Silk fibroin salt-resistant film as well as preparation method and application thereof

Abstract

The invention provides a silk fibroin salt-resistant film as well as a preparation method and application thereof, and relates to the technical field of biological materials, and the silk fibroin biomass salt-resistant film provided by the invention comprises silk fibroin, allantoin and sodium alginate. The silk fibroin biomass salt-resistant film can be used for seed coating, the water absorption rate and the germination rate of alfalfa seeds can be remarkably improved under normal conditions, the early water absorption capacity and the germination rate of the seeds can be enhanced under salt stress conditions, and damage of salt stress to the seeds is relieved. Important theoretical basis and practical guidance are provided for improving germination and growth of the seeds in the saline-alkali soil.

Description

Technical Field

[0001] The invention belongs to the technical field of biomaterials, and in particular relates to a silk fibroin salt-blocking membrane and a preparation method and application thereof. Background Art

[0002] Soil salinization has always been a major problem that has plagued agricultural production. Land salinization can cause soil compaction, reduce soil fertility, and seriously threaten the growth and development of crops. Therefore, methods to improve crop tolerance to salt and alkali are of great significance to the development of my country's agriculture.

[0003] Salt-alkali stress can have many adverse effects on plants, including ion exosmosis, metabolic blockage, oxidative stress, etc. caused by salt stress, which inhibits their growth. In addition, it will further damage crops due to the increase in pH. Under salt-alkali stress, plants mainly reduce the damage caused by adverse environments to their growth and development by synthesizing osmotic regulating substances, improving the antioxidant capacity of enzymes, selectively absorbing ions, balancing nutrition, changing metabolic types, and adjusting the distribution of biomass.

[0004] The harm of saline-alkali soil to crops is mainly reflected in two aspects. On the one hand, it indirectly changes the physical and chemical properties of the soil, causing plants to lose a good living environment and nutritional conditions; saline-alkali soil has a salinization layer or an alkalization layer, and the soil contains a large amount of soluble salts. Various soluble salts have different harms to crops. The more soluble ions are in water, the stronger their ability to penetrate cell walls is, and the greater the harm is. When the salt concentration in the soil is too high, it will cause ion poisoning. Ion poisoning refers to the Na in biochemical reactions. + Replace K + and Na + and Cl - This causes changes in protein conformation. On the other hand, it directly harms crop cells through soil solution, affecting the normal absorption and metabolic functions of crops. When the osmotic pressure of crops is more than twice that of soil solution, they can continuously absorb water from the soil. When the soil contains more soluble salts, the concentration of soil solution increases, and the osmotic pressure also increases accordingly, affecting the plant roots' absorption of nutrients and water, and thus affecting plant growth.

[0005] The harm of saline-alkali soil to plant growth will cause some effects, such as low germination rate and uneven germination. Saline-alkali soil is divided into salinization layer and alkalization layer. Excessive salt in the soil will cause crop seeds to be subjected to multiple stresses such as ions and osmosis during germination, thus affecting crop seed germination and plant morphology.

[0006] Seed coating has been listed as a key agricultural technology project promoted in my country. After sowing, seeds coated with seed coating agents can quickly absorb water and swell. As the embryo in the seed gradually develops and the seedlings continue to grow, the seed coating agent slowly releases the various active ingredients contained in it, which are gradually absorbed into the body by the seed seedlings, thereby achieving the purpose of promoting growth and development and increasing crop yields. Therefore, it is necessary to develop a salt-barrier film that can regulate seed germination, growth and development, and physiological characteristics under salt stress.

[0007] Patent CN115956559A provides a seed coating agent for improving the germination rate under drought and salt stress, and its preparation method and application. The seed coating includes pantothenic acid polysaccharide, polylysine, amino oligosaccharide, polyethylene glycol 400, fruit green, polyvinyl pyrrolidone, etc. The use of the coating can make the seed germination rate reach more than 90% under drought or salt stress conditions; Patent CN114868769A provides a seed coating, including Bacillus subtilis liquid, film-forming agent, coating agent and sodium hydroxymethyl cellulose solution. Under salt stress conditions, the germination rate of coated seeds is increased from 50% to 90%. However, as shown in the above two patents, the coating agents currently developed have complex ingredients, too many steps in the preparation method, and are time-consuming and labor-intensive. Summary of the invention

[0008] In order to solve the above problems, the present invention provides a silk fibroin salt-blocking membrane and a preparation method and application thereof.

[0009] On the one hand, the present invention provides a silk fibroin salt-blocking membrane, which comprises silk fibroin, allantoin and sodium alginate, and the ratio of silk fibroin, allantoin and sodium alginate is 0.2-4mL: 0.2-0.6g: 1-3g.

[0010] Specifically, the preparation steps of the silk fibroin salt-blocking membrane are as follows: (1) Degumming; (2) dissolving silk fibroin using a ternary solvent system or a urea / sodium hydroxide dissolution system to obtain a silk fibroin solution; (3) Dialysis; (4) Centrifugation to obtain regenerated silk fibroin solution.

[0011] Further specifically, in step (1), in step (1), the degumming process conditions are: using 0.2-0.8% (w / v) Na2CO3 solution as the degumming liquid, and boiling the degumming.

[0012] More specifically, in step (2), the ternary solvent system comprises calcium chloride, water and ethanol, and the molar ratio of the three is calcium chloride: water: ethanol = 0.5-1.5: 7.5-8.5: 1.5-2.5.

[0013] More specifically, in step (2), the urea / sodium hydroxide dissolution system comprises: urea and sodium hydroxide, and the mass ratio of the two is: 10-15g:5-9g.

[0014] More specifically, in step (3), the dialysis process is as follows: the silk fibroin solution is transferred to a treated dialysis bag and dialyzed with pure water at 4° C. for 2.5-3.5 days.

[0015] In certain specific embodiments of the present invention, the dialysis is terminated when calcium ions, sodium ions, chloride ions and / or small solvent molecules are substantially removed.

[0016] Preferably, the dialysis bag is treated by placing it in a mixed solution of 1-3% (w / v) NaHCO3 and 0.5-1.5mmol / L EDTA and boiling it, then taking out the dialysis bag and placing it in a 0.5-1.5mmol / L EDTA solution and continuing to boil it.

[0017] More specifically, in step (4), the centrifugation conditions are: 3000-5000 rpm, 15-25 min.

[0018] On the other hand, the present invention provides the use of silk fibroin salt barrier film in the preparation of seed coating.

[0019] Specifically, the steps of seed coating are: (a) mixing a regenerated silk fibroin solution, sodium alginate and allantoin in proportion to prepare a coating agent; (b) mixing the seeds and the coating agent in a ratio of 0.5-1.5 g: 5-15 mL and stirring; (c) After drying, a seed coating is obtained.

[0020] More specifically, in step (c), the drying includes but is not limited to: freeze drying, natural shade drying, natural air drying and / or spray drying.

[0021] In another aspect, the present invention provides a seed coating comprising the silk fibroin salt-blocking film.

[0022] Compared with the prior art, the present invention has the following advantages: The seed coating prepared by the silk fibroin salt barrier film provided by the present invention can significantly improve the water absorption rate and germination rate of alfalfa seeds under normal conditions, and can also enhance the early water absorption capacity and germination rate of seeds under salt stress conditions, and reduce the damage of salt stress to seeds. It provides an important theoretical basis and practical guidance for improving the germination and growth of alfalfa seeds in saline-alkali land. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1It is the water absorption rate of the coated seeds in Example 4.

[0024] Figure 2 It is the water absorption rate of the coated seeds in Example 4.

[0025] Figure 3 It is the germination rate test result of Example 5.

[0026] Figure 4 This is the effect of coating on alfalfa seedling length under salt stress in Example 5. The lowercase letters a, b, c, and d in the figure represent: those with the same letter have no significant difference, and those with different letters have significant difference.

[0027] Figure 5 This is the effect of coating on hypocotyls of alfalfa seeds under salt stress in Example 5. The lowercase letters a, b, c, d, and e in the figure represent: those with the same letter have no significant difference, and those with different letters have significant difference.

[0028] Figure 6 This is the effect of coating on root length of alfalfa seeds under salt stress in Example 5. The lowercase letters a, b, c, d, and e in the figure represent: those with the same letter have no significant difference, and those with different letters have significant difference.

[0029] Figure 7 This is the effect of coating on the malondialdehyde content of alfalfa seeds after germination under salt stress in Example 5. The lowercase letters a, b, c, and d in the figure represent: those with the same letter have no significant difference, and those with different letters have significant difference. DETAILED DESCRIPTION

[0030] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0031] Example 1 Extraction of silk fibroin 1. Washing: Take about 30 silk cocoons of uniform size, cut them into pieces with scissors, and then use pure water to oscillate and wash them in an ultrasonic cleaner for 8-10 minutes to remove impurities on the surface of the cocoons.

[0032] 2. Drying: Dry the cleaned cocoons at 50℃ for 12 hours and weigh them as m0.

[0033] 3. Degumming: Divide the dried silkworm cocoons into 4 equal parts, dissolve them in 0.5% (w / v) Na2CO3 solution respectively, and boil them for 30 minutes for degumming.

[0034] 4. Washing and drying: After degumming, place the silk fibroin in a culture dish to cool, rinse with ultrapure water for 5-7 minutes to remove the remaining sericin and Na2CO3, then place the rinsed silk fibroin in a culture dish and dry in an oven at 60°C to a constant weight of m1.

[0035] Calculate the silk fibroin extraction rate:

[0036] Judgment criteria: An extraction rate of about 70% and a degumming rate of 30%±3% (1-extraction rate) indicate that the silk has been completely degummed and usable silk fibroin has been obtained.

[0037] 5. Dissolution: ① Ternary solvent dissolution system a. Prepare a ternary mixed solvent of calcium chloride (CaCl2): ultrapure water: ethanol (molar ratio 1:8:2): dissolve 110.98g CaCl2 in an appropriate amount of water, add 144.12mL H2O to the CaCl2 solution, add 116.81mL ethanol to the CaCl2 solution, mix well, and dilute to 1L with deionized water.

[0038] b. Dissolve the Na2CO3-treated silk protein into the ternary solvent at a ratio of 1:10 and 1:20 (v / v).

[0039] ②Urea / sodium hydroxide (NaOH) dissolution system This method uses an incomplete dissolution method to prepare a regenerated silk fibroin aqueous solution at the level of a single-layer silk fibroin ribbon (equivalent to the thickness of a single-layer β-fold) in order to retain the multi-level structural structure of the silk fibroin after degumming.

[0040] a. Prepare solvent: Dissolve 7 g NaOH and 12 g urea crystals in 81 mL ultrapure water and precool in a -12°C refrigerator for 1 h.

[0041] b. Add 3 g of silk fibroin into the urea / NaOH system and dissolve it for 3 days. Stir with a glass rod during the dissolution process and take it out and stir it every 12 hours.

[0042] 6. Dialysis bag pretreatment: Pre-treat the dialysis bag according to the dialysis bag manual to change its permeability. Cut a 15cm section of the dialysis bag with scissors, place it in a mixed solution of 500mL 2% (w / v) NaHCO3 and 1mmol / L EDTA (pH 8.0) and boil it for 10min. Then remove the dialysis bag and place it in a 500mL 1mmol / L EDTA (pH 8.0) solution and continue to boil it for 10min. Remove the pre-treated dialysis bag and immerse it in ultrapure water for later use.

[0043] 7. Dialysis: Pour the dissolved regenerated silk fibroin into a dialysis bag, seal it with a sealing clip, place it in a beaker of 2L ultrapure water and dialyze for 3 days to remove the residual solvent. Replace the ultrapure water every 12 hours during the dialysis process.

[0044] 8. Ultrasound: Use an ultrasonic disruptor (power: 200W) to ultrasonicate the solution poured out after dialysis for 30 minutes. Stop the ultrasound when the solution changes from a flocculent turbid liquid to a milky white transparent solution.

[0045] 9. Centrifugation: The regenerated silk fibroin solution after ultrasound is centrifuged at 4500 rpm for 20 min. After centrifugation, it is stored in a refrigerator at 4°C for later use.

[0046] 10. Concentration determination: Take 10 mL of the centrifuged silk fibroin solution and place it in a culture dish. The mass of the empty culture dish is m3. Place them in a 50℃ oven and dry them to a constant weight of m.

[0047] The concentrations of the three silk fibroin aqueous solutions (CRSF) were calculated:

[0048] The extraction rate of silk fibroin fiber in this study was 67.25% ± 1.19%, which was slightly lower than the theoretical value of 70% of silk fibroin in silk. This shows that the extraction rate of silk fibroin in this study was high. According to the concentration determination formula, the concentrations of the regenerated silk fibroin aqueous solution after the silk fibroin was dissolved in the ternary solvent at a ratio of 1:10 and 1:20 (g / mL) were calculated to be 4 ± 0.3% and 2% ± 0.1%, respectively; while the concentration of the regenerated silk fibroin aqueous solution obtained after the silk fibroin was dissolved in the urea / NaOH solvent at a ratio of 3:100 (g / mL) was 0.5% ± 0.03%.

[0049] Example 2 Preparation of Allantoin Functionalized Silk Fibroin / Sodium Alginate Composite Coating Agent Physical mixing and chemical cross-linking were used to organically combine regenerated silk fibroin solution (SF), sodium alginate (SA) and allantoin (AT). Different treatments were performed by mixing three concentrations of regenerated silk fibroin solutions. The specific experimental steps are as follows: The three prepared SF solutions with different concentrations were mixed with 2% (w / v) sodium alginate solution prepared in ultrapure water at a volume ratio of 1:1 to obtain three equal-volume mixed solutions with different SF / SA composition ratios, namely, SA 2% +SF 0.5% ,SA 2% +SF 2% and SA 2% +SF 4% Finally, the bioactive substance allantoin was added into the mixed solution at 0.4% (g / mL), and stirred with a magnetic stirrer to obtain three different mixed solutions: SA 2% +SF 0.5% +AT 0.4% ,SA 2% +SF 2% +AT 0.4% and SA 2% +SF 4% +AT 0.4% . Further abbreviated as, SA+SF 0.5% 、SA+SF 2% and SA 2% +SF 4% .

[0050] Example 3 Seed treatment and coating operation Select alfalfa seeds of uniform size and full grains ( Medicago sativa .), first soak the seeds in 70% alcohol for 1 min, then soak them in 3% sodium hypochlorite for 10 min, rinse the soaked seeds with sterile water, wipe the surface moisture with sterile filter paper and weigh them.

[0051] Stir and coat the sterilized seeds with three different mixed solution coating agents in a clean beaker in batches at a ratio of 1:10 (g / mL). Select the coated alfalfa seeds with distinct particles and uniform size and roundness, and then dry them naturally in the shade on gauze.

[0052] Example 4 Water absorption test of coated seeds under salt stress Place 25 seeds of the three coatings into a Petri dish containing two layers of filter paper and add 100 mmol·L -1 NaCl solution and distilled water were used as two treatments, and the uncoated seeds were treated with 100 mmol·L -1NaCl solution and distilled water were used as double controls. There were 8 treatments in total, and each treatment was repeated 4 times. The culture dish containing seeds and solution was placed in a constant temperature and humidity incubator at 25°C and 80% relative humidity to conduct a seed water absorption test. During the test, the seeds of each treatment were taken out every 8 hours, and the residual water on the surface of the seeds was gently absorbed with absorbent paper and weighed. During the test, the solution in the culture dish was changed every 8 hours to ensure consistent salt concentration, and the total time was 48 hours.

[0053] The grouping and group naming are shown in Table 1: Table 1

[0054] Calculate the water absorption (Y) and water absorption rate (Y1):

[0055]

[0056] Where m0 represents the original mass of 25 alfalfa seeds; m t represents the mass of 25 seeds at the time t of the experiment; m t-1 It represents the mass of 25 seeds at (t-1) of the experiment.

[0057] The experimental results are shown in Figure 1-Figure 2 Under normal conditions, during the germination of uncoated alfalfa seeds, the seeds absorb water rapidly during 0-8h and slowly during 8-24h; and the water absorption rate of seeds increases significantly during 24-48h. Under normal conditions, compared with the uncoated treatment, the three coating treatments all increased the water absorption rate during the 4-24h germination period and reduced the water absorption rate during the 24-48h period. SA+SF 4% The water absorption rate of the treated coated seeds was the highest during the 0-4h and 4-24h periods.

[0058] During the germination process of uncoated alfalfa seeds under salt stress, the rapid water absorption period of the seeds was 0-24h, and the slow water absorption period was 24-48h. Under salt stress, compared with the uncoated treatment, except for SA+SF 2% The water absorption rate of the treated coated seeds during 24-36h was higher than that of the control, except for SA+SF 0.5% With SA+SF 2% Treatment reduced the water absorption rate of alfalfa seeds during the period of 0-48h. 4% The treated coated seeds increased the water absorption rate during the rapid water absorption period of 0-16h, the water absorption rate during 16-24h decreased by only 1.82%, and the water absorption rate during 24-48h decreased significantly compared with the uncoated control.

[0059] In summary, SA+SF4% The treatment increased the water absorption rate of alfalfa seeds during the period of 0-16h under normal conditions and salt stress.

[0060] Under normal conditions, the water absorption rates of alfalfa seeds at 4h, 8h, 16h, 24h, 36h and 48h were 0.43%, 0.90%, 0.33%, 0.35%, 0.64% and 0.76%, respectively. It can be seen that the water absorption rate of uncoated alfalfa seeds treated with clear water was high in the first 8h, then fluctuated, and increased again at 36h and 48h. In clear water, compared with the uncoated treatment, the three coating treatments increased the water absorption rate of seeds at 8, 16 and 24h, and reduced the water absorption rate of seeds at 36h and 48h. SA+SF 0.5% With SA+SF 2% The treatment also reduced the water absorption rate at 4 h, while SA+SF 4% The treatment increased the water absorption rate at 4 h, and the water absorption rates were highest at 8, 16 and 24 h.

[0061] The results showed that coating improved the early water absorption capacity of alfalfa seeds under normal conditions.

[0062] The water absorption rates of uncoated seeds at the same time points under salt stress were 0.74%, 1.13%, 0.64%, 0.80%, 0.33% and 0.28%, respectively. It can be seen that the water absorption rate of uncoated seeds within 24 hours is relatively high, especially after 8 hours and 24 hours, reaching 1.13% and 0.80%, respectively. However, after 36 hours and 48 hours, the water absorption rate dropped sharply to 0.33% and 0.28%, which are lower than the levels under normal conditions. This shows that the early water absorption capacity of alfalfa seeds under salt stress conditions is enhanced, probably to cope with the water demand under stress conditions. However, long-term salt stress may lead to a decrease in the water absorption capacity of seeds, which may be related to the decrease in cell stability or limited cell osmotic regulation ability caused by salt stress. Under salt stress, compared with the uncoated treatment, SA+SF 0.5% With SA+SF 2% The water absorption rate of the treated coated seeds decreased overall within 48 hours; on the contrary, SA+SF 4% The treated coated seeds increased the water absorption rate within 24 hours, and the water absorption rate dropped sharply to 0.08% and 0.06% after 36 hours and 48 hours.

[0063] The results show that SA+SF 4% The treated coated seeds enhanced the early water absorption capacity of alfalfa seeds under salt stress conditions.

[0064] Example 5 Germination test of coated seeds under salt stress The test procedure refers to the coated seed water absorption test in Example 4. There are 8 treatments in total, and each treatment is repeated 4 times. The photoperiod is 12 hours of light / 12 hours of darkness, the temperature is 25°C, and the relative humidity is 80%. During the experiment, the treatment solution is changed every 48 hours to ensure the consistency of salt concentration. The germination standard is that the radicle of the seed breaks through the seed coat by 2 mm. The number of germinations is recorded every 12 hours, and the number of seeds germinating in each dish is counted to calculate the germination rate. Various indicators are measured on the 7th day of germination. Including root length, hypocotyl length, seedling length, that is, the malondialdehyde content of the plant.

[0065] Malondialdehyde content determination method: Take 0.5g of alfalfa plant and grind it, add 5mL of trichloroacetic acid (note its corrosiveness), add appropriate amount of borax and grind it thoroughly, centrifuge at 3000rpm, 4℃ for 10min, take 2mL of the supernatant after centrifugation, add 2mL of 0.67% thiobarbituric acid and shake well, use 2mL of distilled water as a blank control, boil in a water bath for 30min, put into an ice pool and cool quickly, centrifuge (3000rpm, 4℃) for 10min after cooling, take 2mL of the supernatant, measure the absorbance at 400nm, 520nm, and 632nm, and calculate the malondialdehyde content.

[0066] The test results are shown in Figure 3-Figure 7 Compared with normal conditions, salt stress reduced the germination rate of uncoated seeds at various time periods during germination. 0.5% The germination rate of alfalfa seeds treated with SA+SF was also reduced under salt stress; on the contrary, 2% Treatment increased the germination rate of alfalfa seeds under salt stress; SA+SF 4% The treatment increased the germination rate of alfalfa seeds under salt stress during the period of 48-120 hours, and the germination rate during the period of 48-120 hours was higher than that of seeds of other coating and uncoated treatments under salt stress.

[0067] After germination for 7 days, the morphological indexes of several groups of alfalfa seedlings were measured under normal conditions and salt stress conditions with different coating treatments. 2% Under normal conditions, the length of alfalfa seedlings, hypocotyl and root length were significantly optimal, which were 31.62 cm, 7.06 cm and 22.08 cm respectively; SA+SF 4% The length of alfalfa seedlings treated with SA+SF under salt stress (32.01 cm) was significantly different from that of SA+SF 2% There was no significant difference in the seedling length between the treated seedlings under normal conditions. 2% The morphological indices of alfalfa seedlings treated with SA+SF 0.5%In the treated alfalfa seedlings, only the hypocotyl increased significantly under salt stress, while the seedling length and root length were not significantly longer than those under normal conditions; SA+SF 4% The seedling length, hypocotyl and root length of the treated alfalfa seedlings increased significantly under salt stress conditions, and the values ​​of seedling length and root length were significantly higher than those of the other two groups of coating treatments under salt stress.

[0068] MDA content is used to indicate the degree of damage to plant cell membranes after stress. Excessive MDA content indicates a high degree of membrane damage. 2% The MDA content of alfalfa plants under normal conditions was significantly the highest, at 9.73 nmol / g; 2% The MDA content of alfalfa plants under salt stress was significantly the lowest, at 3.8 nmol / g, which was 60.95% lower than that under normal conditions. 4% Treatment under normal conditions and SA+SF 0.5% Treatment under salt stress conditions and SA + SF 2% There was no significant difference in the content of MDA among the treatments under salt stress. 4% Processing and SA+SF 0.5% There was no significant difference in the malondialdehyde content between the treatments under salt stress conditions. 4% Processing and SA+SF 0.5% Treatment can alleviate the damage of alfalfa caused by salt stress.

[0069] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A silk fibroin salt-blocking membrane, characterized in that: The silk fibroin salt-blocking membrane comprises silk fibroin, allantoin and sodium alginate, and the ratio of the silk fibroin, allantoin and sodium alginate is: 0.2-4 mL: 0.2-0.6 g: 1-3 g.

2. The silk fibroin salt-blocking membrane according to claim 1, characterized in that: The preparation steps of the silk fibroin include: (1) Degumming; (2) dissolving silk fibroin using a ternary solvent system or a urea / sodium hydroxide dissolution system to obtain a silk fibroin solution; (3) Dialysis; (4) Centrifugation to obtain regenerated silk fibroin solution.

3. The silk fibroin salt-blocking membrane according to claim 2, characterized in that: In step (1), the degumming conditions are: using 0.2-0.8% (w / v) Na2CO3 solution as the degumming solution and boiling the degumming.

4. The silk fibroin salt-blocking membrane according to claim 2, characterized in that: In step (2), the ternary solvent system comprises calcium chloride, water and ethanol, and the molar ratio of the three is calcium chloride: water: ethanol = 0.5-1.5: 7.5-8.5: 1.5-2.

5.

5. The silk fibroin salt-blocking membrane according to claim 2, characterized in that: In step (2), the urea / sodium hydroxide dissolution system comprises: urea and NaOH, and the mass ratio of the two is: 10-15g:5-9g.

6. The silk fibroin salt-blocking membrane according to claim 2, characterized in that: In step (3), the dialysis comprises the following steps: the silk fibroin solution is transferred to a treated dialysis bag and dialyzed with pure water for 2.5-3.5 days.

7. The silk fibroin salt-blocking membrane according to claim 6, characterized in that: The dialysis in step (3) is terminated when calcium ions, sodium ions, chloride ions and / or small solvent molecules are fully removed.

8. The silk fibroin salt-blocking membrane according to claim 6, characterized in that: In step (3), the dialysis bag is treated by placing it in a mixed solution of 1-3% (w / v) NaHCO3 and 0.5-1.5mmol / L EDTA and boiling it, then removing the dialysis bag and placing it in a 0.5-1.5mmol / L EDTA solution and continuing to boil it.

9. The silk fibroin salt-blocking membrane according to claim 2, characterized in that: In step (4), the centrifugation condition is 3000-5000 rpm, 15-25 min.

10. Use of the silk fibroin salt barrier film according to any one of claims 1 to 9 in preparing seed coating.

11. The use according to claim 10, characterized in that: The steps of seed coating are: (a) mixing a regenerated silk fibroin solution, sodium alginate and allantoin in proportion to prepare a coating agent; (b) mixing the seeds and the coating agent in a ratio of 0.5-1.5 g: 5-15 mL and stirring; (c) After drying, a seed coating is obtained.

12. The use according to claim 11, characterized in that: In step (c), the drying includes: freeze drying, natural shade drying, natural air drying and / or spray drying.

13. A seed coating, characterized in that: The seed coating comprises the silk fibroin salt-blocking film according to any one of claims 1 to 9.

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