Submitted by Erika Demachki (erikademachki@gmail.com) on 2014-10-13T20:47:06Z No. of bitstreams: 2 Dissertacão - Clistiane dos Anjos Mendes - 2010.pdf: 3556172 bytes, checksum: 0afdcb2cfe1cae06f8e21fb23ad19fba (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Jaqueline Silva (jtas29@gmail.com) on 2014-10-13T21:16:57Z (GMT) No. of bitstreams: 2 Dissertacão - Clistiane dos Anjos Mendes - 2010.pdf: 3556172 bytes, checksum: 0afdcb2cfe1cae06f8e21fb23ad19fba (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-10-13T21:16:57Z (GMT). No. of bitstreams: 2 Dissertacão - Clistiane dos Anjos Mendes - 2010.pdf: 3556172 bytes, checksum: 0afdcb2cfe1cae06f8e21fb23ad19fba (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2010-08-16 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Rice is consumed by more than half of the world population. The expectation of substantial population growth and consequently the increase of the consumption has demontrated that the present rice production will not supply the future’s demand. As a result, studies that aim at the increase of the yield must be prioritized by scientific co&unity. Aiming to identify genomic regions related to grain yield and its component traits (panicle number per meter, grain panicle number and hundred grain mass), it was carried out the associative mapping analysis. The analysis of association used two types of SSR markers: a) structural genome derived markers, and b) transcriptome derived markers, i.e. developed from transcripts from marker-anchored genomic regions previously related to yield and its components from 27 different QTL maps already published. The associative mapping methodology used included the whole genome scanning and candidate genes approaches. The molecular characterization used 186 accessions from Embrapa Rice Core Collection (ERiCC), whereas 76 accessions from lowland system of cultivation, and 113 upland system of cultivation. The experimental design was the randomized block with 4 repetitions, and included the cultivars BR Irga 409, Metica 1 and BRS Caiapó as controls. It were evaluated the following traits: Panicle number per meter (NPM), panicle (NGP), Hundred grain mass (PCG) and productivity. The 186 markers were genotyped by 29 transcript-derived SSR markers and 86 SSR markers derived from structural genome. For the irrigated accessions was verified positive correlation for panicle number per meter and productivity (0,2424; p<0,01) and by productivity and hundred grain mass (p<0,05; 0,1457) and negative correlation for panicle number per meter and grain number per panicle (-0,457; p<0,01). For the upland accessions it was observed positive correlation for grain number per panicle and productivity (0,4243; p<0,01). Negative correlations were observed for panicle number per meter and hundred grain weight (p<0,01; -0,2114). Based on 44 SSR markers developed in this work, 181 alelles were detected, an average of 5.5 alelles per locus, average PIC of 0.44 and 25 private alleles. The analysis with 115 SSR markers identified 43 significant associations for grain number per panicle considering the cultivation system upland and three associations for lowland system; 7 significant associations for panicle number per meter for lowland system, and 9 were significant for upland cultivation systems; 14 associations were significant for hundred grain mass in lowland system of cultivarion, and 43 were significant for upland system of cultivarion; 3 associations were significant for grain yield in upland system of cultivarion. The association analysis identified 61 SSR markers consistently associated to yield and its components. It were detected 33 associations statiscally significant to one or more traits, which validated the previous results obtained by QTL mapping. The markers RM125, RM152 e Q69JE3 showed the higher number of associations per trait. The association productivity and PCG, and NCP, respectively, were previously found in the literature. The markers associated to the evaluated traits permit to initiate a scientific program to identify the candidate genes and to proceed the marker assisted selection The accessions CNA0001107, CNA0005478, CNA0010533, CNA0003287, IR 36, CNA0003602, CNA0006413, CNA0006174, CNA0003289, CNA0002253, CNA0004098, CNA0001416, CNA0003490 e CNA0000994 showed the highest number of favorable alleles considering 16 the markers associated to yield and its components. The identified accessions from this work as having the higher number of favorable alleles from the evaluated traits will be recommended to be used as genitors in Brazilian rice breeding programs. The mixed approaches to the associative mapping used in the present work was interesting to permit that, even using a low marker density, in couple with the linkage disequilibrium found in rice, some previously mapped markers from QTL analyses were again associated to traits evaluated in field experiments. This strategy can be used to other traits of interest for rice, such as drought and cold tolerance, and disease resistance. / O arroz é consumido por mais da metade da população mundial. As expectativas de aumento substancial da população, e consequentemente aumento do consumo, têm demonstrado que a produção atual da cultura não suprirá a demanda. A produção de grãos do arroz é uma característica complexa determinada pelos seus três componentes: número de panículas, número de grãos por panícula e peso de grãos, os quais são típicos caracteres quantitativos. A evolução no mapeamento do genoma, o sequenciamento e as pesquisas em genômica funcional têm fornecido ferramentas poderosas para estudar as bases genética e molecular desses caracteres quantitativos. Buscando identificar regiões genômicas responsáveis pela produtividade e componentes de produção (número de panículas por metro, número de grãos por panícula e peso de 100 grãos), foi realizada a análise de mapeamento associativo. O mapeamento associativo foi realizado com dois tipos de marcadores SSR: a) marcadores fluorescentes, e b) marcadores desenvolvidos a partir de transcritos de regiões genômicas ancoradas por marcadores previamente relacionados à produção e seus componentes por 27 análises de QTLs publicadas na literatura. Dessa forma, nesse trabalho foi utilizada uma metodologia híbrida de mapeamento associativo incluindo as técnicas de whole genome scanning e de genes candidatos. A caracterização molecular foi realizada em 186 acessos da coleção nuclear de arroz da embrapa (CNAE), sendo 76 acessos do sistema de cultivo irrigado e 113 do sistema de cultivo de sequeiro, selecionados por não apresentarem vínculo genético. Destes três acessos foram utilizados como testemunha em ambos experimentos, sendo BR Irga 409, Metica 1 e BRS Caiapó. Os acessos foram avaliados em dois experimentos de campo, um para os genótipos do sistema de cultivo irrigado, e outro para os do sistema de cultivo de sequeiro, no delineamento de blocos casualizados com 4 repetições. Foram avaliados os seguintes caracteres: Número de panículas por metro (NPM), Número de grãos por panícula (NGP), Peso de 100 grãos (PCG) e Produtividade em kg.ha-1 (Prod). Para a análise de associação foram utilizados 29 marcadores SSR derivados de transcritos e 86 marcadores SSR derivados de sequências do DNA estrutural. Em relação as análises fenotípicas, os coeficientes de variação obtidos foram consistentes de acordo com o sistema de cultivo. Para os acessos de arroz irrigado foi verificada correlação positiva para NPM e Prod (0,2424; p<0,01) e Prod e PCG (0,1457; p<0,05) e correlação negativa para NPM e NGP (-0,457; p<0,01). Para os acessos do sistema de cultivo de sequeiro foi observada correlação positiva para NGP e Prod (0,4243; p<0,01). Correlação negativa foi observada para NPM e PCG (-0,2114; p<0,01). Em relação aos dados moleculares, para este trabalho foram desenvolvidos 44 marcadores SSR baseados em 27 mapas de QTLs. Destes, foram obtidos 181 alelos e uma média de 5,5 alelos por loco. Resultante da análise dos 44 marcadores SSR foram obtidos 29 marcadores que apresentaram padrão de bandas polimórfico, destes foram obtidas um PIC médio de 0,44 e 25 alelos privados. Para a análise de associação foram usados 29 marcadores polimórficos juntamente aos 86 marcadores fluorescentes (previamente avaliados por Borba et al. 2009), foram identificadas 43 associações significativas para NGP nos acessos de sequeiro e três associações significativas nos acessos de irrigado; 7 associações significativas para NPM nos acessos de irrigado e 9 associações significativas nos acessos de sequeiro; 14 associações foram significativas para PCG nos acessos de irrigado e 43 associações foram significativas nos acessos de sequeiro; 14 3 associações significativas para Prod foram encontradas nos acessos de sequeiro. A análise de associação identificou 61 marcadores SSR associados de forma consistente à produção e aos seus componentes. Foram detectadas 33 associações estatisticamente significativas a um ou mais caracteres, as quais validaram os resultados encontrados por meio das análises de mapeamento de QTLs realizadas em trabalhos anteriores. Os marcadores RM125, RM152 e Q69JE3 apresentaram maior número de associações por caracteres, sendo que algumas associações já foram previamente descritas na literatura. Os marcadores associados aos caracteres avaliados podem ser usados em programa de melhoramento visando a identificação de genes candidatos e seleção assistida. Os acessos CNA0001107, CNA0005478, CNA0010533, CNA0003287, IR 36, CNA0003602, CNA0006413, CNA0006174, CNA0003289, CNA0002253, CNA0004098, CNA0001416, CNA0003490 e CNA0000994 foram os que apresentaram o maior número de alelos favoráveis nos locos marcadores associados aos caracteres produção e seus componentes. Os acessos identificados por este trabalho como tendo o maior número de alelos favoráveis para os caracteres avaliadas serão recomendados para uso como genitores em programas de melhoramento de arroz do Brasil. A metodologia híbrida de mapeamento associativo utilizado por este trabalho foi interessante por permitir que, mesmo utilizando baixa resolução, juntamente com o desequilíbrio de ligação encontrado em arroz, determinados locos previamente mapeados em análises de QTLs foram novamente associados aos caracteres avaliados nos ensaios de campo. Esta estratégia pode ser repetida para outros caracteres de interesse em arroz, como tolerância à seca e frio e resistência a doenças.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2017-05-23T19:49:38Z No. of bitstreams: 1 2014 - Rafael Passos Rangel.pdf: 2601166 bytes, checksum: 8cd9cbb0ff858a2d7c7c31027eaf3efb (MD5) / Made available in DSpace on 2017-05-23T19:49:38Z (GMT). No. of bitstreams: 1 2014 - Rafael Passos Rangel.pdf: 2601166 bytes, checksum: 8cd9cbb0ff858a2d7c7c31027eaf3efb (MD5) Previous issue date: 2014-02-26 / CAPES / Root morphology is a major factor related to the efficiency of nutrient acquisition. For this reason, this work compared this characteristic in three contrasting rice varieties: IAC 47 (improved variety) and two traditional varieties cultivated in the state of Maranh?o (Piau? and Manteiga originated in an environment with low nutrient input). These varieties were subjected to a low N-NH4+ supply and analyzed for the root morphology parameters, expression of ammonium high affinity transport system (OsAMT1.1~1.3) and GS activity in three segments of the root system. It was also determined the soluble fractions and total nitrogen in the different segments of the plants (leaves, sheaths and roots) to verify the metabolic alterations as a result of low ammonium input. The GS activity was higher in the second segment in the Manteiga and IAC-47 varieties compared to Piau?, in the other segments. A similar result was observed in the Manteiga variety in the third segment (bottom of the root system) submitted to NH4+ resupply. When grown without NH4+, the Manteiga variety showed higher OsAMT1.3 expression in the lower segment of the root system, and greater root growth, indicating a possible involvement of this transporter in the control of root morphology in rice plants. There was lower expression of OsAMT1.1 and OsAMT1.2 unlike OsAMT1.3 but being distributed to other segments of the root system. In resupply with NH4+ (38 DAG) there was repression of ammonium transporters in the bottom (PI) of the root system and increased expression of OsAMT1.2 the median part (PM) in Piau? variety. In the upper part of the roots (PS) higher levels of ammonium transporter expression was observed in the IAC-47, and Manteiga. This difference in expression between varieties originated from regions with low N availability (Piau? and Manteiga) and improved IAC-47 is an indication that under conditions of limited N, the highest expression of OsAMT1.3 may be associated with an increase in morphological parameters. / A morfologia radicular ? uma das principais caracter?sticas relacionadas ? efici?ncia de aquisi??o de nutrientes em plantas. Deste modo, variedades de arroz contrastantes: uma melhorada, (IAC-47) e duas variedades locais do Maranh?o: (Piau? e Manteiga, origin?rias de cultivos com baixa disponibilidade de nutrientes) foram submetidas a um baixo suprimento de N-NH4+ e avaliadas quanto aos par?metros de morfologia radicular, express?o dos transportadores de alta afinidade para am?nio (OsAMT1.1~1.3) e atividade de GS em tr?s segmentos do sistema radicular. Tamb?m foram determinadas as fra??es sol?veis e o nitrog?nio total nas diferentes partes da planta (folha, bainha e raiz) de forma a acompanhar altera??es metab?licas em resposta a baixa disponibilidade de am?nio. A atividade da GS foi maior na segunda coleta, nas variedades Manteiga e IAC-47 em rela??o a Piau?, nas demais coletas. Resultado semelhante foi observado na parte inferior do sistema radicular, para a variedade Manteiga na terceira coleta com ressuprimento com am?nio. Quando cultivada sem suplementa??o amoniacal, a variedade Manteiga apresentou maior express?o do OsAMT1.3 na parte inferior (PI) do sistema radicular, e maior crescimento de ra?zes, indicando um poss?vel envolvimento desse transportador com o controle da morfologia radicular de plantas de arroz. Houve menor express?o do OsAMT1.1 e OsAMT1.2 diferentemente do OsAMT1.3, por?m distribuindo-se a outros segmentos do sistema radicular. No ressuprimento com am?nio aos 38 dias ap?s a germina??o (38 DAG) houve repress?o dos transportadores de am?nio na parte inferior (PI) do sistema radicular e aumento de express?o do OsAMT1.2 na parte mediana (PM) para a variedade Piau?. Na parte superior das ra?zes (PS) os maiores n?veis de express?o dos transportadores de am?nio de alta afinidade foram observados para as variedades IAC-47 e Manteiga. Esta diferen?a de express?o entre variedades oriundas de regi?es com baixa disponibilidade de N (Piau? e Manteiga) e a variedade melhorada IAC-47 ? um indicio que sob condi??es limitantes de N, a maior express?o do OsAMT1.3 pode estar associado com incremento nos par?metros morfol?gicos.

Yang, Fan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 111-132). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.iii / ABSTRACT --- p.iv / TABLE OF CONTENTS --- p.viii / LIST OF FIGURES --- p.xii / LIST OF TABLES --- p.xv / LIST OF ABBREVIATIONS --- p.xvi / Chapter Chatper 1. --- General Introduction --- p.1 / Chapter Chapter 2. --- Literature Review --- p.5 / Chapter 2.1 --- Facts on food allergy --- p.5 / Chapter 2.1.1 --- Food allergy and its prevalence --- p.5 / Chapter 2.1.2 --- Pathogenesis of food allergy --- p.6 / Chapter 2.1.3 --- Clineal disorders caused and diagnosis of food allergy --- p.8 / Chapter 2.2 --- Allergenicity assessment of genetically engineered food --- p.13 / Chapter 2.2.1 --- The structural and sequence homology of proteins as a criterion for food allergenicity assessment --- p.14 / Chapter 2.2.2 --- Digestion stability as a criterion for food allergenicity assessment --- p.15 / Chapter 2.2.3 --- Animal models for Food Allergenicity Assessment --- p.21 / Chapter 2.3 --- The importance of rice and its nutritional facts --- p.27 / Chapter 2.3.1 --- The importance of rice --- p.27 / Chapter 2.3.2 --- Rice nutritional facts and its relationship with malnutrition --- p.28 / Chapter 2.4 --- Food allergenicity research in rice --- p.30 / Chapter 2.5 --- Glutelin overexpression transgenic rice GT3 --- p.33 / Chapter 2.6 --- Recent and future perspectives for treatment of food allergy --- p.36 / Chapter Chapter 3. --- Materials and Methods --- p.39 / Chapter 3.1 --- Rice Seed Protein Extraction --- p.39 / Chapter 3.1.1 --- Rice varieties for protein extraction --- p.39 / Chapter 3.1.2 --- Protein extraction from rice seeds --- p.39 / Chapter 3.1.3 --- Fractionation of major rice seed storage proteins --- p.40 / Chapter 3.1.4. --- Protein quantification --- p.41 / Chapter 3.1.5 --- Tricine SDS-PAGE --- p.42 / Chapter 3.2 --- Simulated Gastric Digestibility Assay --- p.43 / Chapter 3.2.1 --- Assay System --- p.43 / Chapter 3.2.2 --- Preparation of Simulated Gastric Fluid --- p.43 / Chapter 3.2.3 --- Assay Procedures --- p.44 / Chapter 3.2.4 --- Results Interpretation --- p.44 / Chapter 3.3 --- Construction of Mouse Models --- p.45 / Chapter 3.3.1 --- Mouse strain and reagents used --- p.45 / Chapter 3.3.2 --- Mouse Model I --- p.46 / Chapter 3.3.3 --- Mouse Model II --- p.50 / Chapter 3.3.4 --- Mouse Model III --- p.51 / Chapter 3.4 --- Bioinformatic Analysis of Glutelin Sequence --- p.52 / Chapter 3.5 --- Epitope Mapping of Glutelin --- p.55 / Chapter 3.5.1 --- Bioinformatic Analysis --- p.55 / Chapter 3.5.2 --- Direct and Competitive ELISA --- p.56 / Chapter 3.5.3 --- Western Blot Analysis --- p.57 / Chapter 3.5.4 --- IgE-binding assay --- p.58 / Chapter Chapter 4. --- Results and Discussion --- p.60 / Chapter 4.1 --- Rice Seed Protein Extraction --- p.60 / Chapter 4.1.1 --- Rice Protein Extraction --- p.60 / Chapter 4.1.2 --- Extraction of rice major seed storage protein fractions --- p.62 / Chapter 4.2 --- Simulated Gastric Digestibility Assay --- p.64 / Chapter 4.2.1 --- Pepsin Digestibility of total protein from GT3 and WT rice seeds --- p.64 / Chapter 4.2.2 --- Pepsin Digestibility of major storage protein fractions in GT3 and WT rice --- p.68 / Chapter 4.2.3 --- Summary of Pepsin Digestibility Assay --- p.74 / Chapter 4.3 --- Mouse Model I --- p.75 / Chapter 4.3.1 --- Protein-specific IgE levels --- p.75 / Chapter 4.3.2 --- Protein-specific IgG1 and IgG2a levels --- p.77 / Chapter 4.3.3 --- Allergic Response Test --- p.79 / Chapter 4.3.4 --- Summary from Mouse Model I --- p.81 / Chapter 4.4 --- Mouse Model II --- p.83 / Chapter 4.4.1 --- Proteins specific IgE levels --- p.84 / Chapter 4.4.2 --- Proteins specific IgG1 and IgG2a levels --- p.85 / Chapter 4.4.3 --- Allergic Response Test --- p.87 / Chapter 4.4.4 --- Summary from Mouse Model II --- p.88 / Chapter 4.5 --- Mouse Model III --- p.90 / Chapter 4.5.1 --- Protein-specific IgE levels --- p.90 / Chapter 4.5.2 --- Proteins specific IgG1 and IgG2a levels --- p.91 / Chapter 4.5.3 --- Allergic Response Test --- p.93 / Chapter 4.5.4 --- Summary from Mouse Model III --- p.93 / Chapter 4.6 --- Potential allergenicity of rice glutelin by bioinformatics and epitope mapping --- p.94 / Chapter 4.6.1 --- Bioinformatic analysis --- p.94 / Chapter 4.6.2 --- ELISA analysis of synthesized epitopes --- p.97 / Chapter 4.6.3 --- Western Blot Analysis --- p.99 / Chapter 4.6.4 --- IgE-binding assay --- p.103 / Chapter Chapter 5. --- Conclusion and Future Perspectives --- p.109 / References --- p.111

Ho, Wing Ho. / On t.p. "beta" appears as the Greek letter. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 157-183). / Abstracts in English and Chinese. / Thesis committee --- p.ii / Statement --- p.iii / Acknowledgements --- p.iv / Abstract --- p.vi / 摘要 --- p.vii / Table of Contents --- p.viii / List of Tables --- p.xv / List of Figures --- p.xvii / List of Abbreviations --- p.xxiii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter Chapter 2 --- Literature Review --- p.4 / Chapter 2.1 --- Vitamin A --- p.4 / Chapter 2.1.1 --- Genral and properties --- p.4 / Chapter 2.1.2 --- Biological importance of vitamin A --- p.6 / Chapter 2.1.3 --- Dietary source of vitamin A --- p.12 / Chapter 2.1.3.1 --- Plant-derived provitamin A and animal-derived vitamin A --- p.12 / Chapter 2.1.3.2 --- Dependence on the plant-derived provitamin A by the poor --- p.14 / Chapter 2.1.3.2.1 --- Plant-derived provitamin A --- p.14 / Chapter 2.1.3.2.1.1 --- General and properties --- p.14 / Chapter 2.1.3.2.1.2 --- Biosynthesis of provitamin A in plants --- p.17 / Chapter 2.1.3.2.1.2.1 --- Assembly of C40 backbone … --- p.17 / Chapter 2.1.3.2.1.2.2 --- Desaturation and cyclization --- p.26 / Chapter 2.1.3.2.1.2.3 --- Oxygenation --- p.29 / Chapter 2.1.3.2.1.2.4 --- Carotenogenic enzymes --- p.31 / Chapter 2.1.4 --- Metabolism of dietary vitamin A and provitamin A in human system --- p.35 / Chapter 2.1.4.1 --- Digestion and absorption --- p.35 / Chapter 2.1.4.2 --- Biocon version --- p.37 / Chapter 2.1.4.2.1 --- "Beta, beta '-carotene 15, 15'-monooxygenase (BCMO)" --- p.40 / Chapter 2.1.4.3 --- "Transport, uptake and storage" --- p.43 / Chapter 2.1.4.4 --- Provision or excretion --- p.46 / Chapter 2.2 --- Vitamin A deficiency (VAD) --- p.48 / Chapter 2.2.1 --- Green revolution --- p.48 / Chapter 2.2.2 --- Rice as the major staple food for feeding the poor --- p.49 / Chapter 2.2.3 --- Provitamin A content in processed rice seeds --- p.49 / Chapter 2.2.4 --- Symptoms of VAD --- p.51 / Chapter 2.2.5 --- Global prevalence of VAD --- p.53 / Chapter 2.3 --- Previous efforts for dealing with the deficiency --- p.55 / Chapter 2.3.1 --- The key for dealing with the deficiency --- p.55 / Chapter 2.3.2 --- Selective plant breeding --- p.55 / Chapter 2.3.3 --- Supplementation and post-harvesting fortification --- p.56 / Chapter 2.3.4 --- Bio-fortification by genetic engineering --- p.57 / Chapter 2.3.4.1 --- Advantages of genetic engineering --- p.57 / Chapter 2.3.4.1.1 --- Genetic engineering of non-cereal crops --- p.58 / Chapter 2.3.4.1.2 --- Genetic engineering of cereal crops --- p.62 / Chapter 2.3.4.1.2.1 --- Golden Rice 1 --- p.62 / Chapter 2.3.4.1.2.2 --- Golden Rice 2 --- p.64 / Chapter 2.4 --- Motivation for striking forward --- p.67 / Chapter 2.4.1 --- Recommended Dietary Amount of vitamin A --- p.67 / Chapter 2.4.2 --- Factors affecting the bioefficacy of provitamin A in human body --- p.68 / Chapter 2.4.2.1 --- Bioavailability --- p.68 / Chapter 2.4.2.2 --- Bioconvertibility --- p.69 / Chapter 2.4.2.3 --- Health and nutritional status --- p.71 / Chapter 2.4.3 --- Further improvement for dealing with the deficiency --- p.73 / Chapter 2.5 --- Hypothesis --- p.75 / Chapter Chapter 3 --- Materials and Methods --- p.78 / Chapter 3.1 --- Chemicals --- p.78 / Chapter 3.2 --- Bacterial strains --- p.78 / Chapter 3.3 --- Transient expression of BCMOs in plant system --- p.79 / Chapter 3.3.1 --- Choice of BCMOs --- p.79 / Chapter 3.3.2 --- Plasmids and genetic material --- p.79 / Chapter 3.3.3 --- Construction of chimeric genes for transient expression --- p.82 / Chapter 3.3.4 --- Microprojectile bombardment and GUS assay --- p.83 / Chapter 3.4 --- Construction of chimeric genes for rice co-transformation --- p.84 / Chapter 3.4.1 --- Choice of carotenogenic genes --- p.84 / Chapter 3.4.2 --- Choice of promoters --- p.84 / Chapter 3.4.3 --- Necessities and choice of transit peptide (TP) --- p.85 / Chapter 3.4.4 --- Arrangement of chimeric gene-cassettes --- p.86 / Chapter 3.4.5 --- Plasmids and genetic materials --- p.87 / Chapter 3.4.6 --- Construction of chimeric gene expressing PSY and PDS coordinately --- p.87 / Chapter 3.4.7 --- "Construction of chimeric gene expressing PSY, PDS and TP equipped CHBCMO coordinately" --- p.92 / Chapter 3.4.8 --- "Construction of chimeric gene expressing PSY, PDS and TP equipped ZEBCMO coordinately" --- p.98 / Chapter 3.4.9 --- Construction of chimeric gene expressing ZDS and LYCB coordinately --- p.103 / Chapter 3.4.10 --- Confirmation of sequence fidelity --- p.108 / Chapter 3.5 --- Rice co-transformation --- p.109 / Chapter 3.5.1 --- Plant materials --- p.109 / Chapter 3.5.2 --- Preparation of Agrobacterium tumefaciens --- p.109 / Chapter 3.5.3 --- Agrobacterium mediated co-transformation --- p.110 / Chapter 3.5.3.1 --- Callus induction from mature rice seeds --- p.110 / Chapter 3.5.3.2 --- Callus induction from immature rice seeds --- p.110 / Chapter 3.5.3.3 --- "Co-cultivation, selection and regeneration" --- p.111 / Chapter 3.6 --- Detection of transgene expression --- p.112 / Chapter 3.6.1 --- Detection at DNA level --- p.112 / Chapter 3.6.1.1 --- Genomic DNA extraction --- p.112 / Chapter 3.6.1.2 --- PCR screening --- p.112 / Chapter 3.6.1.3 --- Synthesis of DIG-labeled DNA probes --- p.114 / Chapter 3.6.1.4 --- Southern blot analysis --- p.115 / Chapter 3.6.2 --- Detection at RNA level --- p.116 / Chapter 3.6.2.1 --- Total RNA extraction --- p.116 / Chapter 3.6.2.2 --- Northern blot analysis --- p.116 / Chapter 3.6.3 --- Detection at product level --- p.117 / Chapter 3.6.3.1 --- Phenotypic identification --- p.117 / Chapter 3.6.3.2 --- HPLC analysis --- p.117 / Chapter 3.6.3.2.1 --- Extraction of total carotenoids and retinoids --- p.117 / Chapter 3.6.3.2.2 --- HPLC identification --- p.118 / Chapter 3.6.3.2.3 --- HPLC quantification --- p.118 / Chapter Chapter 4 --- Results --- p.119 / Chapter 4.1 --- Transient expression of BCMOs in plant system --- p.119 / Chapter 4.1.1 --- Construction of chimeric genes for transient expression --- p.119 / Chapter 4.1.2 --- Microprojectile bombardment and GUS assay --- p.120 / Chapter 4.2 --- Construction of chimeric genes for rice co-transformation --- p.121 / Chapter 4.3 --- Rice co-transformation --- p.123 / Chapter 4.3.1 --- Callus induction from mature and immature rice seeds --- p.123 / Chapter 4.3.2 --- "Co-cultivation, selection and regeneration" --- p.124 / Chapter 4.4 --- Detection of transgene expression --- p.126 / Chapter 4.4.1 --- Detection at DNA level --- p.126 / Chapter 4.4.1.1 --- PCR screening --- p.126 / Chapter 4.4.1.2 --- Southern blot analysis --- p.129 / Chapter 4.4.2 --- Detection at RNA level --- p.133 / Chapter 4.4.2.1 --- Northern blot analysis --- p.133 / Chapter 4.4.3 --- Detection at product level --- p.135 / Chapter 4.4.3.1 --- Phenotypic identification --- p.135 / Chapter 4.4.3.2 --- HPLC identification --- p.137 / Chapter 4.4.3.3 --- HPLC quantification --- p.147 / Chapter Chapter 5 --- Discussion --- p.150 / Chapter Chapter 6 --- Conclusion --- p.156 / References --- p.157

The objectives of this investigation have been to characterize black gram (Phaseolus Mongo L.) and rice (Oryza sativa L.) proteins and to study changes in their nutritional value due to fermentation. black gram, the legume chosen for this work, is one of the most important legume crops throughout a large part of the tropics. The protein content of 60 mesh, dehydrated, defatted black gram meal was 28.5 percent. Sodium carbonate (0.5- 1.0 percent), tetra-sodium pyrophosphate (0.5 percent), and sodium dodecyl sulfate (SDS) (0.5-5 percent) proved to be the potential protein solubilizers as they extracted more than 76 grams of Lowry's protein per 100 grams Kjeldahl protein. On the considerations of contaminating residue in the final product and disruption of native structure of the proteins, these chemical agents were unsuitable. Sodium sulfate at the 10 percent level was judged to be the best protein solubilizer. Proteins separated on polyacrylamide gel using a phenol-acetic acid-mercaptoethanol-urea (PAMU) system were run on the flat bed gel containing SDS. The proteins were separated in 13 constituents and the molecular weights of the major ones were 140,000 and 55,000. Solubilized proteins contained 81 percent globulins, 13 percent albumins, 4 percent prolamins, and 2 percent glutelins . Sulfur containing amino acids and threonine were deficient in total proteins xv of the seeds with 27. 6 and 78.8 as their respective chemical scores . Chemical scores of the albumin, globulin, prolamin, and glutelin fractions were 64, 0, 56, and 70.7, respectively. The predicted biological values in human nutrition varied from O for globulins to 110 for glutelins, and was 14.9 for total proteins in the seeds. The constituents of the protein fractions were isoelectrically focused in the acidic pH range with the exception of two globulins for which the isoelectric points were 8.42 and 8.65. The trypsin inhibitor from black gram was isolated using affinity chromatography gel with 19. 5 fold purification. The inhibitor had 75 amino acid residues and contained one disulfide bridge. Chemical studies assigned an important role for the hydrogen bonds and demonstrated vital importance of the disulfide bridge in retaining the inhibiting activity. The inhibitor was stable and retained 35 percent of the activity when heated at 100°C for 60 minutes at pH 11. Chemical modification of amino acid residues suggested the involvement of lysine and arginine residues at the active site of the inhibitor. Lysine and arginine moieties at the active site have been proposed to be present as alanyllysine and histidylarginine. Inhib i tion of bovine pancreatic trypsin by the inhibitor was kinetically studied . The kinetic constants Km and Vm ax were 2.7 x l0- 5M and 6 x l0 - 3M/min, respectively. The dissociation constant for the enzyme-inhibitor complex (Ki) was 4 x l0- 7M, whereas that for the enzyme-inhibitor-substrate complex (K. 1 , ) was 1.89 x l0- 6M. The inhibition was a mixture of partial competitive and pure-noncompetitive systems. Rice and black gram form the integral parts of a fermented snack food of the Indian subcontinent~idli. Amino acid composition of black gram and rice were complementary. Leucine, lysine, and sulfur containing amino acids were the most limiting amino acids in rice with 65. 1, 66.3, and 67.9 as their respective scores. The estimates of biological values of rice proteins in human nutrition qualified albumins as superior and prolamins as inferior proteins. The PAMUsy stem in polyacrylamide gel electrophoresis was more efficient in resolving protein subunits than the SOS gel system. The PAMUsy stem was not sensitive to the ionic strength of the sample. Mobilities of rice and black gram proteins in SOS and PAMUsy stems were based on the related parameters. In the PAMUsy stem, the mobilities of most proteins seemed to depend on their molecular size. The PAMUsy stem on gel electrophoresis was judged superior to the SOS system. Fermentation of the black gram-rice blend was kinetically studied for changes in physicochemical characteristics and nutritional functionality. Trypsin inhibiting activity was unaffected, but chymotrypsin inhibiting activity was reduced to 3 percent after 20 hours fermentation , Significant increases were noted in the contents of sulfur containing amino acids during fermentation . These amino acids seemed to be bioavailable. In vitro digestion with pepsin and pancreatin indicated improvement in digestibility of proteins after fermentation. The digestibility was further enhanced by steaming.

Imazethapyr was recently approved for use in rice, but limited information is available regarding its efficacy, environmental fate or potential red rice resistance. Therefore, experiments were conducted to 1) determine the effect of flooding time, and stage of imazethapyr application in red rice control, 2) assess the acetolactate synthase resistance to imazethapyr on red rice ecotypes, 3) determine the relative photolysis of imazethapyr, and 4) determine the effect of soil and moisture on imazethapyr adsorption and availability. When imazethapyr was applied in sequential application of PRE followed by a POST application, to achieve >95% red rice control, flood needed to be established within 14 DAT when imazethapyr was applied EPOST, and 7 DAT when imazethapyr was applied LPOST. Delaying the flood up to 21 DAT reduced rice grain yield for both EPOST and LPOST application timings. Based on enzymatic activity, the mean I50 values were 1.5, 1.1, 1.5, 1.6, 20.8, and 590.6 mM of imazethapyr, respectively, for LA 5, MS 5, TX 4, ??Cypress??, ??CL-121??, and ??CL-161??. CL-161 was 32 times more resistant than CL-121, and at least 420 times more resistant than the average of the red rice ecotypes and ??Cypress??. Results from the ALS assay showed that red rice ecotypes and Cypress had high susceptibility to imazethapyr when compared with the tolerant CL-121 and the resistant CL-161. Measurable enzymatic tolerance to ALS-inhibiting herbicides has not yet developed in these red rice ecotypes. Imazethapyr quantum yield (fI ) was 0.023 ?? 0.002 while the hydroxyl radical rate constant ( I OH k?? ) was 2.8 ?? 0.44 x 1013 M-1 h-1. These results show that imazethapyr is susceptible to both direct and indirect photolysis. The results also show that imazethapyr photolysis in paddy water will be affected by turbidity due to its impact on the availability of sunlight to drive direct and indirect photolysis reactions. Imazethapyr was more available and more concentrated in sandy soil. With higher amounts of water in soil there was greater amount of imazethapyr in soil solution and a lower concentration of herbicide due to dilution. The double centrifuge method provided a better estimate of plant available herbicide.

Objetivo general: Estudiar las proteínas de arroz de la variedad Nutriar desde el punto de vista estructural y funcional provenientes de grano pulido e integral. Compararlas con las de una variedad de amplio uso local, de calidad tropical. Objetivos específicos: Determinar la composición proteica, cuali y cuantitativamente de la variedad Nutriar en comparación con la variedad de referencia. Explorar distintos métodos de preparación de aislados proteicos mediante la variación del pH de extracción de proteínas. Elección del método más adecuado. Caracterización estructural, nutricional y funcional de los aislados obtenidos con la metodología de elección.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv. / Härtill 4 uppsatser.

Mestre -- Sao Paulo (Estado). Secretaria da Saude. Coordenacao dos Institutos de Pesquisa. Programa de Pos-Graduacao em Ciencias, Sao Paulo, 2003.

Made available in DSpace on 2014-06-11T19:28:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-11-10Bitstream added on 2014-06-13T20:18:27Z : No. of bitstreams: 1 goria_mm_me_botfca.pdf: 385248 bytes, checksum: aedd73597ba4895b9c4451da9ed36770 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O impacto da elevação da concentração de CO2 do ar sobre a brusone do arroz foi avaliado em estufas de topo aberto (OTCs) na Embrapa Meio Ambiente, Jaguariúna/SP, por dois anos. Foram realizados ensaios com cultivares de arroz em estufas com injeção de CO2, estufas sem injeção de CO2, e campo aberto, sem injeção de CO2 e sem estufa. Avaliaram-se as características de desenvolvimento das plantas, a incidência e a severidade da brusone do arroz, a caracterização química e microbiológica da rizosfera de plantas de arroz, e o teor de silício acumulado na parte aérea das plantas. No primeiro ensaio foi avaliada também a ocorrência de bactérias diazotróficas endofíticas nas raízes das plantas. A concentração média de CO2 atmosférico do tratamento em campo aberto foi 459,4 e 447,4 μmol mol-1 na safra 2007/08 e safra 2008/09, respectivamente. Por outro lado, as concentrações médias de CO2 foram 490,1 e 480,4 μmol mol-1 para o tratamento em estufa sem injeção de CO2 e 531,9 e 608,6 μmol mol-1 para o tratamento com estufa com injeção de CO2 na safra 2007/08 e safra 2008/09, respectivamente. Nos resultados obtidos, verificou-se o aumento significativo na altura de plantas das cultivares Agulha Precoce e Shao Tiao Tsao, na safra 2008/09, no tratamento com injeção do gás. Nas cultivares Caloro e Agulha Precoce, nas safras 2007/08 e 2008/09, respectivamente, o ambiente com a concentração de CO2 do ar elevada aumentou a severidade da brusone nas folhas das plantas. A análise química e microbiológica da rizosfera não apresentou diferenças entre os ambientes com e sem injeção do gás. A massa seca da parte aérea das plantas, a massa das panículas e a massa dos grãos não sofreram alteração devido à elevação do CO2 atmosférico. O aumento da concentração de CO2 do ar pode alterar o crescimento das plantas e a severidade da brusone, acarretando... / The impact of elevated atmospheric CO2 concentration on rice blast disease was evaluated in open-top chambers (OTCs) in Embrapa Meio Ambiente, Jaguariúna /SP, for two years. Trials were developed under OTCs with injection of CO2, OTCs without injection of CO2, and field, without injection of CO2 and without OTC. The characteristics of rice plants growth, the incidence and severity of rice blast, chemical and microbial characterization of rizosphere of rice plants, and leaf silicon content were evaluated. On the first trial it was also examined the occurrence of diazotrophic bacteria in rice plant´s root. Actual season-long average CO2 concentration in field without injection of CO2 and without OTC were 459,4 e 447,4 μmol mol-1 in 2007/08 and 2008/09, respectively. For the other hand, actual season-long average CO2 concentration were 490,1 and 480,4 μmol mol-1 in OTCs without injection of CO2 and 531,9 and 608,6 μmol mol-1 for the treatment under OTCs with CO2 enrichment in 2007/08 and 2008/09, respectively. As results, Agulha Precoce and Shao Tiao Tsao, in 2008/09, it was found a significant increase on rice growth, on treatment with CO2 injection. On Caloro and Agulha Precoce, in 2007/08 and 2008/09, respectively, the atmosphere with elevated CO2 increased the severity of leaf blast. No significant difference was detected on rizhosphere chemical and microbiological analysis in the atmosphere with injection of the gas. CO2 enrichment resulted in a non-significant increase in grain weight, plant dry weight and the panicles weight. The increase of CO2 atmospheric concentration may alter the rice plant´s growth and the severity of rice blast, and consequently, the strategies of disease management.