Santalum album is an economically important plant in the craft, spices and medicine industries. The main chemical constituents found in sandalwood essential oils are sesquiterpenes. 3-Hydroxy-3-methylglutaryl monoacyl-coenzyme A reductase (HMGR) is one of the rate-limiting enzymes required for the synthesis of sandal sesquiterpenes, but there are no studies on the HMGR gene in S. album. In this study, the full-length ORFs of the upper rate-limiting enzyme genes SaHMGR1 and SaHMGR2, which lie upstream of the MVA metabolic pathway of sandal sesquiterpenes, were cloned for the first time. Bioinformatics and phylogenetic analyses were conducted. The results showed that SaHMGR1 and SaHMGR2 had typical domains of HMGR class I enzymes in the HMGR superfamily, including four catalytic sites, six NADPH-binding sites, five substrate binding regions, four inhibitor binding sites, and several dimer interface regions. A phylogenetic analysis showed that SaHMGR1 and SaHMGR2 were highly conserved relative to corresponding genes in other plants. An analysis of subcellular localization showed that these SaHMGR genes were located in the endoplasmic reticulum. SaHMGR1 and SaHMGR2 were detected by real-time PCR in roots, sapwood, heartwood, young leaves, mature leaves and twigs. Highest expression was in roots. SaHMGR1 expression was higher in mature leaves than in heartwood while SaHMGR2 expression was lower in mature leaves than in heartwood. Expression in Escherichia coli strain DH5α with plasmid pET-32a (+) was also used to verify the functionality of both HMGR proteins, which catalyzed the formation of MVA from HMG-CoA. In E. coli, the enzymatic activity of SaHMGR1 was higher than that of SaHMGR2. These findings provide a basis for further studies on the function of SaHMGR genes and the regulation of sesquiterpene biosynthesis in S. album.
Keywords: MVA pathway; Prokaryotic protein expression; SaHMGRs; Sandalwood; Sesquiterpenes; qRT-PCR.
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