Wheat grain weight gene WGY1 as well as encoded protein and application thereof

Abstract

The invention discloses a wheat grain weight gene WGY1 as well as an encoded protein and application thereof, and belongs to the technical field of biological breeding. The technical problem to be solved by the invention is how to regulate and control the grain weight of wheat. To this end, the present application provides proteins and related biomaterials that modulate plant yield. The invention also provides application of the protein and related biological materials thereof in regulation and control of plant yield. According to the application, the WGY1 gene is finally obtained through gene localization and map-based cloning. The gene and the protein coded by the gene can be widely applied to the plant fields of wheat genetic breeding, germplasm resource improvement, transgenosis, genome editing breeding and the like, and play an important role in improving germplasm resources of crops such as wheat and the like.

Description

Technical Field

[0001] This application belongs to the field of biological breeding technology, specifically involving the wheat grain weight gene WGY1 and its encoded protein and its applications. Background Technology

[0002] Wheat (Triticum aestivum L.), as one of the major food crops, meets the needs of approximately 40% of the world's population. my country is the world's largest producer and consumer of wheat, holding a significant position in the global wheat market. Developing high-yield and high-quality varieties to increase wheat production helps ensure national food security and promotes sustainable agricultural development. Thousand-grain weight is one of the three key factors in wheat yield, highly influenced by genetic characteristics and less affected by environmental factors, indicating significant potential for improvement. Cloning genes controlling thousand-grain weight, identifying superior allelic variations, and elucidating their regulatory mechanisms will help elucidate the genetic and molecular mechanisms of yield formation, which is of great significance for high-yield wheat breeding.

[0003] Wheat grain weight is primarily determined by grain size and density, and is a quantitative trait (QTL) controlled by multiple genes. These genes regulate grain shape and density through their involvement in grain formation and grain filling, thus affecting grain weight. For many years, scientists both domestically and internationally have conducted extensive research on the mapping of QTLs related to wheat grain weight genes. Mapped wheat grain weight QTLs are distributed across all 21 chromosomes of the wheat genome. Although the initial mapping of most QTL loci was completed relatively early, the conversion from mapped genes to cloned genes has been extremely difficult. To date, only a few grain weight-related QTL loci have been finely mapped. Currently reported cloned wheat thousand-grain weight genes are mostly obtained through homologous cloning techniques based on grain weight genes already cloned in rice and Arabidopsis thaliana. Summary of the Invention

[0004] The technical problem this application aims to solve is: how to increase plant yield, for example, how to increase wheat yield.

[0005] Therefore, this application provides a protein, which may be any of the following proteins:

[0006] A1) The amino acid sequence is that of the protein shown in SEQ ID No. 3 or SEQ ID No. 6;

[0007] A2) Proteins obtained by substituting and / or deleting and / or adding amino acid residues to the amino acid sequence shown in A1) that have more than 80% identity with the amino acid sequence shown in A1) and are related to plant yield.

[0008] A3) is a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of A1) or A2).

[0009] Furthermore, the protein is derived from wheat.

[0010] SEQ ID No. 3 and SEQ ID No. 6 each consist of 245 amino acid sequences. The protein with the amino acid sequence of SEQ ID No. 3 is named WGY1. BN Protein. The protein with the amino acid sequence of SEQ ID No. 3 is named WGY1. YD protein.

[0011] The proteins mentioned above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0012] The connection described in A3) can be made via peptide bonds.

[0013] The protein tag refers to a polypeptide or protein fused with a target protein using in vitro DNA recombination technology for expression, detection, tracing, and / or purification of the target protein. The protein tag may be a Flag protein tag, His protein tag, MBP protein tag, HA protein tag, myc protein tag, GST protein tag, and / or SUMO protein tag, etc.

[0014] This application also provides biomaterials related to the above-mentioned proteins, said biomaterials may be any of the following:

[0015] B1) Nucleic acid molecules that encode the above proteins;

[0016] B2), an expression cassette containing the nucleic acid molecule described in B1);

[0017] B3) A recombinant vector containing the nucleic acid molecule described in B1) or a recombinant vector containing the expression cassette described in B2);

[0018] B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3);

[0019] B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2), or a transgenic plant cell line containing the recombinant vector described in B3);

[0020] B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2), or transgenic plant tissue containing the recombinant vector described in B3);

[0021] B7) Transgenic plant organs containing the nucleic acid molecules described in B1), or transgenic plant organs containing the expression cassette described in B2), or transgenic plant organs containing the recombinant vector described in B3).

[0022] Furthermore, in the aforementioned application, the expression cassette described in B2) refers to a cassette capable of expressing the WGY1 in host cells. BN Protein and / or WGY1 YD The protein's DNA, which may include the initiator WGY1 BN Protein and / or WGY1 YD The promoter of protein-coding gene transcription may also include a terminator that terminates WGY1. BN Protein and / or WGY1 YD Terminator and / or enhancer sequences of protein-coding gene transcription.

[0023] In one embodiment of this application, the recombinant vector described in B3) may be an overexpression vector pUbi::WGY1. BN -OE and / or overexpression vector pUbi::WGY1 YD -OE.

[0024] Overexpression vector pUbi::WGY1 BN The structure of -OE is as follows: the fragment between the BamHI and SacI restriction sites of the overexpression vector LGY-OE3 is replaced with the full-length CDS sequence of the WGY1 gene from the parental parent WGY1. BN -CDS (nucleotide sequence of the DNA molecule shown in SEQ ID No. 2), and keeping the other sequences of the LGY-OE3 vector unchanged, the overexpression vector pUbi::WGY1 was obtained. BN -OE. Overexpression vector pUbi::WGY1 BN In the OE, the transcription and translation of DNA molecules driven by the maize Ubiquitin promoter is the nucleotide sequence SEQ ID No. 2, and the expressible amino acid sequence is WGY1 of SEQ ID No. 3. BN protein.

[0025] Overexpression vector pUbi::WGY1 YD The structure of -OE is as follows: the fragment between the BamHI and SacI restriction sites of the overexpression vector LGY-OE3 is replaced with the full-length CDS sequence of the WGY1 gene from the Yanda 1817 parent (WGY1). YD -CDS (nucleotide sequence of the DNA molecule shown in SEQ ID No. 5), and keeping the other sequences of the LGY-OE3 vector unchanged, the overexpression vector pUbi::WGY1 was obtained. YD -OE. Overexpression vector pUbi::WGY1YD In the OE, the transcription and translation of DNA molecules driven by the maize Ubiquitin promoter is the nucleotide sequence of SEQ ID No. 5, and the expressible amino acid sequence is WGY1 of SEQ ID No. 6. YD protein.

[0026] Furthermore, in the aforementioned applications, the recombinant microorganisms described in B4) can specifically be yeast, bacteria, algae, and fungi.

[0027] Furthermore, in the aforementioned applications, the plant tissue described in B6) may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos, and anthers.

[0028] Furthermore, in the aforementioned applications, the transgenic plant organs described in B7) can be the roots, stems, leaves, flowers, fruits, and seeds of the transgenic plant.

[0029] Furthermore, in the aforementioned applications, the transgenic plant cell lines, transgenic plant tissues, and transgenic plant organs may or may not include propagation material.

[0030] Furthermore, in the aforementioned biological material, the nucleic acid molecule in B1) may be a DNA molecule as described in any of the following:

[0031] g1) The coding sequence (CDS) of the coding strand is the DNA molecule of SEQ ID No. 2;

[0032] g2) The nucleotide sequence (genomic gene sequence) of the coding strand is the DNA molecule of SEQ ID No. 1;

[0033] g3), DNA molecules that have more than 80% identity with the DNA molecules described in g1) or g2), and that regulate plant yield;

[0034] g4) The coding sequence (CDS) of the coding strand is the DNA molecule of SEQ ID No. 5;

[0035] g5) The nucleotide sequence (genomic gene sequence) of the coding strand is the DNA molecule of SEQ ID No. 4;

[0036] g6) is a DNA molecule that has more than 80% identity with the DNA molecules described in g4) or g5) and regulates plant yield.

[0037] This application also provides the use of the above-mentioned biomaterials in any of the following:

[0038] C1) Application in regulating plant yield;

[0039] C2) Its application in the preparation of products that regulate plant yield;

[0040] C3) Application in regulating plant grain weight;

[0041] C4) Application in the preparation of products that regulate the weight of plant seeds;

[0042] C5) Application in regulating plant seed size;

[0043] C6) Application in the preparation of products that regulate plant weight;

[0044] C7) Applications in plant breeding or plant-assisted breeding;

[0045] C8) Application in the preparation of plant breeding or plant-assisted breeding products.

[0046] Furthermore, in the aforementioned application, the plant may be selected from monocotyledonous plants.

[0047] Furthermore, in the aforementioned application, the monocotyledonous plant may be selected from grasses.

[0048] Furthermore, in the aforementioned application, the grass plant may be selected from wheat plants.

[0049] Furthermore, in the aforementioned application, the wheat plant may be selected from wheat (Triticum aestivum L.).

[0050] Furthermore, in the aforementioned applications, the regulation may be to increase, promote, or upregulate.

[0051] Furthermore, in the aforementioned applications, the regulation may also be a reduction, suppression, or downregulation.

[0052] Furthermore, in the aforementioned applications, the indicators for plant breeding may include plant yield.

[0053] Furthermore, in the aforementioned applications, the purpose of plant breeding may include cultivating plants with increased yields.

[0054] Furthermore, the plant yield can be the economic yield of the plant, that is, the amount of product harvested for the purpose of cultivation.

[0055] In this application, the yield may be the amount of wheat seeds harvested.

[0056] In this application, the weight of the plant seeds can be evaluated by the numerical values ​​of the weight of 100 seeds or the weight of 1000 seeds.

[0057] In this application, the size of the plant seeds can be evaluated by seed phenotypes such as seed length (GL), seed width (GW), and seed thickness (GT).

[0058] This application also provides a method for regulating plant yield, the method comprising regulating the aforementioned protein (WGY1) in the recipient plant. BN Protein and / or WGY1 YD The expression level of the gene encoding the protein or the activity or content of the protein can regulate plant yield.

[0059] Furthermore, the method includes introducing the gene encoding the aforementioned protein into the recipient plant to reduce the yield of the recipient plant.

[0060] Furthermore, in the method, the encoding gene may be any of the following:

[0061] g1) The coding sequence of the coding strand is the DNA molecule of SEQ ID No. 2;

[0062] g2) The nucleotide sequence of the coding strand is the DNA molecule of SEQ ID No. 1;

[0063] g3), DNA molecules that have more than 80% identity with the DNA molecules described in g1) or g2), and that regulate plant yield;

[0064] g4) The coding sequence of the coding strand is the DNA molecule of SEQ ID No. 5;

[0065] g5) The nucleotide sequence of the coding strand is the DNA molecule of SEQ ID No. 4;

[0066] g6) is a DNA molecule that has more than 80% identity with the DNA molecules described in g4) or g5) and regulates plant yield.

[0067] Furthermore, in the method, the plant may be selected from monocotyledonous plants.

[0068] Furthermore, in the method, the monocotyledonous plant may be selected from grasses.

[0069] Furthermore, in the method, the grass plant can be selected from wheat plants.

[0070] Furthermore, in the method, the wheat plant may be selected from wheat (Triticum aestivum L.).

[0071] This application also provides a method for plant breeding, the method comprising regulating the above-mentioned protein (WGY1) in the recipient plant. BN Protein and / or WGY1 YD The expression level of the gene encoding the protein or the activity or content of the protein can be used to obtain plants with increased yield.

[0072] Furthermore, in the method described, the regulation may be to increase, promote, or upregulate.

[0073] Furthermore, in the method described, the regulation may also be a reduction, suppression, or downregulation.

[0074] Furthermore, in the method, the indicators for plant breeding may include plant yield.

[0075] Furthermore, in the method, the purpose of plant breeding may include cultivating plants with increased yields.

[0076] Furthermore, the plant yield can be the economic yield of the plant, that is, the amount of product harvested for the purpose of cultivation.

[0077] Furthermore, the method may include reducing the expression level of the gene encoding the protein or the activity or content of the protein in the recipient plant to obtain a plant with increased yield.

[0078] This application also provides a DNA molecule, wherein the nucleotide sequence of the DNA molecule may be any of the following:

[0079] D1) The nucleotide sequence of the coding strand is the DNA molecule of SEQ ID No. 1;

[0080] D2) The nucleotide sequence of the coding strand is the DNA molecule of SEQ ID No. 4;

[0081] D3) The nucleotide sequence of the coding strand is the DNA molecule of SEQ ID No. 7;

[0082] D4) The nucleotide sequence of the coding strand is the DNA molecule of SEQ ID No. 8;

[0083] D5) The nucleotide sequence of the coding strand is the DNA molecule of SEQ ID No. 9;

[0084] D6) The nucleotide sequence of the coding strand is the DNA molecule of SEQ ID No. 10.

[0085] In this application, identity refers to the identity of amino acid sequences or nucleotide sequences. The identity of amino acid sequences (or nucleotide sequences) can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences, the identity value (%) can be obtained.

[0086] The aforementioned 80% or higher degree of identity can be 80%, 85%, 90%, or 95% or higher degree of identity.

[0087] The 80% or more identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The 85% or more identity can be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The 90% or more identity can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The 95% or higher level of identity can be at least 95%, 96%, 97%, 98%, or 99% identity.

[0088] The beneficial technical effects achieved by this application are as follows:

[0089] This application obtained the WGY1 gene through gene localization and map-based cloning. This gene has a negative regulatory effect on the thousand-grain weight of wheat. This gene and its superior alleles can be widely used in plant fields such as high-yield wheat genetic breeding, germplasm resource improvement, and genome editing breeding, and play an important role in improving and enhancing the germplasm resources of crops such as wheat. Attached Figure Description

[0090] Figure 1 Fine mapping of the QTgw-6B locus. (a) Preliminary mapping of the QTgw-6B locus, (b) Genotypes of recombinant families in the target region, (c) Physical map of the Chinese spring reference genome.

[0091] Figure 2Differential expression analysis of genes annotated within the QTgw-6B localization region. (a) Cluster analysis of gene expression within the QTgw-6B localization region. (b) Differential expression analysis of the QTgw-6B candidate gene WGY1 among parents. Values ​​represent mean ± SEM (n=3), and four asterisks indicate highly significant differences at the Tukey test level (α(0.05)). 7DBF, 7 days before flowering; F, flowering period; 7DAF-28DAF, 7-28 days after flowering.

[0092] Figure 3 For WGY gene sequence analysis. (a) Correspondence and coding region sequence differences of the WGY tandem repeat gene among the Chinese Spring reference genome (CS), YD1817, and BN6. (b) Differences in WGY1 protein sequence between large-particle and small-particle parents. Figure 4 Haplotype analysis of the WGY1 gene. (a) Association analysis between different WGY1 haplotypes and thousand-grain weight phenotypes in microcore germplasm. Box plots of different colors represent the thousand-grain weight composition of different haplotype varieties, and the size of the box plot represents the dispersion of thousand-grain weight among varieties of the same haplotype. Values ​​represent mean ± SEM, and the statistical quantity is the number of varieties for each haplotype. Ns indicates no significant difference, and 1-4 asterisks indicate Tukey's test at α. (0.05) Significant differences in level (b) Specific detection markers for haplotype Hap V (the first 7 samples represent other haplotype compositions, and the last 9 samples represent Hap V type alleles).

[0093] Figure 5 This study analyzed gene expression levels and phenotypes in the T1 generation of the WGY1 transgenic wheat. (a) Gene expression analysis of the T1 generation of the WGY1 transgenic wheat. (b) Thousand-grain weight analysis of the T1 generation of the WGY1 transgenic wheat. Values ​​in Figures (a) and (b) represent mean ± SEM (n=3), with "+" representing positive plants and "-" representing negative plants. (c) Significance analysis of differences in mean wheat expression levels among the T1 generation groups of the WGY1 transgenic wheat. (d) Significance analysis of differences in thousand-grain weight of the T1 generation of the WGY1 transgenic wheat. Values ​​in Figures (c) and (d) represent mean ± SEM, and the statistical count is the number of T1 generation plants obtained in each transgenic event. ns indicates no significant difference, and 1-4 asterisks represent Tukey's test at α. (0.05) The differences in levels are significant. WGY1 FD This serves as a blank control. Detailed Implementation

[0094] The present application will now be described in further detail with reference to specific embodiments. The embodiments given are merely illustrative of the present application and are not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the present application in any way.

[0095] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0096] The overexpression vector LGY-OE3 was kindly provided by Professor Li Genying's research group at the Shandong Academy of Agricultural Sciences and is published in the literature "Niaz et al. (2023) Identification of TaGL1-B1 gene controlling grain length through regulation of jasmonic acid in common wheat. Plant Biotechnology Journal. 21: 979-989". The public can obtain the above-mentioned biological material from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. The obtained biological material is only for repeating the experiments of this application and may not be used for other purposes.

[0097] The transgenic recipient material Fielder is preserved in our laboratory and published in the literature "Lu et al., A rare gain of function mutation in a wheat tandem kinase confers resistance to powderymildew. Nature Communications, 2020, 11, 680". The public can obtain the above-mentioned biological material from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. The obtained biological material is only for repeating the experiments of this application and may not be used for other purposes.

[0098] Unless otherwise specified, the quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0099] Example 1: The role of the WGY1 gene in regulating wheat grain weight

[0100] 1.1. Fine mapping of the WGY1 gene

[0101] In our previous work, using the small-grained local variety Yanda 1817 (YD1817) and the large-grained high-yield line Beinong 6 (BN6), our laboratory constructed recombinant inbred lines and SNP genetic linkage maps, locating a major QTL locus, QTgw-6B, controlling thousand-grain weight in wheat on the short arm of chromosome 6BS. Based on this, using the Chinese spring genome as a reference sequence, we developed molecular markers, obtaining 38 molecular markers linked to QTgw-6B (rows 2-39 in Table 1). These markers cover approximately 32.43 Mb of the Chinese spring reference genome. Based on the QTgw-6B mapping results, we analyzed the genotype data of the recombinant inbred line population. The families BY92 and BY93, containing only the major QTL QTgw-6B, were selected as donor parents, and the family BY210, containing no known major QTL locus for grain weight, was selected as the recurrent parent. These were then hybridized and backcrossed for multiple generations to obtain a high-generation segregating population. Recombinant plants and families were screened from the BC4F2, BC4F3, and BC4F4 populations using molecular markers Xzp607 and TB3, yielding 765 recombinant plants. These plants and families were then planted in the field to obtain hybrid progeny families, and the thousand-grain weight of homozygous recombinant scaffolds was measured. Using phenotypic data from these 765 homozygous recombinant families combined with the developed molecular markers, QTgw-6B was mapped to a genetic region of approximately 0.15 cM between molecular markers M1407 and M1702, corresponding to a 4.2 Mb physical region in the Chinese spring reference genome, and annotated to 25 genes. Figure 1 ).

[0102] 1.2. Cloning of the WGY1 gene

[0103] Throughout the entire panicle development process (from the booting stage to the grain-filling stage), panicle samples were taken from the parents Yanda 1817 and Beinong 6 at one-week intervals: one week before flowering, during flowering, and on days 7, 14, 21, and 28 after flowering. On day 25 after flowering, panicle samples were taken from the donor parents BY92 and BY93, and the recurrent parent BY210. Three biological replicates were set up for each sample at each time point for transcriptome sequencing analysis. A candidate region was identified with a gene, TraesCS6B01G140200, encoding a cysteine ​​protease, which showed significantly differential expression between high-grain-weight and low-grain-weight parents. This gene was highly expressed in the low-grain-weight parent Yanda 1817 throughout panicle development, but its expression level was low in the high-grain-weight parent Beinong 6 throughout panicle development. Figure 2 It was expressed at low levels in the donor parents BY92 and BY93 25 days after flowering, but at high levels in the recurrent parent BY210. As a candidate gene for QTgw-6B, it was named WGY1 (Wheat grainyield 1).

[0104] Analysis of the *Spring* genome sequence in China revealed three genes with identical coding regions located adjacent to each other on the chromosome, forming a tandem repeat gene group. The other two genes adjacent to WGY1 were named WGY2 and WGY3. Using primer design and PCR amplification, the three tandem repeat genes WGY1 from the low-grain-weight parent *Yanda 1817* were obtained. YD WGY2 YD WGY3 YD WGY1 from the high-grain-weight parent Beinong 6 BN WGY2 BN and WGY3 BN Analysis of the transcriptome sequencing data in this study revealed that only WGY1 was found in Beinong 6. BN Gene expression, while WGY2 BN and WGY3 BN In contrast, in Yanda 1817, the coding sequences of the three tandem repeat copies were completely identical, failing to distinguish them at the transcriptome level. Primers were designed using the *Cyclocarya paliurus* genome sequence as a reference sequence. Using cDNA obtained from reverse transcription of total RNA from both low-grain-weight and high-grain-weight parents as templates, 3'-RACE and 5'-RACE cloning were performed to obtain two alleles of the candidate gene WGY1 (WGY1...). YD and WGY1 BN The full-length CDS sequence of WGY1 was obtained. Experimental results showed that the WGY1 gene exhibits a splicing pattern, consisting of two exons and one intron. Sequence alignment revealed that WGY2... YD WGY3 YD WGY2 BN and WGY3 BN Four genes and WGY1 YD The sequences are consistent, while WGY1 BN With WGY1 YD There are 3 SNPs in the intron region and 5 SNPs in the coding region, and all 5 SNP variations in the coding region can lead to changes in amino acids. Figure 3 ).

[0105] WGY1 genotype BN The genomic sequence of WGY1 is SEQ ID No. 1, where positions 1-247 and 1040-1530 are exons. BN The coding sequence (CDS) is SEQ ID No. 2, and the encoded protein is named WGY1. BN Protein, WGY1 BN The amino acid sequence of the protein is SEQ ID No. 3.

[0106] WGY1 genotypeYD The genomic sequence of WGY1 is SEQ ID No. 4, where positions 1-247 and 1041-1531 are exons. YD The coding sequence (CDS) is SEQ ID No. 5, and the encoded protein is named WGY1. YD Protein, WGY1 YD The amino acid sequence of the protein is SEQ ID No. 6.

[0107] The alleles of the QTgw-6B candidate gene WGY1 in different varieties (lines) were amplified using 262 wheat microcore germplasms, and the allelic variation of this gene was analyzed based on the sequencing results. The results showed that six major haplotypes (Hap I-Hap VI) existed in the 262 wheat microcore germplasms. Among them, 47 varieties (lines) were identical to the genotype of Yanda 1817 and were named Hap I; 13 varieties (lines) expressed the haplotype of Beinong 6 and were named Hap II (this gene is identical to WGY1). BN The genomic gene sequences are identical to those of WGY1 in wheat microcore germplasm. The remaining haplotypes Hap III, Hap IV, Hap V, and Hap VI are alleles different from those of Yanda 1817 and Beinong 6, accounting for 4.96%, 47.71%, 3.44%, and 8.4% of the MCC, respectively. Haplotype Hap V, except for an additional 163bp insertion in the intron region, has identical sequence variation sites to haplotype Hap IV. Association analysis using thousand-grain weight data from wheat microcore germplasm in 2001-2002, 2004-2005, 2009-2010, 2017-2018, and 2018-2019 with different haplotypes of the WGY1 gene showed that the thousand-grain weight of haplotype Hap II was significantly higher than that of haplotype Hap I, indicating a close correlation between the WGY1 gene and the thousand-grain weight phenotype. Figure 4 Furthermore, haplotype Hap V exhibits a significant synergistic effect on thousand-grain weight, and given its low application rate in various varieties, it represents a novel and superior haplotype. Based on the specific 163bp short fragment insertion in haplotype Hap V, a specific molecular marker, HapV-1 (Table 1), was developed. Figure 4 It can be used to detect superior alleles of WGY1 of the Hap V type.

[0108] 1.3. Functional verification of the WGY1 gene

[0109] To verify the functionality of WGY1, WGY1 was built separately. YD and WGY1 BNTransgenic overexpression vector. The full-length CDS sequences of the WGY1 gene from the two parental cultivars, Beinong 6 and Yanda 1817, were amplified and ligated into the maize Ubiquitin promoter-driven overexpression vector LGY-OE3 using a one-step ligation method, constructing the overexpression vector pUbi::WGY1 for two haplotypes of WGY1. BN -OE and pUbi::WGY1 YD -OE.

[0110] Overexpression vector pUbi::WGY1 BN The structure of -OE is as follows: the fragment between the BamHI and SacI restriction sites of the overexpression vector LGY-OE3 is replaced with the full-length CDS sequence of the WGY1 gene from the parental parent WGY1. BN -CDS (nucleotide sequence of the DNA molecule shown in SEQ ID No. 2), and keeping the other sequences of the LGY-OE3 vector unchanged, the overexpression vector pUbi::WGY1 was obtained. BN -OE. Overexpression vector pUbi::WGY1 BN In the OE, the transcription and translation of DNA molecules driven by the maize Ubiquitin promoter is the nucleotide sequence SEQ ID No. 2, and the expressible amino acid sequence is WGY1 of SEQ ID No. 3. BN protein.

[0111] Overexpression vector pUbi::WGY1 YD The structure of -OE is as follows: the fragment between the BamHI and SacI restriction sites of the overexpression vector LGY-OE3 is replaced with the full-length CDS sequence of the WGY1 gene from the Yanda 1817 parent (WGY1). YD -CDS (nucleotide sequence of the DNA molecule shown in SEQ ID No. 5), and keeping the other sequences of the LGY-OE3 vector unchanged, the overexpression vector pUbi::WGY1 was obtained. YD -OE. Overexpression vector pUbi::WGY1 YD In the OE, the transcription and translation of DNA molecules driven by the maize Ubiquitin promoter is the nucleotide sequence of SEQ ID No. 5, and the expressible amino acid sequence is WGY1 of SEQ ID No. 6. YD protein.

[0112] After verification, the overexpression vector pUbi::WGY1 was transformed using Agrobacterium-mediated genetic transformation. BN -OE or pUbi::WGY1 YD-OE was introduced into callus tissue of the recipient wheat variety Fielder. Seedlings differentiated from the callus tissue were transferred to growth selection medium and cultured under light at 22-24℃. Transgenic pUbi::WGY1 was obtained. BN -OE or pUbi::WGY1 YD -OE regenerated wheat plants (transferred from WGY1) BD or WGY1 YD The T0 generation transgenic plants were transplanted into flowerpots (20cm × 20cm) and placed in an artificial climate chamber for growth. The artificial climate chamber was maintained at 25℃ / 18℃ (16 hours light / 8 hours dark, 65% relative humidity) for 16 hours of light / 8 hours of darkness. DNA was extracted from the T0 generation transgenic wheat plants. Using the DNA as a template, PCR amplification was performed using the detection primer LGY-OE3-1 to express the recombinant expression plasmid pUbi::WGY1. BN -OE or pUbi::WGY1 YD -OE was used as a positive control, and wheat Fielder DNA was used as a negative control for PCR amplification. The PCR reaction program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 10 min. PCR products were detected by 1% agarose gel electrophoresis, photographed under UV light, and the results were recorded. Four T0 generation transgenic positive plants were obtained by PCR detection and named WGY1. BN -OE-1、WGY1 YD -OE-1、WGY1 YD -OE-2 and WGY1 YD -OE-3.

[0113] T0 generation transgenic positive plants were self-crossed to obtain T1 generation transgenic plants. DNA was extracted from the T1 generation transgenic wheat plants. Using the DNA as a template, PCR amplification was performed using primers LGY-OE3-1 to screen for transgenic positive plants (referring to the screening method for T0 generation transgenic positive plants). A total of plants were obtained as follows, where positive plants (Positive) represent transgenic positive events and negative plants (Negative) represent transgenic negative events.

[0114] WGY1 YD The positive plants of the T1 generation wheat obtained by self-pollination of -OE-1 were numbered Y1-1, Y1-2, Y1-3, Y1-4, Y1-6, Y1-7, Y1-8, Y1-10, Y1-11, and Y1-12, respectively; the negative plants were Y1-5 and Y1-9.

[0115] WGY1 YDThe positive plants of the T1 generation wheat obtained by self-pollination of -OE-2 were numbered Y2-3 and Y2-4, respectively; the negative plants were numbered Y2-1 and Y2-2, respectively.

[0116] WGY1 YD The positive plants of the T1 generation wheat obtained by self-pollination of -OE-3 were numbered Y3-1, Y3-3, Y3-4, and Y3-5, respectively; the negative plants were numbered Y3-2.

[0117] WGY1 BN The positive plants of the T1 generation wheat obtained by self-pollination of -OE-1 were numbered B1-1, B1-2, B1-3, B1-5, and B1-6, respectively; the negative plants were numbered B1-4.

[0118] The T1 generation wheat plants were planted in flowerpots and then transferred to a greenhouse for growth. After harvest, each T1 generation wheat plant was phenotypically identified, with the wheat variety Fielder serving as a control.

[0119] Gene expression in the T1 generation transgenic single plant WGY1 was analyzed by qRT-PCR, with TaACTIN (TraesCS5B01G124100) as the internal reference gene and upstream and downstream primers of Rt1 as amplification primers.

[0120] The specific steps of qRT-PCR: Real-time quantitative PCR reaction is performed using a Roche Light Cycler 480 II and TB PCR instrument. Premix Ex Taq TM The reaction was performed using fluorescent dye II. A 15 μL reaction mixture was prepared on ice (cDNA, 2 μL; 2×TB Green Premix Ex Taq II, 7.5 μL; qRT-F primer (10 μM), 0.25 μL; qRT-R primer (10 μM), 0.25 μL; ddH2O, 5 μL). The reaction program was: 95 °C pre-denaturation for 3 min, 95 °C denaturation for 15 sec, 60 °C annealing for 15 sec, 72 °C extension for 15 sec, for a total of 40 cycles; followed by a final extension at 72 °C for 5 min. The obtained Ct values ​​were analyzed using 2... -ΔΔCT Method calculation.

[0121] The results are as follows Figure 5 a and c, WGY1 BN or WGY1 YD The expression level in transgenic positive plants was significantly higher than that in transgenic negative plants and the recipient wheat Fielder. Figure 5 In the figure, c represents the result of the significance analysis of the relative expression levels in each group of plants.

[0122] Meanwhile, using a negative transgenic event and the recipient variety Fielder as controls, the thousand-grain weight (converted to hundred-grain weight) of T1 generation individual plants was measured and analyzed. The correlation between WGY1 gene expression levels in transgenic individual plants and TGW indicates that the WGY1 gene has a negative regulatory effect on wheat thousand-grain weight, and overexpression of WGY1... YD and WGY1 BN Both genes lead to a decrease in grain weight, and the function of both is closely related to the expression level of the WGY1 gene. Figure 5 (b and d).

[0123] Regarding the GMO incident WGY1 YD -OE-1 and WGY1 BN A phenotypic survey of grain length (GL), grain width (GW), and grain thickness (GT) was conducted on T1 generation transgenic positive plants of -OE-1, with transgenic negative plants serving as controls. Data showed that the transgenic positive plants involved in both transgenic events exhibited a certain degree of decrease in grain length, width, and thickness, suggesting that WGY1 regulates thousand-grain weight by affecting grain size.

[0124] Table 1 Primers used in this application

[0125]

[0126]

[0127] Table 2. Some sequences in this application

[0128]

[0129]

[0130]

[0131]

[0132]

[0133] The present application has been described in detail above. Those skilled in the art will recognize that the present application can be implemented in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments are given in this application, it should be understood that further modifications can be made to the present application. In summary, in accordance with the principles of this application, this application is intended to include any changes, uses, or improvements to the present application, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A protein, characterized by: The protein is any one of the following: A1) The amino acid sequence is that of the protein shown in SEQ ID No. 3 or SEQ ID No. 6; A2) Proteins obtained by substituting and / or deleting and / or adding amino acid residues to the amino acid sequence shown in A1) that have more than 80% identity with the amino acid sequence shown in A1) and are related to plant yield. A3) is a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of A1) or A2).

2. A biomaterial related to the protein of claim 1, characterized in that: The biomaterial is any one of the following: B1) A nucleic acid molecule encoding the protein of claim 1; B2), an expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1) or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2), or a transgenic plant cell line containing the recombinant vector described in B3); B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2), or transgenic plant tissue containing the recombinant vector described in B3); B7) Transgenic plant organs containing the nucleic acid molecules described in B1), or transgenic plant organs containing the expression cassette described in B2), or transgenic plant organs containing the recombinant vector described in B3).

3. The biomaterial according to claim 2, characterized in that: B1) The nucleic acid molecule is any one of the following DNA molecules: g1) The coding sequence of the coding strand is the DNA molecule of SEQ ID No. 2; g2) The nucleotide sequence of the coding strand is the DNA molecule of SEQ ID No. 1; g3), DNA molecules that have more than 80% identity with the DNA molecules described in g1) or g2), and that regulate plant yield; g4) The coding sequence of the coding strand is the DNA molecule of SEQ ID No. 5; g5) The nucleotide sequence of the coding strand is the DNA molecule of SEQ ID No. 4; g6) is a DNA molecule that has more than 80% identity with the DNA molecules described in g4) or g5) and regulates plant yield.

4. The use of the biomaterial according to claim 2 or 3 in any of the following: C1) Application in regulating plant yield; C2) Its application in the preparation of products that regulate plant yield; C3) Application in regulating plant grain weight; C4) Application in the preparation of products that regulate the weight of plant seeds; C5) Application in regulating plant seed size; C6) Application in the preparation of products that regulate plant weight; C7) Applications in plant breeding or plant-assisted breeding; C8) Application in the preparation of plant breeding or plant-assisted breeding products.

5. The application according to claim 4, characterized in that: The plants are selected from monocotyledonous plants.

6. A method for regulating plant yield, characterized in that: The method includes regulating plant yield by controlling the expression level of the gene encoding the protein of claim 1 or the activity or content of the protein in the recipient plant.

7. The method according to claim 6, characterized in that: The method includes introducing the gene encoding the protein of claim 1 into a recipient plant to reduce the yield of the recipient plant.

8. The method according to claim 7, characterized in that: The encoding gene is any one of the following: g1) The coding sequence of the coding strand is the DNA molecule of SEQ ID No. 2; g2) The nucleotide sequence of the coding strand is the DNA molecule of SEQ ID No. 1; g3), DNA molecules that have more than 80% identity with the DNA molecules described in g1) or g2), and that regulate plant yield; g4) The coding sequence of the coding strand is the DNA molecule of SEQ ID No. 5; g5) The nucleotide sequence of the coding strand is the DNA molecule of SEQ ID No. 4; g6) is a DNA molecule that has more than 80% identity with the DNA molecules described in g4) or g5) and regulates plant yield.

9. A method for plant breeding, characterized in that: The method includes regulating the expression level of the gene encoding the protein of claim 1 or the activity or content of the protein in the recipient plant to obtain plants with increased yield.

10. A DNA molecule, characterized by: The nucleotide sequence of the DNA molecule is any of the following: D1) The nucleotide sequence of the coding strand is the DNA molecule of SEQ ID No. 1; D2) The nucleotide sequence of the coding strand is the DNA molecule of SEQ ID No. 4; D3) The nucleotide sequence of the coding strand is the DNA molecule of SEQ ID No. 7; D4) The nucleotide sequence of the coding strand is the DNA molecule of SEQ ID No. 8; D5) The nucleotide sequence of the coding strand is the DNA molecule of SEQ ID No. 9; D6) The nucleotide sequence of the coding strand is the DNA molecule of SEQ ID No. 10.

Images