Recombinant vibrio natriureticus as well as construction method and application thereof

Abstract

The invention discloses a recombinant vibrio natrio as well as a construction method and application thereof, and belongs to the technical field of bioengineering.The recombinant vibrio natrio is an engineering strain for overexpressing vibrio natrio MtrC, MtrA, MtrB, PdsA and CymA proteines.The vibrio natrio is selected as a chassis microorganism for synthesizing selenium nanoparticles, expression of heterologous genes is enhanced by introducing plasmid vectors, and the recombinant vibrio natrio is obtained. The Mtr pathway expression of the sodium selenite reduced by the vibrio natrio is enhanced, and the synthesis of biological nano-selenium by metabolism of organic waste glycerol by the vibrio natrio is realized, so that the production cycle of the nano-selenium can be remarkably shortened, and the production cost can be greatly reduced by using the organic waste as a carbon source to carry out a production experiment. The economic and environmental benefits in the synthesis process of the biological nano-selenium are realized.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a recombinant Vibrio natrii and a construction method and application thereof. Background Art

[0002] As a metalloid, selenium is widely used in electronics, cosmetics, coatings and packaging due to its semiconductor and optoelectronic properties. In addition, in biomedicine, selenium, as an essential trace element for the human body, is widely used as a food supplement; and nano-selenium has antioxidant properties and has important application value in the fields of anti-tumor and cancer treatment.

[0003] Nano-selenium can be obtained in a variety of ways, such as laser ablation, ultraviolet radiation, hydrothermal technology, precipitation catalytic reduction, acid decomposition, etc. However, these physical and chemical methods involve the use of extreme pH and toxic compounds, which makes the use of nano-selenium obtained by these methods in the medical field a safety hazard. In addition, nano-selenium produced by physical and chemical methods requires the addition of stabilizers during the synthesis process to ensure a smaller particle size to ensure its activity, which leads to health risks to the human body and limited application scenarios for the synthesized nano-selenium. In comparison, nano-selenium synthesized based on biological methods not only has simple and mild conditions, but also has high biocompatibility and lower biotoxicity, making it more suitable as an industrial-scale synthesis method for nano-selenium.

[0004] Biosynthesized nano-selenium is mainly obtained through the reduction of selenium oxyanions, such as selenite and selenate. However, selenium oxyanions are toxic to cells and will affect the cell viability of the strain. Therefore, it is very necessary to find strains with strong tolerance to adapt to the synthesis of biological nano-selenium. Summary of the invention

[0005] In response to the above technical problems, the present invention provides a recombinant Vibrio natrii and a construction method and application thereof. Experiments have shown that the recombinant Vibrio natrii is used as a chassis cell to reduce selenium oxyanions to synthesize nano-selenium, with mild reaction conditions and simple operation, and has potential industrial application value in the field of biosynthetic selenium nanomaterials.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] In a first aspect, the present invention provides a recombinant Vibrio natrix, which is an engineered strain that overexpresses Vibrio natrix MtrC, MtrA, MtrB, PdsA, and CymA proteins. The amino acid sequence of MtrC is shown in SEQ ID NO: 1, the amino acid sequence of MtrA is shown in SEQ ID NO: 2, the amino acid sequence of MtrB is shown in SEQ ID NO: 3, the amino acid sequence of PdsA is shown in SEQ ID NO: 4, and the amino acid sequence of CymA is shown in SEQ ID NO: 5.

[0008] In a second aspect, the present invention also provides a method for constructing the above-mentioned recombinant Vibrio natriuresis, comprising the following steps:

[0009] 1) Strain culture: preparing activated Escherichia coli and Vibrio natriuresis bacterial liquids;

[0010] 2) Plasmid construction: synthesize the gene sequences encoding MtrC, MtrA, MtrB, PdsA, and CymA proteins, the IPTG-inducible promoter gene sequence, and the p15A plasmid backbone gene sequence, and then transform the ligation product into Escherichia coli Turbo through Gibson ligation, screen positive clones with chloramphenicol, and finally extract the plasmids from the positive clones to obtain the plasmid p15A-EET;

[0011] 3) Construction of engineered strains of Vibrio natrii: Add the activated Vibrio natrii bacterial solution into LBv2 medium and culture until OD 600 =0.4, centrifuge to remove the supernatant, collect the bacteria and wash them three times with cold 1M sorbitol solution, then add 1M sorbitol solution to obtain competent cells, package and store them to obtain Vibrio natriuresis competent cells;

[0012] The plasmid p15A-EET was electroporated into competent cells of Vibrio natriuresis, and positive clones were selected with chloramphenicol to obtain recombinant Vibrio natriuresis.

[0013] Furthermore, the Vibrio natriuresis is Vibrio natriuresis ATCC 14048.

[0014] Furthermore, the IPTG-inducible promoter gene sequence is shown in SEQ ID NO:6.

[0015] Furthermore, the p15A plasmid backbone gene sequence is shown in SEQ ID NO:7.

[0016] Furthermore, the plasmid p15A-EET was electroporated into the competent cells of Vibrio natriuresis, and the positive clones were screened by chloramphenicol. The specific process is as follows:

[0017] The competent cells of Vibrio natrii were taken out from the -80°C refrigerator and melted in ice. The p15A-EET plasmid and the competent cells of Vibrio natrii were mixed and transferred to a sterile electroporation cuvette, electroporated at 2.5 kV for 5 ms, and then immediately transferred to LBv2 medium and incubated at 37°C for 1 h. Then, the cells were screened on LBv2 agar plates containing 10 μg / mL chloramphenicol to obtain the positive clone V.NA-p15A-EET strain, which was the recombinant Vibrio natrii.

[0018] In a third aspect, the present invention also provides the use of the above-mentioned recombinant Vibrio natrii in synthesizing nano-selenium, comprising the following steps:

[0019] A selenium-containing medium was prepared using mineral salt medium and sodium selenite, and the V.NA-p15A-EET strain was added to the selenium-containing medium to make the initial OD600 = 1.0, and then an IPTG aqueous solution was added to make the final IPTG concentration reach 0.1 mM, and the culture was statically cultured in an incubator at 30°C to obtain a fermented culture solution, the fermented culture solution was centrifuged to remove the supernatant, the bacterial cells were collected, and the bacterial cells were added to deionized water, and the cells were repeatedly frozen and thawed three times, and then ultrasonically disrupted, washed, and resuspended to obtain a nano-selenium solution.

[0020] Further, the freeze-thaw temperatures were -80°C and 37°C.

[0021] Furthermore, ultrasonic crushing was performed using an ultrasonic crusher at a power of 35% with a cycle of 2s on and 2s off for 10 minutes.

[0022] Further, the specific operations of washing and resuspension are as follows:

[0023] The cells were washed twice with 1.5 M Tris-HCl (pH = 8.3) containing 1% SDS solution, each time at 15000 g, centrifuged for 10 min. n-octanol was added to the sediment, and after vigorous shaking, it was allowed to stand at 4 ° C for 1 h. The aqueous phase was drawn, and chloroform, anhydrous ethanol, 75wt% ethanol solution and water were used in sequence to centrifuge at 15000 g for 10 min, washed twice with ultrapure water, and finally resuspended in ultrapure water to obtain a nano-selenium solution.

[0024] Furthermore, the concentration of sodium selenite in the selenium-containing culture medium is 10 mM.

[0025] Furthermore, the mineral salt medium composition is: carbon source 10g / L, yeast extract 2g / L, Na2HPO4 6g / L, NaCl 30g / L, NH4Cl 1g / L, KH2PO4 3.5g / L, MgSO4·7H2O 246.5mg / L, CaCl2·2H2O 14.7mg / L, FeSO4·7H2O 27.8mg / L

[0026] Furthermore, the carbon source is glucose or glycerol or waste glycerol.

[0027] Beneficial effects of the present invention:

[0028] 1. The present invention utilizes recombinant Vibrio natrii as a chassis microorganism to synthesize nano-selenium through a biological method, with mild reaction conditions, simple operation, high biocompatibility and lower biological toxicity, and is more suitable as an industrial-scale synthesis method for nano-selenium.

[0029] 2. The present invention selects Vibrio natrii as the chassis microorganism for synthesizing selenium nanoparticles, strengthens the expression of heterologous genes by introducing plasmid vectors, strengthens the expression of the Mtr pathway for reducing sodium selenite by Vibrio natrii, and realizes the metabolism of organic waste glycerol by Vibrio natrii to synthesize biological nano-selenium, which can not only significantly reduce the production cycle of nano-selenium, but also can use organic waste as a carbon source for production experiments to greatly reduce production costs, thereby achieving economic and environmental benefits in the process of biological nano-selenium synthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The present invention will be further described below in conjunction with the accompanying drawings.

[0031] Figure 1 is a graph showing the growth curve of Vibrio natriuresis under different concentrations of sodium selenite during the sodium selenite tolerance verification process in Example 1 of the present invention;

[0032] Figure 2 This is a diagram showing the reduction results of sodium selenite by Vibrio natrii in the nano-selenium synthesis experiment in Example 1 of the present invention;

[0033] Figure 3 is a scanning electron microscope image of nano-selenium in Example 1 of the present invention;

[0034] Figure 4 is the map of plasmid p15A-EET in Example 2 of the present invention;

[0035] Figure 5 is a growth curve diagram of the V.NA-p15A-EET strain in Example 3 of the present invention in LBv2 medium with different concentrations of waste glycerol;

[0036] Figure 6 It is a graph showing the reduction results of sodium selenite by different strains under different carbon source conditions in Example 3, Example 4, Example 5 and Comparative Example 2 of the present invention. DETAILED DESCRIPTION

[0037] The following will be combined with 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.

[0038] In this application:

[0039] LBv2 medium is LB medium containing 204 mM NaCl, 23.14 mM MgCl2 and 4.2 mM KCl.

[0040] The composition of LB medium is: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl.

[0041] The components of M9 mineral salt culture medium are: glucose 10g / L, yeast extract 2g / L, Na2HPO46g / L, NaCl 30g / L, NH4C l 1g / L, KH2PO43.5g / L, MgSO4·7H2O 246.5mg / L, CaCl2·2H2O14.7mg / L, FeSO4·7H2O27.8mg / L.

[0042] The main components of waste glycerol are glycerol, methanol and salt, which was obtained from Anhui Tianyi Environmental Protection Technology Co., Ltd., with the concentrations of glycerol and methanol being 1056 g / L and 162 g / L, respectively.

[0043] Example 1

[0044] The application of Vibrio natrii in synthesizing nano-selenium includes the following steps:

[0045] 1) Vibrio natrium culture required

[0046] The freezing tube of Vibrio natrium ATCC 14048 was taken out from the -80°C incubator, and then 40 μL of Vibrio natrium bacterial solution was taken and placed in 4 mL of LBv2 culture medium, and cultured at 37°C and 200 rpm for 12 hours to obtain an activated Vibrio natrium bacterial solution.

[0047] 2) Sodium selenite tolerance verification

[0048] Sodium selenite and LBv2 medium were used to prepare selenium-containing LBv2 medium with a gradient of sodium selenite concentration of 0, 1, 5, 10, 50, and 100 mM, respectively, and then placed in an anaerobic serum bottle. After aeration for 15 minutes, the anaerobic selenium-containing LBv2 medium was obtained after high-pressure steam sterilization at 121°C for 20 minutes. The activated Vibrio natriuresis bacterial liquid was added to the selenium-containing LBv2 medium using a syringe to make the initial OD600 = 0.1, cultured in a shaker at 30°C, 200 rpm, and 200 μL was taken at regular intervals to measure OD 600 , draw the strain growth curve, such as Figure 1 shown.

[0049] Figure 1 The growth curve showed that when 1, 5, and 10 mM sodium selenite was added, there was no significant difference in the growth of the strain compared with the control group. When 50 and 100 mM sodium selenite was added, the growth of the strain was slightly affected. Overall, within the appropriate sodium selenite concentration range, Vibrio natrii has a strong tolerance to selenite and can be used as a chassis strain for the production of bio-nanoselenium.

[0050] Taking into account the bacterial growth and the production cycle of nano-selenium, a culture medium with a sodium selenite concentration of 10 mM was selected for the biological nano-selenium synthesis experiment.

[0051] 3) Nano-selenium synthesis experiment

[0052] M9 mineral salt medium and sodium selenite were used to prepare a selenium-containing fermentation medium with a sodium selenite concentration of 10 mM. The activated Vibrio natrii strain was added to 50 mL of the selenium-containing fermentation medium to an initial OD of 600 =1.0. Incubate in an anaerobically static incubator at 30°C, take samples regularly, and test the sodium selenite content. Test results Figure 2 shown.

[0053] Figure 2 The selenite concentration during the fermentation process is shown. After 10 hours of anaerobic fermentation, more than 50% of sodium selenite can be reduced; after 58 hours of fermentation, the content of sodium selenite is less than 10%.

[0054] In summary, Vibrio natrii can achieve the reduction of selenite.

[0055] 4) Extraction and purification of nano-selenium: 2 mL of culture broth after fermentation was placed in an EP tube, centrifuged to remove the supernatant, and the bacteria were collected. The bacteria were added to 200 uL of deionized water, and frozen and thawed repeatedly at -80°C and 37°C for 3 times. The ultrasonic cell disruptor was set at 35% power, with a cycle of 2s on and 2s off for 10 min to further disrupt the cells. The bacteria were washed twice with 1.5M Tris-HCl (pH=8.3) containing 1% SDS solution, each time at 15000g, centrifuged for 10 min, n-octanol was added to the sediment, and after vigorous shaking, it was allowed to stand at 4°C for 1 h, the bottom aqueous phase was absorbed, and chloroform, anhydrous ethanol, 75wt% ethanol solution and water were used in turn to wash by centrifugation at 15000g for 10 min, and finally resuspended in 100 μL of ultrapure water to obtain a nano-selenium solution; the nano-selenium solution was dropped on a silicon wafer, and after the water was completely evaporated, the surface morphology of biological nano-selenium was photographed using a scanning electron microscope, as shown in FIG. Figure 3 shown.

[0056] After sodium selenite reduction, the fermented strain was crushed to extract nano-selenium, and the physical characteristics of nano-selenium such as morphology and particle size were observed by scanning electron microscopy. Figure 3 It is shown that the generation of bio-nanoselenium particles can be clearly seen.

[0057] Among them, the method for detecting the content of sodium selenite in the culture solution after fermentation is as follows:

[0058] Use M9 mineral salt culture medium and sodium selenite to prepare standard sample solutions with sodium selenite concentrations of 0, 0.5, 1, 2, 3, and 4 mM, respectively, dilute the standard sample solution and the sample to be tested with deionized water to 10 times the original volume, add 80 μL of 0.5 M hydrochloric acid solution, 40 μL of 0.1 M EDTA-2Na solution, 20 μL of 0.1 M NaF solution, 20 μL of 0.1 M sodium oxalate solution, 250 μL of standard sample solution or sample to be tested, and 100 μL of 0.1% 2,3-diaminonaphthalene anhydrous ethanol solution (add in dark, prepare now). After 40° C. water bath for 40 min, draw 100 μL in a 96-well plate and place in an ELISA reader to detect the absorbance at a wavelength of 377 nm. Then calculate the sodium selenite content of the system according to the absorbance.

[0059] Example 2

[0060] The invention discloses a recombinant Vibrio natrii, wherein the recombinant Vibrio natrii is an engineered strain that overexpresses Vibrio natrii MtrC, MtrA, MtrB, PdsA, and CymA proteins, wherein the amino acid sequence of MtrC is shown in SEQ ID NO: 1, the amino acid sequence of MtrA is shown in SEQ ID NO: 2, the amino acid sequence of MtrB is shown in SEQ ID NO: 3, the amino acid sequence of PdsA is shown in SEQ ID NO: 4, and the amino acid sequence of CymA is shown in SEQ ID NO: 5.

[0061] The method for constructing a recombinant Vibrio natriuresis comprises the following steps:

[0062] 1) Strain culture

[0063] Take out the Escherichia coli Turbo cryopreservation tube and the Vibrio natrii ATCC 14048 cryopreservation tube from the -80°C incubator, thaw and take out 40uL of Escherichia coli liquid and 40uL of Vibrio natrii liquid, respectively, and put them into 4mL LB medium and 4mL LBv2 medium, and culture them at 37°C and 200rpm for 12h to obtain activated Escherichia coli liquid and Vibrio natrii liquid. Escherichia coli is cultured in LB medium, and Vibrio natrii is cultured in LBv2 medium.

[0064] 2) Plasmid construction

[0065] The extracellular electron transfer-related gene cluster of Vibrio natriuresis (gene sequences encoding MtrC, MtrA, MtrB, PdsA, and CymA proteins), IPTG-inducible promoter gene sequence, and p15A plasmid backbone gene sequence (chloramphenicol resistance) were synthesized, and the above gene fragments were connected by Gibson ligation, and then the ligation product was transferred into Escherichia coli Turbo, and positive clones were screened by chloramphenicol. Finally, the plasmids in the positive clones were extracted to obtain the target plasmid p15A-EET, as shown in FIG. Figure 4 The IPTG-inducible promoter gene sequence is shown in SEQ ID NO: 6, and the p15A plasmid backbone gene sequence is shown in SEQ ID NO: 7.

[0066] 3) Construction of engineered strains of Vibrio natriuresis

[0067] Add the activated Vibrio natriuresis bacterial solution into LBv2 medium and culture until OD 600 =0.4, centrifuge to remove the supernatant, collect the bacteria and wash three times with cold 1M sorbitol solution, then add 1M sorbitol solution to obtain competent cells, and package into 100 μL / tube. All operations are carried out at 4°C and stored at -80°C to obtain competent cells of Vibrio natrii.

[0068] 100 μL of Vibrio natrii competent cells were taken out from the -80°C refrigerator and melted in ice, 1 μg of p15A-EET plasmid was mixed with the Vibrio natrii competent cells and transferred to a 1 mm sterile electroporation cuvette, electroporated at 2.5 kV for 5 ms to obtain a mixed solution, and then the mixture was immediately added to 1 mL of LBv2 medium and incubated at 37°C for 1 h, and then the cells were selected on an LBv2 agar plate containing 10 μg / mL chloramphenicol to obtain the positive clone V.NA-p15A-EET strain, i.e., the recombinant Vibrio natrii.

[0069] Comparative Example 1

[0070] The construction method of the recombinant Vibrio natrii is different from that of Example 2, except that the p15A-EET plasmid is replaced by the p15A empty plasmid. The specific steps are as follows:

[0071] The competent cells of Vibrio natrii were taken out from the -80°C refrigerator and melted in ice, 1 μg of p15A plasmid was mixed with 100 μL of competent cells of Vibrio natrii and transferred to a 1 mm sterile electroporation cuvette, and electroporated at 2.5 kV for 5 ms to obtain a mixed solution, and then the mixture was immediately added to 1 mL of LBv2 medium and incubated at 37°C for 1 h, and then the cells were selected on an LBv2 agar plate containing 10 μg / mL chloramphenicol to obtain the positive clone strain V.NA-p15A strain, i.e., the recombinant Vibrio natrii.

[0072] Example 3

[0073] Testing the effect of waste glycerol on the growth of Vibrio natriuresis:

[0074] Waste glycerol was added to LBv2 medium to prepare composite culture medium containing waste glycerol at concentrations of 0, 5 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L, and 30 g / L, respectively. Then, the culture medium was placed in an anaerobic serum bottle, aerated for 15 min, and sterilized by high pressure steam at 121°C for 20 min to obtain an anaerobic composite culture medium. The activated Vibrio natriuresis liquid was added to the anaerobic composite culture medium using a syringe to make the initial OD 600 =0.1, cultured in a shaker at 30°C and 200 rpm. Take 200 μL samples at regular intervals and measure the OD in the culture system using an enzyme-labeled instrument. 600 , draw the strain growth curve, the curve is as follows Figure 5 As shown. Figure 5 It can be seen that the growth of Vibrio natriuresis after adding 5-30g / L crude glycerol is better than that of the zero addition group. Therefore, Vibrio natriuresis has good environmental adaptability, and the complex components in crude glycerol do not affect its normal growth.

[0075] Example 4

[0076] The application of the recombinant Vibrio natrii was to inject the V.NA-p15A-EET strain obtained in Example 2 into M9 mineral salt medium to carry out a sodium selenite reduction experiment, and the specific steps were as follows:

[0077] M9 mineral salt medium [denoted as M9 (G lu)] and sodium selenite were used to prepare a selenium-containing medium with a sodium selenite concentration of 10 mM. The V.NA-p15A-EET strain was added to 50 mL of the selenium-containing medium to an initial OD of 600 =1.0, then add 5 μL of 1 M IPTG aqueous solution, and culture it in an incubator at 30°C to obtain the fermented culture solution. Samples are taken at regular intervals to detect the sodium selenite content.

[0078] Example 5

[0079] The application of the recombinant Vibrio natrii was compared with Example 4, in which the 10 g / L glucose in the M9 mineral salt medium in Example 3 was replaced with 10 g / L glycerol, as a new mineral salt medium, recorded as M9 (G ly), and the remaining raw materials, processes, and parameters were the same as in Example 4.

[0080] Example 6

[0081] The application of recombinant Vibrio natrii was compared with Example 5, in which the 10 g / L pure glycerol in the M9 (G ly) mineral salt medium was replaced with 10 g / L waste glycerol as a new mineral salt medium, recorded as M9 (Crude-G ly), and the other raw materials, processes and parameters were the same as in Example 5.

[0082] Comparative Example 2

[0083] The application of recombinant Vibrio natrii is compared with Example 4, in which the "V.NA-p15A-EET strain obtained in Example 2" in Example 4 is replaced with the "V.NA-p15A strain obtained in Comparative Example 1", and the remaining raw materials, processes and parameters are the same as in Example 4.

[0084] The test results of sodium selenite content in the application of recombinant Vibrio natrii in Example 4, Example 5, Example 6 and Comparative Example 2 are as follows Figure 6 As shown by Figure 6 It can be seen that the V.NA-p15A-EET strain has a higher nano-selenium synthesis rate than the V.NA-p15A strain. In the mineral salt fermentation culture containing 10mM sodium selenite, the overall reduction rate of sodium selenite with glycerol as the carbon source and glucose as the carbon source is equivalent, but in the first 9.5h, the average reduction rate of sodium selenite in the glycerol group was 0.72mM / h, which was 11% higher than the average reduction rate of 0.65mM / h in the glucose group. Waste glycerol was used instead of pure glycerol for nano-selenium fermentation production. In the mineral salt fermentation medium containing 10mM sodium selenite, the biological nano-selenium synthesis rate of V.NA-p15A-EET was not significantly different from that of pure glycerol as an external carbon source.

[0085] In summary, Vibrio natriuresis can efficiently utilize crude glycerol to produce bio-nanoselenium, achieving a win-win situation in economic and environmental benefits.

[0086] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0087] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A recombinant Vibrio natriuresis, characterized in that: The recombinant Vibrio natrii is an engineered strain that overexpresses Vibrio natrii MtrC, MtrA, MtrB, PdsA, and CymA proteins. The amino acid sequence of MtrC is shown in SEQ ID NO: 1, the amino acid sequence of MtrA is shown in SEQ ID NO: 2, the amino acid sequence of MtrB is shown in SEQ ID NO: 3, the amino acid sequence of PdsA is shown in SEQ ID NO: 4, and the amino acid sequence of CymA is shown in SEQ ID NO:

5.

2. The method for constructing a recombinant Vibrio natriuresis according to claim 1, characterized in that: The following steps are involved: 1) preparing activated Escherichia coli bacterial solution and Vibrio natriuresis bacterial solution; 2) synthesizing the gene sequences encoding MtrC, MtrA, MtrB, PdsA, and CymA proteins, the IPTG-inducible promoter gene sequence, and the p15A plasmid backbone gene sequence, and then transferring the ligation products into Escherichia coli Turbo by Gibson ligation, screening positive clones with chloramphenicol, and finally extracting the plasmids from the positive clones to obtain the plasmid p15A-EET; 3) Preparing Vibrio natriuresis competent cells; 4) The plasmid p15A-EET was electroporated into competent cells of Vibrio natriuresis, and positive clones were selected with chloramphenicol to obtain recombinant Vibrio natriuresis.

3. The method for constructing a recombinant Vibrio natriuresis according to claim 2, wherein: The IPTG inducible promoter gene sequence is shown in SEQ ID NO:

6.

4. The method for constructing a recombinant Vibrio natriuresis according to claim 2, characterized in that: The p15A plasmid backbone gene sequence is shown in SEQ ID NO:

7.

5. The method for constructing a recombinant Vibrio natriuresis according to claim 2, characterized in that: Step 4) The specific process is as follows: The competent cells of Vibrio natrii were taken out from the -80°C refrigerator and thawed in ice. The p15A-EET plasmid and the competent cells of Vibrio natrii were mixed and transferred to a sterile electroporation cuvette, electroporated at 2.5 kV for 5 ms, and then transferred to LBv2 medium and incubated at 37°C for 1 h. The recombinant Vibrio natrii was then screened on an LBv2 agar plate containing 10 μg / mL chloramphenicol to obtain recombinant Vibrio natrii.

6. Use of the recombinant Vibrio natriuresis according to claim 1 in synthesizing nano-selenium.

7. A method for synthesizing nano-selenium, characterized in that: The following steps are involved: A selenium-containing medium is prepared by using a mineral salt medium and sodium selenite, and the recombinant Vibrio natrii described in claim 1 is added to the selenium-containing medium to control the initial OD 600 Then, IPTG aqueous solution was added to make the final concentration of IPTG reach 0.1 mM. The culture was statically cultured in an incubator at 30°C to obtain the fermented culture solution, which was centrifuged to collect the bacteria, and then ultrasonically broken after freezing and thawing, washed and resuspended to obtain the nano-selenium solution.

8. The method for synthesizing nano-selenium according to claim 7, characterized in that: Initial OD 600 =1.

0.

9. The method for synthesizing nano-selenium according to claim 7, characterized in that: The components of mineral salt culture medium are: carbon source 10g / L, yeast extract 2g / L, Na2HPO46g / L, NaCl 30g / L, NH4Cl 1g / L, KH2PO43.5g / L, MgSO4·7H2O246.5mg / L, CaCl2·2H2O 14.7mg / L, FeSO4·7H2O27.8mg / L.

10. The method for synthesizing nano-selenium according to claim 8, characterized in that: The carbon source is glucose or glycerol or waste glycerol.

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