Dgfe2 gene for cultivating salt-tolerant microorganism

Abstract

This invention discloses a Gobi abnormal coccus ( Deinococcus gobiensis Genes encoding ferritin in I-0 DgfE2 The nucleotide sequence of this gene is shown in SEQ ID NO. 1. The protein DgfE2 encoded by this gene has salt-tolerant function and can be used to cultivate salt-tolerant microorganisms, thus providing a reference for cultivating plants with salt-tolerant traits. The amino acid sequence of this protein is shown in SEQ ID NO. 2. After being subjected to 0.6 mol / L NaCl stress for 5 h, it contains... DgfE2 Salt-tolerant recombinant strain pET28a- DgfE2 The growth of the / BL21 strain was better than that of the control pET28a / BL21 strain containing only an empty plasmid, indicating that this recombinant strain has the ability to tolerate salt stress. DgfE2 DgfE2 The gene has the function of improving the salt tolerance of E. coli.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and genetic engineering, and more specifically, this invention relates to a method for cultivating salt-tolerant microorganisms. DgfE 2 genes. Background Technology

[0002] Gobi abnormal cocci ( Deinococcus gobiensis I-0 was isolated from the cold desert and Gobi habitat of Xinjiang, China, and exhibits exceptional resistance to abiotic stresses such as radiation, oxidation, and salinity. Previous studies have identified the gene encoding the cold shock protein in this bacterium. Csp1 It can enhance the ability of Escherichia coli to withstand abiotic stresses such as high temperature, high salt, and drought. Some researchers have also found that under freezing and high salt stress conditions, the Dgl5 protein in Gobi anomala can protect the activity of related intracellular enzymes, thereby reducing the damage of the external environment to the host Escherichia coli.

[0003] However, the source of the discovery in this invention Deinococcus gobiensis I-0 gene DgfE 2. No research reports have been found on its ability to improve cell salt tolerance. Summary of the Invention

[0004] One object of the present invention is to solve at least the above-mentioned problems and / or defects, and to provide at least the advantages described below.

[0005] To achieve these objectives and other advantages of the present invention, a method for cultivating salt-tolerant microorganisms is provided. DgfE 2 genes, the DgfE The nucleotide sequence of gene 2 is shown in SEQ ID NO. 1.

[0006] One as described above DgfE 2. A protein encoded by a gene, the amino acid sequence of which is shown in SEQ ID NO. 2.

[0007] One as described above DgfE Application of 2 genes in the cultivation of salt-tolerant microorganisms.

[0008] Application of a protein as described above in the cultivation of salt-tolerant microorganisms.

[0009] A recombinant expression plasmid containing the ingredients described above for cultivating salt-tolerant microorganisms. DgfE 2 genes.

[0010] Preferably, the recombinant expression plasmid is obtained by... DgfE The two genes were constructed by linking them to the pET28a expression vector.

[0011] Application of a recombinant expression plasmid as described above in the cultivation of salt-tolerant microorganisms.

[0012] A salt-tolerant recombinant engineered strain containing the recombinant expression plasmid as described above.

[0013] Preferably, the salt-tolerant recombinant engineered strain is obtained by transferring the recombinant expression plasmid into the engineered bacteria.

[0014] Preferably, the engineered bacteria include Escherichia coli.

[0015] This invention includes at least the following beneficial effects: This invention provides a Gobi aberrant cocci ( Deinococcus gobiensis Genes encoding ferritin in I-0 DgfE2 The nucleotide sequence of the gene is shown in SEQ ID NO. 1. The protein DgfE2 encoded by this gene has salt tolerance function, and its amino acid sequence is shown in SEQ ID NO. 2. It can be used to cultivate salt-tolerant microorganisms, thereby providing a reference for cultivating plants with salt tolerance traits.

[0016] Gobi aberrant cocci I-0 were subjected to different NaCl concentrations for 12 h. DgfE2 The relative expression level of the gene increased with increasing NaCl concentration, and was 2-5 times higher than that of the unstressed strain, indicating that... DgfE2 Genes are involved in the process of cellular salt tolerance.

[0017] This invention uses the recombinant expression plasmid pET28a- DgfE2 The salt-tolerant recombinant engineered strain pET28a- was obtained by transferring the strain into the prokaryotic host cell recipient *Escherichia coli* BL21. DgfE2 / BL21, after being subjected to 0.6 mol / L NaCl stress for 5 h, contains DgfE2 pET28a- gene DgfE2 The recombinant strain / BL21 grew better than the control strain pET28a / BL21 containing only an empty plasmid, indicating that the recombinant strain has the ability to tolerate salt stress. DgfE2 The gene has the ability to increase the salt tolerance of E. coli.

[0018] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0019] Figure 1 The abnormal Gobi cocci under NaCl stress in Example 1 DgfE2 The relative expression level of genes; Figure 2 In Example 2 DgfE2 Electrophoresis diagram confirming the PCR product of the gene; Figure 3 Example 2 contains DgfE2 Electrophoresis image verifying the prokaryotic recombinant expression plasmid of the gene; lane 1 is pET28a- DgfE2 Recombinant plasmid, lane 2 is pET28a- DgfE2 Enzyme digestion verification results; Figure 4 The samples in Example 3 before the NaCl stress test contained empty plasmids and plasmids containing... DgfE2 Colony diagram showing the growth status of E. coli with the gene expression plasmid, where: a) is the control E. coli (pET28a / BL21) containing the empty expression plasmid; b) is the colony of E. coli containing the gene expression plasmid. DgfE2 Recombinant engineered E. coli strain (pET28a-) expressing gene plasmid DgfE2 / BL21); Figure 5 Example 3 contains DgfE2 Colony images showing the growth of *E. coli* containing the prokaryotic expression plasmid and the empty plasmid in LB solid medium after treatment with 0.6 mol / L NaCl for 5 h. In image a, control *E. coli* (pET28a / BL21) containing the empty expression plasmid after treatment with 0.6 mol / L NaCl is shown; in image b, colony images showing the growth of *E. coli* containing the empty expression plasmid after treatment with 0.6 mol / L NaCl is shown. DgfE2 Recombinant engineered E. coli strain (pET28a-) expressing gene plasmid DgfE2 / BL21) Colony diagram after treatment with 0.6 mol / L NaCl. Detailed Implementation

[0020] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0021] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0022] The plasmids, bacterial strains, and reagents used in the following examples are as follows: (1) Plasmid: The expression vector pET28a is preserved in our laboratory. DgfE2 ( DgfE2 The recombinant plasmid (gene fragment and cloning vector pET28a ligated by enzyme digestion) was constructed by this invention.

[0023] (2) Experimental strains: Gobi abnormal cocci ( Deinococcus gobiensisI-0 was preserved in our laboratory; E. coli Top10 and BL21 strains were purchased from Beijing TransGen Biotech Co., Ltd.; pET28a / BL21 (recombinant strain obtained by transforming E. coli BL21 with empty vector pET28a), pET28a- DgfE2 / BL21 (recombinant plasmid pET28a- DgfE2 The recombinant strain obtained by transforming Escherichia coli BL21 was constructed by this invention. Both Escherichia coli and the recombinant strain were grown in LB medium at a temperature of 37 °C.

[0024] (3) Biochemical reagents: Prime Script™ RT reagent Kit with gDNA Eraser was purchased from Novizan Biotechnology Co., Ltd.; ChamQ SYBR qPCRMaster Mix was purchased from Takara Bio Inc.; TRizol (AG RNAex Pro RNA extraction reagent) was purchased from Acery Biotech Co., Ltd.; RNA prep Pure culture cell / bacterial total RNA extraction kit, agarose gel DNA recovery kit, and ordinary plasmid miniprep kit were all purchased from Tiangen Biotech Co., Ltd.; restriction endonucleases and T4 DNA ligase were purchased from NEB Biotechnology Co., Ltd.; dNTPs and high-fidelity transStartFastPFu DNA polymerase were purchased from TransGen Biotech Co., Ltd.; IPTG was purchased from Aladdin; kanamycin was purchased from VWR Biotechnology Co., Ltd.; primer synthesis and sequencing used in the experiment were all completed by BGI Genomics Co., Ltd.

[0025] (4) Culture medium: LB medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH adjusted to 7.0, with 15 g / L agar added to the solid medium. High-pressure steam (121℃, 1.034×10⁻⁶) 5 Sterilize for 30 minutes (Pa).

[0026] (5) Preparation of antibiotics and main solutions: Kanamycin (Km) (50 mg / mL): Weigh 1 g of kanamycin powder, dissolve it in 20 mL of sterile deionized water, filter to remove impurities, dispense into aliquots, and store at -20 ℃. IPTG (100 mmol / L): Weigh 0.24 g IPTG, dissolve it in 10 mL of double-distilled water, filter it through a membrane for sterilization, dispense it into individual containers, and store it at -20 ℃.

[0027] This invention provides a gene for cultivating salt-tolerant microorganisms. DgfE2This can be used to cultivate salt-tolerant microorganisms, thus providing a reference for cultivating plants with salt-tolerant traits; genes DgfE2 The nucleotide sequence is shown in SEQ ID NO. 1, with a nucleotide length of 810 bp, as follows: GTCGAGCCGGTCAAGCCGAACTATGACGCCAAGATCGGCAACTTCGCCCTGAACCTCGAGTACCTCGAAGCGGCCTTCTCTCGCCGCCGTGGGACGCATCAACGAACTCAAGGCCATCGGCGGCAGCGCCCAGATCATCCTGCCCAGCGGTTTCGACGGCACCAGCAGCATCGCCTTCTCCAGCCCGGCGGTCGCCCAGT ACGCTCAGGAAATCGCTCAGGACGAGCTGAACCACGTCATCGCTCTGCGCGCCAAGCTCGGCAGCGCCGCCGTCGACCGTCCTGTGCTCGACATCGGCCCGGCCTTTGCGGCGGCGGCCAACGGCGGCGGCGGGCGACCCTCAGCCCCTCGTTCAATCCCTACCTCAACGATCTGTTCTTCCTGCACGGCGCCTTCATTTTT GAAGACGTGGGTGTCACCGCGTACAAGGGCGCCGCGCGCCTGATCGTGGACTACAGCGAAGGCGGCATCCTCGATTCGGCGGCTGGCATCCTGTCGGTCGAGGCCTACCATGCGGGCGAAATCCGTACCCTCCTGTACGCCCAGAAGGACGTCGTGACCCCCTACGGCGTCACTGTCGAGCAGCTCATCCAGAAGATCAGCG ATCTGCGCGCGGCGGTCGGCGGCGGCAAGGACGAAGGCCTCACCAAGAACGGCAAGGCCAACATCGTCGTGGCCGATTCCAACAGCGTGGCCTACGGCCGCAGCCCCCGCGAAGTCCTGAACATCGTGTACCTGGGGGCCAACGCCAGCAAGGGCGGCTTCTTCCCCAACGGCCTCAACGGGGACTTCAGCGGCCTCGTCTGA This invention also provides a gene DgfE2The encoded protein DgfE2 possesses salt-tolerant capabilities and can be used to cultivate salt-tolerant microorganisms, thereby providing a reference for cultivating plants with salt-tolerant traits. The amino acid sequence of this protein is shown in SEQ ID NO. 2, and is as follows: VEPVKPNYDAKIGNFALNLEYLEAAFYLAAVGRINELKAIGGSAQIILPSGFDGTSSIAFSSPAVAQYAQEIAQDELNHVIALRAKLGSAAVDRPVLDIGPAFAAAANAAAGATLSPSFNPYLNDLFFLHGAFI FEDVGVTAYKGAARLIVDYSEGGILDSAAGILSVEAYHAGEIRTLLYAQKDVVTPYGVTVEQLIQKISDLRAAVGGGKDEGLTKNGKANIVVADSNSVAYGRSPREVLNIVYLGANASKGGFFPNGLNGDFSGLV The amino acid sequence of the DgfE2 protein was predicted online using http: / / web.expasy.org / protparam / . This protein consists of 269 amino acids and has the molecular formula C2. 1268 H 1978 N 332 O 383 Its molecular weight is 28 kDa, its theoretical isoelectric point is 4.89, and its instability coefficient is 30.69, making it a stable protein. Analysis of the DgfE2 protein sequence revealed that the protein is rich in Ala (15.6%), Gly (10.8%), and Leu (9.7%), with a total of 27 negatively charged amino acid residues (Asp+Glu) and a total of 19 positively charged amino acid residues (Arg+Lys). The total average hydrophobicity index (GRAVY) is 0.223. NCBI analysis predicted that the DgfE2 protein contains a ferritin domain and belongs to the Ferritin_2 superfamily.

[0028] Example 1 Salt stress DgfE2 Gene expression induction experiment I. Experimental Methods 1. Store in a -80℃ refrigerator Deinococcus gobiensisAfter activation by streaking on LB solid medium, single colonies of strain I-0 were inoculated into LB liquid medium and cultured at 30°C with shaking for 24 h. Then, 1% of the culture was inoculated into 50 mL of medium and cultured at 30°C with shaking until the early logarithmic growth phase. 20 mL of the bacterial suspension was taken, and different concentrations of NaCl were added to achieve NaCl concentrations of 0.1, 0.2, and 0.3 mol / L. The suspensions were cultured at 30°C with shaking for 12 h, with a control group containing no NaCl. The bacterial suspensions under different stress treatments were centrifuged at 4°C and 8000 r / min for 10 min in a refrigerated centrifuge. The cells were washed three times with PBS, collected by centrifugation, and then rapidly frozen in liquid nitrogen.

[0029] 2. Total RNA was extracted from bacterial cells under different stress treatments using TRizol (AG RNAex Pro RNA extraction reagent) in conjunction with the RNAprep Pure Cell / Bacteria Kit. RNA reverse transcription was performed using the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara).

[0030] 3. According to Deinococcus gobiensis I-0 genome DgfE2 Gene sequence amplification products were designed using specific primers of 200 bp, with 16S rRNA as an internal reference gene. 16S-F: 5'AATGGCGAAGGCAGGTTA3'; 16S-R: 5'CGTGCATCGTTTAGGGTGT3'; DgfE2-F: 5'CAGCATCGCCTTCTCCAGC3'; DgfE2-R: 5'GCGGTGACACCCACGTCTT3'.

[0031] qRT-PCR reactions were performed using the Novizan 2 × ChamQ SYBR qPCR Master Mix kit, and the results were analyzed. Deinococcus gobiensis I-0 DgfE2 Gene expression under salt stress.

[0032] II. Experimental Results from Figure 1 It can be seen that, DgfE2 The relative expression level of the gene increased by 2-5 times under salt stress, and increased with increasing NaCl concentration.

[0033] III. Experimental Conclusions DgfE2The expression of the gene was induced by salt stress, indicating that the DgfE2 protein is involved in the salt tolerance process of Gobi abnormal cocci.

[0034] Example 2 A recombinant expression plasmid pET28a- DgfE2 Applications in the cultivation of salt-tolerant microorganisms and salt-tolerant recombinant engineered strain pET28a- DgfE2 / BL21 1) From PCR Deinococcus gobiensis I-0 amplification DgfE2 The gene has a nucleotide length of 810 bp and encodes 269 amino acids; 2) DgfE2 The gene was linked to the pET28a expression vector to construct a vector containing the complete gene. DgfE2 Recombinant expression plasmid pET28a- DgfE2 ; 3) Import DgfE2 Recombinant expression plasmid pET28a- DgfE2 The salt-tolerant recombinant engineered strain pET28a- was obtained by transferring the strain into the prokaryotic host cell recipient *Escherichia coli* BL21. DgfE2 / BL21.

[0035] Specifically, it includes: according to Deinococcus gobiensis I-0 genome DgfE2 Design a pair of PCR-specific primers from the gene sequence. Deinococcus gobiensis Complete nucleotide sequences amplified from I-0 genomic DNA: F: 5'AGCCGCCCATACTGACGGCACGGTATCGATAAGCTTGATAT3'; R: 5'TAGAGGATCCTGATTAGAAAAACTCATCGAGCATCAAATGAAAC3'; From PCR method Deinococcus gobiensis The target gene fragment was amplified from the genome of I-0. The reaction conditions were: 98 ℃ for 10 min, [95 ℃ for 30 sec, 68 ℃ for 30 sec, 72 ℃ for 45 sec] for 35 cycles, followed by 72 ℃ for 10 min. The PCR product was then recovered from the gel. Bam HI / Hin dIII double digestion yielded pET28a expression vector containing sticky ends and DgfE2 Gene fragments, DgfE2 The fragment was ligated into the pET28a vector to construct the recombinant expression plasmid pET28a- DgfE2The recombinant expression plasmid was transformed into E. coli BL21, and the inserted sequence was verified to be correct by PCR, plasmid digestion, and sequencing (see [link to article]). Figure 2 and Figure 3 The recombinant engineered strain was named pET28a- DgfE2 / BL21.

[0036] The Escherichia coli BL21 containing the pET28a empty plasmid was named pET28a / BL21.

[0037] Example 3 Recombinant Escherichia coli engineered strain pET28a- DgfE2 Salt tolerance test of BL21 I. Experimental Methods 1. The sample containing the substance obtained in Example 2 Deinococcus gobiensis I-0 DgfE2 pET28a- gene DgfE2 The / BL21 strain and the pET28a / BL21 strain containing the empty plasmid were inoculated separately into 4 mL of LB liquid medium (containing Km antibiotic), and cultured overnight on a shaker (37 ℃). Then, they were cultured according to OD... 600 =0.1 was transferred to 20 mL of LB liquid medium, cultured for 30 min, and then IPTG was added to a final concentration of 0.1 mmol / L. Cultured further until OD was reached. 600 A value between 0.5 and 0.6 is acceptable.

[0038] 2. Take 1 mL of bacterial culture, centrifuge at 4000 r / min for 4 min to collect bacterial cells, resuspend the cells in 1 mL of 0.6 mol / L NaCl solution, and resuspend the control group in 1 mL of sterile water. Incubate at 37 ℃ and 220 r / min for 5 h. Dilute each sample serially with sterile deionized water to a concentration of 10. -4 Take 10 μL and spot it on the surface of LB solid medium. Incubate at 37 ℃ and 220 r / min for 12-16 h, observe the colony formation and take pictures.

[0039] II. Experimental Results Figure 4 This is a colony diagram showing the growth status of *E. coli* before NaCl stress treatment, where: a) is the pET28a / BL21 strain containing the empty expression plasmid; b) is the colony of *E. coli* containing... DgfE2 pET28a- gene expression plasmid DgfE2 / BL21 strain. Figure 5 These are colony images showing the growth status of *E. coli* after NaCl stress treatment. In image a, the colony image of strain pET28a / BL21 containing the empty expression plasmid after treatment with 0.6 mol / L NaCl is shown; in image b, the colony image of strain pET28a / BL21 containing the empty expression plasmid is shown. DgfE2pET28a- gene expression plasmid DgfE2 Colony diagram of strain BL21 after treatment with 0.6 mol / L NaCl. The diagram shows that: Before NaCl treatment: contain DgfE2 pET28a- gene DgfE2 The growth ability of strain / BL21 is consistent with that of strain pET28a / BL21 containing the empty plasmid (see...). Figure 4 ); After NaCl treatment: pET28a- DgfE2 The growth ability of strain / BL21 is significantly stronger than that of strain pET28a / BL21, with a difference in growth performance of 1-2 orders of magnitude (see [link to relevant documentation]). Figure 5 ).

[0040] III. Experimental Conclusions contain DgfE2 Recombinant engineered strain pET28a- DgfE2 / BL21 exhibits significantly higher resistance to salt stress than strains containing only an empty plasmid, and the expression of this gene enhances the salt tolerance of Escherichia coli.

[0041] Salt stress leads to the accumulation of reactive oxygen species in cells, disrupting the balance between reactive oxygen species and their scavenging systems, damaging membrane systems, and inactivating enzymes, ultimately resulting in metabolic imbalance in the organism. This invention has discovered... DgfE2 Genes can enhance pET28a- DgfE2 / BL21 strain's tolerance to NaCl-simulated salt stress.

[0042] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A method for cultivating salt-tolerant microorganisms DgfE 2 genes, characterized in that, The DgfE The nucleotide sequence of gene 2 is shown in SEQ ID NO.

1.

2. A device as described in claim 1 DgfE The protein encoded by 2 genes is characterized by, The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. A device as described in claim 1 DgfE Application of 2 genes in the cultivation of salt-tolerant microorganisms.

4. The use of the protein as described in claim 2 in the cultivation of salt-tolerant microorganisms.

5. A recombinant expression plasmid, characterized in that, Containing the salt-tolerant microorganisms as described in claim 1 DgfE 2 genes.

6. The recombinant expression plasmid as described in claim 5, characterized in that, By the DgfE The two genes were constructed by linking them to the pET28a expression vector.

7. The use of a recombinant expression plasmid as described in any one of claims 5 or 6 in the cultivation of salt-tolerant microorganisms.

8. A salt-tolerant recombinant engineered strain, characterized in that, Contains the recombinant expression plasmid as described in any one of claims 5 or 6.

9. The salt-tolerant recombinant engineered strain as described in claim 8, characterized in that, The recombinant expression plasmid was obtained by transferring it into engineered bacteria.

10. The salt-tolerant recombinant engineered strain as described in claim 9, characterized in that, The engineered bacteria include Escherichia coli.

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