Prediction,Prokaryotic Expression And Related Functions Analysis Of Azorhizobium Caulinodans ORS571 Type ? Secretion System Effector Proteins In Sesbania Cannabina

Rhizobia can establish symbiosis with leguminous plants,but aslo can built endosymbiosis with non-leguminous plants such as wheat and maize.During the process of symbiosis,rhizobia provide nitrogen source for the growth and development of host plants.The formation of the symbiotic relationship involves complex signal communication between rhizobia and its hosts,among them the type III secretion system?T3SS?effector proteins of rhizobia play important roles.Azorhizobium caulinodans ORS571?hereafter A.caulinodans ORS571?,the microsymbionts of tropical legume Sesbania cannabina,who can fix nitrogen under the condition of authigenic and form nitrogen-fixing root-and stem-nodules on Sesbania cannabina.In addition to establish symbiosis with Sesbania cannabina,A.caulinodans ORS571 can built endosymbiosis with gramineous plants wheat and maize under natural condition and increase dry weight and total nitrogen of the plants above.However,there were no reports about the T3 SS effector proteins of A.caulinodans ORS571.Based on the above characteristics,this study chosen A.caulinodans ORS571 as the experimental strain.The objective of this study was to predict,express and analysis related functions of A.caulinodans ORS571 T3 SS effector proteins,and this research would lay a foundation for the application of the A.caulinodans ORS571 T3 SS effector proteins in symbiotic nitrogen fixation of non-leguminous plants.The results of the study were as follows.?1?The whole proteome of A.caulinodans ORS571 was analyzed by bioinformatics software Effective T3 and BPBAac with related literature reports,seven candidate effector proteins of A.caulinodans ORS571 were obtained,and they were AZC₀308,AZC₀649,AZC₀667,AZC₂699,AZC₂928,AZC₃165 and AZC₄087,respectively.?2?The 7 gene fragments were successfully cloned into the prokaryotic expression vector pMAL-c4 X,respectively.Detection of fusion proteins expression were completed by Western blot using anti-maltose binding protein?MBP?monoclonal antibody?horse radish peroxidase conjugated?,and the results implied that these 7 fusion proteins expressed correctly and the effect of protein expression was better.?3?Based on purification results,fusion proteins AZC₀667,AZC₂928 and AZC₄087 were chosen to prepare polyclonal antibodies,and the results of ELISA implied that the polyclonal antibodies were prepared successfully.With the above polyclonal antibodies as primary antibodies respectively,the AZC₂928 was detected in supernatant proteins of A.caulinodans ORS571.The results revealed AZC₂928 was a A.caulinodans ORS571 T3 SS effector protein.?4?pK18mobsacB was used as a gene knock-out vector,the AZC₂928 gene deletion mutant was constructed and infected wheat?Triticum aestivum?Xiao Yan 22,and the results showed that AZC₂928 gene had inhibitory effect on the growth of wheat.