CN110669850A - Method for detecting colonization ability of bacteria in salvia miltiorrhiza - Google Patents
Method for detecting colonization ability of bacteria in salvia miltiorrhiza Download PDFInfo
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Abstract
The invention relates to a method for detecting the colonization ability of bacteria in salvia miltiorrhiza, which comprises the following steps: 1) inoculating bacteria to be detected into the sterilized salvia miltiorrhiza seedlings for culture so as to colonize the inoculated bacteria; 2) the colonized salvia miltiorrhiza seedlings are respectively treated and grouped by the following steps: w) sterile water washing; SD) sterilized saline solution is washed for 10min to 30min and then is washed by sterile water; B) washing with sterilized saline solution, washing with sodium hypochlorite, and washing with Na2S2O3Washing with a solution; 3) amplifying the characteristic nucleic acid fragments of the bacteria to be detected which are colonized in each group, dyeing the amplification result, and determining the colonization ability of the bacteria to be detected according to W, SD and whether a target band appears in group B, wherein the target band appearing in group B has the strongest quantification ability, and the group SD has the second time, and the group W has the second time. The method can be moreThe colonization ability of the bacteria was objectively evaluated.
Description
Technical Field
The invention relates to the field of microorganism-plant interaction research, in particular to a method for detecting the colonization ability of bacteria in salvia miltiorrhiza.
Background
The existing scientific research and the microbial inoculum sold on the market can influence the growth, development and metabolic activity of plants to a certain extent, and especially plant growth-promoting rhizobacteria (PGPR) can promote the growth of plants, prevent and control diseases, increase crop yield and the like. Successful colonization of the plant root system by bacteria plays an extremely important role in the interaction between bacteria and plants. Because a plurality of microorganisms with remarkable growth promoting effect under laboratory conditions can not play a good role when being applied to a field, the strong and weak colonization ability is the key to influence the action of the microorganisms. Therefore, whether beneficial bacteria are inoculated to successfully colonize the root circumference or the leaf circumference of the host plant or not can be realized, and the method has the characteristics of strong colonizing capacity, large colonizing quantity and the like and is a key factor for determining the direct action effect, the application dosage, the application conversion and the like of the biocontrol bacteria.
Currently, the most common methods for detecting bacterial colonization include microscopy and colony counting, but are time-consuming, labor-consuming and highly affected by human manipulation. The molecular biology can further reveal the special rule of the colonization of the bacterial roots, the detection result is more accurate, and the existing detection methods for researching the colonization capability of the biocontrol bacteria on the salvia miltiorrhiza by using molecular markers, fluorescent markers, special primers and the like are relatively few.
Disclosure of Invention
The invention relates to a method for detecting the colonization ability of bacteria in salvia miltiorrhiza, which comprises the following steps:
1) inoculating bacteria to be detected into the sterilized salvia miltiorrhiza seedlings for culture so as to colonize the inoculated bacteria;
2) the colonized salvia miltiorrhiza seedlings are respectively treated and grouped by the following steps:
w) sterile water washing;
SD) sterilized saline solution is washed for 10min to 30min and then is washed by sterile water;
B) washing with sterilized saline solution for 10-30 min, and washing with 3-7% (g-100ml) sodium hypochlorite washing for 1.5 min-2.5 min, Na2S2O3Washing with a solution;
wherein the salt solution comprises the following components:
127mM～147mM NaCl，1.7mM～3.7mM KCl，5mM～15mM Na2HPO4，1.3mM～2.3mMKH2PO4，0.3mM～0.7mM MgSO4，0.7mM～1.3mM CaCl20.07-0.13% (v/v) Triton-X100, and the pH of the salt solution is 7.0-7.8;
3) amplifying the characteristic nucleic acid fragments of the bacteria to be detected which are colonized in each group, dyeing the amplification result, and determining the colonization ability of the bacteria to be detected according to W, SD and whether a target band appears in group B, wherein the target band appearing in group B has the strongest quantification ability, and the group SD has the second time, and the group W has the second time.
W, SD and B have different elution/sterilization abilities, wherein W has the weakest ability and B has the strongest ability, so if B has amplification products of target bands, it indicates that the bacteria to be detected corresponding to the target have stronger colonization ability. By the method, the bacteria colonization ability can be evaluated more objectively.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 shows the detection results of the bacteria Sp7 and Sp245 colonizing the root system of Salvia miltiorrhiza Bunge in one embodiment of the present invention;
sp7& Sp245: isolated from wheat, early studies have identified epiphytic and endophytic bacteria; IGS, a 16S-23S rRNA intergenic region of bacteria, Sp7 of-562 bp and Sp245 of-547 bp; LFY, Salvia miltiorrhiza LEAFY gene-257 bp; controls: PCR control; no TC: no DNA template; -: sterile red sage root; +: bacterial DNA;
FIG. 2 is a fluorescent staining pattern of two bacteria inoculated to Salvia miltiorrhiza Bunge after three different chemical treatments in one embodiment of the invention; the length of the scale is 32 μm.
Detailed Description
Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.
It is therefore intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present invention are disclosed in or are apparent from the following detailed description. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present invention.
The invention relates to a method for detecting the colonization ability of bacteria in salvia miltiorrhiza, which comprises the following steps:
1) inoculating bacteria to be detected into the sterilized salvia miltiorrhiza seedlings for culture so as to colonize the inoculated bacteria;
2) the colonized salvia miltiorrhiza seedlings are respectively treated and grouped by the following steps:
w) sterile water washing;
SD) sterilized saline solution is washed for 10min to 30min and then is washed by sterile water;
B) washing with sterilized saline solution for 10-30 min, washing with 3-7% (g/100ml) sodium hypochlorite for 1.5-2.5 min, and washing with Na2S2O3Washing with a solution;
wherein the salt solution comprises the following components:
127mM～147mM NaCl，1.7mM～3.7mM KCl，5mM～15mM Na2HPO4，1.3mM～2.3mMKH2PO4，0.3mM～0.7mM MgSO4，0.7mM～1.3mM CaCl20.07-0.13% (v/v) Triton-X100, and the pH of the salt solution is 7.0-7.8;
3) amplifying the characteristic nucleic acid fragments of the bacteria to be detected which are colonized in each group, dyeing the amplification result, and determining the colonization ability of the bacteria to be detected according to W, SD and whether a target band appears in group B, wherein the target band appearing in group B has the strongest quantification ability, and the group SD has the second time, and the group W has the second time.
The invention can be suitable for the research of rhizosphere microorganism colonization condition. Rhizosphere (rhizosphere) refers to an area whose biological and physical characteristics are closely affected by root systems, and the rhizosphere and a microbial community form a very complex ecological system together, which is a habitat for ensuring normal growth and development of plant root systems and is also a main place for exchanging substances and energy between plants and the external environment, wherein microorganisms are rich in diversity and have strong rhizosphere effect. Therefore, the microbial environment formed by rhizosphere microorganisms plays an extremely important role in plant growth.
In some embodiments, the salt solution comprises the following components:
132mM～142mM NaCl，1.9mM～3.5mM KCl，8mM～12mM Na2HPO4，1.5mM～2.1mMKH2PO4，0.4mM～0.6mM MgSO4，0.9mM～1.1mM CaCl20.08-0.12% (v/v) Triton-X100, and the pH of the salt solution is 7.2-7.6.
In some embodiments, the nucleic acid fragment characteristic of the bacterium is a fragment in the 16S rDNA of the bacterium.
The 16S rDNA has high conservation in structure and function, is commonly used for bacterial isolation, and can be used as a simple fingerprint sequence of bacterial species in the invention.
In some embodiments, the method of staining the amplification results is selected from the group consisting of silver staining, methylene blue staining, ethidium bromide staining, and acridine orange staining.
In some embodiments, the forward primer and the reverse primer used for amplification of the nucleic acid fragment characteristic of the bacterium are shown in SEQ ID NO 1 and 2, respectively.
The sequences shown in SEQ ID NO 1 and 2 are designed aiming at the evolutionary conserved region of 16S rDNA, and the segment can be amplified.
In some embodiments, the method further comprises the step of amplifying a gene whose expression is stable endogenously in salvia miltiorrhiza as an internal reference gene.
In some embodiments, the endogenous expression-stable gene of salvia miltiorrhiza is the LEAFY gene.
In some embodiments, the upstream and downstream primers used to amplify the LEAFY gene are shown in SEQ ID Nos. 3 and 4, respectively.
In some embodiments, the method for preparing the sterilized young salvia miltiorrhiza seedlings comprises the following steps:
and culturing and germinating the salvia miltiorrhiza seeds for 10-15 days to obtain the salvia miltiorrhiza seedlings.
In some embodiments, the salvia miltiorrhiza seeds are subjected to a sterilization cleaning treatment selected from the group consisting of:
70-85% ethanol water solution cleaning, 40-60% (g/100ml) sodium hypochlorite cleaning and sterile water cleaning.
In some embodiments, the salvia miltiorrhiza seeds are further subjected to low temperature vernalization prior to germination in culture:
culturing in 1/2MS solid culture medium at 0-8 deg.c in dark for 2-4 days.
In some embodiments, the culture conditions for 5-10 days of germination are: culturing for 12-16 h under illumination at 23-27 deg.C for 8-12 h under dark condition at 20-24 deg.C.
In some embodiments, the method further comprises:
staining the bacteria in the colonized salvia miltiorrhiza seedlings to assist in judging the bacteria colonization ability.
In some embodiments, the dye used to stain the bacteria is SYTO 9 and propidium iodide.
Embodiments of the present invention will be described in detail with reference to examples.
Examples
1. Sterilizing and seedling culturing of salvia miltiorrhiza seeds
Putting 8-10 seeds of Saviae Miltiorrhizae radix into a centrifuge tube, adding 1mL 80% ethanol, and placing in an oscillator at 1000rpm for 3 min. After discarding the supernatant, 1mL of 50% NaClO was added and shaken for 5 min. After discarding the supernatant, 1mL of sterile water was added and washed 4 times. The seeds were transferred to 1/2MS solid medium and sealed with Parafilm sealing film and placed in a dark room at 4 ℃ for vernalization at low temperature. After three days, the seeds are moved to a light incubator, the illumination is carried out at 25 ℃ (14h), the illumination is carried out, the darkness is carried out at 22 ℃ (10h), and the inoculation treatment is carried out after the seeds germinate and grow for one week.
2. Inoculation of bacteria
Using Sp7 and Sp245 isolated from wheat, both of Azospirillum, earlier studies have identified epiphytic (epiphytic) and endophytic (endophytic) bacteria as reference strains for the study of bacterial colonization characteristics in plant-microorganism interactions.
Two target strains are inoculated in 6mL LB culture solution, and are cultured in a constant temperature incubator at 28 ℃ for 48h by shaking at 250 rpm. Centrifuging the bacteria culture solution at 10,000rpm for 5min, discarding the supernatant, diluting the bacteria solution with sterile water, and measuring the absorbance of the bacteria solution at 600nm with spectrophotometer to be about 0.7. 100 mu L of diluted bacterial liquid is evenly coated on 1/2MS plant culture medium in a clean bench, red sage root seedlings are transferred to square culture dishes (the specification is 9cm multiplied by 9cm), sealing is carried out by using sealing films, six seedlings can be placed in each culture medium, and then the culture medium is placed in a constant temperature culture room (25 ℃, 12h day and 12h night) for culture for one week. Meanwhile, the medium was coated with 100. mu.L of sterile water, and six Salvia miltiorrhiza seedlings were placed for blank control.
3. Nucleic acid extraction
For the salvia plant inoculated with bacteria, nucleic acid was extracted seven days after inoculation. Before that, three different types of chemical treatments are adopted to detect the strong and weak ability of the inoculated bacteria to colonize the root system of the salvia miltiorrhiza, namely, the bacteria can be symbiotic with plants in the form of epiphyte or endophyte. Three chemical treatments of root system tissue of salvia miltiorrhiza include: 1) sterile water was washed twice, and the treated sample was labeled as W; 2) sterile saline solution (137mM NaCl,2.7mM KCl,10mM Na)2HPO4,1.8mM KH2PO4,0.5mMMgSO4,1mM CaCl20.1% Triton-X100, pH 7.4) for 20min, and then washed twice with sterile water, and the sample is recorded as SD; 3) the sterilized salt solution was washed for 20min, then 5% sodium hypochlorite for 2min, and finally washed twice with Na2S2O3 solution, sample B.
Transferring 3-5 radix Salviae Miltiorrhizae plants into centrifuge tubes labeled with W, SD and B, performing chemical treatment in steps 1, 2 and 3, transferring radix Salviae Miltiorrhizae into centrifuge tubes pre-filled with grinding magnetic beads (14mm silica beads), adding 750 μ L of 2 × CTAB and 300 μ L of chloroform, and grinding for 3 min. Centrifuge at 16,000g for 10min at room temperature and transfer the supernatant to a new centrifuge tube. mu.L of ammonium acetate and 750. mu.L of absolute ethanol were added, and the mixture was centrifuged at 16,000g for 30min at 4 ℃. The supernatant was discarded, 500. mu.L of 75% ethanol was added, centrifuged at 16,000g for 10min at 4 ℃ and the procedure was repeated. After discarding the supernatant, the nucleic acid was dried, and the DNA was diluted to 100 ng/. mu.L with sterile double distilled water, and the purity and concentration of the DNA were examined by Nanodrop 2000 and 1% agarose gel electrophoresis.
Identification of bacterial genes by PCR
Bacterial 16S-23S IGS primers, forward primer 16S-e1390f (SEQ ID NO: 1: 5 '-TGYACACACCGCCCGTCA-3', wherein Y represents T or C), reverse primer 23S-e130R (SEQ ID NO: 2: 5 '-GGGTTBCCCATTCRG-3', wherein B represents G or C or T, and R represents G or A) were used. A25. mu.L PCR reaction consisted of 2.5. mu.L of 10 XPCR buffer, 0.5mM MgCl20.2mM dNTPs, 0.8. mu.M of forward and reverse primers, 0.5. mu.L of 5U/. mu.L Taq polymerase, about 100ng DNA template, and finally sterile ddH2O to 25. mu.L. The PCR reaction procedure included pre-denaturation at 95 ℃ for 5min, 30 cycles including denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 30s and extension at 72 ℃ for 90s, and finally extension at 72 ℃ for 5 min. After being stained by smelling ethidium, the PCR product is electrophoresed for about one hour through 1% agarose gel, and then the PCR amplification result is detected by a gel imager and photographed. Whether the samples W, SD and B have the fingerprint of the target bacteria or not is judged by the result of PCR amplification of the bacterial fingerprint.
Meanwhile, sterile salvia miltiorrhiza bunge which is not inoculated with bacteria is used as a negative control, no bacterial fingerprint is theoretically generated by PCR amplification, if the bacterial fingerprint exists, salvia miltiorrhiza bunge seedlings are polluted, and the experiment needs to be repeated. When the PCR results of the samples W, SD and B both have obvious bacterial fingerprint spectra, the bacteria inoculated to the salvia miltiorrhiza are considered to be colonized in an endophyte form, and the colonizing capacity is strong. When only the PCR result of the sample W has a bacterial fingerprint, or the sample SD also has a weak fingerprint, but the sample B does not have the bacterial fingerprint, the bacteria inoculated with the salvia miltiorrhiza is considered to be colonized in the form of epiphyte, and the colonization capability is weak. The results are shown in FIG. 1.
Identification of plant genes by PCR method
In the PCR amplification, in addition to designing a bacteria-specific primer to amplify a specific bacterial gene, a plant-specific primer is specially designed to amplify a salvia miltiorrhiza specific gene LEAFY gene based on the LEAFY gene of Arabidopsis thaliana. The forward and reverse primers designed from the Salvia miltiorrhiza genome are SmLFY-F (SEQ ID NO: 3: 5'-GTTCCGGTACGCGAAGAAG-3') and SmLFY-R (SEQ ID NO: 4: 5'-GGACGTACCAAATGGAGAGG-3'), respectively. 25 μ L of PCR reaction system comprises 2.5 μ L of 10 XPCR buffer, 0.2mM dNTPs, 0.4 μ M of forward and reverse primers, 0.4 μ L of 5U/. mu.L Taq polymerase, about 200ng DNA template, and finally sterile ddH2O to 25. mu.L. The PCR reaction procedure included pre-denaturation at 95 ℃ for 1min, 28 cycles including denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 15s and extension at 72 ℃ for 45s, and finally extension at 72 ℃ for 5 min. After being stained by smelling ethidium, the PCR product is electrophoresed for about one hour through 1 percent agarose gel, and then the PCR amplification result is detected by a gel imager and photographed and recorded. The results are shown in FIG. 1.
6. Fluorescence microscope double validation
In order to observe the infection condition of bacteria in the interaction between the bacteria and plants, a small part of plant tissue is cut after one week of bacteria inoculation, the plant tissue is washed by sterile water, the plant tissue is combined to pass through three different types of chemical treatment modes, the plant tissue is respectively placed on different glass slides, and about 20 mu L of green fluorescent nucleic acid dye SYTO 9 (SYTO) is drippedTM9Green fluorescent nucleic Acid Stain, ThermoFisher, USA) and the Green Red fluorescent nucleic Acid dye PI (Propidium IodideReady probes)TMReagent, ThermoFisher, USA) for 3min, and adding appropriate amount of sterile water to wash offAnd (4) adding redundant dye solution on the glass slide, and finally covering the glass slide. SYTO 9green fluorescent nucleic acid dye was demonstrated to stain both live and dead cells of gram positive and gram negative bacteria. The fluorescent dye PI (propidium iodide) is a nuclear staining reagent that stains DNA and is commonly used in apoptosis detection. Then, the slide glass was placed under an Olympus (FSX100) fluorescence microscope for observation, and the bacterial population stained in the plant tissue was observed looking for a suitable field of view, and observed and photographed using magnifications of 10X, 20X and 30X, respectively. The manner in which the bacteria colonize the plant (either epiphytes or endophytes) and the magnitude of the colonization ability were further verified by observing the staining results of the bacteria infecting the plant, in contrast to the PCR results of samples W, SD and B. The result of fluorescence microscopy is shown in fig. 2, the fluorescence staining result is consistent with the result of PCR detection, and for the epiphytic bacterium Sp7, the bacterium cannot be detected after the salvia miltiorrhiza tissue is washed by a chemical mode B. For endophytic bacterium Sp245, after the salvia miltiorrhiza tissue is washed by a chemical method B, the bacterium can still be detected. Furthermore, the combination of PCR detection based on two special primers and a specific chemical treatment mode of plant tissues has strong research significance and practical value for the research on the capability of bacteria to colonize salvia miltiorrhiza and the function of bacteria.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
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Claims (10)
1. A method for detecting the colonization ability of bacteria in salvia miltiorrhiza is characterized by comprising the following steps:
1) inoculating bacteria to be detected into the sterilized salvia miltiorrhiza seedlings for culture so as to colonize the inoculated bacteria;
2) the colonized salvia miltiorrhiza seedlings are respectively treated and grouped by the following steps:
w) sterile water washing;
SD) sterilized saline solution is washed for 10min to 30min and then is washed by sterile water;
B) washing with sterilized saline solution for 10-30 min, washing with 3-7% (g/100ml) sodium hypochlorite for 1.5-2.5 min, and washing with Na2S2O3Washing with a solution;
wherein the salt solution comprises the following components:
127mM～147mM NaCl，1.7mM～3.7mM KCl，5mM～15mM Na2HPO4，1.3mM～2.3mM KH2PO4，0.3mM～0.7mM MgSO4，0.7mM～1.3mM CaCl20.07-0.13% (v/v) Triton-X100, and the pH of the salt solution is 7.0-7.8;
3) amplifying the characteristic nucleic acid fragments of the bacteria to be detected which are colonized in each group, dyeing the amplification result, and determining the colonization ability of the bacteria to be detected according to W, SD and whether a target band appears in group B, wherein the target band appearing in group B has the strongest quantification ability, and the group SD has the second time, and the group W has the second time.
2. The method of claim 1, wherein the nucleic acid fragment characteristic of the bacterium is a fragment of the bacterium in 16S rDNA.
3. The method for detecting the colonization ability of bacteria in Salvia miltiorrhiza Bunge according to claim 2, wherein the upstream primer and the downstream primer for amplifying the characteristic nucleic acid fragment of the bacteria are shown in SEQ ID NO 1 and SEQ ID NO 2, respectively.
4. The method for detecting the colonization ability of salvia miltiorrhiza bunge bacteria according to claim 1, further comprising the step of amplifying a gene with stable endogenous expression of salvia miltiorrhiza bunge as an internal reference gene.
5. The method for detecting the colonization ability of salvia miltiorrhiza bunge by bacteria according to claim 4, wherein the gene with stable endogenous expression of salvia miltiorrhiza bunge is LEAFY gene.
6. The method for detecting the colonization ability of bacteria in Salvia miltiorrhiza Bunge according to claim 5, wherein the upstream primer and the downstream primer for amplifying the LEAFY gene are shown in SEQ ID NO 3 and SEQ ID NO 4, respectively.
7. The method for detecting the colonization ability of bacteria on salvia miltiorrhiza according to any one of claims 1 to 6, wherein the preparation method of the sterilized salvia miltiorrhiza seedlings comprises the following steps:
and culturing and germinating the salvia miltiorrhiza seeds for 10-15 days to obtain the salvia miltiorrhiza seedlings.
8. The method for detecting the colonization ability of salvia miltiorrhiza bunge, according to claim 7, wherein the salvia miltiorrhiza bunge seeds are subjected to a sterilization and cleaning treatment, wherein the sterilization and cleaning treatment is selected from the following treatments:
70-85% ethanol water solution cleaning, 40-60% (g/100ml) sodium hypochlorite cleaning and sterile water cleaning.
9. The method for detecting the colonization ability of bacteria in salvia miltiorrhiza according to claim 7, wherein the salvia miltiorrhiza seeds are further subjected to low-temperature vernalization treatment before culture and germination:
culturing in 1/2MS solid culture medium at 0-8 deg.c in dark for 2-4 days.
10. The method for detecting the colonization ability of bacteria on salvia miltiorrhiza according to claim 7, wherein the culture conditions for 5-10 days of culture germination are as follows: culturing for 12-16 h under illumination at 23-27 ℃ and 8-12 h under dark at 20-24 ℃;
preferably, the method further comprises:
dyeing bacteria in the colonized salvia miltiorrhiza seedlings to assist in judging the colonization ability of the bacteria;
preferably, the dyes used for staining bacteria are SYTO 9 and propidium iodide.
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| CN113416793A (en) * | 2021-06-16 | 2021-09-21 | 广东省科学院生物工程研究所 | Method for detecting colonization ability of bacteria in duckweed |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113416793A (en) * | 2021-06-16 | 2021-09-21 | 广东省科学院生物工程研究所 | Method for detecting colonization ability of bacteria in duckweed |
| CN113416793B (en) * | 2021-06-16 | 2024-04-12 | 广东省科学院南繁种业研究所 | Method for detecting colonization ability of bacteria in duckweed |
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