請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/49191
標題: | 建構胡瓜嵌紋病毒載體於基因靜默及其在病害防治上的應用 Construction of a Cucumber mosaic virus vector and its application on gene silencing and disease control |
作者: | Hsiao-Hsuan Jan 詹孝軒 |
指導教授: | 葉信宏(Hsin-Hung Yeh) |
關鍵字: | 胡瓜嵌紋病毒,菸草嵌紋病毒,轉錄前基因靜默,病毒誘導基因靜默,交叉保護, Cucumber mosaic virus,Tobacco mosaic virus,virus induced gene silencing,transcriptional gene silencing,cross-protection, |
出版年 : | 2016 |
學位: | 碩士 |
摘要: | 病毒誘導基因靜默(virus induced gene silencing, VIGS)是一種藉由病毒感染來誘導生物體內發生基因靜默的技術,可藉由轉錄後基因靜默 (post transcriptional gene silencing) 或轉錄前基因靜默 (transcriptional gene silencing, TGS) 靜默特定基因,協助基因功能之研究。胡瓜嵌紋病毒 (Cucumber mosaic virus, CMV) 為一廣泛分布於世界的植物病毒,隸屬於Cucumovirus病毒屬,其寄主範圍橫跨上百科,超過1200種的植物。由於CMV的寄主範圍廣泛,因此有相當的潛力可發展成為一個可應用於各類植物上之病毒載體。於本研究中,我們參考國外文獻來構築實驗室前人所留下來的CMV感染性選殖株 (infectious clones),將之建構成新的病毒載體。我們在CMV病毒RNA2上的2b基因序列中以重疊延伸聚合酶鏈鎖反應 (overlapping polymerase chain reaction)的方式構築出一個限制酶切位,以此切位嵌入目標基因序列於病毒之中,再以此病毒載體感染植物來誘導VIGS的發生。為了確定此病毒載體的基因靜默效能,我們利用能產生綠螢光蛋白的16c轉基因菸草(35S::GFP)來做為實驗材料,並以其轉基因的35S啟動子為目標,試圖誘導其轉錄發生變化。實驗中可以看到受到CMV 病毒載體 (CMV-35S)感染的植物並未產生明顯的病徵,使用UV燈照射來觀察螢光可觀察到明顯弱化之螢光,且其綠螢光蛋白的基因較少。我們也試圖以此載體在菸草誘導穀胺酸-1-半醛轉胺酶 (glutamate-1-semialdehyde aminotransferase, GSA)的基因靜默。GSA是一個葉綠素生合成的前期酵素,實驗中接種了病毒載體而產生VIGS的植物因為葉綠素無法合成的關係出現了葉片白化的現象,且其GSA基因產物較少。此外我們也將菸草嵌紋病毒(Tobacco mosaic virus, TMV)的序列片段插到CMV病毒載體(pCMV-TMVCP),嘗試分析pCMV-TMVCP接種植物是否可同時對於CMV及TMV同時具有保護效果。實驗結果可以看到嵌有TMV之pCMV-TMVCP不會引起明顯病徵,且71%的接種植物對於CMV和TMV具有保護效果,然而仍在少數試驗植物上發現其接種的CMV會剔除外接基因而引起嚴重病徵,這是未來這個病毒載體還可以改進的一個研究方向。 Virus induced gene silencing (VIGS) is to knockdown gene(s) with interests through virus vector. VIGS can induce post transcriptional gene silencing (PTGS) or transcriptional gene silencing (TGS), and can be applied in gene functional analysis. Cucumber mosaic virus (CMV) belongs to the genus Cucumovirus, and it is important plant virus widespread around the world. CMV has a wide host range of over 1200 species in more than 100 families. Thus, it has good potential to become a favorable virus vector which can be applied on various plants. In this research, we try to modify our previously constructed CMV infectious clone to a viral vector. We used overlapping polymerase chain reaction (overlapping PCR) to introduce an additional restriction site on the 2b open reading frame on CMV RNA2, and used this restriction enzyme site for the insertion of foreign sequences. To test the viral vector in silencing inducing, we inoculated the CMV carrying a fragment of 35S promoter (pCMV-35Spro) on a transgenic Nicotiana benthamiana (35S::GFP), line 16c. We observed that plant infected with pCMV-35Spro only showed mild symptoms, decreased expression of green florescence expression under UV illumination and decreased expression of GFP gene on the inoculated plants. We also tried our vector to induce VIGS of glutamate-1-semialdehyde aminotransferase (GSA) in N. benthamiana. GSA is an enzyme involved in early biosynthesis of chlorophyll (Matters and Beale, 1994). Plants induced GSA silencing by our vectors were expected to show chlorotic phenotypes and decrease GSA gene expression. We also inserted a fragment of Tobacco mosaic virus (TMV) into the CMV vector p(CMV-TMVCP) in an attempt to see whether the plants inoculated with pCMV-TMVCP can protect later wildtype CMV and TMV infection. The results showed that pCMV-TMVCP inoculated plants show mild symptoms, and 71% of inoculated plants did not show severe symptoms after inoculation with wildtype TMV or CMV. However, some pCMV-TMVCP inoculated plants showed severe symptoms and PCR analysis revealed that some TMV gene was disappeared from pCMV-TMVCP. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/49191 |
DOI: | 10.6342/NTU201602919 |
全文授權: | 有償授權 |
顯示於系所單位: | 植物病理與微生物學系 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-105-1.pdf 目前未授權公開取用 | 1.56 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。