臺灣博碩士論文加值系統

　　在台灣冬季時溫度和鹽度適合烏魚(Mugil cephalus)生殖腺的發育，因此會有自大陸沿岸的烏魚向南洄游至台灣西部、西南部產卵，烏魚子、烏魚膘、烏魚肫皆是台灣烏魚的重要經濟價值之因。本研究中希望以分子生物學為基礎，利用分子標誌(molecular markers)測定生物體核酸序列之變異，以期找出可清楚判定烏魚子來源的方法，藉以改善市場中辨別烏魚子的問題，並透過發展快速鑑別技術，協助種苗生產業者之種魚選別。
　　實驗中以 cyt b基因進行序列分析，結果可將烏魚區分成養殖族群、罕見族群及洄游族群，而利用multiplex PCR則可呈現分子量大小分別為G1(549 bp)、G2(362 bp)、G3(283 bp)三種產物，對照cyt b序列分析結果， G1落在罕見養殖群、G2落在洄游群及G3落在主要養殖群；PCR-RFLP方式擴增16S rRNA片段大小約為636 bp，以限制酶作用後可分為洄游型(M-type)(無切位)、本地第一型(R1-type)會有約380 bp和260bp條帶；第二型(R2-type)則顯示約500 bp和130 bp條帶；在脂肪酸組成分析中發現，養殖烏魚的C18:1與18:2的脂肪酸含量較野生、進口者為高，此現象可能與飼料之脂肪酸來源與生長環境有關。
　　台灣地區冬至期間洄游至台灣西部沿海之烏魚族群應屬於特定族群，但由於各族群均於台灣沿岸產卵，因此河口所採集之魚苗混有不同族群，若能以DNA分析方法輔以脂肪酸分析，則較能具有足夠之鑑別力。

The mullet (Mugil cephalus) from coast of China migrate to the west and southwestern of Taiwan during winter, because of the water temperature and salinity suitable for spawning. Salted mullet roe,spermary of mullet and stomach of mullet were the economic sector in Taiwan. Based on molecular biology, this study used molecular markers to measure the variation of nucleic acid sequence. The research was to clearly identify the source of mullet in the market and to develop rapid identification technology to help sort the species of fish seed production industry sorting.
Sequence analysis with cyt b gene, revealed that mullet were divided into aquaculture group, rare group and migratory group. There were three molecular size of multiplex PCR, G1 (549 bp), G2 (362 bp), G3 (283 bp). Comparised with cyt b gene, G1 was equal to rare group, G2 equal to migratory group and G3 equal to main aquaculture group. Amplified by PCR-RFLP methods 16S rRNA fragment size was about 636 bp. By reaction with restriction enzyme, mullet could be divided into migratory type (M-type) (no cleavage), local first type (R1-type) with about 380 bp and 260 bp bands, and local second type (R2-type) with about 500 bp and 130 bp bands. Aquaculture group had the highest fatty acid content in wild and import mullet, especially of 18:1 and 18:2 of fatty acid. This phenomenon might be related to fatty acid source in feed and growing environment.
The mullet migration to the west coast of Taiwan during the winter should belong to the specific groups. However, due to various groups were spawning around Taiwan coast, the larvae were mixed with different groups. Using DNA analysis compled with fatty acid analysis would be reliable to identify group.

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中文摘要--------------------------------------------------------------------- Ⅰ
英文摘要--------------------------------------------------------------------- Ⅱ
目錄--------------------------------------------------------------------------- Ⅲ
表目錄------------------------------------------------------------------------ Ⅴ
圖目錄------------------------------------------------------------------------ Ⅵ
第壹章　前言--------------------------------------------------------------- 1
一、烏魚的簡介--------------------------------------------------------- 1
二、烏魚與台灣漁業經濟的關聯------------------------------------ 4
三、台灣地區烏魚子的生產------------------------------------------ 5
四、台灣地區烏魚族群之分析方法--------------------------------- 7
(一) 定序法------------------------------------------------------------ 7
(二) PCR-RFLP分析法--------------------------------------------- 8
(三) AFLP指紋圖譜------------------------------------------------- 11
(四) 多套式聚合酶連鎖反應（Multiplex PCR）--------------- 12
(五) 脂肪酸組成分析------------------------------------------------ 12
(六) 其他--------------------------------------------------------------- 14
五、研究動機------------------------------------------------------------- 16
第貳章　材料與方法------------------------------------------------------ 17
一、樣品的採集---------------------------------------------------------- 17
(一) 台灣各區樣品的採集------------------------------------------ 17
(二) 脂肪酸含量組成分析------------------------------------------ 17
二、樣品的處理---------------------------------------------------------- 17
(一) DNA分析-------------------------------------------------------- 17
1. 萃取烏魚組織DNA--------------------------------------------- 17
2. PCR擴增---------------------------------------------------------- 18
3. 序列分析---------------------------------------------------------- 18
4. 粒線體16s rRNA PCR-RFLP---------------------------------- 18
5. 多套式聚合酶連鎖反應（Multiplex PCR）---------------- 19
(二) 脂肪酸含量組成分析------------------------------------------ 20
1. 樣品採樣----------------------------------------------------------- 20
2. 樣品分析方法----------------------------------------------------- 20
第參章　結果--------------------------------------------------------------- 23
一、DNA分析----------------------------------------------------------- 23
1. 序列分析------------------------------------------------------------ 23
2. 粒線體16s rRNA PCR-RFLP------------------------------------ 24
3. 多套式聚合酶連鎖反應（Multiplex PCR）------------------ 24
4. 各區採樣地之結果整理------------------------------------------- 25
二、脂肪酸含量組成分析---------------------------------------------- 25
1. 不同來源之烏魚子脂肪酸組成---------------------------------- 25
2. 隨機採樣之烏魚子樣品分析結果------------------------------- 25
3. 烏魚子脂肪酸組成成份------------------------------------------- 26
4. 留烏(魚肉)脂肪酸分析結果-------------------------------------- 26
第肆章　討論---------------------------------------------------------------- 27
一、cyt b基因------------------------------------------------------------ 27
二、multiplex PCR------------------------------------------------------- 27
三、PCR-RFLP----------------------------------------------------------- 27
四、脂肪酸---------------------------------------------------------------- 28
五、未來研究方向------------------------------------------------------- 29
第伍章　參考文獻---------------------------------------------------------- 30

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