Bacterial cadmium response element screening method

Abstract

The invention discloses a bacterial cadmium response element screening method, and belongs to the technical field of biology. According to the invention, a strain of Microbacterium oxydans SC-30 is screened, the tolerance to Cd < 2 + > is relatively high, and the minimum inhibitory concentration (MIC) value is 5.72 mmol / L, so that the cadmium resistance of microorganisms is greatly broken through. Degenerate primers of bacterium sources cadR and cadC are further designed to be used for obtaining a Cd < 2 + > response element in a microbacterium oxydans SC-30 strain, then cadR and cadC gene sequences in the strain SC-30 are obtained, the sizes of the cadR and cadC gene sequences are 438 bp and 2097 bp, and the cadR and cadC gene sequences have the highest homology with cadR4 and cadC1 gene sequences in Pseudomonas putida, and the cadR4 and cadC1 gene sequences have the highest homology with the cadR4 and cadC1 gene sequences in Pseudomonas putida, and the cadR4 and cadC1 gene sequences are 54.90% and 55.85% respectively.

Description

Technical Field

[0001] This invention relates to a method for screening bacterial cadmium-responsive elements, belonging to the field of biotechnology. Background Technology

[0002] Cadmium (Cd) is a highly toxic and carcinogenic heavy metal, and its permitted limits in the environment are very low. When the cadmium content in a plant exceeds a certain concentration, the plant will suffer serious damage, such as wilting, yellowing, and weak growth. Plant roots easily absorb cadmium and then transport it to the above-ground parts, which can lead to plant poisoning. Cadmium also damages root structure, affecting the absorption of nutrients and thus inhibiting plant growth.

[0003] Microorganisms and plants are closely related, and microorganisms have positive or negative effects on plant growth and development. The cadmium-resistant bacteria reported in existing literature, such as *Comamonas testosteroni*, *Burkholderia ambifaria*, and *Bacillus paranthracis* NT14.9, all possess cadmium-resistant properties (references are as follows: 1. Chi Y, Huang Y, Wang J, Chen X, Chu S, Hayat K, et al. Two plant growth promoting bacterial Bacillus strains possess different mechanisms in adsorption and resistance to cadmium. *Sci Total Environ.* 2020; 741:140422.; 2. Shi Z, Qi X, Zeng XA, Lu Y, Zhou J, Cui K, et al. A newly isolated bacterium *Comamonas* sp. XL8 alleviates the toxicity of cadmium exposure in rice seedlings by accumulating cadmium. *J Hazard Mater.* 2021; 403:123824.; 3. Zeng G, Qiao S, Wang X, Sheng M, Wei M, Chen Q, et al. Immobilization of cadmium by Burkholderia sp. QY14 through modified microbially induced phosphate precipitation. J Hazard Mater. 2021; 412:125-156.). However, the cadmium tolerance of the aforementioned microorganisms still needs improvement, and the mechanism of cadmium tolerance in microorganisms is not clear. Therefore, it is urgent to develop new highly cadmium-tolerant microorganisms. Summary of the Invention

[0004] To address the shortcomings of the prior art, this invention provides a method for screening bacterial cadmium-responsive elements, aiming to solve the technical problems that the performance of microbial cadmium resistance still needs to be improved and the mechanism of microbial cadmium resistance is not clear.

[0005] The first technical solution provided by this invention is a strain of Microbacterium oxydans SC-30, which was deposited at the China Center for Type Culture Collection on June 19, 2024, with accession number CCTCC NO:M20241296.

[0006] The second technical solution provided by the present invention is a microbial preparation containing the oxidizing microbacterium SC-30 described in the first technical solution.

[0007] In some embodiments, the amount of *Microbacterium oxyphylla* SC-30 added to the microbial preparation is not less than 1 × 10⁻⁶. 6 CFU / g or 1×10 6 CFU / mL.

[0008] Furthermore, in the microbial preparation, the amount of *Microbacterium oxyphylla* SC-30 added is not less than 1×10⁻⁶. 8 CFU / g or 1×10 8 CFU / mL.

[0009] The third technical solution provided by the present invention is a cadmium response gene of Microbacterium oxidans SC-30, wherein the cadmium response gene includes a cadR gene and a cadC gene, the nucleotide sequence of the cadR gene is shown in SEQ ID NO:1, and the nucleotide sequence of the cadC gene is shown in SEQ ID NO:2.

[0010] The fourth technical solution provided by this invention is the application of the oxidizing microbacterium SC-30 described in the first technical solution, or the microbial preparation described in the second technical solution, or the gene-based anti-cadmium poisoning stress described in the third technical solution.

[0011] Furthermore, the resistance to cadmium toxicity stress includes at least one of the following effects:

[0012] (1) Cultivating cadmium-resistant plants;

[0013] (2) Reduces the inhibition of plant growth by cadmium;

[0014] (3) Prepare cadmium-resistant microbial pesticides for plants.

[0015] The fifth technical solution provided by the present invention is a method for cloning the gene described in the third technical solution. The method is to use the DNA of Microbacterium oxysporum SC-30 as a template, and use degenerate primer pair A to perform PCR amplification to obtain the cadR gene, and use degenerate primer pair B to perform PCR amplification to obtain the cadC gene.

[0016] The degenerate primer pair A includes the upstream primer cadR-F: 5'-ATGAAGATCGGWGARCTGGCSAA-3' (SEQ ID NO:3); and the downstream primer cadR-R: 5'-CGSCCCACRTGVGAATGYTC-3' (SEQ ID NO:4); wherein W is A / T, R is A / G, S is C / G, V is A / C / G, and Y is C / T;

[0017] The degenerate primer pair B includes the upstream primer cadC-F: 5'-CCCCGGCCTGGTACCTCG-3' (SEQ ID NO:5); and the downstream primer cadC-R: 5'-GCTTGATCGGCAGCCAGTGG-3' (SEQ ID NO:6).

[0018] In some implementations, the conditions for PCR amplification of A using degenerate primers are as follows: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 40 s, repeated 30 times; final extension at 72°C for 10 min, and termination of the reaction at 12°C.

[0019] In some implementations, the conditions for PCR amplification of B using degenerate primers are as follows: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 40 s, repeated 30 times; final extension at 72°C for 10 min, and termination of the reaction at 12°C.

[0020] The sixth technical solution provided by the present invention is a degenerate primer pair for cloning the gene described in the third technical solution, wherein the degenerate primer pair includes a degenerate primer pair A for cloning the cadR gene and / or a degenerate primer pair B for cloning the cadC gene;

[0021] The degenerate primer pair A includes the upstream primer cadR-F: 5'-ATGAAGATCGGWGARCTGGCSAA-3' (SEQ ID NO:3); and the downstream primer cadR-R: 5'-CGSCCCACRTGVGAATGYTC-3' (SEQ ID NO:4); wherein W is A / T, R is A / G, S is C / G, V is A / C / G, and Y is C / T;

[0022] The degenerate primer pair B includes the upstream primer cadC-F: 5'-CCCCGGCCTGGTACCTCG-3' (SEQ ID NO: 5); and the downstream primer cadC-R: 5'-GCTTGATCGGCAGCCAGTGG-3' (SEQ ID NO: 6).

[0023] The technical effects of this invention are as follows:

[0024] This invention screened out a strain of oxidizing microbacterium SC-30, which is effective against Cd. 2+ The bacteria exhibited high tolerance, with a minimum inhibitory concentration (MIC) of 9.4 mmol / L, representing a significant breakthrough in the cadmium resistance of microorganisms. Furthermore, degenerate primers for bacterial cadR and cadC were designed for the detection of Cd in *Microbacterium oxysporum* strain SC-30. 2+ The responsive elements were obtained, and the cadR and cadC gene sequences of strain SC-30 were obtained, with sizes of 438bp and 2097bp, respectively. They showed the highest homology with the cadR4 and cadC1 gene sequences of Pseudomonas putida, at 54.90% and 55.85%, respectively.

[0025] Preservation of biological materials

[0026] A strain of Microbacterium oxydans, SC-30, was deposited on June 19, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCCNO:M 20241296. Attached Figure Description

[0027] Figure 1 For the determination of MIC values ​​of cadmium-resistant strains; A: strain SC-30; B: strain S-4-35; C: strain S-4-37; D: strain S-4-45.

[0028] Figure 2 Gel electrophoresis image of the PCR amplification product of the 16S rRNA gene of strain SC-30; M: Marker.

[0029] Figure 3 Molecular and morphological identification of strain SC-30; A is a phylogenetic tree of strain SC-30 based on the 16S rRNA gene sequence; B is the morphology of a single colony; C is the microscopic morphology. The scale bar represents 20 μm.

[0030] Figure 4 Electrophoresis images of cadR and cadC gene amplification in Microbacterium oxysporum SC-30; A: cadR gene amplification electrophoresis image, B: cadC gene amplification electrophoresis image, M: Marker. Detailed Implementation

[0031] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0032] Test method:

[0033] Minimum inhibitory concentration (MIC) determination: Single colonies were picked and inoculated into liquid LB medium and cultured at 28°C and 180 rpm until the OD600 reached 2. Inoculation was then performed at a 2% (v / v) inoculum into LB liquid medium containing different Cd²⁺ concentrations, and incubated at 28°C and 180 rpm for 24 h. The OD600 value was measured to determine bacterial growth. When the OD600 value of the strain was 0 after 24 h of culture, the corresponding Cd²⁺ concentration was determined. 2+ Concentration is the MIC value.

[0034] Raw materials used in the examples:

[0035] LB medium: 1% tryptone (Shanghai Bowei Microbial Technology Co., Ltd.), 0.5% yeast extract (OXOID, UK), 1% NaCl (Shanghai Bowei Microbial Technology Co., Ltd.), 1.5% agar (Beijing Solarbio Science & Technology Co., Ltd.). Example 1: Screening and identification of cadmium-resistant colonies in soil.

[0036] 1.1 Screening of cadmium-resistant bacterial colonies in soil

[0037] Weigh 3.0g of cadmium-contaminated soil sample from Guiyang City into 25mL of sterile water, vortex for 2 minutes, and spread the bacterial suspension onto a Cd-containing substrate. 2+ LB medium was incubated at 28℃ for 3-5 days, and the growth of microorganisms on the plates was observed to preliminarily screen the cadmium resistance of the strains. 2+ When the concentration reached 20 mmol / L, highly cadmium-tolerant strains SC-30, S-4-35, S-4-37 and S-4-45 were obtained through screening.

[0038] 1.2 Determination of minimum inhibitory concentration for bacteria

[0039] Strains SC-30, S-4-35, S-4-37, and S-4-45 were inoculated into liquid LB medium and cultured at 28°C and 180 rpm until the bacterial concentration reached 4 × 10⁻⁶. 8 CFU / mL. Inoculated with Cd at a 2% (v / v) inoculum. 2+ In LB liquid medium with a concentration of 0-6 mmol / L, incubate at 28°C and 180 rpm for 24 h, and then measure the OD. 600 The value is used to determine bacterial growth. For example... Figure 1 As shown, strain SC-30 is effective against Cd. 2+ The strains exhibited the highest tolerance, with a minimum inhibitory concentration (MIC) of 5.72 mmol / L. Strains S-4-35, S-4-37, and S-4-45 showed resistance to Cd. 2+ Tolerance decreased sequentially, with MIC values ​​of 4.80 mmol / L, 3.18 mmol / L, and 2.72 mmol / L, respectively. When Cd... 2+At a concentration of 1 mmol / L, the growth of all four strains decreased significantly. The MIC value of strain SC-30 was higher than that of the previously reported cadmium-resistant bacteria Burkholderia ambifaria and Bacillus paranthracis NT (2.49 mmol / L), indicating that it exhibits higher cadmium resistance.

[0040] PCR amplification of strain SC-30 was performed using universal primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-GGTTACCTTG TTACGACTT-3′). Figure 2 The PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were compared and analyzed using the NCBI database to find highly similar gene sequences. A phylogenetic tree was constructed using MEGA11.0. Figure 3 A). Based on the phylogenetic tree and BLAST results from the NCBI database, strain SC-30 showed the highest homology (77%) with *Microbacterium oxydans* NBRC. Single colonies of strain SC-30 ( Figure 3 B) It appears pale yellow, round, with smooth edges; under an optical microscope, strain SC-30 appears rod-shaped. Figure 3 C). Molecular and morphological identification results indicate that SC-30 is M. suwonense.

[0041] Example 2: Obtaining Bacterial Cadmium Response Elements

[0042] The genes cadR and cadC are bacterial responses to Cd. 2+ The cadR gene is an important gene. It is a member of the MerR family of metalloprotein regulatory proteins and regulates Cd. 2+ Expression of the cadA transporter gene. The cadC gene is one of the genes in the cadC operon, encoding the cadC repressor protein, which can bind Cd. 2+ Lower Cd 2+ Toxicity to bacteria. Since strain SC-30 is a bacterium, reported bacterial cadR and cadC gene sequences were searched in the NCBI database and literature (Table 1). Gene homology was analyzed, and degenerate primers for bacterial cadR and cadC were designed (Table 2) for Cd toxicity in strain SC-30. 2+ Acquisition of response elements.

[0043] Table 1. cadR and cadC gene sequences from different sources.

[0044]

[0045]

[0046] NF: Not found

[0047] Table 2. Degenerate primer sequences for bacterial genes cadR and cadC

[0048]

[0049] W:A / TR:A / GS:C / GV:A / C / GY:C / T

[0050] Amplification primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. using corresponding degenerate primers. Single colonies of strain S-4-30 were picked and placed in PCR tubes as PCR amplification templates. PCR amplification systems were prepared using degenerate primer pair A and degenerate primer pair B: 1 μL upstream primer, 1 μL downstream primer, 20 μL 2×Taq PCR StarMix (Dye), and ddH2O to a final volume of 40 μL. The PCR program was as follows: pre-denaturation at 94℃ for 2 min; denaturation at 94℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 40 s, repeated 30 times; final extension at 72℃ for 10 min, and termination at 12℃. The cadR and cadC gene sequences of strain SC-30 were obtained, with sizes of 438 bp and 2097 bp, respectively. Figure 4 The sequence showed the highest homology with the cadR4 and cadC1 gene sequences in Pseudomonas putida, at 54.90% and 55.85%, respectively.

[0051] Example 3: Application of Bacterial Cadmium Response Element and Strains SC-30

[0052] The oxidizing microbacterium SC-30 provided by this invention, or the microbial preparation thereof, or the cadR and cadC genes obtained therefrom, can be used in the treatment of cadmium poisoning stress.

[0053] Specifically, resistance to cadmium toxicity stress includes at least one of the following effects:

[0054] (1) Cultivating cadmium-resistant plants;

[0055] (2) Reduces the inhibition of plant growth by cadmium;

[0056] (3) Prepare cadmium-resistant microbial pesticides for plants.

[0057] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A strain of Microbacterium oxydans SC-30, characterized in that, It was deposited at the China Center for Type Culture Collection on June 19, 2024, with accession number CCTCC NO:M 20241296.

2. A microbial preparation containing the Oxidizing Microbacterium SC-30 of claim 1.

3. The microbial preparation according to claim 2, characterized in that, In the microbial preparation, the amount of *Microbacterium oxyphylla* SC-30 added is not less than 1×10⁻⁶. 6 CFU / g or 1×10 6 CFU / mL.

4. The cadmium-responsive gene of *Microbacterium oxidans* SC-30, characterized in that, The cadmium response genes include the cadR gene and the cadC gene, the nucleotide sequence of the cadR gene is shown in SEQ ID NO:1, and the nucleotide sequence of the cadC gene is shown in SEQ ID NO:

2.

5. A method for cloning the gene according to claim 4, characterized in that, The method involves using the DNA of Microbacterium oxysporum SC-30 as a template, performing PCR amplification on degenerate primer pair A to obtain the cadR gene, and performing PCR amplification on degenerate primer pair B to obtain the cadC gene. The degenerate primer pair A includes the upstream primer cadR-F, with the nucleotide sequence shown: 5'-ATGAAGATCGGWGARCTGGCSAA-3'; and the downstream primer cadR-R: 5'-CGSCCCACRTGVGAATGYTC-3'; wherein W is A / T, R is A / G, S is C / G, V is A / C / G, and Y is C / T; The degenerate primer pair B includes the upstream primer cadC-F: 5'-CCCCGGCCTGGTACCTCG-3'; and the downstream primer cadC-R: 5'-GCTTGATCGGCAGCCAGTGG-3'.

6. The method according to claim 5, characterized in that, The conditions for PCR amplification of A using degenerate primers are as follows: pre-denaturation at 94℃ for 2 min; denaturation at 94℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 40 s, repeated 30 times; final extension at 72℃ for 10 min, and termination of the reaction at 12℃.

7. The method according to claim 5, characterized in that, The conditions for PCR amplification of B using degenerate primers are as follows: pre-denaturation at 94℃ for 2 min; denaturation at 94℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 40 s, repeated 30 times; final extension at 72℃ for 10 min, and termination of the reaction at 12℃.

8. A degenerate primer pair for cloning the gene of claim 4, characterized in that, The degenerate primer pairs include degenerate primer pair A for cloning the cadR gene and / or degenerate primer pair B for cloning the cadC gene; The degenerate primer pair A includes the upstream primer cadR-F: 5'-ATGAAGATCGGWGARCTGGCSAA-3'; and the downstream primer cadR-R: 5'-CGSCCCACRTGVGAATGYTC-3'; wherein W is A / T, R is A / G, S is C / G, V is A / C / G, and Y is C / T; The degenerate primer pair B includes the upstream primer cadC-F: 5'-CCCCGGCCTGGTACCTCG-3'; and the downstream primer cadC-R: 5'-GCTTGATCGGCAGCCAGTGG-3'.

9. The application of the oxidizing microbacterium SC-30 according to claim 1, or the microbial preparation according to claim 2 or 3, or the gene-based anti-cadmium poisoning stress according to claim 4.

10. The application according to claim 9, characterized in that, The resistance to cadmium poisoning stress includes at least one of the following effects: (1) Cultivating cadmium-resistant plants; (2) Reduces the inhibition of plant growth by cadmium; (3) Prepare cadmium-resistant microbial pesticides for plants.

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