flowers of R.agastum and R.irroratum were qualitatively analyzed.The results showed that a total of 7 anthocyanins were detected in the flowers of R.agastuYyi,while no anthocyanins were detected in R.irroratum.Subsequently,the flower development process of the R.agastum was divided into four stages,and quantitative analysis of total anthocyanins during flower development process was completed by using the external standard curve method.It was found that the accumulation level of total anthocyanins gradually decreased with the opening of the R.agastum.According to the results of qualitative and quantitative analysis of anthocyanins,the metabolic pathway of anthocyanins in the flowers of R.agastuYyi was preliminarily determined.Then,based on the transcriptome sequencing results,RaDFRl and Ra3GT2,two key structural genes that regulate the color of R.agastuYyi flowers were screened through the correlation analysis between gene expression and total anthocyanin content.2.Cloning and bioinformatics analysis of RaDFR1 and Ra3GT2The cDNA sequence of RaDFRl was successfully cloned using RT-PCR.But the 5’ end of Ra3GT2 sequence measured by the transcriptome was too short to design primers for cloning,so it was sent to the company for synthesis.The open reading frame lengths of RaDFRl and Ra3GT2 were 1035 bp and 1395 bp,encoding 344 and 464 amino acids,and the predicted protein sizes were 38.34 kDa and 50.31 kDa,respectively.Both RaDFRl and Ra3GT2 are hydrophilic proteins and have no signal peptides.There are highly conserved NADPH binding sites and substrate binding regions at the N-terminal of RaDFRl,and the phylogenetic tree shows that it is closely related to NtDFR.Ra3GT2 belongs to the GT-B superfamily of glycosyltransferases,containing the PSPG conserved domain unique to the glycosyltransferases,as well as has two catalytic active sites.It is closely related to Rd3GT1 of Rhododendron delavayi.3.Eukaryotic expression vectors construction and genetic transformation ofRaDFRl and Ra3GT2Using pBI121 as an overexpression vector,the overexpressed recombinants of RaDFR1 and Ra3GT2 were constructed and transformed into Agrobacterium tumefaciens GV3101 to infect Arabidopsis thaliana tt3 and ugt78d2 mutants,respectively.The results showed that both RaDFR1 and Ra3GT2 could restore the anthocyanins synthesis of mutant.To verify the effect of two genes on flower color formation,they were also overexpressed in tobacco.The results displayed that the petal color of transgenic tobacco was deepened to varying degrees,and the anthocyanins in the petals of Ra3GT2 overexpressing tobacco increased from 2 to 3,indicating that both genes have the function for regulating anthocyanin synthesis and can be used for improving plant flower color.4.Construction of prokaryotic expression vectors of RaDFRl and Ra3GT2 and detection of enzyme activity in vitroThe prokaryotic expression vectors TpET32a(+)-RaDFRl and pET32a(+)-Ra3GT2 were successfully constructed and expressed inductively in E.coli strain BL21(DE3).The recombinant protein was prepared and the enzyme activity was detected.The results showed that RaDFRl could catalyze dihydroquercetin and dihydromyricetin to produce corresponding products,but could not catalyze dihydrokaempferol,indicating that this specific selection of substrates may be the direct reason for the absence of pelargonidin anthocyanins in R.agastuYyi.Ra3GT2 can catalyze the formation of corresponding glycosides from pelargonidin,cyanidin,delphinidin,peonidin,petunidin,malvinidin when using UDP-galactose as a sugar donor.When UDP-glucose is used as the sugar donor,it can catalyze the formation of corresponding glycosides from cyanidin,delphinidin,and malvinidin.The recombinant Ra3GT2 protein cannot undergo glycosylation reactions using UDP-arabinose and UDP-rhamnose as glycosyl donors.At the same time,the recombinant Ra3GT2 protein can also catalyze the formation of corresponding glycosides from quercetin,myricetin,and kaempferol using UDP-galactose and UDP-glucose as sugar donors.The catalytic efficiency analysis revealed that whether Ra3GT2 catalyzed anthocyanins or flavonols,the catalytic efficiency of UDP-galactose was higher than that of UDP-glucose.Therefore,the substrate preference of Ra3GT2 directly affects the anthocyanins diversity in flowers of R.agastumn.