Abstract
The quality of alfalfa, a main legume forage worldwide, is of great importance for the dairy industry and is affected by the content of triterpene saponins. These natural terpenoid products of triterpene aglycones are catalyzed by squalene synthase (SQS), a highly conserved enzyme present in eukaryotes. However, there is scare information on alfalfa SQS. Here, an open reading frame (ORF) of SQS was cloned from alfalfa. Sequence analysis showed MsSQS had the same exon/intron composition and shared high homology with its orthologs. Bioinformatic analysis revealed the deduced MsSQS had two transmembrane domains. When transiently expressed, GFP-MsSQS fusion protein was localized on the plasma membrane of onion epidermal cells. Removal of the C-terminal transmembrane domain of MsSQS improved solubility in Escherichia coli. MsSQS was preferably expressed in roots, followed by leaves and stems. MeJA treatment induced MsSQS expression and increased the content of total saponins. Overexpression of MsSQS in alfalfa led to the accumulation of total saponins, suggesting a correlation between MsSQS expression level with saponins content. Therefore, MsSQS is a canonical squalene synthase and contributes to saponin synthesis in alfalfa. This study provides a key candidate gene for genetic manipulation of the synthesis of triterpene saponins, which impact both plant and animal health.
Highlights
In model legume Medicago truncatula, the triterpene saponins, important terpenoid natural products, are glycosides of at least five different triterpene aglycones catalyzed by squalene synthase (SQS/SS), squalene epoxidase (SE) and beta-amyrin synthase [1]
Protein BLAST search demonstrated that it encodes squalene synthase, which converts two molecules of farnesyl diphosphate (FPP) into squalene via an intermediate: presqualene diphosphate (PSPP) (Figure 1a)
It was designated as MsSQS, a squalene synthase encoding gene first identified from forage crop alfalfa
Summary
In model legume Medicago truncatula, the triterpene saponins, important terpenoid natural products, are glycosides of at least five different triterpene aglycones catalyzed by squalene synthase (SQS/SS), squalene epoxidase (SE) and beta-amyrin synthase (beta-AS) [1]. SQS encoding genes have been identified from a wide range of species, including model plants (e.g., Arabidopsis and barrel clover) [1,7], crops (e.g., rice, soybean, barley and potato) [8,9,10,11], the economically or pharmaceutically important plants (e.g., tobacco and ginseng) [12,13] and trees [14,15]. Most of these SQS genes have been characterized by bioinformatics approaches to evaluate the physicochemical properties and structural characteristics, and some were explored by molecular techniques to analyze their biological functions
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