CN116656586A - Method for applying metallothionein surface display bacteria in prevention and treatment of ligusticum chuanxiong hort cadmium pollution - Google Patents
Method for applying metallothionein surface display bacteria in prevention and treatment of ligusticum chuanxiong hort cadmium pollution Download PDFInfo
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- CN116656586A CN116656586A CN202310258793.6A CN202310258793A CN116656586A CN 116656586 A CN116656586 A CN 116656586A CN 202310258793 A CN202310258793 A CN 202310258793A CN 116656586 A CN116656586 A CN 116656586A
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- 108090000157 Metallothionein Proteins 0.000 title claims abstract description 55
- 102000003792 Metallothionein Human genes 0.000 title claims abstract description 54
- DIGQNXIGRZPYDK-WKSCXVIASA-N (2R)-6-amino-2-[[2-[[(2S)-2-[[2-[[(2R)-2-[[(2S)-2-[[(2R,3S)-2-[[2-[[(2S)-2-[[2-[[(2S)-2-[[(2S)-2-[[(2R)-2-[[(2S,3S)-2-[[(2R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[2-[[(2S)-2-[[(2R)-2-[[2-[[2-[[2-[(2-amino-1-hydroxyethylidene)amino]-3-carboxy-1-hydroxypropylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxybutylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1,5-dihydroxy-5-iminopentylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxybutylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxyethylidene]amino]hexanoic acid Chemical compound C[C@@H]([C@@H](C(=N[C@@H](CS)C(=N[C@@H](C)C(=N[C@@H](CO)C(=NCC(=N[C@@H](CCC(=N)O)C(=NC(CS)C(=N[C@H]([C@H](C)O)C(=N[C@H](CS)C(=N[C@H](CO)C(=NCC(=N[C@H](CS)C(=NCC(=N[C@H](CCCCN)C(=O)O)O)O)O)O)O)O)O)O)O)O)O)O)O)N=C([C@H](CS)N=C([C@H](CO)N=C([C@H](CO)N=C([C@H](C)N=C(CN=C([C@H](CO)N=C([C@H](CS)N=C(CN=C(C(CS)N=C(C(CC(=O)O)N=C(CN)O)O)O)O)O)O)O)O)O)O)O)O DIGQNXIGRZPYDK-WKSCXVIASA-N 0.000 title claims abstract description 53
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- 229910052793 cadmium Inorganic materials 0.000 title claims abstract description 28
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- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
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- 230000009286 beneficial effect Effects 0.000 description 2
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 2
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- 229910052698 phosphorus Inorganic materials 0.000 description 2
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- 239000011591 potassium Substances 0.000 description 2
- 229910052700 potassium Inorganic materials 0.000 description 2
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- QJZYHAIUNVAGQP-UHFFFAOYSA-N 3-nitrobicyclo[2.2.1]hept-5-ene-2,3-dicarboxylic acid Chemical compound C1C2C=CC1C(C(=O)O)C2(C(O)=O)[N+]([O-])=O QJZYHAIUNVAGQP-UHFFFAOYSA-N 0.000 description 1
- 101100295756 Acinetobacter baumannii (strain ATCC 19606 / DSM 30007 / JCM 6841 / CCUG 19606 / CIP 70.34 / NBRC 109757 / NCIMB 12457 / NCTC 12156 / 81) omp38 gene Proteins 0.000 description 1
- 206010027439 Metal poisoning Diseases 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- 241001619461 Poria <basidiomycete fungus> Species 0.000 description 1
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- 241000347485 Silurus glanis Species 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
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- WLZRMCYVCSSEQC-UHFFFAOYSA-N cadmium(2+) Chemical compound [Cd+2] WLZRMCYVCSSEQC-UHFFFAOYSA-N 0.000 description 1
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- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
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- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
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- 101150087557 omcB gene Proteins 0.000 description 1
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- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
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- NNMHYFLPFNGQFZ-UHFFFAOYSA-M sodium polyacrylate Chemical compound [Na+].[O-]C(=O)C=C NNMHYFLPFNGQFZ-UHFFFAOYSA-M 0.000 description 1
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- 239000013598 vector Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/825—Metallothioneins
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P21/00—Plant growth regulators
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
- C07K2319/43—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a FLAG-tag
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Plant Pathology (AREA)
- Biotechnology (AREA)
- Environmental Sciences (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Pest Control & Pesticides (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Gastroenterology & Hepatology (AREA)
- Mycology (AREA)
- Soil Sciences (AREA)
- Environmental & Geological Engineering (AREA)
- Agronomy & Crop Science (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Virology (AREA)
- Genetics & Genomics (AREA)
- Dentistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Botany (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for applying metallothionein surface display bacteria in prevention and treatment of ligusticum wallichii cadmium pollution, and particularly relates to the field of genetic engineering. The surface display bacteria contains a fusion expression vector of the metallothionein, and the fusion expression vector carries the gene sequence of the metallothionein, so that the metallothionein can be displayed on the surface of the bacteria. The invention uses bioengineering means to display the cell surface of metallothionein, and is used in environment of treating cadmium pollution of Ligusticum chuanxiong, and has the following advantages: (1) has the capability of improving the binding capacity of metal ions; (2) Among various heavy metals, metallothionein exhibits excellent cadmium adsorption ability.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a method for applying metallothionein surface display bacteria in prevention and treatment of cadmium pollution of ligusticum wallichii.
Background
Ligusticum wallichii is used as a medicinal material commonly used in clinic, and in recent years, the problem of exceeding heavy metal, especially cadmium, is faced. Cadmium exceeding the standard affects the export trade of Ligusticum chuanxiong, and cadmium is a toxic heavy metal element and also has great harm to human body. Therefore, it is necessary to solve the problem of cadmium overstepping of Ligusticum chuanxiong.
At present, the treatment method for the cadmium content of the ligusticum wallichii is less, and the improvement on the cultivation method is mainly focused, such as: mixing humic acid and sodium polyacrylate with surface soil, cultivating rhizoma Ligustici Chuanxiong (Zhou Jin, 2015), mixing quicklime and potassium dihydrogen phosphate with surface soil, and cultivating rhizoma Ligustici Chuanxiong (Li Min, 2015).
The method for repairing heavy metal pollution by utilizing microorganisms is a green, environment-friendly and efficient method, and common microorganisms also have a certain repression effect on the enrichment of ligusticum chuanxiong hort cadmium, but the enrichment of wild microorganism cells on metals is limited, and the selectivity on heavy metal ions is not strong. Therefore, the microorganism is treated by adopting the gene recombination technology, so that the heavy metal inhibition effect of the microorganism can be improved in terms of adsorption effect and heavy metal specificity. The cell surface display technology is characterized in that a foreign protein or polypeptide and a misidentification protein are fused and expressed through a genetic engineering technology, and are fixed on the cell surface in a fusion protein mode, and the foreign protein or polypeptide has independent structural characteristics and bioactivity. Therefore, proteins with heavy metal binding capacity are selected for microorganism cell surface display, and the heavy metal adsorption capacity of the product can be enhanced.
Metallothionein (MT) is a protein with low molecular weight, high cysteine (mercapto) content, zero aromatic oxyacid content and good thermal stability, exists in organisms, is secreted by cells in an excessive amount of metal ions, and is combined with the metal ions, so that the purpose of detoxification is achieved. In addition, the metallothionein also has certain advantages in the aspects of free radical removal, anti-inflammatory, anti-radiation and the like. Based on these capabilities of metallothionein, it is often used in the preparation of pharmaceutical formulations, such as the manufacture of heavy metal antidotes, as an adjunct to tumor therapy to reduce cytotoxicity of heavy metal components in tumor drugs (Yu Long et al, 2002); or preparing health products containing metal sulfur proteins for diabetes and complications thereof (Zhou Guojie, etc., 2003); or the use of metallothionein antagonism to treat intestinal inflammation (De Vos Martine et al 2012). It is also commonly used in skin care preparation processes, such as adding metallothionein to cosmetics, stimulating organism resistance to free radicals (wels Thomas, 2021), or using Saccharomyces cerevisiae to express long-acting recombinant metallothionein, enhancing the efficacy of cosmetics in degrading heavy metal poisoning, scavenging free radicals, activating zinc regulatory proteins (Zhao Jun, 2021).
Due to the excellent characteristics of metallothionein in terms of heavy metal binding, antioxidation stress and the like, the metallothionein is also gradually applied to the field of environmental protection in recent years, particularly the field of heavy metal pollution control. For example, the extracted and purified metallothionein is used directly for heavy metal removal, or the organism is stimulated to produce metallothionein for heavy metal removal (Roger AAce et al, 2007; huang Zhiyong et al, 2018). Multiple studies have shown that metallothionein for Cd 2+ 、Cu 2+ 、Cr 6+ The adsorption removal of various heavy metal ions has better effect, and meanwhile, a large number of microorganisms secreting metallothionein can excrete Cd 2+ Is also more tolerant (Cao Qiufen, 2010; mao Xinfang, 2019).
Although metallothionein can be produced after organism stress, the expression position of the metallothionein has a great influence on heavy metal adsorption capacity, and when expressed outside cells, the metallothionein has a great influence on Cd 2+ The adsorption rate of (a) can reach several times of intracellular expression. Therefore, the invention applies the cell surface display technology to display and express metallothionein genes outside recipient bacteria cells so as to improve Cd of microorganisms 2+ Adsorption capacity.
Disclosure of Invention
In view of the above problems, it is an object of the present invention to provide a surface-displaying bacterium, wherein the surface-displaying bacterium comprises a fusion expression vector of a metallothionein, and the fusion expression vector carries the gene sequence of the metallothionein, so that the metallothionein can be displayed on the surface of the bacterium.
Further, the metallothionein is Saccharomyces cerevisiae metallothionein.
Further, the metallothionein comprises or has the gene sequence of SEQ ID NO. 1.
Further, the fusion expression vector is composed of two parts of escherichia coli lipoprotein (Lpp) and outer membrane protein a (OmpA).
Further, the surface display bacteria is E.coli DH5 alpha.
A method of producing metallothionein, comprising the steps of:
s1, constructing a fusion expression vector of metallothionein;
s2, transforming surface display bacteria by using the fusion expression vector;
furthermore, the surface display bacteria are applied to the prevention and treatment of the cadmium pollution of ligusticum wallichii.
The beneficial effects of the invention are as follows:
the invention uses bioengineering means to display the cell surface of metallothionein, and is used in environment of treating cadmium pollution of Ligusticum chuanxiong, and has the following advantages: (1) The metal ion binding capacity is achieved, and the binding effect can be obviously improved when the metal ion binding capacity is expressed outside cells; (2) Among various heavy metals, metallothionein exhibits excellent cadmium adsorption capacity; (3) The metallothionein has other characteristics of oxidation resistance, inflammation resistance and the like, and has certain promotion effect on the growth of the ligusticum chuanxiong under the condition of being cultivated together with the ligusticum chuanxiong. Therefore, compared with the existing Ligusticum wallichii cadmium enrichment and suppression technology, the product grows in the rhizomes of Ligusticum wallichii, and can suppress enrichment of the rhizomes of Ligusticum wallichii to cadmium to a certain extent. Moreover, the preparation and application processes of the bacterial liquid are relatively more convenient and simpler, the harm of secondary pollution to soil is avoided temporarily, and partial nutritional metabolites of microorganisms are beneficial to the growth of ligusticum wallichii plants. Proved by field experiments, compared with the Ligusticum wallichii which is co-cultivated by adding the metallothionein surface display bacterial liquid and not adding any cadmium repressor, the cadmium content of the rhizome part is reduced by 16.7%; compared with rhizoma Ligustici Chuanxiong with chemical passivating agent, the cadmium content of rhizome is reduced by 13.9%. Meanwhile, after the bacterial liquid is added, the contents of organic matters such as nitrogen, phosphorus, potassium and the like in the rhizosphere soil of the ligusticum chuanxiong are increased to different degrees, the plant height of the ligusticum chuanxiong is increased by 5%, and the fresh weight is also increased by 6%.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following brief description of the drawings of the embodiments will make it apparent that the drawings in the following description relate only to some embodiments of the present invention and are not limiting of the present invention.
FIG. 1 is a schematic diagram showing the PCR verification of bacterial liquid of the recombinant plasmid pBSD-LCF of the present invention;
FIG. 2 is a diagram showing verification of expression of the fusion protein Lpp-OmpA-MT-Flag of the present invention;
FIG. 3 is a Cd of recombinant strain E.coli DH 5. Alpha (pBSD-LCF) of the present invention 2+ Tolerance verification graph;
FIG. 4 is a graph showing the growth of recombinant strain E.coli DH 5. Alpha (pBSD-LCF) of the present invention;
FIG. 5 is a scanning electron microscope image of recombinant strains of the present invention after adsorption in liquids at concentrations of 0mg/L and 100 mg/L;
FIG. 6 is a graph showing cadmium content of Ligusticum wallichii for 5 months and 7 months according to the present invention;
FIG. 7 is a graph (from left to right) showing plant height, fresh weight and dry weight of Ligusticum chuanxiong Hort
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. All other embodiments, which can be made by a person skilled in the art without creative efforts, based on the described embodiments of the present invention fall within the protection scope of the present invention.
The invention will be further described with reference to the drawings and examples.
The invention provides a surface display bacterium for expressing metallothionein, which comprises a fusion expression vector of the metallothionein, wherein the fusion expression vector carries a gene sequence of the metallothionein, so that the metallothionein can be displayed on the surface of the bacterium; the metallothionein is Saccharomyces cerevisiae metallothionein; the metallothionein comprises or has the gene sequence of SEQ ID NO. 1; the fusion expression vector consists of two parts, namely escherichia coli lipoprotein (Lpp) and outer membrane protein A (ompA); the surface display bacteria is E.collDH5α.
It is another object of the present invention to provide a method for producing metallothionein, comprising the steps of:
1. constructing a fusion expression vector of metallothionein;
2. transforming a surface display bacterium with the fusion expression vector;
the invention also provides application of the surface display bacteria in prevention and treatment of cadmium pollution of ligusticum wallichii.
Example 1
Construction of recombinant plasmid pBSD-LCF
Plasmid pUC57-Lpp-OmpA-CUP1-Flag was synthesized according to the sequence published in SEQ ID NO. 1, and the Lpp-OmpA-CUP1-Flag gene was amplified by PCR. After cleavage with NdeI and EcoRI, the PCR amplified Lpp-OmpA-CUP1-Flag was ligated with vector pBSD, yielding plasmid pBSD-LCF.
Example 2
Transformation of recombinant Strain E.coli DH 5. Alpha (pBSD-LCF)
The recombinant plasmid was transformed into E.coli DH 5. Alpha. By heat shock transformation. After transformation, recombinant strain E.coli DH 5. Alpha (pBSD-LCF) was obtained, and the bacterial solution was spread on LB solid medium containing 100. Mu.g/mL chloramphenicol and cultured at 37℃for 16 hours. Single colonies were inoculated into LB liquid medium containing 100. Mu.g/mL chloramphenicol, and cultured at 150rpm and 37 ℃.
Recombinant plasmid pBSD-LCF for surface display was extracted from recombinant E.coli and its correct structure was verified using double enzyme digestion, DNA fragment sequencing and bacterial liquid PCR.
The result of the bacterial liquid PCR verification experiment of the recombinant plasmid pBSD-LCF is shown in figure 1, and lane M is DL2000 DNA Marker. Lanes 1-4 are CUP1-Flag fragments (350 bp) obtained by bacterial liquid PCR. Lanes 5-8 are Lpp-OmpA-CUP1-Flag fragments (850 bp) obtained by bacterial liquid PCR. The result of enzyme digestion electrophoresis and bacterial liquid PCR shows that the recombinant plasmid is constructed successfully.
Example 3
Expression verification of fusion protein Lpp-OmpA-MT-Flag
1. Flow cytometry analysis
FITC-labeled anti-Flag antibody was added to E.coli DH 5. Alpha (pBSD-LCF) protoplasts containing recombinant plasmid pBSD-LCF and flow cytometry analysis was performed after 1 hour of incubation. The experimental results are shown in FIG. 2a, which shows that the target protein MT is successfully expressed and anchored on the bacterial surface by the surface display system Lpp-OmpA.
2. Western blot analysis of recombinant bacteria E.coli DH 5. Alpha (pBSD-LCF)
Western Blot analysis was performed on all lysates of E.coli DH 5. Alpha. Containing pBSD-LCF, with untransformed host bacteria E.coli DH 5. Alpha. As negative control. As shown in FIG. 2b, the "+" represents fusion protein Lpp-OmpA-MT-Flag in recombinant bacteria E.coli DH 5. Alpha (pBSD-LCF), and "-" represents E.coli DH 5. Alpha. Without recombinant plasmid as negative control. Bacterial cells were broken up and centrifuged to collect the different fractions, and the total cell lysate was assayed with anti-Flag-HPR diluted 1:1000, wherein: 1 is an inner/outer membrane fraction, 2 is a soluble fraction, and 3 is a supernatant fraction. The arrow indicates the position (35 kDa) of the fusion protein Lpp-OmpA-MT-Flag. The results showed that E.coli DH 5. Alpha. Carrying the recombinant plasmid pBSD-LCF (pBSD-LCF) did synthesize the fusion protein MT-Flag, and that the fusion protein MT-Fag was successfully displayed on the strain surface by LPP-OmpA cell surface display system while maintaining its specificity.
Example 4
Cd of recombinant strain E.coli DH 5. Alpha (pBSD-LCF) 2+ Tolerance verification
Inoculating recombinant strain E.coli DH5 alpha (pBSD-LCF) into strain containing different concentration Cd 2+ To determine the tolerance of cells to cadmium toxicity. Cd (cadmium sulfide) 2+ Is set at 50, 75, 100, 125, 150, 175 and 200mg/L. Coli DH 5. Alpha. Strain served as control. After incubation at 150rpm and 37℃for 36 hours, the OD of each group of bacteria was determined 600 。
The experimental results are shown in FIG. 3, and the E.coli DH5 alpha without recombinant plasmid is used as a control, cd 2+ The Minimum Inhibitory Concentration (MIC) for this was 50ml/L, cd 2+ For recombinant strain EThe minimum inhibitory concentration of the coli DH5 alpha (pBSD-LCF) is 150ml/L.
Example 5
Growth curve of recombinant strain E.coli DH5 alpha (pBSD-LCF) and Cd 2+ Adsorption experiment
Firstly, taking E.coli DH5 alpha without recombinant plasmid as a control, respectively inoculating the recombinant strain and the control strain into the initial Cd 2+ In medium at a concentration of 0 and 100 mg/L. Then for Cd in the medium with initial concentration of 100mg/L 2+ The concentration was monitored and recombinant strain was in Cd 2+ And (3) carrying out scanning electron microscope result analysis after adsorption in the liquid with the concentration of 0 and 100 mg/L.
The experimental results are shown in FIG. 4, which shows that Cd is not present 2+ Under the conditions, the recombinant strain and the control strain have the same growth curve, and the original Cd is the same 2+ The growth of the control strain is obviously inhibited under the condition of 100mg/L concentration, and along with the growth of the recombinant strain, cd in the culture medium 2+ The concentration began to drop rapidly and the recombinant strain was responsible for the Cd in the medium 2+ The highest adsorption rate of the catalyst can reach 95.2 percent; the experimental result is shown in figure 5, and the scanning electron microscope can be used for observing that the surface of the strain is obviously enriched with cadmium ions after the strain is adsorbed in 100mg/L of bacterial liquid.
Example 6
Application of recombinant strain E.coli DH5 alpha (pBSD-LCF) in planting Ligusticum wallichii in field
1. Inoculating engineering bacteria into LB culture medium, and culturing and increasing the bacteria for 24h at 170 r/m.
2. When the bacterial liquid is applied, the bacterial liquid volume is as follows: soil mass was 100ml:3kg, applying the bacterial liquid into soil, turning the soil and mixing uniformly. The chemical passivating agent is applied in equal amounts.
3. Normally sowing rhizoma Ligustici Chuanxiong, wherein the seed of rhizoma Ligustici Chuanxiong is seed of rhizoma Ligustici Chuanxiong and Poria in Wenchuan county.
4. Digging up the whole plant during sampling, and measuring the plant height of the ligusticum chuanxiong hort plant by using a tape measure; removing non-medicinal parts, cleaning rapidly, air drying, and measuring fresh weight of rhizoma Ligustici Chuanxiong; and (5) putting the mixture into an electrothermal blowing type drying oven to be dried to constant weight at 50 ℃, taking out the mixture, cooling the mixture, and weighing the mixture.
5. The method for measuring the cadmium content of the ligusticum wallichii adopts a graphite furnace atomic absorption method, and a sample is subjected to pretreatment by adopting a microwave digestion method. Namely, accurately weighing about 0.1g of sample powder, putting the sample powder into a digestion inner tank, adding 0ml of nitric acid, selecting a microwave digestion temperature main control mode, setting the digestion temperature program to 130 ℃, 10min,150 ℃, 5min,180 ℃ and 10min, and setting the digestion power according to the number of the digestion tanks. After microwave digestion, transferring the digestion liquid to a conical flask, heating by an electric furnace to remove acid, transferring to a 100m volumetric flask, and fixing the volume to obtain a sample solution, and waiting for on-machine measurement. The instrument requires wavelength 228.8nm, slit 0.5nm, lamp current 0.3mA, zeeman correction. The temperature programming of the graphite furnace is that the graphite furnace is dried at 95 ℃ for 40S, 160 ℃ for 10S, ashed at 550 ℃ for 9S and atomized at 1800 ℃ for 3S; the concentration of the mother solution of the standard solution is 1.0ug/L, the instrument automatically dilutes the mother solution into each concentration of the standard series, and the standard series range is set to be 0.2, 0.4, 0.8, 1.0ug/L and 1.5ug/L according to the concentration of the sample. The matrix modifier is 1.0g/L phosphoric acid.
As shown in FIG. 6, the cadmium content of rhizome parts of Ligusticum wallichii co-cultivated with the bacterial liquid of the metallothionein-added surface display is reduced by 16.7% compared with that of Ligusticum wallichii without any cadmium repressor; compared with rhizoma Ligustici Chuanxiong with chemical passivating agent, the cadmium content of rhizome is reduced by 13.9%. As shown in FIG. 7, the contents of organic nitrogen, phosphorus, potassium and the like in the rhizosphere soil of the Ligusticum wallichii plant are increased to different degrees after the bacterial liquid is added, the plant height of the Ligusticum wallichii plant is increased by 5%, and the fresh weight is also increased by 6%.
The present invention is not limited to the above-mentioned embodiments, but is intended to be limited to the following embodiments, and any modifications, equivalents and modifications can be made to the above-mentioned embodiments without departing from the scope of the invention.
Claims (7)
1. The surface display bacterium is characterized by comprising a fusion expression vector of metallothionein, wherein the fusion expression vector carries a gene sequence of the metallothionein, and the metallothionein can be displayed on the surface of the bacterium.
2. The surface displaying bacterium of claim 1, wherein said metallothionein is Saccharomyces cerevisiae metallothionein.
3. The surface displaying bacterium according to claim 1, wherein said metallothionein-encoding nucleic acid molecule has the nucleotide sequence of SEQ ID NO. 1.
4. The surface display bacterium according to claim 1, wherein said fusion expression vector is composed of two parts of E.coli lipoprotein, lpp and outer membrane protein A (OmpA).
5. The surface displaying bacterium according to claim 1, wherein the surface displaying bacterium is e.coli DH5 a.
6. A method for producing metallothionein comprising the steps of:
s1, constructing a fusion expression vector of metallothionein;
s2, transforming the surface display bacteria by using the fusion expression vector.
7. The surface display bacterium according to any one of claims 1 to 6, wherein the use of the surface display bacterium in the prevention and treatment of cadmium pollution of ligusticum chuanxiong hort.
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