Desenvolvimento de substrato e adequação de metodologia para detecção de β-1,3 glucanase em soja Glycine max (L.) Merril
The current food production model based on the use of chemical pesticides, has been discussed and questioned, considering the risks to the environment and food security. Many efforts have been made for alternative methods to be used in disease control, such as using resistance inductors. The inducer...
| Autor principal: | Bertoldo, Edson |
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| Formato: | Tese |
| Idioma: | Português |
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Universidade Tecnológica Federal do Paraná
2019
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riut-1-45242019-11-07T06:00:53Z Desenvolvimento de substrato e adequação de metodologia para detecção de β-1,3 glucanase em soja Glycine max (L.) Merril Substrate development and adaptation methodology for the detection of β-1,3 glucanase in soybean - Glycine max (L.) Merril Bertoldo, Edson Mazaro, Sergio Miguel http://lattes.cnpq.br/4271660992059925 Busso, Cleverson http://lattes.cnpq.br/5986131313813011 Leite, Deborah Catharine De Assis http://lattes.cnpq.br/5891155336419190 Sirtoli, Luchele Furlan http://lattes.cnpq.br/7939841668966282 Rubert, Marceléia http://lattes.cnpq.br/5065670903691241 Ernandes, Samara http://lattes.cnpq.br/7521000161016435 Mazaro, Sergio Miguel http://lattes.cnpq.br/4271660992059925 Fitopatologia Fisiologia Vegetal Bioquímica Plant diseases Plant physiology Biochemistry CNPQ::CIENCIAS AGRARIAS::AGRONOMIA::FITOTECNIA Engenharia/Tecnologia/Gestão The current food production model based on the use of chemical pesticides, has been discussed and questioned, considering the risks to the environment and food security. Many efforts have been made for alternative methods to be used in disease control, such as using resistance inductors. The inducers (elicitors) that may be biotic or abiotic, inorganic, organic or synthetic nature, are capable of inducing a defense response when they bind to receptors of the plasma membrane of the plant cell. They can induce systemic acquired resistance (SAR), which is one of the most important forms of resistance, because it confers systemic protection to the plant to a broad spectrum of microorganisms. Among the defense mechanisms of SAR, are the cell wall modifications, the production of phytoalexins, and concomitantly increased expression of pathogenesis related genes, which express the pathogenicity-related proteins (PR proteins). Among the PR proteins, the most important are the chitinase (PR-3) and β-1,3 glucanases (PR-2), which have hydrolytic activity, often synergistic, cleaving structural chitin and glucan polymers, respectively, present in the wall of pathogens, especially fungi. The activity of these enzymes is enhanced when plants are treated with specific inducers or when attacked by their natural enemies. However, research of new inductors require a series of tests to effective proof that plant defense response has been activated. Biochemical analysis of PR-proteins, such as β-1,3-glucanase, are needed to support the discovery of new and efficient inductors. However, the current enzyme substrate available in the market to evaluate this enzyme has shown discontinuous production. In this scenario, this study aimed to develop substrate and matching methodology for detecting β-1,3 glucanase in soybean plants - Glycine max (L.) Merr, main culture produced in Brazil. It has been developed two substrates glucan base: Curdlan-RBB and Scleroglucan-RBB. The efficiency of the substrates was compared with other currently available methods of determination, and the substrates were tested produced through β-1,3 glucanase purified action. Since the tests were positive biochemical analysis was performed with induced systemically and soybean plants inoculated with Phakopsora pachyrhizi fungus Syd. & P. Syd. The results showed that all substrates enables to quantify the enzymatic activity of β-1,3 glucanase, hence one of the substrates developed in this research, the scleroglucan-RBB had higher efficiency to commercial substrate AZCLCurdlan®. Conselho Nacional do Desenvolvimento Científico e Tecnológico (CNPq) O atual modelo de produção de alimentos, baseado no uso de defensivos químicos, vem sendo discutido e questionado, considerando os riscos ao meio ambiente e a segurança alimentar. Muitos esforços têm sido realizados para que métodos alternativos sejam utilizados no controle de doenças, como o uso de indutores de resistência. Os indutores (elicitores ou eliciadores), que podem ser de origem biótica ou abiótica, de natureza inorgânica, orgânica ou sintética, são capazes de induzir respostas de defesa quando se ligam a receptores da membrana plasmática da célula vegetal. Eles podem induzir a resistência sistêmica adquirida (RSA), a qual é uma das mais importantes formas de resistência, pois confere proteção sistêmica à planta a um amplo espectro de microrganismos. Dentre os mecanismos de defesa da RSA, estão as modificações de parede celular, a produção de fitoalexinas, e concomitantemente, o aumento da expressão de genes relacionados a patogênese, os quais expressam as Proteínas Relacionadas a Patogenicidade (proteínas PR). Dentre as proteínas-PR, as mais importantes são as quitinases (PR-3) e as β-1,3 glucanases (PR-2), as quais apresentam atividade hidrolítica, muitas vezes sinérgica, clivando polímeros estruturais de quitina e glucana, respectivamente, presentes na parede de patógenos, principalmente de fungos. A atividade dessas enzimas é aumentada quando plantas são tratadas com indutores específicos ou quando atacadas pelos seus inimigos naturais. No entanto, pesquisas de novos indutores requerem uma série de testes para comprovação efetiva que a resposta de defesa vegetal tenha sido ativada. Análises bioquímicas de proteínas-PR, como a β- 1,3 glucanase, são necessárias para auxiliar a descoberta de novos e eficientes indutores. Entretanto, o atual substrato enzimático disponível no mercado para avaliar essa enzima tem apresentado produção descontínua. Diante desse cenário, o presente estudo teve por objetivo desenvolver substrato e adequar metodologia para detecção de β-1,3 glucanase em plantas de soja - Glycine max (L.) Merr., principal cultura produzida no Brasil. Foram desenvolvidos dois substratos a base de glucana - Curdlana-RBB e Escleroglucana-RBB. A eficiência dos substratos foi comparada com outros métodos de determinação atualmente disponíveis e os substratos produzidos foram testados por meio da ação de β-1,3 glucanase purificada. Como os testes foram positivos, realizou-se análises bioquímicas com plantas de soja induzidas sistemicamente e inoculadas com o fungo Phakopsora pachyrhizi Syd. & P. Syd. Os resultados demonstraram que todos os substratos testados possibilitam quantificar a atividade enzimática de β-1,3 glucanase, sendo que um dos substratos desenvolvidos nesta pesquisa, a Escleroglucana-RBB, apresentou eficiência superior ao substrato comercial AZCL-Curdlan®. 2019-11-06T14:04:23Z 2019-11-06T14:04:23Z 2019-08-19 doctoralThesis BERTOLDO, Edson. Desenvolvimento de substrato e adequação de metodologia para detecção de β-1,3 glucanase em soja Glycine max (L.) Merril. 2019. Tese (Doutorado em Agronomia) - Universidade Tecnológica Federal do Paraná, Pato Branco, 2019. http://repositorio.utfpr.edu.br/jspui/handle/1/4524 por openAccess application/pdf Universidade Tecnológica Federal do Paraná Pato Branco Brasil Programa de Pós-Graduação em Agronomia UTFPR |
| institution |
Universidade Tecnológica Federal do Paraná |
| collection |
RIUT |
| language |
Português |
| topic |
Fitopatologia Fisiologia Vegetal Bioquímica Plant diseases Plant physiology Biochemistry CNPQ::CIENCIAS AGRARIAS::AGRONOMIA::FITOTECNIA Engenharia/Tecnologia/Gestão |
| spellingShingle |
Fitopatologia Fisiologia Vegetal Bioquímica Plant diseases Plant physiology Biochemistry CNPQ::CIENCIAS AGRARIAS::AGRONOMIA::FITOTECNIA Engenharia/Tecnologia/Gestão Bertoldo, Edson Desenvolvimento de substrato e adequação de metodologia para detecção de β-1,3 glucanase em soja Glycine max (L.) Merril |
| description |
The current food production model based on the use of chemical pesticides, has been discussed and questioned, considering the risks to the environment and food security. Many efforts have been made for alternative methods to be used in disease control, such as using resistance inductors. The inducers (elicitors) that may be biotic or abiotic, inorganic, organic or synthetic nature, are capable of inducing a defense response when they bind to receptors of the plasma membrane of the plant cell. They can induce systemic acquired resistance (SAR), which is one of the most important forms of resistance, because it confers systemic protection to the plant to a broad spectrum of microorganisms. Among the defense mechanisms of SAR, are the cell wall modifications, the production of phytoalexins, and concomitantly increased expression of pathogenesis related genes, which express the pathogenicity-related proteins (PR proteins). Among the PR proteins, the most important are the chitinase (PR-3) and β-1,3 glucanases (PR-2), which have hydrolytic activity, often synergistic, cleaving structural chitin and glucan polymers, respectively, present in the wall of pathogens, especially fungi. The activity of these enzymes is enhanced when plants are treated with specific inducers or when attacked by their natural enemies. However, research of new inductors require a series of tests to effective proof that plant defense response has been activated. Biochemical analysis of PR-proteins, such as β-1,3-glucanase, are needed to support the discovery of new and efficient inductors. However, the current enzyme substrate available in the market to evaluate this enzyme has shown discontinuous production. In this scenario, this study aimed to develop substrate and matching methodology for detecting β-1,3 glucanase in soybean plants - Glycine max (L.) Merr, main culture produced in Brazil. It has been developed two substrates glucan base: Curdlan-RBB and Scleroglucan-RBB. The efficiency of the substrates was compared with other currently available methods of determination, and the substrates were tested produced through β-1,3 glucanase purified action. Since the tests were positive biochemical analysis was performed with induced systemically and soybean plants inoculated with Phakopsora pachyrhizi fungus Syd. & P. Syd. The results showed that all substrates enables to quantify the enzymatic activity of β-1,3 glucanase, hence one of the substrates developed in this research, the scleroglucan-RBB had higher efficiency to commercial substrate AZCLCurdlan®. |
| format |
Tese |
| author |
Bertoldo, Edson |
| author_sort |
Bertoldo, Edson |
| title |
Desenvolvimento de substrato e adequação de metodologia para detecção de β-1,3 glucanase em soja Glycine max (L.) Merril |
| title_short |
Desenvolvimento de substrato e adequação de metodologia para detecção de β-1,3 glucanase em soja Glycine max (L.) Merril |
| title_full |
Desenvolvimento de substrato e adequação de metodologia para detecção de β-1,3 glucanase em soja Glycine max (L.) Merril |
| title_fullStr |
Desenvolvimento de substrato e adequação de metodologia para detecção de β-1,3 glucanase em soja Glycine max (L.) Merril |
| title_full_unstemmed |
Desenvolvimento de substrato e adequação de metodologia para detecção de β-1,3 glucanase em soja Glycine max (L.) Merril |
| title_sort |
desenvolvimento de substrato e adequação de metodologia para detecção de β-1,3 glucanase em soja glycine max (l.) merril |
| publisher |
Universidade Tecnológica Federal do Paraná |
| publishDate |
2019 |
| citation |
BERTOLDO, Edson. Desenvolvimento de substrato e adequação de metodologia para detecção de β-1,3 glucanase em soja Glycine max (L.) Merril. 2019. Tese (Doutorado em Agronomia) - Universidade Tecnológica Federal do Paraná, Pato Branco, 2019. |
| url |
http://repositorio.utfpr.edu.br/jspui/handle/1/4524 |
| _version_ |
1805307740123299840 |
| score |
10,814766 |