Organ-specific modulation of gene expression in transgenic plants using antisene RNA

We have shown leaf-specific inhibition GUS gene expression in transgenic Nicotiana plants using an antisense RNA with a 41-base homology spanning the translation start codon of the gene. GUS was expressed from the nominally constitutive 35S promoter and the antisense RNA was expressed from the light-regulated ca/b promoter of Arabidopsis thaliana. A range of GUS inhibition from 0 to 100% was obtained by screening a small population of transgenic plants and the specific levels of inhibition observed were stably inherited in two generations. An antiGUS gene dosage effect was observed in plants which were homozygous for antiGUS. RNA detection results suggest that duplex formation with the 41 base pair antiGUS RNA destabilized the GUS mRNA and that an excess of antisense. RNA was not required. Our results demonstrate the potential of antisense RNA as a strategy for obtaining plant mutants, especially down mutations in essential genes where only a short 5 sequence of the mRNA is required. They also suggest that the position effect on gene expression could be used in conjunction with an antisense RNA strategy to provide a versatile approach for crop improvement.     

Commercial production of β-glucuronidase (GUS): a model system for the production of proteins in plants

We have generated transgenic maize seed containing -glucuronidase(GUS) for commercial production. While many other investigators have demonstrated the expression of GUS as a scoreable marker, this is one of the first cases where a detailed characterization of the transgenic plants and the protein were performed which are necessary to use this as a commercial source of GUS. The recombinant -glucuronidase was expressed at levels up to 0.7% of water-soluble protein from populations of dry seed, representing one of the highest levels of heterologous proteins reported for maize. Southern blot analysis revealed that one copy of the gene was present in the transformant with the highest level of expression. In seeds, the majority of recombinant protein was present in the embryo, and subcellular localization indicated that the protein was dispersed throughout the cytoplasm. The purified recombinant -glucuronidase (GUS) was compared to native -glucuronidase using SDS-PAGE and western blot analysis. The molecular mass of both the recombinant and native enzymes was 68 000 Da. N-terminal amino acid sequence of the recombinant protein was similar to the sequence predicted from the cloned Escherichia coli gene except that the initial methionine was cleaved from the recombinant GUS. The recombinant and native GUS proteins had isoelectric points (pI) from 4.8 to 5.0. The purified proteins were stable for 30 min at 25, 37, and 50 ° C. Kinetic analysis of the recombinant and native GUS enzymes using 4-methylumbelliferyl glucuronide (MUG) as the substrate was performed. Scatchard analysis of these data demonstrated that the recombinant enzyme had a K_m of 0.20 mM and a V_max of 0.29 mM MUG per hour, and the native enzyme had a K_m and V_max of 0.21 mM and 0.22 mM/h respectively. Using D-saccharic acid 1,4-lactone, which is an inhibitor of -glucuronidase, the K_i of the native and recombinant enzymes was determined to be 0.13 mM. Thus, these data demonstrate that recombinant GUS is functionally equivalent to native GUS. We have demonstrated the expression of high levels of GUS can be maintained in stable germlines and have used an efficient recovery system where the final protein product, GUS, has been successfully purified. We describe one of the first model systems for the commercial production of a foreign protein which relies on plants as the bioreactor.     

The rice metallothionein gene promoter does not direct foreign gene expression in seed endosperm

We generated transgenic tobacco and rice plants harboring a chimeric gene consisting of the 5-upstream sequence of the rice metallothionein gene (ricMT) fused to the -glucuronidase (GUS) gene. The activity and tissue-specific expression of the ricMT promoter were demonstrated in these transgenic plants. In the transgenic rice plants, despite substantial levels of GUS activity in the shoot and root, almost no GUS signal was detected in the endosperm. Thus, the ricMT promoter could be useful in avoiding accumulation of undesired proteins in the seed endosperm.     

A pistil-specific thaumatin/PR5-like protein gene of Japanese pear (Pyrus serotina): sequence and promoter activity of the 5′ region in transgenic tobacco

The genomic clone encoding the pistil-specific thaumatin/PR5-like protein (PsTL1) was isolated from Japanese pear (Pyrus serotina). Sequence analysis showed that the genomic clone contained the 5-flanking sequence of 2.4 kb, the 3-flanking sequence of 648 bp and the coding region interrupted by a intron of 351 bp. A sequence motif conserved in some pistil self-incompatibility gene promoters of solanaceous and brassicaceous species was located in the 5-flanking region of the PsTL1 gene. The 2.4 kb 5-flanking region was fused to the GUS coding sequence and transferred to tobacco. Transgenic tobacco showed GUS activity in pistil and, at low level, in anther, but not in other floral organs and leaf. Histochemical analysis localized GUS activity to stigma, transmitting tissue, anther and pollen of transgenic tobacco.     

A/T-rich sequences act as quantitative enhancers of gene expression in transgenic tobacco and potato plants

The role of an A/T-rich positive regulatory region (P268, -444 to -177 from the translation start site) of the pea plastocyanin gene (PetE) promoter has been investigated in transgenic plants containing chimeric promoters fused to the -glucuronidase (GUS) reporter gene. This region enhanced GUS expression in leaves of transgenic tobacco plants when fused in either orientation to a minimal pea PetE promoter (-176 to +4) and in roots when fused in either orientation upstream or downstream of a minimal cauliflower mosaic virus 35S promoter (-90 to +5). The region was also able to enhance GUS expression in microtubers of transgenic potato plants when placed in either orientation upstream of a minimal class I patatin promoter (-332 to +14). Dissection of P268 revealed that cis elements responsible for enhancing GUS expression from the minimal PetE promoter were distributed throughout P268. Multiple copies of a 31 bp A/T-rich sequence from within P268 and of a 26 bp random A/T sequence were able to enhance GUS expression from the minimal PetE promoter, indicating that A/T-rich sequences are able to act as quantitative, non-tissue-specific enhancer elements in higher plants. Abbreviations: CaMV, cauliflower mosaic virus; GUS, -glucuronidase; HMG, high-mobility group; MAR, matrix-associated region; MU, methylumbelliferone; SAR, scaffold-associated region.     

Expression of a Synthetic Porcine α-Lactalbumin Gene in the Kernels of Transgenic Maize

The main nutritional limitation of maize used for feed is the content of protein that is digestible, bioavailable and contains an amino acid balance that matches the requirements of animals. In contrast, milk protein has good digestibility, bioavailability and amino acid balance. As an initial effort to create maize optimized as a source of swine nutrition, a codon-adjusted version of a gene encoding the milk protein porcine -lactalbumin was synthesized. Maize expression vectors containing this gene under the control of the Ubi-1 promoter and nos 3 terminator were constructed. These vectors were used to transform maize callus lines that were regenerated into fertile plants. The -lactalbumin transgenes were transmitted through meiosis to the sexual progeny of the regenerated plants. Porcine -lactalbumin was detected in callus and kernels from transgenic maize lines that were transformed by two constructs containing the 27-kDa maize gamma-zein signal sequence at the 5 end of the synthetic porcine -lactalbumin coding sequence. One of these constructs contained an ER retention signal and the other did not. Expression was not observed in kernels or callus from transgenic maize lines that were transformed by a construct that does not contain an exogenous protein-targeting signal. This suggests that the signal peptide might play an important role in porcine -lactalbumin accumulation in transgenic maize kernels.     

Semi-constitutive expression of an Arabidopsis thaliana α-tubulin gene

In Arabidopsis tissues, the pool of tubulin protein is provided by the expression of multiple -tubulin and -tubulin genes. Previous evidence suggested that the TUA2 -tubulin gene was expressed in all organs of mature plants. We now report a more detailed analysis of TUA2 expression during plant development. Chimeric genes containing TUA2 5-flanking DNA fused to the -glucuronidase (GUS) coding region were used to create transgenic Arabidopsis plants. Second-generation progeny of regenerated plants were analyzed by histochemical assay to localize GUS expression. GUS activity was seen throughout plant development and in nearly all tissues. The blue product of GUS activity accumulated to the highest levels in tissues with actively dividing and elongating cells. GUS activity was not detected in a few plant tissues, suggesting that, though widely expressed, the TUA2 promoter is not constitutively active.     

Evidence for non-proteinaceous inhibitor(s) of β-glucuronidase in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis>L.) leaf and root tissues

The GUS gene of E. coli, encoding -glucuronidase, has been widely used as a reporter gene in plant transformation. However, -glucuronidase activity in transgenic wheat leaf or root tissue is rarely observed or reported. To address this question, we investigated three wheat lines transformed with the GUS reporter gene. We found all three lines expressed GUS mRNA as well as -glucuronidase protein in their leaf and root tissues as detected by RNA gel blot, ELISA, and immunoblot analyses. However, -glucuronidase enzyme activity was only detected in pollen grains from the transgenic plants. Fluorometric and histochemical assays performed in the presence of wheat tissue extracts indicated that wheat leaf and root tissues contain inhibitor(s) of -glucuronidase activity, but pollen grains contain much lower concentrations. Further characterizations indicated that the inhibitor(s) is of low molecular weight (<10 kDa) and is non-proteinaceous.     

Quantitative analysis of the transgene variability among primary tobacco transformants

The aim of this study was to develop a model for the quantitative estimation of the genetic and environmental variance components in the first generation (T₁) of transgenic plants, in which the transgene effect is considered as a source of genetic variation. The experimental population consisted of T₁ independent transgenic plants (ITPs). Forty-two ITPs of tobacco were generated, containing a chimaeric gene comprising the cauliflower mosaic virus (CaMV) 35S promoter and the reporter gene -glucuronidase (GUS). From each ITP, four cuttings were grown in a randomized block design, and GUS activity in the leaves was determined. The mean GUS activity of the ITPs ranged from 0.55 to 167.9 pmol MU per mg protein per min. Testing of the statistical assumptions of the model revealed a significant scale effect, resulting from correlation between the intra-ITP variance and the average GUS activity of the ITPs. Log GUS activity (LGA) and power of –0.15 of GUS activity (TGA) scale transformations eliminated the scale effect. For GUS activity, the inter-ITP variance was only 28% of the total variance in the experiment, whereas for LGA and TGA it was 72% and 76%, respectively. The opposite was true for the intra-ITP variance, which was reduced from 58% to 18% and 16%, respectively. The experimental design allowed partitioning of the phenotypic variance in T₁ transgenic plants into genetic and environmental components. According to the original scale GUS activity, most of the phenotypic variance was due to environmental variance; the common tendency to interpret this variance as an outcome of position effect and other genetic changes due to transformation leads to incorrect findings. In the present example, after scale transformation the genetic component was 80% of the phenotypic variance.     

Expressing multiple genes in a single open reading frame with the 2A region of foot-and-mouth disease virus as a linker

The Food-and-mouth disease virus (FMDV) 2A proteinis only 16–20 amino acid long. It is responsible for thecleavage of the FMDV polyprotein at its own carboxyl-terminus. Weused the cleavage property of the 2A protein to processartificial polyproteins produced in transgenic plants. In our system, single or multiplecopies of the reporter CAT and GUS genes were fused into a single open readingframe (ORF) with a copy of the FMDV 2A protein gene placed between the reportergenes. Expression of various constructs in transgenic tobacco resulted inconsistent detection of freed CAT and/or GUS proteins, suggesting that FMDV 2Aprotein functioned properly in plant cells. Cleavage efficiencyranged from 80% to 100% depending on the constructs. The variability incleavage efficiency suggested that the contexts flanking a 2Aprotein might modulate its activity. We further expressed constructs wheremultiple copies of the 2A and reporter genes were fused into one ORF. Thepresence of freed GUS protein together with partially processed polyproteinintermediates in the transgenic plants indicated that multiple copies of the 2Aprotein in a single ORF function independently. Our data demonstrate that usingthe FMDV 2A protease as a linker, multiple genes could be easily expressed in asingle ORF.     

Xylem-specific expression of a GRP1.8 promoter::4CL gene construct in transgenic tobacco

Lignin is a complex aromatic polymer of vascular plants that provides mechanical strength to the stem and protects cellulose fibres from chemical and biological degradation. 4-Coumarate:CoA ligases (EC 6.2.1.12) are key enzymes for the biosynthetic pathway of monolignols which is an important complex aromatic polymer for lignin biosynthesis and tree growth. Recently, 4-coumarate:CoA ligase has been used as exogenous gene in transgenic plants to genetically modify the lignin biosynthesis pathway. Since most lignin is produced in the vascular cells, a tissue-specific-expressed promoter in the vascular cell would be important and useful to change and modify the content of lignin. Here we report the existence of a promoter of GRP1.8 (the glycine-rich protein 1.8) in Sopho japonica L. (GenBank accession number AF250149) and studies on its function in transgenic tobacco. The promoter activity was analyzed in transgenic tobacco plants by histochemical staining of GUS gene expression driven by a 613-bp sjGRP1.8p promoter sequence. In sjGRP1.8p-GUS transgenic plants, intense GUS staining was detected in the xylem of the stem. To further investigate the regulation of the tissue-specific expression of the 4CL1 gene, we analyzed the activity of the 4CL1 gene which is sense orientated with the sjGRP1.8p promoter in transgenic tobacco. The Pto4CL1 gene was expressed in the stem of transgenic tobacco. The activity of the 4CL1 enzyme was increased 1–2-fold in the stem but not increased in the leaves of transgenic tobacco. In comparison with the control plants, the content of lignin was increased 25% in the stem but there was no increase in the leaves of transgenic tobacco.     

Intron position affects expression from thetpi promoter in rice

A series of promoter-GUS fusion constructs containing a portion of the rice triosephosphate isomerase (tpi) promoter, the firsttpi intron, and the gene encoding bacterial -glucuronidase (GUS) were made. These constructs were electroporated into rice protoplasts and transient expression was monitored. Inclusion of the first intron from the ricetpi gene enhanced expression of the GUS gene from thetpi promoter when it was placed 5 of the GUS gene. When thetpi intron was placed in the 3-untranslated region no enhancement of GUS gene expression was observed, indicating the importance of position in intron-mediated enhancement of gene expression.     

Genetic characterization of a new splice variant of the β2 subunit of the voltage-dependent calcium channel

This study reports a novel splice variant form of the voltage-dependent calcium channel ₂ subunit (_2g). This variant is composed of the conserved amino-terminal sequences of the _2a subunit, but lacks the -subunit interaction domain (BID), which is thought essential for interactions with the ₁ subunit. Gene structure analysis revealed that this gene was composed of 13 translated exons spread over 107 kb of the genome. The gene structure of the ₂ subunit was similar in exon-intron organization to the murine ₃ and human ₄ subunits. Electrophysiological evaluation revealed that _2a and _2g affected channel properties in different ways. The _2a subunit increased the peak amplitude, but failed to increase channel inactivation, while _2g had no significant effects on either the peak current amplitude or channel inactivation. Other subunits, such as ₃ and ₄, significantly increased the peak current and accelerated current inactivation.     

Analysis of kafirin promoter activity in transgenic tobacco seeds

Sequences corresponding to 855 bp of 5 promoter region and the transit peptide from GK.1, a genomic clone encoding a 22 kDa -kafirin seed protein from sorghum, were translationally fused to a cloned -glucuronidase (GUS) coding sequence from uidA and transferred to tobacco via Agrobacterium tumefaciens-mediated transformation. No GUS expression was detectable at any stage of growth in stems or leaves of these plants. However, GUS expression was detected in both embryo and endosperm tissues of resulting tobacco seeds 10–15 days after flowering. Dissected tissues indicate endosperm expression was localized within the bulk endosperm and not within the parenchyma cell layer underlying the integument. These studies also demonstrate that within dissected tobacco embryos, expression from the kafirin promoter was restricted to the mesocotyl region.     

Introduction of a chimeric gene encoding an oryzacystatin-β-glucuronidase fusion protein into rice protoplasts and regeneration of transformed plants

In order to construct transgenic rice plant with an introduced oryzacystatin (OC)--glucuronidase (GUS) fusion gene, we first introduced it into rice protoplasts by electroporation, together with a marker gene conferring hygromycinresistance (pUC-HPH). In a transient assay using the transfected protoplasts, both OC and GUS activities were detected. The GUS activity was higher when the OC-GUS fusion protein was expressed than when only a single GUS protein was expressed. Next, to isolate stable transformants, hygromycin-resistant calli were selected. Forty one out of 116 hygromycin-resistant calli expressed a 2.2 kb mRNA transcribed from the chimeric gene and their extracts exhibited the activities of both OC and GUS. Finally, the transgenic calli were regenerated into rice plants whose tissues (leaves, roots and seeds) exhibited GUS activity probably derived from the fusion protein.     

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 -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.     

Expression of the rice Osgrp1 promoter-Gus reporter gene is specifically associated with cell elongation/expansion and differentiation

To study the expression and regulation of a rice glycine-rich cell wall protein gene, Osgrpl, transgenic rice plants were regenerated that contain the Osgrpl promoter or its 5 deletions fused with the bacterial -glucuronidase (GUS) reporter gene. We report here a detailed histochemical analysis of the Osgrpl-Gus expression patterns in transgenic rice plants. In roots of transgenic rice plants, GUS expression was specifically located in cell elongation and differentiation regions, and no GUS expression was detectable in the apical meristem and the mature region. In shoots, GUS activity was expressed only in young leaves or in the growing basal parts of developing leaves, and little GUS activity was expressed in mature leaves or mature parts of developing leaves. In shoot apices, GUS activity was detected only in those leaf cells which were starting to expand and differentiate, and GUS expression was not detected in the apical meristem and the young meristematic leaf primordia. GUS activity was highly expressed in the young stem tissue, particularly in the developing vascular bundles and epidermis. Thus, the expression of the Osgrpl gene is closely associated with cell elongation/expansion during the post-mitotic cell differentiation process. The Osgrpl-Gus gene was also expressed in response to wounding and down-regulated by water-stress conditions in the elongation region of roots. Promoter deletion analysis indicates that both positive and negative mechanisms are involved in regulating the specific expression patterns. We propose a simple model for the developmental regulation of the Osgrpl gene expression.     

Transgenic plantlets of ‘Chancellor’ grapevine (Vitis sp.) from biolistic transformation of embryogenic cell suspensions

Transgenic plantlets of Chancellor grapevine (Vitis L. complex interspecific hybrid) were produced via biolistic transformation. Embryogenic cell suspensions were bombarded with 1 m tungsten particles coated with pBI426 which encodes a fusion peptide between -glucuronidase (GUS) and neomycin phosphotransferase II (NPTII). The fusion peptide is under the control of a double 35S Cauliflower Mosaic Virus promoter and a leader sequence from Alfalfa Mosaic Virus. The cells were placed on kanamycin-containing media (10, 25 or 50 mg/l) 2 d after bombardment. Activated charcoal reduced cell browning. Embryos were first observed on selective media 14–29 weeks after bombardment. More than 1600 clusters of embryos were germinated and/or assayed for GUS. Of 621 embryos assayed for GUS expression, 182 (29.3%) were positive. PCR confirmed the presence of the NPTII gene in all 5 GUS-positive and 2 GUS-negative (bombarded) embryos tested. In germination experiments, 15% of the embryo clusters produced at least one plant with normal shoot growth. Of 164 normal plants assayed for GUS expression, 37 (22.6%) were positive. The NPTII gene was amplified by PCR in 1 (of 1) GUS-positive and 4 (of 5) GUS-negative bombarded plants, but not in non-bombarded control plants. Southern blotting confirmed integration of the NPTII gene in all 3 of the GUS and PCR-NPTII positive plants tested. Biolistics is an efficient method for transformation of Chancellor and should be applicable to other important grape cultivars.Abbreviations AC activated charcoal - GUS -glucuronidase - 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzylaminopurine - NAA -naphthalene acetic acid - TDZ thidiazuron - NPTII neomycin phosphotransferase II - Km kanamycin - MS Murashige and Skoog (1962) medium - WPM Woody Plant Medium of Lloyd and McCown (1980)     

Expression of a chimeric stilbene synthase gene in transgenic wheat lines

A chimeric stilbene synthase (sts)gene was transferred into wheat. Stilbene synthases play a role in the defence against fungal diseases in some plant species (e.g. groundnut or grapevine) by producing stilbenetype phytoalexins like resveratrol. Resveratrol is also claimed to have positive effects to human health. Embryogenic scutellar calli derived from immature embryos of the two commercial German spring wheat cultivars Combi and Hanno were used as target tissue for cotransformation by microprojectile delivery. The selectable marker/reporter gene constructs contained the bargene either driven by the ubiquitinpromoter from maize (pAHC 25, also containing the uidAgene driven by the ubiquitinpromoter), or by the actinpromoter (pDM 302) from rice. The cotransferred plasmid pStil 2 consisted of a grapevine stscoding region driven by the ubiquitin promoter. Eight transgenic Combi and one Hanno T_Oplant were obtained and, except one Combi T_Oplant, found to be cotransformants due to the integration of both the stsgene and the selectable marker or reporter genes. Expression of the stsgene was proven by RTPCR, and, for the first time, by detection of the stilbene synthase product resveratrol by HPLC and mass spectrometry. The stsgene was expressed in four of the seven transgenic Combi T_{_o}plants. Two of the respective T₁progenies segregated in a Mendelian manner were still expressing the gene. Investigations into methylation of the stsgene showed that in three nonexpressing progenies inactivation was paralleled by methylation.