跳到主要內容

臺灣博碩士論文加值系統

(44.222.64.76) 您好!臺灣時間:2024/06/14 07:41
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

: 
twitterline
研究生:林堯泓
研究生(外文):Yau-Hung Lin
論文名稱:海鱺禁食差異性基因表現研究
論文名稱(外文):Fasting induced differential gene expression
指導教授:陳榮祥陳榮祥引用關係
指導教授(外文):Ron-Shan Cheng
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:水產養殖學系
學門:農業科學學門
學類:漁業學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:83
中文關鍵詞:抑制性扣減雜交禁食差異性基因表現海鱺
外文關鍵詞:SSHFastingDifferential gene expressionCobia
相關次數:
  • 被引用被引用:0
  • 點閱點閱:190
  • 評分評分:
  • 下載下載:18
  • 收藏至我的研究室書目清單書目收藏:0
生物體短期禁食時會調整體內能量的運用,增加代謝效率,並且強化生物的生存功能。海鱺於禁食的代謝機制目前尚未明瞭,實驗首先以退化性聚合酶連鎖反應找出禁食指標基因(天門冬胺酸合成酶和解偶聯蛋白2),用來發展出改良抑制性扣減雜交(標誌基因輔助-抑制性扣減雜交),以此技術篩選海鱺肝臟禁食9天的差異性表現基因。藉由標誌基因輔助可以確認抑制性扣減雜交產物的品質,增加篩選到差異性表現基因的機會。定序比對分析後一共篩選到 33 個基因,依功能可以分為以下種類:醣類代謝相關、脂質代謝相關、蛋白質代謝相關、免疫相關、緊迫相關、解毒相關、未知功能…等。接著再以即時定量聚合酶連鎖反應來量化篩選出的基因,觀察其在禁食 0 、 3 、 6 、 9 天的表現變化。其中20 個基因在禁食時表現明顯上升,而這些差異表現基因中又以解偶聯蛋白2和3-磷酸甘油脫氫酶的表現差距最大。解偶聯蛋白2的基因表現量在禁食 3 天增加至 16.2 倍, 6 天增加至6.7 倍, 9 天增加至 4.8 倍。3-磷酸甘油脫氫酶(G3PD)的基因表現量在禁食 3 天增加至 19.2 倍, 6 天增加至 15.2 倍, 9 天增加至 8.3 倍。為了避免聚合酶連鎖反應的複製偏差影響,實驗中以核醣核酸點墨雜交反應佐證解偶聯蛋白2在即時定量聚合酶鍊反應的結果。結果顯示解偶聯蛋白2的基因表現量在禁食 3 天增加至 5.38 倍, 6 天增加至 2.88 倍, 9 天增加至 2.91 倍。兩者的表現趨勢一致,在此證實解偶聯蛋白2為禁食的差異表現基因,即時定量聚合酶連鎖反應的結果是可信的,其餘基因的差異表現是有生理意義的。
After short term of fasting, animals will regulate energy expenditure. It can enhance metabolism efficiency and strengthen animal’s livability. The effect of metabolism change by fasting in cobia(Rachycentron canadum) is not clear now. In the experiment, fasting marker genes(Asparagine synthetase and uncoupling protein 2)were cloned to improve suppression subtractive hybridization(SSH). The improved SSH:marker-assisted suppression subtractive hybridization(MA-SSH) is developed for identifing the genes up-regulated in cobia liver after 9 days of fasting. Fasting marker genes were used for identing the quality of SSH product. It increases the chance to find the differential expression genes. In this experiment, thirty-three genes were found in SSH product. The genes can be assigned to seven major functional classes. There are carbohydrate metabolism related genes, lipid metabolism related genes, protein metabolism related genes, immune related genes, genes that respond to stress, genes that respond to detoxification and unknown function genes. These genes were quantitated by RT-PCR to observe the gene expression after 3, 6, 9 days of fasting. In thirty-three genes there were twenty genes up-regulated significantly after 9 days of fasting. Among these differential expression genes uncoupling protein 2(UCP2)and Glycerol 3-Phosphate Dehydrogenase(G3PD)were up-regulated most. The mRNA expression of UCP2 after 3, 6 and 9 days fasting was up-regulated to 16.2 fold, 6.7 fold and 4.8 fold, respectively. The mRNA expression of G3PD after 3, 6 and 9 days fasting was up-regulated to 19.2 fold, 15.2 fold and 8.3 fold, respectively. To prevent PCR bias RNA-dot blot was used to confirm the RT-PCR data of UCP2. The gene expression trend from RT-PCR is simiar to that of dot blot hybridization. It means that the RT-PCR data is credible. The other genes are up-regulated significantly.
頁次
謝辭………………………………………………………………………i
中文摘要…………………………………………………………………ii
英文摘要………………………………………………………………iii
目錄………………………………………………………………………v
壹、前言…………………………………………………………………1
一、禁食引發的生理變化………… ……………………………………1
二、差異性表現基因……………………………………………………5
三、研究目的……………………………………………………………8
貳、實驗材料與方法……………………………………………………9
一、海鱺禁食實驗………………………………………………………9
二、海鱺禁食指標基因(Fasting Maker Gene)的選殖……………9
三、抑制性扣減雜交(Suppression Subtractive Hybridization, SSH)……………………………………………………………………16
四、Reverse Screening………………………………………………22
五、即時定量PCR(Real-time quantitative PCR, Q PCR)…………25
六、RNA dot blot………………………………………………………26
參、結果…………………………………………………………………27
一、海鱺禁食實驗………………………………………………………27
二、退化性PCR尋找禁食指標基因……………………………………27
三、標誌基因輔助-抑制性扣減雜交(marker-assisted suppression subtractive hybridization, MA-SSH)……………28
四、即時定量 PCR 證實基因差異性表現……………………………31
五、RNA dot blot 佐證 UCP2 的即時定量 PCR 結果………………33

肆、討論…………………………………………………………………34
一、差異表現基因的篩選……………………………………………34
二、禁食差異表現基因………………………………………………38
三、後續發展…………………………………………………………42
伍、參考文獻……………………………………………………………43
陸、圖表…………………………………………………………………48
漆、附錄…………………………………………………………………74
Araki, S. and Goto, S.. 2003. Age-associated changes in the serum level of apolipoproteins A-I and A-IV and the gene expression as revealed by fasting and refeeding in mice. Exp Gerontol. 38 : 499-506.

Berg, J. M., Tymoczko, J. L. and Stryer L.. 2001. Biochemistry fifth edition. W. H. Freeman and Company.

Beauloye, V., Willems, B., de Coninck, V., Frank, S. J., Edery, M. and Thissen, J. P.. 2002. Impairment of liver GH receptor signaling by fasting.
Endocrinology. 143 : 792-800.

Brown, B. L., Allis, J. W., Simmons, J. E. and House, D. E.. 1995. Fasting for less than 24 h induces cytochrome P450 2E1 and 2B1/2 activities in rats. Toxicology Letters. 81 : 39-44.

Coulibaly, I., Gahr, S. A., Palti, Y., Yao, J. and Rexroad, C. E. 3rd.. 2006. Genomic structure and expression of uncoupling protein 2 genes in rainbow trout (Oncorhynchus mykiss). BMC Genomics. 203.

Diatchenko, L., Lau, Y.F., Campbell, A. P., Chenchik, A., Moqadam, F., Huang, B., Lukyanov, S., Lukyanov, K., Gurskaya, N., Sverdlov, E. D. and Siebert, P. D.. 1996. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci U S A. 93 : 6025-6030.

Ehrenfried, J. A., Evers, B. M., Chu, K. U., Townsend, C. M. Jr and Thompson, J. C.. 1996. Caloric restriction increases the expression of heat shock protein in the gut. Annals of Surgery. 223 : 592-599.

Goldsmith, Z.G. and Dhanasekaran, N.. 2004. The microrevolution: applications and impacts of microarray technology on molecular biology and medicine (review). International Journal of Molecular Medicine. 13 : 483-495.

Gong, S. S., Guerrini, L. and Basilico, C.. 1991. Regulation of asparagine synthetase gene expression by amino acid starvation. Molecular and Cellular Biology. 11 : 6059-6066.

Hall, J. R., Short, C. E. and Driedzic, W. R.. 2006. Sequence of Atlantic cod (Gadus morhua) GLUT4, GLUT2 and GPDH: developmental stage expression, tissue expression and relationship to starvationinduced changes in blood glucose. The Journal of Experimental Biology. 209 : 4490-4502.

Harding, J. W., Pyeritz, E. A. Jr., Copeland, E. S. and White, H. B.. 1975. Role of glycerol 3-phosphate dehydrogenase in glyceride metabolism. Effect of diet on enzyme activities in chicken liver. Biochem. J. 146 : 223-229.

Huggett, J., Dheda, K., Bustin, S. and Zumla, A.. 2005. Real-time RT-PCR normalisation; strategies and considerations. Genes and Immunity. 6 : 279–284.


Jastroch, M., Wuertz, S., Kloas, W. and Klingenspor, M.. 2005. Uncoupling protein 1 in fish uncovers an ancient evolutionary history of mammalian nonshivering thermogenesis. Physiol Genomics. 22 : 150-156.

Kivioja, T., Arvas, M., Saloheimo, M., Penttila, M. and Ukkonen, E.. 2005. Optimization of cDNA-AFLP experiments using genomic sequence data. Bioinformatics. 21 : 2573-2579.

Klover, P. J. and Mooney, R. A.. 2004. Hepatocytes: critical for glucose homeostasis. The International Journal of Biochemistry & Cell Biology. 36 : 753-758.

Laine, R. O., Shay, N. F. and Kilberg, M. S.. 1994. Nuclear retention of the induced mRNA following amino acid-dependent transcriptional regulation of mammalian ribosomal proteins L17 and S25. The Journal of Biological Chemistry. 269 : 9693-9697.

Ledesma, A., de Lacoba, M. G. and Rial, E.. 2002. The mitochondrial uncoupling proteins. Genome Biology. 3(12) : 3015.1–3015.9.
Liang, X. F., Ogata, H. Y., Oku, H., Chen, J. and Hwang, F.. 2003. Abundant and constant expression of uncoupling protein 2 in the liver of red sea bream Pagrus major. Comp Biochem Physiol A Mol Integr Physiol. 136 : 655-661.

Liao, W. Q., Liang, X. F., Wang, L., Fang, L., Lin, X., Bai, J. and Jian, Q.. 2006. Structural conservation and food habit-related liver expression of uncoupling protein 2 gene in five major Chinese carps. Journal of Biochemistry and Molecular Biology. 39 : 346-354.
Madden, S. L., Wang, C. J. and Landes, G.. 2000. Serial analysis of gene expression: from gene discovery to target identification. Drug Discov Today. 5 : 415-425.

Matz, M. V. and Lukyanov, S. A.. 1998. Different strategies of differential display: areas of application. Nucleic Acids Research. 26 : 5537-5543.

Norrelund, H., Frystyk, J., Jorgensen, J. O., Moller, N., Christiansen, J. S., Orskov, H. and Flyvbjerg, A.. 2003. The effect of growth hormone on the insulin-like growth factor system during fasting. The Journal of Clinical Endocrinology and Metabolism. 88 : 3292-3298.

Pascual, P., Pedrajas, J. R., Toribio, F., Lopez-Barea, J. and Peinado, J.. 2003. Effect of food deprivation on oxidative stress biomarkers in fish (Sparus aurata). Chemico-Biological Interactions. 145 : 191-199.

Patsouris, D., Mandard, S., Voshol, P. J., Escher, P., Tan, N. S., Havekes, L. M., Koenig, W., Marz, W., Tafuri, S., Wahli, W., Muller, M. and Kersten, S.. 2004. PPARα governs glycerol metabolism. The Journal of Clinical Investigation. 114 : 94-103.

Sahin, T., Akbulut, B. and Aksungur, M.. 2000. Compensatory Growth in Sea bass (Dicentrarchus labrax), Sea bream (Sparusaurata) and Rainbow trout (Oncorhynchus mykiss). Turk J Zool. 24 : 81–86.

Soengas, J. L., Strong, E. F. and Andres, M. D.. 1998. Glucose, lactate, and hydroxybutyrate utilization by rainbow trout brain: changes during food deprivation. Physiological Zoology. 71 : 285–293.
Uchida, K., Kajimura, S., Riley, L. G., Hirano, T., Aida, K. and Grau, E. G.. 2003. Effects of fasting on growth hormone/insulin-like growth factor I axis in the tilapia, Oreochromis mossambicus. Comp Biochem Physiol A Mol Integr Physiol. 134 : 429-39.

Van Gilst, M. R., Hadjivassiliou, H. and Yamamoto, K. R.. 2005. A Caenorhabditis elegans nutrient response systempartially dependent on nuclear receptor NHR-49. Proc Natl Acad Sci USA. 102 : 13496-13501.

Yamada, K. and Noguchi, T.. 1999. Nutrient and hormonal regulation of pyruvate kinase gene expression. Biochemical Journal. 337 : 1-11.

Zhang, J., Underwood, L. E. and DErcole, A. J.. 2001. Hepatic mRNAs up-regulated by starvation: an expression profile determined by suppression subtractive hybridization. The FASEB Journal. 15 : 1261-3.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top