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Doctoral Thesis
DOI
https://doi.org/10.11606/T.9.2017.tde-26012017-141922
Document
Author
Full name
Priscila Robertina dos Santos Donado
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2016
Supervisor
Committee
Finardi Filho, Flavio (President)
Fabi, João Paulo
Palmisano, Giuseppe
Labate, Carlos Alberto
Souza, Gisele Monteiro de
Title in Portuguese
Estudo proteômico de variedades de milho (Zea mays L.) obtidas por melhoramento clássico e por recombinação genética
Keywords in Portuguese
Espectrometria de massas
Milho com qualidade proteica melhorada
Milho geneticamente modificado
Proteômica
Segurança de alimentos geneticamente modificados
Zea mays L.
Abstract in Portuguese
O melhoramento genético clássico de sementes milho (Zea mays L.) permitiu desenvolver inúmeras variedades, incluindo o milho com qualidade proteica melhorada (Quality Protein Maize, QPM), que visava aumentar os teores proteicos e as propriedades nutricionais. Por outro lado, novas variedades comerciais foram obtidas por vegetais geneticamente modificados (GM), com foco em parâmetros agronômicos. Em ambos os casos, a segurança dessas variedades para uso como alimento é uma das principais preocupações dos desenvolvedores e dos órgãos de regulamentação. A Equivalência Substancial é a base do sistema de avaliação da segurança de culturas geneticamente modificadas, no entanto alterações na expressão de proteínas não são devidamente analisadas e esclarecidas. As abordagens proteômicas complementam as técnicas de avaliação de biossegurança para alimentos GM, bem como permitem investigar possíveis efeitos indesejáveis derivados do melhoramento clássico. Os objetivos do presente estudo foram caracterizar e comparar os perfis proteicos de variedades de milhos convencionais melhorados (QPM) e geneticamente modificados (GMs), contra suas respectivas linhas convencionais utilizando técnicas proteômicas como eletroforese bidimensional (2-DE) e bottom up shotgun (gel-free). Num primeiro estudo, foram utilizadas três amostras de milho, sendo duas variedades convencionais com QPM (QP1 e QP2) e uma variedade convencional normal (CN). No segundo estudo, foram analisadas duas cultivares de milho GM (GM1 e GM2) e seus respectivos convencionais genitores (CG1 e CG2). As composições químicas de todas as amostras também foram avaliadas quanto a Equivalência Substancial. O extrato bruto proteico foi submetido à análise de eletroforese unidimensional (1-DE), bidimensional (2-DE) e bottom up shotgun (gel-free). As imagens dos mapas proteicos foram analisadas pelo software Image Master 2D Platinum 7.0 (GE). Os spots diferencialmente expressos e selecionados foram sequenciados por MS. Pela composição química das principais frações das amostras de milho foi possível identificar a equivalência substancial entre as amostras convencionais e GMs, bem como QPMs e sua convencional dentro das faixas de variabilidade esperadas da espécie. Nos géis 1-DE foram observadas bandas proteicas com perfis similares entre os grupos de amostras avaliadas para ambos estudos. Nas imagens dos géis 2-DE não houveram alterações extremas entre as amostras de milhos GMs e seus respectivos convencionais genitores (CGs), mas apenas diferenças na intensidade dos spots proteicos. As variedades QPMs e CN apresentaram diferenças devido à distribuição dos spots. Os mapas proteicos das amostras CG1 x GM1 e CG2 x GM2 apresentaram maior semelhança com porcentagens de matchings superiores a 70 %, enquanto as porcentagens de matchings entre variedades diferentes (QPMs e CN) foram menores. No total foram identificadas 219 proteínas das amostras CGs x GMs e QPMs x CN, classificadas quanto aos seus processos biológicos e função molecular. Em conclusão, foram encontradas diferenças entre os cultivares GMs e CGs, indicando uma variação normal entre variedades de milho, que não comprometem a segurança alimentar das amostras estudadas. Quanto às amostras com QPM e CN as diferenças encontradas são devido à sua distância nas linhagens ou germoplasma.
Title in English
Proteomic study of maize (Zea mays L.) varieties obtained by classical breeding and genetic recombination.
Keywords in English
Genetically modified organisms
Mass spectrometry
Proteomic
Quality protein maize
Safety of genetically modified foods
Zea mays L.
Abstract in English
The classic genetic breeding of corn seeds (Zea mays) has enabled the development of many varieties, including corn with improved protein quality (Quality Protein Maize, QPM), which aimed to increase protein levels and nutritional properties. On the other hand, new commercial varieties have been obtained out of genetically modified (GM) vegetables, with a focus in agronomic parameters. In both cases, the safety of these varieties for food use is one of the main concerns for the developers and for the regulatory agencies. Substantial Equivalence is the basis of the safety evaluation system for genetically modified crops, however, alterations in the protein expressions are not been properly analyzed and clarified. The protein approaches complement the techniques of biosafety evaluation for GM foods, as well as allow for possible undesirable effects derived from classic improvement to be investigated. The goals of the current studies were to characterize and compare the protein profiles of the different varieties of conventionally improved (QPM) and genetically modified (GM) corn, against their respective conventional lines using proteomic techniques, such as, two-dimensional electrophoresis (2-DE), bottom up shotgun (gel-free) and masses spectrometry (MS). In a first instance of the study, three samples of corn were used, two of conventional varieties with QPM (QP1 and QP2) and one conventional normal variety (CN). In a second instance of the study, two cultures of GM corn (GM1 and GM2) were analyzed and their respective conventional genitors (CG1 and CG2). The chemical compositions of all the samples were also evaluated for their Substantial Equivalence. The protein raw extract was submitted to analysis of one-dimensional (1-DE), two-dimensional (2-DE) electrophoresis, and bottom up shotgun (gel-free). The protein image maps were analyzed by the Image Master 2D Platinum 7.0 (GE) software. The spots which were expressed and selected differentially were sequenced by MS. By the chemical composition of the main fractions of the samples of corn, it was possible to identify the substantial equivalence between the conventional samples and GMs, likewise with OPMs and their conventional in the ranges of variability which were expected for the species. On the 1-DE gel, it was observed protein bands with similar profiles amongst the groups of evaluated samples for both studies. In the images of the 2-DE gel, there were no alterations between the GM corn and their respective conventional genitors (CGs), but only differences in intensity of the protein spots. The OPM and CN varieties presented differences due to the distribution of the spots. The protein maps of samples CG1 vs. GM1 and CG2 vs. GM2 presented greater similarities with the percentages of matchings superior to 70%, while the percentage of matchings among different varieties (QPMs and CN) were smaller. In total, there were 219 proteins identified in the samples CGs vs. GMs and QPMs vs. CN, classified by the biologic processes and molecular function. In conclusion, there were found differences between the cultures of GMs and CGs, indicating a normal variation among the corn varieties, which do not affect the food security of the studied samples. As per the samples with QPM and CN, the differences found were due to the line distances or germplasm.
 
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Publishing Date
2017-03-13
 
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