臺灣博碩士論文加值系統

雙生病毒(geminiviruses) 感染植物後，經常會造成葉片捲曲，生長受阻，導致產量銳減，造成經濟重大的損失。本實驗室先前研究發現，圓葉菸草(Nicotiana benthamiana)感染霍香薊黃脈病毒(Ageratum yellow vein virus, AYVV)或番茄捲葉病毒(Tomato leaf curl virus, TLCV)後，其葉片分別會出現特定向上或向下捲曲的捲葉病徵，且此病徵與AYVV及TLCV之C4蛋白有關；因此，本實驗之目的即為以TLCV之C4蛋白為對象，探討其調控葉片捲曲向性的關鍵胺基酸，以期望未來可應用於病徵的緩解與治療。先前研究顯示，感染AYVV南投株系(AYVV-NT)的圓葉菸草其葉片會出現向上捲曲的病徵，且AYVV-NT之C4蛋白中的第八及第十四個胺基酸確實具有影響葉片捲曲向性的功能。本研究利用可造成圓葉菸草葉片下捲的TLCV台南株(TLCV-TN)為材料，將其C4蛋白上的第八及第十四個胺基酸做突變，製作出C4蛋白的2種感染性突變株，分別命名為A、B突變，並利用農桿菌接種於圓葉菸草，觀察其突變對於葉片捲曲方向與程度的影響。結果顯示A突變株與B突變株所產生的病徵均較野生型病毒緩和，接種A突變株植株的新生葉下捲角度較野生型病毒少，而接種B突變株植株的新生葉僅出現彎曲下垂，葉片的邊緣並未像接種野生型病毒的植株產生捲曲的外觀。此結果證實TLCV-TN C4蛋白中的第八及第十四個胺基酸確實具有影響葉片捲曲向性的功能，未來將進一步利用其他突變株探討不同雙生病毒C4蛋白調控植物葉片捲曲之趨向性的分子機制，並可供日後研發雙生病毒病害之管理策略。

Geminiviruses usually inflict typical leaf curling symptoms on infected plants, with severe growth retardation and yield reduction, resulting in serious losses in agricultural economy. Previous studies in our lab have revealed that the C4 proteins of geminiviruses are involved in the determination of the direction of leaf curling symptoms. However, the underlying mechanisms remained to be explored. Therefore the objectives of this study are to identify the key amino acid residues involved in the determination of leaf curling directions. Previously, it has been shown by our group that two amino acids in the C4 protein of Ageratum yellow vein virus (AYVV) played key roles in the determination of leaf curling direction and severity. In this study, the C4 protein of Tomato leaf curl virus Tai-Nan Strain (TLCV-TN), which caused severe downward leaf curl symptom on Nicotiana benthamiana, was used as the primary material for mutational analysis. Since the C4 gene open reading frame overlaps that of the Rep protein and at least two copies of origin of replication (Ori) are required for the infectivity of the clones, different constructs were designed and tested in this study. Accordingly, infectious clones of two mutants, designated A-, and B-mut, were constructed and assayed in N. benthamiana. The results revealed that two amino acids in A-mut and B-mut could attenuate the downward leaf curling symptoms. Thus, the results elucidated key amino acids in C4 proteins that are involved in the modulation of leaf curling symptoms. It is expected that the results from this study may contribute to the knowledge on the modulation of leaf curling symptom, and may be applied in the development of effective management strategy for viral diseases.

前言 1
一、雙生病毒簡介 1
(一) 經濟重要性 1
(二) 分類地位 2
(三) 基因體結構 2
二、影響葉片發育之植物因子 3
(一) 基因異常 4
(二) 小分子RNA異常 4
(三) 賀爾蒙異常 5
三、雙生病毒影響病徵表現之因子 5
(一) C1基因 6
(二) C2基因 6
(三) C4基因 7
(四) V2基因 8
(五) βC1基因 8
實驗室前人研究 8
本研究目的 9
材料與方法 10
一、試驗材料 10
二、試驗方法 10
(一) 構築番茄捲葉病毒定點突變質體 10
(二) 構築番茄捲葉病毒感染性突變株 12
(三) 接種圓葉菸草(Nicotiana bethamiana)試驗 14
(四) 植株接種病毒後的測試 15
(五) 接種病毒之C4基因分析 16
結果 18
一、設計C4蛋白胺基酸定點突變引子 18
二、構築番茄捲葉病毒定點突變的質體 18
三、構築番茄捲葉病毒之感染性突變株 18
四、病徵觀察與分析 19
(一) 接種病毒之植物外觀的表現 19
(二) 接種病毒後植物的檢測 20
討論 23
參考文獻 27
圖表與附錄 33

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