Application of dihydroquercetin to improvement of drought resistance of wheat

Abstract

The invention discloses application of dihydroquercetin to improvement of drought resistance of wheat. The technical problem to be solved by the invention is how to improve the drought resistance of gramineous plants. The invention specifically discloses application of dihydroquercetin or salts thereof, or solvates of dihydroquercetin and salts thereof in any one of the following aspects. A1) application in improving the drought resistance of gramineous plants; a2) preparing a product for improving the drought resistance of the gramineous plants to be neutral. Dihydroquercetin is applied to the roots of the gramineous plants, the drought resistance of the gramineous plants can be improved, and the method can be used for industrial production.

Description

Technical Field

[0001] The invention specifically relates to the application of dihydroquercetin in improving the drought resistance of wheat. Background Art

[0002] Wheat is a highly adaptable and widely distributed world food crop, providing about 21% of food calories and 20% of protein for humans. Wheat has high nutritional value and can be used to make a variety of foods. It is the staple food of about 35%-40% of the world's population. Drought is one of the main abiotic stresses that restrict wheat production and has a great impact on wheat yield. Drought usually affects processes such as seed germination, seedling growth and accumulation of organic matter, causing irreversible losses to plants.

[0003] Dihydroquercetin is a dihydroflavonol that, as a product of flavonoid metabolism, also has strong antioxidant properties. Dihydroquercetin can protect cells from the harmful effects of free radicals and protect cell membranes from damage. Currently, most reports on dihydroquercetin are focused on animals, and there are few reports on the effects of dihydroquercetin in plants. Summary of the invention

[0004] The technical problem solved by the invention is how to improve the drought resistance of Gramineae plants, especially wheat.

[0005] In order to solve the above technical problems, the present invention provides the following applications.

[0006] Use of dihydroquercetin or its salt or solvate thereof in any of the following:

[0007] A1) Application in improving drought resistance of grass plants;

[0008] A2) Use of the neutral method for preparing a product for improving drought resistance of gramineous plants.

[0009] In the above, the grass plant may be a two-leaf, one-heart stage grass plant.

[0010] In the above, the drought resistance index may be root length, seedling length, fresh weight, dry weight, relative water content and / or survival rate.

[0011] In the above, the fresh weight may be the above-ground fresh weight of the plant or the below-ground fresh weight of the plant.

[0012] In the above, the dry weight may be the above-ground dry weight of the plant or the underground dry weight of the plant.

[0013] In the above, the relative water content may be the relative water content of leaves.

[0014] In the above, the drought condition for drought resistance may be 25g / 1000ml PEG 6000.

[0015] In the above application, the product described in A2) is a medicine.

[0016] In the above, the product or the drug described in A2) may include a pharmaceutically acceptable carrier. The material of the carrier includes, but is not limited to, a water-soluble carrier material (such as polyethylene glycol, polyvinyl pyrrolidone, organic acid, etc.), a poorly soluble carrier material (such as ethyl cellulose, cholesterol stearate, etc.), and an enteric carrier material (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). Among them, a water-soluble carrier material is preferred. These materials can be used to make a variety of dosage forms, including but not limited to tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, microspheres, transdermal agents, buccal tablets, suppositories, freeze-dried powder injections, etc. It can be a common preparation, a sustained-release preparation, a controlled-release preparation, and various microparticle delivery systems. In order to make a unit dosage form into a tablet, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; wetting agents and binders, such as water, glycerol, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, acacia paste, gelatin paste, sodium carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinyl pyrrolidone, etc.; disintegrants. , such as dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid esters, sodium lauryl sulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors, such as sucrose, tristearate, cocoa butter, hydrogenated oil, etc.; absorption promoters, such as quaternary ammonium salts, sodium lauryl sulfate, etc.; lubricants, such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. The tablets can also be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets. In order to make the unit dosage form into a pill, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents, such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinyl pyrrolidone, kaolin, talc, etc.; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or flour paste, etc.; disintegrants such as agar powder, dry starch, alginate, sodium lauryl sulfate, methylcellulose, ethylcellulose, etc. In order to prepare the unit dosage form into a suppository, various carriers known in the art can be widely used. Examples of carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, etc. In addition, colorants, preservatives or other materials can also be added to the pharmaceutical preparations as needed.

[0017] In order to solve the above technical problems, the present invention also provides a method for improving the drought resistance of Gramineae plants.

[0018] The method comprises applying the dihydroquercetin or its salt, or their solvate or the drug to gramineous plants to improve the drought resistance of gramineous plants.

[0019] In the above, the method of application may be root application.

[0020] In the above, the grass plant can be a grass plant in the seedling stage. The seedling stage can be a two-leaf one-heart stage.

[0021] In the above, the grass plant may be a two-leaf, one-heart stage grass plant.

[0022] In the above method, the application is root application.

[0023] In the above method, the concentration of dihydroquercetin in the liquid solution applied to the roots is 10 g / ml-20 g / ml.

[0024] In the above, the concentration of dihydroquercetin may be 11 g / ml-19 g / ml, 12 g / ml-18 g / ml, 13 g / ml-17 g / ml or 14 g / ml-16 g / ml, and may be specifically 15.2 g / ml.

[0025] In the above method, the concentration of dihydroquercetin in the liquid solution applied to the roots is 50 μM.

[0026] In the above application or any of the methods described in the figure, the Gramineae plant can be any of the following:

[0027] D1) Triticum plants;

[0028] D2) Wheat.

[0029] In the above, the wheat may be the wheat variety Chang 6878.

[0030] In order to solve the above technical problems, the present invention also provides a product.

[0031] The product contains the above-mentioned dihydroquercetin or its salt, or their solvate or the above-mentioned drug.

[0032] In order to solve the above technical problems, the present invention also provides a kit.

[0033] The kit contains the above-mentioned dihydroquercetin or its salt, or their solvate or the drug according to claim 2.

[0034] In order to solve the above technical problems, the present invention also provides a liquid preparation.

[0035] The dihydroquercetin content in the liquid is 10g / ml-20g / ml.

[0036] In the above, the concentration of dihydroquercetin can be 11g / ml-19g / ml, 12g / ml-18g / ml, 13g / ml-17g / ml or 14g / ml-16g / ml. Specifically, it can be 15.2g / ml

[0037] In the above, the content of dihydroquercetin in the liquid may be 50 μM.

[0038] Beneficial Effects

[0039] The problem to be solved by the present invention is to overcome the technical deficiencies and provide a flavonoid substance - dihydroquercetin, which can improve the drought resistance of wheat and alleviate the damage caused by drought stress to wheat.

[0040] The technical solution adopted by the present invention to solve the technical problem is: in order to achieve the above purpose, exogenous dihydroquercetin is used to improve the drought resistance of wheat.

[0041] In the above application, the characteristic of improving the drought resistance of wheat is to alleviate the growth inhibition of wheat variety Chang 6878 caused by indoor drought stress.

[0042] In the above application, the characteristic of improving wheat drought resistance is to alleviate the inhibition of stress-related physiological indicators of wheat variety Chang 6878 under indoor drought stress.

[0043] In the above application, the characteristic of improving the drought resistance of wheat is to alleviate the inhibition of indoor drought stress on the antioxidant capacity of the wheat variety Chang 6878.

[0044] In the above application, the characteristic of improving the drought resistance of wheat is to alleviate the damage of indoor drought stress to the cell membrane stability of the wheat variety Chang 6878.

[0045] The present invention discloses the application of dihydroquercetin to improve drought resistance of wheat. The technical problem solved by the present invention is how to improve drought resistance of gramineous plants. Specifically disclosed is the application of dihydroquercetin or its salts, or their solvates in any of the following: A1) application in improving drought resistance of gramineous plants; A2) application in preparing products for improving drought resistance of gramineous plants. The present invention can increase drought resistance of gramineous plants by applying dihydroquercetin to the roots of gramineous plants, and can be used for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1Figure 1 shows the phenotype of Chang 6878 treated with dihydroquercetin under normal and drought stress conditions; A shows the phenotype of Chang 6878 seedlings under different treatments; B shows the survival rate; C shows the relative water content; D shows the fresh weight of the aboveground part; E shows the fresh weight of the underground part; F shows the dry weight of the aboveground part; G shows the dry weight of the underground part; H shows the seedling length; I shows the root length; W, control group; WD, dihydroquercetin applied under normal conditions; D, drought treatment; DD, dihydroquercetin applied under drought conditions. Scale bar = 5 cm.

[0047] Figure 2 This is a graph showing the results of stress-related physiological values ​​measurement of Chang 6878 with and without dihydroquercetin under normal and drought stress conditions; A is CAT activity; B is SOD activity; C is POD activity; D is MDA content; E is superoxide anion content; F is H2O2 content; G is proline content; H is soluble protein content; I is chlorophyll a concentration; J is chlorophyll b concentration; K is total chlorophyll concentration; L is carotenoid concentration; W, control group; WD, dihydroquercetin applied under normal conditions; D, drought treatment; DD, dihydroquercetin applied under drought conditions.

[0048] Figure 3 Overview and Venn diagram of DEGs identified by transcriptome analysis for comparison of the four treatments using |log2(fold change)|>1 and FDR<0.05; A is principal component analysis of transcriptomic data of the four treatment groups, B is Pearson correlation analysis of transcriptomic data from 12 different samples, and C is the total number of DEGs in the four treatments (DD vs. D, WD vs. W); orange represents upregulated genes, while blue represents downregulated genes; D is the Venn diagram of DEGs identified in DD vs. D and WD vs. W; overlapping areas represent co-expressed DEGs between different datasets, and numbers in single circles represent DEGs expressed in only one pool; W, control group; WD, dihydroquercetin applied under normal conditions; D, drought treatment; DD, dihydroquercetin applied under drought conditions.

[0049] Figure 4 Figure 2 is a GO and KEGG enrichment analysis of differentially expressed genes in different treatment groups; A is a statistical graph of the GO annotation classification of differentially expressed genes between group D and group DD; B is a statistical graph of the GO annotation classification of differentially expressed genes between group W and group WD; C is a scatter plot of KEGG pathway enrichment of differentially expressed genes between group D and group DD; D is a scatter plot of KEGG pathway enrichment of differentially expressed genes between group W and group WD.

[0050] Figure 5The effect of dihydroquercetin on phenylpropanoid biosynthesis, linoleic acid and α-linolenic acid metabolism, and glycerophospholipid metabolism in wheat seedlings under drought stress; A is phenylpropanoid biosynthesis, B is linoleic acid and α-linolenic acid metabolism, C is glycerophospholipid metabolism, and D is the expression level of DEGs in important pathways; the horizontal axis in the figure is different treatments, and the vertical axis is the gene name; red indicates up-regulated gene expression, while blue indicates down-regulated gene expression. D, drought treatment; DD, dihydroquercetin applied under drought conditions.

[0051] Figure 6 Comparison of qRT-PCR and RNA-Seq of important differentially expressed genes in phenylpropanoid biosynthesis, linoleic acid and α-linolenic acid metabolism, and glycerophospholipid metabolism, where A, B, C, D, E, F, G, H, and I correspond to different genes or different transcripts of the same gene, respectively; W, control group; WD, dihydroquercetin applied under normal conditions; D, drought treatment; DD, dihydroquercetin applied under drought conditions. DETAILED DESCRIPTION

[0052] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0053] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0054] The following examples used GraphPad Prism 9.0 software to analyze morphological data, physiological indicators, and qRT-PCR data. Statistical significance was set at P < 0.05.

[0055] Example 1 Effect of exogenous dihydroquercetin on the growth of wheat variety Chang 6878 under drought stress

[0056] Materials and Methods

[0057] The molecular formula of dihydroquercetin is C 15 H 12 O7, CAS number is 480-18-2.

[0058] The composition of wheat nutrient solution is shown in Table 1.

[0059] Table 1 Wheat nutrient solution composition

[0060]

[0061] Wheat nutrient solution mother solution: weigh the corresponding mass of substances according to the unit volume (Table 1), and prepare the mother solutions of each substance according to the mother solution multiples in Table 1 (wherein, the mother solution of iron-EDTA mixed solution needs to first weigh the corresponding mass of iron-containing substances, add water and heat to fully dissolve, and at the same time weigh disodium EDTA and dissolve it in water, then add the sodium EDTA solution to the iron-containing solution and make up the volume).

[0062] Wheat nutrient solution: Dilute each substance in ddH2O at a concentration of 1 using the above method to obtain wheat nutrient solution.

[0063] Wheat nutrient solution containing dihydroquercetin: weigh 0.0152 g of dihydroquercetin, add 300 μL of anhydrous ethanol to dissolve it, and then add the prepared wheat nutrient solution to make the volume to 1 L to obtain the dihydroquercetin wheat nutrient solution.

[0064] Wheat nutrient solution containing 25% PEG 6000: weigh 25 g PEG 6000, add the prepared wheat nutrient solution to make the volume to 1 L, and obtain wheat nutrient solution containing 25% PEG 6000.

[0065] Wheat nutrient solution containing dihydroquercetin-25% PEG 6000: Weigh 0.0152 g of dihydroquercetin, add 300 μL of anhydrous ethanol to dissolve it, and then add wheat nutrient solution containing 25% PEG 6000 to make the volume to 1 L to obtain wheat nutrient solution containing dihydroquercetin-25% PEG 6000.

[0066] Pick an appropriate amount of wheat variety Chang 6878 seeds in a culture dish, use 2% H2O2 for disinfection, soak for 30 minutes, and wash with clean water 3-6 times. Germinate the seeds at room temperature for 3 days, pick the germinated seeds with the same growth status, and move them to a black light-proof plant hydroponic box 96-well plate, with one hole between every two seeds, and plant them in a cross-type, that is, plant 48 wheat seeds of the same strain on each plate. The transplanted wheat plants were moved to the greenhouse for culture, and the culture conditions were set to 20℃ light for 14h (light intensity 600μmoles / m 2 / s), 18℃ dark for 10h, start to culture with tap water for 3 days, change to wheat nutrient solution for 4 days, wheat seedlings grow to two leaves and one heart stage (total culture for 7 days), start to process on the 7th day of culture, the operation is as follows:

[0067] The above wheat was randomly divided into 4 groups, namely W group, WD group, D group and DD group, with 48 wheat plants in each group. Each treatment was repeated three times, and the phenotypes were observed and photographed, which were recorded as before treatment.

[0068] Group W:

[0069] The cells were transferred to wheat nutrient solution (should be wheat nutrient solution, cultured in water for the first three days and replaced with nutrient solution on the fourth day) and cultured under 20°C light (light intensity 600 μmoles / m 2 / s) for 14h, 18℃ dark for 10h, and after 7 days of culture, samples were taken to test various physiological indicators, including CAT, SOD, POD activity, malondialdehyde content, proline and soluble protein content, and chlorophyll concentration of each component. Phenotypic observation and photography were performed for 20 days of culture, which was recorded as treatment 20d; then the seeds were transferred to wheat nutrient solution for culture, and the culture conditions were set to 20℃ light (light intensity 600μmoles / m 2 / s) for 14 h, 18 ℃ in the dark for 10 h, and cultured for 5 days for phenotypic observation and photography, which was recorded as 5 days after rehydration. Plants were randomly selected for testing of survival rate, aboveground fresh weight, underground fresh weight, aboveground dry weight, underground dry weight, seedling length, and root length.

[0070] WD Group:

[0071] The only difference from the W group was that the wheat nutrient solution was replaced with wheat nutrient solution containing dihydroquercetin, and the rest of the operations were the same as the W group.

[0072] Group D:

[0073] The only difference from the W group was that the wheat nutrient solution was replaced with a wheat nutrient solution containing 25% PEG 6000, and the rest of the operations were the same as the W group.

[0074] DD Group:

[0075] The only difference from the W group was that the wheat nutrient solution was replaced with a wheat nutrient solution containing dihydroquercetin-25% PEG 6000, and the rest of the operations were the same as the W group.

[0076] Root length: Use scissors to separate the seedlings from the roots, and use a ruler to directly measure the length of the roots.

[0077] Seedling length: Use scissors to separate the seedling from the root, and use a ruler to directly measure the length of the seedling.

[0078] Fresh weight of above-ground part: Use scissors to separate the seedlings from the roots, and weigh the seedlings with a thousandth electronic scale.

[0079] Dry weight of above-ground part: Use scissors to separate the seedlings from the roots, then place them in a 60℃ oven for 72h, and weigh the seedlings again after the moisture is completely dried.

[0080] Fresh weight of underground part: Use scissors to separate the seedlings from the roots, and weigh the roots with a thousandth electronic scale.

[0081] Dry weight of underground part: Use scissors to separate the seedlings from the roots, then place them in a 60℃ oven for 72h, and weigh the roots again after the moisture is completely dried.

[0082] Survival rate: 5 days after rehydration, the number of surviving seedlings in each treatment was counted, and the survival rate of each treatment was calculated according to the following formula: Survival rate (%) = (number of surviving seedlings / total number of seedlings) × 100%

[0083] Relative water content: Cut plant tissue (about 0.1g) and weigh the fresh weight W F Place it in a conical flask filled with distilled water, seal it and leave it for 24 hours, take out the leaves, wipe off the water on the surface of the leaves, and weigh its weight as W. T The leaves were dried at 105°C for 15 min, and then dried at 80°C for 72 h until constant weight was reached. After natural cooling, the leaves were weighed and their mass was recorded as W. D The relative water content of leaves was calculated according to the following formula:

[0084] Relative water content (RWC) = (W F -W D ) / (W T -W D )*100%.

[0085] The root length refers to the part of the wheat seedling excluding the leaves and stems.

[0086] The seedling length is the leaves and stems of wheat seedlings.

[0087] The above-ground parts are leaves and stems of wheat seedlings.

[0088] The underground part is the part of the wheat seedling excluding leaves and stems.

[0089] The experimental results are as follows Figure 1 ( Figure 1 In the figure, W is W group, WD is WD group, D group is D group, and DD is DD group; A is the phenotype of Chang 6878 seedlings under different treatments, before treatment is the phenotype before treatment, 20d after treatment is the phenotype after 20 days of treatment, and 5d after rehydration is the phenotype after 5 days of recovery; B is the survival rate; C is the relative water content; D is the fresh weight of the aboveground part; E is the fresh weight of the underground part; F is the dry weight of the aboveground part; G is the dry weight of the underground part; H is the seedling length; I is the root length). Figure 1 The results showed that the seedlings of Chang 6878 at the two-leaf-one-heart stage were treated with four different treatments. On the 20th day, the application of dihydroquercetin under drought conditions could alleviate the inhibition of wheat growth caused by drought ( Figure 1Middle A). By measuring the root length, seedling length, fresh weight, dry weight and other indicators, we found that under normal conditions, exogenous dihydroquercetin significantly reduced the aboveground dry weight of Chang 6878 and increased the fresh weight and dry weight of the underground part, but had no significant effect on the survival rate, relative water content, seedling length and root length. Under drought conditions, exogenous dihydroquercetin significantly increased the survival rate, relative water content, aboveground fresh weight, aboveground dry weight, underground fresh weight and underground dry weight of Chang 6878, but had no significant effect on seedling length and root length ( Figure 1 These experimental results indicate that exogenous application of dihydroquercetin can alleviate the growth inhibition of wheat variety Chang 6878 under indoor drought conditions.

[0090] Example 2 Effect of exogenous dihydroquercetin on stress-related physiological parameters of wheat variety Chang 6878 under drought stress

[0091] Materials and Methods

[0092] When the wheat seedlings of group W, group WD, group D and group DD in Example 1 began to be cultured to the 7th day, the wheat leaves were taken to measure the CAT, SOD, POD, MDA content, superoxide anion content, H2O2 content, proline content, soluble protein content, etc.

[0093] CAT: On the 7th day after treatment, samples were taken from each treatment, about 0.1 g of leaves were weighed, frozen in liquid nitrogen, and then ground into powder using an automatic sample grinder. 1 ml of extract was added, and the mixture was shaken and mixed, then centrifuged at 8000 g at 4 °C for 10 min and placed on ice for testing. Subsequent steps were carried out according to the instructions. Catalase (CAT, BC0205) activity assay kit was purchased from Beijing Solebow Technology Co., Ltd.

[0094] SOD: On the 7th day after treatment, samples were taken from each treatment, about 0.1 g of leaves were weighed, frozen in liquid nitrogen, and then ground into powder using an automatic sample grinder. 1 ml of extract was added, and the mixture was shaken and mixed, then centrifuged at 8000 g at 4 °C for 10 min and placed on ice for testing. Subsequent steps were carried out according to the instructions. The superoxide dismutase (SOD, BC0175) activity assay kit was purchased from Beijing Solebow Technology Co., Ltd.

[0095] POD: On the 7th day after treatment, samples were taken from each treatment, about 0.1 g of leaves were weighed, frozen in liquid nitrogen, and then ground into powder using an automatic sample grinder. 1 ml of extract was added, and the mixture was shaken and mixed, then centrifuged at 8000 g at 4 °C for 10 min and placed on ice for testing. Subsequent steps were carried out according to the instructions. The peroxidase (POD, BC0095) activity assay kit was purchased from Beijing Solebow Technology Co., Ltd.

[0096] MDA content: On the 7th day after treatment, samples were taken from each treatment, about 0.1 g of leaves were weighed, frozen in liquid nitrogen, and then ground into powder using an automatic sample grinder. 1 ml of extract was added, and the mixture was shaken and mixed, then centrifuged at 8000 g at 4 °C for 10 min and placed on ice for testing. Subsequent steps were carried out according to the instructions. Malondialdehyde content (MDA, BC0025) assay kit was purchased from Beijing Solebow Technology Co., Ltd.

[0097] Superoxide anion content: On the 7th day after treatment, samples were taken for each treatment, about 0.1g of leaves were weighed, frozen in liquid nitrogen, and then ground into powder using an automatic sample grinder. 1ml of extract was added, and after shaking and mixing, centrifuged at 8000g at 4℃ for 10min and placed on ice for testing. Subsequent steps were carried out according to the instructions. The superoxide anion (SA-1-G) kit was purchased from Suzhou Keming Biotechnology Co., Ltd.

[0098] H2O2 content: On the 7th day after treatment, samples were taken from each treatment, about 0.1g of leaves were weighed, frozen in liquid nitrogen, and then ground into powder using an automatic sample grinder. 1ml of extract was added, and after shaking and mixing, the samples were centrifuged at 8000g and 4℃ for 10min, and placed on ice for testing. Subsequent steps were carried out according to the instructions. The H2O2 (BC3595) content determination kit was purchased from Beijing Solebow Technology Co., Ltd.

[0099] Proline content: The proline content (Proline, G0111F96) kit was purchased from Suzhou Keming Biotechnology Co., Ltd. Weigh about 0.1g of leaves, grind them with an automatic sample grinder, then add 1ml of extract, shake and mix, centrifuge at 1000g, 4℃ for 10min, and place on ice for testing. Then follow the instructions of the kit for subsequent steps.

[0100] Soluble protein content: The soluble protein content (ADS-F-SP002-96) kit was purchased from Jiangsu Jingmei Biotechnology Co., Ltd. Weigh about 0.1g of leaves, grind them with an automatic sample grinder, then add 1ml of extract, shake and mix, centrifuge at 1000g, 4℃ for 10min, and place on ice for testing. Then follow the instructions of the kit for subsequent steps.

[0101] The experimental results are as follows Figure 2 ( Figure 2In the figure, W is W group, WD is WD group, D group is D group, and DD is DD group; A is CAT activity; B is SOD activity; C is POD activity; D is MDA content; E is superoxide anion content; F is H2O2 content; G is proline content; H is soluble protein content; I is chlorophyll a concentration; J is chlorophyll b concentration; K is total chlorophyll concentration; L is carotenoid concentration). As shown in the figure, the Chang 6878 seedlings at the two-leaf and one-heart stage were treated with four treatments. On the 7th day, the exogenous application of dihydroquercetin under drought conditions can alleviate the inhibition of drought on the physiological condition of wheat. The results showed that under normal nutrient solution conditions, the exogenous application of dihydroquercetin had little effect on the physiological condition of wheat. Under drought conditions, the application of dihydroquercetin significantly increased the activity of antioxidant enzymes CAT, SOD, and POD, reduced the content of MDA, superoxide anion, and H2O2 in leaf cells, and increased the content of osmotic regulators proline and soluble protein. At the same time, the use of dihydroquercetin under drought stress also increased the concentrations of chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids in leaves ( Figure 2 ). These results indicate that exogenous application of dihydroquercetin can improve the antioxidant capacity of wheat by increasing the activity of antioxidant enzymes, reducing the content of reactive oxygen species in cells, and maintaining the stability of cell membranes.

[0102] Example 3 Effect of exogenous dihydroquercetin on antioxidant capacity and cell membrane stability of wheat variety Chang 6878 under drought stress

[0103] Materials and Methods

[0104] After the wheat seedlings of group W, group WD, group D and group DD in Example 1 were cultured for 3 days, leaves at the same position were selected for sampling, 3 replicates were taken for each treatment, a total of 12 samples, which were quickly frozen in liquid nitrogen and then transferred to a refrigerator at -80°C for storage. RNA was extracted from the sample tissues for subsequent transcriptome sequencing.

[0105] Experimental Results

[0106] The results are as follows Figure 3 and Figure 4 As shown in the figure, principal component analysis and the Person correlation coefficient between different samples show that each sample has good repeatability and reliability ( Figure 3 A and B). Using Fold Change ≥ 2 and P value < 0.05 as the criteria for screening differentially expressed genes, the results showed that 101 genes were up-regulated and 469 genes were down-regulated in DD vs D. 137 genes were up-regulated and 149 genes were down-regulated in WD vs W ( Figure 3C and D). The differentially expressed genes were analyzed by GO and KEGG enrichment analysis. The results showed that the differentially expressed genes after dihydroquercetin application were enriched in metabolism, cell, single organic process, membrane, organelle, and binding and catalytic activity in the biological process, cellular component and molecular function of GO, respectively ( Figure 4 Middle A). The KEGG pathways were mainly enriched in phenylpropanoid biosynthesis, linoleic acid metabolism, α-linolenic acid metabolism and lipid metabolism ( Figure 4 (C). The differentially expressed genes in WD vs W were mainly enriched in KEGG pathways of phenylpropanoid biosynthesis, plant hormone signal transduction, glyoxylate and dicarboxylate metabolism, carbon fixation in photosynthetic organisms, and carbon metabolism ( Figure 4 (middle D).

[0107] Further analysis of differentially expressed genes in phenylpropanoid biosynthesis, linoleic acid metabolism, α-linolenic acid metabolism, and glycerophospholipid metabolism revealed that exogenous dihydroquercetin under drought conditions could affect the expression of β-glucosidase BGLU2, aldehyde dehydrogenase ALDH, lipoxygenase LOX, nonspecific phospholipase C NPC, and glycerophosphodiester phosphodiesterase GDPD genes ( Figure 5 )

[0108] The expression of these genes was verified by qRT-PCR as follows:

[0109] 1. RNA Extraction

[0110] RNA was extracted using the KK Ultrafast Plant Total RNA Extraction Kit (ZP405K-2) from Zhuangmeng Biotechnology. Total RNA was extracted according to the instructions in the kit. The clean bench was sprayed with RNase-free reagent in the early stage to reduce RNase interference in plant RNA extraction. The centrifuge tubes and gun tips used were RNase-free treated. The operation steps are as follows:

[0111] (1) Take 0.1 g of fresh plant leaves and place them in a centrifuge tube filled with steel balls. Immediately freeze them in liquid nitrogen and grind them thoroughly with a tissue grinder under low temperature conditions until the leaves are in powder form.

[0112] (2) Take out the pre-cooled plant lysate R from the refrigerator, pipette 1 mL of the lysate into the ground sample, and immediately mix quickly using a vortexer (QL-901, Haimen) to form a uniform solution.

[0113] (3) Add 200 μL of chloroform to the centrifuge tube and quickly vortex to mix.

[0114] (4) Centrifuge at 12,000 rpm at 4°C for 2 min.

[0115] (5) Take out the centrifuge tube and separate the sample into three layers. Carefully transfer the upper water layer containing RNA to a new 1.5 mL centrifuge tube, about 600-700 μL.

[0116] (6) Add 1 / 2 volume of anhydrous ethanol (about 300 μL) to a new RNase-free centrifuge tube, invert to mix, and transfer to the adsorption column with a collection tube.

[0117] (7) Centrifuge at 4°C, 12,000 rpm for 1 min. Discard the waste liquid and return the adsorption column to the collection tube.

[0118] (8) Add 650 μL of rinse solution to the adsorption column, centrifuge at 12,000 rpm and 4°C for 1 min, and discard the waste solution.

[0119] (9) Repeat step (8).

[0120] (10) Place the adsorption column back into the empty collection tube, centrifuge at 13,000 rpm and 4°C for 2 min, remove the rinse solution, and place the adsorption column into a clean RNase-free centrifuge tube.

[0121] (11) Add 60 μL of RNase-free water to the center of the adsorption membrane and leave it at room temperature for 2 minutes to allow the RNA to

[0122] The mixture was fully dissolved in water and centrifuged at 12,000 rpm at 4°C for 2 min to collect the RNA solution.

[0123] (12) The quality of RNA extraction was tested by electrophoresis. 5 μL of RNA was analyzed by electrophoresis using 1.2% agarose gel. The bands were clear and were used for the next experiment.

[0124] 2. Synthesis of cDNA

[0125] Use Quanshijin One-Step gDNA Removal and cDNA Synthesis SuperMix (AT311) was used to complete the cDNA synthesis. The reaction system is shown in Table 2. The reaction system needs to be centrifuged briefly to mix. The PCR reaction procedure is shown in Table 3.

[0126] Table 2 cDNA synthesis reaction system

[0127]

[0128] Table 3 PCR reaction procedure

[0129] temperature time 25℃ 10min 42℃ 30min 85℃ 5s

[0130] 3 Reaction system

[0131] qRT-PCR assay was performed using Quanshijin FastGreen qPCR SuperMix (AQ611), the instrument used was a real-time quantitative PCR instrument (ABI7500, USA), the reaction system is shown in Table 4, and the reaction program is shown in Table 5.

[0132] Table 4 qRT-PCR reaction system

[0133]

[0134] Table 5 qRT-PCR reaction procedure

[0135]

[0136]

[0137] 4qRT-PCR primers

[0138] Nine important differentially expressed genes obtained from the transcriptome and key genes for phenylpropanoid and flavonoid biosynthesis were selected for qRT-PCR analysis to verify the accuracy and repeatability of the transcriptome sequencing data. RNase was used as the internal reference gene, and the primers used in the experiment were all from Table 6.

[0139] Table 6 qRT-PCR primers

[0140]

[0141] The results are as follows Figure 6 ( Figure 6 In the figure, W is the W group, WD is the WD group, D is the D group, and DD is the DD group; AH represent the expression levels of different genes, respectively), which is consistent with the transcriptome results.

[0142] BGLU2 is a β-glucosidase. The application of dihydroquercetin under drought conditions can upregulate the expression of this gene. Studies have shown that high expression of BGLU2 can improve the drought resistance of plants. In the phenylpropanoid biosynthesis pathway, BGLU2 can catalyze the production of coumarin, which is an antioxidant that can inhibit lipid peroxidation and scavenge free radicals to promote growth. As an aldehyde dehydrogenase, the upregulation of ALDH can reduce the toxic effects of aldehydes on cells, maintain the level of ROS in plants, and thus improve the drought resistance of plants ( Figure 5A). LOX is a key enzyme in the metabolic pathway of linoleic acid and α-linolenic acid. It can catalyze the oxidation of unsaturated fatty acids to produce peroxidation products, thereby destroying the integrity of cell membranes and causing irreversible damage to plants. Application of dihydroquercetin under drought stress can significantly downregulate the expression of LOX genes, inhibit the degree of cell membrane lipid peroxidation, increase the ratio of unsaturated fatty acids to saturated fatty acids, maintain the fluidity and stability of cell membranes, and thus make plants show higher drought resistance ( Figure 6 Middle B). The use of dihydroquercetin under drought stress can also increase the expression of NPC and GDPD in the glycerophospholipid metabolic pathway ( Figure 6 C). NPC and GDPD are important enzymes in the lipid metabolism pathway of plants. The activity of NPC affects the stability of cell membranes. NPC is responsible for producing DAG and phosphatidylcholine, which enter the cell metabolic pathway. DAG can serve as a precursor to replace the production of sulfur and glycolipids, thereby maintaining the integrity of the membrane. GDPD can participate in lipid remodeling and maintain cell membrane stability under phosphorus-deficient conditions. GDPD hydrolyzes glycerophosphodiester into glycerol-3-phosphate (G-3-P) and the corresponding alcohol. The accumulation of glycerol triphosphate can enhance the drought resistance of plants. G-3-P can be converted into DAG by acyltransferases and phospholipid phosphatases to maintain the stability of cell membranes.

[0143] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Use of dihydroquercetin or its salt, or its solvate in any of the following: A1) Application in improving drought resistance of grass plants; A2) Use of the neutral method for preparing a product for improving drought resistance of gramineous plants.

2. The use according to claim 2, characterized in that A2) The product is a drug.

3. A method for improving drought resistance of Gramineae plants, characterized in that: The method comprises applying the dihydroquercetin or its salt, or their solvate as claimed in claim 1 or the drug as claimed in claim 2 to gramineous plants to improve the drought resistance of gramineous plants.

4. The method according to claim 3, characterized in that The application is a root application.

5. The method according to claim 4, characterized in that The concentration of dihydroquercetin in the liquid preparation for root application is 10 g / ml-20 g / ml.

6. The method according to claim 4, characterized in that The concentration of dihydroquercetin in the root-applied solution was 50 μM.

7. The use according to claim 1 or 2 or the method according to any one of claims 3 to 6, characterized in that: The grass plant may be any of the following: D1) Triticum plants; D2) Wheat.

8. A product, characterized in that The product contains the dihydroquercetin or its salt according to claim 1, or their solvates or the drug according to claim 2.

9. A kit, characterized in that The kit contains the dihydroquercetin or a salt thereof, or a solvate thereof according to claim 1 or the drug according to claim 2.

10. A liquid preparation, characterized in that The dihydroquercetin content in the liquid is 10g / ml-20g / ml.

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