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研究生(外文):Chee ShinChua
論文名稱(外文):Molecular characterization of autophagy marker LC3 in orange-spotted grouper (Epinephelus coioides)
指導教授(外文):Tzong-Yueh Cheng
外文關鍵詞:LC3autophagyEpinephelus coioidesorange-spotted grouperautophagosomenervous necrosis virus
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細胞自噬是細胞內一種重要的機制,主要透過形成自噬小體和自噬溶酶體,降解細胞質中非必需蛋白質,減少外來物質與壓力之干擾,進而促進細胞正常之生長與發展。LC3蛋白對於自噬小體形成過程中扮演關鍵角色,並被廣泛視為是否產生細胞自噬之重要標記。點帶石斑魚過去相關研究,並無選殖及開發該物種之LC3作為生物標記。本研究嘗試選殖點帶石斑魚的LC3基因並針對該基因特性進行探討。該基因全長1440 bp可轉譯 126氨基酸長之蛋白。此外,LC3基因可在各組織表現。為了解 LC3 於石斑魚生長期表現情形,研究中觀察其石斑魚卵孵化後不同時間點之基因表現,結果顯示基因能在不同批的魚苗有相同的趨勢。經Hank’s solution培養及rapamycin處理GF-1細胞下,証實點帶石斑LC3會轉換成LC3-II形成自噬小體和自噬溶酶體。再者,無論是細胞亦或是動物實驗,顯示經病毒感染後皆可誘發點帶石斑LC3之表現,進一步經研究証實點帶石斑LC3及Hsp90AB彼此間具有交互作用,未來可深入研究LC3蛋白與 Hsp90AB於病毒感染機制上所扮演之角色為何。
Autophagy is a conserve and important cellular mechanism to sustain cell survival during stress, promote healthy growth and development by degrade and recycle unwanted cytosolic proteins and organelles in autophagosome and autolysosome. LC3, involved in autophagosome formation is widely used as marker. However, currently there is no orange-spotted grouper (Epinephelus coioides) LC3 cloned. In this study, a grouper’s LC3 (osgLC3) mRNA had been cloned. Its size is 1440 bp DNA sequence and can be translated to 126 amino acid long proteins. Besides, expression level of osgLC3 found constitutively expressed in all tissues. For the investigation of osgLC3 expression level during grouper’s larvae development, sample from different days of post-hatchery were examined found similar pattern of expression from 3 different batches. Moreover, treatments of Hank’s solution and rapamycin on GF 1 cell line, the osgLC3 was able to form osgLC3-II and synthesizes autophagosome and autolysosome. Furthermore, it has found that autophagy was induced during virus infection, not only in vitro, but also in vivo experiments. Interesting discovery of protein-protein interaction between osgLC3 and Hsp90AB allows future study on their roles and mechanisms during NNV infection in orange-spotted grouper.
中文摘要 I
Abstract II
Acknowledgements IV
Table of contents VI
Contents of Figures, Tables and Appendices X
Research Background 1
1. Autophagy 1
2. Types of autophagy 2
3. Biogenesis of autophagosome 2
4. Regulation of autophagy 3
5. Roles of autophagy 4
6. Roles of autophagy in development 4
7. Roles of autophagy in immunity 5
8. Role of LC3 in autophagy 5
9. Development of detection methods for autophagy 6
10. Autophagy studies on fishes 9
11. Autophagy and betanodavirus 9
12. Research objectives 9
Materials and Methods 11
A) Materials 11
1. Biological materials 11
2. Plasmids 11
3. Reagent kits 11
4. Antibodies 12
5. Commercial chemicals 12
6. Self-prepared buffers and mediums 13
B) Methods 16
1. RNA extraction 16
2. RNA electrophoresis analysis 17
3. Reverse transcription polymerase chain reaction (RT-PCR) 17
4. Quantitative reverse transcription PCR (qRT-PCR) 18
5. DNA electrophoresis analysis 18
6. Gel elution 19
7. TA cloning 20
8. Phylogenetic analysis 20
9. Plasmid construction 20
10. Competent cells preparation 21
11. Transformation 22
12. Plasmid extraction 22
13. Large scale plasmid extraction 23
14. Expression of LC3 recombinant protein 24
15. Purification of osgLC3 recombinant protein 25
16. Rabbit immunization 26
17. Western blot analysis 26
18. GF-1 cell culture 27
19. Cryopreservation of GF-1 cell 28
20. GF-1 cell plasmid transfection 28
21. Preparation of microscope slide sample 29
22. Immunofluorescence staining 30
23. Virus particle purification 31
24. Virus infection 31
25. Acceptor photobleaching FRET (Förster resonance energy transfer) 32
Results 33
1. Gene cloning of osgLC3 33
2. osgLC3 gene expression on various tissues and days post-hatchery 33
3. osgLC3 functional cleavage site 34
4. Punctate formation of transient-expressed osgLC3 in cell 35
5. Punctate formation of transient-expressed osgLC3 under virus infection 36
6. Punctate formation of endogenous osgLC3 under virus infection 37
7. Endogenous osgLC3 expression during in vivo virus infection 37
8. In vitro protein-protein interaction of osgLC3 and Hsp90AB 38
Discussion 39
1. Molecular characterization of osgLC3 gene 39
2. Gene expression of osgLC3 on various tissues and days post-hatchery 39
3. Functional characterization of osgLC3 40
4. Regulation of autophagy during in vitro virus infection 43
5. Regulation of autophagy during in vivo virus infection 44
6. In vitro protein-protein interaction of osgLC3 and Hsp90AB 45
7. Future prospective 46
References 48
Figures 55
Table 80
Appendices 81

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