利用CRISPR/Cas9系统建立内生链霉菌SAT1的基因簇敲除体系

doi:10.13560/j.cnki.biotech.bull.1985.2018-1080

生物技术通报 ›› 2019, Vol. 35 ›› Issue (6): 1-8.doi: 10.13560/j.cnki.biotech.bull.1985.2018-1080

• 研究报告 • 下一篇

利用CRISPR/Cas9系统建立内生链霉菌SAT1的基因簇敲除体系

田文佳¹, 窦桂铭², 王莎¹, 孙靖雅¹, 马玉超¹

1. 北京林业大学生物科学与技术学院,北京 100083;
2. 北京林业大学林学院,北京 100083

收稿日期:2018-12-21 出版日期:2019-06-26 发布日期:2019-07-08
作者简介:田文佳,女,硕士,研究方向：链霉菌天然产物及调控基因;E-mail：tianwenjia@163.com
基金资助:
中央高校基本科研业务费（2017ZY14）,国家林业局公益项目（201304409）,北京市科技新星项目（2011033）

Establishment of a CRISPR/Cas9-mediated Gene Cluster-knockout System in the Endophytic Streptomyces SAT1

TIAN Wen-jia¹, DOU Gui-ming², WANG Sha¹, SUN Jing-ya¹, MA Yu-chao¹

1. College of Biological Sciences and Technology,Beijing Forestry University,Beijing 100083;
2. College of Forestry,Beijing Forestry University,Beijing 100083

Received:2018-12-21 Published:2019-06-26 Online:2019-07-08

摘要/Abstract

摘要： hyBl（hygB-like）基因簇是内生链霉菌SAT1中的次级代谢基因簇,经分析它与潮霉素B基因簇具有52%的相似性,可能在SAT1抑制栗疫菌（Cryphonectria parasitica）的过程中发挥主要作用。为了深入研究该菌的抑菌机制和hyBl基因簇的功能,以hyBl为目标,利用CRISPR/Cas9技术建立SAT1的基因簇敲除体系。在实验过程中,通过protospacer的筛选、敲除载体的构建、接合转移供体菌、Ca²⁺和Mg²⁺的选择以及接合时间等多个条件的探索,建立了SAT1的基因簇敲除体系。最终以E. coliET12567（pUZ8002/pCRISPomyces2-CB）为供体菌,在MS培养基（含10 mmol Mg²⁺）上接合16-18 h,成功获得突变株SAT1△hyBl（缺失14 kb 的hyBl基因簇）。突变株对栗疫菌的抑制带宽较野生株降低了77%,这说明该基因簇指导合成的次级代谢产物在抑制栗疫菌方面具有重要的功能。本研究证明了利用CRISPR/Cas9系统可以对内生链霉菌SAT1进行基因簇编辑,也为深入研究该菌的抑菌机制、基因工程法验证未知基因簇功能提供了良好的技术支撑。

关键词: CRISPR/Cas9系统, pCRISPomyces-2, 内生链霉菌SAT1, 接合, 基因簇敲除

Abstract: The hyBl（hygB-like）gene cluster,a secondary metabolic gene cluster in endophytic Streptomyces SAT1,is 52% similar to the hygromycin B gene cluster,and may play a key role in SAT1 inhibiting Cryphonectria parasitica. In order to further study the antibacterial mechanism of the strain and the function of the hyBl gene cluster,hyBl gene cluster was used to establish the CRISPR/Cas9-mediated gene cluster-knockout system in SAT1. A series of conditions were optimized,including the screening of protospacers,the construction of knockout vectors,the conjugation and transformation of donor bacteria,the selection of Ca²⁺and Mg²⁺,and conjugation time,etc.,and the knockout system of gene cluster for SAT1 was established. Finally,a mutant SAT1△hyBl（14 kb deleted）was obtained with Escherichia coli ET12567（pUZ8002/pCRISPomyces2-CB）as a donor and conjugating on the MS（containing 10 mmol Mg²⁺）for 16-18 h. The antibacterial activity of SAT1△hyBl was 77% lower than that of wild strain,indicating the synthesized secondary metabolites guided by hyBl gene cluster indeed played an important role in inhibiting chestnut blight. This study demonstrates that the CRISPR/Cas9 system can be used for gene cluster editing in endophytic Streptomyces SAT1,and provides promising technical support in further studying the antimicrobial mechanism in SAT1 and verifying the functions of unknown gene clusters.

Key words: CRISPR/Cas9 system, pCRISPomyces-2, endophytic Streptomyces SAT1, conjugation, knockout of gene cluster

田文佳, 窦桂铭, 王莎, 孙靖雅, 马玉超. 利用CRISPR/Cas9系统建立内生链霉菌SAT1的基因簇敲除体系[J]. 生物技术通报, 2019, 35(6): 1-8.

TIAN Wen-jia, DOU Gui-ming, WANG Sha, SUN Jing-ya, MA Yu-chao. Establishment of a CRISPR/Cas9-mediated Gene Cluster-knockout System in the Endophytic Streptomyces SAT1[J]. Biotechnology Bulletin, 2019, 35(6): 1-8.

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利用CRISPR/Cas9系统建立内生链霉菌SAT1的基因簇敲除体系

Establishment of a CRISPR/Cas9-mediated Gene Cluster-knockout System in the Endophytic Streptomyces SAT1

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