Use of protein ZmRtn16 in regulating plant drought resistance

Abstract

The application discloses application of a protein ZmRtn16 in regulating drought resistance of plants and belongs to the technical field of genetic engineering breeding. The application discloses application of the protein ZmRtn16 or an expression material of a gene coding the protein or a material for regulating activity or content of the protein in regulating drought resistance of plants, wherein the protein is a protein with an amino acid sequence of SEQ ID No. 1. The application further discloses a method for improving drought resistance of plants, which comprises introducing a coding gene of the protein into a receptor plant to obtain a target plant with higher drought resistance than the receptor plant. The application verifies, through experiments, that the protein ZmRtn16 has a regulating function on drought resistance of plants, and the protein ZmRtn16 and related biological materials can be applied to regulation of drought resistance of plants.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering breeding technology, specifically to the application of protein ZmRtn16 in regulating plant drought resistance. Background Technology

[0002] Drought is a major agricultural natural disaster. About 43% of the world's arable land is located in arid and semi-arid regions, and drought-induced food losses account for 60% of the total losses caused by all natural disasters. As a dryland crop with high water requirements and sensitivity to drought stress, maize's yield will be severely affected once water shortage occurs. Therefore, research on genetic improvement of maize drought resistance is particularly important.

[0003] Traditional breeding techniques suffer from drawbacks such as long cycles, high degree of uncertainty, and large workload. Moreover, the use of traditional breeding to increase grain yield has reached a certain bottleneck. In recent years, with the development of plant molecular biology and genetics, and in-depth research on the molecular mechanisms of plant stress resistance, the introduction of stress-resistance-related genes into plants through genetic engineering to improve crop stress resistance has become increasingly mature, providing possible genetic resources and theoretical basis for the genetic improvement of maize drought 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 increasing its yield. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to regulate the drought resistance of plants.

[0006] To address the aforementioned technical problems, in a first aspect, the present invention provides the application of a protein or a substance regulating the expression of the protein-coding gene or a substance regulating the activity or content of the protein, wherein the application may be any of the following:

[0007] D1) The application of proteins or substances that regulate the expression of the protein-encoding genes or substances that regulate the activity or content of the proteins in regulating plant drought resistance.

[0008] D2) The application of proteins or substances that regulate the expression of the protein-encoding genes or substances that regulate the activity or content of the proteins in the preparation of products that regulate plant drought resistance.

[0009] D3) The application of proteins or substances that regulate the expression of the protein-encoding genes or substances that regulate the activity or content of the proteins in the preparation of products for cultivating drought-resistant plants;

[0010] D4) The application of proteins or substances that regulate the expression of the protein-encoding genes or substances that regulate the activity or content of the proteins in plant breeding.

[0011] The protein may be A1), A2), or A3):

[0012] A1) The amino acid sequence of this protein is that of SEQ ID No. 1;

[0013] A2) Proteins obtained by substituting and / or deleting and / or adding one or more amino acid residues to the amino acid sequence shown in A1) have more than 80% identity with the protein shown in A1) and are related to plant drought resistance.

[0014] A3) is a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of A1) or A2).

[0015] In this invention, SEQ ID No.1 consists of 228 amino acid residues.

[0016] The proteins mentioned above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0017] The protein tag refers to a polypeptide or protein fused with a target protein using in vitro DNA recombination technology for expression, detection, tracing, and / or purification of the target protein. The protein tag may be a Flag protein tag, His protein tag, MBP protein tag, HA protein tag, myc protein tag, GST protein tag, and / or SUMO protein tag, etc.

[0018] Furthermore, 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, which may be any one of B1) to B9) below:

[0019] B1) Nucleic acid molecules that encode the above proteins;

[0020] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0021] B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0022] 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);

[0023] 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);

[0024] 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);

[0025] 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);

[0026] 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;

[0027] B9) Expression cassettes, recombinant vectors, recombinant microorganisms, or transgenic plant cell lines containing the nucleic acid molecules described in B8).

[0028] Furthermore, in the above applications, the nucleic acid molecule described in B1) can be any of the following DNA molecules as shown in b1) to b3):

[0029] b1) The coding sequence of the coding strand is the DNA molecule shown in SEQ ID No. 2;

[0030] b2) The nucleotide sequence of the coding strand is the DNA molecule shown in SEQ ID No. 2;

[0031] b3) The DNA molecule that has 80% or more identity with the nucleotide sequence defined by b1) or b2) and encodes the above-mentioned protein;

[0032] (B8) The nucleic acid molecule may be a DNA molecule expressing gRNA that targets the protein-coding gene of claim 1, or gRNA that targets the protein-coding gene of claim 1.

[0033] In the above applications, identity refers to the similarity between amino acid sequences or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambdaratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences, the identity value (%) can then be obtained.

[0034] In the above applications, the 80% or more of identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0035] Furthermore, in the above applications, the target sequence of the gRNA can be the nucleotide shown at positions 1757-1775 of SEQ ID No. 3 (i.e., 5'-GGCGAGCGCGTGTTACATT-3').

[0036] In the aforementioned biological materials, the expression cassette described in B2) refers to DNA capable of expressing the aforementioned proteins in host cells. This DNA may include not only promoters that initiate gene transcription but also terminators that terminate gene transcription. Furthermore, the expression cassette may also include enhancer sequences. Promoters that can be used in this invention include, but are not limited to: constitutive promoters, tissue-, organ-, and development-specific promoters, and inducible promoters. Examples of promoters include, but are not limited to: constitutive promoter 35S of cauliflower mosaic virus; wound-inducible promoters from tomatoes, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiol 120:979-992); chemically induced promoters from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiohydroxy acid S-methyl ester)); tomato protease inhibitor II promoter (PIN2) or LAP promoter (both induced by methyl jasmonic acid); heat shock promoters (US Patent 5,187,267); tetracycline-inducible promoters (US Patent 5,057,422); seed-specific promoters, such as the millet seed-specific promoter pF128 (CN101063139B (Chinese Patent 2007 1)). 0099169.7)), seed storage protein-specific promoters (e.g., promoters of beta-conglycin, napin, oleosin, and soybean beta-conglycin (Beachy et al. (1985) EMBO J.4:3047-3053). They can be used alone or in combination with other plant promoters. All references cited herein are cited in full. Suitable transcription terminators include, but are not limited to: Agrobacterium carmine synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator, and carmine and octopine synthase terminator (see, e.g., Odell et al. (I 985 Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; Guerineau et al. (1991) Mol. Gen. Genet, 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5:141; Mogen et al. (1990) Plant Cell, 2:1261; Munroe et al. (1990) Gene, 91:151; Ballad et al. (1989) Nucleic Acids Res. 17:7891; Joshi et al. (1987) Nucleic Acid Res. 15:9627.

[0037] In the aforementioned biological materials, the recombinant vector described in B3) may contain the DNA molecule shown in SEQ ID No. 2 for encoding the aforementioned protein.

[0038] Recombinant vectors containing the above-mentioned gene expression cassettes can be constructed using plant expression vectors. These plant expression vectors can be Gateway system vectors or binary Agrobacterium vectors, such as pGWB411, pGWB412, pGWB405, pBin438, pCAMBIA1300, pCAMBIA1300-35S, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1301, pBI121, pCAMBIA1391-Xa, or pCAMBIA1391-Xb. When constructing recombinant vectors using coding genes, any type of enhancing, constitutive, tissue-specific, or inducible promoter can be added before the transcription initiation nucleotide, such as the cauliflower mosaic virus (CAMV) 35S promoter, the ubiquitin gene Ubiqutin promoter (pUbi), etc. These can be used alone or in combination with other plant promoters. Furthermore, when constructing plant expression vectors using the genes of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are wide-ranging; they can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes.

[0039] To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as by adding genes that can be expressed in plants that encode enzymes or luminescent compounds that produce color changes (GUS gene, luciferase gene, etc.), antibiotic resistance markers (gentamicin marker, kanamycin marker, etc.), or chemical reagent resistance marker genes (such as herbicide resistance genes).

[0040] In this invention, when the recipient is a plant, the recombinant expression vector contains the Zmubi1 promoter, which initiates transcription of the encoding gene. More specifically, the recombinant vector is a recombinant plasmid (named pCU) obtained by inserting the encoding gene of the ZmRtn16 protein into the multiple cloning site (such as XcmI, located downstream of the Zmubi1 promoter) of the pCUNm vector. When the recipient is tobacco, the recombinant plasmid is specifically obtained by inserting the coding gene of the ZmRtn16 protein into the multiple cloning site (such as SalI and KpnI) of the pCAMBIA1300-NLuc / CLuc vector. When the recipient is yeast, the recombinant plasmid is specifically obtained by inserting the coding gene of the ZmRtn16 protein into the multiple cloning site (such as EcoRI) of the pBT3-STE vector. When the recipient is Escherichia coli, the recombinant plasmid is specifically obtained by inserting the coding gene of the ZmRtn16 protein into the multiple cloning site (such as SmaI) of the pGreenII-GFP vector.

[0041] In the above method, the recombinant expression vector is introduced into the recipient plant, specifically by transforming plant cells or tissues using conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, or Agrobacterium-mediated transformation, and then culturing the transformed plant tissues into plants. The transformed cells, tissues, or plants are understood to include not only the final product of the transformation process but also its transgenic progeny.

[0042] Among the aforementioned biological materials, the recombinant microorganisms mentioned in B4) can specifically be yeast, bacteria, algae, and fungi.

[0043] Among the aforementioned biological materials, the plant tissues described in B6) may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos, and anthers.

[0044] Among the aforementioned biological materials, the transgenic plant organs described in B7) can be the roots, stems, leaves, flowers, fruits, and seeds of transgenic plants.

[0045] Among the aforementioned biological materials, the transgenic plant cell lines, transgenic plant tissues, and transgenic plant organs may or may not include propagation material.

[0046] Furthermore, 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 can be any one of the biological materials from B1) to B7), and the regulation of plant drought resistance can be to improve the drought resistance of the plant; or,

[0047] The substance that regulates the expression of the protein-coding gene or the substance that regulates the activity or content of the protein may be the biological material described in B8) or B9), and the regulation of plant drought resistance may be to reduce the drought resistance of the plant.

[0048] In further applications described above, the plant may be any of the following:

[0049] P1) Monocotyledons,

[0050] P2) Plants of the order Poales,

[0051] P3) Gramineae plants,

[0052] P4) Plants of the genus *Zea*.

[0053] P5) Corn.

[0054] 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).

[0055] In the above applications, the regulation of gene expression can be achieved by inhibiting or reducing gene expression, which can be accomplished through gene knockout or gene silencing. Gene knockout refers to the phenomenon of inactivating a specific target gene through homologous recombination. Gene knockout inactivates a specific target gene by altering its DNA sequence. Gene silencing refers to the phenomenon of preventing or reducing gene expression without damaging the original DNA. Gene silencing requires no alteration of the DNA sequence to prevent or reduce gene expression. Gene silencing can occur at two levels: transcriptional gene silencing due to DNA methylation, heterochromatinization, and position effects; and post-transcriptional gene silencing, which inactivates the gene at the post-transcriptional level through specific inhibition of target RNA, including antisense RNA, co-suppression, gene quelling, RNA interference (RNAi), and microRNA (miRNA)-mediated translational repression.

[0056] 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.

[0057] To address the aforementioned technical problems, in a second aspect, the present invention provides a method for improving plant drought resistance, comprising introducing the coding gene of the protein described in the above-mentioned application into a recipient plant to obtain a target plant with higher drought resistance than the recipient plant.

[0058] To address the aforementioned technical problems, in a third aspect, the present invention provides a method for reducing drought resistance in maize, comprising reducing drought resistance in maize by knocking out the gene encoding the protein described in the above-mentioned application.

[0059] Furthermore, in the above method, the gene encoding the protein can be any one of the genes shown in b1)-b3) below:

[0060] b1) The coding sequence of the coding strand is the DNA molecule shown in SEQ ID No. 2;

[0061] b2) The nucleotide sequence of the coding strand is the DNA molecule shown in SEQ ID No. 2;

[0062] b3) has 80% or more identity with the nucleotide sequence defined by b1) or b2) and is a DNA molecule encoding the aforementioned protein.

[0063] Furthermore, in the above method, the plant can be any of the following:

[0064] P1) Monocotyledons,

[0065] P2) Plants of the order Poales,

[0066] P3) Gramineae plants,

[0067] P4) Plants of the genus *Zea*.

[0068] P5) Corn.

[0069] In order to solve the above-mentioned technical problems, in a fourth aspect, the present invention provides the above-mentioned protein or the above-mentioned biological material.

[0070] In this invention, the improved drought resistance can be manifested in any of the following types:

[0071] 1) The survival rate of transgenic plants under drought stress is higher than that of wild-type plants;

[0072] 2) Under drought stress, the yield of transgenic plants is higher than that of wild-type plants;

[0073] 3) Under drought stress, the transgenic plants had more fully closed and partially open stomata than the wild type.

[0074] The beneficial technical effects achieved by this invention are as follows:

[0075] 1. This invention experimentally verifies that the protein ZmRtn16 has a regulatory function on plant drought resistance, and that the protein ZmRtn16 and its related biomaterials can be applied to the regulation of plant drought resistance.

[0076] 2. This invention provides a method for regulating plant drought resistance. Based on the technical solution provided by this invention, transgenic homozygous lines with improved drought resistance and gene-edited homozygous lines with reduced drought resistance can be obtained respectively. Attached Figure Description

[0077] Figure 1 Association analysis of ZmRtn16 gene variation with maize seedling survival rate (SR) under drought stress.

[0078] Figure 2 ZmRtn16 positively regulates drought resistance in maize.

[0079] Figure 3 Stomatal observation and subcellular localization of proteins in ZmRtn16 transgenic material.

[0080] Figure 4 The interaction between ZmRtn16 protein and ZmVHA-A / ZmVHA-E3 protein.

[0081] Figure 5 The results show the localization of ZmVHA-A and ZmVHA-E3 proteins by ZmRtn16 protein regulation.

[0082] Figure 6 Statistical results of field drought phenotype of ZmRtn16 transgenic material. Detailed Implementation

[0083] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0084] 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.

[0085] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0086] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0087] The maize transgenic overexpression vector pBCXUN was derived from the pCXUN (NCBI GenBank: FJ905215) vector. The modification involved replacing the selection marker gene Hyg with the Bar gene at the XhoI site. The above-mentioned biological materials are available to the public from the applicant and are intended solely for replicating the experiments of this invention; they may not be used for any other purpose.

[0088] The transgenic maize recipient material LH244 (PI 612589) is available from GRIN-Global (https: / / npgsweb.ars-grin.gov / gringlobal / search).

[0089] The maize gene-editing vector pBUE411 was kindly provided by Professor Chen Qijun of China Agricultural University. It was used in the literature "Hui-LiXing, Li..." The biological material described above is disclosed in Zhi-Ping Wang, Hai-Yan Zhang, Chun-Yan Han, Bing Liu, Xue-Chen Wang and Qi-Jun Chen (2014). A CRISPR / Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biology 2014, 14:327. (in the literature, it is named pBUE411(Bar)). The public can obtain the above biological material from the applicant. The obtained biological material is only for repeating the experiments of this invention and cannot be used for other purposes.

[0090] 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).

[0091] Example 1: The genetic variation of the ZmRtn16Rtn16 gene is related to drought resistance in maize seedlings.

[0092] GWAS analysis was performed after normalizing the expression levels of the ZmRtn16 gene under three drought conditions, indicating that the gene expression has strong local regulation, such as... Figure 1 As shown in AC. Figure 1 In the table, A, B, and C represent GWAS analyses of gene loci associated with ZmRtn16 expression levels under conditions of sufficient water (WW), moderate drought (WS1), and severe drought (WS2), respectively.

[0093] Association analysis was performed on a 1.5 Mb variant site in the ZmRtn16 gene region with SR under drought stress. Magnification revealed a 28-bp insertion (S2290) in the 3' UTR downstream of ZmRtn16, which was significantly associated with plant survival after drought stress. Significant variants (p<10⁻⁷) were linked to each other in the LD plot, with red asterisks indicating strong LDs between variant sites. Figure 1 As shown in D. Figure 1 The upper figure in the middle D plot shows the correlation between ZmRtn16 gene variants and survival rates after drought treatment within a 1.5 Mb region. The lower figure shows the LD plot of ZmRtn16 variant sites. Significant variants (p<10⁻⁷) are connected to each other in the LD heatmap by lines. Red asterisks indicate strong LDs between variants, circles represent SNPs, and triangles represent InDels.

[0094] Based on the presence or absence of S2290, the expression levels and survival rates were divided into two groups. The results showed that compared to the group carrying S2290, the group lacking S2290 had higher expression levels and survival rates. Figure 1 As shown in EF, Figure 1 The figure in Figure E shows the grouping of ZmRtn16 expression levels under WS2 based on the presence or absence of S2290. The numbers in parentheses represent the number of inbred lines for the two genotypes. Figure 1 The F-plot shows the grouping of drought survival rates based on the presence or absence of S2290; + indicates the presence of S2290, and - indicates the absence of S2290, indicating that this variation may affect gene expression and drought resistance.

[0095] Two representative inbred lines, B73 and CIMBL55, were selected. B73 carries the S2290 gene, while CIMBL55 lacks the S2290 gene. The 3' UTR sequences of the ZmRtn16 CDS fusion of the two genotypes were constructed, and SNP sites were selected for mutation. Furthermore, the ZmRtn16... B73 The genotype mutation is ZmRtn16 CIMBL55 Genotype, results as follows Figure 1 As shown in G, Figure 1 The left side of the G-graph represents ZmRtn16. B73 and ZmRtn16 CIMBL55Genotypic diagram of downstream sequences. The right side represents the expression level of ZmRtn16 for the corresponding genotype. Different letters indicate significant differences in expression when tested with a two-tailed t-test (p<0.05). The results show that the expression level of ZmRtn16 in the absence of S2290 is higher than that of ZmRtn16 carrying S2290, while SNP2181 does not affect the expression level of ZmRtn16. This indicates that it is S2290, not S2181, that affects the expression level of the ZmRtn16 gene, and S2290 is a functional variant site of the ZmRtn16 gene.

[0096] Example 2: Obtaining protein ZmRtn16 and its encoding gene

[0097] 1. Cloning of protein ZmRtn16 and its encoding gene

[0098] 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, PCR amplification was performed with primers F1 and R1. The amplified product was subjected to 1% agarose gel electrophoresis, yielding a 687 bp PCR amplification product (SEQ ID No. 2).

[0099] Sequencing revealed that the PCR product derived from the sensitive maize inbred line B73 contained nucleotides 164-850 of SEQ ID No. 2 (this reference sequence can be found in...). Search for Zm00001d047517 on the website https: / / www.gramene.org / (Obtained), the gene shown in SEQ ID No. 3 was named ZmRtn16, and the protein encoded by this gene was named ZmRtn16. In SEQ ID No. 3, positions 1-600 are the 5' untranslated region, positions 601-2624 are the open reading frame region, and positions 2625-3224 are the 3' untranslated region.

[0100] The primer sequences mentioned above are as follows:

[0101] F1: 5'-ATGGACGCTGCCGGCGGG-3';

[0102] R1: 5'-TCAGTCCCTCTTTATCTCTGATAA-3'.

[0103] The coding sequence of the protein ZmRtn16 is named the ZmRtn16 gene. The nucleotide sequence of the ZmRtn16 gene is shown in positions 164-850 of SEQ ID No. 2, and the nucleotide sequence of the ZmRtn16 genome is shown in SEQ ID No. 3.

[0104] 2. Construction of the recombinant vector pBCXUN

[0105] The coding sequence of the ZmRtn16 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 to a pBCXUN vector fragment that had also been digested with the same enzyme. This yielded a recombinant vector containing the ZmRtn16 coding sequence, named pBCXUN-ZmRtn16. pBCXUN-ZmRtn16 is a recombinant expression vector 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 ZmRtn16 gene in the recombinant vector pBCXUN-ZmRtn16 is Zmubiquitin1.

[0106] 3. Obtaining recombinant Agrobacterium

[0107] The recombinant vector pBCXUN-ZmRtn16 was transformed into Agrobacterium EHA105 to obtain recombinant Agrobacterium EHA105 / pBCXUN-ZmRtn16 containing the recombinant vector pBCXUN-ZmRtn16 (after colony PCR, plasmid was extracted by shaking and sequencing to verify that the recombinant Agrobacterium was a positive clone).

[0108] 4. Obtaining the genetically modified homozygous ZmRtn16 maize

[0109] Recombinant Agrobacterium EHA105 / pBCXUN-ZmRtn16 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 532 bp 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 ZmRtn16 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 ZmRtn16 gene was detected using specific primers QF2 and QR2, with wild-type LH244 as a control. The sequences of the primers are as follows:

[0110] F2: 5'-TTTTAGCCCTGCCTTCATACGC-3';

[0111] R2: 5'-AGTAATGTCATGCGCTATCA-3'.

[0112] QF1: 5'-TGGTTGTGGCTTCGTTGGTT-3';

[0113] QR1: 5'-GCTGCAGAAGAGTTTTGGGTACA-3'.

[0114] QF2: 5'-TTGGTGCTACTCTGGCATGG-3';

[0115] QR2: 5'-CCGCAGCATTGCTAACCATC-3'.

[0116] 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 testing. T1 represents the seeds produced by the current generation of the transformation recipient plant and the plants that grow from it; T2 generation represents the seeds produced by self-pollination of the T1 generation and the plants that grow from it; T3 generation represents the seeds produced by self-pollination of the T2 generation and the plants that grow from it, and so on.

[0117] Total RNA was extracted from the T3 generation ZmRtn16 transgenic maize line and the 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 ZmRtn16 gene was analyzed using the method (Methods. 25:402-408). The results are as follows: Figure 2 As shown in Figure A, the relative expression levels of the T3 generation ZmRtn16 maize lines named OE#3 and OE#6 are more than 40 times higher than those of wild-type maize, indicating that the T3 generation ZmRtn16 maize lines OE#3 and OE#6 are positive transgenic maizes.

[0118] Example 3: Functional study of protein ZmRtn16 and its encoding gene

[0119] 1. Obtaining the ZmRtn16 gene mutant in maize

[0120] A gRNA target (5'-GGCGAGCGCGTGTTACATT-3') was designed on the first exon of ZmRtn16 using CRISPR-P (http: / / crispr.hzau.edu.cn / CRISPR2 / ). The target was constructed into the pBUE411 vector. After identifying positive clones by colony PCR, plasmids were extracted and sequenced. 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 primers. Self-crossing yielded T2 generation seeds of maize ZmRtn16 gene mutants with two edited types. CRISPR Cas9 free was identified in the F4 and R4 generations of materials from these two genotypes, with wild-type LH244 as a control. PCR-negative plants were CRISPR Cas9 free. Seeds of Cas9-free T2 generation homozygous edited lines from the self-crossing of the two edited maize ZmRtn16 gene mutants were named KO#1 and KO#2. Figure 2 As shown in B, it is used for subsequent experiments.

[0121] The sequences of the primers mentioned above are as follows:

[0122] F3: 5'-TTTTAGCCCTGCCTTCATACGC-3';

[0123] R3: 5'-AGTAATGTCATGCGCTATCA-3'.

[0124] F4: 5'-GACAGGCGTCTTCTACTGGTGCTAC-3';

[0125] R4: 5'-TATTCACTAGCTCGGGATAGTTGGC-3'.

[0126] like Figure 2The mutant KO#1 shown in Figure B, compared with wild-type maize LH244, has the following mutation in the ZmRtn16 gene in the maize genome on its two homologous chromosomes: 5'-TGGAGGCGAGCGCGTGTTTGGTGTTATATTTGGTGC-3' is replaced in the ZmRtn16 gene in the maize genomic DNA (positions 1753-1792 of SEQ ID No. 3), resulting in a frameshift mutation after position 161 of the CDS of the ZmRtn16 gene, thereby knocking out the ZmRtn16 gene (wild type). The mutated gene was named ZmRtn16-1. The coding sequence (CDS) of the ZmRtn16-1 gene is obtained by replacing nucleotide T with G at position 324 of the DNA molecule shown in SEQ ID No. 2, deleting nucleotides (TACA) at positions 325-328 of sequence 2 in the sequence listing, and keeping the other nucleotide sequences of SEQ ID No. 2 unchanged. The DNA molecule encodes a protein ZmRtn16-1 composed of 75 amino acid residues, the amino acid sequence of which is shown in SEQ ID No. 4 in the sequence listing.

[0127] like Figure 2 The mutant KO#2 shown in Figure B, compared with wild-type maize LH244, has the following mutation in the ZmRtn16 gene in the maize genome on its two homologous chromosomes: 5'-TGGAGGCGAGCGCGTGTGTTATATTTGGTGC-3' replaces 5'-TGGAGGCGAGCGCGTGTTACATTTGGTGTTATATTTGGTGC-3' in the ZmRtn16 gene in the maize genomic DNA (positions 1753-1792 of SEQ ID No. 3), resulting in a frameshift mutation after position 159 of the CDS of the ZmRtn16 gene, thereby knocking out the ZmRtn16 gene (wild type). The mutated gene was named ZmRtn16-2 gene. The coding sequence (CDS) of the ZmRtn16-2 gene is obtained by deleting the nucleotides (GTTACATTTG) shown at positions 323-332 of sequence 2 in the sequence listing, while keeping the other nucleotide sequences of SEQ ID No.2 unchanged. It encodes a protein ZmRtn16-2 composed of 73 amino acid residues, the amino acid sequence of which is shown in SEQ ID No.5 in the sequence listing.

[0128] 2. Phenotypic analysis of ZmRtn16's role in drought resistance in maize

[0129] Five-day-old T3 generation ZmRtn16 maize lines (OE#3 and OE#6) 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. Three to 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 from each line in each replicate. The average value was used for statistical analysis.

[0130] The results are shown in Table 1. Figure 2 C and Figure 2 As shown in D, Figure 2 Image C shows photos of OE#3 and OE#6 before drought and after rehydration; image D shows the drought survival rate statistics for OE#3 and OE#6. It can be seen that after 20 days of drought treatment, the leaf shriveling degree of T3 generation ZmRtn16 transgenic maize (OE#3 and OE#6) was less than that of wild-type maize LH244(WT), and their survival rate was significantly higher than that of wild-type maize LH244(WT).

[0131] Table 1. Survival rate (%) of transgenic maize plants after drought treatment.

[0132] WT 5.56 11.11 11.11 9.26±3.21 OE#3 27.78 38.89 38.89 33.33±5.56** OE#6 33.33 33.33 44.44 38.89±5.56**

[0133] The same method was used to statistically analyze the survival rates of WT, KO#1, and KO#2, and the results are shown in Table 2. Figure 2 China F and Figure 2 As shown in G, Figure 2 The image in the middle (F) shows KO#1 and KO#2 before drought and after re-watering. Figure 2 G represents the drought survival rate statistics for KO#1 and KO#2. The results show that after 20 days of drought treatment, the leaf driestness of ZmRtn16 mutants KO#1 and KO#2 was greater than that of wild-type maize, and their survival rate was significantly lower than that of wild-type maize.

[0134] Table 2. Survival rate (%) of mutant plants after drought treatment.

[0135] WT 55.55 55.55 61.11 57.40±3.21 KO#1 16.67 11.11 11.11 12.96±3.21** KO#2 16.67 22.22 22.22 20.37±3.2**

[0136] 3. Statistical analysis of water loss from detached leaf blades

[0137] Wild-type LH244 (WT), transgenic LH244 (OE#3 and OE#6), and maize ZmRtn16 gene mutants (KO#1 and KO#2) were germinated in small red pots. After 5 days of germination, the lines were transplanted into white boxes, with WT, KO#1, and KO#2 planted in one box, and WT, OE#3, and OE#6 planted in another box. Each line was planted in two rows, with six plants per row. At stage V2, the second true leaf (four leaves per group, divided into three groups) was selected for an in vitro leaf water loss experiment. The cut leaves were immediately weighed as the weight at 0h. Thereafter, they were weighed every hour. The weight at 0h minus the weight at each time point was the water loss weight for that time period. The water loss weight divided by the weight at 0h was the water loss rate. Figure 2 China E and Figure 2 As shown in H, Figure 2 E represents the statistical data on the water loss rate of detached blades of OE#3 and OE#6; Figure 2 H represents the statistical data on the water loss rate of detached leaves of KO#1 and KO#2. The water loss rate of WT was significantly faster than that of OE#3 and OE#6, while the water loss rate of WT was significantly slower than that of KO#1 and KO#2.

[0138] Therefore, the expression level of the ZmRtn16 protein-encoding gene is significantly correlated with the drought resistance of maize seedlings. Decreasing the expression level of the ZmRtn16 gene can significantly reduce the survival rate of seedlings and accelerate the rate of water loss, while increasing the expression level of the ZmRtn16 gene can significantly improve the survival rate of maize seedlings, slow down the rate of water loss, and increase the drought resistance of maize.

[0139] Example 4: Stomatal aperture analysis of ZmRtn16 transgenic material

[0140] Wild-type LH244 (WT), transgenic LH244 (OE#3 and OE#6), and maize ZmRtn16 gene mutants (KO#1 and KO#2) were germinated in small red pots. After 5 days, WT and two transgenic LH244 materials were transplanted into a white box, and WT and two maize ZmRtn16 gene mutant materials were transplanted into another white box. Each line was planted in two rows with six plants per row. When the plants reached stage V2 (the second true leaf was fully expanded), the middle leaves of the second true leaf were taken and their stomata were photographed using a TM3000 / TM4000 (desktop scanning electron microscope). For the drought treatment, the entire V2 stage leaf was cut off and placed on a table for 5 hours of drought. The observation method was the same as under normal conditions. Stomatal opening was classified into three states: fully open, partially open, and fully closed. At least 12 plants were observed for each line. The results are as follows: Figure 3 As shown in A, Figure 3Figure A shows the statistical analysis of the number of stomata in three stomatal opening states for WT, OE#3 and OE#6, and KO#1 and KO#2 under normal conditions (WW) and 5 hours of terrace drought (WS). The results indicate that the number of half-open and fully closed stomata in ZmRtn16 overexpressing materials was higher than that in WT under WW and WS, while the number of half-open and fully open stomata in ZmRtn16 mutant materials was higher than that in WT under WW and WS.

[0141] Example 5: Study on subcellular localization of ZmRtn16 protein

[0142] 1. Construction of recombinant vectors

[0143] The ZmRtn16 sequence, represented by positions 164-850 of SEQ ID No. 2, was inserted into the SmaI space of the vector pGreenⅡ-ubi:GFP (located downstream of the ubi promoter), while keeping the rest of the sequence unchanged. The resulting recombinant vector was named pGreenⅡ-ubi:ZmRtn16-GFP.

[0144] The pGreenⅡ-ubi:GFP vector is created by replacing the 35S terminator of the pGreenⅡ-GFP vector with the ubi promoter.

[0145] 2. Observation of subcellular localization in transformed maize protoplasts

[0146] The correctly sequenced plasmid was co-transformed with the endoplasmic reticulum localization marker protein (HDEL-mCherry nucleotide sequence SEQ ID No. 6) into (LH244) maize protoplasts. After overnight culture, subcellular localization was observed using Zeiss 710 microparticles. Figure 3 As shown in B, Figure 3 In Figure B, the subcellular localization of ZmRtn16 protein was observed. Ubi:ZmRtn16-GFP was co-transformed with an endoplasmic reticulum marker (HDEL-mCherry), and ZmRtn16-GFP was localized in the endoplasmic reticulum. GFP was detected using an excitation wavelength of 488 nm, and RFP was detected using an excitation wavelength of 561 nm.

[0147] Example 6: Interaction between ZmRtn16 protein and ZmVHA-A and ZmVHA-E3

[0148] 1. Mass spectrometry identification of ZmRtn16 interacting proteins

[0149] To better understand the biological function of the ZmRtn16 protein, we attempted to identify its potential interacting proteins by purifying pGreenⅡ-ubi:ZmRtn16-GFP via co-immunoprecipitation (co-IP) and mass spectrometry analysis, using the empty vector pGreenⅡ-ubi:GFP as a control. We transformed LH244 maize protoplasts with pGreenⅡ-ubi:ZmRtn16-GFP, cultured overnight, and then collected the cells. 7 ml of 2×Extration Buffer (50 mM Na-Phosphate, pH 7.4; 150 mM NaCl; 1 mM DTT; 1×protease inhibitor cocktail tablets; 0.1% Nonidet P-40) was added and transferred to a 1.5 ml centrifuge tube. Cells were lysed using sonication (30 s on, 30 s off, 3 min) and then centrifuged at 13,000 g, 4 °C for 5 min. Transfer the supernatant to 1.5 ml centrifuge tubes, add 15 μL of Anti-GFP mAb-Magneticbeads (MBL) pre-washed 3 times with 2×Extration Buffer to each tube, and incubate at 4°C for 2 h. Collect all the beads using a magnetic rack, wash 3-4 times with 2×Extration Buffer to remove non-specifically bound proteins, and then wash 3-4 times with 2×Extration Buffer without NP-40. Finally, elute with 100 μL of Elution buffer containing 0.1 M Gly-HCl (pH 3.0), and quickly add 1 / 10 volume of 1 M Tris-HCl (pH 6.7) to neutralize the proteins to prevent denaturation. Submit the eluent to the mass spectrometry platform of China Agricultural University for interaction protein detection. Mass spectrometry results revealed ZmVHA-A and ZmVHA-E3 (vacuole membrane H... + The subunit of ATPase was detected multiple times.

[0150] 2. Co-IP verification of the interaction between ZmRtn16 protein and ZmVHA-A and ZmVHA-E3

[0151] To further confirm whether the ZmRtn16 protein interacts with ZmVHA-A and ZmVHA-E3, the ZmVHA-A protein was fused with a Myc tag, the ZmVHA-E3 protein with an HA tag, and the ZmRtn16 protein with a GFP tag. These were then constructed into the vector pGreenⅡ-ubi:GFP and transformed into maize B73 protoplasts. The results are as follows: Figure 4 As shown in Figure A, the results indicate that the ZmRtn16 protein interacts with ZmVHA-A and ZmVHA-E3.

[0152] 3. Luciferase complementation assay to verify the interaction between ZmRtn16 protein and ZmVHA-A and ZmVHA-E3.

[0153] Insert ZmRtn16, represented by bits 164-850 of SEQ ID No. 2, into the KpnI space of the vector pCAMBIA-35S:CLuc to obtain a recombinant vector named pCAMBIA-35S:CLuc-ZmRtn16. Insert ZmVHA-A and ZmVHA-E3 into the SalI space of the vector pCAMBIA-35S:NLuc to obtain recombinant vectors named pCAMBIA-35S:ZmVHA-A-NLuc and pCAMBIA-35S:ZmVHA-E3-NLuc. Two plasmids were transformed into Agrobacterium GV3101+pSoup. Colonies that tested positive by PCR were shaken. The two bacterial suspensions were mixed with P19 bacterial suspension (helper plasmid, p19 is a gene silencing repressor) at a ratio of 1:1:0.6, centrifuged at 5000g for 5 min, the supernatant was discarded, and the suspension was resuspended in 1 mL of infection buffer containing 150 μM acetylsyleugenol (if 10 mL of infection solution is needed, increase the volume of bacterial suspension and infection buffer proportionally). After resuspending, the bacterial suspension was incubated at 30℃ for 3-5 h before injecting into tobacco (Nicotiana benthamiana, stored in the laboratory). After culturing for 48-54 h, the substrate (D-Luciferin stock solution) was sprayed for fluorescence imaging. Figure 4 As shown in Figure B, the results indicate that ZmRtn16 interacts with ZmVHA-A and ZmVHA-E3 in the luciferase complementation experiment.

[0154] 4. Yeast two-hybrid verification of the interaction between ZmRtn16 protein and ZmVHA-E3 in membrane systems

[0155] The ZmRtn16 sequence, represented by positions 164-850 of SEQ ID No. 2, was inserted into the EcoRI space of the vector pBT3-STE, and the ZmVHA-E3 coding sequence was inserted into the EcoRI space of the vector pPPR3-SUC. Both recombinant plasmids were transformed into yeast strain NMY51 and cultured upside down at 30°C for 2-3 days. Single colonies were then incubated overnight by shaking. OD 600 The value is approximately 1.0; adjust each bacterial culture to 10. 0 10 -1 10 -2 Yeast growth was uniformly spotted onto two- and three-deficient culture media and cultured for 1-2 days. The results are as follows: Figure 4 As shown in Figure C, the results indicate that ZmRtn16 protein interacts with ZmVHA-E3 in the membrane yeast system.

[0156] Example 7: ZmRtn16 protein regulates the vacuolar membrane localization of ZmVHA-A and ZmVHA-E3 proteins.

[0157] To verify the effect of ZmRtn16 protein on VH + The influence of the A and E3 subunits of ATPase was investigated. ZmVHA-A and ZmVHA-E3 were labeled with antibodies AtVHA-A and AtVHA-E3, respectively. AtVHA-A antibody indicated the purity of the vacuolar membrane in the whole membrane composition, and AtVHA-A antibody also indicated consistent protein loading. Actin antibody further confirmed consistent protein loading. Chemiluminescence was used for development. The developed images were analyzed for grayscale using ImageJ software to quantify the ZmVHA-A / E3 protein on the vacuolar membrane. Results are as follows: Figure 5 As shown in Figure A, the amount of ZmVHA-A / E3 protein in the vacuolar membrane protein component of the ZmRtn16 mutant material is significantly less than that in the WT material, such as... Figure 5 As shown in Figure B, the amount of ZmVHA-A / E3 protein in the vacuolar membrane protein component of the ZmRtn16 transgenic material is significantly higher than that in the WT material, indicating that the ZmRtn16 protein affects the vacuolar membrane localization of ZmVHA-A and ZmVHA-E3.

[0158] We also detected VH in the ZmRtn16 transgenic material. + -ATPase activity and vacuolar membrane H + -PPase(VH + -PPase activity, results as follows Figure 5 As shown in Figure C, VH in the ZmRtn16 transgenic material + -PPase showed no significant difference from WT, but VH was observed in ZmRtn16 transgenic material. + -ATPase activity is higher than WT, and VH in ZmRtn16 mutant materials + -ATPase activity is lower than WT.

[0159] In summary, ZmRtn16 may affect VH by influencing the vacuolar membrane localization of ZmVHA-A and ZmVHA-E3. + -ATPase activity, but does not affect VH + -PPase activity.

[0160] Example 8: Field drought phenotypic analysis

[0161] Under field experimental conditions, the tolerance of wild-type maize LH244 (WT), ZmRtn16 transgenic homozygous lines (OE#3 and OE#6), and ZmRtn16 mutant homozygous lines (KO#1 and KO#2) to drought treatment was tested. This experiment was conducted at two locations using a randomized block design with two treatments: normal irrigation and drought treatment. WT, OE#3, OE#6, KO#1, and KO#2 were planted in a dry greenhouse in Zhuozhou. The normal irrigation group received sufficient water throughout the growth period to ensure normal maize growth (water potential maintained between 0 and -20 kPa). The drought treatment group stopped watering 30 days after sowing, and soil water potential was automatically monitored using a buried Watermark 200SS sensor. During normal irrigation, the water potential was maintained between 0 and -20 kPa, while during the drought treatment, the soil water potential gradually decreased to approximately -150 kPa. After pollen shedding, plant height, pollen shedding time (DTA), silking time (DTS), and pollen shedding-silking interval (ASI) were statistically analyzed. Normal watering was repeated twice, and drought treatment was repeated three times. Each line had at least 17 plants, and the average values ​​were used for statistical analysis.

[0162] like Figure 6 Photograph A shows the growth status of WT, OE#3, OE#6, KO#1, and KO#2 in the normal treatment group and the drought treatment group. Figure 6 Figure B shows the comparison of the silking interval (ASI) of WT, OE#3, OE#6, KO#1 and KO#2 in the normal treatment group and the drought treatment group. The results show that in the normal treatment group, there is no significant difference in the ASI of each maize. In the drought treatment group, the ASI of OE#3 and OE#6 is significantly smaller than that of WT, while the ASI of KO#1 and KO#2 is significantly larger than that of WT. Figure 6 C represents the pollen shedding time (DTA) comparison results of WT, OE#3, OE#6, KO#1, and KO#2 in the normal treatment group and the drought treatment group. The results show that, except for the DTA of OE#3 being significantly delayed compared to WT under drought conditions, there were no significant differences between them and WT. Figure 6 D represents the time to silk (DTS) of WT, OE#3, OE#6, KO#1, and KO#2 in the normal treatment group and the drought treatment group. The results show that there was no significant difference in DTS in the normal treatment group. In the drought treatment group, the DTS of OE#3 and OE#6 was significantly earlier than that of WT, while the DTS of KO#1 and KO#2 was significantly delayed compared to WT. Figure 6The results of comparing plant height of WT, OE#3, OE#6, KO#1, and KO#2 in the normal treatment group and the drought treatment group (E represents plant height) showed that under normal irrigation conditions, the plant height of OE#3, OE#6, KO#1, and KO#2 was not significantly different from that of WT, and there was also no difference in single-ear yield. Under drought conditions, only the plant height of KO#1 and KO#2 was significantly lower than that of WT, while the plant height of OE#3 and OE#6 was not significantly different from that of WT.

[0163] At the Zhangye experimental base, WT, OE#3, and OE#6 varieties were planted. From the V5 stage (the fifth leaf visible at the ligule) to the completion of silking, the plants were subjected to drought treatment. Soil water potential was maintained between -20 kPa and -220 kPa. Irrigation was carried out approximately every 10 days, with each application of 45-450 tons of water per hectare, depending on soil moisture. The total irrigation amount for the drought-affected plots was approximately 40% of that for the normally growing plots. After pollination, the yield per spike was statistically analyzed, with at least 50 plants per line. The average yield was used for statistical analysis. Figure 6 As shown in F. Figure 6 The results from F indicate that the yields of OE#3 and OE#6 in the drought treatment group were significantly higher than those in WT.

[0164] The above results indicate that overexpression of the gene encoding the protein ZmRtn16 in plants advances the silking time and increases the yield per ear, while reducing the expression level of the gene encoding the protein ZmRtn16 delays the silking time and significantly reduces the yield per ear.

[0165] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims. sequence list <110> China Agricultural University <120> Application of protein ZmRtn16 in regulating plant drought resistance <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> 228 <212> PRT <213> Zea mays <400> 1 Met Asp Ala Ala Gly Gly Val Glu Ala Ala Ala Asn Gly Gly Ile Glu 1 5 10 15 Gly Ser Ala Asp Pro Cys Ser Ser Gly Gly Ala Gly Gly Tyr Arg Arg 20 25 30 Ser Val His Gln Ile Ala Gly Gly Gly Lys Ala Ala Asp Ile Ile Leu 35 40 45 Trp Arg Arg Ala Arg Val Thr Phe Gly Val Ile Phe Gly Ala Thr Leu 50 55 60 Ala Trp Trp Leu Phe Glu Lys Ser Gly Leu Ser Leu Leu Thr Ile Cys 65 70 75 80 Ser Asp Ile Leu Leu Ile Leu Ile Val Leu Gln Phe Ile Arg Val Lys 85 90 95 Ile Ala Gly Leu Leu Asp Arg Gln Pro Arg Pro Leu Pro Glu Leu Val 100 105 110 Leu Ser Glu Glu Met Val Ser Asn Ala Ala Ala Ser Phe Arg Val Lys 115 120 125 Val Asn Ser Met Leu Met Ile Ala His Asp Ile Thr Leu Gly Lys Asp 130 135 140 Phe Arg Leu Phe Phe Gln Val Val Ile Val Leu Trp Leu Leu Ser Val 145 150 155 160 Ile Gly Asn Phe Cys Ser Ser Ile Thr Leu Ala Tyr Ile Gly Thr Val 165 170 175 Ala Leu Val Thr Met Pro Ala Leu Tyr Asn Lys Tyr Gln Arg Pro Val 180 185 190 Asp Arg Tyr Ala Gly Met Val Tyr Arg Asn Ile Ser Arg His Tyr Lys 195 200 205 Ile Val Asp Glu Asn Val Ile Ser Arg Leu Pro Arg Arg Phe Ile Arg 210 215 220 Asp Lys Glu Asp 225 <210> 2 <211> 1123 <212> DNA <213> Artificial Sequence <400> 2 gttcggcttc ggtcctaccg ccgccgcgtc gtgggaggat aatcgaactg cttcccttct 60 cgtcttccct aacctttccg tccttcccgg attatttcca tcaccagagc gtatgcgtc 120 gtctcctctc tatccgattc cgcccgcgga cccaggctcc cgcatggacg ctgccggcgg 180 ggtggaggcc gccgccaacg gcggatcga gggctccgcc gacccctgca gctcaggcgg 240 tgccggcggg tacaggcgct ccgtccacca gatcgccggc ggcgggaaag ctgctgatat 300 catcttatgg aggcgagcgc gtgttacatt tggtgttata tttggtgcta ctctggcatg 360 gtggttgttt gagaagtctg gactatcatt attaactatc tgctctgata tacttctcat 420 cttgatagtt ctgcagttca taagggttaa aatagctgga ctgctagata 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ccgatcgatt tttagattct aatcgtaacc gttgtaattt 180 tcgtaccgaa ctttcgtttc cgatatttct gaattaccga tatcgtttct gttttcggaa 240 ttaccgtttc gatctgaaaa aatatgaaaa ctaaaataat tttagtgttt accgatcttt 300 tccatcgttt tcaacactag ccgaaagtac ggacccgcta atcatccaac ggcctaatta 360 atcatcacaa tccagcttaa tttacgggaa aacactgagg aatgaataaa gaatggcggc 420 gcactcgtgc tgacctcgtt cggcttcggt cctaccgccg ccgcgtcgtg ggaggataat 480 cgaactgctt cccttctcgt cttccctaac ctttccgtcc ttcccggatt atttccatca 540 ccagagcgta tcgcgtcgtc tcctctctat ccgattccgc ccgcggaccc aggctcccgc 600 atggacgctg ccggcggggt ggaggccgcc gccaacggcg ggatcgaggg ctccgccgac 660 ccctgcagct caggcggtgc cggcgggtac aggcgctccg tccaccagat cgccggcggc 720 gggaaaggtc tgctgctctc ctgcccgaac tctccgctcc ccggtttgcg aattgcgata 780 ggttctgcgc cctgttgtca tgtccgtgcg attagcagcg cggattggtt agtgacgtcg 840 ttacgtcgat gcagttttgg ccttctcgat cagataagtg ccgtgcttgt cccagctgta 900 gtagattagt actgcaggat tggaaaacgg tggctcgcga ggttttgatt tgtgcaacta 960 caactactac aagcaaatgt tgtgttccgt gtagttcaaa actttggagt ttggactaca 1020 tcatgtacgg aagctacggt tctgtgtgat gtggacttgt agccagagag tatcgtgctg 1080 ttttaaatga aattatagtt attactgtga acccaatagg gcttgaaata cttgtgctgg 1140 aatttgacct gataattgct gcaacgagcc catttcaaag ataagagtca tgtaagataa 1200 gaattggtag catttgaatt tgggtctaat tgggctgtaa agtgcatggt tgttgtactt 1260 aatctcagat gttagtcaat ttcttttatc accccgccta aactagttga tcatcttagc 1320 ttcaagtggt ttgacaattc atattttc tgcgcaagga ttgcttgctt gtcattatgg 1380 atgtttctag agtctcggct gctcatgtgg cgcttgcttg tcactatgga tgttctaga 1440 ctctcgacta ctcatgtggc gccggcctat gatgccaggt tgcctcttgc attggctagt 1500 gcctggctgc gtatggcact ttacgatgca tctttttgtgt ggaaccatgg tttggggctt 1560 ttctcgacca gcagagttaa ggttctacta cctacatgaa atgtcctagg gaagctttta 1620 ctggctggtc aattgtaatt tagagatgga taaacggtgc tacttttatg ccctattaaa 1680 gtgcagctga acatctatct gtcttgttcc tgctaatgtt aattgccgtt ttcagctgct 1740 gatatcatct tatggaggcg agcgcgtgtt acatttggtg ttatatttgg tgctactctg 1800 gcatggtggt tgtttgagaa gtctggacta tcattattaa ctatctgctc tgatatactt 1860 ctcatcttga tagttctgca gttcataagg gttaaaatag ctggactgct agataggtaa 1920 gcaacccgga tcctccttcc attgtatctc aaatttggat gaattgccta tatgttatca 1980 tactggttta actatttgca ggcagcctag gccactacct gagttagttt tatcagagga 2040 gatggttagc aatgctgcgg cttcattccg tgtgaaagtg aacagcatgc tgatgatagc 2100 gcatgacatt actcttggca aggacttcag gctatttttc caggttcact gttatgcatg 2160 tactttgtgt tgattttttt ggtattcctg tcattgctga gacattttct cttcacaggt 2220 cgtgattgtc ttgtggctgt tatctgtcat tggaaatttc tgctcgtcta tcactcttgc 2280 ttacataggt atatattatc ctgtacaggt catactttac cctttgttcc ttacacgttt 2340 aggaatatac agacactcag acagtaccac caaagtgcag gcagaatctt atctctgttt 2400 cgtcttgagt tattatttct gaatttcatt gctaatttta aaatgggtgc acgacaggaa 2460 ccgttgcttt ggtcacaatg cctgcattgt acaacaagta ccagaggcct gttgataggt 2520 atgctgggat ggtctaccgg aacatttcga ggcattacaa gatagttgat gagaatgtga 2580 tcagtagact accgagaagg tttatcagag ataaagagga ctgatactct gaaaaatgaa 2640 ctcgctaagc tagtcaggta atcttttacc tacattgtca gaccttacat ggtgtgaaac 2700 tatagagcta cgaaaacaga gaacggcatg gaacctacag tagataccgt tgcagtgtac 2760 tcttgggcaa aggtttgaga cgaaattaaa atgtatagct ttctgtgtta gctctttgca 2820 acgaactggc taaatgttgt taaaaataac aaaagcaatt gtcatgggaa gttgaaaatg 2880 aagtggttgt aacttgtgaa agcgagtgct gtagcaggta ccagcaatgg caacagactg 2940 aatctactgc agggtacttt ggttgcggct tgcagtcaga tgcatggcta cgtctcactc 3000 cgaagtcagc tccgaacatg ttacccccct ggtatcattt cactagtaat tctgttaaat 3060 ctgacaggtc ctactcctac ccttcatgat aaaataagat gactgacagg tgcaagtgct 3120 cgctgctcag tatcgctaaa ccttactcct tcagcattat gccacaagtg caatacatgg 3180 ttcaaaataa aggcgctgtt tggactttgg acccattagc taac 3224 <210> 4 <211> 75 <212> PRT <213> Artificial Sequence <400> 4 Met Asp Ala Ala Gly Gly Val Glu Ala Ala Ala Asn Gly Gly Ile Glu 1 5 10 15 Gly Ser Ala Asp Pro Cys Ser Ser Gly Gly Ala Gly Gly Tyr Arg Arg 20 25 30 Ser Val His Gln Ile Ala Gly Gly Gly Lys Ala Ala Asp Ile Ile Leu 35 40 45 Trp Arg Arg Ala Arg Gly Leu Val Leu Tyr Leu Val Leu Leu Trp His 50 55 60 Gly Gly Cys Leu Arg Ser Leu Asp Tyr His Tyr 65 70 75 <210> 5 <211> 73 <212> PRT <213> Artificial Sequence <400> 5 Met Asp Ala Ala Gly Gly Val Glu Ala Ala Ala Asn Gly Gly Ile Glu 1 5 10 15 Gly Ser Ala Asp Pro Cys Ser Ser Gly Gly Ala Gly Gly Tyr Arg Arg 20 25 30 Ser Val His Gln Ile Ala Gly Gly Gly Lys Ala Ala Asp Ile Ile Leu 35 40 45 Trp Arg Arg Ala Arg Val Leu Tyr Leu Val Leu Leu Trp His Gly Gly 50 55 60 Cys Leu Arg Ser Leu Asp Tyr His Tyr 65 70 <210> 6 <211> 648 <212> DNA <213> Artificial Sequence <400> 6 atgaacattt tcagattagc tggtgatatg actcacctag ccagtgttct tgtcttgctt 60 ctcaagatcc acaccatcaa atcctgcgct ggtgtttcat tgaagactca agaactctat 120 gccattgtct ttgcgacgcg ttatttggat attttcacga gttttgtgtc tctgtacaac 180 acctctatga agttggtgtt tttaggaagt tctttttcga ttgtgtggta catgaagtat 240 cataaggccg tccacaggac ttacgacaga gagcaagata cgtttcgtca ttggttcctt 300 gtgcttcctt gctttctctt agctcttttg atacatgaaa agtttacctt tcttgaggta 360 ttgtggacgt cttcattgta cttggaggct gttgccatat tacctcagct tgtcttgttg 420 caaaggacta gaaatattga caacttgacc ggacaatata tatttctcct tggggggtac 480 cgtggattat acatcctcaa ctggatctac cgttacttca ctgagccgca ctttgttcac 540 tggataacat ggatcgccgg gtttgttcaa acactgctct atgccgactt cttctattat 600 tatttcctaa gctggaaaa caacaaaaaag ctccaattac cagcttaa 648

Claims

1. The application of proteins, characterized by: The application is any one of the following: D1) Application in improving the drought resistance of maize; Application of D2 in the preparation of products that improve the drought resistance of maize; Application of D3 in the preparation of products for cultivating drought-resistant maize; Application of D4 in drought-resistant breeding of maize; The protein is either A1) or A2): A1) The amino acid sequence of this protein is that of SEQ ID No. 1; A2) is a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of A1).

2. The application of biomaterials related to the protein of claim 1, characterized in that: The application is any one of the following: D1) Application in improving the drought resistance of maize; Application of D2 in the preparation of products that improve the drought resistance of maize; Application of D3 in the preparation of products for cultivating drought-resistant maize; Application of D4 in drought-resistant breeding of maize; The biomaterial is any one of B1) to B7) below: B1) A nucleic acid molecule encoding the protein described in claim 1; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2), or a transgenic plant cell line containing the recombinant vector described in B3); B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2), or transgenic plant tissue containing the recombinant vector described in B3); B7) A transgenic plant organ containing the nucleic acid molecule described in B1), or a transgenic plant organ containing the expression cassette described in B2), or a transgenic plant organ containing the recombinant vector described in B3).

3. The application according to claim 2, characterized in that: B1) The nucleic acid molecule described below is a DNA molecule as shown in b1) or b2): b1) The coding sequence of the coding strand is the DNA molecule shown in SEQ ID No. 2; b2) The nucleotide sequence of the coding strand is the DNA molecule shown in SEQ ID No.

2.

4. A method for improving the drought resistance of maize, comprising introducing the gene encoding the protein of claim 1 into a recipient maize to obtain a target maize with higher drought resistance than the recipient maize.

5. The method according to claim 4, characterized in that: The gene encoding the protein is the gene shown in b1) or b2) below: b1) The coding sequence of the coding strand is the DNA molecule shown in SEQ ID No. 2; b2) The nucleotide sequence of the coding strand is the DNA molecule shown in SEQ ID No. 2.

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