Mutagenesis of Slow Growing Rhizobium japonicum by Transposon Tn5

Transposon Tn5를 이용한 Slow growing Rhizobium japonicum의 돌연변이 유도

  • Kim, Sung-Hoon (Genetic engineering Center, KAIST) ;
  • Rhee, Yoon (Genetic engineering Center, KAIST) ;
  • Sun, Dae-Kyu (Genetic engineering Center, KAIST) ;
  • Yoo, Ick-Dong (Genetic engineering Center, KAIST)
  • 김성훈 (한국과학기술원 유전공학센터) ;
  • 이윤 (한국과학기술원 유전공학센터) ;
  • 선대규 (한국과학기술원 유전공학센터) ;
  • 유익동 (한국과학기술원 유전공학센터)
  • Published : 1988.12.01

Abstract

The spectinomycin resistant strain of slow growing R. japonicum R-168 was selected to be participated in conjugation with E. coli WA803/pGS9. Tn5 was introduced from suicide vector pGS9 into R. japonicum R-168 $spr^{r}$ chromosome at the frequency of $1.0\times 10^{-5}-5.0\times 10^{-7}$ and the transconjugante were selected on the yeast extract-mannitol plate containing kanamycin ($50{\mu}$g/ml) and spectinomycin ($100{\mu}$g/ml) after 8-9 days incubation. All transconjugants we tested were found to contain Tn 5 DNA on their genome, which was confirmed by Southern hybridization experiments. R. japonicum RNa75, which had been selected through plant test, was found to be defective in symbiotic nitrogen fixing ability and the production of leghemoglobin in soybean nodules formed by the inoculation of this mutant. In addition, this mutant strain hardly developed nitrogenase activity asymbiotically in contrast with the wild type strain, indicating that some nitrogen fixing gene might be blocked in this strain and the production of leghemoglobin could be decreased by the interference in nitrogen fixing genes.

Keywords

Transposon Tn5;Mutagenesis;Rhizobium japonicum