Construction method and application of Saccharomyces cerevisiae gsh1 deleted mutant strain
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A Saccharomyces cerevisiae, deletion mutation technology, applied in the construction of Δgshl mutant strains, in the field of Δgshl mutant strains of Saccharomyces cerevisiae, can solve the problems of in vitro cytotoxicity studies such as not outstanding performance
Inactive Publication Date: 2013-08-28
WUHAN UNIV
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[0004] Saccharomyces cerevisiae is a single-celled eukaryotic model organism that plays an important role in biological research. However, because Saccharomyces cerevisiae is more resistant to heavy metals than animal culture cells, although Saccharomyces cerevisiae is easy to culture and operate, it is cytotoxic in vitro Didn't stand out in the study
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Embodiment 1
[0035] The present invention will be further described below by taking the construction of the Saccharomyces cerevisiae Δgshl mutant strain and the detection of cadmium chloride cytotoxicity as examples, referring to the accompanying drawings. Saccharomyces cerevisiae BY47421 strain and plasmid pFA6a-Kan MX6 were obtained from China Center for Type Culture Collection.
[0036] A method for constructing a Saccharomyces Δgshl mutant strain, the steps of which are:
[0037]Construction of Saccharomyces cerevisiae Δgshl mutant strain: Saccharomyces cerevisiae gene homologous recombination knockout principle, primer design, knockout fragment transformation method was carried out according to the method described in the literature (Yeast, 1998, 14:953-962), this method is the current Saccharomyces cerevisiae General method for gene knockout in China;
[0038] 1) Primer design: Design primers based on the gshl gene sequence of Saccharomyces cerevisiae (GenBank: BK006943.1):
[0039...
Embodiment 2
[0046] An application of the Δgshl mutant strain utilizing Saccharomyces cerevisiae in improving the cytotoxicity sensitivity of heavy metals such as cadmium chloride to yeast cells, the steps are: (Others commonly used in heavy metal cytotoxicity detection also include V79 cells, CHL cells, L929 cells, etc.)
[0047] 1) Cadmium chloride treatment: the Δgshl mutant strain and the BY47421 strain were inoculated in 5mLYPD medium from the slant, 200r / min, 30°C shaking culture for 24h; cadmium chloride was added to the final concentration of 1, 5 and 10mmol / L, the sample without cadmium chloride was used as the control; all samples were shaken at 200r / min, 30°C for 24h;
[0048] The YPD components are as follows: every liter contains 20g of peptone, 10g of yeast extract and 20g of glucose; adding agar to a final concentration of 2% (w / v) is a solid medium;
[0049] 2) Viable cell count: in step 1), the Δgshl mutant strain and the wild-type BY47421 strain were not added with cadm...
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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 [0001] The invention belongs to the technical fields of microbiology, biochemistry and toxicological detection, and more specifically relates to a method for constructing a Δgshl mutant strain of Saccharomyces cerevisiae, and also relates to a use of a Δgshl mutant strain of Saccharomyces cerevisiae, which can improve the detection sensitivity of heavy metal cytotoxicity It is suitable for the detection of cytotoxicity of other substances. Background technique [0002] With the social production activities of human beings, heavy metal pollution such as cadmium (Cd), lead (Pd), mercury (Hg) and chromium (Cr) in the environment is becoming more and more severe. Because heavy metals can combine with biological macromolecules such as nucleic acids and proteins, causing irreversible changes in the structure of biological macromolecules, or generating free radicals that cause damage to cell structure and function, and even cell death, heavy metals have obvious cyto...
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