A method and application for improving wheat drought stress resistance based on transcription factor TaHY5.

Abstract

This invention discloses a method and application for improving wheat drought stress resistance based on the transcription factor TaHY5, relating to the fields of plant genetic engineering, wheat genetic breeding, and crop stress resistance improvement. This invention involves constructing a TaHY5 overexpression vector or a CRISPR / Cas9 editing vector, and transforming wheat immature embryos / callus tissue using Agrobacterium-mediated transformation or gene gun methods to obtain transgenic and gene-edited materials. After drought stress treatment, indicators such as biomass, malondialdehyde (MDA) content, and electrolyte permeability are measured to comprehensively evaluate drought resistance. Results show that TaHY5 overexpression significantly increases wheat biomass, APX, CAT and other antioxidant enzyme activities, as well as soluble protein and GSH content under drought stress, while reducing MDA accumulation and electrolyte permeability, and enhancing photosynthetic maintenance, reactive oxygen species scavenging, osmotic regulation, and cell membrane stability. This invention is the first to demonstrate that TaHY5 can synergistically improve wheat drought resistance, providing a simple and comprehensive improvement scheme, and offering new gene resources and practical technical pathways for wheat drought-resistant breeding.

Description

Technical Field

[0001] This invention relates to the fields of plant genetic engineering, wheat genetic breeding and crop stress resistance improvement, specifically a method and application for improving wheat drought stress resistance based on transcription factor TaHY5. Background Technology

[0002] Wheat is a crucial food crop in my country and globally. Drought stress, as a frequent abiotic adverse event, has become a core factor limiting wheat growth, development, yield, and quality improvement. Enhancing wheat's drought stress resistance is of significant practical importance for ensuring food security. Currently, wheat drought-resistant breeding mainly relies on conventional hybridization breeding and molecular marker-assisted selection breeding. At the same time, genetic engineering technology is also used to introduce exogenous drought-resistant genes into wheat to achieve targeted improvement of drought-resistant traits.

[0003] Traditional wheat drought-resistance breeding relies on hybridization and marker-assisted selection. However, wheat is an allohexaploid with a complex genome structure. Drought resistance is a quantitative trait controlled by multiple genes, and there is a genetic negative correlation between high yield, quality, disease resistance, and other desirable traits and drought resistance. This makes it difficult to achieve efficient aggregation of desirable traits using traditional methods, resulting in low breeding efficiency and long cycles. While genetic engineering methods can introduce exogenous drought-resistance genes, they suffer from low wheat transformation efficiency, uncertain integration site effects of exogenous genes, and severe segregation of traits in transgenic plant offspring. Furthermore, limited public acceptance of genetically modified crops hinders their large-scale application in wheat drought-resistance breeding.

[0004] Existing research has not revealed the direct functional link between the transcription factor TaHY5 and wheat drought stress resistance, nor has it established a technical solution to comprehensively improve wheat drought resistance by TaHY5 through regulating physiological indicators such as photosynthetic characteristics, osmotic regulators, antioxidant enzyme activity, and cell membrane stability. There is a lack of feasible genetic improvement pathways for wheat drought resistance centered on TaHY5, and there is an urgent need to discover new drought-resistant genes and construct supporting breeding technologies. Summary of the Invention

[0005] 1. The technical problem to be solved by the present invention

[0006] The purpose of this invention is to propose a method and application for improving wheat drought stress resistance based on the transcription factor TaHY5, in order to solve the following problems existing in the prior art: (1) Traditional wheat drought-resistant breeding is inefficient, has a long cycle, and is difficult to aggregate traits. Wheat is an allohexaploid with a complex genome. Drought resistance is a quantitative trait controlled by multiple genes. Furthermore, high yield, high quality, and other excellent traits are genetically negatively correlated with drought resistance. Conventional hybridization and molecular marker-assisted breeding are difficult to efficiently aggregate excellent traits, resulting in low breeding efficiency and a long cycle.

[0007] (2) Existing genetic engineering improvements have limitations in transformation and application, and lack TaHY5-related drought resistance technology pathways; the transformation of wheat with exogenous drought resistance genes has problems such as low transformation efficiency, uncertain integration position effect, and severe segregation of offspring traits, and public acceptance limits its large-scale application; at the same time, the direct link between TaHY5 and wheat drought resistance has not been revealed, and no wheat drought resistance genetic improvement technology scheme with TaHY5 as the core has been established.

[0008] 2. Technical Solution To achieve the above objectives, the present invention provides the following technical solution: a method for improving wheat drought stress resistance based on transcription factor TaHY5, comprising the following steps: S1, clone the full-length coding sequence of the wheat-derived TaHY5 gene; S2, construct an overexpression vector for the TaHY5 gene or a CRISPR / Cas9 gene editing vector; S3, the vector is transformed into wheat embryos or callus tissue by Agrobacterium-mediated transformation or gene gun method, and TaHY5 overexpressing plants or gene knockout mutants are obtained by screening and identification. In S4, under drought stress, the TaHY5 overexpressing plants described in S3 showed significantly increased biomass, chlorophyll content, antioxidant enzyme activity, and osmotic regulator content compared to wild-type wheat, while malondialdehyde content and electrolyte permeability were significantly reduced.

[0009] Preferably, the transformation recipient is the immature embryo of the wheat variety Fielder, and resistance screening is performed using hygromycin after transformation.

[0010] Preferably, the drought stress is a simulated drought treatment with 20% (w / v) mannitol under hydroponic conditions for 10-14 days.

[0011] Preferably, the antioxidant enzymes include APX, CAT, SOD, and POD; the osmotic regulating substances include soluble proteins and reduced glutathione (GSH).

[0012] The application of transcription factor TaHY5 in improving wheat drought stress resistance: TaHY5 enhances wheat's tolerance to drought stress by regulating its photosynthetic characteristics, osmotic regulation content, antioxidant enzyme activity and cell membrane stability. The application also includes the genetic improvement of drought resistance of HY5 homologous genes in gramineous crops such as barley, rice and maize.

[0013] Preferably, the wheat includes allohexaploid common wheat, and the application can be extended to drought resistance improvement of HY5 homologous genes in barley, rice and other gramineous crops.

[0014] A biomaterial for improving wheat drought stress resistance includes at least one of the TaHY5 gene, a TaHY5 overexpression vector, and a CRISPR / Cas9 gene editing vector. The biomaterial can significantly improve wheat drought stress resistance after being transformed into wheat.

[0015] Preferably, the TaHY5 transgenic wheat line obtained after the biomaterial is transformed into wheat possesses a stable and heritable drought-resistant phenotype under drought stress.

[0016] Compared with existing technologies, the present invention provides a method and application for improving wheat drought stress resistance based on transcription factor TaHY5, which has the following beneficial effects: This invention is simple to operate, has a short transformation cycle, and high screening efficiency, making it suitable for large-scale implementation and promotion in conventional plant genetic breeding laboratories. This invention systematically measures multiple physiological indicators, including fresh weight of aboveground and underground parts, chlorophyll content, antioxidant enzyme activity, osmotic regulation substance content, membrane lipid peroxidation products, and electrolyte permeability. It can comprehensively evaluate the drought resistance of wheat from multiple dimensions, such as growth, photosynthetic capacity, reactive oxygen species scavenging capacity, osmotic regulation capacity, and cell membrane integrity, providing a comprehensive and objective evaluation system.

[0017] This invention is the first to apply the TaHY5 core transcription factor, a light signaling gene, to the genetic improvement of drought resistance in wheat. It provides a novel genetic resource and an operational improvement pathway for wheat drought-resistant breeding, effectively complementing existing breeding strategies that primarily rely on conventional hybridization or a few known drought-resistant genes. The TaHY5 transgenic wheat material created using this invention exhibits significantly enhanced growth advantages and physiological tolerance under drought stress, demonstrating direct application value in improving the stable yield of wheat in arid and semi-arid regions. It also provides a technical reference for improving drought resistance in other gramineous crops (such as barley and rice) using HY5 homologous genes. Attached Figure Description

[0018] Figure 1 This is a transformation vector spectrum from Example 1 of the present invention; Figure 2 This is a schematic diagram illustrating the phenotypic and physiological and biochemical index analysis of mutant plant lines under drought stress according to an embodiment of the present invention. Figure 3 This is a schematic diagram of the process for improving wheat drought stress resistance based on the transcription factor TaHY5 according to the present invention. Detailed Implementation

[0019] 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, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0020] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments.

[0021] Example 1, please refer to Figures 1 to 3 As shown: To address the problems mentioned in the technical solutions, this application provides a method and application for improving wheat drought stress resistance based on the transcription factor TaHY5. This embodiment evaluates the drought resistance effect by measuring a series of physiological and biochemical indicators.

[0022] Specifically, such as Figure 1 As shown, this invention constructs a TaHY5 gene vector and a CRISPR / Cas9 gene editing vector, transforms wheat immature embryos and callus tissue using Agrobacterium-mediated transformation or gene gun method, and successfully obtains TaHY5 gene-edited mutant materials after resistance screening and PCR identification. To clarify the effect of the TaHY5 gene on wheat drought stress resistance, the obtained TaHY5 transgenic wheat and wild-type control were subjected to drought stress treatment. The specific operation procedure is as follows: First, the wheat seeds were placed in an artificial climate chamber for hydroponics. After reaching a suitable growth stage, they were subjected to drought stress treatment for 10–14 days. The specific steps for obtaining transgenic wheat material were as follows: 1. Select wheat grains 14 days after pollination, remove the embryos under aseptic conditions, and infect them with Agrobacterium EHA105 strain. 2. The infected embryos and Agrobacterium were co-cultured in liquid culture medium at 22–25°C in the dark for 2 days; 3. Transfer the co-cultured embryos to a culture medium containing callus inducing agent and continue culturing for 5–7 days; 4. Transfer to selective culture medium and culture for 2 weeks to further induce callus formation; 5. Transfer the callus tissue back into the selective medium and culture for 3 weeks to perform a second callus induction. 6. Under 25℃ light conditions, the dense callus tissue was transferred to the differentiation medium and cultured for about 14 days until fresh shoots emerged. 7. Transfer the regenerated shoots to the rooting medium to promote normal root development; 8. Transplant plants with well-developed root systems into flowerpots, and place plants that have been identified as positive in a growth chamber for further cultivation.

[0023] Plant samples were collected on the 10th day of drought stress, and the following physiological and biochemical indicators were systematically measured to analyze the regulatory role of the TaHY5 gene in wheat drought resistance: Fresh weight of above-ground and underground parts: The above-ground parts and roots of the plant were cut separately and weighed accurately using an electronic balance; Chlorophyll content: Chlorophyll was extracted using 80% acetone extraction and the content was determined by spectrophotometer. Antioxidant enzyme activity: Ascorbate peroxidase (APX) activity was determined by ascorbate oxidation method, and catalase (CAT) activity was determined by ultraviolet absorption method; Osmotic conditioning substance content: Soluble protein content was determined by Coomassie Brilliant Blue G-250 staining method, and reduced glutathione (GSH) content was determined by dithiodinitrobenzic acid (DTNB) method. Cell membrane stability related indicators: malondialdehyde (MDA) content was determined by the thiobarbituric acid (TBA) method, and electrolyte permeability was measured by the conductivity meter of leaf leachate, expressed as relative conductivity.

[0024] By comparing the differences in the above-mentioned indicators between TaHY5 transgenic wheat and wild-type control under drought stress, the effect of the TaHY5 gene on enhancing wheat's resistance to drought stress was clarified. Experimental results showed that, compared with wild-type wheat, TaHY5 transgenic wheat under drought stress exhibited significantly increased aboveground and underground fresh weight, chlorophyll content, APX and CAT enzyme activities, soluble protein content, and GSH content, while significantly decreased MDA content and relative electrolyte permeability. Conversely, the TaHY5 gene knockout mutant showed the completely opposite physiological phenotype.

[0025] The above experimental results indicate that the transcription factor TaHY5 can significantly enhance wheat's drought stress resistance through a synergistic effect of increasing the activity of wheat antioxidant enzymes, improving the content of osmotic regulatory substances, maintaining cell membrane stability, and protecting photosynthetic pigments. The technical solution provided by this invention is simple to operate and has a comprehensive and systematic indicator system, enabling precise evaluation of the application value of the TaHY5 gene in wheat drought resistance improvement, and providing new gene resources and feasible technical pathways for wheat drought resistance breeding.

[0026] Example 2: Based on Example 1, but with some differences, the following description, in conjunction with specific examples and accompanying drawings, illustrates the method and application of the present invention for improving wheat drought stress resistance based on the transcription factor TaHY5. The specific details are as follows: This example demonstrates the application of the TaHY5 gene in improving wheat drought stress resistance. Specifically, by constructing TaHY5 wheat mutant materials and combining them with mannitol-simulated drought treatment under hydroponic conditions, it was confirmed that the application of the TaHY5 gene can significantly enhance wheat's tolerance to drought stress. This method can be applied to wheat stress resistance breeding.

[0027] 1. Construction of the TaHY5 wheat mutant; (1) Cloning of the target gene: The full-length coding sequence (CDS) of the TaHY5 gene was cloned using the wheat genome as a template. Primers were designed based on wheat homologous genes, and the amplified fragment was verified by sequencing.

[0028] (2) Genetic transformation: Wheat embryos were transformed using Agrobacterium-mediated transformation. The constructed vector was transformed into Agrobacterium strain EHA105 and infected the embryos of wheat variety “Fielder” (12-14 days after pollination). After co-culturing for 2 days, the embryos were transferred to a selection medium containing 50 mg / L hygromycin. After 3 rounds of selection, resistant callus tissue was obtained and differentiated into seedlings.

[0029] (3) Identification of positive plants: Genomic DNA was extracted from the leaves of transgenic wheat and PCR detection was performed using TaHY5 specific primers.

[0030] 2. Hydroponic cultivation and mannitol-simulated drought stress treatment; Wild-type wheat (WT) and the TaHY5 mutant wheat seeds constructed above were subjected to hydroponics and drought treatment according to the following steps: (1) Seed disinfection and germination: Soak seeds in 75% ethanol for 5 min and rinse 3 times with sterile water; disinfect with 2.5% sodium hypochlorite for 15 min and rinse 5 times with sterile water. Place in a petri dish lined with moist filter paper and germinate in the dark at 25℃ for 48 h.

[0031] (2) Hydroponic culture: Select seeds with uniform germination and transfer them to a hydroponic box containing Hoagland nutrient solution, fixing them with sponges. Culture conditions: 16h light / 8h dark, light intensity 200μmol·m - ²·s - ¹, Temperature 22±1℃, Relative Humidity 65%. Nutrient solution changed every 3 days.

[0032] (3) Mannitol simulated drought stress treatment: 20% (w / v) mannitol was added to the nutrient solution and treated for 10 days. During the stress period, the nutrient solution containing the same concentration of mannitol was replaced every 2 days to ensure osmotic stress stability. The control group plants were cultured normally in Hoagland nutrient solution without mannitol.

[0033] 3. Evaluation indicators for application effectiveness; After the stress treatment was completed, tissues from wild-type and mutant wheat were collected, and the following indicators were measured to evaluate... TaHY5 The effects of gene application: (1) Biomass: Fresh weight of aboveground parts and fresh weight of underground parts.

[0034] (2) Chlorophyll content: determined by 95% ethanol extraction method, A 663 and A 645 Calculate the total chlorophyll content.

[0035] (3) Antioxidant enzyme activity and ascorbic acid content: (4) Malondialdehyde (MDA) content: TBA method.

[0036] (5) Electrolyte permeability: conductivity method.

[0037] 4. Results: The application of the TaHY5 gene significantly improved the drought resistance of wheat; like Figure 2 As shown, A represents the plant phenotype comparison between the TaHY5 gene-edited mutant and wild-type wheat after drought treatment (WT represents wild-type); B to I represent the statistical results of aboveground fresh weight, underground fresh weight, chlorophyll content, relative leaf water content, APX activity, CAT activity, MDA content, and relative electrolyte permeability, respectively. All data are expressed as mean ± standard error, and different lowercase letters indicate significant differences between groups at the P < 0.05 level.

[0038] Under salt stress, the TaHY5 gene knockout mutant showed significant abnormalities in all physiological indicators compared with wild-type wheat: aboveground fresh weight decreased by 40.6% and 44.1%, respectively; underground fresh weight decreased by 31.8% and 33.8%, respectively; chlorophyll content decreased by 29.4% and 25.0%, respectively; leaf water content decreased by 18.6% and 21.4%, respectively; APX activity decreased by 38.6% and 24.5%, respectively; CAT activity decreased by 12.6% and 10.0%, respectively; MDA content increased by 59.6% and 55.4%, respectively; and electrolyte permeability increased by 35.9% and 40.1%, respectively.

[0039] The above results indicate that loss of function of the TaHY5 gene significantly reduces biomass accumulation under drought stress, promotes chlorophyll degradation, weakens antioxidant defense systems (APX and CAT), and increases membrane lipid peroxidation products MDA and membrane permeability damage. Therefore, the TaHY5 gene can serve as an effective genetic resource for drought tolerance and can be used to create new drought-resistant wheat germplasm or varieties.

[0040] Please refer to the above work process. Figures 1 to 3 .

[0041] It should be noted that the term "comprising" or any other variation thereof is intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0042] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for improving wheat drought stress resistance based on transcription factor TaHY5, characterized in that, Includes the following steps: S1, clone the full-length coding sequence of the wheat-derived TaHY5 gene; S2, construct an overexpression vector for the TaHY5 gene or a CRISPR / Cas9 gene editing vector; S3, the vector is transformed into wheat embryos or callus tissue by Agrobacterium-mediated transformation or gene gun method, and TaHY5 overexpressing plants or gene knockout mutants are obtained by screening and identification. In S4, under drought stress, the TaHY5 overexpressing plants described in S3 showed significantly increased biomass, chlorophyll content, antioxidant enzyme activity, and osmotic regulator content compared to wild-type wheat, while malondialdehyde content and electrolyte permeability were significantly reduced.

2. The method for improving wheat drought stress resistance based on transcription factor TaHY5 according to claim 1, characterized in that, The transformation recipient was the immature embryo of the wheat variety Fielder, and resistance screening was performed using hygromycin after transformation.

3. The method for improving wheat drought stress resistance based on transcription factor TaHY5 according to claim 1, characterized in that, The drought stress was simulated by 20% (w / v) mannitol for 10-14 days under hydroponic conditions.

4. The method for improving wheat drought stress resistance based on transcription factor TaHY5 according to claim 1, characterized in that, The antioxidant enzymes include APX and CAT; the osmotic regulators include soluble proteins and reduced glutathione (GSH).

5. The application of transcription factor TaHY5 in improving wheat drought stress resistance is applicable to the method for improving wheat drought stress resistance based on transcription factor TaHY5 as described in any one of claims 1-4, characterized in that, The transcription factor TaHY5 enhances wheat's tolerance to drought stress by regulating its photosynthetic properties, osmotic regulation content, antioxidant enzyme activity, and cell membrane stability.

6. The application of the transcription factor TaHY5 according to claim 5 in improving wheat drought stress resistance, characterized in that, The wheat mentioned includes allohexaploid common wheat, and the application can be extended to drought resistance improvement of HY5 homologous genes in barley, rice, and other gramineous crops.

7. A biomaterial for improving wheat drought stress resistance, applicable to the method for improving wheat drought stress resistance based on transcription factor TaHY5 as described in any one of claims 1-4, characterized in that, The biomaterial contains at least one of the TaHY5 gene, a TaHY5 overexpression vector, and a CRISPR / Cas9 gene editing vector, and the transformation of wheat with the biomaterial can significantly improve the drought stress resistance of wheat.

8. A biomaterial for improving wheat drought stress resistance according to claim 7, characterized in that, The TaHY5 transgenic wheat line obtained after the biomaterial was transformed into wheat exhibits a stable and heritable drought resistance phenotype under drought stress.

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