【作者】 俞丽丽；

【导师】 顾福康；

【作者基本信息】 华东师范大学 ， 动物学， 2011， 硕士

【摘要】 腹毛目纤毛虫是纤毛虫中最高等的一个类群,其最典型的特征即具有纤毛聚集成的复杂的纤毛器结构。这些纤毛器结构主要包括纤毛器微管及基部附属微管,即口围带及其附属微管(小膜托架、小膜附属微管)、波动膜及其附属微管(波动膜托架)、额腹横棘毛及其附属微管(前纵微管束、后纵微管、横微管)、左右缘棘毛及其附属微管(前纵管束、后纵微管束和横微管)、尾棘毛和背触毛。且纤毛器结构的主要成分是微管蛋白,因此腹毛目纤毛虫是用以研究纤毛虫皮层微管胞器和微管蛋白的常用材料。根据纤毛形态结构及发生等特征的不同,可将腹毛目纤毛虫划分为不同的种类。腹毛目侧毛虫科纤毛虫在分类地位上属于尾柱虫科和尖毛虫科之间的过渡类群。该类群纤毛虫种类及数量较少,且相关研究报道不多见,导致其科内分类较为混乱。本文以一种侧毛虫科(Pleurotrichidae)纤毛虫拟翁口虫(Onychodromopsissp)为研究材料,应用荧光紫杉醇(FLUTAX)直接荧光标记法、透射电镜术、扫描电镜术、SDS-PAGE电泳和生化抽提等方法,就本种纤毛虫的皮层微管胞器的形态及其形态发生、射出胞器的形态结构及发生、微管蛋白等方面展开研究,初步得到了相应的研究结果,从显微和亚显微水平上为侧毛虫科纤毛虫的种间分类提供材料和证据。所得结果如下：1拟翁口虫纤毛器微管的形态及形态发生应用荧光紫杉醇直接荧光标记方法显示本种纤毛虫的腹皮层纤毛器微管胞器及形态发生。其中,口围带含30～35片小膜；波动膜一片；额腹横棘毛为典型的8-5-5模式,左缘棘毛2列,右缘棘毛3列；背面含6列背触毛；额、腹棘毛基部的前纵微管束与后纵微管束均较发达,但未见横微管束；横棘毛前纵微管束发达,前4根横棘毛基部前纵微管束向前伸展并汇聚成一个三角结构；左缘棘毛横微管束极不发达。形态发生过程中,细胞缘棘毛起始于最外侧右缘棘毛,且每列老缘棘毛的前、后部棘毛都瓦解,并在相应位置产生的新棘毛不断延伸代替老棘毛。据该纤毛虫的皮层纤毛模式和纤毛器基部微管的形态,将其归为侧毛虫科(Pleurotrichidae)拟翁口虫属(O.sp)；并据后仔虫口原基的发生、左右缘棘毛原基的发生不同于已报道的柔软拟翁口虫(Onychodromopsis flexilis)的情况,认为该纤毛虫可能为另一种拟翁口虫。2拟翁口虫细胞及微管类细胞骨架的超微结构应用透射电镜术显示本种纤毛虫皮层细胞骨架的超微结构。结果显示拟翁口虫的表膜具高度发达的实质状表膜泡；纤毛杆轴纤丝排列成“9+2”模式,相邻两根纤毛基体之间具有连接结构；在同一虫体中每个口围带小膜单元的组成模式有两种：2列长度相当的纤毛组成和3列长度相当的纤毛；围棘纤维篮围着一层纤维,且向周围发出许多微管束,其中前纵微管束及其发达；表膜下一般具3-5层微管层,口区附近具由数十上百根微管紧密叠加在一起成为的加厚的微管层；大核复制带为透明均一状,未出现染色质状态不一的前部区、后部区。结果表明拟翁口虫表膜及表膜下纤维结构、大核复制带均具不同于其他腹毛目纤毛虫的特征,且其特征是符合腹毛目纤毛虫由尾柱虫类(Urostylidae)—侧毛虫类(Pleurotrichidae)—尖毛虫类(Oxytrichidae)—游仆虫类(Euplotidae)的进化规律的,从亚显微水平上为侧毛虫科内纤毛虫种群划分提供资料。3拟翁口虫射出胞器的超微结构应用扫描电镜和透射电镜术对腹毛目纤毛虫拟翁口虫细胞中射出胞器的超微结构进行了研究。结果显示该射出胞器扫描电镜下为颗粒状突起,口区分布较少：透射电镜下散布在整个细胞中,成熟后定位于表膜下,且顶部中间(靠近表膜的一端)的纤维物质密度较少,有的几乎出现空洞；射出胞器的发生过程中形成可高低电子密度相间的同心圆结构,且囊泡结构中间出现缢痕,随后一分为二,形成两个具电子密度相间的同心圆结构的囊泡。射出胞器体外及成熟定位后的形态特征表明该射出胞器可能为一种类粘液泡;囊泡随缢痕一分为二的现象可能为射出胞器的一种新型起源方式；该射出胞器可能行使一种防御或应激功能,对捕食行为无直接作用。4拟翁口虫全蛋白和γ-微管蛋白基因初步分析应用SDS-PAGE电泳技术对拟翁口虫全蛋白组分进行了分析,并对其γ-微管蛋白基因片段进行了扩增。电泳结果显示：116KD与66.2KD之间、45KD与35KD之间的蛋白条带密集度高,在约30KD、20KD、16KD附近的条带蛋白表达量高达7.0%-7.9%；特异性地扩增出一条约为1200bp的γ-微管蛋白条带。结果表明本种纤毛虫具有不同于其他纤毛虫的全蛋白和γ-微管蛋白基因含量。更多还原

【Abstract】 Hypotrichous ciliate are highest group in ciliates, it’s most typical groups with characteristics namely complicated ciliature apparatus. These ciliature apparatus consist of microtubular organelles and the base associated microtubules of these ciliatures:adoral zone of membranelles (AZM) and its base associated microtubules (membranelle brackets, membranelle associated microtubules), undulating membranes (UM) and its base associated microtubules, frontal-ventral-transverse cirri (FVTC) and its base associated microtubules(anterior longitudinal microtubules (ALM), posterior longitudinal microtubules (PLM), transverse microtubules (TM)), marginal cirri (MC) and its base associated microtubules(ALM, PLM, TM), caudal cirri (CC), dorsal kineties (DK). The main ingredients of ciliature are microtubulins, therefore ciliated protozoa are common materials for research of microtubular organelles and microtubulins of ciliates.According to different characteristics on morphology and morphogenetic, ciliated protozoa can be classified into different groups. In taxonomy, Pleurotrichidae is a transition gronp between Urostylidae and Oxytrichidae. For Pleurotrichidae have low category and quantity, and few related researches, its classification are out of order. In this study, by using direct-fluorescent labeling、TEM、SEM、SDS-PAGE、biochemical extraction, the strucutures and functional components of microtubular organelles, morphological structure of extrosomes, and microtubulin were discussed. Expect to provide materials and evidence for taxonomic research of Pleurotrichidae. Results are as follows:1 The morphology and morphogenetic of the ciliature microtubular organelles in Onychodromopsis Sp The morphology and morphogenetic of the ciliature microtubular organelles in the ventral cortex of Onychodromopsis Sp were visualized using FLUTAX direct fluorescent labeling. In the front of cell, the AZM possesses 30～50 adoral membranelles; UM possesses 2 rows of cilia; FVTC distribute into 8 frontal,5 ventral and 5 transverse cirri; 2 left marginal cirri (LMC) and 3 right marginal cirri (RMC),6 DK rows; Developed ALM and PLM of FVC, and developed ALM of TC, the first four ALM of TC extend forward together into a triangle structure. TM of LMC are extreme underdevelopment. During morphogenesis, the new MC primordium first originate from the outest RMC, and then MC primordium presents in the front and the middle part of th degenerated site if the old marginal cirri. On the features of its ciliature and the base-associated microtubules, this species qualifies to be the genus Onychodromopsis. However, the morphogenetic study shows the originating position of oral primordium of the opisthe and differentiation of the marginal rows were different from other related species Onychodromopsis flexilis, this species was probably another species in the genus Onychodromopsis.2 The ultrastructure of cytoskeleton in Onychodromopsis SpThe results of TEM showed that ultrastructure of microtubular cytoskeleton of Onychodromopsis Sp. It showed that a layer of alveolar membrane beneath the pellicle; ciliary shafts possess "9+2" doublet microtubules; and basal bodies of the cirri are also linked by different kinds of connections. Each membranelle of AZM has two types of compositional model:2 rows of cilia and 3 rows of cilia; A layer fiber is cover around FCB, and lots of microtubules send forth from FCB, developed ALM. There has 3-5 microtubules layers beneath the pellicle. The pellicle closed to AZM has thicken microtubules layers, which was consisted of hundreds of layers. During morphogenesis, Macronucleus appeared replication band, it’s a homogeneous structure. Reasults shows compared with others ciliates, Onychodromopsis Sp have different microtubules layers beneath the pellicle and republication band of Macronucleus. These characters are correpond with evolutionary discipline from Urostylidae, Pleurotrichidae, Oxytrichidae to Euplotidae. To provide the mateials for taxonomic research of Pleurotrichidae at sub-microscopy level.3 The ultrastructure of the extrusomes in Onychodromopsis SpBy using SEM and TEM, the ultrastructure of extrusomes in Onychodromopsis Sp to be observed. SEM shows there has granulate structure on surface of the body, and less in vicinity of AZM; TEM shows that extrusomes distributed in the cortical cytoplasm of Onychodromopsis Sp Mature organelles are located beneath pellicle,the top part of extrusome have low density fibrous matter, even appear cavity; In the process of morphogenesis of extrusomes, it appear concentric circles structure with high-low electron-dense matter. And vesicle extended to be divided in two parts, which also has concentric circles structure. The morphology and morphogenetic of extrusomes indicated this extrusome was probably a kind of mucocyst, the divide of its probably a new way of origin. And this extrusome might be a protective or defensive response to the environment changes, irrelevant with predatory behavior.4 The protein composition of Onychodromopsis Sp and cloning analysis of gramma-tubuliln gene fragment from Onychodromopsis SpBy using the methods of SDS-PAGE analyse the protein composition of Onychodromopsis Sp, meanwhile cloning of gamma-tubulin gene fragment from Onychodromopsis Sp. The result of SDS-PAGE shows that the molecular weight of protein range from 66.2-116kD and 35-45KD was rather higher, the protein composition of 30kD,20kD, and 16kD bands come to 7.0-7.9%; The result of clonging showed that the molecular weight of gamma-tubulin is 1000bp approximately. It demonstrated that Onychodromopsis Sp has different characters in the protein composition and gamma-tubulin gene fragment.更多还原