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研究生:林順啟
論文名稱:斑馬魚hoxa1a基因啟動子及上游調控DNA序列的選殖與其表現的分析
論文名稱(外文):Cloning and expression analysis zebrafish hoxala promoter and upatream regulation DNA sequence
指導教授:胡清華胡清華引用關係黃聲蘋
學位類別:碩士
校院名稱:國立海洋大學
系所名稱:水產生物技術研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:70
中文關鍵詞:斑馬魚
外文關鍵詞:hoxa1a
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中文摘要
斑馬魚(Danio rerio) hoxa1a基因為一homeobox基因,所轉譯之蛋白質有一60個氨基酸之homeodomain其中有7個特異(diagnostic)氨基酸其功能可能與DNA結合之特異性有所關聯。另外在homeodomain之N端區域的第19或第20個胺基酸則具有大段高度保守性之六個氨基酸(WMKVR)存在於所有paralogus group (PG) 1 Hox蛋白質中。斑馬魚hoxa1a不同於其他PG1基因在於其因為兩種splicing方式而具有兩種不同長度之mRNA。經由全覆式原位雜合反應可以得知hoxa1a主要表現在中腦及後腦前端腹面之一些神經細胞上。目前對於hoxa1a基因上游區域的調控並不清楚,故本論文利用基因轉殖的技術來詳細研究Hoxa1a基因上游區域的cis-acting element。將不同長度之上游調控區DNA片段與綠螢光蛋白cDNA(報導基因)相連,以顯微注射之方式,將其注射至斑馬魚受精卵中,觀察到從轉譯起始點之上游片段-2946/-54、-2482/-54、-2031/-54和-1611/-54之hoxa1a基因上游序列在轉殖胚胎中水母綠色螢光之表現率分別為31.97%、32.32%、33.68%和30.21%,但所表現之位置均只在於卵黃囊中;而利用coinjection方式將上述四個質體分別與hoxa1a基因3΄端下游5kb DNA序列一起注射至斑馬魚胚胎中,觀察其綠螢光表現率為33.15%、32.83%、33.52%和30.09%,雖然其表現率並無任何差別但其表現模式可分為2種;-2946/-54和-2482/-54除了在卵黃囊中有表現外,在神經、肌肉及表皮等地方亦有表現,其中在神經細胞主要是表現在後腦前端之神經中,而注射-2031/-54和-1611/-54質體時兩者則與單獨注射相同僅僅表現在卯黃囊中,以上結果顯示hoxa1a基因之上游調控區域能指使水母綠色螢光表現在特定神經細胞上為位於轉譯起始點之上游2482bp至2031bp這一段DNA內,並且顯示hoxa1a基因調控機制除了需要本身之調控區域外尚需要其基因3΄端下游DNA序列來幫助調控其基因表現。另外利用5΄RACE來確認hoxa1a mRNA轉錄之起啟點目前已得到hoxa1a之5΄UTR(untranslation region)有1.4kb長。
Abstract
Zebrafish hoxa1a is a homeobox gene. It encodes a polypeptide including a 60 amino acid homeodomain. Seven diagnostic residues that confering specific DNA binding locate in the homedomain. In addition, there are conserved hexapeptide sequence, WMKVR, that lies 19 or 20 amino acids N-terminal of the homeodoamin. Zebrafish hoxa1a has a splice variant resulting two different length mRNA. Results of whole-mount in-situ hybridization showed that hoxa1a was expressed in the ventral region of both the midbrain and anterior hindbrain neuronal cells. Because of gene regulation mechanism of hoxa1a is not clear, we used transient transgenic analysis to dissect cis-regulatory elements of hoxa1a promoter and 5΄upstream region in this thesis. Four different lengths of 5΄upstream region of hoxa1a were fused with the GFP reporter gene and microinjected into one-cell zebrafish zygotes. Results showed that GFP expression rate of embryos injected with different length of 5΄ upstream sequence including nucleotide -2946/-54bp、-2482/-54bp、-2031/-54bp and -1611/-54bp were 31.97%、32.32%、33.68% and 30.21%, respectively. However, green fluorescence was only observed in the yolk sac. While 5kb hoxa1a 3΄untranslation region was coinjection with GFP constructs containing different length of 5΄upstream region, GFP expression rates were 33.15%、32.83%、33.52% and 30.09%, respectively. There are two different GFP expression pattern:(1)when -2946/-54bp or-2482/-54bp constructs were injected, green fluorescence was observed not only in the yolk sac but also expressed in the neuron、muscle and skin.(2)when- 2031/-54bp or-1611/-54bp constructs were injected, GFP expression was only observed in the yolk sac. These results suggest that DNA segment between —2482bp and —2031bp of hoxa1a 5΄upstream region in combination of hoxa1a 3΄ untranslation region may confer some neuron-specific GFP expression. In addition 5΄-RACE was used to find mRNA transcription start site and we have identified 1.4kb long 5΄ untranslated region.
目錄
中文摘要………………………………………………………………….I
英文摘要………………………………………………………………...II
目錄…………………………………………………………...………...IV
表目錄………………………………………………………….………..V
圖目錄…………………………………………………………..………VI
緒論………………………………………………………………………1
材料與方法………………………………………………………………9
結果……………………………………………………………………..29
討論……………………………………………………………………..33
參考文獻……………………………………………………………..…37
圖表…………………………………………………………………….44
附錄……………………………………………………………………..67
表目錄
Tabel 1…………………………………………………………………..64
Table 2…………………………………………………………………..65
Tabel 3………………………………………………………………..…66
圖目錄
圖一、Location of probe used for BAC library screening。……………..44
圖二、ZlabRT550TOPO#4 probe specificity test。…………………..….45
圖三、Zebrafish BAC library screening。……………………………..…46
圖四、Southern blot analyses of BAC 154-L-6 clone。……………….…47
圖五、Structure of hoxa1a translation start site 5΄ upstream 10kb region/EGFP-ITR。…………………………………...…………48
圖六、BLASTX result of hoxa1a translation start site 5΄upstream 10kb region from NCBI。…………………………………………...…49
圖七、Structure of hoxa1a translation start site 5΄upstream 3kb region 6c(3k)/LacZ-ITR。………………………………………………50
圖八、5΄upstream sequences (-3006~+116) of the translation start site of zebrafish hoxa1a gene。…………………………………………51
圖九、Structure of different length of 5΄upstream region of translation start site of Hoxa1a/EGFP-1。…………………………………..53
圖十、GFP expression in the yolk sac of the Hoxa1a/pEGFP-1#9 injected 24hpf and 48hpf embryos。……………………………………54
圖十一、GFP expression in the yolk sac of the Hoxa1a/pEGFP-1#1-15 injected 24hpf and 48hpf embryos。…………………………….55
圖十二、GFP expression in the yolk sac of the Hoxa1a/pEGFP-1#2-2 injected 24hpf and 48hpf embryos。…………………………….56
圖十三、GFP expression in the yolk sac of the Hoxa1a/pEGFP-1#3-6 injected 24hpf and 48hpf embryos。…………………………….57
圖十四、Structure of different length of 5΄upstream region of translation start site of Hoxa1a/EGFP-1 and hoxa1a 3΄UTR downstream 5kb region。…………………………………………………58
圖十五、GFP expression pattern in 24 and 48hpf embryos when coinjected with Hoxa1a/pEGFP-1#9 and hoxa1a downstream ~5kb DNA。…………………………………………………..59
圖十六、GFP expression pattern in 24 and 48hpf embryos when coinjected with Hoxa1a/pEGFP-1#1-15 and hoxa1a downstream ~5kb DNA。……………………………………..60
圖十七、GFP expression pattern in 24 and 48hpf embryos when coinjected with Hoxa1a/pEGFP-1#2-2 and hoxa1a downstream ~5kb DNA。…………………………………………………..61
圖十八、GFP expression pattern in 24 and 48hpf embryos when coinjected with Hoxa1a/pEGFP-1#3-6 and hoxa1a downstream ~5kb DNA。…………………………………………………..62
圖十九、Confirmation of hoxa1a 5΄RACE end by RT-PCR and Southern blot analysis。…………………………………………………63
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