Cloning And Sequcing Of Squalene Synthase Gene And Cycloartenol Synthase Gene From Paris Polyphylla Smith Var. Yunnanensis

Sterol saponin is a large class of natural plant secondary metabolites, which is synthesized by the isoprenoid pathway, and has a wide range of physiological activity and pharmacological effects on human healthy. As a traditional Chinese medicine plant, Rhizoma Paridis are rich in sterol saponin, which is the main biologically active component. However, the biosynthetic pathway of sterol saponin in Rhizoma Paridis is still relatively week and limited the usage of biological engineering technology in this plant material. In this study, we focused on the cloning and characterization of squalene synthase gene (PpSQS) and cycloartenol synthase gene (PpCAS), which are the key enzyme genes of isoprenoid pathway in Paris polyphylla Smith var. yunnanensis. Using homology cloning and RACE techniques, the full-length cDNA sequence of a PpSQS and a PpCAS were obtained and characterized. This work will provide a deep view on gene function, molecular evolution, and the biosynthetic pathway of sterol saponin in Rhizoma Paridis.The main results are as follows:1. According to the conserved sequence of SQS genes and CAS genes in other plant, two pairs of degenerate primers were designed and synthesized. Using the first cDNA strand as template, which was synthesized from total RNA of Rhizoma Paridis, the conserved fragments of PpSQS (330bp) and PpCAS (869bp) were obtained. The nucleotide and amino acid sequence alignment results showed that these two fragments belonged to the plant’s SQS and CAS.2. In this study, RACE technology was used to amplify the5’-ends and3’-ends of PpSQS and PpCAS from Rhizoma Paridis. The full-length cDNA of PpSQS is1489bp in length, and contains a24bp5’-UTR, a262bp3’-UTR, and a completed ORF (1212bp). The full-length cDNA of PpCAS is2763bp in length, and contains a244bp5’-UTR, a208bp3’-UTR, and a completed ORF (2283bp). The results of Blast analysis indicated that the PpSQS gene and PpCAS gene were successfully isolated from Paris polyphylla Smith var. yumianensis.3. The ORF of PpSQS encoded a403amino acid protein, with a predicted molecular mass of46.37KDa and pI of6.39. The homology of PpSQS amino acid ranged from42%to85%to the other genes from GenBank, and its amino acid sequence in the phylogenetic closer to monocots. The secondary structure and3D modeling results showd that the PpSQS was rich in a-helix, and had a transmembrane region in the C-terminal300amino acids at the C-terminus contains a hydrophobic region. The3D structure of PpSQS showed a typical SQS with other species and had two catalytic active center pockets, which consisted by a-helixes, and conservative amino acid residue sites.4. The ORF of PpCAS encoded a760amino acid protein, with a predicted molecular mass of86.90KDa and pI of6.54. The homology of PpSQS amino acid ranged from60%to83%to the other genes from GenBank, and its amino acid sequence in the phylogenetic belonged to the Cluster II in monocots, matching to the classical plant taxonomy. The secondary structure and3D modeling results showed that, PpCAS contained a typical CAS domain, an active pocket, and some catalytic sites, which were all consisted by the a-helixes.