Cloning, bioinformatics, and expression analysis of MpF2H gene in Microcos paniculata / 中草药

ABSTRACT

Objective To obtain the key enzyme gene involved in flavone C-glycosides biosynthesis pathway, a flavanone 2-hydroxylase (F2H) gene was cloned from Microcos paniculata, and its bioinformatics analysis and gene expression pattern were also performed. Methods The specific primers were designed according to Unigene in F2H annotated in the transcriptome data of M. paniculata. The open reading frame (ORF) of MpF2H gene was amplified by PCR. Then the PCR product was purified and ligated to pET30a, and finally a prokaryotic expression vector pET30a-MpF2H was constructed. The bioinformation of F2H gene cDNA sequences was analyzed by some online tools. Using RT-qPCR with suitable primers, the quantitative expression analysis of MpF2H gene in different tissues, namely, buds, leaves, twigs, flowers and fruits was carried out. Results The length of MpF2H gene ORF was 1 557 bp (GenBank accession number KY652921), which encoded a protein with 518 amino acid residues, relative molecular weight of 54 500, theory isoelectric point of 5.49. In which was no transmembrane domain. It was hypothesized that this protein located in chloroplast. MpF2H gene was expressed in different tissues, with the highest expression in leaves and the lowest expression in twigs and flowers. Conclusion The expression of MpF2H gene varied widely in different tissues. The MpF2H gene was cloned from M. paniculata based on pET30a-MpF2H expression vector. This study will provide the fundamental information for the further preparation and functional research of MpF2H protein in flavone C-glycosides biosynthesis pathway.