CN117965562A - Wheat TaZF-B1 gene and application thereof - Google Patents

Abstract

The invention belongs to the field of plant breeding, and particularly relates to a wheat TaZF-B1 gene and application thereof. According to the invention, a new gene TaZF-B1 affecting the characteristics of wheat ears is obtained through GWAS and meta-QTL analysis. Manipulation of TaZF-B1 gene can improve wheat ear properties.

Description

Wheat TaZF-B1 gene and application thereof

Technical Field

The invention belongs to the field of plant breeding, and particularly relates to a wheat TaZF-B1 gene and application thereof.

Background

Wheat yield is affected by several main characteristics of effective ears per unit area, grain number per ear and grain weight. Because the grain number and grain weight characteristics of each spike can be directly influenced by the spike structure, analysis of the spike structure forming mechanism, cloning of key genes and improvement of characteristics play an important role in improving the wheat yield.

The heading period is an important character in the process of crop evolution and adaptation, understanding the genetic basis of the character in the heading period of crops and cloning candidate genes can improve the environmental adaptability and plasticity of crops, so that the heading period has important significance for cultivating excellent crop varieties adapting to different ecological areas, and simultaneously, the genetic improvement process of important production characters, such as yield and the like, closely related to the heading period is promoted.

Disclosure of Invention

The invention aims to provide a key gene affecting the characteristics of wheat ears.

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

the invention provides an application of a gene in regulating and controlling wheat spike length and/or heading period, which is characterized in that the gene comprises any one of the following components:

(1) A gene of the sequence shown in SEQ ID NO. 1;

(2) A gene encoding the sequence shown in SEQ ID NO. 2;

(3) The gene numbered TraesCS B02G480400 in the wheat gene database.

The invention also provides an application of the biological material in regulating and controlling the wheat ear length and/or the heading period, which is characterized in that the biological material comprises any one of the following components:

(1) An expression cassette comprising the above gene;

(2) An expression vector containing the above gene;

(3) Host cells comprising the above genes, said host cells being bacterial cells or non-regenerable plant cells.

The invention also provides a method for shortening the wheat ear length and/or the heading period, which is characterized by comprising the following steps:

(1) Increasing the expression of the above genes in the wheat material to be modified;

(2) Wheat plants with reduced ear length and/or heading time are selected.

In some embodiments, the above method of increasing gene expression is the use of a highly active promoter to drive expression of the gene of claim 1.

In some embodiments, the above-described high activity promoter is SEQ ID No.3 or SEQ ID No.5.

The invention also provides a promoter which is characterized in that the sequence of the promoter is shown as SEQ ID NO. 3.

The invention also provides a biological material, which is characterized in that the biological material comprises any one of the following components:

(1) An expression cassette comprising the above promoter;

(2) An expression vector comprising the above promoter;

(3) A host cell comprising the above promoter, said host cell being a bacterial cell or a non-regenerable plant cell.

The invention also provides the application of the method, the promoter or the biological material in regulating the wheat spike length and/or the heading period.

The invention also provides a method for increasing the wheat spike length, which is characterized in that wheat germplasm containing a sequence shown in SEQ ID NO.4 is used as a donor, a fragment of the sequence shown in SEQ ID NO.4 is introduced into the wheat germplasm containing the sequence shown in SEQ ID NO.3 by a sexual hybridization method, and wheat plants with increased spike length are selected.

The invention has the advantages and beneficial effects as follows: according to the invention, a new gene TaZF-B1 affecting the characteristics of wheat ears is obtained through GWAS and meta-QTL analysis. The wheat spike property can be improved by manipulating TaZF-B1 gene and increasing the expression level of TaZF-B1 gene. The invention also provides a novel promoter which can be used in the field of plant genetic engineering.

Drawings

FIG. 1 shows a wheat germ plasm population for association analysis. A: separation according to germplasm genetic relationship; b: genetic relationship analysis of 290 wheat germplasm.

FIG. 2 is a diagram showing the morphology of young ear tissues subjected to gene activity analysis. DRS and FPS represent young ear tissue in the double-ridged and flower primordial phases, respectively.

FIG. 3TaZF-B1 gene (TraesCS B02G 480400) transcriptional signal, chromatin opening degree and histone modification signal.

FIG. 4 structure and activity of two TaZF-B1 promoters. A: the structure and differences of the two TaZF-B1 promoters; b: schematic representation of the expression cassette of the double Luciferase (LUC) assay. C: LUC activity represents P <0.01.

FIG. 5TaZF phenotype of the B1-OE strain.

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.

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.

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", "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.

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. The hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group such as 32P or other detectable marker. Thus, for example, hybridization probes can be prepared by labeling synthetic oligonucleotides based on the sequences of the embodiments. Methods for preparing hybridization probes and constructing cDNA and genomic libraries are generally known in the art. Hybridization of the sequences may be performed under stringent conditions. As used herein, the term "stringent conditions" or "stringent hybridization conditions" refers to conditions under which a probe will hybridize to its target sequence to a detectably greater extent (e.g., at least 2-fold, 5-fold, or 10-fold over background) relative to hybridization to other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the hybridization stringency and/or controlling the washing conditions, target sequences 100% complementary to the probes can be identified (homologous probe method). Alternatively, stringent conditions can be adjusted to allow for some sequence mismatches in order to detect lower similarity (heterologous probe method). Typically, the probe is less than about 1000 or 500 nucleotides in length. Typically, stringent conditions are those in which the salt concentration is less than about 1.5M Na ion, typically about 0.01M to 1.0M Na ion concentration (or other salt) at a pH of 7.0 to 8.3, and the temperature conditions are: when used with short probes (e.g., 10 to 50 nucleotides), at least about 30 ℃; when used with long probes (e.g., greater than 50 nucleotides), at least about 60 ℃. Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization at 37 ℃ with 30% to 35% formamide buffer, 1M NaCl, 1% sds (sodium dodecyl sulfate), washing in 1 x to 2 x SSC (20 x SSC = 3.0M NaCl/0.3M trisodium citrate) at 50 ℃ to 55 ℃. Exemplary moderately stringent conditions include hybridization in 40% to 45% formamide, 1.0M NaCl, 1% SDS at 37℃and washing in 0.5 XSSC to 1 XSSC at 55℃to 60 ℃. Exemplary high stringency conditions include hybridization in 50% formamide, 1M NaCl, 1% sds at 37 ℃ and a final wash in 0.1 x SSC at 60 ℃ to 65 ℃ for at least about 20 minutes. Optionally, the wash buffer may comprise about 0.1% to about 1% sds. The duration of hybridization is typically less than about 24 hours, typically from about 4 hours to about 12 hours. Specificity generally depends on post-hybridization washing, the key factors being the ionic strength and temperature of the final wash solution. The Tm (thermodynamic melting point) of DNA-DNA hybrids can be approximated from the formula Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284: tm=81.5 ℃ +16.6 (log) +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. Tm is the temperature (at a defined ionic strength and pH) at which 50% of the complementary target sequence hybridizes to a perfectly matched probe. Washing is typically performed at least until equilibrium is reached and a low hybridization background level is reached, such as 2 hours, 1 hour, or 30 minutes. Each 1% mismatch corresponds to a decrease in Tm of about 1 ℃; thus, tm, hybridization, and/or wash conditions can be adjusted to hybridize to sequences of desired identity. For example, if sequences with ≡90% identity are desired, the Tm can be reduced by 10 ℃. Typically, stringent conditions are selected to be about 5 ℃ lower than the Tm for the specific sequence and its complement at a defined ionic strength and pH. However, under very stringent conditions, hybridization and/or washing may be performed at 4℃below the Tm; hybridization and/or washing may be performed at 6 ℃ below the Tm under moderately stringent conditions; hybridization and/or washing can be performed at 11℃below the Tm under low stringency conditions.

Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about". The term "about," as used herein, when referring to a measurable value, such as a mass, weight, time, volume, concentration, or amount of percent, is meant to encompass a change of ± 20% from a specified amount in some embodiments, a change of ± 10% from a specified amount in some embodiments, a change of ± 5% from a specified amount in some embodiments, a change of ± 1% from a specified amount in some embodiments, a change of ± 0.5% from a specified amount in some embodiments, and a change of ± 0.1% from a specified amount in some embodiments, as such changes are suitable for performing the disclosed methods and/or using the disclosed compositions, nucleic acids, polypeptides, and the like. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.

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: 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.

Examples

Example 1 wheat ear developmental Gene localization

The inventors performed GWAS-related analysis of wheat ear traits using a population of 290 wheat germplasm with wide genetic diversity, and identified 356 SNPs as affecting ear length (SPIKE LENGTH, SL), small ear per ear (spikelet number per spike, SNS), and grain per ear (grain number per spike, GNS) traits (see fig. 1 for results). These SNPs were analyzed to be located within 356 QTL intervals, including some genes known to affect wheat head development, such as WAPO-A1, FUL3-2B, VRN1-5A, and the like.

The inventors further gathered some of the published GWAS data and analyzed transcriptome, histone modification group (H3K 9ac, H3K4me3, H3K27me 3) and chromatin patency data of Double RIDGE STAGE (DRS) and floral primordial (floret primordium stage, FPS) young ear tissue (fig. 2). Finally, approximately 10 ten thousand genes with higher activity in the spikelet meristem and the flower meristem are obtained.

Because eQTL has a great relationship with genomic variation and gene transcription dynamics, the inventors also detected eQTL data in young ear tissue at the double-ridged stage in 90 wheat germplasm.

By combining the above data, the inventors obtained 489 genes, which were not only located in the ear development QTL interval, but also exhibited high activity in the eQTL, chromatin opening degree, and histone modification signal in the gene interval. Of these 489 genes, 25 were noted as transcription factors. One of the transcription factors encodes the Zinc FINGER CCCH domain-containing (ZF) protein, whose promoter region has a very high chromatin opening state and histone activation modification signals (fig. 3). The gene was designated TaZF-B1 for intensive studies. The sequence of TaCYP A-6B gene is shown as SEQ ID NO.1, and the coded protein sequence is shown as SEQ ID NO. 2.

EXAMPLE 2TaZF-B1 Gene function Studies

The inventors first amplified and analyzed the promoter sequence of TaZF-B1 gene in different wheat germplasm, found that the promoter of TaZF-B1 gene was divided into two different haplotypes proZF-I and proZF-II, wherein proZF-II lacks the sequence of nearly 600bp near the 3' end of the initiation codon than proZF-I. The inventors constructed proZF-I and proZF-II promoters (shown as SEQ ID NO.3 and SEQ ID NO.4, respectively) onto a dual luciferase LUC reporter vector to test the activity of the promoters. The results show that proZF-I promoter was significantly more active than proZF-II (FIG. 4). This indicates that the TaZF-B1 gene carrying proZF-I promoter is expressed in higher amounts.

The inventors further overexpress TaZF-B1 gene in wheat variety Fielder, and the promoters and terminators used in the overexpressing vectors are maize ubiquitin promoter and nos terminator, respectively, commonly used in the art, and found that overexpression of TaZF-B1 gene can reduce spike length and shorten heading time of wheat (see FIG. 5 and Table 1).

TABLE 1TaZF-B1 Gene overexpression phenotype

Data are expressed as mean ± standard deviation. * Indicating significant differences compared to the control (P <0.05, student's t-test). 3. 17, 19 represent 3 overexpressing transformants, respectively.

Example 3 wheat germplasm trait comparison of different promoter genotypes

Since different wheat germplasm contained promoters of both genotypes proZF-I and proZF-II and proZF-I and proZF-II were different in activity, the inventors analyzed the promoter sequences of TaZF-B1 genes in several representative wheat germplasm and compared their ear length traits, which showed that germplasm ear lengths containing proZF-II promoters were significantly longer than those containing proZF-I (Table 2).

TABLE 2 ear trait comparison of wheat germplasm of different promoter types

Data are expressed as mean ± standard deviation.

Thus, the grain length of wheat can be increased by introducing proZF-II genotype into wheat germplasm containing proZF-I genotype using means of backcross transformation.

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 (9)

1. An application of a gene in regulating the length of wheat ears and/or the heading date, which is characterized in that the gene comprises any one of the following components:

(1) A gene of the sequence shown in SEQ ID NO. 1;

(2) A gene encoding the sequence shown in SEQ ID NO. 2;

(3) The gene numbered TraesCS B02G480400 in the wheat gene database.

2. Use of a biological material for regulating the length of wheat ears and/or the heading stage, wherein the biological material comprises any one of the following:

(1) An expression cassette comprising the gene of claim 1;

(2) An expression vector comprising the gene of claim 1;

(3) A host cell comprising the gene of claim 1, which is a bacterial cell or a non-regenerable plant cell.

3. A method of reducing the length of a wheat ear and/or the heading time, the method comprising the steps of:

(1) Increasing the expression of the gene of claim 1 in a wheat material to be improved;

(2) Wheat plants with reduced ear length and/or heading time are selected.

4. The method of claim 3, wherein the method of increasing gene expression is to drive expression of the gene of claim 1 using a highly active promoter.

5. The method of claim 4, wherein the high activity promoter is SEQ ID NO.3 or SEQ ID NO.5.

6. The promoter is characterized in that the promoter sequence is shown in any one of SEQ ID NO.3 or SEQ ID NO. 4.

7. A biomaterial, characterized in that the biomaterial comprises any one of the following:

(1) An expression cassette comprising the promoter of claim 6;

(2) An expression vector comprising the promoter of claim 6;

(3) A host cell comprising the promoter of claim 6, said host cell being a bacterial cell or a non-regenerable plant cell.

8. Use of the method according to any one of claims 3-5, or the promoter according to claim 6, or the biological material according to claim 7 for regulating the ear length and/or heading stage of wheat.

9. A method for increasing the length of wheat ears is characterized in that wheat germplasm containing a sequence shown in SEQ ID NO.4 is used as a donor, a fragment of the sequence shown in SEQ ID NO.4 is introduced into the wheat germplasm containing a sequence shown in SEQ ID NO.3 by a sexual hybridization method, and wheat plants with increased ear length are selected.