The Prokaryotic Expression Analysis of CBF Genes in the two Early Spring Ephemeral Plants
[Objective]To verify the reality of the sequence and explore the condition of the top expression of the CBF genes in the prokaryotic expression system for the purpose of functional verification of transcription factors.[Method]The two early spring ephemeral plants which have the ability to resist the low temperature stress are taken as the testing materials.The full length of CDS containing restriction enzyme site of Nco Ⅰ and Hind Ⅲ was harvested by PCR amplification.Prokaryotic expression vectors pET32a-LlCBF and pET32a-MsCBF were successfully constructed.The vector was transferred in Rosetta(DE3) to induce the expression of protein with different contents of IPTG at 15℃, 25℃, 30℃ and 37℃.Ultrasonic wave was used to break the efficiently expressed bacterium precipitation, then the supematant precipitate was centrifuged and boiled respectively, finally SDS-PAGE was used to analyze the solubility of protein.[Result]Prokaryotic expression results showed that efficient expression of LlCBF protein could be realized after being induced with 1 mmol/L IPTG for 16 h at 30℃ and efficient expression of MsCBF protein could be realized after induced with 2 mmol/L IPTG for 16 h at 30℃.[Conclusion]The optimal conditions were explored for the top expression of the target proteins in prokaryotic expression system.Solubility analysis showed that the fused protein mainly existed as inclusion bodies.
国家科技期刊平台
NSTL
万方数据
维普
