CN110577957B

CN110577957B - Application of straw mushroom manganese superoxide dismutase VMn-SOD in improving stress tolerance of microorganisms

Info

Publication number: CN110577957B
Application number: CN201910933458.5A
Authority: CN
Inventors: 赵妍; 杨焕玲; 陈明杰; 游华芳; 余昌霞; 李正鹏; 奚莉萍; 冯爱萍
Original assignee: Shanghai Academy of Agricultural Sciences
Current assignee: Shanghai Academy of Agricultural Sciences
Priority date: 2019-09-29
Filing date: 2019-09-29
Publication date: 2020-08-07
Anticipated expiration: 2039-09-29
Also published as: CN110577957A

Abstract

The invention discloses an application of straw mushroom manganese superoxide dismutase VMn-SOD in improving the stress tolerance of microorganisms, wherein the amino acid sequence of the straw mushroom manganese superoxide dismutase VMn-SOD is shown as SEQ ID NO.1, and the nucleotide sequence of the coding gene thereof is shown as SEQ ID NO. 2. The volvariella volvacea manganese superoxide dismutase VMn-SOD gene is transferred into a proper microbial host to enable the host to express the manganese superoxide dismutase, so that the heat stress resistance, the cold stress resistance and the salt stress resistance of the host are improved. The volvaria volvacea manganese superoxide dismutase VMn-SOD is derived from fungi, is transferred into a suitable microbial host, and can improve the temperature stress resistance and salt stress resistance of the host.

Description

Application of straw mushroom manganese superoxide dismutase VMn-SOD in improving stress tolerance of microorganisms

Technical Field

The invention belongs to the technical field of antioxidant enzymes, and relates to application of straw mushroom manganese superoxide dismutase (VMn-SOD) in improving stress tolerance of microorganisms.

Background

Volvariella volvacea (Bull.) Singer is a delicious edible fungus native to tropical and subtropical regions of China, belonging to Basidiomycetes, Agaricales, Pholiopsidae and Hypsizygus marmoreus, and has an optimal growth temperature of 32-35 deg.C. As the volvariella volvacea belongs to high-temperature mushroom species, the mycelium or the fruit body of the volvariella volvacea can generate the phenomenon of low-temperature autolysis under the conventional refrigeration condition of 0-4 ℃, and the phenomenon is manifested as softening, liquefaction, rotting and the like of tissues. The low temperature intolerance of the straw mushroom seriously affects the low-temperature preservation of strains and the postharvest storage and transportation of sporocarp, and hinders the rapid development of the straw mushroom industry.

When the organism is stressed by heat, the dynamic balance of active oxygen is destroyed, the ROS in the cell is excessively accumulated and generates toxicity, and the excessive ROS in the cell is very necessary to be removed in time. Organisms generally have both enzymatic and non-enzymatic scavenging mechanisms for scavenging ROS. Superoxide dismutase (SOD), Catalase (CAT), Ascorbate Peroxidase (APX), Glutathione Reductase (GR), Glutathione Peroxidase (GPX) and the like belong to enzymatic mechanisms. The enzyme in the antioxidant enzyme system that first scavenges ROS is SOD. SOD can be divided into Cu/Zn-SOD, Mn-SOD and Fe-SOD according to different metal coenzyme factors. Fe-SOD is mainly present in prokaryotic cells and some plants; Cu/Zn-SOD mainly exists in cytoplasm of eukaryotic cells, chloroplast and peroxidase body; while Mn-SOD exists mainly in mitochondria and prokaryotic cells of eukaryotic cells. SOD catalyzes O under the action of adversity stress^2·-Disproportionation to H₂O₂And O₂Thereby removing O^2·-The probability of producing OH & is reduced. When an organism is stressed by adversity, Mn-SOD positioned in mitochondria is used as a first defense line for eliminating superoxide anions, and the expression and regulation in the organism play an important role in maintaining the redox stable state of the organism.

In agriculture, Mn-SOD is found to improve stress resistance of crops, such as cold resistance, drought resistance and other characteristics, Bowler C and the like improve Mn-SOD expression level of tobacco and corn plants through a transgenic method, and Mn-SOD overexpression in tobacco and corn chloroplasts is found to enhance protection effect of transgenic tobacco and corn on plasma membranes and tolerance to oxygen stress caused by herbicides (Bowler C, Slooten L, Vandenbranden S, et al. Manganese Suspenide mutant strain cell and gene expression of Mn-SOD in tobacco chloroplast is found to be obvious after Bowler C, Slooten, Vandenbranden strain S, et al. Manganese Suspend mutant strain cell and gene expression of Titeng Nu and the like, Cugdong wood can quickly induce Mn-SOD expression under adverse conditions such as low temperature, aluminum, salt stress and the like (Mitsuga strain, Japan strain, Sponghuo J. Tougo et al., Japan, Mitsuga and the Mn-SOD expression of Ducheng strain, Mn-SOD is found to be obviously induced by Ducheng algal genes such as Mn-SOD expression of Mn-SOD in cold resistance, Skawasp C, Skawasp et al, Skol et al, Skohlia strain, Skohlrabi et al, Skohlia strain, et al, research, et al, Skohlia strain genes are found to be obviously induced by cold resistance and SOD expression under adverse conditions such as drought resistance and SOD expression of Mn-SOD strain, Skohlia strain, Skohl.

Disclosure of Invention

The invention aims to provide application of straw mushroom manganese superoxide dismutase (VMn-SOD) in improving stress tolerance of microorganisms. The nucleotide sequence of the volvariella volvacea manganese superoxide dismutase VMn-SOD is transferred into a host, and the heat stress resistance, the cold stress resistance and the salt stress resistance of the host are improved by enabling the host to express the manganese superoxide dismutase.

The invention provides application of straw mushroom manganese superoxide dismutase (VMn-SOD) in improving stress tolerance of microorganisms. The microorganism of the present invention may be a fungus or a bacterium. In a particular embodiment of the invention, the microorganism employed is Escherichia coli. The stress tolerance of the invention comprises heat stress tolerance, cold stress tolerance and salt stress tolerance.

The straw mushroom manganese superoxide dismutase VMn-SOD has the amino acid sequence shown in SEQ ID No. 1.

Specifically, the stress tolerance of the microorganism is improved by transferring an expression vector containing the straw mushroom manganese superoxide dismutase VMn-SOD gene into a microorganism host cell.

The coding gene of the volvaria volvacea manganese superoxide dismutase VMn-SOD has a nucleotide sequence shown in SEQ ID No. 2.

In the specific implementation mode of the invention, the expression vector containing the VMn-SOD gene of the straw mushroom manganese superoxide dismutase is pBAR GPE1/VMn-SOD, and the expression vector is constructed by connecting the nucleotide fragment containing the VMn-SOD gene of the straw mushroom manganese superoxide dismutase with pBAR GPE1 plasmid which is subjected to double enzyme digestion by BamH I and EcoRI.

In the specific implementation mode of the invention, the expression vector pBAR GPE1/VMn-SOD containing the straw mushroom manganese superoxide dismutase VMn-SOD gene is transferred into escherichia coli, so that the heat stress resistance, the cold stress resistance and the salt stress resistance of the escherichia coli are improved.

Compared with the prior art, the invention has the following advantages:

the volvariella volvacea manganese superoxide dismutase VMn-SOD gene is transferred into a proper microbial host, so that the manganese superoxide dismutase is expressed by the host, and the heat stress resistance, the cold stress resistance and the salt stress resistance of the host are improved. Experiments prove that the heat stress resistance, cold stress resistance and salt stress resistance of escherichia coli with the transferred straw mushroom manganese superoxide dismutase VMn-SOD gene are improved after the manganese superoxide dismutase is overexpressed. The straw mushroom manganese superoxide dismutase VMn-SOD is derived from fungi, and because the fungi and the bacteria share the same set of codons, the fungi and the bacteria are transferred into a suitable microbial host, so that the stress tolerance of the host is improved.

Drawings

FIG. 1 is a map of overexpression vector pBAR GPE 1;

FIG. 2 is a diagram of the result of predicting the signal peptide of volvaria volvacea manganese superoxide dismutase VMn-SOD;

FIG. 3 is a diagram showing the result of predicting phosphorylation sites of Volvariella volvacea manganese superoxide dismutase VMn-SOD;

FIG. 4 is a diagram showing the result of predicting the transmembrane structure of Volvariella volvacea manganese superoxide dismutase VMn-SOD;

FIG. 5 is the PCR identification electropherogram of pBAR GPE1/VMn-SOD recombinant plasmid;

FIG. 6 is an SDS-PAGE electrophoresis of pBAR GPE1/VMn-SOD recombinant expression protein product, wherein lane 1 is an electropherogram of Escherichia coli expression protein product of control group containing pBAR GPE1 empty vector without IPTG induction; lane 2 is an electropherogram of the control E.coli expression protein product after IPTG induction, containing pBAR GPE1 empty vector; lane 3 is protein Marker; lane 4 is an E.coli protein expression electropherogram containing pBAR GPE1/VMn-SOD recombinant plasmid without IPTG induction; lane 5 is an electropherogram of E.coli proteins containing pBAR GPE1/VMn-SOD recombinant plasmid after IPTG induction;

FIG. 7 is a comparison of growth rates of Escherichia coli before and after transferring into Volvariella volvacea VMn-SOD gene under heat stress (50 ℃);

FIG. 8 is a comparison of growth rates of Escherichia coli before and after transferring into Volvariella volvacea VMn-SOD gene under cold stress (4 ℃);

FIG. 9 shows the comparison of the growth rate of Escherichia coli before and after transferring into Volvariella volvacea VMn-SOD gene under salt stress (1%).

Detailed Description

The invention is further illustrated below with reference to specific embodiments and the accompanying drawings. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

The invention takes straw mushroom strain V23 as a material, constructs a local database through straw mushroom genome, finds out straw mushroom manganese superoxide dismutase VMn-SOD gene conserved sequence through local Blast, uses CE Design V1.04 to Design a primer of straw mushroom VMn-SOD gene, amplifies VMn-SOD whole gene segment from straw mushroom genome DNA, connects and converts a target segment and a pBAR GPE1 carrier into escherichia coli after PCR identification, and sends the escherichia coli to a biological engineering (Shanghai) limited company for sequencing, wherein the segment length is 537bp, as shown in SEQ ID NO.2, and codes 178 amino acids, as shown in SEQ ID NO. 1.

The bioinformatics analysis shows that the quantity of the straw mushroom manganese superoxide dismutase VMn-SOD amino acid is 178, the molecular weight of the protein is about 19.53kDa, and the isoelectric point (pI) is 6.75; the instability coefficient is 33.84, belonging to stable protein; the hydrophobicity coefficient is-0.204, which indicates that the protein has hydrophilicity. The NetPhos 3.1 software predicts that the phosphorylation sites of Ser of Volvariella volvacea VMn-SOD protein are 6, the phosphorylation sites of Thr are 4 and the phosphorylation sites of Tyr are 3 (figure 3). Signal peptide prediction is carried out on straw mushroom VMn-SOD by using SignalP 5.0 (figure 2), and the result shows that the probability of the protein signal peptide is 0.0003, and the probability of other protein signal peptides is 0.9997, so that the straw mushroom manganese superoxide dismutase VMn-SOD can be judged to have no signal peptide and does not belong to secretory protein. According to prediction of an EMBnet Tmpred transmembrane structure (figure 4), firstly, from the N end, the membrane is transmembrane from inside to outside and then from outside to inside, and then the membrane is transmembrane for 3 times, and according to a prediction result, the Volvaria volvacea VMn-SOD is a bidirectional transmembrane protein which is possibly related to membrane positioning and transmembrane transport.

The invention connects the obtained Volvaria volvacea manganese superoxide dismutase VMn-SOD gene sequence, the nucleotide sequence shown as SEQ ID NO.2 and an expression vector pBAR GPE1 to construct a prokaryotic expression vector pBAR GPE1/Mn-SOD, transfers the prokaryotic expression vector pBAR GPE1/Mn-SOD into escherichia coli, carries out heterologous expression by IPTG induction, extracts expression protein and analyzes by SDS-PAGE electrophoresis, and the result shows that an obvious protein band appears between 15kDa and 25kDa, which is consistent with the expected result.

After escherichia coli over-expressing Volvariella volvacea VMn-SOD is subjected to salt stress (1%) at high temperature (50 ℃) and low temperature (4 ℃), the growth rate of the escherichia coli is measured, and the results show that the heterologous expression of Volvariella volvacea manganese superoxide dismutase VMn-SOD protein can obviously improve the heat resistance, cold resistance and salt tolerance of the escherichia coli, and are respectively shown in fig. 7, fig. 8 and fig. 9.

Test materials

1.1 E.coli strains: stbl3 strain.

1.2 vectors

The expression vector was pBAR GPE1 (purchased from Ghman Biotech Co., Ltd.) having a total length of 5518bp and carrying an ampicillin (Amp) resistance gene.

2. Reagent

TABLE 1 reagents and sources

3. Instrument for measuring the position of a moving object

TABLE 2 instruments and sources

Name of instrument	Source of instruments
		Voltage-stabilizing electrophoresis apparatus	Bio-Rad
Gel imager	Haimen Kylin-Bell Lab Instruments Co.,Ltd.
		Bacteria shaking table	SHANGHAI YIHENG INSTR Co.,Ltd.
Bacteria incubator	SHANGHAI YIHENG INSTR Co.,Ltd.
		PCR instrument	Eppendorf
High-speed centrifugal machine	Thermo
		Disposable plate	Dust living things in Shanghai
50ml polypropylene tube	Corning
		Liquid transfer device	Gilson

4. Preparation of commonly used solutions

4.1 ampicillin stock:

l g ampicillin was dissolved in 10m L deionized water to a final concentration of 100mg/m L, sterilized by filtration through a 0.22 μm microporous membrane, and stored at-20 ℃ until use.

4.2IPTG：

IPTG was prepared as a 24mg/m L (100mM) aqueous solution, which was sterilized by filtration through a 0.22 μm microfiltration membrane and aliquoted for storage at-20 ℃ until use.

4.3PBS buffer:

NaCl 137mmol/L，KCl 2.7mmol/L，Na₂HPO₄10mmol/L，KH₂PO₄1.76 mmol/L, adding distilled water to reach 1000m L, and adjusting pH to 7.2-7.4.

4.4 electrode Buffer (Running Buffer):

3.1g of Tris, 18.8g of glycine, l g of SDS, and adding distilled water to make the volume to be 1L.

Example 1 construction of manganese superoxide dismutase VMn-SOD overexpression vector pBAR GPE1/VMn-SOD in Volvariella volvacea V23

Firstly, cutting out a target fragment through enzyme cutting sites at two ends of a VMn-SOD gene of a target fragment straw mushroom manganese superoxide dismutase, and connecting the target fragment to an overexpression vector pBAR GPE1 which is subjected to enzyme cutting; then transferring the ligation product into a prepared competent cell of escherichia coli Stbl3, sequencing the grown monoclonal colony by a sequencing company, and obtaining a clone with correct comparison, namely the successfully constructed target gene overexpression vector pBAR GPE 1/VMn-SOD.

1. Double digestion of vector pBAR GPE1

(1) Stbl3 bacterial liquid containing pBAR GPE1 vector is cultured overnight, and 3-5m L of fresh bacterial liquid is taken to extract plasmid.

(2) Mu.g of fresh plasmid was digested simultaneously with the restriction enzymes BamH I and EcoRI. The enzyme digestion system is as follows:

carrier	1μg
		green Buffer	3μL
BamH I	1.5μL
		EcoRI	1.5μL
ddH2O	Complement
		30 μ L

The cleavage was carried out at 37 ℃ for about 3 h.

(3) Cutting off an adhesive tape containing a target fragment under an ultraviolet lamp, calculating the weight of the gel by using the balance total weight and subtracting the weight of an empty tube, calculating the volume of the gel according to 100mg to 100 mu L, adding a Binging Solution with the volume being 1 time of the volume of the gel, putting the Binging Solution into a water bath kettle at 65 ℃ to completely melt the gel, and appropriately shaking an EP tube during the process to accelerate the dissolution of the gel.

(4) Transferring all the above liquid into a filter column, centrifuging at 13000rpm for 30S (repeated once), then discarding the liquid in the tube, adding WASolution of 500 mu L into the column, centrifuging at 13000rpm for 30S, discarding the liquid in the tube, adding Wash Solution of 500 mu L into the column, centrifuging at 13000rpm for 30S (repeated once)Times) then empty for 3min, the column was placed in a new 1.5m L EP tube and allowed to air dry at room temperature, finally 35 μ L ddH was added to the column₂O, standing for 5min, and centrifuging at 13000rpm for 1.5 min. To increase the recovery rate, the dissolved DNA can be added to the column again and centrifuged for 1 min. The column was discarded, the recovered vector fragment, and the concentration was determined.

2. Enzyme digestion of plasmid containing volvariella volvacea manganese superoxide dismutase VMn-SOD gene

(1) The plasmid containing the VMn-SOD gene is subjected to double enzyme digestion by restriction enzymes BamH I and EcoRI, and the enzyme digestion system is as follows:

VMn-SOD plasmid	0.7μg
		green Buffer	5μL
BamH I	2.5μL
		EcoRI	2.5μL
ddH₂O	Complement 50 μ L

The cleavage was carried out at 37 ℃ for about 1 h.

(2) The enzyme digestion product is subjected to agarose electrophoresis and the target fragment is recovered, the method is the same as the above.

3. Ligation of overexpression vectors to fragments of interest

(1) Determining the concentration of the recovered vector pBAR GPE1 and the target fragment straw mushroom manganese superoxide dismutase VMn-SOD, and according to the vector: the molar ratio of the target fragment to 1:7 was used to calculate the required volume ratio of the vector to the target fragment.

(2) And (3) connecting the overexpression vector pBAR GPE1 with a target fragment straw mushroom manganese superoxide dismutase (VMn-SOD).

The linking system is as follows:

recovery carrier	70ng
		Segment of interest	Calculated value
T4 DNA ligation buffer	2μL
		T4 DNA ligase	1μL
ddH₂O	Complement 20 μ L

Ligation was carried out at 22 ℃ for 60 min.

4. Transformation of

(1) The competent cells Stbl3 were placed on ice (4 ℃ C.) and allowed to thaw spontaneously, and 10. mu. L of the ligation product was added to the competent cells and allowed to stand on ice (4 ℃ C.) for 30 min.

(2) Then, the mixture was heat-shocked for 90 seconds in a water bath at 42 ℃ and then rapidly placed on ice (4 ℃) for 2-3 min.

(3) 500 μ L antibiotic-free L B medium was added and the mixture was cultured at 37 ℃ for 45min with shaking at 225 rpm.

(4) Centrifuging at 3000rpm for 2min, discarding supernatant of 900 μ L, blowing the bacterial liquid at the bottom of the tube, adding into culture plate containing carrier corresponding resistance (ampicillin), coating with sterilized coater (the temperature of the coater cannot be too high to avoid killing thallus), and culturing overnight in 37 deg.C constant temperature incubator.

5. Sequencing

(1) And picking a plurality of single colonies, and carrying out a small amount of shake culture.

(2) PCR verification is carried out, and 3 positive clones are selected and sent to the company for sequencing.

The primer sequences are as follows:

VMn-SOD-F(SEQ ID NO.3):5'-ATGGCCCACACTCTCCCTG-3'；

VMn-SOD-R(SEQ ID NO.4):5'-TCAACTTACATCGGCCTTGACA-3'。

(3) the correctly sequenced bacterial solution was added to a sterile EP tube at a ratio of 1:1 with 15% glycerol and bacterial solution, and then stored at-80 ℃.

Example 2 prokaryotic expression of Volvariella volvacea VMn-SOD Gene

1. A single colony of Escherichia coli Stbl3 inoculated with pBAR GPE1/VMn-SOD was cultured in L B liquid medium 100m L containing 50. mu.g/m L Amp at 220rpm 37 ℃ until OD600 reached 0.6-0.8, then IPTG at a concentration of 1mM was added to 100m L liquid L B, and culturing was continued at 220rpm 37 ℃ for 4 hours for the purpose of optimizing the induction culture conditions, while Stbl3 was transformed with the empty vector pBAR GPE1 as a control.

2. Extracting prokaryotic expression protein to carry out SDS-PAGE electrophoresis

① protein sample preparation

(1) Adding 1m L of the bacterial liquid obtained in the step 1 into 100m L of L B liquid culture medium containing Amp, culturing at 37 ℃ and 220r/min for 2.5-3h, and adding IPTG (isopropyl-beta-thiogalactoside) with the concentration of 1mM for induction when OD600 reaches 0.4-0.6 by using an ultraviolet spectrophotometer;

(2) adding IPTG, continuing culturing for 6h, transferring the bacterial liquid into a 50m L sterile centrifuge tube, and centrifuging for 10min at 4 ℃ under the condition of 5000 r/min;

(3) adding PBS (3 m L) into a sterile centrifuge tube, sucking and uniformly mixing the thalli by using a gun head, adding 2.4 mu L Protease Inhibitor Cocktail to protect protein from being damaged, adding 200 mu L lysate, standing for 30min at minus 4 ℃, and centrifuging the crushed thalli for 10min at 4 ℃ and 8000 r/min;

(4) and (3) putting 50 mu L of the centrifuged supernatant into a sterile centrifuge tube, adding 12.5 mu L of 5 × protein L oadingBuffer, boiling in a metal water bath at 100 ℃ for 10min, and storing at-80 ℃.

② SDS-PAGE gel electrophoresis

(1) Preparing a glass plate, washing an electrophoresis rack, an electrophoresis tank and the like by using clear water, wiping the glass plate clean by using filter paper, and then installing an electrophoresis device;

(2) determining the concentration of gel, preparing gel according to the kit, and preparing 15% separation gel and 3% concentrated gel; after coagulation, inserting a comb, placing in a constant temperature incubator at 37 ℃ for 30min, and taking care to avoid generating bubbles during insertion;

(3) after the concentrated gel is fixed, pulling out the comb, cleaning the gel plate by using double distilled water, fixing the gel plate in an electrophoresis tank, injecting the prepared electrophoresis buffer solution into an upper electrophoresis tank and a lower electrophoresis tank, and checking whether the electrophoresis tank leaks or not;

(4) sequentially injecting 20 mu L protein samples into the loading holes;

(5) when the gel is concentrated, the voltage is 80V; the voltage of the separation gel is 120V;

(6) after electrophoresis, putting the gel into a clean box, adding Coomassie brilliant blue dye solution, and dyeing for 60 min;

(7) after dyeing, placing the Coomassie brilliant blue dye solution in a recovery bottle;

(8) adding a destaining solution, covering the surface of the gel, and changing the destaining solution for 1h until the gel is transparent, so that clear protein bands can be observed;

(9) the protein gel was photographed and then the results were analyzed.

The electrophoresis result is shown in figure 6, an obvious protein band appears at the position close to 20kDa, and the expression quantity of the protein band is obviously higher than that of an empty vector control group, which indicates that the volvariella volvacea VMn-SOD is successfully expressed heterologously in escherichia coli.

Example 3 study of Volvariella volvacea VMn-SOD protein Heat resistance function

1. Sucking bacterial liquid with the concentration of 50 mu L to verify the correctness, inoculating the bacterial liquid into a liquid culture medium with the concentration of 50m L L B and containing Amp, culturing the bacterial liquid at the temperature of 37 ℃ and the speed of 150r/min for 2.5 to 3 hours, and adding 1mM IPTG for induction when OD600 is detected to reach 0.4 to 0.6 by an ultraviolet spectrophotometer;

2. placing in 50 deg.C incubator, and heat-shocking for 0min, 30min, 60min, 90min and 120min respectively;

3. taking the thermally excited bacterial liquid 3m L every 30min, detecting the absorbance under 600nm by using an ultraviolet spectrophotometer, and recording data, wherein 3 times of experiments are set in a gradient mode.

4. The absorbance of the treated bacteria liquid OD600 at 0min was used as a control group, and the growth rate of escherichia coli was calculated, and a line graph of the growth rate after heat stress (i.e., the growth rate of treated escherichia coli OD 600/the control group OD600) was prepared, as shown in fig. 7.

As can be seen from FIG. 7, the growth rates of Escherichia coli containing pBARGPE1/VMn-SOD at 30min, 60min, 90min and 120min of heat stress were respectively increased by 11.52%, 12.43%, 16.47% and 18.06% compared with the growth rates of Escherichia coli containing pBAR GPE1 empty vector control group, which indicates that the introduction and expression of Volvaria volvacea manganese superoxide dismutase VMn-SOD gene actually increases the heat resistance of Escherichia coli Stbl 3.

Example 4 study of Volvariella volvacea VMn-SOD protein Cold-tolerant function

2. placing in an incubator at 4 ℃ and carrying out cold stress for 2d, 4d, 6d and 8 d;

3. taking the bacterial liquid 3m L after cold stress treatment every 2d, detecting the absorbance under 600nm by using an ultraviolet spectrophotometer, and recording data, wherein 3 times of the experiment gradient are set.

4. The growth rate of escherichia coli was calculated using the absorbance of the treated 2d bacterial suspension OD600 as a control group, and a line graph of the growth rate after cold stress was prepared (note: growth rate ═ treated escherichia coli OD 600/control group OD600) as shown in fig. 8.

As can be seen from FIG. 8, the growth rates of Escherichia coli containing pBAR GPE1/VMn-SOD were 8.13% and 8.74% higher than that of Escherichia coli containing pBAR GPE1 empty vector control group respectively under cold stress of 6d and 8d, which indicates that the introduction and expression of Volvaria volvacea manganese superoxide dismutase VMn-SOD gene indeed improves the cold tolerance of Escherichia coli Stbl 3.

Example 5 study of salt tolerance of Volvariella volvacea VMn-SOD protein

1. Sucking a bacterial liquid with the concentration of 50 mu L to verify the correctness, inoculating the bacterial liquid into a liquid culture medium with the concentration of 1 percent and the concentration of Amp of 50m L L B, culturing the bacterial liquid at 37 ℃ for 2.5 to 3 hours at the speed of 150r/min, and adding 1mM IPTG for induction when the OD600 is detected to reach 0.4 to 0.6 by an ultraviolet spectrophotometer;

2. culturing in an incubator at 37 deg.C for 4h, 8h, 10h, 12h, and 14 h;

3. taking the bacterial liquid 3m L after salt stress treatment, detecting the absorbance under 600nm by using an ultraviolet spectrophotometer, and recording data, wherein 3 times of experiments are set in a gradient mode.

4. The absorbance of the treated 4h bacterial suspension OD600 was used as a control group, the growth rate of Escherichia coli was calculated, and a line graph of the growth rate after salt stress was prepared as shown in FIG. 9 (Note: growth rate ═ treated Escherichia coli OD 600/control group OD 600).

As can be seen from FIG. 9, the growth rate of Escherichia coli containing pBAR GPE1/VMn-SOD during 1% salt stress was increased by 23.90%, 18.18%, 20.29% and 13.05% respectively compared with that of Escherichia coli containing pBAR GPE1 empty vector control group, which indicates that the introduction and expression of Volvaria volvacea manganese superoxide dismutase VMn-SOD gene indeed increases the salt tolerance of Escherichia coli Stbl 3.

Sequence listing

<110> Shanghai city academy of agricultural sciences

Application of <120> straw mushroom manganese superoxide dismutase (VMn-SOD) in improving stress tolerance of microorganisms

<160>4

<170>SIPOSequenceListing 1.0

<210>1

<211>178

<212>PRT

<213>Volvariella volvacea

<400>1

Met Ala His Thr Leu Pro Asp Leu Pro Tyr Asp Tyr Asn Ala Leu Glu

1 5 10 15

Pro Phe Ile Ser Glu Gln Ile Met Thr Leu His His Lys Lys His His

20 25 30

Gln Thr Tyr Val Asn Ala Leu Asn Ala Ala Glu Glu Ala Tyr Ala Arg

35 40 45

Ala Ser Thr Pro Lys Glu Arg Ile Ala Leu Gln Ala Ala Leu Lys Phe

50 55 60

Asn Gly Gly Gly His Ile Asn His Ser Leu Phe Trp Lys Asn Leu Ala

65 70 75 80

Pro Ser Ser Ser Lys Gly Gly Asn Gly Gly Val Leu Lys Asp Gly Pro

85 90 95

Leu Lys Asp Ala Ile Ile Arg Ala Phe Gly Ser Val Glu Ala Phe Lys

100 105 110

Lys Glu Phe Asn Thr Thr Thr Ala Ala Ile Gln Gly Ser Gly TrpGly

115 120 125

Trp Leu Gly Leu Asn Pro Ser Thr Lys Val Leu Glu Ile Val Thr Thr

130 135 140

Ala Asn Gln Asp Pro Leu Leu Thr His Ile Pro Ile Ile Gly Val Asp

145 150 155 160

Ile Trp Glu His Ala Phe Tyr Leu Gln Tyr Leu Asn Val Lys Ala Asp

165 170 175

Val Ser

<210>2

<211>537

<212>DNA

<213>Volvariella volvacea

<400>2

atggcccaca ctctccctga tctcccatac gactacaatg ctctcgagcc cttcatctcg 60

gagcagatca tgaccctgca ccacaagaaa caccatcaga cttacgtcaa tgccctcaat 120

gcagccgagg aggcatacgc aagggcttcc acccctaagg agcgcatcgc cctccaggct 180

gctctcaagt tcaacggtgg tggacacatc aaccactccc tcttctggaa gaaccttgcc 240

ccctcctcca gcaagggcgg caacggtggt gttctcaagg atggcccctt gaaggacgct 300

atcattcgcg cattcggaag tgttgaggcc ttcaagaagg agttcaacac caccaccgct 360

gcgatccagg gttctggatg gggctggctc ggtcttaacc catccaccaa ggtcctcgag 420

atcgttacca ccgccaacca agaccctctc ctcacccaca tccccattat cggtgtcgac 480

atctgggagc acgccttcta cctccaatac ttgaatgtcaaggccgatgt aagttga 537

<210>3

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

atggcccaca ctctccctg 19

<210>4

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

tcaacttaca tcggccttga ca 22

Claims

1. The application of straw mushroom manganese superoxide dismutase VMn-SOD in improving the stress tolerance of escherichia coli is disclosed, wherein the amino acid sequence of the straw mushroom manganese superoxide dismutase VMn-SOD is shown as SEQ ID NO. 1; the nucleotide sequence of the coding gene of the volvaria volvacea manganese superoxide dismutase VMn-SOD is shown as SEQ ID NO. 2; the stress tolerance is heat stress tolerance, cold stress tolerance and salt stress tolerance.

2. The use as claimed in claim 1, characterized by the use of a straw mushroom manganese-containing superoxide dismutaseVMn-SODThe expression vector of the gene is transferred into the escherichia coli, so that the stress tolerance of the escherichia coli is improved.

3. The use as claimed in claim 2, wherein the straw mushroom manganese-containing superoxide dismutaseVMn-SODThe expression vector of the gene is pBAR GPE1/VMn-SOD, and the superoxide dismutase containing straw mushroom manganese is prepared byVMn-SODNucleotide fragments and polypeptides of genesBamHI andEcoand (3) carrying out plasmid connection construction on pBAR GPE1 subjected to RI double enzyme digestion.