【作者】 解志红；

【导师】 林敏； 王国英；

【作者基本信息】 中国农业大学 ， 遗传学， 2005， 博士

【摘要】 斯氏假单胞菌（Pseudomonas stutzeri）A1501分离自非豆科作物水稻根际,在无铵和微好氧的条件下能将空气中氮气固定为植物可以吸收利用的铵,是一种潜在的微生物肥料,具有重要的理论研究和实际应用价值。本研究采用基因敲除、酵母双杂交系统及蛋白体外结合技术,对斯氏假单胞菌A1501固氮调节基因nifLA进行了功能鉴定和表达调控机制的研究。 构建了斯氏假单胞菌A1501的粘粒基因组文库,筛选到两个阳性克隆,分别为XL1,XL2。然后以pUC19为载体,从中筛选到7.7Kb包含nifLA-like的阳性克隆,并完成了核苷酸序列分析（EMBL/GenBank登陆号为:AJ297529）。遗传学分析表明该菌nifL-like及nifA-like与棕色固氮菌（A.vinelandii）在核苷酸序列上的同源性分别为78%、85%,在氨基酸序列上的同源性分别为72%、82%,暂分别命名为nifA及nifL。利用Southern杂交证实nifL在P.stutzeri A1501中为单拷贝。 将nifHp-lacZ融合基因表达载体转入A1501野生性菌株中,在不同的铵和氧浓度诱导后测定其β—半乳糖苷酶活性,结果表明斯氏假单胞菌A1501的固氮基因表达受铵和氧的调控。为了研究nifLA的固氮调节功能,构建了nifL及nifA突变株及其互补质粒。结果显示nifA突变株及nifL极性突变株的固氮酶活性丧失;nifL非极性突变株仍有部分固氮活性,且对铵和氧不敏感,在高铵下,仍有一定的固氮活性。nifA的组成性表达质粒pVA3仅恢复nifA突变株的三分之一的固氮活性,增强了A1501及nifL非极性突变株的固氮活性。而nifL的组成性表达质粒pVL抑制了A1501及nifL非极性突变株中固氮酶的表达。过量表达的NifA增强了nifHp-lacZ的表达,而过量表达的NifL对nifHp-lacZ的表达没有大的影响,表明在A1501中存在固氮正调节蛋白NifA和负调节因子NifL。 采用酵母双杂交系统研究NifL和NifA蛋白在表达调控中的相互作用。构建了nifL和nifA全长序列及其不同结构域区段的酵母双杂交质粒。酵母双杂交实验证明NifA的中间结构域与NifL的羧基端之间在酵母体内存在直接的相互作用。构建了pET28a-nifL及pGEX2T-nifA融合表达载体。利用体外沉降方法证实NifL与NifA在细胞外也可以结合,表明斯氏假单胞菌A1501中可能存在一个通过NifL与NifA相互作用关闭nif基因表达的调控机制。 构建了A1501 nifLAp-lacZ融合表达载体,分别将其转入A1501野生型及ropN^-和ntrC^-突变菌株中。β半乳糖苷酶活性的测定结果表明,nifLA基因的启动子为σ⁵⁴-依赖型,nifLA正常水平的表达需要σ⁵⁴因子及NtrC的参与。 在上述研究的基础上,初步提出了P.stutzeri固氮基因转录调控模式,即固氮负调节因子NifL感受外界氮和氧的信号,与固氮正调节因子NifA互作,控制nif基因系统的表达。这将为今后采用基因工程手段打破联合固氮的氧和铵限制,获得稳定高效的联合固氮效率奠定重要的理论基础。更多还原

【Abstract】 Pseudomonas stutzeri A1501 (formerly Alcaligenes faecalis A1501), is an associative nitrogen-fixing bacteria, which colonizes the rhizosphere of rice. This strain can fix nitrogen under microaerobic conditions in the free-living state. In this study, we present data on the mechanism of regulation of nitrogen fixation by NifLA in pseudomonas stutzeri A1501.A cosmid library of A1501 was constructed using SuperCosl Cosmid Vector kit, and two transformants containing nifLA-like were screened by colony PCR and Southern hybridization. The 7.7kb DNA fragment containing nifLA-like was subcloned( EMBL/GenBank: AJ297529). The sequence analysis revealed that the highest identity of 72% was observed with NifL of A.vinelandii and 82% with NifA of A.vinelandii. nifL gene is single copy in P.stutzeri A1501.P. stutzeri A1501 nifHp-lacZ fusion vector was transfered into the wild type strain A1501 by tri-parental mating assay, then 6-galactosidase activity was assayed after induced at different ammonium and oxygen concentration. The result suggested that the expression of nifH was repressed by ammonium and oxygen. In order to study the regulation function of nifLA of Pseudomonas stutzeri A1501, nifL or nifA mutants and recombinant plasmids of nifA or nifL (pVA3 and pVL) were constructed, respectively. The nifA mutant and nifL polar-mutant were Nif negative, whereas non-polar nifL mutant displayed some residual nitrogenase activity even under high ammonium and oxygen concentration. By assaying acetylene reduction activity of transconjugants, the results showed that plasmid which expressed nifA from the Km promoter restored 30% nitrogenase activity to the nifA mutant strain, and this plasmid also increased nitrogenase activity of the wild type A1501 and the nifL non-polar mutant. The plasmid which constitutively expressed nifL descreased nitrogenase activity of A1501 and the nifL non-polar mutant. Also overexpressed NifA increased expression of nifHp-lacZ and overexpressed NifL couldn’t work on nif gene expression.In order to study the interaction between NifL and NifA, full-length nifA and nifL and different regions encoding the open reading frame of nifLA in P. stutzeri A1501 were amplified by PCR with specific primers and cloned into plasmid pGAD-C(1)and pGBD-C(1). Recombinant plasmids were co-transformed into the expression host S. cerevisiae PJ69-4A by the lithium acetate method, and selected transformants were grown on SD plates lacking His/Ade, Leu, and Trp. An interaction between NifL and NifA in vivo was detected by yeast two-hybrid system, and it was shown that only the C-terminal domain of NifL displayed binding activity to NifA.Contraction of pET28a-nifA and pGEX2T-nifLc fusion expression vector were performed and transformed it into E. coli BL21(DE3) cells. Overexpression in soluble form was achieved with isopropyl β-D-thiogalactoside(IPTG). Interaction between NifA and NifL in vitro was proved using purified NifA and NifLc by binding assay. The result suggested that there existed a regulation mode that nif genes were inactivated by interaction between NifL and NifA.The nifLAp-lacZ fusion vector was constructed and transfered into the wild type strain A1501 and different mutants. The result of β-galactosidase assays suggested the nifLA promoter was dependent onNtrC and σ54, not absolutely on NifA and NifL.更多还原