Application of bHLH18 transcription factor in regulation and control of plant stress resistance

Abstract

The invention discloses application of a bHLH18 transcription factor in regulation and control of plant stress resistance, and belongs to the technical field of biology. The invention reports the drought resistance positive regulation related protein ZmbHLH18 and the coding gene thereof for the first time. A corn knockout strain and an overexpression strain of the gene are obtained, drought-related physiological indexes are measured, the overexpression of the ZmbHLH18 can improve the drought tolerance of corn, and the overexpression strain shows higher leaf water content, lower water loss rate, higher antioxidant enzyme activity and stronger photosynthesis stability. The physiological indexes directly reflect the survivability of the plants under the drought condition, so that the drought resistance of the plants is enhanced, the positive regulation effect on the drought resistance of the plants is shown, the technical scheme provides a basis for cultivating resistant plants, and a more accurate and quicker way is provided for drought-resistant breeding of corns.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically involving bHLH18 Application of transcription factors in regulating plant stress resistance. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Maize is a water-intensive crop and is highly sensitive to drought stress. Global warming has led to more frequent extreme weather events such as droughts, seriously threatening stable maize yields. Enhancing maize's drought resistance is a core breeding strategy. Currently, research on plant stress resistance has gradually penetrated to the cellular and molecular levels and is combined with genetics and genetic engineering research. Biotechnology can be used to improve plant growth characteristics, thereby enhancing plants' ability to adapt to adverse conditions.

[0004] Transcription factors are regulatory genes and important molecules in regulatory networks. They participate in almost all life processes of organisms through signal transduction and the regulation of stress-responsive genes. Among them, bHLH transcription factors are an important class of transcription factors discovered in recent years. They not only participate in plant growth, development, and signal transduction, but also regulate plant responses to abiotic stresses such as drought. Some bHLH genes respond positively to drought stress, some negatively, and others exhibit irregular expression changes. Further analysis of bHLH gene function is needed to elucidate the molecular mechanisms of drought resistance and discover new and effective gene resources, providing crucial support for breeding new drought-resistant maize varieties with broad adaptability. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this invention is to provide... bHLH18 Application of transcription factors in regulating plant stress resistance.

[0006] This invention, through the ZmbHLH18 Genetic studies revealed that this gene responds to drought induction, with its expression level upregulated after drought treatment. The encoded protein belongs to the bHLH transcription factor family and possesses transcriptional activation activity. Knockout and overexpression lines of this gene were obtained in maize, and drought-related physiological indicators were measured. Overexpression of this gene improved maize drought tolerance, while knockout reduced drought tolerance, indicating that this gene plays a positive regulatory role in maize drought resistance. Subsequently, transcriptome sequencing and yeast one-hybrid sequencing were used to preliminarily elucidate the mechanism of action of this gene, which has important guiding significance for enhancing the production potential of maize under drought conditions and promoting agricultural development.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a protein, named ZmbHLH18, which is any one of the following a1)-a3): a1) Proteins as shown in SEQ ID NO. 1; a2) Proteins derived from SEQ ID NO. 1 that have the same function as the amino acid sequence shown in SEQ ID NO. 1, but with one or more amino acid residues substituted and / or deleted and / or added; a3) Other genes encoding proteins that have an amino acid sequence similarity of more than 80% to that shown in SEQ ID NO. 1 and have the activity of the protein shown in SEQ ID NO. 1.

[0008] In a2) above, the “substitution and / or deletion and / or addition of one or more amino acid residues” refers to the substitution and / or deletion and / or addition of no more than 5 amino acid residues.

[0009] The proteins mentioned above (a1)-a3) can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0010] In a second aspect, the present invention provides a nucleic acid molecule that encodes the aforementioned protein.

[0011] In some embodiments of the present invention, the above-mentioned nucleic acid molecule is a DNA molecule of any one of the following b1)-b4); b1) The coding region is the DNA molecule shown in SEQ ID NO. 2; b2) The nucleotide sequence is the DNA molecule shown in SEQ ID NO. 2; b3) has 80% or more identity with the nucleotide sequence defined by b1) or b2) and is a DNA molecule encoding the protein ZmbHLH18; b4) Hybridizes under stringent conditions to the nucleotide sequence defined by b1) or b2) and to a DNA molecule encoding the protein ZmbHLH18. The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0012] A third aspect of the present invention provides a recombinant vector, expression cassette, transgenic cell line, or host bacterium containing a nucleic acid molecule encoding the aforementioned protein. In some embodiments of the present invention, the host bacteria may be eukaryotic bacteria or prokaryotic bacteria.

[0013] In a fourth aspect, the present invention provides primers for amplifying the full length of a nucleic acid molecule encoding the above-mentioned protein or any fragment thereof. A fifth aspect of the present invention provides the use of the above-described protein, the above-described nucleic acid molecule, the above-described recombinant vector, expression cassette, transgenic cell line or host bacterium in any one or more of the following c1)-c13); c1) Improve plant drought resistance; c2) Improve the water retention capacity of plants; c3) Increase the water content of plants under drought stress; c4) Reduce the rate of water loss from detached leaves of plants under drought stress; c5) Increase the proline content in plants under drought stress; c6) Enhances the antioxidant capacity of plants; c7) Reduce malondialdehyde (MDA) content in plants under drought stress; c8) Enhances the plant's ability to maintain cell membrane integrity; c9) Reduce the electrical conductivity of plants under drought stress; c10) enhances the activity of antioxidant enzymes in plants under drought stress; c11) Increase the chlorophyll content of plants under drought stress; c12) Increases starch content in plants under drought stress; c13) regulates plant stress resistance.

[0014] In some embodiments of the present invention, the regulation of plant stress resistance involves either reducing or increasing plant stress resistance; preferably, it involves increasing plant stress resistance. The present invention has discovered through research that promoting or enhancing plant stress resistance in plants... ZmbHLH18 Gene expression can enhance a plant's resistance to adverse conditions, specifically by improving its drought resistance.

[0015] In some embodiments of the present invention, the plant may be a dicotyledonous plant (such as Arabidopsis thaliana, cotton, castor bean, peanut, cassava, etc.) or a monocotyledonous plant (such as sweet sorghum, sorghum, corn, wheat, rice, etc.).

[0016] In some embodiments of the present invention, the antioxidant enzyme includes any one or more of superoxide dismutase, catalase, peroxidase, and ascorbate peroxidase.

[0017] A sixth aspect of the present invention provides a method for improving the drought resistance of plants, comprising the following steps: Will ZmbHLH18 Genes are constructed into expression vectors to form recombinant expression vectors, which are then transformed into plants, enabling the plants to carry the gene. ZmbHLH18 Genes, and ultimately throughZmbHLH18 High expression of genes regulates drought resistance in plants. In some embodiments of the present invention, the plant may be a dicotyledonous plant (such as Arabidopsis thaliana, cotton, castor bean, peanut, cassava, etc.) or a monocotyledonous plant (such as sweet sorghum, sorghum, corn, wheat, rice, etc.).

[0018] In some embodiments of the present invention, the step is transformed into: introducing a recombinant expression vector into plant cells or tissues using biological methods; The biological methods include any one of Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electrocoagulation, Agrobacterium-mediated transformation, gene gun, pollen tube introduction, and liposome fusion.

[0019] The beneficial effects of this invention are as follows: This invention reports for the first time a drought resistance-positive regulatory protein, ZmbHLH18, and its encoding gene. Specifically, this invention screened from maize... ZmbHLH18 The gene, whose expression level was upregulated after drought treatment, encodes a protein belonging to the bHLH transcription factor family, which possesses transcriptional activation activity. Knockout and overexpression lines of this gene were obtained in maize, and drought-related physiological indicators were measured, indicating... ZmbHLH18 Overexpression of this substance can enhance the drought resistance of maize. Overexpressing lines exhibit higher leaf water content, lower water loss rate, higher antioxidant enzyme activity, and stronger photosynthetic stability. These physiological indicators directly reflect the plant's ability to survive under drought conditions, thus enhancing its drought resistance. This indicates that it plays a positive regulatory role in the plant's drought resistance. The above technical solution provides a foundation for cultivating resistant plants, has important guiding significance for enhancing the production potential of maize under drought conditions and promoting agricultural development, and provides a more precise and faster approach for maize drought-resistant breeding, with good practical application value. Attached Figure Description

[0020] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0021] Figure 1 The tertiary structure of the ZmbHLH18 protein.

[0022] Figure 2 Phylogenetic analysis of ZmbHLH18 homologous proteins.

[0023] Figure 3 For drought treatment at different times ZmbHLH18 Gene expression levels in maize roots (A) and leaves (B).

[0024] Figure 4 for ZmbHLH18 Localization in tobacco leaf cells.

[0025] Figure 5 for ZmbHLH18 Autotoxicity (A) and transcriptional activity (B) analysis; and in the auxotrophic culture media shown in A and B, the left column shows yeast competent cells AH109 transformed with pGBKT7 empty vector, and the right column shows cells transformed with pGBKT7- ZmbHLH18 Yeast competent cells AH109 expressing the vector.

[0026] Figure 6 Seedling phenotypes of each line before, after, and after rehydration: A represents the untreated phenotype of the overexpression line; B represents the phenotype of the overexpression line after 7 days of drought treatment; C represents the phenotype of the overexpression line after 72 hours of rehydration; D represents the untreated phenotype of the knockout line; E represents the phenotype of the knockout line after 7 days of drought treatment; and F represents the phenotype of the knockout line after 72 hours of rehydration.

[0027] Figure 7 The relative leaf water content of maize overexpression line (A) and knockout line (B) before and after drought treatment.

[0028] Figure 8 The values ​​represent the detached leaf water loss rate of each line before and after drought treatment; where A represents the detached leaf water loss rate of the overexpression line and wild type before treatment, B represents the detached leaf water loss rate of the overexpression line and wild type after drought treatment, C represents the detached leaf water loss rate of the knockout line and wild type before treatment, and D represents the detached leaf water loss rate of the knockout line and wild type after drought treatment.

[0029] Figure 9 The values ​​represent the free proline content in the leaves of each strain after drought treatment; where A represents the proline content in the overexpression strain and WT after drought treatment, and B represents the proline content in the knockout strain and WT after drought treatment (the values ​​are the mean ± standard deviation of 4 replicates, and different letters indicate significant differences between the same index in different treatments, i.e., P < 0.05).

[0030] Figure 10 The values ​​represent the leaf MDA content of each line after drought treatment; where A represents the MDA content of the overexpressing line and wild type after drought treatment, and B represents the MDA content of the knockout line and wild type after drought treatment (the values ​​are the mean ± standard deviation of 4 replicates, and different letters indicate that the same indicator is significantly different in different treatments, i.e., P < 0.05).

[0031] Figure 11The values ​​represent the relative electrical conductivity of leaves of each line after drought treatment; where A represents the relative electrical conductivity of the overexpression line and wild type after drought treatment; and B represents the relative electrical conductivity of the knockout line and wild type after drought treatment (the values ​​are the mean ± standard deviation of 4 replicates, and different letters indicate significant differences in the same index under different treatments, i.e., P < 0.05).

[0032] Figure 12 The values ​​represent the SOD activity of each line after drought treatment; where A represents the SOD activity of the overexpressing line and wild type after drought treatment; and B represents the SOD activity of the knockout line and wild type after drought treatment (the values ​​are the mean ± standard deviation of 4 replicates, and different letters indicate significant differences in the same indicator under different treatments, i.e., P < 0.05).

[0033] Figure 13 The values ​​represent the CAT activity of each line before and after drought treatment; where A represents the CAT activity of the overexpressing line and wild type after drought treatment; and B represents the CAT activity of the knockout line and wild type after drought treatment (the values ​​are the mean ± standard deviation of 4 replicates, and different letters indicate significant differences between the same index in different treatments, i.e., P < 0.05).

[0034] Figure 14 POD activity of each line after drought treatment; where A represents POD activity of overexpressing lines and wild type after drought treatment; B represents POD activity of knockout lines and wild type after drought treatment (the values ​​are the mean ± standard deviation of 4 replicates, and different letters indicate significant differences between the same index in different treatments, i.e., P < 0.05).

[0035] Figure 15 The values ​​represent the APX activity of each line before and after drought treatment; where A represents the APX activity of the overexpressing line and wild type after drought treatment; and B represents the APX activity of the knockout line and wild type after drought treatment (the values ​​are the mean ± standard deviation of 4 replicates, and different letters indicate significant differences between the same index in different treatments, i.e., P < 0.05).

[0036] Figure 16 The values ​​represent the chlorophyll content of each line after drought treatment; where A represents the chlorophyll content of the overexpression line and wild type after drought treatment; and B represents the chlorophyll content of the knockout line and wild type after drought treatment (the values ​​are the mean ± standard deviation of 4 replicates, and different letters indicate significant differences in the same indicator under different treatments, i.e., P < 0.05).

[0037] Figure 17The values ​​represent the total starch content of each strain before and after drought treatment; where A represents the starch content of the overexpression strain and wild type after drought treatment; and B represents the starch content of the knockout strain and wild type after drought treatment (the values ​​are the mean ± standard deviation of three replicates, and different letters indicate significant differences in the same indicator under different treatments, i.e., P < 0.05). Detailed Implementation

[0038] This invention focuses on bHLH family transcription factors in maize. ZmbHLH18 To investigate its function and regulatory mechanism under drought stress, we first screened transcriptome data from the drought-resistant maize inbred line 287M. ZmbHLH18 Genes were analyzed, including their amino acid sequences, protein structures, and physicochemical properties, and detection was performed. ZmbHLH18 Expression at different time points under drought stress showed that the expression level of this gene was significantly upregulated under drought conditions. Further transient transformation of tobacco cells confirmed that the ZmbHLH18 protein is located in the cell nucleus. This was verified using a yeast two-hybrid system. ZmbHLH18 The transcriptional activity. And respectively constructed ZmbHLH18 Gene overexpression and knockout vectors were used in Agrobacterium-mediated transformation to transfer these vectors into wild-type maize lines, successfully obtaining and identifying transgenic maize lines with overexpression and knockout. Under drought stress conditions, the wild-type and knockout maize lines showed positive results. ZmbHLH18 Phenotypic observations and physiological index measurements were performed on overexpression and knockout lines. During drought treatment, physiological indicators such as leaf water content, detached leaf water loss rate, proline content, malondialdehyde (MDA) content, relative conductivity, and antioxidant enzyme activity were measured for each line. Results showed that the overexpression lines were superior to the wild type in these drought resistance indicators, while the drought resistance of the knockout lines was significantly reduced, further validating the results. ZmbHLH18 It plays a positive regulatory role in drought resistance. Transcriptome sequencing (RNA-seq) analysis was performed on wild-type and overexpression lines under drought stress. The results showed that... ZmbHLH18 The drought-regulated genes are mainly enriched in pathways related to photosynthesis, carbon metabolism, and photosynthesis. This indicates that... ZmbHLH18 It may help plants adapt to drought stress by regulating pathways such as photosynthesis and carbon metabolism. Further verification was conducted using yeast one-hybrid experiments. ZmbHLH18 Interactions with some potential downstream target genes. Experimental results show that... ZmbHLH18 and ZmGAPA There is an interaction between genes involved in glyceraldehyde-3-phosphate dehydrogenase, which is involved in photosynthesis, indicating that... ZmbHLH18 It may enhance the drought resistance of plants by maintaining the stability of photosynthesis.

[0039] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0040] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0041] Example 1: Corn ZmbHLH18 Bioinformatics analysis of genes Transcriptome data from the drought-resistant maize inbred line 287M were screened to select... ZmbHLH18 The gene (GRMZM2G155043) was analyzed, including its amino acid sequence, protein structure, and physicochemical properties.

[0042] ZmbHLH18 Gene: GRMZM2G155043 Amino acid sequence: MEDSSLFMOWAMDTLQHENNPAPASAAAVHGGFSEATFPSLKALREASHAAEMVQELIADADVVRHAPNSWNSGDNTTANYNVPAGWGFGGATAASAALPGSHGMMEAPPAMATRGRPPPGLVYRLPPTRRAGLKSLGSMAAAYAKDHIIAERKRREKI NORFIELSTVIPGLKKMDKATILLDATRYLKELQEKLKDLEQRKEAGGGSIETLVLYKKPCLHAAAARDDDGGSSLPASPPAGTPTEGKRLPEIEVQFSELEKTVAVRVHCENRKGVVVHVLAELEDLHLSNIHANVMPFTACTRIITITAKASFPCPAF (SEQ ID NO.1).

[0043] ZmbHLH18Gene sequence: atggaggactcgagcttattcatgcagtgggcgatggacacgctgcagcacgagaacaaccctgcgcctgccagcgccgccgccgtccacggcggcttcagcgaggctactttcccctcgctcaaagcgctccgcgaggcctcgcatgcagccgaaatggtccaggagttgatcgcggacgccgacgtcgtccgccatgcgccaaacagctggaactccggcgacaacaccaccgcgaactacaacgtgcccgcgggctggggcttcggcggtgccactgcagcctccgcgctgccaggcagccacggcatgatggaggccccgcctgccatggccacacgcggtcggccgccgccgggtctggtgtacaggcttccgccgacgaggagagccggcctaaagagcttgggctccatggcggcggcgtacgcgaaggaccacatcatcgcagagcggaaacggcgagagaagatcaaccagcgcttcatcgagctctccactgttatcccaggcctcaagaagatggacaaggcgaccatccttctggacgcgacaaggtacctgaaggagctccaagagaagctgaaggatctggagcagcggaaggaggccggcggcgggagcatcgagaccctggtgctcgtcaagaagccatgcctccatgccgcggctgcgcgggatgacgacggtggctcctcgctgccggcgtcgccgccggcaggcacaccaacagaggggaaacggctgccggagatcgaggtccagttctcggagttggagaagaccgtggcggtgagggtgcactgcgagaacaggaagggggtggtcgtccacgtgctcgccgagcttgaagatctccacctcagtaacatccacgccaatgttatgccgttcacggcttgcactcggatcataaccatcacggcaaaggctagttttccttgcccggccttttaa (SEQ ID NO.2).

[0044] The tertiary structure of the ZmbHLH18 protein ( ​ ZmbHLH18 is composed of two α-helices linked by a loop. This protein is hydrophilic, lacks a transmembrane domain, and has no signal peptide. The phylogenetic tree of the ZmbHLH18 amino acid sequence is as follows: ​ As shown, the ZmbHLH18 protein in maize is most closely related to the homologous protein in sorghum. In addition, the protein is also closely related to the homologous protein in millet.

[0045] Example 2: Corn ​ Functional research of genes 1. Test materials Maize: inbred lines 287M, C01, and B104, ​ Overexpression of maize lines OE-1, OE-4 and ​ Knockout strains cris-1 and cris-3.

[0046] Tobacco: Tobacco Bunsenii, used in Agrobacterium transient transformation experiments.

[0047] Build ​ The gene cloning vector used is pMD TM 18 T Vector (TaKaRa); pMUE411 was used to construct the knockout vector, pMWB-110 was used for the overexpression vector, pCAMBIA1300 was used for Agrobacterium transient transformation, and pGBKT7, pGADT7, and pAbAi vectors were used for yeast-related experiments. The *E. coli* DH5α and yeast AH109 cells used in this experiment were from Weidi Biotechnology, the yeast Y1HGold competent cells were from Yushuo Biotechnology, and the *Agrobacterium* GV3101 cells were preserved in our laboratory.

[0048] 2. Test methods 2.1 Acquisition and Cultivation of Corn and Tobacco (1) Corn planting Select C01, B104, 287M, and others with uniform shape and size and full-bodied particles. ​ Overexpression and knockout maize seeds were disinfected by soaking in 75% alcohol for 1 minute, then rinsed with distilled water and soaked overnight. The soaked seeds were then evenly placed in round dishes lined with absorbent paper. After germination, the seeds were transferred to seedling trays containing a 1:1 ratio of nutrient soil and vermiculite. The trays were then watered and placed in an artificial climate chamber (maize growing environment: 28℃ - 16 h light / 20℃ - 8 h darkness, humidity around 30%), watering every 2-3 days. When the seeds reached the three-leaf stage, uniformly growing maize seeds were selected for drought treatment. The corn plants to be drought-treated were transferred to clean and dry trays. Watering was stopped when the corn reached the three-leaf stage, and natural drought treatment was initiated. A photograph was taken at this point to record the first day of drought treatment. During the drought treatment, the tray positions were adjusted daily to reduce the positional effect. A photograph was taken after seven days of drought treatment to record the post-drought condition, and the corn growth was observed. Rehydration was initiated when the corn was near death, and a photograph was taken three days after rehydration to record the post-rehydration condition. Corn samples were collected at 0 h, 24 h, 48 h, 72 h, and 7 days after drought treatment and stored at -80℃ for later use. (2) Tobacco cultivation Sow an appropriate amount of tobacco seeds in a flowerpot filled with nutrient soil. After the seedlings emerge, transplant them. When the tobacco seedlings have grown 4-5 true leaves, conduct an Agrobacterium-mediated instantaneous transformation experiment.

[0049] 2.2 Corn ​ Cloning of genes (1) RNA extraction and reverse transcription: Total RNA was extracted from the roots and leaves of maize and flash-frozen with liquid nitrogen. RNA was extracted using the Huayueyang Plant Total RNA Extraction Kit.

[0050] (2) Maize cDNA synthesis: The concentration of RNA was determined and diluted to 200 ng / μL, and Evo was used. ​ The reverse transcription premixed kit (Science Technology) is used to reverse transcribe cDNA. The process includes gDNA removal (42℃, 2 min) and reverse transcription (37℃, 15 min; 85℃, 5 s). Use immediately or store at -20℃ for later use. (3) ​ Gene amplification Using synthesized maize leaf cDNA as a template, ​ -F、 ​ -R is a primer used for PCR to synthesize the target gene CDS sequence. The reaction system contains 2×Taq Plus Master Mix, primers, and cDNA. The program is 95℃ pre-denaturation for 5 min, 33 cycles (95℃ for 30 s, 55℃ for 1 min, 72℃ for 45 s), and 72℃ extension for 7 min. The product is recovered after detection by agarose gel electrophoresis.

[0051] Primer sequences (from 5' to 3'): ​ -F:ATGGAGGACTCGAGCTTAT(SEQ ID NO.3); ​ -R:TTAAAAGGCCGGGCAAGG (SEQ ID NO. 4).

[0052] 2.3​ Gene expression pattern analysis Using qRT-PCR technology to ​ The expression patterns of the gene under different drought treatments were quantitatively analyzed to observe the changes in gene expression under stress.

[0053] The specific method is as follows: using cDNA obtained from reverse transcription as a template, design quantitative primers for qRT-PCR, and... ​ -F and ​ -R is the internal reference primer. ​ -qF and ​ -qR is the quantitative primer for gene sequencing. The target gene was obtained using the Akerui quantitative PCR kit. ​ Excerpt.

[0054] The primer sequences (from the 5' end to the 3' end) are as follows: ​ -F: TGGGCCTACTGGTCTTACTACTGA (SEQ ID NO.5); ​ -R: ACATACCCACGCTTCAGATCCT (SEQ ID NO.6); ​ -qF: CTCAAGAAGATGGACAAG (SEQ ID NO.7); ​ -qR:GATCCTTCAGCTTCTCTT (SEQ ID NO. 8). 2.4 Ligation and Transformation of Target Fragment and Cloning Vector target gene ​ The fragment was ligated to the cloning vector pMD18-T Vector (16℃ for 30 min). The ligation product was transformed into competent DH5α cells (Weidi Bio DH5α Chemically Competent Cell), and single colonies were selected after ampicillin screening for colony PCR verification and sequencing.

[0055] 2.5 Colony PCR and Plasmid Extraction After cloning vector colony PCR, agarose gel electrophoresis was performed to detect the PCR reaction. Single colonies of the correct size were then shaken and sent for sequencing. Plasmids that were correctly sequenced were extracted using the Tiangen Plasmid Mini-Prep Kit. The primer sequences (from the 5' end to the 3' end) are as follows: ​ -F:ATGGAGGACTCGAGCTTAT(SEQ ID NO.3); ​-R:TTAAAAGGCCGGGCAAGG (SEQ ID NO. 4).

[0056] 2.6 Construction of the Expression Carrier Using the extracted plasmid as a cDNA template, respectively... ​ -1300-F, ​ -1300-R; ​ -BD-F、 ​ -BD-R and ​ -AD-F、 ​ Using -AD-R as primers, the full-length target gene containing Kpn I, SaII; EcoRI, SaII and EcoRI, BamHI restriction sites was amplified, and the PCR product was purified by gel extraction. The primer sequences (from the 5' end to the 3' end) are as follows: ​ -1300-F:GGGTACCATGGAGGACTCGAGCTTAT(SEQ ID NO.9); ​ -1300-R: GCGTCGACTTAAAAGGCCGGGCAAGG (SEQ ID NO.10); ​ -BD-F: GGAATTCATGGAGGACTCGAGCTTAT (SEQ ID NO.11); ​ -BD-R: GCGTCGACTTAAAAGGCCGGGCAAGG (SEQ ID NO. 12); ​ -AD-F: GAATTC ATGGAGGACTCGAGCTTAT (SEQ ID NO. 13); ​ -AD-R:GGATCCTTAAAAGGCCGGGCAAGG (SEQ ID NO. 14).

[0057] The PCR product, along with pCAMBIA1300, pGBKT7, and pGADT7, were double-digested with DNA endonucleases. The PCR reaction was then purified and recovered. The target gene and expression vector were ligated using T4 DNA ligase. The ligation product was transformed into competent E. coli DH5α cells, followed by colony PCR, plasmid extraction, and preservation of the bacterial strain.

[0058] 2.7 Transformation of Agrobacterium GV3101 competent cells Add 5 µL of recombinant plasmid to 50 µL of Agrobacterium competent cells, mix gently, and then incubate sequentially on ice, in liquid nitrogen, in a 37°C water bath, and on ice for 5 min each, keeping the cells still. Under aseptic conditions, take an appropriate amount of sterile LB liquid culture medium based on the transformed cells and incubate with shaking in a 28°C Agrobacterium incubator for 2 h. After the transformed Agrobacterium has amplified, centrifuge at 5000 rpm for 150 s. Under aseptic conditions, retain 100 µL of supernatant, resuspend the bacterial cells at the bottom of the EP tube by pipetting, and then evenly spread them on medium containing antibiotics (Kana, Rif), and incubate overnight upside down at 28°C.

[0059] 2.8 Transformed yeast AH109 competent cells Carrier DNA pretreatment: Heat in a 95°C metal bath for 5 min, immediately then quickly freeze, and repeat once. Thaw 50 µL of AH109 competent cells in an ice bath, and add pGBKT7- to a centrifuge tube sequentially. ​ 2.5 µg of vector plasmid, 5 µL of carrier DNA, and 250 µL of PEG / LiAc were gently mixed. The centrifuge tubes were placed in a 30°C water bath for 30 min, and inverted 6 times at 15 min to mix. Then, the tubes were placed in a 42°C water bath for 15 min, and inverted 6 times at 7 min to mix. The tubes were centrifuged at 5000 rpm for 40 s, the pellet was resuspended in 400 µL of ddH2O, and centrifuged again for 30 s. The supernatant was then aspirated. The bacterial cells were resuspended in 50 µL of ddH2O, plated onto the corresponding defect-specific culture medium plates, and incubated at 29°C for 72 h.

[0060] 2.9 ​ Subcellular localization Building pCAMBIA1300- ​ The GFP expression vector was ligated to the target fragment via double digestion (KpnⅠ and SalⅠ) and T4 ligase. The recombinant vector was transformed into Agrobacterium GV3101 and mixed with an equal volume of Agrobacterium containing the P19 plasmid (OD). 600 =0.9), injected into tobacco leaves, cultured in the dark for 36-48 h, GFP fluorescence was observed using a laser confocal microscope, and cell nuclei were stained with DAPI. 2.10 ​ Obtaining overexpression lines Target fragment amplification: Using maize cDNA as a template, primers with restriction enzyme sites were used ( ​ -1300-F and ​ -1300-R) amplification ​ The full-length gene CDS was obtained, the target fragment was obtained by PCR reaction, and then recovered and purified after detection by agarose gel electrophoresis.

[0061] Vector digestion and ligation: The overexpression vector pMWB-110 and the target fragment were digested with BamHI (37℃ for 2.5 h). After purification of the digestion products, ligation was performed overnight at 16℃ using T4 DNA ligase to construct pMWB-110- ​ Recombinant vector.

[0062] Vector transformation and identification: The recombinant vector was transformed into E. coli DH5α competent cells, and colony PCR was performed (primers were as follows). ​ -F and ​ After screening for positive clones and verifying the sequence correctness through sequencing, plasmids are extracted.

[0063] Maize transformation and screening: The correct recombinant vector was sent to a professional institution (Life Science and Technology Center of China Seed Group Co., Ltd.) for transformation of wild-type maize C01 to obtain transgenic plants. Positive plants were identified by PCR (primers UBI-F and OE-R) combined with qRT-PCR detection. ​ Expression levels were used to screen for homozygous overexpression lines with high expression (OE-1, OE-4).

[0064] The primer sequences (from the 5' end to the 3' end) are as follows: UBI-F: GTTTCTTTTGTCGATGCTCA (SEQ ID NO.15); OE-R: AACTAGCCTTTGCCGTGAT (SEQ ID NO. 16).

[0065] 2.11 ​ Obtaining knockout strains Target design and vector construction: In ​ Three mutation targets were designed at nucleotides 15-37, 138-160, and 235-257 of the gene's CDS region to... ​ -F and ​ -R represents the primers for amplifying the target fragment, which is then ligated to the knockout vector pMUE411 via BamHI digestion (37℃, 2.5 h) to construct pMUE411- ​ Recombinant vector.

[0066] Vector transformation and identification: The recombinant vector was transformed into Escherichia coli DH5α. After verification by colony PCR and sequencing, the plasmid was extracted.

[0067] Maize transformation and screening: The recombinant vector was transformed into wild-type maize B104 to obtain transgenic plants. Plant DNA was extracted and PCR amplification was performed using primers cris-F and cris-R. The sequencing products were compared with the wild-type sequence to screen for knockout lines (cris-1, cris-3) with base insertions or deletions at the target site.

[0068] The primer sequences (from the 5' end to the 3' end) are as follows: cris-F: CTTCGGATTAATGGTCTGCTTGT (SEQ ID NO. 17); cris-R: CTCTGCGATGATGTGGTCCTT (SEQ ID NO. 18). Obtaining homozygous lines: Positive transgenic plants were bagged and self-crossed, and the seeds were harvested and sown. Homozygous knockout lines were screened by molecular identification for subsequent experiments.

[0069] 2.12. Analysis of drought phenotypes in wild-type, overexpression lines, and knockout lines. Select maize inbred lines C01 and B104 with uniform morphology and size and full kernels, as well as maize seeds from over-exposed lines OE-1 and OE-4 and knockout lines cris-1 and cris-3. Treat them according to the method in "Maize Cultivation". The drought treatment time is 0 and 7 days, and the rehydration time is 72 hours. Record the maize phenotype after each treatment. 2.13. Determination of water loss rate and relative water content of detached leaves Quickly cut corn leaves of similar size and location, weigh them using an analytical balance, and record the weight as 0h. Simultaneously, start a timer. Every two minutes, cut a different sample and weigh it. During the first hour, weigh the fresh weight every half hour, and then every hour thereafter, continuing for 5-6 hours. Calculate the water loss rate (%) at each time point: the difference between the fresh weight at each time point and the fresh weight at 0h is recorded as the water loss at that time point. Divide the water loss at each time point by the fresh weight at 0h to obtain the water loss rate at that time point. At least three replicates were set up for each strain.

[0070] Quickly cut corn leaves at the 7-8 leaf stage and weigh them using an analytical balance to obtain their fresh weight (FW). After weighing, completely immerse the leaves in water overnight, remove them, wipe off any remaining moisture, and immediately weigh them again using an analytical balance to obtain their saturated fresh weight (SFW). Place the corn leaves with the saturated fresh weight in a paper envelope and put them in a 100℃ oven for half an hour. Then, reduce the oven temperature to about 70℃ and continue drying for 3-4 hours. Remove the corn leaves, cool them to room temperature, and weigh them to obtain their dry weight (DW). The formula for calculating the relative moisture content (%) of the leaves is: (fresh weight - dry weight) / (saturated weight - fresh weight) × 100%, which is (FW - DW) / (SFW - FW) × 100%. 2.14 Determination of proline content The proline (Pro) content of wild-type, overexpression and knockout lines under drought stress was determined. For specific steps, please refer to the Solarbio Proline (Pro) Content Detection Kit (BC0290). 2.15 Determination of malondialdehyde (MDA) content Malondialdehyde (MDA) content was determined in wild-type, overexpression, and knockout lines under drought stress. 0.1% TCA and 0.5% TBA solutions were prepared in advance. 0.2 g of maize leaves were weighed and ground in a mortar with an appropriate amount of quartz sand. During grinding, 1 mL of TCA solution was added twice. After grinding, the liquid was transferred to a 15 mL centrifuge tube, and 5 mL of TBA solution was added. The centrifuge tube was placed in a water bath and heated to boiling. Timing began when small bubbles slowly rose from the bottom of the tube. After 10 minutes, the timing ended, and the centrifuge tube was immediately removed and allowed to cool to room temperature. It was then centrifuged at 3000 rpm for 15 minutes. The supernatant was poured into a clean centrifuge tube, and its volume was measured. The absorbance at 532 nm and 600 nm was measured (zeroed with TBA). The formula for calculating the malondialdehyde (MDA) content in plant tissue is: MDA content (mmol / gFw) = (A532-A600) × supernatant volume / 155 × leaf mass × 1000.

[0071] 2.16. Determination of relative conductivity Relative conductivity was determined using a conductivity meter: Uniformly sized corn leaves were cut and rinsed thoroughly with ddH2O. 0.2 g of the leaves were weighed and placed in a test tube, and 4 mL of ddH2O was added (ensuring the leaves were completely submerged). The tube was shaken for 2 hours, and the original conductivity value was measured. The test tube was then placed in a boiling water bath for 20 minutes, removed, and allowed to cool to room temperature before measuring the total conductivity value. The ratio of the original conductivity value to the total conductivity value is the relative conductivity.

[0072] 2.17. Determination of antioxidant enzyme activity in different strains under drought treatment Enzyme extraction: Weigh an appropriate amount of leaf tissue, add pre-cooled extraction buffer (prepared according to the kit instructions), grind in an ice bath to form a homogenate, centrifuge, and take the supernatant as the crude enzyme extract.

[0073] Enzyme activity assays: SOD activity: The ability to inhibit the photoreduction of nitroblue tetrazolium (NBT) was measured by measuring the absorbance at 560 nm and calculating the SOD activity.

[0074] CAT activity: The rate of CAT decomposition of H2O2 was calculated based on the rate of decrease of absorbance of hydrogen peroxide (H2O2) at 240 nm.

[0075] POD activity: Using guaiacol as a substrate, the rate of increase in absorbance at 470 nm was measured to reflect the ability of POD to catalyze the oxidation of the substrate.

[0076] APX activity: The rate of APX oxidation of ASA was calculated by measuring the rate of decrease in absorbance of ascorbic acid (ASA) at 290 nm.

[0077] 2.18 Determination of chlorophyll content Cut plant leaves into 1 mm wide segments, weigh 0.2 g and place them in a test tube; add 5 mL DMSO and 5 mL 80% acetone sequentially to completely submerge the plant material. Place the test tube in a 65℃ water bath in the dark for 24 h, until the green color of the plant leaves fades or becomes transparent. Remove the tube and allow it to cool to room temperature in the dark. Filter the extract into a 25 mL volumetric flask, dilute to volume with 80% acetone and mix well; using 80% acetone as a blank control, measure the absorbance at 663 nm and 645 nm. Calculate the chlorophyll content.

[0078] Total chlorophyll Ct (mg / L) -1 ) = (8.02A 663 +20.21 A 645) *Immediate volume (L) / Fresh weight (g) Chlorophyll content (mg / g Fw) = (Ct × total extract volume × dilution factor) / fresh weight of sample 2.19 Transcriptome sequencing (RNA-seq) RNA extraction and sequencing: Total RNA was extracted from each sample, and the quality was tested with NanoDrop. Lianchuan Biotechnology performed RNA-seq sequencing. The sequencing results were compared with the maize genome (Zm-B73-REFERENCE-NAM-5.0) and filtered to obtain Clean Reads.

[0079] Differential gene analysis: Differentially expressed genes (DEGs) were screened using |log2FoldChange|≥1 and qvalue≤0.05 as thresholds. GO and KEGG enrichment analyses were then performed to further screen... ​ Regulated drought response genes (DEG intersection). Under drought stress ​ GSEA analysis was performed on gene expression profiles in phenotypic and WT cells to comprehensively characterize the genes. ​ The regulatory effect of all genes on a specific regulatory pathway.

[0080] 2.20 Bioinformatics Analysis ( ​ (The interaction between the gene and its downstream potential target genes) Predict using PlantCARE and other online tools ​ Promoter elements of potential target genes were identified to analyze the location and potential regulatory role of these genes in the maize genome.

[0081] Genes and vectors: Downstream candidate genes (related to photosynthesis) ​ , ​ , ​ Related to ABA signals ​ , ​ , ​ ), vectors pAbAi and pGADT7, yeast strain Y1HGold.

[0082] Promoter element prediction: The PlantCARE website was used to analyze the 2 kb promoter sequence upstream of the start codon of candidate genes to predict cis-acting elements (such as E-box and G-box).

[0083] Yeast one-hybrid validation: pBait-AbAi bait vector (containing target gene promoter fragment) and pGADT7-ZmbHLH18 prey vector were constructed; the bait vector was transformed into yeast Y1HGold, and the optimal AbA concentration for inhibiting self-activation was screened; the bait vector and prey vector were co-transformed, and the growth of yeast on AbA-deficient medium was observed.

[0084] Yeast one-hybrid assay (Y1H): Yeast one-hybrid technology was used to verify the interaction between ZmbHLH18 and potential target genes, helping to confirm the role of the gene in regulating downstream genes.

[0085] 2.21 Determination of starch content in different maize lines under drought stress The starch content of wild-type, overexpression, and knockout lines under drought stress was determined using the Solarbio Starch Content Detection Kit (BC0700).

[0086] 3. Results and Analysis 3.1​ Analysis of gene induction expression patterns Roots were found after drought treatment ( ​ (Figure A) and Ye Zhong ( ​ (Figure B) The relative expression levels of this gene are all increased, indicating that... ​ It may have been involved in the regulation of corn's response to drought.

[0087] 3.2 ​ Gene cloning and expression vector construction by ​ -F、 ​ -R indicates primer amplification. ​ The full-length target gene was amplified by PCR to obtain a length of 957 bp. ​ The gene CDS sequence was sequenced, and the results showed that it was consistent with the expected sequence.

[0088] Colony PCR verification showed that the recombinant plasmid ligating the pCAMBIA1300-35S-sGFP and pGBKT7 vectors was successfully constructed.

[0089] 3.3 Subcellular localization analysis of ZmbHLH18 like ​ As shown, green fluorescence was observed throughout the entire cell in tobacco cells injected with the empty pCAMBIA1300 vector, while that injected with pCAMBIA1300-... ​ In tobacco cells stained with -GFP, GFP fluorescence is present only in the cell nucleus and overlaps with the nuclear location stained by DAPI, indicating that... ​ It is located in the cell nucleus.

[0090] 3.4 ​ Autotoxicity and transcriptional activity analysis pGBKT7- ​ The expression vector was transformed into competent yeast cells AH109, ​​with the pGBKT7 empty vector used as a blank control. The cells were plated on SD / -Trp monoculture medium and cultured for 3-5 days, after which yeast growth was observed. ​ As shown in Figure A, both groups of yeast grew well, and the single yeast colonies were of uniform size, indicating that the ZmbHLH18 protein is not toxic to yeast. Single yeast colonies from successfully transformed monodeficient media were picked, serially diluted 0, 10, and 100 times with ddH2O, and then spotted onto monodeficient SD / -Trp, triple-deficient SD / -Trp-His-Ade, and SD / -Trp-His-Ade+X-α-ga solid media, and cultured for approximately 3 days. ​As shown in Figure B, both the experimental and control groups grew normally and with consistent growth on SD / -Trp solid medium. However, only the pGBKT7- transformed group grew on SD / -Trp-His-Ade medium. ​ The yeast expressing the vector grew, and only the experimental group grew normally and turned blue on the triple-deficient medium containing X-α-gal, indicating that... ​ As a transcription factor, it has transcriptional activation activity.

[0091] 3.5 Phenotypic observation of wild-type, overgrown, and knockout lines under drought treatment Before drought treatment, the growth of the overgrown lines, wild-type lines, and knockout lines was consistent, with no significant differences. ​ (Figures A and D). After 7 days of drought, the overexpression lines showed less leaf wilting and curling than the wild-type C01, and were also taller. ​ (Figure B in the middle); the knockout strains were weaker and had more severe leaf drying than the wild type B104. ​ (Figure E). After 72 h of rehydration, the overexpression lines recovered better ( ​ (Figure C) Knockout strains showed almost no recovery ( ​ (See Figure F). This indicates overexpression. ​ The gene enhances the drought resistance of maize lines, and knockout ​ The drought resistance of maize lines with this gene is weakened.

[0092] 3.6 Relative water content of leaves and rate of water loss from detached leaves in different maize lines before and after drought treatment By comparing the relative water content of leaves ( ​ ) and the rate of water loss from detached leaves ( ​ It was found that, compared to the wild type, ​ Overexpressing maize plants exhibited higher relative leaf water content and lower leaf water loss rates, while knockout lines showed the opposite. Under water deficit conditions, overexpressing lines maintained a more stable water state and had stronger water retention capacity compared to the wild type; knockout lines had a higher leaf water loss rate and poorer drought resistance. 3.7 Determination of free proline content, MDA content, and relative conductivity in leaves of different maize lines under drought treatment Before drought treatment, there was no significant difference in proline content among the different strains. After drought treatment, the overexpression strains had higher proline content compared to the wild type. ​ Figure A in the middle), the knockout strain is the opposite ( ​ (See Figure B). This indicates that the accumulation of free proline in the leaves of the over-exposed strain is higher than that of the knockout strain, and the over-exposed strain has stronger drought resistance. After drought treatment, the MDA content in the leaves of all maize plants increased, indicating that all lines suffered varying degrees of oxidative damage. Compared to the wild type, the over-exposed lines had lower MDA content after drought treatment. ​ Figure A in the middle), the knockout strain is the opposite ( ​ Figure B shows that the knockout strains experienced more severe oxidative stress.

[0093] After drought treatment, the relative electrical conductivity of all strains increased to varying degrees. Compared with the wild type, the increase was less pronounced in the over-surface strains. ​ Figure A in the middle), the knockout strain is the opposite ( ​ Figure B shows that the cell membrane damage in the knockout line is more severe than that in the overexpression line. 3.8 Determination of antioxidant enzyme activity and chlorophyll content in leaves of different maize lines under drought treatment Compared with the wild type, the overexpressing strain of SOD ( ​ (Figure A), CAT ( ​ (Image A) POD ( ​ (Figure A), APX ( ​ (Figure A) has higher activity and higher chlorophyll content. ​ Figure A); the knockout strain is the opposite ( ​ China B map ​ China B map ​ China B map ​ China B map ​ (See Figure B). This indicates that after drought treatment, the knockout strains suffered more severe leaf damage than the over-surface strains.

[0094] 3.9. RNA-seq screening for differentially expressed genes under drought stress Number of differentially expressed genes: 9271 DEGs were observed in the wild type after drought (5086 upregulated and 4185 downregulated); compared with the wild type, the overexpressing lines had 4094 DEGs under CK (2059 upregulated and 2035 downregulated) and 3645 DEGs under drought (1361 upregulated and 2284 downregulated).

[0095] Functional enrichment: ​ The drought-responsive genes regulated by the organisms are mainly enriched in pathways related to photosynthetic carbon fixation, carbon metabolism, and photosynthetic components, as well as biological signal transduction and cellular metabolic pathways. This indicates that... ​ It may help plants adapt to drought stress by regulating pathways such as photosynthesis and carbon metabolism.

[0096] GSEA analysis showed that genes involved in photosynthesis, chlorophyll metabolism, and carotenoid biosynthesis were significantly upregulated in the overexpression lines.

[0097] 3.9 ZmbHLH18Screening of genes regulating drought stress response In order to determine ZmbHLH18 Potential target genes regulated under drought stress, obtained from transcriptome data. ZmbHLH18 Further screening was conducted on genes regulating drought stress responses. Based on gene function and relative gene expression before and after treatment, the selected genes were further screened. ZmbHLH18 Six potential downstream target genes were selected from the drought stress response genes that regulate the response, and these are related to photosynthesis. ZmGAPA , ZmOHP2 and ZmSPS2 Related to ABA signal transduction and resilience ZmCUE1 , ZmLrgB and ZmHSFA6B Regardless of whether under conditions of ample irrigation or drought treatment, the relative expression levels of these genes in the overexpression lines increased to varying degrees, consistent with the results of transcriptome sequencing.

[0098] Promoter elements: Candidate gene promoters contain a large number of elements such as MYC (ABA, drought response), G-box (light response), and ABRE (ABA response).

[0099] Interaction verification (yeast one-hybrid results): In order to find the interaction with... ZmbHLH18 Six potential target genes were selected based on their direct binding downstream target genes. ZmGAPA , ZmOHP2 , ZmSPS2 , ZmCUE1 , ZmLrgB and ZmHSFA6B Yeast heterosis validation was performed. Only... ZmbHLH18 and ZmGAPA The promoter of the gene (related to the synthesis of glyceraldehyde-3-phosphate dehydrogenase subunit) can grow on 400 ng / mL and 600 ng / mL AbA medium, indicating that the two interact. ZmbHLH18 and ZmGAPA There is an interaction between genes involved in glyceraldehyde-3-phosphate dehydrogenase, which is involved in photosynthesis, indicating that... ZmbHLH18 It may enhance the drought resistance of plants by maintaining the stability of photosynthesis.

[0100] 3.10 Total starch content of each strain before and after drought treatment.

[0101] Under drought stress, the total starch content of maize overgrown lines was higher than that of wild-type lines. Figure 17 In the middle A line, the starch content of the knockout strain was lower than that of the wild type. Figure 17 (B) This indicates that under drought stress, the over-surfaced lines accumulate more photosynthetic products and have a stronger ability to resist drought stress, while the knockout lines show the opposite. It also indicates... ZmbHLH18It can affect the accumulation of photosynthetic products.

[0102] In summary, the table ZmbHLH18 The corn plants exhibited more drought-resistant characteristics, while ZmbHLH18 The knockout strains exhibited drought sensitivity. This allows for a preliminary judgment... ZmbHLH18 Genes play a positive regulatory role in the drought resistance of maize, possibly by enhancing the water retention capacity of leaves, regulating the activity of antioxidant enzymes, the synthesis of chlorophyll and osmotic substances, and the expression of photosynthesis-related genes, thereby enhancing the drought resistance of maize.

[0103] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A protein, characterized in that, It is any one of the following: a1)-a3) a1) Proteins with the amino acid sequence shown in SEQ ID NO.1; a2) Proteins derived from SEQ ID NO.1 with the same function as the amino acid sequence shown in SEQ ID NO.1, by substitution and / or deletion and / or addition of one or more amino acid residues; a3) Proteins encoded by other genes that have an amino acid sequence similarity of more than 80% to that shown in SEQ ID NO.1 and have the activity of the protein shown in SEQ ID NO.

1.

2. A nucleic acid molecule encoding the protein of claim 1; Furthermore, the nucleic acid molecule is a DNA molecule of any one of the following (b1)-b4); b1) The coding region is the DNA molecule shown in SEQ ID NO.2; b2) The nucleotide sequence is the DNA molecule shown in SEQ ID NO.2; b3) has 80% or more identity with the nucleotide sequence defined by b1) or b2) and is a DNA molecule encoding the protein of claim 1; b4) Hybridizes under stringent conditions to the nucleotide sequence defined in b1) or b2) and to a DNA molecule encoding the protein of claim 1.

3. A recombinant vector, expression cassette, transgenic cell line, or host bacterium containing a nucleic acid molecule encoding the protein of claim 1.

4. Primers for amplifying the full length of the nucleic acid molecule encoding the protein of claim 1 or any fragment thereof.

5. The use of the protein of claim 1, the nucleic acid molecule of claim 2, the recombinant vector, expression cassette, transgenic cell line or host bacterium of claim 3 in any one or more of the following c1)-c13); c1) Improve plant drought resistance; c2) Improve the water retention capacity of plants; c3) Increase the water content of plants under drought stress; c4) Reduce the rate of water loss from detached leaves of plants under drought stress; c5) Increase the proline content in plants under drought stress; c6) Enhances the antioxidant capacity of plants; c7) Reduce malondialdehyde (MDA) content in plants under drought stress; c8) Enhances the plant's ability to maintain cell membrane integrity; c9) Reduce the electrical conductivity of plants under drought stress; c10) enhances the activity of antioxidant enzymes in plants under drought stress; c11) Increase the chlorophyll content of plants under drought stress; c12) Increases starch content in plants under drought stress; c13) regulates plant stress resistance.

6. The application as described in claim 5, characterized in that, The plant is a dicotyledonous or monocotyledonous plant; preferably, the plant includes corn. Preferably, the regulation of plant stress resistance is to reduce or increase plant stress resistance; further, it is to increase plant stress resistance; preferably, the stress resistance is drought resistance.

7. The application as described in claim 5, characterized in that, The antioxidant enzymes include any one or more of superoxide dismutase, catalase, peroxidase, and ascorbate peroxidase.

8. A method for improving plant drought resistance, characterized in that, Includes the following steps: Will ZmbHLH18 Genes are constructed into expression vectors to form recombinant expression vectors, which are then transformed into plants, enabling the plants to carry the gene. ZmbHLH18 Genes, and ultimately through ZmbHLH18 High expression of genes regulates drought resistance in plants.

9. The method as described in claim 8, characterized in that, The plant is a dicotyledonous or monocotyledonous plant; preferably, the plant includes corn.

10. The method as described in claim 8, characterized in that, The transformation refers to: introducing a recombinant expression vector into plant cells or tissues using biological methods; The biological methods include any one of Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electrocoagulation, Agrobacterium-mediated transformation, gene gun, pollen tube introduction, and liposome fusion.

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