【作者】 陈季武；

【导师】 顾福康；

【作者基本信息】 华东师范大学 ， 动物学， 2003， 博士

【摘要】 纤毛虫中经常发生形成包囊和脱包囊的过程，期间，细胞结构经历了不同水平的分化和重组，细胞内发生了一系列生理生化变化。目前，对纤毛虫包囊现象的研究不仅是探讨真核细胞的结构与功能、细胞模式形成和控制机理的一个重要方面，并已成为揭示细胞于特殊生理条件下生命活动规律及其细胞在不同水平调控的一个重要内容。对包囊现象的研究在细胞及胞器水平已经积累了大量的资料，但从生物化学和分子水平上的工作则刚开始，所见报道甚少。本文以形成“毛基体吸收型包囊”的膜状急纤虫和形成“毛基体非吸收型包囊”的包囊游仆虫的休眠包囊和营养细胞为材料，应用多项生物学技术，从细胞水平、酶水平、基因水平对大核DNA、线粒体DNA、全蛋白质组成和等电点、包囊壁蛋白质组成、同工酶组成和活性、能量代谢以及抗氧化能力等进行了比较研究，实验结果和结论如下： 一．大核DNA和线粒体DNA的RAPD 应用RAPD技术对急纤虫、游仆虫休眠包囊和营养细胞大核DNA、线粒体DNA进行了比较研究。结果表明，利用35条随机引物，游仆虫大核DNA共扩增出220条片段，其中以休眠包囊大核DNA为模板扩增出97条片段，以营养细胞大核DNA为模板扩增出123条片段，两者有79条共享片段，共享度为72％；休眠包囊大核DNA扩增出18条特异性片段，营养细胞大核DNA扩增出44条特异性片段，两者存在28％的差异。利用34条随机引物，急纤虫大核DNA共扩增出203条片段，其中以休眠包囊大核DNA为模板扩增出106条片段，以营养细胞大核DNA为模板扩增出97条片段，两者有61条共享片段，共享度为60％；休眠包囊大核DNA扩增出45条特异性片段，营养细胞大核DNA扩增出36条特异性片段，两者存在40％的差异。这些结果表明，急纤虫休眠包囊和营养细胞选择性表达的大核基因在很大程度上是一致的，但也存在差异。游仆虫休眠包囊与营养细胞选择性表达的大核基因也是如此。这些差异提示，游仆虫和急纤虫休眠博士学位论文不同生理条件下两种纤毛虫细胞的比较研究包囊大核DNA的基因表达与营养细胞并不完全一致，且在某些方面可能发生明显的变化，这些变化与细胞在营养状态下的代谢活动等向相对静止状态的剧烈变化以及休眠的生理生化变化密切相关。相似地，利用32条随机引物，游仆虫线粒体DNA扩增出巧4条片段，其中以休眠包囊线粒体DNA为模板扩增出74条片段，以营养细胞线粒体DNA为模板扩增出80条片段，两者有55条共享片段，共享度为71%;休眠包囊线粒体DNA扩增出19条特异性片段，营养细胞线粒体DNA扩增出25条特异性片段，两者有29%的差异。利用34条随机引物，急纤虫线粒体DNA扩增出216条片段，其中以休眠包囊线粒体DNA为模板扩增出102条片段，以营养细胞线粒体DNA为模板扩增出114条片段，两者有67条共享片段，共享度为62%;休眠包囊线粒体DNA扩增出35条特异性片段，营养细胞线粒体DNA扩增出47条特异性片段，两者存在38%的差异。这些结果表明，急纤虫休眠包囊和营养细胞的选择性表达的线粒体基因在很大程度上是一致的，但也存在差异。游仆虫休眠包囊和营养细胞的选择性表达的线粒体基因也是如此。这些差异提示，在包囊形成过程中，游仆虫和急纤虫线粒体DNA的基因表达都发生了相应的变化，这些变化与包囊形成及其休眠状态下对能量的需求密切相关。 所得结果表明，与营养细胞相比，两种休眠包囊的大核DNA和线粒体DNA表达均发生了变化，但两种包囊的DNA变化程度有差异，急纤虫休眠包囊的变化更大。这可能是由于急纤虫形成“毛基体吸收型包囊”时全部吸收了毛基体、纤毛杆和微管结构，在细胞结构发生一系列剧烈变化的同时，起细胞调控作用的基因组DNA的功能活动变化(例如相应的基因开放和关闭)相对较大;而游仆虫形成“毛基体非吸收型包囊”时仅吸收了部分纤毛结构和胞质微管结构，大部分毛基体和部分微管结构被保留下来。与前者相比，与细胞结构分化相联系的细胞基因组DNA的功能活动变化(例如相应的基因开放和关闭)相对较小，因此，“毛基体吸收型包囊”脱包囊过程可能比“毛基体非吸收型包囊”更为复杂。 从电泳图谱中可以看出，不仅有共享条带和特异性条带，而且条带存在明亮或暗淡的差异。推测其原因是:在纤毛虫形成包囊过程中，大核基因组和线粒体基因组不仅会激活或关闭某些基因，使RAPD中扩增出共享片段和特异性片段，而且大核DNA和线粒体DNA可能也会通过基因扩增，在短期内大量产生出某些特定基因拷贝以适应特殊需要，因而使RAPD中扩增出明亮条带;而没有扩增的表达基因拷贝数少，因而使RAPD中扩增出暗淡条带。博士学位论文不同生理条件下两种纤毛虫细胞的比较研究二.细胞全蛋白和包囊壁蛋白 应用SDS一PAGE技术和IEF技术以及高灵敏度银染色技术，对急纤虫、游仆虫休眠包囊和营养细胞进行了蛋白组成以及蛋白等电点比较分析;对两者休眠包囊壁与脱包囊后的包囊壁进行了蛋白组成比较分析。结果显示，急纤虫休眠包囊有27条蛋白质条带，营养细胞有34条蛋白质条带，共享条带有23条，共享度为75.4%;休眠包囊和营养细胞?更多还原

【Abstract】 The encystment and excystment often occurs in ciliates. In the meantime, the cell structures go the different differentiation and recombination. A series of physiological and biochemical changes occur in the cell. At present, the research on these phenomenon is not only an important aspect for exploring the structure and function, formation pattern and regulation mechanism of the eukaryotic cell, but also becomes an important content for revealing the life activity law in the specific physiological condition as well as the cell regulation in the different levels. A lot of research data of phenomenon of cyst in cell level and organelle level had been accumulated. But the investigations on cyst in biochemical level and molecular level just began and were very limitedly reported. Therefore, the resting cyst and the vegetative cell in Tachysoma pellionella, Euplotes encysticus, forming "kinetosome-resorbing cyst" and "non kinetosome-resorbing cyst " respectively, were used as experimental materials. By using many kinds of biological techniques, the comparative study of the macronucleus DNA , mitochondria DNA, whole protein composition and isoelectric point, cyst wall protein composition, activity and composition of isozyme, energy metabolism and antioxidation ability were carried hi cell level, enzyme level and gene level.1 Macroucleus DNA and mitochondria DNAThe macronucleus DNA and mitochondria DNA in Tachysoma pellionella, Euplotes encysticus were comparatively studied by using RAPD technique. The results showed that 220 fragments were amplified in the resting cyst and the vegetative cell DNA from Euplotes encysticus by using 35 random primers. In these fragments, the resting cyst and the vegetative cell macronucleus DNA had 97 fragments and 123 fragments, respectively. Both had 79 similar fragments. The similarity values is 72%;The resting cyst and the vegetative cell macronucleus DNA had 18 and 44 specific fragments, respectively. The variance values is 28%. 203 fragments were amplified in the resting cyst and the vegetative cell macronucleus DNA from Tachysoma pellionella by using 34 random primers. In which, the resting cyst and the vegetative cell macronucleus DNA had 97 and 106 fragments, respectively. Both had 61 similar fragments. The similarity values is 60%; The resting cyst and the vegetative cell macronucleus DNA had 45 and 36 specific fragments, respectively. The variance values is 40%. These facts implied that the choice expressing macronucleus genes of the resting cyst and the vegetative cell in Tachysoma pellionella are similar to a high degree but both had some variances. So do the resting cyst and the vegetative cell macronucleus genes in Euplotes encysticus. These variances suggested that macronucleus genes expression of the resting cyst and the vegetative cell in Tachysoma pellionella, Euplotes encysticus was not identical. The significant changes in some sides perhaps happened. These changes were closely related to the acute alteration for transferring the metabolism activities in the vegetative state into related motionless state and closely related to physiological and biochemical alteration in the resting cyst. Similarly, 154 fragments were amplified in the resting cyst and the vegetative cell mitochondria DNA from Euplotes encysticus by using 34 random primers. Among them, the resting cyst and the vegetative cell mitochondria DNA had 74 and 80 fragments, respectively . Both had 55 similar fragments. The similarity values is 71 %. The resting cyst and the vegetative cell mitochondria DNA had 19 and 25 specific fragments , respectively . The variance values is 29 % . 216 fragments were amplified in the resting cyst and the vegetative cell mitochondria DNA from Tachysoma pellionella by using 32 random primers . Among them, the resting cyst and the vegetative cell mitochondria DNA had 102 and 114 fragments, respectively . Both had 67 similar fragments. The similarity values is 62 %. The resting cyst and the vegetative cell mitochondria DNA had 35 and 47 specific fr更多还原