Academic literature on the topic 'Transgenic tobacco'
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Journal articles on the topic "Transgenic tobacco"
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Kanthang, Supha, and Kanokporn Sompornpailin. "Increasing Plant Flavonoid Biomaterials in Response to UV-A Light." Advanced Materials Research 802 (September 2013): 74–78. http://dx.doi.org/10.4028/www.scientific.net/amr.802.74.
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Flavonoid biomaterials have a protecting function from various stresses. We examined the flavonoid biosynthesis in plant treated under visible light (VL) and additional UV-A light. The transgenic tobacco containing PRODUCTION OF ANTHOCYANIN PIGMENT 1 (PAP1) cDNA, involved in flavonoid biosynthesis from Arabidopsis thaliana, were used for studying the flavonoid biosynthesis under both light conditions comparing to non transgenic tobacco. The flavonoid biomaterials were extracted with acidic methanol and water solvent from treated plant leaves. The absorbance of each biomaterial in the extract was measured under specific wavelength using a spectrophotometer. Additional UV-A radiated to non transgenic and transgenic tobacco affect the increasing of p-coumaric acid, naringenin, apigenin and kaempherol biomaterials from themselves grown under VL (approximately 120-130%). However, PAP1 transgenic tobaccos under additional UV-A radiation enhance the accumulation of these biomaterials up to160-180% higher than non transgenic tobaccos grown under VL condition. Moreover, PAP1 transgenic tobacco radiated with UVA light also significantly increased pelargonidin biomaterial. PAP1 transgenic tobaccos had a similar phenotype with non transgenic tobaccos but the color of fully expanding flower was more pink intensity than non transgenic.
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Franco-Lara, Liliana F., Kara D. McGeachy, Uli Commandeur, Robert R. Martin, Mike A. Mayo, and Hugh Barker. "Transformation of tobacco and potato with cDNA encoding the full-length genome of Potato leafroll virus: evidence for a novel virus distribution and host effects on virus multiplication." Journal of General Virology 80, no. 11 (November 1, 1999): 2813–22. http://dx.doi.org/10.1099/0022-1317-80-11-2813.
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A full-length cDNA copy of the genome of Potato leafroll virus (PLRV) was introduced into the genome of tobacco and potato plants by Agrobacterium tumefaciens-mediated transformation. Transgenic lines were obtained in which the transgene was readily detected by PCR with DNA extracted from T1 tobacco seedlings and clonally multiplied potato plants. PLRV-specific genomic and sub- genomic RNAs, coat protein antigen and virus particles were detected in transgenic plants. Aphids fed on the transgenic tobacco plants readily transmitted PLRV to test plants. Infected transgenic tobacco plants, like non-transgenic (WT) PLRV-infected plants, displayed no symptoms of the infection but transgenic plants of potato were severely stunted. In parallel tests, the mean PLRV titres in WT tobacco plants and transgenic tobacco plants were 600 and 630 ng virus/g leaf, respectively, although differences in PLRV titres among transgenic plants were much greater than those among infected WT plants. In similar tests with potato, the mean PLRV titre of WT plants was 50 ng virus/g leaf whereas higher concentrations (up to 3400 ng virus/g leaf) accumulated in transgenic potato plants. In tissue prints of stems, PLRV was detected in similar proportions of phloem cells in transgenic and infected WT plants. In transgenic tobacco and potato plants, but not in infected WT plants, a few stem epidermal cells also contained virus. From tissue prints of transgenic tobacco leaves, it was estimated that about one in 40000 mesophyll cells contained virus, but in transgenic potato, a greater proportion of mesophyll cells was infected.
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Thủy, Lê Thị, Triệu Thị Hằng, Lâm Đại Nhân, and Lê Văn Sơn. "Assessment of the mannose inhibition on tobacco regeneration for establishment of a mannose selection system for transgenic plants." Vietnam Journal of Biotechnology 14, no. 1 (March 30, 2016): 131–38. http://dx.doi.org/10.15625/1811-4989/14/1/9303.
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Recently, the application of environmentally friendly methods in transgenic-plant selection has been exploited with an interest to contribute to the commercialization of transgenic products. In this study, mannose was chosen in selecting transgenic tobacco. The ability of mannose to inhibit tobacco regeneration was assessed through three stages: creating multi-shoot, elongating shoot, and rooting of tobacco cultivars K326 and C9-1. The results showed that mannose concentrations at 30 g/l and 20 g/l are suitable for creating multi-shoot of K326 and C9-1, respectively. In addition, mannose concentration at 30 g/l in combination with 7,5 g/l sucrose is fit for elongating shoot while its concentration at 30 g/l is appropriate for rooting transgenic tobacco in both K326 and C9-1 cultivars. The mannose selection of transgenic tobacco using gus gene showed that the transgenic effectiveness is ranging 16-17% of K326 and 9-11% of C9-1 cultivars. The successful establishment of mannose selection system in transgene tobacco contributes to generate bio-safety transgenic plants.
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Santoso, Tri Joko, Muhammad Herman, Sri H. Hidayat, Hajrial Aswidinnoor, and Sudarsono Sudarsono. "Molecular Analysis and Effectiveness Assay of AV1 Gene in Transgenic Tobacco for Resistance to Begomovirus." Jurnal AgroBiogen 8, no. 2 (August 15, 2016): 45. http://dx.doi.org/10.21082/jbio.v8n2.2012.p45-53.
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<p>Genetic transformation<br />of tobacco plant using AV1 gene was conducted at<br />the previously experiment and generated transgenic tobacco<br />plants positively carrying the selectable marker nptII gene.<br />The objectives of this experiment were to (1) analyze the<br />presence of Begomovirus AV1 gene in T0 generation putative<br />transgenic tobacco plants using PCR technique with specific<br />primers and its correlation with resistance phenotype, (2)<br />analyze the integration and copy number of the transgene in<br />T0 generation putative transgenic tobacco plants and its<br />correlation with resistance response, (3) screen the T0<br />generation putative transgenic tobacco plants with the target<br />virus infection and to detect the presence of the virus in the<br />transgenic plant tissue using universal primers. PCR<br />detection of AV1 gene in tobacco transgenic was conducted<br />by using specific primer for Begomovirus AV1 gene.<br />Meanwhile, Southern Blot analysis was conducted by using<br />the AV1 gene probe. The effectiveness of AV1 gene in<br />tobacco transgenic was tested by inoculation of target virus<br />using whiteflies vector. Result of the experiments showed<br />that there was a positive correlation between the presence<br />of the AV1 transgene in T0 generation putative transgenic<br />tobacco plants and the resistant phenotype. Transgenic<br />plants with a single copy integration of the transgene<br />exhibited more resistant than the multiple copy one. and<br />non transgenic plant. The resistance as a result of AV1 gene<br />expression was indicated with no symptom in T0 generation<br />transgenic tobacco plants and the accumulation of the virus<br />in the transgenic plants tissue. Northern and Western<br />hybridization analysis need to be perfomed for investigating<br />the presence of mRNA or protein accumulation so that the<br />resistance mechanism of the AV1 gene could be explained<br />more detail.</p>
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Duan, Yong-Ping, Charles A. Powell, Dan E. Purcifull, Peter Broglio, and Ernest Hiebert. "Phenotypic Variation in Transgenic Tobacco Expressing Mutated Geminivirus Movement/Pathogenicity (BC1) Proteins." Molecular Plant-Microbe Interactions® 10, no. 9 (December 1997): 1065–74. http://dx.doi.org/10.1094/mpmi.1997.10.9.1065.
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Tobacco plants were transformed with the movement protein (pathogenicity) gene (BC1) from tomato mottle gem-inivirus (TMoV), using Agrobacterium-mediated transformation. Different transgenic tobacco lines that expressed high levels of the BC1 protein had phenotypes ranging from plants with severe stunting and leaf mottling (resembling geminivirus symptoms) to plants with no visible symptoms. The sequence data for the BC1 transgene from the transgenic plants with the different phenotypes indicated an association of spontaneously mutated forms of the BC1 gene in the transformed tobacco with phenotype variations. One mutated transgene associated with an asymptomatic phenotype had a major deletion at the C terminus of 119 amino acid residues with a recombination resulting in the addition of 26 amino acid residues of unidentified origin. This asymptomatic, mutated BC1 attenuated the phenotypic expression of the symptomatic BC1 in a tobacco line containing both copies of the BC1 gene. Another mutated form of the BC1 gene amplified from an asymptomatic, multicopy transgenic tobacco plant did not induce symptoms when transiently expressed in tobacco via a virus vector. The symptom attenuation in the transgenic tobacco by the asymptomatic BC1 may involve trans-dominant negative interference.
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Pavlíková, D., T. Macek, M. Macková, M. Surá, J. Száková, and P. Tlustoš. "The evaluation of cadmium, zinc and nickel accumulation ability of transgenic tobacco bearing different transgenes." Plant, Soil and Environment 50, No. 12 (December 10, 2011): 513–17. http://dx.doi.org/10.17221/4067-pse.
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Tobacco, Nicotiana tabacum L., var. Wisconsin 38 as the control (WSC), and four genetically modified lines of the same variety, were tested for Cd, Zn and Ni accumulation. Genetically modified lines of the same variety, bearing the transgene CUP1 (gene coding a yeast metallothionein), GUS (reporter gene for ß-glucuronidase), HisCUP (CUP combined with a polyhistidine tail), and HisGUS (reporter gene for ß-glucuronidase, combined with a polyhistidine tail) under a constitutive promoter, enabling it to follow the heavy metal tolerance and uptake changes as a function of the transgene present. Control and transgenic lines were tested for accumulation of risk elements on sand nutrient medium with the addition of cadmium, zinc and nickel. The results showed high Cd accumulation ability of HisCUP line. The Cd content in aboveground biomass was increased by 90% compared to the non-transformed control and Cd content in roots was decreased by 49%. Determination of Zn content in aboveground biomass did not confirm higher uptake by transgenic plants significant for phytoremediation. The Ni content was significantly increased in aboveground biomass of HisGUS construct. GUS construct introduced the ability to accumulate all investigated metals; the others accumulated only one in extended amount.
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Jan, Fuh-Jyh, Carmen Fagoaga, Sheng-Zhi Pang, and Dennis Gonsalves. "A minimum length of N gene sequence in transgenic plants is required for RNA-mediated tospovirus resistance." Microbiology 81, no. 1 (January 1, 2000): 235–42. http://dx.doi.org/10.1099/0022-1317-81-1-235.
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We showed previously that transgenic plants with the green fluorescent protein (GFP) gene fused to segments of the nucleocapsid (N) gene of tomato spotted wilt virus (TSWV) displayed post-transcriptional gene silencing of the GFP and N gene segments and resistance to TSWV. These results suggested that a chimeric transgene composed of viral gene segments might confer multiple virus resistance in transgenic plants. To test this hypothesis and to determine the minimum length of the N gene that could trans-inactivate the challenging TSWV, transgenic plants were developed that contained GFP fused with N gene segments of 24–453 bp. Progeny from these plants were challenged with: (i) a chimeric tobacco mosaic virus containing the GFP gene, (ii) a chimeric tobacco mosaic virus with GFP plus the N gene of TSWV and (iii) TSWV. A number of transgenic plants expressing the transgene with GFP fused to N gene segments from 110 to 453 bp in size were resistant to these viruses. Resistant plants exhibited post-transcriptional gene silencing. In contrast, all transgenic lines with transgenes consisting of GFP fused to N gene segments of 24 or 59 bp were susceptible to TSWV, even though the transgene was post-transcriptionally silenced. Thus, virus resistance and post-transcriptional gene silencing were uncoupled when the N gene segment was 59 bp or less. These results provide evidence that multiple virus resistance is possible through the simple strategy of linking viral gene segments to a silencer DNA such as GFP.
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Cho, Eun Jin, Quynh Anh Nguyen, Yoon Gyo Lee, Younho Song, Bok Jae Park, and Hyeun-Jong Bae. "Enhanced Biomass Yield of and Saccharification in Transgenic Tobacco Over-Expressing β-Glucosidase." Biomolecules 10, no. 5 (May 23, 2020): 806. http://dx.doi.org/10.3390/biom10050806.
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Here, we report an increase in biomass yield and saccharification in transgenic tobacco plants (Nicotiana tabacum L.) overexpressing thermostable β-glucosidase from Thermotoga maritima, BglB, targeted to the chloroplasts and vacuoles. The transgenic tobacco plants showed phenotypic characteristics that were significantly different from those of the wild-type plants. The biomass yield and life cycle (from germination to flowering and harvest) of the transgenic tobacco plants overexpressing BglB were 52% higher and 36% shorter than those of the wild-type tobacco plants, respectively, indicating a change in the genome transcription levels in the transgenic tobacco plants. Saccharification in biomass samples from the transgenic tobacco plants was 92% higher than that in biomass samples from the wild-type tobacco plants. The transgenic tobacco plants required a total investment (US$/year) corresponding to 52.9% of that required for the wild-type tobacco plants, but the total biomass yield (kg/year) of the transgenic tobacco plants was 43% higher than that of the wild-type tobacco plants. This approach could be applied to other plants to increase biomass yields and overproduce β-glucosidase for lignocellulose conversion.
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Wang, B., H. Shen, X. Yang, T. Guo, B. Zhang, and W. Yan. "Effects of chitinase-transgenic (McChit1) tobacco on the rhizospheric microflora and enzyme activities of the purple soil ." Plant, Soil and Environment 59, No. 6 (May 22, 2013): 241–46. http://dx.doi.org/10.17221/704/2012-pse.
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In order to evaluate the bio-security of genetically modified (GM) plants in the purple soil, we carried out a pot experiment about rhizospheric microflora at different development stages of a chitinase-transgenic (McChit1) tobacco (T-Chit), a plasmid-transgenic tobacco (T-Vi), and a non-transgenic tobacco (Nt-X) that were grown in the same purple soil, and surveyed the growth of three tobaccos and the properties of soil (i.e. the dynamic changes of the cultivable rhizospheric bacteria and fungi, soil enzyme activity and pH). The results showed that, compared with Nt-X plant as a control, T-Chit and T-Vi at the stages of flowering and mature significantly decreased the number of cultivable rhizospheric bacteria, but at their stubble stage the bacteria number returned to the same levels. Moreover, there were no significant differences about the number of cultivable rhizospheric fungi and the ratio of fungi to bacteria (F/B) among three treatments. It was of interest that soil catalase activities of T-Chit and T-Vi were lower than that of Nt-X during the same period, and urease activities of T-Vi and T-Chit were also lower than that of Nt-X at the stages of budding and stubble. Protease activity and the biomass of tobacco, however, showed no significant difference. This indicated that 1-year-old transgenic tobacco plants (T-Vi and T-Chit) inhibited the catalase and urease activities of the purple soil. In conclusion, the results revealed that 1-year-old T-Chit and T-Vi plants were non-toxic to the colony-forming units of cultivable bacteria and fungi in the studied purple soil during tobacco growth.
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Qin, Li-Jun, Dan Zhao, Yi Zhang, and De-Gang Zhao. "Selectable marker-free co-expression of Nicotiana rustica CN and Nicotiana tabacum HAK1 genes improves resistance to tobacco mosaic virus in tobacco." Functional Plant Biology 42, no. 8 (2015): 802. http://dx.doi.org/10.1071/fp14356.
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The viral disease caused by tobacco mosaic virus (TMV) is the most prevalent viral disease in many tobacco production areas. A breeding strategy based on resistance genes is an effective method for improving TMV resistance in tobacco. Also, the physiological status of plants is also critical to disease resistance improvement. Potassium ion is one of the most abundant inorganic nutrients in plant cells, and mediates plant responses to abiotic and biotic stresses. Improving K+ content in soil by fertilising can enhance diseases resistance of crops. However, the K+ absorption in plants depends mostly on K+ transporters located in cytoplasmic membrane. Therefore, the encoding genes for K+ transporters are putative candidates to target for improving tobacco mosaic virus resistance. In this work, the synergistic effect of a N-like resistance gene CN and a tobacco putative potassium transporter gene HAK1 was studied. The results showed that TMV-resistance in CN-HAK1-containing tobaccos was significantly enhanced though a of strengthening leaf thickness and reduction in the size of necrotic spots compared with only CN-containing plants, indicating the improvement of potassium nutrition in plant cells could increase the tobacco resistance to TMV by reducing the spread of the virus. Quantitative real-time polymerase chain reaction (qRT–PCR) analysis for TMV-CP expression in the inoculated leaf of the transgenic and wild-type plants also supported the conclusion. Further, the results of defence-related determination including antioxidative enzymes (AOEs) activity, salicylic acid (SA) content and the expression of resistance-related genes demonstrated CN with HAK1 synergistically enhanced TMV-resistance in transgenic tobaccos. Additionally, the HAK1- overexpression significantly improved the photosynthesis and K+-enriching ability in trans-CN-HAK1 tobaccos, compared with other counterparts. Finally, this work provides a method for screening new varieties of marker-free and safe transgenic antiviral tobacco.
Dissertations / Theses on the topic "Transgenic tobacco"
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Champanis, Reinette. "Aspects of sucrose metabolism in transgenic tobacco." Thesis, Stellenbosch : Stellenbosch University, 2004. http://hdl.handle.net/10019.1/49854.
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Dissertation (PhD) -- University of Stellenbosch, 2004.ENGLISH ABSTRACT: In most plants the efficiency of sucrose production and the systemic distribution thereof are the major determinants of growth, development and yield. The factors governing sugar partitioning co-ordinate its distribution in response to intrinsic and environmental signals. These factors include sugar transporters and invertases as well as metabolites, including sucrose and glucose, which function as signalling molecules to modulate gene expression. The genetic transformation of plants and the subsequent development of transgenic lines with disturbed sugar metabolism have made an unprecedented impact on the study of sugar translocation and -partitioning. For instance, the transformation of plants with a yeast-derived invertase targeted to different subcellular compartments has led to the elucidation of several key aspects of sugar metabolism, including phloem loading mechanisms, the regulation of photosynthesis by sugars, the importance of sugar-metabolism compartmentation with regards to sucrose biosynthesis, storage and distribution, as well as the role of cell-wall invertase in phloem unloading and sink strength. In this study, a similar strategy of transgenic plant analysis was employed to expand our insight into the regulation of sugar partitioning. The yeast-invertase Suc2 gene, from Saccharomyces cere visiae , was overexpressed in either the cytosol, vacuole or apoplast of transgenic tobacco plants. These transgenic lines displayed varying increases in invertase activity, altered sugar levels and consequently disturbed sink-source interactions and sugar partitioning. Transgenic lines overproducing the yeast-derived invertase in either the vacuole (Vac-Inv) or apoplast (Apo-Inv) were utilised to analyse the effect of the altered sugar levels in sink and source organs on the expression of sugar transporters, as well as the endogenous cell wall invertase and inhibitors in these plants. Transcript levels of the sucrose transporter NtSUT1 and hexose transporter NtMST1 encoding genes increased significantly in the source leaves and roots of Vac-Inv lines, whereas increased NtMst1 transcript levels were also detected in the roots of Apo-Inv lines. The increased mRNA levels could be correlated to the altered invertase activities and sugar levels in these tissues. It is concluded that NtSUT1 and NtMST1 are differentially regulated by sucrose and/or hexose content on a transcriptional level. Furthermore, the regulatory effect of the altered sugar levels on transporter expression depended on the subcellular compartment in which the yeast invertase was expressed. It would seem that the subcellular compartmentation of sugar metabolism is also fundamental to the regulation of sugar partitioning. The transcription levels of the endogenous cell wall invertase (CWt) and cell wall invertase inhibitor (Cwi-Inh) genes were examined in the various tissues of Apo-Inv and Vac-Inv lines at both the vegetative and flowering growth stages. In comparison with the control lines, the various tissues of the Apo-Inv and Vac-Inv lines displayed altered Cwi and Cwi-Inh expression levels, depending on the sink-source status and growth stage. However, no obvious correlation between the Cwi and Cwi-Inh expression levels and soluble sugar content of these tissues was found. It is suggested that the post-transcriptional and post-translation control of these proteins by sugars might play an important role in their regulation. Analysis of the Cwi:Cwi-lnh mRNA ratio and growth observations of the various tissues of control as well as Apo-Inv and Vac-Inv lines indicated that this transcription ratio could be an accurate indicator of the sink strength of sink organs. In addition, the influence of sink-source interactions on sugar partitioning was investigated. Reciprocal grafting between Apo-Inv and control lines resulted in scions with an altered sucrose metabolism in either the sink or source organs. These scions were subjected to biomass distribution, soluble sugar quantification and C4C]- radiolabelling experiments. The latter revealed an unaltered state of sugar partitioning from the above-ground tissues of the Apo/GUS scions and a significant shift in sugar partitioning towards the roots of the GUS/Apo scions in comparison to the control GUS/GUS scions. Phenotypic changes, opposite to those observed in Apo-Inv lines expressing the heterologous invertase in both sink and source organs, could initially be observed in the GUS/Apo and Apo/GUS scions. However, no significant differences in phenotype or biomass distribution could be observed between the mature GUS/Apo, Apo/GUS and GUS/GUS scions seven weeks postgrafting. This inconsistency between phenotype and sugar partitioning might be explained by an increase in the respiration rate of the tissues as supported by the soluble sugar content. These results highlight the complexity and adaptability of sucrose metabolism and sugar partitioning. In addition, it confirms that sugar partitioning can be modulated by sink-source interactions and emphasise the importance of invertases in the regulation of sugar partitioning through its ability to alter sink strength. This study forms part of the rapidly expanding initiative to unravel the control mechanisms of sugar partitioning. The results obtained in this study confirmed again that the introduction and expression of a single heterologous gene in transgenic plants could provide significant insight into the regulation of this process. It was shown here that the expression of sugar transporters is closely regulated by sugar levels and therefore fulfils a vital function in sugar sensing and consequently the regulation of sugar partitioning. The data presented in this study also demonstrated the intricate and flexible nature of the relationship that exists between sugar metabolism, partitioning and growth phenomena.
AFRIKAANSE OPSOMMING: Die doeltreffendheid van sukroseproduksie, tesame met die sistemiese verspreiding daarvan, is die vernaamste faktore wat die groei, ontwikkeling en opbrengsvermoë van die meeste plante bepaal. Die faktore wat suikerverdeling beheer, funksioneer om suikerverspreiding te koordineer in reaksie op beide inherente- en omgewingsseine. Hierdie faktore sluit suikertransporters en invertases in, asook metaboliete soos sukrose en glukose wat funksioneer as seinmolekule in die modulering van geenuitdrukking. Die genetiese transformasie van plante en die gevolglike daarstelling van transgeniese lyne met veranderde suikermetabolismes het 'n beduidende inwerking op die bestudering van suikervervoer en -verdeling gehad. Byvoorbeeld, die transformasie van plante met 'n gis-invertase geteiken na verskillende sub-sellulêre kompartemente, het tot die toeligting van verskeie aspekte van suikermetabolisme gelei, insluitende dié van floëemladingsmeganismes, die regulering van fotosintese deur suikers, die belang van kompartementalisering ten opsigte van sukrosebiosintese, -opberging en -verspreiding, en die rol van selwand-invertases in floëemontlaaiing en swelgpuntkrag. In hierdie studie is van soortgelyke transgeniese plantontledings gebruik gemaak om 'n dieper insig tot die regulering van suikerverdeling te verkry. Die gis-invertase Suc2 geen, afkomstig van Saccharomyces cerevisiae, is ooruitgedruk in óf die sitosol, vakuool óf apoplastiese ruimte van transgeniese tabakplante. Hierdie transgeniese lyne het wisselende toenames in invertase-aktiwiteite en veranderde suikervlakke getoon, asook gevolglike versteurde bron-swelgpunt interaksies en suikerverdeling. Transgeniese lyne met ooruitdrukking van die gis-invertase in óf die vakuool (Vac-Inv) óf die apoplast (Apo-Inv) is gebruik om die gevolg van die veranderde suikervlakke in bron- en swelgpuntorgane op die uitdrukking van suikertransporters, asook die endogene selwand-invertase en invertase-inhibitor in hierdie plante te bepaal. Transkripsievlakke van die sukrosetransporter NtSut1 en die heksosetransporter, NtMst1, het beduidend toegeneem in die bron-blare en wortels van die Vac-Inv lyne; 'n toename in NtMst1 transkripsievlakke is ook in die wortels van Apo-Inv lyne bevestig. Die toenames in boodskapper RNA kon gekorreleer word met die veranderde invertase-aktiwiteite en suikervlakke in hierdie weefsels. Die gevolgtrekking word gemaak dat NtSUT1 en NtMST1 differensieël gereguleer word op transkripsionele vlak deur die sukrose en/of heksose inhoud van weefsels. Meer nog, die regulerende effek van die veranderde suikervlakke op transporteruitdrukking het afgehang van die subsellulêre kompartement waarin die gis-invertase uitgedruk is. Dit wil dus voorkom dat die subsellulêre kompartementalisering van suikermetabolisme fundamenteel tot die deurgee en waarneming van suikerseine is, met In gevolglike eweneens belangrike rol in die regulering van suikerverdeling. Die transkripsievlakke van beide die endogene selwand-invertase (CWI) en die selwand-invertase-inhibitor (CWI-Inh) enkoderende gene is in verskeie weefsels van die Apo-Inv en Vac-Inv lyne, tydens beide die vegetatiewe- en blomstadia, bestudeer. Die onderskeie weefsels van die Apo-Inv en Vac-Inv lyne het, in vergelyking met die kontrole lyne, veranderde Cwi en Cwi-inh transkripsievlakke getoon wat bepaal is deur bron-swelgpunt status en groeistadium. Geen duidelike korrelasie kon tussen beide Cwi en Cwi-inh uitdrukkingsvlakke en oplosbare suiker inhoud gevind word nie. Daar word voorgestel dat post-transkripsionele en posttranslasionele beheer deur suikers 'n belangrike rol in die regulering van hierdie proteïne speel. Bestudering van die Cwi:Cwi-lnh mRNA verhouding, asook groei verskynsels van die onderskeie weefsels van kontrole en Apo-Inv en Vac-Inv lyne, dui daarop dat hierdie transkripsievlak-verhouding moontlik 'n akkurate aanwyser van die swelgpuntkrag van 'n swelgpuntorgaan kan wees. Voorts is die invloed van bron-swelgpuntorgaan interaksies op suikerverdeling ondersoek. Omgekeerde enting tussen Apo-Inv en kontrole lyne het entlote met gemodifiseerde suikermetabolisme in óf hul bron- óf hul swelgpuntorgane tot gevolg gehad. Hierdie entlote is aan biomassaverspreidings-, oplosbare suiker kwantifisering en C4C]-radiomerking eksperimente onderwerp. Hierdie resultate het gewys dat, in vergelyking met die kontrole (GUS/GUS) ente, daar geen verandering in die status van suikerverdeling vanaf die bogrondse plantdele in die Apo/GUS ente is nie, maar wel 'n beduidende verskuiwing in suikerverdeling na die wortels van die GUS/Apo ente. Fenotipiese veranderinge, wat teenoorgesteld van dié teenwoordig in die Apo- Inv lyne waar die heteroloë invertase in beide bron en swelgpuntorgane uitgedruk word, is aanvanklik in die GUS/Apo en Apo/GUS ente waargeneem. Geen verskille in fenotipe of biomassa-verspreiding kon egter sewe weke na die entings prosedures tussen die GUS/Apo, Apo/GUS and GUS/GUS ente gevind word nie. Dit mag verduidelik word deur 'n moontlike toename in respirasietempo in die betrokke weefsels; die oplosbare suikervlakke wat in die verskillende ente aangeteken is ondersteun dié moontlikheid. Hierdie resultate as geheelonderstreep die kompleksiteit en aanpasbaarheid van suikermetabolisme en -verdeling. Verder bevestig dit dat suikerverdeling beïnvloed kan word deur bron-swelgpunt interaksies, asook die belang van invertases in die regulering van suikerverdeling gegewe die vermoë om swelgpuntkrag te verander. Hierdie studie vorm deel van 'n vinnig groeiende inisiatief om die beheermeganismes van suikerverdeling te ontrafel. Die resultate verkry in hierdie studie bekragtig die belang van rekombinante DNA tegnologie in die bestudering van fundamentele plantprosesse. Die invoeging en uitdrukking van 'n geteikende gisinvertase in transgeniese plante het gelei tot veranderde suikervlakke en bronswelgpunt interaksies in hierdie lyne met die gevolglike ontginning van waardevolle inligting ten opsigte van die regulering van suikerverdeling in reaksie tot interne seine. Daar is aangetoon dat suikertransporters onlosmaakbaar gekoppel is aan die deurgee en waarneming van suikerseine, spesifiek op die vlak van transkripsionele regulering, en dus ook die regulering van suikerverdeling. Voorts wys die resultate op die komplekse en aanpasbare aard van die verhouding wat bestaan tussen suikermetabolisme, -verdeling en groeiverskynsels.
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Ahmad, Kafeel. "Molecular farming : production of pharmaceuticals in transgenic tobacco." Thesis, University of Leicester, 2011. http://hdl.handle.net/2381/10241.
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Molecular farming is an experimental application of biotechnology to modify crops in order to produce proteins and chemicals for medicinal and commercial interests. The vast majority in the developing world cannot afford the high cost of therapeutics produced by existing methods. We not only need to produce new therapeutics but also need to produce cheaper versions of the existing ones. Molecular farming could offer a viable option for this growing need for biopharmaceuticals. Part of the thesis deals with investigating ways to produce DesB30 form of human insulin in transgenic tobacco. The human insulin was synthesized in vitro as strep-tag II-mini-insulin fusion protein. Expression of mini-insulin by transgenic tobacco was confirmed by RT-PCR, western blotting and ELISA. However, sufficient levels of purified insulin could not be obtained to carry out further functional assays. Strategies for increasing the yield of insulin by transgenic tobacco are discussed and further increases in yield would need to be developed for this to become a viable and cost effective source of this important pharmaceutical. The second part of the thesis describes the production of a recombinant microbial polysaccharide in tobacco. Seven type 2 pneumococcal polysaccharide biosynthetic genes were expressed in a single tobacco plant, utilizing the plant Kex2 (Kexin protease 2) like protease system for multiple gene expression. Expression of these genes in transgenic tobacco was confirmed by RT-PCR and western blotting. Correct processing of the expressed proteins by the Kex2 protease system was confirmed. However, In planta production of type 2 polysaccharide could not be confirmed mainly as a result of high background from the wild type plant polysaccharide extracts. Strategies to overcome these issues are described. The usefulness of Kex2 protease system for multiple gene expression and metabolic pathways engineering is also discussed.
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Cherukumilli, Sri. "Expression of Human Interferon in Transgenic Tobacco Chloroplasts." Honors in the Major Thesis, University of Central Florida, 2005. http://digital.library.ucf.edu/cdm/ref/collection/ETH/id/747.
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This item is only available in print in the UCF Libraries. If this is your Honors Thesis, you can help us make it available online for use by researchers around the world by following the instructions on the distribution consent form at http://library.ucfBachelors
Burnett College of Biomedical Sciences
Molecular Biology and Microbiology
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Ni, Hao II. "Expression of Human Protein C in Transgenic Tobacco." Thesis, Virginia Tech, 1997. http://hdl.handle.net/10919/33367.
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Human Protein C (hPC) is a vitamin K-dependent serine protease that has a critical role in the naturally-occurring anticoagulant pathway. Upon activation of the zymogen by thrombin at the endothelial cell surface, the active form of hPC has anticoagulant activity in hemostasis due to its ability to inactivate factors Va and VIIIa. For biological activity, hPC requires several post-translational modifications including proteolytic cleavage, disulfide bond formation, b-hydroxylation, g-carboxylation, and N-linked glycosylation. Plants have the eukaryotic protein modifying mechanisms required for many human proteins and may provide a safe, cost-effective system for producing hPC on a large-scale basis. Tobacco (Nicotiana tabacum L.) is particularly well suited for use as a bioreactor for high-value recombinant proteins. Tobacco is one of the easiest plants to transform, it is an excellent biomass producer and can produce up to a million seeds from a single genetically engineered plant. Previous attempts to produce hPC in tobacco were limited by expression levels.
The overall goal of the research was to develop transgenic plants that express hPC at higher levels. A cDNA encoding hPC was fused to an enhanced constitutive 35S promoter (35SDE ) and introduced into a plant transformation vector. The hPC construct was introduced into tobacco leaf disks using Agrobacterium tumefaciens-mediated transformation, and 30 transgenic plants were generated.
Stable integration of the hPC gene construct into the tobacco genome and transgene copy number were determined by genomic Southern hybridization and segregation analyses. The majority of transgenic plants expressed the hPC transgene based on RNA analyses by northern hybridization. Plants utilizing the enhanced 35S promoter had equivalent levels of expression to previously generated hPC-containing plants. A variety of polyclonal and monoclonal antibodies raised against hPC were tested for detection of hPC standards and tobacco-synthesized hPC by western immunoblotting. Novel proteins in the size range of hPC heavy chain cross-reacted with anti-heavy chain hPC antibodies in 35SDE:hPC plants. Thus, plants may be capable of synthesizing hPC and proteolytically processing it to light and heavy chains. Although further experiments will be required to confirm the identity of these putative hPC proteins in tobacco, these result suggest that analyses of hPC expressed in plants have been limited by effective tools for detecting the hPC gene product rather than expression levels determined by the transgene promoter.Master of Science
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馮景良 and King-leung Fung. "Purification of Brassica juncea chitinase BJCHI1 from transgenic tobacco." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2001. http://hub.hku.hk/bib/B31224374.
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Hamdollah-Zadeh, Akram. "Transgenic resistance to pollen transmission of tobacco ringspot virus." Thesis, University of Bristol, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.364912.
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Holler, Christopher J. "Purification of an acidic recombinant protein from transgenic tobacco." Thesis, Virginia Tech, 2007. http://hdl.handle.net/10919/32379.
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Tobacco has been studied as a host for producing recombinant therapeutic proteins on a large-scale, commercial basis. However, the proteins expressed in tobacco usually need to be purified to high yield and purity from large amounts of biomass in order for their production to be commercially viable. The methods needed to purify proteins from tobacco are very challenging and not well studied. The objective of this research was to develop a process for the purification of the acidic model protein, recombinant β-glucuronidase (rGUS), from transgenic tobacco leaf tissue to high yield and purity.
Polyelectrolyte precipitation with polyethyleneimine (PEI) was identified as an initial purification step for purifying acidic recombinant proteins from tobacco. Polyethyleneimine precipitation allowed for high recovery and concentration of the target protein while removing large amounts of impurities from the initial extract. At dosages of 700-800 mg PEI/g total protein, nearly 100% of the rGUS activity was precipitated with generally more than 90% recovered from the pellet. In addition, more than 60% of the native tobacco proteins were removed in the process, resulting in a purification factor near 4.
Recombinant GUS was further purified by a step of hydrophobic interaction chromatography (HIC) followed by a step of hydroxyapatite chromatography (HAC). The HIC step served to remove PEI and other contaminants such as nucleic acids that were accumulated during the precipitation step, while the HAC step served to separate rGUS from the remaining native tobacco proteins, most notably ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco). Nearly 40% of the initial rGUS activity was recovered as a near homogeneous fraction based on SDS-PAGE analysis after the three step process.
The main steps used in this process are anticipated to be scalable and do not rely on affinity separations, making the process potentially applicable to a wide variety of acidic recombinant proteins expressed in tobacco as well as other leafy crops.
Master of Science
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Fung, King-leung. "Purification of Brassica juncea chitinase BJCHI1 from transgenic tobacco." Hong Kong : University of Hong Kong, 2001. http://sunzi.lib.hku.hk/hkuto/record.jsp?B22956347.
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Tame, Joanna Catherine. "Aspects of transgenic resistance to Tospoviruses." Thesis, University of Birmingham, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.369202.
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Carelse, Orseline. "Molecular studies of carotenoid biosynthesis in transgenic tomato and tobacco." Thesis, Royal Holloway, University of London, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.252061.
Full textBooks on the topic "Transgenic tobacco"
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Ordog, Sandi Helga. Comparative analysis of disease resistance responses to tobacco mosaic virus in transgenic tobacco plants with altered levels of mitochondrial alternative oxidase. 2002.
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Gordon, Jacqueline. In vitro import charateristics of transgenic tobacco plants over-expressing PHSP1, the mtHSP70 from PEA. 2001.
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Zhao, Yuan. Transcriptional analysis of the rice glutelin Gt3 gene. 1993.
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Lindbo, John A. Virus resistance in transgenic plants expressing translatable and untranslatable forms of the tobacco etch virus coat protein gene sequence. 1993.
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Broeckling, Corey D. Comparative Metabolomics of Transgenic Tobacco Plants (Nicotiana tabacum var. Xanthi) Reveals Differential Effects of Engineered Complete and Incomplete Flavonoid Pathways on the Metabolome. INTECH Open Access Publisher, 2012.
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Shao, Jiahong. Identification of peptide substrates of calcium-dependent protein kinase from random peptide phage display libraries and phosphorylation studies of the peptide substrate in transgenic tobacco cells. 1999.
Find full textBook chapters on the topic "Transgenic tobacco"
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Jäger, A. K., and J. van Staden. "Genetic Transformation of Solanum mauritianum Scop. (Tobacco Tree)." In Transgenic Trees, 283–90. Berlin, Heidelberg: Springer Berlin Heidelberg, 2000. http://dx.doi.org/10.1007/978-3-642-59609-4_20.
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Miki, B. L. A., S. G. Mcttugh, H. Labbe, T. Ouellet, J. H. Tolman, and J. E. Brandle. "Transgenic Tobacco: Gene Expression and Applications." In Transgenic Medicinal Plants, 336–54. Berlin, Heidelberg: Springer Berlin Heidelberg, 1999. http://dx.doi.org/10.1007/978-3-642-58439-8_25.
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Pospíšilová, J., H. Synková, I. Macháčková, and J. Čatský. "Photosynthesis of Transgenic Tobacco Plants." In Photosynthesis: from Light to Biosphere, 4411–14. Dordrecht: Springer Netherlands, 1995. http://dx.doi.org/10.1007/978-94-009-0173-5_1037.
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Worrall, Dawn. "PCR Analysis of Transgenic Tobacco Plants." In Plant Virology Protocols, 417–23. Totowa, NJ: Humana Press, 1998. http://dx.doi.org/10.1385/0-89603-385-6:417.
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Worrall, Dawn. "Southern Analysis of Transgenic Tobacco Plants." In Plant Virology Protocols, 425–36. Totowa, NJ: Humana Press, 1998. http://dx.doi.org/10.1385/0-89603-385-6:425.
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Baldewijns, L., and R. Valcke. "Photosystem II Electron Transport in Transgenic Tobacco." In Photosynthesis: Mechanisms and Effects, 1173–76. Dordrecht: Springer Netherlands, 1998. http://dx.doi.org/10.1007/978-94-011-3953-3_280.
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Dunsmuir, Pamela, William Howie, Ed Newbigin, Larry Joe, Eva Penzes, and Trevor Suslow. "Resistance to Rhizoctonia Solani in Transgenic Tobacco." In Advances in Molecular Genetics of Plant-Microbe Interactions, Vol. 2, 567–71. Dordrecht: Springer Netherlands, 1993. http://dx.doi.org/10.1007/978-94-017-0651-3_63.
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Knoester, M., L. C. Loon, J. F. Bol, and H. J. M. Linthorst. "Modulation Of Ethylene Production in Transgenic Tobacco." In Biology and Biotechnology of the Plant Hormone Ethylene, 347–54. Dordrecht: Springer Netherlands, 1997. http://dx.doi.org/10.1007/978-94-011-5546-5_42.
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Szeto, Tim H., Pascal M. W. Drake, Audrey Y.-H. Teh, Nicole Falci Finardi, Ashleigh G. Clegg, Mathew J. Paul, Rajko Reljic, and Julian K.-C. Ma. "Production of Recombinant Proteins in Transgenic Tobacco Plants." In Recombinant Proteins in Plants, 17–48. New York, NY: Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2241-4_2.
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Kares, Christa, Ann Depicker, Peter Debreyne, Philippe Crouzet, and Leon Otten. "Transgenic tobacco plants with heat-inducible IAA synthesis genes." In Progress in Plant Growth Regulation, 713–23. Dordrecht: Springer Netherlands, 1992. http://dx.doi.org/10.1007/978-94-011-2458-4_87.
Full textConference papers on the topic "Transgenic tobacco"
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Sidik, Nik Marzuki, and Noor Farhan Othman. "Accumulation of nickel in transgenic tobacco." In THE 2013 UKM FST POSTGRADUATE COLLOQUIUM: Proceedings of the Universiti Kebangsaan Malaysia, Faculty of Science and Technology 2013 Postgraduate Colloquium. AIP Publishing LLC, 2013. http://dx.doi.org/10.1063/1.4858672.
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Shirokikh, I. G., and Ya I. Nazarova. "Variability of actinomycete complexes in the rhizosphere of transgenic and intact tobacco lines." In 2nd International Scientific Conference "Plants and Microbes: the Future of Biotechnology". PLAMIC2020 Organizing committee, 2020. http://dx.doi.org/10.28983/plamic2020.223.
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The results on the detection of changes in model microbial complexes formed in the rhizosphere of transgenic tobacco plants with enhanced antioxidant (FeSOD 1) and osmoprotective (codA) protection are presented.
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Chen, Yafei, Yong Zhan, Peng Guo, Hui Liu, Yingrong Han, Yuhong Zhang, Liya Gao, Xiaoming Zhao, and Yuguang Du. "Comparison of Defense Related Enzyme between Oligochitosan Induced Protein Kinase Gene Silenced Transgenic Tobacco and Wild Type Tobacco." In 2008 2nd International Conference on Bioinformatics and Biomedical Engineering (ICBBE '08). IEEE, 2008. http://dx.doi.org/10.1109/icbbe.2008.263.
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Li, Liya, Qiuyi Cai, Kai Jia, Meng Zhang, and Changhong Guo. "Molecular and Phenotypic Characterization of Transgenic Tobacco Expressing the Arabidopsis IRT1 Gene." In 2010 4th International Conference on Bioinformatics and Biomedical Engineering (iCBBE 2010). IEEE, 2010. http://dx.doi.org/10.1109/icbbe.2010.5516422.
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Guo, Bei, Yanzhai Song, Xuwen Guo, Wenping Wang, and Ning Ding. "Inhibition of Gene Expression of Trehalase Enhances Drought Resistance in Transgenic Tobacco." In 2010 4th International Conference on Bioinformatics and Biomedical Engineering (iCBBE 2010). IEEE, 2010. http://dx.doi.org/10.1109/icbbe.2010.5516954.
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Kim, Yonggyun. "Transgenic tobacco expressing a viral cystatin gene, CpBV-CST1, exhibits insect resistance." In 2016 International Congress of Entomology. Entomological Society of America, 2016. http://dx.doi.org/10.1603/ice.2016.93511.
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Han, Qiang, Jing Liu, Wei Yao, Hongwei Liang, Dechun Zhang, Faju Chen, and Zhengquan He. "Studies on expression of rabbit defensin gene (NP-1) in transgenic tobacco." In International Conference on Medical Engineering and Bioinformatics. Southampton, UK: WIT Press, 2014. http://dx.doi.org/10.2495/meb140241.
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WANG, XIN-GUO, GUO-HUA ZHANG, RONG-XIANG FANG, CHUAN-XUAN LIU, YAN-HONG ZHANG, and CHENG-ZU XIAO. "PURIFIED CHOLERA TOXIN B SUBUNIT FROM TRANSGENIC TOBACCO PLANTS POSSESSES AUTHENTIC ANTIGENICITY." In International Seminar on Nuclear War and Planetary Emergencies 25th Session. Singapore: World Scientific Publishing Co. Pte. Ltd., 2001. http://dx.doi.org/10.1142/9789812797001_0012.
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Desagani, Dayananda, Aakash Jog, Adi Avni, and Yosi Shacham-Diamand. "In-Vivo Dehydration Sensing in Transgenic Tobacco Plants using an Integrated Electrochemical Chip." In 2020 IEEE International Symposium on Circuits and Systems (ISCAS). IEEE, 2020. http://dx.doi.org/10.1109/iscas45731.2020.9181292.
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Guo, Bei. "Physiological Identification of Salt Tolerance in Transgenic Tobacco Expressing Genes Related to Plant Trehalose Metabolism." In 2009 3rd International Conference on Bioinformatics and Biomedical Engineering (iCBBE). IEEE, 2009. http://dx.doi.org/10.1109/icbbe.2009.5163193.
Full textReports on the topic "Transgenic tobacco"
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Aly, Radi, James H. Westwood, and Carole L. Cramer. Novel Approach to Parasitic Weed Control Based on Inducible Expression of Cecropin in Transgenic Plants. United States Department of Agriculture, May 2003. http://dx.doi.org/10.32747/2003.7586467.bard.
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Our overall goal was to engineer crop plants with enhanced resistance to Orobanche (broomrape) based on the inducible expression of sarcotoxin-like peptide (SLP). A secondary objective was to localize small proteins such as SLP in the host-parasite union in order to begin characterizing the mechanism of SLP toxicity to Orobanche. We have successfully accomplished both of these objectives and have demonstrated that transgenic tobacco plants expressing SLP under control of the HMG2 promoter show enhanced resistance to O. aegyptiaca and O. ramosa . Furthermore, we have shown that proteins much larger than the SLP move into Orobanche tubercles from the host root via either symplastic or apoplastic routes. This project was initiated with the finding that enhanced resistance to Orobanche could be conferred on tobacco, potato, and tomato by expression of SLP (Sarcotoxin IA is a 40-residue peptide produced as an antibiotic by the flesh fly, Sarcophaga peregrina ) under the control of a low-level, root-specific promoter. To improve the level of resistance, we linked the SLP gene to the promoter from HMG2, which is strongly inducible by Orobanche as it parasitizes the host. The resulting transgenic plants express SLP and show increased resistance to Orobanche. Resistance in this case is manifested by increased growth and yield of the host in the presence of the parasite as compared to non-transgenic plants, and decreased parasite growth. The mechanism of resistance appears to operate post-attachment as the parasite tubercles attached to the transgenic root plants turned necrotic and failed to develop normally. Studies examining the movement of GFP (approximately 6X the size of SLP) produced in tobacco roots showed accumulation of green fluorescence in tubercles growing on transformed plants but not in those growing on wild-type plants. This accumulation occurs regardless of whether the GFP is targeted to the cytoplasm (translocated symplastically) or the apoplastic space (translocated in xylem). Plants expressing SLP appear normal as compared to non-transgenic plants in the absence of Orobanche, so there is no obvious unintended impact on the host plant from SLP expression. This project required the creation of several gene constructs and generation of many transformed plant lines in order to address the research questions. The specific objectives of the project were to: 1. Make gene constructs fusing Orobanche-inducible promoter sequences to either the sarcotoxin-like peptide (SLP) gene or the GFP reporter gene. 2. Create transgenic plants containing gene constructs. 3. Characterize patterns of transgene expression and host-to-parasite movement of gene products in tobacco ( Nicotiana tabacum L.) and Arabidopsis thaliana (L.). 4. Characterize response of transgenic potato ( Solanum tuberosum L.) and tomato ( Lycopersicon esculentum Mill .) to Orobanche in lab, greenhouse, and field. Objectives 1 and 2 were largely accomplished during the first year during Dr. Aly's sabbatical visit to Virginia Tech. Transforming and analyzing plants with all the constructs has taken longer than expected, so efforts have concentrated on the most important constructs. Work on objective 4 has been delayed pending the final results of analysis on tobacco and Arabidopsis transgenic plants. The implications of this work are profound, because the Orobanche spp. is an extremely destructive weed that is not controlled effectively by traditional cultural or herbicidal weed control strategies. This is the first example of engineering resistance to parasitic weeds and represents a unique mode of action for selective control of these weeds. This research highlights the possibility of using this technique for resistance to other parasitic species and demonstrates the feasibility of developing other novel strategies for engineering resistance to parasitic weeds.
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Wolf, Shmuel, and William J. Lucas. Involvement of the TMV-MP in the Control of Carbon Metabolism and Partitioning in Transgenic Plants. United States Department of Agriculture, October 1999. http://dx.doi.org/10.32747/1999.7570560.bard.
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The function of the 30-kilodalton movement protein (MP) of tobacco mosaic virus (TMV) is to facilitate cell-to-cell movement of viral progeny in infected plants. Our earlier findings have indicated that this protein has a direct effect on plasmodesmal function. In addition, these studies demonstrated that constitutive expression of the TMV MP gene (under the control of the CaMV 35S promoter) in transgenic tobacco plants significantly affects carbon metabolism in source leaves and alters the biomass distribution between the various plant organs. The long-term goal of the proposed research was to better understand the factors controlling carbon translocation in plants. The specific objectives were: A) To introduce into tobacco and potato plants a virally-encoded (TMV-MP) gene that affects plasmodesmal functioning and photosynthate partitioning under tissue-specific promoters. B) To introduce into tobacco and potato plants the TMV-MP gene under the control of promoters which are tightly repressed by the Tn10-encoded Tet repressor, to enable the expression of the protein by external application of tetracycline. C) To explore the mechanism by which the TMV-MP interacts with the endogenous control o~ carbon allocation. Data obtained in our previous project together with the results of this current study established that the TMV-MP has pleiotropic effects when expressed in transgenic tobacco plants. In addition to its ability to increase the plasmodesmal size exclusion limit, it alters carbohydrate metabolism in source leaves and dry matter partitioning between the various plant organs, Expression of the TMV-MP in various tissues of transgenic potato plants indicated that sugars and starch levels in source leaves are reduced below those of control plants when the TMV-MP is expressed in green tissue only. However, when the TMV-MP was expressed predominantly in PP and CC, sugar and starch levels were raised above those of control plants. Perhaps the most significant result obtained from experiments performed on transgenic potato plants was the discovery that the influence of the TMV-MP on carbohydrate allocation within source leaves was under developmental control and was exerted only during tuber development. The complexity of the mode by which the TMV-MP exerts its effect on the process of carbohydrate allocation was further demonstrated when transgenic tobacco plants were subjected to environmental stresses such as drought stress and nutrients deficiencies, Collectively, these studies indicated that the influence of the TMV-MP on carbon allocation L the result of protein-protein interaction within the source tissue. Based on these results, together with the findings that plasmodesmata potentiate the cell-to-cell trafficking of viral and endogenous proteins and nucleoproteins complexes, we developed the theme that at the whole plant level, the phloem serves as an information superhighway. Such a long-distance communication system may utilize a new class of signaling molecules (proteins and/or RNA) to co-ordinate photosynthesis and carbon/nitrogen metabolism in source leaves with the complex growth requirements of the plant under the prevailing environmental conditions. The discovery that expression of viral MP in plants can induce precise changes in carbon metabolism and photoassimilate allocation, now provide a conceptual foundation for future studies aimed at elucidating the communication network responsible for integrating photosynthetic productivity with resource allocation at the whole-plant level. Such information will surely provide an understanding of how plants coordinate the essential physiological functions performed by distantly-separated organs. Identification of the proteins involved in mediating and controlling cell-to-cell transport, especially at the companion cell-sieve element boundary, will provide an important first step towards achieving this goal.
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Zilinskas, Barbara A., Doron Holland, Yuval Eshdat, and Gozal Ben-Hayyim. Production of Stress Tolerant Plants by Overproduction of Enzymatic Oxyradical Scavengers. United States Department of Agriculture, May 1993. http://dx.doi.org/10.32747/1993.7568751.bard.
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Most of the objectives that were outlined in the original proposal have been met with two exceptions. Briefly, our goals were to: (1) constract transgenic tobacco plants which overproduce one or more of the enzymatic oxyradical scavengers and associated ancillary enzymes, including superoxide dismutase, ascorbate peroxidase, glutathione peroxidase, glutathione reductase, and monodehydrascorbate reductase; (2) evaluate the tolerance of these transgenic plants to oxidative stress; and (3) extend these studies to an agronomically important crop such as citrus. As can be seen i the following pages, our objectives (1) and (2) have been achieved, although transgenic lines overexpressing phospholipid hydroperoxidase glutathione peroxidase (PHGPX) were not obtained and our evidence to date suggests that constitutive overexpressing of the enzyme is probably lethal. Howeever, transgenic tobacco expressing the antisense construct for PHGPX were obtained. Tobacco plants overexpressing ascorbate peroxidase and those sensesuppressing monodehydroascorbate reductase are more tolerant to oxidative stress, as mediated by the redox-cycling agent paraquant; in contrast, plants expressing the PHGPX-antisense construct are more sensitive to paraquat. Additional research is warranted on each of the six types of transgenic lines which we generated with regard to their tolerance to saline stress. Until recently, attempts to transform citrus were not very successful, and thus additional attention is currently being directed at objective (3). We are optimistic that use of the plant transformation vector, pBIN, will lead to stable transgenic citrus, as preliminary experiments demonstrate stable expression of the GUS reporter gene. Other important contributions resulting from this BARD project include the biochemical characterization of the first plant phospholipid glutathione peroxidase and the biochemical and molecular analysis of another key antioxidant enzyme, monodehydroascorbate reductase. Overall this BARD-supported project was quite successful, and the biological resource of numerous transgenic lines which have altered levels of antioxidant enzymes should be valuable for years to come.
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Sengupta-Gopalan, Champa, Shmuel Galili, and Rachel Amir. Improving Methionine Content in Transgenic Forage Legumes. United States Department of Agriculture, February 2001. http://dx.doi.org/10.32747/2001.7580671.bard.
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Leguminous forage crops are high in proteins but deficient in S- amino acids. It has been shown that both wool quality and milk production can be limited by the post-ruminal supply of sulfur-containing amino acids. Efforts to use conventional plant breeding and cell selection techniques to increase the S-amino acid content of alfalfa have met with little success. With the objective to increase the S-amino acid content of forage legumes, the goal of this project was to co- express the methionine rich zein genes from corn along with a gene for a key enzyme in methionine biosynthesis, aspartate kinase(AK). The zeins are seed storage proteins from corn and are groupec into four distinct classes based on their amino acid sequence homologies. The b-zein (15kd) and the 6zein (10kD and 18kD) have proportionately high levels of methionine (10%, 22% and 28%, respectively). Initial studies from our lab had shown that while the 15kD zein accumulated to high levels in vegetative tissues of transgenic tobacco the l0kD zein did not. However, co-expression of the 10kD zein with the 15kD zein genes in tobacco showed stabilization of the 10kD zein and the co-localization of the 10kD and 15kD zein proteins in unique ER derived protein bodies. AK is the key enzyme for producing carbon skeletons for all amino acids of the aspartate family including methionine. It is, however, regulated by end-product feedback inhibition. The specific objectives of this proposal were: i. to co-express the 15kD zein with the 10/18kD zein genes in alfalfa in order to enhance the level of accumulation of the 10/18kD zein; ii. to increase methionine pools by expressing a feedback insensitive AK gene in transformants co-expressing the 15kD and 10/18kD zein genes. The Israeli partners were successful in expressing the AK gene in alfalfa which resulted in an increase in free and bound threonine but not in methionine (Galili et al., 2000). Since our target was to increase methionine pools, we changed our second objective to replace the AK gene with the gene for cystathionine gamma synthase (CGS) in the co-expression studies. The first methionine specific reaction is catalyzed by CGS. An additional objective was to develop a transformation system for Berseem clover, and to introduce the appropriate gene constructs into it with the goal of improving their methionine content. Genes for the 15kD zein along with the genes for either the 10kD or 18kD zein have been introduced into the same alfalfa plant both by sexual crosses and by re-transformation. Analysis of these zein co-expressors have shown that both the IOkD and 18kD zein levels go up 5 to 10 fold when co-expressed with the 15kD zein (Bagga et al., MS in preparation). Incubation of the leaves of transgenic alfalfa co-expressing the 15kD and 10kD zein genes, in the rumen of cows have shown that the zein proteins are stable in the rumen. To increase the level of zein accumulation in transgenic alfalfa different promoters have been used to drive the zein genes in alfalfa and we have concluded that the CaMV 35S promoter is superior to the other strong leaf -specific promoters. By feeding callus tissue of alfalfa plants co-expressing the 15kD and 10kD zein genes with methionine and its precursors, we have shown that the zein levels could be significantly enhanced by increasing the methionine pools. We have now introduced the CGS gene (from Arabidopsis; kindly provided to us by Dr. Leustek), into the 15kD zein transformants and experiments are in progress to check if the expression of the CGS gene indeed increases the level of zein accumulation in alfalfa. We were not successful in developing a transformation protocol for Berseem clover.
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Lee, Richard, Moshe Bar-Joseph, K. S. Derrick, Aliza Vardi, Roland Brlansky, Yuval Eshdat, and Charles Powell. Production of Antibodies to Citrus Tristeza Virus in Transgenic Citrus. United States Department of Agriculture, September 1995. http://dx.doi.org/10.32747/1995.7613018.bard.
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Citrus tristeza virus (CTV) is the most important virus disease of citrus in the world. CTV causes death of trees on sour orange rootstock and/or stem pitting of scions regardless of rootstock which results in trees of low vigor, reduced yield with reduction in size and quality of fruit. The purpose of this project was to produce monoclonal antibodies (MABs) to CTV coat protein (CP), develop single domain antibodies (dAbs) or Fab fragments which neutralize the infection by binding to the virus, and to produce transformed plants which express the dAbs. The objectives of this research have been met and putative transgenic tobacco and citrus plants have been developed. These putative transgenic plants are presently undergoing evaluation to determine the level of dAbs expression and to determine their resistance to CTV. Additionally, the CTV genome has been sequenced and the CP gene of several biologically characterized CTV strains molecular characterized. This has indicated a correlation between CP sequence homology and biological activity, and the finding of DI RNAs associated with some CTV strains. Several MABs have been produced which enable broad spectrum identification of CTV strains while other MABs enable differentiation between mild and severe strains. The use of selected MAbs and determination of the CP gene sequence has enabled predictions of biological activities of unknown CTV isolates. The epitopes of two MABs, one reacting selectively with severe CTV strains and the other reacting with all strains, have been characterized at the molecular level.
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Epel, Bernard, and Roger Beachy. Mechanisms of intra- and intercellular targeting and movement of tobacco mosaic virus. United States Department of Agriculture, November 2005. http://dx.doi.org/10.32747/2005.7695874.bard.
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To cause disease, plant viruses must replicate and spread locally and systemically within the host. Cell-to-cell virus spread is mediated by virus-encoded movement proteins (MPs), which modify the structure and function of plasmodesmata (Pd), trans-wall co-axial membranous tunnels that interconnect the cytoplasm of neighboring cells. Tobacco mosaic virus (TMV) employ a single MP for cell- cell spread and for which CP is not required. The PIs, Beachy (USA) and Epel (Israel) and co-workers, developed new tools and approaches for study of the mechanism of spread of TMV that lead to a partial identification and molecular characterization of the cellular machinery involved in the trafficking process. Original research objectives: Based on our data and those of others, we proposed a working model of plant viral spread. Our model stated that MPᵀᴹⱽ, an integral ER membrane protein with its C-terminus exposed to the cytoplasm (Reichel and Beachy, 1998), alters the Pd SEL, causes the Pd cytoplasmic annulus to dilate (Wolf et al., 1989), allowing ER to glide through Pd and that this gliding is cytoskeleton mediated. The model claimed that in absence of MP, the ER in Pd (the desmotubule) is stationary, i.e. does not move through the Pd. Based on this model we designed a series of experiments to test the following questions: -Does MP potentiate ER movement through the Pd? - In the presence of MP, is there communication between adjacent cells via ER lumen? -Does MP potentiate the movement of cytoskeletal elements cell to cell? -Is MP required for cell-to-cell movement of ER membranes between cells in sink tissue? -Is the binding in situ of MP to RNA specific to vRNA sequences or is it nonspecific as measured in vitro? And if specific: -What sequences of RNA are involved in binding to MP? And finally, what host proteins are associated with MP during intracellular targeting to various subcellular targets and what if any post-translational modifications occur to MP, other than phosphorylation (Kawakami et al., 1999)? Major conclusions, solutions and achievements. A new quantitative tool was developed to measure the "coefficient of conductivity" of Pd to cytoplasmic soluble proteins. Employing this tool, we measured changes in Pd conductivity in epidermal cells of sink and source leaves of wild-type and transgenic Nicotiana benthamiana (N. benthamiana) plants expressing MPᵀᴹⱽ incubated both in dark and light and at 16 and 25 ᵒC (Liarzi and Epel, 2005 (appendix 1). To test our model we measured the effect of the presence of MP on cell-to-cell spread of a cytoplasmic fluorescent probe, of two ER intrinsic membrane protein-probes and two ER lumen protein-probes fused to GFP. The effect of a mutant virus that is incapable of cell-to-cell spread on the spread of these probes was also determined. Our data shows that MP reduces SEL for cytoplasmic molecules, dilates the desmotubule allowing cell-cell diffusion of proteins via the desmotubule lumen and reduces the rate of spread of the ER membrane probes. Replicase was shown to enhance cell-cell spread. The data are not in support of the proposed model and have led us to propose a new model for virus cell-cell spread: this model proposes that MP, an integral ER membrane protein, forms a MP:vRNAER complex and that this ER-membrane complex diffuses in the lipid milieu of the ER into the desmotubule (the ER within the Pd), and spreads cell to cell by simple diffusion in the ER/desmotubule membrane; the driving force for spread is the chemical potential gradient between an infected cell and contingent non-infected neighbors. Our data also suggests that the virus replicase has a function in altering the Pd conductivity. Transgenic plant lines that express the MP gene of the Cg tobamovirus fused to YFP under the control the ecdysone receptor and methoxyfenocide ligand were generated by the Beachy group and the expression pattern and the timing and targeting patterns were determined. A vector expressing this MPs was also developed for use by the Epel lab . The transgenic lines are being used to identify and isolate host genes that are required for cell-to-cell movement of TMV/tobamoviruses. This line is now being grown and to be employed in proteomic studies which will commence November 2005. T-DNA insertion mutagenesis is being developed to identify and isolate host genes required for cell-to-cell movement of TMV.
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Aly, Radi, and John I. Yoder. Development of resistant crop plants to parasitic weeds based on trans-specific gene silencing. United States Department of Agriculture, January 2013. http://dx.doi.org/10.32747/2013.7598146.bard.
Full textAbstract:
Broomrapes (Orobanche/Phelipanchespp.) are holo parasitic plants that subsist on the roots of a variety of agricultural crops and cause severe losses to the yield quality and quantity. Effective methods for controlling parasitic weeds are scarce, with only a few known cases of genetic resistance. In the current study, we proposed an improved strategy for the control of parasitic weeds based on trans-specific gene-silencing of three parasite genes at once. We used two strategies to express dsRNA containing selected sequences of three Phelipancheaegyptiacagenes PaACS, PaM6PR and PaPrx1 (pma): transient expression using Tobacco rattle virus (TRV:pma) as a virus-induced gene-silencing (VIGS) vector and stable expression in transgenic tomato Solanumlycopersicum(Mill.) plants harboring a hairpin construct (pBINPLUS35:pma). siRNA-mediated transgene-silencing (20–24 nt) was detected in the host plants. Our results demonstrate that the quantities of PaACSand PaM6PR transcripts from P. aegyptiacatubercles grown on transgenic tomato or on Tobacco rattle virus-infected Nicotianabenthamianaplants were significantly reduced. However, only partial reductions in the quantity of PaPrx1 transcripts were observed in the parasite tubercles grown on tomato and on N. benthamianaplants. Concomitant with the suppression of the target genes, there were significant decreases in the number and weight of the parasite tubercles that grew on the host plants, in both the transient and the stable experimental systems. The results of the work carried out using both strategies point to the movement of mobile exogenous siRNA from the host to the parasite, leading to the impaired expression of essential parasite target genes. In light of the importance of parasitic weeds to world agriculture and the difficulty of obtaining resistance by conventional methods, we assume that genetic resistance based on the silencing of key metabolic genes in the parasite is now feasible. BARD Report - Project4622 Page 2 of 60
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Watad, Abed A., Paul Michael Hasegawa, Ray A. Bressan, Alexander Vainstein, and Yigal Elad. Osmotin and Osmotin-Like Proteins as a Novel Source for Phytopathogenic Fungal Resistance in Transgenic Carnation and Tomato Plants. United States Department of Agriculture, January 2000. http://dx.doi.org/10.32747/2000.7573992.bard.
Full textAbstract:
The goal of this project is to enhance fungal resistance of carnation and tomato through the ectopic expression of osmotin and other pathogenesis-related (PR) proteins. The research objectives were to evaluate in vitro antifungal activity of osmotin and osmotin and other PR protein combinations against phytopathogens (including Fusarium oxysporum, Verticillium dahliae, Botrytus cinerea or Phytophthora infestans), develop protocols for efficient transformation of carnation and tomato, express PR proteins in transgenic carnation and tomato and evaluate fungal resistance of transgenic plants. Protocols for microprojectile bombardment and Agrobacterium-mediated transformation of carnation were developed that are applicable for the biotechnology of numerous commercial cultivars. Research established an efficient organogenetic regeneration system, optimized gene delivery and transgene expression and defined parameters requisite to the high frequency recovery of transgenic plants. Additionally, an efficient Agrobacterium-mediated transformation protocol was developed for tomato that is applicable for use with numerous commercial varieties. Rigorous selection and reducing the cytokinin level in medium immediately after shoot induction resulted in substantially greater frequency of adventitious shoots that developed defined stems suitable for rooting and reconstitution of transgenic plants. Transformation vectors were constructed for co-expression of genes encoding osmotin and tobacco chitinase Ia or PR-1b. Expression of osmotin, PR-1 and/or chitinase in transgenic carnation mediated a high level resistance of cv. White Sim (susceptible variety) to F. oxysporum f. sp. dianthi, race 2 in greenhouse assays. These plants are being evaluated in field tests. Comprehensive analysis (12 to 17 experiments) indicated that germination of B. cinerea conidia was unaffected by PR protein expression but germ tube elongation was reduced substantially. The disease severity was significantly attenuated by PR protein expression. Constitutive expression of osmotin in transgenic tomato increased resistance to B. cinerea, and P. infestans. Grey mold and late blight resistance was stable through the third selfed generation. The research accomplished in this project will have profound effects on the use of biotechnology to improve carnation and tomato. Transformation protocols that are applicable for efficient stable gene transfer to numerous commercial varieties of carnation and tomato are the foundation for the capacity to bioengineer these crops. The research further establishes that PR proteins provide a measure of enhanced disease resistance. However, considerations of PR protein combinations and conditional regulation and targeting are likely required to achieve; sustained level of resistance.
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Mevarech, Moshe, Jeremy Bruenn, and Yigal Koltin. Virus Encoded Toxin of the Corn Smut Ustilago Maydis - Isolation of Receptors and Mapping Functional Domains. United States Department of Agriculture, September 1995. http://dx.doi.org/10.32747/1995.7613022.bard.
Full textAbstract:
Ustilago maydis is a fungal pathogen of maize. Some strains of U. maydis encode secreted polypeptide toxins capable of killing other susceptible strains of U. maydis. Resistance to the toxins is conferred by recessive nuclear genes. The toxins are encoded by genomic segments of resident double-strande RNA viruses. The best characterized toxin, KP6, is composed of two polypeptides, a and b, which are not covalently linked. It is encoded by P6M2 dsRNA, which has been cloned, sequenced and expressed in a variety of systems. In this study we have shown that the toxin acts on the membranes of sensitive cells and that both polypeptides are required for toxin activity. The toxin has been shown to function by creating new pores in the cell membrane and disrupting ion fluxes. The experiments performed on artificial phospholipid bilayers indicated that KP6 forms large voltage-independent, cation-selective channels. Experiments leading to the resolution of structure-function relationship of the toxin by in vitro analysis have been initiated. During the course of this research the collaboration also yielded X-ray diffracion data of the crystallized a polypeptide. The effect of the toxin on the pathogen has been shown to be receptor-mediated. A potential receptor protein, identified in membrane fractions of sensitive cells, was subjected to tryptic hydrolysis followed by amino-acid analysis. The peptides obtained were used to isolate a cDNA fragment by reverse PCR, which showed 30% sequence homology to the human HLA protein. Analysis of other toxins secreted by U. maydis, KP1 and KP4, have demonstrated that, unlike KP6, they are composed of a single polypeptide. Finally, KP6 has been expressed in transgenic tobacco plants, indicating that accurate processing by Kex2p-like activity occurs in plants as well. Using tobacco as a model system, we determined that active antifungal toxins can be synthesized and targeted to the outside of transgenic plant cells. If this methodology can be applied to other agronomically crop species, then U. maydis toxins may provide a novel means for biological control of pathogenic fungi.
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Dolja, Valerian V., Amit Gal-On, and Victor Gaba. Suppression of Potyvirus Infection by a Closterovirus Protein. United States Department of Agriculture, March 2002. http://dx.doi.org/10.32747/2002.7580682.bard.
Full textAbstract:
The plant virus family Polyviridae is the largest and most destructive of all plant viruses. Despite the continuous effort to develop resistant plant varieties, there is a desperate need for novel approaches conferring wide-range potyvirus resistance. Based on experiments with the tobacco etch potyvirus (TEV)-derived gene expression vector, we suggested approach for screening of the candidate resistance genes. This approach relies on insertion of the genes into a virus vector and evaluation of the phenotypes of the resulting recombinant viruses. The genes which suppress infection by the recombinant virus are selected as candidates for engineering transgenic resistance. Our analysis of the TEV variants expressing proteins of the beet yellows closterovirus (BYV) revealed that one of those, the leader proteinase (L-Pro), strongly and specifically interfered with the hybrid TEV infection. Since closterovirus L-Pro is evolutionary related to potyviral helper component-proteinase (HC-Pro), we suggested that the L-Pro interfered with HC-Pro function via a trans-dominant inhibitory effect. Based on these findings, we proposed to test two major hypotheses. First, we suggested that L-Pro-mediated suppression of potyvirus infection is a general phenomenon effective against a range of potyviruses. The second hypothesis stated that the suppression effect can be reproduced in transgenic plants expressing L-Pro, and can be utilized for generation of resistance to potyviruses. In accord with these hypotheses, we developed two original objectives of our proposal: A) to determine the range of the closterovirus-derived suppression of potyviral infection, and B) to try and utilize the L-Pro-mediated suppression for the development of transgenic resistance to potyviruses. In the first phase of the project, we have developed all major tools and technologies required for successful completion of the proposed research. These included TEV and ZYMV vectors engineered to express several closteroviral L-Pro variants, and generation of the large collection of transgenic plants. To our satisfaction, characterization of the infection phenotypes exhibited by chimeric TEV and ZYMV variants confirmed our first hypothesis. For instance, similar to TEV-L- Pro(BYV) chimera, ZYMV-L-Pro(LIYV) chimera was debilitated in its systemic spread. In contrast, ZYMV-GUS chimera (positive control) was competent in establishing vigorous systemic infection. These and other results with chimeric viruses indicated that several closteroviral proteinases inhibit long-distance movement of the potyviruses upon co-expression in infected plants. In order to complete the second objective, we have generated ~90 tobacco lines transformed with closteroviral L-Pro variants, as well as ~100 lines transformed with BYV Hsp70-homolog (Hsp70h; a negative control). The presence and expression of the trans gene in each line was initially confirmed using RT-PCR and RNA preparations isolated from plants. However, since detection of the trans gene-specific RNA can not guarantee production of the corresponding protein, we have also generated L-Pro- and Hsp70h-specific antisera using corresponding synthetic peptides. These antisera allowed us to confirm that the transgenic plant lines produced detectable, although highly variable levels of the closterovirus antigens. In a final phase of the project, we tested susceptibility of the transgenic lines to TEV infection. To this end, we determined that the minimal dilution of the TEV inoculum that is still capable of infecting 100% of nontransgenic plants was 1:20, and used 10 plants per line (in total, ~2,000 plants). Unfortunately, none of the lines exhibited statistically significant reduction in susceptibility. Although discouraging, this outcome prompted us to expand our experimental plan and conduct additional experiments. Our aim was to test if closteroviral proteinases are capable of functioning in trans. We have developed agroinfection protocol for BYV, and tested if co- expression of the L-Pro is capable of rescuing corresponding null-mutant. The clear-cut, negative results of these experiments demonstrated that L-Pro acts only in cis, thus explaining the lack of resistance in our transgenic plants. We have also characterized a collection of the L-Pro alanine- scanning mutants and found direct genetic evidence of the requirement for L-Pro in virus systemic spread. To conclude, our research supported by BARD confirmed one but not another of our original hypotheses. Moreover, it provided an important insight into functional specialization of the viral proteinases and generated set of tools and data with which we will be able to address the molecular mechanisms by which these proteins provide a variety of critical functions during virus life cycle.