Wheat taZNF10 gene and application thereof

Abstract

The application belongs to the field of plant breeding, and particularly relates to a wheat TaZNF10 gene and application thereof. Through combined analysis of chromatin states, gene expression and GWAS after vernalization, a new gene TaZNF10 affecting the heading date trait of wheat is located. Manipulation of the TaZNF10 gene can improve the heading date trait of wheat.

Description

Technical Field

[0001] The present invention belongs to the field of plant breeding, and in particular relates to a wheat TaZNF10 gene and an application thereof. Background Art

[0002] Wheat yield is influenced by several key traits: effective ears per unit area, number of grains per ear, and grain weight. Since ear structure directly influences both grain number and grain weight, understanding the mechanisms of ear structure formation, cloning key genes, and improving these traits are crucial for increasing wheat yield.

[0003] Heading date is an important trait in the evolution and adaptation of crops. Understanding the genetic basis of heading date traits and cloning candidate genes can improve the environmental adaptability and plasticity of crops. This is of great significance for cultivating excellent crop varieties adapted to different ecological zones. It will also promote the genetic improvement process of important production traits closely related to heading date, such as yield. Summary of the Invention

[0004] The purpose of the present invention is to provide a key gene that affects the heading period traits of wheat.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention provides an application of a gene in regulating the heading period of wheat, characterized in that the gene comprises any one of the following:

[0007] (1) A gene having a sequence shown in SEQ ID NO. 1, SEQ ID NO. 3 or SEQ ID NO. 5;

[0008] (2) A gene encoding the sequence shown in SEQ ID NO. 2, SEQ ID NO. 4, or SEQ ID NO. 6;

[0009] (3) in the wheat gene database with the number TraesCS5B02G406000 or

[0010] gene of TraesCS5A02G401200 or TraesCS5D02G411400.

[0011] The present invention also provides an application of a biological material in regulating the heading period of wheat, characterized in that the biological material comprises any one of the following:

[0012] (1) an expression cassette containing the above-mentioned gene;

[0013] (2) an expression vector containing the above-mentioned gene;

[0014] (3) A host cell containing the above gene, wherein the host cell is a bacterial cell or a non-renewable plant cell.

[0015] The present invention also provides a method for extending the heading period of wheat, characterized in that the expression of the above-mentioned gene is increased in the wheat material to be improved, and wheat plants with an extended heading period are selected;

[0016] In some embodiments, the method for increasing gene expression is to use a high-activity promoter to drive the expression of the gene;

[0017] In some embodiments, the high-activity promoter sequence is shown as SEQ ID NO.5.

[0018] The present invention also provides a method for shortening the heading period of wheat, characterized in that the expression of the above-mentioned gene or encoded protein is knocked out or inhibited in the wheat material to be improved, and wheat plants with a shortened heading period are selected;

[0019] In some embodiments, the above-mentioned method of knocking out or inhibiting the expression of a gene or encoded protein is a gene editing method;

[0020] In some embodiments, the target sequence selected by the above gene editing method is shown in SEQ ID NO.4 or SEQ ID NO.5

[0021] The present invention also provides a kit, characterized in that it comprises any one of the following:

[0022] (1) an RNA molecule capable of recognizing the target sequence shown in SEQ ID NO. 8 or SEQ ID NO. 9;

[0023] (2) a DNA molecule encoding the RNA described in (1);

[0024] (3) a vector for expressing the RNA described in (1);

[0025] In some embodiments, the above-mentioned kit further comprises a Cas9 protein or a nucleic acid molecule encoding a Cas9 protein or a vector expressing a Cas9 protein;

[0026] In some embodiments, the RNA molecule sequence is shown as SEQ ID NO.10 or SEQ ID NO.11.

[0027] The present invention also provides a mutant gene, characterized in that the sequence of the mutant gene is shown in any one of SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16 or SEQ ID NO.18.

[0028] The present invention also provides a mutant protein, characterized in that the sequence of the mutant protein is shown in any one of SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17 or SEQ ID NO.19.

[0029] The present invention also provides the use of the above method, or the above kit, or the above mutant gene, or the above mutant protein in shortening the heading period of wheat.

[0030] The advantages and beneficial effects of the present invention are as follows: Through combined analysis of post-vernalization chromatin states, gene expression, and GWAS, the present invention locates a novel gene, TaZNF10, affecting wheat heading date traits. These genes include TaZNF10-5A, TaZNF10-5B, and TaZNF10-5D. Manipulating the TaZNF10 gene can improve wheat heading date traits. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Omic identification process of vernalized wheat.

[0032] Figure 2 Regulatory network of differentially expressed genes before and after vernalization.

[0033] Figure 3 Some differentially expressed genes are shown.

[0034] Figure 4 Results of genome-wide association analysis for heading time traits.

[0035] Figure 5 Effects of overexpression and knockout of TaZNF10-5B gene on wheat heading time. DETAILED DESCRIPTION

[0036] The following definitions and methods are provided to better define this application and to guide those skilled in the art in practicing this application. Unless otherwise noted, terms are to be understood according to conventional usage by those skilled in the relevant art. All patent documents, academic papers, industry standards, and other publications cited herein are hereby incorporated by reference in their entirety.

[0037] In the present application, the words “comprise”, “include” or variations thereof should be understood as including other elements, numbers or steps in addition to the elements, numbers or steps described.

[0038] Unless otherwise indicated, nucleic acids are written from left to right in a 5' to 3' direction; amino acid sequences are written from left to right in an amino to carboxyl direction. 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 Commission. Similarly, nucleotides may be represented by commonly accepted single-letter codes. Numerical ranges include numbers defining the ranges. As used herein, "nucleic acid" includes deoxyribonucleotides or ribonucleotide polymers in single-stranded or double-stranded form, and, unless otherwise limited, includes known analogs (e.g., peptide nucleic acids) having the basic properties of natural nucleotides, which hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides. As used herein, the terms "encoding" or "encoded" are used in the context of a specific nucleic acid to refer to the nucleic acid containing the necessary information for directing the translation of the nucleotide sequence into a specific protein. Codons are used to represent information encoding proteins. As used herein, the "full-length sequence" of a specific polynucleotide or its encoded protein refers to the entire nucleic acid sequence or the entire amino acid sequence having a natural (non-synthetic) endogenous sequence. The full-length polynucleotide encodes the full-length, catalytically active form of the specific protein. The terms "polypeptide," "polypeptide," and "protein" are used interchangeably herein to refer to polymers 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 acids. The term is also used for naturally occurring amino acid polymers. The terms "residue" or "amino acid residue" or "amino acid" are used interchangeably herein to refer to an amino acid that is incorporated into a protein, polypeptide, or peptide (collectively, "protein"). Amino acids can be naturally occurring amino acids and, unless otherwise limited, can include known analogs of naturally occurring amino acids that can function in a manner similar to naturally occurring amino acids.

[0039] In some embodiments, the nucleotide sequences of the present application can be altered to make conservative amino acid substitutions. The principles and examples of conservative amino acid substitutions are further described below. In certain embodiments, the nucleotide sequences of the present application can be substituted without changing the amino acid sequence according to the disclosed monocot codon preferences, for example, codons encoding the same amino acid sequence can be replaced with codons preferred by monocots without changing the amino acid sequence encoded by the nucleotide sequence. In some embodiments, part of the nucleotide sequence in the present application is replaced with different codons encoding the same amino acid sequence, thereby not changing the amino acid sequence encoded by the nucleotide sequence while changing the nucleotide sequence. Conservative variants include those sequences that encode the amino acid sequence of one of the proteins of the embodiments due to the degeneracy of the genetic code. In some embodiments, part of the nucleotide sequence in the present application is replaced according to the monocot codon preference. Those 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, for example, the hydrophobicity, charge, size, etc. of the substituents. Exemplary amino acid substitution groups with various aforementioned 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. Guidance on appropriate 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 (Natl. Biomed. Res. Found., Washington, DC) (incorporated herein by reference). Conservative substitutions such as replacing one amino acid with another amino acid having similar properties can be performed. Sequence identity identification 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 cloned genomic DNA fragments or cDNA fragment groups (i.e., genomic libraries or cDNA libraries) from a selected organism. The hybridization probe can be a genomic DNA fragment, a cDNA fragment, an RNA fragment or other oligonucleotide, and can be marked with a detectable group such as 32P or other detectable markers. Thus, for example, a hybridization probe can be prepared by marking a synthetic oligonucleotide based on the embodiment sequence. The method for preparing hybridization probes and building cDNA and genomic libraries is generally known in the art. The hybridization of the sequence can be carried out under stringent conditions. As used herein, the term "stringent conditions" or "stringent hybridization conditions" represents following conditions, i.e., under these conditions, relative to hybridizing with other sequences, the probe will hybridize with its target sequence to a greater extent (e.g., at least 2 times, 5 times or 10 times of background) that can be detected.Stringent conditions are sequence-dependent and vary in different environments. By controlling hybridization stringency and / or controlling washing conditions, a target sequence that is 100% complementary to the probe can be identified (homologous probe method). Alternatively, stringent conditions can be adjusted to allow some sequence mismatches in order to detect lower similarities (heterologous probe method). Typically, the probe length is less than about 1000 or 500 nucleotides. Typically, stringent conditions are those in which the salt concentration is less than about 1.5 M Na ions, typically about 0.01 M to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is: when used for short probes (e.g., 10 to 50 nucleotides), at least about 30°C; when used for long probes (e.g., greater than 50 nucleotides), at least about 60°C. Stringent conditions can also be achieved by adding destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization at 37°C using 30% to 35% formamide buffer, 1M NaCl, 1% SDS (sodium dodecyl sulfate), and washing in 1× to 2× SSC (20× SSC = 3.0M NaCl / 0.3M trisodium citrate) at 50°C to 55°C. Exemplary moderate stringency conditions include hybridization at 37°C in 40% to 45% formamide, 1.0M NaCl, 1% SDS, and washing in 0.5× to 1× SSC at 55°C to 60°C. Exemplary high stringency conditions include hybridization at 37°C in 50% formamide, 1M NaCl, 1% SDS, and a final wash in 0.1× SSC at 60°C to 65°C for at least about 20 minutes. Optionally, the wash buffer may contain about 0.1% to about 1% SDS. Duration of hybridization is typically less than about 24 hours, typically about 4 hours to about 12 hours. Specificity generally depends on post-hybridization washes, with the key factors being the ionic strength and temperature of the final wash solution. The Tm (thermodynamic melting point) of a DNA-DNA hybrid can be approximated by the formula of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284: Tm = 81.5°C + 16.6 (log M) + 0.41 (% GC) - 0.61 (% formamide) - 500 / L, where M is the molar concentration of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, "% formamide" is the percentage of formamide in the hybridization solution, and L is the base pair length of the hybrid. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the complementary target sequence hybridizes to a perfectly matched probe. Washes are typically performed at least until equilibrium is reached and low background levels of hybridization are achieved, such as for 2 hours, 1 hour, or 30 minutes. Each 1% mismatch should reduce the Tm by about 1°C; thus, the Tm, hybridization, and / or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with ≥90% identity are desired, the Tm can be reduced by 10°C.Generally, stringent conditions are selected to be about 5°C lower than the Tm of the specific sequence and its complement at a defined ionic strength and pH. However, under very stringent conditions, hybridization and / or washing can be performed at 4°C below the Tm; under moderately stringent conditions, hybridization and / or washing can be performed at 6°C below the Tm; and under low stringency conditions, hybridization and / or washing can be performed at 11°C below the Tm.

[0040] Unless otherwise indicated, all numbers used in the specification and claims expressing amounts of ingredients, reaction conditions, and the like should be understood as being modified in all instances by the term "about." As used herein, the term "about," when referring to a measurable value such as an amount of mass, weight, time, volume, concentration, or percentage, is meant to encompass variations of ±20% from the stated amount in some embodiments, ±10% from the stated amount in some embodiments, ±5% from the stated amount in some embodiments, ±1% from the stated amount in some embodiments, ±0.5% from the stated amount in some embodiments, and ±0.1% from the stated amount in some embodiments, as such variations are suitable for performing the disclosed methods and / or using the disclosed compositions, nucleic acids, polypeptides, and the like. Therefore, unless indicated to the contrary, the numerical parameters set forth in the specification and appended claims are approximate values ​​that may vary depending upon the desired properties sought to be obtained by the subject matter disclosed herein.

[0041] The following examples are intended to illustrate the present invention but are not intended to limit the scope of the present invention. Without departing from the spirit and substance of the present invention, modifications or replacements made to the inventive method, steps or conditions are intended to fall within the scope of this application. Unless otherwise specified, the examples are based on conventional experimental conditions, such as Sambrook et al.'s Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular cloning: a laboratory manual, 2001), or the conditions recommended by the manufacturer's instructions. Unless otherwise specified, the chemical reagents used in the examples are conventional commercially available reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

[0042] Example

[0043] Example 1 Construction of a regulatory network for differentially expressed genes before and after vernalization in wheat

[0044] Vernalization is a key factor influencing flowering and heading in wheat. To identify new genes regulating heading time, the inventors analyzed the chromatin state and gene expression at the genomic level of wheat after vernalization. This analysis allowed them to identify genes with altered chromatin environments and expression levels, thereby further identifying new genes regulating heading time.

[0045] The inventors used the elite wheat variety Aikang 58 (AK58) as material to analyze the effects of vernalization. First, the effect of vernalization on heading date was tested. The results showed that without vernalization, AK58 could not head and flower within 130 days of sowing. Both 28-day and 14-day vernalization promoted heading, but the 28-day vernalization resulted in an earlier heading date. Therefore, the inventors used three treatments for AK58 at the three-leaf stage: V0 (no vernalization), V28N (treated at 4°C for 28 days), and V28N6 (treated at 4°C for 28 days followed by 6 days of normal growth). The transcriptome, epigenetic modification group, and chromatin interaction group were detected after treatment. The transcriptome was analyzed using RNA sequencing, and the epigenetic modification group was analyzed using chromatin immunoprecipitation sequencing (ChIP-seq). The antibodies used for immunoprecipitation were H3K4me3, H3K27me3, and H3K9ac, respectively. Chromatin interaction was analyzed using H3K4me3-mediated ChIA-PET (chromatin interaction by paired-end tag sequencing). Chromatin accessibility was also identified using ATAC-seq (transposase-accessible chromatin using sequencing) (process as shown in the following example). Figure 1 shown).

[0046] The results of the above experiments were analyzed together to construct a regulatory network of differentially expressed genes before and after vernalization (V0 vs V28N6) (e.g. Figure 2 Finally, 6703 differentially expressed genes were obtained in the network, including 39 C2H2-type zincfinger transcription factors, 24 HSF transcription factors, 50 WRKY transcription factors and 48 ERF transcription factors (as shown in Figure 2). Figure 3 The analysis of the expression regulatory network showed that these genes may be regulated by vernalization-related transcription factors (such as VRN1, VRT2, TaFDL2, WAG1, WAG2, TaAGL17 and TaSTK) (as shown). Figure 2 ), so it is possible that it is related to the wheat heading period.

[0047] Example 2 Genome-wide association analysis of wheat heading period traits

[0048] In order to further clarify the genes related to the heading period of wheat, the inventors further conducted a genome-wide association analysis of the heading period traits and investigated the changes in the heading time between different haplotypes of wheat. The association analysis used laboratory resequencing of natural variant wheat germplasm resources and detected the SNPs of the target gene to divide the materials into different ploidies. The significance of the differences in the heading time of different haplotypes was analyzed. The results of the genome-wide association analysis showed that there was a significant signal enrichment peak in the 595.351-595.955Mb region of chromosome 5A. There were 3 wheat genes in this linkage region, of which only TraesCS5A02G401200 (TaZNF10-5A) had changes in expression during the vernalization process (such as Figure 4 Further haplotype analysis showed that different haplotypes of TaZNF10-5A had significant differences in wheat flowering / heading time.

[0049] The results of Example 2 and Example 1 were further analyzed together. The inventors further identified TaZNF10-5A and its homologous genes on 5B and 5D, and named them TaZNF10-5B (TraesCS5B02G406000) and TaZNF10-5D, respectively.

[0050] (TraesCS5D02G411400). The sequence of the TaZNF10-5B gene in the reference genome is shown in SEQ ID NO. 1, and the encoded protein sequence is shown in SEQ ID NO. 2; the sequence of the TaZNF10-5A gene in the reference genome is shown in SEQ ID NO. 3, and the encoded protein sequence is shown in SEQ ID NO. 4; the sequence of the TaZNF10-5D gene in the reference genome is shown in SEQ ID NO. 5, and the encoded protein sequence is shown in SEQ ID NO. 6. The inventors also isolated and identified the gene sequence in AK58, which is identical to the reference genome sequence.

[0051] Example 3 Overexpression and gene editing knockout of TaZNF10-5B gene

[0052] The inventors further performed overexpression and gene editing knockout operations on the TaZNF10-5B gene in the wheat variety KN199 to verify its function.

[0053] The promoter and terminator used in the overexpression vector are the maize ubiquitin promoter (SEQ ID NO.7) and nos terminator commonly used in the art, respectively. It was found that overexpression of the TaZNF10-5B gene can prolong the heading period of wheat (see Figure 5 and Table 1). Similar effects were observed after overexpression of TaZNF10-5A and TaZNF10-5D.

[0054] Table 1 TaZNF10-5B gene overexpression phenotype

[0055]

[0056] Gene editing selected double targets (sequences as SEQ ID NO. 8 and SEQ ID NO. 9), used TaU3 to drive the expression of Cas9 and gRNAs, constructed editing vectors, transformed wheat KN199, and identified the genotypes of the obtained edited plants.

[0057] Two successfully edited wheat plants were identified, one of which had a 15bp deletion in the TaZNF10-5B gene at the target site shown in SEQ ID NO. 8 (the mutant gene sequence is shown in SEQ ID NO. 12), and this plant showed an early heading date phenotype; the other plant had mutations in the TaZNF10-5A, TaZNF10-5B and TaZNF10-5D genes, and the inventors obtained various mutant gene combinations such as ABD triple mutation, AB double mutation, BD double mutation, B single mutation and D single mutation in the T1 generation segregation lines, and the heading date investigation results showed that these different mutations could all make the wheat heading date advance. The data of the heading date phenotypes of the mutants are shown in Table 2.

[0058] Table 2 TaZNF10-5A / B / D gene editing phenotype

[0059]

[0060]

[0061] Although the present application has been described in detail with general description and specific embodiments above, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, all belong to the scope of the present application claimed.

Claims

1. Application of a gene in regulating the heading period of wheat, characterized in that: The gene includes any one of the following: (1) The gene with the sequence shown in SEQ ID NO.1; (2) a gene encoding the sequence shown in SEQ ID NO. 2; (3) The gene numbered TraesCS5B02G406000 in the wheat gene database.

2. Application of a biomaterial in regulating the heading period of wheat, characterized in that: The biological material includes any one of the following: (1) An expression cassette containing the gene according to claim 1; (2) an expression vector containing the gene according to claim 1; (3) A host cell containing the gene according to claim 1, wherein the host cell is a bacterial cell or a non-regenerable plant cell.

3. A method for extending the heading period of wheat, characterized in that: The expression of the gene according to claim 1 is increased in the wheat material to be improved, and wheat plants with prolonged heading period are selected.

4. The method according to claim 3, characterized in that The method for increasing gene expression is to use a high-activity promoter to drive the expression of the gene according to claim 1.

5. The method according to claim 4, characterized in that The high-activity promoter sequence is shown in SEQ ID NO.

7.

6. A method for shortening the heading period of wheat, characterized in that: Knock out or inhibit the expression of genes or coded proteins in the wheat material to be improved, and select wheat plants with shortened heading period; The gene includes any one of the following: (1) The gene with the sequence shown in SEQ ID NO.1 or the gene encoding the sequence shown in SEQ ID NO.2 or the gene numbered TraesCS5B02G406000 in the wheat gene database; (2) The gene with the sequence shown in SEQ ID NO.5 or the gene encoding the sequence shown in SEQ ID NO.6 or the gene numbered TraesCS5D02G411400 in the wheat gene database; (3) A combination of the gene described in (1) and the gene described in (2); (4) a gene having a sequence shown in SEQ ID NO. 3 or a gene encoding a sequence shown in SEQ ID NO. 4 or a gene numbered TraesCS5A02G401200 in the wheat gene database and a combination of the gene described in (1); (5) A combination of the gene described in (4) and the gene described in (2).

7. The method according to claim 6, characterized in that The method of knocking out or inhibiting the expression of a gene or encoded protein is a gene editing method.

8. The method according to claim 7, characterized in that The target sequence selected by the gene editing method is shown in SEQ ID NO.8 or SEQ ID NO.

9.

9. Use of a kit for shortening the heading period of wheat, characterized in that: The kit includes any one of the following: (1) an RNA molecule capable of recognizing the target sequence shown in SEQ ID NO. 8 or SEQ ID NO. 9; (2) a DNA molecule encoding the RNA described in (1); (3) a vector for expressing the RNA described in (1); The kit further comprises a Cas9 protein or a nucleic acid molecule encoding the Cas9 protein or a vector expressing the Cas9 protein; The RNA molecule sequence is shown as SEQ ID NO.10 or SEQ ID NO.

11.

10. A mutant gene, characterized in that The sequence of the mutant gene is shown in any one of SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16 or SEQ ID NO.

18.

11. A mutant protein, characterized in that The sequence of the mutant protein is shown in any one of SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17 or SEQ ID NO.

19.

12. Use of the method according to claims 3-5, or the method according to claims 6-8, or the mutant gene according to claim 10, or the mutant protein according to claim 11 in shortening the heading period of wheat.

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