A Novel Vpb4 Gene and Its Mutants Exhibiting High Insecticidal Activity Against the Monolepta hieroglyphica

<i>Monolepta hieroglyphica</i> Motschulsky, a major agricultural pest in China, causes considerable economic damage to crops, such as maize. In this study, a <i>Bacillus thuringiensis</i> (Bt) strain was discovered to exhibit insecticidal activity against <i>M. hieroglyphica</i>. A novel Bt gene, <i>vpb4Fa1</i>, with toxicity against both adults and larvae of <i>M. hieroglyphica</i> was cloned. The Vpb4Fa1 protein causes damage to the midgut of adult <i>M. hieroglyphica</i>, disrupting their normal growth and development and ultimately leading to death. To further enhance the insecticidal activity of the <i>vpb4Fa1</i> gene, a random mutation library was established. A total of 75 mutants with amino acid mutations were generated, among which 7 mutants demonstrated significantly enhanced activity relative to the wild-type gene. Notably, three mutants, C9, 6C2, and 6A7, exhibited the highest activity, with LC₅₀ values for adult <i>M. hieroglyphica</i> of 10.21, 9.45, and 9.83 µg/g, respectively. The mutants C9, 6C2, and 6A7 each harbored nine, three, and six amino acid mutations, respectively, mainly located in Domains I, II, and III. The novel insecticidal gene <i>vpb4Fa1</i> and its mutants offer valuable genetic resources for the biological control of <i>M. hieroglyphica</i> and the development of Bt transgenic maize.