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研究生:楊士弘
研究生(外文):Yang shih-hung
論文名稱:絲瓜簇葉病植物菌質體胸酸激脢(tmk)基因之選殖及定性
論文名稱(外文):Cloning and characterization of the Thymidylate kinase(tmk)gene from the phytoplasma associated with the loofah witches' broom
指導教授:何國傑何國傑引用關係
指導教授(外文):Ho kuo-chieh
學位類別:碩士
校院名稱:國立中央大學
系所名稱:生命科學研究所
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:1999
畢業學年度:87
語文別:中文
論文頁數:54
中文關鍵詞:絲瓜簇葉病植物菌質體胸酸激脢南方氏轉漬雜合分析
外文關鍵詞:clone 138tmkribosomal binding site10 and -35 regions
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中文摘要
利用內含Mycoplasma genitalium rRNA基因的clone 138片段作為探針,對絲瓜簇葉病植物菌質體 EcoRI部份酵解基因庫作篩選(screening),得到8個構築於λZAP載體中的溶菌斑,分別為clone 3-1, clone 3-2, clone 3-3, clone 3-4, clone 4-1, clone 4-2, clone 4-3, clone 4-4,其中clone 4-2溶菌斑內含16S rRNA基因後半段、16S-23S ITS及23S rRNA基因前段。本論文承接其餘7個溶菌斑進行生體內剪接作用,經EcoRI 酵解後,得到 4個載體帶有插入DNA(insert)之選殖株,命名為Clone Y2、Clone Y3、Clone Y5、Clone Y8。
DNA序列完整定序的Clone Y3和Clone Y5經DNA及胺基酸序列與基因庫雙重比對的結果,得知Clone Y5可能內含oligopeptide ABC transporter permease,protein B、protein C整段及protein D前段的基因;而Clone Y3於其插入片段之第1,206 bp至1,835 bp之間為一完整的 tmk 基因段,共含209個胺基酸,分子量為24,791 Da。其上游區域有一個ribosomal binding site (rbs),並具有putative -10和-35 regions,同時亦符合胸酸激脢 三個保守性區域的特性。將絲瓜簇葉病植物菌質體胸酸激脢與各物種胸酸激脢胺基酸序列相比較,顯示絲瓜簇葉病植物菌質體胸酸激脢和原核生物的胸酸激脢較相似。此外,經由南方氏轉漬雜合分析的結果推斷,tmk基因在絲瓜簇葉病植物菌質體的genome中可能只有single copy。

Abstract
Using insert DNA fragment of clone 138, which contained Mycoplasma genitalium rDNA gene, as probe to screen theλZAP/EcoRI genomic library of loofah witches' broom (LfWB) phytoplasma, 8 clones were obtained. Thy were clone 3-1, 3-2, 3-3, 3-4, 4-1, 4-2, 4-3, and clone 4-4. The clone 4-2 contained part of 16S rDNA, 16S-23S internal transceibed spacer (ITS), and first one-third of 23S rDNA. The other 7 clones were converted into phagemid by in vivo excision, and 4 colonies were obtained: clone Y2, Y3, Y5, and Y8.
The complete nucleotide sequences of clone Y3 and clone Y5 were determined and compared to Mycoplasma genitalium genomic DNA databank. It revealed that the clone Y5 contains protein B gene, protein C gene, and part of protein D gene of oligopeptide ABC transporter permease, and the clone Y3 contained an impact thymidylate kinase (tmk) gene. The opening reading frame of tmk, from nucleotides 1,206 to 1,835 encoded a protein of 209 amino acids with a molecular weight of 24,791 Da. This ORF was preceded by a strong consensus sequence for ribosomal binding site (rbs) and the putative -10 and -35 regions. The protein contains three conserved motifs of TMK. The deduced amino acid sequence of tmk was compared with previously determined sequence of several cellular and viral TMKs. The TMK of LfWB was more similar to prokaryotic TMK. The result of Southern hybridization analysis indicated that there was only a single copy of tmk gene in phytoplasma genome.

目錄
縮寫表…………………………………………………………………A
中文摘要…………………………………………………………….. Ⅰ
英文摘要…………………………………………………………….. Ⅱ
壹、 緒言……………………………………………………………… 1
貳、 材料與方法………………………………………………………9
一、材料來源與繁殖………………………………………….. .9
二、絲瓜簇葉病植物菌質體之抽取…………………………… 9
1. 健康及罹病植株總 DNA 之抽取…………………….. 9
2. 植物菌質體DNA之純化…………………………….. .10
三、植物菌質體 Eco RI基因庫( genomic library )之建構….. .11
1. 包被用細胞萃取物之製備……………………………... 11
2. EcoRI 酵解菌質體DNA ……………………………… 13
3. 接合反應…………………………………………………13
4. 包被反應………………………………………………. ..14
5. 基因庫大小的測定…………………………………… …14
四、植物菌質體基因庫之篩選…………………………15
1. 基因庫的轉印…………………………………………… 15
2. DNA 探針的來源…………………………………….. ...15
3. DNA 探針的標識……………………………………….16
4. 雜合反應…………………………………………………16
5. 噬菌體 DNA 的抽取……………………………………17
五、EcoRI部份酵解基因庫之獲得………………………17
六、生體內剪接作用………………………………………18
七、轉型株之確認………………………………………. .19
八、DNA序列分析…………………………………………….. 20
1. 引子的設計……………………………………………… 20
2.模板DNA之製備……………………..…………………..20
3. Autosequence…………………………………………... ...21
4. DNA 序列分析………………………………………… ..21
九、南方轉漬雜合分析法………………………………….. ..22
1. 酵素酵解……………………………………………….. .22
2. 南方氏轉漬……………………………………………… 22
3. DNA 探針的來源………………………………………..23
4. DNA 探針的標識………………………………………..24
5. 雜合反應………………………………………………….24
參、 結果……………………………………………………… .26
一、 絲瓜簇葉病植物菌質體於日日春上的病徵……………...26
二、 生體內剪接作用………………………………………… .26
三、 DNA 序列定序……………………………………………26
四、 選殖株插入之DNA片段的特性分析……………………27
五、絲瓜簇葉病植物菌質體DNA及健康日日春總DNA南方
氏轉漬雜合分析……………………………………………27
肆、 圖表……………………………………………………………… 29
伍、 討論……………………………………………………………… 40
陸、 未來展望………………………………………………………… 43
柒、 參考文獻………………………………………………………… 44

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