葉信宏博士臺灣大學：植物病理與微生物學研究所周俊男Chou, Chun-NanChun-NanChou2007-11-272018-06-292007-11-272018-06-292007http://ntur.lib.ntu.edu.tw//handle/246246/58057摘要 本論文根據小室康雄(1973)與吳(1994)之分類模式，將自田間採集之香蕉嵌紋病，以圓葉煙草(Nicotiana glutinosa)、豇豆(Vigna sinensis)為鑑別寄主進行病毒系統鑑定。依照病徵表現，可將CMV分成三大病徵型：第一型可在圓葉煙草上造成嚴重嵌紋且葉片變形，並在造成蕉葉嚴重嵌紋，而造成畸形葉；第二型可在圓葉煙草上造成嚴重嵌紋並產生壞疽斑，且在蕉葉上造成黃化嵌紋並產生壞疽；第三型可在圓葉煙草上造成輕微病徵但在蕉葉上產生嚴重嵌紋，但並不產生葉片畸形的現象。以豇豆病徵進行系統鑑定時發現，第一型其中一分離株(sm1)與第三型病毒(由皇帝豆嵌紋病分離來)能在豇豆上造成系統性感染，屬於豆科CMV系統群。而其餘之第一型與第二型病毒皆只能在豇豆接種葉上造成局部壞疽斑點，無法造成系統感染，屬茄科CMV普通系統群。選殖所有分離株之鞘蛋白序列，定序後之序列與臺灣所發現CMV分離株及世界其它各地之分離株進行類源分析。其結果發現，茄科普通系統群與豆科系統群明顯屬於兩不同分類群而均屬subgroup I。豆科系統群歸類於subgroup IA，而所有之茄科系統群歸類於自Subgroup I中形成一新分類群(IT)。此分類群IT中包含在臺灣自其他寄主所分離出之CMV，顯示拓荒者效應(founder effect)扮演重要的角色。經由雙股RNA電泳分析，發現一造成香蕉嚴重嵌紋分離株(22)中具有一衛星核酸。在進行鑑別寄主鑑定中發現，其中一分離株(20)，於感染N. benthamiana時能造成植株生長不良且矮化。經由建立感染性系統進行病毒重組交換，發現其病徵決定因子位於RNA 2。隨後以Potato Virus X感染性載體表現出2a與2b蛋白接種於N. benthamiana後證明，此病徵之產生，為2a與2b蛋白共同調控所致。Abstract Banana samples with mosaic symptoms were collected from southern Taiwan, and 12 type isolates of Cucumber mosaic cucumovirus associated with banana mosaic disease were established. The 12 CMV isolates were characterized into three types, (I-III), by the symptoms induced by CMV on bananas. The type I-infected bananas initially showed chlorosis with little necrosis streak, and later the full expanded leaves showed severe green mosaic with distortion either becoming slender leaf. Type II induced yellow mosaic with severe necrosis on leaves. The severe necrosis on pseudostem will eventually cause heart rot in field. Type III induced leaf chlorosis, however, the full expanded leaves show severe green mosaic without leaf deformation. For biological characterization of the collected CMV, we followed the methods described by Wahyuni (1992) and Wu (1994) using cowpea and N. glutinosa as differential hosts. When Nicotiana. glutinosa was used as differential hosts, Type I CMV induce severe green mosaic and parkling, or fern leaf-like leaf distortion. Type II CMV induce chlorosis on newly emerging leaves, and later the newly expanded leaves were deformed and caused necrosis spot on shoot tip and leaves. Type III only induce mild mottling and did not induce leaf distortion. When cowpea was used as the differential hosts, most Type I and Type II isolates can not systematically infect cowpea (belonging to as Solanaceae common strains). One isolate of type I (sm1) and the only isolate of type III (PL) can systematically infect the cowpea showing mosaic, (belonging to Legume strain). The phylogenetic analysis using CMV-CP sequences revealed that Legume and Solanaceae common strains were distinct groups. In addition to previously identified subgroups CMV IA, IB and II, new subgroups IS, IC and IT were confined. Members within these groups were geographic related, which suggest founder effect play roles in CMV evolution. One satellite RNA associated with isolate 22 without causing attenuate symptom on banana and N, glutinosa was identified. During characterization of CMV by differential host, isolates 20 caused stunting by repressing the internodes growth of N. benthamiana. By pseudorecombination between CMV 20 and 25 isolates the symptom determinater was found locating on RNA 2 of CMV 20. Following express the CMV proteins 2a and 2b protein individually or in combination, we found that the symptom determinater was controlled synergistically by both 2a and 2b encoded by RNA2 of 20.Table of Content Abstract (in Chinese) i Abstract ii Chapter I introduction 1 Chapter II. Material and methods 8 2.1 Health plant material 8 2.2 Sample collection and preservation 8 2.3 Inoculation method 2.3-1. From banana to indicator plant 8 2.3-2. From indicator plant to differential hosts 9 2.3-3. From tobacco to banana 9 2.4 RNA extraction 9 2.5 CMV coat protein (CP) cloning by RT-PCR and TA cloning 10 2.6 dsRNA precipitation 11 2.7 Cloning CARNA 5 of CMV isolate by RT-PCR and TA cloning 11 2.8 Phylogenetic analysis 12 2.9 CMV transcripts and infectious clone construction 2.9-1. RNA 3 5’ RACE 14 2.9-2. Construction of pCMV20R1, pCMV25R1 15 2.9-3. Construction of pCMV20R2, pCMV25R2 16 2.9-4 Construction of pCMV20R3, pCMV25R3 17 2.9-5 in vitro transcription 18 2.10 Expression CMV 2a and 2b protein with PVX vector 2.10-1. Construction of pPVX-202a and pPVX-252a 18 2.10-2. Construction of pPVX-202b and pPVX-252b 19 2.10-3. in vitro transcription 20 2.11 Pseudorecombination of CMV infectious transcripts 20 2.12 PVX expression vector transcript inoculation 21 Chapter III. Result 3.1 Sample collection 22 3.2 Characterization of CMV by symptoms induced on banana 22 3.3 Characterization of CMV based by differential hosts 23 3.4 Identification and characterization of CMV satellite RNA 24 3.5 Phylogenetic analysis of CMV 25 3.6 Construction of CMV infectious clones 26 3.7 Identification of symptom determinaters by pseudorecombination and by recombinant PVX expression vector 27 Chapter IV. Discussion 29 Reference 34 Tables and Figures 40 Appendix 601156443 bytesapplication/pdfen-US香蕉嵌紋病胡瓜嵌紋病毒banana mosaiccucumber mosaic cucumovirussymptom determinaterotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/58057/1/ntu-96-R93633003-1.pdf