Identification and functional characterization of 2,3-oxidosqualene cyclase genes family in Tripterygium wilfordii / 药学学报

italic>Tripterygium wilfordii Hook. f. is a valuable medicinal plant, with anti-tumor, anti-inflammatory, immunosuppressive and other pharmacological activities. Triterpenoids are one of the main active components that exert pharmacological effects. However, the content of triterpenoids dominated by triptolide is very low in Tripterygium wilfordii, and the analysis of the biosynthetic pathway of triterpenoids in Tripterygium wilfordii provides an effective new idea for obtaining these compounds. 2,3-Oxidosqualene cyclases (OSCs) are the key enzyme that catalyzes the formation of triterpene skeleton diversity. Based on the genome and transcriptome data of Tripterygium wilfordii, 16 OSC genes were identified and analyzed. Phylogenetic analysis showed that 16 TwOSC proteins could be mainly classified as four groups. They are β-amyrin synthase group, friedelin synthase group, multifunctional amyrin synthase and cycloartenol synthase group. TwOSC6 was successfully cloned. Functional characterization analysis revealed that TwOSC6 can catalyze the formation of α-amyrin and β-amyrin. This indicates that TwOSC6 is a multifunctional amyrin synthase. This provides new gene resources for the diversity of Tripterygium wilfordii triterpenoids, as well as new gene elements for biosynthesis triterpenoids.

Effect of GR24 on accumulation of diterpenoids in Tripterygium wilfordii suspension cells / 中国中药杂志

Terpenoids are main bioactive components in Tripterygium wilfordii,but the contents of some terpenoids are relatively low. In order to provide scientific evidence for the regulation of terpenoids in T. wilfordii,this research explored the effect of GR24 on accumulations of four diterpenoids( triptolide,tripterifordin,triptophenolide,and triptinin B) in T. wilfordii suspension cells by biological technology and UPLC-QQQ-MS/MS. The results indicated that 100 μmol·L-1 GR24 inhibited the accumulations of triptolide,tripterifordin,triptophenolide,and triptinin B to different degrees. Compared with the control group,the contents of 4 diterpenoids( in the induced group) were down to 96.59%,63.80%,61.02% and 33.59% in 240 h,respectively. Among them,the accumulation of triptinin B iswas significantly inhibited. In addition,the key time point of inhibitory effect was 120 h after induction with GR24 in some diterpenoids. This is the first systematic study focusing on the effect of GR24 on the accumulations of diterpenoids in T. wilfordii suspension cells. The dynamic accumulation ruleregularity of four diterpenoids after induced by GR24 was summarized,which laid a foundation for further study on the chemical response mechanism of terpenoids to GR24.

Cloning and protein expression analysis of geranyl diphosphate synthase genes in Tripterygium wilfordii / 中国中药杂志

Based on the transcriptome data, the study cloned full-length cDNA of TwGPPS1 and TwGPPS2 genes from Tripterygium wilfordii suspension cells and then analyzed the bioinformation of the sequence and protein expression. The cloned TwGPPS1 has a 1 278 bp open reading frame (ORF) encoding a polypeptide of 425 amino acids. The deduced isoelectric point (pI) was 6.68, a calculated molecular weight was about 47.189 kDa. The full-length cDNA of the TwGPPS2 contains a 1 269 bp open reading frame (ORF) encoding a polypeptide of 422 amino acids. The deduced isoelectric point (pI) was 6.71, a calculated molecular weight was about 46.774 kDa.The entire reading frame of TwGPPS1,2 was cloned into the pET-32a(+) vector and expressed in E. coli BL21 (DE3) cells to obtain the TwGPPS protein, which laid a basis for further study on the regulation of terpenoid secondary metabolism and biological synthesis.

Molecular authentication of Jinyinhua formula granule by using allele-specific PCR / 中国中药杂志

Traditional authentication method is hard to identify herb's authenticity of traditional Chinese medicine(TCM) formula granules because they have lost all their morphological characteristics. In this study, a new allele-specific PCR method was established for identifying the authentication of Jinyinhua formula granule (made from Lonicerae Japonicae Flos) based on an SNP site in trnL-trnF fragment. Genomic DNA was successfully extracted from Lonicerae Japonicae Flos and its formula granules by using an improved spin column method and then PCR was performed with the designed primer. Approximately 110 bp specific bands was obtained only in the authentic Lonicerae Japonicae Flos and its formula granules, while no bands were found in fake mixed products. In addition, the PCR product sequence was proved from Lonicerae Japonicae Flos trnL-trnF sequence by using BLAST method. Therefore, DNA molecular authentication method could make up the limitations of character identification method and microscopic identification, and quickly identify herb's authenticity of TCM formula granules, with enormous potential for market supervision and quality control.