International Journal of Industrial Entomology and Biomaterials

Korean Society of Sericultural Science (한국잠사학회)
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Characterization of a Novel cry1-Type Gene from Bacillus thuringiensis subsp. alesti Strain LY-99

  • Qi, Xu Feng (Laboratory of Insect Microbiology, College of Plant Science and Technology, Huazhong Agricultural University) ;
  • Li, Ming Shun (College of Science and Technology, Huazhong Agricultural University) ;
  • Choi, Jae-Young (Research Institute for Agriculture and Life Sciences, Seoul National University) ;
  • Roh, Jong-Yul (Department of Agricultural Biotechnology, College of Agriculture & Life Sciences, Seoul National University) ;
  • Song, Ji Zhen (Zhengzhou Tobacco Research Institute, China National Tobacco Corporation) ;
  • Wang, Yong (Department of Agricultural Biotechnology, College of Agriculture & Life Sciences, Seoul National University) ;
  • Jin, Byung-Rae (College of Natural Resources and Life Science, Dong-A University) ;
  • Je, Yeon-Ho (Department of Agricultural Biotechnology, College of Agriculture & Life Sciences, Seoul National University) ;
  • Li, Jian Hong (Laboratory of Insect Microbiology, College of Plant Science and Technology, Huazhong Agricultural University)
  • Published : 2009.03.31
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Abstract

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.

Keywords

References

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