Metabolic Engineering of for High-Level Friedelin Genetic Manipulation.

Friedelin, the most rearranged pentacyclic triterpene, also exhibits remarkable pharmacological and anti-insect activities. In particular, celastrol with friedelin as the skeleton, which is derived from the medicinal plant , is a promising drug due to its anticancer and antiobesity activities. Although a previous study achieved friedelin production using engineered , strains capable of producing high-level friedelin have not been stably engineered.

[Discussion on research idea of quality marker of Salvia miltiorrhiza based on biosynthetic pathway of tanshinone compounds].

Based on the theory of Q-marker, the hairy root of Salvia miltiorrhiza and S. miltiorrhiza in many provinces were studied. The relative expressions of SmCPS, SmKSL and CYP76AH1 genes in hairy roots were detected by real-time fluorescence quantitative PCR and the contents of tanshinoneⅡ_A, cryptotanshinone, tanshinoneⅠ, 1,2-dihydrotanshinone, ferruginol and miltiradiene were detected by UPLC and GC-MS, respectively.

[Effect of GR24 on accumulation of diterpenoids in Tripterygium wilfordii suspension cells].

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.

Differential expression of the TwHMGS gene and its effect on triptolide biosynthesis in Tripterygium wilfordii.

3-Hydroxy-3-methylglutaryl-CoA synthase (HMGS) is the first committed enzyme in the MVA pathway and involved in the biosynthesis of terpenes in Tripterygium wilfordii. The full-length cDNA and a 515 bp RNAi target fragment of TwHMGS were ligated into the pH7WG2D and pK7GWIWG2D vectors to respectively overexpress and silence, TwHMGS was overexpressed and silenced in T. wilfordii suspension cells using biolistic-gun mediated transformation, which resulted in 2-fold increase and a drop to 70% in the expression level compared to cells with empty vector controls.

Isolation and characterization of a glycosyltransferase with specific catalytic activity towards flavonoids from .

Flavonoids are important secondary metabolites that exist in many medicinal plants. Flavonoid glycosyltransferases can transfer sugar moieties to their parent rings, producing various flavonoid glycosides with significant pharmacological activities. Here, we report the molecular cloning of the O-glycosyltransferase TwUGT2 from and its catalytic activity was explored by heterologous expression in .

Rapid discovery and functional characterization of diterpene synthases from basidiomycete fungi by genome mining.

Basidiomycete fungi are a rich source of bioactive diterpenoid secondary metabolites. However, compared with the large number of diterpene synthases (di-TPSs) identified in plants and ascomycete fungi, only three di-TPSs have been described from basidiomycete fungi. Large scale genome sequencing projects combined with the development of synthetic biology techniques now has enabled the rapidly discovery and characterization of di-TPSs from basidiomycete fungi.

Eudesmane-type sesquiterpene diols directly synthesized by a sesquiterpene cyclase in .

Cryptomeridiol, a typical eudesmane diol, is the active principle component of the antispasmodic Proximol. Although it has been used for many years, the biosynthesis pathway of cryptomeridiol has remained blur. Among terpenoid natural products, terpenoid cyclases are responsible for cyclization and generation of hydrocarbon backbones.

[Cloning and protein expression analysis of monoterpene synthase gene TwMS in Tripterygium wilfordii].

In this study, we cloned a monoterpene synthases, TwMS from Tripterygium wilfordii suspension cells. TwMS gene contained a 1 797 bp open reading frame (ORF), encoding a polypeptide of 579 amino acids, which deduced isoelectric point (pI) was 6.10 and the calculated molecular weight was 69.

[Cloning and protein expression analysis of geranyl diphosphate synthase genes in Tripterygium wilfordii].

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.

[Cloning and expression analysis of kaurenoic acid oxidase gene in Tripterygium wilfordii].

Kaurenoic acid oxidase involved in biosynthesis pathway of gibberellin. According to the transcriptome database, the specific primers were designed and used in cloning the full-length cDNA of TwKAO, the bioinformatic analysis of the sequence was performed. The qRT-PCR were used to detect the expression level of TwKAO after MeJA treatment.

[Prospecting application of CRISPR/Cas9 genome editing technology in research of medicinal plants].

CRISPR/Cas9 genome editing technology is a newly discovered genome editing technology in recent years.It has been widely used in many fields such as gene therapy, gene function research, animal model-making,cropvariety improvement and so on. This article briefly introduces the CRISPR/Cas9 genome editing system, and discusses its potential applications in the research of medicinal plants, including functional genomics studies, secondary metabolism and synthetic biology research of important active components and molecular breeding research.

[Effect of PDK1 on Neutrophil Differentiation].

Objective: To knock out PDK1 by using Vav-Cre and observe the effects of PDK1 knock out on the ratio, number and differentiation of neutrophil.

Methods: The PDK1 expression level of Vav-Cre;PDK1 mouse in the bone marrow cells was analyzed by RT-PCR. The effect of PDK1 on hematopoietic progenitor was observed by CFU-C assay, and the effect of PDK1 on the ratio and number of neutrophil was detected by flow cytometric analysis.

Cloning and functional analysis of two sterol-C24-methyltransferase 1 (SMT1) genes from Paris polyphylla.

The biosynthetic pathways of phytosterols and steroidal saponins are located in two adjacent branches which share cycloartenol as substrate. The rate-limiting enzyme S-adenosyl-L-methionine-sterol-C24-methyltransferase 1 (SMT1) facilitates the metabolic flux toward phytosterols. It catalyzes the methylation of the cycloartenol in the side chain of the C24-alkyl group, to generate 24(28)-methylene cycloartenol.

[Cloning and protein expression of sterol-C-24-methyl transferase 2 in Tripterygium wilfordii].

24-Alkyl sterols are the major players in the control of membrane component and plant growth. In this paper, we cloned an important rate-limiting enzyme: sterol-C-24-methyl transferase (SMT) in the sterol biosynthetic pathway according to the transcriptome data of Tripterygium wilfordii. suspension cells, whose full-length c DNA was 1 631 bp with an open reading frame of 1 080 bp, encoding a protein of 359 amino acids.

[Cloning and protein expression of sterol-C-24-methyl transferase 2 in Tripterygium wilfordii].

24-Alkyl sterols are the major players in the control of membrane component and plant growth. In this paper, we cloned an important rate-limiting enzyme: sterol-C-24-methyl transferase (SMT) in the sterol biosynthetic pathway according to the transcriptome data of Tripterygium wilfordii. suspension cells, whose full-length cDNA was 1 631 bp with an open reading frame of 1 080 bp, encoding a protein of 359 amino acids.

[ADAR1 Knockout Inhibits Notch1-induced T-ALL in Mice].

Objective: To investigate the effect of ADAR1 on the occurrence and development of mouse T cell acute lymphoblastic leukemia (T-ALL).

Methods: Lck-Cre; ADAR1lox/lox mice were generated through interbreeding. The lineage-cells of Lck-Cre; ADAR1lox/lox mice and the control were enriched respectively by the means of MACS, and the lin- cells were transfected with retrovirus carrying MSCV-ICN1-IRES-GFP fusion gene.

[Effect of PDK1 on Notch1-Induced Mouse T-cell Acute Lymphoblastic Leukemia].

Objective: To explore the role of PDK1 in T-ALL development through establishing the Notch1-induced T-ALL mouse model by using Mx1-cre; LoxP system to knock-out PDK1.

Methods: Cell cycle and apoptosis of leukemic cells were detected by flow cytometry, and relative expression of tumor-related genes and transcription factors of leukemic cells were determined by quantitative real-time PCR.

Results: Notch1-induced T-ALL mouse model with inducible knock-out of PDK1 was established successfully.

[Cloning and expression analysis of 4- (cytidine-5-diphospho) -2-C-methyl-D-erythritol kinase gene in Tripterygium wilfordii].

4-(Cytidine-5-diphospho) -2-C-methyl-D-erythritol kinase is a key enzyme in the biosynthesis pathway of terpenoids. According to the transcriptome database, the specific primers were designed and used in PCR. The bioinformatic analysis of the sequenced TwCMK gene was performed in several bioinformatics software.

[Cloning and expression analysis of squalene synthase gene in Tripterygium wilfordii].

In this paper, we cloned the full-length cDNA of TwSQS from Tripterygium wilfordii suspension cells(Gen Bank: KR401220) and performed the bioinformation and m RNA expression analysis. The expression after methyl jasmonate(MJ) treatment of the gene was detected by RT-PCR. The full-length cDNA of TwSQS was 1 800 bp containing a 1 242 bp open reading frame(ORF) encoding a polypeptide of 413 amino acids.

The MVA pathway genes expressions and accumulation of celastrol in Tripterygium wilfordii suspension cells in response to methyl jasmonate treatment.

Celastrol is an important bioactive triterpenoid in traditional Chinese medicinal plant, Tripterygium wilfordii. Methyl Jasmonate (MJ) is a common plant hormone which can regulate the secondary metabolism in higher plants. In this study, the mevalonate (MVA) pathway genes in T.

Research progress of synthetic biology for tanshinones.

Synthetic biology research methods which design and build a new artificial biological systems (medicinal plants or microorganisms system) with specific physiological functions through clarifying and simulating the basic law of the biosynthesis of active components of traditional Chinese medicine, is considered to be a potential method to produce an abundant resources of bioactive components. Tanshinones is a kind of diterpene quinone compounds with important pharmacological activities from traditional Chinese medicine Salvia miltiorrhiza. This article systematically introduced the research progress of the synthetic biology of S.

Identification of geranylgeranyl diphosphate synthase genes from Tripterygium wilfordii.

We found triptolide synthesis is correlated with the expressions of TwGGPPS1 and TwGGPPS4 . This lays the foundation for future studies of biosynthetic pathways for triptolide and other diterpenoids in T. wilfordii.

[Cloning and expression analysis of a acetyl-CoA U-acetyltransferase gene (TwAACT) from Tripterygium wilfordii].

In this study, based on the transcriptome data, we cloned the full-length cDNAs of TwAACT gene from Tripterygium wilfordii suspension cells, and then analyzed the bioinformation of the sequence, detected the genetic differential expression after being induced by methyl jasmonate (MeJA) by RT-PCR. The full-length cDNA of the TwAACT was 1 704 bp containing a 1 218 bp open reading frame (ORF) encoding a polypeptide of 405 amino acids (GeneBank accession No. KP297934).

Surgical treatment of incidental gallbladder cancer discovered during or following laparoscopic cholecystectomy.

Background: The optimal surgical management of patients with incidental gallbladder cancer (IGBC) and their long-term survival remains unclear.

Objective: The purpose of this study was to examine the long-term prognosis of patients with IGBC diagnosed during or after LC.

Methods: Between January 2002 and January 2012, a total of 7,582 consecutive patients underwent LC for presumed gallbladder benign disease in the Chinese PLA General Hospital, China.