若尔盖高原湿地泥炭沼泽土亚硝酸盐还原酶(nirK)反硝化细菌群落结构分析

摘要

若尔盖泥炭湿地是世界少有的低纬度永久冻土湿地,具有高海拔、高紫外辐射、高有机质的特点.该区域N2O的排放量对全球气候变暖有重要影响.对若尔盖高原湿地泥炭沼泽土中的亚硝酸盐还原酶(nirK)反硝化细菌群落结构多样性进行分析,以期揭示该区域N,O释放的微生物调控机制.基于若尔盖高原湿地泥炭沼泽土的理化性质和反硝化活性(PDA),结合限制性酶切片段长度多态性(Restriction fragment length polymorphism,RFLP)技术、克隆文库及分子测序对该生态系统中的nirK反硝化细菌群落结构及多样性进行分析.反硝化活性测定结果显示:阿西地区＞麦溪地区＞分区地区,反硝化活性与土壤有机碳、总氮和丰富度呈显著正相关(P＜0.05).Shannon-wiener多样性指数以阿西最高、分区最低.3个样品中共测序15条nirK基因代表序列,系统发育表明若尔盖高原湿地优势nirK反硝化菌群为变形门菌群.其中,阿西地区主要为α-变形菌门,麦溪地区主要为β-变形菌门,分区地区无法确定优势种群.冗余分析(Redundancy analysis,RDA)显示:有效钾和有效磷是影响nirK反硝化细菌群落结构的关键环境因子.本论文显示,若尔盖高原湿地存在着明显的反硝化作用,调控这些反硝化作用的nirK反硝化细菌多样性较高,且与土壤有效钾和有效磷密切相关.%The Zoige Wetland,which is located in the northeast of the Tibetan Plateau,is a typical low latitude permanent permafrost wetland,with high altitude intensive ultraviolet radiation and high soil organic matter content.The nitrous oxide fluxes in this area are critical to global warming.The main objective of this study was to elucidate the diversity and structure of the nitrate reductase (nirK) gene denitrifier community,and to further explore the microbial mediated mechanism of N2O release in this climatically extreme area.Based on the soil physicochemical properties and denitrifying activity (PDA) analysis,restriction fragment length polymorphism (RFLP),clone library and sequencing were further used to analyze the diversity and structure of the nirK gene in the denitrifier community.The results showed that the highest activity of PDA was detected in the Axi soil,while the lowest activity was in the Fenqu soil.The PDA was significantly and positively correlated with soil organic carbon,total nitrogen and nirK gene richness (P ＜ 0.05).The highest and lowest Shannon-Wiener diversity indices were detected in Axi and Fenqu soils,respectively.Based on the RFLP patterns,15 different nirK gene clones were selected for sequencing and further phylogenetic analysis.The phylogenetic analysis showed that the majority of the nirK-denitrifiers in the peat soil of the Zoige Wetland belonged to Proteobacteria,and the dominant species in the Axi soil were Alphaproteobacteria,whereas Betaproteobacteria species dominated in the Maixi soil.The dominant nirK denitrifiers remained unidentified in the Fenqu soil.Redundancy analysis (RDA) was used to explore the possible relationship between the nirK denitrifiers community and soil physicochemical parameters,and the results showed that soil available potassium and available phosphorus were the two most important factors in shaping nirK denitrifier communities.In conclusion,the current study revealed obvious denitrifying activities in the Zoige Plateau Wetland.The nirK gene denitrifying community,which plays an important role in mediating the denitrifying process,was relatively diverse and positively influenced by soil available potassium and available phosphorus.