A method and engineering bacteria for improving the rhizosphere chemotaxis and colonization ability of PGPR

Abstract

The present invention belongs to the field of molecular biology, and relates to plant growth-promoting rhizobacteria (PGPR), and in particular to a method and an engineered bacterium for improving the rhizosphere chemotaxis and colonization ability of PGPR. The present application discovered that at least one of the bacterial chemotaxis receptors has a pentapeptide in its C-terminal extension region, and the pentapeptide can mediate the interaction between the methyltransferase CheR and the methylesterase CheB involved in chemotaxis, and reversibly methylate the chemotaxis receptor. Grafting the pentapeptide onto the C-terminus of the chemotaxis receptor of a strong chemoattractant in the chemosphere of the PGPR can enhance its chemotactic response to the strong chemoattractant, thereby improving the rhizosphere chemotaxis and colonization ability of the PGPR.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology, relates to plant growth-promoting rhizobacteria (PGPR), and in particular refers to a method and an engineered bacterium for improving the rhizosphere chemotaxis and colonization ability of PGPR. Background Art

[0002] The plant growth promoting rhizobacteria (PGPR) used in microbial fertilizers promote plant growth through multiple functions, including nitrogen fixation, phosphorus and potassium solubilization, synthesis of various plant hormones and biocontrol, as well as degradation of ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) synthesized by plants, reducing plant ethylene synthesis and enhancing plant adaptability to stress conditions. Therefore, the use of microbial fertilizers is an effective way to reduce the use of chemical fertilizers and pesticides.

[0003] However, there is a problem of unstable growth-promoting effect of microbial fertilizer in field application, which greatly limits the promotion and application of microbial fertilizer. An important reason for the unstable growth-promoting effect of microbial fertilizer in field application is the weak rhizosphere chemotactic competitiveness of the strain. Identifying the strong chemoattractants of PGPR in root secretions and using genetic engineering technology to transform PGPR to improve its chemotactic competitiveness in the rhizosphere of plants are the main means to solve the unstable growth-promoting effect of PGPR. Once the strong chemoattractants of PGPR to the rhizosphere are determined, we can use genetic engineering technology to improve the ability of PGPR to attract this component, such as enhancing the affinity of the chemoattractant receptor of the chemoattractant, enhancing the methylation modification efficiency of the chemoattractant receptor of the chemoattractant, increasing the abundance of the chemoattractant receptor and increasing the metabolic rate of the chemoattractant, thereby improving the rhizosphere chemotactic competitiveness of PGPR and improving and stabilizing the use effect of PGPR. Patent CN110218736A replaces the promoter by homologous recombination to increase the expression of the AcdS gene, AcdS enzyme activity and ACC metabolic rate of PGPR bacteria, thereby increasing the chemotaxis intensity of PGPR bacteria to ACC; Patent CN107205403A improves the growth-promoting effect of PGPR bacteria by physically detecting pectin or pectin-related sugars during plant growth; and our research group found during the research that the chemotactic behavior of bacteria is closely related to the methylation modification of their chemotactic receptors. In order to further study methods to improve the rhizosphere chemotactic colonization ability of PGPR bacteria, our research group conducted the following research. Summary of the invention

[0004] The present invention proposes a method and an engineered bacterium for improving the rhizosphere chemotaxis and colonization ability of PGPR. It is found that the C-terminal extension region of at least one chemotaxis receptor of the bacteria contains a pentapeptide, which can mediate the interaction between the methyltransferase CheR and the methylesterase CheB involved in chemotaxis, and reversibly methylate the chemotaxis receptor. The pentapeptide is grafted onto the C-terminus of the chemotaxis receptor of the strong chemotaxis of the PGPR rhizosphere, which can enhance its chemotaxis response to the strong chemotaxis, thereby improving the rhizosphere chemotaxis and colonization ability of PGPR.

[0005] The technical solution of the present invention is achieved in this way:

[0006] A method for improving the rhizosphere chemotaxis and colonization ability of PGPR, comprising the following steps:

[0007] (1) Query the chemotactic receptor of a certain PGPR from the genome annotation of the bacteria, and then screen the domains in the chemotactic receptor that are related to enhancing the efficiency of methylation modification based on the amino acid sequence of the chemotactic receptor;

[0008] (2) screening pentapeptides that may bind to methyltransferases involved in chemotaxis from the C-terminal extension region of the domain in step (1);

[0009] (3) Molecular modeling of the pentapeptides screened in step (2) and homology modeling of the methyltransferase involved in chemotaxis are performed, and pentapeptides with low binding free energy to the methyltransferase involved in chemotaxis are screened based on the binding free energy of the two;

[0010] (4) Grafting the pentapeptide of step (3) onto the C-terminus of a chemotactic receptor that is a strong chemoattractant of the rhizosphere and does not contain the pentapeptide screened in step (3), thereby obtaining an engineered bacterium with improved chemotactic and colonization ability.

[0011] In the above step (1), the screening of domains in chemotactic receptors related to enhancing the efficiency of methylation modification is achieved through smart online analysis tools.

[0012] The above-mentioned domain related to enhancing the efficiency of methylation modification is referred to as MA domain.

[0013] In the above step (3), molecular modeling was performed using chemoffice19.0_win software, and homology modeling was performed using the SWISS-MODEL tool.

[0014] The PGPR mentioned above is Pseudomonas UW4, and the methyltransferase involved in chemotaxis is CheR2, whose GenBank accession number is AFY21330.

[0015] The pentapeptide sequence in the above step (4) is shown as SEQ ID No. 1, and the chemotactic receptor without the pentapeptide is McpACC, and its sequence is shown as SEQ ID No. 7.

[0016] The engineered bacteria of Pseudomonas UW4 prepared by the above method is a gene-edited Pseudomonas UW4 obtained by grafting a pentapeptide with a sequence as shown in SEQ ID No. 1 to the C-terminus of the Pseudomonas UW4 chemotaxis receptor.

[0017] The Pseudomonas UW4 chemotaxis receptor is a Pseudomonas UW4 chemotaxis receptor that does not contain or has knocked out the pentapeptide that binds to CheR2.

[0018] The first construction method of the above-mentioned engineered bacteria is to use the Pseudomonas UW4 chemotactic receptor that does not contain the pentapeptide that binds to CheR2 as the target fragment, and graft the screened pentapeptide sequence that binds to CheR2 to the C-terminus of the target fragment. The steps are: design the homologous fragment HRF1 sequence as shown in SEQ ID No. 2, connect it with the plasmid pEX18Gm to obtain pEX18Gm-McpACC+PEKPR, then first transfer it into the competent state of Escherichia coli, and then transform it into UW4 through double parental hybridization. The successfully identified one is the UW4-1 engineered bacteria.

[0019] The second construction method of the above-mentioned engineered bacteria is based on the engineered bacteria constructed by the first method, and the chemotactic receptor containing the pentapeptide that binds to CheR2 is used as the target fragment, and the C-terminal pentapeptide of the target fragment is knocked out. The steps are: design a homologous fragment HRF2 sequence as shown in SEQ ID No.3, connect it with the plasmid pEX18Gm to obtain pEX18Gm-Mcp14ΔPEKPR, and then transform it into UW4-1 engineered bacteria through double parental hybridization. The successfully identified one is UW4-2 engineered bacteria.

[0020] The present invention has the following beneficial effects:

[0021] 1. Our research group discovered that at least one of the bacterial chemotaxis receptors has a pentapeptide in its C-terminal extension region, which can mediate the interaction between methyltransferase and methylesterase, reversibly methylate the chemotaxis receptor, and enhance the chemotaxis response. The method and ideas of this application can be used to recombinantly identify the chemotaxis receptors of various PGPR bacteria, and ultimately obtain engineered bacteria with strong rhizosphere chemotaxis and colonization abilities. There are relatively few measures reported to improve the rhizosphere chemotaxis and colonization abilities of PGPR. Our research group previously invented a method to improve the rhizosphere chemotaxis and colonization abilities of PGPR by increasing the metabolic rate of PGPR for strong chemotaxis in root secretions, and this method is limited by the amount of strong chemotaxis secreted by plant roots. Compared with this method, the present invention is not affected by the amount of root secretions, and has a more significant effect in improving the rhizosphere chemotaxis and colonization abilities of PGPR.

[0022] 2. Pseudomonas UW4 ( Pseudomonassp. UW4) is a typical PGPR. Previous studies have found that the strong chemoattractant of the bacteria in the rhizosphere is ACC, the chemotactic receptor of ACC is McpACC (GenBank accession number is WP_015093116), and the methyltransferase involved in chemotaxis is CheR2 (GenBank accession number is AFY21330). Increasing its metabolic rate of ACC can increase its chemotactic colonization and growth-promoting effect in the wheat rhizosphere. The present invention introduces that on the basis of identifying the terminal pentapeptide of the UW4 chemotactic receptor, the pentapeptide is grafted to the end of McpACC to enhance the methylation modification efficiency of McpACC, thereby improving the chemotactic response of UW4 to ACC, and improving its rhizosphere chemotactic competitiveness and growth-promoting effect. Pot experiments show that the colonization amount in the wheat rhizosphere is 62.0% higher than that of the starting strain, the root length is 6.3% higher than that of the starting strain, the root dry weight is 10.3% higher than that of the starting strain, the aboveground height is 16.0% higher than that of the starting strain, and the aboveground dry weight is 16.5% higher than that of the starting strain. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 Molecular docking diagram of CheR2 in Pseudomonas UW4 and the C-terminal pentapeptide PEKPR of Mcp14, where (A) The overall structure of the docking complex of CheR2 and pentapeptide PEKPR. The stick figure represents the pentapeptide. (B) The residues of CheR2 binding to pentapeptide PEKPR. (C) Ligplot of CheR2 docking with pentapeptide. Hydrogen bonds are represented by green dashed lines between the atoms involved, and hydrophobic bonds are represented by red arcs, with the spokes facing the ligand atoms that interact with it. (D) Amino acid residues in CheR2 involved in pentapeptide binding.

[0025] Figure 2The invention discloses an electrophoresis analysis of the PCR detection products of the UW4-1 and UW4-2 strains; wherein 1. the primer pair is UW4-1-F1 and UW4-1-R1A, and the template is the UW4 genome; 2. the primer pair is UW4-1-F2 and UW4-1-R2B, and the template is the UW4 genome; 3. the primer pair is UW4-1-F1 and UW4-1-R1A, and the template is the UW4-1 genome; 4. the primer pair is UW4-1-F2 and UW4-1-R2, and the template is UW4-1; 5. the primer pair is UW4-1-F1 and UW4-1-R1, and the template is UW4-2; 6. the primer pair is UW4-1-F2 and UW4-1-R2, and the template is UW4-2.

[0026] Figure 3 The colonization amount of Pseudomonas UW4 strain in the wheat rhizosphere and its growth-promoting effect on wheat; A, the colonization amount of bacteria in the wheat rhizosphere; B, wheat root length; C, wheat root dry weight; D, wheat aerial part height; E, wheat aerial part dry weight. Ctr, control; different lowercase letters indicate significant differences ( P <0.05). DETAILED DESCRIPTION

[0027] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] A method for improving the rhizosphere chemotaxis and colonization ability of PGPR, comprising the following steps:

[0029] (1) Query the chemotactic receptor of a certain PGPR from the genome annotation of the bacteria, and then screen the domains in the chemotactic receptor that are related to enhancing the efficiency of methylation modification based on the amino acid sequence of the chemotactic receptor;

[0030] (2) screening pentapeptides that may bind to methyltransferases involved in chemotaxis from the C-terminal extension region of the domain in step (1);

[0031] (3) Molecular modeling of the pentapeptides screened in step (2) and homology modeling of the methyltransferase involved in chemotaxis are performed, and pentapeptides with low binding free energy to the methyltransferase involved in chemotaxis are screened based on the binding free energy of the two;

[0032] (4) Grafting the pentapeptide of step (3) onto the C-terminus of the chemotactic receptor of a strong chemoattractant in the chemotactic root secretions that do not contain the pentapeptide, thereby obtaining an engineered bacterium with improved chemotactic colonization ability.

[0033] In the above step (1), the screening of domains in chemotactic receptors related to enhancing the efficiency of methylation modification is achieved through smart online analysis tools.

[0034] The above-mentioned domain related to enhancing the efficiency of methylation modification is referred to as MA domain.

[0035] In the above step (3), molecular modeling was performed using chemoffice19.0_win software, and homology modeling was performed using the SWISS-MODEL tool.

[0036] In the vast majority of PGPR bacteria, at least one chemotactic receptor has a pentapeptide at its C-terminus that can bind to the methyltransferase involved in chemotaxis. Therefore, this method is applicable to the vast majority of PGPR.

[0037] Below is the Pseudomonas UW4 ( Pseudomonas sp.UW4): deposited in the Agricultural Research Culture Collection of the United States (deposit number is NRRL B-50193, deposit date is June 9, 2008) as an example for explanation: Example

[0038] Material

[0039] 1.1 Experimental strains

[0040] Pseudomonas sp. UW4 ( Pseudomonas sp.UW4): Deposited at the Agricultural Research Service Culture Collection (NRRL B-50193, deposit date June 9, 2008). The Agricultural Research Service Culture Collection is located in Picheria, Illinois, and is a government-run collection of fungi supported by the Agricultural Research Center of the United States Department of Agriculture. Its full name is Agricultural Research Service Culture Collection, or NRRL for short.

[0041] 1.2 Culture medium

[0042] LB liquid medium: weigh 5 g of yeast extract, 10 g of tryptone, and 10 g of sodium chloride, dissolve in 1000 mL of distilled water, dispense into 250 mL Erlenmeyer flasks, and sterilize under high pressure at 121°C for 30 min.

[0043] Preparation of LB solid culture medium: weigh 5 g yeast extract, 10 g tryptone, and 10 g sodium chloride, dissolve in 1000 mL distilled water, add 20 g agar powder to the solid culture medium, dispense into 250 ml Erlenmeyer flasks, and sterilize at 121°C for 30 min.

[0044] Prediction of the terminal pentapeptide of Pseudomonas UW4 chemotactic receptor

[0045] There are 28 chemotactic receptors annotated in the Pseudomonas UW4 genome. The amino acid sequences of these 28 chemotactic receptors were analyzed using the smart online analysis tool (http: / / smart.embl-heidelberg.de / index2.cgi) to analyze the MA domain (Methyl-Accepting domain) in the receptor. The C-terminal extension region of this domain may contain a pentapeptide sequence that binds to CheR2. At the same time, the 28 receptor amino acid sequences were aligned and analyzed using DNAMAN software to find out the chemotactic receptors with an extension region at the C-terminus of the receptor. The pentapeptide sequence may exist in this C-terminal extension region. It was found that the C-termini of four receptors contained extended regions. Among them, Mcp11 had only four amino acids in the extended region, which was not enough to form a pentapeptide sequence. Therefore, the remaining three receptors (Mcp14 (sequence shown in SEQ ID No.4), Mcp27 (sequence shown in SEQ ID No.5) and Mcp26 (sequence shown in SEQ ID No.6)) were preliminarily predicted to contain pentapeptide sequences that could bind to CheR2 protein. The three predicted pentapeptide sequences were PEKPR, VVDKA and NQLTN, respectively (Table 1).

[0046] Table 1 Alignment of C-terminal sequences of Pseudomonas UW4 chemotactic receptor

[0047]

[0048] a The underlined characters represent hypothetical pentapeptides that bind to CheR and CheB.

[0049] 3. Docking of the pseudopentapeptide molecule with CheR2 molecule

[0050] The pseudo-pentapeptide molecular structure was modeled using chemoffice19.0_win software, and the online modeling analysis tool SWISS-MODEL (https: / / swissmodel.expasy.org / ) was used to model the CheR2 homology. The template number used was SMTLID: 5y4r.1. Among the three pentapeptides, only PEKPR can bind to the β-subdomain of CheR2. The amino acid residues in CheR2 that bind to the pentapeptide are Leu173, Gln174, Pro179, Lys180, Gly181, Pro182, and Trp185 ( Figure 1 A, B), the binding force is mainly hydrogen bond, hydrophobic bond and van der Waals force ( Figure 1 C, D), the binding free energy is -13.17 kcal / mol. The other two pentapeptides cannot bind to the β-subdomain of CheR2, indicating that the pentapeptide PEKPR may be the pentapeptide that mediates the binding of CheR and CheB.

[0051] Construction of Pentapeptide-grafted Engineering Bacteria of Pseudomonas UW4 ACC Chemotactic Receptor

[0052] In order to improve the chemotactic response of Pseudomonas UW4 to ACC, two engineered bacteria were constructed. One was to graft the pentapeptide PEKPR to the C-terminus of the ACC chemotactic receptor McpACC, named UW4-1; the other was to truncate the pentapeptide PEKPR of Mcp14 of UW4-1, named UW4-2. The construction method was that two homologous recombination fragments HRF1 and HRF2 were synthesized by BGI. The HRF1 fragment (sequence as shown in SEQ ID No.2) contained 206 bp upstream homology arm, 221 bp downstream homology arm, restriction enzyme EcoRI, KpnI restriction site and pentapeptide PEKPR base sequence CCGGAAAAACCGCGC. HRF1 was connected with plasmid pEX18Gm to obtain pEX18Gm-McpACC+PEKPR. The constructed plasmid was transformed into Escherichia coli S17-1 by conventional heat shock method and transformed into UW4 by double parental hybridization. Colony PCR was performed using primer pairs UW4-1-F1, UW4-1-R1, UW4-1-F2, and UW4-1-R2 (Table 2) to obtain UW4-1 ( Figure 2 ). The pentapeptide of Mcp14 of UW4-1 was knocked out using HRF2 homologous recombination. HRF2 (sequence shown in SEQ ID No.3) contains 206 bp upstream homologous arm, 206 bp downstream homologous arm, restriction enzyme EcoRI and KpnI restriction sites. HRF2 was ligated with plasmid pEX18Gm to obtain pEX18Gm-Mcp14ΔPEKPR. The plasmid was transformed into Escherichia coli S17-1 by conventional heat shock method and then transformed into UW4-1 by biparental hybridization to produce UW4-2. Colony PCR was performed using primer pairs UW4-1-F1 and UW4-1-R1, UW4-1-F2 and UW1-1-R2 (Table 2) to identify UW4-2 ( Figure 2 ).

[0053] Table 2 Primer list

[0054]

[0055] 4.1 Biparental hybridization method

[0056] (1) 10 μL of each of the verified E. coli S17-1 strains containing the plasmids pEX18Gm-McpACC+PEKPR and pEX18Gm-Mcp14ΔPEKPR were inoculated into 10 ml of liquid LB medium and cultured at 37°C in a shaker at 220 rpm for 12-15 hours;

[0057] (2) Take 10 μL of Pseudomonas UW4 stored in a glycerol tube and inoculate it into 10 ml of liquid LB medium, place it in a shaker at 30°C and culture it at 220 rpm for 12-15 hours;

[0058] (3) After the culture, collect 1.5 mL of each of the two S17-1 bacterial cultures into a centrifuge tube and centrifuge at 12,000 rpm for 1 min, then discard the supernatant;

[0059] (4) Resuspend the two S17-1 bacterial strains in 1 ml of 0.85% NaCl solution, centrifuge at 12,000 rpm for 1 min, discard the supernatant, add 1.5 ml of UW4 bacterial solution to each of the centrifuge tubes containing the two S17-1 bacterial strains, centrifuge at 12,000 rpm, and discard the supernatant;

[0060] (5) Resuspend the cells in 1 ml of 0.85% NaCl solution, centrifuge at 12,000 rpm, discard the supernatant, and repeat this step 5-6 times;

[0061] (6) Resuspend the cells in 100 μL of 0.85% NaCl solution;

[0062] (7) Cut the nitrocellulose membrane to the size of the culture dish and spread it on the LB solid plate;

[0063] (8) The two resuspended bacterial solutions were added dropwise onto a plate covered with a nitrocellulose membrane and incubated at 30°C for 24 hours;

[0064] (9) After the culture is completed, the bacteria are washed off the nitrocellulose membrane with 800 μL of 0.85% NaCl solution;

[0065] (10) After resuspending the bacterial solution, 100 μL was applied to a LB solid plate containing 25 μg / mL gentamicin and 100 μg / mL ampicillin, and then incubated at 30°C for 12-16 hours to allow single exchange of Pseudomonas UW4 or UW4-1;

[0066] (11) Pick a single exchanger and subculture it in liquid LB medium at 30°C, 220 rpm for 5 hours. Then spread it on a solid LB plate with a sucrose content of 10% and 100 μg / mL ampicillin, and culture it at a constant temperature of 30°C for 24-35 hours.

[0067] The colonies that can grow are double exchangers, and the double exchangers corresponding to the two Escherichia coli S17-1 containing different plasmids are UW4-1 strain and UW4-2 strain respectively.

[0068] 4.2 Colony PCR Identification of Strains UW4-1 and UW4-2

[0069] The primers used for PCR are listed in Table 2.

[0070] The reaction system and reaction procedure are as follows:

[0071]

[0072] The results are as follows Figure 2 shown.

[0073] Potted experiment of Pseudomonas UW4 ACC chemotactic receptor grafted with pentapeptide engineering bacteria

[0074] Wheat seeds were soaked in 70% alcohol for 1 min and then rinsed with sterile water 3 times. Then, they were soaked in 2% sodium hypochlorite for 5 min and then rinsed with sterile water 5 times. 2 After soaking for 5 minutes, rinse with sterile water for 6 times. Soak the sterilized wheat seeds in the suspension of UW4, UW4-1 and UW4-2 with OD600=1, and imbibition treatment for 24 hours under dark conditions. The control is soaked in sterile water. After germination, potted plants are planted.

[0075] The soil was prepared by mixing peat soil and vermiculite in a ratio of 1:1, watering with enough water, and sterilizing at 121°C for 3 hours. The obtained soil was divided into polyethylene seedling trays, and the treated seeds were sown in pots, 3 seeds per hole, about 1 cm deep. After sowing, it was placed in a rhythmic culture at 25°C, 16 hours of light, and 8 hours of darkness. In the first two days, the plants were watered with 5mL of different bacterial suspensions with an OD600 of 1, and 5mL of sterile water was used as a control. After the wheat grew for about 15 days, the roots were taken for tissue grinding and gradient dilution and coating on LB solid plates containing 100μg / mL of ampicillin resistance to count the number of rhizosphere colonizing bacteria in potted wheat ( Figure 3 A). Cut the root and the aboveground part to measure the root length and the aboveground part length. After drying the root and the aboveground part to constant weight, measure the root dry weight and the aboveground part dry weight respectively ( Figure 3 BCDE).

[0076] Depend on Figure 3 It can be seen that wheat was inoculated with Pseudomonas UW4, UW4-1 and UW4-2. Compared with UW4-2 and UW4, the colonization of UW4-1 in the wheat rhizosphere increased by 14.3% and 62.0%, respectively, and the colonization of UW4-2 increased by 41.8% compared with UW4 ( Figure 3 A). Compared with the uninoculated control, the root length of wheat inoculated with UW4, UW4-1 and UW4-2 increased by 32.3%-40.7%. The root length of wheat inoculated with UW4-1 increased by 4.3% and 6.3% compared with that inoculated with UW4-2 and UW4, respectively. There was no difference in root length between wheat inoculated with UW4-2 and UW4 ( Figure 3B). The root dry weight of wheat inoculated with UW4, UW4-1 and UW4-2 increased by 64.8% to 81.8% compared with the control. The root dry weight of wheat inoculated with UW4-1 increased by 5.5% and 10.3% compared with wheat inoculated with UW4-2 and UW4, respectively, and the root dry weight of wheat inoculated with UW4-2 increased by 4.5% compared with that inoculated with UW4 ( Figure 3 C). Compared with the control, the height of the aboveground part of wheat inoculated with UW4, UW4-1 and UW4-2 increased by 14.9%-33.2%. The height of the aboveground part of wheat inoculated with UW4-1 increased by 5.1% and 16.0% compared with wheat inoculated with UW4-2 and UW4, respectively, and the height of the aboveground part of wheat inoculated with UW4-2 increased by 10.3% compared with that inoculated with UW4 ( Figure 3 D). The aboveground dry weight of wheat inoculated with UW4, UW4-1 and UW4-2 increased by 26.7% to 47.6% compared with the control. The aboveground dry weight of wheat inoculated with UW4-1 increased by 13.2% and 16.5% compared with wheat inoculated with UW4-2 and UW4, respectively. The aboveground dry weight of wheat inoculated with UW4-2 increased by 2.9% compared with wheat inoculated with UW4 ( Figure 3 E).

[0077] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for improving the rhizosphere chemotaxis and colonization ability of PGPR, It is characterized in that The steps are: (1) Query the chemokine receptor of a PGPR from the genome annotation of the PGPR, and then screen the domains in the chemokine receptor that are related to enhancing the efficiency of methylation modification based on the amino acid sequence of the chemokine receptor; (2) screening pentapeptides that may bind to methyltransferases involved in chemotaxis from the C-terminal extension region of the domain in step (1); (3) Molecular modeling of the pentapeptides screened in step (2) and homology modeling of the methyltransferase involved in chemotaxis are performed, and pentapeptides with low binding free energy to the methyltransferase involved in chemotaxis are screened based on the binding free energy of the two; (4) grafting the pentapeptide of step (3) onto the C-terminus of a chemotactic receptor that is a strong chemoattractant of the rhizosphere that does not contain the pentapeptide screened in step (3), thereby obtaining an engineered bacterium with improved chemotactic and colonization ability; The PGPR is Pseudomonas UW4, the methyltransferase involved in chemotaxis is CheR2, and its GenBank accession number is AFY21330; the pentapeptide sequence in step (4) is shown in SEQ ID No. 1, and the chemotactic receptor without pentapeptide is McpACC, and its sequence is shown in SEQ ID No.

7.

2. The method for improving the rhizosphere chemotaxis and colonization ability of PGPR according to claim 1, Features: In step (1), screening of the structural domains in the chemotactic receptor related to enhancing the efficiency of methylation modification is achieved through a smart online analysis tool.

3. The method for improving the rhizosphere chemotaxis and colonization ability of PGPR according to claim 2, Features: The structural domain associated with enhancing the efficiency of methylation modification is referred to as the MA domain.

4. The method for improving the rhizosphere chemotaxis and colonization ability of PGPR according to claim 3, Features: In step (3), molecular modeling was performed using chemoffice19.0_win software, and homology modeling was performed using the SWISS-MODEL tool.

5. An engineered bacterium prepared by the method according to any one of claims 1 to 4, Features: The engineered bacteria is a gene-edited Pseudomonas UW4 obtained by grafting a pentapeptide with a sequence as shown in SEQ ID No. 1 onto the C-terminus of the Pseudomonas UW4 chemotactic receptor.

6. The engineered bacteria according to claim 5, Features: The Pseudomonas UW4 chemotaxis receptor is a Pseudomonas UW4 chemotaxis receptor that does not contain or has knocked out the pentapeptide that binds to CheR2.

7. The method for constructing the engineered bacteria according to claim 6, It is characterized in that The steps are as follows: design a homologous fragment HRF1 sequence as shown in SEQ ID No. 2, connect it with the plasmid pEX18Gm to obtain pEX18Gm-McpACC+PEKPR, then first transfer it into the competent state of Escherichia coli, and then transform it into UW4 through parental hybridization. The one that is successfully identified is the UW4-1 engineered bacterium.

8. The method for constructing the engineered bacteria according to claim 7, It is characterized in that The steps are: designing a homologous fragment HRF2 sequence as shown in SEQ ID No. 3, connecting it with the plasmid pEX18Gm to obtain pEX18Gm-Mcp14ΔPEKPR, and then transforming it into the UW4-1 engineered bacteria constructed in claim 7 through biparental hybridization, and the one successfully identified is the UW4-2 engineered bacteria.

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