CN116406590A - Method for improving resistance of tomato plants and preventing and controlling gray mold by utilizing fragmented tomato esDNA and application - Google Patents
Method for improving resistance of tomato plants and preventing and controlling gray mold by utilizing fragmented tomato esDNA and application Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G13/00—Protecting plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/05—Fruit crops, e.g. strawberries, tomatoes or cucumbers
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/20—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
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Abstract
The invention provides a method for improving resistance of tomato plants and preventing and treating gray mold by utilizing fragmented tomato esDNA and application thereof, belonging to the technical field of biology. The method provided by the invention is to treat tomato plant leaves by fragmenting tomato esDNA, and to improve the resistance of tomato plants and prevent gray mold by activating the jasmonic acid pathway of tomato and stimulating the generation of active oxygen. Through detection, the tomato plants are treated by fragmented tomato esDNA, the up-regulated expression of jasmonic acid synthesis genes can be induced, the accumulation of jasmonic acid isoleucine (JA-Ile) is promoted, and a JA signal pathway is activated; and the segmented tomato esDNA treatment can obviously promote the generation of active oxygen of tomatoes and increase the activity of peroxidase and superoxide dismutase. The tomato gray mold can be inhibited by the fragmented tomato esDNA treatment, and the average inhibition rate is 45%.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for improving resistance of tomato plants and preventing gray mold by utilizing fragmented tomato esDNA and application thereof.
Background
Tomato (Solanum lycopersicum l.) is an annual or perennial herb of the genus solanaceae, a vegetable crop cultivated most widely worldwide and consumed most. China is the largest country for tomato production and consumption in the world, and tomato production is an important industry for peasants to increase income and enrich and export and create foreign exchange. However, year-round cultivation causes tomato diseases to become more serious. The tomato gray mold can be infected at each stage of growth, flowering and fruiting of the tomato, which can generally lead to 20% -30% yield reduction of the tomato, and can reach 50% when serious, which seriously affects the yield and quality of the tomato, and the control of the tomato gray mold is difficult, so that the improvement of the resistance of the tomato to the gray mold is very important.
At present, the control method for the gray mold of the tomato is mainly chemical control, and because chemical agents are used for a long time, the gray mold has drug resistance to most agents, the use amount of the chemical agents is often increased by growers, so that the drug resistance is increasingly prominent, the pesticide residues in fruits and soil are increased, the food safety is threatened, and the environment is polluted. Therefore, many scholars can realize the control of the gray mold of the tomato through the biological control by screening antagonistic bacteria with better antibacterial performance. However, the antagonistic bacteria play a role in preventing and controlling depends on stable and long-term colonization, and is influenced by factors such as plant varieties, cultivation modes, pesticide application, ecological environment in fields and the like, so that the preventing and controlling effect is unstable. Therefore, there is a need for a simple and effective method for inducing resistance of tomato plants, which utilizes the plant's own defense mechanism to control gray mold of tomato.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for improving resistance of tomato plants and preventing and treating gray mold by using fragmented tomato esDNA, and the method provided by the present invention can induce a systematic defense reaction to pathogenic bacteria by treating receptor plants with fragmented tomato esDNA, thereby improving resistance to gray mold.
In order to achieve the above object, the present invention provides the following technical solutions:
a method for improving resistance of tomato plants and preventing gray mold by using fragmented tomato esDNA, wherein the fragmented tomato esDNA is a tomato plant extracellular self DNA fragment; the method is to treat tomato plants by fragmenting tomato esDNA, activate tomato jasmonic acid signal pathway and stimulate active oxygen generation so as to improve the resistance of the tomato plants and prevent gray mold.
Preferably, the preparation method of the fragmented tomato esDNA comprises the following steps:
s1, extracting DNA of a tomato plant;
s2, carrying out ultrasonic treatment on the tomato plant DNA obtained in the S1 under the ice bath condition;
s3, carrying out integrity detection on the tomato plant DNA subjected to ultrasonic treatment, and obtaining fragmented tomato esDNA if the sizes of DNA fragments are less than 1000 bp; if the DNA has fragments with the size of more than 1000bp, carrying out second ultrasonic treatment and integrity detection until the sizes of the DNA are less than 1000bp, and obtaining the fragmented tomato esDNA.
Preferably, the ultrasonic power of the ultrasonic treatment in the step S2 is 120W-180W, and the total ultrasonic time is 6 min-10 min; the ultrasonic treatment is a batch ultrasonic treatment in which the gap time/ultrasonic time is (2 s to 4 s)/(2 s to 4 s).
Preferably, the ultrasonic power of the second ultrasonic treatment in the step S3 is 120W-180W, and the total ultrasonic time is 3 min-5 min; the second sonication is a batch sonication, wherein the gap time/sonication time is (2 s-4 s)/(2 s-4 s).
Preferably, the treatment of tomato plants is spraying tomato plant leaves with a solution of fragmented tomato esDNA to make them completely moist or culturing at tomato seedling stage using a medium containing fragmented tomato esDNA.
Preferably, the concentration of the fragmented tomato esDNA solution is 100-200 mug/mL; the final concentration of the fragmented tomato esDNA in the culture medium is 100-200 mug/mL.
Preferably, the culture time of the culture medium is 30-120 min.
The invention also provides an application of the fragmented tomato esDNA in preparing a medicine for preventing and treating tomato gray mold.
The invention also provides an application of the fragmented tomato esDNA in preparing a medicine for activating the JA signal pathway of tomatoes.
The invention also provides an application of the fragmented tomato esDNA in preparing a medicament for improving tomato resistance.
The beneficial technical effects are as follows: the invention provides a method for improving the resistance of tomato plants and preventing and controlling gray mold by utilizing fragmented tomato esDNA and application thereof. Through detection, the tomato plants are treated by fragmented tomato esDNA, the up-regulated expression of JA synthetic genes can be induced, the accumulation of jasmonic acid isoleucine (JA-Ile) is promoted, and a JA signal pathway is activated; and the segmented tomato esDNA treatment can obviously promote the generation of active oxygen of tomatoes and increase the activity of peroxidase and superoxide dismutase. The fragmented tomato esDNA treatment can inhibit the gray mold disease condition of the tomato, and the average inhibition rate is 45%.
Drawings
FIG. 1 shows the results of gray mold resistance experiments for different tomato treatment groups; ". Times." indicates p < 0.05, the difference is significant;
FIG. 2 shows the results of JA gene expression in different tomato seedlings; wherein "×" indicates that p < 0.05, the difference is significant;
FIG. 3 is a graph showing the results of ROS content in various tomato leaves;
FIG. 4 shows the results of jasmonic acid synthesis precursor (OPDA) and isoleucine jasmonic acid (JA-lle) content of different tomato seedlings; wherein "×" indicates that p < 0.05, the difference is significant;
FIG. 5 is a graph showing the results of antioxidant enzyme activity in various tomato leaves; wherein "×" indicates that p < 0.05, the difference is significant;
Detailed Description
The invention provides a method for improving the resistance of tomato plants and preventing and controlling gray mold by utilizing fragmented tomato esDNA, wherein the fragmented tomato esDNA is a tomato plant extracellular self DNA fragment; the method is to treat tomato plants by fragmenting tomato esDNA, activate tomato jasmonic acid signal pathway and stimulate the generation of active oxygen of tomato so as to improve the resistance of the tomato plants and prevent gray mold.
In the invention, the activation of the tomato jasmonic acid signal pathway is to induce the up-regulated expression of the jasmonic acid synthesis gene, the response gene and the signal gene, so that the accumulation of jasmonic acid synthesis precursor (OPDA) isoleucine jasmonic acid (JA-Ile) is promoted; the active oxygen of the stimulated tomato is generated to promote the content of hydrogen peroxide and increase the activities of peroxidase and superoxide dismutase.
In the present invention, the fragment size of the fragmented tomato esDNA is preferably < 1000bp, more preferably 50bp to 1000bp, most preferably 750bp. When a plant is damaged or infected by a pathogenic bacterium, its innate immune system can recognize elicitors derived from the pathogenic bacterium or endogenous injury-related signaling molecules (DAMPs) themselves through receptors and make a series of defensive responses, thereby blocking or slowing down infection by the pathogenic bacterium. The fragmented plant extracellular self DNA fragment (esDNA) is taken as a DAMP, can promote the generation of plant active oxygen (such as hydrogen peroxide) in a concentration-dependent and species-specific manner, triggers plant self-specific immune response, and improves the resistance, wherein the plant hormone jasmonic acid is one of important signal molecules for activating the system resistance and participates in immune response of invasion of dead nutritional pathogenic bacteria.
In the present invention, the preparation method of the fragmented tomato esDNA preferably comprises the following steps:
(1) Extracting DNA of tomato plants; the DNA of the tomato plant is preferably DNA of stems or leaves of the tomato plant, more preferably DNA of leaves of the tomato plant; the extraction is preferably carried out by adopting a CTAB method;
(2) Carrying out ultrasonic treatment on the tomato plant DNA obtained in the step (1) under ice bath conditions; the ultrasonic power of the ultrasonic treatment is preferably 120W-180W, more preferably 150W; the total ultrasonic time is preferably 6-10 min, more preferably 8min; the ultrasonic treatment is a batch ultrasonic treatment in which the gap time/ultrasonic time is preferably (2 s to 4 s)/(2 s to 4 s), more preferably 3s/3s. The intermittent ultrasonic method and parameters can ensure that the size of the finally obtained segmented tomato esDNA is 50 bp-1000 bp, and if the ultrasonic power is too high, a large amount of foam can be generated in an ultrasonic system; if the ultrasound power is too small, fragmentation is incomplete.
(3) Carrying out integrity detection on the tomato plant DNA after ultrasonic treatment, and obtaining fragmented tomato esDNA if the sizes of DNA fragments are less than 1000 bp; if the DNA has fragments with the size of more than 1000bp, carrying out second ultrasonic treatment and integrity detection until the sizes of the DNA are less than 1000bp, and obtaining the fragmented tomato esDNA. In the present invention, the integrity test is preferably performed by 1% agarose gel electrophoresis; the ultrasonic power of the second ultrasonic treatment is preferably 120W to 180W, more preferably 150W; the total ultrasonic time is preferably 3-5 min, more preferably 4min; the second ultrasonic treatment is preferably a batch ultrasonic treatment in which the gap time/ultrasonic time is preferably (2 s to 4 s)/(2 s to 4 s), more preferably 3s/3s. The size of the fragmented tomato esDNA is set to be 50-1000 bp, so that the fragmented tomato esDNA can enter the tomato plant, and the specific immune response of the tomato plant can be effectively triggered, the resistance to gray mold is improved, and gray mold is prevented.
In the present invention, the treatment of tomato plants is preferably spraying tomato plant leaves with a solution of fragmented tomato esDNA to make them completely moist or culturing in a medium containing fragmented tomato esDNA at the tomato seedling stage; the concentration of the fragmented tomato esDNA solution is preferably 100-200 mug/mL, more preferably 200 mug/mL; the spraying is preferably tomato plants growing for 3-5 weeks; the concentration of the fragmented tomato esDNA in the culture medium is preferably 100-200 mug/mL, more preferably 200 mug/mL; the tomato seedling stage is preferably tomato seedlings growing for 5-10 days; the culture medium is preferably a liquid culture medium suitable for growing tomato seedlings, more preferably a 1/2MS liquid culture medium; the culture time of the culture medium is preferably 30min to 120min, more preferably 60min.
The invention also provides an application of the fragmented tomato esDNA in preparing a medicine for preventing and treating tomato gray mold.
The fragmented tomato esDNA of the present invention is preferably the fragmented tomato esDNA described in the above technical scheme. The invention is not particularly limited in the form of the drug, and the segmented tomato esDNA can be used as a pharmaceutically acceptable form. The preparation method of the medicine is not particularly limited, and the preparation method of the corresponding dosage form is adopted. The content of fragmented tomato esDNA in the medicine is preferably 100-200 mug/mL.
The invention also provides an application of the fragmented tomato esDNA in preparing a medicine for activating the JA signal pathway of tomatoes.
The fragmented tomato esDNA of the present invention is preferably the fragmented tomato esDNA described in the above technical scheme. In the present invention, the dosage form, the preparation method and the content of the active ingredient of the medicine are the same as those described above, and are not described herein. In the present invention, the active ingredients and the content of the fragmented tomato esDNA are the same as the above, and are not described herein.
The invention also provides an application of the fragmented tomato esDNA in preparing a medicament for improving tomato resistance.
The fragmented tomato esDNA of the present invention is preferably the fragmented tomato esDNA described in the above technical scheme. In the present invention, the dosage form, the preparation method and the content of the active ingredient of the medicine are the same as those described above, and are not described herein. In the present invention, the active ingredients and the content of the fragmented tomato esDNA are the same as the above, and are not described herein.
For a better understanding of the present invention, the following examples are further illustrated, but are not limited to the following examples. Materials, reagents and the like used in the examples and test examples of the present invention can be obtained commercially unless otherwise specified; the methods used in the examples and test examples of the present invention are conventional methods unless otherwise specified.
Tomato plants used in the present invention:
the tomato variety tested in the present invention was M82 (source: university of California, davis, calif., USA-university of California in the United states) and CM (center for research on genetics and developmental biology at the national academy of sciences).
esDNA is extracellular self DNA of tomato plants, and Mock is distilled water treatment tomato plant leaves.
Example 1 preparation of fragmented tomato esDNA:
(1) Extracting tomato plant DNA: fresh tomato leaves are collected, and DNA in the tomato leaves is extracted by using a CTAB method:
(1) tomato leaves were ground in liquid nitrogen, weighed and placed in 50mL pre-chilled centrifuge tubes, 10mL sorbitol buffer (100 mM Tris-HCl, pH 8.0;0.35M sorbitol; 5mM EDTA,pH 8.0;1% (w/v) PVP-40 (polyvinylpyrrolidone, average molecular weight 40000), 1% (v/v) beta-mercaptoethanol (added prior to use) was added, the tubes were thoroughly mixed on a vortex shaker, centrifuged at 4500g room temperature for 5min, and the supernatant was discarded.
(2) 15mL of a CTAB lysis buffer (100 mM Tris-HCl, pH 8.0;3M NaCl;2%CTAB (cetyltrimethylammonium bromide)), 20mM EDTA and 1% (w/v) PVP-40 (polyvinylpyrrolidone, average molecular weight 40000) and 1% (v/v) beta mercaptoethanol (added prior to use) preheated at 65℃were added to the centrifuge tube containing the pellet.
(3) The centrifuge tube is watered in 65 ℃ water for 1h, and is gently shaken once every 10min.
(4) The tube was removed and cooled to room temperature, centrifuged at 13000rpm for 10min, the supernatant was aspirated, and an equal volume of 24:1, evenly mixing the chloroform isoamyl alcohol and standing for 10min. Centrifuge at 13000rpm for 10min.
(5) The procedure (4)1) was repeated.
(6) The supernatant was taken and degraded by adding 3. Mu.L of RNase in a water bath at 37℃for 2 hours.
(7) Equal volume of 24:1 chloroform isoamyl alcohol, mixing well and standing for 10min. Centrifuging at 13000rpm for 10min, and collecting supernatant.
(8) Adding isopropanol with equal volume and cooling, storing at-20deg.C for 20min, centrifuging at 13000rpm for 10min, washing with 80% absolute ethanol twice, and blow drying with a clean bench.
(9) An appropriate amount of preheated sterile deionized water was added to dissolve the DNA.
(2) Intermittently performing ultrasonic treatment on the obtained tomato plant DNA under ice bath conditions; the ultrasonic power is 120W-180W; the total ultrasonic time is preferably 6-10 min; wherein the gap time/ultrasonic time is (2 s-4 s)/(2 s-4 s).
(3) Detecting extracellular DNA of the tomato plant after ultrasonic treatment by adopting 1% agarose gel electrophoresis, wherein the sizes of DNA fragments are less than 1000bp, namely fragmented tomato esDNA.
EXAMPLE 2 Gray mold resistance test
Tomato seedling plants grown in the medium for 4 weeks were selected for resistance experiments. 200. Mu.g.mL -1 The fragmented tomato esDNA of (A) was sprayed onto CM and M82 tomato plant leaves, respectively, distilled water was used as a control (Mock), after 24 hours of treatment, the plant leaves not treated with eDNA solution and the plant leaves treated with eDNA solution were removed, and placed in 1% agar plates, respectively, and inoculated with 5. Mu.L of 5X 10 concentration 5 After inoculation, the plates were sealed to prevent evaporation of water. Each treatment was repeated 5 times, three seedlings each time. After plates were incubated at 22℃for 72h, the plaque area was counted by Image J software.
The gray mold resistance test results are shown in FIG. 1. As can be seen from fig. 1, the esDNA treatment can significantly reduce the gray mold spot area of tomatoes CM, M82. The average inhibition rate was 45%.
Example 3 Effect of esDNA treatment on tomato ROS and antioxidant enzyme content
Tomato seedling plants grown in the medium for 4 weeks were selected for resistance experiments. The CM and M82 tomato leaves were sprayed with 200 μg/mL of fragmented tomato esDNA, respectively, and distilled water was used as a control (Mock), 3 biological replicates were set for each treatment, 3 seedlings were replicated for each treatment, and 3 leaves of uniform size were selected for each seedling for treatment. Samples were taken after 60min of treatment, and the ROS (hydrogen peroxide) content and antioxidant enzyme activity in the tomato leaf were determined, respectively.
(1) Determination of ROS (hydrogen peroxide) content
The tomato leaves were weighed and thoroughly ground to a homogenate in a pre-chilled mortar with 2mL of acetone and then centrifuged at 5000g for 10min at 4 ℃. To 1mL of the supernatant was added 0.1mL of 5% (w/v) TiSO 4 And 0.1mL of cold ammonia water, centrifuge for 10min at 3000g, discard supernatant, use 4mL of 2M H 2 SO 4 The precipitate was dissolved and absorbance was measured at 415 nm. At a known concentration of H 2 O 2 And (5) making a standard curve.
The results of ROS content in various tomato leaves are shown in FIG. 3. As can be seen from fig. 3, both esDNA treatments significantly increased the hydrogen peroxide content in tomato plants.
(2) Determination of peroxidase and superoxide dismutase Activity
0.3g of leaf was added to 5mL of phosphate buffer (50 mM, pH7.8 phosphate buffer, 0.1mM EDTA-Na) 2 2% pvp), grinding under ice bath conditions, centrifuging 12000g of the leaf homogenate at 4 ℃ for 20min, and measuring the activity of peroxidase and superoxide dismutase by using the supernatant.
The results of antioxidant enzyme activity in different tomato leaves are shown in figure 5. As can be seen from fig. 5, the esDNA treatment significantly increased the activity of peroxidase and superoxide dismutase in the tomato plants,
example 4 effect of esDNA on expression of tomato seedlings JA Gene and jasmonic acid isoleucine JA-lle), the content of JA Synthesis precursor substances
Taking CM and M82 tomato seeds for surface disinfection, respectively sowing the seeds in a 1/2MS culture medium, culturing the seedlings in an illumination incubator under the conditions that the temperature in the incubator is 23 ℃ per 18 ℃ (day/night) and the illumination intensity is 400 mu mol.m -2 ·s -1 Photoperiod 12h/12h (day/night), air humidity 65%. 4 tomato seedlings grown 7d old were carefully removed from the medium and transferred to 1/2MS liquid medium containing 200. Mu.g/mL of fragmented tomato esDNA, respectively, to treat with sterile water as a control. Each treatment was performed with 3 biological replicates of 12 seedlings each. After treatment for 0min, 30min, 60min and 120min, tomato seedlings are taken out of the culture medium, quickly frozen by liquid nitrogen and stored in a refrigerator at-80 ℃ for standby use for measuring JA gene expression. At the same time, tomato seedlings sampled after 60min of treatment were also used for the determination of JA content.
(1) Determination of JA Gene expression was determined by qPCR analysis and quantitative primers were designed using primer5.0 software, and the determined genes and primers are shown in Table 1.
TABLE 1 qPCR determination genes and primers
qPCR reaction system: each of the upstream and downstream primers (10. Mu.M) was 0.3. Mu.L, the cDNA template was 0.6. Mu.L, and 10. Mu.L of 2 XPlus SYBR real-time PCR mixture, 8.8. Mu.L of deionized water. qPCR reaction conditions: pre-denaturation at 95℃for 10min, denaturation at 95℃for 10s, annealing at 58℃for 30s,42 cycles, extension at 72℃for 40s.
The results of JA gene expression in different tomato seedlings are shown in FIG. 2. As can be seen from FIG. 2, after 60min of treatment, the esDNA significantly increased the relative expression levels of the JA synthetic genes SlMOX11 and TomLoxD, slAOC in tomato seedlings, the relative expression levels of the JA signal genes MYC2, JAZ1 and JAZ8, and the relative expression levels of the JA response genes SlPI-1 and SlMPK1, compared with the control. It was demonstrated that esDNA treatment can significantly affect the JA signaling pathway.
(2) Determination of JA synthesis precursor substance (OPDA) and jasmonic acid isoleucine (JA-lle):
the method for measuring the content of the JA synthesis precursor substance and the JA comprises the following steps:
grinding the sample stored in the ultralow temperature refrigerator to powder by using a grinder, weighing 50mg of sample powder, respectively adding 10 mu L of an internal standard mixed solution with the concentration of 100ng/mL, 1mL of methanol/water/formic acid (15:4:1, v/v/v) extracting solution, uniformly mixing by vortex for 10min, centrifuging at 12000rpm and 4 ℃ for 5min, sucking the supernatant into a new centrifuge tube, concentrating, re-dissolving the concentrated sample by using 100 mu L of 80% methanol solution, filtering by using a 0.22 mu m filter membrane, and placing the sample into a sample injection bottle for LC-MS/MS analysis.
Drawing a standard curve: preparing standard substance solutions with different concentrations of 0.01ng/mL, 0.05ng/mL, 0.1ng/mL, 0.5ng/mL, 1ng/mL, 5ng/mL, 10ng/mL, 50ng/mL, 100ng/mL, 200ng/mL and 500ng/mL, obtaining mass spectrum peak intensity data of corresponding quantitative signals of the standard substances with different concentrations, taking the concentration ratio of an external standard to an internal standard as an abscissa, the peak area ratio of the external standard to the internal standard as an ordinate, and drawing a standard curve.
Substituting the detected integral peak area ratio of all samples into a standard linear equation for calculation, and further substituting into a calculation formula for calculation to finally obtain the actual content of the substance in the sample.
Hormone content in sample (ng/g) =c×v/1000/m
Wherein: c is a concentration value (ng/mL) obtained by substituting the integrated peak area ratio in the sample into a standard curve; v is the volume of solution (μL) used in reconstitution; m is the mass (g) of the sample being weighed.
The results of the content of the synthesized precursor substances of the different tomato seedlings JA are shown in FIG. 4. As can be seen from fig. 4, the esDNA treatment significantly increased the content of the Jasmonic Acid (JA) synthesis precursor substance 12-oxodienoic acid (OPDA) and jasmonic acid isoleucine (JA-Ile) in tomato seedlings. It was demonstrated that esDNA treatment can significantly affect the synthesis of JA precursor species.
The results of the different tomato seedlings JA synthesis of the precursor substance 12- -oxodienoic acid (OPDA) content are shown in FIG. 4. As can be seen from fig. 4, the esDNA treatment significantly increased the levels of OPDA and isoleucine jasmonate (JA-lle) in tomato seedlings. It was demonstrated that esDNA treatment can significantly promote JA synthesis.
In conclusion, the tomato leaf treated by the fragmented tomato esDNA can activate the tomato jasmonic acid signal pathway and stimulate the generation of active oxygen of the tomato to improve the resistance of the tomato plant and prevent and treat gray mold. The fragmented tomato esDNA treatment can inhibit the gray mold disease condition of the tomato, and the average inhibition rate is 45%.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A method for improving resistance of tomato plants and preventing gray mold by using fragmented tomato esDNA is characterized in that the fragmented tomato esDNA is a tomato plant extracellular self DNA fragment; the method is to treat tomato plants by fragmenting tomato esDNA, activate tomato jasmonic acid signal pathway and stimulate active oxygen generation to improve tomato plant resistance and prevent and treat tomato gray mold.
2. The method for improving resistance of tomato plants and preventing and treating gray mold by using fragmented tomato esDNA according to claim 1, wherein the preparation method of the fragmented tomato esDNA comprises the following steps:
s1, extracting DNA of a tomato plant;
s2, carrying out ultrasonic treatment on the tomato plant DNA obtained in the S1 under the ice bath condition;
s3, carrying out integrity detection on the tomato plant DNA subjected to ultrasonic treatment, and obtaining fragmented tomato esDNA if the DNA fragment is less than 1000 bp; if the DNA has fragments with the size of more than 1000bp, carrying out second ultrasonic treatment and integrity detection until the sizes of the DNA are less than 1000bp, and obtaining the fragmented tomato esDNA.
3. The method for improving the resistance of tomato plants and preventing and treating gray mold by utilizing fragmented tomato esDNA according to claim 2, wherein the ultrasonic power of the ultrasonic treatment in the S2 is 120-180W, and the total ultrasonic time is 6-10 min; the ultrasonic treatment is a batch ultrasonic treatment in which the gap time/ultrasonic time is (2 s to 4 s)/(2 s to 4 s).
4. The method for improving the resistance of tomato plants and preventing and treating gray mold by utilizing fragmented tomato esDNA according to claim 2, wherein the ultrasonic power of the second ultrasonic treatment in the step S3 is 120-180W, and the total ultrasonic time is 3-5 min; the second sonication is a batch sonication, wherein the gap time/sonication time is (2 s-4 s)/(2 s-4 s).
5. The method for improving resistance of tomato plants and preventing and treating gray mold by using fragmented tomato esDNA according to claim 1, wherein the tomato plants are treated by spraying tomato plant leaves with the fragmented tomato esDNA solution to be completely moist or culturing in a medium containing the fragmented tomato esDNA at the stage of tomato seedlings.
6. The method for improving the resistance of tomato plants and preventing and treating gray mold by using fragmented tomato esDNA according to claim 5, wherein the concentration of the fragmented tomato esDNA solution is 100-200 mug/mL; the final concentration of the fragmented tomato esDNA in the culture medium is 100-200 mug/mL.
7. The method for improving the resistance of tomato plants and preventing and treating gray mold by utilizing fragmented tomato esDNA according to claim 5, wherein the culture time of the culture medium is 30-120 min.
8. Application of fragmented tomato esDNA in preparing medicine for preventing and treating tomato gray mold is provided.
9. Use of fragmented tomato esDNA in the preparation of a medicament for activating the JA signaling pathway of tomato.
10. Use of fragmented tomato esDNA in the preparation of a medicament for improving tomato resistance.
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