Application of corn gene Zm00001d034479 in improving drought resistance of plants

Abstract

The present application relates to the field of biotechnology, and particularly relates to the application of a maize gene Zm00001d034479 in improving plants, crops belonging to the plants, and particularly in improving the drought resistance of maize; particularly in the application of maize germplasm improvement, in the application of maize genetic breeding, or in the application of corresponding drought-resistant transgenic crops of maize. The present application provides a new drought-resistant gene resource, and provides a new target gene for drought-resistant molecular breeding of maize and other monocotyledonous crops, and a transgenic drought-resistant maize breeding method based on the gene; the present application expands the application range, and the function verification of the gene in a model plant provides a theoretical reference for the application of the gene in other crops. The present application proves that the gene has a function of improving the drought resistance of maize, thereby providing a new technical means for the cultivation of new strains of drought-resistant maize and the innovation of germplasm resources, and thus the present application has important agricultural application value and a wide practical prospect.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of the maize gene Zm00001d034479 in improving the drought resistance of plants, especially the crop maize. Background Technology

[0002] Drought severely affects the growth, development, and yield of plants, especially crops, and has become one of the central topics of plant stress.

[0003] As the world's number one crop, the yield and quality of corn are also seriously threatened by drought and water shortage.

[0004] Current technology predicts that the maize gene Zm00001d034479 encodes histone H1, but its function in plant abiotic stress response has not been reported. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to reduce the impact of drought stress on plants and monocotyledonous crops, especially on the growth, development and yield of maize, that is, how to improve the drought stress resistance of maize.

[0006] The technical solution of the present invention is to provide the application of overexpression of maize gene Zm00001d034479 in improving the drought resistance of maize, and preferably in improving the drought resistance of maize B73-329. The nucleotide sequence of the maize gene Zm00001d034479 is shown in SEQ ID NO. 1.

[0007] This invention also provides the application of the maize gene Zm00001d034479 in the improvement of maize germplasm resources, genetic breeding, or in the preparation of drought-resistant transgenic maize crops.

[0008] Furthermore, the present invention also provides a method for improving the drought resistance of maize crop by integrating the maize gene Zm00001d034479 into the cells, tissues or organs of maize crop B73-329 and overexpressing it.

[0009] This invention also provides a method for breeding highly drought-resistant maize, comprising the following steps: constructing a plant expression vector containing the maize gene Zm00001d034479; transforming the expression vector into the immature embryo of maize B73-329; obtaining T0 generation transgenic maize through co-culture, resistance screening, callus induction, subculture and differentiation; and obtaining stable homozygous transgenic lines through continuous self-pollination and molecular detection, which are highly drought-resistant maize. The nucleotide sequence of the maize gene Zm00001d034479 is shown in SEQ ID NO. 1 of the sequence listing.

[0010] Finally, this invention also provides the application of overexpressed maize gene Zm00001d034479 in drought resistance in Arabidopsis thaliana. Specifically, heterologous expression of the maize gene Zm00001d034479 in Arabidopsis thaliana can improve the drought resistance of transgenic Arabidopsis thaliana, indicating that the function of this gene is conserved in plants, providing a theoretical basis for its application in other crops.

[0011] This invention reveals the function of gene Zm00001d034479 through the following research: 1. Expression pattern analysis: By creating GUS and RUBY reporter gene materials driven by the Zm00001d034479 promoter, it was found that this gene is expressed in maize root, stem, and leaf tissues, exhibiting constitutive expression characteristics; further analysis showed that this gene has a relatively high expression level in guard cells. 2. Functional verification: By creating gene-silenced maize material of Zm00001d034479, experiments revealed that gene-silenced maize material of Zm00001d034479 exhibited drought stress sensitivity phenotypes such as increased leaf temperature and larger stomatal conductance, indicating that this gene is essential for normal drought resistance response in maize; and overexpression of Zm00001d034479 in transgenic maize promoted enhanced drought tolerance in transgenic maize. This indicates that the gene plays an important role in plant responses to drought stress, thus providing a new technical means to utilize this gene to breed superior crop lines with enhanced drought tolerance. 3. Cross-species verification: Heterologous expression of this gene in the model plant Arabidopsis thaliana resulted in transgenic Arabidopsis thaliana also exhibiting enhanced drought resistance, indicating that the function of this gene is conserved in plants.

[0012] This invention reveals for the first time the positive regulatory function of the maize histone H1 encoding gene Zm00001d034479 in plant drought stress response.

[0013] The beneficial effects of this invention are mainly reflected in the following three aspects: 1. This invention provides new drought-resistant gene resources, offering new target genes for drought-resistant molecular breeding of monocotyledonous crops such as maize. 2. This invention establishes an effective technical method, providing a method for breeding transgenic drought-resistant maize based on the Zm00001d034479 gene. 3. This invention expands the scope of application; the functional verification of this gene in model plants provides a theoretical reference for its application in other crops.

[0014] More specifically, by providing the application of the maize gene Zm00001d034479 in improving the drought resistance of plants and their related crop maize, especially in improving the drought resistance of maize B73-329, in germplasm resource improvement, in genetic breeding, or in the preparation of corresponding drought-resistant transgenic crops, and by providing methods for improving the drought resistance of maize and methods for breeding highly drought-resistant maize, this invention provides new technical means for breeding new maize varieties with high drought resistance and for the innovation of excellent maize germplasm resources. Therefore, this invention has important agricultural application value and broad practical prospects. Attached Figure Description

[0015] Figure 1 This document describes the constitutive expression validation of the maize gene Zm00001d034479 (expression in the whole plant of ProZmH1A::RUBY). A represents the accumulation of betaine red in maize seeds in the ProZmH1A::RUBY maize material; B represents the accumulation of betaine red in roots and stems in the ProZmH1A::RUBY maize material; C represents the accumulation of betaine red in leaves in the ProZmH1A::RUBY maize material; and D represents the observation of the whole plant of the ProZmH1A::RUBY positive maize material.

[0016] Figure 2 This study describes the GUS staining results of guard cells in leaves of ProZmH1A::GUS transgenic maize and the accumulation of betaine red in guard cells of the lower epidermis of ProZmH1A::RUBY transgenic maize. In this study, A represents the epidermal strip staining analysis of ProZmH1A::GUS transgenic material, and B represents the observation and analysis of betaine red in the lower epidermis of ProZmH1A::RUBY maize material.

[0017] Figure 3 To confirm that the ZmH1A protein is located in the cell nucleus, A represents tobacco leaves after the empty vector was transformed into competent Agrobacterium cells and co-injected with a nuclear marker, and the fluorescence signal was observed using a laser scanning confocal microscope; B represents tobacco leaves after the recombinant vector was transformed into competent Agrobacterium cells and co-injected with a nuclear marker, and the fluorescence signal was observed using a laser scanning confocal microscope.

[0018] Figure 4This document describes the identification of drought tolerance phenotypes in Arabidopsis thaliana genetic materials overexpressing ZmH1A. A shows camera images of the ZmH1A transgenic Arabidopsis thaliana materials ZmH1A (OE-1, OE-2) and wild-type Col-0 under normal growth conditions; B shows far-infrared images of the ZmH1A transgenic Arabidopsis thaliana materials ZmH1A (OE-1, OE-2) and wild-type Col-0 under normal growth conditions; and C shows leaf surface temperature statistics for the ZmH1A transgenic Arabidopsis thaliana materials ZmH1A (OE-1, OE-2) and wild-type Col-0 under normal growth conditions.

[0019] Figure 5 This study describes the identification of drought tolerance phenotypes in ZmH1A overexpressing maize lines during the seedling stage. A and B show phenotypic comparisons of ZmH1AOE-1, ZmH1AOE-2, and WT materials; C shows leaf temperature measurements of ZmH1AOE-1, ZmH1AOE-2, and WT; D shows the transgenic overexpression material and WT material before drought treatment; E shows the transgenic overexpression material and WT material after 5 days of drought treatment; F shows the transgenic overexpression material and WT material after rehydration following severe drought treatment; G shows stomatal conductance measurements of ZmH1AOE-1, ZmH1AOE-2, and WT; H shows moisture content measurements of ZmH1AOE-1, ZmH1AOE-2, and WT; and I shows MDA content measurements of ZmH1AOE-1, ZmH1AOE-2, and WT under normal and drought conditions.

[0020] Figure 6 This document describes the identification of drought sensitivity in ZmH1A-VIGS-silenced maize seedlings. A and B show phenotypic comparisons of the positive control ISPH, the negative control LUC, and the experimental group's ZmH1A-VIGS material with transient ZmH1A expression after virus infection. C shows a phenotypic comparison of the positive control ISPH, the negative control LUC, and the experimental group's ZmH1A-VIGS material with transient ZmH1A expression under normal growth conditions. D shows a phenotypic comparison of the positive control ISPH, the negative control LUC, and the experimental group's ZmH1A-VIGS material with transient ZmH1A expression after drought treatment. E represents the positive control. Comparison of ZmH1A expression levels in ISPH, negative control LUC, and experimental ZmH1A transient expression material ZmH1A-VIGS; F is a comparison of leaf temperature in positive control ISPH, negative control LUC, and experimental ZmH1A transient expression material ZmH1A-VIGS; G is a comparison of stomatal conductance in leaves in positive control ISPH, negative control LUC, and experimental ZmH1A transient expression material ZmH1A-VIGS; H is a comparison of leaf transpiration rate in positive control ISPH, negative control LUC, and experimental ZmH1A transient expression material ZmH1A-VIGS. Detailed Implementation

[0021] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Unless otherwise specified, the methods used in the embodiments are conventional methods known to those skilled in the art, and the reagents and materials used are commercially available products. The maize material B73-329 used in this invention was provided by the Maize Molecular Breeding and Gene Editing Platform of the State Key Laboratory of Crop Stress Adaptation and Improvement, Henan University; the Arabidopsis material was the Columbia-0 ecotype preserved in the laboratory.

[0023] For ease of description and concise experimental records, the maize gene Zm00001d034479 is simply represented as ZmH1A in this invention; the transgenic overexpression materials of maize Zm00001d034479 are labeled as ZmH1AOE-1 and ZmH1AOE-2, respectively.

[0024] Example 1. Confirmation that ZmH1A is a constitutively expressed gene.

[0025] (1) First, we created RUBY maize genetic material. Since the RUBY gene is activated and expressed in plant cells, a large amount of red betaine accumulates within the cells, and the red pigment deposition can be observed with the naked eye, we created ProZmH1A::RUBY material. Using B73-329 maize genomic DNA as a template, we amplified the promoter region of the ZmH1A gene (2000 bp upstream of the transcription start site) by PCR. After enzyme digestion, the amplified fragment was ligated into the pro-RUBY betaine gene expression vector to construct the ProZmH1A::RUBY recombinant plasmid. After sequencing verification, the correct recombinant plasmid was introduced into Agrobacterium LBA4404 competent cells and cultured at 28℃ for 2 days. Single colonies were picked for PCR identification, and positive clones were used for subsequent transformation. The above recombinant Agrobacterium was submitted to the laboratory maize genetic transformation platform to transform maize embryos. Transgenic plants were obtained through co-culture, screening, and regeneration. Positive plants were detected using Bar test strips.

[0026] (2) Tissue localization detection:

[0027] ProZmH1A::RUBY positive plants were simultaneously planted in a greenhouse with wild-type B73-329. Root, stem, and leaf tissues were collected at the three-leaf stage of maize for observation. Results showed that stable accumulation of red betaine (a substance) was observed in all tissues of the transgenic plants. Figure 1This indicates that ZmH1A is a constitutively expressed gene.

[0028] Example 2. Confirmation of specific expression of ZmH1A in stomatal guard cells.

[0029] (1) To verify that ZmH1A can participate in the regulation of stomatal movement, this study constructed a ZmH1A promoter-driven GUS reporter system. The promoter fragment of the ZmH1A gene was ligated into the β-glucuronide acetyltransferase (GUS) gene expression vector to obtain the ProZmH1A::GUS recombinant vector. This vector was transformed into Agrobacterium LBA4404 competent cells and cultured in a constant temperature incubator at 28℃ for about 2 days. Positive clones were screened by colony PCR, and after expansion culture, they were submitted to the maize genetic transformation platform to obtain transgenic plants. Seedlings obtained after culture were used after testing positive with bar test strips.

[0030] (2) GUS histochemical staining: Positive seeds were planted in boxes and cultured in a greenhouse. The lower epidermis of the leaves of transgenic GUS plants that had grown to the three-leaf stage was carefully dissected. The upper epidermis was cut open with a blade but not cut through, and then the upper epidermis was peeled off with pointed tweezers. Two drops of deionized water were placed on a flat plastic plate, and the lower epidermis was placed in it. The mesophyll tissue on the lower epidermis was removed with a brush. The lower epidermis was placed in a centrifuge tube containing GUS staining solution (preparation method as follows), the centrifuge tube was wrapped with aluminum foil, and placed in a 37°C incubator for staining overnight. The next day, the lower epidermis was destained in 75% ethanol. The lower epidermis was then prepared into a slide and observed using an Olympus CX-43 optical microscope. The results showed that ZmH1A was mainly found in the guard cells and subsidiary cells of maize ( Figure 2 (A), which corresponds to its function as a histone in regulating stomatal opening and closing in response to drought stress.

[0031] GUS staining solution preparation: Use the Beijing Solarbio GUS staining solution kit to prepare the solution. Add 229 μL of X-Glu A solvent from the kit to an 80 mg X-GlcA centrifuge tube and mix gently to obtain the X-Glu A working solution. Aliquot 20 μL into 200 mL centrifuge tubes and store at -20°C protected from light. Take a sterile 10 mL centrifuge tube and add 2.5 mL of GUS buffer A, 10 μL of GUS buffer B, 10 μL of GUS buffer C, 2 mL of methanol, 20 μL of X-Glc A working solution, and 5.5 mL of deionized water. Mix well and aliquot 1 mL into 1.5 mL centrifuge tubes. Store at 4°C.

[0032] (3) Observation of the lower epidermis of maize leaves using ProZmH1A::RUBY

[0033] Simultaneously, verification was performed using the ProZmH1A::RUBY system. Previously obtained RUBY-positive seeds were planted in boxes, and when they reached the three-leaf stage, the lower epidermis of the positive seedlings was peeled off. The accumulation of betaine red pigment in various cells, including the epidermis, was observed under an optical microscope. The results showed that betaine red pigment accumulation was significant in the guard cells of maize. Figure 2 The B result indicates that ZmH1A is expressed relatively specifically in maize guard cells, which is consistent with the accumulation of GUS staining in maize guard cells, both of which can prove that ZmH1A is expressed relatively specifically in maize guard cells.

[0034] Example 3. Confirmation that the ZmH1A protein is located in the cell nucleus.

[0035] As a member of the histone family, we hypothesized that ZmH1A resides in the cell nucleus and plays a role in inducing stomatal opening and closing. To investigate the subcellular localization of this protein, a fusion expression vector of ZmH1A and green fluorescent protein (GFP) driven by the constitutive expression promoter 35S was constructed, and the expression localization of ZmH1A was detected using a tobacco expression system. After transforming the fusion expression vector (Pro35S::ZmH1A-GFP) into Agrobacterium GV3101 competent cells, correctly selected single colonies were picked, and after shaking, they were injected into tobacco plants that had grown for 4-5 weeks in good condition. The results showed that the GFP fluorescence of the ZmH1A-GFP recombinant protein co-localized with the fluorescence signal of the nuclear marker (pr35S::H2A:mCherry). Figure 3 The above experimental results indicate that ZmH1A exists in the maize cell nucleus, suggesting that it may function as a histone to regulate stomatal opening and closing in response to drought stress.

[0036] Example 4. Confirmation of drought tolerance phenotype overexpression in Arabidopsis Zm00001d034479 transgenic strain.

[0037] To preliminarily confirm the functional mechanism of ZmH1A in regulating plant response to drought stress, particularly in stomatal closure and drought tolerance, this study first created transgenic Arabidopsis materials that overexpress ZmH1A. By creating heterologous Arabidopsis materials, we can more quickly explore the relevant phenotypes of the ZmH1A gene.

[0038] A method using Agrobacterium-mediated transformation of Arabidopsis thaliana (method as follows) was employed to create Arabidopsis thaliana transgenic overexpressing the ZmH1A gene. Normal Arabidopsis thaliana seeds (wild-type and transgenic overexpression) were sown on MS solid medium and vernalized at 4°C for 2-4 days, then transferred to a suitable temperature for 10 days of cultivation. Plants with uniform growth were selected and transplanted into nutrient soil, where they were cultured under strictly controlled environmental conditions (temperature 22°C, light 16 hours, humidity 75%). Under normal growth conditions, the leaf temperature was higher than that of wild-type Arabidopsis thaliana materials. Figure 4Preliminary evidence suggests that ZmH1A is related to moisture loss and the degree of stomatal opening.

[0039] Transgenic Arabidopsis thaliana infection and transformation: The recombinant Agrobacterium tumefaciens to be transformed was inoculated into YEP liquid medium containing the corresponding antibiotic and cultured overnight for about 16 h at 220 rpm in a constant temperature shaker at 28℃. When the OD600 of the bacterial suspension reached 1.0-1.2, the bacterial suspension was poured into a 50 mL sterile centrifuge tube and centrifuged at 5,000 rpm for 5 min at room temperature. The supernatant was discarded. An appropriate amount of resuspension (1 / 2 MS + 5% sucrose) was added and mixed thoroughly. The OD600 of the mixture was adjusted to 0.8-1.0, and surfactant Silwet-L77 was added at a ratio of 5,000:1. The mixture was thoroughly mixed to prepare the infection bacterial suspension. Unopened flower buds of wild-type Arabidopsis thaliana were placed in the infection bacterial suspension for 1 min and cultured in a humid and dark environment for 24 h before returning to normal culture conditions. After culturing under normal conditions for 5-7 days, newly formed flower buds were infected again. After the seeds matured, they were collected and further identified.

[0040] Screening and identification of transgenic materials: Arabidopsis transgenic materials were screened for resistance. Seeds of Arabidopsis infected with Agrobacterium were sterilized in a clean bench with 10% NaClO solution for 5 min, with continuous shaking during the process. They were then rinsed 5-8 times with sterile water. The seeds were transferred to MS solid medium containing the corresponding hygromycin. After the surface of the medium dried, it was sealed with sealing film and vernalized in a refrigerator at 4°C for 2-4 days. The culture dishes were then wrapped with aluminum foil and cultured in the dark in an incubator. After 3 days, the seedlings were cultured under normal light and their growth was observed. Positive plants showed good growth, while negative plants showed poor growth.

[0041] Example 5. Creation of ZmH1A overexpression maize materials and phenotypic identification

[0042] (1) Creation and expression detection of transgenic overexpression genetic germplasm

[0043] Using cDNA from wild-type maize B73-329 as a template, specific primers were designed, and the ZmH1A gene was amplified by PCR. The amplified fragment was purified and recovered. The purified and recovered amplified fragment was ligated with the transgenic overexpression empty vector pCMBIA3300, which was digested with restriction endonucleases (Novizan one-step cloning ligation). The ligation product was transformed into competent E. coli cells and cultured overnight on solid LB medium containing kanamycin to screen for positive transformants. The recombinant plasmid DNA of the successfully ligated cells was extracted and sequenced. The recombinant vector was transformed into Agrobacterium EHA105 strain and submitted to the Maize Molecular Breeding and Gene Editing Platform of the State Key Laboratory of Crop Stress Adaptation and Improvement, Henan University, for the creation of transgenic overexpression germplasm.

[0044] Single colonies of recombinant Agrobacterium were inoculated into 2-3 mL of liquid medium containing 100 μg / mL kanamycin and 50 μg / mL rifampin, and cultured overnight at 28°C with shaking. The next day, the colonies were transferred to 50 mL of liquid medium containing the same antibiotics and cultured with shaking. After centrifugation, the bacterial cells were collected, resuspended, and the OD600 was adjusted to 0.8-1.0 to prepare the recombinant Agrobacterium infection solution. This infection solution was used to transform B73-329 maize embryos. After co-culture, resistance was selected using glufosinate as a selection marker. Subsequently, pre-differentiation, differentiation, and rooting cultures were completed sequentially to obtain T0 generation regenerated plants. T0 generation transgenic positive plants were self-pollinated to harvest T1 generation seeds. T1 generation plants were further self-pollinated to the T3 generation to obtain stable overexpression transgenic lines, named OE-1 and OE-2, respectively. In the above transformation process, untransformed maize B73-329 was used as a wild-type control (WT).

[0045] (2) In order to further confirm the role of ZmH1A in the drought stress response of maize, the inventors tested the phenotype and physiological indicators of transgenic maize materials overexpressing ZmH1A.

[0046] ① Far-infrared thermal imaging experiment of transgenic maize materials

[0047] Stomata are the main channels for water and gas exchange between leaves and the atmosphere, and stomatal movement directly controls over 90% of plant water loss through transpiration. Water loss through transpiration effectively reduces leaf surface temperature. Therefore, abnormal stomatal opening and closing directly leads to changes in leaf water status and temperature. Far-infrared imaging technology for leaves provides a powerful tool for efficiently screening mutants with abnormal stomatal function. We conducted seedling phenotypic identification by planting WT wild-type maize and transgenic maize materials overexpressing ZmH1A in a constant-temperature culture room.

[0048] The specific method is as follows: In the material room, mix vermiculite and nutrient soil in a 1:3 ratio, add an appropriate amount of water and carbendazim, and mix evenly; evenly fill the mixed nutrient soil into the box; select 4 plump wild-type WT and 4 overexpression maize materials each; place the wild-type seeds on the left and the seeds of the two overexpression lines on the right, stuff the seeds into the soil and level the soil; place a flat plate under the box and pour an appropriate amount of water into the plate; place it in a constant temperature incubation room at 25℃, with suitable light conditions, and a light duration of 8:00-22:00; when the maize materials have grown for 8-13 days, use a FLIRT865 infrared thermal imaging system camera to take infrared photos, observe the far-infrared thermal imaging of wild-type and overexpression maize materials and take photos.

[0049] The temperature of maize seedling leaves was monitored using a far-infrared thermal imaging system during drought treatment. The results showed that the leaf surface temperature of ZmH1A overexpressing lines was significantly higher than that of WT plants. Figure 5 (A, B, C).

[0050] ② Determination of stomatal conductance in maize leaves

[0051] Plants effectively regulate leaf surface temperature through stomatal transpiration, which reflects their ability to retain internal water and their tolerance to drought. To investigate whether ZmH1A affects drought stress response in maize by regulating stomatal movement, we further conducted physiological phenotypic measurements on the overexpressing lines. Water supply was stopped at 10 days of growth, and the materials were subjected to drought treatment. After 5 days of drought treatment, comparisons between the transgenic overexpressing ZmH1A maize lines ZmH1AOE-1 and ZmH1AOE-2 and the wild-type material WT revealed that the transgenic overexpressing lines exhibited stronger drought tolerance than the wild-type WT. After severe drought and rehydration, the wild-type WT material died, while most of the transgenic overexpressing maize materials recovered to normal growth status. Figure 5 The results (D, E, F) indicate that overexpression of ZmH1A enhances drought tolerance in maize seedlings, which corresponds to higher leaf temperature and lower stomatal conductance in the transgenic overexpression material. The results also show that the stomatal conductance of the ZmH1A overexpression lines was significantly lower than that of the wild-type WT (D, E, F). Figure 5 The G indicates that overexpression of ZmH1A leads to a decrease in stomatal conductance and less water loss from leaves, which means stronger water retention capacity and higher drought tolerance.

[0052] ③ Determination of malondialdehyde (MDA) content:

[0053] This study used Solarbio's visible spectrophotometry method to determine malondialdehyde (MDA) content. First, the MDA working solution was prepared by dissolving one bottle of reagent II in 20 mL of reagent I. 0.1 g of corn leaves were weighed, flash-frozen in liquid nitrogen, and thoroughly ground. 1 mL of extract was added and homogenized in an ice bath. After centrifugation at 8,000 rpm and 4℃ for 10 min, the supernatant was collected. 200 μL of the supernatant was mixed with 600 μL of the MDA working solution and 200 μL of reagent III to form a test tube; another 600 μL of the working solution, 200 μL of distilled water, and 200 μL of reagent III were mixed to form a blank tube. After sealing both tubes, they were incubated in a water bath at 100℃ for 60 min, cooled in an ice bath, and centrifuged at 12,000 rpm at room temperature for 10 min. The supernatant was collected, and the tubes were zeroed with deionized water. The absorbance of the blank tube and the test tube was measured at 532 nm and 600 nm, respectively. The final MDA content (nmol / g mass) was calculated using the formula ΔA = (A532 measurement - A532 blank) - (A600 measurement - A600 blank) = 32.258 × ΔA ÷ 0.1. Three replicates were set up for wild-type and overexpression maize materials under normal growth conditions and drought conditions, respectively.

[0054] Relative water content (RWC) in leaves directly reflects the degree of water deficit and water retention capacity of plants under drought stress, while malondialdehyde (MDA) content characterizes the degree of lipid peroxidation damage to cell membranes. Together, they constitute the core physiological indicators for assessing plant water retention capacity and membrane system stability. To investigate the effects of ZmH1A on water status and membrane system stability in maize, the RWC and MDA contents of different lines under drought stress were measured. The results showed that drought stress significantly reduced RWC in maize leaves, but the RWC of overexpressing lines was significantly higher than that of WT plants (…). Figure 5 Meanwhile, MDA content increased significantly under drought stress, while the MDA content of the overexpressing lines was significantly lower than that of the WT plants (H); Figure 5 The above results indicate that ZmH1A positively regulates the drought resistance of maize by enhancing the water-holding capacity of the plant and reducing membrane lipid peroxidation damage, further revealing the important role of ZmH1A in maize's response to drought stress and regulation of stomatal movement.

[0055] Example 6. Creation and Phenotypic Identification of ZmH1A Gene-Silenced Maize Materials

[0056] To investigate the function of ZmH1A in the regulation of stomatal movement and drought tolerance in maize, the inventors created a transient expression material of ZmH1A based on the VIGS system of cucumber mosaic virus (CMV), namely the pCMVZ-2bN81::ZmH1A fusion vector containing a partial fragment of the CDS of ZmH1A, and transformed this fusion vector into Agrobacterium GV3101 competent cells. Three infection combinations (positive control: pCMVZ-2bN81::ISPH, pCMV-F1, pCMV-FZ3; negative control: pCMVZ-2bN81::LUC, pCMV-F1, pCMV-FZ3; experimental group: pCMVZ-2bN81::ZmH1A, pCMV-F1, pCMV-FZ3) were used to infect tobacco plants that were in good growth condition for 4-5 weeks. The plants were then treated in the dark for 24-48 hours and cultured under normal light conditions for 3 days. The virus was extracted from the tobacco and injected into the endosperm of B73 maize seeds using the needle prick method. After 2 days of dark culture, the plants were transferred to nutrient soil for culture, and the phenotype was observed for 5-10 days.

[0057] After three trials, the results were as follows: Figure 6 As shown, compared with the positive control ISPH and the negative control LUC, the ZmH1A gene transcript level was significantly reduced in the ZmH1A-VIGS experimental group maize materials, exhibiting a phenotype of lower leaf temperature, higher stomatal conductance, and drought sensitivity. This corroborates the role of ZmH1A in influencing stomatal movement and drought tolerance in maize, as demonstrated by the transgenic overexpression materials.

[0058] The above description is only a preferred embodiment of the present invention. It should be noted that any modifications made by those skilled in the art without departing from the principle of the present invention are considered to be within the scope of protection of the present invention.

Claims

1. Application of overexpression of maize gene Zm00001d034479 in improving drought resistance of maize crop, wherein the nucleotide sequence of maize gene Zm00001d034479 is shown in SEQ ID NO.1 of the sequence listing.

2. The application as described in claim 1, characterized in that, The crop in question is corn variety B73-329.

3. The application as described in claim 1 or 2, characterized in that, It is applied to the improvement of maize germplasm resources.

4. The application as described in claim 1 or 2, characterized in that, It is applied to maize genetic breeding.

5. The application as described in claim 1 or 2, characterized in that, It is used to prepare drought-resistant genetically modified corn.

6. A method for improving the drought resistance of maize, characterized in that, The maize gene Zm00001d034479 described in claim 1 is integrated into the cells, tissues, or organs of the crop maize B73-329 and overexpressed.

7. A method for cultivating highly drought-resistant maize, characterized in that, The procedure includes the following steps: constructing a plant expression vector containing the maize gene Zm00001d034479; transforming the expression vector into the immature embryo of maize B73-329; obtaining T0 generation transgenic maize through co-culture, resistance screening, callus induction, subculture, and differentiation; and obtaining stable, homozygous transgenic lines, i.e., drought-resistant maize, through continuous self-pollination and molecular detection. The nucleotide sequence of the maize gene Zm00001d034479 is shown in SEQ ID NO.1 of the sequence listing.

8. The application of overexpression of the maize gene Zm00001d034479 in improving drought resistance in Arabidopsis thaliana, characterized in that, The nucleotide sequence of the maize gene Zm00001d034479 is shown in SEQ ID NO.1.

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