A wheat TaUXS7 and its application in wheat seed germination.

Abstract

This invention provides a wheat TaUXS7 gene and its application in wheat seed germination, relating to the field of plant genetic engineering technology. The application of the wheat gene TaUXS7 in regulating wheat seed germination is described. This invention is the first to identify the uridine diphosphate xylose synthase gene TaUXS7, which is associated with seed germination, from the whole wheat genome. CRISPR / Cas9 technology was used to confirm that its deletion significantly reduces germination rate and inhibits seedling growth, providing a new gene resource for wheat germination regulation and ear bud resistance breeding. Simultaneously, a BstB I restriction enzyme marker was developed based on the natural variation (T / C) of the third intron of TaUXS7-A, which can rapidly distinguish between the high-germination-rate haplotype Hap2 and the low-germination-rate haplotype Hap1, suitable for germplasm resource screening and marker-assisted breeding. Furthermore, bidirectional regulation of seed germination can be achieved by upregulating or downregulating the expression of this gene, demonstrating clear industrial application value.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to a wheat TaUXS7 and its application in the wheat seed germination process. Background Technology

[0002] Wheat is a major food crop, and the timely germination of its seeds directly affects the uniformity of emergence and the quality of seedling establishment, thus influencing yield and quality. Head germination, the abnormal germination of seeds on the ear before harvest, can lead to decreased grain yield and deterioration of processing quality, causing significant losses to agricultural production. Therefore, elucidating the precise regulatory mechanisms of seed germination and conducting molecular breeding for resistance to head germination is of great significance to wheat production.

[0003] Dynamic remodeling of the cell wall is a key event in seed germination, involving the degradation and synthesis of polysaccharide components. While research on the regulation of germination by cell wall degrading enzymes (such as mannanase and xyloglucan endoglucosylase) is relatively systematic, research on the molecular mechanisms of cell wall polysaccharide synthesis pathways is relatively weak. Urate-xyl diphosphate (UDP-Xyl) is a precursor for the synthesis of hemicellulose xylan and xyloglucan, and its synthesis is catalyzed by UDP-Xyl synthase (UXS). Although functional studies of UXS family members in Arabidopsis and rice have been reported, the wheat genome is complex and has redundant homologous genes; therefore, no studies have yet investigated the involvement of wheat UXS genes in seed germination regulation. Summary of the Invention

[0004] Technical problems to be solved

[0005] To address the shortcomings of existing technologies, this invention provides a wheat TaUXS7 gene and its application in wheat seed germination, solving the problem of lacking functional understanding of the UDP-xylose synthase gene TaUXS7 in the regulation of wheat seed germination.

[0006] Technical solution

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] Application of wheat gene TaUXS7 in regulating wheat seed germination.

[0009] Preferably, the TaUXS7 gene is TaUXS7-A, TaUXS7-B, or TaUXS7-D.

[0010] An isolated wheat uridine diphosphate xylose synthase, TaUXS7, wherein the amino acid sequence of TaUXS7 is shown in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6.

[0011] Preferably, its nucleotide sequence is shown in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5.

[0012] Recombinant expression vectors.

[0013] Biomaterials.

[0014] A CRISPR / Cas9 gene editing construct comprising sgRNAs targeting conserved regions of the wheat TaUXS7-A, TaUXS7-B, and TaUXS7-D genes, wherein the targeting sequences of the sgRNAs are shown in SEQ ID NO:7 or SEQ ID NO:8.

[0015] A method for detecting wheat TaUXS7 haplotype includes the following steps:

[0016] S1. Extract wheat genomic DNA;

[0017] S2. PCR amplification was performed using the primer pair shown in SEQ ID NO:9 and SEQ ID NO:10 to obtain a fragment containing nucleotide 516 of the third intron of the wheat TaUXS7-A gene;

[0018] S3. The amplification product was digested with the restriction endonuclease Bst BI;

[0019] S4. Determine the haplotype based on the size of the enzyme digestion product fragments: those that are not digested are of type Hap1, and those that are digested to produce two fragments are of type Hap2.

[0020] A method for regulating wheat seed germination by modulating the expression level of the TaUXS7 gene in wheat.

[0021] Beneficial effects

[0022] This invention provides a wheat TaUXS7 nutrient and its application in the wheat seed germination process. It has the following beneficial effects:

[0023] 1. This invention provides a wheat TaUXS7 gene and its application in wheat seed germination. This invention is the first to identify the uridine diphosphate xylose synthase gene TaUXS7, which is related to seed germination, from the whole wheat genome. By creating mutants using CRISPR / Cas9 technology, it was confirmed that the deletion of this gene leads to a decrease in germination rate and inhibition of seedling growth, providing a new gene resource for wheat germination regulation.

[0024] 2. This invention provides a wheat TaUXS7 gene and its application in wheat seed germination. This invention reveals the correlation between natural variation of the TaUXS7 gene and seed germination rate. Based on the single nucleotide polymorphism of the third intron, a BstB I restriction enzyme molecular marker was developed, which can distinguish between two haplotypes and is suitable for the identification of germination characteristics of wheat germplasm resources and molecular marker-assisted breeding. Attached Figure Description

[0025] Figure 1 This figure shows the results of a multi-transcriptome integration analysis of the environmental response during wheat spike germination and sprouting. A represents the identification of differentially expressed genes in five pairs of spike-germination resistant and susceptible materials under different seed water absorption times or under normal and spike germination induction conditions; B represents the comparative analysis of differentially expressed genes under the condition of greatest difference in expression levels between the five pairs of spike-germination resistant and susceptible materials; C represents the identification of differentially expressed genes under different seed water absorption times, salt stress, and ABA treatment conditions; D represents the comparative analysis of conserved differentially expressed genes in the five pairs of spike-germination resistant and susceptible materials with genes responding to germination salt stress and ABA; and E represents the GO enrichment analysis results of 992 common differentially expressed genes.

[0026] Figure 2 This is a diagram analyzing the expression patterns of the TaUXS7 gene. A shows the phylogenetic analysis results of the UXS family members in wheat, Arabidopsis, and rice; B shows the differential expression of 24 UXS gene family members in wheat transcriptome data (orange indicates differentially expressed genes, gray indicates non-differentially expressed genes); C shows the expression levels of three homologous genes of TaUXS7 at different water absorption times during wheat grain germination, before and after salt stress and ABA treatment during germination, and between five pairs of germination-resistant and susceptible materials under different seed water absorption times or normal and spikelet germination induction conditions.

[0027] Figure 3The images show the germination rate and seedling growth phenotypes of the uxs7-cr mutant. A is a schematic diagram of CRISPR / Cas9-mediated TaUXS7 homologous gene editing sites and mutation types (black squares represent exons, horizontal lines represent introns, red underlines indicate PAM sequences, and sequence alignment results marked "WT" indicate wild-type Fielder, with "WT" in parentheses indicating no editing). B shows the germination phenotypes of wild-type Fielder and four uxs7-cr mutant lines after 3 days of seed imbibition and the phenotypes of seedlings after 7 days of imbibition (scale bar = 1 cm). C shows the statistical results of the germination rate of wild-type Fielder and four uxs7-cr mutant lines after 3 days of imbibition and the root and aboveground length of seedlings after 7 days of imbibition (values ​​represent the mean ± standard deviation of three biological replicates; significance was determined by Student's t-test, "*" indicates P < 0.05, "**" indicates P < 0.01, "***" indicates P < 0.001, and "ns" indicates no significant difference).

[0028] Figure 4 This is a haplotype analysis diagram of TaUXS7. A shows the number and types of SNP sites in the homologous gene region of TaUXS7 and its upstream and downstream 2 kb regions; B shows the minor allele frequency (MAF) statistics of SNP sites in the TaUXS7-A / B / D gene region and its upstream and downstream 2 kb regions; C shows a schematic diagram of the two haplotypes formed by the three SNPs located in the intron of TaUXS7-A (vertical lines represent variant sites, and numbers represent the corresponding nucleotide positions); D shows the frequency distribution of haplotypes Hap1 and Hap2 in 166 local varieties and 163 improved varieties; E shows the association analysis results between the TaUXS7-A haplotype and the seed germination rate after 3 days of imbibition (the values ​​are the average germination rates of each material, and the significance was verified by the Mann-Whitney U-test); F shows the sequence comparison of the third intron of TaUXS7-A between the two haplotype materials (the blue box indicates BstB). I represents the identification sequence, with red triangles indicating the base differences between the two haplotypes; G represents the electrophoresis results of the PCR products of Chinese Spring (Hap2 type) and Dwarf Antibody 58 (Hap1 type) after BstB I digestion. Detailed Implementation

[0029] 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Example 1: Identification of members of the wheat TaUXS7 family

[0031] Using the protein sequences of Arabidopsis thaliana AtUXS1 (AT3G53520), AtUXS2 (AT3G62830), AtUXS3 (AT5G59290), AtUXS4 (AT2G47650), AtUXS5 (AT3G46440), and AtUXS6 (AT2G28760), and rice OsUXS1 (LOC_Os03g17230), OsUXS2 (LOC_Os01g21320), OsUXS3 (LOC_Os03g16980), OsUXS4 (LOC_Os01g62020), OsUXS5 (LOC_Os05g29990), and OsUXS6 (LOC_Os07g47700) as query sequences, the BLASTP program was used to search the wheat (Triticum aestivum) reference genome database (IWGSC). (RefSeq v1.1). With an E-value threshold of 1e-10, candidate sequences were initially obtained. The SMART online tool was used to analyze conserved protein domains, retaining sequences containing the UDP-GlcA decarboxylase domain (PFAM: PF16363). After manual correction and redundant sequence removal, 24 UXS family members were identified. Based on phylogenetic relationships, they were divided into three subfamilies.

[0032] Specifically, in the above-described embodiment, the wheat genome consists of three subgenomes: A, B, and D, with high sequence similarity among homologous genes. To avoid phenotypic masking due to functional redundancy of homologous genes, subsequent functional studies need to target genes on all three subgenomes simultaneously. Among the 24 UXS genes identified in this embodiment, only the three partial homologous genes of TaUXS7 (TaUXS7-A, TaUXS7-B, and TaUXS7-D) showed differential expression in the ear-germination-related transcriptome data; therefore, they were selected as candidate genes for further research.

[0033] It should be noted that: the coding region nucleotide sequence of the TaUXS7-A gene is shown in SEQ ID NO:1, and its encoded amino acid sequence is shown in SEQ ID NO:2; the coding region nucleotide sequence of the TaUXS7-B gene is shown in SEQ ID NO:3, and its encoded amino acid sequence is shown in SEQ ID NO:4; the coding region nucleotide sequence of the TaUXS7-D gene is shown in SEQ ID NO:5, and its encoded amino acid sequence is shown in SEQ ID NO:6.

[0034] Example 2: Differential expression analysis of the TaUXS7 gene in the budding transcriptome.

[0035] To overcome the limitations of a single genetic background, transcriptome data from five pairs of wheat varieties (covering China, the United States, and South Korea) with significant differences in pre-sprout resistance were integrated and analyzed at different stages of seed water absorption and under pre-sprout induction conditions. The five pairs of varieties include: the Chinese varieties Jimai 5265 (highly resistant to pre-sprout) and HMC21 (sensitive), Jimai 22 (resistant) and Zhoumai 18 (sensitive); the American varieties UC1110 (resistant) and Anza (sensitive); and the South Korean varieties Keumgang (resistant) and Jokyoung (sensitive).

[0036] Specifically, in the above-mentioned specific embodiments, embryos or whole seeds were collected after 0h, 6h, 12h, and 24h of seed water absorption, as well as under the conditions of induction of ear germination (mature seeds were placed on moist filter paper and cultured at 25℃ and 100% relative humidity for 72 hours). Total RNA was extracted, and a transcriptome library was constructed and then subjected to Illumina high-throughput sequencing. Differentially expressed genes were identified using EdgeR software, with the screening criteria being |log2 fold change| ≥ 1 and a corrected P-value < 0.05.

[0037] The results are as follows Figure 1 As shown in Figure A, the number of downregulated genes was greater than the number of upregulated genes in the budding resistance material, and the number of differentially expressed genes increased significantly after 12 hours of water absorption and under budding induction conditions. Comparative analysis identified 3353 differentially expressed genes conserved across all genetic backgrounds. Figure 1 B). Further integration of transcriptome data from salt stress (150 mM NaCl treatment for 24 hours) and ABA treatment (50 μM ABA treatment for 12 hours) revealed that both significantly suppressed gene transcription. Figure 1 C, and from this, 992 core genes that may synergistically participate in pre-budding resistance and germination period stress response were screened out (C), Figure 1 D). GO enrichment analysis results showed that these genes were significantly enriched in biological processes such as "cell wall polysaccharide catabolism", "D-xylose transport", and "chromatin assembly". Figure 1 E), where the significant enrichment of “D-xylose metabolism” suggests that UXS, a key enzyme in UDP-xylose synthesis, may be involved in regulating wheat ear germination.

[0038] Among the 24 UXS gene family members, only the three partial homologs of TaUXS7 showed significant differential expression across all transcriptome data. Figure 2B). Using Jimai 5265 as material, the expression level of TaUXS7 was detected after seed water absorption for 0 h, 6 h, and 12 h. The results showed that its expression increased in a gradient with the extension of water absorption time. After salt stress treatment (180 mM NaCl, 10 h, 14 h, and 18 h) and ABA treatment (200 μM, 10 h, 14 h, and 18 h), the expression level of TaUXS7 decreased to 17%–23% of the control, respectively. In the high germination rate variety J411, the expression level of TaUXS7 was 4.6 times that of the low germination rate variety HMC21. After ear sprouting induction treatment, the expression level of TaUXS7 increased by 2.8 times, and the expression level in ear sprouting sensitive materials was significantly higher than that in resistant materials. Figure 2 C).

[0039] It is important to note that the expression pattern described above indicates that TaUXS7 is induced during seed germination, while abiotic stress (salt, ABA) inhibits its expression. This is consistent with the expression characteristics of germination-resistant materials. Specifically, TaUXS7 expression levels are lower in germination-resistant materials and higher in germination-sensitive materials. Therefore, TaUXS7 may act as a negative regulator of germination resistance.

[0040] Example 3: Creation of CRISPR / Cas9-mediated TaUXS7 gene editing mutants

[0041] Using the wheat variety "Fielder" as the genetic background, two specific sgRNAs were designed targeting highly conserved regions of the TaUXS7-A, TaUXS7-B, and TaUXS7-D genes. The targeting sequence of sgRNA1 is shown in SEQ ID NO:7, and the targeting sequence of sgRNA2 is shown in SEQ ID NO:8.

[0042] Specifically, in the above-described embodiments, the sgRNA coding sequence was cloned into a CRISPR / Cas9 editing vector (containing a Cas9 expression cassette and selection markers) to construct a dual-target editing vector. The editing vector was introduced into immature embryo cells of the wheat variety "Fielder" using Agrobacterium-mediated transformation. T0 generation transgenic plants were obtained after resistance selection and regeneration culture.

[0043] Hi-TOM high-throughput sequencing technology was used to detect gene editing mutations in T3 generation plants. The results showed that multiple homozygous lines were obtained with simultaneous editing of three partial homologous genes (TaUXS7-A / B / D). Figure 3 A). Sequencing verification was performed on four independent lines (uxs7-cr-1, -2, -3, -4), confirming that frameshift mutations occurred at all three sites, leading to the premature appearance of the stop codon.

[0044] It is important to note that, due to the high degree of repetition in the wheat genome and the sequence similarity of homologous genes, simultaneously editing three partial homologous genes is crucial for overcoming functional redundancy and obtaining a heritable phenotype. The edited lines obtained in this example provide a material basis for subsequent functional studies.

[0045] Example 4: Phenotypic analysis of seed germination and seedling growth of uxs7-cr mutant

[0046] Seed germination experiments were conducted on wild-type Fielder and four uxs7-cr mutant lines. Plump and uniform seeds were selected, surface-sterilized with 2% sodium hypochlorite for 15 minutes, rinsed three times with distilled water, and placed in petri dishes lined with two layers of moistened filter paper. The seeds were then incubated in the dark at 20°C. Germination was defined as the radicle breaking through the seed coat. Germination counts were recorded daily for 7 days.

[0047] The results are as follows Figure 3 As shown in B and 3C, after 3 days of imbibition, the germination rate of the wild type reached 81.5% ± 4.2%, while the germination rates of uxs7-cr-1, -2, -3, and -4 were 22.3% ± 3.1%, 18.6% ± 2.8%, 35.4% ± 4.0%, and 28.7% ± 3.5%, respectively, all significantly lower than that of the wild type (Student's t-test, P < 0.001). After 7 days of imbibition, observation of seedling phenotypes revealed that the mutant seedlings had significantly reduced uniformity and exhibited significant differences in plant height. Figure 3 B). Compared with the wild type, the root and aboveground lengths of the uxs7-cr mutant were both suppressed. Taking uxs7-cr-1 as an example, the root length was 42.6% of the wild type, and the aboveground length was 67.8% of the wild type. Figure 3 C).

[0048] Specifically, in the above-described embodiments, the results indicate that the deletion of the TaUXS7 gene leads to a significant decrease in wheat seed germination rate, while also inhibiting the growth of seedling roots and aboveground parts. This suggests that TaUXS7 not only participates in the germination initiation process but also affects subsequent seedling morphogenesis.

[0049] It is worth noting that root growth is more severely inhibited than that of the aboveground parts. This may be because root tip cells are more sensitive to cell wall remodeling during division and elongation, and insufficient UDP-Xyl supply leads to inhibited hemicellulose synthesis, thereby limiting root cell expansion.

[0050] Example 5: TaUXS7 Haplotype Analysis and Association Between Natural Variation and Seed Germination

[0051] Sequence polymorphisms in the TaUXS7-A, TaUXS7-B, and TaUXS7-D gene regions and their upstream and downstream 2kb sequences were analyzed using the wheat genome variation website (http: / / wheatvariation.com / ). The results showed that the number of SNPs in TaUXS7-A (132) was higher than that in TaUXS7-B (78) and TaUXS7-D (37). Figure 4 A), while the minor allele frequency (MAF) of the TaUXS7-A SNP site is also higher than that of TaUXS7-B / D ( Figure 4 (B), which indicates that the sequence polymorphism of TaUXS7-A is higher than that of TaUXS7-B / D.

[0052] Based on resequencing data from 330 wheat varieties (including 166 local varieties and 163 improved varieties), three naturally occurring SNP sites located in introns 3, 4, and 10 of the TaUXS7-A region were identified. Figure 4 C). Based on the combinations of these SNPs, all materials were divided into two haplotypes: haplotype 1 (Hap1, 156 accessions) and haplotype 2 (Hap2, 174 accessions). Among the 166 local varieties, the Hap2 type accounted for as high as 84.94% (141 accessions); while among the 163 improved varieties, the Hap2 type accounted for only 20.25% (33 accessions). Figure 4 D).

[0053] The seed germination rate of these materials after 3 days of imbibition was statistically analyzed. The results are as follows: Figure 4 As shown in E, there was no significant difference between the two haplotypes in the local varieties (P=0.179). However, in the improved varieties, the germination rate of Hap2 was significantly higher than that of Hap1 (P=0.001).

[0054] Further sequence alignment revealed a difference between T (Hap1) and C (Hap2) at position 516 bp of the third intron of TaUXS7-A. Figure 4 F). This mutation endows the Hap2 material with a specific restriction endonuclease BstB I recognition sequence (TTCGAA). PCR amplification using the primer pairs shown in SEQ ID NO:9 and SEQ ID NO:10 yielded a product length of 621 bp. The Hap1 product, after BstB I digestion, remained a single 621 bp band; the Hap2 product was cleaved into two fragments of 516 bp and 105 bp (F). Figure 4 G).

[0055] It is important to note that the haplotype analysis results above indicate that single nucleotide variants in the third intron of TaUXS7-A are significantly associated with seed germination rates in improved varieties, with the Hap2 type being a haplotype associated with high germination rates. The difference in frequency distribution of this locus between local and improved varieties suggests that it may have been subject to artificial selection during breeding.

[0056] Example 6: Molecular marker detection method for TaUXS7 haplotypes

[0057] This embodiment provides a procedure for detecting the haplotype of the wheat TaUXS7-A gene.

[0058] Step 1: Extract wheat genomic DNA. Take approximately 100 mg of young wheat leaves, grind them into powder using liquid nitrogen, add 400 μL of LCTAB extraction buffer (2% CTAB, 1.4 M NaCl, 20 mM EDTA, 100 mM Tris-HCl (pH 8.0), 1% PVP-40), and incubate at 65°C for 30 minutes. Add 400 μL of chloroform, mix thoroughly, centrifuge at 12000 rpm for 5 minutes at room temperature, transfer the supernatant to a new tube, add an equal volume of isopropanol, mix well, incubate at -20°C for 30 minutes, and centrifuge at 12000 rpm for 5 minutes. Discard the supernatant, add 600 μL of 75% ethanol, mix well, and incubate at 12000 rpm for 3 minutes at room temperature. Repeat once, air dry, and dissolve in 50 μL of ddH2O.

[0059] Step 2: PCR Amplification. PCR reaction system (30 μL): DNA template 1 μL, forward primer (10 μM) 1.2 μL, reverse primer (10 μM) 1.2 μL, dNTP 0.6 μL, 2×phanta Max Buffer 15 μL, Phanta Max Super Fidelity Poiymerase 0.6 μL, ddH2O to 30 μL. The nucleotide sequence of the forward primer is shown in SEQ ID NO:9, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO:10. PCR program: 95℃ pre-denaturation for 3 minutes; 95℃ denaturation for 15 seconds, 59℃ annealing for 15 seconds, 72℃ extension for 40 seconds, for a total of 30 cycles; final extension at 72℃ for 5 minutes. 5 μL of the PCR product was subjected to 1.5% agarose gel electrophoresis to confirm the amplified fragment size was 621 bp.

[0060] Step 3: Enzyme digestion identification. Enzyme digestion reaction system (20 μL): 5 μL PCR product, 2 μL 10×FastDigest Green Buffer, 1 μL BstB I restriction endonuclease, ddH2O to 20 μL. Digest at 37°C for 30 min, then inactivate at 80°C for 5 min. Perform 2% agarose gel electrophoresis (120V, 15 min) on all digested products. Result determination: Hap1 type is characterized by only a 621 bp band; Hap2 type is characterized by two bands, 516 bp and 105 bp.

[0061] The 105bp fragment migrates relatively quickly in a 2.5% agarose gel, requiring an extended electrophoresis time or the use of a high-concentration gel for clear differentiation.

[0062] Example 7: Application of the TaUXS7 gene in regulating wheat seed germination

[0063] Based on the results of the above embodiments, those skilled in the art can realize the application of the TaUXS7 gene in regulating wheat seed germination in the following ways.

[0064] (a) Inhibiting TaUXS7 gene expression to reduce the risk of panicle sprouting.

[0065] The CRISPR / Cas9 dual-target editing vector described in Example 3, containing the sgRNA targeting sequences shown in SEQ ID NO:7 and SEQ ID NO:8, was used. This vector was transformed into wheat immature embryos, and homozygous lines with simultaneous loss-of-function mutations in three partial homologous genes (TaUXS7-A, TaUXS7-B, and TaUXS7-D) were obtained through screening. Figure 3 As shown, the germination rate of this type of mutant was significantly lower than that of the wild type under normal germination conditions. Under rain conditions at harvest (simulating ear germination treatment), the ear germination rate of the mutant material was lower than that of the wild type. Therefore, knocking out the TaUXS7 gene can reduce the risk of wheat ear germination.

[0066] (ii) Enhancing TaUXS7 gene expression to promote seed germination

[0067] A TaUXS7 overexpression vector was constructed. Using cDNA from the wheat variety Fielder as a template, the full-length TaUXS7-A coding region (SEQ ID NO:1) was amplified using specific primers. The amplified product was cloned into a plant expression vector containing a strong promoter. Overexpressing transgenic wheat was obtained by Agrobacterium transformation. After detecting the expression level by qRT-PCR, lines with elevated expression levels were selected for seed germination experiments. The germination rate and final germination rate of the overexpressing lines were higher than those of the wild type.

[0068] Specifically, in the above specific embodiments, the two application directions are aimed at different production needs: inhibiting expression is suitable for areas with high incidence of ear sprouting. By reducing the transcription level of TaUXS7, the supply of UDP-Xyl is reduced, thereby weakening the synthesis of hemicellulose in the cell wall, hindering the moderate softening of the endosperm cell wall, and delaying the time for the radicle to break through the seed coat; promoting expression is suitable for scenarios that require rapid and uniform germination, such as factory seedling production and planting in saline-alkali land.

[0069] It should be noted that the promoters used in the overexpression strategy can be constitutive strong promoters (such as the maize Ubiquitin promoter), or seed-specific promoters can be selected to achieve spatiotemporally specific expression as needed. The specific primers used to amplify the full-length TaUXS7-A coding region can be designed based on SEQ ID NO:1, and can be determined by those skilled in the art without inventive effort. The specific construction methods and transformation steps of the above vectors are conventional techniques in this field and will not be elaborated here.

[0070] Example 8: Exploration of the mechanism of cell wall remodeling in wheat seed germination using the TaUXS7 gene

[0071] Although the focus of this invention is on the function and application of the TaUXS7 gene, this embodiment provides an analysis of the mechanism by which the TaUXS7 gene may participate in cell wall remodeling in order to further illustrate its mechanism of action.

[0072] Specifically, in the above-described embodiments, UDP-Xyl is a precursor for the synthesis of hemicellulose xylan and xyloglucan. The uridine diphosphate xylose synthase encoded by TaUXS7 catalyzes the irreversible decarboxylation of UDP-glucuronic acid (UDP-GlcA) to form UDP-Xyl. During seed germination, the endosperm cell wall needs to undergo moderate softening to facilitate radicle breakthrough. Insufficient UDP-Xyl content leads to inadequate hemicellulose synthesis, resulting in a loose cell wall structure and decreased mechanical strength; conversely, excessive UDP-Xyl content leads to excessive hemicellulose deposition, making the cell wall overly rigid and hindering cell elongation. Therefore, TaUXS7 maintains a balance in cell wall remodeling by regulating the supply of UDP-Xyl, thereby influencing the seed germination process.

[0073] Table 1 summarizes the main SEQ ID NOs and their corresponding sequence types involved in this invention.

[0074] Table 1 Summary of Sequence Information

[0075] 1 Nucleotide (CDS) TaUXS7-A encoding area 2 amino acids TaUXS7-A protein 3 Nucleotide (CDS) TaUXS7-B encoding area 4 amino acids TaUXS7-B protein 5 Nucleotide (CDS) TaUXS7-D encoding area 6 amino acids TaUXS7-D protein 7 Nucleotides sgRNA1 target sequence 8 Nucleotides sgRNA2 target sequence 9 Nucleotides Forward primers (for haplotype detection) 10 Nucleotides Reverse primers (for haplotype detection)

[0076] 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 alterations 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. Application of wheat gene TaUXS7 in regulating wheat seed germination.

2. The application according to claim 1, characterized in that, The TaUXS7 gene is TaUXS7-A, TaUXS7-B, or TaUXS7-D.

3. An isolated wheat uridine diphosphate xylose synthase, TaUXS7, characterized in that, The amino acid sequence of the TaUXS7 is shown in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:

6.

4. The gene encoding TaUXS7 as described in claim 3, characterized in that, Its nucleotide sequence is shown in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:

5.

5. A recombinant expression vector comprising the gene of claim 4.

6. Biological material comprising the recombinant expression vector of claim 5.

7. A CRISPR / Cas9 gene editing construct, characterized in that, The sgRNA contains sgRNAs that target conserved regions of the wheat TaUXS7-A, TaUXS7-B, and TaUXS7-D genes, and the targeting sequences of the sgRNAs are shown in SEQ ID NO:7 or SEQ ID NO:

8.

8. A method for reducing wheat ear germination, characterized in that, The wheat TaUXS7 gene was knocked out using the gene editing construct described in claim 7.

9. A method for detecting wheat TaUXS7 gene haplotypes, characterized in that, Includes the following steps: S1. Extract wheat genomic DNA; S2. PCR amplification was performed using the primer pair shown in SEQ ID NO:9 and SEQ ID NO:10 to obtain a fragment containing nucleotide 516 of the third intron of the wheat TaUXS7-A gene; S3. The amplification product was digested with the restriction endonuclease Bst BI; S4. Determine the haplotype based on the size of the enzyme digestion product fragments: those that are not digested are of type Hap1, and those that are digested to produce two fragments are of type Hap2.

10. A method for regulating wheat seed germination, characterized in that, Regulate the expression level of the TaUXS7 gene in wheat.

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