Molecular Cloning and Functional Identification of a Squalene Synthase Encoding Gene from Alfalfa (Medicago sativa L.)

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.

Structural and Molecular Characterization of Squalene Synthase Belonging to the Marine Thraustochytrid Species Aurantiochytrium limacinum Using Bioinformatics Approach

“…The 3-D model generated SQS of selected species was predicted to majorly contain α- helixes and was made of a monomer with large space in the center as a binding site. The active site at the center provides space to interact with NAD(P)H and Mg 2+ via hydrogen bonding [ 32 ]. The 3-D structure was similar to many predicted models reported earlier for the SQS from different species [ 16 , 17 , 26 , 30 ].…”

Transcriptome analysis to identify genes involved in lignan, sesquiterpenoid and triterpenoid biosynthesis in medicinal plant Kadsura heteroclita

“…In Salvia miltiorrhiza, the gene expression profiles of SmSQS2 in roots is almost twice the gene expression level of leaves samples (Rong et al, 2016). Similarly, previous studies conducted on Tripterygium wilfordii, Medicago sativa and Dryopteris fragrans, TwSQS, MsSQS and DfSQS1 have reported highest gene expression patterns in roots, followed by leaves and stem samples respectively (Zhang et al, 2018;Gao et al, 2019;Kang et al, 2019). Interestingly in K. heteroclita transcriptome, KhSQS gene exhibited almost the same expression pattern in roots, stems and leaves samples (Figure 14B), suggesting the significance of KhSQS gene during K. heteroclita seedling development.…”

“…Recent studies have implemented transgenic technology for successfully enhancing the production of triterpenoid in plants. For example, over expression of MsSQS in alfalfa led to the accumulation of total saponins, suggesting a correlation between MsSQS expression level with saponins content respectively (Kang et al, 2019). Similarly, in Eleutherococcus Senticosus gene expression of the SQS and SE genes are positively correlated with the saponin content (Wang et al, 2019), Ganoderma lingzhi, and overexpression of SE doubled the production of ganoderic acid (Zhang et al, 2017).…”

Production of Squalene in Bacillus subtilis by Squalene Synthase Screening and Metabolic Engineering

“…Limited efforts have been given to study prokaryotic SQSs, and only those from M. capsulatus, Thermosynechococcus elongates, and Bradyrhizobium japonicum have been cloned and expressed. ,, SQSs from multiple other microbial species have yet to be comprehensively and systematically investigated. Therefore, four SQS candidates from both prokaryotic and eukaryotic organisms were selected to compare and explore their capacity to synthesize squalene in B.…”