Fine-tuning levels of heterologous gene expression in plants by orthogonal variation of the untranslated regions of a nonreplicating transient expression system.
Fine-tuning levels of heterologous gene expression in plants by orthogonal variation of the untranslated regions of a nonreplicating transient expression system.
复制标题
DOI：
10.1111/pbi.12175
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发表时间：
2014-08
影响因子：
13.8
通讯作者：
Lomonossoff GP
中科院分区：
文献类型：
--
作者：
Meshcheriakova YA;Saxena P;Lomonossoff GP
基于豇豆花叶病毒 (CPMV) RNA-2 删除版本的瞬时表达系统（称为 CPMV-HT），其中待表达的序列位于修饰的 5' UTR 和 3' UTR 之间，已成功用于多种蛋白质（包括异源多聚体复合物）的植物表达。虽然之前的工作已经证明 5'UTR 序列的改变可以显着影响表达水平，但 3'UTR 在增强表达中的作用尚未确定。在这项工作中，我们使用表达载体 pEAQexpress-HT-GFP 编码的报告蛋白 GFP 检查了 CPMV RNA-2 3'UTR 中不同突变对表达水平的影响。结果表明，CPMV-HT 系统中 3' UTR 的存在对于实现最大表达水平非常重要。删除整个 3' UTR 可使表达量减少至其存在时的表达量的约 30%。研究发现3'UTR的125-165位核苷酸形成的Y形二级结构对其功能起着关键作用；破坏这种 Y 形结构的突变具有相当于删除整个 3' UTR 的效果。我们的结果表明，Y 形二级结构通过增强 mRNA 积累而不是直接影响 RNA 翻译来发挥作用。本文描述的工作表明 CPMV-HT 中的 5' 和 3' UTR 正交作用，并且引入它们的突变允许对蛋白质表达水平进行精细调节。
A transient expression system based on a deleted version of Cowpea mosaic virus (CPMV) RNA-2, termed CPMV-HT, in which the sequence to be expressed is positioned between a modified 5′ UTR and the 3′ UTR has been successfully used for the plant-based expression of a wide range of proteins, including heteromultimeric complexes. While previous work has demonstrated that alterations to the sequence of the 5′ UTR can dramatically influence expression levels, the role of the 3′ UTR in enhancing expression has not been determined. In this work, we have examined the effect of different mutations in the 3′UTR of CPMV RNA-2 on expression levels using the reporter protein GFP encoded by the expression vector, pEAQexpress-HT-GFP. The results showed that the presence of a 3′ UTR in the CPMV-HT system is important for achieving maximal expression levels. Removal of the entire 3′ UTR reduced expression to approximately 30% of that obtained in its presence. It was found that the Y-shaped secondary structure formed by nucleotides 125–165 of the 3′ UTR plays a key role in its function; mutations that disrupt this Y-shaped structure have an effect equivalent to the deletion of the entire 3′ UTR. Our results suggest that the Y-shaped secondary structure acts by enhancing mRNA accumulation rather than by having a direct effect on RNA translation. The work described in this paper shows that the 5′ and 3′ UTRs in CPMV-HT act orthogonally and that mutations introduced into them allow fine modulation of protein expression levels.
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通过复制和非复制载体在植物中快速瞬时生产产生高质量的功能性抗 HIV 抗体。
影响因子：
3.7
作者：
Sainsbury F;Sack M;Stadlmann J;Quendler H;Fischer R;Lomonossoff GP
通讯作者：
Lomonossoff GP
影响因子：
10.5
作者：
GALLIE, DR;WALBOT, V
通讯作者：
WALBOT, V
影响因子：
13.8
作者：
Thuenemann, Eva C.;Meyers, Ann E.;Lomonossoff, George P.
通讯作者：
Lomonossoff, George P.
影响因子：
4.8
作者：
Fabian, MR;White, KA
通讯作者：
White, KA
影响因子：
13.8
作者：
Sainsbury, Frank;Thuenemann, Eva C.;Lomonossoff, George P.
通讯作者：
Lomonossoff, George P.