CN114164078A - Bacterial chemotactic substance screening glass slide and application thereof - Google Patents
Bacterial chemotactic substance screening glass slide and application thereof Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/04—Flat or tray type, drawers
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/38—Caps; Covers; Plugs; Pouring means
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/06—Quantitative determination
Abstract
The invention discloses a bacterial chemotactic substance screening glass slide and application thereof. The glass slide surface on be equipped with 1 columniform central groove, still be equipped with 1 to a plurality of columniform peripheral recess around central groove, all be connected through a spread groove between central groove and every peripheral recess. Using the slide of the present invention as a chemotaxis assay device can reduce a large number of complicated steps. And through multiple comparison of experimental results, the results of chemotaxis experiments carried out by using the device are consistent with those of the traditional electrophoresis plate method, and multiple defects of the traditional electrophoresis plate method can be overcome. For example, the error of plate counting is reduced, the experimental period is greatly shortened, the cost of experimental materials is reduced, and the like. The invention provides a new technical means for screening the bacterial chemotactic substances.
Description
Technical Field
The invention relates to a bacterial chemotactic substance screening glass slide, which can rapidly identify chemotactic substances capable of recruiting bacteria in plant rhizosphere secretions, and also relates to application of the bacterial chemotactic substance screening glass slide in the aspect of plant-microorganism interaction research.
Background
The chemotaxis of plant rhizosphere-promoting bacteria (PGPR) has important implications for providing beneficial functions for plant-microorganism interactions to the chemotaxis of plant rhizosphere secretions (Shenyifei, jaw Yao Yao, Yangyang. influence of amino acid components in watermelon root secretions on the chemotaxis and rhizosphere colonization of Paenibacillus polymyxa SQR-21 [ J ]. Nanjing agricultural university report, 2017,40(01): 101-108; Belas, R.Biofilms, Flagenlla, and mechanosense, bacteria by bacteria. trends Microbiol 2014,22(9): 517-.
A class of low molecular weight compounds in plant root exudates induces the chemotactic movement of PGPR towards the root surface. Individual components of root exudates that stimulate chemotaxis of Bacillus include malic acid and citric acid. For example, glucose, citric acid and fumaric acid in corn root exudates can absorb rhizobacteria. Velezensis SQR9 was shown to be chemotactic for the root secreted chemotactic agent D-galactose. Organic acids such as fumaric acid, malic acid and succinic acid affect chemotaxis and colonization of various plant growth-promoting rhizobacteria in the pod-corn intercropping system. Thus, chemotactic agents which induce chemotactic movement of rhizosphere-promoting bacteria are highly necessary (Haichar, Z. et al. root extracts media interactions biochemical and biochemical 2014,77(7) 69-80; Zhang, N. et al. influence of microbial organisms and organic acids on chemicals, biofilm formation and catalysis by molecular Biology strain 2014,374(1-2) 689. BMC 700; Zhang, N. et al. genome vector microorganisms-expression tissue engineering, 11. 9. faecal. lysate and biochemical analysis 798. 9. and 22. molecular tissue culture medium, 11. 9. agar, 2. molecular tissue-expression and biochemical strain, 9. agar, 2. expression and biochemical strain, 9. 12. yeast strain, biochemical strain, and biochemical strain, 9. 12. yeast strain, biochemical strain, strain, 18(12):100331).
Currently, the chemotaxis analysis methods of bacteria are more: the electrophoresis plate method, the capillary-like method, the microfluidic chip device and the like, wherein the electrophoresis plate method comprises the steps of adding chemotactic buffer solution containing 0.2% of agar on a plate, heating the buffer solution, and then mixing with a chemotactic agent. The culture medium is prepared from semisolid culture medium, 8 mu L of bacterial drops are added into a dish, and the dish is placed in an incubator at 28 ℃ and is regularly observed and photographed. However, the electrophoresis plate method has a long experimental period, a large error in the capillary-like method, and a large cost of the microfluidic chip device (Link, N.et al. identification and role of organic acids in water droplets ex-dates for recovery of Paenibacillus polymyxa SQR-21in the rhinosphere. European Journal of Soilbiology.2011,47(6): 374) 379).
Therefore, the invention establishes a bacterial chemotactic substance screening slide comprising 1 to a plurality of channels, which can reduce a large number of complicated steps as a device for chemotaxis experiments.
Disclosure of Invention
The invention aims to provide a bacterial chemotactic substance screening slide glass comprising 1 to a plurality of channels, and the device has the remarkable advantages of intuition, simplicity, short experimental period, reduction of experimental cost and the like.
In order to achieve the purpose, the invention adopts the following technical means:
the invention discloses a bacterial chemotactic substance screening glass slide which is characterized in that 1 cylindrical central groove is arranged on the surface of the glass slide, 1 to a plurality of cylindrical peripheral grooves are arranged around the central groove, and the central groove is connected with each peripheral groove through a connecting groove.
Preferably, the glass slide is made of glass.
Wherein preferably, 1 to 6 cylindrical peripheral grooves are provided around said central groove. More preferably, 3 to 6 cylindrical peripheral grooves are provided around the central groove.
Preferably, the depth of the connecting groove is the same as that of the central groove and the peripheral groove.
Preferably, the thickness of the glass slide is 1cm, the diameters of the central groove and the peripheral grooves are 0.5cm, the depth of the central groove and the peripheral grooves is 0.5cm, the width of a connecting groove between the central groove and each peripheral groove is 0.1cm, the depth of the connecting groove is 0.5cm, and the length of the connecting groove is 1 cm.
Preferably, the slide is placed in a volatilization prevention cover with a push-pull cover for preventing liquid from volatilizing during operation.
Furthermore, the invention also provides application of the glass slide in screening of bacterial chemotactic substances.
Furthermore, the invention also provides a method for screening the bacterial chemotactic substances, which comprises the following steps:
(1) culturing the bacteria to OD6000.5, obtaining a bacterial solution;
(2) preparing 1 or more kinds of aqueous solutions of the chemoattractant to be tested, and filtering the solutions by using a 0.22 mu m bacterial filter;
(3) respectively and sequentially dripping 1 to a plurality of sterile chemotactic substance aqueous solutions into peripheral grooves of the bacterial chemotactic substance screening glass slide, preferably, respectively dripping sterile water and 0.9% w/v NaCl solution into two peripheral grooves to serve as a negative control and a positive control;
(4) dripping a bacterial solution into a central groove of the bacterial chemotactic substance screening glass slide, standing for 10-20 minutes at room temperature without air drying, respectively sucking the liquid in the groove in which the chemotactic substance aqueous solution is dripped, transferring the liquid into a sterile centrifuge tube containing safranin dye, sucking the mixed solution into a blood counting chamber after 2-5 minutes, and observing by a microscope;
(5) if the concentration of the bacteria is obviously higher than that of the negative control and the positive control, the chemotactic effect of the chemotactic compound aqueous solution on the bacteria is shown.
Compared with the prior art, the invention has the beneficial effects that:
the invention establishes a bacterial chemotactic substance screening slide glass comprising 1 to a plurality of channels, which can reduce a large number of complicated steps as a chemotaxis experiment device. In the invention, the results of chemotaxis experiments performed by using the device are consistent with those of the traditional electrophoresis plate method through multiple comparison of experimental results, and the multiple defects of the traditional electrophoresis plate method can be overcome. For example, the error of plate counting is reduced, the experimental period is greatly shortened, the cost of experimental materials is reduced, and the like.
Drawings
FIG. 1 is a schematic structural diagram of a multichannel bacterial chemotactic substance screening slide;
wherein: 1-peripheral grooves; 2-a central recess; 3-connecting groove 3; 4-glass slide; 5-an anti-volatilization cover;
FIG. 2 is a graph showing the results of the electrophoresis plate method;
FIG. 3 is a schematic view of the bacterial chemotactic substance screening slide experiment of the present invention.
FIG. 4 is a graph showing the results of the verification experiment in the present invention.
Detailed Description
The invention will be further described with reference to specific embodiments and drawings, the advantages and features of which will become apparent as the description proceeds. The examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
Example 1 preparation of bacterial chemotactic substance screening slide glass
The bacterial chemotactic substance screening slide glass 4 provided by the embodiment is made of glass, the length is 8cm, the width is 5.5cm, the thickness is 1cm, seven cylindrical grooves are formed in the surface, the seven cylindrical grooves comprise a central groove 2 and 6 peripheral grooves 1 surrounding the central groove 2, the diameter of each groove is 0.5cm, the height of each groove is 0.5cm, the central groove 2 and each peripheral groove 1 are connected through a connecting groove 3 with the width of 0.1cm, and the depth of each connecting groove 3 is 0.5 cm. Wherein the central groove 2 is used for dripping bacterial solution in the experiment, four peripheral grooves 1 are used for dripping chemotactic solution, and the remaining two peripheral grooves 1 are respectively used for dripping sterile water and 0.9% w/vNaCl as negative control and positive control. The slide glass 4 is provided with a push-pull volatilization prevention cover 5, so that liquid volatilization caused by external factors is avoided in an experiment, and the specific structural schematic diagram is shown in figure 1.
Example 2 Collection of Rice rhizosphere secretions
1. Test material
The strain is Bacillus altitudinis LZP02 separated from rhizosphere soil of rice in northeast China, and is preserved in China center for type culture Collection, wherein the address is Wuhan university, the microbial preservation number is CCTCC NO: M2018598, and the preservation date is 09/06/2018. The strain is described in the patent application with the publication number of CN109536401A and the name of the invention is a compound microbial fertilizer and a preparation method thereof and application in promoting the growth of rice. Albitudinis LZP02 in LB Medium (peptone, 10g L)-1(ii) a Sodium chloride, 8g L-1And yeast extract 5g L-1) Culturing for 10h, centrifuging at 10000rpm and 4 deg.C for 2min, and storing with 15% glycerol at-80 deg.C. The rice seeds (46 rice in Longjiang agriculture) were provided by the Rice research institute of Heilongjiang academy of agriculture.
2. Test method
2.1 cultivation of Rice seedlings
The rice seeds were sterilized with 30% hydrogen peroxide for 10min, rinsed five times with sterile water and soaked in water overnight. The rice seeds are randomly distributed in a growth chamber for accelerating germination, and the illumination is controlled to be (16/8h light/dark period), the temperature is controlled to be (22 +/-2 ℃) and the relative humidity is controlled to be (70%). Sterile water was added twice a week until the seedling stage. Similar sized rice seedlings were selected and planted in 50mL of MS liquid medium. Incubate under sterile conditions for 48 h.
2.2 Mass spectrometric analysis of root secretions by liquid chromatography
50mL of sample was weighed into an EP tube, and after adding 1000. mu.L of extraction solvent (acetonitrile-methanol-water 2: 2: 1, containing internal standard), the sample was vortexed for 30s, homogenized at 45Hz for 4min, and sonicated in an ice water bath for 5 min. The homogenization and sonication cycles were repeated 3 times, followed by incubation at-20 ℃ for 1h, followed by centrifugation at 12000rpm and 4 ℃ for 15 min. The supernatant was transferred to LC-MS vials and stored at-80 ℃ until UHPLCQE Orbitrap/MS analysis.
LC-MS/MS analysis: LC-MS/MS analysis was performed using a UHPLC system (1290, Agilent technologies) coupling a UPLC HSS T3 column (2.1 mm. times.100 mm, 1.8 μm) with Q active (Orbitrap MS, Thermo). Mobile phase a was 0.1% formic acid solution (positive ion index) and 5mmol/L ammonium acetate solution (negative ion index) and mobile phase B was acetonitrile. The elution gradient was set as follows: 0min, 1% B; 1 minute, 1% B; 8 min, 99% B; 10 minutes, 99% B; 10.1min, 1% B; 12 min, 1% B. The flow rate was 0.5mL/min and the amount of sample was 2. mu.L. The gas source conditions for ESI are as follows: the capillary temperature was 320 ℃ and the full millisecond resolution and MS/MS resolution were 70,000 and 17500, respectively. The collision energy was 20/40/60 eV. The spray voltage was 3.8kV (positive) or-3.1 kV (negative).
3. Results and analysis
Data were acquired using acquisition software (xcalibur4.0.27, Thermo). 584 and 937 known metabolites were detected in positive and negative ion index modes, respectively. As the chemoattractant generally belongs to organic acids, amino acids, carbohydrates and the like. The following experiments with 16 chemoattractants selected from the rice rhizosphere secretion in the present invention are shown in table 1.
Table 1 16 chemoattractants were selected from among the rice rhizosphere exudate secretions.
POS and NEG in the table represent positive ion index and negative ion index, respectively. The third column shows that the database matches were scored using the secondary spectra and the unscaled chemotactic agents were matched using the first order spectra. The fraction ranges from 0 to 1.
Example 3 chemotaxis assay of 16 chemoattractants by use of the phoresis plate method
1. Test materials: the strains are the same as example 2, and the reagents are all standard products with the content of more than 98 percent and are purchased from Nanjing institute of bioengineering.
2. Test method
A swimming medium (peptone, 10 g/L; sodium chloride, 8 g/L; agar, 4%) was prepared. The medium was sterilized at 121 ℃ for 20min and then cooled to 40-50 ℃. In the experimental group, different dilutions of the aqueous solution of the chemoattractant were added 1:1 to the medium to final concentrations of 25. mu.M, 50. mu.M, 100. mu.M. The control group was prepared with an equal amount of sterile water. After mixing well, the medium was dispensed into a petri dish and kept upside down and allowed to stand at room temperature. Then, the culture was carried out to OD600Bacterial suspension 5 μ L of 0.5 b.altitudinis LZP02 was plated on prepared agar plates in triplicate for each treatment. After absorption, the plates were placed in a 30 ℃ incubator. After 12h-14h, the observation and the photographing are carried out.
3. Results and analysis
The experimental results are shown in fig. 2: to compare the magnitude of chemotaxis, we introduced a chemotaxis index (RCI) showing the ratio of the mean treatment aggregate area diameter to the corresponding control diameter. A higher RCI indicates a stronger attraction for bacteria. The results show that 100 μ M citric acid (RCI ═ 2.34) and 100 μ M caffeic acid (RCI ═ 1.85) have the highest chemotaxis indices. Citric acid, caffeic acid induced significant chemotactic motility. 100 μ M galactose was of inferior chemotactic index.
Example 4 results verification Using bacterial chemotactic Material screening slide glass of the present invention
1. Test materials: same as in example 2
2. Test method
The material of the bacterial chemotactic substance screening slide glass that this embodiment provided is glass, and length is 8cm, and the width is 5.5cm, and thickness is 1cm, and there are 4 cylindrical recesses on the surface, including 1 central recess and around 3 peripheral recesses of central recess, the recess diameter is 0.5cm, highly is 0.5cm, all be connected through a width connecting groove that is 0.1cm between central recess and every peripheral recess, the degree of depth of connecting groove is 0.5cm equally.
As shown in FIG. 3, the bacteria were first cultured to OD6000.5. The aqueous chemoattractant solution was filtered through a 0.22 μm bacterial filter. Firstly, respectively and sequentially dripping a plurality of 30-80 mu L sterile chemotactic substance aqueous solutions into 1 peripheral groove. The other two concaveThe cells were dropped with equal amounts of sterile water (negative control) and 0.9% w/v NaCl solution (positive control) in order to eliminate the effect of intermolecular forces on the experiment. And next, dropwise adding an equal amount of bacterial solution into the central groove, wherein a connecting groove in the device can automatically connect the central groove with the peripheral groove. And (3) pushing an anti-volatilization cover, standing for 20 minutes under the condition of room temperature without wind drying, respectively sucking the liquid in the grooves in which the chemoattractant aqueous solution is dropwise added, transferring the liquid into a 1.5ml sterile centrifuge tube containing safranin dye, sucking the mixed solution into a blood counting chamber after 2 minutes, and observing the mixed solution by a microscope.
3. Results and analysis
The number of viable microorganisms attracted is determined according to the equation.
Note: NBC: total number of bacterial cells; n: number of bacteria in 80 grids
Statistical analysis of the data was performed using SPSS24.0 software. The error is based on three different replicate experimental values. Based on a one-way posterior analysis followed by a posterior multiple comparison analysis. P is less than or equal to 0.05, and the difference has statistical significance.
The present inventors first collected rice rhizosphere secretions and performed qualitative and quantitative analysis using LC-MS (1290, Agilent Technologies). The subsequent multiple comparisons of the experimental results confirm the accuracy of the experimental results using the device of the present invention. The results are shown in fig. 4, and the experiment shows that: 584 and 937 known metabolites were detected in positive and negative ion index modes, respectively. Previous studies have shown that chemoattractants generally belong to the group of organic acids, amino acids and carbohydrates. The invention selects 16 chemoattractants in the rice rhizosphere exudate for subsequent experiments. Chemotaxis Index (RCI) of 16 chemoattractants was determined by the electrophoresis plate method, with higher RCI indicating greater attraction to bacteria. The results show that 100 μ M citric acid (RCI ═ 2.34) and 100 μ M caffeic acid (RCI ═ 1.85) have the highest chemotaxis indices. Citric acid, caffeic acid, and galactose induced significant chemotactic movement. The bacterial chemotactic substances are used for screening the glass slide, and the experimental result shows that when the citric acid and the caffeic acid are used as chemotactic substances, the bacterial concentration in the glass slide is obviously higher than that in a negative control group and a positive control group. And citric acid is most chemotactic. The experimental results were consistent with those of the conventional method of example 3.
In conclusion, the novel single-channel bacterial chemotactic substance screening glass slide device established by the invention has accurate and reliable experimental results, overcomes multiple defects of the traditional experimental method, and is simple in material, so that the multi-channel bacterial chemotactic substance screening glass slide is deeply developed, has important significance for researching plant-microorganism interaction, and can be produced in large scale and applied to experimental research.
Claims (8)
1. The bacterial chemotactic substance screening glass slide is characterized in that 1 cylindrical central groove is formed in the surface of the glass slide, 1 to a plurality of cylindrical peripheral grooves are further formed around the central groove, and the central groove is connected with each peripheral groove through a connecting groove.
2. The bacterial chemotactic substance screening slide of claim 1, wherein said slide is glass.
3. The bacterial chemotactic material screening slide of claim 1, wherein 1 to 6 cylindrical peripheral grooves are provided around said central groove, preferably 3 to 6 cylindrical peripheral grooves are provided around said central groove.
4. The bacterial chemotactic material screening slide of claim 1 wherein the depth of said attachment grooves is the same as the depth of said central groove and said peripheral groove.
5. The bacterial chemotactic substance screening slide as claimed in claim 1, wherein said slide has a thickness of 1cm, said central groove and said peripheral grooves have a diameter of 0.5cm and a depth of 0.5cm, and the connecting groove between the central groove and each peripheral groove has a width of 0.1cm, a depth of 0.5cm and a length of 1 cm.
6. The bacterial chemotactic material screening slide of claim 1, wherein said slide is placed in a push-pull cap to prevent evaporation of liquid during operation.
7. Use of a slide according to any one of claims 1 to 6 for screening for bacterial chemotactic substances.
8. A method for screening a bacterial chemotactic substance, which comprises the following steps:
(1) culturing the bacteria to OD6000.5, obtaining a bacterial solution;
(2) preparing 1 or more kinds of aqueous solutions of the chemoattractant to be tested, and filtering the solutions by using a 0.22 mu m bacterial filter;
(3) sequentially dropping 1 to more kinds of sterile chemotactic agent aqueous solutions into the peripheral grooves of the bacterial chemotactic substance screening slide according to any one of claims 1 to 6, preferably, further dropping sterile water and 0.9% w/v NaCl solution into two of the peripheral grooves as a negative control and a positive control;
(4) dripping a bacterial solution into a central groove of the bacterial chemotaxis substance screening slide glass of any one of claims 1 to 6, standing for 10 to 20 minutes under the condition of room temperature without air drying, respectively sucking the liquid in the groove dripping the chemotaxis substance aqueous solution, transferring the liquid into a sterile centrifuge tube containing safranin dye, sucking the mixed solution into a blood counting chamber after 2 to 5 minutes, and observing by a microscope;
(5) if the concentration of the bacteria is obviously higher than that of the negative control and the positive control, the chemotactic effect of the chemotactic compound aqueous solution on the bacteria is shown.
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