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標題: 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
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
作者: 卓育秀
Cho, Yu-Hsiu
關鍵字: BaMV
gene silencing suppressor
transgenic N. benthamiana
出版社: 生物科技學研究所
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摘要: 以攜帶外源基因的病毒複製體建立基因轉殖植物,是現今表現外源蛋白的一種方式;然而植物體內後轉錄基因靜默機制 (post-transcriptional gene silencing) 的影響,使得此策略結果不如預期。前人研究中顯示,將帶有 HC-Pro 突變基因的轉殖菸草與攜帶外源基因的病毒載體轉殖菸草進行雜交的子代,其外源蛋白表現量明顯增加。在本實驗中,利用馬鈴薯Y屬病毒 (Potyvirus) 菸草蝕刻病毒 (Tobacco etch virus)的 P1/HC-Pro 基因片段,其中含有基因靜默抑制子 (gene silencing suppressor) HC-Pro 基因,以農桿菌轉殖方式建立 HC-Pro 基因轉殖菸草 (Nicotiana benthamiana)。此外進行了在 P1 部分插入 3 個胺基酸構築的 P1/HC-Pro 基因突變株。以農桿菌注射方式將含有野生型或突變型 P1/HC-Pro 構築的農桿菌,分別與含有竹嵌紋病毒 (Bamboo mosaic virus, BaMV),攜帶綠螢光蛋白(Green florescent protein,GFP) 或口蹄疫病毒(Foot-and-mouth disease virus) VP1 蛋白抗原片段序列的 BaMV 嵌合株 (pKB、pKBG 及 pKBVP1) 構築的農桿菌共同感染菸草,分析接種葉及系統葉,發現與含有野生型 P1/HC-Pro 構築的農桿菌共同感染的葉片,病毒累積量及外源蛋白表現量均較單獨接種量高,其他突變株的影響則無明顯差異。將含有 pKB、pKBG 及 pKBVP1 構築的農桿菌接種於 P1/HC-Pro 基因轉殖菸草,在轉殖品系 37-5-9 與 37-5-11 中接種葉和系統葉的病毒累積量及外源蛋白表現量均比非轉殖菸草中高。利用含有野生型或突變型 P1/HC-Pro 構築的農桿菌接種無病徵表現的竹嵌紋病毒全長基因轉殖株 B2B12-1,於十天接種葉病毒累積均高於未接種的基因轉殖株,但在系統性葉片卻無明顯差異,推論對於基因轉殖株 B2B12-1 而言,P1/HC-Pro 僅能原位抑制接種葉而無法抑制系統葉中基因靜默的發生。以上結果推測野生型 P1/ HC-Pro 藉由抑制基因靜默作用的影響,增加病毒在植物體中的複製並提高外源蛋白產量;但在突變型 P1/HC-Pro 中,因 P1 蛋白酵素活性被破壞或是 HC-Pro 蛋白 C 端缺失而影響了 HC-Pro 作為基因靜默抑制子的功能。
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.
其他識別: U0005-2307200819472700
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