Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31540
標題: 西瓜銀斑病毒複製酶高保留性區域轉基因煙草抗性及利用具感染力矮南瓜黃化嵌紋病毒載體表現其各部位基因之研究
Broad-spectrum resistance to tospoviruses in transgenic Nicotiana benthamiana plants carrying the conserved RNA polymerase motifs of Watermelon silver mottle virus and expression of its individual genes using the Zucchini yellow mosaic virus vector
作者: 陳宗祺
Chen, Tsung-Chi
關鍵字: 西瓜銀斑病毒
transgenic Nicotiana benthamiana plants
保留性區域
轉基因煙草
矮南瓜黃化嵌紋病毒
病毒載體
Watermelon silver mottle virus
Zucchini yellow mosaic virus
Broad-spectrum resistance
plant viral vector
RNA polymerase motifs
出版社: 植物病理學系
摘要: 西瓜銀斑病毒複製酶高保留性區域轉基因煙草抗性及利用具感染力矮南瓜黃化嵌紋病毒載體表現其各部位基因之研究 論文摘要 西瓜銀斑病毒 (Watermelon silver mottle virus,WSMoV) 為Bunyaviridae科,番茄斑萎病毒屬 (Tospovirus) 之一員,是台灣瓜類作物栽培的主要限制因子之一。WSMoV之基因體近晚已由本實驗室解序完成。將其L RNA對應產生的大蛋白 (L protein) 與Bunyaviridae科中之其他成員進行比對,發現胺基酸序列中含有已報導之RNA複製酶的高保留性區域。本研究建構了此WSMoV的複製酶保留性區域之可轉譯性與非轉譯性之不同構築,並以農桿菌感染的方式將所構築之基因片段導入煙草 (Nicotiana benthamiana) 中,以期獲得具廣泛抗性之轉基因煙草植物。所育成之轉基因煙草於溫室條件下評估其抗性表現,結果得到一可轉譯性之轉基因煙草株系WLm3-6,與四個非轉譯性之轉基因煙草株系WLmAs1-7、WLmAs1-14、WLmAs1-17及WLmt1-2可廣泛地抵抗WSMoV和另一不同種之番茄斑萎病毒 (Tomato spotted wilt virus,TSWV) 之感染。經由康黴素 (kanamycin) 抗性分析其轉基因之存在,可測得轉入的基因在這些抗病株系中具有3:1或15:1之分離率,顯示這些轉基因煙草株系之染色體中含有一或二個轉基因。且廣泛性抗性亦由接種評估證明其性狀可遺傳至子代。由於無適當之血清可偵測轉基因於抗性煙草中之表現,故利用北方轉漬法分析其轉基因轉錄體RNA之表現量來得知,此等高度抗性表現乃由RNA所誘導之基因沉寂作用 (RNA-mediated gene silencing) 所產生。這也是第一篇利用RNA 複製酶的保留性區域對番茄斑萎病毒屬之病毒提供高度廣效抗性的報導。 本論文另一研究方向為利用矮南瓜黃化嵌紋病毒 (Zucchini yellow mosaic virus,ZYMV) 作為載體表現WSMoV之各部位基因,如L RNA所對應產生的L 蛋白,M RNA所對應產生的非結構性 (nonstructural,NSm) 蛋白和醣蛋白 (glycoproteins,G1/G2),以及由S RNA所對應產生的核鞘 (nucleocapsid,N) 蛋白與非結構性 (nonstructural,NSs) 蛋白等,以對映轉譯 (in frame) 的方式插入ZYMV載體之協同性蛋白 (HC-Pro protein) N端,並加入額外的組織胺基酸標定 (histidine tag) 及NIa蛋白裂解酶切位序列,以利游離態蛋白之裂解與純化。含有WSMoV之N基因轉譯架構 (open reading frame,ORF) 的重組病毒ZYMV-WN,在矮南瓜上引起相當嚴重的黃化嵌紋、葉部變形,及葉柄彎曲等病徵。含有WSMoV之完整的NSs ORF的重組病毒ZYMV-WNSs,亦在矮南瓜上造成非常嚴重的黃化嵌紋及葉部變形等病徵。利用RT-PCR及免疫分析法證實罹病植物乃由重組病毒ZYMV-WN和ZYMV-WNSs感染所致,且可分別測得N及NSs蛋白表現。此外,NSs蛋白之部分ORFs包括N端,中間部位及C端亦被導入ZYMV載體,其中含有NSs之N端的重組病毒ZYMV-WNSsN及含有NSs之C端的重組病毒ZYMV-WNSsC在矮南瓜上引起較輕微之病徵。而含有NSs中間區域之重組病毒ZYMV-WNSsM則僅能於單斑寄主Chenopodium quinoa上產生單斑,無法在系統性的寄主矮南瓜上造成系統性的病徵。於重組病毒ZYMV-WN感染之植物萃取液中可測得與WSMoV感染煙草相當之大量含有組織胺基酸 (histidine) 標定之N蛋白。且由重組病毒表現之游離態的N和 NSs蛋白亦可從罹病植物材料中利用Ni2+-NTA親和性交換樹脂 (Ni2+-NTA affinity chromatography) 將之純化出來。由此推論,利用ZYMV載體表現其他Tospoviruse屬或Bunyaviridae科之其他病毒的N及NSs proteins有其之可行性,可作為血清製備、疫苗或其他醫療所需之用。 此外,WSMoV之L蛋白的不同部位,包括N端、C端和複製酶保留性部位與其各區域 (motifs),以及M RNA所對應產生之NSm與G1/G2 ORFs亦被導入ZYMV載體,以期於植物體內表現。大部分的重組病毒皆可於單斑寄主C. quinoa上產生單斑病徵,但是於系統性寄主矮南瓜上卻造成多種不同的病徵型態。罹病植物亦經由RT-PCR和免疫分析證實重組病毒之存在。然而,以抗組織胺基酸標定之單元抗體偵測這些植物卻無法測得蛋白表現,且這些重組病毒在矮南瓜上所造成的病徵通常較重症病毒所引起的病徵輕微,此病徵之改變於本文中有所討論。
Broad-spectrum resistance to tospoviruses in transgenic Nicotiana benthamiana plants carrying the conserved RNA polymerase motifs of Watermelon silver mottle virus and expression of its individual genes using the Zucchini yellow mosaic virus vector Abstract Watermelon silver mottle virus (WSMoV), a member of the genus Tospovirus, is one of the major limiting factors for growing watermelon, melon, wax gourd and other cucurbits in Taiwan. Comparison of the deduced L protein encoded by the L RNA of WSMoV with those of other members of the family Bunyaviridae revealed that its amino acid sequence includes the reported conserved motifs of RNA-dependent RNA polymerase (RdRp). In this investigation, the translatable and untranslatable constructs of the conserved region containing the polymerase motifs of WSMoV were introduced into plants of Nicotiana benthamiana by Agrobacterium-mediated transformation. One translatable line WLm3-6 and four untranslatable lines WLmAs1-7, WLmAs1-14, WLmAs1-17, and WLmt1-2 were found resistant to both WSMoV and an unrelated tospovirus, Tomato spotted wilt virus (TSWV). Segregation ratios of transgene in 3:1 or 15:1 were found in the self-pollinated R1 progeny derived from the resistant lines when they were analyzed by kanamycin-resistance assay, indicating that one or two copies of the transgene were inserted in the chromosome of the resistant lines. The broad-spectrum resistance was inherited as nuclear traits in the R1 progeny, as evidenced by inoculation with TSWV and WSMoV. In northern blot analyses the message of the transgene was not detected in the highly resistant transgenic lines, suggested that the resistance is RNA-mediated gene silencing. This is the first report for using the conserved region of the RNA polymerase to generate high degrees of broad-spectrum resistance to different tospoviruses. On the other investigations, the individual open reading frames (ORFs) of WSMoV, including the portions of the L protein encoded by L RNA, the nonstructural (NSm) protein and glycoproteins (G1/G2) encoded by M RNA, and the nucleocapsid (N) protein and nonstructural (NSs) protein encoded by S RNA, were in frame inserted into the Zucchini yellow mosaic virus (ZYMV) vector at the N-terminal region of the HC-Pro protein, with additional six histidine residues and a NIa protease cleavage site to facilitate the purification and to process the free form proteins. The recombinant ZYMV-WN carrying the N ORF of WSMoV induced severe symptoms of yellow mosaic, leaf distortion, and hooky petioles on zucchini squash (Cucurbita pepo L. var. Zucchini). The recombinant ZYMV-WNSs carrying the entire NSs ORF of WSMoV also induced severe symptoms of yellow mosaic and leaf distortion on squash plants. On the other hand, the partial ORFs including the reading frames corresponding to N-terminal, middle, and the C-terminal regions of the NSs protein were also introduced into the ZYMV vector. Milder symptoms with mosaic on squash plants were induced by the recombinants ZYMV-WNSsN and ZYMV-WNSsC carrying the N-terminal and the C-terminal regions of the NSs gene, respectively. The recombinant ZYMV-WNSsM carrying the middle portion of the NSs gene induced local lesions on Chenopodium quinoa but failed to infect zucchini squash. Fidelity of the recombinants ZYMV-WN and ZYMV-WNSs, and the expressed protein in the infected plants were confirmed by RT-PCR and immunoassays, respectively. Large quality of the histidine-tagged N protein was detected in the extract of squash plants infected with the recombinant ZYMV-WN, similar to that produced in WSMoV-infected plants of N. benthamiana. The free form N and NSs proteins expressed by the ZYMV recombinants in squash plants were readily purified by the Ni2+-NTA affinity chromatography. The possibilities to utilize the ZYMV vector to express the N and NSs proteins of other tospoviruses or viruses belong to the family Bunyaviridae for antisera production, vaccines, and clinical applications are discussed. In addition, the cDNA fragments encoding portions of the L protein of WSMoV, including the N and C-terminal regions, and the conserved domain and the individual motifs of the polymerase, and the ORFs of the NSm and G1/G2 genes encoded by the M RNA of WSMoV were also inserted into the ZYMV vector for expression in plants. Most of the ZYMV recombinants induced local lesions on the local lesion host Chenopodium quinoa but various symptom types developed on the systemic host zucchini squash plants. The fidelity of the recombinants infecting plants was confirmed by RT-PCR and immunoassays. However, the recombinants carrying the ORFs derived from L and M RNA of WSMoV did not express detectable proteins in infected host plants, as monitored by the monoclonal antibody to the histidine tag. Also, the expected proteins of WSMoV were not obtained by the Ni2+-NTA affinity chromatography. Nevertheless, symptom attenuation of the ZYMV recombinants by the inserted fragments derived from the L and M RNAs of WSMoV is discussed.
URI: http://hdl.handle.net/11455/31540
Appears in Collections:植物病理學系

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