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研究生:陳昱樺
研究生(外文):Yu-Hua Chen
論文名稱:東方果實蠅膜蛋白型鳥苷酸環化酶基因之選殖及其在蟲體內之表現
論文名稱(外文):Cloning, characterization, and developemental expression of a membrane form guanylyl cyclase gene in the oriental fruit fly, Bactrocera dorsalis (Hendel)
指導教授:路光暉
學位類別:碩士
校院名稱:國立中興大學
系所名稱:昆蟲學系
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:71
中文關鍵詞:東方果實蠅膜蛋白型鳥苷酸環化酶基因選殖保留性的半胱胺酸殘基天冬醯胺聯結的糖基化作用位
外文關鍵詞:oriental fruit flyBactrocera dorsalis (Hendel)membrane form guanylyl cyclaseCloningconserved cysteine residuesAsn-linked glycosylation site
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利用設計之簡併引子對(degenerate primer pair),對東方果實蠅(Bactrocera dorsalis (Hendel))頭部之總量RNA進行反轉錄作用(reverse transcription)及聚合酶鏈鎖反應(polymerase chain reaction),得到一膜蛋白型鳥苷酸環化酶基因序列,稱為Bdmgc1。Bdmgc1具有4,673 bp,內含一完整開放讀架(open reading frame),可推測出1,251個胺基酸序列。此胺基酸序列具有一個穿過膜一次的構型 (transmembrane domain),將此蛋白分成一N端的細胞膜外與配體結合的構型(extracellular ligand binding domain)及C端的細胞膜內的構型,而細胞膜內又可分成似蛋白質激酶構型(kinase homologous domain)及環化酶催化構型(cyclase catalytic domain)。BdMGC1在細胞膜外與配體結合的構型具有5個保留性的半胱胺酸殘基(conserved cysteine residues)及4個與天冬醯胺聯結的糖基化作用位(Asn-linked glycosylation sites);似蛋白質激酶構型具有22個在蛋白質激酶(protein kinase)中的保留性殘基,且具有一類似與ATP結合的作用位─Gly-X-X-Gly-X-X-Gly。將BdMGC1與哺乳動物的膜蛋白型鳥苷酸環化酶(GC-A~F)進行系統分類分析(phylogenetic analysis),發現BdMGC1歸類為鈉尿多肽受器(natriuretic peptide receptor)。利用專一性引子觀察Bdmgc1在東方果實蠅體內之表現情形,發現此一從頭部選殖出的基因在整個生活史(卵、幼蟲、蛹、成蟲)及成蟲的各體區(頭、胸、腹)皆有表現,其中又以幼蟲期及蛹期的表現量較多。BdMGC1在胚胎發育期間皆有表現;在幼蟲期的表現以第4日齡及第5日齡的表現量較多;在蛹期的表現則是隨著日齡的增加而增強。
In this study, we cloned a membrane form guanylyl cyclase gene from the head RNA of Bactrocera dorsalis (Hendel), named Bdmgc1. Bdmgc1 has 4763 bp which contains an open reading frame with 1,251 deduced residues. The deduced protein contains a signal transmembrane domain that divides the protein into an amino acid-terminal extracellular ligand binding domain and a carboxyl-terminal intracellular domain, which further divides in to a kinase homologous domain and a cyclase catalytic domain. Five conserved cysteine residues and four Asn-linked glycosylation sites were indentified in the extracellular ligand binding domain of Bdmgc1. The kinase homologous domain contains 22 out of 33 residues that are invariant or conserved in protein kinases and a likely ATP binding site─Gly-X-X-Gly-X-X-Gly. Phylogenetic analysis of BdMGC1 and other membrane form guanylyl cyclases suggests that BdMGC1 might be a member of natriuretic peptide receptors. Reverse transcription-polychain reaction analysis demonstrated that Bdmgc1 transcripts was present throughout life cycle of B. dorsalis, including egg, larva, pupa and adult stages, however there were higher expression levels in the larva and pupal stages than other stages. Bdmgc1 transcripts expressed throughout the embryo and larval development periods. In the pupal stage, Bdmgc1 experssed at the highest level in the pharate adults.
中文摘要-----------------------------------------1
英文摘要-----------------------------------------3
前言---------------------------------------------4
前人研究-----------------------------------------6
材料與方法---------------------------------------17
結果---------------------------------------------28
討論---------------------------------------------35
參考文獻-----------------------------------------41
圖表---------------------------------------------49
附錄---------------------------------------------70

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