水稻土细菌硝化作用基因（amoA和hao）多样性组成与长期稻草还田的关系研究

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、克隆文库构建以及序列测定等分子生物学技术分析稻草还田对亚硝化功能基因amoA和hao多样性的影响.结果表明,水稻土稻草还田处理(氮磷钾+水稻秸秆,SR)降低了amoA和hao基因的多样性,其Shannon指数分别为3.7和3.2;而氮磷钾处理(CK)的Shannon指数达到4.0和3.7.LIBSHUFF分析比较CK和SR处理克隆文库的差异,结果显示amoA和hao基因处理间群落结构p值分别为0.002和0.001,均达到极显著水平.序列分析结果表明,对于amoA基因,只检测到与亚硝化螺菌属(Nitrosospira)相似的以及与未知的amoA基因相似的基因,并且与Nitrosospira相似的只出现在SR处理中,相似率达96%以上,而与未知的amoA基因相距最近的已知菌属也是Nitrosospira;获得的hao基因则分布于变形菌门(Proteobacteria)中的3个纲(α、β、γ),其中CK处理获得的hao基因主要与Silicibacter、甲基球菌属(Methylococcus)相似,SR处理获得的基因主要与亚硝化螺菌属(Nitrosospira)、亚硝化单胞菌属(Nitrosomonas)亲缘关系较近.系统发育树分析显示亚硝化基因(amoA、hao)在这2个处理中可被分为4个基因簇(Cluster),稻草还田使亚硝化细菌群落发生了明显的分异,出现了基因聚类现象,并且在amoA基因树图中出现了只由SR处理构成的分支(ClusterⅣ).总体来说,长期稻草还田降低了亚硝化基因amoA和hao的多样性,明显改变了亚硝化细菌群落结构.

Effects of Rice Straw on the Diversity of Nitrifying Genes (amoA and hao) in Paddy Soil

The effects of long-term (16 years) fertilization on the diversity and community structure of soil ammonia-oxidizing gene (amoA) and hydroxylamine-oxidizing gene (hao) in paddy soil were evaluated using the methods of polymerase chain reaction, cloning and sequencing. The soil samples were collected from the treatments of NPK (CK) and NPK plus rice straw (SR) of the long-term field fertilization experiment in Taoyuan Agro-ecological Experimental Station. The Shannon Indices showed that the diversity of amoA and hao in SR treatment was lower than that in CK, and LUBSHUFF statistical analyses demonstrated that the sequence compositions of both amoA and hao libraries were significantly different between CK and SR. The phylogenetic trees indicated that some clusters appeared in SR treatment but were not detected in CK treatment. As to amoA, only Nitrosospira besides the uncultured amoA sequences were cloned from the two treatments, while no Nitrosomonas species were detected. As to hao, the strains from Silicibacterand Methylococcus were dominant in CK, while in SR the strains from Nitrosospira and Nitrosomonas were dominant. Sum up, the long-term rice straw application has caused a remarkable impact on the diversity and community structure on Nitrosobacteria.