KSBB Journal

The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
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Construction of Transformation Method for Streptomyces scabiei ATCC 49173 Producing Phytotoxin

식물독소를 생산하는 Streptomyces scabiei ATCC 49173의 형질전환법 구축

  • Jang, Bo-Youn (Department of Food Science and Biotechnology, Kyungnam University) ;
  • Ha, Heon-Su (Department of Food Science and Biotechnology, Kyungnam University) ;
  • Choi, Sun-Uk (Department of Food Science and Biotechnology, Kyungnam University)
  • 장보연 (경남대학교 식품생명학과) ;
  • 하헌수 (경남대학교 식품생명학과) ;
  • 최선욱 (경남대학교 식품생명학과)
  • Received : 2009.10.30
  • Accepted : 2010.02.25
  • Published : 2010.04.28
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Abstract

Streptomyces scabiei producing phytotoxin called thaxtomin, which cause scab disease on economically important crops such as potato. For molecular genetics study of S. scabiei an effective transformation method was established based on conjugal transfer from Escherichia coli ET12567 (pUZ8002) using a phiC31-derived integration vector, pSET152, containing oriT and attP fragments. The high frequency was obtained on MS medium containing 50 mM $MgCl_2$. In addition, the sequence and location of the chromosomal integration attB site of S. scabiei was identified for the first time in the strains producing thaxtomin by the southern blot analysis of exconjugants and the sequencing of plasmid containing DNA flanking the insertion sites from exconjugant chromosome. Similar to the case of Streptomyces species, a single phiC31 attB site of S. scabiei is present within an ORF encoding a pirin-homolog.

농작물에 심각한 피해를 주는 phytotoxin을 생산하는 S. scabiei ATCC 49173의 분자 유전학적인 연구를 위해 대장균으로부터 S. scabiei로 plasmid DNA를 도입하는 접합 전달법을 이용한 형질전환법을 확립하였다. 본 연구를 통해 확인된 S. scabiei의 접합전달용 최적배지는 50 mM의 $MgCl_2$를 첨가한 MS배지이며 접합전달에 사용되는 DNA 수용체인 포자는 $45^{\circ}C$의 열처리와 $5{\times}10^7$이상의 plasmid DNA 공여체가 필요하다는 것을 확인하였다. 또 얻어진 접합전달체에 대하여 Southern blot hybridization과 벡터가 삽입된 염색체부분의 염기서열분석을 통해 attB site의 특성을 분석한 결과 S. scabiei 염색체의 pirin 상동체를 코드하는 ORF내에 단일위치로 존재하고 있으며 이미 밝혀진 다른 방선균유래 attB site의 염기서열에 대해 86.3%~96.1%의 상동성을 보였다.

Keywords

References

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