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Lentinus edodes S-alkyl-L-cysteine sulfoxide lyase gene and application

A technology of cysteinyl sulfoxide and lyase, which is applied in the field of cysteinyl sulfoxide lyase gene and application in shiitake mushrooms, can solve the problem of no cloning gene, difficulty in purification of cysteinyl sulfoxide lyase from shiitake mushrooms, and Unpublished genome data and other issues

Inactive Publication Date: 2016-04-13
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no research report on the identification of cysteinyl sulfoxide lyase at the molecular level of Lentinus edodes, and no gene encoding cysteinyl sulfoxide lyase has been cloned. The reasons are mainly divided into two aspects: one is Due to the difficulty in purifying cysteinyl sulfoxide lyase from Lentinus edodes, the second is that the genome data of Lentinus edodes has not been published

Method used

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  • Lentinus edodes S-alkyl-L-cysteine sulfoxide lyase gene and application
  • Lentinus edodes S-alkyl-L-cysteine sulfoxide lyase gene and application
  • Lentinus edodes S-alkyl-L-cysteine sulfoxide lyase gene and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Cloning of Cysteinyl Sulfoxide Lyase Gene (Csl) from Lentinus edodes

[0029] 1) Extract sample RNA. Weigh 0.5g mushroom fruiting body and put it in a pre-cooled mortar, pour an appropriate amount of liquid nitrogen, grind it into powder quickly, collect the powder in a 1.5mL RNase-free centrifuge tube; add 300μL extraction buffer and 600μL PCI Solution (a solution obtained by mixing water-saturated phenol, chloroform, and isoamyl alcohol at a volume ratio of 25:24:1), shake vigorously on an oscillator for 30s, centrifuge at 12000g for 15min at 4°C; draw the supernatant in another Add an equal volume of PCI solution to a clean 1.5mL RNase-free centrifuge tube, shake gently for 30s, centrifuge at 12000g for 15min at 4°C. After centrifugation, if there is too much solid material in the middle layer, repeat this step once; pipette the supernatant into another clean 1.5mL RNase-free centrifuge tube, add 1 / 10 volume of 3mol / L sodium acetate solution, After mixing, add 2.5 ...

Embodiment 2

[0036] Application of mushroom cysteinyl sulfoxide lyase gene in preparing recombinant protein of mushroom cysteinyl sulfoxide lyase:

[0037] 1) Construction of PMD18-T-Csl plasmid: Use Beijing Biotek DNA Agarose Gel Recovery Kit to recover the full-length cDNA fragment of the cysteinyl sulfoxide lyase gene by gel cutting; mix 4 μL of the DNA fragment with 1 μL PMD18 -T vector was mixed, and then 5 μL of solutionI was added, and ligated overnight at 16°C to construct the PMD18-T-Csl plasmid.

[0038] 2) Plasmids PMD18-T-Csl and PET28a(+) were extracted using the plasmid extraction kit of Beijing Aisijin Company.

[0039] 3) Use NheI and XhoI nucleases to carry out a double enzyme digestion reaction on the plasmid PMD18-T-Csl and the Escherichia coli expression vector PET28a(+). The reaction condition is 8h at 37°C. The enzyme digestion results were detected by electrophoresis, and the enzyme digestion products were gel-cut and recovered.

[0040] 4) Using T4 DNA ligase to c...

Embodiment 3

[0044] Determination of enzyme activity of recombinant cysteinyl sulfoxide lyase by pyruvate method:

[0045] 1) Preparation of pyruvate standard curve. To prepare 1mmol / L pyruvic acid solution, take 0.2, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0mL pyruvic acid solution into 50mL volumetric flasks and dilute to volume; absorb 2mL pyruvic acid solution from each volumetric flask, Add to 7 stoppered test tubes, then add 1mL of 0.1% (m / v) 2,4-dinitrophenylhydrazine solution, keep warm at 25°C for 5min, then add 2.5mL of 2.5mol / L sodium hydroxide Shake the solution well, react for 10 minutes, and compare the color at a wavelength of 520nm, use distilled water instead of pyruvic acid solution as a blank, and record the OD measured by each tube 520nm The absorbance value, and the concentration of pyruvate (μmol / L) as the abscissa, the absorbance value as the ordinate, draw a standard curve.

[0046] 2) Determination of the enzyme activity of recombinant cysteinyl sulfoxide lyase. 0.5 mL of en...

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Abstract

The invention discloses a lentinus edodes S-alkyl-L-cysteine sulfoxide lyase gene and application. The sequence of the lentinus edodes S-alkyl-L-cysteine sulfoxide lyase gene is as indicated by SEQ ID No. 1, and the coded protein is as indicated by SEQ ID No. 2. By means of the gene sequence, a prokaryotic expression vector containing the gene is constructed, and through escherichia coli heterogenous expression, recombined lentinus edodes S-alkyl-L-cysteine sulfoxide lyase having enzymatic activity is obtained. The lentinus edodes S-alkyl-L-cysteine sulfoxide lyase gene obtained through clone is obviously different from other S-alkyl-L-cysteine sulfoxide lyase genes which are reported at present, is a new type of S-alkyl-L-cysteine sulfoxide lyase gene and provides conditions for further research of the evolutionary relationship of the gene family. By means of the lentinus edodes S-alkyl-L-cysteine sulfoxide lyase gene and application, more gene resources are provided for regulating and controlling generation of lentinus edodes endogenous formaldehyde through the genetic engineering technology, and wider thinking is provided for researching food safety problems caused by biological endogenous formaldehyde.

Description

technical field [0001] The invention relates to the technical field of molecular biology of shiitake mushrooms, in particular to a cysteinyl sulfoxide lyase gene of shiitake mushrooms and its application. Background technique [0002] Shiitake mushrooms are deeply loved by consumers due to their delicious taste, strong aroma, and anti-cancer properties. People in more than 60 countries and regions in the world have the habit of consuming shiitake mushrooms. my country is a big country in the production and export of shiitake mushrooms, and the development of the shiitake mushroom industry is related to the overall development of the edible fungus industry in my country. However, the food safety and "green barrier" problems caused by excessive formaldehyde in shiitake mushrooms have plagued the development of shiitake mushroom industry in China. Studies have found that formaldehyde in shiitake mushrooms is gradually formed during the growth and drying of shiitake mushrooms. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/60C12N9/88C12N15/11
CPCC12N9/88C12Y404/01004
Inventor 刘莹黄文雷霄宇高双双王益
Owner HUAZHONG AGRI UNIV
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