Isolation And Functional Characterization Of MYB Genes Related To Anthocyanin Biosynthesis In Rhododendron Delavayi

Rhododendron delavayi is one of the world’s famous ornamental plants.Widely distributed in southwest China,it is an important dominant species in nature reserves.The flowers of R.delavayi are red in color,and the inflorescence consists of more than10 small flowers and the branches are also red.We used bioinformatics to analyze the MYB gene family and transcriptomic data of R.delavayi to investigate the molecular basis of the formation of floral color glycosides.At the same time,we constructed a genetic transformation system to homologously transform the target genes and verify the gene functions.The main results are as follows:（1）184 MYB genes were identified based on the released genome of R.delavayi.These genes included 78 1R-MYB,101 R2R3-MYB,4 3R-MYB,and 1 4R-MYB.The RdMYBs were divided into 35 subgroups using phylogenetic analysis of the MYBs of Arabidopsis thaliana.The members of the same subgroup in R.delavayi had similar conserved domains and motifs,gene structures,and promoter cis-acting elements,which indicate their relatively conserved function.（2）Transcriptome based on unique molecular identifier strategy and color difference of the spotted petals,unspotted petals,spotted throat,unspotted throat,and branchlet cortex were detected.Results showed significant differences in the expression levels of R2R3-MYB genes.Weighted co-expression network analysis between transcriptome and chromatic aberration values of five types of red samples showed that the MYBs were the most important TFs involved in the color formation,of which seven were R2R3-MYB,and three were 1R-MYB.Two R2R3-MYB（DUH019226.1and DUH019400.1,were named RdMYB8 and RdMYB24）had the highest connectivity in the whole regulation network,and they were identified as hub genes for red color formation.（3）The optimum transformation conditions for agrobacterium tumefaciens infestation of leaf of R.delavayi were pre-culture time 0 day,agrobacterium tumefaciens concentration OD₆₀₀=0.7,infestation time 60 min,As concentration 20 mg/L,total incubation time 6 days;After decolonization,the first screening culture was incubated for 30d in medium supplemented with Kan 30 mg/L、Cef 500 mg/L;For the second screening culture for 60d,the medium was supplemented with Kan 30 mg/L、Cef 400mg/L;For the third screening culture for 90d,the medium was supplemented with Kan40 mg/L、Cef 300 mg/L.Identification of positive transgenic material callus after 120 d of incubation.（4）RdMYB8 and RdMYB24 genes were cloned and overexpression vectors were successfully transformed into agrobacterium tumefaciens GV3101 to infest leaf of R.delavayi,and transgenic positive material was successfully obtained.Compared with the control,the transgenic healing tissues became red and the content of total anthocyanin increased.The results indicated that RdMYB8 and RdMYB24 genes were involved in the synthesis of anthocyanin in R.delavayi.