Optimization Of Purification Process And Modification For Ga-Regulated Nitrogen Fixation-Related Important Protein GID1-DELLA In Legumes

Nitrogen fixation by legume-rhizobia symbiosis is an efficient biological nitrogen fixation system,and the analysis of the molecular regulation mechanism of legume-rhizobia symbiosis nitrogen fixation is not only of scientific importance,but also an important guarantee for the sustainable development of agriculture,which has been a hot spot for research.Among them,the key transcription factor DELLA protein can be regulated by gibberellin(GA)to affect the formation of rhizobia and thus the efficiency of nitrogen fixation.It has been reported that the activity of DELLA protein is dependent on the gibberellin signalling pathway.Following GA binding by the receptor GID1,DELLA protein binds tightly to GA-GID1 and is further degraded by ubiquitination in combination with GID2,thereby regulating plant growth and development.At present,the function of DELLA protein is well studied,but the structure of DELLA is poorly understood.The mechanism of GA-mediated interaction between DELLA and GID1 and GID2 is not clear,so it is necessary to analyze the structure of the full-length complex protein containing GA,GID1 and DELLA.The purpose of this study is to provide high-quality protein machine material for subsequent structural studies of the complex,and in the future,on this basis,it can be combined with relevant biochemical experiments will further elaborate a comprehensive clarification of the specific involvement of DELLA in the signal transduction of GAs.If we can analyze its assembly mechanism as well as play the activity regulation mechanism,and then modify the complex activity,we can lay the theoretical foundation for breeding nitrogen-fixing efficient strains,achieving high crop yield and developing green agriculture.The following results have been achieved: the target protein was recombinantly expressed by E.coli expression system,and the high purity GA-GID1-DELLA complex was successfully obtained by co-purification,co-expression and co-incubation strategies,among which the co-expression strategy was the most economical and efficient,and the most complexes were obtained by co-expression in the same bacterial amount of 20 g,reaching 5mg,and the time required was short,taking 4 days;the protein was modified and state optimized by serine Finally,by means of cryoelectron microscopy,we successfully collected a set of cryoelectron microscopy data of about 7 ? complexes,and initially analyzed and verified that DELLA and The C-terminal region of the DELLA protein is also involved in the GID1-DELLA interaction.In the future,we need to continue to optimize the complex proteins to improve the resolution,and then provide more data support for the subsequent structural studies.