Cloning and expression analysis of squalene epoxidase genes from Siraitia grosvenorii / 中国中药杂志

ABSTRACT

Siraitia grosvenorii, vine plant of Cucurbitaceae family, has been used as natural sweetener and folk medicine. The major components and sweet substances are both known as mogrosides which are cucurbitane-type tetra-triterpenoids. Squalene epoxidase (SQE) has been generally recognized as the common rate-limiting enzyme in triterpenes and phytosterols, catalyzing into their common precursor 2,3-oxidosqualene (OS); however, in the biosynthesis of mogrosides, the precursor was 2,3,22,23-dioxidosqualene (DOS) instead of OS. To explore the specific SQE in S. grosvenorii, we cloned two full-length SQEs (SgSQE1, SgSQE2), performed bioinformatic analysis, analyzed the expression patterns in different periods of fruits by Real-time PCR, and induced the prokaryotic expressions. Finally, the interactive sites between SQE and substrate were predicted by docking, which would provide evidence for SQE gene function study of mogrosides and also lay foundation for triterpene biosynthesis in other plants. SgSQE1 and SgSQE2 both encoded predicted proteins of 524 amino acids, and shared 84% identity to each other at residues level, but had high specificity at N-terminal region. They both accumulated in fruits, but with different patterns, SgSQE1 increased rapidly and reached the highest level at 15 d, which had identical co-expression pattern with cucurbitadienol synthase (CS). SgSQE2 had a relatively constant level. The docking results showed that predicted proteins of SgSQE1 and SgSQE2 can interact with OS, with different contact sites (R348 for SgSQE1, H349 for SgSQE2). The recombinant proteins had no activities by prokaryotic expression, which were caused by transmembrane regions. However, all the results strongly suggested that SgSQEs were both involved in secondary metabolites biosynthesis in S. grosvenorii. SgSQE1 might be involved in mogrosides biosynthesis and SgSQE2 might participate in other cucurbitane-type triterpenes or phytosterols biosynthesis.