Expression And Prification Of JA Signaling Pathway Receptor COI1 In Arabidopsis

As a plant hormone, JA is required in plant growth and defences processes. It is well-known that JA can take part in roots growth, leaf senescence, pollen development, male fertility, trichome formation and inducing the expression of defence genes. In JA signaling pathway, COI1, the receptor of JA, can form a SCF complex with ASK1, Cullinl and Rbxl proteins, that interacts with JAZ repressors after combining with JA leading to ubiquitination and degradation of JAZ protein by 26S proteosome, then the JA-related transcriptional factors are released to control the gene expression in downstream. Identification of COI1 is an essential process to reveal JA signaling pathway, however, expression and purification of COI1 protein is extremely difficult. For these reasons, we constructed expression systems of COI1 and exproled the way which is better for its purification.First we built an E.coli expression system, but the results were not good enough, most of the proteins were in the sediment while there had little in supernatant. After that, we built an eucaryotic expression system. Though we can get some of the protien, it is not enough to adapt to our desire. So we transformed COIl with the proteins of which can interact with it. As a result, the ASK1 protein not only can improve COI1’s expression but also can make it more stable and do not influnce COI1’s bioactivity.On the basis of it, we fumbled the purification methods of COI1. As the protein contained two different tags,6*His tag and Flag tag, we compared the effects of purifications using the affine beads according to the tags, finally we choosed Ni-NTA Agarose. For the further purification, the AKTA system was applied to get more purify protieins. Using these methods, we also built some of the homologous proteins of AtCOI1, and we can get the same resuilts.