Wheat grain hardness regulating protein and related biological material and application thereof

Abstract

The invention discloses a wheat grain hardness regulating protein as well as a related biological material and application thereof. One technical scheme protected by the invention is the application of the protein from wheat in improving the grain hardness, the name of the protein is HA2, and the protein can be a protein with an amino acid sequence as a sequence 2 in a sequence table. The wheat gene HA2 is cloned from wheat, and it is found that an HA2 mutant plant obtained after the 27th amino acid residue of a sequence 2 is changed from Gly to Asp has higher grain hardness than a wild type, so that the wheat HA2 protein has the effect of regulating and controlling the grain hardness of wheat and can be applied to breeding or high-grain-hardness wheat.

Description

Technical Field

[0001] This patent mainly relates to the fields of molecular biology and crop genetics and breeding, specifically to wheat grain hardness regulating proteins and their related biomaterials and applications. Background Technology

[0002] Wheat is one of the world's three major food crops, consistently ranking among the top three globally in terms of sown area and yield. Wheat grain hardness is a key trait determining wheat milling quality and is also an important factor in determining the market value and grading of wheat seeds. Previous studies have found that wheat grain hardness is related to environmental factors such as climate and soil, but is primarily determined by genetic factors. The Ha locus was the first key site identified regulating grain hardness, located in a specific region at the end of the short arm of wheat chromosome 5D, encoding the Friabilin protein complex. Genes involved in regulating the synthesis of this complex mainly include pina, pinb, and Gsp-1. Numerous studies have shown that pina and pinb are key genes determining wheat grain hardness, encoding PINA and PIB proteins respectively, collectively known as PIN proteins. Only when both pina and pinb are wild-type sequences does the wheat grain exhibit softness, displaying a relatively singular genetic characteristic. Compared to pina (pina, pinb), research on Gsp-1 is relatively limited. Since the cloning of the pina gene in the last century, scientists have discovered numerous mutation types of pina, mostly involving single-base substitutions or deletions leading to loss of gene function. However, apart from the three genes mentioned above, no other genes that affect wheat grain hardness have been discovered so far.

[0003] Wheat hardness has a significant impact on wheat quality. Firstly, wheat with higher hardness generally has lower starch content and higher gluten quality, resulting in better processing performance in foods such as noodles. Secondly, wheat hardness affects the moisture content of wheat flour in dough; generally, harder wheat has a higher water absorption rate, while softer wheat has a lower water absorption rate. Furthermore, wheat hardness also influences the color, texture, and taste of wheat flour; harder wheat flour has larger particles and a lighter color, while softer wheat flour has the opposite characteristics. Therefore, wheat hardness has a crucial impact on wheat quality, especially in wheat processing and food production, where its influence is more pronounced. Conducting thorough wheat hardness testing and selecting appropriate varieties is of great importance for optimizing processing techniques and improving product quality in wheat processing and food production. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to regulate the hardness of wheat kernels and / or how to improve the hardness of wheat kernels.

[0005] To address the aforementioned technical problems, the present invention first provides the application of a protein, a substance regulating the expression of the protein-encoding gene, or a substance regulating the activity or content of the protein, wherein the application may be any of the following:

[0006] P1. The application of the protein or the substance that regulates the expression of the protein-encoding gene or the substance that regulates the activity or content of the protein in regulating plant grain hardness.

[0007] P2. The application of the protein or the substance that regulates the expression of the protein-encoding gene or the substance that regulates the activity or content of the protein in the preparation of products that regulate the hardness of plant seeds.

[0008] P3. The application of the protein or the substance that regulates the expression of the protein-encoding gene or the substance that regulates the activity or content of the protein in improving the hardness of plant seeds or in the preparation of products that improve the hardness of plant seeds.

[0009] P4. The application of the protein or the substance that regulates the expression of the protein-encoding gene or the substance that regulates the activity or content of the protein in the breeding or quality improvement of plant grain hardness traits.

[0010] The protein may be one of the following:

[0011] A1) The amino acid sequence is that of sequence 2 in the sequence listing;

[0012] A2) The amino acid sequence is that of the protein in sequence 4 of the sequence listing;

[0013] A3) A protein derived from A1) or A2) or having the same function as the protein shown in A1) or A2) by substitution and / or deletion and / or addition of one or more amino acid residues.

[0014] A5) is a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1), A2) or A3).

[0015] In the above applications, the protein may be derived from wheat.

[0016] In the above applications, the substance that regulates the activity or content of the protein may be a substance that knocks out the coding gene of the protein and / or a substance that regulates the expression of the coding gene of the protein.

[0017] In the above applications, the substance regulating gene expression can be a substance that performs at least one of the following six types of regulation: 1) regulation at the gene transcription level; 2) post-transcriptional regulation of the gene (i.e., regulation of splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (i.e., regulation of mRNA transport of the gene from the nucleus to the cytoplasm); 4) regulation of gene translation; 5) regulation of mRNA degradation of the gene; and 6) post-translational regulation of the gene (i.e., regulation of the activity of the protein translated from the gene).

[0018] In the above applications, the regulation of gene expression can be achieved by inhibiting or reducing gene expression, which can be achieved by gene knockout or gene silencing.

[0019] Gene knockout refers to the phenomenon of inactivating a specific target gene through homologous recombination. Gene knockout inactivates a specific target gene by altering its DNA sequence.

[0020] Gene silencing refers to the phenomenon of preventing or reducing gene expression without damaging the original DNA. Gene silencing presupposes no change in the DNA sequence, resulting in the absence or reduction of gene expression. Gene silencing can occur at two levels: transcriptional silencing due to DNA methylation, heterochromatinization, and position effects; and post-transcriptional gene silencing, which inactivates the gene at the post-transcriptional level through specific inhibition of target RNA. This includes antisense RNA, co-suppression, quelling, RNA interference (RNAi), and microRNA (miRNA)-mediated translational repression.

[0021] In the above applications, the substance regulating gene expression can be a reagent that inhibits or reduces the expression of the gene. The reagent that inhibits or reduces the expression of the gene can be a gene knockout reagent, such as a reagent that knocks out the gene through homologous recombination or a reagent that knocks out the gene through CRISPR-Cas9. The reagent that inhibits or reduces the expression of the gene can contain a polynucleotide that targets the gene, such as siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0022] In the above applications, the 80% or more of identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, or 99% identity.

[0023] To address the aforementioned technical problems, the present invention also provides any of the following applications of biomaterials related to the proteins described above:

[0024] Q1. Application of the biomaterial in regulating plant seed hardness;

[0025] Q2. Application of the biomaterial in the preparation of products that regulate the hardness of plant seeds;

[0026] Q3. The application of the biomaterial in improving the hardness of plant seeds or in the preparation of products that improve the hardness of plant seeds;

[0027] Q4. Application of the biomaterials in breeding or quality improvement of plant grain hardness traits;

[0028] The biomaterial may be any of the following:

[0029] B1) Nucleic acid molecules that encode the proteins described above;

[0030] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0031] B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0032] 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);

[0033] 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);

[0034] B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2);

[0035] B7) Transgenic plant organs containing the nucleic acid molecules described in B1), or transgenic plant organs containing the expression cassette described in B2);

[0036] B8) Nucleic acid molecules that inhibit or reduce the expression of genes encoding the proteins described above or the activity of the proteins described above;

[0037] B9) Expression cassettes, recombinant vectors, recombinant microorganisms, or transgenic plant cell lines containing the nucleic acid molecules described in B8).

[0038] In the above applications, the nucleic acid molecule can be a DNA molecule as shown below:

[0039] b1) The coding sequence is the DNA molecule shown in sequence 3 of the sequence listing;

[0040] b2) The nucleotide sequence is the DNA molecule shown in sequence 5 of the sequence listing;

[0041] b3) has 90% or more identity with the nucleotide sequence defined by b1) or b2) and is a DNA molecule encoding the protein of claim 1;

[0042] b4) hybridizes under stringent conditions with the nucleotide sequence defined by b1), b2), or b3) and encodes a DNA molecule that encodes the protein of claim 1.

[0043] The term "identity" refers to sequence similarity to a natural nucleic acid sequence. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences. Having 90% or more identity can mean at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity.

[0044] In the aforementioned biological materials, the expression cassette containing nucleic acid molecules described in B2) refers to DNA capable of expressing the proteins described in the above applications in host cells. This DNA may include not only promoters that initiate transcription of protein-coding genes but also terminators that terminate transcription of protein-coding genes. Furthermore, the expression cassette may also include enhancer sequences. Promoters that can be used in this invention include, but are not limited to: constitutive promoters, tissue-, organ-, and development-specific promoters, and inducible promoters.

[0045] Recombinant expression vectors containing the protein-coding gene expression cassettes can be constructed using existing plant expression vectors.

[0046] In the aforementioned biological materials, the recombinant microorganisms may specifically be yeast, bacteria, algae, and fungi.

[0047] In the above applications, the plant may be any of the following:

[0048] D1) Dicotyledons;

[0049] D2) Monocotyledons,

[0050] D3) Plants of the Poales order,

[0051] D4) Gramineae plants,

[0052] D5) Plants of the genus *Wheat*,

[0053] D6) Wheat.

[0054] To address the aforementioned technical problems, the present invention also provides a method for improving the hardness of plant seeds, wherein the method may be M or N; wherein M may include improving the hardness of plant seeds by inhibiting or reducing the expression level of the gene encoding the protein described in A1) above or the activity of the protein described in A1) above.

[0055] In the above method, the method includes changing the 27th amino acid residue of sequence 2 in the sequence listing of the plant genome from Gly to Asp.

[0056] In the above method, changing the 27th amino acid residue of sequence 2 in the sequence listing of the plant genome from Gly to Asp can be achieved by changing the 80th nucleotide of sequence 3 in the sequence listing of the plant genome to G or A.

[0057] The N may include enhancing or increasing the expression level of the gene encoding the protein described in A2) above in wheat or the activity of the protein described in A2) above to improve wheat grain hardness; the enhancement or increase of the activity of the protein described in A2) above in the target plant or / and the expression level of the gene encoding the protein described in A2) above is achieved by introducing the gene encoding the protein described in A2) above into the target plant.

[0058] The reduction or inhibition of the expression level of the protein-coding genes mentioned above in plants can be achieved by any method in the existing technology, so as to induce deletion mutations, insertion mutations or base transformation mutations in the genes, thereby reducing or losing gene function. Specifically, this can be achieved by chemical mutagenesis, physical mutagenesis, RNAi, site-directed genome editing or homologous recombination, etc.

[0059] Among the aforementioned site-specific genome editing methods, zinc finger nuclease (ZFN) technology, transcription activator-like effector nuclease (TALEN) technology, clustered regularly interspaced short palindromic repeats / CRISPR-associated (CRISPR / Cas9 system) technology, and other technologies capable of site-specific genome editing can be employed. Regardless of the method used, the entire coding gene of the aforementioned proteins can be targeted, or individual elements regulating the expression of the coding gene can be targeted, as long as gene function loss or reduction is achieved. For example, exons or 5' UTRs of the coding genes of the aforementioned proteins can be used as targets.

[0060] The plant mentioned above could be wheat.

[0061] The protein mentioned in the method described above may be the protein in A1 mentioned above.

[0062] The above method can produce plants that have all chromatids edited at specific points.

[0063] To address the aforementioned technical problems, this invention also provides a method for identifying or assisting in the identification of wheat grain hardness, comprising detecting the genotype or polymorphism of the SNP1 locus in the genome of the wheat to be tested, and identifying or assisting in the identification of the hardness of the wheat grains to be tested based on the genotype or polymorphism of the SNP1 locus; wherein the grain hardness of wheat with SNP1 being A is greater than or candidate to be greater than that of wheat with SNP1 being G.

[0064] The SNP1 is a SNP in the wheat genome, which is the 80th nucleotide of sequence 1 in the sequence listing, and it is either G or A.

[0065] In the above applications, the substance for detecting the SNP1 polymorphism or genotype, or the substance for detecting the haplotype, is as follows: D1), D2), or D3):

[0066] D1) Contains PCR primers that amplify crop genomic DNA fragments including the SNP1 site;

[0067] D2) PCR reagents containing the PCR primers described in D1);

[0068] D3) A kit containing the PCR primers described in D1) or the PCR reagents described in D2).

[0069] In the above applications, the PCR primers may be a primer set consisting of the single-stranded DNA shown in sequence 6, sequence 7, sequence 8, and sequence 9 of the sequence listing.

[0070] The proteins and / or biological materials described above are also within the scope of protection of this invention.

[0071] This invention screens wheat hardness mutants and clones new genes that affect wheat grain hardness;

[0072] Developing molecular markers for the corresponding genes will provide new gene resources and effective molecular markers for wheat quality breeding.

[0073] The advantages of this invention compared to the prior art are as follows:

[0074] 1) Compared with the previously reported pina and pinb genes, the new wheat grain hardness gene HA2 cloned in this invention is a novel gene that affects wheat grain hardness.

[0075] 2) At present, the regulatory mechanism of wheat grain hardness is still unclear. The new wheat grain hardness gene HA2 cloned in this invention can provide a basis for studying the mechanism of wheat grain hardness.

[0076] 3) This invention also discloses corresponding PCR primers that can amplify wheat genomic DNA fragments, including the 80th nucleotide of the wheat HA2 gene. This marker can be used in marker-assisted breeding and has important theoretical and practical significance for improving wheat grain firmness. Attached Figure Description

[0077] Figure 1 The grain characteristics of mutant ha2 were investigated. A is a bar chart comparing the grain hardness of WT and ha2, with the ordinate representing the grain hardness index; B is a scanning electron microscope image of the cross-section of the grains of WT and ha2; C is a photograph of the grain appearance of WT and ha2; D is a bar chart comparing the thousand-grain weight of WT and ha2, with the ordinate representing the thousand-grain weight in grams (g); E is a bar chart comparing the grain length of WT and ha2, with the ordinate representing the grain length (mm); F is a bar chart comparing the grain width of WT and ha2, with the ordinate representing the grain width (mm).

[0078] Figure 2 The initial localization of the mutant gene HA2 is shown in Figure A. Figure A shows the partial genotypes of marker 1D-457965316 linked to the mutant phenotype on an extreme flocculent plant; Figure B shows the partial genotypes of marker 1D-472301120 on the right side of the initial localization region on an extreme flocculent plant.

[0079] Figure 3 Fine mapping of the mutant gene HA2.

[0080] Figure 4 The base sequences of the HA2 gene signal peptide region are those of wild-type WT and mutant ha2.

[0081] Figure 5 Comparative observation of protein bodies in grain slices from wild-type WT and mutant ha2 after 20 days of development. The red arrows indicate protein bodies in the endosperm cells.

[0082] Figure 6 Transmission electron microscopy observation and comparison of wild-type WT and mutant ha2 grains after 20 days of development.

[0083] Figure 7 The endoplasmic reticulum of wild-type WT and mutant ha2 grains was observed and compared after 20 days of development. The red arrow points to the endoplasmic reticulum in the endosperm cells.

[0084] Figure 8 Immunoelectron microscopy observation of HA2 antibody in grains of wild-type WT and mutant ha2 after 16 days of development. The red arrows indicate the immunogold labeling of HA2. The top image shows the immunoelectron microscopy observation of endosperm cells from WT and ha2 grains; the middle image shows the immunoelectron microscopy observation of endosperm cells from WT and ha2 grains; the bottom image shows the immunoelectron microscopy observation of endosperm cells from WT and ha2 grains.

[0085] Figure 9 The image shows the co-localization of HA2 with the endoplasmic reticulum. The top image is a laser confocal microscopy image of the co-localization of wild-type HA2 protein with the endoplasmic reticulum; the bottom image is a laser confocal microscopy image of the co-localization of mutant HA2 protein with the endoplasmic reticulum.

[0086] Figure 10 This study illustrates the endoplasmic reticulum stress response during seed development in the mutant ha2. A shows the BiP protein expression level in seeds 25 days after pollination with WT and ha2, as detected by Western blot. B is a heatmap comparing the expression levels of eight endoplasmic reticulum stress-related genes in RNA-seq data from seeds 20 days after pollination with WT and ha2.

[0087] Figure 11 Western blot analysis was performed to detect the reduced PIN protein content in the grains of the mutant ha2. Jinying8 (genome AABB) lacks the wheat D genome, therefore this material does not contain the PIN gene and can be used as a negative control. Detailed Implementation

[0088] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and 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 invention in any way.

[0089] 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.

[0090] The following examples used SPSS 26 statistical software to process the data. The experimental results are expressed as mean ± standard deviation. One-way ANOVA was used. P < 0.05 (*) indicates a significant difference, P < 0.01 (**) indicates a highly significant difference, and P < 0.001 (***) indicates a highly significant difference.

[0091] Example 1: The effect of the wheat HA2 gene on wheat grain hardness

[0092] 1. Screening of wheat grain hardness mutants

[0093] Using a high-generation EMS-mutated population of Ruomai 15 (wild type, WT, hardness 35.03) as material, the hardness of 2787 families was tested using an SKCS-4100 single-kernel hardness tester, and a mutant with increased hardness, ha2 (hardness 70.73), was screened. Figure 1 (A) The grain hardness of this mutant has reached the standard for hard wheat (based on the SKCS soft wheat and hard wheat testing standard definition method DOI:10.3390 / plants9121631). Compared with wild-type wheat grain hardness, the endosperm texture of mutant ha2 grains is from loose starch grains to a floury texture (a typical characteristic of soft wheat). Figure 1 The left image of B in the middle section shows a transformation into a corneous type with tightly bound starch granules (a typical characteristic of hard wheat). Figure 1 (Right image of B in the middle). Compared with wild-type wheat, the thousand-grain weight of mutant ha2 ( Figure 1 (D), Particle length ( Figure 1 (E), Particle width (( Figure 1 Both F and WT decreased ( Figure 1 (C)

[0094] 2. Map-based cloning of the mutant gene HA2

[0095] Using a laboratory soft wheat breeding material ND4020 crossed with the mutant ha2 to generate an F2 segregating population, the F2 growth rate of 561 F2 individual plants was analyzed. 2:3 Grain hardness was measured in 119 F2 individual plants. 2:3 The endosperm texture of the grains is similar to that of WT, and the F2 individual plants of 143 F2 are similar. 2:3 The endosperm texture of the grains is similar to that of ha2, with the remaining 299 F2 individual plants having F2... 2:3 The endosperm texture of the grains is intermediate between the two. The population exhibits a segregation ratio of 1:2:1 (χ2=4.393<χ2(0.05,2)=5.991).

[0096] Using resequencing data from ND4020 and ha2, 41 polymorphic InDel markers were developed on the long and short arms of wheat chromosome 21. DNA was extracted from 51 extreme hard-grain F2 single plants. Using these 41 polymorphic markers, linkage analysis revealed a marker 1D-457965316 (χ²) linked to the hardness mutation phenotype on the long arm of wheat chromosome 1D. 2 =56.53>χ 2 0.05,2=5.99). Subsequently, several pairs of markers (1D-401410476, 1D-450755782, 1D-467806482, 1D-472301120, and 1D-482834134) were developed on both sides of marker 1D-457965316 to determine the gene region. The initial mapping region was then preliminarily determined to be at marker 1D-457965316 ( Figure 2 (A) and 1D-472301120 ( Figure 2 The reference genome for Chinese Spring 1.0 between B and C is 457.97 Mb and 472.30 Mb on the long arm of chromosome 1D, with a physical distance of 14.34 Mb.

[0097] DNA was extracted from the remaining 1936 F2 single plants. Genotyping of these F2 single plants was performed using these markers, identifying 61 single plants exchanging within the initial genome mapping intervals. Based on these initial mapping intervals, the markers were further refined, resulting in the development of 5 InDel markers and 3 KASP markers: 1D-461539153, 1D-466126674, 1D-467806482, 1D-470076736, 1D-471125262, SNP469.8, SNP469.9, and SNP470.2. These markers were used to identify the phenotypes and genotypes of the exchanging single plants. The HA2 gene was ultimately located between markers SNP469.9 and 1D-470076736, corresponding to the Chinese Spring 1.0 version reference genome. This fine-mapping interval has a physical distance of 206.6 kb and contains 7 annotated high-confidence genes. Figure 3 ).

[0098] To identify candidate genes associated with the low gluten protein phenotype, specific primers were designed based on the exons, introns, and approximately 2 kb promoter regions of seven genes (Table 1). Sequence variations within the fine-mapping regions of WT and ha2 were analyzed. Only the coding region of the TraesCS1D02G405700 gene showed a GA mutation at position 80 after the translation initiation site (this mutation was named SNP1, corresponding to nucleotide 80 of sequence 1 in the sequence listing, where it is either G or A, and R in sequence 1 represents G or A). This gene encodes an alpha-2-purothionin protein abundant in wheat endosperm. This nucleotide mutation caused the 27th amino acid of the encoded protein (corresponding to amino acid residue 27 of sequence 2 in the sequence listing) to change from Gly (glycine) to Asp (aspartic acid). Figure 4TraesCS1D02G405700 is speculated to be a candidate gene for HA2. Signal peptide cleavage site prediction revealed that the Gly residue is the last amino acid of the signal peptide, which is crucial for the cleavage recognition by signal peptidase during protein synthesis.

[0099] Table 1. Primer sequences for amplification of promoters and coding regions of 7 genes within the fine-mapping region.

[0100]

[0101]

[0102] Sequence 1 (5'-3'):

[0103] ATGGGAAGCAAGGGCCTCAAGGGTGTGATGGTGTGTTTACTCATACTGGGGTTGGTTCTGGAACAGGTGCAAGTAGAAGRCAAGAGTTGCTGCAGGACCACCCTGGGAAGAAACTGCTACAACCTTTGCCGCTCCCGTGGTGCTCAGAAGTTATGCTCAACCGTCTGTAGGTGTAAACTCACAAGTGGCCTAAGCTGCCCCAAAGG TTTCCCTAAATTGGCCCTTGAGTCCAACTCAGATGAACCAGACACCATTGAGTATTGCAACTTGGGATGTAGGTCTTCCGTGTGTGACTACATGGTCAACGCAGCTGCTGACGATGAAGAGATGAAACTCTATGTGGAAAATTGTGGCGATGCTTGTGTCAATTTCTGTAACGGTGATGCTGGCCTCACTTCCCTTGATGCGTAA.

[0104] 3. The mutant ha2 protein body is smaller, and the PIN protein content is lower.

[0105] 3.1 Observation of protein body development using an optical microscope

[0106] Optical microscopy was performed on wheat grains from WT and the mutant ha2 20 days after pollination. After staining with toluidine blue, semi-thin sections of the samples were observed, revealing the presence of the ha2 protein body (…). Figure 5 (Right image) Compared to WT ( Figure 5 (Left image) They cannot aggregate into large globular protein bodies, are more dispersed, and have a significantly reduced protein body content. Figure 5 Transmission electron microscopy was used to observe the protein morphology of WT and ha2 seeds 20 days after pollination. It was found that ha2 protein bodies (… Figure 6(middle right figure) compared to WT ( Figure 6 The middle left image is noticeably smaller and has an irregular appearance. Figure 6 (PB). These results indicate that the mutant ha2 protein body development is abnormal.

[0107] 3.2 Observation of endoplasmic reticulum morphology using transmission electron microscopy

[0108] Purine thiocyanate proteins in plants may be synthesized via the endoplasmic reticulum (ER), suggesting that ER stress also occurred during seed development in the mutant ha2. To verify this hypothesis, ultrathin sections of seeds from WT and the mutant ha2 20 days after pollination were observed using transmission electron microscopy. The results showed that the WT ER exhibited a double-layered, continuous, stacked structure. Figure 7 (Left image, middle section) The endoplasmic reticulum of mutant ha2 exhibits a broken, abnormally looped structure. Figure 7 (Right image in the middle)

[0109] Immunoassay was performed on ultrathin sections of WT and ha2 seeds 16 days after pollination using HA2 antibody, and the sections were then observed under a transmission electron microscope. Colloidal gold particles were observed in the endoplasmic reticulum of both WT and ha2 seeds with abnormal endoplasmic reticulum. Figure 8 (Indicated by the red arrow in the middle). In addition, this invention also found that some ha2 protein bodies are attached to abnormal endoplasmic reticulum, and colloidal gold particles can also be observed within the protein bodies. Figure 8 (Right image from the top middle section, right image from the middle section, and bottom image). These results suggest that abnormally cleaved HA2 proteins may remain in the abnormal endoplasmic reticulum.

[0110] 3.3 Fluorescence microscopy observation of co-localization of HA2 protein and endoplasmic reticulum localizing proteins

[0111] Overexpression vectors were constructed by fusing GFP with the wild-type full-length HA2 gene (HA2-G) and the mutant gene (HA2-A), and then co-transformed into wheat protoplasts with an endoplasmic reticulum localization protein (RFP-HDEL) labeled with red fluorescence. The results showed that both proteins co-localized with the endoplasmic reticulum, but the localization signal of wild-type HA2 (HA2-G protein) was more uniform in the cytoplasm. Figure 9 (See the image above). The localization signal of mutant HA2 (HA2-A protein) accumulates in part of the endoplasmic reticulum (ER). Figure 9 (See the figure below). These results indicate that the mutant HA2 protein cannot be properly cleaved during synthesis, and the abnormal HA2 protein residues accumulate in the endoplasmic reticulum.

[0112] Among them, the coding sequence of the full-length HA2 gene (HA2-G, TraesCS1D02G405700) in wild-type wheat WT is sequence 3 in the sequence listing, and the amino acid sequence of the HA2-G protein it encodes is sequence 2 in the sequence listing. SNP1 corresponds to the 80th nucleotide of sequence 3 in the sequence listing. The coding sequence of the HA2 mutant gene (HA2-A) in the ha2 mutant is sequence 5 in the sequence listing, and the amino acid sequence of the HA2 protein HA2-A it encodes is sequence 4 in the sequence listing.

[0113] Sequence 2 (5'-3'):

[0114] MGSKGLKGVMVCLLILLGLVLEQVQVEGKSCCRTTLGRNCYNLCRSRGAQKLC STVCRCKLTSGLSCPKGFPKLALESNSDEPDTIEYCNLGCRSSVCDYMVNAAADDE EMKLYVENCGDACVNFCNGDAGLTSLDA;

[0115] Sequence 3 (5'-3'):

[0116] ATGGGAAGCAAGGGCCTCAAGGGTGTGATGGTGTGTTTACTCATACTGGGGTTGGTTCTGGAACAGGTGCAAGTAGAAGGCAAGAGTTTGCTGCAGGACCACCCTGGGAAGAAACTGCTACAACCTTTGCCGCTCCCGTGGTGCTCAGAAGTTATGCTCAACCGTCTGTAGGTGTAAACTCACAAGTGGCCTAAGCTGCCCCAAAGG TTTCCCTAAATTGGCCCTTGAGTCCAACTCAGATGAACCAGACACCATTGAGTATTGCAACTTGGGATGTAGGTCTTCCGTGTGTGACTACATGGTCAACGCAGCTGCTGACGATGAAGAGATGAAACTCTATGTGGAAAATTGTGGCGATGCTTGTGTCAATTTCTGTAACGGTGATGCTGGCCTCACTTCCCTTGATGCGTAA;

[0117] Sequence 4 (5'-3'):

[0118] MGSKGLKGVMVCLLILLGLVLEQVQVEDKSCCRTTLGRNCYNLCRSRGAQKLC STVCRCKLTSGLSCPKGFPKLALESNSDEPDTIEYCNLGCRSSVCDYMVNAAADDE EMKLYVENCGDACVNFCNGDAGLTSLDA;

[0119] Sequence 5 (5'-3'):

[0120] ATGGGAAGCAAGGGCCTCAAGGGTGTGATGGTGTGTTTACTCATACTGGGGTTGGTTCTGGAACAGGTGCAAGTAGAAGACAAGAGTTGCTGCAGGACCACCCTGGGAAGAAACTGCTACAACCTTTGCCGCTCCCGTGGTGCTCAGAAGTTATGCTCAACCGTCTGTAGGTGTAAACTCACAAGTGGCCTAAGCTGCCCCAAAGG TTTCCCTAAATTGGCCCTTGAGTCCAACTCAGATGAACCAGACACCATTGAGTATTGCAACTTGGGATGTAGGTCTTCCGTGTGTGACTACATGGTCAACGCAGCTGCTGACGATGAAGAGATGAAACTCTATGTGGAAAATTGTGGCGATGCTTGTGTCAATTTCTGTAACGGTGATGCTGGCCTCACTTCCCTTGATGCGTAA.

[0121] 3.4 Expression analysis of the endoplasmic reticulum chaperone protein BiP

[0122] The expression level of the endoplasmic reticulum chaperone protein BiP can reflect the level of endoplasmic reticulum stress in cells. Western blot analysis of BiP expression levels in seeds of WT and mutant ha2 20 days after pollination revealed that the BiP protein content in ha2 was significantly higher than that in WT. Figure 10 Transcriptome analysis of seeds from WT and mutant ha2 20 days post-pollination showed that eight endoplasmic reticulum stress-related genes were upregulated in ha2. Figure 10 BiP antibody information: anti-BiP (AS09481, Agrisera).

[0123] Total protein was extracted from mature seeds of WT and ha2. The PIN protein content of both was detected using PINA and PINB protein antibodies (PINA and PINB antibodies are synthesized from peptides; the PINA peptide is C-QRDRASKVIQEAKNL, and the PINB peptide is RGEVFKQLQRAQSLPSKC; both are rabbit antibodies, synthesized by ABclonal; please provide the selling company and catalog number). It was found that the mutant ha2 had a higher PINA content than WT. Figure 11 (Top and middle images) and PINB protein content ( Figure 11 (The middle figure) All decreased (Jinying8 is a tetraploid wheat and does not contain the D genome, so it is used as a negative control for PIN protein). Internal control antibody information: anti-eEF1α (AS10934, Agrisera).

[0124] The results in summary indicate that the wheat HA2 gene (TraesCS1D02G405700) mutant ha2 grains cannot be cleaved normally. The HA2 protein trapped on the endoplasmic reticulum exerts pressure on the endoplasmic reticulum, causing an endoplasmic reticulum stress response in the cell. As a result, the endoplasmic reticulum function is impaired, PIN protein synthesis is affected, and grain hardness increases.

[0125] Therefore, the wheat HA2 gene regulates wheat grain hardness. By reducing or inhibiting the expression of the wheat HA2 gene, wheat grain hardness can be improved, thus enabling breeding and quality improvement of wheat grain hardness-related traits.

[0126] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Use of a protein or of a substance which modulates the expression of the gene coding for said protein or of a substance which modulates the activity or the content of said protein, characterized in that: The application is any one of the following: P1. Use of the protein or a substance that regulates expression of the gene encoding the protein or a substance that regulates activity or content of the protein in regulating hardness of plant seeds; P2. Use of the protein or a substance that regulates expression of the gene encoding the protein or a substance that regulates activity or content of the protein in the preparation of a product for regulating hardness of plant seeds; P3. Use of the protein or a substance that regulates expression of the gene encoding the protein or a substance that regulates activity or content of the protein in increasing hardness of plant seeds or in the preparation of a product for increasing hardness of plant seeds; P4. Use of the protein or a substance that regulates expression of the gene encoding the protein or a substance that regulates activity or content of the protein in breeding or quality improvement of the hardness trait of plant seeds; The protein is a protein as follows: A1) a protein with the amino acid sequence of SEQ ID NO: 2 in the sequence listing; A2) a protein with the amino acid sequence of SEQ ID NO: 4 in the sequence listing; A3) a protein derived from A1) or A2) by substitution, deletion and / or addition of one or more amino acid residues and having the same function, or a protein having more than 80% identity to the protein represented by A1) or A2) and having the same function; A5) a fusion protein obtained by linking a protein tag to the N-terminal or / and C-terminal end of A1), A2) or A3).

2. Use according to claim 1, characterized in that: The protein is derived from wheat.

3. Use of a biological material related to the protein as claimed in claim 1 or 2 in any one of the following: Q1. Use of the biological material in regulating hardness of plant seeds; Q2. Use of the biological material in the preparation of a product for regulating hardness of plant seeds; Q3. Use of the biological material in increasing hardness of plant seeds or in the preparation of a product for increasing hardness of plant seeds; Q4. Use of the biological material in breeding or quality improvement of the hardness trait of plant seeds; The biological material is any one of the following: B1) a nucleic acid molecule encoding the protein as claimed in claim 1; B2) an expression cassette containing the nucleic acid molecule as claimed in B1); B3) a recombinant vector containing the nucleic acid molecule as claimed in B1) or the expression cassette as claimed in B2); B4) a recombinant microorganism containing the nucleic acid molecule as claimed in B1) or the expression cassette as claimed in B2) or the recombinant vector as claimed in B3); B5) a transgenic plant cell line containing the nucleic acid molecule as claimed in B1) or the expression cassette as claimed in B2); B6) a transgenic plant tissue containing the nucleic acid molecule as claimed in B1) or the expression cassette as claimed in B2); B7) a transgenic plant organ containing the nucleic acid molecule as claimed in B1) or the expression cassette as claimed in B2); B8) a nucleic acid molecule that inhibits or reduces expression of the gene encoding the protein as claimed in claim 1 or activity of the protein as claimed in claim 1; B9) an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic plant cell line containing the nucleic acid molecule as claimed in B8).

4. Use according to claim 3, characterized in that: The nucleic acid molecule is a DNA molecule as shown below: b1) the coding sequence is a DNA molecule as represented in SEQ ID NO: 3 of the sequence listing; b2) the nucleotide sequence is a DNA molecule as represented in SEQ ID NO: 5 of the sequence listing; b3) a DNA molecule having 90% or more identity with the nucleotide sequence defined in b1) or b2) and encoding the protein as defined in claim 1; b4) a DNA molecule hybridizing under stringent conditions with the nucleotide sequence defined in b1), b2) or b3) and encoding the protein as defined in claim 1.

5. Use according to any one of claims 1 to 4, characterized in that: said plant is any one of: D1) a dicotyledonous plant; D2) a monocotyledonous plant, D3) a plant of the order Poales, D4) a plant of the family Poaceae, D5) a plant of the genus Triticum, D6) a plant of the species Triticum aestivum.

6. A method of increasing the hardness of wheat kernels, characterized by: said method is M or N; said M comprises increasing the hardness of the wheat grain by inhibiting or reducing the expression level of the gene encoding the protein as defined in A1) of claim 1 or the activity of the protein as defined in A1) of claim 1 in the wheat; said method comprises changing the 27th amino acid residue of SEQ ID NO: 2 of the sequence listing from Gly to Asp in the genome of said plant; changing the 27th amino acid residue of SEQ ID NO: 2 of the sequence listing from Gly to Asp in the genome of said plant is achieved by changing the 80th nucleotide of SEQ ID NO: 3 of the sequence listing from G or A; said N comprises increasing the hardness of the wheat grain by enhancing or increasing the expression level of the gene encoding the protein as defined in A2) of claim 1 or the activity of the protein as defined in A2) of claim 1 in the wheat; said enhancing or increasing the activity of the protein as defined in A2) of claim 1 or the expression level of the gene encoding the protein as defined in A2) of claim 1 in the plant of interest is achieved by introducing the gene encoding the protein as defined in A2) of claim 1 into said plant of interest.

7. A method for identifying or assisting in identifying the hardness of the grain of wheat, comprising detecting the genotype or polymorphism of SNP1 in the genome of the wheat to be tested, and identifying or assisting in identifying the hardness of the grain of the wheat to be tested according to the genotype or polymorphism of SNP1; the grain of the wheat whose SNP1 is A is harder or is a candidate for being harder than the grain of the wheat whose SNP1 is G; said SNP1 is a SNP in the genome of wheat, which is the 80th nucleotide of SEQ ID NO: 1 of the sequence listing, which is G or A.

8. Use according to claim 7, characterized in that: the substance for detecting the polymorphism or genotype of SNP1 or the substance for detecting the haplotype is any one of D1), D2) or D3): D1) PCR primers for amplifying a fragment of the genome of the crop comprising the SNP1 site; D2) PCR reagents containing the PCR primers of D1); D3) a kit containing the PCR primers of D1) or the PCR reagents of D2).

9. Use according to claim 7 or 8, characterized in that: said PCR primers are a primer set consisting of the single-stranded DNA as represented in SEQ ID NO: 6 of the sequence listing, the single-stranded DNA as represented in SEQ ID NO: 7 of the sequence listing, the single-stranded DNA as represented in SEQ ID NO: 8 of the sequence listing and the single-stranded DNA as represented in SEQ ID NO: 9 of the sequence listing.

10. The protein as defined in claim 1 or 2 and / or the biological material as defined in claim 3 or 4.

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