Abstract
An efficient genetic transformation system and suitable promoters are essential prerequisites for gene expression studies and genetic engineering in streptomycetes. In this study, firstly, a genetic transformation system based on intergeneric conjugation was developed in Streptomyces rimosus M527, a bacterial strain which exhibits strong antagonistic activity against a broad range of plant-pathogenic fungi. Some experimental parameters involved in this procedure were optimized, including the conjugative media, ratio of donor to recipient, heat shock temperature, and incubation time of mixed culture. Under the optimal conditions, a maximal conjugation frequency of 3.05×10-5 per recipient was obtained. Subsequently, based on the above developed and optimized transformation system, the synthetic promoters SPL-21 and SPL-57, a native promoter potrB, and a constitutive promoter permE" commonly used for gene expression in streptomycetes were selected and their activity was analyzed using gusA as a reporter gene in S. rimosus M527. Among the four tested promoters, SPL-21 exhibited the strongest expression activity and gave rise to a 2.2-fold increase in (3-glucuronidase (GUS) activity compared with the control promoter permE*. Promoter SPL-57 showed activity comparable to that of permE*. Promoter potrB, which showed the lowest activity, showed a 50% decrease in GUS activity compared with the control permE*. The transformation system developed in this study and the tested promotors provide a basis for the further modification of S. rimosus M527.
概要
目 的
建立并优化适用于龟裂链霉菌 (Streptomyces rimosus) M527 的属间接合转移体系, 并在此基础上分析四个启动子的表达活性。
创新点
有效的遗传转化系统和合适的启动子是链霉菌基因表达和基因工程的必要前提。 由于链霉菌的遗传背景复杂, 接合转移实验参数对菌种具有高度特异性。 因此, 本研究建立并优化了一套适用于 S. rimosus M527 的属间接合转移体系, 并在此基础上分析比较了四个启动子的表达活性。 这将为进一步遗传修饰 S. rimosus M527 奠定基础。
方 法
以 S. rimosus M527 为研究对象, 通过对接合转移体系中的一些重要的实验参数 (如接合转移培养基、 供受体比例、 热激温度和混合培养时长等) 进行了优化。 在此基础上, 构建以 gusA 为报告基因的质粒, 通过直观显色反应以及 GUS 酶活的定量检测, 分析比较了四个启动子的表达活性。
结 论
建立了一种有效的适用于 S. rimosus M527 接合转移体系, 优化后的接合效率最高达 3.05×10−5。分析测试的四个启动子中, 合成启动子 SPL-21 表现出最高表达活性, 比常用强组成型启动子 permE*活性高出 2.2 倍, 合成启动子 SPL-57 与 permE*的活性无显著差异, 内源启动子 potrB 的活性比 permE*降低了 50%。
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Zhang-qing SONG, Zhi-jun LIAO, and Ye-feng HU participated in the design. Zheng MA and Zhang-qing SONG wrote this article. Andreas BECHTHOLD revised the manuscript. Zheng MA and Xiao-ping YU checked the final version. All authors approved the final manuscript and take responsibility for the integrity and security of the data.
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Zhang-qing SONG, Zhi-jun LIAO, Ye-feng HU, Zheng MA, Andreas BECHTHOLD, and Xiao-ping YU declare that they have no conflict of interest.
This article does not contain any studies with human or animal subjects performed by any of the authors.
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Project supported by the National Natural Science Foundation of China (Nos. 31772213 and 31972320) and the Excellent Youth Fund of Zhejiang Province, China (No. LR17C140002)
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Song, Zq., Liao, Zj., Hu, Yf. et al. Development and optimization of an intergeneric conjugation system and analysis of promoter activity in Streptomyces rimosus M527. J. Zhejiang Univ. Sci. B 20, 891–900 (2019). https://doi.org/10.1631/jzus.B1900270
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DOI: https://doi.org/10.1631/jzus.B1900270