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Query: EC:3.2.1.31 (beta-glucuronidase)
 7,680 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Chitinase gene expression has been shown to be transcriptionally regulated by a number of inducers, including ethylene, elicitors, and pathogen attack. To investigate the mechanism(s) responsible for induction of chitinase gene expression in response to various stimuli, we have developed a transient gene expression system in bean (Phaseolus vulgaris) protoplasts that is responsive to ethylene and elicitor treatment. This system was used to study the expression of a chimeric gene composed of the 5' flanking sequences of a bean endochitinase gene fused to the reporter gene beta-glucuronidase linked to a 3' fragment from nopaline synthase. Addition of 1-aminocyclopropane-1-carboxylic acid, the direct precursor of ethylene, or elicitors such as chitin oligosaccharides or cell wall fragments derived from Colletotrichum lagenarium, to transformed protoplasts resulted in a rapid and marked increase in the expression of the chimeric gene. The kinetics and dose response for these treatments were similar to those observed for the native gene in vivo. Analyses of 5' deletion mutants in the protoplast system indicated that DNA sequences located between -305 and -236 are important for both ethylene and elicitor induction of the reporter gene.
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PMID:Regulation of a chitinase gene promoter by ethylene and elicitors in bean protoplasts. 1666 5

We developed a new series of binary vectors useful for Gateway cloning to facilitate transgenic experiments in plant biotechnology. The new system, Gateway Binary Vectors (pGWBs) realized efficient cloning, constitutive expression using the cauliflower mosaic virus (CaMV) 35S promoter and the construction of fusion genes by simple clonase reaction with an entry clone. The reporters employable in this system are beta-glucuronidase (GUS), synthetic green fluorescent protein with S65T mutation (sGFP), luciferase (LUC), enhanced yellow fluorescent protein (EYFP), and enhanced cyan fluorescent protein (ECFP). The tags available are 6xHis, FLAG, 3xHA, 4xMyc, 10xMyc, GST, T7-epitope, and tandem affinity purification (TAP). In total, 13 kinds of reporter or tag were arranged and were almost applicable to both N- and C-fusions. The pGWBs could be used for many purposes, such as promoter::reporter analysis, observation of subcellular localization by the expression of proteins fused to a reporter or tag, and analysis of protein-protein interaction by copurification and immunodetection experiments. The pGWBs were constructed with modified pBI101 containing a CaMV35S promoter-driven hygromycin phosphotransferase (HPT) gene as the second selection marker. We also constructed pGWBs with the marker HPT driven by the nopaline synthase promoter. By using the pGWB system, the expression of tagged proteins, and the localization of GFP-fused proteins were easily analyzed. Moreover, tissue-specific and inducible gene expression using a promoter was also monitored with pGWBs. It is expected that, the pGWB system will serve as a powerful tool for plasmid construction in plant research.
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PMID:Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation. 1769 81

The constitutive promoter of cauliflower mosaic virus 35S (CaMV 35S) is a polar unidirectional promoter and is widely used in plant genetic engineering. In the present study, the unidirectional CaMV 35S promoter has been modified to a bi-directional promoter by fusing its minimal promoter element to the 5' end of CaMV 35S promoter in the opposite orientation. To qualitatively and quantitatively estimate its bi-directional transcriptional function and activity, two visible reporter genes, gusA (beta-glucuronidase, GUS) and gfp (green fluorescent protein, GFP), were fused to the two ends of the promoter in bi-orientations ending with NOS terminator sequences, respectively. Stable expression of gusA and gfp genes in transgenic tobacco (Nicotiana tabacum L.) was visualized by histochemically staining for GUS and fluorescence microscopic observation under UV for GFP in transgenic plants. The expression of two reporter genes showed that the constructed bi-directional promoter did have the bi-directional transcriptional function in both expected orientations. The quantitative estimation of GUS and GFP were determined on a HITACHI F1000 Fluorescence Spectrophotometer with various wavelengths of excitation and emission. The GUS activity varied from 8 to 250 pmol 4-MU/min/mg protein and the GFP content varied from 0.9 to 1.8 microg/mg protein in various lines of transgenic tobacco plants. Higher GUS activity generally coupled with lower GFP content, and vice versa.
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PMID:Construction and analysis of a plant transformation binary vector pBDGG harboring a bi-directional promoter fusing dual visible reporter genes. 1843 82

Transient expression of foreign genes in plant tissue is a valuable tool for testing the efficacy of transformation methods. In this work, we present, for the first time, the utilization of agroinjection as an efficient transformation system for gene delivery in date palm fruit. The research utilized Agrobacterium tumefaciens strain LBA4404 harboring the binary vector pRI201-AN-GUS carrying the beta-glucuronidase (GUS) gene, under the control of a CaMV 35S and kanamycin (NPTII) as an antibiotic gene under the control of a NOS promoter. Based on histochemical assay of agroinjected fruit for the GUS gene expressions, this protocol has proved to be an efficient and reliable tool for transgene expression in date palm. PCR for plasmid DNA, extracted from the transformed Agrobacterium, demonstrated the generation of the expected amplicon, corresponding to the GUS gene using GUS primers.
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PMID:Transient GUS Gene Expression in Date Palm Fruit Using Agroinjection Transformation Technique. 2875 54


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