【作者】 滕齐辉； 曹慧； 孙波； 李顺鹏； 崔中利；

【Author】 Teng Qihui~1 Cao Hui~1 Sun Bo~2 Li Shunpeng~1 Cui Zhongli~1 1 Key Laboratory of Microbiological Engineering of agricultural environment, MOA, Nanjing Agricultural University, Nanjing 210095; 2 Institute of Soil Science,Chinese Academy of Sciences,Nanjing 210008

【机构】 南京农业大学农业部农业环境微生物工程重点开放实验室； 中国科学院南京土壤研究所；

【摘要】 固氮酶基因 nifH 广泛存在于固氮微生物体中,是研究固氮微生物群落结构的良好标记。采用 PCR-RFLP 的未培养方法, 扩增固氮微生物中固氮酶的保守基因 nifH,以酶切分型与基因序列测定揭示不同有机肥施用量对固氮微生物多样性的影响。结果表明,施加低量有机肥(LM)的红壤中固氮酶基因 nifH 酶切类型数量最多,达13种类型,其次为对照土壤(CK),酶切类型数量为9种,高量有机肥(HM)和高量有机肥混施石灰(ML)酶切类型数量较少,仅有7种和6种;低量有机肥施用的土壤与对照、高量有机肥和高量有机肥混施石灰处理土壤中的固氮酶基因 nifH 均有一些共有类群;测序结果表明,高量有机肥处理土壤中的固氮微生物种类主要是光能自养、好氧性的自生固氮菌—念珠蓝细菌,序列相似性为96%,约占克隆数量的90%。在对照土壤中有64%类群与共生固氮菌—鱼腥蓝细菌属相似性为94%,在对照土壤和低量有机肥的土壤中分别发现了10%和20%类型与固氮根瘤菌或伯克氏菌群相似性90%左右。该结果揭示出在红壤生态系统中投加适量有机肥有利于增加固氮微生物的多样性,但过量有机肥施用反而造成红壤生态系统中的固氮微生物多样性减少。这对改良红壤生态系统微生物群落结构、提高红壤肥力、指导红壤生态系统下的农业生产具有新的启示。更多还原

【Abstract】 All nitrogen-fixing organisms have the nifH gene encoding nitrogenase reductase in their genome, and this gene is a good molecular marker for researching population construction of nitrogen-fixing bacteria. Adopting culture-independent method, the extracted DNA was amplified by using specific primers.PCR amplifications were ligated into the pMD 18-T vector and transformed into E.coli DH 10B, thus the nifH gene library’ was constructed. 161 clones from the library were screened and its nifH gene fragments were re-amplified. These PCR products were digested by the enzymes Rsal and Hhal respectively and their fingerprints on agarose gels were analyzed. The results indicated that the numbers of nifH gene type in soil applicating low organic manure (LM) was the most, have 13 restriction endonuclease types. The second in control soil (CK), have 9 gene types. While in soils applicating high organic manure (HM) and high organic manure mixed with lime (ML), the numbers of nifH gene type are only 7 and 6 types respectively. There are some common nitrogen-fixing genotypes between LM and CK, HM, ML treatment soil.The nifH gene sequencing showed that the primary, nitrogen-fixing bacteria in HM treatment soil, is Anabaena, this gene is identical to that of Anabaena at 96% level, and its clones number account for 90% to the total clones. In control treatment soil, the gene sequencing of 64% clones is identical to that of Cyanobacteria at 94% level. The nifH gene similarity in CK and LM soils to Rhizobia or Burkholderia at 90% level were found, its clone numbers account for 10% and 20% respectively.更多还原