Application of over-expression perR gene in improvement of biological manganese oxidation effect of bacillus thuringiensis

Abstract

The invention relates to the field of gene engineering, in particular to application of an overexpressed perR gene to improvement of the biological manganese oxidation effect of bacillus thuringiensis. According to the invention, a strain Bt 97-27 is used as an original strain, and the strain can oxidize Mn (II), form spores and generate a biological membrane to resist external adverse environmental factors. The novel genetic engineering strain OEperR is successfully constructed through overexpression of the perR gene in the Bt 97-27 strain, compared with an original strain, the OEperR strain is remarkably improved in the aspects of manganese oxidation, manganese removal, biological membrane generation and the like, the culture and domestication periods of the strain are shortened, and the yield of the strain is increased. And the environmental adaptability and tolerance of the strain are greatly enhanced, a new way is opened up for the application of a microbial technology in the field of manganese polluted environment restoration, and an important basis is provided for deeply exploring how microorganisms respond to manganese elements and regulating and controlling the oxidation process of the manganese elements.

Description

Technical Field

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

[0002] In recent years, due to the mining of manganese ore and the mass production of manganese-containing industries, a large amount of manganese has 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 toxic sludge will be produced. The filtration adsorption method is simple to operate, but the filter material needs to be cleaned regularly, and the maintenance and management cost is high. Biological manganese removal refers to the oxidation of soluble Mn (II) into insoluble Mn (III) or Mn (IV) manganese oxide precipitation 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 groundwater Mn (II) pollution remediation due to their fast growth and reproduction, high manganese removal efficiency, low treatment cost and environmental friendliness.

[0003] Bacillus thuringiensis (Bt) is a Gram-positive bacterium that is rod-shaped under a microscope and arranged in short or long chains. It can form endogenous spores. The spores are oval and grow close to the middle. They are widely found in soil, insects and corrupted organic matter. They are insect pathogens. Bt is a member of the Bacillus cereus group. It has two growth periods: the vegetative period and the spore period. The vegetative period is mainly characterized by secondary division, and the bacteria continue to reproduce. When the bacteria are in an unfavorable environment such as lack of nutrients or strong acid and alkali, they can form spores. Once the nutrients in the environment are sufficient, the spores will automatically enter the vegetative period and grow back into Bt.

[0004] Peroxide resistance regulator (PerR) is a transcription factor present in many bacteria. It has a molecular weight of 20.35 kDa and belongs to the large family of iron uptake regulation proteins (Fur). It mainly responds to intracellular H 2 O 2 concentration, and participates in regulating the expression of a series of genes related to hydrogen peroxide stress. Research on PerR mainly focuses on the antioxidant effect of bacteria, the dynamic balance of metal ions in cells, quorum sensing, pathogenicity and other complex biological effects. However, it is worth noting that, up to now, there is no research on the perR gene as a key gene for regulating manganese oxidation. Summary of the invention

[0005] In view of this, the present invention uses strain Bt 97-27 as the starting strain, which can oxidize Mn (II), form spores and produce biofilm to resist adverse external environmental factors. The present invention successfully constructed a new genetically engineered strain OEperR by overexpressing the perR gene in the Bt 97-27 strain. The OEperR showed significant improvement in manganese oxidation and manganese removal capabilities. Compared with the starting strain, it not only shortened the culture and domestication period of the strain, but also greatly enhanced the environmental adaptability and tolerance of the strain.

[0006] The technical solution of the present invention is achieved in this way:

[0007] The present invention provides an application of a perR gene in improving the manganese oxidation of Bacillus thuringiensis. Overexpression of the perR gene improves the manganese oxidation of Bacillus thuringiensis. The manganese oxidation 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] The Bacillus thuringiensis strain with significantly improved manganese oxidation function, wherein 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 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 used to specifically amplify the target fragment, wherein the target fragment includes 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 more manganese oxides than the starting strain in an LB 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 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 in an LB medium containing 5 mM Mn (II) ions compared with the starting strain.

[0017] The Bacillus thuringiensis strain with significantly improved manganese oxidation is used to produce manganese oxide ramsdalite MnO 2 and rhodochrosite MnCO 3 .

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

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

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

[0021] The manganese oxidation product of the present invention is ramsdellite MnO 2 and rhodochrosite MnCO 3 The significantly increased biofilm formation of strain OEperR further promoted the efficient adsorption of Mn(II) and manganese oxidation products. This enhanced adsorption capacity not only improves the stability of the biofilm itself, but also makes strain OEperR more adaptable and tolerant under various environmental conditions, laying a solid foundation for its widespread promotion in practical applications.

[0022] The present invention shortens the culture and domestication period of bacterial strains, and utilizes genetically engineered strains to oxidize and remove manganese with higher efficiency and environmental friendliness, thus opening up a new path for the application of microbial technology in the field of manganese-polluted environmental remediation, and also providing 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 creative work.

[0024] Figure 1 It is a schematic diagram of the structure of the homologous recombination plasmid pHT315-perR of the present invention;

[0025] Figure 2 The results are the results of the determination of the concentration of manganese oxides generated by the starting strain Bt 97-27 of the present invention under different Mn(II) concentrations. Different letters represent significant differences between the groups.

[0026] Figure 3 The results of the determination of the manganese oxide concentration generated 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 in FIG. 1 , where * indicates a significant difference between the two groups; ns indicates no significant difference between the two groups.

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

[0028] Figure 5 The SEM images of the cell surface of Bt 97-27 and OEperR in Example 4 of the present invention are as follows: (A) SEM image of Bt 97-27 strain plus 5mM Mn(II); (B) SEM image of OEperR strain plus 5mM Mn(II). The red arrows are biofilms, and the blue arrows are 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 scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not 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 of the present invention. 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 "including" and "having" and any variations thereof of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, device, product or equipment comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to these processes, methods, products or equipment.

[0034] In the present invention, the term "multiple" refers to two or more than two. "And / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B can represent the following three situations: A exists alone, A and B exist at the same time, and B exists alone. The character " / " generally indicates that the associated 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 fragment

[0037] Bacillus thuringiensis 97-27 (hereinafter referred to as Bt97-27) in the present application example is shown in the reference (Su Jianmei. Mechanism of action of bacterial manganese oxidation and characteristics of biological manganese oxide [D]. Huazhong Agricultural University, 2015.), which can be obtained by the public from Hubei University. The applicant promises to release biological materials to the public within 20 years from the date of application. The GenBank number of the Bt97-27 genome sequence is CP010088.1. Using the Bt97-27 genome as a template, PCR specifically amplifies the target fragment, i.e., the promoter region of the perR gene (peroxide resistance regulator) in Bt97-27 (sequence as shown in SEQ ID NO: 2) and its coding sequence (sequence as shown in SEQ ID NO: 1).

[0038] The homologous sequence of the expression vector pHT315 plasmid was introduced into the primer design. The pHT315 plasmid has been disclosed in non-patent literature before the application date of the present invention, see reference (Li Chaorui, Du Lixin, Peng Qi, et al. Construction of high-efficiency expression vector of Bacillus thuringiensis [J]. Microbiology Bulletin, 2013. DOI: CNKI: SUN: WSWT.0.2013-02-029.), and the public can obtain it from Hubei University. The applicant promises to distribute biological materials to the public within 20 years from the application 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 Biotech Co., Ltd.) was used, and the reaction program was: 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] The amplified product was subjected to 1.5% (w / v) agarose gel electrophoresis to identify the product fragment size, and the target fragment was purified using a DNA gel recovery kit (agarose gel) and the restriction enzyme QuickCut was used to extract the target fragment. TM EcoRI and restriction enzyme QuickCut TM HindⅢ (purchased from Takara) was used to double-digest the target fragment and plasmid pHT315, respectively. The digested products were recovered from the gel, and the fragments amplified by PCR and the linearized vector were homologously recombined using the 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, and both use the sticky ends of DNA for complementary pairing. However, 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 digestion. Seamless cloning can efficiently and directionally 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 restriction sites of the vector, and being able to insert multiple target fragments at the same time. Studies have shown that seamless cloning technology can be used to insert multiple fragments into recombinant vectors to construct vectors that express fusion proteins. Seamless cloning technology is faster and more efficient than double digestion cloning for the construction of multi-fragment cloning vectors. Figure 1As shown. The constructed recombinant plasmid was transformed into DH5α, screened with solid LB medium containing chloramphenicol (final concentration 25 μg / mL), and sequenced and compared with the genome sequence. The recombinant Escherichia coli pHT315-perR / DH5α was obtained. After culturing and screening the positive transformants, the plasmid was extracted and stored at -20°C for future use.

[0044] 3. Construction of recombinant strain OEperR

[0045] Preparation of competent state of 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 bacterial 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 bacterial 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 light for 20 min; (2) Add the plasmid to the competent cells, mix well, and precool on ice for 15 min; (3) Then transfer the mixture of bacterial liquid and plasmid to the precooled electroporation cup and use an electroporator (purchased from Bio-Rad, Gene Pulser Xcell TM ) 2500V electric shock for 5ms. After the electric shock, add 800μL of antibiotic-free LB medium to the electroporation cup, transfer to a 1.5mL centrifuge tube, and revive at 200rpm and 28℃ for 3h; (4) Collect the bacteria by centrifugation, remove most of the supernatant and use the remaining liquid to resuspend the precipitate and apply it to a plate containing erythromycin (final concentration 25μg / mL Ery r ) on LB plates, a perR gene overexpression strain was successfully screened and named OEperR.

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

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

[0049] Leucoberbelin blue (LBB) is a synthetic triphenyl complex used for qualitative and quantitative detection of manganese oxide formation. It can specifically reduce high-valent manganese Mn(III) and Mn(IV) while being oxidized. 2 The sample was reacted with LBB in the dark for 15-20 min at 620 nm, and the absorbance of the mixture was calculated based on the concentration of KMnO 4 The corresponding concentration value can be found from the standard curve, and then multiplied by 2.5 to obtain the corresponding MnO 2 concentration.

[0050] (1) Build A 620 / KMnO 4 Standard curve and linear fitting formula of concentration relationship: Prepare KMnO with a concentration of 1mM 4 Mother liquor, KMnO 4 The concentrations were diluted 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; 50μL of the above dilution solutions were respectively taken into 96-well plates, and 250μL of LBB solution was added to the wells, and the mixture was reacted in the dark for 15-20min after mixing; the absorbance of the reaction mixture at 620nm was measured on an ELISA instrument; KMnO 4 The concentration is the vertical axis, A 620 Plot A as the horizontal axis. 620 / KMnO 4 The standard curve corresponding to the concentration was obtained, and the linear fitting formula was obtained;

[0051] Determination of the manganese oxide content generated by Bt 97-27 at different manganese ion concentrations: a single colony of Bacillus thuringiensis Bt97-27 was picked up 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+ MnCl 2 Take a sample every 24 hours and react with LBB in the dark for 15 minutes, 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 finally the corresponding manganese oxide concentration is obtained. Figure 2 As shown, when the 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+ To generate manganese oxide content: add MnCl to a final concentration of 5 mM in LB medium. 2 , inoculate Bt 97-27 strain and OEperR strain into the culture medium at a volume ratio of 0.1%, and culture at 200rpm and 28℃; take 1mL of bacterial solution every 24h, and measure the absorbance of the reaction solution at 620nm by LBB method, and convert the absorbance into the concentration of oxidized Mn(IV) using the standard curve, and 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 manganese removal ability of 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 treated with 5mM Mn(II) on the 0th and 14th days were selected. Take 10mL of bacterial solution, centrifuge at 12000rpm for 10min, take the supernatant and precipitate and use concentrated nitric acid with a final concentration of 10% to digest at 121℃ for 30min, dilute the nitric acid concentration to 1% with deionized water, and then filter 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, the manganese removal rate of Bt 97-27 was 31.9±3.65%, and the manganese removal rate of OEperR was 60.95±0.49%. The results showed that the manganese removal capacity of the engineered bacteria after overexpressing the perR gene was significantly improved.

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

[0056] The morphology of the samples was determined by SEM (Scanning Electron Microscope): each single colony was picked up in a conical flask containing LB liquid culture 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. The initial OD 600 =0.01 transferred to 5mMMn 2+ 50mL LB medium. After 3 days of culture, 1mL of bacterial solution was centrifuged at 6000rpm for 3-4min, and the supernatant was removed to obtain the bacteria, which were washed with PBS at pH7.2 for 3-4 times, and then fixed with 2.5% glutaraldehyde fixative at 4°C for 12-24h, washed again with PBS at pH7.2, and finally dehydrated step by step with 30%, 50%, 70%, 80%, 90% and 100% alcohol, 2-3 times per level, and 10-15min per dehydration. The sample was then freeze-dried in a vacuum freeze dryer for 12h. After the sample powder was sprayed with gold, it was observed and imaged using a resolution cold field emission scanning electron microscope (HITACHI Regulus 8100). The electron microscope photos were magnified 10,000 times in the experiment to scan the morphological characteristics of the strain, as well as the manganese attachment and biofilm conditions of the sample.

[0057] The morphological appearance of Bt 97-27 and OEperR observed by SEM imaging is shown in Figure 5 As shown, after overexpression of perR, 5 mM Mn 2+ Compared with the starting strain Bt97-27, the biofilm on the bacterial surface (indicated by the red arrow) has a significant increase, and the manganese oxide (indicated by the blue arrow) has 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 promoted 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 generated by the OEperR strain during manganese oxidation, the biological manganese oxides on the 14th day were centrifuged at 12000rpm for 10min, the precipitate was taken and washed three times with deionized water, the sample was placed in a -80℃ refrigerator for 12h, and then freeze-dried for 8h, and the powder sample was obtained by grinding. 3Powder instrument was used to perform X-ray diffraction (XRD) to determine the composition of manganese oxide, using Cu-Kα (λ = 0.15418 nm) with nickel filtering. The tube voltage of the diffractometer 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 oxide produced by the oxidation catalyzed by the OEperR strain is Ramsdellite-MnO 2 and Rhodochrosite,syn-MnCO 3 The composite has five broad peaks at 20.935°, 31.85°, 45.62°, 54.75° and 63.37°, corresponding to ramsdellite MnO 2 (PDF#42-1316) with crystal face indices (101) and (402), rhodochrosite MnCO 3 (PDF#44-1472) with crystal plane indices (104), (202) and (1010).

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

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

[0063] The Mn2p binding energy has two main peaks: Mn2p 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 The peak fitting at Figure 7 ), where Mn 2p 3 / 2 The electron binding energies of 642.80 eV, 641.50 eV, and 640.82 eV 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 protection scope 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 of Bacillus thuringiensis, wherein the manganese oxidation includes oxidation of Mn(II) ions to form manganese oxides. The nucleotide sequence of the perR gene is shown in SEQ ID NO:

1.

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: The Bacillus thuringiensis strain with significantly improved manganese oxidation function, wherein 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, wherein the target fragment includes 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 more manganese oxides than the starting strain in an LB medium containing 5 mM Mn(II) 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 Mn(II) ions than the starting strain in an LB medium containing 5 mM Mn(II) 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 in an LB medium containing 5 mM Mn (II) ions compared with the starting strain.

8. The use according to any one of claims 2 to 7, characterized in that: The Bacillus thuringiensis strain with significantly improved manganese oxidation is used to produce manganese oxides ramsdalite MnO2 and rhodochrosite MnCO3.

9. The use according to any one of claims 2 to 7, characterized in that: The Bacillus thuringiensis strain with significantly improved manganese oxidation is used to oxidize and remove Mn(II) pollution in water environments.

10. The use according to claim 9, characterized in that: The removal includes the adsorption of Mn(II) and manganese oxides by the biofilm formed by the bacteria.

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