Application of ZmAGO4b protein in regulation and control of plant drought resistance

Abstract

The invention relates to an application of a ZmAGO4b protein in regulation and control of drought resistance of plants. The amino acid sequence of the ZmAGO4b protein disclosed by the invention is shown as SEQ ID NO. 1, and the nucleotide sequence of the coding gene of the protein is shown as SEQ ID NO. 2. Through overexpression of the ZmAGO4b gene in a plant, the drought resistance of a transgenic plant after drought stress is improved compared with that of a control group plant. The invention further discloses a method for improving the drought resistance of the corn, the coding gene of the ZmAGO4b protein is introduced into a receptor plant, and the corn with high drought resistance can be obtained. The invention verifies that the protein ZmAGO4b has a positive regulation function on the drought resistance of the corn, the ZmAGO4b protein and related biological materials thereof can be applied to regulation of the drought resistance of the corn, and excellent candidate gene resources are provided for cultivation and improvement of new varieties of drought-resistant corn. Compared with a traditional breeding mode, the provided drought-resistant plant breeding method has the advantages that the breeding process is more efficient, reliable, accurate and controllable, and the method has important theoretical and practical significance for improving crop resistance and accelerating the corn breeding process.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering breeding technology, specifically to the application of ZmAGO4b protein in regulating plant drought resistance. Background Technology

[0002] As a vital source of food, fuel, feed, and chemical raw materials worldwide, maize production is continuously subjected to biotic and abiotic stresses. Drought stress, as a major abiotic stress, significantly impacts maize yield and quality. However, the mechanisms by which maize responds to drought stress are not yet fully understood, making drought-resistant maize breeding a promising field.

[0003] With the advancement of genetically modified technology, the country's acceptance of genetically modified crops has increased, and the scope of market promotion has expanded. Compared with the disadvantages of traditional breeding, such as long time consumption and inability to accurately control the introduction of a certain trait, genetically modified breeding can directly change the target gene and accurately introduce the desired trait, thereby improving trait improvement in a short period of time and increasing crop yield, nutritional value, or stress resistance.

[0004] As an important feed and food crop widely cultivated in my country, cloning drought-resistant genes in maize (Zea mays L.) is of great significance for improving its drought resistance and yield. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide an application of the ZmAGO4b protein in regulating plant drought resistance. This invention improves drought resistance in maize by overexpressing the ZmAGO4b gene, providing an excellent candidate gene resource for breeding and improving new drought-resistant maize varieties. Breeding drought-resistant plants through ZmAGO4b protein overexpression is more efficient, reliable, precise, and controllable than traditional breeding methods, and has significant theoretical and practical implications for improving crop resistance and accelerating maize breeding.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] Applications of ZmAGO4b protein, its encoding gene, or biological materials containing its encoding gene in the following directions:

[0008] Improve plant drought resistance;

[0009] Genetic breeding for drought resistance in maize;

[0010] Improvement of drought-resistant plant resources;

[0011] The amino acid sequence of the ZmAGO4b protein is as follows:

[0012] (1) The amino acid sequence shown in SEQ ID No. 1;

[0013] (2) 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.

[0014] The nucleotide sequence of the ZmAGO4b protein encoding gene is as follows:

[0015] (1) As shown in SEQ ID No. 2; the gene sequence encodes the protein shown in SEQ ID No. 1;

[0016] (2) A nucleotide sequence of the nucleotide sequence shown in SEQ ID No. 2 that has been substituted, deleted and / or added with one or more nucleotides and expresses a protein with the same function as the protein shown in SEQ ID No. 1;

[0017] The biomaterials containing the ZmAGO4b protein-coding gene include:

[0018] Expression cassettes, vectors, host cells, or recombinant bacteria containing the ZmAGO4b protein-coding gene.

[0019] Primers used for amplifying the ZmAGO4b gene are characterized by:

[0020] The nucleotide sequences of the primers are shown in SEQ ID No. 4-5.

[0021] A ZmAGO4b gene mutant, characterized in that: the coding sequence of the ZmAGO4b gene mutant is shown in SEQ ID No. 18, and the ZmAGO4b gene mutant encodes the protein shown in SEQ ID No. 19.

[0022] A ZmAGO4b gene mutant, characterized in that: the coding sequence of the ZmAGO4b gene mutant is shown in SEQ ID No. 21, and the ZmAGO4b gene mutant encodes the protein shown in SEQ ID No. 22.

[0023] A gRNA target for obtaining ZmAGO4b gene mutants, characterized in that:

[0024] The nucleotide sequence of the target is shown in SEQ ID No. 12.

[0025] A method for breeding drought-resistant maize, characterized by increasing the expression level and / or activity of ZmAGO4b protein in maize; wherein the ZmAGO4b protein is any of the following proteins:

[0026] (1) The amino acid sequence is shown in SEQ ID No. 1 of the sequence listing;

[0027] (2) 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.

[0028] A method for cultivating transgenic plants includes the following steps: linking a gene encoding the ZmAGO4b protein to a pBCXUN vector to obtain a recombinant vector pBCXUN-ZmAGO4b containing the ZmAGO4b coding sequence; transforming the recombinant vector into Agrobacterium EHA105 to obtain recombinant Agrobacterium EHA105 / pBCXUN-ZmAGO4b containing the recombinant vector pBCXUN-ZmAGO4b; infecting maize with recombinant Agrobacterium EHA105 / pBCXUN-ZmAGO4b and introducing it into recipient plants to obtain transgenic plants with increased drought resistance.

[0029] The ZmAGO4b protein is any of the following proteins:

[0030] (1) The amino acid sequence is shown in SEQ ID No. 1 of the sequence listing;

[0031] (2) 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.

[0032] Based on the above plan,

[0033] The primer sequences for detecting the ZmAGO4b gene are shown in SEQ ID No. 6-11.

[0034] The beneficial effects of the ZmAGO4b protein described in this invention in regulating plant drought resistance are as follows:

[0035] 1. This invention verifies that the ZmAGO4b protein has a regulatory function on plant drought resistance, and that the ZmAGO4b protein and its related biomaterials can be applied to the regulation of plant drought resistance.

[0036] 2. This invention provides a method for regulating plant drought resistance. 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 drought resistance breeding.

[0037] 3. This invention uses transgenic technology to obtain transgenic overexpression homozygous lines with improved drought resistance and gene-edited homozygous lines with reduced drought resistance, providing gene resources for breeding and improving new drought-resistant plant varieties.

[0038] 4. This invention provides a theoretical basis for elucidating the molecular mechanism of ZmAGO4b protein in plant drought resistance. Attached Figure Description

[0039] The present invention includes the following figures:

[0040] Figure 1 The identification results of ZmAGO4b gene overexpression and mutant materials are as follows: A shows the expression level of ZmAGO4b gene in the ZmAGO4b gene overexpression material; B is a schematic diagram of the target site editing effect of ZmAGO4b-knock out material (KO).

[0041] Figure 2 The ZmAGO4b protein positively regulates drought resistance in maize: A represents the seedling drought resistance test results of the ZmAGO4b gene overexpression material (OE); B represents the survival rate statistics of the ZmAGO4b gene overexpression material in the seedling drought experiment; C represents the seedling drought resistance test results of the ZmAGO4b-knockout material; and D represents the survival rate statistics of the ZmAGO4b-knockout material in the seedling drought experiment.

[0042] Figure 3 The following are the field drought yield statistics for ZmAGO4b transgenic materials: A represents a photograph of a single ear of ZmAGO4b overexpression material in the field drought experiment, and B represents the statistical results of the single ear yield of ZmAGO4b overexpression material in the field drought experiment. In the figure, WW represents normal irrigation conditions, and WS represents drought conditions. Detailed Implementation

[0043] The present invention will be further described in detail 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 invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0044] 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.

[0045] The transgenic maize recipient material LH244 (PI 612589) was obtained from GRIN-Global (https: / / npgsweb.ars-grin.gov / gringlobal / search).

[0046] 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)).

[0047] In this invention, one-way ANOVA was used to determine statistical significance. * indicates a significant difference (P < 0.05), and ** indicates an extremely significant difference (P < 0.01).

[0048] The sequence involved in this invention, specifically the maize ZmAGO4b gene number GRMZM2G141818, is as follows:

[0049] The amino acid sequence of the ZmAGO4b protein is shown in SEQ ID No. 1, consisting of 910 amino acid residues; the coding sequence of the ZmAGO4b gene is shown in SEQ ID No. 3; the nucleotide sequence of the ZmAGO4b genome is shown in SEQ ID No. 1. As shown in No. 3, it consists of 7990 bases. Positions 1-1659 are the 5' non-coding region, positions 1659-6848 are the open reading frame region, positions 1659-1899 are the first exon, positions 2008-2214 are the second exon, positions 2299-2524 are the third exon, positions 2614-2751 are the fourth exon, positions 2859-2957 are the fifth exon, positions 3065-3163 are the sixth exon, positions 3375-3538 are the seventh exon, positions 3774-3891 are the eighth exon, positions 3996-4104 are the ninth exon, positions 4378-4441 are the tenth exon, and positions 45... Exon 37-4651 is the eleventh exon, exon 4791-4885 is the twelfth exon, exon 4962-5075 is the thirteenth exon, exon 5162-5297 is the fourteenth exon, exon 5421-5549 is the fifteenth exon, exon 5636-5702 is the sixteenth exon, exon 5874-5938 is the seventeenth exon, exon 6028-6140 is the eighteenth exon, exon 6221-6295 is the nineteenth exon, exon 6375-6470 is the twentieth exon, exon 6552-6584 is the twenty-first exon, exon 6669-6848 is the twenty-second exon, and the rest are introns.

[0050] The proteins mentioned above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0051] Example 1: Obtaining the ZmAGO4b protein and its encoding gene

[0052] 1. Cloning of ZmAGO4b protein and its encoding gene

[0053] Seeds of the sensitive maize inbred line LH244 were grown at 24℃ for 14 days. The second true leaf of V2 stage seedlings was 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, PCR amplification was performed with primers F1 and R1. The amplified product was subjected to 1.5% agarose gel electrophoresis, yielding a 2733 bp PCR amplification product (SEQ ID No. 2).

[0054] Sequencing revealed that the PCR product derived from the sensitive maize inbred line B73 has the nucleotide sequence shown in positions 91-2823 of SEQ ID No. 2 (this reference sequence can be obtained by searching GRMZM2G141818 on https: / / www.maizegdb.org / / ).

[0055] The primer sequences mentioned above are as follows:

[0056] F1: 5'-ATGGGCTCTCATGATGGC-3'; (SEQ ID No. 4)

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

[0058] 2. Construction of the recombinant vector pBCXUN-ZmAGO4b

[0059] The coding sequence of the ZmAGO4b gene (SEQ ID No. 2) was modified by adding a homologous arm to the XcmI restriction enzyme site (downstream of the ubi promoter). The vector was then digested with XcmI, and the digestion product was ligated with a similarly digested pBCXUN vector fragment using T4 ligase to obtain a recombinant vector containing the ZmAGO4b coding sequence, named pBCXUN-ZmAGO4b. pBCXUN-ZmAGO4b is obtained by inserting a DNA molecule with the nucleotide sequence shown in SEQ ID No. 2 between the restriction endonuclease XcmI sites of the pBCXUN vector, while keeping the other nucleotide sequences of the pBCXUN vector unchanged. The promoter for initiating the ZmAGO4b gene in the recombinant vector pBCXUN-ZmAGO4b is Zmubiquitin1.

[0060] 3. Obtaining recombinant Agrobacterium

[0061] The recombinant vector pBCXUN-ZmAGO4b was transformed into Agrobacterium EHA105 to obtain recombinant Agrobacterium EHA105 / pBCXUN-ZmAGO4b containing the recombinant vector pBCXUN-ZmAGO4b (after colony PCR, plasmid was extracted by shaking and sequencing to verify that the recombinant Agrobacterium was a positive clone).

[0062] 4. Obtaining genetically modified homozygous ZmAGO4b maize

[0063] Recombinant Agrobacterium EHA105 / pBCXUN-ZmAGO4b was used to infect the immature embryos of wild-type maize LH244, and T1 generation seeds were harvested. The whole genome DNA of the T1 generation transgenic plants was extracted and PCR was performed to identify transgenic positivity. The primers used were F2 and R2. The positive plants (PCR products of 1084 bp were obtained by PCR with F2 and R2) were harvested to obtain T2 generation seeds. The T2 generation seeds were germinated (at least 24 seeds) and genomic DNA was extracted. PCR was performed again using primers F2 and R2. If the PCR test of at least 24 seeds in a package was positive, it indicates that the package of seeds may be homozygous transgenic ZmAGO4b seeds. RNA was extracted from the seed plants of the package and reverse transcribed to obtain cDNA. The gene ZmUbi2 (Zm00001d053838) in maize was used as an internal control, and the primers were QF1 and QR1. The expression level of the ZmAGO4b gene was detected using specific primers QF2 and QR2, with wild-type LH244 as a control.

[0064] The sequences of the primers mentioned above are as follows:

[0065] F2:5'-AAGATCTCCAATAATGTACCCATACGATGTTCCAGATTACGCGATGGGCTCTCATGATGGC-3'(SEQ ID No.6)

[0066] R2: 5'-CGGATCCCCAATACTCTAGCAGAAGAACATGGAGCTCCT-3'

[0067] (SEQ ID No.7)

[0068] QF1: 5'-TGGTTGTGGCTTCGTTGGTT-3' (SEQ ID No. 8)

[0069] QR1: 5'-GCTGCAGAAGAGTTTTGGGTACA-3' (SEQ ID No.9)

[0070] QF2: 5'-TGAGGCAGAGAAATGGTAGC-3' (SEQ ID No. 10)

[0071] QR2: 5'-AGGGATGACCTTTGTAGCG-3' (SEQ ID No. 11)

[0072] T2 generation homozygous seeds or seeds produced by self-pollination of homozygous T2 generation plants (T3 generation) are used for experiments such as drought phenotype. T1 represents the seeds and plants produced by the current generation of the transformation recipient plant; T2 generation represents the seeds and plants produced by self-pollination of T1 generation; T3 generation represents the seeds and plants produced by self-pollination of T2 generation, and so on.

[0073] Total RNA was extracted from T3 generation ZmAGO4b transgenic maize lines and wild-type LH244 (control) and reverse transcribed to obtain cDNA. Using the maize gene Zmubiquitin1 (Zm00001d015327) as an internal control, real-time quantitative PCR (RT-qPCR) analysis was performed using specific primers QF2 and QR2. -△△CT Method (Livak KJ,Schmittgen TD.2001.Analysis of relative gene expression data using real-time quantitative PCR and the 2 -△△CT The expression level of the ZmAGO4b gene was analyzed using the method (Methods. 25:402-408). The results are as follows: Figure 1 As shown in Figure A, the relative expression levels of the T3 generation ZmAGO4b transgenic maize lines named OE1 and OE2 are more than 50 times higher than those of wild-type maize, indicating that the T3 generation ZmAGO4b transgenic maize lines OE1 and OE2 are positive transgenic maizes.

[0074] Example 2: Functional study of ZmAGO4b protein and its encoding gene

[0075] 1. Obtaining the ZmAGO4b gene mutant in maize

[0076] A gRNA target (5'-TCTTGGAGGACGTATCTAC-3') (SEQ ID No. 12) was designed on the second exon of ZmAGO4b 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 F3 and R3 primer pairs, respectively. Self-crossing yielded T2 generation seeds of maize ZmAGO4b gene mutants with two edited types. The materials of these two mutant genotypes were CRISPR Cas9 free using F4 and R4 cells, with wild-type LH244 as a control. PCR-negative plants were CRISPR Cas9 free. Seeds of the T2 generation homozygous edited lines without Cas9, obtained from self-crossing of the two edited maize ZmAGO4b gene mutants, were named zmago4b-KO1 and zmago4b-KO2. Figure 1 As shown in B, it is used for subsequent experiments.

[0077] The sequences of the primers mentioned above are as follows:

[0078] F3: 5'-GACCCGTTGATGTAAAGG-3' (SEQ ID No. 13)

[0079] R3: 5'-GTTCTCAGATTCTTCGCCTCTA-3' (SEQ ID No. 14)

[0080] F4: 5'-GACAGGCGTCTTCTACTGGTGCTAC-3' (SEQ ID No. 15)

[0081] R4: 5'-TATTCACTAGCTCGGGATAGTTGGC-3' (SEQ ID No. 16)

[0082] like Figure 1The mutant zmago4b-KO1 shown in B, compared with wild-type maize LH244, has the following mutation in the ZmAGO4b gene in the maize genome on its two homologous chromosomes: 5'-GTCTTGGAGGACGTACTGGAAAGTTAG-3' (bases 2189-2219 of SEQ ID No. 3) is replaced by 5'-GTCTTGGAGGACGTATCTACTGGAAAGTTAG-3', resulting in a frameshift mutation after base 525 of the CDS of the ZmAGO4b gene, thereby knocking out the ZmAGO4b gene (wild type). The mutated gene was named zmago4b-KO1 (SEQ ID No. 17); the coding sequence (CDS) of the ZmAGO4b-KO1 gene is obtained by deleting 4 nucleotides after the 681st nucleotide of the DNA molecule shown in SEQ ID No. 2, while keeping the other nucleotide sequences of SEQ ID No. 2 unchanged (SEQ ID No. 18); it encodes the protein ZmAGO4b, which consists of 168 amino acid residues, and its amino acid sequence is shown in SEQ ID No. 19 of the sequence listing.

[0083] like Figure 1 The mutant zmago4b-KO2 shown in B, compared with wild-type maize LH244, has the following mutation in the ZmAGO4b gene in the maize genome on its two homologous chromosomes: 5'-GTCTTGGAGGACGTATCTACTGGAAAG-3' (positions 2189-2219 of SEQ ID No. 3) is replaced by 5'-TCTTGGAGGACGTATCATACTGGAAAG-3', resulting in a frameshift mutation after position 527 of the CDS of the ZmAGO4b gene, thereby knocking out the ZmAGO4b gene (wild-type). The mutated gene was named zmago4b-KO2 gene (SEQ ID No. 20); the coding sequence (CDS) of the ZmAGO4b-KO2 gene is obtained by inserting an A base after position 527 of the DNA molecule shown in SEQ ID No. 2, while keeping the other nucleotide sequences of SEQ ID No. 2 unchanged (SEQ ID No. 21); it encodes the protein ZmAGO4b, which consists of 158 amino acid residues, and its amino acid sequence is shown in SEQ ID No. 22 of the sequence listing.

[0084] 2. Phenotypic analysis of ZmAGO4b's role in drought resistance in maize

[0085] Five-day-old T3 generation ZmAGO4b transgenic maize lines (OE1 and OE2) and wild-type maize LH244 (WT) plants were transferred to white boxes containing 3L of nutrient soil:vermiculite:imported soil in a 1:1:1 ratio. Six seedlings were placed in each row, with three rows of wild-type and three rows of ZmAGO4b transgenic maize lines per box. After seven days of normal growth, a 25-day drought treatment (i.e., watering was stopped) was applied, 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 six times, with at least 18 plants in each replicate. The average value was used for statistical analysis.

[0086] result Figure 2 A and Figure 2 As shown in B, Figure 2 A shows photos of OE1 and OE2 before drought and after rehydration; Figure 2 B represents the drought survival rate statistics for WT, OE1, and OE2. The figure shows that after drought treatment, the leaf shriveling degree of T3 generation ZmAGO4b transgenic maize (OE1 and OE2) was less than that of wild-type maize LH244 (WT), and the survival rate was significantly higher than that of wild-type maize LH244 (WT).

[0087] The same method was used to statistically analyze the survival rates of WT, KO1, and KO2 lines. The experiment was repeated four times, with at least 18 plants from each line in each replicate. The average values ​​were then used for statistical analysis. The results are as follows: Figure 2 C and Figure 2 As shown in D; Figure 2 C shows photos of KO1 and KO2 before drought and after rehydration; Figure 2 D represents the drought survival rate statistics for KO1 and KO2.

[0088] The results showed that after drought treatment, the leaves of the ZmAGO4b mutants KO1 and KO2 were more severely withered than those of wild-type maize, and their survival rate was significantly lower than that of wild-type maize.

[0089] 3. Field drought phenotypic analysis

[0090] Under field experimental conditions, the tolerance of wild-type maize LH244 (WT) and ZmAGO4b overexpression pure lines (OE1 and OE2) to drought treatment was tested.

[0091] This experiment employed a randomized block design with two treatments: normal irrigation and drought. In 2023, at the Zhangye Water-Saving Experimental Farm in Gansu Province, WT, OE1, and OE2 maize varieties were planted. The normal irrigation group received adequate water throughout the growth period to ensure normal maize growth (water potential maintained between 0 and -20 kPa). From stage V5 (the fifth leaf visible at the ligule) until silking, the plants were subjected to drought treatment. Soil water potential was maintained between -80 kPa and -120 kPa. The total irrigation amount for the drought-stressed (WS) plot was approximately 80% of that for the well-watered (WW) plot. After harvest, the yield per ear was statistically analyzed, with at least 50 plants per line. The average yield was taken for statistical analysis. The results are as follows: Figure 3 As shown, Figure 3 A shows photographic results of wild-type and overexpressed maize ears; Figure 3 B represents the statistical results of single-ear yield for wild-type and overexpression materials. The results show that under mild drought conditions, the single-ear yield of both OE1 and OE2 was significantly higher than that of WT.

[0092] The contents not described in detail in this specification are existing technologies known to those skilled in the art.

Claims

1. Applications of ZmAGO4b protein, its encoding gene, or biological materials containing its encoding gene in the following areas: Improve plant drought resistance; Genetic breeding for drought resistance in maize; Improvement of drought-resistant plant resources; The amino acid sequence of the ZmAGO4b protein is as follows: (1) The amino acid sequence shown in SEQ ID No. 1; (2) 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. The nucleotide sequence of the ZmAGO4b protein encoding gene is as follows: (1) As shown in SEQ ID No. 2; the gene sequence encodes the protein shown in SEQ ID No. 1; (2) A nucleotide sequence of the nucleotide sequence shown in SEQ ID No. 2 that has been substituted, deleted and / or added with one or more nucleotides and expresses a protein with the same function as the protein shown in SEQ ID No. 1; The biomaterials containing the ZmAGO4b protein-coding gene include: Expression cassettes, vectors, host cells, or recombinant bacteria containing the ZmAGO4b protein-coding gene.

2. Primers for amplifying the ZmAGO4b gene, characterized in that: The nucleotide sequences of the primers are shown in SEQ ID No. 4-5.

3. A ZmAGO4b gene mutant, characterized in that: The coding sequence of the ZmAGO4b gene mutant is shown in SEQ ID No. 18, and the ZmAGO4b gene mutant encodes the protein shown in SEQ ID No.

19.

4. A ZmAGO4b gene mutant, characterized in that: The coding sequence of the ZmAGO4b gene mutant is shown in SEQ ID No. 21, and the ZmAGO4b gene mutant encodes the protein shown in SEQ ID No.

22.

5. A gRNA target for obtaining ZmAGO4b gene mutants, characterized in that: The nucleotide sequence of the target is shown in SEQ ID No.

12.

6. A method for breeding drought-resistant maize, characterized in that, To increase the expression level and / or activity of ZmAGO4b protein in maize; wherein the ZmAGO4b protein is any of the following proteins: (1) The amino acid sequence is shown in SEQ ID No. 1 of the sequence listing; (2) 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.

7. A method for cultivating transgenic plants, comprising the following steps: linking a gene encoding the ZmAGO4b protein to a pBCXUN vector to obtain a recombinant vector pBCXUN-ZmAGO4b containing the ZmAGO4b coding sequence; transforming the recombinant vector into Agrobacterium EHA105 to obtain recombinant Agrobacterium EHA105 / pBCXUN-ZmAGO4b containing the recombinant vector pBCXUN-ZmAGO4b; infecting maize with recombinant Agrobacterium EHA105 / pBCXUN-ZmAGO4b and introducing it into recipient plants to obtain transgenic plants with increased drought resistance; The ZmAGO4b protein is any of the following proteins: (1) The amino acid sequence is shown in SEQ ID No. 1 of the sequence listing; (2) 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.

8. The method for cultivating transgenic plants according to claim 7, characterized in that, The primer sequences for detecting the ZmAGO4b gene are shown in SEQ ID No. 6-11.

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