Marker-free Transgenic Tobacco Lines with Concurrent Resistance to Whitefly-Borne DNA Geminivirus and Thrips-Borne RNA Tospovirus
| Autor: | Ching-Fu Yang, 楊景富 |
|---|---|
| Rok vydání: | 2014 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 102 Aphids-borne Potyvirus, thrips-borne Tospovirus and Whitefly-borne Begomovirus cause severe damages on crops worldwide. Incorporation of natural resistance gene into commercial cultivars is difficult by conventional breeding and requires many years to eliminate unfavorable characteristics, even if natural resistance source is available. Therefore, using genetic engineering to generate transgenic resistance provides an effective approach to control the diseases caused by plant viruses. In this study, we attempted to use post-transcriptional gene silencing (PTGS) or in combination of PTGS with transcriptional gene silencing (TGS) to generate transgenic resistance to Potyvirus and Tospovirus or concurrent resistance to a ssDNA Begomovirus and a Tospovirus on economic crops. This dissertation is divided into three chapters as described below. Chapter 1,“Literature review and research objectives”describes references are relevant to this study and the objectives of investigations. Chapter 2,“Generation of marker-free transgenic plants concurrently resistant to a DNA geminivirus and a RNA tospovirus” We tried to develop an RNA-directed DNA methylation (RdDM) transgenic system using a hairpin construct (int-hpIGR) of intergenic region (IGR) of Ageratum yellow vein virus (AYVV) residing in an intron of Arabidopsis to mediate TGS in tobacco plants. Furthermore, the int-hpIGR of AYVV IGR coupled with an untranslatable nucleocapsid protein (NP) fragment of Melon yellow spot virus (MYSV) was constructed to induce PTGS against MYSV. The resistant lines showed better resistance to AYVV, when challenged with MYSV, one of the AYVV-resistant lines was immune to MYSV infection, in which the transgenic NP transcript was silenced and the corresponding siRNAs were detected. Hence, we provide a novel way for concurrent control of noxious DNA and RNA viruses by TGS and PTGS with less biosafety concerns. Chapter 3,“Generation transgenic tobacco plants conferring resistance to a DNA geminivirus and broad-spectrum resistance to distinct RNA tospovirus species” Using the int-hpIGR of AYVV IGR coupled with an untranslatable antisense large (L) gene fragment containing the highly conserved RNA-dependent RNA polymerase (RdRp) region of WSMoV was created to induce PTGS against tospoviruses. When challenged with WSMoV and Tomato spotted wilt virus (TSWV), the TSWV-resistant lines were also highly resistant to WSMoV and AYVV. Hence, our novel approach provides a valuable way for concurrent control of ssDNA AYVV and broad-spectrum resistance to different distinct (-)ssRNA tospoviruses. Appendix I,“Generation of transgenic watermelon lines with resistance to two aphid-borne viruses and one thrips-borne tospovirus”thrips-borne Watermelon silver mottle virus (WSMoV), and aphid-borne Zucchini yellow mosaic virus (ZYMV) and Papaya ringspot virus type W (PRSV-W) are major limiting factors for watermelon production worldwide. An untranslatable construct containing truncated ZYMV coat protein (CP), PRSV-W CP and WSMoV nucleocapsid protein (NP) genes by genetic engineering to generation transgenic watermelon to control these viruses. A total of 13 transgenic watermelon lines were obtained. Among them, three transgenic lines conferred immunity to ZYMV and PRSV-W, of which the transgenic transcripts were not detected by northern blotting, but the accumulation of small interfering RNAs (siRNAs) were detected, indicating that the transgenic resistance was mediated by PTGS. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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