Transfer of RP4:Mu cts from E. coli to Rhizovium leguminosarum

RP4:Mu cts의 E.coli로 부터 Rhizovium leguminosarum으로의 전달

In order to use for recipient strains of RP4:Mu cts, 5 strainsof Rhizobium were selected among 32 strains, which were isolated and identified in this study. Hybrid plasmin RP4::Mu cts, which, is temperature sensitive and confers resistance to ampicillin, kanamycin and tetracycline was transfered by conjugation from E. coli to other atrains of C. coli and the symbiotic nitrogen fixer, Rhizobium leguminosarum. Transfer frequencies of RP4::Mu cts plasmid from E. coli to Rhizobium were about $10^{-8}-10^{-7}$ in LB agar and YMA media. The transconjugants were confirmed by demonstrating that the drug-resistant and temperature-sensitive clones isolated were drug-resistant and temperature-sensitive clones isolated were capable of releasing phage and forming plaques. The plaque-forming units of transconjugants were about $10^2\;to\;10^3$. Stability test of RP4::Mucts in Rhizobium represented that most of the transconjugants had drug resistance and produce phage Mu cts.

우리나라 자연환경으로부터 분리한 Rhizob ium 32권주로부터 카나마이신, 세트라싸이클린 등에는 예민하고, 클로람페니콜, 젠타마이신 등에는 강한 내성을 나타내는 5 균주를 선별하고, 이를 수용세포로하여 RP4::Mu cts를 접함에 의해 E. coli로부터 Rhizobium leguminosarum으로 전달 시켰다. 그 전달빈도는 $5.8{\times}10^{-7}$의 빈도를 나타내었다. 접합체에서의 RP4::Mu cts플라스미드의 존재는 암피실린과 카나마이신, 테트라싸이클린에 대한 내정과 $42^{\circ}C$에서의 플라크 형성으로 확인하였다. 접합체들은 $10^2~10^3$단위로 플라크를 형성하였고, 안정성를 조사한 신파 4주 후에도 대부분이 RP4: :Mu cts의 성질을 유지하고 있는 것으로 나타났다.