Iaam gene overexpression pseudomonas nitroreductase engineering bacterium as well as construction method and application of iaam gene overexpression pseudomonas nitroreductase engineering bacterium

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to an iaam gene overexpression pseudomonas nitroreductase engineering bacterium as well as a construction method and application of the iaam gene overexpression pseudomonas nitroreductase engineering bacterium. The construction method comprises the following steps: carrying out PCR (Polymerase Chain Reaction) amplification on an iaam gene in genomic DNA (Deoxyribose Nucleic Acid) of the pseudomonas nitroreducens LSF-6 by taking genomic DNA of the pseudomonas nitroreducens LSF-6 as a template; a PCR amplification product is connected to a pBBR1MCS vector, and an iaam gene overexpression vector is constructed; and transforming the iaam gene overexpression vector into the nitroreductive pseudomonas LSF-6 to obtain the nitroreductive pseudomonas engineering bacteria for overexpressing the iaam gene. Compared with the original strain, the auxin secretion capability of the pseudomonas nitroreducens engineering strain is obviously improved. When the pseudomonas nitroreducens engineering bacteria are prepared into bacterial suspension to be applied to rice, the plant height, the root length, the overground part fresh weight, the underground part fresh weight and the chlorophyll SPAD value of the rice can be obviously increased. The invention lays a foundation for promoting rice growth by a microbiological method.

Description

Technical Field

[0001] The present invention belongs to the technical field of microorganisms, and in particular relates to an engineering bacterium of nitroreducing Pseudomonas that overexpresses an iaam gene, and a construction method and application thereof. Background Art

[0002] In modern agricultural production, improving crop yield and stress resistance is a core issue of continuous concern. Rice, as one of the most important food crops in the world, has a direct impact on global food security due to its yield and growth stability. Traditional agricultural practices rely on chemical fertilizers and plant growth regulators to improve crop production, but these methods are often accompanied by high costs and environmental pollution.

[0003] Auxin is a key plant hormone, especially IAA, which regulates plant cell division and expansion, and has a profound impact on physiological processes such as root development, stem growth, and fruit development. However, the natural synthesis of IAA in plants is limited by gene expression regulation and environmental conditions, which limits its potential application in agricultural production.

[0004] Microorganisms are the second genome of plants and are closely related to plant growth. Some rhizosphere microbial bacteria can naturally produce IAA and interact with plants through the root system, thereby promoting plant growth. However, the microbial preparations with efficient IAA production capacity for rice on the market are still very limited. By using gene editing technology to increase the expression of the bacterial auxin synthesis gene iaam, the bacterial IAA production can be increased, promoting rice growth more efficiently, while reducing the use of chemical fertilizers and enhancing the sustainability of agricultural production. Summary of the invention

[0005] The object of the present invention is to provide an engineered nitroreducing Pseudomonas bacterium overexpressing the iaam gene and a construction method and application thereof.

[0006] To achieve the above object, the present invention adopts the following technical solutions: The first aspect of the present invention provides a method for constructing an engineered nitroreducing Pseudomonas bacterium that overexpresses the iaam gene, which comprises using the genomic DNA of the nitroreducing Pseudomonas LSF-6 as a template, performing PCR amplification on the iaam gene in the genomic DNA of the nitroreducing Pseudomonas LSF-6; connecting the PCR amplification product to the pBBR1MCS vector to construct an iaam gene overexpression vector; transforming the iaam gene overexpression vector into the nitroreducing Pseudomonas LSF-6 to obtain an engineered nitroreducing Pseudomonas bacterium that overexpresses the iaam gene; The nucleotide sequence of the iaam gene is shown in SEQ ID NO.1; The nitroreduced Pseudomonas LSF-6 is classified and named as Pseudomonas nitroreducens, which has been deposited in the China Center for Type Culture Collection on May 12, 2022, with the deposit address at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number is CCTCC NO: M2022607; Furthermore, the nucleotide sequences used for PCR amplification are shown as SEQ ID NO.2 and SEQ ID NO.3.

[0007] The second aspect of the present invention provides an engineered nitroreducing Pseudomonas bacterium that overexpresses the iaam gene, which is constructed by the above-mentioned construction method.

[0008] The third aspect of the present invention provides the use of the above-mentioned nitroreducing Pseudomonas engineered bacteria in preparing plant growth hormones and / or promoting rice growth; Further, the plant growth hormone is indoleacetic acid; Furthermore, the promotion of rice growth is to increase the plant height, root length, aboveground fresh weight, underground fresh weight and chlorophyll SPAD value of rice.

[0009] The fourth aspect of the present invention provides an application of overexpressing the iaam gene in improving the auxin secretion ability of nitroreducing Pseudomonas LSF-6, the nucleotide sequence of the iaam gene is shown in SEQ ID NO.1; the classification name of the nitroreducing Pseudomonas LSF-6 is Pseudomonas nitroreducens, which has been deposited in the China Center for Type Culture Collection on May 12, 2022, with a deposit address of No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and a deposit number of CCTCC NO: M2022607; Furthermore, the plant growth hormone is indoleacetic acid.

[0010] The fifth aspect of the present invention provides a method for promoting rice growth, which comprises preparing the above-mentioned nitroreducing Pseudomonas engineered bacteria into a bacterial suspension and applying it to rice; Furthermore, the application method is seed soaking.

[0011] The significant advantages of the present invention are: The present invention provides an engineered nitroreducing Pseudomonas bacteria overexpressing the iaam gene, and a construction method and application thereof. The engineered nitroreducing Pseudomonas bacteria has a significantly improved auxin secretion capacity compared to the starting strain. The engineered nitroreducing Pseudomonas bacteria is prepared into a bacterial suspension and applied to rice, which can significantly increase the plant height, root length, aboveground fresh weight, underground fresh weight and chlorophyll SPAD value of the rice. The present invention lays a foundation for promoting rice growth by microbial methods, has broad application prospects in increasing rice yield, etc., and helps to stabilize or increase rice yield and ensure food security. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 : Colony PCR results of the starting strain and the engineered nitroreducing Pseudomonas bacteria overexpressing the iaam gene.

[0013] Figure 2 :Plant auxin secretion ability of the starting strain and the engineered nitroreducing Pseudomonas bacteria overexpressing the iaam gene.

[0014] Figure 3 :Application of engineered nitroreducing Pseudomonas overexpressing the iaam gene to rice increases rice plant height.

[0015] Figure 4 :Application of engineered nitroreducing Pseudomonas overexpressing the iaam gene to rice increases rice root length.

[0016] Figure 5 :Application of engineered nitroreducing Pseudomonas overexpressing iaam gene to rice increases the fresh weight of aboveground rice parts.

[0017] Figure 6 :Application of engineered nitroreducing Pseudomonas overexpressing iaam gene to rice increases the fresh weight of underground rice.

[0018] Figure 7 :Application of engineered nitroreducing Pseudomonas overexpressing the iaam gene to rice increases the chlorophyll SPAD value.

[0019] Note: Figure 1 to Figure 2 In the figure, WT indicates the starting strain Pseudomonas nitroreducens LSF-6, and iaamOE indicates the engineered strain of Pseudomonas nitroreducens that overexpresses the iaam gene. Figures 3 to 7 In the table, CK means that the seeds were soaked in sterile water, and iaamOE means that the seeds were soaked in a bacterial suspension of engineered nitroreducing Pseudomonas overexpressing the iaam gene. DETAILED DESCRIPTION

[0020] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0022] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0023] The nitroreduced Pseudomonas LSF-6 used in the following examples is classified and named Pseudomonas nitroreducens, which was deposited in the China Center for Type Culture Collection on May 12, 2022, with a preservation address of No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and a preservation number of CCTCC NO: M2022607.

[0024] Embodiment 1: The steps for constructing the nitroreducing Pseudomonas engineered bacteria overexpressing the iaam gene are as follows: S1: Using the genomic DNA of Pseudomonas nitroreducens LSF-6 as a template, PCR amplification of the iaam gene in the genomic DNA of Pseudomonas nitroreducens LSF-6 was performed using primers containing the XbaI restriction enzyme site; Wherein, the nucleotide sequence of the iaam gene in the genomic DNA of Pseudomonas nitroreducens LSF-6 is shown as SEQ ID NO.1; The nucleotide sequence of the primer containing the XbaI restriction enzyme site is: F (SEQ ID NO.2): 5'-gggggatccactagttctagaTCAGTCCGGCAGTTCCATCG-3', R (SEQ ID NO.3): 5'-cgcggtggcggccgctctagaATGCACAAGAACCGCCATCC-3'; S2: The PBBR1MCS vector (Ubao Biotech, VT8175) was digested with restriction endonuclease XbaI to obtain the linearized PBBR1MCS vector; the seamless cloning technology ( -Basic Seamless Cloning and Assembly Kit, Beijing Quanshijin Biotechnology Co., Ltd.) to connect the PCR amplification product of the iaam gene with the linearized PBBR1MCS vector to obtain the iaam gene overexpression vector; Wherein, the nucleotide sequence of the iaam gene overexpression vector is shown in SEQ ID NO.4; S3: The iaam gene overexpression vector was transformed into the nitroreduced Pseudomonas LSF-6 competent cells by electroporation, and a single clone was selected for colony PCR using identification primers. The positive clone was the one showing a 1918 bp band after agarose gel electrophoresis ( Figure 1 ); verifying the positive clones by sequencing, and verifying that the correct positive clones are the nitroreducing Pseudomonas engineered bacteria that overexpress the iaam gene; Among them, the nucleotide sequence of the identification primer is: F (SEQ ID NO.5): 5'-AGTAAAACGACGGCCAGT-3', R (SEQ ID NO. 6): 5'-GTCATAGCTGTTTCCTG-3'.

[0025] Embodiment 2: The nitroreduced Pseudomonas LSF-6 and the nitroreduced Pseudomonas engineered bacteria overexpressing the iaam gene were inoculated into LB liquid culture medium, shaken and cultured at 30°C and 200rpm for 1 day, centrifuged at 6000rpm, and 50μL of the supernatant was added to 50mL of Salkowskis colorimetric solution (Salkowskis colorimetric solution, Feijing Biology, PH1941). After 30 minutes of color development in the dark, if pink appears, it is positive, indicating that the strain can secrete IAA. The darker the color, the greater the intensity of IAA secretion; no color change is negative, indicating that IAA cannot be secreted. Quantitatively measure the absorbance of the reaction solution at 530nm, compare it with the standard curve, and calculate the IAA concentration (mg / L) in the reaction solution. The results are as follows: Figure 2 shown.

[0026] Embodiment 3: Select complete and full rice seeds. After selection, sterilize with 1% NaClO for 3-5 minutes, wash with sterile water for 3-5 times, and air-dry for later use. Activate and culture the nitroreduced Pseudomonas engineered bacteria overexpressing the iaam gene in LB liquid medium for 24 hours, centrifuge at 6000 rpm for 10 minutes, collect the bacterial precipitate, and resuspend it with sterile water to OD 600=2.0 bacterial suspension. Rice seeds were soaked in a bacterial suspension of nitroreducing Pseudomonas engineered bacteria that overexpressed the iaam gene for 24 hours, with the volume of bacterial suspension: seed mass = 10:1 (mL: g). During this period, the seeds were gently shaken every 4 hours to ensure that the seeds were in full contact with the bacterial suspension and could absorb water evenly. After soaking, the seeds were quickly rinsed with tap water, the surface moisture was wiped dry, and then a germination test was conducted. The seeds were placed in a germination basin (12cm×12cm×6cm) padded with three layers of moist filter paper for germination on paper. The control group was treated in the same way using sterile water instead of bacterial suspension. After the seeds germinated, 6 seedlings of the same growth were taken for hydroponic culture. After 30 days of culture, samples were taken, and the plant height, root length, plant fresh weight and chlorophyll SPAD value were measured and analyzed. The results are as follows Figures 3 to 7 shown.

[0027] Depend on Figure 2 It can be seen that the secretion concentration of the auxin IAA of the nitroreducing Pseudomonas engineered bacteria overexpressing the iaam gene increased by 37%; soaking seeds with the bacterial suspension of the nitroreducing Pseudomonas engineered bacteria overexpressing the iaam gene increased the plant height of rice by 9%, the root length by 43%, the fresh weight of the aboveground part and roots of the plant by 48% and 54% respectively, and the chlorophyll SPAD by 30%.

[0028] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

[0029] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. Overexpression iaam A method for constructing a nitro-reducing Pseudomonas engineering bacterium, characterized in that: Using the genomic DNA of nitro-reduced Pseudomonas sp. LSF-6 as a template, the genomic DNA of nitro-reduced Pseudomonas sp. LSF-6 was purified by Western blotting. iaam The gene was amplified by PCR; The PCR amplification product was connected to the pBBR1MCS vector to construct iaam Gene overexpression vector; iaam The gene overexpression vector was transformed into nitroreducing Pseudomonas LSF-6 to obtain overexpression iaam Genetic engineering of nitroreducing Pseudomonas; Said iaam The nucleotide sequence of the gene is shown in SEQ ID NO.1; The nitroreducing Pseudomonas LSF-6 is named as Pseudomonas nitroreducing ( Pseudomonas nitroreducens ), which was deposited in the China Center for Type Culture Collection on May 12, 2022, with the deposit address at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number is CCTCC NO: M2022607.

2. The construction method according to claim 1, characterized in that: The nucleotide sequences used for the PCR amplification are shown in SEQ ID NO.2 and SEQ ID NO.

3.

3. Overexpression iaam The nitroreducing Pseudomonas engineered bacteria of the gene is characterized by: The method is constructed by any one of claims 1 to 2.

4. Use of the nitroreducing Pseudomonas engineered bacteria as claimed in claim 3 in preparing plant growth hormones and / or promoting rice growth.

5. The use according to claim 4, characterized in that: The plant growth hormone is indoleacetic acid.

6. The use according to claim 4, characterized in that: The rice growth promotion is to increase the plant height, root length, aboveground fresh weight, underground fresh weight and chlorophyll SPAD value of the rice.

7. Overexpression iaam The application of the gene in improving the secretion capacity of auxin of nitroreducing Pseudomonas LSF-6 is characterized by: Said iaam The nucleotide sequence of the gene is shown in SEQ ID NO.1; the classification of the nitroreducing Pseudomonas LSF-6 is named as nitroreducing Pseudomonas ( Pseudomonas nitroreducens ), was deposited in the China Center for Type Culture Collection on May 12, 2022. The deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number is CCTCC NO: M2022607.

8. The use according to claim 8, characterized in that: The plant growth hormone is indoleacetic acid.

9. A method for promoting rice growth, characterized in that: The engineered nitroreducing Pseudomonas bacteria according to claim 3 is prepared into a bacterial suspension and applied to rice.

10. The method according to claim 9, characterized in that: The application method is seed soaking.

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