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Application of lentinus edodes manganese superoxide dismutase LeMn-SOD to improving stress resistance of microorganisms

A superoxide and dismutase technology, applied in the directions of microorganism-based methods, microorganisms, oxidoreductases, etc., can solve problems such as limited improvement ability, achieve the improvement of cold stress tolerance and salt stress tolerance, improve stress tolerance, The effect of improving cold stress tolerance and salt stress tolerance

Active Publication Date: 2020-11-03
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the heterologous expression of manganese superoxide dismutase VMn-SOD in variegated mushroom can improve the cold tolerance and salt tolerance of Escherichia coli, its ability to improve is still limited.

Method used

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  • Application of lentinus edodes manganese superoxide dismutase LeMn-SOD to improving stress resistance of microorganisms
  • Application of lentinus edodes manganese superoxide dismutase LeMn-SOD to improving stress resistance of microorganisms
  • Application of lentinus edodes manganese superoxide dismutase LeMn-SOD to improving stress resistance of microorganisms

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Construction of manganese superoxide dismutase LeMn-SOD overexpression vector pBARGPE1 / LeMn-SOD in Lentinus edodes 18

[0049] First, design primers to amplify the LeMn-SOD fragment of the target gene from the original plasmid, and then recombine it into the overexpression vector after digestion through the seamless cloning recognition sites at both ends of the primer; transfer the ligated product into the preparation For the competent cells of Escherichia coli Stbl3, the grown monoclonal colonies were sent to Sangon Bioengineering (Shanghai) Co., Ltd. for sequencing and identification, and the correct clone sequence was the successful construction of the overexpression plasmid pBAR GPE1 / LeMn -SOD, after successful construction, the map is as follows Figure 5 shown.

[0050] 1. Design and synthesize primers

[0051] (1) Design PCR amplification fragment primers, and introduce homologous sequences at the ends of the linearized cloning vector at the 5' end of...

Embodiment 2

[0087] The prokaryotic expression of embodiment 2 mushroom LeMn-SOD gene

[0088] 1. Inoculate a single colony of Escherichia coli Stbl3 containing pBAR GPE1 / LeMn-SOD in 100mL LB liquid medium containing 100μg / 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.

[0089] 2. Extract prokaryotic expressed protein for SDS-PAGE electrophoresis

[0090] ①Protein sample preparation

[0091] (1) Take 1 mL of the bacterial solution from step 1 and add it to 100 mL of LB liquid medium containing 100 μg / mL Amp, incubate at 37°C and 220 r / min for 2.5-3 hours, and detect with a UV spectrophotometer when the OD600 reaches 0.4-0.6, Add IPTG at a concentration of 1 mM for induction;

[0092] (2) Aft...

Embodiment 3

[0106] Example 3 Research on cold-resistant function of Lentinus edodes LeMn-SOD protein

[0107] 1. Take 50μL of the verified bacterial solution, inoculate it into 50mL LB liquid medium containing 100μg / mL Amp, incubate at 37°C and 150r / min for 2.5-3h, detect with a UV spectrophotometer when the OD600 reaches 0.4-0.6, Add 1mM IPTG for induction;

[0108] 2. Placed in an incubator at 4°C, subjected to cold stress for 24h, 36h, 48h, 60h, 72h;

[0109] 3. Take 3 mL of the bacterial liquid after cold stress treatment, detect the absorbance at 600 nm with an ultraviolet spectrophotometer, and record the data. Three replicates were set for each experimental gradient.

[0110] 4. Take the absorbance of the OD600 of the bacterium solution at 0h as a control, calculate the growth rate of Escherichia coli containing pBAR GPE1 / LeMn-SOD and Escherichia coli containing pBAR GPE1, and make a line graph of growth rate after cold stress (note: growth rate=cold-treated Escherichia coli OD6...

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Abstract

The invention discloses an application of lentinus edodes manganese superoxide dismutase LeMn-SOD to improving the stress tolerance of microorganisms. The amino acid sequence of the lentinus edodes manganese superoxide dismutase LeMn-SOD is shown as SEQ ID NO.1, and the nucleotide sequence of a coding gene of the lentinus edodes manganese superoxide dismutase LeMn-SOD is shown as SEQ ID NO.2. Thelentinus edodes manganese superoxide dismutase LeMn-SOD gene is transferred into a suitable microbial host, so that the host expresses manganese superoxide dismutase, and the cold stress resistance and salt stress resistance of host cells are improved. The lentinus edodes manganese superoxide dismutase LeMn-SOD disclosed by the invention is derived from fungi and can be transferred into a suitablemicrobial host, so that the stress resistance of the host is improved.

Description

technical field [0001] The invention belongs to the technical field of antioxidant enzymes, and relates to the application of mushroom manganese superoxide dismutase LeMn-SOD in improving the stress resistance ability of microorganisms. Background technique [0002] Lentinula edodes (Berk.) Pegler belongs to Basidiomycota, Agaricaceae, Agaricales, Phytophthora, and Lentinula genus in taxonomy. Lentinus edodes belongs to wood rot fungi, and its cultivation and production require the degradation of lignin to provide sufficient nutrition for mycelial growth and fruiting body development, and it is a medium-low temperature edible fungus, and the growth rate of mycelium decreases significantly at 30°C (Wang Bo, Tang Limin , Xiong Ying, Jiang L, Xian Ling. Experimental study on the growth of mycelia and fruiting bodies of Lentinus edodes strains against high temperature. Journal of Jilin Agricultural University, 2004, 26(2): 145-147), high temperature directly leads to the reducti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/70C12N1/21C12R1/19
CPCC12N9/0089C12N15/70C12Y115/01001
Inventor 赵妍陈明杰杨焕玲游华芳余昌霞李治平黄波常婷婷
Owner SHANGHAI ACAD OF AGRI SCI
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