阅读说明：本技术 草菇过氧化氢酶vcat及其编码基因和应用 (Volvariella volvacea (Volvariella volvacea) catalase VCAT as well as coding gene and application thereof ) 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：本发明公开了一种草菇过氧化氢酶VCAT及其编码基因和应用。所述的草菇过氧化氢酶VCAT的氨基酸序列如SEQ ID NO.1所示,核苷酸序列如SEQ ID NO.2所示。将草菇过氧化氢酶VCAT的核苷酸序列转入适合的宿主体内,通过使宿主表达过氧化氢酶,能够提高宿主的耐热性及耐冷性。本发明以大肠杆菌为模式生物,实验结果表明,转入草菇过氧化氢酶VCAT基因的大肠杆菌过量表达过氧化氢酶后获得了耐热性及耐冷性。本发明的草菇过氧化氢酶VCAT来源于真菌,能够转入适合的微生物宿主中,提高宿主的耐温度胁迫能力。(The invention discloses straw mushroom catalase VCAT and an encoding gene and application thereof. The amino acid sequence of the straw mushroom catalase VCAT is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO. 2. The nucleotide sequence of straw mushroom catalase VCAT is transferred into a proper host body, and the heat resistance and the cold resistance of the host can be improved by enabling the host to express the catalase. The invention takes escherichia coli as a model organism, and experimental results show that heat resistance and cold resistance are obtained after the escherichia coli with the straw mushroom catalase VCAT gene is transferred over to express catalase. The straw mushroom catalase VCAT is derived from fungi, can be transferred into a suitable microbial host, and improves the temperature stress resistance of the host.)

1. Straw mushroom catalase VCAT is characterized in that the amino acid sequence is shown as SEQ ID NO. 1.

2. The coding gene of straw mushroom catalase VCAT is characterized in that the nucleotide sequence is shown in SEQ ID NO. 2.

3. The coding gene of Volvariella volvacea (Volvariella Volvacea) Catalase (VCAT) of claim 2, wherein the primer sequence of the Volvariella volvacea (Volvariella Volvacea) Catalase (VCAT) gene is as follows:

VCAT-F:5'-ATGAGCGGTGTAGCCTCTACTGC-3'；

VCAT-R:5'-TTAGTACGCGACACCACTAGTCAAT-3'。

4. An expression vector comprising the volvaria volvacea catalase VCAT gene of claim 2.

5. the expression vector of claim 4, wherein the expression vector containing Volvariella volvacea (Volvariella volvacea) catalase VCAT gene is pBAR GPE1/VCAT, and is constructed by connecting a nucleotide fragment containing Volvariella volvacea (Volvariella volvacea) catalase VCAT gene with pBAR GPE1 plasmid subjected to double enzyme digestion by BamH I and EcoRI.

6. Use of the straw mushroom catalase VCAT according to claim 2 for improving heat and cold tolerance of microorganisms.

7. The use according to claim 6, wherein the microorganism is a fungus or a bacterium.

8. The use according to claim 6, wherein the microorganism is Escherichia coli.

9. the use according to claim 6, wherein the heat and cold tolerance of the microorganism is improved by transferring an expression vector containing the Volvariella volvacea (Volvariella volvacea) catalase VCAT gene into a host cell of the microorganism.

10. The use according to claim 6, wherein the heat and cold tolerance of Escherichia coli is improved by transferring the expression vector pBAR GPE1/VCAT containing Volvariella volvacea (Volvariella volvacea) catalase VCAT gene into Escherichia coli.

Technical Field

The invention belongs to the technical field of antioxidant enzymes, and relates to straw mushroom catalase VCAT and an encoding gene and application thereof.

Background

volvariella volvacea (Bull.) Singer belongs to Basidiomycetes, Agaricales, Pholiopsidae and Hypsizygus marmoreus, is native to tropical and subtropical regions of China, and has delicious taste and wide market. The straw mushroom belongs to high-temperature edible mushrooms, the optimal growth temperature of mycelium is 32-35 ℃, and the mycelium or fruiting body can generate low-temperature autolysis phenomenon under the conventional refrigeration condition of 0-4 ℃, which is characterized by softening, liquefying, rotting and the like of tissues. The low temperature resistance of the straw mushroom seriously affects the low-temperature preservation of the strain and the postharvest storage and transportation of the fruiting body, and hinders the rapid development of the straw mushroom industry.

Catalase (CAT) is one of antioxidant enzymes widely existing in animals and plants, is tetrameric heme enzyme consisting of 4 identical peptide chain subunits, has a conserved activation center and a ferrohemoglobin binding site, is mainly present in peroxisomes, glyoxylate cycle bodies and cytoplasm of cells, and is partially distributed in mitochondria and chloroplasts.

In plants, CAT primarily eliminates hydrogen peroxide (H2O2) produced during mitochondrial electron transport, beta-fatty acid oxidation, and photo-respiration to prevent damage to plants from reactive oxygen radicals. When plants are subjected to biotic or abiotic stress, the receptor substances on the cell membrane receive and transmit stress signals, promoting Reactive Oxygen Species (ROS). H2O2, as the main active oxygen species, has a dual role in plants. A proper amount of H2O2 can be used as a signal molecule to participate in responding to various stress signal pathways, so that the expression of related genes is started, and the tolerance of plants is generated; when the concentration of H2O2 is too high, oxidative damage may occur, causing apoptosis. CAT plays an important role in plant stress resistance as a specific scavenger of H2O 2.

researches show that CAT plays an important role in physiological processes of plant growth and development, adversity stress defense response, oxidative senescence and the like, and CAT belongs to a multigene family, and the expression activity of CAT is influenced by various biological and non-biological factors, such as illumination, temperature, high salt, drought, phytohormone and other pathogenic microorganisms.

The plant tolerance is improved by enhancing the activity of antioxidant enzymes and enhancing the metabolism of active oxygen, and the plant tolerance is widely applied in the field of agricultural production. Chua Yongzhi et al transfer catalase gene KatG into cotton, and the transgenic cotton shows better drought resistance (Chua Yongzhi, build wave, Hao Xiao Yun, etc. transfer catalase gene KatG cotton drought resistance [ J ]. West North agricultural science, 2013,22(12): 56-61.). Kupffer and the like transform dragon fruit CAT into tobacco, and the transgenic tobacco can resist drought and low temperature stress (Kupffer, genetic transformation and functional analysis of dragon fruit CAT gene in tobacco [ D ]). Leeni introduces wheat CAT gene into rice, finds that the wheat CAT gene has higher CAT activity, improves the low temperature resistance of the rice, and can be used for cultivating low temperature rice (introduces wheat catalase to cultivate low temperature resistant rice [ J ] Biotechnology report, 2001(3): 48-49.). The transformation of maize CAT2 into tobacco by POLIOROS AN et al reduced the toxicity of herbicides to tobacco (Polidors A N, Mylona P V, Scandalios J G. Transgenic tobacco plants expressing the main Cat2 gene having altered levels of enzymes that are not present at plant-pathogenic organisms and reactive to oxidative stress [ J ] Transgenic Research,2001,10(6): 555) 569.). The methods improve the stress resistance of plants by over-expressing CAT, cultivate high-quality resistant crop varieties, are only suitable for plant systems, and do not see the research on the edible fungi catalase at present.

Disclosure of Invention

The invention aims to provide straw mushroom catalase VCAT and a coding gene and application thereof. The nucleotide sequence of the straw mushroom catalase VCAT is transferred into a host body, and the heat resistance and the cold resistance of the host are improved by enabling the host to express the catalase, so that the straw mushroom catalase VCAT is suitable for the variety culture of microorganisms.

The amino acid sequence of the straw mushroom catalase VCAT is shown in SEQ ID NO. 1.

the nucleotide sequence of the coding gene of the straw mushroom catalase VCAT is shown in SEQ ID NO. 2.

the invention provides an expression vector containing straw mushroom catalase VCAT gene.

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

the invention also provides application of the straw mushroom catalase VCAT in improving heat resistance and cold resistance 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.

Specifically, the heat resistance and cold resistance of the microorganism are improved by transferring an expression vector containing a straw mushroom catalase VCAT gene into a microorganism host cell. In the specific embodiment of the invention, the expression vector pBAR GPE1/VCAT containing straw mushroom catalase VCAT gene is transferred into escherichia coli, so that the heat resistance and the cold resistance of the escherichia coli are improved.

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

The invention takes the straw mushroom strain V23 as a material to obtain the nucleotide sequence of the straw mushroom catalase VCAT gene and the amino acid sequence of the straw mushroom catalase VCAT. The nucleotide sequence of straw mushroom catalase VCAT is transferred into a proper host body, and the heat resistance and the cold resistance of the host can be improved by enabling the host to express the catalase. The invention takes escherichia coli as a model organism, and experimental results show that heat resistance and cold resistance are obtained after the escherichia coli with the straw mushroom VCAT gene is transferred over to express catalase. The straw mushroom catalase VCAT is derived from fungi, and because the fungi and the bacteria share the same set of codons, the straw mushroom catalase VCAT can be transferred into a proper microbial host, so that the temperature stress resistance of the host can be improved.

Drawings

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

FIG. 2 is a graph showing the predicted result of the signal peptide of Volvariella volvacea catalase VCAT;

FIG. 3 is a graph showing the result of predicting phosphorylation sites of Volvariella volvacea catalase VCAT;

FIG. 4 is a graph showing the results of prediction of transmembrane structure of Volvariella volvacea catalase VCAT;

FIG. 5 is a PCR identification electropherogram of pBAR GPE1/VCAT recombinant plasmid;

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

FIG. 7 is a graph showing comparison of growth rates of Escherichia coli before and after transformation of a VCAT gene into Volvariella volvacea under heat stress (50 ℃);

FIG. 8 is a graph showing the comparison of growth rates of E.coli before and after transformation of the VCAT gene of Volvariella volvacea under cold stress (4 ℃ C.).

Detailed Description

The invention will be further elucidated with reference to a specific embodiment and a drawing. 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 a volvariella volvacea strain V23 as a material, constructs a local database through volvariella volvacea genome, finds a volvariella volvacea catalase VCAT gene conserved sequence through local Blast, uses CE Design V1.04 to Design a primer of volvariella volvacea VCAT gene, amplifies VCAT whole gene segment from volvariella volvacea genome DNA, converts a target segment into escherichia coli after being connected with a pBAR GPE1 carrier after being identified by PCR, sends the escherichia coli to a maritime biological engineering (Shanghai) limited company for sequencing, has the segment length of 2274bp, is shown as SEQ ID NO.2, and codes 757 amino acids, is shown as SEQ ID NO. 1.

As analyzed by bioinformatics, the straw mushroom catalase VCAT amino acid number is 757, the molecular weight of the protein is about 84kDa, and the isoelectric point (pI) is 6.40; the instability coefficient is 29.88, belonging to stable protein; the hydrophobicity coefficient is-0.370, which indicates that the protein has hydrophilicity. The NetPhos 3.1 software predicts that the phosphorylation sites of Ser, Thr and Tyr of Volvaria volvacea (Volvaria volvacea) catalase VCAT protein are 30, 23 and 8 respectively (FIG. 3). The signal peptide of straw mushroom VCAT is predicted by using SignalP 5.0 (figure 2), and the result shows that the probability of the signal peptide of the protein is 0.0017, and the other probabilities are 0.9983, so that the straw mushroom catalase VCAT can be judged to have no signal peptide and not belong to secretory protein. As predicted by the transmembrane structure of the EMBnet Tmpred (FIG. 4), the protein has no transmembrane region from the N terminal, and the probability of being positioned outside the membrane is 100%, so that the straw mushroom catalase VCAT is not a transmembrane protein.

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	Marigold instruments manufacturing Ltd
		Bacteria shaking table	Shanghai-Heng scientific instruments Co., Ltd
Bacteria incubator	Shanghai-Heng scientific instruments 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 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.

4.2 IPTG：

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, Na2HPO 410 mmol/L, KH2PO41.76mmol/L, adding distilled water to reach volume of 1000mL, and adjusting pH 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.