Diversity Of Thaumarchaeal Nitrite Reductase (nirK)-Like Genes And Ammonia Monooxygenase Gene(amoA) In The Pearl River,China

In recent years, a amount of agricultural, industrial and urban sewage waste pour into thePearl River and the urbanization intensifies make the concentration of soluble inorganicnitrogen in the water growing higher, which result in environmental issues, such asgreenhouse, acid rain and eutrophication. The ammonia oxidation process is a key step innitrogen cycle, in which ammonia oxidizing archaea (Ammonia-oxidizing archaea, AOA)plays an important role the ammonia monooxygenase encoded by AOA-amoA is responsibefor the ammonia oxidation, and the AOA-nirK that encodes nitrite reductase, is diverse inmarine ecosystem and supposed to be mainly responsible for the marine nitrogen gas nitrousoxide emissions.However in freshwater, the diversity of AOA-nirK genes are seldom studied.In this paper, we took the Pearl River water as research objectto study the diversity andabundance of AOA-nirK and AOA-amoA, in this ecosystem by clone library, DGGE, andreal-time quantitative PCR (RT-PCR),the main results are as follows:1. The AOA-nirK gene clone libraries were construted on the water samples from Sui shi,Bai xian ke, Hai xin sha, and Nansha sections of the Pearl River. According to the restrictionfragment length (RFLP) analysis and BLASTx comparison: Sui shi, Bai xian ke, Hai xin sha,and Nansha AOA-nirK gene clone libraries were divided into17OTUs, in which, the OTU4and OTU5are the dominant clones of Hai xin sha nirK gene clone libraries, both account for14.06%of the total clones, the OTU10is the dominant in Sui shi nirK gene library andaccount for13.29%, the OTU16is the dominant clones in Nansha water nirK gene clonelibrary and account for17.35%, the OTU16is the dominant clones in Bai xian ke nirK geneclone library and account for13.08%of the total clones. The amino acid sequences analysissuggested the similarity of AOA-nirK from the Pearl River clone libraries are low (62%-76%)with the sequences from the NCBI database.2The AOA-amoA gene clone libraries were construted on the water samples from Suishi, Bai xian ke, Hai xin sha, and Nansha sections of the Pearl River. According to therestriction fragment length (RFLP) analysis and BLASTx comparison: Sui shi, Bai xian ke,Hai xin sha, and Nansha AOA-amoA gene clone libraries were divided into16OTUs, inwhich, OTU2is the dominant OUT in Hai xin sha AOA-amoA gene clone library, account for17.27%of the total clones, the OTU2is the dominant in Sui shi AOA-amoA gene library andaccount for24.24%, the OTU2is the dominant clones in Bai xian ke AOA-amoA geneclone library and account for17.35%, the OTU2is the dominant clones in Bai xian keAOA-amoA gene clone library and account for52.69%of the total clones. the OTU1is the dominant clones in Nansha AOA-amoA gene clone library and account for17.95%of the totalclones. The amino acid sequences analysis suggested the similarity of AOA-amoA from thePearl River clone libraries are high (99%-100%) with the sequences from the NCBI database.3． Phylogenetic analysis suggested the AOA-amoAgenes are closely related to theAOA-amoA genes from oceans, hot springs and soils. However, the similarity of AOA-nirKfrom Pearl River clone libraries are only62%-76%with the closest nirK sequences, andPhylogenetic analysis suggested the nirK sequences from the Pearl River water were clusteredin a monophyletic lineage distinct from the Crenarchaeota and Thaumarchaeota clusters. Infour water samples, the results of RT-PCR indicated the abundance of AOA-nirK gene lowerthan the AOA-amoA gene with a10-20times divergence, the abundance of AOB are higherthan AOA by16S rRNA gene analysis, and the the abundance of AOA-amoA, AOA-nirK, and16S rRNA gene in Nansha was significantly higher than the other points.4. The AOA-nirK gene diversity and abundance in different depth of Sui shi were studiedby DGGE and RT-PCR. The DGGE results suggested the number of bands at S0is most.electrophoresis band at W150is7,the other is4or5. the electrophoresis bands of S0and W0are relatively light. RT-PCR results suggested that the abundance of thaumarchaeal nirK were10-20times lower than thaumarchaeal amoA and AOB-amoA,but S8. The abundance ofthaumarchaeal AOA-amoA is gradually reduced from the meter to the deep sediment, butreaches a maximum at S0.The abundance of thaumarchaeal nirK gene is high in surface waterand S0, but lowest in the deep sediment.