Application of zmdapf1 protein in regulating drought resistance in plants

Abstract

The present invention relates to the application of a ZmDapF1 protein in regulating drought resistance in plants. The protein is as shown in SEQ ID No. 1. Further disclosed is a method for improving drought resistance in plants, the method comprising: introducing a coding gene of the protein into a recipient plant to obtain a target plant having a higher drought resistance than that of the recipient plant. Experiments have verified that the ZmDapF1 protein has the function of regulating controlling drought resistance in plants; thus, said protein and related biomaterials thereof can be applied in regulating plant drought resistance.

Description

Application of ZmDAPF1 protein in regulating drought resistance of plants TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering breeding, in particular to an application of ZmDAPF1 protein in regulating drought resistance of plants. BACKGROUND

[0002] Drought is a major agricultural natural disaster, about 43% of the global arable land is located in arid and semi-arid regions, and the loss of grain caused by drought accounts for 60% of the total loss caused by all natural disasters. As a crop with high water requirement and sensitive to drought stress, corn will be seriously affected in yield once water deficiency occurs, so it is particularly important to genetically improve the drought resistance of corn.

[0003] Traditional breeding techniques have the disadvantages of long cycle, high blindness and large workload, and the improvement of grain yield through traditional breeding has reached a certain bottleneck. In recent years, with the development of plant molecular biology and genetics and the in-depth study of plant stress resistance molecular mechanism, it has become mature to introduce stress resistance related genes into plants to improve the stress resistance of crops through genetic engineering, which provides possible gene resources and theoretical basis for genetic improvement of drought resistance of corn.

[0004] Corn (Zea mays L.) is an important feed and food crop widely planted in China, and it is of great significance to clone drought resistance genes of corn, improve drought resistance of corn and increase yield of corn. SUMMARY

[0005] In view of the defects in the prior art, the purpose of the present application is to provide an application of ZmDAPF1 protein in regulating drought resistance of plants. The ZmDAPF1 protein and related biological materials provided by the present application can be applied to the regulation of drought resistance of plants, and the transgenic homozygous lines with improved drought resistance and the gene edited homozygous lines with reduced drought resistance can be obtained by using the above biological materials.

[0006] To achieve the above purpose, the technical scheme adopted by the present application is:

[0007] A ZmDAPF1 protein, characterized in that it comprises:

[0008] (1) the amino acid sequence of the ZmDAPF1 protein is shown as SEQ ID No. 1;

[0009] (2) an amino acid sequence having 80% or more identity with the amino acid sequence of item (1);

[0010] (3) a fusion protein obtained by connecting a tag to the N and / or C terminus of the amino acid sequence according to item (1) or item (2).

[0011] A ZmDAPF1 gene, characterized in that the sequence of the ZmDAPF1 gene comprises:

[0012] (1) a nucleotide sequence as shown in SEQ ID No. 2 for encoding the ZmDAPF1 protein according to item (1) of claim 1;

[0013] (2) a nucleotide sequence for encoding the ZmDAPF1 protein according to item (2) of claim 1;

[0014] (3) a nucleotide sequence for encoding the ZmDAPF1 protein according to item (3) of claim 1;

[0015] A biological material, characterized in that the biological material is:

[0016] (1) the ZmDAPF1 gene according to claim 2;

[0017] (2) an expression cassette, a recombinant vector, a recombinant microorganism, a transgenic plant cell line, a transgenic plant tissue or a transgenic plant organ containing the ZmDAPF1 gene according to item (1);

[0018] (3) a recombinant vector, a recombinant microorganism, a transgenic plant cell line, a transgenic plant tissue or a transgenic plant organ containing the expression cassette according to item (2);

[0019] (4) a recombinant microorganism, a transgenic plant cell line, a transgenic plant tissue or a transgenic plant organ containing the recombinant vector according to item (3);

[0020] (5) a gRNA or DNA for inhibiting or reducing the protein activity of the ZmDAPF1 protein or for inhibiting or reducing the expression of the ZmDAPF1 gene,

[0021] the gRNA is a gRNA targeting the ZmDAPF1 gene according to claim 2, and the target sequence of the gRNA is:

[0022] 5'-CGCCGCGCCGTCGCTTCGA-3';

[0023] 5'-CCCCACTGTGTCACATTTG-3';

[0024] the DNA is a DNA for expressing the above-mentioned gRNA, and the nucleotide sequence of the DNA is as shown in SEQ ID No. 24-25;

[0025] 13117 bp 13120 bp

[0026] (6) An expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line containing the gRNA or DNA of item (5).

[0027] The application of a ZmDAPF1 gene in improving the drought resistance of plants, characterized in that the biological material of item (5) or item (6) is used to knockout the ZmDAPF1 gene as described in claim 2 in plants to improve the drought resistance of plants.

[0028] The application of a ZmDAPF1 gene in breeding drought-resistant plants, characterized in that the biological material of item (5) or item (6) is used to knockout the ZmDAPF1 gene as described in claim 2 in plants to breed drought-resistant plants.

[0029] On the basis of the above scheme, the plant is a plant of the family Poaceae.

[0030] On the basis of the above scheme, the plant of the family Poaceae is corn.

[0031] The application of a ZmDAPF1 protein in regulating the drought resistance of plants, which has the beneficial effect that:

[0032] 1. The application verifies through experiments that the ZmDAPF1 protein has a regulating function on the drought resistance of plants, and the ZmDAPF1 protein and related biological materials can be applied to the regulation of the drought resistance of plants.

[0033] 2. The application provides a method for regulating the drought resistance of plants, and the transgenic homozygous line with reduced drought resistance and the gene editing homozygous line with improved drought resistance are obtained according to the technical scheme provided in the application.

[0034] BRIEF DESCRIPTION OF DRAWINGS

[0035] The application has the following drawings:

[0036] FIG. 1 is a correlation analysis of ZmDAPF1 gene variation and corn seedling survival rate (SR) under drought stress;

[0037] FIG. 2 is that ZmDAPF1 positively regulates the drought resistance of corn;

[0038] FIG. 3 is the statistical result of the field drought phenotype of ZmDAPF1 mutant material (2022);

[0039] FIG. 4 is the statistical result of the field drought plant height of ZmDAPF1 mutant material (2023);

[0040] Figure 5 is the field drought yield statistics of ZmDAPF1 mutant materials (2023). DETAILED DESCRIPTION

[0041] The application will be further described in conjunction with the specific embodiments. The examples given are only to illustrate the application, and are not intended to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not in any way constitute a limitation on the application.

[0042] The experimental methods in the following examples are conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0043] The experimental methods used in the following examples are conventional methods, unless otherwise specified.

[0044] The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0045] The maize transgenic overexpression vector pBCXUN is modified from pCXUN (NCBI GenBank: FJ905215) vector. The modification process is to replace the selection marker gene Hyg with the Bar gene through the XhoI site. The above biological material can be obtained from the applicant, and the obtained biological material is only for repeating the experiments of the application and cannot be used for other purposes.

[0046] The maize transgenic recipient material LH244 (PI 612589) can be obtained from GRIN-Global (https: / / npgsweb.ars-grin.gov / gringlobal / search).

[0047] The maize gene editing vector pBUE411 vector is donated by Professor Chen Qijun of China Agricultural University, which is disclosed in the literature "Hui-Li Xing, Li Dong†, 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." (the name of the literature is pBUE411 (Bar)), the above biological material can be obtained from the applicant, and the above biological material is only used for repeating the experiments of the application and cannot be used for other purposes.

[0048] In the present application, bilateral t-test is used to determine statistical significance, indicates that the difference is significant (P < 0.05), indicates that the difference is extremely significant (P < 0.01).

[0049] Example 1, Genetic variation of ZmDAPF1 gene is related to drought resistance of maize seedling stage

[0050] The candidate gene association analysis found that the genetic variation of the diaminopimelic acid isomerase gene ZmDAPF1 located on chromosome 1 was significantly related to the drought resistance of maize seedlings, as shown in Figure 1A. The only non-synonymous mutation SNP530 in the coding region is the most significant variation. It changes the 120th amino acid from valine to isoleucine (V120I). The inventors resequenced the upstream 2k of the ZmDAPF1 promoter and gene region of 359 maize inbred lines, and found that Indel-1509 and SNP-1454 were also significantly related to the survival rate, and the two sites were in complete linkage disequilibrium (r 2 = 1), and were strongly linked to SNP530, as shown in Figure 1A. Significant variations (p < 10 -5 ) are connected by lines in the pairwise LD map, and the red blocks represent strong LD between the variation sites, as shown in Figure 1A. The upper panel of Figure 1A is the association map of ZmDAPF1 gene variation in the 16 kb region and the survival rate after drought treatment, and the lower panel is the LD map of ZmDAPF1 variation sites, and the significant variation (p < 10 -5) are connected to the two-by-two LD heat map by lines. Circles represent SNPs and triangles represent InDels. Based on the three mutation sites of SNP530, Indel-1509, and SNP-1454, 359 corn germplasms are divided into two haplotypes, as shown in Figure 1B. Hap1 is represented by the drought-sensitive germplasm B73, and Hap2 is represented by the drought-tolerant germplasm CML118. Under conditions of sufficient water (WW) and drought stress (WS), the survival rate of Hap2 is higher than that of Hap1, and the expression level of the ZmDAPF1 gene is lower than that of Hap1, as shown in Figures 1C-E. At the same time, under drought stress conditions, the expression of the ZmDAPF1 gene in B73 and CML118 is down-regulated, and the expression in CML118 is lower than that in B73, as shown in Figure 1F. It is indicated that ZmDAPF1 may negatively regulate the drought resistance of corn. Confocal microscope images show that ZmDAPF1 SNP530=G and ZmDAPF1 SNP530= A are both located in chloroplasts, indicating that SNP530 has no effect on the subcellular localization of ZmDAPF1, as shown in Figure 1G.

[0051] Example 2, Obtaining of Protein ZmDAPF1 and Its Encoding Gene

[0052] 1. Cloning of Protein ZmDAPF1 and Its Encoding Gene

[0053] Seeds of the sensitive corn inbred line B73 were taken, and after 5 days of germination at 24°C, the germinated seeds were transferred to nutrient solution for 1 week of culture. The whole seedlings were quickly frozen in liquid nitrogen and ground, total RNA was extracted, and cDNA was obtained by reverse transcription. The cDNA was used as a template, and primers F1 and R1 were used as primers for PCR amplification. The amplification product was subjected to 1% agarose gel electrophoresis, and a 1059 bp PCR amplification product (SEQ ID No. 2) was obtained.

[0054] After sequencing, the PCR product derived from the sensitive corn inbred line B73 has the nucleotides shown in SEQ ID No. 2 from position 24 to 1082 (the reference sequence can be searched for Zm00001d030677 on the website https: / / www.gramene.org / ). The genome shown in SEQ ID No. 3 is named ZmDAPF1 genome, and the protein encoded by the genome is named ZmDAPF1. In SEQ ID No. 3, positions 1-84 are the 5' non-coding region, positions 85-12073 are the open reading frame region, and positions 12074-13119 are the 3' non-coding region.

[0055] The primer sequences are as follows:

[0056] F1: 5'- ATGTCGTCCGCCGCCG-3' (SEQ ID No. 6); F2: 5'- ATGTCGTCCGCCGCCG-3' (SEQ ID No. 7).

[0057] R1: 5'- CTAGTGAACAACAGATCCATAAAAG-3' (SEQ ID No. 7).

[0058] The coding sequence of the protein ZmDAPF1 is named as ZmDAPF1 gene, the nucleotide sequence of the ZmDAPF1 gene is shown as SEQ ID No. 2, 24-1082, and the nucleotide sequence of the ZmDAPF1 genome is shown as SEQ ID No. 3.

[0059] 2. Construction of the recombinant vector pBCXUN

[0060] The coding sequence of the ZmDAPF1 gene (SEQ ID No. 2) is added with the homologous arm of the XcmI (downstream of the ubi promoter) enzyme cutting site, and then the vector is subjected to XcmI single enzyme cutting, and the enzyme cutting product is connected with the pBCXUN vector fragment subjected to the same single enzyme cutting, to obtain the recombinant vector containing the ZmDAPF1 coding sequence, which is named as pBCXUN-ZmDAPF1. The pBCXUN-ZmDAPF1 is a recombinant expression vector obtained by inserting the DNA molecule (24-1082) with the nucleotide sequence shown as SEQ ID No. 2 between the enzyme cutting sites of the restriction endonuclease XcmI of the pBCXUN vector, while keeping the other nucleotide sequences of the pBCXUN vector unchanged. The promoter for starting the ZmDAPF1 gene in the recombinant vector pBCXUN-ZmDAPF1 is Zmubiquitin1.

[0061] 3. Obtaining of the recombinant Agrobacterium

[0062] The recombinant vector pBCXUN-ZmDAPF1 is transformed into Agrobacterium EHA105, to obtain the recombinant Agrobacterium EHA105 / pBCXUN-ZmDAPF1 containing the recombinant vector pBCXUN-ZmDAPF1 (after colony PCR, the bacteria are shaken to extract the plasmid, and the correct recombinant Agrobacterium verified by sequencing is the positive clone).

[0063] 4. Obtaining of the transgenic homozygous ZmDAPF1 corn

[0064] The recombinant Agrobacterium EHA105 / pBCXUN-ZmDAPF1 is used to infect the immature embryo of corn wild type LH244, and T1 generation seeds are obtained. The whole genome DNA of the T1 generation transgenic plant is extracted, and PCR identification of transgenic positive is carried out. The primers used are F2 and R2. The positive plant (PCR product of 1142 bp is obtained by using F2 and R2 for PCR) is used to obtain T2 generation seeds. The T2 generation seeds are germinated (at least 24 seeds) for genome DNA extraction. The primers F2 and R2 are also used for PCR identification. If at least 24 seeds of a bag of seeds are all positive in PCR identification, it is indicated that the bag of seeds may be transgenic ZmDAPF1 homozygous seeds. The RNA of the plant of the bag of seeds is extracted for reverse transcription to obtain cDNA. The gene ZmUbi2 (Zm00001d053838) in corn is used as an internal reference, and the primers are QF1 and QR1. The expression amount of ZmDAPF1 gene is detected by using specific primers QF2 and QR2, and wild type LH244 is used as a control. The sequences of the above primers are as follows:

[0065] F2: 5'-TTTTAGCCCTGCCTTCATACGC-3' (SEQ ID No. 8);

[0066] R2: 5'-GTGAACAACAGATCCATAAAAG-3' (SEQ ID No. 9).

[0067] QF1: 5'-TGGTTGTGGCTTCGTTGGTT-3' (SEQ ID No. 10);

[0068] QR1: 5'-GCTGCAGAAGAGTTTTGGGTACA-3' (SEQ ID No. 11).

[0069] QF2: 5'-GAAATGTTTCCTGCTCGCACA-3' (SEQ ID No. 12);

[0070] QR2: 5'-ACAGGCAAGAGTTGCTCCAG-3' (SEQ ID No. 13).

[0071] The T2 generation homozygous seeds or the seeds (T3 generation) generated by selfing of the homozygous T2 generation plants are used for drought phenotype and other experiments. T1 represents the seeds and plants grown from the seeds of the transformation receptor plant; T2 represents the seeds and plants grown from the seeds generated by selfing of the T1 generation; T3 represents the seeds and plants grown from the seeds generated by selfing of the T2 generation, and the like.

[0072] The total RNA of T3 generation of transgenic ZmDAPF1 corn lines and wild type LH244 (control) was extracted to obtain cDNA by reverse transcription. The gene Zmubiquitinl (Zm00001d015327) in corn was used as an internal reference, and specific primers QF2 and QR2 were used for real-time fluorescent quantitative PCR (RT-qPCR) analysis. The expression amount of ZmDAPF1 gene was analyzed by the 2 -△△CT -△△CT method (Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2 -△△CT method. Methods. 25: 402-408). The results are shown in Figure 2A, which shows that the relative expression amount of T3 generation of transgenic ZmDAPF1 corn lines named OE#1 and OE#5 is more than 50 times higher than that of wild type corn, indicating that T3 generation of transgenic ZmDAPF1 corn OE#1 and OE#5 are positive transgenic corn.

[0073] Example 3, Function Research of Protein ZmDAPF1 and Its Coding Gene

[0074] 1. Obtaining of Corn ZmDAPF1 Gene Mutant

[0075] The gRNA target site (5'-CGCCGCGCCGTCGCTTCGA-3', SEQ ID No. 23) was designed on the first exon of ZmDAPF1 and the gRNA target site (5'-CCCCACTGTGTCACATTTG-3', SEQ ID No. 20) was designed on the fifth exon of ZmDAPF1 by CRISPR-P (http: / / crispr.hzau.edu.cn / CRISPR2 / ). The target sites were constructed into the pBUE411 vector, and after positive clones were identified by colony PCR, the plasmids were extracted for sequencing. The plasmids with correct sequencing were transformed into Agrobacterium EHA105, and the Agrobacterium with positive colony PCR infected the maize LH244 immature embryos to obtain T0 generation plants. The T0 generation plants were selfed to obtain T1 generation seeds, and the T1 generation seeds were germinated and planted to obtain maize cotyledons, and the genomic DNA was extracted. The F3 and R3 primer pairs and the F4 and R4 primer pairs were used for genotyping, and three types of edited maize ZmDAPF1 gene mutant T2 generation seeds were obtained by selfing. The F5 and R5 were used for CRISPR Cas9 free identification of the two genotypes, and wild type LH244 was used as a control. The PCR negative was a CRISPR Cas9 free plant, and the three types of edited maize ZmDAPF1 gene mutants were selfed to obtain Cas9-free T2 generation homozygous edited line seeds, named KO#1, KO#2 and KO#3, as shown in Figure 2B, for subsequent experiments.

[0076] The sequences of the above primers are as follows:

[0077] F3: 5'-CAGGTTTATGTACCGGTGAC-3' (SEQ ID No. 14);

[0078] R3: 5'-CCCTGTTGTCCACCTACG-3' (SEQ ID No. 15).

[0079] F4: 5'-GCCTCCTATTTAACTTGGACC-3' (SEQ ID No. 16);

[0080] R4: 5'-GGATCACAGGGGTCACTG-3' (SEQ ID No. 17).

[0081] F5: 5'-GACAGGCGTCTTCTACTGGTGCTAC-3' (SEQ ID No. 18);

[0082] R5: 5'-TATTCACTAGCTCGGGATAGTTGGC-3' (SEQ ID No. 19).

[0083] As shown in Figure 2B, mutant KO#1 and KO#2, compared with wild type corn LH244, the ZmDAPF1 gene in the corn genome in the two homologous chromosomes of KO#1 and KO#2 has the following mutations: 5'- CCCCACTGTGTCACATTTG-3' (SEQ ID No. 20) is replaced in the ZmDAPF1 gene in the corn genome DNA 5'- CCCCACTGTGTCACATTTTG-3' (SEQ ID No. 21, 7900-7918 of SEQ ID No. 3), resulting in a frameshift mutation after the 728th position of the CDS of the ZmDAPF1 gene, thereby knocking out the ZmDAPF1 gene (wild type). The mutated gene is named zmdapf1-ko1 and zmdapf1-ko2 gene; the coding sequence (CDS) of the ZmDAPF1 gene is a DNA molecule (SEQ ID No. 24) obtained by inserting a nucleotide T after the 728th nucleotide of the DNA molecule shown in SEQ ID No. 2, keeping other nucleotide sequences of SEQ ID No. 2 unchanged; encoding a protein ZmDAPF1 composed of 255 amino acid residues, the amino acid sequence is shown in SEQ ID No. 4 of the sequence listing.

[0084] As shown in Figure 2B, mutant KO#1 and KO#2, compared with wild type corn LH244, the ZmDAPF1 gene in the corn genome in the two homologous chromosomes of KO#1 and KO#2 has the following mutations: 5'- CCCCACTGTGTCACATTTG-3' (SEQ ID No. 20) is replaced in the ZmDAPF1 gene in the corn genome DNA 5'- CCCCACTGTGTCACATTTTG-3' (SEQ ID No. 21, 7900-7918 of SEQ ID No. 3), resulting in a frameshift mutation after the 728th position of the CDS of the ZmDAPF1 gene, thereby knocking out the ZmDAPF1 gene (wild type). The mutated gene is named zmdapf1-ko1 and zmdapf1-ko2 gene; the coding sequence (CDS) of the ZmDAPF1 gene is a DNA molecule (SEQ ID No. 24) obtained by inserting a nucleotide T after the 728th nucleotide of the DNA molecule shown in SEQ ID No. 2, keeping other nucleotide sequences of SEQ ID No. 2 unchanged; encoding a protein ZmDAPF1 composed of 255 amino acid residues, the amino acid sequence is shown in SEQ ID No. 4 of the sequence listing.

[0085] 2, Phenotypic analysis of ZmDAPF1 involved in drought resistance of corn

[0086] The T3 generation of the transgenic ZmDAPF1 maize lines (OE#1 and OE#5) and the wild type maize LH244 (WT) plants which were sowed for 5 days were transplanted into white boxes filled with 2 Kg of nutrient soil:vermiculite:imported soil = 1:1:1. After growing for 7 days under normal conditions, the plants were subjected to drought treatment (i.e. stopping watering) for 25 days, and then re-watered. After 5 days of re-watering, the survival rate of the plants of each line was calculated (the plants with normal leaf color and normal growth were defined as survival plants, and the plants with scorched leaves and abnormal growth were defined as dead plants; the survival rate was the percentage of the number of survival plants in each line to the total number of plants). The experiment was set with 6 replicates, and the number of plants in each line in each replicate was not less than 18, and the average value was taken for statistical analysis.

[0087] As shown in Table 1, Figure 2A and Figure 2E, Figure 2A is the photograph of OE#1 and OE#5 before drought and after re-watering; and Figure 2E is the drought survival rate of OE#1 and OE#5. It can be seen that after 20 days of drought treatment, the leaf wilting degree of the T3 generation of the transgenic ZmDAPF1 maize (OE#1 and OE#5) was greater than that of the wild type maize LH244 (WT), and the survival rate was significantly lower than that of the wild type maize LH244 (WT).

[0088] Table 1 Survival rate (%) of transgenic maize plants after drought treatment

[0089]

[0090] The survival rate of WT, KO#1, KO#2 and KO#3 was statistically analyzed by the same method as above, and the results are shown in Table 2, Figure 2A and Figure 2C, Figure 2A is the photograph of KO#1, KO#2 and KO#3 before drought and after re-watering; and Figure 2C is the drought survival rate of KO#1, KO#2 and KO#3. The results show that after 20 days of drought treatment, the leaf wilting degree of the ZmDAPF1 mutants KO#1, KO#2 and KO#3 was weaker than that of the wild type maize, and the survival rate was significantly higher than that of the wild type maize.

[0091] Table 2 Survival rate (%) of mutant plants after drought treatment

[0092]

[0093] Example 3, Field drought phenotype analysis

[0094] Under field experimental conditions, the drought tolerance of maize LH244 wild type (WT), ZmDAPF1 mutant homozygous lines (KO#1, KO#2 and KO#3) under drought treatment was detected. In this experiment, normal watering and drought treatment were set up for randomized block experiment, which was repeated twice in Zhangye in 2022 and Zhangye in 2023. In the Zhangye experimental base in 2022, WT, KO#1 and KO#2 were planted. The normal watering treatment group (Normal) was irrigated with sufficient water throughout the growing season to ensure the normal growth of maize (water potential maintained at 0 to -20 kPa). The drought treatment (Drought) was carried out from the V5 (fifth leaf ligule visible leaf) stage to the completion of spinning. The soil water potential was maintained between -20 kPa and -220 kPa. Irrigation was carried out approximately every 10 days, with 45-450 tons of water per hectare each time, depending on the soil moisture. The total irrigation amount of drought plot was about 40% of that of normal growth plot. After the end of scattering powder, the plant height, yield per plant and ear grain number were counted respectively. The number of plants of each strain was not less than 50, and the average value was taken for statistical analysis.

[0095] As shown in Figure 3A, the plant height of WT, KO#1 and KO#2 in the normal treatment group and the drought treatment group. Figure 3B is the comparison results of yield per plant of WT, KO#1 and KO#2 in the normal treatment group and the drought treatment group. Figures 3C and 3D are the comparison results of ear grain number of WT, KO#1 and KO#2 in the normal treatment group and the drought treatment group. The above results show that in the normal treatment group and the drought treatment group, there is no significant difference in the plant height of each maize; under normal watering conditions and under drought conditions, the yield per plant and ear grain number of KO#1 and KO#2 are significantly higher than those of WT.

[0096] In order to verify whether ZmDAPF1#KO can stably increase the yield of corn, WT, KO#1, KO#2 and KO#3 were planted in Zhangye experimental base in 2023, and this experiment added a slightly drought plot. From the V5 (fifth visible leaf ligule) stage to the completion of silk, the plants were subjected to drought treatment. The soil water potential was maintained between-20 kPa and-220 kPa. Irrigation was performed approximately every 10 days, with 45-450 tons of water per hectare each time, depending on the soil moisture. The total irrigation amount of the slightly drought plot (moderate water-stressed, WS0.5) was about 80% of that of the normal growth plot (well-watered, WW), and the total irrigation amount of the severe drought plot (severe water-stressed, WS1) was about 40% of that of the normal growth plot. After the end of the scattering powder, the plant height and single ear yield were counted, respectively, and the number of plants of each strain was not less than 50, and the average value was taken for statistical analysis, as shown in FIG. 4 and FIG. 5. FIG. 4 shows that there is no significant difference in the height of each corn in the normal treatment group. FIG. 5 shows that under normal watering conditions and under mild and severe drought conditions, the single plant yield and ear grain number of KO#1, KO#2 and KO#3 are significantly higher than that of WT.

[0097] The above results show that under normal conditions and mild and severe drought conditions, knocking out the coding gene of ZmDAPF1 in plants can significantly increase the single ear yield without affecting the height of corn plants. The yield-increasing results of ZmDAPF1#KO are reproducible and have stable drought-resistant yield-increasing advantages.

[0098] The above has been described in detail. For those skilled in the art, without departing from the purpose and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wider range under equivalent parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that further improvements can be made to the present application. In general, according to the principle of the present application, this application intends to include any change, use or improvement of the present application, including changes made by conventional techniques known in the art, which deviates from the scope disclosed in the present application. Some basic features can be applied within the scope of the following attached claims.

[0099] The contents not described in detail in the specification belong to the prior art known to those skilled in the art.

Claims

1. A ZmDAPF1 protein, characterized in that, Comprise: (1) the amino acid sequence of the ZmDAPF1 protein is shown as SEQ ID No. 1; (2) the amino acid sequence having 80% or more identity with the amino acid sequence described in item (1); (3) the fusion protein obtained by connecting a tag to the N and / or C terminal of the amino acid sequence described in item (1) or item (2).

2. A ZmDAPFl gene, characterized in that, The sequence of the ZmDAPF1 gene comprises: (1) the nucleotide sequence shown as SEQ ID No. 2 for encoding the ZmDAPF1 protein described in item (1) of claim 1; (2) the nucleotide sequence for encoding the ZmDAPF1 protein described in item (2) of claim 1; (3) the nucleotide sequence for encoding the ZmDAPF1 protein described in item (3) of claim 1.

3. A biomaterial, characterized by: The biological material is: (1) the ZmDAPF1 gene described in claim 2; (2) the expression cassette, recombinant vector, recombinant microorganism, transgenic plant cell line, transgenic plant tissue or transgenic plant organ containing the ZmDAPF1 gene described in item (1); (3) the recombinant vector, recombinant microorganism, transgenic plant cell line, transgenic plant tissue or transgenic plant organ containing the expression cassette described in item (2); (4) the recombinant microorganism, transgenic plant cell line, transgenic plant tissue or transgenic plant organ containing the recombinant vector described in item (3); (5) gRNA or DNA for inhibiting or reducing the protein activity of the ZmDAPF1 protein described in claim 1 or for inhibiting or reducing the expression of the ZmDAPF1 gene described in claim 2, The gRNA is: the gRNA targeting the ZmDAPF1 gene described in claim 2, and the target sequence of the gRNA is: 5'-CGCCGCGCCGTCGCTTCGA-3'; 5'-CCCCACTGTGTCACATTTG-3'; The DNA is: the DNA for expressing the above gRNA, and the nucleotide sequence is shown as SEQ ID No. 24-25; (6) the expression cassette, recombinant vector, recombinant microorganism or transgenic cell line containing the gRNA or DNA described in item (5).

4. The use of ZmDAPF1 gene in improving drought resistance of plants, characterized in that: The biological material described in item (5) or item (6) of claim 3 is used to knockout the ZmDAPF1 gene described in claim 2 in plants to improve the drought resistance of plants.

5. Use of a ZmDAPF1 gene in breeding drought-resistant plants, characterized in that: The biological material described in item (5) or item (6) of claim 3 is used to knockout the ZmDAPF1 gene described in claim 2 in plants to breed drought-resistant plants.

6. Use according to claim 4 or 5, characterized in that: The plant is a plant of the family Poaceae.

7. Use according to claim 6, wherein The plant of the family Poaceae is corn.

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