短小芽孢杆菌质粒pCJ转化枯草杆菌的研究

本文采用我国自己分离的短小芽孢杆菌抗四环素质粒pCJ3转化枯草杆菌的各种突变体.质粒DNA经氯化铯—溴化乙锭密度梯度离心纯化后转化枯草杆菌的感受态细胞.结果表明枯草杆菌BR151,SB202,168,QB1130及QB1133都可作为质粒pCJ3的受体,转化效率在10~3左右,其中以SB202这株突变体为最高,从各种转化体中提取的质粒及经BamHI消化后的质粒的电泳图均与原质粒pCJ3及其BamHI酶切片段相同,并具有pCJ3的抗四环素转化活性.将pCJ3DNA经BamHI酶切后重新用T_4-DNA连接酶连接再转化枯草杆菌细胞,其转化效率比原质粒高1—2个数量级.研究了二价金属离子、pH及培养基成分对转化的影响,结果证明:Ca~(++)对枯草杆菌转化有促进作用,Cu~(++),Zn~(++)则有抑制作用,转化的最适pH在7.2—7.5之间;营养丰富培养能提高转化效率.

Transformation of Bacillus subtilis with Plasmid pCJ3 from Bacillus pumilus

The Tc~R plasmid pCJ3 from B. pumilus was used to transform competent cells of different B. subtilis mutants. It was shown that B. subtilis BR151, SB202, 168, QB1130 and QB1133 are suitable as the recipient of plasmid pCJ3 with a transformation efficiency of 10~3 transformants/ug DNA. Plasmids isolated from different transformants and their BamHI fragments have same electrophoresis pattern as plasmid pCJ3 and its BamHI fragment and have the Tc~R activity as pCJ3. The religated plasmid from BamHI fragment of pCJ3 has a transformation efficiency 1-2 magnitude higher than the original plasmid. The effect of divalent cations, pH and medium composition on transformation was investigated. Ca~(++) promotes while Cu~(++) and Zn~(++) inhibit the transformation The optimun pH for transformation is between 7.2-7.5 and rich medium can increase the transformation efficiency.