Application of protein and related biological materials thereof in wheat grain hardness breeding

Abstract

The invention discloses application of protein and related biological materials thereof in wheat grain hardness breeding. According to the method, a homozygous novel pinb haplotype wheat material is created by editing a wheat pinb gene by utilizing a guide editing means, and the wheat material containing the novel pinb haplotype is backcrossed with wheat with different grain hardness, so that wheat lines with different grain hardness can be obtained; the novel pinb gene haplotype and a small part containing the haplotype can be applied to genetic breeding and quality improvement of wheat.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically to the application of proteins and their related biological materials in wheat grain hardness breeding. Background Art

[0002] Wheat is one of the world's three major food crops, and its sown area and yield have always ranked among the top three globally. Wheat grain hardness is one of the key traits determining wheat milling quality and is also an important aspect determining the market value and grading of wheat seeds. The wheat hardness index is a comprehensive index calculated based on data obtained by measuring the hardness of wheat grains by applying a certain pressure in a fixed environment. Generally, the higher the wheat hardness index, the harder the texture of the wheat, and vice versa. The wheat gluten index is an index calculated based on the gluten strength value determined by processing and operating wheat dough to measure the strength of its gluten. Generally, the higher the wheat gluten index, the stronger the gluten strength of the wheat, and better pasta products can be made.

[0003] Previous studies have found that wheat grain hardness is related to environmental factors such as climate and soil, but is mainly determined by genetic factors. The Ha locus is the first key locus mapped to regulate grain hardness and is located in a specific region at the end of the short arm of wheat chromosome 5D, encoding the Friabilin protein complex. The 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 the key genes determining wheat grain hardness, encoding PINA and PINB proteins respectively, collectively called PIN proteins. Only when pina and pinb are both wild-type sequences, the wheat grains show soft texture and exhibit relatively single genetic characteristics. Compared with pin (pina, pinb), the research on Gsp-1 is relatively less. Since the cloning of the pin gene in the last century, scientists have discovered a large number of mutant types of pin, mostly single-base substitutions or deletions resulting in the loss of gene function, and there are no haplotypes related to base insertions. Although different haplotypes contribute differently to grain hardness, any missense mutation in either pina or pinb will cause an increase in wheat grain hardness.

[0004] There are many hypotheses about the regulation of grain hardness. One hypothesis holds that there is a Friabilin protein complex on the surface of starch granules in wheat endosperm. The component PIN of this protein complex is rich in tryptophan and easily binds to polar lipids on the amyloplast membrane, affecting the interaction between starch granules inside the amyloplast membrane and proteins outside the membrane, thereby affecting grain hardness. During the grain maturation process of soft wheat, the PIN subunit enhances the stability of the amyloplast membrane, enabling the starch inside the membrane to fully separate from the proteins outside the membrane. In hard wheat, due to changes in the PIN subunit sequence, the PIN protein cannot normally maintain the stability of the amyloplast membrane, and the binding between starch granules and proteins is stronger.

[0005] Both PINA and PINB contain a backbone composed of ten cysteine residues, eight of which have similar structural characteristics, making this protein similar to non-specific lipid transfer protein (ns-LTP). It consists of four α-helices, with the helixes interrupted by loops in the middle, and the protein stabilizes this structure through five disulfide bonds.

[0006] In recent years, the CRISPR / Cas9 system has been widely and deeply studied and applied by scientists. Due to its high specificity, high editing efficiency, and low off-target rate, it has become one of the essential tools in molecular biology research. However, because the repair of DNA after cleavage in vivo by this system is uncertain, it sometimes cannot precisely edit a certain sequence as expected by scientists. In 2019, the David R. Liu laboratory developed the prime editing system, which can precisely achieve any conversion, transversion of bases, and insertion and deletion of small fragments. Compared with CRISPR / Cas9, although the efficiency is lower, it overcomes the disadvantage of its inability to precisely edit, opening up a new path for gene editing. Summary of the Invention

[0007] The technical problems to be solved by the present invention are how to cultivate wheat with different grain hardness or gluten index and / or how to create wheat containing new haplotypes related to wheat grain hardness or gluten index and / or how to improve the grain hardness or gluten index of wheat.

[0008] To solve the above technical problems, the present invention first provides the following applications of a protein or a substance that regulates the expression of the gene encoding the protein or a substance that regulates the activity or content of the protein:

[0009] P1. Application in regulating wheat grain hardness or gluten index;

[0010] P2. Application in preparing a product for regulating wheat grain hardness or gluten index;

[0011] P3. Application in wheat breeding;

[0012] P4. Use in wheat quality improvement;

[0013] The protein may be the following protein:

[0014] A1) A protein with an amino acid sequence that is SEQ ID NO: 2 in the sequence listing;

[0015] A2) A protein with an amino acid sequence that is SEQ ID NO: 4 in the sequence listing;

[0016] A3) A protein with an amino acid sequence that is SEQ ID NO: 6 in the sequence listing;

[0017] A4) A protein obtained by substitution and / or deletion and / or addition of more than one amino acid residue to the amino acid sequence shown in A1), A2), or A3), and having the same function, which is derived from A1), A2), or A3), or has an identity of more than 80% with the protein shown in A1), A2), or A3) and has the same function;

[0018] A5) A fusion protein obtained by linking a protein tag to the N-terminus and / or C-terminus of A1), A2), or A3).

[0019] To solve the above technical problems, the present invention also provides any one of the following applications of biological materials related to the above-mentioned protein:

[0020] P1. Use in regulating wheat grain hardness or gluten index;

[0021] P2. Use in preparing a product for regulating wheat grain hardness or gluten index;

[0022] P3. Use in wheat breeding;

[0023] P4. Use in wheat quality improvement;

[0024] The biological material may be any one of the following:

[0025] B1) A nucleic acid molecule encoding the above-mentioned protein;

[0026] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0027] B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0028] B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

[0029] 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);

[0030] B6) A transgenic plant tissue containing the nucleic acid molecule described in B1), or a transgenic plant tissue containing the expression cassette described in B2);

[0031] B7) A transgenic plant organ containing the nucleic acid molecule described in B1), or a transgenic plant organ containing the expression cassette described in B2);

[0032] B8) A nucleic acid molecule that inhibits or reduces the expression of the coding gene of the protein described in A1) above or the activity of the protein described in A1) above;

[0033] B9) An expression cassette, recombinant vector, recombinant microorganism or transgenic plant cell line containing the nucleic acid molecule described in B8).

[0034] In the above applications, the nucleic acid molecule may be the coding gene of the protein shown below:

[0035] b1) The nucleotide of the coding strand is a cDNA molecule or DNA molecule of Sequence 1 in the Sequence Listing;

[0036] b2) The nucleotide of the coding strand is a cDNA molecule or DNA molecule of Sequence 3 in the Sequence Listing;

[0037] b3) The nucleotide of the coding strand is a cDNA molecule or DNA molecule of Sequence 5 in the Sequence Listing;

[0038] b4) A DNA molecule that has 90% or more identity with the nucleotide sequence defined in b1), b2) or b3) and encodes the protein described above;

[0039] b5) A DNA molecule that hybridizes with the nucleotide sequence defined in b1), b2), b3) or b4) under stringent conditions and encodes the protein described above;

[0040] The nucleic acid molecule of B8) is a DNA molecule encoding a gRNA targeting the protein coding gene described in A1) above or a gRNA targeting the protein coding gene described in A1) above.

[0041] 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.

[0042] In the above application, the substance for regulating gene expression may be a substance that performs at least one of the following six regulations: 1) regulation at the gene transcription level; 2) regulation after gene transcription (i.e., regulation of the splicing or processing of the primary transcript of the gene); 3) regulation of the RNA transport of the gene (i.e., regulation of the transport of the gene's mRNA from the nucleus to the cytoplasm); 4) regulation of the translation of the gene; 5) regulation of the degradation of the gene's mRNA; 6) regulation after translation of the gene (i.e., regulation of the activity of the protein translated from the gene).

[0043] In the above application, the regulation of gene expression may be to inhibit or reduce the gene expression, and the inhibition or reduction of the gene expression may be achieved by gene knockout or by gene silencing.

[0044] The gene knockout (gene knockout) refers to the phenomenon of inactivating a specific target gene through homologous recombination. Gene knockout inactivates a specific target gene by changing the DNA sequence.

[0045] Gene silencing refers to the phenomenon of gene non-expression or low-expression without damaging the original DNA. Gene silencing occurs on the premise of not changing the DNA sequence, resulting in gene non-expression or low-expression. Gene silencing can occur at two levels. One is transcriptional gene silencing caused by DNA methylation, heterochromatinization, and position effects, etc. The other is post-transcriptional gene silencing, that is, at the post-transcriptional level of the gene, the gene is inactivated by specifically inhibiting the target RNA, including antisense RNA, co-suppression, quelling, RNA interference (RNAi), and translation inhibition mediated by microRNA (miRNA), etc.

[0046] In the above application, the substance for regulating gene expression may be a reagent for inhibiting or reducing the gene expression. The reagent for inhibiting or reducing the gene expression may be a reagent for knocking out the gene, such as a reagent for knocking out the gene by homologous recombination, or a reagent for knocking out the gene by CRISPR-Cas9, or a reagent for knocking out the gene by a prime editing system. The reagent for inhibiting or reducing the gene expression may contain polynucleotides targeting the gene, such as siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0047] The term "identity" refers to the sequence similarity with a natural nucleic acid sequence. Identity can be evaluated by the naked eye or by 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. The identity of 90% or more can be at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity.

[0048] Among the above biological materials, the expression cassette containing the nucleic acid molecule described in B2) refers to a DNA that can express the protein described in the above application in a host cell. This DNA may not only include a promoter that initiates the transcription of the protein-coding gene, but also include a terminator that terminates the transcription of the protein-coding gene. Further, the expression cassette may also include enhancer sequences. Promoters that can be used in the present invention include, but are not limited to: constitutive promoters, tissue-, organ- and development-specific promoters, and inducible promoters.

[0049] An existing plant expression vector can be used to construct a recombinant expression vector containing the expression cassette of the protein-coding gene.

[0050] Among the above biological materials, the recombinant microorganism can specifically be yeast, bacteria, algae and fungi.

[0051] In the above application, the plant can be any one of the following:

[0052] D1) Monocotyledonous plants,

[0053] D2) Plants of the order Poales,

[0054] D3) Gramineous plants,

[0055] D4) Plants of the genus Triticum,

[0056] D5) Wheat.

[0057] To solve the above technical problems, the present invention also provides a method for regulating or changing the hardness of plant grains and / or the gluten index, including regulating or changing the hardness of plant grains and / or the gluten index by inhibiting or reducing the expression level of the coding gene of the protein described in A1) above or the activity of the protein described in A1) above.

[0058] The above method may include introducing a substance that reduces or inhibits the expression of the protein-coding gene described in A1) above into the plant; the substance that reduces or inhibits the expression of the protein-coding gene described in A1) above is any one of the following substances:

[0059] c1) A nucleic acid molecule that inhibits or reduces the expression of the protein-coding gene described in A1) above;

[0060] c2) An expression cassette containing the nucleic acid molecule described in c1);

[0061] c3) A recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2);

[0062] c4) A recombinant microorganism containing the nucleic acid molecule described in c1), or a recombinant microorganism containing the expression cassette described in c2), or a recombinant microorganism containing the recombinant vector described in c3).

[0063] The reduction or inhibition of the expression level of the above-mentioned protein-coding gene in plants can be achieved by any method in the prior art, so as to cause deletion mutations, insertion mutations or base substitution mutations in the gene, thereby reducing or losing the gene function. Specifically, it can be chemical mutagenesis, physical mutagenesis, RNAi, genome site-directed editing or homologous recombination, etc.

[0064] In the above-mentioned genome site-directed editing methods, Zinc finger nuclease (ZFN) technology, Transcription activator-like effector nuclease (TALEN) technology or Clustered regularly interspaced short palindromic repeats / CRISPR associated (CRISPR / Cas9 system) technology, as well as other technologies that can achieve genome site-directed editing, can be used. No matter which method is adopted, either the entire coding gene of the above-mentioned protein can be used as a target, or each element regulating the expression of the above-mentioned protein-coding gene can be used as a target, as long as the gene function can be lost or reduced. For example, the exons or 5'UTR of the coding gene of the above-mentioned protein can be used as targets.

[0065] The above-mentioned method can specifically be to perform site-directed editing on the above-mentioned A1) protein-coding gene by the CRISPR / Cas9 method or the prime editing system, so as to reduce or inhibit the expression level of the above-mentioned A1) protein-coding gene in plants. For example, the recombinant plasmid vector expressing Cas9 protein and gRNA is used to achieve the transfer and integration of foreign genes into plant cells through the infection of Agrobacterium.

[0066] In the above-mentioned method, the plant can be any one of the following:

[0067] D1) Monocotyledonous plants,

[0068] D2) Plants of the order Poales,

[0069] D3) Gramineous plants,

[0070] D4) Triticum plants,

[0071] D5) Wheat.

[0072] c1) The nucleic acid molecule may be Sequence 7 in the Sequence Listing.

[0073] To solve the above technical problems, the present invention also provides the use of wheat containing a new pinb haplotype in the breeding of wheat grain hardness and gluten index traits or quality improvement; the new pinb haplotype may correspond to a variation on a chromosome of the wheat genome, and the variation may be at least one of the following mutations of the protein-coding gene shown in Sequence 1 in the Sequence Listing of the wheat genome:

[0074] 1) Inserted 3 nucleotides "TGT" between the 102nd and 103rd nucleotides of Sequence 1 in the Sequence Listing, and at the same time inserted 3 nucleotides "TGC" between the 189th and 190th nucleotides of Sequence 1, and at the same time inserted 6 nucleotides "TGTTGT" between the 306th and 307th nucleotides;

[0075] 2) Inserted 6 nucleotides "TGTTGT" between the 306th and 307th nucleotides of Sequence 1 in the Sequence Listing.

[0076] To solve the above technical problems, the present invention also provides a method for breeding wheat grain hardness and / or gluten index traits, the method may include crossing Wheat A containing the new pinb haplotype with Wheat B to obtain F 1 -generation wheat, using the Wheat B as the recurrent parent, backcrossing the F 1 -generation wheat with the Wheat B to obtain backcross progeny, selecting wheat containing the new pinb haplotype from the backcross progeny for self-crossing to obtain self-cross progeny, and selecting wheat with the new pinb haplotype being homozygous from the self-cross progeny to obtain target wheat with grain hardness and / or gluten index different from that of the Wheat B.

[0077] The new pinb haplotype may correspond to a variation on a chromosome of the wheat genome, and the variation may be at least one of the following mutations of the nucleotide sequence shown in Sequence 1 in the Sequence Listing of the wheat genome:

[0078] 1) Inserted 3 nucleotides "TGT" between the 102nd and 103rd nucleotides of Sequence 1 in the Sequence Listing, and at the same time inserted 3 nucleotides "TGC" between the 189th and 190th nucleotides of Sequence 1, and at the same time inserted 6 nucleotides "TGTTGT" between the 306th and 307th nucleotides;

[0079] 2) Six nucleotides "TGTTGT" were inserted between nucleotides 306 and 307 of Sequence 1 in the Sequence Listing.

[0080] The A wheat containing the new pinb haplotype can be obtained by introducing a substance that reduces or inhibits the expression of the protein-coding gene described in A1) above into wheat. The substance can be any of the following:

[0081] c1) A nucleic acid molecule that inhibits or reduces the expression of the protein-coding gene described in A1) above;

[0082] c2) An expression cassette containing the nucleic acid molecule described in c1);

[0083] c3) A recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2);

[0084] c4) A recombinant microorganism containing the nucleic acid molecule described in c1), or a recombinant microorganism containing the expression cassette described in c2), or a recombinant microorganism containing the recombinant vector described in c3).

[0085] The nucleic acid molecule described in c1) can be Sequence 7 in the Sequence Listing.

[0086] The protein and / or the biological material described above also belong to the protection scope of the present invention. The present invention creates a homozygous new pinb haplotype wheat material by means of prime editing and applies it to wheat genetic breeding.

[0087] All the reported pin haplotypes in nature are base substitutions or deletions. Compared with the wild type, usually the encoded amino acid sequence will cause different degrees of loss or weakening of its function, resulting in an increase in wheat grain hardness. The present invention aims to use the method of the prime editing system to introduce a base sequence capable of encoding cysteine at a new different position of the pinb gene to create a new transgenic wheat with the pinb haplotype. On the one hand, it provides germplasm resources for cultivating new wheat varieties with different grain hardnesses and gene resources for creating special wheat; on the other hand, it provides materials for further studying the molecular regulation mechanism of the pinb gene.

[0088] Beneficial effects compared with the prior art:

[0089] 1) Compared with the reported pinb haplotypes, the haplotype created by gene editing in the present invention is a haplotype not found in nature, enriching the variation types of pin and also providing a material basis for the creation of special wheat.

[0090] 2) At present, the regulation mechanism of pin is still unclear. The new pinb haplotype material created in the present invention can facilitate the study of the function of the PINB protein and the mechanism of the pinb gene regulating grain hardness.

[0091] 3) The guiding editing system overcomes the shortcoming of the traditional CRISPR / Cas9 technology being not precise enough, and can accurately improve any position of the pinb gene as expected, breaking through the previous breeding limitations and facilitating the development of molecular design breeding. Description of the Drawings

[0092] Figure 1 It is the editing type of the homozygous pinb transgenic material.

[0093] Figure 2 It is the detection of the grain hardness and gluten index of the gene-edited material. "****" represents P≤0.0001, "***" represents P≤0.001, and "**" represents P≤0.01. Detailed Implementation Modes

[0094] The present invention will be further described in detail below in combination with the detailed implementation modes. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0095] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0096] The following embodiments use GraphPad Prism 10.1.2 statistical software to process the data. The experimental results are expressed as the mean ± standard deviation. The T-test is used. P < 0.05 (*) indicates a significant difference, P < 0.01 (**) indicates a highly significant difference, P < 0.001 (***) indicates a highly significant difference, and P < 0.0001 (****) indicates a highly significant difference.

[0097] In the embodiments of the present invention, the TaU3-esgRNA vector was a generous gift from the research group of Professor Zong Yuan at China Agricultural University. Related literature: Efcient and versatile multiplex prime editing in hexaploid wheat; The guide protein vector ePPEmax was a generous gift from the research group of Professor Zong Yuan at China Agricultural University. Related literature: Efcient and versatile multiplex prime editing in hexaploid wheat; The pBUE414-ePPEmax-V223A vector was a generous gift from the research group of Professor Zong Yuan at China Agricultural University. Related literature: Efcient and versatile multiplex prime editing in hexaploid wheat.

[0098] Example 1. Introducing new sequences at different positions of the pinb gene using the prime editing system

[0099] 1. Designing gene editing targets

[0100] The CDS sequence of the pinb gene (sequence 1 in the sequence listing) was downloaded through the website EnsemblPlants (https: / / plants.ensembl.org / index.html). Then, the target sequence corresponding to the pinb gene sequence was designed using the website (http: / / www.plantgenomeediting.net / ). Combining with the amino acid sequence of the PINB protein (sequence 2 in the sequence listing), some targets that caused frameshift mutations were excluded. Then, non-specific targets were excluded through blast analysis.

[0101] Sequence 1 (5’-3’)

[0102] ATGAAGACCTTATTCCTCCTAGCTCTCCTTGCTCTTGTAGCGAGCACAACCTTCGCGCAATACTCAGAAGTTGGCGGCTGGTACAATGAAGTTGGCGGAGGAGGTGGTTCTCAACAATGTCCGCAGGAGCGGCCGAAGCTAAGCTCTTGCAAGGATTACGTGATGGAGCGATGTTTCACAATGAAGGATTTTCCAGTCACCTGGCCCACAAAATGGTGGAAGGGCGGCTGTGAGCATGAGGTTCGGGAGAAGTGCTGCAAGCAGCTGAGCCAGATAGCACCACAATGTCGCTGTGATTCTATCCGGCGAGTGATCCAAGGCAGGCTCGGTGGCTTCTTGGGCATTTGGCGAGGTGAGGTATTCAAACAACTTCAGAGGGCCCAGAGCCTCCCCTCAAAGTGCAACATGGGCGCCGACTGCAAGTTCCCTAGTGGCTATTACTGGTGA。

[0103] Sequence 2:

[0104] MKTLFLLALLALVASTTFAQYSEVGGWYNEVGGGGGSQQCPQERPKLSSCKDYVME RCFTMKDFPVTWPTKWWKGGCEHEVREKCCKQLSQIAPQCRCDSIRRVIQGRLGGFLGIW RGEVFKQLQRAQSLPSKCNMGADCKFPSGYYW。

[0105] 2. Test the efficiency of the target

[0106] Design a fusion fragment (5’-ACGACGAAAACAGGTAAGAGCTAAGCTTCCTGCAGGTTCACTGCCGTATAGGCAG GGAGCGATGTTTCACAATGAGTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCGTGACTGGAAAGCAATCCTTCATTGTGAAACATCCGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAAGTTCACTGCCGTATAGGCAGGCCTGCCTTGGATCACTCGCGTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCATTCTATCCGGTGTTGTCGAGTGATCCAAGCGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAAGTTCACTGCCGTATAGGCAGAATGAAGTTGGCGGAGGAGGGTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTGAGAACCACCACATCCTCCGCCCGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAAGTTCACTGCCGTATAGGCAGGGATCCTTTCTCCATAATAATGTGTGAGT AGTTCC-3’, Sequence 7 in the Sequence Listing) according to the target designed in Step 1, synthesize it at Beijing Tianyi Huiyuan Co., Ltd., construct the fusion fragment onto the guide editing small vector TaU3-esgRNA vector, then transform Escherichia coli. After detecting by colony PCR and Sanger sequencing, extract the plasmid from the correctly sequenced bacterial solution in large quantity, and co-transfer it with the guide protein vector ePPEmax vector into wheat protoplasts. After culturing for 48 h, extract the genomic DNA of the protoplasts. Using this DNA as a template, design primers for different targets and amplify and construct a library, and detect the editing efficiency of each target by next-generation sequencing. After efficiency detection, the sequences of three targets (Target 1 - Target 3) with higher gene editing efficiency are shown in Table 1.

[0107] Table 1. Sequences of the finally screened targets

[0108]

[0109] Table 2. Partial amplification and sequencing primers

[0110]

[0111] 3. Obtaining recombinant vectors and genetic transformation

[0112] After efficiency detection, the targets with higher efficiency (Table 1) were concatenated onto the gene editing large vector pBUE414-ePPEmax-V223A vector to obtain the recombinant vector pBUE414-ePPEmax-V223A-gRNA123. Subsequently, the constructed vector was transformed into Agrobacterium tumefaciens EHA105 and sent to the transgenic platform for genetic transformation. The specific steps are as follows:

[0113] 3.1 PCR amplification

[0114] Using the amplification primer pair 2 in Table 2 as the PCR amplification primers, and the TaU3-esgRNA vector constructed in step 2 as the template, a PCR amplification product containing the concatenated sequence of the three targets in Table 1 was obtained by PCR amplification.

[0115] The PCR reaction system was as follows:

[0116]

[0117] 3.2 Obtaining the gene-guided editing recombinant vector

[0118] The PCR amplification product obtained in 3.1 was ligated to the pBUE414-ePPEmax-V223A vector by homologous recombination to obtain the gene-guided editing recombinant vector pBUE414-ePPEmax-V223A-gRNA123.

[0119] 3.3 Genetic transformation

[0120] The recombinant vector pBUE414-ePPEmax-V223A-gRNA123 obtained in step 3.2 was transformed into the competent cells of Escherichia coli Mach1-T1 (Shanghai Weidi Biotechnology Co., Ltd.) to obtain monoclonal transformed bacteria. The monoclonal transformed bacteria were propagated and the plasmid was extracted to obtain the recombinant vector plasmid pBUE414-ePPEmax-V223A-gRNA123.

[0121] The recombinant vector plasmid pBUE414-ePPEmax-V223A-gRNA was transformed into the competent cells of Agrobacterium tumefaciens EHA105 (Shanghai Weidi Biotechnology Co., Ltd.) to obtain recombinant Agrobacterium, named EHA105 / pBUE414-ePPEmax-V223A-gRNA123.

[0122] The recombinant Agrobacterium tumefaciens EHA105 / pBUE414-ePPEmax-V223A-gRNA123 was transformed into the wheat material Fielder to obtain transgenic T 0 generation wheat seedlings.

[0123] 4. Positive detection of transgenic seedlings and generation advancement of transgenic positive seedlings

[0124] The transgenic T0 generation seedlings obtained in step 3.3 were transferred to greenhouse culture soil for cultivation (cultivation conditions: 16 h light and 8 h dark). After 30 days, leaves were taken to extract DNA, and then PCR amplification was performed to identify the vector transformation situation and the target editing situation (primers are shown in Table 2).

[0125] The seeds from the T0 generation seedlings detected as positive (PCR product size is 765 bp) were sown, and the target editing situation was identified for each individual plant. The individual plants that met the expected editing and were homozygous were tagged and harvested, obtaining two homozygous transgenic editing lines, named type1 and type2 respectively. The editing types of the two homozygous transgenic editing lines are as Figure 1 shown:

[0126] In the type1 line, 12 nucleotides were inserted into the pinb gene (i.e., 3 nucleotides "TGT" were inserted between nucleotides 102 and 103 of Sequence 1 in the sequence listing, and at the same time, 3 nucleotides "TGC" were inserted between nucleotides 189 and 190 of Sequence 1, and at the same time, 6 nucleotides "TGTTGT" were inserted between nucleotides 306 - 307 of Sequence 1), obtaining the mutant gene pinb-m1 (a new haplotype of the pinb gene), thereby inserting four cysteine residues into the PINB protein to obtain the PINB-m1 mutant protein ( Figure 1 in type1).

[0127] The mutated pinb-m1 gene sequence in the above type1 mutant is as shown in Sequence 3, and the amino acid sequence of the PINB-m1 protein encoded by it is as shown in Sequence 4.

[0128] In the type2 line, 6 nucleotides were inserted into the pinb gene (i.e., 6 nucleotides "TGTTGT" were inserted between nucleotides 306 and 307 of Sequence 1 in the sequence listing), obtaining the mutant gene pinb-m2 (a new haplotype of the pinb gene), thereby inserting two cysteine residues into the PINB protein to obtain the PINB-m2 mutant protein ( Figure 1 in type2).

[0129] The mutated pinb-m2 gene sequence in the above type 2 mutant is shown in Sequence 5, and the amino acid sequence of the encoded PINB-m2 protein is shown in Sequence 6.

[0130] Sequence 3 (5’-3’):

[0131] ATGAAGACCTTATTCCTCCTAGCTCTCCTTGCTCTTGTAGCGAGCACAACCTTCGCGCAATACTCAGAAGTTGGCGGCTGGTACAATGAAGTTGGCGGAGGATGTGGTGGTTCTCAACAATGTCCGCAGGAGCGGCCGAAGCTAAGCTCTTGCAAGGATTACGTGATGGAGCGATGTTTCACAATGAAGGATTGCTTTCCAGTCACCTGGCCCACAAAATGGTGGAAGGGCGGCTGTGAGCATGAGGTTCGGGAGAAGTGCTGCAAGCAGCTGAGCCAGATAGCACCACAATGTCGCTGTGATTCTATCCGGTGTTGTCGAGTGATCCAAGGCAGGCTCGGTGGCTTCTTGGGCATTTGGCGAGGTGAGGTATTCAAACAACTTCAGAGGGCCCAGAGCCTCCCCTCAAAGTGCAACATGGGCGCCGACTGCAAGTTCCCTAGTGGCTATTACTGGTGA;

[0132] Sequence 4:

[0133] MKTLFLLALLALVASTTFAQYSEVGGWYNEVGGGCGGSQQCPQERPKLSSCKDYVME RCFTMKDCFPVTWPTKWWKGGCEHEVREKCCKQLSQIAPQCRCDSIRCCRVIQGRLGGFL GIWRGEVFKQLQRAQSLPSKCNMGADCKFPSGYYW;

[0134] Sequence 5 (5’-3’):

[0135] ATGAAGACCTTATTCCTCCTAGCTCTCCTTGCTCTTGTAGCGAGCACAACCTTCGCGCAATACTCAGAAGTTGGCGGCTGGTACAATGAAGTTGGCGGAGGAGGTGGTTCTCAACAATGTCCGCAGGAGCGGCCGAAGCTAAGCTCTTGCAAGGATTACGTGATGGAGCGATGTTTCACAATGAAGGATTTTCCAGTCACCTGGCCCACAAAATGGTGGAAGGGCGGCTGTGAGCATGAGGTTCGGGAGAAGTGCTGCAAGCAGCTGAGCCAGATAGCACCACAATGTCGCTGTGATTCTATCCGGTGTTGTCGAGTGATCCAAGGCAGGCTCGGTGGCTTCTTGGGCATTTGGCGAGGTGAGGTATTCAAACAACTTCAGAGGGCCCAGAGCCTCCCCTCAAAGTGCAACATGGGCGCCGACTGCAAGTTCCCTAGTGGCTATTACTGGTGA;

[0136] Sequence 6:

[0137] MKTLFLLALLALVASTTFAQYSEVGGWYNEVGGGGGSQQCPQERPKLSSCKDYVMER CFTMKDFPVTWPTKWWKGGCEHEVREKCCKQLSQIAPQCRCDSIRCCRVIQGRLGGFLGI WRGEVFKQLQRAQSLPSKCNMGADCKFPSGYYW。

[0138] 5. Phenotypic detection of gene-edited wheat

[0139] 5.1 Kernel hardness detection

[0140] Harvest the kernels of the wild-type Fielder lines of the pinb gene, and the gene-edited lines type1 and type2, and use the single-kernel characterization system (SKCS) method to determine the kernel hardness.

[0141] The detection results show that compared with the wild-type line Fielder, the kernel hardness of the new pinb haplotypes (the pinb gene haplotypes carried by type1 and type2) created by the present invention ( Figure 2 left middle figure) is significantly increased, as Figure 2 .

[0142] 5.2 Gluten index detection

[0143] The grains of the wild-type Fielder line of the pinb gene, the gene-edited lines type1 and type2 obtained from the harvest were ground into flour, and the gluten index of the flour was measured. The measurement method refers to the national standard GB / T14608-93.

[0144] The test results showed that compared with the wild-type line Fielder, the gluten index of the grains of the new pinb haplotype materials (the pinb gene haplotypes carried by type1 and type2) created by the present invention ( Figure 2 the right figure in the middle) also increased significantly.

[0145] Therefore, the newly created pinb gene-edited lines of the present invention and the pinb haplotypes carried by these lines can be applied to the genetic breeding of wheat grain hardness and / or gluten index traits. For example, taking Jimai 22 with relatively high grain hardness as an example, using Jimai 22 as the recurrent parent and the new pinb haplotype lines type1 and type2 of the present invention as the non-recurrent parents, continuous backcrossing was carried out. During the backcrossing process, the sequence differences of the pin genes were used as molecular markers in each backcross generation. Plants containing the new pinb type haplotypes of the present invention were screened as one of the parents for the next backcross generation. After multiple generations of backcrossing, they were allowed to self-cross. Plants containing homozygous new pinb haplotypes were screened by sequencing. Finally, new germplasms with the background of Jimai 22 and the new pinb haplotypes of the present invention were obtained, which can achieve the effect of singly improving grain hardness and gluten index without changing other excellent characteristics of Jimai 22, so as to meet the diverse processing needs of humans for wheat.

[0146] At the same time, wheat materials with relatively low grain hardness can be used as the recurrent parent, and the new pinb haplotype lines type1 and type2 of the present invention can be used as the non-recurrent parents for continuous backcrossing. During the backcrossing process, the sequence differences of the pinb gene are used as molecular markers in each backcross generation. Plants containing the new pinb type haplotypes of the present invention are screened as one of the parents for the next backcross generation. After multiple generations of backcrossing, they are allowed to self-cross. Plants containing homozygous new pinb haplotypes are screened by sequencing. Finally, new germplasms with the background of wheat materials with relatively low grain hardness and the new pinb haplotypes of the present invention are obtained, which can achieve the effect of singly improving grain hardness and gluten index without changing other excellent characteristics of the wheat materials with relatively low grain hardness, so as to meet the diverse processing needs of humans for wheat.

[0147] The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention and without the need for unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses, or improvements to the present invention, including changes made using conventional techniques known in the art that depart from the scope disclosed in this application.

Claims

1. Any of the following uses of a protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein: P1. Application in regulating wheat grain hardness or gluten index; P2. Application in the preparation of products for regulating wheat grain hardness or gluten index; P3, Application in wheat breeding; P4. Application in wheat quality improvement; The protein is the following protein: A1) The amino acid sequence is the protein of sequence 2 in the sequence list; A2) The amino acid sequence is the protein of sequence 4 in the sequence list; A3) The amino acid sequence is the protein of sequence 6 in the sequence list; A4) A protein derived from A1), A2) or A3) or having 80% or more identity with the protein shown in A1), A2) or A3) and having the same function, obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in A1), A2) or A3); A5) A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A1), A2) or A3).

2. Any of the following uses of the biological material related to the protein of claim 1: P1. Application in regulating wheat grain hardness or gluten index; P2. Application in the preparation of products for regulating wheat grain hardness or gluten index; P3, Application in wheat breeding; P4. Application in wheat quality improvement; The biological material is any of the following: B1) a nucleic acid molecule encoding the protein according to 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) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism 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); B6) transgenic plant tissue containing the nucleic acid molecule described in B1), or transgenic plant tissue containing the expression cassette described in B2); B7) a transgenic plant organ containing the nucleic acid molecule described in B1) or a transgenic plant organ containing the expression cassette described in B2); B8) a nucleic acid molecule that inhibits or reduces the expression of a gene encoding a protein described in A1) of claim 1 or the activity of a protein described in A1) of claim 1; B9) An expression cassette, a recombinant vector, a recombinant microorganism or a transgenic plant cell line containing the nucleic acid molecule described in B8).

3. The use according to claim 2, characterized in that: The nucleic acid molecule is a gene encoding the protein as shown below: b1) the nucleotide of the coding strand is a cDNA molecule or a DNA molecule of sequence 1 in the sequence list; b2) the nucleotide of the coding strand is a cDNA molecule or a DNA molecule of sequence 3 in the sequence list; b3) the nucleotide of the coding strand is a cDNA molecule or a DNA molecule of sequence 5 in the sequence list; b4) a DNA molecule having 90% or more identity with the nucleotide sequence defined in b1), b2) or b3), and encoding the protein described in claim 1; b5) a DNA molecule that hybridizes with the nucleotide sequence defined in b1), b2), b3) or b4) under stringent conditions and encodes the protein described in claim 1; B8) The nucleic acid molecule is a DNA molecule expressing a gRNA targeting the protein-coding gene described in A1) of claim 1 or a gRNA targeting the protein-coding gene described in A1) of claim 1.

4. The use according to any one of claims 1 to 3, characterized in that: The plant is any of the following: D1) Monocots, D2) Gramineae, D3) Gramineae plants, D4) Triticum, D5) Wheat.

5. A method for regulating or changing plant seed hardness and / or gluten index, comprising regulating or changing plant seed hardness and / or gluten index by inhibiting or reducing the expression level of a gene encoding the protein described in A1) of claim 1 or the activity of the protein described in A1) of claim 1 in the plant.

6. The method according to claim 5, characterized in that: The method comprises introducing into the plant a substance that reduces or inhibits the expression of the protein encoding gene described in A1) of claim 1; the substance that reduces or inhibits the expression of the protein encoding gene described in A1) of claim 1 is any of the following substances: c1) a nucleic acid molecule that inhibits or reduces the expression of the gene encoding the protein described in A1) of claim 1; c2) an expression cassette containing the nucleic acid molecule described in c1); c3) a recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2); c4) a recombinant microorganism containing the nucleic acid molecule described in c1), or a recombinant microorganism containing the expression cassette described in c2), or a recombinant microorganism containing the recombinant vector described in c3).

7. The method according to claim 5 or 6, characterized in that: The plant is any of the following: D1) Monocots, D2) Gramineae, D3) Gramineae plants, D4) Triticum, D5) Wheat.

8. Application of wheat containing a new pinb haplotype in wheat grain hardness and gluten index trait breeding or quality improvement; the new pinb haplotype corresponds to a variation on a chromosome of the wheat genome, the variation being at least one of the following mutations in the protein coding gene shown in sequence 1 in the sequence table of the wheat genome: 1) Three nucleotides "TGT" were inserted between nucleotides 102 and 103 of Sequence 1 in the sequence list, and three nucleotides "TGC" were inserted between nucleotides 189 and 190 of Sequence 1, and six nucleotides "TGTTGT" were inserted between nucleotides 306 and 307 of Sequence 1; 2) Six nucleotides "TGTTGT" were inserted between nucleotides 306 and 307 of sequence 1 in the sequence list.

9. A method for breeding wheat grain hardness and / or gluten index traits, characterized in that: The method comprises hybridizing wheat A containing a new pinb haplotype with wheat B to obtain F1 generation wheat, using the wheat B as a recurrent parent, backcrossing the F1 generation wheat with the wheat B to obtain backcross offspring, selecting wheat containing a new pinb haplotype from the backcross offspring for self-pollination to obtain self-pollination offspring, selecting wheat homozygous for the new pinb haplotype from the self-pollination offspring, and obtaining target wheat having a grain hardness and / or a gluten index different from that of the wheat B. The new pinb haplotype corresponds to a variation on a chromosome of the wheat genome, and the variation is to perform at least one of the following mutations on the nucleotide sequence shown in sequence 1 in the sequence table of the wheat genome: 1) Three nucleotides "TGT" were inserted between nucleotides 102 and 103 of Sequence 1 in the sequence list, and three nucleotides "TGC" were inserted between nucleotides 189 and 190 of Sequence 1, and six nucleotides "TGTTGT" were inserted between nucleotides 306 and 307 of Sequence 1; 2) Six nucleotides "TGTTGT" were inserted between nucleotides 306 and 307 of sequence 1 in the sequence list.

10. The protein according to claim 1 and / or the biomaterial according to claim 2 or 3.

Images