Construction of sRNA-deficient and-overexpressing strains of Yersinia pestis
10.7644/j.issn.1674-9960.2017.10.004
- VernacularTitle:鼠疫耶尔森菌中长度在100nt以内的小RNA缺失株及过表达菌株的构建
- Author:
Xiao-Fang GAO
1
;
Zi-Zhong LIU
;
Wen-Liang LI
;
Hong-Duo WANG
;
Rui-Fu YANG
;
Yan-Ping HAN
Author Information
1. 安徽医科大学
- Keywords:
Yersinia pestis;
small RNA;
λ-Red homologous recombination;
overexpression
- From:Military Medical Sciences
2017;41(10):800-804
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct small RNA deletion and overexpression strains with a length of less than 100 nt in Yersinia pestis.Methods Deletion mutants of the target sRNAs were constructed by increasing the length of homologous regions.Meanwhile, the high copy plasmid pBAD/HisA was modified into an inducible transcriptional vector as an sRNA-overexpression plasmid by using QuikChange lightning site-directed mutagenesis kit .The presence , size, and transcription-al initiation sites of the indicated sRNA were predicted by transcriptome sequencing , primer extension , and previous stud-ies.The full-length DNA fragments of target sRNAs were transformed into the transcriptional vector .The overexpressing strains of sRNAs were identified by Northern Blot .Results and Conclusion Four sRNAs deletion mutants of sR01, sR02, sR03 and HmsA and three sRNAs overexpression mutants MicF , HmsA and CpxQ were successfully constructed .A method of construction of sRNA deficient and overexpressing strains of Y.pestis has been quickly and efficiently established by λ-Red homologous recombination technology and QuikChange ? lightning site-directed mutagenesis kit.
