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Assessment of Genetic Variability, Characterization of Genome Organization, Construction of Infectious Transcripts, Development of Viral Vector, and Generation of Valuable Attenuated Strains of a Taiwan Strain of Zucchini yellow mosaic virus
|關鍵字:||Zucchini yellow mosaic virus|
heteroduplex mobility assay
|摘要:||矮南瓜黃化嵌紋病毒分離株 (Zucchini yellow mosaic virus; ZYMV) 屬於馬鈴屬 Y 群病毒 (the genous Potyvirus)，此病毒為瓜類作物生長期間最大為害因子，因此本研究以分子生物學方法來研究鑑定台灣 ZYMV 的分類特性，構築台灣本土 ZYMV 之全長基因體及核酸定序，建構 ZYMV 具感染力轉錄體，並將其發展成植物病毒載體。另外，此病毒載體也被研發為可供交護保護的輕症型病毒。本論文第一章主要是整理近幾年內與本研究相關的參考文獻。
本論文第二章主要研究台灣不同瓜類產區所分離的五個矮南瓜黃化嵌紋病毒分離株 (Zucchini yellow mosaic virus; ZYMV)，TW-TC1、TW-CY2、TW-TN3、TW-TNML1 及 TW-NT1，以異質結合性移動分析法 (heteroduplex mobility assay; HMA) 檢測其鞘蛋白 (coat protein; CP) 的N 端變化區基因片段彼此間的親源關係。HMA 結果顯示，TW-TC1、TWCY2、TW-TN3及 TW-TNML1 的親源關係較接近，而 TW-NT1與其它四個台灣分離株較疏遠。這五個分離株的完整鞘蛋白及 3'' 端基因序列亦被解序完畢，並且與其它六個世界上不同地區的分離株鞘蛋白進行序列比對。從鞘蛋白演化關係圖上顯示 TW-TC1、TW-CY2、TW-TN3 及 TW-TNML1 屬於第一基因型，TW-NT1 與美國分離株屬第二基因型，雷優尼島 (Resunion Island) 與新加坡分離株分別屬第三與第四基因型。此外，在第一基因型的鞘蛋白胺基酸序列之第 73、102、109、149 位置胺基酸分別為 lysine、serine、arginine 以及 aspartic acid，這些胺基酸為第一基因型所特有，可作為分類判別的依據。
本論文第三章主要研究 TW-TN3 分離株，作為台灣典型 ZYMV 的代表，使用 RT-PCR 與 cDNA library 等技術，共選殖出五個互相重疊並且涵蓋病毒全長基因體之 cDNA clones。將其解序完畢後發現，TW-TN3 全長共 9591 個核甘酸，預期可轉譯出一個含帶 3080 個胺基酸的大轉譯讀架 (open reading frame; ORF)。經由電腦比對 TW-TN3、CA、RU 與 S 分離株之蛋白序列發現，P1 蛋白為所有 ZYMV 蛋白中變化最大的蛋白，其胺基酸相同度為 59-93.2%。將TW-TN3 的 P1 蛋白與其它已被報導之 ZYMV 分離株 (CA, MD, SV, FL/AT, RU, S) 的 P1 蛋白進行演化分析，發現其演化關係亦可以區分為四個基因型，而台灣分離株 TW-TN3 屬於第一基因型，美國分離株 (CA, MD, SV, FL/AT) 為第二基因型，其演化分析結果與鞘蛋白的演化分析結果 (第二章) 相符。P1 的演化距離 (distances) 顯示其第一基因型與第二基因型的演化距離較為接近，顯示兩者可能從共同的始組所演化而來。此外，比較 TW-TN3、CA、RU 與 S 的 NIa 蛋白裂解酵素切位發現其共同之辨識區為 V-x-x-(Q,E)/(S,A,G)，分別由NIa 蛋白裂解酵素切割在 Q-S、E-S、Q-A 或 Q-G 上。
本論文第四章之研究是依據 TW-TN3 病毒全長基因體序列將五段含有部份片段之 cDNA clones 連接成一個完整的 cDNA。並且分別以 T7 噬菌體啟動子 (T7 promoter) 或 Cauliflower mosaic virus 的 35S 啟動子 (35S promoter) 啟動轉錄出全長病毒的 RNA，前者產生生體外 (in vitro ) 轉錄體，稱為 pT7ZYMV2-5，後者產生生體內 (in vivo)轉錄體，稱為p35SZYMV2-26。此二轉錄體都具有感染植物的能力，使用粒子槍將轉錄體送入矮南瓜發現確可產生系統性病徵，而以機械接種轉錄體於奎藜植物則產生局部病斑 (local lesion)。
本論文第五章進一步改造生體外具感染力轉錄體，p35SZYMV2-26 質體，使成為病毒載體，可應用於瓜類作物中表現外源蛋白。本研究將綠螢光蛋白（GFP）報導基因構築於 p35SZYMV2-26 具感染力轉錄體之 HC-Pro 之 N 端，預期表現一個GFP/Hc-Pro的融合蛋白，稱此質體為 p35SZYMVGFP；另外藉由 GFP C 端插入一組 ZYMV NIa 蛋白裂解酵素切位序列，則預期可表現單一游離的 GFP 蛋白，稱為 p35SSZYMVGFPQS。此外，構築數組限制酵素切位與一個 histidine tag 於 GFP 蛋白 C 端，前者可方便用於置換它種外來基因片段，後者則用於自感染重組病毒之植物體中蛋白。藉由感染力分析、螢光顯微鏡及免疫墨點分析顯示，本文中所創造之病毒載體確實可以大量表現單一或融合之 GFP 蛋白，以及塵之過敏原表面抗原 DerpV 蛋白，由親合性管柱層析分析亦證實可純化植物體中的外源蛋白。因此，我們所構築之 ZYMV 病毒載體可以於瓜類植物上以穩定及高效率方示表現外源蛋白。另外，此病毒載體可以在植物體表現融合型 GFP 蛋白 120 天，而表現單一游離型 GFP 的病毒載體則可以在植物體超過一年以上，顯示此病毒載體極具穩定表現外源蛋白的能力。
本論文第六章依據以前研究比對輕徵型木瓜輪點病毒（PRSV HA5-1）與輕徵型 ZYMV (ZYMV-WK) 之協同性蛋白（Hc-Pro）之氨基酸序列顯示，其 Arg180、Ph205 及 Glu396 三個胺基酸扮演由強系病毒轉變為弱系病毒之關鍵位置。將此這三個氨基酸 Arg180、Phe205及Glu396 分別置換成 Ile、Leu 及Asn，產生單一、雙重及三重突變株病毒共七株，將之分別以機械接種之方式接種於產生局部病斑寄主奎藜或系統性寄主矮南瓜上，分析其致病性。結果顯示單一突變株病毒 GA、GB、GC 以及雙重突變株病毒 GBC 於矮南瓜上產生第二型嵌紋病徵；雙重突變株病毒 GAC於矮南瓜植物上產生第三型輕微嵌紋病徵；此外，另一個雙重突變病毒 GAB 在矮南瓜上產生第四型無病徵。然而三重突變株病毒 GABC 則失去奎藜及矮南瓜的感染能力。另一方面，所有的突變株在奎藜上的局部病斑型態均會改變，GA、GC及GBC 突變株在奎藜上產生黃化扇斑狀的病斑； GAB 及 GAC 則不會產生任何局部病斑；並且 GB 突變株則產生與野生型病毒相似的局部病斑。經 9 個月的植物連續接種植物 9 次後發現，GAC 突變株穩定的表現輕微的嵌紋病徵，而沒有回變成嚴重型病徵的現象。GAC 若病毒接種於矮南瓜植物，5天後即可產生抵抗強系病毒的交互保護能力；然而GAC 弱系病毒保護時間若不足 5天，則無法完全抵抗強系病毒發病。針對 GAC 遺傳工程突變之弱系病毒所誘導產生的交互保護可能機制也在此探討。|
Zucchini yellow mosaic virus (ZYMV), a member of the genus Potyvirus in the family Potyviridae, caused devastating epidemics in commercial cucurbits worldwide. In this study, the molecular characteristics of ZYMV, were analyzed by the assessment of genetic variability on the coat protein (CP) gene and the complete nucleotide sequence of the genomic viral RNA was determined. The infectious clones were constructed and further developed as an efficient viral vector to express foreign protein in cucurbit species. Finally, the generations of valuable attenuated strains were also created by site-directed mutagenesis. The chapter 1 reviews all the relevant reference for this study. This chapter is written in Chinese to meet the official requirement. The other chapters are written in English for publication in journal. In the chapter 2, five isolates of ZYMV, TW-TC1, TW-CY2, TW-TN3, TW-TNML1, and TW-NT1, collected from cucurbit fields in different areas of Taiwan and the variable region of the N terminal fragments of the CP gene were analyzed by heteroduplex mobility assay (HMA). The HMA results indicated that, TW-TC1, TW-CY2, TW-TN3, and TW-TNML1 share a high degree of sequence homology, while the TW-NT1 isolate is more distinct. The complete nucleotide sequences of CP gene and 3'' non-coding regions of the five isolates were compared with another six ZYMV isolates from different geographical areas and the phylogenetic tree of CP amino acid sequence indicated that the TW-TC1, TW-CY2, TW-TN3, and TW-TNML1 were in genotype I; while TW-NT1 and USA isolates were in genotype II. The Resunion Island (RU) and Singapore (S) isolates were separated in genotypes III and IV, respectively. In addition, amino acids Lys73, Ser102, Arg109, Asp149 of the CP gene, were found uniquely conserved for genotype I. In the chapter 3, the complete nucleotide sequence of TW-TN3 was determined from five overlapping cDNA clones as 9591 nucleotides in length excluding the poly(A) tail. Computer analysis of the sequence revealed a large open reading frame (ORF) that encodes a polyprotein of 3080 amino acids. Comparison of the gene products of the TW-TN3 with those of the reported California (CA), RU, and S isolates of ZYMV revealed that P1 protein is most variable, with amino acid identities of 59-93.2%. A phylogenetic tree derived from the sequences of P1 proteins of TW-TN3 and other six reported isolates also revealed four major genotypes that TW-TN3 was classified in genotype I, and US isolates were in genotype II. The distance relationships of P1 protein of genotype I were closer to genotype II indicating that the Taiwan and US isolates may have evolved from the same ancestor. Moreover, analyses of the cleavage sites of NIa protease of TW-TN3, CA, RU, and S isolates revealed that NIa protease cleaves at Q-S, and E-S dipeptide sequences, with a consensus sequence of V-x-x-(Q,E)/(S, A, G). In the chapter 4, in vitro transcript of pT7ZYMV2-5 containing the full-length cDNA clone of TW-TN3 with the bacteriophage T7 promoter in pBluescript II SK(-). The plassmid was able to generate an in vitro transcript corresponding to TW-TN3 (9591 nt) with one extra guanosine residue at the 5'' terminus and a poly(A) 95 tract at the 3'' end. In addition, the pT7ZYMV2-5 was used for the construction of p35SZYMV2-26 containing the full-length cDNA of TW-TN3 with a Cauliflower mosaic virus (CaMV) 35S promoter and a nopaline synthase (nos) polyadenylation signal terminator. Capped in vitro transcription products generated from pT7ZYMV2-5 and purified DNA of p35SZYMV2-26 were introduced into zucchini squash plants by mechanical inoculation and particle bombardment, respectively. Both in vitro and in vivo transcripts induced systemic symptoms on zucchini squash at 4 to 6 days after inoculation. In addition, both transcripts also induced local lesions on Chenopodium quinoa by mechanical inoculation. The results of infectivity assay, symptomatology, and serologically specific electron microscopy indicated that in vitro and in vivo TW-TN3 transcripts derived from pT7ZYMV2-5 and p35SZYMV2-26, respectively, are infectious. In the chapter 5, the highly infectious cDNA clone, p35SZYMV2-26, was engineered as a viral vector to express foreign proteins in cucurbit species. The coding region for the green fluorescent protein (GFP) was inserted in frame at the N-terminal region of the HC-Pro gene of TW-TN3 in p35SZYMV2-26 to create p35SZYMVGFP, which generated recombinant virus expressing GFP fused with HC-Pro protein of ZYMV, and p35SZYMVGFPQS, which generated recombinant virus expressing the free form GFP processed by an inserted NIa protease cleavage site of ZYMV at its C-terminus. In addition, convenient multiple cloning sizes were created at both 5''and 3'' termini of the GFP reading frame to facilitate the replacement with other foreign proteins and a histidine tag was created at the C-terminus of the GFP to facilitate the purification of the expressed proteins by affinity chromatography. Through this design, GFP was successfully replaced by an allergen Der p V protein that was isolated from the mite, Dermatophagoides pteronyssinus. Our results of infectivity assays, fluorescence microscopy, and immunblotting indicated that large quantity of fused GFP, free form GFP, and free form Der p V protein was produced in squash plants infected with the corresponding recombinants. The free form GFP and Der p V protein, both carrying the histidine tag, was successfully purified by the Ni-NTA affinity column. Furthermore, the results of bioassay and investigation in cell-to-cell movement, systemic translocation, and pathogenicity indicated that the recombinant viruses behaved similarly to the wild type virus ZYMV TW-TN3. The recombinants carrying the fused GFP and the free form GFP remained unchanged after several passages in squash plants for a period of 120 days and one year, respectively. Thus, the constructed ZYMV vector is considered a highly efficient, stable viral vector for expressing foreign proteins in cucurbit species. In the chapter 6, three conserved amino acids, Arg180, Phe205, and Glu396 of a severe of ZYMV, TW-TN3, were substituted with Ile180 (GA), Leu205 (GB), Asn396 (GC), respectively, different combinations (single, double, and triple mutations) at the full-length cDNA clone that carried a reported GFP. The virulence of the corresponding in vivo transcripts of the mutants was tested by mechanical inoculation on plants of C. quinoa and zucchini squash. Among them, the three single-mutated viruses (GA, GB, GC) and one double-mutated virus (GBC) caused type II symptoms with mosaic in zucchini squash plants. The double-mutated virus of GAC induced type III symptoms with symptoms dramatic change from severe to slightly mild mosaic in zucchini squash plants. In addition, another double-mutated virus (GAB) caused the type IV symptoms that was symptomless on zucchini squash plants. However, the triple-mutated virus (GABC) did not infect plants of C. quinoa and zucchini squash plants. Furthermore, all the mutated viruses changed the local lesion types on C. quinoa leaves. The GA, GC, and GBC mutants caused chlorotic spots, however, the mutants GAB and GAC produced no local lesions on C. quinoa plants, and the GB mutant caused the local lesions similar to those induced by the wild type. When the severe strain TW-TN3 was used to challenge the zucchini squash plants that protected with the GAC mutant ≧ 5 days after protective inoculation, 100% of cross-protection against the severe strain ZYMV was observed. However, the incomplete protection occurred when the severe strain was introduced within 5 days after the protective inoculation. In addition, the GAC mutant was stable, no revertant virus was found after 9 passages through plants after a period of 9 months. On the contrary, the GAB mutant did not provide any protection against the challenge virus. The possible mechanisms of attenuation and the cross-protection effectiveness related to the mutations are discussed.
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