Xanthomonas sp. Capable of producing exopolysaccharides at high yield, preparation method and application of Xanthomonas sp.

Abstract

The invention discloses Xanthomonas sp. Capable of producing exopolysaccharides at high yield as well as a preparation method and application thereof, and relates to the technical field of genetic engineering. According to the Xanthomonas oryzae for high-yield production of the exopolysaccharides, wild type rice bacterial Xanthomonas oryzae is taken as a starting bacterium, ACU1210520 genes are knocked out or knocked down, and the nucleotide sequence of the ACU1210520 genes is shown as SEQ ID NO: 3. According to the mutant strain obtained after the ACU1210520 gene is knocked out or knocked down, the exopolysaccharide yield of the strain is remarkably improved compared with that of a wild strain. Therefore, the Xanthomonas sp. Capable of producing the exopolysaccharides at high yield, provided by the invention, has important significance for improving the yield of xanthan gum. In addition, the method is also used for screening an antibacterial agent for preventing and treating rice bacterial streak pathogen.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, in particular to a pathogenic leaf streak pathogen with high extracellular polysaccharide production, a preparation method and application thereof. Background Art

[0002] Xanthomonas oryzae pv. oryzicola (Xoc) is a rod-shaped, Gram-negative bacterium that infects rice and causes rice bacterial leaf streak. A single bacterium is 0.7-2 μm long and 0.4-0.7 μm wide. It has a single polar flagellum that aids in movement. On nutrient-rich, glucose-containing solid media, the colony morphology presents a yellow, circular shape with irregular shapes. It also produces a large amount of sticky extracellular polysaccharides, which help the bacteria cause disease and resist adversity.

[0003] Exopolysaccharides refer to macromolecular compounds released into the outer layer of bacteria or into the environment. Their main component is a heteropolysaccharide composed of repeating pentose units, known industrially as xanthan gum. It is a commonly used thickener, and the bacterial leaf streak pathogen of rice can produce large amounts of exopolysaccharides. In Xanthomonas bacteria, exopolysaccharides are synthesized by 12 highly conserved genes (gumB-gumM) in the gum gene cluster. Due to their high hydrophilicity, the production of exopolysaccharides not only facilitates bacterial adhesion to host surfaces but also helps them withstand dry environments. For most microtubule-bundling Xanthomonas pathogens, exopolysaccharides also have the effect of blocking microtubules. Furthermore, studies have shown that exopolysaccharides play an important role in inhibiting plant defense responses and in the formation of bacterial biofilms.

[0004] Screening the leaf streak pathogen with high extracellular polysaccharide production is of great significance for the production of xanthan gum.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of the present invention is to provide a pathogenic leaf streak pathogen with high extracellular polysaccharide production, a preparation method and application thereof to solve the above technical problems.

[0007] The present invention is achieved in that:

[0008] In a first aspect, the present invention provides a high-exopolysaccharide leaf streak fungus. The high-exopolysaccharide leaf streak fungus uses wild-type rice bacterial leaf streak fungus as a starting strain, and the ACU12_10520 gene is knocked out or knocked down. The nucleotide sequence of the ACU12_10520 gene is shown in SEQ ID NO: 3.

[0009] In a second aspect, the present invention also provides a method for preparing a high-yield extracellular polysaccharide leaf streak fungus, which comprises the following steps: making the ACU12_10520 gene in the rice bacterial leaf streak fungus not expressed or inhibiting its expression, and the nucleotide sequence of the ACU12_10520 gene is shown in SEQ ID NO: 3.

[0010] In a preferred embodiment of the present invention, the ACU12_10520 gene in the leaf streak pathogen is not expressed or its expression is inhibited by gene knockout.

[0011] In a preferred embodiment of the present invention, the gene knockout adopts a gene editing technology selected from at least one of LEAPER technology, CRISPR / Cas gene editing technology, ZFN technology, TALEN technology and homologous recombination;

[0012] In a preferred embodiment of the present invention, the editing tool used in the CRISPR / Cas gene editing technology is selected from any one of CRISPR / Cas9, CRISPR / Cas13 technology, CRISPR / Cas12 and CRISPR / Cas14;

[0013] In a preferred embodiment of the present invention, the gene editing technology used for knockout is selected from homologous recombination technology.

[0014] In a preferred embodiment of the present invention, when the homologous recombination method is used, a recombinant vector including the upstream homologous arm gene and the downstream homologous arm gene of the ACU12_10520 gene is constructed and transduced into the wild-type rice bacterial leaf streak fungus, and mutant strains are screened to obtain; the nucleotide sequences of the upstream homologous arm gene and the downstream homologous arm gene are shown in SEQ ID NO: 1-2, respectively.

[0015] In a preferred embodiment of the present invention, the wild type Xanthophyllomyces oryzae is Xanthophyllomyces oryzae Rs105.

[0016] In a preferred embodiment of the present invention, the recombinant vector is pK18mobsacB.

[0017] In a third aspect, the present invention also provides a recombinant vector, which includes an upstream homologous arm gene and a downstream homologous arm gene of the ACU12_10520 gene, and the nucleotide sequences of the upstream homologous arm gene and the downstream homologous arm gene are shown in SEQ ID NO: 1-2, respectively.

[0018] In a fourth aspect, the present invention further provides a recombinant bacterium comprising the above-mentioned recombinant vector.

[0019] In a fifth aspect, the present invention also provides the use of the high-exopolysaccharide-producing rice leaf streak pathogen or the above-mentioned recombinant bacteria in the fermentation production of xanthan gum or in the screening of antibacterial agents for controlling rice bacterial leaf streak pathogen.

[0020] In a sixth aspect, the present invention also provides a method for producing xanthan gum by fermentation, which comprises culturing the above-mentioned high-exopolysaccharide-producing pathogenic leaf streak fungus or the above-mentioned recombinant bacteria.

[0021] In a preferred embodiment of the present invention, the culture conditions are 25°C-28°C;

[0022] In a preferred embodiment of the present invention, the culture is carried out on NA medium containing 10%-12% sucrose.

[0023] The present invention has the following beneficial effects:

[0024] The present invention discovered that mutants obtained by knocking out or downregulating the ACU12_10520 gene, using wild-type Pseudomonas oryzae var. oryzae as a starting strain, significantly increased exopolysaccharide production compared to the wild-type strain. Therefore, the high-exopolysaccharide-producing Pseudomonas oryzae var. oryzae var. oryzae provided by the present invention are of great significance for increasing xanthan gum production. Furthermore, the method can be used to screen for antimicrobial agents for controlling Pseudomonas oryzae var. oryzae var. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0026] Figure 1 Comparison of colony morphology between the ACU12_10520 gene mutant and the wild-type strain;

[0027] Figure 2 This is the statistical result of EPS production of ACU12_10520 gene mutant and wild-type strain;

[0028] Figure 3 Figure 2 is the colony PCR result of ACU12_10520 gene mutation mutant and wild-type strain Rs105;

[0029] Figure 4 This is a standard curve of glucose content and absorbance. DETAILED DESCRIPTION

[0030] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0031] In a first aspect, the present invention provides a high-exopolysaccharide leaf streak fungus. The high-exopolysaccharide leaf streak fungus uses wild-type rice bacterial leaf streak fungus as a starting strain, and the ACU12_10520 gene is knocked out or knocked down. The nucleotide sequence of the ACU12_10520 gene is shown in SEQ ID NO: 3.

[0032] The gene editing technology used for gene knockout is selected from at least one of LEAPER technology, CRISPR / Cas gene editing technology, ZFN technology, TALEN technology and homologous recombination.

[0033] The function of the ACU12_10520 gene is Fis family transcriptional regulator.

[0034] By knocking out or knocking down the ACU12_10520 gene in wild-type Psoralea oryzae oryzae, thereby eliminating or suppressing its expression, the ACU12_10520 gene significantly increases the exopolysaccharide production of the bacteria compared to the wild-type strain. Therefore, the high-exopolysaccharide-producing Psoralea oryzae oryzae provided by the present invention is of great significance for increasing xanthan gum production. Furthermore, the invention can also be used to screen for antimicrobial agents for controlling Psoralea oryzae oryzae oryzae.

[0035] The present invention provides a new target for the production of exopolysaccharides, which is not limited to the gum gene cluster.

[0036] In a second aspect, the present invention also provides a method for preparing a high-yield extracellular polysaccharide leaf streak fungus, which comprises the following steps: making the ACU12_10520 gene in the rice bacterial leaf streak fungus not expressed or inhibiting its expression, and the nucleotide sequence of the ACU12_10520 gene is shown in SEQ ID NO: 3.

[0037] As long as the expression level (mRNA level and / or protein level) of the ACU12_10520 gene in Pseudomonas oryzae var. oryzae is decreased, significantly decreased or markedly decreased compared to the wild type, it is within the scope of protection of the present invention.

[0038] In an optional embodiment, the expression regulation of the ACU12_10520 gene can be achieved by targeting the ACU12_10520 gene through siRNA, shRNA, pre-miRNA, miRNA, and pri-miRNA.

[0039] In a preferred embodiment of the present invention, gene knockout is used to prevent or inhibit the expression of the ACU12_10520 gene in the pathogenic bacteria Pseudomonas streak.

[0040] In a preferred embodiment of the present invention, the gene editing technology used for gene knockout is selected from at least one of LEAPER technology, CRISPR / Cas gene editing technology, ZFN technology, TALEN technology and homologous recombination.

[0041] LEAPER technology involves introducing an RNA (arRNA) sequence complementary to the target site into cells, recruiting ADAR proteins to edit the target RNA site. The RNA used in this technology, which is complementary to the target site sequence, can stimulate highly efficient RNA editing at the target site without any modification.

[0042] In a preferred embodiment of the present invention, the editing tool used in the CRISPR / Cas gene editing technology is selected from any one of CRISPR / Cas9, CRISPR / Cas13 technology, CRISPR / Cas12 and CRISPR / Cas14; as long as the knockout of the ACU12_10520 gene can be achieved, it is within the scope of protection of the present invention.

[0043] In a preferred embodiment of the present invention, the gene editing technology used for knockout is selected from homologous recombination technology.

[0044] In a preferred embodiment of the present invention, when the homologous recombination method is used, a recombinant vector including the upstream homologous arm gene and the downstream homologous arm gene of the ACU12_10520 gene is constructed and transduced into the wild-type rice bacterial leaf streak fungus, and mutant strains are screened to obtain; the nucleotide sequences of the upstream homologous arm gene and the downstream homologous arm gene are shown in SEQ ID NO: 1-2, respectively.

[0045] In a preferred embodiment of the present invention, the wild type Xanthophyllomyces oryzae is Xanthophyllomyces oryzae Rs105.

[0046] In a preferred embodiment of the present invention, the recombinant vector is pK18mobsacB. In other embodiments, the recombinant vector may also be other shuttle vectors, as long as they can achieve the transfer of the target gene into the wild-type rice bacterial leaf streak fungus.

[0047] In a third aspect, the present invention also provides a recombinant vector, which includes an upstream homologous arm gene and a downstream homologous arm gene of the ACU12_10520 gene, and the nucleotide sequences of the upstream homologous arm gene and the downstream homologous arm gene are shown in SEQ ID NO: 1-2, respectively.

[0048] The recombinant vector can also be provided with a selection marker gene for screening positive clones, such as antibiotic genes such as AmpR, KanR, Chl, Spe, Tet (tetracycline resistance gene), Zeo / ble, Str, Hyg, Gen, Apr, and Ery.

[0049] The recombinant vector also includes a regulatory sequence, which includes regulatory elements such as promoters and terminators.

[0050] In a fourth aspect, the present invention further provides a recombinant bacterium comprising the above-mentioned recombinant vector. The recombinant bacterium is Pseudomonas oryzae leaf streak pathogen.

[0051] The recombinant bacteria are prepared by transforming a recombinant expression vector into a host cell (e.g., a microorganism) using conventional methods in the art. The host microorganism can be any of a variety of conventional host microorganisms in the art, as long as the recombinant expression vector can stably replicate and the exogenous gene carried by it can be effectively expressed. The host microorganism is a bacterium or a fungus.

[0052] In a fifth aspect, the present invention also provides the use of the high-exopolysaccharide-producing rice leaf streak pathogen or the above-mentioned recombinant bacteria in the fermentation production of xanthan gum or in the screening of antibacterial agents for controlling rice bacterial leaf streak pathogen.

[0053] The application of screening antimicrobial agents for controlling bacterial leaf streak of rice includes but is not limited to: when the target biocontrol bacteria has a good inhibitory effect on bacterial leaf streak of rice, the biocontrol bacteria is evaluated to have a good control effect, and has the prospect of preparing antimicrobial agents for controlling bacterial leaf streak of rice.

[0054] The present invention quantitatively measured EPS production by the anthrone method and found that the production of extracellular polysaccharides of the mutated rice bacterial leaf streak pathogen was 2.6 times higher than that of the wild-type strain.

[0055] In a sixth aspect, the present invention also provides a method for producing xanthan gum by fermentation, which comprises culturing the above-mentioned high-exopolysaccharide-producing pathogenic leaf streak fungus or the above-mentioned recombinant bacteria.

[0056] In a preferred embodiment of the present invention, the culture conditions are 25°C-28°C;

[0057] In a preferred embodiment of the present invention, the culture is carried out on NA medium containing 10%-12% sucrose.

[0058] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0059] Example 1

[0060] This example provides a method for constructing a deletion mutant or a preparation method of Xanthophyllomyces syringae with high exopolysaccharide production, specifically utilizing a homologous recombination method.

[0061] The Xoc wild-type strain RS105 (Xanthomonas oryzae pv. oryzicola strain RS105) was used as the starting strain, which was collected by the Plant Pathology Laboratory of Shanghai Academy of Agricultural Sciences, and pK18mobsacB was used to construct deletion mutants.

[0062] The specific steps for knocking out the ACU12_10520 gene are as follows: First, using Rs105 genomic DNA as a template, two pairs of primers, ACU12_10520-1F and ACU12_10520-1R, and ACU12_10520-2F and ACU12_10520-2R, were used to amplify the upstream (i.e., upstream homology arm gene) and downstream fragments (i.e., downstream homology arm gene) of the ACU12_10520 gene, respectively. The nucleotide sequences of the upstream homology arm gene and the downstream homology arm gene are shown in SEQ ID NOs: 1-2, respectively.

[0063] The two fragments were then ligated into pMD19-T and heat-transferred into E. coli DH5α to generate the pMD19-upstream fragment and pMD19-downstream fragment plasmids. The fragments were then sent to the company for sequencing to verify the correctness of the fragments. Once verified, the upstream and downstream fragments were digested with the corresponding restriction endonucleases and then ligated into pK18mobsacB, which had been double-digested with the corresponding enzymes. Finally, through repeated heat-transfer, plasmid extraction, and enzyme digestion verification, the recombinant vector containing the upstream and downstream homology arm genes was obtained.

[0064] The recombinant vector was electroporated into Rs 105 electrocompetent cells by electroporation. The primary exchange products were screened using Km 25 μg / mL and NA plates without sucrose. The single colonies that grew were then verified by PCR. The correctly verified primary exchange products were cultured in NBWS (NB without sucrose) liquid medium with shaking at 28°C, 200 rpm, and cultured for 7-9 hours. About 100 μL was spread on NA plates containing 10% sucrose and cultured at 28°C for 2-3 days. After a single colony grew, PCR was performed to verify whether it was a deletion mutant. The results of colony PCR were referenced to Figure 3 As shown, Figure 3The red mark in the figure indicates the wild type, and the green box indicates the mutant, indicating that the mutant was obtained. The results of the ACU12_10520 gene mutant and the wild type strain Rs105 produced extracellular polysaccharides by culturing at 28℃ for 4 days refer to the figure. Figure 1 As shown, the results showed that the mutant strain significantly increased the production of exopolysaccharides.

[0065] ACU12_10520-1F <![CDATA[5′-CG GGATCC ACCACCAAACCGCTGCTTGC-3′]]> ACU12_10520-1R <![CDATA[5′′-CCC AAGCTT TCACCGCCATCCACTCATCGT-3′]]> ACU12_10520-2F <![CDATA[5′′-CCC AAGCTT TCAACGAAGCACTGGAACGCA-3′]]> ACU12_10520-2R <![CDATA[5′-GC TCTAGA GCGGTGGCAACGAAGGAGTA-3′]]>

[0066] Example 2

[0067] Determination of extracellular polysaccharide content (sulfuric acid-anthrone method).

[0068] (1) Preparation of anthrone reagent and glucose standard solution:

[0069] Anthrone reagent: Dissolve 0.2 g of anthrone in 100 ml of concentrated sulfuric acid to prepare the glucose standard solution: 100 μg / ml.

[0070] (2) Preparation of glucose standard curve: Take 7 test tubes and add 1 ml of glucose solution with concentrations of 0, 10, 20, 30, 40, 60, and 80 μg / ml respectively. Add 4 ml of anthrone reagent to the test tubes and then immerse them in an ice water bath for cooling. After adding the anthrone reagent to each tube, shake them evenly on an oscillator. Then immerse all the test tubes in a boiling water bath and plug them with test tube stoppers to prevent evaporation. Start timing from the time the water bath is boiled again. Remove the test tubes from the water bath after 10 minutes.

[0071] Cool the test tube and the liquid in it to room temperature in an ice water bath. At a wavelength of 620nm, use the tube with a glucose content of 0μg / ml as a blank and measure the absorbance of the remaining tubes. Use the glucose content (μg) as the horizontal axis and the absorbance value as the vertical axis to draw a standard curve ( Figure 4 shown).

[0072] (3) Determination of total sugar in the sample: The mutant strain and wild-type Rs105 bacterial culture cultured in Example 1 were diluted so that the final measured absorbance value was within the range of the standard curve; 1 ml of sample was added to the test tube, and then 4 ml of anthrone reagent was immediately added, and the subsequent operation was consistent with step (2); then, the glucose content (μg) was found on the standard curve based on the measured absorbance value.

[0073] Figure 2 The results showed that the EPS production of one of the mutant strains constructed in the embodiment reached 4.83ugEPS / ug protein, while the wild type only produced 1.58ugEPS / ug protein. The EPS production of the mutant strain was 2.6 times higher than that of the wild type strain.

[0074] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A pathogenic bacterium with high production of extracellular polysaccharides, characterized in that: The high-exopolysaccharide leaf streak fungus uses wild-type rice bacterial leaf streak fungus as a starting strain, knocks out or knocks down the ACU12_10520 gene, and the nucleotide sequence of the ACU12_10520 gene is shown in SEQ ID NO:

3.

2. A method for preparing a pathogenic leaf streak bacteria with high extracellular polysaccharide production, characterized in that: The method comprises the following steps: making the ACU12_10520 gene in oxysporum leaf streak pathogenic bacteria of oryzae not expressed or inhibiting its expression, wherein the nucleotide sequence of the ACU12_10520 gene is shown in SEQ ID NO:

3.

3. The method for preparing the pathogenic leaf streak bacteria with high extracellular polysaccharide production according to claim 2, characterized in that: By knocking out the gene, the ACU12_10520 gene in the leaf streak fungus is not expressed or its expression is inhibited; Preferably, the gene knockout adopts a gene editing technology selected from at least one of LEAPER technology, CRISPR / Cas gene editing technology, ZFN technology, TALEN technology and homologous recombination; Preferably, the editing tool used in the CRISPR / Cas gene editing technology is selected from any one of CRISPR / Cas9, CRISPR / Cas13 technology, CRISPR / Cas12 and CRISPR / Cas14; Preferably, the gene editing technology used for the knockout is selected from homologous recombination technology.

4. The method for preparing the pathogenic leaf streak bacteria with high extracellular polysaccharide production according to claim 3, characterized in that: When the homologous recombination method is used, a recombinant vector including the upstream homologous arm gene and the downstream homologous arm gene of the ACU12_10520 gene is constructed, transduced into the wild-type rice bacterial leaf streak fungus, and a mutant strain is obtained by screening; the nucleotide sequences of the upstream homologous arm gene and the downstream homologous arm gene are shown in SEQ ID NO: 1-2, respectively.

5. The method for preparing the pathogenic leaf streak bacteria with high extracellular polysaccharide production according to claim 4, characterized in that: The wild type oxysporum leaf streak of oryzae is oxysporum leaf streak of oryzae Rs105.

6. The method for preparing the pathogenic leaf streak bacteria with high extracellular polysaccharide production according to claim 4, characterized in that: The recombinant vector is pK18mobsacB.

7. A recombinant vector, characterized in that: It includes an upstream homologous arm gene and a downstream homologous arm gene of the ACU12_10520 gene, and the nucleotide sequences of the upstream homologous arm gene and the downstream homologous arm gene are shown in SEQ ID NO: 1-2, respectively.

8. A recombinant bacterium, characterized in that: It comprises the recombinant vector according to claim 7.

9. Use of the high-exopolysaccharide leaf streak pathogen as claimed in claim 1 or the recombinant bacterium as claimed in claim 8 in fermentation production of xanthan gum or in screening antimicrobial agents for controlling bacterial leaf streak pathogen of rice.

10. A method for producing xanthan gum by fermentation, characterized in that: Cultivating the pathogenic bacteria of claim 1 that produces high amounts of exopolysaccharides or the recombinant bacteria of claim 8; Preferably, the culture condition is 25°C-28°C; Preferably, the culture is carried out in NA medium containing 10%-12% sucrose.

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