链霉菌S10遗传操作系统的优化

doi:10.16409/j.cnki.2095-039x.2023.02.029

摘要/Abstract

摘要： 链霉菌Streptomyces pratensis S10是分离自番茄叶霉病异常菌落上的一株生防菌，具有良好的生防应用价值。建立高效稳定的遗传操作系统对该生防菌进行基因定向改造、提高基因表达水平及深入研究其生防分子机制具有重要意义。本试验以具有安普霉素抗性基因标记的温敏型质粒pKC1139为载体，大肠杆菌Escherichia coli ET12567（pUZ8002）为供体菌，链霉菌S10为受体菌，通过接合转移的方法对其遗传操作系统进行优化。结果表明，选择高氏一号培养基为链霉菌S10接合转移的最适培养基，孢子50 ℃热激10 min，最适供受体比为1:100，安普霉素添加的最适浓度为25 μg/mL，接合转移16 h后覆盖抗生素，添加MgCl₂终浓度为15 mmol/L时接合转移效率最高，达到 8.3×10^-7。利用该遗传转化系统成功得到了一个调控基因的双交换突变株。获得链霉菌S10稳定高效的遗传操作系统，为进一步构建高产菌株和研究抑菌活性物质合成机理奠定了基础。

关键词: 链霉菌S10, 遗传操作, pKC1139, 基因敲除

Abstract: Streptomyces pratensis S10 was isolated from tomato leaf mould, exhibiting a potential application in biological control. It is of great significance to establish an efficient and stable genetic manipulation system to carry out gene-directed modification, improve the level of gene expression, and further explore the biocontrol molecular mechanism of S. pratensis S10. In this study, a genetic transformation system based on intergeneric conjugation was optimized in S. pratensis S10, the temperature sensitive vector pKC1139 was used as the plasmid, Escherichia coli ET12567 (pUZ8002) used as the donor strain, and S. pratensis S10 was the recipient. The results show that the optimum conjugation transformation with a conjugation efficiency of 8.3×10^-7was developed under following conditions: Gauze’s medium No.1 as S. pratensis S10 and E. coli junction transfer medium, spores were heat shocked at 50 ℃ for 10 min, 1:100 for donor-recipient ratio, 25 μg/mL apramycin coverage 16 h post transconjugation, and adding MgCl₂ till final concentration of 15 mmol/L. Subsequently, based on the above optimized genetic manipulation system, a double-crossover mutant of a regulating gene was successfully obtained. A stable and efficient genetic manipulation system for S. pratensis S10 was developed, which lays a solid foundation for the further constructing high-yield strain and studying the active substance biosynthesis mechanism.

Key words: Streptomyces pratensis S10, genetic manipulation, pKC1139, gene knockout

中图分类号:

S476

胡礼芳, 陈婧, 贾瑞敏, 王阳. 链霉菌S10遗传操作系统的优化[J]. 中国生物防治学报, 2023, 39(3): 624-635.

HU Lifang, CHENG Jing, JIA Ruimin, WANG Yang. Optimization of Genetic Manipulation System for Streptomyces pratensis S10[J]. Chinese Journal of Biological Control, 2023, 39(3): 624-635.

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