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

苏云金芽孢杆菌新型宿主载体系统的开发。

• 批准号: 05660053
• 负责人: KANDA Kohzo
• 金额: $ 1.41万
• 依托单位: Saga University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05660053/
• 关键词: Bacillus thuringiensis; Phage vector; J7W-1; Electropolation; Cat gene; Homologous recombination; ファージJ7W-1; Bacillus cereus; クロラムフェニコール耐性

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.

苏云金芽孢杆菌是众所周知的生产虫草毒素。在大多数情况下，苏云金B.thuringiensis的毒素基因已被报道位于该细菌的质粒上。利用温带噬菌体J7 W-1基因组整合在含毒素基因的质粒中的特点，尝试建立一种新的苏云金芽孢杆菌分子育种的宿主-载体系统。用溴化乙锭处理的模式菌株中发现了低水平的噬菌体诱导。通过对J7 W-1及其突变体噬菌体基因组物理图谱的比较，发现J7 W-1基因组中存在外源DNA的插入区，其长度约为13 kb。此外，尝试通过同源重组将cat基因添加到J7 W-1基因组中。将cat基因与J7 W-1的部分DNA导入pUC 18构建嵌合质粒，将该质粒电转化到具有J7 W-1原噬菌体的菌株AF 101中。虽然通过噬菌体诱导重组细胞制备了含有cat基因的J7 W-1噬菌体，但在噬菌体感染以色列亚种宿主细胞的过程中，该噬菌体基因组上的cat基因被删除。这被认为是限制可能发生在宿主细胞中，因为限制酶：AvaII isosizomer，被确定在亚种。