CN111893099B - Azo-reductase BslM _2044, coding gene thereof, recombinant bacterium containing gene and application thereof - Google Patents
Azo-reductase BslM _2044, coding gene thereof, recombinant bacterium containing gene and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of genetic engineering, in particular to azoreductase BsLM _2044, a coding gene thereof, a recombinant bacterium containing the gene and application thereof. The amino acid sequence of the azoreductase BsLM _2044 is shown in SEQ ID NO. 1. The nucleotide sequence of the BslM _2044 gene encoding the BslM _2044 is shown in SEQ ID No. 2. The recombinant strain is obtained by introducing BslM _2044 gene into an Escherichia coli receptor strain through an expression vector pET28 a-azoR. The result of the IPTG induced expression under the action of molybdenum stress shows that the azo reductase BsLM _2044 is actually induced and expressed, and Mo in the culture medium is used6+The molybdenum blue is reduced to a low-valence state and shows reduction characters, so the azo reductase and the recombinant bacteria have good application prospects in molybdenum pollution remediation.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to azoreductase BsLM _2044, a coding gene thereof, a recombinant bacterium containing the gene and application thereof.
Background
Bioremediation (Bioremediation) is a technique for removing or neutralizing pollutants in a polluted site by decomposing and converting harmful substances into less toxic or nontoxic treatment processes using the metabolism of organisms themselves. The bioremediation technology accelerates the degradation speed of pollutants, remarkably reduces the concentration of pollutants, does not cause secondary pollution, has little interference to the surrounding environment, and has high efficiency, safety, operability and low cost on the basis of not damaging organic matters of soil. In addition, the direct contact between the operator and pollutants is reduced, and the human body is not damaged.
The molybdenum metal has wide application in the fields of metallurgy, chemistry, agriculture and biology, is often used for manufacturing alloy components, corrosion-resistant parts, additives, lubricants, pigments and the like, and has high economic benefit. But at the same time, the molybdenum can cause serious pollution to the environment in the process of mining, smelting and processing, and the content of molybdenum in soil and water is continuously increased. When a human or an animal is exposed to a high-concentration molybdenum environment, the blood plasma uric acid level is increased, so that gout is caused; animal experiments show that the molybdenum content exceeding 10ppm can seriously affect the digestion of most ruminants; even low levels of molybdenum can be severely toxic to mouse embryos and fish sperm. Therefore, how to treat the molybdenum metal in the polluted site becomes an environmental protection problem to be solved urgently.
The microorganism can reduce the toxicity of heavy metal to the microorganism through biological adsorption, reduction, transformation, a transport system and the like, realize the purpose of detoxification, and live in the environment containing metal ions by virtue of the stress resistance of the microorganism. Some heavy metal elements in soil are generally low in solubility when existing in a high valence state and are more easily migrated when existing in a low valence state, and the valence state of the heavy metal elements can be reduced by the redox action of some microorganisms on the heavy metal elements. Therefore, bioremediation of heavy metal contamination can be deeply performed using microorganisms having such characteristics. It is reported that some of the currently isolated ferrous oxide thiobacillus, sulfolobus, etc. can reduce hexavalent molybdenum ions into molybdenum blue, so that free molybdate ions form a multiphase colloidal complex, thereby reducing the residue of molybdenum ions in a polluted site and reducing the biological toxicity of the molybdenum ions. However, the bioremediation effect of the strain is not ideal, and researches show that under the culture condition, the oxidation capability of the thiobacillus ferrooxidans can be obviously inhibited when the molybdenum ion reaches 1mmol/L, so that the strain has great limitation in practical application.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides azoreductase BsLM _2044, a coding gene thereof, a recombinant bacterium containing the gene and application thereof.
The invention adopts the following technical scheme:
the application of azoreductase BsLM _2044 in preparing a preparation for repairing molybdenum pollution is disclosed, wherein the amino acid sequence of BsLM _2044 is shown in SEQ ID NO. 1.
Further, the nucleotide sequence of BslM _2044 gene encoding BslM _2044 is shown in SEQ ID No. 2.
The recombinant bacterium is recombinant escherichia coli containing the BslM _2044 gene, and the BslM _2044 gene is introduced into an escherichia coli receptor bacterium through an expression vector pET28a-azoR to obtain the recombinant escherichia coli.
Further, the recipient bacterium is Escherichia coli BL21(DE 3).
Furthermore, the introduction method is chemical conversion, in particular to CaCl2The method comprises preparing BL21(DE3) competent cells, adding 10 μ L recombinant plasmid into 100 μ L competent cells, standing on ice for 30min, heat shocking for 90s, ice-cooling for 2min, adding 900mL LB Broth for resuscitation for 1h, spreading on LB plate containing kan resistance, and incubating for 12-16 h.
The invention also provides application of the recombinant bacterium in repairing molybdenum pollution.
Compared with the prior art, the invention has the following beneficial effects:
the invention introduces a recombinant vector containing BslM _2044 gene into escherichia coli BL21(DE3) for heterologous expression, and when the obtained recombinant escherichia coli grows in a solid LMP (low-phosphorus culture medium) culture medium containing sodium molybdate, the thalli is blue, which shows that the recombinant strain can lead Mo in the culture medium to be treated6+Reduced to molybdenum blue in a low valence state, and shows a reduction character. IPTG induction shows that the expression level of azoreductase BslM _2044 is gradually increased along with the extension of induction time. Therefore, the azoreductase BslM _2044 and the recombinant bacterium containing BslM _2044 gene have good application prospects in molybdenum pollution remediation.
Drawings
FIG. 1 is a plasmid map of expression vector pET28a (+).
FIG. 2 shows the molybdenum tolerance of recombinant E.coli BsLM _2044-BL21(DE3) and control strain E.coli BL21(DE3) under molybdenum stress.
FIG. 3 shows the phenotype of recombinant E.coli BsLM _2044-BL21(DE3) grown in solid LMP containing sodium molybdate.
FIG. 4 shows the expression of azoreductase in recombinant E.coli BsLM _2044-BL21(DE3) detected by Western Blot.
FIG. 5 shows the expression of azoreductase in recombinant E.coli BslM _2044-BL21(DE3) detected by SDS-PAGE.
Detailed Description
The invention is described in detail below with reference to the figures and the specific embodiments, but the invention should not be construed as being limited thereto. The technical means used in the following examples are conventional means well known to those skilled in the art, and materials, reagents and the like used in the following examples can be commercially available unless otherwise specified.
Example 1
Screening for Azo-reductase
LM4-2((Bacillus subtilis LM4-2, the strain is preserved in China general microbiological culture Committee center with the preservation number of CGMCC 1.15213.) and is inoculated in an MS culture medium and an MS culture medium containing 80,000ppm of sodium molybdate according to the inoculation amount of 1% and 5%, the thalli are collected for 8 hours and 32 hours respectively, a transcriptome of the thalli is determined after the collection is finished, the gene expression difference under the stress action of the sodium molybdate is detected, 11 genes are selected to verify transcriptome data by utilizing qPCR, the result shows that the transcriptome data is consistent with the transcriptome data, the transcriptome data shows that the BsLM 2044 gene expression (encoding azoreductase) is extremely obviously upregulated, and therefore the BsLM 2044 is selected to carry out heterologous expression.
Example 2
Construction of recombinant bacterium
(1) Cloning of azo reductase Gene
According to the sequencing result, an upstream primer and a downstream primer of BslM _2044 gene (627bp) are designed, an enzyme cutting site is added at the 5' end of the primer, and the upstream primer BslM _2044F (shown in SEQ ID NO. 3) and the downstream primer BslM _2044R (shown in SEQ ID NO. 4) are both synthesized by Shanghai biological Limited. And (3) extracting a genome from LM4-2, and using the extracted genome as a template for subsequent azoreductase gene cloning according to a conventional bacterial genome extraction method. The template is amplified by a conventional PCR method, and a PCR reaction system is 25 mu L, wherein the template is 1 mu L, 2U/. mu.L Taq polymerase is 0.5 mu L, the upstream primer and the downstream primer are respectively 1 mu L, 10mM dNTPs are 0.5 mu L, 10 XPCR Buffer is 2.5 mu L, dd H2O18.5. mu.L, the procedure was as follows:
pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 40s, extension at 72 ℃ for 60s, reaction for 30 cycles, final extension at 72 ℃ for 10min, after the reaction is finished, loading all target fragments to gel electrophoresis, cutting off gel containing target bands, performing gel recovery by using an agarose gel DNA rapid recovery kit of Omega company, and storing the recovered DNA fragments at-20 ℃.
(2) Plasmid extraction of recombinant vectors
The pET28a (+) expression vector was used, and the plasmid map is shown in FIG. 1. Escherichia coli carrying pET28a (+) expression vector is inoculated into LB culture medium containing 50 mug/mL kanamycin according to 1% inoculation amount, the culture is carried out in an electrothermal constant temperature shaking table under the culture condition of 37 ℃ and 180r/min for 12h, Plasmid extraction is carried out by using TIANPrep Rapid Mini Plasmid Kit, and the extracted Plasmid is stored at-20 ℃.
(3) Construction of recombinant vectors
And carrying out double digestion and ligation reaction on the target fragment and the expression vector plasmid respectively. Double digestion 20. mu.L system: mu.L of the gene or plasmid of interest, 1. mu.L of BamH I endonuclease, 1. mu.L of Xho I endonuclease, 2. mu.L of 1 XK Buffer, 10. mu.L of dd H2O 10, and endonucleases and buffers were purchased from Takara. And (3) placing the uniformly mixed centrifuge tube into a water bath kettle at 37 ℃ for digestion for 2h, and performing gel recovery in the same manner as above after double enzyme digestion. The recovered target fragment and the digested expression vector were ligated with T4 ligase overnight in a refrigerator at 4 ℃ as follows: 2. mu.L of vector, 6. mu.L of target fragment, 1. mu.L of T4 buffer, and 1. mu.L of T4 ligase.
(4) Introduction of recombinant vector
Removing frozen BL21(DE3) from refrigerator, inoculating BL21(DE3) into LB medium, adding CaCl2The preparation method of competent cell can be used for preparing competent cell. The constructed ligation product is transformed into a BL21(DE3) strain, and Escherichia coli BL21(DE3) is selected as an expression strain mainly because the strain is a protease-deficient strain and can not degrade the protein after heterologous expression, and the specific method comprises the following steps: adding 10 μ L of the ligation product into 100 μ L of BL21(DE3) competent cells taken out of a refrigerator, uniformly mixing, standing on ice for 30min, placing a centrifuge tube in a 42 ℃ water bath kettle, thermally shocking for 90s, rapidly placing the centrifuge tube on ice, carrying out ice bath for 2min, adding 900 μ L of LB liquid culture medium into the centrifuge tube after the ice bath is finished, culturing at 37 ℃ for 1.5h, coating the mixture on an LB solid plate containing kanamycin, and placing the mixture in a 37 ℃ incubator for inverted culture for 12-16 h. Selecting a single clone to be added into an LB culture medium of the kanamycin so as to obtain the recombinant strain containing the target plasmid.
Example 3
Detection of molybdenum reduction capability of recombinant bacteria
Under the condition that a culture medium contains the same sodium molybdate concentration, after IPTG induction expression (firstly, heterologous expression is carried out on recombinant protein, namely, when the recombinant bacteria constructed in the example 2 are cultured to the logarithmic growth phase, 1mM IPTG inducer is added, and a strain (BL21-DE3) containing empty vector is used as a control group, and then the control group is addedCarrying out molybdenum tolerance detection, namely measuring the growth amount under the condition of molybdenum stress), wherein the growth amount of the detected recombinant strain is obviously higher than that of a control, and the recombinant strain shows better molybdenum resistance, as shown in figure 2; when grown in solid LMP (low phosphorus medium) medium containing 2.4g/L sodium molybdate, the cells appeared blue (FIG. 3), indicating that the recombinant strain could remove Mo from the medium6+Reduced to molybdenum blue in a low valence state, and shows a reduction character.
In addition, the expression of azoreductase in the recombinant strain was examined by SDS-PAGE and Western Blot, and the results are shown in FIGS. 4 and 5. The size of the recombinant Escherichia coli BL21(DE3) azoreductase is about 18.4Kda, the expression quantity is gradually increased along with the prolonging of the induction time, and Western Blot results show that the azoreductase is really induced to express.
It should be noted that the BslM _2044 gene of the present invention can be expressed by electrotransformation or transformation into other strains, such as Bacillus, Escherichia coli of other species, etc.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including the preferred embodiment and all changes and modifications that fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Sequence listing
<110> university of Henan science and technology
<120> azoreductase BslM _2044, coding gene thereof, recombinant bacterium containing gene and application thereof
<141> 2020-08-11
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 208
<212> PRT
<213> Bacillus subtilis LM 4-2
<400> 1
Met Ser Thr Val Leu Phe Val Lys Ser Ser Asp Arg Thr Ala Glu Glu
1 5 10 15
Gly Val Ser Thr Lys Leu Tyr Glu Ala Phe Leu Ala Ala Tyr Lys Glu
20 25 30
Asn Asn Pro Asn Asp Glu Val Val Glu Leu Asp Leu His Lys Glu Asn
35 40 45
Leu Pro Tyr Leu Gly Arg Asp Met Ile Asn Gly Thr Phe Lys Ala Gly
50 55 60
Gln Gly Met Glu Met Thr Glu Asp Glu Lys Lys Gln Ala Ala Ile Ala
65 70 75 80
Asp Lys Tyr Leu Asn Gln Phe Val Lys Ala Asp Lys Val Val Phe Ala
85 90 95
Phe Pro Leu Trp Asn Phe Thr Val Pro Ala Val Leu His Thr Tyr Val
100 105 110
Asp Tyr Leu Ser Arg Ala Gly Val Thr Phe Lys Tyr Thr Gln Glu Gly
115 120 125
Pro Val Gly Leu Met Gly Gly Lys Lys Val Ala Leu Leu Asn Ala Arg
130 135 140
Gly Gly Val Tyr Ser Glu Gly Pro Met Ala Ala Leu Glu Met Ser Leu
145 150 155 160
Asn Phe Met Lys Thr Val Leu Gly Phe Trp Gly Val Gln Asp Leu His
165 170 175
Thr Val Val Ile Glu Gly His Asn Ala Ala Pro Asp Gln Ala Gln Glu
180 185 190
Ile Val Glu Lys Gly Leu Gln Glu Ala Lys Asp Leu Ala Ala Lys Phe
195 200 205
<210> 2
<211> 627
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<213> Bacillus subtilis LM 4-2
<400> 2
ttagaatttt gcagcaagat cttttgcttc ttgtaaacct ttttcaacga tttcttgcgc 60
ttgatcaggt gctgcgttat gtccttcgat gacaactgtg tgcaagtctt gaacacccca 120
gaaaccaaga actgttttca tgaagtttaa tgacatttca agtgcagcca ttggtccttc 180
tgagtagaca ccgccgcgag cgttaagaag cgcaactttt ttgccgccca ttaaaccgac 240
tggtccttct tgtgtgtatt tgaatgtaac gcctgcgcga gacagataat caacataagt 300
atgaagcact gctggcactg tgaagttcca aagcgggaat gcgaaaacaa ctttgtcagc 360
ttttacaaac tggttcagat atttgtcagc aattgctgct tgttttttct catcttctgt 420
catttccatt ccttgacctg ctttaaatgt tccgttaatc atatctctgc caaggtaagg 480
aaggttttcc ttatgaagat ctaattcaac cacttcatca ttagggttgt tttctttata 540
agcagctaag aaagcttcgt aaagtttagt tgaaacgcct tcttcagctg tacggtcgct 600
tgattttaca aataaaactg tagacat 627
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<213> Artificial sequence
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<213> Artificial sequence
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ccgctcgagt tagaattttg cagcaag 27
Claims (5)
1. The application of azoreductase BsLM _2044 in preparing a preparation for repairing molybdenum pollution is characterized in that the amino acid sequence of BsLM _2044 is shown in SEQ ID No. 1.
2. The use of the azoreductase BslM _2044 of claim 1 in the preparation of a formulation to remediate molybdenum contamination, wherein BslM _2044 is encodedBsLM_2044The nucleotide sequence of the gene is shown in SEQ ID NO. 2.
3. The use of a recombinant bacterium in the remediation of molybdenum pollution, wherein the recombinant bacterium comprises the recombinant bacterium of claim 2BsLM_2044Recombinant Escherichia coli of the geneBsLM_2044The gene is introduced into an escherichia coli receptor bacterium through an expression vector pET28a-azoR to obtain recombinant escherichia coli.
4. The use of the recombinant bacterium of claim 3 for remediating molybdenum contamination, wherein the recipient bacterium is E.coli BL21(DE 3).
5. Use of the recombinant bacterium of claim 4 for repairing molybdenum contamination by chemical transformation, in particular with CaCl2The method comprises preparing BL21(DE3) competent cells, adding 10 μ L recombinant plasmid into 100 μ L competent cells, standing on ice for 30min, heat shocking for 90s, ice-cooling for 2min, adding 900mL LB Broth for resuscitation for 1h, spreading on LB plate containing kan resistance, and incubating for 12-16 h.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104560815A (en) * | 2014-12-24 | 2015-04-29 | 大地绿源环保科技(北京)有限公司 | Bacillus licheniformis with azo compound degradation activity and application thereof |
| CN111187728A (en) * | 2019-10-22 | 2020-05-22 | 汕头大学 | Clostridium bifidum ST12 and application thereof in azo dye degradation |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US20100064393A1 (en) * | 2006-11-29 | 2010-03-11 | Novozymes, Inc. | Bacillus liceniformis chromosome |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104560815A (en) * | 2014-12-24 | 2015-04-29 | 大地绿源环保科技(北京)有限公司 | Bacillus licheniformis with azo compound degradation activity and application thereof |
| CN111187728A (en) * | 2019-10-22 | 2020-05-22 | 汕头大学 | Clostridium bifidum ST12 and application thereof in azo dye degradation |
Non-Patent Citations (4)
| Title |
|---|
| "Exclusive Extracellular Bioreduction of Methyl Orange by Azo Reductase-Free Geobacter sulfurreducens";Yi-Nan Liu 等;《ENVIRONMENTAL SCIENCE & TECHNOLOGY》;20170703;第1-8页 * |
| "MULTISPECIES: FMN-dependent NADH-azoreductase [Bacillus]，ACCESSION:WP_003231260.1";NCBI;《NCBI》;20200128;第1页 * |
| "Regulation of quinone detoxification by the thiol stress sensing DUF24/MarR-like repressor, YodB in Bacillus subtilis";Montira Leelakriangsak 等;《Molecular Microbiology》;20081231;第67卷(第5期);第1108-1124页 * |
| "偶氮还原酶AZR的结构及其K109的定点突变研究";柳广飞 等;《微生物学通报》;20080520;第35卷(第5期);第661-665页 * |
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