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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2019, Vol. 39 Issue (11): 78-86    DOI: 10.13523/j.cb.20191109
技术与方法     
反向筛选标记基因upp在杀真菌链霉菌遗传改造中的应用 *
吴果果1,宋淑婷1,岳荣1,张晶1,关莹1,王玥1,刘宝爱2,吕学敏2,魏建军2,张会图1,**()
1 天津科技大学生物工程学院 天津 300457
2 天津市新星兽药厂 天津 300402
Application of Counterseletable Gene upp in Genetic Manipulation of Streptomyces fungicidicus
WU Guo-guo1,SONG Shu-ting1,YUE Rong1,ZHANG Jing1,GUAN Ying1,WANG Yue1,LIU Bao-ai2,LV Xue-min2,WEI Jian-jun2,ZHANG Hui-tu1,**()
1 Laboratory of Enzyme and Applied Microbiology, College of Biotechnology,Tianjin University of Science and Technology,Tianjin 300457, China
2 Tianjin Xinxing Veterinary Pharmaceutical Factory, Tianjin 300402, China
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摘要：

为进一步简化放线菌的遗传操作流程,缩短重组菌株的筛选周期,在对杀真菌链霉菌(Streptomyces fungicidicus ATCC 21013)进行遗传操作的过程中引入了一个反向筛选标记基因——尿嘧啶磷酸核糖转移酶基因(upp)。并通过原始菌株中upp基因的敲除,以及带有upp基因的自杀型基因敲除载体的构建,开发了一套完整的针对杀真菌链霉菌的无痕敲除系统。通过载体的整合、二次交换及反向筛选实现了杀真菌链霉菌基因组中StrR基因的快速无痕敲除。upp反向筛选标记基因的引入使得链霉菌重组菌株的平均筛选周期缩短了2周左右,并进一步减少了假阳性重组菌株出现的概率,可实现放线菌中目的基因的连续无痕敲除,因此值得进一步推广和应用。
关键词： 杀真菌链霉菌;  反向筛选标记;  尿嘧啶磷酸核糖转移酶;  基因敲除    
Abstract:

In order to further simplify the genetic operation process of actinomycetes and shorten the screening cycle of the recombinant strains, a counter selectable marker gene —— uracil phosphoribosyl transferase gene (upp) was introduced into the genetic operation of Streptomyces fungicidicus ATCC 21013. By deletion of the upp gene in the wild type strain and construction of a suicide vector that carries the upp gene expression cassette, a markerless inframe deletion system was developed, and the StrR gene in S. fungicidicus genome was successfully knocked out after vector integration, plasmid excision and counterselection. The introduction of upp gene shortened the average screening cycle of Streptomyces recombinant strains by about 2 weeks, and further reduced the probability of false positive recombinant strains. The simplicity of this system should make it adaptable for continuous markerless gene deletion in other actinomycetes, so it is worth further promotion and application.
Key words: Streptomyces fungicidicus    Counterselectable marker    Uracil phosphoribosyl transferase    Gene knockout
收稿日期: 2019-04-03 出版日期: 2019-12-17
ZTFLH:  Q78  
基金资助: * 国家自然科学基金(81373309)
通讯作者: 张会图     E-mail: hzhang@tust.edu.cn
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吴果果
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关莹
王玥
刘宝爱
吕学敏
魏建军
张会图

引用本文:

吴果果,宋淑婷,岳荣,张晶,关莹,王玥,刘宝爱,吕学敏,魏建军,张会图. 反向筛选标记基因upp在杀真菌链霉菌遗传改造中的应用 *[J]. 中国生物工程杂志, 2019, 39(11): 78-86.

WU Guo-guo,SONG Shu-ting,YUE Rong,ZHANG Jing,GUAN Ying,WANG Yue,LIU Bao-ai,LV Xue-min,WEI Jian-jun,ZHANG Hui-tu. Application of Counterseletable Gene upp in Genetic Manipulation of Streptomyces fungicidicus. China Biotechnology, 2019, 39(11): 78-86.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20191109    或    https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I11/78

Strains or plasmids Description Source
S.fun ATCC 21013 Wild type stain producing enramycin Stored in lab
E. coli JM109 Host strain Stored in lab
E. coli ET12567 dam dcm hsdS cat tet [8]
pKC1139 ori-pSG5, aac( 3) IV,oriT [9]
pSET152 PCR template of promoter ermE* Stored in lab
pKC1139-upp Knockout upp from S.fun ATCC 21013 This study
pKC1139-E*upp Express upp in S.fun Δupp This study
pKC1139-StrR Knockout StrR from S.fun Δupp This study
表1  本研究中使用的菌株与质粒
Primer name Primer sequence (5'-3')
upp-LF AAGCTTCTTCCCACCCGTCTTCGGTG
upp -LR TCTAGACTTGTGGGCGACCAGAGGGT
upp -RF GGATCC CGAGCGTCTGAACGAGCAGG
upp -RR GAATTCCCGGATCACGGGTGACAGGT
upp OUT-F GTTTGACGCCAGCGAAGGAG
upp OUT-R CCGACAAGGCCCTGGCCGTA
ermE*-F ACGACGGCCAGTGCCAAGCTTGCTGCGCAACTGTTGGGAAG
ermE*-R CTCCTTCGCTGGCGTCAAAC CCTCCGTTCCGCTAGATCCT
upp-F GTAGGATCTAGCGGAACGGA GTTTGACGCCAGCGAAGGAG
upp-R CTCGTTCAGACGCTCGGGATCCGACAAGGCCCTGGCCGTACT
StrR -LF GGATCCCTCGTCGAGGCGGACGGCTC
StrR -LR TCTAGA CCCTTTCGAGCGCAGCGACTC
StrR -RF TCTAGA GTCCGACGCCGTGCCGTC
StrR -RR GAATTCGACCACGCGGTGGCCCTCG
StrR OUT-F CCGGCTGCACTACTCCGTCG
StrR OUT-R CCGCCCTGGAGATGGTGTCG
表2  本研究所用的引物
图1  重组载体
Types of antibiotics Concentration of 5-fluorouracil (μg/ml)
5-fluorouracil 0 1 5 10
++ + - -
表3  S. fun ATCC 21013在梯度浓度 5-FU平板上的生长情况
图2  S. fun ATCC 21013在5-FU和无抗平板上的生长状况
图3  S. fun Δupp 在5-FU平板上的筛选
图4  S.fun Δupp 菌株的PCR鉴定
图5  同源双交换原理示意图
图6  upp基因缺失对菌株的影响
图7  PCR鉴定upp基因回补菌株S.fun Δupp/upp
图8  upp基因回补菌株在5-FU平板上的生长状况
图9  利用S.fun Δupp进行基因无痕敲除原理图
图10  S. fun Δupp ΔStrR菌株的PCR鉴定
图11  测活平皿
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