Zm00001eb247080 gene with drought resistance and application thereof

Abstract

This invention discloses a drought-resistant material. Zm00001eb247080 Genes and their applications belong to the field of genetic engineering technology. Zm00001eb247080 The nucleotide sequence of the gene is shown in SEQ ID NO.1. This invention provides a drought-resistant gene. Zm00001eb247080 Genes. Overexpression lines under drought stress Zm00001eb247080 The gene expression level in the overexpression lines was significantly higher than that in the non-transgenic lines, while the expression level in the gene knockout lines was significantly lower than that in the non-transgenic lines. Simultaneously, the root biomass and lateral root number of the overexpression lines were significantly increased, as were the antioxidant enzyme activities and the contents of ABA and IAA. This invention provides… Zm00001eb247080 Genes have a significant effect on improving drought resistance in maize, providing important genetic resources and theoretical basis for molecular breeding of drought-resistant maize.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a material with drought resistance. Zm00001eb247080 Genes and their applications. Background Technology

[0002] Maize (Zea mays L.) is one of the most widely planted and highest-yielding food crops globally, playing an irreplaceable role in ensuring food security, supporting livestock development, and providing industrial raw materials. However, global climate change has led to more frequent and intensified extreme drought events, making drought the most significant abiotic stress factor limiting maize growth, development, and yield. In arid and semi-arid regions, water scarcity and seasonal droughts severely threaten the stability of maize production. Drought stress not only inhibits photosynthesis and growth rate but also disrupts key physiological processes such as nutrient absorption, assimilate transport, and grain filling, ultimately resulting in a significant drop in yield. Therefore, in-depth analysis of the molecular mechanisms of drought resistance in maize and the discovery of key drought-resistant functional genes are of significant theoretical and practical value for cultivating new high-yielding, stable-yielding, and drought-resistant germplasm.

[0003] The root system is the primary organ for plants to absorb water and mineral nutrients, and a key tissue for sensing changes in soil moisture and initiating systemic stress responses. Compared to the aboveground parts, the root system can directly sense soil drought signals and coordinate the overall drought resistance response of the plant through hormone synthesis and translocation, accumulation of osmotic regulatory substances, and systemic signal transduction. Root architecture (including root length, root depth, lateral root density, and root biomass) and root activity are core traits determining a plant's drought resistance. Drought-tolerant genotypes typically exhibit deeper root distribution, larger root biomass, and higher water use efficiency. In recent years, a series of key genes regulating root development and drought resistance have been identified. However, a comprehensive understanding of the systemic response characteristics and regulatory networks of the whole transcriptome and whole proteome of different drought-tolerant genotypes under drought stress is still lacking. There is an urgent need to identify key drought-resistant regulatory genes through multi-omics integrated analysis. Summary of the Invention

[0004] The purpose of this invention is to provide a drought-resistant material. Zm00001eb247080 Genes and their applications, to address the problems of existing technologies, overexpression Zm00001eb247080 Genes can improve a plant's drought resistance.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides a drought-resistant material. Zm00001eb247080 Genes, the ones mentioned Zm00001eb247080 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0006] The present invention also provides the aforementioned Zm00001eb247080 A gene-encoded protein, the amino acid sequence of which is shown in SEQ ID NO.2.

[0007] The present invention also provides the overexpression described above. Zm00001eb247080 The application of gene-based preparations in any of the following: (1) Improve plant drought resistance; (2) Prepare products that improve plant drought resistance; (3) Cultivate drought-resistant plant varieties; (4) Breeding of drought-resistant plants.

[0008] Optionally, the formulation includes... Zm00001eb247080 A recombinant vector of the gene or a host bacterium containing the recombinant vector.

[0009] The present invention also provides the use of formulations that increase the expression level of said protein in any of the following: (1) Improve plant drought resistance; (2) Prepare products that improve plant drought resistance; (3) Cultivate drought-resistant plant varieties; (4) Breeding of drought-resistant plants.

[0010] Optionally, the plant includes corn.

[0011] The present invention also provides a method for improving plant drought tolerance, comprising overexpressing in plants Zm00001eb247080 Genes are used to improve plant drought resistance. The Zm00001eb247080 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0012] Optionally, the plant includes corn.

[0013] The present invention also provides a method for breeding drought-resistant plant varieties, comprising overexpressing in plants Zm00001eb247080 Genes, Enhance Zm00001eb247080 Gene expression levels are a key step in obtaining drought-resistant plant varieties. The Zm00001eb247080 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0014] Optionally, the plant includes corn.

[0015] The present invention discloses the following technical effects: (1) This invention provides a drought-resistant material. Zm00001eb247080 Genes, the Zm00001eb247080The nucleotide sequence of the gene is shown in SEQ ID NO.1. The protein it encodes is located in the cell nucleus and is significantly induced to express under drought stress, positively regulating the drought resistance of maize.

[0016] (2) Under drought stress, transformation Zm00001eb247080 overexpression lines of the gene Zm00001eb247080 Gene expression levels in all overexpression lines were significantly higher than in non-transgenic lines, while expression levels in gene knockout lines were significantly lower than in non-transgenic lines. Overexpression lines showed significantly better results than wild-type lines in terms of root biomass, lateral root number, antioxidant enzyme activity, and hormone content, confirming... Zm00001eb247080 Genes are positive regulators of maize's response to drought stress.

[0017] (3) After drought stress, Zm00001eb247080 The knockout strains were more wilted than the control wild-type plants, and Zm00001eb247080 Overexpression lines showed significantly less wilting than wild-type plants. The ABA and IAA contents of overexpression lines were significantly higher than those of the wild type, while the ABA and IAA contents of gene knockout lines were significantly lower than those of the wild type, indicating that under drought stress, maize... Zm00001eb247080 The gene can increase the content of ABA and IAA, which is beneficial for inducing the expression of downstream stress resistance genes and promoting root development.

[0018] (4) Under drought stress, maize Zm00001eb247080 The gene can enhance the activity of SOD, POD, and CAT antioxidant enzymes, effectively alleviating drought-induced oxidative damage. Meanwhile, Zm00001eb247080 It can interact directly with multiple stress response proteins, forming a drought resistance regulatory network with this gene at its core, thereby coordinating root development and stress defense processes and improving the ability of maize plants to resist drought stress. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 A comparative graph of phenotypic indices of KF and PH6WC maize inbred lines under drought stress; A: plant height; B: root length; C: stem diameter; D: root volume; E: 100-kernel weight; Figure 2 for Zm00001eb247080 Protein subcellular localization map; Figure 3 For drought stress Zm00001eb247080Effects of transgenic plants (KO or OE) and wild-type plants (CK / WT); A: Plant phenotype; B: Plant root phenotype; C: Number of lateral roots; D: Zm00001eb247080 Gene expression level; E: SOD activity; F: CAT activity; G: POD activity; H: IAA content; I: ABA content; J: root activity; Figure 4 Figure 1 shows the results of yeast two-hybrid library screening; a: Y2Hgold[pGBKT7]SD / -Trp; b: Y2Hgold[pGBKT7-GAL4]SD / -Trp; c: Y2Hgold[pGBKT7-Zm00001eb247080]SD / -Trp; d: Y2Hgold[pGBKT7]SD / -Trp / -His / -Ade / Xa-gal; e: Y2Hgold[pGBKT7-GAL4]SD / -Trp / -His / -Ade / Xa-gal; f: Y2Hgold [pGBKT7-Zm00001eb247080]SD / -Trp / -His / -Ade / Xa-gal; g: SD-Trp; h: SD -Trp-His-Ade; i: SD-Trp-His-Ade+5mM3-AT+100ngAbA; j: SD-Trp-His-Ade+10mM3-AT+200ngAbA; k: SD-Trp-His-Ade+20mM3-AT+400ngAbA ; l: SD-Trp-His-Ade+30mM3-AT+600ngAbA; m: SD-Trp-His-Ade+40mM3-AT+800ngAbA; n: SD-Trp-His-Ade+50mM+3-AT1000ngAbA; o: Primary screening QDO (SD / -Leu / -Trp); p: Secondary screening QDO (SD / -Leu / -Trp); q: Tertiary screening QDO(SD / -Leu / -Trp); r: SD / -Leu / -Trp / -His / -Ade / 5mM3-AT / 100ngAbA; s: SD / -Leu / -T rp / -His / -Ade / 5mM3-AT / 100ngAbA; t: SD / -Leu / -Trp / -His / -Ade / 5mM3-AT / 100ngAbA; Figure 5Figure 1 shows the results of spot-to-spot validation of yeast two-hybrids; A: Y2Hgold[pGBKT7-Zm00001eb247080&pGADT7]; B: Y2Hgold[pGBKT7-Zm00001eb247080&pGADT7-Zm00001eb027730]; C: Y2Hgold[pGBKT7-Zm00001eb247080&pGADT7-Zm00001eb038960]; D: Y2Hgold[pGBKT7-Zm00001eb247080&pGADT7-Zm00001eb078440]; E: Y 2Hgold[pGBKT7-Zm00001eb247080&pGADT7-Zm00001eb220750]; F:Y 2Hgold[pGBKT7-53&pGADT7-T]; H: Y2Hgold[pGBKT7-Lam&pGADT7-T]; I: SD-Leu-Trp; J: SD-Leu-Trp-His-Ade+Xa-gal+5mM3AT-100ngAbA; Figure 6 For WGCNA results; A: Sample clustering tree; B: Determination of soft threshold for gene co-expression network; C: Number of gene clusters and module cutting in gene co-expression network; Figure 7 A heatmap showing the association between gene co-expression network modules and physiological traits; Figure 8 KEGG enrichment results for genes in the black module; Figure 9 KEGG enrichment results for genes in the brown module; Figure 10 KEGG enrichment results for genes in the magenta module; Figure 11 The results show the KEGG enrichment of genes in the turquoise module. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] This invention utilizes multi-omics integrated analysis (combined transcriptomics and proteomics analysis) to systematically analyze the differential response characteristics of root systems to drought stress in two genotypes. Using drought-resistant maize inbred line PH6WC and drought-sensitive inbred line KF as materials, root samples were collected during two key growth stages: pre-flowering and mid-flowering. Differential expression analysis, functional enrichment analysis, and WGCNA co-expression network construction were employed to screen core candidate genes significantly associated with drought resistance. Zm00001eb247080 This gene was significantly induced by drought in the drought-resistant material PH6WC, and its encoded protein is located in the cell nucleus. Further verification of its function through transgenic overexpression and CRISPR / Cas9 knockout, as well as yeast two-hybrid screening, confirmed its efficacy. Zm00001eb247080 It is a positive regulator of drought resistance in maize.

[0027] Example 1 1. Experimental materials Plant materials: Maize inbred lines PH6WC (drought-tolerant) and KF (drought-sensitive), as well as Nicotiana benthamiana L., were provided by the Maize Genetics, Breeding and Cultivation Innovation Team of the Crop Research Institute, Xinjiang Academy of Agricultural Sciences. A comparison of phenotypic indices between the KF and PH6WC maize inbred lines under drought stress can be found in [link to relevant documentation]. Figure 1 .

[0028] Vectors: Plant overexpression vector pCAMBIA2300s-35S-GFP; CRISPR / Cas9 vector pRGEB32; Escherichia coli strain DH5α and Agrobacterium strain GV3101 were used for gene cloning and genetic transformation. Yeast two-hybrid vectors pGBKT7 and pGADT7 were used for protein interaction analysis.

[0029] The primers used and their sequences are shown in Table 1.

[0030] Table 1 Primer Sequences 2. Experimental Methods 2.1 Experimental treatment of plant materials (1) Gene cloning: Take root, stem and leaf tissues of maize plants, freeze the samples rapidly with liquid nitrogen and store them in a -80℃ freezer. Take 3 biological replicates for each tissue.

[0031] (2) Drought stress treatment of transgenic maize plants: Transgenic maize tissue culture seedlings were transplanted and hardened off, and then cultivated in a greenhouse. Transgenic maize plants that had grown to the three-leaf-one-heart stage were selected, and drought stress conditions were simulated using artificial water control. Root samples were collected in the pre-flowering and mid-flowering stages, with three biological replicates set up for each time point. The samples were immediately frozen in liquid nitrogen after collection and stored in a -80℃ freezer.

[0032] 2.2 Total RNA extraction and cDNA synthesis Total RNA was extracted from each sample using TRIzol reagent (Invitrogen, USA). RNA integrity and concentration were assessed by agarose gel electrophoresis and Nanodrop spectrophotometry. mRNA was enriched using Oligo(dT) magnetic beads, and cDNA libraries were constructed. Paired-end 150 bp sequencing was performed on an Illumina Novaseq 6000 platform. Simultaneously, the extracted RNA was reverse transcribed into cDNA using a cDNA synthesis kit (TOYOBO), and the products were stored at -20°C for later use.

[0033] 2.3 Zm00001eb247080 Expression vector construction and Agrobacterium-mediated transformation 2.3.1 Cloning of the target gene PCR amplification was performed using cDNA from the maize inbred line PH6WC as a template. The target gene was amplified using KOD FX high-fidelity enzyme according to the instructions. The amplification system and procedure are shown in Tables 2 and 3 below.

[0034] Table 2 PCR reaction system for target gene cloning Table 3 PCR reaction procedure for target gene cloning 2.3.2 Gel recovery of PCR products and T-cloning After the PCR reaction, the PCR products were detected by 1.0% agarose gel electrophoresis, and the target band was recovered. Following the cloning reaction system shown in Table 4, the recovered PCR products were gently mixed with the cloning vector pEASY-Blunt Cloning Vector (purchased from Beijing Bio-Innovation Technology Co., Ltd.), and incubated at 37°C for 5 minutes. After the reaction, the centrifuge tubes were placed on ice. The ligation product was transformed into *E. coli* DH5α competent cells, plated on LB agar containing the appropriate antibiotics, and incubated overnight at 37°C inverted position. Single colonies were picked for PCR identification (system and procedure are shown in Tables 5 and 6, respectively) and sequencing verification to obtain positive clones containing the correct sequence.

[0035] Table 4 Cloning reaction system Table 5 Colony PCR Identification System Table 6. Colony PCR Identification Procedure 2.3.3 Construction of overexpression vectors Construct using homologous recombination technology Zm00001eb247080 Overexpression vector. (This will be used to generate the correctly sequenced vector.) Zm00001eb247080 The target gene fragment was ligated with the double-digested linearized vector pCAMBIA2300s-35S-GFP to construct a 35S:: Zm00001eb247080 -GFP fusion expression vector (plant overexpression vector pCAMBIA2300s-35S-) Zm00001eb247080 -GFP).

[0036] 2.3.4 Construction of CRISPR / Cas9 Knockout Vectors according to Zm00001eb247080CRISPR / Cas9 target sequences were designed based on the gene's CDS sequence, and primers containing the target sequence were synthesized. The synthesized target sequence was annealed to form a double strand, which was then ligated into the pRGEB32 vector, which had been linearized by BsaI restriction, to construct the CRISPR / Cas9 knockout vector pRGEB32- Zm00001eb247080 .

[0037] 2.3.5 Agrobacterium-mediated transformation The correctly sequenced overexpression vector and CRISPR / Cas9 knockout vector were transformed into Agrobacterium GV3101 competent cells using standard methods. The cells were then plated on YEB solid medium containing the corresponding antibiotics and incubated upside down at 28°C for 2-3 days. Single colonies were picked for PCR identification to obtain positive Agrobacterium strains containing the target vector.

[0038] 2.4 Zm00001eb247080 Subcellular localization The constructed 35S::GFP (empty vector control) and 35S:: Zm00001eb247080 - The GFP fusion expression vector was transformed into tobacco leaf epidermal cells using an Agrobacterium-mediated transient expression system. After culturing for 48-72 h, the distribution of GFP green fluorescence signal in tobacco leaf epidermal cells was observed and photographed using a laser confocal microscope.

[0039] 2.5 Maize genetic transformation The plant overexpression vector pCAMBIA2300s-35S- was introduced using Agrobacterium-mediated transformation. Zm00001eb247080 -GFP and CRISPR / Cas9 vector pRGEB32- Zm00001eb247080 Transformation of maize inbred lines. Maize embryo explants were immersed in Agrobacterium suspension containing the target vector, co-cultured, and then subjected to callus induction, shoot differentiation and rooting culture. Transgenic positive plants were obtained through resistance screening.

[0040] 2.6 Detection of transgenic plants DNA was extracted from the preliminarily screened transgenic maize and used as a template for PCR amplification with identification primers. Wild-type maize DNA was used as a negative control and recombinant plasmid as a positive control to perform molecular identification of transgenic maize plants.

[0041] 2.7 Drought stress treatment and physiological index measurement Transgenic maize tissue culture seedlings were transplanted for hardening-off and then cultivated in a greenhouse. Transgenic maize plants that had reached the three-leaf-one-heart stage were selected, and drought stress conditions were simulated using artificial water control. During the drought stress treatment, the growth status and wilting degree of each transgenic line and wild-type control plant were observed and recorded daily. After the stress treatment ended, all lines were photographed and their phenotypic differences were compared between the overexpression lines, knockout lines, and wild-type plants. Emphasis was placed on indicators such as leaf wilting degree, plant uprightness, and overall growth vigor.

[0042] After the drought stress treatment ended, aboveground parameters such as plant height, stem diameter, and aboveground biomass were measured for each line. Simultaneously, the plant roots were completely removed, and the attached soil was carefully washed away. Root images were acquired using a root scanner (Epson Expression 11000XL), and root architecture parameters were analyzed using WinRHIZO software. Six biological replicates were set up for each line.

[0043] Weigh 0.5 g of fresh root sample, add 5 mL of pre-cooled phosphate buffer (50 mM, pH 7.8), homogenize in an ice bath, centrifuge at 12000 rpm for 20 min at 4℃, and use the supernatant for enzyme activity assay. Superoxide dismutase (SOD) activity was determined using the nitroblue tetrazolium (NBT) photochemical reduction method; peroxidase (POD) activity was determined using the guaiacol method; and catalase (CAT) activity was determined using the ultraviolet absorption method. Three technical replicates were set up for each sample.

[0044] Weigh 0.2 g of fresh root sample, grind with liquid nitrogen, add 1 mL of pre-cooled extraction buffer (methanol:water:formic acid = 75:20:5, v / v / v), and extract by ultrasonication at 4℃ for 30 min. Centrifuge at 12000 rpm for 10 min and collect the supernatant. Simultaneously determine the contents of abscisic acid (ABA) and auxin (IAA) using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). Prepare a standard curve using standards and quantify using the external standard method.

[0045] Root activity was determined using the TTC (triphenyltetrazolium chloride) reduction method.

[0046] Simultaneously, using the Actin gene as an internal reference, qPCR was used for detection. Zm00001eb247080 Gene expression levels under drought treatment.

[0047] 2.8 Combined Transcriptome and Proteome Analysis Transcriptome sequencing and proteome identification were performed on root samples from maize inbred lines PH6WC and KF under drought treatment. Raw transcriptome data, after quality control and filtering, were aligned to the maize B73 reference genome using HISAT2 software. Transcript assembly and expression quantification were performed using StringTie, and differential expression analysis was conducted using DESeq2. The selection criteria were |log2(Fold Change)| > 1 and P < 0.05. Peptides were analyzed by LC-MS / MS, and proteins were identified and quantified using Spectronaut v14.2 software based on the maize reference protein database. Nine-quadrant association analysis and Pearson correlation analysis were performed on the transcriptome and proteome data. A co-expression network was constructed using the R package WGCNA (soft threshold power = 12) to screen key modules and hub genes related to drought stress response and root development.

[0048] 2.9 Screening and peer-to-peer validation of yeast two-hybrid libraries by Zm00001eb247080 The protein encoding the bait is used as a bait, and the bait plasmid pGBKT7- Zm00001eb247080 The yeast strain Y2HGold was transformed into the yeast Y2HGold strain, and self-activation and toxicity tests were performed. After confirming the absence of self-activation activity and toxicity, the bait strain was fused with a maize drought-stressed root cDNA library for screening. The positive clones obtained were sequenced and sequence alignment analyzed to identify the... Zm00001eb247080 Candidate "prey" protein genes involved in protein-protein interactions were identified. Subsequently, the coding sequences of the candidate interacting proteins were cloned into the pGADT7 vector, co-transformed with the bait plasmid into yeast cells, and validated through point-to-point yeast two-hybrid experiments.

[0049] 2.10 KEGG pathway enrichment analysis KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis was performed on key co-expression module genes identified in the WGCNA analysis. Enrichment analysis was conducted using the clusterProfiler R package, with P < 0.05 used as the screening criterion for significant enrichment pathways. Emphasis was placed on metabolic pathways related to drought response, including plant hormone signal transduction, carbon metabolism, cell wall modification, reactive oxygen species scavenging, and osmotic regulation.

[0050] 2.11 Data Analysis Basic statistical analysis of the raw data was performed using Excel 2021. One-way ANOVA and Duncan's multiple comparisons (P < 0.05) were conducted using SPSS 22.0 software. WGCNA co-expression network analysis, KEGG enrichment analysis, and data visualization were performed using R. In the bar charts, different letters indicate significant differences between treatments (P < 0.05), * indicates P < 0.05, and ** indicates P < 0.01.

[0051] 3. Experimental Results 3.1 Corn Zm00001eb247080 Cloning of genes Cloning was achieved using maize leaf tissue as a template. Zm00001eb247080 The gene was cloned and transformed into E. coli DH5α competent cells, cultured overnight at 37°C, and single colonies were picked for PCR detection and sequencing. The results showed that the cloned gene sequence was correct.

[0052] SEQ ID NO.1 ( Zm00001eb247080 Gene CDS sequence (2040 bp): SEQ ID NO.2 (Amino acid sequence of the protein encoded by SEQ ID NO.1): *

[0053] 3.2 Construction of overexpression vectors and CRISPR / Cas9 vectors Homologous recombination technology was used to construct Zm00001eb247080 The overexpression vector was identified by double enzyme digestion, yielding the target band. Zm00001eb247080 Plant expression vector 35S- Zm00001eb247080 -GFP. A targeted assay was also constructed. Zm00001eb247080 The CRISPR / Cas9 knockout expression vector for the gene was sequenced and verified to be correct.

[0054] 3.3 Zm00001eb247080 Subcellular localization 35S:GFP and 35S:GFP were constructed. Zm00001eb247080 The plant expression vector GFP was introduced into tobacco leaves using Agrobacterium-mediated transient transformation. Fluorescence confocal microscopy revealed that the positive control 35S:GFP showed detectable green fluorescence signals in both the nucleus and cell membrane of tobacco mesophyll cells, while 35S:GFP... Zm00001eb247080 In GFP-transformed tobacco leaves, a distinct green fluorescence signal was observed only at the cell nucleus. Figure 2 ).show Zm00001eb247080 The protein is mainly located in the cell nucleus and may play an important role in nuclear signal transduction.

[0055] 3.4 Obtaining and Identifying Positive Transgenic Maize Plants Maize was transformed using Agrobacterium-mediated transformation with overexpression vectors and CRISPR / Cas9 vectors. DNA from preliminarily screened transgenic maize was extracted and amplified by PCR, using wild-type maize DNA as a negative control and recombinant plasmids as a positive control. Molecular identification of the transgenic maize plants was then performed. Figure 3 As shown, overexpressing transgenic lines (OE-3, OE-4, OE-6) and gene knockout lines (KO-2, KO-4, KO-5) were obtained. qPCR was used to detect... Zm00001eb247080 Gene expression levels under drought treatment. Under drought stress, the expression levels of overexpressing lines (OE-3, OE-4, OE-6)... Zm00001eb247080 Gene expression levels in all gene knockout lines (KO-2, KO-4, KO-5) were significantly higher than in the non-transgenic line (WT), while the expression levels in these lines were significantly lower than in the non-transgenic line (WT), indicating that... Zm00001eb247080 Genes can respond to drought stress and are regulated at the transcriptional level. RT-qPCR validation further confirmed the reliability of the transcriptome data.

[0056] 3.5 Phenotypic and physiological analysis of transgenic maize under drought stress After drought stress, Zm00001eb247080 The knockout strains were more wilted than the control wild-type plants, and Zm00001eb247080 The overexpression lines showed significantly less wilting than the wild-type plants, indicating that... Zm00001eb247080 Genes are positive regulators of maize's response to drought stress. Meanwhile, overexpression lines showed significantly higher root biomass and lateral root number than wild-type lines, while root development was significantly inhibited in knockout lines, confirming... Zm00001eb247080 Its key role in promoting root development.

[0057] 3.5.1 Analysis of hormone indicators in transgenic plants under drought stress Under drought stress, the ABA and IAA contents of overexpression lines (OE-3, OE-4, OE-6) were significantly higher than those of non-transgenic lines (WT), while the ABA and IAA contents of gene knockout lines (KO-2, KO-4, KO-5) were significantly lower than those of wild type. This indicates that under drought stress, maize... Zm00001eb247080 The gene can increase ABA content, which is beneficial for inducing the expression of downstream stress-resistance genes; at the same time, it increases IAA content, promotes cell elongation and root development, thereby improving the ability of maize plants to resist drought stress. Figure 3 (China HI).

[0058] 3.5.2 Analysis of antioxidant enzyme activity in transgenic plants under drought stress Under drought stress, the activities of SOD, POD, and CAT in overexpression lines (OE-3, OE-4, OE-6) were significantly higher than those in the non-transgenic line (WT), while the activities of these antioxidant enzymes in gene knockout lines (KO-2, KO-4, KO-5) were significantly lower than those in the wild type. This indicates that under drought stress, maize... Zm00001eb247080 Genes can enhance the plant's antioxidant defense capabilities, effectively alleviate drought-induced oxidative damage, and thus improve the corn plant's ability to resist drought stress. Figure 3 (Zhong EG).

[0059] 3.5.3 Root Vitality Analysis of Transgenic Plants under Drought Stress Under drought stress, the root activity of overexpression lines (OE-3, OE-4, OE-6) was significantly higher than that of non-transgenic lines (WT), while the root activity of gene knockout lines (KO-2, KO-4, KO-5) was significantly lower than that of wild type. This indicates that under drought stress, maize... Zm00001eb247080 Genes can enhance root vigor, thereby improving the ability of corn plants to resist drought stress. Figure 3 (J).

[0060] 3.6 Multi-omics integration analysis and construction of WGCNA co-expression network Combined transcriptomic and proteomic analysis revealed that drought stress induced thousands of differentially expressed genes (DEGs) and proteins (DEPs) in both genotypes, primarily enriched in pathways related to plant hormone signal transduction, carbon metabolism, cell wall modification, reactive oxygen species scavenging, and osmotic regulation. WGCNA analysis identified several co-expression modules significantly associated with root development and drought resistance. Figure 6 Among them, the MEblack module showed a significant positive correlation with SOD, POD, CAT activity and ABA, IAA content, while the MEmagenta and MEturquoise modules were closely related to root length, root volume and yield-related traits. Figure 7 ).

[0061] 3.7 Results of KEGG pathway enrichment analysis KEGG pathway enrichment analysis was performed on the key co-expression module genes identified in the WGCNA analysis, and the results are as follows: Figures 8-11 As shown, the MEblack module is mainly associated with antioxidant defense and protein turnover, the MEbrown module is associated with carbon metabolism and biosynthesis, the MEmagenta module is associated with hormone signal transduction and the MAPK signaling pathway, and the MEturquoise module is associated with protein synthesis and vesicle transport. These modules together constitute a multilayer co-expression regulatory network related to drought resistance in maize.

[0062] 3.8 Results of yeast two-hybrid screening and point-to-point validation Results of yeast two-hybrid library screening ( Figure 4 )show, Zm00001eb247080 It can directly interact with multiple stress-related proteins, including bZIP transcription factor, NAC transcription factor, WRKY transcription factor, and reactive oxygen species scavenging-related proteins. Point-to-point rotational validation ( Figure 5 This further confirms the reliability of the aforementioned interaction relationship. These results indicate that... Zm00001eb247080 It may play a central role in regulating drought tolerance in maize by participating in the construction of complex protein-protein interaction networks and coordinating root development and stress defense processes.

[0063] In summary, this invention provides a drought-resistant material. Zm00001eb247080 Gene, Zm00001eb247080 The gene-encoded protein is located in the cell nucleus and plays an important role in nuclear-related signal transduction. Under drought stress, the overexpressing lines... Zm00001eb247080 Gene expression levels in overexpression lines were significantly higher than in non-transgenic lines, while gene knockout lines showed significantly lower expression levels than non-transgenic lines. Simultaneously, overexpression lines exhibited significantly increased root biomass and lateral root number, as well as significantly elevated activities of antioxidant enzymes (SOD, POD, CAT) and ABA and IAA levels. Yeast two-hybrid screening and point-to-point validation further revealed… Zm00001eb247080 It can directly interact with multiple stress-response proteins, forming a drought resistance regulatory network centered on this gene. This invention provides... Zm00001eb247080 Genes have a significant effect on improving drought resistance in maize, providing important genetic resources and theoretical basis for molecular breeding of drought-resistant maize.

[0064] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A drought-resistant variety Zm00001eb247080 Genes, characterized by, The Zm00001eb247080 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The claim 1 Zm00001eb247080 Gene-encoded proteins are characterized by, The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. Overexpression of the expression described in claim 1 Zm00001eb247080 The application of gene-based preparations in any of the following: (1) Improve plant drought resistance; (2) Prepare products that improve plant drought resistance; (3) Cultivate drought-resistant plant varieties; (4) Breeding of drought-resistant plants.

4. The application as described in claim 3, characterized in that, The preparation includes containing Zm00001eb247080 A recombinant vector of the gene or a host bacterium containing the recombinant vector.

5. The use of a formulation that increases the expression level of the protein of claim 2 in any of the following: (1) Improve plant drought resistance; (2) Prepare products that improve plant drought resistance; (3) Cultivate drought-resistant plant varieties; (4) Breeding of drought-resistant plants.

6. The application as described in any one of claims 3-5, characterized in that, The plant mentioned includes corn.

7. A method for improving plant drought resistance, characterized in that, Including overexpression in plants Zm00001eb247080 Genes are used to improve plant drought resistance. The Zm00001eb247080 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

8. The method as described in claim 7, characterized in that, The plant mentioned includes corn.

9. A method for cultivating drought-resistant plant varieties, characterized in that, Including overexpression in plants Zm00001eb247080 Genes, Enhance Zm00001eb247080 Gene expression levels are a key step in obtaining drought-resistant plant varieties. The Zm00001eb247080 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

10. The method as described in claim 9, characterized in that, The plant mentioned includes corn.

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