南美白对虾养殖底泥中氨氧化细菌与氨氧化古菌多态性分析

Diversity of β-Proteobacterial ammonia-oxidizing bacteria and ammonia-oxidizing archaea in shrimp farm sediment

【目的】本研究皆在了解虾养殖底泥中氨氧化细菌与氨氧化古菌群落多态性。【方法】以功能基因为基础,构建氨氧化细菌(AOB)与氨氧化古菌(AOA)的氨单加氧酶α亚基基因(amoA)克隆文库。利用限制性片段长度多态性(Restriction Fragment Length Polymorphism,RFLP)技术将克隆文库阳性克隆子进行归类分析分成若干个可操作分类单元(Operational Taxa Units,OTUs)。【结果】通过序列多态性分析,表明AOB amoA基因克隆文库中所有序列都属于变形杆菌门β亚纲(β-Proteobacteria)中的亚硝化单细胞菌属(Nitrosomonas)及Nitrosomonas-like,未发现亚硝化螺旋菌属(Nitrosospira)。AOA amoA基因克隆文库中只有一个OTU序列属于未分类的古菌(Unclassified-Archaea),其余序列都属于泉古菌门(Crenarchaeote)。AOA群落结构单一且存在一个绝对优势类群OTU3,其克隆子数目占克隆文库的57.45%。AOB和AOA amoA基因克隆文库分别包括13个OTUs和9个OTUs,其文库覆盖率分别为73.47%和90.43%。AOB amoA基因克隆文库的Shannon-Wiener指数、Evenness指数、Simpson指数、Richness指数均高于AOA。【结论】虾养殖塘底泥中存在氨氧化古菌的amoA基因,且多态性低于氨氧化细菌,表明氨氧化古菌在虾养殖塘底泥的氮循环中可能具有重要的作用。

[Objective]In order to study the diversity of ammonia-oxidizing bacteria （AOB） and ammonia-oxidizing archaea （AOA） in shrimp farm sediment.[Methods]Total microbial DNA was directly extracted from the shrimp farm sediment.The clone library of amoA genes were constructed with β-Proteobacterial-AOB and AOA specific primers.The library was screened by PCR-restriction fragment length polymorphism （RFLP） analysis and clones with unique RFLP patterns were sequenced.[Results]Phylogenetic analyses of the amoA gene fragments showed that all AOB sequences from shrimp farm sediment were affiliated with Nitrosomonas （61.54%） or Nitrosomonaslike （38.46%） species and grouped into Nitrosomonas communis cluster、Nitrosomonas sp.Nm148 cluster、 Nitrosomonas oligotropha cluster.All AOA sequences belonged to the kingdom Crenarchaeote except that one Operational Taxa Unit（OTU）sequence was Unclassified-Archaea and fell within cluster S （soil origin）.AOB and AOA species composition included 13 OTUs and 9 OTUs.The clone coverage of bacterial and archaeal amoA genes was 73.47% and 90.43%.The Shannon-Wiener index,Evenness index,Simpson index and Richness index of AOB were higher than those of AOA.[Conclusion]These findings represent the first detailed examination of archaeal amoA diversity in shrimp farm sediment and demonstrate that diverse communities of Crenarchaeote capable of ammonia oxidation are present within shrimp farm sediment,where they may be actively involved in nitrification.