Abstract

The crystal (Cry) proteins produced by Bacillus thuringiensis determine a particular strain's toxicity profile. This study was focused on cloning a cry1Ia gene based on amplicon restriction fragment length polymorphism (ARFLP) profile which would be helpful in developing new biopesticides with broader and higher spectrum of toxicity against Lepidoptera and Coleoptera insect pests. The present paper describes cry1Ia gene from a local isolate of Bacillus thuringiensis (B.t) CFE20(3). A PCR-restriction fragment length polymorphism method for identification of cry1I-type genes from Bacillus thuringiensis was established by designing a pair of primers based on the conserved regions of the genes to amplify 2,169 bp cry1I-type gene fragments. Amplification products were digested with the KpnI and XbaI, and new kind of cry1I-type genes was successfully identified. Escherichia coli DH5α was transformed with recombinant DNA comprising pTZ57R/T and Bt cry1I (2169 bp) amplified from a native isolate CFE20(3) for cloning. The cloned 2169 bp was sequenced and then ligated in the expression vector pQE30 for transformation of E. coli M15 and SG13009 for expression analysis. The sequence obtained shows 99% homology with known cry1Ia from B. thuringiensis subsp. Kurstaki. An expected band size of 81 KDa was observed after sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicating the expression of cry1I. The toxicity of crude recombinant Cry1I proteins was determined against third instar larvae of Diamond back moth, Plutella xylostella and Spodoptera litura. Cry1I protein was found to be effective against Plutella xylostella.

Key words: Bacillus thuringiensis, cry1I, SDS-PAGE, Plutella xylostella, Spodoptera litura.