Gene cloning of squalene synthase in Alisma orientalis and its bioinformatic analysis / 中草药

Objective: To clone the full-length cDNA encoding squalene synthase (SS), a key enzyme of protostane type triterpenes biosynthesis, from Alisma orientalis and to perform bioinformatic analysis. Methods: With the total RNA as template, the full-length cDNA of SS in A. orientalis was cloned via homology-based cloning approach and rapid amplification of cDNA ends technique. The bioinformatics of the cloning SS gene was analyzed by DNAMAN and ExPASy online analysis. Results: The full-length cDNA (1 577 bp) of SS gene was obtained (GenBank accession number JX866770), with an open reading frame of 1 230 bp, encoding 409 amino acid polypeptides, which had higher homology with the known SS in other medicinal species. The calculated relative molecular mass was 4.68 × 104, the isoelectric point was 5.97, and there was no signal peptide in SS. The deduced protein sequence exhibited two conserved domains rich in Asp (DXXDD). Conclusion: The cDNA encoding SS from A. orientalis is cloned and reported for the first time. This work provides a foundation for exploring the biosynthetic pathway of protostane type triterpenes in A. orientalis and their applications in bioengineering.

Molecular cloning of farnesyl pyrophosphate synthase from Alisma orientale (Sam.) Juzep. and its distribution pattern and bioinformatics analysis / 药学学报

Triterpenes, which have large application potential in the treatment of cancer, are the main active components of genuine medicinal material Alisma orientale (Sam.) Juzep. Farnesyl pyrophosphate synthase (FPPS) is one of the important rate-limiting enzymes in the synthetic pathway of triterpenes. In this study the FPPS full length cDNA of the A. orientale, was cloned via homology-based cloning approach and rapid amplification of cDNA ends (RACE). The full length of the FPPS cDNA was 1 531 bp (accession no. HQ724508), which contained a full 1 032 bp ORF that encoded 343 amino acids. The deduced protein sequence exhibited five conserved motifs, two of which is riched of Asp (DDXXD). The result of real-time quantitative PCR (QRT-PCR) showed that FPPS gene was expressed in different organs of A. orientale. The expression increased from October to the first ten-day period of December, and then decreased. The FPPS gene expression was higher in leaves but lower in leafstalk, tuber and root. HPLC analysis of active components 23-acetyl-alismol B of A. orientale. during different periods indicated that its change trend should be consistent with FPPS gene expression. It can be primarily deduced that FPPS gene should be an important control point in the synthetic pathway of Alisma terpenes. This study may facilitate the quality of medicinal plants through gene engineering in the future.