臺灣博碩士論文加值系統

矮南瓜黃化嵌紋病毒(Zucchini yellow mosaic virus, ZYMV)是造成瓜類作物上嚴重危害的病毒之一。利用輕症病毒來感染植物以保護其不被強系病毒感染稱之為交互保護作用，利用此方法能夠有效的降低ZYMV對於作物的影響。篩選亞硝酸誘變後的ZYMV突變株能夠獲得有實際應用價值的輕症病毒株，但在傳統的方式上篩選突變株過程繁雜，費時費力。我們在過去的研究中發現輕症多種馬鈴薯Y屬病毒(potyviruses)在奎藜葉片上不會誘發過敏性反應(hypersensitive reaction , HR)的壞死病斑，因此我們推測可以利用此現象來加速馬鈴薯Y屬病毒的輕症病毒株的篩選。為了方便偵測，我們利用帶有綠色螢光蛋白的ZYMV做為病毒來源以進行亞硝酸誘變。利用螢光照相系統追蹤接種在奎藜葉片上的所有處理過的病毒感染，把在葉片上不會產生過敏性反應的螢光感染點分離出來後，再接種至ZYMV的系統性寄主以測試突變株的病原性。經過三次獨立試驗後得到10個突變株能夠成功感染矮南瓜、刺角瓜和小胡瓜，並且皆僅引起輕微的病徵。經過病毒的病徵分析、病毒量累積變化、基因體穩定性測試後，篩選出兩個理想的突變株ZG-4-3和ZG-4-10。在溫室的交互保護試驗中，矮南瓜接種輕症突變株後15天進行挑戰接種強系病毒ZYMV-TN3，試驗結果顯示兩個突變株皆能夠提供完全對抗此強系病毒感染的交互保護能力。我們進一步利用重疊延伸聚合酶連鎖反應(overlapping extension polymerase chain reaction)去除了ZG-4-10的GFP基因，ZG-4-10其他部分則利用聚合酶連鎖反應(polymerase chain reaction, PCR)來擴增，再利用限制酶的切位與p35SZYMV-TN3進行置換，獲得了不帶有螢光基因的具感染性cDNA 克隆 Z-4-10。如此我們順利獲得了一個非基因改造的輕症病毒株Z-4-10，具有被應用於控制瓜類上ZYMV的危害的優越能力。經過定序後得知Z-4-10突變株在基因體核酸上有6的突變點，在胺基酸層次則是有5個改變。藉此，我們利用輕症病毒在奎藜上不產生過敏性反應的特性，建立了一種方法能夠快速獲得經過亞硝酸誘變並具有實用性價值的輕症病毒株，可提供完全的交互保護作用來防治ZYMV。

Zucchini yellow mosaic virus (ZYMV) causes severe damage on cucurbits. Cross protection, a phenomenon describes that a mild virus strain is used to protect a host against infection by the related severe viruses, is an effective control measure for plant viruses. Useful mild strains can be obtained from nitrous-acid induction; however, the process is time consuming, tedious and difficult. Our previous studies have revealed that effective mild strains of several potyviruses for cross protection do not induce hypersensitive reaction (HR) in Chenopodium quinoa plants and causing infection without formation of local lesion. Thus, we hypothesize that a potential mild virus mutant can be readily identified based on its inability to cause HR in C. quinoa plants after nitrous acid mutagenesis. Accordingly, a reporter gene expressing green fluorescent protein (GFP) was inserted into ZYMV genome for monitoring virus mutants in C. quinoa leaves after nitrous acid induction. The infected leaves were examined under an imaging system to identity infection spots with fluorescence but without HR reaction. Viruses present in individual identified spots were then transferred to ZYMV systemic hosts of squash to test their pathogenicity. From three independent experiments, ten mutants were successfully transferred to plants of squash (Cucurbita pepo L.), horned melon (Cucumis metuliferus L.) and cucumber (Cucumis sativus L.), on which all induced mild symptoms. After analyses of their symptomatology, virus titer and genetic stability, two ideal mild mutants of ZG-4-3 and ZG-4-10 were selected, both induced inconspicuous symptoms on three cucurbits. Under greenhouse conditions, these two mild mutants provided compete cross protection in squash plants against the severe strain ZYMV TN3. GFP gene in the genome of ZG-4-10 was removed by overlapping extension polymerase chain reaction. The infectious cDNA clone of Z-4-10 without GFP gene was constructed using p35SZYMV-TN3 as backbone, by replacing the cDNA fragments amplified from the genome of the mild mutant. Thus, Z-4-10 becomes a pure nitrous acid-induced mild strain and can be used for control of ZYMV in cucurbits similar to the conventional mild protectant. Genomic sequencing of Z-4-10 revealed that there are six nucleotides mutated, resulting in 5 amino acid changes. Here, we have established a novel way to promptly select effective nitrous-acid induced mild strains for control of ZYMV by cross protection.

中文摘要……………………………………………………………………………………………………………………………i
Abstract…………………………………………………………………………………………………………………………ii
前人研究……………………………………………………………………………………………………………………………1
矮南瓜黃化嵌紋病毒之特性……………………………………………………………………………………1
針對矮南瓜黃化嵌紋病毒的防治策略…………………………………………………………………2
交互保護之背景與輕症病毒之特性………………………………………………………………………3
交互保護的機制……………………………………………………………………………………………………………3
獲取有交互保護潛力的輕症病毒之策略……………………………………………………………4
亞硝酸誘導突變的機制……………………………………………………………………………………………5
本研究的假設、策略與目的……………………………………………………………………………………5
Introduction………………………………………………………………………………………………………………7
The features of Zucchini yellow mosaic virus…………………………7
The strategies for control of ZYMV……………………………………………………9
Background of cross-protection and the feature of an effective mild virus strain……………………………………………………………………10
The mechanism of cross-protection……………………………………………………11
The strategies to obtain effective mild strain virus for cross-protection…………………………………………………………………………………………………12
The mechanism of mutation induced by nitrous-acid…………14
Hypothesis, objectives and the strategy use in this study ……………………………………………………………………………………………………………………………………………15
Materials and Methods……………………………………………………………………………………16
Virus sources and nitrous acid induction…………………………………16
Screen of mild mutants from leaves of C. quinoa………………17
Accumulation of ZYMV mutants in plants………………………………………18
Cross-protection assay of the selected ZYMV mutants……18
Construction of infectious clone of ZYMV mild mutants and removal of GFP gene…………………………………………………………………………………………19
Results…………………………………………………………………………………………………………………………21
Screening for ZYMV-GFP mutants with inability to induce necrotic lesions in Chenopodium quinoa leaves……………………21
Symptoms induced by mutants in squash plants………………………21
Symptoms induced by mild mutants on cucurbit plants……22
Accumulation of ZYMV mild mutants in squash plants………22
Cross-protection effectiveness of attenuated mutants against severe strain ZYMV infection……………………………………………23
Construction of infectious clone from mild mutants………24
Genomic sequence of ZYMV mutant Z-4-10………………………………………25
Discussion…………………………………………………………………………………………………………………27
References…………………………………………………………………………………………………………………32
Table 1. Primers used in study……………………………………………………………44
Table 2. Symptoms of cucurbit plants infected by mild mutants…………………………………………………………………………………………………………………………45
Table 3. Cross-protection effectiveness of ZG-4-3 and ZG-4-10 against zucchini yellow mosaic virus severe strain ZYMV-GFPhis3…………………………………………………………………………………………………………………………46
Figure 1. GFP expression in Chenopodium quinoa leaves inoculated with ZYMV-GFP mutants………………………………………………………47
Figure 2. Symptoms on cucurbit plants inoculated with different attenuated mutants of zucchini yellow mosaic virus (ZYMV)……………………………………………………………………………………………………………48
Figure 3. Accumulation of ZYMV mutants in squash plants at different time course as determined by indirect ELISA.…49
Figure 4. The symptoms on squash plants after challenge inoculation.……………………………………………………………………………………………………………50
Figure 5. The amino acid sequence comparison of helper-component proteinase (HC-Pro) of zucchini yellow mosaic (ZYMV) mild mutants ZG-4-3 and ZG-4-10………………………………………51
Figure 6. Construction of infectious cDNA clone of mild mutant Z-4-10 without GFP gene using severe strain ZYMV-TN3 as a backbone…………………………………………………………………………………………………………53
Figure 7. The formation of local infection and molecular detection after introduction of Z-4-10 infectious clones to Chenopodium quinoa leaves…………………………………………………………………………55
Figure 8. Schematic representation of sequence comparison with mutant ZG-4-10 and ZYMV-TN3………………………………………………………57

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