Application of ZmC2DP1 protein in regulation and control of drought resistance and salt resistance of plants

Abstract

The invention relates to the technical field of genetic engineering breeding, in particular to application of ZmC2DP1 protein in regulation and control of drought resistance and salt tolerance of plants, the amino acid sequence of the ZmC2DP1 protein is as shown in SEQ ID NO.1, and the nucleotide sequence of a gene for coding the ZmC2DP1 protein is as shown in SEQ ID NO.2. After the ZmC2DP1 gene is knocked out, the drought resistance and the salt resistance of a mutant plant under the drought treatment or salt treatment condition are improved compared with those of a control group plant, and it is verified that the ZmC2DP1 protein has a negative regulation function on the drought resistance and the salt resistance of the plant. Compared with a traditional breeding mode, the method for regulating and controlling the drought resistance and the salt resistance of the plants has the advantages that the breeding time is short, the purposiveness is high, the drought resistance breeding period is remarkably shortened, and the crop stress resistance breeding efficiency is improved; a gene editing homozygous strain with improved drought resistance and salt tolerance is obtained by adopting a gene knockout technology, and gene resources are provided for cultivating and improving new varieties of stress-resistant plants; and a theoretical basis is provided for clarification of a molecular mechanism of the ZmC2DP1 protein in drought resistance and salt resistance of plants.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering breeding technology, specifically to the application of ZmC2DP1 protein in regulating plant drought resistance and salt tolerance. Background Technology

[0002] Studies have shown that drought is the leading meteorological disaster threatening agricultural production. Given the current context of rapid global climate change, ensuring food security is of paramount importance. Developing new varieties of major crops that are resilient to adverse conditions and offer stable and increased yields is a crucial requirement for ensuring food security.

[0003] Maize is one of the three traditional major crops, and the maize industry has developed rapidly. However, its production is often affected by various natural disasters. Currently, the identified genes for drought and salt stress tolerance in maize are still limited, greatly restricting the breeding of drought- and salt-tolerant maize varieties based on molecular design. Therefore, identifying key genes for drought and salt tolerance in maize and elucidating their mechanisms of action is of significant practical importance. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide the application of ZmC2DP1 protein in regulating plant drought resistance and salt tolerance. By knocking out the ZmC2DP1 gene, mutant plants under drought or salt treatment conditions showed increased resistance compared to the control group, verifying that the protein ZmC2DP1 has a negative regulatory function on plant drought resistance and salt tolerance.

[0005] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows:

[0006] Application of ZmC2DP1 protein, its encoding gene, or biological materials containing its encoding gene in improving plant drought resistance, wherein the ZmC2DP1 protein is any of the following proteins:

[0007] A1) The amino acid sequence is that of the protein listed as SEQ ID No. 1 in the sequence listing;

[0008] 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 in the sequence listing.

[0009] Application of ZmC2DP1 protein, its encoding gene, or biological materials containing its encoding gene in salt tolerance of maize, wherein the ZmC2DP1 protein is any of the following proteins:

[0010] A1) The amino acid sequence is that of the protein listed as SEQ ID No. 1 in the sequence listing;

[0011] 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 in the sequence listing.

[0012] Preferably, the gene encoding the ZmC2DP1 protein is any one of the following:

[0013] A1) The nucleotide sequence shown in SEQ ID No. 2;

[0014] A2) 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. 2.

[0015] Preferably, in the above applications, the substance regulating the expression of the protein-coding gene or the substance regulating the activity or content of the protein is a biological material, and the biological material may be any one of B1) to B9) below:

[0016] B1) The nucleic acid molecule encoding the ZmC2DP1 protein mentioned above;

[0017] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0018] B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0019] 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);

[0020] 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);

[0021] 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);

[0022] 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);

[0023] B8) Nucleic acid molecules that inhibit or reduce the expression of the genes encoding the above proteins or nucleic acid molecules that inhibit or reduce the activity of the above proteins;

[0024] B9) Expression cassettes, recombinant vectors, recombinant microorganisms, or transgenic plant cell lines containing the nucleic acid molecules described in B8).

[0025] Preferably, this is achieved by inhibiting or reducing the expression level and / or activity of the ZmC2DP1 protein in plants.

[0026] In the above applications, the substance regulating gene expression can be a substance that performs at least one of the following six types of regulation: 1) regulation at the gene transcription level; 2) post-transcriptional regulation 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 of the gene from the nucleus to the cytoplasm); 4) regulation of gene translation; 5) regulation of mRNA degradation of the gene; and 6) post-translational regulation of the gene (i.e., regulation of the activity of the protein translated from the gene).

[0027] In the above applications, the substance regulating gene expression can be a reagent that inhibits or reduces the expression of the gene. The reagent that inhibits or reduces the expression of the gene can be a gene knockout reagent, such as a reagent that knocks out the gene through homologous recombination or a reagent that knocks out the gene through CRISPR-Cas9. The reagent that inhibits or reduces the expression of the gene can contain a polynucleotide that targets the gene, such as siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0028] A method for improving plant drought resistance is achieved by reducing or inhibiting the expression level and / or activity of the ZmC2DP1 protein in maize; the ZmC2DP1 protein is any of the following proteins:

[0029] A1) The amino acid sequence is that of the protein listed as SEQ ID No. 1 in the sequence listing;

[0030] 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 in the sequence listing.

[0031] A method for improving salt tolerance in plants is achieved by reducing or inhibiting the expression level and / or activity of the ZmC2DP1 protein in maize; the ZmC2DP1 protein is any of the following proteins:

[0032] A1) The amino acid sequence is that of the protein listed as SEQ ID No. 1 in the sequence listing;

[0033] 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 in the sequence listing.

[0034] The application of ZmC2DP1 protein, its encoding gene, or biological materials containing its encoding gene in genetic breeding for drought resistance or salt tolerance in maize, wherein the ZmC2DP1 protein is any one of the following proteins:

[0035] A1) The amino acid sequence is that of the protein listed as SEQ ID No. 1 in the sequence listing;

[0036] 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 in the sequence listing.

[0037] Preferably, the inhibition or reduction of gene expression is achieved by gene knockout or gene silencing.

[0038] Preferably, the gene encoding the ZmC2DP1 protein in maize is edited using CRISPR / Cas9 technology to suppress the expression level and / or activity of the ZmC2DP1 protein in maize; the gRNA target sequence is shown in SEQ ID No. 9.

[0039] The primers for amplifying the ZmC2DP1 gene have nucleotide sequences shown in SEQ ID No. 3 and SEQ ID No. 4.

[0040] The beneficial effects of this invention are:

[0041] 1. This invention verifies that the ZmC2DP1 protein has a regulatory function on plant drought resistance and salt tolerance, and that the ZmC2DP1 protein and its related biomaterials can be applied to the regulation of plant drought resistance and salt tolerance.

[0042] 2. This invention provides a method for regulating plant drought resistance and salt tolerance. Compared with traditional breeding methods, it has a shorter breeding time, stronger purpose, significantly shortens the cycle of drought resistance breeding, and improves the efficiency of crop stress resistance breeding.

[0043] 3. This invention uses gene knockout technology to obtain gene-edited homozygous lines with improved drought resistance and salt tolerance, providing gene resources for breeding and improving new stress-resistant plant varieties.

[0044] 4. This invention provides a theoretical basis for elucidating the molecular mechanism of ZmC2DP1 protein in plant drought resistance and salt tolerance. Attached Figure Description

[0045] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0046] Figure 1 Figure A shows the results of detecting the transcriptional level of the ZmC2DP1 gene in maize roots under salt stress treatment. Figure 1 Figure B shows the detection results of ZmC2DP1 protein levels in maize roots under salt stress treatment.

[0047] Figure 2 A is a diagram showing the mutation sites of the mutant Zmc2dp1; Figure 2 B shows the phenotypic diagrams of WT and Zmc2dp1 before drought and after rehydration; Figure 2 C represents the drought survival rate statistics for WT and Zmc2dp1; Figure 2 D shows the results of DAB and NBT staining of WT and Zmc2dp1 under drought treatment (Water-stressed, WS) and normal watering (Well-watered, WW) conditions; Figure 2 E represents the MDA content determination results of WT and Zmc2dp1 under drought and normal irrigation conditions.

[0048] Figure 3 Figure A shows the results of salt stress treatments (WT and Zmc2dp1) and the control treatment. Figure 3 B represents the percentage reduction in biomass after WT and Zmc2dp1 salt stress treatments; Figure 3 C represents the SPAD values ​​measured under the WT and Zmc2dp1 control treatments and salt stress treatments; Figure 3 D represents the Na content in the underground parts under the control and salt stress conditions (WT and Zmc2dp1 treatments). + Ion content; Figure 3 E represents the Na+ content in the aboveground parts under the control treatments of WT and Zmc2dp1 and the salt treatment conditions. + Ion content Detailed Implementation

[0049] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the following embodiments are given for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0050] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0051] The maize transgenic overexpression vector pBCXUN was modified from the pCXUN (NCBI GenBank: FJ905215) vector. By replacing the selection marker gene Hyg with the Bar gene at the XhoI site, this vector was kindly provided by Professor Chen Qijun of China Agricultural University.

[0052] The maize transgenic recipient material LH244 (PI 612589) was obtained from GRIN-Global (https: / / npgsweb.ars-grin.gov / gringlobal / search).

[0053] The maize gene editing vector pBUE411 was kindly provided by Professor Qijun Chen of China Agricultural University and was disclosed in the literature "Hui-LiXing,Li Dong,Zhi-Ping Wang,Hai-Yan Zhang,Chun-Yan Han,Bing Liu,Xue-Chen Wangand Qi-Jun Chen (2014).A CRISPR / Cas9 toolkit for multiplex genome editing inplants.BMC Plant Biology 2014,14:327." (in the literature, it is named pBUE411(Bar)).

[0054] 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).

[0055] The amino acid sequence of the ZmC2DP1 protein is shown in SEQ ID No. 1, and the nucleotide sequence of its encoding gene is shown in SEQ ID No. 2. It consists of 996 bases and has no introns.

[0056] Example 1: Obtaining the ZmC2DP1 protein and its encoding gene

[0057] 1. Cloning of ZmC2DP1 protein and its encoding gene

[0058] Seeds of the sensitive maize inbred line B73 were germinated at 24℃ for 5 days. Germinated seeds were then transferred to a nutrient solution and cultured for one week. Whole seedlings were then flash-frozen in liquid nitrogen, ground, and total RNA was extracted. Reverse transcription was performed to obtain cDNA. Using this cDNA as a template and F1 and R1 primers, PCR amplification was performed. The amplified product was subjected to 1% agarose gel electrophoresis, yielding a 996 bp PCR product.

[0059] Sequencing revealed that the PCR product derived from the sensitive maize inbred line B73 has the nucleotide sequence shown in positions 136-1137 of SEQ ID No. 2 (this reference sequence can be obtained by searching Zm00001d005315 on https: / / www.maizegdb.org / / ).

[0060] The primer sequences mentioned above are as follows:

[0061] F1: 5'-ATGGGTTCCCGCTACGAGGTGGAGGTCAC-3'; (SEQ ID No. 3)

[0062] R1: 5'-GTAGTCGTCGTCGCCGCCATAGTCGTCGTT-3' (SEQ ID No. 4)

[0063] 2. Construction of the recombinant vector pBCXUN-ZmC2DP1-GFP

[0064] The 3' end of the coding sequence of the ZmC2DP1 gene (SEQ ID No. 2) was fused with the coding sequence of green fluorescent protein (GFP), and a homologous arm of the XcmI restriction enzyme site (downstream of the ubi promoter) was added. The vector was then digested with XcmI, and the digestion product was ligated with the pBCXUN vector fragment, which had also been digested with the same enzyme, using T4 ligase to obtain a recombinant vector containing the ZmC2DP1-GFP coding sequence, named pBCXUN-ZmC2DP1-GFP. The promoter for the ZmC2DP1 gene in the recombinant vector pBCXUN-ZmC2DP1-GFP is Zmubiquitin1.

[0065] 3. Obtaining recombinant Agrobacterium

[0066] The recombinant vector pBCXUN-ZmC2DP1-GFP was transformed into Agrobacterium EHA105 to obtain recombinant Agrobacterium EHA105 / pBCXUN-ZmC2DP1-GFP containing the recombinant vector pBCXUN-ZmC2DP1-GFP (after colony PCR, plasmid was extracted by shaking and sequencing to verify that the recombinant Agrobacterium was a positive clone).

[0067] 4. Obtaining transgenic homozygous ZmC2DP1 maize

[0068] Recombinant Agrobacterium EHA105 / pBCXUN-ZmC2DP1-GFP was used to infect the immature embryos of wild-type maize LH244 to obtain transgenic positive T1 generation seeds, which were used for subsequent experiments to detect ZmC2DP1 protein.

[0069] Example 2: The transcriptional and protein levels of the ZmC2DP1 encoding gene can be induced by salt stress.

[0070] 1. Detection of the transcriptional level of the ZmC2DP1 gene in maize roots under salt stress treatment

[0071] Wild-type LH244 maize, grown hydroponically for 5 days, was treated with 80 mM NaCl. Samples were taken at 0.5 h, 1 h, 6 h, 12 h, and 24 h after treatment, and RNA was extracted from the root tissue. cDNA was obtained by reverse transcription. Using the cDNA as a template and the maize gene ZmUbi2 (Zm00001d053838) as an internal control, primers QF1 and QR1 were used. The expression level of the ZmC2DP1 gene was detected using specific primers QF2 and QR2, with wild-type LH244 as a control.

[0072] The sequences of the primers mentioned above are as follows:

[0073] QF1: 5'-TGGTTGTGGCTTCGTTGGTT-3' (SEQ ID No. 5)

[0074] QR1: 5'-GCTGCAGAAGAGTTTTGGGTACA-3' (SEQ ID No. 6)

[0075] QF2: 5'-CGGGGTACCTTCCACCTA-3' (SEQ ID No. 7)

[0076] QR2: 5'-GCTCCTCGAACTTGTCCTCC-3' (SEQ ID No. 8)

[0077] The results are as follows Figure 1 As shown in Figure A, after 0.5 h of salt stress treatment, the transcriptional level of the ZmC2DP1 gene was induced, and after 24 h of treatment, the transcriptional level of the ZmC2DP1 gene was significantly higher than that of the untreated sample. This indicates that the ZmC2DP1 gene can be induced by salt stress.

[0078] 2. Detection of ZmC2DP1 protein levels in maize roots under salt stress treatment

[0079] Root tissues from ZmC2DP1-GFP T1 transgenic plants treated with 80 mM NaCl for 0 h, 1 h, 6 h, and 12 h were used as experimental materials. Total protein was extracted using 2×SDS loading buffer, and the accumulation of ZmC2DP1 protein in the two types of inbred lines was detected by Western blot. The specific procedures are as follows:

[0080] Root tissues from ZmC2DP1-GFPT1 transgenic plants subjected to salt stress for 0h, 1h, 6h, and 12h were 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, and 100 μL of 2×SDS loading buffer containing β-mercaptoethanol was added. The mixture was vigorously vortexed to ensure thorough mixing, heated in a 95°C metal bath for 10 min, and then centrifuged at 10,000 g for 10 min at room temperature. The supernatant was collected as total protein and subjected to SDS-PAGE gel electrophoresis.

[0081] 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 the transfer buffer. Cut an ECL membrane slightly larger than the gel and two pieces of thick filter paper. Soak them in the transfer buffer. On a semi-dry transfer apparatus, place one piece of filter paper first, then place 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 for 90 minutes (determine the transfer time based on protein size). After transfer, 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 recycled primary antibody directly), and react at room temperature for 2 hours or overnight at 4°C. Recycle the primary antibody, wash the membrane 3-4 times with TBST, 5 minutes each time. For the last wash, discard the TBST wash 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 (iBrightCL1500, Life Technologies Holdings Pte Ltd.) for imaging.

[0082] Reagents required for the experiment:

[0083] 1) 2×SDS loading buffer 10mL: glycerol 2mL, bromophenol blue 0.0202g, 1M Tris-HCl (pH 6.8) 1mL, β-mercaptoethanol 0.14mL, 10% SDS 4mL, add ddH2O to make up to 10mL, store at -20℃.

[0084] 2) Transfer buffer 1L: 39mM glycine, 2.9g, Tris 5.8, SDS 0.37g, methanol 200mL, add ddH2O to make up to 1L.

[0085] 3) TBST (pH 7.6) 1L: Tris 2.42g, NaCl 8.8g, Tween 201ml; ddH2O 900mL; adjust pH and bring volume to 1L.

[0086] 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). ZmC2DP1-GFP protein levels were detected using GFP antibody (mouse-derived, Abclonal, catalog number: AE012), with mouse anti-HRP-conjugated Goat anti-Mouse (Abclonal, catalog number: AS003) as the secondary antibody.

[0087] The results are as follows Figure 1 As shown in Figure B, the protein level of ZmC2DP1-GFP gradually increased after salt stress treatment, indicating that salt stress treatment can promote the accumulation of ZmC2DP1 protein.

[0088] Example 3: Functional study of ZmC2DP1 protein and its encoding gene

[0089] 1. Obtaining the ZmC2DP1 gene mutant in maize

[0090] A gRNA target (5'-GACCTCAAGCCCTACGCCGTGG-3') (SEQ ID No. 9) was designed on the third exon of ZmC2DP1 using CRISPR-P (http: / / crispr.hzau.edu.cn / CRISPR2 / ). The target sequence was constructed into the pBUE411 vector. After identifying positive clones by colony PCR, plasmids were extracted and sequenced. The correctly sequenced plasmids were transformed into Agrobacterium EHA105. Agrobacterium colony PCR-positive bacteria infected maize LH244 embryos to obtain T0 generation plants. T0 generation plants were self-crossed 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 F2 and R2 primer pairs, respectively. Self-crossing yielded T2 generation seeds of a maize ZmC2DP1 gene mutant with an edited type. The mutant material was identified as CRISPR Cas9 free using F3 and R3 cells, with wild-type LH244 as a control. PCR-negative plants were CRISPR Cas9 free. Seeds of the T2 generation homozygous edited line without Cas9, obtained by self-pollination of the maize ZmC2DP1 gene mutant, were named Zmc2dp1. Figure 2As shown in A, it is used for subsequent experiments.

[0091] The sequences of the primers mentioned above are as follows:

[0092] F2: 5'-ATGGGTTCCCGCTACGAG-3' (SEQ ID No. 10)

[0093] R2: 5'-GCTCCTCGAACTTGTCCTCC-3' (SEQ ID No. 11)

[0094] F3: 5'-GACAGGCGTCTTCTACTGGTGCTAC-3' (SEQ ID No. 12)

[0095] R3: 5'-TATTCACTAGCTCGGGATAGTTGGC-3' (SEQ ID No. 13)

[0096] like Figure 2 The mutant Zmc2dp1 shown in Figure A, compared to wild-type maize LH244, has a deletion of one base (C) at position 93 in the coding sequence of the ZmC2DP1 gene on both homologous chromosomes of the maize genome. This results in a frameshift of the ZmC2DP1 coding sequence starting from amino acid 31, effectively knocking out the ZmC2DP1 gene. This mutated gene is named the ZmC2DP1-1 gene. The coding sequence (CDS) of the ZmC2DP1-1 gene is obtained by deleting one base (C) at position 93 of the nucleotide molecule shown in SEQ ID No. 2, while keeping the other nucleotide sequences of SEQ ID No. 2 unchanged.

[0097] 2. Phenotypic analysis of ZmC2DP1's role in drought resistance in maize

[0098] Five-day-old T2 generation gene knockout mutant Zmc2dp1 and wild-type maize LH244(WT) plants were transferred to white boxes containing 2 kg of nutrient soil: vermiculite: imported soil in a 1:1:1 ratio. After 7 days of normal growth, a drought treatment (i.e., cessation of watering) was applied for 25 days, followed by rewatering. Five days after rewatering, the survival rate of each line was recorded (plants with normal leaf color and normal growth were defined as surviving plants, while those with scorched leaves and abnormal growth were defined as dead plants; the survival rate was the percentage of surviving plants in each line out of the total number of plants). The experiment was repeated three times, with at least 18 plants in each line per replicate, and the average value was used for statistical analysis. Twelve days after the drought treatment, samples were taken for DAB and NBT staining to detect reactive oxygen species levels; simultaneously, malondialdehyde (MDA) content was measured to determine the redox level of the plants in response to drought stress.

[0099] The results are as follows Figure 2 As shown in BE, Figure 2 B shows photos of WT and Zmc2dp1 before drought and after rehydration, with a scale bar of 10cm. Figure 2 C represents the drought survival rate statistics for WT and Zmc2dp1; Figure 2 D shows the results of DAB staining and NBT staining of WT and Zmc2dp1 under drought treatment (Water-stressed, WS) and normal watering (Well-watered, WW) conditions, with a scale bar of 1 cm. Figure 2 E represents the MDA content of WT and Zmc2dp1 under drought and normal irrigation conditions. Significance analysis was performed using one-way ANOVA combined with Turkey's post-hoc test; different letters indicate significance at the P < 0.05 level. The figure shows that after drought treatment, the Zmc2dp1 mutant exhibited less leaf wilt than wild-type maize (WT), and its survival rate was significantly higher. Under drought stress, the Zmc2dp1 mutant had lower reactive oxygen species (ROS) and MDA levels than the wild type. These results indicate that the ZmC2DP1 protein negatively regulates drought resistance in maize, and knocking out the gene encoding the ZmC2DP1 protein enhances drought resistance in maize.

[0100] 3. Phenotypic analysis of the ZmC2DP1 gene's role in maize salt tolerance

[0101] Experimental materials: T2 generation gene knockout mutant Zmc2dp1 and wild-type maize LH244(WT) plants were used for salt stress phenotype identification. The experimental procedures are as follows:

[0102] 1) Filling with soil: Sift the nutrient soil and mix it well. Take an equal amount of soil and fill it into a 12cm x 12cm black round plastic pot, and compact it.

[0103] b. Pouring salt solution: Add 80mM NaCl solution to the bottom of the pot and let the soil in the pot fully absorb it until the surface soil absorbs the salt water.

[0104] c. Sowing: Select plump seeds and sow 10 seeds in each pot. Cover the seeds with an equal amount of sand (a mixture of nutrient soil and sand in equal proportions). After the surface soil absorbs the salt water, discard the excess salt water at the bottom.

[0105] d. Salt phenotype identification: After growing in the culture room for about 12 days, the salt phenotype of the maize material was determined based on seedling size and leaf chlorophyll retention (SPAD value), and the Na content in the aboveground (shoot) and underground (root) parts was measured. +Ion content was confirmed; incubation conditions: temperature 24±1℃, humidity approximately 40%, 14h light, 10h darkness. The experiment was repeated in triplicate, and the average value was used for statistical analysis.

[0106] The results are as follows Figure 3 As shown in AE, Figure 3 A shows photographs of WT and Zmc2dp1 salt stress treatments (80 mM NaCl) and the control treatment (0 mM NaCl), with a scale bar of 10 cm. Figure 3 B represents the percentage reduction in biomass after WT and Zmc2dp1 salt stress treatments. Figure 3 C represents the SPAD values ​​measured under WT and Zmc2dp1 control treatments and salt stress treatments. Figure 3 D represents the Na content in the underground parts under the control and salt stress conditions (WT and Zmc2dp1 treatments). + Ion content. Figure 3 E represents the Na+ content in the aboveground parts under the control treatments of WT and Zmc2dp1 and the salt treatment conditions. + Ion content. Among them... Figure 3 The significance analysis of CE was performed using one-way ANOVA combined with Turkey's post-hoc test, with different letters indicating significance at the P < 0.05 level. The figure shows that after salt stress treatment, the Zmc2dp1 mutant plants were larger, had less biomass reduction, higher leaf chlorosis (SPAD value), and higher Na+ levels in the aboveground parts compared to wild-type maize LH244(WT). + The ion content was significantly lower than that of wild-type maize LH244(WT). These results indicate that the ZmC2DP1 protein negatively regulates the salt tolerance of maize, and knocking out the gene encoding the ZmC2DP1 protein can enhance the salt tolerance of maize.

[0107] The contents not described in detail in this specification are existing technologies known to those skilled in the art.

[0108] 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 application of ZmC2DP1 protein, its encoding gene, or biological materials containing its encoding gene in improving plant drought resistance, characterized in that, The ZmC2DP1 protein is any of the following proteins: A1) The amino acid sequence is that of the protein listed as SEQ ID No. 1 in the sequence listing; 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 in the sequence listing.

2. The application of the ZmC2DP1 protein, its encoding gene, or biological materials containing its encoding gene in the salt tolerance of maize, characterized in that, The ZmC2DP1 protein is any of the following proteins: A1) The amino acid sequence is that of the protein listed as SEQ ID No. 1 in the sequence listing; 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 in the sequence listing.

3. The application according to claim 1 or 2, characterized in that, The gene encoding the ZmC2DP1 protein is any one of the following: A1) The nucleotide sequence shown in SEQ ID No. 2; A2) 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.

2.

4. The application according to claim 1 or 2, characterized in that, The biomaterial is any one of B1) to B9) below: B1) The nucleic acid molecule encoding the ZmC2DP1 protein mentioned above; 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) Nucleic acid molecules that inhibit or reduce the expression of the genes encoding the above proteins or nucleic acid molecules that inhibit or reduce the activity of the above proteins; 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 claim 1 or 2, characterized in that, This is achieved by inhibiting or reducing the expression level and / or activity of the ZmC2DP1 protein in plants.

6. A method for improving plant drought resistance, characterized in that, This is achieved by reducing or inhibiting the expression level and / or activity of the ZmC2DP1 protein in maize; the ZmC2DP1 protein is any of the following proteins: A1) The amino acid sequence is that of the protein listed as SEQ ID No. 1 in the sequence listing; 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 in the sequence listing.

7. A method for improving the salt tolerance of plants, characterized in that, This is achieved by reducing or inhibiting the expression level and / or activity of the ZmC2DP1 protein in maize; the ZmC2DP1 protein is any of the following proteins: A1) The amino acid sequence is that of the protein listed as SEQ ID No. 1 in the sequence listing; 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 in the sequence listing.

8. The method according to claim 6 or 7, characterized in that: The suppression or reduction of gene expression is achieved through gene knockout or gene silencing.

9. The method according to claim 6 or 7, characterized in that: The gene encoding the ZmC2DP1 protein in maize was edited using CRISPR / Cas9 technology to suppress the expression and / or activity of the ZmC2DP1 protein in maize; the gRNA target sequence is shown in SEQ ID No.

9.

10. Primers for amplifying the ZmC2DP1 gene, the nucleotide sequences of which are shown in SEQ ID No. 3 and SEQ ID No. 4.

Images