VIRUS RESISTANCE OF TRANSGENIC TOBACCO PLANTS EXPRESSING TOBACCO MOSAIC VIRUS COAT PROTEIN GENE

An intermediate expressing vector carrying the tobacco mosaic virus (TMV, Chinese common strain) coat protein (CP) gene was constructed by recombinant DNA techniques. The TMV-CP gene was transferred into the tobacco genome via Ti plasmid and a large number of regenerated plants, including both systemic and local lesion hosts for TMV, were obtained. Southern blot analysis revealed that 1-5 copies of the CP gene were integrated into the tobacco genome. RNA and protein analysis demonstrated that the TMV-CP gene was correctly expressed in the transgenic plants. The abundance of TMV-CP mRNA in total leaf RNA accounted for 0.005-0.01%, while the amount of coat proteins reached 0.05-0.2% of the total leaf soluble proteins. Virus challenge experiments showed that the symptom development of virus infection was markedly delayed and the replication as well as the spread of the virus was significantly inhibited in the transgenic plants expressing the TMV-CP gene. Three of these plants were completely protected afte     

Enhanced expression of B-subunit of Escherichia coli heat-labile enterotoxin in tobacco by optimization of coding sequence

Escherichia coli heat-labile toxin (LT) is a potent mucosal immunogen and immunoadjuvant for coadministered antigens. We synthesized a gene encoding the B-subunit of LT(LTB) adapted to the coding sequence of tobacco plants and fused to the endoplasmic reticulum retention signal SEKDEL to enhance its level of expression in plants. The synthetic LTB gene was cloned into a plant expression vector adjacent to the CaMV 35S promoter and was introduced into tobacco by Agrobacterium-mediated transformation. The amount of LTB protein detected in transgenic tobacco leaves was 2.2% of the total soluble plant protein, which is approx 200-fold higher than in previous reports of native LTB gene expression in transgenic plants. Enzyme-linked immunosorbent assay indicated that plant-synthesized LTB protein bound specifically to G_M1-ganglioside, suggesting that the LTB subunits formed active pentamers.     

Transgenic Tobacco Expressing <Emphasis Type="Italic">Zephyranthes grandiflora</Emphasis> Agglutinin Confers Enhanced Resistance to Aphids

Plant lectins have been reported as transgenic resistance factors against a variety of insect pests. Herein, homologous analysis demonstrated that Zephyranthes grandiflora agglutinin (ZGA) exhibited high similarity with other monocot mannose-binding lectins (MBLs). Phylogenetic analysis revealed that it had taxonomical relationships with insecticidal MBLs. Subsequently, a plasmid expression vector pBI121 containing zga gene (pBIZGA) was constructed using the zga sequence, under the control of CaMV35S promoter and nos terminator. pBIZGA was then integrated into the genome of Nicotiana tabacum L. Polymerase chain reaction and Southern blot analysis demonstrated that this zga gene was integrated into the plant genome. Western blotting and agglutinating activity analysis also showed that transgenic tobacco plants expressed different levels of ZGA. Carbohydrate inhibition analysis indicated that recombinant ZGA and the native shared the same carbohydrate-binding specificity. Moreover, genetic analysis confirmed Mendelian segregation (3:1) of the transgenic in T1 progenies. In planta bioassays on T0 plants and their progenies indicated that expressed ZGA had an effect on reducing the survivability and fecundity of tobacco aphids (Myzus nicotianae). These findings demonstrate that the novel zga gene of ZGA can be expressed in crop plants susceptible to various sap-sucking insects.     

Overexpression of the Formaldehyde Dehydrogenase Gene from Brevibacillus brevis to Enhance Formaldehyde Tolerance and Detoxification of Tobacco

The faldh gene coding for a putative Brevibacillus brevis formaldehyde dehydrogenase (FALDH) was isolated and then transformed into tobacco. A total of three lines of transgenic plants were generated, with each showing 2- to 3-fold higher specific formaldehyde dehydrogenase activities than wild-type tobacco, a result that demonstrates the functional activity of the enzyme in formaldehyde (HCHO) oxidation. Overexpression of faldh in tobacco confers a high tolerance to exogenous HCHO and an increased ability to take up HCHO. A ¹³C-nuclear magnetic resonance technique revealed that the transgenic plants were able to oxidize more aqueous HCHO to formate than the wild-type (WT) plants. When treated with gaseous HCHO, the transgenic tobacco exhibited an enhanced ability to transform more HCHO into formate, citrate acid, and malate but less glycine than the WT plants. These results indicate that the increased capacity of the transgenic tobacco to take up, tolerate, and metabolize higher concentrations of HCHO was due to the overexpression of B. brevis FALDH, revealing the essential function of this enzyme in HCHO detoxification. Our results provide a potential genetic engineering strategy for improving the phytoremediation of HCHO pollution.     

EXPRESSION OF A ZEIN GENE (Z4) INTRODUCED INTO DICOTYLEDONOUS Solanum nigrum

A maize genomic clone containing a zein gene (Z4) is inserted into the T-DNA of the Ti plasmid pTiT37. Agrobacterium tumefaciens strain harboring this modified Ti plasmid is used to infect stem sections of young plants or explants of dicotyledonous Solanum nigrum. Axenic transformed calli active in nopaline synthesis are obtained and transgenic plants are differentiated from them DNA Southern hybridization and RNA dot-hybridization analyses show that the zein gene is really transferred and integrated into the nuclear genome of transformed Solanum nigrum and that the zein gene can be transcribed into mRNA in the transformed calli and shoots. But the presence of the zein protein cannot be detected in either the transformed calli or the transgenic shoots. The results of thte experiments demonstrate that the promoter of a gene from monocotyledonous plants can function normally in transgenic dicots. The possibility of developmentally-regulated expression of the zein gene in transformed dicots is discussed in     

Chip-Based CE for Avidin Determination in Transgenic Tobacco and Its Comparison with Square-Wave Voltammetry and Standard Gel Electrophoresis

Avidin transgenic plants are a potential tool for providing resistance against various species of insect pests due to the sequestration of vitamin H (biotin) in the plant from the insect pests. In this project we compared three techniques for avidin determination in transgenic tobacco plants, a novel chip-based capillary electrophoretic method (Experion), classical polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE) and a square wave voltammetric method using a carbon paste electrode. We determined that the automated chip-based capillary electrophoretic method is rapid, sensitive and the results obtained are well repeatable. The avidin content measured in transgenic tobacco leaves using chip-based capillary electrophoresis varied from 15 to 854 ng per mg of fresh mass depending on the individual plant.     

Silica Nanoparticles-mediated Stable Genetic Transformation in Nicotiana tabacum

Nanoparticles as gene carriers become popular in the mammalian cells, whereas the application of them in plant cells is still very limited. Herein lies a report on silica nanoparticles(SiNPs) modified with positively charged poly-L-lysine(PLL) successfully delivering plasmid-encoded β-glucuronidase(GUS) gene into tobacco with the help of gene gun. The stable transgenic tobacco plants mediated by SiNPs can be obtained. Furthermore, we revealed the quantity of gene and types of receptor materials could affect the expression efficiency. In comparison to conventional gold particles-mediated transformation, the silica nanoparticles-mediated stable genetic transformation enhances transformation efficiency, potentially overcoming transgenic silencing. Our results demonstrate the great potential of SiNPs as gene carrier in plant genetic transformation and prove a novel approach for plant genetic decoration.     

Purification of a human immunoglobulin G1 monoclonal antibody from transgenic tobacco using membrane chromatographic processes

Efficient purification of protein biopharmaceuticals from transgenic plants is a major challenge, primarily due to low target protein expression levels, and high impurity content in the feed streams. These challenges may be addressed by using membrane chromatography. This paper discusses the use of cation-exchange and Protein A affinity-based membrane chromatographic techniques, singly and in combination for the purification of an anti-Pseudomonas aerugenosa O6ad human IgG1 monoclonal antibody from transgenic tobacco. Protein A membrane chromatography on its own was unable to provide a pure product, mainly due to extensive non-specific binding of impurities. Moreover, the Protein A membrane showed severe fouling tendency and generated high back-pressure. With cation-exchange membrane chromatography, minimal membrane fouling and high permeability were observed but high purity could not be achieved using one-step. Therefore, by using a combination of the cation-exchange and Protein A membrane chromatography, in that order, both high purity and recovery were achieved with high permeability. The antibody purification method was first systematically optimized using a simulated feed solution. Anti-P. aeruginosa human IgG1 type monoclonal antibody was then purified from transgenic tobacco juice using this optimized method.     

Cloning of Promoter of Chinese Bean GRP 1.8 Gene and Characterization of Its Function in Transgenic Tobacco Plants

IntroductionVascular cells that are determined to becometracheary elements undergo a very distinct celldifferentiation.In contrast to many other types ofplant cells that can dedifferentiate and evenultimately form a new plant,cells determined tobecome tracheary elements are programmed to die.Glycine- rich proteins belong to a class ofstructural cell wall proteinsthatcontain about60 %glycine on the molar basis. Glycine- rich proteinsare localized in cell walls of bean[1] .Glycine- richprotein1…     

Expression of Trichoderma reesei exo-cellobiohydrolase I in transgenic tobacco leaves and calli

Expression of Trichoderma reesei exo-cellobiohydrolase I (CBHI) gene in transgenic tobacco was under the control of CaMV 35S promoter. In transgenic leaf tissues, CBHI activity up to 66.1 μmol/h/g total protein was observed. In transgenic calli, the highest CBHI activity was 83.6 μmol h/g total protein. Protein immunoblot analysis confirms the presence of CBHI enzyme in both transgenic calli and leaf tissues. CBHI expression levels accounted for about 0.11% and 0.082% of total protein in transgenic leaf tissues and calli, respectively, Furthermore, expression of CBHI gene did not affect normal growth and development of transgenic plants.     

Molecular cloning and transgenic expression of a synthetic human erythropoietin gene in tobacco

Erythropoietin (EPO) is a hormone belonging to a group of hematopoietic growth factors that control the proliferation and differentiation of bone marrow cells. It induces the production of erythrocytes, thereby increasing the amount of circulating hemoglobin and oxygen. Previous attempts to transgenically express human EPO in plants failed to succeed because the plants exhibited abnormal morphology and infertility. In the present work, we describe the generation of fertile transgenic tobacco plants able to express a synthetic version of human EPO. A 582-bp fragment of the human EPO gene was synthesized using a PCR-based method and ligated into pCR-Blunt. After sequencing, the human EPO fragment was transferred to pWUbi.tm1 and the expression cassette was then transferred to the binary vector pWBVec4a. After Agrobacterium-mediated transformation of Nicotiana tabacum SR1 plants, integration of the transgene into T₀ and T₁ plant genomes was confirmed by PCR. The human EPO gene was found to be expressed in tobacco leaves at the mRNA and protein levels. Self-crossing allowed us to obtain T₁ plants exhibiting Mendelian segregation of the transgene. None of the plants presented any kind of malformation or deformity.     

Heterologous Acidothermus cellulolyticus 1,4-β-endoglucanase E1 produced within the corn biomass converts corn stover into glucose

Commercial conversion of lignocellulosic biomass to fermentable sugars requires inexpensive bulk production of biologically active cellulase enzymes, which might be achieved through direct production of these enzymes within the biomass crops. Transgenic corn plants containing the catalytic domain of Acidothermus cellulolyticus E1 endo-1,4-beta glucanase and the bar bialaphos resistance coding sequences were generated after Biolistic (BioRad Hercules, CA) bombardment of immature embryo-derived cells. E1 sequences were regulated under the control of the cauliflower mosaic virus 35S promoter and tobacco mosaic virus translational enhancer, and E1 protein was targeted to the apoplast using the signal peptide of tobacco pathogenesis-related protein to achieve accumulation of this enzyme. The integration, expression, and segregation of E1 and bar transgenes were demonstrated, respectively, through Southern and Western blotting, and progeny analyses. Accumulation of up to 1.13% of transgenic plant total soluble proteins was detected as biologically active E1 by enzymatic activity assay. The corn-produced heterologous E1 could successfully convert ammonia fiber explosion-pretreated corn stover polysaccharides into glucose as a fermentable sugar for ethanol production, confirming that the E1 enzyme is produced in its active form.     

Cloning and Sequencing of Trichosanthin Gene and Its Expression in Escherichia coli and Tobacco Plant

A Trichosanthin gene was cloned from Trichosanthes kirilowii genomic DNA by polymerase chain reaction (PCR). Nucleotide sequence data indicated that we obtained the coding region of the mature Trichosanthin peptide as well as its signal peptide at the N-terminus. Comparisons of our sequence with the previously reported nucleotide sequences of this gene showed 99.25% homology, yet there were notable differences between the previously reported amino acid sequence and our deduced result. This gene was subcloned into a highlevel expression plasmid (pJLA502) of E. coli under the control of a P_RP_L promoter, and we observed the gene product after temperature induction. The gene was further cloned into plant intermediate vector pE3 under the control of a CaMV 35S promoter, and transferred into a tobacco genome using the agrobacterium-mediated gene transfer system. Western blotting analysis of the protein extracted from Escherichia coli and transgenic tobacco plants proved that the Trichosanthin gene has been     

Effects of ammonia fiber explosion treatment on activity of endoglucanase from Acidothermus cellulolyticus in transgenic plant

A critical parameter affecting the economic feasibility of lignocellulosic bioconversion is the production of inexpensive and highly active cellulase enzymes in bulk quantity. A promising approach to reduce enzyme costs is to genetically transform plants with the genes of these enzymes, thereby producing the desired cellulases in the plants themselves. Extraction and recovery of active proteins or release of active cellulase from the plants during bioconversion could have a significant positive impact on overall lignocellulose conversion economics. The effects of ammonia fiber explosion (AFEX) pretreatment variables (treatment temperature, moisture content, and ammonia loading) on the activity of plant-produced heterologous cellulase enzyme were individually investigated via heat treatmett or ammonia treatment. Finally, we studied the effects of all these variables in concert through the AFEX process. The plant materials included transgenic tobacco plants expressing E1 (endoglucanase from Acidothermus cellulolyticus). The E1 activity was measured in untreated and AFEX-treated tobacco leaves to investigate the effects of the treatment on the activity of this enzyme. The maximum observed activity retention in AFEX-treated transgenic tobacco samples compared with untreated samples was approx 35% (at 60°C, 0.5∶1 ammonia loading, and 40% moisture). Based on these findings, it is our opinion that AFEX pretreatment is not a suitable option for releasing cellulase enzyme from transgenic plants.     

Transgenic Expression and Functional Characterization of Human Platelet Derived Growth Factor BB (hPDGF-BB) in Tobacco (Nicotiana tabacum L.)

Recombinant human platelet derived growth factor BB (rhPDGF-BB) is clinically approved for treating diabetic neuropathic ulcers. Plant-based expression systems offer less expensive ways of producing recombinant drugs, which do not require purification for clinical use. From this perspective, rhPDGF-BB is an ideal candidate for expression in plants as it can be applied topically. Here, we report a proof of concept study, in which rhPDGF-BB was expressed in tobacco plants, and its biological activity was tested in vitro. The mature human platelet derived growth factor BB (hPDGF-BB) gene was codon-optimized for tobacco and fused with ER targeting and retention signals, 5′ and 3′ UTRs of arc5-1 gene along with CaMV 35S promoter, and then, transferred by Agrobacterium-mediated transformation. Gene and protein expression of hPDGF-BB were confirmed by PCR and immunoblot studies. Bioactivity of hPDGF-BB expressed protein was determined by in vitro assays such as proliferation and migration in NIH3T3 cells. Our data reveals that total soluble proteins containing hPDGF-BB from transgenic plants showed a 4.5-fold increase in fibroblast proliferation compared to non-transgenic plants. Furthermore, plant-made rhPDGF-BB induced chemotaxis of treated cells and promoted wound healing in vitro. These results clearly demonstrate that functionally active rhPDGF-BB protein can be produced in plants and might have therapeutic benefits.     

Functional analysis of polyphenol oxidases by antisense/sense technology

Polyphenol oxidases (PPOs) catalyze the oxidation of phenolics to quinones, the secondary reactions of which lead to oxidative browning and postharvest losses of many fruits and vegetables. PPOs are ubiquitous in angiosperms, are inducible by both biotic and abiotic stresses, and have been implicated in several physiological processes including plant defense against pathogens and insects, the Mehler reaction, photoreduction of molecular oxygen by PSI, regulation of plastidic oxygen levels, aurone biosynthesis and the phenylpropanoid pathway. Here we review experiments in which the roles of PPO in disease and insect resistance as well as in the Mehler reaction were investigated using transgenic tomato (Lycopersicon esculentum) plants with modified PPO expression levels (suppressed PPO and overexpressing PPO). These transgenic plants showed normal growth, development and reproduction under laboratory, growth chamber and greenhouse conditions. Antisense PPO expression dramatically increased susceptibility while PPO overexpression increased resistance of tomato plants to Pseudomonas syringae. Similarly, PPO-overexpressing transgenic plants showed an increase in resistance to various insects, including common cutworm (Spodoptera litura (F.)), cotton bollworm (Helicoverpa armigera (Hübner)) and beet army worm (Spodoptera exigua (Hübner)), whereas larvae feeding on plants with suppressed PPO activity had higher larval growth rates and consumed more foliage. Similar increases in weight gain, foliage consumption, and survival were also observed with Colorado potato beetles (Leptinotarsa decemlineata (Say)) feeding on antisense PPO transgenic tomatoes. The putative defensive mechanisms conferred by PPO and its interaction with other defense proteins are discussed. In addition, transgenic plants with suppressed PPO exhibited more favorable water relations and decreased photoinhibition compared to nontransformed controls and transgenic plants overexpressing PPO, suggesting that PPO may have a role in the development of plant water stress and potential for photoinhibition and photooxidative damage that may be unrelated to any effects on the Mehler reaction. These results substantiate the defensive role of PPO and suggest that manipulation of PPO activity in specific tissues has the potential to provide broad-spectrum resistance simultaneously to both disease and insect pests, however, effects of PPO on postharvest quality as well as water stress physiology should also be considered. In addition to the functional analysis of tomato PPO, the application of antisense/sense technology to decipher the functions of PPO in other plant species as well as for commercial uses are discussed.     

IN ACTIVATION OF A STRAIN OF TOBACCO NECROSIS VIRUS AND OF THE RNA ISOLATED FROM IT, BY ULTRAVIOLET RADIATION OF DIFFERENT WAVE-LENGTHS

Abstract— A train of tobacco necrosis virus (TNV) and infective nucleic acid isolated from it (TNV-RNA) are equally susceptible to inactivation by U.V. radiation at all wave-lengths tested (230-290 m_μ) and can be photoreactivated to the same extent by exposing inoculated host plants to daylight. The shape of the action spectrum for inactivation by U.V. of TNV and of TNV-RNA follows that of the absorption spectrum of TNV-RNA. Thus, unlike the RNA of tobacco mosaic virus, the RNA of TNV behaves in all these respects in the same way irrespective of whether it is inside or outside the virus particle. To inactivate TNV or TNV-RNA to 50 per cent of their original infectivities, each mg of RNA must absorb about 0.27 joules of radiation energy of any wave-length between 230 and 290 mp, which corresponds to a quantum yield of about 0.65 ×10^-3 at 260 mμ.     

Sensitization and allergic response and intervention therapy in animal models

A review is presented of 3 murine models and a swine neonatal model used to investigate immunotherapeutic options. In Model 1, mutation of linear IgE-binding epitopes of Ara h 1 for the preparation of a hypoallergenic Ara h 1 is discussed with respect to expression in transgenic tobacco plants and correct folding following expression in the pET16b construct. In Model 2, the mutations of Ara h 1 were assessed for use as an immunotherapeutic agent. Although some protective benefit was observed with the modified Ara h 1 protein, animals desensitized with heat-killed E. coil preparations showed increased protection to challenge. In Model 3, soybean homologs to peanut proteins were investigated to determine if soybean immunotherapy can potentially provide benefit to peanut-allergic subjects. Although some protection was provided, additional experimentation with respect to optimal doses for sensitization and challenge will need to be investigated. In Model 4, the neonatal swine model was used to profile different foods (low to moderate to high sensitizing) similar to food allergies in humans. Evidence suggests such feasiblity; however, threshold levels for sensitization and allergic responses will need additional study. In summary, murine and swine animal models are being used to address immunotherapeutic avenues and investigation into the mechanisms of food-allergic sensitization.