Dataset Information

Molecular cloning, functional characterization and expression of the β-amyrin synthase gene involved in saikosaponin biosynthesis in Bupleurum chinense DC.

ABSTRACT: Bupleurum chinense DC. is a commonly used plant in traditional Chinese medicine, and saikosaponins(SSs) are the main active oleanane-typetriterpene saponins in B. chinense. β-Amyrin synthase (β-AS) is an important enzyme in oleanane-type triterpenoid saponin synthesis, but its role in saikosaponin synthesis has rarely been studied. Here, the putative β-AS gene BcBAS1(Accession No.ON890382) selected according to metabolomic and transcriptomic analyses was cloned and functionally characterized by heterologous expression in Escherichia coli and Pichia pastoris, and its subcellular localization and expression patterns were examined. The molecular weight of the BcBAS1 recombinant protein was approximately 87 kDa, and this protein could catalyse the production of β-amyrin, the precursor of SSs. Furthermore, BcBAS1 was located in the cytosol, and relative expression in four tissues of the four genotypes was positively correlated with SSa and SSd contents. Our results indicate that BcBAS1 is a β-AS gene and may play an important role in saikosaponin biosynthesis and regulation. This study sheds light on the role of β-AS genes in the synthesis of SSs and provides insights for the metabolic engineering of SSs.

SUBMITTER: Mao Y

PROVIDER: S-EPMC9483273 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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Molecular cloning, functional characterization and expression of the β-amyrin synthase gene involved in saikosaponin biosynthesis in Bupleurum chinense DC.

Mao Yanping Y Chen Hua H Zhao Jun J Li Yuchan Y Feng Liang L Yang Yuping Y Zhang Yiguan Y Wei Ping P Hou Dabin D

Journal of plant biochemistry and biotechnology 20220916 2

Bupleurum chinense DC. is a commonly used plant in traditional Chinese medicine, and saikosaponins(SSs) are the main active oleanane-typetriterpene saponins in B. chinense. β-Amyrin synthase (β-AS) is an important enzyme in oleanane-type triterpenoid saponin synthesis, but its role in saikosaponin synthesis has rarely been studied. Here, the putative β-AS gene BcBAS1(Accession No.ON890382) selected according to metabolomic and transcriptomic analyses was cloned and functionally cha ...[more]

PMID: 36160316

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Project description:BackgroundBupleurum chinense DC. is a widely used traditional Chinese medicinal plant. Saikosaponins are the major bioactive constituents of B. chinense, but relatively little is known about saikosaponin biosynthesis. The 454 pyrosequencing technology provides a promising opportunity for finding novel genes that participate in plant metabolism. Consequently, this technology may help to identify the candidate genes involved in the saikosaponin biosynthetic pathway.ResultsOne-quarter of the 454 pyrosequencing runs produced a total of 195, 088 high-quality reads, with an average read length of 356 bases (NCBI SRA accession SRA039388). A de novo assembly generated 24, 037 unique sequences (22, 748 contigs and 1, 289 singletons), 12, 649 (52.6%) of which were annotated against three public protein databases using a basic local alignment search tool (E-value ≤1e-10). All unique sequences were compared with NCBI expressed sequence tags (ESTs) (237) and encoding sequences (44) from the Bupleurum genus, and with a Sanger-sequenced EST dataset (3, 111). The 23, 173 (96.4%) unique sequences obtained in the present study represent novel Bupleurum genes. The ESTs of genes related to saikosaponin biosynthesis were found to encode known enzymes that catalyze the formation of the saikosaponin backbone; 246 cytochrome P450 (P450s) and 102 glycosyltransferases (GTs) unique sequences were also found in the 454 dataset. Full length cDNAs of 7 P450s and 7 uridine diphosphate GTs (UGTs) were verified by reverse transcriptase polymerase chain reaction or by cloning using 5' and/or 3' rapid amplification of cDNA ends. Two P450s and three UGTs were identified as the most likely candidates involved in saikosaponin biosynthesis. This finding was based on the coordinate up-regulation of their expression with β-AS in methyl jasmonate-treated adventitious roots and on their similar expression patterns with β-AS in various B. chinense tissues.ConclusionsA collection of high-quality ESTs for B. chinense obtained by 454 pyrosequencing is provided here for the first time. These data should aid further research on the functional genomics of B. chinense and other Bupleurum species. The candidate genes for enzymes involved in saikosaponin biosynthesis, especially the P450s and UGTs, that were revealed provide a substantial foundation for follow-up research on the metabolism and regulation of the saikosaponins.

Dataset Information

Molecular cloning, functional characterization and expression of the β-amyrin synthase gene involved in saikosaponin biosynthesis in Bupleurum chinense DC.

Publications

Molecular cloning, functional characterization and expression of the β-amyrin synthase gene involved in saikosaponin biosynthesis in <i>Bupleurum chinense</i> DC.

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