Application of β-Tyr as an elicitor in regulating the synthesis of jasmonates and plant insect resistance

Abstract

The present invention discloses the application of β-Tyr as an elicitor in regulating the synthesis of jasmonates and plant insect resistance. After treating plants by the root absorption method and spraying treatment method, β-Tyr can be detected in the tested plants, and it can be detected that the content of JAs substances increases significantly, and the resistance to pests also increases, indicating that exogenous addition of β-Tyr can be absorbed and transported by different types of plants, can activate the synthesis of JAs in plant tissues, and can also increase the resistance of rice to pests. Therefore, the exogenous addition of β-Tyr can be used to activate the biosynthesis of jasmonates in plants and improve the insect resistance of plants.

Description

Technical Field

[0001] The present invention relates to the field of plant metabolism, and particularly to the application of β-Tyr as an elicitor in regulating the synthesis of jasmonates and plant insect resistance. Background Art

[0002] An elicitor refers to a general term for substances that can induce plant defense responses. It usually has the following characteristics: the elicitor itself has relatively low bactericidal or insecticidal activity, and its action mechanism is relatively complex, with diverse action targets. The induced resistance has persistence, broad-spectrum, and lag; in addition, compared with traditional chemical pesticides, it does not generate selection pressure on pests and diseases and is not prone to drug resistance. After being stimulated by an elicitor, plants will produce various defense responses, mainly including: (1) at the physical level, causing the deposition of lignin and the formation of polyphenols such as callose and flavonoids in the cell wall to prevent the invasion of pathogenic bacteria; (2) activation and accumulation of plant endogenous hormones; (3) triggering reactive oxygen species (ROS) resistance and promoting programmed cell death in tissues; (4) inducing the expression of defense enzymes such as peroxidase, chitinase, polyphenol oxidase, and phenylalanine lyase in host plant cells, and causing the accumulation of defense compounds such as phytoalexins and flavonoids and defense-related toxic proteins. These defense responses indicate that elicitors play an important role in the process of plant resistance.

[0003] Non-protein amino acids (NPAAs) refer to the general term for amino acids that do not participate in protein synthesis. Generally, non-coding amino acids have the following characteristics: (1) they are toxic themselves and participate in the plant's defense process when the plant is damaged by the outside world; (2) they constitute the precursors and intermediates of plant secondary metabolism, such as the precursors of alkaloids; (3) they act as plant-derived elicitors to activate the plant's defense response. Some non-protein amino acids play extremely important roles in aspects such as plant salt tolerance, drought tolerance, cold tolerance, disease resistance, and antioxidant capacity. At present, except for the functions of a few NPAAs being confirmed, the physiological functions of the vast majority of non-coding amino acids in plants have not been confirmed, including non-protein amino acid β-Tyr. Therefore, it is of great significance to deeply explore the physiological function of β-Tyr in plants. Summary of the Invention

[0004] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide the application of β-Tyr as an elicitor in regulating the synthesis of jasmonates and / or plant insect resistance.

[0005] The purpose of the present invention is achieved by the following technical solutions:

[0006] The application of β-Tyr as an elicitor in regulating the synthesis of jasmonates (JAs) and / or plant insect resistance.

[0007] The chemical structural formula of the described β-Tyr (3-amino-3-(4-hydroxyphenyl) propionic acid) is shown in Formula I:

[0008]

[0009] The synthesis of the described jasmonates (JAs) is regulated by exogenously applying β-Tyr to activate the biosynthesis of jasmonates (JAs) in plants, so as to increase the jasmonates in plants.

[0010] The regulation of plant insect resistance is achieved by exogenously applying β-Tyr to activate the biosynthesis of jasmonates (JAs) in plants, so as to improve the insect resistance (resistance to insect pests) of plants.

[0011] The method of exogenously applying β-Tyr includes at least one of root absorption method, spraying method and smearing method; preferably the root absorption method or the spraying method.

[0012] The root absorption method is achieved by the following way: adding β-Tyr into the plant culture medium.

[0013] The culture medium is preferably a liquid culture medium.

[0014] The treatment concentration of the described β-Tyr is 0 - 50 mg / L (excluding 0); preferably 5 - 50 mg / L.

[0015] The described jasmonates (JAs) include jasmonic acid (JA) and / or jasmonic acid-isoleucine (JA-Ile).

[0016] The described plants include monocotyledonous plants and dicotyledonous plants; preferably crops, flowers or tobacco; further preferably at least one of rice, corn, wheat, sorghum, tomato, narcissus, tulip and Nicotiana benthamiana; more preferably at least one of rice, celery, tomato, Nicotiana benthamiana and narcissus.

[0017] The insect resistance is the resistance to pests.

[0018] The described pests are at least one of planthoppers and stem borers; preferably at least one of brown planthopper, white-backed planthopper, small brown planthopper and Chilo suppressalis; more preferably brown planthopper.

[0019] The present invention has the following advantages and effects compared with the prior art:

[0020] (1) The present invention takes non-coding protein amino acid (β-Tyrosine, β-Tyr) and conventional crops as objects. By using the root absorption method and spraying treatment method to treat test materials such as rice, tomato, tobacco, narcissus and celery, β-Tyr can be detected in the test plants after a period of time, indicating that β-Tyr can be absorbed and transported by different types of plants and then participate in the plant physiological metabolism process.

[0021] (2) For the plants treated with β-Tyr in the present invention, while β-Tyr can be detected in the plant tissues, a significant increase in the content of JAs substances can be detected, but the content of salicylic acid (SA) remains unchanged, and the change trend of the content of JAs is the same as that of β-Tyr, indicating that after being absorbed, β-Tyr can activate the synthesis of JAs in plant tissues, and this activation mode is universal and not affected by plant species.

[0022] (3) In the present invention, the insect resistance of rice treated with β-Tyr is evaluated. The resistance of rice treated with β-Tyr to the brown planthopper increases, and the oviposition of the brown planthopper is inhibited, but the treatment of rice with α-Tyr has no significant effect on the resistance of rice and the oviposition of the brown planthopper. The experimental results also show that the exogenous addition of β-Tyr can increase the resistance of rice to pests, and also indicate that β-Tyr may have the function of a plant elicitor.

[0023] (4) In the present invention, through the gastric toxicity experiment of β-Tyr on pests, there is no significant difference in the mortality rate of brown planthoppers fed with different concentrations of β-Tyr compared with the control, indicating that β-Tyr has no gastric toxicity effect on brown planthoppers and has the characteristics of an elicitor.

[0024] (5) Through the research on the activation of the synthesis of JAs in plant tissues by β-Tyr after being absorbed by plants, the influence of rice treated with β-Tyr on the resistance to the brown planthopper, and the gastric toxicity to the brown planthopper, the present invention reveals the mechanism of the action of β-Tyr on plant resistance and the role of β-Tyr as a new plant defense elicitor in increasing the resistance of plants. Therefore, this β-Tyr can be used as a new elicitor to stimulate the synthesis of Jas and increase the resistance of plants. Description of the Drawings

[0025] Figure 1is the content of β-Tyr in different tissue parts of rice at different time points (root absorption method); among them, a is the chromatogram of β-Tyr and α-Tyr in rice leaves treated with exogenous β-Tyr for 0-72 h; b is the content of β-Tyr in rice roots, leaf sheaths and leaves treated with exogenous β-Tyr for 0-72 h; c is the content of JA and JA-Ile in rice leaf sheaths at 0 h and 24 h after treatment with exogenous β-Tyr; d is the content of JA and JA-Ile in rice leaves at 0 h, 24 h and 48 h after treatment with exogenous β-Tyr; e is the content of SA in rice leaf sheaths at 0 h and 24 h after treatment with exogenous β-Tyr; f is the content of SA in rice leaves at 0 h and 24 h after treatment with exogenous β-Tyr.

[0026] Figure 2 are the chromatograms of β-Tyr and α-Tyr in the seed stems of different plants at 0 h and 24 h before and after β-Tyr treatment, and the content diagrams of JA and JA-Ile in the seed stems of different plants at 0 h, 24 h and 48 h (spraying method); among them, a is celery (Apium graveolens L.); b is tomato (Lycopersicon esculentum Mill); c is Nicotiana benthamiana; d is Tulipa gesneriana L.; e is rice (Oryza sativa BBS.L); f is rice (Oryza sativa lemont.L).

[0027] Figure 3 is the result diagram of the resistance response of rice to the brown planthopper by exogenous β-Tyr; among them, a is the phenotype of rice before exogenous tyrosine treatment; b is the phenotype of rice on the 10th day after being damaged by the brown planthopper after exogenous tyrosine treatment; c is the dynamic change diagram of the number of brown planthoppers in rice from the 2nd h to the 96th h after BPH damage; d is the statistical chart of the survival number of rice after BPH treatment.

[0028] Figure 4 is the diagram of the effect of exogenous β-Tyr treatment on the egg-laying number of the brown planthopper; among them, a is the experimental model of the brown planthopper egg-laying after exogenous tyrosine treatment; b is the statistical chart of the egg-laying number of the brown planthopper after different tyrosine treatments.

[0029] Figure 5 is the diagram of the effect of β-Tyr on the mortality rate of the brown planthopper; among them, a is the diagram of the brown planthopper feeding mode; b is the statistical chart of the mortality rate of the brown planthopper after treatment with different β-Tyr concentrations. Specific implementation manners

[0030] The present invention will be further described in detail below in conjunction with embodiments, but the embodiments of the present invention are not limited thereto. It should be noted that the following detailed descriptions are all illustrative and are intended to provide further explanations for the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application belongs. The test materials and reagents used in the following embodiments without specific description are conventional test materials and reagents in the art and can all be purchased through commercial channels. The test methods without specific experimental conditions noted in the following embodiments are generally carried out under conventional experimental conditions or according to the experimental conditions recommended by the manufacturer.

[0031] In the embodiments of the present invention, the structural formulas of the non-coding protein amino acids α-Tyr (L-tyrosine) and β-Tyr (3-amino-3-(4-hydroxyphenyl)propionic acid) involved are shown as follows and are purchased from InnoChem - High-end Chemical and Biological Reagents (inno-chem.com.cn).

[0032]

[0033] The rice variety PI312777 (abbreviation: PI) involved in the embodiments of the present invention has been disclosed in the reference (Qiu Zhenguo, Chen Xionghui, Liu Zeqiang, Peng Haifeng, Wan Banghui. Field evaluation of allelopathic potential and yield comparison of new antigenic rice line PI312777 [J]. Journal of Yunnan Agricultural University (Natural Science), 2012, 27(04): 461 - 466 + 474.).

[0034] The rice varieties BBS (Oryza sativa BBS.L) and lemont (Oryza sativa lemont.L) involved in the embodiments of the present invention are conventional rice varieties in the art and have been disclosed in the National Rice Data Center (https: / / www.ricedata.cn / ).

[0035] Example 1 Effect of exogenous β-Tyr on the synthesis of jasmonates (JAs) in plants

[0036] 1.1 Exogenous tyrosine treatment of rice (rice tyrosine root absorption method): Solutions of α-Tyr and β-Tyr with concentration gradients of 0 mg / L, 25 mg / L, and 50 mg / L were prepared respectively. Germinated PI rice seeds were sown into 96-well plates and cultured with complete rice nutrient solution until they had four leaves and one heart. Then, after being starved with deionized water for 48 h, they were replaced with rice basal culture solution (complete rice culture solution without nitrogen source), and α-Tyr and β-Tyr with concentrations of 0 mg / L and 25 mg / L were added respectively. Three replicates were set for each concentration, and each replicate had 96 plants. Roots, stems, leaves, and culture solutions of rice at 0 h before treatment and 0.5 h, 2 h, 6 h, 12 h, 24 h, 48 h, and 72 h after treatment were frozen in liquid nitrogen and stored in a -80 °C ultra-low temperature refrigerator for later use. Among them, the preparation method of the rice basal culture solution is as follows:

[0037] (1) P: Dissolve 10.08 g of NaH2PO4·2H2O in 200 mL of water; add 5 mL to every 4 L of water when in use;

[0038] (2) K: Dissolve 17.86 g of K2SO4 in 200 mL of water; add 5 mL to every 4 L of water when in use;

[0039] (3) Ca: Dissolve 6.328 g of CaCl2 in 200 mL of water; add 10 mL to every 4 L of water when in use;

[0040] (4) Mg: Dissolve 81 g of MgSO4·7H2O in 200 mL of water; add 5 mL to every 4 L of water when in use;

[0041] (5) Trace elements: Dissolve 0.7045 g of MnSO4·4H2O, 0.0507 g of Na2MoO4·2H2O, 0.467 g of H3BO3, 0.0175 g of ZnSO4·7H2O, and 0.0155 g of CuSO4·5H2O in 500 mL of water; add 5 mL to every 4 L of water when in use;

[0042] (6) Iron salt: Dissolve 3.725 g of disodium ethylenediaminetetraacetate (EDTA-Na2) and 2.785 g of FeSO4·7H2O together in 500 mL of water; add 8 mL to every 4 L when in use;

[0043] (7) Adjust the pH of the rice basal culture solution to 5.8.

[0044] 1.2. Exogenous tyrosine spraying method: After germination, the commercially available glass crisp celery (Apium graveolens L.), millennium tomato (Lycopersicon esculentumMill), Nicotiana benthamiana, Aladdin tulip (Tulipa gesneriana L.) and rice seeds (variety: BBS, lemont) were cultivated in seedling trays until 3 leaves and 1 heart, and then transplanted into pots and nutrient pots respectively. After the plants grew stably for a period of time, rice BBS and lemont with similar growth, celery, tomato, tulip and Nicotiana benthamiana with a seedling age of 50 days were set up as control group and treatment, respectively, with three replicates for each treatment. The treatment group was sprayed with 1 mL of β-Tyr with a concentration of 25 mg / L, and each plant in the control group was sprayed with an equal volume of deionized water, and samples were taken at 0h, 6h and 24h for later use. The leaf sheaths of rice, the fleshy leaves of tulip, the stems of tobacco, tomato and celery were taken. Before sampling, the β-Tyr remaining in the leaves and stems was rinsed clean as much as possible, and 100 mg of the sample was quickly weighed and frozen in liquid nitrogen, and then stored in a -80℃ refrigerator for later use.

[0045] 1.3. Tyrosine detection: To prove whether exogenous β-Tyr can be absorbed by plants and transported to different tissues, we tested the rice roots, stems, and leaves before and after the exogenous β-Tyr treatment in the above step 1.1, as well as the stems, leaves or leaf sheaths of different plants sprayed with β-Tyr in the above step 1.2. After treating the samples with 0.1% dilute hydrochloric acid, β-Tyr was quantitatively detected by LC-MS / MS method.

[0046] Chromatographic column: Mobile phase: Chromatographic column: Zorbax XDB-C18 (4.6 mm×250 mm, 5 μm); mobile phase A: 0.1% (v / v) acetic acid in water, mobile phase B: 0.1% (v / v) acetic acid in acetonitrile, flow rate is 300 μL / min, gradient elution program is shown in Table 1; column temperature is 42°C, injection volume is 1 μL.

[0047] Mass spectrometry conditions: electrospray ion source (ESI), negative ion scanning mode, multiple reaction monitoring, sample chamber temperature of 16 °C, capillary voltage of 3.5 kV, cone voltage of 15 V, ion source temperature of 150 °C, desolvation temperature of 100 °C, desolvation gas flow rate of 800 L / h, cone flow rate of 50 L / h.

[0048] Table 1

[0049]

[0050] 1.4 Determination of JA and SA contents: To prove the activation effect of exogenous β-Tyr on JAs in different crops, accurately weigh 200 mg of rice leaves and sheaths at 0 h, 6 h, 24 h, and 48 h treated by the β-Tyr root absorption method in step 1.1 above, and 200 mg of rice sheaths, leaves, or stems at 0 h, 6 h, and 24 h treated by exogenous spraying in step 1.2 above. Each treatment is repeated 3 times. After adding 1000 mL of 0.1% hydrochloric acid solution by mass and quickly grinding, shake in a metal bath at 6 °C and 1500 rpm for 4 h, centrifuge at 12000 rpm for 5 min in a 4 °C centrifuge, and then filter through a 0.22 μm filter membrane for measurement. JAs (jasmonic acid JA and jasmonic acid-isoleucine JA-Ile) and salicylic acid (SA) are quantitatively detected by LC-MS / MS method before and after using 0.1% dilute hydrochloric acid sample by mass. The liquid phase and mass spectrometry conditions are detected with reference to the tyrosine detection method in step 1.3.

[0051] 1.5 Experimental results: The results are as Figure 1 and Figure 2 shown. The experimental results show that, similar to the root absorption method, spraying exogenous β-Tyr can also be absorbed by plant leaves and transported to other tissue parts without being affected by the plant species. By detecting the JA content in the roots and stems of plants at different time periods after treatment with β-Tyr, it shows that β-Tyr can be absorbed and transported by plants, activating the biosynthesis of JAs in plants and promoting the increase of JAs in plants within a certain time range. The SA quantitative results show that β-Tyr has no activation effect on the synthesis of SA, indicating that after adding β-Tyr, the increase in plant resistance has nothing to do with the synthesis of SA.

[0052] Example 2 Effect of β-Tyr-treated rice on resistance to brown planthopper

[0053] 2.1 Brown planthopper treatment experiment: Refer to 1.1 above for the treatment of rice with exogenous tyrosine. Take PI rice at the tillering stage and transplant it into a large-hole hydroponic box, with 6 plants in each box and a total of 15 boxes. After the growth is stable, replace it with clear water for starvation treatment for 48 h, and then change to the basic culture solution (prepared in the same way as 1.1) and culture for 24 h. Randomly divide the rice into 5 groups, with 3 replicates in each group, and add α-Tyr and β-Tyr at concentrations of 0 mg / L (as a control), 25 mg / L, and 50 mg / L respectively for treatment for 12 h. Then, treat with 5 second-instar nymphs of brown planthopper per plant. During this period, count the dynamic changes of brown planthoppers in each treatment at each time period (2, 4, 6, 48, 72, 96 h), and count the mortality rate of rice plants on the 10th day after treatment.

[0054] 2.2 Experimental results: The results are as Figure 3As shown, the results showed that the number of Nilaparvata lugens treated with β-Tyr was significantly lower than that of the control and the rice treated with the addition of α-Tyr. With the increase of the treatment time, the difference in the number of Nilaparvata lugens on the rice treated with tyrosine at different concentrations became more obvious. The survival rate of the rice treated with β-Tyr on the tenth day was significantly higher than that of the CK and the rice treated with α-Tyr. Figure 3 a). It shows that the rice treated with β-Tyr can increase the resistance to Nilaparvata lugens, and the increase in β-Tyr resistance is related to the activation of JAs substances.

[0055] Example 3 Exogenous addition of β-Tyr affected the oviposition number of Nilaparvata lugens

[0056] 3.1 Nilaparvata lugens oviposition experiment: Place PI rice seedlings at the tillering stage (about 50 days) in 100 mL glass tubes. After the growth is stable, replace the clear water and starve for 48 h, and then replace it with the basic culture solution (prepared in the same way as 1.1) and culture for 1 day. Add α-Tyr and β-Tyr with concentrations of 0 mg / L (CK) and 25 mg / L respectively. After 12 h of treatment, inoculate 1 female Nilaparvata lugens at the oviposition stage in each test tube. After 5 days of culture, observe and count the oviposition number of Nilaparvata lugens on each rice plant under a stereomicroscope. Each treatment has 5 replicates. Figure 4 a).

[0057] 3.2 Experimental results: There was no significant difference in the oviposition amount of Nilaparvata lugens between the rice treated with the addition of α-Tyr and the control. At the same time, there was also no significant change in the oviposition number of Nilaparvata lugens treated with the addition of α-Tyr and β-Tyr. By comparing and analyzing the oviposition amount between the addition of β-Tyr and the control, it was found that the oviposition amount of Nilaparvata lugens was significantly lower than that of the control, and the oviposition amount decreased by 20% compared with the control. Figure 4 b). The above results indicate that exogenous β-Tyr affects the oviposition behavior of Nilaparvata lugens on rice.

[0058] Example 4 β-Tyr has no stomach toxicity to Nilaparvata lugens

[0059] 4.1 Experiment on feeding Nilaparvata lugens with exogenous β-Tyr: To verify the elicitor characteristics of β-Tyr, we first prepared a stock solution by adding β-Tyr to pure water. The artificial diet for BPH was formulated according to the D-97 recipe ("Wang, S., M. Tedesco, P. Alexander, M. Xu, and X. Fettweis, 2020: Quantifying spatiotemporal variability of ice algal blooms and the impact on surface albedo in southwest Greenland. The Cryosphere, 14, no. 8, 2687-2713, doi: tc-14-2687-2020."). The prepared artificial diet for Nilaparvata lugens was added with β-tyrosine at concentrations of 0 mg / L, 5 mg / L, 25 mg / L, and 50 mg / L respectively. The Nilaparvata lugens feeder was made of two layers and the tensile area was four times the original. The artificial diet was placed between the two layers of film. A 2.5 cm × 15 cm centrifuge tube was used as the feeding chamber, and 15 second-instar Nilaparvata lugens nymphs were placed in each feeding chamber. The artificial diet was placed at one end of the cavity, and the diet was changed every 2 days. The other end of the feeding chamber was covered with a layer of black nylon net to ensure air circulation in the chamber ( Figure 5 a). Each treatment had 6 replicates, and the experiment was set at 26 ± 2 °C, 80% relative humidity (RH), and a 12:12 L:D photoperiod. The number of dead Nilaparvata lugens was recorded once a day for 4 consecutive days.

[0060] 4.2 Experimental results: The stomach toxicity experiment on Nilaparvata lugens fed with β-Tyr showed that as the number of treatment days increased, the mortality rate of Nilaparvata lugens gradually increased. However, within a unit time, the mortality rate of Nilaparvata lugens decreased with the increase in the concentration of β-Tyr, indicating that β-Tyr has no direct stomach toxicity effect on Nilaparvata lugens ( Figure 5 b). This further shows that the resistance of β-Tyr to plants is that β-Tyr activates the biosynthesis of JAs in plants in the form of an elicitor, JAs transduction, and activates the increase of plant resistance, thereby improving the resistance of plants to insect pests.

[0061] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Application of β-Tyr as an elicitor in regulating the synthesis of jasmonates, characterized in that: The regulation of the synthesis of jasmonates is achieved by exogenously applying β-Tyr to activate the biosynthesis of jasmonates in plants, so as to increase the jasmonates in plants; The jasmonates are jasmonic acid and / or jasmonic acid-isoleucine; The plants are at least one of rice, celery, tomato, Nicotiana benthamiana and tulip; The treatment concentration of β-Tyr is 5-50 mg / L.

2. The application according to claim 1, wherein: The method of exogenously applying β-Tyr is at least one of root absorption method, spraying method and smearing method.

3. The application according to claim 2, characterized in that The root absorption method is achieved by the following way: adding β-Tyr to the plant culture medium.

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