• Journal of Microbiology and Biotechnology
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• v.8 no.5
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• pp.517-522
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• 1998

The cry1 gene content of a collection of Bacillus thuringiensis strains, which include new isolates from Malaysia and Indonesia, was determined by a multiplex PCR using a set of eight oligonucleotide forward primers specific to cry1Aa, cry1Ab, cry1Ac, cry1Ba, cry1Ca, cry1Da, cry1Ea, and cry1Fa genes, and two reverse primers, one specific to cry1Ab and the other common to the remaining cry1 genes. Two-thirds of the 59 strains screened were cry1 positive and contained one to four different genes. The cry gene profiles correlated well with toxicities of the strains to lepidopteran insects. The method can be used for rapid screening of a large number of new isolates as the total DNA extracted by boiling cells from single colonies can be used directly in the PCR. However, it is not suitable for follow-up monitoring of specific commercial strains after application in the field as the PCR product profiles of these strains could not be differentiated from those of new isolates.

• International Journal of Industrial Entomology and Biomaterials
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• v.18 no.1
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• pp.18-27
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• 2009

B. thuringiensis strain LY-99 belonging to subsp. alesti (H3a3c), was isolated from Chinese tobacco warehouse and showed significantly high toxicity to Plutella xylostella. For the identification of the cry1-type genes from B. thuringiensis LY-99, an extended multiplex PCRrestriction fragment length polymorphism (PCRRFLP) method was established by using two pairs of universal primers based on the conserved regions of the cry1-type genes to amplify around 2.4 kb cry1-type gene fragments. Then the DNA fragment was cloned into pGEM-T Easy vector and digested with EcoRI and EcoRV enzymes. Through this method, a known cry1-type gene was successfully identified from the reference strain, B. thuringiensis subsp. alesti. In addition, the RFLP patterns revealed that B. thuringiensis LY-99 included a novel cry1A-type gene in addition to cry1Aa, cry1Ac, cry1Be and cry1Ea genes. The novel cry1A-type gene was designated cry1Ah2 (Genbank accession No DQ269474). An inverse PCR method was used to amplify the flank regions of cry1Ah2 gene. Finally, 3143 bp HindIII fragment from B. thuringiensis LY-99 plasmid DNA including 5' region and partial ORF was amplified, and sequence analysis revealed that cry1Ah2 gene from LY-99 showed 89.31% of maximum sequence similarity with cry1Ac1 crystal protein gene. In addition, the deduced amino acid sequence of Cry1Ah2 protein shared 87.80% of maximum identity with that of Cry1Ac2. This protein therefore belongs to a new class of B. thuringiensis crystal proteins.