聚磷酸激酶基因的阻断对刺糖多孢菌生长及多杀菌素合成的影响.pdfVIP

30(5)703-712 中国生物防治学报 Chinese Journal of Biological Control 2014 年 10 月 聚磷酸激酶基因的阻断对刺糖多孢菌生长及 多杀菌素合成的影响 杨 燕，罗林根，唐斯佳，徐 妙，丁学知，胡胜标，孙运军，夏立秋* （湖南师范大学生命科学学院/微生物分子生物学国家重点实验室培育基地，长沙 410081 ） 摘要:聚磷酸激酶基因（Polyphosphate kinase gene ，ppk ）是放线霉菌中的一种影响抗生素合成的全局 性负调控因子，阻断该基因能显著提高其次级代谢产物产量。本文利用PCR 扩增了刺糖多孢菌中的 ppk 基因中间片段，经酶切连接技术将其克隆到大肠杆菌-链霉菌穿梭载体pOJ260 上，构建阻断型载 体pOJ260-ppk ；通过接合转移将该功能质粒导入刺糖多孢菌中，获得了遗传性能稳定的重组菌株S. sp-△ppk 。对工程菌株的PCR 检测结果显示，ppk 基因片段已整合到刺糖多孢菌染色体上并成功阻断 了该基因的表达。摇瓶发酵结果显示，工程菌株多杀菌素产量较原始菌株提高了122%。阻断聚磷酸 激酶基因的表达对刺糖多孢菌的菌丝形态及生长发育产生了影响，并有效地促进该菌多杀菌素的生 物合成。 关 键 词：刺糖多孢菌；聚磷酸激酶基因；阻断；多杀菌素；接合转移 中图分类号：S476 文献标识码：A 文章编号：1005-9261 (2014)05-0703-010 Disruption of Polyphosphate Kinase Gene Affects Phenotypes and Spinosyns Production of Saccharopolyspora spinosa * YANG Yan, LUO Lingen, TANG Sijia, XU Miao, DING Xuezhi, HU Shengbiao, SUN Yunjun, XIA Liqiu (Hunan Provincial Key Laboratory of Microbial Molecular Biology-State Key Laboratory Breeding Base of Microbial Molecular Biology/College of Life Science, Hunan Normal University, Changsha 410081, China) Abstract ：Previous report revealed that polyphosphate kinase of Streptomyces played a negative role in antibiotic production. Here, we investigated whether polyphosphate kinase of Saccharopolyspora spinosa also negatively regulates the production of spinosad. Polyphosphate kinase encoding gene (ppk ) was disrupted by homologous recombination. Primer pair used for amplification of ppk gene from Saccharopolyspora spinosa was designed according to the homologous ppk gene of S. spinosa. PCR product of partial sequence of ppk gene in S. spinosa was cloned into Escherichia coli-Streptomyces shuttle vector p