Application of lentinus edodes glutathione reductase LeGR in improving temperature stress resistance of microorganism

A glutathione and reductase technology, applied in microorganism-based methods, microorganisms, oxidoreductases, etc., can solve the problem of glutathione reductase not seen in mushrooms, and improve cold stress tolerance and heat stress. ability, the effect of improving the ability to withstand temperature stress

Active Publication Date: 2020-12-11
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The GR used in the above methods are all derived from plants, and there is no application of heterologous expression of glutathione reductase LeGR in Lentinus edodes to improve the stress tolerance of microorganisms.

Method used

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  • Application of lentinus edodes glutathione reductase LeGR in improving temperature stress resistance of microorganism
  • Application of lentinus edodes glutathione reductase LeGR in improving temperature stress resistance of microorganism
  • Application of lentinus edodes glutathione reductase LeGR in improving temperature stress resistance of microorganism

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Construction of glutathione reductase LeGR overexpression vector pET30a / LeGR in Example 1 Lentinus edodes 18

[0047] First, design primers to amplify the LeGR fragment of the target gene from the original plasmid, and then recombine it into the digested overexpression vector pET30a through the seamless cloning recognition sites at both ends of the primer; transfer the ligated product into the prepared The competent cells of Escherichia coli LB21 were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing and identification of the grown monoclonal colonies. The correct clone sequence was the successful construction of the overexpression plasmid pET30a / LeGR, and the map was successfully constructed. Such as Figure 5 shown.

[0048] 1. Design and synthesize primers

[0049] (1) Design PCR amplification fragment primers, and introduce homologous sequences at the ends of the linearized cloning vector at the 5' end of the primers, so that the 5' and 3' end sequence...

Embodiment 2

[0085] Prokaryotic expression of embodiment 2 Lentinus edodes LeGR gene

[0086] 1. Inoculate a single colony of Escherichia coli LB21 containing pET30a / LeGR in 100mL LB liquid medium containing 100μg / mL Kan, culture at 220rpm and 37°C until the OD600 reaches 0.6-0.8, and then add IPTG with a concentration of 1mM 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 pET30a was transformed into LB21 as a control.

[0087] 2. Extract prokaryotic expressed protein for SDS-PAGE electrophoresis

[0088] ①Protein sample preparation

[0089] (1) Take 1mL of the bacterial solution from Step 1 and add it to 100mL LB (containing 100μg / mL Kan) liquid medium, incubate at 37°C and 220r / min for 2.5-3h, and use a UV spectrophotometer to detect when the OD600 reaches 0.4-0.6 , adding IPTG with a final concentration of 1 mM for induction;

[0090] (2) After adding...

Embodiment 3

[0104] Example 3 Research on cold-resistant function of Lentinus edodes LeGR protein

[0105] 1. Take 50μL of the verified bacterial solution, inoculate it into 50mL LB (containing 100μg / mL Kan) liquid medium, incubate at 37°C and 150r / min for 2.5-3h, and detect it with a UV spectrophotometer until the OD600 reaches 0.4-0.6 , 1 mM IPTG was added for induction.

[0106] 2. Placed in an incubator at 4°C, and subjected to cold stress for 12h, 24h, 36h, 48h, 60h.

[0107] 3. Take 2 mL of the bacterial liquid after the 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.

[0108] 4. Treat the absorbance of 0h bacterial liquid OD600 as a control group respectively, calculate the growth rate of Escherichia coli containing pET30a / LeGR and pET30a Escherichia coli, and make a line graph of growth rate after cold stress (note: growth rate=treatment Escherichia coli OD...

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Abstract

The invention discloses application of lentinus edodes glutathione reductase LeGR in improving the temperature stress resistance of a microorganism. The amino acid sequence of the lentinus edodes glutathione reductase LeGR is shown as SEQ ID NO.1; and the nucleotide sequence of a coding gene of the lentinus edodes glutathione reductase LeGR is shown as SEQ ID NO.2. According to the invention, thelentinus edodes glutathione reductase LeGR gene is transferred into a suitable microbial host, so that the host expresses glutathione reductase, and the cold stress resistance and heat stress resistance of host cells are improved. The lentinus edodes glutathione reductase LeGR in the invention is derived from fungi and can be transferred into the suitable microbial host; and the temperature stressresistance of the host is improved.

Description

technical field [0001] The invention belongs to the technical field of antioxidant enzymes, and in particular relates to the application of a mushroom glutathione reductase LeGR in improving the ability of microorganisms to withstand temperature stress. Background technique [0002] 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 reduction of yield and quality of Lentinus edodes. Nutrition and temperature are the key factors to improve the yield an...

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
CPCC12N9/0051C12N15/70C12Y108/01007
Inventor 赵妍杨焕玲陈明杰查磊李正鹏黄波宋盼盼常婷婷
Owner SHANGHAI ACAD OF AGRI SCI
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