Spraying liquid for increasing zinc and selenium content of wheat as well as preparation method and spraying method of spraying liquid

Abstract

The invention relates to a spraying liquid for increasing the zinc and selenium content of wheat and a preparation method and a spraying method of the spraying liquid, and belongs to the field of optimization of the zinc and selenium content of the wheat. 0.0021% to 0.0024% of a selenium source substance; 0.02% of a surfactant; and the balance of water. By optimizing the concentration of Zn and Se sprayed on leaf surfaces, the antagonism between Zn and Se can be effectively compensated, and safe and synchronous enrichment of Zn and Se in wheat grains is realized; according to the present invention, the daily nutrition requirements of the human body can be met, and the Se content is controlled to be less than the Se toxicity threshold value; the proportion of organic Se is increased, and the related risk of inorganic Se is reduced, so that the food safety is improved; the Zn / Se response protein based on proteomics analysis provides a molecular marker for cultivating a wheat variety with synchronously enriched Zn-Se; the method can be compatible with traditional agricultural practice, extra large equipment or significant cost input is not needed, yield loss is avoided, and a feasible scheme is provided for solving the hidden hunger problem.

Description

Technical Field

[0001] This invention belongs to the field of optimizing the zinc and selenium content of wheat, specifically a spray solution for simultaneously increasing the zinc and selenium content of wheat, its preparation method, and its application method. Background Technology

[0002] Zinc (Zn) and selenium (Se) are essential trace elements for life, playing a vital role in maintaining health. Zinc is the second most abundant transition metal in the human body, participating in approximately 10% of protein synthesis and playing an indispensable role in many aspects, including immune function, DNA synthesis, and the nervous system. Zinc deficiency can lead to a range of health problems, such as growth retardation, weakened immunity, and nervous system abnormalities. Selenium, as an important antioxidant, can scavenge free radicals, alleviate oxidative stress, and enhance immunity. Selenium deficiency can cause health risks such as growth retardation, thyroid dysfunction, and infertility (Diet al., 2023; Feng & Ma, 2021; Ikram et al., 2024; Yang et al., 2023). Globally, approximately 30% and 15% of the population are affected by zinc and selenium deficiencies, respectively, posing a significant threat to public health (Dinh et al., 2018; Sigalas et al., 2024; Wang, Ali, et al., 2020).

[0003] In agronomic biofortification, soil-applied Zn and Se need to be transported long distances through the roots to the aboveground parts, while foliar spraying involves short-distance transport, enabling more effective accumulation of Zn and Se in wheat grains. Spraying Zn or Se alone can significantly increase the content of the corresponding elements in wheat grains, achieving the fortification target. In some cases, the Se content in the grains even exceeds the safe upper limit (>1.0 mg·kg⁻¹). -1 Compared to selenates, selenites are less toxic, more easily absorbed and utilized by organisms, and exhibit better Se enrichment effects after foliar spraying (Aiqing et al., 2022; Cakmak & Kutman, 2018; Zou et al., 2012). However, although single-element spraying can increase the content of individual elements, it not only increases production costs but also fails to bring additional economic benefits and cannot meet the needs of populations deficient in both Zn and Se. Furthermore, research on the combined application of Zn and Se is relatively limited. Therefore, conducting research on simultaneous Zn-Se enhancement is particularly important.

[0004] Zn and Se belong to the same periodic element and have similar atomic structures, thus exhibiting certain similarities and competition at the molecular level. This characteristic may lead to a variety of outcomes, including synergistic, antagonistic, or non-influencing effects, during their absorption, translocation, and accumulation in plants. When applied in combination, this complex interaction is significantly influenced by the type, concentration, and application method of Se fertilizer, as well as the crop type and variety (Gui, 2022). Notably, the range between Se accumulation and toxicity in wheat grains is relatively narrow (Han et al. 2013; Hawrylak-Nowak et al. 2015; Zhu et al. 2017). Furthermore, Zn is unevenly distributed in wheat grains, with a higher concentration in the outer layer than in the central endosperm, while Se is more evenly distributed (Persson et al., 2016). During flour processing, Zn loss is typically as high as 30-40%, while Se loss is relatively low (Brouns et al., 2012). Therefore, accurately determining the concentration conditions for Zn-Se combined spraying is crucial to ensuring that the Zn and Se content in wheat grains, especially flour, is within a safe range and meets human nutritional needs.

[0005] In biological and nutritional studies, inorganic and organic forms of Zn and Se exhibit different bioactivities, toxicities, and bioavailability. Inorganic Zn and Se have low bioavailability in the human body, and excessive intake can lead to chronic poisoning; therefore, many countries have banned the addition of inorganic Se to food. Organic Zn and Se, on the other hand, have low toxicity, high bioavailability, and a longer retention period in the body (Hadrup & Ravn-Haren, 2020; Sun et al., 2013; Yang et al., 2023). Se typically exists in an organic form by binding with amino acids through biotransformation. Therefore, the conversion ratio of organic Se in wheat grains after combined foliar spraying still needs further clarification.

[0006] With the rapid development of modern molecular biology and bioinformatics, omics analysis has provided new perspectives on revealing the molecular mechanisms by which plants respond to external fertilization. Proteins, as executors of gene function, play a crucial role. Previous studies have identified multiple Zn-binding proteins in wheat and barley grains (Dionisio et al., 2018); furthermore, another study, through transcriptomic and proteomic association analysis, revealed the molecular mechanism of wheat seedling response to Se spraying (Feng & Ma, 2021). However, the changes in proteins in wheat grains after combined Zn-Se spraying, particularly the metabolic and regulatory mechanisms of their interaction, remain unclear. Summary of the Invention

[0007] This invention provides a spray solution that simultaneously increases the zinc and selenium content of wheat, as well as its preparation and application methods, to overcome the deficiencies in the prior art.

[0008] This invention is achieved through the following technical solution:

[0009] The spray solution for increasing the zinc and selenium content of wheat comprises the following substances by mass percentage: 0.4% zinc source; 0.0021%-0.0024% selenium source; 0.02% surfactant; and the balance is water.

[0010] In the above-mentioned spray solution for increasing the zinc and selenium content of wheat, the zinc source in step one is zinc sulfate heptahydrate.

[0011] In the above-mentioned spray solution for increasing the zinc and selenium content of wheat, the selenium source in step two is sodium selenite.

[0012] The surfactant in the spray solution for increasing the zinc and selenium content of wheat as described above is Tween-20.

[0013] The preparation method of a spray solution for increasing the zinc and selenium content of wheat includes the following steps:

[0014] Step 1: Add the zinc source material to water until it is completely dissolved;

[0015] Step 2: Add the selenium source substance to the solution obtained in Step 1 until it is completely dissolved;

[0016] Step 3: Add the surfactant to the mixed solution obtained in Step 2 and mix to obtain a mixed spray solution that increases the zinc and selenium content of wheat.

[0017] In the preparation method of the spray solution for increasing the zinc and selenium content of wheat as described above, in step one, the zinc source substance is dissolved by ultrasonication for 10 minutes after being added to water.

[0018] In the preparation method of the spray solution for increasing the zinc and selenium content of wheat as described above, in step two, the selenium source substance is added to the solution obtained in step one and then dissolved by sonication for 10 minutes.

[0019] In the preparation method of the spray solution for increasing the zinc and selenium content of wheat as described above, in step three, the surfactant is added to the mixed solution obtained in step two and mixed by ultrasonication at 25°C for 30 minutes.

[0020] The application method for the foliar spray solution to increase the zinc and selenium content of wheat includes the following steps: Evenly spray the mixed foliar spray solution onto the wheat ears and leaves, twice per wheat growth cycle. The application rate of the mixed foliar spray solution for each field operation is 1250 L·ha. -1 .

[0021] The spraying method for increasing the zinc and selenium content of wheat as described above involves spraying twice, on the 7th and 14th day after wheat flowering or 7 days after the early grain filling stage of wheat. Each spraying should be avoided during periods of direct sunlight.

[0022] The advantages of this invention are: by optimizing the concentrations of Zn and Se applied through foliar spraying, this invention can effectively compensate for the antagonistic effect between the two, achieving safe and simultaneous enrichment of Zn and Se in wheat grains; this invention can meet the daily nutritional needs of the human body while controlling the Se content below the Se toxicity threshold; this invention also increases the proportion of organic Se and reduces the risks associated with inorganic Se, thereby improving food safety; the Zn / Se responsive protein based on proteomics analysis provides a molecular marker for cultivating wheat varieties with simultaneous Zn-Se enrichment; this invention is compatible with traditional agronomic practices, requires no additional large-scale equipment or significant cost investment, and results in no yield loss, providing a practical solution to the problem of hidden hunger. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This is a schematic diagram of the six spray solutions prepared according to embodiments of the present invention: CK (distilled water), Se2, Se3, Zn, ZnSe2, and ZnSe3.

[0025] Figure 2 This diagram illustrates the effects of combined zinc and selenium foliar spraying on wheat yield (A), zinc carryover from grains (B), and selenium carryover from grains (C) under field conditions in 2021-2022 and 2022-2023, as described in this invention. Wherein, CK represents sprayed distilled water; Zn, 0.4% w / v ZnSO4; Se1 / Se2 / Se3, 0.001% / 0.002% / 0.003% w / v Na2SeO3; error bars represent the standard error of four replicates, where ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, no significant difference. Different lowercase letters indicate significant differences between treatments (P < 0.05).

[0026] Figure 3 This is a schematic diagram illustrating the effects of combined zinc and selenium spraying on the zinc and selenium content (C and F) of whole grains (A, D), flour (B, E), and bran under field conditions in 2021-2022 and 2022-2023, as described in the embodiments of the present invention.

[0027] Figure 4 This is a schematic diagram of a univariate linear fit of the correlation analysis between the concentration of selenium sprayed and the selenium content of whole wheat grains under the field spraying zinc conditions in this embodiment of the invention;

[0028] Figure 5 This is a schematic diagram showing the estimated daily total zinc (TAZ) (A) and daily selenium intake (EDI) (B) of wheat flour under field conditions in 2021-2022 and 2022-2023, and the percentage of selenium forms in whole wheat grains after foliar spraying of zinc and selenium in the soil culture experiment in 2022-2023 (C).

[0029] Figure 6 This is a schematic diagram illustrating the effects of zinc and selenium spraying on the crude protein content of whole grain (A), flour (B), and bran (C) and the protein component content of whole grain (D and E) and flour (F and G) under field conditions in 2021-2022 and 2022-2023 in the embodiments of the present invention.

[0030] Figure 7 The Pearson correlation coefficients between the whole-grain Zn content and Se content, protein composition, Fe content, and Cu content in embodiments of the present invention with and without zinc or selenium spraying and with zinc or selenium spraying are represented by (A)-Zn, (C)-Se, (B)+Zn, and (D)+Se, respectively; wherein the upper and lower triangular matrices represent the 2021-2022 and 2022-2023 growth cycles, respectively, ***, P<0.001; **, P<0.01; *, P<0.05. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, 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.

[0032] The spray solution for increasing the zinc and selenium content of wheat comprises the following substances by mass percentage: 0.4% zinc source; 0.0021%-0.0024% selenium source; 0.02% surfactant; and the balance is water.

[0033] Preferably, the zinc source substance in step one of this embodiment is zinc sulfate heptahydrate.

[0034] Preferably, the selenium source substance in step two of this embodiment is sodium selenite.

[0035] Preferably, the surfactant described in this embodiment is Tween-20.

[0036] The preparation method of a spray solution for increasing the zinc and selenium content of wheat includes the following steps:

[0037] Step 1: Add the zinc source material to water until it is completely dissolved;

[0038] Step 2: Add the selenium source substance to the solution obtained in Step 1 until it is completely dissolved;

[0039] Step 3: Add the surfactant to the mixed solution obtained in Step 2 and mix to obtain a mixed spray solution that increases the zinc and selenium content of wheat.

[0040] Preferably, in step one of this embodiment, the zinc source material is dissolved by sonication for 10 minutes after being added to water.

[0041] Preferably, in step two of this embodiment, the selenium source substance is added to the solution obtained in step one and then sonicated for 10 minutes to dissolve it.

[0042] Preferably, in step three of this embodiment, the surfactant is added to the mixed solution obtained in step two and then ultrasonically mixed at 25°C for 30 minutes.

[0043] The application method for the foliar spray solution to increase the zinc and selenium content of wheat includes the following steps: Evenly spray the mixed foliar spray solution onto the wheat ears and leaves, twice per wheat growth cycle. The application rate of the mixed foliar spray solution for each field operation is 1250 L·ha. -1 .

[0044] Preferably, the two spraying times described in this embodiment are the 7th and 14th day after wheat flowering, or the two sprayings are carried out 7 days apart during the early grain-filling stage of wheat; each spraying should be carried out during periods of direct sunlight.

[0045] Example

[0046] The chemical analysis experiment included two research factors: ZnSO4·7H2O concentration and Na2SeO3 concentration. ZnSO4·7H2O concentrations were 0 and 0.4% (w / v) for Zn, and 0.002% (w / v) for Na2SeO3 for Se2, and 0.003% (w / v) for Se3. A complete protocol was used, repeated four times, with six treatments: CK (sprayed with distilled water), Se2, Se3, Zn, ZnSe2, and ZnSe3. All reagents were analytical grade. ZnSO4·7H2O was completely dissolved in 500 mL of distilled water, followed by the addition of Na2SeO3 and 0.02% (w / v) Tween-20 as a surfactant. The spray solution was sonicated at 25°C for 30 min (40 kHz, 320 W), filtered through a 0.22 μm filter, and 10 mL of the filtered solution was stored at 4°C for later use. Figure 1 (As shown).

[0047] The field experiment was conducted from October 2021 to June 2023 at the Northwest A&F University Crop Station 1 (34°17′56″N, 108°4′7″E) in Yangling, Shaanxi Province, China. The experimental area is at an altitude of 525m, with an average annual temperature of 13℃, belonging to a typical semi-humid and arid region with an average annual precipitation of approximately 600mm. The soil in this area belongs to the loess parent material, brown soil class, loam subclass, red oil soil genus, and is a type of soil-matured dryland man-made soil. Before the experiment, the basic physicochemical properties of the 0-20cm topsoil layer were: pH 8.32, SOC 9.80 g·kg⁻¹. -1 Total nitrogen 0.73 g·kg -1 NO3 - -N 11.30mg·kg -1 NH4 + -N 2.32 mg·kg -1 16.64 mg / kg of readily available phosphorus -1 171.42 mg / kg of readily available potassium -1 Total Zn 75.63 mg·kg -1 DTPA-Zn 0.72 mg·kg -1 Total Se 0.30 mg·kg -1 Available selenium 0.008 mg·kg -1 The experimental site adopted a winter wheat-summer fallow planting system.

[0048] The field trial included two research factors: two levels of ZnSO4·7H2O concentration (0, 0.4% (Zn) w / v) and three levels of Na2SeO3 concentration (0, 0.001% (Se1), 0.002% (Se2) w / v in 2021-2022, and 0, 0.002% (Se2), 0.003% (Se3) w / v in 2022-2023). The trial employed a completely randomized block design with four blocks and six treatments: CK (distilled water spray), Se1, Se2, Zn, ZnSe1, ZnSe2 in 2021-2022, and CK (distilled water spray), Se2, Se3, Zn, ZnSe2, ZnSe3 in 2022-2023.

[0049] The tested wheat (Triticum aestium L. Xiaoyan 22) was sown on October 21, 2021 and October 18, 2022, at a sowing rate of 210-225 kg·ha. -1 The nitrogen fertilizer used is urea, applied at a rate of 120 kg / ha of N. -1The phosphate fertilizer used is superphosphate, with an application rate of 100 kg·ha of P₂O₅. -1 Wheat straw returned to the field: 7500 kg·ha -1 All fertilizers were applied as base fertilizer, spread on the soil surface once before sowing and then incorporated into the soil using a rotary tiller. All foliar sprays included 0.02% (w / v) Tween-20 as a surfactant. Spraying was performed using a 330mL small manual sprayer (8cm×22cm), applied evenly to wheat ears and leaves in the evening on the 7th and 14th days after wheat flowering (April 30th and May 7th, 2022, and April 27th and May 4th, 2023). Each spraying micro-plot was 1.45m×1.45m, with 250mL applied each time, for a total application rate of 1250L·ha. -1 All fertilizers used for spraying were analytical grade, and other field management practices were carried out according to local conventions.

[0050] The wheat full-growth-cycle soil culture experiment was conducted in a dry greenhouse at the South Campus of Northwest A&F University from October 2022 to May 2023. The soil used was collected from the 0-20cm topsoil layer of the field experiment.

[0051] The soil cultivation experiment included two research factors. The concentration of ZnSO4·7H2O sprayed was set at two levels: 0 and 0.4% (denoted as Zn) (w / v); the concentration of Na2SeO3 sprayed was set at two levels: 0 and 0.002% (denoted as Se) (w / v). A completely randomized block design was adopted, with three blocks and a total of four treatments: CK (sprayed with distilled water), Se, Zn, and ZnSe.

[0052] Fresh soil was sieved through a 5mm sieve and adjusted to 70% of field capacity. Pots (240cm inner diameter, 360cm height) were filled with the equivalent of 7kg of dry soil. 0.33g urea and 0.43g KH2PO4 were added per kg of dry soil as base fertilizer and mixed thoroughly. Wheat (Triticum aestium L. Xiaoyan 22) was sown on October 20, 2022, with 10 seeds per pot at a depth of approximately 2cm. Seedlings were thinned to 5 plants per plant at the two-leaf stage. Watering was done by weighing to maintain soil moisture at 70% of field capacity. All foliar treatments included 0.02% (w / v) Tween-20 as a surfactant. Spraying was performed using a 330mL small manual sprayer (8cm x 22cm) on the wheat ears and leaves in the evening of the 7th and 14th day after flowering. The spraying rate was 125ml / m³ per application. -2 All base fertilizers and foliar fertilizers used were of analytical grade. Imidacloprid and triadimefon were used to control aphids and powdery mildew during wheat growth.

[0053] Methods for detecting various substances in wheat

[0054] In field trials, wheat was harvested from each microplot at maturity, and yield was measured manually after threshing. Whole grains were ground using a high-throughput tissue grinder (Beijing Dinghaoyuan Technology Co., Ltd., TL2020) and stored. Another 100g of grains was ground into flour and bran (approximately 60% flour yield) using a small-scale experimental mill (Quadrumat Junior mill, Brabender, Duisburg, Germany). In soil-culture trials, whole pots of wheat were harvested at maturity. A portion of the grains from the middle of the main ear was flash-frozen in liquid nitrogen at -80℃ for proteomics analysis, and the remaining grains were ground for later use.

[0055] Zn, Fe, and Cu content determination: Weigh 0.5 g of sample, ashed in a muffle furnace at 600℃ for 6 h, dissolve in HNO3, and determine by atomic absorption spectrophotometry (PE-PinAAcle 900F flame atomic absorption spectrometer). A blank and national standard reference material (GWB08503c, Zn = 41.1 ± 2.0 mg·kg⁻¹) were added during the analysis. -1 Quality control was implemented, and the Zn recovery rate was between 95% and 105%.

[0056] Se content determination: Weigh 0.5g of sample and digest with HNO3:HClO4 (4:1 volume ratio) at 165℃. After digestion, bring the volume to 25mL with ultrapure water. Add 5mL of HCl (1:1 volume ratio) to 5mL of the digestion solution and incubate at 95℃ for 30min. Se content is determined using hydride generation-atomic fluorescence spectrometry (LC-AFS-8530, Beijing Haiguang). A blank and national standard reference material (MCS-10294, Se = 0.439 ± 0.046 mg·kg⁻¹) are added during the analysis. -1 Quality control was performed, and the Se recovery rate was between 95% and 105%.

[0057] Crude protein content determination: After digestion with H2SO4-H2O2, the nitrogen content was determined using a flow analyzer (Auto Analyzer 3-AA3 continuous flow analyzer). The crude protein content of whole grains, flour, and bran was calculated by multiplying the nitrogen content by 5.83, 5.7, and 6.31, respectively.

[0058] Protein components: Albumin, globulin, prolamins, and gluten were extracted sequentially using ultrapure water, 5% NaCl solution, 75% ethanol solution, and 0.2% NaOH solution. The nitrogen content of the extracted protein components was determined by the semi-micro Kjeldahl method (K9860). The protein content of the whole grain and flour was calculated by multiplying the nitrogen content of each component by 5.83 and 5.7, respectively (Osborne & Voorhees, 1894).

[0059] Phytic acid content determination: using 10% Na₂SO₄ and 0.2 mol·L⁻¹ -1Phytic acid was extracted with HCl solution, and FeCl3 was used to precipitate ferric phytate. The Fe concentration in the extract was determined by atomic absorption spectrometry (PE-PinAAcle 900F flame atomic absorption spectrometer), and the phytic acid content was calculated.

[0060] Selenium speciation determination: Weigh 0.1000g of sample, add 15mg of proteinase XIV and 10mL of ultrapure water for microwave extraction. Extraction program: 200W, heat to 37℃ for 10min, maintain temperature at 37℃ for 30min, 6000r·min -1 After centrifugation for 5 min, the sample was filtered through a 0.45 μm aqueous filter membrane, and the selenium speciation was determined by HPLC-ICP-MS. The HPLC conditions were: column: Hamilton PRP-X100; mobile phase: 20 mmol / L citric acid solution (pH 6.0); flow rate: 1.5 mL / min; injection volume: 100 μL. The ICP-MS conditions were: sampling depth: 10 mm; RF power: 1550 W; collision gas: He; gas flow rate: 5 mL / min; Se integration time: 0.5 s. 4+ Se 6 + The recoveries of the standard substances for selenocysteine ​​(SeCys2), methylselenocysteine ​​(SeMeCys), and selenomethionine (SeMet) were all between 90% and 110%.

[0061] Verification of the solubility of Zn and Se in the spray solution

[0062] The recovery rates of Zn and Se in CK (sprayed distilled water), Se2, Se3, Zn, ZnSe2, and ZnSe3 were statistically analyzed, and the results are shown in Table 1. Table 1 shows that the measured concentrations of Zn and Se in the spray solution were lower than the theoretical values. The recovery rate of Zn in the spray solution was >80%, and the addition of Se did not affect the recovery rate of Zn. The addition of Zn significantly reduced the recovery rate of Se. There was no significant difference in the recovery rate of Se between ZnSe2 and Se2, but the recovery rate of ZnSe3 was significantly lower than that of Se3.

[0063] Table 1. Concentrations and recoveries of Zn and Se in the spray solution

[0064]

[0065]

[0066] Note: Numerical values ​​represent the mean ± standard error of the four replicates for each treatment. Different lowercase letters indicate significant differences between treatments (P < 0.05).

[0067] Enrichment of zinc and selenium in wheat grains after foliar spraying and verification of selenium speciation

[0068] In studies conducted over two consecutive wheat growing cycles, all spraying treatments did not significantly affect wheat yield, with yield variations ranging from 4.43 to 5.37 t·ha. -1 (like Figure 2 (As shown in A). Spraying Zn alone or in combination with Se significantly increased the zinc carryover from wheat grains, with Zn spraying increasing zinc carryover by 78.6%. Spraying Se significantly increased selenium carryover from wheat grains, but compared with Se spraying alone, the Zn-Se combination reduced selenium carryover, and the reduction was more pronounced with increasing Se concentration (e.g., ...). Figure 2 (As shown in BC).

[0069] The effects of foliar application of zinc and selenium on the zinc and selenium content of wheat grains in soil cultivation experiments were studied (the results are shown in Table 2 and 3). Figure 3 As shown in the figure, the Zn-Se combination significantly increased the Zn and Se content in various parts of wheat grains, which is consistent with the results of the soil culture experiment. Spraying Zn alone significantly increased the Zn content in wheat grains, while spraying in combination with Se did not have any effect on the Zn content (e.g., ...). Figure 3 (As shown in AC). After Zn spraying, the Zn content in whole grains, flour, and bran reached 43.7-54.7 mg·kg⁻¹, respectively. -1 17.3-20.8 mg·kg -1 and 89.5-115.5 mg·kg -1 The levels were increased by 82.5%, 96.6%, and 102.4% respectively compared to the control (CK), and the Zn content in the whole grain has reached the zinc-rich standard (40-60 mg / kg). -1 However, Zn is unevenly distributed in the grain, preferentially accumulating in the outer layer, with the Zn content in flour being only 33%-48% of the whole grain. On the other hand, spraying with Se significantly increases the Se content in the grain, and all treatments did not exceed the upper limit of selenium toxicity in grains (>1.0 mg·kg⁻¹). -1 However, when sprayed in combination with Zn, the Se content in the grains will decrease (e.g., Figure 3 (As shown in DF). Compared with Se spraying alone, the decrease in Se content in Zn-Se combined spraying increased dramatically with increasing Se concentration. At Se concentrations of 0.001%, 0.002%, and 0.003%, the total Se content decreased by 31%, 41%, and 51%, respectively. This indicates an antagonistic effect in Zn-Se combined spraying, which prevents some treatments from achieving the Se-enriched standard of 0.25-0.30 mg / kg Se content when spraying Se alone. -1 However, when sprayed in combination with Zn, this standard was not achieved.

[0070] Table 2. Effects of foliar application of zinc and selenium on the zinc and selenium content of wheat grains in soil cultivation experiments from 2022 to 2023.

[0071]

[0072] Note: The data shown in the table are the mean ± standard error (SE) of three independent and repeated samples.

[0073] Nevertheless, by increasing the concentration of Se when spraying in combination with Zn, the Se content in various parts of the grain can still achieve the same effect as when spraying a low concentration of Se alone. A univariate linear fit was performed on the Se concentration and total Se content of the grain after Zn-Se combined spraying, yielding the equation y = 167.52x - 0.0995. This means that, in addition to foliar Zn spraying, to ensure a total Se content of 0.25-0.30 mg·kg⁻¹ in the grain, further adjustments can be made. -1 The concentration of Se used for spraying should be 0.0021-0.0024% (e.g., ...). Figure 4 (As shown).

[0074] In conclusion, the combined application of 0.4% (w / v) ZnSO4·7H2O and 0.0021%-0.0024% (w / v) Na2SeO3 can achieve simultaneous Zn-Se biofortification in grains without affecting yield, meeting the daily zinc and selenium intake requirements of the human body without exceeding the safety threshold.

[0075] Estimated daily human intake of Zn and Se and verification of selenium speciation in wheat grains after foliar spraying

[0076] The estimated daily intake of Zn and Se in humans is based on the adult daily intake of flour and its products of 173.2g (Chinese Center for Disease Control and Prevention). The estimated trends in daily zinc and selenium intake from flour are consistent with the trends in Zn and Se content (e.g., ...). Figure 5 (As shown in AB); After Zn spraying, the Zn intake from flour increased from 0.84-1.15 mg / day. -1 Increase to 1.62-2.52 mg / day -1 After spraying with Se alone, the Se intake in flour increased to 38.81-146.85 μg / day. -1 However, when sprayed in combination with Zn, the intake decreased by 66.7%-110.7%, down to 22.22-75.55 μg·d. -1 .

[0077] Daily zinc intake (TAZ, mg / person) -1 ·day -1 Estimate using the following formula:

[0078]

[0079] Assuming an average daily intake of 0.1732 kg / person of flour and its products. -1 ·day-1 TDZ is the total daily Zn intake of adults (mmol Zn·day). -1 TDP is the total daily intake of phytic acid (PA) for adults (mmol PA·day). -1 ), A max K R and K P These represent the maximum uptake (0.091), the equilibrium dissociation constant of the zinc acceptor binding reaction (0.680), and the equilibrium dissociation constant of the Zn-PA binding reaction (0.033), respectively.

[0080] Estimated daily selenium intake (SeI, μg / person) -1 ·day -1 Calculate according to the following formula:

[0081] EDI=C grain ×W grainintake

[0082] Among them, EDI (μg·person) -1 ·day -1 C represents the estimated daily selenium intake for adults. grain (μg·kg -1 ) represents the Se content of wheat grains, W grainintake (kg·person -1 ·day -1 The average daily intake of flour and its products by adults is 0.1732 kg per person. -1 ·day -1 ).

[0083] Regardless of foliar spraying, 67.7-69.6% of the total selenium in whole wheat grains was SeMet, while inorganic selenium accounted for only 2.9-5.5%, and selenium was not detected in any of the treatments. 4+ (like Figure 5 As shown in Figure C, compared with the control (CK), the Zn-Se combination increased the proportion of SeMet in whole wheat grains; compared with spraying Se alone, the Zn-Se combination spray reduced Se content. 6+ This proportion promotes the conversion of inorganic selenium to organic selenium.

[0084] Study on protein quality of wheat grains after foliar spraying

[0085] To investigate the effects of combined Zn-Se spraying on wheat grain protein quality, the crude protein and protein component contents in whole grains and flour were measured. In a two-year experiment, compared with the control (CK), combined Zn spraying with 0.002% and 0.003% Se significantly increased the crude protein content in various parts of the grain. The increases in crude protein, prolamin, and gluten content in flour were 12.9%-20.8%, 13.8%-25.6%, and 22.3%-35.0%, respectively (e.g., ...). Figure 6 (As shown).

[0086] Correlation analysis revealed that without Zn and Se spraying, the whole grain Zn and Se content was not related to the crude protein and protein component content. However, with Zn or Se spraying, the whole grain Zn and Se content showed a positive correlation with the crude protein, prolysin, and glutenin content (e.g., ...). Figure 7 (As shown).

[0087] Pearson correlation analysis was performed pairwise on the contents of whole-grain Zn, Se, protein, prolamin, glutenin, Fe, and Cu. If the effects of spraying Zn fertilizer and spraying Se fertilizer are both significant, then η was calculated. 2 This indicates the contribution of each factor to the total effect.

[0088] η 2 =SS A / SS total

[0089] SS A SStotal represents the variance between treatments caused by factor A, and the total sum of squares is SStotal.

[0090] Analysis of DEPs in wheat grains at maturity after foliar spraying

[0091] To further elucidate the molecular response mechanism of mature wheat grain proteins to Zn-Se spraying, 4D label-free proteomics analysis technology was used to detect the expressed proteins in mature wheat grains from soil culture experiments. A total of 5443 proteins were identified across the four treatments.

[0092] There were 66 common DEPs between Zn vs CK and ZnSe vs CK, of which 64 were not differentially expressed in ZnSe vs Zn, and 52 proteins were detectable in ZnSe vs Zn. This indicates that there was no difference in Zn accumulation in grains when Zn was sprayed alone versus when Zn-Se was sprayed in combination, but both were significantly higher than CK. This phenomenon may be closely related to these proteins. To investigate the biological functions of these DEPs, GO functional enrichment analysis was performed. 37 GO entries co-enriched between Zn vs CK and ZnSe vs CK and not differentiating in ZnSe vs Zn were found. These mainly function as chitin binding and histone-lysine N-methyltransferase activity, participating in biological processes such as histone lysine methylation and ncRNA metabolic processes, and are located in the small-subunit processome. To more systematically and comprehensively analyze the biological metabolic pathways and signal transduction pathways involved in DEPs, KEGG enrichment analysis was performed, and the top 20 most significant pathways were selected for presentation. Compared with the control (CK), DEPs sprayed alone and in combination with Zn-Se were significantly enriched in pathways such as lysine degradation, fatty acid degradation, porphyrin and chlorophyll metabolism, aminoacyl-tRNA biosynthesis, glycerophospholipid metabolism, and ribosome biogenesis in eukaryotes, with no difference between ZnSe and Zn. To understand the interactions among DEPs, interaction analysis was conducted, and heat shock protein 70 (A0A3B6JMW4) may play an important role in the accumulation of Zn in grains. Therefore, the Zn-responsive proteins identified in this study are mostly involved in metabolic and genetic information processing pathways.Candidate proteins were screened based on the function, metabolic pathways involved, and expression levels of DEPs. DEPs with no difference in Zn content between Zn and Zn-Se treatments but significantly higher Zn content than CK were closely related to redox reactions, structural proteins, stress proteins, and storage proteins.

[0093] Compared with the control (CK) level, 76 common DEPs were found when Se was sprayed alone or in combination with other treatments. For Se accumulation in grains, Se alone > Zn-Se > CK, a phenomenon likely closely related to the expression levels of these proteins. Among the DEPs in ZnSe vs Zn, 15 GO entries were identical to those in Zn vs CK and ZnSe vs CK, primarily involved in ribosome biogenesis. Furthermore, three KEGG pathways enriched in the ZnSe vs Zn DEPs were identical to those in Zn vs CK and ZnSe vs CK, primarily involved in tyrosine metabolism, related to amino acid metabolism. In summary, the Se content in grains was higher than CK but significantly lower when Se was sprayed alone during combined application. This may be closely related to DEPs such as transferases, proteins containing specific domains, kinases, and transaminases.

[0094] This invention optimizes the concentrations of Zn and Se during foliar spraying (0.4% ZnSO4·7H2O + 0.0021%-0.0024% Na2SeO3) to effectively compensate for their antagonistic effects, achieving safe and simultaneous enrichment of Zn and Se in wheat grains. This strategy meets the daily nutritional needs of the human body while controlling Se content below the Se toxicity threshold. Furthermore, it increases the proportion of organic Se, reducing the risks associated with inorganic Se, thereby improving food safety. Zn / Se-responsive proteins based on proteomics analysis provide molecular markers for breeding wheat varieties with simultaneous Zn-Se enrichment. This method is compatible with traditional agronomic practices, requires no additional large-scale equipment or significant cost investment, and results in no yield loss, providing a practical solution to the problem of hidden hunger.

[0095] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A spray solution for increasing the zinc and selenium content of wheat, characterized in that: It includes the following substances by mass percentage: 0.4% zinc source; 0.0021%-0.0024% selenium source; 0.02% surfactant; and the balance is water.

2. The spray solution for increasing the zinc and selenium content of wheat according to claim 1, characterized in that: The zinc source substance in step one is zinc sulfate heptahydrate.

3. The spray solution for increasing the zinc and selenium content of wheat according to claim 1, characterized in that: The selenium source substance in step two is sodium selenite.

4. The spray solution for increasing the zinc and selenium content of wheat according to claim 1, characterized in that: The surfactant mentioned is Tween-20.

5. A method for preparing a spray solution to increase the zinc and selenium content of wheat, characterized in that: Includes the following steps: Step 1: Add the zinc source material to water until it is completely dissolved; Step 2: Add the selenium source substance to the solution obtained in Step 1 until it is completely dissolved; Step 3: Add the surfactant to the mixed solution obtained in Step 2 and mix to obtain a mixed spray solution that increases the zinc and selenium content of wheat.

6. The method for preparing the spray solution for increasing the zinc and selenium content of wheat according to claim 5, characterized in that: In step one, the zinc source material is dissolved by sonication for 10 minutes after being added to water.

7. The method for preparing the spray solution for increasing the zinc and selenium content of wheat according to claim 5, characterized in that: In step two, the selenium source substance is added to the solution obtained in step one and then sonicated for 10 minutes to dissolve it.

8. The method for preparing the spray solution for increasing the zinc and selenium content of wheat according to claim 5, characterized in that: In step three, the surfactant is added to the mixed solution obtained in step two and mixed by sonication at 25°C for 30 minutes.

9. A spraying method for increasing the zinc and selenium content of wheat, characterized in that: The procedure includes the following: Evenly spray the mixed spray solution onto the wheat ears and leaves, twice per wheat growth cycle. The application rate of the mixed spray solution for each field operation is 1250 L·ha. -1 .

10. The spraying method for the spray solution to increase the zinc and selenium content of wheat according to claim 1, characterized in that: The two spraying times are 7 days and 14 days after wheat flowering, or 7 days apart during the early grain-filling stage of wheat. Each spraying should be carried out during periods of direct sunlight.

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