Molecular Cloning and Sequence Analysis of C4H Gene of Mangifera indica L.

摘要

Cinnamate4hydroxylase (C4H) is a key enzyme in phenylpropanoid pathway in plants. Its activity and abundance directly affect the biosynthesis of flavonoids and aromatic compounds. In this study, degenerate primers were designed according to previously reported C4H gene sequences to clone C4H cDNA sequence with 3 and 5 RACEPCR from mango (Mangifera indica L). The fulllength cDNA of M. indica C4H is 1 680 bp long. Its open reading frame (ORF) is 1 518 bp, encoding a protein of 505 amino acids with a predicted molecular weight of 58.08 kDa. The isoelectric point of the predicted protein is 9.52. Functional prediction showed that this gene is mainly located in mitochondria. In addition, the tertiary structure of the protein was built using SWISSMODEL, and the results showed that the protein has three possible conformations. Phylogenetic analysis based on C4H protein sequences revealed that M. indica has a close genetic relationship with olive (Canarium album) and cocoa (Theobroma cacao). By analyzing the expression level of C4H gene in three colored mango cultivars, we found that that the expression level of C4H gene in Guifei (with red peel) was the highest, and that in Guiqi (with green peel) was the lowest. The results provide a theoretical basis for studying the molecular mechanism of anthocyanin biosynthesis and C4Hs impact on the color of mango fruit.