張百恩2006-07-262018-07-092006-07-262018-07-092002http://ntur.lib.ntu.edu.tw//handle/246246/22660本研究計畫之主要目的在選殖模式動 物斑馬魚crystallin-βB1 基因cDNA，並探 討其與先天性白內障生成之關係。利用 RT-PCR (reverse transcription-polymerase chain reaction) 之方法配合退化引子 (degenerate primers) 獲致一段斑馬魚 crystallin-βB1 gene 之cDNA片段。以RACE (Rapid Amplification of cDNA Ends)方法獲 致 cDNA 5’及3’端cDNA 序列，合併三片 段得知cDNA 之全長序列。其胺基酸之序 列與其他物種之此基因序列非常接近，僅 在蛋白質N 端歧異度較大。以原位雜合反 應(whole mount in situ hybridization)來了解 此基因在斑馬魚胚胎發育初期之時空動態 變化，得知此基因在斑馬魚胚胎20 小時開 始在水晶體表現。由北方墨點法，得知此 基因在48 小時胚胎有大量的表現，而且僅 有一種mRNA 表現。另一方面，從斑馬魚 之genomic DNA library 中，篩選crystallin- βB1gene，以了解其基因之結構(Exon-Intron composition)，經細部分析其中一篩選的基 因體基因片段，得知此基因含有6 個表現 序列（Exon）及5 個插入序列（Intron）。 而上游啟動子（promoter region）亦被成功 的選殖。以PCR 方法構築缺失型態之 cDNA (truncated form cDNA)，再利用試管 內合成部分缺失型態的mRNA (truncated form mRNA)，將此有缺陷之mRNA 直接以 顯微注射(microinjection)方式注入正在發 育的斑馬魚胚胎，並沒有明顯的白內障現 象。The aim of the project was focused on the cloning of zebrafish crystallin-βB1 and the study of its implication in the congenital cataractogenesis. A fragment of zebrafish crystallin-βB1 cDNA was cloned by RT-PCR using degenerate primers. The 5’ and 3’ cDNA were obtained by 5’RACE and 3’RACE methods (Rapid Amplification of cDNA Ends). Compilation of the three fragments resulted in the full-length cDNA sequence. The zebrafish crystallin-βB1 sequence shows high homology in amino acid sequence when compared with those of other vertebrates with some varied residues in the N-terminal region. The spatialtemporal expression of the gene in the developing embryo was revealed by whole mount in situ hybridization. The results shows that this gene is initially expressed in the developing lens at about 20 hr stage. The transcripts of the gene are abundant in the embryos at 48 hr stage and a single kind of transcripts is expressed as revealed by Northern blot analysis. On the other hand, the genomic DNA fragment of the gene was isolated by screening its cognate genomic DNA library. After analyses by restriction enzyme mapping and subcloning, the structure of crystallin-βB1 gene was unraveled. This gene is composed of 6 exons and 5 introns. The upstream promoter region was also cloned. Concerning the relation between the cataractogenesis and the mutation of the gene, mutated form of cDNA was constructed by PCR. The mutant form of the transcripts was micro-injected, however, no evident cataract was formed in the embryo.application/pdf1199643 bytesapplication/pdfzh-TW國立臺灣大學醫學院口腔生物科學研究所晶體蛋白水晶體白內障基因體cataractcDNAcrystallingenomelensjournal articlehttp://ntur.lib.ntu.edu.tw/bitstream/246246/22660/1/902320B002146.pdf