苏云金芽胞杆菌幕虫亚种YBT-020具有典型的晶胞粘连表型。在前期的研究中, 通过质粒消除实验, 推测晶胞粘连现象与YBT-020内生质粒pBMB28有关。为了定位质粒pBMB28上控制晶胞粘连表型的基因, 首先对质粒pBMB28进行克隆。利用穿梭载体pEMB0557, 成功构建了苏云金芽胞杆菌YBT-020的基因组人工染色体(BAC)文库。前期的研究表明晶体蛋白基因cry28Aa定位在质粒pBMB28上, 根据cry28Aa基因序列设计引物, 从文库中筛选到含有cry28Aa的重组质粒pBMB231。镜检和SDS-PAGE证明质粒pBMB231转化无晶体突变株BMB171形成的重组子BMB231可以产生Cry28Aa晶体蛋白, 但不能恢复晶胞粘连表型。对重组质粒pBMB231的插入片段末端序列测定并设计引物筛选文库, 通过染色体步移方式得到4个可以重叠覆盖质粒pBMB28不同区域的克隆子, 从而克隆了该质粒。对这4个克隆子末端测序和酶切分析, 测算出该质粒的大小约为140 kb。进一步确定应用基因组BAC文库以及重叠片段筛选的方法, 可以快速有效的克隆苏云金芽胞杆菌大质粒。
Bacillus thuringiensis serovar. finitimus strain YBT-020 is a typical strain with the spore-crystal association (SCA) phenotype. In our previous studies, plasmid curing experiment suggested that native plasmid pBMB28 of strain YBT-020 might contribute to the SCA phenotype. Thus, plasmid pBMB28 was cloned in order to isolate the genes related to SCA on pBMB28. Using shuttle vector pEMB0557, a shuttle genomic bacterial artificial chromosome (BAC) library of B. thuringiensis strain YBT-020 was constructed. The plasmid pBMB231 containing crystal protein gene cry28Aa, which was located on plasmid pBMB28, was screened out. By SDS-PAGE analysis and microscopic observation, we discovered the recombinant strain BMB231 that originated from the electrotransfer strain BMB171 with pBMB231 could produce Cry28Aa protein. With the chromosome walking strategy and terminal sequencing of pBMB231, four clones covering the full length of plasmid pBMB28 were screened out from this BAC library. With pulsed gel analysis of the four BAC clones and terminal sequencing, the size of the plasmid was calculated to be 140 kb. This study additionally revealed that we could clone a large plasmid from B. thuringiensis by genomic BAC library con-struction and overlaping fragment screening.
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