Optimization of Genetic Manipulation System for Streptomyces pratensis S10

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

Abstract

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

CLC Number:

S476

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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