Identification of cry1I-type genes from Bacillus thuringiensis strains and characterization of a novel cry1I-type gene

Abstract

A PCR-restriction fragment length polymorphism method for identification of cry1I-type genes from Bacillus thuringiensis was established by designing a pair of universal primers based on the conserved regions of the genes to amplify 1,548-bp cry1I-type gene fragments. Amplification products were digested with the Bsp119I and BanI enzymes, and four kinds of known cry1I-type genes were successfully identified. The results showed that cry1I-type genes appeared in 95 of 115 B. thuringiensis isolates and 7 of 13 standard strains. A novel cry1I-type gene was found in one standard strain and six isolates. The novel cry1I gene was cloned from B. thuringiensis isolate Btc007 and subcloned into vector pET-21b. Then it was overexpressed in Escherichia coli BL21(DE3). The expressed product was shown to be toxic to the diamondback moth (Plutella xylostella), Asian corn borer (Ostrinia furnacalis), and soybean pod borer (Leguminivora glycinivorella). However, it was not toxic to the cotton bollworm (Helicoverpa armigera), beet armyworm (Spodoptera exigua), or elm leaf beetle (Pyrrhalta aenescens) in bioassays. Subsequently, the Cry protein encoded by this novel cry gene was designated Cry1Ie1 by the B. thuringiensis delta-endotoxin nomenclature committee.

FIG. 1.

PCR-RFLP patterns of cry1I-type genes from B. thuringiensis standard strains and isolates. Lanes: 1 and 10 to 17, B. thuringiensis isolates; 2, B. thuringiensis isolate Btc007; 3, Bacillus thuringiensis subsp. aizawai (H7); 4, B. thuringiensis subsp. morrisoni (H8a8b); 5, Bacillus thuringiensis subsp. entomocidus (H6); 6, B. thuringiensis subsp. pakistani (H13); 7, B. thuringiensis subsp. thuringiensis (H1); 8, Bacillus thuringiensis subsp. thompsoni (H13); 9, B. thuringiensis subsp. galleriae (H5a5b); M, Molecular mass marker (pUC mix).

FIG. 2.

Electrophoretic analysis of proteins from recombinant E. coli BL21(DE3) and B. thuringiensis isolate Btc007 on an SDS-10% polyacrylamide gel. Lanes: M, high-molecular-mass protein marker; 1, total proteins from B. thuringiensis isolate Btc007 after the formation of the crystal; 2, purified inclusions from recombinant E. coli BL21(DE3) harboring the cry1Ie1 gene; 3, total proteins from recombinant E. coli BL21(DE3) harboring the cry1Ie1 gene; 4, total proteins from E. coli BL21(DE3).