论文部分内容阅读
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.
Taking the soil samples from long-term experiment of Taoyuan Agro-Ecological Experimental Station of Chinese Academy of Sciences as object, the effects of rice straw returning on the diversity of nitrosomonas functional genes amoA and hao were analyzed by PCR amplification, clone library construction and sequence determination molecular biology techniques. The results showed that the rice straw soil treatment (NPK + rice straw, SR) reduced the diversity of amoA and hao genes with Shannon’s index of 3.7 and 3.2 respectively, while the Shannon’s index of NPK treatments reached 4.0 and 3.7.LIBSHUFF analysis showed that the p value of the community structure between amoA and hao gene was 0.002 and 0.001, respectively, and reached extremely significant levels.The sequence analysis showed that for amoA gene only A gene similar to Nitrosospira and similar to the unknown amoA gene was detected and similar to Nitrosospira only appeared in the SR treatment with a similarity rate of over 96% and closest to the unknown amoA gene Is also known as Nitrosospira. The obtained hao genes are distributed in three classes (α, β, γ) in Proteobacteria. Among them, hao gene obtained by CK treatment mainly Similar to Silicibacter and Methylococcus, the genes obtained by SR treatment are closely related to Nitrosospira and Nitrosomonas.The phylogenetic tree analysis showed that the nitrite gene (amoA, hao) can be divided into four gene clusters in these two treatments. The rice straw returned to the field and the nitrosated bacterial community was obviously differentiated, and the phenomenon of gene clustering occurred. In amoA gene tree In the figure, only ClusterⅣ was formed by SR treatment.In general, long-term straw application reduced the diversity of nitrosated genes amoA and hao, and significantly changed the structure of nitrosated bacterial communities.