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標題: | 全身性及組織專一性基因在模式魚種的研究 Studying on the Non-specific and Tissue-specific Genes of Model Fish |
作者: | 洪龍賢 |
出版年 : | 2001 |
學位: | 碩士 |
摘要: | 由於日本稻田魚(medaka )擁有下列諸多優點:卵徑大且透明、易於顯微注射、體色淺而易於觀察、產卵時間可用光週期自由控制、胚胎發育快速、行體外受精、全年皆可產卵等等,所以非常適合用以研究轉殖基因在活體脊椎動物上的功能、調控與遺傳等各種表現情況。然而,送入基因轉殖魚體內的外來 DNA ,常常有表現率低、且多為差異性(variegated)及鑲嵌性(mosaic)表現、以及不易嵌入染色體中導致遺傳率較低等重大缺點存在。為瞭解決這些問題,本研究利用第二型腺聯病毒(adeno- associated virus , AAV)的末端重複序列 ( inverted terminal repeats , ITR )置於外來 DNA 兩端,並用細胞質顯微注射的方式將 DNA 送入一細胞期的受精卵中, DNA的構築則使用稻田魚本身的全身性表現?動子(β一actin ) ,並以綠螢光蛋白( green fluorescent protein , GFP )當作報導基因。結果發現加了 AAV ITR 之後,可大幅提升親代的均勻表現率達15.1 %。同時也從冷凍切片進一步證實其身體內部亦為均勻表現全身性螢光的情形,且在這些均勻表現的轉殖魚親代中,有 44 %能把外來 DNA 穩定遺傳給子代。接著利用南方轉印法,證實外來 DNA 在染色體中的嵌入情形為多重拷貝嵌入。綜以上所言, AAV ITR 或可應用於提升各種外來 DNA 的均勻表現率,並有利於?動子的分析和轉殖魚的篩選,故本論文第一章的部份主要著重於轉殖技術的建立。 而在第二章的部份則鎖定斑馬魚的 TnT1 gene 作為研究的對象。所謂 T 型肌鈣蛋白(Troponin T , TnT )是肌肉收縮單元中極為重要的結構性蛋白質,在哺乳動物中依表現位置可分為 slow muscle isoform ( TnT1) , cardiac isoform ( TnT2)與 fast muscle isoform ( TnT3 ) ,主要生理功能是調節粗肌絲( thick filament)與細肌絲(thin filament)之間的滑動,完成收縮運動。可是這麼重要的基因群,在魚類的研究卻相當有限,許多的TnT isoforms 仍然不知或尚未被鑑定,至於在基因結構與分子調控方面上的研究更是完全闕如。本研究以斑馬魚(Danio rerio)為實驗魚種,嘗試去瞭解TnT基因的異質性,分子結構,在胚胎發育過程時的動態表現,以及基因調節機制。本研究自斑馬魚中選殖出完整的 slow TnT ( ZF-TnT1 ) isoform , cDNA 含有基因全長為 1432bP 個核甘酸並可轉譯出 290 個胺基酸,和已知物種之TnT序列相較下,發現與雞的TnT2有85%的相似度,但和鮭魚TnT 1 isoform , 鮭魚TnT3 isoform,與斑馬魚TnT3 isoform的相似度則只有65 % , 54 % 與 60%。以 whole- mount in situ hybridization 檢視其時空表現情形,發現 ZF-TnT1 isoform 最早表現於 12 hours post-ferterlizatin (hpf) 的somites ,隨著發育至24 hpf ,表現量達到高峰,但36hpf 時較早形成的 somites 逐漸停止表現,只剩下末端新形成之somites 有表現,至48 hpf時,在軀幹部的體節已不復表現,但轉移至 fin bud 與頭部肌肉。由冷凍切片的結果證實, ZF-TnT1 基因,早期(15 hpf)主要表現在 adaxial cell ( slow muscle precursor ) ,在 24 hpf,隨著肌肉的分化慢慢擴散至快肌的部分,到了36 hpf則主要在慢肌表現,推斷ZF-TnT1基因是一種在快慢肌過渡型表現的基因。 A green fluorescent protein (GFP) cDNA flanked by inverted terminal repeats (ITR) of adeno-associated virus was constructed. The construct sharply improved the efficiency and specificity of the transient expression of genes driven by general promoters (medaka β-actin) and muscle-specific promoter (zebrafish α-actin) in transgenic medaka. In addition, treatment with ITR sequence-containing constructs resulted in a dramatic increase in the number of embryos showing uniform GFP-expression at F0. Of the GFP-positive embryos, showed homogenous GFP-expression for the derivative constructs of the α-actin and β-actin promoters, respectively. As a result of uniform GFP-expression, green fluorescence in founders was a) extended for an entire lifetime without degradation, and b) transmitted as a genetic trait to Fl and F2 progeny of some transgenic lines via Mendelian inheritance. A Southern blot analysis revealed a random integration of the transgene into the genome of founders and progeny in both head-to-tail and tail-to-tail concatemerization patterns. Interestingly, some transgenic medaka with uniform and strong fluorescence could be visually noticeable to the unaided eye. On the other way, in chapter 2 I focused my research on the zebrafish TnT 1 gene. At first, what is called Troponin complex One of muscle structural proteins, is composed of three subunits: Troponin C (TnC)Troponin T (TnT) and Troponin I (TnI) . Based on the expressional locations, TnT is subdivided into slow muscle type (TnT 1), cardiac muscle type (TnT2) and fast muscle type (TnT3). Although heterogeneities of TnT have been studied in mammal and chicken, extremely few fish TnT genes are known in terms of gene structure and regulation. We cloned a full-length of zebrafish TnT1 (ZF-TnT1) cDNA from 24 hpf embryos. This ZF-TnT1 cDNA was around 1.4 kb and encoded 290 amino acid residues, in which glutamic acids were rich in the N-terminus. Compared with chicken TnT2, salmon TnT1, zebrafish TnT3 and salmon TnT3, ZF-TnT1 shared 84, 65, 60 and 54﹪amino acid identity, respectively. Using whole-mount in situ hybridization, I found that ZF-TnT1 transcripts were first detectablen at 12 hpf embryos, increased substantially at 24 hpf, and then declined gradually until 48 hpf stage. ZF-TnT1 signals were totallyundetectable in the trunk muscle of 48 hpf embryos, but, interestingly enough, ZF-TnT1 transcripts were found in fin buds and head muscle. Using cryosection, I found that ZF-TnT1 transitional expression in slow and fast muscles. These all evidences strongly suggest that ZF-TnT1 is a muscle and stage specific TnT isoform, which may be involved in the early development of muscle cells. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75254 |
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顯示於系所單位: | 漁業科學研究所 |
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