Application of overexpression of perR gene in improving manganese oxidation in Bacillus thuringiensis

Abstract

The present invention relates to the field of genetic engineering, and in particular to the application of overexpressing the perR gene in improving the biological manganese oxidation of Bacillus thuringiensis. The present invention uses the strain Bt 97-27 as the starting strain, which is capable of oxidizing Mn (II), forming spores and producing biofilms to resist adverse external environmental factors. The present invention successfully constructs a new genetically engineered strain OEperR by overexpressing the perR gene in the Bt 97-27 strain. Compared with the starting strain, the OEperR strain achieves significant improvements in manganese oxidation, manganese removal and biofilm formation. It not only shortens the culture and acclimation cycle of the strain, but also greatly enhances the environmental adaptability and tolerance of the strain, opens up a new way for the application of microbial technology in the field of manganese-contaminated environmental remediation, and provides an important basis for in-depth exploration of how microorganisms respond to manganese and regulate its oxidation process.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, in particular to application of overexpressing perR gene in improving biological manganese oxidation of Bacillus thuringiensis. Background Art

[0002] In recent years, due to human mining of manganese ore and large-scale production of manganese-containing industries, large amounts of manganese have been released into the air, soil and water environment. Environmental Mn pollution poses a potential health threat to organisms and humans. At present, common methods for removing manganese from groundwater mainly include oxidation precipitation, filtration adsorption, and biological manganese removal. The oxidation precipitation method requires the addition of chemical oxidants and precipitants, and the cost of treating manganese wastewater is low, but it produces toxic sludge. The filtration adsorption method is simple to operate, but the filter media needs to be cleaned regularly, and the maintenance and management costs are high. Biological manganese removal refers to the oxidation of soluble Mn(II) into insoluble Mn(III) or Mn(IV) manganese oxide precipitates under the enzymatic catalysis and metabolism of microorganisms such as bacteria and fungi. These manganese-oxidizing microorganisms have become one of the main technologies for remediation of groundwater Mn(II) pollution due to their rapid growth and reproduction, high manganese removal efficiency, low treatment cost and environmental friendliness.

[0003] Bacillus thuringiensis (Bt) is a Gram-positive bacterium that appears rod-shaped under a microscope, arranged in short or long chains. It can form endospores, which are oval and grow close together. It is widely found in soil, insects, and decaying organic matter, and is an insect pathogen. Bt, a member of the Bacillus cereus group, has two growth phases: the vegetative phase and the spore phase. The vegetative phase is primarily characterized by secondary division, during which the bacteria continue to reproduce. The bacteria can form spores when exposed to adverse conditions such as nutrient deficiency or strong acid or alkaline conditions. Once sufficient nutrients are present, the spores automatically enter the vegetative phase and regenerate into Bt.

[0004] Peroxide resistance regulator (PerR) is a 20.35kDa transcription factor found in a variety of bacteria. It belongs to the large family of iron uptake regulation proteins (Fur). It primarily responds to intracellular H2O2 concentrations and regulates the expression of a range of genes associated with hydrogen peroxide stress. Research on PerR has primarily focused on bacterial antioxidant activity, intracellular metal ion homeostasis, quorum sensing, pathogenicity, and other complex biological effects. However, it is worth noting that, to date, research on the perR gene as a key gene directly regulating manganese oxidation has been lacking. Summary of the Invention

[0005] In light of this, the present invention uses the Bt 97-27 strain as the starting strain. This strain is capable of oxidizing Mn(II), forming spores, and producing biofilms to protect against adverse environmental factors. By overexpressing the perR gene in the Bt 97-27 strain, the present invention successfully constructed a new genetically engineered strain, OEperR. OEperR exhibits significant improvements in both manganese oxidation and removal capabilities. Compared to the starting strain, this not only shortens the culture and acclimation period but also greatly enhances the strain's environmental adaptability and tolerance.

[0006] The technical solution of the present invention is achieved as follows:

[0007] The present invention provides an application of the perR gene in improving the manganese oxidation function of Bacillus thuringiensis. Overexpression of the perR gene improves the manganese oxidation function of Bacillus thuringiensis. The manganese oxidation function includes oxidation of Mn(II) ions. The nucleotide sequence of the perR gene is shown in SEQ ID NO: 1.

[0008] Furthermore, the present invention provides the use of the perR gene in constructing a Bacillus thuringiensis strain with significantly improved manganese oxidation.

[0009] In the Bacillus thuringiensis strain with significantly improved manganese oxidation, the overexpression of perR is regulated by its endogenous promoter perR promoter, and the nucleotide sequence of the endogenous promoter is shown in SEQ ID NO: 2.

[0010] Furthermore, the method for constructing the Bacillus thuringiensis strain with significantly improved manganese oxidation activity comprises the following steps:

[0011] (1) Using the genome of Bacillus thuringiensis strain Bt 97-27 as a template and the sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 as upstream and downstream primers, PCR was performed to specifically amplify the target fragment, which included the promoter region of the perR gene and its coding sequence;

[0012] (2) The target fragment and plasmid pHT315 were digested with EcoRI and HindIII, and then homologous recombination was performed to construct the recombinant plasmid pHT315-perR;

[0013] (3) The recombinant plasmid pHT315-perR was electroporated into competent cells of Bt 97-27 strain, and the perR gene overexpressing strain was selected by erythromycin resistance and named OEperR.

[0014] The Bacillus thuringiensis strain with significantly improved manganese oxidation generates significantly higher manganese oxides than the starting strain in an LB culture medium containing 5 mM Mn(II) ions.

[0015] The Bacillus thuringiensis strain with significantly improved manganese oxidation has a significantly better ability to remove Mn(II) ions than the starting strain in an LB culture medium containing 5 mM Mn(II) ions.

[0016] The Bacillus thuringiensis strain with significantly improved manganese oxidation has significantly increased biofilm and manganese oxide production on the bacterial surface compared to the starting strain in an LB culture medium containing 5 mM Mn(II) ions.

[0017] The Bacillus thuringiensis strain with significantly improved manganese oxidation is used to produce manganese oxides ramsdellite MnO2 and rhodochrosite MnCO3.

[0018] The Bacillus thuringiensis strain with significantly improved manganese oxidation is used to oxidize and remove Mn(II) pollution in water environments. The removal includes adsorption of Mn(II) and manganese oxides by a biofilm formed by the bacteria.

[0019] The advantages and effects of the present invention include at least the following:

[0020] The present invention overexpresses the perR gene in the Bacillus thuringiensis strain Bt 97-27 to obtain the genetically engineered strain OEperR. Compared with the starting strain, the OEperR strain achieves significant improvements in manganese oxidation, manganese removal, and biofilm formation in LB culture medium containing 5mM Mn(II) ions.

[0021] The manganese oxidation product of the present invention is a complex of ramsdellite (MnO2) and rhodochrosite (MnCO3). The significantly increased biofilm formation of the strain OEperR further promotes the efficient adsorption of Mn(II) and the manganese oxidation product. This enhanced adsorption capacity not only improves the stability of the biofilm itself but also makes the strain OEperR more adaptable and tolerant to various environmental conditions, laying a solid foundation for its widespread promotion in practical applications.

[0022] The present invention shortens the culture and acclimation cycle of the bacterial strain, and uses genetically engineered strains to oxidize and remove manganese with higher efficiency and environmental friendliness, opening up new avenues for the application of microbial technology in the field of manganese-contaminated environmental remediation. It also provides an important basis for in-depth exploration of how microorganisms respond to manganese and regulate its oxidation process. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 Schematic diagram of the structure of the homologous recombination plasmid pHT315-perR of the present invention;

[0025] Figure 2 The results show the concentration of manganese oxides produced by the starting strain Bt 97-27 under different Mn(II) concentrations. The values between different letters indicate significant differences between the groups.

[0026] Figure 3 The results of the manganese oxide concentration measurement every 24 hours after adding 5 mM Mn(II) to the culture medium of the perR gene overexpressing strain OEperR of the present invention and the starting strain Bt 97-27 are shown. The * indicates a significant difference between the two groups; the ns indicates no significant difference between the two groups.

[0027] Figure 4 The results of manganese removal efficiency determination of the perR gene overexpressing strain OEperR and the starting strain Bt 97-27 after 14 days of culture with 5 mM Mn(II) added to the culture medium. The * indicates a significant difference between the two groups.

[0028] Figure 5 These are SEM images of the cell surfaces of Bt 97-27 and OEperR in Example 4 of the present invention: (A) SEM image of the Bt 97-27 strain plus 5 mM Mn(II); (B) SEM image of the OEperR strain plus 5 mM Mn(II). Red arrows indicate biofilms, and blue arrows indicate manganese oxides.

[0029] Figure 6 This is the X-ray diffraction pattern of the crystal structure of biological manganese oxide produced by catalytic oxidation of the OEperR strain.

[0030] Figure 7 This is the X-ray photoelectron spectrum of the manganese valence state distribution in the manganese oxidation products of the OEperR strain. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solutions and advantages of this application more clearly understood, the present application is further described in detail below with reference to the following examples. It should be understood that the specific examples described herein are merely for the purpose of explaining this application and are not intended to limit this application. Reagents not described in detail in this application are all conventional reagents and can be obtained from commercial channels; methods not specifically described in detail are all conventional experimental methods and can be obtained from the prior art.

[0032] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0033] The terms "comprise," "comprising," and "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, product, or device comprising a series of steps is not limited to the listed steps or modules but may optionally include steps not listed, or other steps inherent to the process, method, product, or device.

[0034] In this application, "plurality" refers to two or more. "And / or" describes the relationship between related objects, indicating that three possible relationships exist. For example, "A and / or B" can mean: A exists alone, A and B exist simultaneously, or B exists alone. The character " / " generally indicates that the related objects are in an "or" relationship.

[0035] Example 1 Construction of perR gene overexpression strain OEperR

[0036] 1. Primer design and PCR amplification of target fragments

[0037] Bacillus thuringiensis 97-27 (hereinafter referred to as Bt97-27) in the examples of this application is described in reference (Su Jianmei. Mechanism of action of bacterial manganese oxide and characteristics of biological manganese oxide [D]. Huazhong Agricultural University, 2015). It is publicly available from Hubei University. The applicant has pledged to release biological materials to the public within 20 years from the date of application. The Bt97-27 genome sequence has a GenBank accession number of CP010088.1. Using the Bt97-27 genome as a template, PCR was performed to specifically amplify the target fragment, namely the promoter region of the perR gene (peroxide resistance regulator) in Bt97-27 (sequence shown in SEQ ID NO: 2) and its coding sequence (sequence shown in SEQ ID NO: 1).

[0038] The primers were designed to incorporate homologous sequences from the expression vector pHT315 plasmid. This plasmid had been previously published in non-patent literature prior to the filing date of this application, as described in Li Chaorui, Du Lixin, Peng Qi, et al. Construction of a High-Efficiency Expression Vector for Bacillus thuringiensis [J]. Microbiology Bulletin, 2013. DOI: CNKI: SUN: WSWT.0.2013-02-029. The plasmid is available to the public from Hubei University. The applicants pledge to release biological materials to the public within 20 years of the filing date. The upstream and downstream primer sequences are as follows:

[0039]

[0040] Among them, the shaded part of the sequence is the homologous sequence of the expression vector pHT315 plasmid.

[0041] A high-fidelity PCR reaction system (2×Phanta Max Master Mix, purchased from Nanjing Novozymes Biotechnology Co., Ltd.) was used, and the reaction program was as follows: 94°C, 5 min; 94°C, 45 s; 60°C, 60 s; 72°C, 90 s; 30 cycles, and a final extension of 10 min.

[0042] 2. Construction of recombinant plasmid

[0043] After the amplified product was identified by 1.5% (w / v) agarose gel electrophoresis, the target fragment was purified using a DNA gel recovery kit (agarose gel) and the restriction enzyme QuickCut was used. TM EcoRI and restriction enzyme QuickCut TM HindⅢ (purchased from Takara) was used to double-enzyme digest the target fragment and plasmid pHT315, respectively. The products after enzyme digestion were recovered from the gel, and the fragments amplified by PCR and the linearized vector were homologously recombined using recombinase to construct the recombinant plasmid pHT315-perR. Homologous recombination technology is a technology that uses the principle of homologous sequence recombination to perform seamless cloning. Seamless cloning (infusion-cloning) is similar to traditional PCR cloning in that both use the sticky ends of DNA for complementary pairing, but the difference is that the sticky ends of seamless cloning are formed by degrading DNA along the 5'→3' direction by T5 nuclease, rather than double enzyme digestion. Seamless cloning can efficiently clone PCR products into linearized vectors. Compared with traditional methods of constructing recombinant vectors, seamless cloning has the advantages of being able to replace DNA ligase, not needing to consider the enzyme cutting sites of the vector, and being able to insert multiple target fragments at the same time. Studies have shown that using seamless cloning technology, multiple fragments can be inserted into recombinant vectors to construct vectors expressing fusion proteins. Seamless cloning technology is faster and more efficient than double enzyme digestion cloning for the construction of multi-fragment cloning vectors. Figure 1The constructed recombinant plasmid was transformed into DH5α and screened using solid LB medium containing chloramphenicol (final concentration 25 μg / mL). Sequencing and alignment with the genomic sequence were performed to obtain recombinant E. coli pHT315-perR / DH5α. Positive transformants were cultured and the plasmid was extracted and stored at -20°C until further use.

[0044] 3. Construction of recombinant strain OEperR

[0045] Preparation of competent recipient bacteria Bt97-27 strain: (1) multiple transfer activation strains; (2) overnight culture, transfer to 100 mL of LB medium (250 mL conical flask) at a volume ratio of 1%, and culture at 200 rpm and 28 ° C to the early logarithmic growth stage (about 2-3 hours); (3) pre-cool the culture solution to the early logarithmic growth stage in an ice bath for 15 minutes, and collect the bacteria by centrifugation at 4 ° C and 7000 rpm for 8 minutes; (4) discard the supernatant, resuspend the bacteria with 5 mL of SG Buffer per 100 mL of bacteria solution, and collect the bacteria by centrifugation at 4 ° C and 7000 rpm for 8 minutes; (5) repeat step (4) three times; (6) discard the supernatant, resuspend the bacteria with 2 mL of SG Buffer (SG Buffer: 272 mM sucrose, 15% glycerol, sterilized at 115 ° C for 30 minutes) per 100 mL of bacteria solution, and then divide into 100 μL / centrifuge tube; finally, divide into 1.5 mL centrifuge tubes. After cooling with liquid nitrogen, store at -80°C.

[0046] Electroporation: (1) Dry the electroporation cup in a clean bench and sterilize with UV for 20 min; (2) Add the plasmid to the competent cells, mix well, and pre-cool on ice for 15 min; (3) Transfer the mixture of bacterial solution and plasmid to the pre-cooled electroporation cup and use an electroporator (purchased from Bio-Rad, Gene Pulser Xcell) to transfer the plasmid to the competent cells. TM ) 2500V electric shock for 5ms, after the end of the electric shock, add 800μL of antibiotic-free LB medium to the electroporation cup, transfer to a 1.5mL centrifuge tube, and recover at 200rpm and 28℃ for 3h; (4) collect the bacteria by centrifugation, remove most of the supernatant and resuspend the precipitate with the remaining liquid and apply it to a plate containing erythromycin (final concentration 25μg / mL Erythromycin). r ) LB plate, a perR gene overexpression strain was successfully screened and named OEperR.

[0047] Example 2 Analysis of the manganese oxidation capacity of the perR gene overexpressing strain OEperR

[0048] The detection method of manganese oxide in the embodiment of this application:

[0049] Leucoberbelin blue (LBB) is a synthetic triphenyl complex used for the qualitative and quantitative detection of manganese oxide formation. It specifically reduces high-valent manganese (Mn(III)) and (IV) while being oxidized. Manganese oxide is converted to MnO2 concentration by reacting the sample with LBB in the dark at 620 nm for 15-20 minutes. The absorbance of the mixture is then compared to a KMnO4 standard curve to determine the corresponding concentration. This is then multiplied by 2.5 to obtain the corresponding MnO2 concentration.

[0050] (1) Build A 620 / KMnO4 concentration relationship standard curve and linear fitting formula: prepare KMnO4 stock solution with a concentration of 1mM, and dilute KMnO4 to 0.01mM, 0.02mM, 0.03mM, 0.04mM, 0.05mM, 0.06mM, 0.07mM, 0.08mM, 0.09mM, 0.10mM, 0.11mM and 0.12mM; take 50μL of the above diluted solutions into 96-well plates, add 250μL of LBB solution to the wells, mix and react in the dark for 15-20min; measure the absorbance of the reaction mixture at 620nm on a microplate reader; use KMnO4 concentration as the vertical axis, A 620 Plot A as the horizontal axis. 620 / KMnO4 concentration corresponding standard curve, and obtain the linear fitting formula;

[0051] Determination of the manganese oxide content produced by Bt 97-27 at different manganese ion concentrations: a single colony of Bacillus thuringiensis Bt97-27 was picked and placed in a conical flask containing LB liquid medium and activated overnight in a constant temperature shaker at 28°C and 200 rpm for 12 hours. The bacterial solution was transferred at 1% (volume ratio) and the initial OD was calculated after overnight culture. 600 = 0.01 transferred to 0, 2.5, 5, 10 and 20 mM Mn 2+ (In the present embodiment, Mn 2+ Take MnCl2 as an example) in 50mL LB liquid culture medium. Take a sample every 24h and react with LBB in the dark for 15min, then centrifuge and take the supernatant for A 620 The determination of A 620 The value is brought into the standard curve for conversion and the corresponding manganese oxide concentration is obtained. Figure 2 As shown, when the culture medium contains Mn 2+The initial concentrations were 0, 2.5, 5, 10, and 20 mM. As the culture time increased, the strain began to oxidize Mn(Ⅱ) at 72 h. At 168 h, the manganese oxide concentration produced by 5 mM reached 1.1 mM and was significantly higher than the manganese oxide concentrations produced by other concentrations (P<0.05). Therefore, 5 mM Mn was used to test the strain's ability to oxidize Mn(Ⅱ). 2+ .

[0052] (2) Determination of the activity of Bt 97-27 and OEperR strains after addition of 5 mM Mn 2+ Manganese oxide content: Add MnCl2 to a final concentration of 5mM in LB medium, inoculate Bt 97-27 strain and OEperR strain into the medium at a volume ratio of 0.1%, and culture at 200rpm and 28℃; take 1mL of bacterial solution every 24 hours, measure the absorbance of the reaction solution at 620nm by LBB method, and convert the absorbance into the concentration of oxidized Mn(IV) using a standard curve to analyze and compare the ability of different strains to oxidize Mn(II). Figure 3 As shown, the content of manganese oxide produced by the OEperR strain was significantly higher than that of the starting strain Bt 97-27.

[0053] Example 3 Analysis of the manganese removal ability of the perR gene overexpressing strain OEperR

[0054] In order to study the removal of Mn(II) by the strains, the Bt 97-27 and OEperR strains were treated with 5mM Mn(II) on the 0th and 14th days. 10mL of bacterial solution was taken and centrifuged at 12000rpm for 10min. The supernatant and precipitate were digested with concentrated nitric acid at a final concentration of 10% at 121℃ for 30min. The nitric acid concentration was diluted to 1% with deionized water and then filtered through 0.22μm. The total content of intracellular and extracellular Mn(II) was determined using an Agilent 7850 ICP-MS plasma emission mass spectrometer; manganese removal rate = (initial manganese content - manganese content after treatment) / initial manganese content × 100%. The results are shown in Figure 4 As shown in the figure, the manganese removal efficiency of Bt 97-27 was 31.9±3.65%, and the manganese removal efficiency of OEperR was 60.95±0.49%. The results showed that the manganese removal ability of the engineered bacteria after overexpressing the perR gene was significantly improved.

[0055] Example 4 Analysis of the biofilm formation ability of the perR gene overexpressing strain OEperR

[0056] The morphology of the samples was determined using SEM (Scanning Electron Microscope): Each single colony was picked and placed in a conical flask containing LB liquid medium and activated overnight in a constant temperature shaker at 28°C and 200 rpm for 12 hours. The bacterial solution was transferred at a 1% volume ratio and cultured overnight, and the initial OD was calculated. 600 =0.01 transferred to 5mMMn 2+ 50 mL of LB medium was used. After 3 days of culture, 1 mL of the bacterial culture was centrifuged at 6000 rpm for 3-4 minutes. The supernatant was removed to obtain the bacteria, which were then rinsed 3-4 times with PBS (pH 7.2). The samples were then fixed with 2.5% glutaraldehyde at 4°C for 12-24 hours, washed again with PBS (pH 7.2), and finally dehydrated in a stepwise manner using 30%, 50%, 70%, 80%, 90%, and 100% alcohol, with each step dehydrating 2-3 times for 10-15 minutes. The samples were then freeze-dried in a vacuum freeze dryer for 12 hours. After powder gold coating, the samples were observed and images were collected using a high-resolution cold-field emission scanning electron microscope (HITACHI Regulus 8100). Electron micrographs were taken at a magnification of 10,000x to examine the morphological characteristics of the strains, as well as the manganese adhesion and biofilm content of the samples.

[0057] The morphological appearance of Bt 97-27 and OEperR observed by SEM imaging is shown in Figure 2. Figure 5 As shown, after overexpression of perR, 5 mM Mn 2+ The biofilm on the bacterial surface (indicated by the red arrow) was significantly increased compared with the starting strain Bt97-27, and the manganese oxide (indicated by the blue arrow) had better adhesion. The good adhesion of manganese oxide may further enhance the stability of the biofilm.

[0058] It can be seen that the OEperR strain was treated with 5 mM Mn 2+ Finally, its enhanced biofilm system promotes the improvement of the strain's environmental adaptability and tolerance, as well as the adsorption of the generated manganese oxides.

[0059] Example 5 X-ray diffraction pattern of biological manganese oxide produced by catalytic oxidation of OEperR strain

[0060] In order to determine the composition of manganese oxides produced by the OEperR strain during manganese oxidation, the biological manganese oxides on the 14th day were centrifuged at 12000 rpm for 10 min, the precipitate was taken and washed three times with deionized water, the sample was placed in a -80℃ freezer for 12 h, and then freeze-dried for 8 h. The powder sample was obtained by grinding. 3The composition of the manganese oxide was determined by X-ray diffraction (XRD) using a nickel-filtered Cu-Kα (λ = 0.15418 nm). The diffractometer tube voltage was 45 kV, the tube current was 40 mA, the scanning range was wide angle 2θ = 5-90°, and the scanning speed was 10° / min. According to the XRD diffraction results ( Figure 6 ), the manganese oxides catalyzed by the OEperR strain are complexes of Ramsdellite-MnO2 and Rhodochrosite,syn-MnCO3, with five broad peaks at 20.935°, 31.85°, 45.62°, 54.75° and 63.37°, corresponding to the crystal plane indices (101) and (402) of ramsdellite MnO2 (PDF#42-1316) and the crystal plane indices (104), (202) and (1010) of rhodochrosite MnCO3 (PDF#44-1472), respectively.

[0061] Example 6 X-ray photoelectron spectroscopy of the valence state distribution of manganese in the manganese oxidation product of the OEperR strain

[0062] In order to determine the elemental composition of manganese oxides produced by the OEperR strain during manganese oxidation, the biological manganese oxides on the 14th day were centrifuged at 12000 rpm for 10 min, the precipitate was taken and washed three times with deionized water, the sample was placed in a -80℃ freezer for 12 h, and then freeze-dried for 8 h. The powder sample was obtained by grinding. The elemental valence state of the sample was determined by X-ray photoelectron spectroscopy (XPS, Thermo Scientific TM ESCALAB TM Xi+, UK). A monochromated Al target (E = 1486.68 eV) was used, with a voltage of 13.4 keV, a current of 6 mA, a vacuum of P < 10-9 mBar, a pass energy of 150 eV (full spectrum), 30 eV (fine spectrum), a step size of 0.05 eV, a dwell time of 50 ms, and charge correction using a binding energy of C1s = 284.80 eV. The spot size was 500 μm, and the work function was 4.20 eV.

[0063] There are two main peaks in the Mn2p binding energy: 1 / 2 and Mn2p 3 / 2 , the electron binding energies are 653.41eV and 643.3eV respectively, among which Mn2p 3 / 2 The peaks in the spectrum are asymmetric. 1 / 2 and Mn 2p 3 / 2 Peak fitting at ( Figure 7 ), where Mn 2p 3 / 2The electron binding energies of 642.80eV, 641.50eV, and 640.82eV correspond to the characteristic peaks of Mn(IV), Mn(III), and Mn(II), respectively. 1 / 2 The corresponding Mn(IV) is 64.27% of the manganese oxide valence ratio, Mn(III) is 22.42% of the manganese oxide valence ratio, and Mn(II) is 13.31% of the manganese oxide valence ratio.

[0064] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Use of the perR gene in improving manganese oxidation in Bacillus thuringiensis, characterized in that: Overexpression of the perR gene improves the manganese oxidation function of Bacillus thuringiensis, wherein the manganese oxidation function includes the oxidation of divalent manganese ions to form manganese oxides, wherein the manganese oxides are ramsdellite MnO2 and rhodochrosite MnCO3, and the nucleotide sequence of the perR gene is shown in SEQ ID NO:1; the Bacillus thuringiensis strain overexpressing the perR gene oxidizes divalent manganese ions in the water environment and utilizes the biofilm formed by the bacteria to adsorb divalent manganese ions and manganese oxides, thereby removing divalent manganese ion pollution in the water environment.

2. The use according to claim 1, characterized in that The perR gene is used in constructing a Bacillus thuringiensis strain with significantly improved manganese oxidation.

3. The use according to claim 2, characterized in that In the Bacillus thuringiensis strain with significantly improved manganese oxidation, the overexpression of perR is regulated by its endogenous promoter perR promoter, and the nucleotide sequence of the endogenous promoter is shown in SEQ ID NO:

2.

4. The use according to claim 3, characterized in that The method for constructing the Bacillus thuringiensis strain with significantly improved manganese oxidation comprises the following steps: (1) Using the genome of Bacillus thuringiensis strain Bt 97-27 as a template and the sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 as upstream and downstream primers, PCR was performed to specifically amplify the target fragment, which included the promoter region of the perR gene and its coding sequence; (2) The target fragment and plasmid pHT315 were digested with EcoRI and HindIII, and then homologous recombination was performed to construct the recombinant plasmid pHT315-perR; (3) The recombinant plasmid pHT315-perR was electroporated into competent cells of Bt 97-27 strain, and the perR gene overexpressing strain was selected by erythromycin resistance and named OEperR.

5. The use according to claim 4, characterized in that The Bacillus thuringiensis strain with significantly improved manganese oxidation generates significantly higher manganese oxides than the starting strain in an LB culture medium containing 5 mM divalent manganese ions.

6. The use according to claim 4, characterized in that The Bacillus thuringiensis strain with significantly improved manganese oxidation has a significantly better ability to remove divalent manganese ions than the starting strain in an LB culture medium containing 5 mM divalent manganese ions.

7. The use according to claim 4, characterized in that The Bacillus thuringiensis strain with significantly improved manganese oxidation has significantly increased biofilm and manganese oxide production on the bacterial surface compared to the starting strain in an LB culture medium containing 5 mM divalent manganese ions.

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