臺灣博碩士論文加值系統

有毒蟹在臺灣沿岸珊瑚礁與岩礁水域常被發現，且由目前國外毒蟹研究資料得知，蟹類科別中以扇蟹科的有毒種類較多，毒性較強。而海洋生物的毒性與環境息息相關，即使是同種生物，在不同水域也具有不相同的毒性，甚至同一水域的每個個體毒性都不相同。然而，蟹類為雜食性的動物，其攝食的範圍非常廣，包括海藻及魚蝦蟹貝等小型動物，甚至是底泥中的生物碎屑或有機質，皆可能是牠的攝食範圍，而如果該環境具有海洋生物毒，只要是對於海洋生物毒具有抵抗性的蟹類就有可能經由食物鏈，將海洋生物毒蓄積於體內。為了鑑定有毒蟹類及建立基因圖譜資料，乃以生物毒性檢驗法 (mouse bioassay)、高效能液相層析 (high performance liquid chromatography; HPLC) 檢測毒性及毒成分，並利用直接定序法和聚合?鏈鎖反應限制片段長度多型性 (polymerase chain reaction-restriction fragment length polymorphism; PCR-RFLP) 技術鑑定物種基因相關性，針對臺灣蟹類花紋愛潔蟹 (Atergatis floridus)、正直愛潔蟹 (A. integerrimus)、廣闊愛潔蟹 (A. latissimus)、絨毛仿銀杏蟹 (Actaeodes tomentosus)、隆背瓢蟹 (Carpilius convexus)、紅斑瓢蟹 (C. maculates)、鈍額曲毛蟹 (Camposcia retusa)、光手酋婦蟹 (Eriphia sebana)、灰青鄰扇蟹 (Juxtaxanthias lividus)、鏽花脊熟若蟹 (Lophozozymus pictor)、羅氏毛足蟹 (Lachnopodus rodgersi) 及銅鑄熟若蟹 (Zosimus aeneus) 等 12 種蟹類物種建立近年來蟹類毒性、毒成分資料及其基因圖譜。
其中 11 種蟹類，由生物毒性檢驗法研究結果得知，J. lividus 最高個體毒性值為 167 老鼠單位 (mouse unit; MU)；平均個體毒性值為 97 ± 52 (mean ± SD) MU，而 Z. aeneus 的最高個體毒性值則為 3,503 MU；平均個體毒性值為 897 ± 1,306 MU，而其它 9 種蟹類則未檢測出毒性；而由 HPLC 進行進一步的毒成分鑑定的結果則發現 J. lividus及 Z. aeneus 皆含有河魨毒 (tetrodotoxin; TTX) 及其衍生物和麻痺性貝毒 (paralytic shellfish poisons; PSP，包含 gonyautoxin; GTX 及 saxitoxins; STX) 等毒成分。在基因實驗方面，首先，利用文獻中設計之引子對 (HCO2198 & CrustF2)，成功增幅生鮮及 100℃, 15 mim 和100℃, 30 min 加工處理條件之 9 種蟹類部份 cytochrome oxidase I (COI) 基因 715 bp 特異性片段，但以高溫高壓滅菌 (121℃, 15 mim) 的蟹肉組別，因加工條件過於嚴苛，使 DNA 發生嚴重斷裂的情況，因此自行設計適用於增幅短片段COI 基因之引子對 (COI-403L/ COI-664H) 進行 PCR 反應，即可成功增幅出部份 COI 基因 301 bp。最後篩選出 DdeI 及 HaeIII 兩種具有特異性切位之限制? (restriction enzyme) 進行 715 bp 片段之切位分析，可確實將生鮮及加工條件 100℃, 30 mim 處理之 9 種蟹類區分出來。綜合顯示臺灣珊瑚礁蟹類毒性範圍大，毒成分主要為 TTX 及 PSP；利用直接定序法配合 RFLP 法可成功鑑別出 9 種生鮮蟹類樣品，並建立9 種蟹類之限制?圖譜。

The toxic crabs are often found on coral reefs and coasts of Taiwan. The related researches pointed out that toxic crabs mostly belong to the family Xanthidae. In order to investigate the toxicity, toxin and specific species-gene characteristics of coral reef crab species in Taiwan, the mouse bioassay, high performance liquid chromatography (HPLC), direct sequencing and the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technology were used in Atergatis floridus, A. integerrimus, A. latissimus, Actaeodes tomentosus, Carpilius convexus, C. maculates, Camposcia retusa, Eriphia sebana, Juxtaxanthias lividus, Lophozozymus pictor, Lachnopodus rodgersi and Zosimus aeneus.
Toxicity was determined by mouse bioassay in 11 species of crab. Except for J. lividus and Z. aeneus, other 9 crabs species all are non- toxic. The mean value of the toxicity of J. lividus and Z. aeneus, was 97 ± 52 (mean ± SD) and 897 ± 1,306 MU/specimen, respectively. The highest toxicity was 167 and 3,503 MU/specimen, respectively. In HPLC-FLD analysis for the toxins, J. lividus and Z. aeneus contained tetrodotoxin (TTX) and paralytic shellfish poisons (PSP). In gene aspect, at beginning, the utility of designed primers (HCO2198 & CrustF2) was successfully amplified 715 bp of cytochrome oxidase I (COI) gene of 9 coral reef crab species from fresh and cooked (100℃ heating for 15 - 30 min) meat of crabs. Because DNA extracted from steam sterilized (121℃ heating for 15 min) meats has serious damage, the another primer pair COI-403L/ COI-664H were designed to successfully amplify smaller fragments of 301 bp from this meat. Finally, the two restriction enzymes DdeI and HaeIII with specific cutting sites that were applied to identify species of 9 fresh and cooked (100℃ heating for 30 min) crabs successfully. Conclusively, the coral reef crab species contain TTX and PSP as the the major toxin in Taiwan, and these species identification could be conducted by using gene sequencing and PCR-PFLP method.

中文摘要...................................................................................................iii
Abstract.....................................................................................................v
文獻整理................................................................................................1
一、蟹類之分類與實驗之蟹類簡介……………………………………….2
二、歷年研究蟹類毒成分與其毒性...……………………………………..6
三、麻痺性貝毒和河魨毒之介紹…...……………………………………15
四、有毒蟹類毒成分之分析方法…….…………………………………..22
五、利用mtDNA 鑑定物種之特性………………………………………27
六、分子生物技術鑑定物種……………………………………………...31
研究內容.....................................................................................................38
第一章、臺灣扇蟹科蟹類毒性及毒成分分析...........................................39
一、前言....................................................................................................39
二、材料與方法........................................................................................40
三、結果....................................................................................................45
四、討論....................................................................................................47
附表與附圖..............................................................................................50
第二章、扇蟹科蟹類之粒線體部份 COI 基因序列分析及利用 PCR-RFLP 鑑定不同溫度處理蟹類之種類........................ 60
一、前言....................................................................................................60
二、材料與方法........................................................................................61
三、結果....................................................................................................68
四、討論....................................................................................................71
附表與附圖..............................................................................................74
參考文獻.....................................................................................................97
謝辭........................................................................................................115
附錄........................................................................................................117

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