Application of Maize ZmEREB131 Gene in Regulating Maize Growth

Abstract

The present application relates to the field of genetic engineering, and more specifically to the use of the ZmEREB131 gene in regulating maize growth and maize breeding methods utilizing the same. The ZmEREB131 gene comprises a polynucleotide sequence as set forth in SEQ ID NO: 1 and / or SEQ ID NO: 2; and / or a polynucleotide sequence encoding an amino acid sequence as set forth in SEQ ID NO: 3. The gene is capable of (i) regulating maize flowering time; and / or (ii) improving at least one of maize ear length, kernel number per ear, and kernel size.

Description

Technical Field

[0001] The present application relates to the field of genetic engineering, and specifically to the application of the ZmEREB131 gene in regulating corn growth and a corn breeding method using the same. Background Art

[0002] Maize, a core crop, exhibits significant regional imbalance in its cultivation. Maize distribution in China is primarily concentrated in Northeast China, North China, and Southwest China, forming a long, oblique maize-growing belt stretching from Northeast China to Southwest China. Under these diverse environmental conditions, adjusting flowering time can influence maize's regional adaptability and stress tolerance. Adjusting flowering time not only significantly enhances maize's regional adaptability to diverse ecological environments, thereby improving its yield stability and stress tolerance, but also influences multiple key traits such as yield, quality, and nutritional value, making it a crucial parameter in maize breeding and production. Maize ear length is another crucial trait. Ear length is directly related to the number and quality of maize kernels. Longer ear length generally means more kernels, which significantly contributes to improving maize yield and production efficiency. Although some studies have examined genes regulating maize flowering time and ear length, their number is far less than that of the model plant Arabidopsis thaliana and the important monocot rice, and the establishment of their regulatory networks is relatively underdeveloped.

[0003] Given the important effects of flowering time and ear length on corn growth and yield, it is necessary to further develop and research genes that can effectively regulate these traits and breeding methods that utilize them. Summary of the Invention

[0004] The present invention aims to solve one of the technical problems in the related art at least to a certain extent.

[0005] To this end, an embodiment of the first aspect of the present invention provides a use of a nucleic acid in regulating corn growth, so as to effectively regulate corn flowering time, promote female ear development, optimize female ear structure, increase the number of ears and kernels per ear, and thus improve corn yield through genetic engineering technology. The nucleic acid is used to (i) regulate corn flowering time; and / or (ii) improve at least one of corn ear length, kernel number per ear, and kernel size; wherein the nucleic acid:

[0006] a. having a polynucleotide sequence as shown in SEQ ID NO: 1 and / or SEQ ID NO: 2;

[0007] b. a polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 3;

[0008] c. A polynucleotide sequence capable of hybridizing to the polynucleotide sequence described in a or b under stringent hybridization conditions, which still has the function of (i) regulating corn flowering period; and / or (ii) improving at least one of corn ear length, number of kernels per ear, and kernel size; or

[0009] d. A polynucleotide sequence that is at least 90%, at least 95% or at least 99% identical to the polynucleotide sequence described in any one of ac, which still has the function of (i) regulating the flowering period of corn; and / or (ii) improving at least one of the length of corn ears, the number of kernels per ear, and the size of kernels per ear.

[0010] In some embodiments, corn growth is regulated by overexpressing and / or inhibiting expression of the nucleic acid.

[0011] In some embodiments, the nucleic acid is the ZmEREB131 gene, whose gene ID is Zm00001d003884.

[0012] An embodiment of the second aspect of the present invention provides the use of a protein in promoting early maturity of corn, wherein the protein is used to (i) regulate the flowering period of corn; and / or (ii) improve at least one of corn ear length, number of kernels per ear, and kernel size, wherein the protein:

[0013] a. having an amino acid sequence as shown in SEQ ID NO: 3, or

[0014] b. An amino acid sequence that is at least 90%, 95% or 99% identical to the amino acid sequence shown in SEQ ID NO: 3, which has the function of (i) regulating the flowering period of corn; and / or (ii) improving at least one of corn ear length, number of kernels per ear, and kernel size per ear.

[0015] An embodiment of the third aspect of the present invention provides an expression cassette, a recombinant expression vector or a recombinant cell, wherein the expression cassette or the recombinant expression vector comprises a targeting sequence, and the targeting sequence targets the nucleic acid as defined in the embodiment of the first aspect of the present invention to inhibit its expression, wherein the expression of the nucleic acid as defined in the embodiment of the first aspect of the present invention in the recombinant cell is inhibited.

[0016] In some embodiments, the expression cassette, recombinant expression vector or recombinant cell is a recombinant eukaryotic expression cassette, a recombinant eukaryotic expression vector or a recombinant plant cell, respectively; preferably, the recombinant eukaryotic expression cassette or recombinant eukaryotic expression vector is a recombinant plant expression cassette or a recombinant plant expression vector.

[0017] In some embodiments, the expression cassette, recombinant expression vector or recombinant cell is used to (i) advance or delay the flowering period of corn; and / or (ii) increase at least one of corn ear length, kernel number, and kernel size.

[0018] An embodiment of the fourth aspect of the present invention proposes a corn breeding method, characterized in that the method includes: reducing the expression level of the nucleic acid as defined in the embodiment of the first aspect of the present invention or the protein as defined in the embodiment of the second aspect of the present invention in corn and / or reducing the activity of the protein to promote early maturity of corn and improve corn yield.

[0019] In some embodiments, the method comprises: (i) advancing the flowering period of corn; and / or (ii) increasing at least one of ear length, kernel number, and kernel size of corn.

[0020] In some embodiments, the nucleic acid sequence in corn as defined in the embodiments of the first aspect of the invention is mutated by CRISPR / Cas9 technology to reduce the expression level of the nucleic acid as defined in the embodiments of the first aspect of the invention or the protein as defined in the embodiments of the second aspect of the invention in corn and / or reduce the activity of the protein.

[0021] In some embodiments, the mutating of the nucleic acid sequence in corn as defined in the embodiments of the first aspect of the present invention by CRISPR / Cas9 technology comprises:

[0022] The nucleic acid sequence defined in the embodiment of the first aspect of the present invention is mutated in corn by introducing a vector comprising a targeting sequence into corn,

[0023] The targeting sequence targets the nucleic acid as defined in the embodiment of the first aspect of the present invention to inhibit its expression, and the targeting sequence is the sgRNA coding sequence shown in SEQ ID NO: 4.

[0024] In some embodiments, the corn mutant obtained by the method has the nucleic acid sequence defined in the embodiment of the first aspect of the present invention compared to the wild-type corn.

[0025] (i) the CG bases at positions 294-298 are deleted; or

[0026] (ii) The sequence shown in SEQ ID NO: 5 at positions 241-299 was deleted.

[0027] The advantages and technical effects brought about by the independent claims according to the embodiments of the present invention are:

[0028] (1) By genetic engineering technology, the expression of the ethylene signal response factor ZmEREB131 provided in the embodiment of the present invention is inhibited in corn, which can promote early flowering of corn under suitable conditions; overexpression of ZmEREB131 in corn can delay corn flowering under suitable conditions, thereby achieving the technical effect of effectively regulating the flowering time of corn.

[0029] (2) By using genetic engineering technology, the expression of ZmEREB131 provided by the embodiments of the present invention is inhibited in corn, which not only promotes the development of female ears, increases the number of ears and the number of grains per ear, thereby improving yield, but also optimizes the structure of the female ears, increases the ear length, makes the corn more adaptable to the corresponding environmental conditions, and improves the fruit set rate.

[0030] (3) The ZmEREB131 provided in the embodiment of the present invention provides a new natural variation site and its application in regulating corn growth, which can efficiently, conveniently and economically achieve the regulation of corn growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of the ZmEREB131 overexpression vector according to the embodiment of the present application;

[0032] Figure 2 Shown is an agarose gel electrophoresis diagram of PCR products of ZmEREB1311 overexpression strains OE1 and OE2 according to an embodiment of the present application;

[0033] Figure 3 The relative expression levels of ZmEREB131 in the ZmEREB131 overexpression strains OE1 and OE2 according to the examples of the present application are shown;

[0034] Figure 4 Shown is a schematic diagram of the knockout sites of the ZmEREB131 mutant strains zmereb131-1 and zmereb131-2 according to the examples of the present application;

[0035] Figure 5 Schematic diagram showing the sequencing results of ZmEREB131 mutant strains zmereb131-1 and zmereb131-2 according to the examples of the present application;

[0036] Figure 6 The flowering time statistics of the wild type ND101, the ZmEREB131 mutant strain zmereb131-1, and the overexpression strains OE1 and OE2 according to the examples of the present application are shown;

[0037] Figure 7 The tassel differentiation of the wild-type ND101, ZmEREB131 mutant strains zmereb131-1 and zmereb131-2 at the V7, V9, and V11 stages according to the examples of the present application is shown;

[0038] Figure 8 The figures show the development of female ears at the V11 stage of the wild-type ND101 and the ZmEREB131 mutant strains zmereb131-1 and zmereb131-2 according to the examples of the present application;

[0039] Figure 9 The ear and grain phenotypes of the wild-type ND101 and the ZmEREB131 mutant strains zmereb131-1 and zmereb131-2 according to the examples of the present application are shown. DETAILED DESCRIPTION

[0040] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0041] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. All reagents or instruments used without specifying the manufacturer are commercially available conventional products.

[0042] This application is made based on the following knowledge of the inventors:

[0043] Corn cultivation in China is unevenly distributed across regions, primarily concentrated in Northeast China, North China, and Southwest China, forming a long, oblique corn-growing belt stretching from Northeast China to Southwest China. Corn adapts to different environmental conditions by adjusting its flowering time, and the regulation of flowering time has long been an active area of ​​research. Flowering time not only affects corn's adaptability to growing regions and its resistance to various stresses, but also has a significant impact on multiple traits, including yield, quality, and nutritional value. Flowering time is influenced by complex factors, including both internal genomic genetics and external environmental factors. By studying and analyzing the flowering time of corn varieties under different environmental conditions, we can better understand and utilize flowering time traits, thereby selecting superior varieties that are more adaptable to different environments. Furthermore, by accurately measuring and comparing flowering time, breeders can understand the genetic differences between varieties and provide important insights for selective breeding. Therefore, in-depth research and utilization of flowering time can not only help corn varieties adapt to different growing environments but also provide important guidance for corn breeding. Research on flowering time can not only improve corn yield and quality but also accelerate breeding processes, thereby better meeting agricultural production needs and promoting sustainable agricultural development.

[0044] Corn ear length, as a key trait in corn, plays a key role in corn breeding and production. Yield impact: Corn ear length directly affects the quantity and quality of corn kernels. Longer ear length generally means more kernels, which helps increase corn yield and production efficiency. In corn breeding, ear length is often an important selection criterion. By selecting superior varieties with longer ear lengths for subsequent generations, corn ear length performance can be gradually improved. The variability in corn ear length provides breeders with greater options to develop corn varieties that better meet market demand, such as large-eared corn varieties suitable for mechanized harvesting. In summary, corn ear length, as a key corn trait, is of great significance in increasing yield, adapting to the environment, optimizing breeding selection, and meeting market demand. Therefore, the study and utilization of corn ear length is crucial in corn breeding and production.

[0045] While some studies have examined genes regulating flowering time and ear length in maize, the number is far less than that of the model plant Arabidopsis and the important monocot rice, and the establishment of their regulatory networks is relatively underdeveloped. Given the crucial impact of flowering time and ear length on maize growth and yield, the development of genes and breeding methods that can effectively regulate these traits is crucial.

[0046] To this end, the inventors of this application, after extensive experiments, screened and located a gene in maize that is involved in regulating ear length gain and flowering time. This gene is located at Chr2:65020854..65025676 in the maize reference genome Zm-B73-REFERENCE-GRAMENE-4.0. The gene ID in the B73 genome is Zm00001d003884 (https: / / maizegdb.org). This application names it ZmEREB131, and its sequence is shown in SEQ ID NO: 1. The examples of this application further disclose methods for manipulating ZmEREB131 using genetic engineering techniques to (i) regulate maize flowering time; and / or (ii) increase at least one of maize ear length, kernel number per ear, or kernel size.

[0047] In the examples of the present application, unless otherwise indicated, nucleic acids are written from left to right in the 5' to 3' direction; amino acid sequences are written from left to right in the amino to carboxyl direction. Amino acids can be represented herein by their commonly known three-letter symbols or the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Similarly, nucleotides can be represented by commonly accepted single-letter codes. Numerical ranges include numbers that define the ranges. As used herein, "nucleic acid" includes deoxyribonucleotides or ribonucleotide polymers in single-stranded or double-stranded form, and unless otherwise limited, includes known analogs (e.g., peptide nucleic acids) having the basic properties of natural nucleotides, which hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides. As used herein, the term "encoding" or "encoded" when used in the context of a specific nucleic acid refers to the nucleic acid containing the necessary information to guide the translation of the nucleotide sequence into a specific protein. Codons are used to represent protein-encoding information. As used herein, the "full-length sequence" of a specific polynucleotide or its encoded protein refers to the entire nucleic acid sequence or the entire amino acid sequence having a natural (non-synthetic) endogenous sequence. The full-length polynucleotide encodes the full-length, catalytically active form of the specific protein. The terms "polypeptide," "polypeptide," and "protein" are used interchangeably herein to refer to polymers of amino acid residues. The term is used for amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids. The term is also used for naturally occurring amino acid polymers. The terms "residue" or "amino acid residue" or "amino acid" are used interchangeably herein to refer to an amino acid that is incorporated into a protein, polypeptide, or peptide (collectively, "protein"). Amino acids can be naturally occurring amino acids and, unless otherwise limited, can include known analogs of naturally occurring amino acids that can function in a manner similar to naturally occurring amino acids.

[0048] In the present application embodiment, the term " identity percentage " about nucleic acid or peptide sequence is defined as after arranging sequence to obtain maximum identity percentage and introducing breach (if necessary) to realize maximum homology percentage, the nucleotide identical with known polypeptide or the percentage of amino acid residue in candidate sequence.N-terminal or C-terminal insertion or deletion should not be interpreted as affecting homology.Homology or identity on nucleotide or amino acid sequence level can be determined by BLAST (basic local comparison search tool, Basic Local Alignment Search Tool) analysis, described analysis uses the algorithm (Altschul (1997) adopted by program blastp, blastn, blastx, tblastn and tblastx, Nucleic Acids Res [nucleic acids research] 25,3389-3402 and Karlin (1990), Proc.Natl.Acad.Sci.USA [U.S. National Academy of Sciences journal] 87,2264-2268), described program is customized for sequence similarity search.

[0049] In the embodiments of the present application, the nucleic acid has a polynucleotide sequence as shown in any one of SEQ ID NOs: 1-3, or a sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, or 80% identical to any one of SEQ ID NOs: 1-3 (including endpoint values), wherein the percentage of identity generally describes the degree to which the two sequences are identical, that is, it generally describes the percentage of nucleotides that correspond to the same nucleotides in the reference sequence at their sequence positions. In some embodiments, the nucleic acid has a polynucleotide sequence as shown in any one of SEQ ID NOs: 1-3, or a sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, or 80% identical to any one of SEQ ID NOs: 1-3 (including endpoint values), wherein the percentage of identity generally describes the degree to which the two sequences are identical, that is, it generally describes the percentage of nucleotides that correspond to the same nucleotides in the reference sequence at their sequence positions. At least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, or at least 99%, at least 99.1% compared to any one of NOs. 1-3. , at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.91%, at least 99.92%, at least 99.93%, at least 99.94%, at least 99.95%, at least 99.96%, at least 99.97%, at least 99.98%, at least 99.99% or any value therebetween (e.g., sequence identity represented by an infinite number of decimals between two adjacent integers) but less than 100% identical nucleotide sequence, wherein the nucleic acid has one or more single nucleotide mutations compared to any one of SEQ ID NOs: 1-3, and the mutant nucleic acid still has the function of (i) regulating corn kernel moisture content and / or dehydration rate; and / or (ii) regulating corn flowering time.

[0050] In the embodiments of the present application, the protein has the amino acid sequence shown in SEQ ID NO: 4, or a sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, or 80% identical to SEQ ID NO: 4 (including endpoint values). In some embodiments, the protein is identical to SEQ ID NO: %, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, or at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.9%, or ... .4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.91%, at least 99.92%, at least 99.93%, at least 99.94%, at least 99.95%, at least 99.96%, at least 99.97%, at least 99.98%, at least 99.99% or any value therebetween (e.g., sequence identity represented by an infinite number of decimals between two adjacent integers) but less than 100%, said mutant protein still has the function of (i) regulating the moisture content and / or dehydration rate of corn kernels; and / or (ii) regulating the flowering time of corn. In some embodiments, unless otherwise specified, except for the specified mutations, the remaining amino acids of the protein are identical to those of SEQ ID NO: 4 or are conservative substitutions corresponding to the amino acids of SEQ ID NO: 4.

[0051] In the present application embodiment, the term "stringent conditions" or "stringent hybridization conditions" means conditions under which, relative to hybridization with other sequences, the probe will hybridize to its target sequence to a detectable greater extent (e.g., at least 2 times, 5 times, or 10 times the background). Stringent conditions are sequence-dependent and vary in different environments. By controlling hybridization stringency and / or controlling cleaning conditions, a target sequence that is 100% complementary to the probe can be identified (homologous probe method). Alternatively, stringent conditions can be adjusted to allow some sequence mispairings to detect lower similarity (heterologous probe method). Typically, the probe length is less than approximately 1000 or 500 nucleotides. Typically, stringent conditions are those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 M to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions can also be achieved by adding destabilizing agents such as formamide.

[0052] The first embodiment of the present invention provides a nucleic acid for use in regulating corn growth, thereby effectively regulating corn flowering time, promoting ear development, optimizing ear structure, increasing ear number and kernel number per ear, and thus improving corn yield through genetic engineering technology. The nucleic acid is used to (i) regulate corn flowering time; and / or (ii) improve at least one of corn ear length, kernel number per ear, and kernel size; wherein the nucleic acid:

[0053] a. having a polynucleotide sequence as shown in SEQ ID NO: 1 and / or SEQ ID NO: 2;

[0054] b. a polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 3;

[0055] c. A polynucleotide sequence capable of hybridizing to the polynucleotide sequence described in a or b under stringent hybridization conditions, which still has the function of (i) regulating corn flowering period; and / or (ii) improving at least one of corn ear length, number of kernels per ear, and kernel size; or

[0056] d. A polynucleotide sequence that is at least 90%, at least 95% or at least 99% identical to the polynucleotide sequence described in any one of ac, which still has the function of (i) regulating the flowering period of corn; and / or (ii) improving at least one of the length of corn ears, the number of kernels per ear, and the size of kernels per ear.

[0057]

[0058]

[0059] Among them, the amino acid sequence shown in SEQ ID NO: 3 is the amino acid sequence of the protein encoded by the ZmEREB131 gene (Zm00001d003884) provided in the present application, specifically as follows: MSSAPRPTNSRRSRPPACTAPPARRSCGRERETIRNRKRLRTTIPVATSPPRRSKDAAIAYDEAALRFKGTKAKLNFPERVQGRTDLGFVVTRGIPDHHRHPRAAAVNLAAMPQAQAQPHLQHGRPTVMPYPYPYPDLMQYAQLLQGGRGGGDHAAAVQQQLMMMGGRGGNLPFSFSPPSSWSAPPQILDFSARQLITQPGPPSSPAAPGGAAPSTPSSTTTASSPSASASGSAWPYGGEHHRNKKDA (SEQID NO: 3).

[0060] In some embodiments, corn growth is regulated by overexpressing and / or inhibiting expression of the nucleic acid.

[0061] In some embodiments, the nucleic acid is the ZmEREB131 gene, whose gene ID is Zm00001d003884.

[0062] An embodiment of the second aspect of the present invention provides the use of a protein in promoting early maturity of corn, wherein the protein is used to (i) regulate the flowering period of corn; and / or (ii) improve at least one of corn ear length, number of kernels per ear, and kernel size, wherein the protein:

[0063] a. having an amino acid sequence as shown in SEQ ID NO: 3, or

[0064] b. An amino acid sequence that is at least 90%, 95% or 99% identical to the amino acid sequence shown in SEQ ID NO: 3, which has the function of (i) regulating the flowering period of corn; and / or (ii) improving at least one of corn ear length, number of kernels per ear, and kernel size per ear.

[0065] An embodiment of the third aspect of the present invention provides an expression cassette, a recombinant expression vector or a recombinant cell, wherein the expression cassette or the recombinant expression vector comprises a targeting sequence, and the targeting sequence targets the nucleic acid as defined in the embodiment of the first aspect of the present invention to inhibit its expression, wherein the expression of the nucleic acid as defined in the embodiment of the first aspect of the present invention in the recombinant cell is inhibited.

[0066] In some embodiments, the expression cassette, recombinant expression vector or recombinant cell is a recombinant eukaryotic expression cassette, a recombinant eukaryotic expression vector or a recombinant plant cell, respectively; preferably, the recombinant eukaryotic expression cassette or recombinant eukaryotic expression vector is a recombinant plant expression cassette or a recombinant plant expression vector.

[0067] In some embodiments, the expression cassette, recombinant expression vector or recombinant cell is used to (i) advance or delay the flowering period of corn; and / or (ii) increase at least one of corn ear length, kernel number, and kernel size.

[0068] An embodiment of the fourth aspect of the present invention proposes a corn breeding method, characterized in that the method includes: reducing the expression level of the nucleic acid as defined in the embodiment of the first aspect of the present invention or the protein as defined in the embodiment of the second aspect of the present invention in corn and / or reducing the activity of the protein to promote early maturity of corn and improve corn yield.

[0069] In some embodiments, the method comprises: (i) advancing the flowering period of corn; and / or (ii) increasing at least one of ear length, kernel number, and kernel size of corn.

[0070] In some embodiments, the nucleic acid sequence in corn as defined in the embodiments of the first aspect of the invention is mutated by CRISPR / Cas9 technology to reduce the expression level of the nucleic acid as defined in the embodiments of the first aspect of the invention or the protein as defined in the embodiments of the second aspect of the invention in corn and / or reduce the activity of the protein.

[0071] It is understood that in the field of plant biotechnology, various technical means are used to inhibit gene expression, including gene silencing, RNAi, and CRISPR-Cas9. These methods precisely regulate the transcription and translation of specific genes in plants through different mechanisms, achieving fine control of growth and development. Those skilled in the art will understand that as long as the expression of the ZmEREB131 gene can be effectively inhibited, the technical effects of regulating corn flowering period and improving growth traits such as corn ear length described in this application can be achieved.

[0072] In some embodiments, the mutating of the nucleic acid sequence in corn as defined in the embodiments of the first aspect of the present invention by CRISPR / Cas9 technology comprises:

[0073] The nucleic acid sequence defined in the embodiment of the first aspect of the present invention is mutated in corn by introducing a vector comprising a targeting sequence into corn,

[0074] The targeting sequence targets the nucleic acid as defined in the embodiment of the first aspect of the present invention to inhibit its expression, and the targeting sequence is the sgRNA coding sequence shown in SEQ ID NO: 4.

[0075] Among them, the sequence of SEQ ID NO: 4 is specifically: GACCCTCGGCCACCGCTAA.

[0076] In some embodiments, the corn mutant obtained by the method has the nucleic acid sequence defined in the embodiment of the first aspect of the present invention compared to the wild-type corn.

[0077] (i) the CG bases at positions 294-298 are deleted; or

[0078] (ii) The sequence shown in SEQ ID NO: 5 (TCAGGAGGAGAAGCATCACGTTGTCGTCTCGCAGCTGCAAGCAGGAGCTACCTTTA GC) at positions 241 to 299 was deleted.

[0079] The maize inbred line ND101 was used as the recipient material in the following examples of this application. The experimental methods in the following examples, unless otherwise specified, are conventional methods and were performed according to the techniques and conditions described in literature in the field or according to product specifications. The materials and reagents used in the following examples, unless otherwise specified, are commercially available. Unless otherwise specified, the quantitative analysis experiments in the following examples were performed in triplicate, and the results were averaged.

[0080] Example

[0081] Example 1 - Acquisition and identification of ZmEREB131 overexpressing transgenic maize lines

[0082] In this example, a ZmEREB131 overexpression vector was constructed and the transgenic maize lines OE1 and OE2 were obtained using the vector.

[0083] 2.1 Construction of vector

[0084] (1) Synthesize DNA template and primers and perform PCR amplification

[0085] The DNA sequence of the ZmEREB131 gene (DNA template) was amplified using a high-fidelity enzyme kit (Nanjing Novozymes Biotech Co., Ltd.; Cat. No. P505-d1) based on specific primers (F1: GCACTGCTGCACCGAGCC, SEQ ID NO: 6; R1: TCACGCGTCCTTTTTATTCC, SEQ ID NO: 7). The reaction system was carried out according to the reagent manufacturer's instructions, and the PCR amplification program was as follows: 95°C for 3 min; 35 cycles of 95°C for 15 s, 55°C for 15 s, and 72°C for 1 min 30 s; and 72°C for 5 min.

[0086] (2) Agarose gel electrophoresis

[0087] The PCR products were electrophoresed on 1.5% agarose gel.

[0088] (3) DNA recovery

[0089] The gel was cut and the purified gene fragments were recovered using the Kangwei DNA Recovery Kit (Jiangsu Kangwei Century Biotechnology Co., Ltd., catalog number: CW2302M).

[0090] (4) Construction of vector

[0091] The purified gene fragment was recombined with the overexpression tag vector pBECXUN-Myc at 37°C for 30 minutes to complete the ligation. The 5μl recombination reaction system consists of: 1μl 5×CE II Buffer, 0.5μl Exnase II, 2μl pBECXUN-Myc vector, and 1.5μl ZmEREB131 gel-recovered fragment product.

[0092] (4) Conversion

[0093] Take 5 μL of the ligation product and transform Escherichia coli DH5α using the heat shock method. Screen the positive clones on an LB solid plate containing 50 mg / L kanamycin, pick 5 monoclonal plaques for sequencing, and perform gene sequence comparison based on the sequencing results to obtain the correct monoclonal colony.

[0094] Figure 1 Schematic diagram of the ZmEREB131 overexpression vector according to the embodiment of the present application.

[0095] 2.2 Obtaining ZmEREB131-overexpressing transgenic maize lines

[0096] (1) Obtaining ZmEREB131-overexpressing transgenic maize lines OE1 and OE2

[0097] The overexpression vector constructed in Example 2.1 was extracted using the Tiangen Plasmid Extraction Kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.). The overexpression vector was transferred into the Agrobacterium EHA105 strain according to conventional methods in the art. The ZmEREB131 gene was transferred into the immature embryos of the maize inbred line ND101 through the mediation of Agrobacterium, and T0 generation transgenic plants were obtained through tissue culture. The identified T0 generation plants were transplanted into the greenhouse of the West Campus of China Agricultural University, and the positive transgenic plants were identified by PCR amplification. Strict self-pollination was performed to obtain seeds. According to the above method, three generations of self-pollination were performed to obtain T3 generation homozygous overexpression strains OE1 and OE2 (PCR amplification results are shown in Figure 2 ).

[0098] (2) Verification of ZmEREB131 overexpressing transgenic maize lines OE1 and OE2

[0099] To examine the gene expression efficiency of ZmEREB131 in the obtained strains, leaves from seedlings of the background material ND101 and the overexpression strains OE1 and OE2 were extracted, snap-frozen in liquid nitrogen, and ground. Total RNA was then extracted using the Sigma Total RNA Extraction Reagent (Sigma-Aldrich (Shanghai) Trading Co., Ltd.). First-strand cDNA was synthesized using the Adlay Reverse Transcription Kit (Adlay Biotechnology), and expression levels were determined by fluorescent quantitative PCR using the Xerix RT-qPCR Kit (Xerix Biotechnology).

[0100] Figure 2 Shown is an agarose gel electrophoresis diagram of PCR products of ZmEREB1311 overexpression strains OE1 and OE2 according to an embodiment of the present application; Figure 3 The relative expression levels of ZmEREB131 in the ZmEREB131 overexpression strains OE1 and OE2 according to the examples of the present application are shown.

[0101] Figure 2 and Figure 3 The verification results showed that the expression levels of ZmEREB131 in the two overexpression lines were significantly increased compared with the background material ND101 ( Figure 3 , "***", p < 0.001; One-Way ANOVA). T3 generation transgenic homozygous seeds were used for subsequent experiments.

[0102] Example 2 - Acquisition and identification of ZmEREB131 knockout mutants

[0103] In this example, the ZmEREB131 pXUE411C-BG vector was constructed and the knockout mutant maize lines zmereb131-1 and zmereb131-2 were obtained using the vector.

[0104] (1) Construction of vector

[0105] The ZmEREB131 pXUE411C-BG vector was obtained by conventional methods in the art.

[0106] (2) Obtaining ZmEREB131 knockout mutants

[0107] The vector was transformed into Agrobacterium tumefaciens strain EHA105 using conventional methods in the art. The target sequence was knocked out in immature embryos of the maize inbred line ND101 using Agrobacterium-mediated transfection, and T0-generation transgenic plants were obtained through tissue culture. The identified T0-generation plants were transplanted to a greenhouse on the West Campus of China Agricultural University and rigorously selfed to obtain seeds. Following the above method, three generations of selfing were performed to obtain the T3-generation homozygous mutant lines zmereb131-1 and zmereb131-2.

[0108] (3) Use sequencing method to identify mutant plants.

[0109] Figure 5 A schematic diagram of the sequencing results of the ZmEREB131 mutant strains zmereb131-1 and zmereb131-2 according to the examples of the present application is shown. Figure 5 The results showed that the ZmEREB131 knockout mutants, zmereb131-1 and zmereb131-2, were successfully generated. Compared to wild-type maize ND101, zmereb131-1 had the CG bases at positions 294-298 deleted, while zmereb131-2 had the sequence shown in SEQ ID NO:5 deleted at positions 241-299. Homozygous T3 mutant seeds were used for subsequent experiments.

[0110] Example 3 - Effect of ZmEREB131 on flowering time

[0111] Experimental steps: The wild type and ZmEREB131 overexpression lines OE1 and OE2 and the zmereb131 mutant material two lines zmereb131-1 and zmereb131-2 were planted at the Shangzhuang Experimental Station in Beijing. Sampling and identification were carried out at the two-leaf and one-heart stage. Positive materials were selected and thinned to one plant per hole. The flowering time was further observed and counted.

[0112] Test results:

[0113] Figure 6 The flowering time statistics of wild type ND101, ZmEREB131 mutant strain zmereb131-1, overexpression strains OE1 and OE2 according to the embodiment of the present application are shown. Figure 6 It can be seen that compared with the wild type ND101, the ZmEREB1311 overexpression lines OE1 and OE2 flower later than ND101, and the peak flowering period is 2-3 days later than ND101; zmereb131-1 (ereb131-d58) flowers earlier than the wild type ND101, and the peak flowering period is 2-3 days earlier than ND101.

[0114] Example 4 - Effect of ZmEREB131 on Tassel Differentiation

[0115] Experimental Procedure: Wild-type and zmereb131 mutant strains zmereb131-1 and zmereb131-2 were planted at the Shangzhuang Experimental Station in Beijing. Samples were collected and tested at the two-leaf, one-heart stage. Positive seedlings were thinned to one plant per hole. The fifth unfolding leaf was spray-marked at V6-7 to determine the subsequent leaf expansion period. At V7, V9, and V11, wild-type and zmereb131 mutant strains zmereb131-1 and zmereb131-2 were sampled, tassel tissue removed, and phenotypic observations were performed using cryo-electron microscopy.

[0116] Test results:

[0117] Figure 7 Figure 2 shows the tassel differentiation of wild-type ND101, ZmEREB131 mutant strains zmereb131-1 and zmereb131-2 at the V7, V9, and V11 stages according to the examples of the present application; the effect of ZmEREB131 on tassel differentiation is shown in Figure 2. Figure 7 As shown, the tassel development, IM (inflorescence meristem) elongation and floral organ establishment of the zmereb131 mutant material were earlier than those of the wild type ND101.

[0118] Example 5 - Effect of ZmEREB131 on corn production traits (ear length, number of kernels per ear, and kernel size)

[0119] Experimental Procedure: Wild-type and zmereb131 mutant strains, zmereb131-1 and zmereb131-2, were planted at the Shangzhuang Experimental Station in Beijing. Samples were collected and identified at the two-leaf, one-heart stage. Positive seedlings were selected and thinned to one plant per hole. The male spikes were bagged at the time of pollen shedding and flowering to ensure self-pollination. Female spikes of the wild-type and zmereb131 mutant strains were collected at the V11 stage and observed using cryo-electron microscopy. Approximately 50 days after pollination, spikes of the wild-type and zmereb131 mutant strains were harvested and analyzed for spike length, kernel number, and kernel size.

[0120] Test results:

[0121] Figure 8 and Figure 9 The female ear development and ear-grain phenotype of the wild-type ND101, ZmEREB131 mutant strain zmereb131-1 (also referred to as eb131-1 mutant or 131-CR-1 in the figure) and zmereb131-2 (also referred to as eb131-2 mutant or 131-CR-2 in the figure) at the V11 stage according to the examples of the present application are shown.

[0122] It can be seen that compared with ND101, the development speed of the female ear in the ereb131 mutant is accelerated ( Figure 8 At V11, the ereb131 single mutant showed earlier development of the spikelet meristem (SM) in the female ear compared to the wild-type. Statistical analysis of the mature ear phenotypes of the ereb131 mutant and wild-type revealed that the ereb131 mutant had significantly longer ears, larger grains, and increased single ear weight.

[0123] In summary, the use of ZmEREB131 in the embodiments of the present invention can not only advance or delay the flowering of corn under suitable conditions, thereby achieving the technical effect of effectively regulating the flowering time of corn; but also promote the development of female ears, increase the number of ears and the number of grains per ear, and increase the ear length, so that corn can better adapt to the corresponding environmental conditions and improve the fruit set rate, thereby achieving the beneficial technical effect of increasing yield.

[0124] In the present invention, the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" mean that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and the features of different embodiments or examples without contradiction.

[0125] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. The application of nucleic acid in regulating corn growth, characterized in that: The nucleic acid is used for (i) advancing the flowering period of corn; and / or (ii) improving at least one of corn ear length, number of kernels per ear and kernel size; The nucleic acid is a polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 3, and the growth of corn is regulated by inhibiting the expression of the nucleic acid.

2. The use according to claim 1, characterized in that wherein the nucleic acid is ZmEREB131 gene, whose gene ID is Zm00001d003884.

3. The application of protein in promoting early maturity of corn, characterized in that: The protein is used for (i) advancing the flowering period of corn; and / or (ii) improving at least one of corn ear length, kernel number, and kernel size, wherein the amino acid sequence of the protein is shown in SEQ ID NO:

3. The early maturity of corn is promoted by reducing the expression level of the protein.

4. A corn breeding method, characterized in that: The method comprises: reducing the expression level of the nucleic acid as defined in claim 1 or 2 or the protein as defined in claim 3 in corn, so as to promote early maturity of corn and improve corn yield, wherein the method further comprises: (i) advancing the flowering period of corn; and / or (ii) increasing at least one of the length of corn ears, the number of kernels per ear, and the size of kernels per ear.

5. The method according to claim 4, characterized in that The nucleic acid sequence as defined in claim 1 or 2 in corn is mutated by CRISPR / Cas9 technology to reduce the expression level of the nucleic acid as defined in claim 1 or 2 or the protein as defined in claim 3 in corn.

6. The method according to claim 5, characterized in that The method of mutating the nucleic acid sequence as defined in claim 1 or 2 in corn by using CRISPR / Cas9 technology comprises: The nucleic acid sequence as defined in claim 1 or 2 is mutated in corn by introducing a vector comprising a targeting sequence into corn, wherein the targeting sequence targets the nucleic acid as defined in claim 1 or 2 to inhibit its expression, and the targeting sequence is an sgRNA coding sequence as shown in SEQ ID NO: 4.

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