Identification and analysis of CYP450 supergene family members from the transcriptome of Aralia elata (Miq.) Seem reveal candidate genes for triterpenoid saponins biosynthesis

Abstract

BackgroundMembers of the cytochrome P450 (CYP450) gene superfamily have been shown to play essential roles in regulating secondary metabolites biosynthesis. However, the systematic identification and bioinformatics analysis of CYP450s have not been reported in Aralia elata (Miq.) Seem, a highly valued medicinal plant. ResultsIn the present study we conducted the RNA-sequencing (RNA-seq) analysis of the leaves, stems, and roots of A. elata, yielding 66,713 total unigenes. Following the annotation and classification of these unigenes, we were able to identify two pathways and 19 putative genes associated with the synthesis of triterpenoid saponins in these plants, with qRT-PCR subsequently being used to validate these gene expression patterns. Scanning with the CYP450 model from Pfam resulted in the identification of 111 full-length and 143 partial-length CYP450s, with the full-length CYP450s being further clustered into 7 clans and 36 families. Through phylogenetic and conserved motif analyses, we were further able to group these CYP450 proteins into two primary branches: A-type (53%) and non-A type (47%). We further conducted representative protein sequence alignment for these CYP450 family members, with secondary elements being assigned in light of the recently published Arabidopsis CYP90B1 structure. Using the available sequence information, we further identified predicted substrate recognition sites (SRSs) and substrate binding sites within these putative proteins.We further assessed the expression patterns of these 111 CYP450 genes across A. elata tissues, with 12 members of this gene family being selected at random for qRT-PCR validation. From these data, we identified CYP716A295 and CYP716A296 as the candidate genes most likely to be associated with oleanolic acid synthesis, while CYP72A763 was identified as being the most likely to play a role in hederagenin biosynthesis. Finally, we assessed the subcellular localization of these CYP450 proteins within Arabidopsis protoplasts, highlighting the fact that they localize to the endoplasmic reticulum.ConclusionsThis study presents a systematic analysis of the CYP450 gene family in A. elata and provided a foundation for further functional characterization of CYP450 genes.