Preliminary Analysis Of Protein Domain Function Of Receptor-like Protein Kinase DNF5

Legumes are typical plants with the ability of nitrogen fixation.In the process of nitrogen fixation,a complex series of signals are exchanged between the plant host and the rhizobia to form a special organ,the root nodules,so as to obtain the nitrogen elements needed by the plant.In the symbiotic process of nitrogen fixation of the legume model organism Medicago truncatula,the rhizobia will enter the plant cells to form the ultimate differentiated bacteria-like body.Bacterioid is the core unit of plant nitrogen fixation symbiosis.In recent years,researchers have obtained various nitrogen fixation deleterious mutants from legume model plants by fast neutron bombardment,and a series of key genes involved in the regulation of nitrogen fixation symbiosis have been obtained by means of map cloning.In the early stage of this research group,the nitrogen-fixing-deficient mutant dnf5 was used as the research object,and it was found that a small round nodule with no nitrogen-fixing ability was formed on the root.It was found that the mutation of a gene encoding receptor-like protein kinase DNF5 resulted in the formation of phenotypes related to dnf5 mutant.Based on previous studies,we hypothesized that DNF5 is a key gene involved in the regulation of bacterioid differentiation.Therefore,this paper aims to analyze the conservative amino acid sites of the DNF5 protein through bioinformatic analysis of the DNF5protein domain and find its conservative amino acid sites,and to conduct genetic site-directed mutations of the conservative amino acid sites to analyze its participation of the biological function of bacteroid differentiation,which helps to analyze the mechanism of action of the key node DNF5 in the nitrogen fixation symbiotic signal network.Specific research results are as follows:1.Bioinformatic analysis found that:based on the gene sequence of the gene deletion site obtained from the previous map-based cloning,the DNF5(Medtr3g079850)gene was found to consist of 5153 nucleotides,which encodes a receptor-like protein kinase with 655 amino acids.The DNF5 protein has a signal peptide,and it has a single transmembrane domain,an extracellular domain with two cysteine-rich domains,and an intracellular kinase domain.The extracellular domain of DNF5 is rich in cysteine sites.Through conservative comparison analysis with homologous proteins of other species,6 sites with high conservation were selected,namely C95,C98,C213,C123,C225,C251,numbered1-6 in sequence;DNF5 kinase domain has typical Mg²⁺binding sites and ATP binding sites,namely K391 and D484;three conserved self-activation sites in the intracellular kinase region of DNF5 protein,they are S521,S524,and S538,respectively.2.In order to verify the effect of the conserved cysteine site of the extracellular domain of DNF5 protein on the function of DNF5 protein,11 DNF5-E-point mutation-p KGWWRR vectors were successfully constructed(see Appendix II).It was found that the single-site mutations at positions 1,3,4,5 and the double-site mutations at 1,2 of the cysteine site of the extracellular domain of DNF5 can successfully complement dnf5;Four-point mutations at positions 1,2,3,and 4 and six-point mutations at positions 1,2,3,4,5,and 6 failed to successfully complement dnf5.This shows that the conserved cysteine site of the extracellular domain of DNF5affects the function of DNF5 protein.The site-directed mutation of a single cysteine site,that is,the destruction of a pair of disulfide bonds,does not make the DNF5protein loss of function.3.After successfully constructed DNF5 kinase domain Mg²⁺binding site K391and ATP binding site D484 site-directed mutation vectors:DNF5^K391M-p KGWWRR,DNF5^D484A-p KGWWRR,it was found that they could not successfully complement the dnf5 mutant.This indicates that the Mg²⁺binding site and ATP binding site required for the activation of the DNF5 intracellular kinase domain are necessary for the function of the entire DNF5 protein.It is speculated that the function of the DNF5protein may activate downstream substrates through phosphorylation.Detailed analysis of the DNF5 protein signaling pathway provides reference.4.To further determine that DNF5 activates downstream substrates through phosphorylation,this study began to focus on the autonomous activation of the intracellular kinase domain of DNF5 protein.At present,we have successfully constructed three DNF5-E-A-point mutation-p KGWWRR vectors that use six site-directed mutant DNF5 extracellular domains as templates and three DNF5-K-A-point mutation-p KGWWRR vector mutations that use DNF5 with missing extracellular domain as templates.This paper provides a preliminary analysis of the function of the DNF5 protein domain of Medicago truncatula,and provides help for the subsequent detailed analysis of the mechanism of DNF5 protein regulation of bacteroid differentiation,and provides a new idea for the subsequent research on plant nitrogen-fixing symbiosis.