Amylose content regulator and application thereof in increasing amylose content of wheat

Abstract

The invention provides an amylose content regulator and application thereof in improving the amylose content of wheat, and relates to the technical field of plant variety improvement. The amylose content regulator is mainly prepared from 24-brassinolide with the concentration of 0.2 to 25 [mu] g / L, abscisic acid with the concentration of 0.05 to 6.25 mg / L, zinc sulfate with the concentration of 0.1 to 1 wt% and boric acid with the concentration of 0.01 to 0.1 wt%. The invention also provides an application of the amylose content regulator in increasing the amylose content of wheat. The method specifically comprises the step of spraying the amylose content regulator on the surfaces of wheat plants in a wheat filling period so as to increase the amylose content of the wheat. The growth period of wheat is regulated and controlled through the amylose content regulator, so that the amylose content of the mature wheat seeds is increased, the method is easy and convenient to operate, and the amylose content of the treated mature seeds is increased by about 9%-16% compared with a contrast.

Description

Technical Field

[0001] The present invention relates to the technical field of plant variety improvement, and in particular to an amylose content regulator and application thereof in increasing the amylose content of wheat. Background Art

[0002] Wheat (Triticum aestivum L.) is a widely cultivated staple food crop worldwide. The starch content and composition of its seeds significantly impact food processing and nutritional value. Starch is primarily composed of amylose (AM) and amylopectin (AP). The amylose content directly influences the processing properties of wheat flour and the final quality of food. High-amylose wheat has unique applications in the food industry, such as improving texture and extending shelf life in noodles and bread. Resistant starch, also known as enzymatically resistant starch, is primarily found in high-amylose, low-amylose granules, and its content is positively correlated with the amylose content. Studies have shown that resistant starch cannot undergo enzymatic degradation in the healthy small intestine and does not increase blood sugar levels. However, when fermented in the large intestine, resistant starch helps increase beneficial bacteria in the intestine and decrease harmful bacteria, thereby improving intestinal health. Other benefits of resistant starch include increasing satiety, preventing and treating constipation, lowering cholesterol, and reducing the risk of colon cancer. Because resistant starch ferments slowly, it produces less gas and is gentler than other fibers. This helps control high blood pressure, high cholesterol, and high blood sugar, as well as weight, playing an important role in maintaining human health. Therefore, research into increasing wheat amylose content is of great significance and a key research focus in this area.

[0003] Traditional breeding methods improve starch quality by selecting wheat varieties with high amylose content. However, the production of high-amylose wheat varieties relies on natural variation and artificial selection, resulting in long breeding cycles, low efficiency, and limited genetic diversity. Recent advances in biotechnology have provided new approaches, such as the gene-editing technology CRISPR / Cas9 system, for the targeted modification of key genes controlling starch synthesis. However, these approaches involve the technical complexity of transgenic manipulation and face regulatory and public acceptance challenges. Emerging biotechnologies, such as RNA interference (RNAi), are also being used to regulate genes involved in starch synthesis. These techniques require specialized technical implementation and similarly face the multiple effects of gene interference, leading to unexpected side effects. Summary of the Invention

[0004] In view of this, the main purpose of the present invention is to provide an amylose content regulator, which is used to regulate the growth period of wheat, thereby increasing the amylose content of mature wheat seeds.

[0005] To achieve the above-mentioned object, the technical solution provided by the present invention is: an amylose content regulator, mainly composed of 0.2-25 μg / L 24-brassinolide (24-Epibrassinolide), 0.05-6.25 mg / L abscisic acid (ABA), 0.1wt%-1wt% zinc sulfate and 0.01wt%-0.1wt% boric acid.

[0006] Wherein, "%" in the present invention refers to the percentage by mass.

[0007] Furthermore, the amylose content regulator further comprises a surfactant at a concentration of 0.8‰ to 1.2‰ to improve the dispersibility of the components and ensure uniform mixing of the components. Preferably, the surfactant is an organosilicon surfactant Silwet L-77.

[0008] Another object of the present invention is to provide a use of the above-mentioned amylose content regulator in increasing the amylose content of wheat.

[0009] The amylose content regulator can increase the amylose content in wheat leaves or mature wheat seeds, especially increase the amylose content in mature wheat seeds by 9% to 16%, without significantly changing the thousand-grain weight and yield per mu.

[0010] Another object of the present invention is to provide a method for increasing the amylose content of wheat, comprising: spraying the amylose content regulator on wheat plants, wherein the wheat plants include wheat ears and / or wheat leaves.

[0011] Furthermore, during the wheat filling stage, the amylose content regulator is sprayed on the wheat plants.

[0012] Furthermore, the spraying amount of the amylose content regulator is 5 to 7 L / mu.

[0013] Another object of the present invention is to provide an application of the above-mentioned amylose content regulator in reducing the expression level of wheat starch metabolism-related genes, wherein the key genes of wheat starch metabolism include starch branching enzyme (TaSBEIIa1, TaSBEIIb1), starch debranching enzyme (TaISA), glucan hydration dikinase (TaGWD), pyruvate kinase (TaPK), starch synthase (TaSSII, TaSSIV) or starch phosphorylase (TaPHO) encoding genes.

[0014] Another object of the present invention is to provide a use of the above-mentioned amylose content regulator in wheat breeding, wherein the above-mentioned amylose content regulator can regulate the amylose content in wheat seeds. The amylose content regulator can be used to cultivate high-amylose content wheat seeds.

[0015] The present invention regulates the growth period of wheat by regulating the amylose content regulator, wherein the amylose content regulator mainly includes 24-brassinolide, abscisic acid, zinc sulfate and boric acid. 24-Brassinolide and abscisic acid are both key plant growth hormones. The former belongs to the brassinolide hormone class, which plays an important role in plant growth and development. It is a broad-spectrum and highly effective plant growth regulator that is widely used in agricultural production, including wheat cultivation. It has been proven to promote wheat growth and improve plant tolerance to various environmental stresses such as drought, salinity and extreme temperatures, while increasing yield and improving wheat quality. 24-Brassinolide can regulate the accumulation of starch in wheat grains, including the ratio of amylose to amylopectin, to optimize the composition of wheat starch, thereby improving the processing performance of wheat flour and the quality of the final product.

[0016] Abscisic acid plays a core role in the plant's response to stress, seed development and dormancy, and maturation process. It regulates the signal transduction pathways within the plant, affecting gene expression and enzyme activity, thereby changing the plant's metabolic state. It has multiple functions such as promoting metabolism and growth, improving stress and disease resistance, and enhancing the overall quality of crops.

[0017] Zinc sulfate, as a trace mineral nutrient, participates in the active centers of multiple enzymes, affects plant photosynthesis, protein synthesis and cell division, and is essential to plant health.

[0018] Boric acid is one of the essential nutrients for plants and is crucial to the cell wall structure, membrane function and signal transduction of plants. Boron plays the role of a cofactor in many enzymatic reactions in plants, affecting the reproductive development and cell expansion of plants.

[0019] Therefore, the amylose content regulator provided by the present invention increases the amylose content of wheat by more than 9% through the synergistic effect of 24-brassinolide, abscisic acid, zinc sulfate, and boric acid therein, and the amylose content of mature seeds ranges from 23% to 30%. At the same time, it can ensure that the thousand-grain weight and per-acre yield of mature wheat seeds do not change significantly. The method for increasing the amylose content of wheat provided by the present invention is simple and easy to implement. It only needs to be sprayed once during the wheat filling period, which is easy to promote. In addition, the amylose content regulator provided by the present invention can increase the amylose content in wheat leaves by regulating the expression of starch metabolism-related genes in wheat leaves, thereby increasing the amylose content in mature wheat seeds. This is beneficial to the cultivation of wheat with a high amylose content, simplifies the high-quality wheat cultivation process, shortens the breeding cycle, and is simple and has no obvious side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the standard curve of amylose content used in the examples of the present invention;

[0021] Figure 2 Schematic diagram of the expression of key genes for starch synthesis in wheat leaves in Example 4 of the present invention;

[0022] Figure 3 This is a schematic diagram of the amylose content in wheat leaves in Example 4 of the present invention. DETAILED DESCRIPTION

[0023] The technical solution of the present invention is further described in detail below through specific implementation methods.

[0024] Unless otherwise specified, the terms used in this invention are commonly used in the relevant field. The technical means used in the examples, such as preparation processes and testing methods, are conventional means well known to those skilled in the art. The reagents and products used are commercially available. The sources and trade names of the reagents used, as well as their components, are indicated upon their first appearance, where necessary.

[0025] The following describes the embodiments of the present application through specific examples, and those skilled in the art can easily understand other advantages and effects of the present application from the contents disclosed in this specification. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. The present application can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present application. It should be noted that, in the absence of conflict, the features in the following embodiments and embodiments can be combined with each other. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of this application.

[0026] The conditions and methods for measuring the content and yield of amylose in wheat seeds in the following examples are:

[0027] Wheat seeds of the Nongmai 482 variety were sown normally in fields and maintained with normal water and fertilizer management. Once the wheat reached the grain filling stage, approximately one mu (approximately 1 acre) of uniformly growing wheat plants were selected from each field as the treatment group, while another approximately one mu (approximately 1 acre) of uniformly growing wheat plants from a neighboring field served as the control group. Approximately 6 liters (approximately 1 liter) of a premixed amylose content regulator and 1‰ of the silicone surfactant Silwet L-77 were evenly sprayed onto the treatment group using an agricultural sprayer producing droplets smaller than 1 μm. The control group was evenly sprayed with the same volume of water. Both treatment and control groups were then grown to maturity under normal conditions. The seeds were then harvested and analyzed for amylose content and yield in mature wheat grains using the national standard method GB / T 15683-2008.

[0028] Method for constructing the amylose standard curve:

[0029] Weigh 0.1000 g of pure potato-derived amylose standard and corn-derived amylopectin standard respectively, place them in a 100 mL volumetric flask, add 10 mL of 0.5 mol / L KOH, dissolve in hot water, cool and dilute to 100 mL with distilled water to obtain 1 mg / mL amylose standard solution and amylopectin standard mother solution. Measure 0, 50, 100, 200, 400, 600, 800 μL of amylose standard mother solution and 1000, 950, 800, 600, 400, 200 μL of amylopectin standard mother solution to prepare gradient concentration solutions with amylose concentrations of 0%, 5wt%, 10wt%, 20wt%, 40wt%, 60wt%, and 80wt%, respectively, and measure the absorption peak at 570 nm using a microplate reader to construct a standard curve of amylose content and absorbance, as shown in the following example: Figure 1 As shown. The standard curve equation is as follows:

[0030] y=106.84x-0.2261,R 2 =0.9999;

[0031] In the above standard curve equation, x is absorbance, and y is the content of amylose, wt%.

[0032] Main component screening

[0033] This experiment provides different concentrations of 24-brassinolide and abscisic acid combinations, and different concentrations of zinc sulfate and boric acid combinations as amylose content regulators. The specific ingredients are shown in Table 1.

[0034] Table 1. Statistics of main components screening combination of amylose content regulators

[0035]

[0036] Table 2. Amylose content and yield analysis

[0037] sample Amylose content / wt% Increase in amylose / wt% Thousand-grain weight / g Yield per mu / kg control group 25.72±0.015 -- 46.68 595.2 Treatment group 1 26.21±0.024 1.91 46.7 603.12 Treatment group 2 26.33±0.033 2.37 46.71 607.08 Treatment group 3 26.56±0.042 3.27 46.78 599.16 Treatment group 4 26.84±0.051 4.35 46.8 596.52 Treatment group 5 27.22±0.060 5.83 46.73 601.8 Treatment group 6 27.92±0.069 8.55 46.85 604.44 Treatment group 7 27.31±0.078 6.18 46.75 600.48 Treatment Group 8 27.45±0.087 6.73 46.72 605.76 Treatment Group 9 27.12±0.096 5.44 46.83 611.04 Treatment group 10 26.79±0.077 4.16 46.81 608.4 Treatment group 11 26.66±0.060 3.65 46.79 609.72 Treatment group 12 26.53±0.042 3.15 46.74 612.36 Treatment group 13 26.33±0.024 2.37 46.61 597.84 Treatment group 14 26.05±0.006 1.28 46.82 613.68 Treatment group 15 25.92±0.012 0.78 46.77 615 Treatment group 16 25.83±0.030 0.43 46.84 620.28 Treatment group 17 26.81±0.048 4.24 46.23 621.6 Treatment group 18 26.04±0.065 1.25 46.32 624.24 Treatment group 19 25.81±0.021 0.34 46.14 618.96 Treatment group 20 26.02±0.001 1.18 46.36 617.64 Treatment Group 21 27.44±0.019 6.68 46.59 622.92 Treatment Group 22 26.24±0.061 2.02 46.75 626.88 Treatment Group 23 27.16±0.012 5.58 46.77 625.56 Treatment Group 24 26.87±0.017 4.48 46.8 618.2 Treatment group 25 26.59±0.009 3.39 46.85 619.5

[0038] The different treatment combinations shown in Table 1 were used as amylose content regulators and sprayed onto wheat plants in the grain filling stage. The wheat grew normally to maturity. The amylose content and yield of the obtained mature wheat grains were combined with the Figure 1 , the results are shown in Table 2.

[0039] The results shown in Table 2 indicate that the amylose content regulators of different concentration combinations provided in this Example can all increase the amylose content in mature wheat seeds, especially treatment combination 6 consisting of 1 μg / L 24-brassinolide and 0.25 mg / L abscisic acid and treatment combination 21 consisting of 0.2 wt% zinc sulfate and 0.02 wt% boric acid. The amylose content in the seeds obtained from the treatment groups corresponding to the two combinations increased by 8.55% and 6.68%, respectively, but had little effect on the 1000-grain weight and yield per mu.

[0040] Examples 1-3, Plant Growth Regulator Mixture

[0041] Examples 1-3 each provide an amylose content regulator, comprising the main components shown in Table 3 and 1‰ of an organic silicon surfactant Silwet L-77, wherein the components are uniformly mixed.

[0042] Table 3. Main components of plant growth regulator mixtures

[0043]

[0044] The plant growth regulator mixtures provided in the examples were uniformly mixed and then sprayed onto wheat plants in the grain filling stage. The wheat was allowed to grow normally until maturity. The results of the obtained indicators such as the content of amylose in the mature wheat grains and the yield are shown in Table 4.

[0045] Table 4. Analysis of amylose content and yield in mature wheat seeds

[0046] sample Amylose content% Increase in amylose % Thousand-grain weight / g Yield per mu / kg Example 1 29.7±0.066 15.47433904 47.77 635.52 Example 2 28.51±0.013 10.84758942 47.02 610.3 Example 3 28.13±0.022 9.370139969 46.86 614.62

[0047] The "increase in amylose content" in this table is based on the amylose content of the "control group" in Table 2.

[0048] As can be seen from Tables 2 and 4, after treatment with the plant growth regulating mixture provided in Examples 1-3, the amylose content in the mature wheat seeds was between 28% and 30%, an increase of 9.37-15.47%, and the thousand-grain weight and yield per mu did not change significantly. Among them, the amylose content in the mature wheat seeds treated with the plant growth regulating mixture provided in Example 1 was the highest.

[0049] Example 4 Application of Amylose Content Regulator in Wheat Breeding

[0050] Typically, the amylose content in wheat leaves is closely correlated with that in mature wheat seeds. The amylose content in wheat leaves is closely related to the genes encoding amylose synthase. Therefore, during wheat screening and breeding, the expression levels of amylose-related genes in wheat leaves can be used to preliminarily screen and assess the amylose content in mature wheat seeds.

[0051] The following study examines the expression of amylose-related genes in wheat leaves treated with the plant growth regulator mixture provided in Example 1. Specifically, this example provides a method for altering the expression of starch metabolism-related genes in wheat leaves, comprising the following steps:

[0052] The variety of dwarf 58 wheat seeds was hydroponically cultivated until the wheat seedling leaves were about 15 cm long. 150 wheat seedlings with the same growth momentum were selected as the treatment group, and another 150 were used as the control group. 15 mL of the plant growth regulator mixture provided in Example 1 was evenly mixed and evenly sprayed on the treatment group through a household air pressure sprayer with an aperture of 2.0 mm. The control group was evenly sprayed with 15 mL of clean water. The sample was then placed in an open greenhouse with a temperature of about 25 ° C. After treatment for 3 days, the leaves were quickly cut with scissors and placed in liquid nitrogen for quick freezing, and stored in a -80 ° C refrigerator. By means of real-time quantitative PCR (qRT-PCR), the expression of starch metabolism key genes starch branching enzyme TaSBEIIa1 and TaSBEIIb1, starch debranching enzyme TaISA, glucan hydration dikinase TaGWD, pyruvate kinase TaPK, starch synthase TaSSII and TaSSIV, and starch phosphorylase TaPHO genes in the treatment group and the control group were analyzed. The nucleotide sequences of the primers used are shown in Table 5 and SEQ NO. 1-18 below.

[0053] Table 5 Nucleotide sequence of PCR amplification primers for key genes in starch metabolism

[0054]

[0055] Among them, "GAPDH" in the table is the coding gene of glyceraldehyde-3-phosphate dehydrogenase, and its expression level is used as a reference standard for changes in the expression levels of starch metabolism-related genes.

[0056] like Figure 2 The results showed that compared with the control, the expression of the TaSBEIIa1 gene was downregulated by 45%, the TaSBEIIb1 gene was downregulated by 39%, the TaISA gene was downregulated by 31%, the TaGWD gene was downregulated by 23%, the TaPK gene was downregulated by 34%, the TaSSII gene was downregulated by 25%, the TaPHO gene was upregulated by 1.5 times, and the expression level of the TaSSIV gene was not significantly different. The downregulation of TaSBEIIa1 and TaSBEIIb1 expression is consistent with the results of knocking out or interfering with the expression of the starch branching enzyme (SBE) gene, which indicates an increase in amylose content.

[0057] In order to further confirm the effect of treatment on the amylose content in wheat leaves, the national standard method GB / T 15683-2008 was also used to analyze the amylose content in wheat leaves. Figure 1The standard curve shown in FIG. 1 shows that the amylose content in the wheat leaves increased by about 16% after treatment, which proves that the plant growth regulator mixture provided by the embodiment of the present invention, whose main components are 24-brassinolide, abscisic acid, zinc sulfate and boric acid, can increase the amylose content in the leaves after treating the wheat seedlings. The results are shown in FIG. Figure 3 shown.

[0058] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to preferred embodiments, ordinary technicians in the field should understand that the specific implementation methods of the present invention can still be modified or some technical features can be replaced by equivalents without departing from the spirit of the technical solutions of the present invention. They should all be included in the scope of the technical solutions claimed for protection by the present invention.

Claims

1. A straight chain starch content regulator, characterized in that: It is mainly composed of 0.2-25 μg / L 24-brassinolide, 0.05-6.25 mg / L abscisic acid, 0.1 wt%-1 wt% zinc sulfate and 0.01 wt%-0.1 wt% boric acid.

2. The amylose content regulator according to claim 1, characterized in that: It also includes a surfactant with a concentration of 0.8‰ to 1.2‰.

3. Use of the amylose content regulator according to claim 1 or 2 in increasing the amylose content of wheat, wherein: The application is manifested in increasing the amylose content in wheat leaves and / or mature wheat seeds.

4. The use according to claim 3, characterized in that: The amylose content regulator is used to increase the amylose content in mature wheat seeds by 9% to 15%.

5. A method for increasing the amylose content of wheat, comprising: The amylose content regulator according to claim 1 or 2 is sprayed on wheat plants, wherein the wheat plants include wheat ears and / or wheat leaves.

6. The method according to claim 5, characterized in that: The method comprises spraying the amylose content regulator on wheat plants during the wheat filling period.

7. The method according to claim 5 or 6, characterized in that: The spraying amount of the amylose content regulator is 5 to 7 L / mu.

8. Use of the amylose content regulator according to claim 1 or 2 in reducing the expression of key genes in wheat starch metabolism, wherein: The key genes for wheat starch metabolism include genes encoding starch branching enzyme, starch debranching enzyme, glucan hydration dikinase, pyruvate kinase, starch synthase or starch phosphorylase.

9. Use of the amylose content regulator according to claim 1 or 2 in wheat breeding.

10. The use according to claim 9, characterized in that: The application of the amylose content regulator in cultivating wheat seeds with high amylose content.

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