State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, People's Republic of China.
Appl Microbiol Biotechnol. 2010 Jun;87(1):243-9. doi: 10.1007/s00253-010-2479-5. Epub 2010 Feb 18.
A newly engineered strain, denominated BIOT185, was constructed through integrating the cry8Ca2 gene into the endogenous plasmid of BT185 (contains cry8Ea1) by homologous recombination. The thermosensitive plasmid vector was eliminated by the rising temperature of recombinant cultures. No antibiotic gene or other unnecessary genes were introduced to the new strain. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis demonstrated that the cry8Ca2 gene was expressed normally and produced a 130-kDa protein in the BIOT185 strain. Bioassay results showed that the new strain had high toxicity to the pests Anomala corpulenta and Holotrichia parallela, which often damage the same fields.
构建了一株新型工程菌 BIOT185，通过同源重组的方法将 cry8Ca2 基因整合到内源性质粒 BT185（含有 cry8Ea1）中。重组菌经升温处理消除了温度敏感型质粒载体。该新型菌株未引入抗生素基因或其他非必需基因。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和 Western blot 分析表明，cry8Ca2 基因在 BIOT185 菌株中正常表达，产生了 130kDa 的蛋白。生物测定结果表明，该新型菌株对常同时为害同块田地的暗黑鳃金龟和暗黑齿爪鳃金龟具有较高的毒力。
