臺灣博碩士論文加值系統

本研究中以建構與篩選花生簇葉病菌質體次基因體基因庫 (subgenomic library) 之策略，針對其 serS 基因進行選殖與分析。利用本研究室先前獲得之選殖株 clone 22 選殖片段中所含有之部分花生簇葉病菌質體 serS 基因序列，透過聚合酵素連鎖反應 (PCR) 進行探針 WS2-5 之製備，並以該探針應用於由限制酵素 SpeI 構築之次基因體基因庫之篩選，獲得一選殖株 lib_SpeI_4-19, 由於該選殖株之選殖片段中之 serS 基因缺乏 5’ 端序列，故進一步以該選殖株之選殖片段序列，利用 PCR 進行探針 WS8-6 之製備，並將其應用於由 HindIII 構築之次基因體基因庫之篩選，並獲得一選殖株 lib_HindIII_2-36。經序列整合與分析，得知在該二選殖株之選殖片段中包含有兩個完整之開放式讀取架 (open reading frame, ORF)，與兩個不完整之 ORFs，依其由 5’ 端至 3’ 端之順序分別將之命名為 ORF1 至 ORF4。將各 ORFs 所推衍出之胺基酸序列進行比對，結果顯示完整之 ORF2 與 ORF3 分別與其他物種之 serS 基因與 hflB 基因具相似性；ORF1 則與 rpsT 基因具有相似性；而 ORF4 則無明確之比對結果，因此暫定其可能為一未知功能之假設性蛋白質 (hypothetical protein)。為獲得完整之 rpsT 基因，即由植物菌質體之 rpsT 之高保守性區域設計簡併性引子 (degenerate primer)，並利用 PCR 之策略進行花生簇葉病菌質體 rpsT 基因之選殖，並獲得選殖株c_wsf6mr7_rpsT，選殖片段包含完整之 rpsT 基因。以軟體將各選殖株嵌入片段續接，遂獲得一 3,902 bp 花生簇葉病菌質體基因體之片段序列，由其 5’端至 3’ 端依序為 rpsT基因、serS 基因、hflB基因及一不完整之 ORF4。rpsT 基因之轉譯產物為組成核糖體之小次單元 30S subunit中之核糖體蛋白質 S20；serS 基因轉譯產物為絲胺基化 tRNA 合成酵素，兩者都參與於轉譯中，為蛋白質生合成之相關基因；hflB基因其轉譯產物為 ATP-dependent Zn metalloendoprotease，具有分解多種蛋白質之能力，在植物菌質體內為一高套組數基因，其保守性序列主要位於兩個功能性區塊中。以橫跨在 rpsT 與 serS 之基因間區域 (intergenic region) 兩側之兩組引子對，與橫跨在 serS 與 hlfB 之基因間區域兩側之一組引子對，進行反轉錄聚合酵素連鎖反應 (RT-PCR)，皆可獲得 RT-PCR 增幅產物，經選殖與解序分析後，其與本研究中獲得之花生簇葉病菌質體之基因體片段序列 100% 吻合，顯示 rpsT、serS 與 hlfB 均能進行 RNA 表現，且均位於同一轉錄產物上，說明此三者可能位於同一多基因操縱組 (polycistronic operon) 內。

To investigate serS gene in peanut witches’-broom (PnWB) phytoplasma, subgenomic libraries of PnWB phytoplasma were constructed in this study. Clone lib_speI_4-19 was obtained from the PnWB phytoplasma SpeI-restriction subgenomic library by colony hybridization using PCR- amplified probe WS2-5 based on the partial sequence of serS gene of PnWB phytoplasma obtained in our previous study. To obtain the 5’ end sequence of serS, another probe WS8-6 was amplified based on the sequence of cloned fragment of lib_speI_4-19. Clone lib_HindIII_2-36 was then selected from the PnWB phytoplasma HindIII-restriction subgenomic library. Sequences of the cloned fragments of lib_speI_4-19 and lib_HindIII_2-36 were integrated and analyzed to reveal that the sequences may cotain four putative open reading frames (ORFs 1-4). The deduced amino acid sequences of ORF1, ORF2 and ORF3 showed homology with those of rpsT gene, serS gene and hflB gene, respectively. On the other hand, amino acid sequence of the ORF4 probably encodes a hypothetical protein with no significant homology to any known sequences. To clone complete rpsT gene, a degenerate PCR primer was designed according to the 5’ conserved region of phytoplasma rpsT gene, a clone c_wsf6mr7_rpsT with full length of rpsT gene was then obtained using PCR-based cloning strategy. The 3,902 bp genomic DNA sequence of PnWB phytoplasma was determined from the sequences of the cloned fragments of lib_speI_4-19, lib_HindIII_2-36 and c_wsf6mr7_rpsT using SeqMan sequence analysis program. The fragment contains full length of rpsT gene, serS gene, hflB gene and an incomplete ORF4 in order. The rpsT gene encodes a 30S subunit ribosomal protein S20. The serS gene encodes a seryl-tRNA synthetase with three highly conservative motifs. Both proteins were involved in translation and related to protein biosynthesis. The hflB gene encodes an ATP-dependent Zn metalloendoprotease of the AAA family (ATPases associated with diverse celluar activities) with two functional domains responsible for protein degradation. PCR fragments that spanned the rpsT-serS and serS-hflB intergenic region were amplified separately in reverse transcription-PCR (RT-PCR) using the total RNA prepared from PnWB-affected periwinkle as template, and the sequences of PCR products were identical to the corresponding sequences of the 3,902 bp PnWB phytoplasma genomic fragment. RT-PCR experiments indicate that the three genes are expressed as a single transcript, demonstrating that they constitute an operon.

論文口試委員審定書 II
誌謝 III
中文摘要 IV
英文摘要 VI
壹、前言 1
貳、前人研究 3
一、植物菌質體之發現與其植物病理學 (plant pathology) 3
二、植物菌質體之分群 5
三、植物菌質體之生物特性與其分子生物學上的研究 7
四、細菌之 serS 基因之研究及特性 8
(一) serS 基因及其蛋白質產物之特性 8
(二) serS 基因之調控機制 11
(三) serS 基因之演化 13
五、細菌之 rpsT 與 hflB 基因之研究及其特性 14
(一) rpsT 基因及其蛋白質產物之特性 14
(二) hflB 基因及其蛋白質產物之特性 15
參、材料與方法 18
一、研究材料來源與全 DNA (total DNA) 之純化 18
(一) 試驗植物來源及繁殖 18
(二) 健康及受花生簇葉病菌質體感染植物全 DNA (total DNA) 之純化 18
二、花生簇葉病菌質體 serS 基因與 hflB 基因之選殖與分析 19
(一) 花生簇葉病菌質體 serS 基因篩選用核酸探針之製備 19
1. 核酸探針片段序列之增幅與選殖 19
2. 核酸探針之標識 (labeling) 24
(二) 花生簇葉病菌質體次基因體基因庫 (subgenomic library) 之構築與篩選 25
1. 南方氏轉漬 (Southern blot) 及雜配反應 (hybridization) 25
2. 花生簇葉病菌質體次基因體基因庫之構築 27
3. 花生簇葉病菌質體次基因體基因庫之篩選 30
4. 轉形株重組質體特性分析 31
三、花生簇葉病菌質體 serS 基因 5’ 端與 5’ 端上游未轉譯區 (5’-UTR) 之選殖與分析 31
(一) 篩選用核酸探針之製備 32
(二) 次基因體基因庫之構築與篩選 32
四、serS上游基因 rpsT 之選殖與分析 32
(一) 聚合酵素連鎖反應 33
(二) 聚合酵素連鎖反應產物之選殖與分析 33
1. 聚合酵素連鎖反應產物之選殖 33
2. 重組質體之特性分析 33
五、花生簇葉病菌質體serS 基因、rpsT 基因與 hlfB 基因推衍蛋白質之結構預測 33
六、花生簇葉病菌質體 serS 基因之北方氏雜配反應 (Northern hybridization ) 分析 33
(一) 健康及罹病植物全 RNA 之純化 34
(二) 北方氏雜配反應 (Northern hybridization) 34
七、花生簇葉病菌質體 serS 基因轉錄 (transcription) 起始點之分析 35
(一) 反轉錄反應引子及聚合酵素連鎖反應引子對之設計 35
(二) 反轉錄聚合酵素連鎖反應 (reverse transcription PCR) 36
1. 以反轉錄 (reverse transcription) 反應進行serS 基因cDNA 之合成 36
2. 聚合酵素連鎖反應 37
(三) 反轉錄聚合酵素連鎖反應產物之選殖與分析 38
1. 反轉錄聚合酵素連鎖反應產物之選殖 38
2. 重組質體之特性分析 38
肆、結果 39
一、以日日春為宿主繁殖花生簇葉病菌質體及植物全DNA (total DNA) 之純化 39
二、花生簇葉病菌質體 serS 基因與 5’ 端上游未轉譯區之選殖 39
(一) serS 基因之選殖 39
1. 南方氏轉漬與雜配反應 39
2. SpeI 次基因體基因庫篩選 40
3. 選殖株lib_SpeI_4-19 之重組質體序列分析 40
(二) serS 基因5’ 端與 5’ 端上游未轉譯區之選殖 41
1. 南方氏雜配反應 41
2. HindIII 次基因體基因庫篩選 42
3. 選殖株lib_HindIII_2-36之重組質體序列分析 42
三、serS上游基因 rpsT 之選殖 43
(一) rpsT 基因之選殖 43
(二) 選殖株c_wsf6mr7_rpsT之重組質體序列分析 43
四、各選殖株之選殖片段序列整合與特性分析 44
五、花生簇葉病菌質體serS 基因、rpsT 基因與 hlfB 基因推衍蛋白質之結構分析 46
(一) serS 基因推衍蛋白質之結構分析 46
(二) rpsT 基因推衍蛋白質之結構分析 47
(三) hflB 基因推衍蛋白質之結構分析 47
六、花生簇葉病菌質體 serS 基因之RNA 表現分析 49
伍、討論 50
陸、參考文獻 60
柒、圖表 74
捌、附錄 114

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