CN102296033A - Construction method and application of Saccharomyces cerevisiae gsh1 deleted mutant strain - Google Patents
Construction method and application of Saccharomyces cerevisiae gsh1 deleted mutant strain Download PDFInfo
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Abstract
The invention discloses a construction method and application of a Saccharomyces cerevisiae gsh1 deleted mutant strain. The method is performed according to the description in the document (Yeast, 1998, 14:953-962), including designing of primers and transformation of knockout segment according to the principle of homologous recombination and gene knockout of Saccharomyces cerevisiae gene. The method is a conventional method for gene knockout in the Saccharomyces cerevisiae at present. The method specifically comprises the following steps: designing primers (forward primer P1 and reverse primer P2) according to the gene sequence of Saccharomyces cerevisiae gsh1; carrying out PCR (polymerase chain reaction) amplification on the plasmid pFA6a-Kan MX6 by using the forward primer P1 and the reverse primer P2 to obtain a knockout segment containing forward and reverse sequences of the Saccharomyces cerevisiae gsh1 gene; and introducing the knockout segment into a Saccharomyces cerevisiae BY47421 strain cell by a lithium acetate/PEG (polyethylene glycol) transformation method, and screening to obtain the Saccharomyces cerevisiae DELTAgsh1 mutant strain YJL101C. The Saccharomyces cerevisiae cell DELTAgsh1 mutant strain is constructed by a microbial genetic method, and the cytotoxicity of cadmium chloride or any other heavy metal is detected by using the mutant strain, thereby enhancing the cytotoxicity detection sensitivity. The method is easy to implement and simple to operate.
Description
Technical field
The invention belongs to microbiology, biological chemistry, toxicity detection technique field, the construction process that more specifically relates to a kind of Δ gshl mutant strain of yeast saccharomyces cerevisiae, the purposes that also relates to a kind of Δ gshl mutant strain of yeast saccharomyces cerevisiae, application in improving heavy metal cytotoxicity detection sensitivity is applicable to the Cytotoxic detection of other materials.
Background technology
Along with the mankind's social production activity, cadmium in the environment (Cd), plumbous (Pd), mercury (Hg) and chromium heavy metal contaminations such as (Cr) are more and more severe.Because heavy metal can combine with nucleic acid, protein and other, make the biomacromolecule structure that irreversible change take place, perhaps produce the destruction that free radical causes cellularstructure and function, even the death of cell, so heavy metal has significant cytotoxicity.Heavy metal can influence mankind's activity directly or indirectly by approach such as skin contact, food chain, drinking-water, is detrimental to health.As cadmium is common environment and industrial pollutants, has certain carinogenicity and mutagenicity, and long half time in animal body is big because of its toxicity, be widely used and become a kind of main heavy metal contaminants gradually.
Detecting the Cytotoxic method of heavy metal at present has many, as: MTT colorimetry, toluylene red absorption experiment, trypan blue repel methods such as experiment, determining the protein quantity, lactate dehydrogenase activity detection, cell micronucleus detection, dna damage detection, cell survival rate detection, be material wherein with V79 cell, CHL cell, L929 cell and yeast cell, by detect the cell growth, that survival rate is assessed the Cytotoxic experiment of heavy metal is more.
Yeast saccharomyces cerevisiae is a kind of unicellular eucaryon model animals, the important role of performer in biological study, but tolerance is higher than the animal culturing cell because yeast saccharomyces cerevisiae is to heavy metal, though yeast saccharomyces cerevisiae cultivation, simple to operate, performance is outstanding in vitro cytotoxicity research.In order to improve the sensitivity of yeast saccharomyces cerevisiae in the heavy metal cytotoxicity detects, applicant's imagination is started with to the tolerance of heavy metal from reducing brewing yeast cell.
Gsh (GSH), it is γ-L-glutamy-L-cysteinyl glycine, can effectively dispose the intravital free radical of cell, GSH is through gamma-glutamylcysteine synthetase (GSH I) and glutathione synthetase (GSH II) catalysis and synthetic by precursor substance L-L-glutamic acid, L-halfcystine and glycine, wherein gamma-glutamylcysteine synthetase is the rate-limiting enzyme in the GSH route of synthesis, by the gshl genes encoding.Knock out the gshl gene of yeast saccharomyces cerevisiae, will effectively reduce the tolerance of brewing yeast cell, promptly adopt Δ gshl mutant strain to carry out the cytotoxicity detection and can effectively improve detection sensitivity heavy metal.This method and technical thought are applicable to the Cytotoxic detection of other materials.
Summary of the invention
The present invention seeks to be to provide a kind of Δ gshl mutant strain YJL101C of yeast saccharomyces cerevisiae, this mutant strain gshl genetically deficient, effective synthesizing glutathion, mutant strain cell resistance descends, and heavy metal etc. is coerced the factor tolerance reduces, susceptibility raises.
Another object of the present invention is the construction process that has been to provide a kind of yeast saccharomyces cerevisiae Δ gshl mutant strain, after utilizing the microbial genetics method to make up brewing yeast cell Δ gshl mutant strain, again mutant strain is used for the Cytotoxic detections of heavy metal such as Cadmium chloride fine powder.
A further object of the invention is to be to provide the application of a kind of yeast saccharomyces cerevisiae Δ gshl mutant strain in improving heavy metal cytotoxicity detection sensitivity, because the Δ gshl mutant strain of yeast saccharomyces cerevisiae is synthesizing glutathion effectively, the mutant strain cell is lower than wild-type cell to heavy metal tolerances such as Cadmium chloride fine powdeies, and the cytotoxicity of therefore utilizing the Δ gshl mutant strain of yeast saccharomyces cerevisiae to detect heavy metals such as Cadmium chloride fine powder can improve the sensitivity of detection significantly.
In order to realize above-mentioned purpose, the present invention by the following technical solutions:
A kind of construction process of yeast saccharomyces cerevisiae Δ gshl mutant strain, its step is as follows:
Yeast saccharomyces cerevisiae Δ gshl mutant strain makes up: the genes of brewing yeast homologous recombination knocks out principle, design of primers, knock out the fragment method for transformation carries out (Yeast according to the described method of document, 1998,14:953-962), this method is the universal method of gene knockout in the present yeast saccharomyces cerevisiae;
1) design of primers: design primer according to yeast saccharomyces cerevisiae gshl gene order (GenBank:BK006943.1):
Upstream primer P1:
GCAGATTTAGTATAGGGCTACATTGTAGGGTGGTTTAGAGTATCGAAAATATACATATAGAAGAATAAACGGATCCCCGGGTTAATTAA；
Downstream primer P2:
GCTTTTTCAATCACCGTGTCACCCAAATCGATAATGTCAACTTTCTTTCACAACCGAAGTAAAAGGAGTGAATTCGAGCTCGTTTAAAC；
2) pcr amplification: utilize upstream primer P1 and downstream primer P2 that plasmid pFA6a-Kan MX6 (is derived from Chinese typical culture collection center, can obtain by buying) carry out pcr amplification, obtain to contain the fragment that knocks out of yeast saccharomyces cerevisiae gshl gene upstream and downstream sequence;
3) knocking out of gshl gene: the method for transformation by Lithium Acetate/PEG imports the fragment that knocks out that pcr amplification obtains yeast saccharomyces cerevisiae BY47421 strain cell (the BY47421 bacterium source is in China typical culture collection center, can obtain by buying), and obtain having the Δ gshl mutant strain YJL101C (Saccharomyces cerevisiae YJL101C) of the yeast saccharomyces cerevisiae of G418 resistance by G418 resistance marker screening, the preservation of this bacterial strain, depositary institution: Chinese typical culture collection center, address: China. Wuhan. Wuhan University, preservation date: on April 20th, 2011, deposit number CCTCC No:M 2011130, classification name: yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) YJL 101C.
A kind of Δ gshl mutant strain of yeast saccharomyces cerevisiae that utilizes is improving heavy metals such as detecting Cadmium chloride fine powder to the application in the cytotoxicity sensitivity of yeast cell, the steps include: (other are usually used in the clones such as V79 cell, CHL cell, L929 cell in addition that the heavy metal cytotoxicity detects)
1) Cadmium chloride fine powder is handled: Δ gshl mutant strain and BY47421 bacterial strain are inoculated in the 5mLYPD substratum 200r/min, 30 ℃ of shaking culture 24h respectively from the inclined-plane;
Adding Cadmium chloride fine powder to final concentration respectively and be 1,5 and 10mmol/L, is contrast with the sample that does not add Cadmium chloride fine powder; All samples is in 200r/min, 30 ℃ of shaking culture 24h;
2) viable count: get respectively in the step 1), Δ gshl mutant strain and wild-type BY47421 bacterial strain do not add Cadmium chloride fine powder and add final concentration respectively is 1,5 and the Cadmium chloride fine powder of 10mmol/L, and the bacterium liquid through after the 24h shaking culture carries out 10 times of serial dilutions; Get 10 respectively
-3, 10
-4With 10
-5The extent of dilution sample is coated the YPD flat board, 3 flat boards of every extent of dilution, and glue spread is every dull and stereotyped 200 μ L, cultivates the 72h counting for 30 ℃;
3) calculating of cell survival rate and relative survival rate: calculate survival rate and relative survival rate by formula,
Viable cell concentrations after survival rate (%)=Cadmium chloride fine powder is handled/do not handle viable cell concentrations with Cadmium chloride fine powder;
Wild-type BY47421 bacterial strain survival rate under relative survival rate (%)=Δ gshl mutant strain survival rate/same treatment condition;
The Cadmium chloride fine powder final concentration is 1,5 and during 10mmol/L, Δ gshl mutant strain survival rate is respectively 69.8 ± 8.95%, 24.5 ± 2.15% and 0.22 ± 0.08%, wild-type BY47421 bacterial strain survival rate is respectively 85 ± 9.5%, 34 ± 1.3% and 3.9 ± 0.1%, and Δ gshl mutant strain relative survival rate is respectively 82.12 ± 10.52%, 72.06 ± 6.32% and 5.64 ± 2.5%.
4) relatively Cadmium chloride fine powder is handled back brewing yeast cell survival rate, the cytotoxicity of assessment Cadmium chloride fine powder.After the Cadmium chloride fine powder of same concentrations was handled, Δ gshl mutant strain survival rate was lower than the survival rate of wild strain, increased with Cadmium chloride fine powder concentration, and the survival rate gap increases.Δ gshl mutant strain survival rate is lower than the wild strain survival rate of same treatment, illustrate that it descends to the Cytotoxic tolerance of Cadmium chloride fine powder, more responsive to Cadmium chloride fine powder, the cytotoxicity that promptly adopts Δ gshl mutant strain to be used for heavy metals such as Cadmium chloride fine powder detects the sensitivity that can improve detection.
The present invention compared with prior art has the following advantages and effect:
The present invention makes up Δ gshl mutant strain by the gene knockout method and is used for the detection of heavy metal cytotoxicity.Because gsh (GSH) redox system is opposing or the equitoxic important channel of elimination heavy metal in the cell, and the gamma-glutamylcysteine synthetase of gshl genes encoding (GSH I) is the key enzyme in the GSH route of synthesis.Greatly influenced the synthetic of GSH in the cell behind the gshl gene knockout, the toxic resistance of counterweight metal cell weakens, and adopts Δ gshl mutant strain to be used for the heavy metal cytotoxicity and detects and improved the cytotoxicity detection sensitivity.At the Cadmium chloride fine powder final concentration is 1,5 and during 10mmol/L, Δ gshl mutant strain survival rate is equivalent to about 80%, 70% and 5～6% of wild-type respectively, show increase along with the Cadmium chloride fine powder final concentration, with respect to wild-type, Δ gshl mutant strain significantly descends to the Cytotoxic tolerance of Cadmium chloride fine powder, and is more responsive to the cytotoxicity of Cadmium chloride fine powder.
Description of drawings
Fig. 1 is the survival rate synoptic diagram that a kind of different concentrations of cadmium chloride is handled back mutant strain and wild-type.
Along with Cadmium chloride fine powder concentration increases, cell survival rate descends, and Δ gshl mutant strain cell survival rate is lower than the wild-type cell of same treatment.
Fig. 2 is that a kind of different concentrations of cadmium chloride is handled back mutant strain relative survival rate synoptic diagram.
After the Cadmium chloride fine powder of high density (10mmol/L) was handled, mutant strain survival relatively significantly reduced.
Embodiment
Embodiment 1:
Detecting with yeast saccharomyces cerevisiae Δ gshl mutant strain structure and Cadmium chloride fine powder cytotoxicity below is example, further sets forth the present invention with reference to accompanying drawing.Yeast saccharomyces cerevisiae BY47421 bacterial strain and plasmid pFA6a-Kan MX6 derive from Chinese typical culture collection center.
A kind of construction process of yeast saccharomyces cerevisiae Δ gshl mutant strain the steps include:
Yeast saccharomyces cerevisiae Δ gshl mutant strain makes up: the genes of brewing yeast homologous recombination knocks out principle, design of primers, knock out the fragment method for transformation carries out (Yeast according to the described method of document, 1998,14:953-962), this method is the universal method of gene knockout in the present yeast saccharomyces cerevisiae;
1) design of primers: design primer according to yeast saccharomyces cerevisiae gshl gene order (GenBank:BK006943.1):
Upstream primer P1 (SEQ ID NO.1):
GCAGATTTAGTATAGGGCTACATTGTAGGGTGGTTTAGAGTATCGAAAATATACATATAGAAGAATAAACGGATCCCCGGGTTAATTAA；
Downstream primer P2 (SEQ IDNO.2):
GCTTTTTCAATCACCGTGTCACCCAAATCGATAATGTCAACTTTCTTTCACAACCGAAGTAAAAGGAGTGAATTCGAGCTCGTTTAAAC；
2) pcr amplification: utilize upstream primer P1 and downstream primer P2 that plasmid pFA6a-Kan MX6 (is derived from Chinese typical culture collection center, can obtain by buying) carry out pcr amplification, obtain to contain the fragment that knocks out of yeast saccharomyces cerevisiae gshl gene upstream and downstream sequence;
3) knocking out of gshl gene: the method for transformation by Lithium Acetate/PEG imports the fragment that knocks out that pcr amplification obtains yeast saccharomyces cerevisiae BY47421 strain cell (the BY47421 bacterium source is in China typical culture collection center, can obtain by buying), and obtain having the Δ gshl mutant strain YJL101C (Saccharomyces cerevisiae YJL101C) of the yeast saccharomyces cerevisiae of G418 resistance, Chinese typical culture collection center deposit number CCTCC No:M 2011130 by G418 resistance marker screening.
Embodiment 2:
A kind of Δ gshl mutant strain of yeast saccharomyces cerevisiae that utilizes is improving heavy metals such as detecting Cadmium chloride fine powder to the application in the cytotoxicity sensitivity of yeast cell, the steps include: (other are usually used in the clones such as V79 cell, CHL cell, L929 cell in addition that the heavy metal cytotoxicity detects)
1) Cadmium chloride fine powder is handled: Δ gshl mutant strain and BY47421 bacterial strain are inoculated in the 5mLYPD substratum 200r/min, 30 ℃ of shaking culture 24h respectively from the inclined-plane; Adding Cadmium chloride fine powder to final concentration respectively and be 1,5 and 10mmol/L, is contrast with the sample that does not add Cadmium chloride fine powder; All samples is in 200r/min, 30 ℃ of shaking culture 24h;
Described YPD component is: every liter contains peptone 20g, yeast extract 10g and glucose 20g; Adding agar to final concentration is 2% (w/v), is solid medium;
2) viable count: get respectively in the step 1), Δ gshl mutant strain and wild-type BY47421 bacterial strain do not add Cadmium chloride fine powder and add final concentration respectively is 1,5 and the Cadmium chloride fine powder of 10mmol/L, and the bacterium liquid through after the 24h shaking culture carries out 10 times of serial dilutions; Get 10 respectively
-3, 10
-4With 10
-5The extent of dilution sample is coated the YPD flat board, 3 flat boards of every extent of dilution, and glue spread is every dull and stereotyped 200 μ L, cultivates the 72h counting for 30 ℃;
3) calculating of cell survival rate and relative survival rate: calculate survival rate and relative survival rate by formula,
Viable cell concentrations after survival rate (%)=Cadmium chloride fine powder is handled/do not handle viable cell concentrations with Cadmium chloride fine powder;
Wild-type BY47421 bacterial strain survival rate under relative survival rate (%)=Δ gshl mutant strain survival rate/same treatment condition;
The Cadmium chloride fine powder final concentration is 1,5 and during 10mmol/L, Δ gshl mutant strain survival rate is respectively 69.8 ± 8.95%, 24.5 ± 2.15% and 0.22 ± 0.08%, wild-type BY47421 bacterial strain survival rate is respectively 85 ± 9.5%, 34 ± 1.3% and 3.9 ± 0.1%, and Δ gshl mutant strain relative survival rate is respectively 82.12 ± 10.52%, 72.06 ± 6.32% and 5.64 ± 2.5%.After the Cadmium chloride fine powder of same concentrations was handled, Δ gshl mutant strain survival rate was lower than the survival rate of wild strain, increased with Cadmium chloride fine powder concentration, and the survival rate gap increases.
4) relatively Cadmium chloride fine powder is handled back brewing yeast cell survival rate, the cytotoxicity of assessment Cadmium chloride fine powder.After relatively different concentrations of cadmium chloride is handled, the survival rate of wild-type yeast saccharomyces cerevisiae BY47421 bacterial strain and Δ gshl mutant strain (Cadmium chloride fine powder processing back viable cell concentrations/do not handle viable cell concentrations), and Δ gshl mutant strain relative survival rate (wild-type BY47421 bacterial strain viable cell concentrations under Δ gshl mutant strain viable cell concentrations/same treatment condition) with Cadmium chloride fine powder.As can be seen, Δ gshl mutant strain greatly reduces with respect to the survival rate of wild-type when Cadmium chloride fine powder reaches 10mmol/L from the result of Fig. 1 and 2.The height of survival rate has reflected that directly yeast cell is to the Cytotoxic tolerance of Cadmium chloride fine powder, mutant strain descends to the Cadmium chloride fine powder tolerance, more responsive to Cadmium chloride fine powder, the cytotoxicity that promptly adopts Δ gshl mutant strain to be used for heavy metals such as Cadmium chloride fine powder detects the sensitivity that can improve detection.
More than describing is explanation of the invention, is not limitation of the invention, and institute of the present invention restricted portion is referring to claim, and under the situation of spirit of the present invention, the present invention can make any type of modification.
SEQUENCE LISTING
＜110〉Wuhan University
＜120〉a kind of yeast saccharomyces cerevisiae
Gsh1The construction process of deletion mutantion strain and application
＜130〉a kind of yeast saccharomyces cerevisiae
Gsh1The construction process of deletion mutantion strain and application
<160> 2
<170> PatentIn version 3.1
<210> 1
<211> 89
<212> DNA
＜213〉synthetic
<400> 1
gcagatttag tatagggcta cattgtaggg tggtttagag tatcgaaaat atacatatag 60
aagaataaac ggatccccgg gttaattaa 89
<210> 2
<211> 89
<212> DNA
＜213〉synthetic
<400> 2
gctttttcaa tcaccgtgtc acccaaatcg ataatgtcaa ctttctttca caaccgaagt 60
aaaaggagtg aattcgagct cgtttaaac 89
Claims (3)
1. the gshl deletion mutantion strain of a yeast saccharomyces cerevisiae is characterized in that: the Δ gshl mutant strain YJL101C (Saccharomyces cerevisiae YJL101C) of yeast saccharomyces cerevisiae, CCTCC No:M 2011130.
2. the construction process of the gshl deletion mutantion strain YJL101C of the described a kind of yeast saccharomyces cerevisiae of claim 1 the steps include:
A, design of primers: according to the primer of yeast saccharomyces cerevisiae gshl gene order design:
Upstream primer P1:
GCAGATTTAGTATAGGGCTACATTGTAGGGTGGTTTAGAGTATCGAAAATATACATATAGAAGAATAAACGGATCCCCGGGTTAATTAA；
Downstream primer P2:
GCTTTTTCAATCACCGTGTCACCCAAATCGATAATGTCAACTTTCTTTCACAACCGAAGTAAAAGGAGTGAATTCGAGCTCGTTTAAAC；
B, pcr amplification: utilize upstream primer P1 and downstream primer P2 that plasmid pFA6a-Kan MX6 is carried out pcr amplification, obtain to contain the fragment that knocks out of yeast saccharomyces cerevisiae gshl gene upstream and downstream sequence;
Knocking out of C, gshl gene: the method for transformation by Lithium Acetate/PEG imports yeast saccharomyces cerevisiae BY47421 strain cell with the fragment that knocks out that pcr amplification obtains, and screening obtains the Δ gshl mutant strain of yeast saccharomyces cerevisiae.
3. the gshl deletion mutantion strain of the described a kind of yeast saccharomyces cerevisiae of claim 1 is improving detection Cadmium chloride fine powder heavy metal to the application in the cytotoxicity sensitivity of yeast cell.
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| CN107227323A (en) * | 2017-07-17 | 2017-10-03 | 武汉大学 | A kind of structure of high yield selenomethionine saccharomyces cerevisiae and application |
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| CN108753634A (en) * | 2018-06-27 | 2018-11-06 | 江南大学 | A kind of saccharomyces cerevisiae and its application with clotrimazole resistance |
| KR20220098543A (en) * | 2021-01-04 | 2022-07-12 | 씨제이제일제당 (주) | Glutamate-cysteine ligase variant and method of producing glutathione using thereof |
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