Molecular cloning and sequence analysis of the immediate early 1 gene of Anticarsia gemmatalis MNPV

BILEN MARCOS; PILLOFF MARCELA; RIBEIRO BERGMAN MORAIS; ROMANOWSKY VICTOR; LOZANO MARIO ENRIQUE; GHIRINGHELLI DANIEL

Mexico

Congreso; Annual Meeting of the Society for Invertebrate Pathology; 2000

Anticarsia gemmatalis (Lep. Noctuidae), is a major pest of soybean in South America. An indigenous nucleopolyhedrovirus (AgMNPV) isolated in Brazil proved to be a feasible microbial control agent. This baculovirus is currently used in more than one million hectares annually in Brazil. However, the low speed of action (6-8 days after feeding) limits its wider acceptance. The pathogenicity could be altered using recombinant DNA techniques to introduce genetic modifications, such as the insertion of foreign genes coding for toxins, insect hormones, etc. and/or the deletion of specific viral genes. The strategies for the expression of foreign genes could involve early and late promoters. Several early genes have been characterized in other baculoviruses; most of them are associated with the regulation of late and very late genes (e.g. ie1, ie2, etc). The possible use of the ie1 promoter to drive the expression of foreign genes is an interesting approach that has been successful in other baculovirus-insect systems. We identified the genomic restriction fragment containing the ie1, cloned and sequenced the locus of this virus. A 2396 bp BglII/XhoI fragment of the AgMNPV genome contains the 1707 nucleotides long ie1 ORF and regulatory upstream and downstream regions. The IE1 protein has 63% amino acid identity (77% homology) with the IE1 of Orgyia pseudotsugata and 65% amino acid identity (75% homology) with the IE1 of Choristoneura fumiferana. On the other hand, we used the nucleotide sequence to locate conserved regulatory signals in non-coding regions and to conduct phylogenetic studies with the corresponding sequences of other baculoviruses. This initial cloning and characterization will enable the further development of transfer vectors for the expression of foreign genes under the control of the ie1 promoter.