沈湯龍臺灣大學：植物病理與微生物學研究所王麗雅Wang, Li-YaLi-YaWang2007-11-272018-06-292007-11-272018-06-292007http://ntur.lib.ntu.edu.tw//handle/246246/58081轉錄後基因靜默（post-transcriptional gene silencing，PTGS），亦即RNA干擾現象 (RNAi)，會導致目標基因的mRNA被分解，是為一種調節基因表現的機制。近幾年來有許多研究顯示，此現象不但廣泛地存在於生物界，更為真核生物用以抵禦病毒侵染的一套固有防禦機制。然而，病毒也演化出另一種反防禦策略，亦即利用抑制子 (suppressor)，去干擾內生的基因靜默訊息傳遞路徑，以達到成功感染目的。本研究以煙草鑲嵌病毒 (TMV) 為模式，探討TMV p126蛋白如何參與對抗PTGS。首先以GFP煙草轉植株 (Nicotiana benthamiana, line 16c) 建立篩選平台，藉由農桿菌接種法 (agroinfiltration) 送入外來GFP，啟動PTGS機制導致16c中GFP mRNA量降低。我們首先觀察到當煙草株遭受TMV感染，或單獨表現p126蛋白時，GFP不僅可恆常表現，甚至有被助長表現之情況。進一步將p126蛋白依據其不同的功能性區塊 (domain) 進行擷取，共分為methyltransferase (MET)、non-conserved region (nonI)、non-conserved region II (nonII) 和helicase (HEL) 四個區塊。當個別表現MET、nonII和HEL於16c時，皆顯示其有抑制PTGS之功能。為了進一步釐清其參與機制，我們利用點突變破壞MET及HEL之酵素活性區，但結果顯示其酵素功能似乎不參與在抑制PTGS之機制中。令人驚訝的是，當我們替換nonII三個會影響病徵之胺基酸後，便失去抑制PTGS之功能。另外，我們也建立p126煙草轉殖株，期望找出病毒致病性與PTGS抑制功能兩者之間的相關連結，但若要明確了解整個機制則仍有待持續的研究與探討。Post-transcriptional gene silencing (PTGS) is a conservative phenomenon across kingdoms. In plants, it has been demonstrated that PTGS functions as an intrinsic defense mechanism during viral infection. On the other hand, viruses have evolved counter-defense strategies by encoded suppressors to circumvent PTGS. In previous studies, Tobacco mosaic virus (TMV) encoded p126 protein has been exhibited as a suppressor of PTGS although its mechanism remains unknown. In this current study, we found that there are multiple suppressor domains/motifs within the p126 protein, including methyltransferase, helicase and non-conserved region II, implicating that these domains/motifs could function synergistically against PTGS. Nevertheless, these suppressor domains might exhibit differential consequences on local and/or systemic gene silencing. Moreover, the enzymatic activities of methyltransferase and helicase are dispensable in the suppression pathways. Interestingly, several amino acids within the non-conserved region II pivotal for pathological consequence seem to intimately correlate with extend of the ability of gene silencing suppression. Together, these results suggest that a role of gene silencing suppression of TMV determinates its susceptibility on host plants. Furthermore, the MAPK signal cascade(s) was involved in the functioning of the p126-mediated gene silencing in tobacco. Our findings will therefore help elucidate the function and evolution of different virus suppressor and lead to new crop protection strategies.口試委員會審定書………………………………………………………………….. I Abstract…………………………………………………………………………........ II 中文摘要…………………………………………………………………………… IV Content of figures Figure 1. The map of constructs used in this study. ………………………….………... 30 Figure 2. Suppression of PTGS by TMV……………………...……………………. 32 Figure 3. Effects of p126 on systemic GFP silencing………..……………..…………… 34 Figure 4. The different p126 fragments are associated with suppressor activity…………... 37 Figure 5. Transient expression of mutant variants of p126 in N. benthamiana p126 mutate variants………………………………………………………………………………….…..… 39 Figure 6. The phenotype of MII-16………………………………………………….………. 41 Figure 7. MAPKs were affected by TMV infection and overexpression of the p126 protein... 43 Content of tables Table 1. Primers sequences used in this study………………………………………………….45 Table 2. Statistics of systemic silencing suppression by the TMV p126 and others suppressors…………………………………………………………………………………….47 Table 3. Suppression of systemic silencing by TMV p126 and other mutants………………………………………………………………………………………….48 Introduction……………………………………………………………………….. 1 RNA silencing…………………………………………………………….……. 1 RNA silencing pathway…………………………………………………………. 2 Viral suppressors of RNA silencing …………………………………………… 4 Diverse mechanisms of viral suppressors affect RNA silencing……………….. 4 RNA silencing suppressor encoded by TMV………………..…………….……. 6 Materials and Methods …………………………………………………………... 10 Plant material and growth conditions…………………………………………. 10 Plasmid constructs…………………………………………………………….. 10 Agrobacterium mediated the induction of systemic GFP silencing…………….. 12 GFP image of plants under UV illumination………………………………..…... 12 Analysis of proteins extracted from infiltrated N. benthamiana (16c) leaves by pull-down western blotting……………………………………………………. 13 RT-PCR……………………………………………………………………...... 14 Northern blot analysis ………………………………………………………… 15 Results ……………………………………………………………….…………... 17 Suppression of PTGS by TMV………………………………………………... 17 The TMV-encoded p126 protein involves in the PTGS suppression……..…... 18 Dissection of the TMV-encoded p126 protein in the suppression of PTGS….. 19 The enzymatic activities of the p126 are negligible on the PTGS suppression.... 21 Mutational analysis of the non-conserved region II in the PTGS suppression…. 22 The TMV-encoded p126 suppressor affects the MAPK signal transduction…… 24 Discussion………………………………………………………………………...... 25 Figures……………………………………………………………………………. 30 Tables……………………………………………………………………………... 45 References…………………………………………………………………….….. 49 Appendix…………………………………………………………………………. 67 Generation of the p126 transgenic tobacco plants…………………………. 674166764 bytesapplication/pdfen-US基因靜默煙草鑲嵌病毒抑制子p126PTGSsiRNAsuppressorTMVotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/58081/1/ntu-96-R94633005-1.pdf