A method for improving biological stress resistance using dotp gene

Abstract

The application provides a method for improving biological stress resistance by using a dotP gene, which comprises the following steps: transferring the dotP gene into a host organism, and making the host organism express the dotP gene; the host organism is a plant or a microorganism; the dotP gene encodes a protein (a) or (b): (a) a protein consisting of the amino acid sequence shown in SEQ ID NO. 2; (b) a protein derived from (a) by substituting, deleting or adding n amino acids in the amino acid sequence in (a) and having the activity of a quadruplet repeat sequence protein, wherein n is any integer between 1 and 4. By the technical scheme, the biological stress resistance of the plant or the microorganism is improved, and in particular, the dry resistance of the plant or the microorganism is improved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a method for improving the stress resistance of organisms using the dotP gene. Background Technology

[0002] When plants or microorganisms are subjected to stress, some are damaged and die, while others, although their physiological activities are affected to varying degrees, can survive. If they live in such a stressful environment for a long time, through natural selection, advantageous traits are preserved and continuously strengthened, while unfavorable traits are constantly eliminated. In this way, through the long-term evolution and adaptation of plants or microorganisms, plants or microorganisms growing under different environmental conditions will develop the ability to adapt to certain environmental factors, that is, they can adopt different ways to resist various stress factors. The ability of plants or microorganisms to resist various stress (or adverse) factors is called stress resistance, also known as resilience.

[0003] Extremophiles are a class of microorganisms that can survive in extreme environments. Known extremophiles mainly include radiation-resistant bacteria, thermophiles, halophiles, acidophiles, alkaliphiles, psychrophiles, and barophiles. Screening for strains with extreme resistance from specific environments and mining related resistance gene resources can provide a rich source of stress-resistant functional genes for practical applications such as breeding new transgenic crop varieties and lines with superior traits.

[0004] Deinococcus radiodurans bacteria are among the most radiation-resistant microorganisms discovered on Earth to date. Deinococcus radiodurans R1, first identified in 1956, exhibits exceptional resistance to a variety of abiotic stresses, including gamma radiation, UV radiation, drying, high temperatures, and oxidation. The complete genome sequence of Deinococcus radiodurans R1 was completed in 1999. The genome sequence revealed that more than half of the predicted ORFs encode proteins with unknown functions, for which no matching genes exist in other organisms in databases. Summary of the Invention

[0005] The purpose of this invention is to discover genes in the genome of radiation-resistant abnormal cocci that can enhance cellular tolerance to desiccation and further improve the biological stress resistance of plants or microorganisms, especially their desiccation resistance.

[0006] To achieve the above objectives, the present invention provides a method for using dotP Methods to enhance an organism's resistance to stress through gene expression include: dotP The gene is transferred into the host organism, and the host organism expresses the gene. dotP Gene; the host organism is a plant or a microorganism; thedotP The gene encodes the following protein (a) or (b): (a) a protein consisting of the amino acid sequence shown in SEQ ID NO.2; (b) a protein derived from (a) by substitution, deletion or addition of n amino acids in the amino acid sequence of (a) and having a tetrapeptide repeat protein domain, wherein n is any integer between 1 and 4.

[0007] Through the above technical solutions, the present invention improves the biological stress resistance of plants or microorganisms, especially the desiccation resistance of plants or microorganisms.

[0008] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0009] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the following detailed description to explain the invention, but do not constitute a limitation thereof. In the drawings:

[0010] Figure 1 It is to construct a radiation-resistant abnormal cocci deletion mutant strain △ dotP Schematic diagram.

[0011] Figure 2 It involves constructing fusion fragments and verifying △ dotP Electrophoretic pattern of the mutant strain.

[0012] Figure 3 Under sorbitol stress, DR wild-type strains and △ dotP Experiment on the stress phenotype of mutant strains.

[0013] Figure 4 Under drying stress conditions, DR wild-type strains and △ dotP Experiment on the stress phenotype of mutant strains.

[0014] Figure 5 This is a schematic diagram of the construction of the recombinant engineered strain DH5α-dotP.

[0015] Figure 6 This is a diagram showing the results of the drying resistance experiment of the DH5α-dotP recombinant engineered strain. Detailed Implementation

[0016] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0017] This invention provides a method for using dotP Methods to enhance an organism's resistance to stress through gene expression include: dotPThe gene is transferred into the host organism, and the host organism expresses the gene. dotP Gene; the host organism is a plant or a microorganism; the dotP The gene encodes the following protein (a) or (b): (a) a protein consisting of the amino acid sequence shown in SEQ ID NO.2; (b) a protein derived from (a) by substitution, deletion or addition of n amino acids in the amino acid sequence of (a) and having a tetrapeptide repeat protein domain, wherein n is any integer between 1 and 4.

[0018] Preferably, the dotP A gene is a DNA molecule with the nucleotide sequence shown in SEQ ID NO.1.

[0019] in, dotP The gene was cloned from a soil metagenomic sample. The protein encoded by the dotP gene (amino acid sequence shown in SEQ ID NO.2) contains a tetratricopeptide-epeat protein domain, hence the name. dotP . dotP The DNA of the gene (the DNA molecule shown in SEQ ID NO.1) and radiation-resistant abnormal cocci ( Deinococcus radiodurans The genome fragments of this gene are identical to those of *Streptococcus radiodurans*. The inventors of this invention unexpectedly discovered that this gene, when transferred into *Streptococcus radiodurans*, is identical to that of *Streptococcus radiodurans*. Deinococcus radiodurans After being introduced into other organisms (host organisms) besides the host organism, it can bring significantly enhanced desiccation resistance to the host organism.

[0020] Preferably, the method further includes placing the dotP A recombinant vector is obtained by inserting a gene into a vector, and the recombinant vector is then transferred into the host organism.

[0021] Preferably, the recombinant vector is pRADZ3-dotP, that is, it contains... groEL promoter completeness dotP The recombinant plasmid pRADZ3-dotP is used to... dotP The gene fragment was obtained by ligating it into a linearized pRADZ3 shuttle plasmid, which contains components that function in both *E. coli* and *Abnormal cocci*. groEL Promoter.

[0022] Preferably, the host organism is Escherichia coli.

[0023] Preferably, the host organism is Escherichia coli DH5α strain.

[0024] Preferably, the host organism is a fungus.

[0025] Preferably, the host organism is Saccharomyces cerevisiae, Candida albicans, or Pichia pastoris.

[0026] Preferably, the host organism is a plant, preferably Arabidopsis thaliana, rapeseed, corn, rice, wheat, tobacco, soybean or cotton.

[0027] The present invention will be further described in detail below through examples.

[0028] The plasmids and strains mentioned in the following examples are only used to further illustrate the present invention and do not limit the substantive content of the present invention. Unless otherwise specified, all experimental conditions are conventional conditions well known to those skilled in the art, such as those described in Sambrook et al. Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or those recommended by the manufacturer.

[0029] Example 1

[0030] Radiation-resistant abnormal cocci △ dotP Construction of gene deletion mutant strains.

[0031] This embodiment uses primers (SEQ ID NO. 3-12) as shown in Table 1.

[0032] Table 1

[0033]

[0034] D. radiodurans Wild-type strain genome extraction: Bacterial genome extraction was performed according to the instructions of Magen Biotechnology's HiPureBacterial DNA Kit; pKatAPH3 plasmid extraction: Plasmid extraction was performed according to the instructions of Magen Biotechnology's HiPure Plasmid Micro Kit.

[0035] by D. radiodurans Using the genome as a template, the target gene was amplified by PCR. dotP The upstream fragment 517 bp (U) and the downstream fragment 524 bp (D) were obtained. Using pKatAPH3 plasmid as a template, the kanamycin resistance gene fragment 970 bp (K) was amplified by PCR. After detection by 1% agarose gel electrophoresis, the target fragment was excised and recovered. A one-step fusion PCR reaction was performed to obtain the fusion product of the three fragments, 2011 bp UKD. The recovered fragment was sent to a sequencing company for sequencing.

[0036] D. radioduransPreparation of competent cells and transformation of fusion fragments: In a clean bench, the fusion fragments were genetically transformed using the CaCl2 treatment method and cultured upside down in a 30℃ incubator for 2-3 days.

[0037] Mutant resistance screening: Single colonies were picked from transformed TGY plates and inoculated into fresh TGY medium. Deletion mutants were screened on Km (kanamycin) plates containing different resistance pressures. dotP This ensures that the gene is completely mutated.

[0038] Positive clone identification and sequencing verification: From the plates where antibiotic selection was completed, a single colony was picked and inoculated into fresh TGY medium. After culturing for 2-3 days, the bacterial culture was collected, and the genome was extracted for mutant strain verification. First, it was verified whether the mutant strain had completed the mutation using... dotP Internal primer YZ dotP -F / R amplifies DR wild-type and Δ respectively. dotP The product length is 297 bp; secondly, to verify whether the gene mutation location is correct, external primer YZ- dotP UD-F / R amplifies DR wild-type and Δ dotP The product lengths were 2889 bp and 2324 bp, respectively, and the PCR products were sent to BGI Genomics for sequencing.

[0039] Experimental results show that, through homologous recombination, replacing the Km resistance cassette with... dotP Target gene strategy, successfully constructed without dotP Mutant strain of gene △ dotP (See Figure 1 ,and Figure 2 ).

[0040] Example 2

[0041] dotP Experimental analysis of how genes enhance cellular tolerance to sorbitol stress.

[0042] The experimental strain was the radiation-resistant abnormal cocci obtained in Example 1. dotP △ of genes dotP Strains. The control strain was *Radiopyrhabditis elegans* (Radiopyrhabditis Deinococcus radiodurans ) wild-type strain.

[0043] Will D. radiodurans Wild-type strain and the △ strain successfully constructed in Example 1 dotPThe strain was activated by streaking on TGY solid agar plates; single colonies were picked and inoculated into 5 mL of fresh TGY liquid agar (recombinant strain supplemented with 10 µg / mL Km antibiotic) and incubated at 30°C for 2-3 days until mid-exponential growth; then, 1% of the culture was re-transferred into 20 mL of fresh TGY liquid agar (recombinant strain supplemented with 10 µg / mL Km antibiotic) and incubated until early logarithmic growth (OD). 600 =Approximately 0.6~0.8; Take 1 mL of bacterial suspension, centrifuge at 6,000 rpm for 5 min at room temperature to collect bacterial cells, then wash the bacterial cells twice with the same volume of sterile phosphate buffer (pH 7.0), and shake well; High concentration sorbitol stress experiment: serially dilute the resuspended bacterial cells (10... -1 -10 -5 Eight μL of each dilution was spotted onto the surface of TGY solid medium containing different concentrations of sorbitol (0.6 M, 0.8 M, 1.0 M). TGY solid medium without sorbitol was used as a blank control. The samples were incubated at 30°C for approximately 4 days, and colony formation was observed. Three independent replicates were performed for each experiment.

[0044] The results showed that the bacterial cell growth remained largely consistent before stress treatment. After 4 days of osmotic stress treatment in TGY solid petri dishes containing different concentrations of sorbitol, the mutant strain Δ dotP The mutant strain DR was more sensitive to sorbitol shock than the wild-type strain, and the difference between the wild-type and mutant strains gradually increased with increasing sorbitol concentration, especially at 1.0 M sorbitol concentration, where the mutant strain showed almost no colony growth. dotP The deletion of the gene makes the cell extremely sensitive to osmotic stress, indicating that the expression of this gene makes a significant contribution to the bacterial response to osmotic stress.

[0045] This illustrates that: dotP The gene has the function of significantly improving the cell's tolerance to dryness resistance.

[0046] Example 3

[0047] dotP Experimental analysis of gene enhancement of cell resistance to desiccation stress.

[0048] The experimental strain was the radiation-resistant abnormal cocci obtained in Example 1. dotP △ of genes dotP Strains; the control strain was *Radiopyrhabditis elegans* (…). Deinococcus radiodurans ) wild-type strain.

[0049] Will D. radiodurans Wild-type strain and the △ strain successfully constructed in Example 1 dotPThe strain was activated by streaking on TGY solid agar plates; single colonies were picked and inoculated into 5 mL of fresh TGY liquid agar (recombinant strain supplemented with 10 µg / mL Km antibiotic) and incubated at 30°C for 2-3 days until mid-exponential growth; then, 1% of the culture was re-transferred into 20 mL of fresh TGY liquid agar (recombinant strain supplemented with 10 µg / mL Km antibiotic) and incubated until early logarithmic growth (OD). 600 =Approximately 0.6~0.8; Take 20mL of bacterial suspension, centrifuge at 6,000 rpm for 5 min at room temperature to collect bacterial cells, then wash the bacterial cells twice with the same volume of sterile phosphate buffer (pH 7.0), and shake well; Dry stress experiment: Take 100 µL of bacterial suspension at the bottom of a 2 mL round-bottom centrifuge tube, perform 3 replicates for each sample, then place the centrifuge tube containing the bacterial suspension open in a sterile desiccator (the desiccator uses silica gel as a desiccant), and every 10 days, wild-type WT and Δ dotP Take one tube from each mutant strain, add 1 mL of phosphate buffer and incubate for 30 min, then dilute with sterile phosphate buffer at a 1:10 ratio. -1 Up to 10 -5 Eight µL of each dilution was spotted onto the surface of TGY solid medium and incubated at 30°C for 2–3 days. The growth of wild-type and mutant strains on the medium was observed. Three independent replicates were performed for each strain.

[0050] The △ constructed above dotP Mutant and wild-type DR strains were subjected to drying stress treatments for different durations (10 d, 20 d, 30 d, 40 d, 50 d) for analysis. Experiments confirmed that the mutant strain △ dotP Compared to the wild-type DR strain, the mutant strain showed reduced resistance to desiccation, especially in the sampling results after 30 days, where the survival rate of the mutant strain was an order of magnitude lower than that of the wild-type, indicating that... dotP The absence of this gene directly makes the cells sensitive to dry stress, indicating that the expression of this gene contributes to the bacterial response to dry stress.

[0051] The experimental results show that: dotP Genes have the function of enhancing cells' resistance to dryness stress.

[0052] Example 4

[0053] Construction experiment of recombinant engineered strain DH5α-dotP.

[0054] Table 2

[0055]

[0056] Using the primers (SEQ ID NO. 13-14) containing homologous arms as shown in Table 2, the target gene fragment was amplified by PCR, and the target gene sequence was amplified using a metagenomic sample of desert upper soil as a template.

[0057] PCR amplification reaction conditions: 98℃ for 10 min, [95℃ for 30 sec, 60℃ for 30 sec, 72℃ for 2 min] for 35 cycles, 72℃ for 10 min.

[0058] Purification and recovery of target fragment: After the PCR amplification reaction, 1% agarose gel electrophoresis was performed for about 30 minutes. When the bands were clearly separated under UV irradiation, the gel was cut and the product was purified according to the instructions of the HiPure Gel Pure Micro Kit from Meiji Biotechnology.

[0059] Double digestion of plasmids: using... BamH I and Spe The vector pRADZ3 was double-digested with restriction enzyme I. The system was placed in a PCR instrument at 37℃ for 2 h. The digestion product was purified and recovered, and stored at -20℃ for later use.

[0060] Target fragment recombination ligation: The enzyme-digested and purified linearized vector was homologously recombinated with the target gene containing homologous arms. The ligation reaction was carried out in a metal bath at 55°C for 30 min, following the instructions of the Vazyme Clone Express® Ultra One Step Cloning Kit (C115).

[0061] Transformation of ligation products: Cloned strains were directly transformed using the heat shock method in a clean bench. E.coli DH5α competent cells were incubated overnight in an incubator at 37°C.

[0062] Positive clone identification and sequencing verification: Single colonies were picked from the transformed plates for colony PCR and enzyme digestion verification, and then sent to BGI Genomics for sequencing. This strain was named DH5α-dotP. The strain containing the pRADZ3 empty plasmid was named DH5α. - 0.

[0063] Experimental results show that the expression was successfully constructed. dotP The recombinant Escherichia coli engineered strain DH5α-dotP.

[0064] Example 5

[0065] Include dotP Stress resistance experiment of genetically recombinant engineered strains.

[0066] The recombinant engineered strain obtained in Example 1 contains dotP The DH5α-dotP strain containing the gene; the control strain was the DH5α-0 strain containing the empty plasmid described in Example 1.

[0067] The control strain and the recombinant engineered strain were streaked onto LB agar plates for activation. Single colonies were picked and inoculated into 5 mL of fresh LB liquid medium (recombinant strain supplemented with 50 μg / mL ampicillin antibiotic) and incubated overnight at 37°C until the mid-to-late exponential growth phase. The culture was then re-transferred at 1% to 20 mL of fresh LB liquid medium (recombinant strain supplemented with 50 μg / mL ampicillin antibiotic) and incubated until the bacterial growth reached OD500. 600 =Approximately 0.6~0.8; Take 1 mL of bacterial suspension, centrifuge at 6,000 rpm for 5 min at room temperature to collect bacterial cells, then wash the bacterial cells twice with the same volume of sterile phosphate buffer (pH 7.0), and shake well; High concentration sorbitol stress experiment: serially dilute the resuspended bacterial cells (10... -1 -10 -5 Eight μL of each dilution was spotted onto the surface of LB solid medium containing 1.3 M sorbitol. LB solid medium without sorbitol was used as a blank control. The culture was incubated at 37°C for approximately 5 days, and colony formation was observed. Three independent replicates were performed for each dilution.

[0068] Experimental results are as follows Figure 3 As shown, under prolonged (5 days) high-concentration sorbitol stress treatment, the content of dotP The recombinant engineered strain DH5α-dotP showed good growth, with a significantly higher colony count than the DH5α-0 strain containing only an empty plasmid. Furthermore, its resistance to drying was nearly two orders of magnitude higher than the control strain, approximately 100-fold.

[0069] The experimental results show that: dotP The gene significantly enhances the ability of prokaryotic host cells to resist osmotic stress (see...). Figure 6 ).

[0070] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0071] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0072] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A method for improving biological stress resistance using the dotP gene, characterized in that, The method includes: The dotP gene is transferred into a host organism, and the host organism expresses the dotP gene; the host organism is Escherichia coli DH5α; the dotP gene encodes a protein consisting of the amino acid sequence shown in SEQ ID NO.2; The biological stress resistance mentioned above is dryness resistance.

2. The method according to claim 1, wherein, The dotP gene is a DNA molecule with the nucleotide sequence shown in SEQ ID NO.

1.

3. The method according to claim 1 or 2, wherein, The method further includes inserting the dotP gene into a vector to obtain a recombinant vector, and transferring the recombinant vector into the host organism.

4. The method according to claim 3, wherein, The recombinant vector contains groEL promoter completeness dotP Recombinant plasmid pRADZ3-dotP.

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