β-Glucuronidase在植物基因功能研究中的应用

β-Glucuronidase(GUS)是从大肠杆菌K-12菌株分离出的一种酸性水解酶,具有高稳定性、检测的灵敏性与简易性及其对转基因生物体无毒性的特点,是转基因研究中最常用的标识基因。有三种商品化的底物分别用于分光光度定量分析、荧光定量分析与组织化学定位研究。由于植物体内存在着不同程度的GUS本底表达,可以通过在反应体系中加入20%甲醇来抑制。GUS基因有弱的表达特点,可以在其前面加终止子来抑制。利用带有GUS基因的杆菌转化拟南芥具有简单易行的特点,能有效保障学习与了解植物转基因的过程与机制。

Determination of β-glucuronidase in human colorectal carcinoma cell lines

IM To study the relationship between βglucuronidase and the invasiveness of human colorectal carcinoma cell lines.METHODS Six colorectal carcinoma cell lines including three welldifferentiated (CX1, CCL 187, CCL 229) and three poorlydifferentiated ones (CCL 227, CCL 228, Clone A) were analyzed for βglucuronidase in the medium by Fischman′s method.RESULTS Low levels of βglucuronidase (activity range, 129 to 196μg/106 cells·h) were associated with the poorly invasive cells. This was in contrast to the elevated levels of the enzyme (246337μg/106·h) present in the medium derived from the more aggressive cells (CCL 227, CCL 228, Clone A, CCL 229).CONCLUSION The highly invasive colorectal carcinoma cells secreted higher levels of βglucuronidase than the poorly invasive ones, and the determination of secreted βglucuronidase might provide a useful in vitro measurement of the invasiveness of colorectal carcinoma.

Clinical significance of TGF-β1 and β-glucuronidase synchronous detection in human pancreatic cancer

Objective: To investigate the relation of transfergrowth factor （TGF-β1） and β-glucuronidase （β-GCD） on the occurrence and progress of pancreaticcancer.Methods: The expression of TGF-β1 and β-GCD in thepancreatic cancer tissue and normal pancreatic tissuewas determined synchronously using ABC method ofimmunohistochemistry.Results: The percentage of TGF-β1 positive cells wassignificantly higher in pancreatic cancer tissue （43.8%±5.2%） than in adjacent pancreatic tissue （28.7%±3.6%, P【0.01）. The worse the cancer cells differen-tiated and lymph nodes metastasis, the more over-ex-pression of TGF-β1. The percentage of β-GCD positivecells was also significantly higher in the pancreaticcancer tissue （62.5%±4.1%） than in the adjacentpancreatic tissue （33.5%±2.8%, P【0.01）. The de-gree of over-expression of β-GCD was related to thedegree of cancer cells differentiation, but not to thelymph nodes metastasis. The expression of TGF-β1was significantly correlated with the expression of β-GCD in pancreatic cancer tissue.Conclusions: The genesis of pancreatic cancer resultsfrom multi-factor, multi-step and multi-gene varia-tion. The synchronous detection of TGF-β1 and β-GCD helps to determine the malignant degree oftumors and the prognosis of patients with such disease.

Promoting effect of licorice extract on induction of β-glucuronidase in Penicillium purpurogenum Li-3

PeniciUium purpurogenum Li-3, a fungus producing β-glucuronidase （PGUS）, can con- vert glycyrrhizin （GL） to glycyrrhetinic acid monoglucuronide （GAMG） when grown in medium with GL as the sole carbon source. In order to improve the conversion rate of GL and the yield of GAMG, licorice extract （LE） was added as an inducer to enhance the production of GAMG by the PGUS. In this work, the influence of LE on the conversion rate of GL to GAMG was studied. When the Penicil- lium purpurogenum Li-3 was grown in the medium containing LE and GL （ concentration ratio of LE to GL was 2： 3）, the conversion rate of GL was 84. 12% with 38. 18% increase and the yield of GAMG was 80. 47% with 37. 18% increase, comparing with to the medium only containing GL at 48 h. The enzyme activity of ^-glucuronidase was also enhanced from 22. 4 U/mL to 82.3 U/mL, which in- creased up to about 3. 67 fold. The results showed that LE could significantly improve the induced expression level of PGUS.

Detection of β-Glucuronidase Activity within Actinomadura madurae Grains of Human Actinomycetoma

Actinomycetoma syndrome by Actinomadura (A.) madurae is characterized by a subcutaneous chronic lesion that affects fascia, muscle and bone. A. madurae produces colonies that form grains of less than 1 mm in diameter. Grains are surrounded and infiltrated by neutrophils involved in the grain disruption by enzymes like β-glucuronidase released after the neutrophil degranulation. The aim of this work was to evaluate the polysaccharide degradation of grains treated with β-glucuronidase and to detect the presence and activity of β-glucuronidase within the A. madurae grains. Actinomadura madura grains from patients infected were processed to quantify the total content of polysaccharide with the phenol-sulfuric acid reaction. Grains were treated with β-glucuronidase at different conditions to evaluate the optimal polysaccharide degradation. Grains were analyzed to detect the enzyme by using anti-human β-glucuronidase antibody while enzymatic activity was assessed by evaluating the release of reduced sugars and by in situ enzymatic activity. Optimal degradation of polysaccharide in the grains treated with β-glucuronidase was found with 300 units/ml of enzyme and 24 hr of incubation at 37°C. Presence and activity of β-glucuronidase enzyme within the grains were detected. Results suggested that β-glucuronidase present within A. madurae grain resulted from degranulated neutrophils surrounding and/or infiltrated within the grain.

Immobilization of β-glucuronidase in lysozyme-induced biosilica particles to improve its stability

Mesoporous silica particles were prepared for efficient immobilization of the β-glucuronidase （GUS） through a biomimetic mineralization process, in which the solution containing lysozyme and GUS were added into the prehydrolyzed tetraethoxysilane （TEOS） solution. The silica particles were formed in a way of biomineralization under the catalysis of lysozyme and GUS was immobilized into the silica particles simultaneously during the precipitation process. The average diameter of the silica particles is about 200 nm with a pore size of about 4 rim. All the enzyme molecules are tightly entrapped inside the biosilica nanoparticles without any leaching even under a high ionic strength condition. The immobilized GUS exhibits significantly higher thermal and pH stability as well as the storage and recycling stability compared with GUS in free form. No loss in the enzyme activity of the immobilized GUS was found after 30-day＇s storage, and the initial activity could be well retained after 12 repeated cycles.

Enhanced production of β-glucuronidase from Penicillium purpurogenum Li-3 by optimizing fermentation and downstream processes

β-Glucuronidase from Penicillium purpuro- genum Li-3 （PGUS） can efficiently hydrolyze glycyrrhizin into the more valuable glycyrrhetic acid monoglucuronide. However, a low productivity of PGUS and the lack of an effective separation strategy have significantly limited its industrial applications. Therefore, the production of PGUS has been improved by optimizing both the fermentation and purification strategies. A two-stage fermentation strategy was developed where PGUS was first grown with glucose and then PGUS was produced in the presence of glycyrrhizin as an inducer. By using this strategy, the biomass was increased 1.5 times and the PGUS activity increased 5.4 times compared to that when glycyrrhizin was used as the sole carbon source. The amount of PGUS produced was increased another 16.6% when the fermentation was expanded to a 15-L fermenter. An effective protocol was also established to purify the PGUS using a sequential combination of hydrophobic, strong anionexchange and gel filtration chromatography. This protocol had a recovery yield of 6% and gave PGUS that was 39 times purer than the crude PGUS. The purified PGUS had a specific activity of 350 U. mg-1.

Highly sensitive light-up near-infrared fluorescent probe for detection and imaging of β-glucuronidase in human serum,living cells and tumor-bearing mice

β-Glucuronidase(GUS)plays a key role in tumor initiation,metastasis,and progression,and thus,has been proposed as a promising cancer biomarker.In this study,we designed an enzyme-activatable near-infrared(NIR)fluorescent probe(DCM-βGlcA)for the rapid and accurate detection of GUS activity in vitro,in vivo and ex vivo.The DCM-βGlcA was prepared by linking a glucuronic acid residue to dicyanomethylene-4 H-pyran(DCM).This probe exhibited significant light-up NIR fluorescent signals at 680 nm after reacting with GUS and the Stokes shift could reach 150 nm.The DCM-βGlcA showed a high sensitivity toward GUS and an excellent linear relationship at concentrations ranging between 0 and 4 U L^(-1)(R^(2)=0.9974)with the limit of detection as low as 0.19 U L^(-1).We used the DCM-βGlcA to identify GUS serum levels in both cancer patients and healthy individuals with a similar accuracy as that of an enzyme-linked immunosorbent assay(ELISA)while being easier and faster to perform.Moreover,the DCM-βGlcA was used for tracking endogenous GUS in living cells,thereby discriminating GUSoverexpressed liver cancer from normal cells.Additionally,the DCM-βGlcA was able to detect and image endogenous GUS in liver cancer tissue and tumor-bearing mouse models.These findings demonstrate the potential of the DCM-βGlcA as a promising tool for detecting and monitoring GUS activity in preclinical applications.

Immobilization of β-glucuronidase:biocatalysis of glycyrrhizin to 18β-glycyrrhetinic acid and in-silico lead finding

The non-covalent immobilization ofβ-glucuronidase enzyme obtained from Rhizopus oryzae was carried out by entrapment in natural fiber(papaya and coconut).The bioconversion capability of immobilized enzyme was analyzed based on conversion of glycyrrhizin to 18β-glycyrrhetinic acid under different conditions.The hydrolytic activity of theβ-glucuronidase enzyme was highly depended on the microbial source and matrix,in which enzyme was immobilized.R.oryzaeβ-glucuronidase immobilized in papaya fibers produced the highest GA content(13.170μg/mL)at 10 h of reaction.However R.oryzaeβ-glucuronidase immobilized in coconut fibers produced the highest GA content(21.425μg/mL)at 15 h of reaction.Online Molinspiration software was used to predict drug like molecular properties of the 18β-glycyrrhetinic acid,and software suggested that the compounds had potential of becoming the orally active molecules.Therefore,in silico studies were conducted on proposed 18β-glycyrrhetinic acid to select the best possible drug candidates based on drug properties and bioactivity score of the compounds.

Biliary microbiome and gallstones: A silent friendship

With increasing evidence,the biliary tract and the gallbladder mucosa are no longer considered sterile environments devoid of bacteria.Rather a profound biofilm of resident bacterial flora is associated with the mucosal surface.The bile too harbors a resident flora.It is when a dysbiotic process ensues,that this bac-terial flora either becomes opportunist or is replaced by a pathogenic one that has a strong ability to survive the challenges of the biliary environment.Although once believed a metabolic problem,recent evidence indicates a complex intera-ction between different species of bacteria and gallbladder mucosa and bile which may culminate in calculus formation.The resident microbiota and its several enzymes dictate the type of gallstone by the mere interplay of the constituting type of bacteria in the biofilm,even without any evidence of infection.Dysbiosis is often mediated by either intestinal dysbiosis or less probably by oral dysbiosis.The gallstones,in turn,provide a haven for the resident microbiota in which they can form their own defined niche enriched with the biofilm that can resist the biliary defense mechanisms and survive the hostile biliary environment in the background of biliary stasis and local infection.However,this process of silent friendship is more complex than said,and further research is needed to define the relationship between the two.

Trichome-Specific Expression of Amorpha-4,11-Diene Synthase, a Key Enzyme of Artemisinin Biosynthesis in <i>Artemisia annua</i>L., as Reported by a Promoter-GUS Fusion

Artemisia annua L. produces small amounts of the sesquiterpenoid artemisinin, which is used for treatment of malaria. A worldwide shortage of the drug has led to intense research to increase the yield of artemisinin in the plant. In order to study the regulation of expression of a key enzyme of artemisinin biosynthesis, the promoter region of the key enzyme amorpha-4,11-diene synthase (ADS) was cloned and fused with the β-glucuronidase (GUS) reporter gene. Transgenic plants of A. annua expressing this fusion were generated and studied. Transgenic plants expressing the GUS gene were used to establish the activity of the cloned promoter by a GUS activity staining procedure. GUS under the control of the ADS promoter showed specific expression in glandular trichomes. The activity of the ADS promoter varies temporally and in old tissues essentially no GUS staining could be observed. The expression pattern of GUS and ADS in aerial parts of the transgenic plant was essentially the same indicating that the cis-elements controlling glandular trichome specific expression are included in the cloned promoter. However, some cis-element(s) that control expression in root and old leaf appears to be missing in the cloned promoter. Furthermore, qPCR was used to compare the activity of the wild-type ADS promoter with that of the cloned ADS promoter. The latter promoter showed a considerably lower activity than the wild-type promoter as judged from the levels of GUS and ADS transcripts, respectively, which may be due to the removal of an enhancing cis-element from the ADS promoter. The ADS gene is specifically expressed in stalk and secretory cells of glandular trichomes of A. annua.

Role of intestinal barrier in pathogenesis of pigment gallstone in a guinea pig model

BACKGROUND: The function of the intestinal barrier has drawn more and more attention from researchers in recent years for its important role in many diseases such as burns, wounds, and pancreatitis. In our experimental studies on pigment gallstone, we found potential relationships between the function of the intestinal barrier and pigment gallstone formation. This study was undertaken to investigate the possible action and mechanism of the function of the intestinal barrier in the pathogenesis of pigment gallstone. METHODS: Eighty guinea pigs were divided into a normal group (CON), a pigment gallstone group (PS) and an intestinal mucosa protection group (GLN). Normal forage, pigment gallstone-forming forage and pigment gallstoneforming forage with supplemental intestinal mucosa protector (glutamine) were given to each group. In the gallstone-forming rate, morphology of intestinal mucosa, intestinal permeability, serum endotoxin and biliaryβ-glucuronidase were assessed after 8 weeks. RESULTS: The rate of gallstone-formation was 73.9% in the PS group. Damage of intestinal mucosa, endotoxemia (from 77±43×106 EU/L to 1367±525×l06 EU/L, P【0.01) and increased activity of biliaryβ-glucuronidase (endogenousβ-glucuromdase from 122.1±39.5 to 209.8±47.5 Fishman Unit, P【0.01, and exogenous p-glucuronidase from 573.5±476.9 to 2206.6±983.9 Fishman Unit, P【0.01) were observed in the PS group compared with the CON group. The rate gallstone-formation decreased significantly to 44.4% and the other indices except P-glucuronidase were lower in the GLN group than in the PS group. CONCLUSIONS: The function of the intestinal barrier is correlated with pigment gallstone formation. Dysfunction of the intestinal barrier function may promote pigment gallstone formation through bacterial translocation, endotoxemia, and biliaryβ-glucuronidase.

Optimization of Electroporation Parameters for Immature Embryos of indica Rice (Oryza sativa)

To obtain a suitable condition for electroporation transformation in indica rice, the 10-day-old immature embryos were selected for optimization experiments. The results showed that one pulse at 850 V/cm, 950μF capacitance, 200 μL electroporation buffer with 70 mmol/L sodium glutamate, 100 μg/mL plasmid, 50μg/mL carrier DNA, 20 embryos per cuvette, 0℃ treatment and CC medium were the best parameters, which not only improved the transformation efficiency to 30.89%, but also ameliorated the embryo survival ratio to 95.92%. To further verify the practicability of this condition, the embryos from another indica rice variety and a rice type Ⅱ metallothionein-like gene （OsMT2bL） promotec：mgfp5：：gusA construct were tested, and specific GUS expression on the embryos was visualized by histochemical staining. The results showed that the GUS expression on the embryos activated by the OsMT2bL promoter was mainly concentrated on the apical point of the plumule whereas the expression driven by CaMV35S promoter was distributed on nearly all areas of the electroporated tissues. These results indicated that the optimized embryo electroporation conditions could be used not only in genetic transformation of indica rice but also in assay of gene regulation on embryos.

Efficient gusA Transient Expression in Porphyra yezoensis Protoplasts Mediated by Endogenous Beta-tubulin Flanking Sequences

Endogenous tubulin promoter has been widely used for expressing foreign genes in green algae, but the efficiency and feasibility of endogenous tubulin promoter in the economically important Porphyra yezoensis (Rhodophyta) are unknown. In this study, the flanking sequences of beta-tubulin gene from P. yezoensis were amplified and two transient expression vectors were con-structed to determine their transcription promoting feasibility for foreign gene gusA. The testing vector pATubGUS was constructed by inserting 5’- and 3’-flanking regions (Tub5’ and Tub3’) up- and down-stream of β-glucuronidase (GUS) gene (gusA), respectively, into pA, a derivative of pCAT?3-enhancer vector. The control construct, pAGUSTub3, contains only gusA and Tub3’. These con-structs were electroporated into P. yezoensis protoplasts and the GUS activities were quantitatively analyzed by spectrometry. The results demonstrated that gusA gene was efficiently expressed in P. yezoensis protoplasts under the regulation of 5’-flanking sequence of the beta-tubulin gene. More interestingly, the pATubGUS produced stronger GUS activity in P. yezoensis protoplasts when com-pared to the result from pBI221, in which the gusA gene was directed by a constitutive CaMV 35 S promoter. The data suggest that the integration of P. yezoensis protoplast and its endogenous beta-tubulin flanking sequences is a potential novel system for foreign gene expression.

Transient gene expression in western white pine using agroinfiltration

A method for transient gene expression was developed for western white pine(WWP,Pinus monticola Dougl.ex D.Don)using reporter gene uidA encodingβ-glucuronidase(GUS).GUS was transiently expressed in cross sections of primary and secondary needles,cotyledons,and current and second year stems of WWP via vacuum-infiltration with Agrobacterium tumefaciens.Histochemical assays of cross sections of secondary needles showed stronger blue color indicating GUS expression at day 1 and 2 than on other days post agroinfiltration(dpa).GUS activity expressed inside WWP cells was confirmed using light microscopy.In fluorometric assays,GUS expression was high at 1 dpa and lasted until 4 dpa in detached secondary needles,while similarly high expression levels only lasted until 2 dpa in attached secondary needles then dropped significantly.Although the length of GUS-staining zones varied among different WWP organs and between growth and dormant seasons,all tested WWP tissues using the protocol had high levels of transient GUS expression.Thus,heterologous candidate genes or endogenous silencing can be expressed in various WWP tissues or organs using this agroinfiltration approach.The current protocol for efficient transient gene expression will aid functional genomics study of WWP and its pathogens and related conifer species.

Establishment of genetic transformation system via Agrobacterium in tall fescue cultivar

Tall fescue （Festuca arundinacea Schreb.） is a cool-season turfgrass used on fairways in golf courses. The object of this study was to develop a more efficient, reliable, and repeatable approach in transforming the grass using Agrobacterium （EHA105）, where β-glucuronidase gene （uidA） was used as a reporter and hygromycin phosphotransferase gene （hyg） as a selectable marker. An effective expression of transgene was observed in transforming 2-month-old calli derived from mature seeds （cv. Bingo） cultured on MS medium supplemented with 9 mg·L^-1 2, 4-D. A two-step solid medium selection with increasing hygromycin concentration （from 30 to 50 mg· L^-1） was used to obtain resistant calli. Transgenic plants have been produced from many independent transformed calli. The presence of functional β-glucuronidase gene （uidA） was detected in hygromycin-resistant calli. Transgenic plants were regenerated and PCR and Southern blot confirmed transgene integration in the tall fescue genome.

Regulation of OsPRIOa Promoter Activity by Phytohormone and Pathogen Stimulation in Rice

OsPRIOa is one of the well known pathogenesis-related genes in rice,and is induced by multiple plant hormones and pathogens.However,the underlying transcriptional regulation mechanisms in response to differe nt signals and their crosstalks are still largely unknown.In order to find new players participated in the activation of OsPRIOa,we systematically analyzed the basal expression patterns as well as the expression responses of a 2.5 kb OsPRIOa promoter in rice transgenic plants after phytohormone and pathogen stimulations.In agreement with the native gene expression,the OsPRIOa promoter can drive glucuronidase(GUS)gene expressing in spots of leaf cells,leaf trichomes,lemmas and paleae,germinating embryos,calli and root tips.The leaf expression of OsPR10a::GUS was dramatically in creased upon jasm onic acid(JA)and cytoki nin(CK)treatme nts,or challe nges of the pathogen Magnaporthe grisea and Xanthomonas oryzae pv.oryzae.Thus,the OsPRIOa promoter reported here can faithfully reflect its native gene expression.The effects of several JA and CK responsive OsWRKY genes on the regulation of OsPRIOa promoter were then inspected by luciferase transient expression assay,and the JA inducible OsWRKYlO transcription factor was found as a new positive regulator of OsPRIOa.However,the key transcription factors of JA and CK signaling pathways,OsMYC2 and B-type response regulators,were not responsible for the activation of OsPRIOa promote Our findings provided new in sights into the regulation of OsPRIOa expression during plan t-hormone/pathoge n interactions,and the OsPRIOa reporter system can be useful to unravel novel regulators from both pathogen and host.

Agrobacterium tumefaciens-mediated GUS gene transfer to Sophora japonica L.

Agrobacterium-mediated genetic transformation of Sophora japonica was standardized using the Agrobacterium tumefa- ciens strain LBA4404 that harbored the binary vector pBI121 containing genes for fl-glucuronidase （GUS） and neomycin phos- photransterase （npt II）. S. japonica transformants were selected by the ability of the leaf explants to produce kanamycin-resistant calli that regenerated into kanamycin-resistant plantlets. Successful transformation was confirmed by histochemical assay for GUS activity, PCR analysis and Southern blot. The period of nearly two months was required for the regeneration of transgenic plantlets fi＇om the explants. The transformed plants resembled their parents in morphology.

Long term survival and limited migration of genetically modified monocytes/macrophages grafted into the mouse brain

In mammals, myeloid progenitors infiltrate the developing central nervous system (CNS), through the immature blood-brain barrier (BBB), the ventricular layer or the pial surface migrate and give rise to resident microglia. In the mature brain, however, the BBB hampers such recruitment from the blood-stream and long-term establishment of blood borne myeloid cells in the CNS thus appears at best limited. Hematopoietic stem cell-derived microglia, nevertheless, represents a promising tool for the correction of genetic deficits in the brain. We thus investigated the fate of primary human monocytes, and monocyte-derived macrophages, following transplantation into the adult mouse brain overpassing the BBB. Furthermore, we documented the ability of such cells to deliver a lysosomal enzyme into the brain following genetic modification with a recombinant adenoviral vector carrying the human β-glucuronidase cDNA. When implanted into the mouse striatum, the engineered primary cells survived and expressed the transgene for as much as 8 months. Moreover, the donor cells could migrate out of the grafting site and settle along blood vessels or myelin tracts although at limited distance. Migrating donor cells down-regulated the expression of CD14 andHLA DR, suggesting the adoption of a deactivated microglia-like phenotype. Our observations establish the ability of circulating mononuclear phagocytes to integrate into the brain after transplantation and express a transgene on the long term. These cells might thus be employed for autologous transplantation for the delivery of secreted therapeutic proteins in the context of a wide range of brain affections.

Agrobacterium-mediated transient transformation of Pentalinon andrieuxii Müll.Arg.

Sections of hypocotyls, roots and leaves from Pentalinon andrieuxii plantlets were transiently transformed with Agrobacterium tumefaciens LBA4404 bearing the binary plasmid pCAMBIA2301 with an interrupted β-Glucuronidase (GUS) gene. Histochemical GUS assays showed transient gene expression in all infected tissues, being older roots those which displayed the most intense GUS staining. To our knowledge, this is the first report of Pentalinon andrieuxii susceptibility to Agrobacterium tumefaciens-mediated genetic transformation.