Cloning and bioinformatic analysis of β-AS gene from Panax japonicus / 中草药

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.

Over-expressing root-specific β-amyrin synthase gene increases glycyrrhizic acid content in hairy roots of glycyrrhiza uralensis / 中草药·英文版

Objsective: Glycyrrhizia uralensis, one of the most widely-used traditional Chinese medicines, is mainly cropped in China. However, many cultivars are less in glycyrrhizic acid than Chinese Pharmacopoeia requires. In this paper, we improved glycyrrhizic acid by regulating β-amyrin synthase gene (GuBAS). Methods: Tobacco root-specific promoter TobRB7 and GuBAS cDNA were obtained and combined with linearized pCAMBIA1305.1 to construct root-specific plant expression vector which was later transformed into Agrobacterium rhizogenes ACCC10060 by electrotransformation. The cotyledons and hypocotyls of G. uralensis were infected by the recombinant A. rhizogenes ACCC10060 to induce hairy roots. The GA content was quantified by HPLC. Results: The PCR and sequencing results both showed that three transgenic hairy root lines were obtained. The copy number of GuBAS in these transgenic hairy roots was intended by qRT-PCR to be 3, 7, and 4. GA was detected by HPLC, and the results showed that GA was present in the three transgenic hairy roots, while absent in wild hairy roots. Conclusion: Over-expressing GuBAS root-specifically in hairy roots of G. uralensis enhanced GA accumulation.

Gene cloning and expression level of bAS and their correlation with content of saponins in Eleutherococcus senticosus / 中草药

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.

Cloning, prokaryotic expression, and functional identification of β-amyrin synthase cDNA of Psammosilene tunicoides / 中草药

Objective: To clone, express, and characterize the full-length cDNA of β-amyrin synthase provided an important basis for the study on the key role in the biosynthetic pathway of triterpenoid saponins and secondary metabolism engineering applied to Psammosilene tunicoides. Methods: The full-length cDNA fragment of P. tunicoides was isolated by the method of RT-PCR and rapid amplification of cDNA ends (RACE). The fragment was transformed into Escherichia coli expression strain BL21, which was induced by IPTG, and the crude recombinant enzyme was purified from E. coli cell. In the presence of 2, 3-oxidosqualene and other substances, 2, 3-oxidosqualene was converted into β-amyrin efficiently. The catalytic product of P. tunicoides β-amyrin synthase was detected by high-performance liquid chromatography (HPLC) and identified as β-amyrin. Results: The full-length cDNA fragment of P. tunicoides is 2 882 bp and contains an open reading frame of 2 284 bp nucleotides, which codes for 760 amino acids. Conclusion: The full-length cDNA can produce β-amyrin synthase by prokaryotic expression. The expression product has the catalytic activity of 2, 3-oxidosqualene into β-amyrin, which would provide an important basis for the secondary metabolism engineering to P. tunicoides.

Cloning of β-amyrin synthase gene from Panax quinquefolius and its bioinformatics analysis / 中草药

Objective: To obtain the full-length cDNA of β-amyrin synthase (bAS) involved in triterpene saponin biosynthesis in Panax quinquefolius and provide the reference for saponin biosynthesis and the regulation of secondary metabolism in P. quinquefolius. Methods: Based on large-scale ESTs sequencing and RACE technology, the full-length cDNA of P. quinquefolius bAS (PqbAS) was obtained. Results: The full-length cDNA of PqbAS (GenBank No. JX185490) was 2 309 bp which included an open reading frame (ORF) code of 631 amino acid peptide. Common conserved domains of oxidosqualene cyclases (OSCs) were found in PqbAS including active sites and conserved sequence. Singal P4.0 analysis showed that PqbAS was a non-secreted protein. Tmhmm 2.0 analysis showed that PqbAS was a non-transmembrane protein. Real-time fluorescence quantitative PCR analysis showed PqbAS gene expressed in various organs, higher in flowers and stems while relatively lower in roots and leaves. Conclusion: The full-length of bAS is first cloned, which could provide the foundation for the investigation on expression characteristics and its role in synthesis of saponin.