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研究生:曾穗雯
研究生(外文):Sui-wen Zeng
論文名稱:一個黏菌起始發育必需的新穎基因
論文名稱(外文):A novel gene is essential for the initiation of development in dictyostelium
指導教授:張文粲
指導教授(外文):Wen-Tsan Chang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
論文頁數:86
中文關鍵詞:黏菌發育生物學環腺苷單磷酸限制酶插入致突變法
外文關鍵詞:Dictyosteliumdevelopmental biologycAMPREMIPKA
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黏菌 (Dictyostelium)是一種強而有力的生物體模式,可用來研究細胞及發育生物學。當食物供應不足時,細胞進入交替繁殖過程,並且產生細胞外環腺苷單磷酸(extracellular cAMP)。cAMP receptor與cAMP結合後,會活化許多基因,例如ACA (adenylyl cyclase A),使細胞產生高濃度cAMP。cAMP對黏菌主要有兩種影響,一種是趨化作用 (chemotaxis),趨化作用就是細胞朝向cAMP濃度較高的區域移動,使細胞聚集;另一種是活化細胞內PKA途徑,除了與cAMP調控有關,也可改變許多發育相關基因的活性,使黏菌分化成桿原細胞 (prestalk cell)或孢原細胞 (prespore cell)。WTC188是一株利用限制酶插入致突變法 (Restriction enzyme-mediated integration, REMI)產生的突變株,此結果經南方墨點法証實WTC188為載體插入所造成之突變株,且載體並無斷裂或重組之現象。利用質體拯救法所拯救出來的部份WTC188基因序列經由database比對,可以知道被破壞的基因是一個未經發表的基因。此外,利用同源重組可以使野生株細胞表現出與WTC188相同的表現型,且南方墨點法証實同源重組所交換的位置與原始突變株相同,如此可說明WTC188所被破壞的基因的確可造成野生株細胞表現型異常。WTC188的表現型在細菌培養基及不含營養成份的培養基在96小時內都呈現單一阿米巴細胞的型態;水的發育 (submergin aggregation assay)也証實WTC188有細胞聚集的缺陷。在細胞凝集實驗 (cell agglutination assay)則觀察到WTC188細胞與細胞之間的吸附較差。為了解WTC188被破壞的基因可否被拯救 (rescued),故將野生株細胞與WTC188作不同比例,不同時間的發育,在野生株細胞與突變株的比例為9:1時,且發育第24個小時,觀察到仍有少量類似聚集體的結構無法形成子實體;而隨著突變株的比例增加,細胞發育的抑制現象愈明顯。X-gal染色可進一步知道WTC188的缺陷無法被野生株拯救 (野生株細胞與突變株的比例為9:1)。另外利用cAMP pulsing也無法使WTC188聚集,且cAMP pulsing過的WTC188細胞內cAMP濃度很低。由於8-Br-cAMP促使WTC188分化成孢子細胞的比例很低,因此推測WTC188的PKA途徑之下游有所異常。
.Dictyostelium discoideum is the free-living amoebae and widely used as a model organism in cell and developmental biology. When food supply is exhausted, some cells (aggregation centers) start to release cAMP. Neighboring cells sense extracellular cAMP by cell surface cAMP receptors (cARs) and move towards aggregation centers to form a multicellular aggregate. Intracellular cAMP activates PKA (cAMP-dependent protein kinase); PKA is the central regulator that mediates many changes of gene expression and plays an important role during Dictyostelium development.
Using restriction enzyme-mediated integration (REMI), an aggregation minus mutant, WTC188 was isolated. Southern blot demonstrated that WTC188 was created by plasmid insertion. WTC188 failed to initiate cell aggregation both on bacterial lawn agar and nonnutrient agar. Through NCBI Blast, the wtc188 gene was known as a novel gene. The predicded protein encoded by wtc188 was Gln-rich and about 115 kDa. Disruption of wtc188 through homologous recombination demonstrated that wtc188 was responsible for the specific morphological phenotype and suggested that wtc188 encoded an important compound during early development. WTC188 had the cell-cell adhesion defect via cell agglutination assay. Using cell agglutination assay, WTC188 had poor cell-cell adhesion than wild-type. The synergy experiment and X-gal staining experiment demonstrated that wild-type did not rescue aggregation minus defect of WTC188 cells. After cAMP-pulsing, WTC188 did not aggregate. Furthermore, intracellular cAMP concentrations of cAMP pulsed-WTC188 were as low as cells without cAMP pulsing. 8-Br-cAMP induced poorly the spore formation of WTC188, suggesting that downstream of PKA pathway of WTC188 was abnormal.
目錄
摘要 I
Abstract II
誌謝 III
第一章 緒論
A 黏菌之簡介
  A.1 黏菌為一種生物體模式 1
  A.2 黏菌生活史 2
  A.3 黏菌基因組 (genome)特性與定序 2
B 細胞內、外cAMP功能 4
C 細胞早期發育的分子機制及PKA 4
D 限制酶插入法 (REMI) 9
第二章 材料與方法
A 實驗材料
A.1 菌株 11
A.2 限制酶 11
A.3 化學試劑 11
A.4 黏菌用培養液及緩衝液 13
A.5 細菌用培養液 17
A.6 保存plasmid與一般cloning過程所需的緩衝液 18
A.7 南方墨點法所需要的緩衝液 19
A.8 培養基 20
A.9 儀器設備 22
A.10 廠商網址 22
B 實驗方法
B.1 黏菌基因組DNA (genomic DNA)的製備 24
B.2 電擊 (Electroporation) 及限制酶插入突變法 (REMI) 25
B.3 更換培養液 26
B.4 平碟培養法 (Plating) 26
B.5 黏菌細胞再選殖 (Reclone) 27
B.6 黏菌細胞增殖 (Amplify) 27
B.7 將細胞養入培養液中 27
B.8 黏菌細胞的保存 28
B.9 黏菌細胞的發育 28
B.10 不同比例之黏菌細胞的協同發育 (synergy) 29
B.11 染色 (-galactosidase staining) 29
B.12 水的發育 (submerged monolay assay) 30
B.13 孢子生存能力試驗 (spore viability assay) 31
B.14 cAMP pulsing 31
B.15 測細胞內cAMP濃度 (cAMP determination) 32
B.16 細胞凝集分析 (Cell agglutination assay) 32
B.17 南方墨點法 (Southern blot) 32
C. 質體的構築及相關方法
C.1 限制酶切割質體DNA 37
C.2 質體DNA的凝膠電泳 (argarose gel) 37
C.3 質體DNA去磷酸化反應 (dephosphorylation) 37
C.4 質體DNA之補齊反應 (fill in) 38
C.5 質體DNA的回收 38
C.6 接合作用 (ligation) 39
C.7 形質轉移 (transformation) 39
C.8 小量質體製備 (mini-prep of plasmid DNA) 39
C.9 中量質體製備 (midi-prep of plasmid DNA) 40
C.10 去氧核糖核酸、核糖核酸濃度的測定 41
C.11 核酸定序分析 (DNA sequencing) 41
C.12 勝任細胞 (competent cell)的製備 41
C.13 聚合酶連鎖反應 (Polymerase chain reaction, PCR) 42
C.14 苯酚-氯仿萃取法 (Phenol-chloroform extraction) 42
C.15 乾燥DNA 42
第三章 實驗結果 44
第四章 討論 50
第五章 參考文獻 55
圖表 65
圖表目錄
圖一 黏菌的生活史 65
圖二 黏菌發育的早期分子機制 66
圖三 限制酶插入突變法 (REMI)及選殖黏菌發育相關基因流程圖 68
圖四 黏菌野生株、突變株WTC188及重創突變株WTC188/HR1
在細菌培養基之發育型態比較 69
圖五 南方墨點法分析限制酶插入突變株WTC188 70
圖六 南方墨點法分析WTC188與重新創造之突變株 71
圖七 wtc188基因的分子轉殖 (molecular cloning) 72
圖八 黏菌wtc188基因所合成的蛋白質可能序列 73
圖九 突變株及野生株之KK2 agar發育 74
圖十 水的發育分析 (submerged monolayrer assay) 75
圖十一 細胞凝集分析 (cell agglutination assay ) 76
圖十二 野生株與WTC188的混合聚集發育 77
圖十三 野生株細胞與突變株細胞混合聚集體的-半乳醣酶染色 81
圖十四 經cAMP pulsing處理的細胞之KK2 agar發育 82
圖十五 細胞內cAMP濃度的測定 83
表一 黏菌聚集時期基因 84
表二 孢子生存實驗 (spore viability) 85
表三 細胞在水的發育分析之分化現象 86
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