Analysis of Active Regions of Insecticidal Proteins of Bacillus thuringiensis var. israelensis

Among the strains of Bacillus thuringiensis, B. tkuringiensis var. israelensis (BTI) produces crystalline protein bodies (CPBs) consisting of several kinds of insectcidal proteins, molecular masses of which are 130-kDa, 70-kDa, 28-kDa and so on, and which are toxic to diptera such as mosquitoes and black flies. The insecticidal proteins are solubilized and proteolytically activated, and then break the cells in midgut to kill the insect. Two genes for 130-kDa insecticidal proteins, which we named ISRH3 (CryIVB) and ISRH4 (CryIVA), have been cloned and sequenced. To delineate the mosquitocidal regions of the ISRH3 and ISRH4 proteins, a deletion analysis of these protein genes was done. Based on the evidence that each 130-kDa protein had two mosquitocidal regions, N-terminal and C-terminal ones, and these two regions shared a common part in the center of the 130-kDa proteins, deleted genes on this region were constructed. As the protein products which lacked the central region had reduced activities, the central region could be important for the mosquitocidal activity. The mosquitocidal and non-mosquitocidal truncated gene products of 130-kDa protein genes were also applied to cultured insect cell lines. The mosquitocidal proteins caused the swelling and disruption of the cells, but the non-mosquitocidal proteins did not. therefore, the mosquitocidal fragments of 130-kDa proteins of BTI were cytotoxic to two insect cell lines. And an insecticidal fragment of ISRH4 protein actually bound to two insect cell lines