Transgenic Plant Extracts Research Articles

Production of functional recombinant roseltide rT1 antimicrobial peptide in tobacco plants

Plant-derived peptides represent a promising group of natural compounds with broad industrial and pharmaceutical applications. Low-efficiency production level is the major obstacle to the commercial production of such bioactive peptides. Today, recombinant techniques have been developed for fast and cost-effective production of high-quality peptides for various applications in the chemical and food industries. The roseltide rT1 is a plant peptide with different antimicrobial properties and therapeutic applications in the prevention and treatment of inflammatory lung diseases by inhibiting human neutrophil elastases. Here, we report the expression of functional recombinant roseltide rT1 peptide in tobacco plants. Transgenic plants were generated by the Agrobacterium-mediated transformation method followed by molecular analysis of transgenic plants to demonstrate successful integration and expression of recombinant rT1 peptide. Protein extracts of transgenic plants expressing a single-copy rT1 gene showed efficient antimicrobial properties as verified by growth inhibition of different bacterial strains. Our results illustrate that plant-derived recombinant rT1 peptide is a promising alternative for rapid and cost-effective production of this important antimicrobial peptide for application in therapeutic and food industries.

Agrobacterium-Mediated Genetic Transformation of Withania coagulans (Dunal) with rol A Genes and Its Antioxidant Potential.

In ancient times, Withania coagulans Dunal was used as a therapeutic plant for the treatment of several diseases. This report aims to examine the effect of Agrobacterium tumefactions-mediated transformation of W. coagulans with the rolA gene to enhance secondary metabolite production, antioxidant activity, and anticancer activity of transformed tissues. Before transgenic plant production, the authors designed an efficient methodology for in vitro transformation. In this study, leaf explants were cultured on Murashage and Skoog (MS) media containing different amounts of naphthalene acetic acid (NAA) and benzyl adenine (BA). The best performance for inducing embryogenic callus was in MS medium containing 4 μM NAA and 6.0 μM BA, while the best results for shooting (100%) were obtained at 8 μM benzyl adenine. On the other hand, direct shooting was attained by subculturing leaves on MS medium supplemented with 8 μM benzyl adenine. Prolonged shoots showed excellent in vitro rooting results (80%) with 12 μM indole-3-butyric acid (IBA). The samples were precultivated for 3 days and were followed by 48 h infection with A. tumefaciens strain GV3101 having pCV002. Then, a vector expressed the rol A gene of strain Agrobacterium rhizogenes. Furthermore, three independent transgenic shoot lines and one callus line (T2) were produced and exhibited stable integration of transgene rol A genes, as revealed by PCR analysis. Transgenic strains showed a significant increase in antioxidant potential as compared to untransformed plants. Additionally, LC-MS analysis showed that the transformed strains have a higher withanolide content as compared to untransformed ones. Moreover, the reduced proliferation of prostate cancer cells was observed after treatment with extracts of transgenic plants. Furthermore, these transformed plants exhibited superior antioxidant capability and higher withanolide content than untransformed ones. In conclusion, the reported data can be used to select withanolide-rich germplasm from transformed cell cultures.

Accumulation of colicin M protein and its biological activity in transgenic lettuce and mizuna plants.

Food-borne illnesses caused by pathogenic Escherichia coli strains, especially enterohaemorrhagic E. coli (EHEC), are a serious public health problem, as debilitating disease and even death from such food poisonings have been repeatedly reported. Colicin M (ColM), a non-antibiotic antimicrobial protein produced by some strains of E. coli, has shown promising activity in controlling multiple enteropathogenic strains of E. coli and related pathogens. As contaminated green leafy vegetables are a frequent source of pathogenic E. coli infections, we genetically modified (GM) two edible crops, lettuce (Lactuca sativa L.) and mizuna (Brassica rapa subsp. nipposinica var. laciniata), to stably express the ColM gene and assessed the antibacterial activity of tissue extracts from these plants against selected E. coli strains in vitro. Transgenic plants of these species were developed using Agrobacterium-mediated transformation with a vector containing the ColM-coding gene (cma) under the control of the 35S promoter. Western blot analysis of recombinant ColM protein was performed in selected transgenic plants to confirm cma gene expression and quantify ColM accumulation. Extracts of transgenic plants expressing ColM showed significant activity against two major strains of EHEC (O157:H7 and O104:H4) as well as E. coli strains resistant to beta-lactam- and carbapenem-class antibiotics. Importantly, the antibacterial activity persisted in several subsequent generations of transgenic lettuce and mizuna plants that stably expressed the ColM gene. In addition, our results also show that the antibacterial activity of dried (up to 40°C) biomass of transgenic plants remained stable without a decrease for at least three months.

Insights into acylation mechanisms: co-expression of serine carboxypeptidase-like acyltransferases and their non-catalytic companion paralogs.

SUMMARYSerine carboxypeptidase‐like acyltransferases (SCPL‐ATs) play a vital role in the diversification of plant metabolites. Galloylated flavan‐3‐ols highly accumulate in tea (Camellia sinensis), grape (Vitis vinifera), and persimmon (Diospyros kaki). To date, the biosynthetic mechanism of these compounds remains unknown. Herein, we report that two SCPL‐AT paralogs are involved in galloylation of flavan‐3‐ols: CsSCPL4, which contains the conserved catalytic triad S‐D‐H, and CsSCPL5, which has the alternative triad T‐D‐Y. Integrated data from transgenic plants, recombinant enzymes, and gene mutations showed that CsSCPL4 is a catalytic acyltransferase, while CsSCPL5 is a non‐catalytic companion paralog (NCCP). Co‐expression of CsSCPL4 and CsSCPL5 is likely responsible for the galloylation. Furthermore, pull‐down and co‐immunoprecipitation assays showed that CsSCPL4 and CsSCPL5 interact, increasing protein stability and promoting post‐translational processing. Moreover, phylogenetic analyses revealed that their homologs co‐exist in galloylated flavan‐3‐ol‐ or hydrolyzable tannin‐rich plant species. Enzymatic assays further revealed the necessity of co‐expression of those homologs for acyltransferase activity. Evolution analysis revealed that the mutations of the CsSCPL5 catalytic residues may have taken place about 10 million years ago. These findings show that the co‐expression of SCPL‐ATs and their NCCPs contributes to the acylation of flavan‐3‐ols in the plant kingdom.

Developing a platform for production of the oxylipin KODA in plants.

KODA (9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid) is a plant oxylipin involved in recovery from stress. As an agrichemical, KODA helps maintain crop production under various environmental stresses. In plants, KODA is synthesized from α-linolenic acids via 9-lipoxygenase (9-LOX) and allene oxide synthase (AOS), although the amount is usually low, except in the free-floating aquatic plant Lemna paucicostata. To improve KODA biosynthetic yield in other plants such as Nicotiana benthamiana and Arabidopsis thaliana, we developed a system to overproduce KODA in vivo via ectopic expression of L. paucicostata 9-LOX and AOS. The transient expression in N. benthamiana showed that the expression of these two genes is sufficient to produce KODA in leaves. However, stable expression of 9-LOX and AOS (with consequent KODA production) in Arabidopsis plants succeeded only when the two proteins were targeted to plastids or the endoplasmic reticulum/lipid droplets. Although only small amounts of KODA could be detected in crude leaf extracts of transgenic Nicotiana or Arabidopsis plants, subsequent incubation of the extracts increased KODA abundance over time. Therefore, KODA production in transgenic plants stably expressing 9-LOX and AOS requires specific sub-cellular localization of these two enzymes and incubation of crude leaf extracts, which liberates α-linolenic acid via breakdown of endogenous lipids.

Overexpression of LmgshF from Listeria monocytogenes in Indica Rice Confers Salt Stress Tolerance

Glutathione (γ-glutamylcysteinylglycine; GSH), is a multi-functional tri-peptide antioxidant, a key agent in defense against abiotic and biotic stress. A novel bi-functional enzyme LmgshF identified from Listeria monocytogenes, had developed transgenic rice (Oryza sativa L.) plants under the control of cauliflower mosaic virus (CaMV) 35S promoter in indica rice cultivar NAUR-1. LmgshF gene was stably inherited and expressed in T2 plants, as indicated by the results of PCR, ELISA, RT-PCR and qRT-PCR (quantitative real time PCR) analyses. An average expression of LmgshF was 3.03 μg/mg of total soluble protein in leaf extracts of transgenic plants. QRT-PCR analysis of LmgshF showed 5.83- and 6.07-fold higher in gshF-5-7 and gshF-5-8 plants respectively, at 200 mM NaCl treatment in transgenic rice. Under salt stress conditions, transgenic plants possessed higher level of glutathione, proline, photosynthetic pigments and antioxidant enzyme activities, than that of its wild type (WT) plants. It showed better germination, displays long roots, shoots and heavier fresh weights and survival percentage compared to its wild type under salt stress conditions. HPLC (High Performance Liquid Chromatography) analysis showed that the glutathione content was 14-fold higher in transgenic rice at 200 mM NaCl treatment. Transgenic rice plants showed enhanced levels of glutathione to counter the adverse effects of salinity and resulted in improved salt stress tolerance in rice.

IN GEL DETECTION OF A HIS-TAGGED TRANSGENE FOLLOWING THE SEPARATION OF CRUDE PLANT PROTEIN EXTRACTS WITH SDS PAGE

In the present study, we visualised sundew His6-tagged chitinase protein in crude protein extracts of transgenic tobacco plants following protein separation with sodium dodecyl sulfate polyacrylamide gel electrophoresis by detecting chitinase activity as well as the His-tag. A short sequence encoding six histidines was fused downstream of the DNA sequence encoding the last amino acid of the mature protein. Following binary vector construction and plant transformation, a set of 10 transgenic plants was analysed for transgene expression. Except for one, all transgenic plants exhibited the presence of sundew chitinase protein of ~52 kDa, which was different from the calculated molecular weight of ~32 kDa. Clear identification of DrChitHis protein was performed with a Ni2+:NTA complex conjugated to a fluorescent dye and visualized using light laser-based scanner. A subsequent endochitinolytic activity assay using a N-fluorescein-labelled chitin substrate confirmed that the two transgenic lines with the strongest expression of DrChitHis protein had endochitinolytic activity 6.4 and 6.7 times higher than non-transgenic control.

Overexpression of the gibberellin 20-oxidase gene from Torenia fournieri resulted in modified trichome formation and terpenoid metabolities of Artemisia annua L.

Gibberellins (GAs) are diterpenoid hormones, control various physiological developments in plants. The role of gibberellins on morphology and secondary metabolite production was examined in Artemisia annua, a medicinal plant that has been acknowledged as a source of artemisinin, an antimalarial compound. Subsequently, the GA20ox gene from Torenia fournieri (TfGA20ox2) was transferred to A. annua by Agrobacterium-mediated transformation. Compared with wild type plants, all nine transgenic plants showed significantly higher plant heights and artemisinin contents. The highest artemisinin content and yield in TfGA20ox2-overexpressing plants was around two-fold higher than wild type. Moreover, transgenic plants had higher numbers of branches (52.4%) and greater branch lengths (60–203%), but smaller leaf size (77.6%). Interestingly, relative to wild type the number and size of glandular trichomes in transgenic leaves was about 30 and 35% higher, respectively. From GC–MS analysis, the proportion of diterpenes in transgenic plant extracts was 1.5-fold lower than those noticed in wild type, while the proportion of sesquiterpenes was increased about 1.6 times when compared to wild type. However, the content proportion of monoterpenes showed a slightly increase, whereas the level of triterpenes showed no variation. In addition, two monoterpenes (eucalyptol and borneol), four sesquiterpenes (α-caryophyllene, β-guaiene, δ-cadinene and β-cubebene) and one triterpenes (isomultiflorenone) were detected only in transgenic extract, whereas d-α-tocopherol, a diterpenoid compound was found only in wild type but not transgenic plant. These results suggested that gibberellins play a significant role in regards to morphology, trichome formation and terpenoid metabolite production in A. annua.

Improved antioxidant activity in transgenic Perilla frutescens plants via overexpression of the γ-tocopherol methyltransferase (γ-tmt) gene.

The main goal of this study was to generate transgenic Perilla frutescens with enhanced antioxidant properties by overexpressing the γ-tocopherol methyltransferase (γ-tmt) gene. In this study, the antioxidant activity of methanolic crude extracts of transgenic and non-transgenic control plants was investigated using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging method. Free radical scavenging activity was evaluated using α-tocopherol and butylated hydroxyl toluene as standard antioxidants. In general, the ethyl acetate fraction of transgenic P. frutescens showed stronger DPPH radical scavenging activity than the ethyl acetate fraction from non-transgenic control plants (IC50 2.00 ± 0.10 and 5.53 ± 0.40 μg ∙ ml(-1), respectively). High-performance liquid chromatography analysis of phenolic acids in leaf extracts confirmed increased levels of 16 individual phenolic compounds in two transgenic lines (pf47-5 and pf47-8) compared with control plants. Changes in the phenolic compound profile and α-tocopherol content were correlated with the antioxidant properties of transgenic plants, indicating that the introduction of transgene γ-tmt influenced the metabolism of phenolic compounds and subsequently produced biochemical changes in the transformants. There were no significant differences in photosynthetic rate in the transgenic plants as compared to the non-transgenic control plants, suggesting that the alteration of phenolic compounds and tocopherol composition had little impact on photosynthesis.

Engineering temporal accumulation of a low recalcitrance polysaccharide leads to increased C6 sugar content in plant cell walls.

Reduced cell wall recalcitrance and increased C6 monosaccharide content are desirable traits for future biofuel crops, as long as these biomass modifications do not significantly alter normal growth and development. Mixed-linkage glucan (MLG), a cell wall polysaccharide only present in grasses and related species among flowering plants, is comprised of glucose monomers linked by both β-1,3 and β-1,4 bonds. Previous data have shown that constitutive production of MLG in barley (Hordeum vulgare) severely compromises growth and development. Here, we used spatio-temporal strategies to engineer Arabidopsis thaliana plants to accumulate significant amounts of MLG in the cell wall by expressing the rice CslF6 MLG synthase using secondary cell wall and senescence-associated promoters. Results using secondary wall promoters were suboptimal. When the rice MLG synthase was expressed under the control of a senescence-associated promoter, we obtained up to four times more glucose in the matrix cell wall fraction and up to a 42% increase in saccharification compared to control lines. Importantly, these plants grew and developed normally. The induction of MLG deposition at senescence correlated with an increase of gluconic acid in cell wall extracts of transgenic plants in contrast to the other approaches presented in this study. MLG produced in Arabidopsis has an altered structure compared to the grass glucan, which likely affects its solubility, while its molecular size is unaffected. The induction of cell wall polysaccharide biosynthesis in senescing tissues offers a novel engineering alternative to enhance cell wall properties of lignocellulosic biofuel crops.

Antiviral activity of extracts of transgenic chicory and lettuce plants with the human interferon α2b gene

Biological activity of protein extracts from transgenic plants of chicory Cichorium intybus L. and lettuce Lactuca sativa L. with human interferon alpha2b gene was investigated against vesicular stomatitis virus. It was shown that the extracts from the hairy roots of chicory and lettuce transformed by A. rhizogenes possess the antiviral activity 1620...5400 IU/g weight, and the extracts from leaves of the plants transformed by A. tumefaciens--till 9375 IU/g weight. Dependence of plant extract biological activity on the transformation vector was shown.

Transgenic lettuce (Lactuca sativa L.) expressing H1N1 influenza surface antigen (neuraminidase)

Influenza, which occurs globally, is an acute respiratory disease that causes a higher rate of death and hospitalization annually. Although current vaccines are effective against influenza virus infection, alternative strategies must be developed to overcome the complex production processes and global demand for an influenza vaccine. Therefore, we have engineered and produced the full-length neuraminidase (NA) protein from the H1N1 strain of influenza in plants. The gene encoding the H1N1 surface protein antigen was constructed into a plant expression vector, under the control of CaMV 35S promoter, and was introduced into lettuce via Agrobacterium-mediated transformation. The presence of the NA gene in putative transgenic lines was detected by PCR amplification method. Reverse-transcriptase (RT)-PCR was conducted to determine specific NA transcript expression. The results of immunoblot analysis, with anti-NA antibodies, showed H1N1 NA protein expression in transgenic plant extracts. Based on the ELISA results, the amount of NA protein produced in transgenic lettuce leaves constituted approximately 0.018–0.045% of the total soluble plant protein. Mice immunized subcutaneously with the transgenic lettuce extracts showed an immune response against the NA antigen. These results demonstrate that plant-produced NA protein is antigenic, and generate immunogenicity against the highly pathogenic influenza virus.

Improving antioxidant activity in transgenic Codonopsis lanceolata plants via overexpression of the γ-tocopherol methyltransferase (γ-tmt) gene

Codonopsis lanceolata Trautv (Companulaceae) is a folk medicine in Korea. To shift the content of tocopherol and enhance its antioxidant properties, we overexpressed the γ-tocopherol methyltransferase (γ-tmt) gene in C. lanceolata. The antioxidant activity of methanolic crude extracts of the transgenic plants was compared to that of control plants using the 1,1-diphenyl-2-picrylhydrazyl radical scavenging method, with α-tocopherol and butylated hydroxy toluene as standard antioxidants. The antioxidant activity of the leaf and root extracts of transgenic plants was higher (IC 50 12–17.33 and 408–524 μg/ml, respectively) than that of control plant leaf and root extracts (18 and 529 μg/ml, respectively). High-performance liquid chromatography analysis of phenolic compounds confirmed an increase in the levels of 12 major phenolic acids and flavonoids in the leaf and root extracts of transgenic plants compared to control plants. We also found that the rate of photosynthesis was 48% higher in transgenic plants than in control plants. Based on these results, we suggest that increases in the α-tocopherol level in transgenic C. lanceolata plants may result in increases in the photosynthetic performance and antioxidant metabolism of these plants.

Cholera toxin B subunit-domain III of dengue virus envelope glycoprotein E fusion protein production in transgenic plants

Envelope glycoprotein E of the dengue virus, which plays a crucial role in its entry into host cells, has an immunogenic domain III (EIII, amino acids 297–394), which is capable of inducing neutralizing antibodies. However, mice immunized with EIII protein without adjuvant elicited low immune responses. To improve low immune responses, a DNA fragment, consisting of cholera toxin B subunit and EIII gene (CTB–EIII), was constructed and introduced into tobacco plant cells ( Nicotiana tabacum L. cv. MD609) by Agrobacterium tumefaciens-mediated transformation methods. The integration and transcription of CTB–EIII fusion gene were confirmed in transgenic plants by genomic DNA PCR amplification and Northern blot analysis, respectively. The results of immunoblot analysis with anti-CTB and anti-dengue virus antibodies showed the expression of the CTB–EIII fusion protein in transgenic plant extracts. Based on the G M1-ELISA results, the CTB–EIII protein expressed in plants showed the biological activity for intestinal epithelial cell membrane glycolipid receptor, G M1-ganglioside, and its expression level was up to about 0.019% of total soluble protein in transgenic plant leaf tissues. The feasibility of using a plant-produced CTB–EIII fusion protein to generate immunogenicity against domain III will be tested in future animal experiments.

Production of a heat-labile enterotoxin B subunit-porcine epidemic diarrhea virus-neutralizing epitope fusion protein in transgenic lettuce (Lactuca sativa).

Plant-based vaccines have been produced in transgenic plants including tobacco, potatoes, corn, and rice. However, these plants are not suitable for administration without cooking. To overcome this obstacle, a fusion gene encoding the synthetic enterotoxigenic Escherichia coli heat-labile enterotoxin B subunit genetically fused with a synthetic neutralizing epitope of porcine epidemic diarrhea virus (sLTB-sCOE) was introduced into lettuce cells (Lactuca sativa) by Agrobacterium-mediated transformation methods. The integration and expression of the sLTB-sCOE fusion gene was confirmed in transgenic lettuce by genomic DNA PCR amplification and Northern blot analysis, respectively. Synthesis and assembly of the LTB-COE fusion protein into oligomeric structures with pentamer size were observed in transgenic plant extracts by Western blot analysis with anti-LTB or anti-COE antibodies. The binding of plantproduced LTB-COE to intestinal epithelial cell membrane glycolipid receptors was confirmed by GM1-ganglioside enzyme-linked immunosorbent assay (GM1-ELISA). Based on the ELISA results, LTB-COE fusion protein made up about 0.026∼0.048% of the total soluble protein in the transgenic lettuce leaf tissues. The synthesis and assembly of LTB-COE monomers into biologically active oligomers in transgenic lettuce leaf tissues demonstrates the feasibility of using uncooked edible plant-based vaccines for mucosal immunization.

Transgene Stacking and Coordinated Expression of Plant Defensins Confer Fungal Resistance in Rice

Abstract Transgenic rice with cleavable chimeric polyprotein and single-protein gene constructs placed under the control of single maize ubiquitin promoter were generated by Agrobacterium tumefaciens-mediated transformation. The polyprotein precursor consists of a leader peptide and two different antimicrobial proteins (AMPs), Dm-AMP1 and Rs-AFP2, from the seeds of Dahlia merckii and Raphanus sativus, respectively. These genes were linked by a 16 amino acid Ib-AMP linker peptide region isolated from the seeds of Impatiens balsamina. Average expression of Dm-AMP1 and Rs-AFP2 was 8.35 ± 0.56 and 8.1 ± 0.6 2 g/mg of total soluble proteins in the leaf extracts of transgenic plants. Plants transformed with polyprotein construct showed significantly improved disease resistance against Magnaporthe oryzae and Rhizoctonia solani by 90% and 79%, respectively, as compared to untransformed plants. The plants transformed with polyprotein construct were more resistant to M. oryzae compared to plants transformed with control single-gene constructs of Dm-AMP1 and Rs-AFP2. The chimeric polyprotein was cleaved and present in equimolar concentration in transgenic rice and the individual AMPs were secreted into the extracellular space. The results of in vitro, in planta, and microscopic analyses suggest that chimeric polyprotein expression has the potential to provide broad-spectrum disease resistance in rice.

Expression and Assembly of Cholera Toxin B Subunit (CTB) in Transgenic Carrot (Daucus carota L.)

We expressed the cholera toxin B subunit (CTB) fused to an endoplasmic reticulum retention signal (SEKDEL) in carrot roots using an Agrobacterium-mediated transformation method. Fourteen independent transgenic lines were regenerated via somatic embryogenesis after 6 months of culture. The sCTB gene was detected in the genomic DNA of transgenic carrot by PCR amplification. Expressions and assembly of sCTB protein into oligomeric structures of pentameric size were observed in transgenic plant extracts by Western blot analysis. The sCTB produced by transgenic carrot roots demonstrated strong affinity for GM1-ganglioside, suggesting that the sCTB conserved the antigenic sites for binding and proper folding of the pentameric sCTB structure. The expression level of sCTB comprised approximately 0.48% of total soluble protein (TSP) in root of transgenic carrot.

Mucosal immunity induced by orally administered transgenic plants

Oral immunization is an efficient means to induce protection at the portal entrance for many pathogens. Therefore, the design of efficient edible vaccines through transgenic plants represents a challenging alternative to the traditional injectable ones. We have previously reported the construction of transgenic potato plants expressing the genes coding for the immunogenic proteins of Newcastle Disease Virus (NDV) and their immunogenicity in mice. All mice receiving transgenic plant extracts in incomplete Freund's adjuvant produced specific antibodies. Animals fed with transgenic leaves also showed a specific response against NDV. The aim of the present study was to continue the evaluation of the mucosal immune response. Adult Balb/c mice were fed with potato leaves for a month and on day 36 mucosal samples were collected. ELISAs performed on intestinal washes showed that transformed plants elicited the synthesis of NDV-specific IgG and IgA antibodies. In addition, anti-NDV IgA antibodies were detected in supernatants of cultured small intestine fragments of mice fed with the recombinant immunogens, suggesting the presence of NDV-specific IgA secreting plasma cells in the intestinal tissue. Moreover, we detected specific anti-NDV antibodies in intestinal fluids after oral immunization with F and HN transgenic plants. Also, indirect immunofluorescence on intestinal tissue was performed. The present results suggest that these immunogens, F and HN glycoproteins of NDV, when orally administered, would enhance the number of IgA + B cells, and the cytotoxic cellular immune response via CD8 + T cells, found in the gut lamina propria that is in accordance with our first findings.

The Isoenzyme 7 of Tobacco NAD(H)-Dependent Glutamate Dehydrogenase Exhibits High Deaminating and Low Aminating Activities in Vivo

Following the discovery of glutamine synthetase/glutamate (Glu) synthase, the physiological roles of Glu dehydrogenase (GDH) in nitrogen metabolism in plants remain obscure and is the subject of considerable controversy. Recently, transgenics were used to overexpress the gene encoding for the beta-subunit polypeptide of GDH, resulting in the GDH-isoenzyme 1 deaminating in vivo Glu. In this work, we present transgenic tobacco (Nicotiana tabacum) plants overexpressing the plant gdh gene encoding for the alpha-subunit polypeptide of GDH. The levels of transcript correlated well with the levels of total GDH protein, the alpha-subunit polypeptide, and the abundance of GDH-anionic isoenzymes. Assays of transgenic plant extracts revealed high in vitro aminating and low deaminating activities. However, gas chromatography/mass spectrometry analysis of the metabolic fate of (15)NH(4) or [(15)N]Glu revealed that GDH-isoenzyme 7 mostly deaminates Glu and also exhibits low ammonium assimilating activity. These and previous results firmly establish the direction of the reactions catalyzed by the anionic and cationic isoenzymes of GDH in vivo under normal growth conditions and reveal a paradox between the in vitro and in vivo enzyme activities.

Synthesis and assembly of Escherichia coli heat-labile enterotoxin B subunit in transgenic lettuce (Lactuca sativa)

Escherichia coli heat-labile enterotoxin B subunit (LTB) strongly induces immune responses and can be used as an adjuvant for co-administered antigens. Synthetic LTB (sLTB) based on optimal codon usage by plants was introduced into lettuce cells ( Lactuca sativa) by Agrobacterium tumefaciens-mediated transformation methods. The sLTB gene was detected in the genomic DNA of transgenic lettuce leaf cells by PCR DNA amplification. Synthesis and assembly of the sLTB protein into oligomeric structures of pentameric size was observed in transgenic plant extracts using Western blot analysis. The binding of sLTB pentamers to intestinal epithelial cell membrane glycolipid receptors was confirmed by G M1-ganglioside enzyme-linked immunosorbent assay (G M1-ELISA). Based on the results of ELISA, sLTB protein comprised approximately 1.0–2.0% of total soluble protein in transgenic lettuce leaf tissues. The synthesis and assembly of sLTB monomers into biologically active oligomers in transgenic lettuce leaf tissues demonstrates the feasibility of the use of edible plant-based vaccines consumed in the form of raw plant materials to induce mucosal immunity.