Application of corn ZmEREB131 gene in regulation and control of corn growth

Abstract

The invention relates to the field of gene engineering, in particular to application of a ZmEREB131 gene in regulation and control of corn growth and a corn breeding method using the ZmEREB131 gene. The ZmEREB131 gene has a polynucleotide sequence as shown in SEQ ID NO: 1 and / or SEQ ID NO: 2; and / or a polynucleotide sequence encoding the amino acid sequence as shown in SEQ ID NO: 3. The gene can (i) regulate and control the flowering phase of corn; and / or (ii) improve at least one of corn ear length, ear number, and ear 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] As a core crop, the distribution of corn planting regions shows a significant imbalance. The distribution of corn in China is mainly concentrated in the Northeast, North China and Southwest China, forming an oblique long corn planting belt from the Northeast to the Southwest. Under such different environmental conditions, it is necessary to adjust the flowering time to affect the adaptability and stress resistance of corn in the growing area. Adjusting the flowering time can not only significantly enhance the regional adaptability of corn to different ecological environments, thereby improving its yield stability and stress resistance, but also affect multiple key traits such as corn yield, quality and nutritional value. It is an important parameter that cannot be ignored in corn breeding and production. In addition, corn ear length is another crucial trait. Ear length is directly related to the number and quality of corn kernels. Longer ear length often means more kernels, which plays a significant role in improving corn yield and production efficiency. Although there have been some studies on corn flowering period and ear length regulatory genes, their number is far less than that of the model plant Arabidopsis and the important monocot rice, and the establishment of its regulatory network is relatively lagging behind.

[0003] Given the important effects of flowering time and ear length on corn growth and yield, it is necessary to further develop and study 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, the embodiment of the first aspect of the present invention proposes an application 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 the number of kernels per ear, thereby improving corn yield through genetic engineering technology. The nucleic acid is used for (i) regulating corn flowering period; and / or (ii) improving at least one of corn ear length, ear kernel number and ear 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 that can hybridize with 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, ear kernel number, and ear 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] The second aspect of the present invention provides an application of a protein in promoting early maturity of corn, wherein the protein is used for (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, wherein the protein:

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

[0014] b. An amino acid sequence having at least 90%, 95% or 99% identity 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 the length of corn cob, the number of kernels per ear, and the size of kernels 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 the 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, ear kernel number, and ear kernel size.

[0018] An embodiment of the fourth aspect of the present invention proposes a corn breeding method, characterized in that the method comprises: 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 the length of corn ears, the number of kernels per ear, and the size of kernels per ear.

[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 in corn is mutated by introducing a vector comprising a targeting sequence into corn,

[0023] Wherein 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 a nucleic acid sequence defined in the embodiment of the first aspect of the present invention compared to 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 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 embodiment 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 the 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 setting 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 This is a schematic diagram of the ZmEREB131 overexpression vector according to an 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 overexpressing strains OE1 and OE2 according to the examples of the present application are shown;

[0034] Figure 4 A schematic diagram of knockout sites of ZmEREB131 mutant strains zmereb131-1 and zmereb131-2 according to an embodiment of the present application is shown;

[0035] Figure 5 A schematic diagram showing sequencing results of ZmEREB131 mutant strains zmereb131-1 and zmereb131-2 according to an embodiment of the present application is shown;

[0036] Figure 6 The statistical results of flowering time of wild type ND101, ZmEREB131 mutant strain zmereb131-1, 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 development of female ears at the V11 stage of the wild-type ND101, the ZmEREB131 mutant strains zmereb131-1 and zmereb131-2 according to the examples of the present application is shown;

[0039] Fig. 9 The ear-kernel 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 is 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 be used as a guide for further improvements by those of ordinary skill 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 techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

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

[0043] The planting distribution of corn in China is not balanced in various regions. It is mainly concentrated in the Northeast, North China and Southwest China, forming an oblique long corn planting belt from the Northeast to the Southwest. Corn needs to adapt to different environmental conditions by adjusting the flowering time. The regulation of flowering time has long been an active research field. The flowering period not only affects the adaptability of corn to the growing area and its resistance to various adversities, but also has an important impact on multiple traits such as corn yield, quality, and nutritional value. The factors affecting flowering time are very complex, including both internal genomic genetic factors and external environmental factors. By studying and analyzing the flowering time performance of corn varieties under different environmental conditions, we can better understand and utilize the flowering time characteristics, so as to select better varieties that are more adaptable to different environments. In addition, by accurately measuring and comparing the flowering time, breeders can understand the genetic differences between different varieties and provide an important basis for selective breeding. Therefore, in-depth research and utilization of flowering time can not only help corn varieties adapt to different growth environments, but also provide important guidance for corn breeding. The study of flowering time can not only improve corn yield and quality, but also accelerate the breeding process, so as to better meet the needs of agricultural production and promote sustainable agricultural development.

[0044] As an important trait of corn, corn ear length plays a key role in corn breeding and production. Yield impact: Corn ear length directly affects the number and quality of corn grains. Longer ear length usually means more kernels, which helps to improve corn yield and production efficiency. In corn breeding, ear length is usually one of the important selection criteria. By selecting excellent varieties with longer ear length for future generation breeding, the ear length performance of corn can be gradually improved. The variability of corn ear length can provide breeders with a larger selection space to develop corn varieties that better meet market demand, such as large-ear corn varieties suitable for mechanized harvesting. In summary, as one of the important traits of corn, corn ear length is of great significance in increasing yield, adapting to the environment, optimizing breeding selection and meeting market demand. Therefore, in corn breeding and production, the study and utilization of corn ear length is crucial.

[0045] Although there have been some studies on maize flowering time and ear length regulatory genes, their number is far less than that of the model plant Arabidopsis and the important monocot rice, and the establishment of their regulatory network is relatively lagging. Given the important effects of flowering time and ear length on maize growth and yield, it is necessary to develop genes that can effectively regulate these traits and their breeding methods.

[0046] In this regard, the inventors of the present application have screened and located a gene involved in ear length gain and flowering period regulation in corn after a large number of experiments. The gene is located at Chr2:65020854..65025676 of the corn reference genome Zm-B73-REFERENCE-GRAMENE-4.0, and the gene ID in the B73 genome is Zm00001d003884 (https: / / maizegdb.org). The present application names it ZmEREB131, and its sequence is shown in SEQ ID NO: 1. The present application further discloses a method for manipulating ZmEREB131 by genetic engineering to (i) regulate corn flowering period; and / or (ii) increase at least one of corn ear length, ear kernel number, and ear kernel size.

[0047] In the examples of the present application, unless otherwise specified, 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 may be represented herein by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Similarly, nucleotides may be represented by commonly accepted single-letter codes. Numerical ranges include numbers that define the range. As used herein, "nucleic acid" includes deoxyribonucleotides or ribonucleotide polymers involving single-stranded or double-stranded forms, and unless otherwise limited, includes known analogs (e.g., peptide nucleic acids) having the basic properties of natural nucleotides, which hybridize with 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 information encoding proteins. 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 particular 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, known analogs of natural amino acids can be included that can function in a manner similar to naturally occurring amino acids.

[0048] In the present application embodiment, the term "percentage identity" about nucleic acid or peptide sequence is defined as the percentage of nucleotides or amino acid residues identical with known polypeptides in candidate sequence after arranging the sequence to obtain maximum percentage identity and introducing a gap (if necessary) to realize maximum percentage homology. 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 alignment search tool, Basic Local Alignment Search Tool) analysis, the analysis uses the algorithm (Altschul (1997) adopted by program blastp, blastn, blastx, tblastn and tblastx, Nucleic Acids Res [nucleic acid research]. 25, 3389-3402 and Karlin (1990), Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 87, 2264-2268), the 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 (including endpoint values) that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80% identical to any one of SEQ ID NOs: 1-3, 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%, 80% identical to any one of SEQ ID NOs: 1-3, 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. NO: 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 NO: 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 the moisture content and / or dehydration rate of corn kernels; and / or (ii) regulating the flowering period of corn.

[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%, 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%, 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%, at least 99.9%, or at least 99.1%, at least 99.2%, at least 99.3%, at least 99.9%, ...9%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.9%, at least 99.9%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.9%, at least 99.9%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.9 .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% identity, the mutant protein still has the function of (i) regulating corn kernel moisture content and / or dehydration rate; and / or (ii) regulating corn flowering period. In some embodiments, unless otherwise specified, except for the indicated mutations, the remaining amino acids of the protein are identical to 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 with its target sequence to a detectable greater degree (e.g., at least 2 times, 5 times, or 10 times of 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 mismatches to detect lower similarity (heterologous probe method). Typically, the probe length is less than about 1000 or 500 nucleotides. Typically, stringent conditions are those in which the salt concentration is less than about 1.5 M Na ions, 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 aspect of the present invention provides an application 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 the number of kernels per ear, thereby improving corn yield through genetic engineering technology. The nucleic acid is used for (i) regulating corn flowering period; and / or (ii) improving at least one of corn ear length, number of kernels 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 that can hybridize with 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, ear kernel number, and ear 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] The second aspect of the present invention provides an application of a protein in promoting early maturity of corn, wherein the protein is used for (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, wherein the protein:

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

[0064] b. An amino acid sequence having at least 90%, 95% or 99% identity 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 the length of corn cob, the number of kernels per ear, and the size of kernels 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 the 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, ear kernel number, and ear kernel size.

[0068] An embodiment of the fourth aspect of the present invention proposes a corn breeding method, characterized in that the method comprises: 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 the length of corn ears, the number of kernels per ear, and the size of kernels per ear.

[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 understandable 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 to achieve fine control of growth and development. It is understandable to those skilled in the art that as long as the expression of the ZmEREB131 gene can be effectively inhibited, the technical effects of regulating the flowering period of corn 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 in corn is mutated by introducing a vector comprising a targeting sequence into corn,

[0074] Wherein 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 a nucleic acid sequence defined in the embodiment of the first aspect of the present invention compared to 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 acceptor material inbred line used in the following examples of this application is ND101. The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels. Unless otherwise specified, the quantitative analysis tests in the following examples are set up for three repeated experiments, and the results are 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 corn 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] Based on specific primers (F1: GCACTGCTGCACCGAGCC, SEQ ID NO: 6; R1: TCACGCGTCCTTTTTATTCC, SEQ ID NO: 7), the DNA sequence (DNA template) of the ZmEREB131 gene was amplified using a high-fidelity enzyme kit (company Nanjing Novogene Biotech Co., Ltd.; catalog number P505-d1). The reaction system was carried out according to the reagent operating instructions, and the PCR amplification program was: 95°C for 3 min; 95°C for 15 s, 55°C for 15 s, 72°C for 1 min 30 s, 35 cycles; 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 recovered purified gene fragment was recombined with the overexpression tag vector pBECXUN-Myc at 37°C for 30 minutes to complete the connection. The 5μl recombination reaction system was: 5×CE II Buffer 1μl, Exnase II 0.5μl, pBECXUN-Myc vector 2μl, ZmEREB131 gel recovered fragment product 1.5μl.

[0092] (4) Conversion

[0093] Take 5 μL of the ligation product, transform Escherichia coli DH5α by heat shock method, screen positive clones on LB solid plate containing 50 mg / L kanamycin, pick 5 monoclonal plaques for sequencing, and compare the gene sequences according to the sequencing results to obtain the correct monoclonal colonies.

[0094] Figure 1 Schematic diagram of the ZmEREB131 overexpression vector map 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 the 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, and the T3 generation homozygous overexpression lines OE1 and OE2 were obtained by self-pollination for 3 generations according to the above method (PCR amplification results are shown in Figure 2 ).

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

[0099] To detect the gene expression efficiency of ZmEREB131 in the obtained strains, the leaves of the seedlings of the background material ND101 and the overexpression strains OE1 and OE2 were extracted, quick-frozen in liquid nitrogen, and ground, and then the total RNA was extracted using Sigma total RNA extraction reagent (Sigma-Aldrich (Shanghai) Trading Co., Ltd.). The first-strand cDNA was synthesized using the Adele reverse transcription kit (Adele Biotechnology), and the expression level was detected by fluorescence quantitative PCR using the Acre RT-qPCR kit (Acre 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 compared with the background material ND101, the expression levels of ZmEREB131 in the two overexpression strains were significantly increased ( Figure 3 , "***", p<0.001; One Way ANOVA). T3 transgenic homozygous seeds were used for subsequent experiments.

[0102] Example 2-Obtaining and identifying 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 transferred into the Agrobacterium EHA105 strain according to conventional methods in the art. The target sequence in the immature embryo of the maize inbred line ND101 was knocked out by the Agrobacterium-mediated knockout, and T0 transgenic plants were obtained by tissue culture. The identified T0 plants were transplanted to the greenhouse of the West Campus of China Agricultural University, and strictly self-pollinated to obtain seeds. According to the above method, three generations of self-pollination were performed to obtain T3 homozygous mutant strains 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 obtained. Compared with the wild-type corn ND101, the CG bases at positions 294-298 of zmereb131-1 were knocked out; the sequence shown in SEQ ID NO:5 at positions 241-299 of zmereb131-2 was knocked out. Subsequent experiments were performed using homozygous seeds of the T3 generation mutants.

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

[0111] Experimental procedures: The wild type and ZmEREB131 overexpression lines OE1 and OE2 and the zmereb131 mutant material two lines zmereb131-1 and zmereb131-2 were planted in 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 the wild type ND101, the ZmEREB131 mutant strain zmereb131-1, and the overexpression strains OE1 and OE2 according to the embodiments 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 steps: The wild type and zmereb131 mutant material strains zmereb131-1 and zmereb131-2 were planted in Beijing Shangzhuang Experimental Station. Samples were taken and identified at the two-leaf and one-heart stage. Positive materials were selected and thinned to one plant per hole. The fifth unfolded leaf was sprayed and marked at V6-7 to determine the subsequent leaf expansion period. At V7, V9, and V11, the wild type and zmereb131 mutant strains zmereb131-1 and zmereb131-2 were taken, the tassel tissue was peeled off, and the phenotypic observation was carried out by cryo-electron microscopy.

[0116] Test results:

[0117] 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; the effect of ZmEREB131 on tassel differentiation is shown in FIG. 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 steps: The wild type and zmereb131 mutant material strains zmereb131-1 and zmereb131-2 were planted in Shangzhuang Experimental Station, Beijing. Samples were taken and identified at the two-leaf and one-heart stage. Positive materials were selected and thinned to one plant per hole. The male spikes were bagged when the pollen was scattered and flowering to ensure self-pollination of the materials. The female spikes of the materials were taken at the V11 stage, and the female spikes of the wild type and zmereb131 mutant materials were observed using a cryo-electron microscope. About 50 days after pollination, the spikes of the wild type and zmereb131 mutant materials were harvested to count the spike length, number of spikelets, and spikelet size.

[0120] Test results:

[0121] Figure 8 and Fig. 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 embodiments of the present application are shown.

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

[0123] In summary, the use of ZmEREB131 in the embodiment 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 setting 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" etc. 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 representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the described specific features, structures, materials or characteristics may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

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

Claims

1. The use of nucleic acid in regulating corn growth, characterized in that: The nucleic acid is used for (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; wherein the nucleic acid: a. having a polynucleotide sequence as shown in SEQ ID NO: 1 and / or SEQ ID NO: 2; b. a polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 3; c. A polynucleotide sequence that can hybridize with 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, ear kernel number, and ear kernel size; or 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.

2. The use according to claim 1, characterized in that: The growth of corn is regulated by overexpressing and / or inhibiting the expression of the nucleic acid, optionally wherein the nucleic acid is the 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) regulating the flowering period of corn; and / or (ii) improving at least one of corn ear length, ear kernel number, and ear kernel size, wherein the protein: a. having an amino acid sequence as shown in SEQ ID NO: 3, or b. An amino acid sequence having at least 90%, 95% or 99% identity 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 the length of corn cob, the number of kernels per ear, and the size of kernels per ear.

4. An expression cassette, a recombinant expression vector or a recombinant cell, characterized in that: wherein the expression cassette or recombinant expression vector comprises a targeting sequence, wherein the targeting sequence targets the nucleic acid as defined in claim 1 or 2 to inhibit its expression, wherein the expression of the nucleic acid as defined in claim 1 or 2 in the recombinant cell is inhibited, Optionally, 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 the recombinant eukaryotic expression vector is a recombinant plant expression cassette or a recombinant plant expression vector.

5. The expression cassette, recombinant expression vector or recombinant cell according to claim 4, characterized in that: The expression cassette, recombinant expression vector or recombinant cell is used for (i) advancing or delaying 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.

6. A corn breeding method, characterized in that: The method comprises: reducing the expression level of the nucleic acid defined in claim 1 or 2 or the protein defined in claim 3 in corn and / or reducing the activity of the protein, so as to promote early maturity of corn and improve corn yield.

7. The method according to claim 6, characterized in that The method 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.

8. The method according to claim 6, 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 and / or reduce the activity of the protein.

9. The method according to claim 8, 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 the sgRNA coding sequence shown in SEQ ID NO:

4.

10. The method according to claim 8, characterized in that The corn mutant obtained by the method has a nucleic acid sequence as defined in claim 1 or 2 compared to wild-type corn, (i) the CG bases at positions 294-298 are deleted; or (ii) The sequence shown in SEQ ID NO: 5 at positions 241-299 was deleted.

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