拟南芥SHORT-ROOT基因稀有密码子分析及其在原核细胞中的表达和纯化

Rare Codon Analysis, Prokaryotic Expression and Purification of SHORT-ROOT Gene from Arabidopsis thaliana

目的:在大肠杆菌中重组表达拟南芥SHORT-ROOT(SHR)蛋白并纯化。方法:将SHR基因编码区克隆至p GEX-4T-2表达载体,构建重组质粒p GEX-4T-2-SHR并转化大肠杆菌Rosetta(DE3),表达产物经谷胱甘肽亲和层析凝胶4B分离纯化,SDS-PAGE分析和Western印迹鉴定。结果:SHR融合蛋白在大肠杆菌Rosetta(DE3)中获得表达,亲和纯化后,SDS-PAGE显示相对分子质量为预期的86×103,且经Western印迹确证。结论:获得了拟南芥SHR融合蛋白,为进一步研究其生物学功能奠定了基础。

Objective： To express and purify the recombinant SHORT-ROOT（SHR） protein of Arabidopsis thali- ana in Escherichia coli Rosseta（DE3）. Methods： The SHR open reading frame（ORF） was cloned into the pGEX- 4T-2 vector, the recombinant plasmid pGEX-4T-2-SHR was verified by nucleotide sequencing, and transformed in- to E.coli Rosseta（DE3）. The expressed products were purified by glutathione Sepharose 4B beads, detected by means of SDS-PAGE and Western blotting analysis. Results： The fusion SHR protein was expressed in E.coli Ro- setta（DE3）. After glutathione Sepharose 4B beads purification, a single 86 kD band was observed in the SDS- PAGE gel. The SHR-fused recombinant protein was confirmed by Western blotting analysis. Conclusion： The fu- sion SHR protein of A.thaliana was successfully expressed and purified, which lays the foundation of further study on its biological function.