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研究生:呂笈歡
研究生(外文):Chi-Huan Lu
論文名稱:臺灣常見之笛鯛科魚類種屬基因鑑定及濱鯛肝臟維生素A含量之分析
論文名稱(外文):Studies on Genetic Identification of Common Lutjanidae in Taiwan and Level of Vitamin A in Liver of Etelis carbunculus
指導教授:黃登福黃登福引用關係
指導教授(外文):Deng-Fwu Hwang
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:114
中文關鍵詞:笛鯛科維生素 A
外文關鍵詞:LutjanidaeVitamin A
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笛鯛科魚類廣泛分佈於世界三大洋之熱帶及亞熱帶海域,屬珊瑚礁區之肉食性魚類,全世界共計有 4 亞科 21 屬 126種,而臺灣產則有 4 亞科 10 屬 52 種,其中如濱鯛 (Etelis carbunculus) 及長尾濱鯛 (Etelis coruscans) 等體長可達 100 cm 以上,其肝臟可能蘊含有大量維生素 A,若誤食則可能引發急性維生素 A 中毒。於 2007 年 12 月基隆地區,一家庭中三人因食用疑似大型笛鯛魚 (約重達 20 kg) 之肝臟而引起急性維生素 A 中毒的案例。為了解引起本次食物中毒之魚種,將臺北榮民總醫院所提供之檢體魚片經直接定序法 (direct sequence analysis) 及聚合?限制片段長度多型性 (polymerase chain reaction-restriction fragment length polymorphism; PCR-RFLP) 之技術針對粒線體 DNA (mitochondrial DNA; mtDNA) 中部份 cytochrome b (Cyt b) 基因序列做分析比對,結果顯示中毒檢體之魚種為濱鯛。接著收集不同魚體大小之濱鯛,利用高效能逆相層析儀 (high performance liquid chromatography; HPLC) 測定維生素 A 含量,得知濱鯛魚肉維生素 A 含量平均為 12 ± 2 IU/g (mean ± SD, n = 8) 而肝臟維生素 A 含量平均為 9,844 ± 7,812 IU/g。經迴歸分析結果得知,濱鯛之魚體及肝臟愈大,其肝臟所累積之維生素 A 含量愈高。
許多笛鯛科魚類因外觀型態而難以辨認,故本研究中將利用聚合?鏈鎖反應 (polymerase chain reaction; PCR) 之技術鑑定 10 種臺灣常見且外觀相似之笛鯛科魚類 (包含 Aphareus rutilans、E. carbunculus、E. coruscans、Lutjanus boutton、L. fulviflamma、L. quinquelineatus、L. rivulatus、L. stellatus、Pristipomoides filamentosus及 P. multidens)。以 6 組引子對建構 1,141 bp 完整 Cyt b 基因序列,再以 neighbor-joining (NJ) 進行演算其種屬演化關係,結果顯示 A. rutilans 與 P. multidens 歸納為同一族群 (group),與傳統型態分類學不同。另外,利用 PCR-RFLP 之技術,搜尋出兩種限制? BsmA I 及 Hsp92 II 進行序列切位分析,可確實將 10 種笛鯛科魚類作區分。綜合上述結果顯示,濱鯛肝臟之維生素 A含量與魚體及肝臟之大小有正相關性;直接定序法及 PCR-RFLP 之技術可應用在笛鯛魚科魚類之魚種鑑定。
The Lutjanidae fish is one of carnivorous coral reef fishes that distributes around the sea area of Taiwan, and it is also the commercially important fish species in Taiwan. Some of large-sized Lutjanidae fish like Etelis carbunculus and E. coruscans, may accumulate high level of vitamin A in the liver and it will cause hypervitaminosis A if ingesting incautiously. In December 2007, one case of hypervitaminosis A was due to ingesting the liver of large-sized Lutjanidae fish in Keelung City. In order to identify the species of large-sized Lutjanidae fish implicated into this food poisoning, 465 bp fragment sequence of cytochrome b gene on 2 species of Lutjanidae fish by using a pair of primers (L14735/H15149ad) was obtained successfully. The species of large-sized Lutjanidae fish implicated into this food was judged E. carbunculus by using direct sequence analysis and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Following, different body weight of E. carbunculus was collected from Ilan and Keelung. The level of vitamin A in the muscle and liver was analyzed by high performance liquid chromatography (HPLC). The average level of vitamin A in E. carbunculus muscle was 12 ± 2 IU/g (mean ± SD, n = 8) and liver was 9,844 ± 7,812 IU/g. Regression model shows that the more body weight and liver weight, the higher vitamin A level in liver of E. carbunculus.
It’s hard to identify Lutjanidae fish by morphological characteristics because of their similar morphology. In order to determine the complete 1,141 bp nucleotide sequence of mitochondrial cytochrome b gene (Cyt b) for 10 species of Lutjanidae (including Aphareus rutilans, Etelis carbunculus, E. coruscans, Lutjanus boutton, L. fulviflamma, L. quinquelineatus, L. rivulatus, L. stellatus, Pristipomoides filamentosus, and P. multidens) in Taiwan, it was found that 6 sets of fish-versatile primer to amplify 4 continuous and overlapping of entire Cyt b sequences. The complete 1,141 bp Cyt b sequences are utilized to establish phylogenetic relationship of 10 species of Lutjanidae in Taiwan. Entire Cyt b sequences from 10 species were evaluated by neighbor-joining (NJ) method to built the phylogenetic trees. The results show that P. filamentosus forms a group with A. rutilans rather than P. multidens and the phylogenetic relationships of other species are quite similar with traditional morphological taxonomy. Furthermore, PCR-RFLP technique was developed to identify the 10 species of Lutjanidae. One set of primer pair (L14735/H15149ad) was amplify 465/466 bp of partial Cyt b sequences, and 2 restriction enzymes (including BsmA I and Hsp92 II) were used to analyze the amplified fragments. The developed PCR-RFLP technique was applied to identify 10 species of Lutjanidae successfully.
中文摘要...................................................................................................1
Abstract.....................................................................................................3
第一章、文獻整理
一、笛鯛科魚類之簡介…………………………………………………..6
二、維生素 A 之簡介………………………………………………….10
三、高量維生素 A 之毒性…………………………………………….16
四、一般鑑定魚種的方法………………………………………………23
五、利用 PCR 技術鑑定物種………………………………………….27
六、利用 mitochondrial DNA 鑑定物種………………………………35
第二章、引起維生素 A 中毒之笛鯛科魚類其魚種之探討及肝臟維生素 A 含量之分析
一、前言…………………………………………………………………42
二、材料與方法…………………………………………………………43三、結果....................................................................................................50
四、討論....................................................................................................52
附表與附圖..............................................................................................54
第三章、臺灣常見之笛鯛科魚種基因鑑定
一、前言…………………………………………………………………68
二、材料與方法…………………………………………………………69
三、結果....................................................................................................72
四、討論....................................................................................................76
附表與附圖..............................................................................................78
參考文獻................................................................................................95
謝辭........................................................................................................112
附錄........................................................................................................114
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