紅龍果X病毒缺失性RNA之選殖與特性分析

Cloning and characterization of Defective RNAs of Pitaya virus X

陳君弢(Chun-Tao Chen)

指導教授 ： 張雅君

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.2012.00917

摘要

紅龍果為仙人掌科(Cactaceae)作物，原產於中美洲地區，為本島近幾年新興之經濟水果作物。在國內，紅龍果可被仙人掌X病毒(Cactus virus X, CVX)、蟹爪蘭X病毒(Zygocactus virus X, ZVX)和本實驗室於2008年發現之紅龍果X病毒(Pitaya virus X, PiVX)所感染。PiVX為Potexvirus屬病毒，病毒顆粒成短絲狀，基因體為正意單股RNA，全長不包含polyA為6677個核苷酸。缺失性RNA (defective RNA, D RNA)是一種RNA病毒基因體經過缺失與重組後所形成的次病毒分子，需輔助病毒(helper virus)協助，才能正常進行複製、包被和移行，因這些獨特的性質，缺失性RNA成為病毒學研究上常用之工具。本研究嘗試以人為及天然篩選的方式選殖PiVX之缺失性RNA，以協助我們進一步探討PiVX之特性。首先利用實驗室先前已建立之p35S-PiVX5之感染性選殖株 (infectious clone)作為構築材料，利用限制酶進行截切，以構築五個人為缺失性RNA (artificial defective RNA, aD RNA)。將五個人為缺失性RNA與輔助病毒共同接種至菸草原生質體中，以北方雜合反應進行分析，可發現五個人為缺失性RNA皆可在輔助病毒的協助下進行增殖。顯示當PiVX人為缺失性RNA保留5’端269與3’端262個核苷酸即具有被輔助病毒複製酶(RNA-dependent RNA polymerase, RdRp)辨識並複製之能力。進一步將五個人為缺失性RNA與輔助病毒共同接種至白藜植株上，則只有PiVX-AB與EB可觀察到增殖累積之情形。顯示PiVX缺失性RNA生物活性不僅受到保留區域，也受到長度及其它因素之影響。另一方面，從重複繼代接種PiVX的白藜植株與長期感染PiVX的紅龍果植株中，選殖出9種類型天然產生的D RNA (naturally occurring D RNA, nD RNA)。將9種PiVX天然缺失性RNA與輔助病毒共同接種至白藜植株上，發現除從白藜選殖出之PiVX-N3與從紅龍果選殖出之PiVX-P2、P3、P4外，其餘皆可在白藜上被輔助病毒所增殖。在比對PiVX天然缺失性RNA所攜帶的轉譯架構與在白藜上增殖能力的測試結果後，可知是否維持原鞘蛋白之轉譯架構對PiVX缺失性RNA增殖能力並無影響。在比較不同缺失性RNA在白藜上增殖能力之差異後，我們推測270-755 nt間存在決定PiVX缺失性RNA是否具有在白藜上增殖能力之序列；經構築不同5’端長度刪除株並測試後，已將此序列範圍縮小至356-562 nt之間。進一步以點突變方式將所攜帶轉譯架構之啟始碼除去並接種測試，發現PiVX缺失性RNA在植物中累積量下降，但仍可進行增殖。顯示轉譯架構的維持對PiVX缺失性RNA增殖能力而言，並非必須。此外我們也嘗試在PiVX缺失性RNA重組蛋白上加上myc及C4蛋白標籤並接種測試，但在西方墨漬法的測試結果中，我們無法偵測到此重組蛋白之累積，顯示PiVX缺失性RNA轉譯架構在寄主細胞中的表現量不高或此重組蛋白較不穩定，可能也暗示了轉譯作用對PiVX缺失性RNA增殖能力影響大於其重組蛋白。而在本研究中，我們不僅成功以人為及天然選殖的方式取得PiVX之缺失性RNA，並且也對其基本特性進行基礎分析，為研究PiVX提供了良好的資訊及材料。

關鍵字

順勢作用子 ； 缺失性RNA ； 馬鈴薯X病毒屬 ； 紅龍果 ； 紅龍果X病毒

並列摘要

Pitaya (Hylocereus spp.), a climbing succulent plant in the family Cactaceae originating in Central and South America, is cultivated in Taiwan as a new tropical fruit. Cactus virus X (CVX), Zygocactus virus X (ZVX), and Pitaya virus X (PiVX) have been reported to infect pitaya in Taiwan. PiVX, a new Potexvirus identified in 2008, is a virus with short filamentous particles containing a single-stranded, positive-sense RNA genome. Genomic RNA of PiVX consists of 6677 nucleotides excluding polyA tail and five open reading frames (ORFs). Defective RNAs (D RNAs) are subviral RNAs produced from RNA virus genome by deletion and recombination. D RNA is dependent on its parental virus (helper virus) for normal replication, encapsidation and movement. Because of this unique character, D RNAs become useful tool in viral research. In this study, we created artificial D RNAs (aD RNAs) from the PiVX infectious clone (p35S-PiVX5) and clone naturally occurring PiVX D RNAs (nD RNAs). All five PiVX aD RNAs could be replicated in Nicotiana benthamiana protoplasts under the help of PiVX transcripts. This result suggested that 5’ 269-nt region and 3’ 262-nt region within PiVX aD RNA possibly contain the replication signal recognized by PiVX RNA-dependent RNA polymerase. When plasmid DNAs of PiVX and its aD RNAs were applied together onto Chenopodium quinoa, only the RNAs of PiVX-AB and EB were detected in the inoculated leaves. The data indicated that not only the domain of PiVX aD RNA but also the length of the domain could affect its biological activity. In addition, we have obtained nine groups of PiVX nD RNA clones from the inoculated leaves of C. quinoa by serial passages of inoculation and also from PiVX-infected pitaya. All nD RNAs could replicate in the presence of its helper virus in the inoculated leaves of C. quinoa except PiVX-N3 from C. quinoa as well as PiVX-P2, P3 and P4 from pitaya. After comparing the predicted ORFs and the infectivity of PiVX nD RNAs, it demonstrated that the presence or absence of CP ORF did not affect the accumulation of PiVX D RNAs in planta. According to the results of inoculation and sequence analyses, we speculated the region from nt 270 to 755 may contain the sequence essential for the accumulation of PiVX D RNAs in C. quinoa. By testing PiVX-N1 deletion mutants, the indispensable sequence for PiVX D RNA accumulation was mapped to the region of nt 356 to 562. On the other hand, we constructed PiVX-N1D, an ORF-dysfunctional mutant of PiVX-N1, and inoculated it to C. quinoa to test the necessity of the PiVX D RNA ORF. Although the RNA accumulation decreased, PiVX-N1D is still detectable in planta. The results indicated that the existence of ORF is not crucial for PiVX D RNA accumulation in C. quinoa. Then, we added myc and C4 protein tags to the putative recombinant protein encoded by the PiVX D RNA ORF. Because this recombinant protein could not be detected by anti-myc and anti-C4 antibodies, suggesting that the expression of PiVX D RNA ORF was too low to be detected, or the recombination protein was very unstable in host cell. It also implied the translation itself is more important than the expressed recombination protein for PiVX D RNA accumulation in C. quinoa. In this study, we have constructed the aD RNA and cloned the nD RNA of PiVX successfully, and have studied their basic properties. These results mat provide useful information and tools for future research of PiVX.

並列關鍵字

cis-acting element ； defective RNA ； Potexvirus ； Pitaya ； Pitaya virus X

參考文獻

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