Characterization of Cry4Aa Toxin from Bacillus thuringiensis JW-1 and Its Insecticidal Activity Against Bradysia difformis

<i>Bradysia difformis</i> is a notorious pest in mushroom production in China. Biological control using <i>Bacillus thuringiensis</i> (Bt) offers an environmentally friendly and effective strategy against this pest. Here, we show that the complete genome of strain JW-1 consists of one circular chromosome and seven circular plasmids. JW-1-Plasmid 4 comprises 127,921 bp with a GC content of 33.9%, and is predicted to contain 131 genes, including six insecticidal genes: <i>cry4Aa</i>, <i>cry4Ba</i>, <i>cry10Ab</i>, <i>cry11Aa</i>, <i>cyt1Aa</i>, and <i>cyt2Ba</i>. A 3542-bp fragment containing the <i>cry4Aa</i> gene was amplified from this strain. Phylogenetic analysis based on Cry4 toxin sequences showed that JW-1 Cry4 toxin belongs to the Cry4Aa toxin cluster. A Cry4Aa fusion protein was subsequently expressed in <i>E. coli</i> and purified using Ni-IDA affinity chromatography. A larval feeding assay showed that purified Cry4Aa was toxic to <i>B. difformis</i> larvae, with an LC₅₀ of 2.71 ng/mL. These results confirmed the identity and bioactivity of Cry4Aa from strain JW-1, offering a promising biological control agent against this major pest.