A method for preparing a culture medium for studying the effect of algal liquid exudates on the growth of arabidopsis

Abstract

The present application relates to a kind of culture medium preparation method for studying the influence of algal liquid secretion on arabidopsis growth, belong to the field of biotechnology.The method includes the preparation of solid medium, extraction of algal liquid secretion, preheating, four major steps of medium mixing.The liquid blue algae extracellular secretion is mixed into solid medium in specific method, the interference of microcystis aeruginosa liquid medium on experimental results is excluded by designing negative control, the "dose-effect" study of secretion to plant is carried out by adjusting the ratio of extracellular secretion and medium, the chemical stability of active component in extracellular secretion is inferred by designing stability experiment.The culture method designed in the application can be widely applied in other similar researches exploring the influence of microbial secretion on arabidopsis growth.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a method for preparing a culture medium for studying the influence of algal liquid exudates on the growth of Arabidopsis thaliana and a method for studying the influence of algal liquid exudates on the growth of Arabidopsis thaliana using the culture medium. BACKGROUND

[0002] Microorganisms in the environment have a great influence on the growth and development of plants, mainly depending on the exudates produced during their growth, which are mostly liquid and complex in composition. At present, most of the researches on the influence of the extracellular exudates of Microcys tis aeruginosa (MaE) produced by Microcys tis aeruginosa belonging to Cyanophyta in the logarithmic growth phase on plant growth need to directly expose the plants to the exudates (for example, Zheng Guilei. 2010. The allelopathy and its mechanism between Microcys tis aeruginosa and Potamogenton malaianus, Ceratophyllum demersum [D]. Yunnan University.). However, this experimental system has many problems. First, since the individual of higher plants is large, directly exposing them to the exudates requires a large amount of MaE, especially when carrying out dose-effect experiments, the demand for exudates and experimental space is higher. This system is extremely wasteful of resources, and the experimental results obtained are relatively unstable, which is not conducive to quickly and effectively obtaining experimental data. For example, using water culture method to plant Potamogenton malaianus, about 3-5 liters of water culture solution and 1-3 liters of MaE are needed for one concentration treatment experiment (Xue Sailian. Water culture method: CN201611170344.2[P]. 2017-05-10.). Second, the plants used in the exposure experiment are mostly not model organisms, which are not conducive to carrying out related mechanism research at the gene and protein levels. Finally, the algal culture medium contained in MaE is easy to interfere with the experimental results. Effectively solving the above problems will help to efficiently and accurately observe the effect of MaE on plants, and is conducive to analyzing the related mechanism of producing the effect from the omics level.

[0003] Arabidopsis thaliana, as a recognized model plant, has the advantages of small individual plant, simple morphological characteristics, short growth and development cycle, etc. (Lavenus J, Goh T, Roberts I, et al. 2013. Lateral Root Development in Arabidopsis: Fifty Shades of Auxin [J]. Trends in Plant Science; Ma Junjie, Guo Fengdan, Wang Xingjun, et al. 2019. Auxin synthesis, transport and signal transduction regulate early embryonic development of plants [J]. Acta Physiologiae Plantarum, 55(05): 547-557.). At present, researchers have conducted in-depth research on the molecular level of Arabidopsis genes and proteins, and have obtained complete gene bank data and various mutants for studying the mechanism of growth and development. Therefore, Arabidopsis has great potential for use in the study of the effects of compounds on plant growth and development, and is an ideal material for studying the effects of MaE on plants and their mechanisms. In addition, due to the small size of Arabidopsis, less MaE is required for exposure experiments. For example, a concentration treatment experiment requires only 0.1-0.3 liters of MaE, and an experimental period of 14 days can be completed, obtaining a large amount of parallel data, which is very conducive to obtaining stable experimental results.

[0004] At present, the liquid compound effect research based on Arabidopsis is often carried out by directly irrigating, spraying, etc. Compound is sprayed to Arabidopsis seedlings planted in soil medium, which changes the bioavailability of the effect component in liquid microbial exudates, and further affects the experiment. For example, in the patent of Zhang Huijuan and Jiang Ming for inducing Arabidopsis resistance to gray mold by azelaic acid, azelaic acid aqueous solution is used to treat Arabidopsis by root irrigation and spraying (Zhang Huijuan, Jiang Ming. Application and method for inducing Arabidopsis thaliana resistance to gray mold by azelaic acid: CN202010393262.4 [P]. 2021-11-19.). Although research based on Arabidopsis can use solid medium. For example, Fu Shu, Yang Guang, Liu Zhaoxia, et al. invented a culture medium for promoting rape rooting (Fu Shu, Yang Guang, Liu Zhaoxia, et al. A culture medium for promoting rape rooting: CN201810874255.9 [P]. 2018-11-20.) and Liu Handong invented a method for inducing Arabidopsis callus (Liu Handong. Method for efficiently inducing Columbia-type Arabidopsis thaliana callus and induction medium: CN201510824482.7 [P]. 2018-05-29.). However, the above methods using solid medium and liquid compounds are difficult to observe the changes of Arabidopsis plant phenotype in early growth and development, especially the changes of root development. Therefore, how to overcome the shortcomings of the prior art is a problem that needs to be solved in the field of biotechnology. SUMMARY

[0005] The present application aims at providing a preparation method of a culture medium for studying the influence of liquid secretion of algae on the growth of Arabidopsis thaliana, and using the culture medium to study the influence of liquid secretion of algae on the growth of Arabidopsis thaliana.

[0006] To achieve the above object, the technical scheme adopted by the present application is as follows:

[0007] The present application provides a preparation method of a culture medium for studying the influence of liquid secretion of algae on the growth of Arabidopsis thaliana, comprising the following steps:

[0008] Step (1), preparation of solid culture medium: prepare a solid culture medium with a solute concentration of 5 times of 1 / 2MS solid culture medium, and adjust the pH to 5.8;

[0009] Step (2), extraction of liquid secretion of algae: centrifuge the algae liquid obtained by culture at 4℃, use a 0.45μm glass fiber filter membrane to extract the supernatant obtained by centrifugation, and then filter sterilization to obtain liquid secretion of algae; the concentration of the liquid secretion of algae is 1×10 7 ~ 2×10 7 cells / ml;

[0010] Step (3), preheating: preheat the solid culture medium obtained in step (1) and the liquid secretion of algae obtained in step (2) respectively;

[0011] If the liquid secretion of algae needs to be diluted, the culture medium for culturing the algae liquid in step (2) is used to dilute to the target concentration, and then preheating is performed;

[0012] The preheating temperature is not higher than 60℃, and the preheating time is less than 1 hour;

[0013] After preheating, the temperature of the liquid secretion of algae should be higher than that of the solid culture medium;

[0014] The preheated solid culture medium should be in a liquid state;

[0015] Step (4), mixing of the culture medium:

[0016] The preheated solid culture medium is added to the preheated diluted or undiluted liquid secretion of algae, and after mixing uniformly, standing, the culture medium for studying the influence of liquid secretion of microorganisms on the growth of Arabidopsis thaliana is obtained;

[0017] Preferably, in step (2), the liquid secretion of algae is extracellular secretion of blue algae.

[0018] Further, preferably, in step (2), the liquid secretion of algae is extracellular secretion of blue algae.

[0019] Further, preferably, in step (2), the algal liquid secretion is Microcystis aeruginosa extracellular secretion.

[0020] The Microcystis aeruginosa is cultured at 25±1°C, the algal liquid is shaken 2-3 times per day, and the algal liquid is transferred to new HGZ-145 medium every 3-5 days.

[0021] The algal liquid is centrifuged at 6000 rpm for 10 minutes at 4°C, and then the supernatant is taken, the supernatant obtained by centrifugation is filtered by using a 0.45 μm glass fiber filter, and then the bacteria are filtered and sterilized by using a 0.22 μm pore size membrane filter, to obtain the algal liquid secretion.

[0022] Further, preferably, in step (2), the light-dark cycle ratio is 12l:12d during the culture of Microcystis aeruginosa, and the extraction of the algal liquid secretion is performed after the algal liquid is transferred more than three times.

[0023] Further, preferably, the HGZ-145 medium is sterilized in a 121°C high-temperature high-pressure sterilization pot for 30 minutes before use.

[0024] Further, preferably, in step (3), the HGZ-145 medium is sterilized in a 121°C high-temperature high-pressure sterilization pot for 30 minutes, and then the sterilized HGZ-145 medium is used to dilute the algal liquid secretion.

[0025] A method for studying the effect of algal liquid secretion on the growth of Arabidopsis thaliana, using the culture medium for studying the effect of algal liquid secretion on the growth of Arabidopsis thaliana prepared by the above-mentioned method for preparing a culture medium for studying the effect of algal liquid secretion on the growth of Arabidopsis thaliana, comprising the following steps:

[0026] In a sterile environment, first wash the Arabidopsis thaliana seeds with sterilized water, then wash them several times with sterile water, and then evenly place the Arabidopsis thaliana seeds in the culture medium, seal and place in a 4°C environment for vernalization, and then place in a culture box for light incubation after two days, observe the phenotype of the light incubated Arabidopsis thaliana, and analyze it, to obtain the results of the study of the effect of algal liquid secretion on the growth of Arabidopsis thaliana.

[0027] Further, preferably, the Arabidopsis thaliana seeds are washed with sterile water 3 times, and the incubation temperature is constant at 22°C, and the light-dark cycle ratio is 16l:8d.

[0028] Further, preferably, the study of the effect of algal liquid secretion on the growth of Arabidopsis thaliana includes dose-effect relationship study of the effect of algal liquid secretion on Arabidopsis thaliana, effect time stability study of the effect of algal liquid secretion on Arabidopsis thaliana, and effect heat stability study of the effect of algal liquid secretion on Arabidopsis thaliana.

[0029] The solutes and their concentrations of the 1 / 2MS solid medium without sugar and agar are as follows: potassium nitrate 950 mg / L, ammonium nitrate 825 mg / L, potassium dihydrogen phosphate 85 mg / L, magnesium sulfate 185 mg / L, calcium chloride 220 mg / L, potassium iodide 0.415 mg / L, boric acid 3.1 mg / L, manganese sulfate 11.15 mg / L, zinc sulfate 4.3 mg / L, sodium molybdate 0.125 mg / L, copper sulfate 0.0125 mg / L, cobalt chloride 0.0125 mg / L, disodium ethylenediaminetetraacetate 18.625 mg / L, ferrous sulfate 13.925 mg / L, myo-inositol 50 mg / L, glycine 1 mg / L, thiamine hydrochloride 0.05 mg / L, pyridoxal hydrochloride 0.25 mg / L, and nicotinic acid 0.25 mg / L; and the pH is 6.0.

[0030] In step (1), the solid medium of Arabidopsis thaliana is prepared at 5 times the actual use concentration, and the concentrations of agar and sucrose are also 5 times the actual concentrations (both 50 g / L), and higher than 5 times is not conducive to keeping the medium in a liquid state after high-temperature sterilization.

[0031] In step (2), in order to ensure that the algal liquid secretion is not inactivated or denatured under the condition of high temperature and high pressure sterilization, the algal liquid secretion needs to be filtered and sterilized in a sterile operation environment using a sterile syringe and a 0.22 μm liquid phase filter head.

[0032] Preferably, in step (2), a 0.45 μm glass fiber filter membrane is used for filtering, and a 0.22 μm pore size membrane filter is used for sterilization.

[0033] In step (3), the preheating time needs to be less than 1 hour, and the temperature needs to be controlled within 60°C, and too long holding time and too high temperature may cause denaturation; the present application does not limit the dilution multiple of step (3), and those skilled in the art can dilute according to actual needs; those skilled in the art should know that the concentration of the diluted algal liquid secretion ranges from 0% to 100%, note: 100% means undiluted.

[0034] In step (3), the medium for culturing algal liquid of step (2) should be sterilized, and the algal liquid secretion should be diluted in proportion, because the medium for culturing algal liquid of step (2) is the medium for culturing algal liquid, and the algal liquid secretion contains components of the medium, so deionized water cannot be used to dilute the algal liquid secretion. The diluted algal liquid secretion can be first placed in an oven for holding, so that its temperature is not too low to affect the uniformity of the components, and then taken out of the oven for use. Note: after dilution, the final concentration of MaE in the medium should not be higher than 80%, otherwise it is easy to cause uneven mixing of the medium and uneven solidification of the medium.

[0035] In step (4), when mixing, the solid medium should be poured into the algal liquid secretion, not the other way around, to prevent the agar component in the solid medium from solidifying when it is cooled, resulting in uneven mixing and affecting the experimental results.

[0036] Steps (1) to (4) of the present application all need to be performed in a clean bench.

[0037] In the present application, to avoid the loss of temperature of the algal liquid secretion and the solid medium due to long-time operation in the clean bench, the time of the above-mentioned materials in the clean bench should be minimized, and the mixing should be done in time (preferably within 3 to 5 minutes to mix the materials and complete the pouring of the plates); to avoid the loss of temperature of the solution due to too low temperature of the weighing container during the process of weighing the volume of the solution, a plastic weighing container or a glass weighing container that is heated in advance should be used; the operator should wear sterilized gloves to avoid being scalded during the mixing of the medium.

[0038] By adjusting the proportions of the solid medium, the algal liquid secretion, deionized water, and the medium, the "dose-effect" relationship of the algal liquid secretion on plants can be studied.

[0039] Key points: When mixing the algal liquid secretion and the solid medium, the medium for culturing the algal liquid in step (2) that has been sterilized is used to dilute the algal liquid secretion in proportion (as shown in Table 1), and then the solid medium is mixed with the diluted liquid in a ratio of 1:4 to obtain the medium with different dilution degrees.

[0040] Table 1: Volume of each component for diluting the algal liquid secretion with the medium for culturing the algal liquid

[0041]

[0042] When studying the influence of the microbial liquid secretion on the growth of Arabidopsis thaliana, a reasonable control group can be designed to exclude the interference of the medium for culturing the algal liquid in step (2) (i.e., HGZ-145 medium) on the experimental results.

[0043] Key points: To exclude the interference of the medium for culturing the algal liquid in step (2) on the experimental results, deionized water should be used as the negative control and compared with the treatment group with the medium for culturing the algal liquid in step (2) added (as shown in Table 2). By comparing the control group with different total amounts of the medium for culturing the algal liquid in step (2) added with the treatment group with the algal liquid secretion added, it can be explored whether the medium for culturing the algal liquid in step (2) has an impact on the growth and development of Arabidopsis thaliana.

[0044] Table 2: Volume of each component in the negative control (algal liquid medium control) of each gradient treatment of the algal liquid secretion concentration

[0045] Algal liquid exudate concentration gradient Algal liquid exudate Culture medium for step (2) algal liquid culture Solid medium Deionized water 80％ - - 20ml 80ml 25％ - 25ml 20ml 55ml 8％ - 8ml 20ml 72ml 2.5％ - 2.5ml 20ml 77.5ml 0.8％ - 0.8ml 20ml 79.2ml

[0046] Designing the stability experiment of liquid exudate of algae, laying the foundation for the separation and identification of effectors in the exudate in the later stage.

[0047] Carrying out the time stability and thermal stability experiments of liquid exudate of algae, observing whether the liquid exudate of algae after the above treatment can still produce effects on Arabidopsis thaliana, and further inferring the possible types of effect compounds in the liquid exudate of algae. For example, poor time stability (lose effect after being stored at-20 DEG C for one month) indicates that the stability of the effect compound is insufficient, and long-term storage cannot be achieved by freezing; poor thermal stability (lose effect after being heated at 60 DEG C for 6 hours) indicates that the compound will degrade, denature or evaporate at high temperature, which may be volatile substances or protein and polypeptide compounds.

[0048] Point 1: Time stability experiment. The liquid exudate of algae is stored in a refrigerator at-20 DEG C for one month under sterile conditions, and then it is observed whether the treated liquid exudate of algae can still produce the same effect on Arabidopsis thaliana as the untreated liquid exudate of algae. If the effect is lost, it indicates that the compound producing the effect in the exudate is prone to hydrolysis or decomposition in water, and cannot be stored for a long time.

[0049] Point 2: Thermal stability experiment. The liquid exudate of algae is heated at 60 DEG C for 6 hours under sterile conditions (the temperature is a commonly used temperature and treatment time for concentrating natural compounds in conventional natural compound effector research), and then it is observed whether the treated liquid exudate of algae can still produce the same effect on Arabidopsis thaliana as the untreated liquid exudate of algae. If the effect is lost, it indicates that the compound producing the effect in the exudate will degrade, denature or evaporate at high temperature, which may be volatile substances or protein and polypeptide compounds, and cannot be separated by conventional natural product separation technology route.

[0050] In the present application, the liquid extracellular exudate of blue-green algae is mixed into a solid culture medium in a specific method, the negative control is designed to exclude the interference of Microcystis aeruginosa liquid culture medium on the experimental results, the ratio of extracellular exudate and culture medium is adjusted to carry out the "dose-effect" study of the effect of exudate on plants, and the stability experiment is designed to infer the chemical stability of the active component in the extracellular exudate. The culture method designed in the present application can be widely applied to other similar researches on the influence of microbial exudate on the growth of Arabidopsis thaliana.

[0051] Compared with the prior art, the present application has the following beneficial effects:

[0052] The solid culture medium used in the preparation method is 1 / 2MS solid culture medium suitable for growth culture of Arabidopsis thaliana and complete in nutritional elements, and by adding the algal liquid exudate into the solid culture medium, the Arabidopsis thaliana seeds can grow directly in the solid culture medium containing the algal liquid exudate, so that researchers can study the effect of the algal liquid exudate on plant growth in the early growth stage of Arabidopsis thaliana, and provide strong guarantee for subsequent mechanism research.

[0053] Firstly, compared with the prior art, the concentration multiple of 1 / 2MS is changed to 5 times of the prior art, so that the solid forming effect is good after the algal liquid exudate is added under the premise that the content of the nutritional elements is unchanged and the types are complete, and further research on the influence of the algal liquid exudate on lateral root development of Arabidopsis thaliana is carried out. Secondly, compared with the prior art, the influence of the Microcystis aeruginosa culture medium on the experimental results is excluded through the negative control algal culture experiment. By comparing the lateral root data of Arabidopsis thaliana on the 7th day of light culture between the treatment groups with different contents of the Microcystis aeruginosa culture medium, and the lateral root data of Arabidopsis thaliana on the 7th day of light culture between the treatment group (Table 1) containing different concentrations of the extracellular exudate of the Microcystis aeruginosa and the treatment group (Table 2) containing the Microcystis aeruginosa, whether the Microcystis aeruginosa culture medium will affect the experimental results can be explored.

[0054] In summary, the influence of the algal liquid exudate on the development of Arabidopsis thaliana is explored by mixing the algal liquid exudate and the solid culture medium, the “dose-effect” research design provides an effective basis for ecological risk evaluation of the exudate, and the experimental design for exploring the stability of the algal liquid exudate provides important guidance for subsequent experiments for further separating the actual effective substances in the exudate. BRIEF DESCRIPTION OF DRAWINGS

[0055] Figure 1 It is a plant hormone dose-effect research result graph of the extracellular exudate of the Microcystis aeruginosa in application example 1, wherein (“blue-green algae exudate” represents the MaE group, “negative control” represents the negative control group, and “*” represents that the number of lateral roots of Arabidopsis thaliana in the MaE group and the corresponding negative control group is significantly different (p<0.05) under the treatment concentration, as shown by the students't test). DETAILED DESCRIPTION

[0056] The application will be further described in detail below in combination with examples.

[0057] Those skilled in the art will appreciate that the following examples are intended to be illustrative only and should not be viewed as limiting the scope of the present application. Unless otherwise indicated, the techniques and conditions used in the examples are as described in the literature documents or as described in the product manuals. Unless otherwise indicated, the materials or apparatuses used are conventional products available on the market.

[0058] Example 1

[0059] A method for preparing a culture medium for studying the effect of algal liquid exudates on the growth of Arabidopsis thaliana, comprising the following steps:

[0060] Step (1), preparation of solid culture medium: prepare a solid culture medium with a solute concentration of 5 times that of 1 / 2MS solid culture medium, and adjust the pH to 5.8;

[0061] Step (2), extraction of algal liquid exudates: centrifuge the algal liquid obtained by culture at 4°C, use a 0.45 μm glass fiber filter to suction filter the supernatant obtained by centrifugation, and then filter sterilize to obtain algal liquid exudates; the concentration of the algal liquid exudates is 1×10 7 cells / ml;

[0062] Step (3), preheating: preheat the solid culture medium obtained in step (1) and the algal liquid exudates obtained in step (2) respectively;

[0063] If the algal liquid exudates need to be diluted, the culture medium for culturing the algal liquid in step (2) is used to dilute to the target concentration, and then preheating is performed;

[0064] The preheating temperature is not higher than 60°C; the preheating time is less than 1 hour;

[0065] After preheating, the temperature of the algal liquid exudates should be higher than that of the solid culture medium;

[0066] The preheated solid culture medium should be in a liquid state;

[0067] Step (4), mixing of the culture medium:

[0068] The preheated solid culture medium is added to the preheated algal liquid exudates which are diluted or not diluted, and after uniform mixing, standing is performed, thereby obtaining a culture medium for studying the effect of microbial liquid exudates on the growth of Arabidopsis thaliana;

[0069] The volume ratio of the preheated solid culture medium to the preheated algal liquid exudates which are diluted or not diluted is 1:4.

[0070] A method for studying the influence of algal liquid secretion on the growth of Arabidopsis thaliana, using the culture medium for studying the influence of algal liquid secretion on the growth of Arabidopsis thaliana prepared by the preparation method of the culture medium for studying the influence of algal liquid secretion on the growth of Arabidopsis thaliana, comprising the following steps:

[0071] In a sterile environment, first wash the Arabidopsis thaliana seeds with sterilized water, then wash them several times with sterile water, then evenly place the Arabidopsis thaliana seeds in the culture medium, seal and place in a 4°C environment for vernalization, then place in a culture box for light cultivation after two days, observe the phenotype of the light cultivated Arabidopsis thaliana, and analyze it, thereby obtaining the results of the study of the influence of algal liquid secretion on the growth of Arabidopsis thaliana.

[0072] Example 2

[0073] A preparation method of a culture medium for studying the influence of algal liquid secretion on the growth of Arabidopsis thaliana, comprising the following steps:

[0074] Step (1), preparation of solid medium: prepare a solid medium with a solute concentration of 1 / 2MS solid medium 5 times, and adjust the pH to 5.8;

[0075] Step (2), extraction of algal liquid secretion: centrifuge the algal liquid obtained by culture at 4°C, then use a 0.45μm glass fiber filter to extract the supernatant obtained by centrifugation, then filter sterilize to obtain algal liquid secretion; the concentration of the algal liquid secretion is 2×10 7 cells / ml;

[0076] Step (3), preheating: preheat the solid medium obtained in step (1) and the algal liquid secretion obtained in step (2) respectively;

[0077] If the algal liquid secretion needs to be diluted, then use the culture medium of step (2) to dilute the algal liquid to the target concentration, and then preheat;

[0078] The preheating temperature is not higher than 60°C; the preheating time is less than 1 hour;

[0079] After preheating, the temperature of the algal liquid secretion should be higher than that of the solid medium;

[0080] The preheated solid medium should be in a liquid state;

[0081] Step (4), mixing of the culture medium:

[0082] Add the preheated solid medium to the preheated algal liquid secretion which is diluted or not diluted, mix evenly, and then stand still, thereby obtaining the culture medium for studying the influence of algal liquid secretion on the growth of Arabidopsis thaliana;

[0083] The volume ratio of the preheated solid culture medium to the preheated diluted or undiluted algal liquid secretion is 1:4.

[0084] In step (2), the algal liquid secretion is blue algae extracellular secretion.

[0085] In step (2), the algal liquid secretion is blue algae extracellular secretion.

[0086] The green microcystis is cultured at 25±1℃, and the algal liquid is shaken 2-3 times per day, and the algal liquid is transferred to new HGZ-145 culture medium every 3-5 days.

[0087] The algal liquid with a concentration of 2×10 7 The algal liquid with a concentration of 2×10

[0088] A method for studying the influence of algal liquid secretion on the growth of Arabidopsis thaliana, using the culture medium for studying the influence of algal liquid secretion on the growth of Arabidopsis thaliana prepared by the preparation method of the culture medium for studying the influence of algal liquid secretion on the growth of Arabidopsis thaliana, comprising the following steps:

[0089] In a sterile environment, first wash the Arabidopsis thaliana seeds with sterilized water, then wash them several times with sterile water, then evenly place the Arabidopsis thaliana seeds in the culture medium, seal and place in a 4℃ environment for vernalization, then place in a culture box for light incubation after two days, observe the phenotype of the light incubated Arabidopsis thaliana, and analyze it to obtain the results of the study of the influence of algal liquid secretion on the growth of Arabidopsis thaliana.

[0090] Wash 3 times with sterile water; the incubation temperature is constant at 22℃, and the light-dark cycle ratio is 16l:8d.

[0091] Example 3

[0092] A method for preparing a culture medium for studying the influence of algal liquid secretion on the growth of Arabidopsis thaliana, comprising the following steps:

[0093] Step (1), preparation of solid culture medium: prepare a solid culture medium with a solute concentration of 1 / 2MS solid culture medium 5 times, and adjust the pH to 5.8;

[0094] Step (2), extraction of the liquid algal secretion: centrifuge the algal liquid obtained by culture at 4℃, and then use a 0.45 μm glass fiber filter to extract the supernatant obtained by centrifugation, and then filter sterilization to obtain the liquid algal secretion; the concentration of the liquid algal secretion is 1.5 x 10 7 cells / ml;

[0095] Step (3), preheating: preheat the solid medium obtained in step (1) and the liquid algal secretion obtained in step (2) respectively;

[0096] If the liquid algal secretion needs to be diluted, the medium for culturing the algal liquid in step (2) is used to dilute it to the target concentration, and then preheating is performed;

[0097] The preheating temperature is not higher than 60℃, and the preheating time is less than 1 hour;

[0098] After preheating, the temperature of the liquid algal secretion should be higher than that of the solid medium;

[0099] The preheated solid medium should be in a liquid state;

[0100] Step (4), mixing of the medium:

[0101] After the preheated solid medium is added to the preheated diluted or undiluted liquid algal secretion, the mixture is mixed uniformly and then left to stand, thereby obtaining the medium for studying the effect of the liquid algal secretion on the growth of Arabidopsis thaliana;

[0102] The volume ratio of the preheated solid medium to the preheated diluted or undiluted liquid algal secretion is 1:4.

[0103] In step (2), the liquid algal secretion is an extracellular secretion of cyanobacteria.

[0104] In step (2), the liquid algal secretion is an extracellular secretion of Microcystis aeruginosa;

[0105] The Microcystis aeruginosa is cultured at 25±1℃ using HGZ-145 culture medium, the algal liquid is shaken 2-3 times a day, and the algal liquid is transferred to new HGZ-145 culture medium every 3-5 days;

[0106] Take the algal liquid cultured to a concentration of 2 x 10 7 cells / ml, centrifuge it at 6000 rpm for 10 minutes at 4℃, then take the supernatant, use a 0.45 μm glass fiber filter to extract the supernatant obtained by centrifugation, and then use a 0.22 μm pore size membrane filter to filter sterilization to obtain the liquid algal secretion.

[0107] In step (2), the light and dark cycle ratio is 12l:12d when cultivating Microcystis aeruginosa, and the algal liquid exudate is extracted after being transferred for more than three times.

[0108] The HGZ-145 culture medium is sterilized in a high-temperature and high-pressure sterilization pot at 121℃ for 30 minutes before use.

[0109] In step (3), the algal liquid exudate is diluted with the sterilized HGZ-145 culture medium after the HGZ-145 culture medium is sterilized in a high-temperature and high-pressure sterilization pot at 121℃ for 30 minutes.

[0110] A method for researching the influence of algal liquid exudate on the growth of Arabidopsis thaliana, which comprises the following steps:

[0111] In a sterile environment, the Arabidopsis thaliana seeds are first washed with sterilized water and then washed with sterile water for multiple times, and then the Arabidopsis thaliana seeds are uniformly placed in the culture medium, sealed and placed in a 4℃ environment for vernalization, and then placed in a culture box for light incubation after two days, the phenotype of the light incubated Arabidopsis thaliana is observed, and the influence of the algal liquid exudate on the growth of Arabidopsis thaliana is obtained by analysis.

[0112] The Arabidopsis thaliana seeds are washed with sterile water for 3 times, and the incubation temperature is constant at 22℃, and the light and dark cycle ratio is 16l:8d.

[0113] The research on the influence of algal liquid exudate on the growth of Arabidopsis thaliana includes the dose-effect relationship research of the effect of algal liquid exudate on Arabidopsis thaliana, the effect time stability research of the effect of algal liquid exudate on Arabidopsis thaliana, and the heat stability research of the effect of algal liquid exudate on Arabidopsis thaliana.

[0114] Comparative Example 1

[0115] The algal liquid exudate is directly added to the solid culture medium with a solute concentration of 1 / 2MS solid culture medium 5 times without preheating by using a traditional method. The method for researching the influence of algal liquid exudate on the growth of Arabidopsis thaliana is the same as that in Example 3.

[0116] Then, compared with Example 3, the number of lateral roots of Arabidopsis thaliana after 7 days of light is observed.

[0117] The amount of the liquid secretion of algae added in Comparative Example 1 is 80 ml, the amount of the preheated solid culture medium added is 20 ml, and the total volume is 100 ml; the amount of the liquid secretion of algae (including the diluted liquid secretion of algae) added in Example 3 is 80 ml, the amount of the preheated solid culture medium added is 20 ml, and the total is 100 ml, so that the concentration of the medium-related substances of Arabidopsis thaliana in the medium obtained by diluting the MS culture medium mother liquor (the solid culture medium with a solute concentration of 5 times the solid culture medium of 1 / 2MS) with the liquid secretion of algae is consistent with that of a typical 1 / 2MS Arabidopsis thaliana culture medium, and meanwhile, the instability of the experimental results caused by uneven mixing of the medium is avoided. The comparison results are shown in Table 3.

[0118] Table 3 Comparison of the number of lateral roots of Arabidopsis thaliana on the seventh day of illumination, the stability of the data, and the results of the significant difference test of ANOVA analysis in the medium prepared in Comparative Example 1 and the medium prepared in Example 3

[0119]

[0120] *“±” represents the plus or minus of the absolute value; “Mean” represents the average value; “S.D.” represents the standard deviation; “CV%” represents the coefficient of variation, i.e. CV% = S.D. / Mean. * indicates that there is a significant difference (p<0.05) in the number of lateral roots of Arabidopsis thaliana in Comparative Example 1 and Example 3 after ANOVA statistical comparison.

[0121] From the above results, compared with Comparative Example 1, the coefficient of variation of the lateral roots of Arabidopsis thaliana in Example 3 is 24.4%, which is significantly lower than that in Comparative Example 1, indicating that the operation method in Example 3 can effectively improve the stability of the experimental results. In addition, although the final dilution concentration of the algae secretion in Comparative Example 1 and Example 3 is both 80%, the number of lateral roots of Arabidopsis thaliana in Example 3 is significantly more than that in Comparative Example 1, which is consistent with the role of algae secretion on root development in the previous experiment. It indicates that Example 3 can better reflect the effect of medium additives on the root system of Arabidopsis thaliana.

[0122] The present application optimizes the optimal mixing method of the liquid secretion of algae and the solid culture medium, ensures the culture effect of the obtained medium, and lays a foundation for subsequent stable Arabidopsis thaliana growth and development phenotype data.

[0123] The excellent effects of the present application are produced by the synergistic effect of each step and parameter, for example, in Example (3), the 5 times concentrated 1 / 2MS solid culture medium prepared in step (1) has a pH value of 3.3-3.7, and if the pH value is not adjusted at this time, the solidification effect of the subsequent agar component will be affected, and the solid form of the culture dish will be affected;

[0124] The algal liquid in step (2) needs to be centrifuged before being filtered to obtain MaE. The centrifugation time and speed will affect the separation effect of algal cells and extracellular secretions. If the centrifuge speed is set too fast, the algal cells will be broken, and the intracellular secretions will flow out, resulting in impure MaE. If the centrifuge speed is set too low, the algal cells and extracellular secretions may not be completely separated, affecting the filtration effect (other algae need to consider different speeds when centrifuging, and the preferred speed is 5500-6500 rpm). At the same time, since MaE will be inactivated at 121°C high temperature sterilization, it must be filtered through a 0.45 μm pore size membrane filter before use to remove bacteria; the algal liquid needs to be activated 1-2 times before cultivation, that is, the algal liquid is transferred every 3-5 days; preheating of the mixed medium in this step is necessary, otherwise it will affect the uniformity of the mixed medium with the added MaE, HGZ, etc., resulting in uneven composition of the medium and affecting the experimental results.

[0125] Application Example 1 (Dose-effect study of plant hormones of extracellular secretions of Microcystis aeruginosa)

[0126] This example constructs an experimental system to explore the effects of cyanobacterial extracellular secretions (Extracellular Exudates of M. aeruginosa, MaE) on the growth and development of Arabidopsis thaliana and its mechanism, which excludes potential interference that may affect the experimental results during the experiment and is successfully used for the development of the experiment, obtaining relatively stable phenotype data of Arabidopsis thaliana. The construction process of the experimental system of the present application is as follows:

[0127] (1) The algae need to be activated and cultured before the experiment. In this example, the algae used are Microcystis aeruginosa, and HGZ-145 medium (Table 4) is used as the basic medium for Microcystis aeruginosa. The algae are cultured in an artificial climate chamber (temperature 25±1°C, light-dark ratio 12h:12h, light intensity level 1, carbon dioxide concentration 4799ppm), and the algal liquid is shaken 2-3 times a day during the cultivation process. The algal liquid is transferred every 3-5 days to ensure that it is in the logarithmic growth phase. When used, the concentration of algal liquid secretions is 2x10 7 cells / ml.

[0128] Table 4 Main components of HGZ medium

[0129] Macronutrients Content (g / L) Micronutrients Content (g / L) NaNO3 0.2 [HBO3] 0.00286 KNO3 0.051 MnCl2.4H2O 0.00181 K2HPO4 0.049 ZnSO4·7H2O 0.000222 Ca(NO3)2·4H2O 0.059 Na2Mo4.2H2O 0.000391 MgSO4.7H2O 0.075 [CuSO4·5H2O] 0.000079 Na2CO3 0.02 Fe-EDTA 0.000932 NH4Cl-EDTA 0.0391

[0130] Step (1), preparation of solid medium: prepare a solid medium with a solute concentration of 1 / 2MS solid medium 5 times, and adjust the pH to 5.8;

[0131] Step (2), extraction of the algal liquid secretion: centrifuge the algal liquid obtained by culture at 4℃, and then use a 0.45 μm glass fiber filter to extract the supernatant obtained by centrifugation, and then filter sterilization to obtain the algal liquid secretion; the concentration of the algal liquid secretion is 2×10 7 cells / ml;

[0132] The algal liquid secretion is the extracellular secretion MaE of cyanobacteria;

[0133] Step (3), preheating: preheat the solid culture medium obtained in step (1) and the algal liquid secretion obtained in step (2) respectively;

[0134] If the algal liquid secretion needs to be diluted, the culture medium of the algal liquid in step (2) is used to dilute it to the target concentration, and then preheating is performed;

[0135] The preheating temperature is not higher than 60℃, and the preheating time is less than 1 hour;

[0136] After preheating, the temperature of the algal liquid secretion should be higher than that of the solid culture medium;

[0137] The preheated solid culture medium should be in a liquid state;

[0138] Step (4), mixing of the culture medium:

[0139] After the preheated solid culture medium is added to the preheated algal liquid secretion which is diluted or not diluted, and mixed uniformly, it is left to stand, and the culture medium for studying the effect of the microbial liquid secretion on the growth of Arabidopsis thaliana is obtained;

[0140] Among them, the volume ratio of the preheated solid culture medium to the preheated algal liquid secretion which is diluted or not diluted is 1:4.

[0141] In step (2), the algal liquid secretion is the extracellular secretion of Microcystis aeruginosa;

[0142] The Microcystis aeruginosa is cultured at 25±1℃ using HGZ-145 culture medium, the algal liquid is shaken 2-3 times a day, and the algal liquid is transferred to new HGZ-145 culture medium every 3-5 days;

[0143] Take the algal liquid cultured to a concentration of 2×10 7 cells / ml, centrifuge it at 4℃ at a speed of 6000 rpm for 10 minutes, then take the supernatant, use a 0.45 μm glass fiber filter to extract the supernatant obtained by centrifugation, and then use a 0.22 μm pore size membrane filter to filter sterilization to obtain the algal liquid secretion.

[0144] In step (2), the light-dark cycle ratio of Microcystis aeruginosa is 12l:12d during culture; the algal liquid secretions are extracted after more than three transfers.

[0145] HGZ-145 culture medium was sterilized in a high-temperature autoclave at 121℃ for 30 minutes before use.

[0146] In step (3), after the HGZ-145 medium is sterilized in a high-temperature and high-pressure autoclave at 121°C for 30 minutes, the sterilized HGZ-145 medium is used to dilute the liquid secretions of algae.

[0147] In a sterile environment, Arabidopsis seeds were first rinsed with disinfectant, then washed multiple times with sterile water. The seeds were then evenly placed in a culture medium, sealed, and placed in a 4°C environment for vernalization. After two days, they were placed in an incubator for light cultivation (constant temperature 22°C, light cycle (day:night = 16:8)). The phenotypes of Arabidopsis on the fourth and seventh days after light cultivation were observed and analyzed to obtain the results of the study on the effect of algal liquid secretions on Arabidopsis growth.

[0148] (3) By using HGZ-145 medium to dilute MaE, dilutions containing different concentrations of MaE can be obtained. Different dilution gradients of medium can be obtained according to the method in (2) (Table 5), and the "dose-effect" relationship of MaE on plants can be studied.

[0149] Table 5. Volume of culture medium components added for different MaE dilution concentration treatment groups

[0150]

[0151] (4) Replace MaE with deionized water according to the same dilution ratio in (3), and add HGZ-145 medium with the corresponding dilution ratio to obtain deionized water negative control medium with different dilution gradients. Compare the experimental results of Arabidopsis thaliana cultured with MaE medium to explore whether HGZ-145 medium has a hormonal effect on the growth and development of Arabidopsis thaliana, so as to eliminate its interference with the experimental results.

[0152] Table 6. Culture medium composition and volume for HGZ negative control treatment groups at different dilutions.

[0153]

[0154] The results are as follows Figure 1 As shown, after 12 days of light exposure, the number of lateral roots in Arabidopsis thaliana plants treated with different concentrations of MaE showed a decreasing trend with decreasing MaE exposure concentration, and the number of lateral roots in the 80% MaE group was significantly higher than that in other groups (p<0.05).

[0155] The number of lateral roots in the root system of Arabidopsis thaliana in the negative control group made by replacing MaE with the same volume of HGZ-145 medium did not change significantly; the asterisk represents that the students't test shows that there is a significant difference in the number of lateral roots of Arabidopsis thaliana between the MaE treatment group and the corresponding negative control group (p<0.05).

[0156] Example 2 (Plant hormone effect time stability of Microcystis aeruginosa extracellular secretion)

[0157] This example explores the stability of the experimental system for exploring the influence of Microcystis aeruginosa extracellular secretion on the development of Arabidopsis thaliana after long-term freezing and thawing, eliminates potential interference that may affect the experimental results during the experiment, and is successfully used for the development of the experiment, obtaining relatively stable phenotype data of Arabidopsis thaliana. The experimental system is as follows:

[0158] (1) Before the experiment, the algae need to be cultured and activated, and the algae are preferably transferred more than three times before MaE extraction. The algae used in this example are Microcystis aeruginosa, and HGZ-145 is used as the basic culture medium for Microcystis aeruginosa. Microcystis aeruginosa is cultured in an artificial climate box (temperature 25±1℃, light-dark ratio 12h:12h, light intensity level 1, carbon dioxide concentration 4790ppm, during the culture process, the bottle is shaken once at a fixed point in the morning, noon and afternoon, to keep Microcystis aeruginosa in suspension growth in the culture medium. Every 3-5 days, the algae liquid is transferred to ensure that it is always in the logarithmic growth phase. When used, the concentration of algal liquid secretion is 2×10 7 cells / ml.

[0159] (2) MaE treatment before medium configuration

[0160] Before the filtration of MaE, the algae liquid needs to be centrifuged at 6000 rpm for 10 minutes at 4℃ in a centrifuge, and then filtered by using a 0.45 μm glass fiber filter membrane. The extracted MaE is filtered by using a 0.22 μm liquid filter head in a clean bench to remove bacteria. Most natural products cannot be stored for a long time, so the low-temperature storage method of conventional natural products is selected in this experiment, that is, frozen storage at -20℃. Because a large amount of MaE is needed for the separation and identification of the effector substance of MaE in the subsequent process, the amount of MaE required can be obtained by culturing and extracting MaE twice. Therefore, the storage time of MaE in this example is set to the time of extracting MaE twice, that is, 30 days. The volume of MaE liquid will increase after being frozen at -20℃, so glass containers cannot be used for storage. Ordinary plastic containers made of polyethylene have poor chemical stability and are easy to contaminate MaE. In order to ensure that the chemical composition of MaE is not affected by the material of the container during storage, polypropylene material cryotubes with excellent chemical stability are selected to store MaE. The sterilized MaE is divided into 50 ml cryotubes, and then stored in a -20℃ refrigerator for 30 days after division.

[0161] (3) Preparation of a culture medium for studying the effect of algal liquid exudates on the growth of Arabidopsis thaliana

[0162] Step (1), preparation of a solid culture medium: a solid culture medium with a solute concentration of 5 times that of a 1 / 2MS solid culture medium is prepared, and the pH is adjusted to 5.8;

[0163] Step (2), extraction of algal liquid exudates: the algae liquid obtained by culturing is centrifuged at 4℃, and the supernatant obtained by centrifugation is filtered by using a 0.45 μm glass fiber filter membrane, and then filtered to remove bacteria to obtain algal liquid exudates; the concentration of the algal liquid exudates is 2×10 7 cells / ml;

[0164] The algal liquid exudates are cyanobacterial extracellular exudates MaE;

[0165] Step (3), preheating: the solid culture medium obtained in step (1) and the algal liquid exudates obtained in step (2) are preheated respectively;

[0166] If the algal liquid exudates need to be diluted, the culture medium of the algae liquid in step (2) is used to dilute the algal liquid exudates to the target concentration (in this example, the dilution degree is 80%, that is, diluted to 1.6×10 7 cells / ml), and then preheated;

[0167] The preheating temperature is not higher than 60℃, and the preheating time is less than 1 hour;

[0168] After preheating, the temperature of the algal liquid exudates should be higher than that of the solid culture medium;

[0169] The preheated solid medium should be in liquid state;

[0170] Step (4), mixing of the medium:

[0171] The preheated solid medium is added to the preheated diluted or undiluted algal liquid secretion, and after being mixed uniformly, the medium for studying the influence of microbial liquid secretion on the growth of Arabidopsis thaliana is obtained by standing.

[0172] The volume ratio of the preheated solid medium to the preheated diluted or undiluted algal liquid secretion is 1:4.

[0173] In step (2), the algal liquid secretion is extracellular secretion of Microcystis aeruginosa.

[0174] The Microcystis aeruginosa is cultured at 25±1℃, and the algal liquid is shaken 2-3 times a day. The algal liquid is transferred to new HGZ-145 medium every 3-5 days.

[0175] The algal liquid cultured to a concentration of 2×10 7 The algal liquid cultured to a concentration of 2×10

[0176] In step (2), the light-dark cycle ratio of the Microcystis aeruginosa culture is 12l:12d. The algal liquid secretion is extracted after the algal liquid is transferred more than three times.

[0177] The HGZ-145 medium is sterilized in a 121℃ high-temperature high-pressure sterilization pot for 30 minutes before use.

[0178] In step (3), the algal liquid secretion is diluted using the sterilized HGZ-145 medium after the HGZ-145 medium is sterilized in a 121℃ high-temperature high-pressure sterilization pot for 30 minutes.

[0179] In a sterile environment, the Arabidopsis thaliana seeds are first washed with sterilized water and then washed several times with sterile water. The Arabidopsis thaliana seeds are uniformly placed in the medium, sealed and placed in a 4℃ environment for vernalization (this operation can make the growth of each plant in the culture dish consistent, and also helps to break seed dormancy and promote growth.). After two days, the seeds are placed in a culture box for light incubation (constant temperature of 22℃, light cycle (day: night = 16:8)). The phenotype of Arabidopsis thaliana on the fifth day after light incubation is observed and analyzed, and the influence of algal liquid secretion on the growth of Arabidopsis thaliana is obtained.

[0180] In step (3) of the experiment, the algae liquid exudates used for preheating were long-term cryopreserved MaE and fresh MaE, which were respectively prepared into culture medium for comparison.

[0181] (4) The observed phenotype data of Arabidopsis thaliana on the seventh day after illumination were collated, and the results of the experiment in which Arabidopsis thaliana was cultured with culture medium containing long-term cryopreserved MaE and fresh MaE were compared to prepare a table (Table 7), and finally the conclusion was drawn that the effective compound composition of MaE did not change after long-term cryopreservation, and MaE can be cryopreserved for a long time after preparation.

[0182] Table 7 Comparison of the number of lateral root primordia and the length of the main root of Arabidopsis thaliana cultured with fresh MaE and time-stable MaE on the seventh day of illumination and significant comparison

[0183]

[0184] Application Example 3 (Thermal stability of the plant hormone effect of extracellular exudates of Microcystis aeruginosa)

[0185] The present example explores the stability of the influence of extracellular exudates of Microcystis aeruginosa on the development of Arabidopsis thaliana after long-term heating, eliminates potential interference that may affect the experimental results during the experiment, and is successfully used for the development of the experiment, and relatively stable phenotype data of Arabidopsis thaliana are obtained. The experimental system of the present application is constructed in the following steps:

[0186] (1) Before the experiment, the algae should be cultured and activated, and the algae should be transferred for more than three times before the extraction of MaE. In the present example, the algae used is Microcystis aeruginosa, and HGZ-145 is used as the basic culture medium for Microcystis aeruginosa. The Microcystis aeruginosa is cultured in an artificial climate chamber (temperature 25±1℃, light-dark ratio 12h:12h, light intensity level 1, carbon dioxide concentration 4790ppm, during the culture process, the bottle is shaken at fixed points in the morning, noon and afternoon once, to keep the Microcystis aeruginosa in suspension growth in the culture medium. The algae liquid is transferred every 3-5 days to ensure that it is always in the logarithmic growth phase. When used, the concentration of the algae liquid exudates is 2×10 7 cells / ml.

[0187] (2) Treatment of MaE before preparation of the culture medium

[0188] The algae liquid needs to be centrifuged at 6000 rpm for 10 minutes at 4°C before being filtered by 0.45 μm glass fiber filter. MaE may contain volatile organic compounds, and the subsequent separation and identification of the effector in MaE may involve rotary evaporation concentration of MaE, which may affect the composition of MaE. Therefore, MaE needs to be heated for pretreatment. The heating temperature and heating time for pretreatment are selected as the conventional heating pretreatment conditions for natural products, i.e. 60°C for 6 hours. Long-time heating can make volatile organic compounds completely volatilize. The extracted MaE is divided into 250 ml conical flasks and placed in a constant temperature heating water bath at 60°C for 6 hours. Water bath heating ensures that MaE is heated at a constant temperature and is evenly heated. The heated MaE needs to be sterilized by 0.22 μm liquid phase filter in a clean bench.

[0189] (3) Preparation of culture medium for studying the effect of algae liquid secretion on the growth of Arabidopsis thaliana

[0190] Step (1), preparation of solid medium: prepare a solid medium with a solute concentration of 5 times the 1 / 2MS solid medium, and adjust the pH to 5.8;

[0191] Step (2), extraction of algae liquid secretion: centrifuge the algae liquid obtained by culture at 4°C, and then filter the supernatant obtained by centrifugation by 0.45 μm glass fiber filter, and then filter sterilization to obtain algae liquid secretion; the concentration of algae liquid secretion is 2×10 7 cells / ml.

[0192] The algae liquid secretion is blue algae extracellular secretion MaE;

[0193] Step (3), preheating: preheat the solid medium obtained in step (1) and the algae liquid secretion obtained in step (2) respectively;

[0194] If the algae liquid secretion needs to be diluted, the culture medium of step (2) is used to dilute the algae liquid to the target concentration (in this example, the dilution is 80%, i.e. diluted to 1.6×10 7 cells / ml), and then preheated;

[0195] The preheating temperature is not higher than 60°C; the preheating time is less than 1 hour;

[0196] After preheating, the temperature of the algae liquid secretion should be higher than that of the solid medium;

[0197] The preheated solid medium should be in a liquid state;

[0198] Step (4), mixing of the medium:

[0199] The preheated solid medium is added to the preheated diluted or undiluted algal liquid secretion, mixed uniformly, and then left to stand, thereby obtaining a medium for studying the effect of microbial liquid secretion on the growth of Arabidopsis thaliana.

[0200] The volume ratio of the preheated solid medium to the preheated diluted or undiluted algal liquid secretion is 1:4.

[0201] In step (2), the algal liquid secretion is extracellular secretion of Microcystis aeruginosa.

[0202] The M. aeruginosa is cultured at 25±1℃, and the algal liquid is shaken 2-3 times a day. The algal liquid is transferred to new HGZ-145 medium every 3-5 days.

[0203] The algal liquid is cultured to a concentration of 2×10 7 The algal liquid is cultured to a concentration of 2×10

[0204] In step (2), the light-dark cycle ratio is 12l:12d when the M. aeruginosa is cultured, and the extraction of algal liquid secretion is performed after the algal liquid is transferred more than three times.

[0205] The HGZ-145 medium is sterilized in a 121℃ high-temperature high-pressure sterilization pot for 30 minutes before use.

[0206] In step (3), the algal liquid secretion is diluted using the sterilized HGZ-145 medium after the HGZ-145 medium is sterilized in a 121℃ high-temperature high-pressure sterilization pot for 30 minutes.

[0207] In a sterile environment, the Arabidopsis thaliana seeds are first washed with sterilized water and then washed several times with sterile water. The seeds are then uniformly placed in the medium, sealed, and placed in a 4℃ environment for vernalization (this operation can make the growth of each plant in the culture dish consistent, and also helps to break seed dormancy and promote growth). After two days, the seeds are placed in a culture box for light incubation (light intensity level 1, carbon dioxide concentration 4790ppm). The phenotype of Arabidopsis thaliana on the fifth day after light incubation is observed and analyzed, thereby obtaining the results of the effect of algal liquid secretion on the growth of Arabidopsis thaliana.

[0208] In step (3) of the experiment, the preheated algal liquid secretion is MaE heated for a long time and fresh MaE, which are used to prepare the medium for comparison.

[0209] (4) collate the observed phenotype data of Arabidopsis thaliana on the seventh day after illumination, and compare the experimental results of cultivating Arabidopsis thaliana with the medium containing MaE after long-time heating and fresh MaE, tabulate (as shown in Table 8), and finally conclude that the effect compounds of MaE are degraded, denatured or evaporated after long-time heating, and MaE may contain volatile substances or protein and polypeptide compounds.

[0210] Table 8 Comparison of the number of lateral root primordia and the length of the main root of Arabidopsis thaliana cultivated by the fresh MaE group and the heat stability group on the seventh day of illumination and the significance comparison

[0211]

[0212] The above shows and describes the basic principles, main features and advantages of the present application. It should be understood by those skilled in the art that the present application is not limited by the above examples, and the above examples and descriptions in the specification are only to illustrate the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the claimed present application. The scope of protection of the present application is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a culture medium for studying the effects of algal liquid secretions on Arabidopsis thaliana growth, characterized in that, Includes the following steps: Step (1), preparation of solid culture medium: prepare solid culture medium with a solute concentration of 5 times that of 1 / 2 MS solid culture medium, and adjust the pH to 5.8; Step (2), extraction of algal liquid secretions: The concentration of the algal liquid secretions is 1×10 7 ~2×10 7 cells / ml; The algal liquid secretion mentioned is the extracellular secretion of Microcystis aeruginosa; The *Microcystis aeruginosa* was cultured in HGZ-145 medium at 25±1℃, with the algal solution shaken 2-3 times daily. Every 3-5 days, the algal solution was transferred to a new HGZ-145 medium. After centrifuging the cultured algal solution at 4°C, the supernatant obtained by centrifugation was filtered through a 0.45μm glass fiber filter membrane, and then filtered to remove bacteria, thus obtaining the liquid secretion of algae. Step (3), preheating: The solid culture medium obtained in step (1) and the liquid secretions of algae obtained in step (2) are preheated respectively; If the algal liquid secretions need to be diluted, the culture medium used in step (2) for culturing algae should be used to dilute them to the target concentration before preheating. Preheating temperature should not exceed 60℃; preheating Duration less than 1 hour; After preheating, the temperature of the liquid algal secretions should be higher than that of the solid culture medium; The preheated solid culture medium should be in a liquid state; Step (4), mixing of the culture medium: Add the preheated solid culture medium to the preheated diluted or undiluted algal liquid secretions, mix well, and let stand to obtain the culture medium for studying the effect of microbial liquid secretions on Arabidopsis thaliana growth. The volume ratio of the preheated solid culture medium to the preheated diluted or undiluted algal liquid secretions is 1:

4.

2. The method for preparing the culture medium for studying the effect of algal liquid secretions on Arabidopsis growth according to claim 1, characterized in that, In step (2), the algal solution is centrifuged at 6000 rpm for 10 minutes at 4°C. The supernatant is then taken and filtered using a 0.45 μm glass fiber filter membrane. Finally, a 0.22 μm pore size membrane filter is used to remove bacteria and obtain the liquid secretion of algae.

3. The method for preparing the culture medium for studying the effect of algal liquid secretions on Arabidopsis growth according to claim 2, characterized in that, In step (2), the light-dark cycle ratio of Microcystis aeruginosa is 12l:12d during culture; the algal liquid secretions are extracted after more than three transfers.

4. The method for preparing the culture medium for studying the effect of algal liquid secretions on Arabidopsis growth according to claim 2, characterized in that, HGZ-145 culture medium was sterilized in a high-temperature autoclave at 121℃ for 30 minutes before use.

5. The method for preparing a culture medium for studying the effects of algal liquid secretions on Arabidopsis growth according to claim 2, characterized in that, In step (3), after the HGZ-145 medium is sterilized in a high-temperature and high-pressure autoclave at 121°C for 30 minutes, the sterilized HGZ-145 medium is used to dilute the liquid secretions of algae.

6. A method for studying the effect of algal liquid secretions on Arabidopsis growth, comprising a culture medium for studying the effect of algal liquid secretions on Arabidopsis growth prepared by the method for preparing the culture medium for studying the effect of algal liquid secretions on Arabidopsis growth according to any one of claims 1 to 5, characterized in that, Includes the following steps: In a sterile environment, Arabidopsis seeds were first rinsed with disinfectant water, then rinsed several times with sterile water. The seeds were then evenly placed in a culture medium, sealed, and placed in a 4°C environment for vernalization. After two days, they were placed in an incubator for light cultivation. The phenotype of Arabidopsis thaliana cultivated under light was observed and analyzed to obtain the results of the study on the effect of algal liquid secretions on Arabidopsis thaliana growth.

7. The method for studying the effect of algal liquid secretions on Arabidopsis growth according to claim 6, characterized in that, Wash three times with sterile water; incubate at a constant temperature of 22℃ with a light-dark cycle ratio of 16l:8d.

8. The method for studying the effect of algal liquid secretions on Arabidopsis growth according to claim 6, characterized in that, The study investigated the effects of algal liquid secretions on Arabidopsis growth, including dose-response studies, time stability studies, and thermal stability studies.

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