綜觀本研究我們成功在菸草原生質體及白藜植株上，以 PiVX 缺失性 RNA 表現 外源基因，且發現結合 p19 之表現，PiVX 可以成功感染菸草葉片，但卻無法協助缺 失性 RNA 載體表現外源基因；而 p19 並無法增加 PiVX 感染白藜之能力，因此無法 協助缺失性 RNA 載體表現外源蛋白。我們發現並選殖 CVX 缺失性 RNA，這是 Potexvirus 屬第五個被發現具有缺失性 RNA 的例子。此外，我們確認 PiVX 缺失性 RNA 保留序列 1-435 nt 即可被 PiVX 複製，顯示其內應具有可被 RdRP 辨識的複製
41
相關的 cis-acting elements。另外，進一步分析發現 PiVX 與 CVX 序列 354-394 nt 具 有一保守性二級結構，似乎與兩者被 PiVX 辨識相關，但需要進一步實驗確認。本 研究使我們更加了解 PiVX 的分子特性，及提供其缺失性 RNA 應用的範例。
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陸、表
表一、本研究所使用之引子序列
Table 1. The primers used in this study.
Primer name Sequence (5’-3’)
PiVX-F GGAAAACCAACCCAAACCAAACCTACCTC
PiVX-dTBam AGCTGGATCCTTTTTTTTTTTTTTTTTTTTCTGAAACTTG CATTAAAACAGAAGGC
PiVX-F-T7-Sal ATCGGTCGACTAATACGACTCACTATAGGAAAACCAACC CAAACCAAACCTACCTC
PiVX-map-F1 AACCACCTCCACTCTGGTGACTAAAGG PiVX-map-R3 TGGTGCATGGAGTTTTCTCCAGAT PiVX-map-R4 TTGATGAACTTGTCGTTCTGGTGTG
T7-Farm-F CGATGGATCCTAATACGACTCACTATAGGAAAACCAAC CCCAACCCAAACC
NTU-dT-R CGTAGGATCCTTTTTTTTTTTTTTTTTTTTTTTTTGAAACT TGCATTAAAAC
P1-HpaI-XhoI-R CTCGAGATCGTTAACAATGGCCTCAACTGGAAGT P1-SacII-R CCGCGGAATGGCCTCAACTGGAAGTACAAT M13-F CGACGTTGTAAAACGACGGCCAGTG
EGFP-R1 TTGTACAGCTCGTCCATGCCGAG
SacII-EGFP-F CCGCGGATGGTGAGCAAGGGCGAGGAGC XhoI-EGFP-R CTCGAGTTACTTGTACAGCTCGTCCATGCCG
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柒、圖
圖一、Pitaya virus X (PiVX)缺失性 RNA 及預測轉譯架構之圖譜。本研究從田間採 集確認之複合感染 PiVX 與 CVX 之紅龍果植株為材料，選殖出 3 類 PiVX (1P、2P 與 5P)缺失性 RNA。(A)定序分析後繪製缺失性 RNA 與 PiVX 基因體之比較示意 圖。P1 為接種 PiVX-37 病毒株之紅龍果植株所選殖出之缺失性 RNA (陳, 2012)。
(B)以 GeneDoc 進行缺失性 RNA 轉譯架構之預測。藍色為保留之 PiVX RdRP 胺基 酸序列；虛線為消失之鞘蛋白胺基酸序列；斜線表示改變後之胺基酸序列。
Fig. 1. The maps of Pitaya virus X (PiVX) defective RNAs and their predicted ORF.
There were three types (1P, 2P and 5P) of PiVX defective RNAs cloned from both PiVX and CVX mixedly infected pitaya plants in this study. (A) The maps of PiVX and its defective RNAs. P1 defective RNA was previously cloned from pitaya plant that was
52
inoculated with PiVX-37 isolate (Chen, 2012). (B) The open reading frames of PiVX defective RNAs predicted by GeneDoc. Blue block represents the region of retained RdRP amino acids; dashed line represents the region of disappeared CP amino acids;
dashed line block represents the region of changed amino acids.
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圖二、以北方雜合反應分析 PiVX 缺失性 RNA 於菸草原生質體中之複製能力。為 測試 PiVX 缺失性 RNA 在單細胞中之複製能力，將 PiVX 與其缺失性 RNA 之生體 外轉錄體，以莫耳數 1:10 共同接種或單獨接種至菸草原生質體。經過 48 小時，抽 取原生質體全 RNA，並以 PiVX 之 5'端專一性探針進行北方雜合分析。Mock 為接 種 T7 transcription buffer；P 為單獨接種 PiVX 生體外轉錄體之處理；P+P1、P+1P、
P+5P、P+2P-1 及 P+2P-2 為共同接種 PiVX 與缺失性 RNA 生體外轉錄體之處理；
P1、1P、5P、2P-1 及 2P-2 為單獨接種 PiVX 與缺失性 RNA 生體外轉錄體之處理。
gRNA 為 PiVX 基因體 RNA；dRNA 代表缺失性 RNA 之大小範圍；rRNA 為 loading control。
Fig. 2. The biological activity analysis of PiVX defective RNAs in Nicotiana benthamiana protoplasts by northern hybridization. For analyzing biological activity of PiVX defective RNAs, N. benthamiana protoplasts were inoculated with PiVX defective RNA transcripts alone, or mixture of PiVX and PiVX defective RNA transcripts (molar ratio 1:10). After 48 hour of protoplast inoculation, northern blot assay was conducted with PiVX 5’ probe. Mock: sample inoculated with T7 transcription buffer; P: sample inoculated with PiVX transcripts alone; P+P1, P+1P, P+5P, P+2P-1 and P+2P-2: samples inoculated with mixture of PiVX and PiVX defective RNA transcripts; P1, 1P, 5P, 2P-1 and 2P-2: samples inoculated with PiVX defective RNA transcripts. gRNA: size of PiVX genomic RNA; dRNA: sizes of PiVX defective RNAs; rRNA: loading control.
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圖三、分析缺失性 RNA 在 PiVX 協助下於白藜系統葉之累積與引起之病徵。將 p35S promoter 驅動的 PiVX 及其缺失性 RNA 之選殖株，各取 20 μg 質體 DNA 接種至白 藜葉片，20 天後收取未接種之系統葉進行觀察與分析。(A)以 PiVX 5’端專一性探 針進行北方雜合分析。(B) 白藜系統葉之病徵。Mock 為接種無菌水之處理；P 為 單獨接種 p35S-PiVX；P+P1 與 P+1P 為共同接種 p35S-PiVX 與缺失性 RNA；P1 與 1P 為單獨接種缺失性 RNA 之處理。gRNA 為 PiVX 基因體 RNA；rRNA 為 loading control。
Fig. 3. Symptom and RNA accumulation of Chenopodium quinoa systemic leaves caused by PiVX and PiVX defective RNA. Plasmid DNAs of PiVX and PiVX defective RNAs driven by p35S promoter were inoculated to C. quinoa plants. After 20 days, the systemic leaves were observed and analyzed. (A) Northern hybridization with PiVX 5’
probe. (B) Symptoms of C. quinoa systemic leaves. Mock: inoculated with ddH2O; P:
inoculated p35S-PiVX alone; P+P1 and P+1P: inoculated with mixture of p35S-PiVX and p35S-P1/p35S-1P; P1 and 1P: inoculated defective RNA alones. gRNA: size of PiVX genomic RNA; rRNA used as loading control.
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圖四、pUC-P1-EF 及 pUC-P1-E 之構築示意圖。首先利用聚合酶鏈鎖反應在 pUC-P1 之 RdRP 與 CP 序列間分別創造 SacII (pUC-P1S)以及 HpaI-XhoI (pUC-P1HX)之切 位。利用 SacII 及 XmaI 酵素將 pGR-PiVX-EGFP 上的外源基因 EGFP 與 PiVX 鞘蛋 白全長序列切下，選殖至以相同酵素處理的 pUC-P1S，以構築 pUC-P1-EF。另外，
利用 HpaI-XhoI 切位，將帶有 PiVX 之鞘蛋白次基因體 RNA 啟動子的 EGFP 基因，
插入 pUC-P1HX 之 RdRP 與鞘蛋白序列之間，以完成 pUC-P1-E 之構築。紅色線段 代表 PiVX 之鞘蛋白次基因體 RNA 啟動子。
Fig. 4. Schematic structure of pUC-P1-EF and pUC-P1-E construction. At first, SacII (pUC-P1S) and HpaI-XhoI (pUC-P1HX) sites between RdRP and CP sequences of pUC-P1 were created by PCR mutagenesis. Fragment of EGFP and PiVX CP genes of pGR-PiVX-EGFP was cloned into pUC-P1S through the help of SacII and XmaI digestion and thus constructed pUC-P1-EF. In addition, EGFP with PiVX CP subgenomic promoter was inserted between RdRP and CP sequences of pUC-P1HX with the help of HpaI and XhoI digestion and ligation for pUC-P1-E construction. Red line represents PiVX CP subgenomic promoter.
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圖五、利用 PiVX 缺失性 RNA 為載體，於菸草原生質體中分別以 PiVX 鞘蛋白次 基因體 RNA 啟動子或融合蛋白方式表現外源基因 egfp 之結果。將 PiVX 與 P1-E 或 P1-EF 之生體外轉錄體共同接種至菸草原生質體，利用 PiVX 鞘蛋白次基因體 RNA 啟動子或融合蛋白的方式表現 EGFP，接種後 48 小時進行觀察與分析。(A) 以 螢光顯微鏡觀察菸草原生質體螢光表現。分別以可見光(bright field)、Chroma 41020 濾鏡(GFP)及 I3 濾鏡觀察。白色線條代表 100 μm。(B) 以 PiVX 5’端探針與 EGFP 專一性探針共同(上圖)或單獨以 EGFP 專一性探針(下圖)進行北方雜合分析之結 果。(C) 以 PiVX 5’端探針進行北方雜合分析之結果。gRNA 為 PiVX 基因體 RNA；
P1、P1-E 與 P1-EF 為 PiVX 缺失性 RNA；rRNA 為 loading control。
Fig. 5. The results of PiVX defective RNA-based vectors expressing foreign gene egfp
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by the ways of CP subgenomic promoter and fusion protein in N. benthamiana protoplasts. Mixture of PiVX and P1-E or P1-EF transcripts were inoculated to N.
benthamiana protoplasts to express EGFP by PiVX CP subgenomic promoter or fusion
benthamiana protoplasts to express EGFP by PiVX CP subgenomic promoter or fusion