CN111493076B

CN111493076B - Application and method for inducing resistance of arabidopsis thaliana to gray mold by 3,5-dichloro anthranilic acid

Info

Publication number: CN111493076B
Application number: CN202010391958.3A
Authority: CN
Inventors: 张译匀; 陆玲悦; 张慧娟
Original assignee: Taizhou University
Current assignee: Taizhou University
Priority date: 2020-05-11
Filing date: 2020-05-11
Publication date: 2022-05-27
Anticipated expiration: 2040-05-11
Also published as: CN111493076A

Abstract

The invention discloses application of 3,5-dichloro anthranilic acid in resistance induction of gray mold in arabidopsis thaliana and a method thereof, belonging to the field of plant resistance induction by compounds; according to the invention, after 3,5-dichloro anthranilic acid aqueous solution is used for root irrigation or page spraying treatment for two days on arabidopsis thaliana cultivated at four weeks, botrytis cinerea is inoculated, and the result shows that the arabidopsis thaliana seedlings treated by the 3,5-dichloro anthranilic acid show obviously increased resistance to botrytis cinerea; the 3,5-dichloro anthranilic acid induced resistance of arabidopsis thaliana to gray mold has the advantages of good water solubility, small using amount, safety, greenness, low price, easy obtaining and the like.

Description

Application and method for inducing resistance of arabidopsis thaliana to gray mold by 3,5-dichloro anthranilic acid

Technical Field

The invention belongs to the field of plant resistance induction by compounds, relates to application of a compound in plant resistance induction and a method thereof, and particularly relates to application of 3,5-dichloro anthranilic acid in induction of gray mold resistance of arabidopsis thaliana and a method thereof.

Background

3,5-Dichloroanthranilic Acid (DCA), also known as 2-amino-3, 5-dichlorobenzoic Acid, is a white crystalline powder, low in toxicity and commonly used as an intermediate in medicines and perfumes. The chemical formula is C7H5Cl2NO2, the molecular weight is 206.026, the density is 1.607g/cm3, the melting point is 227-230 ℃ and the boiling point is 344.4 ℃ Cat760mmHg, and the CAS accession number is 2789-92-6.

When the growth and development of plants are deeply studied, the plants such as corn, tomato, pea, rice, barley, petunia, and snapdragon have been studied as model gene systems, but the results are not satisfactory. Arabidopsis thaliana becomes the first plant with a genome completely sequenced in 2000, has short growth cycle and simple genome, is an ideal material for researching plant physiology, biochemistry and plant molecular biology, is known as fruit fly in plant by scientists, and makes important contribution in the fields of grain yield increase, crop stress tolerance, environmental protection and the like. When it is cultivated as a model plant, care must be taken in pest control in order for the plant to grow normally, to cultivate a representative population and potentially new mutants without losing phenotype due to pest infestation during early screening, wherein one of the most common pathogens is botrytis cinerea, which is present on the surface of arabidopsis thaliana plants, with leaf decay, sometimes with degradation of the material.

If chemical pesticide is directly used to kill botrytis cinerea, the problems of environmental pollution, pesticide residue, drug resistance and the like can be caused. Due to the popularization of greenhouse and greenhouse planting technology, the crop has serious gray mold disease, and becomes a main limiting factor for the production of vegetable, flower and forestry seedling cultivation bases. The induced resistance of the plant has broad spectrum and lasting characteristics. The induction of plant self-production systems by inducers to obtain disease Resistance (SAR) is becoming the direction of development in the control of plant diseases.

Various plant activators have been shown to induce resistance in plants to pathogen invasion. Such as BTH (benzothiadiazole), thiabendazole (PBZ), methyl jasmonate (MeJA), Salicylic Acid (SA) and the like, can induce a defense mechanism in plants, and play a role in resisting diseases and preventing diseases. However, these plant activators (e.g., BTH) have disadvantages of poor water solubility, large use amount, influence on plant growth, high toxicity, high price, and the like.

Therefore, when a chemical inducer is used for inducing resistance to botrytis cinerea in the cultivation process of arabidopsis thaliana, an inducer which has the advantages of good water solubility, small using amount, safety, greenness, low price and the like is urgently needed to be found. The method has great significance for self cultivation and mass propagation of arabidopsis as a model plant, and also has strong reference value for selection of a chemical inducer when inducing other crops to generate resistance to botrytis cinerea.

Disclosure of Invention

The invention aims to provide application and a method for inducing resistance of arabidopsis thaliana to gray mold by 3,5-dichloro anthranilic acid aiming at the problems of high cost, environment pollution and poor water solubility of an inducer in prevention and treatment of gray mold in the prior art. The 3,5-dichloro anthranilic acid induced resistance of arabidopsis thaliana to gray mold has the advantages of good water solubility, small using amount, low toxicity, safety, greenness, low price, easy obtaining and the like.

In order to achieve the above object, the present invention provides the use of 3,5-dichloroanthranilic acid to induce resistance of arabidopsis to gray mold.

As a preferred technical scheme, the application concentration of the 3,5-dichloro anthranilic acid is 5-15 mu M.

As a preferred technical scheme, the application concentration of the 3,5-dichloro anthranilic acid is 10 μ M.

Preferably, the Arabidopsis thaliana is a 4-week-old Arabidopsis thaliana plant.

Preferably, the solvent of the 3, 5-dichloro-anthranilic acid solution is water.

In order to achieve the above object, the present invention also provides a method for inducing resistance of arabidopsis thaliana to gray mold by 3,5-dichloroanthranilic acid, comprising the steps of:

the method comprises the following steps: cultivating an arabidopsis plant;

step two: preparing 5-15 mu M of 3,5-dichloro anthranilic acid solution, and performing root irrigation treatment or foliage spraying on the arabidopsis plants in the step one by using the prepared 3,5-dichloro anthranilic acid solution;

step three: inoculating the arabidopsis thaliana plant treated in the second step with botrytis cinerea.

Preferably, the arabidopsis thaliana treated in the second step is an arabidopsis thaliana plant grown for 4 weeks in the first 4 steps.

Preferably, the solvent used for preparing the 3, 5-dichloro-anthranilic acid solution in the second step is water.

As a preferred technical scheme, the third step is to inoculate the Arabidopsis thaliana plants which are treated for two days in the second step with the botrytis cinerea.

The invention has the beneficial effects that: 1. according to the invention, the 3,5-dichloro anthranilic acid solution is used for root irrigation treatment or leaf surface spraying on an arabidopsis thaliana plant before the botrytis cinerea is inoculated, so that the accumulation of active oxygen can be quickly up-regulated, the expression of plant disease-resistant related genes and the activity of in-vivo resistance related enzymes can be regulated, and the resistance of arabidopsis thaliana to botrytis cinerea can be obviously improved.

2. The 3,5-dichloro anthranilic acid has good water solubility, low toxicity, low cost and easy obtaining, and the dosage of the 3,5-dichloro anthranilic acid for inducing the arabidopsis thaliana to generate resistance to gray mold is small, so that secondary pollution is avoided.

Drawings

FIG. 1 is a graph comparing the number of lesions on leaves of Arabidopsis plants previously treated with 10. mu.M of an aqueous DCA solution 4 days after inoculation with Botrytis cinerea in example three with those of control plants.

FIG. 2 is a graph comparing the number of Botrytis cinerea in Arabidopsis plants previously treated with 10 μ M aqueous DCA solution 48 hours after inoculation with Botrytis cinerea with that in control plants in example four.

FIG. 3 is a graph comparing the expression of disease resistance related genes in Arabidopsis plants treated with 10 μ M DCA aqueous solution in advance with those in control plants in example six.

FIG. 4 is a graph comparing the amount of active oxygen accumulated in Arabidopsis plants previously treated with 10. mu.M aqueous DCA solution in example VII with control plants.

FIG. 5 is a graph comparing the active oxygen scavenging capacity in Arabidopsis plants pre-treated with 10 μ M aqueous DCA solution in example seven with that of control plants.

FIG. 6 is a graph comparing the concentration of hydrogen peroxide accumulated in Arabidopsis plants previously treated with 10. mu.M aqueous DCA solution in example seven with that of control plants.

FIG. 7 is a graph comparing the catalase activity in Arabidopsis plants previously treated with 10. mu.M aqueous DCA solution in example eight with that of control plants.

FIG. 8 is a graph comparing the superoxide dismutase activity in Arabidopsis plants pretreated with 10 μ M aqueous DCA solution compared to control plants in example eight.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The terms used in the specification and claims of the present invention have the following meanings, and other terms not specified are general terms in the art unless otherwise specified.

48 hpi: 48 hours post infection (48 hours post infection).

DCA: 3,5-Dichloroanthranilic Acid (3,5-Dichloroanthranilic Acid).

Plant material: arabidopsis thaliana (Arabidopsis thaliana) Wild type (Columbia, Col).

Strain: botrytis cinerea (A)Botrytis cinerea) Strain BO5.10, supplied by Tesfaye mengite, university of przege, usa.

The main experimental reagents are as follows: DAB and NBT drugs are purchased from Sigama company; reagents related to real-time fluorescent quantitative PCR (qRT-PCR) were purchased from Takara; 3, 5-dichloro-anthranilic acid was purchased from Chishiai (Shanghai) chemical industry development Co., Ltd; the related enzyme activity determination kit is purchased from Nanjing construction company; other conventional reagents were purchased from biotechnology limited liability company in beijing dingguo.

The first embodiment is as follows: cultivation of Arabidopsis plants

Suspending seeds of wild type Arabidopsis (Col-0) in 0.1% agarose solution, vernalizing at low temperature of 4 ℃ for 2-4 days, sowing in mixed nutrient soil (vermiculite: plant ash: perlite =6:2: 1), applying fertilizer, diluting the fertilizer and water according to mass-volume ratio l:1000, and covering for 2-3 days to facilitate seedling emergence. The culture temperature is maintained at 22-24 deg.C, relative humidity is 80%, and illumination intensity is about 85 μmol s^-1·m^-2The light irradiation time was 16 hours. Other water, insect pests and cultivation management are carried out according to the conventional method. 4-week-old Arabidopsis plants were used for experimental treatment.

Example two: treatment of 3, 5-dichloro-anthranilic acid

10 mu M of 3,5-dichloro anthranilic acid aqueous solution is prepared. The Arabidopsis plants were root-irrigated with 10. mu.M aqueous 3,5-dichloroanthranilic acid. Arabidopsis plants were treated by root irrigation with the same volume of water as a control group, and after 2 days, inoculation with Botrytis cinerea was performed.

Example three: the Arabidopsis thaliana obtained in the second example was inoculated with Botrytis cinerea

Using 2 XV 8 solid medium (36% V8 fruit juice, 0.2% CaCO)₃2% agar) at a temperature of 20-25 deg.c. After the dish is full of spores, the hypha mass is placed into a heavy suspension (maltose 4%, tryptone 1%) to be vigorously vortexed to release the spores, the spores are separated by filtration with gauze, and the concentration of the spores is adjusted to 2 × 10 with a hemocytometer⁵spores/mL. During inoculation, the spore suspension is sprayed onto the inoculated seedling uniformly until most of the leaves are full of fine droplets. Placing the inoculated seedling in 24 deg.C, light period of 12 hr/dark period of 12 hr, and light intensity of 200 muE.m^-2S^-1And observing the disease condition after the film is highly moisturized for 4 days. The same volume of the resuspension solution was sprayed onto arabidopsis control plants as a control (MOCK).

As shown in FIG. 1, Arabidopsis plants treated beforehand with 10. mu.M aqueous 3,5-dichloroanthranilic acid for two days in example III showed a smaller number of lesions on the leaves than those on the control plants 4 days after inoculation with Botrytis cinerea.

Example four: growth measurement of Botrytis cinerea

To be provided withBotrytisIs/are as followsActinThe gene expression level is used as an index, and qRT-PCR is adopted to determine the content of inoculated plantsBotrytisThe variation of the growth amount.B.cinerea Actin The primer A is as follows:

BcActin-qRT-1F：5’-CGTCACTACCTTCAACTCCATC-3’；

BcActin-qRT-1R：5’-CGTCACTACCTCAACTCCATC-3’。

using Arabidopsis thalianaActinThe gene expression level is used as an internal reference, and the primers are as follows:

AtActin-qRT-1F：5’-GGCGATGAAGCTCAATCCAAACG-3’；

AtActin-qRT-1R：5’-GGCACGACCAGCAAGATCAAGACG-3’；

the specific operation of qRT-PCR is described in example six.

As shown in FIG. 2, example four Arabidopsis plants previously treated with 10. mu.M aqueous 3,5-dichloroanthranilic acid for two days were inoculated with Botrytis cinerea 48 hours later, and the growth of Botrytis cinerea was measured in the inoculated plants, which was less in the body than the control plants. These show that Arabidopsis plants pretreated with 10. mu.M aqueous 3,5-dichloroanthranilic acid have an increased resistance to Botrytis cinerea compared to the control phase.

Example five: extraction of Total RNA

The extraction of total RNA adopts a TRIzol method, leaves are taken and fully ground in liquid nitrogen, the grinding is rapid, and preferably, the grinding time is not more than 1 minute; then, filling the ground powder into a centrifugal tube; adding 0.2mL of chloroform into 1mLTRIzol, covering a tube cover, violently shaking for 15 seconds, and standing at room temperature for 3 minutes; 4^oC, centrifuging at 10000rpm for 10-15 minutes; transferring the water phase into a new tube, adding isopropanol with the same volume, uniformly mixing, and standing at room temperature for 20-30 minutes; 4^oC, centrifuging at 10000rpm for 10 minutes, and removing a supernatant; washing the precipitate with 1mL75% ethanol (at least 1mL75% ethanol per 1mL LTRNzol); 4^oC, centrifuging at 5000rpm for 3 minutes, and pouring out the liquid; air-dried at room temperature, and added with 30-100L of RNase-free water according to the experiment requirement to dissolve RNA.

Example six: fluorescent real-time quantitative PCR (qRT-PCR)

The total RNA samples extracted were first decontaminated from the RNA by the Takara reverse transcription kit. Then reverse transcription is carried out according to the kit instructions to obtain the corresponding cDNA. The qRT-PCR reaction adopts SYBR Green I as a reaction fluorescent dye and uses arabidopsis thalianaActinThe gene is used as an internal reference gene to carry out reaction. The reaction is carried out in CFX96^TMReal-time System (Bio-Rad, Hercules, Calif., USA) instrument.

The primers used were as follows:

AtPR1-qRT-F：5’-CGTCTTTGTAGCTCTTGTAGGTG-3’；

AtPR1-qRT-R：5’-TAGATTCTCGTAATCTCAGCTCT-3’；

AtPR3-qRT-F：5’-CGTTGTGGCTCTTTACAAACAACAAAAC-3’；

AtPR3-qRT-R：5’-GAAATTAACTTCATACTTAGACTGTCGAT-3’；

AtPR5-qRT-F：5’-ATGGCAAATATCTCCAGTATTCACA-3’；

AtPR5-qRT-R：5’-ATGTCGGGGCAAGCCGCGTTGAGG-3’；

AtPDF1.2-qRT-F：5’-GCTAAGTTTGCTTCCATCATCACCCTT-3’；

AtPDF1.2-qRT-R：5’-AACATGGGACGTAACAGATACACTTGTG-3’。

as shown in figure 3, 10 μ M3, 5-dichloro-anthranilic acid aqueous solution is used for root irrigation of Arabidopsis plants in advance, Botrytis cinerea is inoculated after 2 days, and the expression condition of disease-resistant related genes in vivo is detected after 24 hours. After inoculating Botrytis cinerea to an Arabidopsis thaliana plant treated with 10. mu.M aqueous solution of 3,5-dichloroanthranilic acid,AtPR3and inAtPDF1.2The expression level of (A) is significantly increased compared to that of a control plant, andAtPR5the expression quantity of the plant is obviously reduced compared with that of a plant in a control group,AtPR1the expression level of the gene has no difference with the plant ratio of the control group. It shows that the 3,5-dichloro anthranilic acid aqueous solution induces the resistance of arabidopsis thaliana to gray mold, which is probably realized by the regulation and control of the expression of disease-resistant related genes.

Example seven: DAB and NBT staining for detection of reactive oxygen species production

And respectively detecting the accumulation conditions of hydrogen peroxide and superoxide anions in the arabidopsis thaliana plants treated by the 3,5-dichloro anthranilic acid aqueous solution and the control plants before and after the inoculation of the botrytis cinerea by adopting a DAB dyeing method and an NBT dyeing method.

The specific operation is as follows: the Arabidopsis thaliana plants are treated by root irrigation with 10 μ M aqueous solution of 3,5-dichloroanthranilic acid or water, and then inoculated with Botrytis cinerea respectively after 2 days, and after 24 hours, the leaves are picked up respectively and immersed in a solution containing 1mg/mLDAB (Sigma-Aldrich) (pH 3.8), and placed for 3 hours under dark condition or immersed in 1mg/mLNBT (1 mg/mLNBT dissolved in a solution containing 10 mMNaN)₃In 10mM phosphate buffer, pH 7.8), under dark conditions, at least 1After that, chlorophyll in the leaves was eluted to colorless with boiling water in 95% alcohol, and then stored in 50% ethanol, and photographed.

As shown in FIG. 4, Arabidopsis plants pretreated with 10. mu.M aqueous 3,5-dichloroanthranilic acid accumulated more active oxygen than the control plants.

Example eight: determination of reactive oxygen species scavenging ability (ROS scavenging capacity), Catalase (CAT) activity, superoxide dismutase (SOD) activity, and hydrogen peroxide concentration

The root of Arabidopsis plant is treated by irrigating with 10 μ M3, 5-dichloro-anthranilic acid aqueous solution or water, Botrytis cinerea is inoculated after 2 days, leaves are selected after 24 hours, and active oxygen scavenging ability, catalase activity, superoxide dismutase and hydrogen peroxide concentration are measured by using active oxygen, catalase, superoxide dismutase and hydrogen peroxide detection kits.

As shown in FIG. 5, Arabidopsis plants pretreated with 10. mu.M aqueous 3,5-dichloroanthranilic acid showed a decrease in the scavenging ability of active oxygen in vivo as compared with the control plants. A typical one of these active oxygen-hydrogen peroxide was selected for quantitative determination, and the results are shown in FIG. 6, in which the amount of hydrogen peroxide accumulated in the body of Arabidopsis thaliana plants treated with 10. mu.M aqueous 3,5-dichloroanthranilic acid was larger than that of the control plants. These all show that the previous treatment of the plants with 10. mu.M aqueous 3,5-dichloroanthranilic acid induces the accumulation of active oxygen in the plants.

As shown in FIG. 7 and FIG. 8, there was no change in catalase activity, whereas superoxide dismutase activity was significantly increased, after inoculation with Botrytis cinerea, in Arabidopsis plants that had been root-drenched with 10. mu.M aqueous 3,5-dichloroanthranilic acid, as compared with control plants.

Example nine:

EXAMPLE ninth experiment was conducted by replacing 10. mu.M of 3,5-dichloroanthranilic acid aqueous solution used in the above examples one to eight with 5. mu.M of 3,5-dichloroanthranilic acid aqueous solution, and other steps and experimental conditions were not changed, and the experiment results showed that 5. mu.M of 3,5-dichloroanthranilic acid aqueous solution also exerts a good effect on inducing resistance of Arabidopsis thaliana to gray mold.

Example ten:

EXAMPLE ten the experimental procedure was conducted by replacing 10. mu.M of 3,5-dichloroanthranilic acid aqueous solution used in the above examples one to eight with 15. mu.M of 3,5-dichloroanthranilic acid aqueous solution, and the other steps and experimental conditions were not changed, and the experimental results showed that 15. mu.M of 3,5-dichloroanthranilic acid aqueous solution also exerts a good effect on inducing resistance of Arabidopsis thaliana to gray mold.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

The application of 3,5-dichloro anthranilic acid in inducing arabidopsis thaliana to generate resistance to gray mold is characterized in that the application concentration of the 3,5-dichloro anthranilic acid is 5-15 mu M, and the arabidopsis thaliana is an arabidopsis thaliana plant with the age of 4 weeks.
2. The use of 3,5-dichloroanthranilic acid to induce resistance in arabidopsis thaliana to gray mold as claimed in claim 1, wherein the 3,5-dichloroanthranilic acid is used at a concentration of 10 μ M.
3. A method for inducing resistance of arabidopsis thaliana to gray mold by 3,5-dichloro anthranilic acid, which is characterized by comprising the following steps:

the method comprises the following steps: cultivating an arabidopsis plant;

step two: preparing 5-15 mu M of 3,5-dichloro anthranilic acid solution, and performing root irrigation treatment or foliage spraying on the arabidopsis thaliana plants in the step one by using the prepared 3,5-dichloro anthranilic acid solution;

step three: inoculating the arabidopsis thaliana plants treated in the second step with botrytis cinerea;

the arabidopsis treated in the second step is an arabidopsis plant which is cultivated in the first step and is 4 weeks old;

and step three, inoculating botrytis cinerea to the arabidopsis thaliana plant treated in the step two days later.