木瓜輪點病毒嚴重嵌紋壞疽系統及嚴重嵌紋系統感染性選殖株之構築及不同系統間之比較

Construction of infectious clones from two strains of Papaya ringspot virus (PRSV) and comparative studies among different isolates of PRSV

楊瑞春(Jui-Chun Yang)

指導教授 ： 洪挺軒

國立臺灣大學/生物資源暨農學院/植物病理與微生物學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.01308

摘要

木瓜輪點病由木瓜輪點病毒(Papaya ringspot virus, PRSV)所引起，可視為影響木瓜產業最重要的病害。臺灣於1975年首次發現此病，病毒歷經數十年的演替至今田間所存的PRSV依病徵表現可細分為數種系統(strains)，包括引起葉片出現嚴重嵌紋病徵之嚴重嵌紋系統(severe mottling, SM strain)，致使葉片嚴重嵌紋及畸形扭曲之畸形系統(deformation, DF strain)，以及造成葉片嚴重嵌紋並伴隨嚴重壞疽之嚴重嵌紋壞疽系統(severe mottling wih necrosis, SMN strain)。臺灣田間的PRSV目前以DF系統最常見；而SMN系統則在冷熱交替之際造成木瓜植株快速萎凋，危害性較高而被視為最具威脅性的系統。三種病毒系統雖具相當之序列相似性，但在木瓜上呈現的病徵卻有明顯的差異，在分子學及病理學上頗有研究意義。本論文將三系統之病毒基因體序列以two-step RT-PCR各自增幅出3到4個cDNA片段，配合片段間頭尾重疊區域剪接構築出全長感染性選殖株，並且將三種系統選殖株利用胞外轉錄(in vitro transcription)合成完整的病毒RNA transcripts，機械接種於木瓜及白藜寄主。結果發現人工轉錄的病毒RNA transcripts可感染木瓜並在葉部產生典型病徵，但發病時間比原自然的病毒株約延遲一週；若將病葉汁液進行繼代接種，則發病的時間和產生的病徵都與原自然的病毒株近似。將三種病毒系統在木瓜呈現的病徵與過往記錄相比較，發現SMN系統在後期出現絲狀葉，顯示其病毒可能發生變異而導致病徵改變。經核苷酸定序後將三系統之全長選殖株序列進行比對，發現以SM系統與DF系統相似度97.3%為最高，SMN系統與兩者比對皆為96.5%；各基因核苷酸序列相似度大都約在96-99%，但P3基因的相似度稍低，在95.8-96.7%，P1基因低於95%，5’-UTR則在93.0-94.2%。與各地已發表之PRSV分離株全長核酸序列進行比對，三系統與其他源自臺灣之分離株以及韓國分離株較為近似，並與中國、泰國之分離株歸於同一關係群；與美洲和南亞地區分離株親緣較遠，切合PRSV的序列歧異度與分布地域相關之推論。本論文已成功獲得三種系統具感染力之全長cDNA選殖株，對後續找尋病徵決定因子(symptom determinants)將可提供極大幫助，例如可針對系統間差異性較大的基因序列如5’-UTR、P1、P3進行重組置換，或針對特定基因如HC-Pro或CI進行序列變換，藉以探討其致病機制。另外，由於先前所設計的DF系統之專一性引子對經常也會在SM系統的RT-PCR試驗中出現微量增幅現象，本論文另行設計新的DF系統專一性引子對，靈敏度與專一性俱佳，對日後PRSV系統鑑別將更有助益。

關鍵字

木瓜輪點病毒 ； 感染性選殖株 ； 病毒系統

並列摘要

Papaya ring spot caused by Papaya ringspot virus (PRSV) is considered to be the most important disease impacting papaya industry. This disease has occurred in Taiwan for decades since 1975. After the long-term genomic evolution in the field, there are 3 major pathological strains of PRSV in Taiwan at present time such as SM (severe mottling), DF (severe mottling with leaf-deformation) and SMN (severe mottling with necrosis and quick decline) strains. DF is a predominant strain of PRSV in the field of Taiwan now, and SMN should be the most destructive strain causing quick decline in papaya plants especially when the season changes. SM, DF and SMN strains have highly similar nucleotide sequences, but their incited symptoms in papaya hosts are different. To investigate the relationship between molecular and pathological characters, this thesis was dedicated to construct the infectious clones for SM, DF and SMN strains of PRSV. Each intact infectious clone was derived from the splicing of 3~4 amplified cDNA fragments with two-step RT-PCR. The artificial full-length PRSV RNA transcripts could be harvested from the infectious clones through in vitro transcription, and they were used in the inoculation tests in either papaya or Chenopodium quinoa hosts. The results showed that the PRSV RNA transcripts derived from infectious clones could successfully infect papaya plants. However, the symptoms caused by the transcripts appeared 1 week later than those caused by natural PRSV inocula. The developing time of symptoms produced by the transcript-infected sap was similar to those caused by natural PRSV. The present SMN strain might incite “fern-leaf” in addition to original symptoms, which indicated mutation probably occurred in the genome of PRSV-SMN. Based on the alignment of full-length genomic nucleotide sequences among 3 different PRSV strains, SM and DF have 97.3% homology; SM and SMN have 96.5% homology; and SM and DF have 96.5% homology. Individual gene analyses revealed that they were 95.8-96.7%, 94~95% and 93.0-94.2% similarity of nucleotide sequences in P3, P1 and 5’-UTR respectively. The rest of genes showed 96-99% similarity among 3 strains. The further alignment of full-length genomic nucleotide sequences among several PRSV isolates from different countries demonstrated that all isolates from Taiwan are close to those from Korea and they can be categorized into a phylogenic group with those from China and Thailand. The PRSV isolates from America and South Asia form another phylogenic group. It indicates that strain-diversity of PRSV corresponds with geographical difference. Constructions of infectious clones of 3 PRSV strains will be helpful to study key genes or regions associated with symptom determinants (such as 5’-UTR, P1, P3, HC-Pro and CI) through molecular recombination and substitution. Besides, a new primer pair specific to the DF strain was developed in this thesis to improve the sensitivity and specificity and avoid non-specific amplification in the RT-PCR assay of PRSV-SM. It will provide a more reliable method for the identification among different PRSV strains.

並列關鍵字

papaya ringspot potyvirus ； infectious clone ； virus strain

參考文獻

李宜霞. 2006. 木瓜輪點病毒之Real-Time RT-PCR定量偵測技術之研發與應用. 國立臺灣大學植物病理與微生物學研究所碩士論文. 110pp.

張有朋、蕭吉雄、楊偉正、許秀惠、趙玉珍、黃秋雄. 1987. 五種瓜類病毒在本省及西瓜之發生與分佈. 中華農業研究. 36: 389-397.

蔡素錡. 2006. 木瓜輪點病毒病對番木瓜地上部與根系生長之影響. 國立臺灣大學園藝學研究所碩士論文. 40pp.

陳冠君. 2008. 木瓜輪點病毒感染木瓜及其毒力相關基因之分析. 國立中興大學植物病理學系所博士論文. 125pp.

陳冠君. 2001. 木瓜輪點病毒西瓜型生體外具感染力載體之構築及感染木瓜寄主專一性之分析. 國立中興大學植物病理學系所碩士論文. 40pp.