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研究生:謝喻文
研究生(外文):Hsieh Yu-Wen
論文名稱:利用細胞色素b基因之PCR技術和基因序列作為紅鰭多紀魨和橫紋多紀魨原料及其加工品之魚種鑑定
論文名稱(外文):Application of polymerase chain reaction / direct sequence analysis of cytochrome b gene on identification of species for raw material and products of Puffer Takifugu rubripes and Takifugu oblongus
指導教授:黃登福黃登福引用關係
指導教授(外文):Hwang Deng-Fwu
學位類別:碩士
校院名稱:國立海洋大學
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:2000
畢業學年度:88
語文別:中文
論文頁數:66
中文關鍵詞:鑑定細胞色素b基因聚合脢連鎖反應河豚虎河豚瀧紋河豚
外文關鍵詞:authenticationcytochrome b genepolymerase chain reactionPufferTakifugu rubripesTakifugu oblongus
相關次數:
  • 被引用被引用:12
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  • 下載下載:32
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中文摘要
以PCR技術及直接定序分析新鮮、冷凍、烹煮過之紅鰭多紀魨及橫紋多紀魨和其製品之香魚片中粒線體DNA之cytochrome b gene部分片段,用以鑑別此376 bp序列之差異度。
結果發現,紅鰭多紀魨及橫紋多紀魨各有其獨特之基因型態。而且新鮮狀態、冷凍狀態及烹煮過之紅鰭多紀魨其三種狀態之此段序列並無差異。另外,類似情形之橫紋多紀魨和其製作之香魚片的此段序列亦無差異。
經由序列分析後發現,限制?BstZ I在紅鰭多紀魨之cytochrome b gene中有認識之切位,可將其切割成兩段,而限制?Aat II並無切位。然而限制?EcoR V在橫紋多紀魨之此段cytochrome b gene中具有切位,同樣可將其切割成兩段,相反地限制?BstZ I,Aat II則無切位。
由此二實驗證明了此技術可用以鑑定經過不同加工過程之紅鰭多紀魨和橫紋多紀魨魚肉且限制?切位之分析較直接定序提供了快速且廉價之鑑別方法。
Abstract
The polymerase chain reaction (PCR) technology and sequence analysis were used to determine the amount of genetic variation in a 376 nucleotides region of the mitochondrial cytochrome b gene in fresh, frozen, cooked meat of puffer Takifugu rubripes and fresh, frozen and dried dressed fillets of puffer Takifugu oblongus.
It was found that puffer Takifugu rubripes and T. oblongus had their unique genotypes. The range of sequence divergence between fresh, frozen and cooked meat of puffer Takifugu rubripes was not found and also between fresh, frozen and dried dressed fillets of puffer Takifugu oblongus.
Restriction enzyme BstZ I was found to cut the amplified region of cytochrome b gene of Takifugu rubripes. Restriction enzyme EcoR V was found to cut the amplified region of cytochrome b gene of Takifugu oblongus.
Restriction site analysis of amplified region of cytochrome b gene demonstrated a faster and less expensive method than sequencing for distinguishing PCR products of different processed puffer meat. Thus, restriction site analysis of PCR products can be used in conjunction with sequencing to identify species of different processed products of puffer Takifugu rubripes and T. oblongus.
目 錄
頁次
專有名詞對照表………………………………………….….…..1
中文摘要……………………………….………………………...2
英文摘要………………………………………….……………...3
本論文之發表情形………………………………………………4
文獻整理………………………………………………….……...5
一、前言………………………………………………….…...5
二、紅鰭多紀魨之特徵、分佈及毒性……….……………...6
三、橫紋多紀魨之特徵、分佈及毒性………………….…...6
四、一般鑑別魚種之方法…………….……………………...7
五、以mtDNA鑑定物種之由來及優點…………..…….…..9
六、應用mtDNA差異性鑑別魚種………..…………….…..9
七、應用mtDNA cytochrome b gene鑑別魚種………..……11
八、Cytochrome b gene差異性應用於魚類加工品之魚
種鑑定……………………………………………….…12
第一章、 利用細胞色素b基因之PCR技術和基因序列
作為紅鰭多紀魨原料及其加工品之魚種鑑定……15
一、前言……………………………………………………....15
二、材料與方法……………………………………………..17
(一) 實驗材料…………………………………………….17
(二) 實驗方法…………………………………………….17
1. 粗萃DNA……………………………………………...17
2. 引子設計……………………………………….……...18
3. DNA片段的大量增幅………………………………...18
4. PCR產物之純化與定序……………………………....19
5. 限制?分析確認PCR產物……………………….…..20
三、結果………………………………………………………21
四、討論………………………………………………………23
圖表…………………………………………………………..27
第二章、 利用細胞色素b基因之PCR技術和基因序列作
為橫紋多紀魨原料及其加工品之魚種鑑定………37
一、前言……………………………………………………...37
二、材料與方法……………………………………………..38
(一)實驗材料……………………………………………...38
(二)實驗方法……………………………………………...38
三、結果……………………………………………………...39
四、討論………………………………………………………41
圖表…………………………………………………………..42
參考文獻…………………….…………………………….…....53
謝辭………………………………………………………..…....64
附錄……………………………………………………………..65
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