- 學位論文
台灣產重要食用棘河魨及其加工製品DNA條碼－COI及Cyt b基因圖譜之探討
Studies on DNA barcoding of COI and Cyt b in Taiwanese Monacanthidae and related products
摘要
學者Hebert於2003年提出以粒線體細胞色素c氧化酶I (COI)片段基因作為鑑定物種之指標，並定義為DNA條碼 (DNA barcode)。然而許多魚類相關研究文獻則多以細胞色素b (Cyt b) 片段基因作為各種物種之鑑別指標。魚類經過加工會去除其形態上的特徵造成辨識上的困難，而也因此在國內外許多以低經濟價值魚種取代高經濟價值魚種之摻假情形以及包裝上標示不實之案例層出不窮。故本研究將以單棘魨類作為主要研究對象，建立其完整COI及Cyt b基因，探討此二基因區域之差異性，以挑選最適合做為單棘魨類魚種之DNA barcode指標，並分析其相關加工製品有無摻假及標示錯誤之現象。 本實驗自台灣高雄及台中採集四種單棘魨：單角革單及魨(Aluterus monoceros)、長尾革單棘魨 (A. scriptus)、杜氏刺鼻單棘魨(Cantherhines dumerilii)及單棘魨 (Monacanthus chinensis)。另外，自宜蘭老X林食品廠購得疑似單棘魨之罐頭、香魚片等加工製品。利用七組引子增幅單棘魨之COI (1553 bp) 及Cyt b (1141 bp) 片段，序列經GCG軟體分析比較發現各種單棘魨Cyt b序列之相似度 (74.25~89.06%) 大於COI之序列之相似度 (77.63~ 85.34%)，並且由基因序列圖亦顯示Cyt b基因序列中有較多變異性較大之區域，適用作為鑑別指標，可用來設計專一性引子及限制酶圖譜之分析。另外，COI有較高保留性，可用於設計共通性引子。故本研究分析Cyt b基因序列資料後，由差異度最大之區域設計一組可增幅約136 bp片段之引子 (包含引子)，並實際應用於加工食品，利用PCR增幅此段DNA序列，比對結果顯示罐頭樣品與單棘魨序列之相似度最高為66.54%而香魚片為71.45%，證明罐頭、香魚片之原料非單棘魨，同時亦證明所收集之加工製品有標示不清之現象。因此，本篇論文對未來食品原料物種之檢測技術，尤其是摻雜單棘魨科魚肉製品之檢測，提供了重要之參考依據。
並列摘要
“DNA barcode” was defined in 2003 by Hebert. The cytochrome c oxidase I (COI) was the target gene for species identification as a DNA barcode. However, most studies on fish species identification focused on the cytochrome b (Cyt b) gene as a DNA barcode of fish. Always, morphological characters of fish were partially or completely lost during processing, and it was difficult to identify the species of commercial fish products by externals. Commercial products were sometimes mislabeled or substituted by toxic or lower price fish species. The aim of this study was compared the divergency of COI gene and Cyt b gene of Monacanthidae in Taiwan seacoast, and chosen the suitable target gene as DNA barcode for identifying the commercial products. Aluterus monoceros, Aluterus scriptus, Cantherhines dumerilii and Monacanthus chinensis were collected from the seacoast of Taichung and Kaohsiung county in Taiwan. The commercial products of dry dressed fish fillets and fish cans were purchased from Ilan. 1553 bp of COI gene and 1141 bp of Cyt b gene were amplified by seven sets of primer. The homologous of Cyt b gene among these four species were from 74.25% to 89.06%. Homologous of COI gene among these four species were from 77.63% to 85.34%. The gene map was also shown that there were more point mutation in Cyt b region than COI region. In this study, we desiged a new set of common primer for amplifying 158 bp fragment in Cyt b gene region, and used for amplifying the degraded DNA from commercial products. After comparing the 87 bp of Cyt b gene of 12 dry dressed fish fillets and 6 can samples, the results shown that their fish source were not Monacanthidae which were all mislabeling. Therefore, it was the molecularly based study of Monacanthidae diversity and sheds light on the evolution and taxonomy of this major fish family and their commencial products.