Flexible Tools for Gene Expression and Silencing in Tomato
Flexible Tools for Gene Expression and Silencing in Tomato
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DOI：
10.1104/pp.109.147546
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发表时间：
2009-12-01
期刊：
影响因子：
7.4
通讯作者：
Hilson, Pierre
中科院分区：
文献类型：
--
作者：
Fernandez, Ana I.;Viron, Nicolas;Hilson, Pierre
作为一个遗传平台，番茄（Solanum lycopersicum）受益于丰富的种质资源和易于培养和转化，使生物过程的分析不可能在其他模式物种的调查。为了便于组装一个开放的遗传工具箱，旨在研究茄科，我们发起了一个公开的基因操作工具的联合收集。我们重点研究了水果发育期间在指定时间窗口表达的启动子的特征，以调节感兴趣基因的表达或沉默。捕获五个启动子序列作为与通用MultiSite Gateway格式相容的入口克隆：来自番茄的PPC 2、PG、TPRP和IMA以及来自拟南芥（Arabidopsis thaliana）的CRC。用GUS基因、核定位的GUS-GFP报告基因和嵌合LhG 4转录因子进行相应的转录融合。在转基因番茄品系中证实了在果实发育期间和果实组织中启动子的活性。在这些启动子的控制下，产生了用于人工微小RNA（amiRNA）前体和发夹RNA的转录的新型Gateway目的载体，其中方案仅涉及Gateway BP和LR Clonase反应。在花椰菜花叶病毒35 S或PPC 2启动子下产生匹配amiRNA的转基因番茄品系中证明了内源八氢番茄红素去饱和酶基因的有效沉默。最后，利用的pOP/LhG 4双组分系统，我们发现，良好的特征化的花特异性拟南芥启动子驱动的模式通常与异源表达兼容的报告的表达。番茄品系和质粒将通过一个新的诺丁汉拟南芥股票中心服务单位专门为茄科资源分配。
As a genetic platform, tomato (Solanum lycopersicum) benefits from rich germplasm collections and ease of cultivation and transformation that enable the analysis of biological processes impossible to investigate in other model species. To facilitate the assembly of an open genetic toolbox designed to study Solanaceae, we initiated a joint collection of publicly available gene manipulation tools. We focused on the characterization of promoters expressed at defined time windows during fruit development, for the regulated expression or silencing of genes of interest. Five promoter sequences were captured as entry clones compatible with the versatile MultiSite Gateway format: PPC2, PG, TPRP, and IMA from tomato and CRC from Arabidopsis (Arabidopsis thaliana). Corresponding transcriptional fusions were made with the GUS gene, a nuclear-localized GUS-GFP reporter, and the chimeric LhG4 transcription factor. The activity of the promoters during fruit development and in fruit tissues was confirmed in transgenic tomato lines. Novel Gateway destination vectors were generated for the transcription of artificial microRNA (amiRNA) precursors and hairpin RNAs under the control of these promoters, with schemes only involving Gateway BP and LR Clonase reactions. Efficient silencing of the endogenous phytoene desaturase gene was demonstrated in transgenic tomato lines producing a matching amiRNA under the cauliflower mosaic virus 35S or PPC2 promoter. Lastly, taking advantage of the pOP/LhG4 two-component system, we found that well-characterized flower-specific Arabidopsis promoters drive the expression of reporters in patterns generally compatible with heterologous expression. Tomato lines and plasmids will be distributed through a new Nottingham Arabidopsis Stock Centre service unit dedicated to Solanaceae resources.