Molecular Cloning of Two cDNAs Encoding an Insecticidal Toxin from the Spider, Araneus ventricosus, and Construction of a Recombinant Baculovirus Expressing a Spider Toxin

Abstract

We have cloned cDNAs encoding toxin from the spider, Araneus ventricosus, and constructed a recombinant baculovirus expressing the insecticidal toxin. The cDNAs encoding toxin were cloned from the cDNA library of A. ventricosus. Sequence analysis of the cDNAs encoding the toxin of A. ventricosus revealed that the 240 bp cDNA for AvTox-1 and 192 bp cDNA for AvTox-2 have an open reading frame of 80 and 64 amino acid residues, respectively. The deduced protein sequence of the toxin genes of AvTox-1 and AvTox-2 was aligned to that of the snack Anemonia sulcata and scorpion Centruroides limpidus limpidus, respectively. Northern blot analysis indicated that AvTox-2 toxin gene showed a fat body-spe-cific expression pattern at the transcriptional level. Furthermore, we have explored the possibility of improving baculovirus by incorporating the A. vontricosus toxin gene into Bombyx mori nuclear polyhedrosis virus genome under the control of polyhedrin promoter, The AvTox-2 toxin gene was expressed as approximately 5.8 kDa band in the recombinant baculovirus-injected silkworm larvae. Bioassays with the recombinant virus expressing AvTox-2 on 5th instar silkworm larvae demonstrated a decrease in the time to kill compared to wild-type BmNPV-Kl in the injection of 10 viruses. These results indicate that A. ventricosus toxin is a novel member of the spider toxin family, suggesting that the toxin gene can be used in recombinant baculoviruses to reduce insect feeding damage and increase the speed of insect kill.