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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 王重雄(Chung-Hsiung Wang) | |
dc.contributor.author | Yu-Shin Nai | en |
dc.contributor.author | 乃育昕 | zh_TW |
dc.date.accessioned | 2021-06-15T04:15:16Z | - |
dc.date.available | 2013-01-21 | |
dc.date.copyright | 2010-01-21 | |
dc.date.issued | 2010 | |
dc.date.submitted | 2010-01-12 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/45344 | - |
dc.description.abstract | 黑角舞蛾 (Lymantria xylina Swinhoe) 為台灣林木及果樹類重要害蟲,本實驗室由野外罹病蟲體分離出兩種病毒病原體,皆屬桿狀病毒科,Alphabaculovirus 屬。在野外黑角舞蛾族群中以 LyxyMNPV 感染為主要,將 LyxyMNPV 基因體解序,並進行深入研究。LyxyMNPV 基因體大小為 156,344 bp,GC 百分比為 53.47%,其中包含 157 個開放譯讀區 (open reading frame, ORF),13 個同源區域 (homologus regsions, hrs) 及 14 個桿狀病毒重複開放譯讀區 (baculovirus repeat orf, bro)。親緣關係分析上顯示 LyxyMNPV 屬於 group II NPV 與 LdMNPV 最為接近,唯基因排列上有所差異。LyxyMNPV 157 個 ORFs 中,包括 151 個 ORFs 與 LdMNPV 互為同源基因;2 個 ORFs 與其他桿狀病毒之基因同源;4 個 ORFs為特有基因,而其中一個為桿狀病毒中首次發現之特有基因,為 gag-like 的同源基因。LyxyMNPV 具有兩個非經由複製產生之 odv-e27 同源基因。LyxyMNPV 缺乏 host range factor-1 (hrf-1),但仍可感染 NTU-LY-1 (LY) 及 IPLB-LD-652Y (LD) 細胞;進一步比較三種可感染 LD 細胞之病毒基因體 (LyxyMNPV、LdMNPV 及 OpMNPV),結果顯示其共同擁有三個基因:inhibitor of apoptosis-3 (iap-3)、ribonucleotide reductase 2b (rr2b) 及 dutpase,其中 iap 基因在病毒感染細胞初期扮演非常重要的角色。
LyxyMNPV 具有兩個 iap 基因:iap-2 (687 bp) 與 iap-3 (450 bp),經轉錄本分析結果顯示,iap-2 之轉譯起始點 (ATG) 上游 (upstream) 第15個鹼基對具有一個晚期及非常晚期基因轉錄起始位置 (TAAG),而 iap-2 之轉錄起始點位於 ATG 上游第 16 個鹼基對;iap-3 之轉錄起始點位於 ATG 上游第 44 個鹼基對上之 CTTGT 保守區域,而保守區域下游第 25 個鹼基對接有 TCGT 序列;利用反轉錄-聚合酶鏈鎖反應得知此二基因 mRNA 表現量在 LyxyMNPV 感染寄主 LD 細胞後 6 小時開始上升至 72 小時,但於 72 小時後便開始下降直至 120 小時;而在 SF 細胞中,iap-2 之mRNA 表現量至感染後 120 小時仍相當低,而 iap-3 之mRNA 表現量在感染 48 小時後開始表現,且持續上升至感染後 120 小時。進一步在 SF 細胞株中利用共表現實驗 (co-expression) 進行蛋白質功能截斷分析,結果顯示 IAP-2、IAP-3 及 IAP-2-BIR 均可抑制由果蠅 RPR 蛋白所誘導之細胞凋亡反應 (P<0.05) 而 IAP-3-BIR、IAP-2-RZF 及 IAP-3-RZF在抑制由果蠅 RPR 蛋白所誘導之細胞凋亡反應則無顯著功效,推測 IAP-3 需要全長表現才具抑制由果蠅 RPR 蛋白所誘導之細胞凋亡效果。 另一株由黑角舞蛾分離出的病毒,經由 polyhedrin,lef-8 及 lef-9 基因序列分析,証實此病毒係異於 LyxyMNPV 而近於 LdMNPV的病毒,因此命名為 LdMNPV-like virus,其感染 LD 細胞呈現典型寡多角體 (Few polyhedra, FP) 病徵,經由選殖定發現其 fp25k 基因缺失 44 bp。此病毒是首次野外所發現呈現 FP 的病毒。 | zh_TW |
dc.description.abstract | The casuarina moth, Lymantria xylina Swinhoe (Lepidoptera: Lymantriidae), is an important forest pest in Taiwan. Two nucleopolyhedrovirus (NPV) strains were isolated from L. xylina larvae with an epizootic nucleopolyhedrosis. In the field, LyxyMNPV is the most prevalent vius strain, therefore, the nucleotide sequence was determined and analysed. The genome of LyxyMNPV consists of 156,344 bases with a G+C content of 53.4% and contains 157 putative open reading frames (ORFs). The LyxyMNPV genome encoded 14 bro genes. 13 homologous regions (hrs) were identified. In a phylogenetic analysis, LyxyMNPV is a member of Group II NPV and closely related to LdMNPV but with highly distinct genomic organization. The gene content and gene order of LyxyMNPV were similar to those of LdMNPV, with 151 ORFs identified homologous to those reported in the LdMNPV genome. Two genes were homologous to other baculoviruses and four unique ORFs were identified in LyxyMNPV genome, including a gag-like gene which was not reported in baculoviruses. Two putative odv-e27 homologues were identified in LyxyMNPV, each of which has been acquired independently and not by gene duplication. LyxyMNPV lacks host range factor-1 (hrf-1). However, in vitro host range assay indicated that LyxyMNPV could infect both NTU-LY and IPLB-LD-652Y cell lines with the absence of hrf-1. Therefore, we compared LyxyMNPV gene content to those of LdMNPV and OpMNPV, and found that these three NPVs shared three genes, iap-3, rr2b, and dutpase, that are absent in AcMNPV genome, of these three genes, iap-3 is one of the baculovirus genes that affect viral host range and prevent apoptosis in baculovirus-infected cells.
Two groups of anti-apoptotic genes, p35 and iap (inhibitor of apoptosis) family, have been identified in baculovirus. There are two iap genes, iap-2 (687 bp) and iap-3 (450 bp), in LyxyMNPV genome. The 5’UTR of iap-2 contains a transcriptional initiation site, TAAG, of late and very late gene motif, but 5’UTR of iap-3 contains CTTGT promoter motif plus TCGT in 25 bp spacing context. The mRNA expression profiles of these two genes in permissive cell line LD were evaluated using RT-PCR. The expression levels of iap-2 and iap-3 in the LD cells infected with LyxyMNPV raised from 3 to 72 hours post-infection (p.i.), but declined after 72 to 120 hours. Interestingly, the expression level of iap-2 and iap-3 of the infected SF cells showed significant difference. The mRNA of iap-2 was not detected; in contrary, iap-3 was detectable. During the process of infecting SF cells with LyxyMNPV, iap-3 presented a delayed expression pattern, which was detected at 48 hours p.i. and continued to rise for 120 hours. Functional assay of two iap genes was performed using over-expression method. In SF cells, full length IAP-2, IAP-3 and IAP-2-BIR domain inhibited the apoptosis induced by Drosophila RPR protein (P<0.05), however, IAP-3-BIR domain could not rescue the apoptosis induced by D-RPR. LdMNPV-like virus was nominaded base on the sequences analyses of polyhedrin, lef-8 and lef-9. This virus was closely related to LdMNPV but far from LyxyMNPV. LdMNPV-like virus showed a few polyhedra (occlusion bodies) CPE in the infected LD cells. A significant deletion of a 44 bp sequence found in LdMNPV-like virus was noted in the fp25k sequences of LdMNPV and LyxyMNPV and may play an important role in the few polyhedra CPE. This is the first FP virus isolated from the field-collected larvae infected with NPV. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T04:15:16Z (GMT). No. of bitstreams: 1 ntu-99-D95632003-1.pdf: 8168544 bytes, checksum: 6f6ae6b9eb973771c762388b97bbd417 (MD5) Previous issue date: 2010 | en |
dc.description.tableofcontents | 目 錄
口試委員審定書.............................................i 致謝......................................................ii 中文摘要.................................................iii 英文摘要...................................................v 第壹章、緒言...............................................1 第貳章、文獻探討...........................................3 (一) 黑角舞蛾基本生物特性及為害............................3 (二) 黑角舞蛾之防治綜觀....................................4 1. 化學防治................................................4 2. 費洛蒙及物理防治........................................4 3. 生物防治................................................4 4. 微生物防治..............................................5 (三) 桿狀病毒之生物學及分類學介紹..........................5 1. 桿狀病毒之簡介..........................................5 2. 桿狀病毒之生活史及基因表現..............................6 3. 桿狀病毒之分類..........................................7 (四) 病毒基因體解序之發展..................................8 (五) 桿狀病毒主域之研究....................................8 (六) 細胞凋亡與桿狀病毒抑制細胞凋亡因子....................9 1.細胞凋亡.................................................9 2. 桿狀病毒抑制細胞凋亡因子...............................10 3. p35/p49 基因...........................................11 4. 抑制細胞凋亡基因家族 (iap gene family).................11 (七) 黑角舞蛾桿狀病毒之研究...............................12 第參章、材料方法..........................................15 (一) 黑角舞蛾核多角體病毒 (LyxyMNPV) 基因體解序及分析.....15 1. 供試細胞株.............................................15 2. 病毒來源及其純化.......................................15 3. 病毒 DNA 之製備........................................16 4. 基因體序列解序.........................................16 5. DNA 序列分析...........................................16 6. 親緣關係分析...........................................17 (二) LyxyMNPV 抑制細胞凋亡基因之研究......................18 1. iap-2 及 iap-3 核酸與胺基酸序列分析比對................18 2. 供試細胞株.............................................18 3. 供試病毒株.............................................18 4. 病毒感染...............................................18 5. RNA 之製備.............................................18 6. 基因表現時序檢測.......................................19 (1) cDNA 之製備...........................................19 (2) RT-PCR................................................19 7. 五端與三端未轉譯區段 (Untranslated region, UTR) 鑑定...19 (1) 五端未轉譯區段鑑定 (5’ RACE).........................19 (2) 三端未轉譯區段鑑定 (3’ RACE).........................20 8. DNA 片段選殖...........................................21 (1) 接合反應 (Ligation)...................................21 (2) 轉形作用 (transformation).............................21 (3) 質體 DNA 之萃取.......................................21 9. 基因快速表現及功能分析.................................22 (1) 表現載體建構..........................................22 (2) 表現載體之轉染........................................22 (3) 熱誘導表現............................................22 (4) 果蠅 rpr 誘導細胞凋亡存活率分析.......................22 (5) 統計分析..............................................23 10. 西方墨點法 (Western blot).............................23 (1) 聚丙烯醯胺膠體電泳 (SDS-PAGE).........................23 (2) 蛋白質轉印、雜合及呈色................................23 (三) 類吉普賽舞蛾核多角體病毒 (LdMNPV-like virus) 特徵建立24 1. 供試細胞株.............................................24 2. 供試病毒株.............................................24 3. 病毒超微結構之觀察.....................................24 (1) 病毒感染..............................................24 (2) 穿透式電子顯微鏡超微結構觀察..........................24 4. LdMNP-like virus 出芽病毒之純化........................25 5. 病毒 DNA 之製備........................................25 6. 限制酶圖譜分析.........................................25 7. 南方墨點雜合法 (Southern blot hybridization)...........26 (1) 探針製備..............................................26 (2) 轉印..................................................26 (3) 雜合反應..............................................26 (4) 呈色反應..............................................27 8. 基因選殖...............................................27 9. 序列分析與比較.........................................27 10. 親緣關係分析..........................................28 第肆章、結果..............................................29 (一) 黑角舞蛾核多角體病毒 (LyxyMNPV) 基因體解序及分析.....29 1. LyxyMNPV 基因體特性....................................29 2. ORFs 啟動子分析........................................30 3. LyxyMNPV 之 ORFs 與其他桿狀病毒之比較..................30 4. 結構基因...............................................31 5. 轉錄相關基因...........................................31 6. DNA 複製相關基因.......................................32 7. LyxyMNPV 具輔助功能之基因..............................33 8. 抑制細胞凋亡基因.......................................33 9. 桿狀病毒重複開放譯讀區 (baculovirus repeat orf, bro)...33 10. 同源區域 (homologous regions, hrs)....................34 11. LyxyMNPV 之重複基因...................................35 12. LyxyMNPV 特有 ORFs....................................35 13. LyxyMNPV 之親緣關係演化分析...........................36 14. LyxyMNPV 與 LdMNPV 之比較.............................37 15. 感染 LD 細胞之桿狀病毒 ORFs 比較......................37 (二) LyxyMNPV 抑制細胞凋亡基因之研究......................38 1. 核酸與胺基酸序列比較...................................38 (1) 基因相似度比較........................................38 (2) 胺基酸序列相似度比較..................................38 2. 蛋白質結構分析.........................................38 3. iap-2 與 iap-3 之 5’ 與 3’ 端未轉譯區段分析..........39 4. iap-2 及 iap-3 基因 mRNA 表現偵測......................39 5. IAPs 功能分析..........................................39 (1) 過量表現 (over expression)............................40 (2) 果蠅 RPR 蛋白誘導細胞凋亡存活率分析...................40 (三) 類吉普賽舞蛾核多角體病毒 (LdMNPV-like virus) 特徵化之建立........................................................41 1. 病毒超微結構之觀察.....................................41 2. LdMNPV-like virus 限制酶圖譜分析.......................41 3. 南方墨點雜合法.........................................41 4. DNA 序列分析和比較.....................................42 5. 親源演化分析...........................................42 第伍章、討論..............................................44 (一) 黑角舞蛾核多角體病毒 (LyxyMNPV) 基因體解序及分析.....44 (二) LyxyMNPV 抑制細胞凋亡基因之研究......................48 (三) 類吉普賽舞蛾核多角體病毒 (LdMNPV-like virus) 特徵化之建立........................................................51 第陸章、結論..............................................54 第柒章、參考文獻..........................................55 附表......................................................73 附圖......................................................85 附錄.....................................................107 附錄一、各種桿狀病毒之基本特性...........................107 附錄二、桿狀病毒之抑制細胞基因p35/p49 及 iap gene family.111 附錄三、本論文所使用的引子名稱與序列.....................113 附錄四、桿狀病毒之 Polyhedrin、LEF-8及 LEF-9 胺基酸序列..115 附錄五、已發表論文......................................118 表次 表一、LyxyMNPV 基因體中所預測之開放譯讀區.................73 表二、LyxyMNPV 之 LdMNPV 非同源基因.......................80 表三、LdMNPV 與 OpMNPV之 ORF 中,與 LyxyMNPV 非同源之.....81 表四、LdMNPV-like virus、LyxyMNPV 及 LdMNPV 之 fp25k、polh、 lef-8 及 lef-9 DNA 及胺基酸序列相似度、相同度比較..83 表五、polh、lef-8 and lef-9 基因及三基因合併之 Kimura-2-parameter 演化距離........................................84 圖次 圖一、LyxyMNPV 基因體輿圖.................................85 圖二、LyxyMNPV ORFs 與 6 種桿狀病毒之同源基因相對位置比較........................................................86 圖三、LyxyMNPV 與 LdMNPV 基因體片段之 ORFs 及 hrs 之位置排列比較......................................................87 圖四、LyxyMNPV 迴紋中心序列比較...........................88 圖五、比較 LyxyMNPV、LdMNPV 以及 AcMNPV 各 hrs 在基因體中之位置......................................................89 圖六、桿狀病毒之核心蛋白親緣關係樹........................90 圖七、LdMNPV 胺基酸長度差異之比較.........................91 圖八、 LyxyMNPV、LdMNPV 及 OpMNPV 之 IAP 蛋白結構位置及其比較........................................................92 圖九、iap-2 與 iap-3 之3’ 端及 5’ 端 cDNA 快速放大......93 圖十、iap-2 及 iap-3 之 DNA 序列及胺基酸序列..............94 圖十一、黑角舞蛾核多角體病毒感染LD 與 SF 後 6、9、12、24、48 及 72 小時,iap-2 及 iap-3 RT-PCR 結果.................96 圖十二、黑角舞蛾核多角體病毒感染接LD 與 SF 後 1 至 5 天中, iap-2 及 iap-3 RT-PCR 結果................................97 圖十三、果蠅 rpr 基因誘導細胞凋亡存活率分析結果顯示 IAP-2、IAP-3及IAP-2- BIR 區域可抑制細胞凋亡反應..................98 圖十四、LyxyMNPV與LdMNPV-like virus感染LD細胞7天後之細胞病變.......................................................100 圖十五、LyxyMNPV與LdMNPV-like virus感染LD細胞株7天後之病毒超微結構...................................................101 圖十六、LdMNPV-like virus 基因組 DNA 之限制酶圖譜........103 圖十七、利用南方墨點雜合法偵測多角體基因位置.............104 圖十八、LdMNPV-like virus、LyxyMNPV及LdMNPV之fp25k DNA序列比對.......................................................105 圖十九、利用部分Polyhedrin、LEF-8及LEF-9胺基酸序列建構之親緣關係樹...................................................106 | |
dc.language.iso | zh-TW | |
dc.title | 黑角舞蛾核多角體病毒基因體解序及一種類吉普賽舞蛾核多角體病毒特性之分析 | zh_TW |
dc.title | Genomic Anlyses of LyxyMNPV and the Characterization of an LdMNPV-like Virus | en |
dc.type | Thesis | |
dc.date.schoolyear | 98-1 | |
dc.description.degree | 博士 | |
dc.contributor.coadvisor | 柯俊成(Chiun-Cheng Ko) | |
dc.contributor.oralexamcommittee | 侯豐男(Roger Feng-nan Hou),石正人(Cheng-Jen Shih),趙裕展(Yu-Chan Chao),羅竹芳(Chu-Fang Lo),吳宗遠(Tzong-Yuan Wu),張俊哲(Chun-che Chang) | |
dc.subject.keyword | 黑角舞蛾核多角體病毒,基因體解序,抑制細胞凋亡基因,基因表現,功能分析,類吉普賽舞蛾核多角體病毒, | zh_TW |
dc.subject.keyword | LyxyMNPV,genomic sequence,antiapoptosis genes,gene expression,functional assay,LdMNPV-like virus, | en |
dc.relation.page | 118 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2010-01-13 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 昆蟲學研究所 | zh_TW |
顯示於系所單位: | 昆蟲學系 |
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