Transposon Tn5를 이용한 Slow growing Rhizobium japonicum의 돌연변이 유도

Mutagenesis of Slow Growing Rhizobium japonicum by Transposon Tn5

Slow growing R. japonicum R-l68 균주로부터 spectinomycin 내성 균주를 선발하고 이 Rhizobium내에 Tn-5를 도입시키기 위하여 Tn5가 함유된 E. coli WA 803/pGS9과의 conjugation을 통한 transposon mutagenesis를 실시하였다. 이때 C conjugation을 통한 Tn5 전이 빈도는 $1.0\times 10^{-5}-5.0\times 10^{-7}$ 범위 이였으며, 얻어진 transconjugant들은 spectinomycin (($100{\mu}$g/ml)과 kanamycin ($50{\mu}$g/ml)을 함유한 yeast extract-mannitol 배지에서 8-10일 배양후 colony를 형성하였다. 또한 transconjugant들은 genome상에 Tn-5를 함유하고 있음을 hybridization-을 통하여 확인하였다. 한편 nodule은 형성 하나 질소고정 활성이 없는 돌연변이주 R. japonicum RMa 75 $nod^{+}fix^{-}$ 균주를 선발하였는데 이 균주는 nodule내에 leghemoglobin이 결핍되어 있음이 확인되었다.

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.