| Agrobacterium tumefaciens is a soil-born gram-negative plant pathogen that can infect injured parts of plants and cause tumors.Under natural conditions,it can infect dicotyledonous plants,and introduce the T-DNA from its Ti plasmid into the genome of the injured host to induce the crown gall tumors.When Agrohacterium tumefaciens infects the host,the first step is to perceive the signal substances secreted by the injured plant,which commonly include phenolic compounds,organic acid,amino acids and sugars,and then the next step of infection can be carried out.The behavior of bacteria to recognize and respond to signal substances is called bacterial chemotaxis.Chemotaxis is a directed adaptive behavior of bacteria,which helps bacteria migrate toward a more favorable direction by sensing chemical signals in the environment.Methyl-accepting chemotaxis protein(MCP)plays an important role in recognizing signaling substances during chemotactic process.Methyl-accepting chemotaxis proteins play an important role in bacterial chemotaxis.Most MCPs can sense the chemical substances inside and outside the cell through their ligand binding domain(LBD),and transmit the signal to the downstream functional elements in the cytoplasm,making the chemotactic behavior.Agrobacterium tumefaciens C5 8 contains 20 MCP-encoding genes,and atu0526 is one of them.Methyl-accepting chemotaxis protein encoded by this gene is a typical transmembrane MCP,which contains the basic functional elements of transmembrane region(TM),ligand binding domain(LBD),HAMP and MA.Since methyl-accepting chemotaxis proteins play an important role in bacterial chemotaxis and chemotaxis has an important connection with the formation of bacterial biofilms,we need to study the function of atu0526 encoding proteins,which is of great significance to the chemotaxis study of Agrobacterium tumefaciens.In this project,we explore the function of atu0526 encoding protein from the following aspects,including the construction of the atu0526-deleted mutant strain and complemental strain,measurement of the growth curve to explore whether the gene deletion afects the growth of Agrobacterium tumefaciens,exploring the chemotactic response to leaf exudates by the swimming plate method,verifying the interaction between methyl-accepting chemotaxis proteins and coupling protein CheW,screening protein ligand substances by protein prokaryotic expression and purification in vitro,construction of site-directed mutations in key amino acids of proteins,verification of chemotaxis of ligand substances determined by capillary method and the determination of the biofilm.The main results of this project are as follows:(1)The upstream and downstream fragments of atu0526 were linked with suicide plasmid pEX18Km by overlapping PCR,and transferred to competent state of Agrobacterium tumefaciens C58.The mutant with deletion of this gene was obtained by two screening methods(using a kanamycin resistant gene as the positive selection marker and a suicide gene,sacB,as the counter-selectable marker).The complemental strains were constructed in the form of plasmid.By comparing the growth curves of wild-type,mutant and complemental strains,it was observed that the deletion of this gene did not affect the growth of Agrobacterium tumefaciens.(2)The colony diameters of wild type,mutant strain and complemental strain on semi-solid plate containing only carbon source was compared by swimming plate.It was found that the colony diameter of mutant strain was smaller than that of complemental strain,and much smaller than that of wild type.The results indicated that the deletion of this gene affected the chemotaxis of Agrobacterium tumefaciens.Agrobacterium can infect the injured plant leaves,therefore,we explore the chemotactic response of each bacterium to leaf exudates,the results showed that wild type had the most obvious chemotactic response to leaves,and the chemotactic effect of the gene deletion strain was significantly weaker than that of the wild type,but still higher than that of the CheA-deletion strain,the chemotactic effect on leaves was significantly enhanced after gene-compensation,indicating that the chemotactic effect on leaf secretions was weakened after gene deletion,but the chemotactic response did not completely disappear.(3)To exert chemotactic effect,MCP must form three component complexes with CheA and CheW.Bacterial double hybridization and pull-down experiments were conducted to verify whether Atu0526 interacts with coupling protein CheW in Agrobacterium tumefaciens,which to a certain extent verified that this gene is the gene encoding methyl-accepting chemotaxis proteins(4)By constructing prokaryotic expression strain,the protein was expressed in vitro,and differential fluorescence scanning test(DSF)was used to verify the binding of the chemotactic functional region LBD region of Atu0526 with chemical ligands in vitro.The results of differential fluorescence scanning showed that the protein binding with formic acid and malic acid was more effective.By building site-directed mutations to find key amino acid sites where the ligand binds to the protein,build some 46,47,115,125,154,156,166,191 site-directed mutations,these eight amino acid residues were mutated by replacing them with alanine residues.Differential fluorescence scanning results showed that 115,125 site-directed mutation,the protein no longer binds to the previously obvious substances,indicating that the binding between the protein and ligand substances is affected by mutations at site 115 and 125.(5)It was found that the response to formic acid,malic acid was significantly reduced after the deletion of this gene by capillary method,indicating that this gene affected the chemotactic response of Agrobacterium tumefaciens to these substances.(6)Biofilm is attached to the surface of bacteria,which can help bacteria resist the external environment and harvest nutrients.The biofilm formation ability of the mutant was significantly higher than that of the wild type,indicating that the gene had a negative regulation effect on the formation of biofilm.The functional study of atu0526 gene can provide a basis for the study of other MCP in Agrobacterium tumefaciens C58 and the modification of the function of Agrobacterium tumefaciens. |
PDF Full Text Request