CN116836249A

CN116836249A - Three homologous genes and related proteins related to wheat yield

Info

Publication number: CN116836249A
Application number: CN202210898756.7A
Authority: CN
Inventors: 郭自峰; 刘洋洋; 申立平; 张丽丽
Original assignee: Institute of Botany of CAS
Current assignee: Institute of Botany of CAS
Priority date: 2022-07-28
Filing date: 2022-07-28
Publication date: 2023-10-03

Abstract

The application discloses three homologous genes and related proteins related to wheat yield. The three homologous genes related to wheat yield are HGY1 genes derived from wheat A, B, D genome, and the HGY1 genes respectively code proteins shown in sequence 3, sequence 6 and sequence 9 in a sequence table. Experiments prove that compared with wild plants, the grain number of spikes and thousand seed weight of a transgenic plant obtained by introducing the HGY1 gene related to wheat yield into the plants are obviously increased, and the grain number of spikes and thousand seed weight of the HGY1 gene knocked out are obviously reduced, which proves that the HGY1 gene and the protein encoded by the HGY1 gene can regulate and control the grain number of spikes and thousand seed weight of wheat.

Description

Three homologous genes and related proteins related to wheat yield

Technical Field

The application relates to three homologous genes and related proteins related to wheat yield in the biotechnology field.

Background

The united nations grain and farming organization predicts that in the middle of the 21 st century, the global grain yield needs to be increased by 50% to meet the ever-increasing human demand. Wheat (triticum aestivum l.) is a annual herb of the genus wheat of the family poaceae, has strong adaptability, and is one of the most widely planted crops worldwide. The caryopsis of wheat is rich in nutrition, can be made into bread, steamed bread, biscuit, noodles and other foods after being ground into flour, can be made into beer, alcohol and white spirit after being fermented, and is one of the staple foods for human beings. Wheat yield represents 30% of the global grain yield, providing 20% of the energy and protein required by humans. Increasing wheat yield remains one of the main tasks of current wheat breeding efforts. In recent years, with global climate change, natural disasters and crop disease frequency, great difficulty is faced in improving the yield of wheat. Wheat is one of main grain crops in China, and stable and high yield of wheat is important to guaranteeing grain safety in China.

Grain size, grain number per unit area and grain number per unit area are decisive factors for wheat yield. Thousand kernel weight is an important indicator of kernel size. Wherein the thousand grain weight and the spike grain number of the wheat are generally inversely related, and the spike grain number and the thousand grain weight are difficult to be simultaneously improved in the wheat breeding process.

Molecular breeding can directionally promote specific yield traits and accelerate the cultivation process of new varieties. Cloning of related genes and excavation of excellent sites can provide valuable information for molecular breeding of wheat.

Disclosure of Invention

The technical problem to be solved by the application is how to improve the plant yield.

To solve the above technical problems, the present application provides, first, any one of the following applications of proteins or substances regulating the activity or content of the proteins:

d1 Regulating plant yield;

d2 Preparing a product for regulating and controlling plant yield;

d3 Increasing plant yield;

d4 Preparing a product for increasing plant yield;

d5 Cultivating the yield-enhancing plant;

d6 Preparing a plant product with improved cultivation yield;

the protein is derived from wheat and is named as High Grain Yield1 (HGY 1 for short), wherein HGY1 is as follows A1), A2) or A3):

a1 Amino acid sequence is a protein of sequence 3, sequence 6 or sequence 9;

a2 A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues for the amino acid sequences shown in the sequence 3, the sequence 6 or the sequence 9 in the sequence table and has the same function;

a3 A fusion protein obtained by ligating a tag to the N-terminal or/and the C-terminal of A1) or A2).

HGY1 is HGY-A1, HGY-B1 and/or HGY-D1, and the amino acid sequences of HGY-A1, HGY-B1 and HGY-D1 are respectively sequence 3, sequence 6 and sequence 9.

In order to facilitate purification of the protein of A1), a tag shown in the following table may be attached to the amino-terminal or carboxyl-terminal of a protein consisting of the amino acid sequence shown in sequence 3, sequence 6 or sequence 9 in the sequence listing.

Table: tag sequence

Label (Label)	Residues	Sequence(s)
			Poly-Arg	5-6 (usually 5)	RRRRR
Poly-His	2-10 (usually 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The HGY1 protein in A2) is a protein having 75% or more identity with the amino acid sequence of the protein represented by sequence 3, sequence 6 or sequence 9 and having the same function. The identity of 75% or more is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

The HGY1 protein in the A2) can be synthesized artificially or can be obtained by synthesizing the coding gene and then biologically expressing.

The gene encoding HGY1 protein in the above A2) can be obtained by deleting one or several amino acid residues in the DNA sequence shown in the sequence 2, 5 or 8, and/or performing one or several base pair missense mutation, and/or ligating the coding sequences of the tags shown in the above table at the 5 'end and/or 3' end thereof. Wherein the DNA molecules shown in the sequences 2, 5 and 8 respectively encode HGY1 proteins shown in the sequences 3, 6 and 9.

In the above application, the substance may be any one of the following B1) to B9):

b1 A nucleic acid molecule encoding HGY 1;

b2 An expression cassette comprising the nucleic acid molecule of B1);

b3 A recombinant vector comprising the nucleic acid molecule of B1) or a recombinant vector comprising the expression cassette of B2);

b4 A recombinant microorganism comprising the nucleic acid molecule of B1), or a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism comprising the recombinant vector of B3);

b5 A transgenic plant cell line comprising the nucleic acid molecule of B1) or a transgenic plant cell line comprising the expression cassette of B2);

b6 A transgenic plant tissue comprising the nucleic acid molecule of B1) or a transgenic plant tissue comprising the expression cassette of B2);

b7 A transgenic plant organ comprising the nucleic acid molecule of B1) or a transgenic plant organ comprising the expression cassette of B2);

b8 A nucleic acid molecule that reduces HGY1 expression;

b9 An expression cassette, a recombinant vector, a recombinant microorganism, a transgenic plant cell line, a transgenic plant tissue or a transgenic plant organ comprising the nucleic acid molecule of B8).

In the above applications, the nucleic acid molecule of B1) may be B11) or B12) or B13) or B14) or B15) as follows:

b11 A cDNA molecule or a DNA molecule of which the coding sequence is a sequence 2, a sequence 5 or a sequence 8 in a sequence table;

b12 A DNA molecule shown as a sequence 2, a sequence 5 or a sequence 8 in the sequence table;

b13 A DNA molecule shown as a sequence 1, a sequence 4 or a sequence 7 in the sequence table;

b14 A cDNA molecule or a DNA molecule having 75% or more identity to the nucleotide sequence defined in b 11) or b 12) or b 13) and encoding HGY 1;

b15 Under stringent conditions) with a nucleotide sequence defined by b 11) or b 12) or b 13) or b 14), and a cDNA molecule or DNA molecule encoding HGY 1.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

The nucleotide sequence encoding HGY1 protein of the present application can be easily mutated by those skilled in the art using known methods such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or more identity to the nucleotide sequence of the HGY1 protein isolated according to the present application are derived from the nucleotide sequence of the present application and are equivalent to the sequence of the present application as long as they encode the HGY1 protein and have the function of the HGY1 protein.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences having 75% or more, or 85% or more, or 90% or more, or 95% or more identity to the nucleotide sequence of the protein consisting of the amino acid sequences shown in coding sequence 3, sequence 6 or sequence 9 of the present application. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to evaluate the identity between related sequences.

In the above application, the stringent conditions may be as follows: 50℃in 7% Sodium Dodecyl Sulfate (SDS), 0.5M NaPO ₄ Hybridization with 1mM EDTA, rinsing in2 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M NaPO ₄ Hybridization with 1mM EDTA, rinsing in 1 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M NaPO ₄ Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M NaPO ₄ Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M NaPO ₄ Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃; the method can also be as follows: hybridization was performed in a solution of 6 XSSC, 0.5% SDS at 65℃and then washed once with 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS; the method can also be as follows: hybridization and washing the membrane 2 times at 68℃in a solution of 2 XSSC, 0.1% SDS for 5min each time, and hybridization and washing the membrane 2 times at 68℃in a solution of 0.5 XSSC, 0.1% SDS for 15min each time; the method can also be as follows: hybridization and washing of membranes were performed at 65℃in 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS solution.

The 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

In the above applications, the expression cassette (HGY 1 gene expression cassette) comprising a nucleic acid molecule encoding an HGY1 protein according to B2) refers to a DNA capable of expressing an HGY1 protein in a host cell, which DNA may include not only a promoter for initiating transcription of the HGY1 gene, but also a terminator for terminating transcription of the HGY1 gene. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present application include, but are not limited to: constitutive promoters, tissue, organ and development specific promoters, and inducible promoters. Examples of promoters include, but are not limited to: a constitutive promoter of cauliflower mosaic virus 35S; wound-inducible promoters from tomato, leucine aminopeptidase ("LAP", chao et al (1999) Plant Physiol 120:979-992); a chemically inducible promoter from tobacco, pathogenesis-related 1 (PR 1) (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester); tomato protease inhibitor II promoter (PIN 2) or LAP promoter (both inducible with methyl jasmonate); heat shock promoters (U.S. Pat. No. 5,187,267); tetracycline-inducible promoters (U.S. Pat. No. 5, 057,422); seed-specific promoters, such as the millet seed-specific promoter pF128 (CN 101063139B (China patent 200710099169.7)), seed storage protein-specific promoters (e.g., promoters of phaseolin, napin, oleosin, and soybean beta-cone (Beachy et al (1985) EMBO J. 4:3047-3053)). They may be used alone or in combination with other plant promoters. All references cited herein are incorporated by reference in their entirety. Suitable transcription terminators include, but are not limited to: agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine synthase terminator (see, e.g., odell et al (I) ⁹⁸⁵ ) Nature 313:810; rosenberg et al (1987) Gene,56:125; guerineau et al (1991) mol. Gen. Genet,262:141; proudroot (1991) Cell,64:671; sanfacon et al Genes Dev.,5:141; mogen et al (1990) Plant Cell,2:1261; munroe et al (1990) Gene,91:151; ballad et al (1989) Nucleic Acids Res.17:7891; joshi et al (1987) Nucleic Acid Res., 15:9627).

Recombinant vectors containing the HGY1 gene expression cassette can be constructed using existing expression vectors. The plant expression vector comprises a binary agrobacterium vector, a vector which can be used for plant microprojectile bombardment and the like. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, PSN1301, or pCAMBIA1391-Xb (CAMBIA Co.), etc. The plant expression vector may also comprise the 3' -untranslated region of a foreign gene, i.e., comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The polyadenylation signal may direct the addition of polyadenylation to the 3 'end of the mRNA precursor and may function similarly to the 3' transcribed untranslated regions of Agrobacterium tumefaciens induction (Ti) plasmid genes (e.g., nopaline synthase gene Nos) and plant genes (e.g., soybean storage protein genes). When the gene of the present application is used to construct a plant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancers may be ATG initiation codon or adjacent region initiation codon, etc., but must be identical to the reading frame of the coding sequence to ensure proper translation of the entire sequence. The sources of the translational control signals and initiation codons are broad, and can be either natural or synthetic. The translation initiation region may be derived from a transcription initiation region or a structural gene. To facilitate identification and selection of transgenic plant cells or plants, the plant expression vectors used may be processed, for example by adding genes encoding enzymes or luminescent compounds which produce a color change (GUS gene, luciferase gene, etc.), antibiotic marker genes (such as nptII gene conferring resistance to kanamycin and related antibiotics, bar gene conferring resistance to the herbicide phosphinothricin, hph gene conferring resistance to antibiotic hygromycin, dhfr gene conferring resistance to methotrexate, EPSPS gene conferring resistance to glyphosate) or chemical marker genes, etc. (such as herbicide resistance genes), mannose-6-phosphate isomerase gene providing mannose metabolization ability, etc. From the safety of transgenic plants, transformed plants can be screened directly in stress without adding any selectable marker gene.

In the above applications, the vector may be a plasmid, cosmid, phage or viral vector. The plasmid may specifically be a pMWB110 vector.

B3 The recombinant vector may specifically be pMWB110-HGY-A1 or pMWB110-HGY-D1. The pMWB110-HGY-A1 is a recombinant vector obtained by inserting HGY-A1 genes shown in sequence 2 in a sequence table between multiple cloning sites of the pMWB110 vector. The pMWB110-HGY-D1 is a recombinant vector obtained by inserting HGY-D1 genes shown in sequence 8 in a sequence table between multiple cloning sites of the pMWB110 vector.

B8 The nucleic acid molecule may be a sgRNA in a CRISPR/Cas9 system that targets the nucleic acid molecule of B1). The target sequence of the sgRNA can be a sequence 10 or a sequence 11 in a sequence table.

B9 The recombinant vector may be a recombinant vector that can reduce HGY1 content prepared using CRISPR/Cas9 system. The recombinant vector may express an sgRNA targeting the nucleic acid molecule of B1).

In the above application, the microorganism may be yeast, bacteria, algae or fungi. Wherein the bacterium may be Agrobacterium.

In the above applications, none of the transgenic plant cell lines, transgenic plant tissues and transgenic plant organs include propagation material.

The application also provides any one of the following methods:

x1) a method of growing a yield-increasing plant comprising expressing HGY1 in a recipient plant, or increasing the content or activity of HGY1 in a recipient plant, to obtain a plant of interest having increased yield as compared to said recipient plant;

x1) a method for increasing plant yield comprising expressing HGY1 in a recipient plant or increasing the content or activity of HGY1 in a recipient plant to obtain a plant of interest having increased yield as compared to said recipient plant, whereby an increase in plant yield is achieved.

In the above method, the methods of X1) and X2) may be carried out by introducing a gene encoding HGY1 into the recipient plant and allowing the gene to be expressed.

The coding gene may be the nucleic acid molecule of B1).

In the above method, the coding gene of HGY1 may be modified as follows, and then introduced into a recipient plant to achieve better expression effect:

1) Modification and optimization are carried out according to actual needs so as to enable the genes to be expressed efficiently; for example, the codon of the gene encoding HGY1 of the present application may be changed to conform to plant preferences while maintaining the amino acid sequence thereof, according to the codon preferred by the recipient plant; during the optimization process, it is preferable to maintain a certain GC content in the optimized coding sequence to best achieve high level expression of the introduced gene in the plant, wherein the GC content may be 35%, more than 45%, more than 50% or more than about 60%;

2) Modifying the gene sequence adjacent to the initiation methionine to allow efficient initiation of translation; for example, modifications are made using sequences known to be effective in plants;

3) Ligating to promoters expressed by various plants to facilitate expression thereof in plants; the promoter may include constitutive, inducible, chronologically regulated, developmentally regulated, chemically regulated, tissue-preferred, and tissue-specific promoters; the choice of promoter will vary with the time and space of expression requirements and will also depend on the target species; for example, a tissue or organ specific expression promoter, depending on the desired time period of development of the receptor; although many promoters derived from dicots have been demonstrated to be functional in monocots and vice versa, it is desirable to select dicot promoters for expression in dicots and monocot promoters for expression in monocots;

4) The expression efficiency of the gene of the application can be improved by connecting with a proper transcription terminator; e.g., tml derived from CaMV, E9 derived from rbcS; any available terminator known to function in plants may be ligated to the gene of the present application;

5) Enhancer sequences such as intron sequences (e.g., derived from Adhl and bronzel) and viral leader sequences (e.g., derived from TMV, MCMV and AMV) are introduced.

The gene encoding HGY1 may be introduced into a recipient plant using a recombinant expression vector containing the gene encoding HGY 1. The recombinant expression vector can be specifically the pMWB110-HGY-A1 or the pMWB110-HGY-D1.

The recombinant expression vector may be introduced into plant cells by conventional biotechnological methods using Ti plasmids, plant viral vectors, direct DNA transformation, microinjection, electroporation, etc. (Weissbach, 1998,Method for Plant Molecular Biology VIII,Academy Press,New York,pp.411-463;Geiserson and Corey,1998,Plant Molecular Biology (2 nd Edition)).

The plant of interest is understood to include not only the first generation plants in which the HGY1 protein or gene encoding it has been altered, but also their progeny. For the plant of interest, the gene may be propagated in that species, or may be transferred into other varieties of the same species, including particularly commercial varieties, using conventional breeding techniques. The plants of interest include seeds, calli, whole plants and cells.

The application also provides a product for increasing plant yield, said product comprising HGY1 or said substance modulating HGY1 activity or content.

The product can take HGY1 or the substance for regulating the activity or the content of HGY1 as an active ingredient thereof, and can also take HGY1 or the substance for regulating the activity or the content of HGY1 and other substances with the same function as the active ingredient thereof.

In the above, the yield may be expressed in terms of spike number and/or grain weight. The number of ears may be the number of ears per ear. The grain weight may be thousand grain weight.

The plant may be M1) or M2) or M3):

m1) monocotyledonous or dicotyledonous plants; m2) a gramineous plant; m3) wheat.

HGY1 or the substance for regulating and controlling the activity or content of HGY1 also belongs to the protection scope of the application.

Experiments prove that compared with wild plants, the number of spike grains and thousand grain weight of transgenic plants obtained by introducing the HGY1 gene into the plants are obviously increased, and the number of spike grains and thousand grain weight of the transgenic plants obtained by knocking out the HGY1 gene are obviously reduced, which indicates that the HGY1 gene and the protein encoded by the HGY1 gene can regulate and control the number of spike grains and thousand grain weight of each spike of wheat.

Drawings

FIG. 1. Influence of gene knockout on spike number and grain size. (a) different strains of spike; (b) the aspect ratio of the seeds of different strains is higher; (c) counting the number of grains per spike; (d) thousand grain weight statistics. The white scales in figures a, b represent 4cm,0.5cm, respectively, where the two scales in figure (b) are identical. Data are shown as mean ± standard deviation (n=20), the significance of the difference calculated according to Student's t-test (< 0.05, < 0.001).

FIG. 2. Influence of over-expression of genes on spike number and grain size. (a) snapping of different overexpression lines; (b) the aspect ratio of the different overexpressing lines is relatively high; (c) the results of measuring the expression levels of the wild-type and the over-expression lines; (d) statistics of the number of grains per ear; (e) statistics of thousand kernel weight. The white scales in figures a, b represent 4cm,0.5cm, respectively, where the two scales in figure (b) are identical. Data are shown as mean ± standard deviation (n=20), difference significance calculated according to ANOVA, a, b letters indicate difference significance at p <0.05 level.

Detailed Description

The following detailed description of the application is provided in connection with the accompanying drawings that are presented to illustrate the application and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the application in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents, instruments and the like used in the examples described below are commercially available unless otherwise specified. The quantitative tests in the following examples were all set up in triplicate and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.

The pCBC-MT1T2 vector and the CRISPR/Cas9 vector pBUE411 in the examples described below are both described in the literature (Xing et al, A CRISPR/Cas9 toolkit for multiplex genome editing in plants, BMC Plant biol.2014Nov 29;14:327.doi:10.1186/s 12870-014-0327-y.), which is available to the public from the applicant for use only in the relevant experiments of the repeated application, and not as a further use.

The pMWB110 vector in the examples described below (Liu et al Efficient induction of haploid plants in wheat by editing of TaMTL using an optimized Agrobacterium-mediated CRISPR system, J Exp Bot.2020Feb 19;71 (4): 1337-1349.Doi:10.1093/jxb/erz 529.) the public has access to this biomaterial from the applicant, which is used only for repeated experiments in connection with the application and is not available for other uses.

Example 1,

The present example provides a group of genes capable of increasing thousand kernel weight and spike kernel number of wheat at the same time, which consists of three homologous genes located in A, B, D chromosome group respectively, which are High Grain Yield1 gene located on chromosome 7 of chromosome A (denoted HGY-A1), high Grain Yield1 gene located on chromosome 7 of chromosome B (denoted HGY-B1), and High Grain Yield1 gene located on chromosome 7 of chromosome D (denoted HGY-D1).

In a spring wheat variety Fielder, the genome sequence of the HGY-A1 gene is a sequence 1 in a sequence table, the CDS sequence of the gene is a sequence 2 in the sequence table, and the gene codes HGY-A1 protein shown in a sequence 3 in the sequence table; the genome sequence of the HGY-B1 gene is a sequence 4 in a sequence table, the CDS sequence of the HGY-B1 gene is a sequence 5 in the sequence table, and the HGY-B1 protein shown in a sequence 6 in the sequence table is encoded; the genome sequence of the HGY-D1 gene is sequence 7 in the sequence table, the CDS sequence of the HGY-D1 gene is sequence 8 in the sequence table, and the HGY-D1 protein shown in the sequence 9 in the sequence table is encoded.

The HGY-A1 gene, HGY-B1 gene and HGY-D1 gene functions were detected as follows:

1. construction of recombinant vectors

Construction of CRISPR/Cas 9-based gene editing vector:

two target sequences, namely a target sequence 1 and a target sequence 2, for gene editing by taking a sequence 10 and a sequence 11 in a sequence table as a CRISPR/Cas9 system are selected, and can be used for simultaneously editing HGY-A1 genes, HGY-B1 genes and HGY-D1 genes, wherein the target sequence 1 is 982-1000 th site of the sequence 1, 1096-1114 th site of the sequence 4, 1258-1276 th site of the sequence 7, 4539-4557 th site of the sequence 1, 4145-4163 th site of the sequence 4 and 4356-4374 th site of the sequence 7.

PCR amplification is carried out by using a pair of primers containing a target sequence 1 and a target sequence 2 and using a pCBC-MT1T2 vector as a template, an amplified product contains a fragment of a wheat U3 promoter, the obtained PCR product is digested by BsaI, the obtained digested product is connected with a vector skeleton obtained by BsaI digestion of a CRISPR/Cas9 vector pBUE411, and the obtained recombinant vector with the correct sequence is marked as an sgRNA/Cas9 vector. The sgRNA/Cas9 vector is capable of transcribing sgRNA1 and sgRNA2 that target sequence 1 and target sequence 2, respectively, in the wheat genome and expressing Cas9 protein. The sgRNA/Cas9 vector contains a DNA fragment shown in sequence 12, the 439-533 th site transcribes sgRNA1, and the 1350-1444 th site transcribes sgRNA2.

Construction of the over-expression vector:

the HGY-A1 gene shown in the sequence 2 in the sequence table is inserted between multiple cloning sites of the pMWB110 vector by utilizing BamHI, the obtained recombinant vector is marked as pMWB110-HGY-A1, and the pMWB110-HGY-A1 can express the HGY-A1 protein shown in the sequence 3 in the sequence table.

The HGY-D1 gene shown in the sequence 8 in the list is inserted between the multiple cloning sites of the pMWB110 vector by utilizing BamHI, the obtained recombinant vector is marked as pMWB110-HGY-D1, and the pMWB110-HGY-D1 can express the HGY-D1 protein shown in the sequence 9 in the sequence table.

2. Construction of transgenic plants and Gene knockout lines

And (2) introducing the sgRNA/Cas9 vector, the pMWB110-HGY-A1 and the pMWB110-HGY-D1 obtained in the step (1) into the agrobacterium tumefaciens EHA105 by using an agrobacterium tumefaciens mediated transformation method, and performing genetic transformation by using a spring wheat variety Filer as a receptor.

Identification of the knockout lines:

the inventors identified 20 bialaphos-resistant transgenic lines containing the sgRNA/Cas9 vector T-DNA from the sgRNA/Cas9 vector transformed wheat.

Detecting the DNA editing condition of the target sequence of the HGY-A1 gene in the transgenic strain by using a specific primer of the HGY-A1 gene, detecting the DNA editing condition of the target sequence of the HGY-B1 gene in the transgenic strain by using a specific primer of the HGY-B1 gene, and detecting the DNA editing condition of the target sequence of the HGY-D1 gene in the transgenic strain by using a specific primer of the HGY-D1 gene. Sequencing of the PCR products of the corresponding primers showed that 50% (10/20) of the T0 line had a complex heterozygous mutation at target sequence 2, whereas no mutation was detected at target sequence 1. The detection primers at target sequence 2 were as follows:

HGY-A1 gene: HGY1-seq-FA (5'-AGCAGCTGCTGATTTGCCA-3') and HGY1-seq-RAD (5'-CATGTAGTTACTCGCCATGATGGAG-3');

HGY-B1 gene: HGY1-seq-FB (5'-TCTCCTACCCAAGGGTTCCG-3') and HGY1-seq-RB (5'-ACTGGGGGCGGCATGTAG-3');

HGY-D1 gene: HGY1-seq-FD (5'-CGGTCAAGTTCCATTTGCTGAT-3') and HGY1-seq-RAD (5'-CATGTAGTTACTCGCCATGATGGAG-3').

Seeds of 10 mutant T0 lines with the composite heterozygous mutation at target sequence 2 were collected and planted in the greenhouse, and 10T 1 plants were selected for each T0 line to detect the mutation type at target sequence 2. Most commonly, 1-bp deletions and 1-bp insertional mutations are found at the target site. All mutations lead to frame shifts and to premature translation stop codons. To avoid the impact of potential off-target sites on phenotypic and functional assays, the inventors selected four most likely off-target sites (2-3 mismatches) and amplified and analyzed with specific primers. Sequencing results showed that there were no mutations in the potential off-target sites of all mutants.

One of the homozygous lines (T2 generation knockout line obtained by planting, designated KO-ABD) was selected for subsequent phenotypic identification, which had the following mutations in the HGY-A1 gene: a T is inserted between the 4554 th and 4555 th positions of the sequence 1; the HGY-B1 gene was mutated as follows: deletion of 5 nucleotides 4156-4160 of sequence 4; the HGY-D1 gene was mutated as follows: a C is inserted between the 4371 th and 4372 th positions of the sequence 7.

Identification of transgenic plants:

the inventor identifies transgenic condition of wheat transformed by pMWB110-HGY-A1 vector on RNA level, selects a T2 generation strain (namely over-expression line) from positive transgenic plants by taking spring wheat variety Fielder (WT) as a control for subsequent phenotype identification, marks the strain as OE-A, and the HGY-A1 gene expression condition of the strain is shown in figure 2, wherein the HGY-A1 gene expression quantity is obviously higher than that of the WT. The primers used for HGY-A1 gene using wheat actin gene as reference are as follows: f, ATGGAGATTGGCAGCGGC; r is CTACAGGGACCAGTTGGACGAG.

The inventor identifies transgenic condition on RNA level from wheat transformed by pMWB110-HGY-D1 vector, selects a T2 generation strain (namely over-expression line) from positive transgenic plants by taking spring wheat variety Fielder (WT) as a control for subsequent phenotype identification, marks the strain as OE-D, and the HGY-A1 gene expression condition of the strain is shown in figure 2, wherein the HGY-D1 gene expression quantity is obviously higher than that of the WT. The primers used for HGY-D1 gene using wheat actin gene as reference are as follows: f, ATGGAGATTGGCAGCGGC R and CTACAGAGACCAGTTGGACGAGC.

3. Phenotypic identification

Spring wheat varieties Fielder (WT), KO-ABD and OE-A, OE-D are planted in Beijing test fields of China national academy of agricultural sciences in natural growth seasons, and after the wheat is mature, main stem spikes of 20 plants are randomly selected for each plant line, and the number of spikes per spike and thousand seed weight are measured.

The results (FIG. 1) showed that the knockout lines KO-ABD of the three homologous genes had significantly reduced numbers of ears and thousand kernel weights, respectively, of 64.35.+ -. 4.66, 34.90.+ -. 3.65 and 41.93.+ -. 1.03g and 39.76.+ -. 1.06g, respectively, for the spring wheat variety Fielder and KO-ABD.

The results (FIG. 2) show that OE-A, OE-D had significantly increased numbers of grains per ear and thousand kernel weights, respectively, of 65.10.+ -. 4.33, 73.70.+ -. 3.65, 72.40.+ -. 4.79 and 41.00.+ -. 0.68g, 45.62.+ -. 0.79g and 44.61.+ -. 1.06g, respectively, for spring wheat variety Fielder and OE-A, OE-D, respectively.

In conclusion, the three homologous genes of the High Grain Yield1 and the protein encoded by the same can regulate the spike Grain number and thousand Grain weight of wheat.

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Sequence 1 (genomic sequence of HGY-A1 gene):

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sequence 2 (CDS sequence of HGY-A1 gene):

ATGGAGATTGGCAGCGGCGGCTGCGGCGGCGGCGACGGGAGCGGCGGAGGGGGCGGGGACGACCAGCAGCGCCACGGGCTCAAGTTCGGCAAGAAGATCTACTTCGAGGACAGCACGGGCCCCGGCGGTGGCAGTGGCGGCGTTAATGCGTCCTCGTCCAAGCCGCCCGCGGGCGGCGGGAGGAAGGGGAAGGCCGTGGCCGCCGGGGGAGCGTCCGGCTCCGCGCCGCCGCGGTGCCAGGTGGAGGGGTGCGGCGTGGATCTGAGCGGCGCCAAGCAATACCACTGCCGCCACAAGGTGTGCTCCATGCACACCAAGGAGCCCCGCGTCGTCGTCGCCGGCCTCGAGCAGCGCTTCTGCCAGCAGTGCAGCAGGTTCCACCAGTTGCCTGAATTCGATCAAGGAAAACGCAGCTGCCGCAGACGCCTCGCAGGCCACAACGAACGCCGGAGGAAGGCACCCCCCGGCCCTCTCGCAACACGCTACGGGCGACTCGCTGCATCCTTTGAAGAACCCGGCAGGTTCAGAAGCTACCTGCTGGATTTCTCGTACCCAAGAGTTCCGAGCAGCGTGCGGGATGCCTGGCCGGGGGCTCGACTAGGCTACCGGATGCCTGGTGAAATCCAGTGGCAAGGCAACCTAGACCTGCGTCCTCACACAGGTGCAGGCACGGGATACGGCCACCACCATGCATACAGCAGCCACGGCGGCTTCCCCGGCCCAGAGCTCCCTCCAGGTGGGTGTCTCGCAGGGGTCGCCGCCGACTCCAGCTGTGCTCTCTCTCTTCTGTCAACTCAGCCATGGGACAACACCCCCCACGGTGCCAGCCACGACCACCGGTCCGCGGGCTTCGATGGCCACCCTGTGGGAGTGTCACCCTCCATCATGGCGAGTAACTACATGCCGCCGCCGGCGAGCCCCTGGGGTGGCTCCCGGGGCCATGAAGGCGGCCGGAACGCGCCGCATCAGCAGCTGCCACATGACGTCCAGCTCCACGAGGTGCACCACCCAGCAGGCTCTAGCCAGCACGGCCACTTCTCAGGCGAGCTCGAGCTCGCCCTGCAGGGGAACAGGCCGGCGCCTGGGCCACGCGGCGGCGATCACGGCAGCAGTGGCGGCGCGTTCGACCACCCCGGCAGCTCGTCCAACTGGTCCCTGTAG

sequence 3 (HGY-A1 protein sequence):

MEIGSGGCGGGDGSGGGGGDDQQRHGLKFGKKIYFEDSTGPGGGSGGVNASSSKPPAGGGRKGKAVAAGGASGSAPPRCQVEGCGVDLSGAKQYHCRHKVCSMHTKEPRVVVAGLEQRFCQQCSRFHQLPEFDQGKRSCRRRLAGHNERRRKAPPGPLATRYGRLAASFEEPGRFRSYLLDFSYPRVPSSVRDAWPGARLGYRMPGEIQWQGNLDLRPHTGAGTGYGHHHAYSSHGGFPGPELPPGGCLAGVAADSSCALSLLSTQPWDNTPHGASHDHRSAGFDGHPVGVSPSIMASNYMPPPASPWGGSRGHEGGRNAPHQQLPHDVQLHEVHHPAGSSQHGHFSGELELALQGNRPAPGPRGGDHGSSGGAFDHPGSSSNWSL*

sequence 4 (genomic sequence of HGY-B1 gene):

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sequence 5 (CDS sequence of HGY-B1 gene):

ATGGAGATTGGCAGCGGCGGCTGCGACGGGAGCGGCGGCGACGGGAGCGGCGGAGGGGGCGGGGACGACCAGCAGCGCCACGGGCTCAAGTTCGGCAAGAAGATCTACTTCGAGGACGCCACAGGCACCGGCGGCGTCAATGCGTCCTCGTCCAAGCCGCCCGCCGGTGGCGGGAGGAAGGGGAAGGCCGTGGCCGCCGGGGGAGCGTCCGGCTCCGCGCCGCCGCGGTGCCAGGTGGAGGGGTGCGGCGTGGATCTGAGCGGCGCCAAGCAATACCATTCCCGCCACAAGGTGTGCTCCATGCACACCAAGGAACCCCGCGTCGTCGTCGCCGGCCTCGAGCAGCGCTTCTGCCAGCAGTGCAGCAGGTTCCACCAGTTGCCTGAATTCGATCAAGGAAAACGCAGCTGCCGCAGACGCCTCGCAGGCCACAACGAACGCCGGAGGAAGGCGCCCCCCGGCCCTCTCGCGTCACGCTACGGGCGACTCGCTGCATCCTTTGAAGAACCCGGCAGGTTCAGAAGCTACCTGCTGGATTTCTCCTACCCAAGGGTTCCGAGCAGCGTGCGGGATGCCTGGCCGGGGGCTCGACCAGGCTACCGGATGCCCGGTGAAATCCAGTGGCAAGGGAACCTAGACCTGCGTCCTCACACAGGTGCGGCCACGGGATACCACGGCCACCACGCATACAGCAGCCACGGCGGCGGCTTCCCCGGCCCAGAGCTCCCTCCAGGTGGGTGTCTCGCAGGGGTCGCCGCCGACTCCAGCTGTGCTCTCTCTCTTCTGTCAACTCAGCCATGGGACAATACCCCCCACGGTGCCAGCCACGACCATCGGTCCGCGGGCTTCGATGGCCACCCTGCGGGAGTGTCACCCTCCATCATGGCGAGTAACTACATGCCGCCCCCAGTGAGCCCCTGGGGTGGCTCCCGGGGCCATGAAGGCGGCCGCAACGCGCCACATCAGCAGCTGCCACATGACGTCGCGCTCCACGAGGTGCACCACCCTGCAGGCTCTAGCCAGCACGGCCACTTCTCAGGCGAGCTCGAGCTCGCCCTGCAGGGGAACAGGACGGCGCCTGGGCCACGCGGCGGCGGCGATCACAGCAGCGGTGGCGGGGCATTCGACCACCCCGGCAGCTCGTCCAACTGGTCTCTGTAG

sequence 6 (HGY-B1 protein sequence):

MEIGSGGCDGSGGDGSGGGGGDDQQRHGLKFGKKIYFEDATGTGGVNASSSKPPAGGGRKGKAVAAGGASGSAPPRCQVEGCGVDLSGAKQYHSRHKVCSMHTKEPRVVVAGLEQRFCQQCSRFHQLPEFDQGKRSCRRRLAGHNERRRKAPPGPLASRYGRLAASFEEPGRFRSYLLDFSYPRVPSSVRDAWPGARPGYRMPGEIQWQGNLDLRPHTGAATGYHGHHAYSSHGGGFPGPELPPGGCLAGVAADSSCALSLLSTQPWDNTPHGASHDHRSAGFDGHPAGVSPSIMASNYMPPPVSPWGGSRGHEGGRNAPHQQLPHDVALHEVHHPAGSSQHGHFSGELELALQGNRTAPGPRGGGDHSSGGGAFDHPGSSSNWSL*

sequence 7 (genomic sequence of HGY-D1 gene):

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sequence 8 (CDS sequence of HGY-D1 gene):

ATGGAGATTGGAAGCGGCGGCGACGGGAGTGGCGGAGGGGGCGGGGACGACCAGCAGCGCCACGGGCTCAAGTTCGGCAAGAAGATCTACTTCGAGGACGCCACGGGCACCGGCGGTGGCAGTGGCAGTGGCGGCGTCAATGCGTCCTTGTCCAAGCCGCCCGCGGGCAGCGGGAGGAAGGGGAAGGCCGTGGCCGCCGGGGGAGCGTCCGGCTCCGCGCCGCCGCGGTGCCAGGTGGAGGGGTGCGGCGTGGATCTGAGCGGCGCCAAGCAATACCACTCCCGCCACAAGGTGTGCTCCATGCACACCAAGGAACCCCGCGTCGTCGTCGCCGGCCTCGAGCAGCGCTTCTGCCAGCAGTGCAGCAGGTTCCACCAGTTGCCTGAATTTGATCAAGGAAAACGCAGCTGCCGCAGACGCCTCGCAGGCCACAACGAACGCCGGAGGAAGGCGCCCCCCGGCCCTCTCGCGACACGCTACGGGCGACTCGCCGCATCCTTTGAACCCGGCAGGTTCAGAAGCTACCTGCTGGATTTCTCGTACCCAAGGGTTCCGAGCAGCGTGCGGGATGCCTGGCCGGGCGCTCGACCAGGCTACCGGATGCCCGGTGAAATCCAGTGGCAAGGCAACCTAGACCTGCGTCCTCACACAGGTGCGGCCACGGGATACCACGGCCACCACGCATACAGCAGCCACGGTGGCGGCTTCCCCGGCCCAGAGCTCCCTCCAAGTGGGTGTCTCGCAGGGGTCGCCGCCGACTCCAGCTGTGCTCTCTCTCTTCTGTCAACTCAGCCATGGGACAACACCCCCCACGGTGCCAGCCACGACCACCGGTCCGCGGGCTTCGATGGCCACCCTGTGGGAGTGTCACCCTCCATCATGGCGAGTAACTACATGCCGCCGCCGGCGAGCCCCTGGGGTGGCTCCCGGGGCCATGAAGGCGGCCGGAACGCGCCACATCAGCAGCTGCCACATGACGTCCCGCTCCACGAGGCGCACCACCCTGCAGGCTCTAGCCAGCACGGCCACTTCTCAGGCGAGCTCGAGCTCGCTCTGCAGGGGAACAGGCCGGCGCCTGGGCCACGCGGCGGCGATCACAGCAGCGGTGGCGGCGCGTTCGACCACCCCGGCAGCTCGTCCAACTGGTCTCTGTAG

sequence 9 (HGY-D1 protein sequence):

MEIGSGGDGSGGGGGDDQQRHGLKFGKKIYFEDATGTGGGSGSGGVNASLSKPPAGSGRKGKAVAAGGASGSAPPRCQVEGCGVDLSGAKQYHSRHKVCSMHTKEPRVVVAGLEQRFCQQCSRFHQLPEFDQGKRSCRRRLAGHNERRRKAPPGPLATRYGRLAASFEPGRFRSYLLDFSYPRVPSSVRDAWPGARPGYRMPGEIQWQGNLDLRPHTGAATGYHGHHAYSSHGGGFPGPELPPSGCLAGVAADSSCALSLLSTQPWDNTPHGASHDHRSAGFDGHPVGVSPSIMASNYMPPPASPWGGSRGHEGGRNAPHQQLPHDVPLHEAHHPAGSSQHGHFSGELELALQGNRPAPGPRGGDHSSGGGAFDHPGSSSNWSL*

sequence 10:

GGTGCGGCGTGGATCTGAG

sequence 11:

TAGACCTGCGTCCTCACAC

sequence 12:

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Claims

1. any of the following uses of a protein or a substance that modulates the activity or content of said protein:

d1 Regulating plant yield;

d2 Preparing a product for regulating and controlling plant yield;

d3 Increasing plant yield;

d4 Preparing a product for increasing plant yield;

d5 Cultivating the yield-enhancing plant;

d6 Preparing a plant product with improved cultivation yield;

the protein is A1), A2) or A3) as follows:

a1 Amino acid sequence is a protein of sequence 3, sequence 6 or sequence 9;

2. The use according to claim 1, characterized in that: the substance is any one of the following B1) to B9):

b1 A nucleic acid molecule encoding the protein of claim 1;

b2 An expression cassette comprising the nucleic acid molecule of B1);

b8 A nucleic acid molecule which reduces the expression of the protein of claim 1;

3. The use according to claim 2, characterized in that: b1 The nucleic acid molecule is b 11) or b 12) or b 13) or b 14) or b 15) as follows:

b14 A cDNA molecule or DNA molecule having 75% or more identity to the nucleotide sequence defined in b 11) or b 12) or b 13) and encoding the protein according to claim 1;

b15 A cDNA molecule or DNA molecule which hybridizes under stringent conditions to a nucleotide sequence as defined in b 11) or b 12) or b 13) or b 14) and which codes for a protein according to claim 1.

4. A use according to any one of claims 1-3, characterized in that: the yield is manifested in spike and grain number and/or grain weight.

5. Use according to any one of claims 1-4, characterized in that: the plant is M1) or M2) or M3):

m1) monocotyledonous or dicotyledonous plants;

m2) a gramineous plant;

m3) wheat.

6. The method comprises the following steps:

x1) a method of growing a yield-increasing plant comprising allowing expression of a protein according to claim 1 in a recipient plant or increasing the amount or activity of a protein according to claim 1 in a recipient plant to yield a plant of interest having increased yield compared to said recipient plant;

x1) a method for increasing plant yield comprising allowing expression of a protein according to claim 1 in a recipient plant or increasing the amount or activity of a protein according to claim 1 in a recipient plant to obtain a plant of interest with increased yield as compared to said recipient plant, resulting in increased plant yield.

7. The method according to claim 6, wherein: the methods of X1) and X2) are carried out by introducing into the recipient plant a gene encoding the protein of claim 1 and allowing the gene to be expressed.

8. A product for increasing plant yield comprising a protein according to any one of claims 1 to 3 or said substance.

9. The method according to claim 6 or 7, or the product according to claim 8, characterized in that: the yield is expressed in terms of spike and grain number and/or grain weight;

and/or, the plant is M1) or M2) or M3):

m1) monocotyledonous or dicotyledonous plants;

m2) a gramineous plant;

m3) wheat.

10. A protein or said substance according to any one of claims 1-3.