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

苏云金芽孢杆菌以生产杀虫毒素而闻名。在大多数情况下，苏云金芽胞杆菌的毒素基因已被报道定位在该细菌的质粒(S)上。利用温带噬菌体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含有毒素基因的AF101菌株中。对该噬菌体突变株苏云金芽孢杆菌的J7W-1基因组在模式菌株中的分布进行了研究，发现溴化乙锭对印度亚种模式菌株有低水平的诱导作用。J7W-1与其突变体的物理图谱比较表明，J7W-1基因组具有约13kb的外源DNA插入区。此外，还尝试通过同源重组将CAT基因加入到J7W-1基因组中。将CAT基因与部分J7W-1 DNA构建成pUC18的嵌合表达载体后，将其电转化至含有J7W-1噬菌体的AF101菌株中。虽然重组细胞通过噬菌体诱导获得了含有CAT基因的J7W-1噬菌体，但在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基因是缺失的。这种限制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异构体。

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.