PCR-based strategy for cloning a wide spectrum of new Bacillus thuringiensis toxin genes.

BERÓN, C.M.; CURATTI, L.; SALERNO, G.

Foz do Iguassu, Brasil

Congreso; VIII International Colloquium on Invertebrate Pathology and Microbial Control. XXXV Annual Meeting of the SIP. VI International Conference on Bacillus thuringiensis.; 2002

Society of Invertebrate Pathology

Since the first cloning of the cry1Aa gene from Bacillus thuringiensis (Bt) serovar. kurstaki HD-1, more than one hundred Bt toxin genes have been cloned. The vast majority of the toxins cloned are active against lepidopteran pest in agriculture. In contrast the number of toxins that are active against other pest in agriculture, and in human and animal health are limited. Therefore, the isolation and characterization of novel toxins are critical for increasing the diversity of Bt toxins for biological control of pests and for overcoming potential problems associated with resistance. In order to identify new Bt toxin genes, different strategies were developed taking advantage of PCR methodology. Most of those initiatives involved the design of large number of primer sets directed against specific regions of type-specific cry genes, and later size differentiation of the amplification products. The aim of this work was to design a small set of oligonucleotides that can amplify a wide spectrum of cry genes. After multiple sequence alignments of representative of the most distantly sequences of known cry genes, we designed a set of primers suitable for PCR reactions. Different combinations of four of these primers in nested PCR reactions and with different PCR conditions, allowed us to amplify cry genes of all Bt isolations of our laboratory collection, including serovars. israelensis, tenebrionis and kurstaki strain HD1. Some of these PCR products were identified as cry genes after cloning and sequencing. Remarkably, this set of primers can be consider as universal Bt primers and constitutes an important tool to be used as a straight-forward alternative for the cloning of Bt genes.