Cloning and Expression of an Insecticidal Crystal Protein CryIIA Gene from Bacillus thuringiensis subsp. kurstaki HD-1

Bacillus thuringiensis subsp. kurstaki HD-1 CryIIA의 내독소 단백질 유전자의 클로닝 및 발현

  • 김호산 (서울대학교 농업생물 신소재 연구센터) ;
  • 김상현 (농촌진흥청 잠업시험장) ;
  • 제연호 (서울대학교 농업생물 신소재 연구센터) ;
  • 유용만 (서울대학교 농업생물 신소재 연구센터) ;
  • 서숙재 (경상대학교 자연과학대학) ;
  • 강석권 (서울대학교 농업생물 신소재 연구센터) ;
  • 조용섭 (서울대학교 농업생명과학대학)
  • Published : 1993.09.01


The CryIIA gene encoding the insecticidal crystal protein of Bacillus thuringiens!s subsp. kurstalri HD-l has been cloned in Escherichia col!, and its nucleotide sequences were determined completely. 5kb Hindlli fragment harboring CryIIA gene was screened in the large ca. 225kb plasmid DNA by southern blot. HindlIT digested 5kb fragment was ligated into pUC19 and transformed in E. coli. The 4kb BamHI-HindlIT fragment containing the CryIIA gene was subcloned and named pSKIIA. DNA sequence analysis demonstrates that pSKIIA is the gene of an operon which is comprised of Lhree open reading frames (designated orn, orf2 and or£3). The CrylIA gene is composed of 3,952bp-long BamHI-Hindill DNA restriction fragment. The orf3 code for a polypeptide of 633 amino acid residues. The protoxin protein has a predicted molecular weight of 70,780. The E. coli derived protoxin gene product is biologICally active against three species of Lepidopteran (Plu.lelia maculipennis, He/iolhis assulta, Spodoptera litura) and a species of Dip Leran( Culex pipines) larvae in bioassay.