Identification and functional characterization of eight novel tpp family genes from Bacillus thuringiensis

Bacillus thuringiensis (Bt) and its transgenic crops are widely used as biological control agents for agricultural pests. The tpp family is a branch of Bt insecticidal genes and consists of a few members. Research on the Tpp family proteins has focused on the binary toxins Gpp34Ab/Tpp35Ab and Tpp1/Tpp2, which need to function together to achieve insecticidal activity. However, only a few tpp family genes have been reported to exert insecticidal activity independently. This study aimed to identify and characterize tpp family genes that independently perform insecticidal functions.

A total of 162 nucleotide sequences homologous to the single component Bt insecticidal gene tpp78Aa were obtained from the genome data of 1368 wild-type Bt strains, and 25 new full-length tpp family genes were identified. Eight new tpp family genes were successfully cloned and expressed, and bioassays of the expressed products were performed against five different pests. Bioassay results showed that these proteins exerted high insecticidal activity only against Laodelphax striatellus, a globally important rice pest, and were named Tpp78Ab1, Tpp78Bb1, Tpp78Ca1, Tpp78Da1, Tpp80Aa3, Tpp80Ac1, Tpp80Ad1, and Tpp80Ae1. The LC₅₀ values of Tpp78Ab1, Tpp78Bb1, Tpp78Ca1, and Tpp80Ae1 against L. striatum were 8.1, 8.6, 10.1, and 9.6 μg mL^-1 , respectively. The phylogenetic tree and conserved motifs indicated that the Tpp family had a common evolutionary ancestor. During evolution, the C-terminal pore-forming domain of the Tpp family adopted a similar arrangement; however, the N-terminal conserved motif showed high variability.

Twenty-five full-length tpp family genes were identified. Eight new tpp family genes were cloned successfully, which could independently achieve insecticidal activity against L. striatellus. This provides abundant genetic resources for the biological control of important rice pests. In this study, we found that the relative conservation of the Tpp family proteins in the lengthy evolutionary process and the diversity generated for adapting to the environment can lay a theoretical foundation for an in-depth analysis of the function and evolution of the Tpp family. © 2023 Society of Chemical Industry.