Zhang Liu, Zhao Yang, Liang Haiyan, Li Xugang, Gallagher Kimberly L, Wu Shuang
College of Life Sciences, FAFU-UCR Joint Center and Fujian Provincial Key Laboratory of Haixia Applied Plant Systems Biology, Fujian Agriculture and Forestry University, Fuzhou, China.
College of Resource and Environment, Fujian Agriculture and Forestry University, Fuzhou, China.
Plant Methods. 2020 Jul 6;16:93. doi: 10.1186/s13007-020-00635-z. eCollection 2020.
Genetically encoded fluorescent proteins are often used to label proteins and study protein function and localization in vivo. Traditional cloning methods mediated by restriction digestion and ligation are time-consuming and sometimes difficult due to the lack of suitable restriction sites. Invitrogen developed the Gateway cloning system based on the site-specific DNA recombination, which allows for digestion-free cloning. Most gateway destination vectors available for use in plants employ either the 35S or ubiquitin promoters, which confer high-level, ubiquitous expression. There are far fewer options for moderate, cell-type specific expression.
Here we report on the construction of a Gateway-compatible cloning system (SWU vectors) to rapidly tag various proteins and express them in a cell-type specific manner in plants. We tested the SWU vectors using the HISTONE (H2B) coding sequence in stable transgenic plants.
The SWU vectors are a valuable tool for low cost, high efficiency functional analysis of proteins of interest in specific cell types in the root.
基因编码荧光蛋白常用于标记蛋白质，并在体内研究蛋白质功能和定位。传统的由限制性酶切和连接介导的克隆方法耗时，且由于缺乏合适的限制性酶切位点，有时操作困难。英杰公司基于位点特异性DNA重组技术开发了Gateway克隆系统，该系统允许无酶切克隆。大多数可用于植物的Gateway目的载体采用35S或泛素启动子，可实现高水平的普遍表达。而用于适度的、细胞类型特异性表达的选择则少得多。
本文报道了一种与Gateway兼容的克隆系统（SWU载体）的构建，该系统可快速标记各种蛋白质，并以细胞类型特异性方式在植物中表达。我们在稳定转基因植物中使用组蛋白（H2B）编码序列测试了SWU载体。
SWU载体是在根的特定细胞类型中对感兴趣的蛋白质进行低成本、高效功能分析的有价值工具。
