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研究生:吳佳穎
研究生(外文):Chia-Ying Wu
論文名稱:霍香薊黃脈病毒Rep蛋白與基因體核酸之分子交互作用及利用原核系統分析病毒之複製機制
論文名稱(外文):Molecular interactions among Rep protein and genomic DNA of ageratum yellow vein virus Ping-Tung strain and analyses of replication in prokaryotic system
指導教授:胡仲祺
學位類別:碩士
校院名稱:國立中興大學
系所名稱:農業生物科技學研究所
學門:農業科學學門
學類:農業技術學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:74
中文關鍵詞:霍香薊黃脈病毒雙生病毒
外文關鍵詞:ageratum yellow vein virusgeminivirus
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摘要
植物病毒的複製能力是決定病毒生存的主要關鍵之一。而 Rep 蛋白是雙生病毒感染植物初期最早轉譯的蛋白並且具有起始rolling-circle複製機制、專一性切割及粘合基因體、調控基因體啟動子、自體聚合以及調控細胞週期等功能。本研究以霍香薊黃脈病毒屏東株系 (AYVV-PT) 為材料並利用大腸桿菌外源蛋白表現系統表現具有活性的 Rep 蛋白以進一步了解Rep 蛋白在單基因體之 AYVV-PT 病毒複製系統中所扮演之角色。並利用 E. coli 噬菌體 M13 的複製機制以研發雙生病毒感染系統。首先將Rep 蛋白基因構築於pETBlue-2 外源蛋白表現質體中,並以純化之Rep蛋白產生具專一性之多株抗體;而為保有 Rep 蛋白之活性,另行將 Rep 蛋白基因轉殖於 pGEX-4T-1 外源蛋白表現質體中,以親和性管柱純化方式得到自然狀態 (Native form) 之Rep蛋白,並利用核酸蛋白交互作用暨酵素結合免疫吸附分析法 (NB-ELISA) 及南方-西方墨點分析法偵測證實 Rep 蛋白確可與病毒基因體進行交互作用且對於包含 Rep;TrAP 啟動子片段 (nt. 1522-2731) 具有較高之結合效率。此外,為研發雙生病毒感染系統及建立原核生物複製系統以便於研究雙生病毒之複製狀況,本研究將 AYVV-PT 之全長基因體構築於 M13 噬菌體中再轉染細菌 E. coli strain JM101,經由南方墨點法分析細菌中所萃取之 DNA,發現的確有類似於複製中間體 (Replication intermediate) 的小分子 DNA 片段存在。並利用 time course 實驗測得此 DNA 片段在噬菌體 M13 的系統中有累積的現象。再利用生體外轉錄方法以及單向 PCR 方法製備 AYVV-PT基因體之正負股專一性探針,以南方墨點分析法,PCR 核苷酸序列分析方法以及 RNase 和 S1 nuclease digestion方法在分子層次上證實此小分子 DNA 片段確為 AYVV-PT 單股環狀DNA。由此可推測噬菌體 M13的複製系統的確具有支持 AYVV-PT 產生單股環狀DNA的能力。由上述結果推測 AYVV-PT 應可於 E. coli/M13 的系統中進行複製以產生單股環狀之基因體 DNA。此一原核系統將可簡化雙生病毒複製機制之研究,並可應用於病毒接種系統及外源蛋白表達系統之研發。
Abstract
The replication ability is one of the key determinants for surviving of viruses. The Rep protein, the first expressed by geminiviruses during the early infection stage, is the only virus-encoded protein required for the replication process, including specific cleavage and ligation to initiate and terminate rolling-circle replication, self-interaction, and regulation of the host cell cycles. In this research, the monopartite begomovirus, ageratum yellow vein virus Ping-Tung strain (AYVV-PT) was used to investigate the interactions between Rep proteins and viral DNAs and to develop the prokaryotic replication system based on E.coli/phage M13 to facilitate the study of geminivirus replication. The Rep gene of AYVV-PT was cloned into plasmid pETBlue for expression, and gel-purified Rep proteins were used for producing specific polyclonal antibodies. The Rep gene was further sub-cloned into plasmid pGEX-4T-1 to obtain Rep proteins in native condition by affinity chromatography. The interactions between Rep proteins and viral genomic DNAs were investigated by Nucleoprotein Binding-ELISA (NB-ELISA) and Southwestern blot analyses. Rep protein has the highest affinity to nt. 1522-2731 of AYVV-PT genome under the optimized in vitro binding condition, 20 mM Tris-HCl buffer, pH 7.4 containing 0.4-0.6 % NaCl. The full-length AYVV-PT genome was cloned into phage M13 as a single unit, and specific Digoxygenin-labeled RNA and DNA probes for AYVV-PT were prepared by in vitro transcription and asymmetric PCR. The signals of small DNAs co-migrating with the single-stranded, circular DNAs encpasidated in virus particles were detected by Southern blot analysis, and the accumulation of the small DNAs was revealed by time course analysis. In addition, the small DNAs were further confirmed as single-stranded, circular DNA of AYVV-PT by RNase A, S1 nuclease digestions and PCR followed by nucleotide sequence analysis. The results suggested that the prokaryotic replication machinery of E.coli/phage M13 could support the generation of single-stranded, circular DNA of AYVV-PT in a construct containing only one copy of replication origin and a single unit viral genome. The E.coli/phage M13 system may facilitate the study of geminivirus replication and the development of convenient infection and foreign gene expression systems.
Table of Contents
A).中文摘要………………………………………………1
B).Abstract………………………………………………2
C).Introduction…………………………………………3
1).Economical significance………………………… 4
2).Taxonomy………………………………………………6
(a). Mastrevirus……………………………………6
(b). Curtovirus…………………………………… 7
(c). Begomovirus……………………………………7
3). Biological properties……………………………8
4). Rep protein..………………………………………9
(a). Viral DNA replication and transcription……10
(b). Gene regulations………………………………… 10
(c). Cell cycle regulation……………………………11
5). Abilities for Cross-Kingdom replication…………12
6). The need for an experimental system for the study of
geminivirus replication………………………………………………… 14
D). Objectives……………………………………………… 14
E). Materials and Methods…………………………………15
1). Virus source..………………………………………… 15
2). Virus purification…………………………….………15
3). Total DNA extraction from purified virus particle ……………………… 15
4). Simple extraction method of viral DNA from infected plant…….………… 16
5). Total DNA extracted from infected plant…..………………………… 16
6). Cloning of Rep gene into pETBlue-2 expression vector……………………… 17
7). Sequence analysis………………………………………18
8). Expression and purification of AYVV-PT Rep protein………………………. 18
9). Preparation of antisera……………………………………………………… 19
10). Western blotting analysis…………………………………………………. 19
11). Cloning the Rep gene into pGEX-4T-1 expression vector……………………. 20
12). Purification of the Rep protein under native condition………………..….…… 20
13). Cloning of the full-length AYVV-PT into phage M13…………………...…… 21
14). Screening and preparation of double-stranded bacteriophage M13 DNA.……. 22
15). The preparation of specific DIG-labeled RNA and DNA probes……..………. 22
(a). DIG-labeled RNA probes……………………………………… 22
i). PCR amplification of templates for in vitro transcription……………… 22
ii). In vitro transcription…………………23
(b). DIG-labeled DNA probes…………………… 24
(c). DIG-labeled DNA probes used in NB-ELISA24
16). The characterization of the extra DNAs by PCR with specific primers……… 25
17). Southern blot hybridization analysis……………25
18). Southwestern blot analyses…………………………26
19). The Nucleoprotein Binding-ELISA (NB-ELISA) assay………………… 27
F). Results and Discussion……………………………… 28
1). Expression of AYVV-PT Rep protein in bacteria using
pETBlue-2 vector system…………………………… 28
2). Expression of AYVV-PT Rep protein in bacteria using
pGEX-4T-1 vector system…………………………… 29
3). Production of antisera against Rep protein…………………………………… 29
4). The identification of GST-Rep fusion protein by western blot analysis……… 30
5). Rep protein-DNAs interaction analyses………………………………………… 30
5-1). NB-ELISA analysis……………………………………………………...31
(a). The effect of salt concentration……………………………………… 31
(b). The effect of concentration of Rep proteins………………………… 31
(c). The effect of concentration of various DNAs……………………… 32
5-2). Southwestern analysis…………………… 33
6). Analysis of single-stranded circular DNA genome of AYVV-PT
produced in bacteria E. coli/ phage M13 system………………………… 34
(a). Construction of AYVV-PT/ mp18……………………………………… 34
(b). Small DNAs of AYVV-PT produced in the E. coli /phage M13 system… 34
(c). The accumulation of small DNA in E. coli /phage M13 system………… 35
(d). The characterization of the small DNA derived from AYVV-PT/mp18
by PCR method and nucleotide sequence analysis…………………… 35
(e). Southern blot analysis of the strandedness of the circular DNA………… 36
(f). Detection of M13 sequence by Southern blot analysis………………… 37
(g). Identification of single-stranded circular DNA of AYVV-PT
by RNase A and S1 nuclease digestion analysis……………………… 37
G). Summary……………………………………………………40
H). References……………………………………………… 42
I ).Figures and Tables…………………………………… 53
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