Cloning of cry2Aa and cry2Ab genes from new isolates of Bacillus thuringiensis and their expression in recombinant Bacillus thuringiensis and Escherichia coli strains Shantanu Kumar 1 and V. Udayasuriyan 2, * 1 Centre of Advanced Studies in Agriculture Microbiology, Tamil Nadu Agricultural University, Coimbatore 641 003, Tamil Nadu, India 2 Centre for Plant Molecular Biology, Tamil Nadu Agricultural University, Coimbatore 641 003, Tamil Nadu, India *Author for correspondence: Tel.: þ91-0422-2431222 ext. 262, Fax: þ91-0422-2431672, E-mail: udayvar@hotmail.com Received 5 February 2003; accepted 31 July 2003 Keywords: Bacillus thuringiensis, Cry2Aa, Cry2Ab, E. coli, T7 promoter Summary Bacillus thuringiensis (Bt) is the major source for transfer of genes to impart insect resistance in transgenic plants. Cry2A proteins of Bt are promising candidates for management of resistance development in insects due to their difference from the currently used Cry1A proteins, in structure and insecticidal mechanism. Two insecticidal crystal protein genes of Bt, viz. cry2Aa and cry2Ab were cloned from new isolates of Bt, 22-4 and 22-11, respectively. Expression of both the genes was studied in an acrystalliferous strain of Bt (4Q7) by fusing the cry2Aa and cry2Ab genes downstream of cry2Aa promoter and orf1 + orf2 sequences. Western blot analysis revealed a low level expression of the cloned cry2Aa and cry2Ab genes in the recombinant Bt strains. High-level expression of cry2Aa and cry2Ab genes was achieved in the recombinant E. coli by cloning the cry2A genes under the control of the T7 promoter. Introduction Bacillus thuringiensis (Bt), is a well-known gram-posi- tive, spore-forming soil bacterium that forms parasporal insecticidal crystal proteins during the stationary phase of its growth cycle. Cloning of the first crystal protein gene (cry) of Bt was reported by Schnepf & Whiteley (1981), since then more than 120 cry genes have been cloned, characterized, and their classification based on amino acid sequence similarity of their proteins (Crick- more et al. 1998). Cloning of cry genes provides an opportunity to express the cloned gene in acrystallifer- ous Bt or E. coli to find out the insecticidal activities of their proteins. The reintroduction of cloned genes into Bt also provides a system to study factors regulating the expression of delta endotoxin genes. Using such a system, it has been discovered that two endotoxin genes, cytA and cry2Aa require an accessory protein to be co- expressed in order that their products may form crystalline inclusions (Adams et al. 1989; Crickmore et al. 1990; Crickmore & Ellar 1992). In the natural isolates of Bt the cry2Aa gene is expressed as third orf in the operon model whereas the cry2Ab gene is cryptic in nature. Dankocsik et al. (1990) achieved expression of the cry2Ab gene in an acrystalliferous strain of Bt by using the promoter derived from Bt cry3A gene. Although the gene was expressed there was no crystal inclusion formation. The orf2 of cry2Aa is necessary to achieve crystal formation of Cry2Aa and Cry2Ab proteins in Bt (Crickmore & Ellar 1992; Crickmore et al. 1994). Cry2Aa is an unusual subset of the Cry protein, toxic against two insect orders Lepidoptera and Diptera. The Cry2Aa protein has 633 amino acid residues with molecular mass of 63 kDa, and forms small cubical crystals (Yamamoto & Mclanghlin 1981; Donovan et al. 1988). Cry2Ab, an 87% sequence homologue of Cry2Aa, showed no activity against dipteran species but was more active than Cry2Aa against Helicoverpa zea (Dankocsik et al. 1990). The commercial use of Bt as suspension of spores and inclusions has been limited in part due to the need to spray at rather frequent intervals in order to sustain an effective level of biopesticide. Not only is the Bt crystal protein inclusion readily inactivated, but also the number of spores and/or vegetative cells decreases rather rapidly in the sprayed area. This problem has been circumvented by engineering plants (cotton, corn etc.) to produce the toxin (Schnepf et al. 1998). Trans- genic Bt-cotton expressing Cry1Ac has been registered for commercial cultivation in India during the year 2002 and it primarily targets Helicoverpa armigera. Continuous exposure to a single kind of Bt toxin can lead to resistance development in insects. Kranthi et al. (2000) reported 76-fold resistance development in the laboratory to an Indian population of H. armigera against Cry1Ac after 10 generations of selection. World Journal of Microbiology & Biotechnology 20: 11–17, 2004. 11 Ó 2004 Kluwer Academic Publishers. Printed in the Netherlands.