Siligato Riccardo, Wang Xin, Yadav Shri Ram, Lehesranta Satu, Ma Guojie, Ursache Robertas, Sevilem Iris, Zhang Jing, Gorte Maartje, Prasad Kalika, Wrzaczek Michael, Heidstra Renze, Murphy Angus, Scheres Ben, Mähönen Ari Pekka
Institute of Biotechnology (R.S., X.W., S.R.Y., S.L., R.U., I.S., J.Z., A.P.M.) and Department of Biosciences, Viikki Plant Science Centre (R.S., X.W., S.R.Y., S.L., R.U., I.S., J.Z., M.W., A.P.M.), University of Helsinki, Helsinki 00014, Finland;Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, Indiana 47907 (A.M., G.M.);Molecular Genetics, Department of Biology, Utrecht University, Utrecht 3584 CH, The Netherlands (M.G., R.H., B.S.);School of Biology, Indian Institute of Science Education and Research, Thiruvananthapuram 695016, Kerala, India (K.P.);Plant Developmental Biology, Wageningen University, Wageningen 6708 PB, The Netherlands (R.H., B.S.); andDepartment of Plant Science and Landscape Architecture, University of Maryland, College Park, Maryland 20742 (A.M.).
Institute of Biotechnology (R.S., X.W., S.R.Y., S.L., R.U., I.S., J.Z., A.P.M.) and Department of Biosciences, Viikki Plant Science Centre (R.S., X.W., S.R.Y., S.L., R.U., I.S., J.Z., M.W., A.P.M.), University of Helsinki, Helsinki 00014, Finland;Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, Indiana 47907 (A.M., G.M.);Molecular Genetics, Department of Biology, Utrecht University, Utrecht 3584 CH, The Netherlands (M.G., R.H., B.S.);School of Biology, Indian Institute of Science Education and Research, Thiruvananthapuram 695016, Kerala, India (K.P.);Plant Developmental Biology, Wageningen University, Wageningen 6708 PB, The Netherlands (R.H., B.S.); andDepartment of Plant Science and Landscape Architecture, University of Maryland, College Park, Maryland 20742 (A.M.)
Plant Physiol. 2016 Feb;170(2):627-41. doi: 10.1104/pp.15.01246. Epub 2015 Dec 7.
A powerful method to study gene function is expression or overexpression in an inducible, cell type-specific system followed by observation of consequent phenotypic changes and visualization of linked reporters in the target tissue. Multiple inducible gene overexpression systems have been developed for plants, but very few of these combine plant selection markers, control of expression domains, access to multiple promoters and protein fusion reporters, chemical induction, and high-throughput cloning capabilities. Here, we introduce a MultiSite Gateway-compatible inducible system for Arabidopsis (Arabidopsis thaliana) plants that provides the capability to generate such constructs in a single cloning step. The system is based on the tightly controlled, estrogen-inducible XVE system. We demonstrate that the transformants generated with this system exhibit the expected cell type-specific expression, similar to what is observed with constitutively expressed native promoters. With this new system, cloning of inducible constructs is no longer limited to a few special cases but can be used as a standard approach when gene function is studied. In addition, we present a set of entry clones consisting of histochemical and fluorescent reporter variants designed for gene and promoter expression studies.
研究基因功能的一种强大方法是在可诱导的细胞类型特异性系统中进行表达或过表达,随后观察由此产生的表型变化,并在靶组织中对相关报告基因进行可视化。已经为植物开发了多种可诱导基因过表达系统,但其中很少有系统能同时具备植物选择标记、表达域控制、多种启动子和蛋白质融合报告基因的使用、化学诱导以及高通量克隆能力。在此,我们介绍一种与多位点Gateway兼容的拟南芥植物诱导系统,该系统能够在单个克隆步骤中生成此类构建体。该系统基于严格控制的雌激素诱导型XVE系统。我们证明,用该系统产生的转化体表现出预期的细胞类型特异性表达,类似于组成型表达的天然启动子所观察到的情况。有了这个新系统,诱导型构建体的克隆不再局限于少数特殊情况,而是可作为研究基因功能时的标准方法。此外,我们还展示了一组入门克隆,这些克隆由用于基因和启动子表达研究的组织化学和荧光报告基因变体组成。