Recombinant rhizobium OEuspA with stress resistance, plant salt-resistant microbial inoculum and application of plant salt-resistant microbial inoculum

Abstract

The invention discloses recombinant rhizobium OEuspA with stress resistance, a plant salt-resistant bacterial agent and application of the plant salt-resistant bacterial agent, and belongs to the technical field of genetic engineering. According to the invention, a new uspA gene is identified from azotobacter stenoma ORS571 for the first time. And then carrying the uspA gene into the azotobacter stem nodule by using a plasmid vector, and constructing to obtain the recombinant rhizobium over-expressing the uspA gene. Compared with wild-type rhizobium, the stress resistance of the recombinant rhizobium for overexpressing the uspA gene, which is constructed by the invention, is remarkably improved. After the recombinant rhizobium over-expressing the uspA gene is symbiotic with sesbania eriocalyx, wheat and corn, the growth of plants under the salt stress condition can be promoted. The uspA gene disclosed by the invention can be used for constructing a recombinant rhizobium material with stronger stress resistance and growth promotion capability, and has potential and wide application prospects in the fields of genetic engineering transformation of rhizobium or other bacteria, the rhizobium microbial inoculum preparation industry, the bean planting industry in agricultural production and saline-alkali soil development.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a recombinant rhizobium OE with stress resistance. uspA Plant salt-tolerant bacteria and their applications. Background Technology

[0002] Rhizobia are Gram-negative bacteria that can establish symbiotic relationships with legumes. Through symbiosis, legumes can convert atmospheric nitrogen into nitrogen compounds that are usable by the plant, thus significantly expanding their nitrogen utilization capacity. The stem-nodulating nitrogen-fixing rhizobium ORS571 is a relatively unique rhizobium, possessing both symbiotic and autotrophic nitrogen-fixing capabilities. It can form nodules not only on the roots of its host plant, *Senecio scandens*, but also on its stems. This unique nitrogen-fixing ability allows it to effectively promote the growth of *Senecio scandens* and enhance its adaptability to saline-alkali environments.

[0003] Soil salinization is a significant problem in current agricultural production. Salt stress causes ion toxicity, osmotic stress, nutrient deficiency, and oxidative stress, severely impacting crop yields. Planting salt-tolerant plants can achieve long-term improvement of saline-alkali soils, while methods such as improving microbial inoculants can enhance the salt tolerance of plants. In saline-alkali soil environments, rhizobia, whether in a free state or in symbiotic relationships with plants, are often subjected to various environmental stresses, including salt-alkali stress, oxidative stress, heavy metal stress, and acid-base stress. These environmental pressures reduce the survival ability of rhizobia, thereby affecting their colonization in host plants and their ability to promote host plant growth under stress. Therefore, improving the stress resistance of rhizobia is an urgent problem to be solved. Summary of the Invention

[0004] In view of the above-mentioned prior art, the purpose of this invention is to provide a recombinant rhizobium OE with stress resistance. uspA Plant salt-tolerant bacteria and their applications.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A first aspect of the present invention provides uspA The application of genes in the following (1) or (2): (1) Improve the stress resistance of rhizobia; (2) Constructing engineered rhizobium strains with stress resistance; The uspA A gene is a nucleic acid molecule as shown in (i) or (ii): (i) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.1; (ii) Nucleic acid molecules other than (i) that encode the amino acid sequence shown in SEQ ID NO.2.

[0006] In the above applications, the stress resistance refers to the ability to withstand salt stress, heavy metal stress, and / or oxidative stress.

[0007] This invention is the first to identify a novel rhizobium from stem nodule nitrogen-fixing rhizobium ORS571. uspA Gene, overexpressed in stem-nodulating nitrogen-fixing rhizobia. uspA Genes can significantly enhance the stress resistance of rhizobia.

[0008] A second aspect of the present invention provides the application of UspA protein in enhancing the stress resistance of rhizobia; said UspA protein is a protein as shown in (A1) or (A2) below: (A1) A protein with the amino acid sequence shown in SEQ ID NO.2; (A2) The protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

[0009] In the aforementioned proteins, the protein tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, detection, tracing, and / or purification of the target protein. The protein tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0010] A third aspect of the invention provides a way to promote uspA The substance that enhances gene expression uspA The application of substances with gene activity and / or content in the following (1) or (2): (1) Improve the stress resistance of rhizobia; (2) Constructing engineered strains of rhizobium with stress resistance.

[0011] In some preferred embodiments of the present invention, promoting uspA The substance used for gene expression can be any of the following: C1) contains uspA Gene expression cassettes; C2) contains uspA Recombinant vectors of genes, or recombinant vectors containing the expression cassette described in C1); C3) contains uspA Recombinant microorganisms containing genes, or recombinant microorganisms containing the expression cassette described in C1), or recombinant microorganisms containing the recombinant vector described in C2).

[0012] A fourth aspect of the invention provides overexpression uspA The recombinant rhizobium of the gene is used in at least one of the following (1)-(4): (1) Promote plant growth under salt stress; (2) Increase the number of root nodules in plants under salt stress; (3) Increase the number of stem nodules in plants under salt stress; (4) Preparation of plant salt-resistant bacteria.

[0013] In the above applications, the overexpression uspA The recombinant rhizobium was constructed using the following method: The nucleotide sequence shown in SEQ ID NO.1 was cloned into a plasmid to obtain a recombinant plasmid; the recombinant plasmid was then introduced into stem-nodulated nitrogen-fixing rhizobium host cells to construct an overexpression. uspA Recombinant rhizobia.

[0014] Preferably, the plasmid is pBBR1MCS-2.

[0015] In the above applications, the plant is a legume or a grass; preferably, it is a sesquiterpenoid (Gnaphalium affine). Sesbania rostrata ), corn or wheat.

[0016] In the above application, the salt stress condition is a NaCl concentration of 0.3%.

[0017] The beneficial effects of this invention are: (1) This invention is the first to identify a novel rhizobium from stem nodule nitrogen-fixing rhizobium ORS571. uspA Genes. Then, plasmid vectors are used to... uspA Genes were delivered into stem-nodulated nitrogen-fixing rhizobia to construct an overexpression gene. uspA Recombinant rhizobia with recombinant genes. Compared with wild-type rhizobia, the overexpression gene constructed in this invention... uspA The recombinant genes in rhizobia significantly enhance stress resistance, specifically manifested in the following ways: The growth rate was faster under 1.5% NaCl conditions, and the growth stability period was reached 12 h earlier than that of the wild type; the survival rate was significantly increased under heavy metal stress (100 μM CdCl2); and the inhibition zone generated under H2O2 and CHP oxidant stress was significantly reduced.

[0018] (2) Overexpression uspA When the recombinant rhizobium was symbiotic with Sesbania pubescens, it significantly promoted the growth of the host plant under 0.3% salt stress: plant height increased by 24.6%, root length increased by 13.4%, root nodule number increased by 33.3%, and stem nodule number increased by 5.77%. Plant height and root nodule number were significantly higher than those of the wild type, and the differences between the two were statistically significant. P <0.05).

[0019] In summary, the present invention uspAGenes can be used to construct recombinant rhizobium materials with stronger stress resistance and growth-promoting ability. They have potential and broad application prospects in the field of genetic engineering of rhizobia or other bacteria, in the preparation of rhizobium inoculants, in the legume planting industry in agricultural production, and in the development of saline-alkali land. Attached Figure Description

[0020] Figure 1 The recombinant plasmid pBBR1MCS-2- was constructed. uspA The results of agarose gel electrophoresis of the double enzyme digestion products are as follows: Lane 1 is the product of plasmid pBBR1MCS-2 after double digestion with restriction endonucleases HindIII and XbaI; Lane 2 is the product of recombinant plasmid pBBR1MCS-2-uspA after double digestion with restriction endonucleases HindIII and XbaI. uspA The sequence has been successfully ligated into the vector; Lane 3 is uspA Gene amplification products.

[0021] Figure 2 The wild-type strain WT (ORS571) and the overexpressing strain OE provided for embodiments of the present invention uspA Growth curves in (A) TY liquid medium and (B) TY liquid medium with 1.5% NaCl concentration; where squares represent wild-type strain WT and triangles represent overexpression strain OE. uspA .

[0022] Figure 3 For wild-type strain WT (ORS571) and overexpressing strain OE uspA Serial dilutions were performed, and the colonies were inoculated into TY solid medium supplemented with 100 μC dCl2. The colony states were observed. In the figure, from left to right, the colonies were 10 times the original concentration. 3 10 4 10 5 10 6 10 7 10 8 .

[0023] Figure 4 To investigate the effects of H2O2 and CHP on wild-type WT (ORS571) and overexpressing strain OE. uspA The antibacterial zone is formed.

[0024] Figure 5 Phenotypic comparisons and bar charts of growth indicators were created for potted *Senecio scandens* plants in garden soil with a salt concentration of 0.3% NaCl after 48 days of inoculation with different bacterial strains. Growth indicators included plant height, root length, number of root nodules, and number of stem nodules. The inoculated bacterial strains included: WT (ORS571) and the overexpression strain OE. uspA .

[0025] Figure 6 Bar charts showing the growth indicators of potted wheat and maize plants in garden soil supplemented with 0.3% NaCl after 20 days inoculated with different bacterial strains. Growth indicators included plant height and root length. Inoculated bacterial strains included: WT (ORS571) and the overexpression strain OE. uspA . Detailed Implementation

[0026] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0027] As mentioned earlier, environmental stresses such as salt and alkali stress, heavy metal stress, and oxidative stress can reduce the survival ability of rhizobia, thereby affecting their colonization in host plants and their ability to promote host plant growth under stress. Therefore, improving the stress resistance of rhizobia is an urgent problem to be solved.

[0028] In view of this, the present invention has identified a gene capable of enhancing the stress resistance of rhizobia ORS571 from the whole genome of nitrogen-fixing rhizobia. uspA Gene. Compared with the previously reported universal stress protein (UspA) in Escherichia coli, Methanococcus janthraceae, and soybean, its similarity is less than 12%, making it a novel UspA protein.

[0029] By overexpressing in rhizobia uspA Genes can enhance the resistance of rhizobia to salt stress, heavy metal stress, and oxidative stress.

[0030] To achieve uspA Gene overexpression: This invention constructs an overexpression... uspA The gene expression cassette, the nucleotide sequence of which is shown in SEQ ID NO.1. The expression cassette is composed of... uspA Upstream sequence of the gene (549bp) (positions 1-549 of SEQ ID NO.1), encoding uspA The gene sequence (834 bp) (positions 550-1383 of SEQ ID NO.1) and uspA The downstream sequence (432 bp) (positions 1384-1815 of SEQ ID NO.1) is composed; the amino acid sequence of the encoded UspA protein is shown in SEQ ID NO.2.

[0031] Furthermore, overexpression uspA Recombinant rhizobia live in symbiosis with host plants and can promote the growth of host plants under salt stress.

[0032] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0033] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available. Wherein: The preservation information for *Oryza sativa* ORS571, a nitrogen-fixing rhizobium in stem nodules, is DSMZ (German Scientific Centre for Culture Collections). Azorhizobium caulinodans ORS571, DSM No.: 5975; and described in non-patent literature Dreyfus, B., Garcia, JL, Gillis, M (1988). Characterization of Azorhizobium caulinodans gen. nov. a stem uspA lating nitrogen-fixing bacteriumisolated from Sesbania rostrata Int. J. Syst. Bacteriol. 38:89-98. The results are available from the applicant for use in replicating this experiment.

[0034] Escherichia coli DH5α was purchased from TransGen Biotech; plasmid pBBR1MCS-2 was kanamycin resistant; helper plasmid pRK2013 was kanamycin resistant.

[0035] The culture media used in the embodiments of this invention are as follows: Liquid TY medium (solid medium with an additional 15 g / L agar powder): tryptone 5 g / L, yeast extract 3 g / L, anhydrous calcium chloride 0.6 g / L, pH=7.0, sterilized at 121 ℃ for 20 min.

[0036] LB medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar powder 15 g / L. pH=7.0, sterilize at 121 ℃ for 20 min.

[0037] In the quantitative experiments described below, all experiments were repeated three times. GraphPad Prism 8 statistical software was used to process the data. Experimental results are presented as mean ± standard deviation in line and bar charts, and Student's st-test was used to detect whether there were differences between samples and whether these differences were significant. The representatives showed significant differences. P Values ​​<0.05 are marked above the bar chart.

[0038] Example 1: Construction of recombinant plasmids The genome of *O. 571*, a nitrogen-fixing rhizobium in stem nodules, was extracted using the Tiangen Bacterial Genome Extraction Kit (extraction method as per the included instructions). The concentration of the extracted sample was determined using a Nanodrop 2000 (100-300 ng / μL is preferred), and the sample was stored at -20 ℃ for later use. Using the extracted genomic DNA as a template, primers were applied... uspA -HB-F and uspA -HB-R amplification of the target gene: Table 1: Primers used in the examples Note: The underlined part is the added restriction endonuclease recognition sequence.

[0039] The above primers amplify the sequence shown in SEQ ID NO.1, because uspA Gene expression in the strain is reversed; therefore, to ensure that its expression direction in the recombinant plasmid is consistent with that in the original strain, the amplification direction is adjusted to be from top to bottom. uspA -HB-R amplifies the beginning of the SEQ ID NO.1 sequence. uspA -HB-F amplifies the end of the SEQ ID NO.1 sequence, and the direction is reverse complementary.

[0040] The fragment amplified in Example 1 was confirmed to be the same size as the designed length by 1% agarose gel electrophoresis. The amplified gene fragment was purified using a PCR product purification kit to obtain the specific amplified band, and the target fragment uspA was recovered by gel excision. Plasmid pBBR1MCS-2 was extracted using a plasmid mini-extraction kit. The obtained plasmid pBBR1MCS-2 was cleaned and recovered after double digestion with XbaI and HindIII to obtain a linear vector with sticky ends. The target fragment uspA was also double-digested using the same method to obtain the target fragment with sticky ends.

[0041] The enzyme-digested pBBR1MCS-2 plasmid and uspA The fragment was ligated overnight at 16 °C using Thermofisher Scientific T4 DNA ligase. The ligated system was introduced into DH5α E. coli competent cells and diluted and plated onto LB agar plates containing kanamycin (50 μg / mL) and ampicillin (100 μg / mL). The plates were incubated at 37 °C for 24 h, and positive recombinants were screened. Positive plaques were picked and inoculated into test tubes, incubated at 37 °C for 24 h, and plasmids were extracted using a plasmid mini-extraction kit. After double digestion with XbaI and HindIII, agarose gel electrophoresis was performed to confirm the recombinant plasmid pBBR1MCS-2-.uspA Successfully built ( Figure 1 ).

[0042] Example 2: Construction of overexpression strain OE uspA Recipient bacteria (wild-type ORS571 strain) and donor bacteria (containing pBBR1MCS-2-) were cultured separately. uspA Bacterial suspensions were obtained from the recombinant plasmid (DH5α) and helper bacteria (DH5α containing pRK2013 plasmid). The bacterial cells were collected by centrifugation at 5000 rpm for 3 min, washed twice with antibiotic-free liquid TY medium, and finally resuspended in 1 mL of antibiotic-free liquid TY medium. 300 μL of donor bacteria, 200 μL of helper bacteria, and 100 μL of recipient bacteria were mixed thoroughly and suspended in the center of a TY plate. After drying, the plates were incubated at 37℃ for 2 days. The cultured bacterial cells were scraped off with a sterile pipette tip, resuspended in 1 mL of sterile water, and diluted to 10⁻⁶. 3 The bacterial suspension was obtained by dilution, and 100 μL was spread onto TY solid medium containing kanamycin (50 ug / mL) and ampicillin (100 ug / mL) and incubated at 37 ℃ for 2 days. The single colonies obtained above were cultured in liquid TY medium containing kanamycin (50 ug / mL) and ampicillin (100 ug / mL) at 37 ℃ with shaking at 180 rpm for 24 h. Plasmids were extracted using a plasmid mini-extraction kit, digested with XbaI and HindIII, and then subjected to agarose gel electrophoresis to confirm the transformation of the recombinant plasmid into the overexpressing strain OE. uspA Construction successful. The validated positive strain, OEuspA, was stored in glycerol at -80 °C for later use.

[0043] Example 3: Detection of Rhizobium Stress Resistance 1. Salt tolerance test: Wild-type rhizobium WT (ORS571) and the overexpressing rhizobium OE constructed in Example 2 were respectively used. uspA The cultures were inoculated into test tubes containing liquid TY medium and incubated at 37°C and 180 rpm for 24 h. The OD values ​​of each strain were then calculated. 600 Adjust the pH to 0.6, and take 500 μL of each culture medium to inoculate into Erlenmeyer flasks containing 50 mL of liquid TY medium containing 1.5% NaCl and 5 mL of liquid TY medium without NaCl, respectively. Incubate at 37 ℃ and 180 rpm, and measure the OD at 600 nm every 4 h. After 48 h, plot the growth curve.

[0044] The results are as follows Figure 2 As shown, compared to the wild-type rhizobium WT, the overexpressing strain OE, in NaCl-free liquid TY medium,uspA The growth curves showed no significant differences. Figure 2 A). This indicates that the recombinant rhizobium did not cause growth inhibition compared to wild-type rhizobium in liquid TY medium, ruling out the influence of the recombinant plasmid on the growth rate of the strain. However, in liquid TY medium containing 1.5% NaCl, the growth of wild-type rhizobium WT was significantly inhibited, and the growth rate slowed down, while the overexpression of rhizobium OE... uspA Growth was significantly faster than that of wild-type rhizobium (WT). This indicates overexpression of rhizobium OE. uspA Its salt tolerance is significantly higher than that of wild-type rhizobium WT.

[0045] 2. Test of resistance to heavy metal stress: Wild-type rhizobium WT (ORS571) and the overexpressing rhizobium OE constructed in Example 2 were respectively used. uspA The cultures were inoculated into test tubes containing liquid TY medium and incubated at 37°C and 180 rpm for 24 h. The OD values ​​of each strain were then calculated. 600 Adjust each to 0.6, then use sterile water to serially dilute to 10. 8 10 were absorbed respectively 3 -108 concentrations of 10 μL were spotted onto TY solid plates containing 100 μM CdCl2 and incubated at 37 ℃. Colony growth was observed after 48 h.

[0046] The results are as follows Figure 3 As shown, compared to the wild-type rhizobium WT, the overexpressing strain OE uspA In 10 8 The higher colony count at the dilution concentration indicates that the overexpression strain OE... uspA Its resistance to heavy metal stress is significantly higher than that of wild-type rhizobium WT.

[0047] 3. Oxidation resistance test: Wild-type rhizobium WT (ORS571) and the overexpressing rhizobium OE constructed in Example 2 were respectively used. uspA The cultures were inoculated into test tubes containing liquid TY medium and incubated at 37°C and 180 rpm for 24 h. The OD values ​​of each strain were then calculated. 600 Adjust the pH to 0.6, add 1 mL of each to 100 mL of TY solid plate culture medium, mix well, pour into plates, place a sterile 6 mm filter paper in the center of the plate, and add 5 μL of hydrogen peroxide (H2O2) and cumene hydroperoxide (CHP) respectively. After 2 days, measure the diameter of the inhibition zone.

[0048] The results are as follows Figure 4 As shown, compared to the wild-type rhizobium WT, the overexpressing strain OE uspA The inhibition zone formed under H2O2 stress was smaller, and the difference between the two was extremely significant. P<0.01), under CHP stress, the overexpressing strain OE uspA The inhibition zone formed was also significantly smaller than that of the wild type. P <0.05). This indicates that the overexpression strain OE uspA Its antioxidant capacity is significantly higher than that of wild-type rhizobium WT.

[0049] Example 4: Symbiotic Experiment between Rhizobium and Leguminosae 1. Test method: Seed disinfection: Select sesbania seeds of similar size and weight and place them in a 50 mL sterile Erlenmeyer flask. Soak the seeds in 98% concentrated sulfuric acid for 10 min to soften the seed coat, and then rinse off the sulfuric acid with plenty of sterile water. Next, soak the seeds in 3% sodium hypochlorite solution for 5 min, and then in 75% ethanol for 5 min to disinfect the seed surface. Finally, wash the seeds three times with sterile water and place them on sterile moist filter paper to germinate for 2 days.

[0050] Co-culture: Wild-type rhizobium WT and the overexpressing rhizobium OE constructed in Example 2 were cultured separately. uspA Inoculate into test tubes containing 5 mL of liquid TY medium, incubate at 37 ℃ and 180 rpm for 24 h, collect and centrifuge, and then separate the cultures of each strain. OD 600 Adjust the pH to 0.6, and inoculate 500 μL of each into a conical flask containing 50 mL of liquid TY medium. Incubate at 37 °C and 180 rpm for 24 h, centrifuge at 5000 rpm for 5 min, and resuspend in sterile water. OD 600 =0.6 bacterial solution.

[0051] Thirty seedlings of *Senecio scandens* with uniform growth were selected and placed in each of the following bacterial solutions, soaking for 3 hours at 25°C. Seedlings soaked in sterile water served as the control group. After soaking, the seedlings were planted in gallon pots containing 700 g of sterilized garden soil, with three seedlings per pot and 10 replicates per treatment. Seven days after planting, each pot in the salt treatment group was watered with 80 mL of 150 mM NaCl solution for three consecutive days until the final salt concentration in the soil reached 0.3%. Twenty days after planting, the same bacterial solution used for soaking was applied to the stems of the plants. Observations, photographs, and treatments were conducted 48 days after planting. Student's test results showed significant differences. P <0.05, The differences are significant.

[0052] 2. Test Results: The results are as follows Figure 5 As shown, wild-type rhizobium WT and overexpressing rhizobium OE uspABoth can induce root and stem nodules in Sesbania pubescens, promoting plant growth under salt stress. However, compared to wild-type rhizobium (WT), inoculation with OE... uspA The number of root nodules formed afterward increased significantly, the number of stem nodules increased relatively, and this further promoted the growth of plant height and root length, with the plant height being significantly higher than that of wild-type plants.

[0053] Example 5: Symbiotic Experiment between Rhizobium and Gramineae 1. Test method: Seed disinfection: Select wheat or corn seeds of similar size and weight and place them in a 50 mL sterile Erlenmeyer flask. Rinse the surface with 75% ethanol, then soak in 0.1% HgCl2 for 10 min to disinfect the seed surface. Finally, wash three times with sterile water and place the seeds on sterile moist filter paper to germinate for 2 days.

[0054] Co-culture: Wild-type rhizobium WT and the overexpressing rhizobium OE constructed in Example 2 were cultured separately. uspA Inoculate into test tubes containing 5 mL of liquid TY medium, incubate at 37 ℃ and 180 rpm for 24 h, collect and centrifuge, and then separate the cultures of each strain. OD 600 Adjust the pH to 0.6, and inoculate 500 μL of each into a conical flask containing 50 mL of liquid TY medium. Incubate at 37 °C and 180 rpm for 24 h, centrifuge at 5000 rpm for 5 min, and resuspend in sterile water. OD 600 =0.6 bacterial solution.

[0055] One hundred wheat seedlings or 40 corn seedlings of uniform growth were selected and placed in each bacterial solution, respectively, and soaked at 25 ℃ for 3 hours. Seedlings soaked in sterile water served as the control group. After soaking, the seedlings were planted in gallon pots containing 700 g of sterilized garden soil, with 10 wheat seedlings per pot and 4 corn seedlings per pot, with 10 replicates per treatment. Seven days after planting, each pot in the salt treatment group was watered with 80 mL of 150 mM NaCl solution for 3 consecutive days until the final salt concentration in the soil was 0.3%. Observations, photographs, and treatments were conducted 20 days after planting. The Student's test for significant differences showed that… P <0.05, The differences are significant.

[0056] 2. Test Results: The results are as follows Figure 6 As shown, wild-type rhizobium WT and overexpressing rhizobium OE uspA Both can promote the growth of wheat or corn under salt stress, but compared with wild-type rhizobium (WT), inoculation with OE... uspA This further promoted the growth of plant height and root length, especially after OE inoculation.uspA The height and root length of the wheat plants after inoculation were significantly higher than those of the wild-type plants; after inoculation with OE uspA The maize plants were significantly taller than those inoculated with wild-type plants.

[0057] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. uspA The application of genes in the following (1) or (2): (1) Improve the stress resistance of rhizobia; (2) Constructing engineered rhizobium strains with stress resistance; The uspA A gene is a nucleic acid molecule as shown in (i) or (ii): (i) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.1; (ii) Nucleic acid molecules other than (i) that encode the amino acid sequence shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, The stress resistance refers to the ability to withstand salt stress, heavy metal stress, and / or oxidative stress.

3. Application of UspA protein in enhancing the stress resistance of rhizobia; wherein the UspA protein is a protein as shown in (A1) or (A2) below: (A1) A protein with the amino acid sequence shown in SEQ ID NO.2; (A2) The protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

4. Promote uspA The substance that enhances gene expression uspA The application of substances with gene activity and / or content in the following (1) or (2): (1) Improve the stress resistance of rhizobia; (2) Constructing engineered strains of rhizobium with stress resistance.

5. The application according to claim 4, characterized in that, Promote uspA The substance expressed by the gene is any one of the following: C1) contains uspA Gene expression cassettes; C2) contains uspA Recombinant vectors of genes, or recombinant vectors containing the expression cassette described in C1); C3) contains uspA Recombinant microorganisms containing genes, or recombinant microorganisms containing the expression cassette described in C1), or recombinant microorganisms containing the recombinant vector described in C2).

6. Overexpression uspA The recombinant rhizobium of the gene is used in at least one of the following (1)-(4): (1) Promote plant growth under salt stress; (2) Increase the number of root nodules in plants under salt stress; (3) Increase the number of stem nodules in plants under salt stress; (4) Preparation of plant salt-resistant bacteria.

7. The application according to claim 6, characterized in that, The overexpression uspA The recombinant rhizobium was constructed using the following method: The nucleotide sequence shown in SEQ ID NO.1 was cloned into a plasmid to obtain a recombinant plasmid; the recombinant plasmid was then introduced into stem-nodulated nitrogen-fixing rhizobium host cells to construct an overexpression. uspA Recombinant rhizobia.

8. The application according to claim 7, characterized in that, The plasmid is pBBR1MCS-2.

9. The application according to claim 6, characterized in that, The plant is a legume or a grass; preferably, *Guarnanchi pubescens* (…). Sesbania rostrata ), corn or wheat.

10. The application according to claim 6, characterized in that, The salt stress condition is a NaCl concentration of 0.3%.

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