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

• Main Authors: Yu-Hsiu Cho, 卓育秀
• Other Authors: Yau-Heiu Hsu
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/30132412519406268384

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.