by Fong Man Kim. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 127-137). / Abstracts in English and Chinese. / Thesis committee --- p.i / Acknowledgements --- p.ii / Abstract --- p.iii / List of figures --- p.viii / List of tables --- p.xi / Abbreviations --- p.xii / Table of contents --- p.xiv / Chapter CHAPTER 1 --- GENERAL INTRODUCTION --- p.1 / Chapter CHAPTER 2 --- LITERATURE REVIEW --- p.3 / Chapter 2.1 --- MIH from Penaeus vannamei --- p.3 / Chapter 2.1.1 --- General Introduction to P. vannamei --- p.3 / Chapter 2.1.1.1 --- Morphology --- p.3 / Chapter 2.1.1.2 --- Geographical distribution --- p.5 / Chapter 2.1.1.3 --- Economic value --- p.5 / Chapter 2.1.2 --- Physiology of Molting in Crustacean --- p.7 / Chapter 2.1.2.1 --- The molt cycle --- p.7 / Chapter 2.1.2.2 --- Physiological effects of ecdysone --- p.8 / Chapter 2.1.2.3 --- Regulation of the secretion of ecdysone --- p.9 / Chapter 2.1.2.4 --- Physiological effects of Molt-inhibiting hormone --- p.10 / Chapter 2.1.3 --- Cloning of MIH cDNA from P. vannamei --- p.14 / Chapter 2.1.3.1 --- Molecular identity of MIH --- p.14 / Chapter 2.1.3.2 --- Cloning of MIH cDNA --- p.15 / Chapter 2.1.3.3 --- Comparison of the cloned MIH-like cDNA with the CHH/MIH/VIH peptide family --- p.16 / Chapter 2.2 --- Plants as Bioreactors --- p.20 / Chapter 2.2.1 --- Principles & Techniques --- p.20 / Chapter 2.2.2 --- Advantages of plant bioreactors --- p.21 / Chapter 2.2.3 --- Tobacco expression system --- p.22 / Chapter 2.2.3.1 --- Tobacco as model plants --- p.22 / Chapter 2.2.3.2 --- Transformation methods --- p.23 / Chapter 2.2.4 --- Phaseolin --- p.26 / Chapter CHAPTER 3 --- EXPRESSION OF MIH IN TRANSGENIC TOBACCO --- p.28 / Chapter 3.1 --- Introduction --- p.28 / Chapter 3.2 --- Materials & Methods --- p.29 / Chapter 3.2.1 --- Chemicals --- p.29 / Chapter 3.2.2 --- Plant materials --- p.29 / Chapter 3.2.3 --- Bacterial strains and plasmid vectors --- p.30 / Chapter 3.2.4 --- Construction of chimeric genes - --- p.30 / Chapter 3.2.4.1 --- PCR amplification of MIH --- p.30 / Chapter 3.2.4.2 --- Cloning of PCR-amplified MIH into vector pET --- p.31 / Chapter 3.2.4.3 --- Cloning of MIH into vector pBK/Phas-sp and pTZ/Phas --- p.31 / Chapter 3.2.4.4 --- Cloning of MIH into binary vector pBI121 --- p.32 / Chapter 3.2.5 --- Transformation of Agrobacterium with pBI121/Phas-sp-MIH and pBI121 /Phas-MIH by electroporation --- p.39 / Chapter 3.2.6 --- Transformation of tobacco --- p.40 / Chapter 3.2.7 --- Selection of transgenic plants --- p.41 / Chapter 3.2.8 --- GUS assay --- p.42 / Chapter 3.2.9 --- Extraction of leaf genomic DNA --- p.43 / Chapter 3.2.10 --- Extraction of total RNA from developing seeds --- p.44 / Chapter 3.2.11 --- Synthesis of DIG-labeled DNA and RNA probes --- p.45 / Chapter 3.2.12 --- Southern blot analysis of genomic DNA --- p.47 / Chapter 3.2.13 --- Reverse transcriptase - polymerase chain reaction (RT-PCR) --- p.47 / Chapter 3.2.14 --- Northern blot analysis of total RNA --- p.48 / Chapter 3.2.15 --- Protein extraction and tricine-SDS-PAGE --- p.49 / Chapter 3.2.16 --- Purification of 6xHis-tag proteins --- p.50 / Chapter 3.2.17 --- Western blot analysis --- p.50 / Chapter 3.2.18 --- In vitro transcription & translation --- p.52 / Chapter 3.2.18.1 --- Construction of transcription vector containing the chimeric MIH gene --- p.52 / Chapter 3.2.18.2 --- In vitro transcription --- p.56 / Chapter 3.2.18.3 --- In vitro translation --- p.56 / Chapter 3.2.19 --- Particle bombardment --- p.57 / Chapter 3.2.19.1 --- Construction of MIH-GUSN fusion chimeric genes --- p.57 / Chapter 3.2.19.2 --- Conditions of particle bombardment --- p.63 / Chapter 3.2.20 --- Codon modification of MIH gene --- p.63 / Chapter 3.3 --- Results --- p.73 / Chapter 3.3.1 --- Construction of chimeric MIH genes --- p.73 / Chapter 3.3.2 --- "Tobacco transformation, selection and regeneration" --- p.73 / Chapter 3.3.3 --- Detection of GUS activity --- p.74 / Chapter 3.3.4 --- Southern blot analysis --- p.79 / Chapter 3.3.5 --- Detection of MIH transcript in transgenic tobacco --- p.83 / Chapter 3.3.5.1 --- RT-PCR --- p.83 / Chapter 3.3.5.2 --- Northern blot analysis --- p.86 / Chapter 3.3.6 --- Detection of MIH protein by Tricine-SDS-PAGE --- p.86 / Chapter 3.3.7 --- Detection of MIH protein by western blot analysis --- p.88 / Chapter 3.3.7.1 --- Western blot analysis using Anti-MIH antibody --- p.88 / Chapter 3.3.7.2 --- Western blot analysis using Anti-His antibody --- p.90 / Chapter 3.3.7.3 --- Western blot analysis using Anti-MIHA & Anti-MIHB antibodies --- p.90 / Chapter 3.3.8 --- Purification of 6xHis-tag proteins by Ni-NTA column --- p.94 / Chapter 3.3.8.1 --- Western blot analysis of proteins purified by Ni-NTA column --- p.97 / Chapter 3.3.9 --- In vitro transcription and translation --- p.100 / Chapter 3.3.9.1 --- In vitro transcription --- p.100 / Chapter 3.3.9.2 --- In vitro translation --- p.100 / Chapter 3.3.10 --- Particle bombardments --- p.103 / Chapter 3.3.10.1 --- Transient expression of MIH in soybean & tobacco leaves --- p.103 / Chapter CHAPTER 4 --- DISCUSSION --- p.107 / Chapter 4.1 --- Transient expression of MIH genes --- p.109 / Chapter 4.1.1 --- In vitro transcription and translation --- p.109 / Chapter 4.1.2 --- Particle bombardments --- p.220 / Chapter 4.2 --- Post-transcriptional gene silencing (PTGS) --- p.114 / Chapter 4.2.1 --- Post-transcriptional cis-inactivation --- p.114 / Chapter 4.2.2 --- Post-transcriptional trans-inactivation --- p.116 / Chapter 4.2.3 --- MIH gene and PTGS --- p.118 / Chapter 4.3 --- Codon usage --- p.119 / Chapter 4.3.1 --- Codon usage of MIH in plants --- p.120 / Chapter 4.3.2 --- Codon modification of MIH and further study on MIH expression in plants --- p.122 / Chapter 4.4 --- Post-translational protein degradation --- p.123 / Chapter 4.4.1 --- Construction of LRP-MIH fusion proteins --- p.123 / CONCLUSION --- p.125 / REFERENCES --- p.127

by Lee Juon Kiu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 139-152). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Acknowledgements --- p.iii / Abstract --- p.v / Table of contents --- p.ix / List of figures --- p.xv / List of tables --- p.xvii / List of graphs --- p.xviii / List of abbreviations --- p.xix / Chapter Chapter 1: --- General Introduction --- p.1 / Chapter Chapter 2: --- Literature Review --- p.4 / Chapter 2.1 --- Human granulocyte colony-stimulating factor (hG-CSF) --- p.4 / Chapter 2.1.1 --- Physiological roles --- p.4 / Chapter 2.1.2 --- Molecular properties --- p.8 / Chapter 2.1.3 --- Biochemical properties --- p.9 / Chapter 2.1.4 --- Comparison to G-CSF of other specie --- p.10 / Chapter 2.1.5 --- Clinical application --- p.11 / Chapter 2.1.6 --- Economic value --- p.13 / Chapter 2.2 --- Expression systems producing recombinant hG-CSF --- p.15 / Chapter 2.2.1 --- Bacteria --- p.15 / Chapter 2.2.2 --- Yeasts --- p.17 / Chapter 2.2.3 --- Animal cell lines --- p.18 / Chapter 2.2.4 --- Transgenic animals --- p.19 / Chapter 2.2.5 --- Transgenic plants --- p.20 / Chapter 2.3 --- Plant as bioreactors --- p.21 / Chapter 2.3.1 --- Characteristics of using plant as bioreactors --- p.22 / Chapter 2.3.2 --- Transgenic plants producing hematopoietic growth factors --- p.24 / Chapter 2.3.2.1 --- Granulocyte-macrophage colony-stimulating factor (GM-CSF) --- p.24 / Chapter 2.3.2.2 --- Erythropoietin (Epo) --- p.26 / Chapter 2.3.3 --- Arabidopsis and tobacco as model plants --- p.27 / Chapter 2.3.3.1 --- Arabidopsis --- p.28 / Chapter 2.3.3.2 --- Tobacco --- p.28 / Chapter 2.3.4 --- Phaseolin and its regulatory sequences --- p.29 / Chapter 2.4 --- Plant transformation methods --- p.31 / Chapter 2.4.1 --- Agrobacterium-mediated transformation --- p.31 / Chapter 2.4.1.1 --- Tissue culture methods --- p.31 / Chapter 2.4.1.2 --- Non-tissue culture (In planta) methods --- p.32 / Chapter 2.4.2 --- Direct DNA uptake transformation --- p.33 / Chapter 2.4.2.1 --- Chemical methods --- p.33 / Chapter 2.4.2.2 --- Electrical methods --- p.34 / Chapter 2.4.2.3 --- Physical methods --- p.34 / Chapter Chapter 3: --- Materials and Methods --- p.36 / Chapter 3.1 --- Introduction --- p.36 / Chapter 3.2 --- Chemicals --- p.37 / Chapter 3.3 --- Bacterial strains --- p.37 / Chapter 3.4 --- Chimeric gene construction --- p.37 / Chapter 3.4.1 --- Cloning of pTZ/Phas/His/EK/hG-CSF --- p.41 / Chapter 3.4.2 --- Cloning of pBK/Phas/SP/His/EK/hG-CSF --- p.44 / Chapter 3.4.3 --- Cloning of pBK/Phas/SP/hG-CSF --- p.47 / Chapter 3.4.4 --- Confirmation of sequence fidelity of chimeric genes --- p.50 / Chapter 3.4.5 --- Cloning of chimeric genes into Agrobacterium binary vector --- p.51 / Chapter 3.5 --- Expression in Arabidopsis --- p.52 / Chapter 3.5.1 --- Agrobacterium GV3101/pMP90 transformation --- p.52 / Chapter 3.5.2 --- Arabidopsis transformation --- p.53 / Chapter 3.5.2.1 --- Plant materials --- p.53 / Chapter 3.5.2.2 --- Vacuum infiltration --- p.54 / Chapter 3.5.3 --- Screening of successful R1 transformants --- p.55 / Chapter 3.5.4 --- Screening of hemizygous and homozygous transgenic Arabidopsis --- p.56 / Chapter 3.5.5 --- GUS assay --- p.57 / Chapter 3.5.6 --- Genomic DNA extraction --- p.57 / Chapter 3.5.7 --- Southern blot analysis --- p.58 / Chapter 3.5.8 --- Total RNA extraction from developing siliques --- p.59 / Chapter 3.5.9 --- Northern blot analysis --- p.60 / Chapter 3.5.10 --- Protein extraction and Tricine SDS-PAGE --- p.61 / Chapter 3.5.11 --- Western blot analysis --- p.62 / Chapter 3.5.12 --- Functional analysis --- p.63 / Chapter 3.5.12.1 --- Culture ofNFS-60 cells --- p.64 / Chapter 3.5.12.2 --- MTT assay --- p.65 / Chapter 3.6 --- Expression in tobacco --- p.67 / Chapter 3.6.1 --- Agrobacterium LBA4404/pAL4404 transformation --- p.67 / Chapter 3.6.2 --- Tobacco transformation --- p.68 / Chapter 3.6.2.1 --- Plant materials --- p.68 / Chapter 3.6.2.2 --- Tobacco transformation using leaf-disc technique --- p.68 / Chapter 3.6.3 --- Regeneration of transgenic tobacco --- p.69 / Chapter 3.6.4 --- GUS assay --- p.70 / Chapter 3.6.5 --- Genomic DNA extraction --- p.70 / Chapter 3.6.6 --- Southern blot analysis --- p.70 / Chapter 3.6.7 --- Total RNA extraction from immature seeds --- p.70 / Chapter 3.6.8 --- Northern blot analysis --- p.71 / Chapter 3.6.9 --- Protein extraction and Tricine SDS-PAGE --- p.71 / Chapter 3.6.10 --- Western blot analysis --- p.71 / Chapter 3.6.11 --- Functional analysis --- p.71 / Chapter 3.6.11.1 --- Culture of NFS-60 cells --- p.72 / Chapter 3.6.11.2 --- MTT assay --- p.72 / Chapter Chapter 4: --- Results --- p.73 / Chapter 4.1 --- Chimeric gene construction --- p.73 / Chapter 4.1.1 --- Cloning of pTZ/Phas/His/EK/hG-CSF --- p.73 / Chapter 4.1.2 --- Cloning of pBK/Phas/SP/His/EK/hG-CSF --- p.75 / Chapter 4.1.3 --- Cloning of pBK/Phas/SP/hG-CSF --- p.77 / Chapter 4.1.4 --- Cloning of chimeric genes into Agrobacterium binary vector --- p.79 / Chapter 4.2 --- Expression in Arabidopsis --- p.81 / Chapter 4.2.1 --- Agrobacterium GV3101/pMP90 transformation --- p.81 / Chapter 4.2.2 --- Arabidopsis transformation and screening of R1 transformants --- p.83 / Chapter 4.2.3 --- Screening of hemizygous transgenic R1 Arabidopsis --- p.84 / Chapter 4.2.4 --- Screening of homozygous transgenic R2 Arabidopsis --- p.86 / Chapter 4.2.5 --- GUS assay --- p.88 / Chapter 4.2.6 --- Genomic DNA extraction --- p.89 / Chapter 4.2.7 --- Southern blot analysis --- p.91 / Chapter 4.2.8 --- Total RNA extraction from developing siliques --- p.93 / Chapter 4.2.9 --- Northern blot analysis --- p.94 / Chapter 4.2.10 --- Protein extraction and Tricine SDS-PAGE --- p.96 / Chapter 4.2.11 --- Western blot analysis --- p.99 / Chapter 4.2.12 --- Functional analysis --- p.103 / Chapter 4.3 --- Expression in tobacco --- p.108 / Chapter 4.3.1 --- Agrobacterium LBA4404/pAL4404 transformation --- p.108 / Chapter 4.3.2 --- Tobacco transformation and regeneration of transformants --- p.109 / Chapter 4.3.3 --- GUS assay --- p.111 / Chapter 4.3.4 --- Genomic DNA extraction --- p.112 / Chapter 4.3.5 --- Southern blot analysis --- p.114 / Chapter 4.3.6 --- Total RNA extraction from immature seeds --- p.116 / Chapter 4.3.7 --- Northern blot analysis --- p.116 / Chapter 4.3.8 --- Protein extraction and Tricine SDS-PAGE --- p.118 / Chapter 4.3.9 --- Western blot analysis --- p.120 / Chapter 4.3.10 --- Functional analysis --- p.123 / Chapter Chapter 5: --- Discussion --- p.126 / Chapter 5.1 --- Introduction --- p.126 / Chapter 5.2 --- Successful in producing biologically active rhG-CSF from transgenic plants --- p.128 / Chapter 5.2.1 --- Production level --- p.129 / Chapter 5.2.2 --- O-glycosylation --- p.130 / Chapter 5.2.3 --- Phaseolin signal peptide --- p.131 / Chapter 5.2.4 --- Functional analysis --- p.131 / Chapter 5.3 --- Comparison of the productivity of other expression systems producing rhG-CSF --- p.132 / Chapter 5.4 --- Comparison of the productivity of plants producing different human proteins --- p.135 / Chapter 5.5 --- Future perspectives --- p.137 / Chapter Chapter 6: --- Conclusion --- p.138 / References --- p.139

Les propriétés insecticides du Bacillus thuringiensis, découvert par ShigentaneIshiwatari, ont été utilisées pendant des décennies comme biopesticides et cette utilisation a augmenté rapidement en raison de préoccupations au sujet des effets environnementaux négatifs des pesticides chimiques. Actuellement, la toxine Bt dans la forme de biopesticides et des plantes transgéniques Bt peut compléter ou remplacer les pesticides chimiques. Il y a peu d’indication que la toxine Bt a un effet nocif pour l'environnement ou la santé humaine. Néanmoins, il ya des préoccupations que les cultures transgéniques commerciales peuvent avoir des effets néfastes sur l'environnement. Après son introduction dans le sol l'exsudation racinaire et la dégradation des résidus végétaux, la toxine Bt interagit avec les particules de sol. Les interactions de la toxine Bt avec des particules de sol influencent sa mobilité, sa biodisponibilité, sa persistance et sa toxicité.Dans cette étude, nous visons à établir l'importance relative des facteurs biologiques et physico-chimiques dans la détermination de la dynamique des protéines Cry détectables dans les sols, de clarifier si la protéine adsorbée conserve ses propriétés insecticides et d'identifier les propriétés du sol qui déterminent le devenir des protéines Cry dans le sol. Les résultats montrent que les protéines Cry ont une forte affinité sur la surface du sol. Cependant, il y avait peu de relation entre l'affinité pour le sol ou le rendement d'extraction et les propriétés du sol, y compris la teneur en argile, teneur en carbone organique et le pH du sol. Il y avait peu de rapport entre l'affinité et le rendement d'extraction. Les protéines diffèrent à la fois dans leur affinité pour les sols et leurs rendements d'extraction.Une évaluation du rôle du sol et des facteurs environnementaux dans le sort des protéines Cry de la formulation de biopesticides commerciale a montré un déclin rapide de la protéine Cry détectable soumise aux rayons du soleil sous la condition de laboratoire, alors que peu d'effet a été observé dans des conditions de terrain. La demi-vie des protéines dans le sol dans des conditions naturelles était d'environ 1 semaine. Des effets de la température forts ont été observés, mais ils diffèrent pour les biopesticides et la protéine purifiée, indiquant différentes étapes limitantes. Pour le biopesticide, la baisse observée était ralenties par des facteurs biologiques, y compris éventuellement sporulation. En revanche pour des protéines purifiées, augmentation de la température améliorée des changements conformationnels de la protéine adsorbée du sol, conduisant à une fixation et, par conséquent diminué efficacité d'extraction qui a diminué avec le temps. En outre, l'étude de la persistance de diverses protéines Cry dans les sols contrastés a été réalisée par immuno-détection et dosage biologique a montré que la toxine extractible diminue avec incubation allant jusqu'à quatre semaines. L'activité insecticide était toujours maintenue à l'état adsorbé, mais a disparue après deux semaines d'incubation à 25°C. La baisse de la protéine extractible et la toxicité était beaucoup plus faible à 4°C à 25°C. La stérilisation du sol n'a pas eu d'effet significatif sur la persistance de la toxine Cry indiquant que le déclin observé était provoqué par la fixation en fonction du temps de la protéine adsorbée ce qui diminue la quantité de toxine Cry extractable, la dégradation de la protéine par l’activité microbienne jouant un rôle plus mineur.L’exposition des insectes aux protéines Cry sous la forme adsorbé pourrait avoir un impact significatif sur les insectes cibles et même les insectes non cibles, et devrait être plus étudiée afin de déterminer son impact potentiel. / The insecticidal properties of Bacillus thuringiensis, discovered by Shigentane Ishiwatari, have been used for decades as biopesticides and this use has been increasing rapidly because of concerns about the negative environmental effects of chemical pesticides. Currently, Bt toxin in the form of both biopesticides and Bt transgenic plantsmay supplement or replace chemical pesticide. There is little evidence to demonstrate that Bt toxin has any harmful effect to the environment or to human health. Nevertheless, there are concerns that commercial transgenic crops may have harmful impacts on the environment. After release into soil via root exudation and breakdown of plant residues, Bt toxin interacts with soil particles. The interactions of Bt toxin with soil particles influence its mobility, its bioavailability, its persistence and its toxicity. In this study, we aim to establish the relative importance of biological and physicochemical factors in the determination of the dynamics of detectable Cry proteins in soils, to clarify if adsorbed protein maintains its insecticidal properties and to identify the soil properties that determine the fate of Cry proteins in soil. The results show that Cry proteins have strong affinity on soil surface. However, there was little relationship between affinity for soil or the extraction yield and soil properties including clay content, organic carbon content and soil pH. There was little relationship between the affinity and the extraction yield. The proteins differ in both their affinity for soil and their extraction yields.An assessment of role of soil and environmental factors in the fate of Cry protein from commercial biopesticide formulation showed a rapid decline of detectable Cry protein subjected to direct sunlight under the laboratory condition, whereas, little effect was observed under field conditions. The half-life of proteins in soil under natural conditions was about one week. Strong temperature effects were observed, but theydiffered for biopesticide and purified protein, indicating different limiting steps. For biopesticide, the observed decline was due to biological factors, possibly including sporulation. In contrast for purified proteins, increased temperature enhanced conformationalchanges of the soil-adsorbed protein, leading to fixation and hence extraction efficiency decreased that decreased with time. Moreover, the study of persistence of various Cry proteins in contrasting soils was carried out by immuno-detection and bioassay showed that extractable toxin decreased with incubation of up to four weeks. Insecticidal activity was still retained in the adsorbed state, but lost after two weeks of incubation at 25°C. The decline in extractable protein and toxicity was much lower at 4°C than 25°C. There was no significant effect of soil sterilization to persistence of Cry toxin indicating that decrease in detectable Cry toxin in soil may be time-dependent fixation of adsorbed protein as well as decreasing solubilization in larva midgut, but not microbial breakdown.Exposition to Cry in the adsorbed form could have a significant impact on target and even non target insects and should be investigation to determine the potential impact.

Os elementos celulares que constituem o tecido xilemático de várias espécies vegetais são amplamente utilizados em diversos setores industriais, com inúmeras aplicações, como por exemplo, a geração de energia e produção de celulose e papel. A parede celular das células vegetais é formada basicamente por celulose, hemiceluloses e lignina. A formação dos polímeros de celulose e hemicelulose dependem exclusivamente do suprimento de precursores chamados de nucleotídeos-açúcares, tais como UDP-glicose, UDP-glucuronato, UDP-xilose, UDP-arabinose, UDP-manose e UDP-galactose. A biossíntese da parede celular é altamente regulada do ponto de vista metabólico, e envolve a participação de várias enzimas que catalisam uma série de reações. Estratégias para alterar o fluxo metabólico destes precursores, podem originar modificações na deposição dos polissacarídeos na parede celular. Em particular, para o setor de celulose e papel tal estratégia pode resultar em fibras com determinadas características, melhorando a qualidade da polpa celulósica ou do papel produzido. O UDP-glucuronato é um dos principais precursores de polissacarídeos hemicelulósicos da parede celular, sendo formado a partir de UDP-glucose pela ação da enzima UDP-glicose desidrogenase (EC. 1.1.1.22). Esta enzima é chave na regulação da biossíntese das pentoses e hexoses da parede celular de plantas superiores. Com o objetivo de modular a síntese dos polissacarídeos hemicelulósicos na parede celular, o presente trabalho analisou o impacto da superexpressão do gene ugdh em plantas transgênicas de tabaco. Foram realizadas análises da composição química da parede celular primária e secundária de folhas e caules, bem como avaliações morfológicas das fibras do tecido xilemático, e também cortes histológicos da região basal do caule. Da parede secundária do tecido xilemático determinou-se o conteúdo de lignina klason e solúvel, bem como a concentração dos carboidratos via HPAE-PAD. No tecido xilemático, todas as plantas transgênicas apresentaram aumento do conteúdo de xilose, embora não significativo. Juntamente, ocorreu um aumento de arabinose significativo em três linhagens transgênicas. Paralelamente, todas as plantas transgênicas tiveram redução do conteúdo de lignina klason, embora significativo em apenas uma linhagem. A relação hexoses/pentoses reduziu em todas as linhagens transgênicas, sendo significativa em três linhagens. As análises histológicas do caule mostraram que os transformantes apresentaram um aumento do tecido xilemático em relação às plantas controles. As análises morfológicas das fibras mostraram que todas as plantas transgênicas apresentaram reduções significativas no comprimento, em relação às plantas controles. No tecido foliar, o conteúdo de polissacarídeos da parede celular primária apresentou uma redução significativa em todas as plantas transgênicas. / The cellular elements that constitute the xylematic tissue of various plant species are widely used in diverse industrial sectors, with numerous applications, for example, the generation of energy and production of cellulose and paper. Plant cell walls are basically formed by cellulose, hemicellulose and lignin. The formation of cellulose and hemicellulose polymers depend exclusively on the supply of the precursors called nucleotide sugars, such as UDPglucose, UDP-glucuronate, UPD-xylose, UDP-arabinose, UDP-mannose and UDP-galactose. The biosynthesis of the cell wall is highly regulated from the metabolic point of view and involves the participation of various enzymes that catalyse a series of reactions. Strategies to alter the metabolic flux of these precursors could give rise to modifications in the deposition of polysaccharides in the cell wall. In particular, for the cellulose and paper sector these strategies could result in fibers with determined characteristics, improving the quality of the cellulose pulp or the paper produced. UDP-glucuronate is one of the principal precursors of the hemicellulose polysaccharides of the cell wall, which is formed from UDP-glucose by the action of UDPglucose dehydrogenase (EC1.1.1.22). This is a key enzyme in the regulation of the biosynthesis of the pentoses and hexoses in the cell walls of higher plants. With the objective of modulating the synthesis of the hemicellulose polysaccharides in the cell wall, the present study analysed the impact of the superexpression of the ugdh gene in transgenic tobacco plants. Chemical analyses were performed to determine the chemical composition of the primary and secondary cell walls of leaves and stems, as well as morphological evaluations of the fibers of the xylematic tissue and histological cuts through the base of the stem. The Klason and soluble lignin content as well as the carbohydrate concentrations (using HPAE-PAD) were determined in the secondary cell walls of the xylematic tissue. All of the transgenic plants showed an increase in xylose content, albeit not significant. A significant increase in arabinose content was observed in three transgenic lines. In parallel all the transgenic plants presented a reduction in Klason lignin, but only significant in one line. The ratio hexose/pentose was reduced in all transgenic lines, being significant in three. Histological analyses on the stem showed that the transformants presented an increase in the xylematic tissue when compared to the controls. The morphological analyses of the fibers showed that all the transgenic plants presented significant reductions in length when compared to the controls. In the leaf tissue, the polysaccharide content of the primary cell wall showed a significant reduction in all of the transgenic plants.

Citrus tristeza virus (CTV) ocorre em quase todas as áreas produtoras de citros do mundo. O controle da doença se baseia, principalmente, no uso de porta-enxertos tolerantes e na premunização das copas. A obtenção de copas de laranjas doces ou de porta-enxerto de laranja azeda transgênicos resistentes ao CTV permitiria retornar a um uso mais intensivo deste excelente porta-enxerto. Com isso, esse trabalho buscou avaliar linhagens transgênicas de laranja doce (Citrus sinensis) e obter plantas transgênicas de laranja azeda (Citrus aurantium) para a resistência ao CTV, a fim de oferecer uma outra alternativa para o controle desta doença em citros. Foram avaliadas plantas transgênicas de laranja doce cv. Valência e cv. Hamlin contendo três diferentes construções gênicas. Uma contendo uma sequência sense (684 pb) do gene da capa protéica do CTV (pCTV-CP), outra contendo uma sequência conservada (559 pb) do CTV (pCTV-SC) e uma do tipo hairpin, contendo sequências sense e antisense do gene da capa protéica separadas por um íntron (pCTV-dsCP). Dez linhagens transgênicas de cada construção gênica e de cada cultivar foram previamente confirmadas por análises de Southern blot e RT-PCR, totalizando 60 linhagens transgênicas. Tais linhagens foram clonadas e enxertadas sobre limão Cravo (C. limonia) e laranja azeda (C. aurantium), totalizando 360 plantas. Essas plantas, juntamente com plantas não transgênicas utilizadas como controle, foram inoculadas com o CTV por meio de Toxoptera citricida virulífero. As técnicas de ELISA indireto utilizando anticorpo monoclonal contra a capa protéica do CTV ou de Real-time PCR utilizando primers amplificadores dos genes p20 e p23 do genoma do CTV foram utilizadas para detectar o vírus nas plantas avaliadas, 4 semanas após as inoculações. Ocorreu variação na resistência ao vírus nas diferentes construções transgênicas utilizadas e entre clones de uma mesma planta. Alguns clones não foram infectados com o vírus mesmo após a quarta inoculação, indicando uma possível resistência ao patógeno. Um total de 30 experimentos de transformação genética de laranja azeda foram realizados, utilizando como explantes segmentos internodal, de epicótilo e de cotilédone associado ao hipocótilo. O teste de GUS permitiu a identificação de duas gemas com reação positiva (0,13% de eficiência de transformação). Tais gemas foram enxertadas in vitro sobre citrange Carrizo, mas apenas uma gema se desenvolveu. A planta obtida foi aclimatizada em casa-de-vegetação. / Citrus tristeza virus (CTV) occurs in almost all citrus-growing areas of the world. Control of citrus tristeza relies mainly on the use of tolerant rootstocks and scion cross protection. Obtaining transgenic sweet oranges cultivars or sour orange resistant to CTV would allow a better use of this excellent rootstock. This way, the aim of this work was to evaluate transgenic sweet orange (Citrus sinensis) lines and to obtain transgenic sour orange (Citrus aurantium) for the resistance to CTV, in order to offer another alternative for the control of the disease in citrus. Transgenic sweet orange cv. Valencia and cv. Hamlin containing three different genetic constructs were evaluated. One gene construct contains a sense sequence (684 pb) of the coat protein gene of CTV (pCTV-CP), another contains a conserved sequence (559 pb) of CTV (pCTV-SC), and the last one a hairpin type, containing the sense and antisense sequences of the coat protein gene separated by an intron (pCTV-dsCP). Ten transgenic lines of each gene construct and each cultivar were previously confirmed by Southern blot and RT-PCR analysis, totalizing 60 transgenic lines. These lines were cloned and grafted into C. limonia and into C. aurantium, totaling 360 plants. The plants, along with non-transgenic plants used as control, were challenged four times with the CTV by means of viruliferous Toxoptera citricida. Indirect ELISA using monoclonal antibody against the CTV coat protein or the Real-time PCR using primers to amplify the CTV genes p20 and p23 were used to detect the virus in the tested plants, 4 weeks after inoculation. Variation in the virus resistance was observed among different transgenic constructs and different clones of the same plant. Some clones were not infected with the virus even after the fourth inoculation, indicating a possible resistance to the pathogen. A total of 30 genetic transformation experiments of sour orange were performed, using as explants internodal segments, epicotyl segments and cotyledon fragment with hypocotyl attached. GUS reaction detected two shoots positive (transformation efficiency of 0,13%). These shoots were in vitro grafted in Carrizo citrange, but only one shoot developed. The plant obtained was acclimatized in greenhouse.

A produção vegetal é dependente do processo fotossintético. As técnicas de biologia molecular e transformação genética de plantas trouxeram boas perspectivas para a alteração do metabolismo fotossintético. Plantas transgênicas de tabaco (Nicotiana tabacum, L.) que superexpressam o gene quimérico Lhcb1*2 de ervilha têm sido estudadas por apresentarem uma série de alterações no desenvolvimento e no metabolismo fotossintético, em relação à linhagem selvagem. Vários autores observaram mudanças morfológicas, fisiológicas, bioquímicas e adaptativas que favorecem essas plantas em diversas condições de cultivo. O objetivo desse trabalho foi o de avaliar o impacto da superexpressão desse gene na formação plastidial de plântulas de tabaco germinadas e mantidas no escuro por sete dias e depois transferidas à luz, com coletas periódicas de 0, 6, 18 e 120 horas pós-iluminação. O desenvolvimento plastidial, avaliado por microscopia de luz, mostra um provável adiantamento na formação dos cloroplastos dos materiais vegetais transgênicos (TR1 e TR2) em relação à selvagem (WT). A análise de ultraestrutura dos plastídios por microscopia eletrônica de transmissão, demostrou um real adiantamento na formação dos cloroplastos maduros nas duas linhagens transgênicas A análise de Western blot confimou a presença de proteínas específicas do fotossistema II (Lhcb 1-2 e D1). Este fato implica que a montagem do aparato fotossintético é antecipada nos transgênicos, assim como o desenvolvimento morfológico e estrutural observado nos plastídios. / The vegetal production is strictly dependent on the photosynthetic process. Techniques of molecular biology and genetic transformation of plants brought good perspectives for the alteration of the photosynthetic metabolism. Transgenic tobacco plants (Nicotiana tabacum, L.) which express the chimeric pea Lhcb1*2 gene were pbtained and presenta series of alterations on development and photosynthetic metabolism in relation to the wild type. Previous analysis have demonstrated morphological, physiological, biochemical and adaptative changes that favour these transgenic lines in various conditions of culture. The aim of this work was to evaluate the impact of the expression of this gene in the plastid formation, of tobacco seedlings. Seeds were germinated and kept in darknes for seven days, and transferred to light. The seedlings were then collected after 0, 6, 18 and 120 hours of exposure to continuous ilumination. The plastidial development evaluated by light microscopy, showed an advanced chloroplast formation of the transgenic lines (TR1 and TR2) in relation to the wild type (WTSR1). The ultrastructural analysis of the plastids by electronic microscopy showed, indeed on advanced formation of mature chloroplasts in the transgenic lines. The Western blot analysis confirmed the presence of two specific proteins (CAB and D1), of the photosystem II. This fact implies that the assembly of the photosynthetic apparatus might occurs earlier in the transgenic lines, as well as the morphological and structural development of the plastids.

Os custos adaptativos associados à resistência de insetos a inseticidas podem ser explorados mediante a integração com outras estratégias de controle de pragas em programas de Manejo Integrado de Pragas (MIP). No presente estudo, objetivou-se verificar custos adaptativos associados à resistência de Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) ao inseticida piretroide lambda-cyhalothrin e suas implicações na utilização do hospedeiro pelo parasitoide larval Campoletis aff. flavicincta (Hymenoptera: Ichneumonidae) e as interações com milho geneticamente modificado (MON810) que expressa a proteína Cry1Ab de Bacillus thuringiensis Berliner (milho Bt) e na resposta imunológica ao parasitismo por Campoletis aff. flavicincta. Foram verificados presença de custos adaptativos associados à resistência de S. frugiperda a lambda-cyhalothrin, dado o prolongamento no desenvolvimento larval e duração pupal, redução do peso de pupas fêmeas e longevidade das fêmeas, razão sexual, taxa líquida de reprodução (Ro), taxa intrínseca de aumento (rm) e taxa finita de aumento () de insetos resistentes ao inseticida. Não foi verificada diferença na aceitação de lagartas de S. frugiperda suscetível e resistente a lambda-cyhalothrin por Campoletis aff. flavicincta. Entretanto, o parasitismo de lagartas resistentes foi maior que de suscetíveis em estudos de gaiolas com milho Bt e não-Bt. Posteriormente, foram conduzidos estudos para avaliar, por PCR em tempo real, a expressão diferencial de genes associados ao metabolismo (proteína rica em metionina), resposta imunológica (calreticulina, lisozima, colágeno IV-2, hemócito protease-3, serina protease, imunolectina, receptor scavenger classe C) e detoxificação de xenobióticos (glutationa-S-transferase 145 e as monoxigenases P450 Cyp9A31 e Cyp333B2) expressos em diferentes tecidos (tecido adiposo, hemócitos e/ou mesêntero), na ausência e presença de parasitismo de lagartas das duas linhagens de S. frugiperda por Campoletis aff. flavicincta. No geral, a expressão gênica em lagartas suscetíveis foi superior àquela de lagartas resistentes a lambda-cyhalothrin, independente do período de desenvolvimento, do tecido avaliado e da presença ou não do parasitismo por Campoletis aff. flavicincta. E por fim, foram conduzidos estudos para avaliar o efeito da resistência de S. frugiperda a lambda-cyhalothrin nas respostas imunológica celular (contagem total de hemócitos) e humoral (atividade das fenoloxidases, lisozimática e antimicrobiana e concentração de óxido nítrico) de lagartas, tanto na ausência como na presença do parasitismo por Campoletis aff. flavicincta. A resistência de S. frugiperda a lambda-cyhalothrin induziu somente a pequenas alterações no sistema imunológico do hospedeiro (aumento do número total de hemócitos, redução da atividade antimicrobiana e aumento da atividade lisozimática), as quais não interferem a ponto de resultar em custos adaptativos que leve à maior exploração de lagartas resistentes na presença do parasitismo por Campoletis aff. flavicincta. Portanto, o manejo de S. frugiperda mediante o emprego da tecnologia de milho Bt e do controle biológico via parasitoide Campoletis aff. flavicincta pode favorecer o restabelecimento da suscetibilidade de S. frugiperda a lambda-cyhalothrin. / Fitness costs of insect resistance to insecticides can be exploited by integrating other pest control strategies in Integrated Pest Management (IPM) programs. The objective of this research was to evaluate the existence of fitness costs associated with the resistance of Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) to the pyrethroid insecticide lambda-cyhalothrin and their implications for host use by the larval parasitoid Campoletis aff. flavicincta (Hymenoptera: Ichneumonidae) and interactions with the genetically modified maize (MON810) that expresses Cry1Ab toxin of Bacillus thuringiensis Berliner (Bt maize) and in the immune response to parasitization by Campoletis aff. flavicincta. Fitness costs associated to resistance of S. frugiperda to lambda-cyhalothrin were detected by the delay in larval and pupal development, reduction in the pupal weight and longevity of females, sex ratio, net reproductive rate (Ro), intrinsic rate of natural increase (rm) and the finite rate of increase (). No differences were detected in host acceptance and survival of Campoletis aff. flavicincta in susceptible and lambda-cyhalothrin-resistant larvae of S. frugiperda. However, larval parasitization was higher on the resistant than on the susceptible strain of S. frugiperda in cage studies with Bt and non-Bt maize plants. Then, studies were conducted by using Real time-PCR to evaluate the differential expression of genes associated with metabolism (methionine-rich protein), immune response (calreticulin, lysozyme, collagen IV-2, protease-3 hemocyte, serine protease, immunolectin, scavenger receptor class C) and xenobiotic detoxification (glutathione-S-transferase 145 and P450 monooxygenases Cyp9A31 and Cyp333B2) expressed in different tissues (fat body, hemocytes and/or midgut), in the absence and presence of larval parasitization of both strains of S. frugiperda by Campoletis aff. flavicincta. Overall, gene expression in susceptible larvae was higher than that of lambdacyhalothrin- resistant larvae, regardless of the period of development, tissue evaluated and presence or not of parasitization by Campoletis aff. flavicincta. Finally, studies were conducted to evaluate the effect of the resistance of S. frugiperda to lambda-cyhalothrin on cellular (total hemocyte count) and humoral (phenoloxidases, lysozyme and antimicrobial activities and nitric oxide concentration) immune responses in the absence or presence of parasitization by Campoletis aff. flavicincta. The resistance of S. frugiperda to lambdacyhalothrin conferred only minor changes in the host immune system (increased total hemocyte count, reduced antimicrobial activity and increased lysozyme activity), which may not interfere with fitness costs leading to higher exploitation of resistant larvae in the presence of parasitization by Campoletis aff. flavicincta. Therefore, the management of S. frugiperda by using the Bt maize technology and the biological control via parasitoid Campoletis aff. flavicincta can favor the resetting to susceptibility of S. frugiperda to lambda-cyhalothrin.

Cheung Chun Kai. / Thesis submitted in: December 2003. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 133-146). / Abstracts in English and Chinese. / Acknowledgements --- p.ii / Abstract --- p.iv / 摘要 --- p.vii / Table of Contents --- p.ix / List of Tables --- p.xv / List of Figures --- p.xvi / List of Abbreviations --- p.xxi / Chapter Chapter 1 --- Overview --- p.1 / Chapter Chapter 2 --- Literature Review --- p.3 / Chapter 2.1 --- Historical background --- p.3 / Chapter 2.2 --- Insulin-like growth factor --- p.5 / Chapter 2.2.1 --- Structure and synthesis --- p.5 / Chapter 2.2.2 --- Physiologic role and biological actions --- p.6 / Chapter 2.3 --- Insulin-like growth factor binding protein-3 --- p.8 / Chapter 2.3.1 --- Structure and synthesis --- p.8 / Chapter 2.3.2 --- Physiologic role and biological actions --- p.8 / Chapter 2.4 --- Clinical aspects --- p.10 / Chapter 2.4.1 --- Metabolic effects of IGF-1 --- p.10 / Chapter 2.4.1.1 --- Similarities between IGF-I and insulin --- p.11 / Chapter 2.4.1.2 --- Differences between IGF-I and insulin --- p.13 / Chapter 2.4.2 --- Glucose and protein metabolism --- p.14 / Chapter 2.4.3 --- Therapeutic use of IGF-I --- p.15 / Chapter 2.4.3.1 --- Type 1 diabetes mellitus --- p.16 / Chapter 2.4.3.2 --- Type 2 diabetes mellitus --- p.17 / Chapter 2.4.4 --- Side effects --- p.19 / Chapter 2.5 --- World demands --- p.21 / Chapter 2.5.1 --- Significance of large-scale production --- p.21 / Chapter 2.5.2 --- IGF-I production --- p.21 / Chapter 2.6 --- Plants as bioreactors --- p.24 / Chapter 2.6.1 --- Medical molecular farming --- p.24 / Chapter 2.6.2 --- Advantages of plant bioreactor --- p.24 / Chapter 2.6.3 --- Commercial biopharmaceutical protein --- p.25 / Chapter 2.7 --- Tobacco expression system --- p.26 / Chapter 2.7.1 --- Tobacco model plant --- p.26 / Chapter 2.7.2 --- Transformation methods --- p.26 / Chapter 2.8 --- Hypotheses and aims of study --- p.28 / Chapter Chapter 3 --- Expression of Human IGF-I and IGFBP-3 in Transgenic Tobacco --- p.30 / Chapter 3.1 --- Introduction --- p.30 / Chapter 3.2 --- Materials and methods --- p.31 / Chapter 3.2.1 --- Chemicals --- p.31 / Chapter 3.2.2 --- Plant materials --- p.31 / Chapter 3.2.3 --- Bacterial strains --- p.32 / Chapter 3.2.4 --- Codon modification of IGF-I and IGFBP-3 cDNAs --- p.32 / Chapter 3.2.5 --- Transient assay to study IGF-I or IGFBP-3 translatability --- p.39 / Chapter 3.2.5.1 --- Construction of chimeric genes for particle bombardment --- p.39 / Chapter 3.2.5.2 --- Particle bombardment of GUS fusion constructs --- p.42 / Chapter 3.2.6 --- Construction of chimeric genes for tobacco transformation --- p.44 / Chapter 3.2.6.1 --- Construction of chimeric genes with different promoters --- p.44 / Chapter 3.2.6.1.1 --- Construction of chimeric gene with CaMV 35S promoter --- p.44 / Chapter 3.2.6.1.2 --- Construction of chimeric genes with phaseolin promoter --- p.46 / Chapter 3.2.6.2 --- Construction of fusion constructs --- p.48 / Chapter 3.2.6.2.1 --- Construction of GUS fusion constructs --- p.48 / Chapter 3.2.6.2.2 --- Construction of LRP fusion constructs --- p.51 / Chapter 3.2.6.3 --- Construction of phaseolin targeting constructs --- p.56 / Chapter 3.2.6.3.1 --- Construction of phaseolin targeting constructs without AFVY --- p.56 / Chapter 3.2.6.3.2 --- Construction of phaseolin targeting constructs with AFVY --- p.60 / Chapter 3.2.6.4 --- Cloning of chimeric genes into Agrobacterium binary vector pBI 121 --- p.64 / Chapter 3.2.7 --- Confirmation of sequencing fidelity of chimeric genes --- p.66 / Chapter 3.2.8 --- Transformation of Agrobacterium by electroporation --- p.66 / Chapter 3.2.9 --- Transformation of tobacco --- p.67 / Chapter 3.2.10 --- Selection and regeneration of transgenic tobacco --- p.67 / Chapter 3.2.11 --- GUS assay --- p.68 / Chapter 3.2.12 --- Extraction of leaf genomic DNA --- p.68 / Chapter 3.2.13 --- PCR of genomic DNA --- p.69 / Chapter 3.2.14 --- Synthesis of DIG-labeled double-stranded DNA probe --- p.69 / Chapter 3.2.15 --- Southern blot analysis --- p.70 / Chapter 3.2.16 --- Extraction of total RNA from leaves or developing seeds --- p.70 / Chapter 3.2.17 --- Northern blot analysis --- p.71 / Chapter 3.2.18 --- Extraction of total protein --- p.71 / Chapter 3.2.19 --- Tricine SDS-PAGE --- p.72 / Chapter 3.2.20 --- Western blot analysis --- p.72 / Chapter 3.2.21 --- Enterokinase digestion of fusion protein --- p.73 / Chapter Chapter 4 --- Results --- p.74 / Chapter 4.1 --- Particle bombardment for transient assay --- p.74 / Chapter 4.1.1 --- Construction of GUS fusion genes for particle bombardment --- p.74 / Chapter 4.1.2 --- Transient expression of GUS fusion genes in soybean cotyledons and tobacco leaves --- p.76 / Chapter 4.2 --- Construction of chimeric genes for tobacco transformation --- p.78 / Chapter 4.3 --- "Tobacco transformation, selection and regeneration" --- p.81 / Chapter 4.4 --- Detection of GUS activity --- p.83 / Chapter 4.5 --- Detection of transgene integration --- p.84 / Chapter 4.5.1 --- Extraction of genomic DNA and PCR --- p.84 / Chapter 4.5.2 --- Southern blot analysis --- p.88 / Chapter 4.6 --- Detection of transgene transcription --- p.92 / Chapter 4.6.1 --- Extraction of total RNA --- p.92 / Chapter 4.6.2 --- Northern blot analysis --- p.92 / Chapter 4.7 --- Detection of transgene translation --- p.99 / Chapter 4.7.1 --- Extraction of total protein and Tricine SDS-PAGE --- p.99 / Chapter 4.7.2 --- Western blot analysis --- p.102 / Chapter 4.7.3 --- Enterokinase digestion of fusion protein --- p.109 / Chapter Chapter 5 --- Discussion --- p.111 / Chapter 5.1 --- Codon modification of IGF-I and IGFBP-3 cDNAs --- p.114 / Chapter 5.2 --- Transient expression of IGF-I and IGFBP-3 cDNAs --- p.116 / Chapter 5.3 --- Fusion of IGF-I and IGFBP-3 cDNA with LRP gene --- p.118 / Chapter 5.4 --- Enterokinase digestion --- p.120 / Chapter 5.5 --- Phaseolin targeting signal --- p.122 / Chapter 5.6 --- Gene silencing --- p.124 / Chapter 5.7 --- Future perspectives --- p.128 / Chapter Chapter 6 --- Conclusion --- p.131 / References --- p.133

by Ng Wing Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 156-174). / Abstracts in English and Chinese. / Acknowledgements --- p.iii / Abstract --- p.v / 摘要 --- p.vii / Table of Contents --- p.ix / List of Figures --- p.xiii / List of Tables --- p.xvi / List of Graphs --- p.xvii / List of Abbreviations --- p.xviii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter Chapter 2 --- Literature Review --- p.3 / Chapter 2.1 --- Human granulocyte colony-stimulating factor (hG-CSF) --- p.3 / Chapter 2.1.1 --- Historical background --- p.3 / Chapter 2.1.2 --- Physiological Roles --- p.5 / Chapter 2.1.3 --- Molecular properties --- p.8 / Chapter 2.1.4 --- Biochemical properties --- p.9 / Chapter 2.1.5 --- Comparison to G-CSF of other species --- p.11 / Chapter 2.1.6 --- Biological Activities --- p.12 / Chapter 2.1.7 --- Clinical Applications --- p.14 / Chapter 2.1.7.1 --- Clinical use in myelosuppressive chemotherapy and neutropenic fever --- p.14 / Chapter 2.1.7.2 --- Clinical use in bone marrow transplantation (BMT) and peripheral blood progenitor cell (PBPC) transplantation --- p.14 / Chapter 2.1.7.3 --- Clinical use in HIV infection --- p.16 / Chapter 2.1.7.4 --- Clinical use in diabetes mellitus --- p.17 / Chapter 2.1.7.5 --- Clinical use in severe chronic neutropenia --- p.18 / Chapter 2.1.7.6 --- Future prospects --- p.18 / Chapter 2.1.7.7 --- Dosages and adverse effects --- p.19 / Chapter 2.1.8 --- Economic value --- p.20 / Chapter 2.2 --- Plant as bioractor --- p.20 / Chapter 2.2.1 --- Medical molecular farming --- p.20 / Chapter 2.2.2 --- Commercial biopharmaceutical proteins --- p.25 / Chapter 2.2.3 --- Transgenic plants producing hematopoietic growth factors --- p.25 / Chapter 2.2.3.1 --- Granulocyte-macrophage colony-stimulating factor (GM-CSF) --- p.26 / Chapter 2.2.3.2 --- Interleukin-2 (IL-2) --- p.28 / Chapter 2.3 --- Rice as expression system --- p.29 / Chapter 2.3.1 --- Characteristics --- p.29 / Chapter 2.3.2 --- Advantages of using rice as bioreactor --- p.30 / Chapter 2.3.3 --- Previous studies --- p.31 / Chapter 2.3.4 --- Transformation method --- p.33 / Chapter 2.3.5 --- Super-binary vector --- p.34 / Chapter 2.4 --- Strategies for enhancing protein expression level --- p.36 / Chapter 2.4.1 --- Vacuolar targeting --- p.36 / Chapter 2.4.1.1 --- Protein targeting signals --- p.38 / Chapter 2.4.1.2 --- Binding protein of 80kDa (BP-80) --- p.39 / Chapter 2.4.1.3 --- a-Tonoplast intrinsic protein (α-TIP) --- p.39 / Chapter 2.4.1.4 --- Receptor homology region-transmembrane domain-Ring H2 motif (RMR) --- p.40 / Chapter 2.4.2 --- Fusion with glutelin in rice --- p.41 / Chapter 2.5 --- Hypotheses and aims of this study --- p.43 / Chapter Chapter 3 --- Materials and Methods --- p.45 / Chapter 3.1 --- Introduction --- p.45 / Chapter 3.2 --- Chemicals --- p.45 / Chapter 3.3 --- Bacterial strains --- p.46 / Chapter 3.4 --- Chimeric genes construction --- p.46 / Chapter 3.4.1 --- Protein targeting constructs --- p.51 / Chapter 3.4.2 --- Enterokinase site constructs --- p.60 / Chapter 3.4.3 --- Glutein signal peptide constructs --- p.65 / Chapter 3.4.4 --- Glutelin fusion constructs --- p.70 / Chapter 3.4.5 --- Sequence fidelity of chimeric genes --- p.77 / Chapter 3.4.6 --- Cloning of chimeric genes into rice super-binary vector --- p.77 / Chapter 3.5 --- Rice transformation --- p.79 / Chapter 3.5.1 --- Plant materials --- p.79 / Chapter 3.5.2 --- Agrobacterium transformation --- p.79 / Chapter 3.5.3 --- A grobacterium-mediated transformation of rice --- p.79 / Chapter 3.6 --- Transgenic expression --- p.81 / Chapter 3.6.1 --- Extraction of leaf genomic DNA --- p.81 / Chapter 3.6.2 --- Synthesis of DIG-labeled double-stranded DNA probe --- p.82 / Chapter 3.6.3 --- Southern blot analysis --- p.83 / Chapter 3.6.4 --- Extraction of total RNA from immature rice seeds --- p.84 / Chapter 3.6.5 --- Northern blot analysis --- p.85 / Chapter 3.6.6 --- Protein extraction --- p.86 / Chapter 3.6.7 --- Tricine SDS-PAGE --- p.86 / Chapter 3.6.8 --- Western blot analysis --- p.87 / Chapter 3.6.9 --- Enterokinase digestion of EK fusion proteins --- p.88 / Chapter 3.7 --- Confocal immunoflorescence studies of rhG-CSF in rice grain --- p.89 / Chapter 3.7.1 --- Preparation of sample sections --- p.89 / Chapter 3.7.2 --- Double-labeling of fluorescence probes --- p.89 / Chapter 3.7.3 --- Image collection --- p.90 / Chapter 3.8 --- Functional analysis of rhG-CSF --- p.91 / Chapter 3.8.1 --- Culture of NFS-60 cells --- p.91 / Chapter 3.8.2 --- MTT cell proliferation assay --- p.92 / Chapter 3.9 --- Bacterial expression of anti-hG-CSF --- p.93 / Chapter 3.9.1 --- pET expression in E. coli --- p.93 / Chapter 3.9.2 --- Purification of His-hG-CSF --- p.97 / Chapter 3.9.3 --- Immunization of rabbits --- p.97 / Chapter Chapter 4 --- Results --- p.99 / Chapter 4.1 --- Construction of chimeric genes for rice transformation --- p.99 / Chapter 4.2 --- "Rice transformation, selection and regeneration" --- p.103 / Chapter 4.3 --- Southern blot analysis --- p.105 / Chapter 4.4 --- Northern blot analysis --- p.109 / Chapter 4.5 --- Western blot analysis --- p.114 / Chapter 4.6 --- Enterokinase digestion of EK fusion proteins --- p.125 / Chapter 4.7 --- Confocal immunofluorescence studies of rhG-CSF in transgenic rice grain --- p.128 / Chapter 4.8 --- Functional analysis of rhG-CSF --- p.132 / Chapter 4.9 --- Bacterial expression of anti-hG-CSF --- p.135 / Chapter 4.9.1 --- Expression and purification of recombinant His-hG-CSF in E. coli --- p.135 / Chapter 4.9.2 --- Titer and specificity of the anti-serum --- p.137 / Chapter Chapter 5 --- Discussion --- p.139 / Chapter 5.1 --- Introduction --- p.139 / Chapter 5.2 --- Fusion of hG-CSF with protein sorting determinants --- p.141 / Chapter 5.3 --- Fusion of hG-CSF with rice glutelin --- p.145 / Chapter 5.4 --- Glutelin signal peptide --- p.146 / Chapter 5.5 --- O-glycosylation --- p.148 / Chapter 5.6 --- Enterokinase digestion --- p.148 / Chapter 5.7 --- Expression level of rhG-CSF --- p.149 / Chapter 5.8 --- Functional analysis of rhG-CSF --- p.151 / Chapter 5.9 --- Future perspectives --- p.151 / Chapter Chapter 6 --- Conclusion --- p.155 / References --- p.156

No Brasil a citricultura está entre as culturas de maior importância. A produtividade dessa cultura no país ainda é considerada baixa e esse fato se deve, em parte, a diversas pragas e doenças. Dentre as doenças, tem-se a tristeza, causada pelo Citrus tristeza virus (CTV). Esse patógeno também pode estar relacionado com outra importante doença da cultura, a morte súbita dos citros (MSC). Com isso, o CTV ganhou ainda maior expressão. Uma alternativa para controlar viroses de plantas é a obtenção de plantas transgênicas resistentes a esses patógenos. Este trabalho objetivou caracterizar com análise molecular e avaliar a resistência ao CTV de plantas transgênicas de laranja Valência (Citrus sinensis L. Osbeck), contendo fragmentos do genoma do CTV, em três construções gênicas diferentes. A transgenia das plantas foi confirmada por análises de Southern blot. A transcrição do transgene foi avaliada por RT-PCR. O material foi inoculado com duas borbulhas infectadas pelo isolado Pêra- IAC, enxertadas no porta-enxerto abaixo do ponto de enxertia da copa transgênica, e pelo vetor Toxoptera citricida infectado. Após quatro semanas da inoculação, para avaliar a resistência ao vírus, brotações da copa transgênica foram submetidas ao teste de ELISA sanduíche indireto com anticorpo monoclonal contra a proteína da capa protéica do CTV. Os resultados indicaram variação na resistência à translocação do vírus nas diferentes construções transgênicas utilizadas e entre clones de uma mesma planta. Todas as linhagens transgênicas inoculadas indicaram a presença do vírus em pelo menos uma das três repetições avaliadas, quando inoculadas por enxertia. Quando inoculadas pelo vetor algumas plantas apresentaram todos os seus clones com baixos valores de absorbância, indicando uma possível resistência ao patógeno. / In Brazil, citrus is one of the most important cultures. The productivity of this culture in the country is still considered low and this fact is due to several pests and diseases that affect the crop. Among the diseases there is the tristeza, caused by Citrus tristeza virus (CTV). This pathogen can also be related with another important disease, the citrus sudden death. Therefore, CTV acquired much more significance. This work aimed to characterize with molecular analysis and to evaluate the resistance to CTV of transgenic Valência plants (Citrus sinensis L. Osbeck), containing genomic fragments of CTV, in three different transgenic constructs. The plants were confirmed as transgenic by Southern blot. The transcription of the transgene was evaluated by RT-PCR. The transgenic plants were challenged with a weak strain of CTV, CTV-IAC, by bud inoculation with two infected bubbles, and by the infected vector Toxoptera citricida. After four weeks of inoculation, the evaluation of viral replication in the transgenic seious was done by ELISA indirect sandwich with monoclonal antibody against the CTV coat protein. The results indicated variation of the resistance to the translocation of the virus between the different transgenic constructs used and between clones of the same plant. All the inoculated plants indicated the presence of the virus in, at least, one of the three evaluated clones, when inoculated by grafting. When inoculated by the vector some plants had all their clones with low values of virus, indicating a possible resistance to the pathogen.