【作者】 齐建；

【导师】 李越中； 胡玮；

【作者基本信息】 山东大学 ， 微生物学， 2010， 硕士

【摘要】 钙离子(Ca2+)是重要的信使分子,参与了真核生物的许多细胞行为过程,如DNA合成的起始、有丝分裂、细胞分裂等。细胞胞内游离钙离子浓度([Ca2+]i)受到钙离子通道(Ca2+channels)、钙泵(Ca2+pumps)、钙结合蛋白(Ca2+-binding proteins, CaBPs)的严格调节,使得静息状态下,[Ca2+]i维持在很低水平(10-7 M),远低于胞外液体中Ca2+浓度(10-3 M)。细胞利用形成的Ca2+浓度梯度来传递信息,将信号从细胞表面传递到细胞内部。细胞受到刺激后,受到严格调控的[Ca2+]i发生瞬间变化,引起磷酸化反应、调节蛋白激活、细胞周期调节等一系列反应。目前对Ca2+在细菌细胞活动中的作用还不甚清楚,但已经发现细菌中[Ca2+]i严格控制在和真核生物相似的水平(100-300 nM),细菌细胞也有离子通道、初级和次级转运蛋白、钙结合蛋白,这些都可能参与了Ca2+的动态平衡。同时,有证据表明,[Ca2+]i的变化与细菌的许多细胞行为,如枯草芽孢杆菌(Bacillus subtilis)的孢子形成、大肠杆菌(Escherichia coli)的趋化性、蓝细菌(cyanobacteria)的异形胞分化、粘细菌(myxobacteria)的子实体发育等有关,越来越多的证据表明Ca2+在细菌中起到调节剂的作用。测定细胞中[Ca2+]i对于研究Ca2+作为胞内信使的作用是必需的。Ca2+起到信使作用的观点是基于对环境信号会诱发[Ca2+]i变化的认识,因此,确定细胞受刺激后胞质Ca2+水平,对于研究Ca2+在信号转导中的作用是至关重要的。由于细菌独特的细胞结构特征(细胞小,胞外有细胞壁)、试剂的毒性及活细菌操作的困难,测定细菌细胞[Ca2+]i是一个挑战。虽然如此,钙结合染料fura-2和光蛋白aequorin、obelin已经在原核生物中成功使用。其中,obelin是一种非常好的Ca2+指示剂,对Ca2+浓度变化反应迅速,且与镁离子(Mg2+)的结合力较aequorin更低。通过分子DNA技术在细菌细胞中组成性表达obelin基因,可以在不同种的细菌中连续监测胞质自由Ca2+浓度的变化。粘细菌是一类革兰氏阴性杆状细菌,作为一种高等的原核生物,具有特殊的多细胞行为,主要表现为滑动运动能力、分化发育过程和社会性的细胞行为三个方面,是研究原核生物分化发育和细胞间信号传导的重要模式材料。已有的研究表明,在粘细菌的细胞行为过程中,钙及钙信号(calcium signals)扮演了十分重要的角色,但目前仍没有粘细菌[Ca2+]i测定的报道。虽然S蛋白在粘球菌分化发育末期的粘孢子生成阶段起到钙调蛋白类似组分的功能,但还不清楚粘细菌中还存在其它哪些CaBPs组分。本论文通过在黄色粘球菌DK1622中表达obelin基因来测定[Ca2+]i。首先利用表达载体pET15b、pET22b在大肠杆菌BL21 (DE3)中表达obelin基因,其中利用pET22b有正确表达。荧光显微镜下观察菌体,紫外激发,可以观察到黄绿色荧光。然后利用粘球菌自主复制质粒pZJY41和整合质粒pSWU30在DK1622中表达obelin基因,RT-PCR结果显示,obelin可以在DK1622中表达,但荧光显微镜下观察不到菌体荧光。本论文根据原核生物中已公布的5个CaBPs序列及EF-hand钙离子结合位点的氨基酸序列的保守性,通过序列比对及保守序列搜索的方法,在DK1622预测的7514个ORF中分析出5个可能编码CaBPs的基因-MX AN 0106、MXAN 0962、MXAN 1334、MXAN 2316、MXAN 5194,并将这五个基因在大肠杆菌中异源表达,其中MXAN 0106、MXAN 2316成功表达,为下一步通过Ca2+-dependent electrophoretic motility-shift assay鉴定蛋白的Ca2+结合能力,在DK1622中寻找CaBPs打下基础。更多还原

【Abstract】 Calcium is an important messenger that is involved in many eukaryotic cellular processes, such as the initiation of DNA synthesis, mitosis, and cell division. The intracellular free calcium ion concentration ([Ca2+]i) is tightly regulated through Ca2+ channels, Ca2+pumps, and Ca2+-binding proteins (CaBPs). At rest, [Ca2+]i is maintained at very low levels (10-7 M) compared with the concentration of Ca2+in extracellular fluid (10-3 M). This situation creates a Ca2+ concentration gradient, which is utilized by cells to transmit signals from the cell surface to the interior of the cell. Cells respond to a number of stimuli by transient changes in [Ca2+]i. Calcium fluxes activate a series of events, leading to phosphorylation reactions, activation of regulatory proteins, and cell cycle regulation.In bacteria, the role of Ca2+ in cellular activities is less clear. It has been demonstrated that bacteria tightly regulate their [Ca2+]i, with values similar to those found in eukaryotes (100 to 300 nM). Similar to eukaryotes, bacterial cells have ion channels, primary and secondary transporters, and CaBPs, which may be involved in Ca2+ homeostasis. There is evidence indicating that calcium functions as a regulator in bacteria and that changes in [Ca2+]i are critical to some bacterial cellular processes, such as sporulation of Bacillus, chemotaxis of Escherichia coli, heterocyst differentiation of cyanobacteria, and fruiting body formation in myxobacteria.The ability to measure [Ca2+]i in cells is essential in evaluating the role of calcium as an intracellular messenger. The concept that Ca2+ acts as a messenger is based on the idea that environmental signals induce changes in [Ca2+]i. Therefore, determining [Ca2+]i in response to stimuli has been crucial in establishing the role of calcium in signal transduction. Measuring [Ca2+]i in bacterial cells has been difficult because of the unique structural features of bacteria (including their size and cell wall), toxic effects of reagents and the difficulty of manipulating live bacteria. Nevertheless, the calcium-binding dye fura-2 and the photoproteins aequorin and obelin have been successfully used in bacteria. Obelin is an excellent indicator for Ca2+ because it can respond quickly to allow for following the most rapid transient changes in intracellular calcium and is less sensitive to Mg2+ than aequorin. Using molecular DNA technology to constitutively express the obelin gene in bacteria, it is possible to continuously monitor [Ca2+]i in different genera of bacteria.Myxobacteria are rod-shaped Gram-negative bacteria that are characterized by their complicated multicellular behaviors, including gliding motility, morphogenetic and development processes, and cellular social behaviors, which make them an excellent prokaryotic model for the study of cell-to-cell communication, cell differentiation, and development. It has been reported that calcium plays an important role in cellular behaviors of myxobacteria; however, the [Ca2+]i in myxobacteria has not yet been reported. S protein functions in a manner similar to calmodulin during myxospore formation in Myxococcus xanthus, but it is still not clear whether there are other CaBPs.The obelin gene was inserted into the expression vectors pET15b and pET22b, and the constructed vectors were transformed into BL21 (DE3) to express obelin in E. coli. Obelin was normally expressed in cells transformed with pET22b, and when the cells were observed using fluorescence microscopy with UV light for excitation, yellow-green fluorescence was detected.To measure [Ca2+]i in myxobacteria, the obelin gene was cloned into vectors pZJY41 and pSWU30; resultant vectors were electroporated into M. xanthus DK1622. The total RNA of transformants was isolated, and reverse transcription PCR (RT-PCR) was performed. The results showed that obelin was expressed in DK1622. Unfortunately, fluorescence was not detected when cells were exposed to UV light.Local BLASTP was performed between five published CaBP sequences of bacteria and the predicted amino acid sequences of 7514 ORFs of DK1622 using BioEdit software. The amino acid sequences of the 7514 ORFs of DK1622 were also searched using the consensus sequence of the canonical EF-hand motif. Five ORFs, MXAN 0106, MXAN 0962, MXAN 1334, MXAN 2316 and MXAN 5194, were presumed to encode CaBPs. These five genes were heterogeneously expressed in E. coli, and MXAN 0106 and MXAN 2316 were successfully expressed. These data provided the fundation for identifying the calcium-binding ability of these two proteins using a Ca2+-dependent electrophoretic motility-shift assay.更多还原