苏云金杆菌天门变种晶体蛋白基因的克隆和表达

用改进的Birnboim法检测了苏云金杆菌9个变种的质粒组成,其中我国分离到的天门变种有12条质粒带,用Southern杂交法证明,天门变种的δ-内毒素基因位于1个约60MD的质粒上。该质粒经BglⅡ酶切后与Bam HI酶切的pBR322载体连接,转化大肠杆菌HB101。通过菌落原位放射免疫反应和Southern杂交方法,筛选带有δ-内毒素基因并能在大肠杆菌中表达的转化体。对玉米螟幼虫进行生物测定表明,阳性克隆pTCC65及pTCC18有较强的抑制幼虫生长的作用。

CLONING AND EXPRESSION OF DELTA-ENDOTOXIN OFBacillus thuringiensis IN Escherichia coli

The plasmids contents from 9 varieties including 14 strains of Bacillus thuringiensis have been detected by modified Birnboim's method.Southern blot hybridization analysis has been performed in the 14 strains,using variety subtoxicus plasmid DNA as probe.B.thuringiensis var.tianmensis isola-ted from Tiamen county,China,contains 14 plasmid bands in agarose gel electrophoresis,and the delta-endotoxin was located mainly in 60 MD plasmid.The Bgl Ⅱ restriction fragments of the plasmid were ligated into.the Bam H I site of the vector pBR322,and transformed into E..coli HB.101.Of.1800 transformants,219 Apt Tcs clones have been examined.Reco-mbinants for production of antigen reacted with antibody which was made against B.thuringiensis var.kurstaki HD-1.crystals we're screened by ra-dioimmunoassay.Among them seven strains were shown to produce crystal delta-endotoxin protein antigen and hybridize with pES 1 plasmid;pTCC 65 and pTCC 18 are toxic to larvar of corn borer (Ostrinia furnacatis),suggesting that these recombinants carried the crystal protein gene with have been expressed.