Cloning and identification of the anthocyanin-regulated MsMYB44-likes genes in crabapple

Objective This study aimed to clone and identify the transcription factors （MYB44-likes） that related to the anthocyanin biosynthesis in crabapple， with the goal of providing a reference for elucidating the regulatory mechanism MsMYB44-likes genes in the leaf coloring process of crabapple and improving the ornamental traits of crabapple.Method Using the crabapple cultivar Malus spectabilis as the research object， tissue-cultured seedlings of Malus spectabilis were sprayed with 25 μmol/L abscisic acid（ABA）treatment， while the seedlings sprayed with water as the control （CK）. Three MsMYB44-likes genes in the tender leaves of Malus spectabilis were cloned by PCR （MsMYB44-like1， MsMYB44-like2， and MsMYB44-like3）， respectively. Bioinformatics analysis， encoded protein subcellular localization and phylogenetic analysis were performed on these three MsMYB44-likes genes. Transient overexpression analysis of the MsMYB44-like2 gene was conducted in apple epidermis， and the binding of MsMYB44-like2 protein to the promoter of the structural gene UFGT in the anthocyanin biosynthesis pathway was studied using electrophoretic mobility shift assay （EMSA）.Result Three MsMYB44-likes genes （MsMYB44-like1， MsMYB44-like2， and MsMYB44-like3 ） were cloned， whose full-length cDNA sequences of were 954， 960 and 942 bp， and they encoded 317， 319， and 313 amino acid residues， respectively. In the three MsMYB44-likes protein structures， α-helix and random coil accounted for relatively high proportions. The protein sequences of MsMYB44-like1， MsMYB44-like2， MsMYB44-like3 and the three proteins shared typical MYB-DNA binding domains with protein sequences of Arabidopsis thaliana， wheat， rice， and pear. MsMYB44-like1 and MsMYB44-like2 clustered with the homologous proteins of pear into one clade. Three MsMYB44-likes proteins were localized in the nucleus. The transient transformation experiment showed that after injecting the Agrobacterium suspension contained pCAMBIA2300-MsMYB44-like2， the area around the injection point on the apple epidermis turned yellowish-green， indicating that the gene inhibited the anthocyanins biosynthesis of the apple epidermis. Compared with the leaves of CK， the relative expression of the MsMYB44-like2 gene in leaves after ABA treatment decreased significantly （P<0.05）. The results of EMSA showed that the binding of MsMYB44-like2 protein to the promoter fragment of the MsUFGT gene led to increased mobility.Conclusion The three MsMYB44-likes proteins contain typical MYB-DNA binding domains and are localized in the nucleus. The expression of the MsMYB44-like2 gene is negatively regulated by ABA， and the protein of this gene suppresses anthocyanin biosynthesis in crabapple by directly binding to the promoter of the gene MsUFGT.