大豆GmNAC8基因克隆与原核表达

Cloning and prokaryotic expression of GmNAC8 gene isolated from soybean

为了进一步明确GmNAC8基因的功能,设计带有Nde I和Xba I双酶切位点引物,从大豆根系cDNA中克隆到GmNAC8基因全长ORF(Open reading frame),编码蛋白质分子质量为37 000,理论等电点为6.43。将扩增片段克隆至原核表达载体pCZN1,构建重组质粒pCZN1-GmNAC8,经过酶切和PCR验证,并转化至大肠杆菌BL21(DE3)中,0.2 mmol/L IPTG诱导后,经SDS-PAGE电泳检测获得了目的蛋白质,该蛋白质主要以包涵体的形式存在,通过镍柱子纯化获得纯化蛋白质,然后利用His标签蛋白质作为抗体进行Western-blot分析,证明所纯化蛋白质为目的蛋白质。

In order to further define the function of GmNAC8 gene, the whole open reading frame of GmNAC8 was cloned from soybean roots with the primers carrying Nde I and Xba I restriction sites, encoding 283 amino acids with a calculated molecular mass of 37 000 and a theoretical pI of 6.43. The fragment was transformed into a prokaryotic expression vector pCZN1. After digestion and PCR verification, the recombinant plasmid pCZN1-GmNAC8 was introduced into Escherichia coli BL21 （DE3）. SDS-PAGE analysis showed that the fusion protein induced with 0.2 mmol/ L IPTG was successfully expressed in E. coli BL21（DE3）, and existed mainly in form of inclusion bodies. The specific reaction of Ni resin purified protein expressed in prokaryotic cells with His tag protein revealed by western-blot analysis confirmed the target protein.