Bacillus thuringiensis 살충성 결정단백질 유전자(cry II A)의 형질전환 식물 제작

Generation of Transgenic Plant (Nicotiana tabacum var. Petit Havana SR1) harboring Bacillus thuringiensis Insecticidal Crystal Protein Gene, cry II A

  • 이정민 (성균관대학교 생명자원과학대학 유정공학과) ;
  • 류종석 (웨인주립대학 유전 및 분자생물학과) ;
  • 권무식 (성균관대학교 생명자원과학대학 유정공학과)
  • 발행 : 1997.09.01

초록

Bacillus thuringiensis는 그람 양성 토양 세균으로 포자형성시 결정화된 내포체를 형성하는데, 이 내포체를 구성하는 결정단백질은 각 곤충에 대하여 특이적인 독성을 나타낸다. 살충성 결정단백질 중 Cry II A 결정단백질은 인시류와 쌍시류 곤충에 모두 특이적으로 작용한다. 결정단백질은 살충제로서 불안정하고 포장에서 지속성이 낮은 단점을 가지고 있으므로, 본 연구에서는 Cry II A 결정단백질 유전자가 형질전환된 담배 식물을 제작하고자 하였다. cry IIA 유전자가 삽입된 벡터를 대장균에 형질전환한 후, 알칼리 용액에 대한 용해도의 차이를 이용하여 Cry II A 결정단백질(70 kDa)을 분리하였고, 분리한 Cry II A 결정단백질을 trypsin 처리하여 활성화된 Cry II A (50 kDa)를 확인하였다. 식물 형질전환을 위하여 두 개의 CaMV 35S promoters에 의해 발현이 조절되는 식물 발현 벡터에 cry II A 유전자를 클로닝 하였다. 이 식물 발현 벡터를 Agrobacterium을 이용한 엽편형질 전환을 통해 담배(N. tabacum var. Petit Havana SRI)에 형질전환 시켰으며, 재분화 과정을 거쳐 여섯 개체의 형질전환 식물체를 얻었다. Southern blot을 통하여 분석한 결과 세 개체 내에 cry II A 유전자가 존재하였는데, 한 개체에는 하나의 cry II A 유전자가, 또 한 개체에는 두 개의 cry II A 유전자가, 다른 한 개체에는 잘려진 형태의 cry II A 유전자가 존재함을 확인할 수 있었다. 본 연구를 통하여 얻어진 cry II A 형질전환 식물체는 살충성 검정 등을 거친 후 내충성 식물 생산 및 후대 유전 양상 분석을 위한 기초 자료로 이용될 수 있을 것이다.

Bacillus thuringiensis, a gram-positive soil bacterium, is characterized by its ability to produce crystalline inclusions during sporulation. The crystal proteins exhibit a highly specific insecticidal activity. An insecticidal crystal protein (ICP), Cry II A, is specifically toxic to both lepidopteran and dipteran insects. In this study, tobacco plants transformed by the cry II A gene have been generated. The Cry II A crystal protein was purified from E. coli JM103 harboring cry II A gene by differential solubility. The activated Cry II A was prepared by tryptic digestion. The purified protoxin (70 kDa) and the activated toxin (50 kDa) were analyzed by SDS-PAGE. To generate the transgenic tobacco having cry II A gene, the cry II A gene was subcloned to a plant expression vector, pSRL2, having two CaMV 35S promoters. The recombinant plasmid was transformed into tobacco (N. tabacum var. Petit Havana SR1) by Agrobacterium-mediated leaf disc transformation. Through the regeneration, six putative transgenic tobacco plants were obtained and three transformants were confirmed by Southern blot analysis. It has been found that one plant had single copy of cry II A gene, another had two copies of the gene, and the third had a truncated gene. After the immunochemical confirmation of cry II A expression in plants, the transgenic tobacco plants will be used to study the genetics of future generation with the insecticidal crystal protein gene cry II A.

키워드

참고문헌

  1. J Bacteriol v.177 How does Bacillus thuringiensis produce so much insecticidal crystal protein? Agaisse H;Lereclus D
  2. Plant Molecular Biology Manual Binary vectors An G;Ebert PR;Mitra A;Ha SB;Publishing staff (eds.)
  3. Mol Microbiol v.7 The two faces of Bacillus thuringiensis : insecticidal proteins and post-exponential survival Aronson AI
  4. Microbiol Rev v.50 Bacillis thuringiensis and related insect pathogens Aronson AI;Beckman W;Dunn P
  5. J Bacteriol v.172 Isolation of the second Bacillus thuringiensis RNA polymerase that transcribes from a crystal protein gene promoter Brown KL;Whiteley HR
  6. pHD dissertation Transformation of chinese cabbage (Brassica campestris ssp. pekinensis) with a Bt insecticidal gene Cho HS
  7. Mol Microbiol v.6 Involvement of a possible chaperonin in the efficient expression of a cloned Cry II A ${\delta}$-endotoxin gene in Bacillus thuringiensis Crickmore N;Ellar DJ
  8. Appl Environ Microbiol v.60 Comparison of disulfide contents and solubility at alkaline pH of insecticidal and noninsecticidal Bacillus thuringiensis protein crystals Du C;Martin PAW;Nickerson KW
  9. Anal Biochem v.132 A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity Feinberg AP;Vogelstein B
  10. Proc Natl Acad Sci v.86 Location of the Bombyx mori specificity domain on a Bacillus thuringiensis ${\delta}$-endotoxin protein Ge AZ;Shivarova NI;Dean DH
  11. Annu Rev Entomol v.37 The mode of action of Bacillus thuringiensis endotoxins Gill SS;Cowies EA;Pietrantonio PV
  12. Eur J Biochem v.173 Binding of delta endotoxin from Bacillus thuringiensis to brush-border membrane vesicles of the cabbage butterfly (Pieris brassicae) Hofman C;Luthy P;Hutter R;Pliska V
  13. Microbiol Rev v.53 Insecticidal crystal proteins of Bacillus thuringiensis Hofte H;Whiteley HR
  14. J Econ Entomol v.86 Growth and survival of Heliothis virescens (Lepidoptera: Noctuidae) on transgenic cotton containing a truncated from of the delta endotoxin gene from Bacillus thuringiensis Jenkins JN;Parrott WL;McCarty JCJr;Callahan FE;Berberich SA;Deaton WR
  15. Science v.236 Duplication of CaMV 35S promoter sequences creats a strong enhancer for plant genes Kay R;Chan A;Daly M;McPherson J
  16. Developmental Dynamics v.193 JunB does not inhibit the induction of c-Jun during the retinoic acid induced differentiation of F9 cells Kwon M;Oshima RG
  17. BioScience v.42 insecticidal promise of Bacillus thuringiensis Lambert B;Peferoen M
  18. J Biol Chem v.67 Location of Bombyx mori receptor binding region on a Bacillus thuringiensis ${\delta}$-endotoxin Lee MK;Milne RE;Ge AZ;Dean DH
  19. Plant Physiol v.107 How and why do plants inactivate homologous (trans) genes? Matzke MA;Matzke AJM
  20. Theor Appl Genet v.76 Molecular mapping of rice chromosomes McCouch SR;Kochert G;Yu ZH;Wang ZY;Khush GS;Coffman WR;Tanksley SD
  21. Proc Natl Acad Sci v.88 Modification of the coding sequence enhances plant expression of insect control protein genes Perlak FJ;Fuchs RL;Dean DA;McPherson SL;Fischhoff DA
  22. Plant Mol Biol v.26 Insect resistance of transgenic plants that express modified Bacillus thuringiensis cry I Ab and cry I C genes: a resistance management strategy Salm T;Bosch D;Hon e G;Feng L;Munsterman E;Bakker P;Stiekema WJ;Bert V
  23. Molecular Cloning a Laboratory Manual Preparation and transformation of competent E. coli Sambrook J;Fritsch EF;Maniatis T;Sambrook J (ed.);Fritsch EF (ed.);Maniatis T (ed.)
  24. The Plant Cell v.8 Agrobacterium-plant cell DNA transport: have virulence proteins, will travel Sheng J;Citovsky V
  25. Plant Mol Biol Report v.8 A rapid and inexpensive method for isolation of total DNA from dehydrated plant tissue Tai HT;Tanksley SD
  26. Nature v.328 Transgenic plants protected from insect attack Vaeck M;Reynaerts A;Hofte H;Jansens S;De Beuckeleer M;Dean C;Zabeau M;Van Montagu M;Leemans J
  27. Ann Rev Microbiol v.40 The molecular biology of parasporal crystal body formation in Bacillus thuringiensis Whiteley HR;Schnepf HE
  28. J Bacteriol v.171 Two highly related insecticidal crystal proteins of Bacillus thuringiensis subsp. kurstaki possess different host range specificities Widner WR;Whiteley HR