Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/89341
標題: 番茄斑萎病毒的NSs基因片段可在含有或不含篩選標誌的轉基因植物上增強由L高保留性區域所誘發之廣泛抗病毒性狀
Untranslatable tospoviral NSs fragment enhances the broad resistance conferred by L gene conserved region in transgenic plants with or without a selection marker gene
作者: Yazhisai Uthaman
葉意香
關鍵字: Untranslatable tospoviral NSs fragment
broad resistance
L gene conserved region
transgenic plants
Untranslatable tospoviral NSs fragment
broad resistance
L gene conserved region
transgenic plants
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摘要: ABSTRACT Tospovirus, the only plant-infecting viruses in the family Bunyaviridae, cause serious damages on various economic crops all over the world. Watermelon silver mottle virus (WSMoV) and Melon yellow spot virus (MYSV), the two members of the genus Tospovirus, are the major limiting factors for cucurbits cultivation in Taiwan. In our earlier studies, transgenic tomato lines carrying the conserved region containing the RNA- dependent RNA polymerase (RdRp) motifs within the L gene of WSMoV confer broad-spectrum resistance against different tospoviruses mediated by post-transcriptioned gene silencing (PTGS). In this study, the enhanced broad resistance against distinct tospoviruses, in transgenic tobacco plants conferred by the conserved regions of L, N and NSs genes of tospoviruses were generated. Furthermore, marker-free transgenic tobacco plants carrying the L,N and NSs conserved regions of WSMoV and conferring broad resistance to distinct tospoviruses were produced . Our marker-free approach was further extended to produce marker-free economically important crops such as melon (Cucumis melo L.) carrying the conserved regions of L,N and NSs genes of WSMoV. Chapter 1, 'Literature review and research objective' describes the background informations, references and research objectives of this this study. Chapter 2, 'Untranslatable tospoviral NSs fragment coupled with L conserved region enhances transgenic resistance against the homologous virus and a serologically unrelated tospovirus'. In this study, Nicotiana benthamiana transgenic lines carrying individual untranslatable constructs comprised of the conserved region of the L gene (denoted as L), the 5′ half of NSs coding sequence (NSs) or the antisense fragment of whole N coding sequence (N) of Watermelon silver mottle virus (WSMoV), individually or in combination, were generated. A total of 15 -17 transgenic Nicotiana benthamiana lines carrying individual transgenes were evaluated against the WSMoV and the serologically unrelated Tomato spotted wilt virus (TSWV). Among lines carrying single or chimeric transgenes, the level of resistance ranged from susceptible to completely resistant against WSMoV. From the lines carrying individual transgenes and highly resistant to WSMoV (56-63% of lines assayed), 30% of the L lines (3/10 lines assayed) and 11% of NSs lines (1/9 lines assayed) were highly resistant against TSWV. The chimeric transgenes provided higher degrees of resistance against WSMoV (80-88%), and the NSs fragment showed an additive effect to enhance the resistance to TSWV. Particularly, the chimeric transgenes with the triple combination of fragments, namely L/NSs/N or HpL/NSs/N (a hairpin construct), provided a higher degree of resistance (both 50%, with 7/14 lines assayed ) against TSWV. Our results indicate that the untranslatable NSs fragment is able to enhance the transgenic resistance conferred by the L conserved region. The better performance of L/NSs/N and HpL/NSs/N in transgenic N. benthamiana lines suggests their potential usefulness in generating high levels of enhanced transgenic resistance against serologically unrelated tospoviruses in agronomic crops. Chapter 3, 'Marker-free homozygous transgenic tobacco plants with resistance against tospoviruses of different serogroups and serotype'. In this study, to develop selectable marker-free transgenic plants resistant to different tospoviruses, we adopted a co-transformation system. Agrobacterium harboring pBI2T-based binary vectors, each with two T-DNAs separately carrying the selection marker gene (SMG) nptII and the construct with the aforementioned L fragment, alone or in combination with the NSs and N fragments , were used for transformation of Nicotiana benthamiana plants. Transgenic lines (12-18 for each construct) with high degrees of resistance against two serologically unrelated tospoviruses, Watermelon silver mottle virus (WSMoV) and Tomato spotted wilt virus (TSWV), were obtained. R1 progenies of 7 self-pollinated highly resistant transgenic R0 lines were further analyzed for the segregation of the SMG and the viral construct by PCR. When the individuals of the R3 progeny of the marker-free R2 homozygous transgenic plant were challenged with members of different tospoviruses of WSMoV, TSWV and Impatiens necrotic spot virus ( INSV ), resistance levels of 90-100% were noticed, indicating that the marker-free R2 and R3 homozygous transgenic N. benthamiana plants confer high degrees of broad resistance against tospoviruses of different serogroups and serotype. This approach is currently being extended to commercially important crops such as tomato, watermelon and melon. Supplementary information, 'Generation of transgenic melon plants with marker-free hairpin construct' . In this study, using the co-transformation method, Agrobacterium harboring pBI2T 1.5 binary vector with two T-DNAs carrying the nptII selection marker gene and the gene of interest HpL/NSs/N (constructed using conserved regions of tospoviral L (replicase), N (nucleocapsid) and NSs (gene silencing suppressor) genes of tospoviruses) was used for transformation of melon (cv.Silver light) plant. The regenerated marker-free transgenic 12 melon plant lines showed the presence of transgene and marker gene when checked through PCR. The marker-free transgenic melon plant lines will be challenged with different tospoviruses such as WSMoV and MYSV.
ABSTRACT Tospovirus, the only plant-infecting viruses in the family Bunyaviridae, cause serious damages on various economic crops all over the world. Watermelon silver mottle virus (WSMoV) and Melon yellow spot virus (MYSV), the two members of the genus Tospovirus, are the major limiting factors for cucurbits cultivation in Taiwan. In our earlier studies, transgenic tomato lines carrying the conserved region containing the RNA- dependent RNA polymerase (RdRp) motifs within the L gene of WSMoV confer broad-spectrum resistance against different tospoviruses mediated by post-transcriptioned gene silencing (PTGS). In this study, the enhanced broad resistance against distinct tospoviruses, in transgenic tobacco plants conferred by the conserved regions of L, N and NSs genes of tospoviruses were generated. Furthermore, marker-free transgenic tobacco plants carrying the L,N and NSs conserved regions of WSMoV and conferring broad resistance to distinct tospoviruses were produced . Our marker-free approach was further extended to produce marker-free economically important crops such as melon (Cucumis melo L.) carrying the conserved regions of L,N and NSs genes of WSMoV. Chapter 1, 'Literature review and research objective' describes the background informations, references and research objectives of this this study. Chapter 2, 'Untranslatable tospoviral NSs fragment coupled with L conserved region enhances transgenic resistance against the homologous virus and a serologically unrelated tospovirus'. In this study, Nicotiana benthamiana transgenic lines carrying individual untranslatable constructs comprised of the conserved region of the L gene (denoted as L), the 5′ half of NSs coding sequence (NSs) or the antisense fragment of whole N coding sequence (N) of Watermelon silver mottle virus (WSMoV), individually or in combination, were generated. A total of 15 -17 transgenic Nicotiana benthamiana lines carrying individual transgenes were evaluated against the WSMoV and the serologically unrelated Tomato spotted wilt virus (TSWV). Among lines carrying single or chimeric transgenes, the level of resistance ranged from susceptible to completely resistant against WSMoV. From the lines carrying individual transgenes and highly resistant to WSMoV (56-63% of lines assayed), 30% of the L lines (3/10 lines assayed) and 11% of NSs lines (1/9 lines assayed) were highly resistant against TSWV. The chimeric transgenes provided higher degrees of resistance against WSMoV (80-88%), and the NSs fragment showed an additive effect to enhance the resistance to TSWV. Particularly, the chimeric transgenes with the triple combination of fragments, namely L/NSs/N or HpL/NSs/N (a hairpin construct), provided a higher degree of resistance (both 50%, with 7/14 lines assayed ) against TSWV. Our results indicate that the untranslatable NSs fragment is able to enhance the transgenic resistance conferred by the L conserved region. The better performance of L/NSs/N and HpL/NSs/N in transgenic N. benthamiana lines suggests their potential usefulness in generating high levels of enhanced transgenic resistance against serologically unrelated tospoviruses in agronomic crops. Chapter 3, 'Marker-free homozygous transgenic tobacco plants with resistance against tospoviruses of different serogroups and serotype'. In this study, to develop selectable marker-free transgenic plants resistant to different tospoviruses, we adopted a co-transformation system. Agrobacterium harboring pBI2T-based binary vectors, each with two T-DNAs separately carrying the selection marker gene (SMG) nptII and the construct with the aforementioned L fragment, alone or in combination with the NSs and N fragments , were used for transformation of Nicotiana benthamiana plants. Transgenic lines (12-18 for each construct) with high degrees of resistance against two serologically unrelated tospoviruses, Watermelon silver mottle virus (WSMoV) and Tomato spotted wilt virus (TSWV), were obtained. R1 progenies of 7 self-pollinated highly resistant transgenic R0 lines were further analyzed for the segregation of the SMG and the viral construct by PCR. When the individuals of the R3 progeny of the marker-free R2 homozygous transgenic plant were challenged with members of different tospoviruses of WSMoV, TSWV and Impatiens necrotic spot virus ( INSV ), resistance levels of 90-100% were noticed, indicating that the marker-free R2 and R3 homozygous transgenic N. benthamiana plants confer high degrees of broad resistance against tospoviruses of different serogroups and serotype. This approach is currently being extended to commercially important crops such as tomato, watermelon and melon. Supplementary information, 'Generation of transgenic melon plants with marker-free hairpin construct' . In this study, using the co-transformation method, Agrobacterium harboring pBI2T 1.5 binary vector with two T-DNAs carrying the nptII selection marker gene and the gene of interest HpL/NSs/N (constructed using conserved regions of tospoviral L (replicase), N (nucleocapsid) and NSs (gene silencing suppressor) genes of tospoviruses) was used for transformation of melon (cv.Silver light) plant. The regenerated marker-free transgenic 12 melon plant lines showed the presence of transgene and marker gene when checked through PCR. The marker-free transgenic melon plant lines will be challenged with different tospoviruses such as WSMoV and MYSV.
URI: http://hdl.handle.net/11455/89341
文章公開時間: 10000-01-01
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