Gateway binary vectors with organelle-targeted fluorescent proteins for highly sensitive reporter assay in gene expression analysis of plants.
Gateway binary vectors with organelle-targeted fluorescent proteins for highly sensitive reporter assay in gene expression analysis of plants.
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DOI：
10.1016/j.jbiotec.2019.03.015
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发表时间：
2019-05
影响因子：
4.1
通讯作者：
Mst Momtaz Sultana;A. Dutta;Yuji Tanaka;Mostafa Aboulela;K. Nishimura;S. Sugiura;Tomoko Niwa;K. Maeo;Shino Goto-Yamada;Tetsuya Kimura;S. Ishiguro;S. Mano;T. Nakagawa
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文献类型：
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作者：
Mst Momtaz Sultana;A. Dutta;Yuji Tanaka;Mostafa Aboulela;K. Nishimura;S. Sugiura;Tomoko Niwa;K. Maeo;Shino Goto-Yamada;Tetsuya Kimura;S. Ishiguro;S. Mano;T. Nakagawa
荧光蛋白是生物科学领域中非常有价值的工具，特别是在蛋白质亚细胞定位分析和基因表达分析方面。与细胞器靶向信号的融合在特定细胞器中积累荧光蛋白，增加局部亮度，甚至在发出高背景自身荧光的组织中也能突出荧光蛋白的信号。由于这些优点，细胞器靶向荧光蛋白优选用于启动子：报告子测定，以详细定义基因的器官、组织或细胞特异性表达模式。在这项研究中，我们开发了一系列新的与Gateway克隆技术兼容的双载体，pgwb （attR1-attR2受体位点）和R4L1pGWB （attR4-attL1受体位点），携带细胞器靶向合成的S65T突变绿色荧光蛋白（sGFP） （ER-、核-、过氧化物酶体-和线粒体靶向的sGFP）和细胞器靶向标记红色荧光蛋白（TagRFP）（核-、过氧化物酶体-和线粒体靶向的TagRFP）。这些可用于通过与启动子进入克隆的LR反应制备启动子报告子结构，分别为attl1 -promoter- attl2（用于pgwb）或attl4 -promoter- attr1（用于r4l1pgwb）。利用花菜花叶病毒35S启动子驱动构建体的粒子轰击瞬时表达实验，通过与先前建立的细胞器靶向sgfp共定位分析，证实了新开发的细胞器靶向tagrfp的正确定位。在转基因拟南芥启动子实验中，以核和过氧化物酶体为目标的sGFPs比正常sGFPs检测到更强烈和明显的荧光信号。本研究开发的新型pgwb和r4l1pgwb具有很高的效率，可为植物基因表达分析中更准确地观察GFP和RFP信号提供有用的平台。
Fluorescent proteins are valuable tools in the bioscience field especially in subcellular localization analysis of proteins and expression analysis of genes. Fusion with organelle-targeting signal accumulates fluorescent proteins in specific organelles, increases local brightness, and highlights the signal of fluorescent proteins even in tissues emitting a high background of autofluorescence. For these advantages, organelle-targeted fluorescent proteins are preferably used for promoter:reporter assay to define organ-, tissue-, or cell-specific expression pattern of genes in detail. In this study, we have developed a new series of Gateway cloning technology-compatible binary vectors, pGWBs (attR1-attR2 acceptor sites) and R4L1pGWB (attR4-attL1 acceptor sites), carrying organelle-targeted synthetic green fluorescent protein with S65T mutation (sGFP) (ER-, nucleus-, peroxisome-, and mitochondria-targeted sGFP) and organelle-targeted tag red fluorescent protein (TagRFP) (nucleus-, peroxisome-, and mitochondria-targeted TagRFP). These are available for preparation of promoter:reporter constructs by an LR reaction with a promoter entry cloneattL1-promoter-attL2 (for pGWBs) orattL4-promoter-attR1 (for R4L1pGWBs), respectively. A transient expression experiment with particle bombardment using cauliflower mosaic virus 35S promoter-driven constructs has confirmed the correct localization of newly developed organelle-targeted TagRFPs by a co-localization analysis with the previously established organelle-targeted sGFPs. More intense and apparent fluorescence signals were detected by the nucleus- and peroxisome-targeted sGFPs than by the normal sGFPs in the promoter assay using transgenicArabidopsis thaliana. The new pGWBs and R4L1pGWBs developed here are highly efficient and may serve as useful platforms for more accurate observation of GFP and RFP signals in gene expression analyses of plants.