Expression Of Β-glucuronidase Reporter Gene Research Articles

Isolation and functional characterization of a novel stress inducible promoter from pigeonpea (Cajanus cajan L)

The present study deals with isolation and characterization of a novel hybrid-proline-rich protein gene (CcHyPRP) promoter from pigeonpea. Real time PCR analysis revealed that CcHyPRP expression was strongly induced by dehydration, salt, Abscisic acid (ABA) and Salicylic acid (SA) treatments. The CcHyPRP promoter, isolated by genome-walking method, contained 1112 bp and showed the presence of various cis-regulatory elements necessary for tissue specific expression and stress responsiveness. Different 5′ deletions of the promoter were generated and were used to drive the expression of β-glucuronidase reporter gene (gusA) in Arabidopsis thaliana. Histochemical and fluorometric assays confirmed that GUS expression driven by the full-length fragment (1112 bp) was higher when compared to different deletion fragments. Under normal conditions, GUS expression was predominantly detected in the roots and hypocotyls of transformants, while under mannitol, NaCl, ABA and SA treatment conditions higher GUS expression levels were observed in the roots and leaves. However, the GUS expression was mostly confined to the roots of transformants carrying 477 and 300 bp promoter regions. The results amply indicate that CcHyPRP promoter is regulated by different stress factors, and as such the promoter can be deployed in genetic engineering of crop plants for enhanced abiotic stress tolerance.

Multiple roles of the transcription factor AtMYBR1/AtMYB44 in ABA signaling, stress responses, and leaf senescence.

BackgroundThe transcription factor AtMYBR1 (MYB44) is a member of the MYB family of transcription factors and is expressed throughout the plant life cycle and especially in senescing and wounded leaves. It has previously been shown to be involved in responses to abiotic stress and is regulated by phosphorylation.ResultsWhen MYBR1 was over-expressed under the control of the constitutive 35S promoter in Arabidopsis thaliana (OxMYBR1), leaf senescence was delayed. In contrast loss-of-function mybr1 plants showed more rapid chlorophyll loss and senescence. The MYBR1 promoter strongly drove β-GLUCURONIDASE reporter gene expression in tissues immediately after wounding and many wounding/pathogenesis genes were downregulated in OxMYBR1. OxMYBR1 plants were more susceptible to injury under water stress than wildtype, which was correlated with suppression of many ABA inducible stress genes in OxMYBR1. Conversely, mybr1 plants were more tolerant of water stress and exhibited reduced rates of water loss from leaves. MYBR1 physically interacted with ABA receptor PYR1-LIKE8 (PYL8) suggesting a direct involvement of MYBR1 in early ABA signaling. MYBR1 appears to exhibit partially redundant functions with AtMYBR2 (MYB77) and double mybr1 X mybr2 mutants exhibited stronger senescence and stress related phenotypes than single mybr1 and mybr2 mutants.ConclusionsMYBR1 is a negative regulator of ABA, stress, wounding responses and blocks senescence. It appears to have a homeostatic function to maintain growth processes in the event of physical damage or stress.

Assays of polychlorinated biphenyl congeners and co-contaminated heavy metals in the transgenic Arabidopsis plants carrying the recombinant guinea pig aryl hydrocarbon receptor-mediated β-glucuronidase reporter gene expression system

The transgenic Arabidopsis plant XgD2V11-6 carrying the recombinant guinea pig (g) aryl hydrocarbon receptor (AhR)-mediated β-glucuronidase (GUS) reporter gene expression system was examined for assay of polychlorinated biphenyl (PCB) congeners and co-contaminated heavy metals. When the transgenic Arabidopsis plants were treated with PCB126 (toxic equivalency factor; TEF: 0.1) and PCB169 (TEF: 0.03), the GUS activity of the whole plants was increased significantly. After treatment with PCB80 (TEF: 0), the GUS activity was nearly the same level as that treated with 0.1% dimethylsulfoxide (DMSO) as a vehicle control. After exposure to a 1:1 mixture of PCB126 and PCB169, the GUS activity was increased additively. However, after exposure to a mixture of PCB126 and PCB80, the GUS activity was lower than that of the treatment with PCB126 alone. Thus, PCB80 seemed to be an antagonist towards AhR. When the transgenic plants were treated with each of the heavy metals Fe, Cu, Zn, Cd and Pb together with PCB126, Cd and Pb increased the PCB126-induced GUS activity. On the other hand, Fe, Cu and Zn did not affect the PCB126-induced GUS activity. In the presence of the biosurfactant mannosylerythritol lipid-B (MEL-B) and the carrier protein bovine serum albumin (BSA), the PCB126-induced GUS activity was increased, but the Cd-assisted PCB126-induced GUS activity was not affected. Thus, MEL-B and BSA seemed to increase uptake and transport of PCB126, respectively.

High Frequency β-glucuronidase Reporter Gene Expression in Shoot Tip, Leaf, and Hypocotyl Explants of Jatropha curcas L.

Development of a suitable genetic transformation system is the backbone of transgenic technology for efficient recovery of Jatropha curcas plants with desirable traits. Although in vitro plant regeneration protocols for shoot tip, leaf, and hypocotyl explants of J. curcas have been reported, these protocol have not been translated into efficient genetic transformation systems. The objective of this study was to standardize the parameters influencing transient β-glucuronidase reporter gene expression following Agrobacterium-mediated genetic transformation using shoot tip, leaf, and hypocotyl explants of Jatropha curcas. Disarmed Agrobacterium tumefaciens strain EHA105, harboring pCAMBIA2301 carrying neomycin phosphotransferase (nptII) as a selectable marker and the β-glucuronidase (GUS) as a reporter gene, were used for transformation. Frequencies of GUS gene expression of 86.7%, 78.3%, and 74.2% were obtained for leaf, hypocotyls, and shoot tip explants, respectively. The transgenic nature of the kanamycin-resistant explants was also confirmed by using polymerase chain reaction (PCR) assay. Because, to date, there is no report on Agrobacterium-mediated transformation of shoot tip, leaf, and hypocotyl explants of Jatropha curcas, our findings/protocol should be useful for recovering stable transgenic Jatropha curcas plants from these explants.

Assays of dioxins and dioxin-like compounds in actually contaminated soils using transgenic tobacco plants carrying a recombinant mouse aryl hydrocarbon receptor-mediated β-glucuronidase reporter gene expression system

This article has been retracted.

Assays of dioxins and dioxin-like compounds in actually contaminated soils using transgenic tobacco plants carrying a recombinant mouse aryl hydrocarbon receptor-mediated β-glucuronidase reporter gene expression system

The transgenic tobacco plant XD4V-26 carrying the recombinant mouse aryl hydrocarbon receptor XD4V-mediated β-glucuronidase (GUS) reporter gene expression system was used for assay of dioxins and dioxin-like compounds consisting of polychlorinated dibenzeno-p-dioxins, polychlorinated dibenzofurans, and coplanar polychlorinated biphenyls (Co-PCBs) in actually contaminated soils. The transgenic tobacco plant XD4V-26 showed a significant dose-dependent induced GUS activity when cultured on MS medium containing PCB126 [toxic equivalency factor (TEF) = 0.1]. In contrast, PCB169 and PCB180, which have 0.03 of TEF and unassigned TEF values, respectively, did not significantly induce GUS activity under the same conditions as with PCB126. When the tobacco plants were cultivated for up to 5 weeks on actually contaminated soils with dioxins and dioxin-like compounds collected from the periphery of an incinerator used for disposal of residential and industrial wastes, GUS activity in the leaves was dose-dependently increased. The plants clearly detected 360 pg-TEQ g−1 of dioxins and dioxin-like compounds in this assay. There was a positive correlation between GUS activity and TEQ value of dioxins and dioxin-like compounds in the plants. This assay does not require any extraction and purification processes for the actually contaminated soil samples.

Elicitation of systemic resistance against the bacterial speck pathogen in Arabidopsis thaliana by culture filtrates of plant growth-promoting fungi

The potential for mediation of induced systemic resistance (ISR) and the mechanisms used by nonpathogenic beneficial microbes to mediate ISR are currently under intense investigation but have been overlooked in studies examining plant growth promoting fungi (PGPF) or their metabolites. Here, we examined whether culture filtrates (CFs) of three well-characterized PGPF isolates, which demonstrated different responses in hypersensitive reaction-like cell death and β-glucuronidase reporter gene expression of PR-1a and PDF 1.2 constructs, had the ability to induce systemic resistance against the bacterial leaf speck pathogen Pseudomonas syringae pv. tomato DC3000 (Pst) in Arabidopsis thaliana. Arabidopsis plants treated with CFs of Phoma sp. (GS6-2 and GS7-3) and sterile fungus (GU23-3) elicited ISR against the Pst pathogen, resulting in a reduction in the number of symptomatic leaves, restriction of disease severity, and suppression of pathogen proliferation, with the best results obtained with Phoma sp. To determine the mechanism used to induce systemic resistance, Arabidopsis genotypes expressing the bacterial salicylate hydroxylase gene or containing disruptions in the nonexpressor of pathogenesis-related genes 1 (NPR 1), jasmonic acid JA, and ethylene (ET) signaling were screened. We demonstrate that signal transduction leading to GS6-2-mediated ISR requires salicylic acid (SA) accumulation, whereas CF of GU23-3-mediated ISR stimulates a pathway dependent on JA signaling. However, GS7-3-mediated ISR does not require SA, JA, ET, or NPRl signaling. Our results suggest that ISR mediated by three PGPF isolates studied were divergent in the biochemical pathways affected.

Isolation of a Calmodulin-binding Transcription Factor from Rice (Oryza sativa L.)

Calmodulin (CaM) regulates diverse cellular functions by modulating the activities of a variety of enzymes and proteins. However, direct modulation of transcription factors by CaM has been poorly understood. In this study, we isolated a putative transcription factor by screening a rice cDNA expression library by using CaM:horse-radish peroxidase as a probe. This factor, which we have designated OsCBT (Oryza sativa CaM-binding transcription factor), has structural features similar to Arabidopsis AtSRs/AtCAMTAs and encodes a 103-kDa protein because it contains a CG-1 homology DNA-binding domain, three ankyrin repeats, a putative transcriptional activation domain, and five putative CaM-binding motifs. By using a gel overlay assay, gel mobility shift assays, and site-directed mutagenesis, we showed that OsCBT has two different types of functional CaM-binding domains, an IQ motif, and a Ca(2+)-dependent motif. To determine the DNA binding specificity of OsCBT, we employed a random binding site selection method. This analysis showed that OsCBT preferentially binds to the sequence 5'-TWCG(C/T)GTKKKKTKCG-3' (W and K represent A or C and T or G, respectively). OsCBT was able to bind this sequence and activate beta-glucuronidase reporter gene expression driven by a minimal promoter containing tandem repeats of these sequences in Arabidopsis leaf protoplasts. Green fluorescent protein fusions of two putative nuclear localization signals of OsCBT, a bipartite and a SV40 type, were predominantly localized in the nucleus. Most interestingly, the transcriptional activation mediated by OsCBT was inhibited by co-transfection with a CaM gene. Taken together, our results suggest that OsCBT is a transcription activator modulated by CaM.

AtGA3ox2, a Key Gene Responsible for Bioactive Gibberellin Biosynthesis, Is Regulated during Embryogenesis by LEAFY COTYLEDON2 and FUSCA3 in Arabidopsis

Embryonic regulators LEC2 (LEAFY COTYLEDON2) and FUS3 (FUSCA3) are involved in multiple aspects of Arabidopsis (Arabidopsis thaliana) seed development, including repression of leaf traits and premature germination and activation of seed storage protein genes. In this study, we show that gibberellin (GA) hormone biosynthesis is regulated by LEC2 and FUS3 pathways. The level of bioactive GAs is increased in immature seeds of lec2 and fus3 mutants relative to wild-type level. In addition, we show that the formation of ectopic trichome cells on lec2 and fus3 embryos is a GA-dependent process as in true leaves, suggesting that the GA pathway is misactivated in embryonic mutants. We next demonstrate that the GA-biosynthesis gene AtGA3ox2, which encodes the key enzyme AtGA3ox2 that catalyzes the conversion of inactive to bioactive GAs, is ectopically activated in embryos of the two mutants. Interestingly, both beta-glucuronidase reporter gene expression and in situ hybridization indicate that FUS3 represses AtGA3ox2 expression mainly in epidermal cells of embryo axis, which is distinct from AtGA3ox2 pattern at germination. Finally, we show that the FUS3 protein physically interacts with two RY elements (CATGCATG) present in the AtGA3ox2 promoter. This work suggests that GA biosynthesis is directly controlled by embryonic regulators during Arabidopsis embryonic development.

Genetic transformation of loblolly pine using mature zygotic embryo expiants byAgrobacterium tumefaciens

Agrobacterium tumefaciens strain LBA 4404 carrying pBI121 plasmid was used to transform mature zygotic embryos of three genotypes (E-Hb, E-Ma, and E-Mc) of loblolly pine. The results demonstrated that the expression frequency of β-glucuronidase reporter gene (GUS) varied among genotypes after mature zygotic embryos were infected withAgrobacterium tumefaciens cultures. The highest frequency (27.8%) of GUS expressing embryos was obtained from genotype E-Mc with mean number of 21.9 blue GUS spots per embryo. Expression of β-glucuronidase reporter gene was observed on cotyledons, hypocotyls, and radicles of transformed mature zygotic embryos, as well as on organogenic callus and regenerated shoots derived from co-cultivated mature zygotic embryos. Nineteen regenerated transgenic plants were obtained from GUS expression and kanamycin resistant calli. The presence and integration of the GUS gene was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. These results suggested that an efficientAgrobacterium tumefaciens-mediated transformation protocol for stable integration of foreign genes into loblolly pine has been developed and that this transformation system could be useful for the future studies on transferring economically important genes to loblolly pine.

Specific interactions with TBP and TFIIB in vitro suggest that 14-3-3 proteins may participate in the regulation of transcription when part of a DNA binding complex.

The 14-3-3 family of multifunctional proteins is highly conserved among animals, plants, and yeast. Several studies have shown that these proteins are associated with a G-box DNA binding complex and are present in the nucleus in several plant and animal species. In this study, 14-3-3 proteins are shown to bind the TATA box binding protein (TBP), transcription factor IIB (TFIIB), and the human TBP-associated factor hTAF(II)32 in vitro but not hTAF(II)55. The interactions with TBP and TFIIB were highly specific, requiring amino acid residues in the box 1 domain of the 14-3-3 protein. These interactions do not require formation of the 14-3-3 dimer and are not dependent on known 14-3-3 recognition motifs containing phosphoserine. The 14-3-3-TFIIB interaction appears to occur within the same domain of TFIIB that binds the human herpes simplex virus transcriptional activator VP16, because VP16 and 14-3-3 were able to compete for interaction with TFIIB in vitro. In a plant transient expression system, 14-3-3 was able to activate GAL4-dependent beta-glucuronidase reporter gene expression at low levels when translationally fused with the GAL4 DNA binding domain. The in vitro binding with general transcription factors TBP and TFIIB together with its nuclear location provide evidence supporting a role for 14-3-3 proteins as transcriptional activators or coactivators when part of a DNA binding complex.

Optimisation of procedures for microprojectile bombardment of microspore-derived embryos in wheat

Using the PDS-1000/He Biolistic® Particle Delivery System, the microprojectile travel distance, rupture disk pressure and DNA/gold particle concentrations were assessed in order to optimise short and longer-term β-glucuronidase reporter gene expression in microspore-derived embryos of wheat. The effects were also evaluated of using sterile filter paper to support explants and treatment with a high osmoticum medium (0.2 M mannitol/0.2 M sorbitol or 0.4 M maltose). In the optimised procedure, wheat microspore-derived embryos (MDEs), were placed on filter paper and incubated on medium containing 0.4 M maltose, for 4 h pre- and 45 h post-bombardment. Five μl pAHC25 (0.75 mg ml-1 in TE buffer) was precipitated onto 25 μl gold particles (60 mg ml-1 in sterile water), using 20 μl spermidine (0.1 M) and 50 μl CaCl2 (2.5 M). The particles were centrifuged and resuspended in 75 μl absolute ethanol prior to the preparation of 6 macrocarriers. A microprojectile travel distance of 70 mm, a rupture pressure of 1300 p.s.i., and a vacuum of 29′′ Hg were employed. Maltose at 0.4 M in the support medium was the most important factor influencing GUS activity in bombarded tissues. GUS activity, 1 day post-bombardment, reached 52 ± 17 GUS-positive foci/MDE (mean ± s.e.m, n=3), with 17 ± 4 foci/MDE at 15 days, giving a 3.0-fold increase (p<0.05) compared to expression in MDEs bombarded on medium without a high osmoticum treatment.

Cell-specific expression of the promoters of two nonlegume hemoglobin genes in a transgenic legume, Lotus corniculatus.

The promoters of the hemoglobin genes from the nitrogen-fixing tree Parasponia andersonii and the related nonnitrogen-fixing Trema tomentosa both confer beta-glucuronidase reporter gene expression to the central zone of the nodules of a transgenic legume, Lotus corniculatus. beta-Glucuronidase expression was high in the uninfected interstitial cells and parenchyma of the surrounding boundary layer and was low in the Rhizobium-infected cells. This contrasts with the expression of both the P. andersonii hemoglobin protein in P. andersonii nodules and the endogenous Lotus leghemoglobins that are expressed in the infected cells at very high levels. The expression pattern of the P. andersonii and T. tomentosa hemoglobin promoters in L. corniculatus resembles that of a nonsymbiotic hemoglobin gene from Casuarina glauca, which was introduced into this legume, and suggests that only the nonsymbiotic functions of the P. andersonii promoter are being recognized. Deletion of the distal segments of both the P. andersonii and T. tomentosa promoters identified regions important for the control of their tissue-specific and temporal activity in Lotus. Potential regulatory elements, which enhance nodule expression and suppress nonnodule expression, were also identified and localized to a distal promoter segment. A proximal AAGAG motif is present in the P. andersonii, T. tomentosa, and nonsymbiotic Casuarina hemoglobin genes. Mutation of this motif in the P. andersonii promoter resulted in a significant reduction in both the nodule and root expression levels in L. corniculatus. Some of the regulatory motifs characterized are similar to, but different from, the nodulin motifs of the leghemoglobins.