RESUMEN
Objective: The key enzyme of triterpene saponin metabolism was cloned and its sequence, structure and function were analyzed by bioinformatics. Methods: RNAs were extracted from the leaves of Panax japonicus The full-length cDNA sequences of β-AS were cloned by utilizing RT-PCR method, and the sequence was connected to the pMDTM18-T for cloning and sequencing.β-AS protein characteristics in transplanted species and cultivated species of P. japonicus were predicted and compared by bioinformatics analysis and the phylogenetic tree of β-AS protein was constructed. Results: The β-AS sequences in transplanted species and cultivated species of P. japonicus were obtained, which had 2 286 bp ORF and encoded 761 amino acids. There were little differences between the two varieties of β-AS proteins in physicochemical properties, secondary structure, tertiary structure, and phosphorylation sites, which may lead to show difference in catalytic activity. Conclusion: This work also obtained the full-length of cDNA sequence of β-AS gene in transplanted and cultivated varieties of P. japonicus, and provided a systemic sequence analysis of β-AS proteins, which can provide the useful information for β-AS studies in the future.
RESUMEN
Objective: To clone β-amyrin synthase (bAS) gene from Eleutherococcus senticosus and analyze the effect of its expression on saponin contents. Methods: The sequence of cDNA of E. senticosus bAS was cloned by homologous cloning strategy. Quantitative real time PCR was developed to analyze the expression pattern of E. senticosus bAS gene. And E. senticosus saponin contents were measured by spectrophotometry method. Results: Length 1 223 and 1 226 bp of E. senticosus bAS1 and bAS2 genes were cloned. The results showed that bAS1 and bAS2 were expressed in the each growth period and every organ of E. senticosus, and the expression differed significantly (P < 0.05). bAS1 showed the highest expression when the plants were grown in germination stage, then rapidly depressed, and changed slightly in the end. The expression of bAS2 showed the characteristic of low-high-low. The expression of bAS1 in different organs of E. senticosus was constant, but the highest content of the expression of bAS2 was in the leaves. With the treatment of methyl jasmonate (MeJA), bAS2 expression has been significantly improved and bAS1 without a significant changing. There exists significantly positive correlation (P < 0.01) between the content of E. senticosus saponins and the expression levels of bAS2, and bAS1 without a significant difference. Conclusion: bAS2 may be a key enzyme gene in the biosynthesis of triterpenoid saponins.