Vascular bundle targeting biological nano-selenium and preparation method and application thereof

Abstract

This invention discloses a vascular bundle-targeted bio-selenium nanoparticle, its preparation method, and its application, belonging to the field of plant treatment technology. The invention involves mixing and dissolving bio-selenium nanoparticles, water, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide, followed by activation to obtain an activated bio-selenium nanoparticle solution. A xylem-anchored peptide and MES buffer are then mixed and dissolved to obtain a xylem-anchored peptide solution. The activated bio-selenium nanoparticle solution and the xylem-anchored peptide solution are mixed, and the pH is adjusted to 6.3-6.8 before stirring to obtain the vascular bundle-targeted bio-selenium nanoparticle. The vascular bundle-targeted bio-selenium nanoparticle provided by this invention achieves interception of heavy metal transport pathways by specifically anchoring to xylem vessels. Compared with existing technologies that rely solely on broad-spectrum physiological antagonism, its efficiency in reducing heavy metal content in crop stems and leaves represents a qualitative leap.

Description

Technical Field

[0001] This invention belongs to the field of plant treatment agent technology, specifically relating to a vascular bundle-targeted bio-nano selenium, its preparation method, and its application. Background Technology

[0002] With the rapid development of industry and agriculture, soil heavy metal pollution has become a serious problem threatening global agricultural product safety and ecological health. Among them, toxic heavy metals such as cadmium, lead, and mercury can migrate through the soil-plant system, accumulate in the edible parts of crops (especially leaves and stems), and ultimately harm human health through the food chain. Traditional physicochemical remediation methods are costly and easily damage the soil ecosystem, while simple soil passivation technology, although it can reduce the bioavailability of heavy metals, cannot completely block their absorption and translocation in specific crops (especially leafy vegetables and tea). Therefore, there is an urgent need to develop a targeted technology that can directly and efficiently intercept heavy metals within plants and along key transport pathways to achieve safe agricultural production.

[0003] Currently, the mainstream technologies for reducing the accumulation of heavy metals in plants can be divided into the following three categories, but all of them have significant limitations:

[0004] Soil passivation / fixation technology: This method involves adding passivating agents such as lime, biochar, and clay minerals to the soil to alter the form of heavy metals and reduce their bioavailability. While this method targets the entire soil system and has a general effect on reducing heavy metal absorption by crops, it lacks specificity and is ineffective against heavy metals already present in plant roots. Furthermore, its effectiveness may be unstable due to changes in the soil environment.

[0005] Foliar spraying technology: By spraying elements such as silicon, selenium, and zinc, the antagonistic effects of these elements within the plant or their promotion of cell wall fixation are utilized to reduce the toxicity of heavy metals and their translocation to edible parts. Existing technologies are mostly broad-spectrum, non-targeted physiological regulation. For example, ordinary nano-selenium foliar fertilizers can alleviate toxicity by enhancing antioxidant capacity and competitive absorption, but their effects are dispersed throughout the leaf tissue, lacking the proactive and precise interception capability for heavy metal transport in the vascular bundle system—a "high-speed channel"—resulting in a ceiling on control efficiency.

[0006] Genetic engineering and microbial remediation technologies aim to reduce heavy metal uptake by cultivating low-accumulation varieties or utilizing rhizosphere microorganisms. However, these technologies face challenges in regulation and acceptance, and their effectiveness is limited by environmental factors and the stability of microbial colonization. Furthermore, since their primary site of action is the rhizosphere, they lack effective intervention methods for heavy metals transported long distances through the xylem.

[0007] After being absorbed by plant roots, heavy metal ions are primarily transported to the above-ground parts via xylem vessels through transpiration, which is the core pathway for them to enter the edible parts of stems and leaves. The fundamental limitation of existing technologies lies in their inability to precisely intervene in this crucial transport pathway. Therefore, developing a barrier agent that can specifically target xylem vessels and immobilize heavy metals in situ is a new approach to fundamentally overcome the bottlenecks of existing technologies. Summary of the Invention

[0008] In view of this, the purpose of this invention is to provide a vascular bundle-targeted bio-nano selenium, its preparation method and application. This invention can reduce the heavy metal content in crop stems and leaves and solve the problem of heavy metal pollution in farmland.

[0009] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for preparing vascular bundle-targeted bio-selenium nanoparticles, comprising the following preparation steps: S1. Bio-nano selenium, water, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide are mixed, dissolved, and activated to obtain an activated bio-nano selenium solution. S2. Mix and dissolve the xylem anchoring peptide and MES buffer to obtain a xylem anchoring peptide solution; S3. After mixing the activated bio-nano selenium solution and the xylem-anchored peptide solution, the pH was adjusted to 6.3-6.8 and the mixture was stirred to obtain vascular bundle-targeted bio-nano selenium.

[0010] Preferably, the mass ratio of bio-nano selenium, water, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide in S1 is (8-12):1000:(4-6):(2-4).

[0011] Preferably, the activation temperature in S1 is 25-28℃ and the activation time is 20-40 min.

[0012] Preferably, the mass ratio of xylem-anchored peptide to MES buffer in S2 is (0.5-1):100.

[0013] Preferably, the sequence of the xylem-anchored peptide is: H2N-YYC-WWLPWWLPWWLP-COOH.

[0014] Preferably, the volume ratio of the activated bio-nano selenium solution and the xylem-anchored peptide solution in S3 is 2:(4-6).

[0015] Preferably, the temperature of the stirring reaction in S3 is 20-30℃ and the time is 10-14h.

[0016] The present invention also provides a vascular bundle-targeted bio-selenium nanoparticle, which is prepared by the above-described preparation method.

[0017] This invention also provides the application of the above-mentioned vascular bundle-targeted bio-nano selenium in reducing the heavy metal content in crop stems and leaves.

[0018] As a preferred method, the vascular bundle-targeting bio-nano selenium, potassium sorbate, and organosilicon surfactant are mixed in a mass ratio of 5:0.5:2 and then diluted together before application.

[0019] It contains at least the following beneficial technical effects: The vascular bundle-targeted bio-nano-selenium provided by this invention achieves interception of heavy metal transport pathways by specifically anchoring xylem vessels. Compared with existing technologies that rely solely on broad-spectrum physiological antagonism, its efficiency in reducing heavy metal content in crop stems and leaves represents a qualitative leap. Pot and field trials show that this technology can reduce cadmium and lead content in leafy vegetables and rice stems and leaves by more than 70%. In lightly to moderately polluted farmland, it can increase the safety compliance rate of agricultural products from 0% to 100%, while simultaneously achieving natural selenium enrichment in agricultural products. This provides an efficient, precise, and practical innovative solution to resolving the contradiction between heavy metal pollution in farmland and agricultural product safety. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] Unless otherwise specified, "room temperature" and "normal temperature" in this invention refer to 25±2℃.

[0026] Unless otherwise specified, all raw materials or instruments used in the following embodiments of the present invention are commercially available.

[0027] The sequence of the xylem anchoring peptide is: H2N-YYC-WWLPWWLPWWLP-COOH; it was synthesized by a commissioned biotechnology company; the bio-nano selenium was obtained by fermentation of Bacillus belysin N-8, and other raw materials were commercially available.

[0028] Example 1 This embodiment provides a method for preparing vascular bundle-targeted bio-selenium nanoparticles, the steps of which are as follows: S1. Preparation of activated bio-nano selenium solution Weigh 8.0 g of bio-nano selenium and add it to 1000 g of deionized water. Stir at 25 °C until completely dispersed. Then, add 4.0 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 2.0 g of N-hydroxysuccinimide. Continue stirring at 25 °C for 20 minutes to activate the solution and obtain an activated bio-nano selenium solution.

[0029] S2. Preparation of xylem-anchored peptide solution Weigh 0.5 g of xylem-anchored peptide and dissolve it in 100 g of 0.1 M MES buffer (pH 6.0). Gently shake until completely dissolved to obtain a xylem-anchored peptide solution.

[0030] S3. Coupling reaction and product acquisition Measure 200 mL of the activated bio-nano selenium solution obtained in step S1 and 400 mL of the xylem-anchored peptide solution obtained in step S2, and mix them with stirring. Finely adjust the pH of the mixture to 6.3 using 0.1 M sodium hydroxide solution. Place the reaction system in a constant temperature environment of 20°C and continuously stir gently for 10 hours. After the reaction is complete, transfer the resulting reaction solution to an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa, dialyze and centrifuge three times with deionized water to remove unreacted small molecule reagents and salts, finally obtaining a concentrated, deep red, clear vascular bundle-targeted bio-nano selenium colloidal solution.

[0031] S4. Formulation Take 5.0 g of the vascular bundle-targeted bio-nano selenium colloidal solution prepared above, mix it evenly with 0.5 g of food-grade potassium sorbate and 2.0 g of organosilicon surfactant (Silwet L-77) to obtain a concentrated formulation.

[0032] Example 2 This embodiment provides a method for preparing vascular bundle-targeted bio-selenium nanoparticles, the steps of which are as follows: S1. Preparation of activated bio-nano selenium solution Weigh 10.0 g of commercially available microbial-derived bio-nano selenium powder and add it to 1000 g of deionized water. Stir at 26 °C until completely dispersed. Then, add 5.0 g of EDC and 3.0 g of NHS, and continue stirring at a constant temperature of 26 °C for 30 minutes to activate the solution, thus obtaining an activated bio-nano selenium solution.

[0033] S2. Preparation of xylem-anchored peptide solution Weigh 0.5 g of xylem-anchored peptide and dissolve it in 100 g of 0.1 M MES buffer (pH 6.0). Gently shake until completely dissolved to obtain a xylem-anchored peptide solution.

[0034] S3. Coupling reaction and product acquisition Measure 200 mL of the activated bio-nano selenium solution obtained in step S1 and 500 mL of the xylem-anchored peptide solution obtained in step S2, and mix them with stirring. Finely adjust the pH of the mixture to 6.5 using 0.1 M sodium hydroxide solution. Place the reaction system in a constant temperature environment of 25°C and continuously stir gently for 12 hours. After the reaction is complete, dialyze and concentrate the reaction solution using a tangential flow ultrafiltration system, using deionized water as the replacement fluid, until the conductivity of the filtrate is below 50 μS / cm, obtaining the vascular bundle-targeted bio-nano selenium product solution.

[0035] S4. Formulation Take 5.0 g of the above finished liquid, mix it with 0.5 g of potassium sorbate and 2.0 g of organosilicon surfactant to obtain a concentrated preparation.

[0036] Example 3 This embodiment provides a method for preparing vascular bundle-targeted bio-selenium nanoparticles, the steps of which are as follows: S1. Preparation of activated bio-nano selenium solution Weigh 12.0 g of commercially available microbial-derived bio-nano selenium powder and add it to 1000 g of deionized water. Stir at 28 °C until completely dispersed. Then, add 6.0 g of EDC and 4.0 g of NHS, and continue stirring at a constant temperature of 28 °C for 40 minutes to activate the solution, thus obtaining an activated bio-nano selenium solution.

[0037] S2. Preparation of xylem-anchored peptide solution Weigh 0.5 g of xylem-anchored peptide and dissolve it in 100 g of 0.1 M MES buffer (pH 6.0). Gently shake until completely dissolved to obtain a xylem-anchored peptide solution.

[0038] S3. Coupling reaction and product acquisition Measure 200 mL of the activated bio-nano selenium solution obtained in step S1 and 600 mL of the xylem-anchored peptide solution obtained in step S2, and mix them with stirring. Finely adjust the pH of the mixture to 6.8 using 0.1 M sodium hydroxide solution. Place the reaction system in a constant temperature environment of 30°C and continuously stir gently for 14 hours. After the reaction is complete, purify the reaction solution by gel chromatography, collect the target product peak, and then freeze-dry to obtain a reddish-brown vascular bundle-targeted bio-nano selenium solid powder.

[0039] S4. Formulation Take 5.0 g of the above solid powder and redisperse it in an appropriate amount of deionized water. Mix it evenly with 0.5 g of potassium sorbate and 2.0 g of organosilicon surfactant, and make up to the appropriate volume to obtain a concentrated preparation.

[0040] Experimental Example Potted plant simulation verification experiment This experiment was conducted in a controlled greenhouse environment to accurately evaluate the inhibitory effect of the product of this invention on the accumulation of heavy metals in the stems and leaves of different crops.

[0041] 1. Experimental Materials and Methods The tested soil was collected from a slightly polluted farmland and homogenized. Its basic physicochemical properties and heavy metal content were as follows: pH 6.5, total cadmium (Cd) 1.2 mg / kg, and total lead (Pb) 120 mg / kg.

[0042] Test crops: Two typical crops with different heavy metal absorption characteristics were selected: Chinese cabbage and rice.

[0043] Test formulation: Product group (T) of this invention: Vascular bundle-targeted bio-nano selenium preparation prepared according to the method of Example 2.

[0044] Ordinary nano-selenium control group (C1): Commercially available ordinary biological nano-selenium foliar fertilizer (same selenium content).

[0045] Blank control group (C0): Sprayed with an equal amount of water.

[0046] Experimental Design: Three treatments were set up for each crop, with four replicates per treatment, arranged completely randomly. Soil was filled into pots and seeds / transplanted. During the vigorous growth period of the crops (4-leaf stage for Chinese cabbage, peak tillering stage for rice), a 200-fold diluted solution was applied to the leaves of each treatment evenly, ensuring the leaves were moist but not dripping. Water and fertilizer management was consistent throughout the entire growth period.

[0047] Sample collection and analysis: Above-ground parts of plants from each treatment were collected during the crop harvest period (30 days after the growth of Chinese cabbage, and at the maturity stage of rice). The samples were washed with deionized water, dried, and pulverized. Inductively coupled plasma mass spectrometry (ICP-MS) was used to determine the Cd, Pb, and selenium content in the stem and leaf tissues.

[0048] 2. Experimental Results and Analysis Changes in heavy metal content: As shown in the table below, compared with the blank control group, both selenium spraying treatments significantly reduced the heavy metal content in crop stems and leaves. However, the effect of the product group of this invention far exceeded that of the ordinary nano-selenium control group.

[0049] Table 1. Effects of different treatments on heavy metal content in crop stems and leaves (mg / kg, DW)

[0050] The selenium content in crop tissues of all selenium-treated groups was significantly increased, and there was no statistically significant difference between the product group and the ordinary nano-selenium group, indicating that this product also has good selenium fortification function. However, at the same selenium absorption level, the product of this invention has a heavy metal blocking efficiency that is about 20-30 percentage points higher. This result strongly proves that its superior effect is not only due to the physiological antagonism of selenium, but also mainly due to the "vascular bundle targeted interception" function endowed by xylem anchoring peptides. This targeting allows the bio-nano-selenium to be precisely positioned on the inner wall of xylem vessels that transport heavy metals, thereby achieving efficient in-situ fixation of heavy metal ions and blocking their migration pathway to aboveground soft tissues.

[0051] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing vascular bundle-targeted bio-selenium nanoparticles, characterized in that, The preparation steps include the following: S1. Bio-nano selenium, water, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide are mixed, dissolved, and activated to obtain an activated bio-nano selenium solution. S2. Mix and dissolve the xylem anchoring peptide and MES buffer to obtain a xylem anchoring peptide solution; S3. After mixing the activated bio-nano selenium solution and the xylem-anchored peptide solution, the pH was adjusted to 6.3-6.8 and the mixture was stirred to obtain vascular bundle-targeted bio-nano selenium.

2. The preparation method according to claim 1, characterized in that, The mass ratio of bio-nano selenium, water, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide in S1 is (8-12):1000:(4-6):(2-4).

3. The preparation method according to claim 1, characterized in that, The activation temperature in S1 is 25-28℃, and the activation time is 20-40 min.

4. The preparation method according to claim 1, characterized in that, The mass ratio of xylem-anchored peptides to MES buffer in S2 is (0.5-1):

100.

5. The preparation method according to claim 4, characterized in that, The sequence of the xylem-anchored peptide is: H2N-YYC-WWLPWWLPWWLP-COOH.

6. The preparation method according to claim 1, characterized in that, The volume ratio of the activated bio-nano selenium solution and the xylem-anchored peptide solution in S3 is 2:(4-6).

7. The preparation method according to claim 1, characterized in that, The stirring reaction in S3 is carried out at a temperature of 20-30℃ for 10-14 hours.

8. A vascular bundle-targeted bio-nano selenium nanoparticle, characterized in that, It is prepared by the preparation method according to any one of claims 1-7.

9. The application of the vascular bundle-targeted bio-nano selenium as described in claim 8 in reducing the heavy metal content in crop stems and leaves.

10. The application according to claim 9, characterized in that, The vascular bundle-targeted bio-nano selenium, potassium sorbate, and organosilicon surfactant were mixed in a mass ratio of 5:0.5:2 and then diluted together before application.

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