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 ␦-endotoxin nomenclature committee.Crystal proteins from the gram-positive spore-forming bacterium Bacillus thuringiensis are toxic to a wide variety of insects that are economically important as pests. Many different genes encoding the B. thuringiensis endotoxin have been isolated and characterized. The genes have been classified as cry1 to cry40, cyt1, and cyt2 and are ranked according to their homology (10, 21; see also the B. thuringiensis toxin nomenclature website at http://www.biols.susx.ac.uk/home/Neil_Crickmore /Bt/). Cry1 proteins that are active against lepidopteran insects are produced as crystalline parasporal inclusions during sporulation. Generally, the crystals are composed of protoxins of approximately 130 kDa, but cry1I-type genes are usually silent genes capable of encoding a protein of about 81 kDa in B. thuringiensis strains (9,13,21,24). We decided to screen B. thuringiensis isolates for cry1I genes with the aim of finding novel cry1I genes, which could encode insecticidal proteins toxic to insensitive or resistant insect pests.This screening approach included the development of an analysis protocol based on PCR-restriction fragment length polymorphism (RFLP). PCR-based methods have been developed to detect different cry genes from B. thuringiensis strains (1-8, 11, 13, 15, 17, 23). More than 80 primer pairs have been designed to identify entire groups and individual cry genes (19). Several specific primers and probes for Southern blotting were designed to detect cry1I-type genes. A wide distribution of cry1I-type genes among many different B. thuringiensis strains has been reported (13,22,25). However, the list of cry genes is increasing, and novel PCR primers are needed in order to identify some of the recently described genes (5).The present study establishes a PCR-RFLP method for identify...

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