SARAIVA, Luis Flávio Mendes. Avaliação molecular da biossíntese de ácido ascórbico e possível participação da Oxidase alternativa em dois clones de aceroleira (Malpighia emarginata DC). 2011. 128 f. Tese (Doutorado em bioquímica)- Universidade Federal do Ceará, Fortaleza-CE, 2011. / Submitted by Elineudson Ribeiro (elineudsonr@gmail.com) on 2016-07-20T17:07:55Z No. of bitstreams: 1 2011_tese_lfmsaraiva.pdf: 11676061 bytes, checksum: a4b2ae06988f2e8feb11ebae7bf0d662 (MD5) / Approved for entry into archive by José Jairo Viana de Sousa (jairo@ufc.br) on 2016-08-02T17:55:52Z (GMT) No. of bitstreams: 1 2011_tese_lfmsaraiva.pdf: 11676061 bytes, checksum: a4b2ae06988f2e8feb11ebae7bf0d662 (MD5) / Made available in DSpace on 2016-08-02T17:55:52Z (GMT). No. of bitstreams: 1 2011_tese_lfmsaraiva.pdf: 11676061 bytes, checksum: a4b2ae06988f2e8feb11ebae7bf0d662 (MD5) Previous issue date: 2011 / The acerola, Malpiguia emarginata DC is a fruit small endowed with enviable nutritional qualities, being consumed both fresh and processed. The mail feature that stands out from other species is its enormous capacity to synthesize ascorbic acid. In this sense, Embrapa has developed four commercial clones with phenotypic and genetic characteristics well defined. The clones BRS 235 (Apodi), BRS 236 (Cereja), BRS 237 (Roxinha) e BRS 238 (Frutacor) have characteristics fully standardized. This study aimed to evaluate the clones BRS 236 (Cereja) and BRS 237 (Roxinha) were selected because of huge amount of ascorbic acid synthesized between then, approximately 50% in Cereja clone. For this study we analyzed the gene expression of enzymes of the way Wheeler /Smirnoff as well as the Alternative Oxidase (AOX). The last step this of this pathway ends in the inner mitochondrial membrane, due to the presence of transmembrane enzyme L-Galactono-1,4- Lactone dehydrogenase (L-GalLdh), located between the complex III and IV. Since the alternative oxidase is an enzyme uncoupling, no phosphorylating and insensitive to cyanide, among the complexes II and III of the inner mitochondrial membrane, responsible for alternative pathway of electrons. This study aimed to identify the enzymes that are crucial in this process and generate the difference between the clones, as well as the probable relationship between the biosynthesis of ascorbic acid and the role that the AOX could play. Acerola five years of age were harvested four tissues (flowers, unripe, semi mature and mature fruits). The first step of this work was the determination of ascorbic acid in unripe, semi mature and mature fruits of the two clones by titration of Tillman. Then, we performed the characterization of the AOX gene to define its isoforms. After isolation of DNA and carried out the reactions of PCR with degenerate primers the amplicons were purified and subjected to cloning, transformation and sequencing. The results revealed sequences that correspond to one AOX1 and one AOX2. The next step of work was the study of gene expression of enzymes to the Wheeler/Smirnoff path, AOX1 and AOX2 newly characterized. After the design of pairs of degenerate and specific primers for all the enzymes of the route beyond the AOX1 and AOX2 they were used in semiquantitative PCR reactions, using the elongation factor alpha as a constitutive elemento of reference. The results showed that both clones decrease their levels of ascorbic acid as the fruits develop, revealing that in all three stages of development, the Cereja clone has higher amounts of ascorbic acid that Roxinha clone. However, when comparing the levels of ascorbic acid between green and ripe fruits of both clones is evident that the Roxinha clone has a lower difference between these two stages. The gene expression of three enzymes stood out from others, these showed a synergism with the expression levels of vitamin C contained in each tissue. In addition, gene expression in two clones showed differences in tissues that favor of Cereja clone. They are: Mannose pyrophsphorylase, GDP-Mannose 3’5’ epimerase and GDP-Galactose phosphorylase. With greater emphasis on GDP-Mannose 3’5’ epimerase and GDP-Galactose phosphorylase. The enzymes that stood out showed a higher gene expression in tissues where exactly had more ascorbic acid accumulated, gradually reducing the expression with the degree of fruit development, revealing that the difference occurs not only between clones, but also in tissues clones. There were no differences in gene expression of other enzymes of the route Wheeler/Smirnoff to justfy the levels of ascorbic acid present in the tissues and also between clones. The analysis of gene expression of isoforms of AOX revealed that in both clones the AOX1 gene expression increases as the fruit ripens. However, the clone Roxinha has a higher gene expression than the clone Cereja, in all three stages of development. The analysis of transcripts of AOX2 revealed that in Cereja clone the gene expression decreased with fruit ripening, since in Roxinha clone the expression xx elevated in three developmental stages. The results cearly showed that three enzymes are essential for biosyntesis of ascorbic acid highlighting the GDP-Mannose 3’5’epimerase and GDP-Galactose phosphrylase. Another important conclusion was that the AOX may be owner a function not previously reported in the literature, that of assisting in the biosyntesis of vitamin C. This may be due uncoupling promoted by the activity of AOX causing a gradual rise of the relationship oxidized cytochrome C/rediced cytochrome C, where the cytochrome C oxidized acts of a GalLdh substrates in the syntesis of ascorbic acid. This fact may be explained by not only the highest levels of AOX1 transcripts present in Roxinha clone, but the increasing pattern of gene expression in AOX2 exatly in clone that produces less ascorbic acid and which has the smallest difference between the stages of fruit Green and ripe fruit. In the clone Cereja, one that synthesizes more ascorbic acid and has a huge difference in the levels between the stages of green fruit and ripe fruit of gene expression of AOX2 shown decresing during ripening. Thus, the concentration of products the Wheeler / Smirnoff path is still determining the amount of ascorbic acid produced, and in parallel, the increase in the AOX gene expression appears to contribute at some point to establish the total levels of vitamin C synthesized. / A acerola Malpiguia emarginata DC é uma fruta dotada de qualidades nutricionais invejáveis, sendo consumida tanto in natura quanto processada. A principal característica que destaca a acerola é sua enorme capacidade em sintetizar o ácido ascórbico. A Embrapa desenvolveu quatro clones comerciais com características fenotípicas e genéticas bem definidas denominado BRS 235 (Apodi), BRS 236 (Cereja), BRS 237 (Roxinha) e BRS 238 (Frutacor). Os clones BRS 236 (Cereja) e BRS 237 (Roxinha) foram escolhidos devido a enorme diferença de ácido ascórbico sintetizada entre eles, aproximadamente 50% a mais no clone Cereja. Para esse estudo foi analisada a expressão gênica das enzimas pertencentes a via Wheeler/Smirnoff, reconhecida como a principal via biossintética do ácido ascórbico em plantas bem como a Oxidase alternativa (AOX) uma enzima desacopladora, não fosforilante e insensível ao cianeto, presente entre os complexos II e III da membrana mitocondrial interna, responsável pela via alternativa de elétrons. O objetivo desse trabalho foi identificar quais enzimas são determinantes na diferença do conteúdo de vitamina C entre os clones, bem como avaliar a expressão da AOX nos diferentes tecidos. De aceroleiras com cinco anos de idade foram colhidos quatro tecidos (Flores, Frutos verdes, Frutos semimaduros e Frutos maduros. Inicialmente os teores de ácido ascórbico foram dosados nos frutos verdes, semimaduros e maduros dos dois clones por titulometria de Tillman. Em seguida foi realizada a caracterização gênica da AOX para definição de suas isoformas. Após o isolamento do DNA e executadas as reações de PCR com um par de primers degenerados os amplicons foram purificados e submetidos a clonagem, transformação e seqüenciamento. As dosagens de ácido ascórbico mostraram que ambos os clones decrescem seus níveis de ácido ascórbico a medida que os frutos se desenvolvem, além do que em todos os três estádios de desenvolvimento o clone Cereja apresenta quantidades de ácido ascórbico superiores ao clone Roxinha. Os níveis de ácido ascórbico entre os frutos verdes e maduros de ambos os clones revelaram que o clone Roxinha possui uma menor diferença entre esses dois estádios de desenvolvimento. As análises de expressão gênica revelaram que três enzimas possuem sua expressão destacada das demais, sendo que essas mostraram um sinergismo de expressão com o padrão decrescente dos níveis de vitamina C contida no tecido, apresentando ainda diferenças de expressão favoráveis ao clone Cereja. São elas: Manose pirofosforilase, GDP-Manose 3’5’ epimerase e GDP Galactose fosforilase. Com maior destaque para a GDP-Manose 3’5’ epimerase e GDP Galactose fosforilase. Não existiam diferenças nas expressões gênicas das demais enzimas da via Wheeler/Smirnoff que justificassem as diferenças nos teores de ácido ascórbico presentes nos tecidos e também entre os clones. Quanto a AOX os resultados revelaram duas seqüências, uma relativa a uma AOX1 e outra a uma AOX2. A análise da expressão gênica das isoformas da AOX demonstrou que a AOX1 eleva sua expressão gênica em ambos os clones a medida que os frutos amadurecem entretanto, o clone Roxinha possui uma expressão gênica mais elevada que o clone Cereja, em todos os três estádios de desenvolvimento. A AOX2 possui diferenças de expressão gênica onde no clone Cereja ela se mostrou decrescente, já no clone Roxinha ocorreu a elevação da expressão nos três estádios de desenvolvimento. Três enzimas são essenciais a biossíntese do ácido ascórbico em Malpiguia emarginata DC, a Manose pirofosforilase, GDP-Manose 3’5’ epimerase e GDP Galactose fosforilase, sendo a via Wheeler/Smirnoff determinante na quantidade de ácido ascórbico produzido. As expressões gênicas da AOX1 e AOX2 favorecem o clone Roxinha, aparentemente como um mecanismo compensatório por esse clone sintetizar menos ácido ascórbico que o clone Cereja.