摘要
【目的】建立和优化固氮链霉菌Streptomyces chartreusi WZS021(简称菌株WZS021,下同)的接合转移体系,以丰富固氮链霉菌基因片段转移技术。【方法】以大肠杆菌Escherichia coli ET12567(pUZ8002)为供体菌,携带整合型质粒pSET152的菌株WZS021为受体菌,进行菌株WZS021接合转移。【结果】选择高氏一号培养基为菌株WZS021接合转移培养基,50℃热激10 min,供受比为1∶10,涂布20.0 mg/L安普霉素、接合转移12 h后覆盖抗生素,添加MgCl_2终浓度为5.0 mmol/L时接合转移效率最高,达3.24×10^(-6)。【结论】通过确定适用于菌株WZS021接合转移条件建立的菌株WZS021遗传转化体系,有助于今后插入标记基因确定该菌的固氮定殖位点及导入外源基因的操作。
[Objective ]The present study was conducted to establish and optimize transconjugation system of Streptomyces chartrezsis WZS021 （hereinafter referred to as strain WZS021 ）,in order to enrich strain WZS021 gene fragment transfer technology. [ Method ] As donor strain, Escberichia coli ET12567 （pUZ8002） contained integrative plasmid pSET152. Strain WZS021 was recipient. Transconjugation was conducted on strain WZS021. [ Result]The optimal conditions were as follows：Gauze＇s medium No.1 as the transconjugation medium, heat shock at 50℃ for 10 min, donor-recipient ratio 1：10, 20.0 mg/L apramycin coverage 12 h post transconjugation, and adding MgC12 till final concentration of 5.0 mmol/L. Under these conditions,the transconjugation efficiency was the highest, which was up to 3.24x10-6. [Conclusion]The suitable transconjugation conditions for strain WZS021 are ascertained, and an effective genetic transformation system for strain WZS021 is established based on it. It is helpful to insert marker genes to detect nitrogen fixation sites of the strain and exogenous gene transfer.
出处
《南方农业学报》
CAS
CSCD
北大核心
2017年第4期581-586,共6页
Journal of Southern Agriculture
基金
国家高技术研究发展计划项目(2013AA102604)
国家自然科学基金项目(31471449)
国家现代农业产业技术体系广西甘蔗创新团队建设项目(gjnytxgxcxtd-03)
广西八桂学者和特聘专家专项项目([2013]3号)
作者简介
王震（1991-），研究方向为甘蔗生物固氮，E-mail：wang798110510@163．com
王震（1991-），研究方向为甘蔗生物固氮，E—mail：wang798110510@163．com
李杨瑞（1957-），博士，教授，博士生导师，主要从事甘蔗栽培与育种研究工作，E-mail：liyr@gxu．edu．cn。
