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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.
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Objective To study the cyfluthrin resistance and potential mechanisms of Anopheles sinensis in Nanchang Chang-bei International Airport,Nanchang City,Jiangxi Province. Methods The resistance levels of the local An. sinensis were de-tected by WHO drug resistance bioassay. During the bioassay,the dying mosquitos were classed as sensitive mosquitos,and the survival ones were classed as resistant mosquitos. The P450 monooxygenase activity and glutathione S-transferase activity were detected and compared between the two groups. At the same time,the death time of each sensitive mosquito was recorded,and the correlations between the death time and the P450 monooxygenase activity and glutathione S-transferase activity were ana-lyzed,respectively. Results The bioassay mortality of the local An. sinensis was 59.5%. The differences of the P450 monooxy-genase activities among the resistant mosquitos,sensitive mosquitos and laboratory sensitive mosquitos had statistical signifi-cances(F=151.89,Psensitive mosquitos>laboratory sensitive mosquitos. The differences of glutathione s-transferase activities among the three groups had no statistical significance(F=0.72,P=0.49). There existed positive correlation between the mosquito death time and the P450 monooxygenase activity,and the regression equation was y=79.479+1.512x with the correlation coefficient of 0.88,while there was no correlation between the mosquito death time and the glutathione S-transferaseactivity. Conclusion The An. sinensis in Nanchang Changbei International Airport has been resistant to cyfluthrin,and the promotion of P450 monooxygenase activity maybe one of the reasons for the resistance.
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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.
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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.
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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.
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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
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This study was performed to find out the influences of ethanol on the metabolism of trichloroethylene(TRI) in rats. TRI in corn oil at the dosage of 150, 300, 600 mg/kg was injected peritoneally once a day for two days to two groups. In one group ethanol(4 g/kg) was taken orally 30 minutes before TRI injection, and the other group ethanol was not. The results of experiments are as follows: 1. The contents of cytochrome P-450 and b5 had inverse relationship with in-jected TRI amounts in both groups. 2. The activity of NADPH P-450 reductase was decreased slowly in TRI injected group related with TRI amount, but decreased drastically in the group pretreated with ethanol. 3. The activity of NADH b5 reductase had relationship with injected TRI amount, but the statistical significance was found only in the groups of 300 and 600 mg/kg of TRI injected without relevance to ethanol when compared with the group that was not injected. 4. The activity of ADH was more decreased and ALDH activity was more increased in groups that TRI injected and ethanol was pretreated with ethanol groups than in group without any treatment. These results suggest that ethanol may inhibit epoxide formulation, the first step or TRI metabolism, and change from TCE-OH to TCA also.