Generation of transgenic plants expressing gene silencing suppressor HC-Pro and analyses of the effects of HC-Pro on gene expression using Bamboo mosaic virus-based vector
碩士 === 國立中興大學 === 生物科技學研究所 === 96 === One of the strategies to increase the expression of foreign gene in plants is to generate transgenic lines that express a replicating virus vector carrying a gene of interest. However, productivity in combination of transgenic and viral vector expression system...
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ndltd-TW-096NCHU51110162016-05-09T04:13:39Z http://ndltd.ncl.edu.tw/handle/30132412519406268384 Generation of transgenic plants expressing gene silencing suppressor HC-Pro and analyses of the effects of HC-Pro on gene expression using Bamboo mosaic virus-based vector 建立基因靜默抑制子HC-Pro基因轉殖植物及分析HC-Pro對應用竹嵌紋病毒載體表現外來基因之影響 Yu-Hsiu Cho 卓育秀 碩士 國立中興大學 生物科技學研究所 96 One of the strategies to increase the expression of foreign gene in plants is to generate transgenic lines that express a replicating virus vector carrying a gene of interest. However, productivity in combination of transgenic and viral vector expression system could be greatly reduced by post-transcriptional gene silencing (PTGS). This limitation could be effectively overcome by crossing stably-transformed plants expressing the suppressor of PTGS with a stably-transformed line carrying a replicating virus vector. In this study, we have generated transgenic N. benthamiana lines expressing HC-Pro gene by Agrobacterium-mediated transformation. At meantime, we also construct P1/HC-Pro mutant by inserting three additional amino acid into P1 region. To investigate the effects of HC-Pro on gene expression using Bamboo mosaic virus-based vector (BaMV vector), N. benthamiana plants were co-infiltrated with Agrobacteria harboring the plasmid clones of wild-type or mutant P1/HC-Pro and BaMV (pKB), or BaMV vector carrying GFP (pKBG) or VP1 epitopes of Foot-and-mouth disease virus (pKBVP1). At 7 and 14 days post infiltration (d.p.i.), plants co-infiltrated with pBIN-P1/HC and pKB or pKBG or pKBVP1 showed higher accumulation of viral coat protein or foreign proteins in both infiltrated and systemic leaves than those without pBIN-P1/HC; but there were no significant difference between the infiltration with and without mutant P1/HC-Pro. P1/HC-Pro transgenic lines 37-5-9 and 37-5-11 were agroinfiltrated with pKB、pKBG or pKBVP1, the virus and foreign gene accumulation of infiltrated and systemic leaves were higher than non-transgenic line. Furthermore, symptom-less transgenic N. benthamiana line B2B12-1 expressing full length BaMV-S genome was agroinfiltrated with wild-type or mutant P1/HC-Pro, the virus accumulation of infiltrated leaves was higher than non-infiltrated one; but there was no significant difference between systemic leaves of the plants infiltrated either with wild-type or mutant P1/HC-Pro or without. We hypothesize that HC-Pro could suppress local and systemic gene silencing in plants and improved the replication of virus and the expression of foreign proteins, but in B2B12-1 line, P1/HC-Pro could just improve the virus accumulation in infiltrated leaves by suppressing local gene silencing. Yau-Heiu Hsu 徐堯煇 2008 學位論文 ; thesis 82 zh-TW |
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碩士 === 國立中興大學 === 生物科技學研究所 === 96 === One of the strategies to increase the expression of foreign gene in plants is to generate transgenic lines that express a replicating virus vector carrying a gene of interest. However, productivity in combination of transgenic and viral vector expression system could be greatly reduced by post-transcriptional gene silencing (PTGS). This limitation could be effectively overcome by crossing stably-transformed plants expressing the suppressor of PTGS with a stably-transformed line carrying a replicating virus vector. In this study, we have generated transgenic N. benthamiana lines expressing HC-Pro gene by Agrobacterium-mediated transformation. At meantime, we also construct P1/HC-Pro mutant by inserting three additional amino acid into P1 region. To investigate the effects of HC-Pro on gene expression using Bamboo mosaic virus-based vector (BaMV vector), N. benthamiana plants were co-infiltrated with Agrobacteria harboring the plasmid clones of wild-type or mutant P1/HC-Pro and BaMV (pKB), or BaMV vector carrying GFP (pKBG) or VP1 epitopes of Foot-and-mouth disease virus (pKBVP1). At 7 and 14 days post infiltration (d.p.i.), plants co-infiltrated with pBIN-P1/HC and pKB or pKBG or pKBVP1 showed higher accumulation of viral coat protein or foreign proteins in both infiltrated and systemic leaves than those without pBIN-P1/HC; but there were no significant difference between the infiltration with and without mutant P1/HC-Pro. P1/HC-Pro transgenic lines 37-5-9 and 37-5-11 were agroinfiltrated with pKB、pKBG or pKBVP1, the virus and foreign gene accumulation of infiltrated and systemic leaves were higher than non-transgenic line. Furthermore, symptom-less transgenic N. benthamiana line B2B12-1 expressing full length BaMV-S genome was agroinfiltrated with wild-type or mutant P1/HC-Pro, the virus accumulation of infiltrated leaves was higher than non-infiltrated one; but there was no significant difference between systemic leaves of the plants infiltrated either with wild-type or mutant P1/HC-Pro or without. We hypothesize that HC-Pro could suppress local and systemic gene silencing in plants and improved the replication of virus and the expression of foreign proteins, but in B2B12-1 line, P1/HC-Pro could just improve the virus accumulation in infiltrated leaves by suppressing local gene silencing.
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author2 |
Yau-Heiu Hsu |
author_facet |
Yau-Heiu Hsu Yu-Hsiu Cho 卓育秀 |
author |
Yu-Hsiu Cho 卓育秀 |
spellingShingle |
Yu-Hsiu Cho 卓育秀 Generation of transgenic plants expressing gene silencing suppressor HC-Pro and analyses of the effects of HC-Pro on gene expression using Bamboo mosaic virus-based vector |
author_sort |
Yu-Hsiu Cho |
title |
Generation of transgenic plants expressing gene silencing suppressor HC-Pro and analyses of the effects of HC-Pro on gene expression using Bamboo mosaic virus-based vector |
title_short |
Generation of transgenic plants expressing gene silencing suppressor HC-Pro and analyses of the effects of HC-Pro on gene expression using Bamboo mosaic virus-based vector |
title_full |
Generation of transgenic plants expressing gene silencing suppressor HC-Pro and analyses of the effects of HC-Pro on gene expression using Bamboo mosaic virus-based vector |
title_fullStr |
Generation of transgenic plants expressing gene silencing suppressor HC-Pro and analyses of the effects of HC-Pro on gene expression using Bamboo mosaic virus-based vector |
title_full_unstemmed |
Generation of transgenic plants expressing gene silencing suppressor HC-Pro and analyses of the effects of HC-Pro on gene expression using Bamboo mosaic virus-based vector |
title_sort |
generation of transgenic plants expressing gene silencing suppressor hc-pro and analyses of the effects of hc-pro on gene expression using bamboo mosaic virus-based vector |
publishDate |
2008 |
url |
http://ndltd.ncl.edu.tw/handle/30132412519406268384 |
work_keys_str_mv |
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