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. / 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.

Synthetic auxin, indole-3-butyric acid (IBA), effectively promoted the rooting in Cinnamomum kanehirae cuttings. The easy-to-root genotype, H107, responded to IBA much earlier than the difficult-to-root genotype, L41. On day 5, the POD activity significantly decreased in the IBA-treated tissues as compared with the control. Similar phenomenon was observed in extract of L41 genotype showing that IBA inhibited POD activity. In addition, the variation in POD activity corresponds to an inverse variation in the concentration of free IAA. The levels of IAA in H107 genotype increased dramatically in IBA-treated tissues on day 5; while, in L41 genotype, the raise of IAA in IBA-treated tissues was observed on day 20. Moreover, the lignin content in IBA-treated H107 cuttings decreased is quite correlated with the decline of the POD activity; yet the lignin content in L41 genotype cuttings was almost unchanged. Hence, we suggest that the inhibition on POD may lead to the redifferentiation processes induced by IBA, then produce the new root primordia during the formation of adventitious roots.

Auxin can induce adventitious rooting. Synthetic auxin, indole-3-butyric acid (IBA), effectively promoted the rooting in Cinnamomum kanehirae. The peroxidase (POX) activity significantly decreased in the IBA-treated tissues as compared with the control. Hence, I suggest that the inhibition on POX activity may lead to the redifferentiation processes induced by IBA, which produces the new root primordia during the formation of adventitious roots. On this investigation, I cloned POX cDNA from the young roots. Degenerate primers were designed from the conservative regions of other published POX to amplify the expectant DNA fragment. Full-length cDNA of the POX gene designated CKPX1 and CKPX3 were cloned by the method of 5'and 3' RACE. The deduced amino acid of CKPX1 and CKPX3 were compared with the previously reported POX and showed between 40% and 60% identity with those plant species. Further studies on the promoter elements of CKPX3 were found out that elements related to auxin response, lignification, pathogen invasion and stress response. The results suggest that CKPX3 may be involved in the regulate process of adventitious rooting and defense against pathogens and environment stress.

Auxin can induce adventitious rooting. Synthetic auxin, indole-3-butyric acid (IBA), effectively promoted the rooting in Cinnamomum kanehirae. In Cinnamomum kanehirae, there are high (H) and low (L) rooting cultivar. The peroxidase (POX) activity significantly decreased in the IBA-treated tissues as compared with the control. The inhibition on POX activity may lead to the redifferentiation processes induced by IBA. In this investigation, we cloned POX cDNA from the young roots. Degenerate primers were designed from the conservative regions of other published POXs to amplify the expectant DNA fragment. We found that the H and L line have similar genes (>99%). The Full-length cDNA of the POX genes were cloned by the method of 5'and 3' Rapid Amplification of cDNA Ends (RACE). The deduced amino acid were compared with the previously reported POX and showed between 40% and 70% identity with other plant POXs. Further studies on the promoter elements of POX in High-rooting cultivar and Low-rooting line show that some elements are related to auxin response, lignification, pathogen invasion and stress response. The regulatory elements of the POX gene in High-rooting line contain sugar repression responsiveness (SRS) elements that might repress the expression of POX gene, causing the lower POX activity.

Diss. (sammanfattning). Umeå : Sveriges lantbruksuniv., 2003. / Härtill 4 uppsatser.

Diss. (sammanfattning). Umeå : Sveriges lantbruksuniv., 2003. / Härtill 5 uppsatser.

Diss. (sammanfattning) Umeå : Sveriges lantbruksuniversitet, 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Umeå : Univ., 2007. / Härtill 5 uppsatser.

Made available in DSpace on 2014-06-11T19:32:26Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-11-11Bitstream added on 2014-06-13T18:43:51Z : No. of bitstreams: 1 kohatsu_ds_dr_botfca.pdf: 455943 bytes, checksum: 5e4fc68a9fff388100712c74b410c0a6 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O presente trabalho teve como objetivo avaliar se o cultivo de pepineiro em porta enxertos recomendado e não recomendado para a cultura poderiam diferenciar-se entre si em relação aos processos bioquímicos de lignificação, conferindo assim, mudanças na atividade de enzimas marcadoras de estresse, crescimento, produtividade e qualidade dos frutos em relação ao cultivo de pepineiro não enxertado. Para alcançar os objetivos propostos, o trabalho foi dividido em três experimentos: no primeiro experimento foi avaliada a atividade enzimática durante o pegamento da enxertia. Já no segundo, foi estudado o efeito dos porta enxertos no crescimento das plantas e na atividade das enzimas antioxidantes durante o desenvolvimento. E no terceiro foi estudado o efeito dos porta enxertos na produtividade, número de frutos comercializáveis e qualidade de frutos. Durante o pegamento da enxertia observou-se que as partes inferiores e superiores à região da enxertia mantiveram atividade semelhante às plantas não enxertadas, exceto para a parte inferior à enxertia no híbrido Shelper, o qual apresentou aumento similar à região da enxertia. O porta enxerto ‘Shelper’ proporcionou maior crescimento e menor estresse às plantas, conforme a análise das enzimas antioxidantes, assim como maior produtividade, maior número de frutos comercializáveis, além de influenciar na coloração e brilho dos mesmos. Concluí-se que há diferença na atividade enzimática dos porta enxertos estudados e que o porta enxerto recomendado (‘Shelper’) proporcionou menor estresse às plantas durante o ciclo da cultura e, consequentemente, maior produtividade / This study aimed to evaluate whether the cultivation of cucumber in rootstocks recommended and not recommended for cucumber could differentiate between them in relation to the biochemical processes of lignification, giving thus, changes in the antioxidant enzymes, growth, productivity and fruit quality in relation to the cultivation of cucumber ungrafted. To achieve the proposed objectives, the work was divided in three experiments. In the first experiment, were evaluated biochemical characteristics during grafting. In the second, were studied the rootstock effect on plant growth and antioxidant enzymes activity during development. And in the third, were studied the rootstock effect on yield, commercial fruit number and fruit quality. During tissue union, was observed that enzyme activity below and above the grafting site remained similar to non-grafted plants, except above grafting site in Shelper hybrid, which was similar to the grafted site. The rootstock ‘Shelper’ provided greater growth and less stress to the plants, considering the antioxidant enzymes analysis, higher productivity, higher commercial fruits number, and even changed fruit color and brightness. According to the results, it was concluded that occurred difference in enzyme activity of the studied rootstocks and the recommended rootstock (‘Shelper’) provided the lowest stress to plants during the crop cycle and hence greater productivity

Orientador: Elizabeth Orika Ono / Banca: Rumy Goto / Banca: Carmen Silvia Fernandes Boaro / Banca: Marcos Antonio Bacarin / Banca: Roberto Botelho Ferraz Branco / Resumo: O presente trabalho teve como objetivo avaliar se o cultivo de pepineiro em porta enxertos recomendado e não recomendado para a cultura poderiam diferenciar-se entre si em relação aos processos bioquímicos de lignificação, conferindo assim, mudanças na atividade de enzimas marcadoras de estresse, crescimento, produtividade e qualidade dos frutos em relação ao cultivo de pepineiro não enxertado. Para alcançar os objetivos propostos, o trabalho foi dividido em três experimentos: no primeiro experimento foi avaliada a atividade enzimática durante o pegamento da enxertia. Já no segundo, foi estudado o efeito dos porta enxertos no crescimento das plantas e na atividade das enzimas antioxidantes durante o desenvolvimento. E no terceiro foi estudado o efeito dos porta enxertos na produtividade, número de frutos comercializáveis e qualidade de frutos. Durante o pegamento da enxertia observou-se que as partes inferiores e superiores à região da enxertia mantiveram atividade semelhante às plantas não enxertadas, exceto para a parte inferior à enxertia no híbrido Shelper, o qual apresentou aumento similar à região da enxertia. O porta enxerto 'Shelper' proporcionou maior crescimento e menor estresse às plantas, conforme a análise das enzimas antioxidantes, assim como maior produtividade, maior número de frutos comercializáveis, além de influenciar na coloração e brilho dos mesmos. Concluí-se que há diferença na atividade enzimática dos porta enxertos estudados e que o porta enxerto recomendado ('Shelper') proporcionou menor estresse às plantas durante o ciclo da cultura e, consequentemente, maior produtividade / Abstract: This study aimed to evaluate whether the cultivation of cucumber in rootstocks recommended and not recommended for cucumber could differentiate between them in relation to the biochemical processes of lignification, giving thus, changes in the antioxidant enzymes, growth, productivity and fruit quality in relation to the cultivation of cucumber ungrafted. To achieve the proposed objectives, the work was divided in three experiments. In the first experiment, were evaluated biochemical characteristics during grafting. In the second, were studied the rootstock effect on plant growth and antioxidant enzymes activity during development. And in the third, were studied the rootstock effect on yield, commercial fruit number and fruit quality. During tissue union, was observed that enzyme activity below and above the grafting site remained similar to non-grafted plants, except above grafting site in Shelper hybrid, which was similar to the grafted site. The rootstock 'Shelper' provided greater growth and less stress to the plants, considering the antioxidant enzymes analysis, higher productivity, higher commercial fruits number, and even changed fruit color and brightness. According to the results, it was concluded that occurred difference in enzyme activity of the studied rootstocks and the recommended rootstock ('Shelper') provided the lowest stress to plants during the crop cycle and hence greater productivity / Doutor