- 學位論文
斑馬魚第一型角質蛋白基因keratin 18-like的表現
The Expression of a Zebrafish Type I Keratin Gene, keratin 18-like
摘要
中間絲蛋白(intermediate filament)共分五大類，第一型角質蛋白(type I keratin)及第二型角質蛋白(type II keratin)分屬其中最主要之兩大類別。已知在哺乳動物中，角質蛋白主要表現於上皮細胞，然而目前對於斑馬魚角質蛋白的研究很少。選取一段斑馬魚(Danio rerio)第一型角質蛋白基因keratin 18-like cDNA，以此段序列合成之探針進行原位雜交染色法，觀察斑馬魚胚胎中此型角質蛋白表現情形，發現自受精卵起即有zkrt18-like表現，可知zkrt18-like具母體效應(maternal effect)；當受精後8小時，zkrt18-like在胚胎表層表現，至受精後10小時已遍佈胚胎表層；受精後20小時，zkrt18-like除了在表皮表現外，也在胚胎腹側表現；切片觀察受精後48小時的胚胎，在背大動脈、腸道及原腎管等位置有表現。利用已建立之帶有zkrt18-like上游長度3.3 kb區域連接紅螢光蛋白報導基因的斑馬魚品系zkrt18-like-RFP，比較內生性ZKRT18-LIKE表現與zkrt18-like-RFP品系的紅螢光表現，雌性轉殖魚(zkrt18-like-RFP♀)與野生型雄魚配對所產下之F2，全部於一細胞時期便有紅螢光表現，顯示由zkrt18-like上游片段驅動之紅螢光表現亦具母體效應；雄性轉殖魚(zkrt18-like-RFP♂)與野生型雌魚配對所產下之F2則自受精後14小時起始可觀察到紅螢光在胚胎表層表現，此後持續於表皮表現；切片觀察受精後48小時的zkrt18-like-RFP品系，可見到背大動脈、腸道及原腎管中亦有紅螢光表現，顯示zkrt18-like-RFP品系之紅螢光與zkrt18-like表現一致。同時以紅螢光RNA探針進行原位雜交染色法，偵測受精後8小時及20小時之zkrt18-like-RFP胚胎，證實紅螢光之mRNA與內生性zkrt18-like mRNA表現同步。經反轉錄-聚合酵素鏈鎖反應偵測zkrt18-like-RFP♂ F2之紅螢光基因，得知其紅螢光於受精後3小時開始表現，因此推估內生性zkrt18-like開啟時間約於囊胚期中期，這個現象符合魚類及兩生類的mid-blastula transition現象。此外，觀察zkrt18-like-RFP品系幼魚及成魚，可知ZKRT18-LIKE在魚體終生表現於眼、腸、肝、鱗片上層表皮、脊髓等位置，不同於角質蛋白主要在上皮細胞表現的哺乳動物。
並列摘要
Type I (acidic) and type II (basic) keratins, two major classes of intermediate filament proteins, express mainly in epithelial cells in mammals. However, they are little known in zebrafish. Using a partial cDNA sequence of a zebrafish type I keratin zkrt18-like as probes to observe the mRNA expression pattern of zkrt18-like, the zkrt18-like mRNA signal was detected in one cell stage embryos by whole mount in situ hybridization, suggesting that the zkrt18-like mRNA was inherited through the maternal effect. The zkrt18-like expressed in envelope layer of 8 hours-post-fertilization (hpf) embryos, and it expressed in all surface cells after 10 hpf. Moreover, the zkrt18-like transcripts not only presented in epidermal cells but also presented in the venter of 20 hpf embryos. Through the cross section of 48 hpf embryos, the expression in venter was observed to be in the dorsal aorta, gut and pronephric duct. Comparing the expression profile of the reporter gene and the endogenetic zkrt18-like mRNA by an established transgenic line zkrt18-like-RFP, which contained the upstream 3.3 kb of zkrt18-like fused with red fluorescent protein (rfp), the RFP expression in all the progeny from transgenic F1 female mated with wild-type male in one cell stage suggests that the rfp regulated by zkrt18-like promoter was also inherited through the maternal effect. The RFP expression of the F2 from transgenic F1 male mated with wild-type female (zkrt18-like-RFP♂ F2) was on the surface of 14 hpf embryos, and kept expressing in epidermal cells. Through the cross section of 48 hpf zkrt18-like-RFP embryos, the RFP expression was also found in dorsal aorta, gut and pronephric duct, indicating that the RFP expression was similar to the zkrt18-like expression pattern. Detecting the rfp mRNA of 8 hpf and 10 hpf zkrt18-like-RFP♂ F2 embryos by whole mount in situ hybridization, it has been demonstrated that the rfp transcripts was in accordance with the endogenous zkrt18-like mRNA expression. By RT-PCR analysis, the rfp expression of zkrt18-like-RFP♂ F2 initiated at 3 hpf, suggesting that the zkrt18-like expression initiated during the mid-blastula stage and conformed the mid-blastula transition phenomenon in fish and amphibian embryos. Besides, through the observation on the zkrt18-like-RFP larva and adult, the ZKRT18-LIKE was observed to exist in eyes, intestine, liver, scale epidermis, and spinal cord during its whole life. This result is different from those keratins in mammals, which mainly express in epithelial cell.