Orientador : Paulo Arruda

Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia

Resumo: As proteinas da semente do Coix lacryma-jobi L. var. Adlay foram extraidas e caracterizadas. O endosperma do Adlay contem aproximadamente 21% de proteina. A principal fração proteica do endosperma do Adlay e uma prolamina denominada coixina, cuja porcentagem varia de 10,8% a 77,8%,...

Resumo: As proteinas da semente do Coix lacryma-jobi L. var. Adlay foram extraidas e caracterizadas. O endosperma do Adlay contem aproximadamente 21% de proteina. A principal fração proteica do endosperma do Adlay e uma prolamina denominada coixina, cuja porcentagem varia de 10,8% a 77,8%, dependendo da concentração de 2-ME utilizado no solvente de extração. As analises de aminoacidos revelaram que albuminas e globulinas (F1) do endosperma do Adlay são ricas Acido glutamico, Acido aspartico, glicina e alanina. As prolaminas (F2) são ricas em Acido glutamico, alanina, leucina prolina. As prolaminas não são completamente extraidas, mesmo quando 20% de 2-ME e utilizado no solvente de extração, e permanecem como uma contaminação das proteinas residuais (F3), o que faz com que a composição de aminoacidos da F3 seja similar A da F2. Analise das frações proteicas do Adlay atraves de eletroforese em SDS-PAGE demonstrou que a F1 e F3 possuem um padrão bastante complexo de bandas, enquanto que a F2 se subdividiu em apenas cinco bandas de pesos moleculares distintos. Estas cinco bandas foram denominadas de C1, C2, C3, C4 e C5. Anticorpos policlonais foram produzidos contra as bandas de coixina C2 e C3. Os anticorpos contra C2 reagiram com a banda C2 e apresentaram reação cruzada com proteinas de peso molecular mais elevado que o das coixinas e que so foram extraidas quando 20% de 2-ME foram utilizados no solvente de extração. Anticorpos contra C3 reconheceram a banda C3 e apresentaram reação cruzada com uma proteina de 40.5 kDa extraida quando 2-ME era utilizado no solvente de extração.Da analise das coixinas em focalização isoeletrica (IEF) revelou a presença de sete bandas principais. Quando as bandas individuais de IEF foram submetidas a SDS-PAGE, subdividiram-se em mais de uma classe de peso molecular distinto. As bandas de pI 7,26, 6.91, 6,55, 6,34 e 6,20 apresentaram reaçãoo com ambos os antisoros. As proteinas do endosperma do Adlay foram comparadas com as do Zea mays L. var. Maya. Foi constatado que o Maya possui um total de 8,7% de proteina no endosperma, sendo que 51,7% desta proteina são representados por prolaminas que, no caso do milho, são denominadas de zeina. A analise de aminoacidos da Fl, F2 e F3 do Maya revelou que existe uma similaridade na composição de aminoacidos das frações proteicas do Maya e do Adlay. Quando submetidas a SDS-PAGE, a F1 e F3 do Maya apresentaram um complexo padrão de bandas, sendo que algumas das bandas tinham o mesmo peso molecular de bandas encontradas nos padrões de Fl e F3 do Adlay. A F2 do Maya, do mesmo modo que a F2 do Adlay, apresentou uma baixa heterogeneidade em SDS-PAGE. As sete bandas encontradas no padrão de SDS-PAGE do Maya foram denominadas de Z1, Z2, Z3, Z4, Z5, Z6 e Z7. As prolaminas do Maya não apresentaram reação cruzada com o antisoro contra a classe de coixina C2. Contudo, o antisoro contra C3 apresentou reação cruzada com Z1 e com proteInas de 40,5 e 50 kDa extraIdas na presenCa de 2-ME. Em IEF, a zeina do Maya apresentou oito bandas principais que, quando foram submetidas a SDS-PAGE, revelaram ser principalmente do tipo Z2 e/ou Z3. Apesar da dificil visualização no gel de SDS-PAGE, bandas do tipo Z1 se encontram nos mesmos pis das bandas Z2 e Z3, como foi detectado atraves de analise imunologica com o antisoro contra C3. Os DNAs genomicos do Adlay e do Maya foram digeridos com as enzimas de restrição Eco RI, Bam HI e Hind III e submetidos a hibridizações com as sondas de cDNA da zeina pZ 22,3 e pZ 19,1. Enquanto que o DNA do Maya apresentou homologia com ambas as sondas de zeina, o DNA do Adlay so apresentou homologia com a sonda pZ 22,3. As proteinas do endosperma do Coix lacryma-jobi var. Acre, da linhagem de milho L1038 e dos teosintes Zea mays mexicana e Zea luxurians foram comparadas com as do Adlay e do Maya. Ficou constatado que os padrões de SDS-PAGE da Fl, F2 e F3 do Adlay e do Acre são praticamente iguais. Os padrões de SDS-PAGE da Fl, F2 e F3 dos milhos e teosintes tambem são praticamente iguais. Quando submetida a reações imunologicas, a F2 do Acre apresentou reação cruzada com os antisoros contra as classes de coixina C2 e C3 do Adlay. Da mesma forma que havia sido observado para F2 do Maya, as F2 da L1038, Zea mays mexicana e Zea luxurians nAo apresentaram reação cruzada com o antisoro contra a classe de coixina C2 do Adlay, mas apresentaram reação cruzada com o antisoro contra C3. Da homogeneidade encontrada nos padrões de SDS-PAGE da F2 do Adlay e do Acre e da F2 do Maya, L1038, Zea mays mexicana e Zea luxurians fez com que estas proteinas fossem submetidas a focalização isoeletrica e, subsequentemente, comparadas com os padrões de IEF de prolaminas de outras linhagens de milho e variedades de Coix e teosinte, onde se observou um padrão individual de bandas para cada um dos materiais analisados. Os DNAs genomicos do Zea mays mexicana, Zea luxurians, L1038 foram digeridos com as enzimas de restrição Eco RI e Bam HI e submetidos a hibridizações com as sondas pZ 22,3 e pZ 19,1. Foi constatado que existe homologia entre os DNAs dos milhos e teosintes e ambas as sondas de cDNA da zeina

Abstract: The seed proteins of Coix lacryma-jobi L. var. Adlay were extracted and characterized. The endosperm of Adlay contains ca. 21% of proteins. The major protein fraction of the endosperm of Adlay is a prolamin, known as coixin. The amount of coixin extracted ranges from 10.8% to 77.8%,...

Abstract: The seed proteins of Coix lacryma-jobi L. var. Adlay were extracted and characterized. The endosperm of Adlay contains ca. 21% of proteins. The major protein fraction of the endosperm of Adlay is a prolamin, known as coixin. The amount of coixin extracted ranges from 10.8% to 77.8%, depending upon the concentration of 2-mercaptoethanol in the extraction solvent. Amino acid analysis revealed that albumins and globulins (F1) from Adlay endosperm are rich in glutamic acid, aspartic acid, glycine and alanine. The prolamine (F2) are rich in glutamic acid, alanine, leucine, and proline. The prolamins are not completely extracted, even when 20% of the 2-ME is used in the extraction solvent. They remain as a contamination of residual proteins(F3). Because of the incomplete extraction of coixin, the amino acid content of F3 proteins is similar to that of F2. SDS-PAGE analysis of Adlay protein fractions showed that F1 and F3 have a complex pattern of bands, while F2 has only five bands. Those five bands were denominated C1, C2, C3, C4, and C5. Polyclonal antibodies were raised against the C2 and C3 protein bands. The C2 antibodies recognized the C2 band and cross-reacted with proteins of higher molecular weight that were extracted only when 20% of 2-ME were used in the extraction solvent. The C3 antibodies recognized the C3 band and cross-reacted with a 40.5 kDa protein, extracted when 2-ME was included in the extraction solvent. Isoelectric focusing (IEF) analysis of coixin revealed the presence of seven major bands. When individual IEF bands were submitted to SDS-PAGE, they subdivided into more than one molecular weight class. The IEF bands with pI 7.26, 6.91, 6.55, 6.34, and 6.20 reacted with both antibodies. The endosperm proteins of Adlay were compared with those of Zea mays L. var. Maya. It was observed that Maya has 8.7% of protein in the endosperm. Aproximately 51.7% of that protein are prolamins, which are called zein in corn. Amino acid analysis of Maya F1, F2, and F3 showed that there is a similarity between the amino acid composition of the protein fractions of Maya and Adlay.When submitted to SDS-PAGE, the F1 and F3 of Maya gave a complex band pattern. Some of the bands observed the F1 and F3 of Maya had the same molecular weight of bands found in the F1 and F3 of Adlay. Like the F2 of Adlay, the F2 of Maya showed low heterogeneity in SDS-PAGE. The seven bands observed in Maya F2 SDS-PAGE pattern were named Z1, Z2, Z3, Z4, Z5, Z6, and Z7. No cross-reaction was observed between the prolamins of Maya and C2 antibody. However, a cross-reaction was observed between C3 antibody and Z1. Proteins extracted in the presence of 2-ME with 40.5 and 50 kDa a1so cross-reacted with C3 antibodies. A total of eight major bands were observed for Maya in IEF. When submitted to SDS-PAGE, those bands revealed to be mostly Z2 and Z3. Bands Z1 were detected in SDS-PAGE with C3 antibody in the same pI of Z2 and Z3 bands.The genomic DNAs of Adlay and Maya were digested with the restriction enzymes Eco RI, Bam HI, and Hind III and submitted to hybridizations with zein cDNA probes pZ 22.3 and pZ 19.1. While the DNA of Maya showed homology with both probes, the DNA of Adlay showed homology only with pZ 22.3. The endosperm proteins of Coix lacryma-jobi var. Acre, corn inbred 1ine 1038, and of the teosintes Zea mays mexicana and Zea luxurians were compared with the ones of Adlay and Maya. It was observed that the SDS-PAGE patterns of the F1, F2, and F3 of Adlay and Acre were practically the same. The SDS-PAGE patterns of the F1, F2, and F3 of the corn and teosintes were also practically the same. When submitted to immunological reactions, a cross-reaction was observed between Acre F2 and C2 and C3 antibodies. As it was found for Maya F2, no cross-reaction was observed for L1038, Zea mays mexicana, and Zea luxurians F2 and C2 antibody. However, a cross-reaction was observed for the F2 of those materials and C3 antibody. The lack of heterogeneity observed for the SDS-PAGE pattern of the F2 of Adlay and Acre and the F2 of Maya, L1038, Zea mays mexicana, and Zea luxurians was the reason why those proteins were submitted to isoelectric focusing and after that were compared with the IEF pattern of the prolamins of other corn inbred lines and Coix and teosinte varieties. Those comparisons showed an individual pattern for each material analysed. The genomic DNA of Zea mays mexicana, Zea luxurians, L1038, and Maya were digested with the restriction enzymes Eco RI and Bam HI and submitted to hibridization with pZ 22.3 and pZ 19.1. It was observed that there is a homology between the DNAs of corn and teosinte and both probes