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題名 | Construction and Evaluation of Transgenic Tobacco Plants Expressing the Coat Protein Gene of Papaya Ringspot Virus with Different Translation Leaders=具備不同前導序列木瓜輪點病毒鞘蛋白基因之轉基因菸草之構築及其抗病分析 |
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作者姓名(中文) | 鄭櫻慧; 葉錫東; | 書刊名 | Botanical Bulletin of Academia Sinica |
卷期 | 41:1 2000.01[民89.01] |
頁次 | 頁1-10 |
分類號 | 433.4 |
關鍵詞 | 木瓜輪點病毒; 鞘蛋白基因; 馬鈴薯Y群病毒; 轉譯前導序列; Coat protein gene; Papaya ringspot virus; Potyvirus; Translation leader; |
語文 | 英文(English) |
中文摘要 | 木瓜輪點病毒YK系統為分離自臺南縣永康鄉之嚴重嵌紋型病毒系統,其基因體已被選殖及解序完成。此病毒之鞘蛋白基因及3'非轉譯區利用定點突變後接於gus基因前導序列,構築成質體pGGCP。質體pG5'CP為pGGCP上之gus基因前導序列被PRSV之5'片段(nt 1-347,此片段含P�絨J白N端之9kDa部分)取代後之載體。pGGCP和pG5'CP在生體外轉錄及轉譯分析中,分別產生可與PRSV之抗血清反應之36kDa與45kDa蛋白,而以前者產生之量較後者為多。此二構築之PRSV鞘蛋白基因分別利用農桿菌媒介轉殖至菸草植株(Nicotiana tabacum L. Havana 423),轉基因菸草利用西方漬染法可偵測到表現良好之36kDa蛋白或較微量之 45kDa蛋白。利用聚合連鎖反應可偵測到CP基因存在於菸草之中。分析八個轉基因株系之子代,其分離比顯示CP基因均以單一顯性基因形式存在菸草染色體。四個含有gus基因前導序列及四個含有PRSV 5'前導序列之鞘蛋白轉基因菸草之R0及R�等@代植株經以TEV、PVY及PepMoV挑戰接種,轉基因植株明顯延緩病徵之表現及減輕病徵之嚴重程度,但含有gus基因前導序列之轉基因菸草之抗性優於PRSV 5'前導序列之轉基因菸草,此顯示PRSV之前導序列並未能增加抗病性。 |
英文摘要 | Papaya ringspot virus (PRSV) YK isolate used in this study is a local mosaic strain isolated from Yung-Kang, Tainan, and its genome has been cloned and completely sequenced. A NcoI site before the coat protein (CP) reading frame of PRSV YK was generated by oligonucleotide-directed mutagenesis, and then the CP reading frame with the 3' noncoding region of PRSV YK was ligated with the gus leader sequence from the pGEM vector to create the construct pGGCP. To express the CP with a homologous viral translation sequence, the gus leader was replaced by the cDNA sequence corresponding to the 5' region (nt 1-347) of PRSV genome to generate a protein containing 9 kDa polypeptide of PRSV P1 protein fused with the CP, and the construct was designated as pG5'CP. In vitro translation from the transcripts derived from pGGCP and pG5'CP generated protein probucts of 36 kDa and 45 kDa, respectively. Both proteins reacted with the antiserum to PRSV CP, and the level of 36 kDa protein was higher than that of 45 kDa protein. The CP reading frame with the gus or PRSV 5' leaders was individually subcloned into a Ti binary vector. Transgenic tobacco plants (Nicotiana tabacum L. Havana 423) expressing the PRSV CP gene with the gus leader (GCP lines) or with the viral leader (5'CP lines) were obtained by Agrobacterium-mediated transformation. When the transgenic lines wer analyzed by western blotting, the protein products of 36 kDa and 45 kDa reacting to PRSV CP antiserum were detected in the GCP lines and 5'CP lines, respectively. The presence of the CP gene in the transgenic tobacco was also confirmed by polymerase chain reaction (PCR) using primers specific to the CP gene. Analysis of segregation ratios in the R�� plants of four GCP lines and four 5'CP lines indicated that the CP gene in all of them was nuclearly inherited as a single dominant trait. R0 and R�� plants of the four GCP lines and four 5'CP lines were inoculated with tobacco etch virus (TEV), potato virus Y (PVY), or pepper mottle virus (PepMoV). The transgenic lines showed significant delay in symptom development and the severity of symptoms was attenuated. The GCP lines expressing the PRSV CP gene by the gus leader accumulated high levels of CP and showed higher degrees of resistance than the 5'CP lines with the PRSV 5' leader. Our results indicate that the homologous viral leader does not enhance CP expression either in vitro or in vivo, nor does it provide better resistance in transgenic tobacco. |
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