Título:	Toxinas Cry : potencial uso no controle da broca gigante da cana-de-açúcar (Telchin licus licus)
Autor(es):	Gomes Júnior, José Edilson
Orientador(es):	Sá, Maria Fátima Grossi de
Coorientador(es):	Freitas, Sônia Maria de
Assunto:	Cana-de-açúcar Cana-de-açucar - doenças e pragas Toxinas Cry Mariposa
Data de publicação:	15-Jan-2020
Data de defesa:	Fev-2012
Referência:	GOMES JÚNIOR, José Edilson. Toxinas Cry: potencial uso no controle da broca gigante da cana-de-açúcar (Telchin licus licus). 2012. [123] f., il. Tese (Doutorado em Biologia Molecular)—Universidade de Brasília, Brasília, 2012.
Abstract:	In Brazil, the sugarcane crops (Saccharum officinarum) have great eraphasis, with approximately 40% world production. It presents estimated production about 571 million tonnes for the 2011/2012 harvest, with an interesting and environmentally friendly altemative for the production of renewable energy sources. Although Brazil i ranked among the world’s leading producers of sugarcane, regions like the Northeast and Southeast have economic problems related to damage caused by the attack of insect pests, especially the sugarcane giant borer (Telchin licus licus) (Lepidoptera: Castniidae) and sugarcane borer (Diatraea saccharalis) (Lepidoptera: Crambidae), respectively. The difficulty in controlling these pests is the fact that they have endophytic habit, blocking the chemical insecticides action. In this context, several entomotoxic proteins have been described as potential candidates for the control of insect pests. Cry toxins or ô-endotoxins secreted by Gram-positive bacterium Bacillus thuringiensis (Bt) are effectively used to control insect pests because of their specificity and toxicity of some insect orders. In this work, the goal is creating and selecting Cry toxins effective against T. I. licus, using the DNA shuffling and Phage display molecular techniques. Bioassays results using Bt strains (Bt sor. kurstaki S2195 and SI905 and Bt sor. tolworthi SI35 and S62) and l st instar T. I. licus larvae showed high mortality in treatments with Bt sor. kurstaki. The cry genes were present in that serovars were determined and osmotic swelling assays based on light scattering indicated that CrylAa, CrylAb, CrylAc, Cryllal2 and Cry2Aa toxins were able to form pores in T. I. licus BBMVs. In bioassays with the same Cry toxins (1 mg/mL) and T. I. licus larvae, effectiveness was demonstrated, with a mortality rate of approximately 90%. In order to obtain new molecules for in vitro molecular evolution application, nucleotide sequences analysis between crylAa, cryllal2 and cry2Aa was held and the first two were chosen to be recombined by this technique therefore have higher nucleotide identity. After DNA shuffling, a combinatorial library was obtained containing 1.36 x 106 variant crylAa and cryllal2 genes. Before start selecting Cry variants using Phage display (biopanning), the receptors present in sugarcane giant borer midgut membranes (BBMVs) were tested for their quality and exhibited enrichment from 7.0 to 13.5-fold relative to marker enzymes tested. We selected five genes showing affmity to T. I. licus BBMVs. These were expressed in E. coli, validated by bioassays and BBMVs binding assays. We identified two variant Cryllal2 toxins exhibiting increased activity of 2.5 to 3.5 times compared to wild toxin; further the Cry2Aa toxin activity showed to be effective against the insect pest in question, activities subject to protection under patent. The results presented here will be used for the expression vectors construction in sugarcane genetic transformation, in order to resistance to sugarcane giant borer.
Unidade Acadêmica:	Instituto de Ciências Biológicas (IB) Departamento de Biologia Celular (IB CEL)
Informações adicionais:	Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, Programa de Pós-Graduação em Biologia Molecular, 2012.
Programa de pós-graduação:	Programa de Pós-Graduação em Biologia Molecular
Licença:	A concessão da licença deste item refere-se ao termo de autorização impresso assinado pelo autor com as seguintes condições:Na qualidade de titular dos direitos de autor da publicação, autorizo a Universidade de Brasília e o IBICT a disponibilizar por meio dos sites www.bce.unb.br, www.ibict.br, http://hercules.vtls.com/cgi-bin/ndltd/chameleon?lng=pt&skin=ndltd sem ressarcimento dos direitos autorais, de acordo com a Lei nº 9610/98, o texto integral da obra disponibilizada, conforme permissões assinaladas, para fins de leitura, impressão e/ou download, a título de divulgação da produção científica brasileira, a partir desta data.
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