Wheat character improvement and new germplasm creation method based on apparent modification change

Abstract

The invention discloses a wheat character improvement and new germplasm creation method based on apparent modification change, which is characterized in that a gene H3K27M (the nucleotide sequence is as shown in SEQ ID NO.1, and the coded amino acid sequence is as shown in SEQ ID NO.2) obtained by mutating lysine (K) at the 27th site of rice histone H3 into methionine (M) is introduced into wheat Fielder, so that the modification level of a wheat whole genome H3K27me3 is reduced. Experiments prove that the wheat material obtained by the method can significantly improve the drought tolerance of wheat, improve the grain quality and increase the yield-related traits. The H3K27M gene and related biological materials thereof provided by the invention can be used for improving the yield, quality and drought tolerance of wheat, an efficient technical approach is provided for rapidly cultivating new germplasm of high-yield, high-quality and drought-tolerant wheat, and the H3K27M gene and related biological materials have important significance on guaranteeing grain safety, promoting wheat breeding technology upgrading and accelerating the breeding process.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of genetic engineering, agricultural biotechnology, and crop genetic improvement, specifically relating to a method for improving wheat traits and creating new germplasm based on epigenetic modification changes. This method is a technical approach to improve important agronomic traits of wheat, such as yield, quality, and stress resistance, and to create new high-yielding, high-quality, and drought-resistant wheat germplasm through epigenetic modification changes. Background Technology

[0002] As one of the most important food crops in my country and even globally, improving wheat's yield, quality, and resilience in a coordinated manner is a core issue for ensuring food security and meeting the demands of consumption upgrading. Currently, my country's wheat production faces three major challenges: First, under the constraints of population growth and arable land, yield increases have entered a plateau period, with the average yield in major producing areas still 20%-25% lower than the theoretical potential; second, adverse stresses such as drought and high temperatures lead to an average annual yield reduction of 15%-30%, and the frequency of extreme weather events is on the rise; third, the proportion of high-quality specialty varieties is insufficient, with the self-sufficiency rate of strong and weak gluten wheat at only 60%, and the quality indicators of ordinary varieties, such as protein content and gluten strength, being unstable, making it difficult to meet the refined needs of the food industry.

[0003] Traditional breeding techniques have significant limitations in the synergistic improvement of high yield, high quality, and drought resistance. Hybrid breeding relies on phenotypic selection, has a cycle of 8-10 years, and high yield and high quality are often negatively correlated; the introduction of drought-resistant genes can easily lead to deterioration of yield traits. Although molecular marker-assisted breeding can directionally aggregate a few known genes, it lacks sufficient understanding of the complex quantitative trait regulation mechanisms related to quality and neglects the synergistic regulatory effects of epigenetic modifications on multiple traits.

[0004] In recent years, gene editing technology has made breakthroughs in improving single traits in wheat, such as increasing thousand-grain weight by editing the TaGW2 gene and enhancing disease resistance by knocking out the TaMLO gene. However, these technologies mostly focus on sequence variations in the coding region of genes, with limited potential for regulating epigenetic modifications. Epigenetic modifications (DNA methylation, histone modifications, non-coding RNA, etc.) serve as a key bridge connecting the genome and phenotype, demonstrating unique value in the synergistic regulation of multiple traits in crops: in Arabidopsis, H3K27me3 modification coordinates flowering time and cold resistance by regulating FLC gene expression, and in rice, miR156 simultaneously optimizes yield and cooking quality by targeting the SPL gene. However, in wheat, the multi-dimensional regulatory mechanisms of epigenetic modifications on yield, quality, and drought resistance have not yet been systematically understood.

[0005] The current research faces three major bottlenecks: First, the complexity of the wheat polyploid genome (17 Gb) makes it difficult to map epigenetic modifications, and the epigenetic regulatory network of quality-related genes remains unclear. Second, most of the identified epigenetic regulatory factors target single traits, such as TaCKX2 which regulates grain size or TaERF3 which responds to drought, lacking core nodes that simultaneously regulate yield formation (e.g., number of grains per ear), quality synthesis (e.g., protein accumulation), and stress response. Third, there are antagonistic effects at the epigenetic level in the regulation of yield, quality, and drought resistance. For example, while increased DNA methylation levels under drought stress can enhance drought resistance, they can also inhibit the expression of starch synthesis genes during the grain filling stage, leading to a decrease in grain starch content.

[0006] Existing germplasm resources are insufficient to meet the needs of synergistic improvement: local varieties, while possessing unique quality characteristics (such as the high protein content of wheat), suffer from low yields and weak stress resistance; wild relatives (such as Aegilops scabra) exhibit outstanding drought resistance but carry genes that degrade quality and have poor hybridization compatibility with cultivated varieties. During traditional backcrossing and conversion breeding processes, approximately 30% of introduced lines show segregation of the "yield-quality-drought resistance" trait. The core reason for this lies in the lack of elucidation of the epigenetic network governing the synergistic regulation of multiple traits. Therefore, elucidating the molecular mechanisms of epigenetic modification in the synergistic regulation of high yield, high quality, and drought resistance in wheat, and establishing a multi-trait improvement technology system based on targeted editing of epigenetic modifications, will become a key pathway to overcome breeding bottlenecks, create groundbreaking new germplasm, and accelerate the breeding process. Summary of the Invention

[0007] To address the problems of long breeding cycles and low efficiency in improving wheat traits and quality in existing wheat breeding programs, the present invention aims to provide a method for improving wheat traits and creating new germplasm based on epigenetic modification changes. This method is beneficial for accelerating the achievement of the goal of breeding high-yielding, high-quality, and drought-resistant wheat varieties.

[0008] This invention creates a material with reduced levels of H3K27me3 (trimethylation of lysine at position 27 of histone H3) across the entire genome by mutating lysine (K) to methionine (M) at position 27 of histone H3 and transferring it into wheat Fielder. Through quality determination, drought treatment phenotypic identification, and field agronomic trait surveys, it was found that this material can increase wheat yield, improve wheat quality, and enhance wheat drought resistance. Its application in accelerating breeding for improved wheat yield, quality, and drought resistance is also elucidated.

[0009] The objective of this invention can be achieved through the following technical solutions:

[0010] In a first aspect, the present invention seeks protection for the use of the H3K27M gene or biological materials associated with the H3K27M gene in any of the following:

[0011] (a1) Applications in improving plant seed quality, yield, and drought resistance;

[0012] (a2) Application in the preparation of products for improving the quality, yield and drought resistance of plant seeds;

[0013] (a3) Application in cultivating new plant germplasm with improved seed quality, yield and drought resistance;

[0014] (a4) Reduce the level of H3K27me3 modification in the whole plant genome;

[0015] The gene H3K27M is a DNA molecule with the nucleotide sequence shown in SEQ ID NO.1.

[0016] Furthermore, the biological material associated with the H3K27M gene is at least one of the following (b1)-(b10):

[0017] (b1) An expression cassette containing the H3K27M gene;

[0018] (b2) A recombinant vector containing the H3K27M gene, or a recombinant vector containing the expression cassette described in (b1);

[0019] (b3) Recombinant microorganisms containing the H3K27M gene, or recombinant microorganisms containing the expression cassette of (b1), or recombinant microorganisms containing the recombinant vector of (b2);

[0020] (b4) A transgenic plant cell line containing the H3K27M gene, or a transgenic plant cell line containing the expression cassette described in (b1), or a transgenic plant cell line containing the recombinant vector described in (b2);

[0021] (b5) Transgenic plant tissue containing the H3K27M gene, or transgenic plant tissue containing the expression cassette of (b1), or transgenic plant tissue containing the recombinant vector of (b2);

[0022] (b6) A transgenic plant organ containing the H3K27M gene, or a transgenic plant organ containing the expression cassette of (b1), or a transgenic plant organ containing the recombinant vector of (b2);

[0023] (b7) A transgenic plant containing the H3K27M gene, or a transgenic plant containing the expression cassette of (b1), or a transgenic plant containing the recombinant vector of (b2);

[0024] (b8) Tissue cultures produced from regenerative cells of the transgenic plant described in (b7);

[0025] (b9) Protoplasts produced from the tissue culture described in (b8);

[0026] (b10) The protein encoded by the H3K27M gene.

[0027] Furthermore, the protein encoded by the H3K27M gene described in (b10) is as follows (c1) or (c2):

[0028] (c1) A protein with the amino acid sequence shown in SEQ ID NO.2;

[0029] (c2) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein described in (c1).

[0030] Furthermore, the above application involves overexpressing the H3K27M gene in target plants through genetic engineering methods, reducing the H3K27me3 modification level in the whole plant genome, and improving the grain quality, yield, and drought resistance of the target plants, or cultivating new plant germplasm with improved grain quality, yield, and drought resistance.

[0031] Secondly, this invention seeks to protect a method for improving plant traits and creating new germplasm based on epigenetic modification changes. Through genetic engineering methods, the aforementioned H3K27M gene is overexpressed in the target plant to reduce the H3K27me3 modification level in the whole plant genome, thereby improving the grain quality, yield and drought resistance of the target plant or cultivating new plant germplasm with improved grain quality, yield and drought resistance.

[0032] In the technical solution of this invention, the plant is wheat.

[0033] Thirdly, the present invention seeks protection for an H3K27M gene, which is a DNA molecule with a nucleotide sequence as shown in SEQ ID NO.1 obtained by mutating lysine (K) at position 27 of rice histone H3 to methionine (M).

[0034] Fourthly, the present invention claims protection for the protein encoded by the H3K27M gene, which is as follows (c1) or (c2):

[0035] (c1) A protein with the amino acid sequence shown in SEQ ID NO.2;

[0036] (c2) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein described in (c1).

[0037] Fifthly, the present invention seeks protection for biological materials comprising the H3K27M gene of claim 8, wherein the biological material is at least one of the following (b1)-(b4):

[0038] (b1) An expression cassette containing the H3K27M gene;

[0039] (b2) A recombinant vector containing the H3K27M gene, or a recombinant vector containing the expression cassette described in (b1);

[0040] (b3) Recombinant microorganisms containing the H3K27M gene, or recombinant microorganisms containing the expression cassette of (b1), or recombinant microorganisms containing the recombinant vector of (b2);

[0041] (b4) A transgenic plant cell line containing the H3K27M gene, or a transgenic plant cell line containing the expression cassette described in (b1), or a transgenic plant cell line containing the recombinant vector described in (b2).

[0042] Because the H3 genes in wheat and rice are highly homologous, this invention cloned the histone H3 gene from rice and obtained the target gene H3K27M by artificially mutating lysine (K) at position 27 to methionine (M). Overexpression of this gene in wheat Fielder cells showed that it reduced the H3K27me3 modification level across the entire wheat genome. This modified H3K27me3 has the nucleotide core sequence shown in SEQ ID NO.1 and the amino acid sequence shown in SEQ ID NO.2.

[0043] In a specific embodiment of the present invention, the recombinant vector containing the H3K27M gene is a recombinant overexpression vector. This recombinant overexpression vector is obtained by inserting the aforementioned H3K27M gene (the lysine K at position 27 of rice histone H3 is artificially mutated to methionine M) between the BamH1 restriction sites of the backbone vector using PWM110-GFP as the backbone vector. Its amplification primers are:

[0044] The primer sequences for forward amplification of the inserted fragment CE II are as follows (lowercase sequences are the recombinant sequences added at the 5' end): (Gene-specific primer Tm calculated value: 61.2℃; Homologous arm length: 15bp; Homologous arm GC content: 53.3%; Restriction site: GGATCC)

[0045] 5'- caggtcgactctagaggatccATGGCCCGTACGAAGCAGA -3'

[0046] The primer sequences for reverse amplification of the inserted fragment CE II are as follows (lowercase sequences are the recombinant sequences added at the 5' end): (Gene-specific primer Tm calculated value: 76.9℃; Homologous arm length: 15bp; Homologous arm GC content: 73.3%; Restriction site: GGATCC)

[0047] 5'- gagctcggtacccggggatccCGCGCGCTCGCCGCGGAT -3'

[0048] Studies have shown that the method for wheat trait improvement and new germplasm creation based on epigenetic modification changes (H3K27me3 level regulation) provided by this invention specifically reduces the level of H3K27me3 modification in the whole wheat genome through the transformation of histone H3K27M, thereby enabling the rapid breeding of high-yielding, high-quality, and drought-resistant wheat varieties. It can also be used for basic epigenetic research related to drought resistance, yield, and quality improvement in wheat.

[0049] The beneficial effects of this invention are:

[0050] This invention is the first to clone the histone H3 sequence from the rice genome, and then obtain the target gene H3K27M by artificially mutating lysine (K) at position 27 to methionine (M). Overexpression of this gene in wheat Fielder showed that it could decrease the level of H3K27me3 modification throughout the wheat genome (e.g., ...). Figure 2 (As shown in c). Drought treatment was applied to 8-day-old seedlings of the T3 generation transgenic material. This material improved wheat's drought tolerance; that is, under similar soil drought conditions, the transgenic plants grew better (e.g., ...). Figure 2 (As shown in a and b in the figure). Near-infrared spectroscopy (NIRS) quality analysis of seeds from two lines of the T3 generation transgenic plant revealed a significant improvement in grain quality. Specifically, the protein content of the two lines (H3K27M-1 and H3K27M-2) increased by 8.845% and 10.877%, respectively; the starch content decreased by 4.866% and 3.479%, respectively; the wet gluten content increased by 10.856% and 11.599%, respectively; the hardness increased by 21.210% and 15.797%, respectively; the weakening degree decreased by 24.415% and 8.839%, respectively; and the SDS-sedimentation value increased by 12.345% and 12.556%, respectively (e.g., as shown in a and b in the figure). Figure 3 (As shown). In addition, phenotypic identification of the ears at maturity in the field of its T3 generation revealed that, under the premise that grain traits (length and width) were not significantly affected during field planting, this material exhibited larger and longer ears, and the number of effective florets per ear, the number of grains per ear, and the yield per ear were all increased, specifically, the yield per ear increased by 21.0148% (e.g., ...). Figure 4 (As shown). That is, overexpression of the gene H3K27M can reduce the level of H3K27me3 in the whole wheat genome, thereby improving wheat grain quality, yield and drought tolerance. If the mechanism of this gene or its homologous genes is analyzed in depth, it will be beneficial to the discovery of drought-resistant, yield and quality genes in wheat, and ultimately help to ensure national food production and security.

[0051] This material holds promise for the following applications: By using gene editing technology to mutate the lysine residue at position 27 of histone H3 in plants, a transgenic vector-free wheat material with a decreased H3K27me3 level across the entire genome can be obtained. This will serve as an excellent intermediate material in wheat breeding, accelerating the development of high-yielding, high-quality, and drought-resistant new wheat varieties. Furthermore, this material can be used for basic epigenetic research related to improving wheat yield, quality, and drought resistance, accelerating theoretical research on wheat and ultimately ensuring national food security. Attached Figure Description

[0052] Figure 1 The results are PCR amplification detection results for two transgenic lines, H3K27M-1 and H3K27M-2.

[0053] Figure 2 This study measured the drought-treated phenotype, soil moisture content, and histone modification levels of transgenic materials containing the H3K27M gene. Figure a shows the phenotypes before (CK) and after (Drought) overexpression of the H3K27M gene in Fielder. Figure b shows the soil moisture content in wheat after drought treatment. Figure c shows the whole-genome H3K27me3 modification level of the H3K27M transgenic material as determined by Western blotting.

[0054] Figure 3 Near-infrared spectroscopy (NIRS) data of seeds from the T3 generation H3K27M transgenic materials (H3K27M-1 and H3K27M-2) were used to analyze their grain quality traits under field planting conditions. Among them, a) grain protein content; b) starch content; c) wet gluten content; d) grain hardness; e) SDS-sedimentation value; and f) degree of weakening.

[0055] Figure 4 Data on agronomic traits of the T3 generation H3K27M transgenic material were obtained from field measurements. Where: a) represents the plant phenotype; b) represents the wheat spike phenotype; c) represents the wheat grain phenotype; d) represents the number of effective florets per plant; e) represents the number of grains per wheat spike; and f) represents the yield of wheat grains per spike. Detailed Implementation

[0056] The technical solution of the present invention will be further described in detail below with reference to the embodiments and related drawings. However, it is worth noting that the embodiments of the present invention are not limited thereto, and those skilled in the art can implement them in other different forms without departing from the spirit and purpose of the present invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods; unless otherwise specified, the experimental materials used in the following embodiments were all purchased from conventional biochemical reagent companies.

[0057] Example 1: The rice histone H3 gene sequence was cloned, and the target gene H3K27M was obtained by artificially mutating lysine (K) at position 27 to methionine (M).

[0058] 1. Gene amplification

[0059] First, amplification was performed using rice NIP cDNA as a template via a high-fidelity enzyme. Amplification primer F:

[0060] 5'- caggtcgactctagaggatccATGGCCCGTACGAAGCAGA -3'

[0061] R: 5'- gagctcggtacccggggatccCGCGCGCTCGCCGCGGAT -3'

[0062] 2. By amplifying segments through homologous recombination, the lysine (K) at position 27 was artificially mutated to methionine (M).

[0063] The primers were designed to divide the segment into two parts, and the base A at position 83 was replaced with T, causing the lysine (K) at position 27 to be artificially mutated to methionine (M). The primer design is as follows:

[0064] The primer sequence for forward amplification of insert fragment 1 is as follows: (Primer Tm calculated value: 61.2℃, homologous arm length: 15bp, homologous arm GC content: 53.3%, restriction enzyme site: GGATCC)

[0065] 5'- caggtcgactctagaggatccATGGCCCGTACGAAGCAGA -3'

[0066] The primer sequence for reverse amplification of insert fragment 1 is as follows: (The calculated primer Tm value is 61.5℃)

[0067] 5'- ttcttcacgccgCCGGTGGTCGGAGCAGACA-3' (The bold italicized text indicates a single-base mutation that changes the amino acid from lysine to methionine)

[0068] The primer sequences for forward amplification of insert fragment 2 are as follows: (Primer Tm calculated value: 64.9℃, homologous arm length: 20bp, homologous arm GC content: 65.0%, restriction enzyme sites: none)

[0069] 5'-accaccggCGGCGTGAAGAAGCCCCA-3'

[0070] The primer sequences for reverse amplification of insert fragment 2 are as follows: (Primer Tm calculated value: 76.9℃, homologous arm length: 15bp, homologous arm GC content: 73.3%, restriction enzyme site: GGATCC)

[0071] 5'- gagctcggtacccggggatccCGCGCGCTCGCCGCGGAT -3'

[0072] Then, using the primers described above and the product from step 1 as a template, amplification was performed. The small fragments were then recovered, and both fragments were simultaneously ligated into the vector using ligase. The reaction system is as follows:

[0073]

[0074] The ligation product was placed in a PCR instrument and the program was set to 37℃ for 60 min. After heat shock transformation in competent cells, the PWM110 recombinant vector containing H3K27M was obtained. The recombinant vector was then sent to the transgenic platform of China Agricultural University to transform target plants.

[0075] 3. Identification of positive strains

[0076] After the bacterial culture was sent out, the resulting transgenic seeds were germinated and transplanted into nutrient soil. Then, leaves from each individual plant were cut off, ground into powder using liquid nitrogen, and DNA from each individual plant was extracted using the conventional CTAB method.

[0077] GFP primers were designed, and PCR amplification was performed using single-strain DNA as a template. Two transgenic lines were ultimately detected and named H3K27M-1 and H3K27M-2, respectively. (e.g.) Figure 1 (As shown)

[0078] The amplification primers are as follows:

[0079] GFP-F:ATCGAGCTGAAGGGCATCG

[0080] GFP-R:CTCGATGTTGTGGCGGATC.

[0081] Example 2: Detection of H3K27me3 modification level in constitutive transgenic seedlings

[0082] 1. Western blotting (WB) was used to detect the histone modification levels in transgenic plants.

[0083] The specific steps are as follows:

[0084] 1) Select seeds from plump wheat overexpression plants. Germinate them in the dark at 25℃ for 2 days, then transfer them to nutrient soil and grow them in a 25℃ incubator for 10 days.

[0085] 2) Collect the plant leaves and grind the material into powder using liquid nitrogen.

[0086] 3) Weigh 5-10 g of powder and place it in a pre-cooled 50 ml tube. Add 15-30 ml of Buffer A and shake well until the mixture becomes a flowable viscous liquid. Place on ice for 10 min and shake.

[0087] 4)Miracloth filtration: first filter once with a single layer of membrane cloth, then filter once with a double layer of membrane cloth.

[0088] 5) Centrifuge the obtained supernatant at 4°C, 1100 xg, for 20 min. A green precipitate will be visible.

[0089] 6) Discard the supernatant, resuspend the precipitate in 5 ml of Buffer A, and gently aspirate and mix.

[0090] 7) Centrifuge at 4 ℃, 1100 xg, for 20 min.

[0091] 8) Discard the supernatant, resuspend the precipitate in 1 ml Buffer A, gently aspirate and mix, and transfer to a 1.5 ml tube.

[0092] 9) Centrifuge at 4℃, 2000 xg, for 10 min.

[0093] 10) Discard the supernatant, add 180 μl of Low Salt Buffer, and mix by suction and whisking (it is difficult to whisk).

[0094] 11) Add 270 μl of High Salt Buffer and mix thoroughly by pipetting.

[0095] 12) Wrap the sample in an ice pack and shake it on a shaker for 30 min; or place it on a rotating shaker and shake it at 4℃ for 1 h.

[0096] 13) Centrifuge at 4℃, 14,000 xg, for 20 min.

[0097] 14) Aspirate the supernatant. A lot of turbidity will be aspirated. Centrifuge at 14,000 xg at 4℃ for 5 min.

[0098] 15) Aspirate the supernatant and transfer it to a 1.5 ml tube (store at -20℃ / -70℃). Add 5× Protein Loading Buffer and one-tenth the volume of β-mercaptoethanol to the supernatant.

[0099] 16) Then invert the container to mix thoroughly, place it in a metal bath at 100℃ for 10 min, and immediately place it on ice for 15 min. The Western blot (WB) experiment can then be performed.

[0100] 2. The formulas for the solutions required for the experiment are as follows:

[0101] Note: Use Sigma imported reagents whenever possible.

[0102] (1) Preparation of 500ml 1 M HEPES, pH = 7.0 Stock solution: Dissolve 119.15 g HEPES in 400ml distilled water, add 0.5~1M NaOH aqueous solution to adjust to the required pH (the effective pH range of HEPES is 6.8~8.2), then make up to 500ml with distilled water and store at 4℃.

[0103] (2) Buffer A: 10mM HEPES (pH 7.8), 10mM KCl, 10mM MgCl2, 5mM EDTA,

[0104] 250 mM sucrose, 0.5% (v / v) Triton-X-100.

[0105] Add the following before use: 1 mM DTT; 0.2 mM PMSF. Pre-cool on ice before use.

[0106] (3) Low Salt Buffer: 20mM HEPES (pH 7.8), 20mM KCl, 1.5mM MgCl2, 0.2mMEDTA, 25% (v / v) Glycerol.

[0107] Add the following before use: 0.5 mM DTT; 0.2 mM PMSF. Pre-cool on ice before use.

[0108] (4) High Salt Buffer: 20 mM HEPES (pH 7.8), 1.6 M KCl, 1.5 mM MgCl2, 0.2mM EDTA, 25% (v / v) Glycerol.

[0109] Add the following before use: 0.5 mM DTT; 0.2 mM PMSF. Pre-cool on ice before use.

[0110] The results showed that the whole genome H3K27me3 modification levels of the two transgenic lines (H3K27M-1 and H3K27M-2) were lower than those of the wild type. (e.g.) Figure 2 (as shown in c)

[0111] Example 3: The obtained T3 generation positive seedlings were subjected to drought treatment phenotypic observation and soil moisture content determination.

[0112] Wheat seedlings that were identified as positive were planted in nutrient soil and placed in a 25°C incubator for normal growth for 8 days. Watering was then stopped, and the phenotype was investigated and recorded.

[0113] The detailed steps are as follows:

[0114] 1. Germinate wheat seeds in a petri dish, then plant them in a small pot with a perforated bottom filled with nutrient soil.

[0115] 2. Place in a 25-degree incubator for 8 days, gradually reducing the amount of water.

[0116] 3. Stop watering completely on the 8th day of growth.

[0117] 4. Then continue to grow for 5-7 days.

[0118] 5. Conduct phenotypic observations and use a soil moisture meter to measure and record the moisture content in the soil of each pot.

[0119] The results showed that, under similar moisture content conditions, the two transgenic lines grew well under similar drought conditions, while the wild-type plants exhibited wilting and severely drooping leaves, indicating that this material can significantly improve the drought resistance of wheat. Figure 2 (as shown in a and b)

[0120] Editing the aforementioned sites can improve the drought resistance of wheat, facilitate the discovery of drought-resistant genes in wheat, and improve drought-sensitive varieties.

[0121] Example 4: Investigation of field agronomic traits of transgenic plants and quality determination of transgenic seeds.

[0122] The phenotypes of transgenic wheat plants and grains in the field were recorded by photographing them with a camera. Plant height and ear length were measured and recorded using a ruler. This included plant height, ear type and length, grain length and width, number of effective florets per ear, number of grains per ear, and grain weight per ear. The final results showed that the H3K27M transgenic material had a shorter plant height than the wild type, which is beneficial for improving lodging resistance. It also had longer wheat ears, and the number of effective florets per ear, the number of grains per ear, and the grain weight per ear were significantly higher than the wild type. Ultimately, this increased wheat yield (e.g., ...). Figure 4 (As shown).

[0123] The protein, starch, wet gluten content, hardness, fortification degree, and SDS-sedimentation value of wheat grains were measured using a near-infrared scanner. The results showed that the H3K27M transgenic material had higher protein content, wet gluten content, hardness, and SDS-sedimentation value, and lower starch content and fortification degree (e.g., ...). Figure 3 (As shown).

[0124] From a processing quality perspective, higher protein content is an important indicator of wheat's nutritional quality, providing a richer source of protein for food processing. Increased wet gluten content means enhanced dough elasticity and resilience, which is crucial for the production of bread, noodles, and other wheat-based products. Higher wet gluten content allows bread to ferment to a larger volume and a more fluffy structure, and noodles to be chewier and less prone to breaking.

[0125] Increased hardness makes wheat easier to break into coarser particles during milling, making it suitable for producing high-gluten flour to meet the particle size requirements of various food processing methods. SDS (Sedimentation Value) is related to the quality and quantity of wheat protein; a higher value indicates better protein quality and better processing performance of the flour products.

[0126] The lower starch content gives the H3K27M transgenic material an advantage in the field of low-sugar food processing, meeting the demands of modern consumers for low-sugar diets. Reduced weakening indicates enhanced shear resistance and better stability of the dough during mixing, minimizing the impact of improper processing on dough quality and improving the stability and consistency of flour product production.

[0127] The comprehensive improvement of these quality indicators gives the H3K27M transgenic material both good nutritional characteristics and excellent processing performance, making it promising for wide application in the food industry and enhancing the market competitiveness of this new wheat germplasm.

[0128] The DNA sequence (H3K27M) in which the lysine (K) at position 27 of histone is mutated to methionine (M) is described. Its nucleotide CDS sequence (SEQ ID NO.1) is as follows:

[0129] 5’ATGGCCCGTACGAAGCAGACCGCCCGCAAGTCCACCGGCGGCAAGGCCCCGAGGAAGCAGCTCGCCACCAAGGCGGCGAGGATGTCTGCTCCGACCACCGGCGGCGTGAAGAAGCCCCACCGCTACAGGCCGGGGACGGTGGCGCTCCGCGAGATCCGCAAGTACCAGAAGAGCACGGAGCTCCTGATCCGCAAGCTCCCCTTCCAGCGCCTCGTCCGCGAGATCGCGCAGGACTTCAAGACCGACCTCCGCTTCCAGAGCCACGCGGTGCTCGCCCTCCAGGAGGCCGCCGAGGCCTACCTCGTCGGTCTCTTCGAGGACACCAACCTCTGCGCCATCCACGCCAAGCGCGTCACCATCATGCCCAAGGACATCCAGCTCGCCCGCCGCATCCGCGGCGAGCGCGCGTAA -3’。

[0130] The amino acid sequence of the protein encoded by the gene H3K27M (SEQ ID NO.2) is as follows:

[0131] MARTKQTARKSTGGKAPRKQLATKAARMSAPTTGGVKKPHRYRPGTVALREIRKYQKSTELLIRKLPFQRLVREIAQDFKTDLRFQSHAVLALQEAAEAYLVGLFEDTNLCAIHAKRVTIMPKDIQLARRIRGERA*。

Claims

1. Use of H3K27M gene in any of the following: (a1) improving grain quality, yield and drought tolerance of plants; (a2) preparing products for improving grain quality, yield and drought tolerance of plants; (a3) breeding new germplasm of plants with improved grain quality, yield and drought tolerance; (a4) reducing the level of H3K27me3 modification in whole genome of plants. The gene H3K27M is a DNA molecule with nucleotide sequence as shown in SEQ ID NO.

1.

2. Use of biological material related to the H3K27M gene in any of the following: (a1) improving grain quality, yield and drought tolerance of plants; (a2) preparing products for improving grain quality, yield and drought tolerance of plants; (a3) breeding new germplasm of plants with improved grain quality, yield and drought tolerance; (a4) reducing the level of H3K27me3 modification in whole genome of plants. The biological material related to the H3K27M gene is at least one of the following (b1) - (b10): (b1) an expression cassette containing the H3K27M gene; (b2) a recombinant vector containing the H3K27M gene, or a recombinant vector containing the expression cassette of (b1); (b3) a recombinant microorganism containing the H3K27M gene, or a recombinant microorganism containing the expression cassette of (b1), or a recombinant microorganism containing the recombinant vector of (b2); (b4) a transgenic plant cell line containing the H3K27M gene, or a transgenic plant cell line containing the expression cassette of (b1), or a transgenic plant cell line containing the recombinant vector of (b2); (b5) a transgenic plant tissue containing the H3K27M gene, or a transgenic plant tissue containing the expression cassette of (b1), or a transgenic plant tissue containing the recombinant vector of (b2); (b6) a transgenic plant organ containing the H3K27M gene, or a transgenic plant organ containing the expression cassette of (b1), or a transgenic plant organ containing the recombinant vector of (b2); (b7) a transgenic plant containing the H3K27M gene, or a transgenic plant containing the expression cassette of (b1), or a transgenic plant containing the recombinant vector of (b2); (b8) a tissue culture produced from regenerable cells of the transgenic plant of (b7); (b9) a protoplast produced from the tissue culture of (b8); (b10) a protein encoded by the H3K27M gene.

3. Use according to claim 2, characterized in that, The protein encoded by the H3K27M gene in (b10) is as follows (c1) or (c2): (c1) a protein with amino acid sequence as shown in SEQ ID NO. 2; (c2) a fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of the protein of (c1).

4. Use according to claim 1 or 2, characterized in that, The H3K27M gene is overexpressed in target plants by genetic engineering method, the whole genome H3K27me3 modification level of the plants is reduced, and the grain quality, yield and drought tolerance of the target plants are improved or new plant germplasm with improved grain quality, yield and drought tolerance is bred.

5. Use according to claim 1 or 2 or 3 or 4, characterized in that, The plant is wheat.

6. A method for plant trait improvement and new germplasm creation based on epigenetic modification changes, characterized in that, The H3K27M gene is overexpressed in target plants by genetic engineering method, the whole genome H3K27me3 modification level of the plants is reduced, and the grain quality, yield and drought tolerance of the target plants are improved or new plant germplasm with improved grain quality, yield and drought tolerance is bred.

7. The method of claim 6, wherein, The plant is wheat.

8. An H3K27M gene, which is a DNA molecule with a nucleotide sequence as shown in SEQ ID NO. 1 obtained by mutating lysine (K) at position 27 of histone H3 of rice to methionine (M).

9. The protein encoded by the H3K27M gene according to claim 8, characterized in that, The protein is as follows (c1) or (c2): (c1) a protein with an amino acid sequence as shown in SEQ ID NO. 2; (c2) a fusion protein obtained by connecting a protein tag to the N terminus and / or C terminus of the protein of (c1).

10. Biomaterial comprising the H3K27M gene according to claim 8, characterized in that, The biological material is at least one of the following (b1) to (b4): (b1) an expression cassette containing the H3K27M gene; (b2) a recombinant vector containing the H3K27M gene or containing the expression cassette of (b1); (b3) a recombinant microorganism containing the H3K27M gene or containing the expression cassette of (b1) or containing the recombinant vector of (b2); (b4) a transgenic plant cell line containing the H3K27M gene or containing the expression cassette of (b1) or containing the recombinant vector of (b2).

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