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

Monolepta hieroglyphica Motschulsky, a major agricultural pest in China, causes considerable economic damage to crops, such as maize. In this study, a Bacillus thuringiensis (Bt) strain was discovered to exhibit insecticidal activity against M. hieroglyphica. A novel Bt gene, vpb4Fa1, with toxicity against both adults and larvae of M. hieroglyphica was cloned. The Vpb4Fa1 protein causes damage to the midgut of adult M. hieroglyphica, disrupting their normal growth and development and ultimately leading to death. To further enhance the insecticidal activity of the vpb4Fa1 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 M. hieroglyphica 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 vpb4Fa1 and its mutants offer valuable genetic resources for the biological control of M. hieroglyphica and the development of Bt transgenic maize.