兩種仙人掌X病毒感染性選殖株之研究與紅龍果原生質體系統之建立

Abstract

紅龍果(Hylocereus spp.)為臺灣近年新興之熱帶水果，其於田間受三種Potexvirus屬病毒--仙人掌X病毒(Cactus virus X, CVX)、紅龍果X病毒(Pitaya virus X, PiVX)與蟹爪蘭X病毒(Zygocactus virus X, ZyVX)--單獨或複合感染甚為普遍。其中CVX自2001年首次報導感染紅龍果以來，國內已發表CVX-Hu及CVX-NTU兩個全長基因體序列具一定程度差異的分離株。本研究首先設計可區分上述兩個CVX分離株之專一性引子對，針對臺東池上、屏東佳冬及萬丹等三處果園進行檢測，發現整體受CVX感染比率極高，且植株受CVX分離株複合感染率至少達73%。為了進一步分析兩種CVX分離株的分子與感染特性，以及彼此間潛在的交互作用，我們利用採集自臺中霧峰及南投集集之罹病紅龍果枝條，從中選殖並構築出兩個CVX感染性選殖株--H015及N015，兩者的核酸及胺基酸序列分別與CVX-Hu、CVX-NTU有極高相同度，且與其他Potexvirus屬病毒進行親緣演化樹分析，皆分支於感染仙人掌科植物之一群。將CVX-N015與CVX-H015分別接種於白藜(Chenopodium quinoa)植株，可見感染CVX-N015的接種葉會有褪綠黃斑的形成，CVX-H015則未產生顯著病徵，但以間接型酵素連結抗體免疫吸附分析(indirect enzyme-linked immunosorbent assay, indirect ELISA)接種葉，並未有明顯的病毒累積量差異。進一步於圓葉菸草(Nicotiana benthamiana)原生質體接種兩個選殖株的轉錄體，以北方墨點(Nortehrn blot)分析顯示CVX-N015的RNA累積量遠較CVX-H015為佳。為了探究影響兩個選殖株RNA累積能力的因子，進一步針對兩者的RNA複製酶(RNA-dependent RNA polymerase, RdRP)及鞘蛋白(coat protein, CP)基因進行置換分析，發現以CVX-N015為基礎，置換上CVX-H015的RdRP或CP基因，其在菸草原生質體中的病毒RNA累積量皆顯著下降；反之，保留CVX-H015為骨架而置換CVX-N015的RdRP或CP基因之重組病毒，其病毒RNA累積量皆有所上升；顯示兩個選殖株於RdRP及CP基因的差異，對彼此RNA累積能力的不同扮演重要角色。此外，RdRP似乎是CVX選殖株在白藜上產生黃斑病徵的決定因子。針對RdRP胺基酸序列進行分析，介於methyltransferase domain與RNA helicase domain間的非保守序列，其存在CVX-N015與CVX-H015最主要的差異區塊，可能是造成上述病徵及RNA累積差異的重要因子。另外，將兩個選殖株轉錄體共同接種於菸草原生質體，利用即時定量反轉錄聚合酶連鎖反應 (quantitative real-time reverse transcription-PCR, RT-qPCR)分析，發現兩者的RNA累積量皆較單獨感染時高，似乎有互惠的效果。最後，本研究亦嘗試建立紅龍果原生質體系統，測試結果顯示同時使用1%纖維酶(cellulase)、1%離析酶(macerozyme)及1%蝸牛酶(snailase)，搭配0.7 M的甘露醇(mannitol)溶液、甘胺酸(glycine)溶液及蔗糖(sucrose)溶液，可獲得最佳產量。綜合以上成果，期望本研究中對CVX分子特性的探討與紅龍果原生質體系統的建立，對於未來紅龍果病毒病害之研究提供有用的資訊與研究平台。

Pitaya (Hylocereus spp.), an emerging fruit crop in Taiwan, has been found to be infected singly or mixedly with Cactus virus X (CVX), Pitaya virus X (PiVX), and Zygocactus virus X (ZyVX), all of which belong to the genus Potexvirus. In Taiwan, since the first report of CVX infection on pitaya in 2001, there has been two published CVX isolates, Hu and NTU, of which complete genomes had been sequenced and shown to be notably different in nucleotide and amino acid sequences. We developed isolate-specific primer pairs according to genome sequences of the two isolates, and found high incidence of CVX infection in pitaya fields and that mixed infection was common as at least 73% of pitaya samples collected from three orchards in Pintung and Taitung were positive for both isolates. To further investigate the characteristics of CVX isolates and possible interaction between them, we constructed two CVX cDNA infectious clones, H015 and N015, whose level of complete nucleotide sequence identity are about 97-98% with CVX-Hu and CVX-NTU, respectively. Phylogenetic analyses also show that CVX-H015 and -N015 are classified into the cluster of cactus-infecting potexviruses. When Chenopodium quinoa plants were mechanically inoculated with the infectious clones, chlorotic spots in the inoculated leaves treated with CVX-N015 were observed; meanwhile, those treated with CVX-H015 appeared no apparent symptom. The inoculated leaves were further analyzed by indirect enzyme-linked immunosorbent assay (indirect ELISA) but showed no significant difference in virus accumulation. Moreover, Northern blot revealed much higher viral RNA accumulation of CVX-N015 than that of CVX-H015 by inoculating Nicotiana benthamiana protoplasts with viral transcripts. In view of this, we chose RNA-dependent RNA polymerase (RdRP) and coat protein (CP) genes to perform swapping analyses based on alignment and comparison of amino acid sequences in order to dissect possible factors that affect viral replication. As a result, the differences in RdRP or CP genes between CVX-N015 and CVX-H015 do play vital roles in viral RNA accumulation as RNA accumulation of chimeric CVX-N015 significantly reduced, while increase in RNA accumulation of chimeric CVX-H015 was observed. Besides, the RdRP genes seem to be involved in the determination of chlorotic spot symptom in C. quinoa. Additionally, after N. benthamiana protoplasts were inoculated with CVX-N015 and/or CVX-H015, mutual benefit in viral RNA accumulation were observed by quantitative real-time reverse transcription-PCR (RT-qPCR). Lastly, we tried to establish a pitaya protoplast preparation system, in which the combination of the enzyme system containing 1% cellulase, 1% macerozyme, and 1% snailase as well as 0.7 M solution of mannitol, glycine, or sucrose was found most effective. As a whole, this research in construction and characterization of CVX infectious clones and pitaya protoplast preparation system is expected to be useful for further study in viral diseases of pitaya.