Application of ZmSKI3 gene and ZmSKI2 gene in regulating corn kernel development

Abstract

This invention relates to the field of biotechnology, specifically to the application of the ZmSKI3 and ZmSKI2 genes in regulating maize kernel development. This invention is the first to identify the ZmSKI3 and ZmSKI2 genes in maize and confirms that both genes control maize kernel development. Specifically, inhibiting the expression of ZmSKI3 and ZmSKI2 genes can delay maize kernel development. Therefore, this invention provides two key regulatory factors affecting maize kernel development, offering important molecular targets for improving maize kernel yield. Furthermore, this invention provides a novel gene resource for regulating maize kernel development; future manipulation of these genes can yield new germplasm with improved kernel development, enabling widespread application in production.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the application of the ZmSKI3 and ZmSKI2 genes in regulating maize kernel development. Background Technology

[0002] As one of the world's most important food, feed, and energy crops, maize plays an irreplaceable role in ensuring global food security, promoting economic development, and alleviating the energy crisis. Grain is the most important component of maize yield, and research on genes affecting grain development helps to elucidate the molecular basis of maize grain development, which has significant theoretical and practical implications. Studies have shown that maize grain development is a continuous biological process involving a series of complex physiological and biochemical metabolisms, which can be divided into three main periods: (1) 0-15 days after pollination, during which embryo and endosperm cells rapidly divide, mainly completing the cellular framework of the embryo and endosperm, determining the potential accumulation space for photosynthetic products; (2) 15-40 days after pollination, during which the grain filling rate determines the speed of photosynthetic product accumulation, and the effective filling time determines the time of photosynthetic product accumulation; (3) 40-70 days after pollination is the grain dehydration and maturation period, during which nutrients transformed from gradually aging leaves and stems are transported to the grain, and the grain matures physiologically.

[0003] Maize kernel development is controlled by a complex regulatory network of numerous regulatory and functional genes. Through the interactions between these genes, traits directly related to kernel development, such as cellular structure, grain-filling rate, and effective grain-filling time, are influenced, leading to differences in kernel yield among different varieties. Therefore, elucidating the genetic mechanisms of maize kernel development at the molecular level can provide theoretical support for the genetic improvement of maize kernel yield. Although some genes regulating maize kernel development have been cloned, our understanding of the molecular basis of maize kernel development remains incomplete and requires further exploration and analysis of new genes controlling seed development. Summary of the Invention

[0004] The purpose of this invention is to provide the application of the ZmSKI3 and ZmSKI2 genes in regulating maize kernel development, thereby addressing the problems existing in the prior art. The ZmSKI2 and ZmSKI3 genes described in this invention are related to maize kernel traits, and mutations in these genes can lead to defects in maize kernel development, providing a novel gene resource for regulating maize kernel development.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides the application of the ZmSKI3 gene or the ZmSKI2 gene in regulating maize kernel development, wherein the nucleotide sequence of the ZmSKI3 gene is shown in SEQ ID NO.1; and the nucleotide sequence of the ZmSKI2 gene is shown in SEQ ID NO.2.

[0007] Preferably, the development of maize kernels is delayed by inhibiting the expression of the ZmSKI3 or ZmSKI2 gene in maize.

[0008] Preferably, the delayed development includes reducing the length, width, thickness, and weight of 100 corn kernels.

[0009] More preferably, the protein encoded by the ZmSKI3 gene (ZmSKI3 protein) and the protein encoded by the ZmSKI2 gene (ZmSKI2 protein) are both SKIs cofactors.

[0010] This invention provides the application of the ZmSKI3 gene or the ZmSKI2 gene in the cultivation of transgenic maize with delayed grain development. The nucleotide sequence of the ZmSKI3 gene is shown in SEQ ID NO.1; the nucleotide sequence of the ZmSKI2 gene is shown in SEQ ID NO.2.

[0011] Preferably, the delayed development includes reducing the length, width, thickness, and weight of 100 corn kernels.

[0012] This invention provides a method for breeding maize with delayed kernel development. By inhibiting the expression of the ZmSKI3 gene or the ZmSKI2 gene in maize, the transgenic maize with delayed kernel development is obtained. The nucleotide sequence of the ZmSKI3 gene is shown in SEQ ID NO.1; the nucleotide sequence of the ZmSKI2 gene is shown in SEQ ID NO.2.

[0013] Preferably, the developmental delay includes reducing the length, width, thickness, and weight of 100 corn kernels.

[0014] The present invention discloses the following technical effects:

[0015] This invention identifies the ZmSKI3 and ZmSKI2 genes in maize for the first time and demonstrates that these genes can regulate maize kernel development. In a specific embodiment of this invention, the ZmSKI3 gene in maize was obtained through protein sequence alignment analysis. Two loss-of-function mutants of the ZmSKI3 protein, Zmski3-1 and Zmski3-2, were further screened. Phenotypic observation revealed that randomly distributed defective kernels appeared on the self-pollinated ears of both the Zmski3-1 / + heterozygotes and the Zmski3-2 / + heterozygotes. Allelic testing of these two mutants confirmed that the mutation in the ZmSKI3 gene is the cause of kernel development defects. Paraffin section analysis revealed basal endosperm transfer in the Zmski3 mutant. The mutant cells lacked the characteristic of inward cell wall growth, and the formation rate of starch granules in the kernels was significantly slowed. Yeast two-hybrid and luciferase complementation experiments revealed that another SKIs cofactor protein, ZmSKI2, interacts with ZmSKI3, and the ZmSKI2 gene directly regulates maize kernel development. Furthermore, transcriptome sequencing showed a significant enrichment of differentially expressed genes related to metabolism in the Zmski3-1 mutant kernels, indicating that the mutation of the ZmSKI3 gene directly regulates energy metabolism during maize seed development, leading to kernel development defects. These experiments confirmed that both the ZmSKI3 and ZmSKI2 genes control maize kernel development; specifically, inhibiting the expression of both genes delays kernel development, while overexpression promotes kernel development. Therefore, this invention has obtained two key regulatory factors affecting maize kernel development, providing important molecular targets for the molecular improvement of maize kernel yield. At the same time, this invention provides a new gene resource that regulates maize kernel development. In the future, by manipulating this gene, new germplasm with improved kernel development can be obtained and can be promoted and applied in production. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 Evolutionary analysis of the maize ZmSKI3 protein;

[0018] Figure 2The ZmSKI3 gene controls maize kernel development. A is a schematic diagram of the ZmSKI3 gene structure, with long black arrows indicating mutation sites. B shows the ear phenotypes of Zmski3-1 / + (Zmski3-1 / +, heterozygous), Zmski3-2 / + (Zmski3-2 / +, heterozygous), and Zmski3-1 / +×Zmski3-2 / + (obtained by crossing Zmski3-1 / + heterozygous with Zmski3-1 / + heterozygous). Red arrows indicate mutation sites. C represents the phenotype of wild-type and Zmski3-1 mutant seeds, scale bar = 1 cm; D represents the comparison of seed length, width, and thickness between wild-type and Zmski3-1 mutant seeds, values ​​are mean ± standard deviation, 50 biological replicates (***: P ≤ 0.001, t test); E represents the comparison of 100-seed weight between wild-type and Zmski3-1 mutant seeds, values ​​are mean ± standard deviation, 3 biological replicates (***: P ≤ 0.001, t test). F is a longitudinal section of mature grains of wild type and Zmski3-1 mutant, scale bar = 0.25 cm; G is a longitudinal paraffin section of wild type grains 18 days after pollination, with endosperm cells (upper right) and BETL cells (lower right) magnified, and red arrows indicating intracellular proliferation of BETL cells, scale bar = 1000 μm (left), 100 μm (upper right) and 50 μm (lower right); H is a longitudinal paraffin section of wild type grains 18 days after pollination, with endosperm cells (upper right) and BETL cells (…). (Bottom right) is magnified; red arrows indicate intracellular proliferation of BETL cells. Scale bars = 1000 μm (left), 100 μm (top right), and 50 μm (bottom right). I represents RT-qPCR analysis of BETL-specific genes in wild-type and Zmski3-1 mutant grains, with ZmGAPC1 gene as a control. Values ​​are mean ± standard deviation, with three biological replicates (**: 0.001 < P ≤ 0.01, t-test). J represents the phenotype of wild-type and Zmski3-2 mutant grains. Scale bar = 1 cm. K represents a comparative analysis of grain length, width, and thickness between wild-type and Zmski3-2 mutant grains. Values ​​are expressed as mean ± standard deviation, with 50 biological replicates (***: P ≤ 0.001, t-test); L represents the comparison of 100-kernel weight between wild-type and Zmski3-2 mutant kernels, values ​​are expressed as mean ± standard deviation, with 3 biological replicates (***: P ≤ 0.001, t-test); M represents longitudinal sections of mature kernels from wild-type and Zmski3-2 mutants, scale bar = 0.25 cm; N represents the ear phenotype of Zmski3 mutant 18 days after pollination in different backgrounds, with red arrows indicating mutant kernels, scale bar = 5 cm; O represents longitudinal sections of mature kernels from wild-type and Zmski3 mutants in different backgrounds, scale bar = 0.25 cm, En represents the embryo; Em represents the endosperm; BETL represents the basal endosperm transfer layer;

[0019] Figure 3 Genes that are significantly differentially expressed in the seed transcriptome of the Zmski3-1 mutant;

[0020] Figure 4 GO analysis of differentially expressed genes in the seed transcriptome of Zmski3-1 mutant;

[0021] Figure 5 Heatmap analysis of genes enriched for starch synthesis and metabolism-related entries in the GO analysis of the Zmski3-1 mutant grain transcriptome;

[0022] Figure 6 This study demonstrates the interaction between maize ZmSKI3 and ZmSKI2 proteins. A shows the evolutionary analysis of the maize ZmSKI2 protein; B shows the yeast two-hybrid analysis of ZmSKI3 and ZmSKI2 proteins, where BD-SKI3+AD-SKI2 is the experimental group (positive transformants after co-transfection of yeast Y2H Gold with BD-SKI3 and AD-SKI2), BD+AD-SKI2 is the negative control (positive transformants after co-transfection of yeast Y2H Gold with pGBKT7 vector and AD-ZmSKI2), BD-SKI3+AD is the negative control (positive transformants after co-transfection of yeast Y2H Gold with BD-SKI3 and pGADT7 vector), BD-53+AD-T is the systemic positive control, and BD-Lam+AD-T is the systemic negative control; C shows a schematic diagram of the ZmSKI3 protein structure; and D shows the luciferase complementation experiment of ZmSKI3 and ZmSKI2 proteins.

[0023] Figure 7The ZmSKI2 gene controls maize kernel development. A is a schematic diagram of the ZmSKI2 gene structure; long black arrows indicate mutation sites. B shows the ear phenotypes of Zmski2-1 / +, Zmski2-2 / +, and Zmski2-1 / +×Zmski2-2 / +, with red arrows indicating mutant kernels (scale bar = 1 cm). C shows the kernel phenotypes of wild type and Zmski2-1 mutant (scale bar = 1 cm). D shows the kernel phenotypes of wild type and Zmski2-1 mutant. Comparative analysis of grain length, width, and thickness, values ​​are mean ± standard deviation, 50 biological replicates (***: P ≤ 0.001, t test); E is a comparative analysis of 100-grain weight between wild type and Zmski2-1 mutant grains, values ​​are mean ± standard deviation, 3 biological replicates (***: P ≤ 0.001, t test); F is a longitudinal section of mature grains of wild type and Zmski2-1 mutant, scale bar = 0.25 cm; G is the phenotype of grains of wild type and Zmski2-2 mutant. Scale bar = 1 cm; H is a comparative analysis of grain length, width, and thickness between wild-type and Zmski2-2 mutant grains, with values ​​representing mean ± standard deviation, 50 biological replicates (***: P ≤ 0.001, t test); I is a comparative analysis of 100-grain weight between wild-type and Zmski2-2 mutant grains, with values ​​representing mean ± standard deviation, 3 biological replicates (***: P ≤ 0.001, t test); J is a longitudinal section of mature grains from wild-type and Zmski2-2 mutant, scale bar = 0.25 cm. Detailed Implementation

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0027] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0028] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0029] Unless otherwise specified, the methods used in this invention are all well-known to those skilled in the art.

[0030] Example 1: Identification of ZmSKI3 protein in maize

[0031] Based on previous studies of the SKI3 protein in humans, yeast, and rice, a phylogenetic tree was constructed using protein sequence alignment (using the Muscle algorithm in MEGA X). Figure 1

[0032] CGAGCTCGAAGCATTGATCCATCGTTGGCCTTGCCATGGGCTGGAATGTCGGCGGAAAA

[0033] TTATCATCAATCTGGGAGCAGTCCTGTAAATGAATCTTTTGAAAGCTGCTTGAGGGCTGT

[0034] GCAGATCCTACCTCTGCCAGAGTTCCAGATTGGTCTTGGAACAATTGCAGCCCGTTCTAG

[0035] TAATCTTCTGTCACCTCAGGTATTGATGGCTGTAAGACAGGCTGTTCAACGAGCACCTCA

[0036] TTATCCAGAGTCTCACAATTTAAATGGCTTGATTTCTGAAGTGCGATTAGATTTCCGGTCT

[0037] GCAATCACATTTTACTTGCAAGCGAGATTTGCTTTAGGCATGATGTACAATTCCATGTCAG

[0038] ATAATAGACAAGCCTTTGCTGATGTTTCAGTGAATCTTGCCCGTGCACTATGCAAGGCTG

[0039] GTCTTGCAAGTGATGCAGCGCGGGAGTGTGAAGAGCTGAGATGTCAAGGACTGTTGAG

[0040] CGTTGATGGATTGCAGATATATGCTCTGGCATTGTGGAAAATTGGACAAAGCAAGGAAG

[0041] CTCTTTCTGTATCTGGAAGCTTGGCTGAAAATTTAAGCGGTATAAAGGTGGAGAGTGCC

[0042] ACTGCAGCTTGGGGATTCATATGCACCTTGACGTATGGAATTTCTGGGAAGGACTCTGCA

[0043] GCTGCCATGATTCAGAAGCTTCCTGATCAACTCAATTCAACTCACAGTTGAAATTCATT

[0044] ATCTCTGCATTGGATGCTTTGCATCCAAACCAAACGTCTCCAGCTGCCTCAGTTGAATATG

[0045] CCTCCTAAGCGTACAGCTTATGAAGTGATGAGTGAAGTACACTCGAATATTGCTCTTGGGG

[0046] AAGGCTATTGGTGTGGGATTTGACAAGCCTCTGAGGGTTGATGGTAGTTTGTCTTACCTG

[0047] AAAAAAGTACTGCACATGTATCCTGACTGCAGTTTAGTGAGAAACCAACTTGGATCCCT

[0048] GCTGCTGTGGAGTGGAGATTGGATGGCTTCTCCACAAAGCAATAAGGGTTACCTCTCTGT

[0049] CACATGGGCACACATCCAGTATGGGCCTAAGATCACCACATCACATTCAAGCCTGTGCAA

[0050] TGGTTTCTTGCTATGCTACCTGCTGCACGTACACAAAGTTCTCTTTTGCAACATGTGAAC

[0051] ACCAGTACCTAAGTGGACCTGATGCAATACACCACCTGCAAAGGTGGGTTCATTGTGAA

[0052] CCATGGAACCAAGATGCACGCTACCTGCTTGTACTTGCCATTTTCCAAAAAGCACATGA

[0053] AGAGAAGTACCCCCAAACATACCTGTGTTATTCTGAAGAAGCTCATTATGCAAGTGTTGTC

[0054] CAATATTAGCAATCCACATGAGAAAGAAGCTATGCAGTATCAGGTGTTCCTGCTACTTCT

[0055] TCTGTCATCAGAGGTCTGTTTGCAATCTTTAGACTATGAAAACTGCATCACCGAAGCTAA

[0056] AGAAGCTCTAAAACTCACCCCGTCAAGCCGTGTGGGATACTTTTTTGCAAATTTGCAACT

[0057] GTGTCGGGCCTATGTGGTGCATGGGGATCTTTTGAACTCCAGGAATGAGTACATGAAATG

[0058] CTTGAGAAACCATACAAATACTGAGATTGGCTGGGTAATGCTGAAGCAACTTGAATCTG

[0059] CATGTTCTTTGGAGGGCTTTTCTGACGAAATAGATATAAAACTGCATGAATGTGTTAAAA

[0060] GGAATGGCAGCGACTCATCAAAATGGACATCCCTTTTCAATCTGGCGTGTGCTCAGTGC

[0061] TTTCTGTGGGGTGGAAACTTTGAAAGCGCTGAAAAGCTCTTGCTCAGGCATGTAGTCA

[0062] AGTAGATCCGGATAGCTGCATCTTGTTCCTTAATGGTCAACCTGTATGGAAATCGCTCG

[0063] GAGGTTTGTAGCTCCTCAATTTATATCCCGTGCAGCTTCCAGCCTCAGAAAGGCCCAGCA

[0064] GAAATCACATGCTTCACTACCTCTCGTGTCCCTGCTACTGGCCCAAGCCGAGGGTAGCCT

[0065] TGGCTCCAAACCAAGTGGGAAAGAAACCTTCGCCTGGAATGGTTCTCATGGCCCCCA

[0066] GAGCTGAGGCCTGCGGAGGTGTACTTCCAAATGCATCTGCTGTCGAGGCAGTCGTCTGC

[0067] AGCTGTTTCTCAGCAGAACCAGCTGGTGGAGACGATCCAGAGCCCCGAGTTATGGTTGC

[0068] TTCGGGCGATTCACCTGAACCCGTCCTGCCCCAGGTACTGGAAGGCTCTGCTGCAGCAAATATATGTGTAG (SEQ ID NO. 1).

[0069] Example 2: The ZmSKI3 gene in maize controls the development of maize kernels.

[0070] To investigate the function of the ZmSKI3 gene in maize growth and development, two mutants containing premature termination of the ZmSKI3 protein were obtained by screening the EMS mutant library (http: / / maizeems.qlnu.edu.cn / ): Zmski3-1 (compared to the wild type, the ZmSKI3 gene in this mutant has a G-to-A mutation at position 765 of SEQ ID NO.1, specifically: ATGCCCGAAACCGCGGCGGAGGCCAACCTCCGTAGGCAACTGGAGCAAACCCT, SEQ ID NO.3) and Zmski3-2 (compared to the wild type, the ZmSKI3 gene in this mutant has a G-to-A mutation at position 990 of SEQ ID NO.1).Specifically: ATGCCCGAAACCGCGGCGGAGGCCAACCTCCGTAGGCAACTGGAGCAAACCCTCGCCGCTGAACCCTCCAGCCCGCTCCACCACTACAACCTTGGCGTCTTCCTATGGGACCGCGCCGAGGCAGAGCAGGAGGGTGATGGGGAAGAGGCGCGGAAGCTCCGCGCGGAAGCGTCGGAGCATTTTCTCGCGGCGGCCAAGCTGAATCCCAACGATGGCGTCCCCTTCCGCTTCCTTGGCCACCACTATGCGCGCGGCGGTGACAATCAACGAGCGGTCAAGTGCTATCACCGTGC CGTGACCCTCAACCCTGACGACTCTGAGGCTGGTGACACACTCTGTGGCTTGCTAGATGTTGAAGGGAAGGAGAGCTTGGAGCTTGCTGTCTGCAAGGAGGCAGCTGGCAAGTCACCGCGTGCGTTCTGGGCTTTTCGGAGACTTGGCTATTTACAGGTTCATCAGAGGAAATGGTCAGAGGGTATACAAAGCCTTCAGCATGGAATACGAGGTTACCCAACATGTGCAGATTTATGGGAGGCACTTGGTCTGGCATACCACCGTTTGGGCATGTTCACCGCAGCAGTAAAGTCATATGGACGAGCTATTGAACTTGATAGTTCCAGGGTCTTTGCATTGATAGAAAGTGGAAACATCCAGTTAATGCTTGGTTACTATAGAAAGGGAGTGGAGCAGTTTCGTTCTGCTTTGGAAATGGCTCCATGTAATCATTCAGCATACTTTGGCCTTGCTTCTGCATTGCTTGCATGGGCAAGGAATTGTGTAACTACTGGGGCCTTTGGTTGGGCTGCTAGCCTGTTGAAGGAAGCTTCAGAAGCTTCCAGAATTTGTACTTCTTTGACTGGAAACCTTTCATGCGTCTGGAAATTGCATGGAGATGTTCAGCTCACACTTGCGAGATGCTTTCCATGGGTGGATGGGAAAATCAAAAGGGTCATGGATGCACAGATGTTCAAAAATTCTGTTCAAGAGTGA,SEQ ID NO.4)(, Figure 2 (A) Simultaneously, mutant seeds were purchased. After sowing and self-pollination, randomly distributed defective kernels were observed on the ears of both Zmski3-1 / + heterozygotes and Zmski3-2 / + heterozygotes, indicating that the ZmSKI3 gene is very likely to regulate the kernel development process of maize. Figure 2 (B in the original text). Simultaneously, hybridization was performed after sowing, and allelic analysis confirmed that the mutation in the ZmSKI3 gene was the cause of grain development defects. Figure 2 (B in the original text). Further analysis after the corn kernels matured revealed that the mutant kernels had varying degrees of reduction in kernel length, width, thickness, and 100-kernel weight compared to the wild type. Figure 2 CF in Figure 2 JM). Subsequent paraffin sectioning revealed a lack of significant endocellular cell wall formation in the endosperm transfer layer cells at the base of the mutant grains. Furthermore, chemical staining showed a significant reduction in starch grain aggregation in the mutant compared to the wild-type grains. Figure 2 The expression levels of the genes ZmBETL1 (ZmBETL1-qPCR-F, ZmBETL1-qPCR-R), ZmBETL2 (ZmBETL2-qPCR-F, ZmBETL2-qPCR-R), ZmBETL9 (ZmBETL9-qPCR-F, ZmBETL9-qPCR-R), and ZmBETL10 (ZmBETL10-qPCR-F, ZmBETL10-qPCR-R) in the basal endosperm transfer layer were detected using RT-qPCR with ZmGAPC1 (ZmGAPC1-qPCR-F, ZmBETL1-qPCR-R) as internal controls. The results showed that the expression levels in mutant grains were significantly reduced. Figure 2The nucleotide sequences of ZmGAPC1-qPCR-F are shown in SEQ ID NO.7, specifically CCTTCATCACCACGGACTAC; ZmGAPC1-qPCR-R is shown in SEQ ID NO.8, specifically AACCTTCTTGGCACCACCCT; ZmBETL1-qPCR-F is shown in SEQ ID NO.9, specifically CTGTTGCCATTCTGTCCTCA; ZmBETL1-qPCR-R is shown in SEQ ID NO.10, specifically TCTGGTGGTCCTCGGAATAG; ZmBETL2-qPCR-F is shown in SEQ ID NO.11, specifically ACAAAGTGGGCACACCAAAGAGG; ZmBETL2-qPCR-R is shown in SEQ ID NO.12, specifically ACAAAGTATGTTGCCCGATGCC; and ZmBETL9-qPCR-F is shown in SEQ ID NO. As shown in SEQ ID NO.13, the specific nucleotide sequence is TGGCTACAAGTGCGGGAGTTATAC; the nucleotide sequence of ZmBETL9-qPCR-R is shown in SEQ ID NO.14, specifically TACCACATGTCCCAACCACTCC; the nucleotide sequence of ZmBETL10-qPCR-F is shown in SEQ ID NO.15, specifically TCCTTGTGGCCTATCGTGCG; the nucleotide sequence of ZmBETL10-qPCR-R is shown in SEQ ID NO.16, specifically GCTCATGCATGGGCCGTGAT.

[0071] Furthermore, by hybridization, the mutant Zmski3-1 was introduced into two different genetic backgrounds, Chang7-2 and Huangzao4. The ear phenotype 18 days after pollination was as follows: Figure 2 As shown in N, randomly distributed defective kernels appeared on the ears; at the same time, longitudinal sections were taken of mature kernels of wild type, Chang7-2 introduced with Zmski3-1, and Huangzao4 introduced with Zmski3-1, and the longitudinal section images are shown in Figure 1. Figure 2 As shown in Figure O, compared to the wild type, the embryos and endosperm of Chang7-2 and Huangzao4 introduced with Zmski3-1 were significantly reduced.

[0072] In summary, the effect of Zmski3-1 in regulating grain development is stable, further confirming its important role in maize grain development. Figure 2 (NO in the text).

[0073] Example 3: Differentially expressed genes in the seeds of mutant Zmski3-1 were significantly enriched in starch synthesis and energy metabolism pathways.

[0074] To further elucidate the activated or inhibited molecular pathways in Zmski3-1 mutant seeds, seeds containing the Zmski3-1 mutant were sown and self-pollinated. Transcriptome analysis was performed on Zmski3-1 mutant seeds and wild-type seeds 18 days after pollination. The results showed that 943 genes were significantly upregulated and 929 genes were significantly downregulated in Zmski3-1 mutant seeds. Figure 3 GO analysis of these upregulated and downregulated genes showed that the upregulated genes were mainly enriched in molecular pathways of energy metabolism. Figure 4 This indicates that mutations in the ZmSKI3 gene may affect maize kernel development at the molecular level by regulating the metabolism of matter and energy. Subsequent analysis of differentially expressed genes in the mutant Zmski3-1 revealed that some important genes affecting starch synthesis and metabolism, such as AMY1 and AMY3, were downregulated in the mutant kernels, further confirming the important role of the ZmSKI3 gene in maize kernel development. Figure 5 ).

[0075] Example 4: Interaction between maize ZmSKI3 and ZmSKI2 proteins

[0076] As another subunit of the SKIs cofactor, the inventors wanted to know if the ZmSKI2 protein also had a function in regulating maize kernel development. Therefore, they identified the maize ZmSKI2 protein. First, through protein sequence alignment (using the Muscle algorithm in MEGAX), the inventors obtained the coding gene for the maize ZmSKI2 protein, Zm00001d015314, named the ZmSKI2 gene. The specific nucleotide sequence of the ZmSKI2 gene is: ATGTCCATGGACGGCCCTGCCACCTCGCCGGCAAGCGAGGTGCCGTTCCGCATCAGCTTCTCCGGCCACAGCGGCCACCTCCGCCTCGACCCTACCCCGCACACGCCCAGCCCCATTCCGGACTTC GTCCTGCCGCCGGCATACCCGGCCGAGAGCCCGAGCAGCGTGAAGGAGTACCTCGAGAGGAACTACCTCGACCCCGAGCTGCACCTCCCCACCGCGGCCGATAGCGGGAGGGTGTGGGATGTCGACTGGTTTGCCCTGGCCAGGCCGCCGCTGGAGCCCTCCGCCCCCCGCACCATGCTCGCGCCCGTCTGGGTGCCGCCTTTCCGGCGCGGGCAGGAGAAGTTG

[0077] CAATCCGCGGCAGAGTCGCGAGTGTGGGACCCTGAGTCCGTGCAAATGGAGATGGTCG

[0078] ACGTGTTCGATTCGGGGACCGGGGGGATAGCGCCCCGGATGCCTGGTCCGGCGAAGGA

[0079] CTTCGTCAGGGGGAGCATCAACAACAGACCTTTTCGTCCAGGCGGTCTGCAGGATGAC

[0080] GCCGCTGAGGCGGCTGCGCTGGAAAAGGCGTTCCCAGAGGGTGCAAGGACTGGTGATT

[0081] GGGTGCGTGAGCTCATGAGCGGTGGCCCGGCGCAGGTTGCGCCTCCAGGGTTCCGTAA

[0082] GGGATTGGAGCTGGGGCCAGTTGAAGGGGTATGAAAGCCACTGGAAGTGTTTCCGGGAT

[0083] GGAGAACTTGTAGGGAGCAACCTGCATCATCATCGAATGACACAATGGAGAAGTACTC

[0084] TGTGCAGTTTGATGATCTTTTCAAGATAGCGTGGGAGGAAGATACTGCCAACAAGTTGT

[0085] TGAAGGACGGTGTTGTTCAACAATCTGCTGAAGGTGAAGGAATCAATGAAATTGGCGA

[0086] ACAAAAAGTTGATGCATTGCAGGATGAGTTCGAGAGTATAACAACGCTAGATGACGAGA

[0087] AACAGGAAGTTGATGTCATAAGAAATGTTCCTGAAACTCAAACAGACTTGGATCAGATG

[0088] TTATCTTCTGAAGTACAGGATACAGGCAGGGAACCAGGTGCATCAGGTGATAAGAAGCC

[0089] AACACAAGATGGCATGGTTTGGGCACTTGTTGGTGGGGACGAGGACATAGTGACTAACT

[0090] TCTCCAAACTCGTTCCAGATATGGCAATCGAGTTTCCATTTGAAATTGGATAAGTTCCAGA

[0091] AGGAGGCTATATATTATCTCGAGAAGGGTGAATCAGTCTTTTGTTGCAGCCCATACTTCAG

[0092] CTGGAAAGACGGTTGTTGCTGAGTATGCATTCGCATTAGCAACGAAACATTGCACTAGG

[0093] TCTGTCTATACTGCTCCTATTAAACTATCAGCAACCAGAAATACAGAGATTTTTCTGGG

[0094] AAGTTTGATGTGGGACTTCTGACAGGGAGATGTTAGCATCAGGCCAGAGGGCAACTTGCTT

[0095] AATTATGACTACTGAGATATTGCGTTCAATGCTCTACAGAGGCGCAGACATTATACGTGAT

[0096] ATTGAATGGGTAATCTTTGATGAAGTGCATTATGTAAATGATGCTGAAAGAGGTGTAGTC

[0097] TGGGAGGAGGTCATTATAATGCTCCCGAAGCACATTAACATTGTTCTTCTTTCGGCAACG

[0098] GTCCCAAATACTGTTGAATTTGCTGACTGGATTGGTCGGACAAAGCAGAAGAAAATTCG

[0099] TGTCACATCGACCAACAAAAGGCCTGTTCCACTTGAGCATTGCCTGTTCTACTCTGGAG

[0100] AAGTGTACAAAATATGTGAGAGGGATATGTTTCTTGCTCAAGGATTTAAAGAAGCAAAA

[0101] GATGCTTTCAAAAAAGAAAAATTTGAATAAGTTTGGAGTGAAACCTGGTTCAAAGTCAGG

[0102] AACCCTGCAGTACGTGCTGGAACTCAAGGCAAAAATCCAGATACATCCAACAAGGGG

[0103] AGAGATCAAGTACCCAAAGCACCGCAATTCCAATTCAGGAGTAGCCACAGTTCAAC

[0104] AGAGCTCCTCAGGGCCAAAGAGATTTGAATCTTTATTTTGGATGCCACTTGTGAATAACC

[0105] TTCTGAAGAAATCCCTTGTGCCTGTGGTGATTTTTTGTTTCTCAAAAGAATCGCTGTGATA

[0106] AATCGGCAGATAGTATGTTTGGCACTGATCTCACCAGTAGTTCAGAGAAAAGTGAAATA

[0107] CGTGTCTTCTGTGACAAGGCATTTTCACGTCTTAAAGGATCTGATAGGAACCTTCCCACAG

[0108] GTTGTAGGAATACAAAGCCTTCTGCGAAGAGGAATTGGAGTACACCACGCTGGGCTTCT

[0109] CCCTATTGTGAAGGAAGTTGTTGAGATGCTGTTTTGCCGTGGTGTAATCAAGGTACTGTT

[0110] TTCCACTGAGACATTTGCAATGGGTGTCAATGCACCGGCAAGAACGGATTATGAAGCTA

[0111] GCTCTGAGCCTATCTCAGTTTTGACCCAACAGGTTGTGTTTGATTCTTTAAGAAAGTTTG

[0112] ATGGAAAGAACACCGGAAATTGCTTCCAGGGGAATATATACAAATGGCTGGCGAGCT

[0113] GGTCGGGAGGACTTGATAACATTGGTACTGTGATCATTATTGTGTCGTGATGAAATTCCT

[0114] GAAGAAAGCGATTTGAAAAATTTGATCGTTGGAAACCAACTCGTTTGGAATCTCAATT

[0115] TCGATTAACATACACCATGATACTACATCTTCTGCGTGTGGAGGAACTGAAGGTCGAGGA

[0116] CATGCTCAAGAGAAGTTTTGCTGAATTCCACGCACAAAAGAATTTGCCTGAGAAGGAA

[0117] AAGCTTCTCTGCAAATGCTTCGTCAACCTACAAGGACAATAGAGTGCATAAAAGGAGA

[0118] GCCTTCTATTGAGGAATACTACGAGATGACTTTAGATGCTGAGGCACACAGGGAATACAT

[0119] AACAGAAGCAATTATGCAGCTGCCTAATTCTCAACAGTTTCTTACGCCTGGGAGATTGGT

[0120] GGTTGTTAAATCTGATTCTGATGATGATCACTTGCTTTGGTGTTATACTGAAAAATCCATCT

[0121] GCATTGCTAAAGAAATATGTTGTTCTGGTATTGACTGGTGATTGCAGTTCATCTGCACTAG

[0122] CCCCCTGAGTTCAATAAAAATGAAAAGGGTCCTGTGGATTTTCAAGGAGGACAATTTATT

[0123] GTCCTGAAAGGAAAACGTGGCATGGACGATGAATATTTCTCTTCTGTTAGTTCACGAAA

[0124] AGCTTCAGGTGTAATCAATATCAATCTACCATACAAGGGGGATGCATCTGGAATGGGCTT

[0125] TGAAGTAAGAGCAATTGAGAATAAAGAAATCATTAGTATATGCAGCAGCAAAATAAAGA

[0126] TTGATCAAGTCAGACTTCTTGAGGAGCCTAACAAAACTGCATACTCTAGAACTGTCCAA

[0127] CAGCTTATAAAGGAGCAACCAGATGGAACCAAGTATCCCTCTGCTTTAGATGCAATAAA

[0128] AGATCTAAAAATGAAAGACATGTATCTTGTTGAAAGTTACCGTGCATATCACATACTACT

[0129] GCAAAAAATGTCTGAAAACAAGTGCCATGGTTGTATAAAACTGAAGGAGCATATATCATT

[0130] GATGAGGGAGCAAAAGATGTACAAGGATCAGTTGAATGAATTGAAATTCCAAATGTCCG

[0131] ACGAGGCACTTCAACAAATGCCAGAGTTTCAAGGCAGAATTGATGTACTAAAGGTAATC

[0132] CACTACATTGATTCTGATCTAGTTGTGCAACTTAAGGGTCGGGTAGCATGTGAAATGAAC

[0133] TCCGGTGAGGAGTTAATATCAACAGAATGTCTGTTTGAAAATCAATTGGATGACCTAGAA

[0134] CCCGAAGAAGCTGTGGCTATTATGTCTGCATTCGTCTTCCAACAACGCAATGCTTCAGAA

[0135] CCATCTCTTACTCCAAAACTGGCTGAAGCGAAGAAGAGGCTCTATGATACAGCCATAAA

[0136] ATTAGGGAAGCTCCAATCCGAGTTCAAGGTGCCTGTGGACCCTGAAGAGTATGCACGTG

[0137] ATAATCTCAAGTTTGGCCTTGTTGAGGTCGTCTACGAGTGGGCAAAGGGGACGCCTTTC

[0138] GCAGACATATGCGAGCTGACTGATGTATCCGAAGGGATCATTGTAAGAACAATCGTCCGT

[0139] CTGGACGAAACATGTAGGGAATTCAGGAATGCAGCTTCCATCATGGGGAACTCTGCGCT

[0140] GTTCAAGAAGATGGAGGTCGCGTCTAACGCTATTAAGCGTGACATTGTGTTTGCAGCAAGTTTGTATGTCACAGGAATCTGA (SEQ ID NO.2), and a phylogenetic tree was constructed using MEGAX, such as Figure 6 As shown in A in the diagram. Further, using a yeast two-hybrid experiment, the specific steps were as follows: the full-length CDS sequence of the ZmSKI3 gene (as shown in SEQ ID NO.1) was ligated into the pGBKT7 vector to form BD-ZmSKI3, abbreviated as BD-SKI3; the full-length CDS sequence of the ZmSKI2 gene (as shown in SEQ ID NO.2) was ligated into the pGADT7 vector to form AD-ZmSKI2, abbreviated as AD-SKI2. Both were co-transformed into yeast strain Y2H Gold and plated on growth medium SD / -Leu / -Trp (SD / -LT) and selection medium SD / -Leu / -Trp / -His / -Ade (SD / -LTHA) supplemented with X-α-Gal. BD-53+AD-T and BD--Lam+AD-T were used as positive and negative controls, respectively. Positive transformants grown on SD / -LT medium were serially diluted and spotted onto SD / -LT and SD / -LTHA, and incubated in the dark at 28°C for 4 days before photographing. The inventors discovered an interaction between the ZmSKI3 and ZmSKI2 proteins. Figure 6 (B) The luciferase complementation experiment was conducted as follows: the ZmSKI3 coding sequence containing all TPR domains, ZmSKI3(72-465, i.e., positions 72-465 as shown in SEQ ID NO. 1, were ligated to the vector) was linked to the p1305-nLUC vector to form ZmSKI3(72-465)-nLUC; the full-length CDS sequence of ZmSKI2 was ligated to the p1305-cLUC vector to form cLUC-ZmSKI2. This was then transformed into Agrobacterium GV3101 strain and co-injected into tobacco leaves. nLUC / cLUC, ZmSKI3(72-465)-nLUC / cLUC, and nLUC / cLUC-ZmSKI2 served as negative controls. Forty-eight hours after injection, D-luciferin potassium salt was applied, and the mixture was photographed using a plant imaging system. This further confirmed the interaction between the ZmSKI3 and ZmSKI2 proteins. Figure 6 (CD in the middle).

[0141] The above results indicate that the maize ZmSKI2 and ZmSKI3 proteins interact, suggesting that they may play an important role in regulating maize kernel development.

[0142] Example 5: The ZmSKI2 gene in maize controls the development of maize kernels.

[0143] To investigate the function of the ZmSKI2 gene in maize kernel development, two mutants with premature termination of the ZmSKI2 protein were obtained by screening the EMS mutant library: Zmski2-1 (compared to the wild type, the ZmSKI3 gene in this mutant has a G-to-A mutation at SEQ ID NO. 243, specifically: ATGTCCATGGACGGCCCTGCCACCTCGCCGGCAAGCGAGGTGCCGTTCCGCATCAGCTTCTCCGGCCACAGCGGCCACCTCCGCCTCGACCCTACCCCGCACACGCCCAGCCCCATTCCGGACTTCGTCCTGCCGCCGGCATACCCGGCCGAGAGCCCGAGCAGCGTGAAGGAGTACCTCGAGAGGAACTACCTCGACCCCGAGCTGCACCTCCCCACCGCGGCCGATAGCGGGAGGGTGTGA, SEQ ID NO. 5) and Zmski2-2 (compared to the wild type, the ZmSKI3 gene in this mutant has a G-to-A mutation at SEQ ID NO. 243, specifically: ATGTCCATGGACGGCCCTGCCACCAGAGCCCGAGCAGCGTGAAGGAGTACCTCGAGAGGAACTACCTCGACCCCGAGCTGCACCTCCCCACCGCGGCCGATAGCGGGAGGGTGTGA, SEQ ID NO. 5).At the 937th base of 2, a C to T mutation occurred, specifically: ATGTCCATGGACGGCCCTGCCACCTCGCCGGCAAGCGAGGTGCCGTTCCGCATCAGCTTCTCCGGCCACAGCGGCCACCTCCGCCTCGACCCTACCCCGCACACGCCCAGCCCCATTC CGGACTTCGTCCTGCCGCCGGCATACCCGGCCGAGAGCCCGAGCAGCGTGAAGGAGTACCTCGAGAGGAACTACCTCGACCCCGAGCTGCACCTCCCCACCGCGGCCGATAGCGGGAGGGTGTGGGATGTCGACTGGTTTGCCCTGGCCAGGCCGCCGCTGGAGCCCTCCGCCCCCCGCACCATGCTCGCGCCCGTCTGGGTGCCGCCTTTCCGGCGCGGGCAGGAGAAGTTGCAATCCGCGGCAGAGTCGCGAGTGTGGGACCCTGAGTCCGTGCAAATGGAGATGGTCGACGTGTTCGATTCGGGGACCGGGGGGATAGCGCCCCGGATGCCTGGTCCGGCGAAGGACTTCGTCAGGGGGAGCATCAACAACAGACCTTTTCGTCCAGGCGGTCTGCAGGATGACGCCGCTGAGGCGGCTGCGCTGGAAAAGGCGTTCCCAGAGGGTGCAAGGACTGGTGATTGGGTGCGTGAGCTCATGAGCGGTGGCCCGGCGCAGGTTGCGCCTCCAGGGTTCCGTAAGGGATTGGAGCTGGGCCAGTTGAAGGGGTATGAAAGCCACTGGAAGTGTTTCCGGGATGGAGAACTTGTAGAGGAGCAACCTGCATCATCATCGAATGACACAATGGAGAAGTACTCTGTGCAGTTTGATGATCTTTTCAAGATAGCGTGGGAGGAAGATACTGCCAACAAGTTGTTGAAGGACGGTGTTGTTCAACAATCTGCTGAAGGTGAAGGAATCAATGAAATTGGCGAACAAAAAGTTGATGCATTGCAGGATGAGTTCGAGAGTATAACAACGCTAGATGACGAGAAATAG, SEQ ID NO.6)(. Figure 7(A) and purchased mutant seeds. After sowing and self-pollination, the inventors found randomly distributed defective kernels on the ears of self-pollinated Zmski2-1 / + heterozygotes and Zmski2-2 / + heterozygotes, indicating that the ZmSKI2 gene is very likely to regulate the kernel development process of maize. Figure 7 (B in the original text). Simultaneously, hybridization was conducted after sowing, and allelic analysis confirmed that the mutation in the ZmSKI2 gene was the cause of grain development defects. Figure 7 (B in the original text). Further analysis after the corn kernels matured revealed that the mutant kernels had varying degrees of reduction in kernel length, width, thickness, and 100-kernel weight compared to the wild type. Figure 7 CF in Figure 7 (GJ in the middle).

[0144] In summary, the mutations in the ZmSKI2 and ZmSKI3 genes discovered in this invention lead to defects in maize kernel development, providing a new gene resource for regulating maize kernel development. In the future, by manipulating these genes, new germplasm with improved kernel development can be obtained and promoted for application in production.

[0145] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. ZmSKI3 Gene or ZmSKI2 The application of genes in regulating maize kernel development is characterized by, The ZmSKI3 The nucleotide sequence of the gene is shown in SEQ ID NO.1; ZmSKI2 The nucleotide sequence of the gene is shown in SEQ ID NO.2; By inhibiting the corn's internal... ZmSKI3 Gene or ZmSKI2 Gene expression reduces the length, width, thickness, and weight of 100 corn kernels.

2. ZmSKI3 Gene or ZmSKI2 The application of genes in the breeding of transgenic maize with reduced kernel length, width, thickness, and 100-kernel weight is characterized by, The ZmSKI3 The nucleotide sequence of the gene is shown in SEQ ID NO.1; ZmSKI2 The nucleotide sequence of the gene is shown in SEQ ID NO.2; By inhibiting the corn's internal... ZmSKI3 Gene or ZmSKI2 Gene expression reduces the length, width, thickness, and weight of 100 corn kernels.

3. A method for cultivating corn with reduced kernel length, width, thickness, and 100-kernel weight, characterized in that, By inhibiting the corn's internal... ZmSKI3 Gene or ZmSKI2 Gene expression was delayed, resulting in delayed kernel development in the transgenic maize. ZmSKI3 The nucleotide sequence of the gene is shown in SEQ ID NO.1; ZmSKI2 The nucleotide sequence of the gene is shown in SEQ ID NO.2.

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