CN112646014B - Genes and Methods for Altering Maize Flowering Period - Google Patents

Abstract

The present invention is in the field of molecular genetics. In particular to a gene and a method for changing the flowering period of corn. The invention provides a sequence of a gene for controlling the flowering phase of corn and discloses a method for changing the flowering phase by mutating the gene by using a genetic engineering means. The invention also provides a maize mutant gene sequence with an altered flowering phase, which can be used for improving maize flowering phase traits.

Description

Genes and Methods for Altering Maize Flowering Period

technical field

The present invention belongs to the field of molecular genetics. Specifically, it relates to genes controlling the flowering stage of maize and its application in changing the flowering stage of maize. The present invention provides a sequence of a gene for controlling the flowering period of maize, and discloses a method for mutating the gene by means of genetic engineering to shorten the flowering period. The present invention also provides a maize mutant gene sequence with shortened flowering period, and the mutant gene sequence can be used to improve maize flowering period traits.

Background technique

In higher plants, flowering represents the transition from vegetative growth to reproductive growth, which plays an important role in the entire growth and development stages of the plant. The biological character of when to bloom is affected by the dual effects of the plant's own genetic factors and external environmental factors. Under the influence of these dual effects, a series of flowering induction processes have a general rule in higher plants, that is, plant leaves can produce flower-forming substances (or Florigen), the flower-forming material is transported from the leaves to the shoot apex through the transport tissue, and stimulates the apical meristem to form flowers.

Flowering period is an important trait in the process of crop evolution and adaptation. Understanding the genetic basis of crop flowering period traits and cloning candidate genes can improve the environmental adaptability and plasticity of crops, which is of great significance for cultivating excellent crop varieties that adapt to different ecological regions. At the same time, it will also promote the process of genetic improvement of important production traits such as yield, which are closely related to flowering period.

CCT (CO, COL and TOC1) family genes are widely involved in the regulation process of plant flowering, and play a crucial role in plant growth and development. There are 53 genes with CCT structure in the maize B73 reference genome. The applicant previously used a related group composed of 368 maize inbred lines to map 34 CCT-like genes related to maize flowering (Jin Minliang. Maize Pan-transcriptome). Construction and functional analysis of the maize flowering inhibitor ZmCOL3 [D]. Hubei: Huazhong Agricultural University, 2018; Jin M, Liu X, Jia W, et al. ZmCOL3, a CCT gene represses flowering inmaize by interfering with the circadian clock and activating expression of ZmCCT[J].J Integr Plant Biol.,2018,60(6):465-480.), most of the genes have no specific functional identification results, so which genes will actually control maize flowering Traits are not fully understood. If you want to change the flowering period of maize more precisely, it is necessary to specifically identify which genes control the flowering characteristics of maize and to what extent these genes change the flowering characteristics after genetic engineering.

In order to solve the above problems, the present invention utilizes gene editing technology to mutate 15 genes among the 34 genes, obtains genes and mutant genes that can affect the flowering stage of maize through phenotypic identification, and provides a method for artificially changing the flowering stage of maize. method. These genes and methods can be used to artificially regulate the flowering period of maize and cultivate new maize materials adapted to different ecological environments.

SUMMARY OF THE INVENTION

One of the objects of the present invention is to provide a gene sequence for controlling the flowering period of maize.

The second purpose of the present invention is to disclose a method for changing the flowering period of corn.

The third object of the present invention is to provide a mutant gene for changing the flowering period of corn.

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

The invention provides the application of a protein in controlling the traits of maize flowering period, characterized in that: the amino acid sequence of the protein is shown in SEQ ID NO.1.

The present invention also provides the application of a nucleic acid molecule in the control of maize flowering stage traits, characterized in that: the nucleic acid molecule encodes the above-mentioned protein; in some embodiments, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO. .2 or SEQ ID NO.3.

The sequence of SEQ ID NO. 1 is the amino acid sequence of the GRMZM2G065896 gene in the maize inbred line B73. The sequence of SEQ ID NO. 2 is the genomic sequence of the GRMZM2G065896 gene. The sequence of SEQ ID NO.3 is the cDNA sequence of the GRMZM2G065896 gene.

The present invention also provides a method for early flowering period of maize, which is characterized by: inhibiting the expression and/or activity of the protein encoded by the above gene in maize, and selecting plants with early flowering period of maize.

In some embodiments, the aforementioned methods of inhibiting protein expression and/or activity include any of gene editing, RNA interference, T-DNA insertion, physical or chemical mutagenesis.

In some embodiments, the gene editing described above employs the CRISPR/Cas9 approach.

In some embodiments, the DNA sequence of the genomic target region of the above-mentioned CRISPR/Cas9 method in maize is shown in SEQ ID NO.4.

The present invention also provides a kit for early flowering of corn, characterized in that it includes any of the following:

(1) sgRNA molecule, its sequence is as shown in SEQ ID NO.5;

(2) the coding DNA molecule of the sgRNA;

(3) a vector for expressing the sgRNA.

The present invention also provides a mutant gene for early flowering of maize, characterized in that: the mutant gene sequence is shown in any one of SEQ ID NO.6-SEQ ID NO.9.

Many different editing types can be obtained by gene editing of mutated target genes, and the performance of the plants corresponding to these different editing types is not exactly the same. After screening and identification in the present invention, it is determined that the mutant gene shown in SEQ ID NO.6 or SEQ ID NO.7 or SEQ ID NO.8 or SEQ ID NO.9 can make the flowering period of maize moderately early. The mutant gene can be introduced into maize materials with different genetic backgrounds through sexual hybridization, thereby creating new early-flowering maize varieties.

The present invention also provides a primer pair for detecting the above mutant gene, characterized in that: the primer pair is the sequences shown in SEQ ID NO. 10 and SEQ ID NO. 11 or their complementary sequences.

The present invention also provides the application of the above-mentioned primer pair in detecting the above-mentioned mutant gene. Use the above primers to amplify the genomic DNA of the sample to be tested by PCR, and sequence and analyze the sequence of the amplified product. If the sequence of the amplified product after sequencing is the same as SEQ ID NO. If the partial sequence of the sequence shown in ID NO. 9 is consistent, the sample to be tested contains the above-mentioned mutant gene.

The advantages and beneficial effects of the present invention are as follows: the present invention uses the associated population to locate 34 CCT genes related to maize flowering period traits, but it is unknown which specific genes among these candidate genes will indeed control maize flowering traits and affect flowering. effect of period traits. The present invention uses gene editing technology to mutate 15 of the 34 genes, and confirms through phenotypic identification that GRMZM2G065896 can indeed affect maize flowering period traits. The CRISPR/Cas9 gene editing method can change the flowering period of maize, and the edited mutant gene can be used to create new early-flowering maize varieties. The invention provides a new gene and method for artificially regulating the flowering period of maize to cultivate new maize materials adapted to different ecological environments.

Description of drawings

Figure 1 Gene editing vector diagram. The English and abbreviation meanings of each element are listed as follows:

RB T-DNA repeat T-DNA right border repeat

M13 fwd M13 primer sequence (forward)

p000204_1F target gRNA sequence

Ubi promoter

3×FLAG tag sequence

SV40NLS Simian virus 40 nuclear localization signal

Cas9 cas9 gene sequence

Nucleoplasm in NLS nuclear localization signal

NOS terminator nopaline synthase terminator

lac promoter

M13 rev M13 primer sequence (reverse)

lac operator lactose operon

CAP bidding site CAP binding site

CaMV35S promoter(enhanced) enhanced cauliflower mosaic virus 35S promoter

BlpR encodes a Bar protein that confers glufosinate tolerance in plants

CaMV35S polyA single Cauliflower mosaic virus 35S polyadenylation sequence

LB T-DNA repeat T-DNA left border repeat

Kan R kanamycin resistance sequence

Ori initiation region sequence

Bom framework sequence

pVS1 RepA pVS1 replicon

pVS1 StaA pVS1 transcription initiation region

Detailed ways

The following definitions and methods are provided to better define this application and to guide those of ordinary skill in the art in the practice of this application. Unless otherwise specified, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. All patent documents, academic papers, industry standards and other publications, etc. cited herein are incorporated by reference in their entirety.

As used herein, "maize" is any maize plant and includes all plant species that can be bred with maize, including whole plants, plant cells, plant organs, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant healing Intact plant cells in wound tissue, plants, or plant parts such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, stems, roots, root tips, anthers, and the like. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation. Amino acids may be represented herein by either their commonly known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Likewise, nucleotides can be represented by generally accepted one-letter codes. Numerical ranges include the numbers defining the range. As used herein, "nucleic acid" includes references to deoxyribonucleotides or ribonucleotide polymers in single- or double-stranded form, and, unless otherwise limited, includes known analogs having essential properties of natural nucleotides ( For example, peptide nucleic acids), the analogs hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides. As used herein, the terms "encode" or "encoded" when used in the context of a particular nucleic acid means that the nucleic acid contains the necessary information to direct translation of the nucleotide sequence into a particular protein. Use codons to express the information that encodes the protein. As used herein, "full-length sequence" in reference to a particular polynucleotide or the protein it encodes refers to the entire nucleic acid sequence or the entire amino acid sequence having the native (non-synthetic) endogenous sequence. The full-length polynucleotide encodes the full-length, catalytically active form of this particular protein. The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer 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 acid. 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"). The amino acid can be a naturally occurring amino acid and, unless otherwise limited, can include known analogs of natural amino acids that can function in a similar manner to the naturally occurring amino acids.

As used herein, the terms "isolated" and "purified" are used interchangeably to refer to a nucleic acid or polypeptide, or biologically active portion thereof, that is substantially or essentially free as found in its naturally occurring environment The components of the nucleic acid or polypeptide are usually associated with or reacted to. Thus, when an isolated or purified nucleic acid or polypeptide is produced by recombinant techniques, the isolated or purified nucleic acid or polypeptide is substantially free of other cellular material or culture medium, or when the isolated or purified nucleic acid or polypeptide is chemically synthesized, substantially Contains no chemical precursors or other chemicals. An "isolated" nucleic acid is generally free of sequences (such as protein-encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, an isolated nucleic acid may comprise less than about 0.5 kb of nucleotide sequences that naturally flank the nucleic acid in the genomic DNA of the cell from which the nucleic acid is derived.

In this application, the words "comprising", "comprising" or variations thereof should be understood to include other elements, numbers or steps in addition to the described elements, numbers or steps. "Test plant" or "test plant cell" refers to a plant or plant cell in which a genetic modification has been effected, or a progeny cell of a plant or cell so modified, the progeny cell comprising the modification. A "control" or "control plant" or "control plant cell" provides a reference point for measuring changes in the phenotype of a test plant or plant cell. A control plant or plant cell can include, for example: (a) a wild-type plant or cell, i.e., a plant or cell of the same genotype as the genetically engineered starting material that produced the test plant or cell; (b) a The starting material is of the same genotype but has been transformed with an empty construct (ie, with a construct that has no known effect on the trait of interest, such as a construct comprising a target gene); (c) is subject to A plant or plant cell that is a non-transformed segregant of a test plant or plant cell; (d) a plant or plant that is genetically identical to the test plant or plant cell but has not been exposed to conditions or stimuli that induce expression of the gene of interest cells; or (e) the test plant or the plant cell itself, which is under conditions in which the gene of interest is not expressed.

Those skilled in the art will readily recognize that advances in the field of molecular biology such as site-specific and random mutagenesis, polymerase chain reaction methods, and protein engineering techniques provide a wide range of appropriate tools and procedures, Amino acid sequences and potential gene sequences for engineering or engineering proteins of agricultural interest.

In some embodiments, changes may be made to the nucleotide sequences of the present application to make conservative amino acid substitutions. Principles and examples of conservative amino acid substitutions are described further below. In certain embodiments, substitutions of the nucleotide sequences of the present application that do not alter the amino acid sequence can be made in accordance with the published monocot codon preferences, for example, codons encoding the same amino acid sequence can be replaced with codons preferred by monocots , without changing the amino acid sequence encoded by the nucleotide sequence. In some embodiments, parts of the nucleotide sequences in the present application are replaced with different codons encoding the same amino acid sequence, so that the nucleotide sequence is altered without altering the amino acid sequence it encodes. Conservative variants include those sequences that, due to the degeneracy of the genetic code, encode the amino acid sequence of one of the proteins of the embodiments. In some embodiments, part of the nucleotide sequence in the present application is substituted according to monocot-preferred codons. Those skilled in the art will recognize that amino acid additions and/or substitutions are often based on the relative similarity of amino acid side chain substituents, eg, hydrophobicity, charge, size, etc., of the substituents. Exemplary amino acid substitution groups having various of the aforementioned properties contemplated are well known to those skilled in the art and include arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine. Guidelines for appropriate amino acid substitutions that do not affect the biological activity of the protein of interest can be found in Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C) ( Incorporated herein by reference) models. Conservative substitutions such as replacing one amino acid with another amino acid of similar properties can be made. Identification of sequence identity involves hybridization techniques. For example, all or part of a known nucleotide sequence is used as a probe to selectively hybridize to other corresponding nucleotide sequences present in cloned genomic DNA fragments from the organism of choice Or a population of cDNA fragments (ie, a genomic library or a cDNA library).

In some embodiments, fragments of the nucleotide sequences and the amino acid sequences they encode are also included. As used herein, the term "fragment" refers to a portion of the nucleotide sequence of a polynucleotide of the embodiments or a portion of the amino acid sequence of a polypeptide. Fragments of nucleotide sequences can encode protein fragments that retain the biological activity of the native or corresponding full-length protein, and thus possess protein activity. Mutant proteins include biologically active fragments of the native protein comprising contiguous amino acid residues that retain the biological activity of the native protein. Some embodiments also include transformed plant cells or transgenic plants comprising the nucleotide sequence of at least one embodiment. In some embodiments, a plant is transformed using an expression vector comprising the nucleotide sequence of at least one embodiment and a promoter operably linked thereto that drives expression in a plant cell. Transformed plant cells and transgenic plants refer to plant cells or plants that contain a heterologous polynucleotide within the genome. Generally, the heterologous polynucleotide is stably integrated within the genome of the transformed plant cell or transgenic plant such that the polynucleotide is passed on to progeny. The heterologous polynucleotide can be integrated into the genome alone or as part of an expression vector. In some embodiments, plants contemplated by the present application include plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant masses, and plant cells, which are whole plants or plant Parts such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, nuts, ears, cobs, shells, stalks, roots, root tips, anthers and the like. The present application also includes plant cells, protoplasts, tissues, calli, embryos, and flowers, stems, fruits, leaves and roots.

The following examples are intended to illustrate the present invention, but not to limit the scope of the present invention. Modifications or substitutions made to the methods, steps or conditions of the present invention without departing from the spirit and essence of the present invention all belong to the scope of the present application. Unless otherwise specified, the examples follow conventional experimental conditions, such as the molecular cloning laboratory manual of Sambrook et al. (Sambrook J&Russell D W, Molecular cloning: a laboratory manual, 2001), or the conditions suggested by the manufacturer's instructions. Unless otherwise specified, the chemical reagents used in the examples are all conventional commercially available reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

Example

Example 1 Identification of changes in maize flowering period after gene editing and knocking out candidate genes

The invention utilizes CRISPR-Cas9 gene editing technology to perform site-directed editing on 15 genes among the 34 genes related to the flowering period of maize shown by the results of association analysis.

Embodiments include construction of gene editing vectors, genetic transformation of maize, and functional verification of editing effects. details as follows:

1. Construction of gene editing vector

The gene editing vector of the present invention is G08943-CPB-ZmUbi-hspCas9, and its vector diagram is shown in FIG. 1 . The base vector of this vector is CPB-ZmUbi-hspCas9. The present invention obtains double-target U6-sgRNA through OverlapPCR and then clones it into the base vector through homologous recombination. The specific construction process is as follows:

(1) Cloning of U6 promoter. The U6 promoter was cloned from B73.

(2) Design of target gRNA. The receptor material B73 reference genome sequence was imported into http://cbi.hzau.edu.cn/crispr/ for target design.

(3) U6-sgRNA was obtained by Overlap PCR. The primer pair U6F1/U6R was used to amplify the U6 promoter of the first target, and the length of the product was 515bp; the primer pair gR-1F(3F)/gRR1 was used to amplify the sgRNA of the first target, and the length of the product was 127bp; U6F1/gRR1 was used for the second step of Overlap PCR amplification (U6-sgRNA), and the product length was 634 bp. In the first step of Overlap PCR, U6 and sgRNA were amplified respectively. The PCR products were diluted 50 times and mixed as a template for the second step of Overlap PCR amplification. The amplified products were recovered by gel electrophoresis and sequenced to confirm the sequence. The Overlap PCR system and conditions are as follows: The 15 μL reaction system of the first step of Overlap PCR is as follows, template DNA (U6 or sgRNA, ≥30ng/μL): 0.5 μL, Primer F/R: 1.2 μL each, sterilized ddH ₂ O: 3.7 μL, 2×phanta max Buffer: 7.5 μL, dNTP mix: 0.6 μL, Phanta enzyme (product number: P505-d1/d2/d3): 0.3 μL. The reaction volume of the second step of Overlap PCR is 30 μL system. Aspirate 1 μL of U6, add 49 μL ddH ₂ O to dilute; aspirate 1 μL of sgRNA, add 49 μL ddH 2 O to dilute each by 10 μL, and mix well. The details are as follows: mixed template DNA (U6+sgRNA): 1.5 μL, Primer F/R: 2.4 μL each, sterilized ddH2O: 6.9 μL, 2×phanta maxBuffer: 15 μL, dNTP mix: 1.2 μL, Phanta enzyme: 0.6 μL. The Overlap PCR program is as follows: (1) 94°C for 5 minutes, (2) 94°C for 30 seconds, (3) 62°C for 35 seconds, (4) 72°C for 30 seconds, step (5) is from step (2)-( 4) Step cycle 32 times, (6) 72°C for 10 minutes, (7) 25°C for 5 minutes. The primer sequences required for vector construction are shown in Table 1.

Table 1 Primer sequences required for vector construction

(4) Construction into backbone vector by recombinant cloning. The CPB-Ubi-hspcas9 vector was digested with HindIII and recovered. Both the U6-gRNA and the vector were joined by homologous recombination. Before preparing the reaction solution, ensure that the concentrations of each Overlap product are close to the same. The 20 μL homologous recombination system is as follows: Cas Hind III: 3 μL, T-1F Overlap: 1 μL, sterilized ddH2O: 10 μL, 5×CE MultiS buffer: 4 μL, Exnase MultiS ( Product number: C113-01/02): 2 μL.

2. Maize genetic transformation

The vector was transferred into Agrobacterium EHA105 by electroporation, and identified by PCR. The young embryos of the freshly peeled corn inbred line KN5585 (an inbred line selected by Weimi Biotechnology (Jiangsu) Co., Ltd.) of about 1 mm were used as materials, and the peeled corn embryos were put into 2 mL of the suspension containing 1.8 mL. In a plastic centrifuge tube, about 150 immature immature embryos were processed within 30 minutes; the suspension was aspirated, the remaining corn embryos were placed in the tube, and 1.0 mL of Agrobacterium suspension was added, and left for 5 minutes. The immature embryos in the centrifuge tube were suspended and poured onto the co-culture medium, and the excess Agrobacterium liquid on the surface was removed with a pipette, and co-cultured at 23°C for 3 days in the dark. After co-cultivation, immature embryos were transferred to resting medium, cultured at 28°C in the dark for 6 days, placed on screening medium containing 5 mg/L Bialaphos, and the selection culture was started for 2 weeks, and then transferred to screening medium containing 8 mg/L Bialaphos. The culture medium was screened for 2 weeks. The resistant calli were transferred to differentiation medium 1, 25°C, 5000lx, and cultivated in the light for 1 week. Then transfer the callus to differentiation medium 2, and cultivate in light for 2 weeks; transfer the differentiated seedlings to rooting medium, 25 ° C, 5000lx, and cultivate in light until rooting; transfer the seedlings into small pots and grow, certain growth Transplant in the greenhouse after the stage and harvest offspring seeds 3-4 months later.

3. Characterization of gene-edited plants

The flowering period traits of the obtained gene editing materials were identified, and it was found that some of these genes could indeed cause significant changes in the flowering period of maize after editing, while some gene editing could not observe obvious changes in the flowering period. See Table 2.

Table 2 Changes in flowering period after gene editing

Example 2 In-depth analysis of maize flowering stage characters and identification of mutant genes

Perform more in-depth trait characterization of gene-edited materials with altered flowering traits and analyze specific editing sites. After the gene editing of GRMZM2G065896, the flowering period of maize in the T0 generation material was earlier than that of the recipient control KN5585. The flowering stage of the T1 generation material of this transformant was further characterized and the specific editing sites were analyzed.

The target site of GRMZM2G065896 designed during gene editing is shown in SEQ ID NO.4. The gRNA sequence expressed by the vector containing this target is shown in SEQ ID NO.5.

Seedling-stage DNA was extracted to detect gene editing. Primers were designed, and the primer sequences are shown in SEQ ID NO.10 and SEQ ID NO.11. Amplify the target editing segment. The amplification system is as follows: DNA: 3 μL, 1 μL of bidirectional primers, 2×TaqMix: 7.5 μL, ddH ₂ O: 2.5 μL, and a total volume of 10 μL. PCR reaction conditions are as follows: (1) 94°C for 5 minutes, (2) 94°C for 40 seconds, (3) 57°C for 30 seconds, (4) 72°C for 60 seconds, (5) from step (2) to step (4) Cycle 35 times, (6) 72 ℃ for 7 minutes, (7) 4 ℃ storage. The PCR products were handed over to Wuhan Qingke Biotechnology Co., Ltd. for Sanger sequencing. The PCR amplification and sequencing results of the transformant and the receptor KN5585 were compared, and the material with base substitution, insertion or deletion was the positive editing material, otherwise it was negative material.

After sequence comparison, it was found that there were 4 types of editing materials, among which 37bp was deleted at the target of A1 material and C was changed to T, 1bp was deleted at the target point of A2 material, 2bp was deleted at the target point of A3 material, and the target point of A4 material was deleted. 3 bp was deleted (Table 3). Therefore, after editing, the gene sequences of A1-A4 materials were changed from SEQ ID NO.2 to SEQ ID NO.6-SEQ ID NO.9, respectively.

Table 3 Flowering trait data of gene-edited maize materials

"-" indicates deleted sequences, bold indicates replaced bases, boxes indicate PAM sequences, and underlined indicates editing targets.

In the summer of 2018, the characteristics of the flowering period (including tasseling period, powder loosening period, and silking period) of A1-A4 materials were investigated in the experimental field in Jilin Province. The results are shown in Table 4. The edited materials of A1-A4 all had earlier flowering period than the unedited control material to different degrees, which proved that the gene controls the flowering period traits, and the flowering period was earlier after gene editing.

Table 4. Flowering trait data of gene-edited maize materials

The data of flowering period are expressed as mean ± standard deviation, unit: day. "CK" indicates unedited control material. "**" indicates a very significant difference compared with the control (P<0.01), and "*" indicates a significant difference compared with the control (P<0.05).

Therefore, the mutant gene shown in SEQ ID NO.6 or SEQ ID NO.7 or SEQ ID NO.8 or SEQ ID NO.9 can make the flowering period of maize moderately early. The mutant gene can be introduced into maize materials with different genetic backgrounds through sexual hybridization, thereby creating new early-flowering maize varieties.

In the process of introduction, the primer pair of the sequence shown in SEQ ID NO.10 and SEQ ID NO.11 can be used to detect whether the above-mentioned mutant gene is contained in the maize genome, and the genomic DNA of the sample to be tested is PCR amplified with this primer, And sequence the amplification product, if the amplification product is consistent with the sequence shown in the 3083-3304th position of SEQ ID NO:6, it contains the mutant gene shown in SEQ ID NO.6; if the amplification product is consistent with the sequence shown in the SEQ ID NO:7 If the sequence shown in the 3083-3340 position is consistent, it contains the mutant gene shown in SEQ ID NO.7; if the amplified product is consistent with the sequence shown in the 3083-3339 position of SEQ ID NO:8, it contains the SEQ ID NO.8. If the amplification product is consistent with the sequence shown in position 3083-3338 of SEQ ID NO: 9, it contains the mutant gene shown in SEQ ID NO. 9; if the amplification product is consistent with the sequence shown in position 3083 of SEQ ID NO: 2 If the sequence shown at position -3341 is consistent, it is an unedited genotype.

Although the present invention has been described in detail above with general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention fall within the scope of the claimed protection of the present invention.

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tctagctatt agggaagatt atttggaaag atggaagcaa ctagattagg gaacttctgt 1200

gagtaagaag ttgagatgac ttgcctggat tggtcagagt catagtcata actttcaaaa 1260

tgttactaga gatctaatca tgaaacaaca taaatatttt gtcttcacgg ttacaaggtg 1320

attttgccag tcttccattt gaacgtgttt ttgaaatcta tttggtctat ttttcttcaa 1380

aaaaaaatct atttggtcta tctcaattcc ttatctgctt attagtgaga atgcctataa 1440

atgcaatcat gagccctagt gatgtcgttt gtagtgaaaa gcttcactgg gatgtatgtc 1500

ctaagataaa ccaagaccaa agtaagcatg atgcgaccat actgaaggtt ttcagggtga 1560

ccatatgcct atgtcattat tgatttgttt gagtgcatat ggtgatgaga ttgtttcact 1620

gacacgtgta tcccagggcc acatgcgagc cagcgtgtca gtgagatatg ggagtcacat 1680

gtgcagaaac tcaacctagg ggagctgtat atcctaagta ctttctcttg atcaggttca 1740

ggctgttctt ttggtacttg gagggtacga ggttccgcct ggtttggtaa acatggctgt 1800

ttcttcagca aatgatgaga aggtatccgc atacttactg cagtgaggat aggtatttcc 1860

agcttctcct aaagcattgt ctcatgtgca gaatacaact gtggctgcta gaagggttgc 1920

ttccttaatg agattccgtg agaagagaaa ggaaagatgt ttcgataaaa ggattagata 1980

cagtgtgcgc aaggaggttg cccaaaagta agtacagaat aatgtcactt ctatgtttag 2040

caatgatata attattcttc gcatgggcat aatgaatttt taaaggcaat cttgattaac 2100

agattgtttg ctgtcatttg gaggatttga gatataaata gtaagaagtc tgcatttgtt 2160

agtctccaca ttagataaaa ttatcagctt attgcagact tgattatgtt tgactttgac 2220

attgctttac cacattaata ggatgtgctg taagaccagg atttacttaa ttctctcctt 2280

gtatcactct gtttaaatag gaatacacat gccatattca gattattatc actgttatat 2340

catatccaat cttaggtcac ctttttatct cgaatagttt ctccaagtac taatgccaga 2400

aaaaaactaa ataggataat aatctgaatg tgcaccatgg atctacctat ttggagtact 2460

tgtgcactaa aggtgttatt caaggttacc ataagcgaag tcaatgttta aaagatttat 2520

ttcagtcaga tataaaaatg aacgaggact ttcttctatg ccttttagtc cacgatttgt 2580

aagaagtatc atgtaaaggc ttagatgaac tccgtggaga atggaacaga tgatttctat 2640

tatttccatt tgtcagaact tttccagaac ttatgtgctt atttgcatac ctacctggtt 2700

caatccagtt acctttttttt atatcaatca tgctagaaaa ggtgttttttt tcttcttttc 2760

atcatactgt ttacattctg ttgcaataaa ccctggccaa acaattgctg ctcaccatca 2820

ttcaccactg ggaatatttc ccaggtttat ttaccgtaac cattgcataa ccatattttg 2880

cattcaaatg atcgctactc agtgatgttt ttgttgtttt ttttttttga atcttgcatt 2940

gcacaatctc aataatactt tcaggatgaa acggcgtaaa ggccaatttg ctgggagatc 3000

agattttggc gatggtgcct gttcttctgc agcttgtggc tctccagcta acggcgagga 3060

tgatcatttt cgagaaaccc agtgagttag ttgccatgtg ccaactatca ctttgtgatt 3120

gtgtgtatgt gttacatcag tttcatatgc agaaagttgt tgccttcatg tatggactaa 3180

tgttggcctg ttttgctagt tgccaaaatt gtggtatcag ctcaaggctt actccagcga 3240

tgcgtcgggg cccagctggt ccgaggtccc tctgcaatgc ttgtggctta atgtgggcaa 3300

ataaggtaca actttctcta tgccaatcat cgcacctctt gtccagttgt cctgtttggc 3360

tgtttggtac ctttttcctc atgtcaaata aaaagcatca ttcttcttaa tttgaaaaac 3420

ttcccaatga gatattctaa ttggtcaaaa aatgatctga ttaaagtgtg tctattagca 3480

acattcctac tagttaacct ttgttttttt cagggtactc tacgaagtcc tctaaatgca 3540

cccaaaatga ctcagcagct tcttgccaat ccatgtaaca tggtgcgcta catttgtaat 3600

agaaacttgt ttatttgaac gccttttctt gtagcgaaga ttatctgaac agtacacttg 3660

tgcaggtaga tactgatgac aaaaactcaa atgttcttcc tgtggagcat aatcaagcca 3720

cacccaaaac tgactcgatg taagcatgct ctttactgta cgtgaaatca ttgtctggaa 3780

gctgtgtcat gtacatggtc ggatgtgcaa atctgaactt cgtgttctct tgagttaagc 3840

tcaaactggg agggacaaag gcgctgtttg tttaccctct agattatata atccaactta 3900

aataagttga gaggcaaaca tataactcag attattaggt ggattatata atctagattc 3960

gtacaacaac aacaacaaag cctttttgtc ccaagcacgt tggggtaggc tagagatgaa 4020

accccgtatg aaaacttcag agctcaaccc caaagaaaag aaaagggaaa caatggcaaa 4080

agagaaccga aaaacgacaa aacggggaaa cacataaaaa gagataaaac ccacaagaaa 4140

cagccagatc taaatgggca caagaaaagg tcaaacgatt aaggaggaaa agcgaaacta 4200

tgaatcaagg ttctggcacg tgaattgcac acttccaccc cttcctatcc acggcgagct 4260

ctttatcaat attccactct ttatcaataa tctagattcg tagattatga taattcataa 4320

gcaggttatt aggtgcttct ataatccata agctagatta taataatcct agaaggaaac 4380

aacagggcca aagtctcatg acacagttta gatggatttt ttttcatctg ctttatcagt 4440

gcatatagct gcctaggtaa cttctatttt ttacccaaga ggtctctcca aaactttctt 4500

tataatgaag tacaccaatc actgaacctt aatttattgg tcaggatgcc aaaagaggag 4560

cagaagctgg acatacgctt gcccaccgaa gaagatacga aggcagtttc atgacaaatg 4620

ctcaaacatg tatatagatc gagacaggtt tctgagttta ggatgacata taaatcagta 4680

aaactaacaa aggcagcaca aaaaaaatta catgcattgt gttgccacat ttatctccgt 4740

acccgattca tattgtattg ctaactgatg cagttcttac tccttaccct gtgtcaacct 4800

gaccaggtga cttggcagga actcaaaatg ttttcaagaa tacgacgcat tcttgagaaa 4860

cgttttgttt attgttgtaa aaaccacttc aatagtaatt tgcatcaact ttgatgcaat 4920

ttattttata gaaaaacatg tgatattttg gt 4952

<210> 3

<211> 1325

<212> DNA

<213> Zea mays

<400> 3

cagagtggga ccagcaagcc gcgccagtgt acatgtcttc accgtcacac gtgcgctcct 60

ctcgtccatc ctccacacgt gcgctcaaac cgagcttgtc cacggaccgc accctgtctg 120

cacgcatctc caccgtccga tccccagccg atggcggcgg agcccgcggc ggccgaccac 180

gatctccggc cacccctggc ggatggcgcc gctgctgccg gcgtgggcgc cgcctccttg 240

gcggcggcgg cgggcgcggc agaggcgctg atgagcgcga cgtcggagca gctcacgctg 300

gtgtatcagg gcgacgtcta cgtcttcgac cccgtcccgc cccaaaaggt tcaggctgtt 360

cttttggtac ttggagggta cgaggttccg cctggtttgg taaacatggc tgtttcttca 420

gcaaatgatg agaagaatac aactgtggct gctagaaggg ttgcttcctt aatgagattc 480

cgtgagaaga gaaaggaaag atgtttcgat aaaaggatta gatacagtgt gcgcaaggag 540

gttgcccaaa agatgaaacg gcgtaaaggc caatttgctg ggagatcaga ttttggcgat 600

ggtgcctgtt cttctgcagc ttgtggctct ccagctaacg gcgaggatga tcattttcga 660

gaaacccatt gccaaaattg tggtatcagc tcaaggctta ctccagcgat gcgtcggggc 720

ccagctggtc cgaggtccct ctgcaatgct tgtggcttaa tgtgggcaaa taagggtact 780

ctacgaagtc ctctaaatgc acccaaaatg actcagcagc ttcttgccaa tccatgtaac 840

atggtagata ctgatgacaa aaactcaaat gttcttcctg tggagcataa tcaagccaca 900

cccaaaactg actcgatgat gccaaaagag gagcagaagc tggacatacg cttgcccacc 960

gaagaagata cgaaggcagt ttcatgacaa atgctcaaac atgtatatag atcgagacag 1020

gtttctgagt ttaggatgac atataaatca gtaaaactaa caaaggcagc acaaaaaaaa 1080

ttacatgcat tgtgttgcca catttatctc cgtacccgat tcatattgta ttgctaactg 1140

atgcagttct tactccttac cctgtgtcaa cctgaccagg tgacttggca ggaactcaaa 1200

atgttttcaa gaatacgacg cattcttgag aaacgttttg ttattgttg taaaaaccac 1260

ttcaatagta atttgcatca actttgatgc aatttatttt atagaaaaac atgtgatatt 1320

ttggt 1325

<210> 4

<211> 20

<212> DNA

<213> Zea mays

<400> 4

gcttactcca gcgatgcgtc 20

<210> 5

<211> 103

<212> RNA

<213> unknown (synthetic)

<400> 5

gcuuacucca gcgaugcguc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu uuu 103

<210> 6

<211> 4915

<212> DNA

<213> unknown (synthetic)

<400> 6

cagagtggga ccagcaagcc gcgccagtgt acatgtcttc accgtcacac gtgcgctcct 60

ctcgtccatc ctccacacgt gcgctcaaac cgagcttgtc cacggaccgc accctgtctg 120

cacgcatctc caccgtccga tccccagccg atggcggcgg agcccgcggc ggccgaccac 180

gatctccggc cacccctggc ggatggcgcc gctgctgccg gcgtgggcgc cgcctccttg 240

gcggcggcgg cgggcgcggc agaggcgctg atgagcgcga cgtcggagca gctcacgctg 300

gtgtatcagg gcgacgtcta cgtcttcgac cccgtcccgc cccaaaaggt cgcgcctctt 360

gcacccctga taattccatt ggcccctccc aaattctctc cttggttcct atatgttgcg 420

atggtcgtta atagtcttgg ccggagatta gcgtgcgcgg ggcgccggct atgaattagc 480

aaatattgtt ctgcttttgc gggggcgctc tgctgtcgag ttgcacataa ttaaattctg 540

tcttcatcgt tcttgattga attctaaggg gacttgattg ccagctatgg ctaagttagt 600

cgcagtttct gcttcgctac tgtaaattga ccatattcct ttgttccctt gctgaattgc 660

accaaagaat acgggtgcta tatgatatga ctgtgctttt ggtatcctgt aaggggctga 720

actaatgtta ttatgaattg ctctgtccat ttttcgatca gtatggatat accgttctac 780

tgttcgagtg gaatcgagaa tgcttgatcg ctaggtagac acttttttgt gcgtgttgag 840

atgttttata gttgtgtcgg ggaaaagtcg cgagtttgat gaacagcgga gtagatatct 900

tcgttcttca gctaggattt taacatattt gtgtttttgt tatagttgcc aaataaagtt 960

ccaatagaag catgtatgtt ctttttttcc gatgagaagt ataaattctt tgttccttaa 1020

gttttattag tgttttaaac ccaaatatga aaatttggaa ctatgtttca tctttggtcc 1080

tggactaagc tcactcatat gatttgcttt tctcattggc aagttgagga ttatttttgt 1140

tctagctatt agggaagatt atttggaaag atggaagcaa ctagattagg gaacttctgt 1200

gagtaagaag ttgagatgac ttgcctggat tggtcagagt catagtcata actttcaaaa 1260

tgttactaga gatctaatca tgaaacaaca taaatatttt gtcttcacgg ttacaaggtg 1320

attttgccag tcttccattt gaacgtgttt ttgaaatcta tttggtctat ttttcttcaa 1380

aaaaaaatct atttggtcta tctcaattcc ttatctgctt attagtgaga atgcctataa 1440

atgcaatcat gagccctagt gatgtcgttt gtagtgaaaa gcttcactgg gatgtatgtc 1500

ctaagataaa ccaagaccaa agtaagcatg atgcgaccat actgaaggtt ttcagggtga 1560

ccatatgcct atgtcattat tgatttgttt gagtgcatat ggtgatgaga ttgtttcact 1620

gacacgtgta tcccagggcc acatgcgagc cagcgtgtca gtgagatatg ggagtcacat 1680

gtgcagaaac tcaacctagg ggagctgtat atcctaagta ctttctcttg atcaggttca 1740

ggctgttctt ttggtacttg gagggtacga ggttccgcct ggtttggtaa acatggctgt 1800

ttcttcagca aatgatgaga aggtatccgc atacttactg cagtgaggat aggtatttcc 1860

agcttctcct aaagcattgt ctcatgtgca gaatacaact gtggctgcta gaagggttgc 1920

ttccttaatg agattccgtg agaagagaaa ggaaagatgt ttcgataaaa ggattagata 1980

cagtgtgcgc aaggaggttg cccaaaagta agtacagaat aatgtcactt ctatgtttag 2040

caatgatata attattcttc gcatgggcat aatgaatttt taaaggcaat cttgattaac 2100

agattgtttg ctgtcatttg gaggatttga gatataaata gtaagaagtc tgcatttgtt 2160

agtctccaca ttagataaaa ttatcagctt attgcagact tgattatgtt tgactttgac 2220

attgctttac cacattaata ggatgtgctg taagaccagg atttacttaa ttctctcctt 2280

gtatcactct gtttaaatag gaatacacat gccatattca gattattatc actgttatat 2340

catatccaat cttaggtcac ctttttatct cgaatagttt ctccaagtac taatgccaga 2400

aaaaaactaa ataggataat aatctgaatg tgcaccatgg atctacctat ttggagtact 2460

tgtgcactaa aggtgttatt caaggttacc ataagcgaag tcaatgttta aaagatttat 2520

ttcagtcaga tataaaaatg aacgaggact ttcttctatg ccttttagtc cacgatttgt 2580

aagaagtatc atgtaaaggc ttagatgaac tccgtggaga atggaacaga tgatttctat 2640

tatttccatt tgtcagaact tttccagaac ttatgtgctt atttgcatac ctacctggtt 2700

caatccagtt acctttttttt atatcaatca tgctagaaaa ggtgttttttt tcttcttttc 2760

atcatactgt ttacattctg ttgcaataaa ccctggccaa acaattgctg ctcaccatca 2820

ttcaccactg ggaatatttc ccaggtttat ttaccgtaac cattgcataa ccatattttg 2880

cattcaaatg atcgctactc agtgatgttt ttgttgtttt ttttttttga atcttgcatt 2940

gcacaatctc aataatactt tcaggatgaa acggcgtaaa ggccaatttg ctgggagatc 3000

agattttggc gatggtgcct gttcttctgc agcttgtggc tctccagcta acggcgagga 3060

tgatcatttt cgagaaaccc agtgagttag ttgccatgtg ccaactatca ctttgtgatt 3120

gtgtgtatgt gttacatcag tttcatatgc agaaagttgt tgccttcatg tatggactaa 3180

tgttggcctg ttttgctagt tgccaaaatt gtggtatcag ctcaaggctt actccagcga 3240

tgtttgtggc ttaatgtggg caaataaggt acaactttct ctatgccaat catcgcacct 3300

cttgtccagt tgtcctgttt ggctgtttgg tacctttttc ctcatgtcaa ataaaaagca 3360

tcattcttct taatttgaaa aacttcccaa tgagatattc taattggtca aaaaatgatc 3420

tgattaaagt gtgtctatta gcaacattcc tactagttaa cctttgtttt tttcagggta 3480

ctctacgaag tcctctaaat gcacccaaaa tgactcagca gcttcttgcc aatccatgta 3540

acatggtgcg cttatttgt aatagaaact tgtttatttg aacgcctttt cttgtagcga 3600

agattatctg aacagtacac ttgtgcaggt agatactgat gacaaaaact caaatgttct 3660

tcctgtggag cataatcaag ccacacccaa aactgactcg atgtaagcat gctctttact 3720

gtacgtgaaa tcattgtctg gaagctgtgt catgtacatg gtcggatgtg caaatctgaa 3780

cttcgtgttc tcttgagtta agctcaaact gggagggaca aaggcgctgt ttgtttaccc 3840

tctagattat ataatccaac ttaaataagt tgagaggcaa acatataact cagattatta 3900

ggtggattat ataatctaga ttcgtacaac aacaacaaca aagccttttt gtcccaagca 3960

cgttggggta ggctagagat gaaaccccgt atgaaaactt cagagctcaa ccccaaagaa 4020

aagaaaaggg aaacaatggc aaaagagaac cgaaaaacga caaaacgggg aaacacataa 4080

aaagagataa aacccacaag aaacagccag atctaaatgg gcacaagaaa aggtcaaacg 4140

attaaggagg aaaagcgaaa ctatgaatca aggttctggc acgtgaattg cacacttcca 4200

ccccttccta tccacggcga gctctttatc aatattccac tctttatcaa taatctagat 4260

tcgtagatta tgataattca taagcaggtt attaggtgct tctataatcc ataagctaga 4320

ttataataat cctagaagga aacaacaggg ccaaagtctc atgacacagt ttagatggat 4380

tttttttcat ctgctttatc agtgcatata gctgcctagg taacttctat tttttaccca 4440

agaggtctct ccaaaacttt ctttataatg aagtacacca atcactgaac cttaatttat 4500

tggtcaggat gccaaaagag gagcagaagc tggacatacg cttgcccacc gaagaagata 4560

cgaaggcagt ttcatgacaa atgctcaaac atgtatatag atcgagacag gtttctgagt 4620

ttaggatgac atataaatca gtaaaactaa caaaggcagc acaaaaaaaa ttacatgcat 4680

tgtgttgcca catttatctc cgtacccgat tcatattgta ttgctaactg atgcagttct 4740

tactccttac cctgtgtcaa cctgaccagg tgacttggca ggaactcaaa atgttttcaa 4800

gaatacgacg cattcttgag aaacgttttg ttattgttg taaaaaccac ttcaatagta 4860

atttgcatca actttgatgc aatttatttt atagaaaaac atgtgatatt ttggt 4915

<210> 7

<211> 4951

<212> DNA

<213> unknown (synthetic)

<400> 7

cagagtggga ccagcaagcc gcgccagtgt acatgtcttc accgtcacac gtgcgctcct 60

ctcgtccatc ctccacacgt gcgctcaaac cgagcttgtc cacggaccgc accctgtctg 120

cacgcatctc caccgtccga tccccagccg atggcggcgg agcccgcggc ggccgaccac 180

gatctccggc cacccctggc ggatggcgcc gctgctgccg gcgtgggcgc cgcctccttg 240

gcggcggcgg cgggcgcggc agaggcgctg atgagcgcga cgtcggagca gctcacgctg 300

gtgtatcagg gcgacgtcta cgtcttcgac cccgtcccgc cccaaaaggt cgcgcctctt 360

gcacccctga taattccatt ggcccctccc aaattctctc cttggttcct atatgttgcg 420

atggtcgtta atagtcttgg ccggagatta gcgtgcgcgg ggcgccggct atgaattagc 480

aaatattgtt ctgcttttgc gggggcgctc tgctgtcgag ttgcacataa ttaaattctg 540

tcttcatcgt tcttgattga attctaaggg gacttgattg ccagctatgg ctaagttagt 600

cgcagtttct gcttcgctac tgtaaattga ccatattcct ttgttccctt gctgaattgc 660

accaaagaat acgggtgcta tatgatatga ctgtgctttt ggtatcctgt aaggggctga 720

actaatgtta ttatgaattg ctctgtccat ttttcgatca gtatggatat accgttctac 780

tgttcgagtg gaatcgagaa tgcttgatcg ctaggtagac acttttttgt gcgtgttgag 840

atgttttata gttgtgtcgg ggaaaagtcg cgagtttgat gaacagcgga gtagatatct 900

tcgttcttca gctaggattt taacatattt gtgtttttgt tatagttgcc aaataaagtt 960

ccaatagaag catgtatgtt ctttttttcc gatgagaagt ataaattctt tgttccttaa 1020

gttttattag tgttttaaac ccaaatatga aaatttggaa ctatgtttca tctttggtcc 1080

tggactaagc tcactcatat gatttgcttt tctcattggc aagttgagga ttatttttgt 1140

tctagctatt agggaagatt atttggaaag atggaagcaa ctagattagg gaacttctgt 1200

gagtaagaag ttgagatgac ttgcctggat tggtcagagt catagtcata actttcaaaa 1260

tgttactaga gatctaatca tgaaacaaca taaatatttt gtcttcacgg ttacaaggtg 1320

attttgccag tcttccattt gaacgtgttt ttgaaatcta tttggtctat ttttcttcaa 1380

aaaaaaatct atttggtcta tctcaattcc ttatctgctt attagtgaga atgcctataa 1440

atgcaatcat gagccctagt gatgtcgttt gtagtgaaaa gcttcactgg gatgtatgtc 1500

ctaagataaa ccaagaccaa agtaagcatg atgcgaccat actgaaggtt ttcagggtga 1560

ccatatgcct atgtcattat tgatttgttt gagtgcatat ggtgatgaga ttgtttcact 1620

gacacgtgta tcccagggcc acatgcgagc cagcgtgtca gtgagatatg ggagtcacat 1680

gtgcagaaac tcaacctagg ggagctgtat atcctaagta ctttctcttg atcaggttca 1740

ggctgttctt ttggtacttg gagggtacga ggttccgcct ggtttggtaa acatggctgt 1800

ttcttcagca aatgatgaga aggtatccgc atacttactg cagtgaggat aggtatttcc 1860

agcttctcct aaagcattgt ctcatgtgca gaatacaact gtggctgcta gaagggttgc 1920

ttccttaatg agattccgtg agaagagaaa ggaaagatgt ttcgataaaa ggattagata 1980

cagtgtgcgc aaggaggttg cccaaaagta agtacagaat aatgtcactt ctatgtttag 2040

caatgatata attattcttc gcatgggcat aatgaatttt taaaggcaat cttgattaac 2100

agattgtttg ctgtcatttg gaggatttga gatataaata gtaagaagtc tgcatttgtt 2160

agtctccaca ttagataaaa ttatcagctt attgcagact tgattatgtt tgactttgac 2220

attgctttac cacattaata ggatgtgctg taagaccagg atttacttaa ttctctcctt 2280

gtatcactct gtttaaatag gaatacacat gccatattca gattattatc actgttatat 2340

catatccaat cttaggtcac ctttttatct cgaatagttt ctccaagtac taatgccaga 2400

aaaaaactaa ataggataat aatctgaatg tgcaccatgg atctacctat ttggagtact 2460

tgtgcactaa aggtgttatt caaggttacc ataagcgaag tcaatgttta aaagatttat 2520

ttcagtcaga tataaaaatg aacgaggact ttcttctatg ccttttagtc cacgatttgt 2580

aagaagtatc atgtaaaggc ttagatgaac tccgtggaga atggaacaga tgatttctat 2640

tatttccatt tgtcagaact tttccagaac ttatgtgctt atttgcatac ctacctggtt 2700

caatccagtt acctttttttt atatcaatca tgctagaaaa ggtgttttttt tcttcttttc 2760

atcatactgt ttacattctg ttgcaataaa ccctggccaa acaattgctg ctcaccatca 2820

ttcaccactg ggaatatttc ccaggtttat ttaccgtaac cattgcataa ccatattttg 2880

cattcaaatg atcgctactc agtgatgttt ttgttgtttt ttttttttga atcttgcatt 2940

gcacaatctc aataatactt tcaggatgaa acggcgtaaa ggccaatttg ctgggagatc 3000

agattttggc gatggtgcct gttcttctgc agcttgtggc tctccagcta acggcgagga 3060

tgatcatttt cgagaaaccc agtgagttag ttgccatgtg ccaactatca ctttgtgatt 3120

gtgtgtatgt gttacatcag tttcatatgc agaaagttgt tgccttcatg tatggactaa 3180

tgttggcctg ttttgctagt tgccaaaatt gtggtatcag ctcaaggctt actccagcga 3240

tggtcggggc ccagctggtc cgaggtccct ctgcaatgct tgtggcttaa tgtgggcaaa 3300

taaggtacaa ctttctctat gccaatcatc gcacctcttg tccagttgtc ctgtttggct 3360

gtttggtacc tttttcctca tgtcaaataa aaagcatcat tcttcttaat ttgaaaaact 3420

tcccaatgag atattctaat tggtcaaaaa atgatctgat taaagtgtgt ctattagcaa 3480

cattcctact agttaacctt tgtttttttc agggtactct acgaagtcct ctaaatgcac 3540

ccaaaatgac tcagcagctt cttgccaatc catgtaacat ggtgcgctac atttgtaata 3600

gaaacttgtt tatttgaacg ccttttcttg tagcgaagat tatctgaaca gtacacttgt 3660

gcaggtagat actgatgaca aaaactcaaa tgttcttcct gtggagcata atcaagccac 3720

acccaaaact gactcgatgt aagcatgctc tttactgtac gtgaaatcat tgtctggaag 3780

ctgtgtcatg tacatggtcg gatgtgcaaa tctgaacttc gtgttctctt gagttaagct 3840

caaactggga gggacaaagg cgctgtttgt ttaccctcta gattatataa tccaacttaa 3900

ataagttgag aggcaaacat ataactcaga ttattaggtg gattatataa tctagattcg 3960

tacaacaaca acaacaaagc ctttttgtcc caagcacgtt ggggtaggct agagatgaaa 4020

ccccgtatga aaacttcaga gctcaacccc aaagaaaaga aaagggaaac aatggcaaaa 4080

gagaaccgaa aaacgacaaa acggggaaac acataaaaag agataaaacc cacaagaaac 4140

agccagatct aaatgggcac aagaaaaggt caaacgatta aggaggaaaa gcgaaactat 4200

gaatcaaggt tctggcacgt gaattgcaca cttccacccc ttcctatcca cggcgagctc 4260

tttatcaata ttccactctt tatcaataat ctagattcgt agattatgat aattcataag 4320

caggttatta ggtgcttcta taatccataa gctagattat aataatccta gaaggaaaca 4380

acagggccaa agtctcatga cacagtttag atggattttt tttcatctgc tttatcagtg 4440

catatagctg cctaggtaac ttctattttt tacccaagag gtctctccaa aactttcttt 4500

ataatgaagt acaccaatca ctgaacctta atttattggt caggatgcca aaagaggagc 4560

agaagctgga catacgcttg cccaccgaag aagatacgaa ggcagtttca tgacaaatgc 4620

tcaaacatgt atatagatcg agacaggttt ctgagtttag gatgacatat aaatcagtaa 4680

aactaacaaa ggcagcacaa aaaaaattac atgcattgtg ttgccacatt tatctccgta 4740

cccgattcat attgtattgc taactgatgc agttcttact ccttaccctg tgtcaacctg 4800

accaggtgac ttggcaggaa ctcaaaatgt tttcaagaat acgacgcatt cttgagaaac 4860

gttttgttta ttgttgtaaa aaccacttca atagtaattt gcatcaactt tgatgcaatt 4920

tattttatag aaaaacatgt gatattttgg t 4951

<210> 8

<211> 4950

<212> DNA

<213> unknown (synthetic)

<400> 8

cagagtggga ccagcaagcc gcgccagtgt acatgtcttc accgtcacac gtgcgctcct 60

ctcgtccatc ctccacacgt gcgctcaaac cgagcttgtc cacggaccgc accctgtctg 120

cacgcatctc caccgtccga tccccagccg atggcggcgg agcccgcggc ggccgaccac 180

gatctccggc cacccctggc ggatggcgcc gctgctgccg gcgtgggcgc cgcctccttg 240

gcggcggcgg cgggcgcggc agaggcgctg atgagcgcga cgtcggagca gctcacgctg 300

gtgtatcagg gcgacgtcta cgtcttcgac cccgtcccgc cccaaaaggt cgcgcctctt 360

gcacccctga taattccatt ggcccctccc aaattctctc cttggttcct atatgttgcg 420

atggtcgtta atagtcttgg ccggagatta gcgtgcgcgg ggcgccggct atgaattagc 480

aaatattgtt ctgcttttgc gggggcgctc tgctgtcgag ttgcacataa ttaaattctg 540

tcttcatcgt tcttgattga attctaaggg gacttgattg ccagctatgg ctaagttagt 600

cgcagtttct gcttcgctac tgtaaattga ccatattcct ttgttccctt gctgaattgc 660

accaaagaat acgggtgcta tatgatatga ctgtgctttt ggtatcctgt aaggggctga 720

actaatgtta ttatgaattg ctctgtccat ttttcgatca gtatggatat accgttctac 780

tgttcgagtg gaatcgagaa tgcttgatcg ctaggtagac acttttttgt gcgtgttgag 840

atgttttata gttgtgtcgg ggaaaagtcg cgagtttgat gaacagcgga gtagatatct 900

tcgttcttca gctaggattt taacatattt gtgtttttgt tatagttgcc aaataaagtt 960

ccaatagaag catgtatgtt ctttttttcc gatgagaagt ataaattctt tgttccttaa 1020

gttttattag tgttttaaac ccaaatatga aaatttggaa ctatgtttca tctttggtcc 1080

tggactaagc tcactcatat gatttgcttt tctcattggc aagttgagga ttatttttgt 1140

tctagctatt agggaagatt atttggaaag atggaagcaa ctagattagg gaacttctgt 1200

gagtaagaag ttgagatgac ttgcctggat tggtcagagt catagtcata actttcaaaa 1260

tgttactaga gatctaatca tgaaacaaca taaatatttt gtcttcacgg ttacaaggtg 1320

attttgccag tcttccattt gaacgtgttt ttgaaatcta tttggtctat ttttcttcaa 1380

aaaaaaatct atttggtcta tctcaattcc ttatctgctt attagtgaga atgcctataa 1440

atgcaatcat gagccctagt gatgtcgttt gtagtgaaaa gcttcactgg gatgtatgtc 1500

ctaagataaa ccaagaccaa agtaagcatg atgcgaccat actgaaggtt ttcagggtga 1560

ccatatgcct atgtcattat tgatttgttt gagtgcatat ggtgatgaga ttgtttcact 1620

gacacgtgta tcccagggcc acatgcgagc cagcgtgtca gtgagatatg ggagtcacat 1680

gtgcagaaac tcaacctagg ggagctgtat atcctaagta ctttctcttg atcaggttca 1740

ggctgttctt ttggtacttg gagggtacga ggttccgcct ggtttggtaa acatggctgt 1800

ttcttcagca aatgatgaga aggtatccgc atacttactg cagtgaggat aggtatttcc 1860

agcttctcct aaagcattgt ctcatgtgca gaatacaact gtggctgcta gaagggttgc 1920

ttccttaatg agattccgtg agaagagaaa ggaaagatgt ttcgataaaa ggattagata 1980

cagtgtgcgc aaggaggttg cccaaaagta agtacagaat aatgtcactt ctatgtttag 2040

caatgatata attattcttc gcatgggcat aatgaatttt taaaggcaat cttgattaac 2100

agattgtttg ctgtcatttg gaggatttga gatataaata gtaagaagtc tgcatttgtt 2160

agtctccaca ttagataaaa ttatcagctt attgcagact tgattatgtt tgactttgac 2220

attgctttac cacattaata ggatgtgctg taagaccagg atttacttaa ttctctcctt 2280

gtatcactct gtttaaatag gaatacacat gccatattca gattattatc actgttatat 2340

catatccaat cttaggtcac ctttttatct cgaatagttt ctccaagtac taatgccaga 2400

aaaaaactaa ataggataat aatctgaatg tgcaccatgg atctacctat ttggagtact 2460

tgtgcactaa aggtgttatt caaggttacc ataagcgaag tcaatgttta aaagatttat 2520

ttcagtcaga tataaaaatg aacgaggact ttcttctatg ccttttagtc cacgatttgt 2580

aagaagtatc atgtaaaggc ttagatgaac tccgtggaga atggaacaga tgatttctat 2640

tatttccatt tgtcagaact tttccagaac ttatgtgctt atttgcatac ctacctggtt 2700

caatccagtt acctttttttt atatcaatca tgctagaaaa ggtgttttttt tcttcttttc 2760

atcatactgt ttacattctg ttgcaataaa ccctggccaa acaattgctg ctcaccatca 2820

ttcaccactg ggaatatttc ccaggtttat ttaccgtaac cattgcataa ccatattttg 2880

cattcaaatg atcgctactc agtgatgttt ttgttgtttt ttttttttga atcttgcatt 2940

gcacaatctc aataatactt tcaggatgaa acggcgtaaa ggccaatttg ctgggagatc 3000

agattttggc gatggtgcct gttcttctgc agcttgtggc tctccagcta acggcgagga 3060

tgatcatttt cgagaaaccc agtgagttag ttgccatgtg ccaactatca ctttgtgatt 3120

gtgtgtatgt gttacatcag tttcatatgc agaaagttgt tgccttcatg tatggactaa 3180

tgttggcctg ttttgctagt tgccaaaatt gtggtatcag ctcaaggctt actccagcga 3240

cgtcggggcc cagctggtcc gaggtccctc tgcaatgctt gtggcttaat gtgggcaaat 3300

aaggtacaac tttctctatg ccaatcatcg cacctcttgt ccagttgtcc tgtttggctg 3360

tttggtacct ttttcctcat gtcaaataaa aagcatcatt cttcttaatt tgaaaaactt 3420

cccaatgaga tattctaatt ggtcaaaaaa tgatctgatt aaagtgtgtc tattagcaac 3480

attcctacta gttaaccttt gtttttttca gggtactcta cgaagtcctc taaatgcacc 3540

caaaatgact cagcagcttc ttgccaatcc atgtaacatg gtgcgctaca tttgtaatag 3600

aaacttgttt atttgaacgc cttttcttgt agcgaagatt atctgaacag tacacttgtg 3660

caggtagata ctgatgacaa aaactcaaat gttcttcctg tggagcataa tcaagccaca 3720

cccaaaactg actcgatgta agcatgctct ttactgtacg tgaaatcatt gtctggaagc 3780

tgtgtcatgt acatggtcgg atgtgcaaat ctgaacttcg tgttctcttg agttaagctc 3840

aaactgggag ggacaaaggc gctgtttgtt taccctctag attatataat ccaacttaaa 3900

taagttgaga ggcaaacata taactcagat tattaggtgg attatataat ctagattcgt 3960

acaacaacaa caacaaagcc tttttgtccc aagcacgttg gggtaggcta gagatgaaac 4020

cccgtatgaa aacttcagag ctcaacccca aagaaaagaa aagggaaaca atggcaaaag 4080

agaaccgaaa aacgacaaaa cggggaaaca cataaaaaga gataaaaccc acaagaaaca 4140

gccagatcta aatgggcaca agaaaaggtc aaacgattaa ggaggaaaag cgaaactatg 4200

aatcaaggtt ctggcacgtg aattgcacac ttccacccct tcctatccac ggcgagctct 4260

ttatcaatat tccactcttt atcaataatc tagattcgta gattatgata attcataagc 4320

aggttattag gtgcttctat aatccataag ctagattata ataatcctag aaggaaacaa 4380

cagggccaaa gtctcatgac acagtttaga tggatttttt ttcatctgct ttatcagtgc 4440

atatagctgc ctaggtaact tctatttttt acccaagagg tctctccaaa actttcttta 4500

taatgaagta caccaatcac tgaaccttaa tttattggtc aggatgccaa aagaggagca 4560

gaagctggac atacgcttgc ccaccgaaga agatacgaag gcagtttcat gacaaatgct 4620

caaacatgta tatagatcga gacaggtttc tgagtttagg atgacatata aatcagtaaa 4680

actaacaaag gcagcacaaa aaaaattaca tgcattgtgt tgccacattt atctccgtac 4740

ccgattcata ttgtattgct aactgatgca gttcttactc cttaccctgt gtcaacctga 4800

ccaggtgact tggcaggaac tcaaaatgtt ttcaagaata cgacgcattc ttgagaaacg 4860

ttttgtttat tgttgtaaaa accacttcaa tagtaatttg catcaacttt gatgcaattt 4920

attttataga aaaacatgtg atattttggt 4950

<210> 9

<211> 4949

<212> DNA

<213> unknown (synthetic)

<400> 9

cagagtggga ccagcaagcc gcgccagtgt acatgtcttc accgtcacac gtgcgctcct 60

ctcgtccatc ctccacacgt gcgctcaaac cgagcttgtc cacggaccgc accctgtctg 120

cacgcatctc caccgtccga tccccagccg atggcggcgg agcccgcggc ggccgaccac 180

gatctccggc cacccctggc ggatggcgcc gctgctgccg gcgtgggcgc cgcctccttg 240

gcggcggcgg cgggcgcggc agaggcgctg atgagcgcga cgtcggagca gctcacgctg 300

gtgtatcagg gcgacgtcta cgtcttcgac cccgtcccgc cccaaaaggt cgcgcctctt 360

gcacccctga taattccatt ggcccctccc aaattctctc cttggttcct atatgttgcg 420

atggtcgtta atagtcttgg ccggagatta gcgtgcgcgg ggcgccggct atgaattagc 480

aaatattgtt ctgcttttgc gggggcgctc tgctgtcgag ttgcacataa ttaaattctg 540

tcttcatcgt tcttgattga attctaaggg gacttgattg ccagctatgg ctaagttagt 600

cgcagtttct gcttcgctac tgtaaattga ccatattcct ttgttccctt gctgaattgc 660

accaaagaat acgggtgcta tatgatatga ctgtgctttt ggtatcctgt aaggggctga 720

actaatgtta ttatgaattg ctctgtccat ttttcgatca gtatggatat accgttctac 780

tgttcgagtg gaatcgagaa tgcttgatcg ctaggtagac acttttttgt gcgtgttgag 840

atgttttata gttgtgtcgg ggaaaagtcg cgagtttgat gaacagcgga gtagatatct 900

tcgttcttca gctaggattt taacatattt gtgtttttgt tatagttgcc aaataaagtt 960

ccaatagaag catgtatgtt ctttttttcc gatgagaagt ataaattctt tgttccttaa 1020

gttttattag tgttttaaac ccaaatatga aaatttggaa ctatgtttca tctttggtcc 1080

tggactaagc tcactcatat gatttgcttt tctcattggc aagttgagga ttatttttgt 1140

tctagctatt agggaagatt atttggaaag atggaagcaa ctagattagg gaacttctgt 1200

gagtaagaag ttgagatgac ttgcctggat tggtcagagt catagtcata actttcaaaa 1260

tgttactaga gatctaatca tgaaacaaca taaatatttt gtcttcacgg ttacaaggtg 1320

attttgccag tcttccattt gaacgtgttt ttgaaatcta tttggtctat ttttcttcaa 1380

aaaaaaatct atttggtcta tctcaattcc ttatctgctt attagtgaga atgcctataa 1440

atgcaatcat gagccctagt gatgtcgttt gtagtgaaaa gcttcactgg gatgtatgtc 1500

ctaagataaa ccaagaccaa agtaagcatg atgcgaccat actgaaggtt ttcagggtga 1560

ccatatgcct atgtcattat tgatttgttt gagtgcatat ggtgatgaga ttgtttcact 1620

gacacgtgta tcccagggcc acatgcgagc cagcgtgtca gtgagatatg ggagtcacat 1680

gtgcagaaac tcaacctagg ggagctgtat atcctaagta ctttctcttg atcaggttca 1740

ggctgttctt ttggtacttg gagggtacga ggttccgcct ggtttggtaa acatggctgt 1800

ttcttcagca aatgatgaga aggtatccgc atacttactg cagtgaggat aggtatttcc 1860

agcttctcct aaagcattgt ctcatgtgca gaatacaact gtggctgcta gaagggttgc 1920

ttccttaatg agattccgtg agaagagaaa ggaaagatgt ttcgataaaa ggattagata 1980

cagtgtgcgc aaggaggttg cccaaaagta agtacagaat aatgtcactt ctatgtttag 2040

caatgatata attattcttc gcatgggcat aatgaatttt taaaggcaat cttgattaac 2100

agattgtttg ctgtcatttg gaggatttga gatataaata gtaagaagtc tgcatttgtt 2160

agtctccaca ttagataaaa ttatcagctt attgcagact tgattatgtt tgactttgac 2220

attgctttac cacattaata ggatgtgctg taagaccagg atttacttaa ttctctcctt 2280

gtatcactct gtttaaatag gaatacacat gccatattca gattattatc actgttatat 2340

catatccaat cttaggtcac ctttttatct cgaatagttt ctccaagtac taatgccaga 2400

aaaaaactaa ataggataat aatctgaatg tgcaccatgg atctacctat ttggagtact 2460

tgtgcactaa aggtgttatt caaggttacc ataagcgaag tcaatgttta aaagatttat 2520

ttcagtcaga tataaaaatg aacgaggact ttcttctatg ccttttagtc cacgatttgt 2580

aagaagtatc atgtaaaggc ttagatgaac tccgtggaga atggaacaga tgatttctat 2640

tatttccatt tgtcagaact tttccagaac ttatgtgctt atttgcatac ctacctggtt 2700

caatccagtt acctttttttt atatcaatca tgctagaaaa ggtgttttttt tcttcttttc 2760

atcatactgt ttacattctg ttgcaataaa ccctggccaa acaattgctg ctcaccatca 2820

ttcaccactg ggaatatttc ccaggtttat ttaccgtaac cattgcataa ccatattttg 2880

cattcaaatg atcgctactc agtgatgttt ttgttgtttt ttttttttga atcttgcatt 2940

gcacaatctc aataatactt tcaggatgaa acggcgtaaa ggccaatttg ctgggagatc 3000

agattttggc gatggtgcct gttcttctgc agcttgtggc tctccagcta acggcgagga 3060

tgatcatttt cgagaaaccc agtgagttag ttgccatgtg ccaactatca ctttgtgatt 3120

gtgtgtatgt gttacatcag tttcatatgc agaaagttgt tgccttcatg tatggactaa 3180

tgttggcctg ttttgctagt tgccaaaatt gtggtatcag ctcaaggctt actccagcga 3240

gtcggggccc agctggtccg aggtccctct gcaatgcttg tggcttaatg tgggcaaata 3300

aggtacaact ttctctatgc caatcatcgc acctcttgtc cagttgtcct gtttggctgt 3360

ttggtacctt tttcctcatg tcaaataaaa agcatcattc ttcttaattt gaaaaacttc 3420

ccaatgagat attctaattg gtcaaaaaat gatctgatta aagtgtgtct attagcaaca 3480

ttcctactag ttaacctttg tttttttcag ggtactctac gaagtcctct aaatgcaccc 3540

aaaatgactc agcagcttct tgccaatcca tgtaacatgg tgcgctacat ttgtaataga 3600

aacttgttta tttgaacgcc ttttcttgta gcgaagatta tctgaacagt acacttgtgc 3660

aggtagatac tgatgacaaa aactcaaatg ttcttcctgt ggagcataat caagccacac 3720

ccaaaactga ctcgatgtaa gcatgctctt tactgtacgt gaaatcattg tctggaagct 3780

gtgtcatgta catggtcgga tgtgcaaatc tgaacttcgt gttctcttga gttaagctca 3840

aactgggagg gacaaaggcg ctgtttgttt accctctaga ttatataatc caacttaaat 3900

aagttgagag gcaaacatat aactcagatt attaggtgga ttatataatc tagattcgta 3960

caacaacaac aacaaagcct ttttgtccca agcacgttgg ggtaggctag agatgaaacc 4020

ccgtatgaaa acttcagagc tcaaccccaa agaaaagaaa agggaaacaa tggcaaaaga 4080

gaaccgaaaa acgacaaaac ggggaaacac ataaaaagag ataaaaccca caagaaacag 4140

ccagatctaa atgggcacaa gaaaaggtca aacgattaag gaggaaaagc gaaactatga 4200

atcaaggttc tggcacgtga attgcacact tccacccctt cctatccacg gcgagctctt 4260

tatcaatatt ccactcttta tcaataatct agattcgtag attatgataa ttcataagca 4320

ggttattagg tgcttctata atccataagc tagattataa taatcctaga aggaaacaac 4380

agggccaaag tctcatgaca cagtttagat ggatttttttt tcatctgctt tatcagtgca 4440

tatagctgcc taggtaactt ctatttttta cccaagaggt ctctccaaaa ctttctttat 4500

aatgaagtac accaatcact gaaccttaat ttattggtca ggatgccaaa agaggagcag 4560

aagctggaca tacgcttgcc caccgaagaa gatacgaagg cagtttcatg acaaatgctc 4620

aaacatgtat atagatcgag acaggtttct gagtttagga tgacatataa atcagtaaaa 4680

ctaacaaagg cagcacaaaa aaaattacat gcattgtgtt gccacattta tctccgtacc 4740

cgattcatat tgtattgcta actgatgcag ttcttactcc ttaccctgtg tcaacctgac 4800

caggtgactt ggcaggaact caaaatgttt tcaagaatac gacgcattct tgagaaacgt 4860

tttgtttatt gttgtaaaaa ccacttcaat agtaatttgc atcaactttg atgcaattta 4920

ttttatagaa aaacatgtga tattttggt 4949

<210> 10

<211> 20

<212> DNA

<213> unknown (synthetic)

<400> 10

tgagttagtt gccatgtgcc 20

<210> 11

<211> 20

<212> DNA

<213> unknown (synthetic)

<400> 11

caagaggtgc gatgattggc 20

Claims (11)

1. The application of a protein in controlling maize flowering stage traits, characterized in that: the amino acid sequence of the protein is as shown in SEQ ID NO.1. 2 . The application of a nucleic acid molecule in the control of maize flowering stage traits, wherein the nucleic acid molecule encodes the protein described in claim 1 . 3. The application according to claim 2, wherein the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.2 or SEQ ID NO.3. 4. A method for early flowering of maize, characterized in that: inhibiting the expression and/or activity of the protein described in claim 1 in maize, and selecting plants with early flowering in maize. 5. The method according to claim 4, wherein the method for inhibiting protein expression and/or activity comprises any one of gene editing, RNA interference or T-DNA insertion. 6. The method according to claim 5, wherein the gene editing adopts the CRISPR/Cas9 method. 7 . The method for early flowering of maize according to claim 6 , wherein the DNA sequence of the genomic target region of the CRISPR/Cas9 method in maize is shown in SEQ ID NO. 4. 8 . 8. a test kit for early maize flowering period, characterized in that: comprising any of the following: (1) sgRNA molecule, its sequence is as shown in SEQ ID NO.5; (2) a DNA molecule encoding the sgRNA described in (1); (3) A vector for expressing the sgRNA described in (1). 9. A mutant gene for early flowering of maize, wherein the mutant gene sequence is shown in any one of SEQ ID NO.6-SEQ ID NO.9. 10 . The primer pair for detecting the mutant gene of claim 9 , wherein the primer pair is the sequences shown in SEQ ID NO.10 and SEQ ID NO.11. 11 . 11. Use of the primer pair of claim 10 in detecting the mutant gene of claim 9.

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