摘要
【目的】拟通过构建高效杀虫枯草芽孢杆菌YZ-1的转座突变体库,从中筛选出高产表面活性素的突变体菌株,为后续YZ-1的表面活性素合成调控机制解析和开发利用提供前期材料。【方法】利用携带转座子TnYLB-1和温敏型复制蛋白的穿梭载体pMarA转化YZ-1,通过50℃高温诱导转座子转座,并对转座突变体进行抗性培养复筛,构建YZ-1菌株的转座突变体库。组合使用血平板法初筛,溴百里香酚蓝比色法复筛及HPLC定量检测,从突变体库中筛选表面活性素合成显著增强的突变体。【结果】通过自然转化将pMarA质粒导入枯草芽孢杆菌YZ-1中,获得含有3000株转座突变子的YZ-1突变体库,从中筛选出11株表面活性素合成增强的候选菌株。HPLC检测显示与YZ-1野生型相比,表面活性素合成量增强及相近的突变体有5株(M1、M6、M7、M8、M9),显著下降的有6株(M2、M3、M4、M5、M10、M11),其中表面活性素产量最高的为M8突变体(282.4 mg/L),较野生型增加13.7%,最低的为M2菌株(47.53 mg/L),较野生型下降80.9%。【结论】增设抗性复筛培养可提高YZ-1突变体库的正确率。基于血平板培养和溴百里香酚蓝比色的高通量筛选可初步筛选脂肽类产物显著变化的突变体菌株。
【Objective】The study aimed to construct a transposable mutant library of highly efficient insecticidal Bacillus subtilis YZ-1 and screen for mutant strains with high yields of surfactant, thereby providing preliminary materials for the analysis and development of the regulation mechanism of surfactant synthesis in YZ-1. 【Method】 The strain YZ-1 was transformed using the shuttle vector pMarA carrying the transposon TnYLB-1 and a temperature-sensitive replicative protein. The transposon was induced to transpose at a high temperature of 50 ℃, and the transposon mutants were subjected to resistance culture rescreening to construct a transposon mutant library for the YZ-1 strain. A combination of initial screening with the blood agar plate method, rescreening with bromothymol blue colorimetry, and quantitative detection with HPLC was used to select mutants with significantly enhanced surfactin synthesis from the mutant library. 【Result】 Through natural transformation, the pMarA plasmid was successfully introduced into B.subtilis YZ-1, resulting in a YZ-1 mutant library containing 3000 transposon mutants. From this library, 11 candidate strains with enhanced surfactin synthesis were screened. HPLC detection revealed that compared to the YZ-1 wild type, there were 5 mutants(M1, M6, M7, M8 and M9) with increased or similar levels of surfactin synthesis, and 6 mutants(M2, M3, M4, M5, M10 and M11) with significantly decreased synthesis. Among them, the M8 mutant, which had the highest surfactin yield at 282.4 mg/L, showed a 13.7% increase compared to the wild type, while the M2 strain, with the lowest yield at 47.53 mg/L, experienced an 80.9% decrease compared to the wild type. 【Conclusion】 The addition of a resistance rescreening cultivation step can improve the accuracy of the YZ-1 mutant library. High-throughput screening based on blood agar culture and bromothymol blue assay can preliminarily select mutant strains with significant changes in lipopeptide products.
作者
万莉
何月秋
谢鑫欣
刘迎龙
何鹏搏
Shahzad MUNIR
吴毅歆
何鹏飞
WAN Li;HE Yue-qiu;XIE Xin-xin;LIU Ying-long;HE Peng-bo;Shahzad MUNIR;WU Yi-xin;HE Peng-fei(College of Agronomy and Biotechnology,Yunnan Agricultural University,Kunming 650201,China;College of Plant Protection,Yunnan Agricultural University,Kunming 650201,China)
出处
《西南农业学报》
CSCD
北大核心
2024年第11期2475-2484,共10页
Southwest China Journal of Agricultural Sciences
基金
国家重点研发计划项目(2022YFD1400700)
国家自然科学基金项目(32160672)。
