深海放线菌Marinactinospora thermotolerans SCSIO 00652遗传操作系统的建立

摘要

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 fourgenerations 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.%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(Nonribosomal peptide synthetase,NRPSs)的基因sl02-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.