Analysis Of NifH、amoA、nirK、nosZ Gene Diversity In A Red Paddy Soil Under Long-Term Fertilization

The problem of red soil degeneration in South China had severely limited the sustainable development of agricultural economy. We can evaluate the soil quality from three aspects:physics indicators、 chemical indicators and biological indicators. But now we lack the data about biological indicators on soil quality. Microbial index is an important index of biological indicators. With regard to such research background, we can know the renew of red soil using microbial indicators and the impact of soil microbial community structure under long-term cultivation of rice and fertilizer treatments.We can bypass the difficulties of microbial isolation and culture using these molecular biological techniques. In addition, nitrogen is the key factor in agricultural production and is an important nutrient to plant. Many soil microbes, just like nitrogen-fixing microorganisms, ammonia-oxidizing bacteria and denitrifying bacteria, play a decisive role in the process of nitrogen cycle. The subject want to analyse the impact of nitrogen-fixing microorganisms, ammonia-oxidizing bacteria and denitrifying bacteria under different fertilizer treatment using Restriction Fragment Length Polymorphism (RFLP) methods.We obtained the following results based on this experimental methods. For the nifH gene of nitrogen-fixing microorganisms, we obtained240clones and75RFLP in the four gene library,70positive clones and28different RFLP band types in nifH-CK library;58positive clones and31different RFLP band types in nifH-N library;64positive clones and27different RFLP band types in nifH-NC library;48positive clones and22different RFLP band types in nifH-NPK library. These four libraries have their own dominant RFLP band type, and some RFLP band types are same among the four libraries. The diversity indexes of nitrogen-fixing microorganisms are reduced in the order of N sample, CK sample, NC sample, NPK sample. According to the sequencing results, most clones have a high similarity with the known nitrogen-fixing microorganisms in Proteobacteria.For the amoA gene of ammonia-oxidizing bacteria, we obtained238clones and32RFLP in the four gene library,48positive clones and10different RFLP band types in amoA-CK library;69positive clones and11different RFLP band types in amoA-N library; 57positive clones and16different RFLP band types in amoA-NC library;64positive clones and14different RFLP band types in amoA-NPK library. These four libraries have their own dominant RFLP band type, and these dominant RFLP band types of the four libraries are same. The diversity indexes of ammonia-oxidizing bacteria are reduced in the order of NC sample, NPK sample, N sample, CK sample. According to the sequencing results, besides most clones have a high similarity with unknown microorganisms, some sequences master with Nitrosospira sp..We studied two functional gene(nirK、nosZ) of denitrifying bacteria. For the nirK gene we obtained298clones and24RFLP in the four gene library,74positive clones and14different RFLP band types in nirK-CK library;74positive clones and6different RFLP band types in nirK-N library;75positive clones and8different RFLP band types in nirK-NC library;75positive clones and4different RFLP band types in nirK-NPK library. For the nosZ gene we obtained284clones and76RFLP in the four gene library,75positive clones and19different RFLP band types in nosZ-CK library;61positive clones and32different RFLP band types in nosZ-N library;81positive clones and33different RFLP band types in nosZ-NC library;67positive clones and29different RFLP band types in nosZ-NPK library. These libraries of denitrifying bacteria also have their own dominant RFLP band type, and some RFLP band types are same. For example, the dominant RFLP band types of nirK gene libraries are same. For nirK gene library, the diversity indexes of denitrifying bacteria are reduced in the order of CK sample, NC sample, N sample, NPK sample. While for nosL gene library, the diversity indexes of denitrifying bacteria are reduced in the order of N sample, NC sample, NPK sample, CK sample. A total of13nucleotide sequences of nirK clones and17nucleotide sequences of nosZ clones were sequenced. According to the sequencing results, for nirK gene, most clones have high similarity with α-、β-proteobacteria. While for nosZ gene, besides most clones have a high similarity with unknown microorganisms, some sequences have high similarity with the known denitrifying bacteria in α-、β-、γ-proteobacteria.Nitrogen fertilization maybe can maintain the diversity of nitrogen-fixing bacteria and nitrogen and straw fertilization maybe can maintain the diversity of ammonia-oxidizing bacteria in red paddy soil. While the impact of fertilizer on denitrifying bacteria need further research.