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研究生:郭虹彣
研究生(外文):Hung-Wen Kuo
論文名稱:探討DNA甲基化在Aeolosoma viride的前端再生過程中扮演的角色
論文名稱(外文):The putative role of DNA methylation on anterior regeneration in Aeolosoma viride
指導教授:陳俊宏陳俊宏引用關係
指導教授(外文):Jiun-Hong Chen
口試委員:郭典翰陳柏仰
口試委員(外文):Dian-Han KuoPao-Yang Chen
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2020
畢業學年度:108
語文別:英文
論文頁數:51
中文關鍵詞:瓢體蟲DNA甲基化再生甲基轉移酶甲基CpG結合域
外文關鍵詞:Aeolosoma virideDNA methylationRegenerationDNA methyltransferaseMethyl-CpG-binding domain
DOI:10.6342/NTU202000636
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DNA甲基化是表觀遺傳中進行轉錄調控的重要因子,可以在不改變基因序列的情況下調控基因的表現。在細胞不同的分化過程中,DNA甲基化可以調控特定基因的表現,進而在一些生物中進行生命現象的調節,例如幹細胞自我更新與去分化等過程。瓢體蟲(Aeolosoma viride)是一種具有很強再生能力的環節動物,本研究用它來了解DNA甲基化與再生的關聯,本論文已於瓢體蟲的transcriptome database及體內找到 DNA甲基轉移酶1 (dnmt1)、DNA甲基轉移酶2 (dnmt2)和甲基CpG結合域(Methyl-CpG-binding domain, mbd)的序列,並且針對open reading frame完成cloning。接著利用ELISA及超高效液相層析法(UPLC) 測量瓢體蟲的DNA甲基化程度,根據實驗結果,於瓢體蟲體內確定有低DNA甲基化存在,另外,也利用定量聚合酶鍊反應(qPCR)檢測出Avi-dnmt1、 Avi-dnmt2、 和 Avi-mbd在瓢體蟲再生過程中第48小時具有最高的基因表現量,也藉由全胚體原位雜交技術(Whole-mount in situ hybridization, WISH)發現Avi-dnmt1於前端再生過程中會在再生端表現。此外,瓢體蟲的再生顯著地受到DNA甲基化抑制劑5-Azacytidine影響,檢測其前端再生的甲基化程度時,可以看到再生前期的甲基化程度相較於未再生個體有下降的表現。因此瓢體蟲的再生是會受到DNA甲基化的調控。
DNA methylation is an essential epigenetic factor for transcriptional regulation. It can regulate gene expression without altering the genetic sequences. At different developmental stages, DNA methylation can potentially regulate the expression of specific genes, and further modulate biological process such as the renewal and dedifferentiation in stem cells. Aeolosoma viride, an annelid with strong regeneration capacity was used to study the relationship of DNA methylation and regeneration. In this study, DNA methyltransferase 1 (dnmt1), DNA methyltransferase 2 (dmnt 2) and Methyl-CpG-binding domain (mbd) in A. viride have been identified and cloned. To examine the level of DNA methylation in the genome, DNA methylation level was detected by using both ELISA and Ultra-Performance Liquid Chromatography (UPLC). Accordingly, DNA methylation did exist in A. viride. Furthermore, quantitative polymerase chain reaction (qPCR) was used to detect and found that Avi-dnmt1, Avi-dnmt2, and Avi-mbd all had the highest gene expression level at 48 hour-post-amputation (hpa) in the regeneration process. By WISH result, the gene expression of Avi-dnmt1 clearly showed at regeneration side during anterior regeneration. In addition, regeneration of A. viride was significantly affected by 5-azacytidine, a DNA methylation inhibitor treatment. Therefore, DNA methylation is involved in the anterior regeneration of A. viride.
口試委員審定書 i
誌謝 i
中文摘要 ii
Abstract iii
Introduction 1
DNA methylation and methylation related genes 1
DNA methyltransferase 1 (DNMT1) 2
DNA methyltransferase 2 (DNMT2) 3
Regeneration 4
The relationship between DNA methylation and regeneration 5

Materials and Methods 8
Experiment animals and sample preparation 8
Total RNA extract 9
Reverse transcription (RT) 10
Gene cloning and sequence analysis 10
RNA probes synthesis 11
Whole-mount in situ hybridization (WISH) 12
Quantitative PCR (qPCR) analysis 14
Genomic DNA extraction 15
DNA nucleotides hydrolysis 16
Ultra- performs liquid chromatography (UPLC) 16
UPLC standard preparation 17
5-Azacytidine (5-AZA) 17
Result 19
Investigation of DNA methylation in A. viride 19
Molecular cloning and sequences identification of DNA methylation related genes in A. viride 19
Expression of DNMT during anterior regeneration in A. viride 21
Cytosine methylation regulates anterior regeneration in A. viride 22
Discussion 24
References 27

Table 32
Table 1. Percentage of methylated cytosine for A. viride measured in triplicate. 32

Figure 33
Figure 1. The morphology of Aeolosoma viride 33
Figure 2. Investigation of global DNA methylation level in A. viride 35
Figure 3. Sequence identification and phylogenetic analysis of Avi-dnmt 37
Figure 4. Sequence identification and phylogenetic analysis of Avi-mbd 39
Figure 5. Gene expression level of DNA methylation relative genes in A. viride 41
Figure 6. Qualitative gene expression of Avi-dnmt1 in anterior regenerating A. viride 43
Figure 7. Cytosine methylation in A. viride is inhibited by 5-Azacytidine 45
Figure 8. Inhibition of cytosine methylation reduced the A. viride anterior regeneration 47
Figure 9. 5-aza will affect cytosine methylation during anterior regeneration 49
Figure 10. Percentage of 5-mC methylation in anterior regeneration 50
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