Genetic Transfer of Bacillus pasteurii Urease Gene into Antagonistic Bacillus subtilis YBL-7 against Root Rotting Fungi Fusarium solani

Bacillus parteurii Urease Gene의 생물방제균 Bacillus subtilis YBL-7내에서의 발현

- To investigate the possibility of genetic development for a multi-purpose strain of Bacillus subtilis YBL-7 against Fusat-iurn solani causing root rot of many impotant corps, the plasmid pGU66 inserting urease gene of Bacillus pasteurii had been introduced into Bacillus subtilis YBL-7 by PEG-induced protoplast (PIP) transformation system. Protoplasts of B. subtilis YBL-7 were prepared by treating the cells with lysozyme (200 $\mu g$/ml) in hypertonic buffer (SMMP). The highest transformation frequency was achieved when cells of the strain with lysozyme at $42^{\circ}C$ for 90 minutes. Optimal transformation was obtained using polyethylene glycol (MW 4000) at final concentration of 30% (V/V). The transformation frequency was increased proportionally to 1.2 $\mu g$ of plasmid DNA. At best condition, the transformation frequency (transformants/ regenerants/$\mu g$ of DNA) for pGU66 was appoximately $4 \times 10^{-3}$. Also, the urease gene was strongly expressed in the transformants of B. subtilis YBL-7 and maintained steadily. The antifungal ability of transformant was very similar to that of B. ssubtilis YBL-7.

식물근부병의 방제균으로 선발된 Bacillus subtilis YBL-7의 식물부균 Fusarium solani에 대한 길항력을 유전공학적 조작에 의해 다목적으로 증강시킬 수 있는지를 타진하기 위해 외부유전자인 Bacillus pasteurii의 urease 유전자를 생물방제균 B.subtilis YBL-7내 도입자하고자 시도하였다. 외부 urease 유전자는 B.pasteurii의 urease gene을 shuttle vector인 pGR71의 HindII site에 삽입하여 E.coli내에서 발현시킨 pGU66을 사용하여 형질전환시켰으며 이때의 최적 형질전환조건과 도입된 urease 유전자의 발현을 조사해 보았다.