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| 深海放线菌Marinactinospora thermotolerans SCSIO 00652遗传操作系统的建立 | |
| 引用本文： | 李军,朱清华,张云,马俊英,田新朋,李文均,张长生,鞠建华.深海放线菌Marinactinospora thermotolerans SCSIO 00652遗传操作系统的建立[J].中国抗生素杂志,2012,37(2):105-111. |
| 作者姓名： | 李军 朱清华 张云 马俊英 田新朋 李文均 张长生 鞠建华 |
| 作者单位： | 1. 中国科学院海洋生物资源可持续利用重点实验室,广东省海洋药物重点实验室,中国科学院海洋微生物中心,中国科学院南海海洋研究所,广州510301;中国科学院研究生院,北京100049 2. 中国科学院海洋生物资源可持续利用重点实验室,广东省海洋药物重点实验室,中国科学院海洋微生物中心,中国科学院南海海洋研究所,广州510301;德州学院生物系,德州253023 3. 中国科学院海洋生物资源可持续利用重点实验室,广东省海洋药物重点实验室,中国科学院海洋微生物中心,中国科学院南海海洋研究所,广州510301 4. 云南省微生物研究所,昆明,650091 |
| 基金项目： | 国家自然科学基金，国家自然科学青年基金，中国科学院知识创新工程重要方向项目，中国科学院南海海洋所领域前沿项目 |
| 摘 要： | 目的建立深海放线菌Marinactinospora thermotolerans SCSIO 00652菌株的遗传操作系统,通过基因阻断研究次级代谢产物的生物合成途径。方法在对该菌株的全基因组进行测序的基础上,以基因组序列中编码非核糖体肽合成酶(Non-ribosomal peptide synthetase,NRPSs)的基因s102-A和s63-A为目标基因,利用PCR-targeting介导的基因置换技术构建了重组质粒,在加有3%海盐的培养基上,通过ET12567/pUZ8002属间接合转移导入SCSIO 00652野生菌。结果接合子通过一代、二代松弛培养都无法得到双交换突变菌株,松弛培养四代后经抗性筛选,双交换率明显提高,PCR分析结果显示s102-A基因和s63-A基因均被成功阻断,HPLC分析结果显示突变株的发酵产物与野生型菌株有一定的差异。结论成功建立了深海放线菌M.thermotolerans SCSIO 00652的遗传操作系统,为对其它基因进行遗传学操作奠定了基础。 |
| 关 键 词： | M.thermotolerans SCSIO 00652 NRPSs s102-A s63-A PCR-Targeting 接合转移 松弛培养 |
Development of a genetic system of deep sea marine actinomycete Marinactinospora thermotolerans SCSIO 00652 |
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| Li Jun , Zhu Qing-hua , Zhang Yun , Ma Jun-ying , Tian Xin-peng , Li Wen-jun , Zhang Chang-sheng , Ju Jian-hua.Development of a genetic system of deep sea marine actinomycete Marinactinospora thermotolerans SCSIO 00652[J].Chinese Journal of Antibiotics,2012,37(2):105-111. | |
| Authors: | Li Jun Zhu Qing-hua Zhang Yun Ma Jun-ying Tian Xin-peng Li Wen-jun Zhang Chang-sheng Ju Jian-hua |
| Institution: | 1(1 CAS Key Laboratory of Marine Bio-resources Sustainable Utilization,Guangdong Key Laboratory of Marine Materia Medica, RNAM Center for Marine Microbiology,South China Sea Institute of Oceanology,Chinese Academy of Sciences, Guangzhou 510301;2.Graduate University of Chinese Academy of Sciences,Beijing 100049; 3 Biology Department,Dezhou University,Dezhou 253023; 4 Yunnan Institute of Microbiology,Kunming 650091) |
| Abstract: | Objective To develop a genetic system of the strain,Marinactinospora thermotolerans SCSIO 00652 and study its biosynthetic pathway of secondary metabolites using gene disruption.Method Two gene disruption cosmids targeting the adenylation domains of the s102-A and s63-A genes was constructed using PCR-targeting method.Intergeneric conjugation was followed for the transformation of ET12567/pUZ8002,into M.thermotolerans SCSIO 00652,when grown on modified ISP4 medium.Results The double crossover recombinant strains showing ApramycinRKanamycinS were selected after culturing the single-crossover exconjugants for four generations without adding antibiotics and further verified by Polymerase Chain Reaction(PCR).Mutant strain showed difference in the fermentation product when compared to the wild type,which was further,evaluated using High Performance Liquid Chromatography(HPLC).Conclusion A genetic system of the deep sea marine bacterium M.thermotolerans SCSIO 00652 was successfully developed,setting the stage for future genetic manipulation of this strain to study functional genes to increase the potency of the bioactive metabolites at large scale. |
| Keywords: | M thermotolerans SCSIO 00652 NRPSs s102-A s63-A PCR-Targeting Conjugation Relaxed culture |
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