Cloning, Analysis, and Expression of Flavonoid 3'-hydroxylase Gene inEuphorbia maculata

Objective Expressions of flavonoid 3'-hydroxylase gene (F3'H) and promoter in organs of Euphorbia maculata at growth stages were analyzed to study the related biosynthetic pathways in the plant.

Method Based on the transcriptome sequences, open reading frame (ORF) of F3'H was cloned from E. maculata, and sequence of the gene promoter obtained by Tail-PCR technique with bioinformatics analysis performed. RT-qPCR was applied to determine the expressions in different plant parts at various growth stages. The protein was expressed through prokaryotic system for analysis.

Results EmF3'H (GenBank accession No. OR944655.1) had an ORF of 1,524 bp in length encoded 507 amino acids. It was a hydrophilic transmembrane protein in the cytochrome P450 (CYP450) family and subcellularly localized in endoplasmic reticulum. Its 1,273 bp promoter contained core elements TATA-box and CAAT-box as well as cis-acting elements related to hormone, stress, and light response. The expressions of EmF3'H significantly differentiated in the organs with the highest level found in the fruits and the lowest in the leaves at the reproductive stage. The target protein of approximately 75 kDa was obtained through prokaryotic expression at the highest level observed 4h after IPTG induction.

Conclusion EmF3'H was expressed in various tissues at different growth stages. It played an important role in the biosynthesis of flavonoids in E. maculata.