Cloning And Functional Research Of An Anthocyanin Regulating Gene MwMYB4L From Magnolia Wufengensis

Magnolia wufengensis L.Y.Ma et L.R.Wang is a rare ornamental tree with abundant color variations.However,the mechanism of flower color regulation is still unclear.Previous transcriptome data and phenotypic data analysis illustrated that a MYB family gene MwMYB4L showed significant correlation with the change of M.wufengensis color,suggesting that it may be a key gene in the regulation of M.wufengensis anthocyanin biosynthesis.In this study,MwMYB4L gene was cloned from M.wufengensis 'Jiaohong No.1'.Phylogenetic analysis showed that MwMYB4L belonged to R2R3-MYB subfamily,and amino acid sequence structure analysis further showed that it had typical sequence structure characteristics of R2R3-MYB subfamily.Subcellular localization revealed that the gene was only expressed in the nucleus,suggesting that it might be a transcription factor.Tissue-specific expression analysis showed that the gene was expressed in both leaf and flower organs.In different development stages of flower organ,MwMYB4L showed high expression in early stage(stage S1?S3),while in late stage of flower development(S4-S5 stage,with gradually deepening flower color)the MwMYB4L showed a down-regulated trend.To further reveal the function of MwMYB4L in regulating metabolic pathway of flower color,it was transformed into Arabidopsis thaliana for further analysis.The morphological results of transgenic A.thaliana showed that the gene played a negative role in anthocyanin synthesis and inhibited the development of trichome and root hair.The expression of related genes in the anthocyanin metabolism pathway of transgenic A.thaliana showed that the gene might reduce the accumulation of anthocyanin in A.thaliana by down-regulating the expression of CHI,DFR,ANS and ANR(BAN).This study comprehensively analyzed the function of MwMYB4L in the anthocyanin biosynthesis pathway and laid an important theoretical foundation for the research on the anthocyanin regulation mechanism for M.wufengensis.In addition,it also provided important genetic resources and guidance for the molecular breeding of M.wufengensis in the future.