摘要
本文通过使用USER酶克隆技术,构建一系列用于镰刀菌基因敲除和荧光融合蛋白表达,并且操作简单快速的通用载体。在hph基因两侧引入USER酶特异位点,构建了基因敲除载体pKH-KO,可以一步在hph的两侧同时插入上下游同源重组片段;在gfp基因5′端引入USER位点构建了荧光融合蛋白表达载体pKHGFPE和pKNGFPE。此方法构建的载体在插入目的片段时均不用考虑酶切位点,极大地方便了试验设计,并且步骤简单,操作简便,节约大量时间。在大规模进行基因敲除以及相关蛋白定位等基因功能研究上具有广阔的应用前景。
A series of effective universal vectors for gene knockout and fluorescent expression in Fusarium graminearum were constructed by a USER enzyme based technique.The gene knockout vector pKH-KO were constructed by insertion of two USER cloning sites （UCS） into the two sites of hph gene and the fluorescent expression vectors pKHGFPE and pKNGFPE were constructed by insertion of UCS into the 5＇ side of gfp gene.This method has many advantages,such as a simple experimental design,few procedures,user convenience and less time-consuming,which has a broad application prospect in gene function research such as large scale gene-knockout and fluorescent protein localization.
出处
《植物保护》
CAS
CSCD
北大核心
2014年第3期106-112,共7页
Plant Protection
基金
国家自然科学基金项目(31201477)
国家国际合作专项(2013DFG31930)
公益性行业(农业)科研专项(201303016)
关键词
镰刀菌
基因功能
载体构建
USER酶
Fusarium graminearum
gene function
vector construction
USER enzyme
