Salicylic acid-agaro-oligosaccharide-nano-selenium compound as well as preparation method and application thereof

Abstract

The invention provides a salicylic acid (at) agaro-oligosaccharide-nano-selenium compound (SA (at) AGOS-SeNPs) as well as a preparation method and application thereof, and relates to the technical field of agricultural application. An active group of salicylic acid in the compound is combined with agaro-oligosaccharide nano-selenium through a covalent bond. The preparation method of the SA (at) AGOS-SeNPs mainly comprises the following steps: (1) firstly mixing a sodium selenite solution with an agaro-oligosaccharide solution, then adding a salicylic acid solution, stirring and uniformly mixing, then dropwise adding ascorbic acid under a vigorous stirring condition, and stirring for a period of time at a certain temperature under a dark condition to obtain a reaction solution; and (2) dialyzing the reaction solution in pure water in a dark environment by using a dialysis bag, collecting the solution in the dialysis bag, and freeze-drying to obtain the SA-AGOS-SeNPs. The SAatAGOS-SeNPs prepared by the preparation method disclosed by the invention is small in particle size, can quickly enter plant cells and diffuse and conduct, is easily absorbed and transferred by plants, solves the problems of poor water solubility and low bioavailability of salicylic acid, and can effectively promote germination of soybean seeds under salt stress.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural applications, and specifically relates to a salicylic acid@agar oligosaccharide-nanoselenium complex and a preparation method thereof, and application of the complex in promoting soybean seed germination under salt stress. Background Art

[0002] In recent years, my country's land salinization problem has become more serious, with saline soil area of ​​about 34.6 million hectares, accounting for 10% of the country's cultivated land. Land salinization poses a huge threat to agricultural production and sustainable development. The seed germination stage is critical to the growth and development of crops. Compared with the growth stage, plants are more susceptible to salt stress during the seed germination stage. Salt stress will reduce the germination rate of plant seeds, hinder plant growth and development, and cause crop yield reduction. Therefore, how to alleviate the impact of salt stress on crop germination and growth has become an urgent problem to be solved in current salt marshland agricultural planting.

[0003] Studies have shown that under salt stress conditions, appropriate concentrations of salicylic acid can promote plant seed germination and radicle growth, and enhance seed salt tolerance. However, due to the poor water solubility and low bioavailability of salicylic acid, a large amount of salicylic acid alone is required to achieve the desired effect, which not only greatly increases costs, but also causes salicylic acid to accumulate in seed cells, resulting in side effects such as inhibiting seed germination.

[0004] The application of nanotechnology provides an effective way to solve the above-mentioned shortcomings of salicylic acid. As carriers of active substances, nanomaterials have many advantages such as strong targeting and amplification of their physiological activity. Nanoselenium (SeNPs) is a nanoscale elemental selenium, which is the least toxic form of selenium that has been discovered. Nanoselenium has strong water solubility, can quickly enter plant cells and diffuse and conduct, and is easily absorbed and transported by plants. However, nanoselenium synthesized by chemical methods requires the addition of stabilizers to maintain its stability and activity, and different stabilizers have a great influence on the properties and application effects of nanoselenium. Agar oligosaccharide molecules are rich in hydroxyl groups, sulfate groups and other groups, which can provide a favorable microenvironment for the growth of selenium atom clusters, effectively prevent nanoselenium aggregation, reduce the particle size of nanoselenium complexes, and are more conducive to plant absorption.

[0005] This patent combines salicylic acid, agar oligosaccharide and nano-selenium into a new type of nanomaterial, and applies it to improving soybean seed germination under salt stress. There are currently no related reports at home and abroad. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a salicylic acid@agar oligosaccharide-nanoselenium complex (SA@AGOS-SeNPs) and a preparation method thereof, which solves the problems of poor water solubility and low bioavailability of salicylic acid; the present invention also provides an application of SA@AGOS-SeNPs in promoting soybean seed germination under salt stress.

[0007] In order to achieve the above object, the present invention adopts the following technical solution: The present invention provides a salicylic acid@agar oligosaccharide-nano selenium complex (SA@AGOS-SeNPs), in which the active groups of salicylic acid are combined with agar oligosaccharide nano-selenium through covalent bonds.

[0008] The present invention also provides a method for preparing salicylic acid@agar oligosaccharide-nanoselenium complex (SA@AGOS-SeNPs), comprising the following steps: (1) firstly mix the sodium selenite solution and the agar oligosaccharide solution, then add the salicylic acid solution and stir to mix, then add ascorbic acid dropwise under vigorous stirring, stir for a period of time at a certain temperature and in the dark to obtain a reaction solution; (2) The reaction solution was dialyzed in pure water using a dialysis bag in a light-proof environment, and the liquid in the dialysis bag was collected to obtain SA@AGOS-SeNPs.

[0009] Preferably, the molar concentration of the sodium selenite solution is 20-100 mmol·L -1 .

[0010] Preferably, the preparation method of the agar oligosaccharide is as follows: adding agar powder to water at a mass ratio of 1:25-40, adjusting the pH to 7.5, heating to 40-50°C, adding agarase at 1-5% of the mass of agar powder, performing enzymatic hydrolysis for 5-10 hours under stirring conditions, boiling for 5-10 minutes after the reaction, cooling to room temperature, adding anhydrous ethanol to the supernatant to make the ethanol concentration in the solution reach 60% of the final concentration, allowing to settle and stand at room temperature for 12 hours, taking the supernatant after centrifugation (10000 rpm, 10 min), rotary evaporation and concentration, and freeze-drying to obtain agar oligosaccharides with a degree of polymerization of 3-8.

[0011] Preferably, the mass concentration of the agar oligosaccharide solution is 1-5 mg / mL.

[0012] Preferably, the molar concentration of the ascorbic acid solution is 50-150 mmol·L -1 .

[0013] Preferably, the salicylic acid solution is an ethanol solution of salicylic acid with a molar concentration of 20 to 100 mmol·L -1 .

[0014] Preferably, the volume ratio of the sodium selenite solution, the agar oligosaccharide solution, the salicylic acid solution and ascorbic acid is 1:1:1:1.

[0015] Preferably, stirring is carried out at 20-40° C. in the dark for 1-4 h.

[0016] Preferably, the molecular weight cut-off of the dialysis bag is 3500Da, and the dialysis is performed for 24 to 72 hours.

[0017] The present invention also provides the use of the SA@AGOS-SeNPs described in the above technical solution in promoting seed germination under salt stress.

[0018] The beneficial effects of the present invention are: 1. The preparation method of SA@AGOS-SeNPs provided by the present invention is simple, low-cost, green and pollution-free; 2. The SA@AGOS-SeNPs prepared by the present invention have a small particle size, can quickly enter plant cells and diffuse and conduct, and are easily absorbed and transported by plants, thus solving the problem of poor water solubility and low bioavailability of salicylic acid; 3. The application of SA@AGOS-SeNPs can effectively improve the germination rate, germination index, germination potential and vitality index of soybean seeds under salt stress, enhance the activity of antioxidant enzymes and cell osmotic regulation ability, and can be used to promote the germination of crop seeds in saline soil in agricultural production. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The effects of agar oligosaccharide solution concentration, stirring time and reaction temperature on the particle size of SA@AGOS-SeNPs.

[0020] Figure 2 Particle size diagram of SA@AGOS-SeNPs and AGOS-SeNPs.

[0021] Figure 3 Surface potential diagrams of SA@AGOS-SeNPs and AGOS-SeNPs.

[0022] Figure 4 Infrared spectra of SA@AGOS-SeNPs and AGOS-SeNPs.

[0023] Figure 5 This is the transmission electron microscopy characterization result of SA@AGOS-SeNPs.

[0024] Figure 6 These are the results of the determination of antioxidant enzyme activity and MDA content in soybean seedlings in Example 4.

[0025] Figure 7 The following are the results of measuring the proline content in soybean seedlings in Example 4. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] Unless otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art. The test methods in the following examples are conventional methods unless otherwise specified. Unless otherwise specified, the reagents and materials used can all be purchased from the market.

[0028] The agar oligosaccharide used in the following examples was prepared by enzymatic hydrolysis. The specific preparation method was as follows: agar powder was added to water at a mass ratio of 1:25, the pH was adjusted to 7.5, heated to 50°C, agar lyase was added at 3% of the mass of agar powder, and the enzymatic hydrolysis reaction was carried out under stirring for 8 hours. After the reaction was completed, it was boiled for 5 minutes, cooled to room temperature, and anhydrous ethanol was added to the supernatant to make the ethanol concentration in the solution reach 60% of the final concentration. The solution was allowed to settle and stand at room temperature for 12 hours, and the supernatant was taken after centrifugation (10000 rpm, 10 minutes), concentrated by rotary evaporation, and freeze-dried to obtain agar oligosaccharides with a degree of polymerization of 3-8.

[0029] Example 1 Optimization of preparation conditions of salicylic acid@agar oligosaccharide-nanoselenium (SA@AGOS-SeNPs) 1. Screening of the concentration of agar oligosaccharide solution, the specific steps are as follows: 5 portions of 10 mL 50 mM sodium selenite solution were mixed with 10 mL agar oligosaccharide solution of different concentrations (1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL and 5 mg / mL), and then 10 mL 50 mM salicylic acid solution was added and stirred, and then 10 mL 100 mM ascorbic acid was added dropwise under vigorous stirring, and stirred for 2 h at 30 ° C in the dark to obtain a reaction solution. The reaction solution was then dialyzed in pure water with a 3500 Da dialysis bag for 48 h in a light-proof environment, and the liquid in the dialysis bag was collected to obtain salicylic acid @ agar oligosaccharide-nanoselenium (SA@AGOS-SeNPs) prepared from agar oligosaccharide solutions of different concentrations.

[0030] The particle size of the prepared SA@AGOS-SeNPs was measured. The particle sizes of SA@AGOS-SeNPs prepared from agar oligosaccharide solutions with different concentrations were as follows: Figure 1 It can be seen that when the concentration of agar oligosaccharide is 4 mg / mL, the particle size is the smallest, which is 152.6 nm.

[0031] Second, screen the reaction temperature. The specific steps are as follows: 5 portions of 10 mL 50 mM sodium selenite solution were mixed with 10 mL 4 mg / mL agar oligosaccharide solution, and then 10 mL 50 mM salicylic acid solution was added and stirred, and then 10 mL 100 mM ascorbic acid was added dropwise under vigorous stirring conditions, and stirred for 2 h at different temperatures (20 ° C, 25 ° C, 30 ° C, 35 ° C, 40 ° C) and in the dark to obtain a reaction solution. The reaction solution was then dialyzed in pure water with a 3500 Da dialysis bag for 48 h in a light-proof environment, and the solution in the dialysis bag was collected to obtain SA@AGOS-SeNPs prepared at different reaction temperatures.

[0032] The particle size of the prepared SA@AGOS-SeNPs was measured. The particle sizes of SA@AGOS-SeNPs prepared at different reaction temperatures are as follows: Figure 1 It can be seen that when the temperature is 25°C, the particle size is the smallest, which is 119.2nm.

[0033] 3. Screening of stirring time, the specific steps are as follows: 5 portions of 10 mL 50 mM sodium selenite solution were mixed with 10 mL 4 mg / mL agar oligosaccharide solution, and then 10 mL 50 mM salicylic acid solution was added and stirred, and then 10 mL 100 mM ascorbic acid was added dropwise under vigorous stirring conditions, and stirred at 25 ° C and in the dark for different time (1h, 2h, 3h, 4h and 5h) to obtain the reaction solution. The reaction solution was then dialyzed in pure water with a 3500 Da dialysis bag in a light-proof environment for 48h, and the solution in the dialysis bag was collected to obtain SA@AGOS-SeNPs prepared with different stirring times.

[0034] The particle size of the prepared SA@AGOS-SeNPs was measured. The particle sizes of SA@AGOS-SeNPs prepared with different stirring times are as follows: Figure 1 It can be seen that when the stirring time is 3h, the particle size is the smallest, which is 98.3nm.

[0035] In summary, when preparing SA@AGOS-SeNPs, the agar oligosaccharide concentration should be 4 mg / mL, the temperature should be 25 °C, and the stirring time should be 3 h.

[0036] Example 2 This embodiment provides a method for preparing salicylic acid@agar oligosaccharide-nanoselenium (SA@AGOS-SeNPs), comprising the following steps: 10 mL of 50 mM sodium selenite solution was mixed with 10 mL of 4 mg / mL agar oligosaccharide solution, and then 10 mL of 50 mM salicylic acid solution was added and stirred, followed by adding 10 mL of 100 mM ascorbic acid dropwise under vigorous stirring, and stirred for 3 h at 25 ° C in the dark to obtain a reaction solution. The reaction solution was then dialyzed in pure water with a 3500 Da dialysis bag in a light-proof environment for 48 h, and the solution in the dialysis bag was collected to obtain SA@AGOS-SeNPs.

[0037] Comparative Example 1 Preparation of agar oligosaccharide-selenium nanoparticles (AGOS-SeNPs) The preparation process of SA@AGOS-SeNPs is basically the same as that in Example 1, except that the amount of salicylic acid used is 0 mM, and the prepared product is agar oligosaccharide-nanoselenium (AGOS-SeNPs) without salicylic acid loading.

[0038] Example 3 The SA@AGOS-SeNPs prepared in Example 2 and the AGOS-SeNPs in Comparative Example 1 were characterized. 1. Particle size analysis The particle size of the sample was measured using a 90Plus PALS Zeta potential laser particle size analyzer. Figure 2 Figure 2 shows the particle size of SA@AGOS-SeNPs and AGOS-SeNPs. The average particle size of AGOS-SeNPs is 91.9 nm, and the average particle size of SA@AGOS-SeNPs is 98.5 nm. The increase in particle size indicates that salicylic acid is successfully loaded into AGOS-SeNPs.

[0039] 2. Zeta potential analysis The Zeta potential of the samples was measured using a 90Plus PALS Zeta potential laser particle size analyzer. Figure 3 The Zeta potential diagram of SA@AGOS-SeNPs and AGOS-SeNPs. The Zeta potential of AGOS-SeNPs is 26.91 mV, and the Zeta potential of SA@AGOS-SeNPs is 18.74 mV. Compared with AGOS-SeNPs, the Zeta potential of SA@AGOS-SeNPs is reduced, indicating that salicylic acid is successfully loaded in AGOS-SeNPs.

[0040] 3. Infrared spectroscopy analysis The infrared spectra of the samples were collected using a V70 infrared spectrometer with a wavenumber range of 4000–650 cm. Figure 4 Infrared spectra of SA@AGOS-SeNPs and AGOS-SeNPs. Figure 5It can be seen that the absorption peaks of SA@AGOS-SeNPs and AGOS-SeNPs are basically consistent. In the infrared spectrum of SA@AGOS-SeNPs, 3358 cm -1 、3016cm -1 、1693cm -1 , and 1227cm -1 , a new absorption peak appeared, indicating that a covalent bond was formed between salicylic acid and AGOS-SeNPs.

[0041] 4. TEM analysis The samples were observed using a JEM-1400PLUS transmission electron microscope. Figure 5 This is the TEM image of SA@AGOS-SeNPs. It can be seen that SA@AGOS-SeNPs are spherical, with uniform particle size, a particle size of about 100 nm, and good dispersion, indicating that the elemental selenium generated by the reaction is adsorbed and wrapped in situ by agar oligosaccharides, effectively preventing the agglomeration of the initially formed particles.

[0042] Example 4 Effects of SA@AGOS-SeNPs soaking on soybean seed germination under salt stress Soybean seeds of uniform size, full grains and from the same batch were selected, and the seeds were disinfected with 1% sodium hypochlorite. After 10 minutes, they were rinsed with deionized water and dried naturally. The seeds were immersed in 0.2mM salicylic acid (SA), 0.2mM AGOS-SeNPs, and 0.2mM SA@AGOS-SeNPs, respectively, and distilled water was used as a control (CK); after soaking for 48 hours, they were rinsed with distilled water, and the surface moisture of the seeds was absorbed with sterile filter paper, and then the treated soybean seeds were evenly distributed in a culture dish pre-laid with two layers of filter paper. 100mM NaCl solution was added, and the culture dish was placed in a culture room at 28℃, with 14h of light and 10h of darkness for cultivation. Three parallel experiments were set for each treatment, and the treatment was 7d in total. Multiple indicators of soybean seed germination were detected and comprehensively analyzed.

[0043] In this example, SA@AGOS-SeNPs is the sample prepared in Example 2, and AGOS-SeNPs is the sample prepared in Comparative Example 1.

[0044] The results of soybean seed germination index determination are shown in Table 1. Different lowercase letters in the table indicate significant differences among different treatments ( P <0.05). As can be seen from the table, after soaking seeds with SA@AGOS-SeNPs, the germination rate, germination index, germination potential and vitality index of salt-stressed soybean seeds were significantly higher than those of the SA and AGOS-SeNPs soaking treatment groups.

[0045] The results of the determination of antioxidant enzyme activity and MDA content in soybean seedlings are as follows Figure 6 As shown in the figure, after SA@AGOS-SeNPs soaking, the SOD, CAT, and POD activities of salt-stressed soybean seedlings were significantly higher than those in the SA and AGOS-SeNPs soaking treatment groups, while the MDA content was significantly reduced.

[0046] The results of proline content determination in soybean seedlings are as follows Figure 7 As shown in the figure, after SA@AGOS-SeNPs seed soaking, the proline content in salt-stressed soybean seedlings was significantly higher than that in the SA and AGOS-SeNPs seed soaking treatment groups.

[0047] The above results show that salicylic acid and AGOS-SeNPs can play a synergistic role. SA@AGOS-SeNPs can more effectively promote the germination of soybean seeds after seed soaking, improve the antioxidant capacity and cell osmotic regulation ability of soybean seedlings under salt stress, and reduce the damage caused by salt stress.

[0048] Table 1 Effects of SA@AGOS-SeNPs on soybean seed germination characteristics under salt stress deal with Germination rate (%) Germination Index Germination potential (%) Vitality Index Comparison <![CDATA[48.61±2.72 d ]]> <![CDATA[15.52±0.95 c ]]> <![CDATA[41.84±2.73 c ]]> <![CDATA[0.18±0.02 d ]]> SA <![CDATA[61.83±4.65 c ]]> <![CDATA[24.08±1.37 b ]]> <![CDATA[55.32±3.16 bc ]]> <![CDATA[0.36±0.04 c ]]> AGOS-SeNPs <![CDATA[69.31±5.18 b ]]> <![CDATA[28.45±1.04 b ]]> <![CDATA[58.42±4.65 b ]]> <![CDATA[0.49±0.06 b ]]> SA@AGOS-SeNPs <![CDATA[86.65±5.15 a ]]> <![CDATA[31.69±2.05 a ]]> <![CDATA[83.77±6.79 a ]]> <![CDATA[0.82±0.08 a ]]> It should be noted that although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they are aware of the basic inventive concepts. Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A salicylic acid@agar oligosaccharide-nano-selenium complex (SA@AGOS-SeNPs), in which the active groups of salicylic acid are bound to agar oligosaccharide nano-selenium through covalent bonds.

2. A method for preparing SA@AGOS-SeNPs, characterized in that: The method comprises the following steps: (1) firstly mix the sodium selenite solution and the agar oligosaccharide solution, then add the salicylic acid solution and stir to mix, then add ascorbic acid dropwise under vigorous stirring, stir for a period of time at a certain temperature and in the dark to obtain a reaction solution; (2) The reaction solution was dialyzed in pure water using a dialysis bag in a light-proof environment, and the liquid in the dialysis bag was collected and freeze-dried to obtain SA@AGOS-SeNPs.

3. The method for preparing SA@AGOS-SeNPs according to claim 2, characterized in that: The preparation method of the agar oligosaccharide is as follows: adding agar powder to water at a mass ratio of 1:25-40, adjusting the pH to 7.5, heating to 40-50°C, adding agarase at 1-5% of the mass of the agar powder, performing enzymatic hydrolysis reaction for 5-10 hours under stirring conditions, boiling for 5-10 minutes after the reaction is completed, cooling to room temperature, adding anhydrous ethanol to the supernatant to make the ethanol concentration in the solution reach 60% of the final concentration, allowing to settle and stand at room temperature for 12 hours, taking the supernatant after centrifugation (10000 rpm, 10 min), rotary evaporation and concentration, and freeze-drying to obtain agar oligosaccharides with a degree of polymerization of 3-8.

4. The method for preparing SA@AGOS-SeNPs according to claim 2, characterized in that: The molar concentration of the sodium selenite solution is 20-100 mmol·L -1 The mass concentration of agar oligosaccharide solution is 1~5mg / mL, and the molar concentration of ascorbic acid solution is 50~150mmol·L -1 The salicylic acid solution is an ethanol solution of salicylic acid with a molar concentration of 20~100mmol·L -1 .

5. The method for preparing SA@AGOS-SeNPs according to claim 2, characterized in that: The volume ratio of the sodium selenite solution, the agar oligosaccharide solution, the salicylic acid solution and the ascorbic acid solution is 1:1:1:

1.

6. The preparation method according to claim 2, characterized in that: The stirring time is 1-4 hours, and the reaction temperature is 20-40°C. The molecular weight cut-off of the dialysis bag is 3500Da, and the dialysis is performed for 24-72 hours.

7. The SA@AGOS-SeNPs prepared according to the preparation method according to any one of claims 2 to 5 can be used to promote the germination of soybean seeds under salt stress.

8. The use according to claim 6, characterized in that: The SA@AGOS-SeNPs can improve the germination rate, germination index, germination potential and vitality index of soybean seeds, and improve the activity of antioxidant enzymes and cell osmotic regulating substances.

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