臺灣博碩士論文加值系統

莫三鼻克吳郭魚（Oreochromis mossabicus）屬口孵型吳郭魚，為廣鹽性魚種，屬於高蛋白來源經濟魚種之一；目前國際上有關吳郭魚基因體之研究多集中於genetic mapping方面，至於功能性基因方面研究則較為欠缺；過去對兩生類及比目魚在變態的研究主要集中於外觀型態改變、腸道發育，亦或探討荷爾蒙對其變態發生之影響，有關變態行為在基因層面的研究卻十分有限。故本研究欲以PCR-select subtractive hybridization方法找出與仔魚成長相關之基因，以進一步分析及研究其功能性或與其他基因的交互作用。實驗先以解剖顯微鏡方式觀察仔魚孵化後生長情形，發現仔魚在孵化後第三天開口，第五天開始魚鰭出現再吸收的現象。而pepsin基因在二日齡有微量表現，而trypsin與chymotrypsin基因則在一日齡即具表現，三者皆隨日齡成長而增加表現，另以西方墨點法偵測仔魚胃蛋白酶（pepsin）、胰蛋白酶（trypsin）、胰凝乳蛋白酶（chymotrypsin）蛋白質表現，發現三者皆於仔魚孵化後第一天即出現，測定其酵素活性，結果發現胃蛋白酶在孵化後第三天、胰蛋白酶與胰凝乳蛋白酶於孵化後第五天始具活性，推測此三種消化酵素雖在孵化後第一天即存在，但仍需一段時間經由後修飾作用才具有活性。由仔魚孵化後生長情形、腸胃道消化酵素蛋白表現與活性，推測仔魚孵化後第五天始變態發生，而後萃取吳郭仔魚孵化後2-3及4-5天（pre-metamorphosis and metamorphosis）全身之RNA，進行subtraction PCR，將酵素分切之cDNA選殖後，PCR screening 1440顆菌落確定是否有inserted DNA，並挑選其中163顆insert大小不同之菌落做定序，經序列比對後得到50個已知基因，其中包括10個與能量代謝相關基因，3個與發育直接相關基因，6個與肌肉構造相關基因，及其他ribosomal protein基因；亦獲得41個未知基因，其次藉dot-blot方法確定clone(3)N73等未知基因之真偽，並以northern blotting分析此基因之時間與組織分佈差異，結果發現(3)N73在孵化後二、三天表現量增加而後減少，且組織特異分佈上只在肝臟有基因表現，並發現(3)N73受環境因子（鹽度、溫度）所影響，此外在飢餓狀態下此基因表現亦會降低，推測此基因可能與肝臟形成、代謝或環境適應有關。並進行5’及3’端RACE，已完成其全長之定序工作，其基因長度為1175 bps，轉譯為302氨基酸，爾後將搭配SiRNA或QPCR方式，以研究其功能性與其他基因之間的關係，釐清該基因在吳郭仔魚成長過程中所扮演的角色。

Mossabicus tilapia (Oreochromis mossabicus), euryhaline and mouthbrooder, is easily reared and short reproductive cycle becoming a high economic fish. Most recent researches focus on the genetic mapping in tilapia and lacking in functional genes assay. The study was conducted to find morphological changes, development of digestive tract, and metamorphosis or development related genes by using anatomic microscope and PCR-select subtractive hybridization (subtraction PCR). After microscopic observations, tilapia larvae mouth opened at day 3 after hatching and actinotrichia reabsorbed at day 5. Analysis the expression of larvae pepsin, trypsin and chymotrypsin at various times after hatching by western blotting, enzymatic proteins appear at day 1 and specific activity of these enzymes occurs at day 3 (pepsin) and day 5 (trypsin and chymotrypsin), respectively. These results suggest that the fin formation might be in association with the functional development of gastric intestine tract.
Isolated whole body total RNA from larvae after hatching 2-3 and 4-5day were applied to do subtraction PCR. After the cloning of restriction enzyme digested cDNA and screened 1440 colonies to make sure the DNA inseration. And 163 colonies contained different size of inserted DNA were sequenced. After blast, fifty well-known genes were obtained, including ten energy metabolism related genes, three development related genes, six structure related genes, others were ribosomal protein genes, and forty-one unknown genes. Furthermore, one of unknown genes clone(3)N73 was proved really expressed after dot blotting. Compared northern blotting results of temproal pattern and tissue distribution, clone(3)N73 expression was increased at day 2-3 following decrease and clone(3)N73 was only expressed in adult fish liver. In addition, liver-specific gene clone(3)N73 was affected by environmental factors (salinity and temperature) and nutritional status (starvation). These results imply that the unknown gene might be related to liver development and metabolism or adaptation. After 5’ end and 3’ end of RACE to complete gene sequence, SiRNA or real-time PCR will be used to get a better understanding of its function, interaction with other genes and the role of this gene during tilapia larvae development.

致謝 Ⅰ
目錄 Ⅴ
表目錄 Ⅵ
圖目錄 Ⅶ
中文摘要 Ⅱ
英文摘要 Ⅳ
壹、研究背景
一、吳郭魚及其仔魚發育情形 1
二、影響仔魚成長發育之影響因子 7
三、變態（metamorphosis） 12
四、PCR-select subtractive hybridization 14
貳、實驗目的 16
參、實驗流程、材料與方法 18
肆、實驗結果 38
伍、討論 44
陸、結論 51
柒、參考文獻 53
捌、圖表、附圖 62
玖、附錄 88

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