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题名: 金鱼两个与眼睛发育相关基因的的鉴定和特征分析
作者: 马冬梅
答辩日期: 2006-11-29
导师: 桂建芳
授予单位: 中国科学院水生生物研究所
授予地点: 水生生物研究所
学位: 博士
关键词: 金鱼 (Carassius auratus) ; 同源结构域 ; SIX结构域 ; Prospero 结构域 ; 眼睛突变 ; 眼睛的发育 ; 外核层 ; 定位
其他题名: Identification and Characterization of Two Genes Related to Eye Development in Goldfish (Carassius auratus)
摘要: 金鱼有多种品系,且所有品系均起源于中国的野生鲫鱼(Carassius auratus)。就金鱼眼睛的变化而言,有正常眼、龙睛、望天眼、水泡眼等许多种,所以它是研究眼睛发育的好材料。为了探讨这些突变体形成的分子机理,我们首先以正常眼的草金鱼作对照,以龙睛为材料开展了研究。 对正常眼和龙睛的内部结构比较分析表明,成熟的龙睛比对照正常眼体积大;瞳孔小;晶状体结构疏松;视网膜薄,正常眼视网膜外核层有8层细胞核,内核层有5层细胞核,靠近色素细胞层的细胞核为长椭圆形,而龙睛视网膜外核层和内核层挨在一起,共有5层细胞核,几乎辨认不出外网织层,所有的细胞核近圆形;角膜比正常眼少了本处发生层。与正常眼相比,龙睛色素上皮层的突触和视锥、视杆细胞的外段呈无序的形式排列。正常眼和龙睛的早期的形态发生过程基本相同,与其它硬骨鱼类相比也没有明显的差别。至出苗1到2个月,龙睛的性状才能用肉眼观察出来,眼球开始膨大,并逐渐突出于眼眶外,视网膜和脉络膜也变得很薄,直至成熟的龙睛眼睛形成。基于以上这些形态观察,我们对Six3基因在龙睛和正常眼中的定位和Prox1基因在正常眼中的定位进行了分析。 脊椎动物的six家族转录因子与果蝇的sine oculis基因同源,sine oculis基因在果蝇眼睛发育中起重要作用。我们克隆了正常眼金鱼和龙睛眼金鱼的Six3 cDNAs,推测的氨基酸序列完全相同,且与斑马鱼Six3b的同源性高达 97.3%。在早期的胚胎发育阶段,Six3 蛋白在正常眼和龙睛中的表达模式相似,没有明显的差别。但在出苗2天后,Six3蛋白在龙睛视网膜外核层细胞质中的表达量开始比草金鱼大。这一特点一直持续到龙睛的性状表现出来。这个时期正好处于眼睛发育的生长期之前和之初。此后,龙睛的眼球进一步增大,其视网膜外核层的一些细胞的细胞核中也可以检测到Six3蛋白的明显信号,且这样的细胞占细胞总数的比例有增加的趋势。而在成熟龙睛的视网膜中,Six3蛋白在外核层和内核层一些细胞的细胞核中检测到的信号也比正常眼强。 脊椎动物的Prox1基因,与果蝇的转录因子prospero同源。该基因对多种器官的发育有重要的调控作用。整体原位杂交的结果显示,金鱼Prox1 mRNA首先是在晶体期的晶体原基中有显著的转录。心跳期,在整个未成熟的晶体中和视网膜的幼芽区可以检测到Prox1 mRNA。晶体纤维形成后,它主要定位于视纤维层和内网织细胞层。免疫组化的结果显示,心跳期Prox1蛋白的定位与mRNA相同,晶体纤维形成以后,Prox1蛋白主要定位在晶体上皮细胞内侧的晶体纤维上一个环状区域。在成熟的草金鱼晶体纤维和晶体上皮中仍可以检测到Prox1蛋白的表达。Prox1 基因在晶体发生过程中有一个定位迁移的过程,这个过程与小鼠Prox1 基因由晶体外到晶体内的迁移过程相反。
英文摘要: It is well known that numerous varieties of goldfish originate from wild crucian carp (Carassius auratus) in China. Eye varieties, such as dragon eye, telescope eye, celestial eye, bubble eye, and cinnabar eye, are main types in the current several hundred varieties. In order to research the molecular mechanism, we firstly compared their eye structures. In comparison with normal eye, the volume of dragon eye ball is larger; the pupil is smaller; the lens is looser and the volume of the lens for the whole eyeball is smaller. The autochthonous layer is defected in the cornea of the dragon eye. There are eight layers of cells in outer nuclear layer and five layers of cells in inner nuclear layer for normal eye, but in dragon eye the outer nuclear layer is so closer to the inner nuclear layer that outer plexiform layer is hard to be recognized, and about five layers in outer nuclear layer and in inner nuclear. The shape of the nucleus near to pigmented epithelium in outer nuclear layer is long-ellipsoid for normal eye, but the shape of all nucleus is globoid for dragon eye. In contrast to the normal eye, the extending of the pigmented epithelium and the outer segments of rod cells and cone cells are arrayed in disorder in the dragon eye. It is no distinct difference between normal eye and dragon eye during morphogenesis, and similar to other vertebrates. After 1 or 2 months old, the eyeball of dragon eye is larger, and the retina and the chorioidea are thinner than that of the normal eye. This characteristic persists for the dragon eye formed. Based on the previous observation, Six3 was localized in normal eye and dragon eye, and Prox1 was localized in the normal eye during their morphogenesis. The Six family transcription factors are vertebrate homologues of the Drosophila sine oculis gene, which is required for the development of the Drosophila visual system. Dragon eye goldfish is a goldfish with big size eye extruding out of eye-sockets. The Six3 cDNAs from normal eye and dragon eye goldfishes were cloned, and the predicted amino acid sequences are the same, but exhibiting 97.3% identity to the zebrafish Six3b. During the earlier development stages before hatched, the expression pattern of Six3 protein is quite similar in the two types of eye. But for 2 days old larvae, the expression level of Six3 protein began to increase faster in the cytoplasm of the outer nuclear layer cells in dragon eye than that in normal eye, and the trend persists until the eye expanding. In adult dragon eye, Six3 protein was detected not only in the cytoplasm of the outer nuclear layer, inner nuclear layer and ganglion cell layer, but also remarkable in the nucleus of some cells in retina. The data suggested that dragon eye formation might be related to the higher expression of transcription factor Six3. In vertebrates, Prox1 is a homologous to the Drosophila transcription factor, prospero gene. The expression levels increased as developmental process. Whole-mount in situ hybridization demonstrated that in the eye Prox1 mRNA is first predominately detected in the lens placode at lens stage. Then the Prox1 mRNA is localized in the whole puerile lens cells and retina germinative zone at heartheat stage. After lens fiber forming, it can be detected predominantly in the optic fiber layer and inner plexiform layer. At the same time, the Prox1 protein expression in the lens epithelium is detected by immunofluorescence histochenistry. In adult goldfish, the Prox1 protein is predominately detected in lens fiber in a hemi-ring region under the lens epithelium. In mouse, Prox1 protein expression showed a pattern from outside to inside in the lens, but in goldfish the process is reverse.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.ihb.ac.cn/handle/342005/12008
Appears in Collections:中科院水生所知识产出(2009年前)_学位论文

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Recommended Citation:
金鱼两个与眼睛发育相关基因的的鉴定和特征分析.马冬梅[d].中国科学院水生生物研究所,2006.20-25
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