CN112661823B

CN112661823B - Gene and method for changing flowering period of corn

Info

Publication number: CN112661823B
Application number: CN202110087583.6A
Authority: CN
Inventors: 金敏亮; 刘相国; 许洁婷; 刘海军; 尹悦佳; 黄磊; 郭嘉; 韩宝柱; 严建兵
Original assignee: Weimi Biotechnology Jiangsu Co ltd; Jilin Academy of Agricultural Sciences; Huazhong Agricultural University
Current assignee: Changzhou Xinmi Biotechnology Co ltd
Priority date: 2021-01-22
Filing date: 2021-01-22
Publication date: 2022-04-26
Anticipated expiration: 2041-01-22
Also published as: CN112661823A

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

Gene and method for changing flowering period of corn

Technical Field

The present invention is in the field of molecular genetics. In particular to a gene for controlling the maize florescence and application thereof in changing maize florescence characters. The invention provides a sequence of a gene for controlling the flowering phase of corn and discloses a method for shortening the flowering phase by mutating the gene by using a genetic engineering means. The invention also provides a maize mutant gene sequence with shortened flowering phase, and the mutant gene sequence can be used for improving maize flowering phase characters.

Background

In higher plants, flowering represents a transition from vegetative to reproductive growth, which plays an important role throughout the growth and development stages of the plant. The biological character of when to bloom is subjected to the dual functions of the genetic factors of the plant body and the external environmental factors. Under the influence of the dual action, a series of flowering induction processes are common in higher plants, namely, the leaves of the plants generate flowering substances (or florigen) at proper time by sensing external growth conditions (light, temperature, humidity and the like), and the flowering substances are conveyed to the stem tip growing point from the leaves through the conduction tissues to stimulate the apical meristem to flower.

The flowering phase is an important character in the crop evolution and adaptation process, the understanding of the genetic basis of the character of the crop flowering phase and the cloning of candidate genes can improve the environmental adaptability and plasticity of the crop, which has important significance for cultivating good crop varieties adapting to different ecological regions, and simultaneously, the genetic improvement process of important production characters such as yield and the like closely related to the flowering phase can be promoted.

CCT (CO, COL and TOC1) family genes are widely involved in the regulation and control process of plant flowering phase and play an important role in the growth and development of plants. The maize B73 reference genome has 53 genes with CCT structure, and the applicant previously used a related population composed of 368 maize inbred lines to locate 34 CCT genes related to maize flowering (Jinminghai construction of maize pan transcriptome and functional analysis of maize flowering inhibitor ZmCOL3 [ D ]. Hubei: university of China agriculture, 2018; Jin M, Liu X, Jia W, et al. ZmCOL3, a CCT gene expression in mail flowering by maize flowering with the cyclic approach and activation expression of ZmCCT [ J ]. J Integr Plant biol.,2018,60(6):465 and 480.), most of which have no specific functional identification research results, so that the specific genes can really control maize traits incompletely and completely. If a more precise change in maize flowering phase is desired, it is necessary to specify which genes will control maize flowering phase traits and to what extent these genes have been genetically engineered to produce the changes in flowering phase traits.

In order to solve the problems, the invention utilizes a gene editing technology to mutate 15 genes in the 34 genes, obtains the genes and mutant genes which can influence the traits of the maize in the flowering phase through phenotype identification, and provides a method for artificially changing the maize flowering phase. The genes and the method can be used for artificially regulating the flowering phase of the corn and cultivating new corn materials which are suitable for different ecological environments.

Disclosure of Invention

One of the purposes of the invention is to provide a gene sequence for controlling the flowering character of corn.

The invention also aims to disclose a method for changing the flowering phase of the corn.

The invention also aims to provide a mutant gene for changing the flowering phase trait of the corn.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides an application of protein in controlling the flowering phase character of corn, which is characterized in that: the amino acid sequence of the protein is shown as SEQ ID NO. 1.

The invention also provides an application of the nucleic acid molecule in controlling the flowering phase character of corn, which is characterized in that: the nucleic acid molecule encodes the protein described above; in some embodiments, the nucleotide sequence of the nucleic acid molecule is as set forth in SEQ ID No.2 or SEQ ID No. 3.

The sequence of SEQ ID NO.1 is the amino acid sequence of the AC215811.3_ FG003 gene in a maize inbred line B73, the sequence of SEQ ID NO.2 is the genomic sequence of the AC215811.3_ FG003 gene, and the sequence of SEQ ID NO.3 is the cDNA sequence of the AC215811.3_ FG003 gene.

The invention also provides a method for advancing the flowering phase of corn, which is characterized by comprising the following steps: inhibiting the expression and/or activity of the protein coded by the gene in the corn, and selecting the plant with early flowering phase of the corn.

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

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

In some embodiments, the DNA sequence of the genomic target region in maize of the CRISPR/Cas9 method described above is shown as SEQ ID No.4 or SEQ ID No. 5.

The invention also provides a kit for advancing the flowering phase of corn, which is characterized in that: including any of the following:

(1) the sequence of the sgRNA molecule is shown as SEQ ID NO.6 or SEQ ID NO. 7;

(2) a DNA molecule encoding the sgRNA;

(3) a vector expressing the sgRNA.

The invention also provides a mutant gene for advancing the flowering phase of corn, which is characterized in that: the sequence of the mutant gene is shown in any one of SEQ ID NO.8-SEQ ID NO. 12.

A plurality of different editing types can be obtained by gene editing mutation target genes, and the plants corresponding to the different editing types do not perform completely the same. Through screening and identification, the mutant gene shown by SEQ ID NO.8 or SEQ ID NO.9 or SEQ ID NO.10 or SEQ ID NO.11 or SEQ ID NO.12 is determined to lead the maize to be moderately early in the flowering phase. The mutant gene can be introduced into corn materials with different genetic backgrounds in a sexual hybridization mode, so that a new early-flowering corn variety is created.

The present invention also provides a primer set for detecting the above mutant gene, characterized in that: the primer pair is a sequence shown in SEQ ID NO.13 and SEQ ID NO.14 or a complementary sequence thereof.

The invention also provides application of the primer pair in detecting the mutant gene. And carrying out PCR amplification on the genome DNA of the sample to be detected by using the primer pair, sequencing and analyzing the sequence of the amplification product, and if the sequence of the sequenced amplification product is consistent with a partial sequence of the sequence shown by SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 or SEQ ID NO.12, the sample to be detected contains the mutant gene.

The invention has the following advantages and beneficial effects: the invention utilizes the related population to locate 34 CCT genes related to the maize flowering phase trait, however, the specific genes in the candidate genes are not known to really control the maize flowering trait and influence degree on the flowering phase trait. The invention utilizes gene editing technology to mutate 15 of the 34 genes, and determines that AC215811.3_ FG003 can actually influence the maize flowering phase trait through phenotypic identification. The method for editing the CRISPR/Cas9 gene can change the flowering period of the corn, and the edited mutant gene can be used for creating a new early-flowering corn variety. The invention provides a new gene and a new method for manually regulating the flowering period of corn to culture a new corn material suitable for different ecological environments.

Drawings

FIG. 1 Gene editing vector map. The English and abbreviated 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 ubiquitin promoter

3 × FLAG tag sequence

SV40NLS Simian Virus 40 Nuclear localization Signal

Cas9 Cas9 gene sequence

Nucleoplasm in NLS nuclear localization Signal

NOS terminator of nopaline synthase

lac promoter lactose promoter

M13 rev M13 primer sequence (reverse)

lac operator lactose operon

CAP biding site CAP binding site

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

BlpR-encoded Bar protein 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 region sequence

pVS1 RepA pVS1 replicon

pVS1 StaA pVS1 transcriptional initiation region

Detailed Description

The following definitions and methods are provided to better define the present application and to guide those of ordinary skill in the art in the practice of the present application. Unless otherwise indicated, terms are to be understood in accordance with their ordinary 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 varieties 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 calli, intact plant cells in 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 from left to right in the 5 'to 3' direction; amino acid sequences are written from left to right in the amino to carboxy direction. Amino acids may be referred to herein by their commonly known three letter symbols or by the one letter symbols recommended by the IUPAC-IUB Biochemical nomenclature Commission. Similarly, nucleotides may be represented by commonly accepted single-letter codes. Numerical ranges include the numbers defining the range. As used herein, "nucleic acid" includes reference to deoxyribonucleotide or ribonucleotide polymers in either single-or double-stranded form, and unless otherwise limited, includes known analogs (e.g., peptide nucleic acids) having the basic properties of natural nucleotides that hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides. As used herein, the term "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. The information encoding the protein is represented using a codon. As used herein, "full-length sequence" in reference to a particular polynucleotide or protein encoded thereby refers to the entire nucleic acid sequence or the entire amino acid sequence having a native (non-synthetic) endogenous sequence. The full-length polynucleotide encodes the full-length, catalytically active form of the 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 analogues of the corresponding naturally occurring amino acids. The term is also used for naturally occurring amino acid polymers. The terms "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 as naturally occurring amino acids.

As used herein, the terms "isolated" and "purified" may be used interchangeably to refer to a nucleic acid or polypeptide, or biologically active portion thereof, that is substantially or essentially free of components that normally accompany or react with the nucleic acid or polypeptide as found in its naturally occurring environment. Thus, an isolated or purified nucleic acid or polypeptide produced by recombinant techniques is substantially free of other cellular material or culture medium, or is substantially free of chemical precursors or other chemicals when chemically synthesized. An "isolated" nucleic acid is typically free of sequences (such as sequences encoding proteins) 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 0.5kb 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 "comprise", "comprising" or variations thereof are to be understood as embracing elements, numbers or steps in addition to those described. By "subject plant" or "subject plant cell" is meant a plant or plant cell in which the genetic modification has been effected, or a progeny cell of the plant or cell so modified, which progeny cell comprises the modification. The "control" or "control plant cell" provides a reference point for measuring the phenotypic change of the test plant or plant cell. The control plant or plant cell may include, for example: (a) a wild-type plant or cell, i.e., a plant or cell having the same genotype as the starting material for the genetic alteration that produced the test plant or cell; (b) plants or plant cells having the same genotype as the starting material but which have been transformed with an empty construct (i.e., a construct that has no known effect on the trait of interest, such as a construct comprising a target gene); (c) a plant or plant cell that is a non-transformed isolate of a subject plant or plant cell; (d) a plant or plant cell that is genetically identical to the subject plant or plant cell but that has not been exposed to conditions or stimuli that induce expression of the gene of interest; or (e) the subject plant or plant cell itself, 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 suitable tools and procedures for engineering or engineering amino acid sequences and potential gene sequences of 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. The principles and examples of conservative amino acid substitutions are further described below. In certain embodiments, substitutions that do not alter the amino acid sequence of the nucleotide sequences of the present application can be made in accordance with the codon preferences disclosed for monocots, e.g., codons encoding the same amino acid sequence can be substituted with monocot preferred codons without altering the amino acid sequence encoded by the nucleotide sequence. In some embodiments, a portion of the nucleotide sequence in this application is replaced with a different codon that encodes the same amino acid sequence, such that the nucleotide sequence is not altered while the amino acid sequence encoded thereby is not altered. 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, a partial nucleotide sequence herein is replaced according to monocot preferred codons. One skilled in the art will recognize that amino acid additions and/or substitutions are generally based on the relative similarity of the amino acid side-chain substituents, e.g., hydrophobicity, charge, size, etc., of the substituents. Exemplary amino acid substituent groups having various of the foregoing properties are 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. Guidance regarding suitable amino acid substitutions that do not affect the biological activity of the Protein of interest can be found in the model of the Atlas of Protein sequences and structures (Protein Sequence and Structure Atlas) (Natl. biomed. Res. Foundation, Washington, D.C.) (incorporated herein by reference). Conservative substitutions such as exchanging one amino acid for another with similar properties may be made. Identification of sequence identity includes hybridization techniques. For example, all or part of a known nucleotide sequence is used as a probe for selective hybridization to other corresponding nucleotide sequences present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., a genomic library or cDNA library) from a selected organism.

In some embodiments, fragments of the nucleotide sequences and the amino acid sequences encoded thereby are also included. As used herein, the term "fragment" refers to a portion of the nucleotide sequence of a polynucleotide or a portion of the amino acid sequence of a polypeptide of an embodiment. Fragments of the nucleotide sequences may encode protein fragments that retain the biological activity of the native or corresponding full-length protein, and thus have protein activity. Mutant proteins include biologically active fragments of the native protein that comprise contiguous amino acid residues that retain the biological activity of the native protein. Some embodiments also include a transformed plant cell or transgenic plant comprising the nucleotide sequence of at least one embodiment. In some embodiments, plants are transformed with an expression vector comprising at least one embodiment of the nucleotide sequence and operably linked thereto a promoter that drives expression in plant cells. Transformed plant cells and transgenic plants refer to plant cells or plants that comprise a heterologous polynucleotide within their genome. Generally, the heterologous polynucleotide is stably integrated within the genome of the transformed plant cell or transgenic plant such that the polynucleotide is transmitted to progeny. The heterologous polynucleotide may be integrated into the genome alone or as part of an expression vector. In some embodiments, the plants to which the present application relates include plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells, which are whole plants or parts of plants, such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, nuts, ears, cobs, husks, 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 derived from the transgenic plants of the present application or progeny thereof, and thus comprising at least in part the nucleotide sequences of the present application.

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Modifications or substitutions to methods, steps or conditions of the present invention may be made without departing from the spirit and substance of the invention and are intended to be included within the scope of the present application. Unless otherwise indicated, 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 as recommended 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.

Examples

Example 1 identification of maize flowering phase changes following Gene editing knockout of candidate genes

The invention utilizes CRISPR-Cas9 gene editing technology to carry out site-directed editing on 15 genes in 34 genes related to the flowering phase of corn shown by a correlation analysis result.

The implementation mode comprises the construction of a gene editing vector, the genetic transformation of corn and the functional verification of editing effect. The method comprises the following specific steps:

1. construction of Gene editing vector

The gene editing vector of the invention is G08943-CPB-ZmUbi-hspCas9, and the vector diagram is shown in figure 1. The basic vector of the vector is CPB-ZmUbi-hspCas 9. The invention obtains double target U6-sgRNA through overlapPCR and clones the double target U6-sgRNA into a basic vector through homologous recombination, and the specific construction process is as follows:

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

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

(3) U6-sgRNA was obtained by Overlap PCR. The primer pair U6F1/U6R is used for amplifying the U6 promoter of the first target, and the product length is 515 bp; primer pair gR-1F (3F)/gRR1 was used to amplify a first targetThe product length of the sgRNA of (1) is 127 bp; primer pair U6F1/gRR1 was used to perform the Overlap PCR step 2 amplification (U6-sgRNA) with a product length of 634 bp. U6 and sgRNA are respectively amplified in the 1 st step of the Overlap PCR, and PCR products are respectively diluted by 50 times and then mixed to be used as a template for carrying out the 2 nd step amplification of the Overlap PCR. And (5) electrophoresis gel cutting recovery and sequencing of the amplification product to confirm the sequence. The Overlap PCR system and conditions were as follows: the 15. mu.L reaction in step 1 of the Overlap PCR was as follows, template DNA (U6 or sgRNA,. gtoreq.30 ng/. mu.L): 0.5. mu.L, Primer F/R: 1.2. mu.L each, sterilized ddH₂O: 3.7 μ L,2 × phanta max Buffer: 7.5 μ L, dNTP mix: 0.6. mu.L, Phanta enzyme (product No.: P505-d1/d2/d 3): 0.3. mu.L. The reaction system in step 2 of the Overlap PCR was a 30. mu.L system. U6 pipetted 1. mu.L, 49. mu.L ddH₂Diluting with oxygen; sgRNA aspirated at 1. mu.L, diluted with 49. mu.L of ddH2O, aspirated at 10. mu.L each, and mixed well. The method comprises the following specific steps: mixed template DNA (U6+ sgRNA): 1.5 μ L, Primer F/R: 2.4 μ L each, sterile ddH 2O: 6.9 μ L,2 × phanta max Buffer: 15 μ L, dNTP mix: 1.2. mu.L, Phanta enzyme: 0.6. mu.L. The Overlap PCR program was as follows: (1)94 ℃ for 5 minutes, (2)94 ℃ for 30 seconds, (3)62 ℃ for 35 seconds, (4)72 ℃ for 30 seconds, and the (5) th step is a cycle of 32 times from the (2) step to the (4) step, (6)72 ℃ for 10 minutes, and (7)25 ℃ for 5 minutes. The primer sequences required for vector construction are shown in Table 1.

TABLE 1 primer sequences required for vector construction

(4) The construction into a backbone vector is carried out by recombinant cloning. The CPB-Ubi-hspcas9 vector was digested with HindIII and recovered. Both U6-gRNA and the vector were ligated by homologous recombination. Before reaction liquid preparation, the concentration of each Overlap product is ensured to be close to be consistent, and a 20 mu L homologous recombination system comprises the following steps: cas Hind III: 3 μ L, T-1F Overlap: 1 μ L, sterile ddH 2O: 10 μ L, 5 × CE MultiS buffer: 4 μ L, Exnase MultiS (product No.: C113-01/02): 2 μ L.

2. Genetic transformation of maize

The vector is transferred into agrobacterium EHA105 by an electric shock method, and PCR is carried out for identification. Taking a freshly peeled young embryo of a maize inbred line KN5585 (an inbred line bred by Mimi Biotechnology (Jiangsu) Co., Ltd.) of about 1mm as a material, putting the peeled maize embryo into a 2mL plastic centrifuge tube containing 1.8mL of suspension, and treating about 150 immature young embryos within 30 min; the suspension was aspirated, the remaining corn embryos placed in a tube and then 1.0mL of Agrobacterium suspension was added and left for 5 min. The young embryos in the centrifuge tube are suspended and poured onto a co-culture medium, and the surplus agrobacterium liquid on the surface is sucked by a liquid transfer device and is cultured for 3 days in the dark at the temperature of 23 ℃. After co-cultivation, the young embryos were transferred to a resting medium, cultured in the dark at 28 ℃ for 6 days, placed on a screening medium containing 5mg/L of Bialaphos, and screened for 2 weeks, and then transferred to a screening medium containing 8mg/L of Bialaphos for 2 weeks. The resistant calli were transferred to differentiation medium 1 and cultured at 25 ℃ under 5000lx light for 1 week. Transferring the callus to a differentiation culture medium 2, and culturing for 2 weeks by illumination; transferring the differentiated plantlets to a rooting culture medium, and culturing at 25 ℃ and 5000lx by illumination until the plantlets are rooted; transferring the plantlets into small pots for growth, transplanting the plantlets into a greenhouse after a certain growth stage, and harvesting progeny seeds after 3-4 months.

3. Trait identification of gene-edited plants

The flowering phase traits of the obtained gene editing material are identified, and some of the genes can really cause the significant change of the flowering phase of the corn after being edited, while the obvious flowering phase change cannot be observed after some genes are edited, and the specific change mode is shown in table 2.

TABLE 2 flowering-time Change after Gene editing

Example 2 in-depth analysis of maize flowering-stage traits and identification of mutant genes

And carrying out more deep character identification on the gene editing material with the flowering character change, and analyzing specific editing sites. Maize flowering time for the T0 generation material was earlier after AC215811.3_ FG003 gene editing than the receptor control KN 5585. The flowering time of the T1 generation material of this transformant was more deeply characterized and analyzed for specific editing sites.

The AC215811.3_ FG003 gene is edited by two designed target sites which are respectively shown as SEQ ID NO.4 and SEQ ID NO. 5. gRNA sequences expressed by the vectors containing the two targets are respectively shown as SEQ ID NO.6 and SEQ ID NO. 7.

Extracting DNA in seedling stage to detect gene editing condition. Designing primers, wherein the sequences of the primers are shown as SEQ ID NO.13 and SEQ ID NO. 14. Amplifying a target editing section, wherein an amplification system is as follows: DNA: 3 μ L, 1 μ L each of the bidirectional primers, 2 × TaqMix: 7.5 μ L, ddH₂O: 2.5. mu.L, total volume 10. mu.L. The PCR reaction conditions were as follows: (1)94 ℃ for 5 minutes, (2)94 ℃ for 40 seconds, (3)57 ℃ for 30 seconds, (4)72 ℃ for 60 seconds, (5) cycle 35 times from (2) step (4), (6)72 ℃ for 7 minutes, and (7) storage at 4 ℃. The PCR product was submitted to Sanger sequencing by Wuhan Strongziaceae Biotech Ltd. And comparing the PCR amplification sequencing results of the transformant and the receptor KN5585, wherein the material subjected to base substitution, insertion or deletion is a positive editing material, and the material is a negative material otherwise.

After sequence comparison, 5 different editing types of material were found. Where the a1 material deletes one a at target 1 and GG at target 2, the a2 material deletes TC at target 1 and inserts CC at target 2, the A3 material deletes TC at target 1 and inserts G at target 2, the a4 material deletes TC at target 1 and deletes GG at target 2, and the a5 material deletes TC at target 1 and deletes GG at target 2 and changes a to G (table 3). Thus, after editing, the genomic sequence of the five materials A1, A2, A3, A4, A5 was changed from SEQ ID NO.2 to SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12, respectively.

TABLE 3 Gene editing maize Material flowering time trait data

"-" indicates a deleted sequence, bold indicates an inserted or replaced sequence, boxes indicate a PAM sequence, and underlining indicates an editing target.

The flowering phase traits (including the emasculation phase, the pollen scattering phase and the spinning phase) of the A1-A5 material were experimentally investigated in Guilin province in summer of 2018, and the results are shown in Table 4. The flowering phase of the edited material of A1-A5 is earlier than that of the unedited control material to different degrees, and the fact that the gene controls the flowering phase character and the flowering phase is earlier after gene editing is proved.

TABLE 4 Gene editing maize Material flowering time trait data

Florescence data are expressed as mean ± standard deviation, in units: and (5) day. "CK" represents unedited control material. "x" indicates a very significant difference compared to the control (P < 0.01).

Therefore, the mutant gene shown in SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 or SEQ ID NO.12 can lead the maize to be moderately early in flowering phase. The mutant gene can be introduced into corn materials with different genetic backgrounds in a sexual hybridization mode, so that a new early-flowering corn variety is created.

In the process of introduction, a primer pair with sequences shown in SEQ ID NO.13 and SEQ ID NO.14 can be used for detecting whether the maize genome contains the mutant gene, PCR amplification is carried out on the genome DNA of a sample to be detected by using the primer pair, and the sequence of an amplification product is analyzed by sequencing. If the amplification product corresponds to the sequence shown in position 3419-4210 of SEQ ID NO.8, the mutant gene shown in SEQ ID NO.8 is contained; if the amplification product corresponds to the sequence shown in SEQ ID NO.9, position 3419-4213, the mutant gene shown in SEQ ID NO.9 is contained; if the amplification product corresponds to the sequence shown in the 3419-4212 of SEQ ID NO.10, the mutant gene shown in SEQ ID NO.10 is contained; if the amplification product corresponds to the sequence as shown in SEQ ID NO.11, position 3419-4209, the mutant gene as shown in SEQ ID NO.11 is included; if the amplification product corresponds to the sequence shown in SEQ ID NO.12, position 3419-4209, the amplification product comprises the mutant gene shown in SEQ ID NO. 12; if the amplification product corresponds to the sequence shown in SEQ ID NO 2 at position 3419-4213, the genotype is unedited.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

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tccgtcgctc tgacacagcc gactagcatc tcctcactgg tcgcagctct tgccaccatc 300

cagtccgccg tcaccccgtg acccaagcct gtgagcacaa gcatgctgtc cacactactc 360

taagtctcta accgcttagc tagccaaggc tcaacaactc ttcttcagtc gcatcaattc 420

gcccaccccg gcatcccctg cggtccacat cactactagg ctcattgcca ccaccattgc 480

tcaggcggcc ggagttcaca acatccggtt ccttgtcttc attgtgttgg atccagcgtc 540

accccacttc gcccactggt gtgatcaggt gctcctgatg cttcatcgct acactctcat 600

cgaccacgtc ctcgacaacg tcgtcactca tttggctccc tattggatct agatggacaa 660

catcgttatc acatggatca tcgacaccct caccgtcgag cttttcgaca tcgtccatga 720

gtgaggggca ccactcgcca ggcccgagtc tcccttgagg cttagttcct tgacaatagg 780

gaggcctagg ctctccatct tgatgccttg tccccaccta ttctcctaag gggatctctc 840

catcatcgag tactattggt ggatgaaggg gatggccaac tcccttcacg atctcggcga 900

gcccgttgcc gaccgcaccc tcgtattcaa ccttctacgt ggtcttagtc gttgctacaa 960

ccacctgaaa gctcttatca ggcggaccgt gtgcttcccc tcctttggcg acgtctgtaa 1020

tgagtttctc ctcgaggagt ttaccatgga ggccaagtcc accactgcca cgaacctcta 1080

cgatgcaccc tctagtggct aggtgtcctc tggtggctag gcccctacac tccatcgacg 1140

gggacccctg cacgccctcc caccacccta gccttagagc cccacattcg cctgctagcg 1200

tggatgcctg gatggcagtc gctagtggcg gcccggcgtg gctggcgttc taccaccctt 1260

ggaccaagac catctccatg tggctgggcc cgacctcggg tgctctctcg ccacatcctc 1320

cttagtcgtc cctcttggtg ttgccaacct atggtgtctc tccgttgccc acgacatcgg 1380

ctctgctcct cccacctccg gggacccctg ctccgctacc acgatccccg ctggctggag 1440

gttgggacca ggctgccctt gatgtagctt tcggtaccat ggcgccgaat ccaccccaag 1500

tcgactgggt ggttgactct agcgcctcct accacaccac ctccactgtg agcatgttat 1560

cttgctcaca tcccctacat ctctcccacc cttcctatat cattgtgggg aacaactcca 1620

ctcccccggt cacctcagta ggtgactcga ttctcccggg cccgttccac ctcaacgaag 1680

ttctagtcgc tcctcacatc attcgtaatc ttgtttttgt ttgtcagttc actattgaca 1740

actcttattc cattgagttt gacctgtttg gtttatctat gatctgtcca ccaggaccct 1800

cctcgcccgt tgtgatagct tgaagcccct ctacacgatc cggtcatcca ccttcaccat 1860

cgacgtgtcc tccctgcttg tcctggcctc caccacttcg catcatcgtc tcggctacac 1920

tggactctac gtcatgacca agcttgccag tagtttagag atcacgtagt agaggacatt 1980

ttgagggtat ttgtcatgct tgtctgctag gctgccatac tcgtctctct tttgctacta 2040

tttcttctag ggatgagcag acttttgatc ttttattgtg atctatggac atctctcata 2100

ctcagcctct ttggatacaa atactacttg tgattttgga tgatttctct cattttcttt 2160

ggacttttcc tttacggttg aagtctgaca cctttcacac cctcacacac tttattgcct 2220

aggtctccat ctagttctgt cacttggttc atgctctcca gtgcgattac gaccgcgagt 2280

cccacaactc tgcctctcgc tcatttcttt ctttcctatg gcgtccagtt gtgactctcg 2340

tgaccctaca cctcctcata atggctgggc tgaacggatt attcgcacca caccaacatg 2400

attcactgtc tcttttttca gtcgtctctt cctgctagct attgggtaga ggccctgaac 2460

actgctaccc atcatctcaa ccgccttcct tctaaggcgg tgaagatatc caactctcta 2520

taatcttcta taaagcatgg ctaatcatga gatatgtatg gaataaaggt tatagaatct 2580

tctataacaa ccatgacaag atatgaagct tatatatttg ttaccatggt atattaggaa 2640

ttctctagac ttatatattt gttgagattt tttctagaat atgtacaagt gatactcatc 2700

catggctatg tgcgcatata taagataagt gtcccatttt gataatgatg taacacaaca 2760

taccacctca tcccacatat atctgatagt ctgtcaattg tgtcatattt tatttattta 2820

tttagtgtat taatttagta ttggagaagt ttccaaggta agaaataatt aacaacagag 2880

gtccttttct attctactaa ataaaaaaag tagttggaca tattttttcg cctttttcgc 2940

catagtcaaa tatcactatc ttgatattca gatcatgttg attttcaact tatactttta 3000

atagtatgcc acataagcaa gtatgatgat atgacaagaa tttattaagc tgagagacat 3060

agacttctaa taactaaaac taaacaagta tgccacgtaa gcaaatatga tatgacacaa 3120

atttattaaa caaagagaca tagatagttt aaagagagat gagagaattt catctaaatt 3180

aaactcagga tacacgtttt tatagacaaa aaatgattta acatttttga aaaatgtaca 3240

ataaaactct gcattgggaa tatcctaagg atctgatagg tttaattaaa aatcttggaa 3300

ccggagctat gccaaatgag cgatgttata tgtagttaat ttattaacgt aatgtcatat 3360

gagtcgttta aatttcttgc tggtatatag ctgctcatcc aactgttcga tgttgtagcc 3420

agattcgttg tggtcatcgt ggtcgttgga ttatgatgat atttggccat taaaaccatc 3480

ctcatcatat tctctggaaa ccgtaacccg gtcttctact gatcatgctg tggaatctct 3540

tggtgccgac aatgcagctc ttcggctcaa caattcagtg acaagcactg ctgctgcaaa 3600

cacaagcacc agagcgacgc catacctgga tctgcttgct ccccctgttc gtcaacttcg 3660

tcaagaatgc aactactaca acagcggctt gccggtgtgg tcaccggaga agttcccaag 3720

cgagcacttg ctgctgacgc cgtcggcgtt aggtggcaac caagaccaag acacgcctcc 3780

gcagcaggag gcggcgacgg cgatggcagt gatgctgaag caagtatcga gagcgagccc 3840

agacgtgaag gcgagacaaa ccaaactgca gaagaggcag gaggctaaac agagatacat 3900

ggagaagagg aagaacagga ggtatgtgtt gttcaatatg attaattagt actctcagct 3960

cattgaattg tttttataaa tgaaacatgc tctgaaatga aacagctggt aagaacataa 4020

tgcatctcta tacaaaagat aattttttac aattcaatag tctctttgcg attgcataca 4080

taggtatggc aagacaatta tgtatgcatc acggaaggca cgggcaaaca cacgaaacag 4140

agtaaaaggc agatttgcaa aggcatccag cggcagtggc ggcagccatg gcgacggcga 4200

tcagtctaca caacattgct atggccatgg cggcgatgaa gatgatcact ctacagtcta 4260

cacacgatcc tag 4273

<210> 3

<211> 915

<212> DNA

<213> Zea mays

<400> 3

atggatcctc cgacaacgac cctggcacct ctcatcctcc ccgatgtggc cactgctacg 60

accaccaacg acctcgccct tcccaactac ccggttacca tgaccgacac tgaccttgct 120

gctgctggca tgggtagcat cactgcgccc actagcagtc tctcacccgc ctgcctaccc 180

actcgcttcc tacagcacac ctctattgga gaagtttcca agccagattc gttgtggtca 240

tcgtggtcgt tggattatga tgatatttgg ccattaaaac catcctcatc atattctctg 300

gaaaccgtaa cccggtcttc tactgatcat gctgtggaat ctcttggtgc cgacaatgca 360

gctcttcggc tcaacaattc agtgacaagc actgctgctg caaacacaag caccagagcg 420

acgccatacc tggatctgct tgctccccct gttcgtcaac ttcgtcaaga atgcaactac 480

tacaacagcg gcttgccggt gtggtcaccg gagaagttcc caagcgagca cttgctgctg 540

acgccgtcgg cgttaggtgg caaccaagac caagacacgc ctccgcagca ggaggcggcg 600

acggcgatgg cagtgatgct gaagcaagta tcgagagcga gcccagacgt gaaggcgaga 660

caaaccaaac tgcagaagag gcaggaggct aaacagagat acatggagaa gaggaagaac 720

aggaggtatg gcaagacaat tatgtatgca tcacggaagg cacgggcaaa cacacgaaac 780

agagtaaaag gcagatttgc aaaggcatcc agcggcagtg gcggcagcca tggcgacggc 840

gatcagtcta cacaacattg ctatggccat ggcggcgatg aagatgatca ctctacagtc 900

tacacacgat cctag 915

<210> 4

<211> 20

<212> DNA

<213> Zea mays

<400> 4

gatcatgctg tggaatctct 20

<210> 5

<211> 20

<212> DNA

<213> Zea mays

<400> 5

gtatgcatca cggaaggcac 20

<210> 6

<211> 103

<212> RNA

<213> unknown (Artificial Synthesis)

<400> 6

gaucaugcug uggaaucucu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu uuu 103

<210> 7

<211> 103

<212> RNA

<213> unknown (Artificial Synthesis)

<400> 7

guaugcauca cggaaggcac guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu uuu 103

<210> 8

<211> 4270

<212> DNA

<213> unknown (Artificial Synthesis)

<400> 8

atggatcctc cgacaacgac cctggcacct ctcatcctcc ccgatgtggc cactgctacg 60

accaccaacg acctcgccct tcccaactac ccggttacca tgaccgacac tgaccttgct 120

gctgctggca tgggtagcat cactgcgccc actagcagtc tctcacccgc ctgcctaccc 180

actcgcttcc tacagcacac ctcgtgggtt ccaccccccg gcttcaccgc tccttccagc 240

tccgtcgctc tgacacagcc gactagcatc tcctcactgg tcgcagctct tgccaccatc 300

cagtccgccg tcaccccgtg acccaagcct gtgagcacaa gcatgctgtc cacactactc 360

taagtctcta accgcttagc tagccaaggc tcaacaactc ttcttcagtc gcatcaattc 420

gcccaccccg gcatcccctg cggtccacat cactactagg ctcattgcca ccaccattgc 480

tcaggcggcc ggagttcaca acatccggtt ccttgtcttc attgtgttgg atccagcgtc 540

accccacttc gcccactggt gtgatcaggt gctcctgatg cttcatcgct acactctcat 600

cgaccacgtc ctcgacaacg tcgtcactca tttggctccc tattggatct agatggacaa 660

catcgttatc acatggatca tcgacaccct caccgtcgag cttttcgaca tcgtccatga 720

gtgaggggca ccactcgcca ggcccgagtc tcccttgagg cttagttcct tgacaatagg 780

gaggcctagg ctctccatct tgatgccttg tccccaccta ttctcctaag gggatctctc 840

catcatcgag tactattggt ggatgaaggg gatggccaac tcccttcacg atctcggcga 900

gcccgttgcc gaccgcaccc tcgtattcaa ccttctacgt ggtcttagtc gttgctacaa 960

ccacctgaaa gctcttatca ggcggaccgt gtgcttcccc tcctttggcg acgtctgtaa 1020

tgagtttctc ctcgaggagt ttaccatgga ggccaagtcc accactgcca cgaacctcta 1080

cgatgcaccc tctagtggct aggtgtcctc tggtggctag gcccctacac tccatcgacg 1140

gggacccctg cacgccctcc caccacccta gccttagagc cccacattcg cctgctagcg 1200

tggatgcctg gatggcagtc gctagtggcg gcccggcgtg gctggcgttc taccaccctt 1260

ggaccaagac catctccatg tggctgggcc cgacctcggg tgctctctcg ccacatcctc 1320

cttagtcgtc cctcttggtg ttgccaacct atggtgtctc tccgttgccc acgacatcgg 1380

ctctgctcct cccacctccg gggacccctg ctccgctacc acgatccccg ctggctggag 1440

gttgggacca ggctgccctt gatgtagctt tcggtaccat ggcgccgaat ccaccccaag 1500

tcgactgggt ggttgactct agcgcctcct accacaccac ctccactgtg agcatgttat 1560

cttgctcaca tcccctacat ctctcccacc cttcctatat cattgtgggg aacaactcca 1620

ctcccccggt cacctcagta ggtgactcga ttctcccggg cccgttccac ctcaacgaag 1680

ttctagtcgc tcctcacatc attcgtaatc ttgtttttgt ttgtcagttc actattgaca 1740

actcttattc cattgagttt gacctgtttg gtttatctat gatctgtcca ccaggaccct 1800

cctcgcccgt tgtgatagct tgaagcccct ctacacgatc cggtcatcca ccttcaccat 1860

cgacgtgtcc tccctgcttg tcctggcctc caccacttcg catcatcgtc tcggctacac 1920

tggactctac gtcatgacca agcttgccag tagtttagag atcacgtagt agaggacatt 1980

ttgagggtat ttgtcatgct tgtctgctag gctgccatac tcgtctctct tttgctacta 2040

tttcttctag ggatgagcag acttttgatc ttttattgtg atctatggac atctctcata 2100

ctcagcctct ttggatacaa atactacttg tgattttgga tgatttctct cattttcttt 2160

ggacttttcc tttacggttg aagtctgaca cctttcacac cctcacacac tttattgcct 2220

aggtctccat ctagttctgt cacttggttc atgctctcca gtgcgattac gaccgcgagt 2280

cccacaactc tgcctctcgc tcatttcttt ctttcctatg gcgtccagtt gtgactctcg 2340

tgaccctaca cctcctcata atggctgggc tgaacggatt attcgcacca caccaacatg 2400

attcactgtc tcttttttca gtcgtctctt cctgctagct attgggtaga ggccctgaac 2460

actgctaccc atcatctcaa ccgccttcct tctaaggcgg tgaagatatc caactctcta 2520

taatcttcta taaagcatgg ctaatcatga gatatgtatg gaataaaggt tatagaatct 2580

tctataacaa ccatgacaag atatgaagct tatatatttg ttaccatggt atattaggaa 2640

ttctctagac ttatatattt gttgagattt tttctagaat atgtacaagt gatactcatc 2700

catggctatg tgcgcatata taagataagt gtcccatttt gataatgatg taacacaaca 2760

taccacctca tcccacatat atctgatagt ctgtcaattg tgtcatattt tatttattta 2820

tttagtgtat taatttagta ttggagaagt ttccaaggta agaaataatt aacaacagag 2880

gtccttttct attctactaa ataaaaaaag tagttggaca tattttttcg cctttttcgc 2940

catagtcaaa tatcactatc ttgatattca gatcatgttg attttcaact tatactttta 3000

atagtatgcc acataagcaa gtatgatgat atgacaagaa tttattaagc tgagagacat 3060

agacttctaa taactaaaac taaacaagta tgccacgtaa gcaaatatga tatgacacaa 3120

atttattaaa caaagagaca tagatagttt aaagagagat gagagaattt catctaaatt 3180

aaactcagga tacacgtttt tatagacaaa aaatgattta acatttttga aaaatgtaca 3240

ataaaactct gcattgggaa tatcctaagg atctgatagg tttaattaaa aatcttggaa 3300

ccggagctat gccaaatgag cgatgttata tgtagttaat ttattaacgt aatgtcatat 3360

gagtcgttta aatttcttgc tggtatatag ctgctcatcc aactgttcga tgttgtagcc 3420

agattcgttg tggtcatcgt ggtcgttgga ttatgatgat atttggccat taaaaccatc 3480

ctcatcatat tctctggaaa ccgtaacccg gtcttctact gatcatgctg tggatctctt 3540

ggtgccgaca atgcagctct tcggctcaac aattcagtga caagcactgc tgctgcaaac 3600

acaagcacca gagcgacgcc atacctggat ctgcttgctc cccctgttcg tcaacttcgt 3660

caagaatgca actactacaa cagcggcttg ccggtgtggt caccggagaa gttcccaagc 3720

gagcacttgc tgctgacgcc gtcggcgtta ggtggcaacc aagaccaaga cacgcctccg 3780

cagcaggagg cggcgacggc gatggcagtg atgctgaagc aagtatcgag agcgagccca 3840

gacgtgaagg cgagacaaac caaactgcag aagaggcagg aggctaaaca gagatacatg 3900

gagaagagga agaacaggag gtatgtgttg ttcaatatga ttaattagta ctctcagctc 3960

attgaattgt ttttataaat gaaacatgct ctgaaatgaa acagctggta agaacataat 4020

gcatctctat acaaaagata attttttaca attcaatagt ctctttgcga ttgcatacat 4080

aggtatggca agacaattat gtatgcatca cggaacacgg gcaaacacac gaaacagagt 4140

aaaaggcaga tttgcaaagg catccagcgg cagtggcggc agccatggcg acggcgatca 4200

gtctacacaa cattgctatg gccatggcgg cgatgaagat gatcactcta cagtctacac 4260

acgatcctag 4270

<210> 9

<211> 4273

<212> DNA

<213> unknown (Artificial Synthesis)

<400> 9

atggatcctc cgacaacgac cctggcacct ctcatcctcc ccgatgtggc cactgctacg 60

accaccaacg acctcgccct tcccaactac ccggttacca tgaccgacac tgaccttgct 120

gctgctggca tgggtagcat cactgcgccc actagcagtc tctcacccgc ctgcctaccc 180

actcgcttcc tacagcacac ctcgtgggtt ccaccccccg gcttcaccgc tccttccagc 240

tccgtcgctc tgacacagcc gactagcatc tcctcactgg tcgcagctct tgccaccatc 300

cagtccgccg tcaccccgtg acccaagcct gtgagcacaa gcatgctgtc cacactactc 360

taagtctcta accgcttagc tagccaaggc tcaacaactc ttcttcagtc gcatcaattc 420

gcccaccccg gcatcccctg cggtccacat cactactagg ctcattgcca ccaccattgc 480

tcaggcggcc ggagttcaca acatccggtt ccttgtcttc attgtgttgg atccagcgtc 540

accccacttc gcccactggt gtgatcaggt gctcctgatg cttcatcgct acactctcat 600

cgaccacgtc ctcgacaacg tcgtcactca tttggctccc tattggatct agatggacaa 660

catcgttatc acatggatca tcgacaccct caccgtcgag cttttcgaca tcgtccatga 720

gtgaggggca ccactcgcca ggcccgagtc tcccttgagg cttagttcct tgacaatagg 780

gaggcctagg ctctccatct tgatgccttg tccccaccta ttctcctaag gggatctctc 840

catcatcgag tactattggt ggatgaaggg gatggccaac tcccttcacg atctcggcga 900

gcccgttgcc gaccgcaccc tcgtattcaa ccttctacgt ggtcttagtc gttgctacaa 960

ccacctgaaa gctcttatca ggcggaccgt gtgcttcccc tcctttggcg acgtctgtaa 1020

tgagtttctc ctcgaggagt ttaccatgga ggccaagtcc accactgcca cgaacctcta 1080

cgatgcaccc tctagtggct aggtgtcctc tggtggctag gcccctacac tccatcgacg 1140

gggacccctg cacgccctcc caccacccta gccttagagc cccacattcg cctgctagcg 1200

tggatgcctg gatggcagtc gctagtggcg gcccggcgtg gctggcgttc taccaccctt 1260

ggaccaagac catctccatg tggctgggcc cgacctcggg tgctctctcg ccacatcctc 1320

cttagtcgtc cctcttggtg ttgccaacct atggtgtctc tccgttgccc acgacatcgg 1380

ctctgctcct cccacctccg gggacccctg ctccgctacc acgatccccg ctggctggag 1440

gttgggacca ggctgccctt gatgtagctt tcggtaccat ggcgccgaat ccaccccaag 1500

tcgactgggt ggttgactct agcgcctcct accacaccac ctccactgtg agcatgttat 1560

cttgctcaca tcccctacat ctctcccacc cttcctatat cattgtgggg aacaactcca 1620

ctcccccggt cacctcagta ggtgactcga ttctcccggg cccgttccac ctcaacgaag 1680

ttctagtcgc tcctcacatc attcgtaatc ttgtttttgt ttgtcagttc actattgaca 1740

actcttattc cattgagttt gacctgtttg gtttatctat gatctgtcca ccaggaccct 1800

cctcgcccgt tgtgatagct tgaagcccct ctacacgatc cggtcatcca ccttcaccat 1860

cgacgtgtcc tccctgcttg tcctggcctc caccacttcg catcatcgtc tcggctacac 1920

tggactctac gtcatgacca agcttgccag tagtttagag atcacgtagt agaggacatt 1980

ttgagggtat ttgtcatgct tgtctgctag gctgccatac tcgtctctct tttgctacta 2040

tttcttctag ggatgagcag acttttgatc ttttattgtg atctatggac atctctcata 2100

ctcagcctct ttggatacaa atactacttg tgattttgga tgatttctct cattttcttt 2160

ggacttttcc tttacggttg aagtctgaca cctttcacac cctcacacac tttattgcct 2220

aggtctccat ctagttctgt cacttggttc atgctctcca gtgcgattac gaccgcgagt 2280

cccacaactc tgcctctcgc tcatttcttt ctttcctatg gcgtccagtt gtgactctcg 2340

tgaccctaca cctcctcata atggctgggc tgaacggatt attcgcacca caccaacatg 2400

attcactgtc tcttttttca gtcgtctctt cctgctagct attgggtaga ggccctgaac 2460

actgctaccc atcatctcaa ccgccttcct tctaaggcgg tgaagatatc caactctcta 2520

taatcttcta taaagcatgg ctaatcatga gatatgtatg gaataaaggt tatagaatct 2580

tctataacaa ccatgacaag atatgaagct tatatatttg ttaccatggt atattaggaa 2640

ttctctagac ttatatattt gttgagattt tttctagaat atgtacaagt gatactcatc 2700

catggctatg tgcgcatata taagataagt gtcccatttt gataatgatg taacacaaca 2760

taccacctca tcccacatat atctgatagt ctgtcaattg tgtcatattt tatttattta 2820

tttagtgtat taatttagta ttggagaagt ttccaaggta agaaataatt aacaacagag 2880

gtccttttct attctactaa ataaaaaaag tagttggaca tattttttcg cctttttcgc 2940

catagtcaaa tatcactatc ttgatattca gatcatgttg attttcaact tatactttta 3000

atagtatgcc acataagcaa gtatgatgat atgacaagaa tttattaagc tgagagacat 3060

agacttctaa taactaaaac taaacaagta tgccacgtaa gcaaatatga tatgacacaa 3120

atttattaaa caaagagaca tagatagttt aaagagagat gagagaattt catctaaatt 3180

aaactcagga tacacgtttt tatagacaaa aaatgattta acatttttga aaaatgtaca 3240

ataaaactct gcattgggaa tatcctaagg atctgatagg tttaattaaa aatcttggaa 3300

ccggagctat gccaaatgag cgatgttata tgtagttaat ttattaacgt aatgtcatat 3360

gagtcgttta aatttcttgc tggtatatag ctgctcatcc aactgttcga tgttgtagcc 3420

agattcgttg tggtcatcgt ggtcgttgga ttatgatgat atttggccat taaaaccatc 3480

ctcatcatat tctctggaaa ccgtaacccg gtcttctact gatcatgctg tggaatcttg 3540

gtgccgacaa tgcagctctt cggctcaaca attcagtgac aagcactgct gctgcaaaca 3600

caagcaccag agcgacgcca tacctggatc tgcttgctcc ccctgttcgt caacttcgtc 3660

aagaatgcaa ctactacaac agcggcttgc cggtgtggtc accggagaag ttcccaagcg 3720

agcacttgct gctgacgccg tcggcgttag gtggcaacca agaccaagac acgcctccgc 3780

agcaggaggc ggcgacggcg atggcagtga tgctgaagca agtatcgaga gcgagcccag 3840

acgtgaaggc gagacaaacc aaactgcaga agaggcagga ggctaaacag agatacatgg 3900

agaagaggaa gaacaggagg tatgtgttgt tcaatatgat taattagtac tctcagctca 3960

ttgaattgtt tttataaatg aaacatgctc tgaaatgaaa cagctggtaa gaacataatg 4020

catctctata caaaagataa ttttttacaa ttcaatagtc tctttgcgat tgcatacata 4080

ggtatggcaa gacaattatg tatgcatcac ggaaggccca cgggcaaaca cacgaaacag 4140

agtaaaaggc agatttgcaa aggcatccag cggcagtggc ggcagccatg gcgacggcga 4200

tcagtctaca caacattgct atggccatgg cggcgatgaa gatgatcact ctacagtcta 4260

cacacgatcc tag 4273

<210> 10

<211> 4272

<212> DNA

<213> unknown (Artificial Synthesis)

<400> 10

atggatcctc cgacaacgac cctggcacct ctcatcctcc ccgatgtggc cactgctacg 60

accaccaacg acctcgccct tcccaactac ccggttacca tgaccgacac tgaccttgct 120

gctgctggca tgggtagcat cactgcgccc actagcagtc tctcacccgc ctgcctaccc 180

actcgcttcc tacagcacac ctcgtgggtt ccaccccccg gcttcaccgc tccttccagc 240

tccgtcgctc tgacacagcc gactagcatc tcctcactgg tcgcagctct tgccaccatc 300

cagtccgccg tcaccccgtg acccaagcct gtgagcacaa gcatgctgtc cacactactc 360

taagtctcta accgcttagc tagccaaggc tcaacaactc ttcttcagtc gcatcaattc 420

gcccaccccg gcatcccctg cggtccacat cactactagg ctcattgcca ccaccattgc 480

tcaggcggcc ggagttcaca acatccggtt ccttgtcttc attgtgttgg atccagcgtc 540

accccacttc gcccactggt gtgatcaggt gctcctgatg cttcatcgct acactctcat 600

cgaccacgtc ctcgacaacg tcgtcactca tttggctccc tattggatct agatggacaa 660

catcgttatc acatggatca tcgacaccct caccgtcgag cttttcgaca tcgtccatga 720

gtgaggggca ccactcgcca ggcccgagtc tcccttgagg cttagttcct tgacaatagg 780

gaggcctagg ctctccatct tgatgccttg tccccaccta ttctcctaag gggatctctc 840

catcatcgag tactattggt ggatgaaggg gatggccaac tcccttcacg atctcggcga 900

gcccgttgcc gaccgcaccc tcgtattcaa ccttctacgt ggtcttagtc gttgctacaa 960

ccacctgaaa gctcttatca ggcggaccgt gtgcttcccc tcctttggcg acgtctgtaa 1020

tgagtttctc ctcgaggagt ttaccatgga ggccaagtcc accactgcca cgaacctcta 1080

cgatgcaccc tctagtggct aggtgtcctc tggtggctag gcccctacac tccatcgacg 1140

gggacccctg cacgccctcc caccacccta gccttagagc cccacattcg cctgctagcg 1200

tggatgcctg gatggcagtc gctagtggcg gcccggcgtg gctggcgttc taccaccctt 1260

ggaccaagac catctccatg tggctgggcc cgacctcggg tgctctctcg ccacatcctc 1320

cttagtcgtc cctcttggtg ttgccaacct atggtgtctc tccgttgccc acgacatcgg 1380

ctctgctcct cccacctccg gggacccctg ctccgctacc acgatccccg ctggctggag 1440

gttgggacca ggctgccctt gatgtagctt tcggtaccat ggcgccgaat ccaccccaag 1500

tcgactgggt ggttgactct agcgcctcct accacaccac ctccactgtg agcatgttat 1560

cttgctcaca tcccctacat ctctcccacc cttcctatat cattgtgggg aacaactcca 1620

ctcccccggt cacctcagta ggtgactcga ttctcccggg cccgttccac ctcaacgaag 1680

ttctagtcgc tcctcacatc attcgtaatc ttgtttttgt ttgtcagttc actattgaca 1740

actcttattc cattgagttt gacctgtttg gtttatctat gatctgtcca ccaggaccct 1800

cctcgcccgt tgtgatagct tgaagcccct ctacacgatc cggtcatcca ccttcaccat 1860

cgacgtgtcc tccctgcttg tcctggcctc caccacttcg catcatcgtc tcggctacac 1920

tggactctac gtcatgacca agcttgccag tagtttagag atcacgtagt agaggacatt 1980

ttgagggtat ttgtcatgct tgtctgctag gctgccatac tcgtctctct tttgctacta 2040

tttcttctag ggatgagcag acttttgatc ttttattgtg atctatggac atctctcata 2100

ctcagcctct ttggatacaa atactacttg tgattttgga tgatttctct cattttcttt 2160

ggacttttcc tttacggttg aagtctgaca cctttcacac cctcacacac tttattgcct 2220

aggtctccat ctagttctgt cacttggttc atgctctcca gtgcgattac gaccgcgagt 2280

cccacaactc tgcctctcgc tcatttcttt ctttcctatg gcgtccagtt gtgactctcg 2340

tgaccctaca cctcctcata atggctgggc tgaacggatt attcgcacca caccaacatg 2400

attcactgtc tcttttttca gtcgtctctt cctgctagct attgggtaga ggccctgaac 2460

actgctaccc atcatctcaa ccgccttcct tctaaggcgg tgaagatatc caactctcta 2520

taatcttcta taaagcatgg ctaatcatga gatatgtatg gaataaaggt tatagaatct 2580

tctataacaa ccatgacaag atatgaagct tatatatttg ttaccatggt atattaggaa 2640

ttctctagac ttatatattt gttgagattt tttctagaat atgtacaagt gatactcatc 2700

catggctatg tgcgcatata taagataagt gtcccatttt gataatgatg taacacaaca 2760

taccacctca tcccacatat atctgatagt ctgtcaattg tgtcatattt tatttattta 2820

tttagtgtat taatttagta ttggagaagt ttccaaggta agaaataatt aacaacagag 2880

gtccttttct attctactaa ataaaaaaag tagttggaca tattttttcg cctttttcgc 2940

catagtcaaa tatcactatc ttgatattca gatcatgttg attttcaact tatactttta 3000

atagtatgcc acataagcaa gtatgatgat atgacaagaa tttattaagc tgagagacat 3060

agacttctaa taactaaaac taaacaagta tgccacgtaa gcaaatatga tatgacacaa 3120

atttattaaa caaagagaca tagatagttt aaagagagat gagagaattt catctaaatt 3180

aaactcagga tacacgtttt tatagacaaa aaatgattta acatttttga aaaatgtaca 3240

ataaaactct gcattgggaa tatcctaagg atctgatagg tttaattaaa aatcttggaa 3300

ccggagctat gccaaatgag cgatgttata tgtagttaat ttattaacgt aatgtcatat 3360

gagtcgttta aatttcttgc tggtatatag ctgctcatcc aactgttcga tgttgtagcc 3420

agattcgttg tggtcatcgt ggtcgttgga ttatgatgat atttggccat taaaaccatc 3480

ctcatcatat tctctggaaa ccgtaacccg gtcttctact gatcatgctg tggaatcttg 3540

gtgccgacaa tgcagctctt cggctcaaca attcagtgac aagcactgct gctgcaaaca 3600

caagcaccag agcgacgcca tacctggatc tgcttgctcc ccctgttcgt caacttcgtc 3660

aagaatgcaa ctactacaac agcggcttgc cggtgtggtc accggagaag ttcccaagcg 3720

agcacttgct gctgacgccg tcggcgttag gtggcaacca agaccaagac acgcctccgc 3780

agcaggaggc ggcgacggcg atggcagtga tgctgaagca agtatcgaga gcgagcccag 3840

acgtgaaggc gagacaaacc aaactgcaga agaggcagga ggctaaacag agatacatgg 3900

agaagaggaa gaacaggagg tatgtgttgt tcaatatgat taattagtac tctcagctca 3960

ttgaattgtt tttataaatg aaacatgctc tgaaatgaaa cagctggtaa gaacataatg 4020

catctctata caaaagataa ttttttacaa ttcaatagtc tctttgcgat tgcatacata 4080

ggtatggcaa gacaattatg tatgcatcac ggaagggcac gggcaaacac acgaaacaga 4140

gtaaaaggca gatttgcaaa ggcatccagc ggcagtggcg gcagccatgg cgacggcgat 4200

cagtctacac aacattgcta tggccatggc ggcgatgaag atgatcactc tacagtctac 4260

acacgatcct ag 4272

<210> 11

<211> 4269

<212> DNA

<213> unknown (Artificial Synthesis)

<400> 11

atggatcctc cgacaacgac cctggcacct ctcatcctcc ccgatgtggc cactgctacg 60

accaccaacg acctcgccct tcccaactac ccggttacca tgaccgacac tgaccttgct 120

gctgctggca tgggtagcat cactgcgccc actagcagtc tctcacccgc ctgcctaccc 180

actcgcttcc tacagcacac ctcgtgggtt ccaccccccg gcttcaccgc tccttccagc 240

tccgtcgctc tgacacagcc gactagcatc tcctcactgg tcgcagctct tgccaccatc 300

cagtccgccg tcaccccgtg acccaagcct gtgagcacaa gcatgctgtc cacactactc 360

taagtctcta accgcttagc tagccaaggc tcaacaactc ttcttcagtc gcatcaattc 420

gcccaccccg gcatcccctg cggtccacat cactactagg ctcattgcca ccaccattgc 480

tcaggcggcc ggagttcaca acatccggtt ccttgtcttc attgtgttgg atccagcgtc 540

accccacttc gcccactggt gtgatcaggt gctcctgatg cttcatcgct acactctcat 600

cgaccacgtc ctcgacaacg tcgtcactca tttggctccc tattggatct agatggacaa 660

catcgttatc acatggatca tcgacaccct caccgtcgag cttttcgaca tcgtccatga 720

gtgaggggca ccactcgcca ggcccgagtc tcccttgagg cttagttcct tgacaatagg 780

gaggcctagg ctctccatct tgatgccttg tccccaccta ttctcctaag gggatctctc 840

catcatcgag tactattggt ggatgaaggg gatggccaac tcccttcacg atctcggcga 900

gcccgttgcc gaccgcaccc tcgtattcaa ccttctacgt ggtcttagtc gttgctacaa 960

ccacctgaaa gctcttatca ggcggaccgt gtgcttcccc tcctttggcg acgtctgtaa 1020

tgagtttctc ctcgaggagt ttaccatgga ggccaagtcc accactgcca cgaacctcta 1080

cgatgcaccc tctagtggct aggtgtcctc tggtggctag gcccctacac tccatcgacg 1140

gggacccctg cacgccctcc caccacccta gccttagagc cccacattcg cctgctagcg 1200

tggatgcctg gatggcagtc gctagtggcg gcccggcgtg gctggcgttc taccaccctt 1260

ggaccaagac catctccatg tggctgggcc cgacctcggg tgctctctcg ccacatcctc 1320

cttagtcgtc cctcttggtg ttgccaacct atggtgtctc tccgttgccc acgacatcgg 1380

ctctgctcct cccacctccg gggacccctg ctccgctacc acgatccccg ctggctggag 1440

gttgggacca ggctgccctt gatgtagctt tcggtaccat ggcgccgaat ccaccccaag 1500

tcgactgggt ggttgactct agcgcctcct accacaccac ctccactgtg agcatgttat 1560

cttgctcaca tcccctacat ctctcccacc cttcctatat cattgtgggg aacaactcca 1620

ctcccccggt cacctcagta ggtgactcga ttctcccggg cccgttccac ctcaacgaag 1680

ttctagtcgc tcctcacatc attcgtaatc ttgtttttgt ttgtcagttc actattgaca 1740

actcttattc cattgagttt gacctgtttg gtttatctat gatctgtcca ccaggaccct 1800

cctcgcccgt tgtgatagct tgaagcccct ctacacgatc cggtcatcca ccttcaccat 1860

cgacgtgtcc tccctgcttg tcctggcctc caccacttcg catcatcgtc tcggctacac 1920

tggactctac gtcatgacca agcttgccag tagtttagag atcacgtagt agaggacatt 1980

ttgagggtat ttgtcatgct tgtctgctag gctgccatac tcgtctctct tttgctacta 2040

tttcttctag ggatgagcag acttttgatc ttttattgtg atctatggac atctctcata 2100

ctcagcctct ttggatacaa atactacttg tgattttgga tgatttctct cattttcttt 2160

ggacttttcc tttacggttg aagtctgaca cctttcacac cctcacacac tttattgcct 2220

aggtctccat ctagttctgt cacttggttc atgctctcca gtgcgattac gaccgcgagt 2280

cccacaactc tgcctctcgc tcatttcttt ctttcctatg gcgtccagtt gtgactctcg 2340

tgaccctaca cctcctcata atggctgggc tgaacggatt attcgcacca caccaacatg 2400

attcactgtc tcttttttca gtcgtctctt cctgctagct attgggtaga ggccctgaac 2460

actgctaccc atcatctcaa ccgccttcct tctaaggcgg tgaagatatc caactctcta 2520

taatcttcta taaagcatgg ctaatcatga gatatgtatg gaataaaggt tatagaatct 2580

tctataacaa ccatgacaag atatgaagct tatatatttg ttaccatggt atattaggaa 2640

ttctctagac ttatatattt gttgagattt tttctagaat atgtacaagt gatactcatc 2700

catggctatg tgcgcatata taagataagt gtcccatttt gataatgatg taacacaaca 2760

taccacctca tcccacatat atctgatagt ctgtcaattg tgtcatattt tatttattta 2820

tttagtgtat taatttagta ttggagaagt ttccaaggta agaaataatt aacaacagag 2880

gtccttttct attctactaa ataaaaaaag tagttggaca tattttttcg cctttttcgc 2940

catagtcaaa tatcactatc ttgatattca gatcatgttg attttcaact tatactttta 3000

atagtatgcc acataagcaa gtatgatgat atgacaagaa tttattaagc tgagagacat 3060

agacttctaa taactaaaac taaacaagta tgccacgtaa gcaaatatga tatgacacaa 3120

atttattaaa caaagagaca tagatagttt aaagagagat gagagaattt catctaaatt 3180

aaactcagga tacacgtttt tatagacaaa aaatgattta acatttttga aaaatgtaca 3240

ataaaactct gcattgggaa tatcctaagg atctgatagg tttaattaaa aatcttggaa 3300

ccggagctat gccaaatgag cgatgttata tgtagttaat ttattaacgt aatgtcatat 3360

gagtcgttta aatttcttgc tggtatatag ctgctcatcc aactgttcga tgttgtagcc 3420

agattcgttg tggtcatcgt ggtcgttgga ttatgatgat atttggccat taaaaccatc 3480

ctcatcatat tctctggaaa ccgtaacccg gtcttctact gatcatgctg tggaatcttg 3540

gtgccgacaa tgcagctctt cggctcaaca attcagtgac aagcactgct gctgcaaaca 3600

caagcaccag agcgacgcca tacctggatc tgcttgctcc ccctgttcgt caacttcgtc 3660

aagaatgcaa ctactacaac agcggcttgc cggtgtggtc accggagaag ttcccaagcg 3720

agcacttgct gctgacgccg tcggcgttag gtggcaacca agaccaagac acgcctccgc 3780

agcaggaggc ggcgacggcg atggcagtga tgctgaagca agtatcgaga gcgagcccag 3840

acgtgaaggc gagacaaacc aaactgcaga agaggcagga ggctaaacag agatacatgg 3900

agaagaggaa gaacaggagg tatgtgttgt tcaatatgat taattagtac tctcagctca 3960

ttgaattgtt tttataaatg aaacatgctc tgaaatgaaa cagctggtaa gaacataatg 4020

catctctata caaaagataa ttttttacaa ttcaatagtc tctttgcgat tgcatacata 4080

ggtatggcaa gacaattatg tatgcatcac ggaacacggg caaacacacg aaacagagta 4140

aaaggcagat ttgcaaaggc atccagcggc agtggcggca gccatggcga cggcgatcag 4200

tctacacaac attgctatgg ccatggcggc gatgaagatg atcactctac agtctacaca 4260

cgatcctag 4269

<210> 12

<211> 4269

<212> DNA

<213> unknown (Artificial Synthesis)

<400> 12

atggatcctc cgacaacgac cctggcacct ctcatcctcc ccgatgtggc cactgctacg 60

accaccaacg acctcgccct tcccaactac ccggttacca tgaccgacac tgaccttgct 120

gctgctggca tgggtagcat cactgcgccc actagcagtc tctcacccgc ctgcctaccc 180

actcgcttcc tacagcacac ctcgtgggtt ccaccccccg gcttcaccgc tccttccagc 240

tccgtcgctc tgacacagcc gactagcatc tcctcactgg tcgcagctct tgccaccatc 300

cagtccgccg tcaccccgtg acccaagcct gtgagcacaa gcatgctgtc cacactactc 360

taagtctcta accgcttagc tagccaaggc tcaacaactc ttcttcagtc gcatcaattc 420

gcccaccccg gcatcccctg cggtccacat cactactagg ctcattgcca ccaccattgc 480

tcaggcggcc ggagttcaca acatccggtt ccttgtcttc attgtgttgg atccagcgtc 540

accccacttc gcccactggt gtgatcaggt gctcctgatg cttcatcgct acactctcat 600

cgaccacgtc ctcgacaacg tcgtcactca tttggctccc tattggatct agatggacaa 660

catcgttatc acatggatca tcgacaccct caccgtcgag cttttcgaca tcgtccatga 720

gtgaggggca ccactcgcca ggcccgagtc tcccttgagg cttagttcct tgacaatagg 780

gaggcctagg ctctccatct tgatgccttg tccccaccta ttctcctaag gggatctctc 840

catcatcgag tactattggt ggatgaaggg gatggccaac tcccttcacg atctcggcga 900

gcccgttgcc gaccgcaccc tcgtattcaa ccttctacgt ggtcttagtc gttgctacaa 960

ccacctgaaa gctcttatca ggcggaccgt gtgcttcccc tcctttggcg acgtctgtaa 1020

tgagtttctc ctcgaggagt ttaccatgga ggccaagtcc accactgcca cgaacctcta 1080

cgatgcaccc tctagtggct aggtgtcctc tggtggctag gcccctacac tccatcgacg 1140

gggacccctg cacgccctcc caccacccta gccttagagc cccacattcg cctgctagcg 1200

tggatgcctg gatggcagtc gctagtggcg gcccggcgtg gctggcgttc taccaccctt 1260

ggaccaagac catctccatg tggctgggcc cgacctcggg tgctctctcg ccacatcctc 1320

cttagtcgtc cctcttggtg ttgccaacct atggtgtctc tccgttgccc acgacatcgg 1380

ctctgctcct cccacctccg gggacccctg ctccgctacc acgatccccg ctggctggag 1440

gttgggacca ggctgccctt gatgtagctt tcggtaccat ggcgccgaat ccaccccaag 1500

tcgactgggt ggttgactct agcgcctcct accacaccac ctccactgtg agcatgttat 1560

cttgctcaca tcccctacat ctctcccacc cttcctatat cattgtgggg aacaactcca 1620

ctcccccggt cacctcagta ggtgactcga ttctcccggg cccgttccac ctcaacgaag 1680

ttctagtcgc tcctcacatc attcgtaatc ttgtttttgt ttgtcagttc actattgaca 1740

actcttattc cattgagttt gacctgtttg gtttatctat gatctgtcca ccaggaccct 1800

cctcgcccgt tgtgatagct tgaagcccct ctacacgatc cggtcatcca ccttcaccat 1860

cgacgtgtcc tccctgcttg tcctggcctc caccacttcg catcatcgtc tcggctacac 1920

tggactctac gtcatgacca agcttgccag tagtttagag atcacgtagt agaggacatt 1980

ttgagggtat ttgtcatgct tgtctgctag gctgccatac tcgtctctct tttgctacta 2040

tttcttctag ggatgagcag acttttgatc ttttattgtg atctatggac atctctcata 2100

ctcagcctct ttggatacaa atactacttg tgattttgga tgatttctct cattttcttt 2160

ggacttttcc tttacggttg aagtctgaca cctttcacac cctcacacac tttattgcct 2220

aggtctccat ctagttctgt cacttggttc atgctctcca gtgcgattac gaccgcgagt 2280

cccacaactc tgcctctcgc tcatttcttt ctttcctatg gcgtccagtt gtgactctcg 2340

tgaccctaca cctcctcata atggctgggc tgaacggatt attcgcacca caccaacatg 2400

attcactgtc tcttttttca gtcgtctctt cctgctagct attgggtaga ggccctgaac 2460

actgctaccc atcatctcaa ccgccttcct tctaaggcgg tgaagatatc caactctcta 2520

taatcttcta taaagcatgg ctaatcatga gatatgtatg gaataaaggt tatagaatct 2580

tctataacaa ccatgacaag atatgaagct tatatatttg ttaccatggt atattaggaa 2640

ttctctagac ttatatattt gttgagattt tttctagaat atgtacaagt gatactcatc 2700

catggctatg tgcgcatata taagataagt gtcccatttt gataatgatg taacacaaca 2760

taccacctca tcccacatat atctgatagt ctgtcaattg tgtcatattt tatttattta 2820

tttagtgtat taatttagta ttggagaagt ttccaaggta agaaataatt aacaacagag 2880

gtccttttct attctactaa ataaaaaaag tagttggaca tattttttcg cctttttcgc 2940

catagtcaaa tatcactatc ttgatattca gatcatgttg attttcaact tatactttta 3000

atagtatgcc acataagcaa gtatgatgat atgacaagaa tttattaagc tgagagacat 3060

agacttctaa taactaaaac taaacaagta tgccacgtaa gcaaatatga tatgacacaa 3120

atttattaaa caaagagaca tagatagttt aaagagagat gagagaattt catctaaatt 3180

aaactcagga tacacgtttt tatagacaaa aaatgattta acatttttga aaaatgtaca 3240

ataaaactct gcattgggaa tatcctaagg atctgatagg tttaattaaa aatcttggaa 3300

ccggagctat gccaaatgag cgatgttata tgtagttaat ttattaacgt aatgtcatat 3360

gagtcgttta aatttcttgc tggtatatag ctgctcatcc aactgttcga tgttgtagcc 3420

agattcgttg tggtcatcgt ggtcgttgga ttatgatgat atttggccat taaaaccatc 3480

ctcatcatat tctctggaaa ccgtaacccg gtcttctact gatcatgctg tggaatcttg 3540

gtgccgacaa tgcagctctt cggctcaaca attcagtgac aagcactgct gctgcaaaca 3600

caagcaccag agcgacgcca tacctggatc tgcttgctcc ccctgttcgt caacttcgtc 3660

aagaatgcaa ctactacaac agcggcttgc cggtgtggtc accggagaag ttcccaagcg 3720

agcacttgct gctgacgccg tcggcgttag gtggcaacca agaccaagac acgcctccgc 3780

agcaggaggc ggcgacggcg atggcagtga tgctgaagca agtatcgaga gcgagcccag 3840

acgtgaaggc gagacaaacc aaactgcaga agaggcagga ggctaaacag agatacatgg 3900

agaagaggaa gaacaggagg tatgtgttgt tcaatatgat taattagtac tctcagctca 3960

ttgaattgtt tttataaatg aaacatgctc tgaaatgaaa cagctggtaa gaacataatg 4020

catctctata caaaagataa ttttttacaa ttcaatagtc tctttgcgat tgcatacata 4080

ggtatggcaa gacaattatg tatgcatcac ggaacgcggg caaacacacg aaacagagta 4140

aaaggcagat ttgcaaaggc atccagcggc agtggcggca gccatggcga cggcgatcag 4200

tctacacaac attgctatgg ccatggcggc gatgaagatg atcactctac agtctacaca 4260

cgatcctag 4269

<210> 13

<211> 21

<212> DNA

<213> unknown (Artificial Synthesis)

<400> 13

ccagattcgt tgtggtcatc g 21

<210> 14

<211> 20

<212> DNA

<213> unknown (Artificial Synthesis)

<400> 14

ttgtgtagac tgatcgccgt 20

Claims

1. The application of a protein in controlling the flowering phase character of corn is characterized in that: the amino acid sequence of the protein is shown as SEQ ID NO. 1.

2. The application of a nucleic acid molecule in controlling the flowering stage character of corn is characterized in that: the nucleic acid molecule encodes a protein as claimed in claim 1.

3. Use according to claim 2, characterized in that: the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.2 or SEQ ID NO. 3.

4. A method for advancing the flowering phase of corn is characterized in that: inhibiting the expression and/or activity of a protein according to claim 1 in maize and selecting plants that are early in the flowering stage of maize.

5. The method of advancing the flowering stage of corn of claim 4, wherein: the method of inhibiting protein expression and/or activity comprises any one of gene editing, RNA interference or T-DNA insertion.

6. The method of advancing the flowering stage of corn of claim 5, wherein: the gene editing adopts a CRISPR/Cas9 method.

7. The method of advancing the flowering stage of corn of claim 6, wherein: the genome target region of the CRISPR/Cas9 method in maize is the combination of the sequences shown in SEQ ID NO.4 and SEQ ID NO. 5.

8. A kit for advancing the flowering phase of corn, comprising: including any of the following:

(1) sgRNA molecules, the sequences of which are shown as a combination of SEQ ID NO.6 and SEQ ID NO. 7;

(2) a DNA molecule encoding the sgRNA of (1);

(3) a vector expressing the sgRNA of (1).

9. A mutant gene for early flowering phase of maize, which is characterized in that: the sequence of the mutant gene is shown in any one of SEQ ID NO.8-SEQ ID NO. 12.

10. A primer set for detecting a mutant gene according to claim 9, wherein: the primer pair is a sequence shown in SEQ ID NO.13 and SEQ ID NO. 14.

11. Use of the primer set of claim 10 for detecting the mutant gene of claim 9.