苏云金芽胞杆菌苏云金素缺失突变株的筛选及特性研究

Screening and Characterization of a Thuringiensin Mutant from Bacillus thuringiensis

利用高温和SDS对苏云金素高产菌株CT-43-1c进行了质粒消除,筛选得到一株不产苏云金素的无晶体突变株BMB0806;并通过SDS-PAGE、高效液相色谱(HPLC)和脉冲电泳(PFGE)分析了野生型菌株CT-43、出发菌株CT-43-1c和突变株BMB0806杀虫晶体蛋白的组成、苏云金素的产量和质粒图谱之间的差异.结果表明,突变株BMB0806与菌株CT-43和CT-43-1c相比,分别缺失了3个和2个大质粒,从而不再产生苏云金素和由cry1B基因编码的分子量为140×103的杀虫晶体蛋白.进一步分析三者质粒图谱后发现,突变株BMB0806中苏云金素的缺失和cry1B基因所在的大质粒A直接相关.

Thuringiensin is a non-specific heat-stable insecticidal toxin produced by some Bacillus thuringiensis strains. To investigate the effect of the plasmids of B. thuringiensis on thuringiensin production, the plasmids of strain CT-43-1 c and the mutant of wild-type strain CT-43 were cured by elevating growth temperature and adding SDS in medium. Then, the thuringiensin and acrystalliferous mutant BMB0806 were obtained. The differences in ICPs, plasmids map and thuringiensin yield among the three strains were identified by SDS - PAGE, PFGE and HPLC, respectively. The results showed that mutant BMB0806 became unable to produce both thuringiensin and 140 × 10^3 insecticidal crystal protein coded by crylB gene; and in comparison with strain CT-43 or CT-43-1c, mutant BMB0806 lost three or two large plasmids, respectively. The differences among the three strains indicated that the production of thuringiensin in mutant BMB0806 was associated with the plasmid harboring a crylB gene. These results lay a theory foundation and offer an appropriate recipient for the cloning of thuringiensin gene cluster. Fig 5, Ref 12 .