紅龍果X病毒之cis-acting elements與缺失性RNA之分析與應用

紅龍果為仙人掌科三角柱屬(Hylocereus spp.)之多年生攀附性植物，亦為近年台灣大力推廣之熱帶水果，其田間栽種植株常遭受病毒為害。目前國內已發現紅龍果被仙人掌X病毒(Cactus virus X, CVX)、蟹爪蘭X病毒(Zygocactus virus X, ZyVX)及紅龍果X病毒(Pitaya virus X, PiVX)所感染，且這三種potexvirus病毒普遍存在全台各地紅龍果果園中。PiVX為本實驗室所發現鑑定之Potexvirus屬新病毒，我們從感染PiVX的紅龍果植株萃取總量RNA，以北方雜合分析發現一些小片段RNA與PiVX探針有反應；進一步選殖與序列分析，確認其為源自PiVX的缺失性RNA (defective RNA, dRNA)。dRNA為病毒基因體RNA經過缺失與重組後產生的次病毒分子，需輔助病毒(helper virus)提供複製酵素、鞘蛋白與移動蛋白等，才能在寄主體內複製、包被與移行。從紅龍果所選殖到不同長度之PiVX dRNA選殖株，先製備成RNA轉錄體，再與PiVX共同接種菸草原生質體，以篩選在輔助病毒存在下，複製效率最佳的dRNA。經過數次的原生質體接種試驗，篩選出增殖能力最佳的P1 dRNA，其保留了5’端645 nt與3’端196 nt，總長度為841 nt。為了將PiVX dRNA改造為可應用在植物上的病毒載體，我們以P1 dRNA為基礎，構築兩種以不同方法表現綠色螢光基因(enhanced green fluorescent protein, EGFP)的載體，分別為P1-EF與P1-E。P1-EF以複製酶的轉譯架構與外源基因結合，以形成融合蛋白；P1-E為將PiVX鞘蛋白基因之次基因啟動子(subgenomic promoter, SGP)加入PiVX dRNA中，以SGP表現外源基因。其中，P1-E已成功在菸草原生質體及白藜植株中表現EGFP，但表現效率並不高。故嘗試短暫表現基因靜默抑制子p19於菸草與白藜植株，以期加強P1-E表現EGFP之能力；結果在白藜植株上，p19對EGFP之表現沒有顯著影響。而在非繁殖寄主菸草上，先短暫表現p19後再接種P1-E，可以抗體偵測到PiVX鞘蛋白在菸草中累積，但以GFP抗體仍然無法偵測到訊號。此外，欲研究P1 dRNA可被PiVX辨識並增殖的cis-acting elements序列，我們構築數個序列刪除株，同樣以菸草原生質體分析其活性。目前研究結果顯示PiVX dRNA的5’端只需保留435 nt，即可被PiVX輔助病毒所辨識並增殖。另外，我們也由紅龍果中分離出CVX的缺失性RNA，其保留5’端677 nt及3’端591 nt，總長度為1268 nt。進一步將其RNA轉錄體與PiVX以菸草原生質體測試活性，結果發現CVX dRNA可被PiVX複製，顯示CVX dRNA所保留的序列具有被PiVX複製酶辨識的cis-acting elements。

關鍵字

紅龍果X病毒；仙人掌X病毒； cis-acting elements ；缺失性RNA ；病毒載體；次基因啟動子

並列摘要

Pitaya, a climbing succulent plant in the family Cactaceae, has gradually become an important economic fruit in Taiwan. However, its cultivation is restricted by viral diseases. Cactus virus X (CVX), Zygocactus virus X (ZyVX) and Pitaya virus X (PiVX) have been reported to infect pitaya in Taiwan. These viruses are commonly found in the field. PiVX is a new member of the Potexvirus genus identified in our lab. We extracted total RNA from infected pitaya and discovered some small RNAs reacting to PiVX probe by Northern blot. After cloning and sequence analysis, we confirmed that these small RNAs are defective RNAs (dRNAs) of PiVX. dRNAs are subviral RNAs produced from RNA virus via deletion and recombination. dRNAs depend on their parental virus (helper virus) to replicate, encapsidate and move in host plant. To select the PiVX dRNAs with best infectivity, transcripts of different dRNA clones from pitaya were analyzed with the help of PiVX transcripts in Nicotiana benthamiana protoplasts by Northern blot. After several independent inoculation assays, we found P1 dRNA, which contains 5’ 645 nt region and 3’ 196 nt region with its total length of 841 nucleotides had the best replication ability. In order to develop PiVX dRNA as a virus expression vector, we used P1-based vector to express enhanced green fluorescent protein (EGFP) gene by two different ways and thus constructed P1-EF and P1-E. P1-EF combined the open reading frame of RNA-dependent RNA Polymerase (RdRP) with foreign gene to form a fusion protein. P1-E recruited the subgenomic promoter (SGP) of PiVX into P1 dRNA to express foreign gene through sgRNA expression。According to experimental results, P1-E could successfully express EGFP in N. benthamiana protoplasts and Chenopodium quinoa plants, but the expression efficiency was low. To improve the expression efficacy of P1-E, we tried to express p19, a gene silencing suppressor, in N. benthamiana and C. quinoa plants. The results indicated p19 had no significant effect on the expression of EGFP in C. quinoa plants. However, when P1-E was inoculated to N. benthamiana plant after transient expression of p19, PiVX CP accumulation increased but EGFP expression could not be detected by GFP antibody. In addition, in order to study the cis-acting elements that can be recognized and replicated by PiVX, we constructed and analyzed some deletion mutants of P1 dRNA in N. benthamiana protoplasts. The current result indicated that the dRNA containing only 5’ 435 nt region and 3’196 nt region could still be replicated by helper virus. Furthermore, we also cloned CVX dRNAs from pitaya plant, which contain CVX 5’ 677 nt region and 3’ 591 nt region with its total length of 1268 nucleotides. For testing the biological activity, transcripts of CVX dRNAs together with PiVX were inoculated to N. benthamiana protoplasts, and Northern blot demonstrated CVX dRNAs could be replicated by PiVX. Accordingly, the result suggested that PiVX RdRP can recognize the cis-acting elements of CVX dRNAs.