利用植物基因工程技術將外源基因轉入植物中，以植物做為生物反應器來大量生產所需蛋白質，具有成本低，可大量種植等優點。可食性疫苗、抗體、各種醫療或工業用蛋白質酵素等，皆可藉由基因工程方式利用植物來生產獲得，但由於以植物基因轉殖方式生產外源蛋白質具有表現量不高的情形，所以一直無法被廣泛地應用來大量生產蛋白質。
本研究欲藉由在構築轉基因時加入一段菸草鑲嵌病毒的5’-UTR來提升其後轉基因的蛋白質表現量。菸草鑲嵌病毒RNA 5’-UTR中有一段約68 base的序列，稱為Ω序列，可提升mRNA轉譯效率，而HSP101可以結合在Ω序列上，吸引轉譯因子聚集，進而形成轉譯起始複合物，提升蛋白質轉譯效率，其中HSP101扮演著轉譯活化子的角色。本實驗主要探討證實水稻的HSP101與菸草鑲嵌病毒Ω序列在植物體中是否同樣具有提升轉譯效率的能力。
本論文透過菸草原生質體和轉殖植株授粉雜交兩種層次來進一步證實菸草鑲嵌病毒的Ω序列，提升其後基因轉譯效率的能力是否與HSP101的參與有關。在菸草原生質體實驗部分，我們將帶有Ω序列與luciferase 報導基因（Ω-luciferase）的質體，透過電穿孔(electroporation)的方式，送入野生型或大量表現水稻HSP101之菸草原生質體(protoplast)中，偵測luciferase 表現量與HSP101表現量之相關性，進一步研究菸草鑲嵌病毒Ω序列與HSP101的關係。在轉殖植株授粉雜交部分，我們利用農桿菌轉殖法，將具有Ω-luciferase報導基因的DNA轉殖入菸草中，將三個Ω-luciferase的同質純系轉殖品系與六個可大量表現水稻HSP101之同質純系轉殖品系，與一個品系的家禽里奧病毒S1鞘蛋白轉殖菸草及未轉殖植株，進行授粉雜交實驗，之後測試雜交後子代其luciferase表現量並加以分析，實驗結果顯示，部分雜交後子代其luciferase表現量有提升，說明了水稻HSP101可提升具有Ω序列之報導基因（luciferase）表現量。在基因轉殖菸草植物體in planta層面，證實了在有大量水稻HSP101的存在下，可進一步提升Ω序列增進外源蛋白質轉譯效率之能力。

Transgenic plants have been generated as bioreactors to produce proteins. The advantages of plant bioreactors include low-cost, easily and extensively cultivation. Edible vaccine, antibodies, and the therapeutical or industrial proteins, all of them have been produced by plant genetic engineering. However, the protein expression level in transgenic plant are low, and this is the reason to limit the development of plant as bioreactors.

In previous study, the 68 base of TMV 5’-UTR called the Ω sequence, is able to promote the translational efficiency. The plant HSP101 can bind to the Ω sequence and recruit the translational initiation factors to enhance the translational activity of RNA transcripts. In this study we investigated the HSP101 overexpression lines in regulation of Ω-reporter gene expression in planta as well as in protoplast.

In protoplast aspect, we used the electroporation method to deliver the Ω sequence and luciferase gene to the tobacco protoplasts and study the relationship of reporter gene (luciferase) expression level with HSP101. In our study, Ω sequence enhance luciferase expression level about 7~12 fold in OsHSP101 overexpressed tobacco protoplasts.

In planta aspect, we had transformed tobacco with Ω-luciferase expression cassette by Agrobacterium-mediated transformation. Tree Ω-luciferase transgenic tobacco lines were obtained. We crossed the Ω-luciferase transgenic plants with HSP101 overexpression lines or wild type plants, and analyzed luciferase activity in the progenies. The results shown that OsHSP101 can enhance the reporter gene expression with Ω sequence in the 5’-UTR.

呂俊賢 (2004) 植物熱休克蛋白質HSP101的應用性研究. 國立成功大學生物科技研究所碩士論文

林惠茹 (2005) 大量表現熱休克蛋白質HSP101以提升菸草耐熱性之研究. 國立成功大學生物科技研究所碩士論文

黃亮愷 (2005) 以植物生產家禽里奧病毒之可食性疫苗 國立成功大學生物科技研究所碩士論文

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