一种快速高效构建植物表达载体的方法

植物表达载体是将目的基因转化宿主细胞的媒介,载体构建是通过遗传转化方法开展基因功能鉴定与应用等研究的重要环节。以构建玉米谷氨酰胺合成酶基因(GS1)表达载体pCUbi1390-Ubi-GS1-35S-EPSPS为例,研究简单、通用、高效的构建载体的方法。该方法目的片段和载体不需要酶切产生互补的黏性末端,只需通过在目的基因PCR上下游引物的5’端设计与线性载体两端有15个碱基的同源序列,根据序列的同源性,将目的基因插入载体中,通过PCR程序实现DNA重组。结果表明,正确构建了设计的转化载体,该方法大大简化了载体构建的过程,也适用于任何一个基于单酶切位点把外源DNA重组插入目标序列。

A Rapid and High Efficient Method to Construct Plant Expression Vectors

The plant expression vector is the vehicle that takes target gene into and then expresses the target gene in the host cells. Therefore, expression vector construction is one of the key steps of theoretical and applied studies through genetic modification strategy. The demonstration of constructing GS1 gene, encoding a cytosolic glutamine synthetase, into pCUbi1390-Ubi-GS1-35S-EPSPS, a rapid, simple and high efficient constructing method had been introduced in this paper. After a vector linear step with single restriction digestion reaction, the homolog recombination could be executed through PCR reaction, in which the PCR primers for the target insert sequence were designed to share 15 bases of homology with the sequences at expecting site the end of the linearized vector. It was unnecessary to digest the target gene and vector to produce complementary cohesive ends in this method. The results showed the expecting pCUbi1390-Ubi-GS1-35S-EPSPS was successfully constructed. The procedures were largely simplified, and the method could be widely used in any recombination of insertion of any DNA sequence not very long at the single restriction site of any DNA sequence.