阅读说明：本技术 草菇锰超氧化物歧化酶VMn-SOD在提高微生物耐胁迫能力中的应用 (Application of straw mushroom manganese superoxide dismutase VMn-SOD in improving stress tolerance of microorganisms ) 是由 赵妍 杨焕玲 陈明杰 游华芳 余昌霞 李正鹏 奚莉萍 冯爱萍 于 2019-09-29 设计创作，主要内容包括：本发明公开了草菇锰超氧化物歧化酶VMn-SOD在提高微生物耐胁迫能力中的应用,所述的草菇锰超氧化物歧化酶VMn-SOD的氨基酸序列如SEQ ID NO.1所示,其编码基因的核苷酸序列如SEQ ID NO.2所示。本发明通过将草菇锰超氧化物歧化酶VMn-SOD基因转入适合的微生物宿主体内,使宿主表达锰超氧化物歧化酶,从而提高宿主的耐热胁迫能力、耐冷胁迫能力及耐盐胁迫能力。本发明的草菇过氧化氢酶VCAT来源于真菌,转入适合的微生物宿主中,能够提高宿主的耐温度胁迫及盐胁迫的能力。(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 straw mushroom catalase VCAT 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.)

1. The application of straw mushroom manganese superoxide dismutase VMn-SOD in improving the stress tolerance of microorganisms 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 in SEQ ID NO. 2.

2. The use as claimed in claim 1, wherein the primer sequence of the volvaria volvacea manganese superoxide dismutase VMn-SOD gene is as follows:

VMn-SOD-F:5'-ATGGCCCACACTCTCCCTG-3'；

VMn-SOD-R:5'-TCAACTTACATCGGCCTTGACA-3'。

3. use according to claim 1, wherein the microorganism is a fungus or a bacterium.

4. The use according to claim 1, wherein the microorganism is Escherichia coli.

5. The use according to claim 1, wherein the stress tolerance comprises heat stress tolerance, cold stress tolerance and salt stress tolerance.

6. The use as claimed in claim 1, wherein the stress tolerance of the microorganism is improved by transferring an expression vector containing the VMn-SOD gene of volvaria volvacea manganese superoxide dismutase into a host cell of the microorganism.

7. The application as claimed in claim 6, wherein the expression vector containing VMn-SOD gene of straw mushroom manganese superoxide dismutase is pBAR GPE1/VMn-SOD, which is constructed by connecting nucleotide fragment containing VMn-SOD gene of straw mushroom manganese superoxide dismutase with pBAR GPE1 plasmid which is digested by BamH I and EcoRI.

8. The use as claimed in claim 7, wherein the heat stress resistance, cold stress resistance and salt stress resistance of Escherichia coli are improved by transferring expression vector pBAR GPE1/VMn-SOD containing Volvariella volvacea-Mn superoxide dismutase VMn-SOD gene into Escherichia coli.

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. Mn-S located in mitochondria when organisms are stressed by adversityOD is used as the first defense line for eliminating superoxide anions, and the expression and regulation in organisms play an important role in maintaining the redox homeostasis of the organisms.

In agriculture, researches show that Mn-SOD can improve the stress resistance of crops, such as cold resistance, drought resistance and the like. Bowler C et al found that Mn-SOD overexpression in tobacco and corn chloroplasts enhanced the protective effect of transgenic tobacco and corn on plasma membranes and tolerance to herbicide-induced oxygen stress by transgenic methods of Mn-SOD expression in tobacco and corn chloroplasts (Bowler C, Slooten L, Vanderbranden S, et al., Manganie seed superoxide dismutase prepared cellular data medium by oxidative gene irradiation in transgenic plants [ J ]. Embo Journal,1991,10(7): 1723-; studies of Hoolisha and the like show that China fir can rapidly induce the expression of Mn-SOD under adverse conditions of low temperature, drought, aluminum stress, salt stress and the like (China fir Mn-SOD gene expression under different adverse conditions of Hoolisha, Queenbach, Zhang Jiajun, and the like [ J ]. university report of northeast forestry, 2018,46(6): 19-22.). After Dunaliella salina Mn-SOD gene expression vector is used for transfecting SOD deficient escherichia coli and inducing the SOD deficient escherichia coli to express, such as Dengting, the tolerance of the escherichia coli in the aspects of salt tolerance, radiation tolerance and cold resistance is obviously improved (the gene cloning, expression and function research of Dunaliella salina manganese superoxide dismutase (MnSOD) [ D ]. Chengdu university, 2007 ]. The SOD adopted in the research is derived from animals, plants or microalgae, and no related research on manganese superoxide dismutase in edible fungi is found at present.

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/VCAT 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

4. Preparation of commonly used solutions

4.1 ampicillin stock:

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

4.2IPTG：

IPTG was prepared as a 24mg/mL (100mM) aqueous solution, which was sterilized by filtration through a 0.22 μm microporous membrane, aliquoted and stored at-20 ℃ until needed.

4.3PBS buffer:

NaCl 137mmol/L，KCl 2.7mmol/L，Na₂HPO₄ 10mmol/L，KH₂PO₄1.76mmol/L, adding distilled water to reach a constant volume of 1000mL, and adjusting the pH value to 7.2-7.4.

4.4 electrode Buffer (Running Buffer):

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