Isolation of CYP72A154, a gene involved in glycyrrhizin biosynthesis pathway, in Glycyrrhiza glabra L. (Iranian licorice)

Abstract

Background: Cytochrome P450s have essential roles in oxidative reactions during the biosynthesis of secondary metabolites, such as terpenoids. Objective: This research was aimed to identify the gene CYP72A154 as a gene involved in the glycyrrhizin biosynthesis pathway in Iranian licorice. Methods: CYP72A154 gene was isolated from Iranian licorice and cloned it into the PTG19-T vector. After confirmation of fragment length, the recombination plasmid was sent for sequencing. NCBI BLAST was used to analyze the nucleotide/ protein sequence homology between Glycyrrhiza glabra and other plants. The characterization of predicted amino acid sequences such as sequence homology, protein domains and functional sites, was performed using InterProscan. RT-PCR was performed to improve the relative expression this gene in the licorice root. Results: The query length was 314 aa, which after blasting in NCBI had about 78 to 80 % identity to the cytochrome P450 72A154 and 11-oxo-beta-amyrin 30-oxidase in species of G. glabra, G. uralensis and G. pallidiflora, as well as about 64 to 67 % with other species of the Fabaceae family. TMHMM analysis indicated the Exp number of AAs in TMHs was 22.11931, Exp number and first 60 AAs was 20.013. The results of RT-PCR revealed that the expression of this gene was comparable to the β-AS gene and CYP88D6, both being involved in the glycyrrhizin biosynthesis pathway. Conclusion: According to bioinformatics analysis and RT-PCR, it can be stated that the desired fragment belongs to the CYP72A154 gene and is also involved in the biosynthesis of glycyrrhizin.

Keywords

• glycyrrhiza glabra
• cytochrome p450s
• homology
• bioinformatic analysis
• rt-pcr