WO2021184780A1 - 沼泽红假单胞菌胞外多糖及其制备方法和应用 - Google Patents

沼泽红假单胞菌胞外多糖及其制备方法和应用 Download PDF

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WO2021184780A1
WO2021184780A1 PCT/CN2020/128092 CN2020128092W WO2021184780A1 WO 2021184780 A1 WO2021184780 A1 WO 2021184780A1 CN 2020128092 W CN2020128092 W CN 2020128092W WO 2021184780 A1 WO2021184780 A1 WO 2021184780A1
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rhodopseudomonas palustris
polysaccharide
extracellular polysaccharide
extracellular
preparation
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PCT/CN2020/128092
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French (fr)
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苏品
翟忠英
张卓
杨海星
戴建平
周波
张德咏
刘勇
程菊娥
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湖南省植物保护研究所
长沙艾格里生物科技有限公司
苏州艾科尔化工科技有限公司
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Priority to US17/265,097 priority Critical patent/US11473115B2/en
Publication of WO2021184780A1 publication Critical patent/WO2021184780A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N31/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic oxygen or sulfur compounds
    • A01N31/06—Oxygen or sulfur directly attached to a cycloaliphatic ring system
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
    • A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
    • A01N43/14—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings
    • A01N43/16—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings with oxygen as the ring hetero atom
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P21/00—Plant growth regulators
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P3/00—Fungicides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P17/00—Pest repellants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • the invention relates to the technical field of biological pesticides, in particular to a kind of extracellular polysaccharide of Rhodopseudomonas palustris.
  • the present invention also relates to a preparation method of the above-mentioned Rhodopseudomonas palustris extracellular polysaccharide and the application of the above-mentioned Rhodopseudomonas palustris extracellular polysaccharide in plant growth promotion and disease prevention and control.
  • Microbial extracellular polysaccharides are water-soluble or water-insoluble polysaccharides that are synthesized by certain bacteria and fungi during the growth of various carbon sources and secreted to the outside of the cell. Compared with plant polysaccharides and marine algae polysaccharides, microbial extracellular polysaccharides have the advantages of short growth cycle, unaffected by climate, simple production process, low cost, and convenient mass preparation. Microbial extracellular polysaccharides are generally non-toxic, unique in structure, and stable in physical and chemical properties. Some polysaccharides have antioxidant, immunosuppressive, immune-promoting, and anti-tumor activities. They are widely used in medicine, chemical industry, petroleum exploration, and environmental protection. Application, it would be of great benefit to develop a microbial extracellular polysaccharide that can be used for plant growth promotion and biological control to replace abused chemical pesticides.
  • the present invention provides a Rhodopseudomonas palustris extracellular polysaccharide and a preparation method and application thereof, so as to solve the technical problem that the specific active ingredients of the existing biological control are not clear and the biological control effect is not good enough.
  • an extracellular polysaccharide of Rhodopseudomonas palustris and the structural formula of the extracellular polysaccharide is:
  • extracellular polysaccharide is obtained by alcohol precipitation extraction from Rhodopseudomonas palustris GJ-22 (Rhodopseudomonas palustris GJ-22) fermentation broth.
  • a method for preparing the above-mentioned Rhodopseudomonas palustris extracellular polysaccharide including the following steps:
  • the components of the fermentation medium include: (NH 4 ) 2 SO 4 0.1 g, MgSO 4 0.02 g, Na 2 CO 3 0.5 g, K 2 HPO 4 0.05 g, NaCl 0.02 g, casein Amino acid 0.2g, agar 1.5g, pH value is 7.0 ⁇ 7.5;
  • the fermentation and culture conditions are as follows: the fermentation culture temperature is 30° C., the light intensity is 7000 lx to 8000 lx, and the culture time is 10 days.
  • step 2) the alcohol precipitation includes the following steps: adding anhydrous ethanol of twice the volume of the filtered supernatant, stirring uniformly, and standing at 4°C for 24h,
  • the centrifugal collection adopts a centrifugal force of 10,000 to 13,000 g, preferably 10,000 g, and centrifuges at 4° C. for 20 minutes.
  • the steps of the protease enzymatic hydrolysis method include: dissolving the crude polysaccharide obtained in the step in deionized water, adding papain; adjusting the pH to 6.0-6.2, treating in a 60°C water bath for 6 hours, and every 1 hour during the water bath treatment. Shake once every hour to mix the sample. After cooling, add twice the volume of absolute ethanol, and then centrifuge to collect the precipitate.
  • the dialysis treatment adopts a dialysis bag with a molecular weight cut-off of 8000D to 14000D.
  • step 4 the anion exchange column adopts Hi Trap Q Sepharose High Performance
  • the molecular exclusion chromatography column adopts Seharose CL-6B chromatography column.
  • an application of the above-mentioned Rhodopseudomonas palustris extracellular polysaccharide in promoting the growth of tobacco or rice is also provided.
  • Rhodopseudomonas palustris extracellular polysaccharide in the prevention and treatment of tobacco mosaic virus or rice blast.
  • Rhodopseudomonas palustris GJ-22 extracellular polysaccharide of the present invention obtains Rhodopseudomonas palustris GJ-22 cells by fermentation, culture, alcohol precipitation, separation and purification of Rhodopseudomonas palustris GJ-22
  • the exopolysaccharide has a simple production process, stable expression and easy availability.
  • the present invention is the first to study the extracellular polysaccharides of leaf biocontrol bacteria. The study found that the purified Rhodopseudomonas palustris GJ-22 extracellular polysaccharide can significantly promote plant growth and enhance plant disease resistance, and has excellent growth promotion and disease resistance. Rhodopseudomonas marshes GJ-22 provides a possibility for the development of biocontrol agents, which will play an important application value in the agricultural field.
  • Figure 1 is a schematic diagram of the elution curve of a polysaccharide anion exchange column according to a preferred embodiment of the present invention
  • Fig. 2 is a schematic diagram of the elution curve of a Seharose CL-6B column in a preferred embodiment of the present invention
  • Fig. 3 is a schematic diagram of a molecular weight spectrum of Rhodopseudomonas palustris GJ-22 extracellular polysaccharide according to a preferred embodiment of the present invention
  • FIG. 4 is a schematic diagram of the monosaccharide composition of Rhodopseudomonas palustris GJ-22 extracellular polysaccharide monosaccharide according to a preferred embodiment of the present invention
  • Fig. 5 is a schematic diagram of GC-MS of Rhodopseudomonas palustris GJ-22 extracellular polysaccharide according to a preferred embodiment of the present invention
  • Fig. 6 is a hydrogen spectrum of Rhodopseudomonas palustris GJ-22 extracellular polysaccharide according to a preferred embodiment of the present invention.
  • Fig. 7 is a carbon spectrum chart of Rhodopseudomonas palustris GJ-22 extracellular polysaccharide according to a preferred embodiment of the present invention.
  • Fig. 8 is a NMR spectrum of Rhodopseudomonas palustris GJ-22 extracellular polysaccharide DEPT 135 according to a preferred embodiment of the present invention.
  • Figure 9 is a HH-COSY spectrum of Rhodopseudomonas palustris GJ-22 extracellular polysaccharide according to a preferred embodiment of the present invention.
  • Figure 10 is the HMBC spectrum of Rhodopseudomonas palustris GJ-22 extracellular polysaccharide according to a preferred embodiment of the present invention.
  • Figure 11 is a NOESY spectrum of Rhodopseudomonas palustris GJ-22 extracellular polysaccharide according to a preferred embodiment of the present invention.
  • Figure 12 is a graph showing the fresh weight of plants treated with Rhodopseudomonas palustris GJ-22 exopolysaccharide according to a preferred embodiment of the present invention.
  • Figure 13 is a detection diagram of Rhodopseudomonas palustris GJ-22 extracellular polysaccharide inducing tobacco resistance to TMV virus particles in a preferred embodiment of the present invention.
  • Fig. 14 is a detection diagram of Rhodopseudomonas palustris GJ-22 extracellular polysaccharide inducing rice resistance to rice blast spot in a preferred embodiment of the present invention.
  • the microorganism of the present invention is Rhodopseudomonas palustris GJ-22, which is a foliar biocontrol bacteria, obtained by separation and purification from water, and deposited in the General Microbiology Center of China Microbial Species Collection Management Committee.
  • the deposit number is CGMCC No: 17356.
  • the unit address is located at the Institute of Microbiology, Chinese Academy of Sciences.
  • the preparation method of Rhodopseudomonas palustris GJ-22 extracellular polysaccharide includes the following steps:
  • the conditions of fermentation culture are: inoculate the fermentation medium with 10% inoculum, the fermentation culture temperature is 30°C, the light intensity is 7000lx-8000lx, and the culture time is 10 days.
  • centrifuge the fermentation broth to take the supernatant The conditions for centrifugation are 15000g centrifugal force and 50min at 4°C. The purpose of centrifugation is to separate the remaining bacteria and insoluble impurities; the supernatant is filtered through membrane to remove suspended matter. Alcohol precipitation, centrifugation to collect the precipitate, freeze-dry the precipitate to obtain the crude polysaccharide; the membrane is preferably a 0.45 ⁇ m membrane to filter out very tiny suspended particles in the supernatant after centrifugation.
  • Rhodopseudomonas palustris GJ-22 extracellular polysaccharide of the present invention obtains Rhodopseudomonas palustris GJ-22 cells by fermentation, culture, alcohol precipitation, separation and purification of Rhodopseudomonas palustris GJ-22
  • the exopolysaccharide has a simple production process, stable expression and easy availability.
  • the present invention is the first to study the extracellular polysaccharides of leaf biocontrol bacteria. The study found that the purified Rhodopseudomonas palustris GJ-22 extracellular polysaccharide can significantly promote plant growth and enhance plant disease resistance, and has excellent growth promotion and disease resistance. Rhodopseudomonas marshes GJ-22 provides a possibility for the development of biocontrol agents, which will play an important application value in the agricultural field.
  • the components of the fermentation medium include: (NH 4 ) 2 SO 4 0.1g, MgSO 4 0.02g, Na 2 CO 3 0.5g, K 2 HPO 4 0.05g, NaCl 0.02g,
  • the casamino acid is 0.2g
  • the agar is 1.5g
  • the pH is 7.0-7.5.
  • the fermentation medium of this example is added with casamino acids, which improves the production of extracellular polysaccharides.
  • the alcohol precipitation specifically includes the following steps: adding anhydrous ethanol of twice the volume of the filtered supernatant, stirring uniformly, standing at 4°C for 24 hours, and centrifugation to collect the precipitate after the alcohol precipitation is Use a centrifugal force of 10,000 to 13,000 g, and centrifuge at 4°C for 20 minutes, preferably a centrifugal force of 10,000 g.
  • the precipitate is preferably washed three times with absolute ethanol to remove impurities, and then freeze-dried to obtain crude polysaccharides. By collecting crude extracellular polysaccharides by alcohol precipitation, the extracted polysaccharides have higher purity, more complete and stable structure.
  • the step of deproteinizing by protease enzymatic hydrolysis includes: dissolving the crude polysaccharide obtained in step 2) in an appropriate amount of deionized water (the amount of deionized water needs to dissolve all the crude polysaccharide), and adding papain Adjust the pH to 6.0-6.2, and treat it in a 60°C water bath for 6 hours. During the water bath treatment, shake once every 1 hour to mix the sample. After cooling, add twice the volume of absolute ethanol. After standing, centrifuge to collect the precipitate. Papain can decompose protein under acidic, neutral and alkaline conditions. It has the activity of protease and esterase. It has strong hydrolysis ability on bacterial exopolysaccharides, and will not damage the structure of extracellular polysaccharides. The extracted extracellular polysaccharide has a higher content.
  • a dialysis bag with a molecular weight cut-off of 8000D to 14000D is used for the dialysis treatment.
  • the specific operation is: pack the protein-removed polysaccharide in a dialysis bag with a molecular weight cut-off of 8000 ⁇ 14000D, dialyzed with distilled water for 2 days, change the water every 4h to remove small molecular compounds and organic solvents, after the dialysis, reduce the sample Concentrate by pressure and freeze-dry to obtain a polysaccharide sample.
  • the molecular weight of the extracellular polysaccharide of Rhodopseudomonas palustris is about 10KD, so a dialysis bag with a molecular weight cut-off of 8000D-14000D is selected.
  • step 4 the anion exchange column adopts HiTrap Q Sepharose High Performance, and the molecular exclusion chromatography column adopts the Seharose CL-6B chromatography column.
  • the specific operation of the anion exchange column purification is: dissolve the polysaccharide sample from which the protein has been removed in an appropriate amount of distilled water, and then pass it through the anion exchange column for purification.
  • the anion exchange column is HiTrap Q Sepharose High Performance (1.6 ⁇ 2.5cm, GE Healthcare), the loading volume is 5mL, the loading concentration is 20mg/mL, the flow rate is 5mL/min, and the collection is performed in a 2mL centrifuge tube. First wash two column volumes with Tris-HCl (20Mm, pH 7.60), and then use 0.5M Tris-HCl and 0.5M NaCl gradient elution.
  • the phenol-sulfuric acid method was used to detect the polysaccharide content tube by tube, with the number of tubes collected as the abscissa, and the absorbance of each tube at 490 nm as the ordinate, and the elution curve was drawn, as shown in Figure 1.
  • Seharose CL-6B column chromatography purification The specific operation of Seharose CL-6B column chromatography purification is: the components purified by the anion exchange column are dissolved in a balance solution, and the balance solution is an elution buffer (20mM PBS), prepared as a 20mg/mL polysaccharide solution, and removed by centrifugation For the insoluble part, the supernatant is filtered through a 0.22 ⁇ m membrane, and then pushed into the injection port with a syringe. The loading volume is 2mL. Use elution buffer to elute 2 volumes and automatically collect samples. Distilled water was dialyzed for 2 days and freeze-dried to obtain purified Rhodopseudomonas palustris GJ-22 extracellular polysaccharide. The phenol-sulfuric acid method was used for detection, and the elution curve was obtained, as shown in Figure 2.
  • elution buffer (20mM PBS)
  • the elution peak is a single peak when eluted through the anion exchange column. It can be seen from Figure 2 that the elution peak is also a single peak when eluted through the Seharose CL-6B column. Rhodopseudomonas palustris GJ-22 extracellular polysaccharide purity is relatively high.
  • the molecular weight identification of Rhodopseudomonas palustris GJ-22 extracellular polysaccharide prepared in Example 1 includes the following steps:
  • the peak time of the sample is regressed with the logMW of the standard dextran molecular weight LogMW to obtain the dextran standard curve, and then the molecular weight of the sample is calculated according to the retention time of the sample.
  • Figure 3 is the measured molecular weight spectrum of Rhodopseudomonas palustris GJ-22 extracellular polysaccharide. Substituting the measured retention time of the sample into the standard curve of the monosaccharide standard, the relative molecular weight of the sample is calculated to be 10026D.
  • step (3) Polysaccharide component analysis method: the product obtained in step (2) is analyzed by Shimadzu GCMS-QP 2010 gas chromatography-mass spectrometer to determine the acetylated product sample, GC-MS condition: RXI-5SIL MS chromatographic column 30 *0.25*0.25mm;
  • the programmed temperature rise conditions are: the initial temperature is 120°C, the temperature is increased to 250°C/min at 3°C/min, and the temperature is maintained for 5 minutes, the inlet temperature is 250°C, the detector temperature is 250°C/min, and the load The gas is helium, and the flow rate is 1 mL/min.
  • the monosaccharide composition diagram of the obtained exopolysaccharide is shown in Fig. 4, and Fig. 4 shows that Rhodopseudomonas palustris GJ-22 is composed of two monosaccharides, mannose and glucose.
  • Methylation reaction Take 10 mg of Rhodopseudomonas palustris GJ-22 extracellular polysaccharide and add 2 mL of anhydrous dimethyl sulfoxide to fully dissolve it, fill the reaction flask with nitrogen, let it stand at room temperature for 30 minutes, and then add 10 mg NaOH, filled with nitrogen again, react for 1 hour at room temperature under magnetic stirring conditions, then add 0.5 mL of methyl iodide, fill with nitrogen, and continue to react at room temperature for 1 hour. After the reaction is over, add 0.5 mL of distilled water to the reaction tube to stop the reaction. Use distilled water Dialysis for 24h, freeze-drying, and the dried sample continues to methylate.
  • GC-MS analysis conditions Shimadzu GCMS-QP 2010 gas chromatography-mass spectrometer is used for analysis A sample of the acetylated product was determined; the GC-MS spectrum of the methylation result of GJ-22 extracellular polysaccharide is shown in Figure 5.
  • NMR analysis Take 100mg of Rhodopseudomonas palustris GJ-22 extracellular polysaccharide and dissolve it in 1mL D2O, and analyze by 600MHz nuclear magnetic resonance instrument.
  • the hydrogen spectrum of Rhodopseudomonas palustris GJ-22 extracellular polysaccharide is shown in Figure 6, the carbon spectrum is shown in Figure 7, the DEPT 135 NMR spectrum is shown in Figure 8, and the HH-COSY spectrum is shown in As shown in Figure 9, the HMBC spectrum is shown in Figure 10, and the NOESY spectrum is shown in Figure 11.
  • HH-COSY Through HH-COSY, the signal of H1-2 is 5.23/4.06; the signal of H2-3 is 4.06/3.86; the signal of H3-4 is 3.86/3.79; the signal of H4-5 is 3.79/ 3.65; The signal of H5-6a is 3.65/3.71, and it is inferred that H1, H2, H3, H4, H5, H6a are 5.23, 4.06, 3.86, 3.79, 3.65, 3.71 respectively. H6b is 3.82, and the corresponding carbon spectrum is ⁇ 101. 89, 79.67, 71.62, 67.74, 74.63, 62.16;
  • HMBC The anomeric hydrogen of ⁇ 3-Man-1 ⁇ has a correlation peak with the C2 of ⁇ 2-Man-1 ⁇ , indicating the presence of the polysaccharide ⁇ 3-Man-1 ⁇ 2-Man-1 ⁇ ;
  • the anomeric hydrogen of ⁇ 2-Man-1 ⁇ has a correlation peak with the C2 of ⁇ 2,6-Man-1 ⁇ , indicating the presence of the polysaccharide ⁇ 2-Man-1 ⁇ 2,6-Man-1 ⁇ ;
  • Rhodopseudomonas palustris GJ-22 exopolysaccharide prepared in Example 1 is:
  • the present invention identifies and obtains the structure of the extracellular polysaccharide extracted from Rhodopseudomonas palustris GJ-22, which makes the prevention and control active components of biological control more clear.
  • Rhodopseudomonas palustris GJ-22 Through comprehensive and systematic research on the extracellular polysaccharide of Rhodopseudomonas palustris GJ-22, it provides a basic theory for the structure-activity relationship between the function and structure of Rhodopseudomona palustris GJ-22.
  • Rhodopseudomonas palustris GJ-22 extracellular polysaccharide is convenient to produce, stable in effect, and suitable for large-scale production and preparation.
  • Rhodopseudomonas palustris GJ-22 exopolysaccharide prepared in Example 1 was formulated into a 1g/L solution and sprayed evenly on the tobacco leaves after transplanting for 7 days. Spray once a day in the morning and evening to keep moist. The fresh weight of the plant was measured after days, and the supernatant of the fermentation broth, fermentation medium and water of Rhodopseudomonas palustris GJ-22 were used as blank tests.
  • Rhodopseudomonas palustris GJ-22 exopolysaccharide prepared in the example was formulated into a 1g/L solution and sprayed evenly on the rice leaf surface for 14 days after transplanting. Spray once a day in the morning and evening to keep it moist. After 7 days The fresh weight of the plant was measured, and the supernatant of the fermentation broth, fermentation medium and water of Rhodopseudomonas palustris were used as blank tests.
  • Figure 12 is the fresh weight diagram of R. palustris GJ-22 exopolysaccharide in Example 3 and Example 4 after treatment of tobacco and rice.
  • Rhodopseudomonas palustris GJ-22 exopolysaccharide is represented by EPS.
  • the supernatant of the fermentation broth of Rhodopseudomonas palustris is represented by Fermentation
  • the fermentation medium is represented by medium
  • the water is represented by ddH2O
  • the tobacco is represented by Tobacco
  • rice is represented by Rice. It can be seen from Fig.
  • Rhodopseudomonas palustris GJ-22 extracellular polysaccharide can significantly promote the growth of tobacco and rice, and its growth promotion effect is better than that of the fermentation broth supernatant and fermentation medium of R. palustris.
  • Rhodopseudomonas palustris GJ-22 exopolysaccharide prepared in Example 1 was formulated into a 1g/L solution and sprayed on the surface of healthy tobacco leaves with 6 leaves. On the second day, the TMV virus particles were rubbed and inoculated. After inoculation Plant RNA was extracted on day 1, day 2, day 3, and day 4, and the number of TMV virus particles in the plant was determined. As shown in Figure 13, the supernatant of the fermentation broth of Rhodopseudomonas palustris and water were used as a blank test. .
  • FIG 13 shows that Rhodopseudomonas palustris GJ-22 exopolysaccharide can significantly enhance the resistance of tobacco to the virus TMV.
  • Rhodopseudomonas palustris GJ-22 exopolysaccharide prepared in Example 1 was formulated into a 1g/L solution and sprayed on healthy rice leaves grown for 2 weeks. On the second day, the rice blast spore suspension was sprayed on the rice. The leaves were cultured in an incubator with alternating light and dark, and the number of diseased spots on the rice leaves was observed on the 7th, 9th, 11th, and 13th days when the rice blast spore suspension was sprayed, as shown in Figure 14. Rhodopseudomonas palustris fermentation broth supernatant and water are blank tests.
  • Figure 14 shows that Rhodopseudomonas palustris GJ-22 exopolysaccharide can significantly enhance the inhibitory effect of rice on the rice blast fungus.
  • Rhodopseudomonas palustris GJ-22 extracellular polysaccharide can be applied to promote the growth of tobacco and rice, and can also be applied to the control of tobacco mosaic virus and rice blast.

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Abstract

本发明公开了一种沼泽红假单胞菌胞外多糖及其制备方法和应用，所述制备方法包括以下步骤：(1)、将沼泽红假单胞菌GJ-22的种子液发酵培养，获得发酵液；(2)、将发酵液离心处理取上清液，过滤，醇沉、离心收集沉淀，获得粗多糖；(3)、采用蛋白酶酶解和Sevag法去除蛋白，将去除蛋白的粗多糖进行透析处理以去除小分子物质和有机溶剂，获得多糖样品；(4)、将多糖样品经过阴离子交换柱和分子排阻色谱柱纯化，获得沼泽红假单胞菌GJ-22胞外多糖。本发明的沼泽红假单胞菌GJ-22胞外多糖的制备方法，通过对沼泽红假单胞菌GJ-22进行发酵培养，醇沉，分离纯化获得沼泽红假单胞菌GJ-22胞外多糖，表达稳定，应用于烟草和水稻的促生和病虫防治，效果显著。

Description

沼泽红假单胞菌胞外多糖及其制备方法和应用 技术领域
本发明涉及生物农药技术领域，特别地，涉及一种沼泽红假单胞菌胞外多糖。此外，本发明还涉及一种上述沼泽红假单胞菌胞外多糖的制备方法以及上述沼泽红假单胞菌胞外多糖在植物促生以及病害防控等方面的应用。
背景技术
对于植物病害的防治，目前主要还是以化学防治为主，但由于抗药性增强以及发现不及时，农药施用不科学，农药施用剂量与次数严重超标，引起环境污染和农产品质量安全等重大问题。近年来，生物防治以其无毒、无害、无污染、不易产生抗药性和高效等优点，在植物病虫害防治中越来越受到人们的重视。微生物源生物农药发展较快，但对于微生物中具体是哪部分对植物促生和抑制病虫害起作用的研究很少，这对于开发具有针对性、高效的生物农药来说非常不利。
微生物胞外多糖是由某些细菌、真菌在各种碳源上生长过程中合成的，并分泌到细胞外的水溶性或水不溶性多糖物质。相比于植物多糖和海洋藻类多糖，微生物胞外多糖具有生长周期短、不受气候影响、生产工艺简便、成本低、便于大量制备等优点。微生物胞外多糖普遍具有无毒、结构独特、理化性质稳定等特点，有的多糖具有抗氧化、免疫抑制、免疫促进、抗肿瘤等活性，在医药、化工、石油开采以及环境保护等方面被广泛应用，如果能开发一种可以用于植物促生以及生物防治的微生物胞外多糖去替代被滥用的化学农药将大有裨益。
发明内容
本发明提供了一种沼泽红假单胞菌胞外多糖及其制备方法和应用，以解决现有生物防治具体活性成分不明确导致生防功效尚不够好的技术问题。
根据本发明的一个方面，提供一种沼泽红假单胞菌胞外多糖，所述胞外多糖的结构式为：
Figure PCTCN2020128092-appb-000001
进一步地，所述胞外多糖由沼泽红假单胞菌GJ-22(Rhodopseudomonas palustris GJ-22) 发酵液中醇沉提取得到。
根据本发明的另一个方面，提供了一种上述的沼泽红假单胞菌胞外多糖的制备方法，包括以下步骤：
1)、将沼泽红假单胞菌GJ-22(Rhodopseudomonas palustris GJ-22)的种子液在发酵培养基中发酵培养，获得发酵液；
2)、将发酵液离心处理取上清液，采用膜过滤除去上清液中的悬浮质后进行醇沉，离心收集沉淀，将沉淀冷冻干燥获得粗多糖；
3)、采用蛋白酶酶解法和Sevag法去除粗多糖中的蛋白，将去除蛋白的粗多糖用蒸馏水进行透析处理以去除小分子物质和有机溶剂，获得多糖样品；
4)、将多糖样品依次经过阴离子交换柱和分子排阻色谱柱进行纯化，获得所述沼泽红假单胞菌胞外多糖。
进一步地，步骤1)中，发酵培养基的成分包括：(NH 4) 2SO 4 0.1g，MgSO 4 0.02g，Na 2CO 30.5g，K 2HPO 4 0.05g，NaCl 0.02g，酪蛋白氨基酸0.2g，琼脂1.5g，pH值为7.0～7.5；
所述发酵培养的条件为：发酵培养温度为30℃，光照强度为7000lx～8000lx，培养时间为10天。
进一步地，步骤2)中，所述醇沉包括以下步骤：加入过滤后上清液两倍体积的无水乙醇，搅拌均匀后在4℃条件下静置24h，
所述离心收集采用离心力10000～13000g，优选为10000g，在4℃条件下离心20min。
进一步地，步骤3)中，蛋白酶酶解法的步骤包括：将步骤获得的粗多糖溶于去离子水中，加入木瓜蛋白酶；调节pH为6.0～6.2，60℃水浴下处理6h，水浴处理期间每1个小时振荡一次，混匀样品，冷却后，加入两倍体积无水乙醇，静置后离心收集沉淀。
进一步地，所述透析处理采用截留分子量为8000D～14000D的透析袋。
进一步地，步骤4)中，所述阴离子交换柱采用Hi Trap Q Sepharose High Performance，
所述分子排阻色谱柱采用Seharose CL-6B层析柱。
根据本发明的另一个方面，还提供了一种上述的沼泽红假单胞菌胞外多糖在促进烟草或水稻生长中的应用。
根据本发明的再一个方面，还提供了一种上述的沼泽红假单胞菌胞外多糖在防治烟草花叶病毒或水稻稻瘟病中的应用。
本发明具有以下有益效果：
本发明的沼泽红假单胞菌GJ-22胞外多糖的制备方法，通过对沼泽红假单胞菌GJ-22进行发酵培养，醇沉，分离纯化获得沼泽红假单胞菌GJ-22胞外多糖，其生产工艺简单，表达稳定，易于获得。本发明首次对于叶际生防菌胞外多糖进行研究，研究发现纯化后的沼泽红假单胞 菌GJ-22胞外多糖能显著促进植物生长和增强植物抗病性，且促生长和抗病性优于沼泽红假单胞菌GJ-22，为生防菌剂的开发提供了一种可能性，将在农业领域发挥重要的应用价值。
除了上面所描述的目的、特征和优点之外，本发明还有其它的目的、特征和优点。下面将参照图，对本发明作进一步详细的说明。
附图说明
构成本申请的一部分的附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：
图1是本发明优选实施例的多糖阴离子交换柱洗脱曲线示意图；
图2是本发明优选实施例的Seharose CL-6B柱洗脱曲线示意图；
图3是本发明优选实施例的沼泽红假单胞菌GJ-22胞外多糖分子量谱图示意图；
图4是本发明优选实施例的沼泽红假单胞菌GJ-22胞外多糖单糖组成示意图；
图5是本发明优选实施例的沼泽红假单胞菌GJ-22胞外多糖GC-MS示意图；
图6是本发明优选实施例的沼泽红假单胞菌GJ-22胞外多糖氢谱谱图；
图7是本发明优选实施例的沼泽红假单胞菌GJ-22胞外多糖碳谱谱图；
图8是本发明优选实施例的沼泽红假单胞菌GJ-22胞外多糖DEPT 135核磁谱图；
图9是本发明优选实施例的沼泽红假单胞菌GJ-22胞外多糖HH-COSY谱图；
图10是本发明优选实施例的沼泽红假单胞菌GJ-22胞外多糖HMBC谱图；
图11是本发明优选实施例的沼泽红假单胞菌GJ-22胞外多糖NOESY谱图；
图12是本发明优选实施例的沼泽红假单胞菌GJ-22胞外多糖处理植物后鲜重图；
图13是本发明优选实施例的沼泽红假单胞菌GJ-22胞外多糖诱导烟草抵抗TMV病毒粒子检测图；以及
图14是本发明优选实施例的沼泽红假单胞菌GJ-22胞外多糖诱导水稻抵抗稻瘟病斑检测图。
具体实施方式
以下结合附图对本发明的实施例进行详细说明，但是本发明可以由下述所限定和覆盖的多种不同方式实施。
以下实施例中所采用的材料和仪器均为市售。
实施例1
沼泽红假单胞菌胞外多糖的制备纯化
本发明的微生物为沼泽红假单胞菌GJ-22，是一种叶际生防菌，从水中分离纯化得到，保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No：17356，保藏单位地址位于中国科学院微生物研究所。
本实施例中，沼泽红假单胞菌GJ-22胞外多糖的制备方法，包括以下步骤：
1)、将沼泽红假单胞菌GJ-22的种子液在发酵培养基中发酵培养，获得发酵液；种子液的获得方法为：将沼泽红假单胞菌GJ-22经过平板活化后，挑取单菌落进行种子培养，4天后得到种子液。发酵培养的条件为：以10％接种量接种到发酵培养基，发酵培养温度为30℃，光照强度为7000lx～8000lx，培养时间为10天。
2)、将发酵液离心处理取上清液，离心的条件为离心力15000g，4℃下离心50min，离心的目的是分离残留的菌体和不溶性杂质；上清液采用膜过滤除去悬浮质后进行醇沉，离心收集沉淀，将沉淀冷冻干燥获得粗多糖；膜优选为0.45μm的膜，用于过滤掉离心后的上清液中的非常微小的悬浮颗粒物。
3)、采用蛋白酶酶解法和Sevag法去除粗多糖中的蛋白，将去除蛋白的粗多糖用蒸馏水进行透析处理以去除小分子物质和有机溶剂，获得多糖样品；
4)、将多糖样品依次经过阴离子交换柱和分子排阻色谱柱进行纯化，获得沼泽红假单胞菌胞外多糖。
本发明的沼泽红假单胞菌GJ-22胞外多糖的制备方法，通过对沼泽红假单胞菌GJ-22进行发酵培养，醇沉，分离纯化获得沼泽红假单胞菌GJ-22胞外多糖，其生产工艺简单，表达稳定，易于获得。本发明首次对于叶际生防菌胞外多糖进行研究，研究发现纯化后的沼泽红假单胞菌GJ-22胞外多糖能显著促进植物生长和增强植物抗病性，且促生长和抗病性优于沼泽红假单胞菌GJ-22，为生防菌剂的开发提供了一种可能性，将在农业领域发挥重要的应用价值。
本实施例中，步骤1)中，发酵培养基的成分包括：(NH 4) 2SO 4 0.1g，MgSO 4 0.02g，Na 2CO 30.5g，K 2HPO 4 0.05g，NaCl 0.02g，酪蛋白氨基酸0.2g，琼脂1.5g，pH值为7.0～7.5。本实施例的发酵培养基加入了酪蛋白氨基酸，提高了胞外多糖的产量。
本实施例中，步骤2)中，醇沉具体包括以下步骤：加入过滤后上清液两倍体积的无水乙醇，搅拌均匀后在4℃条件下静置24h，醇沉后离心收集沉淀是采用离心力10000～13000g，在4℃条件下离心20min，优选离心力为10000g。沉淀优选采用无水乙醇洗涤三次，除去其中的杂质，然后冷冻干燥获得粗多糖。通过醇沉的方式收集粗胞外多糖，提取的多糖纯度更高，结构更完整和稳定。
本实施例中，步骤3)中，蛋白酶酶解法去蛋白的步骤包括：将步骤2)获得的粗多糖溶于适量的去离子水中(去离子水用量需使粗多糖全部溶解)，加入木瓜蛋白酶；调节pH为6.0～6.2，60℃水浴下处理6h，水浴处理期间每1个小时振荡一次，混匀样品，冷却后，加入两倍体积无水乙醇，静置后离心收集沉淀。木瓜蛋白酶能够在酸性，中性和碱性条件下分解蛋白质，具有蛋白酶和酯酶的活性，对细菌胞外多糖具有较强的水解能力，且对胞外多糖的 结构不会产生破坏作用，使提取的胞外多糖的含量较高。
Sevag法去蛋白的步骤包括：将经过蛋白酶处理的多糖溶于适量去离子水中(去离子水用量需使多糖全部溶解)，加入1/4(v/v)sevag试剂(氯仿：正丁醇＝5:1)。剧烈震荡2h，10000g离心10min，去除水相和有机相交界处变性蛋白。取水相重复操作，直至交界处无明显蛋白。
本实施例中，步骤3)中，透析处理采用截留分子量为8000D～14000D的透析袋。具体操作为：将去除完蛋白的多糖装于截留分子量为8000～14000D的透析袋中，用蒸馏水透析2天，每4h换一次水，去除小分子化合物和有机溶剂，透析结束后，将样品减压浓缩，冷冻干燥，获得多糖样品。沼泽红假单胞菌胞外多糖的分子量在10KD左右，因此选择截留分子量为8000D-14000D的透析袋。
本实施例中，步骤4)中，阴离子交换柱采用Hi Trap Q Sepharose High Performance，分子排阻色谱柱采用Seharose CL-6B层析柱。
阴离子交换柱纯化的具体操作为：将蛋白脱除的多糖样品用适量蒸馏水溶解后，过阴离子交换柱进行纯化。阴离子交换柱为Hi Trap Q Sepharose High Performance(1.6×2.5cm,GE Healthcare)，上样量为5mL，上样浓度为20mg/mL，流速为5mL/min，2mL离心管进行收集。先用Tris-HCl(20Mm，pH 7.60)洗涤两个柱体积，再用0.5M Tris-HCl和0.5M NaCl梯度洗脱。采用苯酚-硫酸法逐管检测多糖含量，以收集的管数为横坐标，各管在490nm下的吸光度为纵坐标，绘制洗脱曲线，如图1所示。合并含糖量相同的管数，用蒸馏水透析2天去除小分子物质，将透析液减压浓缩，冷冻干燥，得到纯化后的组分。Seharose CL-6B柱层析纯化的具体操作为：将经过阴离子交换柱纯化后的组分采用平衡液溶解，平衡液采用洗脱缓冲液(20mM PBS)，配制为20mg/mL多糖溶液，离心去除不溶部分，上清经过0.22μm膜过滤后，用注射器推入进样口，上样量为：2mL。使用洗脱缓冲液洗脱2个体积，自动收集样品。蒸馏水中透析2天，冷冻干燥，得到纯化后的沼泽红假单胞菌GJ-22胞外多糖。采用苯酚-硫酸法检测，得到洗脱曲线，如图2所示。
由图1可知，通过阴离子交换柱洗脱，洗脱峰为一个单峰，由图2可知，通过Seharose CL-6B柱洗脱，洗脱峰也为一个单峰，由此可以得出获得的沼泽红假单胞菌GJ-22胞外多糖纯度比较高。
实施例2
沼泽红假单胞菌GJ-22胞外多糖的结构鉴定
取实施例1制备得到的沼泽红假单胞菌GJ-22胞外多糖进行分子量鉴定，包括以下步骤：
(1)标准品分子量标准曲线的制作：以不同相对分子质量的葡聚糖(Mw1152、11600、23800、48600、80900、148000、273000、409800)作为标准品(Sigma-Aldrich公司)，用高效液相色谱仪(岛津LC-10A)，示差折光检测器进行检测，色谱柱为BRT105-104-102串联凝胶柱8×300mm(Borui Saccharide,Biotech.Co.Ltd.)；以保留时间为横坐标，相对分子质量为纵坐标，绘制单糖标准品的标准曲线。
(2)样品的分子量的测定：精密称沼泽红假单胞菌GJ-22胞外多糖(简称样品)，将样品 配制成5mg/mL溶液，12000rpm离心10min，上清液用0.22μm滤膜过滤，将样品转置于1.8mL进样小瓶中，进样量20μl，用高效液相色谱仪(岛津LC-10A)，示差折光检测器对多糖分子量及纯度进行测定，色谱柱为BRT105-104-102串联凝胶柱8×300mm(Borui Saccharide,Biotech.Co.Ltd.)。
根据系列葡聚糖标准品的分子量，以标准葡聚糖分子量对数LogMW对样品出峰时间进行回归处理，得到葡聚糖标准曲线，然后根据样品保留时间，计算样品的分子量。
图3是测得的沼泽红假单胞菌GJ-22胞外多糖的分子量谱图，将所测得样品的保留时间代入单糖标准品的标准曲线，计算得到样品的相对分子量为10026D。
取实施例1制备得到的沼泽红假单胞菌GJ-22胞外多糖进行单糖组分分析，包括以下步骤：
(1)多糖的完全水解：准确称取沼泽红假单胞菌GJ-22胞外多糖50mg，溶于3mL 2M三氟乙酸中，在氮气保护下110℃反应6h，水解液旋转蒸发后，加入少量甲醇减压蒸干，重复5次，去除残留的TFA，加入少量蒸馏水溶解，冻干，得到完全水解后的单糖样品；
(2)多糖乙酰化：将步骤(1)得到的水解的单糖样品加入2mL双蒸水，100mg硼氢化钠还原，加入冰醋酸中和，旋蒸，110℃烘箱烘干，然后加入1mL乙酸酐乙酰化，100℃反应1h，冷却后加入3mL甲苯，减压浓缩蒸干，重复4次，以除去多余的醋酐，将乙酰化后的产物用3mL氯仿溶解后转移至分液漏斗，加入少量蒸馏水充分震荡后，除去上层水溶液，重复5次，氯仿层以适量的无水硫酸钠干燥，定容10mL。
(3)多糖组分分析方法：将步骤(2)中得到的产物采用Shimadzu GCMS-QP 2010气相色谱质谱联用仪测定乙酰化产物样品进行分析，GC-MS条件：RXI-5SIL MS色谱柱30*0.25*0.25mm；程序升温条件为：起始温度120℃，以3℃/min升温至250℃/min，保持5min，进样口温度为250℃，检测器温度为250℃/min，载气为氦气，流速为1mL/min。得到的胞外多糖的单糖组成图如图4所示，由图4可知沼泽红假单胞菌GJ-22由甘露糖和葡萄糖两种单糖组成。
取实施例1制备得到的沼泽红假单胞菌GJ-22胞外多糖进行甲基化分析，具体方法包括以下步骤：
(1)甲基化反应：取沼泽红假单胞菌GJ-22胞外多糖10mg加入2mL无水二甲基亚砜充分溶解，将反应瓶内充满氮气，室温下静置30min，后加入10mg NaOH，再次充满氮气，室温下，磁力搅拌条件下反应1h，后加入0.5mL碘甲烷，充入氮气，室温下继续反应1h，反应结束后，向反应管中加入0.5mL蒸馏水终止反应，用蒸馏水透析24h，冷冻干燥，干燥后的样品继续甲基化，反应结束后，加入3mL蒸馏水终止反应，用三氯甲烷萃取三次，每次2mL，后合并萃取液，再用蒸馏水洗涤两次，旋转蒸发至干，后加入少量蒸馏水，冷冻干燥，此步骤重复两次，
(2)糖醇甲基乙酸化：将步骤(1)得到的完全甲基化后的样品5mg溶于2mL 2M TFA中，在氮气保护下100℃反应6h，冷却后，减压蒸干，加入3mL甲醇，再旋蒸至干，重复三次，样品经过氮吹仪吹干，加入0.5mL吡啶，90℃反应0.5h，再加入0.5mL醋酸酐，90℃反 应4h，反应结束后，将样品用氮气吹干，即得到完全酸水解的糖醇甲基乙酸酯，样品经过二氯甲烷溶解，进行GC-MS分析，GC-MS分析条件：分析采用Shimadzu GCMS-QP 2010气相色谱质谱联用仪测定乙酰化产物样品；得到GJ-22胞外多糖甲基化结果的GC-MS谱图如图5所示。
(3)GC-MS条件：RXI-5SIL MS色谱柱30*0.25*0.25mm；程序升温条件为：起始温度120℃，以3℃/min升温至250℃/min；保持5min；进样口温度为250℃，检测器温度为250℃/min，载气为氦气，流速为1mL/min。
取实施例1制备得到的沼泽红假单胞菌GJ-22胞外多糖进行NMR分析，具体包括以下内容：
NMR分析：取沼泽红假单胞菌GJ-22胞外多糖100mg溶于1mL D2O，进行600MHz核磁共振仪分析。得到沼泽红假单胞菌GJ-22胞外多糖氢谱谱图如图6所示，碳谱谱图如图7所示，DEPT 135核磁谱图如图8所示，HH-COSY谱图如图9所示，HMBC谱图如图10所示，NOESY谱图如图11所示。
由图6、7、8、9、10和图11可得出，NMR分析测定的结果如下：
(1) 1H NMR：由样品氢谱谱图分析可知，氢谱信号主要集中在3.0ppm～5.5ppm之间，δ3.2ppm～4.0ppm为糖环质子信号，主要端基质子峰δ5.23、5.10、5.06、5.04、5.01、4.85的信号峰集中分布在4.3ppm～5.5ppm区域内；
(2) 13C NMR：由样品的碳谱谱图分析可知，碳谱信号主要集中在60ppm～120ppm之间，通过观察碳谱，可以看到主要异头碳信号峰，103.45、101.84、99.51，异头碳区域主要在，93ppm～105ppm之间；
(3)2D NMR：DEPT 135：由样品的DEPT 135谱图分析可知，66.86ppm、62.26ppm峰为倒峰，表明为C6的化学位移，且66.86ppm处的峰向低场迁移，表明存在取代；
(4)HH-COSY：通过HH-COSY，H1-2的信号为5.23/4.06；H2-3的信号为4.06/3.86；H3-4的信号为3.86/3.79；H4-5的信号为3.79/3.65；H5-6a的信号为3.65/3.71，推断出H1，H2，H3，H4，H5，H6a分别为5.23、4.06、3.86、3.79、3.65、3.71其H6b为3.82，对应的碳谱为δ101.89、79.67、71.62、67.74、74.63、62.16；
(5)HMBC：→3-Man-1→的异头氢与→2-Man-1→的C2有相关峰，表明该多糖存在→3-Man-1→2-Man-1→；
→2-Man-1→的异头氢与→2,6-Man-1→的C2有相关峰，表明该多糖存在→2-Man-1→2,6-Man-1→；
→2,6-Man-1→的异头氢与自身的C2有相关峰，表明该多糖存在→2,6-Man-1→2,6-Man-1→；
因此我们可以推出：→3-Man-1→2-Man-1→2,6-Man-1→2,6-Man-1→的糖链接方式；
(6)NOESY：Man-1→的异头氢与→6-Man-1→的H6b有相关峰，表明存在Man-1→6-Man-1→；
→6-Man-1→的异头氢与→2,6-Man-1→的H6b有相关峰，表明存在→6-Man-1→2,6-Man-1→；
结合甲基化分析和NMR分析，得出实施例1制备得到的沼泽红假单胞菌GJ-22胞外多糖的结构式为：
Figure PCTCN2020128092-appb-000002
本发明鉴定获得了从沼泽红假单胞菌GJ-22中提取的胞外多糖的结构，使得生物防治的防控活性成分更加明确化。通过对沼泽红假单胞菌GJ-22胞外多糖进行全面、***性的研究，为沼泽红假单胞菌GJ-22功能与结构之间的构效关系提供基础理论。并且沼泽红假单胞菌GJ-22胞外多糖生产方便，效果稳定，适宜大规模生产制备。
沼泽红假单胞菌胞外多糖的生防活性及应用
实施例3
将实施例1制得的沼泽红假单胞菌GJ-22胞外多糖配制成1g/L的溶液，均匀的喷施于移栽7天烟草叶面，每天早晚各喷一次，保持湿润，7天后测量植株鲜重，其中以沼泽红假单胞菌GJ-22的发酵液上清液、发酵培养基和水分别为空白试验。
实施例4
将实施例制得的沼泽红假单胞菌GJ-22胞外多糖配制成1g/L的溶液，均匀的喷施于移栽14天水稻叶面，每天早晚各喷一次，保持湿润，7天后测量植株鲜重，其中以沼泽红假单胞菌的发酵液上清液、发酵培养基和水分别为空白试验。
图12为实施例3和实施例4中的沼泽红假单胞菌GJ-22胞外多糖处理烟草、水稻后的鲜重图，沼泽红假单胞菌GJ-22胞外多糖以EPS表示，沼泽红假单胞菌的发酵液上清以Fermentation表示，发酵培养基以medium表示，水以ddH2O表示，烟草以Tobacco表示，水稻以Rice表示。由图12可知沼泽红假单胞菌GJ-22胞外多糖能显著促进烟草和水稻的生长，其促生效果优于沼泽红假单胞菌的发酵液上清液以及发酵培养基。
实施例5
将实施例1制得的沼泽红假单胞菌GJ-22胞外多糖配制成1g/L的溶液，喷施于6叶的健 康的烟草叶片表面，第2天摩擦接种TMV病毒粒子，接种后第1天，第2天，第3天，第4天提取植物RNA，测定植物中TMV病毒粒子数量，如图13所示，以沼泽红假单胞菌发酵液上清液和水为空白试验。
由图13显示，沼泽红假单胞菌GJ-22胞外多糖能显著增强烟草对于病毒TMV的抵抗能力。
实施例6
将实施例1制得的沼泽红假单胞菌GJ-22胞外多糖配制成1g/L的溶液，喷洒于生长2周的健康的水稻叶片上，第2天喷洒稻瘟孢子悬浮液至水稻叶片上，置于光暗交替的培养箱中培养，观测喷洒稻瘟孢子悬浮液的第7天、第9天、第11天、第13天水稻叶片病斑数量，如图14所示，以沼泽红假单胞菌发酵液上清液和水为空白试验。
由图14显示，沼泽红假单胞菌GJ-22胞外多糖能显著增强水稻对于稻瘟菌的抑制效果。
由实施例3～6可得出：沼泽红假单胞菌GJ-22胞外多糖可应用于烟草和水稻的促生，亦可应用于烟草花叶病毒和稻瘟病的防治。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种沼泽红假单胞菌胞外多糖，其特征在于，所述胞外多糖的结构式为：
    Figure PCTCN2020128092-appb-100001
  2. 根据权利要求1所述的沼泽红假单胞菌胞外多糖，其特征在于，
    所述胞外多糖由沼泽红假单胞菌GJ-22(Rhodopseudomonas palustris GJ-22)发酵液中醇沉提取得到。
  3. 根据权利要求1所述的沼泽红假单胞菌胞外多糖的制备方法，其特征在于，包括以下步骤：
    1)、将沼泽红假单胞菌GJ-22(Rhodopseudomonas palustris GJ-22)的种子液在发酵培养基中发酵培养，获得发酵液；
    2)、将发酵液离心处理取上清液，采用膜过滤除去上清液中的悬浮质后进行醇沉，离心收集沉淀，将沉淀冷冻干燥获得粗多糖；
    3)、采用蛋白酶酶解法和Sevag法去除粗多糖中的蛋白，将去除蛋白的粗多糖用蒸馏水进行透析处理以去除小分子物质和有机溶剂，获得多糖样品；
    4)、将多糖样品依次经过阴离子交换柱和分子排阻色谱柱进行纯化，获得所述沼泽红假单胞菌胞外多糖。
  4. 根据权利要求3所述的沼泽红假单胞菌胞外多糖的制备方法，其特征在于，
    步骤1)中，发酵培养基的成分包括：(NH 4) 2SO 40.1g，MgSO 40.02g，Na 2CO 30.5g，K 2HPO 40.05g，NaCl 0.02g，酪蛋白氨基酸0.2g，琼脂1.5g，pH值为7.0～7.5，
    所述发酵培养的条件为：发酵培养温度为30℃，光照强度为7000lx～8000lx，培养时间为10天。
  5. 根据权利要求4所述的沼泽红假单胞菌胞外多糖的制备方法，其特征在于，
    步骤2)中，所述醇沉包括以下步骤：加入过滤后上清液两倍体积的无水乙醇，搅拌均匀后在4℃条件下静置24h，
    所述离心收集采用离心力10000～13000g，优选为10000g，在4℃条件下离心20min。
  6. 根据权利要求4所述的沼泽红假单胞菌胞外多糖的制备方法，其特征在于，
    步骤3)中，蛋白酶酶解法的步骤包括：将步骤(2)获得的粗多糖溶于去离子水中，加入木瓜蛋白酶；调节pH为6.0～6.2，60℃水浴下处理6h，水浴处理期间每1个小时振荡一次，混匀样品，冷却后，加入两倍体积无水乙醇，静置后离心收集沉淀。
  7. 根据权利要求4所述的沼泽红假单胞菌胞外多糖的制备方法，其特征在于，
    所述透析处理采用截留分子量为8000D～14000D的透析袋。
  8. 根据权利要求4所述的沼泽红假单胞菌胞外多糖的制备方法，其特征在于，
    步骤4)中，所述阴离子交换柱采用Hi Trap Q Sepharose High Performance，
    所述分子排阻色谱柱采用Seharose CL-6B层析柱。
  9. 根据权利要求1所述的沼泽红假单胞菌胞外多糖在促进烟草或水稻生长中的应用。
  10. 根据权利要求1所述的沼泽红假单胞菌胞外多糖在防治烟草花叶病毒或水稻稻瘟病中的应用。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102876615A (zh) * 2012-10-17 2013-01-16 湖南省植物保护研究所 沼泽红假单胞菌菌株及应用、菌剂药剂及其制备方法和应用
CN104498399A (zh) * 2014-12-10 2015-04-08 湖南省植物保护研究所 沼泽红假单胞菌菌株、生防菌剂及其制备方法和应用
CN111363056A (zh) * 2020-03-17 2020-07-03 湖南省植物保护研究所 沼泽红假单胞菌胞外多糖及其制备方法和应用

Family Cites Families (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1261103C (zh) * 2003-12-24 2006-06-28 北京精硒生物工程有限责任公司 一种抗癌用微生物原料药、其生产方法及其用途
DE102007034726A1 (de) * 2007-07-23 2009-01-29 Henkel Ag & Co. Kgaa Entfernung von Nebenprodukten aus vernetzbaren Zubereitungen
CN104403978B (zh) * 2014-12-10 2018-01-12 湖南省植物保护研究所 沼泽红假单胞菌菌株、菌剂及菌剂的制备方法、胞外蛋白及其提取方法和应用
PL415883A1 (pl) 2016-01-22 2017-07-31 Międzynarodowy Instytut Biologii Molekularnej I Komórkowej RNazy MiniIII, sposoby zmiany specyficzności cięcia sekwencji RNA przez RNazy MiniIII oraz ich zastosowania
US20190307818A1 (en) 2016-04-08 2019-10-10 The Trustees Of Princeton University Novel antimicrobial compositions and methods of use
CN105794853B (zh) * 2016-05-19 2018-09-18 湖南省植物保护研究所 沼泽红假单胞菌发酵液在防控稻瘟病中的应用
CN106135285B (zh) * 2016-05-19 2018-05-18 湖南省植物保护研究所 沼泽红假单胞菌生防菌剂在防控稻瘟病中的应用
CN106399216B (zh) 2016-11-16 2019-12-10 江南大学 一种高效合成α-氨基丁酸的单细胞工厂及其构建与应用
CN108409877B (zh) * 2017-07-12 2020-02-18 中国科学院过程工程研究所 浒苔多糖、浒苔寡糖、药物组合物及其用途
KR102105851B1 (ko) * 2017-10-26 2020-04-29 충남대학교 산학협력단 C1 가스 전환효율 향상을 위한 유기물질 기반의 신규 나노유체 소재
CN108543547B (zh) 2018-03-19 2019-06-18 华南理工大学 一种细菌纤维素负载金属粒子与植物纤维复合制备的催化试纸及其方法
CN110294794A (zh) * 2019-07-09 2019-10-01 湖南省植物保护研究所 一种沼泽红假单胞菌GroEL蛋白及其制备方法和应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102876615A (zh) * 2012-10-17 2013-01-16 湖南省植物保护研究所 沼泽红假单胞菌菌株及应用、菌剂药剂及其制备方法和应用
CN104498399A (zh) * 2014-12-10 2015-04-08 湖南省植物保护研究所 沼泽红假单胞菌菌株、生防菌剂及其制备方法和应用
CN111363056A (zh) * 2020-03-17 2020-07-03 湖南省植物保护研究所 沼泽红假单胞菌胞外多糖及其制备方法和应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ZHANG PENG: "Preparation,Structure Characterization and Biological Activities of an Extracellular Polysaccharide from Rhodopseudomonas Palustris", AGRICULTURE, CHINA MASTER’S THESES FULL-TEXT DATABASE, 15 August 2019 (2019-08-15), XP055851720 *

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