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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.
Taking the soil samples from long-term experiment of Taoyuan Agro-Ecological Experimental Station of Chinese Academy of Sciences as object, the molecular biological techniques of PCR amplification and cloning and sequencing were applied to study the effects of long-term nitrogen application on the nirK and nirS diversity of paddy soil. Sequence analysis showed that the sequence of nirK gene cloned from paddy soil had a high similarity to the nirK gene of unknown species in NCBI database with an average of 90.7%. However, the similarity of nirS gene fragment with known nirS gene in the database was low , An average of 74.7% .According to the predicted value of Chao1, there are 58 ± 13 and 49 ± 9 different OTUs of nirK gene in CK and N treatments respectively, The 95% confidence intervals (95% CIs) of 49 ± 10 and 132 ± 43 different OTUs were predicted for the treatment, respectively, showing that the nitrogen application significantly increased the diversity of nirS genes but not the nirK gene diversity .The results of LIBSHUFF analysis showed that there was a significant difference in the structure of nirK gene between the two groups (p <0.022), but there was no significant difference in the structure of nirS gene between the two groups The results showed that the nirK and nirS gene phylogenetic trees could be divided into three and four large clusters, respectively. The application of nitrogen fertilizer resulted in the accumulation of nirK and nirS clones in varying degrees, indicating that nitrogen fertilizer changed the nirK and nirS gene community structure, In general, the application of nitrogen fertilizer had no significant effect on the nirK gene diversity in paddy soil, but significantly increased the diversity of nirS gene community. However, the long-term application of nitrogen fertilizer resulted in the denudation of nirK gene Structure changes significantly, nirS gene community structure has no significant effect.