斯氏油脂酵母基因组Fosmid文库的构建及降解百草枯氮次级代谢相关基因的筛选

Screening genes of secondary nitrogen metabolism involved in paraquat degradation by construction of fosmid library of Lipomyces starkeyi

斯氏油脂酵母在以百草枯作为唯一氮源的培养基中能降解百草枯,但其机制尚不清楚。为分离鉴定斯氏油脂酵母中降解百草枯的相关基因,本研究通过构建斯氏油脂酵母的Fosmid文库,用百草枯作为筛选标记,成功筛选到7个百草枯抗性的大肠杆菌克隆。对阳性克隆插入片段进行了测序分析,并对真核基因进行注释。结果在插入片段中发现了nmrA基因及氮代谢相关基因,nmrA是真菌在限氮的条件下才激活的氮代谢相关基因,能调控激活下游的次级氮代谢基因家族,分解那些不常见的氮源。这提示斯氏油脂酵母可能也具有氮的次级代谢调控机制,在百草枯作为唯一氮源的环境下斯氏油脂酵母能激活次级氮代谢相关基因,分解代谢百草枯。

It was found that paraquat could be degraded by Lipomyces starkeyi in the culture medium with paraquat as the sole nitrogen source,but its mechanism was not clear.In order to separate and screen genes involved in degradation of paraquat,the genomic fosmid library of Lipomyces starkeyi was constructed and the anti-paraquat E.coli clones were screened out by chloromycetin and paraquat in LB agar plates.The inserted fragments were sequenced and ORFs were analyzed by eukaryotic genome annotation.The results revealed that one of inserted fragment in clone 264 had a sequence identified as gene nmrA.The nmrA was a central player in network controlling nitrogen metabolite repression in fungi.It regulated a large set of nitrogen catabolic gene clusters which could catalyze the rare nitrogen source.This indicated that there was probably the same nitrogen metabolism system in Lipomyces starkeyi to degrade paraquat when the herbicide was used as a sole nitrogen source in the medium.