RESUMO
Genetically modified (GM) cotton plants that effectively control cotton boll weevil (CBW), which is the most destructive cotton insect pest in South America, are reported here for the first time. This work presents the successful development of a new GM cotton with high resistance to CBW conferred by Cry10Aa toxin, a protein encoded by entomopathogenic Bacillus thuringiensis (Bt) gene. The plant transformation vector harbouring cry10Aa gene driven by the cotton ubiquitination-related promoter uceA1.7 was introduced into a Brazilian cotton cultivar by biolistic transformation. Quantitative PCR (qPCR) assays revealed high transcription levels of cry10Aa in both T0 GM cotton leaf and flower bud tissues. Southern blot and qPCR-based 2-ΔΔCt analyses revealed that T0 GM plants had either one or two transgene copies. Quantitative and qualitative analyses of Cry10Aa protein expression showed variable protein expression levels in both flower buds and leaves tissues of T0 GM cotton plants, ranging from approximately 3.0 to 14.0 µg g-1 fresh tissue. CBW susceptibility bioassays, performed by feeding adults and larvae with T0 GM cotton leaves and flower buds, respectively, demonstrated a significant entomotoxic effect and a high level of CBW mortality (up to 100%). Molecular analysis revealed that transgene stability and entomotoxic effect to CBW were maintained in T1 generation as the Cry10Aa toxin expression levels remained high in both tissues, ranging from 4.05 to 19.57 µg g-1 fresh tissue, and the CBW mortality rate remained around 100%. In conclusion, these Cry10Aa GM cotton plants represent a great advance in the control of the devastating CBW insect pest and can substantially impact cotton agribusiness.
Assuntos
Proteínas de Bactérias/metabolismo , Endotoxinas/metabolismo , Gossypium/metabolismo , Gossypium/parasitologia , Proteínas Hemolisinas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/parasitologia , Gorgulhos/patogenicidade , Animais , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Endotoxinas/genética , Gossypium/genética , Proteínas Hemolisinas/genética , Imunidade Vegetal/genética , Imunidade Vegetal/fisiologia , Plantas Geneticamente Modificadas/genética , Reação em Cadeia da PolimeraseRESUMO
BACKGROUND: The activity of the major digestive cysteine proteinase detected in the intestinal tract of larvae of the bean weevil, Acanthoscelides obtectus (Say), was efficiently inhibited by the well-characterized cysteine proteinase synthetic inhibitor E-64 and also by a recombinant form of chagasin (r-chagasin), a tight-binding cysteine proteinase inhibitor protein from Trypanosoma cruzi. RESULTS: Incorporation of r-chagasin into an artificial diet system at 0.1 g kg(-1) retarded growth rate, decreased larval survival and led to complete mortality of A. obtectus at the end of the trial. The observed differences in growth rates occurred particularly in the first and second development stages. Artificial seeds containing high levels of r-chagasin (0.5-30 g kg(-1)) completely inhibited larval penetration. CONCLUSION: Together, the results reported in this paper support the hypothesis that the inhibitory activity of r-chagasin towards the major insect gut cysteine proteinase in vitro and in vivo is an accurate prediction of its insecticidal effects. The selectivity of this inhibitor against insect digestive proteinases supports the key role in parasite virulence by affecting the endogenous proteinase activity in its natural host.