Gene Encoding Β-glucuronidase Activity Research Articles

Expression of uidA gene in tobacco transplastomic plants under the control of 5'-untranslated region of genomic RNA of potato virus Y

Regulation of the expression of chloroplast genes mainly occurs at the post-transcriptional level, in which 5'-untranslated regions (5'UTR) of plastid mRNA play a significant role. In transplastomic tobacco plants, we studied the effect of the 5’UTRs of the genomic RNA of potato Y-virus (5’PVY), as well as the 5’UTR of the plastid psbA gene (5’psbA) on the expression of the bacterial reporter uidA gene encoding β-glucuronidase (GUS). Genetic constructs containing the uidA gene were created under the control of identical transcriptional regulatory elements: the promoter and terminator of the psbA gene and two different 5’UTRs — 5’psbA and 5’PVY. Тobacco chloroplasts were transformed with these constructs by bioballistic method. Transplastomic lines showed approximately 24-fold differences in GUS activity. Replacing the natural 5’psbA with the heterologous 5’PVY resulted in a significant decrease in the synthesis of GUS protein. Key words: chloroplasts, protein synthesis, 5’UTR, transplastomic plants, reporter protein.

Mature Luffa Leaves (Luffa cylindrica L.) as a Tool for Gene Expression Analysis by Agroinfiltration.

We exploited the potential of cucurbits for ectopic gene expression. Agroinfiltration is a simple and commonly used method to obtain transient expression of foreign genes in plants. In contrast to in vitro transformation techniques, agroinfiltration can be used for genetic modification of mature plant tissues. Although the cucurbits are commonly used as model plants for molecular biology and biotechnology studies, to date there are no literature sources on the possibility of transient gene expression in mature cucurbit tissues. Our research has shown that mature leaves of Luffa cylindrica L. (luffa), in contrast to other cucurbit species, can be successfully transiently transformed with Agrobacterium tumefaciens. We efficiently transformed luffa leaves with a reporter gene encoding β-glucuronidase (GUS). The GUS activity in transiently transformed leaf tissues was detected within 24 h after the infiltration with bacteria. Additionally, we have shown that the activity of a transiently expressed the GUS gene can be monitored directly in the EDTA-exudates collected from the cut petioles of the agroinfiltrated leaves. The results suggest that luffa leaves can be useful as a plant expression system for studies of physiological and biochemical processes in cucurbits.

Overexpression of a rice heme activator protein gene (OsHAP2E) confers resistance to pathogens, salinity and drought, and increases photosynthesis and tiller number.

Heme activator protein (HAP), also known as nuclear factor Y or CCAAT binding factor (HAP/NF-Y/CBF), has important functions in regulating plant growth, development and stress responses. The expression of rice HAP gene (OsHAP2E) was induced by probenazole (PBZ), a chemical inducer of disease resistance. To characterize the gene, the chimeric gene (OsHAP2E::GUS) engineered to carry the structural gene encoding β-glucuronidase (GUS) driven by the promoter from OsHAP2E was introduced into rice. The transgenic lines of OsHAP2Ein::GUS with the intron showed high GUS activity in the wounds and surrounding tissues. When treated by salicylic acid (SA), isonicotinic acid (INA), abscisic acid (ABA) and hydrogen peroxide (H2 O2 ), the lines showed GUS activity exclusively in vascular tissues and mesophyll cells. This activity was enhanced after inoculation with Magnaporthe oryzae or Xanthomonas oryzae pv. oryzae. The OsHAP2E expression level was also induced after inoculation of rice with M. oryzae and X. oryzae pv. oryzae and after treatment with SA, INA, ABA and H2 O2, respectively. We further produced transgenic rice overexpressing OsHAP2E. These lines conferred resistance to M. oryzae or X. oryzae pv. oryzae and to salinity and drought. Furthermore, they showed a higher photosynthetic rate and an increased number of tillers. Microarray analysis showed up-regulation of defence-related genes. These results suggest that this gene could contribute to conferring biotic and abiotic resistances and increasing photosynthesis and tiller numbers.

Response of an aspartic protease gene OsAP77 to fungal, bacterial and viral infections in rice

BackgroundAspartic protease (APs) plays important roles in plant growth, development and biotic and abiotic stresses. We previously reported that the expression of a rice AP gene (OsAP77, Os10g0537800) was induced by probenazole (PBZ), a chemical inducer of disease resistance. In this study we examined some characteristics of this gene in response to fungal, bacterial and viral pathogens.ResultsTo elucidate the spatial and temporal expression of OsAP77, the chimeric gene was constructed carrying the structural gene encoding β-glucuronidase (GUS) driven by the OsAP77 promoter. This construct was introduced into rice and the transgenic lines were tested to analyze gene expression by fungal, bacterial and viral infections. Inoculation with Magnaporthe oryzae or Xanthomonas oryzae pv. oryzae revealed the enhanced GUS activities in vascular tissues surrounding the symptom sites by each pathogen. Moreover, GUS activity also increased after inoculation with Cucumber mosaic virus (CMV). Transgenic plants immersed in a solution containing salicylic acid (SA), isonicotinic acid (INA), hydrogen peroxide (H2O2) or abscisic acid (ABA) showed an increased level of GUS activity exclusively in vascular tissues. RT-PCR analysis showed that OsAP77 was induced not only by infection with these pathogens, but also after treatment with SA, INA, H2O2 or ABA. A knockout mutant line of OsAP77 by the insertion of Tos17 after inoculation with M. oryzae, X. oryzae pv. oryzae or CMV showed an enhanced susceptibility compared to wild type.ConclusionThese results suggest that the expression of OsAP77 is induced by pathogen infection and defense related signaling molecules in a vascular tissue specific manner and that this gene has a positive role of defense response against fungal, bacterial and viral infections.Electronic supplementary materialThe online version of this article (doi:10.1186/s12284-014-0009-2) contains supplementary material, which is available to authorized users.

Analysis of the spatial and temporal expression pattern directed by the Populus tomentosa 4-coumarate:CoA ligase Pto4CL2 promoter in transgenic tobacco

4-Coumarate:CoA ligase (4CL) is a key enzyme in the phenylpropanoid synthesis pathway. The Pto4CL2 promoter was cloned from Populus tomentosa Carr. and fused to the reporter gene encoding β-glucuronidase (GUS); the complex expression patterns directed by the Pto4CL2 promoter were then characterized in Nicotiana tabacum Xanthi by histochemical assays. The promoter 5'-deletion and histochemical assay conducted on transformants indicated that the -317 to -292 nt region supports Pto4CL2 expression in the epidermis and petals and the deletion of the -266 to -252 nt region resulted in the loss of tissue specificity and a dramatic reduction in GUS activity. Furthermore, electrophoretic mobility shift assays testified that an adenine and cytosine-rich element (-264 to -255 nt) and an abscisic acid-responsive element (-242 to -235 nt) in the Pto4CL2 promoter would have functions for the complex expression profiling and efficient basal expression, respectively. These results further clarify the mode of the regulatory expression of class II 4CL promoters in higher plants.

Cytokinin regulates differentially expression of P AHK -GUS constructs in transgenic Arabidopsis thaliana plants

The expression regulation by cytokinin of genetic constructs PAHK2-GUS, PAHK3-GUS, and PAHK4-GUS in transgenic Arabidopsis thaliana (L.) Heynh plants bearing the gene encoding β-glucuronidase (GUS) under the control of the promoter of one of three genes encoding histidine protein kinases, which are membrane receptors of cytokinin was studied. In 4–5-day-old etiolated A. thaliana seedlings, treatment with cytokinin resulted in the strongest expression activation of the constructs PAHK2-GUS and PAHK3-GUS. The same constructs were activated by cytokinin also at the seedling transit from scoto- to photomorphogenesis. Long-term seedling growing in darkness on medium containing cytokinin resulted in the substantial promoter activation of the gene encoding the histidine kinase AHK2. In the leaves of three-week-old plants with actively functioning chloroplasts, treatment with cytokinin mainly stimulated expression of the construct PAHK3-GUS. In detached senescing leaves, treatment with cytokinin retarded the loss of chlorophyll but did not affect significantly GUS activity under both light and darkness conditions in either of tested lines containing GUS gene under the control of promoters of histidine kinase genes. At the same time, cytokinin activated the promoter of the gene of primary response to cytokinin in the construct PARR5-GUS. Thus, in the studied test-system, treatment with cytokinin of A. thaliana plant grown in darkness or in the light affected differently the expression of histidine kinase genes in dependence of plant age, conditions of plant cultivation, and plant physiological state.

Colonization of Phaseolus vulgaris nodules by Agrobacterium-like strains

Non-nodulating Agrobacterium-like strains identified among root nodule isolates of common bean were labeled with gusA, a reporter gene encoding beta-glucuronidase (GUS). Bean plants were then co-inoculated with an infective Rhizobium strain and labeled transconjugants of Agrobacterium-like strains. Blue staining of nodules showed that Agrobacterium-like strains were able to colonize these symbiotic organs. Isolation and characterization by restriction fragment length polymorphism analysis of PCR-amplified 16S rRNA genes revealed a mixed population of Rhizobium and Agrobacterium-like strains in all nodules showing GUS activity. PCR amplification of the nifH gene and nodulation tests did not show any evidence of acquisition of symbiotic gene by lateral transfer from Rhizobium to Agrobacterium-like strains. Moreover, these strains were able to invade mature nodules. Based on sequencing of the 16S rRNA gene, one of these Agrobacterium-like strains showed 99.4% sequence similarity with Agrobacterium bv. 1 reference strains and 99% similarity with an Agrobacterium bv. 1 strain isolated from Acacia mollisima in Senegal. Agrobacterium tumefaciens C58 and the disarmed variant AT123 did not show any ability to colonize nodules. Co-inoculation of bean seeds with Agrobacterium and Rhizobium strains did not enhance nodulation and plant yield under controlled conditions.

Agrobacterium-mediated transformation of cotton (Gossypium hirsutum L.) via vacuum infiltration

AnAgrobacterium-mediated gene transfer system with recovery of putative transformants was developed for cotton (Gossypium hirsutum L.) cv. Cocker-312. Two-month-old hypocotyl-derived embryogenic calli were infected through agroinfiltration for 10 min at 27 psi in a suspension ofAgrobacterium tumefaciens strain GV3101 carrying tDNA with theGUS gene, encoding β-glucuronidase (GUS), and the neomycin phosphotransferase II (nptII) gene as a kanamycin-resistant plant-selectable marker. Six days after the histochemicalGUS assay was done, 46.6% and 20%GUS activity was noted with the vacuum-infiltration and commonAgrobacterium-mediated transformation methods, respectively. The transformed embryogenic calli were cultured on selection medium (100 mg/L and 50 mg/L kanamycin for 2 wk and 10 wk, respectively) for 3 mo. The putative transgenic plants were developed via somatic embryogenesis (25 mg/L kanamycin). In 4 independent experiments, up to 28.23% transformation efficiency was achieved. PCR amplification and Southern blot analysis fo the transformants were used to confirm the integration of the transgenes. Thus far, this is the only procedure available for cotton that can successfully be used to generate cotton transformants.

Antisense RNA Inhibition of uidA Gene Expression in Transgenic Plants: Evidence for Interaction Between First and Second Transformation Events

The uidA reporter gene encoding β-glucuronidase (GUS) driven by the 35s promoter was introduced into Nicotiana tabacum using a bipartite vector system based on Agrobacterium tumefaciens strain A281. Four primary transformants showing high levels of uidA expression were transformed with a binary vector in which the uidA structural gene was in antisense orientation to the CaMV 35s promoter. The retransformants from each primary transformant showed reduction in GUS activity varying from 5 to 99 %. The average reduction between retransformants varied significantly from 44 to 93 %. Southern blot analysis showed the presence of the sense uidA gene in high copy number (10–20) in both primary and secondary transformants. The secondary transformants contained, in addition, 15–60 copies of the antisense uidA construct. Antisense uidA bands resulting from direct or inverted repeats and truncated forms could be provisionally identified. The banding pattern was characteristic for each family, indicating an interaction between the first and second transformation events. Seedlings raised by selling the primary transformants showed monohybrid segregation for the hygromycin resistance marker; seed set from the antisense plants was disturbed, and of the I1 plants setting sufficient seed for analysis, 6 showed dihybrid segregation for the kanamycin resistance marker.

Systemic induction of an Arabidopsis plant defensin gene promoter by tobacco mosaic virus and jasmonic acid in transgenic tobacco

The PDF1.2 gene of Arabidopsis thaliana encodes a plant defensin that is systemically induced by a SA-independent signaling pathway. Traditionally tobacco has been used to analyse pathogen-induced systemic responses. To determine whether a similar systemic signaling pathway exists in tobacco the promoter region of the PDF1.2 gene was fused to the uidA reporter gene encoding β-glucuronidase (GUS) and introduced into tobacco (cv. Xanthi-nc NN). The transgenic tobacco plants showed no increase in GUS activity after treatment with salicylate but treatment of seedlings and mature leaves with jasmonic acid, methyl viologen and rose bengal led to an induction of GUS activity with jasmonic acid being the strongest inducer. Exposure of mature transgenic plants to ethylene also led to a significant induction of GUS. Wounding resulted in highly localised induction at wound sites. Inoculation of leaves with the compatible pathogens Phytophthora parasitica var . nicotianae, Cercospora nicotianae and the incompatible tobacco mosaic virus (TMV) all led to strong GUS induction. The systemic signaling of the PDF1.2 promoter was investigated by either inoculation of a lower leaf with TMV or treatment of this leaf with jasmonic acid. Increased GUS activity was observed in the non-inoculated upper leaves at 4–6 days after treatment. Treatment of the plants with TMV induced GUS mRNA and PR1a mRNA locally and systemically while jasmonic acid treatment induced GUS mRNA only. These results are consistent with the existence of a pathogen-induced, salicylate-independent systemic signaling pathway, possibly involving ethylene and jasmonate signaling components, in both tobacco and Arabidopsis.

High levels of expression of full-length cryIA(c) gene from Bacillus thuringiensis in transgenic somatic walnut embryos

A full-length synthetic version of the cryIA(c) gene, expressing a Bacillus thuringiensis (Bt) insecticidal crystal protein (ICP), was transferred into walnut somatic embryos. Sixty one transgenic embryo lines or clones were obtained and bioassayed with first instar codling moth larvae. In 34% of these lines, designated as ‘class A’, expression was high enough to obtain 80–100% mortality. A total of 20% were designated ‘class B’, which produced a mortality between 25 and 70% and also caused a decreased rate of larval development. Insect mortality and development from the remaining 46% of the lines were indistinguishable from that of the control. Expression of a chimeric gene encoding β-glucuronidase (GUS) was evaluated to serve as a linked but unselected marker gene. About 62% of the class A embryo lines showed correspondingly high activity for the GUS gene. However, 38% of the class A would have been missed if the level of GUS activity was used as the sole indicator. Detectable levels of protein corresponding to cryIA(c) could be found only in class A, but not in class B or C clones. Southern analysis of border regions revealed single inserts for class A clones and multiple inserts for classes B and C.

Tissue-specificity of maize sucrose synthase gene promoters in maize tissues after particle bombardment

The reporter gene encoding β-glucuronidase (GUS) driven by either of the two maize sucrose synthase gene (Sh1 and Sus 1) promoters was introduced and expressed in various maize tissues via particle bombardment. Transient gene expression was examined by histochemical assays. It was found that the two SS promoters directed differential GUS expression. In the developing kernel, the Sh1 promoter was active only in the upper and central parts of the endosperm. In contrast, strong GUS activity controlled by the Sus1 promoter was detected in various types of cells, including the aleurone cells, the subaleurone endosperm cells, the scutellar cells of the embryo and the pericarp cells. Both promoters showed similar expression patterns in vegetative tissues.

Transformation of Perilla frutescens var. crispa Using an Agrobacterium-Ri Binary Vector System.

Transgenic hairy roots of Perilla frutescens var. crispa were obtained using an Agrobacterium-Ri binary vector system. The chimeric uidA gene encoding β-glucuronidase (GUS) flanked by the TR2′ promoter for mannopine synthase was introduced in the plant genome by Agrobacterium-mediated transformation by means of scratching stems of young plants. The adventitious roots which arose on the infected sites were excised, and the several clones of root tissue could be maintained on hormone-free agar medium. The transgenic states of these clones were confirmed by Southern blot hybridization. Expression of the uidA gene in the transgenic tissues was indicated by in vitro enzyme assay and histochemical staining of GUS activity. More than half of hairy roots which emerged were confirmed to be doubly transformed with T-DNAs from Ri and binary Ti plasmids.

Characterisation of the expression of the glutamine synthetase gln-α gene of Phaseolus vulgaris using promoter-reporter gene fusions in transgenic plants

Approximately 900 bp of the Phaseolus vulgaris cytosolic glutamine synthetase gln-α gene promoter has been cloned and the DNA sequence determined. The transcriptional start site was mapped using RNase protection and primer extension techniques. The promoter fragment was fused to the Escherichia coli uidA reporter gene encoding β-glucuronidase (GUS) and used to transform Nicotiana tabacum (tobacco). The gln-α promoter directed uidA expression in the root-tips, emerging lateral roots and in root hairs. Activity of the gln-α promoter was also associated with transgenic leaf vascular tissue as well as several transgenic flower and fruit structures. Mechanical wounding of transgenic plant leaves increased the level of extractable GUS activity from between 2- and 5-fold, relative to non-wounded leaves. Histochemical analysis of the wounded leaf tissue suggested uidA expression was associated with the leaf vascular tissue and not with tissues adjacent to the wound site. The role and regulation of the gln-α gene is discussed.

Transformation and regeneration of French bean plants by the particle bombardment process

Stable transformation of French bean ( Phaseolus vulgaris L. cv. Goldstar) plants has been achieved using gold particles coated with plasmid pSOG-1016 carrying a chimeric gene encoding β-glucuronidase (GUS) under the control of concanavalin A (Con A) promoter. The bombardment conditions were optimized by the observation of transient expression of the GUS reporter gene bombarded to the shoot apexes of embryonic axes. After bombardment of embryonic axes, many particles were detectable at 20–80 μm of depth from the top of the shoot apex by anatomic observation. The bombarded embryonic axes were cultured for 3 weeks on MS or N6 medium without plant hormones. Regenerated plantlets were transferred to soil and kept in a growth chamber where they subsequently flowered and produced seeds. PCR and Southern blot analysis showed the integration of the Con A::GUS gene. The expression of the GUS activity was observed in the cotyledons and seed coats of the transgenic plants by histochemical staining.

Transformation of citrus embryogenic cells using particle bombardment and production of transgenic embryos

A transformation system was developed for the citrus variety tangelo ( Citrus reticulata Blanco × C. paradisi Macf.) cultivar ‘Page’. Embryogenic cells from suspension cultures were bombarded with tungsten particles coated with plasmid DNA which contained the uidA gene encoding β-glucuronidase (GUS) and the nptII gene conferring kanamycin resistance. More than 600 transient and 15 stable transformants were obtained per bombardment based on the number of GUS expression foci at 1 day and 8 weeks after bombardment. Pretreatment of cells with high osmotic potential (0.3 M sorbitol + 0.3 M mannitol) was found to enhance both transient and stable transformation efficiency. Between 2 and 3 months following bombardment, calli were formed on a medium containing kanamycin (100 mg·l −1). The majority of the kanamycin-resistant calli also expressed GUS activity. From these calli, embryos and plantlets were produced. Integration of the uidA and nptII genes into the tangelo genome was confirmed by polymerase chain reaction (PCR) and Southern analyses.

Field performance of derived generations of transgenic tobacco

Two inbred cultivars of Nicotiana tabacum (tobacco), 'Samsun' and 'Xanthi', were transformed with the plasmid pBI 121 using Bin 19 in Agrobacterium tumefaciens. The plasmid carries the nptII gene conferring kanamycin resistance and the uidA gene encoding β-glucuronidase (GUS). Progeny carrying the genes in the homozygous condition were identified and selfed over several generations. One line homozygous for the introduced genes and one untransformed control from each cultivar were then selected and crossed reciprocally to give four families per cultivar. Seeds from each family were grown in a replicated field trial and all plants scored for a range of morphological and agronomic characters. In addition, leaf samples were taken and GUS activity measured. In the 'Samsun' material, which contained one copy of the introduced gene at a single locus and showed high levels of GUS expression, the transformed homozygote showed twice the level of GUS activity as the hemizygotes, wheareas in the 'Xanthi' line, which had a lower level of GUS, the hemizygotes showed the same level of GUS activity as the transformed homozygote. The agronomic data showed differences between the families, but the source of such differences could not be ascribed unambiguously. The results are discussed in the light of related information on gene expression and field performance from other transgenic material.

Silicon carbide fiber-mediated stable transformation of plant cells

Maize (Zea mays, cv 'Black Mexican Sweet') (BMS) and tobacco (Nicotiana tabacum, cv 'Xanthi') tissue cultures were transformed using silicon carbide fibers to deliver DNA into suspension culture cells. DNA delivery was mediated by vortexing cells in the presence of silicon carbide fibers and plasmid DNA. Maize cells were treated with a plasmid carrying both the BAR gene, whose product confers resistance to the herbicide BASTA, and a gene encoding β-glucuronidase (GUS). Tobacco cells were treated with two plasmids to co-transfer genes encoding neomycin phosphotransferase (NPTII) and GUS from the respective plasmids. Thirty-four BASTA-resistant BMS colonies and 23 kanamycin-resistant tobacco colonies recovered following selection contained intact copies of the BAR gene and NPTII genes, respectively, as determined by Southern blot analysis. Sixty-five percent of the resistant BMS colonies and 50% of the resistant tobacco colonies also expressed GUS activity. Intact copies of the GUS gene were observed in Southern blots of all resistant BMS and tobacco colonies that expressed GUS activity. These results indicate that a simple, inexpensive DNA delivery procedure employing silicon carbide fibers can be used to reproducibly transform cells of both monocotyledonous and dicotyledonous plant species.Mention of a trademark, vendor, or proprietary product does not constitute a guarantee or warranty of the product by the University of Minnesota or the USDA, and does not imply its approval to the exclusion of other products or vendors that may also be suitable.

Expression of a foreign gene in electroporated pollen grains of tobacco

The incorporation of genetically engineered DNA into pollen and subsequent fertilization of eggs by the transformed pollen would be a convenient method for producing genetically engineered seed. This method of pollen transformation would circumvent the need for other types of gene transfer methods such as the use of Agrobacterium tumefaciens, which has a limited host range and thus a limited capability for genetically engineering plants. It would also avoid the problems associated with the regeneration of some plants from tissue, cell, or protoplast culture after receiving foreign DNA. To this end, the genetically engineered plasmid DNA vector pBI221 containing the gene encoding β-glucuronidase (GUS) was introduced by electroporation into germinating pollen grains of tobacco (Nicotiana gossei L.). Transient expression of the GUS gene was demonstrated by the presence of GUS activity in fluorometric assays of pollen extracts 24 h after the introduction of pBI221 via electroporation. Intact pBI221 was detected by Southern blotting procedures as a distinct DNA band in pollen extracts 1 h after electroporation. In addition, pBI221 was detected as a diffuse band of higher molecular weight DNA 24 h after electroporation, suggesting that some of the pBI221 was incorporated into the genome of the pollen.