Generation of transgenic Nicotiana benthamiana lines with broad-spectrum resistance to tospovirus and begomovirus

番茄是世界重要的作物之一，然而其生產被病毒病害所影響，其中又以雙生病毒屬(Begomovirus)的番茄黃化捲葉泰國病毒(Tomato yellow leaf curl Thailand virus, TYLCTHV)和番茄捲葉台灣病毒(Tomato leaf curl Taiwan virus, ToLCTWV)及番茄斑萎病毒(Tospovirus)的番茄斑萎病毒(Tomato spotted wilt virus, TSWV)在台灣最為嚴重。前人利用西瓜銀斑病毒(Watermelon silver mottle virus, WSMoV)之L RNA高度保留區域進行基因改良，讓圓葉菸草(Nicotiana benthamiana)能廣泛性對抗多種不同番茄斑萎病毒。另一研究將藿香薊黃脈病毒(Ageratum yellow vein virus, AYVV)的非轉錄區域(intergenic region, IGR)設計成廻紋結構，放入內含子中可引發DNA甲基化並可對藿香薊黃脈病毒提供抗性。在本研究結合上述兩個研究構築WL-IGRs，黏合番茄黃化捲葉泰國病毒和番茄捲葉台灣病毒的非轉錄區域，藉以增加抗性之廣泛性，將非轉錄區域設計成髮夾廻紋結構放入番茄刺激生長激素蛋白酶抑制子(Auxin-induced proteinase inhibitor)的內含子，再將此迴紋結構之內含子放入作為外顯子的L RNA高度保留區域，藉此讓轉基因圓葉菸草廣泛性對抗不同的番茄斑萎病毒及雙生病毒。構築質體的確認是利用PCR、限制酶剪切和定序驗證。再經由農桿菌轉殖圓葉菸草後，接種番茄斑萎病毒及番茄黃化捲葉泰國病毒選拔出九個不同抗性株系，並以聚合酶連鎖反應(polymerase chain reaction, PCR)確認其轉基因。其中株系28號對番茄斑萎病毒及番茄黃化捲葉泰國病毒具有高程度抗性。經由南方墨漬分析轉基因的拷貝數，株系28號在篩選基因及抗病基因WL-IGRs都具有三個拷貝數。由於實驗中的抗病基因WL-IGRs是放在雙T-DNA載體中，因此可以利用自交的方式將篩選基因剃除。本研究設計對抗薊馬傳播的番茄斑萎病毒及粉蝨傳播的雙生病毒兩屬重要的病毒，未來可應用在蕃茄等其他作物上有重要的應用價值。Tomato is an important crop worldwide. However, the production of tomato is limited by virus diseases, among them the begomoviruses of Tomato yellow leaf curl Thailand virus, TYLCTHV) and Tomato leaf curl Taiwan virus (ToLCTWV), and the tospovirus of Tomato spotted wilt virus (TSWV) are considered the most serious threats in Taiwan. Our previous studies have shown that transgenic Nicotiana benthamiana plants carrying Watermelon silver mottle virus (WSMoV) L RNA conserved region provide broad-spectrum resistance to tospoviruses. Another study showed that a viral intergenic region as a hairpin structure inserted in an intron can induce DNA methylation and generate resistance against Ageratum yellow vein virus (AYVV). In this study, for generating transgenic resistance in N. benthamiana plants against tospoviruses and begomoviruses, we used agrobacteria carrying a construct of WL-IGRs, which contains WSMoV L conserved region as exonic sequences in which an intron (from auxin-induced proteinase inhibitor gene of tomato, DQ:L25128) containing a hairpin structure of TYLCTHV and ToLCTWV intergenic regions, for transformation. The transgene construct was verified by PCR, restriction enzyme digestion and sequencing, and then transferred to Agrobacterium tumefaciens for transformation of N. benthamina plants. Nine lines were selected for PCR analysis and resistance evaluation against Tomato spotted wilt virus (TSWV) and TYLCTHV. Among them line 28 showed high levels of resistance against TSWV and WSMoV. After Southern blotting analysis, line 28 showed three inserts of WL-IGRs transgene and three inserts of NptII selection marker. Because, WL-IGRs construct contains two T-DNAs, each with the selection marker or the transgene, thus marker-free transgenic lines can be obtained after selfing. Our approach is valuable for practical application to control important virus diseases on tomato caused by thrips-borne tospovirus and whitefly-borne begomovirus.中文摘要..............................................i Abstract............................................ii 目次............................................... iii 圖表目次.............................................iv Introduction.........................................1 Materials and methods................................7 Virus sources........................................7 Phylogenetic tree analysis of begomovirus IGR........7 Construction of plant transformation vectors.........8 Agrobacterium transformation.........................9 Detection of intron splicing of pK2T-WL-IGRs........10 Nicotiana benthamiana transformation................10 Resistance evaluation...............................11 Southern hybridization..............................12 Results.............................................13 Phylogenetic tree analysis of begomovirus IGR.......13 Confirmation of the transgene construct............ 13 The intron of pK2T-WL-IGRs construct spliced........14 Confirming the transgenic construct in transgenic lines...15 Evaluation of resistance against TYLCTHV, TSWV and WSMoV...15 Insertion number detected in the resistant transgenic lines....15 Discussion..........................................16 References..........................................22 Tables and figures..................................3