【作者】 李文化；

【导师】 沈萍；

【作者基本信息】 武汉大学 ， 遗传学， 2004， 博士

【摘要】 本文通过测定大肠杆菌感受态细胞中Ca²⁺的含量和分布，并分别以AMP及非选择性DNA分子、La³⁺和量子点为探针，研究Ca²⁺诱导大肠杆菌建立感受态并介导摄取外源DNA的分子机理，得到以下研究结果： 1．实验发现在二价金属离子ca²⁺、Mn²⁺、Mg²⁺中，Ca²⁺诱导大肠杆菌建立感受态并实现质粒DNA转化的作用效率最高：Ca²⁺2可促进大肠杆菌的生长，但生长在含较高浓度（50，100mmol／L）Ca²⁺培养基中的大肠杆菌，其摄取外源DNA的能力并未发生改变；大肠杆菌实现摄取外源DNA的必备条件是大肠杆菌细胞、适当浓度的Ca²⁺和转化DNA同时共处于同一体系；Ca²⁺诱导大肠杆菌摄取外源DNA的作用发生在转化过程中，与转化前的处理无关；转化步骤中的DNA与受体细胞冰浴静置、热激和温育等过程也不是DNA进入受体细胞的必需条件，但这些操作可或多或少地提高DNA的转化效率。 2．建立了用Fura-2／AM作探针测定大肠杆菌感受态细胞内Ca²⁺变化的灵敏方法，也将细胞内Ca²⁺定位的焦亚锑酸钾法借鉴到研究大肠杆菌感受态细胞中Ca²⁺的分布情况。结果表明，用100mmol／LCaCl₂处理大肠杆菌细胞，有少量Ca²⁺可以进入到细胞内，并且这种进入在瞬间内（＜1s）就能完成，而大量Ca²⁺聚集在细胞表面或细胞周质。 3．大肠杆菌在低Ca²⁺条件下（5～20 mmol／L）可以摄取外源DNA，但其效率要比在高C扩十条件下低得多。并且实验发现通过这种方式摄取的质粒DNA与传统转化方法导入的质粒DNA具有相同的生物学效应，即进入细胞内的DNA能正常的复制、表达并表现出遗传效应。4.AMP可轻微刺激大肠杆菌的生长，但对细胞内质粒和染色体上相关基因的表达无影响;单磷酸嗓吟碱基（AMP和GN田）都能抑制大肠杆菌摄取外源DNA，并且这种抑制作用发生在感受态形成过程，AMP抑制作用具有浓度效应和时间效应，作用浓度越高处理时间越长，抑制效果越明显;非选择性DNA分子，无论其大小、结构和G+C含量的高低，都对大肠杆菌感受态的形成和目的DNA的转化无明显影响。5.适当浓度的L扩十可促进大肠杆菌细胞的代谢活动，不影响细胞内质粒和染色体上相关基因的表达，但可使细胞的电子显微镜表像发生改变。尽管L扩+在诸多化学性质上同c扩十类似，但L扩十不能诱导大肠杆菌形成感受态和摄取外源DNA，并且在低浓度条件下（0一10阔mL）就能显著降低DNA的转化效率，在高浓度下（>30林留mL）完全抑制大肠杆菌摄取DNA的能力。这种抑制作用可能发生在DNA的转化过程中，只要转化体系中含有一定的L扩+，就能引起大肠杆菌质粒DNA转化效率的显著降低或使其完全丧失摄取DNA的能力。6.用量子点标记大肠杆菌感受态细胞并用原子力显微镜观察发现，IO0nuno比CaC12诱导处理后的大肠杆菌细胞表面发生了显著变化，细胞表面变得不平整，较非感受态细胞起伏大，并且细胞壁出现损伤，形成直径大小约50一300nm的小孔，直径大小约3一4nm的量子点能够进入大肠杆菌感受态细胞但不能进入非感受态细胞。 综合以上实验结果我们认为，大肠杆菌细胞表面没有结合DNA的特异性受体存在，金属离子诱导大肠杆菌摄取外源DNA具有一定的特异性。高浓度非生理条件下c扩+诱导大肠杆菌摄取外源DNA，可能一方面是高浓度的C扩斗聚集在细胞表面改变了细胞壁的渗透性，使转化DNA更容易接近细胞膜。另一方面少量C扩十进入细胞与胞内的PHB和Pi结合成复合物，在细胞膜上形成新的膜通道，为DNA最终进入细胞内提供了条件。低c扩+诱导大肠杆菌摄取DNA可能主要是后者的作用。因此，c扩十诱导大肠杆菌建立感受态并实现DNA转化，既是一个物理化学过程，也是细胞自身生理调节的反应过程。这些研究结果对于我们探索大肠杆菌摄取外源DNA的分子机理，提高基因工程操作中将重组DNA导入受体细胞的效率，以及正确评估大肠杆菌可能发生的水平基因转移事件具有重要意义。更多还原

【Abstract】 The work of this dissertation is concerned about the study of Escherichia coli competence development induced by Ca2+ and the capacity of absorbing external-DNA. We obtained the following results by determining the Ca2+ concentration and locating intracellular Ca2+ of E. coli competence and exploring the molecular mechanism of competence development using AMP, nonselective DNA and quantum dots (QDs) as probes.1. Studies showed that, for divalent cation Ca2+, Mn2+ and Mg2+, Ca2+ was more efficient in inducing E. coli development of competence and accomplishing DNA transformation than the two others. Ca2+ promoted the growth of E. coli but the cells grown in culture medium containing high concentration Ca2+ haven’t more special capacity absorbing external-DNA than low concentration Ca2+. The simplest requirement for E. coli absorbing external-DNA was that E. coli cells, proper Ca2+ and DNA must be in a same system. The function of Ca2+ is acted on the process transformation of E. coli, but other processes. Heat shock and other operations increased the DNA transformation efficiency but weren’t necessary for E. coli absorbing external-DNA.2. We established a new experiment method for determining the Ca2+ concentration of E. coli competent cells using Fura-2/AM as fluorescent probe, and located the cellular Ca2+ using potassium pyroantimonate [K2H2Sb2O7 4H2O]. The results indicated that, after dealing E. coli with 100mmol/L CaCl2, a little quantity of Ca2+ entered into the cells swiftly and a large quantity of Ca2+ gathered on the surface of cells or periplasm.3. E.coli could take in external-DNA under the conditions of resemble to the nature and the DNA absorbed replicated and expressed regularly. Moreover, the transformation frequency was related to Ca2+ concentration and a proper time was required for accomplishing natural genetic transformation.4. AMP promoted the growth of E. coli lightly. But they have no effects on expression of part of gene in plasmid and chromosome. AMP and GMP prevented E. coli development of competence and then held back E. coli cells absorbing external-DNA. Moreover, AMP displayed more remarkable function when its concentration was higher and the time of dealing with was longer. Others of nonselective DNA didn’t affected E. coli absorbing external-DNA regardless their molecular weight, molecular structure and their content of G+C.5. La3+ at concentration from 50 to 150ug/mL stimulated the endogenic metabolism and ectogenic metabolism of E.coli, changed the image of transmission electron microscopy, but had few effects on gene expression. Through many chemical properties of La3+ are similar to Ca2+, they didn’t induced E. coli development of competence and absorbing of external-DNA. Furthermore, La3+ at lower concentration 0.5 to 30ug/mL decreased intensively the transformation efficiency and inhibited completely E. coli absorbing external-DNA at high concentration (>30ug/mL).6. Exploring the mechanism of competence development in E. coli using quantum dots as fluorescent probes and observing the change of cells by atomic force microscope (AFM), results showed that E. coli competence cells surface suffered many change, appearing rough and presented holes of about 50-300 nm in diameter under AFM, while the surface of non competent cells appeared smooth and continuous. Water-soluble QDs of 3-4nm could go into competent cells by these holes on cell surface, but could not enter noncompetent cells.In conclusion, based on these results, we believed that there wasn’t specific receptor on surface of E. coli. Metal cation exhibited speciality in inducing E. coli development of competence. We suggested that, in process of nonphysiological high concentration Ca2+ inducing E. coli into competent cells, not only Ca2+ had enhanced the penetrability of cell membranes but also part of Ca2+ entered into cells and combined PHB and polyPi into compound on membranes which constituted membrane channels for DNA passage. It is inferred that, as far as Ca2+ promoted E. coli absorbing externa更多还原