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Catalase vcat and its coding gene and application

A technology of hydrogen peroxide and coding genes of straw mushrooms, applied in the fields of oxidoreductase, application, genetic engineering, etc., to achieve the effect of improving temperature stress resistance, heat resistance and cold resistance

Active Publication Date: 2020-07-03
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above-mentioned methods all improve the stress resistance of plants by overexpressing CAT, and cultivate high-quality and resistant crop varieties, which are only applicable to plant systems. At present, there is no research on edible fungus catalase

Method used

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  • Catalase vcat and its coding gene and application
  • Catalase vcat and its coding gene and application
  • Catalase vcat and its coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] The construction of catalase VCAT expression vector pBAR GPE1 / VCAT in the embodiment 1 straw mushroom V23

[0048] Firstly, the target fragment was excised through the enzyme cutting sites at both ends of the V. volvulus volvulus catalase VCAT gene, and connected to the overexpression vector pBAR GPE1 after the same enzyme digestion. Then the ligation product was transferred into the prepared bacterial competent cells, and the grown monoclonal colony was sequenced, and the correct clone was the successfully constructed overexpression vector containing the VCAT gene of V. volvulus catalase.

[0049] 1. Double digestion of pBAR GPE1 vector

[0050] (1) Cultivate the Stbl3 bacterial liquid containing the pBAR GPE1 vector overnight, and take 3-5 mL of fresh bacterial liquid to extract the plasmid. For specific methods, refer to the instruction manual of QIAGEN plasmid mini-pump.

[0051] (2) Take 1 μg of fresh plasmid and perform double digestion with restriction endonucl...

Embodiment 2

[0078] Embodiment 2 The prokaryotic expression of the volvox mushroom catalase VCAT gene

[0079] 1. Inoculate a single colony of Escherichia coli Stbl3 containing pBAR GPE1 / VCAT in 100 mL of LB liquid medium containing 50 μg / mL Amp, cultivate at 220 rpm at 37 ° C until the OD600 reaches 0.6-0.8, and then add IPTG with a concentration of 1 mM to In 100mL liquid LB, continue to culture at 220rpm, 37°C for 4h, in order to achieve the purpose of optimizing the induction culture conditions. At the same time, the empty vector pBAR GPE1 was transformed into Stbl3 as a control.

[0080] 2. Extract prokaryotic expressed protein for SDS-PAGE electrophoresis

[0081] ①Protein sample preparation

[0082] (1) Take 1 mL of the bacterial solution from Step 1 and add it to 100 mL of LB liquid medium containing Amp, incubate at 37°C and 220 r / min for 2.5-3 hours, detect with a UV spectrophotometer and when the OD600 reaches 0.4-0.6, add a concentration of 1mM IPTG for induction;

[0083] ...

Embodiment 3

[0097] Example 3 Research on the heat-resistant function of VCAT protein of straw mushroom

[0098] 1. Take 50μL of the verified bacterial solution, inoculate it into 50mL LB liquid medium containing Amp, incubate at 37°C, 150r / min for 2.5-3h, detect with a UV spectrophotometer when the OD600 reaches 0.4-0.6, add 1mM IPTG is induced;

[0099] 2. Place in an incubator at 50°C, heat stress 0h, 2h, 4h, 6h, 8h, 10h;

[0100] 3. Take 3 mL of the bacterial solution after heat shock treatment every 2 hours, detect the absorbance at 600 nm with an ultraviolet spectrophotometer, and record the data. Three replicates were set for each experimental gradient.

[0101] 4. Treat the absorbance of the 0h bacterial liquid OD600 as a control group respectively, calculate the growth rate of Escherichia coli, and make a line graph of the growth rate after heat stress, as shown in Figure 7 Shown (note: growth rate = treated E. coli OD600 / control group OD600).

[0102] Depend on Figure 7 It...

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Abstract

The invention discloses volvariella volvacea catalase (VCAT) and an encoding gene and application thereof. The amino acid sequence of the VCAT is shown as the SEQ ID NO.1, and the amino acid sequenceof the encoding gene of the VCAT is shown as the SEQ ID NO.2. The amino acid sequence of the VCAT is transferred into bodies of appropriate hosts, and by enabling the hosts to express the CAT, the heat resistance and the cold resistance of the hosts can be improved. Escherichia coli is taken as a model organism, and the experimental result indicates that after the escherichia coli with a transferred VCAT gene overexpresses the CAT, the heat resistance and the cold resistance are achieved; and the VCAT comes from fungi and can be transferred into the appropriate microorganism hosts, and the temperature-resistant forcing capacity of the hosts is improved.

Description

technical field [0001] The invention belongs to the technical field of antioxidant enzymes, and relates to a straw mushroom catalase VCAT and its coding gene and application. Background technique [0002] Straw mushroom [Volvariella volvacea (Bull.) Singer] belongs to Basidiomycetes, Agaricaceae, Phyllosporaceae, and Phytophthora genus. It is native to tropical and subtropical regions of China. It is delicious and has a broad market. Straw mushroom is a high-temperature edible fungus. The optimum growth temperature of its mycelium is 32-35°C. Mycelium or fruiting body will appear low-temperature autolysis under the normal refrigerated conditions of 0-4°C, which is manifested as tissue change. Soft, liquefied, rotten, etc. The low temperature resistance of straw mushroom has seriously affected the cryopreservation of strains, post-harvest storage and transportation of fruit bodies, and hindered the rapid development of straw mushroom industry. [0003] Catalase (CAT) is one...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/08C12N15/53C12N15/70C12N15/63C12N1/21C12N1/15C12R1/19
CPCC12N9/0065C12N15/63C12N15/70C12Y111/01006
Inventor 赵妍杨焕玲陈明杰游华芳余昌霞李正鹏奚莉萍冯爱萍
Owner SHANGHAI ACAD OF AGRI SCI
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