Dissertations / Theses on the topic 'Bacterial genetic transformation'

Thesis (Ph.D)--Biology, Georgia Institute of Technology, 2010.
Committee Chair: Patricia Sobecky; Committee Member: Eric Stabb; Committee Member: Jim Spain; Committee Member: Roger Wartell; Committee Member: Thomas DiChristina. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Horizontal gene transfer (HGT) is probably the most important mechanism for functional novelty and adaption in bacteria. However, a robust understanding of the rates of HGT for most bacterial species and the influence of the ecological settings on the rates remain elusive. Four whole-genome comparative studies of free-living bacteria will be described that integrated physiological and ecological data with novel detection bioinformatic pipelines to elucidate the magnitude of HGT at three distinct levels of genetic relatedness: i) the species level, where overlapping ecological niche among co-occurring bacteria in the water column of the Baltic Sea has caused HGT to have been so rampant that it has served as the force of species cohesion; ii) the genus level, where HGT appeared to predominantly mobilize a limited number of genes with ecological/selective advantage (e.g., antibiotic resistance genes) among distinct pathogenic Campylobacter species and hence, did not lead to species convergence; and iii) the phylum level, where HGT was found to be, in general, less frequent than the genus level but, over evolutionary time, has assembled a large part of the metabolic functions of natural microbial communities, especially within organic matter rich, anaerobic habitats. In conclusion, this work advances the methods to link ecological relationships with HGT and suggests that HGT among very divergent organisms may have been more frequent than previously thought and led to successful adaptation.

The bacterial pathogen Vibrio cholerae is responsible for ongoing cholera outbreaks in Haiti and elsewhere. Association of V. cholerae with the human host is responsible for fatal disease, but the bacteria also reside as natural inhabitants of aquatic environments, commonly attaching as biofilms to chitinous surfaces of copepods and crabs. Prior studies in V. cholerae demonstrated that competence for genetic transformation, a mechanism of horizontal gene transfer (HGT), requires the TfoX regulator protein that is triggered by chitin, and the HapR transcription factor that is made in response to quorum sensing (QS) signals produced by V. cholerae and Vibrios. To define regulatory components connecting extracellular signals to natural competence, I first demonstrated that QS molecules produced by Vibrios within multi-species chitinous biofilms are required for DNA uptake by V. cholerae, confirming the critical role of QS signals in HGT. Second, I identified by transposon-mutagenesis a new positive regulator of competence, CytR (cytidine repressor), only studied prior in E. coli as a regulator of nucleoside scavenging. Specific mutations in V. cholerae CytR impaired expression of competence genes and halted DNA uptake; and the addition of exogenous cytidine had similar affects as predicted in E. coli. V. cholerae and other competent Vibrios encode TfoX, HapR, and CytR, although none of these regulators directly controls genes coding for the DNA uptake apparatus. Thus, these results have uncovered a regulatory network, likely used by many Vibrios, that contains additional factors linking several extracellular chemical molecules (cytidine, chitin, and QS signals) to DNA uptake. My study has begun to define a molecular mechanism by which both environment and genetics contribute to genome evolution for this important marine pathogen.

The Wheeler Orchid Collection is home to some of the most endangered species of orchids in the world. This fantastic reservoir of endangered species has been enhanced and broadened by its function as a plant rescue station for the U.S. customs service. Unfortunately, this responsibility increases the risk of bringing orchids, which harbor contageous diseases, into the greenhouse where sap transmitted diseases such as the Tobacco Mosaic Virus (TMV), can run rampant. Although manipulation of orchid characteristics is typically done by classical plant breeding techniques, genetic engineering is emerging as a useful technique for the introduction of desirable traits into the orchid genome. Through the use of genetic engineering techniques it may be possible to mitigate the symptoms associated with this destructive virus. Virus resistance may be achieved through the expression of either the sense or antisense viral coat protein gene in orchid tissues if an efficient means of orchid transformation is developed. In this research two transformation protocols were examined for their ability to efficiently transform orchid tissue. The first transformation protocol explored utilized the native ability of Aq bacterium tumefaciens to incorporate DNA into host plants to achieve transformation. The second mechanism explored was particle bombardment transformation.Many strains of A. tumefaciens were employed using direct exposure of Cattleya_ orchid protocorm and callus tissue. Particle bombardment using DNA coated 0.5 um diameter tungsten particles and high pressure helium tank acceleration was employed. The particle bombardment procedure employed the pG35barB plasmid which confers herbicide resistance to the herbicide basta when integrated and expressed in plant tissues.GUS fluorescence assays and PCR analysis indicate that T-DNA is present in orchid tissues, while Southern blot analysis was unable to display that integration had occurred. Particle bombardment yielded herbicide resistant orchid tissues which have yet to be analyzed by Southern blot analysis to confirm integration due to limited tissue quantities.
Department of Biology

This research was an attempt to use a dicot transformation vector to transform a monocot. The initial purpose of this thesis was to transform orchids with the sense and antisense coat protein genes from the Odontoglossum ringspot virus (ORSV) in an effort to mitigate viral symptoms in transgenic plants using the transformation vector, Agrobacterium tumefaciens. However, it soon became apparent that much time would be needed to develop a transformation protocol. The transformation vectors used included the Agrobacterium tumefaciens disarmed strain LBA4404 with the binary plasmid pB1121, the disarmed strain At699 with the binary plasmid pCNL65, and the wild-type strain Chry5. The marker gene on the binary plasmids of both disarmed strains was p-glucuronidase (GUS).Several transformation protocols were used in an effort to determine if this transformation system would work on orchids. Transformation was not achieved even though a number of experimental conditions were varied. These included using two different types of orchid tissue, callus and protocorms; using two different species of orchids, Cattleya Chocolate Drop x Cattleytonia Kieth Roth and Cymbidium maudidum; varying the time the plant tissue was exposed to the bacteria from 1 hour to 96 hours; performing experiments with and without the wound signal molecule acetosyringone; and exposing the tissue to the virulent strains of A. tumefaciens mentioned previously.This research also developed GUS assay conditions necessary to decrease the number of false positives due to bacterial contamination. These conditions included chloramphenicol in the GUS assay buffer.
Department of Biology

Horizontal gene transfer enables acquisition and dissemination of novel traits including antibiotic resistance and virulence among bacteria. Frequently such traits are gained through the acquisition of clusters of functionally related genes, often referred to as genomic islands (GIs). Quantifying horizontal flow of GIs and assessing their contributions to the emergence and evolution of novel metabolic traits in bacterial organisms are central to understanding the evolution of bacteria in general and the evolution of pathogenicity and antibiotic resistance in particular, a focus of this dissertation study. Methods for GI detection have also evolved with advances in sequencing and bioinformatics, however, comprehensive assessment of these methods has been lacking. This motivated us to assess the performance of current methods for identifying islands on broad datasets of well-characterized bacterial genomes and synthetic genomes, and leverage this information to develop a novel approach that circumvents the limitations of the current state-of-the-art in GI detection. The main findings from our assessment studies were 1) the methods have complementary strengths, 2) a gene-clustering method utilizing codon usage bias as the discriminant criterion, namely, JS-CB, is most efficient in localizing genomic islands, specifically the well-studied SCCmec resistance island in methicillin resistant Staphylococcus aureus (MRSA) genomes, and 3) in general, the bottom up, gene by gene analysis methods, are inherently limited in their ability to decipher large structures such as GIs as single entities within bacterial genomes. We adapted a top-down approach based on recursive segmentation and agglomerative clustering and developed a GI prediction tool, GEMINI, which combined compositional features with segment context information to localize GIs in the Liverpool epidemic strain of Pseudomonas aeruginosa. Application of GEMINI to the genome of P. aeruginosa LESB58 demonstrated its ability to delineate experimentally verified GIs in the LESB58 genome. GEMINI identified several novel islands including pathogenicity islands and revealed the mosaic structure of several LESB58 harbored GIs. A new GI identification approach, CAFE, with broad applicability was developed. CAFE incorporates biological information encoded in a genome within the statistical framework of segmentation and clustering to more robustly localize GIs in the genome. CAFE identifies genomic islands lacking markers by virtue of their association with genomic islands with markers originating from the same source. This is made possible by performing marker enrichment and phyletic pattern analyses within the integrated framework of recursive segmentation and clustering. CAFE compared favorably with frequently used methods for genomic island detection on synthetic test datasets and on a test-set of known islands from 15 well-characterized bacterial species. These tools can be readily adapted for cataloging GIs in just sequenced, yet uncharacterized genomes.

Les systèmes toxine-antitoxine (TA) sont très répandus au sein des génomes bactériens. Ces opérons bicistroniques de petite taille ont été découverts sur des plasmides à bas nombre de copies. Dans ce contexte génétique, les systèmes TA confèrent un avantage sélectif à leurs molécules-hôtes en tuant les bactéries-filles qui ne les ont pas héritées par le mécanisme de tuerie post-ségrégationnelle (PSK, post-segregational killing). Ces systèmes génétiques sont également appelés modules d’addiction étant donné qu’ils rendent la descendance des bactéries qui les contiennent dépendantes de leur présence. Alors que leur rôle dans les molécules d’ADN épisomiques est relativement bien établi, le sens biologique de la présence d’homologues à ces systèmes épisomiques au sein des chromosomes bactériens est sujet à d’intenses débats. L’idée que les systèmes TA chromosomiques confèrent un avantage sélectif a été mise en évidence dans plusieurs modèles. Selon ces modèles, les systèmes TA permettent aux bactéries de mieux faire face à des conditions environnementales stressantes.

Entre-temps, la compréhension de l’évolution des génomes bactériens a connu des avancées significatives. L’impressionnante capacité d’adaptation des bactéries est aujourd’hui majoritairement attribuée au transfert horizontal de gènes (THG) provoqué par les éléments génétiques mobiles (phages, plasmides, transposons…). Dans le débat du rôle des systèmes TA chromosomiques, très peu d’attention a été accordée aux relations phylogénétiques et interactions entre systèmes plasmidiques et chromosomiques co-existant au sein d’un même hôte ainsi qu’à l’impact du THG sur leur évolution. Notre travail de thèse vise à mieux comprendre la biologie des systèmes TA en tenant compte de ces paramètres. Nous nous sommes intéressés à des systèmes homologues au système plasmidique ccdF. Nous avons étudié expérimentalement les 4 systèmes ccd (ccd1, ccd2, ccd3 et ccd4) qui co-habitent au sein du chromosome d’Erwinia chrysanthemi 3937 (une bactérie phytopathogène), leurs interactions intragénomiques et les interactions de ces systèmes avec le système plasmidique ccdF. Ce cadre expérimental a mené à la construction du modèle d’anti-addiction. Ce modèle propose que certains systèmes chromosomiques puissent conférer un avantage sélectif à leurs hôtes bactériens en interférant avec le PSK médié par leurs homologues plasmidiques. Cet avantage sélectif pourrait permettre la fixation de systèmes TA latéralement acquis au sein des populations bactériennes. Nous avons également recherché de nouveaux systèmes ccd au sein des génomes bactériens afin d’avoir un aperçu de leur distribution, des contextes génétiques dans lesquels ils existent et de l’implication du THG dans leur dispersion. Les réflexions qui ont accompagné notre recherche nous ont mené à proposer une synthèse sur le rôle des systèmes TA (plasmidiques et chromosomiques). Celle-ci se nourrit des avancées qui ont été effectuées, ces dernières années, dans la compréhension de l’évolution des génomes bactériens, de la théorie hiérarchique de la sélection naturelle et des processus non-adaptatifs et contingents qui pourraient expliquer la présence et la propagation des systèmes TA au sein des génomes bactériens sans que ceux-ci en soient les agents causaux.

Doctorat en sciences, Spécialisation biologie moléculaire
info:eu-repo/semantics/nonPublished

Gene transfer plays an important role in bacterial evolution. Especially in an under explored species like Rhodococcus, a type of bacteria found in the soil. Rhodococcus has several applications in the pharmaceutical industry and in the production of antibiotics. Rhodococcus possess several unique sets of properties which makes it beneficial to have a reliable method of producing mutants of Rhodococcus. The goal of the experiment was to find an efficient way of forming Rhodococcus colonies with kanamycin resistant genes. The project began from an unexpected observation from an earlier experiment with Rhodococcus strain MTM3W5.2. where I attempted to transform this strain with a transposon via electro-transformation. The colonies that grew/ appeared transformants were screened to confirm the presence of kanamycin gene, however there was no amplified DNA seen on the PCR gel (i.e. absence of the kanamycin gene). The electro-transformant colonies were selected on LB plates containing different higher concentrations of kanamycin. Then the appeared transformants were again screened via disk diffusion assay and were classified into 3 different kanamycin resistant phenotypes. Majority of the “C” phenotypic colonies (i.e., high level resistance to kanamycin) appear to contain the kanamycin gene, but these colonies were less in numbers. This led us to try another method of gene transfer which is conjugation. Conjugation was carried on a double selection antibiotic plate containing both chloramphenicol (30 µg) and kanamycin (100 µg). The transconjugate colonies that appeared on the double selection plates were also screened by PCR, but none of the colonies had amplified DNA suggesting absence of the kanamycin gene. The colonies seen on the double selection plate were possibly due to spontaneous mutation or some type of unknown phenotypic variation. However, in the future, double selection plates with higher concentrations of antibiotics can possibly give us transconjugants with kanamycin genes.

The PhD thesis presented here has as a main objective the study of antibiotic resistance genes clinically relevant in the DNA fraction of bacteriophage particles isolated from environmental samples of different origin in order to determine the importance of bacteriophages as vehicles for the mobilization of antibiotic resistance genes between bacteria. Specifically, a broad range of antibiotic resistance genes were studied as representative of the main groups recently described in our geographical area belonging to three β-lactamases (blaTEM, blaCTXM-1 and blaCTX-M-9), the mecA gene conferring resistance to methicillin in staphylococci, and the quinolones resistance genes qnrA and qnrS. To achieve these goals samples of urban wastewater, river water and animal faecal wastes were analysed quantifying the antibiotic resistance genes of interest in bacteriophages DNA. During the development of this Thesis, it was attempted to optimize the available methodology for bacteriophage DNA extraction, as well as the necessary controls to guarantee the amplification of encapsidated DNA and to remove any free DNA in the samples and any possible vesicles containing DNA. In addition, the ability of phage-encoded genes to confer antibiotic resistance in bacterial strains was assessed by performing transformation experiments. It was also studied the influence of various compounds involved in the induction of the lytic cycle of temperate bacteriophages, on the abundance of antibiotic resistance genes in DNA from the phage fraction in wastewater samples. Finally, due to the importance of horizontal gene transfer as a mechanism for antibiotic resistance dissemination in clinical and environmental settings, transduction experiments were attempted to reproduce in vitro the process that would take place in nature. The research developed in this Thesis is divided into 5 studies included in 4 chapters: (1) Antibiotic resistance genes in the bacteriophage DNA fraction of water samples (wastewater, river water and animal wastewater); (2) Quinolone resistance genes (qnrA and qnrS) in bacteriophage particles from wastewater samples and the effect of inducing agents on packaged antibiotic resistance genes; (3) Evaluation of ARGs in the DNA of bacterial and bacteriophage fraction in wastewater samples from Tunisia and comparison with results obtained in Barcelona area; (4) Detection of quinolone-resistant Escherichia coli isolates belonging to clonal groups O25b:H4-B2-ST131 and O25b:H4-D-ST69 in water samples from Barcelona area. Each of the studies has given rise to a scientific article already published or submitted for scientific publication.
La tesi doctoral que es presenta a continuació té com a objectiu principal l’estudi de gens de resistència a antibiòtics de rellevància clínica en la fracció de DNA de partícules de bacteriòfags aïllades de diferents tipus de mostres ambientals per tal de determinar la importància dels bacteriòfags com a vehicles de mobilització de gens de resistència a antibiòtics entre bacteris. S’ha estudiat un ampli espectre de gens de resistència a antibiòtics com a representants dels grups principals descrits actualment en la nostra àrea geogràfica corresponent a tres β-lactamases (blaTEM, blaCTXM-1 i blaCTX-M-9), el gen mecA de resistència a meticil•lina en estafilococs, i els gens de resistència a quinolones qnrA i qnrS. Per això s’han analitzat diversos tipus de mostres procedents d’aigua residual municipal, d’aigua de riu i d’aigua residual amb contaminació fecal animal per tal de quantificar els gens de resistència a antibiòtics d’interès en DNA aïllat de bacteriòfags. Durant els diferents estudis s’ha intentat optimitzar la metodologia d’extracció de DNA de bacteriòfags així com els controls corresponents per garantir l’amplificació de DNA encapsidat i l’eliminació de qualsevol DNA lliure present a les mostres i de qualsevol possible vesícula amb DNA al seu interior. Per altra banda, també s’ha determinat la capacitat funcional dels gens de resistència detectats en DNA de fags i per això s’han realitzat experiments de transformació a partir de soques bacterianes sensibles a un determinat antibiòtic amb l’objectiu d’incorporar la resistència i per tant, esdevenir resistents a l’antibiòtic en qüestió. També, s’ha estudiat la influència de determinats compostos implicats en la inducció del cicle lític de bacteriòfags temperats, en l’augment en el nombre de còpies de gens de resistència a antibiòtics en DNA present en la fracció de fags de l’aigua residual. Finalment, degut a la importància de la transferència horitzontal de gens com a mecanisme de dispersió de la resistència a antibiòtics en el medi ambient i en clínica s’han dut a terme experiments de transducció per tal d’intentar reproduir in vitro el procés que tindria lloc de manera natural.

O trabalho teve como objetivo otimizar a metodologia de transformação de plantas de Eucalyptus grandis; uma importante espécie florestal amplamente cultivada no Brasil. Foram estudadas a transformação por agrobiobalística de explantes com acúmulo de gemas axilares e do SAAT ('sonication-assisted Agrobacterium mediated transformation'). Ambas as técnicas causam microferimentos nos explantes aumentando a penetração da Agrobacterium nos tecidos vegetais. Na agrobiobalística o explante é submetido ao bombardeamento com micropartículas seguida por inoculação com a bactéria; enquanto que no SAAT o explante é submetido à sonicação por breves períodos de tempo na presença da bactéria. Os experimentos de transformação foram avaliados pela analise da expressão transitória do gene repórter uidA. Os parâmetros estudados na transformação por agrobiobalística foram: pressão de disparo ( pressão do gás hélio), distância de vôo das micropartículas, estabilidade da expressão do gene uidA e temperatura de co-cultivo. As freqüências mais elevadas da expressão da ß-glucuronidase foram observadas utilizando 1350 PSI de gás hélio, 9,5 cm de distância de vôo dos microprojéteis e temperatura de 26ºC durante o co-cultivo. Para otimizar a transformação de explantes com acúmulo de gemas axilares foi realizado um estudo histológico, o qual permitiu constatar que as áreas meristemáticas estão localizadas na superfície do explante. O meristema é formado pela rediferenciação de células da epiderme e das primeiras camadas do parênquima cortical caracterizando sua origem exógena. A localização histológica do produto da expressão do gene repórter uidA mostrou que esta expressão ocorre apenas em células já diferenciadas, não sendo encontrada durante os tratamentos com células meristemáticas. Para realizar os ensaios com SAAT foi selecionado previamente um clone com características favoráveis à transformação: alta taxa de regeneração, susceptibilidade a agentes seletivos e à transformação por Agrobacterium. A maior taxa de plantas que apresentaram atividade da ß-glucuronidase foram observadas utilizando-se 60 segundos de sonicação; tempos superiores a este causaram um comprometimento da regeneração do explante. A sonicação adicional, após a sonicação preliminar do explante, juntamente com a bactéria, melhora a eficiência do processo de transformação. Os resultados demonstram que o uso de SAAT é viável na transformação de eucalipto com Agrobacterium.
The research project aimed the establishment of a method for genetic transformation of Eucalyptus grandis; an important and widely planted forestry tree in Brazil. Two techniques of transformation were tested: agrobiolistic of explants with accumulation of meristematic cells and SAAT ('sonication-assisted Agrobacterium mediated transformation'). Both systems of transformation aim to produce micro wounds in the explants in order to increase the Agrobacterium penetration in the tissues. In the case of agrolistica the explant was previously submitted to micro projectile bombardment, followed by bacteria inoculation. On the other hand in the SAAT technique the explante is submitted to short periods (few seconds) of sonication together with the bacteria. Transformation was evaluated by measuring the expression of the reporter gene uidA which codes the ß-glucuronidase enzyme. Several parameters were tested for agrobiolistic such as, gas pressure (helium), flight distance of micro projectiles, gene expression of the uidA, co-cultivation temperature. The highest values of ß-glucuronidase were observed for 1350 PSI, 9.5 cm from target and 26O C for co-cultivation. A histological analyze was carried out to check it the meristematic tissues were being transformed. The results showed that the meristematic tissues were localized in the surface of the explante. The meristem is formed by the dedifferentiation of epidermal cells and the first layers of the cortical parenchyma indicating its exogenous origin. The histological location of the reporter gene uidA showed that the expression occurs only in cells already differentiated, and not in meristematic cells. For SAAT experiments a clone was previously selected for favorable characteristic of transformation: high rate of regeneration, susceptibility of selective agents and to Agrobacterium transformation. The highest rate of ß-glucuronidase activity were observed for 60 s of sonication; higher sonication time reduced the efficiency of regeneration. Additional sonication, following a preliminary sonication of explante , in the presence of the bacteria, improved the efficiency of transformation. The results provided evidence the SAAT is a viable technique for Eucalyptus transformation via Agrobacterium.

Huanglongbing (HLB), considerada uma das mais graves doenças dos citros, está associada a Candidatus Liberibacter spp., bactérias endógenas restritas ao floema e de difícil cultivo em meio de cultura. Diferentemente de outras doenças que afetam plantas cítricas, ainda não foram encontradas dentro do gênero Citrus espécies resistentes ao HLB. Genes de interesse agronômico têm sido empregados na transformação genética de citros visando a resistência a doenças. Dentre estes, destacam-se os que conferem resistência a bactérias incluindo attA, que codificam os peptídeos antibacterianos atacinas, e hrpN, que ativam a produção de proteínas harpinas, relacionadas ao sistema de defesa. O objetivo deste trabalho foi avaliar a resistência de plantas de laranja doce contendo o gene da atacina A (attA) ou o gene da harpina (hrpN) à Candidatus Liberibacter asiaticus (CLas) utilizando duas formas de inoculação: borbulhões infectados e o inseto vetor da bactéria, Diaphorina citri. Para as plantas contendo o gene hrpN, apenas o segundo método foi utilizado. Os principais sintomas do HLB foram observados aos quatro e oito meses após a inoculação por borbulhões. A infecção das plantas foi confirmada com a detecção de CLas por PCR (quatro e oito meses) e Rt-qPCR (oito meses). Em plantas inoculadas por D. citri, os sintomas foram observados oito e doze meses após a inoculação, assim como a detecção da bactéria por PCR. Após 15, 17 e 18 meses, foi realizada uma nova avaliação por Rt-PCR a partir de spots (imprints em membrana) de folhas. Rt-PCR foi empregado também em spots dos psilídeos utilizados na inoculação. Não foi possível avaliar a resistência ao HLB em plantas contendo o gene attA ou hrpN a partir da inoculação por D. citri. Os resultados de detecção da bactéria nas plantas e nos psilídeos utilizados para inoculação indicam que, possivelmente, não ocorreu a inoculação devido ao baixo percentual de psilídeos que continham CLas utilizados. Dentre as plantas transgênicas contendo o gene attA inoculadas por borbulhões infectivos, oito eventos (cinco de laranja Pera, dois de laranja Hamlin e um de laranja Valência) apresentaram menores títulos bacterianos e algumas também demonstraram redução dos sintomas do HLB quando comparadas com plantas não transgênicas, oito meses após a inoculação, indicando uma possível ação do peptídeo atacina A contra o agente causal do HLB.
Haunglongbing (HLB), considered one of the most serious diseases of citrus, is associated to Candidatus Liberibacter spp., endogenous and phloem-inhabiting bacteria not easily grown in culture medium No species within the genus Citrus is known to resist this bacterial infection. The use of genes of agronomic interest for genetic transformation aiming disease resistance in citrus has been reported. Among these genes, attA that codes for the antibacterial peptides attacin, and hrpN, that codes for proteins harpin that activate the plant defense system may have potential in searching for HLB resistance. The objective of this study was to evaluate the resistance of sweet orange containing attA or hrpN to Candidatus Liberibacter asiaticus (CLas) inoculated through infected budstick grafting or the insect vector, Diaphorina citri. For the plants containing hrpN, only the second method was used. The most obvious HLB symptoms were observed four and eight months after inoculation by infected budstick when CLas also was detected by PCR (four months) and RT-qPCR (eight months). For those inoculated with D. citri, symptoms were observed and bacteria detected eight and twelve months after inoculation. Fifteen, 17 and 18 months after inoculation, a new attempt was made for CLas detection, now through Rt-PCR from leaf and psyllids imprinting spots on membrane. It was not possible to evaluate the HLB resistance in plants containing attA or hrpN gene from D. citri inoculation. The results of CLas detection in plants and psyllids indicate that possibly there was no inoculation due the low rate of psyllids contained CLas used. Among the plants containing attA, five, two and one event of, respectively, Pera, Hamlin, and Valencia sweet orange had lower bacterial titers than those non transgenic plants and some also showed milder HLB symptoms, eight months after inoculation, suggesting a possible effect of attacin A against the causal agent of HLB.

Le virus de la mosaique du chou-fleur, representant modele des caulimovirus, comprend une molecule circulaire d'adn double brin d'organisation genetique connue. Des fragments de l'adn viral ont ete introduits dans le genome du colza, grace au vecteur naturel de transformation agrobacterium rhizogenes, afin de faire produire par la cellule vegetale un arn antisens permettant d'interferer avec l'expression d'un gene essentiel dans le cycle vital du virus. Des plantes transgeniques ont ete regenerees a partir des racines transformees

碩士
臺灣大學
園藝學研究所
95
The purpose of this study is to enhance the resistance of orchids to bacterial soft rot. Pectate lyase genes ( pelE and pelZ ) isolated from Erwinia chrysanthemi were transformed into calli of Phalaenopsis and leaves of tobacco by Agrobacterium-mediated transformation. Transformed calli were selected by medium containing G418 due to nptII selectable marker gene in pelE construct. PelE transgenic callus lines confirmed by molecular analysis were further transformed with pelZ construct containing hpt and gus genes. Therefore, survival transformed callus were double transformed after selection by hygromycin. After callus was selected with antibiotics and regenerated, transgenic lines were confirmed by β-glucuronidase ( GUS ) activity, polymerase chain reaction ( PCR ), and Southern blot analysis. Expression products of transgenes were detected 43 and 49 kD by Western blotting analysis and enzyme-linked immunosorbent assay ( ELISA ). Hydrogen peroxide staining and pathogen inoculation analysis assay revealed that transgenic lines exhibited hypersensitive response ( HR ) to phytopathogens.

Non-pathogenic corynebacteria including Corynebacterium glutamicum, Brevibacterium flavum, and B. lactofermentum have been used traditionally for industrial production of amino acids. Recently, these strains have been also studied using molecular biology or recombinant DNA approaches. However, a major early limitation in the application of this technology was poor transformation efficiency due to the presence of barriers which prevented entry or survival of incoming DNA. These barriers are both physical (cell surface structures) and enzymatic (restriction and modification) barriers. The research described in this thesis involved investigating the nature of both the physical and enzymatic barriers, includings: (i) determining the effect of the presence of glycine and/or isonicotinic acid hydrazide (INH) and resulting lipid profiles (mycotic acids and fatty acids) of several strains of corynebacteria. (ii) examining the enzymatic barriers to transformation of these corynebacteria with specific reference to methyltransferase (MTase) activity and the sites of methylation.

碩士
國立臺北科技大學
化學工程所
93
In this research, we use linear transformation that takes advantage of the homologous sequence between two ends of linear DNA and the host cell E.coli, to transfer the target genes directly into the chromosome of the strain. Because this method enables expressing recombinant proteins continuously without plasmid, the problem of plasmid instability is solved. In practice, we use ZSC114, a multi-auxotrophic E.coli strain that cannot metabolize glucose, mannose and lactose, as our host cell. First, we unite each of the three metabolism-related genes with lacZ gene, to prepare for a functional linear DNA. Then we put these linear substrates into the chromosome of ZSC114. We can make up for the auxotrophic phenotype of the original strain, and with expression of more lacZ genes, enhance the production of recombinant protein β-galactosidase. Protein gel electrophoresis andβ-galactosidase activity assay are executed to measure the production of recombinant protein LacZ among our newly constructed strains. We find out that the production and activity of the genetic product increase while the copy number of lacZ gene rises in the bacterial chromosome. We use the method of serial dilution culture to investigate genetic stability between the three-lacZ gene-carrying strain, ZSCGMtZ, and the plasmid expression system, ZSC114/pJL. We find out that in serial dilution culture, the ZSC114/pJL system would loss LacZ expression at 35th hour, but new strain ZSCGMtZ still keep quite high recombinant protein expression after 60 hours. The result indicates the genetic stability of new strain is better.