Genetic analysis of plasmid dependent prophage for molecular breeding in Bacillus thuringiensis

苏云金芽孢杆菌能产生对鳞翅目、双翅目和鞘翅目昆虫有毒害作用的伴生包涵体(晶体)，在害虫微生物防治中发挥着重要作用。苏云金芽孢杆菌AF101菌株属于H型4a：4b型，对家蚕低毒，对其他鳞翅目害虫有较强的毒力。结果表明，AF101菌株只有一个含有晶体基因和温带噬菌体J7W-I全基因组的质粒pAF101。为开发苏云金杆菌分子育种的有效工具，本研究对菌株AFIOI中的pAF101进行了遗传分析，并通过限制性内切酶构建了pAF101的物理图谱。结果表明，温带噬菌体KK-88基因组的一部分位于pAF101上，该噬菌体也是AF101中的溶原噬菌体，具有与J7W-L噬菌体不同的诱导系统。此外，对这些噬菌体的诱导系统进行了研究，发现34kdal和50kdal的细胞蛋白分别是J7W-L和KK-88的阻遏蛋白。从pAF101中克隆了晶体基因，定位于pAF101上，发现晶体基因区域位于J7W-L噬菌体基因组和KK-88噬菌体基因组之间。据此推测，pAF101主要由两种类型的噬菌体基因组和晶体基因等三个基因区域构成。

Bacillus thuringiensis which is well known for the production of parasporal inclusions(crystals)toxic for lepidopterous, dipterous and coleopterous insects plays an important role in the microbial control of pest insects. B. thuringiensis strain AF101, belonging to H-serotype 4a : 4b, was selected as the strain of low toxic to silkworm, but highly toxic to other lepidopterous pest insects. Strain AF101 was revealed to possess a single plasmid(pAF101)which contained crystal gene and the entire genome of temperate phage J7W-I. In this study, the genetic analysis of pAF101 in stain AFIOI was carried out for the purpose of developing the effective tool for the molecular breeding of B. thuringiensis.The physical map of pAF101 was constracted by digesting restriction enzymes. It was revealed that a part of the genome of temperate phage KK-88 which was also lysogenized phage in AF101 and had different induction system from phage J7W-l was located on pAF101. Furthermore, the induction system of these phages were examined to reveal that 34kdal and 50kdal of cellar proteins were repressor like proteins for J7W-l and KK-88, respectively. The crystal gene was cloned from pAF101- The location on pAF101 was detected to find that the crystal gene region was located between the regions of J7W-l phage genome' and KK-88 phage genome. According to these mentioned above, pAF101 was constrcted by mainly three genetical regions, such as two types of phage genome and crystal gene.