广藿香青枯病菌Tn5转座子插入突变体的构建
投稿时间:2018-04-27  修订日期:2018-05-23  责任编辑:  点此下载全文
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作者中文名作者英文名单位中文名单位英文名E-Mail
王亚琴 WANG Yaqin 广州中医药大学 中药学院 School of Pharmaceutical Sciences,Guangzhou University of Chinese Medicine 956211566@qq.com 
张宇瑶 ZHANG Yuyao 广州中医药大学 中药学院 School of Pharmaceutical Sciences,Guangzhou University of Chinese Medicine  
贺 红* HE Hong 广州中医药大学 中药学院 School of Pharmaceutical Sciences,Guangzhou University of Chinese Medicine hehong67@hotmail.com 
李 颛 LI Zhuan 广州中医药大学 中药学院 School of Pharmaceutical Sciences,Guangzhou University of Chinese Medicine  
邓志成 DENG Zhicheng 广州中医药大学 中药学院 School of Pharmaceutical Sciences,Guangzhou University of Chinese Medicine  
金 华 JIN Hua 广州中医药大学 中药学院 School of Pharmaceutical Sciences,Guangzhou University of Chinese Medicine  
黎广卫 LI Guangwei 广州中医药大学 中药学院 School of Pharmaceutical Sciences,Guangzhou University of Chinese Medicine  
基金项目:国家自然科学基金资助项目(81373901);高等学校博士学科点专项科研基金资助项目(20134425110012)* 通迅作者:贺红,研究员,主要研究方向:中药生物技术。E-mail:hehong67@hotmail.com
中文摘要:本研究以从感染了青枯病的广藿香植株中分离的青枯菌菌株PRS-84为供试菌株,利用电击转化法,对青枯菌进行Tn5转座子插入突变,在含卡那霉素的培养基上筛选抗性克隆。对抗性克隆进行卡那霉素抗性基因的PCR鉴定,并利用反向PCR技术获得转化子Tn5转座子插入位点的侧翼序列并进行序列测定与分析。结果表明,青枯菌感受态细胞经电击转化后,获得了抗性克隆。经PCR扩增,抗性克隆在预计的约700 bp处出现特异性条带。反向PCR扩增获得了转化子Tn5转座子插入位点的侧翼序列,经序列测定及同源性分析,初步推测3个突变体的Tn5转座子插入位点基因分别为typA基因、recO基因和gidA基因。本研究建立了广藿香青枯病菌Tn5转座子插入突变体系,获得了青枯菌Tn5转座子插入突变体,为构建广藿香青枯菌突变体库及发现青枯菌致病基因奠定了基础。
中文关键词:广藿香  青枯菌  Tn5转座子  电击转化  突变体
 
Construction of Tn5 transposon insertion mutants of Ralstonia solanacearum isolated from Pogostemon cablin
Abstract:Ralstonia solanacearum strain PRS-84 used in this study was isolated from diseased Pogostemon cablin plants in our previous study. The competent cells of R. solanacearum strain PRS-84 were transformed by electroporation with Tn5 transposon and then were plated on TTC agar plates containing kanamycin to select for kanamycin-resistant colonies. Detection of kanamycin resistant gene in kanamycin-resistant colonies was performed by PCR. Further, the flanking fragments of Tn5 transposon insertion site in the mutants were amplified by inverse PCR, and the flanking fragments were sequenced and analyzed. The results indicated that the kanamycin-resistant colonies were obtained in the transformation experiment of R. solanacearum strain PRS-84 by electroporation with Tn5 transposon. A specific band of approximately 700bp was amplified by PCR from kanamycin-resistant colonies. The flanking sequences of Tn5 transposon insertion site in the transformants were obtained by inverse PCR. After sequencing and sequence analysis of Tn5 transposon insertion site in mutants, we preliminarily speculated that the Tn5 transposon inserted in the typA gene, recO gene and gidA gene in three mutants, respectively. A random mutagenesis system of R. solanacearum strain PRS-84 by electroporation with Tn5 transposon has been established, and the Tn5 insertion mutants have been obtained. This study might facilitate the creation of mutant library and the discovery of the virulence gene of R. solanacearum isolated from Pogostemon cablin.
keywords:Pogostemon cablin (Blanco) Benth  Ralstonia solanacearum  Tn5 transposon  Electrotransformation  Mutant
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