CN114395580B

CN114395580B - Gene for controlling maize plant height

Info

Publication number: CN114395580B
Application number: CN202210202870.1A
Authority: CN
Inventors: 罗芸; 刘喻; 严建兵
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2022-03-02
Filing date: 2022-03-02
Publication date: 2024-02-27
Anticipated expiration: 2042-03-02
Also published as: CN114395580A

Abstract

The invention relates to application of ZmTPK2 genes in controlling corn plant height and/or spike height traits, and belongs to the field of molecular genetics. The invention discloses application of ZmTPK2 genes in controlling corn plant height and/or spike height traits. Further, the invention provides methods of reducing corn plant height and/or ear height.

Description

Gene for controlling maize plant height

Technical Field

The invention relates to application of ZmTPK2 gene in controlling corn plant height character, belonging to the field of molecular genetics.

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 and the spike position are controlled by the dominant gene and the micro-effect polygene together, so that the quantitative trait inheritance is typical. Although there are several genes regulating the plant height of maize that are located and cloned (university of agricultural in south China. Maize ZmPIF3s mutant protein, its coding gene and its use in breeding:

CN201910273522.1[ P ]. 2019-08-02; application of CYP78A gene in increasing corn plant height and enhancing plant growth vigor: CN201510230547.5[ P ]. 2016-12-07; genes related to corn plant height, and encoding proteins and applications thereof: CN200410037404.4[ P ] 2005-01-26; the method for creating the maize dwarf material by utilizing the gene editing technology comprises the following steps: CN201910371358.8[ P ] 2019-08-16.) more plant height trait related genes are to be further cloned.

Disclosure of Invention

In order to solve the problems, the invention provides a novel gene capable of controlling the plant height and/or the ear position of corn, and the expression or activity of protein encoded by the gene is inhibited, so that the plant height and/or the ear position of corn can be reduced, and the novel gene can be used for cultivating dwarf corn varieties.

The invention aims at providing a nucleic acid sequence of a gene ZmTPK2 affecting the plant height and/or spike height of corn and an amino acid sequence coded by the same.

The second purpose of the invention is to disclose a method for improving the plant height and/or spike height characteristics of corn.

The invention further aims to disclose a mutant gene of dwarf corn.

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

the invention provides an application of a maize gene in regulating and controlling the plant height and/or spike height traits of maize, which is characterized in that: the gene sequence is shown in any one of SEQ ID NO.2-SEQ ID NO. 3. Wherein SEQ ID NO.2 is the genome sequence of the gene in corn, and SEQ ID NO.3 is the cDNA sequence of the gene. The identification result shows that after the gene is knocked out by utilizing a gene editing means, the corn plant height and/or the ear height is reduced, so that the technical effect of improving the corn plant height and/or the ear height can be achieved by constructing a sequence shown as SEQ ID NO.2 or SEQ ID NO.3 on a super-expression vector, and the technical effect of reducing the corn plant height and/or the ear height can be achieved by designing a corresponding gRNA identification target or RNAi interference sequence according to the sequence shown as SEQ ID NO.2 or SEQ ID NO.3 and interfering the normal expression of the gene in a mode of gene editing or RNAi interference or T-DNA directional insertion.

In another aspect, the invention provides a method of reducing corn plant height and/or ear height, characterized by: and (3) inhibiting the expression and/or activity of the protein shown in SEQ ID NO.1 in corn, and selecting plants with reduced corn plant height and/or spike height.

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

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.4 or SEQ ID No. 5.

In another aspect, the invention also provides a kit for reducing the plant height and/or the ear height of corn, which is characterized in that: comprising any one of the following:

(1) An RNA molecule targeting the sequence shown as SEQ ID NO.4 or SEQ ID NO. 5; the RNA molecule can be an sgRNA molecule comprising the structures of the gRNA, the crRNAs and the tracrRNA, or can be a complex formed by the gRNA, the crRNAs and the tracrRNA singly or a complex comprising the gRNA and the crRNAs;

(2) A DNA molecule encoding the RNA of (1);

(3) A vector for expressing the RNA of (1).

In some embodiments, the sequence of the above RNA molecule is shown as SEQ ID NO.6 or SEQ ID NO. 7.

In another aspect, the invention also provides a maize mutant gene characterized in that: the mutant gene sequence is shown as SEQ ID NO.8 or SEQ ID NO. 9.

In another aspect, the invention also provides application of the corn mutant gene in reducing the corn plant height and/or the ear position height. The mutant gene is introduced into other corn materials by a conventional hybridization transfer method, so that the technical effect of reducing the plant height and/or the spike height of the recipient corn can be achieved.

Compared with the prior art, the ZmTPK2 gene and the protein encoded by the gene have the function of regulating and controlling the plant height and/or the ear position height of corn, and the function is not reported in the prior published materials and has no technical significance. Knocking out the gene by using a CRISPR/Cas9 method can reduce the corn plant height and/or the ear position height. The invention identifies the mutant gene sequence after gene knockout, and can further improve the plant height and/or spike height characters of other corn varieties by utilizing the mutant genes, thereby cultivating dwarf corn varieties and improving lodging resistance of corn.

Drawings

FIG. 1 corn ear length QTL positioning results. 1-10 represent chromosome 1 through chromosome 10, respectively. The vertical axis represents LOD value, and the horizontal axis represents genetic distance on chromosome

FIG. 2 gene editing vector diagram. The main elements are marked on the figures.

Figure 3 gene edited target position, target sequence (grey) and edited sequence display. "-" indicates a deletion.

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 this 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 sequences of the present application can 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 can 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 herein 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 herein 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 ofProtein 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 contemplated herein 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 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 invention locates the QTL affecting the corn ear length character in RIL group constructed by KUI and B77, and finds a main effective site in chromosome 6 58kb interval. Zm00001d037916 is estimated to be a gene controlling the corn ear length character in the QTL interval according to the gene function annotation information and is named ZmTPK2.

The invention further verifies the function of the gene through a gene editing knockout experiment, and unexpectedly discovers that the corn plant height and the ear position height of ZmTPK2 gene after editing are reduced.

The corn with reduced plant height and/or spike height can be used for cultivating dwarf corn varieties, and the lodging resistance of the corn is increased. The invention further determines the mutant gene sequences after gene knockout, and the mutant genes can be transferred to other corn varieties to improve the plant height and/or spike height characters of the receptor, thereby cultivating dwarf varieties and increasing lodging resistance of corn.

The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention 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: alaboratory 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.

Example 1 localization of ear of corn Length QTL

The invention constructs RIL group by taking short ear corn inbred line material KUI and long ear corn inbred line material B77 as parents, and identifies a QTL qEL6.2 for controlling ear length and row grain number in the interval range of No. 134.1-139.3Mb (V2 version) of corn by investigating corn ear length phenotype values of different planting time and place and utilizing parent Illumina Maize SNP Beadchip genotype data and RNA-seq data, thus being capable of explaining 18.92% phenotype variation. According to the positioning result, the highest LOD value, the effect value and the explained phenotype mutation rate of the QTL are different when the QTL is detected at 8 environmental points, but the QTL can be detected at 8 environmental points, which shows that the QTL is very stable and can work at a plurality of environmental points (figure 1).

To fine-localize QTL qel6.2, F6 remaining heterozygous material in RIL population was selected (Heterogeneous inbred family, HIF): HZAU-551-1 is used as an initial positioning material, molecular markers IDP8645, IDP785, B4, B14 and the like are designed to screen background heterozygous sections, and finally, a material heterozygous in the positioning section and homozygous in the background is selected to verify the QTL and perform the next fine positioning. In the fine positioning process, the positioning section is reduced by continuously screening new markers and carrying out single-factor variance analysis on the spike length phenotype of the near isogenic line after breeding of new recombinant single plants. QTL qel6.2 was divided into two smaller QTLs, named qel6.2-1 and qel6.2-2, respectively, according to the results of the ear length phenotypes of the different near isogenic lines.

For cloning the spike length gene into qEL6.2-1, maize isolate progeny of multiple different recombination types within the localization interval were grown in Wuhan in spring 2018 and genotyping was performed. Using a plurality of markers within the qEL6.2-2 interval, NIL homozygous for different families in the same chromosomal segment was obtained therefrom ^KUI3 Genotype and homozygous NIL ^B77 Genotype materials, and also select new chromosome recombinant single plants for selfing. And planting the filial generations of the obtained different recombination types and the new recombination single plant in the spring of 2019 in Hebei' S guard, screening new genotype homozygous materials, and positioning qEL6.2-1 locus in the interval of markers S155-S157 by utilizing further genotyping and fine positioning. Based on maize B73 reference genome (RefGenV 4) annotation information, this interval was approximately 58kb, containing the promoter region of one complete functional annotation gene Zm00001d037915, and Zm00001d 037916.

Example 2 functional verification of Gene

The Zm00001d037916 gene encodes thiamine pyrophosphatase 2, which is named as ZmTPK2. According to annotation information on a corn reference genome, the amino acid sequence of the ZmTPK2 coding protein is shown as SEQ ID NO.1, and the genome sequence is shown as SEQ ID NO. 2. In order to verify the function of the gene, the invention uses CRISPR/Cas9 gene editing technology to knock out the gene. The method comprises the following specific steps:

the gRNA was designed using on-line software CRISPR-P2.0 (http:// CRISPR. Hzau. Edu. Cn/CRISPR2 /) by selecting two target editing sites each on the CDS region of the gene (see Table 1 for sequences). The approximately 2000bp "U6-promter 1-gRNA 1-U6 promter 2-gRNA2-sgRNA" fusion unit sequence was synthesized directly, excised from the intermediate vector PUC57 using a double cleavage system and the cleavage fragments were ligated by homologous recombination to the backbone vector CPB-ZmUbi-hspCas9 (FIG. 2). And (3) transforming the recombinant vector into an escherichia coli competent cell DH5 alpha, detecting a target sequence, and transforming the maize inbred line KN5585 after the sequence detection is error-free. The obtained T0 generation transformed plants were examined for two target sites per gene. The detection is amplified by PCR and then sequenced, and the editing type is analyzed according to the sequencing sequence.

TABLE 1 Gene editing target sequence and detection primer information

A total of 2 successfully edited lines, designated KO1 and KO2, respectively, were obtained and the editing of the target sequence is shown in fig. 3. The sequences of KO1 and KO2 after ZmTPK2 gene mutation are shown in SEQ ID NO.8 and SEQ ID NO. 9.

Further analysis of the agronomic traits of the knocked-out lines, the present invention unexpectedly found that when the gene was knocked out, the maize plant type was significantly smaller, the plant height and ear height were significantly reduced, while the flowering period was unaffected (table 2).

TABLE 2 influence of ZmTPK2 on plant type and flowering phase

KO1, KO2:2 knockdown materials; NT: a receptor control; n: number of materials. The phenotype values are expressed as mean ± standard deviation, and the significance is calculated by single factor analysis of variance.

The above results show that ZmTPK2 mutant genes in KO1 and KO2 can reduce maize plant height and/or ear height. The hybridization of any one of the two mutant sequences can be introduced into the conventional corn materials to reduce the corn plant height and/or the spike height, so as to cultivate dwarf corn varieties.

While the invention 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 invention and are intended to be within the scope of the invention as claimed.

Claims

1. The application of the maize gene in regulating and controlling the plant height and/or the spike height of maize is characterized in that: the gene sequence is shown as SEQ ID NO. 2; the regulation mode is that the corn plant height and/or the ear height are reduced after the genes are inhibited.

2. A method for reducing the plant height and/or the ear height of corn, which is characterized in that: and (3) inhibiting the expression and/or activity of the protein shown in SEQ ID NO.1 in corn, and selecting plants with reduced corn plant height and/or spike height.

3. The method of reducing corn plant height and/or ear height of claim 2, wherein: the method for inhibiting protein expression and/or activity comprises any one of gene editing, RNA interference and T-DNA insertion.

4. A method of reducing corn plant height and/or ear height according to claim 3, wherein: the gene editing adopts a CRISPR/Cas9 method.

5. The method for reducing the plant height and/or the ear height of corn according to claim 4, wherein: the DNA sequence of the genome target region of the CRISPR/Cas9 method in corn is shown as SEQ ID NO.4 or SEQ ID NO. 5.

6. A kit for reducing the plant height and/or the ear height of corn, which is characterized in that: comprising any one of the following:

(1) An RNA molecule targeting the sequence shown as SEQ ID NO.4 or SEQ ID NO. 5; the sequence of the RNA molecule is shown as SEQ ID NO.6 or SEQ ID NO. 7;

(2) A DNA molecule encoding the RNA of (1);

(3) A vector for expressing the RNA of (1).

7. A maize mutant gene characterized in that: the mutant gene sequence is shown as SEQ ID NO.8 or SEQ ID NO. 9.

8. Use of the maize mutant gene of claim 7 for reducing maize plant height and/or ear height.