臺灣博碩士論文加值系統

細胞色素b基因（Cytochrome b gene, Cyt b）為粒線體基因組 (mitochondrial DNA, mtDNA)上的一個功能性基因，位於tRNAGlu及tRNAThr兩基因之間，是目前最常應用於物種分類與親緣演化關係研究的一段核酸序列。在2003年更出現了以粒線體基因組中Cytochrome c oxidase subunit I (COI) 基因作為鑑定各類生物的論述，而刻意將其定義為生命條碼 (DNA barcode)。
而本研究為了鑑定假偽之有毒魨科魚種加工品及生鮮產品的原料物種來源，乃利用聚合酶連鎖反應 (Polymerase chain reaction, PCR）技術，並經測試篩選6組引子對，順利用以增幅並重建14種河魨魚種的完整Cyt b序列 (1137 bp)及COI序列 (1546 bp)。結果顯示魨科魚種在Cyt b 的種間相似度範圍分別為66.94%~95.75%，COI的種間相似度範圍分別為76.49%~99.94%。因此，這些結果表明，Cyt b基因區塊比COI基因區塊更適合作為魨科魚類種間差異的指標。
而加工製品常因加工條件過於嚴苛，容易使 DNA 發生嚴重斷裂，使DNA片段長度約在500 bp 以下，而造成引子組Lnewset-1/H15149難以增幅，因此為建立加工品之原料物種檢測模式，在Cyt b變異度最大區域自行設計增幅較小片段之引子對 (TLS-LC及TLS-HC)，並將引子組增幅之基因片段作為魨科魚類加工品DNA條碼，用以鑑定14種可能作為魨科魚種加工品原料之魚類，包括Lagocephalus gloveri、L. wheeleri、L. lunaris、L. inermis、Takifugu xanthopterus、T. oblongus、T. poecilonotus、T. pardalis、T. vermicularis、T. stictonotus、T. niphobles、Sphoeroides pachygaster、Ostracion cubicus 與 Arothron hispidus等。並且模擬市售加工品加工條件的不同 (約121 ℃，10~90 mim不等) 自製加工品，其中加工條件121 ℃，10~80 mim可明顯判讀，而條件在121 ℃，90 mim下的樣品則無法檢出。本研究亦實際將DNA條碼應用於市售加工品的鑑定，成功判讀多項市售疑似以魨科魚類為原料之加工製品。故推薦以TLS-LC及TLS-HC (144 bp) 作為鑑定假偽之有毒魨科魚種加工品及生鮮產品的原料物種之引子，並以此基因區塊作為DNA條碼驗證市售加工品中有疑問之樣本。本論文亦提供分子遺傳距離之數據分析，以重新檢驗並明確定義適合用來分類的形態特徵，以供未來其他相關研究之用。

關鍵字：DNA條碼、細胞色素b、細胞色素c氧化酶I、河魨、偽製加工品、物種鑑定、聚合酶連鎖反應。

Cytochrome b gene (Cyt b) is a functional gene between tRNAGlu and tRNAThr in mitochondria DNA (mtDNA), and which was usually used to study about phylogeny and evolution in vertebrate. In 2003, the so-called “DNA barcode” was proposed and allocated to identify organisms based on the sequences of cytochrome c oxidase subunit I (COI) gene in the mitochondria.
In order to identify the original species of raw materials and processed products, the complete Cyt b (1137 bp) and COI from the 14 pufferfish species including Lagocephalus gloveri、L. wheeleri、L. lunaris、L. inermis、Takifugu xanthopterus、T. oblongus、T. poecilonotus、T. pardalis、T. vermicularis、T. stictonotus、T. niphobles、Sphoeroides pachygaster、Ostracion cubicus and Arothron hispidus were amplified by PCR (Polymerase chain reaction) technique in this study. Six pairs of primers were successfully used to reconstruct the complete Cyt b sequences and the complete COI from the 14 pufferfish species. The homogeneous of the complete Cyt b and the complete COI among the 14 pufferfish species are from 66.94% to 95.75%, and from 76.49% to 99.94% individually. By the way, these results also shows that the Cyt b gene is more suitable for reconstructing species phylogeny of pufferfish than the COI gene.
In order to apply the DNA barcode technique in identification of various processed products, the primers were designed to amplify the short fragment within the length limitation of the confident sequencing. Than the DNA barcode technique was also applied to identify the species of L. gloveri、L. wheeleri、L. lunaris、L. inermis、T. xanthopterus、T. oblongus、T. poecilonotus、T. pardalis、T. vermicularis、T. stictonotus、T. niphobles、S. pachygaster、O. cubicus and A. hispidus in the simulated producte and the commercial processed products. The results indicated that DNA barcode of the simulated samples could be amplified under the condition 121 ℃ for 10 ~ 80 mim. In contrast, that were not DNA barcode fragament be amplified under the sterilize condition 121 ℃ for 90 mim. Therefore, the new design DNA barcode fragament amplified by primers TLS-LC and TLS-HC were recommended as DNA barcodes for raw materials and processed products.

Keywords: DNA barcode, COI, Cyt b, pufferfish, processed products, species identification, PCR.

目錄

目錄 i
圖目錄 .iv
表目錄 vii
中文摘要 1
Abstract 3
研究背景及目的 .5
文獻整理 10
ㄧ、原料河魨之特徵、分佈及毒性 10
(一) 克氏兔頭魨 11
(二) 懷氏兔頭魨 12
(三) 月尾兔頭魨 13
(四) 光兔頭魨 14
(五) 黃鰭多紀魨 14
(六) 橫紋多紀魨 15
(七) 黑點多紀魨 16
(八) 網狀多紀魨 16
(九) 豹紋東方魨 17
(十) 虫紋東方魨 18
(十一) 密點東方魨 19
(十二) 皺紋河魨 19
(十三) 粒突箱魨 20
(十四) 紋腹叉鼻魨 21
二、mtDNA之結構組成及特徵 22
三、利用cytochrome b gene差異鑑別物種 23
四、COI 和DNA barcoding 之相關研究 25
五、基因鑑別技術應用於加工製品 25
第一章、14種河魨魚種完整Cyt b 及COI 之基因序列及胺基酸序列分析 27
前 言 27
材料與方法 29
ㄧ、 實驗架構 29
二、 實驗材料 30
三、 實驗方法 30
四、 引子設計 31
結果與討論 35
圖表 41
第二章、基因條碼測試仿製及市售加工製品之種別鑑定 101
前 言 101
材料與方法 103
一、 實驗材料 103
二、 實驗方法 105
(一)仿製加工品製作方法 105
(二)粗萃DNA 106
(三)Primer之設計 106
結果與討論 108
圖表 112
總結論 118
參考文獻 120

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