【作者】 亓苏伟；

【导师】 杨苏声；

【作者基本信息】 中国农业大学 ， 微生物学， 2004， 博士

【摘要】 费氏中华根瘤菌(Sinorhizobium fredii)RT19是分白天津盐碱地的快生大豆根瘤菌，能在含0.6mol／LNaCl的TY培养基上生长。本文采用高分辨率的双向电泳技术，得到不同生长时期的蛋白表达谱，应用高效的ImageMaster2D软件进行图谱分析，结果显示：(1)从延滞期到稳定期的末期，所表达的蛋白数量由398个逐渐增加至516个，显示了细胞内的蛋白在不同生长期的动态表达水平。(2)不同生长期的RT19的蛋白表达数量和种类约有80％是相同的，证实RT19在正常的生理过程中大部分蛋白的表达是稳定的，即为持家蛋白(如A73)。采用高通量的质谱技术和生物信息学，分析了持家蛋白A73的一级结构和功能，初步鉴定为DNA修复蛋白RecN，对于RT19正常的遗传重组和重组修复都是必不可少的。 采用差异显示蛋白质组学方法，对对数生长期的RT19(培养20小时)在无盐和盐激5min、50min条件下的蛋白表达谱进行研究。结果表明，在无盐时，该菌株呈现的蛋白数目是481个。随盐激时间的延长，其蛋白质数目减少，分别是465个、424个蛋白质。RT19在盐激后，有82个差异蛋白出现，其中诱导表达的蛋白有26个，阻抑表达的蛋白有23个，表达量上调的蛋白有12个，表达量下调的蛋白点21个。而且不同的盐激时间还出现不同的差异蛋白，说明其细胞内存在复杂的应答盐激的蛋白质网络调控。应用MALDI-TOF／MS对26个盐激诱导蛋白进行分析，将获得的肽指纹图谱经数据库检索，初步确定了20个诱导蛋白的功能，其中包括与盐胁迫、γ-丁酰甜菜碱、脂多糖合成、代谢途径和信号传导等有关的酶。 为了探讨RT19抵御盐激和长期盐胁迫所需的整体调节因子的异同，应用差异显示蛋白质组学方法，分别对对数生长末期的RT19(培养28小时)在盐激和长期盐胁迫下的蛋白表达谱进行分析、研究。结果显示：RT19在长期盐胁迫下，有86个差异蛋白点出现，其中诱导表达的蛋白有25个，阻抑表达的蛋白有34个，表达量上调的蛋白有5个，表达量下调的蛋白点22个。应用MALDI-TOF／MS对30个盐胁迫蛋白进行分析，已初步确定17个诱导蛋白的功能，其中包括与盐胁迫相关的热激蛋白(GroES、Clp)、与代谢途径和信号传导等有关的酶，以及ABC转运蛋白、转录调节蛋白、RNA聚合酶的β亚基等。RT19在盐激后，会有84个差异蛋白出现，其中诱导表达的蛋白有25个，阻抑表达的蛋白有12个，表达量上调的蛋白有29个，表达量下调的蛋白点18个。而且不同的盐激时间还出现不同的差异蛋白。应用MALDI-TOF／MS分析，已初步确定25个诱导蛋白的功能，其中包括亲和溶质脯氨酸合成必需的酶、ABC转运蛋白、热激蛋白(Hsp60)、DnaK及蛋白合成的伸长因子EF-Tu、与代谢途径、信号传导和电子传递系统有关的酶。通过对RT19的盐激和盐胁迫诱导蛋白的比较分析，发现：其中12个蛋白在两种不同的胁迫条件下都可被诱导表达。进一步证实：RT19应答不同盐胁迫刺激采取了不同的反应机制，但这些机制存在共性，交织成复杂的调控网络。 根据差异蛋白质组学的分析和鉴定，发现RT19在盐激和盐胁迫条件下，参与脂多糖的核心成分L-丙三醇-D-甘露庚糖生物合成的两个关键的酶：腺酐二磷酸-L-丙三醇-D-甘露庚糖-6-变位酶和甘露糖转移酶，分别被不程度地诱导。采用SDS-PAGE电泳，对RT19在不同盐激和盐胁迫条件下的脂多糖进行分离，发现RT19的脂多糖的组成发生了显著的变化，并且，盐激导致的脂多糖组成的变化比盐胁迫明显。差异蛋白质组学的分析结合代谢物的研究是探讨RT19耐盐机制的新尝试。更多还原

【Abstract】 Sinorhizobium fredii RT19 is a fast-growing soybea rhizobium strain, which was isolated from saline-alkali region in Tianjin and can normally grow on TY medium containing 0.6 mol/ L NaCl. In this study, the profiles of proteins expesssion in different growth phases of RT19 have been obtained using the technique of proteome analysis with two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). An efficient ImageMaster 2D was used to reveal the number of protein spots corresponding from lag phase to stationary phase, varying from 398 to 516, which manifested kinetic state of proteome in cell. Primary strucure and identification of a home-keeping protein (A73) were obtained by MALDI-TOF/MS and databases. Protein A73 is a DNA repair protein, named RecN, which is indispensable protein in genetic recombination and recombination repair.The protein expression profiles of RT19 growing in exponential phase after being treated with no NaCl and lmol/L NaCl for 5 min and 50 min were analyzed by differentially displayed proteome. The results showed that the number of the resolved proteins were 481, 465 and 424 respectively, which were separated by 2-D PAGE, showing that the amount of the proteins were decreased with the extension of salt shock treatment. All together eighty-two proteins were differentially displayed in response to salt-shock. Among them, 26 proteins were induced while 23 ones were inhibited; 12 ones were up-regulated and 21 ones down-regulated. In addition, the appearance of differential displayed proteins responding to different salt shock periods was also demonstrated, illuminating that complex network of regulation exist in cell. Twenty-six induced proteins were analyzed by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and identified by database searching. Among them, 20 ones were assigned and their functions were known, including salt stress proteins, or enzymes are related to salt stress, y-butyrobetaine and Iipopolysaccharide synthesis, metabolism pathway and signal transduction.In order to study the similarities and differences between long-term salt stress and salt shock of RT19, the differential proteomic approach was used to analyzed the protein expression profiles. The results showed that eighty-six proteins were differentially displayed in response to long-term salt stress, among them , 34 ones were inhibited and 22 ones down-regulated, and 5 ones up-regulated. About 25 salt-induced proteins were identified by MALDI-TOF/MS, and finally the function of 17 induced proteins were determined, including heat-shock proteins (GroES, Clp). ABC transporter, RNA Polymerase (P-subunit) and enzymes involved in signal transduction and metabolism. Meanwhile, 84 proteins were idifferentially expressed after salt shock 5 min and 50 min in late exponential phase. Among them, 29 proteins are up-regulated and 18 ones down-regulated, while 12 proteins are inhibited. 25 induced proteins were identified by means of MALDI-TOF/MS. The results showed that these proteins were closely related to heat-shock proteins (DnaK, Hsp60). ABC transporter, the elongation factors (EF-Tu) and the other enzymes involved electron transduction and metabolism. Finally, comparing long-termed salt stress with transient salt shock, there are 12 proteins induced in common,which means different stress regulatory mechanisms are inextricably linked to each other.According to the analysis and identification of differential displayed proteome, two key enzymes, ADP-L-glycerol-D- mannoheptose-6- epimerase and mannosyltransferase, were induced in different salt stress and salt shock for RT19, which commonly participate in the biosynthesis of L-glycerol-D-manno-heptose, a major component of lipopolysaccharide. By means of SDS-PAGE,the results showed that the constructure of lipopolysaccharide changed in different salt stress and salt shock. It is inferred that the structural change is required due to the adaptation of bacteria to environmental stress.更多还原