Application of exogenous treatment in promotion of soybean germination and enrichment of isoflavone content

Abstract

The invention relates to the technical field of agricultural biology. The invention provides application of exogenous treatment in promotion of plant germination and enrichment of isoflavone content. The exogenous treatment is hydrogen sulfide donor treatment. Preferably, the plant is soybean, and the hydrogen sulfide donor treatment is applied to at least one of the following (b1)-(b28). Preferably, the hydrogen sulfide donor is NaHS (sodium hydrogen sulfide). The invention further provides a method for promoting plant germination. The invention also provides a special culture solution for implementing the method. The invention further provides a method for promoting enrichment of isoflavone in plants. The invention also provides the soybean sprouts produced by the method. According to the method, the NaSH serving as a hydrogen sulfide donor is adopted for treatment, so that soybean germination can be promoted, the extension of the soybean sprouts is promoted, and the fresh weight of the soybean sprouts is also promoted; meanwhile, the enrichment of isoflavone content can be promoted, and a basis is provided for producing functional bean sprout products with higher nutritional value.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, specifically relating to the application of exogenous treatment in promoting soybean germination and enriching isoflavone content. Background Technology

[0002] Soybeans Glycine max L. As a globally important food and economic crop, soybeans are not only rich in protein and lipids, but also contain a series of bioactive substances, such as isoflavones, tocopherols, and soy saponins, thus attracting considerable attention. Despite the numerous benefits of soybeans, anti-nutritional factors such as protease inhibitors, lectins, and soy toxins limit their dietary value. Therefore, improving the dietary value of soybeans has become a hot research topic.

[0003] Existing research has shown that germination is one of the cheapest and most effective methods to improve the nutritional quality of soybeans and reduce the levels of anti-nutritional factors. During soybean germination, the content of isoflavones increases significantly. Soy isoflavones are an important class of natural secondary metabolites, mainly found in bean sprouts and seeds, and possess significant antioxidant, cardiovascular disease prevention, and anti-cancer physiological activities, earning them the title of "phytoestrogens." Therefore, studying the content, types, and accumulation mechanisms of isoflavones in sprouted soybeans under different treatments, and how to safely and effectively increase the isoflavone content in soybeans, especially soybean sprouts, has become a research hotspot in the fields of functional foods and agricultural breeding. However, in the conventional germination process, weak germination growth is often a problem. Furthermore, as an ideal carrier for isoflavone accumulation, the natural isoflavone content in bean sprouts still has considerable room for improvement.

[0004] Therefore, existing technologies urgently need to be addressed. Summary of the Invention

[0005] This invention aims to address the shortcomings of existing technologies by providing an application of exogenous treatment to promote soybean germination and isoflavone enrichment. This invention uses hydrogen sulfide donor NaSH to treat soybeans, which promotes soybean germination, elongation of soybean sprouts, and fresh weight of soybean sprouts; simultaneously, it promotes isoflavone enrichment.

[0006] To address the aforementioned problems in the prior art, the present invention provides the following technical solution: An application of an exogenous treatment to promote plant germination and enrichment of isoflavone content, wherein the exogenous treatment is a hydrogen sulfide donor treatment.

[0007] In the application described above, the plant is any one of the following (a1)-(a3): (a1) Dicotyledonous plants; (a2) Legumes; (a3) soybean.

[0008] The use as described above, the plant is soybean, the hydrogen sulfide donor treatment is applied in at least one of (b1)-(b28): (b1) increasing the soybean sprout length; (b2) preparing a product for increasing the soybean sprout length; (b3) increasing the fresh weight of soybean; (b4) preparing a product for increasing the fresh weight of soybean; (b5) increasing the endogenous hydrogen sulfide content of soybean; (b6) preparing a product for increasing the endogenous hydrogen sulfide content of soybean; (b7) increasing the isoflavone content of soybean; (b8) preparing a product for increasing the isoflavone content of soybean; (b9) increasing the total malonyl glucoside content of soybean; (b10) preparing a product for increasing the total malonyl glucoside content of soybean; (b11) increasing the genistin content of soybean; (b12) preparing a product for increasing the genistin content of soybean; (b13) increasing the malonyl daidzin content of soybean; (b14) preparing a product for increasing the malonyl daidzin content of soybean; (b15) increasing the malonyl genistin content of soybean; (b16) preparing a product for increasing the malonyl genistin content of soybean; (b17) increasing the activity of the key enzymes PAL, C4H and / or 4CL in isoflavone synthesis of soybean; (b18) preparing a product for increasing the activity of the key enzymes PAL, C4H and / or 4CL in isoflavone synthesis of soybean; (b19) reducing the MDA content of soybean; (b20) preparing a product for reducing the MDA content of soybean; (b21) reducing the H2O2 content of soybean; (b22) preparing a product for reducing the H2O2 content of soybean; (b23) increasing the activity of the antioxidant enzymes SOD, POD and / or CAT of soybean; (b24) preparing a product for increasing the activity of the antioxidant enzymes SOD, POD and / or CAT of soybean; (b25) increasing the content of the endogenous hormones GA, IAA and / or ETH of soybean; (b26) preparing a product for increasing the content of the endogenous hormones GA, IAA and / or ETH of soybean; (b27) reducing the content of endogenous hormones ABA, JA in soybean; (b28) preparing a product with reduced content of endogenous hormones ABA, JA in soybean.

[0009] The application as described above, wherein the hydrogen sulfide donor is NaHS.

[0010] The application as described above, wherein the hydrogen sulfide donor is administered at a concentration of 0.1-1000 μM.

[0011] The application as described above, wherein the hydrogen sulfide donor is administered at a concentration of 10 μM.

[0012] Based on the same inventive concept, the present application also provides a method for promoting germination of plants, which comprises the following steps: (1) soaking the seeds of plants in an aqueous sodium hypochlorite solution for disinfection, and then washing with sterile water until the sodium hypochlorite solution is completely removed; (2) soaking the seeds disinfected in step (1) in distilled water for 6-8 hours; (3) evenly spreading the seeds soaked in step (2) in a germination box containing moistened filter paper, adding a hydrogen sulfide donor as a culture solution in the germination box, wherein the hydrogen sulfide donor is NaHS, and promoting germination of plants in the dark at 20-30°C.

[0013] Based on the same inventive concept, the present application also provides a special culture solution for implementing the method as described above, wherein the culture solution is an aqueous NaHS solution with a concentration of 0.1-1000 μM.

[0014] Based on the same inventive concept, the present application also provides a method for promoting enrichment of isoflavone content in plants, which comprises treating with a hydrogen sulfide donor, wherein the hydrogen sulfide donor is NaHS, and the hydrogen sulfide donor is administered at a concentration of 0.1-1000 μM.

[0015] Based on the same inventive concept, the present application also provides a soybean sprout produced by the method as described above, wherein the total isoflavone content of the soybean sprout is higher than 7900 μg / g. Further, the total isoflavone content of the soybean sprout is higher than 7957 μg / g.

[0016] Compared with the prior art, the present application has the following effects and advantages: (1) The present application uses the hydrogen sulfide donor NaHS to promote germination of soybean. Experiments show that the length of the soybean sprout treated with NaHS is significantly higher than that of the distilled water control group, indicating that NaHS treatment can promote the elongation of the soybean sprout. In addition, compared with the distilled water control group, NaHS treatment significantly promotes the fresh weight of the sprout, increasing by 8.12%, indicating that NaHS treatment can promote the fresh weight of the soybean sprout.

[0017] (2) The application adopts NaHS treatment to promote the enrichment of isoflavone content. Experiments show that the content of malonyl genistin in the NaHS group increases significantly, which is significantly increased by 38.63% compared with the distilled water control group. This shows that H2S can increase the content of malonyl genistin in bean sprouts, and provides a basis for producing functional bean sprout products with higher nutritional value.

[0018] (3) The application adopts NaHS treatment to significantly improve the activity of key enzymes PAL 、 C4H and 4CL for isoflavone synthesis (increased by 21.14%, 19.73% and 38.67% respectively), and significantly up-regulate the expression of key genes such as PAL 、 C4H and 4CL at the transcription level (up-regulated by 88%, 82% and 163% respectively), which shows that H2S can stimulate the gene expression of key enzymes in the isoflavone synthesis pathway of germinated soybean, thereby increasing the content of isoflavone and driving the biosynthesis of isoflavone from the root.

[0019] (4) The application adopts NaHS treatment to significantly improve the activity of antioxidant enzymes SOD, POD and CAT (increased by 88.81%, 35.09% and 92.23% respectively), and effectively reduce the content of oxidative damage marker MDA (reduced by 16.79%) and the level of active oxygen H2O2 and O2 - , which creates a better intracellular environment for bean sprouts and is an important physiological basis for the growth promotion and quality improvement of bean sprouts.

[0020] (5) The application discloses the bidirectional regulation of H2S on the metabolism of plant hormones in germinated soybean by exogenous NaHS treatment and HT treatment, that is, NaHS treatment can synergistically regulate the germination process by promoting the content of endogenous GA, IAA, inhibiting the content of endogenous ABA, JA and ETH. In addition, the role of H2S in the hormone regulation network of germinated soybean is clarified, which provides a theoretical basis for using H2S to regulate seed germination. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 The effects of different concentrations of NaHS treatment on the length of germinated soybean sprouts (A), fresh weight (B) and dry weight (C) are shown in the figure. Note that there is a significant difference between the data of different samples ( P <0.05). Figure 2 The effects of NaHS and HT treatment on the appearance of germinated soybean growth (A), sprout length (B), fresh weight (C) and dry weight (D) are shown in the figure. Note that there is a significant difference between the data of different samples ( P <0.05) . Figure 3 Effect of NaHS and HT treatment on endogenous H2S in germinated soybean, Note: There were significant differences between different sample data P <0.05); Figure 4 Effect of NaHS and HT treatment on total isoflavone content in germinated soybean, Note: There were significant differences between different sample data P <0.05); Figure 5 Effect of exogenous NaHS treatment on key enzymes of soybean isoflavone synthesis PAL (A), C4H (B) and 4CL (C) and gene expression PAL (D), C4H (E) and 4CL (F), Note: There were significant differences between different sample data P <0.05); Figure 6 Effect of exogenous NaHS treatment on MDA (A), H2O2 (B) and O2 - (C) and antioxidant enzymes SOD (D), POD (E) and CAT (F) in germinated soybean, Note: There were significant differences between different sample data P <0.05); Figure 7 Effect of exogenous NaHS treatment on ABA (A), GA (B), GA / ABA ratio (C), JA (D), IAA (E) and ETH (F) in germinated soybean, Note: There were significant differences between different sample data P <0.05); Figure 8 PCA based on transcriptome data (A), volcano plot of HS treatment differential expression results (B), volcano plot of HT treatment differential expression results (C), clustering heat map of HS treatment differential expression genes (D), clustering heat map of HT treatment differential expression genes (E), Venn diagram of HS and HT treatment differential genes (F), KEGG enrichment statistics of HS treatment differential expression genes (G) and KEGG enrichment statistics of HT treatment differential expression genes (H) figure; Figure 9 KEGG enrichment pathway analysis figure; Figure 10 Isoflavone synthesis and metabolism pathway analysis figure. DETAILED DESCRIPTION

[0022] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this specification includes any and all combinations of one or more of the associated listed items.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0025] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0026] Example 1: Preparation of plant materials and growth conditions The soybean variety used in the test was Qingnongdou 2316, provided by the Soybean Genetics and Molecular Breeding Laboratory of Qingdao Agricultural University. A method for promoting soybean germination specifically includes the following steps: (1) Soak the seeds in a 0.1% (vol%) sodium hypochlorite aqueous solution for 3 min to disinfect them, and then rinse them with sterile water until the sodium hypochlorite solution is completely removed.

[0027] (2) Soak the disinfected seeds in distilled water for 6 hours; (3) Finally, 60 sterilized seeds were evenly scattered in a germination box containing moistened filter paper and germinated in the dark at 26°C. Different culture media were added to the germination box. Distilled water was used as the control group (CK), and different concentrations of H2S donor NaHS (0.1, 1, 10, 100, and 1000 μM) aqueous solutions were screened. 500 μM taurine (HT) aqueous solution was selected as the H2S scavenger through experiments. Germinated soybeans were harvested at 2 and 4 days. Some samples were subjected to physiological tests immediately after harvest. The remaining germinated soybeans were soaked in liquid nitrogen and finally stored in an ultra-low temperature freezer (-80°C).

[0028] Experimental Example 1: Determination of bud length, fresh weight, and dry weight 1. Experimental Method: The shoot length was measured by measuring the distance from the cotyledon to the root tip of the germinated soybean. The fresh weight of the germinated soybean was measured using an analytical balance. The germinated soybean was freeze-dried in a vacuum freeze dryer, and then its dry weight was measured using an analytical balance. All physiological parameters were repeated three times. The results are as follows: Figure 1 and Figure 2 As shown.

[0029] 2. Experimental Results and Analysis: (1) Effects of different concentrations of H2S donor NaHS on the growth status of germinated soybeans Experiments showed that NaHS treatment had a regulatory effect on the growth status of germinating soybeans. Figure 1 As shown in Figures A and B, neither low nor high concentrations of NaHS promoted nor inhibited root elongation. With increasing NaHS concentration, shoot length and fresh weight initially increased and then decreased, only significantly increasing to 17.44 cm and 0.67 g, respectively, at a concentration of 10 μM. Figure 1 As shown in Figure C, there was no significant difference in dry weight. This indicates that treatment with an appropriate concentration of NaHS can promote soybean germination. Therefore, this invention will use 10 μM NaHS to treat germinated soybeans.

[0030] (2) Exogenous NaHS treatment promotes the elongation of bean sprouts Figure 2 A can visually reflect the growth status of soybean sprouts under NaHS and HT treatments compared to the control group. Compared to the control group, exogenous NaHS and HT altered the morphology of soybean sprouts. This indicates that H2S significantly promotes soybean sprout germination, while the H2S scavenger HT significantly inhibits it. Figure 2 As shown in Figure B, during soybean germination, the sprout length of the control group, NaHS treatment, and HT treatment gradually increased with the increase of germination days. At 4 days of germination, the sprout length of soybeans treated with NaHS was significantly higher than that of the control group, while the sprout length of the HT treatment group was significantly lower than that of the control group. P <0.05%, which was 1.21 times and 0.80 times that of the control group, respectively. The analysis results indicate that NaHS treatment can promote the elongation of soybean sprouts.

[0031] like Figure 2 C and Figure 2 As shown in Figure D, the fresh weight of both the control group and the NaHS treatment group continued to increase during soybean germination. At 4 days of germination, compared with the control group, the NaHS treatment significantly promoted the fresh weight of the sprouts, increasing it by 8.12%, while there was no significant difference in dry weight. P >0.05). The fresh weight and dry weight of the HT-treated sprouts decreased significantly to 78.03% and 87.97% of the control group, respectively. These results indicate that NaHS treatment promotes the fresh weight of soybean sprouts.

[0032] Determination of endogenous H2S content in bean sprouts 1. Experimental method: Take 0.5 g of germinated soybean sample in liquid nitrogen, grind thoroughly, add three times of liquid nitrogen during grinding, and resuspend in 1 mL of 100 mm potassium phosphate buffer (pH 7.0, 10 mM EDTA). Then, add 4 mL of reaction buffer (100 mM potassium phosphate, pH 7.0, 10 mM EDTA, 0.2 mM DTNB) to 200 μL of supernatant, centrifuge at 11000 x g, 4°C for 5 min, incubate at 37°C for 2 min, then take the supernatant and measure the absorbance at 412 nm.

[0033] 2. Experimental results and analysis: Effect of exogenous NaHS treatment on endogenous hydrogen sulfide (H2S): Hydrogen sulfide (H2S) is an important gaseous signaling molecule that plays an active physiological function in living organisms. H2S regulates plant growth and greatly enhances the adaptability of plants to environmental stress. Figure 3 The effect of NaHS and HT treatment on endogenous H2S in germinated soybeans. From Figure 3 It can be seen that, compared with the control group, the addition of exogenous H2S donor NaHS can significantly promote the endogenous H2S content in germinated soybeans (P<0.05), while the endogenous H2S content in the HT group is significantly reduced. The above research results show that NaHS treatment has a positive effect on the endogenous H2S content of bean sprouts. P >0.05), and the endogenous H2S content in the HT group was significantly reduced. The above research results show that NaHS treatment has a positive effect on the endogenous H2S content of bean sprouts.

[0034] Test Example 3: Determination of isoflavone content 1. Experimental method: The U-3000 high performance liquid chromatograph (Thermo Fisher Scientific) and AQ-C18 chromatographic column (Shanghai Yuhua Technology Co., Ltd.) were used to detect the isoflavone content. First, the freeze-dried germinated soybeans were ground into powder using a high-throughput tissue grinder, then the soybean powder (0.12 g) was dissolved in 3 mL of 80% methanol aqueous solution, and ultrasonic treatment was performed at 40°C for 60 min. The mixed solution was centrifuged at 11000 x g at 4°C for 40 min. The supernatant was extracted and filtered through a 0.45 μm microporous filter, and the filtered solution (10 μL) was injected into the high performance liquid chromatograph.

[0035] Mobile phase A: 0.1% (vol%) acetic acid in water, mobile phase B: 0.1% (vol%) acetic acid in acetonitrile in water. Elution gradient: B phase ratio 13-35%, 0-50 min; 35-13%, 50-51 min. Flow rate: 1 mL / min. UV detector wavelength: 260 nm.

[0036] 2. Experimental Results and Analysis: Effects of exogenous NaHS on soybean isoflavone content: (1) Composition of soybean isoflavones and standard working curve The peak times of various metabolites of soybean isoflavones are shown in Table 1. The peak times of daidzein are 14.733 min, daidzein 15.907 min, genistein 22.237 min, malonyl daidzein 23.657 min, malonyl daidzein 24.387 min, malonyl genistein 31.377 min, daidzein 35.293 min, daidzein 37.953 min, and genistein 47.547 min.

[0037] Table 1. Composition and Standard Curve of Soy Isoflavones

[0038] (2) Exogenous NaHS promotes the accumulation of soybean isoflavones Soy isoflavones are important secondary metabolites of soybeans, mainly existing in three forms: free aglycones, glucosides, and malonyl glucosides. The changes in the total isoflavone content and the content of each monomer in soybean sprouts under normal growth conditions and exogenous NaHS growth conditions were determined. Figure 4 As shown in Table 2, the results indicate that NaHS treatment promotes the enrichment of isoflavone content.

[0039] Free aglycones are the active forms of soybean isoflavones (daidzein, genistein, and genistein). Daidzein (7,4'-dihydroxyisoflavone) is one of the most abundant soybean isoflavone aglycones and a precursor to many glycoside forms, possessing antioxidant properties. Genistein (5,7,4'-trihydroxyisoflavone) is the most widely studied isoflavone aglycone, possessing a polyphenolic hydroxyl structure that endows it with strong antioxidant capabilities. It is known as a "natural tyrosine kinase inhibitor" and can broadly intervene in cell signal transduction. The changes in daidzein content in germinated soybeans under normal and hydrogen sulfide growth conditions were measured. Genistein (7,4'-dihydroxy-6-methoxyisoflavone) is a relatively rare isoflavone aglycone. The three treatments had no significant effect on the content of the three monomeric aglycones. P <0.05), however, although NaHS and HT did not affect the content of individual free aglycones, compared to the HT group (438.42±21.10), b NaHS group (547.01±35.48) a It significantly increases the level of total active aglycones.

[0040] Glucosides are the basic conjugated form of isoflavones (daidzin, glycitein, genistin). Daidzin (daidzein-7-O-glucoside) is the main glycoside form of daidzein, which is an important component of soy isoflavones and has pharmacological effects in preventing and treating cardiovascular diseases, anti-tumor, etc. The content of daidzin in the three groups has no significant effect P <0.05), indicating that the synthesis and degradation of DAI may not depend on H2S signal. Glycitein (glycitein-7-O-glucoside) is the glycosylation form of glycitein, which also plays an important role in plant defense. The CK group (99.41±7.57 a ) and the NaHS group (109.63±36.49 a ) have no significant difference, but the HT group (47.91±5.33 b ) is significantly lower than the first two groups, with a decrease of 51.8%. This shows that after the removal of H2S, the synthesis of glycitein glycoside is significantly inhibited, and exogenous NaHS can maintain its content at the control level, indicating that exogenous NaHS may play a role in the synthesis of glycitein glycoside. Genistin (genistin-7-O-glucoside) is the most abundant isoflavone glycoside in soybeans, which is the precursor of genistein and can be hydrolyzed to active aglycone under the action of fermented food or intestinal microorganisms. It has a mild estrogen-like activity. In this example, the content of genistin in each group of materials was determined by high performance liquid chromatography. In the HT group, the content of genistin has no significant difference with the CK group; while the content of genistin in the NaHS group is significantly higher than that in the CK group P <0.05), which is 29.09% higher than the CK group, indicating that H2S may promote the synthesis of genistin.

[0041] Malonyl glucoside is the main storage form of soy isoflavones (more than 60% of the total isoflavone content, such as malonyl daidzin, malonyl glycitein and malonyl genistin). Malonyl daidzin is a heat-labile acidic isoflavone derivative, accounting for 15-40% of the total isoflavones in raw soybeans. As the main storage form, malonyl daidzin accumulates significantly in the late development of soybean seeds. As shown in Table 4, the content of malonyl daidzin in the NaHS group of bean sprouts was 2623.86 μg / g, which was 16.79% higher than that in the CK group. H2S scavenger HT significantly inhibited the content of malonyl daidzin, which decreased by 12.18%; this result shows that H2S can induce the synthesis of malonyl daidzin in bean sprouts to a certain extent. Malonyl glycitein is the acylated form of glycitein. CK (475.06±77.52 a ) and NaHS (511.83±63.42 a ) have no significant difference, but HT (275.57±24.14 b) was significantly lower than the previous two groups, with a decrease of 42.0%. This trend is consistent with GLY, that is, after HT removes hydrogen sulfide, both types of isoflavones are significantly reduced, which may be because the glycosylation and malonylation modification pathways of daidzein share hydrogen sulfide-dependent regulatory factors. Malonyl genistin is one of the most abundant isoflavones in soybeans, accounting for 25-50% of total isoflavones in unprocessed soybeans. The content of malonyl genistin in the NaHS group increased significantly, reaching 4239.74 μg / g, which was significantly higher than that in the CK group by 38.63% (P < 0.05). This shows that H2S can increase the content of malonyl genistin in bean sprouts. P <0.05). This shows that H2S can increase the content of malonyl genistin in bean sprouts.

[0042] Table 2 Soybean isoflavone content under different treatments

[0043] Test Example 4 PAL 、 C4H and 4CL Enzyme activity determination 1. Experimental method: Take 0.2 g of germinated soybean sample, add 2 mL of extraction buffer (pH 8.8, 50 mM sodium borate buffer, 40 g / L PVP, 2 mM EDTA and 5 mM β-mercaptoethanol), and use a cryogenic grinder to grind into a homogenate at 4°C, then centrifuge at 12000 x g for 20 min at 4°C, and take the supernatant for enzyme activity determination. The activity of PAL , C4H and 4CL was detected using an activity assay kit.

[0044] 2. Experimental results and analysis: Effect of exogenous NaHS treatment on key enzymes of soybean isoflavone synthesis and their gene expression: As shown in Figure 5 A, Figure 5 B and Figure 5 C, the 4d germinated soybeans, compared with the control group, H2S alone increased the activity of PAL (21.14%), C4H (19.73%) and 4CL (38.67%). HT alone inhibited the activity of PAL and 4CL (14.43% and 62.13% inhibition compared with the CK group, respectively), and had no significant effect on C4H enzyme activity (P < 0.05). As a scavenger of H2S, the addition of HT inhibited the key enzymes of the isoflavone synthesis pathway P and PAL and 4CLactivity, while H2S alone treatment significantly increased the enzyme activity, which indicated that H2S could regulate the key enzyme activity of isoflavone synthesis pathway.

[0045] As Figure 5 D, Figure 5 E and Figure 5 F showed that, compared with the control group, H2S alone treatment significantly increased the gene expression of PAL , C4H and 4CL by 88%, 82% and 163% respectively after 4 d of germination. While HT treatment significantly inhibited the activity of PAL and 4CL (by 62.13% and 58.33% respectively compared with the CK group), and had no significant effect on the gene expression of C4H (<0.05). These trends were consistent with the activity trends of P , PAL , C4H and 4CL . This indicated that H2S could stimulate the gene expression of key enzymes in the isoflavone synthesis pathway of germinated soybeans at the gene level, thereby increasing the content of isoflavones.

[0046] Determination of SOD, POD, CAT activity, malondialdehyde (MDA), hydrogen peroxide (H2O2) and superoxide anion (O2 - ) content 1. Experimental method: (1) Determination of SOD, POD, CAT activity and malondialdehyde (MDA) content Grind and add phosphate buffer (pH = 7.8, 0.05 mM) to extract 0.15 g of sample. After centrifugation at 4°C, mix the supernatant with 0.5 mL of 0.5% (w / %) aqueous solution of thiobarbituric acid. Then heat the mixture to a temperature of 100°C for 20 min, then perform another round of centrifugation, and take the supernatant for determination of SOD, POD and CAT enzyme activity.

[0047] The activity of SOD enzyme was measured using the NBT reduction method. The reaction mixture consisted of 1.5 mL of 0.05 M PBS with a pH value of 7.8, 0.3 mL of 130 mM L-methionine aqueous solution, 0.3 mL of 750 μM NBT aqueous solution, 0.3 mL of 100 μM EDTA-Na aqueous solution, 0.3 mL of 20 μM riboflavin aqueous solution and 250 μL of distilled water. Mix these reagents with 50 μL of extract solution to form a complete analysis mixture. After thorough mixing, place the control tube in the dark (use boiled enzyme as control), while other test tubes are exposed to sunlight for 20 min. Then record the absorbance at a wavelength of 560 nm.

[0048] POD enzyme activity was measured using the guaiacol method. A mixture containing 1 mL of 0.05 M PBS (pH = 7.0) and 0.95 mL of 0.2% guaiacol was prepared in a test tube. Then the solution was mixed and heated to 34°C in a water bath. After that, 1 mL of 0.3% hydrogen peroxide and 0.05 mL of enzyme extract were added to each sample. The rate of change in optical absorbance at 470 nm within 210 s was measured, with data recorded every 30 s.

[0049] 1 mL of 0.3% H2O2, 1.9 mL of H2O, and 0.1 mL of enzyme solution were added to the reaction system. The absorbance was read every 30 s at 240 nm for 210 s to evaluate CAT enzyme activity.

[0050] Freshly germinated soybean samples (0.2 g) were ground in 2 mL of 100 g / L trichloroacetic acid solution and centrifuged at 10000 x g for 20 min at 4°C. 1 mL of supernatant was reacted with 1 mL of 0.05 M NaOH solution containing 6.7 g / L thiobarbituric acid solution. Then the mixture was heated in a 100°C water bath for 20 min, centrifuged at 10000 x g for 20 min, and immediately cooled. The absorbance of the supernatant was read at 450, 532, and 600 nm using enzyme markers to determine the MDA content.

[0051] (2) Hydrogen peroxide (H2O2) and superoxide anion (O2 - ) content determination Take 0.1 g of germinated soybean and add 1 mL of acetone for ice bath homogenization, then centrifuge the mixture at 10000 x g for 30 min at 4°C. The content of H2O2 is quantified using a hydrogen peroxide (H2O2) content detection kit (Solarbio, BC3595).

[0052] Homogenize 0.1 g of germinated soybean with 1 mL of 50 mM potassium phosphate buffer (pH 7.8) on ice, then centrifuge the mixture at 10000 x g for 30 min at 4°C. The content of O2 - is quantified using a superoxide anion content test kit (Solarbio, BC1290). The results are shown in Figure 6 .

[0053] 2. Experimental results and analysis: Exogenous NaHS treatment regulates the antioxidant system of germinated soybeans: As shown in Figure 6 A, compared with the control group, NaHS treatment significantly reduced the MDA content in bean sprouts at 4 d (P <0.05), and decreased by 16.79% to 16.45 nmol / g compared with the control. The MDA content was significantly increased by 44.84% in the HT treatment group, which indicated that H2S could reduce the membrane damage of the germinated soybean.

[0054] H2O2 has cytotoxicity, and its concentration can directly reflect the response of plants to environmental stress. From Figure 6 It can be seen from Fig. 4A that the H2O2 content of the seedlings treated with NaHS was significantly lower than that of the control (P < 0.05) at 4 d after germination, which was 0.88 times that of the control. The H2O2 content was significantly increased by 14.78% in the HT treatment group, which indicated that H2S could reduce the H2O2 content of the germinated soybean. In addition, from Figure 6 B, it can be seen that the O2 - content was significantly reduced by 24.24% in the NaHS treatment, and the O2 - content in the HT treatment group was 1.30 times that of the control.

[0055] From Figure 6 D, Figure 6 E, Figure 6 F, it can be seen that H2S increased the antioxidant enzyme activity of soybean sprouts at 4 d after germination compared with the control. When the seeds were soaked with NaHS, the SOD, POD and CAT activities of the germinated soybean were significantly higher than those of the control (P < 0.05). P Compared with the control, the SOD, POD and CAT enzyme activities of the NaHS group were increased by 88.81%, 35.09% and 92.23%, respectively.

[0056] Example 6 Determination of endogenous plant hormones 1. Experimental method: The contents of abscisic acid (ABA), gibberellin (GA), jasmonic acid (JA) and auxin (IAA) were quantified using the superoxide anion content test kit (Jiangsu Enzyme Free 0138O2, 0125O2, 0887O2 and 0953O2). The results are shown in Figure 7 .

[0057] 2. Experimental results and analysis: Effect of exogenous NaHS treatment on endogenous auxin (IAA), jasmonic acid (JA), abscisic acid (ABA), gibberellin (GA) and ethylene (ETH) of soybean sprouts: From Figure 8 G and Figure 8As shown in the H2S analysis, KEGG significant enrichment analysis revealed that the differentially expressed genes in the CK and NaHS, and CK and HT groups were mainly enriched in plant hormone signal transduction, thus exploring the effect of H2S treatment on plant hormones. Plant hormones, as endogenous signaling molecules, play a central role in regulating physiological processes such as plant growth, development, and stress adaptation. This invention investigated the regulatory mechanisms of H2S on plant hormone metabolism by measuring the contents of ABA, GA, JA, IAA, and ETH, as well as the ABA / GA ratio, under three treatments: control (CK), NaHS, and HT, thereby elucidating the regulatory mechanism of H2S on hormone metabolism in germinating soybeans.

[0058] ABA is an inhibitory hormone for seed germination; elevated levels are typically associated with delayed germination or maintenance of dormancy. Figure 7 As can be seen from A, there was no significant difference in ABA content between CK and NaHS treatments. P >0.05); while the ABA content increased significantly under HT treatment, reaching 637.1 ng / g. This indicates that moderate H2S did not interfere with the basal metabolic balance of ABA, and HT treatment prompted plants to enhance their adaptation to adversity by upregulating ABA synthesis or inhibiting its decomposition.

[0059] GA is a key hormone promoting plant growth (such as stem elongation and seed germination). It drives the germination process by inducing the synthesis of hydrolytic enzymes and breaking dormancy, and it has an antagonistic regulatory relationship with ABA. Figure 7 As can be seen from B, the GA content under NaHS treatment was significantly higher than that under CK and HT treatment ( P <0.05%, with a concentration of 178.3 ng / g, indicating that NaHS treatment may enhance the growth potential of bean sprouts by promoting cell growth. Compared with the CK group, the GA content was significantly reduced under HT treatment ( P< 0.05), with a content of 135.9 ng / g, which may be due to the plant's preferential response to stress and inhibition of growth consumption.

[0060] ABA / GA is a core indicator regulating the balance between growth and stress resistance; a lower ABA / GA ratio generally promotes germination, while a higher ratio inhibits it. Figure 7 C indicates that the ABA / GA ratio in HT treatment was significantly higher than that in CK and NaHS treatment. P <0.05), the proportion of CK treatment was significantly higher than that of NaHS treatment. Although there was no significant difference in ABA content under NaHS treatment and GA content increased, the ABA / GA ratio was still significantly reduced. This may be because the increase in GA exceeded the decrease in ABA, or there may be synergistic regulation by other hormones (such as ETH). The results indicate that the low ABA / GA ratio in NaHS treatment promotes soybean germination while maintaining metabolic homeostasis.

[0061] JA is involved in plant defense responses (such as anti-insect, anti-oxidative stress) and growth regulation, and the increase of its content is usually associated with stress response activation. Figure 7 In D, the JA content under NaHS treatment was 14.1 ng / g, which was significantly lower than that under CK and HT treatments, while the JA content under HT treatment was 34.1 ng / g, which was significantly increased (P < 0.05). The results showed that H2S can inhibit the synthesis of JA or accelerate its decomposition, thereby eliminating the potential inhibitory effect of JA on germination; while HT treatment can lead to the activation of the JA synthesis pathway, thereby enhancing the JA-dependent defense pathway to enhance stress tolerance. P <0.05). The results showed that exogenous H2S can activate the IAA synthesis pathway (such as the activation of tryptophan metabolism pathway related genes or enzymes), providing hormone support for the early growth of germinated soybeans; while HT treatment inhibits IAA synthesis, reflecting the positive regulation of H2S on IAA metabolism, which is of great significance for the growth and development of germinated soybeans.

[0062] IAA mainly promotes cell elongation and differentiation in early plant growth, and its accumulation can promote hypocotyl elongation and seedling establishment, and is the core hormone for regulating plant growth. IAA is synthesized from tryptophan through a series of metabolic pathways. Figure 7 As can be seen from E, the IAA content under NaHS treatment was significantly higher than that under CK and HT treatments (P < 0.05), and the IAA content under CK treatment was significantly higher than that under HT treatment. The results showed that exogenous H2S can activate the IAA synthesis pathway (such as the activation of tryptophan metabolism pathway related genes or enzymes), providing hormone support for the early growth of germinated soybeans; while HT treatment inhibits IAA synthesis, reflecting the positive regulation of H2S on IAA metabolism, which is of great significance for the growth and development of germinated soybeans. P <0.05). The results showed that exogenous H2S can activate the IAA synthesis pathway (such as the activation of tryptophan metabolism pathway related genes or enzymes), providing hormone support for the early growth of germinated soybeans; while HT treatment inhibits IAA synthesis, reflecting the positive regulation of H2S on IAA metabolism, which is of great significance for the growth and development of germinated soybeans.

[0063] ETH is involved in stress response, fruit ripening and other processes, and its content change is closely related to plant stress perception. Figure 7 As can be seen from F, both NaHS and HT treatments promote ETH synthesis, but the ETH content under HT treatment is significantly higher than that under NaHS treatment (P < 0.05), which suggests that low concentration of ETH can promote germination in cooperation with GA and IAA, while high concentration of ETH (HT treatment) is involved in metabolic reprogramming under stress. P <0.05). The results showed that exogenous H2S can activate the IAA synthesis pathway (such as the activation of tryptophan metabolism pathway related genes or enzymes), providing hormone support for the early growth of germinated soybeans; while HT treatment inhibits IAA synthesis, reflecting the positive regulation of H2S on IAA metabolism, which is of great significance for the growth and development of germinated soybeans.

[0064] By exogenous NaHS treatment and HT treatment, the present application reveals the bidirectional regulation of H2S on hormone metabolism of germinated soybean plants, i.e. NaHS treatment can synergistically regulate the germination process by promoting endogenous GA, IAA, inhibiting endogenous ABA, JA, and ETH content. In addition, the role of H2S in the hormone regulation network of germinated soybeans is clarified, which provides a theoretical basis for using H2S to regulate seed germination.

[0065] Test Example 7 RNA extraction and RNA-Seq analysis 1. Experimental method: RNA-seq was performed by Wuhan igenebook Biotechnology Co., Ltd. (http: / / www.igenebook.com). Briefly, total RNA was extracted by trizol method. The RNA concentration and total amount were detected by qubit, and the integrity of all RNA samples was evaluated by Qsep400. Three groups of samples control (CK), NaHS (HS) and HT were sequenced, and each group of samples was repeated three times. VAHTS Universal V10 RNA-seq Library Prep Kit for MGI was used to construct RNA library, and the total RNA amount was 3 μg. The program included polyA capture, reverse transcription cDNA, A-tailing, PCR to DNA library. Finally, DNBSEQ-T7 was used for PE150 sequencing.

[0066] Adaptor and low-quality reads were filtered by cutadapt (version 1.11). Clean reads were mapped to soybean reference genome by Hisat2 (version 2.1.0). These genes were compared with public protein database NR (RefSeq non-redundant proteins). The transcript expression was statistically analyzed by featucount (v1.6.0), and the expression value was normalized to FPKM (Fragments per kilobase of transcript per million Fragments mapped). Differential analysis was performed by edgeR, and the screening threshold was FDR <0.05 and |log2FoldChange| >1.

[0067] GO (Gene Ontology, http: / / geneontology.org / ) and KEGG (Kyoto Encyclopedia of Genes and Genomes, http: / / www.genome.jp / kegg / ) enrichment analysis was performed using ClusterProfiler in R package (http: / / www.bioconductor.org / packages / release / bioc / html / ClusterProfiler.html). GO and KEGG enrichment analysis used hypergeometric distribution calculation, and the threshold was P <0.05. The results are shown in Figure 8

[0068] 2、Experimental results and analysis: Sample clustering analysis and KEGG enrichment analysis: PCA principal component analysis is shown in Figure 8 ​As shown in Figure A, the three samples (CK, HS, and HT) cluster together, and the small area of ​​the elliptical regions grouping the samples indicates high similarity among the samples. Furthermore, the CK, HS, and HT groups are far apart, clearly separated, and exhibit high separation, indicating significant differences in transcriptional levels between HS and HT treatments and the control. Figure 8 Volcano plots for B and 8C showed that, compared to the CK group, the H2S treatment group had 3386 upregulated genes and 2065 downregulated genes; the HT treatment group had 3684 upregulated genes and 4533 downregulated genes.

[0069] Clustering based on expression levels can reveal previously unknown biological connections between genes. For example... Figure 8 D and Figure 8 Cluster analysis of E showed that CK and HS, and CK and HT treatments clustered into two different categories, respectively. Within the same treatment group, gene expression patterns were similar, while the differences between CK and HS, and CK and HT were significant, indicating that HS and HT treatments had a greater impact on the transcriptional level of bean sprouts. Figure 8 As shown in F, compared with the CK group, Venn diagram intersection analysis of the upregulated genes in the HS treatment group and the downregulated genes in the HT treatment group yielded 269 differentially expressed genes, which were mainly enriched in plant hormone signal transduction, MAPK signaling pathway-plant and plant-pathogen interactions.

[0070] like Figure 8 G and Figure 8 As shown in H, KEGG significant enrichment analysis indicated that the differentially expressed genes in the CK and HS and CK and HT groups were mainly enriched in phenylpropanoid biosynthesis, plant hormone signal transduction, and starch and sucrose metabolism.

[0071] Experiment 8: Reverse transcription and quantitative real-time PCR (qRT-PCR) 1. Experimental Method: cDNA and RT-qPCR were synthesized using TranScript® Green One-Step qRT-PCR SuperMix to validate gene expression levels. ELB1 As an internal reference gene, the comparative threshold cycle (Ct) method was used for quantitative analysis of gene expression to determine the relative expression level of the gene. The expression level of the target gene obtained by qRT-PCR was then used to... ELB1 After standardization using the internal reference gene as a reference, 2 -ΔΔCThe relative expression amount was calculated by formula. The gene expression quantitative analysis was performed in a 20 μL reaction system, and the instrument used was a real-time fluorescence quantitative PCR system (ABI Applied Biosystems Company, USA). For each sample, three PCR repeats were performed for real-time quantitative determination. The results are shown in Figure 5 D-F and Figure 10 .

[0072] 2. Experimental results and analysis: (1) Regulation of exogenous NaHS on the hormone signal pathway of germinated soybean plants To further analyze the molecular mechanism of exogenous NaHS regulating the germination of germinated soybean seeds, the present application combines the plant hormone content data and the expression profile of hormone signal pathway related genes, as shown in Figure 9 .

[0073] GA is the core hormone for promoting seed germination, and its signal pathway depends on GA synthetase (such as KAO) and DELLA protein degradation pathway (such as GID2). From the gene expression, the key genes of GA synthesis KAO ( Glyma.01G199800 ) and GA3ox ( Glyma.15G012100 and Gl yma.08G208300) are significantly up-regulated in CK vs HS, and KAO is significantly down-regulated in CK vs HT. The core negative regulator of GA signal pathway DELLA, its degradation related gene GID2 ( Glyma.17G237800 ) also presents a similar expression pattern. And DELLA is significantly up-regulated in CK vs HT, which enhances the binding with the negative regulator JAZ of JA signal pathway. In addition, the GA content is significantly increased under HS treatment and significantly decreased under HT treatment (Figure 7B). This indicates that exogenous NaHS may promote seed germination by promoting the expression of GA synthesis genes and activating the GA signal pathway; and hydrogen sulfide scavenging (HT) weakens the germination promoting effect of GA by down-regulating GA synthesis genes.

[0074] The results show that the JA receptor protein COI1 ( Glyma.02G25430 0 and Glyma.18G030200 ) is down-regulated in CK vs HS and up-regulated in CK vs HT, indicating that exogenous H2S reduces the inhibition of JAZ, thereby causing JAZ (such as Glyma.01G204400 , etc.) to be significantly up-regulated, which enhances the binding with DELLA and MYC2 proteins. In addition, the significant up-regulation of MYC2 ( Glyma.01G199800 ) inhibits the expression of EIN3 ( Glyma.05G180300 ) of the ethylene signal pathway. As Figure 7D. HS treatment significantly decreased JA content, while HT treatment significantly increased JA content. This indicates that exogenous H2S eliminates the inhibitory effect of JA on germination by suppressing the expression of genes in the JA signaling pathway.

[0075] The ET signaling pathway depends on the EIN3-ERF1 pathway. Results showed that the expression level of the growth inhibitor EIN3 gene was significantly downregulated in CK vs HS and significantly downregulated in CK vs HT. ERF1, a downstream core effector of the ethylene signaling pathway... Glyma.03G162700 Significantly upregulated and downregulated in CK vs HS and CK vs HT, respectively. Figure 7 The ETH content was significantly increased in the F, HS, and HT treatments, with the HT group showing a significantly higher content than the HS group. These results indicate that exogenous H2S may produce low concentrations of ET, which synergistically promotes germination with GA; while the HT treatment group over-activates the ET signaling pathway, leading to high concentrations of ET synergistically inhibiting bean sprout elongation with ABA and JA.

[0076] ABA is an inhibitory hormone for seed germination, and its signaling pathway depends on PYR / PYL receptor-mediated signal transduction. For example... Figure 9 ABA pathway receptor PYR / PYL ( Glyma.13G229300 Gene expression was downregulated in CK vs HS and upregulated in CK vs HT. HS treatment weakened the receptor and PP2C, a core inhibitor of the ABA signaling pathway. Glyma.15G172500 The binding of ) leads to a significant upregulation of its gene expression. For example Figure 7 A. HT treatment significantly increased ABA content. The results indicate that exogenous H2S may weaken the "dormancy maintenance" effect of ABA by inhibiting the expression of ABA signaling pathway genes; while HT enhances the inhibitory effect of ABA by upregulating the ABA signaling pathway, leading to a decrease in seed germination potential.

[0077] The expression level of ARF, a factor involved in the regulation of primary root elongation in the IAA pathway, was upregulated in both CK vs HS and CK vs HT, and the expression level of GH3, a negative regulator of IAA synthesis, was inhibited. Glyma.15G125000 The expression levels of genes involved in this process are downregulated. For example... Figure 7 E and HS treatments significantly increased IAA content, while HT treatment significantly decreased IAA content. The results indicate that exogenous H2S enhances the role of IAA in seedling growth after germination by activating the expression of IAA signaling pathway genes; while hydrogen sulfide scavenging (HT) weakens seedling growth by inhibiting IAA signaling genes.

[0078] This invention, through analysis of endogenous hormone levels and gene expression profiles, reveals that the regulation of soybean seed germination by exogenous H2S (HS) and scavengers (HT) involves the synergistic effect of multiple hormone signaling pathways. Exogenous H2S may promote seed germination by promoting GA synthesis and signaling pathways (accelerating DELLA degradation), inhibiting ABA and JA signaling pathways (eliminating dormancy and stress inhibition), and synergistically activating the IAA signaling pathway and ethylene. From a molecular regulatory perspective, H2S, as a signaling molecule, may regulate multiple hormone signaling pathways and promote seed germination and taproot elongation by modulating hormone synthesis / signaling gene transcription levels and participating in protein thiolation modifications (such as thiolation of proteins like DELLA and COI1).

[0079] (2) Regulation of isoflavone synthesis in germinated soybeans treated with exogenous NaHS Isoflavones, as important products of plant secondary metabolism, play a crucial role in plant stress resistance and human health. This study elucidated the expression patterns of key enzyme genes under control (CK), NaHS (HS), and HT treatments, such as... Figure 10 As shown.

[0080] Isoflavone synthesis begins with phenylalanine metabolism, and phenylalanine is metabolized by phenylalanine ammonia-lyase (PAL) PAL Cinnamic acid is produced under the catalysis of cinnamic acid-4-hydroxylase (Cinnamic acid-4-hydroxylase). C4H ) is converted to p-coumaric acid, and then ligated by 4-coumaric acid-CoA ligase ( 4CL The process of generating p-coumaryl-CoA consists of three steps, which constitute the core upstream link. p-CoA subsequently splits into two branches: one branch generates isoliquiritin chalcone via chalcone synthase (CHS), chalcone reductase (CHR), and chalcone isomerase (CHI), which is then converted to isoliquiritin via CHI. Subsequently, under the action of isoflavone synthase (IFS) and 2-hydroxyisoflavone reductase (HID), it generates aglycones such as daidzein, which are finally modified into malonyl isoflavone glycosides by isoflavone 7-O-glycosyltransferase (IF7GT) and isoflavone 7-O-glycosyl-6″-O-malonyltransferase (IF7MaT). The other branch generates naringenin chalcone via CHS, which is then converted to naringenin via CHI. Naringenin is converted to genistein by IFS and HID, or flows to the flavonoid, flavonol, and dihydroflavonoid branches via flavonoid synthase (FNS), flavonol synthase (FLS), and flavanone 3-hydroxylase (F3H).

[0081] In CK vs HS group, the key genes of isoflavone synthesis pathway were significantly up-regulated. The high expression of upstream PAL (0.88), C4H (0.82), 4CL (1.03) activated the upstream flux of phenylpropanoid metabolism; the downstream CHS (1.78), CHR (1.12), CHI (1.62) promoted the synthesis of chalcone and isoflavone precursor, IFS (1.32), HID (1.32) promoted the formation of aglycone, IF7GT (1.80), IF7MaT (2.33) ensured the accumulation of end product. At the same time, the branch pathway F3H (0.12) was down-regulated, and the metabolism was biased towards isoflavone synthesis. In CK vs HT group, many pathway genes were significantly down-regulated. The upstream PAL (-0.41), C4H (-0.55), 4CL (-0.82) inhibited the supply of phenylpropanoid metabolism; the downstream CHS (-1.47), CHR (-0.78), CHI (-1.61), IFS (-0.62), HID (-0.62) blocked isoflavone synthesis, IF7GT (-0.95), IF7MaT (-0.72) reduced the accumulation of end product. At this time, F3H (0.12) was up-regulated, and the metabolic flow was diverted to flavonoids and other branches, reflecting the metabolic reprogramming of plants under HT treatment, and adapting to the environment by adjusting the allocation of secondary metabolic branches.

[0082] In summary, HS treatment widely up-regulates isoflavone synthesis genes and inhibits branch pathways, promoting the enrichment of isoflavone content; HT treatment inhibits isoflavone synthesis and activates branch pathways, realizing the redistribution of metabolic flow. This differential regulation reflects the metabolic adaptation of plants, and also provides a molecular theoretical support for targeted regulation of isoflavone synthesis, improvement of plant stress resistance or functional component accumulation.

[0083] It should be noted that the specific embodiments are only representative examples of the present application. Obviously, the technical solutions of the present application are not limited to the above examples, and there can be many variations. Those of ordinary skill in the art, based on the disclosure of the present application or according to the written description of the file without any doubt, should be considered as the scope to be protected by the present patent.

Claims

1. An application of exogenous treatment in promoting plant germination and isoflavone enrichment, characterized in that, The exogenous treatment is hydrogen sulfide donor treatment.

2. The application according to claim 1, characterized in that, The plant is any one of the following (a1)-(a3): (a1) Dicotyledonous plants; (a2) Legumes; (a3) Soybean.

3. The application according to claim 2, characterized in that, The plant is soybean, and the hydrogen sulfide donor treatment is applied in at least one of the following (b1)-(b28): (b1) Increase soybean sprout length; (b2) Prepare products that improve soybean sprout length; (b3) Increase the fresh weight of soybeans; (b4) Prepare products that increase the fresh weight of soybeans; (b5) Increase the endogenous hydrogen sulfide content in soybeans; (b6) Prepare products that increase the endogenous hydrogen sulfide content in soybeans; (b7) Increase the content of soybean isoflavones; (b8) Prepare products with increased soybean isoflavone content; (b9) Increase the total malonyl glucoside content in soybeans; (b10) Prepare products that increase the total malonyl glucoside content in soybeans; (b11) Increase the content of soybean genistein; (b12) Prepare products with increased soybean genistein content; (b13) Increase the content of malonyl daidzein in soybeans; (b14) Prepare products that increase the content of soybean malonyl daidzein; (b15) Increase the content of malonyl genistein in soybeans; (b16) Prepare products that increase the content of malonyl genistein in soybeans; (b17) Enhance the activity of key enzymes in soybean isoflavone synthesis, PAL, C4H and / or 4CL; (b18) Prepare products that enhance the activity of key enzymes PAL, C4H and / or 4CL in soybean isoflavone synthesis; (b19) Reduce soybean MDA content; (b20) Prepare products with reduced soybean MDA content; (b21) Reduce the H2O2 content of soybeans; (b22) Prepare products that reduce the H2O2 content of soybeans; (b23) Increase the activity of soybean antioxidant enzymes SOD, POD and / or CAT; (b24) Prepare products that enhance the activity of soybean antioxidant enzymes SOD, POD and / or CAT; (b25) Increase the content of soybean endogenous hormones GA, IAA and / or ETH; (b26) Prepare products that increase the content of soybean endogenous hormones GA, IAA and / or ETH; (b27) Reduce the content of soybean endogenous hormones ABA and JA; (b28) Prepare products that reduce the content of endogenous hormones ABA and JA in soybeans.

4. The application according to claim 1, characterized in that, The hydrogen sulfide donor is NaHS.

5. The application according to any one of claims 1-3, characterized in that, The application concentration of the hydrogen sulfide donor is 0.1-1000 μM.

6. The application according to claim 4, characterized in that, The concentration of the hydrogen sulfide donor is 10 μM.

7. A method for promoting plant germination, characterized in that, Treatment using hydrogen sulfide donors specifically includes the following steps: (1) Soak the plant seeds in a sodium hypochlorite solution for disinfection, and then rinse with sterile water until the sodium hypochlorite solution is completely removed; (2) Soak the disinfected seeds from step (1) in distilled water for 6-8 hours; (3) Spread the seeds soaked in step (2) evenly in a germination box containing moist filter paper. Add hydrogen sulfide donor to the germination box as a culture medium. The hydrogen sulfide donor is NaHS. Under dark conditions at 20-30℃, promote plant germination.

8. A specific culture medium for implementing the method as described in claim 7, characterized in that, The culture medium is a NaHS aqueous solution with a concentration of 0.1-1000 μM.

9. A method for promoting the enrichment of plant isoflavones, characterized in that, Treatment was performed using a hydrogen sulfide donor, namely NaHS, at a concentration of 0.1-1000 μM.

10. A soybean sprout produced by the method as described in claim 7 or claim 9, characterized in that, The total isoflavone content of the soybean sprouts is higher than 7900 μg / g.

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