金鱼Prox1基因cDNA的克隆及其在眼睛发育中的表达分析
Molecular Cloning and Expression Characterization of Prox1 in Goldfish (Carassius auratus) Eye Development
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摘要: 脊椎动物的Prox1基因，与果蝇的转录因子prospero同源。为了探讨Prox1基因在金鱼眼睛发生过程中的表达图式，我们从金鱼眼睛SMART库中克隆了Prox1 cDNA。它全长共2 851bp，编码739个氨基酸。组织分布研究表明，Prox1主要分布于眼、脑、心、肝、脾和肾中。整体原位杂交显示，Prox1 mRNA首先是在晶体期的晶体原基中有转录，心跳期则在未成熟晶体的细胞中和视网膜的幼芽区可以检测到。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，与Prox1 mRNA的定位不同。这说明，Prox1 基因在晶体发生过程中有重要作用，且在晶体的不同发育时期起的作用可能有所不同。另外，Prox1在晶体发育过程中有一个从内向外的变化过程。Abstract: In vertebrates, Prox1 is homologous to the Drosophila transcription factor, prospero gene. In order to explore Prox1 expression pattern in goldfish, the 2 832 bp full-length cDNA was cloned, which encodes a protein with 739 amino acids, and the protein exhibits a 93% similarity to Danio rerio Prox1. The results of RT-PCR and Western-blotting analysis revealed Prox1 expression in eye, brain, heart, liver, spleen and kidney, but not in the muscle of the goldfish. RT-PCR results indicated that there exists a small quantity of maternal Prox1 mRNAs in the mature eggs, and the zygotic Prox1 begins to transcribe from the gastrula embryos, and the expression level increased in the 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 the heartbeat stage. After lens fibers form, 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 is detected by immunofluorescence. At the heartbeat stage, the localization of Prox1 protein is the same as the mRNA. But when the lens fibers are forming, it is detected in a ring-shaped region on lens fibers under lens epithelium, which is different from the mRNA localization. The results suggest that the Prox1 gene plays an important role during lens development, and the roles are different at different developmental stages. In contrast to expression patterns from the outside to the inside of the mouse, the Prox1 localization in goldfish lens exhibits from inside to outside.
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