[1]田平雅,吕苗苗,杨光耀,等.重叠延伸PCR-酶切连接法构建链霉菌表达载体[J].黑龙江农业科学,2021,(01):5-10.[doi:10.11942/j.issn1002-2767.2021.1.0005]
　TIAN Ping-ya,LYU Miao-maio,YANG Guang-yao,et al.Construction of Streptomyces Expression Vector by Overlap Extension PCR-Enzyme Ligation[J].HEILONGJIANG AGRICULTURAL SCIENCES,2021,(01):5-10.[doi:10.11942/j.issn1002-2767.2021.1.0005]
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重叠延伸PCR-酶切连接法构建链霉菌表达载体

《黑龙江农业科学》[ISSN:1002-2767/CN:23-1204/S] 卷: 期数: 2021年01 页码: 5-10 栏目: 出版日期: 2021-01-10
Title:
Construction of Streptomyces Expression Vector by Overlap Extension PCR-Enzyme Ligation
作者:
田平雅; 吕苗苗; 杨光耀; 马次郎; 牛春;  张萍
宁夏泰瑞制药股份有限公司， 宁夏 银川 750101
Author(s):
TIAN Ping-ya; LYU Miao-maio; YANG Guang-yao; MA Ci-lang; NIU Chun; ZHANG Ping
Ningxia Tairui Pharmaceutical Company Limited,Yinchuan 750101，China
关键词:
重叠延伸PCR; pSET152载体; 酶切; 连接
Keywords:
overlap extension PCR;  pSET152 Vector;  restriction enzymes;  ligation
DOI:
10.11942/j.issn1002-2767.2021.1.0005
文献标志码:
A
摘要:
在功能基因组学时代，对基因的功能研究将涉及到大量的载体构建，为建立一种链霉菌外源基因多拷贝表达的载体构建方法，本研究采用重叠延伸PCR与双酶切结合的连接方法，构建链霉菌表达载体，以红霉素工业生产菌红色糖多孢菌(Saccharopolyspora erythraea)基因组DNA为模板，扩增红霉素合成相关的甲基化酶基因 (erythromycin O-methyltransferase,eryG)和羟基化酶基因(erythromycin C-12 hydroxylase,eryK)，以及启动子permE基因，采用重叠延伸PCR及EcoR Ⅴ、NotⅠ和XbaⅠ酶切位点相结合的连接方法构建含有多个基因片段的链霉菌重组表达载体。成功构建了携带外源基因的表达载体pSET001、pSET002和pSET003，最终获得含有多个基因的重组质粒。双酶切载体构建时载体或DNA片段上可用的限制性酶切位点受到限制，通过结合重叠延伸PCR技术，可以利用较少的内切酶快速构建含有多个基因片段的重组载体，为红霉素生产菌的改造提供了重组载体。
Abstract:
In the era of functional genomics,a large number of vectors constructed were invoved.Therefore,in order to establish a multi-copy tandem expression of exogenous genes in Streptomyces,the study use a combination method of overlap extension PCR and double enzyme digestion to construct the expression vector of Streptomyces.Genomic DNA of Saccharopolyspora erythraea was used as a template to amplify synthesis-related methylase genes of erythromycin O-methyltransferase (eryG) and C-12 hydroxylase (eryK),and with the promoter (permE),a combination of overlap extension PCR and EcoR Ⅴ,NotⅠ and XbaⅠ restriction sites use to construct a expression vector in Streptomyces with multiple gene fragments.Expression vectors pSET001,pSET002 and pSET003 which carry with exogenous genes were successfully constructed,and obtained recombinant plasmids containing multiple genes.When constructing a double digestion vector,the available restriction sites on the vector or DNA fragments are limited.By combining overlap extension PCR technology,it is possible to quickly construct recombinant vector containing multiple genes useing less restriction endonuclease,provided a recombinant vector for the transformation of erythromycin- producing bacteria.

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备注/Memo

收稿日期：2020-09-13

第一作者：田平雅（1993-），女，硕士，助理工程师，从事微生物菌种发酵研究。E-mail: 564967039@qq.com。

更新日期/Last Update: 2021-02-23