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ObjectiveAgrobacterium tumefaciens-mediated transformation （ATMT） of Clonostachys rosea， an endophytic fungus of Glycyrrhiza uralensis seeds， was established and optimized， and orthogonal test was designed to optimize the colonization conditions of C. rosea for G. uralensis seeds， so as to lay foundation for the development of biofertilizer and the breeding of high-quality G. uralensis. MethodThe conditions of ATMT were optimized from three aspects， including the concentration of acetosyringone， co-culture time and the concentration of conidia of recipient fungi. Then， high-quality transformants were selected. Orthogonal test was used to optimize the colonization conditions by taking co-culture temperature， co-culture time and spore concentration as factors and colonization rate as index. ResultWhen spore concentration was 1×107 cfu·mL-1， acetosyringone concentration was 150 μmol·L-1 and the co-culture time was 60 h， the transformation efficiency of C. rosea was the highest， which was 135 transformants per 1×107 recipient fungal spores. The accuracy and stability of the transformations were tested by cloning the marker gene green fluorescent protein （GFP） and β-glucuronidase （GUS） staining. When co-culture temperature was 25 ℃， co-culture time was 36 h and the spore concentration was 1×106 cfu·mL-1， the colonizing rate for C. rosea back dyeing into G. uralensis seeds by seed soaking method was the highest， which was 71.11%. ConclusionThis study successfully establishes stable and efficient technical systems not only of ATMT in C. rosea， but also of colonization of the transformants into G. uralensis seeds， which can lay a foundation for the development of biofertilizer of G. uralensis.
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In traditional oral practice, the presystemic interactions with gut microbiota is an important mechanism underlying the holistic health benefits of Chinese herbal medicines (CHMs), making the study of CHMs distinct from the research of Western medicines of which the systemic exposure (level in blood) is the starting point and the core. Gut microbial metabolism complements host metabolism in maintaining metabolic homeostasis of many biologically important endogenous molecules and the disposition of numerous exogenous compounds. Among them, the widely distributed gut bacterial β-glucuronidases (BGUSs) coordinate with host UDP-glucuronosyltransferases (UGTs) to play a role in the occurrence and intervention of diseases by affecting the glucuronidation homeostasis and altering the intestinal local and/or systemic exposure of endogenous compounds and xenobiotics. On one hand, many ingredients of CHMs undergo enterohepatic circulation; On the other hand, CHMs can act on BGUSs directly or indirectly change the distribution and function of BGUSs through reprogramming gut microbiome. The multiple interactions between BGUSs and CHMs may play an important role in the overall therapeutic benefits of CHMs. This work firstly summarizes the latest research progress on BGUSs; then the physiological, pathological and pharmacological significance of BGUSs are exemplified with representative endogenous and exogenous compounds from the aspects of nutrient utilization, metabolic homeostasis, and therapeutic response based on the varied substrate spectra of BGUSs; finally, the scattered data in literature were integrated to summarize the multiple interactions between BGUSs and CHMs, highlighting the important role of BGUSs in the holistic actions of CHMs.
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An efficient genetic transformation system and suitable promoters are essential prerequisites for gene expression studies and genetic engineering in streptomycetes. In this study, firstly, a genetic transformation system based on intergeneric conjugation was developed in Streptomyces rimosus M527, a bacterial strain which exhibits strong antagonistic activity against a broad range of plant-pathogenic fungi. Some experimental parameters involved in this procedure were optimized, including the conjugative media, ratio of donor to recipient, heat shock temperature, and incubation time of mixed culture. Under the optimal conditions, a maximal conjugation frequency of 3.05×10-5 per recipient was obtained. Subsequently, based on the above developed and optimized transformation system, the synthetic promoters SPL-21 and SPL-57, a native promoter potrB, and a constitutive promoter permE" commonly used for gene expression in streptomycetes were selected and their activity was analyzed using gusA as a reporter gene in S. rimosus M527. Among the four tested promoters, SPL-21 exhibited the strongest expression activity and gave rise to a 2.2-fold increase in (3-glucuronidase (GUS) activity compared with the control promoter permE*. Promoter SPL-57 showed activity comparable to that of permE*. Promoter potrB, which showed the lowest activity, showed a 50% decrease in GUS activity compared with the control permE*. The transformation system developed in this study and the tested promotors provide a basis for the further modification of S. rimosus M527.
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Objective:To summarize the structure, properties and biological functions ofβ-glucuronidase. Methods:By referring to the relevant literatures at home and abroad onβ-glucuronidase in recent years, this paper induced, analyzed and drew conclusions. Results:The crystal structure of humanβ-glucuronidase is comprised of three distinct structural domains. The most common mutation of β-glucuronidase is missense mutation, which results in the change of enzyme activity, and then induces a series of pathological chan-ges like MPSⅦ. The researches on β-glucuronidase used in antibody-directed enzyme prodrug drug therapy and enzyme replacement therapy are becoming deeper and deeper. Conclusion:β-Glucuronidase as an important acid hydrolytic enzyme in human has a variety of genetic mutations, and plays an important role in the fields of medicine and disease diagnosis, which has become one of research hotspots.
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Objective:To summarize the structure, properties and biological functions ofβ-glucuronidase. Methods:By referring to the relevant literatures at home and abroad onβ-glucuronidase in recent years, this paper induced, analyzed and drew conclusions. Results:The crystal structure of humanβ-glucuronidase is comprised of three distinct structural domains. The most common mutation of β-glucuronidase is missense mutation, which results in the change of enzyme activity, and then induces a series of pathological chan-ges like MPSⅦ. The researches on β-glucuronidase used in antibody-directed enzyme prodrug drug therapy and enzyme replacement therapy are becoming deeper and deeper. Conclusion:β-Glucuronidase as an important acid hydrolytic enzyme in human has a variety of genetic mutations, and plays an important role in the fields of medicine and disease diagnosis, which has become one of research hotspots.
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Objective To study the mechanisms involved in the regulation of endogenous β-glucuronidase expression and to explore the more effective methods to prevent recurrence of hepatolithiasis formation.Methods The expression levels of c-myc and endogenous β-glucuronidase in the liver specimens of hepatolithiasis were examined by immunohistochemical staining.The expressions of c-myc and endogenous β-glucuronidase in the human intrahepatic biliary epithelial cell line (HiBEpiC),and normal liver cell line (L02) treated with different concentrations of lipopolysaccharide (LPS) were studied using western blot.The c-myc siRNA transfection was utilized to detect the role of c-myc in the regulation of the expression of endogenous β-glucuronidase.Results Compared with normal liver samples,the expressions of endogenous β-glucuronidase and c-myc in the liver specimens of hepatolithiasis were significantly increased,and they were positively correlated with each other.LPS induced increased expressions of endogenous β-glucuronidase and c-myc in a dose-dependent manner.C-myc siRNA transfection effectively inhibited the increased expression of endogenous β-glucuronidase as induced by LPS.Conclusion LPS played a crucial role in the formation of hepatolithiasis by stimulating the endogenous expression of β-glucuronidase in liver and biliary epithelial cells via c-myc.
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The key to maximize the sensitivity of inner filter effect ( IFE)-based assay is to enlarge the overlap between the absorption spectra of the absorber and the excitation or emission spectra of the fluorophore. In this work, Mn-doped ZnS quantum dots ( QDs) were chosen for IFE-based detection of β-glucuronidase ( GUS) , since the excitation of QDs was perfectly overlapped with the absorption of the substrate of GUS, namely 4-nitrophenyl-β-D-glucuronide ( PNPG) . In addition, the phosphorescence emission from Mn-doped ZnS QDs could eliminate the fluorescence background from biological samples. Therefore, a simple turn-on phosphorescent GUS assay was developed, with the linear range of 10-300 U/L and limit of detection of 7 U/L (S/N=3).
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Objective: Trying to find the ways to enhance the expression of cyp71av1 gene encoding cytochrome P450 mono-oxygenase which is a key enzyme in artemisinin biosynthesis pathway accelerating the artemisinin synthesis, the promoter of cyp71av1 was isolated and characterized. Methods: 5′ untranslated regions of cyp71av1 were isolated from Artemisia annua with thermal asymmetric interlaced PCR. For functional characterization, the isolated fragments were fused with β-glucuronidase GUS reporter gene and introduced into Nicotiana tabacum by Agrobacterium-mediated transformation. The GUS expression regulated by 5′ untranslated regions of cyp71av1 in transgenic N. tabacum under the normal or stressed conditions were detected by histochemical staining and quantitative spectrophotometry assay. Results: Two DNA fragments upstream of cyp71av1 coding sequence, a long fragment and a truncated fragment, were isolated from A. annua and introduced into N. tabacum respectively. Histochemical staining showed that two isolated fragments confered stable GUS expression in transgenic plants, and no significant difference was found between the two fragments on the GUS activity. The quantitative results also showed that the GUS activity in transgenic tobacco plants treated by dehydration, low-temperature (4 °C), and ultraviolet irradiation were 1.4 to 2.7 folds higher than that in the controls. Conclusion: It suggests that the isolated fragments has promoter activity and may be responsive to adverse environmental stresses.
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Objective To investigate the relationship between the expression of β-glucuronidase mRNA in hepatocellular carcinoma(HCC)tissue and the clinicopathologic factors.Methods The expressions of β-glucuronidase mRNA in 25 samples of HCC tissue and 10 samples of normal hepatic tissue were detected by RT-PCR,and the relationship between β-glucurenidase mRNA in HCC tissue and clinicopathologic factors was analyzed.Results The expression of β-glucuronidase mRNA in HCC tissue was not influenced by the age of patient,tumor size and alpha-fetoprotein level.The expression of β-glueuronidase mRNA in patients with portal vein tumor thrombus or lymph node metastasis Was significantly higher than those without(t=7.857,9.341,P＜0.05).Conclusions The expression of β-glucuronidase mRNA is closely related to the existence of portal vein tumor thrombus and lymph node metastasis.β-glucuronidase may play a role in the invasion and metastasis of HCC.