CN110616220A - Method for improving hardness of wheat grains - Google Patents

Abstract

The invention discloses a method for improving the hardness of wheat grains. The invention discloses a method for improving the hardness of wheat grains, which comprises the following steps: A1) editing a target DNA fragment in a receptor wheat genome by using a gene editing method to obtain a gene-edited wheat with the target DNA fragment changed; the sequence of the target DNA fragment is sequence 5 in the sequence table; A2) the target wheat with grain hardness increased compared with receptor wheat is obtained by screening from the gene editing wheat. The method for cultivating wheat with improved wheat grain hardness and the used reagent set provide powerful tools for improving wheat processing quality, and have wide application prospects.

Description

Method for improving hardness of wheat grains

Technical Field

The invention relates to the field of biotechnology, and discloses a method for improving the hardness of wheat grains.

Background

The hardness of wheat grains refers to the amount of force required to grind the wheat grains. According to the hardness of grains, common wheat is internationally divided into hard wheat and soft wheat. The hard wheat grains have high hardness, high protein content, high gluten strength, relatively thick flour particles and high water absorption rate, and are suitable for baking bread; the soft wheat has soft endosperm, low protein content, weak gluten, fine flour particles and low water absorption, and is suitable for making various foods such as biscuits, cakes and the like. The semi-hard wheat between the two has the same protein content and gluten strength, can be used for making soft and hard bread with short fermentation time and various types of thin pancakes, and is also suitable for making food such as Chinese steamed bread, noodles and the like. Grain hardness is a main index of international wheat market classification, and the Chinese (GB1351-1999) distinguishes soft from hard by cutin rate, wherein the cutin rate is not less than 70% of hard, and the flour rate is not less than 70% of soft. However, cutin rate and hardness are two different concepts, and wheat hardness in China is a mixed population and is not matched with the quantity and quality of protein. Although the protein content and quality of part of high-quality wheat varieties widely popularized in China are close to the standard of high-quality bread wheat, the grain hardness is soft, the water absorption of flour is low, and the commercial value of the flour is seriously influenced, so that the genetic improvement of hardness becomes one of the main targets of wheat quality improvement in China.

Disclosure of Invention

The invention aims to solve the technical problem of how to improve the hardness of wheat grains.

In order to solve the technical problems, the invention firstly provides a method for cultivating wheat with improved wheat grain hardness, which comprises A1) and A2):

A1) editing a target DNA fragment in a receptor wheat genome by using a gene editing method to obtain a gene-edited wheat with the target DNA fragment changed; the sequence of the target DNA fragment is sequence 5 in the sequence table;

A2) and screening the target wheat with grain hardness increased compared with the receptor wheat from the gene editing wheat.

The invention also provides a method for improving the hardness of wheat grains, which comprises A1) and A2):

A1) editing a target DNA fragment in a receptor wheat genome by using a gene editing method to obtain a gene-edited wheat with the target DNA fragment changed; the sequence of the target DNA fragment is sequence 5 in the sequence table;

A2) and screening the target wheat with grain hardness increased compared with the receptor wheat from the gene editing wheat to realize the improvement of the grain hardness of the wheat.

Above, the gene editing method can be performed using the CRISPR/Cas9 system.

The target sequence of sgRNA in the CRISPR/Cas9 system can be sequence 1 in the sequence table.

The sequence of the sgRNA can be a sequence 2 in a sequence table.

Compared with the acceptor wheat, the target DNA fragment of the target wheat can lack 94 th to 127 th positions of a sequence 5 in a sequence table.

Editing the target DNA fragment in the genome of the receptor wheat by using a CRISPR/Cas9 system can be realized by introducing an expression cassette (marked as a sgRNA expression cassette) containing a coding gene of the sgRNA and an expression cassette (marked as a Cas9 expression cassette) containing a coding gene of a Cas9 protein into the receptor wheat.

The sgRNA expression cassette refers to a DNA capable of transcribing the sgRNA in a host cell, and the DNA may include not only a promoter that initiates transcription of the sgRNA-encoding gene but also a terminator that terminates transcription of the sgRNA-encoding gene. Further, the expression cassette may also include an enhancer sequence. In the sgRNA expression cassette, transcription of a gene encoding the sgRNA can be driven by a wheat U3 promoter TaU 3.

The Cas9 expression cassette refers to DNA capable of expressing Cas9 protein in a host cell, which may include not only a promoter for initiating transcription of the Cas9 protein-encoding gene, but also a terminator for terminating transcription of the Cas9 protein-encoding gene. Further, the expression cassette may also include an enhancer sequence. In the Cas9 expression cassette, the expression of the encoding gene of Cas9 can be specifically driven by the maize ubiqutin promoter.

The invention also provides the following products I or II:

I. the sgRNA;

II. A biomaterial associated with the sgRNA, any of the following B1) to B9):

B1) a nucleic acid molecule encoding the sgRNA;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector;

B5) a transgenic plant cell line comprising B1) the nucleic acid molecule or a transgenic plant cell line comprising B2) the expression cassette;

B6) transgenic plant tissue comprising the nucleic acid molecule of B1) or transgenic plant tissue comprising the expression cassette of B2);

B7) a transgenic plant organ containing B1) the nucleic acid molecule or a transgenic plant organ containing B2) the expression cassette.

In the product, the nucleic acid molecule B1) can be a DNA molecule shown in a sequence obtained by replacing all U in a sequence 2 in a sequence table with T.

B2) The expression cassette can be the sgRNA expression cassette. The sequence of the sgRNA expression cassette can be sequence 6.

The invention also provides a kit of reagents, which kit is III or IV:

III, a kit consisting of the product and a Cas9 protein;

IV, a kit consisting of the product and a biological material associated with Cas9 protein; the biomaterial is any one of the following C1) to C9):

C1) a nucleic acid molecule encoding a Cas9 protein;

C2) an expression cassette comprising the nucleic acid molecule of C1);

C3) a recombinant vector comprising the nucleic acid molecule of C1), or a recombinant vector comprising the expression cassette of C2);

C4) a recombinant microorganism containing C1) the nucleic acid molecule, or a recombinant microorganism containing C2) the expression cassette, or a recombinant microorganism containing C3) the recombinant vector;

C5) a transgenic plant cell line comprising C1) the nucleic acid molecule or a transgenic plant cell line comprising C2) the expression cassette;

C6) transgenic plant tissue comprising the nucleic acid molecule of B1), or transgenic plant tissue comprising the expression cassette of C2);

C7) a transgenic plant organ containing B1) the nucleic acid molecule or a transgenic plant organ containing C2) the expression cassette.

In the kit, the Cas9 protein can be a protein encoded by the DNA fragment shown in the 4530-8630 site of the sequence 3 in the sequence table.

C1) The nucleic acid molecule can be a DNA molecule shown in the 4530-8630 site of the sequence 3 in the sequence table.

C2) The expression cassette may be the Cas9 expression cassette. The sequence of the Cas9 expression cassette can be 2403-9201 of sequence 3.

Existing expression vectors can be used to construct recombinant vectors containing the sgRNA expression cassette and/or the Cas9 expression cassette.

As above, the vector may be a plasmid, cosmid, phage or viral vector. The vector may specifically be a pBUE411 vector.

The recombinant vector can be pBUE411-Cas 9-gR. The pBUE411-Cas9-gR is a recombinant vector obtained by inserting a DNA fragment shown in a sequence 1 in a sequence table between BsaI recognition sequences of a pBUE411 vector by using BsaI, and a T-DNA region of the pBUE411-Cas9-gR contains a Cas9 expression cassette and a sgRNA expression cassette, can express Cas9 and can transcribe the sgRNA shown in a sequence 2 in the sequence table.

The microorganism may be a yeast, bacterium, algae or fungus. Wherein the bacteria can be Agrobacterium.

The transgenic plant cell line, the transgenic plant tissue and the transgenic plant organ do not comprise propagation material.

The invention also provides any of the following applications:

x1) use of the product or the kit for increasing the hardness of wheat grain;

x2) the use of the product or the kit for the preparation of a product for increasing the hardness of wheat grain;

x3) the use of the product or the kit for breeding wheat with increased grain hardness;

x4) the use of the product or the kit for the preparation of a product for breeding wheat with increased grain hardness;

x5) use of the product or the kit in wheat breeding;

x6) the method for improving wheat grain hardness by cultivating wheat or the application of the method for improving wheat grain hardness in wheat breeding.

The method for cultivating wheat with improved wheat grain hardness and the kit used in the method provide a powerful tool for improving wheat processing quality, and have wide application prospects.

Drawings

FIG. 1 is the identification of transgenic lines. Samples 1-10 are T0 generation transgenic lines, 11 is a positive control, 12 is a negative control, wherein two bands appear in samples 1,3,4, 5, 6,7,8 and 10, the result is consistent with the result of the positive control 11, and the samples are positive transgenic lines; 2 and 9, a band appears, which is consistent with a negative control and is a negative transgenic line.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.

High-fidelity enzyme required by PCR amplification, T4 ligase required by DNA fragment connection, gel recovery kit required by enzyme digestion fragment recovery and plasmid extraction kit are all products of Dalibao biological company. The plasmid extraction kit is a biological product of Tiangen, the inorganic salt required by the preparation of the culture medium is a product of a national medicine group, and the vitamins, antibiotics and hormones are products of Sigma company.

Reagents such as T4 ligase, DNA Marker DL2000 and the like are products of Takara company; fastpfu, 2 × Taq Mix, DH5 α and the like are products of Beijing Quanzijin company; restriction enzyme BsaI-HF is a product of NEB.

The wheat variety K35 (early-maturing wheat new germplasm K35 cultured by the easily-flowering medicine; Suisuxia, Huangyan, Chuxisheng, Li-gen, Qiqing, Shandong agricultural science, No. 1-116-plus-117 in 2007) is a new germplasm with good tissue culture capability which is self-bred by crops in the Shandong province academy of farming, the public can obtain the biological material from the applicant, and the biological material is only used for repeating the related experiments of the invention and cannot be used for other purposes.

The pBUE411 vector is represented by a sequence 3 (17431bp) in the sequence table, is provided by Susan Youth task group of Chinese agricultural university, and can be obtained by the public from the institute of crop science of Chinese agricultural academy after the Susan Youth task group of Chinese agricultural university agrees. The pBUE411 vector contains the wheat U3 promoter TaU3 to start sgRNA.

Experimental example 1 preparation of wheat Material with grain hardness increased

First, construction of expression vector

A DNA fragment shown as a sequence 1 in a sequence table is used as a target sequence of sgRNA to prepare a recombinant vector used by a CRISPR/Cas9 system, and the steps are as follows:

1. synthesizing single-stranded DNA: single-stranded DNAs with the names of gR1-TaU3-F and gR1-TaU3-R were synthesized, and the sequences of gR1-TaU3-F and gR1-TaU3-R were as follows:

gR1-TaU3-F：5′-GGCGACTTAGCTTCGGCCGCTCCTG-3′；

gR1-TaU3-R：5′-AAACCAGGAGCGGCCGAAGCTAAGT-3′。

where the underlined positions match the sequence in the pBUE411 vector.

2. Preparation of double-stranded DNA: gR1-TaU3-F and gR1-TaU3-R are respectively phosphorylated and then annealed in the same system to form double-stranded DNA.

3. Linearization of the pBUE411 vector: the pBUE411 vector was digested with BsaI to obtain a linear vector fragment.

4. Preparation of recombinant vector: and (3) connecting the double-stranded DNA obtained in the step (2) with the linear vector fragment obtained in the step (3) to obtain a recombinant vector, marking the recombinant vector as pBUE411-Cas9-gR, inserting the DNA fragment shown in the sequence 1 in the sequence table between BsaI recognition sequences of the pBUE411 vector by using BsaI to obtain the pBUE411-Cas9-gR, wherein a T-DNA region of the recombinant vector contains a bar expression cassette, a Cas9 expression cassette and a sgRNA expression cassette, and can express bar and Cas9 and can transcribe the sgRNA shown in the sequence 2 in the sequence table. The sequence of the Cas9 expression cassette is 2403-9201 th site of the sequence 3 in the sequence table, the 4530-8630 th site of the sequence 3 is a Cas9 coding gene sequence, the upstream of the Cas9 coding gene sequence contains a maize ubiqutin promoter, and the expression of the Cas9 is driven by the maize ubiqutin promoter; the sequence of the sgRNA expression cassette is sequence 6 in the sequence table, the upstream of the coding gene (1750-1846 site of sequence 6) of the sgRNA shown in sequence 2 contains a wheat U3 promoter TaU3, and the transcription of the sgRNA is driven by a wheat U3 promoter TaU 3; the bar expression cassette contains the gene coding for bar.

II, activity of pBUE411-Cas9-gR in transcribing sgRNA

Protoplast was used to detect the sgRNA transcription activity of pBUE411-Cas9-gR, as follows:

1. protoplast transformation: reference wheat protoplast preparation and transformation and appropriate adjustment. Growing wheat K35 seed at 25 deg.C for 7-10 days, preparing protoplast from 15-20 fresh leaves, and culturing with MMG solution (0.4Mmannitol,15mM MgCl)_2,4mM MES, balance water) to obtain a protoplast suspension. Transforming the recombinant vector pBUE411-Cas9-gR obtained in the step one into a protoplast by a PEG mediated method, finally, culturing the protoplast containing the pBUE411-Cas9-gR for 2-3 days at 25 ℃ in the dark or in the weak light, centrifuging, collecting the wheat protoplast, and extracting the genome DNA of the wheat protoplast.

2. Gene editing result detection

The target DNA fragment is amplified using specific primers. Forward and reverse Barcode were added to the 5' end of the PCR primers, respectively, for library construction (primers PF and PR in table 1, underlined Barcode). Regions corresponding to the upstream and downstream regions of the target sequence were amplified by primers with corresponding barcodes. After amplification, equal amounts of PCR products were mixed and a 2X150bp paired-end library was constructed according to the literature (German MA, Luo S, Schroth G, Meyers BC, Green PJ: Construction of Parallel Analysis of RNA Ends (PARE) libraries for the study of the captured miRNA targets and the RNA degradome [ J ]. Nat Protoc 2009,4(3):356-362.) for Illumina sequencing by Novista, and the resulting sequenced sequences were aligned to wheat genomic sequences. Sequence changes (including substitutions, insertions and deletions) that occur at the DNA segment of interest are considered mutations.

TABLE 1 primer sequences

Transforming the wheat protoplast by a PEG method, extracting the wheat genome DNA from the wheat protoplast which is incubated for 2-3 days after transformation by using a plant genome DNA extraction kit, amplifying a target DNA fragment, and then establishing a library for sequencing. Sequencing results showed that the editing types of the target DNA fragments were mainly single nucleotide mutations (SNPs) and indels (insertions/deletions). Among them, the target DNA fragment had a mutation efficiency of 3.86%, including 3.83% of SNPs (21982 single nucleotide mutations) and 0.03% of insertions/deletions (178 indels). The mutation site detection of the target DNA fragment discovers the substitution and deletion of nucleotides at the editing site (the 4 th site before the PAM sequence), including the substitution and deletion of mononucleotide and the like. In addition, mutations were also detected at other sites before PAM.

Identification of common wheat and positive transgenic wheat transformed by pBUE411-Cas9-gR

And (3) after the pBUE411-Cas9-gR obtained in the step one is introduced into agrobacterium EHA105, transforming a wheat variety K35 by using an agrobacterium-mediated wheat genetic transformation method to obtain a T0 generation transgenic strain.

The nontransgenic wheat variety K35 is used as a negative control, and a bar gene detection kit Quickstix of Meitson scientific and technical Co Ltd is utilized^TMAnd (3) detecting the T0 generation transgenic lines, screening to obtain positive transgenic lines, wherein partial detection results are shown in figure 1, and samples 1,3,4, 5, 6,7,8 and 10 in figure 1 are all positive transgenic lines.

Fourth, detection of target DNA fragment mutation condition

Whether the target DNA fragment is successfully edited needs sequencing for identification, and the invention adopts a Hi-TOM gene editing site detection kit produced by Tianjinnuo grass genesis company to detect whether the target DNA fragment of the positive transgenic plant strain obtained by screening in the step three is mutated. The kit completes the high-throughput library building process by a PCR method, and then directly analyzes the variation information of multiple samples and multiple sites by Hi-TOM online software. And (3) amplifying the A by using specific primers on both sides of the target sequence, and sending an amplification product to a Nordheim source for sequencing after a library is built.

A strain with mutation of a target DNA fragment is detected by using a Hi-TOM gene editing site detection kit, and the number of the strain is No. 47 wheat. The sequence of the target DNA fragment of No. 47 wheat is sequence 4 in the sequence table, and the sequence of the target DNA fragment of wheat variety K35 is sequence 5 in the sequence table. Compared with wheat variety K35, wheat No. 47 has 34 nucleotides missing in the target DNA fragment.

And performing PCR amplification and sequencing on the genome DNA of the No. 47 wheat by using the primer pair A, and verifying the mutation condition of the No. 47 wheat at the target DNA fragment by using the wheat variety K35 as a control. The sequences of primer pair a are as follows: 5'-TGTAGCGAGCACAACCTT-3', 5'-GTGGTGCTATCTGGCTCA-3' are provided.

Hardness analysis of No. five and 47 wheat grains

And (3) breeding No. 47 wheat to T2 generation, collecting seeds of a strain 47-1-3 with homozygous mutation of a target DNA fragment, placing the seeds and the seeds of the wheat variety K35 harvested at the same time under the same condition for 3 days, and controlling the water content to be 11-13%. Hardness values were determined for 300 wheat samples of 47-1-3 and wheat variety K35, respectively, using a Perten model 4100 Single grain hardness tester (SKCS). Results show that the average grain hardness of 47-1-3 is 62, while the average grain hardness of the wheat variety K35 is 45, compared with the wheat variety K35, the grain hardness of 47-1-3 is obviously improved by 37.8%, and the wheat with high grain hardness is successfully obtained.

<110> institute of agricultural sciences of Shandong province

<120> method for improving hardness of wheat grains

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<170> PatentIn version 3.5

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acaaccagct cttcgaggag aaccccatta atgcgtcagg cgtcgacgcg aaggctatcc 5160

tgtccgctag gctctcgaag tctcggcgcc tcgagaacct gatcgcccag ctgccgggcg 5220

agaagaagaa cggcctgttc gggaatctca ttgcgctcag cctggggctc acgcccaact 5280

tcaagtcgaa tttcgatctc gctgaggacg ccaagctgca gctctccaag gacacatacg 5340

acgatgacct ggataacctc ctggcccaga tcggcgatca gtacgcggac ctgttcctcg 5400

ctgccaagaa tctgtcggac gccatcctcc tgtctgatat tctcagggtg aacaccgaga 5460

ttacgaaggc tccgctctca gcctccatga tcaagcgcta cgacgagcac catcaggatc 5520

tgaccctcct gaaggcgctg gtcaggcagc agctccccga gaagtacaag gagatcttct 5580

tcgatcagtc gaagaacggc tacgctgggt acattgacgg cggggcctct caggaggagt 5640

tctacaagtt catcaagccg attctggaga agatggacgg cacggaggag ctgctggtga 5700

agctcaatcg cgaggacctc ctgaggaagc agcggacatt cgataacggc agcatcccac 5760

accagattca tctcggggag ctgcacgcta tcctgaggag gcaggaggac ttctaccctt 5820

tcctcaagga taaccgcgag aagatcgaga agattctgac tttcaggatc ccgtactacg 5880

tcggcccact cgctaggggc aactcccgct tcgcttggat gacccgcaag tcagaggaga 5940

cgatcacgcc gtggaacttc gaggaggtgg tcgacaaggg cgctagcgct cagtcgttca 6000

tcgagaggat gacgaatttc gacaagaacc tgccaaatga gaaggtgctc cctaagcact 6060

cgctcctgta cgagtacttc acagtctaca acgagctgac taaggtgaag tatgtgaccg 6120

agggcatgag gaagccggct ttcctgtctg gggagcagaa gaaggccatc gtggacctcc 6180

tgttcaagac caaccggaag gtcacggtta agcagctcaa ggaggactac ttcaagaaga 6240

ttgagtgctt cgattcggtc gagatctctg gcgttgagga ccgcttcaac gcctccctgg 6300

ggacctacca cgatctcctg aagatcatta aggataagga cttcctggac aacgaggaga 6360

atgaggatat cctcgaggac attgtgctga cactcactct gttcgaggac cgggagatga 6420

tcgaggagcg cctgaagact tacgcccatc tcttcgatga caaggtcatg aagcagctca 6480

agaggaggag gtacaccggc tgggggaggc tgagcaggaa gctcatcaac ggcattcggg 6540

acaagcagtc cgggaagacg atcctcgact tcctgaagag cgatggcttc gcgaaccgca 6600

atttcatgca gctgattcac gatgacagcc tcacattcaa ggaggatatc cagaaggctc 6660

aggtgagcgg ccagggggac tcgctgcacg agcatatcgc gaacctcgct ggctcgccag 6720

ctatcaagaa ggggattctg cagaccgtga aggttgtgga cgagctggtg aaggtcatgg 6780

gcaggcacaa gcctgagaac atcgtcattg agatggcccg ggagaatcag accacgcaga 6840

agggccagaa gaactcacgc gagaggatga agaggatcga ggagggcatt aaggagctgg 6900

ggtcccagat cctcaaggag cacccggtgg agaacacgca gctgcagaat gagaagctct 6960

acctgtacta cctccagaat ggccgcgata tgtatgtgga ccaggagctg gatattaaca 7020

ggctcagcga ttacgacgtc gatcatatcg ttccacagtc attcctgaag gatgactcca 7080

ttgacaacaa ggtcctcacc aggtcggaca agaaccgggg caagtctgat aatgttcctt 7140

cagaggaggt cgttaagaag atgaagaact actggcgcca gctcctgaat gccaagctga 7200

tcacgcagcg gaagttcgat aacctcacaa aggctgagag gggcgggctc tctgagctgg 7260

acaaggcggg cttcatcaag aggcagctgg tcgagacacg gcagatcact aagcacgttg 7320

cgcagattct cgactcacgg atgaacacta agtacgatga gaatgacaag ctgatccgcg 7380

aggtgaaggt catcaccctg aagtcaaagc tcgtctccga cttcaggaag gatttccagt 7440

tctacaaggt tcgggagatc aacaattacc accatgccca tgacgcgtac ctgaacgcgg 7500

tggtcggcac agctctgatc aagaagtacc caaagctcga gagcgagttc gtgtacgggg 7560

actacaaggt ttacgatgtg aggaagatga tcgccaagtc ggagcaggag attggcaagg 7620

ctaccgccaa gtacttcttc tactctaaca ttatgaattt cttcaagaca gagatcactc 7680

tggccaatgg cgagatccgg aagcgccccc tcatcgagac gaacggcgag acgggggaga 7740

tcgtgtggga caagggcagg gatttcgcga ccgtcaggaa ggttctctcc atgccacaag 7800

tgaatatcgt caagaagaca gaggtccaga ctggcgggtt ctctaaggag tcaattctgc 7860

ctaagcggaa cagcgacaag ctcatcgccc gcaagaagga ctgggatccg aagaagtacg 7920

gcgggttcga cagccccact gtggcctact cggtcctggt tgtggcgaag gttgagaagg 7980

gcaagtccaa gaagctcaag agcgtgaagg agctgctggg gatcacgatt atggagcgct 8040

ccagcttcga gaagaacccg atcgatttcc tggaggcgaa gggctacaag gaggtgaaga 8100

aggacctgat cattaagctc cccaagtact cactcttcga gctggagaac ggcaggaagc 8160

ggatgctggc ttccgctggc gagctgcaga aggggaacga gctggctctg ccgtccaagt 8220

atgtgaactt cctctacctg gcctcccact acgagaagct caagggcagc cccgaggaca 8280

acgagcagaa gcagctgttc gtcgagcagc acaagcatta cctcgacgag atcattgagc 8340

agatttccga gttctccaag cgcgtgatcc tggccgacgc gaatctggat aaggtcctct 8400

ccgcgtacaa caagcaccgc gacaagccaa tcagggagca ggctgagaat atcattcatc 8460

tcttcaccct gacgaacctc ggcgcccctg ctgctttcaa gtacttcgac acaactatcg 8520

atcgcaagag gtacacaagc actaaggagg tcctggacgc gaccctcatc caccagtcga 8580

ttaccggcct ctacgagacg cgcatcgacc tgtctcagct cgggggcgac aagcggccag 8640

cggcgacgaa gaaggcgggg caggcgaaga agaagaagtg agctcagagc tttcgttcgt 8700

atcatcggtt tcgacaacgt tcgtcaagtt caatgcatca gtttcattgc gcacacacca 8760

gaatcctact gagtttgagt attatggcat tgggaaaact gtttttcttg taccatttgt 8820

tgtgcttgta atttactgtg ttttttattc ggttttcgct atcgaactgt gaaatggaaa 8880

tggatggaga agagttaatg aatgatatgg tccttttgtt cattctcaaa ttaatattat 8940

ttgttttttc tcttatttgt tgtgtgttga atttgaaatt ataagagata tgcaaacatt 9000

ttgttttgag taaaaatgtg tcaaatcgtg gcctctaatg accgaagtta atatgaggag 9060

taaaacactt gtagttgtac cattatgctt attcactagg caacaaatat attttcagac 9120

ctagaaaagc tgcaaatgtt actgaataca agtatgtcct cttgtgtttt agacatttat 9180

gaactttcct ttatgtaatt ttccagaatc cttgtcagat tctaatcatt gctttataat 9240

tatagttata ctcatggatt tgtagttgag tatgaaaata ttttttaatg cattttatga 9300

cttgccaatt gattgacaac gaattcgtaa tcatggtcat agctgtttcc tgtgtgaaat 9360

tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg 9420

ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag 9480

tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 9540

ttgcgtattg gctagagcag cttgccaaca tggtggagca cgacactctc gtctactcca 9600

agaatatcaa agatacagtc tcagaagacc aaagggctat tgagactttt caacaaaggg 9660

taatatcggg aaacctcctc ggattccatt gcccagctat ctgtcacttc atcaaaagga 9720

cagtagaaaa ggaaggtggc acctacaaat gccatcattg cgataaagga aaggctatcg 9780

ttcaagatgc ctctgccgac agtggtccca aagatggacc cccacccacg aggagcatcg 9840

tggaaaaaga agacgttcca accacgtctt caaagcaagt ggattgatgt gataacatgg 9900

tggagcacga cactctcgtc tactccaaga atatcaaaga tacagtctca gaagaccaaa 9960

gggctattga gacttttcaa caaagggtaa tatcgggaaa cctcctcgga ttccattgcc 10020

cagctatctg tcacttcatc aaaaggacag tagaaaagga aggtggcacc tacaaatgcc 10080

atcattgcga taaaggaaag gctatcgttc aagatgcctc tgccgacagt ggtcccaaag 10140

atggaccccc acccacgagg agcatcgtgg aaaaagaaga cgttccaacc acgtcttcaa 10200

agcaagtgga ttgatgtgat atctccactg acgtaaggga tgacgcacaa tcccactatc 10260

cttcgcaaga ccttcctcta tataaggaag ttcatttcat ttggagagga cacgctgaaa 10320

tcaccagtct ctctctacaa atctatctct ctcgagtcta ccatgagccc agaacgacgc 10380

ccggccgaca tccgccgtgc caccgaggcg gacatgccgg cggtctgcac catcgtcaac 10440

cactacatcg agacaagcac ggtcaacttc cgtaccgagc cgcaggaacc gcaggagtgg 10500

acggacgacc tcgtccgtct gcgggagcgc tatccctggc tcgtcgccga ggtggacggc 10560

gaggtcgccg gcatcgccta cgcgggcccc tggaaggcac gcaacgccta cgactggacg 10620

gccgagtcga ccgtgtacgt ctccccccgc caccagcgga cgggactggg ctccacgctc 10680

tacacccacc tgctgaagtc cctggaggca cagggcttca agagcgtggt cgctgtcatc 10740

gggctgccca acgacccgag cgtgcgcatg cacgaggcgc tcggatatgc cccccgcggc 10800

atgctgcggg cggccggctt caagcacggg aactggcatg acgtgggttt ctggcagctg 10860

gacttcagcc tgccggtacc gccccgtccg gtcctgcccg tcaccgagat ttgactcgag 10920

tttctccata ataatgtgtg agtagttccc agataaggga attagggttc ctatagggtt 10980

tcgctcatgt gttgagcata taagaaaccc ttagtatgta tttgtatttg taaaatactt 11040

ctatcaataa aatttctaat tcctaaaacc aaaatccagt actaaaatcc agatcccccg 11100

aattaattcg gcgttaattc agtacattaa aaacgtccgc aatgtgttat taagttgtct 11160

aagcgtcaat ttgtttacac cacaatatat cctgccacca gccagccaac agctccccga 11220

ccggcagctc ggcacaaaat caccactcga tacaggcagc ccatcagtcc gggacggcgt 11280

cagcgggaga gccgttgtaa ggcggcagac tttgctcatg ttaccgatgc tattcggaag 11340

aacggcaact aagctgccgg gtttgaaaca cggatgatct cgcggagggt agcatgttga 11400

ttgtaacgat gacagagcgt tgctgcctgt gatcaccgcg gtttcaaaat cggctccgtc 11460

gatactatgt tatacgccaa ctttgaaaac aactttgaaa aagctgtttt ctggtattta 11520

aggttttaga atgcaaggaa cagtgaattg gagttcgtct tgttataatt agcttcttgg 11580

ggtatcttta aatactgtag aaaagaggaa ggaaataata aatggctaaa atgagaatat 11640

caccggaatt gaaaaaactg atcgaaaaat accgctgcgt aaaagatacg gaaggaatgt 11700

ctcctgctaa ggtatataag ctggtgggag aaaatgaaaa cctatattta aaaatgacgg 11760

acagccggta taaagggacc acctatgatg tggaacggga aaaggacatg atgctatggc 11820

tggaaggaaa gctgcctgtt ccaaaggtcc tgcactttga acggcatgat ggctggagca 11880

atctgctcat gagtgaggcc gatggcgtcc tttgctcgga agagtatgaa gatgaacaaa 11940

gccctgaaaa gattatcgag ctgtatgcgg agtgcatcag gctctttcac tccatcgaca 12000

tatcggattg tccctatacg aatagcttag acagccgctt agccgaattg gattacttac 12060

tgaataacga tctggccgat gtggattgcg aaaactggga agaagacact ccatttaaag 12120

atccgcgcga gctgtatgat tttttaaaga cggaaaagcc cgaagaggaa cttgtctttt 12180

cccacggcga cctgggagac agcaacatct ttgtgaaaga tggcaaagta agtggcttta 12240

ttgatcttgg gagaagcggc agggcggaca agtggtatga cattgccttc tgcgtccggt 12300

cgatcaggga ggatatcggg gaagaacagt atgtcgagct attttttgac ttactgggga 12360

tcaagcctga ttgggagaaa ataaaatatt atattttact ggatgaattg ttttagtacc 12420

tagaatgcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg 12480

tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc 12540

aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc 12600

tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt 12660

agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc 12720

taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact 12780

caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac 12840

agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag 12900

aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg 12960

gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg 13020

tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga 13080

gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt 13140

ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct 13200

ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg 13260

aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac 13320

accgcatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata 13380

cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc 13440

tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt 13500

ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcaggg 13560

tgccttgatg tgggcgccgg cggtcgagtg gcgacggcgc ggcttgtccg cgccctggta 13620

gattgcctgg ccgtaggcca gccatttttg agcggccagc ggccgcgata ggccgacgcg 13680

aagcggcggg gcgtagggag cgcagcgacc gaagggtagg cgctttttgc agctcttcgg 13740

ctgtgcgctg gccagacagt tatgcacagg ccaggcgggt tttaagagtt ttaataagtt 13800

ttaaagagtt ttaggcggaa aaatcgcctt ttttctcttt tatatcagtc acttacatgt 13860

gtgaccggtt cccaatgtac ggctttgggt tcccaatgta cgggttccgg ttcccaatgt 13920

acggctttgg gttcccaatg tacgtgctat ccacaggaaa cagacctttt cgaccttttt 13980

cccctgctag ggcaatttgc cctagcatct gctccgtaca ttaggaaccg gcggatgctt 14040

cgccctcgat caggttgcgg tagcgcatga ctaggatcgg gccagcctgc cccgcctcct 14100

ccttcaaatc gtactccggc aggtcatttg acccgatcag cttgcgcacg gtgaaacaga 14160

acttcttgaa ctctccggcg ctgccactgc gttcgtagat cgtcttgaac aaccatctgg 14220

cttctgcctt gcctgcggcg cggcgtgcca ggcggtagag aaaacggccg atgccgggat 14280

cgatcaaaaa gtaatcgggg tgaaccgtca gcacgtccgg gttcttgcct tctgtgatct 14340

cgcggtacat ccaatcagct agctcgatct cgatgtactc cggccgcccg gtttcgctct 14400

ttacgatctt gtagcggcta atcaaggctt caccctcgga taccgtcacc aggcggccgt 14460

tcttggcctt cttcgtacgc tgcatggcaa cgtgcgtggt gtttaaccga atgcaggttt 14520

ctaccaggtc gtctttctgc tttccgccat cggctcgccg gcagaacttg agtacgtccg 14580

caacgtgtgg acggaacacg cggccgggct tgtctccctt cccttcccgg tatcggttca 14640

tggattcggt tagatgggaa accgccatca gtaccaggtc gtaatcccac acactggcca 14700

tgccggccgg ccctgcggaa acctctacgt gcccgtctgg aagctcgtag cggatcacct 14760

cgccagctcg tcggtcacgc ttcgacagac ggaaaacggc cacgtccatg atgctgcgac 14820

tatcgcgggt gcccacgtca tagagcatcg gaacgaaaaa atctggttgc tcgtcgccct 14880

tgggcggctt cctaatcgac ggcgcaccgg ctgccggcgg ttgccgggat tctttgcgga 14940

ttcgatcagc ggccgcttgc cacgattcac cggggcgtgc ttctgcctcg atgcgttgcc 15000

gctgggcggc ctgcgcggcc ttcaacttct ccaccaggtc atcacccagc gccgcgccga 15060

tttgtaccgg gccggatggt ttgcgaccgc tcacgccgat tcctcgggct tgggggttcc 15120

agtgccattg cagggccggc agacaaccca gccgcttacg cctggccaac cgcccgttcc 15180

tccacacatg gggcattcca cggcgtcggt gcctggttgt tcttgatttt ccatgccgcc 15240

tcctttagcc gctaaaattc atctactcat ttattcattt gctcatttac tctggtagct 15300

gcgcgatgta ttcagatagc agctcggtaa tggtcttgcc ttggcgtacc gcgtacatct 15360

tcagcttggt gtgatcctcc gccggcaact gaaagttgac ccgcttcatg gctggcgtgt 15420

ctgccaggct ggccaacgtt gcagccttgc tgctgcgtgc gctcggacgg ccggcactta 15480

gcgtgtttgt gcttttgctc attttctctt tacctcatta actcaaatga gttttgattt 15540

aatttcagcg gccagcgcct ggacctcgcg ggcagcgtcg ccctcgggtt ctgattcaag 15600

aacggttgtg ccggcggcgg cagtgcctgg gtagctcacg cgctgcgtga tacgggactc 15660

aagaatgggc agctcgtacc cggccagcgc ctcggcaacc tcaccgccga tgcgcgtgcc 15720

tttgatcgcc cgcgacacga caaaggccgc ttgtagcctt ccatccgtga cctcaatgcg 15780

ctgcttaacc agctccacca ggtcggcggt ggcccatatg tcgtaagggc ttggctgcac 15840

cggaatcagc acgaagtcgg ctgccttgat cgcggacaca gccaagtccg ccgcctgggg 15900

cgctccgtcg atcactacga agtcgcgccg gccgatggcc ttcacgtcgc ggtcaatcgt 15960

cgggcggtcg atgccgacaa cggttagcgg ttgatcttcc cgcacggccg cccaatcgcg 16020

ggcactgccc tggggatcgg aatcgactaa cagaacatcg gccccggcga gttgcagggc 16080

gcgggctaga tgggttgcga tggtcgtctt gcctgacccg cctttctggt taagtacagc 16140

gataaccttc atgcgttccc cttgcgtatt tgtttattta ctcatcgcat catatacgca 16200

gcgaccgcat gacgcaagct gttttactca aatacacatc acctttttag acggcggcgc 16260

tcggtttctt cagcggccaa gctggccggc caggccgcca gcttggcatc agacaaaccg 16320

gccaggattt catgcagccg cacggttgag acgtgcgcgg gcggctcgaa cacgtacccg 16380

gccgcgatca tctccgcctc gatctcttcg gtaatgaaaa acggttcgtc ctggccgtcc 16440

tggtgcggtt tcatgcttgt tcctcttggc gttcattctc ggcggccgcc agggcgtcgg 16500

cctcggtcaa tgcgtcctca cggaaggcac cgcgccgcct ggcctcggtg ggcgtcactt 16560

cctcgctgcg ctcaagtgcg cggtacaggg tcgagcgatg cacgccaagc agtgcagccg 16620

cctctttcac ggtgcggcct tcctggtcga tcagctcgcg ggcgtgcgcg atctgtgccg 16680

gggtgagggt agggcggggg ccaaacttca cgcctcgggc cttggcggcc tcgcgcccgc 16740

tccgggtgcg gtcgatgatt agggaacgct cgaactcggc aatgccggcg aacacggtca 16800

acaccatgcg gccggccggc gtggtggtgt cggcccacgg ctctgccagg ctacgcaggc 16860

ccgcgccggc ctcctggatg cgctcggcaa tgtccagtag gtcgcgggtg ctgcgggcca 16920

ggcggtctag cctggtcact gtcacaacgt cgccagggcg taggtggtca agcatcctgg 16980

ccagctccgg gcggtcgcgc ctggtgccgg tgatcttctc ggaaaacagc ttggtgcagc 17040

cggccgcgtg cagttcggcc cgttggttgg tcaagtcctg gtcgtcggtg ctgacgcggg 17100

catagcccag caggccagcg gcggcgctct tgttcatggc gtaatgtctc cggttctagt 17160

cgcaagtatt ctactttatg cgactaaaac acgcgacaag aaaacgccag gaaaagggca 17220

gggcggcagc ctgtcgcgta acttaggact tgtgcgacat gtcgttttca gaagacggct 17280

gcactgaacg tcagaagccg actgcactat agcagcggag gggttggatc aaagtacttt 17340

gatcccgagg ggaaccctgt ggttggcatg cacatacaaa tggacgaacg gataaacctt 17400

ttcacgccct tttaaatatc cgttattcta a 17431

<210> 4

<211> 413

<212> DNA

<213> Artificial sequence

<400> 4

atgaagacct tattcctcct agctctcctt gctcttgtag cgagcacaac cttcgcgcaa 60

tactcagaag ttggcggctg gtacaatgaa gttagcggcc gaagctaagc tcttgcaagg 120

attacgtgat ggagcgatgt ttcacaatga aggattttcc agtcacctgg cccacaaaat 180

ggtggaaggg cggctgtgag catgaggttc gggagaagtg ctgcaagcag ctgagccaga 240

tagcaccaca atgtcgctgt gattctatcc ggcgagtgat ccaaggcagg ctcggtggct 300

tcttgggcat ttggcgaggt gaggtattca aacaacttca gagggcccag agcctcccct 360

caaagtgcaa catgggcgcc gactgcaagt tccctagtgg ctattactgg tga 413

<210> 5

<211> 447

<212> DNA

<213> Artificial sequence

<400> 5

atgaagacct tattcctcct agctctcctt gctcttgtag cgagcacaac cttcgcgcaa 60

tactcagaag ttggcggctg gtacaatgaa gttggcggag gaggtggttc tcaacaatgt 120

ccgcaggagc ggccgaagct aagctcttgc aaggattacg tgatggagcg atgtttcaca 180

atgaaggatt ttccagtcac ctggcccaca aaatggtgga agggcggctg tgagcatgag 240

gttcgggaga agtgctgcaa gcagctgagc cagatagcac cacaatgtcg ctgtgattct 300

atccggcgag tgatccaagg caggctcggt ggcttcttgg gcatttggcg aggtgaggta 360

ttcaaacaac ttcagagggc ccagagcctc ccctcaaagt gcaacatggg cgccgactgc 420

aagttcccta gtggctatta ctggtga 447

<210> 6

<211> 2137

<212> DNA

<213> Artificial sequence

<400> 6

catgaatcca aaccacacgg agttcaaatt cccacagatt aaggctcgtc cgtcgcacaa 60

ggtaatgtgt gaatattata tctgtcgtgc aaaattgcct ggcctgcaca attgctgtta 120

tagttggcgg cagggagagt tttaacattg actagcgtgc tgataatttg tgagaaataa 180

taattgacaa gtagatactg acatttgaga agagcttctg aactgttatt agtaacaaaa 240

atggaaagct gatgcacgga aaaaggaaag aaaaagccat actttttttt aggtaggaaa 300

agaaaaagcc atacgagact gatgtctctc agatgggccg ggatctgtct atctagcagg 360

cagcagccca ccaacctcac gggccagcaa ttacgagtcc ttctaaaagc tcccgccgag 420

gggcgctggc gctgctgtgc agcagcacgt ctaacattag tcccacctcg ccagtttaca 480

gggagcagaa ccagcttata agcggaggcg cggcaccaag aagcggcgtg agaccaaccc 540

agtggacata agcctgttcg gttcgtaagc tgtaatgcaa gtagcgtatg cgctcacgca 600

actggtccag aaccttgacc gaacgcagcg gtggtaacgg cgcagtggcg gttttcatgg 660

cttgttatga ctgttttttt ggggtacagt ctatgcctcg ggcatccaag cagcaagcgc 720

gttacgccgt gggtcgatgt ttgatgttat ggagcagcaa cgatgttacg cagcagggca 780

gtcgccctaa aacaaagtta aacatcatgg gggaagcggt gatcgccgaa gtatcgactc 840

aactatcaga ggtagttggc gtcatcgagc gccatctcga accgacgttg ctggccgtac 900

atttgtacgg ctccgcagtg gatggcggcc tgaagccaca cagtgatatt gatttgctgg 960

ttacggtgac cgtaaggctt gatgaaacaa cgcggcgagc tttgatcaac gaccttttgg 1020

aaacttcggc ttcccctgga gagagcgaga ttctccgcgc tgtagaagtc accattgttg 1080

tgcacgacga catcattccg tggcgttatc cagctaagcg cgaactgcaa tttggagaat 1140

ggcagcgcaa tgacattctt gcaggtatct tcgagccagc cacgatcgac attgatctgg 1200

ctatcttgct gacaaaagca agagaacata gcgttgcctt ggtaggtcca gcggcggagg 1260

aactctttga tccggttcct gaacaggatc tatttgaggc gctaaatgaa accttaacgc 1320

tatggaactc gccgcccgac tgggctggcg atgagcgaaa tgtagtgctt acgttgtccc 1380

gcatttggta cagcgcagta accggcaaaa tcgcgccgaa ggatgtcgct gccgactggg 1440

caatggagcg cctgccggcc cagtatcagc ccgtcatact tgaagctaga caggcttatc 1500

ttggacaaga agaagatcgc ttggcctcgc gcgcagatca gttggaagaa tttgtccact 1560

acgtgaaagg cgagatcacc aaggtagtcg gcaaataatg tctagctaga aattcgttca 1620

agccgacgcc gcttcgcggc gcggcttaac tcaagcgtta gatgcactaa gcacataatt 1680

gctcacagcc aaactatcag gtcaagtctg cttttattat ttttaagcgt gcataataag 1740

ccggtctcga cttagcttcg gccgctcctg gttttagagc tagaaatagc aagttaaaat 1800

aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttttttcgtt 1860

ttgcattgag ttttctccgt cgcatgtttg cagttttatt ttccgttttg cattgaaatt 1920

tctccgtctc atgtttgcag cgtgttcaaa aagtacgcag ctgtatttca cttatttacg 1980

gcgccacatt ttcatgccgt ttgtgccaac tatcccgagc tagtgaatac agcttggctt 2040

cacacaacac tggtgacccg ctgacctgct cgtacctcgt accgtcgtac ggcacagcat 2100

ttggaattaa agggtgtgat cgatactgct tgctgct 2137

Claims (10)

1. A method for raising wheat with improved wheat grain hardness, which comprises A1) and A2):

A1) editing a target DNA fragment in a receptor wheat genome by using a gene editing method to obtain a gene-edited wheat with the target DNA fragment changed; the sequence of the target DNA fragment is sequence 5 in the sequence table;

A2) and screening the target wheat with grain hardness increased compared with the receptor wheat from the gene editing wheat.

2. A method of increasing the hardness of wheat grain comprising a1) and a 2):

A1) editing a target DNA fragment in a receptor wheat genome by using a gene editing method to obtain a gene-edited wheat with the target DNA fragment changed; the sequence of the target DNA fragment is sequence 5 in the sequence table;

A2) and screening the target wheat with grain hardness increased compared with the receptor wheat from the gene editing wheat to realize the improvement of the grain hardness of the wheat.

3. A method according to claim 1 or 2, characterized in that: the gene editing method is carried out by using a CRISPR/Cas9 system.

4. The method of claim 3, wherein: the target sequence of sgRNA in the CRISPR/Cas9 system is sequence 1 in the sequence table.

5. The method of claim 4, wherein: the sequence of the sgRNA is sequence 2 in the sequence table.

6. The method according to any one of claims 1-5, wherein: compared with the acceptor wheat, the target DNA fragment of the target wheat lacks 94 th to 127 th sites of a sequence 5 in a sequence table.

7. The following products I or II:

I. the sgRNA of claim 4 or 5;

II. The biological material related to the sgRNA of claim 4 or 5, which is any one of the following B1) to B9):

B1) a nucleic acid molecule encoding the sgRNA of claim 4 or 5;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector;

B5) a transgenic plant cell line comprising B1) the nucleic acid molecule or a transgenic plant cell line comprising B2) the expression cassette;

B6) transgenic plant tissue comprising the nucleic acid molecule of B1) or transgenic plant tissue comprising the expression cassette of B2);

B7) a transgenic plant organ containing B1) the nucleic acid molecule or a transgenic plant organ containing B2) the expression cassette.

8. The product of claim 7, wherein: B1) the nucleic acid molecule is a DNA molecule shown in a sequence obtained by replacing U in a sequence 2 in a sequence table with T.

9. The kit is the following III or IV:

III, a kit consisting of the product of claim 7 or 8 and a Cas9 protein;

IV, a kit of reagents consisting of the product of claim 7 or 8 and a biological material associated with a Cas9 protein; the biomaterial is any one of the following C1) to C9):

C1) a nucleic acid molecule encoding a Cas9 protein;

C2) an expression cassette comprising the nucleic acid molecule of C1);

C3) a recombinant vector comprising the nucleic acid molecule of C1), or a recombinant vector comprising the expression cassette of C2);

C4) a recombinant microorganism containing C1) the nucleic acid molecule, or a recombinant microorganism containing C2) the expression cassette, or a recombinant microorganism containing C3) the recombinant vector;

C5) a transgenic plant cell line comprising C1) the nucleic acid molecule or a transgenic plant cell line comprising C2) the expression cassette;

C6) transgenic plant tissue comprising the nucleic acid molecule of B1), or transgenic plant tissue comprising the expression cassette of C2);

C7) a transgenic plant organ containing B1) the nucleic acid molecule or a transgenic plant organ containing C2) the expression cassette.

10. Any of the following applications:

x1) use of the product of claim 7 or 8 or the kit of claim 9 for increasing the hardness of wheat grain;

x2) use of the product of claim 7 or 8 or the kit of claim 9 for the manufacture of a product for increasing the hardness of wheat grain;

x3) use of the product of claim 7 or 8 or the kit of claim 9 for breeding wheat with increased grain hardness;

x4) use of the product of claim 7 or 8 or the kit of claim 9 for the preparation of a product for growing wheat having increased grain hardness;

x5) the use of a product according to claim 7 or 8 or a kit according to claim 9 in wheat breeding;

x6) use of the method of any one of claims 1 to 6 in wheat breeding.