玫瑰黄链霉菌Men-myco-93-63 nsdAmgh基因阻断突变株的构建

Construction of nsdAmgh gene disruption mutant in Strempomyces roseoflavus Men-myco-93-63

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nsdA基因同源的nsdAmgh基因的功能，本文构建了nsdAmgh基因破坏型重组质粒pSRNA2500 (pKC1139::1.5 kb nsdAmgh::1.0 kb Kmr)，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AmsKmr 的nsdAmgh基因阻断突变株，通过PCR、Dot bloting和Southern blotting验证了突变株中的nsdAmgh 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。

关键词:玫瑰黄链霉菌Men-myco-93-63，pKC1139，nsdA基因，基因阻断，同源重组

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 AmsKmr, 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.

Key words:Streptomyces roseoflavus Men-myco-93-63, pKC1139, nsdA gene, gene disruption, homologous recombination