The integrative expression of GUS gene driven by FCP promoter in the seaweed Laminaria japonica (Phaeophyta)

In this study, the background activity of β-glucuronidase (GUS) was analyzed histochemically and fluorometrically in the negative control of Laminaria japonica (Phaeophyta) thalli, showing low level of activity. GUS gene transformation without selectable gene in L. japonica was performed using four different promoters, i.e., Cauliflower mosaic virus 35S promoter (CaMV35S) from cauliflower mosaic virus, ubiquitin promoter (UBI) from maize, adenine-methyl transfer enzyme gene promoter (AMT) from virus in green alga Chlorella, and fucoxanthin chlorophyll a/c-binding protein gene promoter (FCP) from diatom Phaeodactylum tricornutum. The GUS transient activity was determined fluorometrically after bombarding sliced parthenogenetic sporophytes explants, and it was found that the activity resulting from CaMV35S and FCP promoters (in 114.3 and 80.6 pmol MU min⁻¹ (mg protein)⁻¹, respectively) was higher than for the other two promoters. The female gametophytes were bombarded and regenerated parthenogenetic sporophytes. FCP was the only promoter that resulted in detectable GUS chimeric expression activity during histochemical staining and polymerase chain reaction. Results of Southern blot showed that GUS gene was integrated with the L. japonica genome.     

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.     

Authentic seed-specific activity of the <Emphasis Type="Italic">Perilla</Emphasis> oleosin 19 gene promoter in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

The Perilla (Perilla frutescens L. cv. Okdong) oleosin gene, PfOle19, produces a 19-kDa protein that is highly expressed only in seeds. The activity of the −2,015 bp 5′-upstream promoter region of this gene was investigated in transgenic Arabidopsis plants using the fusion reporter constructs of enhanced green fluorescent protein (EGFP) and β-glucuronidase (GUS). The PfOle19 promoter directs Egfp expression in developing siliques, but not in leaves, stems or roots. In the transgenic Arabidopsis, EGFP fluorescence and histochemical GUS staining were restricted to early seedlings, indehiscent siliques and mature seeds. Progressive 5′-deletions up to the −963 bp position of the PfOle19 promoter increases the spatial control of the gene expression in seeds, but reduces its quantitative levels of expression. Moreover, the activity of the PfOle19 promoter in mature seeds is 4- and 5-fold greater than that of the cauliflower mosaic virus 35S promoter in terms of both EGFP intensity and fluorometric GUS activity, respectively.     

Promoter of the rolC gene of Agrobacterium rhizogenes can be strongly regulated in glandular cell of transgenic tobacco

A 577-bp promoter segment of Agrobacterium rhizogenes rolC, previously known as the phloem-specific gene expression promoter, was fused to the 5′ end of a reporter gene, β-glucuronidase (GUS), uidA. This rolC-promoter-driven expression of the GUS gene was found to be significantly strong in glandular cells in transgenic tobacco plants. Analysis of this segment of the promoter sequence revealed a myb response element.     

Identification and preliminary analysis of a new PCP promoter from Brassica rapa ssp. chinensis

The promoter of Brassica campestris Male Fertile 5 (BcMF5), a pollen coat protein member, class A (PCP-A) gene family, was isolated from Brassica rapa L. ssp. chinensis Makino (Chinese cabbage-pak-choi) by Thermal Asymmetric Interlaced Polymerase Chain Reaction (TAIL-PCR). Sequence analysis suggested that the 605-bp promoter of BcMF5 appears to be a pollen promoter. In an attempt to confirm the promoter activity of BcMF5 promoter, −609 to +3 bp and −377 to +3 bp fragments of the upstream sequence of BcMF5 were inserted at the site upstream of the coding region of the uidA gene in the sense orientation to construct two deletion expression vectors. Transient expression analysis in onion epidermal cells by particle bombardment showed that both −609 to +3 bp and −377 to +3 bp fragments of BcMF5 promoter were capable of driving β-glucuronidase gene expression. Furthermore, by Agrobacterium-mediated genetic transformation method, Arabidopsis transgenic Kan^R plants were obtained. GUS assay analysis revealed that the promoter of BcMF5 induced gene expression at the early stage of anther development and drove high levels of GUS expression in anther walls, upper regions of petals, pollen, and pollen tubes in the middle and late stage of anther development, but did not drive any expression in sepals and pistils.     

Abscisic acid-regulated Glb1 transient expression in cultured maize P3377 cells

In a study of the 5′-flanking sequence of the Zea mays L. (maize) Glb1 gene in vitro, serial promoter deletions were generated and linked with the β-glucuronidase (GUS) reporter gene. The promoter deletion-GUS fusions were introduced into the maize P3377 cell line by particle bombardment. GUS assays indicated that treatment of the maize cultured cells with abscisic acid (ABA) was required for Glb1-driven GUS transient expression, and that the –272-bp sequence of the Glb1 promoter was sufficient for ABA-regulated expression of GUS. The longest undeleted sequence used, –1391 GUS, showed relatively low expression which could be indicative of an upstream silencer element in the Glb1 promoter between –1391 and –805. Further studies show that the Glb1-driven GUS activity of bombarded maize P3377 cells increases with increasing ABA concentration (up to 100–300 μm). Site-directed mutagenesis of a putative ABA response element, Em1a, abolished GUS expression in P3377 cells. This observation indicated that the Em1a sequence in the Glb1 5′ regulatory region is responsible for the positive ABA regulation of gene expression. Received: 9 May 1997 / Revision received: 9 November 1997 / Accepted: 8 December 1997     

Reporter gene introduction and transient expression in protoplasts of Porphyra yezoensis

The transient expression of foreign genes in the protoplasts of Porphyrayezoensis was examined using three recombinant vectors, pYez-Rub-GUS, pYez-Rub-GFP and pYez-Rub-LUC, which were constructed with the promoter sequence of the ribulose-bisphosphate-carboxylase / oxygenase (Rubisco) gene as a promoter and the bacterial β-glucuronidase (GUS), mutant of green fluorescent protein (S65T-GFP) and firefly luciferase (LUC) genes, respectively, as reporter genes. When the pYez-Rub-GUS was introduced into protoplasts by electroporation, cells stained dark blue by indigotin were observed after the histochemical GUS assay. GUS activity was also detected by quantitative enzyme assays with a chemiluminescent substrate. When the pYez-Rub-GFP was electroporated into protoplasts, the expression of GFP could be detected in vivo observations with fluorescence microscopy. However, the rates of gene expression cells to the total number of cells were different between the GUS and GFP genes. LUC activity was also detected by assay with a chemiluminescent substrate after the introduction of pYez-Rub-LUC into protoplasts, although the activity levels were considerably lower. Relatively high expression rates of introduced GUS genes were observed 3 to 5 days after electroporation. These results show that the promoter sequence of the chloroplast Rubisco gene functions as a promoter of foreign gene expression and that transient expression occurred in protoplasts of P. yezoensis after the introduction of foreign genes. This revised version was published online in August 2006 with corrections to the Cover Date.     

Functional characterization of the pollen-specific <Emphasis Type="Italic">SBgLR</Emphasis> promoter from potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.)

SBgLR (Solanum tuberosum genomic lysine-rich) gene was isolated from a potato genomic library using SB401 (S. berthaultii 401) cDNA as probe. RT-PCR analysis of SBgLR gene expression profile and microscopic analysis of green fluorescent protein (GFP) expression in tobacco plants transformed with SBgLR promoter-GFP reporters indicate that SBgLR is a pollen-specific gene. A series of 5′deletions of SBgLR promoter were fused to the β-glucuronidase (GUS) gene and stably introduced into tobacco plants. Histochemical and quantitative assays of GUS expression in transgenic plants allowed us to localize an enhancer of SBgLR promoter to the region −345 to −269 relative to the translation start site. This 76 bp (−345 to −269) fragment enhanced GUS expression in leaves, stems and roots when fused to −90/+6 CaMV 35S minimal promoter. Deletion analysis showed that a cis-element, which can repress gene expression in root hairs, was located in the region −345 to −311. Further study indicated that the −269 to −9 region was sufficient to confer pollen-specific expression of GFP when fused to CaMV 35S enhancer. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Authors Zhihong Lang and Peng Zhou contributed equally to this work.     

Promoterless gus gene shows leaky β-glucuronidase activity during transformation of tomato with bspA gene for drought tolerance

Transformation of tomato (Lycopersicon esculentum Mill.) was carried out using disarmed Agrobacterium tumefaciens strain EHA 105 harboring a binary vector pBIG-HYG-bspA. The plasmid contains the bspA (boiling stable protein of aspen) gene under the control of a CaMV35S promoter and nopaline synthase (NOS) terminator, hygromycin phosphotransferase gene (hpt) driven by nopaline synthase promoter and polyadenylation signal of Agrobacterium gene7 as terminator and a promoterless gus gene. Very strong β-glucuronidase (GUS) expression was observed in transformed tomato plants but never in non-transformed (control). Since GUS expression was observed only in transformed plants, the possibility of the presence of endogenous GUS enzymes was ruled out. Possibility of false GUS positives was also ruled out because the GUS positive explants reacted positively to polymerase chain reaction (PCR) and PCR-Southern tests carried out for the presence of bspA gene, which indicated the integration of T-DNA in tomato genome. The promoterless GUS expression was hypothesized either due to leaky NOS termination signal of bspA gene or due to different cryptic promoters of plant origin. It was concluded that GUS expression was observed in the putative transgenics either due to the read through mechanism by the strong CaMV35S promoter or due to several cryptic promoters driving the gus gene in different transgenic lines.     

Evaluation of transformation in peach Prunus persica explants using green fluorescent protein (GFP) and beta-glucuronidase (GUS) reporter genes

To determine the optimum conditions for Agrobacterium-mediated gene transfer, peach explants including cotyledons, embryonic axes and hypocotyl slices from non-germinated seeds and epicotyl internode slices from germinating seeds were exposed to Agrobacterium-mediated transformation treatments. The GUS (uidA) marker gene was tested using two different A. tumefaciens strains, three plasmids and four promoters [CaMV35s, (Aocs)3AmasPmas (“super-promoter”), mas-CaMV35s, and CAB]. GFP was tested with six A.␣tumefaciens strains, one plasmid (pLC101) and the doubleCaMV35s (dCaMV35s) promoter. The CaMV35s promoter produced more GUS expression than the CAB promoter. A. tumefaciens strains EHA105 and LBA4404 harboring the same plasmid (pBIN19) differed in their effects on GUS expression suggesting an interaction between A. tumefaciens strain and plasmid. A combination of A. tumefaciens EHA105, plasmid pBIN19 and the CaMV35s promoter produced the highest rates of transformation in peach epicotyl internodes (56.8%), cotyledons (52.7%), leaves (20%), and embryonic axes (46.7%) as evaluated by the percentage of explants expressing GUS 14 days after co-cultivation. GFP expression under the control of the dCaMV35s promoter was highest for internode explants but only reached levels of 18–19%. When GFP-containing plasmid pCL101 was combined with each of five A. tumefaciens strains the highest levels of transformation were 20–21% (internode and cotyledons, respectively). When nine peach genotypes were co-cultivated with A. tumefaciens strain EHA105 and GFP-containing plasmid pCL101 the highest levels of transformation were 26–28% (cotyledons and internodes, respectively). While GFP represents a potentially useful transformation marker that allows the non-destructive evaluation of transformation, rates of GFP transformation under the conditions of this study were low. It will be necessary to optimize expression of this marker gene in peach.     

Functional characterization of a cotton late embryogenesis-abundant D113 gene promoter in transgenic tobacco

Previous studies have shown that mRNA and protein encoded by late embryogenesis-abundant (LEA) gene D113 from Gossypium hirsutum L. accumulate at high levels in mature seeds and also in response to abscisic acid (ABA) in young embryo. In this study, we studied the expression of four promoter 5′ deletion constructs (−1383, −974, −578 and −158) of the LEA D113 gene fused to beta-glucuronidase (GUS). GUS activity analysis revealed that the −578 promoter fragment was necessary to direct seed-specific GUS expression in transgenic tobacco plants (Nicotiana tabacum L.). To further investigate the expression pattern of LEA D113 promoter under environmental stresses, 2-week-old transgenic tobacco seedlings were exposed to ABA, dehydration, high salinity and cold treatments. GUS activity in the seedlings was quantified fluorimetrically, and expression was also observed by histochemical staining. An apparent increase in GUS activity was found in plants harboring constructs −1383, −974 and −578 after 24 h of ABA or high-salinity treatments, as well as after 10 days of dehydration. By contrast, only a slight increase was observed in all the three lines after cold treatment. Virtually no change in expression was found in construct −158 in response to dehydration, salinity and cold, but there was a moderate response to ABA, suggesting that the region between −574 and −158 was necessary for dehydration- and salinity-dependent expression, whereas ABA-responsive cis-acting elements might be located in the −158 region of the promoter.     

Isolation and Characterization of a Curcin Promoter from <Emphasis Type="Italic">Jatropha curcas</Emphasis> L. and Its Regulation of Gene Expression in Transgenic Tobacco Plants

Ribosome-inactivating proteins (RIPs) represent those proteins that universally depurinate conserved α-sarcin loops of large rRNAs. In this study, a 0.6-kb fragment of a 5′ flanking region preceding a curcin gene, encoding a type I RIP curcin, of Jatropha curcas L. endosperm was cloned, and its regulation of expression of the β-glucuronidase (GUS) reporter gene was investigated in transgenic tobacco. Analysis of GUS activities showed that the 0.6-kb flanking fragment of the curcin gene was sufficient to drive the GUS reporter gene expression in tobacco seed. The activity of this flanking fragment was analyzed at different stages of seed development. Histochemical localization of GUS activity indicated that the promoter was specifically active in the endosperm tissue of the dicotyledonous tobacco embryo. Moreover, this activity was first initiated at the heart-shaped embryonic stage during seed development.