长期施用氮肥对水稻土亚硝酸还原酶基因多样性的影响

Effect of Long-term Fertilization on the Diversity of Nitrite Reductase Genes(nirK and nirS) in Paddy Soil

以中国科学院桃源农业生态试验站长期定位试验的土壤样品为对象,采用PCR扩增、克隆测序等分子生物学技术,研究长期施氮肥对水稻土亚硝酸还原酶基因nirK、nirS多样性的影响.序列分析结果表明,从水稻土中克隆的系列nirK基因片段与NCBI数据库中未知菌种的nirK基因相似性较高,平均达90.7%;而nirS基因片段与数据库中已知的nirS基因相似度低,平均74.7%.通过Chao1估计值预测,nirK基因在不施肥处理(CK)、施氮肥处理(N)中分别有58±13和49±9个不同的OTUs,而nirS基因在CK处理、N处理中分别有49±10和132±43个不同的OTUs.Chao1预测曲线95%置信区间(95%CIs)显示,氮肥施用显著提高了nirS基因的多样性,而对nirK基因多样性则无显著影响.LIBSHUFF分析比较N、CK处理克隆文库间的差异,结果显示nirK基因处理间群落结构差异p<0.022,达到显著水平;而nirS基因处理间的群落结构无显著差异.系统发育分析显示,nirK、nirS基因的系统发育树分别可分为3个及4个大簇.施用氮肥导致nirK、nirS克隆有不同程度的聚集,说明氮肥改变了nirK和nirS基因群落结构,其中氮肥对nirK基因群落结构的影响更大.总体来说,氮肥的施用对水稻土nirK基因群落多样性无显著影响,但明显提高nirS基因群落的多样性;而长期施氮肥使含nirK基因的反硝化菌群落结构发生显著变化,对nirS基因群落结构则无显著影响.

In order to investigate the effects of long-term application of nitrogen fertilizer on soil denitrifying communities, the diversities of nir genes （nirK and nitS） were studied using molecular approaches in the long-term paddy field experiment （ started in 1990） located in Taoyuan. Analysis of clone sequences indicated that the nirK fragments from paddy soil Showed close similarity （90.7%） to the nirK sequences registered in GenBank database, but were not related to any known strain. Whereas, most of the nitS clones showed low similarity （74.7%） to the nirS gene fragments registered in GenBank. The Chaol estimates showed that the diversity of nirK gene communitiy appeared to be higher in CK treatment [ （58 ± 13） OTUs] than in N treatment [ （49 ±9） OTUs], but the difference was not significant. However, application of nitrogen fertilizer resulted in significant difference of nitS-community compared to CK. Nitrogen fertilizer had obvious effect on the community structure of nirK-denitrifiers （p 〈 0. 022） , but the nitS-containing community was not affected. Based on phylogenetic analysis, nirK clones grouped into three clusters with aggregations of some OTUs cloned from N treatment. Although nits clones grouped into four clusters, the majority of the clones were attributed in one cluster. The results suggested that application of nitrogen fertilizer had a greater influence on the diversity of nirS-containing bacterial community than that of the nirK. However, the community structure of nirK-containing denitrifiers was more sensitive to nitrogen fertilization than that of the nirS.