An engineered strain of Pseudomonas aeruginosa that regulates rhizosphere colonization and phosphate solubilization, its construction method, and its application.

Abstract

This invention belongs to the field of genetic engineering technology, specifically relating to an engineered strain of *Pseudomonas* that regulates rhizosphere colonization and phosphorus solubilization, its construction method, and its applications. This invention achieves gene-specific expression through a phosphorus starvation-inducible promoter and knocks out… crp The gene reduces metabolic burden, increases colonization rate and phosphorus solubility, and its functional activity does not significantly decrease under extreme environments. The engineered strain constructed in this invention has growth-promoting effects on grain crops such as wheat and corn, as well as specialty economic crops such as strawberries and tomatoes. It is tolerant to extreme environments such as severe salinity, low temperature, strong acidity, low oxygen, and low nutrition. It can activate various insoluble phosphorus compounds such as tricalcium phosphate, phytate phosphorus, and ferric phosphate, breaking through the application limitations of existing strains in single crops, soils, and phosphorus forms.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to an engineered strain of *Pseudomonas* that regulates rhizosphere colonization and phosphorus solubilization, its construction method, and its applications. This engineered strain can be applied to multiple crops and soil types, and is particularly suitable for fertilizer reduction and soil ecological restoration in complex environments such as saline-alkali land, acidic soil, facility agriculture soil, and soil with continuous cropping obstacles. It is suitable for planting various crops, including grain crops and specialty cash crops. Background Technology

[0002] Pseudomonas ( Pseudomonas Phosphate-promoting bacteria are widely present in soil, and their rhizosphere colonization ability and phosphorus solubilization function are the core characteristics that enable them to promote growth. In current agricultural production, the excessive application of chemical phosphate fertilizers has led to ecological problems such as soil degradation, desertification, and water pollution. Moreover, the utilization rate of chemical phosphate fertilizers is less than 30%, and a large amount of phosphorus remains in the soil in the form of inorganic and organic insoluble phosphorus, resulting in resource waste.

[0003] In existing technologies, the field application of *Pseudomonas* faces three main problems: First, its environmental adaptability is narrow. In special environments such as the low-temperature, high-salt, and low-oxygen saline-alkali land of the Yellow River Delta, acidic red soil dominated by iron or aluminum phosphate, and facility agricultural soils with high phytate phosphorus, its growth, reproduction, and functional activity are suppressed, and it lacks adaptability to extreme environments such as severe salinity and strong acidity. Second, its functional synergy is poor. Traditional modified strains often exhibit contradictions such as strong colonization but weak phosphorus solubilization or strong phosphorus solubilization but difficult colonization, and most are single-gene modifications that have not achieved the regulation of chemotaxis, attachment, and phosphorus solubilization. Third, most strains are only effective for specific food crops, but have poor adaptability to specialty economic crops such as strawberries, peppers, and cotton. Fourth, conventional constitutive expression vectors and gene overexpression technologies are mostly used, resulting in a heavy metabolic burden on the strains, and the formulation of inoculants is limited, lacking slow-release and multifunctional formulations. Fifth, research on ecological mechanisms is superficial, with only preliminary verification showing no significant differences in microbial diversity, and the synergistic interaction mechanisms between strains and plants and indigenous microorganisms, as well as the molecular regulatory pathways on soil phosphorus cycling, have not been revealed. Furthermore, the existing strains have unclear phosphorus solubilization and colonization mechanisms, lack theoretical support for targeted modification, and have not undergone in-depth long-term ecological safety verification, which limits their large-scale application.

[0004] Therefore, developing an engineered strain of Pseudomonas that possesses broad-spectrum adaptability to extreme environments, multiple phosphorus-solubilizing capabilities, and adaptability to multiple crops, clarifying its molecular-level regulatory mechanisms and ecological interaction pathways, constructing high-barrier gene modification and formulation preparation technologies, and verifying its long-term application safety are key to achieving stable field application of Pseudomonas and promoting the replacement of excessive fertilizer use. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method that... mcp(methyl-accepting chemotaxisprotein, mcp This is a Pseudomonas engineered strain that combines gene-directed modification with vector optimization and fusion expression to regulate rhizosphere colonization and phosphorus solubilization. This strain achieves simultaneous and efficient enhancement of colonization capacity and phosphorus solubilization function, reduces the metabolic burden of the strain, and enables the strain to adapt to a wide range of extreme environments and various crops.

[0006] Another objective of this invention is to provide a method for constructing engineered strains of Pseudomonas.

[0007] This invention further provides the application of the above-mentioned engineered Pseudomonas strain in plant growth promotion. Using this engineered strain, multi-formulation plant growth-promoting agents containing this strain can be prepared, such as conventional solid form, slow-release microcapsule form, and compound microecological form, providing an efficient solution for fertilizer over-reduction and soil ecological restoration.

[0008] The technical solution adopted by the present invention to achieve the above objectives is as follows:

[0009] This invention provides an engineered strain of Pseudomonas, which... mcp Gene fragments and rhizosphere adhesion protein genes bcsA Phospholyase-enhancing gene phyC Construct a fusion expression cassette and insert it into the prokaryotic expression vector pBBR1-MCS5 to build a fusion recombinant expression vector; then introduce the fusion recombinant expression vector into the carbon metabolism repressor gene knocked out of the original strain via electroporation or conjugation transfer. crp The original Pseudomonas;

[0010] The original *Pseudomonas taiwanensis* strain was deposited on February 27, 2019, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 17268, and was classified as *Pseudomonas taiwanensis*. Pseudomonas taiwanensis .

[0011] Preferably, the mcp The gene sequence is shown in SEQ ID NO.1. mcp The protein sequence is shown in SEQ ID NO.2; the rhizosphere adhesion protein gene. bcsA The gene sequence is shown in SEQ ID NO.5; the phospholytic enzyme enhancing gene phyC The gene sequence is shown in SEQ ID NO. 6; the carbon metabolism repressor gene crp The gene sequence is shown in SEQ ID NO. 11.

[0012] This invention also provides a method for constructing the above-mentioned engineered Pseudomonas strains, comprising the following steps:

[0013] (1) Amplification objective mcp Genes: Using the genomic DNA of the primitive Pseudomonas aeruginosa as a template, PCR amplification was performed using specific primers; simultaneously, the rhizosphere adhesion protein gene was amplified. bcsA Phospholyase-enhancing gene phyC Construct a fusion expression box;

[0014] (2) Gene knockout of the original strain: The carbon metabolism repressor gene of the original Pseudomonas was knocked out using CRISPR / Cas9 gene editing technology. crp ,get crp Knock out the original Pseudomonas;

[0015] (3) Transformation and screening: The recombinant expression vector was introduced into the target cell via electroporation. crp The original Pseudomonas was knocked out, and the transformation solution was plated on LB agar plates containing 50 μg / mL tetracycline for culture. Single colonies were picked and verified by PCR and functional domain activity detection to obtain engineered positive Pseudomonas strains. Pseudomonas taiwanensis JP2-3 / pBBR1-PphoA-MCP.

[0016] Preferably, in step (1), the sequence of the specific primer is:

[0017] Upstream primer (SEQ ID NO:3): 5'-CAGTGGATCCTTATACGCGGAACTGCCGC-3';

[0018] Downstream primer (SEQ ID NO:4): 5'-CAGTAAGCTTATGAAAACCCTGAGATCGATGTC-3'.

[0019] Preferably, in step (1), the PCR amplification reaction system is: 2 μL template DNA, 1 μL each of upstream and downstream primers, 25 μL 2×PCR Master Mix, and 21 μL ddH2O; the PCR amplification conditions are: 95℃ pre-denaturation for 5 minutes; 95℃ denaturation for 30 seconds, 58℃ annealing for 30 seconds, 72℃ extension for 1.5 minutes, 35 cycles; and 72℃ final extension for 10 minutes.

[0020] Preferably, in step (3), the recombinant expression vector is formed by inserting a phosphorus starvation-inducible phoA promoter into the pBBR1-MCS5 vector, linking the MCP-bcsA-phyC fusion expression cassette to the pBBR1-MCS5 vector, transforming Escherichia coli DH5α, screening positive clones, and sequencing verification.

[0021] Preferably, in step (3), the conditions for the electroconversion method are: voltage 2.5 kV, capacitance 25 μF, resistance 200Ω; the culture is carried out at 30℃ for 24 hours.

[0022] This invention also provides the application of the above-mentioned Pseudomonas engineered strains in promoting the growth of multiple crops, activating multi-component insoluble phosphorus, improving the fertility of different types of phosphorus-deficient soils, and constructing stress-resistant phosphorus-solubilizing and growth-promoting microbial communities.

[0023] This invention also provides a plant growth-promoting agent containing the above-mentioned engineered Pseudomonas strain, wherein the active ingredient in the plant growth-promoting agent is the fermentation broth of the engineered Pseudomonas strain; and the viable count of the engineered Pseudomonas strain in the fermentation broth is ≥10⁻⁶. 8 CFU / g.

[0024] The Pseudomonas engineered strains provided by this invention have a growth-promoting effect on grain crops such as wheat and corn, as well as specialty economic crops such as tomatoes and strawberries; the improved soils include, but are not limited to, severely saline-alkali soils containing ≤4% NaCl, acidic red soils, facility agriculture soils, continuous cropping obstacle soils, and desert sandy soils.

[0025] The Pseudomonas engineered strains provided by this invention can be applied by seed dressing, root irrigation, foliar spraying, or compounding with organic fertilizer, adapting to different agricultural operation scenarios; among them, the slow-release microcapsule type is suitable for low-fertility extreme soils such as desert sandy land and severely saline-alkali land, the compound microecological type is suitable for facility agriculture soil and continuous cropping obstacle soil, and the conventional solid type is suitable for conventional phosphorus-deficient soil.

[0026] Beneficial effects of the present invention

[0027] (1) This invention achieves gene-specific expression through a phosphorus starvation-inducible promoter and knocks out crp The gene reduces metabolic burden, increases colonization rate and phosphorus solubility, and its functional activity does not decrease significantly under extreme conditions.

[0028] (2) The engineered strain constructed in this invention has a growth-promoting effect on grain crops such as wheat and corn, as well as specialty economic crops such as strawberries and tomatoes. It is tolerant to extreme environments such as severe salinity, low temperature, strong acidity, low oxygen, and low nutrition. It can activate tricalcium phosphate, phytate phosphorus, iron phosphate and other polyphosphates, breaking through the application limitations of existing strains in single crops, soil and phosphorus forms.

[0029] (3) This invention constructs a multi-dimensional gene modification technology system of inducible expression vector, fusion gene and gene knockout; it develops three special formulations: conventional solid type, slow-release microcapsule type and compound microecological type, which can adapt to different soil types. Among them, the slow-release microcapsule type improves the soil survival time of the strain, and the compound microecological type realizes phosphorus solubilization, nitrogen fixation and disease resistance, and improves soil fertility and strain colonization rate.

[0030] (4) Compared with existing patented strains and commercial microbial agents, the engineered strains provided by this invention have significant advantages in phosphorus solubilization rate, colonization rate and crop yield increase, and can reduce the amount of chemical fertilizer used, thus achieving a stable increase in crop yield; the microbial agent has flexible application methods and can adapt to different agricultural operation scenarios, providing efficient and sustainable solutions for chemical fertilizer over-reduction substitution, extreme soil improvement and green agricultural development. Attached Figure Description

[0031] Figure 1 Gel electrophoresis image of the engineered strain constructed in Example 2;

[0032] Figure 2 This is a colorimetric diagram showing the IAA production capacity of the engineered strain constructed in Example 3;

[0033] Figure 3 This is a comparison diagram of the control group and the inoculum group in the field application trial of Example 5; where A is the control group; and B is the constructed Pseudomonas engineered strain group. Detailed Implementation

[0034] The technical solution of the present invention will be further explained and described below through specific embodiments.

[0035] Example 1: Cloning of a gene

[0036] (1) Strains and reagents: Primitive Pseudomonas (Taiwan Pseudomonas) Pseudomonas taiwanensis (CGMCC No. 17268), PCR kit, DNA extraction kit (TaKaRa), site-directed mutagenesis kit;

[0037] (2) Genomic DNA extraction: Primitive Pseudomonas genomic DNA was extracted using a bacterial genomic DNA extraction kit;

[0038] (3) PCR amplification and cloning: PCR amplification was performed using the original Pseudomonas genomic DNA as a template and specific primers.

[0039] Upstream primer (SEQ ID NO:3): 5'-cagtGGATCCTTATACGCGGAACTGCCGC-3';

[0040] Downstream primer (SEQ ID NO:4): 5'-cagtAAGCTTATGAAAACCCTGAGATCGATGTC-3'.

[0041] PCR reaction system (50μL): template DNA 2μL, forward and reverse primers 1μL each, 2×PCR Master Mix 25μL, ddH2O 21μL;

[0042] PCR reaction conditions: 95℃ pre-denaturation for 5 minutes; 95℃ denaturation for 30 seconds, 58℃ annealing for 30 seconds, 72℃ extension for 1.5 minutes, 35 cycles; 72℃ final extension for 10 minutes; The same PCR system and reaction conditions were used simultaneously to amplify the rhizosphere adhesion protein gene. bcsA (SEQ ID NO:5), Phospholyase Enhancer Gene phyC (SEQ ID NO:6), used to construct a fusion expression box;

[0043] phyC The gene amplification primers are:

[0044] Upstream primer (SEQ ID NO:7): 5' –ATGAAAGCCCATGAAATCGC-3';

[0045] Downstream primer (SEQ ID NO:8): 5'-TTACGCCAAAGGCCGGC-3'.

[0046] bcsA The gene amplification primers are:

[0047] Upstream primer (SEQ ID NO:9): 5'-ATGTCTTCACGTAAATTCGGCCT-3';

[0048] Downstream primer (SEQ ID NO:10): 5'-TCAGGCCTGCTGCGGCT-3'.

[0049] Example 2 Construction and Screening of Engineered Strains

[0050] (1) Construction of inducible vector: The phosphorus starvation-inducible phoA promoter was inserted into the pBBR1-MCS5 vector (commercial vector, purchased from Shanghai Zeye Biotechnology Co., Ltd., product number: ZY8177), and the basic vector containing the phoA promoter was obtained by enzyme digestion and sequencing verification;

[0051] (2) Construction of recombinant expression vector: achieved by overlap extension PCR technology. mcp Gene (SEQ ID NO.1), rhizosphere adhesion protein gene bcsA Phospholyase-enhancing gene phyC Seamless tandem: When designing specific primers, bcsA upstream primers for genes contain mcp The 20 bp homologous sequence downstream of the gene phyC upstream primers for genes contain bcsA The 20bp homologous sequence downstream of the gene was amplified using a three-step PCR method to obtain the complete MCP-. bcsA - phyC The fusion expression cassette (approximately 3980 bp in length) ensures that the three genes are directly spliced ​​in sequence without any additional ligation sequences. The gene fragments obtained by PCR amplification are double-digested with BamHI and HindIII. The digestion system (50 μL) is as follows: 5 μL 10×CutSmart Buffer, 2 μL BamHI (10 U / μL), 2 μL HindIII (10 U / μL), 20 μL PCR product (≥1 μg / μL), and 21 μL enzyme-free water.

[0052] Enzyme digestion conditions: Incubate at 37℃ for 3 h, then heat at 65℃ for 20 min to inactivate restriction endonucleases. The digested target gene fragment is recovered by 1% agarose gel electrophoresis.

[0053] (3) Vector digestion: The pBBR1-MCS5 vector (the basic vector containing the phoA promoter, approximately 5.3 kb in length) containing the phosphorus starvation-inducible phoA promoter was simultaneously double-digested. The digestion system (50 μL) consisted of: 5 μL of 10×CutSmart Buffer, 2 μL of BamHI (10 U / μL), 2 μL of HindIII (10 U / μL), 10 μL of pBBR1-MCS5 vector (50 ng / μL), and 31 μL of enzyme-free water. The reaction conditions were the same as those described in step (2) of the target gene digestion. The linearized vector fragment (approximately 5.3 kb in length) was recovered by electrophoresis. 1 μL of Antarctic Phosphatase was added and incubated at 37℃ for 1 h for dephosphorylation (inactivated at 65℃ for 20 min) to reduce vector self-ligation.

[0054] The enzyme-digested and recovered gene fragments were mixed with the linearized pBBR1-MCS5 vector at a molar ratio of 3:1 and ligated using T4 DNA ligase. The ligation system (20 μL) consisted of: 2 μL of 10×T4 DNA Ligase Buffer, 1 μL of T4 DNA Ligase (5 U / μL), and the digested DNA molecule. mcp 6 μL of gene fragment, 2 μL of enzyme-digested vector fragment, and 9 μL of enzyme-free water were used. Ligation reaction conditions: incubation at 16℃ for 12 h to construct the fusion recombinant vector pBBR1-PphoA-MCP-bcsA-phyC.

[0055] The ligation product was transformed into E. coli DH5α competent cells and plated on LB agar plates containing 50 μg / mL tetracycline (10 g tryptone, 5 g yeast extract, 5 g sodium chloride, 12 g agar, deionized water added and volume adjusted to 1 L). The plates were incubated at 37°C for 12 h. Single colonies were picked and plasmids were extracted and verified by BamHI-HindIII double digestion. Finally, the accuracy of the target gene and fusion expression cassette sequence was verified by Sanger sequencing to ensure that there were no base mutations or frameshift mutations.

[0056] (4) crp Gene knockout: Using CRISPR / Cas9 gene editing technology, designed crp Gene-specific sgRNA was introduced into primitive Pseudomonas bacteria, and obtained through resistance selection and PCR verification. crp Knockout strains;

[0057] sgRNA coding strand sequence (SEQ ID NO:12): 5'-GGTGATGAAGAAGTCGATGG-3';

[0058] sgRNA template strand sequence (SEQ ID NO:13): 5'-CCATCGACTTCTTCATCACC-3'.

[0059] (5) Transformation and screening: The recombinant expression vector pBBR1-PphoA-MCP-bcsA-phyC was introduced into the body via electroconversion (voltage 2.5 kV, capacitance 25 μF, resistance 200 Ω). crp Knockout strain (original Pseudomonas Δcrp) competent cells were immediately added to 1 mL LB liquid medium after electroporation and cultured at 28℃ and 180 rpm for 3 h to restore strain activity. 100 μL of the restored bacterial culture was evenly spread on LB plates containing 50 μg / mL tetracycline (10 g tryptone, 5 g yeast extract, 5 g sodium chloride, 12 g agar, and deionized water to a final volume of 1 L, pH 7.0) and incubated at 30℃ for 24 h. Ten single colonies were randomly selected for PCR verification, using a cross-fragment combination of vector and target gene primers.

[0060] Upstream primer (SEQ ID NO:14): 5'-GCTTGTTCGGCTGGTCGAAAT-3';

[0061] Downstream primer (SEQ ID NO:15): 5'-TCAGTCGATGATGTCGGTGA-3'.

[0062] The PCR reaction system (50 μL) contained 2 μL of single-colony bacterial culture, 1 μL each of forward and reverse primers, 25 μL of 2×PCR MasterMix, and 21 μL of enzyme-free water. The reaction conditions were: 95℃ pre-denaturation for 5 min, 35 cycles (95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 3 min), and a final extension at 72℃ for 10 min. The reaction was detected by 1% agarose gel electrophoresis (120 V, 30 min). Positive clones that amplified a clear, specific band of approximately 6000 bp were selected (e.g., ...). Figure 1 As shown), engineered strains of positive Pseudomonas bacteria were obtained. Pseudomonas taiwanensis JP2-3 / pBBR1-PphoA-MCP-bcsA-phyC.

[0063] Example 3: Functional Verification of Engineered Pseudomonas Strains

[0064] (a) Determination of phosphorus solubilization efficiency:

[0065] Three phosphorus source culture media were prepared: tricalcium phosphate (inorganic phosphorus), phytate phosphorus (organic phosphorus), and ferric phosphate (the main phosphorus form in acidic soils). All three phosphorus source culture media used a phosphorus-free basal medium as a base, with different sparingly soluble phosphorus sources added to each. The specific composition of the phosphorus-free basal medium was as follows: Phosphorus-free basal medium: glucose 10g, (NH4)2SO4 0.5g, MgSO4·7H2O 0.3g, KCl 0.3g, FeSO4·7H2O 0.03g, MnSO4·H2O 0.01g, diluted to 1 liter with deionized water, pH 7.0.

[0066] Based on this, the following phosphorus sources were added respectively: ① Tricalcium phosphate (inorganic phosphorus): 5 g / L; ② Phytate phosphorus (organic phosphorus): 3 g / L; ③ Ferric phosphate (the main phosphorus form in acidic soils): 4 g / L. After sterilization, they were ready for use. Seed cultures of engineered Pseudomonas strains, original strains, and knockout strains were inoculated separately (each strain was cultured to OD200 before inoculation). 600 =1.0, centrifuge to collect bacterial cells, resuspend in sterile physiological saline, and adjust the viable count to 1×10. 8 (CFU / mL), the inoculum size is 2% of the liquid culture medium volume.

[0067] Simultaneously, normal conditions (30℃, pH 7.0), severely saline-alkaline conditions (3% NaCl, pH 8.5), strongly acidic conditions (pH 4.0), and low temperature conditions (10℃) were set up. The culture was carried out under normal conditions and under the extreme saline-alkaline and acidic conditions at 30℃ with shaking at 200 rpm for 3 days, and under the low temperature conditions at 10℃ with shaking at 200 rpm for 5 days. The available phosphorus content in the culture medium was determined using the molybdenum antimony colorimetric method. Specific results are shown in Table 1.

[0068] Table 1

[0069]

[0070] (II) Rhizosphere colonization rate determination:

[0071] Grain crops such as corn, wheat, soybeans, and tomatoes, and specialty cash crops such as strawberries, peppers, and cotton were selected as test crops. Seed dressing and inoculation (10 8 CFU / granule were planted in conventional phosphorus-deficient soil, saline-alkali soil (2% NaCl, pH 8.5; 4% NaCl, pH 9.0), acidic red soil (pH 4.0), facility soil (agricultural) with 60% phytic acid phosphorus content, and strawberry continuous cropping obstacle soil. The number of colonizing bacteria was counted after 14 days and 60 days of pot cultivation. The specific results are shown in Table 2.

[0072] Table 2

[0073]

[0074] (III) IAA and ACC deaminase activities

[0075] The Salkowski colorimetric method was used to qualitatively and quantitatively detect the indoleacetic acid (IAA) secretion capacity of the strain. The engineered strain and the original strain were inoculated into LB liquid medium containing 0.5 g / L tryptophan and cultured at 30℃ and 200 rpm for 48 h with shaking. The supernatant was then collected by centrifugation and mixed with Salkowski colorimetric solution at a 1:2 ratio, and reacted in the dark for 30 min. The colorimetric results showed that the left blank control group (CK) was pale yellow, while the reaction solutions of the original and engineered Pseudomonas strains were distinctly pink, indicating that the strain can secrete IAA (such as...). Figure 2 (As shown). Quantitative analysis revealed that the IAA yield of the original strain was 18.6 ± 1.2 μg / mL, while the IAA yield of the engineered strain of this invention was 19.2 ± 2.3 μg / mL.

[0076] Meanwhile, the 2,4-dinitrophenylhydrazine method was used to detect the ACC deaminase activity. The bacterial cells were inoculated in an induction medium containing 3 mmol / L ACC and induced at 30℃ for 24 h. After ultrasonic disruption to prepare crude enzyme solution, the amount of α-butanone produced was measured. The amount of enzyme required to catalyze the production of 1 nmol of α-butanone in 1 hour was defined as 1 enzyme activity unit (U). The results showed that the ACC deaminase activity of the original strain was 2.36±0.18 U / mg protein, and the ACC deaminase activity of the Pseudomonas engineered strain was 2.82±0.35 U / mg protein, indicating that the genetic modification did not affect the ability of the original strain to secrete auxin IAA and degrade the plant stress ethylene precursor ACC.

[0077] Example 4 Preparation of plant growth-promoting bacteria

[0078] (a) Conventional solid bacterial agents

[0079] 1. Fermentation with engineered bacteria: Inoculate the engineered bacterial strain (1% inoculum) into an LB fermenter and incubate at 30℃ and 200 rpm for 20 hours to obtain the fermentation broth (1.5 × 10⁻⁶ viable cells). 9 (CFU / mL)

[0080] 2. Concentration: Centrifuge the fermentation broth at 8000 rpm for 10 minutes, collect the bacterial precipitate, and resuspend it in sterile water until the viable cell count reaches 5 × 10⁻⁶. 9 CFU / mL;

[0081] 3. Formulation: Mix the bacterial suspension, humic acid, trehalose, and vitamin C in a mass ratio of 10:5:3:0.1, and spray dry (inlet air 130℃, outlet air 70℃) to make a solid bacterial agent;

[0082] 4. Quality testing: viable count 2.3 × 10⁻⁶ 8 CFU / g, contamination rate 2.1%, pH 7.2, meets the standard (viable count ≥10). 8 CFU / g, contamination rate ≤5%, pH 6.0-8.0).

[0083] (ii) Sustained-release microcapsule bacterial agents

[0084] 1. Fermentation and concentration of engineered bacteria: Same as steps 1 and 2 in (I), to obtain a viable count of 5 × 10⁻⁶. 9 CFU / mL bacterial suspension;

[0085] 2. Wall material preparation: Bentonite and chitosan are mixed at a mass ratio of 2:1, dissolved in a 1% acetic acid solution, and stirred until completely dissolved, with a concentration of 5%;

[0086] 3. Microcapsule preparation: The bacterial suspension (core material) and wall material were mixed at a core-to-wall ratio of 1:2, emulsified at high speed (8000 rpm) for 10 minutes, 2% glutaraldehyde crosslinking agent was added, stirred at room temperature for 30 minutes, the microcapsules were collected by centrifugation and freeze-dried;

[0087] 4. Quality testing: viable count 1.8 × 10⁻⁶ 8 CFU / g, encapsulation rate 92.5%, contamination rate 1.8%.

[0088] (III) Compound microecological bacterial agents

[0089] 1. Single-strain fermentation: Following the same method, engineered strains of Pseudomonas, Azotobacter albopictus, and Bacillus subtilis were cultured to obtain fermentation broths for each strain, with a viable cell count of 1.5 × 10⁻⁶ for each strain. 9 CFU / mL;

[0090] 2. Compounding: Mix the three fermentation broths at a live cell count ratio of 5:3:2 to obtain a compound fermentation broth;

[0091] 3. Carrier compounding: The compound fermentation broth and modified biochar (Tianjin Kunhe Biotechnology Group Co., Ltd., KH-MBC-01) were mixed at a mass ratio of 1:1, stirred and adsorbed for 30 minutes, and then freeze-dried.

[0092] 4. Quality testing: Total viable count 2.5 × 10⁻⁶ 8 CFU / g, engineered strains accounted for 51.2%, and the contamination rate was 2.3%.

[0093] Example 5 Field Application Trial

[0094] (I) Experiment 1: Maize Experiment in Phosphorus-Deficient Soil (80% Reduction in Chemical Fertilizer)

[0095] Test site: Phosphorus-deficient soil (available phosphorus 8.5 mg / kg);

[0096] Test crop: Maize (Zhengdan 958);

[0097] Experimental groups: 3 groups, 3 replicates per group, plot area 20 m² 2 .

[0098] ① Control group: No phosphorus and potassium fertilizers were applied, and no inoculants were inoculated;

[0099] ② Conventional fertilizer group: Apply diammonium phosphate (15 kg / mu);

[0100] ③ Group with 80% reduction in fertilizer application: application of diammonium phosphate (3 kg / mu) + conventional solid microbial agent of this invention (prepared in Example 3; usage 1 kg / mu, seed dressing);

[0101] All fields were managed using conventional methods. See the on-site photos below. Figure 3 As shown in A and B, the specific results are shown in Table 3.

[0102] Table 3

[0103]

[0104] (II) Experiment 2: Wheat Experiment in Severely Saline-Alkali Land

[0105] Experimental location: Severely saline-alkali land in the Yellow River Delta (4% NaCl, available phosphorus 6.2 mg / kg); Test crop: Wheat (Jimai 22).

[0106] Experimental groups: 3 groups, 3 replicates per group, plot area 20 m² 2 ;

[0107] ① Control group: No phosphate fertilizer was applied, and no inoculant was inoculated;

[0108] ② Fertilizer group: Apply diammonium phosphate (15 kg / mu);

[0109] ③ Microbial agent group: Apply the slow-release microcapsule microbial agent of this invention (1.5 kg / mu, root irrigation);

[0110] Results: The wheat yield in the microbial agent group was 389.2 kg / mu (compared to 265.3 kg / mu in the control group, an increase of 46.7%), and the grain phosphorus content was 0.29% (compared to 0.22% in the control group). The yield in the fertilizer group was 405.8 kg / mu, and the grain phosphorus content was 0.31%. The effect of the microbial agent group was close to that of the fertilizer group.

[0111] (III) Experiment 3: Soil Experiment on Continuous Crop Obstacles to Tomatoes in Facility Agriculture

[0112] Experimental site: soil with continuous cropping obstacles for tomatoes in protected agriculture (phytic acid phosphorus content 60%, available phosphorus 7.8 mg / kg); test crop: tomato (Zhongza 105).

[0113] Experimental groups: 3 groups, 3 replicates per group, plot area 15 m² 2 ;

[0114] ① Control group: No phosphate fertilizer was applied, and no inoculant was inoculated;

[0115] ② Commercial inoculant group: Apply a certain brand of phosphate-solubilizing inoculant (1 kg / mu, seed dressing);

[0116] ③ The microbial agent group of the present invention: Apply the compound microecological microbial agent of the present invention (1 kg / mu, foliar spray);

[0117] Results: The tomato yield of the inoculant group was 526.3 kg / mu (compared to 418.5 kg / mu in the control group, an increase of 25.8%), and the disease incidence was reduced by 32.5%. The yield of the commercial inoculant group was 472.1 kg / mu (an increase of 12.8%), and the disease incidence was reduced by 10.2%. The strain of the present invention has significant advantages.

[0118] (iv) Experiment 4: Experiment on acidic red soil for specialty cash crop strawberry

[0119] Experimental location: strongly acidic red soil (pH 4.0, available phosphorus 5.5 mg / kg); test crop: strawberry (Hongyan 99);

[0120] Experimental groups: 3 groups, 3 replicates per group, plot area 10 m² 2 ;

[0121] ① Control group: No phosphate fertilizer was applied, and no inoculant was inoculated;

[0122] ② Fertilizer group: Apply diammonium phosphate (8 kg / mu);

[0123] ③ Microbial agent group: Apply the slow-release microcapsule microbial agent of this invention (1 kg / mu, for seed dressing);

[0124] Results: The strawberry yield in the microbial agent group was 1258.3 kg / mu (compared to 985.6 kg / mu in the control group, an increase of 27.7%), and the sugar content of the fruit increased by 1.8%. The yield in the fertilizer group was 1302.5 kg / mu, and the sugar content of the fruit increased by 2.0%.

[0125] Example 6 Antibacterial Test

[0126] The in vitro antibacterial activity of the engineered Pseudomonas strain constructed in this invention was determined using the plate confrontation method. The tested pathogen was activated and cultured on PDA plates. A 5 mm diameter mycelial disc was prepared using a sterile punch and inoculated into the center of the plate. The culture was then carried out until the OD (October Observation Time) reached 5%. 600 Engineered Pseudomonas strains with a concentration of 1.0 and original strains (treatment group) were used to create microbial suspensions. 200 μL of bacterial suspension was added to each microbial suspension in equidistantly spaced wells on agar plates using the Oxford cup method. Sterile physiological saline was used as a blank control. After incubation at 28℃ for 72 h, the diameter of the inhibition zone was measured and recorded using the cross-hatching method, and the inhibition rate was calculated. The in vitro antibacterial test data are shown in Table 4.

[0127]

[0128] Table 4

[0129]

[0130] Example 7: Long-term ecological effect test

[0131] Experimental design: A five-season corn-wheat rotation was conducted to measure soil physicochemical properties, enzyme activity, and microbial diversity. A control group (without microbial agent) and a microbial agent group (1 kg / mu of the compound microbial agent of this invention was applied as basal fertilizer annually) were set up.

[0132] Results: In the fifth season, maize yield still increased by 18.7% (control group 423.5 kg / mu, microbial agent group 502.6 kg / mu); soil organic matter increased by 25.6%, available phosphorus increased by 12.8 mg / kg, and soil urease, phosphatase, and sucrase activities increased by 42.3%, 58.7%, and 35.2%, respectively; the proportion of soil aggregates (>0.25 mm) increased by 32.5%; 16S rRNA sequencing showed that the Shannon index of the soil in the microbial agent group was 12.3% higher than that in the control group, and the abundance of beneficial indigenous microorganisms such as rhizobia and actinomycetes increased by 20.5%-35.8%, without destroying the indigenous microbial community structure, achieving targeted optimization.

Claims

1. An engineered strain of Pseudomonas, characterized in that, The phosphorus starvation-inducible phoA promoter was inserted into the pBBR1-MCS5 vector, and the prokaryotic expression vector pBBR1-MCS5 containing the phoA promoter was obtained by enzyme digestion and sequencing verification; mcp Gene fragments and rhizosphere adhesion protein genes bcsA Phospholyase-enhancing gene phyC A fusion expression cassette was constructed, and a fusion recombinant expression vector was constructed by inserting it into the prokaryotic expression vector pBBR1-MCS5; the fusion recombinant expression vector was then introduced into the original strain that had its carbon metabolism repression gene knocked out. crp primitive Pseudomonas ( Pseudomonas taiwanensis ); The original *Pseudomonas taiwanensis* strain was deposited on February 27, 2019, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 17268 and classified as *Pseudomonas taiwanensis*. Pseudomonas taiwanensis ; The mcp The gene sequence is shown in SEQ ID NO.

1. mcp The protein sequence is shown in SEQ ID NO.2; The rhizosphere adhesion protein gene bcsA The gene sequence is shown in SEQ ID NO.5; the phospholytic enzyme enhancing gene phyC The gene sequence is shown in SEQ ID NO.6; The carbon metabolism repressor gene crp The sequence is shown in SEQ ID NO.

11.

2. A method for constructing an engineered Pseudomonas strain as described in claim 1, characterized in that, Includes the following steps: (1) Amplification objective mcp Genes: Using the genomic DNA of the primitive Pseudomonas aeruginosa as a template, PCR amplification was performed using specific primers; simultaneously, the rhizosphere adhesion protein gene was amplified. bcsA Phospholyase-enhancing gene phyC Construct a fusion expression box; (2) Gene knockout of the original strain: The carbon metabolism repressor gene of the original Pseudomonas was knocked out using CRISPR / Cas9 gene editing technology. crp ,get crp Knock out the original Pseudomonas; (3) Transformation and screening: The recombinant expression vector was introduced into the target cell via electroporation. crp The original Pseudomonas was knocked out, and the transformation solution was plated on LB agar plates containing 50 μg / mL tetracycline for culture. Single colonies were picked and verified by PCR and functional domain activity detection to obtain engineered Pseudomonas positive strains. Pseudomonas taiwanensis JP2-3 / pBBR1-PphoA-MCP.

3. The construction method according to claim 2, characterized in that, In step (1), the sequence of the specific primer is: Upstream primer: 5'-cagtGGATCCTTATACGCGGAACTGCCGC-3'; Downstream primer: 5'-cagtAAGCTTATGAAAACCCTGAGATCGATGTC-3'.

4. The construction method according to claim 3, characterized in that, In step (1), the PCR amplification reaction system is as follows: 2 μL template DNA, 1 μL each of upstream and downstream primers, 25 μL 2×PCR Master Mix, and 21 μL ddH2O. The PCR amplification conditions are as follows: 95℃ pre-denaturation for 5 minutes; 95℃ denaturation for 30 seconds, 58℃ annealing for 30 seconds, 72℃ extension for 1.5 minutes, 35 cycles; and 72℃ final extension for 10 minutes.

5. The construction method according to claim 2, characterized in that, The recombinant expression vector consists of inserting a phosphorus starvation-inducible phoA promoter into the pBBR1-MCS5 vector, linking the MCP-bcsA-phyC fusion expression cassette to the pBBR1-MCS5 vector, transforming it into E. coli DH5α, screening for positive clones, and sequencing verification.

6. The construction method according to claim 2, characterized in that, In step (3), the conditions for the electroconversion method are: voltage 2.5 kV, capacitance 25 μF, resistance 200 Ω; the culture is carried out at 30℃ for 24 hours.

7. The application of a Pseudomonas engineered strain as described in claim 1 in promoting the growth of multiple crops, activating multi-component insoluble phosphorus, and improving the fertility of different types of phosphorus-deficient soils.

8. A plant growth-promoting agent containing the engineered strain of *Pseudomonas* as described in claim 1, characterized in that, The active ingredient in the plant growth promoting bacterial agent is an engineered strain of Pseudomonas fermentation broth; in the engineered strain fermentation broth, the viable cell count of the engineered strain of Pseudomonas is > 10 8 CFU / g.

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