Use of zmddp2 protein and gene encoding same in regulating drought resistance of plant

Abstract

Use of a ZmDDP2 protein and a gene encoding same in regulating the drought resistance of a plant. The present invention relates to the technical field of genetic engineering breeding. Use of the protein ZmDDP2, or a substance regulating the expression of the gene encoding the protein, or a substance regulating the activity or content of the protein, in regulating the drought resistance of a plant. The amino acid sequence of the protein is set forth in SEQ ID No. 1 or SEQ ID No. 2, and the nucleotide sequence encoding the protein is set forth in SEQ ID No. 3 or SEQ ID No. 4. A method for improving the drought resistance of a plant, comprising knocking out the gene encoding the protein in a recipient plant to obtain a target plant whose drought resistance is higher than that of the recipient plant. A genetically engineered plant, wherein the activity or content of the ZmDDP2 protein is reduced, or the expression of the gene encoding the ZmDDP2 protein is reduced, or the gene encoding the ZmDDP2 protein is knocked out. Plant parts or plant cells derived from the described genetically engineered plant. Experiments have verified that the protein ZmDDP2 has the function of negatively regulating the drought resistance of a plant. The protein ZmDDP2 and related biological materials thereof can be applied to the regulation of the drought resistance of a plant.

Description

Application of ZmDDP2 protein and its coding gene in regulating drought resistance of plants TECHNICAL FIELD

[0001] The present application relates to the field of genetic engineering breeding technology, and particularly relates to application of ZmDDP2 protein and its coding gene in regulating drought resistance of plants. BACKGROUND

[0002] With global warming, rapid population growth and increased activity, the available freshwater resources are increasingly scarce, and the frequency and intensity of environmental stress such as drought are increasing year by year. Most parts of the world are threatened by drought, and drought has become the main environmental stress restricting agricultural production and ecological health.

[0003] Maize (Zea mays L.) is one of the three major food crops in the world, and its yield level is the highest in the world. It is not only an important feed source, but also an important industrial processing raw material. China is an important maize producing and consuming country, and maize is widely planted in the northeast and north China plain of China. Maize has higher water requirement than other crops due to its tall plant. Especially, maize is the only food crop with same plant but different location of male and female flowers. Its differentiation of male and female flowers and flowering period is the period with high water requirement and most sensitive to drought. If the flowering period is subjected to drought stress, the interval between male flower shedding and female flower silking (ASI) will significantly increase due to water loss, resulting in that the silk cannot receive the pollen shed by the male flower after extending out of the bract, affecting the pollination rate, causing the seed setting rate to decrease significantly, leading to serious yield reduction, and even absolute loss.

[0004] Therefore, it is very important to genetically analyze the drought resistance of maize at the flowering period, mine and clone the related resistance genes, improve the drought resistance of maize, and maintain the high yield and stable yield of maize under the limited land resource environment. Previous studies have shown that plant flowering and stress response are complex quantitative traits, which are jointly regulated by multiple genes. Therefore, it is very challenging to analyze the regulation of maize male and female flower coordination under drought stress. At the same time, the related research results will lay a foundation for in-depth understanding of the regulation of crop flowering under drought stress.

[0005] Traditional breeding techniques have long cycle, high blindness, and need to invest a large amount of manpower and material resources. In recent years, with the development of bioinformatics, molecular biology, genomics and other disciplines and the in-depth study of plant stress resistance molecular mechanism, the resistance sites can be mined by using omics data, the regulation mode of the gene can be further analyzed by using molecular biology methods, and the resistance of crops can be improved by knocking out the related negative regulation maize resistance genes in the receptor plants by using genetic engineering method, which will provide theoretical support and key target for the precise design of maize drought resistance breeding improvement in the future.

[0006] SUMMARY

[0007] In view of the deficiencies in the prior art, the present application aims to provide the application of ZmDDP2 protein and its encoding gene in regulating the drought resistance of plants, and the improved drought resistance can be embodied in any of the following types:

[0008] 1) The ear flowering time of the transgenic plant under drought stress is earlier than that of the wild type;

[0009] 2) The interval between the male and female ear flowering time of the transgenic plant under drought stress is shorter than that of the wild type;

[0010] 3) The yield of the transgenic plant under drought stress is higher than that of the wild type.

[0011] To solve the above technical problems, the technical scheme provided by the present application is:

[0012] The application of ZmDDP2 protein or its encoding gene or biological material containing the encoding gene is in any of the following aspects:

[0013] D1) in regulating the drought resistance of plants;

[0014] D2) in preparing products for regulating the drought resistance of plants;

[0015] D3) in the breeding of drought-resistant plants;

[0016] The ZmDDP2 protein is any of the following:

[0017] A1) the protein with the amino acid sequence of SEQ ID No. 1 or SEQ ID No. 2;

[0018] A2) the protein with the same biological function obtained by substituting, deleting and / or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 2.

[0019] The present application provides the application of ZmDDP2 protein or its encoding gene or biological material containing the encoding gene in reducing the interval of pollen shedding and silk spinning time of corn; the application of two haplotype proteins of ZmDDP2 or ZmDDP2 gene in regulating the drought resistance of plants in the flowering period, and the regulation is negative regulation, the expression amount of ZmDDP2 protein or ZmDDP2 gene is reduced, and the drought resistance of plants is enhanced.

[0020] The present application also provides the application of ZmDDP2 protein or its encoding gene or biological material containing the encoding gene in improving the yield of corn.

[0021] Further, in the above application, the substance that regulates the expression of the protein-encoding gene or the substance that regulates the activity or content of the protein is a biological material, and the biological material can be any one of the following B1) to B9):

[0022] B1) a nucleic acid molecule encoding the above-mentioned two haplotypes of ZmDDP2 proteins;

[0023] B2) an expression cassette containing the nucleic acid molecule of B1);

[0024] B3) a recombinant vector containing the nucleic acid molecule of B1) or an expression cassette containing the nucleic acid molecule of B2);

[0025] B4) a recombinant microorganism containing the nucleic acid molecule of B1) or an expression cassette containing the nucleic acid molecule of B2) or a recombinant microorganism containing the recombinant vector of B3);

[0026] B5) a transgenic plant cell line containing the nucleic acid molecule of B1) or an expression cassette containing the nucleic acid molecule of B2) or a transgenic plant cell line containing the recombinant vector of B3);

[0027] B6) a transgenic plant tissue containing the nucleic acid molecule of B1) or an expression cassette containing the nucleic acid molecule of B2) or a transgenic plant tissue containing the recombinant vector of B3);

[0028] B7) a transgenic plant organ containing the nucleic acid molecule of B1) or an expression cassette containing the nucleic acid molecule of B2) or a transgenic plant organ containing the recombinant vector of B3);

[0029] B8) a nucleic acid molecule that inhibits or reduces the expression of the above-mentioned protein-encoding gene or a nucleic acid molecule that inhibits or reduces the activity of the above-mentioned protein;

[0030] B9) an expression cassette, a recombinant vector, a recombinant microorganism, or a transgenic plant cell line containing the nucleic acid molecule of B8).

[0031] Further, in the above application,

[0032] the ZmDDP2 protein-encoding gene is any one of the following:

[0033] b1) a nucleotide sequence represented by SEQ ID No. 3 or SEQ ID No. 4;

[0034] b2) a nucleotide sequence represented by SEQ ID No. 3 or SEQ ID No. 4, which has one or more nucleotides substituted, deleted, and / or added and expresses the same functional protein.

[0035] Further, in the above-mentioned application, the regulation of the gene expression can be inhibition or reduction of the gene expression, which can be achieved by gene knockout or by gene silencing.

[0036] The present application also provides a method for breeding drought-resistant plants, which comprises the step of increasing the drought resistance of a plant during its flowering stage by inhibiting or reducing the level of ZmDDP2 protein in the plant.

[0037] The present application also provides a method for increasing the drought resistance of a plant, which comprises the step of obtaining a transgenic plant with higher drought resistance than a receptor plant by using a CRISPR / Cas9 system to perform gene editing on the receptor plant to obtain a plant material with gene knockout.

[0038] The above-mentioned protein can be artificially synthesized, or a gene encoding the protein can be synthesized first and then expressed biologically.

[0039] B8) The nucleic acid molecule can be a DNA molecule expressing a gRNA targeting the gene encoding the protein, or a gRNA targeting the gene encoding the protein.

[0040] Further, in the above-mentioned application, the target sequence of the gRNA can be the nucleotides shown in SEQ ID No. 3 or SEQ ID No. 4 (i.e. 5'-ACATTGGACGTAGCGCTCC-3') at positions 635-653.

[0041] In the present application, when the receptor is a plant, the promoter in the recombinant expression vector for initiating the transcription of the encoding gene is the Zmubi1 promoter. More specifically, the recombinant vector is a recombinant plasmid obtained by inserting the gene encoding the ZmDDP2 protein into the multiple cloning site (e.g. XcmI, downstream of the Zmubi1 promoter) of a pCUNm vector. When the receptor is tobacco, the recombinant vector is specifically a recombinant plasmid obtained by inserting the gene encoding the ZmDDP2 protein into the multiple cloning site (e.g. SalI and KpnI) of a pCAMBIA1300-nLuc / cLuc vector, when the receptor is yeast, the recombinant vector is specifically a recombinant plasmid obtained by inserting the gene encoding the ZmDDP2 protein into the multiple cloning site (e.g. EcoRI) of a pBT3-STE vector, and when the receptor is Escherichia coli, the recombinant vector is specifically a recombinant plasmid obtained by inserting the gene encoding the ZmDDP2 protein into the multiple cloning site (e.g. SmaI) of a pGreenII-GFP vector.

[0042] In the above method, the recombinant expression vector is introduced into the recipient plant, which can be specifically transformed by using a Ti plasmid, a Ri plasmid, a plant virus vector, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated transformation, and the like. The transformed cells, tissues or plants are understood to include not only the final product of the transformation process, but also the transgenic progeny thereof.

[0043] In the above-mentioned related biological materials, the recombinant microorganism of B4) can be yeast, bacteria, algae and fungi; the plant tissue of B6) can be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos and anthers; the transgenic plant organ of B7) can be roots, stems, leaves, flowers, fruits and seeds of a transgenic plant; the transgenic plant cell line, transgenic plant tissue and transgenic plant organ can or can not include propagation material.

[0044] In the above application, the substance for regulating gene expression can be a substance for regulating at least one of the following six regulations: 1) regulation at the transcription level of the gene; 2) regulation after the transcription of the gene (i.e. regulation of splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (i.e. regulation of mRNA transport from the nucleus to the cytoplasm of the gene); 4) regulation of translation of the gene; 5) regulation of mRNA degradation of the gene; and 6) post-translational regulation of the gene (i.e. regulation of the activity of the translated protein of the gene).

[0045] In the above application, the substance for regulating gene expression can be an agent for inhibiting or reducing the expression of the gene. The agent for inhibiting or reducing the expression of the gene can be an agent for knocking out the gene, such as an agent for knocking out the gene by homologous recombination, or an agent for knocking out the gene by CRISPR-Cas9. The agent for inhibiting or reducing the expression of the gene can comprise a polynucleotide targeting the gene, such as siRNA, shRNA, sgRNA, miRNA or antisense RNA.

[0046] The primer pair for amplifying the ZmDDP2 gene has the nucleotide sequence shown in SEQ ID No. 12, SEQ ID No. 13 or SEQ ID No. 14, SEQ ID No. 15.

[0047] In the above application, the plant can be any one of the following:

[0048] P1) a monocotyledonous plant,

[0049] P2) a plant of the order Poales,

[0050] P3) a plant of the family Poaceae,

[0051] P4) a plant of the genus Zea,

[0052] P5) corn.

[0053] The present application also provides a genetically modified plant, in which the activity or content of ZmDDP2 protein is reduced, or the expression of the gene encoding ZmDDP2 protein is reduced, or the gene encoding ZmDDP2 protein is knocked out. The plant can be a monocotyledon or a dicotyledon. The monocotyledon can be a plant of the family Poaceae. The plant of the family Poaceae can be a plant of the genus Zea. The plant of the genus Zea can be corn.

[0054] The present application also provides a plant part or plant cell derived from the genetically modified plant described above, in which the activity or content of ZmDDP2 protein is reduced, or the expression of the gene encoding ZmDDP2 protein is reduced, or the gene encoding ZmDDP2 protein is knocked out.

[0055] The present application has the following beneficial effects:

[0056] 1. The present application verifies through experiments that the protein ZmDDP2 has a regulatory function on the drought resistance of plants in the flowering stage, and the biological materials related to the protein ZmDDP2 and its encoding gene can be applied to the drought resistance regulation of plants in the flowering stage.

[0057] 2. The present application provides a method for regulating the drought resistance of plants, which has a short breeding time and strong purpose compared with the traditional breeding method, significantly shortens the period of drought resistance breeding, and improves the efficiency of drought resistance breeding.

[0058] 3. The present application uses transgenic mutation technology to obtain a transgenic homozygous line with improved drought resistance and a gene editing homozygous line with reduced drought resistance, which provides gene resources for cultivating and improving new varieties of drought-resistant plants.

[0059] 4. The present application provides a theoretical basis for elucidating the molecular mechanism of ZmDDP2 protein in the drought resistance of plants. BRIEF DESCRIPTION OF DRAWINGS

[0060] The accompanying drawings are included to provide a further understanding of the present application, and constitute a part of the specification, which together with the embodiments of the present application, serve to explain the present application, and do not constitute a limitation of the present application.

[0061] FIG. 1 is a diagram of the correlation analysis of ZmDDP2 gene variation and corn ASI under drought stress.

[0062] FIG. 2 is a diagram of ZmDDP2 protein level detection.

[0063] Figure 3 is a graph showing that ZmDDP2 negatively regulates the coordination of corn powder spinning under drought stress in Zhu Zhou shed experiment.

[0064] Figure 4 is a graph showing that ZmDDP2 negatively regulates the coordination of corn powder spinning under drought stress in Zhangye experimental field experiment.

[0065] Figure 5 is a graph of subcellular localization analysis of two haplotype proteins of ZmDDP2.

[0066] Figure 6 is a graph of yield detection of ZmDDP2 gene editing mutant material after field drought. DETAILED DESCRIPTION

[0067] The preferred examples of the present application are described below in conjunction with the accompanying drawings, it should be understood that the following examples are given only for the purpose of illustration and are not intended to limit the scope of the present application. Those skilled in the art can make various modifications and replacements to the present application without departing from the spirit and principles of the present application.

[0068] The experimental methods in the following examples are all conventional methods, unless otherwise specified, according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained commercially. In the following examples, unless otherwise specified, quantitative tests were set up with three repeated experiments, and the results were averaged. In the examples, unless otherwise specified, the greenhouse conditions were as follows: the environmental temperature control range was 16-29℃, the light cycle was 16h light / 8h dark, and the environmental humidity was 40%±5%. The composition of the culture medium: 140kg of domestic soil, 5kg of vermiculite, 5kg of perlite, 40g of calcium hydroxide, and 10kg of chicken manure. After mixing the components of the culture medium, they were divided into 20 pots, which were the pots with culture medium.

[0069] The corn transgenic overexpression vector pBCXUN was modified from pCXUN (NCBI GenBank: FJ905215) vector. The modification process was to replace the selection marker gene Hyg with Bar gene through XhoI site. The above biological materials can be obtained from the applicant, and the obtained biological materials are only for repeating the experiments of the present application and cannot be used for other purposes.

[0070] Maize ND101 (maize inbred line ND101) is described in the following literature (i.e., the “inbred ND101 line” in the literature): High-Throughput and Accurate Determination of Transgene Copy Number and Zygosity in Transgenic Maize: From DNA Extraction to Data Analysis; Int. J. Mol. Sci. 2021, 22, 12487. https: / / doi.org / 10.3390 / ijms222212487.

[0071] The maize gene editing vector pBUE411 was kindly provided by Professor Chen Qijun of China Agricultural University. It was used in the literature "Hui-Li Xing, Li Dong". The biological material described above is disclosed in Zhi-Ping Wang, Hai-Yan Zhang, Chun-Yan Han, Bing Liu, Xue-Chen Wang and Qi-Jun Chen (2014). A CRISPR / Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biology 2014, 14:327. (in the literature, it is named pBUE411(Bar)). The public can obtain the above biological material from the applicant. The obtained biological material is only for repeating the experiments of this invention and cannot be used for other purposes.

[0072] In this invention, a two-tailed t-test is used to determine statistical significance. * indicates a significant difference (P < 0.05), and ** indicates an extremely significant difference (P < 0.01).

[0073] Example 1: The genetic variation of the ZmDDP2 gene is related to drought resistance during the flowering period of maize.

[0074] Genome-wide association analysis (GWA) was performed on 228 temperate maize inbred lines to investigate the ASI phenotype under drought stress at flowering stage. Multiple loci on chromosome 10 were found to be significantly associated with the ASI trait under drought stress, as shown in Figures 1A and 1B. Association analysis was performed on variant loci within a 0.5 Mb region to the left and right of the lead SNP with ASI under drought stress. Upon magnification, two SNP variants were found to be located in the coding region of the ZmDDP2 gene. These two variants were significantly associated with ASI in maize under drought stress (P < 10). -5 Lines connect the variant sites to each other in the LD diagram, and red asterisks indicate strong LD(r) between the variant sites.2 >0.6), as shown in Figures 1C and 1D. Analysis of the changes in two significant SNPs revealed that both SNPs led to amino acid variations. SNP347 caused the amino acid at position 116 to change from glycine (G) to glutamate (E), and SNP2405 caused the amino acid at position 736 to change from lysine (K) to isoleucine (I), as shown in Figure 1E.

[0075] Based on resequencing data and haplotype classification using two significant SNP sites, four haplotypes were identified, as shown in Figure 1F. Among these, Hap2 and Hap4 accounted for a relatively small proportion of the 228 inbred lines, less than 10% of the total. Therefore, subsequent studies mainly focused on analyzing Hap1 and Hap3. The Hap1 haplotype carries SNP347G and SNP2405A, and exhibits low ASI after drought stress; it was named ZmDDP2. S (small); while the Hap3 monomer carries SNP347A and SNP2405T, and has a large ASI after drought stress, and is named ZmDDP2. L (large). The above results indicate that ZmDDP2 S It is an excellent monomer type, as shown in Figure 1F.

[0076] Naturally mutant haplotype ZmDDP2 S The amino acid sequence of the ZmDDP2 protein is shown in SEQ ID No. 1, consisting of 1903 amino acid residues, and the coding nucleotide sequence of its gene is shown in SEQ ID No. 3, consisting of 5715 bases; the mutant monomeric ZmDDP2 L The amino acid sequence of the ZmDDP2 protein is shown in SEQ ID No. 2, consisting of 1902 amino acid residues, and the nucleotide sequence encoding its gene is shown in SEQ ID No. 4, consisting of 5712 bases.

[0077] Example 2: Detection of ZmDDP2 protein levels

[0078] 1. Detection of ZmDDP2 protein levels in representative inbred lines

[0079] To carry ZmDDP2 L Inbred lines and carrying ZmDDP2 S The leaves of the inbred lines were used as experimental materials. Total protein was extracted using 2×SDS loading buffer, and the accumulation of ZmDDP2 protein in the two types of inbred lines was detected by Western blot.

[0080] The specific steps are as follows:

[0081] Corn leaf tissue was collected, flash-frozen in liquid nitrogen, and then thoroughly ground using a mortar and pestle. 100 μL of the powder was transferred to a 1.5 mL centrifuge tube. 100 μL of 2×SDS loading buffer with β-mercaptoethanol was added to the sample. The mixture was vigorously vortexed to ensure thorough mixing. The mixture was heated in a 95°C metal bath for 10 min. At room temperature, the centrifuge speed was set to 10,000 g for 10 min. The supernatant was collected as total protein and used for loading and SDS-PAGE gel electrophoresis.

[0082] After electrophoresis, remove the gel plate and pry it open with a gel pryer. Trim off any excess gel beyond the target band. Place the gel to be transferred into transfer buffer. Cut an ECL membrane slightly larger than the gel and two pieces of thick filter paper. Soak them in transfer buffer. On a semi-dry transfer apparatus, place one piece of filter paper first, then the ECL membrane, protein gel, and the other piece of filter paper in sequence. During placement, use a glass rod to remove air bubbles. Close the transfer tank lid and set the transfer conditions to a constant voltage of 16V and a transfer time of 90 minutes (determine the transfer time based on the protein size).

[0083] After the transfer is complete, take a square dish, pour in blocking buffer (5% milk prepared with TBST), place the membrane in the dish, and block at room temperature for 1 hour or overnight at 4°C. Replace with fresh milk, add the corresponding primary antibody (or use the recovered primary antibody directly), and react at room temperature for 2 hours or overnight at 4°C. Recover the primary antibody, wash the membrane 3-4 times with TBST, 5 minutes each time. For the last wash, discard the TBST washing buffer, add fresh milk containing the corresponding secondary antibody, and react at room temperature for 1 hour. Wash the membrane 3-4 times with TBST, 5 minutes each time. Develop the substrate kit corresponding to the enzyme coupled to the secondary antibody (Immobilon™ Western: MILLIPORE Shanghai Trading Co., Ltd., catalog number: 1305701), add it to the membrane, react for 1 minute, and then place the membrane in a chemiluminescence imaging system for imaging and photography.

[0084] Reagents required for the experiment:

[0085] 1) 2×SDS loading buffer 10mL: glycerol 2mL, bromophenol blue 0.0202g, 1M Tris-HCl (pH 6.8)

[0086] 1 mL of β-mercaptoethanol, 0.14 mL of 10% SDS, and 4 mL of ddH2O were added to bring the volume to 10 mL. The mixture was then stored at -20°C.

[0087] 2) Transfer buffer 1L: 39mM glycine, 2.9g, Tris 5.8, SDS 0.37g, methanol 200mL, add ddH2O to make up to 1L.

[0088] 3) TBST (pH 7.6) 1L: Tris 2.42g, NaCl 8.8g, Tween 20 1ml; ddH2O 900mL; adjust pH and bring volume to 1L.

[0089] Western blot experiments used Actin protein as an internal control. The primary antibody was Actin monoclonal antibody (mouse-derived, Abclonal, catalog number: AC004); the secondary antibody was mouse anti-HRP-conjugated Goat anti-Mouse (Abclonal, catalog number: AS003).

[0090] The results are shown in Figure 2A, carrying ZmDDP2 L The protein accumulation level of ZmDDP2 in the inbred lines was higher than that of ZmDDP2 carriers. S The high abundance in the inbred lines indicates that the variation in amino acids affects the protein abundance.

[0091] 2. Detection of ZmDDP2 protein levels after drought stress treatment

[0092] Wild-type leaves under normal watering and drought treatments were used as experimental materials. Total protein was extracted using 2×SDS loading buffer, and the accumulation of ZmDDP2 protein under normal watering and drought treatments was detected by Western blot.

[0093] The results are shown in Figure 2B, where WW represents normal watering conditions and WS represents drought conditions. The ZmDDP2 protein accumulation level in the drought-treated wild-type material was higher than that in the leaves of the normally watered wild-type material, indicating that drought stress induces the accumulation of ZmDDP2 protein.

[0094] Example 3: Functional study of ZmDDP2 protein and its encoding gene

[0095] 1. Obtaining the ZmDDP2 gene-edited mutant in maize

[0096] A gRNA target (5'-CTACATTGGACGTAGCGCTCCAGG-3') (SEQ ID No. 7) was designed on the first exon of the ZmDDP2 gene using CRISPR-P (http: / / crispr.hzau.edu.cn / CRISPR2 / ). The target was constructed into the pBUE411 vector. After identifying positive clones by colony PCR, plasmids were extracted and sequenced. Plasmids with correct sequencing were transformed into Agrobacterium tumefaciens EHA105. Agrobacterium tumefaciens that were positive by colony PCR infected maize ND101 embryos to obtain T0 generation plants. T0 generation plants were self-pollinated to obtain T1 generation seeds. T1 generation seeds were germinated, planted, and maize cotyledons were harvested. Genomic DNA was extracted, and genotyping was performed using F1 and R1 primers. Self-pollination yielded T2 generation seeds of two edited types of maize ZmDDP2 gene-edited mutants. CRISPR Cas9 free was identified using F2 and R2 cells for both genotypes, with wild-type ND101 as a control. PCR-negative plants were CRISPR Cas9 free. Seeds of T2 generation homozygous edited lines without Cas9 were obtained by self-crossing the two edited maize ZmDDP2 gene-editing mutants and named zmddp2#1 and zmddp2#2 for subsequent experiments.

[0097] The sequences of the primers mentioned above are as follows:

[0098] F1: 5'-ATGGTGGTCTGGCGAATG-3' (SEQ ID No. 8)

[0099] R1: 5'-TGACGACGGAGGCAACTC-3' (SEQ ID No. 9)

[0100] F2: 5'-GACAGGCGTCTTCTACTGGTGCTAC-3' (SEQ ID No. 10)

[0101] R2: 5'-TATTCACTAGCTCGGGATAGTTGGC-3' (SEQ ID No. 11)

[0102] As shown in Figure 3A, the mutant zmddp2#1, compared with wild-type maize ND101, has an insertion of a base (T) at position 651 of the coding sequence of the ZmDDP2 gene in the maize genome on both homologous chromosomes. This results in a premature stop codon in the ZmDDP2 coding sequence, thus knocking out the ZmDDP2 gene. The mutated gene is named ZmDDP2-1. The coding sequence (CDS) of the ZmDDP2-1 gene is obtained by inserting a base (T) at position 651 of the nucleotide molecule shown in SEQ ID No. 3 of the sequence listing, while keeping the other nucleotide sequences of SEQ ID No. 3 unchanged. It encodes a protein ZmDDP2-1 composed of 231 amino acid residues, the amino acid sequence of which is shown in SEQ ID No. 5 of the sequence listing. Compared to wild-type maize ND101, the mutant zmddp2#2 has a 10-base deletion of 5'-CGTAGCGCTC-3' in the ZmDDP2 gene on both homologous chromosomes, resulting in a premature stop codon in the ZmDDP2 coding sequence, thus knocking out the ZmDDP2 gene. This mutated gene is named ZmDDP2-2. The coding sequence (CDS) of the ZmDDP2-2 gene is obtained by deleting nucleotides 643 to 652 (5'-CGTAGCGCTC-3') of the nucleotide molecule shown in SEQ ID No. 2, while keeping the other nucleotide sequences of SEQ ID No. 3 unchanged. It encodes a protein ZmDDP2-2 composed of 263 amino acid residues, the amino acid sequence of which is shown in SEQ ID No. 6 of the sequence listing.

[0103] 2. ASI phenotypic analysis of gene-edited maize with maize germplasm ND101 as background under greenhouse and field drought conditions.

[0104] 1) Zhuozhou Dry Shed

[0105] Year: 2022

[0106] The tested seeds were: maize ND101 seeds, and Cas9-free homozygous mutant seeds of the ZmDDP2 gene-edited mutants zmddp2#1 and zmddp2#2.

[0107] This experiment employed a randomized block design with two treatments: normal irrigation and drought. Both treatments consisted of three plots, each with four rows, 3 meters long, and 0.5 meters apart. Normal irrigation followed sowing. Approximately 35 days later, when the plants reached the V5-V6 stage, differential irrigation began. Soil water potential was automatically monitored using a buried Watermark 200SS sensor. The irrigation amount in the normal irrigation group maintained a soil water potential of 0 to -20 kPa, while the irrigation amount in the drought-stressed group was one-third that of the water-treated group, maintaining a soil water potential of -150 to -180 kPa. During flowering, pollen shedding time (DTA) and silking time (DTS) were recorded, and the average time between pollen shedding and silking (ASI) was calculated. Once 80% of the drought-treated group had completed silking, they were irrigated. Thereafter, the irrigation amount in the drought-treated group remained consistent with that in the normal irrigation group. The irrigation amount for the drought-stressed plots was 60% of that for the normal plots. Pollen shedding time (DTA) was the date when the maize tassel sheds pollen to half-cob length, and silking time (DTS) was the date when the maize silks emerged approximately 1 cm from the husk. The pollen shedding time and silking time for each individual plant were recorded, and the average value was taken. The average interval between pollen shedding and silking (ASI) was the number of days obtained by subtracting the pollen shedding time from the silking time; this was calculated per individual plant, and the average value was taken.

[0108] As shown in Figure 3B, after drought treatment, the pollen shedding time (DTA) of the ZmDDP2 gene-edited mutants zmddp2#1 and zmddp2#2 was not significantly different from that of the wild-type ND101 plants, but the silking time (DTS) was significantly earlier. Therefore, the ASI of the mutants was significantly smaller than that of the wild type, indicating that the ZmDDP2 protein negatively regulates the coordination of flowering of maize male and female ears.

[0109] 2) Experimental fields of Zhangye Experimental Station of Gansu Academy of Agricultural Sciences.

[0110] Years: 2022 and 2023.

[0111] The tested seeds were: maize ND101 seeds, and Cas9-free homozygous mutant seeds of the ZmDDP2 gene-edited mutants zmddp2#1 and zmddp2#2, with the 2022 generation being the T2 generation and the 2023 generation being the T3 generation.

[0112] In 2022, a randomized block experiment was conducted with two treatments: normal watering and drought. Both treatments consisted of three plots, each with four rows, a row length of 3 meters, and a row spacing of 0.5 meters. In 2023, the same randomized block experiment was conducted with two treatments: normal watering and drought. Both treatments consisted of three plots, each with two rows, a row length of 3 meters, and a row spacing of 0.5 meters. The water control conditions for both years were consistent with those in the Zhuozhou dry greenhouse experiment (1). The tested materials were subjected to drought stress and normal watering treatments. The pollen shedding time (DTA), silking time (DTS), and the pollen shedding-silking interval (ASI) were recorded.

[0113] Photographs of plants in R2 stage in 2022 are shown in Figure 4A, magnified photos of female and male ears are shown in Figure 4B, and statistical results of pollen shedding time (DTA), silking time (DTS), and the calculated pollen shedding-silking interval (ASI) are shown in Figure 4C. Statistical results of pollen shedding time (DTA), silking time (DTS), and the calculated pollen shedding-silking interval (ASI) for 2023 are shown in Figure 4D.

[0114] The results of the field experiment in 2022 are shown in Figure 4C. Under normal conditions, the pollen shedding time of the mutant line zmddp2#1 was delayed compared to maize ND101, while the silking time of the zmddp2#1 mutant line was not significantly different from that of wild-type maize ND101, resulting in a lower ASI for the zmddp2#1 mutant material compared to maize ND101. However, the pollen shedding and silking times of the mutant line zmddp2#2 were both earlier than those of wild-type maize ND101, with the silking time being significantly earlier, thus resulting in a lower ASI for the zmddp2#2 line compared to wild-type maize ND101. After drought stress, the pollen shedding time of the mutant line zmddp2#1 was not significantly different from that of maize ND101, but the silking time was significantly earlier. Conversely, the pollen shedding and silking times of the mutant line zmddp2#2 were both significantly earlier than those of maize ND101, with the silking time being significantly earlier. Therefore, the ASI of both mutant lines was significantly reduced compared to maize ND101 after drought stress.

[0115] The results of the field experiment in 2023 are shown in Figure 4D. Under normal conditions, the pollen shedding and silking times of the mutant lines zmddp2#1 and zmddp2#2 were earlier than those of maize ND101, with the silking time being significantly earlier. Therefore, the ASI of the two mutant lines was lower than that of wild-type maize ND101. After drought stress, the pollen shedding time of the mutant line zmddp2#1 was not significantly different from that of maize ND101, but the silking time was significantly earlier. The pollen shedding and silking times of the mutant line zmddp2#2 were both significantly earlier than those of maize ND101, with the silking time being significantly earlier. Therefore, after drought stress, the ASI of both mutant lines was significantly lower than that of maize ND101.

[0116] Two years of experimental data indicate that the ZmDDP2 protein plays a crucial role in regulating the pollen-silking interval of maize, and knocking out ZmDDP2 can effectively reduce the ASI (Advanced Pollen Segregation Intake) of maize under drought stress. In particular, field data from Zhangye showed that ZmDDP2 knockout not only reduced ASI in maize under drought stress but also shortened the growth period of maize (the mutant exhibited earlier pollen segregation) and reduced ASI in maize under normal growth conditions.

[0117] Example 4: ZmDDP2 protein is located in the cell nucleus

[0118] 1. Cloning of the gene encoding protein ZmDDP2

[0119] The coding sequence for the protein ZmDDP2 is named the ZmDDP2 gene, and the nucleotide sequence of the ZmDDP2 gene is shown in SEQ ID No. 3 (ZmDDP2). S ) and SEQ ID No.4 (ZmDDP2) L As shown in the figure, ZmDDP2 is encoded respectively. S (SEQ ID No. 1) and ZmDDP2 L (SEQ ID No.2) Two monomeric proteins.

[0120] Two representative inbred lines, R15 (carrying ZmDDP2), were selected. S ) and GEMS14 (carrying ZmDDP2) L Leaf materials from inbred line V5 were flash-frozen and ground in liquid nitrogen to extract total RNA, which was then reverse transcribed to obtain cDNA. Since the coding region of the ZmDDP2 gene contains 5715 (ZmDDP2...) S ) or 5712(ZmDDP2 LSince direct amplification of ZmDDP2 nucleotides cannot yield a satisfactory full-length fragment, a segmented cloning method was employed. First, using cDNA carrying two monomeric forms as a template, primer pair F3+R33 (containing SmaI and BamHI restriction sites) was used to amplify the fragment. S F4+R4 to ZmDDP2 L (Containing BamHI restriction sites) were subjected to PCR amplification. The amplification products were subjected to 1% agarose gel electrophoresis to obtain PCR amplification products of 2427bp and 3324bp, respectively.

[0121] The primer sequences described above are as follows:

[0122] F3: 5'-TCCCCCGGGATGGACGCCGAGCCCCGC-3' (SEQ ID No. 12)

[0123] R3: 5'-CGCGGATCCATTCTGATGAGAAACTGAGG-3' (SEQ ID No. 13)

[0124] F4: 5'-CGCGGATCCATTGTAACAGGTGGGTCTAAC-3' (SEQ ID No. 14)

[0125] R4: 5'-CGCGGATCCTCATCGCCACTCCCGGCAAC-3' (SEQ ID No. 15)

[0126] The two fragments were then recovered using a gel extraction kit and further ligated using the restriction enzyme sites on both sides to obtain SEQ ID No. 3 (ZmDDP2). S ) and SEQ ID No.4 (ZmDDP2) L The full-length sequence shown is shown in the figure.

[0127] 2. Construction of the expression carrier

[0128] ZmDDP2 as shown in SEQ ID No. 3 S And ZmDDP2 shown in SEQ ID No. 4 L The recombinant vectors were inserted into the SmaI space (downstream of the ubi promoter) of the vector pGreenⅡ-ubi:GFP, keeping the rest of the vector unchanged. The resulting recombinant vectors were named pGreenⅡ-ubi:ZmDDP2. S -GFP and pGreenⅡ-ubi:ZmDDP2 L -GFP.

[0129] The pGreenⅡ-ubi:GFP vector is created by replacing the 35S terminator of the pGreenⅡ-GFP vector with the ubi promoter.

[0130] 3. Observation of subcellular localization in transformed maize protoplasts

[0131] The correctly sequenced plasmid was co-transformed with the nuclear localization marker protein (SV40-mCherry) into ND101 maize protoplasts. After overnight culture, subcellular localization was observed using a Zeiss 880. GFP was detected by excitation at 488 nm and mCherry was detected by excitation at 561 nm.

[0132] As shown in Figure 5A, both monomeric forms of ZmDDP2 protein co-localize with the nuclear localization marker (SV40-mCherry), preliminarily indicating that ZmDDP2-GFP is localized in the cell nucleus.

[0133] 4. Nucleocytoplasmic separation experiment to analyze the subcellular localization of ZmDDP2

[0134] Leaf samples from wild-type ND101 and the ZmDDP2 gene-edited mutant zmddp#1 were used as experimental materials to conduct nucleocytoplasmic separation experiments. Total protein, cytoplasmic components, and nuclear components were extracted, and the ZmDDP2 protein content in different components was detected. Actin protein was used as the cytoplasmic component, and H3 protein was used as the nuclear component to evaluate the effectiveness of nucleocytoplasmic separation.

[0135] As shown in Figure 5B, the ZmDDP2 protein was detected only in the total sample and the nucleoplasm sample, while no target band was found in the mutant. This indicates that the antibody can specifically recognize the ZmDDP2 protein, and that the ZmDDP2 protein is located in the nucleus in plants.

[0136] Example 5: Knocking out ZmDDP2 can reduce drought-induced yield losses.

[0137] Processing years: 2022 and 2023

[0138] Location: Zhangye Experimental Field, Gansu Academy of Agricultural Sciences

[0139] The tested seeds were: maize ND101 seeds, and Cas9-free homozygous mutant seeds of the ZmDDP2 gene-edited mutants zmddp2#1 and zmddp2#2, with the 2022 generation being the T2 generation and the 2023 generation being the T3 generation.

[0140] 1) 2022:

[0141] In 2022, this experiment was conducted as a randomized block design with two treatments: normal irrigation and drought. Both treatments consisted of three plots, each with four rows, a row length of 3 meters, and a row spacing of 0.5 meters. After harvest, the number of kernels and kernel weight of the main ears of maize with relatively uniform growth within each plot were counted, and the number and weight of kernels per ear were calculated.

[0142] Figure 6A shows photographs of maize ears and kernels of WT, zmddp2#1, and zmddp2#2 in the normal and drought treatment groups. The kernel chart on the left and right images shows a 5cm scale, while the kernel chart on the right image shows a 2cm scale. Figure 6B shows the number of kernels per ear and kernel weight of zmddp2#1 and zmddp2#2 in the normal and drought treatment groups. Under normal growth conditions, there was no significant difference in the number of kernels per ear and kernel weight between the mutants and the wild type. However, under drought stress, the number of kernels per ear and kernel weight of both mutant lines were significantly higher than those of the wild type, reducing drought-induced yield loss by approximately 10%.

[0143] 1) 2023:

[0144] In 2023, a randomized block experiment was conducted with two treatments: normal irrigation and drought. Both treatments consisted of three plots, each with two rows, a row length of 3 meters, and a row spacing of 0.5 meters. After harvest, the number of kernels and kernel weight of the main ears of maize with relatively uniform growth in each plot were counted, and the kernel weight per ear was calculated with each plot as a replicate.

[0145] Figure 6C shows photographs of maize ears of WT, zmddp2#1, and zmddp2#2 in the normal and drought treatment groups, with a scale bar of 5 cm. Figure 6D shows the single ear grain weight of WT, zmddp2#1, and zmddp2#2 in the normal and drought treatment groups. Under normal growth conditions, there was no significant difference in single ear grain weight between the mutants and the wild type. However, under drought stress, the single ear grain weight of both mutant lines was significantly higher than that of the wild type, reducing the yield loss caused by drought by approximately 10–15%.

[0146] The above results indicate that reducing or knocking out the gene encoding the protein ZmDDP2 in plants can increase the yield per ear of maize in the drought-treated group without affecting the yield per ear of maize under normal growth conditions.

[0147] The contents not described in detail in this specification are existing technologies known to those skilled in the art.

[0148] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The use of the ZmDDP2 protein or its encoding gene, or biological material containing its encoding gene, in any of the following aspects: D1) Application in regulating plant drought resistance; Application of D2 in the preparation of products that regulate plant drought resistance; Application of D3 in drought-resistant plant breeding; The ZmDDP2 protein is any one of the following: A1) The amino acid sequence of the protein is SEQ ID No. 1 or SEQ ID No. 2; A2) Proteins with the same biological function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No.

2.

2. Application of ZmDDP2 protein or its encoding gene or biological materials containing its encoding gene in reducing the time interval between pollen shedding and silking in maize; The ZmDDP2 protein is any one of the following: A1) The amino acid sequence of the protein is SEQ ID No. 1 or SEQ ID No. 2; A2) Proteins with the same biological function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No.

2.

3. Application of ZmDDP2 protein or its encoding gene or biological materials containing its encoding gene in increasing maize yield; The ZmDDP2 protein is any one of the following: A1) The amino acid sequence of the protein is SEQ ID No. 1 or SEQ ID No. 2; A2) Proteins with the same biological function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No.

2.

4. The application according to any one of claims 1-3, characterized in that: The biomaterial is any one of B1) to B9) below: B1) A nucleic acid molecule encoding the protein described in claim 1; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2), or a transgenic plant cell line containing the recombinant vector described in B3); B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2), or transgenic plant tissue containing the recombinant vector described in B3); B7) A transgenic plant organ containing the nucleic acid molecule described in B1), or a transgenic plant organ containing the expression cassette described in B2), or a transgenic plant organ containing the recombinant vector described in B3); B8) A nucleic acid molecule that inhibits or reduces the expression of the gene encoding the protein of claim 1 or a nucleic acid molecule that inhibits or reduces the activity of the protein of claim 1; B9) Expression cassettes, recombinant vectors, recombinant microorganisms, or transgenic plant cell lines containing the nucleic acid molecules described in B8).

5. The application according to any one of claims 1-3, characterized in that: The gene encoding the ZmDDP2 protein is any one of the following: b1) The nucleotide sequence shown in SEQ ID No. 3 or SEQ ID No. 4; b2) A nucleotide sequence that expresses the same function protein by substituting, deleting and / or adding one or more nucleotides to the nucleotide sequence shown in SEQ ID No. 3 or SEQ ID No.

4.

6. The application according to any one of claims 1-3, characterized in that: This is achieved by inhibiting or reducing the expression level and / or activity of the ZmDDP2 protein.

7. A method for improving plant drought resistance, characterized in that: Plants with improved drought resistance can be obtained by inhibiting or reducing the expression level and / or activity of ZmDDP2 protein through transgenic technology. The ZmDDP2 protein is any one of the following: A1) The amino acid sequence of the protein is SEQ ID No. 1 or SEQ ID No. 2; A2) Proteins with the same biological function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No.

2.

8. The method according to claim 7, characterized in that: The suppression or reduction of gene expression can be achieved by gene knockout or gene silencing.

9. The method according to claim 7, characterized in that: The gene encoding the ZmDDP2 protein in maize was edited using CRISPR / Cas9 technology to suppress the expression and / or activity of the ZmDDP2 protein in maize; the gRNA target sequence is shown in SEQ ID No.

7.

10. Primer pairs for amplifying the ZmDDP2 gene, the nucleotide sequences of which are shown in SEQ ID No. 12, SEQ ID No. 13 or SEQ ID No. 14, SEQ ID No.

15.

11. A genetically modified plant in which the activity or content of the ZmDDP2 protein is reduced, or the expression of the gene encoding the ZmDDP2 protein is reduced, or the gene encoding the ZmDDP2 protein is knocked out, wherein the ZmDDP2 protein is any one of the following: A1) The amino acid sequence of the protein is SEQ ID No. 1 or SEQ ID No. 2; A2) Proteins with the same biological function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No.

2.

12. The plant according to claim 11, wherein the gene encoding the ZmDDP2 protein is any one of the following: b1) The nucleotide sequence shown in SEQ ID No. 3 or SEQ ID No. 4; b2) A nucleotide sequence that expresses the same function protein by substituting, deleting and / or adding one or more nucleotides to the nucleotide sequence shown in SEQ ID No. 3 or SEQ ID No.

4.

13. The plant according to claim 11 or 12, wherein the plant is a monocotyledonous or dicotyledonous plant, preferably a monocotyledonous plant, more preferably a grass, even more preferably a maize plant, and even more preferably maize.

14. A plant part or plant cell derived from a plant according to any one of claims 11-13, wherein the activity or content of the ZmDDP2 protein is reduced, or the expression of the gene encoding the ZmDDP2 protein is reduced, or the gene encoding the ZmDDP2 protein is knocked out.

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