我国主产区水稻内生固氮菌多样性、nifH基因信息数据库构建与固氮活跃菌群研究

Mass production and utilization of chemical nitrogen fertilizers consumes a lot of energy and resources, and also lead to serious ecological and environmental problem，hindering the sustainable development of agriculture. Endophytic nitrogen-fixing bacteria and its diversity play an important role for plant growth and nutrition supply, as long as disease resistance. Research and application of endophytic nitrogen-fixing bacteria open up a way to efficient use of biological nitrogen fixation techniques in crops. Rice has an important strategic position in China’s grain security and econony. In this project, a great quantity of endophytic nitrogen-fixing bacteria will be isolated from rice, and strain 16S rRDA and nifH sequences will be determined, a database of nifH sequences information and their corresponding strain indentication information will be established. Diversity of endophytic nitrogen-fixing bacteria and active nitrogen-fixing bacterial group in rice will be revealed by using experimental techniques of nifH high-throughput sequencing, DGGE and reverse transcript PCR, et al. Furthermore, the relationship of rice varieties and diversity of endophytic nitrogen-fixing bacteria will be explored. Implementation of this project will collect nitrogen-fixing bacterial resources, increase the undstanting of diversity of endophytic nitrogen-fixing bacteria and active nitrogen-fixing bacteria in rice, and promote diazotrophic diversity research progress and biological nitrogen fixateon utilization in rice production.

化学氮肥的大量生产使用消耗大量资源，污染生态环境，不利于农业持续发展。禾本科作物内生固氮菌的发现和研究开辟了一条通过生物固氮为农作物提供氮素营养的途径。水稻作为主要粮食作物在国民经济中起着举足轻重的作用。本项目以我国南方和北方两大主产区水稻为研究对象，大量分离、培养、保藏、鉴定水稻内生固氮菌，测定菌株nifH序列，构建“水稻内生固氮菌nifH基因信息数据库”，完善实验室现有的“固氮菌nifH基因信息数据库”；以nifH为标记，通过高通量测序、梯度电泳、RT-PCR等非培养技术研究揭示我国主产区水稻内生固氮菌多样性、优势种群、固氮活跃菌群，明确其与我国主栽水稻品种的关系；分离、鉴定水稻体内的活跃固氮和稀有(难培养)菌株，鉴定、发现新种。项目实施将获得大量水稻内生固氮菌资源、信息，建立、完善“固氮菌nifH基因信息数据库”，促进固氮微生物多样性研究领域发展，促进生物固氮技术在水稻上的应用。

化学氮肥的大量生产使用消耗大量资源，污染生态环境，不利于农业持续发展。禾本科作物内生固氮菌的发现和研究开辟了一条通过生物固氮为农作物提供氮素营养的途径。水稻作为主要粮食作物在国民经济中起着举足轻重的作用。本项目分离、鉴定了水稻等作物内生固氮菌885株，分类地位涉及40多属150余种。建立了“植物内生固氮菌nifH基因序列与对应菌株分类信息数据库”。该数据库在原有476条玉米内生固氮菌nifH基因序列的基础上，新增nifH基因序列790条，达到了1266条；覆盖物种67属303种, 是目前国际上唯一的植物内生固氮菌nifH基因数据库，对于固氮微生物资源研究具有重要意义。揭示了我国主产区水稻内生固氮菌多样性全貌、优势种群及其与水稻品种的关系。优势种群依次为Anaeromyxobacter（属）、Archangiaceae（科）、Rectinema（属）、Methylobacterium aquaticum（种）。发现了Kosakonia sacchari、Bradyrhizobium denitrificans是水稻体内处于活跃转录固氮酶nifH基因的菌群。分离、鉴定了活跃固氮菌2株和稀有固氮菌21株，发现了微生物新种8个。为作物内生固氮菌多样性研究和农业应用提供了菌株保障和理论参考。