Os resultados mostraram a obtenção de plantas transgênicas de Coffea arabica contendo o gene para o inibidor α-AI1 de P. vulgaris. Os genes nptII e α-AI1, além de terem sido inseridos no genoma do café, passaram para a descendência das plantas avaliadas. Essa transformação levou à expressão do inibidor ativo nas sementes de cafeeiro, sendo capazes de bloquear in vitro as -amilases do H.
hampei. Isto torna essas plantas promissoras como método de controle da broca-do-café.
Bioensaios serão necessários para comprovar a eficiência da expressão do inibidor α-AI1 no controle do H. hampei. No entanto, para que essa avaliação seja feita de forma adequada, é necessária a obtenção de plantas transgênicas homozigotas para o transgene bem como a avaliação do número de cópias inserido em cada planta. Essas plantas homozigotas podem expressar o inibidor em concentrações maiores do que as observadas nesse trabalho, o que deve aumentar a eficiência da planta no controle do inseto. Porém, essa obtenção é demorada, devido ao longo ciclo de vida do cafeeiro, que necessita de pelo menos 3 anos para começar a produzir as primeiras sementes.
Obviamente, um longo caminho ainda será percorrido, caso essa planta se mostre eficiente no controle da broca-do-café. Avaliações de segurança, como equivalência substancial, testes de alergenicidade, avaliação de progênie e testes ambientais ainda serão necessários antes que essa planta seja introduzida no mercado. Porém, todo o esforço e investimentos feitos foram válidos, pois essa planta de C. arabica transgênica poderá controlar o principal inseto-praga do cafeeiro, responsável por centenas de milhões de dólares de prejuízo anualmente, e a sua utilização diminuirá os custos de produção do café e o impacto ambiental causado pela utilização de inseticidas.
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Anexo 1. Seqüência de proteínas e de DNA do inibidor αAI-1, e seqüência de DNA do gene nptII.
Seqüência de proteínas do αAI-1
MIMASSKLLSLALFLALLSHANSATETSFIIDAFNKTNLILQGDATVSSNGNLQLSYNSYDS MSRAFYSAPIQIRDSTTGNVASFDTNFTMNIRTHRQANSAVGLDFVLVPVQPESKGDTVTVE FDTFLSRISIDVNNNDIKSVPWDVHDYDGQNAEVRITYNSSTKVFSVSLSNPSTGKSNNVST TVELEKEVYDWVSVGFSATSGAYQWSYETHDVLSWSFSSKFINLKDQKSERSNIVLNKIL Seqüência de DNA do gene αAI-1
ATGGCTTCCTCCAAGTTACTCTCCCTAGCTCTCTTCCTTGCGCTTCTCAGCCACGCAAACTC AGCCACCGAAACCTCCTTCATCATCGATGCGTTCAACAAAACCAACCTTATCCTTCAAGGCG ATGCCACCGTCTCATCCAACGGCAACTTACAACTATCCTATAATTCATACGACTCTATGAGC AGAGCCTTCTACTCCGCCCCCATCCGAATCAGGGACAGCACCACCGGCAACGTCGCCAGCTT CGACACCAACTTCACAATGAATATCCGCACTCACCGCCAAGCAAATTCCGCCGTTGGCCTTG ACTTTGTTCTCGTCCCCGTCCAGCCCGAATCCAAAGGCGATACTGTGACTGTGGAGTTCGAC ACCTTCCTCAGCCGTATTAGCATCGACGTGAACAACAACGATATCGAAAGCGTGCCTTGGGA TGTACACGACTACGACGGACAAAACGCCGAGGTTCGGATCACCTATAACTCCTCCACGAAGG TCTTCTCGGTTTCTCTGTCAAACCCCTCTACGGGAAAGAGCAACAACGTCTCTACCACAGTG GAGCTGGAGAAAGAAGTTTACGACTGGGTGAGCGTTGGGTTCTCTGCCACCTCAGGGGCTTA TCAATGGAGCTATGAAACGCACGACGTCCTCTCTTGGTCTTTTTCTTCCAAGTTCATCAATC TTAAGGACCAAAAATCTGAACGTTCCAACATCGTCCTCAACAAAATCCTTTAG
Seqüência de DNA do gene nptII
ATGGGGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATT CGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAG CGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTCCAG GACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGA CGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCC TGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTG CATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGC ACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGC TCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTC GTGACACATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATT CATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTG ATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCC GCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGA
Anexo 2. Soluções para Southern blot (BRASILEIRO, A. C. M., 1998).
Solução de desnaturação
Componente Quantidade Concentração final
NaoH 10 N 25 mL 0,5 N
NaCl 5 M 150 mL 1,5 M
H2O q.s.p. 500 mL
Solução de Neutralização
Componente Quantidade Concentração final
Tris-HCl 1M, pH 7,2 250 mL 0,5 M
NaCl 5 M 150 mL 1,5 M
H2O q.s.p. 500 mL
SSC 20X
Componente Quantidade Concentração final
NaCl 175,3 g 3 M
Citrato de sódio 88,2 g 0,3 M
H2O q.s.p. 1000 mL
Solução de pré-hibridização
Componente Quantidade Concentração final
Componente Quantidade Concentração final