Activation domains for controlling plant gene expression using designed transcription factors
Activation domains for controlling plant gene expression using designed transcription factors
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DOI：
10.1111/pbi.12057
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发表时间：
2013-08-01
影响因子：
13.8
通讯作者：
Petolino, Joseph F.
中科院分区：
文献类型：
--
作者：
Li, Jianquan;Blue, Ryan;Petolino, Joseph F.
通过设计的转录因子进行靶向基因调控在精确的表型修饰和加速作物新性状的开发方面具有巨大的潜力。为此，设计的转录激活因子已经通过将转录激活结构域融合到DNA结合蛋白来构建。在这项研究中，从单纯疱疹病毒，VP16的转录激活因子，用于确定植物调节蛋白。从每种蛋白质中鉴定转录激活结构域，并与锌指DNA结合蛋白（ZFP）融合以产生设计的转录激活因子。此外，每个转录激活结构域内的特定序列被修饰以模拟直接与RNA聚合酶II核心转录因子相互作用的VP16接触基序。为了评估这些设计的转录激活因子，在酵母和烟草中构建了测试系统，所述测试系统包含由在转录起始位点上游含有ZFP结合位点的启动子驱动的报告基因。在酵母中，来自植物蛋白ERF 2和PTI4的转录结构域激活MEL 1报告基因表达至与VP16相似的水平，并且修饰的序列显示出更高水平的激活。在用来自ERF 2的转录激活因子稳定转化烟草报告系统后，GUS报告基因转录本积累等于或大于来自VP 16的那些。此外，与VP16和未修饰的ERF2序列相比，修饰的ERF2结构域显示出显著增强的转录激活。这些结果表明，可以发现能够促进转录激活的植物序列，并且当与DNA结合蛋白融合时，可以增强基因表达。
Targeted gene regulation via designed transcription factors has great potential for precise phenotypic modification and acceleration of novel crop trait development. To this end, designed transcriptional activators have been constructed by fusing transcriptional activation domains to DNA-binding proteins. In this study, a transcriptional activator from the herpes simplex virus, VP16, was used to identify plant regulatory proteins. Transcriptional activation domains were identified from each protein and fused with zinc finger DNA-binding proteins (ZFPs) to generate designed transcriptional activators. In addition, specific sequences within each transcriptional activation domain were modified to mimic the VP16 contact motif that interacts directly with RNA polymerase II core transcription factors. To evaluate these designed transcriptional activators, test systems were built in yeast and tobacco comprising reporter genes driven by promoters containing ZFP-binding sites upstream of the transcriptional start site. In yeast, transcriptional domains from the plant proteins ERF2 and PTI4 activated MEL1 reporter gene expression to levels similar to VP16 and the modified sequences displayed even greater levels of activation. Following stable transformation of the tobacco reporter system with transcriptional activators derived from ERF2, GUS reporter gene transcript accumulation was equal to or greater than those derived from VP16. Moreover, a modified ERF2 domain displayed significantly enhanced transcriptional activation compared with VP16 and with the unmodified ERF2 sequence. These results demonstrate that plant sequences capable of facilitating transcriptional activation can be found and, when fused to DNA-binding proteins, can enhance gene expression.