Reference genes screening and spatio-temporal expression analyses of cytochrome P450 monoxoygenase genes in Polygala tenuifolia by qRT-PCR / 中草药

Objective: To provide a suitable expression analysis of Polygala tenuifolia in different growth years and tissues, so as to select the stable internal control genes. The spatial and temporal expression of four cytochrome P450 monooxygenase genes in the above-mentioned samples was analyzed. Methods: The soluble curve and Ct value of eight candidate internal reference genes including Tubulin 1, Tubulin 2, Elongation 1, Elongation 2, Actin 1, Actin 2 and cdc-42 in different growth years (1-3 years old) and tissues (root,stem, leaf, and flower) of P. tenuifolia were obtained by real-time quantitative PCR (qRT-PCR). The expression stability of candidate reference genes was assessed by geNorm and NormFinder. Then, the qRT-PCR technique was also used to identify the temporal and spatial expression of four P450 genes including CYP709B2, CYP71AP39, CYP88A85 and CYP714E38 in P. tenuifolia. Results: The geNorm results showed that the stable internal reference genes expressed in P. tenuifolia were Tubulin 2 and Elongation 1. The NormFinder results showed that the most stable and suitable internal reference gene for expression analysis was Elongation 1. The mRNA expression levels of CYP709B2 and CYP71AP39 genes in stems and leaves (1-3 years old) were higher than that in roots and flowers. The CYP88A85 and CYP714E38 genes had got a higher mRNA expression level in roots (1-3 years old). Conclusion: Elongation 1 is suitable as an ideal internal control gene for qRT-PCR analysis of P. tenuifolia. The expression trend of P450s in roots, stems, leaves and flowers of cultivated P. tenuifolia is inconsistent.

HMGR, SQS, β-AS, and Cytochrome P450 Monooxygenase Genes in Glycyrrhiza uralensis / 中草药·英文版

Glycyrrhiza uralensis is frequently used in traditional Chinese medicine. This plant contains a large amount of effective constituents, including triterpenoids and flavonoids. Among them, glycyrrhizin is believed to be the marker compound to evaluate the quality of G. uralensis based on Chinese Pharmacopoeia. Many studies showed that glycyrrhizin possesses various pharmacological activities, such as antibacterial, antiviral, antitumor, anti-inflammatory, and immune-stimulating activities. In this paper, we summarized the cloning, characterization, expression, and polymorphism analysis of several functional genes involved in glycyrrhizin biosynthesis in G. uralensis.

Research Advances on Triterpenoid Saponins Biosynthesis and Its Key Enzymes / 世界科学技术-中医药现代化

As an important kind of plant secondary metabolites and widely distributed in the plant kingdom, triter-penoid saponins have a variety of biological activities. The constitution and content of triterpenoid saponin are deter-mined by some key enzymes and their expressing level in triterpenoid saponins biosynthesis pathway. So, it is very important to illuminate the molecular mechanism of triterpenoid saponins biosynthetic pathway. In recent years, illu-mination of the entire biosynthetic pathway especially the confirmation and cloning of the key enzymes, such as squa-lene synthase, squalene epoxidase, cytochrome P450 monooxygenase and UDP-glycosyltransferase, had become one of the hot spots by many scholars. In this paper, the entire biosynthetic pathway and some kinds of key enzymes in-volved in the synthesis of carbon skeleton, and its oxidation, and glycation were reviewed for further demonstrating the biosynthetic pathway of triterpenoid saponins and providing a theoretical basis for artificial biosynthesis.

Construction of a Saccharopolyspora erythraea mutant producing erythromycin B as its major fermentation product / 军事医学科学院院刊

Objective:To obtain a large amount of erythromycin B and to investigate the activity site in eryK. Methods:The key sequence of the BC loop region in eryK gene was knocked out and the eryK gene with 101 bp deleted was amplified by overlapping PCR,and cloned into vector pWHM3 to construct recombinant plasmid. The Saccharopolyspora erythraea mutant AK17 was constructed through chromosomal homologous recombination technique.Results and Conclusions:The S.erythraea mutant AK17 was constructed. The results of TCL and MS analysis showed that the major fermentation product of AK17 is erythromycin B.

Distribution of CYP2J3 in rats of gene transfection through vena dorsalis penis / 基础医学与临床

Objective To detect CYP2J3 gene expression and contents of 11,12-EET in heart,liver,lung,kidney and aorta thoracalis after CYP2J3 gene transfection.Methods The rat transgenic model was developed by injecting plasmid through vena dorsalis penis.The animals were divided into control group、 pcDNA3.1 transgenic group and pcDNA3.1-CYP2J3 transgenic group.The expression of CYP2J3 mRNA was detected by RT-PCR and content of 11,12-EET was examined by the HPLC at 14 days and 28 days after injection.Results Twenty eight days after injection,both expression of CYP2J3 mRNA and the content of 11,12-EET were significantly increased as compared with that of control and pcDNA3.1 transgenic group(P