Objective To clone the cycloartenol synthase (CAS) gene PcCAS from Polygonatum cyrtonema, perform bioinformatic and recombinant protein expression analyses, and determine its subcellular localization etc, thereby providing essential resources for further elucidating the pivotal role of PcCAS in saponin biosynthesis. Methods Based on the transcriptome data of P. cyrtonema, the PcCAS gene was screened and cloned, and bioinformatics approaches were employed to analyze the physicochemical properties, secondary structure, and tertiary structure of its encoded protein. qRT-PCR was used to analyze the tissue-specific expression of PcCAS. A prokaryotic expression vector was constructed and transferred into Escherichia coli for prokaryotic expression analysis. Yeast expression vectors were constructed and transformed into Pichia pastoris for subsequent yeast expression analysis. A subcellular-localization vector was built and infiltrated into Nicotiana benthamiana leaves for the analysis of heterologous expression in tobacco. Results The open reading frame (ORF) of PcCAS is 2 286 bp in length and encodes 759 amino acids. The protein is hydrophilic and lacks a signal peptide. The total saponin content and PcCAS expression level in the rhizome are significantly higher than those in the stem, leaf, and flower, indicating the expression level of PcCAS is positively related with the total saponin content. Yeast-expressed recombinant protein matched the expected molecular weight of 86 400. Subcellular-localization assays in tobacco leaves demonstrated that the PcCAS protein is predominantly localized to the endoplasmic reticulum. Conclusion PcCAS protein belongs to the OSC superfamily of enzymes and is localized in the cytoplasm, positively regulating the total saponin biosynthesis.

Polygonatum cyrtonema Hua  /  cycloartenol synthase  /  gene clone  /  total saponin  /  subcellular-localization