versão ISSN 1027-2852
ARMAS, Raúl et al. A barley cystatin stably expressed in rice exhibits strong in vitro inhibitory activity against gut proteinases of rice water weevil. Biotecnol Apl [online]. 2009, vol.26, n.4, pp. 321-327. ISSN 1027-2852.
Rice water weevil, Lissorhoptrus brevirostris Suffrian (Coleoptera: Curculionidae), is one of the most important rice pests in Cuba. Here, we describe the production of transgenic rice plants expressing barley cystatin HvCPI-1 (Icy1 gene) to explore the potential of this protein for the control of rice water weevil. Rice plants (Oryza sativa L. cv IACuba-28) were transformed via Agrobacterium tumefaciens with a plasmid carrying the Icy1 gene fused to the 35S promoter and the first exon/intron/exon from rice actin-1 gene. From 65 independent transgenic lines, 62 were positive in the PCR-Southern blot analyses. The transgene was correctly translated as indicated by western- and dot-blot assays with level of expression in T1 plants of up to 2% of the total extracted protein. The functional integrity of the protein was confirmed in vitro by a reduction of up to 90% of the cysteine-proteinase activity in the gut of rice water weevils exposed to rice leaf extracts. Moreover, proteins extracted from T2 transgenic rice roots showed a significant inhibition of up to 70% at pH 4.5 and 45% at pH 6.0 of the cathepsin B-like activity in the L. brevirostris larvae gut. These results demonstrate the potential of barley cystatin as an effective compound that may be combined with other pest control methods as an alternative in the struggle against insect resistance.
Palavras-chave : Lissorhoptrus brevirostris; cystatin; transgenic rice; gut proteinase activity; curculionids.