生活在熱帶及亞熱帶地區的魚類因為適應了溫暖的環境，因此在冬天時容易因為低溫而造成傷害。為了改善寒害所帶來的重大經濟損失，著手研究改善魚類對於低溫的耐受度是一個重要的課題。在本次的研究中，我們分析了鯉魚第三肌肉型肌酸激酶的生化功能，發現在低溫下，第三肌肉型肌酸激酶仍然穩定以及具有活性，因此第三肌肉型肌酸激酶可能扮演幫助鯉魚在低溫下生活時，能量代謝的一個重要的角色。因此我們分別製備了以三種不同的啟動子表現鯉魚第一型和第三肌肉型肌酸激酶的質體，並使用螢光蛋白作為篩選的標記。
藉著基因轉殖技術將鯉魚第三肌肉型肌酸激酶轉殖到實驗模式魚種（斑馬魚）中，它可以藉由表現持續且足夠強的抗寒能力，而使魚體在攝氏13℃仍能正常的泳動及生活，但是轉殖鯉魚第一肌肉型肌酸激酶並沒有改善基因轉殖斑馬魚在攝氏13℃水溫中的泳動能力。因此可知，此為面臨寒害影響的熱帶及亞熱帶魚類，欲在低溫下維持其代謝能力一個重要的指標。而斑馬魚是一個作為熱帶及亞熱帶地區魚類寒害影響改善研究的良好酵素轉殖基因平台之模式魚種。
本次研究結果顯示：在低溫下，野生型斑馬魚和基因轉殖斑馬魚的泳動能力有明顯的差異，而冷誘導啟動子和鯉魚第三肌肉型肌酸激酶啟動子更可以適時的表現鯉魚第三肌肉型肌酸激酶，增加基因轉殖斑馬魚的泳動能力。第三肌肉型肌酸激酶確實可以改善魚類對於寒冷低溫的耐受性，此外，應用於斑馬魚的成功範例可做為日後應用於國內各種易受寒害影響的經濟魚種之重要指標。

The fish that lives in the tropical or subtropical area is adapted in the temperature of the warm environment and damaged by the low temperature in the winter. In order to confront the economic loss caused by the cold stress, the improvement of the cold tolerance of fish has been studied. We analyzed the biochemical function of the M3-CK sub-isozyme from carp (Cyprinus carpio, a eurythermic fish) and found out the M3-CK was stable and active in low temperature. The M3-CK is the key enzyme of energy metabolism of carp in low temperature.
We constructed a dual functional expression vector that constitutively expressed the carp MCK and a GFP marker gene. The CMV promoter drove the GFP marker and the CMV promoter, cold-inducible promoter and carp M3-CK promoter drove the carp MCK isozymes, M1-CK and M3-CK, respectively.
By transgenesis of the key enzyme, carps M3-CK, into the model fish, zebrafish (Danio rerio), we found that the transgenic fish can express M3-CK constitutively and enhance its cold tolerance from demonstrating a substantial improvement in swimming ability in 13℃. But transgenesis of the carps M1-CK into the model fish zebrafish (Danio rerio); we found that the transgenic fish don’t improve its cold tolerance in swimming ability in 13℃.
It is a proper target to help the tropical or subtropical fish confront the cold stress by maintaining the metabolic capability in low temperature. The zebrafish is a good model system for the transgenesis of the enzyme to improve the cold tolerance of tropical and subtropical fishes.
Statistically, the results in Table 7 revealed significant differences in the swimming velocity between the wild type and the transgenic zebrafish at the lower temperature. The cold-inducible promoter and M3CK promoter can synthesis carp M3-CK that increase swimming ability of transgenic zebrafish at the right moment. The results show that it is work for the assumption that M3-CK is able to improve the target fish’s cold tolerance ability. Consequently, this application of the zebrafish is a clue for the application of economic species of fish to confront the low temperature.

Acknowledgements --------------------------------------------------------------------- i
Contents --------------------------------------------------------------------------------- ii
Figure Content --------------------------------------------------------------------------- iv
Table Content --------------------------------------------------------------------------- vi
Abbreviations ---------------------------------------------------------------------------vii
Chinese Abstract ---------------------------------------------------------------------------viii
English Abstract --------------------------------------------------------------------------- ix
I. Introduction ------------------------------------------------------------------ ------ 1
1. The influence of climate on aquaculture fishes ------------------------------1
2. Providing assistance in energy regulation and adaptation in low temperature by creatine kinase ------------------------------------------------3
3. The problems which transgenic technique confronting recently ------5
II. Purposes ------------------------------------------------------------------------------7
III. Materials and Methods ------------------------------------------------------------8
1. Materials ------------------------------------------------------------------------8
2. Methods ---------------------------------------------------------------------- 11
(1). Zebrafish maintenance ---------------------------------------------- 11
(2). Transgenic vectors preparation ---------------------------------------- 11
(2-1). Isolation of 5’-Flanking Region from Carp (Cyprinus carpio) M3CK Genomic DNA ---------------------------------------- 11
(2-2). Promoter expression analysis ---------------------------------- 13
(2-3). Plasmid construction ---------------------------------------- 13
(3). Generation of transgenic zebrafish ---------------------------------- 14
(3-1). Microinjection ---------------------------------------------------- 14
(3-2). Green fluorescence protein (GFP) expression analysis ---- 15
(3-3). mRNA expression analysis ---------------------------------- 15
(3-4). Immunoblot analysis ---------------------------------------- 15
(3-5). Determination of swimming speeds ---------------------- 16
(4). Statistical analysis ---------------------------------------------------- 16
IV. Results ---------------------------------------------------------------------------- 17
1. Construction of plasmid ---------------------------------------------------- 17
(1). Isolation of 5’-Flanking Region from
Carp (Cyprinus carpio) M3CK Genomic DNA ---------------------- 17
(2). Promoter expression analysis ---------------------------------------- 17
(3). Plasmid construction ---------------------------------------------------- 18
2. Generation of transgenic zebrafish ---------------------------------------- 19
(1). Microinjection ---------------------------------------------------------- 19
(2). Green fluorescence protein (GFP) expression analysis and
statistical analysis ---------------------------------------------------- 19
(3). mRNA expression analysis ---------------------------------------------- 20
(4). Immunoblot analysis ---------------------------------------------------- 20
(5). Determination of swimming speeds ---------------------------------- 21
3. Conclusions ---------------------------------------------------------------------- 23
V. Discussions ---------------------------------------------------------------------- 24
VI. Conclusions and Future work ---------------------------------------------- 27
VII. References ---------------------------------------------------------------------- 28
VIII. Figures and Tables ---------------------------------------------------------------- 37
1. Figure ---------------------------------------------------------------------------- 34
2. Table ---------------------------------------------------------------------------- 58

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