若尔盖高原湿地土壤氨氧化古菌的多样性

Diversity of ammonia-oxidizing archaea in Tibetan Zoige plateau wetland

摘要:

摘要:【目的】研究自然界中氨氧化古菌(ammonia-oxidizing archaea，AOA)对于理解全球氮循环起着至关重要的作用，但人们对高原湿地AOA种群生态还知之甚少。本研究旨在了解若尔盖高原湿地土壤AOA群落组成及多样性。【方法】从若尔盖高原阿西(A'xi)、麦西(Maixi)和分区(Fenqu)3个典型牧区采集土壤样品，提取土壤总DNA，利用AOA 氨单加氧酶(ammonia monooxygenase，amoA)基因通用引物扩增amoA基因，构建amoA基因克隆文库。从每个克隆文库中随机挑选80个阳性克隆子用于后续限制性酶切片段长度多态性(restriction fragment length polymorphism，RFLP)分析，挑选不同酶切类型的克隆子进行测序、比对，利用MEGA 5.0软件构建amoA基因系统发育树。【结果】从3个克隆文库共240个AOA amoA基因阳性克隆中得到15条代表序列，通过Mothur软件进行OTUs (operational taxonomic units)分类得到7个不同的分类单元。其中OTU 6 为优势类群，在3个克隆文库均有发现，约占所有特异性克隆子的27%。15条amoA 基因序列分属于Zoige Wetland Clade 1 (4 OTUs)、Zoige Wetland Clade 2 (2 OTUs)和Zoige Wetland Clade 3 (1OTU)3个系统发育分支。BLAST分析显示所有OTUs均归于泉古菌门(Crenarchaeota)。相关性分析表明，若尔盖高原湿地AOA多样性指数与土壤铵态氮和硝态氮含量存在显著的相关性(P＜0.05)。【结论】若尔盖高原湿地中AOA 多样性较低，均属于泉古菌，且与土壤中氨态氮和硝态氮密切相关。

Abstract:

Abstract:［Objective］ Investigation of ammonia-oxidizing archaea (AOA) in nature environments is important to understand the global nitrogen cycling．However，little is known about the AOA community in plateau wetland．Therefore，we studied the composition and diversity of AOA in Zoige plateau wetland swamp soil．［Methods］ Total DNA was extracted from the swamp soil of three typical wetlands including A' xi pastoral area，Maixi pastoral area and Fenqu pastoral area locate in Zoige plateau wetland，and amoA gene was amplified with universally AOA amoA gene primers and then cloned．Then 80 positive clones for each clone library were chosen for further restriction fragment length polymorphism (RFLP) analysis，and the typical RFLP types were selected for sequencing and clustered into operational taxonomic units (OTUs) at 98% cutoff using the Mothur software． The MEGA 5.0 software was used for the amoA gene phylogeny analysis．［Results］ A total of 240 positive clones for all 3 libraries were used for RFLP analysis，and 15 specific amoA sequences were sequenced and clustered into 7 OTUs at 98% cutoff． Among them，OTU6 was detected in all of the 3 libraries and included 27% of the total specific clones． The phylogeny analysis showed that the 15 amoA sequences were grouped into 3 subgroups consisted of Zoige Wetland Clade 1 (4 OTUs)，Zoige Wetland Clade 2 (2 OTUs) and Zoige Wetland Clade 3 ( 1 OTU)．BLAST analysis showed that all OTUs were affiliated with the phylum Crenarchaeota． Correlation analysis showed that the Shannon diversity index (H') was significantly correlated with ammonia，nitrate/nitrite (P＜0.05)．［Conclusion］AOA in the Zoige plateau wetland swamp soil are all belonged to the Crenarchaeota，and their diversity is significantly correlated with soil ammonia，nitrate/nitrite content．