CN114230681A

CN114230681A - A kind of extracellular polysaccharide extract, extracellular polysaccharide and application thereof

Info

Publication number: CN114230681A
Application number: CN202111422635.7A
Authority: CN
Inventors: 耿丽丽; 王美玲; 张�杰; 束长龙; 王泽宇
Original assignee: Institute of Plant Protection of CAAS
Current assignee: Institute of Plant Protection of CAAS
Priority date: 2021-11-26
Filing date: 2021-11-26
Publication date: 2022-03-25

Abstract

The invention provides an extracellular polysaccharide extract, an extracellular polysaccharide and application thereof. The extract is extracted from bacillus thuringiensis, mainly comprises extracellular polysaccharide and can induce plants to generate system disease resistance to plant fungal diseases.

Description

Extracellular polysaccharide extract, extracellular polysaccharide and application thereof

Technical Field

The invention provides an extracellular polysaccharide extract, in particular to an extracellular polysaccharide extract extracted from bacillus thuringiensis, which can induce rape to generate disease resistance.

Background

The extracellular polysaccharide of bacteria is a polysaccharide mixture which is synthesized by bacteria in the process of growth and metabolism and secreted to the extracellular part, and is a long-chain high-molecular polymer which is polymerized by the same or different monosaccharide units through glycosidic bonds. Exopolysaccharides (EPSs) produced by bacteria exist mainly in two forms, Capsular Polysaccharides (CPS) tightly bound to the cell surface and mucopolysaccharides ("Slime"), also called Free Exopolysaccharides (EPS), secreted extracellularly as mucus, with a small amount of Free exopolysaccharides being the main constituent of the biofilm as a water-soluble matrix. The way of distinguishing capsular polysaccharide from free exopolysaccharide is not absolute by the tight degree of exopolysaccharide binding with cells, and a small amount of capsular polysaccharide is not stably connected with cell surface phospholipid and can be released into a culture medium to become free exopolysaccharide, and the free exopolysaccharide can become a component of capsular polysaccharide due to the close contact with cells. The distribution of these extracellular polymeric substances is influenced by the natural growth environment of the cells, such as the chemical properties of the culture solution, the abundance of nutrients, the growth period of the cells, and the like.

The presence of the outermost capsular polysaccharide or extracellular free polysaccharide of bacteria results in an additional barrier between the cell membrane and the external environment. The diversity of chemical compositions of polysaccharides, such as monosaccharide compositions, results in differences in the structures of different species of the same flora, and the diversity of biological functions of the polysaccharides. In addition, some physicochemical properties of the exopolysaccharide, such as stability, consistency, flocculation, suspension, film forming property, water holding capacity and the like, make the exopolysaccharide widely applied to different industrial industries, such as food industry, cosmetic industry, pharmaceutical industry, petroleum industry, mining industry and the like.

Except for a few gram-positive bacteria, most bacterial exopolysaccharides are transported intracellularly for synthesis. Its biosynthesis is carried out in stages: first, sugar nucleotides are assembled into "chemical" oligosaccharide repeat units under the action of glycosyltransferases; the chemical repeat units are subsequently polymerized in the presence of a polymerase to form "biological" repeat units such as macromolecular polysaccharides. A large number of sugar residues make up the backbone of the polysaccharide and in addition include some non-sugar substituents, such as acetate, pyruvate, succinate, phosphate, and the like. The composition, chemical and physical properties of extracellular polysaccharides of bacteria determine the main conformation of the polysaccharides, and these properties are quite different from one bacterial species to another. Different types of capsular polysaccharides can be produced by the same species of bacterial flora. Thus, different biological functions are gradually discovered, and the extracellular polysaccharide can be hidden on the surface of bacteria under natural conditions, can be used as a bonding agent to interact with other bacteria surface or lower substances, can protect the bacteria against the stress of the external environment, can be used as a matrix for bacteria to aggregate at the rhizosphere to form a biofilm and maintain the stability of the biofilm, and can be used as a signal molecule for the bacteria to exchange with the outside. In a severe growth environment, bacteria can utilize extracellular polysaccharide secreted by the bacteria as a carbon source. The production of extracellular polysaccharide is low, so that the market application of the polysaccharide is limited.

In recent years, with the rapid development of chemistry, molecular biology and imaging technology, the potential functions of extracellular polysaccharides of bacteria still need to be explored.

Disclosure of Invention

The invention provides an exopolysaccharide extract, which is obtained by the following steps:

firstly), adjusting the pH value of fermentation liquor of the Bacillus thuringiensis IPPBiotSR045 to 8-9, then carrying out first centrifugation, and collecting first supernatant after the first centrifugation;

secondly) adding trichloroacetic acid or trichloroacetic acid water solution into the first supernatant, thereby treating the first supernatant with trichloroacetic acid to obtain a first treatment solution, then carrying out second centrifugation, and collecting a second supernatant after the second centrifugation;

and thirdly) adjusting the pH value of the second supernatant to 6-7, adding ethanol into the second supernatant to obtain a second treatment solution, precipitating the second treatment solution, then carrying out third centrifugation, and collecting precipitate after the third centrifugation, wherein the precipitate is the wet crude extracellular polysaccharide extract.

In a specific embodiment, the method further comprises the steps of removing residual protein, desalting, ion exchange chromatography and gel filtration chromatography of the crude exopolysaccharide extract in sequence.

In a specific embodiment, the residual protein is removed using proteinase K; using HiPrep^TM26/10Desalting the crude extracellular polysaccharide extract with residual protein removed with Desalting column to obtain desalted chromatography liquid; purifying the desalted chromatographic solution by using a HiTrap Q HP Column anion exchange Column to obtain an ion chromatographic solution;purifying the ion chromatography liquid by using a HiLoad 26/600 Sephacryl S-100column gel filtration layer column to obtain gel chromatography liquid with a plurality of hole sites; mixing the gel chromatography liquid containing the extracellular polysaccharide hole sites, and concentrating by using a freeze drying instrument to obtain a wet pure extracellular polysaccharide extract; or drying into powder with freeze drying instrument to obtain dried pure exopolysaccharide extract.

In one embodiment, HiPrep is utilized^TM26/10 desaling Desalting column for crude exopolysaccharide extract with residual protein removed comprises the following steps:

1) centrifuging the crude extracellular polysaccharide extract water solution with residual protein removed at 10000r/min for 20min to remove impurities;

2) subjecting the desalting column HiPrep^TM26/10 desaling loading protein purification system

In the method, the ultra-pure water is used for cleaning the HiPrep containing desalting column^TM26/10 desaling-containing protein purification system

3) Filtering the crude extracellular polysaccharide extract water solution with residual protein removed by using a bacterial membrane to obtain a filtered extract water solution, and loading 7mL of the filtered extract water solution to a desalting column at the flow rate of 10mL/min each time;

4) according to UV₄₉₀Respectively starting and ending the peak value and the peak position of the salt peak, and collecting samples flowing out of the desalting column to obtain a desalting chromatographic solution;

the purification of the desalted chromatography liquid by using a HiTrap Q HP Column anion exchange Column comprises the following steps:

I) installing the anion exchange column in a protein purification system

In addition, the ultrapure water is utilized to clean the protein purification system

At the same time, 2mol/L sodium chloride aqueous solution is used for balancing the anion exchange column;

II) after the concentration of the extracellular polysaccharide in the desalted chromatographic solution is 10-15mg/mL, loading the desalted chromatographic solution to an anion exchange column by using an S1 loading pump, wherein the loading amount is 15-20mL each time, the flow rate is 2.0mL/min, and meanwhile, manually collecting by using a 96-hole deep-hole plate, namely 1.7 mL/tube;

III) after the sample loading is finished, washing the anion exchange column by ultrapure water with 5 times of the column volume of the anion exchange column;

IV) carrying out linear elution with the concentration of 0-100% by using 2mol/L sodium chloride aqueous solution for 20min at the flow rate of 5mL/min to obtain an eluent;

v) detecting the hole site of the eluent containing the exopolysaccharide by using a phenol-sulfuric acid method, mixing the eluent containing the hole site of the exopolysaccharide, and concentrating by using a freeze dryer to obtain an ion chromatography liquid;

the purification of the ion chromatography liquid by using HiLoad 26/600 Sephacryl S-100column gel filtration chromatography comprises the following steps:

A) installing the gel filtration chromatography column in a protein purification system

In the above, the protein purification system was washed with 0.15mol/L aqueous sodium chloride solution

And a gel filtration chromatography column;

B) taking 5mL of ion chromatography liquid each time, manually loading the sample to a gel filtration chromatography column, immediately collecting a sample flowing out of the gel filtration chromatography column by using a 96-well plate, wherein the flow rate of a system is 0.5mL/min, 0.5mL is collected in each hole, and stopping collecting 180mL in total to obtain gel chromatography liquid;

C) detecting the hole position of the gel chromatographic solution containing the extracellular polysaccharide by using a phenol-sulfuric acid method, and mixing the gel chromatographic solution containing the hole position of the extracellular polysaccharide; concentrating with freeze drying instrument to obtain wet pure extracellular polysaccharide extract; or drying into powder with freeze drying instrument to obtain dried pure exopolysaccharide extract.

In a specific embodiment, in step 2), the mass/volume content of the trichloroacetic acid and the first supernatant is between 0.4% and 0.5%.

In a specific embodiment, in step 3), the volume of ethanol added to the second supernatant is 3 to 3.5 times the volume of the second supernatant.

In one embodiment, in step 2), the first treatment liquid is left at a temperature of 25 to 35 ℃ for 1.5 to 3 hours.

In one embodiment, in step 3), the second treatment solution is left at 2 to 8 ℃ for 12 to 20 hours.

In a specific embodiment, the centrifugation speed of the first centrifugation, the second centrifugation, and the third centrifugation is independently 6000 to 8000r/min, the centrifugation temperature is independently 3 to 10 ℃, and the centrifugation time is independently 30 to 40 min.

In one embodiment, the culture medium for culturing the bacillus thuringiensis is one of an LB culture medium, a glucose-producing culture medium with glucose as a substrate, and a sucrose-producing culture medium with sucrose as a substrate.

In one embodiment, the precipitate is freeze-dried to obtain a dry crude exopolysaccharide extract.

The second invention provides an exopolysaccharide, wherein the Mw is 66.2kDa, the Mn is 42.7kDa, the Mz is 87.6kDa and the polydispersity is 1.55, the exopolysaccharide is composed of mannose, glucuronic acid, glucose and galactose, wherein the relative molar ratio of the mannose, the glucuronic acid, the glucose and the galactose is 55.77: 5.67: 3.70: 1. the relative molar ratio of the monosaccharides can be converted into the mass percentage content, and after the conversion, the mass of the exopolysaccharide is taken as 100%, the content of mannose is 83.77%, the content of glucuronic acid is 9.18%, the content of glucose is 5.56%, and the content of galactose is 1.49%.

The third invention provides the application of the extract or the exopolysaccharide in the second invention in inducing plant disease resistance.

In a specific embodiment, the plant is canola.

In one embodiment, the disease resistance is against a disease caused by sclerotinia sclerotiorum.

The invention has the beneficial effects that:

it is found for the first time that exopolysaccharides produced by Bt can induce plants to produce systemic disease resistance to plant fungal diseases, and the disease resistance is generated by the activation of jasmonic acid and ethylene signal pathways.

Drawings

FIG. 1 shows OD of extracellular polysaccharide-containing eluents at different pore sites after anion exchange chromatography₄₉₀The value is obtained.

FIG. 2 shows OD of extracellular polysaccharide-containing eluents at different pore sites after gel filtration chromatography₄₉₀The value is obtained.

Fig. 3 shows the full wavelength scan results of pure exopolysaccharides purified from IPPBiotSR045 strain.

Fig. 4 shows the results of ion chromatography detection of standard monosaccharides and exopolysaccharides purified from IPPBiotSR045 strain. Wherein the upper curve is the ion chromatography result of a standard monosaccharide; the lower curve is the ion chromatography result of exopolysaccharides purified from IPPBiotSR045 strain.

Fig. 5 shows exopolysaccharide-induced disease resistance of canola systems purified from IPPBiotSR045 strain.

FIG. 6 shows the transcription of jasmonic acid signal pathway related gene BnHEL.

FIG. 7 shows the transcription of jasmonic acid/ethylene signal pathway related gene BnPDFF 1.2.

FIG. 8 shows the transcription of the salicylic acid signal pathway related gene BnWRKY 70.

Detailed Description

The present invention is further illustrated by the following examples, which are intended to be purely exemplary of the invention and are not to be construed as limiting the invention in any way.

The reagents in the examples of the present invention were all commercially available unless otherwise specified.

Peptone, yeast extract, PDA, MS medium solid powder were purchased from Beijing Bingda Biotech Ltd.

Glucose, sucrose, aniline blue, bengal red were purchased from sigma.

Other biochemical reagents are all commercially available domestic or imported analytical pure or electrophoretic pure chemical reagents.

Liquid LB medium: 10.0g peptone, 5.0g yeast extract, 10.0g sodium chloride, 1.0L volume by volume with distilled water, and sterilizing at 121 deg.C for 20 min.

A sugar production culture medium using glucose as a substrate: 10.0g glucose, 5.0g yeast extract, and distilled water with volume value of 1.0L, sterilizing at 115 deg.C for 30 min.

Sugar production medium with sucrose as substrate: 20.0g sucrose, 5.0g yeast extract, and distilled water with volume value of 1.0L, sterilizing at 115 deg.C for 30 min.

Aniline blue selective medium: 0.05g/L aniline blue solid and 15.0g/L agar powder are respectively added into the two sugar production culture mediums.

PDA solid medium: 39.0g PDA powder was dissolved in 1.0L distilled water, and sterilized with distilled water at 115 ℃ for 15min to give a volume value of 1.0L.

Solid culture medium of Bengal red: 0.5 wt% of peptone, 1 wt% of sodium chloride, 1 wt% of glucose, 0.1 wt% of monopotassium phosphate, 0.05 wt% of magnesium sulfate, 2 wt% of agar, 3mL of 1 wt% of Bengal mother liquor, 1.0L of volume value by using distilled water, and sterilizing for 15min at 115 ℃. Before use, streptomycin is coated by 50 mug/mL according to requirements.

MS culture medium: the MS culture medium solid powder is subjected to volumetric value of 1.0L by using distilled water and sterilized for 15min at 121 ℃.

Extracting and quantifying reagent for extracellular polysaccharide:

1mol/L sodium hydroxide: 4.0g of sodium hydroxide was dissolved in 100mL of sterile distilled water and stored at room temperature.

2mol/L hydrochloric acid: 500mL of sterile distilled water was added to 100mL of concentrated hydrochloric acid, and the mixture was stored at room temperature.

95% ethanol: 950mL of absolute ethanol was added with 50mL of sterile distilled water and stored at room temperature.

80% phenol: 80.0g of phenol was dissolved in 50mL of sterile distilled water, and the volume was adjusted to 100mL with sterile distilled water, and the solution was stored at 4 ℃ in the dark.

The main apparatus comprises: a full-automatic moist heat sterilizer, SANYO, Japan, model MLS-3780/MAC 350P; superclean bench, Beijing Hardong corporation, model DL-CJ-2N; a constant temperature incubator, Shanghai laboratory Instrument Master, model DHP 120; temperature controlled shaker, model MAXQ5000, manufactured by Thermo corporation, USA; a bench centrifuge, eppendorf, germany, model 5415C; high speed centrifuge, Beckman, USA, model Avanti J-26 xp; freeze dryer VirTis, usa.

The cabbage type rape (Brassica campestris L.) is Shanghai green.

The rape planting site is as follows: greenhouse of pilot plant base in corridor of institute of plant protection of Chinese academy of agricultural sciences. The planting mode is as follows: the rape seeds are subjected to surface disinfection by using 75% alcohol, 5% sodium hypochlorite and sterile water in sequence, and are uniformly sowed on an MS (Murashige and Skoog, MS) solid culture medium plate. After about 10 days, the seedlings were transplanted into seedling pots (diameter 6.0cm, depth 10.0cm), and planting soil included nutrient soil and vermiculite (ratio 2: 1). The culture conditions are as follows: the illumination time is 14h, and the illumination intensity is 7,000 Lux; the dark time was 10 h. Growth temperature: about 22 deg.c.

The IPPBiotSR045 strain is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.13302, the preservation date of 2016, 11 and 15 days, and the preservation addresses of: the institute of microbiology, national academy of sciences No.3, Xilu No.1, Beijing, Chaoyang, Beijing. The system is classified as Bacillus thuringiensis (Bacillus thuringiensis).

Example 1

1. Extraction of crude exopolysaccharides

(1) Inoculating 10 mu L of glycerol strain of Bacillus thuringiensis IPPBiotSR045 into 5mL of liquid LB culture medium, and activating at 30 ℃ and 220r/min for 10h to obtain activated bacteria liquid;

(2) transferring the activated bacterium liquid into 100mL of liquid LB culture medium by the inoculation amount of 1%, and culturing in a 500mL triangular flask for 30h to obtain fermentation liquid;

(3) collecting the obtained fermentation liquor, adjusting the pH of the fermentation liquor to 8.5, 4 ℃ and 7000r/min by using 1mol/L NaOH, centrifuging for 30min, and collecting a first supernatant;

(4) adding 1/10 volume of 5 wt% trichloroacetic acid aqueous solution into the first supernatant, and denaturing the protein at 25 deg.C for 2 hr;

(5) centrifuging at 4 deg.C for 30min at 7000r/min, collecting second supernatant, and adjusting pH to 6.5;

(6) adding 3 times volume of absolute ethyl alcohol to precipitate for 12 hours in a refrigerator at 4 ℃ overnight to generate brown precipitate;

(7) centrifuging at 4 deg.C for 30min at 7000r/min, discarding supernatant, and freeze drying brown precipitate to obtain crude exopolysaccharide powder.

2. Quantification of exopolysaccharides

2.1 preparation of Standard Curve

Firstly, preparing a glucose mother solution: 0.1g of glucose is dissolved in 250mL of sterile distilled water and stirred evenly to prepare a glucose mother liquor.

Then glucose mother liquor is utilized to prepare glucose standard liquor with different concentration gradients, then 0.1mL of 98% concentrated sulfuric acid and 0.5mL of 6 wt% phenol aqueous solution are respectively added into the glucose standard liquor with different concentration gradients, and sterile distilled water is added to make up to a system of 0.8 mL. Slowly mixing, cooling, standing at room temperature for 20min, measuring light absorption value at 490nm in an enzyme labeling plate at 200 μ L, replacing sample with sterile distilled water as blank control, and repeating three times for each treatment group to draw a standard curve.

2.2 phenol-sulfuric acid method of quantitation

Diluting with different gradients (to make OD)_490nmValue between 0.2 and 1.0), adding 0.1mL of concentrated sulfuric acid and 0.5mL of 6 wt% of phenol aqueous solution into a centrifuge tube, then supplementing to 0.8mL of system with sterile distilled water, mixing uniformly, cooling, standing at room temperature for 20min, taking 200 mu L of the mixture in an enzyme label plate, measuring the light absorption value at 490nm, and calculating the content of the extracellular polysaccharide in the sample according to a standard curve drawn by 2.1.

The amount of the exopolysaccharide in the crude exopolysaccharide extracted from the IPPBiotSR045 strain is 210mg/L of bacterial liquid.

Example 2

Crude exopolysaccharide extraction was the same as in subsection 1 of example 1, except that:

in the step (2), respectively transferring the activated bacterium liquid into 100mL of liquid culture medium of a sugar production culture medium taking glucose as a substrate by using the inoculation amount of 1%, and culturing in a 500mL triangular flask for 30h to obtain fermentation liquid;

in the step (3), respectively collecting the obtained fermentation liquor, adjusting the pH of the fermentation liquor to 8, 3 ℃ and 6000r/min by using 1mol/L NaOH, centrifuging for 40min, and collecting a first supernatant;

in the step (4), 1/10 volumes of 4 wt% trichloroacetic acid aqueous solution is added into the first supernatant, and the protein is denatured for 3 hours at 35 ℃;

centrifuging at 3 deg.C and 8000r/min for 30min in step (5), collecting second supernatant, and adjusting pH to 6;

in the step (6), adding 3 times of volume of absolute ethyl alcohol to precipitate for 16 hours at 2 ℃ in a refrigerator;

and (7) centrifuging at 3 ℃ and 6000r/min for 40min, discarding supernatant, and freeze-drying the precipitate to obtain crude exopolysaccharide powder.

The amount of the exopolysaccharide in the crude exopolysaccharide extracted from the IPPBiotSR045 strain is 200mg/L of bacterial liquid.

Example 3

in the step (2), respectively transferring the activated bacterium liquid into 100mL of liquid culture medium of a sugar production culture medium taking sucrose as a substrate by using the inoculation amount of 1%, and culturing in a 500mL triangular flask for 30h to obtain fermentation liquid;

in the step (3), respectively collecting the obtained fermentation liquor, adjusting the pH of the fermentation liquor to 9, 10 ℃ and 8000r/min by using 1mol/L NaOH, centrifuging for 30min, and collecting a first supernatant;

in the step (4), 1/10 volumes of 5 wt% trichloroacetic acid aqueous solution is added into the first supernatant, and the protein is denatured at 25 ℃ for 1.5 hours;

centrifuging at 3 deg.C and 6000r/min for 40min in step (5), collecting second supernatant, and adjusting pH to 7;

in the step (6), adding 3 times of volume of absolute ethyl alcohol to precipitate for 20 hours at 8 ℃ in a refrigerator;

in the step (7), centrifuging at 10 ℃ and 8000r/min for 30min, discarding supernatant, and freeze-drying precipitate to obtain crude exopolysaccharide powder.

Example 4

Extracellular polysaccharide was extracted from a bacterial solution obtained by culturing IPPBiotSR045 strain in 7L LB medium.

Extraction and quantification of crude exopolysaccharide as in example 1, the powder of crude exopolysaccharide was then dissolved in 50mL of ultra pure water.

Purification of exopolysaccharides

1.1 digestion of residual proteins

(1) Quantifying residual protein in the sample by using a BCA total protein quantification kit (Thermo);

(2) adding protease K with total residual protein mass of 1/10;

(3) carrying out enzymolysis for 2h in a water bath at 50 ℃, and analyzing whether protein is completely removed by SDS-PAGE;

(4) if the residual protein is not completely removed, adding protease K again for treatment until the protein removes the bacillus; and after the protein is completely removed, carrying out boiling water bath treatment for 15min, and inactivating the protease to obtain a crude extracellular polysaccharide solution with the protein removed for later purification.

1.2 desalination

The desalting column is HiPrep^TM26/10 desaling, the purification procedure for deproteinized crude exopolysaccharide solution is as follows:

(1) centrifuging the crude extracellular polysaccharide solution with protein removed at 10000r/min for 20min to remove impurities;

(2) subjecting the desalting column HiPrep^TM26/10 desaling loading protein purification system

(3) Filtering the crude exopolysaccharide solution from which the protein is removed by using a 0.22 mu m filter membrane to obtain a filtered crude exopolysaccharide solution, and sampling 7mL of the filtered crude exopolysaccharide solution onto a desalting column at the flow rate of 10mL/min each time;

(4) according to UV₄₉₀Respectively starting and ending the peak value and the peak position of the salt peak, and collecting samples flowing out of the desalting column to obtain a desalting chromatographic solution;

(5) finally, the protein purification system is cleaned by sequentially utilizing ultrapure water and 20% (v/v) ethanol

And desalting column HiPrep^TM 26/10 Desalting；

(6) The content of the extracellular polysaccharide in the desalted chromatographic solution is accurately detected by using a phenol-sulfuric acid method, and the concentration is about 20 mg/mL.

1.3 anion exchange chromatography

The anion exchange chromatography Column used was a 5mL HiTrap Q HP Column (Q-HP), and the purification procedure for the desalted chromatography solution was as follows:

(1) installing an anion exchange chromatography column Q-HP in a protein purification system

Protein purification system cleaned by ultrapure water

At the same time, 2.0mol/L sodium chloride water solution is used for balancing the anion exchange chromatographic column Q-HP;

(2) according to the concentration of exopolysaccharide in the desalted chromatography liquid measured by phenol-sulfuric acid method (making the concentration between 10-15 mg/mL), a protein purification system is used

The S1 sample pump is used for loading the desalted chromatographic solution to the anion exchange chromatographic column Q-HP, the sample loading amount is 15-20mL each time, the flow rate is 2.0mL/min, and meanwhile, a 96-hole deep-hole plate is used for manual collection, namely 1.7 mL/tube;

(3) after the sample loading is finished, washing the anion exchange chromatography column Q-HP by using ultrapure water with 5 times of column volume;

(4) linearly eluting with 2mol/L sodium chloride water solution at concentration of 0-100% for 20min at flow rate of 5mL/min to obtain eluate;

(5) finally, respectively cleaning the protein purification system by sequentially utilizing ultrapure water and 20% ethanol

And an anion exchange chromatography column Q-HP;

(6) after the collection, detecting the hole position of the extracellular polysaccharide-containing eluent by using a phenol-sulfuric acid method (see figure 1), wherein the result shows that the single polysaccharide absorption peak appears, the salt ion concentration of the single polysaccharide absorption peak is between 5 and 10 percent, mixing the extracellular polysaccharide-containing hole position eluents, and concentrating the mixed eluents to 1/10 (about 30mL) of the original volume by using a freeze dryer to obtain the ion chromatography liquid.

1.4 gel filtration chromatography

The gel filtration chromatography column HiLoad 26/600 Sephacryl S-100column (Sephacryl S-100column, GE Healthcare Life Sciences) was used, and the purification procedure for the ion chromatography liquid was as follows:

(1) installing gel filtration chromatography column HiLoad 26/600 Seperdex 200 prep grade column in protein purification system

In the middle, 0.15mol/L sodium chloride aqueous solution is used for cleaning the protein purification system

And a gel filtration chromatography column;

(2) taking 5mL of ion chromatography liquid each time, manually loading the sample to a gel filtration chromatography column, immediately collecting the sample, wherein the flow rate of the system is 0.5mL/min, collecting by using a 96-well plate, collecting 0.5mL of ion chromatography liquid in each well, collecting 180mL (namely 1.5 column volumes) in total, and stopping collecting to obtain gel chromatography liquid;

(3) finally, respectively cleaning the protein purification system and the gel filtration chromatographic column by sequentially utilizing ultrapure water and 20% ethanol;

(4) detecting the hole position of the gel chromatographic solution containing the extracellular polysaccharide by using a phenol-sulfuric acid method (see figure 2), wherein the result shows that the gel chromatographic solution is a single absorption peak, and combining the gel chromatographic solutions of the extracellular polysaccharide at the hole position of the single absorption peak to obtain the pure extracellular polysaccharide. And according to OD_490nmAbsorbance values elution curves were plotted and single major peak positions were collected and either freeze-concentrated to 1/10 in bulk or freeze-dried to powder.

2 determination of molecular weight

And (3) determining the total sugar content, the protein content, the reducing sugar content and the uronic acid content of the pure extracellular polysaccharide obtained after gel filtration chromatography. The measurement method and the results are shown in Table 1:

TABLE 1

The results from table 1 show that the total sugar content of the purified exopolysaccharide purified from IPPBiotSR045 is 96.31%, and the uronic acid, protein and reducing sugar contents are all low, 1.38%, 0.13% and 2.18%, respectively. The full wavelength scan showed that the absorbance at 260nm and 280nm was less than 0.1 (fig. 3), i.e. the pure exopolysaccharide contained almost no nucleic acids and proteins, further indicating the high purity of the exopolysaccharide sample.

Molecular weight analysis Gel exclusion chromatography column Ohpak SB-805HQ (300X 8mm), Ohpak SB-804HQ (300X 8mm) and Ohpak SB-803HQ (300X 8mm) were connected in series according to the molecular weight of the extracellular polysaccharide preliminarily judged by the purification step, to obtain a series column, using a Gel chromatography-differential-multiangle laser light scattering system (GPC-RI-MALS), wherein the laser light scattering detector was DAWN HELEOS II (Wyatt technology, CA, USA), and the differential detector was Optilab T-rEX (Wyatt technology, CA, USA). The specific experimental procedures are as follows:

(1) accurately weighing extracellular polysaccharide powder 10.0mg after gel filtration chromatography and freeze drying, and dissolving in 1.0mL of 0.1mol/L NaNO₃In the water solution, then 14000r/min, centrifuging for 10min to obtain supernatant;

(2) filtering the supernatant with 0.22 μm filter membrane to load the serial column;

(3) the sample loading amount to the series column is 100 mu L in one time, and the mobile phase is 0.1mol/L NaNO₃The flow rate is 0.4mL/min, and the column temperature is kept at 45 ℃;

(4) data were collected and analyzed using ASTR6.1(Wyatt technology corporation, USA) software.

The results show that: the extracellular polysaccharide has Mw of 66.2kDa, Mn of 42.7kDa and Mz of 87.6 kDa. The Polydispersity (Mw/Mn) is 1.55. In addition, the change trend of the multi-angle laser light scattering signal is completely consistent with that of the differential signal, which indicates that the molecular weight analysis of the extracellular polysaccharide is more accurate and indicates that the extracellular polysaccharide is single-component homogeneous polysaccharide.

3 analysis of monosaccharide composition

The monosaccharide composition analysis adopts a Thermo-ion exchange chromatography system (High-performance and-exchange chromatography, model ICS5000+, USA), and the liquid chromatography column is Dionex^TMCarboPac^TMPA10(250 × 4.0mm, 10 μm), the specific experimental procedure was as follows:

(1) preparing a standard substance: preparing to weigh 100mg of different monosaccharide standards, which comprise: glucose, rhamnose, arabinose, galactose, xylose, mannose, fructose and glucuronic acid;

(2) drawing a standard curve: diluting the standard substance into 1, 10, 20, 30, 40 and 50 mu g/mL aqueous solution respectively, wherein the loading amount is 20 mu L, the A mobile phase is water, the B mobile phase is 100mM NaOH, the column temperature is kept at 30 ℃, and a curve is drawn according to the retention time and the peak height of each component in the system;

(3) weighing 5mg (+ -0.05 mg) of extracellular polysaccharide powder subjected to gel filtration chromatography and freeze drying, adding trifluoroacetic acid, hydrolyzing at 121 ℃ for 2h, blow-drying with nitrogen, then cleaning with methanol, blow-drying again, and repeating methanol cleaning-blow-drying for 2-3 times. Adding sterile water to dissolve to be detected;

(4) comparing the retention time of the exopolysaccharide with the retention time of the standard monosaccharide to determine the monosaccharide composition of the exopolysaccharide.

The monosaccharide composition analysis results of exopolysaccharides are shown in fig. 4, the retention time of each component in exopolysaccharides is compared with that of a standard sample, and the monosaccharide components of exopolysaccharides are mainly mannose, glucuronic acid, glucose and galactose, and the relative molar ratio of the monosaccharide components to the monosaccharides is 55.77: 5.67: 3.70: 1. thus, exopolysaccharide is a heteropolysaccharide.

Example 3

Respectively dissolving 40 micrograms and 80 micrograms of purified exopolysaccharide in 1mL of sterile water to obtain exopolysaccharide aqueous solution, spraying the exopolysaccharide aqueous solution on the soil surface near the roots of the rapes by 40 micrograms/strain and 80 micrograms/strain, and taking the sterile water as a control group. Three biological replicates per treatment. The sclerotinia sclerotiorum cake was inoculated to rape leaves 5 days after the rape roots were treated with exopolysaccharide or sterile water, and the spread of the disease was observed on the 0 th, 2 th, 5 th and 7 th days of the inoculation, and the spread of the disease of the leaves after the rape roots were treated with exopolysaccharide 40. mu.g/strain and sterile water is shown in FIG. 5. According to the figure 5, after 7 days of exopolysaccharide treatment, the growth state of the rape is good, the leaf spots are not enlarged, the rape leaf spots after sterile water treatment rapidly spread two days after receiving diseases, and the leaf curling phenomenon appears 7 days. The lesion diameter of rape leaves after being inoculated with sclerotinia sclerotiorum for 7 days after being treated by 40 mu g/strain and 80 mu g/strain of exopolysaccharide is measured, and the induced disease-resistant effect is calculated by a formula of (lesion diameter of a control group-lesion diameter of an experimental group)/lesion diameter of the control group multiplied by 100 percent, and the result is shown in table 2. LSD analysis shows that the two groups of extracellular polysaccharide treatment groups have significant difference with the control group, and the difference is more than 50%. In conclusion, the purified extracellular polysaccharide of the IPPBiotSR045 strain has obvious disease resistance induction capability.

TABLE 2 statistical results of the lesion diameter of rape leaves when inoculating sclerotinia sclerotiorum after EPS treatment

Note: each set of data represents the mean ± standard error of the three sets of duplicate data; lower case letters in the graph indicate significant differences at the p <0.05 level for one-way ANOVA analysis between treatments.

Example 4

Fluorescence quantitative PCR standard curve:

respectively amplifying an internal reference gene actin (using act35F and act143R primers, wherein the nucleotide sequence of act35F is shown as SEQ ID No.1, and the nucleotide sequence of act143R is shown as SEQ ID No. 2) and a salicylic acid signal path related gene BnWRKY70 (using wrk592F and wrk693R primers for amplification, wherein the nucleotide sequence of wrk592F is shown as SEQ ID No.3, the nucleotide sequence of wrk693R is shown as SEQ ID No. 4), a jasmonic acid signal path related gene BnHEL (using hel431R and hel549R primers for amplification, wherein the nucleotide sequence of hel431R is shown as SEQ ID No.5, and the nucleotide sequence of hel549R is shown as SEQ ID No. 6) by taking a rape genome as a template, and amplifying the internal reference gene actin, wherein the nucleotide sequence of act35 and the nucleotide sequence of the hel549R are shown as SEQ ID No.6 respectively, jasmonic acid and ethylene signal pathway related gene BnPDFF 1.2 (amplified by using pdf10F and pdf107R primers, wherein the nucleotide sequence of pdf10F is shown as SEQ ID No.7, and the nucleotide sequence of pdf107R is shown as SEQ ID No. 8.) was enriched and purified by 10 cycles of ordinary PCR amplification. And diluting the enriched and purified product in a gradient manner and then using the diluted product as a template for drawing a fluorescent quantitative PCR standard curve. The reaction system and conditions refer to the instructions of a SuperReal PreMix Plus (SYBR Green) kit, and the reference gene and the target gene are drawn into a double-standard curve and subjected to related gene transcription level analysis. And when the R value of the standard curve of the gene is more than 0.99, the requirement of subsequent experiments can be met.

Dissolving 40 mu g of exopolysaccharide in 1mL of sterile water to obtain an exopolysaccharide aqueous solution, spraying 40 mu g of exopolysaccharide per strain on the soil surface near the rape roots, and taking the sterile water as a control group. After the roots of the rapes are treated by exopolysaccharides or sterile water for 5 days, respectively taking rape leaves 0 day, 2 days, 5 days and 7 days after the treatment, extracting total RNA of the rape leaves by using a Trizol method, synthesizing cDNA by using a Promega reverse transcription kit, analyzing the transcription conditions of BnHEL, BnWRKY70 and BnPDF1.2 genes in the rape leaves 0 day, 2 days, 5 days and 7 days after the exopolysaccharides are applied by using fluorescence quantitative PCR (qRT-PCR), and taking actin as an internal reference gene.

The transcription conditions of the gene BnWRKY70 related to the salicylic acid signal pathway, the gene BnHEL related to the jasmonic acid signal pathway and the gene BnPDF1.2 related to the jasmonic acid and ethylene signal pathway after the roots of the rape are treated with the exopolysaccharide for 0 day, 2 days, 5 days and 7 days are shown in FIGS. 6 to 8. All data in the figure are mean ± standard error of three sets of duplicate data. And LSD in the one-way variance ANOVA is used for carrying out statistical analysis, and the result shows that the transcription level of the BnWRKY70 gene is basically unchanged after the rape roots are treated by the extracellular polysaccharide, and has no significant difference (p is more than 0.05) with the transcription level of the day 0; the BnHEL gene started up-regulated transcription 5 days after exopolysaccharide treatment (p ═ 0.1990), with more pronounced up-regulation at day 7 (p ═ 0.009), which was 1.63 and 2.96 times the relative transcription level at day 0, respectively; the bnpdf1.2 gene was up-regulated at day 2 after exopolysaccharide treatment (p 0.102), and at day 7, the transcript level was significantly different from that at day 0 (p 0.005), which was 2.22 times that at day 0. In summary, the jasmonic acid and ethylene signaling pathways were activated after treatment of canola roots with exopolysaccharides.

While the invention has been described with reference to specific embodiments, those skilled in the art will appreciate that various changes can be made without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, and method to the essential scope and spirit of the present invention. All such modifications are intended to be included within the scope of the present invention as defined in the appended claims.

Sequence listing

<110> institute of plant protection of Chinese academy of agricultural sciences

<120> an exopolysaccharide extract, exopolysaccharide and uses thereof

<130> LHA2160512

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gtgccgatct acgaaggtta tg 22

<210> 2

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gtaccctctc tcggtgagaa t 21

<210> 3

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

aagcaagtgc agaagctaga g 21

<210> 4

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ggcttgatct tgggtgatac tt 22

<210> 5

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ggtgaggaac acaaggacta at 22

<210> 6

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gccatcacca tcggtatcta tt 22

<210> 7

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ttcctcacac accacacata c 21

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

acaagagcag cgaagagaag 20

Claims

1. An extracellular polysaccharide extract, which is extracted by the following steps:

1) the pH value of the fermentation broth of Bacillus thuringiensis IPPBiotSR045 is adjusted to 8 to 9, then the first centrifugation is performed to collect the first supernatant after the first centrifugation;

2) adding trichloroacetic acid or an aqueous solution of trichloroacetic acid to the first supernatant, thereby treating the first supernatant with trichloroacetic acid to obtain the first treatment solution, and then performing a second centrifugation to collect the first Second supernatant after centrifugation;

3) After the pH value of the second supernatant is adjusted to 6 to 7, ethanol is added to the second supernatant to obtain a second treatment solution, the second treatment solution is precipitated, and then the first treatment solution is carried out. The third centrifugation, the precipitate after the third centrifugation was collected, and the precipitate was the wet crude exopolysaccharide extract.

2. The extracellular polysaccharide extract according to claim 1, further comprising the steps of successively removing residual protein, desalting, ion exchange chromatography and gel filtration chromatography to the crude extracellular polysaccharide extract ;

Preferably, the residual protein is removed using proteinase K;

The crude exopolysaccharide extract from which residual protein was removed was desalted by using a HiPrep ^TM 26/10 Desalting desalting column to obtain a desalted chromatographic solution;

Utilize HiTrap Q HP Column anion exchange column to purify the desalting chromatographic liquid to obtain ionic chromatographic liquid;

Utilize HiLoad 26/600 Sephacryl S-100column gel filtration layer column to purify the ion chromatographic fluid to obtain gel chromatographic fluid with multiple pores;

The gel chromatography liquid containing the pores of the exopolysaccharide is mixed and concentrated by a freeze dryer to obtain a wet pure exopolysaccharide extract; or dried to a powder by a freeze dryer to obtain a dry pure exopolysaccharide extract.

3. exopolysaccharide extract according to claim 2, is characterized in that, utilizes HiPrep ^TM 26/10 Desalting desalting column to carry out desalting to the crude exopolysaccharide extract that removes residual protein and comprises the following steps:

1) Centrifuge the crude exopolysaccharide extract aqueous solution with residual protein removed at 10000r/min for 20min to remove impurities;

2) Load the desalting column HiPrep ^TM 26/10 Desalting into the protein purification system

, wash the protein purification system including the desalting column HiPrep ^TM 26/10 Desalting with ultrapure water

3) filter the described crude exopolysaccharide extract aqueous solution for removing residual protein with a bacterial filter membrane to obtain the filtered aqueous extract solution, and take 7 mL of the filtered aqueous extract solution each time and load the sample onto the desalting column at a flow rate of 10 mL/min ;

4) according to the peak value of UV ₄₉₀ and the peak position of the salt peak, respectively start and end to collect the samples flowing out from the desalting column to obtain the desalting chromatographic solution;

Purification of desalted chromatographic fluid using HiTrap Q HP Column anion exchange column includes the following steps:

1) Install the anion exchange column in a protein purification system

, using ultrapure water to clean the protein purification system

Equilibrate the anion exchange column with 2mol/L sodium chloride aqueous solution at the same time;

II) After the concentration of the exopolysaccharide in the desalting chromatographic solution is in the range of 10 to 15 mg/mL, the desalting chromatographic solution is loaded onto the anion exchange column using the S1 loading pump, and the amount of each loading is 15-15 mg/mL. 20mL, the flow rate is 2.0mL/min, and the 96-well deep-well plate is used for manual collection, 1.7mL/tube;

III) until the end of sample loading, wash the anion exchange column with ultrapure water that is 5 times the column volume of the anion exchange column;

IV) utilizing 2mol/L sodium chloride aqueous solution to carry out concentration is 0-100% linear elution, elution 20min, flow velocity is 5mL/min, obtains eluent;

V) utilize the phenol-sulfuric acid method to detect the pore position of the eluent containing the exopolysaccharide, mix the eluent containing the pore position of the exopolysaccharide, and concentrate using a freeze dryer to obtain an ion chromatography solution;

Purification of ion chromatography using HiLoad 26/600 Sephacryl S-100 column gel filtration chromatography includes the following steps:

A) Install the gel filtration column in a protein purification system

On, wash the protein purification system with 0.15mol/L sodium chloride aqueous solution

and gel filtration columns;

B) Take 5mL of ion chromatography solution to manually load the gel filtration chromatography column each time, and immediately use a 96-well plate to collect the samples flowing out from the gel filtration chromatography column, and the system flow rate is 0.5mL/min, Collect 0.5mL per well, and stop collecting after a total of 180mL is collected to obtain gel chromatography liquid;

C) use the phenol-sulfuric acid method to detect the pore position of the gel chromatography liquid containing exopolysaccharide, mix the gel chromatography liquid containing the exopolysaccharide pore position; use a freeze dryer to concentrate to obtain wet pure exopolysaccharide extraction Or use freeze dryer to dry into powder to obtain dry pure extracellular polysaccharide extract.

4. The extracellular polysaccharide extract according to claim 1, wherein in step 2), the mass/volume content of the trichloroacetic acid and the first supernatant is 0.4% to 0.5%; and / or

In step 3), the volume of ethanol added to the second supernatant is 3 to 3.5 times the volume of the second supernatant.

5. The extracellular polysaccharide extract according to claim 1, wherein in step 2), the first treatment solution is placed at a temperature of 25 to 35°C for 1.5 to 3 hours; and/or

In step 3), the second treatment solution is placed at 2 to 8° C. for 12 to 20 hours.

6. The extracellular polysaccharide extract according to claim 1, wherein the centrifugal speeds of the first centrifugation, the second centrifugation, and the third centrifugation are independently 6000 to 8000 r/min, and the centrifugation temperature Independently 3 to 10°C, and centrifugation time independently 30 to 40 min.

7. extracellular polysaccharide extract according to claim 1, is characterized in that, the substratum that cultivates described bacillus thuringiensis is LB substratum, the sugar-producing substratum that takes glucose as substrate and the substratum that takes sucrose as substrate One of the sugar-producing media.

8. The exopolysaccharide extract according to any one of claims 1 and 4 to 7, wherein the precipitate is freeze-dried to obtain a dry crude exopolysaccharide extract.

9. An exopolysaccharide having Mw=66.2kDa, Mn=42.7kDa, Mz=87.6kDa, and a polydispersity of 1.55, said exopolysaccharide consisting of mannose, glucuronic acid, glucose and galactose, wherein , the relative molar ratio of the mannose, the glucuronic acid, the glucose and the galactose is 55.77:5.67:3.70:1.

10. Use of the extract according to any one of claims 1 to 8 or the exopolysaccharide described in claim 9 in inducing plant disease resistance;

Preferably, the plant is rape;

Preferably, the disease resistance is resistance to diseases caused by Sclerotinia sclerotiorum.