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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 <i>SQS</i>. Here, an open reading frame (ORF) of <i>SQS</i> was cloned from alfalfa. Sequence analysis showed <i>MsSQS</i> 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 <i>Escherichia coli</i>. <i>MsSQS</i> was preferably expressed in roots, followed by leaves and stems. MeJA treatment induced <i>MsSQS</i> expression and increased the content of total saponins. Overexpression of <i>MsSQS</i> in alfalfa led to the accumulation of total saponins, suggesting a correlation between <i>MsSQS</i> expression level with saponins content. Therefore, <i>MsSQS</i> 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.