DolR gene for improving biological stress resistance and application thereof

Abstract

The invention provides a dolR gene for improving biological stress resistance and application of the dolR gene, and belongs to the technical field of biology, the nucleotide sequence of the dolR gene is shown as SEQ ID NO: 1, by expressing the dolR gene, the biological oxidation resistance, salt resistance and osmotic stress resistance can be remarkably improved, and the biological stress resistance is greatly enhanced.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a dolR gene capable of improving biological stress resistance and an application thereof. Background Art

[0002] The design and construction of microbial cell factories using synthetic biology technology has been widely used to produce a wide variety of chemicals, foods, medicines and energy, and is the core link of green biomanufacturing. The construction of strain cell factories is completed by introducing new enzymes into microbial chassis cells, constructing new pathways and adapting to chassis cells. However, the current low-version chassis strains have bottleneck problems in stress resistance and production performance. How to further improve the chassis' tolerance to substrates and products, increase growth rate, increase substrate utilization and increase product yield still needs to be studied. Summary of the invention

[0003] In view of this, the object of the present invention is to provide a dolR gene for improving biological stress resistance and its application, which can further improve the tolerance of chassis strains to substrates and products, increase growth rate, increase substrate utilization and increase product yield.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a dolR gene for improving biological stress resistance. The nucleotide sequence of the dolR gene is shown in SEQ ID NO:1.

[0006] The present invention provides a protein for improving biological stress resistance, wherein the protein is encoded by the dolR gene;

[0007] The amino acid sequence of the protein is shown in SEQ ID NO:2.

[0008] The present invention provides a primer pair for amplifying the dolR gene, and the nucleotide sequences of the primer pair are shown in SEQ ID NO:3 and SEQ ID NO:4.

[0009] The present invention provides a recombinant expression vector, which comprises the dolR gene and an empty vector;

[0010] The empty vector includes pRADZ3.

[0011] The present invention provides a recombinant bacterium, wherein the recombinant bacterium comprises the recombinant expression vector and an empty vector bacterium;

[0012] The empty vector bacteria include Escherichia coli BW25113.

[0013] The present invention provides the use of the dolR gene, the protein, the primer pair, the recombinant expression vector or the recombinant bacteria in improving biological stress resistance.

[0014] Preferably, the organism includes plants or microorganisms.

[0015] Preferably, the biological stress resistance includes the biological resistance to oxidation, high salt and / or osmotic stress.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention provides a dolR gene for improving biological stress resistance and its application. The nucleotide sequence of the dolR gene is shown in SEQ ID NO: 1. By expressing the dolR gene, the antioxidant, salt and osmotic stress resistance of the organism can be significantly improved, and the biological stress resistance can be greatly enhanced. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This graph shows the effects of oxidation, high salt and osmotic stress on the growth of the strain before and after shock. DETAILED DESCRIPTION

[0019] The present invention provides a dolR gene for improving biological stress resistance, wherein the dolR gene is derived from Deinococcus radiodurans, and the nucleotide sequence of the dolR gene is shown in SEQ ID NO: 1;

[0020] SEQ ID NO: 1:

[0021] ATGGCCGGGCACCCCAACACACCGCCAGAGACCCTGGCCGCGCTGGGGGCCGAGTTTCCCGCCGAGGTGCTGGGCAACCCGGCGCTGCCGCTGCTGCGCCTCGCCCAGCCGGGGTTGCTGGCCCGCT GGCCTGTGCGGACGCTCGAAGCTCTGGCCGCGGCCCCGGCAGCCCCCGTCTGGTTGCTGCGGCTCGCGGCGGCGCACGAGAAGATTGACGTGCAACTCGCCGCCGTGACCCGCCCTGACCTGCCGGG CGACGTGCTGTTAAAGCTCTCGCACTCGCCCTTCTGGACCATCCGCGAGGCGGTGGCGCGCAAACCGCAGTTGCCGCCGGGCGTGCTGGGGCAGCTCGGGCACGACCTCGACTACGGCGTGCGGCTG TCGGTGGCGAACCGCAGCGACCTGTCCCGCGAATTGCGCGGGGCGCTGCGCCGTGACCCGCATCCGCTGGTGCGGGCGGTGGTCATTCTGGGTGAGCCGCAGGTCAGCGAACTCAGTCTCTCTTGA.

[0022] The present invention provides a protein for improving biological stress resistance, wherein the protein is encoded by the dolR gene;

[0023] The amino acid sequence of the protein is shown in SEQ ID NO: 2:

[0024] SEQ ID NO:2:

[0025] MAGHPNTPPETLAALGAEFPAEVLGNPALPLLRLAQPGLLARWPVRTLEALAAAPAAPVWLLRLAAAHEKIDVQLAAVTRPDLPGDVLLKLSHSPFWTIREAVARKPQLPPGVLGQLGHDLDYGVRLSVANRSDLSRELRGALRRDPHPLVRAVVILGEPQVSELSLS.

[0026] The present invention provides a primer pair for amplifying the dolR gene, and the nucleotide sequences of the primer pair are shown in SEQ ID NO: 3 and SEQ ID NO: 4:

[0027] SEQ ID NO: 3: CCCACTAGTATGGCCGGGCACCCCAACACA;

[0028] SEQ ID NO: 4: CCCCATATGTCAAGAGAGACTGAGTTCGCT.

[0029] The SEQ ID NO: 3 is a forward primer, and SEQ ID NO: 4 is a reverse primer.

[0030] The present invention provides a recombinant expression vector, which comprises the dolR gene and an empty vector;

[0031] The empty vector is preferably pRADZ3, and the nucleotide sequence of the recombinant expression vector is shown in SEQ ID NO: 5;

[0032] SEQ ID NO:5:

[0033]

[0034] The present invention provides a recombinant bacterium, wherein the recombinant bacterium comprises the recombinant expression vector and an empty vector bacterium;

[0035] The empty vector bacteria is preferably Escherichia coli BW25113.

[0036] The present invention provides the use of the dolR gene, the protein, the primer pair, the recombinant expression vector or the recombinant bacteria in improving biological stress resistance.

[0037] In the present invention, the organism includes plants or microorganisms; the biological stress resistance includes the resistance of the organism to oxidation, high salt and / or osmotic stress.

[0038] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0039] Sources

[0040] Deinococcus radiodurans was purchased from China General Microbiological Culture Collection Center with the collection number of CGMCCNO.1.633.

[0041] Cloning vector pJET: a commercial product from Thermo Fisher;

[0042] Shuttle plasmid pRADZ3: Institute of Biotechnology, Chinese Academy of Agricultural Sciences;

[0043] Escherichia coli BW25113: Wuhan Miaoling Biotechnology Co., Ltd., product number T0035;

[0044] Restriction endonucleases Spe I / Nde I: NEB (Beijing) Co., Ltd.;

[0045] DNA recovery kit: Beijing Juhemei Biotechnology Co., Ltd. (Cat. No. MF029-04);

[0046] Gene recombination kit: Nanjing Novozymes Biotech Co., Ltd. (C115-01 recombinase).

[0047] Example 1 Construction of a recombinant engineering strain expressing the functional gene dolR

[0048] The full-length nucleic acid sequence of the functional gene dolR was obtained from Deinococcus radiodurans using the PCR method. The PCR amplification system was calculated in 20 μL, including 10 μL 2×PhantaMax MasterMix, 1 μL DNA template, 1 μL upstream primer SEQ ID NO:3 with an initial concentration of 10 μmol / L, 1 μL downstream primer SEQ ID NO:4 with an initial concentration of 10 μmol / L, and 7 μL ddH2O. The PCR amplification program was 95°C denaturation for 3 min→(95°C denaturation for 30 s→60°C renaturation for 30 s→72°C extension for 1 min)×35 cycles→72°C extension for 5 min→4°C storage. Its size was 507 bp, and the nucleotide sequence was shown in SEQ ID NO:1. The gene encoded 168 amino acids, and the amino acid sequence was shown in SEQ ID NO:2. It was cloned into the vector pJET to construct a recombinant cloning plasmid pJET-dolR containing the complete functional gene dolR, and sequenced for verification; then, the Spe I / Nde I double enzyme digestion to obtain the functional gene dolR and pRADZ3 vector fragment containing sticky ends, connect the functional gene dolR to the pRADZ3 vector, construct the E. coli expression vector pRADZ-dolR, and use the expression vector to transform E. coli BW25113. The transformation method is: add 10 μL of the expression vector to 50 μL of competent cells, mix the liquid upside down, and place it on ice for 30 minutes. Place the centrifuge tube containing the mixture in a 42°C heating block and heat shock for 60-90 seconds. Quickly place the centrifuge tube on ice and cool for 2 minutes. Add 800 μL of LB liquid culture medium to the clean bench, and culture it on a shaker at 37°C and 150-180rpm for 45-60 minutes. Take 200 μL of bacterial solution and spread it on an LB plate containing the corresponding antibiotics, and culture it in a 37°C incubator for 14 hours to obtain recombinant engineered bacteria.

[0049] Example 2 Analysis of stress resistance of recombinant engineering strains

[0050] Experimental Grouping

[0051] dolR group: prepared as in Example 1, the strain was named BW-dolR;

[0052] Blank control group: Escherichia coli BW25113 was transformed with the pRADZ3 vector without the functional gene dolR, and the strain was named BW-Z3.

[0053] 1) Experimental methods:

[0054] 1.1) Streak the BW-dolR strain and the BW-Z3 strain on LB solid medium plates respectively;

[0055] 1.2) Pick a single colony and inoculate it into liquid LB medium supplemented with 50 μg / mL ampicillin, and culture it at 37°C for 12 h until the mid-to-late exponential growth period;

[0056] 1.3) Collect the cells by centrifugation at 6,000 rpm for 5 min at room temperature, then wash the cells twice with the same volume of phosphate buffer (pH 7.0) and shake evenly to obtain BW-dolR bacterial solution and BW-Z3 bacterial solution;

[0057] Next, control experiments, oxidative resistance, high salt and dryness stress resistance experiments were carried out.

[0058] 2) Control experiment

[0059] 2.1) Take the bacterial solution prepared in step 1), centrifuge at 25°C, 6,000 rpm for 5 min, and collect the bacterial cells.

[0060] 2.2) Resuspend the cells in LB medium to an OD of 0.8 and dilute 10-fold to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 200 μL of bacterial solution of different concentrations were taken to spread on LB solid culture medium plates, cultured at 37°C for 2 days, and the number of colonies was recorded as untreated control experiments. The experiment was repeated 3 times.

[0061] 3) Oxidation resistance analysis

[0062] 3.1) Take the bacterial solution prepared in step 1), centrifuge at 25°C, 6,000 rpm for 5 min, and collect the bacterial cells;

[0063] 3.2) The cells were resuspended in LB liquid medium containing hydrogen peroxide at a final concentration of 20 mM, with a cell concentration of OD 0.8, and cultured at 30°C and 220 rpm on a shaking platform for 20 min;

[0064] 3.3) Take the bacterial solution cultured on a shaker for 20 minutes and dilute it 10-fold to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 200 μL of bacterial solution of different concentrations were taken to spread on LB solid culture medium plates, cultured at 37°C for 2 days, the number of colonies was recorded, and the survival rate of the strain was calculated. The experiment was repeated 3 times.

[0065] 4) High salt resistance analysis:

[0066] 4.1) Take the bacterial solution prepared in step 1), centrifuge at 25°C, 6,000 rpm for 5 min, and collect the bacterial cells;

[0067] 4.2) The cells were resuspended in LB liquid medium with a final concentration of 5 M sodium chloride, the cell concentration was OD 0.8, and cultured in a shaking incubator at 30°C and 220 rpm for 2 h;

[0068] 4.3) Take the bacterial solution cultured on the shaking table for 2 hours and dilute it 10 times to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 200 μL of bacterial solution of different concentrations were taken to spread on LB solid culture medium plates, cultured at 37°C for 2 days, the number of colonies was recorded, and the survival rate of the strain was calculated. The experiment was repeated 3 times.

[0069] 5) Penetration resistance analysis:

[0070] 5.1) Take the bacterial solution prepared in step 1), centrifuge at 25°C, 6,000 rpm for 5 min, and collect the bacterial cells;

[0071] 5.2) The cells were resuspended in LB liquid medium with a final concentration of 2 M sorbitol, the cell concentration was OD 0.8, and cultured in a shaking incubator at 30°C and 220 rpm for 2 h;

[0072] 5.3) Take the bacterial solution cultured on the shaking table for 2 hours and dilute it 10 times to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 200 μL of bacterial solution of different concentrations were taken to spread on LB solid culture medium plates, cultured at 37°C for 2 days, the number of colonies was recorded, and the survival rate of the strain was calculated. The experiment was repeated 3 times.

[0073] 6) Experimental results

[0074] like Figure 1 The results showed that before the shock of oxidative, high salt and osmotic stress, the growth status of the engineered strain BW-dolR containing the functional gene dolR was basically the same as that of the strain BW-Z3 containing the pRADZ3 vector without the functional gene dolR.

[0075] After oxidative, high salt and osmotic stress shocks, the engineered strain BW-dolR containing the stress resistance gene dolR grew well, and the number of colonies was significantly higher than that of the control strain BW-Z3 containing only the pRADZ3 vector without the functional gene dolR. Under the oxidative stress condition of 20mM hydrogen peroxide shock for 20 minutes, the resistance of the engineered strain BW-dolR expressing the stress resistance gene dolR was significantly improved. Compared with the control BW-Z3 strain, the oxidative resistance was improved by 1 order of magnitude and the survival rate was increased by 5 times. Under the high salt stress condition of 5M sodium chloride, the survival rate of the control strain BW-Z3 strain decreased by 5 orders of magnitude, while the engineered strain BW-dolR expressing the stress resistance gene dolR was 2 orders of magnitude higher than the control strain, and the salt resistance was increased by about 100 times. Under the osmotic stress conditions simulated by 2M sorbitol, the survival rate of the control strain BW-Z3 was 2.69%, while the survival rate of the engineered strain BW-dolR expressing the stress resistance gene dolR was 18.53%, and its anti-osmotic ability was about 5 times higher than that of the control.

[0076] It can be seen from the above experiments that the functional gene dolR of the present invention can significantly enhance the resistance of cells to oxidation, high salt and osmotic stress.

[0077] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A dolR gene for improving biological stress resistance, characterized in that: The nucleotide sequence of the dolR gene is shown in SEQ ID NO:

1.

2. A protein for improving biological stress resistance, characterized in that The protein is encoded by the dolR gene according to claim 1; The amino acid sequence of the protein is shown in SEQ ID NO:

2.

3. A primer pair for amplifying the dolR gene according to claim 1, characterized in that: The nucleotide sequences of the primer pair are shown in SEQ ID NO:3 and SEQ ID NO:

4.

4. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the dolR gene according to claim 1 and an empty vector; The empty vector includes pRADZ3.

5. A recombinant bacterium, characterized in that: The recombinant bacteria include the recombinant expression vector according to claim 7 and empty bacteria; The empty vector bacteria include Escherichia coli BW25113.

6. Use of the dolR gene according to claim 1, the protein according to claim 2, the primer pair according to claim 3, the recombinant expression vector according to claim 4 or the recombinant bacteria according to claim 5 in improving biological stress resistance.

7. The use according to claim 6, characterized in that: The organisms include plants or microorganisms.

8. The use according to claim 7, characterized in that: The biological stress resistance includes the resistance of organisms to oxidation, high salt and / or osmotic stress.

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