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The application of manganese superoxide dismutase vmn-sod of variegated mushroom in improving the stress tolerance of microorganisms

A technology of superoxide and dismutase, applied in the directions of oxidoreductase, application, biochemical equipment and methods, etc., can solve the problems of unseen manganese superoxide dismutase research, etc. The effect of improving stress capacity, cold stress tolerance and salt stress tolerance

Active Publication Date: 2020-08-07
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The SOD used in the above studies are all derived from animals, plants or microalgae, and there is no related research on manganese superoxide dismutase in edible fungi

Method used

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  • The application of manganese superoxide dismutase vmn-sod of variegated mushroom in improving the stress tolerance of microorganisms
  • The application of manganese superoxide dismutase vmn-sod of variegated mushroom in improving the stress tolerance of microorganisms
  • The application of manganese superoxide dismutase vmn-sod of variegated mushroom in improving the stress tolerance of microorganisms

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Construction of Manganese Superoxide Dismutase VMn-SOD Overexpression Vector pBAR GPE1 / VMn-SOD in Volvariella volvacea V23

[0049] Firstly, cut out the target fragment through the restriction sites at both ends of the target fragment Mn-SOD VMn-SOD gene, and connect it into the overexpression vector pBAR GPE1 after the same digestion; then connect the The product was transferred into the prepared Escherichia coli Stbl3 competent cells, and the grown monoclonal colonies were sent to the sequencing company for sequencing. The correct clone was the successful construction of the target gene overexpression vector pBAR GPE1 / VMn-SOD.

[0050] 1. Double digestion of vector pBAR GPE1

[0051] (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.

[0052] (2) Take 1 μg of fresh plasmid and ...

Embodiment 2

[0080] The prokaryotic expression of embodiment 2 Volvariella volvacea VMn-SOD gene

[0081] 1. Inoculate a single colony of Escherichia coli Stbl3 containing pBAR GPE1 / VMn-SOD in 100mL LB liquid medium containing 50μg / mL Amp, cultivate at 220rpm and 37℃ until the OD600 reaches 0.6-0.8, and then add IPTG with a concentration of 1mM Into 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.

[0082] 2. Extract prokaryotic expressed protein for SDS-PAGE electrophoresis

[0083] ①Protein sample preparation

[0084] (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;

[0085] (2) After add...

Embodiment 3

[0099] Example 3 Research on Heat-resistant Function of Volvariella volvacea VMn-SOD Protein

[0100] 1. Take 50μL of the verified bacterial solution, inoculate it into 50mL LB liquid medium containing Amp, incubate at 37°C and 150r / min for 2.5-3h, use a UV spectrophotometer to detect when the OD600 reaches 0.4-0.6, add 1mM IPTG is induced;

[0101] 2. Place in an incubator at 50°C, heat shock for 0min, 30min, 60min, 90min and 120min respectively;

[0102] 3. Take 3 mL of the heat-shock treated bacterial liquid every 30 minutes, detect the absorbance at 600 nm with an ultraviolet spectrophotometer, and record the data. Three replicates were set for each experimental gradient.

[0103] 4. Treat the absorbance of 0min bacterium solution OD600 as a control group respectively, calculate its Escherichia coli growth rate, make the growth rate (note: growth rate=treatment Escherichia coli OD600 / control group OD600) line graph after heat stress, as Figure 7 shown.

[0104] Depend...

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Abstract

The invention discloses the application of a straw mushroom manganese superoxide dismutase VMn-SOD in improving the stress resistance ability of microorganisms. According to the straw mushroom manganese superoxide dismutase VMn-SOD, an amino acid sequence is shown as SEQ ID NO.1, and a nucleotide sequence of a coding gene is shown as SEQ ID NO.2. The straw mushroom manganese superoxide dismutase VMn-SOD gene is transferred into a suitable microbial host body, so that a host expresses the manganese superoxide dismutase, the heat stress resistance, cold stress resistance and salt stress resistance ability of the host are improved. Straw mushroom catalase VCAT is derived from fungi and transferred into the suitable microbial host, and the temperature stress resistance ability and the salt stress resistance ability of the host are improved.

Description

technical field [0001] The invention belongs to the technical field of antioxidant enzymes, and relates to the application of manganese superoxide dismutase VMn-SOD of straw mushroom in improving the stress resistance ability of microorganisms. Background technique [0002] Straw mushroom [Volvariella volvacea (Bull.) Singer] is a delicious edible fungus native to tropical and subtropical regions of China. The optimum growth temperature of the body is 32-35°C. Because straw mushroom is a high-temperature mushroom species, its mycelium or fruiting body will appear low-temperature autolysis under normal refrigerated conditions of 0-4°C, which manifests as tissue softening, liquefaction, and decay. 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] When organisms are subjected to heat stress, the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N15/70C12N15/66
CPCC12N9/0089C12N15/66C12N15/70C12Y115/01001
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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