Objective The squalene synthase (SQS), the first key rate-limited enzyme in the triterpenoids biosynthetic pathway, from Clematis florida Thunb. var. plena D. Don was cloned, and its expression analyzed in preparation for an investigation to increase the medicinal value of the plant by means of genetic engineering.
Method Primers were designed based on the transcriptome data on the SQS genes in C. florida and in other plants. Total RNA was extracted from C. florida leaves, and full-length cDNA obtained by RT-PCR. Bioinformatics and spatiotemporal expression of the gene were analyzed.
Result The ORF of the SQS, designated as CfSQS, was 1 227 bp encoded 408 amino acids. It was an unstable hydrophobic protein comprised 69.61% of α helix and 22.06% of random coil. The deduced amino acid sequence predicted the presence of 4 highly conserved domains with 17–23 amino acids residues (I–IV) and one variable hydrophobic region (V). The predicted 3D structure of CfSQS by various programs was similar to those of SQSs from other species. It had a close phylogenetic relationship and in the same clade with the SQS of Nigella sativa. A spatiotemporal expression analysis indicated the gene to be expressed in all tissues.
Conclusion The full-length cDNA sequence and bioinformatics of the squalene synthase gene CfSQS of C. florida were determined for the first time. The information helped to further clarify the synthesis of the saponin compounds and regulatory mechanism that would pave the way for future studies on the biosynthetic pathways of triterpene saponins in C. florida.
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