Dissertations / Theses on the topic 'Transgenic tobacco'

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.

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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Bachelors
Burnett College of Biomedical Sciences
Molecular Biology and Microbiology

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

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

Thesis (MScAgric (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2010.
ENGLISH ABSTRACT: Necrotrophic fungi infect many economically important crop plants. This results in great losses in the agricultural sector world-wide. Understanding the nature by which plants respond to pathogens is imperative for genetically enhancing disease resistance in plants. Research tools have significantly contributed to our understanding of how the plant responds to pathogen attack, identifying an array of defence mechanisms used by plants upon attack. Many fungal pathogens secrete endopolygalacturonases (endoPGs) when infecting plants. These hydrolytic enzymes are inhibited by polygalacturonase-inhibiting proteins (PGIPs) associated with plant cell walls. PGIPs are well characterised and their current known functions are all linked to endoPG inhibition and the subsequent upregulation of plant defence pathways. Work on grapevine PGIPs have shown that apart from being efficient antifungal proteins, leading to protection of the plant against Botrytis cinerea when overexpressed, PGIPs might also have additional functions linked to cell wall strengthening. This working hypothesis formed the motivation of this study where a cinnamyl alcohol dehydrogenase (CAD) (1.1.1.195) gene was targeted for functional analysis in tobacco (Nicotiana tabacum). Some previous work and genetic resources obtained is relevant to this study, specifically previously characterized transgenic tobacco lines overexpressing the Vitis vinifera pgip1 (Vvpgip1) gene. These lines have confirmed PGIP-specific resistance phenotypes against B. cinerea, as well as increased levels of CAD transcripts in healthy plants. Moreover, preliminary evaluations indicated increased lignin levels as well as differential expression of several other cell wall genes in these overexpressing lines (in the absence of infections). In this study we generated a transgenic tobacco population, overexpressing the native CAD14 gene, via Agrobacterium transformations. The transgene was overexpressed with the Cauliflower Mosaic Virus promoter (CaMV 35Sp). The CAD transgenic population was analyzed for transgene integration and expression and showed active transcription, even from leaves that normally don’t express CAD to high levels. These lines, together with the untransformed control, and a representative transgenic VvPGIP1 tobacco line previously characterized with elevated expression of CAD were used for all further analyses, specifically CAD activity assays of stems and leaves, as well as whole plant infections with B. cinerea. CAD enzyme activity assays were performed on healthy uninfected plant lines, without inducing native CAD expression or resistance phenotypes (i.e. without Botrytis infection). CAD activity was detected in leaves and stems, but a statistically sound separation between the CAD population and the untransformed control was only observed in the stems. The CAD assays also confirmed previous results that indicated that CAD transcription was upregulated in the PGIP line in the absence of infection. Overall, in all plant lines the stems exhibited 10-fold higher levels of CAD activity than the leaves, but the transgenic VvPGIP1 line showed a further 2-3-fold increase in CAD activity in the stems, when compared to the untransformed control and the majority of the CAD overexpressing lines. Disease assessment by whole plant infections with B. cinerea of the CAD transgenic plants revealed reduced disease susceptibility towards this pathogen. A reduction in disease susceptibility of 20 – 40% (based on lesion sizes) was observed for a homologous group of transgenic lines that was statistically clearly separated from the untransformed control plants following infection with Botrytis over an 11-day-period. The VvPGIP1 transgenic line displayed the strongest resistance phenotype, with reduction in susceptibility of 47%. The reduction in plant tissue maceration and lesion expansion was most pronounced in the VvPGIP1 line compared to the CAD transgenic plants, while the CAD transgenic plants showed more reduction than the untransformed control. In combination, the data confirms that CAD upregulation could lead to resistance phenotypes. Relating this data back to the previously observed upregulation of CAD in the VvPGIP1-overexpressing lines, the findings from this study corroborates that increased CAD activity contributes to the observed resistance phenotypes, possibility by strengthening the cell wall. In conclusion, this study yielded a characterized transgenic population overexpressing the CAD14 gene; this overexpression contributed to increased RNA transcription compared to the untransformed control plant, increased CAD activity (most notably in the stems) and a disease resistance phenotype against Botrytis. These findings corroborates the current working hypothesis in our group that PGIPs might have a role in preparing the plant cell for attack by contributing to specific cell wall changes. The exact mechanisms are still currently unknown and under investigation. The transgenic lines generated in this study will be invaluable in the subsequent analyses where these various phenotypes will be subjected to profiling and accurate cell wall analyses.
AFRIKAANSE OPSOMMING: Nekrotrofiese swamme infekteer en beskadig verskeie ekonomies belangrike gewasse. Dit lei wêreldwyd tot groot verliese vir die landbousektor. Dit is noodsaaklik om te verstaan hoe plante reageer teenoor patogene, sodat die siekteweerstand van plante verbeter kan word. Navorsingshulpbronne het beduidend bygedra tot die kennis van plantreaksies tydens patogeniese aanvalle, en het sodoende ‘n reeks van weerstandmeganismes, wat die plant inspan tydens ‘n aanval, geïdentifiseer. Verskeie patogeniese swamme skei endopoligalakturonases (endoPGs) af tydens plantinfeksie. Hierdie hidrolitiese ensieme word geïnhibeer deur poligalakturonase-inhiberende proteïene (PGIPs) wat met die plantselwand geassosieerd is. PGIPs is goed gekarakteriseerd en al hulle huidiglik bekende funksies is gekoppel aan endoPG inhibisie en die daaropvolgende opregulering van plant weerstandspaaie. Navorsing op wingerd PGIPs het gewys dat, afgesien van die feit dat PGIPs goeie antifungiese proteïene is wat lei tot beskerming van die plant teen Botrytis cinerea wanneer dit ooruitgedruk word, PGIPs ook moontlik addisionele funksies verrig wat verwant is aan selwandversterking. Hierdie werkshipotese vorm die motivering vir hierdie studie waarin ‘n sinnamiel alkohol dehidrogenase (SAD) (1.1.1.195) geen geteiken is vir funksionele analise in tabak (Nicotiana tabacum). Vorige navorsing en genetiese hulpbronne daardeur verkry is belangrik vir hierdie studie, spesifiek die gekarakteriseerde transgeniese tabaklyne wat die Vitis vinifera pgip1 (Vvpgip1) geen ooruitdruk. Hierdie lyne besit bevestigde PGIP-spesifieke weerstandsfenotipes teen B. cinerea, sowel as hoër vlakke van SAD transkripte in gesonde plante. Voorlopige analises het ook aangedui dat hierdie ooruitdrukkende lyne hoër lignien vlakke het, asook differensiële uitdrukking van verskeie ander selwandgene (in die afwesigheid van infeksie). In hierdie studie is ‘n transgeniese tabakpopulasie gegenereer wat die natuurlike tabak SAD14 geen ooruitdruk, deur middel van Agrobacterium transformasie. Die transgeen is ooruitgedruk deur die Blomkool Mosaïek Virus promoter (CaMV 35Sp). Die SAD transgeniese populasie is geanaliseer vir transgeen integrasie en uitdrukking en het aktiewe transkriptering getoon, selfs in blare waar daar normaalweg nie hoë vlakke van SAD uitgedruk word nie. Hierdie lyne, die ongetransformeerde wilde-tipe kontrole sowel as ’n verteenwoordigende transgeniese VvPGIP1 tabaklyn wat vooraf gekarakteriseerd was met hoë SAD uitdrukking, is gebruik vir alle verdere analises, spesifiek SAD aktiwiteitstoetse in stingels en blare, asook heelplantinfeksies met B. cinerea. Aktiwiteitsanalises van die SAD ensiem is gedoen op gesonde ongeinfekteerde plantlyne, sonder om natuurlike tabak SAD uitdrukking of weerstandsfenotipes te induseer (dus sonder Botrytis infeksie). SAD aktiwiteit is waargeneem in beide die blare en stingels, maar ‘n statisties betekenisvolle skeiding is slegs gevind tussen die SAD populasie en die ongetransformeerde kontrole in die stingels. Die SAD toetse het ook vorige resultate bevestig wat aangedui het dat SAD transkripsie opgereguleer word in die PGIP lyn in die afwesigheid van infeksie. Die stingels het oor die algemeen ‘n 10-voudige vermeerdering in SAD aktiwiteitsvlakke getoon in vergelyking met die blare, maar die transgeniese VvPGIP1 lyn het ‘n verdere 2-3-voudige verhoging in SAD aktiwiteit gehad in die stingels ,in vergelyking met die ongetransformeerde kontrole en die meerderheid van die SADooruitdrukkende lyne. Siekteweerstand ondersoeke deur middel van heelplantinfeksies met B. cinerea van die SAD transgeniese plante, het verminderde vatbaarheid aangedui ten opsigte van hierdie patogeen. ‘n Afname in siekte-vatbaarheid van 20 – 40% (gebaseer op wondgroottes) is waargeneem vir ‘n homoloë groep transgeniese lyne wat statisties betekenisvol geskei kon word van die ongetransformeerde kontrole plante na infeksie met Botrytis in ‘n infeksietoets wat 11 dae geduur het. Die VvPGIP1 transgeniese lyn het die mees weerstandbiedende fenotipe gehad, met ‘n afname in siekte-vatbaarheid van 47%. Die afname in plantweefselafbreking en wondgrootte was die duidelikste in die VvPGIP1 lyn in vergelyking met die SAD transgeniese plante, terwyl die SAD transgeniese plante ‘n groter afname aangedui het as die ongetransformeerde kontrole. In kombinasie het die data bevestig dat SAD opregulasie kan lei tot weerstandbiedende fenotipes. Hierdie data, in vergelyking met die vorige bevinding van opregulasie van SAD in die VvPGIP1-ooruitdrukkende lyne, bevestig dat hoër SAD aktiwiteit bydra tot die waargenome weerstandbiedende fenotipes, moontlik deur versterking van die plantselwand. Ter afsluiting, hierdie studie het ‘n gekarakteriseerde transgeniese populasie wat die SAD14 geen ooruitdruk gelewer; hierdie ooruitdrukking het bygedra tot hoër RNA transkripsie in vergelyking met die kontrole, verhoogde SAD aktiwiteit (veral in die stingels) en siekteweerstandbiedende fenotipes teenoor Botrytis. Hierdie bevindinge ondersteun die huidige werkshipotese in ons groep dat PGIPs moontlik ‘n rol speel in die voorbereiding van die plantsel teen infeksie deur spesifieke selwandveranderinge te veroorsaak. Die spesifieke meganismes is steeds onbekend en word verder ondersoek. Die transgeniese lyne wat tydens hierdie studie gegenereer is, sal baie belangrik wees in opvolgende analises waar hierdie verskillende fenotipes gebruik kan word om die profiel van selwandkomponente, maar ook die akkurate selwandsamestelling te bestudeer.

Thesis (Ph. D.)--University of Florida, 2001.
Title from first page of PDF file. Document formatted into pages; contains x, 99 p.; also contains graphics. Vita. Includes bibliographical references (p. 81-98).

The cellular prion protein (PrPC) is a glycoprotein with unknown function constitutively expressed in mammalian neurons. PrPC converts to a pathogenic misfolded isomer (PrPSc) through a poorly understood process, resulting in a group of fatal neurodegenerative diseases collectively known as transmissible spongiform encephalopathy or prion disease. Elucidating the molecular mechanisms behind prion conversion requires production of PrPC in recombinant systems. This study was designed to generate transgenic tobacco plants expressing recombinant mouse prion protein (mPrP). Using a synthetic gene encoding the mouse prion protein, plant expression vectors were constructed for constitutive mPrP expression in the apoplast (pGreen35SmPrP-Apo), cytosol (pGreen35SmPrP-Cyto) and endoplasmic reticulum (pGreen35SmPrP-ER). Putative transgenic plants transformed with either pGreen35SmPrP-Cyto or pGreen35SmPrP-ER were analysed by PCR, ELISA and immunoblot for transgene integration and expression. However, no viable plants were obtained from the pGreen35SmPrP-Apo transformants. ELISA analysis showed that recombinant mPrP accumulated up to 0.0024% of total soluble leaf protein in transgenic tobacco leaves transformed with the pGreen35SmPrP-Cyto construct and 0.0016% of total soluble leaf protein in plants designed to sequester recombinant mPrP to the ER. Furthermore, immunoblot analysis showed that ER-targeted recombinant mPrP was mainly unglycosylated, although a glycosylated mPrP isoform was observed indicating that transgenic tobacco plants process ER-targeted recombinant mPrP in a manner analogous to mammalian systems. The nutrient composition of several transgenic plants were analysed to determine the phenotypic effect of expressing recombinant mPrP in tobacco plants. The analysis revealed that transgenic lines expressing cytosolic-mPrP had elevated average levels of Mn2+ and Fe2+. In addition, kanamycin-treated transgenic plants expressing cytosolic-mPrP developed a non-rooting phenotype. Conversely, the average Cu2+ level was increased in analysed transgenic plants designed to sequester recombinant mPrP in the ER. Furthermore, the plants developed no visible phenotype upon kanamycin treatment. This result support studies that suggest that the PrPC has functional role in metal homeostasis and loss of its thermodynamic structure leads to metal dyshomeostasis which in turn has been linked to prion disease associated neurotoxicity. Finally, the recombinant mPrP was purified via affinity chromatography facilitated by the presence of a C-terminal polyhistidine tag on the synthetic gene constructs.

From a cDNA library of potato tuber, cDNAs for the potato carboxypeptidase inhibitors were isolated and characterised by DNA sequencing. One full length clone of each type was used to make plant expression constructs, and these constructs used to transform tobacco. An insect bioassay, conducted using the self crossed progeny from the highest expressing transgenic line, revealed that the expression of potato carboxypeptidase inhibitor in tobacco increased the susceptibility of the plants to attack by Heliothm virescens .A cDNA library of whole Manduca sexta larvae was constructed in XZAP II. Oligos were designed to fit the strongly conserved region of insect haemolymph Q-ypsin inhibitor protein sequences and to a region of published protein sequence from Manduca haemolymph trypsin inhibitor A ( MHTl A ) and these were used to PGR a fragment of a MHTI A cDNA. This fragment was used to screen the cDNA library and a number of clones for MHTI A were isolated, along with cDNAs for a previously unknown related protein. These cDNAs were characterised by DNA sequencing. One of the MHTI A cDNAs was used to make plant and E. coli expression constructs and these were sent, for subsequent bioassays of the resultant transgenic plants and of bacterially expressed protein, to Horticulture Research International. While these assays were seriously flawed, there were strong indications from both the plant and the artificial diet bioassays that MHTI expression did enhance insect resistance. A cDNA library of whole Diabrotica undecimpunctata larvae was constructed in XZAP II. Oligos were designed to fit each of the three strongly conserved regions of protein sequence of mammalian and nematode microsomal aminopeptidases. These were used to PGR fragments from both Manduca and Diabrotica cDNA templates. These PGR products were characterised by DNA sequencing and the Diabrotica PGR products used to screen the cDNA library. Two cDNAs were isolated, neither of which were full length, but which were of sufficient length for protein expressed from them to be likely to be functional as an aminopeptidase. E. coli expression constructs were made from each cDNA and bacterial expression was demonstrated. Pilot work on the feasibility of using antibodies as anti-insect proteins was conducted and the antibodies shown to be reasonably resistant to Diabrotica gut proteases. It was also demonstrated that antibodies could be produced that were active at the extreme pHs (3.5- 11) found in insect guts. While many questions have been left unanswered, this project has successfully demonstrated the viability of such novel approaches to the enhancement of insect resistance in plants by genetic engineering.

Thesis (Ph. D.)--Ohio State University, 2005.
Title from first page of PDF file. Document formatted into pages; contains xv, 104 p.; also includes graphics (some col.). Includes bibliographical references (p. 93-104). Available online via OhioLINK's ETD Center

The seed storage proteins of oat are mainly represented by the globulins (75%) and the prolamins (10%). These proteins are only found in the endosperm and accumulate within protein bodies during the maturation of the oat seeds. Three genomic sequences encoding globulin polypeptides and one avenin genomic sequence encoding an oat prolamin (avenin) have been isolated and characterized. The respective promoters of these genomic clones were fused to the coding sequence of the $\beta$-glucuronidase reporter gene (GUS). These constructs, together with the entire sequence of a globulin gene, were transferred into tobacco via Agrobacterium tumefaciens. Analysis of the transgenic tobacco seeds showed for the first time that the promoters of the oat globulin and avenin genes are able to regulate the expression of the GUS sequence in an endosperm-specific and developmentally controlled manner in a dicot plant, as in oat seeds with the original seed storage protein gene. One of the globulin promoters was shown to be probably inactive, whereas two other promoters appear to direct strong expression in the seeds of transgenic tobacco plants. A deletion analysis on one of the functional promoters demonstrated that a portion of the promoter upstream of nucleotide -259 (relative to the start of transcription as determined in oat) was required for expression. Sequence analysis of the globulin promoters showed the lack of conserved elements which are found in other storage protein gene promoters, and believed to play an important role in the regulation of seed storage protein genes. It was nonetheless demonstrated in this study that the absence of such elements did not prevent a correct functionality of the oat globulin promoters in transgenic tobacco seeds. The avenin promoter, when fused to the GUS sequence, also showed strong expression in the endosperm of transgenic tobacco seeds. Sequence analysis of the upstream region of the avenin gene confirmed the presence of a highly conserved 'prolamin box'. The possible role of this element was therefore further demonstrated in this work. This work has also showed for the first time a difference in the choice of transcriptional start sites of two monocot (oat) seed storage protein genes after transfer into a dicot species (tobacco).

An extensive investigation into and charactaisation of factors influencing transgene expression following introduction of the transgoie into tobacco via Agrobacterium- mediated transformation was carried out. Characterisation of material supplied at the outset of this project revealed that this material was unacceptable for further analysis. It was thus deemed necessary to obtain large populations of transgenic tobacco heterogenous for levels of transgene expression. Characterisation of these populations (CaMV-lecA and ssRubisco-lecA plants) showed that all plants fell into one of four segregation classes based on segregation of the kanamycin-resistance selectable marker. Results showed that the majority of regenerants contained multiple nptII-containing inserts, while the presence of one or two such inserts was also found, albeit at a much lower frequency. Segregation analysis based on detection of the lecA transgene agreed, in the majority of cases, with these results. However, in a few cases it was found that data obtained from both segregation analyses did not agree, with the presence of a single lecA-containing transgene being detected in plants shown to contain two copies of the nptII-contaning transgene. This result indicates the occurrence of T-DNA rearrangement either within the tobacco genome or during T-DNA transfer and integration. Southern blot analyses allowed a detailed characterisation of T-DNA structure, copy number and number of integration sites to be undertaken. Results from these analyses revealed a higher frequency of T-DNA rearrangement within plants containing multiple inserts. However, such rearrangements did not correlate with a significant reduction in levels of transgene expression since all detected rearrangements were found to occur at or towards the left hand border of the T-DNA, that border distant to the lecA transgene. Plants containing more than one T-DNA were also frequently found to contain these T-DNAs arranged as an inverted repeat at a single locus although no significant relationship between copy number and the presence of such structures was found. Correlating transgene expression levels, as determined by radioimmunoassay-based quantitation of lectin protein in tissues of transgenic plants, with T-DNA copy number, organisation and structure revealed no significant relationship. It is thus feasible to conclude that the major contributory factor influencing levels of transgene expression is the location of T-DNA integration within the plant genome. Subsequent work concerned with investigating the nature of those integration site-specific factors i.e. 'position effect' indicated a possible role for methylation-induced modulation of gene expression. Results presented in this thesis provide an insight into the fate of transgenes following introduction into the plant genome and clearly demonstrate the importance of further exploring the molecular mechanisms underlying transgene expression variability.

Protein degradation poses a significant challenge for the efficient production of recombinant proteins in plants, affecting the stability and yield of the recombinant protein. In this study the E. coli-derived enzyme glutathione reductase (GR) was transiently expressed in transgenic tobacco plants constitutively expressing the cysteine protease inhibitor OC-I and non-transgenic plants. A protein resembling the GR was detected in infiltrated leaves. Transiently expressing GR in transgenic N tabacum plants resulted in almost two fold significant increases in GR activity. Transgenic tobacco plants expressing the rice cysteine protease inhibitor OC-I had significantly lower cysteine protease activity when compared to non-transgenic tobacco plants. Lower cysteine protease activity in transgenic plants was directly related to higher GR activity and also higher GR amounts in transgenic plants. The study has demonstrated that OC-I is an effective companion protease inhibitor candidate with the potential to protect other high value proteins such as GR, from cysteine protease degradation.
Dissertation (MSc)--University of Pretoria, 2012.
Plant Science
unrestricted

Monoterpene secoiridoid biosynthesis contributes the terpene component of terpenoid indole alkaloids produced in the medicinal plant Catharanthus roseus. The thesis focuses on the development of fundamental knowledge and methodology to reconstruct the monoterpene secoiridoid pathway in tobacco plants using metabolic engineering. The thrust of the thesis concentrated on remaining challenges to engineer complex secondary metabolic pathways in plants. We focused on the early part of the pathway and we recovered a population of transgenic tobacco plants expressing different combinations of transgenes. Analysis at metabolomic and transcriptomic levels provided novel insights into remaining bottlenecks for the effective engineering of secondary metabolic pathways in plants. The experimental part of the thesis is complemented by the development of a systematic patent search to create an IP database with all relevant patents related with the engineering of terpenoid indole alkaloid pathway in plants.
La biosíntesi dels monoterpens secoiridoïdes aporta el compost terpè dels terpens índole alcaloides produïts per la planta medicinal Catharanthus roseus. La tesi es centra en el desenvolupament dels coneixements fonamentals i la metodologia per reconstruir la ruta metabòlica dels monoterpens secoiridoïdes en plantes de tabac mitjançant enginyeria metabòlica. La idea central de la tesi enfoca els reptes pendents de l'enginyeria de rutes metabòliques complexes en plantes. Centrant-nos en la primera part de la ruta, es va regenerar una població de plantes de tabac transgèniques expressant diferents combinacions de transgens. L'anàlisi a nivell metabolòmic i transcriptòmic ens proporciona nous coneixements per resoldre les dificultats que encara hi ha en l’enginyeria efectiva de les rutes metabòliques secundàries a les plantes. La part experimental de la tesi es complementa amb el desenvolupament d'una recerca sistemàtica de patents, per crear una base de dades de IP, amb totes les patents pertinents relacionades amb l'enginyeria de la ruta metabòlica dels terpens índole alcaloides en plantes.
La biosíntesis de monoterpenos secoiridoides aporta el compuesto terpeno de los terpenos índole alcaloides producidos por la planta medicinal Catharanthus roseus. La tesis se centra en el desarrollo de los conocimientos fundamentales i la metodología para reconstruir la ruta metabólica de los monoterpenos secoiridoides en plantas de tabaco mediante ingeniería metabólica. La idea central de la tesis enfoca en los retos pendientes de la ingeniería de rutas metabólicas complejas en plantas. Centrándonos en la primera parte de la ruta, regeneramos una población de plantas de tabaco transgénicas expresando diferentes combinaciones de transgenes. El análisis a nivel metabolómico y transcriptómico nos proporciona nuevos conocimientos para resolver las dificultades que todavía encuentra la ingeniería efectiva de las rutas metabólicas secundarias en las plantas. La parte experimental de la tesis se complementa con el desarrollo de una búsqueda sistemática de patentes, para crear una base de datos de IP, con todas las patentes pertinentes relacionadas con la ingeniería de la ruta metabólica de los terpenos índole alcaloides en plantas.

Relaxin is a small peptide hormone that has demonstrated potential therapeutic actions for cardiovascular disease and fibrosis. Additionally, relaxin has demonstrated the ability to protect the heart from injuries caused by ischemia and reperfusion, promote the healing of ischemic ulcers, and counteract allergic responses. The objective of this research was to express fully processed porcine relaxin in transgenic tobacco plants, as an alternative to current methods of producing relaxin.

Two recombinant relaxin genes were constructed that contained the patatin signal peptide cDNA fused in frame to prorelaxin cDNA, which was codon-optimized for expression in Nicotiana tabacum, under the control of either the â superâ promoter or the dual enhanced cauliflower mosaic virus 35S promoter. Eighteen transgenic tobacco plants were generated that were transformed with the above recombinant genes. Preprorelaxin, mRNA was detected in 12 of the transgenic plants. Fully processed relaxin protein was not found in any tobacco plants that had demonstrated gene expression by northern blot analysis. Preprorelaxin was only identified in extracts from transgenic plants that contained the insoluble protein fraction, as determined by western blot analysis. Additionally, an increased yield of preprorelaxin was identified after incubation of tobacco leaves in an ubiquitin inhibitor.
Master of Science

Human protein C (HPC) is a vitamin-K dependent plasma glycoprotein which is one of the major components regulating anticoagulation. HPC injection is a promising therapy for several diseases but a heterologous production system would be preferred over purifying HPC from human plasma because of its low concentration (4-5 $ mu$g/ml). A cDNA clone coding for HPC was inserted downstream of the CaMV 35S promoter and of a dimer of the CaMV 35S promoter. Tobacco plants were transformed using Agrobacterium and a binary vector strategy. Kanamycin resistant plants were regenerated and enzyme linked immunosorbent assay determined that HPC, in crude plant extracts, accounted for up to 0.03% of plant soluble proteins. HPC was found to be expressed by R$ sb1$ seedlings suggesting successful integration of the T-DNA into plant genome. A partial protein purification system was developed in order to enrich the protein mixture for HPC. HPC was found to bind tightly at pH 6.0 to Fast Flow Q Sepharose resin.

Plastid division, sustained by the equilibrium expression and coordination of plastid division genes is vital for the maintenance of plastid populations in dividing plant cells. Macrochloroplasts (MCP), the occurrence of one or a few chloroplasts per cell is due to the imbalance in the expression of plastid division genes. Because of the MCP size and number it was proposed that they may provide better targets for the plastid transformation than the normal (WT) chloroplasts and result in better plastid transformation frequencies. The objective of this research was to produce transgenic plants containing macrochloroplasts by nuclear transformation and then to use these plants as a model for the development of plastid transformation of crop species. By using AtFtsZ1-1 and AtMinD1 as query sequences in the TIGR (U.S.A) and ASTRA (Australia) Brassica oleracea EST databases, this project resulted in the isolation of cauliflower FtsZ1-1 (EU684588) and MinD (EU684589) genes. In addition, AtFtsZ1-1 was used as a control gene for comparison to the cauliflower FtsZ1-1. Binary vectors were constructed to express these genes in tobacco and cauliflower either by Agrobacterium tumefaciens-mediated or PEG-mediated transformation methods. Transgenic tobacco and cauliflower plants with abnormal chloroplasts (MCP, minichloroplasts, honeycomb or doughnut shaped chloroplasts, uneven surface membrane chloroplasts) were developed. Furthermore, the transgenic tobacco and cauliflower plants were examined by PCR, RT-PCR and Southern blotting. In addition, th ese plants were also analysed for the different abnormal chloroplast phenotypes by fluorescence microscopy. This project also generated the first plastid transformants from macrochloroplast bearing tobacco plants via biolistics. After one round of regeneration homoplasmic plastid transformants were obtained from both WT chloroplast and MCP tobacco plants. The homoplasmic nature of plastid transformants were confirmed by PCR and Southern blotting. Plastid expression of GFP in WT and MCP was confirmed by fluorescence/confocal microscopy and western blot analysis. This project showed for the first time the characterisation of cauliflower FtsZ1-1 and MinD plastid division genes in homologous and heterologous systems (cauliflower and tobacco). Moreover, obtaining homoplasmic plastid transformant shoots from one round of regeneration from the MCP containing tobacco plants is reported for the first time in this study. In addition this study explored the effect of transgene expression level on the chloroplast abnormality, highlighting the importance of analysing transgenic tobacco and cauliflower plants at the protein lev el specifically with regard to plastid division genes. The maintenance of MCP phenotype in the regenerated shoots and the requirement of standardisation of MCP containing plants via biolistics for increasing the plastid transformation frequency were also examined.

Oxalate oxidase is a water soluble, thermolabile, homo-oligomeric glycoprotein the synthesis of which marks the onset of germination In wheat and barley embryos. The protein Is also highly abundant In barley roots. The enzyme has an average oligomer molecular mass of about 115 kDa and about 22.8 kDa for the monomers, as determined by mass spectrometry. The ollgomeric cereal oxalate oxidases are resistant to dissociation In SDS containing media and to digestion by pepsin. The cereal organs produce two oxalate oxidase Isoforms (G and G') which possess the same apoprotein but are differentially glycosylated. The oligosaccharide side chain(s) has a molecular mass of about 2-3 kDa. Barley root also contains a third active oxalate oxidase isoform with a mass of about 22.5 kDa, which was not detected in germinating embryos of the same cultlvar. All of the cereal oxalate oxidases were shown to have identical N-terminal amino acid sequences and almost identical kinetic properties This thesis describes the characterisation of oxalate oxidases Isolated from three transgenic plants lines, expressing chimeric CaMV 35S-oxalate oxidase genes. SGS5 tobacco was expressing a gene with the native oxalate oxidase signal peptide and 3S1 oilseed rape and C26 tobacco were expressing a gene containing a foreign extensin signal peptide. Transgenic SGS5 tobacco produced an oxalate oxidase which was almost indistinguishable from the native cereal protein, in terms of Its structure, stability, enzyme activity and resistance to dissociation In SDS containing media and digestion by pepsin. This work Illustrated the ability of a dicotyledonous plant (tobacco) to recognised and correctly process a transgenic monocotyledon protein (wheat).Transgenic 3S1 oilseed rape and C26 tobacco were shown to produce active oligomeric oxalate oxidases, which did not exhibit any of the unusual resistance properties normally associated with these proteins. Instead the 3S1 and C26 oxalate oxidases were unstable and exhibited significantly altered kinetic properties compared with the native cereal and transgenic SGS5 enzymes. The instability was thought to have arisen from the Incorrect processing of the 3S1 and C26 oxalate oxidases, resulting in the partial cleavage of the extensin signal peptide, which in turn gave rise to a mature oxalate oxidase with an altered N- terminal sequence compared with the native cereal enzyme. The use of vacuum infiltration confirmed the association of the transgenic enzymes with the extracellular spaces, although the majority of the enzyme was shown to be intracellular. The main objective for producing the transgenic oilseed rape expressing oxalate oxidase was to Improve fungal pathogen resistance against oxalic acid secreting pathogens. The results described in this thesis are concerned with a direct comparison of the structure, stability and kinetics between the native cereal and transgenic oxalate oxidases and the possible consequences for pathogen resistance In plants expressing unstable yet active transgenic enzymes.

Biopharmaceutical manufacturing is a rigorous and expensive process. Due to the medicinal nature of the product, a high purity level is required and several expensive purification steps must be utilized. Cost-effective production and purification is essential for any biopharmaceutical product to be successful and development of the fastest, most economical, and highest-yielding purification scheme is a constant engineering challenge. Commercial-scale purification schemes currently revolve around the use of multiple chromatography steps for the purification of biopharmaceutical products. Chromatography has many shortcomings including high cost, limited throughput, and complex scale up. The goal of this research was to develop an alternative, non-chromatography purification step for the separation of an acidic model protein, recombinant β-glucuronidase (rGUS), from transgenic tobacco with high yield and purity. Aqueous two-phase extraction (ATPE) is a powerful technique for separation and purification of proteins, and has the potential to replace an expensive chromatography step for the initial purification of recombinant proteins. ATPE enables high levels of target protein recovery and concentration while removing large amounts of impurities from the initial extract. Fractional factorial designs and response surface methodology were used to determine an optimized aqueous two-phase system for the purification of rGUS from transgenic tobacco. In a 13.4 % (w/w) PEG/18% (w/w) potassium phosphate system, 74% of the rGUS was recovered in the top PEG-rich phase while 90% of the native tobacco proteins were removed in the interphase and the bottom phase. A purification factor of about 20 was achieved in this process.
Master of Science

The cystic fibrosis transmembrane conductance regulator (CFTR) is one of the most studied membrane protein models because of its clear clinical significance. Mutations within the CFTR gene lead to cystic fibrosis, the most common autosomal recessive genetic disorder in the Caucasian population. CFTR, a large 160 kDa glycoprotein, is a chloride ion channel in the ABC superfamily of transporter proteins. Due to low natural abundance of CFTR and difficulties producing sufficient amounts in heterologous systems, the exact protein function/structure relationship is unknown. Expression of CFTR in E. coli is lethal and mammalian culture systems are expensive and low yielding. However, successful bioproduction of many complex human proteins has been shown in transgenic plants. Our research objective is to develop tobacco as a model system for expressing human CFTR. Constructs of full-length CFTR fused to the 35S double enhanced promoter could not be propagated in E. coli, suggesting that the CFTR product generated by â leakyâ expression was detrimental to bacteria. Two strategies were undertaken to address the problem: 1) a plant intron was introduced into CFTR sequence and 2) a more tightly regulated wound-inducible promoter MeGATM was used. Tobacco was transformed with all constructs. CFTR presence was determined by polymerase chain reaction (PCR). Expression and intron splicing was analyzed by reverse transcriptase-PCR. Splicing did not occur presumably due to intron /exon contexts. In tobacco expressing MeGA:CFTR, however, novel high-molecular-weight membrane-associated proteins were immunodetected using anti-CFTR antibodies suggesting that tobacco may be capable of producing human CFTR.
Master of Science

Hemorrhagic enteritis virus (HEV) causes an acute viral disease in turkeys characterized by bloody diarrhea and death. Current live HEV virus vaccines are immunosuppressive and predispose turkeys to secondary bacterial infections. Data indicates that the capsid proteins (fiber, penton base, hexon) of HEV are capable of stimulating protective antibodies against an HEV challenge. Using tobacco as a model, we sought to determine if a plant could be used to synthesize the HEV fiber protein and produce sufficient antigen to stimulate protective antibodies. To introduce the fiber gene into plants, the coding region of the HEV fiber gene was fused to either a constitutive plant promoter (35S) or a wound inducible promoter (hmg2) on plasmids adapted for Agrobacterium-mediated transformation. Approximately sixty transgenic plants of each construct were generated and determined to contain the HEV fiber gene based on amplification of specific HEV DNA sequences by the polymerase chain reaction. Plants were screened by Northern dot blot to identify lines expressing high levels of fiber mRNA. Expression of fiber protein was observed in selected lines of transgenic tobacco by Western blot analysis using turkey anti - HEV serum. The accumulation of fiber protein in leaves of tobacco transformants was quantified by Sandwich ELISA. Fiber protein from these plants has undergoing large - scale purification and concentration for a turkey immunization trials to determine if plant expressed fiber antigen is capable of inducing protective antibodies against HEV in turkeys.
Master of Science

The objective of this study was to transform sweet pepper ferredoxin-like protein (PFLP) gene, which has antimicrobial properties, to tobacco and investigate the disease resistance abilities of transgenic tobacco. This protein interacts with another protein, harpin that is produced by the bacteria which is invading the plant tissues, and stimulates hypersensitivity response in plants, thus the spreading of disease is limited. Gene transfer was achieved to tobacco by Agrobacterium- mediated method and with indirect organogenesis
the explants were grown on selective media and then transferred to jars and pots respectively. Molecular and genetic analyses such as PCR, RT-PCR, Sequence Analysis and Northern Blot, were performed with plants which their seeds survived and grew on selective medium and also gave positive reactions for GUS histochemical assay. Finally, with putative transgenic plants, some hypersensitive response assays were carried out with Pseudomonas syringae and it was observed that the recovered plants showed hypersensitive response (HR) in the preliminary tests. These results indicated that putative transgenic tobacco plants which carry pflp transgene, can be used in disease resistance studies.

As atmospheric concentrations of N2O, a potent greenhouse gas and destructor of ozone, continue to increase, it is important to explore a range of solutions for this issue. The bacterial enzyme responsible for catalyzing the final step of the denitrification pathway, conversion of N 2O to N2, is nitrous oxide reductase (N2OR). It is the aim of this study to produce model tobacco plants expressing the nitrous oxide reductase gene (nosZ) with the ultimate goal of supplying additional N2OR to the plant-microbial-soil environment for more efficient reduction of N2O and mitigation of agricultural N 2O emissions. Two nitrous oxide reductase expression cassettes under the control of (1) the d35S constitutive promoter and, (2) the rolD root-specific promoter were constructed and used to transform Nicotiana tabacum tissue. Integration and expression of the nosZ transgene in T0 and T1 generation plants was demonstrated by PCR and RT-PCR. Production of recombinant N2OR in T0 plants was shown by western immunoblot.

Transketolase is a TPP dependent enzyme that affects the availability of intermediates in both the Calvin cycle and non-oxidative pentose phosphate pathway. Previous studies have indicated that changes to the activity level of transketolase can limit growth and development as well as the production of isoprenoids, starch, amino acids and thiamine. The overall aim of this project was to further advance the understanding of the mechanism linking increased TK activity and thiamine metabolism. Nicotiana tabacum mutants with increased total transketolase activity ~ 2 to 2.5 fold higher than WT plants were shown to have a reduced growth and chlorotic phenotype. In seedlings, these phenotypes were attributed to a reduction in seed thiamine content. Imbibition of TKox seeds in a thiamine solution produced plants that were comparable to WT plants. However, the chlorotic but not growth phenotype was found to return unless the plants underwent irrigation with a thiamine solution indicating that TKox plants are unable to produce sufficient quantities of thiamine to meet demand. Furthermore, the application of deoxy-xylulose-5-phosphate was also found to be able to partially complement the phenotype suggesting that flux from the C3 cycle into the non-mevalonate pathway is being affected. Analysis of thiamine and TPP levels demonstrated that TKox plants were deficient in thiamine but not TPP in the majority of cases. In plants that had begun to flower, TKox lines had reduced thiamine and TPP levels in the 20th fully open leaf compared to the same leaf in WT plants. Furthermore, sampling of leaf tissue from both WT and TKox seedlings at the same developmental stage indicated that high levels of TK protein may lead to the accumulation of TPP in these areas causing a reduction in the levels of thiamine and TPP in the rest of the plant thereby limiting growth and development.

Bibliography: pages 108-128.
Cucumber mosaic virus (CMV) and tobacco necrosis virus (TN V) often occur in mixed virus infections in South Africa. Both viruses are of economic importance because of their world-wide distribution, extensive host range and their effects on yields of agriculturally important crop plants. The complete cDNA sequences of CMV-Wemmershoek (CMV-Wem) coat protein (CP) and TNV-F5P CP genes were cloned and subjected to sequence analysis. CMV-Wem is closely related to CMV-WL and CMV-Q, and therefore falls into CMV subgroup II. Similar analysis showed that TNV-F5P is closely related to TNV-A. By characterizing and sequencing these clones the authenticity of the CMV and TNV CP genes was also determined, prior to sub cloning into the appropriate vectors for expression in E. coli and tobacco. Constructs containing both the full-length CP genes of CMV-Wem and TNV-F5P were subcloned in frame with the malE gene, encoding the maltose binding protein (MBP), in the IPTG-inducible pMALTM vector system, and expressed in E. coli. Through immunological detection the authenticity of both CPs was confirmed. The CMV CP translation product expressed in E.coli was used as an antigen to raise antiserum free from contaminating plant host-specific antibodies. The CP genes of both viruses were individually cloned in both orientations (sense and antisense) in Agrobacterium tumefaciens Ti-plasmid-based binary and cointegrate vectors. The study was then extended to include engineering doubly transgenic plants. In order to determine whether the full-length CP is required to mediate virus resistance, a truncated form of the TNV CP was generated by deleting 83 amino acids from the C-terminus. Transgenic Nicotiana tabacum cv Petit Havana SRl plants containing one of a number of different forms of CMV and TNV CP nucleotide sequence were generated. In whole plant studies, mechanical inoculation of Ro lines with CMV-Wem resulted in more than 50% of the CMV CP-sense (CP+) and CP-antisense plants not developing visible systemic disease symptoms. In both the CMV CP+ and doubly transgenic plants CMV-Wem accumulation was delayed, but virus was found to accumulate in the inoculated leaves over time. The CMV CP+ lines showed excellent protection against CMV-Q, but showed only a delay in symptom production when inoculated with CMV -Y, from subgroup I.

The spread amongst humans of viral diseases such as acquired immunodeficiency syndrome (AIDS), hepatitis and severe acute respiratory syndrome (SARS) is alarming. A plant-based high fidelity production system is being developed with emphasis on producing antigens capable of being orally delivered to humans in plant packets. To test whether transgenic tobacco, carrot and rice plants can correctly process and assemble the hepatitis-B virus (HBV) core particle/antigen (HBcAg), they were transformed with a C-terminal truncated version of the HBcAg subunit coding sequence. Transgenic tobacco, carrot and rice plants processed the HBV subunits accurately indicating that these recombinant expression systems can be extended to produce other proteins at reduced costs. In the wild-type expression construct (H1); the enhanced cauliflower mosaic virus double 35S (CaMV-d35S) promoter was fused to the alfalfa mosaic virus RNA 4 (AMV-RNA4) sequence to achieve greater translation of a C-terminal truncated HBV core particle subunit. A second expression construct (H2) was plant-codon optimized to match the Arabidopsis thaliana plant genome codon preferences. A third codon-optimized expression construct (H3) had a KDEL (lysyl-aspartyl-glutamyl-leucine) encoded sequence. While a fourth expression construct (H4) included an extensin signal sequence in place of the AMV-RNA4 sequence. Western blotting analysis showed the presence of the HBcAg in transgenic tobacco, carrot and rice plants. The HBcAg levels increased from the H1 to the H4 transgenic tobacco lines. Plant codon-optimization of the HBcAg sequence and addition of the KDEL encoded sequence led to higher levels of HBcAg. The most effective modification was observed when the extensin signal sequence replaced the AMV-RNA4 translation enhancer sequence resulting in the highest observed yields of HBcAg in both the leaves and seeds of the best H4 tobacco plant. In edible plants, higher levels of HBcAg were observed in carrot roots as opposed to carrot leaves and in rice seeds as opposed to rice leaves. Further analyses via electron microscopy indicated that the HBV subunits had assembled into virus-like particles of 25--30 nm diameter in all three plant systems. Therefore, these studies may aid in the global quest to develop cheap, safe and effective vaccines.

Soil salinity negatively affects agricultural production in Turkey by decreasing the yield and quality. Direct introduction of stress related genes by genetic engineering is one of the most rapid approaches to develop stress tolerant crops. In this study, TaNHX1 gene was isolated from bread wheat and three different local wheat cultivars were transformed with overexpression vectors containing TaNHX1 gene by using Agrobacterium-mediated and particle bombardment gene transfer techniques. Immature embryo and inflorescence of Triticum durum cv. Kiziltan-91 and Triticum aestivum cv. Yü
regir-89 and mature embryo of Triticum durum cv. Mirzabey-2000 were used as an explant. In this manner, totally 8960 and 5650 explants were used during particle bombardment and Agrobacterium-mediated transformation, respectively. Moreover, leaves of Nicotiana tabacum cv. Petit Havana were transformed by TaSTR gene to develop salt resistant transgenic tobacco plants by using Agrobacterium-mediated transformation. Stable expression and inheritance of the transgenes was confirmed by both genetic and molecular analyses. T1 progeny showed segregation of the transgenes in a typical Mendelian fashion in most of the plants. Expression of TaSTRG in tobacco was evaluated by physiological and biochemical analysis, such as germination test, root length and MDA analysis. In addition to the nuclear transformation, chloroplast transformation of tobacco was performed with Xyl10B gene responsible for the synthesis of hyperthermostable xylanase enzyme. Stable integration of transgenes and homoplasmy were confirmed with PCR and Southern blotting.

The feasibility of producing recombinant (rt) and biologically active granulocyte-macrophage colony stimulating factor (GM-CSF) in the seeds of transgenic tobacco plants was investigated. The rice seed-specific glutelin promoter (Gt1) was used to direct the expression of the human (h) GM-CSF coding sequence in tobacco seeds. Transgenic tobacco plants producing rthGM-CSF were compared in biological assays with tobacco plants expressing a glutelin/rthGM-CSF fusion protein, driven by the Gt3 promoter. The T7 Sequencing kit from Pharmacia was used to sequence and confirm the authenticity of the Gt1 expression construct. The glutelin-1 signal sequence was fused in the correct orientation to the hGM-CSF cDNA. The rthGM-CSF expression cassette (2.5 kb) was subcloned in a plant binary vector pRD400, which contained a kanamycin resistance gene. The pRD400 vector containing the 2.5 kb construct was used to transform Agrobacterium tumefaciens cells. Tobacco (Nicotiana tabacum cv. Xanthi) leaf sections were transformed by A. tumefaciens carrying the complete 2.5 kb construct. (Abstract shortened by UMI.)

Mucosal surfaces are constantly challenged with a variety of microorganisms. The pathogen invasion of mucosal surfaces is initially countered by the innate immune system via recognition of microbe-specific motifs. Of these microbial motifs, lipopolysaccharide (LPS) is known to be one of the most powerful bacterial virulence factors. This thesis is an attempt to understand the roles of the LPS receptor complex at mucosal surfaces and the ways by which it discriminates between commensal and pathogenic Gram-negative bacteria. The LPS receptor components studied were the LPS binding protein (LBP), cluster of differentiation 14 (CD14), toll-like receptor 4 (TLR4) and myeloid differentiation protein 2 (MD-2). In the first section of this thesis, biological analyses of the LPS receptor complex at the tear-corneal interface suggested that the LPS receptor components are strategically and spatially expressed to fine-tune and even restrain their LPS response to invasive pathogenic Gram-negative bacteria. To study its function in the developing gastrointestinal mucosal surface, soluble CD14 in breast milk was tracked in human neonates. The lack of detection of CD14 in stools of breast-fed neonates and the in vitro proteolysis assays suggested that CD14 is likely to survive its gastrointestinal passage in the low bacteria density lumen of the upper digestive system, but is likely to be absent from the LPS-rich environment of the distal gastrointestinal tract. This controlled and limited spatial distribution could be a strategy to prevent an overzealous immune response against the commensal flora of the distal bowel. The results obtained from these two studies concluded that the function and fate of CD14 at mucosal surfaces was dynamic enough to merit further investigations on a much larger scale. For this to happen, recombinant expression systems needed to be first explored. The successful production of human CD14 in transgenic tobacco proved to be a promising source of a stable and active CD14 to further elucidate the mechanisms of the mucosal LPS response system.

The expression of antibody fragments has been suggested as a possible mechanism for the introduction of disease resistance into transgenic plants. Important pathogenicity factors of certain phytopathogens are secreted cell wall degrading enzymes. Pectate lyases and polygalacturonase are important cell wall degrading enzymes secreted by Erwinia carotovora and Botrytis cinerea respectively. The blocking of activities of these enzymes could offer an opportunity for reducing the ingress of these pathogens. Pectate lyase C from E. carotovara was purified to near homogeneity by cation exchange chromatography following overexpression in E.coli. Hybridoma lines recognising this enzyme were obtained and an scFv (single chain variable fragment) isolated from one of these lines. This scFv did not recognise pectate lyase.;The purified enzyme was used to inoculate mice to construct libraries of scFvs derived from spleen mRNA. These libraries did not yield any scFvs recognising pectate lyase. A synthetic human scFv library was then screened with pectate lyase and Botrytis cinerea polygalacturonase (PG), and scFvs recognising PG isolated. These scFvs were tested against a range of antigens to determine specificity. One scFv, C1, was found to bind PG but did not bind to control antigens bovine serum antigen, hen egg lysozyme or pectate lyase. Neither did this scFv bind to PG isolated from another fungal pathogen, Cryphonectria parasistica. scFv C1 was found to bind to a carbohydrate epitope on the B. cinerea polygalacturonase.

Bibliography: pages 80-90.
The aim of this project was to construct a high level plant expression vector from the RNA 3 of cucumber mosaic virus strain Y (CMV Y). The 5'- and 3'-untranslated regions (UTRs) of this genome segment were reverse transcribed, cloned and sequenced. The chloramphenicol acetyl transferase gene (CAT) was inserted between the two UTRs. This artificial viral cDNA (5'cat3') was cloned immediately downstream of the cauliflower mosaic virus 35 S promoter at the transcription initiation site to make a DNA vector. An RNA vector construct was made by placing the 5'cat3' segment under the control of a T7 RNA promoter sequence. In vitro transcripts, as well as linearised DNA vector constructs were inoculated onto CMV infected plants. Inoculated plants were monitored for CAT expression. No CAT could be detected in total protein extracts of inoculated plants. No CAT mRNA could be detected in northern blots of total RNA extracted from inoculated plants. The vector constructed from the 5' - and 3' -UTR of the RNA 3 of CMV Y did not appear to contain all the necessary attributes for a viral expression vector. To study the expression of a foreign antigen in tobacco, the L 1 capsid protein of human papillomavirus type 16 was cloned into Agrobacterium tumefaciens and used to make transgenic Nicotiana tabacum. Kanamycin resistant tobacco plants were shown to carry the L 1 capsid gene using PCR screening, but western blots on total protein extracts of the transformed plants were indeterminate. Further studies are needed to determine whether the antigen is produced and if it is correctly processed.

This thesis examines the size and distribution of benefits from the use of transgenic tobacco as a production vehicle for pharmaceutical proteins. Ex-ante welfare benefits are estimated for the introduction of two biotech innovations. In both cases economic surplus model with imperfect competition is employed to assess the size and distribution of benefits from these alternative uses of tobacco. An introductory chapter presents an overview of the topic followed by chapters 2 and 3 which contain the two case studies. The first paper (chapter 2) examines the case of Human Serum Albumin production from transgenic tobacco. The second paper (chapter 3) examines the case of Glucocerebrosidase Enzyme from transgenic tobacco. Results demonstrate that new products from bio-pharming applications stand to generate significant social benefits. The introduction of Human Serum Albumin generates average annual gains of $46 million and the introduction of Glucocerebrosidase Enzyme generates average annual gains of $500 to $600 million.
Master of Science