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標題: | 淡水長臂大蝦β-微管蛋白基因的選殖與應用 Molecular Cloning and Application of the β-tubulin genes of Giant Freshwater Prawns(Mscrobrachirm rosenbergii) |
作者: | 陳炳宏 |
出版年 : | 2000 |
學位: | 碩士 |
摘要: | 所有的真核生物都有組成細胞骨架的微管(Microtubule),它是由α-微管蛋白(α-tubulin)及β-微管蛋白(β-tubulin)兩種主要的微管蛋白所組成。β-微管蛋白基因以多基因族系的形式廣泛存在於各種真核細胞中,且其序列在演化的過程中具有極高的保留性。迄今,雖然脊椎動物透過基因選殖的方式解讀大量基因譯碼,幫助我們更充份地瞭解此一基因產物的功能,然而海洋無脊椎動物仍然缺少這一方面訊息的研究。 本研究利用分子生物技術選殖淡水長臂大蝦(Macrobrachium rosenbergii)的β-微管蛋白基因並進行定序,以便瞭解基因的組成與分子的演化。首先設計引子,以聚合酵素連鎖反應擴增並選殖出淡水長臂蝦的β-微管蛋白基因片段;結果選殖出五種不同的淡水長臂大蝦β-微管蛋白基因片段。因此,淡水長臂大蝦的β-微管蛋白基因至少包括五個成員(Type1、2、3、4及5)的多基因族系。我們也應用延伸多聚合酵素連鎖反應(Extender Polymerase Chain Reaction)進行長臂蝦β-微管蛋白基因5'端與3'端序列的選殖,結果選殖出二個全長的β-微管蛋白基因,譯碼區序列全長為1359 bp與1368 bp,可轉譯出452個與455個氨基酸,他們譯碼區域的核甘酸序列與氨基酸序列相似度為81%與77%,但在3'端的歧異性很大。由聚類分析法(neighbor-joining method)分析淡水長臂大蝦β-微管蛋白基因之親源演化關係,顯示長臂蝦與美洲龍蝦(Homarus americanus)的β-微管蛋白基因最為相似。 我們構築含有不同的長臂蝦β-微管蛋白基因(Type 1)的啟動子連接水母綠螢光基因(EGFP cDNA)的片段以細胞質顯微注射的方式進行活體表現實驗,結果證實長臂蝦β-微管蛋白基因啟動子的確在日本稻田魚的胚胎中可以表現,其中以pEGFPSβ0.4(含Type 1的5'上游序列-102 bp至轉錄起始後+322 bp)的表現比pEGFPSβ0.1(只含Type 1上游序列-102 bp)好。此外,並成功的應用Tu-f6與Tu-r7此組引子增殖所有32種臺灣常見之蝦類與無脊椎動物的β-微管蛋白基因片段。未來將可透此組引子來選殖無脊動物β-微管蛋白基因而對無脊動物β-微管蛋白基因有更多的瞭解。 Microtubules is found in all eukaryotic cells which is majorly formed by polymerization of α-and β-tumulin (tub) subunits.β-tubulin,a multifunctional filament, is abundent in organelles and subcellular structures. β-tubulin gene, is highly conserved among eukaryotes. Despite the nucleotide and aminio acid sequences of tub from higher eukaryotes is well studied, few studies have been involved the marine invetebrate tub gene families, including arthropod. This study is to understand the organization structures of β-tubulin genes of giant freshwater prawns, macro brachium rosenbergii. Primers were designed and used for the β-tubulin genomic sequence PCR amplification. Five clones containing β-tubulin sequence of M.rosenbergii were isolated and sequenced. Results revealed that β- tubulin gene family may comprise at least five members (Type 1.2.3.4.5). We also used extender PCR (Extender Polymerase Chain Reaction) method to complete the 5' and 3' end of tub genomic sequence. Two types of M. rosenbergii tub genomic clones were isolated. Type 1 (1359 bp) and Type 5 (1368 bp). Type 1 and Type 5 tub encoded for 452 and 455 amino acid sequences, respectively. They exhited 81% and 71% similarities between nucleotide and amino acid sequences, respectively. But there existed high divergency at 3' region. Phylogenetic analysis of M. rosenbergii β-tubulin genes by neighbor-joining method analysis suggest that M. rosenbergii β-tubulin gene is closely related to American lobster (Homarus americanus). We microinjection two DNA constracts into the fertilized egg of medaka : pEFGPSβ0.4 (Upstream -102 bp to +322 bp) and pEFGPSβ0.1 (only contains upstream -102 bp). Data showed that reporter gene (EGFP cDNA) could be driven by the promoter sequence we cloned at the medaka embryos. The GFP expression of embryos injected with pEFGPSβ0.4 was stronger than that of pEFGPSβ0.1. PCR-based strategy with one set of specific primers : Tu-f6 and Tu-r7. The predicted 426 bp fragment was always amplified from 32 different invertebrate species. This primer pair may be widely applicable for amplifying invertebrate β-tubulin genes and may also provide a useful tool for genome analyses of β-tubulin in other species of invertebrate. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75092 |
全文授權: | 未授權 |
顯示於系所單位: | 漁業科學研究所 |
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