CN112646016A - Gene and method for changing flowering period of corn - 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

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 GRMZM2G095598 gene in maize inbred line B73. The sequence of SEQ ID NO.2 is the genome sequence of GRMZM2G095598 gene. The SEQ ID NO.3 sequence is the cDNA sequence of GRMZM2G095598 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.

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. 5;

(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 as SEQ ID NO. 6.

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.6 is determined to lead the flowering phase of the corn to be properly advanced. 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.7 and SEQ ID NO.8 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, wherein if the sequence of the sequenced amplification product is consistent with a partial sequence of the sequence shown in SEQ ID NO.6, 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 the GRMZM2G095598 can actually affect the maize flowering phase character through phenotype 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; the primer pair gR-1F (3F)/gRR1 is used for amplifying the sgRNA of the first target, and the product length 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. After the gene was edited for GRMZM2G095598, maize flowering time was earlier for the T0 generation material than for 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 target site designed during the editing of the GRMZM2G095598 gene is shown as SEQ ID No. 4. The gRNA sequence expressed by the vector containing the target is shown in SEQ ID NO. 5.

Extracting DNA in seedling stage to detect gene editing condition. Designing a primer, wherein the sequence of the primer is shown as SEQ ID NO.7 and SEQ ID NO. 8. 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, only 1 edit type of material was found, and GG was deleted at the target site (Table 3). Thus, after editing, the gene sequence of the material A1 was changed from SEQ ID NO.2 to SEQ ID NO. 6.

TABLE 3 Gene editing maize Material flowering time trait data

"-" indicates the deleted sequence, boxes indicate the PAM sequence, and underlines indicate the editing target.

The flowering stage traits (including the emasculation stage, the pollen scattering stage and the silking stage) of the a1 material were experimentally investigated in the jilin province in summer 2018, and the results are shown in table 4. The flowering phase of the A1 edited material is obviously earlier than that of the unedited control material, and the gene is proved to control the flowering phase trait, and the flowering phase is advanced after gene editing.

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.6 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.7 and SEQ ID NO.8 can be used for detecting whether the maize genome contains the mutant gene, the primer pair is used for carrying out PCR amplification on the genome DNA of a sample to be detected, an amplification product is sequenced, and if the amplification product is consistent with the sequence shown in 1 st-1140 th positions of SEQ ID NO.6, the mutation gene shown in SEQ ID NO.6 is contained; and if the amplification product is identical to the sequence shown in the 1 st-1142 nd positions of SEQ ID NO.2, the gene type 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

<110> university of agriculture in Huazhong, college of agricultural sciences of Jilin province, Miami Biotechnology (Jiangsu) Co., Ltd

<120> genes and methods for altering flowering phase of maize

<130> 1

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 320

<212> PRT

<213> Zea mays

<400> 1

Met Glu Gly Asp Glu Lys Ser Ala Gly Gly Ala Pro Ala Tyr Trp Gly

1 5 10 15

Leu Gly Ala Arg Pro Cys Asp Ala Cys Gly Ala Glu Ala Ala Arg Leu

20 25 30

Tyr Cys Arg Ala Asp Ala Ala Phe Leu Cys Ala Gly Cys Asp Ala Arg

35 40 45

Ala His Gly Ala Gly Ser Arg His Ala Arg Val Trp Leu Cys Glu Val

50 55 60

Cys Glu His Ala Pro Ala Ala Val Thr Cys Arg Ala Asp Ala Ala Ala

65 70 75 80

Leu Cys Ala Ser Cys Asp Ala Asp Ile His Ser Ala Asn Pro Leu Ala

85 90 95

Arg Arg His Glu Arg Leu His Val Ala Pro Phe Phe Gly Ala Leu Ala

100 105 110

Asp Ala Pro Lys Pro Phe Ala Ser Ala Ala Pro Pro Lys Ala Thr Asp

115 120 125

Asp Asp Gly Ser Asn Glu Asp Glu Ala Ala Ser Trp Leu Leu Pro Glu

130 135 140

Pro Asp His Gly Gln Lys Glu Gly Ala Thr Thr Glu Val Phe Phe Ala

145 150 155 160

Asp Ser Asp Pro Tyr Leu Asp Leu Asp Phe Ala Arg Ser Met Asp Glu

165 170 175

Ile Lys Thr Ile Gly Val Gln Gln Ser Gly Ser Pro Glu Leu Asp Leu

180 185 190

Ala Gly Thr Lys Leu Phe Tyr Ser Asp His Ser Val Asn His Ser Val

195 200 205

Ser Ser Ser Glu Ala Ala Val Val Pro Asp Ala Ala Ser Gly Met Ala

210 215 220

Pro Met Val Ala Val Val Ser Arg Gly Leu Glu Arg Glu Ala Arg Leu

225 230 235 240

Met Arg Tyr Arg Glu Lys Arg Lys Ser Arg Arg Phe Glu Lys Thr Ile

245 250 255

Arg Tyr Ala Ser Arg Lys Ala Tyr Ala Glu Thr Arg Pro Arg Ile Lys

260 265 270

Gly Arg Phe Ala Lys Arg Thr Pro Gly Ala Gly Glu Asp Thr Leu Glu

275 280 285

Glu His Glu Glu Met Tyr Ser Ser Ala Ala Ala Ala Val Ala Ala Leu

290 295 300

Met Ala Pro Gly Gly Ala Asp Ala Asp Tyr Gly Val Val Pro Thr Tyr

305 310 315 320

<210> 2

<211> 1518

<212> DNA

<213> Zea mays

<400> 2

gcacgggcga acaaattgac ccgggagccg ggacggcgac ttccgtggtc agtcggtcct 60

ccacgtcgct cgttcgcgcc gaagacccgc ccaggccgcc ccacacggcc acagctcaaa 120

tcctgagcag agtgggaaag cttggctgag aatcgcttcg tcccgacagc cactactagg 180

aattgccgag cgagcgagct tgcgccgtgc gcgatggagg gtgacgagaa gtcggcgggc 240

ggggcccctg cttactgggg cctgggcgcg cggccctgcg acgcgtgcgg cgccgaggcg 300

gcgcgcctct actgccgcgc ggacgcggcg ttcctgtgcg ccgggtgcga cgcgcgggcg 360

cacggcgccg ggtcgcgcca cgcgcgggtc tggctctgcg aggtctgcga gcacgcgccg 420

gcggcggtca cgtgccgcgc ggacgctgcc gcgctctgcg cctcctgcga cgccgacatc 480

cactcggcga acccgctcgc gcgccgccac gagcgcctcc acgtggcgcc cttcttcggc 540

gcgctggccg acgcgcccaa gcccttcgcc tcggcggcgc cgcccaaagc aaccgacgac 600

gacgggagca acgaggacga ggcggcgtcg tggctcctcc ccgagcccga ccacgggcag 660

aaagaaggcg ccacgacgga ggtgttcttc gcggactctg acccgtacct cgacctcgac 720

ttcgcgcgtt ccatggacga aatcaagacc atcggcgtcc agcagagcgg gtcaccagag 780

ctcgacctcg ccggcaccaa gctcttctac tccgatcact ccgtgaacca cagtgtgagc 840

tagcatctct gatctgggca ctatctattt atctatcatt ctatctacgc acgtcccagc 900

actacttact tgaagttgaa gttaagccat gacgtgacgt cctatatctc actctcagtg 960

ttctgaaaat aaagacacgg atacgattgc aggtgtcatc gtcggaggca gcggttgtgc 1020

ccgacgcggc gtctggcatg gcgcccatgg tggcagtggt cagcaggggc ctggagcgag 1080

aggcgcggct gatgcggtac cgggagaagc gcaagagcag gcggttcgag aagacgatcc 1140

ggtacgcgtc ccgcaaggcg tacgcggaga cgcggccgcg catcaagggc cggttcgcca 1200

agcgcacgcc cggggctggg gaggacacgc tggaggagca cgaggagatg tactcctccg 1260

ccgctgccgc cgtggctgcg ctcatggccc ccggcggagc cgatgcggac tacggcgtcg 1320

tgcccacata ttgatctatg caattgcaac gccgacatgt acactagtta gcctcgtgct 1380

ctggggctgt aatttttgct gcatgcattg catgcaaagc tcttattgat tgcctgtatg 1440

attaacggaa gccattacaa gcaaggactt cctccctcct tttcaattcc tatgtatatt 1500

actatcatca ctacgagc 1518

<210> 3

<211> 1349

<212> DNA

<213> Zea mays

<400> 3

caaattgacc cgggagccgg gacggcgact tccgtggtca gtcggtcctc cacgtcgctc 60

gttcgcgccg aagacccgcc caggccgccc cacacggcca cagctcaaat cctgagcaga 120

gtgggaaagc ttggctgaga atcgcttcgt cccgacagcc actactagga attgccgagc 180

gagcgagctt gcgccgtgcg cgatggaggg tgacgagaag tcggcgggcg gggcccctgc 240

ttactggggc ctgggcgcgc ggccctgcga cgcgtgcggc gccgaggcgg cgcgcctcta 300

ctgccgcgcg gacgcggcgt tcctgtgcgc cgggtgcgac gcgcgggcgc acggcgccgg 360

gtcgcgccac gcgcgggtct ggctctgcga ggtctgcgag cacgcgccgg cggcggtcac 420

gtgccgcgcg gacgctgccg cgctctgcgc ctcctgcgac gccgacatcc actcggcgaa 480

cccgctcgcg cgccgccacg agcgcctcca cgtggcgccc ttcttcggcg cgctggccga 540

cgcgcccaag cccttcgcct cggcggcgcc gcccaaagca accgacgacg acgggagcaa 600

cgaggacgag gcggcgtcgt ggctcctccc cgagcccgac cacgggcaga aagaaggcgc 660

cacgacggag gtgttcttcg cggactctga cccgtacctc gacctcgact tcgcgcgttc 720

catggacgaa atcaagacca tcggcgtcca gcagagcggg tcaccagagc tcgacctcgc 780

cggcaccaag ctcttctact ccgatcactc cgtgaaccac agtgtgtcat cgtcggaggc 840

agcggttgtg cccgacgcgg cgtctggcat ggcgcccatg gtggcagtgg tcagcagggg 900

cctggagcga gaggcgcggc tgatgcggta ccgggagaag cgcaagagca ggcggttcga 960

gaagacgatc cggtacgcgt cccgcaaggc gtacgcggag acgcggccgc gcatcaaggg 1020

ccggttcgcc aagcgcacgc ccggggctgg ggaggacacg ctggaggagc acgaggagat 1080

gtactcctcc gccgctgccg ccgtggctgc gctcatggcc cccggcggag ccgatgcgga 1140

ctacggcgtc gtgcccacat attgatctat gcaattgcaa cgccgacatg tacactagtt 1200

agcctcgtgc tctggggctg taatttttgc tgcatgcatt gcatgcaaag ctcttattga 1260

ttgcctgtat gattaacgga agccattaca agcaaggact tcctccctcc ttttcaattc 1320

ctatgtatat tactatcatc actacgagc 1349

<210> 4

<211> 20

<212> DNA

<213> Zea mays

<400> 4

gttgtgcccg acgcggcgtc 20

<210> 5

<211> 103

<212> RNA

<213> unknown (Artificial Synthesis)

<400> 5

guugugcccg acgcggcguc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu uuu 103

<210> 6

<211> 1516

<212> DNA

<213> unknown (Artificial Synthesis)

<400> 6

gcacgggcga acaaattgac ccgggagccg ggacggcgac ttccgtggtc agtcggtcct 60

ccacgtcgct cgttcgcgcc gaagacccgc ccaggccgcc ccacacggcc acagctcaaa 120

tcctgagcag agtgggaaag cttggctgag aatcgcttcg tcccgacagc cactactagg 180

aattgccgag cgagcgagct tgcgccgtgc gcgatggagg gtgacgagaa gtcggcgggc 240

ggggcccctg cttactgggg cctgggcgcg cggccctgcg acgcgtgcgg cgccgaggcg 300

gcgcgcctct actgccgcgc ggacgcggcg ttcctgtgcg ccgggtgcga cgcgcgggcg 360

cacggcgccg ggtcgcgcca cgcgcgggtc tggctctgcg aggtctgcga gcacgcgccg 420

gcggcggtca cgtgccgcgc ggacgctgcc gcgctctgcg cctcctgcga cgccgacatc 480

cactcggcga acccgctcgc gcgccgccac gagcgcctcc acgtggcgcc cttcttcggc 540

gcgctggccg acgcgcccaa gcccttcgcc tcggcggcgc cgcccaaagc aaccgacgac 600

gacgggagca acgaggacga ggcggcgtcg tggctcctcc ccgagcccga ccacgggcag 660

aaagaaggcg ccacgacgga ggtgttcttc gcggactctg acccgtacct cgacctcgac 720

ttcgcgcgtt ccatggacga aatcaagacc atcggcgtcc agcagagcgg gtcaccagag 780

ctcgacctcg ccggcaccaa gctcttctac tccgatcact ccgtgaacca cagtgtgagc 840

tagcatctct gatctgggca ctatctattt atctatcatt ctatctacgc acgtcccagc 900

actacttact tgaagttgaa gttaagccat gacgtgacgt cctatatctc actctcagtg 960

ttctgaaaat aaagacacgg atacgattgc aggtgtcatc gtcggaggca gcggttgtgc 1020

ccgacgccgt ctggcatggc gcccatggtg gcagtggtca gcaggggcct ggagcgagag 1080

gcgcggctga tgcggtaccg ggagaagcgc aagagcaggc ggttcgagaa gacgatccgg 1140

tacgcgtccc gcaaggcgta cgcggagacg cggccgcgca tcaagggccg gttcgccaag 1200

cgcacgcccg gggctgggga ggacacgctg gaggagcacg aggagatgta ctcctccgcc 1260

gctgccgccg tggctgcgct catggccccc ggcggagccg atgcggacta cggcgtcgtg 1320

cccacatatt gatctatgca attgcaacgc cgacatgtac actagttagc ctcgtgctct 1380

ggggctgtaa tttttgctgc atgcattgca tgcaaagctc ttattgattg cctgtatgat 1440

taacggaagc cattacaagc aaggacttcc tccctccttt tcaattccta tgtatattac 1500

tatcatcact acgagc 1516

<210> 7

<211> 20

<212> DNA

<213> unknown (Artificial Synthesis)

<400> 7

gcacgggcga acaaattgac 20

<210> 8

<211> 20

<212> DNA

<213> unknown (Artificial Synthesis)

<400> 8

ccggatcgtc ttctcgaacc 20

Claims (10)

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 the protein of claim 1; optionally, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.2 or SEQ ID NO. 3.

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

4. The method of advancing the flowering stage of corn of claim 3, wherein: the method for inhibiting the expression and/or activity of the protein comprises any one of gene editing, RNA interference, T-DNA insertion, physical or chemical mutagenesis.

5. The method of advancing the flowering stage of corn of claim 4, wherein: the gene editing adopts a CRISPR/Cas9 method.

6. The method of advancing the flowering stage of corn of claim 5, wherein: the DNA sequence of the genome target region of the CRISPR/Cas9 method in maize is shown in SEQ ID No. 4.

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

(1) the sequence of the sgRNA molecule is shown as SEQ ID NO. 5;

(2) a DNA molecule encoding the sgRNA;

(3) a vector expressing the sgRNA.

8. A mutant gene for early flowering phase of maize, which is characterized in that: the sequence of the mutant gene is shown as SEQ ID NO. 6.

9. A primer set for detecting a mutant gene according to claim 8, wherein: the primer pair is a sequence shown in SEQ ID NO.7 and SEQ ID NO.8 or a complementary sequence thereof.

10. Use of the primer set of claim 9 for detecting the mutant gene of claim 8.

Images