| 固氮链霉菌Streptomyces chartreusi WZS021接合转移系统的建立及优化 |
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| 引用本文： | 王震,庞妃,顾彩彩,王露蓉,邢永秀,杨丽涛,李杨瑞. 固氮链霉菌Streptomyces chartreusi WZS021接合转移系统的建立及优化[J]. 南方农业学报, 2017, 48(4): 581-586. DOI: 10.3969/j.issn.2095-1191.2017.04.003 |
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| 作者姓名： | 王震 庞妃 顾彩彩 王露蓉 邢永秀 杨丽涛 李杨瑞 |
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| 作者单位： | 广西大学 农学院/亚热带农业生物资源保护与利用国家重点实验室,南宁,530004广西大学 农学院/亚热带农业生物资源保护与利用国家重点实验室, 南宁 530004广西农业科学院 甘蔗研究所/中国农业科学院 甘蔗研究中心/农业部广西甘蔗生物技术与遗传改良重点实验室/广西甘蔗遗传改良重点实验室, 南宁 530007 |
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| 基金项目： | 国家高技术研究发展计划项目，国家自然科学基金项目，国家现代农业产业技术体系广西甘蔗创新团队建设项目，广西八桂学者和特聘专家专项项目 |
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| 摘 要： | [目的]建立和优化固氮链霉菌Streptomyceschartreusi 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Escherichiacoli 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MgCl2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.
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| 关 键 词： | 固氮链霉菌WZS021 接合转移 优化 pSET152 |
Establishment and optimization of Streptomyces chartreusi WZS021 transconjugation system |
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| Abstract: | [Objective]The present study was conducted to establish and optimize transconjugation system of Strepto-myces chartreusis WZS021(hereinafter referred to as strain WZS021),in order to enrich strain WZS021 gene fragment trans-fer technology. [Method]As donor strain,Escherichia 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 MgCl2 till final concentration of 5.0 mmol/L. Under these conditions,the transconjugation efficiency was the highest, which was up to 3.24×10-6. [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. |
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| Keywords: | Streptomyces chartreusi WZS021 transconjugation optimization pSET152 |
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