【作者】 何健；

【导师】 李顺鹏；

【作者基本信息】 南京农业大学 ， 微生物学， 2005， 博士

【摘要】 以模式菌株Pseudomonas putida KT2440为材料，研究了营养及各种环境条件对该菌株盐份渗透胁迫适应性的影响，探讨了外源添加各种相容性溶质提高KT2440对盐份渗透胁迫适应性及甜菜碱和四氢嘧啶对KT2440渗透胁迫保护机理。在此基础上对利用基因工程手段提高KT2440对盐份渗透胁迫适应性的可行性和策略进行了研究。研究结果表明(1)外源添加相容性溶质提高KT2440对盐份渗透胁迫适应性的机理在于盐份渗透胁迫时可以显著缩短KT2440的延滞期；显著降低这一时期KT2440细胞的死亡率。(2)盐份渗透胁迫时，KT2440对四氢嘧啶的快速积累是一种“临时性”的应急策略，当细胞适应渗透胁迫后会自身合成甜菜碱来代替四氢嘧啶；但如果KT2440基因组中甜菜碱合成基因被敲除，则KT2440细胞以四氢嘧啶作为细胞内主要的相容性溶质。利用转座插入突变获得了不能降解四氢嘧啶的突变株，并首次克隆到四氢嘧啶降解基因，发现四氢嘧啶降解阻断后，四氢嘧啶对KT2440渗透保护效果没有明显的变化。(3)首次发现接种物培养液上清能提高KT2440对盐份渗透胁迫的适应性，上清中含有一定量的甜菜碱是产生渗透保护的主要因素。(4)采用全新的策略(使甜菜碱合成基因组成型表达，不受渗透压诱导)，首次获得了一株对盐份渗透胁迫适应性显著提高的工程菌株KT2440dgbtbet。 Pseudomonas putida KT2440对盐份渗透胁迫适应性受到各种因素如营养、接种物和接种量等的影响。发现培养基丰富程度影响KT2440对盐份的盐份渗透胁迫适应性，在完全培养基中KT2440对盐份的耐受性要比在基础盐培养基中高。加大接种量可以提高KT2440对盐份渗透胁迫适应性。加大接种量可以显著缩短KT2440细胞的延滞期，但对代时影响不显著。碳源影响KT2440对盐份的耐受性，在以葡萄糖为碳源的培养基中KT2440对盐份的耐受性要比在以苯甲酸钠为碳源的培养基中高。氮素和钾素含量影响KT2440对盐份的耐受性，提高氮素和钾素浓度有利于提高Pseudomonas putida KT2440对盐份的耐受性。接种物培养基盐浓度影响KT2440对盐份渗透胁迫适应性，于0.8mol／L NaCl中培养的接种物对盐份渗透胁迫适应性要比于0mol／L NaCl中培养的要高。接种物培养液上清影响KT2440对盐份渗透胁迫适应性，去除接种物培养液上清使KT2440对盐份渗透胁迫适应性下降。接种物培养更多还原

【Abstract】 The main purpose of this research encompass on: The salt tolerance characteristic of Pseudomonas putida KT2440 and the environmental factors influenced the salinity tolerance of KT2440, The osmprotection of exogenously supplied compatible solutes to KT2440 and its mechanisms, the feasibility and genetic engineering strategies to promote the salinity tolerance of KT2440. The results show that: (1)The mechanisms of the salinity tolerance promotion of KT2440 by exogenous compatible solutes were that exogenous compatible could reduce the lag time and promote the survival rate under salt osmotic stress. (2)Ectoine provided a temporary osmprotection as an emergency strategy when there were no other compatible solutes available under salt osmotic strass. The gene govern the ectoine degradation were cloned , and the block in ectoine degradation pathway did not promote the osmprotection effect to KT2440. (3)The medium supernatant could promote the salt tolerance of KT2440 under salt osmotic stress, and the presence of glycine betaine in the medium supernatant was the main factor of osmprotection effect of medium supernatant. (4)A genetic engineering strain KT2440dgbtbet were constructed by a new genetic engineering strategy, the salt tolerance of KT2440dgbtbet promoted significantly compared with KT2440.The salinity tolerance characteristic of Pseudomonas putida KT2440 and the environmental factors such as the nourishment, inoculant culture and initial biomass effect the salinity tolerance of KT2440 were studied. The enrichment of culture medium influcenced the growth of KT2440 under high salinity stress. In complete medium KT2440 could survive in higher salinities than in chemically defined medium. Augmentation of initial biomass could reduced the lag time and enhanced the salinity tolerance of KT2440, but no significant effected on the generation time of KT2440. Carbon recourses influcenced the salinity tolerance of KT2440, in chemically defined medium used glucose as sole carbon KT2440 could survive in higher salinities than in chemically defined medium used toluate as sole carbon. The level of nitrogen and potassium influcenced the更多还原