CN117344040A - In-situ detection method for key active microorganisms of seeds and dynamic changes of key active microorganisms - Google Patents
In-situ detection method for key active microorganisms of seeds and dynamic changes of key active microorganisms Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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Abstract
The invention provides an in-situ detection method for key active microorganisms of seeds and dynamic changes thereof, and relates to the technical field of biology. The invention provides an in-situ detection method of key active microorganisms of seeds, which comprises the following steps: culturing the seeds with aseptic surfaces in an aseptic environment to obtain germinated seeds, collecting lateral roots or adventitious roots of single germinated seeds, extracting and detecting microbial DNA of the lateral roots or adventitious roots, and determining key active microorganisms of the single seeds according to detection results. The detection method is simple and convenient, can realize the detection of key active microorganisms of a single seed on the premise of not damaging plant growth, can be further applied to the detection of the dynamic change of microorganisms of individual plants under biotic or abiotic stress, can be widely applied to the research of microorganisms of various plant seeds, and has important significance for researching the microorganisms of the seeds.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an in-situ detection method for key active microorganisms of seeds and dynamic changes of the key active microorganisms.
Background
Seed microorganism means a microorganism present on the surface of the seed, in the interior of the seed or in the soil surrounding the seed, and includes bacteria, fungi, actinomycetes, and the like. These microorganisms have an effect on germination, growth and development of the seeds and can interact with the seeds by means of adhesion to the surface of the seeds, internal parasitics or symbiosis in the soil surrounding the seeds. In addition, during germination and growth of seeds, seed microorganisms can positively affect growth and development of seeds by producing hormones, promoting nutrient absorption, preventing invasion of pathogenic microorganisms, and the like. Researches show that the seed microorganism plays an important role in plant growth and development, seed quality control, pest control, plant breeding, ecological environment protection and the like. However, research into seed microorganisms has been overlooked in the past, and the development of related knowledge and techniques has also been relatively delayed. With the rapid development of high-throughput technology, microbiology and molecular biology in recent years, seed microorganism research has received extensive attention from researchers, primarily by measuring seed microorganism associations of their phenotypic effects on plants. Due to the small seeds of most plants, current scholars are primarily studying through a seed mix sample weighing model. However, more and more studies have shown significant individual differences in plants, which may be related to individual seed microorganisms. Thus, the research on the determination of single seed microorganisms is particularly important.
Based on the plant individual difference phenomenon, it has been demonstrated that there is a significant difference in single seed microorganisms. In order to reveal the influence mechanism of seed microorganisms on plant growth, development and the like, it is more significant to discover key active seed microorganisms and observe dynamic changes of the key active seed microorganisms in the plant growth and development process. However, current seed microbiological studies fail to better differentiate the activity of microorganisms, and lack analysis of their dynamic changes. Based on this, it is necessary to propose a method for detecting key active microorganisms of seeds and their dynamic changes, especially a microbiological assay method for individual seeds. This is of great importance for the investigation of seed microorganisms.
In view of this, the present invention has been made.
Disclosure of Invention
A first object of the present invention is to provide an in situ detection method of key active microorganisms of seeds, which is particularly focused on the detection of single seeds, so as to solve at least one of the above problems.
The second object of the invention is to provide the application of the detection method in the detection of the dynamic change of the microorganism of a single seed under biotic or abiotic stress.
The third object of the invention is to provide an in situ detection method for dynamic change of seed microorganism under abiotic stress.
In order to achieve the above object, the following technical solutions are proposed:
in a first aspect, the invention provides a method for in situ detection of a seed-critical active microorganism, comprising the steps of:
culturing the seeds with aseptic surfaces in an aseptic environment to obtain germinated seeds, collecting lateral roots or adventitious roots of single germinated seeds, extracting and detecting microbial DNA of the lateral roots or adventitious roots, and determining key active microorganisms of the single seeds according to detection results.
As a further technical scheme, the seeds are subjected to surface sterilization to obtain the seeds with aseptic surfaces.
As a further technical scheme, the sterilization method comprises the following steps: sterilizing with sodium hypochlorite.
As a further technical scheme, the number of lateral roots or adventitious roots of the single seed after germination is collected to be within 3.
As a further aspect, the detecting comprises gene sequencing.
As a further technical scheme, the seeds comprise rice seeds, wheat seeds, barley seeds or corn seeds.
In a second aspect, the invention provides application of the detection method in detection of dynamic changes of single seed microorganisms under biotic or abiotic stress.
In a third aspect, the invention provides an in-situ detection method for dynamic changes of seed microorganisms under abiotic stress, which comprises the steps of obtaining germinated seeds according to the detection method, determining key active microorganisms of single seeds, carrying out abiotic stress treatment on the germinated seeds, collecting lateral roots or adventitious roots of corresponding single plants after abiotic stress treatment, extracting and detecting microbial DNA of the lateral roots or adventitious roots, and determining dynamic changes of key active microorganisms of the single seeds according to detection results.
As a further technical scheme, the abiotic stress comprises water stress, salt stress, temperature stress or light stress.
As a further technical scheme, the water stress treatment method includes: culturing the germinated seeds in sterile soil with different water contents;
the method for salt stress treatment comprises the following steps: culturing the germinated seeds in sterile soil with different salt concentrations;
the temperature stress treatment method comprises the following steps: culturing the germinated seeds in sterile soil at different temperatures;
the method for light stress treatment comprises the following steps: the germinated seeds are cultured in sterile soil under different illumination times.
Compared with the prior art, the invention has the following beneficial effects:
the in-situ detection method of the key active microorganisms of the seeds is simple and convenient, can realize the detection of the key active microorganisms of the single seeds on the premise of not damaging the growth of plants, can analyze how the seed microorganisms mediate individual growth differences of the plants, can be further applied to the detection of the dynamic changes of the microorganisms of the individual plants under biotic stress or abiotic stress, can be widely applied to the research of the seed microorganisms of various plants, and has important significance for researching the seed microorganisms.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the DNA extraction results of single rice seeds of different varieties;
FIG. 2 shows the result of 1 lateral root DNA amplification of a single rice seed;
FIG. 3 shows the DNA extraction results of single wheat seeds of different varieties;
FIG. 4 shows DNA enrichment of single rice seeds under salt stress;
FIG. 5 shows DNA enrichment of single rice seeds under temperature stress;
FIG. 6 shows DNA enrichment of individual rice seeds at different humidity levels.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to embodiments and examples, but it will be understood by those skilled in the art that the following embodiments and examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The specific conditions are not specified, and the process is carried out according to conventional conditions or conditions suggested by manufacturers. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
In a first aspect, the invention provides a method for in situ detection of a seed-critical active microorganism, comprising the steps of:
culturing the seeds with aseptic surfaces in an aseptic environment to obtain germinated seeds, collecting lateral roots or adventitious roots of single germinated seeds, extracting and detecting microbial DNA of the lateral roots or adventitious roots, and determining key active microorganisms of the single seeds according to detection results.
The in-situ detection method of the key active microorganisms of the seeds is simple and convenient, can realize the detection of the key active microorganisms of the single seeds on the premise of not damaging the growth of plants, can analyze how the seed microorganisms mediate individual growth differences of the plants, can be further applied to the detection of the dynamic changes of the microorganisms of the individual plants under biotic stress or abiotic stress, can be widely applied to the research of the seed microorganisms of various plants, and has important significance for researching the seed microorganisms.
In some alternative embodiments, the seed is surface sterilized to obtain the surface sterile seed.
The invention avoids the influence of microorganisms on the surface of seeds through sterilization treatment.
The sterilization method in the present invention is not particularly limited, and methods well known to those skilled in the art may be employed. In some alternative embodiments, the method of sterilizing comprises: sterilizing with sodium hypochlorite.
In some alternative embodiments, the sterilization process may further include a step of detecting microorganisms on the surface of the seed, for example, the seed surface may be washed with sterile water, and the washed water may be applied to the culture medium to observe whether colonies are growing on the surface of the culture medium.
In some alternative embodiments, the culture medium employed includes, but is not limited to, LB medium, NB medium, MB medium, TSA medium or R2A medium, or other medium suitable for germination of plant seeds as would be known to one of skill in the art.
It should be noted that, in the invention, the germination time, temperature condition and humidity condition of the seeds can be different within a reasonable range, and the shearing length and position of the roots after the germination of the seeds can be different due to different plant varieties.
In some alternative embodiments, the collection of germinated individual seed has a number of lateral or adventitious roots within 3.
The lateral roots or adventitious roots of the germinated seeds are not suitable to be cut too much, so that the growth of plants is prevented from being damaged, and DNA extraction is ensured.
In some alternative embodiments, the detecting comprises gene sequencing. Identification of seed microorganisms is accomplished by sequencing.
The detection methods provided herein are applicable to all plant seeds, including, but not limited to, rice seeds, wheat seeds, barley seeds, or corn seeds, among other plant seeds in some alternative embodiments.
In a second aspect, the invention provides application of the detection method in detection of dynamic changes of single seed microorganisms under biotic or abiotic stress.
The in-situ detection method of the key active microorganisms of the seeds can realize the detection of the key active microorganisms of the seeds on the premise of not damaging the growth of plants, can analyze how the seed microorganisms mediate individual growth differences of the plants, and can be further applied to the detection of dynamic changes of the seed microorganisms under biotic stress or abiotic stress.
In a third aspect, the invention provides an in-situ detection method for dynamic changes of seed microorganisms under abiotic stress, which comprises the steps of obtaining germinated seeds according to the detection method, determining key active microorganisms of single seeds, carrying out abiotic stress treatment on the germinated seeds, collecting lateral roots or adventitious roots of corresponding single plants after abiotic stress treatment, extracting and detecting microbial DNA of the lateral roots or adventitious roots, and determining dynamic changes of key active microorganisms of the single seeds according to detection results.
The detection method is simple and convenient, is suitable for detecting dynamic changes of the seed microorganism under various abiotic stresses, and is accurate in detection.
In some alternative embodiments, the abiotic stress includes, but is not limited to, water stress (e.g., different soil humidity), salt stress, temperature stress, or light stress, or abiotic stress known to those of skill in the art.
In some alternative embodiments, the method of water stress treatment comprises: culturing the germinated seeds in sterile soil with different water contents;
the method for salt stress treatment comprises the following steps: culturing the germinated seeds in sterile soil with different salt concentrations;
the temperature stress treatment method comprises the following steps: culturing the germinated seeds in sterile soil at different temperatures;
the method for light stress treatment comprises the following steps: the germinated seeds are cultured in sterile soil under different illumination times.
The invention is further illustrated by the following specific examples, however, it should be understood that these examples are for the purpose of illustration only in greater detail and are not to be construed as limiting the invention in any way.
In the following examples or test examples, the soil sterilization method may be performed by high temperature, high pressure, gamma rays, or the like.
Example 1
An in situ detection method of key active microorganisms of plant seeds comprises the following steps:
in order to ensure the environmental microorganism interference factor, the seeds are surface sterilized, and all operations ensure no exogenous microorganism pollution. Sterilizing with 2% sodium hypochlorite for 5min, and washing the surface sterilized seeds with sterilized water for 5-10 times to ensure complete sodium hypochlorite washing. 100. Mu.L of the last washing water was spread on the bacterial medium and incubated at 25℃for 3d, with sterile colony growth as a standard for complete seed surface disinfection.
The sterilized seeds are gently placed into a sterile and breathable container by using sterilized forceps, wet sterilized filter paper is paved at the bottom of the container in advance, and the seeds are uniformly placed and a certain space is reserved between the two seeds. Subsequently, the container is covered with a lid having ventilation holes to prevent foreign contamination and to ensure wetting of the interior of the container. It was placed in a 25℃environment for 3 days for seed germination. After aseptic colony growth on an observation plate, opening a container in an ultra-clean bench, shearing one or two lateral roots of germinated seeds by using sterile forceps and scissors, collecting the sheared roots by using a sterile centrifuge tube, immediately placing the roots into liquid nitrogen for quick freezing, and preserving the roots in an ultra-low temperature refrigerator for extracting microorganism DNA, thereby representing key microorganisms during seed germination. Tweezers and scissors are thoroughly washed and sterilized by alcohol after each seed is used, so that cross contamination among seeds is avoided.
Test example 1
After the surface of single Nippon Ningqing, china No. 1, beijing rice No. 62, japonica rice 4119 and Pingyou No. three rice seeds were sterilized according to the detection method provided in example 1, 100 seeds were repeated for each variety. After complete surface sterilization, the germinated seed side roots were collected 1 (50 replicates) or 2 (50 replicates) after germination at a constant temperature of 25 ℃ for 3d under aseptic conditions. The above samples were subjected to a trace DNA extraction and DNA concentration and content determination (fig. 1). Amplification by bacterial high throughput sequencing commonly used V4 variable region primers (515F/806R) confirmed that the method of the invention successfully collected single seed microorganisms (fig. 2).
Test example 2
The rice variety of test example 1 was changed to a wheat variety of Harvest 366, zhengmai 161, sumai 8, jimai 22, yunmai 3, and other methods were identical. The results show that the invention is equally applicable to single wheat seed microorganisms (fig. 3).
Example 2
An in-situ detection method for dynamic change of seed microorganisms of single rice seeds under salt stress comprises the following steps:
the pretreatment was consistent with the key active microorganism detection method for seeds of example 1, and when rice seeds germinated and a part of the roots was cut off, the part of the roots was marked as the seed microorganism before treatment. The partially root deleted germinated seeds were gently transferred to pre-sterilized artificial soil. The soil contains NaCl as salt stress at different concentrations, mainly comprising 0-400mM. The specific method for preparing the gradient concentration soil comprises the following steps: firstly, the soil is treated for 3 hours at a high temperature of 280 ℃, on one hand, the water is removed to reach constant weight, and on the other hand, the soil microorganisms are thoroughly removed. The soil was aliquoted into sterile tissue culture flasks and all treated soil was made to have a water content of 70% by calculation with the addition of sterile water, or NaCl solution at each concentration and with a film (0.22 μm). After the seeds are transferred to soil uniformly and with a sufficient distance, the seeds are placed in a 16/8h day-night illumination alternate greenhouse for culture. After 7d, the rice seedlings are gently taken out together with roots, the soil on the surfaces of the roots is washed off by clean water, after the water is absorbed, the roots are sheared by sterile scissors, the sheared roots are quickly frozen by liquid nitrogen, then the cut roots are preserved at ultralow temperature until microbial DNA extraction is carried out, and the concentration and the content of DNA are recorded (figure 4), wherein 100 seeds are treated as repetition. The result shows that the invention is suitable for the microorganism dynamic change detection of single seeds. Based on example 2, the results show that the invention can successfully extract microbial DNA of a single seed before and after salt stress.
Example 3
An in-situ detection method for dynamic change of seed microorganisms of single rice seeds under temperature stress comprises the following steps:
the pretreatment was consistent with the key active microorganism detection method for seeds of example 1, and when rice seeds germinated and a part of the roots was cut off, the part of the roots was marked as the seed microorganism before treatment. Transferring part of the germination seeds with the missing roots into sterile soil, wherein the water content of the soil is 70%, and respectively culturing for 7d under the conditions of 4, 15, 20, 30 and 40 ℃ in a light-dark mode for 16/8h alternately, wherein 100 seeds are treated as repetition. Subsequently, as in example 2, the seed side roots of each treatment were collected and subjected to DNA extraction, respectively. The results show that the invention can perform DNA extraction of single rice seeds under temperature stress (FIG. 5).
Example 4
An in-situ detection method for dynamic change of seed microorganisms of single rice seeds under different humidity of soil comprises the following steps:
the pretreatment was consistent with the key active microorganism detection method for seeds of example 1, and when rice seeds germinated and a part of the roots was cut off, the part of the roots was marked as the seed microorganism before treatment. The germinated seeds with partial root loss were transferred to sterile soil with water contents of 10%,20%,40%,70% and 100% respectively, 100 seeds per treatment were repeated, and the remaining conditions were the same as in example 2. The seed side roots of each treatment were collected and subjected to DNA extraction, respectively. The results show that the invention is applicable to the microbial dynamic detection of single seeds under drought stress (figure 6).
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (9)
1. An in situ detection method of a key active microorganism of seeds is characterized by comprising the following steps:
culturing the seeds with aseptic surfaces in an aseptic environment to obtain germinated seeds, collecting lateral roots or adventitious roots of single germinated seeds, extracting and detecting microbial DNA of the lateral roots or adventitious roots, and determining key active microorganisms of the single seeds according to detection results.
2. The method of claim 1, wherein the seeds are surface sterilized to obtain the surface sterile seeds.
3. The method according to claim 1, wherein the number of lateral roots or adventitious roots of the single seed after germination is within 3.
4. The method of detection according to claim 1, wherein the detection comprises gene sequencing.
5. The method of claim 1, wherein the seed comprises rice seed, wheat seed, barley seed, or corn seed.
6. Use of the detection method according to any one of claims 1 to 5 for the detection of dynamic changes in single seed microorganisms under biotic or abiotic stress.
7. An in-situ detection method for dynamic changes of seed microorganisms under abiotic stress is characterized in that germinated seeds are obtained and key active microorganisms of single seeds are determined according to the detection method described in any one of claims 1-5, then the germinated seeds are subjected to abiotic stress treatment, corresponding plant lateral roots or adventitious roots of the single plants after the abiotic stress treatment are collected, microbial DNA extraction and detection are carried out on the lateral roots or the adventitious roots, and then dynamic changes of key active microorganisms of the single seeds are determined according to detection results.
8. The detection method of claim 7, wherein the abiotic stress comprises water stress, salt stress, temperature stress, or light stress.
9. The method for detecting according to claim 8, wherein the method for water stress treatment comprises: culturing the germinated seeds in sterile soil with different water contents;
the method for salt stress treatment comprises the following steps: culturing the germinated seeds in sterile soil with different salt concentrations;
the temperature stress treatment method comprises the following steps: culturing the germinated seeds in sterile soil at different temperatures;
the method for light stress treatment comprises the following steps: the germinated seeds are cultured in sterile soil under different illumination times.
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