Font Size: a A A

Functional Study On Outer Membrane Protein MhOpa22 Of Mesorhizobium Huakuii In Symbiotic Nitrogen Fixation

Posted on:2024-08-20Degree:MasterType:Thesis
Country:ChinaCandidate:J WuFull Text:PDF
GTID:2530307160969929Subject:Microbiology
Abstract/Summary:PDF Full Text Request
In addition to phospholipids and lipopolysaccharides,there are also proteins integrated or embedded in the outer membrane of Gram-negative bacteria,which are called outer membrane proteins(OMPs).In recent years,there has been increasing research on the interaction between pathogenic bacterial OMPs and animal host cells,indicating that OMPs play an important role in mediating the invasion of pathogenic bacteria into hosts.However,there are currently few reports on the function and mechanism of rhizobial OMPs in symbiotic nitrogen fixation.In the former work,a new outer membrane protein gene Mhopa22 was reported in Mesorhizobium huakuii 7653 R,and the legume receptor protein As GLP1 was screened for interaction with MhOpa22 by yeast two-hybrid system.Therefore,in this study,the functional pathway of MhOpa22 and the key Loop was explored in symbiotic nitrogen fixation.The results are summarized as follows:Firstly,the truncated fragments of different Loop domains of MhOpa22 were constructed,and Loop-2-3 has been identified as a key Loop domain for the interaction between MhOpa22 and As GLP1 by yeast two-hybrid system and Bi FC.Secondly,the expression of Mhopa22 with the deletion of different Loops inΔMhopa22 and the symbiotic phenotype experiments showed that deletion of Loop2 domain significantly affected the symbiosis and nodulation between M.huakuii 7653 R and Astragalus sinicus.The number of bacteriocyte was significantly reduced,and the morphology of bacteroid was abnormal in nodules.This result is consistent with the yeast two-hybrid and Bi FC analysis,further confirming that the key Loop domain of MhOpa22 is the Loop2.And the experiment of applying inorganic nitrogen confirmed that the symbiotic defect phenotype with missing Loop2 domain was caused by nitrogen deficiency.Under hydroponics culture,in vitro treatment with synthesized peptides showed that Loop2 peptides could partially compensate for the impact of Loop2 domain deletion on symbiosis.Thirdly,transcriptome analysis showed that the deletion of Loop2 domain led to down-regulation of genes related to early symbiosis and nodulation,and partial up-regulation and down-regulation of genes related to defense and disease resistance in host plants,and three key genes affecting early symbiosis were identified: nodule factor hydrolase gene,chalcone synthase gene and chalcone isomerase gene,which were significantly down-regulated,indicating that the deletion of Loop2 domain has a effect on the expression of early symbiotic and defense genes in host plants.Finally,the extracted membrane protein of M.huakuii 7653 R was hybridized with MhOpa22 antibody by Western blot,showing that MhOpa22 is a rhizobial membrane protein.And proteome analysis of outer membrane vesicles(OMVs)showed that MhOpa22 exists in OMVs,indicating that MhOpa22 is secreted by OMVs and interacts with the A.sinicus receptor protein As GLP1,thereby affecting early infection and nodulation.In this study,the important functions and pathways of outer membrane protein MhOpa22 were revealed in symbiotic nitrogen fixation,and Loop2 has been identified as the key Loop domain.This study provides new ideas and insights for further research on the specific functions and mechanisms of rhizobial OMPs in bacteria-plant interactions.
Keywords/Search Tags:Mesorhizobium huakuii 7653R, OMPs, MhOpa22, Extracellular loops, Symbiotic nitrogen fixation, Functional pathway
PDF Full Text Request
Related items