CN117778417A - ScHSP78 gene for regulating and controlling microbial mercury resistance and mercury accumulation and application thereof - Google Patents
ScHSP78 gene for regulating and controlling microbial mercury resistance and mercury accumulation and application thereof Download PDFInfo
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- CN117778417A CN117778417A CN202311677078.2A CN202311677078A CN117778417A CN 117778417 A CN117778417 A CN 117778417A CN 202311677078 A CN202311677078 A CN 202311677078A CN 117778417 A CN117778417 A CN 117778417A
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- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 title claims abstract description 56
- 229910052753 mercury Inorganic materials 0.000 title claims abstract description 56
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 49
- 238000009825 accumulation Methods 0.000 title claims abstract description 18
- 230000000813 microbial effect Effects 0.000 title claims abstract description 9
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- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 6
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- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
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Abstract
The invention relates to a ScHSP78 gene for regulating and controlling microbial mercury resistance and mercury accumulation and application thereof, and belongs to the technical field of genetic engineering. The nucleotide sequence of the ScHSP78 gene is shown as SEQ ID NO. 1. The invention discovers that the ScHSP78 gene of the over-expression yeast can effectively reduce the mercury resistance of microorganisms, enhance the absorption and accumulation of the microorganisms on mercury, and provide gene resources and technical support for bioremediation of mercury pollution in soil. The applicant finds through the comparative experiment of mercury-treated yeast that the microorganism over-expressed by the ScHSP78 gene shows the phenomenon of reduced mercury resistance and the mercury content in the microorganism is obviously increased, which indicates that the ScHSP78 gene relates to the regulation and control of the mercury resistance of the microorganism and the absorption and accumulation of the microorganism to mercury, and can be used for the microbial remediation of mercury contaminated soil.
Description
Technical Field
The invention relates to a ScHSP78 gene for regulating and controlling microbial mercury resistance and mercury accumulation and application thereof, belonging to the technical field of biological genetic engineering.
Background
In recent years, due to unreasonable application of agricultural chemicals such as pesticides and fertilizers, the accumulation amount of heavy metal pollutants in farmland and facility agriculture land soil is increased year by year, and serious threat is brought to the life and physical health of people. Heavy metal pollution has concealment, long-term property and irreversibility, and the severity of the heavy metal pollution is not paid much attention compared with pesticide residues. Heavy metal mercury itself is naturally occurring, but in recent years, due to industrialization, it is released into the environment in various ways, such as burning coal, mining, etc., and the content in the environment has been increasing. Mercury is a naturally occurring toxic metal that accumulates in the body and is readily absorbed by the skin and respiratory and digestive tracts. Mercury damages the central nervous system, adversely affecting the mouth, mucous membranes and teeth; eventually leading to brain damage and death. The well-known water disease in japan is a manifestation of mercury poisoning.
According to the literature report related to the heavy metal pollution of the soil and the crops published at home and abroad for decades, the method for treating the soil pollution and preventing the heavy metal pollution of the crops mainly comprises the following steps: (1) physical method, mainly comprising: a soil-alien and soil-changing method, a separation and repair method, an isolation method, a thermal repair method and the like; (2) chemical method, mainly comprising: chemical solidification, soil leaching, electrokinetic remediation, etc.; (3) biological methods, mainly comprising: plant stabilization, plant volatilization, plant extraction, microbial remediation, and the like. Compared with the other two methods, the bioremediation can achieve the purposes of purifying the soil or passivating the mercury in the soil, can effectively treat the soil polluted by the mercury, has wider application range and has larger potential value. In the face of the increasingly serious heavy metal soil pollution problem, the bioremediation gene for the super accumulation of heavy metal is searched and the function of the bioremediation gene is clarified, so that the bioremediation gene has important significance for the treatment and restoration of the heavy metal pollution in China. Yeast is the simplest eukaryotic organism and is also a common ancestor for other eukaryotic organisms. We found that genes affecting mercury resistance and mercury accumulation are of great biological significance in yeast.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a ScHSP78 gene for regulating and controlling the mercury resistance and mercury accumulation of microorganisms and application thereof.
The technical scheme of the invention is as follows:
the nucleotide sequence of the ScHSP78 gene in regulating and controlling the mercury resistance and mercury accumulation of microorganisms is shown as SEQ ID NO. 1.
According to the invention, the ScHSP78 gene overexpression can reduce the mercury resistance of microorganisms and promote the absorption and accumulation of mercury by the microorganisms.
According to a preferred aspect of the invention, the use is for the microbial remediation of mercury contaminated soil by means of a microorganism overexpressing the ScHSP78 gene.
Preferably, according to the present invention, the microorganism is a yeast.
According to a preferred embodiment of the invention, the application comprises: inserting the ScHSP78 gene into a plasmid vector to construct an overexpression vector of the ScHSP78 gene, introducing the ScHSP78 gene overexpression vector into a receptor microorganism, and screening to obtain a functional transgenic microorganism.
Further preferably, the plasmid vector is pRS416.
The invention has the beneficial effects that:
the invention discovers for the first time that the ScHSP78 gene of the over-expressed yeast heat shock protein 78 can effectively reduce the mercury resistance of microorganisms, enhance the absorption and accumulation of the microorganisms on mercury, and provide gene resources and technical support for bioremediation of mercury pollution in soil. The applicant finds that the microorganisms over-expressed by the ScHSP78 genes show the phenomenon of reduced mercury resistance and the mercury content in the microorganisms is obviously increased through the comparative experiment of mercury-treated yeast, which shows that the ScHSP78 genes relate to the regulation and control of the mercury resistance of the microorganisms and the absorption and accumulation of the microorganisms, and can be used for the microbial remediation of mercury-contaminated soil and the detection of mercury contamination of the soil.
Description of the drawings:
FIG. 1 shows agarose gel electrophoresis of the ScHSP78 gene.
FIG. 2 shows the ScHSP78 gene transferred yeast and wild type yeast in the presence of HgCl 2 (50. Mu.M) of the results of growth on SC solid screening medium and normal SC solid screening medium;
the triangle symbols in the figure indicate the decrease in concentration of the ScHSP 78-transferred yeast and wild-type yeast by 10-fold.
FIG. 3 shows the mercury content of yeasts and wild-type yeasts transformed with the ScHSP78 gene under the same mercury treatment conditions; error bars represent + -SD statistics of three independent replicates; p value of T test: comparison of mercury content of yeast transformed with the ScHSP78 gene with wild-type yeast, P <0.05.
Detailed Description
The invention is further illustrated below with reference to examples. However, the scope of the present invention is not limited to the following examples. Those skilled in the art will appreciate that various changes and modifications can be made to the invention without departing from the spirit and scope thereof. The present invention generally and/or specifically describes the materials used in the test as well as the test methods. Although many materials and methods of operation are known in the art for accomplishing the objectives of the present invention, the present invention will be described in as much detail herein.
The JRY472 yeasts described in the examples are disclosed in the document A Mitochondrial Pyruvate Carrier Required for Pyruvate Uptake in Yeast, drosophila, and Humans.
Example 1
1. RNA extraction from Saccharomyces cerevisiae (Saccharomyces cerevisiae)
5g of budding saccharomyces cerevisiae (Saccharomyces cerevisiae) cell powder is weighed, suspended in 30mL of NaOH solution with the concentration of 0.04M and uniformly ground in a mortar, the suspension is transferred into an Erlenmeyer flask, heated in a boiling water bath for 30min, cooled and transferred into a centrifuge tube, centrifuged for 15min at 3000r/min, the supernatant is slowly poured into 10mL of acid ethanol while stirring, and after the addition is completed, the mixture is left stand, after RNA precipitation, the mixture is centrifuged for 3min at 3000r/min,the supernatant was discarded and the pellet was washed twice with 95% ethanol. After washing the precipitate once more with diethyl ether, the precipitate was transferred to a Buchner funnel with diethyl ether, and the precipitate was dried in air and ddH treated with 50. Mu.L of DEPC 2 O dissolves the RNA sample, and the prepared RNA sample is preserved in an ultralow temperature refrigerator at-80 ℃ for standby.
The total RNA of Saccharomyces cerevisiae (Saccharomyces cerevisiae) was obtained by this method.
2. Reverse transcription PCR
Carrying out reverse transcription PCR amplification (two-step method) by taking a total RNA sample as a template, wherein the specific reverse transcription PCR amplification system and conditions are as follows:
Step1：
reaction conditions: the mixture is incubated at 42 ℃ for 2min and stored at 4 ℃.
Step2：
Reaction conditions: the reaction was carried out at 37℃for 15min and 85℃for 5s.
The total cDNA of Saccharomyces cerevisiae (Saccharomyces cerevisiae) was obtained by this method, and the reagents in Step1 and Step2 were derived from TAKALA (PrimeScript TM II 1st Strand cDNA Synthesis Kit) kit.
Example 2
1. The construction of the ScHSP78 gene overexpression vector comprises the following specific steps:
(1) PCR amplification is carried out by taking the total cDNA of the budding saccharomyces cerevisiae (Saccharomyces cerevisiae) as a template, the ScHSP78 gene sequence (SEQ ID NO. 1) is obtained by amplification, and the primers for PCR amplification are as follows:
HSP78-F：5'-ATGTTAAGACAAGCTACAAAAGCAC-3'，
HSP78-R：5'-TTACTTTTCAGCTTCCTCTTCAAC-3'。
the PCR system is as follows:
the PCR conditions were as follows: pre-denaturation, 98 ℃,30s; denaturation, 98 ℃,10s; annealing at 60 ℃ for 30s; extending at 72deg.C for 1min (35 cycles); stopping extending at 72deg.C for 10min; finally preserving heat at 4 ℃;
10. Mu.L of the PCR product was analyzed by agarose gel electrophoresis at 1%, and the results are shown in FIG. 1. As can be seen from FIG. 1, the ScHSP78 gene was successfully obtained by PCR amplification in this example.
(2) The pRS416 plasmid (containing pGPD promoter) was digested with restriction enzyme BamH1, and the ScHSP78 gene was ligated into the pRS416 plasmid with homologous recombinase (In-Fusion) to obtain an overexpressed vector of the ScHSP78 gene;
the connection system is as follows:
connection reaction conditions: 50 ℃ for 15min; preserving at 4 ℃.
2. Preparation of ScHSP78 Gene-transferred Yeast
Streaking JRY472 saccharomycetes stock solution stored at-80 ℃ on YPDA solid culture medium, and inversely culturing at 30 ℃ for 4 days; selecting a single colony of the JRY472 saccharomycetes into 5mL of YPDA liquid culture medium, and culturing for 10h on a shaking table at 30 ℃ and 250 rpm; 3mL of JRY472 saccharomycete liquid is taken to be expanded and cultured in 50mL of YPDA liquid culture medium, and the culture is carried out on a shaking table at the temperature of 30 ℃ and the speed of 250rpm until the OD600 is less than or equal to 0.5; then centrifuging the JRY472 yeast liquid at 3000rpm and 25 ℃ for 8min, removing the supernatant, and re-suspending with sterile water; continuing to centrifuge at 3000rpm and 25 ℃ for 3min, and re-suspending with sterile water; continuing to centrifuge at 6000rpm at 25 ℃ for 2min, removing the supernatant, and re-suspending with TE-LiAC buffer; continuing to centrifuge at 6000rpm,25 ℃ for 2min, removing the supernatant, and re-suspending with TE-LiAC solution; sucking 100. Mu.L of heavy suspension, mixing with 2. Mu.L of vector DNA (heated at 100deg.C for 5min, placed on ice, and repeated twice) and 10. Mu.L of ScHSP78 gene overexpression vector, mixing well, and placing at 25deg.C for 10min; adding 260 μl of 40% PEG/TE-LiAC solution, mixing, water-bathing at 30deg.C for 1 hr, adding 43 μl of 37 deg.C preheated DMSO, mixing, heat-shock at 42deg.C for 5min, centrifuging the heat-shock mixed solution at 12000rpm for 30s, and re-suspending with sterile water; the resuspended solution was centrifuged at 12000rpm for 30s, resuspended in sterile water, 100. Mu.L of the resuspended solution was spread on SC solid screening medium, and cultured upside down at 30℃for 6 days to obtain the ScHSP78 gene-transferred JRY472 yeast.
Example 3
The single colony of the JRY472 microzyme transferred with the ScHSP78 gene in the example 2 is picked into 5mL of SC liquid screening culture medium, dispersed and mixed uniformly, and shake-cultured for 10 hours at 250rpm and 30 ℃ until the OD600 is more than 0.3, thus obtaining the microzyme liquid transferred with the ScHSP78 gene. The yeast liquid of the ScHSP78 gene is diluted to OD600 = 0.1, shake cultivation is continued for 6 hours at 250rpm and 30 ℃, then the yeast liquid of the ScHSP78 gene is diluted to OD600 = 0.3, and the yeast liquid of the ScHSP78 gene is taken as a mother liquid. The mother liquor was diluted to a concentration gradient of 1/10,1/100,1/1000 times, respectively.
Normal JRY472 yeast (empty vector) was cultured to od600=0.3 in the same manner, and diluted in the same ratio.
Respectively collecting 3 μl of yeast mother liquor transformed with ScHSP78 gene and its gradient dilution, and dripping into HgCl-containing solution 2 (50. Mu.M) SC solid screening medium and normal SC solid screening medium were cultured in an inverted state at 30℃for 4 days. And dripping normal JRY472 yeast mother liquor and its gradient dilution as control to the liquid containing HgCl 2 (50. Mu.M) of the SC solid screening medium and the normal SC solid screening medium were cultured upside down at 30℃for 4 days, and the results are shown in FIG. 2. Wherein wt+schsp78 represents the yeast mother liquor of the transgenic ScHSP78 gene and its gradient dilutions, and wt+ev represents the normal JRY472 yeast mother liquor and its gradient dilutions.
As can be seen from fig. 2, under the condition of mercury stress, the growth vigor of the yeast cells transformed with the ScHSP78 gene is obviously weaker than that of the normal yeast empty vector control, which indicates that the ScHSP78 gene has the effect of increasing the mercury sensitivity of the yeast cells, and the ScHSP78 gene can effectively reduce the mercury tolerance of the yeast cells and can also be used for prompting whether mercury pollution exists in soil.
Example 4
Culturing the ScHSP78 gene-transferred JRY472 yeast and the normal JRY472 yeast (empty vector) according to the method described in example 3 to obtain bacterial solutions, inoculating into YPDA liquid medium, shaking culture at 250rpm and 30℃until OD600 = 0.3, and adding HgCl 2 To a final concentration of 50. Mu.M, hgCl was added 2 The samples were taken at 14h later, and Hg was measured in the cells of the ScHSP78 gene-transferred JRY472 yeast and normal JRY472 yeast (empty vector) 2+ The content and the result are shown in FIG. 3. Wherein WT represents a yeast transformed with the ScHSP78 gene, and EV represents a normal JRY472 yeast.
As can be seen from FIG. 3, the mercury content in the JRY472 yeast cells transformed with the ScHSP78 gene after mercury treatment is significantly improved compared with that of the JRY472 yeast cells containing normal JRY472 yeast (empty vector), which shows that the absorption and accumulation capacity of mercury in the JRY472 yeast transformed with the ScHSP78 gene under heavy metal mercury stress is obviously higher than that of the normal JRY472 yeast, and the ScHSP78 gene can promote the absorption and accumulation of mercury by microorganisms and can be used for the microorganism repair of mercury contaminated soil.
Claims (6)
- The application of the ScHSPS 78 gene in regulating and controlling the mercury resistance and mercury accumulation of microorganisms is characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO. 1.
- 2. The use according to claim 1, wherein the overexpression of the ScHSP78 gene reduces the mercury resistance of the microorganism and promotes the absorption and accumulation of mercury by the microorganism.
- 3. The use according to claim 1, wherein the use is for the microbial remediation of mercury contaminated soil by means of a microorganism overexpressing the ScHSP78 gene.
- 4. The use according to claim 1, wherein the microorganism is a yeast.
- 5. The application of claim 1, wherein the application comprises: inserting the ScHSP78 gene into a plasmid vector to construct an overexpression vector of the ScHSP78 gene, introducing the ScHSP78 gene overexpression vector into a receptor microorganism, and screening to obtain a functional transgenic microorganism.
- 6. The use according to claim 5, wherein the plasmid vector is pRS416.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000047761A2 (en) * | 1999-02-12 | 2000-08-17 | Phase-1 Molecular Toxicology, Inc. | High-throughput toxicological testing using cultured organisms and cells |
| EP1887081A2 (en) * | 1999-02-25 | 2008-02-13 | Ceres Incorporated | DNA Sequences |
| US20150329853A1 (en) * | 2012-12-19 | 2015-11-19 | Helge Zieler | Compositions and methods for creating altered and improved cells and organisms |
| WO2018038234A1 (en) * | 2016-08-24 | 2018-03-01 | 国立研究開発法人産業技術総合研究所 | Stress response promoter |
| JP2019506150A (en) * | 2015-12-22 | 2019-03-07 | アルブミディクス リミティド | Improved protein expression strain |
-
2023
- 2023-12-08 CN CN202311677078.2A patent/CN117778417B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000047761A2 (en) * | 1999-02-12 | 2000-08-17 | Phase-1 Molecular Toxicology, Inc. | High-throughput toxicological testing using cultured organisms and cells |
| EP1887081A2 (en) * | 1999-02-25 | 2008-02-13 | Ceres Incorporated | DNA Sequences |
| US20150329853A1 (en) * | 2012-12-19 | 2015-11-19 | Helge Zieler | Compositions and methods for creating altered and improved cells and organisms |
| JP2019506150A (en) * | 2015-12-22 | 2019-03-07 | アルブミディクス リミティド | Improved protein expression strain |
| WO2018038234A1 (en) * | 2016-08-24 | 2018-03-01 | 国立研究開発法人産業技術総合研究所 | Stress response promoter |
Non-Patent Citations (3)
| Title |
|---|
| ACCESSION: NM_001180566.3: "Saccharomyces cerevisiae S288C chaperone ATPase HSP78 (HSP78), partial mRNA", 《NCBI》, 15 September 2023 (2023-09-15) * |
| YUKO MOMOSE等: "Bioassay of cadmium using a DNA microarray: Genome-wide expression patterns of Saccharomyces cerevisiae response to cadmium", 《ENVIRONMENTAL TOXICOLOGY》, vol. 20, no. 10, 31 October 2001 (2001-10-31), pages 2353 - 2360, XP009043984, DOI: 10.1897/1551-5028(2001)020<2353:BOCUAD>2.0.CO;2 * |
| YUKO MOMOSE等: "Comparison of Genome-wide Expression Patterns in Response to Heavy Metal Treatment in Saccharomyces cerevisiae 1) Cadmium and mercury", 《CHEM-BIO INFORMATICS JOURNAL》, vol. 1, no. 1, 31 March 2001 (2001-03-31), pages 41 - 50 * |
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