Construction of nsdAmgh gene disruption mutant in Strempomyces roseoflavus Men-myco-93-63.
- Author:
Fengying SHEN
;
Weigang WU
;
Yanjie ZHANG
;
Hongda KOU
;
Hongliu JI
;
Yaning LI
;
Daqun LIU
- Publication Type:Journal Article
- MeSH:
Genes, Bacterial;
Genetic Vectors;
Mutagenesis, Insertional;
Polymerase Chain Reaction;
Streptomyces;
genetics
- From:
Chinese Journal of Biotechnology
2015;31(12):1741-1752
- CountryChina
- Language:Chinese
-
Abstract:
Insertional mutagenesis is a widely used method to determine the function(s) of a gene. To study the function(s) of the gene nsdAmgh in Streptomyces roseoflavus, a homologous recombination vector pSRNA2500 was structured in this paper. The recombination donor vector was then transformed into Strempomyces roseoflavus strain Men-myco-93-63 by conjugative transfer. The transformants were subjected to selection under the pressure of high temperature and appropriate antibiotics. As a result, several disrupted mutants of nsdAmgh gene, with a phenotype of Am(s)Km(r), were isolated and verified using PCR and Dot-blotting and Southern blotting hybridization methods. Functional analysis showed that the disrupted mutants of nsdAmgh had a two-fold higher inhibition against Verticillium dahlia Kleb than that of the wild strain Men-myco-93-63, which all will provide a new study route for future research about positive and negative regulator in Men-myco-93-63.
