Development of plant vectors carrying translational enhancers

携带翻译增强子的植物载体的开发

• 批准号: 09554054
• 负责人: OBOKATA Junichi
• 金额: $ 6.4万
• 依托单位: Nagoya University (1999)Hokkaido University (1997-1998)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09554054/
• 关键词: Protein synthesis; translational enhancer; Plant vector; 5' untranslated region; 3' untranslated region; 5' cap; in vitro evolution; mRNA; invitro; 分子進化; 高等植物; 高発現; 5′非翻訳領域; タバコ; psaDb; ポリ(U)

Translational enhancers are mRNA motifs that enhance translational efficiency of encoded proteins. In advance of this research project, we had found two translational enhancer elements of plants from the studies with transgenic tobacco. Hence, the initial plan of this project was to apply those motifs in establishing new plant expression vectors of high translation efficiency. However, halfway the research period, we found that the translational enhancer effects of above motifs greatly alter according to the flanking sequences including those within the 5' UTR and in the coding region. Thus, it became unlikely to establish reliable expression vectors using above motifs. Our ultimate goal was to establish a reliable method to improve translation efficiency in plant gene engineering. We altered the course of this project in 1999 for pursuing the method that enables us to obtain translational enhancers against any given mRNAs. After two years of trial and error, we finally created a novel method fulfilling these requirements. This method is based on the in vitro evolution theory and PCR technology, and is the first reliable method to isolate translational enhancers.

翻译增强子是增强编码蛋白质翻译效率的 mRNA 基序。在这个研究项目之前，我们从转基因烟草的研究中发现了两种植物的翻译增强子元件。因此，该项目的初步计划是将这些基序应用于建立新的高翻译效率的植物表达载体。然而，研究进行到一半时，我们发现上述基序的翻译增强子效应根据侧翼序列（包括5'UTR内和编码区的序列）而发生很大变化。因此，使用上述基序建立可靠的表达载体变得不太可能。我们的最终目标是建立一种可靠的方法来提高植物基因工程的翻译效率。 1999 年，我们改变了该项目的进程，以寻求能够获得针对任何给定 mRNA 的翻译增强子的方法。经过两年的反复试验，我们终于创建了一种满足这些要求的新颖方法。该方法基于体外进化理论和PCR技术，是第一个可靠的分离翻译增强子的方法。

项目成果

Nakamura, M., T. Tsunoda and J. Obokata: "Photosynthesis nuclear genes generally lack TATA-box : a tobacco photosystem I gene respond to light through an initiator"Plant J.. (in press).

Nakamura, M.、T. Tsunoda 和 J. Obokata：“光合作用核基因通常缺乏 TATA-box：烟草光系统 I 基因通过引发剂对光做出反应”Plant J..（出版中）。

Miyamoto, T., T. Nakamura, I. Nagao, and J. Obokata: "Quantitative analysis of the transiently expressed mRNA level in particle bombarded tobacco seedlings"Plant Mol. Biol. Rep.. 18-2. 101-107 (2000)

Miyamoto, T.、T. Nakamura、I. Nagao 和 J. Obokata：“粒子轰击烟草幼苗中瞬时表达 mRNA 水平的定量分析”Plant Mol。

Nakamura, M. and J. Obokata: "Analysis of the light-responsive elements of tobacco psaDb gene using a transient expression system"Photosynthesis : Mechanisms and Effects (ed. By G. Garab). IV. 2797-2800 (1998)

Nakamura, M. 和 J. Obokata：“使用瞬时表达系统分析烟草 psaDb 基因的光响应元件”光合作用：机制和效果（G. Garab 编辑）。

Nakamura M.T.Tsunoda and J.Obokata: "Photosynthesis nuclear genes generally lack TATA-box: a tobacco photosystem I gene responds to light through an initiator"Plant Journal. (発表予定).

Nakamura M.T.Tsunoda 和 J.Obokata：“光合作用核基因通常缺乏 TATA-box：烟草光系统 I 基因通过引发剂对光作出反应”《植物杂志》（待出版）。

Yamamoto Y.Y.,Y.Kondo,A.Kato,H.Tsuji and J.Obokata: "Light responsive elements of tobacco PSI-D gene are located both upstream and within the transcribed regionl"Plant Journal. 12・2. 255-265 (1997)

Yamamoto Y.Y.、Y.Kondo、A.Kato、H.Tsuji 和 J.Obokata：“烟草 PSI-D 基因的光响应元件位于转录区域的上游和内部”Plant Journal 12・2（ 1997）