菸草微綠嵌紋病毒之研究與病毒載體之構築

Abstract

番椒作物源自南美洲，屬於茄科番椒屬(Capsicum spp.)，在世界各地皆為重要經濟作物。據報導至少有8種tobamovirus能感染番椒；目前已從台灣田間分離出TMV及PMMoV，2003年本實驗室也首次在台灣分離出TMGMV-HP，為了解目前TMGMV在台對番椒為害之程度，本研究針對常見感染茄科作物之tobamovirus設計廣效性引子進行初步篩檢，再針對TMGMV及兩種台灣常見感染番椒之TMV及PMMoV分別設計專一性引子，加上植物粒線體nad5專一性引子對，研發出multiplex RT-PCR之檢測法。從宜蘭、花蓮、台北、桃園、台中等地採集番椒樣本，其中有4個樣本出現預期tobamovirus之片段，進一步以mutiplex RT-PCR檢測後，並未出現目標病毒之條帶，推測樣本可能遭受其他tobamovirus感染。雖然本次研究所採集之樣本中並未測到TMGMV，未來可利用此mutiplex RT-PCR檢測更多地方之番椒樣本，除可了解TMV及PMMoV之發生情形，更可確定TMGMV是否已對台灣之番椒造成威脅。另一方面，病毒載體已普遍應用於醫藥、基因功能研究及病毒與寄主間之基礎研究等領域。由於TMGMV尚未被報導研究作為病毒載體，因此本研究將TMGMV野生型p5-6以去除鞘蛋白基因，以及外加一段鞘蛋白subgenomic promoter等兩種不同方式，構築TMGMV表現載體pCRII-TG2和PCRII-TG3；並加入EGFP作為報導基因，構築出pCRII-TG2E及pCRII-TG3E。將TMGMV p5-6及其載體之生體外轉錄體分別接種至菸草原生質體及植株，結果顯示在菸草原生質體中能偵測出TMGMV載體的RNA累積及綠色螢光；其中pCRII-TG3能在植株中移動並表現鞘蛋白，雖然量較p5-6少。但帶有EGFP基因的pCRII-TG2E及pCRII-TG3E，在原生質體中RNA累積量變少，在單斑寄主上病徵出現時間延遲，其中pCRII-TG3E所引起之單斑周圍組織可觀察到綠色螢光，但pCRII-TG2E則無。兩載體在系統性寄主植株的接種葉中RNA累積量降低，也無法在植株上觀察到綠色螢光，顯示pCRII-TG3E失去預期的系統性移動能力。為使TMGMV載體能順利應用在相關研究上，必須將載體構築方式加以改良，以期未來能在植株上進行正常複製、移動及表現外源基因，以作為研究TMGMV與寄主植物間，或與其他病毒間交互作用之工具。

Peppers, belonging to the genus Capsicum in the family Solanaceae, originated from South America and now become important crops worldwide. According to the literatures, there are at least 8 tobamovirus species infecting peppers. TMV and PMMoV have been isolated from the field in Taiwan. In addition, our lab first reported a pepper disease caused by TMGMV in 2003. To understand the disease incidence caused by TMGMV, the degenerate primers for pepper-infecting tobamoviruses were designed for preliminary detection. After that, we separately designed specific primers for TMGMV and two frequently found viruses in pepper (TMV and PMMoV). A multiplex RT-PCR was developed using the specific primers for these three viruses and plant mitochondria nad5 gene. Pepper samples collected from Ilan, Hualian, Taipei, Taoyuan and Taichung areas were first detected by tobamovirus degenerate primers. Four out of 108 samples were infected by tobamovirus. According to the following multiplex RT-PCR, the target viruses were not detected in these 4 samples. This result suggested that these samples may be infected by other tobamovirus. Although we did not detect TMGMV in the survey, the tobamovirus specific primers and multiplex RT-PCR can still be used in the future. Not only the occurrence of TMV and PMMoV can be investigated, but also the infection of TMGMV in peppers can be confirmed by this detection method. Viral vectors have been constructed for medical application, identifying unknown gene function and studying the interaction between virus and its host. Since TMGMV has never been reported as a viral vector, we used two different ways for vector construction. One (pCRII-TG2) is to delete most of the CP gene, and the other (pCRII-TG3) is to insert an additional CP subgenomic promoter (sgp). The EGFP gene was inserted into the viral vectors to construct pCRII-TG2E and pCRII-TG3E. RNA transcripts of TMGMV clones (p5-6) and its derived vectors were inoculated to tobacco protoplasts and plants. The results showed that TMGMV vectors could accumulate their RNAs and expressed green fluorescence in tobacco protoplasts. pCRII-TG3 could move systemically and also expressed CP in plant. On the contrary, the RNA accumulation of pCRII-TG2E and pCRII-TG3E carrying EGFP gene were lower than pCRII-TG2 and pCRII-TG3 in protoplasts, and the symptoms induced by pCRII-TG2E and pCRII-TG3E were delayed in local lesion host. The green fluorescence signal could be observed around the local lesions only in the plant inoculated by pCRII-TG3E. The RNA accumulation in the inoculated leaves of pCRII-TG2E and pCRII-TG3E were lower than that of pCRII-TG2 and pCRII-TG3 and green fluorescence failed to be observed in systemic host plant. These results indicated pCRII-TG3E lost its ability of systemic movement in this host. In the future, we have to improve the TMGMV-based vectors in order to express foreign gene in planta and to use them in studying the interaction between TMGMV and host or other viruses.