Use of trX gene and mutant M81 in expression of siderophore of nitrogen-fixing bacteria

Abstract

This invention relates to the field of bioengineering technology, and in particular to the application of the trX gene and the mutant strain M81 in the expression of siderophores in nitrogen-fixing bacteria. This invention utilizes Tn5 transposon technology to construct a mutant library and screen for the mutant strain M81, which exhibits a significantly increased siderophore synthesis capacity compared to the wild-type strain GXGL-4A. Whole-genome sequencing confirmed that the insertion site of the mutant strain M81 is a transcription factor TrX with an unknown function. Experiments showed that the siderophore production of the mutant strain M81 was significantly increased compared to the wild-type strain, and bacterial growth was not affected. Growth-promoting experiments on cucumbers showed that the obtained high-siderophore-producing mutant strain had a significant growth-promoting effect on cucumbers. Compared with existing technologies, this invention significantly improves the siderophore yield of nitrogen-fixing bacteria by mutating the trX gene. The resulting mutant strain can be used as a plant growth-promoting bacterium, increasing the absorption of iron from the soil while maintaining nitrogen-fixing activity.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to the application of the trX gene and the mutant strain M81 in the expression of ferrophilic compounds in nitrogen-fixing bacteria. Background Technology

[0002] Iron is an essential element for microbial growth and a component of many key enzymes that play crucial roles in electron transport, RNA synthesis, and resistance to reactive oxygen species (ROS) stress. Under physiological conditions, iron typically exists as Fe(OH)3, which has poor solubility and cannot meet the iron requirements of microorganisms. In low-iron environments, bacteria synthesize a class of iron-dependent enzymes... 3+ A high-affinity, low-molecular-weight chelating factor (1-2 kDa) called a ferrophile is secreted extracellularly or onto the cell surface to acquire Fe. 3+ The formation of a heptaphilin-iron complex, which binds to receptor proteins on the cell membrane, transports iron from the extracellular space to the intracellular space to meet the iron requirements of microbial growth. Heptaphilin secretion is an important biocontrol mechanism of plant rhizosphere growth-promoting bacteria. These bacteria can compete for limited iron in the soil by producing heptaphilin, causing pathogens to fail to grow or even die due to iron deficiency, thus achieving biocontrol. In recent years, scholars at home and abroad have conducted in-depth research on the biosynthesis of heptaphilin and its mechanisms of iron absorption and transport. Some progress has been made in improving the ability of microorganisms to produce heptaphilin by improving bacterial culture conditions, screening high-heptaphilin-producing strains, introducing exogenous heptaphilin receptors, and regulating heptaphilin biosynthesis. However, related studies have mostly focused on *Escherichia coli* and *Pseudomonas aeruginosa*. Using genetic engineering to enhance the biosynthesis of heptaphilin to further improve the biocontrol capabilities of growth-promoting or biocontrol bacteria has broad application prospects. Furthermore, many studies have confirmed that heptaphilin plays an important role in promoting plant growth and development and improving crop resistance.

[0003] Associative nitrogen-fixing bacteria possess excellent biological nitrogen-fixing capabilities, and the genetic engineering development of nitrogen-fixing bacteria from non-leguminous plants is currently a research hotspot. These nitrogen-fixing bacteria have advantages such as a wide host range, convenient culture, and abundant sources. Developing them into high-producing heparin strains can fully utilize their nitrogen-fixing and iron-absorbing capabilities, enhancing growth promotion and disease prevention, thereby contributing to the growth and development of host plants. Heparin biosynthesis belongs to the nonribosomal peptide synthesis pathway, involving a class of multifunctional protein complexes, namely nonribosomal peptide synthetases (NRPS), which recognize, activate, and transport amino acid substrates to synthesize nonribosomal peptides (NRPS) in a specific sequence. Heparin is an important secondary metabolite produced by many bacteria and fungi, synthesized by heparin synthesis gene clusters. Determining the complete genome sequence of bacteria or fungi, analyzing the complete heparin synthesis gene clusters using bioinformatics methods, and modifying the core heparin synthase gene is one way to improve the heparin production capacity of microorganisms. However, the biosynthesis of heptaphores is influenced not only by gene regulation but also by multiple factors such as bacterial culture conditions, iron ion concentration, heptaphoric receptors, and iron signal transduction and transport. Therefore, improving the heptaphoric ability of microorganisms by modifying certain genes in the heptaphoric synthesis gene cluster is not an easy task. To better develop and utilize plant growth-promoting bacteria, including nitrogen-fixing bacteria, and to develop them into multifunctional biological agents, finding ways to enhance bacterial heptaphoric production is a novel approach. Summary of the Invention

[0004] To address the aforementioned problems, the purpose of this invention is to provide the application of the trX gene and the mutant strain M81 in the expression of siderophores in nitrogen-fixing bacteria. This invention utilizes Tn5 transposon technology to construct a mutant library and screen for the mutant strain M81, which exhibits significantly enhanced siderophore synthesis compared to the wild-type strain GXGL-4A. Whole-genome sequencing confirmed that the insertion site in mutant strain M81 is a transcription factor of unknown function, named TrX. CAS plate assays and relative siderophore content determination confirmed that the target mutant strain M81 significantly increased siderophore production compared to the wild-type strain, without affecting bacterial growth. Growth-promoting experiments on cucumbers showed that the obtained high-siderophore-producing mutant strain significantly promoted cucumber growth. Compared to existing technologies, this invention, by mutating the trX gene of nitrogen-fixing bacteria, significantly improves the siderophore yield of the strain. The resulting mutant strain can be used as a plant growth-promoting bacterium, increasing iron absorption from the soil while maintaining nitrogen-fixing activity, thus benefiting plant roots by acquiring more iron and increasing plant biomass. Meanwhile, the mutant strains inhibited the growth of soil pathogens by competing for iron nutrition in the soil, which helped improve the plant's disease resistance.

[0005] The objective of this invention can be achieved through the following technical solutions:

[0006] The first objective of this invention is to provide the application of a target gene in the expression of ferrophilic compounds in nitrogen-fixing bacteria, wherein the target gene is the trX gene, and the DNA sequence of the trX gene is shown in SEQ ID NO.1.

[0007] A second objective of this invention is to provide the application of mutant strain M81 in expressing nitrogen-fixing bacteria ferrophiles, wherein the mutant strain M81 is deposited under the accession number CGMCC No. 24400.

[0008] In one embodiment of the present invention, the method of applying the mutant strain M81 to promote plant growth by expressing nitrogen-fixing bacteria ferrophile includes the following steps:

[0009] (1) The mutant strain M81 was inoculated into LB medium and cultured overnight. The cell pellet of mutant strain M81 was then collected.

[0010] (2) After precipitation and treatment of the mutant M81 cells collected in step (1), the cells were released into the plant rhizosphere.

[0011] In one embodiment of the present invention, in step (2), the post-processing is to resuspend the mutant M81 cell pellet in sterile water and then centrifuge it. This process is repeated multiple times, and the mutant M81 cell pellet obtained from the last centrifugation is resuspended in sterile water to obtain a mutant M81 cell suspension.

[0012] In one embodiment of the present invention, a suspension of mutant strain M81 cells is applied to the plant when it is in the two-leaf-one-heart stage.

[0013] In one embodiment of the present invention, each plant releases 5-10 mL of a suspension of mutant strain M81 cells, with a bacterial count of 1 × 10⁻⁶. 8 CFU / mL; the frequency was once every 5 days, for a total of 5 treatments.

[0014] A third objective of this invention is to provide the application of a substance that knocks out or replaces a target gene in the preparation of a high-yielding ferrophilic nitrogen-fixing bacterium agent or a ferrophilic growth-promoting bacterium agent, wherein the target gene is the trX gene, and the DNA sequence of the trX gene is shown in SEQ ID NO.1.

[0015] The fourth objective of this invention is to provide the application of mutant strain M81 in the preparation of high-yield ferrophilic nitrogen-fixing bacteria agents or ferrophilic growth-promoting bacteria agents, wherein the preservation number of mutant strain M81 is CGMCC No. 24400.

[0016] The fifth objective of this invention is to provide a target gene, wherein the target gene is a trX gene, and the DNA sequence of the trX gene is shown in SEQ ID NO.1.

[0017] The sixth objective of this invention is to provide a mutant strain M81, the mutant strain M81 having the accession number CGMCC No. 24400.

[0018] This invention employs a gene mutation method, using the insertion and inactivation of a target gene to enhance the siderophile production capacity of a bacterial strain—a controllable and efficient approach. This method is simple to use, and the inactivation of the target gene is easily manipulated, making it universally applicable and possessing significant potential for application in regulating siderophile production in plant growth-promoting bacteria and nitrogen-fixing bacteria.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) This invention achieves a significant increase in the biosynthesis of siderophores by inserting a mutation to inactivate the target gene trX. The operation is simple, the results are reliable, and the effect is obvious. Studies have found that this gene is closely related to the yield of siderophores synthesis, which is speculated to be a negative regulatory mechanism, but the specific regulatory mechanism of the gene is currently unclear.

[0021] (2) The high expression of heptaphilin in this invention does not cause any harm to the bacterial cells themselves, and the growth and reproduction of the bacteria are not affected. No significant changes were observed in the morphological characteristics and physiological and biochemical processes of the bacteria. The obtained transformed strains, as growth-promoting bacteria, significantly promoted plant growth compared with the control wild strains.

[0022] (3) This invention is the first to clarify that the trX gene can regulate the biosynthesis of heptaphilin.

[0023] (4) The mutant strain created in this invention can be used to prepare high-ferophilic nitrogen-fixing bacteria inoculants or ferophilic growth-promoting bacteria inoculants. High-ferophilic mutant strains constructed by genetic operations such as gene knockout can be used as highly efficient growth-promoting bacteria, especially when the soil is iron-deficient or low in iron, they can significantly improve the absorption of iron by plants. After being made into inoculants, they can be used in agricultural production or ecological environmental protection. Attached Figure Description

[0024] Figure 1 This is the morphology of competent cells of nitrogen-fixing bacteria GXGL-4A after hyperosmolar culture, used for transposon mutation; Figure 1 A: Routine LB culture; Figure 1 B: LB culture containing 15 g / L NaCl; Figure 1 C: HO-2 medium (replace NaCl in conventional LB medium with 7.5 g / L CaCl2).

[0025] Figure 2 This is a qualitative and quantitative detection of the ferrophilic biosynthetic capacity of the mutant strain M81.

[0026] Figure 3 This is a schematic diagram of the transposition insertion site of the mutant strain M81.

[0027] Figure 4 This is a PCR clone of the trX gene; M: DNA molecular weight marker; 4A: amplification of the target gene trX using the genomic DNA of the wild-type nitrogen-fixing bacterium GXGL-4A as a template; M81: amplification of the mutated target gene trX using the genomic DNA of the mutant strain M81 as a template.

[0028] Figure 5 This is a phylogenetic tree of the trX gene; where K: Kosakonia; the numbers in parentheses are the strain accession numbers in GenBank.

[0029] Figure 6 This is a quantitative real-time PCR (qPCR) assay of the mutant M81 gene at the mutation site; the trX gene within GXGL-4A is represented by a relative quantification value of 1, and M81 indicates the relative expression level of the mutated target gene trX. The rpoB gene was used as an internal reference gene in the qPCR experiment.

[0030] Figure 7 This is the growth curve of the target strain.

[0031] Figure 8 This involves detecting the growth-promoting effect of the target strain on cucumber. Figure 8 A represents the effect of treatment with mutant strain M81 on cucumber plant height; Figure 8 B represents the effect of treatment with mutant strain M81 on the fresh weight of cucumber roots; Figure 8 C represents the effect of the mutant strain M81 treatment on the fresh weight of cucumber plants; Figure 8 D represents the effect of the mutant strain M81 treatment on cucumber root length.

[0032] Figure 9 This is a photograph of the target strain's effect on cucumber growth.

[0033] Figure 10 This test measures the inhibitory effect of the target strain on the growth of the pathogen causing maize leaf spot. Detailed Implementation

[0034] This invention provides the application of a target gene in the expression of ferrophilic compounds in nitrogen-fixing bacteria, wherein the target gene is the trX gene, and the DNA sequence of the trX gene is shown in SEQ ID NO.1.

[0035] This invention provides the application of mutant strain M81 in expressing nitrogen-fixing bacteria ferrophile, wherein the preservation number of mutant strain M81 is CGMCC No.24400.

[0036] In one embodiment of the present invention, the method of applying the mutant strain M81 to promote plant growth by expressing nitrogen-fixing bacteria ferrophile includes the following steps:

[0037] (1) The mutant strain M81 was inoculated into LB medium and cultured overnight. The cell pellet of mutant strain M81 was then collected.

[0038] (2) After precipitation and treatment of the mutant M81 cells collected in step (1), the cells were released into the plant rhizosphere.

[0039] In one embodiment of the present invention, in step (2), the post-processing is to resuspend the mutant M81 cell pellet in sterile water and then centrifuge it. This process is repeated multiple times, and the mutant M81 cell pellet obtained from the last centrifugation is resuspended in sterile water to obtain a mutant M81 cell suspension.

[0040] In one embodiment of the present invention, a suspension of mutant strain M81 cells is applied to the plant when it is in the two-leaf-one-heart stage.

[0041] In one embodiment of the present invention, each plant releases 5-10 mL of a suspension of mutant strain M81 cells, with a bacterial count of 1 × 10⁻⁶. 8 CFU / mL; the frequency was once every 5 days, for a total of 5 treatments.

[0042] This invention provides the application of substances that knock out or replace a target gene in the preparation of agents for high-yielding nitrogen-fixing bacteria or agents for growth-promoting bacteria that produce nitrogen-fixing bacteria, wherein the target gene is the trX gene, and the DNA sequence of the trX gene is shown in SEQ ID NO.1.

[0043] This invention provides the application of mutant strain M81 in the preparation of high-yield ferrophilic nitrogen-fixing bacteria agents or ferrophilic growth-promoting bacteria agents, wherein the preservation number of mutant strain M81 is CGMCC No.24400.

[0044] This invention provides a target gene, the target gene being the trX gene, the DNA sequence of which is shown in SEQ ID NO.1.

[0045] This invention provides a mutant strain M81, the preservation number of which is CGMCC No.24400.

[0046] This invention employs a gene mutation method, using the insertion and inactivation of a target gene to enhance the siderophile production capacity of a bacterial strain—a controllable and efficient approach. This method is simple to use, and the inactivation of the target gene is easily manipulated, making it universally applicable and possessing significant potential for application in regulating siderophile production in plant growth-promoting bacteria and nitrogen-fixing bacteria.

[0047] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0048] Unless otherwise specified, all materials used in the following embodiments are commercially available; and all techniques or testing methods described are conventional techniques or testing methods in the art unless otherwise specified.

[0049] Example 1

[0050] This embodiment provides a mutant strain M81.

[0051] (1) Preparation of competent cells of nitrogen-fixing bacteria GXGL-4A

[0052] The nitrogen-fixing bacterium GXGL-4A was isolated from the roots of maize in Guilin, Guangxi. Its whole genome sequence in GenBank is accessed under the number CP015113.1; and its accession number at the China General Microbiological Culture Collection Center (CGMCC) is CGMCC No. 12588.

[0053] The method for preparing competent cells using GXGL-4A includes the following steps:

[0054] 1) Streak culture on a regular LB plate, pick a single colony, and inoculate it into a regular LB medium for overnight culture;

[0055] 2) Inoculate with 1% of the culture medium into HO-2 medium (modified LB medium, without NaCl) containing 7.5 g / L CaCl2 and culture until the logarithmic growth phase.

[0056] 3) Collect bacterial pellet by centrifugation at low temperature, wash away impurities and bacterial fragments with PEB electroporation buffer to obtain competent bacterial cells.

[0057] (2) Morphological observation of competent cells of nitrogen-fixing bacteria GXGL-4A

[0058] When the nitrogen-fixing bacterium GXGL-4A reached an OD600 of 0.6-0.8 in HO-2 solution, 1 mL of the bacterial suspension was centrifuged at 8000 rpm for 5 min, the supernatant was discarded, and the bacteria were washed with an equal volume of sterile water. This process was repeated twice. Finally, the suspension was resuspended in sterile water, and 10 μL of the resuspended bacterial suspension was dropped onto a copper plate and air-dried overnight. Competent cells prepared in conventional LB and HO-1 (LB medium containing 15 g / NaCl) media were used as controls, and the cells were observed under a transmission electron microscope. The results showed that the capsule and cytoplasm of nitrogen-fixing bacterium GXGL-4A cultured in conventional LB medium were tightly and uniformly bound together, with no clear boundary. Nitrogen-fixing bacterium GXGL-4A cultured in HO-1 showed cytoplasmic shrinkage, darker cytoplasm, uneven capsule thickness, and a clear boundary between the capsule and cytoplasm. The shrinkage of nitrogen-fixing bacterium GXGL-4A cultured under HO-2 conditions was more severe, and the morphology was distorted. This shows that the bacterial morphology changed significantly under different culture methods. Figure 1 ).

[0059] (3) Electroporation transformation, screening for Tn5 mutants

[0060] 1) Add Tn5 transposon complex to a PEB solution containing competent GXGL-4A nitrogen-fixing bacteria cells to a final concentration of 2.5 μg / mL, and incubate on ice for 5 minutes. Then, add 200 μL of this mixture to a pre-chilled Bio-Rad electroporation cuvette at 0°C, with the electrodes 0.2 cm apart. Electroporate using a Bio-Rad MicroPulser electroporator at 2.0 kV for approximately 2 ms. Immediately after electroporation, add 800 μL of SOC medium and incubate at 37°C and 150 rpm for 1 hour.

[0061] 2) Plate plating and transformant screening steps: Spread 100 μL of recovered growth cells onto a plate containing Km (50 μg / mL) antibiotic, incubate at 37℃ for 16 h, pick transformants, and identify them after pure culture. SOC recovery medium: containing 0.5% (w / v) yeast extract, 2% (w / v) tryptone, 10 mmol / L NaCl, 2.5 mmol / L KCl, 10 mmol / L MgCl2, 20 mmol / L MgSO4, and 20 mmol / L glucose. Sterilize at 8 psi for 20 min and store at 4℃ for later use.

[0062] PEB electroshock buffer: contains 272 mmol / L sucrose and 1 mmol / L MgCl2.

[0063] 3) Identification of positive transformants: Four primer pairs were designed to amplify the anfD gene and the gene fragment on the Tn5 transposon element of *GXGL-4A*, a nitrogen-fixing bacterium. Primer pair anfD / anfR was used for amplification to ensure the transformants originated from *GXGL-4A*. The remaining three primer pairs, Screen 1-F / Screen 1-R, Screen 2-F / Screen 2-R, and Screen 3-F / Screen 3-R, were designed based on the Tn5 gene sequence. Amplification of the expected size target fragment by all three primer pairs ensured the insertion of the Tn5 element into the transformant genome, thus confirming the transformant as a transposon mutant. Total DNA from the bacterial transformants was used as a template for PCR amplification. The PCR program was: pre-denaturation at 94℃ for 10 min, denaturation at 94℃ for 30 s, annealing at 54℃ for 30 s, extension at 72℃ for 1 min, for a total of 35 cycles, with a final extension at 72℃ for 7 min. The four primer pairs are as follows:

[0064] anfD: 5′-CGGGCAATCTCTTCATCAAT-3′ (SEQ ID NO. 2),

[0065] anfR: 5′-ATACCTTCGCGACCGATATG-3′ (SEQ ID NO.3), target product 655bp;

[0066] Screen 1-F: 5′-CAGGGATCTGCCATTTCATT-3′ (SEQ ID NO.4),

[0067] Screen 1-R: 5′-GCCTGAGCGAGACGAAATAC-3′ (SEQ ID NO.5), target product 922bp;

[0068] Screen 2-F: 5′-GGACCGATGGATATGTTCT-3′ (SEQ ID NO.6),

[0069] Screen 2-R: 5′-GCCTGAGCGAGACGAAATAC-3′ (SEQ ID NO.7), target product 602bp;

[0070] Screen 3-F: 5′-ATTCAACGGGAAACGTCTTG-3′ (SEQ ID NO.8),

[0071] Screen 3-R: 5′-ATTCCGACTCGTCCAACATC-3′ (SEQ ID NO.9), target product 656bp.

[0072] Example 2

[0073] This embodiment provides a method for detecting the ferrophilic synthesis ability of nitrogen-fixing bacterial mutant strains.

[0074] (1) Preparation of CAS detection plates

[0075] 1) Prepare 200mL phosphate buffer solution: 1.924g Na2HPO4, 0.9082g NaH2PO4, 0.15g KH2PO4, 0.5g NH4Cl, 0.25g NaCl, and adjust the pH to 6.8-7.0 with 1mol / L NaOH.

[0076] 2) Preparation of CAS solid detection culture medium: Prepare solutions A and B respectively.

[0077] Solution A: Take two beakers and dissolve 0.0605g CAS-S and 0.0729g CTAB (HTDMA) in 40mL and 50mL of double-distilled water, respectively. Heat CTAB until dissolved and clear. Then pour CAS-S into the CTAB solution while stirring (this step order cannot be reversed, otherwise a large amount of precipitation may occur). Mix well and autoclave for later use. Prepare 1mmol / L FeCl3 (dissolved in 10mmol / L HCl) and filter it through a 0.22μm microporous membrane for sterilization (iron salt solutions are prone to denaturation during autoclaving). After cooling CTAB-CAS, add 10mL of FeCl3 solution and shake well to obtain 100mL of blue, precipitate-free CAS staining agent.

[0078] Solution B: 50 mL phosphate buffer, 60 mL acid-hydrolyzed casein, 2 mL 1 mmol / L CaCl2, 2 mL 10 mmol / L MgSO4, 1 mol / L NaOH to adjust pH to 6.9, 20 g agar, and bring the volume to 1 L.

[0079] After sterilization, add 10 mL of CAS staining agent to 100 mL of solution B, shake well, and then measure 30 mL into a 9 cm diameter petri dish to obtain blue CAS solid detection medium. After cooling and solidification, it can be used for later use.

[0080] (2) CAS plate screening of heptaphilic synthesis capacity

[0081] A 9cm inner diameter petri dish was marked and divided into 6 regions. Three parallel samples of the GXGL-4A wild-type strain and one mutant strain were inoculated into each of the 6 regions. After 24 hours of culture, the size of the yellow halo produced by the mutant and wild-type strains was observed. Mutants with significantly altered heparin production capacity were initially screened by comparing halo size.

[0082] (3) Quantitative detection of ferrophilic synthesis

[0083] After initial screening using the CAS plate assay, the target mutant strains were further screened using a quantitative assay. Mutant strains and wild-type strains were picked up using an inoculation loop and cultured separately in liquid LB at OD. 600 The value is 0.7-0.8. Take 600 μL of bacterial culture into a 1.5 mL centrifuge tube, centrifuge at 8000 rpm for 5 min, take the supernatant, mix it with CAS detection solution at a volume ratio of 1:1, mix well and let stand for 1 h, measure the absorbance (As) at a wavelength of 630 nm, and zero the instrument with double-distilled water as a control. Take a blank culture medium and mix it with CAS detection solution, use its absorbance as the reference value (Ar), and calculate the relative amount of ferrophosphate produced by the mutant strain according to [(Ar-As) / Ar]×100%.

[0084] CAS quantitative detection solution (100mL): The preparation steps are the same as those for solution B. The amounts of each reagent are: 0.009g CAS-S, 0.0218g CTAB, and 1.5mL 1mmol / L FeCl3.

[0085] Example 3

[0086] This embodiment provides the identification of the high-ferrophosphate-producing mutant strain M81.

[0087] (1) Quantitative and qualitative identification of high-yield heparin

[0088] After multiple rounds of initial screening using CAS plates and quantitative screening using CAS detection solution, a high-ferophile-producing mutant strain M81 (also known as Kosakonia radicincitans M81) was finally selected from 1633 mutant strains. Its ferophile-producing capacity was significantly improved compared with the wild-type strain GXGL-4A. Figure 2 The mutant strain M81 has been deposited indefinitely at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC No. 24400.

[0089] (2) Identification of the mutant gene in mutant strain M81

[0090] Whole-genome sequencing of the target mutant strains confirmed that both mutants were single-copy insertions, with the insertion sites located within the coding region of gene trX (DNA sequence shown in SEQ ID NO.1) (locus tag: A3780_19720) (the coding sequence of this gene was mutated from SEQ ID NO.1 to SEQ ID NO.16). Figure 3 To further identify the obtained mutant strain as a single-copy transposon mutation of the target gene, forward and reverse primers were designed based on the flanking sequences of the mutant gene obtained from genome sequencing. The amplification product obtained using genomic DNA from the wild-type strain GXGL-4A as a template served as a control. The amplification product from the mutant strain showed an increase of 2019 bp. If the results matched the expectations, the mutant gene could be confirmed. The primer pairs used are as follows:

[0091] M81-tr-F: 5′-aggggtacaatatggccact-3′ (SEQ ID NO. 10);

[0092] M81-tr-R:5′-tctgcattattccgccattaaca-3′ (SEQ ID NO. 11);

[0093] PCR amplification yielded target products of approximately 0.8 Kb and 2.8 Kb in size, respectively. The trX gene was mutated to the DNA sequence shown in SEQ ID NO. 16, which perfectly matched the theoretical expectation, proving that the genome sequencing of the mutant strain was completely correct and the identification of the mutated gene was accurate. Figure 4 ).

[0094] Example 4

[0095] This embodiment provides the construction of a phylogenetic tree for the trX gene.

[0096] Based on the DNA sequence of the trX gene, it was submitted to GenBank for BLAST alignment. Sequences with a sequence similarity of over 80% were selected, and a phylogenetic tree was constructed using the neighbor-joining method with MEGA7 software. Figure 5 The results showed that this gene is significantly species-specific, highly conserved at the genus level, and exhibits large sequence differences and evolutionary distances with gene species outside the Kosakonia genus.

[0097] Example 5

[0098] This embodiment provides a real-time quantitative PCR of the mutant strain M81.

[0099] Using nitrogen-fixing bacterium GXGL-4A as a control, total RNA was extracted from the strain using the UNIQ-10 Trizol total RNA extraction kit. After reverse transcription, the mutant genes of the M81 mutant strain were analyzed by quantitative real-time PCR. The housekeeping gene rpoB was used as an internal reference gene. The primer sequences for quantitative real-time PCR are as follows:

[0100] rpoB-F3: 5'-GGTGCGTGTAGAGCGTGC-3' (SEQ ID NO. 12),

[0101] rpoB-R3: 5'-ATCTCGGACAGCGGGTTG-3' (SEQ ID NO.13), PCR product: 168bp;

[0102] tr-F2: 5'-GATAAGGATACCCAGCCGAATA-3' (SEQ ID NO.14),

[0103] tr-R2: 5'-GACCAGAAAGTGCGAAACAGTT-3' (SEQ ID NO.15), PCR product: 203bp

[0104] (1) cDNA first-strand synthesis

[0105] RNA was reverse transcribed at a rate of 800 ng.

[0106] 1) Add the following reagents to a nuclease-free PCR tube placed in an ice bath:

[0107] Random Primer p(dN)6(100pmol)1μL

[0108] dNTP Mix(0.5mM final concentration)1μL

[0109] Rnase-free ddH2O was added to a final volume of 14.5 μL.

[0110] 2) After gently mixing, centrifuge for 3 seconds. Incubate the reaction mixture at 65°C for 5 minutes, then in an ice bath for 2 minutes, and finally centrifuge for 3 seconds.

[0111] 3) Place the test tube in an ice bath, then add the following reagents:

[0112] 5X RT Buffer 4μL

[0113] Thermo Scientific RiboLock RNase Inhibitor(20U)0.5μL

[0114] Maxima Reverse Transcriptase(200U)1μL

[0115] 4) Gently mix and then centrifuge for 3 seconds;

[0116] 5) Perform the reverse transcription reaction on a PCR instrument under the following conditions:

[0117] 25℃, 10min; 50℃, 30min; 85℃, 5min.

[0118] 6) Store the above solution at -20℃.

[0119] (2) Real-time PCR

[0120] The cDNA sample was diluted 10 times and used as a template for instrumental detection.

[0121] Quantitative PCR reagent: 2×SG Fast qPCR Master Mix (B639271, BBI, Roche).

[0122] Quantitative PCR instrument: LightCycler 480 II Real-Time PCR Instrument (Roche, Rotkreuz, Switzerland).

[0123] 1) Preparation of reaction mixture

[0124] SybrGreen qPCR Master Mix(2×)5μL

[0125] Primer F (10 μM) 0.2 μL

[0126] Primer R (10 μM) 0.2 μL

[0127] ddH2O 3.6μL

[0128] Template (cDNA) 1μL

[0129] 2) PCR cycling conditions

[0130] 95℃, 3 min; 45 cycles, each cycle consisting of 95℃, 5 s; 60℃, 30 s.

[0131] The 96 / 384-well plate with the sample added was placed in a LightCycler 480 II (Roche) for reaction.

[0132] Quantitative real-time PCR results showed that the transcriptional level of the inserted mutant gene trX was significantly increased after insertion. Figure 6 The target gene has undergone a mutation due to the Tn5 transposition, resulting in a change in its expression product.

[0133] Example 6

[0134] This embodiment provides the determination of the growth curve of the mutant strain M81.

[0135] After purification, single colonies of nitrogen-fixing bacteria GXGL-4A and mutant strain M81 were picked and cultured overnight at 37°C and 180 rpm in 20 mL LB solution on a shaker. The activated strains were then inoculated into 100 mL LB solution at a 1% (v / v) inoculation rate, and OD was measured every 1 hour. 600 The values ​​were measured three times for each treatment.

[0136] The results showed that the growth of the target mutant was almost unaffected after gene mutation, and there was no significant difference in growth rate compared with the wild-type plant. Figure 7 ).

[0137] Example 7

[0138] This embodiment provides the determination of the growth-promoting effect of mutant strain M81 on cucumber.

[0139] After treating cucumber seedlings with the target bacterial strain, physiological indicators such as plant height, plant fresh weight, root length, and root fresh weight were measured. The results showed that the biomass of cucumbers in the mutant treatment group (CK) was significantly higher than that in the wild-type treatment group; except for root length, the other three biomass indicators in the wild-type treatment group were significantly higher than those in the blank control group (sterile water treatment). Figure 8 ), Figure 9 The overall condition of cucumber plants under each treatment is shown. Overall, nitrogen-fixing bacteria treatment has a significant growth-promoting effect on cucumber plants, and the mutant strain treatment is more effective in improving the growth of cucumber plants than the wild strain treatment.

[0140] Example 8

[0141] This embodiment provides an assay of the inhibition of the supernatant of the fermentation broth of mutant strain M81 against Bipolaris maydis, the pathogen of maize leaf spot.

[0142] The strain was inoculated into LB medium and incubated overnight at 37°C with shaking. 50 mL of the bacterial culture was centrifuged at 8000 rpm for 5 min. The supernatant was aspirated with a syringe, filtered through a microporous membrane, and the filtrate was transferred to a 4 mL centrifuge tube and stored at 4°C for later use.

[0143] 600 μL of the filtrate was spread onto PDA medium. An equal volume of liquid LB was added to the blank control and spread evenly. Then, the *B. maydis* bacterial growth was inverted and placed in the center of the PDA medium, gently pressed to ensure close contact with the medium surface, and incubated upside down at 28°C for 4 days. The diameter of the bacterial growth was measured using the cross-sectional method. The results showed that the diameter of the bacterial growth on the control group plate was approximately 8 cm, significantly higher than that of the mutant strain M81 treatment group. Nitrogen-fixing bacteria GXGL-4A and the mutant strain M81 significantly inhibited the growth of *B. maydis*. The ferrophilic acid in the supernatant competed for the iron needed for fungal growth, thus limiting the growth of the small spot pathogen. The higher the ferrophilic acid content, the slower the fungal growth. Figure 10 ).

[0144] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. Use of mutant M81 in the expression of a siderophore of nitrogen-fixing bacteria, characterized in that, The mutant strain M81 is Kosakonia radicincitans M81 was deposited on February 16, 2022, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 24400.

2. The application of the mutant strain M81 according to claim 1 in expressing the siderophile in nitrogen-fixing bacteria, characterized in that, The application method of the mutant strain M81 in expressing nitrogen-fixing bacterial siderophiles to promote plant growth includes the following steps: (1) The mutant strain M81 was inoculated into LB medium and cultured overnight. The cell pellet of mutant strain M81 was then collected. (2) After precipitating and treating the mutant M81 cells collected in step (1), the cells were released into the plant rhizosphere.

3. The application of the mutant strain M81 according to claim 2 in expressing the siderophile in nitrogen-fixing bacteria, characterized in that, In step (2), the post-processing involves resuspending the mutant M81 cell pellet in sterile water and then centrifuging it. This process is repeated multiple times. The mutant M81 cell pellet obtained from the last centrifugation is then resuspended in sterile water to obtain a mutant M81 cell suspension.

4. The application of the mutant strain M81 according to claim 3 in expressing the siderophile in nitrogen-fixing bacteria, characterized in that, The mutant strain M81 cell suspension was applied to the plant when it was in the two-leaf-one-heart stage.

5. The use of the mutant strain M81 according to claim 4 for the expression of siderophores in diazotrophs, characterized in that, 5-10 mL of the mutant M81 cell suspension with a bacterial number of 1 x 10 8 CFU / mL was released per plant; the frequency was once every 5 days, and the treatment was performed for 5 times in succession.

6. The application of mutant strain M81 in the preparation of high-yield ferrophilic nitrogen-fixing bacteria agents or ferrophilic growth-promoting bacteria agents, characterized in that, The mutant strain M81 is Kosakonia radicincitans M81 was deposited on February 16, 2022, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 24400.

7. A mutant strain M81, characterized in that, The mutant strain M81 is Kosakonia radicincitans M81 was deposited on February 16, 2022, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 24400.

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