Application of ZmEMF1 gene in regulation and control of corn kernel endosperm development

Abstract

The invention discloses application of a ZmEMF1 gene in regulation and control of corn kernel endosperm development, and belongs to the technical field of plant genetic engineering. It is found for the first time that the mutant zmemf1 can regulate and control early development of corn endosperm, compared with a wild type, the number of endosperm cells of the mutant zmemf1 is larger, the endosperm is larger, and the mutant zmemf1 has the function of promoting cell division in the early development stage of the endosperm. From the perspective of agronomic traits, the length, width and hundred-grain weight of corn grains can be regulated and controlled through overexpression of the ZmEMF1 gene, so that the yield of corn is increased. According to the invention, by researching the function of the ZmEMF1 gene, the cytological basis of the gene for regulating the development of corn endosperm and the regulation network for influencing the size of corn kernels are analyzed, and a new gene resource is provided for breeding.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and in particular to the application of the ZmEMF1 gene in regulating the development of corn kernel endosperm. Background Art

[0002] Cereals provide approximately 50% of human dietary protein and are a key source of protein. Corn is a major cereal and my country's largest crop. Its production has increased annually over the past decade, accounting for approximately 40% of total cereal production. Corn is highly nutritious and a high-quality food crop, widely used in food processing, healthcare, animal feed, and other industries. Furthermore, corn is a major raw material for clean energy.

[0003] The nutritional and economic value of corn lies primarily in the kernel. The primary component of the kernel is the endosperm, which accounts for approximately 80-85% of the kernel's weight. As the primary storage organ, the development of the endosperm directly impacts corn yield and quality. The number of endosperm cells is a key factor influencing corn yield potential. Research has shown that cell number and size in plants, as crucial factors influencing tissue and organ morphology, significantly influence plant growth, development, and physiological processes.

[0004] Maize endosperm formation occurs through three main stages: early endosperm development, mid-stage storage material synthesis and grain filling, and late dehydration and maturation. While early endosperm development only accounts for a small portion of the entire kernel development timeline (from double fertilization to maturity), the duration of this stage, as well as the number of cells and endosperm structure developed, significantly influence subsequent storage material accumulation and are closely related to seed size.

[0005] Maize has developed numerous endosperm mutations throughout its evolution. Currently, over 20 maize endosperm mutation genes have been discovered, with the main ones showing some or potential application value, including su1, sh2, bt1, bt2, ae1, du1, wx1, se1, and o2. Studies have shown that these mutations can affect the carbohydrate or protein content and composition of the endosperm during the grain-filling and milk-ripening stages, thereby affecting maize quality. Therefore, studying the effects of maize endosperm mutations and their interactions is of great significance for maize breeding and improvement ("Research on the Genetic Basis and Related Genes of Maize Endosperm," Li Shuiqin et al., 2015). However, due to the difficulty in obtaining genetic material, it is difficult to obtain mutants that specifically affect early endosperm development through forward genetics. Furthermore, homozygous mutants that affect early endosperm development are often lethal. Consequently, research on early maize endosperm development is rare. Summary of the Invention

[0006] In response to the above-mentioned prior art, the present invention aims to provide an application of the ZmEMF1 gene in regulating endosperm development in maize kernels. The present invention found that a mutant of the ZmEMF1 gene, zmemf1, can promote cell division in the early stages of endosperm development, providing a new regulatory gene for the study of early maize endosperm development.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] In a first aspect of the present invention, a mutant zmemf1 is provided for use in regulating early endosperm development of maize; the mutant zmemf1 is a protein as shown in any one of the following (1) to (3):

[0009] (1) a protein consisting of the amino acid sequence shown in SEQ ID No. 3;

[0010] (2) a protein consisting of the amino acid sequence shown in SEQ ID No. 4;

[0011] (3) A protein consisting of the amino acid sequence shown in SEQ ID No. 5.

[0012] In the above application, the regulation of early endosperm development of corn specifically includes: promoting cell division, increasing the number of endosperm cells, and increasing the volume of endosperm at 0-6 DAP.

[0013] The second aspect of the present invention provides the use of the ZmEMF1 gene in any one of the following (1)-(3):

[0014] (1) Regulate corn endosperm development;

[0015] (2) regulating corn kernel development;

[0016] (3) Corn breeding;

[0017] The ZmEMF1 gene is a DNA molecule as shown in the following i) or ii) or iii):

[0018] i) the nucleotide sequence is the DNA molecule shown in SEQ ID NO.1;

[0019] ii) a DNA molecule encoding the amino acid sequence shown in SEQ ID NO. 2 other than i);

[0020] iii) a DNA molecule that has 90% or more identity with the DNA fragment defined in i) or ii), and encodes a protein that is functionally equivalent to the protein shown in SEQ ID NO. 2.

[0021] The term "identity" as used herein refers to sequence similarity to a natural nucleic acid sequence. Homology can be evaluated using computer software, for example, the BLAST algorithm (Altschul et al. 1990. Journal of Molecular Biology 215: 403-410; Karlin and Altschul. 1993. Proceedings of the National Academy of Sciences 90: 5873-5877).

[0022] In the above nucleic acid molecules, the 90% or greater homology may be at least 90%, 92%, 93%, 95%, 96%, 98% or 99% homology.

[0023] In the above application, the regulation of corn kernel development is to regulate the kernel length, kernel width and / or 100-kernel weight of the corn kernels.

[0024] The third aspect of the present invention provides the use of the protein encoded by the ZmEMF1 gene in the following (1) or (2):

[0025] (1) Regulate corn endosperm development;

[0026] (2) Regulate corn kernel development.

[0027] In the above application, the amino acid sequence of the protein encoded by the ZmEMF1 gene is shown as SEQ ID No. 2.

[0028] The fourth aspect of the present invention provides the use of a recombinant expression vector, a transgenic cell line or a genetically engineered bacterium containing the ZmEMF1 gene in any one of the following (1) to (3):

[0029] (1) Regulate corn endosperm development;

[0030] (2) regulating corn kernel development;

[0031] (3) Corn breeding.

[0032] A fifth aspect of the present invention provides a method for regulating corn kernel development, comprising the following steps:

[0033] Upregulating the expression of the ZmEMF1 gene in maize plants to obtain transgenic maize plants with increased kernel length, kernel width, and 100-kernel weight;

[0034] Alternatively, the protein encoded by the ZmEMF1 gene in corn plants is mutated to obtain corn mutant plants with reduced grain length, grain width and 100-grain weight.

[0035] In the above method, upregulating the expression of the ZmEMF1 gene in corn plants is achieved by the following means:

[0036] Exogenously transfer the ZmEMF1 gene; or introduce a DNA fragment that can activate or increase the transcription level, translation level or protein activity of the corn ZmEMF1 gene.

[0037] In the above method, the protein encoded by the ZmEMF1 gene is mutated into a protein shown in any one of SEQ ID No. 3 to SEQ ID No. 5.

[0038] The sixth aspect of the present invention provides the use of a downstream regulatory gene of the ZmEMF1 gene in any one of the following (1)-(3):

[0039] (1) Regulate corn endosperm development;

[0040] (2) regulating corn kernel development;

[0041] (3) Corn breeding.

[0042] Preferably, in the above application, the downstream regulatory gene of the ZmEMF1 gene is Zm00001d011323 or Zm00001d004230; the CDS sequence of Zm00001d011323 is shown as SEQ ID No. 6, and the CDS sequence of Zm00001d004230 is shown as SEQ ID No. 7.

[0043] Beneficial effects of the present invention:

[0044] (1) The present invention discovered for the first time that the mutant zmemf1 can regulate the early development of maize endosperm. Compared with the wild type, the mutant zmemf1 has more endosperm cells and a larger endosperm from 0 to 6 DAP, and has the function of promoting cell division in the early stage of endosperm development.

[0045] (2) From the perspective of agronomic traits, compared with the wild type, the mutant zmemf1 has reduced grain length, grain width and 100-grain weight. Therefore, the ZmEMF1 gene can regulate the development of corn kernels. Accordingly, it is inferred that overexpression of the ZmEMF1 gene will increase the grain length, grain width and 100-grain weight of corn kernels, thereby increasing corn yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1Phenotypes of the maize mutant zmemf1; Figures (AC) B73, emf1 / +, and emf1 ear phenotypes. Scale bar: 1 cm. (DE) Kernel length and width phenotypes of B73 and emf1. Scale bar: 1 cm. (FH) Kernel length, width, and 100-kernel weight statistics of B73 and emf1. ****, p < 0.001; ***, p < 0.01.

[0047] Figure 2 Kernel and endosperm phenotypes and statistics at successive maize developmental stages; Figures A-C: Kernel phenotypes and kernel length and width statistics for B73 and emf1 at successive developmental stages. Scale bar: 1 cm. (D-F) Endosperm phenotypes and kernel length and width statistics for B73 and emf1 at successive developmental stages. Scale bar: 1 cm. (G) Paraffin sections of B73 and emf1 at successive developmental stages. (H-I) Endosperm area and cell number statistics for B73 and emf1. ****, p < 0.001; *, p < 0.05, ns, no significant difference.

[0048] Figure 3 : Cytological section observation results; In the figure, (A) Cytological sections of the endosperm of B73 and emf1 3-6 days after pollination and the basal transport cell layer and aleurone layer 12 and 24 days after pollination, scale: 200um; (BC) Statistics of the number and area of ​​endosperm cells of B73 and emf1 5 and 6 days after pollination; (DE) Expression levels of the aleurone layer and basal transport layer-specific expression genes AL9 and BETL9 in the endosperm of B73 and emf1 12 days after pollination.

[0049] Figure 4 : Endoreplication of zmemf1 in the endosperm at 18 DAP.

[0050] Figure 5 : Gene cloning and genetic verification results; in the figure, (A) SNP allele frequencies in BSA analysis (candidate genes are circled in red) (B) ZmEMF1 gene structure and mutation sites; (C) ear phenotypes of emf1-2, emf1-2×emf1-1 / +, emf1-3 and emf1-3×emf1-1 / +; (DG) Allelic verification separates grain length and width in the ear.

[0051] Figure 6 : Expression pattern (A) and subcellular localization (B) of ZmEMF1 gene.

[0052] Figure 7: Transcriptome analysis and H3K27me3 ChIP-seq data analysis of emf1 endosperm; in the figure, (A) metaplot of average read coverage and cross-gene distribution of H3K27me3 ChIP-seq data; (B) volcano plot of differential peaks; (C) Venn diagram analysis of differentially expressed genes and peak genes lost in emf1; (D) box plot of expression levels of genes containing peaks and genes lost in emf1 up-regulated genes; (E) GO enrichment analysis of up-regulated genes with lost peaks in emf1; (F) IGV screenshot of downstream candidate genes selected based on joint analysis.

[0053] Figure 8 : Effect of overexpression of ZmEMF1 gene on 1000-grain weight of plant seeds. DETAILED DESCRIPTION

[0054] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0055] As mentioned earlier, early endosperm development in maize directly impacts yield and quality, making research on endosperm mutations crucial. The EMF1 gene is unique to plants. In Arabidopsis, this gene is essential for vegetative growth. When mutated, it skips the vegetative phase and directly initiates germination and flowering. While most research on the EMF1 gene has focused on vegetative growth and flowering, relatively little research has examined the function of EMF genes in maize.

[0056] In view of this, the present invention has conducted an in-depth study on the function of the ZmEMF1 gene in corn. The CDS sequence of the ZmEMF1 gene is shown in SEQ ID No. 1, which is as follows:

[0057]

[0058] The amino acid sequence of the protein encoded by the ZmEMF1 gene is shown in SEQ ID No. 2, and is as follows:

[0059]

[0060] The present study found that the mutant zmemf1 of the ZmEMF1 gene can affect the division and differentiation of corn endosperm cells. Through the phenotypic study of the mutant zmemf1, it was found that in the early stage of endosperm development, the number of endosperm cells of zmemf1 was greater than that of the wild type, and the endosperm was also larger. Zmemf1 has the function of promoting cell division; in the middle and late stages of endosperm development, zmemf1 has the phenotype of basal transport layer and aleurone layer proliferation, thereby affecting endosperm development.

[0061] There are three mutants of zmemf1 studied in the present invention, namely zmemf1-1, zmemf1-2 and zmemf1-3. The amino acid sequence of zmemf1-1 is shown in SEQ ID No. 3, which is as follows:

[0062] MEAAALARRTRAAVEELRRLSTVAESDDTPEQRLSTVAKSDGMPAEEQECDHFSLRGYVAMLQKKDPKLCSPHIFHSQPQYDEHHGSSPLLVSKYRRWDCSKCLERVKVSGHRPTSGNVSIEQD GMNDGCSISIVRILPNRVDPRRMFYCKQQSSQGNDQLTLSKTAQECNSKCSSPGNKAITAMNIPVAEENVLEALVERSVPATEDLQASPNNIDVSANILNVVPKDASDLPDDVQTISTIEENG TKNPCSPKLRVMPNEDESNIVQDVPNFDPNESNVCKPLSCHKGKQISGHKSSQVCNKGPRRASLKRKVGSDGKKKRDKSTDLPDISGLKFCQRKPKKMRLLSELIDADQVGISANAVQVDRTD SVDLCEGGKRKRHLEVGKDNDTPNQKLDKIQSRAVKNKAKHTAVDKVDDGPSLMNWLKNTHKKIRTDRKDSEHKNLDSSAISRSYPDIVASDDMYHDFIPSVGDVDQVKLPSTTTSAKHGKVNA*

[0063] The amino acid sequence of zmemf1-2 is shown in SEQ ID No. 4, and is as follows:

[0064] MEAAALARRTRAAVEELRRLSTVAESDDTPEQRLSTVAKSDGMPAEE*

[0065] The amino acid sequence of zmemf1-3 is shown in SEQ ID No. 5, and is as follows:

[0066] MEAAALARRTRAAVEELRRLSTAESDDTPEQRLSTVAKSDGMPAEEQECDHFSLRGYVAMLQKKDPKLCSPHIFHSQPQYDEHHGSSPLLVSKYRRWDCSKCLERVKVSGHRPTSGNVSIEQDGMNDGCSISIVRILPNRVDPRR MFYCKQQSSQGNDQLTLSKTAQECNSKCSSPGNKAITAMNIPVAEENVLEALVERSVPATEDLQASPNNIDVSANILNVVPKDASDLPDDVQTISTIEENGTKNPCSPKLRVMPNEDESNIVQDVPNFDPNESNVCKPLSCHKGK*

[0067] The present invention further identified Zm00001d011323 (ZmAGL62) and Zm00001d004230 (ZmHDZIV11) as downstream genes of ZmEMF1 through combined RNA-seq and H3K27me3 CHIP-seq analysis. The CDS sequence of Zm00001d011323 is shown in SEQ ID No. 6, which is as follows:

[0068]

[0069] The CDS sequence of Zm00001d004230 is shown in SEQ ID No. 7, and is as follows:

[0070] .

[0071] They may be involved in the early cell division and late cell differentiation of the endosperm, respectively, providing genetic resources for constructing the corn endosperm development regulatory network.

[0072] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below with reference to specific embodiments.

[0073] All experimental materials used in the examples of the present invention that are not specifically described are conventional experimental materials in the art and can be purchased through commercial channels. Where specific experimental conditions and methods are not specified in the examples of the present invention, conventional conditions, such as those in J. Sambrook et al., ed., Molecular Cloning Laboratory Manual (3rd Edition), Science Press, 2002; and D.L. Spector et al., ed., Cell Experiment Manual, Science Press, 2001, were generally followed, or the conditions recommended by the manufacturer.

[0074] Example 1: Creation and phenotypic analysis of mutants

[0075] Maize kernels are composed of the embryo, endosperm, and seed coat. The development of the endosperm is a key factor influencing kernel size. Therefore, to identify mutants involved in maize endosperm development and to understand the regulatory network underlying endosperm development, we used EMS mutagenesis to generate a mutant, zmemf1, that exhibits reduced kernel size and darker color.

[0076] Since EMS mutagenesis can cause a large number of point mutations on DNA molecules, in order to purify the background and exclude this phenotype caused by multiple unlinked gene mutation sites, we backcrossed the small-grain homozygous mutant zmemf1 with its parent B73 for three generations and obtained segregated ears after two generations of self-pollination.

[0077] We compared the phenotypes of wild-type (B73), heterozygous (zmemf1 / +), and zmemf1 ears. The kernels of zmemf1 were significantly smaller than those of wild-type ( Figure 1 AC). Compared with the wild type, the grain length, grain width and 100-grain weight of zmemf1 were significantly reduced ( Figure 1 DH).

[0078] To investigate whether the mutant was recessively inherited by a single gene, we used B73 X zmemf1 BC3F2 to separate ears and calculate the segregation ratio. The results are shown in Table 1.

[0079] Table 1: Separation ratio statistics of emf1 separated ears

[0080]

[0081] The results showed that the segregation ratio of normal and mutant phenotypes was 3:1, which was consistent with Mendel's law of genetic segregation.

[0082] Example 2: Cytological changes during the continuous development of the maize zmemf1 endosperm

[0083] To further explore the relationship between maize kernel size and endosperm development, we observed intact kernels and endosperm of B73 and zmemf1 at 0-35 days after pollination (DAP) using a stereomicroscope and semi-thin and paraffin sections, and also counted the dynamic changes in kernel and endosperm size and cell number.

[0084] Statistical observations revealed that the grains of the zmemf1 mutant were larger than those of the wild type at 0-6 DAP, and the grains began to shrink after 8 DAP. Figure 2 A), and the grain length and width also follow this trend ( Figure 2 B, C) The endosperm of the zmemf1 mutant develops normally, but its size and the developmental trend of the length and width of the endosperm are the same as those of the kernel ( Figure 2 DF).

[0085] Since 3-6DAP is the stage of endosperm cellularization and rapid cell proliferation, in order to study how zmemf1 affects early endosperm development at the cytological level during this stage, we paraffin-embedded sections and semi-thin sections of B73 and zmemf1 kernels. At 3DAP, zmemf1 began the cellularization stage and produced about 4 layers of endosperm cells, while the wild type was still in the syncytium stage. At 4DAP, zmemf1 had completed cellularization and began to proliferate, while the wild type had just entered the cellularization stage. At 5DAP and 6DAP, both the wild type and zmemf1 entered the early rapid mitosis stage, and the endosperm morphology was normal, but zmemf1 was larger than the wild type ( Figure 3 A). Similarly, the cell number and endosperm area of ​​zmemf1 were significantly larger than those of wild type ( Figure 3 B, C) showed that during the early 3-6 days of endosperm development, the cell division rate of zmemf1 was faster than that of the wild type.

[0086] 8DAP is the time point of early cell differentiation in endosperm development. The endosperm area of ​​zmemf1 is significantly larger than that of wild type. However, after 8DAP, the endosperm area of ​​zmemf1 is significantly smaller than that of wild type ( Figure 2 G, H), indicating that in the late stage of endosperm development, the mechanisms regulating endosperm growth may differ between zmemf1 and wild-type. The basal endosperm transport layer (BETL) is located at the junction of maternal and daughter tissues, transferring photosynthetic products and nutrients from maternal tissue to daughter tissue. Paraffin section observation revealed that in 12 and 24 DAP kernels, zmemf1 BETL cells overproliferated, the number of cell layers increased, and the cell morphology was abnormal ( Figure 3 A). BETL9, which is specifically expressed in BETL, is significantly upregulated in zmemf1 ( Figure 3D). Aleurone cells are regular cubical cells on the surface of the endosperm and are the main storage site for corn kernels. Paraffin sections showed that the number of aleurone cell layers increased in zmemf1. The aleurone-specific gene AL9 was also significantly upregulated in zmemf1 ( Figure 3 E). Flow cytometry analysis showed that at 18 DAP, the level of high-ploidy nuclei increased in the zmemf1 endosperm, especially the proportion of 12C and 24C cells increased significantly ( Figure 4 ), indicating that ZmEMF1 affects the endoreduplication cell cycle.

[0087] Example 3: Cloning and genetic verification of the ZmEMF1 gene

[0088] 1. Cloning of the ZmEMF1 gene

[0089] To clone the ZmEMF1 gene, we hybridized a homozygous zmemf1 mutant obtained by EMS mutagenesis with wild-type B73 to obtain F1 heterozygous ears. The F1 seeds were then planted and self-pollinated to obtain F2 segregated ears. We selected wild-type and mutant leaves from the segregated ears, pooled them, extracted DNA, and performed BSA sequencing. By calculating the differences in polymorphic nucleotides (SNPs) between the wild-type and mutant, we located the gene on chromosome 10. Zm00001d024813 has a single-base mutation from C to T in exon 3, which creates a stop codon and prematurely terminates protein translation. Figure 5 A, B).

[0090] 2. Genetic validation of candidate genes

[0091] To confirm that the zmemf1 phenotype was caused by a mutation in this gene, we screened two alleles from the EMS mutant library (http: / / maizeems.qlnu.edu.cn), EMS5-0af7fc and EMS5-0af81d, and named them zmemf1-2 and zmemf1-3, respectively. The mutants screened in Example 1 were named zmemf1-1. Both zmemf1-2 and zmemf1-3, located in exons 1 and 3, respectively, had a single C-to-T mutation, resulting in a stop codon and replacing glutamine (E) at positions 47 and 291. The amino acid sequence of mutant zmemf1-1 is shown in SEQ ID No. 3; the amino acid sequence of mutant zmemf1-2 is shown in SEQ ID No. 4; and the amino acid sequence of mutant zmemf1-3 is shown in SEQ ID No. 5. The homozygous grain phenotypes of zmemf1-2 and zmemf1-3 were the same as that of zmemf1-1, with smaller grains and darker color.

[0092] To verify allele inheritance, we crossed zmemf1-2 and zmemf1-3 homozygous plants with zmemf1-1 heterozygous plants to obtain segregating ears ( Figure 5 C), the grain length, grain width and 100-grain weight of the zmemf1-1 / zmemf1-2 and zmemf1-1 / zmemf1-3 mutant phenotypes were significantly smaller than those of the wild-type zmemf1-2 / + and zmemf1-3 / + grains. The segregation ratio of the zmemf1-1 / +X zmemf1-2 and zmemf1-1 / +X zmemf1-3 separated ears was close to 1:1, and the segregation ratio of the zmemf1-1 / +X zmemf1-3 / + separated ears was close to 3:1 ( Figure 5 DG), these results indicate that the mutation of Zm00001d024813 leads to the phenotype of zmemf1.

[0093] Zm00001d024813 was named ZmEMF1 gene, and the CDS sequence of the ZmEMF1 gene was shown in SEQ ID No. 1.

[0094] Example 4: Subcellular localization of the ZmEMF1 gene

[0095] We used RT-qPCR to detect the expression of ZmEMF1 gene in endosperm at successive stages after pollination and found that the expression level was higher in the early endosperm ( Figure 6 A).

[0096] To determine the subcellular localization of ZmEMF1, we constructed a fusion expression vector containing ZmEMF1 and GFP. This vector, along with the nuclear localization vector H2B-mCherry, was co-injected into tobacco leaves to observe transient expression in tobacco epidermal cells. The fusion expression vector was constructed by simultaneously cutting the maize ZmEMF1 gene and the pPZP211 vector with KpnI and BamHI and then ligating them using homologous recombination. The upstream and downstream primers used for this ligation are as follows:

[0097] ZmEMF1-pPZP211-F:5'-GAACACGGGGGACGAGCTCGGTACCATGGAAGCAGCTGCTCTTGC-3';

[0098] ZmEMF1-pPZP211-R:5'-TCCTTTACTCATGTCGACTCTAGAGGATCCAAGGGTTCTCATGTACTCGTTCTTG-3'.

[0099] The results are as follows Figure 6As shown in B, the results showed that ZmEMF1-GFP only had fluorescent signals in the cell nucleus ( Figure 6 B) This indicates that ZmEMF1 is localized in the cell nucleus.

[0100] Example 5: Analysis of downstream regulatory genes of the ZmEMF1 gene

[0101] We analyzed the endosperm phenotype of the zmemf1 mutant and determined that the mutant promoted early cell proliferation and later BETL and AL development. We inferred that ZmEMF1 might repress key genes by depositing H3K27me3, so we used ChIP-seq and RNA-seq for combined analysis to try to find downstream target genes.

[0102] By analyzing the EMF1 ChIP signal, it was found that the ZmEMF1 ChIP signal was enriched near the transcription start site, less enriched at the transcription termination sites (TTSs), and even less enriched in the gene body ( Figure 7 A). Analysis of differential peaks in the CHIP-seq results showed that 5504 peaks were obtained in zmemf1, while 4841 peaks were lost ( Figure 7 B). A Venn diagram of the genes with lost peaks and differentially expressed genes in RNA-seq was drawn. It was found that 352 of the upregulated genes in the mutant had lost peaks, and 137 of the downregulated genes had lost peaks ( Figure 7 C). We analyzed the expression levels of genes with and without peaks among the upregulated genes and found that the expression levels of upregulated genes with peaks were significantly higher than those without peaks. This indicates that the degree of ZmEMF1 occupancy in gene DNA has an intrinsic coordination relationship with the active state of gene transcription ( Figure 7 D). Because H3K27me3 is associated with transcriptional repression, we selected 352 genes that were upregulated and had lost peaks among the differentially expressed genes for GO enrichment analysis and found that most of the gene-enriched items were related to transcription ( Figure 7 E). We then analyzed the expression patterns of these 352 genes and screened out 36 genes with high expression levels 6 days after pollination. The analysis predicted ZmAGL62 of the MADS family and ZmHDZIV11 containing the HOX domain as genes downstream of the ZmEMF1 gene ( Figure 7 F).

[0103] Example 6: Acquisition and Functional Verification of ZmEMF1 Transgenic Arabidopsis

[0104] 1. Obtaining ZmEMF1 gene-transgenic Arabidopsis thaliana:

[0105] The CDS sequence of the ZmEMF1 gene shown in SEQ ID No. 1 was ligated into the pBI121 vector to generate the recombinant vector pBI121-ZmEMF1. The recombinant vector pBI121-ZmEMF1 was then transformed into Agrobacterium tumefaciens GV3101. The exogenous ZmEMF1 gene was then transferred into wild-type Arabidopsis thaliana (Col-0) using the Agrobacterium-mediated inflorescence infection method. Resistance-positive plants were screened and serially subcultured to obtain T3 generation homozygous transgenic lines.

[0106] 2. Functional verification of transgenic Arabidopsis thaliana with ZmEMF1 gene:

[0107] The thousand-grain weights of three transgenic ZmEMF1 Arabidopsis lines (OE 1, OE2, and OE3) were measured, with wild-type Arabidopsis (Col-0) as a control. Figure 8 shown.

[0108] The results showed that overexpression of the ZmEMF1 gene could increase the grain weight of plants and thus increase yield.

[0109] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. Application of mutant zmemf1 in regulating early endosperm development of maize; the mutant zmemf1 is a protein as shown in any one of the following (1)-(3): (1) a protein consisting of the amino acid sequence shown in SEQ ID No. 3; (2) a protein consisting of the amino acid sequence shown in SEQ ID No. 4; (3) A protein consisting of the amino acid sequence shown in SEQ ID No.

5.

2. The use according to claim 1, characterized in that The regulation of corn endosperm early development specifically includes: promoting cell division, increasing the number of endosperm cells, and increasing the endosperm volume at 0-6 DAP.

3. Use of the ZmEMF1 gene in any of the following (1)-(3): (1) Regulate corn endosperm development; (2) regulating corn kernel development; (3) Corn breeding; The ZmEMF1 gene is a DNA molecule as shown in the following i) or ii) or iii): i) the nucleotide sequence is the DNA molecule shown in SEQ ID NO.1; ii) a DNA molecule encoding the amino acid sequence shown in SEQ ID NO. 2 other than i); iii) a DNA molecule that has 90% or more identity with the DNA fragment defined in i) or ii), and encodes a protein that is functionally equivalent to the protein shown in SEQ ID NO.

2.

4. The use according to claim 3, characterized in that The regulating corn kernel development is regulating the kernel length, kernel width and / or 100-kernel weight of the corn kernels.

5. Use of the protein encoded by the ZmEMF1 gene in the following (1) or (2): (1) Regulate corn endosperm development; (2) Regulate corn kernel development.

6. The use according to claim 5, characterized in that The amino acid sequence of the protein encoded by the ZmEMF1 gene is shown in SEQ ID No.

2.

7. Use of a recombinant expression vector, transgenic cell line or genetically engineered bacteria containing the ZmEMF1 gene in any of the following (1)-(3): (1) Regulate corn endosperm development; (2) regulating corn kernel development; (3) Corn breeding.

8. A method for regulating corn kernel development, characterized in that: The following steps are involved: Upregulating the expression of the ZmEMF1 gene in maize plants to obtain transgenic maize plants with increased kernel length, kernel width, and 100-kernel weight; Alternatively, the protein encoded by the ZmEMF1 gene in corn plants is mutated to obtain corn mutant plants with reduced grain length, grain width and 100-grain weight.

9. The method according to claim 8, characterized in that The protein encoded by the ZmEMF1 gene is mutated into a protein shown in any one of SEQ ID No. 3 to SEQ ID No.

5.

10. Use of a downstream regulatory gene of the ZmEMF1 gene in any of the following (1)-(3): (1) Regulate corn endosperm development; (2) regulating corn kernel development; (3) Corn breeding; The CDS sequence of the downstream regulatory gene is shown as SEQ ID No. 6 or SEQ ID No. 7.

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