The develeopment of novel host-vector system in Bacillus thuringiensis.

苏云金芽孢杆菌以产生杀虫剂毒素而闻名。在大多数情况下，据报道苏云金芽孢杆菌的毒素基因位于这种细菌的质粒上。利用温控噬菌体J7W-1构建苏云金芽孢杆菌分子育种的宿主-载体系统，将该噬菌体基因组整合到菌株AF101中含有毒素基因的质粒中。研究了J7W-1基因组在苏云金芽孢杆菌型菌株中的分布情况；用溴化乙锭处理后，在印第安纳亚种菌株中发现了低水平的噬菌体诱导。J7W-1与突变体噬菌体基因组物理图谱的比较表明，J7W-1基因组具有外源DNA的插入区，长度约为13kb。此外，还尝试通过同源重组将cat基因添加到J7W-1基因组中。通过导入cat基因和部分J7W-1 DNA构建pUC18嵌合质粒后，将该质粒电接到具有J7W-1噬菌体的菌株AF101中。虽然通过噬菌体诱导重组细胞制备了含有cat基因的J7W-1噬菌体，但在噬菌体感染以色列亚种宿主细胞的过程中，发现该噬菌体基因组上的cat基因缺失。考虑到限制性内切酶可能发生在宿主细胞中，因为在以色列亚种中发现了限制性内切酶：AvaII同大小体。

Bacillus thuringiensis is well known for the production of insecticidaltoxin. In the most of case, toxin genes in B.thuringiensis have been reported to be located on plasmid(s) of this bacterium. The developement of a novel host-vector system for molecular breeding in B.thuringiensis was attempted by using temperate phage J7W-1, since this phage genome was revealed to be integrated in a plasmid containing toxin gene in strain AF101.When the distribution of J7W-1 genome was investigated among type strains of B.thuringiensis, a mutant strain of this phage ; the low level phage induction by ethidium bromide treatment, was found in the type strain of subsp.indiana. The comparison of physical maps of these phage genomes between J7W-1 and its mutant suggested that J7W-1 genome possessed the insertive region for foreign DNA at the length of around 13kb. Furthermore, the addition of cat gene to J7W-1 genome was attempted by homologous recombination. After the chimeric plasmid of pUC18 was constructed by intoducing cat gene and a part of J7W-1 DNA,this plasmid was electropolated into strain AF101 possessing J7W-1 prophage. Although J7W-1 phage containing cat gene was prepared by phage induction of the recombinant cell, cat gene on this phage genome was revealed to be deleted during the phage infection to the host cell of subsp.israelensis. This was considered that the restriction might be occurred in the host cell, since the restriction enzyme : AvaII isosizomer, was identified in subsp.israelensis.