固氮类芽孢杆菌基因组分析、固氮基因功能及固氮酶异源表达研究

发布时间：2018-05-04 04:12

  本文选题：固氮类芽孢杆菌 + 基因组　； 参考：《中国农业大学》2016年博士论文

【摘要】：固氮类芽孢杆菌是一类革兰氏阳性、产芽孢细菌,在农业生产中具有潜在的应用价值。开展对固氮类芽孢杆菌的基因组学研究及发掘其菌种资源,具有重要的学术价值和现实意义。本文首先完成了Paenibacillus sabinae T27的全基因组序列分析,并用生物信息学分析发现,该菌的染色体上含有了一个约11 kb、由10个基因(nifB nifH nifD nifK nijE nifE nifX hesA orf1 nifV)组成的固氮基因簇。此外,还含有多个nif(固氮)基因(3个nifB基因,2个nijH基因,1个nijE基因,1个nifN基因)和多个nif-like基因(2个nifH-like基因,5个nifD-like基因及5个nifK-like基因)。qRT-PCR研究表明,nifHDK基因只在固氮条件(无氧和无铵)下表达,在非固氮条件(有铵和有氧)下不表达；功能互补试验表明,nifHD基因具有固氮功能,而nif-like基因并没有固氮功能。大肠杆菌78-7是携带Paenibacillus固氮基因簇(nifB nifHnifD nifK nifE nifN nifXhesA nifV)的重组菌株,其固氮酶活性只有原宿主Paenibacillus polymyxa WLY78的10%。为了提高固氮酶在异源宿主的活性,从Paenibacillus polymyxa WLY78和产酸克氏杆菌(Klebsiella oxytocd)中选择了28个基因,并将这些基因与Paenibacillus nif启动子组合后,导入到重组固氮大肠杆菌78-7中。结果表明,Paenibacillus polymyxa WLY78的sufCBSUD基因簇(编码铁硫簇合成)和固氮酶电子传递因而pfoAB, fldA1及fer能够提高固氮酶活性；发现K. oxytoca中的nifSU(铁硫簇合成)及nifFJ(固氮酶电子传递)也能够提高固氮酶活性。进一步将Paenibacillus中的pfoABfldA与K. oxytoca中的nifSU基因共同导入到重组固氮大肠杆菌78-7,固氮酶活性由原来的10%提高到50.1%。发现K. oxytoca中的nifWZM(参与固氮酶FeMo-cofactor合成)和nifQ(固氮酶合成过程中负责钼的运输)并不能提高固氮酶活性。并通过基因突变分析表明,pfoAB和fldA在Paenibacillus固氮作用中起电子传递作用。此外,还探索性地研究了固氮基因簇在真核酵母中的表达。
[Abstract]:Nitrogen-fixing Bacillus is a kind of Gram-positive bacteria, which has potential application value in agricultural production. It is of great academic value and practical significance to study the genomics of nitrogen-fixing Bacillus and to explore its resources. In this paper, the whole genome sequence analysis of Paenibacillus sabinae T27 was carried out. By bioinformatics analysis, it was found that the chromosome of Paenibacillus sabinae T27 contained a nitrogen-fixing gene cluster of about 11 kb, composed of 10 genes, nifB nifH nifD nifK nijE nifE nifX hesA orf1 nifV. In addition, There were also several niff genes (3 nifB genes, 2 nijH genes, 1 nijE gene, 1 nifN gene) and multiple nif-like genes (2 nifH-like genes, 5 nifD-like genes and 5 nifK-like genes). Oxygen and ammonium free, No expression was found in non-nitrogen-fixing condition (ammonium and aerobic), and functional complementation test showed that the NIF HD gene had nitrogen-fixing function, while nif-like gene had no nitrogen-fixing function. Escherichia coli 78-7 is a recombinant strain carrying Paenibacillus nitrogen-fixing gene cluster nifB nifHnifD nifK nifE nifN nifXhesA nifV). Its nitrogenase activity is only 10% of that of the original host Paenibacillus polymyxa WLY78. In order to improve the activity of nitrogenase in heterologous hosts, 28 genes were selected from Paenibacillus polymyxa WLY78 and Klebsiella oxytocd. These genes were combined with Paenibacillus nif promoter and then transferred into recombinant nitrogen-fixing Escherichia coli 78-7. The results showed that the sufCBSUD gene cluster of Paenibacillus polymyxa WLY78 (encoding iron-sulfur cluster synthesis) and electron transport of nitrogenase could increase the activity of nitrogenase. It was found that nifSUand nifFJ (electron transport of nitrogenase) in K. oxytoca could also increase the activity of nitrogenase. The pfoABfldA gene in Paenibacillus and nifSU gene in K. oxytoca were further introduced into recombinant nitrogen-fixing Escherichia coli 78-7, and the activity of nitrogenase increased from 10% to 50.1%. It was found that nifWZM (involved in the synthesis of nitrogenase FeMo-cofactor) and nifQ (responsible for the transport of molybdenum in the synthesis of nitrogenase) in K. oxytoca could not increase the activity of nitrogenase. The gene mutation analysis showed that pfoAB and fldA played an important role in the electron transport of Paenibacillus nitrogen fixation. In addition, the expression of nitrogen-fixing gene cluster in eukaryotic yeast was studied.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q933
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本文编号：1841529