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表一、木瓜輪點病毒(Papaya ringspot virus)基因體核酸片段之選殖所用引子對及其 基因產物。
Table 1. The primers used for amplification of the genome of Papaya ringspot virus (PRSV) and their gene products.
Primer name Primer sequence Product
T3Fa 5'-ATT AAC CCT CAC TAA AGA AAT AAA ACA TCT CAA CAC AAC ACA A-3
T3F-HC
R1968 5'-CGT AAA CGT TCA AGC TCA CAA TTC AAG TCC TCA C-3'
F1748 5'-TGG CTC GGT TTC AAC AGG GCT TTC TTA C-3' HC-Nb R8546 5'-CAA TCG TCC CGT CAG GTG TCA AAA TAG G-3'
F6727 5'-TAA TCA AAG CCT ACT ACG TCC GGA ACT CTG C-3'
Na-AX
Poly(A)-XbaIb 5'-CCC GGG TCT AGA TTT TTT TTT TTT TTT TTT TTT TTT T-3'
NcoI+F1577
b 5'-CTC CAT GGC CTC ATC TTT AAA CCA AAA TTC GCT GA-3'NP1-Na
R7359 5'-GAT ATC CAA TGG CAA CCA AAT TCT CCC ATA CCC TC-3'
F6518 5'-ATG GGT GTT AAA ACA AGA AAA TTT GTC GCA ACG TAC GGA T-3'
Na-CP
R9629 5'-TAA GGC CAT AAT CAC TTC TCA CTC CCT CAT ACC ACT-3'
F8353 5'-ATT TCC TGA TGG CTG GGT TTA TTG TGA TGC-3' Nb-AX Poly(A)-XbaIb 5'-CCC GGG TCT AGA TTT TTT TTT TTT TTT TTT TTT
TTT T-3'
NotI+T3F
ab 5’-CGA CCT GCA GGC GGC CGC ACT AGT GAT TATTAA CCC TCA CTA AAG AAA T-3’
T3F-Na
R7359 5'-GAT ATC CAA TGG CAA CCA AAT TCT CCC ATA CCC TC-3'
a引子T3F 之 5’端 T3 promoter 序列以斜體標示，外加 G 為粗體
b限制酶切位序列以底線標示
aItalic nucleotides in Primer T3F represent the sequence of T3 promoter at 5’ end, followed with an additional G in bold.
bNucleotides underlined indicate the recognition sites of restriction enzymes.
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表二、由木瓜輪點病毒 cDNA 選殖株所合成的 RNA transcripts 機械接種木瓜寄生 之感染率分析。
Table 2. Infectivity assays of the in vitro transcripts derived from the cDNA clones of Papaya ringspot virus (PRSV) through mechanical inoculation tests on papaya hosts.
Treatments The batch of exp.
No. of plants infected/ No. of plants inoculation PRSV-SMN
(prT3-AX-NP)
PRSV-SM (prT3-AX-MP)
PRSV-DF (pfT3-AX-DP)
Exp. 1 5/5 6/6 5/5
Exp. 2 (conducted at 26˚C) 4/6 3/6 4/6
Exp. 3 4/4 4/4 2/4
Infectivity 13/15(86.7%) 13/16(81.3%) 11/15(73.3%)
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表三、由木瓜輪點病毒 cDNA 選殖株所合成的 RNA transcripts 機械接種木瓜寄主 後之病徵發展。
Table 3. The symptom development of infected papayas incited by the in vitro transcripts derived from the cDNA clones of Papaya ringspot virus (PRSV) through mechanical inoculation tests.
DPIa
Treatments
PRSV-SMN PRSV-SM PRSV-DF
21 1b 1 1
35 2 3 3 49 3 3 3 63 4 3 4 77 5 4 5 91 5 4 5
a DPI: Days post inoculation
bSymptom index: 0, no symptom; 1, 30% systemic leaves showing mild mottling and vein-clearing; 2, 70% systemic leaves showing mottling and vein-clearing; 3, systemic leaves showing apparent mottling, vein-clearing and yellowing; 4, leaves showing severe mottling, apparent yellowing, necrosis and deformation; 5, leaves showing severe mottling, necrosis, deformation and decline. Records made every two weeks after visible symptom appearing on systemic leaves
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表四、由木瓜輪點病毒 cDNA 選殖株所合成的 RNA transcripts 感染木瓜後，再將 染病汁液繼代接種於木瓜寄主之病徵發展。
Table 4. The symptom development of the papayas inoculated with virus saps derived from the diseased papaya leaves infected by the in vitro transcripts of the cDNA clones of Papaya ringspot virus (PRSV).
DPIa
Treatments
PRSV-SMN PRSV-SM PRSV-DF
21 0b 1 0
35 2 3 3
49 3 3 3
63 4 3 4
77 5 4 5
a DPI: Days post inoculation
bSymptom index: 0, no symptom; 1, 30% systemic leaves showing mild mottling and vein-clearing; 2, 70% systemic leaves showing mottling and vein-clearing; 3, systemic leaves showing apparent mottling, vein-clearing and yellowing; 4, leaves showing severe mottling, apparent yellowing, necrosis and deformation; 5, leaves showing severe mottling, necrosis, deformation and decline. Records made every two weeks after visible symptom appearing on systemic leaves.
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表五、木瓜輪點病毒機械接種木瓜寄主後之病徵發展。
Table 5. The symptom development of papayas infected by Papaya ringspot virus (PRSV) through mechanical inoculation tests.
DPIa
Treatments
PRSV-SMN PRSV-SM PRSV-DF
14 1b 1 1
21 3 2 2 28 4 2 4 35 4 3 4 42 5 4 4
a DPI: Days post inoculation
bSymptom index: 0, no symptom; 1, 30% systemic leaves showing mild mottling and vein-clearing; 2, 70% systemic leaves showing mottling and vein-clearing; 3, systemic leaves showing apparent mottling, vein-clearing and yellowing; 4, leaves showing severe mottling, apparent yellowing, necrosis and deformation; 5, leaves showing severe mottling, necrosis, deformation and decline. Records made every week after visible symptom appearing on systemic leaves.
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表六、PRSV 三系統全長 cDNA 選殖株之全長基因體與個別基因的核苷酸及胺基 酸序列相似度分析。
Table 6. Comparative similarity of nucleotide and amino acid sequences either in full-length genome or in individual genes among the cDNA clones of different PRSV strains.
PRSV strains
Genomic portiona)
Similarity of nucleotideb) sequence (%)
Similarity of amino acid sequence (%) DF vs.
SM
DF vs.
SMN
SM vs.
SMN
DF vs.
SM
DF vs.
SMN
SM vs.
SMN
5’-UTR
94.2 93.0 93.0 - - -P1
94.5 94.3 94.3 92.9 91.6 92.2HC-Pro
97.3 96.1 96.9 98.3 97.8 97.4P3
96.7 96.0 95.8 97.4 97.1 96.86K1
98.7 96.8 98.1 100 100 100CI
97.7 98.2 97.4 99.2 99.8 99.16K2
97.1 95.3 94.8 100 96.6 96.6NIa
98.2 96.4 96.3 99.1 98.1 97.2VPg
98.2 96.7 96.3 99.5 97.9 97.4NIa-Pro
98.2 96.2 96.5 98.7 98.3 97.1NIb
98.4 96.6 97.3 99.0 98.5 99.0CP
98.9 97.7 98.2 99.4 96.8 97.43’-UTR
99.5 96.6 96.6 - - -Total
97.3 96.5 96.5 97.8 97.2 97.0a)The pairwise sequence similarity between three strains presents in individual viral untranslated regions (UTRs) and functional genes.
b)The data were analyzed by MegAlignTM of the Lasergene Package (DNASTAR) using J. Hein method.
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(A)
(B)
圖一、木瓜輪點病毒(PRSV)各系統分離株全長基因體序列選殖之策略。
Fig. 1. The cloning strategy of different strains of Papaya ringspot virus (PRSV) including PRSV-SMN, PRSV-SM and PRSV-DF.
(A) The insert of final plasmids consist of T3 promoter plus nontemplated G residue preceded full-length PRSV cDNA sequence, and followed with XbaI restriction site. (B) The organization of viral genome is shown to scale, with schematic represents the PCR fragments and restriction sites used for cloning purposes. PRSV-SMN full-length cDNA clone generated with Advantage 2 Polymerase is named prT3-AX-NA; full-length cDNA clone of PRSV-SMN, PRSV-SM and PRSV-DF with Phusion DNA polymerase are named prT3-AX-NP, prT3-AX-MP and pfT3-AX-DP, respectively.
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(A) (B)
圖二、PCR 增幅之木瓜輪點病毒基因體核酸片段。
Fig. 2. The RT-PCR assay for amplification of genomic fragments of different strains of Papaya ringspot virus (PRSV).
RT-PCR amplification of PRSV specific fragments. (A) lane 1: T3F-HC-NA (2.0 kb), lane 2: HC-Nb-NA(6.8 kb), lane 3: Na-AX-NA(3.6 kb). (B) lane 1: T3F-HC (2.0 kb), lane 2: NP1-Na (5.8 kb), lane 3: Na-CP (3.1 kb), lane 4: Nb-AX (2.0 kb). Lane M: DNA 1kb marker. These two-step RT-PCR products were analysed with 1% agarose gel electrophoresis.
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圖三、木瓜輪點病毒(PRSV)全長基因體 cDNA 選殖片段以 XbaI 進行酵解後之電 泳分析。
Fig. 3. Gel eletrophoresis of full-length cDNA cloned fragments of Papaya ringspot virus (PRSV) treated with the XbaI restriction enzyme.
Five cDNA clones were digested with XbaI and analysed with 1% agarose gel
electrophoresis. Lane 1: prT3-AX-NP; lane 2: prT3-AX-MP; lane 3: pfT3-AX-DP; lane 4: prT3-AX-NA; lane 5: pT3-NP. Lane M: DNA 1kb marker.
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圖四、木瓜輪點病毒(PRSV)全長 cDNA 選殖株之胞外轉錄 RNA 產物電泳分析。