苏云金杆菌δ-内毒素基因在大肠杆菌中的克隆及表达  

CLONING AND EXPRESSION OF δ-ENDOTOXIN GENE OF BACILLUS THURINGIENSIS IN E COLI

作　　者：田颖川[1] 蔡发兴[1] 王瑛[2] 张斌贤 沙槎云[2] 陈受宜 莽克强[1] 

机构地区：[1]中国科学院微生物研究所 [2]中国科学院动物研究所 [3]中国科学院生物物理研究所

出　　处：《生物工程学报》1989年第1期11-18,共8页Chinese Journal of Biotechnology

基　　金：国家科委资助

摘　　要：分离了苏云金杆菌肯尼亚亚种7404(Bacillus thuringiensis subsp. kenyae 7404)和库斯塔克亚种(Bacillus thuringiensis subsp. kurstaki HD-1)的质粒。经凝胶原位杂交证明苏云金杆菌肯尼亚亚种7404的δ-内毒素基因位于约47Md大小的一个质粒上。用蔗糖密度梯度离心法从Sau 3 A1部分酶解的上述两种苏云金杆菌质粒DNA酶解片段中分离出大于4kb的DNA片段,将这些片段克隆到pBR332的Bam HI位点上并转化大肠杆菌HB101。通过菌落原位杂交、菌落原位放射免疫试验及Western blet分析等方法,选到了带有δ-内毒素基因并能在大肠杆菌中表达此毒蛋白的转化体。初步的生物学试验表明,在四个试验过的转化体中带有肯尼亚亚种δ-内毒素基因的转化体TK89及带有库斯塔克亚种δ-内毒素基因的转化体TH12和TH48对烟青虫(Heliothis assulta)有毒杀活性。Fragments larger than 4 kb from Sau 3A1 partial digested plasmid DNA of Bacillus thuringiensis subsp. kenyae 7404 as well as subsp. kurstaki HD -1 were cloned respectively into the BamH I site of pBR 322. Based on the results of in situ colony hybridization, radioimmune screening and Western blot analysis,four transformants containing the corresponding δ-endotoxin gene and producing proteins reacted with crystal protein antibody were selected. Upon biological toxicity test, out of three transformants tested, the lysate of one transformant TK89 carrying δ-endotoxin gene of B.T. 7404 and two transformants TH12 and TH48 carrying δ-endotoxin gene of HD-1 were toxic to caterpillars of tobacco budworm (Heliothis assulta). This is the first time to have cloed the δ-endotoxin gene of B.T. subsp, kenyae different in serotype with the well studied subsp. kurstaki.

关 键 词：苏云金杆菌 δ-内毒素 DNA克隆 

分 类 号：Q785[生物学—分子生物学]