本文選題：固氮類芽孢桿菌 + 基因組��； 參考：《中國(guó)農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：固氮類芽孢桿菌是一類革蘭氏陽(yáng)性、產(chǎn)芽孢細(xì)菌,在農(nóng)業(yè)生產(chǎn)中具有潛在的應(yīng)用價(jià)值。開(kāi)展對(duì)固氮類芽孢桿菌的基因組學(xué)研究及發(fā)掘其菌種資源,具有重要的學(xué)術(shù)價(jià)值和現(xiàn)實(shí)意義。本文首先完成了Paenibacillus sabinae T27的全基因組序列分析,并用生物信息學(xué)分析發(fā)現(xiàn),該菌的染色體上含有了一個(gè)約11 kb、由10個(gè)基因(nifB nifH nifD nifK nijE nifE nifX hesA orf1 nifV)組成的固氮基因簇。此外,還含有多個(gè)nif(固氮)基因(3個(gè)nifB基因,2個(gè)nijH基因,1個(gè)nijE基因,1個(gè)nifN基因)和多個(gè)nif-like基因(2個(gè)nifH-like基因,5個(gè)nifD-like基因及5個(gè)nifK-like基因)。qRT-PCR研究表明,nifHDK基因只在固氮條件(無(wú)氧和無(wú)銨)下表達(dá),在非固氮條件(有銨和有氧)下不表達(dá)；功能互補(bǔ)試驗(yàn)表明,nifHD基因具有固氮功能,而nif-like基因并沒(méi)有固氮功能。大腸桿菌78-7是攜帶Paenibacillus固氮基因簇(nifB nifHnifD nifK nifE nifN nifXhesA nifV)的重組菌株,其固氮酶活性只有原宿主Paenibacillus polymyxa WLY78的10%。為了提高固氮酶在異源宿主的活性,從Paenibacillus polymyxa WLY78和產(chǎn)酸克氏桿菌(Klebsiella oxytocd)中選擇了28個(gè)基因,并將這些基因與Paenibacillus nif啟動(dòng)子組合后,導(dǎo)入到重組固氮大腸桿菌78-7中。結(jié)果表明,Paenibacillus polymyxa WLY78的sufCBSUD基因簇(編碼鐵硫簇合成)和固氮酶電子傳遞因而pfoAB, fldA1及fer能夠提高固氮酶活性；發(fā)現(xiàn)K. oxytoca中的nifSU(鐵硫簇合成)及nifFJ(固氮酶電子傳遞)也能夠提高固氮酶活性。進(jìn)一步將Paenibacillus中的pfoABfldA與K. oxytoca中的nifSU基因共同導(dǎo)入到重組固氮大腸桿菌78-7,固氮酶活性由原來(lái)的10%提高到50.1%。發(fā)現(xiàn)K. oxytoca中的nifWZM(參與固氮酶FeMo-cofactor合成)和nifQ(固氮酶合成過(guò)程中負(fù)責(zé)鉬的運(yùn)輸)并不能提高固氮酶活性。并通過(guò)基因突變分析表明,pfoAB和fldA在Paenibacillus固氮作用中起電子傳遞作用。此外,還探索性地研究了固氮基因簇在真核酵母中的表達(dá)。
[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.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q933
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本文編號(hào)：1841529