CN111172171B

CN111172171B - Gene for controlling plant height and flowering phase of corn and application thereof

Info

Publication number: CN111172171B
Application number: CN202010080086.9A
Authority: CN
Inventors: 金敏亮; 刘海军; 刘相国; 许洁婷; 严建兵
Original assignee: Weimi Biotechnology Jiangsu Co ltd; Huazhong Agricultural University
Current assignee: Changzhou Xinmi Biotechnology Co ltd; Huazhong Agricultural University
Priority date: 2020-02-04
Filing date: 2020-02-04
Publication date: 2023-09-12
Anticipated expiration: 2040-02-04
Also published as: CN111172171A

Abstract

The present application belongs to the field of molecular genetics. In particular to a gene for controlling the plant height and the flowering phase of corn and an application thereof in changing the plant height or the flowering phase of the corn. The application provides a sequence for controlling corn plant height and flowering phase genes, and discloses a method for reducing corn plant height or delaying flowering phase by mutating the genes by using a genetic engineering means.

Description

Gene for controlling plant height and flowering phase of corn and application thereof

Technical Field

The present application belongs to the field of molecular genetics. In particular to a gene for controlling the plant height and the flowering phase of corn and an application thereof in changing the plant height or the flowering phase of the corn. The application provides a sequence for controlling corn plant height and flowering phase genes, and discloses a method for reducing corn plant height or delaying flowering phase by mutating genes by using a genetic engineering means.

Background

The dwarfing character brings breakthrough of crop yield, and the method for reducing the plant height of the crops has great application potential. In the main agronomic traits of corn, plant height affects lodging resistance, photosynthetic efficiency and harvest index of the corn, and is closely related to corn yield. Therefore, the plant height character has important value in corn breeding practice and germplasm resource improvement work.

The plant height is controlled by the dominant gene and the micro-effect polygene together, and is typical quantitative trait inheritance. Although some genes regulating the plant height of corn were located and cloned (Huanan agricultural university. Corn ZmPIF3s mutant protein, its coding gene and its application in breeding: CN201910273522.1[ P ].2019-08-02.; hangzhou Ruifeng Biotechnology Co., ltd., CYP78A gene was used to increase the plant height of corn and enhance the plant growth, china agricultural university. Genes related to the plant height of corn and their coding proteins and applications: CN200410037404.4[ P ].2005-01-26.; china agricultural academy of crop science, methods of creating dwarf materials by gene editing techniques: CN201910371358.8[ P ]. 2019-08-16.; more plant height trait related genes were required to be further cloned.

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 body. The biological property of flowering is under the double effect of the genetic factors of the plant body and the external environment factors. Under the influence of the dual effects, a series of flowering induction processes have a general rule in higher plants that plant leaves produce flower substances (or florigens) at a proper time by sensing external growth conditions (illumination, temperature, humidity and the like), and the flower substances are transported from the leaves to stem tip growth points through a delivery tissue to stimulate apical meristems to form flowers.

The flowering period is an important character in the process of crop evolution and adaptation, understanding the genetic basis of the flowering period character of crops and cloning candidate genes can improve the environmental adaptation capacity and plasticity of crops, so that the method has important significance for cultivating excellent crop varieties adapting to different ecological areas, and simultaneously promotes the genetic improvement process of important production characters, such as yield and the like, closely related to the flowering period. In addition, the whole growth period of crops is mainly determined by the length of the flowering period, and the whole growth period can be reduced by moderately shortening the flowering period, so that the planting cost is reduced.

Disclosure of Invention

It is an object of the present application to provide a sequence of a gene controlling maize plant height and flowering phase traits.

The second purpose of the application is to disclose a method for reducing the plant height of corn or delaying the flowering phase.

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

the application provides a gene for controlling the plant height and the flowering phase of corn and application thereof in changing the plant height or the flowering phase of the corn, which is characterized in that: the nucleic acid sequence of the gene is shown in any one of SEQ ID NO.1-SEQ ID NO. 4. Wherein, SEQ ID NO.1 sequence is the genome sequence in maize inbred line B73, SEQ ID NO.2 sequence is the genome sequence in maize inbred line KN5585, SEQ ID NO.3 sequence is the gene coding region sequence in maize inbred line B73, and SEQ ID NO.4 sequence is the gene coding region sequence in maize inbred line KN 5585. The nucleic acid sequence of the above gene is substituted and/or deleted and/or added with one or more amino acids, and the sequence having the same function also belongs to the present application.

In another aspect, the application also provides a method for reducing the plant height or delaying the flowering phase of corn, which is characterized by: and (3) inhibiting the expression and/or activity of the protein encoded by the above genes in corn, and selecting plants with reduced corn plant height or delayed flowering phase.

In some embodiments, the amino acid sequence of the above protein is shown as SEQ ID NO.5 or SEQ ID NO. 6. Wherein the sequence of SEQ ID NO.5 is the gene coding protein sequence in the maize inbred line B73, and the sequence of SEQ ID NO.6 is the gene coding protein sequence in the maize inbred line KN 5585.

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

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

In some embodiments, the DNA sequence of the genomic target region in maize of the above CRISPR/Cas9 method is shown as SEQ ID No.7 or SEQ ID No. 8.

In another aspect, the application also provides a kit for reducing the plant height and flowering phase of maize, characterized in that: comprising any one of the following:

(1) The sgRNA molecule has a sequence shown as SEQ ID NO.9 or SEQ ID NO. 10;

(2) A DNA molecule encoding the sgRNA;

(3) A vector expressing said sgRNA.

In another aspect, the application also provides a maize mutant genotype obtained by using the kit, which is characterized in that: the mutant genotype sequence is shown as any one of SEQ ID NO.11-SEQ ID NO. 16.

The application has the advantages and beneficial effects as follows: the application utilizes the multi-parent high-generation inbred line group to locate a genome region for controlling the plant height character of corn, the region does not have a known gene affecting the plant height, and only has one annotation gene GRMZM2G331652 with unknown functions. The CRISPR/Cas9 method is utilized to mutate GRMZM2G331652 genes, so that the genes are proved to be capable of controlling the plant height traits of corn, and the genes are unexpectedly found to be capable of controlling the flowering phase traits of corn. By using the CRISPR/Cas9 gene editing method and the edited mutant genotype sequence, the plant height of the corn or the flowering period can be reduced, and dwarf or flowering period delayed corn varieties can be created, so that the corn yield is improved, the lodging rate is reduced, the planting efficiency is improved, the mechanized harvesting is facilitated, and the ecological adaptability of the corn is improved.

Drawings

FIG. 1 plant height QTL positioning results. The vertical axis represents p-value for each marker association analysis test, -log10 is taken and the horizontal axis represents chromosome position.

FIG. 2 gene editing vector diagram. The English and abbreviations of the elements are listed below:

figure 3 plant height and flowering performance after mutation of maize genes using CRISPR-Cas9 technology. Left: before mutation; right: after mutation.

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 construed according to conventional usage by those of ordinary skill in the relevant art. All patent documents, academic papers, industry standards, and other publications 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, whole 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 in the 5 'to 3' direction from left to right; the amino acid sequence is written in the amino to carboxyl direction from left to right. Amino acids may be represented herein by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB biochemical nomenclature committee. Likewise, nucleotides may be referred to by commonly accepted single letter codes. The numerical range includes 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 codons. As used herein, reference to a "full-length sequence" of a particular polynucleotide or protein encoded thereby refers to an entire nucleic acid sequence or an entire amino acid sequence having a natural (non-synthetic) endogenous sequence. The full length polynucleotide encodes the full length, catalytically active form of the particular protein. The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term is used for amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acid. The term is also used for naturally occurring amino acid polymers. The terms "residue" or "amino acid" are used interchangeably herein to refer to an amino acid that is incorporated into a protein, polypeptide, or peptide (collectively, "protein"). Amino acids may be naturally occurring amino acids, and unless otherwise limited, may include known analogs of natural amino acids, which analogs may function in a similar manner to 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, when isolated or purified nucleic acids or polypeptides are produced by recombinant techniques, the isolated or purified nucleic acids or polypeptides are substantially free of other cellular material or culture medium, or when isolated or purified nucleic acids or polypeptides are chemically synthesized, are substantially free of chemical precursors or other chemicals. An "isolated" nucleic acid is generally 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 a nucleotide sequence of less than about 0.5kb naturally flanking the nucleic acid in genomic DNA of the cell from which the nucleic acid is derived.

In the present application, the terms "comprises," "comprising," or variations thereof, are to be understood to encompass other elements, numbers, or steps in addition to those described. "subject plant" or "subject plant cell" refers to a plant or plant cell in which genetic engineering has been effected, or a progeny cell of a plant or cell so engineered, which progeny cell comprises the engineering. "control" or "control plant cell" provides a reference point for measuring phenotypic changes in a subject plant or plant cell. Control plants or plant cells may include, for example: (a) Wild-type plants or cells, i.e., plants or cells having the same genotype as the genetically engineered starting material, which genetic engineering produces the subject plant or cell; (b) A plant or plant cell having the same genotype as the starting material but which has been transformed with an empty construct (i.e., with a construct that has no known effect on the trait of interest, such as a construct comprising a marker gene); (c) A plant or plant cell that is a non-transformed isolate of the subject plant or plant cell; (d) A plant or plant cell genetically identical to the test plant or plant cell but not 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 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 potentially genetic sequences of proteins of agricultural interest.

In some embodiments, the nucleotide sequences of the present application may be altered to make conservative amino acid substitutions. The principles and examples of conservative amino acid substitutions are described further below. In certain embodiments, the nucleotide sequence of the present application may be subjected to substitutions in accordance with the disclosed monocot codon preferences that do not alter the amino acid sequence, e.g., codons encoding the same amino acid sequence may be replaced 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 the present application is replaced with a different codon encoding the same amino acid sequence, such that the amino acid sequence encoded thereby is not changed while the nucleotide sequence is changed. Conservative variants include those sequences that encode the amino acid sequence of one of the proteins of an embodiment due to the degeneracy of the genetic code. In some embodiments, a portion of the nucleotide sequences of the present application are substituted according to monocot preference codons. Those skilled in the art will recognize that amino acid additions and/or substitutions are generally based on the relative similarity of amino acid side chain substituents, e.g., hydrophobicity, charge, size, etc., of the substituents. Exemplary amino acid substituents having various of the aforementioned contemplated properties are well known to those skilled in the art and include arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine. Guidelines for suitable amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model of Dayhoff et al (1978) Atlas of Protein Sequence and Structure (protein sequence and structure atlas) (Natl. Biomed. Res. Foundation, washington, D.C.), incorporated herein by reference. Conservative substitutions, such as substitution of 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 with other corresponding nucleotide sequences present in a cloned genomic DNA fragment or population of cDNA fragments (i.e., a genomic library or cDNA library) from a selected organism.

In some embodiments, fragments of the nucleotide sequence and the amino acid sequence 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 a nucleotide sequence 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 a native protein that comprise consecutive 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, the plant is transformed with an expression vector comprising the nucleotide sequence of at least one embodiment and operably linked thereto a promoter that drives expression in a plant cell. Transformed plant cells and transgenic plants refer to plant cells or plants comprising a heterologous polynucleotide within the genome. In general, the heterologous polynucleotide is stably integrated within the genome of the transformed plant cell or transgenic plant, such that the polynucleotide is delivered to the offspring. The heterologous polynucleotide may be integrated into the genome, either 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, hulls, stalks, roots, root tips, anthers, and the like. The application also includes plant cells, protoplasts, tissues, calli, embryos and flowers, stems, fruits, leaves and roots derived from the transgenic plants of the application or progeny thereof, and thus comprising at least in part the nucleotide sequences of the application.

The following examples are illustrative of the application and are not intended to limit the scope of the application. Modifications and substitutions to methods, procedures, or conditions of the present application without departing from the spirit and nature of the application are intended to be within the scope of the present application. 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 conditions recommended by the manufacturer's instructions, unless otherwise indicated. Unless otherwise indicated, all chemical reagents used in the examples were conventional commercial reagents, and the technical means used in the examples were conventional means well known to those skilled in the art.

Examples

Example 1 positioning procedure for maize plant height QTL

1. Population construction

According to the application, 24 Chinese fine maize inbred lines are subjected to two-generation double-row hybridization, six-generation open pollination and six-generation selfing to obtain a multi-parent high-generation inbred line group (CUBIC) consisting of 1404 generations. These 24 chinese elite maize inbred lines included LV28 (travel 28), E28, DAN340 (DAN 340), F349, ZI330 (self 330), ZONG3 (heddle 3), ZONG31 (heddle 31), HUANGC (yellow C), HZS (yellow early four), HYS (Huang Yesi), TY4 (skyline 4), YUANGFH (primordial yellow), CHANG7-2 (CHANG 7-2), K12, XI502 (west 502), LX9801, H21, shang 741 (double 741), Q1261, JI853 (Ji 853), JI53 (Ji 53), 5237, 81515, NX110 (agro 110), and 24 parent and offspring inbred lines of this population all performed genomic sequencing, yielding SNP and InDel variations exceeding 14M.

2. Phenotypic analysis

All 24 parents and 1404 offspring inbred lines were planted in 5 places in Huang-Huai-Hai and northeast, and the phenotype variation was abundant. The examined agronomic phenotypes include plant height, flowering phase, spike weight and other yield traits. The Best Linear Unbiased Predictor (BLUP) value for each inbred line was calculated repeatedly for all environments for subsequent analysis, including basic phenotype statistics, correlation analysis, and GWAS. The phenotypic variance or genetic power interpreted by the QTL for a given trait is estimated by linear regression in combination with estimating the total variance of the interpretation.

3. Univariate based GWAS analysis (sGWAS)

After filtration according to Minimal Allele Frequency (MAF), a total of 1180 ten thousand high quality SNPs were used for downstream analysis. The first ten Principal Components (PCs) together explain the 8.76% genetic variance while integrating the genetic relationship K matrix as a random effect for sGWAS analysis. The associated significance threshold was set to 1.23E-8, equal to 0.05/Ne, where Ne is the effective number of independent tests calculated from all variations. The criteria for determining QTL intervals are: the significant SNP of each trait is extracted first, only at least two loci with continuous SNP less than 20Kb are reserved and simultaneously combined as basic interval units, and SNPs with single significant loci within 20KB are excluded in the next analysis; if any significant SNP between adjacent interval units has a higher linkage disequilibrium relationship (LD, r 2. Gtoreq.0.2), the adjacent interval units are further merged into a QTL candidate interval. To reduce the likelihood that QTL intervals obtained from smaller QTL effects are smaller without the inclusion of a functional gene, for those QTLs (i.e., 1.23E-10) that are less than 50Kb apart and that have a significance of the most significant SNP that is 100 times less than the threshold, the QTL interval will continue to extend 25Kb to both sides to obtain a final QTL interval. The most significant SNP in the interval was taken as significance of the sQTL, and all genes in the interval were taken as candidate genes.

4. Obtaining QTL intervals

By using the positioning method, the application finds 1 most obvious SNP of the maize plant height character, and the SNP is positioned in the region 25107834-25110547 of chromosome 1 (figure 1). The query via the MaizeGDB database (https:// www.maizegdb.org /) within this interval contains 1 annotated gene, numbered GRMZM2G331652, the function of which is not well defined in the database. The genome sequence of the gene is shown as SEQ ID NO.1, the full length is 2714bp, the coding region sequence is shown as SEQ ID NO.3, and the coding amino acid sequence is shown as SEQ ID NO. 5. The sequence of the region in the maize inbred line KN5585 bred by the non-rice biotechnology (Jiangsu) limited company is determined simultaneously, and is shown as SEQ ID NO. 2. The coding region sequence is shown as SEQ ID NO.4, and the coded amino acid sequence is shown as SEQ ID NO. 6. The sequence similarity between SEQ ID NO.1 and SEQ ID NO.2 is more than 99%.

Example 2 Gene editing knockout candidate Gene analysis Gene function

The application uses CRISPR-Cas9 gene editing technology to carry out site-directed mutation on the genes in the interval. Embodiments include construction of gene editing vectors, genetic transformation of maize, and functional verification of editing effects.

The method comprises the following steps:

1. construction of Gene editing vector

The gene editing vector of the application is G01652-CPB-ZmUbi-hspCas9, and the vector diagram is shown in FIG. 2. The basic carrier of the carrier is CPB-ZmUbi-hspCas9. The application obtains double-target U6-sgRNA through overlay PCR and then clones the double-target U6-sgRNA into a basic vector through homologous recombination, and the specific construction flow is as follows:

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

(2) Design of target gRNA. The genomic sequence of the acceptor material KN5585 was entered into http:// cbi.hzau.edu.cn/crispr/targeting. The DNA sequences of the two target regions are shown in SEQ ID NO.7 or SEQ ID NO. 8. The sgRNA skeleton sequence is obtained through artificial synthesis, and the sequence information of the sgRNA skeleton sequence is shown as SEQ ID NO.9 and SEQ ID NO. 10.

(3) U6-sgRNA was obtained by overlay PCR. The primer pair pU6F1/pU6R is used for amplifying the U6 promoter of the first target, and the product length is 515bp; primer pair p1652-1F (3F)/pgRR 1 is used for amplifying sgRNA of the first target, and the product length is 127bp; the primer pair pU6F1/gRR1 was used for the 2 nd amplification of the overlay PCR (U6-sgRNA) and the product length was 634bp. Step 1 of overlay PCR, namely amplifying U6 and sgRNA respectively, diluting PCR products by 50 times respectively, and mixing the diluted PCR products as templates to carry out step 2 of overlay PCR. And (5) electrophoresis gel cutting and recovery of amplified products and sequencing to confirm sequences. The overlay PCR system and conditions were as follows: the 15. Mu.L reaction system for the 1 st step of overlay PCR is as follows, template DNA (U6 or sgRNA,. Gtoreq.30 ng/. Mu.L): 0.5 μl, primer F/R: 1.2. Mu.L each, sterilized ddH2O: 3.7. Mu.L, 2X phanta max Buffer: 7.5. Mu.L, dNTP mix: 0.6. Mu.L of Phanta enzyme (product number: P505-d1/d2/d 3): 0.3. Mu.L. The reaction system of the 2 nd step of the overlay PCR is a 30 mu L system. U6 was 1. Mu.L by pipetting and diluting with 49. Mu.L ddH 2O; the sgRNA was blotted 1. Mu.L, diluted 10. Mu.L each with 49. Mu.L ddH2O, and mixed well. The method comprises the following steps: mixed template DNA (u6+sgrna): 1.5. Mu.L Primer F/R: 2.4. Mu.L each, sterilized ddH2O: 6.9. Mu.L, 2X phanta max Buffer:15 μL, dNTP mix: 1.2. Mu.L, phanta enzyme: 0.6. Mu.L. The overlay PCR procedure was as follows: (1) 94℃for 5 minutes, (2) 94℃for 30 seconds, (3) 62℃for 35 seconds, (4) 72℃for 30 seconds, and (5) is a cycle of 32 times from (2) step- (4), (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) Construction into the backbone vector by recombinant cloning. The CPB-Ubi-hspcas9 vector was digested with HindIII and recovered. Both the U6-gRNA and the vector were ligated by homologous recombination. Before the reaction solution is prepared, the concentration of each overlay product is ensured to be nearly consistent, and a 20 mu L homologous recombination system is as follows: cas Hind III:3 μL, T-1F overlay: 1 μl, sterilized ddH2O:10 μL,5 XCE MultiS buffer: 4. Mu.L, exnase MultiS (product number: C113-01/02): 2. Mu.L.

2. Maize genetic transformation

The vector was transferred into agrobacterium EHA105 by electric shock and PCR was identified. Taking young embryo of freshly stripped corn inbred line KN5585 (inbred line bred by non-rice biotechnology (Jiangsu) limited company) of about 1mm as a material, placing the stripped corn embryo into a 2mL plastic centrifuge tube containing 1.8mL suspension, and treating 150 immature young embryos within 30 min; the suspension was aspirated, and the remaining maize embryos were placed in the tube and then 1.0mL of Agrobacterium suspension was added and allowed to stand for 5min. After the young embryo in the centrifuge tube is suspended, the young embryo is poured into a co-culture medium, and the surface excess agrobacterium tumefaciens liquid is sucked by a pipettor, and the young embryo is co-cultured for 3 days in the dark at the temperature of 23 ℃. After co-cultivation, the young embryos are transferred to a resting medium, and after 6 days of dark cultivation at 28 ℃, placed on a screening medium containing 5mg/L biamphos, screening cultivation is started for 2 weeks, and then transferred to a screening medium containing 8mg/L biamphos, screening cultivation is started for 2 weeks. The resistant calli were transferred to differentiation medium 1 and incubated at 25℃with 5000lx for 1 week with light. Transferring the callus to a differentiation medium 2, and culturing for 2 weeks under illumination; transferring the differentiated seedlings to a rooting culture medium, and carrying out illumination culture at 25 ℃ and 5000lx until rooting; transferring the young seedling into a small basin for growth, transplanting the young seedling into a greenhouse after a certain growth stage, and harvesting offspring seeds after 3-4 months.

3. Character identification of gene editing plants

For the condition of extracting seedling stage DNA detection gene editing of T1 generation materials, designing primers (the primer sequence is HR2-2F:5'-AGCGCGTCTTCGCACAT-3', HR2-1R:5 '-CTTGTAGAACTGCCCACTGC-3') and amplifying target editing sections, wherein an amplification system is as follows: DNA: 3. Mu.L of each of the two-way primers, 1. Mu.L of each of the two-way primers, 2 XTaqmix: 7.5. Mu.L, ddH ₂ O: 2.5. Mu.L, total volume 10. Mu.L. The PCR 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) 35 cycles from (2) step- (4) step, (6) 72℃for 7 minutes, and (7) 4 ℃. PCR products were submitted to Sanger sequencing by wuhan qing biotechnology limited. And comparing the sequencing result of the transformant with the genome of the wild KN5585, wherein the material with base substitution, insertion or deletion is a positive editing material, and otherwise, the material is a negative material.

PCR amplifies the target editing segment and the PCR product is sequenced. Comparing the PCR product sequence with the original sequence (SEQ ID NO. 2) in KN5585, the amplified products (the sequences are shown as SEQ ID NO.15 and SEQ ID NO. 16) with 2 independent transformation events A1 and A2 are found to be edited at a gene target point to be a positive plant, and the genotype sequence at the mutated target point is shown as SEQ ID NO.11-SEQ ID NO. 14. The plant height of the T1 generation material of the positive transformation event in the late pollination stage is investigated, and a significant difference is found between the plant height of the positive mutant and the plant height of the receptor KN5585 (Table 2), so that the gene control plant height character is proved, and the plant height is reduced after the gene mutation.

In addition, the application surprisingly found that there was also a significant difference in flowering phase traits between the positive mutant and the receptor KN5585 (table 2), indicating that the gene also controls flowering phase traits. Post-gene mutation flowering phase delay.

TABLE 2 Gene-edited plant height and flowering phase trait data for maize materials

Data are expressed as "mean ± standard deviation", different letters representing significant differences for each material at the p=0.05 level. "-" indicates a base deletion; bold indicates base insertion or variation, and boxes indicate PAM sequence.

While the application has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the application and are intended to be within the scope of the application as claimed.

Sequence listing

<110> Oryza Glutinosa (Jiangsu) Limited, university of agriculture in China

<120> Gene controlling plant height and flowering phase of maize and use thereof

<130> 1

<160> 16

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2714

<212> DNA

<213> Zea mays L.

<400> 1

atgcgagcat tcaacggccg ccgcctccac gatccgaagc ccccctcatt cctccccctc 60

cccttagccc cacgcaccac aacgccgcca ccgccgcccc cctcctcgcc gccgcacgcc 120

atcccgccgc tcaacttccg ctacctcacc ccctgtccgc gctggtctgc ctgggtcgcc 180

accgcactca gcgaccccgt tttcgctccc atcctcgcct cgtccgccat ctccggcgcc 240

gtcgccgcct ccaccgccgt cgtctccccc gaccgtgcgg ccctctccgc tctgctctcg 300

ctctgggacc cggataccca cgcgttccgt ctcccagcgg gccccgccac cttcacactc 360

gaggatgcgc tcgtcctcgc tggtctaccg cccgcaggcg cgccgctgga ccggaccctc 420

acgccagagg aggatgacct ccgcgttcgc ctcgttgtcg agagggagaa gatcaaggag 480

ctccacccct gtgcccgcgc cgcgcgccgc gtgtctgcgg aggtgtggct cgagtggttc 540

gacggcggtg gcatacgccc cggcgaggac gacgaactgc ggtggcttgg gttcctcgct 600

tactggctcg ctttttttgt tacgccaagg ctccggtcaa ggggtggcga gctgccggag 660

cgcgtcttcg cacatgctgc gaggcttagt cttggcgagc gcattgccct tggccagggg 720

atggtggcaa atctatatgc ggaaatggat aagattgtta cttgcactgt ggccgatggt 780

gtgtgtggcc gtctagatgt ctgcgtgcca gtctggatgc tgcaggtgtg gatgtgggag 840

cgctacaaga ggctgtgccc gccggagctg aaggcaccgc agttccctgt ctccaatgtc 900

agggtattgt attggagccg gaggaagaag aagagcacat ctgaagaggc tctaaagatt 960

ttgctggacg aagtatgctt tgagtggagg ccctatcggc acaactccct caactggatg 1020

gaacccaagt ggttcaacaa ggacaccatt ttggtgacct gtcatggtaa agataagcca 1080

gagtggttgt tggattacat tgctgttatc agtcaaacaa tgttgaccgg gtttcatagt 1140

gatgacaccg acaattcagt actgtacaat ccacagcttg ttgcgagaca gtttggttat 1200

gatcaggctg ctcctgtgtc cattgtcagg gaaatccact ttgagggaat cgagttgtgg 1260

ataccaagta tcggtagata tgggatgcct ggtgaagatt atgttgcatg gtgtagcagc 1320

agtgggcagt tctacaagca tcaaaatgat gtccaatatg gttgctcggt attacgaaat 1380

catgaaaatg gggctatttc gtcgcaactg aatgtaaatg agaaatgtgt tgtagtgcca 1440

acacttgatc aatttatcac ccaagtcact aggagagacc acatcaatta cattgtagaa 1500

gggcagccgg agaaaatgga taatggaagt catgaggatg aaacagaggt gattgtctgt 1560

ggccttgaag cttgtgtcaa ggatagcaga actacttcag tgaaacagaa tgtgcagaaa 1620

caaagagata aatttgctga agatggaggt gatagtaaga agaaaagaaa agtagaaagt 1680

aacacgaaaa ggagttcgtt gcaattggaa ggtcagaagt attcttcact ccagaaagct 1740

ctaaattctg attctaagaa atgtgaggag ttggctcaag ttgacagtga tgatgaatgc 1800

attgttctcg aacagcccaa gaataaatgt gaagtaataa acctggatga tgatgaggaa 1860

cagagtgttt ctaatcgaga acatcataac atgcaacttg tcctagaact ggaagagttt 1920

gtccgctctg gtcttctctc gcaatgggag gaaagctcag atgaagatga tgtaagtgga 1980

agcaagcaag aaacccagaa gaaaagcaac aatgacccct atgctgaagc agccatgagg 2040

gagtaccctc tgttctttga gttcattcct cagaaaccac attacagagg cttcgtgaac 2100

tatgatgaaa ctttaggaga tctaccttac agtggattat ggtttttgct aattggcttg 2160

gctaaggagg tgctcaagac gtcatgtgac acagatgctt ctgaaattgc atatctgatg 2220

aaaaaagccc agcatttaga gcaactgggt ttcaacgtga cacatctcat tgcccgctta 2280

aaggagccac aaattaggct taagaagctt caagattcta gagcaaggct tgatcatgcc 2340

cgtgagaaag aggagggcaa cgtagtcgag tctctttcaa gccatctgaa taaactgaag 2400

ggcaacataa ggatgatgga tagtcatctg gatggaacga agcaagcttt tatttcaaat 2460

atacaagata aattgaatga aggaattaat cttgccagtt tggaaaagga agtagaaact 2520

gctgaaaaat gttgtcaggc aatgaaggat gaagtagctg caatgagaat gaggtactcg 2580

gaccgtggaa tttagattgt tccagtgtcc gctatcttca tttaagaagt tactttcttc 2640

ctgacataga gcttgcttga tatctgtctg tatcatcaag agtctgatca atagaatata 2700

accatcttac tttc 2714

<210> 2

<211> 2712

<212> DNA

<213> Zea mays L.

<400> 2

atgcgagcat tcaacggccg ccgcctccac gatccgaagc ccccctcatt cctccccctc 60

cccttagccc cacgcaccac aacgccgcca ccgccgcccc cctcctcgcc gccgcacgcc 120

atcccgccgc tcaacttccg ctacctcacc ccctgtccgc gctggtctgc ctgggtcgcc 180

accgcactca gcgaccccgt tttcgctccc atcctcgcct cgtccgccat ctccggcgcc 240

gtcgccgcct ccaccgccgt cgtctccccc gaccgtgcgg ccctctccgc tctgctctcg 300

ctctgggacc cggataccca cgcgttccgt ctcccagcgg gccccgccac cttcacactc 360

gaggacgcgc tcgtcctcgc tggtctaccg cccgcaggcg cgccgctgga ccggaccctc 420

acgccagagg aggatgacct ccgcgttcgc ctcgtcgtcg agagggagaa gatcaaggag 480

ctccacccct gtgcccgcgc cgcgcgccgc gtgtctgcgg aggtgtggct cgagtggttc 540

gacggcggtg gcatacgccc cggcgaggac gacgaactgc ggtggcttgg gttcctcgct 600

tactggctcg ctttttttgt cacgccaagg ctccggtcaa ggggtggcga gctgccggag 660

cgcgtcttcg cacatgctgc gaggcttagt cttggcgagc gcattgccct tggccagggg 720

atggtggcaa atctatatgc ggaaatggat aagattgtta cttgcactgt ggccgatggt 780

gtgtgtggcc gtctagatct ctgcgtgcca gtctggatgc tgcaggtgtg gatgtgggag 840

cgctacaaga ggctgtgccc gccggagctg aaggcgccgc agttccctgt ctccaatgtc 900

agggtattgt attggagccg gaggaagaag aagagcacat ctgaggaggc tctaaagatt 960

ttgctggacg aagtatgctt tgagtggagg ccctatcggc acaactccct caactggatg 1020

gaacccaagt ggttcaacaa ggacaccatt ttggtgacct gtcatggtaa agataagcca 1080

gagtggttgt tggattacat tgctgttatc agtcaaacaa tgttgactgg gtttcatagt 1140

gatgacaccg acaattcagt actgtacaat ccacagcttg ttgcgagaca atttggttat 1200

gatcaggctg ctcctgtgtc cattgtcagg gaaatccact ttgagggaat cgagttgtgg 1260

ataccaagta tcggtagata tgggatgcct ggtgaagatt atgttgcatg gtgcagcagc 1320

agtgggcagt tctacaagca tcaaaatgat gtccaatacg gttgctcggt attacgagat 1380

cacgaaaatg gggctatttc gtcgcaactg aatgtaagtg agaaatgtgt tgtagtgcca 1440

acacttgatc aatttatcac ccaagtcact aggagagacc acatcaatta cattgtagaa 1500

gggcagctgg agaaaatgga taatggaagt catgaggatg aaacagaggt gattgtctgt 1560

ggccttgaag cttgtgtcaa ggatagcaga actacttcag tgaaacagaa tgtgaagaaa 1620

caaagagata aatttgctga agatggaggt aatagtaaga agaaaagaaa agtagaaagt 1680

aacacggaaa ggagttcgtt gcaattggaa ggtcagaagt attcttcact ccagaaagct 1740

ctaaattctg attctaaaaa atgtgaggag ttggctcaag ttgacagtga tgatgaatgc 1800

attgttctcg aacagcccaa gaataaatgt gaagtaataa acctggatga tgatgaggaa 1860

cagagtgttt ctaatcgaga acatcataac atgcaacttg tcctagaact ggaagagttt 1920

gttcgctctg gtcttctctc gcaatgggag gaaagctcag atgaagatga tgtaagtgga 1980

agcaagcaag aaacccagaa gaaaagcaac aatgacccct atgctgaagc agccatgagg 2040

gagtaccctc tgttctttga gttcattcct cagaaaccac attacagagg cttcatgaac 2100

tatgatgaaa ctttaggaga tctaccttac agtggattat ggtttttgct aattggcttg 2160

gctaaggagg tgctcaagac gtcatgtgac acagatgctt ctgaaattgc atatctgatg 2220

aaaaaagcac agcatttaga gcaactgggt ttcaacgtga cacatctcat tgcccgctta 2280

aaggagccac aaattaggct taagaagctt caagattcta gagcaaggct tgatcatgcc 2340

cgtgagaaag aggagggcaa cgtagtcgag tctctttcaa gccatctgaa taaactgaaa 2400

ggcaacataa ggatgatgga tagtcatctg gatggaacga agcaagcttt tatttcaaat 2460

atacaagata aattgaatga aggaattaat cttgccagtt tggaaaagga agtagaaact 2520

gctgaaaaat gttgtcaggc aatgaaggat gaagtagctg caatgagaat gaggtactcg 2580

gaccgtggaa tttagattgt tccagtgtcc gctatcttca tttaagaagt tactttcttc 2640

ctgacataga gcttgcttga tatctgtctg tcatcaagag tctgatcaat agaatataac 2700

catcttactt tc 2712

<210> 3

<211> 2595

<212> DNA

<213> Synthesis (unown)

<400> 3

atgcgagcat tcaacggccg ccgcctccac gatccgaagc ccccctcatt cctccccctc 60

cccttagccc cacgcaccac aacgccgcca ccgccgcccc cctcctcgcc gccgcacgcc 120

atcccgccgc tcaacttccg ctacctcacc ccctgtccgc gctggtctgc ctgggtcgcc 180

accgcactca gcgaccccgt tttcgctccc atcctcgcct cgtccgccat ctccggcgcc 240

gtcgccgcct ccaccgccgt cgtctccccc gaccgtgcgg ccctctccgc tctgctctcg 300

ctctgggacc cggataccca cgcgttccgt ctcccagcgg gccccgccac cttcacactc 360

gaggatgcgc tcgtcctcgc tggtctaccg cccgcaggcg cgccgctgga ccggaccctc 420

acgccagagg aggatgacct ccgcgttcgc ctcgttgtcg agagggagaa gatcaaggag 480

ctccacccct gtgcccgcgc cgcgcgccgc gtgtctgcgg aggtgtggct cgagtggttc 540

gacggcggtg gcatacgccc cggcgaggac gacgaactgc ggtggcttgg gttcctcgct 600

tactggctcg ctttttttgt tacgccaagg ctccggtcaa ggggtggcga gctgccggag 660

cgcgtcttcg cacatgctgc gaggcttagt cttggcgagc gcattgccct tggccagggg 720

atggtggcaa atctatatgc ggaaatggat aagattgtta cttgcactgt ggccgatggt 780

gtgtgtggcc gtctagatgt ctgcgtgcca gtctggatgc tgcaggtgtg gatgtgggag 840

cgctacaaga ggctgtgccc gccggagctg aaggcaccgc agttccctgt ctccaatgtc 900

agggtattgt attggagccg gaggaagaag aagagcacat ctgaagaggc tctaaagatt 960

ttgctggacg aagtatgctt tgagtggagg ccctatcggc acaactccct caactggatg 1020

gaacccaagt ggttcaacaa ggacaccatt ttggtgacct gtcatggtaa agataagcca 1080

gagtggttgt tggattacat tgctgttatc agtcaaacaa tgttgaccgg gtttcatagt 1140

gatgacaccg acaattcagt actgtacaat ccacagcttg ttgcgagaca gtttggttat 1200

gatcaggctg ctcctgtgtc cattgtcagg gaaatccact ttgagggaat cgagttgtgg 1260

ataccaagta tcggtagata tgggatgcct ggtgaagatt atgttgcatg gtgtagcagc 1320

agtgggcagt tctacaagca tcaaaatgat gtccaatatg gttgctcggt attacgaaat 1380

catgaaaatg gggctatttc gtcgcaactg aatgtaaatg agaaatgtgt tgtagtgcca 1440

acacttgatc aatttatcac ccaagtcact aggagagacc acatcaatta cattgtagaa 1500

gggcagccgg agaaaatgga taatggaagt catgaggatg aaacagaggt gattgtctgt 1560

ggccttgaag cttgtgtcaa ggatagcaga actacttcag tgaaacagaa tgtgcagaaa 1620

caaagagata aatttgctga agatggaggt gatagtaaga agaaaagaaa agtagaaagt 1680

aacacgaaaa ggagttcgtt gcaattggaa ggtcagaagt attcttcact ccagaaagct 1740

ctaaattctg attctaagaa atgtgaggag ttggctcaag ttgacagtga tgatgaatgc 1800

attgttctcg aacagcccaa gaataaatgt gaagtaataa acctggatga tgatgaggaa 1860

cagagtgttt ctaatcgaga acatcataac atgcaacttg tcctagaact ggaagagttt 1920

gtccgctctg gtcttctctc gcaatgggag gaaagctcag atgaagatga tgtaagtgga 1980

agcaagcaag aaacccagaa gaaaagcaac aatgacccct atgctgaagc agccatgagg 2040

gagtaccctc tgttctttga gttcattcct cagaaaccac attacagagg cttcgtgaac 2100

tatgatgaaa ctttaggaga tctaccttac agtggattat ggtttttgct aattggcttg 2160

gctaaggagg tgctcaagac gtcatgtgac acagatgctt ctgaaattgc atatctgatg 2220

aaaaaagccc agcatttaga gcaactgggt ttcaacgtga cacatctcat tgcccgctta 2280

aaggagccac aaattaggct taagaagctt caagattcta gagcaaggct tgatcatgcc 2340

cgtgagaaag aggagggcaa cgtagtcgag tctctttcaa gccatctgaa taaactgaag 2400

ggcaacataa ggatgatgga tagtcatctg gatggaacga agcaagcttt tatttcaaat 2460

atacaagata aattgaatga aggaattaat cttgccagtt tggaaaagga agtagaaact 2520

gctgaaaaat gttgtcaggc aatgaaggat gaagtagctg caatgagaat gaggtactcg 2580

gaccgtggaa tttag 2595

<210> 4

<211> 2595

<212> DNA

<213> Synthesis (unown)

<400> 4

atgcgagcat tcaacggccg ccgcctccac gatccgaagc ccccctcatt cctccccctc 60

cccttagccc cacgcaccac aacgccgcca ccgccgcccc cctcctcgcc gccgcacgcc 120

atcccgccgc tcaacttccg ctacctcacc ccctgtccgc gctggtctgc ctgggtcgcc 180

accgcactca gcgaccccgt tttcgctccc atcctcgcct cgtccgccat ctccggcgcc 240

gtcgccgcct ccaccgccgt cgtctccccc gaccgtgcgg ccctctccgc tctgctctcg 300

ctctgggacc cggataccca cgcgttccgt ctcccagcgg gccccgccac cttcacactc 360

gaggacgcgc tcgtcctcgc tggtctaccg cccgcaggcg cgccgctgga ccggaccctc 420

acgccagagg aggatgacct ccgcgttcgc ctcgtcgtcg agagggagaa gatcaaggag 480

ctccacccct gtgcccgcgc cgcgcgccgc gtgtctgcgg aggtgtggct cgagtggttc 540

gacggcggtg gcatacgccc cggcgaggac gacgaactgc ggtggcttgg gttcctcgct 600

tactggctcg ctttttttgt cacgccaagg ctccggtcaa ggggtggcga gctgccggag 660

cgcgtcttcg cacatgctgc gaggcttagt cttggcgagc gcattgccct tggccagggg 720

atggtggcaa atctatatgc ggaaatggat aagattgtta cttgcactgt ggccgatggt 780

gtgtgtggcc gtctagatct ctgcgtgcca gtctggatgc tgcaggtgtg gatgtgggag 840

cgctacaaga ggctgtgccc gccggagctg aaggcgccgc agttccctgt ctccaatgtc 900

agggtattgt attggagccg gaggaagaag aagagcacat ctgaggaggc tctaaagatt 960

ttgctggacg aagtatgctt tgagtggagg ccctatcggc acaactccct caactggatg 1020

gaacccaagt ggttcaacaa ggacaccatt ttggtgacct gtcatggtaa agataagcca 1080

gagtggttgt tggattacat tgctgttatc agtcaaacaa tgttgactgg gtttcatagt 1140

gatgacaccg acaattcagt actgtacaat ccacagcttg ttgcgagaca atttggttat 1200

gatcaggctg ctcctgtgtc cattgtcagg gaaatccact ttgagggaat cgagttgtgg 1260

ataccaagta tcggtagata tgggatgcct ggtgaagatt atgttgcatg gtgcagcagc 1320

agtgggcagt tctacaagca tcaaaatgat gtccaatacg gttgctcggt attacgagat 1380

cacgaaaatg gggctatttc gtcgcaactg aatgtaagtg agaaatgtgt tgtagtgcca 1440

acacttgatc aatttatcac ccaagtcact aggagagacc acatcaatta cattgtagaa 1500

gggcagctgg agaaaatgga taatggaagt catgaggatg aaacagaggt gattgtctgt 1560

ggccttgaag cttgtgtcaa ggatagcaga actacttcag tgaaacagaa tgtgaagaaa 1620

caaagagata aatttgctga agatggaggt aatagtaaga agaaaagaaa agtagaaagt 1680

aacacggaaa ggagttcgtt gcaattggaa ggtcagaagt attcttcact ccagaaagct 1740

ctaaattctg attctaaaaa atgtgaggag ttggctcaag ttgacagtga tgatgaatgc 1800

attgttctcg aacagcccaa gaataaatgt gaagtaataa acctggatga tgatgaggaa 1860

cagagtgttt ctaatcgaga acatcataac atgcaacttg tcctagaact ggaagagttt 1920

gttcgctctg gtcttctctc gcaatgggag gaaagctcag atgaagatga tgtaagtgga 1980

agcaagcaag aaacccagaa gaaaagcaac aatgacccct atgctgaagc agccatgagg 2040

gagtaccctc tgttctttga gttcattcct cagaaaccac attacagagg cttcatgaac 2100

tatgatgaaa ctttaggaga tctaccttac agtggattat ggtttttgct aattggcttg 2160

gctaaggagg tgctcaagac gtcatgtgac acagatgctt ctgaaattgc atatctgatg 2220

aaaaaagcac agcatttaga gcaactgggt ttcaacgtga cacatctcat tgcccgctta 2280

aaggagccac aaattaggct taagaagctt caagattcta gagcaaggct tgatcatgcc 2340

cgtgagaaag aggagggcaa cgtagtcgag tctctttcaa gccatctgaa taaactgaaa 2400

ggcaacataa ggatgatgga tagtcatctg gatggaacga agcaagcttt tatttcaaat 2460

atacaagata aattgaatga aggaattaat cttgccagtt tggaaaagga agtagaaact 2520

gctgaaaaat gttgtcaggc aatgaaggat gaagtagctg caatgagaat gaggtactcg 2580

gaccgtggaa tttag 2595

<210> 5

<211> 864

<212> PRT

<213> Zea mays L.

<400> 5

Met Arg Ala Phe Asn Gly Arg Arg Leu His Asp Pro Lys Pro Pro Ser

1 5 10 15

Phe Leu Pro Leu Pro Leu Ala Pro Arg Thr Thr Thr Pro Pro Pro Pro

20 25 30

Pro Pro Ser Ser Pro Pro His Ala Ile Pro Pro Leu Asn Phe Arg Tyr

35 40 45

Leu Thr Pro Cys Pro Arg Trp Ser Ala Trp Val Ala Thr Ala Leu Ser

50 55 60

Asp Pro Val Phe Ala Pro Ile Leu Ala Ser Ser Ala Ile Ser Gly Ala

65 70 75 80

Val Ala Ala Ser Thr Ala Val Val Ser Pro Asp Arg Ala Ala Leu Ser

85 90 95

Ala Leu Leu Ser Leu Trp Asp Pro Asp Thr His Ala Phe Arg Leu Pro

100 105 110

Ala Gly Pro Ala Thr Phe Thr Leu Glu Asp Ala Leu Val Leu Ala Gly

115 120 125

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

130 135 140

Asp Asp Leu Arg Val Arg Leu Val Val Glu Arg Glu Lys Ile Lys Glu

145 150 155 160

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

165 170 175

Leu Glu Trp Phe Asp Gly Gly Gly Ile Arg Pro Gly Glu Asp Asp Glu

180 185 190

Leu Arg Trp Leu Gly Phe Leu Ala Tyr Trp Leu Ala Phe Phe Val Thr

195 200 205

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

210 215 220

His Ala Ala Arg Leu Ser Leu Gly Glu Arg Ile Ala Leu Gly Gln Gly

225 230 235 240

Met Val Ala Asn Leu Tyr Ala Glu Met Asp Lys Ile Val Thr Cys Thr

245 250 255

Val Ala Asp Gly Val Cys Gly Arg Leu Asp Val Cys Val Pro Val Trp

260 265 270

Met Leu Gln Val Trp Met Trp Glu Arg Tyr Lys Arg Leu Cys Pro Pro

275 280 285

Glu Leu Lys Ala Pro Gln Phe Pro Val Ser Asn Val Arg Val Leu Tyr

290 295 300

Trp Ser Arg Arg Lys Lys Lys Ser Thr Ser Glu Glu Ala Leu Lys Ile

305 310 315 320

Leu Leu Asp Glu Val Cys Phe Glu Trp Arg Pro Tyr Arg His Asn Ser

325 330 335

Leu Asn Trp Met Glu Pro Lys Trp Phe Asn Lys Asp Thr Ile Leu Val

340 345 350

Thr Cys His Gly Lys Asp Lys Pro Glu Trp Leu Leu Asp Tyr Ile Ala

355 360 365

Val Ile Ser Gln Thr Met Leu Thr Gly Phe His Ser Asp Asp Thr Asp

370 375 380

Asn Ser Val Leu Tyr Asn Pro Gln Leu Val Ala Arg Gln Phe Gly Tyr

385 390 395 400

Asp Gln Ala Ala Pro Val Ser Ile Val Arg Glu Ile His Phe Glu Gly

405 410 415

Ile Glu Leu Trp Ile Pro Ser Ile Gly Arg Tyr Gly Met Pro Gly Glu

420 425 430

Asp Tyr Val Ala Trp Cys Ser Ser Ser Gly Gln Phe Tyr Lys His Gln

435 440 445

Asn Asp Val Gln Tyr Gly Cys Ser Val Leu Arg Asn His Glu Asn Gly

450 455 460

Ala Ile Ser Ser Gln Leu Asn Val Asn Glu Lys Cys Val Val Val Pro

465 470 475 480

Thr Leu Asp Gln Phe Ile Thr Gln Val Thr Arg Arg Asp His Ile Asn

485 490 495

Tyr Ile Val Glu Gly Gln Pro Glu Lys Met Asp Asn Gly Ser His Glu

500 505 510

Asp Glu Thr Glu Val Ile Val Cys Gly Leu Glu Ala Cys Val Lys Asp

515 520 525

Ser Arg Thr Thr Ser Val Lys Gln Asn Val Gln Lys Gln Arg Asp Lys

530 535 540

Phe Ala Glu Asp Gly Gly Asp Ser Lys Lys Lys Arg Lys Val Glu Ser

545 550 555 560

Asn Thr Lys Arg Ser Ser Leu Gln Leu Glu Gly Gln Lys Tyr Ser Ser

565 570 575

Leu Gln Lys Ala Leu Asn Ser Asp Ser Lys Lys Cys Glu Glu Leu Ala

580 585 590

Gln Val Asp Ser Asp Asp Glu Cys Ile Val Leu Glu Gln Pro Lys Asn

595 600 605

Lys Cys Glu Val Ile Asn Leu Asp Asp Asp Glu Glu Gln Ser Val Ser

610 615 620

Asn Arg Glu His His Asn Met Gln Leu Val Leu Glu Leu Glu Glu Phe

625 630 635 640

Val Arg Ser Gly Leu Leu Ser Gln Trp Glu Glu Ser Ser Asp Glu Asp

645 650 655

Asp Val Ser Gly Ser Lys Gln Glu Thr Gln Lys Lys Ser Asn Asn Asp

660 665 670

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

675 680 685

Ile Pro Gln Lys Pro His Tyr Arg Gly Phe Val Asn Tyr Asp Glu Thr

690 695 700

Leu Gly Asp Leu Pro Tyr Ser Gly Leu Trp Phe Leu Leu Ile Gly Leu

705 710 715 720

Ala Lys Glu Val Leu Lys Thr Ser Cys Asp Thr Asp Ala Ser Glu Ile

725 730 735

Ala Tyr Leu Met Lys Lys Ala Gln His Leu Glu Gln Leu Gly Phe Asn

740 745 750

Val Thr His Leu Ile Ala Arg Leu Lys Glu Pro Gln Ile Arg Leu Lys

755 760 765

Lys Leu Gln Asp Ser Arg Ala Arg Leu Asp His Ala Arg Glu Lys Glu

770 775 780

Glu Gly Asn Val Val Glu Ser Leu Ser Ser His Leu Asn Lys Leu Lys

785 790 795 800

Gly Asn Ile Arg Met Met Asp Ser His Leu Asp Gly Thr Lys Gln Ala

805 810 815

Phe Ile Ser Asn Ile Gln Asp Lys Leu Asn Glu Gly Ile Asn Leu Ala

820 825 830

Ser Leu Glu Lys Glu Val Glu Thr Ala Glu Lys Cys Cys Gln Ala Met

835 840 845

Lys Asp Glu Val Ala Ala Met Arg Met Arg Tyr Ser Asp Arg Gly Ile

850 855 860

<210> 6

<211> 864

<212> PRT

<213> Zea mays L.

<400> 6

Met Arg Ala Phe Asn Gly Arg Arg Leu His Asp Pro Lys Pro Pro Ser

1 5 10 15

Phe Leu Pro Leu Pro Leu Ala Pro Arg Thr Thr Thr Pro Pro Pro Pro

20 25 30

Pro Pro Ser Ser Pro Pro His Ala Ile Pro Pro Leu Asn Phe Arg Tyr

35 40 45

Leu Thr Pro Cys Pro Arg Trp Ser Ala Trp Val Ala Thr Ala Leu Ser

50 55 60

Asp Pro Val Phe Ala Pro Ile Leu Ala Ser Ser Ala Ile Ser Gly Ala

65 70 75 80

Val Ala Ala Ser Thr Ala Val Val Ser Pro Asp Arg Ala Ala Leu Ser

85 90 95

Ala Leu Leu Ser Leu Trp Asp Pro Asp Thr His Ala Phe Arg Leu Pro

100 105 110

Ala Gly Pro Ala Thr Phe Thr Leu Glu Asp Ala Leu Val Leu Ala Gly

115 120 125

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

130 135 140

Asp Asp Leu Arg Val Arg Leu Val Val Glu Arg Glu Lys Ile Lys Glu

145 150 155 160

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

165 170 175

Leu Glu Trp Phe Asp Gly Gly Gly Ile Arg Pro Gly Glu Asp Asp Glu

180 185 190

Leu Arg Trp Leu Gly Phe Leu Ala Tyr Trp Leu Ala Phe Phe Val Thr

195 200 205

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

210 215 220

His Ala Ala Arg Leu Ser Leu Gly Glu Arg Ile Ala Leu Gly Gln Gly

225 230 235 240

Met Val Ala Asn Leu Tyr Ala Glu Met Asp Lys Ile Val Thr Cys Thr

245 250 255

Val Ala Asp Gly Val Cys Gly Arg Leu Asp Leu Cys Val Pro Val Trp

260 265 270

Met Leu Gln Val Trp Met Trp Glu Arg Tyr Lys Arg Leu Cys Pro Pro

275 280 285

Glu Leu Lys Ala Pro Gln Phe Pro Val Ser Asn Val Arg Val Leu Tyr

290 295 300

Trp Ser Arg Arg Lys Lys Lys Ser Thr Ser Glu Glu Ala Leu Lys Ile

305 310 315 320

Leu Leu Asp Glu Val Cys Phe Glu Trp Arg Pro Tyr Arg His Asn Ser

325 330 335

Leu Asn Trp Met Glu Pro Lys Trp Phe Asn Lys Asp Thr Ile Leu Val

340 345 350

Thr Cys His Gly Lys Asp Lys Pro Glu Trp Leu Leu Asp Tyr Ile Ala

355 360 365

Val Ile Ser Gln Thr Met Leu Thr Gly Phe His Ser Asp Asp Thr Asp

370 375 380

Asn Ser Val Leu Tyr Asn Pro Gln Leu Val Ala Arg Gln Phe Gly Tyr

385 390 395 400

Asp Gln Ala Ala Pro Val Ser Ile Val Arg Glu Ile His Phe Glu Gly

405 410 415

Ile Glu Leu Trp Ile Pro Ser Ile Gly Arg Tyr Gly Met Pro Gly Glu

420 425 430

Asp Tyr Val Ala Trp Cys Ser Ser Ser Gly Gln Phe Tyr Lys His Gln

435 440 445

Asn Asp Val Gln Tyr Gly Cys Ser Val Leu Arg Asp His Glu Asn Gly

450 455 460

Ala Ile Ser Ser Gln Leu Asn Val Ser Glu Lys Cys Val Val Val Pro

465 470 475 480

Thr Leu Asp Gln Phe Ile Thr Gln Val Thr Arg Arg Asp His Ile Asn

485 490 495

Tyr Ile Val Glu Gly Gln Leu Glu Lys Met Asp Asn Gly Ser His Glu

500 505 510

Asp Glu Thr Glu Val Ile Val Cys Gly Leu Glu Ala Cys Val Lys Asp

515 520 525

Ser Arg Thr Thr Ser Val Lys Gln Asn Val Lys Lys Gln Arg Asp Lys

530 535 540

Phe Ala Glu Asp Gly Gly Asn Ser Lys Lys Lys Arg Lys Val Glu Ser

545 550 555 560

Asn Thr Glu Arg Ser Ser Leu Gln Leu Glu Gly Gln Lys Tyr Ser Ser

565 570 575

Leu Gln Lys Ala Leu Asn Ser Asp Ser Lys Lys Cys Glu Glu Leu Ala

580 585 590

Gln Val Asp Ser Asp Asp Glu Cys Ile Val Leu Glu Gln Pro Lys Asn

595 600 605

Lys Cys Glu Val Ile Asn Leu Asp Asp Asp Glu Glu Gln Ser Val Ser

610 615 620

Asn Arg Glu His His Asn Met Gln Leu Val Leu Glu Leu Glu Glu Phe

625 630 635 640

Val Arg Ser Gly Leu Leu Ser Gln Trp Glu Glu Ser Ser Asp Glu Asp

645 650 655

Asp Val Ser Gly Ser Lys Gln Glu Thr Gln Lys Lys Ser Asn Asn Asp

660 665 670

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

675 680 685

Ile Pro Gln Lys Pro His Tyr Arg Gly Phe Met Asn Tyr Asp Glu Thr

690 695 700

Leu Gly Asp Leu Pro Tyr Ser Gly Leu Trp Phe Leu Leu Ile Gly Leu

705 710 715 720

Ala Lys Glu Val Leu Lys Thr Ser Cys Asp Thr Asp Ala Ser Glu Ile

725 730 735

Ala Tyr Leu Met Lys Lys Ala Gln His Leu Glu Gln Leu Gly Phe Asn

740 745 750

Val Thr His Leu Ile Ala Arg Leu Lys Glu Pro Gln Ile Arg Leu Lys

755 760 765

Lys Leu Gln Asp Ser Arg Ala Arg Leu Asp His Ala Arg Glu Lys Glu

770 775 780

Glu Gly Asn Val Val Glu Ser Leu Ser Ser His Leu Asn Lys Leu Lys

785 790 795 800

Gly Asn Ile Arg Met Met Asp Ser His Leu Asp Gly Thr Lys Gln Ala

805 810 815

Phe Ile Ser Asn Ile Gln Asp Lys Leu Asn Glu Gly Ile Asn Leu Ala

820 825 830

Ser Leu Glu Lys Glu Val Glu Thr Ala Glu Lys Cys Cys Gln Ala Met

835 840 845

Lys Asp Glu Val Ala Ala Met Arg Met Arg Tyr Ser Asp Arg Gly Ile

850 855 860

<210> 7

<211> 20

<212> DNA

<213> Zea mays L.

<400> 7

gtcagggaaa tccactttga 20

<210> 8

<211> 20

<212> DNA

<213> Zea mays L.

<400> 8

gttacttgca ctgtggccga 20

<210> 9

<211> 103

<212> RNA

<213> Synthesis (unown)

<400> 9

gucagggaaa uccacuuuga guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu uuu 103

<210> 10

<211> 103

<212> RNA

<213> Synthesis (unown)

<400> 10

guuacuugca cuguggccga guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu uuu 103

<210> 11

<211> 21

<212> DNA

<213> Synthesis (unown)

<400> 11

gttacttgca ctgtggcgcg a 21

<210> 12

<211> 19

<212> DNA

<213> Synthesis (unown)

<400> 12

gtcagggaaa tccacttga 19

<210> 13

<211> 19

<212> DNA

<213> Synthesis (unown)

<400> 13

gttacttgca ctgcgatgg 19

<210> 14

<211> 21

<212> DNA

<213> Synthesis (unown)

<400> 14

tccattgtca ggggagggaa t 21

<210> 15

<211> 680

<212> DNA

<213> Synthesis (unown)

<400> 15

agcgcgtctt cgcacatgct gcgaggctta gtcttggcga gcgcattgcc cttggccagg 60

ggatggtggc aaatctatat gcggaaatgg ataagattgt tacttgcact gtggcgcgat 120

ggtgtgtgtg gccgtctaga tctctgcgtg ccagtctgga tgctgcaggt gtggatgtgg 180

gagcgctaca agaggctgtg cccgccggag ctgaaggcgc cgcagttccc tgtctccaat 240

gtcagggtat tgtattggag ccggaggaag aagaagagca catctgagga ggctctaaag 300

attttgctgg acgaagtatg ctttgagtgg aggccctatc ggcacaactc cctcaactgg 360

atggaaccca agtggttcaa caaggacacc attttggtga cctgtcatgg taaagataag 420

ccagagtggt tgttggatta cattgctgtt atcagtcaaa caatgttgac tgggtttcat 480

agtgatgaca ccgacaattc agtactgtac aatccacagc ttgttgcgag acaatttggt 540

tatgatcagg ctgctcctgt gtccattgtc agggaaatcc acttgaggga atcgagttgt 600

ggataccaag tatcggtaga tatgggatgc ctggtgaaga ttatgttgca tggtgcagca 660

gcagtgggca gttctacaag 680

<210> 16

<211> 671

<212> DNA

<213> Synthesis (unown)

<400> 16

agcgcgtctt cgcacatgct gcgaggctta gtcttggcga gcgcattgcc cttggccagg 60

ggatggtggc aaatctatat gcggaaatgg ataagattgt tacttgcact gcgatggtgt 120

gtgtggccgt ctagatctct gcgtgccagt ctggatgctg caggtgtgga tgtgggagcg 180

ctacaagagg ctgtgcccgc cggagctgaa ggcgccgcag ttccctgtct ccaatgtcag 240

ggtattgtat tggagccgga ggaagaagaa gagcacatct gaggaggctc taaagatttt 300

gctggacgaa gtatgctttg agtggaggcc ctatcggcac aactccctca actggatgga 360

acccaagtgg ttcaacaagg acaccatttt ggtgacctgt catggtaaag ataagccaga 420

gtggttgttg gattacattg ctgttatcag tcaaacaatg ttgactgggt ttcatagtga 480

tgacaccgac aattcagtac tgtacaatcc acagcttgtt gcgagacaat ttggttatga 540

tcaggctgct cctgtgtcca ttgtcagggg agggaatggg aatcgagttg tggataccaa 600

gtatcggtag atatgggatg cctggtgaag attatgttgc atggtgcagc agcagtgggc 660

agttctacaa g 671

Claims

1. An application of a gene for controlling the plant height and the flowering phase of corn in changing the plant height or the flowering phase of the corn, which is characterized in that: the nucleic acid sequence of the gene is shown as any one of SEQ ID NO.1-SEQ ID NO. 4.

2. A method for reducing the plant height or delaying the flowering phase of corn, comprising the steps of: a method of inhibiting the gene of claim 1 in corn, selecting a plant with reduced maize plant height or delayed flowering phase.

3. The method for reducing the plant height or delaying the flowering phase of corn according to claim 2, wherein: the method for inhibiting the gene comprises any one of gene editing or RNA interference.

4. A method of reducing maize plant height or delaying flowering phase according to claim 3, wherein: the gene editing adopts a CRISPR/Cas9 method.

5. The method for reducing the plant height or delaying the flowering phase of maize of claim 4, wherein: the DNA sequence of the genome target region of the CRISPR/Cas9 method in corn is shown as SEQ ID NO.7 and SEQ ID NO. 8.

6. A kit for reducing the plant height or delaying the flowering phase of corn, characterized in that: comprising any one of the following:

(1) The sgRNA molecules have the sequences shown in SEQ ID NO.9 and SEQ ID NO. 10;

(2) A DNA molecule encoding the sgRNA;

(3) A vector expressing said sgRNA.

7. An application of a maize mutant genotype in reducing maize plant height or delaying flower phase, characterized in that: the mutant genotype sequence is represented by SEQ ID NO.2, and 659 th to 1338 th bases are replaced by any one of SEQ ID NO.15 to SEQ ID NO. 16.