Study On The Interaction Between Rice Ethylene Receptors OsERS1and OsERS2

Ethylene and ethylene signal way mediated by ethylene receptors play an important role in process of plant growth and development, regulate and response to environmental stress. At present, with the method of molecular biology and genetics means, a linear transcription regulation signal way has established, which percept the ethylene signal in the membrane and response to ethylene signal in the cell nucleus through researching Arabidopsis mutants about response to ethylene. Arabidopsis ethylene receptors can form any homologous or heterologous complex in endoplasmic reticulum. They exist in the forms of highly ordered noncovalent interaction or disulfide linkage. Experiments have proved the GAF domain play an important role in mediating the dimerization of ethylene receptors and the interactions among ethylene receptors.Basing the importance of the interactions among ethylene receptors and GAF domain in Arabidopsis thaliana, in this study, by means of analyzing the structure domains and protein homology between rice and Arabidopsis. The interactions between rice ethylene receptors OsERSland OsERS2are examined by the GAL4screen system in yeast two-hybrid system, using the reportor genes ADE2, HIS3and MEL1. The mutants of different domains (transmembrane domain structure, GAF and HisKA domain) of OsERS2are constructed, to future identify the specific domain that OsERS2interacts with OsERSl using yeast two-hybrid system. The corresponding express vectors of OsERSland OsERS2are constructed in GST pull-down, it prepares for future research to the subsequent experiment in vitro. This research helps to understand the biochemical mechanism of rice ethylene signal way and signal output, it also lays the foundation on research of function of rice ethylene receptors and the signal transmission between them.The initial research results are as follows:(1) Two yeast expression vectors of rice ethylene receptors, including pGBKT7-OsERS1and pGBKT7-OsERS2, are successfully constructed, which are bait plasmids. Analysis of the self activation of fusion proteins indicate that pGBKT7- OsERS1, pGBKT7-OsERS2, pGADT7-OsERS1and pGADT7-OsERS2do not have the self activation using yeast two-hybrid system. Using the nutrition defects medium SD/-Trp-Leu-His-Ade and the X-α-Gal color experiment, it proves that the bacterial colonies grow while they appear blue the same as the positive control, which indicates that OsERS2interact with OsERS1strongly.(2) To further identify the specific domains of OsERS2that interact with OsERS1, the analysis of SMART software showes that OsERS2contained three domains including transmembrane domain, GAF domain and HisKA domain. According to this information, three yeast expression vectors containing different domains of OsERS2are constructed:pGBKT7-OsERS2-1, pGBKT7-OsERS2-2and pGBKT7-OsERS2-3. Experiment proves that the GAF domain of OsERS2interacts with OsERS1.(3) Two expression vectors, pET-30a-OsERS1and pET-30a-OsERS2, which contain His label and two expression vectors including pGEX-6P-1-OsERS1and pGEX-6P-1-OsERS2which contain GST label are successfully constructed; These tasks lay the foundation for further research on the interaction between OsERS1and OsERS2in rice protoplasts by GST pull-down.