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

Plant hormones are responsible for plant growth and adaptation to the environment. Among them the most important are cytokinins. Plants undergo gene silencing processes called homology-dependent gene silencing processes. In Arabidopsis there are two homology-dependent gene silencing genes that were chosen for further study, namely AtHOG1 and AtHOG2. Transgenic plants were generated previously with ten different constructs containing AtHOG1 or AtHOG2 genes and were used in this study. Some of the constructs had GFP attached so that the protein expressed could be visualised in a confocal microscope. Transgenic plants generated were T1 and T2 generations. Their DNA was extracted from leaves. By means of PCR transgenic plants were identified. There were 147 samples. Among them there were 39 positiveswith BAR primers and 32 positives with construct specific primers. The localisation of the HOG2 protein was observed in a confocal microscope. Seeds used were T3 generation and were obtained from the lab. HOG2 protein was found to be localised in cell membrane, root tip and chloroplasts.

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. Y&uuml / 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.

Thesis (Ph. D.)--West Virginia University, 2007. / Title from document title page. Document formatted into pages; contains viii, 143 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Thesis (Ph. D.)--Michigan State University. Dept. of Plant Biology, 2008. / Title from PDF t.p. (viewed on Mar. 27, 2009). Includes bibliographical references (p. 120-135). Also issued in print.

Thesis (M.S.)--Oregon State University, 2008. / Printout. Includes bibliographical references. Also available on the World Wide Web.

Missense single-nucleotide polymorphisms (mSNPs) in titin are emerging as a main causative factor of heart failure. However, distinguishing between benign and disease-causing mSNPs is a substantial challenge. Here, we research the question of whether a single mSNP in a generic domain of titin can affect heart function as a whole and, if so, how. For this, we studied the mSNP T2850I, seemingly linked to arrhythmogenic right ventricular cardiomyopathy (ARVC). We used structural biology, computational simulations and transgenic muscle in vivo methods to track the effect of the mutation from the molecular to the organismal level. The data show that the T2850I exchange is compatible with the domain three-dimensional fold, but that it strongly destabilizes it. Further, it induces a change in the conformational dynamics of the titin chain that alters its reactivity, causing the formation of aberrant interactions in the sarcomere. Echocardiography of knock-in mice indicated a mild diastolic dysfunction arising from increased myocardial stiffness. In conclusion, our data provide evidence that single mSNPs in titin's I-band can alter overall muscle behaviour. Our suggested mechanisms of disease are the development of non-native sarcomeric interactions and titin instability leading to a reduced I-band compliance. However, understanding the T2850I-induced ARVC pathology mechanistically remains a complex problem and will require a deeper understanding of the sarcomeric context of the titin region affected.

Abstract not available

No description available.

Thesis (PhD)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: The aim of this study was toe stablish a reliable protocol for the production 0 f transgenic pearl millet as this will open new avenues for augmenting the gene pool of this crop. This was achieved by identifying a highly regenerabie genotype and optimisation of a tissue culture system, and biolistic protocol f or stable integration of selected transgenes. Both a negative, herbicide resistance selectable marker gene, bar, and a positive selectable marker gene, manA, were individually introduced in order to identify and establish a reliable transformation protocol. The optimised transformation protocol was then used to introduce an antifungal gene in the genome of pearl millet to enhance resistance to the biotrophic fungus Sclerospora graminicola. S. graminicola, an obligate oomycetous fungal phytopathogen, is the causal agent of downy mildew in pearl millet plants and a major constraint in the production of pearl millet. A single component of antifungal resistance was introduced into the genome of pearl millet, as preliminary work towards determining its role in the total plant defence system. The approach chosen was to introduce a hydrolytic enzyme, 13-1,3- glucanase, from Trichoderma atroviride (formerly T. harzianum), a soil-borne filamentous fungus, capable of parasitizing several plant pathogenic fungi. It was anticipated that introducing this glucanase gene from T. atroviride which degrades glucan in the fungal cell walls, would significantly contribute to the improvement of resistance against downy mildew. Constructs were prepared containing the gene (gluc78) encoding a 78 kDa beta-1,3- glucanase. The constructs were prepared containing the gluc78 gene driven either by a strong constitutive promoter (ubiquitin promoter, exon and intron) or a wound inducible promoter, the potato proteinase inhibitor ilK gene promoter. The wound inducible promoter includes either an AMV leader' sequence or the rice Act1 intron to obtain higher expression levels in the monocotyledonous plant. The transformation efficiency using the particle inflow gun and the herbicide resistance gene, bar, was improved from 0.02% on a MS based medium, to 0.19 or 0.72% with manA as selectable marker gene on MS or L3 based medium, respectively. However, individual experiments, introducing manA as selectable marker gene, resulted in frequencies of 1.2 and 3%. This translated to one transformation event per plate, which contains on average 31-35 pre-cultured immature zygotic embryos. This is the first report of t he successful introduction and expression of a 13-1,3-glucanase encoding gene from a biocontrol fungus not only under constitutive expression but also under wound inducible expression in a plant. Optimisation of genetic engineering of pearl millet, a cereal crop recalcitrant to transformation, and the introduction of an antifungal transgene, was accomplished in this study. Initial results hint that expression of this transgene enhances resistance to S. graminicola. / AFRIKAANSE OPSOMMING: Die doel van die studie was om 'n betroubare genetiese transformeringsprotokol vir pêrel manna te ontwikkel. Hiervoor moes eerstens 'n regenereerbare genotipe geidentifiseer word. Twedens moes 'n betroubare weefselkultuur en biolistiese transformeringssisteem ontwikkel word. Beide die onkruiddoder bestandheidsgeen, bar, en 'n positiewe selektiewe geen, manA, is vir die doel van die projek onafhanklik in die genoom van pêrel manna in gekloneer. Die optimale sisteem is vervolgens aangewend om 'n geen wat potensieël verbeterde bestandheid teen die biotrofiese swam Sclerospora graminicola wat donsige meeldou by plante veroorsaak, in pêrel manna in te kloneer. 'n Enkele komponent van bestandheid is in die genetiese material van pêrel manna in gekloneer as inleidende werk om die rol van hierdie geen in die totale verdedigingsisteem te bepaal. Die benadering wat gekies was, behels die klonering van 'n hidrolitiese ensiem 13-1,3-glukanase, van Trichoderma atroviride (voorheen T. harzianum), 'n grondgedraagde swam, wat op 'n aantal ander plantpatogene fungus kan parasiteer. Die verwagting is dat klonering van hierdie 13- 1,3-glukanase geen van T. atroviride wat die glukaan verteer in die selwande van swamme, 'n groot verbetering tot die bestandheid teen donsige meeldou sal meebring. Konstrukte is voorberei wat die gluc78 geen bevat wat kodeer vir die 78 kDa beta-1,3-glukanase protein. Die konstrukte wat voorberei is bevat die gluc78 geen geinduseer deur of 'n sterk konstituwe promoter (ubiquitin promoter, exon en intron) of deur 'n wond geinduseerde promoter, die aartappel proteinase inhibeerder ilK geen promoter. Hierdie promoter word gevolg deur of 'n AMV leier volgorde of die rys Act1 intron om verhoogde uitdruk vlakke in monokotiele plante te verseker. As die partikel invloei geweer in kombinasie met die onkruiddoderbestandheidsgeen gebruik word, was die doeltreffendheid van transformasie 0.02% op 'n MS gebasseerde groeimedium. 'n Transformasie doeltreffendheid van onderskeidelik 0.19 en 0.72% is verkry wanneer die manA as selektiewe geen gebruik is op MS of L3 gebasseerde medium. Twee individual eksperimente, waar die manA geen as selektiewe geen gebruik is, het gelei tot 'n transformasie doeltreffendheid van 1.2 of 3%. Dit gee 'n gemiddelde van een transformasie per plaat wat 31 tot 35 voorafgekweekte onvolwasse embrios bevat. Hierdie is d ie eerste verslag van d ie suksesvolle klonering en uitdrukking van 'n 13-1,3-glukanasekoderende geen van 'n swam wat as 'n biologiese beheeragent gebruik word. Hierdie is nie alleenlik onder konstitutiewe uitdrukking nie, maar ook 0 nder wond g einduseerde u itdruk in' n p lant. In hierdie studie is die 0 ptimisering van genetiese verbetering van pêrel manna, 'n graan gewas wat gehard is teen transformasie, deur die klonering van 'n bestandheidsgeen in die genoom van hierdie gewas gedoen. Aanvanklike resultate dui daarop dat die uitdruk van hierdie geen lei tot verbeterde bestandheid teen S. graminicola.