| Aralia elate (Miq.) Seem, which is a deciduous small arbor belongs to Aralia genus in the family Araliaceae. is a traditional Chinese medicine and its root bark, stem bark, leaves and tender bud can be used as medicines. Aralia elate (Miq.) Seem has many effects including invigorating qi for tranquilization, nourishing yin and tonifying kidney, expelling wind and activating blood flow, and dispelling dampness and relieving pain, and can be used for the treatment of neurasthenia, arthritis cancer and other diseases. New research shows that:stems and leaves of Aralia elate (Miq.) Seem have the function of strong anti-liver injury, and based on which, new drugs had been studied and developed for treating hepatitis. According to the research, Aralia elate (Miq.) Seem is a kind of plant containing the highest content of oleanane. which is main medical composition of Aralia elate (Miq.) Seem. The biosynthetic pathway of oleanane is known and Farnesyl diphosphate Synthase (FPS) andβ-Amyrin Synthase (β- AS) are key enzymes of this pathway. The study aims to clone FPS andβ-AS genes from Aralia elata (Miq.) Seem, and transfer the two key genes to E. coli and Yeast for Heterologous expression. The object of the study is to obtain adequate anti hepatitis drugs oleanolic acid from transgenic microorganisms for clinical need.With RT-PCR technology, FPS andβ- AS genes were firstly cloned from Aralia elate (Miq.) Seem, and accession numbers in GenBank were respectively HM219226 and HM219225, and then, structure analysis and functional identification were also performed.Farnesyl pyrophosphate synthase (FPS:EC 2.5.1.1/EC 2.5.1.10) is located at the midstream stage of biosynthetic pathway of triterpene saponin and catalyzes IPP and DMAPP producing FPP containing 15 carbon by continuous condensation of head-to-tail. The full-length cDNA of FPS was 1040bp and contained a 1029bp open reading frame (ORF) encoding a polypeptide of 342 amino acids. The alignment results showed that FPS amino acid sequence shared above 80% identities to Panax ginseng. Panax quinquefolius by DNAman. Furthermore, Prokaryote expression vectors of pET-2%a-FPS was constructed and 39.6kD fusion protein detected by SDS-PAGE and Western blot showed that FPS gene was successfully expressed in E. coli BL21.(3-amyrin synthase (p-AS:EC5.4.99.B1) which belongs to oxido-squalene cyclases (OSCs) family is located at the downstream stage of biosynthetic pathway of triterpene saponin and catalyzes 2.3- oxido-squalene producing pentacyclic triterpene skeletonβ-Amyrin. The full-length cDNA of fi-AS was 2292bp and contained a 2292bp open reading frame (ORF) encoding a polypeptide of 763 amino acids. The alignment results showed thatβ-AS amino acid sequence shared above 70% identities to Panax ginseng. Medicago sativa and Artemisia carvifolia by DNAman. Prokaryote expression vectors of pET-28a-AS was constructed and 87.8kD fusion protein detected by SDS-PAGE and Western blot showed thatβ-AS gene was successfully expressed in E. coli BL21.Theβ-AS gene was transferred into Pichia pastoris for function analysis. Eukaryotic expression vector of pPIC9k-AS was constructed and 87.6kD fusion protein detected by SDS-PAGE and Western blot showed thatβ-AS gene was successfully expressed in Pichia pastoris GS115. Metabolic target product of yeast transferredβ-AS gene was detected with HPLC technology. The results showed thatβ-AS gene was expressed in yeast with activities and it could catalyze 2.3-oxido-squalene producingβ-Amyrin.In this study, expression differences in transcription level of FPS andβ-AS genes from lateral roots, stems, leaves and floral of Aralia elate (Miq.) Seem were analyzed by Real-time RT-PCR. The results demonstrated that the two genes were expressed in lateral roots, stems, leaves and floral and the relative expression level of stems were the highest. of lateral roots were lowest.
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