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马铃薯 SBgLR 基因 5'非翻译区有助于花粉特异性表达。

The 5' untranslated region of potato SBgLR gene contributes to pollen-specific expression.

机构信息

State Key Laboratory of Agricultural Biotechnology, College of Biological Sciences, China Agricultural University, No. 2 Yuanmingyuan West Road, Haidian District, 100193, Beijing, China.

Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, 100193, Beijing, China.

出版信息

Planta. 2017 Sep;246(3):389-403. doi: 10.1007/s00425-017-2695-7. Epub 2017 Apr 25.

Abstract

The 5'UTR of SBgLR enhances gene expression by regulating both its transcription and translation. SBgLR (Solanum tuberosum genomic lysine rich) is a pollen-specific gene in Solanum tuberosum that encodes a microtubule-associated protein. The region from -85 to +180 (transcription start site at +1) was determined to be critical for specific expression in pollen grains. Transient and stable expression assays showed that the 5'UTR (from +1 to +184) enhanced gene expression in all detected tissues of transgenic tobacco. Deletion analysis demonstrated that the secondary structure of the 5'UTR had no effect on pollen-specific SBgLR expression, while the region from +31 to +60 was crucial. Further investigation indicated that mRNA expression was slightly decreased when the +31 to +60 region was deleted, but the mRNA decay rate remained unchanged. Mutation analysis also confirmed that the pollen-specific element TTTCT, located at +37, played an important role in pollen-specific expression. Using yeast one-hybrid screening, we isolated a DNA-binding with one finger (Dof) protein gene (StDof23) and an AT-hook motif nuclear-localized (AHL) protein gene (StAHL) from potato pollen. Further investigation indicated that StDof23 interacted with and positively regulated the +31 to +60 region; moreover, StAHL interacted with and negatively regulated the -49 to +60 region. These results demonstrate that the 5'UTR not only enhanced gene expression but also altered the tissue-specific expression pattern by regulating both transcription and translation.

摘要

SBgLR 的 5'UTR 通过调节转录和翻译来增强基因表达。SBgLR(马铃薯基因组富含赖氨酸)是马铃薯花粉特异性基因,编码微管相关蛋白。从 -85 到 +180(转录起始位点为+1)的区域被确定对花粉粒中的特异性表达至关重要。瞬时和稳定表达分析表明,5'UTR(从+1 到+184)增强了转基因烟草所有检测组织中的基因表达。缺失分析表明,5'UTR 的二级结构对花粉特异性 SBgLR 表达没有影响,而+31 到+60 区域是关键的。进一步的研究表明,当删除+31 到+60 区域时,mRNA 表达略有下降,但 mRNA 衰减率保持不变。突变分析也证实,位于+37 的花粉特异性元件 TTTCT 在花粉特异性表达中起重要作用。通过酵母单杂交筛选,我们从马铃薯花粉中分离出一个具有一个手指的 DNA 结合蛋白(StDof23)和一个 AT 钩模体核定位(AHL)蛋白基因(StAHL)。进一步的研究表明,StDof23 与+31 到+60 区域相互作用并正向调节该区域;此外,StAHL 与-49 到+60 区域相互作用并负向调节该区域。这些结果表明,5'UTR 不仅增强了基因表达,而且通过调节转录和翻译改变了组织特异性表达模式。

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