Buplewrum falcatum is a traditional Chinese medicine,which is mainly used for the treatment of cold and liver protection. B. falcatum is dominantly cultivated in Japan as well as planted in China,Korea and other countries and regions. In order to determine the appropriate sequencing strategy,the genome survey before large-scale genome sequencing is needed. This survey can provide information about the size and complexity of the whole genome of the target species. In the present study,the next generation sequencing technology( Illumina Hiseq 2000) was used to analyze the genome size and complexity of B. falcatum. In addition,SSR loci were analyzed from the sequenced data. Primer 3 was used to design specific primers and 33 pairs of primers were randomly selected for PCR with template DNA of B. falcatum,and the PCR system and optimal annealing temperature were screened. A total of 288. 64 G genome sequence data was obtained,and the estimated genome size of B. falcatum was 2 119. 58 Mb. The measured genome data depth was138×; the rate of heterozygosity was 1. 84%; and the ratio of repeat sequence was 83. 89%. It is speculated that the genome of B. falcatum is complex. The preliminary assembly was performed with K-mer = 41,and the contig N50 was 224 bp,the total length 896. 97 Mb,the scaffold N50 313 bp,and the total length was 922. 67 Mb. A total of 91 377 SSR sequences were detected in the sequenced genome data which were distributed in 70 809 unigenes.The main type is dinucleotide repeats,with 49 680 sequences,accounting for70. 16%. Among the 33 pairs of primers randomly synthesized according to the obtained SSR sequences,21 pairs were successfully amplifying the target sequences. The results will be helpful for later large scale genome sequencing and SSR molecular markers development for germplasm identification and trait mapping.
Bupleurum/genetics* ; Genome, Plant ; Microsatellite Repeats ; Plants, Medicinal/genetics* ; Polymorphism, Genetic

The tissue-specific and MeJA-induced transcriptional levels of BcUGT3 and BcUGT6 in Bupleurum chinense were analyzed in the present study. The transcriptional levels of BcUGT3 in root, leaf, flower and fruit were similar and they all were higher than those in stem. The transcriptional level of BcUGT6 was the highest in leaf and the lowest in flower among in all tested tissues. With non-treated adventitious roots as control, BcUGT6's transcriptional levels were elevated to nearly 2 folds for 2 h, 8 h, 24 h, 2 d and 4 d in MeJA-treated adventitious roots of B. chinense. It showed that the transcriptional level of BcUGT6 was slightly affected by MeJA. While, BcUGT3's transcriptional levels were gradually elevated, and till 4 d after MeJA treatment, the expression level was about 7 folds than that of non-treated control. Using pET-28a (+), the expressions of two genes was investigated. Induced by IPTG, the target proteins were expressed in E. coli and then purified. All the results obtained in the present study will be helpful for follow-up bio-function analysis of BcUGT3 and BcUGT6.
Acetates ; pharmacology ; Bupleurum ; cytology ; enzymology ; genetics ; Cell Membrane ; metabolism ; Cyclopentanes ; pharmacology ; Escherichia coli ; genetics ; Gene Expression ; Gene Expression Regulation, Plant ; drug effects ; Hexosyltransferases ; chemistry ; genetics ; isolation & purification ; metabolism ; Intracellular Space ; metabolism ; Oxylipins ; pharmacology ; Protein Sorting Signals ; Protein Structure, Secondary ; Protein Transport ; Sequence Analysis ; Transcription, Genetic ; drug effects

In this study, ITS2 barcode was used to identify Bupleurum chinense and B. longiradiatum. The ITS2 regions of 48 samples were amplified and sequenced. The sequences obtained above were aligned and the K2P distances were calculated. We used three methods, BLAST1, nearest distance and phylogenetic tree (NJ-tree), to test the identification ability. The results showed that the maximum intraspecific genetic distance of B. chinense was 0.013, and the minimum interspecific genetic distance between B. chinense and B. longiradiatum was 0.049. The NJ-tree can easily identify B. chinense and B. longiradiatum. Therefore, the ITS2 barcode is suitable to identify B. chinense and B. longiradiatum.
Bupleurum ; classification ; genetics ; DNA Barcoding, Taxonomic ; methods ; DNA, Plant ; genetics ; DNA, Ribosomal Spacer ; genetics ; Drugs, Chinese Herbal ; chemistry ; classification ; Molecular Sequence Data ; Phylogeny ; Quality Control

In this study, the induction of hairy roots of Bupleurum chinense DC. was explored and established after experiments at different conditions: A. rhizogenes A4 was used to infect the leaves bases of B. chinense tube seedlings. The explants were co-cultured on Phytagel-solidified media for 3 days and then, were turned into solid media, similar with the co-culture media except that bacteriostat was added. After 10 days, rootlets began to appear and after 4 to 5 weeks, rootlets can be converted into liquid shaking culture stage. Plants regeneration from hairy root was useful for the research of new germplasm production and the variety improvement breeding. In the present study, the regenerated plants were obtained. One approach was to continuously culture under light conditions the seedlings which parting off spontaneously from the hairy roots during liquid shaking culture. The other approach was to culture the callus-like tissues produced by hairy roots with the optimized regeneration media for the induction of regenerated plants. The results of present study provide a technique to induce hairy roots and plantlet regeneration of B. chinense and this technique is helpful for the researches on metabolism, especially on the Agrobacterium-mediated genetic transformation of B. chinense.
Agrobacterium ; genetics ; Bupleurum ; genetics ; growth & development ; Coculture Techniques ; Plant Leaves ; genetics ; growth & development ; Plant Roots ; genetics ; growth & development ; Plants, Genetically Modified ; genetics ; growth & development ; Plants, Medicinal ; genetics ; growth & development ; Regeneration ; Transformation, Genetic

The ORF sequence of glycosyltransferase gene BcUGT1 cloned from Bupleurum chinense DC. was analyzed and its three dimentional structure was predicted. Using qRT-PCR method, the expression characteristics of BcUGT1 after methyl jasmonate (MeJA) induction and in different plant tissues were investigated. The results showed that BcUGT1 may be involved in saikosaponin biosynthesis in B. chinense. Thereafter, the recombinant vectors of BcUGT1 were constructed for its expression in E. coli. The target protein was successfully expressed and purified. In the present study, three vectors, pRSET-A, pET-28a (+) and pET-30a (+), and three isolates of E. coli, BL21 (DE3) plysS, BL21A1 and BL21-CodonPlus (DE3)-RIPL were used under different induction conditions, such as different concentrations and during times of inducers (L-arabinose and IPTG) and different inducing temperatures. The results showed that in the condition of 0.5 or 1 mmol x L(-1) IPTG, 16 degrees C, 20 h, target protein expressed in BL21-CodonPlus (DE3)-RIPL with pET-28a (+) or pET-30a (+) as vector. Using PrepEase His-tagged protein purification kit, the target protein was purified. The present work will be helpful for follow-up bio-function analysis of BcUGT1.
Amino Acid Sequence ; Base Sequence ; Bupleurum ; chemistry ; Cloning, Molecular ; DNA, Complementary ; genetics ; DNA, Plant ; genetics ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Glycosyltransferases ; genetics ; isolation & purification ; metabolism ; Oleanolic Acid ; analogs & derivatives ; biosynthesis ; Open Reading Frames ; genetics ; Phylogeny ; Plants, Medicinal ; chemistry ; Protein Structure, Secondary ; Recombinant Fusion Proteins ; genetics ; metabolism ; Saponins ; biosynthesis

OBJECTIVETo clone the full-length cDNA of a uridine diphosphate glycosyltransferase (UGT) gene which may be involved in saikosaponin biosynthesis of Bupleurum chinense, and construct the transgenic vectors for over expression and RNAi of the cloned UGT. These works will provide foundation for further its function analysis by transgene study.

METHODRAGE and LD-PCR were used to clone the full-length cDNA of the UGT, on the basis of its partial cDNA sequence obtained from our previous 454-sequenced dataset. The ORF and partial sequences of the UGT were PCR cloned using primers with corresponding restriction enzymes cutting sites. The PCR products were digested with corresponding restriction enzymes and then were inserted into pCAMBIA-SUPER 1 300 and pHANNIBAL. The recombinant pHANNIBAL were digested with Not I and then were inserted into a binary vector, pART27. Finally, the transgenic vectors for over expression and RNAi of the cloned UGT were constructed.

RESULTThe full-length cDNA of a UGT were cloned from B. chinense. The recombinant vectors for over expression and RNAi of the UGT were obtained.

CONCLUSIONOur works on full-length cDNA cloning and transgenic vectors construction provide a substantial foundation for follow-up biofunction analysis of the UGT through transgenic research.

Amino Acid Sequence ; Bupleurum ; genetics ; Cloning, Molecular ; Genetic Vectors ; Glucuronosyltransferase ; chemistry ; genetics ; Molecular Sequence Data ; RNA Interference ; Transgenes

OBJECTIVETo explore the genetic evolutionary distance between plants by using karyotype parameters identification of medicinal plants.

METHODThe cluster analysis of karyotype resemblance-near coefficient and evolutionary distance was used for 6 Bupleurum species.

RESULTThe results showed that there were the biggest karyotype resemblance-near coefficient (0.9920) and the smallest evolutionary distance (D(e) = 0.0080) between B. scorzonerifolium and B. chinense, indicating the closest relationship, and the minimum karyotype resemblance-near coefficient (0.4794) and the maximum evolutionary distance (D(e) = 0.7352) between B. smityii and B. falcatum, indicating the most distant relationship.

CONCLUSIONKaryotype was an important parameter for identification of medicinal plants because karyotype was stabilized for species. The genetic distance between in 6 species of Bupleurum species was obtained by karyotype clustering analysis of karyotype resemblance-near coefficient. There was the bigger evolutionary distance between the species which had different chromosome number.

Bupleurum ; classification ; genetics ; Cluster Analysis ; Karyotype

OBJECTIVETo breed new varieties with better uniformity and commercial quality as well as higher saikosaponin contents.

METHODThe excellent germplasm resources were selected from "zhongchai no. 1" population. Single plant method was applied to get better varieties. All the breeding material was investigated according to morphological characters, agronomic characters and the contents of saikosaponin a and saikosaponin d. The experiments of comparative test and varieties regional test were carried out.

RESULTThe bred new varieties of "zhongchai No. 2" and "zhongchai No. 3" had better uniformity. The dark brown roots ratios of the two varieties were 83.2%, 89.9%, respectively. The contents of saikosaponins (a + d) of the two varieties reached 1.31%, 1.02%, respectively.

CONCLUSION"zhongchai No. 2" and " zhongchai No. 3" both had the advantages of better uniformity, darker brown roots and higher saikosaponin contents.

Breeding ; Bupleurum ; chemistry ; genetics ; growth & development ; Oleanolic Acid ; analogs & derivatives ; analysis ; Plant Extracts ; analysis ; Saponins ; analysis

Molecular genetic map is a fundamental organizational tool for genomic research. However, a genetic linkage map for Bupleurum chinense DC. has not been developed. In this study, with the theory of pseudo-testcross, 96 F1 plants from an intraspecific cross of B. chinense were used as mapping populations. Twenty eight ISSR (inter-simple sequence repeat) primers and 44 SSR (simple sequence repeat) primers were used to detect the polymorphisms between the parental plants, and of them, 28 ISSRs and 14 SSRs were selected to analyze the F1 populations. The map consisted of 13 linkage groups which included 80 (72 ISSRs and 8 SSRs) loci, and covered 2 633.9 cM with an average density of 33.4 cM. All 13 linkage groups consisted of 2-31 loci ranging in length from 15.4-1295.7 cM. This map will provide a basis for studies on gene mapping, map-based cloning and maker-assisted selection of important traits in B. chinense.
Bupleurum ; genetics ; Chromosome Mapping ; methods ; DNA, Plant ; genetics ; Genetic Linkage ; Microsatellite Repeats ; Plants, Medicinal ; genetics ; Polymorphism, Genetic

The different growing habitats of Bupleurum chinense were investigated in Donglin mountain & Wuling mountain areas, the saikosaponin a and d in samples of B. chinense collected from different habitats were determined by HPLC. Results showed that B. chinense distributed in various habitats, such as meadow, understory and brushy. Significant differences of saikosaponin contents were observed. The higher saikosaponins contents were showed in samples from meadow habitats, while the lower saikosaponins contents in samples from understory and brushy habitats. The ventilation situation and light condition showed positive correlation with the saikosaponins accumulation in B. chinense. It could be concluded that growing habitats play an important role in accumulation of saikosaponins in B. chinense.
Bupleurum ; chemistry ; China ; Chromatography, High Pressure Liquid ; DNA Primers ; genetics ; Ecosystem ; Oleanolic Acid ; analogs & derivatives ; analysis ; Saponins ; analysis