A method for mining excellent haplotype of wheat drought-related gene TaE2Fa-6A and developing CASP marker

Abstract

This invention belongs to the field of wheat genetics and breeding technology, and discloses a wheat drought resistance-related gene. TaE2Fa‑6A This invention presents a superior haplotype mining and CASP marker development method. It employs bioinformatics methods to systematically analyze the evolutionary relationships, gene structures, and motifs of family proteins. Secondly, under drought stress and other conditions, gene expression analysis is used to screen candidate genes responding to drought and other abiotic stresses. Finally, key candidate genes are identified through transgenic methods. TaE2Fa‑6A The study aimed to investigate the drought resistance function of the gene and elucidate its molecular mechanism regulating drought resistance. Finally, through correlation analysis between gene sequences and phenotypes, the study obtained... TaE2Fa‑6A This invention investigates the effects of different allelic variations on wheat traits such as drought resistance and yield, aiming to identify superior allelic genotypes. This research provides a theoretical foundation for elucidating the mechanism by which wheat E2F / DP transcription factors regulate wheat drought resistance and offers important gene resources and molecular markers for breeding drought-resistant and high-yielding wheat varieties.

Description

Technical Field

[0001] This invention belongs to, but is not limited to, the field of wheat genetics and breeding technology, and particularly relates to a wheat drought resistance-related gene. TaE2Fa-6A A superior haplotype mining and CASP tagging development method. Background Technology

[0002] Wheat, a member of the Poaceae family, is a vital global food source and plays a crucial role in ensuring food security. Drought is one of the major abiotic stress factors affecting wheat production. Under drought conditions, wheat plant growth and development are significantly inhibited, often manifesting as slow growth, reduced grain number per ear, smaller grains, and decreased yield. Therefore, analyzing wheat drought-resistance-related genes and developing corresponding molecular markers is of great significance for breeding drought-resistant varieties.

[0003] In plant growth and development, cell division and DNA replication are crucial biological processes determining tissue growth and organ formation. The E2F or DP transcription factor family is a class of transcriptional regulators widely found in eukaryotes. These proteins typically participate in cell proliferation and differentiation by regulating the expression of cell cycle-related genes. Existing research has shown that E2F or DP transcription factors play important roles in regulating the cell cycle, DNA replication, and cell division, and also have certain regulatory functions in plant responses to abiotic stresses. With the development of plant genomics research, more and more plant genome data are being made public, providing an important data foundation for the systematic mining of functional genes. Currently, in model plants such as Arabidopsis thaliana and rice, members of the E2F or DP family have been systematically identified, and some functional studies have been conducted. However, wheat is a complex allohexaploid crop with a large and complex genome. Compared with model plants, systematic studies of the E2F or DP gene family in wheat remain relatively limited. Existing research mainly focuses on the expression analysis or preliminary functional inference of individual genes; systematic research on the systematic identification, evolutionary relationships, and potential functions of wheat E2F or DP family members under drought stress is still lacking.

[0004] Furthermore, molecular marker-assisted selection (MMR) technology is considered an important means to improve breeding efficiency in wheat drought resistance breeding. However, existing research on the development of molecular markers targeting the relationship between the E2F or DP gene families and wheat drought resistance is still limited, especially in the area of ​​mining allelic variations of key genes and establishing stable detection methods. Therefore, how to screen key genes related to drought resistance from the wheat E2F or DP family, further mine their allelic variations, and develop stable molecular markers has become a pressing technical problem to be solved in the field of wheat drought resistance molecular breeding. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a wheat drought resistance-related gene. TaE2Fa-6A A superior haplotype mining and CASP tagging development method.

[0006] This invention first screens wheat E2F / DP family members using whole-genome analysis; then, it systematically analyzes the evolutionary relationships, gene structures, and motifs of the family proteins using bioinformatics methods; secondly, it screens candidate genes responding to drought and other abiotic stresses through gene expression analysis under drought and other treatments; and finally, it uses transgenic methods to demonstrate the key candidate genes. TaE2Fa-6A The study aimed to investigate the drought resistance function of the gene and elucidate its molecular mechanism regulating drought resistance. Finally, through correlation analysis between gene sequences and phenotypes, the study obtained... TaE2Fa-6A The effects of different allelic variations on wheat traits such as drought resistance and yield can be investigated to identify superior allelic genotypes.

[0007] This invention is achieved through a wheat drought resistance-related gene. TaE2Fa-6A A superior haplotype mining and CASP marker development method, which includes: S1: Identification of family members and phylogenetic analysis of the wheat E2F / DP family; S2: Gene structure, conserved protein motifs, and gene duplication analysis of wheat E2F / DP family members; S3: Analysis of expression patterns of wheat E2F / DP genes under different wheat tissues and abiotic stresses; S4: Sequence characteristics and transcriptional activity analysis of wheat TaE2Fa-6A; S5: Subcellular localization of wheat TaE2Fa-6A protein; S6: Identification of growth, development and drought resistance of wheat TaE2Fa-6A gene overexpression in Arabidopsis thaliana; S7: Drought resistance analysis of wheat TaE2Fa-6A silent wheat lines; S8: Identification of drought resistance in wheat lines overexpressing TaE2Fa-6A; S9: Screening of wheat TaE2Fa-6A interacting proteins; S10: Allelic variant type of wheat TaE2Fa-6A; S11: The relationship between superior allelic variations of the wheat TaE2Fa-6A gene and relative germination rate and agronomic traits; Furthermore, S1 specifically includes: Family member identification and phylogenetic analysis methods: First, download wheat (…) from the EnsemblPlants website (http: / / plants.ensembl.org / ). Triticum aestivum L.), Arabidopsis thaliana ( Arabidopsis thaliana L.) and rice ( Oryza sativa The genome data of L. were obtained; secondly, the HMM configuration file (ID: PF02319) of the E2F / DP family was downloaded from the PFAM database (http: / / pfam.xfam.org); then, HMMER3.0 (http: / / hmmer.org) was used to search and compare the results in the local wheat protein database; further, the obtained protein sequences were used to identify the domains in the NCBI-CDD database (https: / / www.ncbi.nlm.nih.gov / cdd) to ensure the presence or integrity of the domains; finally, the molecular weight, amino acid number, and isoelectric point of TaE2F / DP family proteins were predicted using the ExPASy tool (https: / / web.expasy.org / protparam / ). Multiple sequence alignments were performed on 27 TaE2F / DP, 9 OsE2F / DP, and 8 AtE2F / DP proteins using ClustalW. Subsequently, a phylogenetic tree was constructed using the neighbor-joining (NJ) method (Bootstrap value 1000) with MEGA7 software (https: / / www.megasoftware).

[0008] Family member identification results: Wheat E2F / DP family member identification To identify the whole-genome members of the wheat E2F / DP family, the HMM file was downloaded, and the HMMER program was used to search and compare the results in a local wheat protein database. The NCBI-CDD tool was used to further confirm the retrieved E2F / DP protein domains, identifying a total of 27 wheat E2F / DP proteins. Based on chromosomal location and evolutionary relationships, these 27 family members were renamed, and the naming results are shown in Table 1. Table 1 includes the gene name, gene ID, amino acid length, isoelectric point (PI), protein molecular weight (MW), chromosomal location (Chr), gene location, and subcellular localization information for each family member. The amino acid lengths of wheat E2F / DP family members range from 261 (TaDP1III-10, TaDP1III-11, and TaDP1III-12) to 462 aa (TaE2F4I-6), the PI ranges from 4.58 (TaE2F1I-21) to 9.62 (TaDP1III-10), and the molecular weight ranges from 29.39 (TaDP1III-10) to 50.55 kDa (TaE2F4I-6). Subcellular localization prediction results showed that three TaDP proteins were located in chloroplasts (TaDP1III-10, TaDP1III-11, and TaDP1III-12), three TaDEL proteins (TaDEL1II-22, TaDEL2II-25, and TaDEL2II-27) were located in the nucleus and chloroplasts, and the remaining 21 TaE2F / DP proteins were all located in the nucleus (Table 1). These results suggest that different TaE2F genes may have functional differences.

[0009]

[0010]

[0011]

[0012] Phylogenetic analysis of wheat E2F / DP family members: To reveal the phylogenetic relationships of TaE2F / DP proteins, a neighbor-joining (NJ) phylogenetic tree was constructed using 44 E2F / DP proteins from monocots and dicots (Table 2). TaE2F / DP genes in the same subfamily and adjacent branches represent different homologous copies of the TaE2F / DP member. In this study, each wheat E2F / DP member contained three homologous copies (distributed in genomes A, B, or D). Therefore, 27 homologous genes were identified in the wheat genomes A, B, and D. Based on previous classification rules for E2F / DP proteins in Arabidopsis and rice, the 27 wheat E2F / DP proteins were divided into three subfamilies (I-III). Figure 2Subgroup I contains the most proteins, with 12, while the remaining E2F / DP proteins belong to the other two subgroups (subgroup II has 6, and subgroup III has 9). Figure 2 Compared to the dicotyledonous plant Arabidopsis thaliana, the monocotyledonous plants rice and wheat are more closely related in terms of evolution.

[0013] S2 specifically includes: Methods for analyzing the gene structure, conserved protein motifs, and gene duplication of wheat E2F / DP family members: The gene structure of TaE2F / DP was constructed using GSDS 2.0 (http: / / gsds.gao-lab.org / ). Conserved motifs in the wheat E2F / DP protein sequences were identified using the online MEME tool (http: / / meme-suite.org / ) (the number of motifs was set to 11, and other parameters were left at their default values). Gene duplication analysis was performed using MCScanx (http: / / chibba.pgml.uga.edu / mcscan2 / mcscan.zip), with an E-value set to 1 × 10⁻⁶. -10 Use Circos-0.69 software (http: / / circos.ca / software / ) to draw a gene replication map.

[0014] Analysis of gene structure, conserved protein motifs, and gene duplication of wheat E2F / DP family members: To gain a deeper understanding of the gene structure and conserved domains of wheat E2F / DP family members, a phylogenetic tree was constructed using the amino acid sequences of these members. Figure 3 A). Wheat E2F / DP proteins are divided into three subfamilies (I-III). The exon-intron structure of the wheat E2F / DP gene was plotted using the GSDS 2.0 online tool. Figure 3 B). The exon-intron structures of E2F / DP genes within the same subfamily are highly similar, but differences exist between different subfamilies, manifested in variations in the number and length of introns across the three subfamilies. Most E2F / DP genes in subfamilies II and III contain 8–9 introns, while those in subfamilies I contain 10–13 introns. Eleven conserved motifs were identified in the E2F / DP family, with motif 1, motif 7, and motif 8 being the most conserved and present in all E2F / DP proteins, while other motifs are found only in certain specific proteins. Figure 3 C).

[0015] To further investigate the evolutionary relationship of E2F / DP genes, chromosome localization and gene duplication analysis were performed. Twenty-seven E2F / DP genes from three subtribes were randomly distributed across 21 chromosomes. Twelve E2F / DP genes were distributed across six chromosomes (2A, 2B, 2D, 6A, 6B, and 6D), while the remaining 15 E2F / DP genes were evenly distributed across another 15 chromosomes (Table 1). Furthermore, the number of E2F / DP genes on the chromosomes of wheat A, B, and D genomes was the same. To investigate the relationship between wheat E2F / DP members and gene duplication, collinearity analysis was performed on E2F / DP genes. Twenty-six cloned gene pairs were found, distributed across different chromosomes, including 23 homologous genes (…). Figure 4 ).

[0016] Furthermore, S3 specifically includes: Methods: Gene expression data of the TaE2F / DP gene in different wheat tissues were obtained from the WheatOmics 1.0 database (http: / / 202.194.139.32 / expression / wheat.html) and visualized using TBtools (http: / / cj-chen.github.io / tbtools / ). Chinese spring wheat seedlings were cultured in a light incubator (day / night: 25 / 20℃, 16 / 8h) (GDN-300D-4, Ningbo, China) until the seedlings reached the two-leaf stage. Various stress treatments were applied: 20% PEG6000 (drought), 200 mmol / L NaCl (salt), 180 mmol / L mannitol, and 4℃ (cold). Wheat leaves were collected at 0, 1, 6, 12, and 24 h of stress treatment, with leaves at 0h serving as a control. In a greenhouse at 15-20℃ with a light / dark cycle of 16 / 8h, root, stem, leaf, spike, and grain tissue or organ samples of the wheat variety *Chinese Spring* were collected until 7 days after flowering. All wheat tissue samples were rapidly frozen in liquid nitrogen and stored at -80℃. RNA was then extracted, and tissue-specific gene expression analysis was performed on mature wheat tissue or organ samples using qRT-PCR.

[0017]

[0018] Total RNA was extracted according to the instructions of the TRIzol reagent from Tiangen Pharmaceuticals, and the first strand of cDNA was synthesized using the Takara reverse transcription kit according to the instructions (Table 3).

[0019] Quantitative PCR was performed using a Takara fluorescence quantitative PCR kit (RR820A) and an ABI Pirism 7500 Fast real-time fluorescence analyzer. The reaction system is shown in Table 4.

[0020] Each experiment consisted of three biological replicates, using 23... -ΔΔCt The method is to conduct data analysis.

[0021] Results: Gene expression patterns are often closely related to gene function. To study the expression patterns of the wheat E2F / DP gene in different wheat tissues (roots, stems, leaves, spikes, and grains), data from a public wheat database were submitted to TBtools software for visualization analysis. Figure 5 Most E2F / DP genes are expressed in different wheat tissues, but the expression levels vary. A few E2F genes (TaE2F4I-4, TaE2F4I-6, TaE2F4I-8) are not expressed in any of the five tissues. TaE2F1I, TaE2F2I, TaDP2III, and TaDP3III genes are highly expressed in the spike; TaDEL2II gene is highly expressed in both the spike and root. Five genes (TaE2F1I-19, TaE2F3I-5, TaDP2III-3, TaDP3III-15, and TaDEL2II-27) from three different subgroups and widely expressed in at least four different wheat tissues were randomly selected for further analysis.

[0022] The expression levels of the wheat E2F / DP gene under drought, salt, mannitol, and cold stress were detected using qRT-PCR. Figure 6Under drought stress, the expression levels of the five E2F / DP genes varied at different time points in leaves. The expression levels of TaE2F1I-19 and TaDP3III-15 were significantly upregulated, while the expression levels of TaE2F3I-5, TaDP2III-3, and TaDEL2II-27 were downregulated. TaE2F1I-19 and TaDP3III-15 were initially upregulated, peaking at 1 h, and then downregulated. Under mannitol stress, TaE2F1I-19 expression was significantly upregulated, peaking at 6 h, while the other four genes showed slight downregulation or upregulation. Under drought and mannitol stress, TaE2F1I-19 was significantly upregulated, suggesting that this gene may positively regulate the drought stress response in wheat. Under salt stress, except for TaDP2III-3, the expression levels of the other genes were downregulated. Under cold stress, TaE2F1I-19 and TaDP3III-15 were significantly upregulated, peaking at 12 h, while TaDEL2II-27 peaked at 1 h. Under different stresses, the expression trends of some E2F and DP genes were similar: for example, TaE2F1I-19 and TaDP3III-15 showed an initial upregulation followed by downregulation under drought and mannitol stress. In summary, the wheat E2F / DP gene expression is upregulated under any stress treatment. The typical E2F transcription factor gene TaE2F1I-19, which is significantly upregulated under drought and mannitol stress, will be selected as the subject of further in-depth research.

[0023] S4 specifically includes:

[0024] (1) Amplification and product purification of wheat TaE2Fa-6A gene Based on the cDNA sequence of the wheat variety *Chinese Spring*, specific primers were designed for conserved regions. The cDNA sequence of TaE2Fa-6A was amplified using a high-fidelity enzyme from Novizan. The upstream primer sequence was 5'-AGATCGGGCTTATGTCGGGG-3', and the downstream primer sequence was 5'-GTCTGAACTTGCTGCCCTGATC-3'. The PCR products were purified using a full-gold rapid gel extraction kit.

[0025] (2) Ligation of TaE2Fa-6A gene cloning vector and transformation in E. coli The purified product was ligated into the pEASY-T1 full-gold cloning vector. The optimal total reaction volume was 3-5 μL, with 0.5-4 μL of purified product (1 Kb 20 ng) and 1 μL of pEASY-T1 vector. The volume was brought up to 5 μL with dd H2O, and ligation was performed at 37°C for 5 min.

[0026] Add the ligation product to DH5α competent cells, mix gently, and incubate on ice for 25 min. Heat shock at 42°C for 45 s, then quickly return to ice and incubate for 2 min. Add 700 μL of antibiotic-free LB medium to a centrifuge tube and incubate at 37°C and 200 rpm for 1 h. Centrifuge at 6000 rpm for 1 min to collect the cells, resuspend the bacterial block in 90 μL of supernatant, and spread it onto ampicillin LB medium. Invert the plate and incubate overnight at 37°C for 12–14 h.

[0027] (3) Detection of positive clones by bacterial culture PCR In a clean bench, select several single colonies and place them into 2.0 mL centrifuge tubes. Add 1 mL of ampicillin-resistant LB medium to each tube and incubate at 37°C and 200 rpm for 4-6 h. After the bacterial culture becomes turbid, use it as a template to perform bacterial PCR detection using M13 F / R primers. Select bands that match the size of the target fragment for sequencing.

[0028] (4) Plasmid extraction Plasmids were extracted using the TransGen EM101 plasmid extraction kit according to the EM101 instructions, and the plasmid DNA was stored at -20°C.

[0029] Methods for analyzing the transcriptional activity of wheat TaE2Fa-6A protein: To determine the transcriptional activity of TaE2Fa-6A protein, the full-length TaE2Fa-6A ORF sequence (457 aa) and truncated fragments (including the N-terminus (1-144 aa and 1-334 aa) and the C-terminus (345-457 aa)) were ligated into the pGBKT7 vector via homologous recombination, generating recombinant vectors pGBKT7-TaE2Fa-6A (1-457 aa), pGBKT7-TaE2Fa-6A (1-144 aa), pGBKT7-TaE2Fa-6A (1-334 aa), and pGBKT7-TaE2Fa-6A (345-457 aa). After confirming correct sequencing, the recombinant plasmids were transformed into Y2H yeast cells according to the instructions of the Protech Biotech Y2H competent cells.

[0030] Common wheat is a hexaploid species, and allopolyploidy leads to the production of homologous genes. Therefore, hexaploid wheat often contains three homologous copies. The wheat TaE2F1I-19 gene contains three homologous copies in wheat: TaE2F1I-19, TaE2F1I-21, and TaE2F1I-23. Gene structure analysis shows that there is no difference in the number of introns and exons between TaE2F1I-19 and TaE2F1I-23. Figure 3B), TaE2F1I-21 has fewer introns and exons than TaE2F1I-19 and TaE2F1I-23. Figure 3 B). The intron lengths of TaE2F1I-19, TaE2F1I-21, and TaE2F1I-23 differ significantly: for example, the third intron is the longest in TaE2F1I-19 and TaE2F1I-23 (TaE2F1I-19 > 5 kb, TaE2F1I-23 > 14 kb), while the sixth intron is the longest in TaE2F1I-21 (< 1 kb). Figure 3 (B) Existing research indicates that changes in gene structure may affect gene function, and there may be functional differences among the three TaE2Fa-6 homologs. Cis-acting element analysis shows that the TaE2F1I-19 promoter contains 5 ABRE elements, 1 W-box element, 3 MYB elements, and 5 MYC elements (Table 1); TaE2F1I-21 lacks ABRE elements but has 2 W-box elements (Table 5); and TaE2F1I-23 contains 2 ABRE elements, 1 DRE element, and lacks MYB elements (Table 5). Therefore, it is speculated that TaE2F1I-19 may respond to abiotic stress through the ABA pathway and may be regulated by stress-related transcription factors such as WRKY, MYB, and MYC.

[0031]

[0032] Transcriptional activity analysis results: Using cDNA from leaves of the wheat variety *Traessica rapa* as a template, the ORF (open reading frame) sequence of TaE2F1I-19 (TraesCS6A02G195500) was cloned by RT-PCR. Sequence analysis showed that the full-length ORF was 1377 bp, encoding 458 amino acids, with a molecular weight (MW) of 49.85 kDa and an isoelectric point (PI) of 5.08 (Table 2). Alignment of the TaE2F1I-19 protein sequence with the NCBI database revealed that it is most closely related to wild emmer wheat (AteE2Fa). For ease of understanding, TaE2F1I-19 was renamed TaE2Fa-6A. Overall, the TaE2Fa-6A protein is more closely related to monocotyledonous plants and less closely related to dicotyledonous plants. Figure 7 A). In different plants, most genes are of the E2Fa type. Further analysis of conserved domains in E2F proteins from different plants in the phylogenetic tree revealed that they all possess conserved DBD, DD, and RB domains. Figure 7 B, where DBD: DNA binding domain; DD: dimerizing domain; RB: RB domain).

[0033] For subsequent yeast two-hybrid studies, the transcriptional activity of TaE2Fa-6A needs to be analyzed. Full-length and truncated bait recombinant vectors of TaE2Fa-6A (pGBKT7-TaE2Fa-6A (1-144 aa), pGBKT7-TaE2Fa-6A (1-334 aa), pGBKT7-TaE2Fa-6A (1-457 aa), and pGBKT7-TaE2Fa-6A (335-457 aa)) were constructed. Yeast cells transformed with the recombinant vectors and the control pGBKT7 empty vector, respectively, showed that yeast strains transformed with pGBKT7-TaE2Fa-6A (1-144 aa) and pGBKT7-TaE2Fa-6A (1-334 aa) could only grow on SD / -Trp medium. However, yeast strains transformed with pGBKT7-TaE2Fa-6A (1-457 aa) and pGBKT7-TaE2Fa-6A (335-457 aa) grew well on SD / -Trp / -His / -Ade medium. Figure 7 C). These results indicate that the full-length TaE2Fa-6A possesses transcriptional activation activity, and that the C-terminus (335-457 aa) is the transcriptional activation region.

[0034] S5 specifically includes: Subcellular localization methods: (1) Wheat TaE2Fa-6A Gene adapter fragment amplification according to TaE2Fa-6A Gene and 16318-GFP subcellular localization vector sequence, designed to contain Bam Primers for the HI restriction site. The upstream and downstream primer sequences are 5'-gacgatatctctaga. ggatcc ATGTCGGGGGGCGGCAGG-3' and 5'-gcccttgctcaccatggatccTGGTTGATCCATGCTTGGA-3' were used to amplify wheat vector linkers containing the 16318-GFP vector from plasmids. TaE2Fa-6A Gene fragments are amplified, and the amplified products are purified after amplification.

[0035] (2) Linearization of the 16318-GFP vector Using NEB Company Bam The 16318-GFP vector plasmid was digested with HI endonuclease, and the digestion system is shown in Table 6.

[0036]

[0037] After adding the sample according to the above system, digest the enzyme at 37℃ for 3 h, and then purify the digested product after digestion.

[0038] (3) Carrier recombination reaction Configure the reaction system according to Table 7, react at 37℃ for 30 min to complete the recombinant vector ligation, and place on ice immediately after the reaction is complete.

[0039]

[0040] (4) Identification of recombinant products Recombinant product conversion DH5 α Escherichia coli competent cells were transformed as described in S4(2). After overnight culture, single clones were picked and shaken, and the bacterial culture was sequenced after PCR.

[0041] (5) High concentration TaE2Fa-6A Extraction of subcellular localization recombinant plasmids Use the Tiangen endotoxin-free plasmid small-scale extraction kit (DP118) to extract plasmids according to the instructions. The plasmid concentration should be greater than 1 μg / μL.

[0042] (6) Preparation and transformation of Arabidopsis protoplasts The preparation and transformation of Arabidopsis protoplasts were carried out in accordance with previous studies (Li et al. 2020). After the above process was completed, the protoplasts were cultured in the dark for 48 h and then observed under an Olympus laser confocal microscope (GFP wavelength: 488 nm; chloroplast autofluorescence wavelength: 633 nm).

[0043] Subcellular localization results: As a transcription factor, TaE2Fa-6A protein should be localized in the cell nucleus. To verify this hypothesis, a transient expression vector for TaE2Fa-6A was constructed and transfected into Arabidopsis protoplast cells for transient expression, with an empty vector as a control. Laser confocal microscopy was used to track the GFP fluorescence signal of TaE2Fa-6A protein, showing that the GFP signal in the control was uniformly distributed in the nucleus, cytoplasm, and cell membrane, while the fluorescence signal of TaE2Fa-6A protein was mainly distributed in the nucleus and cytoplasm. Figure 8 A). Therefore, the TaE2Fa-6A protein is mainly located in the nucleus and cytoplasm.

[0044] Furthermore, S6 specifically includes: Method for creating pure lines of Arabidopsis thaliana overexpressing the wheat TaE2Fa-6A gene: Primers containing BamHI and SacI restriction sites were designed based on the TaE2Fa-6A gene and pBI121 overexpression vector sequences. The upstream and downstream sequences of the primers are 5'-acgggggactctaga... ggatccATGTCGGGGGGCGGCAGG-3' and downstream 5'-cgatcggggaaattc gagctc TTATGGTTGATCCATGCTTGGA-3'. The wheat TaE2Fa-6A gene containing the pBI121 overexpression vector adapter was amplified from the plasmid, and the amplification product was purified after amplification. The pBI121 vector plasmid was digested with BamHI and SacI restriction enzymes from NEB. The digestion reaction system consisted of: plasmid N μL (1 μg), CutSmart Buffer 5 μL, Bam 1 μL of HI enzyme and 44-N of RNA-free water were added, and the mixture was digested at 37°C for 3 h. The digestion product was then purified. The TaE2Fa-6A fragment with the adapter was cloned into the pBI121 vector using homologous recombination to generate a recombinant vector. Plasmids were extracted from the correctly sequenced overnight culture and then transformed into Agrobacterium according to the instructions of the Protech GV3101 competent cells.

[0045] Arabidopsis thaliana was infected using the flower-dipping method. After the seeds matured, T0 generation seeds were collected. The T0 generation transgenic Arabidopsis were then screened on MS medium (kanamycin). T1 green positive seedlings grew normally, while negative seedlings failed to grow normally. The positive seedlings were transferred to soil and allowed to mature, at which point T2 generation seeds were collected. The T2 generation seeds were then further screened on MS medium (kanamycin), and the T2 transgenic lines with a segregation ratio of 3:1 were selected and transferred to soil. After maturation, T3 generation single-copy transgenic seeds were collected.

[0046] Methods for determining physiological parameters and analyzing gene expression in transgenic Arabidopsis thaliana: In the survival rate analysis, 40 3-week-old TaE2Fa-6A transgenic Arabidopsis thaliana seedlings were subjected to drought stress (water shortage) for 13 days and rehydrated for 3 days. The number of surviving seedlings was recorded, and the survival rate was calculated. TaE2Fa-6A transgenic Arabidopsis thaliana seedlings were collected before and 8 days after drought stress. The expression levels of stress-related genes (AtDREB2A, AtDREB2B, AtDREB2C) and antioxidant enzyme genes (AtPOD, AtCAT, and AtSOD) were determined by qRT-PCR. Each experiment included three biological replicates.

[0047] Creation of pure lines of wheat TaE2Fa-6A gene overexpressing Arabidopsis: To reveal the function of the wheat TaE2Fa-6A gene, a TaE2Fa-6A overexpression vector was constructed ( Figure 9A) The recombinant vector was infected with Arabidopsis thaliana using the Agrobacterium-mediated flower-dipping method. After harvesting T0 generation seeds, T1 generation lines were obtained by screening on Kansas-resistant MS medium. T1 generation lines were cultured individually, and T2 generation seeds were collected. T2 generation seeds were further screened on antibiotic-containing MS medium, and T2 transgenic lines with a segregation ratio of 3:1 were selected. These were then transferred to soil and cultured until maturity, ultimately yielding homozygous T3 homozygous transgenic lines. RT-PCR was used to detect whether the TaE2Fa-6A gene was successfully overexpressed in Arabidopsis thaliana. Figure 9 B). RT-PCR results showed that wheat TaE2Fa-6A was expressed in Arabidopsis overexpression lines (OE) but not in wild-type (WT), indicating that the wheat TaE2Fa-6A gene was successfully transferred into Arabidopsis.

[0048] Results of physiological index determination and gene expression analysis of transgenic Arabidopsis thaliana: Compared with wild type, after 9 days of culture, the leaves of transgenic Arabidopsis thaliana seedlings were significantly larger than those of wild type. Figure 9 C). Subsequently, the diameter of its rosette leaves was measured, and the diameter of the rosette leaves of the transgenic Arabidopsis was significantly longer than that of the wild type. Figure 9 D). Statistical analysis of the flowering period of transgenic Arabidopsis showed that the flowering period of transgenic Arabidopsis lines was significantly earlier than that of wild-type ( Figure 9 E, Figure 9 F). Statistical analysis of silique length, number, and seed number at maturity showed that the transgenic Arabidopsis had significantly more siliques than the wild-type Arabidopsis, while the length and seed number did not differ significantly. The above analysis indicates that overexpression of the TaE2Fa-6A gene in Arabidopsis affects leaf development, flowering time, and silique number in transgenic Arabidopsis. Before drought treatment, the stomatal distribution and water loss rate of leaves in 3-week-old Arabidopsis under normal water supply conditions were analyzed. The OE Arabidopsis line had fewer stomata than the wild-type Arabidopsis (F). Figure 10 A, among which Figure 10 **Asterisks indicate highly significant differences (P < 0.01); the water loss rate was also lower than that of wild-type Arabidopsis thaliana. Figure 10 B); After 13 days of drought treatment, wild-type lines accumulated large amounts of anthocyanins and severely withered, while drought stress had little effect on transgenic overexpression (OE) lines. Three days after rehydration, although growth was slower, most OE lines continued to grow, while most WT plants died and their leaves faded. Figure 10 C). Furthermore, the survival rate of Arabidopsis thaliana lines overexpressing drought stress was investigated. The survival rate of the OE Arabidopsis thaliana lines was approximately 70.83-75%, significantly higher than the 52.5% of the WT lines. Figure 10 D). These results indicate that overexpression of the wheat TaE2Fa-6A transcription factor gene enhances the drought resistance of overexpressing Arabidopsis lines.

[0049] Furthermore, S7 specifically includes: Methods for silencing the wheat TaE2Fa-6A gene: Common wheat is an allohexaploid, and most wheat genes contain three homologous copies. TaE2Fa-6A, along with its two homologous genes 6B and 6D, exhibit high homology and may have complementary functions. Therefore, using barley stripe mosaic virus-induced gene silencing technology (BSMV-VIGS), TaE2Fa-6A and its two homologous genes 6B and 6D were silenced to verify the function of TaE2Fa-6A under drought stress.

[0050] BSMV silencing vectors used in the VIGS system include pCaBS-α, pCaBS-β, pCaBS-γ, and pCaBS-γ:γ-PDS. Based on the sequences of the TaE2Fa-6A gene and the pCaBS-γ vector, upstream and downstream primers were designed as 5'-AAGGAAGTTTAAAAACAGCACCAGAAGTGCAGTGGGACACC-3' and 5'-AACCACCACCACCGTACTCCAGGAATTCCATATGTCAAGTTTTC-3' (as shown in Table 1). The wheat TaE2Fa-6A gene was amplified from the TaE2Fa-6A plasmid using these primers, and the amplified product was purified after amplification. The pCaBS-γ vector plasmid was digested with NEB's ApaI restriction enzyme, and the digested product was purified after digestion. The purified products were treated with T4 DNA polymerase at room temperature, followed by heat treatment at 75°C for 10 min to inactivate the T4 DNA polymerase. The PCR product (70 ng) treated with T4 DNA polymerase was mixed with the pCaBS-γ linearized vector (200 ng), incubated at 66°C for 2 min, and then slowly cooled to room temperature. The mixture was then transformed into DH5α Escherichia coli. Single clones were picked, cultured, and sequenced. After successful sequencing, plasmids were extracted and transformed into Agrobacterium (EHA105).

[0051] In LB broth containing rifampicin and kanamycin, colonies were incubated overnight at 28°C and 200 rpm with continuous shaking. The activated Agrobacterium-positive culture was centrifuged at 12000 rpm for 1 min, the supernatant was discarded, and the bacterial pellet was resuspended in buffer (0.1 mM AS, 10 mM MES, and 10 mM MgCl2) to allow OD to reach the target concentration. 600The value was 0.7. After placing the resuspension in the dark for 3 hours, tobacco (approximately 6 weeks old) was injected. The underside of the tobacco leaf was gently scratched with a syringe needle, and then the resuspension was slowly injected into the entire leaf using a syringe without the needle. The leaves were cultured in an incubator for 10 days. Leaves with obvious disease were collected and soaked in a buffer solution containing 1% diatomaceous earth (20 mM Na-phosphate buffer, pH 7.2). The leaves were then mechanically inoculated onto the leaves of the two-leaf stage wheat variety Xinong 979. Xinong 979 inoculated with BSMV-TaE2Fa-6A served as the experimental group, while those inoculated with BSMV-PDS (positive control) and BSMV:γ (negative control) served as controls. Symptoms were observed and photographed 10 days after inoculation. Subsequently, the TaE2Fa-6A silent wheat lines were treated with drought (water shortage) for 8 days, and the transcriptional level of TaE2Fa-6A under drought stress was detected using qRT-PCR.

[0052] Physiological parameters and gene expression analysis of transgenic wheat silencing lines: After 8 days of drought stress (water shortage), the expression of BSMV0 and BSMV0 was measured. TaE2Fa-6A Physiological parameters of the plants were measured, and the expression abundance of stress-related genes (TaP5CS, TaDREB1, TaERF3) and antioxidant enzyme genes (TaPOD, TaCAT, and TaSOD(Fe)) was assessed using qRT-PCR. Physiological parameters included the determination of MDA content, proline content, O2.- content, and the activities of antioxidant enzymes (SOD, CAT, and POD).

[0053] Silent results of the wheat TaE2Fa-6A gene: The drought resistance function of the wheat TaE2Fa-6A gene in wheat was further verified using BSMV-VIGS technology. Ten days after inoculation with the virus, BSMV-VIGS was detected in wheat seedlings of the Xinnong 979 variety. PDS All wheat lines showed leucism, while BSMV0 and BSMV... TaE2Fa-6 Wheat strains show symptoms of chlorosis virus (ST). Figure 11 A, among which Figure 11 The error bars represent the standard deviation (mean ± SD, n = 3), and the asterisk indicates significance (*). P < 0.05) or highly significant difference (**) P < 0.01) indicates that the TaE2F1-6A gene was successfully silenced in wheat. Eight days after drought treatment, TaE2F1-6 silenced wheat lines showed severe leaf wilting, while BSMV0 wheat plants exhibited relatively mild symptoms. Figure 11 B); At this time, the expression level of the Ta2F1-6A gene in the silent wheat lines was significantly lower than that in the BSMV0 lines (B); Figure 11C). Under normal water supply conditions, there were no differences in physiological parameters between BSMV0 and TaE2Fa-6 silent wheat lines. Figure 11 D); Under drought conditions, all physiological indicators showed an increasing trend, but the magnitude of the increase varied: for example, proline content increased by approximately 2-3 times compared to before drought stress, while MDA content increased by approximately 1-2 times. Figure 11 D). The MDA and O2.- content in the TaE2Fa-6 silent wheat lines was significantly higher than that in BSMV0, while the proline content and antioxidant enzyme (SOD, CAT, and POD) activity were lower. Figure 11 D). In summary, silencing the TaE2Fa-6 gene in wheat significantly reduced the drought resistance of wheat lines.

[0054] To elucidate the drought resistance mechanism of wheat TaE2Fa-6A, the transcriptional levels of the genes TaP5CS1, TaDREB1, TaERF3, TapOD, TaCAT, and TaSOD (Fe) in TaE2Fa-6 silenced wheat lines under drought stress were further analyzed. Figure 12 In the BSMV-VIGS experiment, the wheat variety Xinong 979 was selected as the virus infection receptor. Six physiological indicators were measured: malondialdehyde (MDA) content, proline content, O2.- content, and the activities of SOD, CAT, and POD enzymes. Error bars represent standard deviation (mean ± SD, n = 3), and asterisks indicate significant (*P < 0.05) or highly significant (**P < 0.01). TaP5CS1, TaDREB1, TaERF3, TapOD, TaCAT, and TaSOD (Fe) are all recognized genes closely related to wheat drought stress tolerance. Compared with the BSMV0 line, the transcription levels of TaP5CS1, TaDREB1, TaERF3, TapOD, TaCAT, and TaSOD (Fe) were reduced in the TaE2Fa-6 silenced wheat line under drought stress.

[0055] Furthermore, S8 specifically includes: Genetic transformation method for TaE2Fa-6A transgenic wheat: The overexpression vector used for transgenic wheat was PLGY-OE3, and the restriction enzyme site was AvrII-SacI. The TaE2Fa-6A gene (containing a 3×FLAG tag at the N-terminus) was ligated into the PLGY-OE3 vector via homologous recombination, with the forward and reverse sequences being 5'-tgttacttctgcagc. cctagg ATGGATTACAAGGATGACG-3' and 5'-acgaacgaaagctct gagctcTTATGGTTGATCCATGC-3'. The recombinant vector plasmid was transformed into Agrobacterium (EHA105), and the Agrobacterium genetically transformed explants into wheat embryos. The genetic transformation process of the wheat variety Fielder involved embryo inoculation, Agrobacterium infection, recovery culture, callus induction, differentiation culture, and regenerated plant culture. The above process yielded T0 generation transgenic plants. Individual T0 plants were subjected to PCR testing, and positive seedlings were transferred to a greenhouse for propagation, eventually reaching the T2 generation of transgenic lines. The genetic transformation of the wheat variety Fielder was completed by the transgenic wheat platform of Researcher Li Genying at the Shandong Academy of Agricultural Sciences.

[0056] Methods for determining physiological indicators and analyzing gene expression in transgenic wheat overexpression lines: Physiological indicators were measured in a light incubator (light / dark: 16 / 8 h, 25℃) under drought stress (water shortage) for 10 days. These indicators included wheat leaf fresh weight, proline content, and MDA content. Simultaneously, qRT-PCR was used to detect the transcriptional levels of drought-stressed genes (TaP5CS1, TaDREB1, TaWRKY33, TaERF1-6A, TaERF3) and antioxidant enzyme genes (TaPOD, TaCAT, and TaSOD(Fe)). Results of physiological parameters of transgenic wheat overexpression lines: To identify the drought resistance function of the wheat TaE2Fa-6A gene in wheat, the wheat TaE2Fa-6A gene was overexpressed in wheat, and its function in regulating drought resistance was further verified in stable TaE2Fa-6A overexpression wheat lines. The expression level of the TaE2Fa-6A gene in wheat lines overexpressing TaE2Fa-6A at the two-leaf stage (OE) was detected using qRT-PCR. Figure 13 A, error bars represent standard deviation (mean ± SD, n = 3), asterisks indicate significant (*P < 0.05) or highly significant (**P < 0.01) differences. Compared with the control, the expression level of the TaE2Fa-6A gene was significantly upregulated in all three overexpression lines, with the OE14 line showing the most significant upregulation. Before drought treatment, the WT and OE lines grew well, with no phenotypic differences observed. After 10 days of drought stress, the WT line wilted severely, while the drought stress had a relatively smaller effect on the wheat OE line. Figure 13 B). The effects of drought stress on the physiological indicators of transgenic wheat showed that the fresh weight of wheat OE lines under drought stress ( Figure 13 C) and proline content ( Figure 13 D) was significantly higher than WT, while the MDA content ( Figure 13E) was significantly lower than WT. In summary, overexpression of the TaE2Fa-6A gene in wheat significantly enhanced the drought resistance of overexpressing wheat lines. To elucidate the drought resistance mechanism of wheat TaE2Fa-6A, the transcriptional levels of TaP5CS1, TaDREB1, TaWRKY33, TaERF1-6A, TaERF3, TapOD, TaCAT, and TaSOD (Fe) genes in TaE2Fa-6A overexpressing wheat lines were further analyzed before and after drought stress. Figure 14 TaP5CS1, TaDREB1, TaWRKY33, TaERF3, TapOD, TaCAT, and TaSOD (Fe) are all recognized genes closely related to wheat drought stress tolerance; our research group's study shows that TaERF1-6A plays an important role in regulating wheat drought resistance. Before drought stress, except for the TaCAT gene, other stress-related genes in the TaE2Fa-6A transgenic wheat lines were significantly upregulated. Under drought stress, all stress-related genes were significantly upregulated, with TaERF1-6A showing the most significant upregulation, with a transcriptional level approximately 24 times that of the control.

[0057] S9 specifically includes: Methods for yeast two-hybrid assay of wheat TaE2Fa-6A protein: Seedlings of drought-resistant wheat variety Jinmai 47 were cultured to the three-leaf stage in a light incubator (light / dark: 25 / 20℃, 16 / 8 h). Drought stress was simulated using 20% ​​PEG6000. Wheat leaves after 0, 6, and 9 h of treatment were submitted to Shanghai Ouyi Biotechnology Co., Ltd. to construct a yeast cDNA library. A bait vector was constructed using the CDS sequence with the TaE2Fa-6A activation region removed, yielding the pGBKT7-TaE2Fa-6A (1-334) recombinant plasmid. This recombinant plasmid was then transformed into yeast cells (AH109). To identify the interacting proteins of TaE2Fa-6A, the yeast cDNA library was screened. Single clones were picked and centrifuged overnight at 200 rpm in 2 mL centrifuge tubes (containing 1 mL SD / -Trp liquid medium). Then, the culture was scaled up by adding 100 μL to 40 mL of SD / -Trp liquid medium and culturing for 15 h to allow OD to increase. 600Approximately 0.8. Centrifuge at 5000 rpm for 5 min at room temperature, discard the supernatant, and resuspend the bacterial block in SD / -Trp liquid medium (4 mL). Add 4 mL of the resuspended bacterial strain, 1 mL of the library bacterial strain, and 45 mL of 2×YPDA to a 2 L sterile Erlenmeyer flask. Incubate at 30℃ with gentle shaking at 50 rpm for 22 h. Subsequently, observe under an optical microscope for the appearance of yeast conjugates. If conjugates are present, proceed to the next step; otherwise, continue shaking incubation. Centrifuge at 4500 rpm for 4 min, discard the supernatant, and resuspend the pellet in 50 mL of 0.5×YPDA. Repeat the above centrifugation and supernatant removal operations, resuspend the bacterial cells in 10 mL of 0.5×YPDA, and spread the resuspended bacterial solution on SD / -Trp / -His / -Leu / -Ade solid medium. Incubate at 30℃ for 4 days. Single colonies were picked and cultured in SD / -Trp / -His / -Leu / -Ade liquid medium at 30°C for 4 days, and positive colonies were selected.

[0058] Yeast plasmids were extracted using the Tiangen Yeast Plasmid Extraction Kit. The collected plasmids were transformed into DH5α *E. coli*, and single clones were picked, cultured, and sequenced. Sequencing results showed that the genes screened from the cDNA library possessed complete CDS sequences. Recombinant plasmids containing pGBKT7-TaE2Fa-6A-19-bait (1-334 aa) and pGADT7-prey were co-transformed into yeast strain Y2H, and cultured on SD / -Trp / -Leu and SD / -Trp / -Leu / -His / -Ade / +X-α-Gal solid media for one-to-one yeast transformation verification. The interaction relationships between proteins were determined based on yeast growth and color reactions.

[0059] Bimolecular fluorescence complementation (BiFC) assay: The interaction between TaE2Fa-6A and TaDPB proteins was further verified in plant cells using a bimolecular fluorescence complementation assay. Gene fragments containing the vector adapters for pCAMBIA1302-nYFP and pCAMBIA1302-cYFP were amplified from the TaE2Fa-6A and TaDPb plasmids, respectively, using specific primers (primers are shown in Appendix Table 1). After amplification, the amplified products were purified. The pCAMBIA1302-nYFP and pCAMBIA1302-cYFP vectors were digested with NEB's BstBI restriction enzyme, respectively. Homologous recombination was used to ligate the TaE2Fa-6A and TaDPb gene fragments containing adapter sequences into the vectors pCAMBIA1302-nYFP and pCAMBIA1302-cYFP, respectively, to obtain the recombinant vectors pCAMBIA1302-nYFP-TaE2Fa-6A and pCAMBIA1302-cYFP-TaDPB. Subsequently, the two recombinant plasmids were co-transformed into Agrobacterium tumefaciens (GV3101) and transiently co-expressed in tobacco. Fluorescence signals were observed using an Olympus laser confocal microscope after 48 h. The presence of YFP fluorescence signals indicated an interaction between the TaE2Fa-6A and TaDPB proteins in tobacco.

[0060] Dual-luciferase assay: To investigate the activation activities of TaE2Fa-6A and TaE2Fa-6A-TaDPb in regulating the promoters of TaMCM5, TaCDC6, and TaCYCA2;1, promoter fragments of the TaMCM5, TaCDC6, and TaCYCA2;1 genes were cloned into the LUC transient expression vector pGreen II 0800 via homologous recombination. This vector contains the Renilla luciferase (REN) gene driven by the CaMV35S promoter, resulting in the recombinant vectors pGreen II 0800-TaMCM5, pGreen II 0800-TaCDC6, and pGreen II 0800-TaCYCA2;1. The TaE2Fa-6A and TaDPb CDS sequences were cloned into pGreenII 62-SK via homologous recombination, resulting in the recombinant vectors pGreenII 62-SK-TaE2Fa-6A and pGreenII 62-SK-TaDPb. The plasmids pGreen II 0800-TaMCM5, pGreen II 0800-TaCDC6, pGreen II 0800-TaCYCA2;1, pGreenII 62-SK-TaE2Fa-6A, and pGreenII 62-SK-TaDPb were transformed into Agrobacterium (strain: GV3101 containing pSoup-19). After centrifugation and mixing in the specified proportions, the Agrobacterium was resuspended in osmosis buffer (150 mm Acetosyringone, 10 mm MgCl2, and 10 mm MES, pH 5.6) and injected into leaves of *Nicotiana benthamiana* for transient expression. Using a dual-luciferase assay system, the ratio of LUC to REN fluorescence was measured using a microplate reader 2 days after transfection, with at least three biological replicates for each combination.

[0061] Protein Interaction Results: To further investigate the potential molecular mechanism of wheat TaE2Fa-6A's involvement in drought stress response, yeast two-hybrid technology was used to screen for proteins that may interact with TaE2Fa-6A in a wheat cDNA library. Some yeast colonies could grow on two- and four-deficient media, but after transforming E. coli with plasmids extracted by shaking, they could not grow on ampicillin medium, possibly due to contamination or false positives. Therefore, after ruling out these possibilities, single clones that grew normally on ampicillin medium were selected for sequencing. After removing proteins encoding repetitive genes, proteins that may interact with TaE2Fa-6A were finally identified and annotated in the NCBI database. Five proteins that may interact with TaE2Fa-6A were ultimately obtained: TaDPb, TaG6PE, TaVDAC, TaCBSX3, and TaOEP24. The genes encoding the interacting proteins screened from the cDNA library have complete ORFs and can be ligated into pGADT7 to construct prey vectors for yeast two-hybrid inversion verification. Five pGADT7 recombinant vectors were co-transformed with pGBKT7-TaE2Fa-6A (1-334 aa) into Y2H yeast strains. All transformed strains were able to grow on SD / -Trp / -Leu and SD / -Trp / -Leu / -His / -Ade / +X-α-Gal media. Figure 15A). In summary, the wheat TaE2Fa-6A protein in yeast may interact with five candidate interacting proteins. Among the five interacting proteins TaDPb, TaG6PE, TaVDAC, TaCBSX3, and TaOEP24, TaDPb is a member of the wheat E2F / DP transcription factor family. Studies have shown that in Arabidopsis, E2Fa can form a dimer with DP protein to regulate target genes in response to abiotic stress. These target genes mainly include WRKY transcription factor family genes (AT5G1089 and AT5G26170), AP2 / ERF transcription factor family genes (AT1G75490, AT2G40340, and AT3G11020), NAC transcription factor family genes (AT3G01600), and bZIP transcription factor family genes (AT2G36270). It can be inferred that in wheat, TaE2Fa-6A may interact with TaDPb to regulate the transcriptional level of drought stress response genes in order to cope with drought stress; glucose-6-phosphate-1-epimerase (G6PE) is an isomerase in sugar metabolism that can catalyze changes in the α or β conformation of glucose-6-phosphate and plays a role in maintaining normal cellular metabolic pathways. Under drought stress, plant G6PE may enhance plant drought resistance by maintaining normal cellular metabolism; VDAC proteins are widely present in the mitochondrial extramedullary model of animals and plants and play a crucial role in regulating plant development and coping with abiotic stress; CBSX3 is a regulator of ROS production in mitochondria and plays an important role in regulating plant development and redox systems; studies have shown that the transcriptional level of OEP genes is significantly increased under ABA, heat, and drought stress, and overexpression of the wheat TaOEP16-2-5B gene in Arabidopsis thaliana can enhance the heat tolerance and drought resistance of overexpressing Arabidopsis thaliana lines. Therefore, it is speculated that the wheat TaE2Fa-6A protein may interact with five proteins involved in sugar metabolism, ion transport, redox reactions and the ABA hormone pathway, thereby regulating wheat drought resistance.

[0062] TaDPb protein is an indispensable cofactor in the regulation of downstream target genes by TaE2Fa-6A. TaDPb was chosen as the subject of further research. Furthermore, the interaction between TaE2Fa-6A and TaDPb was verified using BiFC. YFP fluorescence was observed when TaE2Fa-6A and TaDPb were co-transferred to tobacco, while no YFP fluorescence was observed in the control group, indicating an interaction between TaE2Fa-6A and TaDPb in tobacco cells. Figure 15 B).

[0063] Furthermore, S10 specifically includes: Methods for analyzing wheat TaE2Fa-6A gene sequence polymorphism: To analyze the polymorphism of wheat TaE2Fa-6A gene, the TaE2Fa-6A gene sequences of eight wheat varieties (Claire, Paragon, Cadenza, Chinese Spring, Landmark, Weebil, Mace, and Julius) were downloaded from the Ensemble Plants database, and gene sequence alignment analysis was performed using DNAMAN.

[0064] Development method of wheat CAPS molecular marker: After polymorphism analysis, a CAPS molecular marker was designed and developed based on the -727-bp (SNP, C / T) in the TaE2Fa-6A promoter region. The upstream and downstream primer sequences were 5'-TCGCGACAAGGACTTCAAAACTAG-3' and 5'-GCTGAATTTCGAATGGGCCACT-3', respectively. To verify the effectiveness of the primers, PCR amplification was performed using DNA templates from sequenced varieties such as Chinese Spring, Weimai 8, Jing 411, Taishan 1, and Jingdong 8 to check whether the band size was consistent with the expectation. The PCR reaction system is shown in Table 8.

[0065] The PCR program was as follows: the first cycle was 95℃ for 5 min, 95℃ for 30 s, 62℃ for 30 s, and 72℃ for 1 min; then 95℃ for 30 s, 60℃ for 30 s, and 72℃ for 1 min, for 35 cycles, with a final cycle of 72℃ for 5 min. After one round of PCR and treatment of the PCR products with NlaIV restriction endonuclease, the effects on wheat were investigated. TaE2Fa-6A Genotyping based on allelic variant type (C locus) TaE2Fa-6A PCR products can be digested with enzymes, while those at the T site cannot. The enzyme digestion system consists of: 0.1 μL NlaIV restriction enzyme, 5 μL Cutsmart buffer, 3 μL PCR product, and 1.9 μL RNA-free water.

[0066] Results of TaE2Fa-6A allelic variation in wheat: Polymorphism analysis of the TaE2Fa-6A gene sequence (covering the promoter and coding regions) from eight wheat varieties in the database revealed nine SNPs and two 1-bp InDels in the promoter region of the TaE2Fa-6A gene. The wheat TaE2Fa-6A gene consists of 14 exons and 13 introns. In the eight wheat varieties, the exon sequences remained unchanged, while the intron sequences showed variations; the first intron contained two SNPs; the second intron contained one SNP; the third intron contained four SNPs; two 1-bp InDels and two 2-bp InDels. TaE2Fa-6A formed five haplotypes in the eight wheat varieties. Studies have shown that mutations in the CTCC element in the promoter sequence affect the expression of the DYT1 gene in the tapetum, thereby affecting anther development and ultimately yield traits (Zhou et al. 2017). In the PlantCare database, a CTCC cis-acting element was predicted in the promoter region of the wheat TaE2Fa-6A gene, and a SNP at -727 resulted in a mutation (CTTC) in this element. It can be inferred that mutations in the CTCC element may affect the expression of the wheat TaE2Fa-6A gene, thus influencing yield traits. Therefore, based on this SNP mutation, the CAPS molecular marker was developed, classifying the five haplotypes into two allelic variant types, named Hap-C (CTCC) and Hap-T (CTTC), respectively. Figure 16 , Figure 17 A, where Figure 17 The varieties listed are: Taishan No. 1, Jingdong No. 8, Jing 411, Weimai No. 8, Motuo Wheat, Bianba Spring Wheat, White Mang Wheat, Wujiangzhuo, Muzongzhuoga, Zangdong No. 4, Shigatse No. 54, Shigatse No. 8, Shanmai, Yizhimai, Dabaimai (Qinghai), Ga Laohan, Hongmangmai, Dabaimai (Gansu), Baiqitou, Baimazha, Laotutou, Ganmai No. 8, Gaoyuan 506, and Qingchun 28. Using DNA from Taishan No. 1, Jingdong No. 8, Jing 411, Weimai No. 8, Motuo wheat, Bianba spring wheat, white awn wheat, Wujiangzhuo, Muzongzhuoga, Zangdong No. 4, Shigatse No. 54, Shigatse No. 8, mountain wheat, Yizhi wheat, white wheat, Ga Laohan, red awn wheat, white wheat, Baiqitou, Baimazha, Laotutou, Ganmai No. 8, Gaoyuan 506, and Qingchun 28 as templates, molecular marker amplification PCR was performed. The PCR product length was 1144 bp. The PCR product of the allelic variant containing CTCC elements was successfully digested with enzymes, and the digestion products were 698 bp and 447 bp, respectively. Figure 17 B), achieving the expected results; however, the PCR products of allelic variants containing the mutant element "CTTC" could not be digested by enzymes. Figure 17B) confirmed the effectiveness of the CAPS molecular marker.

[0067] S11 specifically includes: Methods for determining relative germination rate and classifying drought resistance levels in wheat with different allelic variants: The drought resistance of 121 wheat varieties (lines) at the germination period was analyzed. The wheat seed treatment process and relative germination rate calculation were based on previous research (Yu Ming 2022). According to GB / T21127-2007 Technical Specification for Identification and Evaluation of Drought Resistance in Wheat, the drought resistance level of relative germination rate was divided into 5 categories: very weak (0-29.9%), weak (30-49.9%), moderate (50-69.9%), strong (70-89.9%), and very strong (90-100%).

[0068] From 2021 to 2022, the recombinant inbred line population was planted in Yangling District, Xianyang City, Shaanxi Province (34°16′N, 108°4′E). Compound fertilizer and nitrogen fertilizer were applied before sowing. Each line was planted in a 1 m row with a plant spacing of 6 cm and a row spacing of 25 cm. The sown land was irrigated using a flood irrigation method in December. For each line in the recombinant inbred line population, five plants were randomly selected to investigate the following parameters: number of grains per spike (GN), number of effective spikelets (ESN), 1000-grain weight (TKW), spike length (SL), plant height (PH), flag leaf length (FLL), flag leaf width (FLW), harvest index (HI), heading date (HD), and flowering date (FD).

[0069] Genotyping was performed on all wheat populations using CAPS molecular markers. The correlation between allelic combinations and agronomic traits in the recombinant inbred line population was analyzed using IBM SPSS software (*p < 0.05; **p < 0.01). A natural population consisting of 262 wheat microcore germplasms and 121 wheat varieties (lines) was used to analyze the geographical distribution of allelic combinations and their frequency during the breeding years.

[0070]

[0071] Relationship between TaE2Fa-6A allelic variant types and relative germination rate in wheat: Based on the developed CAPS molecular markers, TaE2Fa-6A genotyping was performed on 121 varieties (lines) from a natural population. The average relative germination rate of wheat varieties (lines) containing the Hap-C allelic variant (54.01%) was higher than that of wheat varieties (lines) containing the Hap-T allelic variant (49.93%). Furthermore, wheat varieties (lines) containing the Hap-C allelic variant had a higher number of varieties in the extremely high (HR) and high (R) drought resistance categories than those containing the Hap-T type (Table 9), indicating that Hap-C can improve drought resistance during wheat germination.

[0072] Relationship between TaE2Fa-6A allelic variants and agronomic traits in wheat: Based on the developed CAPS molecular markers, TaE2Fa-6A genotyping was performed on two sets of recombinant inbred lines. TaE2Fa-6A was classified into two allelic variants: Hap-C (CTCC) and Hap-T (CTTC), with a small number of heterozygous variants, Hap-T / C (CTCC / CTTC). Figure 17 C). Subsequently, association analysis was performed on the two allelic variation types in the two sets of recombinant inbred lines with agronomic traits (grain number per spike (GN), effective spikelet number (ESN), 1000-grain weight (TKW), spike length (SL), plant height (PH), flag leaf length (FLL), flag leaf width (FLW), harvest index (HI), heading date (HD), and flowering date (FD)). Preliminary results showed significant associations between the two allelic variation types and GN, PH, HI, and FD. In the two sets of recombinant inbred lines, lines containing the Hap-C (CTCC) allelic variation type had more GN, lower PH, higher HI, and shorter HD and FD (Table 10). Lines containing the Hap-T allelic variation type had higher TKW. In conclusion, Hap-C is a relatively superior allelic variation type.

[0073]

[0074] Based on the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solution to be protected by this invention are as follows: This invention first screens wheat E2F / DP family members using whole-genome analysis; then, it systematically analyzes the evolutionary relationships, gene structures, and motifs of the family proteins using bioinformatics methods; secondly, it screens candidate genes responding to drought and other abiotic stresses through gene expression analysis under drought and other treatments; and finally, it uses transgenic methods to demonstrate the key candidate genes. TaE2Fa-6A The study aimed to investigate the drought resistance function of the gene and elucidate its molecular mechanism regulating drought resistance. Finally, through correlation analysis between gene sequences and phenotypes, the study obtained... TaE2Fa-6AThis invention investigated the effects of different allelic variations on drought resistance and yield in wheat, thereby identifying superior allelic genotypes and developing CAPS markers. This research provides a theoretical foundation for elucidating the mechanism by which wheat E2F / DP transcription factors regulate wheat drought resistance and offers important genetic resources for breeding drought-resistant and high-yielding wheat varieties.

[0075] To address the current shortcomings in research on wheat E2F / DP family genes, including unclear key drought-resistance genes, ambiguous molecular mechanisms, and a lack of superior haplotypes and functional markers, this invention provides a wheat drought-resistance-related gene. TaE2Fa-6A An excellent haplotype mining and CAPS marker development method was developed. Twenty-seven wheat E2F / DP family members were identified through genome-wide identification, and key genes showing significant responses to drought stress were screened using expression profiling. TaE2Fa-6A Cloning, subcellular localization, transcriptional activity assays, Arabidopsis and wheat genetic transformation, VIGS silencing, yeast two-hybrid assays, BiFC assays, and dual-luciferase assays confirmed that TaE2Fa-6A is located in the cell nucleus and possesses transcriptional activation function, positively regulating drought resistance by influencing stomatal development, osmotic regulation, and antioxidant capacity. Based on the promoter-727 bp C / T polymorphism site, a CAPS molecular marker was developed, and association analysis showed that superior haplotypes were significantly associated with high drought resistance and excellent yield traits. This invention systematically elucidates for the first time... TaE2Fa-6A The molecular mechanisms of drought resistance provide gene resources and functional markers that can be directly used for breeding, offering efficient technical support for molecular breeding of drought-resistant and high-yield wheat.

[0076] (1) The expected benefits and commercial value of the technical solution of this invention after transformation are as follows: The superior haplotype TaE2Fa-6A and CAPS functional marker obtained in this invention can be directly applied to drought-resistant and high-yield molecular breeding of wheat. This enables high-throughput, low-cost, and precise genotype screening in early generations of breeding, significantly shortening the breeding cycle and improving selection efficiency. Wheat varieties bred using this marker can significantly improve emergence rate, tiller number, thousand-grain weight, and yield stability in arid and semi-arid regions, reducing the risk of drought-induced yield reduction. The related markers and genes can be used for industrial applications such as variety rights protection, molecular-assisted breeding (MAS), and gene editing improvement, possessing significant food security and market promotion value.

[0077] (2) The technical solution of this invention fills a technical gap in the industry both domestically and internationally: This invention marks the first time that the whole genome of the E2F / DP transcription factor family has been systematically identified in hexaploid wheat; the first time that TaE2Fa-6A has been cloned and confirmed as an important positive regulatory gene for drought resistance in wheat, elucidating its molecular mechanism of regulating drought resistance through cell cycle and stress pathways; and the first time that superior haplotypes of this gene have been discovered and CAPS functional molecular markers have been developed, filling the research gap in the field of molecular breeding for drought resistance of the wheat E2F / DP family.

[0078] (3) The technical solution of the present invention solves a technical problem that people have long wanted to solve but have never been able to solve successfully: This invention solves the long-standing problem in wheat drought-resistant breeding where "drought resistance and yield are difficult to improve in a coordinated manner"; it addresses the challenges of complex gene families in hexaploid wheat and difficulties in screening key drought-resistant genes; it resolves the industry pain point of drought-resistant phenotypic identification being greatly affected by the environment and having low selection efficiency; and it also solves... TaE2Fa-6A Key technical challenges include unknown function, unclear mechanism, and lack of available molecular markers.

[0079] (4) The technical solution of the present invention overcomes technical bias: Traditional research has largely considered the E2F / DP family to regulate only cell division and growth, with insufficient attention paid to its role in abiotic stresses, especially drought. This invention, through systematic functional verification, demonstrates that TaE2Fa-6A not only participates in growth and development but also positively regulates wheat drought resistance. This breaks the technical bias of viewing it merely as a cell cycle regulator, broadening the genetic resources and theoretical understanding for wheat stress resistance breeding. Attached Figure Description

[0080] Figure 1 This is a flowchart of a method for mining superior haplotypes of the wheat drought-resistant gene TaE2Fa-6A, provided in an embodiment of the present invention. Figure 2 This is a neighbor-joining phylogenetic tree constructed from 44 E2F / DP proteins from Arabidopsis thaliana, rice, and wheat, as provided in this embodiment of the invention. Figure 3 This invention provides an analysis of the phylogenetic relationships (A), exon-intron structure (B), and distribution of conserved protein motifs (C) of wheat E2F / DP members. Figure 4 This is the wheat E2F / DP gene collinearity relationship provided in the embodiments of the present invention; Figure 5 This is the expression profile of 27 wheat E2F / DP genes in root, stem, leaf, spike and grain tissues provided in the embodiments of the present invention; Figure 6 Expression profiles of wheat E2F / DP gene under drought, salt, mannitol and cold stress treatments; Figure 7 This invention provides an analysis of the evolutionary relationship, protein domains, and transcriptional activity of the wheat TaE2Fa-6A protein. A: Phylogenetic tree constructed from the amino acid sequences of wheat TaE2Fa-6A and 10 other plant E2F proteins; B: Distribution of protein domains of wheat TaE2Fa-6A and 10 other plant E2F proteins; C: Transcriptional activity analysis of wheat TaE2Fa-6A protein. Figure 8 This invention provides subcellular localization and tissue-specific expression analysis of wheat TaE2Fa-6A protein, A: Subcellular localization of wheat TaE2Fa-6A protein; B: Tissue-specific expression analysis of wheat TaE2Fa-6A gene. Figure 9 The wheat provided in the embodiments of the present invention TaE2Fa-6A Functional analysis of transgenic Arabidopsis thaliana: A: Map of the pBI121 overexpression vector containing the CaMV35S promoter; restriction enzyme sites are BamHI and SacI; B: RT-PCR verification of the Arabidopsis thaliana TaE2Fa-6A transgenic line; C: Phenotypic analysis of transgenic Arabidopsis thaliana seedlings; D: Statistical analysis of rosette leaf diameter during this period; E: Phenotypic analysis of transgenic Arabidopsis thaliana during flowering; F, G, H, and I represent the statistical analysis of flowering period, silique length, silique number, and seed number, respectively. Figure 10 This is a description of wheat under drought stress provided in an embodiment of the present invention. TaE2Fa-6A Functional analysis of Arabidopsis thaliana overexpression: A: Stomatal distribution in leaves of Arabidopsis thaliana lines; B: Statistical analysis of water loss rate in Arabidopsis thaliana leaves; C: Phenotype of Arabidopsis thaliana under drought stress; D: Statistical analysis of survival rate of Arabidopsis thaliana lines. Figure 11 This study focuses on the phenotypic analysis and physiological index determination of TaE2Fa-6 silent wheat lines under drought stress. A: Phenotypic characteristics of wheat leaves 10 days after inoculation with the virus; B: Phenotypic analysis of TaE2Fa-6 silent lines under drought stress; C: Detection of TaE2Fa-6 line silencing efficiency; D: Determination of physiological indexes of TaE2Fa-6 silent wheat lines. Figure 12 It represents the transcriptional levels of the TaP5CS, TaDREB2A, TaERF3, TapOD, TaCAT, and TaSOD (Fe) genes in the TaE2Fa-6 silent wheat line under drought stress. Figure 13This study presents a functional analysis of TaE2Fa-6A transgenic wheat under drought stress. A: qRT-PCR validation of TaE2Fa-6A transgenic wheat lines; B: Drought resistance phenotype analysis of transgenic wheat seedlings; C: Statistical comparison of wheat leaf fresh weight after drought stress; D: Statistical comparison of wheat leaf proline content after drought stress; E: Statistical comparison of wheat leaf MDA content after drought stress. Figure 14 This study compares the transcriptional levels of TaP5CS1, TaDREB1, TaWRKY33, TaERF1-6A, TaERF3, TapOD, TaCAT, and TaSOD (Fe) genes in TaE2Fa-6A overexpressing wheat lines before and after drought stress. Figure 15 This includes point-to-point verification of wheat TaE2Fa-6A's interaction with yeast and bimolecular fluorescence complementation (BiFC) assay for its interaction with TaDPb; A: Point-to-point confirmation of the interaction between yeast two-hybrid and candidate proteins; B: Verification of the interaction between TaE2Fa-6A and TaDPb in tobacco using BiFC. Figure 16 This refers to the sequence information of the TaE2Fa-6A gene in different wheat varieties; Figure 17 This is a schematic diagram of agarose gel electrophoresis of wheat TaE2Fa-6A sequence variation (A), molecular marker detection variation (B), and genotyping (C). Detailed Implementation

[0081] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0082] like Figure 1 As shown in the embodiment of the present invention, a method for mining superior haplotypes of the wheat drought-resistance related gene TaE2Fa-6A includes: S101, obtain genomic data of wheat, Arabidopsis thaliana and rice, and use E2F or DP family to perform homology search on wheat protein database to obtain wheat E2F or DP family candidate protein sequences. S102, perform domain identification on candidate protein sequences, screen for proteins containing E2F or DP domains, and identify wheat E2F or DP family members. S103, perform multiple sequence alignment on the E2F or DP family members and construct a phylogenetic tree, and determine the target transcription factor gene TaE2Fa-6A based on the phylogenetic relationship; S104, the TaE2Fa-6A gene was sequence cloned and its open reading frame sequence was obtained; S105, the transcriptional activation region of TaE2Fa-6A protein was determined by transcriptional activity analysis; S106, the localization characteristics of TaE2Fa-6A protein in the nucleus and cytoplasm were determined by subcellular localization analysis; S107 was used to verify the function of TaE2Fa-6A in regulating plant drought resistance through transgenic overexpression and gene silencing experiments.

[0083] The E2F or DP family used in S101 provided in this embodiment of the invention is the HMM model with the PFAM database number PF02319.

[0084] The phylogenetic tree provided in this embodiment of the invention is constructed using the adjacency method, and the phylogenetic relationships are determined through 1000 bootstrapping tests.

[0085] The transcriptional activity analysis provided in this embodiment of the invention is achieved by constructing full-length TaE2Fa-6A protein and truncated protein bait vectors and detecting their transcriptional activation ability in a yeast system.

[0086] This invention provides a method for developing CAPS molecular markers based on the TaE2Fa-6A gene, comprising the following steps: Step 1: Polymorphism analysis was performed on the promoter sequences of the TaE2Fa-6A gene from multiple wheat varieties to identify single nucleotide mutation sites; Step 2: Design specific PCR primers based on the mutation site to amplify the TaE2Fa-6A gene promoter fragment; Step 3: Perform restriction endonuclease digestion on the PCR amplification products; Step 4: Classify the TaE2Fa-6A allelic variants based on the differences in the size of the enzyme digestion product fragments.

[0087] The mutation site provided in this embodiment of the invention is a single nucleotide polymorphism located at position -727 of the promoter region of the TaE2Fa-6A gene.

[0088] The PCR amplification fragment provided in this embodiment of the invention is 1144 bp in length.

[0089] The restriction endonuclease provided in this embodiment of the invention is NlaIV.

[0090] This invention provides a method for screening wheat haplotypes with excellent drought resistance, comprising the following steps: (1) Genotyping of wheat populations using the CAPS molecular markers described in claim 5; (2) Determine the relative germination rate of wheat materials under drought conditions during the germination period; (3) Classify wheat materials into drought-resistant grades based on their relative germination rate; (4) Correlation analysis was performed on different allelic variation types and agronomic traits to screen for the TaE2Fa-6A haplotype with excellent drought resistance.

[0091] The superior haplotype provided in this embodiment of the invention is the TaE2Fa-6A allelic variant containing the CTCC cis-acting element in the promoter sequence.

[0092] The present invention provides a method for mining superior haplotypes of wheat drought-resistance-related gene TaE2Fa-6A and developing CASP markers, the method comprising: S1: Identification of family members and phylogenetic analysis of the wheat E2F / DP family; S2: Gene structure, conserved protein motifs, and gene duplication analysis of wheat E2F / DP family members; S3: Analysis of expression patterns of wheat E2F / DP genes under different wheat tissues and abiotic stresses; S4: Sequence characteristics and transcriptional activity analysis of wheat TaE2Fa-6A; S5: Subcellular localization of wheat TaE2Fa-6A protein; S6: Identification of growth, development and drought resistance of wheat TaE2Fa-6A gene overexpression in Arabidopsis thaliana; S7: Drought resistance analysis of wheat TaE2Fa-6A silent wheat lines; S8: Identification of drought resistance in wheat lines overexpressing TaE2Fa-6A; S9: Screening of wheat TaE2Fa-6A interacting proteins; S10: Allelic variant type of wheat TaE2Fa-6A; S11: The relationship between superior allelic variations of the wheat TaE2Fa-6A gene and relative germination rate and agronomic traits; Identification of wheat E2F / DP family members: To identify the whole-genome members of the wheat E2F / DP family, the HMM file was downloaded, and the HMMER program was used to search and compare the results in the local wheat protein database. The NCBI-CDD tool was used to further confirm the retrieved E2F / DP protein domains, identifying a total of 27 wheat E2F / DP proteins. Based on chromosomal location and evolutionary relationships, the 27 family members were renamed, and the naming results are shown in Table 1. Table 1 includes the gene name, gene ID, amino acid length, isoelectric point (PI), protein molecular weight (MW), chromosomal location (Chr), gene location, and subcellular localization information for each family member. The amino acid lengths of wheat E2F / DP family members range from 261 (TaDP1III-10, TaDP1III-11, and TaDP1III-12) to 462 aa (TaE2F4I-6), the PI ranges from 4.58 (TaE2F1I-21) to 9.62 (TaDP1III-10), and the molecular weight ranges from 29.39 (TaDP1III-10) to 50.55 kDa (TaE2F4I-6). Subcellular localization prediction results showed that three TaDP proteins were located in chloroplasts (TaDP1III-10, TaDP1III-11, and TaDP1III-12), three TaDEL proteins (TaDEL1II-22, TaDEL2II-25, and TaDEL2II-27) were located in the nucleus and chloroplasts, and the remaining 21 TaE2F / DP proteins were all located in the nucleus (Table 1). These results suggest that different TaE2F genes may have functional differences.

[0093] Phylogenetic analysis: To reveal the phylogenetic relationships of TaE2F / DP proteins, a neighbor-joining (NJ) phylogenetic tree was constructed using 44 E2F / DP proteins from monocots and dicots (Table 2). TaE2F / DP genes in the same subtribe and adjacent branches represent different homologous copies of the TaE2F / DP member. In this study, each wheat E2F / DP member contained three homologous copies (distributed in genomes A, B, or D). Therefore, 27 homologous genes were identified in wheat genomes A, B, and D. Based on previous classification rules for E2F / DP proteins in Arabidopsis and rice, the 27 wheat E2F / DP proteins were divided into three subtribes (I-III). Figure 2 Subgroup I contains the most proteins, with 12, while the remaining E2F / DP proteins belong to the other two subgroups (subgroup II has 6, and subgroup III has 9). Figure 2 Compared to the dicotyledonous plant Arabidopsis thaliana, the monocotyledonous plants rice and wheat are more closely related in terms of evolution.

[0094] Gene structure, conserved protein motifs, and gene duplication analysis of wheat E2F / DP family members: To gain a deeper understanding of the gene structure and conserved domains of wheat E2F / DP family members, a phylogenetic tree was constructed using the amino acid sequences of these members. Figure 3 A). Wheat E2F / DP proteins are divided into three subfamilies (I-III). The exon-intron structure of the wheat E2F / DP gene was plotted using the GSDS 2.0 online tool. Figure 3 B). The exon-intron structures of E2F / DP genes within the same subfamily are highly similar, but differences exist between different subfamilies, manifested in variations in the number and length of introns across the three subfamilies. Most E2F / DP genes in subfamilies II and III contain 8–9 introns, while those in subfamilies I contain 10–13 introns. Eleven conserved motifs were identified in the E2F / DP family, with motif 1, motif 7, and motif 8 being the most conserved and present in all E2F / DP proteins, while other motifs are found only in certain specific proteins. Figure 3 C).

[0095] To further investigate the evolutionary relationship of E2F / DP genes, chromosome localization and gene duplication analysis were performed. Twenty-seven E2F / DP genes from three subtribes were randomly distributed across 21 chromosomes. Twelve E2F / DP genes were distributed across six chromosomes (2A, 2B, 2D, 6A, 6B, and 6D), while the remaining 15 E2F / DP genes were evenly distributed across another 15 chromosomes (Table 1). Furthermore, the number of E2F / DP genes on the chromosomes of wheat A, B, and D genomes was the same. To investigate the relationship between wheat E2F / DP members and gene duplication, collinearity analysis was performed on E2F / DP genes. Twenty-six cloned gene pairs were found, distributed across different chromosomes, including 23 homologous genes (…). Figure 4 ).

[0096] Wheat E2F / DP gene expression patterns in different wheat tissues: Gene expression patterns are often closely related to gene function. To study the expression patterns of the wheat E2F / DP gene in different wheat tissues (roots, stems, leaves, spikes, and grains), data from a public wheat database were submitted to TBtools software for visualization analysis. Figure 5Most E2F / DP genes are expressed in different wheat tissues, but the expression levels vary. A few E2F genes (TaE2F4I-4, TaE2F4I-6, TaE2F4I-8) are not expressed in any of the five tissues. TaE2F1I, TaE2F2I, TaDP2III, and TaDP3III genes are highly expressed in the spike; TaDEL2II gene is highly expressed in both the spike and root. Five genes (TaE2F1I-19, TaE2F3I-5, TaDP2III-3, TaDP3III-15, and TaDEL2II-27) from three different subgroups and widely expressed in at least four different wheat tissues were randomly selected for further analysis.

[0097] Expression pattern analysis under abiotic stress: The expression levels of the wheat E2F / DP gene under drought, salt, mannitol and cold stress were detected by qRT-PCR. Figure 6 Under drought stress, the expression levels of the five E2F / DP genes varied at different time points in leaves. The expression levels of TaE2F1I-19 and TaDP3III-15 were significantly upregulated, while the expression levels of TaE2F3I-5, TaDP2III-3, and TaDEL2II-27 were downregulated. TaE2F1I-19 and TaDP3III-15 were initially upregulated, peaking at 1 h, and then downregulated. Under mannitol stress, the expression level of TaE2F1I-19 was significantly upregulated, peaking at 6 h, while the expression of the other four genes showed a slight downregulation or upregulation trend. Under drought and mannitol stress, the expression level of TaE2F1I-19 was significantly upregulated, indicating that this gene may positively regulate the drought stress response in wheat. Under salt stress, the expression levels of the other genes, except for TaDP2III-3, were downregulated. Under cold stress, the expression levels of TaE2F1I-19 and TaDP3III-15 were significantly upregulated, peaking at 12 h, while TaDEL2II-27 peaked at 1 h. Under different stresses, the expression trends of some E2F and DP genes were similar: for example, TaE2F1I-19 and TaDP3III-15 showed an initial upregulation followed by downregulation under drought and mannitol stress. In summary, the wheat E2F / DP gene expression is upregulated under any stress treatment. The typical E2F transcription factor gene TaE2F1I-19, which is significantly upregulated under drought and mannitol stress, will be selected as the subject of further in-depth research.

[0098] Sequence characteristics of wheat TaE2Fa-6A: Common wheat is a hexaploid species, and allopolyploidy leads to the production of homologous genes. Therefore, three homologous copies are often present in hexaploid wheat. The wheat TaE2F1I-19 gene contains three homologous copies in wheat, including TaE2F1I-21 and TaE2F1I-23 in addition to TaE2F1I-19. Gene structure analysis shows that there is no difference in the number of introns and exons between TaE2F1I-19 and TaE2F1I-23. Figure 3 B), TaE2F1I-21 has fewer introns and exons than TaE2F1I-19 and TaE2F1I-23. Figure 3 B). The intron lengths of TaE2F1I-19, TaE2F1I-21, and TaE2F1I-23 differ significantly: for example, the third intron of TaE2F1I-19 and TaE2F1I-23 is the longest (TaE2F1I-19 > 5 kb, TaE2F1I-23 > 14 kb), while the sixth intron of TaE2F1I-21 is the longest (< 1 kb). Figure 3 (B) Existing research indicates that changes in gene structure may affect gene function, and there may be functional differences among the three TaE2Fa-6 homologs. Cis-acting element analysis shows that the TaE2F1I-19 promoter contains 5 ABRE elements, 1 W-box element, 3 MYB elements, and 5 MYC elements (Table 1); TaE2F1I-21 lacks ABRE elements but has 2 W-box elements (Table 5); and TaE2F1I-23 contains 2 ABRE elements, 1 DRE element, and lacks MYB elements (Table 5). Therefore, it is speculated that TaE2F1I-19 may respond to abiotic stress through the ABA pathway and may be regulated by stress-related transcription factors such as WRKY, MYB, and MYC.

[0099] Using cDNA from the leaves of the wheat variety *Traes CS6A02G195500* as a template, the ORF (open reading frame) sequence of TaE2F1I-19 (TraesCS6A02G195500) was cloned by RT-PCR. Sequence analysis showed that the full-length ORF was 1377 bp, encoding 458 amino acids, with a molecular weight (MW) of 49.85 kDa and an isoelectric point (PI) of 5.08 (Table 1). Alignment of the TaE2F1I-19 protein sequence with the NCBI database revealed that it is most closely related to wild emmer wheat (AteE2Fa). For ease of understanding, TaE2F1I-19 was renamed TaE2Fa-6A. Overall, the TaE2Fa-6A protein is more closely related to monocotyledonous plants and less closely related to dicotyledonous plants. Figure 7 A). In different plants, most genes are of the E2Fa type. Further analysis of conserved domains in E2F proteins from different plants in the phylogenetic tree revealed that they all possess conserved DBD, DD, and RB domains. Figure 7 B).

[0100] Transcriptional activity analysis: Full-length and truncated bait recombinant vectors of TaE2Fa-6A (pGBKT7-TaE2Fa-6A (1-144 aa), pGBKT7-TaE2Fa-6A (1-334 aa), pGBKT7-TaE2Fa-6A (1-457 aa), and pGBKT7-TaE2Fa-6A (335-457 aa)) were constructed. Yeast cells transformed with the recombinant vector and the control pGBKT7 empty vector, respectively, showed that yeast strains transformed with pGBKT7-TaE2Fa-6A (1-144 aa) and pGBKT7-TaE2Fa-6A (1-334 aa) could only grow on SD / -Trp medium. However, yeast strains transformed with pGBKT7-TaE2Fa-6A (1-457 aa) and pGBKT7-TaE2Fa-6A (335-457 aa) grew well on SD / -Trp / -His / -Ade medium. Figure 7 C). These results indicate that the full-length TaE2Fa-6A possesses transcriptional activation activity, and that the C-terminus (335-457 aa) is the transcriptional activation region.

[0101] Subcellular localization of wheat TaE2Fa-6A protein: Subcellular localization and tissue-specific analysis of wheat TaE2Fa-6A protein suggest that, as a transcription factor, TaE2Fa-6A protein should be located in the cell nucleus. To verify this hypothesis, a transient expression vector for TaE2Fa-6A was constructed and transformed into Arabidopsis protoplast cells for transient expression, with an empty vector used as a control. Laser confocal microscopy was used to track the GFP fluorescence signal of TaE2Fa-6A protein, showing that the GFP signal in the control was uniformly distributed in the nucleus, cytoplasm, and cell membrane, while the fluorescence signal of TaE2Fa-6A protein was mainly distributed in the nucleus and cytoplasm. Figure 8 A). Therefore, the TaE2Fa-6A protein is mainly located in the nucleus and cytoplasm.

[0102] The expression level of the TaE2Fa-6A gene was detected in different tissues or organs of mature wheat using qRT-PCR. Figure 8 B). The results showed that the TaE2Fa-6A gene was expressed in all tissues or organs, with higher expression levels in the stem and spike.

[0103] Identification of growth, development, and drought resistance function of wheat TaE2Fa-6A gene overexpression in Arabidopsis thaliana: To reveal the function of the wheat TaE2Fa-6A gene, a TaE2Fa-6A overexpression vector was constructed (…). Figure 9 A, where *** indicates highly significant difference (P < 0.01)**, recombinant vectors were infected with Arabidopsis thaliana using the Agrobacterium-mediated flower-dipping method. After harvesting T0 generation seeds, T1 generation lines were obtained by screening on Kansas-resistant MS medium. T1 generation lines were cultured individually, and T2 generation seeds were collected. T2 generation seeds were further screened on antibiotic-containing MS medium, and T2 transgenic lines with a segregation ratio of 3:1 were selected. These were then transferred to soil and cultured until maturity, ultimately yielding homozygous T3 homozygous transgenic lines. RT-PCR was used to detect whether the TaE2Fa-6A gene was successfully overexpressed in Arabidopsis thaliana. Figure 9 B). RT-PCR results showed that wheat TaE2Fa-6A was expressed in the Arabidopsis overexpression line (OE) but not in the wild type (WT), indicating that the wheat TaE2Fa-6A gene was successfully transferred into Arabidopsis.

[0104] Compared to the wild type, transgenic Arabidopsis seedlings, after 9 days of culture, had significantly larger leaves than the wild type. Figure 9 C). Subsequently, the diameter of its rosette leaves was measured, and the diameter of the rosette leaves of the transgenic Arabidopsis was significantly longer than that of the wild type. Figure 9 D). Statistical analysis of the flowering period of transgenic Arabidopsis showed that the flowering period of transgenic Arabidopsis lines was significantly earlier than that of wild-type ( Figure 9 E, Figure 9 F). Statistical analysis of the length, number, and seed number of mature siliques showed that the transgenic Arabidopsis had a significantly higher number of siliques than the wild-type Arabidopsis, while the length and seed number did not differ significantly. The above analysis indicates that overexpression of the TaE2Fa-6A gene in Arabidopsis affects leaf development, flowering time, and silique number in transgenic Arabidopsis.

[0105] Before drought treatment, the stomatal distribution and water loss rate of 3-week-old Arabidopsis thaliana under normal water supply conditions were analyzed. The OE Arabidopsis thaliana lines had fewer stomata than the wild-type Arabidopsis thaliana. Figure 10 A, where the error bars represent the standard deviation (mean ± SD, n = 3), and the asterisk indicates significant (*P < 0.05) or highly significant difference (**P < 0.01); the water loss rate was also lower than that of wild-type Arabidopsis thaliana. Figure 10B); After 13 days of drought treatment, wild-type lines accumulated large amounts of anthocyanins and severely withered, while drought stress had little effect on transgenic overexpression (OE) lines. Three days after rehydration, although growth was slower, most OE lines continued to grow, while most WT plants died and their leaves faded. Figure 10 C). Furthermore, the survival rate of Arabidopsis thaliana lines overexpressing drought stress was investigated. The survival rate of the OE Arabidopsis thaliana lines was approximately 70.83-75%, significantly higher than the 52.5% of the WT lines. Figure 10 D). These results indicate that overexpression of the wheat TaE2Fa-6A transcription factor gene enhances the drought resistance of overexpressing Arabidopsis lines.

[0106] Drought resistance analysis of wheat TaE2Fa-6 silent lines: The drought resistance function of the wheat TaE2Fa-6A gene in wheat was further verified using BSMV-VIGS technology. Ten days after inoculation with the virus, BSMV-VIGS was observed in wheat seedlings of the Xinnong 979 variety. PDS All wheat lines showed leucism, while BSMV0 and BSMV... TaE2Fa-6 Wheat strains show symptoms of chlorosis virus (ST). Figure 11 In the BSMV-VIGS experiment, the wheat variety Xinong 979 was selected as the virus infection receptor. Six physiological indicators were measured: malondialdehyde (MDA) content, proline content, O2.- content, SOD, CAT, and POD enzyme activity. Error bars represent standard deviation (mean ± SD, n = 3), and asterisks indicate significant (*P < 0.05) or highly significant (**P < 0.01) differences, indicating successful silencing of the TaE2F1-6A gene in wheat. After 8 days of drought treatment, TaE2F1-6 silenced wheat lines showed severe leaf wilting, while BSMV0 wheat plants showed relatively mild symptoms. Figure 11 B); At this time, the expression level of the Ta2F1-6A gene in the silent wheat lines was significantly lower than that in the BSMV0 lines (B); Figure 11 C). Under normal water supply conditions, there were no differences in physiological parameters between BSMV0 and TaE2Fa-6 silent wheat lines. Figure 11 D); Under drought conditions, all physiological indicators showed an increasing trend, but the magnitude of the increase varied: for example, proline content increased by approximately 2-3 times compared to before drought stress, while MDA content increased by approximately 1-2 times. Figure 11 D). The MDA and O2.- content in the TaE2Fa-6 silent wheat lines was significantly higher than that in BSMV0, while the proline content and antioxidant enzyme (SOD, CAT, and POD) activity were lower. Figure 11D). In summary, silencing the TaE2Fa-6 gene in wheat significantly reduced the drought resistance of wheat lines.

[0107] To elucidate the drought resistance mechanism of wheat TaE2Fa-6A, the transcriptional levels of the genes TaP5CS1, TaDREB1, TaERF3, TapOD, TaCAT, and TaSOD (Fe) in TaE2Fa-6 silenced wheat lines under drought stress were further analyzed. Figure 12 TaP5CS1, TaDREB1, TaERF3, TapOD, TaCAT, and TaSOD (Fe) are all recognized genes closely related to wheat drought stress tolerance. Compared with the BSMV0 line, the transcription levels of TaP5CS1, TaDREB1, TaERF3, TapOD, TaCAT, and TaSOD (Fe) were reduced in the TaE2Fa-6 silenced wheat line under drought stress.

[0108] Identification of drought resistance function in wheat lines overexpressing TaE2Fa-6A: To identify the drought resistance function of the wheat TaE2Fa-6A gene, the wheat TaE2Fa-6A gene was overexpressed in wheat, and its function in regulating drought resistance was further verified in stable TaE2Fa-6A overexpression wheat lines. The expression level of TaE2Fa-6A gene in wheat lines overexpressing TaE2Fa-6A at the two-leaf stage (OE) was detected using qRT-PCR. Figure 13 A). Compared with the control, the expression level of the TaE2Fa-6A gene was significantly upregulated in all three overexpression lines, with the OE14 line showing the most significant upregulation. Before drought treatment, the WT and OE lines grew well, with no phenotypic differences observed. After 10 days of drought stress, the WT line wilted severely, while the drought stress had a relatively smaller impact on the wheat OE line. Figure 13 B). The effects of drought stress on the physiological indicators of transgenic wheat showed that the fresh weight of wheat OE lines under drought stress ( Figure 13 C) and proline content ( Figure 13 D) was significantly higher than WT, while the MDA content ( Figure 13 E) was significantly lower than WT. In summary, overexpression of the TaE2Fa-6A gene in wheat significantly enhanced the drought resistance of overexpressing wheat lines. To elucidate the drought resistance mechanism of wheat TaE2Fa-6A, the transcriptional levels of TaP5CS1, TaDREB1, TaWRKY33, TaERF1-6A, TaERF3, TapOD, TaCAT, and TaSOD (Fe) genes in TaE2Fa-6A overexpressing wheat lines were further analyzed before and after drought stress. Figure 14TaP5CS1, TaDREB1, TaWRKY33, TaERF3, TapOD, TaCAT, and TaSOD(Fe) are all recognized genes closely related to wheat drought stress tolerance; our research group's study shows that TaERF1-6A plays an important role in regulating wheat drought resistance. Before drought stress, except for the TaCAT gene, other stress-related genes in the TaE2Fa-6A transgenic wheat lines were significantly upregulated. Under drought stress, all stress-related genes were significantly upregulated, with TaERF1-6A showing the most significant upregulation, with a transcriptional level approximately 24 times that of the control.

[0109] Screening of wheat TaE2Fa-6A interacting proteins: To further investigate the potential molecular mechanism of wheat TaE2Fa-6A in drought stress response, yeast two-hybrid technology was used to screen for proteins that may interact with TaE2Fa-6A in a wheat cDNA library. Some yeast colonies could grow on two- and four-deficient media, but after transforming E. coli with plasmids extracted by shaking, they could not grow on ampicillin medium, possibly due to contamination or false positives. Therefore, after ruling out these possibilities, single clones that could grow normally on ampicillin medium were selected and sequenced. After removing proteins encoded by repetitive genes, proteins that may interact with TaE2Fa-6A were finally identified and annotated in the NCBI database. Ultimately, five proteins that may interact with TaE2Fa-6A were obtained: TaDPb, TaG6PE, TaVDAC, TaCBSX3, and TaOEP24. Genes encoding interacting proteins selected from the cDNA library possessed complete ORFs and could be ligated into pGADT7 to construct prey vectors for yeast two-hybrid transformation validation. Five pGADT7 recombinant vectors were co-transformed into Y2H yeast strains with pGBKT7-TaE2Fa-6A (1-334 aa). All transformed strains were able to grow on SD / -Trp / -Leu and SD / -Trp / -Leu / -His / -Ade / +X-α-Gal media. Figure 15A). In summary, the wheat TaE2Fa-6A protein in yeast may interact with five candidate interacting proteins. Among the five interacting proteins TaDPb, TaG6PE, TaVDAC, TaCBSX3, and TaOEP24, TaDPb is a member of the wheat E2F / DP transcription factor family. Studies have shown that in Arabidopsis, E2Fa can form a dimer with DP protein to regulate target genes in response to abiotic stress. These target genes mainly include WRKY transcription factor family genes (AT5G1089 and AT5G26170), AP2 / ERF transcription factor family genes (AT1G75490, AT2G40340, and AT3G11020), NAC transcription factor family genes (AT3G01600), and bZIP transcription factor family genes (AT2G36270). It can be inferred that in wheat, TaE2Fa-6A may interact with TaDPb to regulate the transcriptional level of drought stress response genes in order to cope with drought stress; glucose-6-phosphate-1-epimerase (G6PE) is an isomerase in sugar metabolism that can catalyze changes in the α or β conformation of glucose-6-phosphate and plays a role in maintaining normal cellular metabolic pathways. Under drought stress, plant G6PE may enhance plant drought resistance by maintaining normal cellular metabolism; VDAC proteins are widely present in the mitochondrial extramedullary model of animals and plants and play a crucial role in regulating plant development and coping with abiotic stress; CBSX3 is a regulator of ROS production in mitochondria and plays an important role in regulating plant development and redox systems; studies have shown that the transcriptional level of OEP genes is significantly increased under ABA, heat, and drought stress, and overexpression of the wheat TaOEP16-2-5B gene in Arabidopsis thaliana can enhance the heat tolerance and drought resistance of overexpressing Arabidopsis thaliana lines. Therefore, it is speculated that the wheat TaE2Fa-6A protein may interact with five proteins involved in sugar metabolism, ion transport, redox reactions and the ABA hormone pathway, thereby regulating wheat drought resistance.

[0110] TaDPb protein is an indispensable cofactor in the regulation of downstream target genes by TaE2Fa-6A. TaDPb was chosen as the subject of further research. Furthermore, the interaction between TaE2Fa-6A and TaDPb was verified using BiFC. YFP fluorescence was observed when TaE2Fa-6A and TaDPb were co-transferred to tobacco, while no YFP fluorescence was observed in the control group, indicating an interaction between TaE2Fa-6A and TaDPb in tobacco cells. Figure 15 B).

[0111] Allelic variants of wheat TaE2Fa-6A: This invention discloses the drought-resistance-related gene TaE2Fa-6A through bioinformatics, and verifies its biological function and molecular mechanism in regulating drought resistance through transgenic analysis. The invention aims to protect the CASP marker developed based on the TaE2Fa-6A gene, including the marker sequence, PCR reaction system and procedure, and enzyme digestion system. An example is an association analysis of drought resistance among different haplotypes using a natural population consisting of 262 wheat microcore germplasms and 121 wheat varieties.

[0112] CASP marker development process: (1) Superior haplotype mining: Through polymorphism analysis of the TaE2Fa-6A gene sequence (genomic sequence covering promoter and coding regions) in 8 wheat varieties in the database, it was found that the TaE2Fa-6A gene promoter region has 9 SNPs and 2 1-bp InDels. The wheat TaE2Fa-6A gene consists of 14 exons and 13 introns. In the 8 wheat varieties, the exon sequences did not change, but the introns changed; the first intron had 2 SNPs; the second intron had 1 SNP; the third intron contained 4 SNPs; 2 1-bp InDels and 2 2-bp InDels. TaE2Fa-6A formed 5 types of haplotypes in the 8 wheat varieties ( Figure 16 Studies have shown that mutations in the CTCC element in the promoter sequence affect the expression of the DYT1 gene in the tapetum, thereby affecting anther development and ultimately yield traits. In the PlantCare database, a CTCC cis-acting element was predicted in the promoter region of the wheat TaE2Fa-6A gene, and a SNP at -727 resulted in a mutation (CTTC) in this element. It can be inferred that mutations in the CTCC element may affect the expression of the wheat TaE2Fa-6A gene, thus influencing yield traits. Therefore, based on this SNP mutation, the CAPS molecular marker was developed, classifying the five haplotypes into two allelic variant types, named Hap-C (CTCC) and Hap-T (CTTC), respectively. Figure 16 , Figure 17 A).

[0113] CASP marker sequences developed based on superior haplotypes: the upstream and downstream primer sequences are 5'-TCGCGACAAGGACTTCAAAACTAG-3' and 5'-GCTGAATTTCGAATGGGCCACT-3', respectively; The PCR reaction system and PCR procedure for CASP labeling are as follows: CASP-labeled PCR reaction system Components Volume (μL) 2×Rapid Taq Master Mix 10 DNA template 1 (100 ng / µL) Upstream / downstream primers (10 µM) 1 RNA-free Water 7 The CASP-labeled PCR program is as follows: the first cycle is 95℃ for 5 min, 95℃ for 30 s, 62℃ for 30 s and 72℃ for 1 min, followed by 95℃ for 30 s, 60℃ for 30 s and 72℃ for 1 min, for 35 cycles, and finally 72℃ for 5 min.

[0114] The CASP-labeled restriction enzyme digestion system consisted of 0.1 μL NlaIV restriction enzyme, 5 μL Cutsmart buffer, 3 μL PCR product, and 1.9 μL RNA-free water. Allelic variants in the wheat TaE2Fa-6A gene were genotyped by treating the PCR product with NlaIV restriction enzymes after one round of PCR (TaE2Fa-6A PCR products at the C site were digestible, while those at the T site were not).

[0115] CASP Marker Example 1: Using DNA from Taishan No. 1, Jingdong No. 8, Jing 411, Weimai No. 8, Motuo Wheat, Bianba Spring Wheat, White Mang Wheat, Wujiangzhuo, Muzongzhuoga, Zangdong No. 4, Shigatse No. 54, Shigatse No. 8, Shanmai, Yizhimai, Dabaimai, Ga Laohan, Hongmangmai, Dabaimai, Baiqitou, Baimazha, Laotutou, Ganmai No. 8, Gaoyuan 506, and Qingchun 28 as templates, molecular marker amplification PCR was performed. The PCR product length was 1144 bp. The PCR product of the allelic variant material containing the CTCC element was successfully digested with enzymes, and the digestion products were 698 bp and 447 bp, respectively. Figure 2-2 0B), achieving the expected results; however, PCR products from materials with allelic variants containing the mutant element "CTTC" could not be digested with enzymes. Figure 17 B) confirmed the effectiveness of the CAPS molecular marker.

[0116] CASP marker example 2: Relationship between wheat TaE2Fa-6A allelic variant type and relative germination rate Based on the developed CAPS molecular markers, the TaE2Fa-6A genotypes were performed on 121 wheat varieties (lines) from a natural population. The average relative germination rate of wheat varieties (lines) containing the Hap-C allele (54.01%) was higher than that of wheat varieties (lines) containing the Hap-T allele (49.93%). Furthermore, wheat varieties (lines) containing the Hap-C allele had a higher number of varieties in the extremely high (HR) and high (R) drought resistance categories than those containing the Hap-T type (Table 2-10), indicating that Hap-C can improve drought resistance during wheat germination.

[0117] CASP marker example 3: Relationship between wheat TaE2Fa-6A allelic variants and agronomic traits Based on the developed CAPS molecular markers, the TaE2Fa-6A genotypes were performed on two sets of recombinant inbred lines. TaE2Fa-6A was classified into two allelic variants: Hap-C (CTCC) and Hap-T (CTTC), with a small number of heterozygous variants, Hap-T / C (CTCC / CTTC). Figure 2-2 (0C). Subsequently, association analysis was performed on the two allelic variation types in the two sets of recombinant inbred line populations with agronomic traits (grain number per spike (GN), effective spikelet number (ESN), 1000-grain weight (TKW), spike length (SL), plant height (PH), flag leaf length (FLL), flag leaf width (FLW), harvest index (HI), heading date (HD), and flowering date (FD)). Preliminary results showed significant associations between the two allelic variation types and GN, PH, HI, and FD. In the two sets of recombinant inbred line populations, lines containing the Hap-C (CTCC) allelic variation type had more GN, lower PH, higher HI, and shorter HD and FD (Table 2-11). Lines containing the Hap-T allelic variation type had higher TKW. In conclusion, Hap-C is a relatively superior allelic variation type.

[0118] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for mining superior haplotypes of the wheat drought resistance-related gene TaE2Fa-6A, characterized in that, Includes the following steps: Step 1: Obtain genomic data of wheat, Arabidopsis thaliana, and rice, and use the E2F or DP family to perform homology search on wheat protein databases to obtain candidate protein sequences of wheat E2F or DP family. Step 2: Identify the domains of candidate protein sequences, screen for proteins containing E2F or DP domains, and identify members of the wheat E2F or DP family. Step 3: Perform multiple sequence alignment on the E2F or DP family members and construct a phylogenetic tree to determine the target transcription factor gene TaE2Fa-6A based on the phylogenetic relationship; Step 4: The TaE2Fa-6A gene was sequence cloned to obtain its open reading frame sequence; Step 5: Identify the transcriptional activation region of the TaE2Fa-6A protein through transcriptional activity analysis; Step 6: Determine the localization characteristics of TaE2Fa-6A protein in the nucleus and cytoplasm through subcellular localization analysis; Step 7: Verify the function of TaE2Fa-6A in regulating plant drought resistance through transgenic overexpression and gene silencing experiments.

2. The method for mining superior haplotypes of wheat drought-resistance related gene TaE2Fa-6A according to claim 1, characterized in that, The E2F or DP family used in step one is the HMM model with the number PF02319 in the PFAM database.

3. The method for mining superior haplotypes of wheat drought-resistance related gene TaE2Fa-6A according to claim 1, characterized in that, The phylogenetic tree was constructed using the adjacency-joining method, and the evolutionary relationships were determined through 1000 bootstrapping tests.

4. The method for mining superior haplotypes of wheat drought-resistance related gene TaE2Fa-6A according to claim 1, characterized in that, The transcriptional activity analysis was performed by constructing TaE2Fa-6A Full-length and truncated protein decoy vectors were developed, and their transcriptional activation capacity was tested in a yeast system.

5. A method for mining superior haplotypes of the wheat drought resistance-related gene TaE2Fa-6A as described in any one of claims 1-4, based on... TaE2Fa-6A A method for developing CAPS molecular markers for genes, characterized in that, Includes the following steps: Step one, for multiple wheat varieties TaE2Fa-6A Polymorphism analysis of the gene promoter sequence was performed to identify single nucleotide mutation sites. Step 2: Design specific PCR primers for amplification based on the mutation site. TaE2Fa-6A Gene promoter fragments; Step 3: Perform restriction endonuclease digestion on the PCR amplification products; Step 4: Based on the differences in the size of the enzyme digestion product fragments... TaE2Fa-6A Allelic variant types are used for typing.

6. The CAPS molecular marker development method according to claim 5, characterized in that, The mutation site is located at TaE2Fa-6A A single nucleotide polymorphism at position -727 in the gene promoter region.

7. The CAPS molecular marker development method according to claim 5, characterized in that, The PCR amplification fragment is 1144 bp in length.

8. The CAPS molecular marker development method according to claim 5, characterized in that, The restriction endonuclease is NlaIV.

9. A method for screening superior haplotypes of wheat drought resistance, implementing the superior haplotype mining method for the wheat drought resistance-related gene TaE2Fa-6A as described in any one of claims 1-4, characterized in that, Includes the following steps: Step 1: Genotyping of wheat populations using the CAPS molecular markers described in claim 5; Step 2: Determine the relative germination rate of wheat materials under drought conditions during the germination period; Step 3: Classify wheat materials into drought-resistant grades based on their relative germination rates; Step four: Conduct correlation analysis between different allelic variation types and agronomic traits to screen for traits with excellent drought resistance. TaE2Fa-6A Haplotype.

10. The method for screening superior haplotypes of drought resistance in wheat according to claim 9, characterized in that, The superior haplotype is the TaE2Fa-6A allelic variant type containing the CTCC cis-acting element in the promoter sequence.

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