Abstract
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.
Highlights
Bupleurum chinense DC. is a widely used traditional Chinese medicinal plant
Sequencing and de novo assembly A one-quarter plate run using the 454 GS FLX Titanium platform was carried out on the cDNA that was generated by SMART technology from the equivalent pooled total RNA from B. chinense roots, germinating seeds, and seedlings
In the present study, a 454 dataset of B. chinense was analyzed. These data represent a substantial contribution to the functional genetic studies of B. chinense
Summary
Bupleurum 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. Bupleurum chinense DC., a perennial herb native to China, belongs to the Umbelliferae family and the genus Bupleurum L This herb is used worldwide for medicinal purposes, but is especially common in China, Japan, and. In traditional Chinese medicine, the roots of B. chinense and other Bupleurum species are known as Chinese thorowax roots (Radix bupleuri), or “chaihu” in Chinese. More than 75 monomer SSs have been isolated from Radix bupleuri [4,5], only SS-a, SS-b2, SS-c, and SS-d have been pharmacologically examined [6,7,8,9,10], because of the low SS content Roots derived from various Bupleurum species such as B. chinense, B. scorzonerifolium, B. falcatum, and B. kaoi, have been widely used in various medicinal decoctions. The concentration and composition of the SSs in the roots is even more complex when studied in combination with diverse planting and harvesting environments and different management methods
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