CN118028319A - Application of over-expressed oxidoreductase gene PtPrx in improving oxidation resistance of chlorella vulgaris in selenium production process - Google Patents
Application of over-expressed oxidoreductase gene PtPrx in improving oxidation resistance of chlorella vulgaris in selenium production process Download PDFInfo
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- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 title claims abstract description 57
- 229910052711 selenium Inorganic materials 0.000 title claims abstract description 52
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 29
- 240000009108 Chlorella vulgaris Species 0.000 title claims abstract description 12
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- 238000007254 oxidation reaction Methods 0.000 title description 2
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses an application of an over-expressed oxidoreductase gene PtPrx in improving the antioxidant capacity of chlorella vulgaris in the selenium production process, and belongs to the technical field of genetic engineering. Introducing PtPrx genes into a common chlorella genome by adopting an electroporation transformation method to obtain transgenic chlorella; the antioxidant capacity and selenium yield of the transgenic chlorella are better than those of common chlorella; the PtPrx gene sequence is shown as SEQ ID NO. 1. The beneficial effects are that: the invention provides an application of an over-expressed oxidoreductase gene PtPrx in improving the antioxidation capability of common chlorella in the efficient selenium production process under the stimulation of exogenous stimulin, wherein PtPrx5 over-expressed common chlorella is obtained through a genetic engineering technology, and the over-expressed common chlorella can help to improve the enzyme activity of an antioxidation system of algae cells and reduce membrane damage in the efficient selenium production process under the stimulation of exogenous stimulin, so that adverse external stress can be coped with, and the self-protection capability of common chlorella and the yield of selenium element are improved.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to application of an over-expressed oxidoreductase gene PtPrx in improving antioxidation capability of common chlorella in a high-efficiency selenium production process under the stimulation of exogenous stimulin.
Background
Selenium (Se) is a nonmetallic trace element, which is vital to human health and is one of the important chemopreventive agents for cancer. Microalgae is used as a biological carrier, is a potential producer of organic selenium with high bioavailability, can be used as feed to improve the selenium content of animal organisms, and is used as fertilizer to enrich selenium in crops and reduce the health risk of selenium intake by human bodies. However, too high selenium concentration can cause algae cell toxicity, cell lysis and death. Peroxiredoxin (Prx) is a class of peroxiredoxin which eliminates hydrogen peroxide (H 2O2), hydroperoxide (ROOH) and peroxynitroso anion (ONOO-) in plant chloroplasts by redox of the sulfhydryl group of the cysteine residue (Cys). The redox process of Prx relies on the thioredoxin (Trx) system, which is widely conserved in organisms, can receive signals from Fdx and NADPH, and uses thiol to undergo redox reactions to form disulfide bonds to alter protein conformation, the thioredoxin-peroxiredoxin (Trx-Prx) pathway being one of the important pathways for plant clearance of H 2O2 in vivo. Overexpression of the Trx-Prx pathway related genes can improve the antioxidant stress capability of plants to a certain extent.
The Chinese patent application publication No. CN107653255A discloses a cDNA of the Penaeus monodon peroxide reductase gene Prx5, and the nucleic acid sequence of the cDNA is shown as SEQ ID NO. 1. The patent also discloses an expression vector comprising the cDNA. And application of the penaeus monodon peroxide reductase Prx5 recombinant protein in preparing medicines with shrimp antibacterial effect or shrimp immunity enhancing effect. However, the patent does not disclose the application of the oxidoreductase gene PtPrx in improving the antioxidant capacity of chlorella vulgaris in the selenium production process.
Disclosure of Invention
The technical problem to be solved by the invention is how to provide an application of over-expressed PtPrx gene in improving the antioxidation capability of the common chlorella in the efficient selenium production process under the stimulation of the common chlorella exogenous stimulin aiming at the oxidative stress reaction generated in the efficient selenium production process under the stimulation of the common chlorella exogenous stimulin.
The invention solves the technical problems by the following technical means:
The first aspect of the invention provides application of an over-expressed oxidoreductase gene PtPrx in improving the antioxidant capacity of chlorella vulgaris in the selenium production process, wherein the gene sequence of PtPrx is shown as SEQ ID NO. 1.
Preferably, the PtPrx gene is introduced into the chlorella vulgaris genome by electroporation transformation.
The second aspect of the invention provides a method for cultivating transgenic chlorella, which comprises the following steps: introducing PtPrx genes into a common chlorella genome by adopting an electroporation transformation method to obtain transgenic chlorella; the antioxidant capacity and selenium yield of the transgenic chlorella are better than those of common chlorella; the PtPrx gene sequence is shown as SEQ ID NO. 1.
The beneficial effects are that: the invention provides an application of an over-expressed oxidoreductase gene PtPrx in improving the antioxidation capability of common chlorella in the efficient selenium production process under the stimulation of exogenous stimulin, wherein PtPrx5 over-expressed common chlorella is obtained through a genetic engineering technology, and the over-expressed common chlorella can help to improve the enzyme activity of an antioxidation system of algae cells and reduce membrane damage in the efficient selenium production process under the stimulation of exogenous stimulin, so that adverse external stress can be coped with, and the self-protection capability of common chlorella and the yield of selenium element are improved.
In a third aspect of the present invention, there is provided a transgenic chlorella obtained by the above cultivation method.
The fourth aspect of the invention provides the application of the transgenic chlorella obtained by the cultivation method in preparing selenium element.
A fifth aspect of the present invention proposes a method for producing elemental selenium, comprising the steps of: inoculating the transgenic chlorella to a growth culture medium for culturing, adding exogenous hormone with the final concentration of 15 mug/L, adding sodium selenite with the final concentration of 200mg/L, continuously culturing to obtain algae cells with high selenium content, and extracting to obtain selenium element.
Preferably, the growth medium composition is (mg/L)：NaNO3：1500,K2HPO4·3H2O：40,MgSO4·7H2O：75,CaCl2·2H2O：36, ferric ammonium citrate: 6, citric acid ：6,EDTA：1,Na2CO3：20,H3BO3：180,MnCl2·4H2O：1.8,ZnSO4·7H2O：0.22,CuSO4·5H2O：0.0784,NaMoO4·2H2O：0.0378,CoCl2·6H2O：0.05.
Preferably, the culture conditions are: humidity 20%, illumination intensity 3000lx, culture temperature 25+ -1deg.C, and light-dark period 12:12h.
Preferably, the exogenous stimulus is quercetin.
Preferably, the time period for continuing the culture is 7 days or longer.
The invention has the advantages that:
The invention provides an application of an over-expressed oxidoreductase gene PtPrx in improving the antioxidation capability of common chlorella in the efficient selenium production process under the stimulation of exogenous stimulin, wherein PtPrx5 over-expressed common chlorella is obtained through a genetic engineering technology, and the over-expressed common chlorella can help to improve the enzyme activity of an antioxidation system of algae cells and reduce membrane damage in the efficient selenium production process under the stimulation of exogenous stimulin, so that adverse external stress can be coped with, and the self-protection capability of common chlorella and the yield of selenium element are improved.
Drawings
FIG. 1 is a schematic diagram of the construction of an overexpression vector of Chlorella vulgaris in example 1 of the present invention;
FIG. 2 is a solid plate of vector construction in example 1 of the present invention (left: negative control, right: ptPrx ligation transformations);
FIG. 3 is a diagram showing the culture of transformed algae on Ble resistance plates in example 1 of the present invention (left: wild type, right: ptPrx positive selection);
FIG. 4 is a diagram showing the molecular biological analysis of the Ble gene in PCR analysis of different algal strains according to example 1 of the present invention;
FIG. 5 is a graph showing the relative expression levels of the gene PtPrx of interest in different algal strains stimulated by exogenous stimulus according to qPCR analysis in example 1 of the present invention;
FIG. 6 is a graph showing the comparison of PRDX5 content during selenium production by different algal strains according to example 1 of the present invention;
FIG. 7 is a graph showing the comparison of total SOD content during selenium production by different algal strains in example 1 of the present invention;
FIG. 8 is a graph showing the comparison of ROS content during selenium production by different algal strains according to example 1 of the present invention;
FIG. 9 is a graph showing the comparison of selenium accumulation amounts of different algal strains in example 1 of the present invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described in the following in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The test materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Those of skill in the art, without any particular mention of the techniques or conditions, may follow the techniques or conditions described in the literature in this field or follow the product specifications.
The common chlorella Chlorella vulgaris used in the invention is stored for passage in the laboratory.
Example 1: (PtPrx Gene is the gene of Phaeodactylum tricornutum (Phaeodactylum tricornutum CCAP 1055/1), cloned from Phaeodactylum tricornutum, and transferred into Chlorella for expression
1. Constructing an ordinary chlorella overexpression vector;
Taking a proper amount of fresh Phaeodactylum tricornutum Phaeodactylum tricornutum, fully grinding in liquid nitrogen, adding 1mLRNAiso Plus, standing at room temperature for 5min, centrifuging at 12000g at 4 ℃ for 5min, sucking supernatant in a new EP tube, adding 200 mu L of chloroform, shaking and mixing uniformly, standing at room temperature for 5min, centrifuging at 12000g at 4 ℃ for 15min, sucking supernatant in a new EP tube, adding 1mL of isopropanol, standing at room temperature for 10min, centrifuging at 12000g at 4 ℃ for 10min, adding 1mL of 75% ethanol for washing precipitate, centrifuging at 7500g at 4 ℃ for 5min, discarding supernatant, repeatedly washing once, standing at room temperature for 3min for drying precipitate, and dissolving in 35 mu LRNase-free water. Reverse transcription to cDNA was performed using Vazyme HISCRIPT III.sup.1: 1st Strand cDNA Synthesis Kit (+ GDNA WIPER) kit.
According to PtPrx gene sequence (NCBI GeneID: 7203681), i.e.the following SEQ ID NO:1:
ATGAAGTTGCGTCTTCTTGCTTTTGTAGCCACCTCCCTTTCGCTTTGCTCGCTCACGACCAGCTTCTCGTCGATCCATAGTTCTTTCGTTTCTACTGCGCTTTTTGCAGCCCCCGAAACAAGTACCATGCCCGAAGTATCGGTGGGTGACACCATTCCTGACGTAACTTTAACTGAACTTTCCAGTGGCGAAGACAAGCCTGTGGACGTAAAGATTGTCGATCTTATCGCTGGTAAGAAAGTTGCAATCTTTGGAGTTCCGGGCGCCTTCACCCCCGGATGCAGCAAATCGCACCTTCCCTCTTTTATGGAAGCTCAGGAGGAACTCAAGGGGAGAGGCGTCGATATGACTATTTGCGTTGCTACGAATGACGCTTACACTATGGAGGCGTGGGGGCGTACGTCCGGAGGTTCGGATGTAGGTATCCGTTTCCTGGCCGACAACTCTGGTACCCTGACGAAAGAACTGGGTCTGGTCATGGAGACTCCGGTTGGTATCCGCACCAAGCGATTCTCTCTTATCGCTGAAGACGGAAAGGTAACAAAGTATTTTAGCTCGGCCAAGGATAGCTCCGATACTTGGGCTCCGAATGTCTTGTCCGCTCTCTAA,
the PRIMER PREMIER is utilized to design a target gene amplification primer,
PtPrx5-F：ATGAAGTTGCGTCTCCTTGCGTTT(SEQ ID NO:2)
PtPrx5-R：TAGAGCGGACAATACATTCGGTGCC(SEQ ID NO:3)
The synthesis of the principal bioengineering (Shanghai) limited was performed using Vazyme 2 × RAPID TAQ MASTER Mix polymerase and the amplification procedure was: pre-denaturation at 95℃for 3min, 15s at 95℃for 60 s, 15s at 72℃for 35 cycles, complete extension at 72℃for 5min, and storage at 4 ℃.
After the PCR amplification, 1% agarose gel electrophoresis was performed, and after the electrophoresis was completed, the gel containing the target gene fragment was rapidly cut off under an ultraviolet lamp, and the excess gel was removed as much as possible, using VazymeGel DNA Extraction Mini Kit the kit was subjected to gel recovery.
The recovered product is used as template to amplify target gene with JPtPrx5-F：ACAATTACAATCCAGTGGTACC ATGAAGTTGCGTCTCCTTGCGTTT(SEQ ID NO:4)、JPtPrx5-R：GTCCTTGT GTCCAGGTG TAGAGCGGACAATACATTCGGTGCC(SEQ ID NO:5) primer, added with homologous recombination joint, electrophoresed and recovered in gel, and the recovered product is the target gene segment for homologous recombination connection. The vector plasmid is subjected to enzyme digestion and purification by using BstXI, then is subjected to connection transformation with a target gene, a connection transformation kit is Vazyme ClonExpress MultiS One Step Cloning Kit, a connection product is transferred into escherichia coli DH5 alpha, the obtained product is coated on an LB solid plate containing Amp (50 mg/L), is cultured for 16 hours at 37 ℃, single colony shaking bacteria are selected, sequencing is carried out by entrusted to a biological engineering (Shanghai) limited company, and a sequencing result is compared with the sequence on NCBI to extract the recombinant plasmid from the correct bacterial liquid for subsequent experiments.
2. Constructing and positively identifying an over-expressed chlorella vulgaris strain;
Recombinant vector plasmid was extracted using Axygen plasmid miniextraction kit, linearized using restriction enzyme BspQI, and digested product was digested with Vazyme Gel DNAExtraction Mini Kit purification. And respectively setting the plasmid content of 200ng to 800ng, and performing electric shock transformation. After the electric shock recovery of growth, the culture was plated on a solid plate medium containing bleomycin (2.5. Mu.g/mL) and subjected to screening culture for 3 weeks. After single algae grow out of the plate, picking up the monoclonal algae cells in the plate by using a gun head, and placing the monoclonal algae cells into a 50mL triangular flask containing liquid BG11 culture medium of bleomycin (2.5 mug/mL) for 3 weeks. After the algae grow green again, the method comprises the following steps of: 5 transfer to fresh liquid medium containing bleomycin (2.5. Mu.g/mL) and continuing the selection. Screening every 2 weeks, and after 5 times screening, obtaining transformed algae P41 and P51, wherein the growth condition of the algae strain is basically stable, and culturing the algae strain by using a culture medium without bleomycin. Taking a proper amount of algae sample, repeatedly freezing, thawing and cracking for 6 times, and designing exogenous gene bleomycin gene primer Ble-F: TCTGCCGTTCCTGTTCTCA (SEQ ID NO: 6), ble-R: TGCTCCTCAGCCACAAAGT (SEQ ID NO: 7), and carrying out PCR identification of positive algae strains, wherein the target band is about 360bp.
FIG. 1 is a schematic diagram of the construction of an overexpression vector of Chlorella vulgaris in the present example;
FIG. 2 is a solid plate of vector construction in this example (left: negative control, right: ptPrx ligation transformations);
FIG. 3 is a diagram showing the culture of transformed algae on Ble resistance plates in this example (left: wild type, right: ptPrx positive selection);
FIG. 4 is a diagram showing the molecular biological analysis of the Ble gene in different algal strains by PCR in this example.
Extracting single exogenous stimulus (quercetin) and 2d transformed algae strain and wild RNA after selenium production treatment of exogenous stimulus (quercetin), performing reverse transcription into cDNA by using Vazyme HISCRIPT III ALL-in-one RT SuperMix Perfect for qPCR as templates, respectively, and designing fluorescent quantitative primers by PRIMER PREMIER according to the gene sequences of Actin and PtPrx searched by NCBI, thus entrusting the synthesis of biological engineering (Shanghai) limited company. AceQ qPCR SYBR GREEN MASTER Mix kit for quantification. The results were analyzed using the 2- ΔΔct method.
qActin-F：TTGACGGAAGGGCACCA(SEQ ID NO:8)
qActin-R：CACCACCCATAGAATCAAGAAAGAG(SEQ ID NO:9)
qPtPrx5-F：CCAGCTTCTCGTCGATCCATAGTTC(SEQ ID NO:10)
qPtPrx5-F：TACTTCGGGCATGGTACTTGTTTCG(SEQ ID NO:11)
As shown in fig. 5, after 2d treatment with a single exogenous stimulus (quercetin), the PtPrx gene expression level of transformed alga P41 was significantly increased by 1.47 times compared with wild type WT, and the PtPrx gene expression level of transformed alga P51 was significantly increased by 3.18 times compared with wild type WT; after 2d of co-treatment of exogenous hormone (quercetin) and sodium selenite, the PtPrx gene expression quantity of transformed alga P41 is remarkably improved by 3.26 times compared with wild type WT, and the PtPrx gene expression quantity of transformed alga P51 is remarkably improved by 6.95 times compared with wild type WT.
3. Measuring the related index of an antioxidant system in the selenium production process of algae cells under the treatment of exogenous estrogen (quercetin);
expanding the culture of transformed strain and wild strain, inoculating at 1×10 6 cells/mL, culturing for 3 days, adding exogenous hormone (quercetin) with final concentration of 15 μg/L, adding sodium selenite with final concentration of 200mg/L after 1d, continuously culturing for 1d,3d,5d, and 7d after sodium selenite treatment, and sampling.
The growth conditions of the common chlorella are as follows: humidity 20%, illumination intensity 3000lx, culture temperature 25+ -1deg.C, shaking the container 2-3 times per day, and light-dark period 12:12h. The growth medium comprises (mg/L)：NaNO3：1500,K2HPO4·3H2O：40,MgSO4·7H2O：75,CaCl2·2H2O：36, ferric ammonium citrate: 6, citric acid ：6,EDTA：1,Na2CO3：20,H3BO3：180,MnCl2·4H2O：1.8,ZnSO4·7H2O：0.22,CuSO4·5H2O：0.0784,NaMoO4·2H2O：0.0378,CoCl2·6H2O：0.05.
Proper amount of algae culture solution is centrifuged at 4500rpm and 4 ℃ for 10min, and the supernatant is discarded. 200 mu LPBS,1mm glass grinding beads, tissue mill 70HZ, 60s were added and ground four times. And (3) centrifuging 12000g at 4 ℃ for 10min, and taking the supernatant as the extracting solution to be detected. PRDX5 enzyme activity was determined using the plant peroxiredoxin 5 (PRDX 5) elisa kit (Boshen, china). Total SOD enzyme activity was measured using Total Superoxide Dismutase ASSAY KIT WITH WST-8 (Beyotime, china), absorbance at 450nm with an enzyme-labeled instrument, calculated according to the kit instructions.
1ML of algae liquid is taken, 1 mu L of DCFH-DA solution with 10mM dissolved in DMSO is added into the microalgae sample, and the mixture is uniformly mixed and is shaded for dyeing for 30min. Centrifuge at 4500rpm for 10min at 4 ℃. The collected stained algal cells were centrifuged and washed three times with 1mL of fresh medium. 200. Mu.L of fresh medium was added and mixed evenly, and transferred to a flow tube for detection by an on-line machine. Undyed samples were used as controls. Fluorescence was measured using a flow cytometer at excitation wavelength 488nm and emission wavelength 500-600 nm.
As shown in FIG. 6, the overall change trend of the enzyme activity of PRDX5 in the control CK group and the single sodium selenite treated Se group is basically the same, the wild type WT is continuously reduced, and the transformed algae P41 and P51 are in a rising and falling trend. In the single exogenous hormone treatment Q group, the enzyme activity of PRDX5 of two transformed algae P41 and P51 shows a trend of rising and then falling, the enzyme activity of the transformed algae PRDX5 is higher than that of the wild type WT from 3d, the enzyme activity of the transformed algae P41 at 3d and 5d is obviously higher than that of the wild type, and the enzyme activity of the transformed algae P51 at 5d and 7d is obviously higher than that of the wild type, wherein the enzyme activity at 5d is extremely obvious. (transformation of algae P41 and P51 into two strains screened after transgenic operation) exogenous stimulation hormone treatment to produce selenium QSe group, PRDX5 enzyme activity of 3d transformation algae P41 and P51 is obviously higher than that of wild type, and PRDX5 enzyme activity is continuously increased in the whole efficient selenium production process.
As shown in FIG. 7, the total variation trend of SOD enzyme activities of the control CK group and the Se group treated with single sodium selenite is the same, and the SOD enzyme activities reach the maximum value in 3 d. The SOD enzyme activity reaches the maximum when the single exogenous hormone is used for treating the Q group and the SOD enzyme activity of the transformed algae P41 and P51 is slightly higher than that of the wild type WT. The exogenous estrogen selenium producing treatment QSe group had higher PRDX5 enzyme activity at 3d transformed algae P41 and P51 than wild type WT, where transformed algae P51 were of great significance.
As shown in FIG. 8, the control CK group showed substantially the same general trend of change in ROS as the Se-treated group, and the P41, P51-transformed algae P41 at 1d had a higher ROS content than the wild-type WT, and 3d had reached a similar level to that of the wild-type WT, and it was presumed that the PtPrx gene overexpression was able to correspond to the exogenous stimulator treatment and showed an effect against the treatment. The selenium production treatment of exogenous thorns is QSe groups, the ROS content of transformed algae P41 and P51 is obviously lower than that of wild type WT at 3d, and the ROS is effectively reduced by nearly one time.
4. Measuring selenium production of algae cells under the treatment of exogenous hormone (quercetin);
The algae culture solution was centrifuged at 6000rpm at 4℃for 10min, the supernatant was collected, and after 1% (w/v) nitric acid was immobilized, the supernatant was filtered through a 0.22 μm filter, and the residual sodium selenite content was measured using iCAP7000 SERIES (7400 DUO), and the algae cell accumulation selenium content per dry weight was calculated.
As shown in fig. 9, selenium content transformed algae of exogenous spike selenium producing treatment QSe groups remained higher than wild type levels, and transformed algae P41 had significantly higher selenium content at 3d, 5d and 7d than wild type WT. The QSe groups had a lower early accumulated selenium unit dry weight content and continued to rise at 7d far above the Se group compared to the single sodium selenite treated Se group.
According to the invention, ptPrx over-expressed common chlorella engineering alga is obtained by a genetic engineering technology, and under the treatment of exogenous stimulus, the antioxidant enzyme activity and selenium accumulation amount of the high-efficiency selenium production process of alga cells are improved, so that the oxidative stress of the selenium production process is dealt with, and the selenium accumulation capacity of common chlorella is improved.
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. The application of the over-expressed oxidoreductase gene PtPrx in improving the antioxidant capacity of chlorella vulgaris in the selenium production process is characterized in that the gene sequence of PtPrx5 is shown as SEQ ID NO. 1.
2. The use according to claim 1, wherein the PtPrx gene is introduced into the chlorella vulgaris genome by electroporation transformation.
3. The method for cultivating the transgenic chlorella is characterized by comprising the following steps of: introducing PtPrx genes into a common chlorella genome by adopting an electroporation transformation method to obtain transgenic chlorella; the antioxidant capacity and selenium yield of the transgenic chlorella are better than those of common chlorella; the PtPrx gene sequence is shown as SEQ ID NO. 1.
4. A transgenic Chlorella obtained by the cultivation method as claimed in claim 3.
5. The use of the transgenic chlorella as claimed in claim 4 for preparing selenium element.
6. A method for producing elemental selenium comprising the steps of: inoculating the transgenic chlorella to the growth medium for culturing, adding exogenous hormone with the final concentration of 15 mug/L, adding sodium selenite with the final concentration of 200mg/L, continuously culturing to obtain the algae cells with high selenium content, and extracting to obtain selenium element.
7. The method of claim 6, wherein the growth medium composition is (mg/L)：NaNO3：1500,K2HPO4·3H2O：40,MgSO4·7H2O：75,CaCl2·2H2O：36, ferric ammonium citrate: 6, citric acid ：6,EDTA：1,Na2CO3：20,H3BO3：180,MnCl2·4H2O：1.8,ZnSO4·7H2O：0.22,CuSO4·5H2O：0.0784,NaMoO4·2H2O：0.0378,CoCl2·6H2O：0.05.
8. The method for producing elemental selenium according to claim 6, wherein the culturing conditions are: humidity 20%, illumination intensity 3000lx, culture temperature 25+ -1deg.C, and light-dark period 12:12h.
9. The method of producing elemental selenium according to claim 6, wherein the exogenous estrogen is quercetin.
10. The method for producing elemental selenium according to claim 6, wherein the period of time for continuing the culture is 7 days or longer.
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