Study And Application Of Split Ubiquitin System In Interaction Of Symbiotic Relative Membrane Proteins Of Lotus Japonicus

In recent years, researchers had isolated and identified a series of genes related to symbiosis from the mode legume Lotus japonicus, such as NFR1, NFR5, SYMRK and LHK1. In order to study the function of these genes in the symbiosis signal transduction pathway, it is necessary to study the physiological and biochemical function of the corresponding proteins, and look for the upstream and downstream partners of these proteins. By constructing the Lotus japonicus AD-cDNA yeast two-hybrid library and screening the library with corresponding bait proteins, researchers had found a number of partners of these proteins. However, conventional yeast two-hybrid system had some defects, such as that protein-protein interactionsoccurred in the nucleus, and proteins with transcriptional activation and repression function could not be studied. Here we introduced the Split-Ub system. Split-Ub system had been proved to be a suitable and reliable research method to study interactions between membrane proteins and cytoplasmic proteins. Split-Ub system had, to some extent, made up for the defects of the conventional yeast two-hybrid system. The protein interactions could occur in the cell membrane or cell matrix, and glycosylation and other post-translational modification could be carried out. This study contains the following main contents:1.15entry clones, that is pDONR221-NFR1/NFR5/ROP6/ROP6CA/ROP6DN/SymRK/SIP1L/SIP1/SIP2/SIE3/LHK1/C3HC4/MPK6/CBL1/CIPK24were constructed using the Gateway technology. Subsequently28yeast expression clones were constructed using these entry clones and Gateway technology. Introduction of Gateway technology would facilitate the work of vector construction in our lab.2. Split-Ub system based on RURA3reporter was successfully applied to verify some interactions between membrane proteins and cytoplasmic proteins within the symbiosis signal transduction pathway. Interactions between NFR5and ROP6, ROP6CA, ROP6DN were verified, so were the interactions between SymRK and SIP1L, SIP1, SIP2, SIE3and interactions between LHK1and C3HC4, MPK6.3. We further explored the interactions between membrane proteins. Our result did not suggest the interactions between SymRK and NFR1or NFR5, further work would be needed to confirm the speculation that NFR1, NFR5and SymRK function as perotein complexes in the early molecular conversation between host plant and rhizobia.4. Problems encountered in the experiment were analysed in detail, and some feasible solutions were proposed. This work would facilitate the wide application of Split-Ub yeast two-hybrid system in our lab.