固氮斯氏假单胞菌A1501泌铵突变株的构建及其理化特性
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摘要
联合固氮菌与宿主根系形成松散的结合方式,所固定的氮主要供自身生长所需,只能为植物供给有限的氮素。因此,探索联合固氮菌的泌铵机制,提高其固氮和泌铵能力,在理论和实际应用上有着重要意义。本研究在完成了斯氏假单胞菌A1501全基因组测序和注释信息,以及开展联合固氮菌固氮基因调控表达的研究基础上,构建了具有能高效固氮的泌铵工程菌,并进行了生理生化特性分析。
根据斯氏假单胞菌A1501的基因组序列,克隆了固氮酶正调节基因(nifA),将含有完整的nifA的DNA片段连接到pVK100,构建了nifA组成型表达质粒pVA3,通过三亲结合的方法将pVA3转化进铵转运载体蛋白AmtB1AmtB2双缺失突变株(1561)和野生型菌株中,经卡那霉素(Km)和四环素(Tc)抗性筛选,分别获得1561/pVA3和A1501/pVA3重组菌。研究结果表明增强nifA基因的表达提高了固氮条件下斯氏假单胞菌的固氮酶活,而且在一定铵浓度(10mM)下也能保持固氮能力(1.53U/mg·protein)。但是,未观测到A1501/pVA3培养物中铵离子的存在。尽管铵转运载体缺失突变株(1561)在固氮条件下的固氮酶活略高于野生型菌株,但无显著性差异,在10mM铵浓度下均未测定到固氮酶活。然而,在以硝酸盐为唯一氮源时,A1501培养液中测定不到铵离子的存在,而菌株1561在含8mM,12mM硝酸盐的培养基中经33h培养后,培养液中的铵浓度分别达到0.60mM和1.32mM。结果表明硝酸盐进入细胞后转化为铵,经自由扩散到胞外,当铵转运载体蛋白缺失时,不能迅速将铵吸收到胞内,从而导致胞外含有铵离子。
nifA组成型表达且铵转运载体蛋白缺失的斯氏假单胞菌突变株(1561/pVA3)在固氮条件下,其固氮酶活(14.24U/mg·protein)是野生型的1.95倍,在10mM铵浓度下仍然保持一定的固氮酶活(4.24U/mg·protein),高于A1501/pVA3的酶活。在无氮的半固体限制性培养基中培养15天后,培养基呈弱碱性(pH=7.42),测定培养基的铵含量达5.15 mM。研究结果表明1561/pVA3具有较强的固氮能力,并且能向胞外泌铵。
1561/pVA3和野生型菌株的水稻接种实验显示,接种工程菌的水稻株高(19.4 cm)、地上、地下部分干重(12.85mg和2.96mg)等指标都要高于接种野生型A1501的水稻(14.8cm,11.70mg和2.11mg)。根表竞争实验表明1561/pVA3在水稻根际的定殖和根际竞争能力上优于野生型。本研究结果表明工程菌对水稻具有较好的促生作用。
上述结果显示1561/pVA3菌株是具有较强固氮能力和具有泌铵能力的工程菌。为进一步探讨1561/pVA3的泌铵作用,我们对工程菌及野生型菌株进行了铵冲击试验,铵离子、硝酸盐、氨基酸含量和GS酶活的测定,以及固氮和非固氮条件下固氮基因转录表达测定,结果表明1) nifA的组成型质粒pVA3增强了nifA基因的表达效率,从转录水平上增强了固氮基因的表达;2)铵转运载体基因amtB1amtB2的缺失使得1561/pVA3失去了向细胞内部主动运输铵的能力,使固氮合成的铵通过自由扩散在胞外积累;3) AmtB1AmtB2可能参与了固氮酶的后修饰和调控固氮基因的表达,并在蛋白的翻译水平上增强了固氮酶的合成效率。
Associative nitrogen-fixing bacteria can colonize on rhizoplane of the host crops for growth and nitrogen fixation. Associative nitrogen-fixing bacteria could supply host plants with nitrogen, but the supply is not sufficient. Pseudomonas stutzeri A1501, isolated from rhizosphere of rice, is an associative nitrogen-fixing bacterium. Recently, the genome sequence and annotation of A1501 have been completed. On the basis of the genetic information and the previous study on expression and regulation of nitrogen fixation systems in P. stutzeri A1501, we construct, identify and characterize an ammonium-excreting strain by improving the ability of nitrogen fixation of P. stutzeri A1501.
The complete nifA gene amplified from A1501 genomic DNA was ligated into the plasmid pVK100, which encodes the key activator NifA of nif gene expression, and then the resulting plasmid, pVA3, was transformed into the wild type strain A1501 and the ammonium transporter AmtBl/AmtB2 deficient mutant (1561) by tri-parental conjugation, to generate the strain A1501/pVA3 and 1561/pVA3, respectively. The data showed that the overexpression of nifA gene enhanced the nitrogenase activity (7.3U/mg·protein) in P. stutzeri under nitrogen fixation conditions and remained nitrogenase activity (1.53U/mg·protein) under non-nitrogen-fixing conditions. But no ammonium was detected in the culture of A1501 or 1501/pVA3; no significant differences of growth rate and nitrogenase activity were observed between the strain 1561 and the wild type strain. However, when the strain 1561 was incubated in the N-free medium containing 8 or 12 mM NO_3~- for 33h, the ammoniumn (0.60 or 1.32mM) in the extracellular milieu was detected, suggesting that disruption of ammonium transporter AmtB1/AmtB2 caused the cells leakage of ammoniumn.
The nitrogenase activity of the strain 1561/pVA3 was about as 1.95-fold as that of the wild type strain under nitrogen fixation conditions, and maintained 30% of nitrogenase activity in presence of 10mM ammonium, which was higher than that of the strain 1501/pVA3. After 15-day incubation in the N-free medium, the medium pH became slightly alkaline, and also the ammoniumn concentration was as high as 5.15 mM. The results showed that the overexpression of nifA and disruption of ammonium transporter could make nitrogenase activity enhanced and amount of ammonium released into the medium.
Rice plant was inoculated with strain 1561/pVA3. The plant height (19.4 cm), dry weights of shoot and root (12.85mg and 2.96mg) of the rice plant treated with 1561/pVA3 were significnatly higher than those of the wild type strain (14.8cm, 11.70mg and 2.11mg). 1561/pVA3 showed a strongly enhanced competitive root-colonizing ability compared with wild type strain. These results indicated that 1561/pVA3 strain obviously improved rice plant growth.
To further understand the ammonium-excretion of the strain 1561/pVA3, ammonium shock experiments were performed, and the concentrations of ammonium, nitrate, amino acid and GS activities were measured. These results indicated that 1) constitutive plasmid of nifA, pVA3, can enhance the expression of nif A and nifH; 2) disruption of AmtB1/B2 deprives function of transporting ammonium, leads to ammonium release into extracellular culture; 3) AmtB1AmtB2 might be involved in post-translational modification of nitrogenase and control of nif gene expression.
根据斯氏假单胞菌A1501的基因组序列,克隆了固氮酶正调节基因(nifA),将含有完整的nifA的DNA片段连接到pVK100,构建了nifA组成型表达质粒pVA3,通过三亲结合的方法将pVA3转化进铵转运载体蛋白AmtB1AmtB2双缺失突变株(1561)和野生型菌株中,经卡那霉素(Km)和四环素(Tc)抗性筛选,分别获得1561/pVA3和A1501/pVA3重组菌。研究结果表明增强nifA基因的表达提高了固氮条件下斯氏假单胞菌的固氮酶活,而且在一定铵浓度(10mM)下也能保持固氮能力(1.53U/mg·protein)。但是,未观测到A1501/pVA3培养物中铵离子的存在。尽管铵转运载体缺失突变株(1561)在固氮条件下的固氮酶活略高于野生型菌株,但无显著性差异,在10mM铵浓度下均未测定到固氮酶活。然而,在以硝酸盐为唯一氮源时,A1501培养液中测定不到铵离子的存在,而菌株1561在含8mM,12mM硝酸盐的培养基中经33h培养后,培养液中的铵浓度分别达到0.60mM和1.32mM。结果表明硝酸盐进入细胞后转化为铵,经自由扩散到胞外,当铵转运载体蛋白缺失时,不能迅速将铵吸收到胞内,从而导致胞外含有铵离子。
nifA组成型表达且铵转运载体蛋白缺失的斯氏假单胞菌突变株(1561/pVA3)在固氮条件下,其固氮酶活(14.24U/mg·protein)是野生型的1.95倍,在10mM铵浓度下仍然保持一定的固氮酶活(4.24U/mg·protein),高于A1501/pVA3的酶活。在无氮的半固体限制性培养基中培养15天后,培养基呈弱碱性(pH=7.42),测定培养基的铵含量达5.15 mM。研究结果表明1561/pVA3具有较强的固氮能力,并且能向胞外泌铵。
1561/pVA3和野生型菌株的水稻接种实验显示,接种工程菌的水稻株高(19.4 cm)、地上、地下部分干重(12.85mg和2.96mg)等指标都要高于接种野生型A1501的水稻(14.8cm,11.70mg和2.11mg)。根表竞争实验表明1561/pVA3在水稻根际的定殖和根际竞争能力上优于野生型。本研究结果表明工程菌对水稻具有较好的促生作用。
上述结果显示1561/pVA3菌株是具有较强固氮能力和具有泌铵能力的工程菌。为进一步探讨1561/pVA3的泌铵作用,我们对工程菌及野生型菌株进行了铵冲击试验,铵离子、硝酸盐、氨基酸含量和GS酶活的测定,以及固氮和非固氮条件下固氮基因转录表达测定,结果表明1) nifA的组成型质粒pVA3增强了nifA基因的表达效率,从转录水平上增强了固氮基因的表达;2)铵转运载体基因amtB1amtB2的缺失使得1561/pVA3失去了向细胞内部主动运输铵的能力,使固氮合成的铵通过自由扩散在胞外积累;3) AmtB1AmtB2可能参与了固氮酶的后修饰和调控固氮基因的表达,并在蛋白的翻译水平上增强了固氮酶的合成效率。
Associative nitrogen-fixing bacteria can colonize on rhizoplane of the host crops for growth and nitrogen fixation. Associative nitrogen-fixing bacteria could supply host plants with nitrogen, but the supply is not sufficient. Pseudomonas stutzeri A1501, isolated from rhizosphere of rice, is an associative nitrogen-fixing bacterium. Recently, the genome sequence and annotation of A1501 have been completed. On the basis of the genetic information and the previous study on expression and regulation of nitrogen fixation systems in P. stutzeri A1501, we construct, identify and characterize an ammonium-excreting strain by improving the ability of nitrogen fixation of P. stutzeri A1501.
The complete nifA gene amplified from A1501 genomic DNA was ligated into the plasmid pVK100, which encodes the key activator NifA of nif gene expression, and then the resulting plasmid, pVA3, was transformed into the wild type strain A1501 and the ammonium transporter AmtBl/AmtB2 deficient mutant (1561) by tri-parental conjugation, to generate the strain A1501/pVA3 and 1561/pVA3, respectively. The data showed that the overexpression of nifA gene enhanced the nitrogenase activity (7.3U/mg·protein) in P. stutzeri under nitrogen fixation conditions and remained nitrogenase activity (1.53U/mg·protein) under non-nitrogen-fixing conditions. But no ammonium was detected in the culture of A1501 or 1501/pVA3; no significant differences of growth rate and nitrogenase activity were observed between the strain 1561 and the wild type strain. However, when the strain 1561 was incubated in the N-free medium containing 8 or 12 mM NO_3~- for 33h, the ammoniumn (0.60 or 1.32mM) in the extracellular milieu was detected, suggesting that disruption of ammonium transporter AmtB1/AmtB2 caused the cells leakage of ammoniumn.
The nitrogenase activity of the strain 1561/pVA3 was about as 1.95-fold as that of the wild type strain under nitrogen fixation conditions, and maintained 30% of nitrogenase activity in presence of 10mM ammonium, which was higher than that of the strain 1501/pVA3. After 15-day incubation in the N-free medium, the medium pH became slightly alkaline, and also the ammoniumn concentration was as high as 5.15 mM. The results showed that the overexpression of nifA and disruption of ammonium transporter could make nitrogenase activity enhanced and amount of ammonium released into the medium.
Rice plant was inoculated with strain 1561/pVA3. The plant height (19.4 cm), dry weights of shoot and root (12.85mg and 2.96mg) of the rice plant treated with 1561/pVA3 were significnatly higher than those of the wild type strain (14.8cm, 11.70mg and 2.11mg). 1561/pVA3 showed a strongly enhanced competitive root-colonizing ability compared with wild type strain. These results indicated that 1561/pVA3 strain obviously improved rice plant growth.
To further understand the ammonium-excretion of the strain 1561/pVA3, ammonium shock experiments were performed, and the concentrations of ammonium, nitrate, amino acid and GS activities were measured. These results indicated that 1) constitutive plasmid of nifA, pVA3, can enhance the expression of nif A and nifH; 2) disruption of AmtB1/B2 deprives function of transporting ammonium, leads to ammonium release into extracellular culture; 3) AmtB1AmtB2 might be involved in post-translational modification of nitrogenase and control of nif gene expression.
引文
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