ABSTRACT
Aralia elata is an important medicinal plant in China; it produces large amounts of oleanane type triterpene saponins. A full-length cDNA encoding ß-amyrin synthase (designated as AeAS) was isolated from young leaves of A. elata by reverse transcription-PCR. The full-length cDNA of AeAS was found to have a 2292-bp open reading frame, encoding a protein with 763 amino acid residues. The deduced amino acid sequence of AeAS showed the highest identity (97%) to Panax ginseng ß-amyrin synthase. When AeAS cDNA was expressed in Escherichia coli, an 87.8-kDa recombinant protein was detected by SDS-PAGE and Western blotting. The sequence was also heterologously expressed in the yeast Pichia pastoris, and production of ß-amyrin was detected by HPLC. Tissue expression pattern analysis by real-time reverse transcription-PCR revealed that AeAS is strongly expressed in leaves and stems, and weakly expressed in roots and flowers.
Subject(s)
Aralia/enzymology , Aralia/genetics , Genes, Plant/genetics , Intramolecular Transferases/genetics , Plants, Medicinal/enzymology , Plants, Medicinal/genetics , Trees/enzymology , Amino Acid Sequence , Base Sequence , Blotting, Western , Chromatography, High Pressure Liquid , Cloning, Molecular , DNA, Complementary/genetics , Electrophoresis, Polyacrylamide Gel , Gene Expression Regulation, Plant , Intramolecular Transferases/chemistry , Molecular Sequence Data , Phylogeny , Saponins/biosynthesis , Sequence Alignment , Sequence Analysis, DNA , Trees/genetics , Triterpenes/metabolismABSTRACT
OBJECTIVE: To clone Aralia elata squalene synthase gene (designated as AeSS) and construct plant expression vector for transgenic research. METHODS: Isolated squalene synthase from Aralia elata with specific primers by RT-PCR and inserted AeSS gene into the plant expression vector pBI121. RESULTS: The full-length cDNA of AeSS (Genebank accession Number: GU354313) was 1 261 bp and contained a 1 245 bp open reading frame (ORF) encoding a polypeptide of 414 amino acids. The plant expression vector pAeSS was constructed by inserted AeSS gene into the downstream of 35 S promoter of plant expression vector pBI121. CONCLUSION: AeSS gene was cloned and plant expression vector was constructed for future research.