Tissue specific expression of β-glucuronidase gene driven by heterologous promoters in transgenic cauliflower plants

In this paper we compare five heterologous promoters fused to β-glucuronidase gene in their influence on localization of GUS activity in cauliflower (Brassica oleracea var. botrytis) tissues: roots, leaves, petioles and curds. A constitutive promoter CaMV 35S and four tissue specific promoters were used: extAP from rape, PsMTAP from pea, RBCS3CP from tomato and SRS1P from soybean, and introduced into cauliflower seedling explants using Agrobacterium rhizogenes mediated transformation. Quantitative and histochemical GUS assays confirmed tissue specific gus expression. It was found that extAP promoter was the most active in petioles but also caused a significant gus expression in curds. GUS activity was hardly observed in curd and restricted only to its epidermis when PsMTAP promoter drove the gene. RBCS3CP and SRS1P promoters controlled similar expression of the gus gene throughout the plant except for curd where RBCS3CP was almost inactive.     

Stability of β-glucuronidase gene expression in transgenic Tricyrtis hirta plants after two years of cultivation

Transgenic plants of Tricyrtis hirta carrying the intron-containing β-glucuronidase (GUS) gene under the control of the CaMV35S promoter have been cultivated for two years. Four independent transgenic plants produced flowers 1–2 years after acclimatization, and all of them contained one copy of the transgene as indicated by inverse polymerase chain reaction (PCR) analysis. All the four transgenic plants showed stable expression of the gus gene in leaves, stems, roots, tepals, stamens and pistils as indicated by histochemical and fluorometric GUS assays, although differences in the GUS activity were observed among different organs of each transgenic plant. No apparent gus gene silencing was observed in transgenic T. hirta plants even after two years of cultivation.     

Tissue-specific expression of the β-glucuronidase reporter gene in transgenic strawberry (Fragaria × ananassa) plants

The strawberry ( Fragaria spp) is regarded as a false fruit because it originates from the receptacle, which is a non-ovarian tissue. For this reason, fruit-specific promoters isolated from plant species in which the fruit is derived from the ovary wall might not be suited to control gene expression in a fruit-specific way in strawberry. In order to achieve (false) fruit-specific expression in strawberry, we tested the petunia FBP7 (floral binding protein7) promoter, which proved to be active in the receptacles of petunia flowers, in transgenic strawberry fruits. In strawberry plants containing the FBP7 promoter fused to the ß-glucuronidase (GUS) reporter gene ( gus), GUS activity was found in floral and fruit tissues of all developmental stages tested but not in leaf, petiole and root tissue . Surprisingly, Northern blot analysis showed the presence of gus-derived mRNAs in root (strong) and petiole (weak) tissue of fbp7- gus plants in addition to the floral and fruit tissues. Therefore, it is concluded that the histological GUS phenotype does not necessarily correspond with expression at the mRNA level. mRNA quantification using the TaqMan polymerase chain reaction technology confirmed the Northern results and showed that in red strawberry tissue the cauliflower mosaic virus 35S promoter is at least sixfold stronger than the FBP7 promoter.     

Endoplasmic reticulum targeting of active modified β-glucuronidase (GUS) in transgenic tobacco plants

We have investigated targeting to the endoplasmic reticulum (ER) of wild-type GUS and a modified form (GUS S358) by making an N-terminal fusion of the -glucuronidase (GUS) enzyme with the wheat -amylase signal peptide.In vitro studies demonstrated that the modified GUS (S358) lacked the glycosylation site present within the wild-type enzyme. Analysis of transgenic tobacco plants revealed that the modified GUS enzyme retained activity upon passage to the ER. When further experiments were carried out to determine the cellular location of the modified GUS enzyme, it was found that (contrary to expectation) the majority of GUS activity was retained within the cell and was not secreted to the cell surface via the default pathway. The data indicated that the modified GUS enzyme is an unsuitable reporter enzyme for studying protein secretion.     

Tomato prosystemin promoter confers wound-inducible, vascular bundle-specific expression of the β-glucuronidase gene in transgenic tomato plants

Tomato (Lycopersicon esculentum Mill. cv. Better Boy) plants were transformed with a fused gene containing a 2.2-kb promoter fragment of the tomato prosystemin gene and the coding region of the β-glucuronidase (GUS) reporter gene. The transgenic plants exhibited a low constitutive level of prosystemin-β-glucuronidase gene expression, assayed by histochemical staining and GUS enzyme activity, that was associated in the vascular bundles of leaf main veins, petiolules, petioles and stems. The GUS activity in the vascular bundles in each tissue was increased by wounding and by treatment of the plants with methyl jasmonate, similar to the induction of prosystemin in wild-type plants. The increase in GUS activity in the vascular bundles of leaves in response to wounding correlated with the wound-inducible increase in prosystemin mRNA. Tissue printing, using rabbit anti-serum prepared against prosystemin, confirmed that inducible prosystemin protein was localized in vascular bundles of petiolules, petioles and stems of wild-type tomato plants. The evidence indicates that the 2.2-kb promoter region of the tomato prosystemin gene contains elements conferring its correct temporal and spatial expression in the vascular bundles of transgenic tomato plants. Received: 7 January 1997 / Accepted: 2 April 1997     

Leaf developmental stage and tissue location affect the detection of β-glucuronidase in transgenic tobacco plants

Expression in Nicotiana tabaccum L. plants containing the -glucuronidase (GUS) gene under the control of the 35S (CaMV promoter) was affected by tissue type and ontogenic development of the leaves. GUS activity in ontogenetically younger leaves was 1003–1022 nmol 7-hydroxy-4-methylcoumarin (MU) formed mg^–1 (protein) min^–1 and in ontogenetically older leaves was only 140–198 nmol (MU) mg^–1 (protein) min^–1.     

Isolation of the promoter of a cotton β-galactosidase gene (GhGal1) and its expression in transgenic tobacco plants

β-galactosidases (GUS, EC 3.2.1.23) are character- ized by their ability to hydrolyze terminal, non-re- ducing β-D-galactosyl residues from β-D-galactosides and are widely distributed in microbes, plants and animals. To date, the primary structures of …     

Lethality of inducible,meristem-localized ectopic β-glucuronidase expression in plants

GUSA fromEscherichia coli, encoded by theuidA gene, has been successfully used as a plant reporter system for more than a decade with no reported deleterious effects. However, when expressed in coordination with a UDP-glucuronosyltransferase isolated from the root cap meristem ofPisum sativum (PsUGT1) at the onset of mitosis, GUSA expression was lethal in pea, alfalfa, andArabidopsis thaliana. These unexpected results indicate that, under some circumstances, using GUSA in plants is incompatible with life and suggest that the cell-specific lethal phenotype might be useful in selecting for genes specifically involved in regulating the G2-M phase of the cell cycle.     

A thermostable β-glucuronidase obtained by directed evolution as a reporter gene in transgenic plants

A β-glucuronidase variant, GUS-TR3337, that was obtained by directed evolution exhibited higher thermostability than the wild-type enzyme, GUS-WT. In this study, the utility of GUS-TR337 as an improved reporter was evaluated. The corresponding gus-tr3337 and gus-wt genes were independently cloned in a plant expression vector and introduced into Arabidopsis thaliana. With 4-MUG as a substrate, plants containing the gus-wt gene showed no detectable β-glucuronidase activity after exposure to 60°C for 10 min, while those hosting the gus-tr3337 gene retained 70% or 50% activity after exposure to 80°C for 10 min or 30 min, respectively. Similarly, in vivo β-glucuronidase activity could be demonstrated by using X-GLUC as a substrate in transgenic Arabidopsis plants hosting the gus-tr3337 gene that were exposed to 80°C for up to 30 min. Thus, the thermostability of GUS-TR3337 can be exploited to distinguish between endogenous and transgenic β-glucuronidase activity, which is a welcome improvement in its use as a reporter.     

Tapetum-specific expression of theOsg6B promoter-β-glucuronidase gene in transgenic rice

The promoter of an anther tapetum-specific gene,Osg6B, was fused to a-glucuronidase (GUS) gene and introduced into rice byAgrobacterium-mediated gene transfer. Fluorometric and histochemical GUS assay showed that GUS was expressed exclusively within the tapetum of anthers from the uninucleate microspore stage (7 days before anthesis) to the tricellular pollen stage (3 days before anthesis). This is the first demonstration of an anther-specific promoter directing tapetum-specific expression in rice.Abbreviations GUS ßGlucuronidase     

Separation of recombinant β-glucuronidase from transgenic tobacco by aqueous two-phase extraction

Transgenic plants hold many promises as viable production hosts for therapeutic recombinant proteins. Many efforts have been devoted to increase the expression level of the proteins, but the efforts for developing economic processes to purify those proteins are lacking. In this report, aqueous two-phase extraction (ATPE) was investigated as an alternative for the separation of an acidic recombinant protein, β-glucuronidase (rGUS), from transgenic tobacco. Screening experiments by fractional factorial designs showed that PEG concentration and ionic strength of the system significantly affected the partitioning of native tobacco proteins and GUS. Response surface methodology was used to determine an optimized aqueous two-phase system for the purification of rGUS from transgenic tobacco. In a 13.4% (w/w) PEG 3400/18% (w/w) potassium phosphate system, 74% of the rGUS was recovered in the top PEG-rich phase while more than 90% of the native tobacco proteins were removed in the interphase and the bottom phase. A purification factor of about 20 was achieved in this process. The most important impurity from tobacco, Rubisco, was largely removed from the rGUS in the recovered phase.     

High-level expression of sucrose inducible sweet potato sporamin gene promoter: β-glucuronidase fusion gene in transgenic Nicotiana plumbaginifolia hairy roots

In Vitro Cellular & Developmental Biology - Plant - We developed transgenic Nicotiana plumbaginifolia hairy roots with sucrose-inducible minimal promoter (Spomin)-β-glucuronidase (GUS)...     

Plant endogenous β-glucuronidase activity: how to avoid interference with the use of theE. coli β-glucuronidase as a reporter gene in transgenic plants

We have detected a plant β-glucuronidase activity, present in several tissues and organs of plant species belonging to different families. The fluorimetric β-glucuronidase assay was used to partially characterize this activity in post-ribosomal supernatants of tobacco leaves. The tobacco activity is very stable at low temperatures, but quickly inactivated above 45°C. It is relatively resistant to proteases and insensitive to-SH group reagents and to ionic conditions. It does not require, nor is it inhibited by, divalent cations. Although these properties are shared by theEscherichia coli β-glucuronidase, the two activities can be distinguished by: (i) their different sensitivity to the specific inhibitor saccharic acid-1,4-lactone; (ii) their different thermal stability (iii) their different pH optima (5.0 for the plant activity and close to neutral for the bacterial enzyme). Therefore, under appropriate experimental conditions, it should be possible to assay theE. coli β-glucuronidase in transgenic plants without interference from the endogenous plant activity.     

The sucrose synthase-1 promoter from Citrus sinensis directs expression of the β-glucuronidase reporter gene in phloem tissue and in response to wounding in transgenic plants

Interest in phloem-specific promoters for the engineering of transgenic plants has been increasing in recent years. In this study we isolated two similar, but distinct, alleles of the Citrus sinensis sucrose synthase-1 promoter (CsSUS1p) and inserted them upstream of the β-glucuronidase (GUS) gene to test their ability to drive expression in the phloem of transgenic Arabidopsis thaliana and Nicotiana tabacum. Although both promoter variants were capable of conferring localized GUS expression in the phloem, the CsSUS1p-2 allele also generated a significant level of expression in non-target tissues. Unexpectedly, GUS expression was also instigated in a minority of CsSUS1p::GUS lines in response to wounding in the leaves of transgenic Arabidopsis. Deletion analysis of the CsSUS1p suggested that a fragment comprising nucleotides −410 to −268 relative to the translational start site contained elements required for phloem-specific expression while nucleotides −268 to −103 contained elements necessary for wound-specific expression. Interestingly, the main difference between the two CsSUS1p alleles was the presence of a 94-bp insertion in allele 2. Fusion of this indel to a minimal promoter and GUS reporter gene indicated that it contained stamen and carpel-specific enhancer elements. This finding of highly specific and separable regulatory units within the CsSUS1p suggests that this promoter may have a potential application in the generation of constructs for the use in the development of transgenic plants resistant to a wide variety of target pests.