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Mémoire de Maîtrise
DOI
https://doi.org/10.11606/D.10.2011.tde-27092012-182653
Document
Auteur
Nom complet
Daniela Maria Gerônimo
Adresse Mail
Unité de l'USP
Domain de Connaissance
Date de Soutenance
Editeur
Pirassununga, 2011
Directeur
Jury
Silva, Luis Felipe Prada e (Président)
Fukumasu, Heidge
Pereira, Angélica Simone Cravo
Titre en portugais
Caracterização molecular de três genes da via da lignificação em plantas forrageiras tropicais
Mots-clés en portugais
Brachiaria brizantha
Panicum maximum
Lignificação
Sequenciamento
Resumé en portugais
O rápido declínio na digestibilidade da parede celular, devido à lignificação, dificulta o uso eficiente das gramíneas de clima tropical. Objetivou-se detectar e sequenciar o cDNA completo dos principais genes responsáveis pelo processo de biossíntese da lignina, CCoAOMT, C4H e PAL nas gramíneas Brachiaria brizantha Stapf. cv. Marandu e Panicum maximum Jacq. cv. Tanzânia. As amostras das espécies de plantas forrageiras foram coletadas do Campo Agrostológico e levadas, imediatamente, para o laboratório para a extração do RNA total. Após tratamento do RNA total com o kit GeneRacer (Invitrogen), obteve-se o comprimento total 3' e 5' do cDNA das forrageiras B. brizantha e P. maximum. Os primers específicos foram delineados com base nas sequências de B. brizantha, P. maximum e milho, depositadas no GenBank, e foram aneladas com os primers 3 e 5 do kit GeneRacer para obtenção do comprimento total do gene. As amplificações foram feitas através PCR qualitativo e os produtos foram purificados, clonados e seqüenciados em ambas direções. Foram encontrados alguns problemas com a otimização na reação de seqüenciamento e obteve-se 67,25 % e 82,22% do gene CCoAOMT, 13,29% e 26,16% do gene PAL e 13,22% do gene C4H em B. brizantha e P. maximum, respectivamente. O sequenciamento dos genes codificantes das enzimas envolvidas no processo de lignificação, apresentaram alta similaridade com as sequências nucleotídicas do milho, e foi possível identificar e classificá-los de acordo com as classes que compõem cada gene. B. brizantha e P. maximum apresentaram 89,6% com CCoAOMT 1 e 88,3% com CCoAOMT 3, 96% e 95% com PAL1 e 96% com C4H1, respectivamente. Ao analisar as similaridades das sequências nucleotídicas dos fragmentos sequenciados, para os genes CCoAOMT, PAL e C4H, entre as demais gramíneas amplamente estudadas, encontrou-se 91% e 91,14% CCoAOMT do arroz, 79,6% e 82% CCoAOMT do trigo, 94,33 e 94,2% CCoAOMT do sorgo, 87 e 86% CCoAOMT da aveia, 90% e 94,25% PAL do arroz, 82% e 93,33% PAL do trigo, 92 % e 89,5% PAL da aveia, 94,33 e 95% PAL do sorgo, 97% e 91% PAL cana-de-açúcar, 90% e 93% PAL da cevada, 76% C4H do arroz, 94% C4H do sorgo, 89% C4H do trigo e 87% C4H da cevada, em B. brizantha e P. maximum, respectivamente. A altíssima similaridade encontrada entre os fragmentos dos genes da via dos fenilpropanóides em B. brizantha e P. maximum, principalmente com o milho e arroz que possuem seu genoma em avançado estudo e conhecimento, possibilitam o uso do banco de dados destas espécies para estudos futuros de biologia molecular.
Titre en anglais
Molecular characterization of three genes of the lignification pathway in tropical forage
Mots-clés en anglais
Brachiaria brizantha
Panicum maximum
Lignification
Sequencing
Resumé en anglais
The rapid decline in digestibility of cell wall, due to lignification, hinders the efficient use of tropical grasses. The objective was to detect and sequence the complete cDNA of the major genes responsible for lignin biosynthesis process, CCoAOMT, PAL and C4H in grasses Brachiaria brizantha Stapf. cv. Marandu and Panicum maximum Jacq. cv. Tanzania. Samples of fodder plant species were collected from the field agrostology and taken immediately to the laboratory for extraction of total RNA. After treatment of total RNA with the Generac kit (Invitrogen), we obtained the full length 3'and 5' cDNA of forage B. brizantha and P. maximum. The specific primers were designed based on sequences of B. brizantha, P. maximum and corn, deposited in GenBank, and were ringed with primers 3 'and 5' of the Generac kit to obtain the total length of the gene. The amplifications were performed by qualitative PCR and the products were purified, cloned and sequenced in both directions. We found some problems with optimization in the sequencing reaction and obtained 67.25% and 82.22% of the CCoAOMT gene, 13.29% and 26.16% of PAL gene and 13.22% of the C4H gene in B. brizantha and P. maximum, respectively. The sequencing of genes encoding enzymes involved in lignification, showed high similarity with the nucleotide sequences of maize, and it was possible to identify and classify them according to the classes that make up each gene. B. brizantha and P. maximum 89.6% presented with CCoAOMT 1 and 88.3% with CCoAOMT 3, 96% and 95% to 96% with PAL1 and C4H1, respectively. By analyzing the similarities of the nucleotide sequences of the fragments sequenced, the genes for CCoAOMT, PAL and C4H, among the other grasses studied extensively, met 91% and 91.14% of the rice CCoAOMT, 79.6% and 82% of the CCoAOMT wheat, 94.33% and 94.2 CCoAOMT sorghum, 87 and 86% oat CCoAOMT, 90% and 94.25% of the rice PAL, 82 PAL% and 93.33% for wheat, 92% and 89.5 PAL% oats, 95% and 94.33 PAL sorghum, 97% and 91% PAL cane sugar, 90% and 93% PAL barley, rice 76% C4H, C4H 94% sorghum, 89% the C4H C4H wheat and barley 87% in B. brizantha and P. maximum, respectively. The high similarity found among the fragments of the genes of the phenylpropanoid pathway in B. brizantha and P. maximum, especially with corn and rice that have their genome in advanced study and knowledge can enable the use of the database of these species for future studies of molecular biology.
 
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Date de Publication
2013-04-12
 
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