Molecular Cloning of nod Genes from Bradyrhizobium sp. SNU001

Bradyrhizobium sp. SNU001 nod 유전자 클로닝

Molccular cloning of nod genes from Bradvrhizobium sp. SNU001, a nitrogen-fixing symbiont isolated from thc root nodules of soybean (Clycine trim) . was carried out. nod genes were found to be located on thc genome of the symbiont by gcnomic hybridization with 4.5 kb EcoRI/HndIII fragment (nod DABC) of Rhizohium meliloti as probe. Genomic library of this symbiont was constructed using h phage EMBL3-BanlHI vector. from which five nod positive clones were sclectcd by primary and secondary screening methods. The partial restriction map of inserted genomic DNA of h CNS-l(c1one 2) was constructed. and 3.9 kh Bun7HI fragment. which showed strong hybridization signal to the probe, was subcloned into pBS KS(+) plasmid vector. Partial restriction inap ot' a selected subclone (pBjCNS-I) was constructed and nod DABC was found to be located on the 1.8 kb KpnI/Sacl fragment of this subclone.

대두 (Glycine max) 뿌리혹의 질소고정 공생균주 Bradyrhizobium japonicum SNU001 의 nod 유전자를 클로닝하였다. Rhizobium meliloti 의 4.5 kb EcoRI/ HindIII 절편을 탐침으로 한 게놈 혼성화 반응을 분리균주의 게놈상에 nod 유전자가 존재함을 확인하고, lambda EMBL3-BamHI vector 를 이용하여 genomic library 를 작성하였다. 작성된 library 로부터 1, 2 차 선별과정을 통해 nod 유전자가 있는 클론 1-5 를 선별하고, 클론 2로부터 nod DABC 탐침과 lambda DNA 탐침을 사용한 혼성화반응을 수행하여 삽입된 genomic DNA 에 대한 부분적인 제한효소 지도를 작성하였다. nod DABC 탐침과 가장 강한 혼성화반응을 보인 phage 클론 lambda CNS-1 의 3.9 kb BamHI 절편을 pBS KS(+) vector 에 subcloning 하고 동일한 탐침을 이용한 혼성화반응을 통해 subclone pBjCNS-1 을 선별하였다. 이 subclone 에 대한 부분적인 제한효소 지도를 작성하여 nod DABC 가 1.8 KpnI/SacI 절편에 존재함을 확인하였다.