斑馬魚βB1-crystallin 之選殖，分子特性，胚胎表現及激素調控之研究

Abstract

水晶體蛋白(Crystallins)是眼球水晶體最主要的構造性蛋白質。水晶體色呈透明且具有反射光線的功能，而這樣一個重要的視覺功能是來自於Crystallins之間的構形及交互作用。Crystallins在脊椎動物主要有a-、β-與γ-crystallin三個主要的families。本研究以β-crystallin family中的β-crystallin為主要研究對象。目的在於選殖出斑馬魚βB1-crystallin基因，探討其表現位置與胚胎時期mRNA的表現動態，並研究飢餓與激素調控對於βB1-crystallin表現的影響。 由已知物種的βB1-crystallin序列設計退化引子，以反轉錄聚合?連鎖反應獲得片段的斑馬魚βB1-crystallin cDNA序列。利用3’RACE取得3’端的序列，並以此片段序列作為探針，自斑馬魚cDNA基因庫中篩選出全長的βB1-crystallin cDNA序列。經過軟體分析得知斑馬魚βB1-crystallin全長943個核?酸，可轉譯成233個胺基酸。比對後亦發現斑馬魚βB1-crystallin與其他物種具有很高的同源性。由北方雜合反應及全體原位雜交法中發現，斑馬魚胚胎發育過程中，βB1-crystallin初期出現在20小時，並於48小時大量表現。觀察其表現位置得知，βB1-crystallin專一且大量的表現於眼睛，證實βB1-crystallin是一個具有組織專一性的基因。 在調控的實驗中，注射insulin family包括胰島素、類胰島素第一、第二型以及成長激素於斑馬魚成魚，另一方面，將斑馬魚進行數天的斷食。兩組均萃取不同時期的Total RNA，並設計此基因專一的引子，進行即時定量反轉錄聚合?連鎖反應(real-time quantitative。RT-PCR)。結果顯示，飢餓作用對斑馬魚體內βB1-crystallin的表現量影響不大，相較之下，激素的刺激確實會影響βB1-crystallin的表現，且有顯著性之差異。因此推測insulin family確實在斑馬魚βB1-crystallin表現上具有調控的功能，進而影響眼睛水晶體的發育與組成，亦在視覺上扮演重要的角色。

Crystallins have been recognized as the main structural protein of eye lens in vertebrates. The transparency and refractive properties of the lens are presumably linked to the high concentration of lens crystallin. However, the structure and function of β-crystallin is the least understood to date among the three major crystallin families. In this study,βB1-crystallin is molecularly cloned and sequenced from zebrafish. The expression and distribution of βB1-crystallin during embroygenesis is investigated, and the effects of starvation and various hormones on βB1-crystallin expression in zebrafish are further examined. βB1-crystallin was cloned from total RNA of zebrafish. With degenerate primers, we first obtained a partial cDNA sequence of 405 bp βB1-crystallin by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). The C-terminal end sequence with 680bp was cloned by 3’Rapid Amplification of cDNA Ends (3’RACE). Following by 24 hr zebrafish cDNA library screening, a complete (βB1-crystallin cDNA sequence of 943 bp in length was cloned. It contains 233 deduced amino acids and shares 57% to 68% identity with other vertebrates. The level of βB1-crystallin mRNA expression pattern in zebrafish embryos was substantially increased through early developmental stages in Northern blot. The result of whole-mount in situ hybridization showed that βB1-crystallin is abundant in the lens, but absent from other tissues. It suggests the tissue-specific expression of the gene concerned. To further analyze the effects of hormones and starvation on the expression of zebrafish βB1-crystallin, we intraperitoneally inject insulin and IGFs, and starved zebrafish for days. Real-time quantitative RT-PCR shows that insulin and IGFs evidently regulate zebrafish βB1-crystallin gene expression, and may play an important physiological role in the fish lens.