玫瑰黄链霉菌Men-myco-93-63 nsdA_(mgh)基因阻断突变株的构建  

Construction of nsdA_(mgh) gene disruption mutant in Strempomyces roseoflavus Men-myco-93-63

作　　者：沈凤英[1,2] 吴伟刚[2] 张艳杰[1] 寇宏达[3] 冀红柳[1] 李亚宁[1] 刘大群[1] 

机构地区：[1]国家北方山区农业工程技术研究中心河北省植物病虫害生物防治工程技术研究中心河北农业大学植物保护学院,河北保定071001 [2]河北北方学院,河北张家口075000 [3]河北农业大学国际合作处,河北保定071001

出　　处：《生物工程学报》2015年第12期1741-1752,共12页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(No.31171894);河北省中药材产业技术体系(No.1004029)资助~~

摘　　要：为研究从玫瑰黄链霉菌Men-myco-93-63中克隆到的,与天蓝色链霉菌M 145中的一个重要负调控基因nsd A基因同源的nsdA_(mgh)基因的功能,本文构建了nsd A_(mgh)基因破坏型重组质粒pSRNA2500(pKC1139::1.5 kb nsdA_(mgh)::1.0 kb Km^r),转化ET12567(pUZ8002)获得接合转移供体菌ET12567(pUZ8002,pSRNA2500),通过接合转移将重组质粒导入玫瑰黄链霉菌Men-myco-93-63中。在高温和抗生素双重筛选压力下,筛选得到表型为Am^sKm^r的nsd A_(mgh)基因阻断突变株,通过PCR、Dot bloting和Southern blotting验证了突变株中的nsdA_(mgh)基因已被正确阻断。与出发菌株相比,突变株在摇瓶水平上对棉花黄萎病菌的抑制能力提高了一倍。Insertional mutagenesis is a widely used method to determine the function（s） of a gene. To study the function（s） of the gene nsdA（mgh） 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 nsdA（mgh） gene, with a phenotype of Am-sKm-r, were isolated and verified using PCR and Dot-blotting and Southern blotting hybridization methods. Functional analysis showed that the disrupted mutants of nsdA（mgh） 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.

关 键 词：玫瑰黄链霉菌MEN-MYCO-93-63 pKC1139 nsd A基因 基因阻断 同源重组 

分 类 号：S476.1[农业科学—农业昆虫与害虫防治]