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研究生:柯雅雯
研究生(外文):Ya-Wen Ka
論文名稱:南美白蝦多巴胺β羥化酶之cDNA分子選殖與免疫抗病之關聯性
論文名稱(外文):Molecular cloning of dopamine beta hydroxylase cDNA and its relation with immune response and disease resistance on the white shrimp , Litopenaeus vannamei
指導教授:鄭文騰鄭文騰引用關係張欽泉
指導教授(外文):Win-ton ChengChin-chyuan Chang
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:水產養殖系所
學門:農業科學學門
學類:漁業學類
論文種類:學術論文
論文出版年:2012
畢業學年度:101
語文別:中文
論文頁數:108
中文關鍵詞:南美白蝦多巴胺β羥化酶兒茶酚胺抗病能力
外文關鍵詞:Litopenaeus vannameidopamine beta hydroxylasecatecholaminesdisease resistance
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南美白蝦 (Litopenaeus vannamei) 多巴胺β羥化酶 (dopamine beta hydroxylase, DBH) 之序列選殖利用反轉錄聚合酶連鎖反應及快速擴增cDNA 末端法。基因全長為2419 bp,包含2241 bp之可轉譯區,2 bp之5端不可轉譯區,173 bp包含有停止碼與多腺嘌呤尾之3端不可轉譯區。可轉譯成747個氨基酸,分子量為84.4 kDa,估計等電點為5.15。南美白蝦DBH序列中發現2個功能區,分別為DOMON及Cu2-monooxygen domain以及3個醣基化位置。使用real-time PCR進行組織表現分析,發現南美白蝦胸神經之DBH有最高的表現量。低溫緊迫試驗結果顯示,南美白蝦經低溫緊迫後之胸神經、血球以及肝胰臟組織中DBH mRNA表現量皆有顯著上升的趨勢,而血淋巴中多巴胺、DBH以及正腎上腺素含量也顯著提升。disulfiram 注射試驗結果顯示,抑制DBH含量後之胸神經、血球以及肝胰臟組織中DBH mRNA表現量與血淋巴中之DBH與正腎上腺素含量皆有顯著下降的趨勢,而血淋巴中多巴胺含量是顯著性上升,顯示蝦類神經內分泌系統調節類似脊椎動物受disulfiram調控。免疫因子方面,抑制DBH含量後之總血球細胞數、半顆粒球細胞數、酚氧化酵素活性、超氧離子、超氧歧化酵素、吞噬活性、清除能力與存活率皆顯著降低。由上述結果顯示南美白蝦抑制DBH含量不但影響神經內分泌系統,同時也降低免疫與抗病能力。
Complementary (c) DNA encoding dopamine beta hydroxylase (DBH) messenger (m) RNA of the white shrimp, Litopenaeus vannamei, was obtained by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The 2419-bp cDNA contained an open reading frame (ORF) of 2241 bp, a 2-bp 5’-untranslated region (UTR), and a 173-bp 3’-UTR containing a stop codon and a poly A tail. The molecular mass of the deduced amino acid (aa) sequence (747 aa) was 84.4 kDa with an estimated pI of 5.15. The L. vannamei DBH contains a DOMON domain, a Cu2-monooxygen domain and three glycosylation sites. The mRNA expressions of DBH in various tissue with the highest expression level in thoracic ganglia by quantitative real-time PCR. The effect of hypothermal stress showed that the expression level of DBH in thoracic ganglia, haemocyte and hepatopancreas, and dopamine, DBH and norepinephrine contents in haemocyte were up-regulated. The effect of disulfiram showed that the expression level of DBH in thoracic ganglia, haemocyte and hepatopancreas, and DBH and norepinephrine contents in haemocyte were down-regulated, but dopamine was up-regulated. The results showed that the immunological parameters of the total haemocyte count, semiganular cell, phenoloxidase activity, superoxide anion, superoxide dismutase activity, phagocytosis activity, and cleareance efficiency were significantly decreased and reduced the resistance to disease resistance.
目錄
摘要 I
Abstract III
誌謝 V
目錄 VI
圖表目錄 XIII
第1章 前言 1
第2章 文獻回顧 4
2.1南美白蝦之分類地位 4
2.1.1南美白蝦之簡介 4
2.1.2臺灣之南美白蝦養殖概況 5
2.1.3臺灣之南美白蝦疾病問題 5
2.2甲殼類的免疫機制 6
2.2.1體液性免疫反應 6
2.2.1.1沈澱素 (precipitins) 6
2.2.1.2殺菌素 (bactericidins) 6
2.2.1.3凝集素 (agglutinins or lectins) 6
2.2.1.4抗微生物胜肽 (antimicrobial pepitides, AMPs) 7
2.2.1.5血球凝集素 (hemagglutinin) 7
2.2.1.6調理素 (opsonins) 7
2.2.2細胞性免疫 7
2.2.2.1透明球 8
2.2.2.2半顆粒球 8
2.2.2.3顆粒球 8
2.2.2.4吞噬作用 (phagocytosis) 9
2.2.2.5包膜作用 (encapsulation) 9
2.2.2.6反應氧族群 (Reactive oxygen intermediates, ROIs) 9
2.2.2.7原酚氧化酵素系統 (prophenoloxidase, proPO) 10
2.2.2.8 76kDa蛋白質 (peroxinecein) 10
2.2.2.9 β-1-3-glucan binding protein (BGBP) 11
2.2.2.10原酚氧化酵素活化酵素 (prophenoloxidase activating enzyme, ppA) 12
2.2.2.11 α2巨球蛋白 (α2-macroglobulin, α2M) 12
2.3甲殼類緊迫反應 12
2.4兒茶酚胺之合成 ..15
第3章 材料與方法 18
3.1南美白蝦DBH基因選殖 18
3.1.1實驗動物 18
3.1.2血球收集處理 18
3.1.3 Total RNA 的萃取 18
3.1.4多巴胺β羥化酶 (DBH) 基因引子 19
3.1.4.1引子 (primer) 之設計 19
3.1.4.2退化性引子 (degenerated primer) 之設計 19
3.1.4.3專一性引子之設計 19
3.1.5反轉錄聚合酶連鎖反應 (reverse transcription polymerase chain reaction, RT-PCR).. 20
3.1.5.1互補去氧核醣核酸之反轉錄之合成 (cDNA synthesis by reverse transcription, RT).. 20
3.1.5.2聚合酶鏈鎖反應 (polymerase chain reaction, PCR) 21
3.1.6 DNA凝膠電泳分析 22
3.1.7勝任細胞之製備 (competent cell preparation) 22
3.1.8 DNA重組接合 (ligation) 23
3.1.9質體轉型作用 (transformation) 23
3.1.10選殖株篩選 (screening) 23
3.1.11核酸序列之分析整理和比對 24
3.1.12 DBH基因親緣關係分析 (phylogeny) 24
3.2南美白蝦DBH基因之組織分佈 25
3.2.1 RT-PCR分析DBH基因組織分佈 25
3.2.2 Real-time PCR分析DBH基因組織表現 25
3.2.3相對定量PCR (Relative real-time quantitation PCR) 25
3.2.3.1資料分析彙整 26
3.3南美白蝦溫度緊迫對DBH、CHH基因與兒茶酚胺之影響 27
3.3.1實驗設計 27
3.3.2多巴胺、正腎上腺素含量分析 27
3.3.3 DBH含量分析 28
3.3.4基因表現分析 28
3.4南美白蝦DBH含量抑制對其DBH與兒茶酚胺之影響 28
3.4.1實驗設計 28
3.4.2多巴胺、正腎上腺素含量分析 29
3.4.3 DBH含量分析 29
3.4.4 DBH基因表現分析 29
3.5南美白蝦DBH含量抑制對其免疫與抗病能力之影響 29
3.5.1實驗設計 29
3.5.2總血球數之計數方法 29
3.5.3不同血球數之計數方法 29
3.5.4酚氧化酵素活性之測定方法 30
3.5.5超氧離子之測定方法 30
3.5.6超氧歧化酵素活性之分析 31
3.5.7蛋白質之測定方法 32
3.5.8吞噬活性及清除作用之分析 32
3.5.8.1吞噬活性之分析 32
3.5.8.2清除作用之測定 33
3.5.9攻擊試驗 33
3.6南美白蝦DBH含量抑制對其生理因子之影響 33
3.6.1實驗設計 33
3.6.2血淋巴葡萄糖含量之測定 34
3.6.3血淋巴乳酸含量之測定 34
3.7統計分析 35
第4章 結果 36
4.1南美白蝦DBH基因選殖及序列分析 36
4.2南美白蝦DBH基因親緣關係分析 40
4.3南美白蝦DBH基因之組織分布 42
4.4南美白蝦溫度緊迫對其基因與兒茶酚胺之影響 44
4.4.1溫度緊迫對南美白蝦基因之影響 44
4.4.1.1溫度緊迫對DBH基因之影響 44
4.4.1.2溫度緊迫對CHH基因之影響 44
4.4.2溫度緊迫對兒茶酚胺之影響 47
4.5南美白蝦DBH含量抑制對其DBH基因與兒茶酚胺之影響 49
4.5.1 DBH含量抑制對DBH基因之影響 49
4.5.2 DBH含量抑制對兒茶酚胺之影響 51
4.6南美白蝦DBH含量抑制對其免疫與抗病能力之影響 53
4.6.1 DBH含量抑制對總血球數之影響 53
4.6.2 DBH含量抑制對不同血球數之影響 54
4.6.3 DBH含量抑制對酚氧化酵素活性之影響 56
4.6.4 DBH含量抑制對超氧離子活性之影響 58
4.6.5 DBH含量抑制對超氧歧化酵素活性之影響 59
4.6.6 DBH含量抑制對吞噬活性及清除能力之影響 60
4.6.7 DBH含量抑制對抗病能力之影響 62
4.7 DBH含量抑制對生理因子之影響 64
4.7.1 DBH含量抑制對葡萄糖含量之影響 64
4.7.2 DBH含量抑制對乳酸含量之影響 65
第5章 討論 67
5.1南美白蝦DBH基因選殖 67
5.2南美白蝦DBH之組織表現 68
5.3南美白蝦溫度緊迫對其基因與兒茶酚胺之影響 68
5.4南美白蝦DBH含量抑制對其DBH與兒茶酚胺之影響 69
5.5南美白蝦DBH含量抑制對其免疫與抗病能力之影響 70
5.6南美白蝦DBH含量抑制對其生理因子之影響 74
第6章 結論 75
參考文獻 76
附錄一 臺灣近十年南美白蝦產量 99
附錄二 甲殼類原酚氧化酵素系統之訊息傳遞過程 100
附錄三 實驗儀器及器材 101
附錄四 反應試劑 102
作者介紹 108

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