Cloning,Sequence Analysis and Interaction Analysis of MiUFGT43 Promoter and MiMYB1 Genes in Mango Fruit
Flavonoids 3-O-glycosyltransferase(UFGT)and MYB TF are the key regulators of anthocyanin bio-synthesis.The purpose of this study was to clarify the sequence characteristics and interaction between MiUFGT43 promoter and MiMYB1 gene and provide theoretical basis for color regulation of postharvest mango.MiUFGT43 promoter and MiMYB1 gene sequence were cloned from mango transcriptome and genome data,and the relative expression levels of MiMYB1 and MiUFGT43 were detected by RT-qPCR.Bioinformatics was used to predict the core region,transcription initiation site and cis-acting element of MiUFGT43 promoter,the domain and physico-chemical properties of MiMYB1.Construction of phylogenetic tree using MEGA-X.The expression vectors pHis-Mi-UFGT43-pro and MiMYB1-AD were constructed and transferred into Y1H Gold strain.The interaction between MiUFGT43 promoter and protein MiMYB1 was verified by Y1H experiment.The MiUFGT43 promoter contains a core region at-532～-582 bp,transcription initiation site G and multiple MYB binding sites.MiMYB1 gene with MYB domain CDs full length 846 bp encoding 282 amino acids,and the encoded protein is an unstable hydrophilic protein.Phylogenetic analysis showed that PvMYB1 had the highest homology with MiMYB1.The expression of MiMYB1 increased during storage,but decreased on the 12th day.The expression of MiUFGT43 increased on the 6th day,then decreased significantly on the 12th day and then increased.The concentration of 3-AT which inhibited the self-activation of MiUFGT43 promoter was 10 mmol/L.Y1H results showed that the interaction between DNA binding protein MiMYB1 and its target sequence in MiUFGT43 promoter could stimulate the transcription of His.This study confirmed that MiMYB1 can activate the transcription of MiUFGT43 by directly binding to MiUFGT43 promoter,thus affecting the anthocyanin synthesis of postharvest mango.
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