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Characterization of a Novel cry1-Type Gene from Bacillus thuringiensis subsp. alesti Strain LY-99

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초록

B. thuringiensis strain LY-99 belonging to subsp. alesti(H3a3c), was isolated from Chinese tobacco warehouseand showed significantly high toxicity to Plutella xylostella. For the identification of the cry1-type genes fromB. thuringiensis LY-99, an extended multiplex PCRrestrictionfragment length polymorphism (PCRRFLP)method was established by using two pairs ofuniversal primers based on the conserved regions ofthe cry1-type genes to amplify around 2.4 kb cry1-typegene fragments. Then the DNA fragment was clonedinto pGEM-T Easy vector and digested with EcoRIand EcoRV enzymes. Through this method, a knowncry1-type gene was successfully identified from the referencestrain, B. thuringiensis subsp. alesti. In addition,the RFLP patterns revealed that B. thuringiensisLY-99 included a novel cry1A-type gene in addition tocry1Aa, cry1Ac, cry1Be and cry1Ea genes. The novelcry1A-type gene was designated cry1Ah2 (Genbankaccession No DQ269474). An inverse PCR method wasused to amplify the flank regions of cry1Ah2 gene. Finally, 3143 bp HindIII fragment from B. thuringiensisLY-99 plasmid DNA including 5’ region andpartial ORF was amplified, and sequence analysisrevealed that cry1Ah2 gene from LY-99 showed89.31% of maximum sequence similarity with cry1Ac1crystal protein gene. In addition, the deduced aminoacid sequence of Cry1Ah2 protein shared 87.80% ofmaximum identity with that of Cry1Ac2. This proteintherefore belongs to a new class of B. thuringiensiscrystal proteins.

Abstract

B. thuringiensis strain LY-99 belonging to subsp. alesti(H3a3c), was isolated from Chinese tobacco warehouseand showed significantly high toxicity to Plutella xylostella. For the identification of the cry1-type genes fromB. thuringiensis LY-99, an extended multiplex PCRrestrictionfragment length polymorphism (PCRRFLP)method was established by using two pairs ofuniversal primers based on the conserved regions ofthe cry1-type genes to amplify around 2.4 kb cry1-typegene fragments. Then the DNA fragment was clonedinto pGEM-T Easy vector and digested with EcoRIand EcoRV enzymes. Through this method, a knowncry1-type gene was successfully identified from the referencestrain, B. thuringiensis subsp. alesti. In addition,the RFLP patterns revealed that B. thuringiensisLY-99 included a novel cry1A-type gene in addition tocry1Aa, cry1Ac, cry1Be and cry1Ea genes. The novelcry1A-type gene was designated cry1Ah2 (Genbankaccession No DQ269474). An inverse PCR method wasused to amplify the flank regions of cry1Ah2 gene. Finally, 3143 bp HindIII fragment from B. thuringiensisLY-99 plasmid DNA including 5’ region andpartial ORF was amplified, and sequence analysisrevealed that cry1Ah2 gene from LY-99 showed89.31% of maximum sequence similarity with cry1Ac1crystal protein gene. In addition, the deduced aminoacid sequence of Cry1Ah2 protein shared 87.80% ofmaximum identity with that of Cry1Ac2. This proteintherefore belongs to a new class of B. thuringiensiscrystal proteins.

참고문헌 (34)

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