Transcriptome analysis of Bupleurum chinense focusing on genes involved in the biosynthesis of saikosaponins
© Sui et al; licensee BioMed Central Ltd. 2011
Received: 6 July 2011
Accepted: 2 November 2011
Published: 2 November 2011
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. 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.
One-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 (P450 s) and 102 glycosyltransferases (GT s) unique sequences were also found in the 454 dataset. Full length cDNAs of 7 P450 s and 7 uridine diphosphate GT s (UGT s) were verified by reverse transcriptase polymerase chain reaction or by cloning using 5' and/or 3' rapid amplification of cDNA ends. Two P450 s and three UGT s 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.
A 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 P450 s and UGT s, that were revealed provide a substantial foundation for follow-up research on the metabolism and regulation of the saikosaponins.
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 South Korea . 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. For more than 2, 000 years these roots have been used for their anti-inflammatory, anti-pyretic, and anti-hepatotoxic effects in the treatment of common colds, fever, influenza, hepatitis, malaria, and menoxenia [2, 3]. The major bioactive components of Radix bupleuri are the saikosaponins (SSs), which belong to the oleanane-type triterpene saponins. Although 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 [6–10], because of the low SS content (usually ca. 1% w/w in dried roots) . Different monomer SSs have been reported to exhibit different predominant pharmacological effects. For example, among the SSs isolated from B. falcatum, SS-a and SS-d, but not SS-c, have anti-inflammatory activities . Whereas SS-c has no correlation with cell growth inhibition, other SSs can inhibit cell growth, as well as induce cancer cell differentiation and apoptosis. Hence, SS-c may have the potential for therapeutic angiogenesis, but is unsuitable for cancer therapy . 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 content and proportion of the monomer SSs are extremely diverse in these medicinal materials. 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.
Studies have shown that the sequencing and analysis of expressed sequence tags (ESTs), combined with genetic and phytochemical methods are effective tools for discovering novel genes in non-model plants [32–34]]. Some genes involved in natural product biosynthetic pathways have been identified via EST analyses [24–26, 28, 35, 36]]. The 454 pyrosequencing technique, with its advantages of throughput, read length, and accuracy, can greatly accelerate the discovery of novel genes in non-model organisms [37–39]]. Candidate genes involved in the metabolic pathways of natural products have been identified using the 454 pyrosequencing technique. These natural products usually have diverse and important functions in plant growth, and are also invaluable as pharmaceuticals and agrochemicals. Examples of such important natural products include triterpene saponins in American ginseng  and Glycyrrhiza uralensis, flavonoids in Artemisia annua, alkaloids in Huperzia serrata and Phlegmariurus carinatus, and cyanogenic glucosides in Zygaena filipendulae. The specific functions of the candidate genes reported in the abovementioned studies are still being validated. Even so, the 454 pyrosequencing is still the preferred choice for novel gene discovery, especially for members of known gene families.
In the present study, 195, 088 high-quality (HQ) reads from a cDNA library of B. chinense were obtained using the Roche GS FLX Titanium platform. The reads were assembled into 24, 037 unique sequences comprising 22, 748 contigs and 1, 289 singletons. Only 864 ESTs were identical with those derived from the 3, 111 ESTs generated in our previous study from a B. chinense root cDNA library using the Sanger sequencing method  and the Bupleurum sequences from NCBI. A total of 246 P450 s and 102 glycosyltransferases (GT s) including 49 UGT s were screened. The assembled full-length cDNAs of the P450 s and UGT s were verified. Several partial cDNAs of the P450 s and UGT s were extended to full length by 5' and/or 3' rapid amplification of cDNA ends (RACE). The candidate P450 s and UGT s that may participate in SS biosynthesis were screened via methyl jasmonate (MeJA) inducibility and tissue-specific expression pattern experiments. These P450 s and UGT s will be the targets of further research on SS biosynthesis.
Sequencing and de novo assembly
Summary of B. chinense 454 sequencing and assembly
Total bases (bp)
Range of length
A sum of 10, 734 (44.7%) of the total unique sequences were further annotated based on their similarity with The Arabidopsis Information Resource (TAIR) proteins, before gene ontology (GO) terms were assigned. The categories of molecular function, biological process, and cellular component are shown in additional file 2: Functional annotations of the 454 unique sequences of B. chinense based on GO categories. A high percentage of the unique sequences were annotated to hydrolases, kinases, and transferases in the molecular function category. For the biological process category, a large number of genes were annotated to metabolic processes, response to abiotic or biotic stimulus, and response to stress. Hence, the 454 dataset should substantially aid the discovery of novel genes involved in the metabolism of SSs and other secondary natural products. A total of 10, 277 unique sequences were annotated using KEGG; 2, 849 of them were related to metabolism, 36 to the metabolism of terpenoids polyketides, and 101 to the biosynthesis of other secondary metabolites (see additional file 3: Summary of metabolic pathway assignments of the 454 assembled unique sequences based on KEGG).
Candidate genes related to SS backbone biosynthesis
Numbers of annotated unique sequences and 454 reads involved in saikosaponin skeleton biosynthesis
Number of unique sequences
Number of 454 reads
Farnesyl diphosphate synthase
Full-length cDNA cloning of P450 s and UGT s
GTs are a superfamily of enzymes in plants. GTs catalyze the transfer of sugar moieties from activated donor molecules to specific acceptor molecules, forming glycosidic bonds. Currently, there are 92 families and some non-classified sequences at the superfamily level http://www.cazy.org/GlycosylTransferases.html. Glycosylation is one of the major factors that determine the bioactivity and bioavailability of natural plant products, such as flavonoids and terpenoids.
Expression characteristics of P450 s and UGT s
The 454 pyrosequencing technology is regarded as a prime choice for novel gene discovery in non-model organisms. In the present study, this technology was applied with the main goal of identifying the P450 s and UGT s involved in the biosynthesis of SSs in B. chinense. In previous reports, CYP93E1 from Glycine max was shown to hydroxylate β-amyrin and sophoradiol with the formation of olean-12-ene-3β, 24-diol, and soyasapogenol B, respectively . CYP88D6 from Glycyrrhiza uralensis was identified as a β-amyrin 11-oxidase . UGT73K1 and UGT71G1 from Medicago truncatula[26, 27] and UGT74M1 from Saponaria vaccaria have been identified to be involved in triterpene biosynthesis. Thus far, no P450 or UGT were identified in SS-producing plant species. All known triterpenes and sterol hydroxylases have been classified into the CYP71 and CYP85 clans [24, 25, 51, 52]. In our 454 dataset, 114 unique sequences in 8 families and 52 unique sequences in 4 families belong to the CYP71 and CYP85 clans, respectively. Of these 49 were UGT s representing nine families, namely, UGT71, UGT72, UGT73, UGT74, UGT76, UGT84, UGT85, UGT91, and UGT94. Our data provide a promising opportunity for identifying the P450 s and UGT s involved in SS biosynthesis. In the present study, 14 unique sequences of P450 s and 20 UGT s were screened. Two P450 s and three UGT s that may be involved in the biosynthesis of SSs, based on MeJA inducibility and tissue-specific expression patterns, were found. They are currently being identified by their heterologous expression in Escherichia coli or yeast, as well as by their overexpression and gene silencing in transgenic B. chinense plants. More candidate SS-related P450 s and UGT s may be found among the annotated P450 s and UGT s. Along with the identified P450 s and UGT s, our results may also be helpful in revealing the formation mechanism of diverse monomer SSs and in elucidating other saponin biosynthetic pathways.
In the present study, the full-length cDNA clones of seven P450 s and seven UGT s were obtained. Two of the P450 s belong to the CYP736 family and the other five P450 s belong to the CYP82, CYP712, CYP90, CYP707, and CYP716 families. The catalytic function of the CYP736 family is still unknown. Recent reports have shown that CYP736B in grapes may be involved in the host response to Xylella fastidiosa infection . CYP736A34 in soybean is also highly co-expressed with genes involved in root and Rhizobium-induced nodule development [; review in ]. CYP82 and CYP712 are part of the CYP71 clan family. Some members of the CYP82 family were found to mediate plant-specific alkaloid pathways, for example, CYP82E4 and CYP82E5v2 in tobacco were identified with nicotine N-demethylase activity [56, 57]. Arabidopsis CYP82C2 and CYP82C4 are 8-methoxypsoralen hydroxylases that mediate modifications of toxic furanocoumarin . However, a recent study has shown that CYP82G1 functions in the terpene pathway as a DMNT/TMTT (C11-homoterpene (E)-4, 8-dimethyl-1, 3, 7-nonatriene/C16-homoterpene (E, E)-4, 8, 12-trimethyltrideca-1, 3, 7, 11-tetraene) homoterpene synthase . CYP712 and the CYP93s may catalyze successive steps in the same pathway(s) in different plants . One of the CYP93s, CYP93E1, was found to participate in the triterpene pathway . CYP90, CYP707, and CYP716 are part of the CYP85 clan family. CYP90 is the first family of CYPs required for brassinosteroid synthesis. CYP90Bs, -As, -Ds, and -Cs successively act in the brassinosteroid pathway [60, 55]. The CYP707s inactivate ABA via 8'-hydroxylation to form phaseic acid, and thereby, play a key role in the regulation of ABA-mediated physiological processes . The CYP716s do not have a known function, but their closest non-plant relatives, CYP26As, are involved in the hydroxylation of retinoic acid . Based on sequence similarity, CYP716 was close to CYP725 in the neighbor-joining tree (Figure 4). A previous study using a broader range of plants also showed some overlap in CYP716 and CYP725. This overlap is evidence of the extensive divergence occurring within this subset of genes in the CYP85 clan. CYP725A has been shown to act on taxane diterpenoids . However, it is still unclear whether these two families share similar functions. The seven UGT s for which full-length cDNAs were generated in the present study have sequence similarities with members of different UGT families. This finding implied that the UGT s identified in the present study may be members of these different UGT families. Based on the neighbor-joining tree (Figure 5), BcUGT3 was found to be close to members of the UGT73 family, in particular to GmSGT2 (UGT73P2), MtGT3 (UGT73F3) and GeGT (UGT73F1); BcUGT6 was close to a UGT709 member. BcUGT2 and BcUGT7 were also close to UGT73 members and to other UGTs without definite family ascriptions. BcUGT1 was close to a UGT90 member. Previous studies [62, 63] have indicated that UGT73 and UGT90 belong to the same orthologous group, OG1 . UGT73B2 was shown to exhibit flavonoid 7-O-glucosyltransferase activity , UGT73A7 has been reported to exhibit 4, 2', 4', 6'-tetrahydroxy chalcone 4'-glucosyltransferase activity , and UGT90A7 was shown to exhibit luteolin 4'/7-O-glucosyltransferase activity . BcUGT4 and BcUGT5 may belong to the UGT94 family because they have sequence similarities with UGT94s. Previous studies have shown that UGT94D1 has UDP-glucose: sesaminol 2'-O-glucoside-O-glucosyltransferase activity and UGT94F1 has anthocyanin 3-O-glucoside-2''-O-glucosyltransferase activity . Although the definite functions of the seven P450 s and seven UGT s from B. chinense identified in the present study still have to be verified by further experiments, the isolation of their full-length cDNAs will be significant for elucidating their biofunctions in the growth and development of B. chinense.
The biosynthesis and regulation of bioactive components was the main focus of the present study on B. chinense. In addition to SSs isolated from members of the genus Bupleurum that exhibit pharmacological activity, several other groups of secondary metabolites with relevant biological activity have been characterized, for example, polysaccharides with anti-ulcer activity and lignans with anti-proliferative activity . Genes involved in polysaccharides and lignans were searched for in the present 454 dataset. For example, enzymes encoded by genes related to polysaccharides include (1, 3)-beta-D-glucan synthase, alpha-1, 6-xylosyltransferase, alpha-(1, 4)-galacturonosyltransferase, xylan 1, 4-beta-xylosidase, etc.  and enzymes encoded by the genes related to lignans, are phenylalanine ammonia lyase, cinnamate 4-hydroxylase, 4-coumarate-CoA ligase, hydroxycinnamoyl CoA: shikimate/quinate hydroxycinnamoyltransferase, caffeoyl-CoA O-methyltransferase, isoeugenol synthase, and dirigent protein oxidase . Therefore, the present 454 dataset is valuable not only in the exploration of genes involved in SS biosynthesis, but also for the discovery of genes involved in other bioactive secondary metabolites derived from the genus of Bupleurum. Additionally, the agronomical traits of B. chinense, such as drought resistance, have been investigated [71, 72]. In our 454 dataset, 2, 933 and 3, 280 unique sequences were annotated as related to responses to abiotic or biotic stimulus and to stress, respectively. These annotations were based on the GO terms. These sequence data may be beneficial to further molecular studies on the stress response of B. chinense. Further, a total of 415 and 209 unique sequences were annotated with transcription factor activity and signal transduction, respectively. Some of these sequences may play roles in regulating SS metabolism and the stress response. These unique sequences deserve to be cloned and functionally analyzed in future studies.
Currently the 454 pyrosequencing technology is considered as a rapid and economical method to generate high-quantity sequence data. Although a large number of 454 reads were obtained by a quarter run in the present study, nearly a quarter of the ESTs from the Sanger-sequenced 3, 111 clones from our previous cDNA library were not sequenced. The different cDNA libraries (the Sanger sequenced root cDNA library and the 454 sequenced combined cDNA library with roots, seeds, and seedlings) and the fact that only the 5' end of the cDNA was sequenced in the Sanger sequencing may explain, to some extent, this difference. In some reports that compared 454 pyrosequencing and traditional Sanger sequencing, bias was found because of differences in the two sequencing methods . Combinations of these two methods have been used in some studies: (1) to generate a high number of good-quality ESTs with improved clustering analysis and with more full-length sequences ; (2) to obtain a less biased method for the identification and diversity analysis of microbes and fungi [74, 75]; and (3) to assemble genome sequences . Recently, Radix bupleuri has aroused global interest, especially in Europe [review in ]. However, studies on the molecular biology of Bupleurum are still limited. More transcriptome data will facilitate a deeper understanding and enable the rapid development of Radix bupleuri applications.
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. The identification of enzymes involved in SS biosynthesis may enable the regulation and improvement of SS production levels in plants or in microbial hosts by metabolic engineering. Almost all of the known genes that encoded enzymes involved in the biosynthesis of the SS backbones were explored. A total of 246 P450 and 102 GT unique sequences containing 49 UGT s were obtained. These sequences will be invaluable to the elucidation of the SS biosynthetic pathway and to the exploration of the molecular mechanism underlying the biosynthesis of different monomer SSs. The full-length cDNAs of seven of the P450 s and seven of the UGT s from our present 454 dataset and previous Sanger's sequencing data were cloned using the RACE method. This procedure may help in elucidating the functions of the P450 s and UGT s. MeJA inducibility and tissue-specific expression pattern experiments were used to screen two P450 s and three UGT s that may be involved in SS biosynthesis.
Plant material and adventitious root preparation
The roots of one-year old plants of "Zhongchai No. 1", a mass-selected cultivar of B. chinense field-grown in IMPLAD, were collected during the flowering stage because more SSs were found to be contained in the roots during this period . Further, a previous study showed that the SS-d and SS-c content significantly changed in germinating seeds and the content of SS-d peaked on the fourth day . Hence, to acquire a high number of unique candidate genes involved in SS biosynthesis the experimental material used in the present study was 4-day geminating seeds, 12-day seedlings, and the roots of one-year-old Zhongchai No. 1 plants during flowering. The germination was performed in germination boxes under 25°C/15°C, 8L/16D conditions. Before germination, the seeds were soaked for 24 h in tap water, which was changed four times. After harvest, all materials were immediately frozen in liquid nitrogen and stored in a -80°C freezer for RNA extraction.
To analyze the MeJA inducibility of P450s and UGTs, the adventitious roots of Zhongchai No. 1 were cultivated as described earlier . Similar to the results of our previous experiment, the SS content was approximately doubled in 8 h MeJA-treated (200 μM, dissolved in ethanol) adventitious roots of B. chinense, assayed by high performance liquid chromatography . MeJA (200 μM) was then added to the cultivation media; an equal quantity of ethanol was used as the control. After 8 h, the treated and control adventitious roots were collected and immediately stored in liquid nitrogen for RNA extraction. For the tissue-specific expression pattern experiments, five tissues (roots, stems, leaves, flowers, and fruits) were collected and similarly restored as described in our previous report .
RNA extraction, cDNA library construction and 454 sequencing
Total RNA was isolated using an RNA purification kit (Norgen Biotek Corp., ON, Canada). RNA purity and degradation were checked on 1% agarose gels. Equivalent RNAs from roots, germinating seeds, and seedlings were pooled. Approximately 1 μg of RNA was reverse transcribed using a Super SMART™ PCR cDNA synthesis kit (Clontech Laboratories, Inc., Mountain View, CA, USA). This kit was used in combination with a modified poly (T) primer to overcome the limitation of long poly (A/T) tails in cDNA for the 454 sequencing . Double-stranded (ds) cDNA was synthesized using an Advantage® 2 PCR kit (Clontech Laboratories, Inc.) and was then digested overnight with Bsg I (New England Biolabs, Ipswich, MA, USA). The ds cDNA was finally purified using a PureLink™ PCR purification kit (Invitrogen Life Science Technologies, Carlsbad, CA, USA). About 5 μg of ds cDNA was sent to the Roche 454 Company (Branford, CT, USA) for pyrosequencing using a GS FLX titanium kit.
The 454 EST assembly and annotation
A pretreatment process that involved trimming the adapter and poly (A/T), as well as removing short sequence (< 50 bp) and low quality files (quality score threshold = 20) was performed. The Mira 3.0.5 software was used for sequence assembly using the default parameters. Reads that did not fit into a contig were defined as singletons. A total of 195, 088 HQ reads assembled in 22, 748 contigs and 1, 289 singletons were finally obtained for further functional annotation with the BLASTX program. The databases KEGG http://www.genome.jp/kegg/, Nr http://www.ncbi.nlm.nih.gov, and UniProt http://www.expasy.ch/sprot were used for the search. GO terms were assigned to the assembled unique genes based on similarities with A. thaliana protein sequences (TAIR9, http://www.arabidopsis.org). A cut-off value of E < 1.0-10 was used in all BLASTX searches. The newly assembled unique genes were compared against the Bupleurum EST/protein encoding sequences in GenBank. The ESTs were derived from a B. chinense root cDNA library that was sequenced by our group using an ABI 3730 sequencer .
Searching for candidate genes involved SS biosynthesis
The candidate genes HMGR, IPPI, FPS, SQS, SE, β-AS, P450, and UGT that are known to be involved in the biosynthesis of SSs were searched for within the text of the annotated unique genes based on their gene names and synonyms. The items from different annotation databases that were repeated were manually erased.
Full-length cDNA verification and cloning of P450s and UGTs
The assembled full-length P450 and UGT cDNA sequences were verified by RT-PCR. Some partial sequences were extended to full length using 5' and/or 3' RACE. The amino acid sequence alignments of the full-length P450 and UGT cDNAs were created in MEGA 4 using CLUSTALW with default settings. Phylogenetic neighbor-joining trees were constructed and bootstrapped with 1000 iterations in MEGA 4. Corresponding sequences from other plants with the most similarity to each full-length P450 and UGT cDNA (obtained both in our present and previous studies)  were identified and downloaded from GenBank. These sequences were used for the alignments and tree constructions.
Real-time PCR analysis
Actin was chosen as the internal reference gene for the real-time PCR gene expression analysis of MeJA-treated B. kaoi. Similar to our previous report, β-tubulin was the most suitable reference gene for the real-time PCR analysis of tissue-specific gene expression patterns in B. chinense. According to one of our previous experiments (data unpublished), EF1α was also a suitable internal reference gene for real-time PCR analysis in MeJA-treated B. chinense. In the present study, the suitability of actin, β-tubulin, and EF1α as internal reference genes in the MeJA-treated adventitious roots of B. chinense was first determined. Based on the results (see additional file 7: Screening of internal reference genes for real-time PCR analysis of MeJA inducibility), actin was selected as the internal reference gene for the MeJA inducibility experiment. For the tissue-specific expression pattern experiment, β-tubulin was selected as the internal reference gene based on our previous research . All real-time PCR analyses were performed according to our previous report  with the following modifications: the RNA was extracted using an RNA purification kit (Norgen Biotek Corp.); the quantification of cDNA was performed on a NanoDrop ND 2000 spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, DE, USA); and a SYBR® PrimeScript® RT-PCR kit II (Perfect Real-Time; TAKARA Bio Inc., Shiga, Japan) was used for the reverse transcription and real-time PCR. Two-step amplification conditions were used: 3 min at 95°C, 40 cycles of 30 s at 95°C, and 20 s at 58°C. For the analyses of the tissue-specific expression patterns, the expression in the root was arbitrarily chosen as the calibrator for each gene. For the MeJA inducibility experiment, the expression of each gene in the control was used as the calibrator. All primers used are listed in additional file 8: The primers used in the present study.
The authors gratefully acknowledge Huazong Zeng at the Shanghai Sensichip Tech@infor Co. Ltd. for his assistance with bioinfomatics. The present work was supported by the National Natural Science Foundation of China (grant No. 81072994), the Beijing Municipal Natural Science Foundation of China (No. 5102033), and the Research Fund of State Administration of Traditional Chinese Medicine of People's Republic of China (No. 201107011).
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