Prokaryotic Expresion Vector Construction And Protein Expression And Purification Of AtIAA1

Phytohormones are some trace organic compounds in vivo synthesized by plants, which play important roles in plant growth and development and response to environment signaling. Precise quantitative determination of phytohormone in trace materials is one of bottleneck in plant hormone researches since concentration of phytohormones in plants is very low and phytohormones are unstable. Some important proteins in phytohormones signaling show a highly specific affinity to plant hormones. This characeristic indicates the brighter prospect for application of them in phytohormones extraction and purification. For the goal, IAA1 were isolated from Arabidopsis through RT-PCR. Expression vectors pGEX-KG-IAA1 was constructed based on pGEX-KG containing GST tag. Then prokaryotic expression vectors were transformed into three E.coli strains named Rosetta, BL21 and Tuner. Expression and purification conditions of recombinant protein were developed. The results are followed:(1) Isolation and amplification of IAA1 gene. Using Arabidopsis thaliana as material, total RNA was extracted and then cDNA was synthetized through reverse transcription. Finally, the IAA1 sequences were acquired by PCR using the cDNA as the templates after designing specific primers.(2) Construction of IAA1 prokaryotic expression vector. PCR products were ligated with vector T and positive clones were screened through sequencing. Then the target gene ligated with pGEX-KG after double enzyme digestion and the right clone was screened through double enzyme digestion.(3) Deciding the best bacterial strain for IAA1 protein expression. Recombined pGEX-KG-IAAl expression vector was transformed into three different strains of expression bacterial cells and IPTG was used to induce the protein expression. Results showed that IAA1 protein was better expressed in Rosetta (DE3) strain.(4) Deciding the best inducting temperature for IAA1 protein. The Rosetta transformed pGEX-KG-IAAl were cultured in a series of inducing temperatures. The expression was analyzed by SDS-page electrophoresis. The results showed that the IAA1 protein was expressed at 25℃.(5) Deciding the best concentration of IPTG. The results showed that IAA1 protein was expressed in the 0.6mmol/L IPTG.