跳到主要內容

臺灣博碩士論文加值系統

(44.210.83.132) 您好!臺灣時間:2024/05/29 12:48
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

我願授權國圖
: 
twitterline
研究生:慮永修
論文名稱:比較聚合脢鎖鏈反應及圓點雜合技術在辨識浮游藻類特定基因與藻種上的專一性
指導教授:張正張正引用關係黃生蘋
學位類別:碩士
校院名稱:國立海洋大學
系所名稱:海洋生物研究所
學門:自然科學學門
學類:海洋科學學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:63
中文關鍵詞:聚合脢鎖鏈反應圓點雜合浮游藻類專一性
相關次數:
  • 被引用被引用:0
  • 點閱點閱:160
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:1
摘要
本篇論文主要目的在測試定量反轉錄聚合酶連鎖反應以及圓點雜合技術在偵測浮游藻類基因表現量時的準確度,測試項目包括了對引子及目標圓點專一性的檢驗,由純種藻種以及混合藻種偵測mRNA的一致性,以及在正常及缺磷培養的樣本中偵測基因表現之差異情形。最後也對少數野外樣本進行測試,並討論如何利用實驗室中的測試結果來解釋野外所得的數據。所測試的基因有Skeletonema costatum的rbcL、PHO、與MCM,Tetraselmis chui的rbcL與PHO,以及Isochrysis sp.的rbcL。其中除S. costatum MCM與T. chui PHO外均依據保守性序列自行選殖。定量反轉錄聚合酶連鎖反應的實驗結果顯示我們所設計引子對的專一性很高,對於不同綱的藻株幾乎看不出增殖的訊號,即使在是同屬一綱的種類間,對非目標藻株的測值也要比目標藻株的測值低67倍以上;而在圓點雜合技術的測試上也有類似的專一性。在Q-RT-PCR對於生理狀態的測試上,經過了t-test的比較之後僅僅只有T. chui PHO mRNA的表現在缺磷及正常培養環境下有明顯的不同,兩者相差一倍左右。至於圓點雜合的部分,則無法清楚的分辨藻株基因表現在缺磷及正常培養環境下的差異。在測試純種及混合藻種樣本間的結果中,S. costatum的MCM、Isochrysis的rbcL、和T. chui的PHO引子對藉由Q-RT-PCR所測得混合藻種中mRNA的相對含量與單一藻種的測值相符,但對於其他引子對而言就有差異存在。野外的測試中,Q-RT-PCR以及圓點雜合技術均只能偵測到Sta. 11的矽藻基因,其餘各測站以及其他的藻種都沒有被偵測到。綜觀實驗的結果,real-time Q-RT-PCR技術的靈敏度的確要比圓點雜合技術要高,即使連細胞內含量很少的RNA都能夠清楚的偵測出來。至於圓點雜合技術的靈敏度及定量能力雖然不像Q-RT-PCR那麼高,不過應可對於野外浮游藻類的各種基因表現同時進行初步的測量,篩選出有變化的基因表現進行定量分析。
Abstrat
We tested the accuracy of two techniques that were used to detect gene expression in phytoplankton. These techniques were quantitative reverse transcription -polymerase chain reaction(Q-RT-PCR)and dot blotting. Items tested included specificity of primer pairs and target dots, consistency of results when RNA was extracted from pure and mixed algal cultures, and differential gene expression in low-phosphate and normal growth conditions. In addition, several field samples were tested and how experiences gained in laboratory tests can help to explain the field data was discussed. The genes and algal species used in the tests were rbcL、PHO and MCM of Skeletonema costatum, rbcL and PHO of Tetraselmis chui, and rbcL of Isochrysis sp. Fragments of these genes were cloned as part of this thesis except MCM of S. costatum and PHO of T. chui. Results of Q-RT-PCR indicated that all primers designed showed high degrees of specificity. Primers designed for one algal class did not generate any signal amplification from phytoplankton of different classes. Even for species of the same class, amplification strength of target alga are 67 fold higher than that of a non-target alga. The technique of dot blotting have the same level of specificity. For algae grown at different physiological situations, only the mRNA expression of T. chui PHO showed statistically significant between low-phosphate and. Using dot blotting mRNA expressions of algae grown in low-phosphate and nutrient replete conditions were indistiguishable. Whether the total RNA was extracted from pure or mixed cultures, Q-RT-PCR detected similar mRNA content for S. costatum MCM , Isochrysis sp. rbcL, and T. chui PHO, but detected inconsistent results for others. In field test, both Q-RT-PCR and dot blotting detected gene expression of diatoms at Sta. 11 in the East China Sea, but failed to obtain signals at others stations for other phytoplankton. In conclusion, our results indicated that the sensitivity of real-time Q-RT-PCR is higher than that of dot blotting. Q-RT-PCR can detect in RNA with very low content in cells. Although the sensitivity and quantification power of dot blotting can not match that of Q-RT-PCR, expressions of multiple genes in natural phytoplankton can be detected at the same time. Dot blotting is suitable for preliminary screening of gene expressions, and identification of interested genes for further quantifications.
目錄
摘要……………………………………………………………………..I
目錄……………………………………………………………………III
表目錄…………………………………………………………………IV
圖目錄………………………………………………………………….V
前言……………………………………………………………………..1
材料方法………………………………………………………………..7
結果……………………………………………………………………19
討論……………………………………………………………………26
參考文獻………………………………………………………………31
圖表……………………………………………………………………35
附錄……………………………………………………………………53
參考文獻
Aparicio, O.M., D.M. Weinstein, and S.P. Bell. 1997. Components and dynamics of DNA replication complexes in S. cerevisiae: redistribution of MCM proteins and Cdc45p during S phase. Cell 91:59-69.
Chung, C.C., S.P.L. Hwang, and J. Chang. 2003. Identification of a high-affinity phosphate transporter gene in a prasinophyte alga, Tetraselmis chui, and its expression under nutrient limitation. Applied and Environmental Microbiology. 69:754-759.
Curry, J., C. McHale, and M.T. Smith. 2002. Low efficiency of the Moloney murine leukemia virus reverse transcriptase during reverse transcription of rare t (8;21) fusion gene transcripts. Biotechniques 32:768-775.
Carpenter, E.J., and J.S. Lively. 1980. Review of estimates of algal growth using 14C tracer techniques. In Falkoski, P.G. (ed). Primary productivity in the Sea. Plenum press, New York, pp.161-178.
Dyhrman, S.T., and P. Brian. 2001. A single-cell immunoassay for phosphate stress in the dinoflagellate Prorocentrum minimum (Dinophyceae). Journal of Phycology 37:400-410.
Erdner, D.L., and D.M. Anderson. 1999. Ferredoxin and flavodoxin as biochemical indicators of iron limitating during open-ocean iron enrichment. Limnology and Oceanography 44:1609-1615.
Eberhard, S., D. Drapier, and F.A. Wollman. 2002. Searching limiting steps in the
expression of chloroplast-encoded proteins: relations between gene copy number,
transcription, transcript abundance and translation rate in the chloroplast of
Chlamydomonas reinhardtii. Plant Journal for Cell and Molecular Biology 31:149-
160.
Frank, I.B., L. Pernilla, C. Yi-Bu, K. Hendrik, K. Zobigniew, B. Birgitta, and F. Paul. 2001. Segregation of nitrogen fixation and oxygenic photosynthesisi in the marine Cyanobacterium Trichodesmium. Science 16:1534-1536.
Furnas, M.J. 1990. In situ growth rates of marine phytoplankton approaches to
measurement, community and species growth rates. Journal of Plankton Research
12:1117-1151.
Guillard, R.R.L. 1973. Division rates. In F. R Stein (ed). Handbook of phycological methods. Cambridge University Press, Cambridge ; New York, pp. 289-311.
Hein, J.J. 1990. Unified approach to alignment and phylogenesis. Methods in Enzymology 183:626-645.
Heid, C.A., J. Stevens, K.J. Livak, and P.M. Williams. 1996. Real time quantitative
PCR. Genome research 6:986-994.
Kumazawa, S., and A. Mitsui. 1992. Phytosynthetic activities of a synchronously grown aerobic N2-fixing cyanobacterium, synechococcus sp. Miami BG 043511. Journal of General Microbiology 18:467-72.
La Roche, J., P.W. Boyd, R.M.L. McKay, and R.J. Geider. 1996. Flavodoxin as an in situ marker for iron stress in phytoplankton. Nature 382:802-804.
Lin, S., J. Chang, and E.J. Carpenter. 1994. Detection of proliferating cell nuclear
antigen analog in four species of marine phytoplankton. Journal of Phycology
30:449-456.
Lin, S., and E.J. Carpenter. 1995. Growth characteristics of phytoplankton
determined by cell cycle protein: the cell cycle Ethmodiscus rex (Bacillariophyceae) in the southwestern North Altantic Ocean and Caribbean Sea. Journal of Phycology 31:778-785.
Lalli C.M., and T.R. Parsons. 1993. Biological oceanography : an introduction. Pergamon press, New York, pp.115-120.
Landry, M.R., and R.P. Hassett. 1982. Estimating the grazing impact of marine microzooplankton. Marine Biology 67:283-288.
Leatherwood, J. 1998. Emerging mechanism of eukaryotic DNA replication initation. Current Opinion in Cell Biology 10:742-748.
Martinez, P., and B.L. Persson. 1998. Identification, cloning and characterization of
a derepressible Na+-coupled phosphate transporter in Saccharomyces cerevisiae.
Molecular and GeneralGgenetics 258:628-38.
Mann, B.J., B.J. Bowman, J. Grotelueschen, and R.L. Metzenberg. 1989.
Nucleotide sequence of pho-4+, encoding a phosphate-repressible phosphate
permease of Neurospora crassa. Gene 83:281-289.
Nielsen, T.H., A. Krapp, U. Röper-Schwarz, and M. Stitt. 1998. The sugar-mediated regulation of genes encoding the small subunit of rubisco and the regulatory subunit of ADP glucose pyrophosphorylase is modified by phosphate and nitrogen. Plant, Cell and Environment 21:443-454.
Orellana, M.V., and M.J. Perry. 1995. Optimization of an immunofluorescent assay of the internal enzyme ribulose-1,5-bisphosphate carboxylase (Rubisco) in single phytoplankton cells. Journal of Phycology 31:785-794.
Orellana, M.V., and M.J. Perry. 1992. An immunoprobe to measure RuBisCO
concentrations and maximal photosynthetic rates of individual phytoplankton cells.
Limnology and Oceanography 37:478-490.
Paul, J.H., J.B. Kang, and F.R. Tabita. 2000. Diel Patterns of Regulation of rbcL
Transcription in a Cyanobacterium and a Prymnesiophyte. Molecular Marine
Biology and Biotechnology 2:429-436.
Pichard, S.L., M.E. Frischer, and J.H. Paul. 1993.Rubisco bisphosphate
carboxylase gene expression in subtropical marine phytoplankton populations.
Marine Ecology Progress Series 101:55-65.
Paul, J.H., A. Albin, J.B. Kang, R.A. Stokes, D.Griffin, L. Campbell, and E. Ornolfsdottir. 2000. Form IA rbcL transcripts associated with a low salinity/ high chlorophyll plume (‘Green River’) in the eastern Gulf of Mexico. Marine Ecology Progress Series 198:1-8.
Paul, J.H., S.L. Pichard, and B. Kang. 1999. Evidence for a clade-specific temporal and spatial separation in ribulose bisphosphate carboxylase geneexpression in phytoplankton populations off Cape Hatteras and Bermuda. Limnology and Oceanography 44:12-23.
Persson, B.L., J. Petersson, U. Fristedt, R. Weinander, A. Berhe and J. Pattison. Phosphate permeases of Saccharomyces cerevisiae : structure, function and regulation. Biochimica et Biophysica Acta 1422:255-272.
Pichard, S.L., L. Campbell, K. Carder, J.B. Kang, J. Patch, F.R. Tabita, and J.H.
Paul. 1997. Analysis of ribulose bisphosphate carboxylase gene expression in
natural phytoplankton communities by group-specific probing. Marine Ecology
Progress Series 149:239-253.
Smith, S.M., and R.J. Ellis, 1981. Light stimulated accumulation of transcripts of nuclear and chloroplast genes for ribulose bisphosphate carboxylase. Journal of Molecular and Applied Genetics 1:127-37.
Singh, A.K., and L.A. Sherman. 2000. Identification of iron-responsible, differential gene expression in the Cyanobacterium Synechocystis sp. Stran PCC6803 with a customized amplification library. Journal of Bacteriology 182:3536-3543.
Suzuki, M.T., and S.J. Giovannoni. 1996. Bias cause by template annealing in the amplification of mixture of 16S rRNA genes in PCR. Applied and Environmental Microbiology 62:625-630.
Wyman, M., J.T. Davies, D.W. Crawford, and D.A. Purdie. 2000. Molecular and physiological responses of two classes of marine Chromophytic phytoplankton
(Diatoms and Prymnesiophytes) during the deveopment of nutrient-stimulated blooms. Applied and Environmental Microbiology 66:2349-2357.
Wawrik, B., J.H. Paul, and F.R. Tabita. 2002. Real-time PCR quantification of rbcL
(Ribulose-1,5-bisphosphate carboxylase / oxygenase ) mRNA in diatoms and
pelagophytes. Applied and Environmental Microbiology 68:3771-3779.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top