Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation
Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation
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DOI：
10.1263/jbb.104.34
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发表时间：
2007-07-01
影响因子：
2.8
通讯作者：
Kimura, Tetsuya
中科院分区：
文献类型：
--
作者：
Nakagawa, Tsuyoshi;Kurose, Takayuki;Kimura, Tetsuya
我们开发了一系列新的用于Gateway克隆的双元载体，以促进植物生物技术中的转基因实验。新系统Gateway Binary Vectors（pGWBs）实现了花椰菜花叶病毒（CaMV）35 S启动子的高效克隆、组成型表达和融合基因的构建。可用于该系统的报道基因是β-葡糖醛酸酶（GUS）、具有S65 T突变的合成绿色荧光蛋白（sGFP）、荧光素酶（LUC）、增强型黄色荧光蛋白（EYFP）和增强型青色荧光蛋白（ECFP）。可用的标签有！6xHis、FLAG、3xHA、4xMyc、10 xMyc、GST、T7表位和串联亲和纯化（TAP）。总共安排了13种报告基因或标签，几乎适用于N-和C-融合。pGWB可用于许多目的，如启动子：：报告基因分析，通过融合到报告基因或标签的蛋白质的表达观察亚细胞定位，以及通过共纯化和免疫检测实验分析蛋白质-蛋白质相互作用。用含有CaMV 35 S启动子驱动的潮霉素磷酸转移酶（HPT）基因作为第二选择标记的修饰的pBI 101构建pGWB。我们还构建了具有由胭脂碱合酶启动子驱动的标记HPT的pGWB。通过使用pGWB系统，标记蛋白的表达和GFP融合蛋白的定位容易地进行分析。此外，还用pGWB监测使用启动子的组织特异性和诱导型基因表达。预计pGWB系统将成为植物研究中质粒构建的有力工具。
We developed a new series of binary vectors useful for Gateway, cloning to facilitate transgenic experiments in plant biotechnology. The new system, Gateway Binary Vectors (pGWBs) realized efficient cloning, constitutive expression using the cauliflower mosaic virus (CaMV) 35S promoter and the construction of fusion genes by simple clonase reaction with an entry clone. The reporters employable in this system are beta-glucuronidase (GUS), synthetic green fluorescent protein with S65T mutation (sGFP), luciferase (LUC), enhanced yellow fluorescent protein (EYFP), and enhanced cyan fluorescent protein (ECFP). The tags available are! 6xHis, FLAG, 3xHA, 4xMyc, 10xMyc, GST, T7-epitope, and tandem affinity purification (TAP). In total, 13 kinds of reporter or tag were arranged and were almost applicable to both N- and C-fusions. The pGWBs could be used for many purposes, such as promoter:: reporter analysis, observation of subcellular localization by the expression of proteins fused to a reporter or tag, and analysis of protein-protein interaction by copurification and immunodetection experiments. The pGWBs were constructed with modified pBI101 containing a CaMV35S promoter-driven hygromcin phosphotransferase (HPT) gene as the second selection marker. We also constructed pGWBs with the marker HPT driven by the nopaline synthase promoter. By using the pGWB system, the expression of tagged proteins, and the localization of GFP-fused proteins were easily analyzed. Moreover, tissue-specific and inducible gene expression using a promoter was also monitored with pGWBs. It is expected that, the pGWB system will serve as a powerful tool for plasmid construction in plant research.