CN115747358A - Method for identifying chemotactic rate of PGPR chemotactic root secretion reaching rhizosphere and application thereof - Google Patents
Method for identifying chemotactic rate of PGPR chemotactic root secretion reaching rhizosphere and application thereof Download PDFInfo
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Abstract
A method for identifying the chemotaxis rate of PGPR (PGPR) for chemotactic root exudates to reach rhizosphere and application thereof. The invention belongs to the field of microorganisms, relates to a chemotaxis of plant growth promoting rhizobacteria, and particularly relates to a method for identifying the arrival rate of different chemotaxis arriving rhizomes of PGPR (protein-coupled plasma) and application thereof. The method can distinguish the chemotaxis rate of a certain chemotaxis object of PGPR from the reproduction rate of the certain chemotaxis object on a root surface, and can determine that the chemotaxis object is a strong chemotaxis object of PGPR in the rhizosphere by comparing the chemotaxis rate difference of a chemotaxis receptor deletion strain of the certain chemotaxis object of PGPR with the chemotaxis rates of a wild-type strain and a replenisher strain and if the chemotaxis rate of the deletion strain is obviously lower than that of the wild-type strain and the replenisher strain.
Description
Technical Field
The invention belongs to the field of microorganisms, relates to a chemotactic substance of plant growth promoting rhizobacteria, and particularly relates to a method for identifying the chemotactic rate of a PGPR chemotactic root secretion reaching the rhizosphere and application thereof.
Background
Plants secrete up to 30% of their fixed carbon through the root system, interacting closely with rhizobacteria. Bacteria that promote plant growth in the rhizosphere of plants are called Plant Growth Promoting Rhizobacteria (PGPR). Rhizobium, azotobacter, phosphate solubilizing bacteria, potassium solubilizing bacteria, growth-promoting bacteria, biocontrol bacteria and the like applied to the microbial fertilizer (bacterial fertilizer) are PGPR. A large number of researches show that the PGPR bacterial manure can reduce the using amount of a chemical fertilizer by 20-30 percent, reduce the using amount of pesticides by more than 30 percent and increase the yield of crops by 10-80 percent, and the function of the PGPR bacterial manure in developing sustainable agriculture is more and more emphasized, so that the PGPR bacterial manure is an important way for realizing the double reduction targets of the chemical fertilizer and the pesticides.
However, the use effect of the PGPR in practical application is not stable, the effects of a laboratory and a greenhouse are obvious, the field effect is poor, and the popularization and the application of the PGPR microbial inoculum are seriously restricted. PGPR functions, depending on whether it can gain advantage in competition with indigenous microorganisms, to colonize the plant rhizosphere (around roots, root surface, root tissue). The first step in the colonization of the root zone by PGPR is the recognition of root secretions as chemoattractants, reaching the root zone by chemoattractant movement, propagating in the root zone and forming a pellicle. The colonization amount of PGPR at the rhizosphere is obviously and positively correlated with the growth promoting effect. Plant root exudates comprise nearly one hundred species of organic acids, amino acids, saccharides and the like, which are strong chemotactic substances for PGPR rhizosphere colonization? Many studies have found that PGPR has strong chemotactic response to malic acid, oxalic acid or glutamic acid, arginine and the like, and is probably a key chemotactic substance for rhizosphere colonization of PGPR. However, these secretory components also attract the chemotaxis of indigenous microorganisms to reach rhizosphere colonization, and thus, may not be a key chemoattractant for PGPR in rhizosphere chemotactic colonization. On the basis of identifying PGPR (poly (propylene glycol) receptor) strong chemotactic substances in root exudates, the genetic engineering technology is applied to improve the chemotactic colonization competitiveness of PGPR (poly (propylene glycol) receptor) at the plant rhizosphere, and the method is an important way for solving the problem of unstable growth promotion effect of PGPR.
Determining key chemotactic matters of PGPR in rhizosphere chemotactic colonization by in vitro plate chemotaxis and capillary chemotaxis experiments, and comparing chemotactic responses of PGPR to the chemotactic matters; experiments of PGPR colonization in the rhizosphere compare the influence of adding certain root secretion on the colonization amount of PGPR in the rhizosphere, or construct deletion mutant strains of certain chemotactic receptor(s) of PGPR, and examine the colonization amount and the colonization rate of the chemotactic receptor(s) deletion mutant strains in the rhizosphere. The conditions of in vitro plate chemotaxis and capillary chemotaxis experiments are greatly different from rhizosphere colonization conditions, and the obtained results can only be used for primary screening. The rhizosphere colonization experiment is the result of measuring the long-time colonization of the PGPR in the rhizosphere, and the obtained colonization rate is the sum of the chemotactic rate of the PGPR reaching the rhizosphere and the reproduction rate on the surface of the root, so that the chemotactic rate of the PGPR reaching the rhizosphere and the reproduction rate on the surface of the root can not be distinguished. Comparing the rate at which PGPR chemotactic different chemoattractants reach the rhizosphere (abbreviated as chemotactic rate) is the gold standard that identifies key chemoattractants for PGPR chemotactic colonization at the rhizosphere; however, the existing method cannot accurately identify the rate of different chemoattractants reaching the rhizosphere, and further identify key chemoattractants of PGPR in chemotactic colonization of the rhizosphere.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for identifying the chemotaxis rate of PGPR chemotactic root exudates to the rhizosphere and application thereof.
The technical scheme of the invention is realized as follows:
a method for identifying the chemotactic rate of PGPR chemotactic root exudates to the rhizosphere comprises the following steps:
(1) Searching an extracellular ligand binding domain of the microorganism from the annotation of the microorganism genome, screening a chemotactic receptor capable of identifying extracellular chemoattractant, performing molecular docking on the chemotactic receptor and root exudate components by using a molecular docking technology, calculating binding free energy, and screening the root exudate components with low free energy values as candidate chemoattractants;
(2) Determining a receptor protein corresponding to the candidate chemoattractant according to the candidate chemoattractant, and constructing an expression vector containing an extracellular ligand binding domain fragment of the receptor protein and a recombinant vector for knocking out the receptor protein by taking the genomic DNA of the microorganism as a template;
(3) Transferring the Escherichia coli containing the recombinant vector in the step (2) into a microorganism by a conjugation method to obtain a chemotactic receptor knockout mutant strain;
(4) Constructing anaplerotic plasmid and anaplerotic no-load plasmid of receptor protein corresponding to the candidate chemoattractant in the step (2) by taking genome DNA of the microorganism as a template, and respectively transferring the anaplerotic plasmid and anaplerotic no-load plasmid into the chemotactic receptor knockout mutant strain in the step (3) to obtain an anaplerotic strain and an anaplerotic no-load strain of the chemotactic receptor knockout mutant strain;
(5) Carrying out a flat-plate chemotaxis experiment on the microbes, the chemotactic receptor knockout mutant strain, the anaplerosis strain and anaplerosis empty strain of the chemotactic receptor knockout mutant strain and the candidate chemotactic substance to verify the correspondence between the receptor protein and the chemotactic substance, and then measuring the chemotactic rate and the proliferation rate of the microbes, the chemotactic receptor knockout mutant strain and the anaplerosis strain of the chemotactic receptor knockout mutant strain at the rhizosphere of wheat by using a microenvironment system.
Further, the root secretion components in the step (1) comprise amino acids, organic acids and saccharides; the molecular docking is realized by molecular docking software AutoDock 4.2; the binding free energy was calculated by the software Amber 17.
Further, in the step (2), the receptor protein corresponding to the candidate strong chemoattractant is determined according to the candidate strong chemoattractant by firstly measuring the chemotaxis threshold concentration value of the microorganism by using a capillary chemotaxis method, determining the response intensity of the microorganism to the chemoattractant and then further identifying the receptor protein of the candidate strong chemoattractant by using a fluorescence thermal drift method and a flat chemotaxis method.
Furthermore, the fluorescence thermal drift value of the chemoattractant and the receptor protein screened by adopting a fluorescence thermal drift method is higher than 2.0 ℃.
Further, in the step (3), the Escherichia coli is Escherichia coli S17-1.
Further, the specific operation of utilizing the microenvironment system in the step (5) is as follows:
a. adding 0.5 XMS culture solution of the inoculation bacterial suspension into the test tube with the cover, and fixing the rhizome combination part of the sterile wheat seedling at the liquid level by using a filter screen;
b. b, placing the wheat seedling sample treated in the step a in 16h illumination for 8h dark culture, taking out half of the wheat seedling after inoculation culture for 2h, transferring the wheat seedling to a microenvironment system without inoculation suspension for culture, and taking the wheat seedling as a transfer group, wherein the other half of the wheat seedling is an untransferred group;
c. b, sampling two groups of wheat seedlings in the step b at 2h, 6h, 10h, 14h, 18h, 22h, 26h, 30h, 34h and 38h respectively, and determining the number of bacteria colonized in the rhizosphere;
d. and (3) fitting a growth kinetic equation of the bacteria in the rhizosphere by using a Logistic equation, solving the first derivative of the growth kinetic equation to obtain a growth rate value, and calculating to obtain the chemotactic rate and the proliferation rate of the rhizosphere of the wheat of different inoculated bacteria.
Further, the inoculated strain in the step a refers to a microorganism, a chemotactic receptor knockout mutant strain and a complementation strain of the chemotactic receptor knockout mutant strain.
Further, the wheat seedling sample in the step b needs shading treatment when placed in 16h illumination and 8h dark culture.
Further, in the step d, the wheat rhizosphere proliferation rate is the growth rate of the transferred group, and the wheat rhizosphere chemotaxis rate is the difference value between the growth rate of the non-transferred group and the growth rate of the transferred group.
Preferably, the microorganism is pseudomonas UW4.
The method is applied to identifying the chemotactic substance as the key chemotactic substance of PGPR in rhizosphere chemotactic colonization, and the specific method comprises the following steps: if the chemotactic rate of the chemotactic receptor knockout mutant strain is obviously lower than that of a anaplerotic strain of the microorganism and the chemotactic receptor knockout mutant strain, the chemotactic substance can be determined to be a key chemotactic substance for the rhizosphere chemotactic colonization of PGPR.
The invention has the following beneficial effects:
1. the method can distinguish the chemotaxis rate of a certain chemotactic substance of PGPR from the reproduction rate of the certain chemotactic substance on the root surface, and can determine that the chemotactic substance is a strong chemotactic substance for chemotactic colonization of the PGPR at the root zone by comparing the chemotactic receptor deletion strain of the certain chemotactic substance of the PGPR with the chemotaxis rate difference of a wild-type strain and a anaplerosis strain if the chemotaxis rate of the deletion strain is obviously lower than that of the wild-type strain and the anaplerosis strain.
2. Taking pseudomonas UW4 as an example, firstly, performing molecular docking and binding free energy analysis on pseudomonas UW4 chemotactic receptors and root secretion components, and finding that the binding free energy of the root secretion components and the pseudomonas UW4 chemotactic receptors is in positive correlation with the logarithm of chemotactic threshold values of the UW4 capillary chemotactic root secretion components; further identifying 3 chemotactic receptors of pseudomonas UW4, namely 1-aminocyclopropane-1-carboxylic acid chemotactic receptor, cysteine chemotactic receptor and malic acid chemotactic receptor; then a microenvironment system is used for measuring the chemotactic rate of pseudomonad UW4 and 3 chemotactic receptor deletion mutant strains and anaplerosis strain chemotactic colonized wheat rhizosphere; finally, according to the chemotactic rate of pseudomonas UW4 and 3 chemotactic receptor deletion mutants thereof in chemotactic colonizing the rhizosphere of wheat, the strong chemotactic compound of the UW4 chemotactic wheat rhizosphere is 1-aminocyclopropane-1-carboxylic acid. The chemotaxis and receptor binding free energy, chemotaxis and growth rule of the microorganism have universality, and the universality and feasibility of the method are verified by utilizing pseudomonas UW4.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a graph of the correlation between the binding free energy of Pseudomonas UW4 family I chemoattractant receptors and partial root exudate components and the chemotactic threshold concentration at which they chemotactic these root exudate components.
FIG. 2 shows a fluorescence thermal drift reaction sequence.
FIG. 3 shows the results of a fluorescence thermal shift analysis of the receptor binding domain (LBD) of the chemoattractant receptor wp116 and the root exudate component.
FIG. 4 shows the fluorescence thermal drift analysis results of the components of the chemotactic receptor wp375-LBD protein and root exudates; wherein ACC: 1-aminocyclopropane-1-carboxylic acid; a05: succinic acid; b09: l-lactic acid; b10: formic acid; c03: d, L-malic acid; c08: acetic acid; e02: tartaric acid; f02: citric acid; f05: fumaric acid; f07: propionic acid; f08: acid sticking; f09: glycolic acid F10: glyoxylic acid; g07: an acetoacetic acid; g11: d-malic acid; g12: l-malic acid; h08: pyruvic acid.
FIG. 5 shows the results of fluorescence thermal drift analysis of the components of the chemotactic receptor wp616-LBD protein and root exudate; wherein ACC: 1-aminocyclopropane-1-carboxylic acid; a07: l-alanine; a08: l-arginine; a09: l-asparagine; a10: l-aspartic acid; a11: l-cysteine; a12: l-glutamic acid; b01: l-glutamine; b02: glycine; b04: l-isoleucine; b05: l-leucine; b06: l-lysine; b07: l-methionine; b08: l-phenylalanine; b09: l-proline; b11: l-threonine; b12: l-tryptophan; c02: l-valine; c03: d-alanine; c04: d-asparagine; c05: d-aspartic acid; c06: d-glutamic acid; c07: d-lysine; c08: d-serine; c09: d-valine.
FIG. 6 shows chemotactic reactions of 3 acceptor mutants of Pseudomonas UW4 and their anaplerosis and anaplerosis null strains to 1-aminocyclopropane-1-carboxylic acid, cysteine and malic acid.
Fig. 7 is a schematic illustration of the microenvironment.
FIG. 8 shows the maximum colonization rate, proliferation rate and chemotaxis rate of wheat rhizosphere of Pseudomonas UW4, UW4 Δ wp116+ wp 116; the different capital letters marked on the data bars indicate significant differences (p < 0.01).
FIG. 9 shows the maximum colonization rate, proliferation rate and chemotaxis rate of wheat rhizosphere of Pseudomonas UW4, UW4 Δ wp616+ wp 616.
FIG. 10 shows the maximum colonization rate, proliferation rate and chemotaxis rate of wheat rhizosphere of Pseudomonas UW4, UW4 Δ wp375 and UW4 Δ wp375+ wp 375.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.
A method for identifying the chemotactic rate of PGPR chemotactic root exudates to the rhizosphere comprises the following steps:
(1) Searching an extracellular ligand binding domain of the microorganism from the annotation of the microorganism genome, screening a chemotactic receptor capable of identifying an extracellular chemoattractant, carrying out molecular docking on the chemotactic receptor and root exudate components by utilizing a molecular docking technology, calculating binding free energy, and screening the root exudate components with low free energy values as candidate strong chemoattractants;
(2) Determining a receptor protein corresponding to the candidate chemotactic substance according to the candidate chemotactic substance, and constructing an expression vector containing an extracellular ligand binding domain fragment of the receptor protein and a recombinant vector for knocking out the receptor protein by taking the genomic DNA of the microorganism as a template;
(3) Competent transfer of Escherichia coli containing the recombinant vector of step (2) into microorganism to obtain chemotactic receptor knockout mutant strain;
(4) Constructing anaplerotic plasmid and anaplerotic no-load plasmid of receptor protein corresponding to the candidate strong chemoattractant in the step (2) by taking genome DNA of the microorganism as a template, and respectively transferring the anaplerotic plasmid and anaplerotic no-load plasmid into the chemotactic receptor knockout mutant strain in the step (3) to obtain an anaplerotic strain and an anaplerotic no-load strain of the chemotactic receptor knockout mutant strain;
(5) Carrying out a flat plate chemotaxis experiment on the microorganism, the chemotactic receptor knockout mutant strain, the anaplerosis strain and anaplerosis empty strain of the chemotactic receptor knockout mutant strain and the candidate strong chemotaxis compound to verify the correspondence between the receptor protein and the chemotaxis compound, and then measuring the chemotaxis rate and the proliferation rate of the microorganism, the chemotactic receptor knockout mutant strain and the anaplerosis strain of the chemotactic receptor knockout mutant strain at the rhizosphere of wheat by using a microenvironment system.
Further, the root secretion components in the step (1) comprise amino acids, organic acids and saccharides; the molecular docking is realized by molecular docking software AutoDock 4.2; the binding free energy was calculated by the software Amber 17.
Further, in the step (2), the receptor protein corresponding to the candidate compound is determined according to the candidate compound by determining the chemotaxis threshold concentration value of the microorganism by using a capillary chemotaxis method, determining the response intensity of the microorganism to the compound, and then further identifying the receptor protein of the candidate compound by using a fluorescence thermal drift method and a flat chemotaxis method.
Furthermore, the fluorescence thermal drift value of the chemoattractant and the receptor protein screened by adopting a fluorescence thermal drift method is higher than 2.0 ℃.
Further, in the step (3), the Escherichia coli is Escherichia coli S17-1.
Further, the specific operation of utilizing the microenvironment system in the step (5) is as follows:
a. adding 0.5 XMS culture solution of the inoculated bacterial suspension into the test tube with the cover, and fixing the rhizome combination part of the sterile wheat seedling at the liquid level by using a filter screen;
b. b, placing the wheat seedling sample treated in the step a in 16h illumination for 8h dark culture, taking out half of the wheat seedling after inoculation culture for 2h, transferring the wheat seedling to a microenvironment system without inoculation suspension for culture, and taking the wheat seedling as a transfer group, wherein the other half of the wheat seedling is an untransferred group;
c. b, sampling two groups of wheat seedlings in the step b at 2h, 6h, 10h, 14h, 18h, 22h, 26h, 30h, 34h and 38h respectively, and determining the number of bacteria colonized in the rhizosphere;
d. and (3) fitting a growth kinetic equation of the bacteria in the rhizosphere by using a Logistic equation, solving the first derivative of the growth kinetic equation to obtain a growth rate value, and calculating to obtain the chemotactic rate and the proliferation rate of the rhizosphere of the wheat of different inoculated bacteria.
Further, the inoculated strain in the step a refers to a microorganism, a chemotactic receptor knockout mutant strain and a replenisher strain of the chemotactic receptor knockout mutant strain; wherein the MS culture solution formula (mg/L): potassium nitrate 1900, ammonium nitrate 1650, monopotassium phosphate 170, magnesium sulfate 370, calcium chloride 440, potassium iodide 0.83, boric acid 6.2, manganese sulfate 22.30, zinc sulfate 8.6, sodium molybdate 0.25, copper sulfate 0.025, cobalt chloride 0.025, ferrous sulfate 27.8, inositol 100, glycine 2, thiamine hydrochloride 0.1, pyridoxine hydrochloride 0.5, nicotinic acid 0.5 and disodium ethylenediaminetetraacetate 37.3. The pH value is 5.7, and the product is stored at normal temperature.
Furthermore, the root of the wheat seedling needs shading treatment when the wheat seedling sample in the step b is placed in 16h of illumination and 8h of dark culture.
Furthermore, the wheat rhizosphere proliferation rate in the step d is the growth rate of the metastatic group, and the wheat rhizosphere chemotaxis rate is the difference value between the growth rate of the non-metastatic group and the growth rate of the metastatic group.
Preferably, the microorganism is pseudomonas UW4.
Taking pseudomonas UW4 as an example, the materials adopted in the application are all purchased and obtained in commercial grade without special description, and the specific operation is as follows:
examples
1. Molecular docking technique for analyzing binding free energy of root secretion components (chemotactic substances) and chemotactic receptors
Taking Pseudomonas UW4 as an example, pseudomonas UW4 (Pseudomonas sp. UW 4): a commonly used strain with the American agricultural Research Culture Collection accession number NRRL B-50193 (the collection date is 2008/6/9) and is publicly available (the American agricultural Research Culture Collection, english full name is agricultural Research Culture Collection, NRRL for short, the center is located in Pickia of Illinois, and is a government-nature Culture Collection supported by the American agricultural Research center).
A total of 15 group I chemoattractant receptors with extracellular ligand-binding domains capable of recognizing extracellular chemoattractants are co-annotated in the genome of this strain (genome sequence GenBank accession No.: CP 003880). 59 common typical root system secretion components are selected, wherein 26 amino acids, 21 organic acids and 12 saccharides are selected. And (3) performing molecular docking on chemotactic receptors and root secretion components by adopting molecular docking software AutoDock 4.2, and calculating the binding free energy by using software Amber 17. The smaller the binding free energy value, the higher the affinity of the receptor for the chemoattractant, and the stronger the chemotactic response. The lower the binding free energy of a certain chemoattractant to a certain chemoattractant receptor, the lower the binding free energy of both, indicating that they may be the relationship of the ligand (chemoattractant) to the receptor.
2. Relationship between binding free energy of chemoattractant and chemoattractant receptor and chemotactic response intensity
According to the fact that the binding free energy of chemotactic substance and chemotactic receptor is low, medium and high, representative compounds 1-aminocyclopropane-1-carboxylic acid, lysine, threonine, glutamic acid and serine are selected from amino acid compounds, representative compounds glycolic acid, guava acid, propionic acid and fumaric acid are selected from organic acid compounds, galactose, rhamnose, maltose, sucrose, oligosaccharide and glucose are selected from carbohydrate compounds (table 1), 18 compounds are respectively subjected to capillary chemotaxis analysis, and the chemotaxis threshold concentration value of pseudomonas UW4 is determined. The chemotactic threshold concentration value of Pseudomonas UW4 for a certain chemotactic substance indicates that the chemotactic response of the bacterium to the chemotactic substance is stronger. Correlation analysis of the absolute value of the lowest binding free energy of the 18 compounds with pseudomonas UW4 group I chemoattractant receptors with the logarithm of the UW4 capillary chemotaxis threshold concentration value showed a significant positive correlation (p < 0.0001) (fig. 1), indicating that the lower the free energy of chemoattractant binding to receptor, the lower the chemotaxis threshold concentration of UW4 for chemoattractant, i.e. the stronger the chemotactic response of UW4.
Capillary chemotaxis method: the UW4 cultured in liquid LB for 24h is inoculated into TB liquid culture medium according to the ratio of 1 (v/v) to 100 (v/v) for 24h, 20ml of bacteria liquid is respectively taken to collect bacteria, the bacteria are washed twice by sterilized ultrapure water and then inoculated into two bottles of 250ml of glycerol salt culture medium (0.5% of glycerol and 3.0mM of chemoattractant) for overnight culture until OD660= 0.3-0.45, the collected bacteria are washed twice by chemotactic buffer pre-cooled at 4 ℃ and then resuspended to 15ml, 300ul of bacteria liquid is respectively sucked and placed in each small hole of a 96 micro-well plate.
The chemoattractant was formulated separately into 5X 10 -1 、1×10 -1 、5×10 -2 、1×10 -2 、5×10 -3 、1×10 -3 、5×10 -4 、1×10 -4 、 5×10 -5 、1×10 -5 、5×10 -6 、1×10 -6 、5×10 -7 、1×10 -7 、5×10 -8 Sucking 100ul of chemotactic substance solution in mol/L, placing in another 96-micro-well plate, sterilizing with 3cm length, inner diameter of about 0.2mm and volume of 1ulOne capillary is sealed by melting and then rapidly passes over the flame for several times, the capillary is inserted into chemotactic substance solution to enable the chemotactic substance solution to absorb about 1/3 of the volume of the capillary, a gun head is used for absorbing chemotactic buffer solution to wash the outer wall of the capillary clean, the sealed end of the capillary is inserted into a 96 micro-porous plate containing 3% agar, then the plate is inserted into a micro-hole filled with bacterial liquid in a turnover mode, and chemotaxis is carried out for 2 hours at room temperature. The chemotactic capillary was removed from the upper plate and the outer wall was rinsed clean and the contents were decanted into a 2ml centrifuge tube. After the content of PBS buffer solution is appropriately gradient dilution, containing Amp 100u g/mL solid LB plate, 30 degrees C were cultured for 36h after colony count. And making a log-log curve of the colony number and the concentration of the chemotaxis buffer solution, and taking a concentration value corresponding to the intersection point of the curve extension of the sudden increase of the colony number in the curve and the colony number line in the chemotaxis buffer solution as a chemotaxis threshold concentration value.
TABLE 1 binding free energy of Pseudomonas UW4 group I chemotactic receptors with partial root exudate component and chemotactic threshold concentration of Pseudomonas UW4 with partial root exudate component
3. Selecting candidate strong chemoattractant of pseudomonas UW4 and constructing receptor deletion mutant strain thereof
According to the conclusion that the chemotactic response of the strain is stronger as the free energy of the binding of the chemoattractant to a receptor is lower, 1-aminocyclopropane-1-carboxylic acid and cysteine are selected from amino acid compounds, and malic acid is selected from organic acid compounds to be candidate chemoattractants. Since many PGPR strains have a weak chemotactic response to carbohydrates, candidate chemoattractants are not selected from among carbohydrates. The binding energy of 1-aminocyclopropane-1-carboxylic acid to wp116 was-9.51 kcal/mol, which was the lowest compound of all root exudate components that could bind to the receptor. Cysteine bound to the receptor protein WP616 (GenBank accession No. WP _ 015096616) with the lowest binding energy, 7.47kcal/mol. The binding energy of malic acid to the receptor protein WP375 (GenBank accession WP-015093375) was the lowest among the organic acids, at-7.29 kcal/mol.
To construct candidate chemoattractant receptor mutants, it was necessary to identify the 3 candidate chemoattractant receptors. The 3 candidate receptors for the chemoattractant were identified by fluorescence thermal drift and plate chemotaxis.
3.1 heterologous expression of ligand binding domains of candidate Strong chemoattractant receptors
3.1.1 construction of expression vectors for ligand binding domains of candidate Strong chemoattractant receptors
Pseudomonas UW4 genome DNA is used as a template, the primer sequence is shown in a table 2, and a PCR reaction is carried out to obtain a ligand binding domain DNA fragment of a candidate strong chemoattractant receptor. The PCR reaction conditions are shown in Table 3.
TABLE 2 primer Table
Underlined sequences are BamHI and HindIII restriction site sequences, respectively
TABLE 3PCR reaction System and procedure
The PCR product was enzymatically ligated with plasmid pET-28a after double digestion to obtain expression plasmids pET-28a-LBD116, pET-28a-LBD616 and pET-28a-LBD375 for the ligand binding domains of 3 candidate chemoattractant receptors. The reaction system and reaction conditions of the enzyme digestion and enzyme ligation are shown in tables 4 and 5.
TABLE 4 digestion System and reaction conditions
TABLE 5 recombination systems and reaction conditions
3.1.2 ligand binding Domain protein expression and purification of candidate Strong chemotactic receptors
E.coli BL21 (DE 3) was transformed with the expression vectors pET-28a-LBD116, pET-28a-LBD616 and pET-28a-LBD375, respectively, the correctly verified BL21 transformant was inoculated in LB liquid medium containing 100. Mu.L/mL Kana resistance, shake-cultured at 37 ℃ and 220rpm until OD600 was 0.5, protein expression was induced by adding 0.3mM IPTG inducer, and protein expression was induced at 16 ℃ and 180rpm for 8 hours. The protein purification method is as follows:
1) Centrifuging the induced BL21 bacterial solution 250mL,12000rpm at 4 ℃ for 10min to collect thalli, abandoning supernatant, resuspending and washing the thalli with sterile water for three times, finally resuspending the thalli with 20mL PBS buffer, filtering the thalli with a 1mL disposable sterile syringe, and preventing cell clusters from blocking a cell disruption instrument (the purification operation is performed on ice to prevent protein denaturation);
2) Crushing the re-suspended and filtered thallus for 5 times by using a low-temperature ultrahigh-pressure crusher until the liquid is clear and transparent, wherein the pressure value of the low-temperature ultrahigh-pressure crusher is set to be 1000pa;
3) Centrifuging the crushed sample at 12000rpm and 4 ℃ for 20min, taking out supernatant to obtain crude enzyme solution, and resuspending the precipitate with 20mL of sterile water;
4) Cleaning the pre-filled nickel ion protein purification column by using 10mL of sterile water;
5) Washing the nickel column with 5mL of 20mM imidazole;
6) Filtering the supernatant with a 0.22 μm microporous membrane, slowly passing the filtered supernatant through a nickel column, collecting flow-through liquid, and repeatedly passing the column for 3 times;
7) Eluting nickel column with 20mM, 50mM, 100mM, 250mM and 500mM imidazole at different concentrations, collecting 10mL of each concentration, and determining protein concentration with nucleic acid protein detector;
8) Regenerating the nickel column with 20mM imidazole and washing the nickel column with 10mL sterile water;
9) Sealing and storing the nickel column by using 5mL of 20% ethanol;
10 Supernatant pellet and each eluted concentration sample were subjected to SDS-PAGE.
11 10mL of eluent containing a single target protein band is added into an inner tube of the ultrafiltration tube, and the mixture is centrifuged at 4000g at 4 ℃ until 1.5mL of inner tube liquid is remained;
12 Adding sterile water to 10mL, centrifuging at 4 ℃ and 4000g until 1.5mL of inner tube liquid remains, and repeating for three times;
13 The concentrated protein solution in the inner tube is sucked by a pipette gun and transferred to a centrifuge tube for preservation at 4 ℃.
3.2 fluorescence thermal Drift analysis of melting temperature Change value (. DELTA.Tm) of LBD bound to root exudate component
Using biolg ecoplates as ligands, each plate had 95 ligand species and one negative control. Using a Saimeifei Protein Thermal Shift TM The dye kit was subjected to a fluorescence thermal drift experiment, and the reaction system is shown in Table 6.
TABLE 6 fluorescent thermal Drift reaction System
Fluorescence thermal drift detection was performed using a QuantStudio7Flex fluorescence quantifier with the program set-up shown in figure 2.
Data were analyzed using Protein Thermal Shift Software 1.3 Software with the parameter setting Δ Tm threshold of 2.0 ℃. The Δ Tm of the LBD protein of wp116 with 1-aminocyclopropane-1-carboxylic acid, gamma-aminobutyric acid, L-arginine, L-asparagic acid, L-phenylalanine, L-serine, gamma-hydroxybutyric acid, L-threonine, itaconic acid and glycyl-L-glutamic acid were each greater than 2.0 ℃, indicating that the receptor protein had a strong affinity with each of the above ligand species, and in particular that the Δ Tm value for the reaction with 1-aminocyclopropane-1-carboxylic acid was 7.99 ℃, from which it was presumed that wp116 should be a chemoattractant receptor for 1-aminocyclopropane-1-carboxylic acid (FIG. 3).
The fluorescence thermal drift value of the wp375-LBD domain protein exceeds the threshold value of 2.0 ℃ and is 3.545 ℃ only with D, L-malic acid (FIG. 4). The wp375-LBD domain protein is proved to have stronger affinity with D, L-malic acid, so the D, L-malic acid is presumed to be a receptor chemotactic receptor of the UW4 wp375 receptor protein. The fluorescence thermal drift value of the wp616-LBD domain protein only exceeds the threshold value of 2.0 ℃ and is 3.15 ℃ (FIG. 5), which indicates that the wp616-LBD domain protein has stronger affinity with L-cysteine, so the L-cysteine is presumed to be the receptor protein chemotactic receptor of UW4 wp 616.
3.3 construction of Pseudomonas UW4 chemokine receptor mutants and complementation strains
3 mutant strains of candidate strong chemoattractant receptors of pseudomonas UW4 are constructed by adopting the parental hybridization technology.
3.3.1 construction of homologous recombinant traceless knockout chemokine receptor plasmids
Using pseudomonas UW4 genome DNA as a template, adopting receptor upstream and downstream homologous arm primers, wherein the primer sequences are shown in table 7, and carrying out PCR reaction to obtain 3 candidate strong chemoattractant receptor homologous recombination knocked-out upstream and downstream homologous arm DNA fragments. The PCR reaction conditions are shown in Table 3. The PCR product is enzymatically linked with a suicide vector pEX18Gm after double enzyme digestion to obtain 3 candidate strong chemotaxis receptor homologous recombination traceless knockout chemotaxis receptor plasmids pEX18Gm-116, pEX18Gm-616 and pEX18Gm-375. The reaction system and reaction conditions of the enzyme digestion and enzyme ligation are shown in tables 4 and 5. The 3 plasmids were individually transformed into E.coli S17-1.
TABLE 7 upstream and downstream homology arms of each receptor protein and gene expression cassette amplification primer sequences
Note: underlined is the restriction site and the name is annotated in the following brackets.
3.3.2 Dual parent hybridization
Correctly verified Escherichia coli S17-1 containing knockout plasmid and Pseudomonas putida UW4 are respectively inoculated into liquid LB culture medium according to the inoculation amount of 1% (v/v), and are respectively placed in shaking tables at 37 ℃ and 30 ℃ for overnight culture at 220 rpm. After the culture is finished, collecting 1mL S17-1 bacterial liquid in a centrifuge tube (several parallel cells can be made), centrifuging at 12000rpm for 1min, discarding the supernatant, re-suspending the bacterial liquid by using 500 mu L of 0.85% NaCl solution, centrifuging at 12000rpm for 1min, discarding the supernatant, adding 1.5mL UW4 bacterial liquid into the centrifuge tube, centrifuging at 12000rpm, discarding the supernatant, re-suspending the bacterial liquid by using 500 mu L of 0.85% NaCl solution, repeating the step for 5 times, finally re-suspending the bacterial liquid by using 100 mu L of 0.85% NaCl solution, dropwise adding the bacterial liquid on a cellulose membrane which is flatly paved on an LB solid plate, and standing and culturing at 30 ℃ for 24h. After the culture is finished, the thalli are washed off the nitrocellulose membrane by 750 mu L of 0.85 percent NaCl solution, after the bacterial liquid is resuspended, 50 mu L of the bacterial liquid is sucked and coated on an LB solid plate containing 25 mu g/mL gentamicin and 100 mu g/mL ampicillin, and the LB solid plate is placed at the constant temperature of 30 ℃ for static culture for 12-16h to generate single exchange.
Selecting single exchanger, subculturing in liquid LB at 30 deg.C and 220rpm for 5h, coating solid LB plate containing 10% aminobenzyl 100 μ g/mL sucrose, and standing at 30 deg.C for 24-36h. The colony that can grow out is the double-exchanger. The double exchangers are chemotactic receptor knockout mutants and are respectively named as UW4 delta wp116, UW4 delta wp616 and UW4 delta wp 375. 3.3.3 construction of complementation Strain of chemotactic receptor mutant strains
Pseudomonas UW4 genome DNA is used as a template, a receptor anaplerosis primer is adopted, the sequence of the primer is shown in a table 7, and 3 protein expression cassettes of candidate strong chemoattractant receptors are obtained through PCR reaction. The PCR reaction conditions are shown in Table 3. The PCR product is enzymatically linked with a broad host expression plasmid pBBR1MCS2 after double enzyme digestion to obtain the anaplerotic plasmids pBBR1MCS2-116, pBBR1MCS2-616 and pBBR1MCS2-375 for expressing 3 candidate chemoattractant receptors. The reaction systems and reaction conditions of the enzyme digestion and enzyme ligation are shown in tables 4 and 5. The 3 plasmids and the empty plasmid were separately transferred into E.coli S17-1. The complementation plasmid and the empty-load plasmid are respectively transferred into UW4 delta wp116, UW4 delta wp616 and UW4 delta wp375 by adopting a double-parent hybridization method, and are named as UW4 delta wp116-wp116, UW4 delta wp616-wp616 and UW4 delta wp375-wp375, and the complementation empty-load strains UW4 delta wp116-pBBR1MCS2, UW4 delta wp616-pBBR1MCS2 and UW4 delta wp375-pBBR1MCS2.
The parental hybridization method comprises the following steps: inoculating the constructed Escherichia coli S17-1 (donor bacteria) and chemotactic receptor knockout mutant (recipient bacteria) containing anaplerotic plasmid and anaplerotic no-load plasmid into liquid LB for overnight culture at 30 ℃, and inoculating the cultured bacteria liquid into LB liquid according to 1% (v/v) for culture for 3-4h. After the culture is finished, respectively collecting 2mL of donor bacterium liquid in a centrifuge tube, centrifuging at 12000rpm for 1min to discard a supernatant, resuspending the bacterium with 500 μ L of 0.85% NaCl solution, centrifuging at 12000rpm for 1min to discard the supernatant, adding 2mL of acceptor bacterium liquid, centrifuging at 12000rpm for 1min to discard the supernatant, resuspending the bacterium with 500 μ L of 0.85% NaCl solution, centrifuging at 12000rpm for 1min to discard the supernatant, finally resuspending the bacterium with 75 μ L of 0.85% NaCl solution, dripping the bacterium liquid on a nitrocellulose membrane, placing on an LB solid plate, and culturing at the constant temperature of 30 ℃ for 24h. After the culture, the cells were washed off the nitrocellulose membrane with 750. Mu.L of 0.85% NaCl solution, 50. Mu.L of the resuspended bacterial suspension was applied to LB solid plate containing kana 100. Mu.g/mL and ampicillin 100. Mu.g/mL, and the cells were incubated at 30 ℃ for 12-16 hours. The grown colonies are anaplerotic strains and anaplerotic no-load strains of the chemotactic receptor mutant strains.
3.3.4 chemotactic receptor mutant plate chemotaxis assay
Inoculating the strain into a glycerol salt culture medium, culturing at 28 deg.C and 200rpm to OD 600 Centrifuging at 8000rpm and 4 deg.C for 5min, discarding supernatant, washing thallus with 4 deg.C precooled chemotaxis buffer solution about 20mL for 2 times, suspending in 200mL bacterial chemotaxis medium containing 0.2% agarose, and adjusting OD 600 About 1.0 or so, pour plates, add about 10mL of medium to each plate, after coagulation, aspirate 10 μ l0.5m 1-aminocyclopropane-1-carboxylic acid, malic acid or cysteine, spot in the center of the plate, observe the bacterial chemotactic response at 30 ℃ and record by taking pictures in the dark.
The formula of the glycerolate culture medium comprises: glycerol 5g (Sterilization alone), K 2 HPO 4 ·3H 2 O 14.7g，KH 2 PO 4 4.8g，MgSO 4 ·7H 2 O 0.25g，Fe 2 (SO 4 ) 3 0.5mg, 1000mL ultrapure water, sterilized at 121 deg.C for 30min, and 3mM ACC mother liquor (N source can be replaced by (NH) before use 4 ) 2 SO 4 2.0g)。
Chemotaxis buffer (CMB): (pH7.0 phosphate buffer 10) -1 M，EDTA10 -4 M) weighing K 2 HPO 4 14.03g， KH 2 PO 4 5.24g, EDTA 0.0372g, dissolved in 1000mL of ultrapure water, and sterilized at 121 ℃ for 30min.
Bacterial chemotaxis medium: (pH7.0 phosphate buffer 10) -1 M，EDTA10 -4 M) weighing K 2 HPO 4 14.03g，KH 2 PO 4 5.24g, EDTA 0.0372g, agarose 2g, dissolved in 1000mL of ultrapure water and sterilized at 121 ℃ for 30min.
The chemotactic results of the pseudomonas UW4 and the chemotactic receptor mutant strain, anaplerotic strain and anaplerotic unloaded strain plate thereof are shown in figure 6. 3. The receptor mutant strain and the anaplerotic unloaded strain can not chemotact the corresponding chemoattractant, and the anaplerotic strain and the original strain can chemotaxis the corresponding chemoattractant, which shows that the 3 receptors and the corresponding chemoattractant are the relationship of the receptor and the ligand.
4. Determination of pseudomonad UW4 and receptor mutant strain and anaplerosis strain chemotaxis colonization wheat rhizosphere chemotaxis rate thereof
A microenvironment system is adopted to determine chemotaxis rate and proliferation rate of UW4 and receptor knockout strains and receptor knockout anaplerosis strains thereof in wheat rhizosphere. The microenvironment system is 0.5 × MS culture solution containing inoculation bacterial suspension in test tube with cover, the rhizome joint of aseptic wheat seedling is fixed on the liquid surface with filter screen (fig. 7), and the seedling is cultured in dark for 16h under light and 8h, and the root part is shaded. And (4) inoculating a half of the wheat seedlings, culturing for 2 hours, taking out, and transferring to a microenvironment system without inoculated bacterial suspension for culturing. Two groups of wheat seedlings are sampled at 2h, 6h, 10h, 14h, 18h, 22h, 26h, 30h, 34h and 38h respectively, and the number of bacteria colonized in the rhizosphere is determined. And (3) fitting a growth kinetic equation of the bacteria at the rhizosphere by using a Logistic equation, and solving the first derivative of the growth kinetic equation to obtain a growth rate value. The growth rate of the nonmetastatic group is the sum of the chemotactic rate and the proliferation rate, the growth rate of the metastatic group is the proliferation rate, and the difference between the growth rate of the nonmetastatic group and the growth rate of the metastatic group is the chemotactic rate. The maximum rate value can be found from the rate equation. The maximum chemotactic rate of UW 4-delta wp116 is significantly lower than that of UW4 and UW 4-delta wp116+ wp116, and the maximum chemotactic rate of UW4 is not different from that of UW 4-delta wp116+ wp 116. The maximal chemotaxis rate and the sum of the proliferation rates and the maximal proliferation rate were not different for the three strains (FIG. 8). No difference was found between the sum of the maximum chemotactic rate and the proliferation rate, the maximum proliferation rate and the maximum chemotactic rate among three strains of UW4, UW4- Δ wp616 and UW4- Δ wp616+ wp616 (FIG. 9). The sum of the maximum chemotaxis rate and the proliferation rate, the maximum proliferation rate and the maximum chemotaxis rate are not different among three strains of UW4, UW 4-delta wp375 and UW 4-delta wp375+ wp375 (figure 10). The deletion of ACC chemotactic receptor is shown to remarkably reduce the rate of UW4 chemotactic rhizosphere, while the deletion of cysteine chemotactic receptor and malic acid chemotactic receptor has no influence on the rate of UW4 chemotactic rhizosphere, and ACC is a strong chemotactic object for the UW4 chemotactic rhizosphere colonization.
MS culture solution formula (mg/L): potassium nitrate 1900, ammonium nitrate 1650, monopotassium phosphate 170, magnesium sulfate 370, calcium chloride 440, potassium iodide 0.83, boric acid 6.2, manganese sulfate 22.30, zinc sulfate 8.6, sodium molybdate 0.25, copper sulfate 0.025, cobalt chloride 0.025, ferrous sulfate 27.8, inositol 100, glycine 2, thiamine hydrochloride 0.1, pyridoxine hydrochloride 0.5, nicotinic acid 0.5 and disodium ethylenediaminetetraacetate 37.3. The pH value is 5.7, and the product is stored at normal temperature.
Preparing wheat seedlings: selecting full wheat seeds without mildew points and damage as experimental materials, selecting 200 wheat seeds, cleaning the wheat seeds in a workbench for three times by using sterile water, and then using 0.1% HgCl 2 Soaking for 5min, washing with sterile water for three times to remove residual HgCl 2 Wheat was placed cord down on 1.5% water agar plates (providing continuous moisture) with 10 seeds placed on each plate to prevent the roots from sticking to each other after germination. Sealing the plate, and culturing in 25 deg.C incubator in dark for 3d.
And (3) analyzing the imprint: and (3) taking the seeds with the sterilized surfaces, placing the seeds on an antibiotic-free LB culture medium for 30s, taking the wheat away, and placing a flat plate at 25 ℃ for culturing for 24h to check the sterilization effect.
Microenvironment establishment: cutting a filter screen made of polyvinyl chloride into a 20cm long strip-shaped inverted U-shaped shape, putting the inverted U-shaped shape into a 50mL glass test tube with a cover, adding 20mL and 0.5 XMS culture medium to prepare a microenvironment, putting a test tube screw cover (not too tightly screwing to prevent a thermal expansion and cold contraction explosion bottle) filled with the MS culture medium into a sterilization pot for sterilization at 121 ℃ for 20min, after completely cooling, putting germinating seeds on the filter screen in an ultraclean workbench by using sterile tweezers, and enabling the roots to penetrate through the holes of the filter screen, so that the liquid level just contacts the seeds, and the roots are completely soaked below the liquid level. The bottom of the glass test tube is wrapped by tinfoil to prevent illumination. The strain is cultured with shaking at 30 deg.C and 220rpm for 5h to logarithmic phase, OD600 of each strain liquid is adjusted to 0.5 with 0.5 × MS culture medium, and 200 μ L of each microenvironment is inoculated.
Root sampling and bacterial count: the plants were removed from the microenvironment with sterile forceps, washed 3 times with sterile PBS buffer, the phyllospheric part (the part above the basal part of the hypocotyl) was removed with an ethanol sterilized single-sided knife, the part was weighed in 1.5mL centrifuge tubes, each centrifuge tube was weighed individually, the total weight was subtracted from the weight of the neat centrifuge tube to obtain the root weight, 500 μ L of PBS buffer was added to the 1.5mL centrifuge tubes and the roots were ground with a glass pestle to homogenize, releasing the bacteria colonized in the roots.
Adding 100 μ L of the homogenized liquid into the first row of sterile 96-well plate, adding 180 μ L of PBS buffer solution into the last five rows, adding 20 μ L of the solution from the first row into the last row with a discharge gun, blowing, mixing, and repeating until the sample is diluted to 10% -5 . 10 μ L of each diluted gradient solution was spotted counterclockwise onto LB solid plates containing 100 μ g/mL Amp resistance, and one plate was spotted with six gradients for one sample. Note that the distance of each spotting is not too close to prevent colonies from clumping together and failing to count. And (3) after sample application, standing for 30min, sealing and inverting the flat plate after the bacterial liquid is dried, and putting the flat plate into an incubator for incubation at 30 ℃ for 24h.
And (3) counting colonies: plates after 24h incubation were removed and the sample dilution gradient and colony counts between 3-30 were recorded.
Performing an Effect analysis
By utilizing the identification method of the application, 15 annotated group I chemotactic receptors which have extracellular ligand binding domains and can recognize extracellular chemoattractant in a pseudomonas UW4 genome and 59 common typical root secretion components are subjected to molecular docking and binding free energy analysis, 18 compounds including partial amino acids, organic acids and saccharides are selected according to the fact that the binding free energy of the chemoattractant and the chemoattractant receptor is low, medium and high, capillary chemotaxis analysis is respectively carried out, and the lower the free energy of the chemoattractant and the receptor binding is, the lower the chemotaxis threshold concentration of the UW4 to the chemoattractant is, namely, the stronger the chemotactic response of the UW4 is. Accordingly, 1-aminocyclopropane-1-carboxylic acid, cysteine and malic acid are screened out to be candidate chemoattractant of UW4 and corresponding pseudochemoattractant receptors. Through the analysis of fluorescence thermal drift experiments of the pseudochemoattractant receptor ligand binding domain recombinant protein and root system secretion components, the delta Tm values of the 3 pseudochemoattractant receptors and the 3 candidate strong chemoattractants are the highest in all root system secretion components and exceed the threshold value of 2.0 ℃. All of the 3 chemotactic receptor mutant strains of the constructed UW4 have no chemotactic response to the corresponding candidate chemoattractant, which indicates that the 3 chemotactic receptors and the corresponding candidate chemoattractant are in a receptor-ligand relationship. The chemotactic rate of UW4 and 3 chemotactic receptor mutant strains and anaplerosis strain chemotactic colonized wheat rhizosphere is measured by applying a microenvironment system, and the chemotactic rate of only the 1-aminocyclopropane-1-carboxylic acid chemotactic receptor mutant strain is obviously lower than that of UW4, which indicates that 1-aminocyclopropane-1-carboxylic acid is a strong chemotactic substance of the chemotactic colonized rhizosphere of UW4. By applying the molecular docking and binding free energy analysis of the group I chemotactic receptors capable of recognizing extracellular chemotactic substances and the components of root secretion, the strong chemotactic substances of bacteria chemotactic colonizing plants at the rhizosphere can be quickly and simply identified.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the scope of the present invention, which is intended to cover any modifications, equivalents, improvements, etc. within the spirit and scope of the present invention.
Claims (10)
1. A method for identifying the chemotaxis rate of PGPR (PGPR) for chemotactic root exudates to reach rhizosphere is characterized by comprising the following steps of:
(1) Searching an extracellular ligand binding domain of the microorganism from the annotation of the microorganism genome, screening a chemotactic receptor capable of identifying an extracellular chemoattractant, carrying out molecular docking on the chemotactic receptor and root exudate components by utilizing a molecular docking technology, calculating binding free energy, and screening the root exudate components with low free energy values as candidate chemoattractants;
(2) Determining a receptor protein corresponding to the candidate chemoattractant according to the candidate chemoattractant, and constructing an expression vector containing an extracellular ligand binding domain fragment of the receptor protein and a recombinant vector for knocking out the receptor protein by taking the genomic DNA of the microorganism as a template;
(3) Transferring the Escherichia coli containing the recombinant vector in the step (2) into a microorganism by a conjugation method to obtain a chemotactic receptor knockout mutant strain;
(4) Constructing anaplerotic plasmid and anaplerotic no-load plasmid of receptor protein corresponding to the candidate chemoattractant in the step (2) by taking genome DNA of the microorganism as a template, and respectively transferring the anaplerotic plasmid and anaplerotic no-load plasmid into the chemotactic receptor knockout mutant strain in the step (3) to obtain an anaplerotic strain and an anaplerotic no-load strain of the chemotactic receptor knockout mutant strain;
(5) The method comprises the steps of firstly carrying out a flat chemotaxis experiment on a microorganism, a chemotactic receptor knockout mutant strain, a anaplerosis empty strain and a candidate chemotactic substance to verify the correspondence between a receptor protein and the chemotactic substance, and then measuring the chemotaxis rate and the proliferation rate of the microorganism, the chemotactic receptor knockout mutant strain and the anaplerosis strain of the chemotactic receptor knockout mutant strain at the rhizosphere of wheat by using a microenvironment system.
2. The method of claim 1 for identifying the rate of arrival of PGPR chemotactic diverse chemoattractants at the rhizosphere, wherein: the root secretion components in the step (1) comprise amino acids, organic acids and saccharides; the molecular docking is realized by molecular docking software AutoDock 4.2; the binding free energy was calculated by the software Amber 17.
3. The method of claim 2 for identifying the rate of arrival of PGPR chemotactic different chemoattractants at the rhizosphere, comprising: in the step (2), the receptor protein corresponding to the candidate chemotaxis is determined according to the candidate chemotaxis, namely, a capillary chemotaxis method is used for determining the chemotaxis threshold concentration value of the microorganism, the response intensity of the microorganism to the chemotaxis is determined, and then the receptor protein of the candidate chemotaxis is further identified by adopting a fluorescence thermal drift method and a flat chemotaxis method.
4. The method of claim 3 for identifying the rate of arrival of PGPR chemotactic diverse chemoattractants to the rhizosphere, wherein: the fluorescence thermal drift value of the chemoattractant and the receptor protein screened by adopting a fluorescence thermal drift method is higher than 2.0 ℃.
5. The method of claim 4 for identifying the rate of arrival of PGPR chemotactic diverse chemoattractants to the rhizosphere, wherein: in the step (3), the Escherichia coli is Escherichia coli S17-1.
6. The method for identifying the rate of arrival of PGPR chemotactic different chemoattractants to the rhizosphere according to claim 5, wherein the specific operation of utilizing the microenvironment system in step (5) is:
a. adding 0.5 XMS culture solution of the inoculation bacterial suspension into the test tube with the cover, and fixing the rhizome combination part of the sterile wheat seedling at the liquid level by using a filter screen;
b. b, placing the wheat seedling sample treated in the step a in 16h illumination for 8h dark culture, taking out half of the wheat seedling after inoculation culture for 2h, transferring the wheat seedling to a microenvironment system without inoculation suspension for culture, and taking the wheat seedling as a transfer group, wherein the other half of the wheat seedling is an untransferred group;
c. b, sampling two groups of wheat seedlings in the step b at 2h, 6h, 10h, 14h, 18h, 22h, 26h, 30h, 34h and 38h respectively, and determining the number of bacteria colonized in the rhizosphere;
d. and (3) fitting a growth kinetic equation of the bacteria in the rhizosphere by using a Logistic equation, solving the first derivative of the growth kinetic equation to obtain a growth rate value, and calculating to obtain the chemotactic rate and the proliferation rate of the rhizosphere of the wheat of different inoculated bacteria.
7. The method of claim 6 for identifying the rate of arrival of PGPR chemotactic different chemoattractants at the rhizosphere, comprising: the inoculation strain in the step a refers to a microorganism, a chemotactic receptor knockout mutant strain and a anaplerotic strain of the chemotactic receptor knockout mutant strain.
8. The method of claim 7 for identifying the rate of arrival of PGPR chemotactic diverse chemoattractants to the rhizosphere, wherein: and c, placing the wheat seedling sample in the step b in 16h illumination for 8h dark culture, wherein the root of the wheat seedling needs shading treatment.
9. The method of claim 7 for identifying the rate of arrival of PGPR chemotactic diverse chemoattractants to the rhizosphere, wherein: and d, the rhizosphere proliferation rate of the wheat in the step d is the growth rate of the transferred group, and the rhizosphere chemotaxis rate of the wheat is the difference value of the growth rate of the non-transferred group and the growth rate of the transferred group.
10. Use of the method according to any one of claims 1 to 9 for identifying a chemoattractant that is a strong chemoattractant for PGPR in rhizosphere chemotactic colonization, characterized in that: if the chemotactic receptor knockout mutant strain has a significantly lower chemotactic rate than the anaplerotic strains of the microorganism and the chemotactic receptor knockout mutant strain, the chemoattractant is determined to be a strong chemoattractant for the PGPR to chemocolonize in the rhizosphere.
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