CN113717982A - Method for increasing wheat grain length by knocking out grain development related protein TaGSR1 through gene editing - Google Patents

Abstract

The invention discloses a method for using gene editing to knock out the grain development-related protein TaGSR1 to increase the length of wheat grains. The single-stranded guide RNA targeting the TaGSR1 gene is designed to construct a plant binary expression vector pBUE411- which knocks out the TaGSR1 gene in wheat. TaGSR1; The plant binary expression vector is transformed into common wheat by Agrobacterium, and the transgenic wheat with TaGSR1 gene knockout is obtained, and the grain length of wheat seeds is increased; wherein the nucleotide sequence of the TaGSR1 gene is as SEQ ID NO. 4 shown. The experiments confirmed that the TaGSR1 gene knockout transgenic wheat achieved increased grain length and increased 1000-grain weight of wheat seeds. The technical scheme of the present invention provides a feasible method for realizing rapid wheat breeding and improving wheat yield by using genetic engineering technology, and has important breeding application value and broad market application prospect.

Description

Method for increasing wheat grain length by knocking out grain development related protein TaGSR1 through gene editing

Technical Field

The invention relates to a method for increasing the length of wheat grains, in particular to a method for increasing the length of wheat grains by knocking out a grain development related protein TaGSR1 through gene editing; belongs to the technical field of plant genetic engineering and molecular breeding.

Background

Wheat is the second largest food crop in the world, providing 20% carbohydrate and 23% protein to humans. The human food safety problem faces the problems of reduction of cultivated land area, global climate warming, continuous population growth, aggravation of environmental pollution and the like, and how to improve the wheat yield under the condition of limited resources has important significance on food safety.

Brassinolide (BR) as the sixth plant hormone plays an important role in the processes of cell elongation and division, leaf senescence, plant morphogenesis, plant stress resistance and the like. Due to the wide role of BR in the growth and development of plants, its application in agricultural production is also gaining increasing attention. BRI1 (Brassicasteroid-inducing 1) is used as a BR receptor located on a cell membrane, after being combined with BR, the BR receptor is mutually phosphorylated with a co-receptor BAK1(BRI1-associated receptor) to initiate downstream signal transduction and further phosphorylate BSK/CDG1 kinase, the phosphorylated BSK/CDG1 further activates BSU1(BRI 1-repressisor 1) downstream through phosphorylation, the BSU1 is used as a phosphatase, and negative regulatory factors in the BR signal channel of BIN2 are inactivated through dephosphorylation BIN2 (Brassicasteroid-inducing 2) to further regulate the expression of downstream genes.

The applicant researches and discovers that TaGSR1(Grain Size Regulator 1 of Triticum aestivum L.) is a transcription factor downstream of BR signals and has the function of regulating and controlling the Size of wheat grains. The regulation and control function provides an important theoretical basis for researching the growth and development of BR in wheat and high-yield breeding. However, the search shows that the coding sequence of the wheat TaGSR1 gene and the grain development related protein TaGSR1 coded by the coding sequence and the method for increasing the length of wheat grains by knocking out the grain development related protein TaGSR1 through gene editing are not reported.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a method for increasing the length of wheat grains by knocking out a grain development related protein TaGSR1 through gene editing.

The invention discloses a method for increasing the length of wheat grains by knocking out a grain development related protein TaGSR1 through gene editing, which is characterized by comprising the following steps: designing a single-stranded guide RNA (sgRNA) of a targeted TaGSR1 gene, and constructing an expression vector for knocking out a TaGSR1 gene in wheat, namely a plant binary expression vector pBUE411-TaGSR 1; transforming the plant binary expression vector into common wheat through agrobacterium to obtain transgenic wheat with TaGSR1 gene knocked out, so as to increase the seed length of wheat seeds; wherein the nucleotide sequence of the TaGSR1 gene is shown in SEQ ID NO. 4.

Wherein, in the above method: the nucleotide sequence of the single-stranded guide RNA of the targeted TaGSR1 gene is SEQ ID NO.5, or SEQ ID NO.6, or SEQ ID NO.7, or SEQ ID NO.8, or sgRNA conforming to the characteristics; sgRNA which is preferably the nucleotide sequence shown in SEQ ID NO. 5; the nucleotide sequence of the plant binary expression vector pBUE411-TaGSR1 is shown in SEQ ID NO. 3; the method is further a method for infecting wheat immature embryo callus by utilizing agrobacterium, an expression element of a specific sgRNA and Cas9 element of a targeted TaGSR1 gene contained in a binary recombinant vector pBUE411-TaGSR1 is transferred into wheat, the TaGSR1 gene is specifically knocked out, and a wheat mutant with increased seed length and yield is obtained.

The invention also discloses a binary vector with the function of knocking out TaGSR1 gene, which is characterized in that: the binary vector is named as pBUE411-TaGSR1, and the nucleotide sequence of the binary vector is shown as SEQ ID NO. 3; the binary vector pBUE411-TaGSR1 consists of an expression cassette E1 and an expression cassette E2; wherein the nucleotide sequence of the expression cassette E1 is shown as SEQ ID NO.1, which is sequentially from upstream to downstream: TaU3 promoter from wheat, single stranded guide rna (sgrna) targeting the TaGSR1 gene, terminator T1; the nucleotide sequence of the expression cassette E2 is shown as SEQ ID NO.2, and the expression cassette E2 comprises the following components in sequence from upstream to downstream: ubiquitin promoter Ubi from maize, maize Cas9 coding sequence, terminator T2.

Wherein: the nucleotide sequence of the wheat TaU3 promoter is shown as SEQ ID NO. 9; the nucleotide sequence of the single-stranded guide RNA of the targeted TaGSR1 gene is SEQ ID NO.5, or SEQ ID NO.6, or SEQ ID NO.7, or SEQ ID NO. 8; the nucleotide sequence of the terminator T1 is shown as SEQ ID NO. 10; the nucleotide sequence of the ubiquitin promoter Ubi is shown in SEQ ID NO. 11; the nucleotide sequence of the maize Cas9 coding sequence is shown as SEQ ID NO. 12; the nucleotide sequence of the terminator T2 is shown as SEQ ID NO. 13.

The invention further discloses a specific guide single-stranded RNA for obtaining TaGSR1 gene knockout mutant wheat, which is characterized in that: the nucleotide sequence of the specific guide single-stranded RNA is SEQ ID NO.5, or SEQ ID NO.6, or SEQ ID NO.7, or SEQ ID NO. 8; the deoxynucleotide sequence belongs to a complementary sequence of a TaGSR1 coding sequence or a TaGSR1 coding sequence, is 22-23 deoxynucleotides in length, is NGG in the sequence of three deoxynucleotides which are immediately adjacent to the 3' end of the deoxynucleotide sequence, and N is adenine, guanine, thymine or cytosine.

Wherein: the nucleotide sequence of the specific guide single-stranded RNA is preferably shown as SEQ ID NO. 5.

The method for increasing the length of wheat seeds by knocking out the seed development related protein TaGSR1 through gene editing comprises the steps of constructing an expression vector pBUE411-TaGSR1 with the function of knocking out the TaGSR1 gene, converting the expression vector into common wheat to knock out the TaGSR1 gene, and increasing the length of the wheat seeds by reducing the expression of the TaGSR1 gene. The nucleotide sequence of the coding sequence of the wheat TaGSR1 gene provided by the invention is shown in SEQ ID NO.4 and is published for the first time. The wheat TaGSR1 is a transcription factor of BR signal downstream, and has the function of regulating and controlling the size of wheat grains.

The invention has the following outstanding effects: an expression vector capable of knocking out TaGSR1 gene in wheat is constructed by designing guide single-stranded RNA (sgRNA) of the TaGSR1 gene; wherein the sgRNA sequence is capable of targeted disruption of an exon region of a TaGSR1 gene, and the targeting domain sequence of the sgRNA sequence is selected from a coding sequence of a TaGSR1 gene and is capable of specifically targeted disruption of a coding region of a TaGSR1 gene to effectively reduce protein levels of TaGSR1. Through genetic transformation of wheat, offspring with TaGSR1 gene knockout is obtained, and the wheat mutant with increased grain length and yield is obtained. Experiments prove that the transgenic wheat with the TaGSR1 gene knockout realizes the increase of the seed length and the improvement of thousand seed weight of wheat seeds. The technical scheme of the invention provides a feasible method for realizing rapid breeding of wheat and improving the yield of the wheat by utilizing a genetic engineering technology, and has important breeding application value and wide market application prospect.

Drawings

FIG. 1 is the electrophoresis diagram of TaGSR1 gene amplification of Shixin 828 wheat.

Wherein: lane 1 is Transgen 2K plus marker, and lane 2 is TaGSR1 band.

FIG. 2 the coding region of the TaGSR1 gene of wheat variety Shixin 828.

Wherein: underlined sequences are sgRNA sequences used for gene editing.

FIG. 3 sequencing results of the binary expression vector pBUE411-TaGSR 1.

Wherein: the sgRNA is underlined, and the remaining sequence is the vector backbone sequence of vector pBUE 411.

FIG. 4T 0 generation wheat transgenic line transformation identification result.

Wherein: lane 1 is Transgen 2K plus marker, lane 2 is the result of amplification of recombinant plasmid pBUE411-TaGSR1, lanes 3-15 are the result of identification of T0 generation transgenic wheat strain, and lane 16 is the result of amplification of wild type JW 1.

FIG. 5 sequencing results for the strain tagsr1-ko in which gene editing occurred for all three homologous copies.

FIG. 6 protein coding results for strain tagsr1-ko in which gene editing occurred in all three homologous copies.

FIG. 7 results of three copy-specific amplification of tagsr1-ko strain.

Wherein: 1 is Transgen 2K plus marker, 2 is a specific PCR fragment for detecting TaGSR1.2A editing condition, 3 is a specific PCR fragment for detecting TaGSR1.2B editing condition, and 4 is a specific PCR fragment for detecting TaGSR1 editing condition.

FIG. 8 shows the results of enzyme digestion identification of the descendant edited by TaGSR1 gene.

Wherein: 1 is a Transgen 2K marker, 2-6 are the enzyme cutting results of three homologous copies of different selfing progenies, 7 is the enzyme cutting results of three homologous copies of wild type JW1, and the box indicates that the three homologous copies of the plant are edited and are homozygous knockout strains.

FIG. 9 shows the comparison of sizes of seeds of OE-TaGSR1 over-expressed transgenic and homozygous gene knockout lines TaGSR 1-ko.

Wherein: a & B, compared with wild type JW1 wheat, the length and width of seeds of OE-TaGSR1 overexpression lines are obviously reduced; c & D, compared with wild JW1 wheat, the homozygous gene knockout strain tagsr1-ko has obviously increased kernel length and no obvious change in kernel width. Bar is 1 cm.

Detailed Description

The present invention will be described in detail with reference to the following detailed drawings and examples. The following examples are only preferred embodiments of the present invention, and it should be noted that the following descriptions are only for explaining the present invention and not for limiting the present invention in any form, and any simple modifications, equivalent changes and modifications made to the embodiments according to the technical spirit of the present invention are within the scope of the technical solution of the present invention.

The experimental procedures in the following examples are conventional unless otherwise specified.

The test materials used in the following examples were purchased from conventional biochemicals, unless otherwise specified.

The high fidelity enzyme required for PCR amplification is KOD-FXNEO (Toyobo); restriction enzymes BsaI and T4 ligase required for Gibbson assembly were purchased from NEB; restriction enzyme CpoI, a gel recovery kit required for enzyme digestion fragment recovery, and a plasmid extraction kit were purchased from Saimer Feishel scientific Co. The inorganic salts required for the preparation of the culture medium were purchased from the national pharmaceutical group, and the vitamins and antibiotics were purchased from Sigma. The plant CRISPR/Cas9 gene editing vector is pBUE411, contains a wheat U3 promoter TaU3 for starting sgRNA, and Cas9 simulates the characteristic that gramineous plant genes have high GC content at the 5' end and is a gene for designing and synthesizing plant codon optimization. The plasmid pBUE411 is publicly available from the university of agriculture in China, and the sequence of the plasmid is known and is 17430 bp. Coli Transgen5 α, purchased from tokyo holotype gold, inc; the primers used were synthesized by Qingdao Optimaki Biotechnology Limited, and the sequences of the relevant primers are shown in Table 1:

table 1: primers used in the present invention

The wheat variety JW1 used in the invention is a new germplasm with good tissue culture capability, which is self-bred by crops of agricultural academy of Shandong province, and can be obtained by the public from the crop research institute of agricultural academy of Shandong province; shixin 828 is a wheat variety which is bred in 2001 by 8 generations of pedigree breeding method at New wheat New breed New technology institute in Shijiazhuang City, examined and rated by the variety examination committee of Hebei province in 2005 and has excellent properties of cold resistance, drought resistance, lodging resistance and the like, and can be obtained by Jiafeng variety Co Ltd.

EXAMPLE 1 construction of expression vectors

1. Design of sgrnas targeting TaGSR1

In order to design sgRNA which can be edited in a coding region of a TaGSR1 gene, the sequence of a TaGSR1 gene is firstly amplified in the wheat variety Shixin 828 in the experiment, primers used are TaGSR1-F and TaGSR1-R, and a PCR reaction system is as follows: KOD-FX NEO buffer: 25 μ L of dNTP (2 mM): 10 μ L, TaGSR1-F (10 μ M): 1.5 μ L, TaGSR1-F (10 μ M): 1.5 μ L, Shixin 828gDNA (50 ng/. mu.L): 1 μ L, KOD-FXNEO: 1 μ L, ddH₂The total amount of O is 50 μ L. The PCR reaction program is: pre-denaturation at 98 ℃ for 2min, and denaturation at 98 ℃ for 12 sec; annealing at 58 ℃ for 20sec, extension at 68 ℃ for 25sec, and reacting for 35 cycles; renaturation at 68 ℃ for 5 min.

The PCR results are shown in FIG. 1, lane 1 is Transgen 2K plus marker, and lane 2 is TaGSR1 band.

The PCR product was sent to Qingdao Hingxi Biotechnology Limited to sequence, and the sequence of TaGSR1 gene in Shixin 828 wheat is shown in FIG. 2, which is similar to the sequence 93.79% of wheat in Ensembl Plants database (http:// Plants. ensemblel. org/index. html). The gene has the full length of 612 basic groups, does not contain introns, and has a nucleotide sequence shown in SEQ ID NO. 4.

A suitable targeting site is searched for in a coding region of a TaGSR1 gene through a website CRISPRC (http:// crispr. dbcls. jp /), a 20bp sequence fragment is found before a PAM structure and is set as a target sequence, in the embodiment, a knockout experiment is carried out by taking one sgRNA as an example, the nucleotide sequence is GCGGCGCGTCGTCGCCGGACCGG, the nucleotide sequence is shown as SEQ ID No.5, namely, an underlined marker sequence in a TaGSR1 gene sequence shown in figure 2 is taken as an sgRNA sequence of a targeted TaGSR1 gene, and CGG at the 3' end of the sgRNA is taken as a PAM sequence.

2. Obtaining fragments containing sgRNA

2 primers TaGSR1-gR1-F and TaGSR1-gR1-R (Table 1) for constructing a pBUE411-TaGSR1 binary expression vector are synthesized by Hippocastanaceae Cathi Biotechnology Limited. Respectively phosphorylating the two primers, and directly annealing to form double chains, wherein the reaction system is as follows: TaGSR1-gR1-F (10. mu.M), 4. mu.L, TaGSR1-gR1-R (10. mu.M): 4 μ L, 10 XT 4 PNK buffer: 1.5. mu.L, PNK: 1 μ L, ATP: 1 μ L, ddH₂O make up to 15. mu.L. Reaction in a PCR instrument: 30min at 37 ℃; 95 ℃ for 5 min; ramp to 25 ℃ at 5 ℃/min.

Ligation of the pBUE411 vector to the sgRNA

The connection of pBUE411 and sgRNA is completed by adopting Gibbson assembly, and the specific reaction system comprises the following steps: pBUE411 plasmid (100 ng/. mu.L): 2 μ L, product fragment of step 2: 2 μ L, 10 XNEB T4 Buffer: 1.5 μ L, 10 × BSA: 1.5 μ L, BsaI: 1 μ L, T4 Ligase: 1 μ L, ddH₂And the content of O is filled up to 15 mu L. The reaction system reacts for 5 hours in a water bath kettle at 37 ℃ to obtain a product of connecting the pBUE411 vector and the sgRNA.

4. Transformation and characterization

The ligation product was transformed into E.coli, specifically: adding the ligation product into Escherichia coli competent Transgen5 alpha, performing ice bath for 20min, performing heat shock reaction at 42 ℃ for 60sec, performing ice bath for 2min, adding non-resistant LB, putting into a shaker at 37 ℃ for resuscitation for 1h, then using an applicator to spread on an LB (containing kanamycin) plate, performing inverted culture at 37 ℃ until clones grow out, selecting 3 monoclones for sequencing, wherein the primers are pBUE411-F and pBUE 411-R.

The sequencing result (as shown in fig. 3) detects the target sequence of the sgRNA, and simultaneously detects TaU3 promoter sequence at the upstream of the target sequence, and the sequencing result shows that the expression cassette E1 containing the sgRNA is successfully assembled into a pBUE411 binary expression vector, which proves that the CRISPR/Cas9 gene editing vector of TaGSR1, i.e. the recombinant binary expression vector pBUE411-TaGSR1, is successfully constructed. The nucleotide sequence of the recombinant binary expression vector pBUE411-TaGSR1 is shown in SEQ ID NO. 3.

Example 2 transgenic progeny acquisition and characterization

Obtaining transgenic progeny of TaGSR1

The recombinant binary expression vector pBUE411-TaGSR1 constructed in the experimental example 1 is transformed into agrobacterium EHA105 competent cells, specifically: adding the recombinant binary expression vector pBUE411-TaGSR1 into agrobacterium EHA105 competence, performing ice bath for 5min, performing liquid nitrogen quick freezing for 5min, performing heat shock reaction for 5min at 37 ℃, performing ice bath for 5min, adding nonresistant LB, putting into a shaker at 28 ℃ for recovery for 2h, then coating on an LB (containing rifampicin, streptomycin and kanamycin) plate by using an applicator, and performing inverted culture at 28 ℃ until clone grows out. The single clone was selected and inoculated into LB medium containing the corresponding resistance, shaken at 28 ℃ and cultured at 160rpm for 24 hours.

Taking JW1 wheat seeds about 15 days after pollination, and stripping young embryos. The Agrobacterium suspension was prepared by placing 1ml of the suspension in a 1.5ml centrifuge tube, adding 1.4ul acetosyringone (0.1M), and mixing. Adding prepared bacterial liquid to infect for 5 minutes, placing on a co-culture medium, and culturing in dark at 23 ℃ for 3 days. After co-cultivation, the cells were cultured in the dark at 25 ℃ for 5 days in a resting medium. The callus was transferred to the screening medium 1, sealed with a sealing film, and cultured in the dark in an incubator at 25.5 ℃ for 2 weeks. The callus was cut and transferred to screening medium 2, the petri dish was sealed again with a sealing film, and dark culture was continued in an incubator at 25.5 ℃ for 2 weeks. After 2 weeks of callus cutting and selection, resistant calli exhibiting green shoots were transferred to regeneration medium. The dishes were sealed and placed in an incubator at 25 ℃ for 2 weeks in light/dark (16h/8 h). After 2 weeks of regeneration, healthy growing plantlets were transferred to new resistant regeneration pods. When the seedlings grow to a certain size, sampling detection can be carried out.

The various culture media involved in the genetic transformation of wheat and the preparation thereof are disclosed in the following documents:

Kan Wang(ed.),Agrobacterium Protocals:Volume 1,Methods in Molecular Biology,vol.1223DOI10.007/978-1-4939-1695-5_15,Spring Science+Businedd Media New York 2015。

taking young regenerated wheat leaves, extracting genome DNA by a CTAB method, and performing PCR identification by using primers on two carriers, namely BUE-DF1 and BUE-DR1, wherein the PCR reaction program is as follows: 2 × PCR master mix: 10 μ L, pBUE411-F (10 μ M): 0.5 μ L, pBUE411-R (10 μ M): 0.5. mu.L, gDNA (50 ng/. mu.L): 1 μ L, ddH₂O: 8 μ L. The PCR reaction program is: pre-denaturation at 95 ℃ for 5min, and denaturation at 95 ℃ for 30 sec; annealing at 58 ℃ for 30sec, extension at 72 ℃ for 30sec, and reacting for 32 cycles; renaturation at 72 ℃ for 5 min. The results are shown in FIG. 4, the bands represent the T0 generation wheat transgenic lines with successful transformation, and 12 positive transgenic lines are obtained.

Identification of transgenic knock-out progeny of TaGSR1

Wheat is an allohexaploid, TaGSR1 gene is copied on a 2D chromosome, homologous copies on chromosomes 2A and 2B are TaGSR1.2A and TaGSR1.2B, the homology of DNA sequences of the three copies is high, the similarity of amino acid sequences reaches 88.6%, and the gene editing condition of the three homologous copies needs to be detected simultaneously. The experiment adopts a Hi-TOM gene editing site detection kit purchased from Xian' qing xue biotechnology limited company, the kit completes the high-throughput library building process through PCR, and Hi-TOM online software is used for directly analyzing the variation information of multiple samples and multiple sites. Specific primers (Seq-F and Seq-R in Table 1) on both sides of the target sequence are used for simultaneously amplifying TaGSR1.2A, TaGSR1.2B and TaGSR1, and amplified products are sent to the Xianqingxue biotechnology limited company for sequencing after being stored in a library.

The gene editing results of the target genes TaGSR1.2A, TaGSR1.2B and TaGSR1 are shown in FIG. 5, and the coded amino acid sequences are shown in FIG. 6. By alignment, the mutant protein tagsr1.2a appears frame-shifted from the 112 th amino acid, the protein tagsr1.2b appears frame-shifted from the 111 th amino acid, and the protein tagsr1 appears frame-shifted from the 109 th amino acid. The result shows that the sgRNA and the Cas9 element are successfully transformed and play a role, and the TaGSR1 gene is edited, so that three proteins of TaGSR1.2A, TaGSR1.2B and TaGSR1 are subjected to frame shift mutation, a conserved HLH functional domain cannot be correctly expressed, and the corresponding function of the gene is lost.

Wherein: first round PCR reaction System: 1 μ L of pBUE411-TaGSR1 transgenic wheat plant leaf DNA was used as a template, and 0.5 μ L of each of 2 XTAQA Master Mix, Seq-F and Seq-R (Table 1) (10 μ M) in the kit was used to complement the volume to 20 μ L. The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 30s, annealing at 64 ℃ for 30s, and extension at 72 ℃ for 20s for 32 cycles; finally, extension is carried out for 5min at 72 ℃. After the PCR is finished, 5 mu L of agarose gel electrophoresis is taken to detect the PCR product, so that the existence of the target product is ensured and the specificity is good. Then, a second round of PCR reaction was performed, in which 12. mu.L of Hi-TOM Mix in the kit was added to make up the volume to 20. mu.L using the first round of PCR product as a template, and 1. mu.L of nucleic-free Water was added. PCR reaction procedure: denaturation at 94 deg.C for 2 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 25s for 33 cycles; finally, extension is carried out for 5min at 72 ℃. And mixing amplification products for gel recovery, wherein the gel recovery product is a library-establishing sequencing sample, and then sending the library-establishing sequencing sample to the SiAnqingxue Biotech limited company for sequencing, and the gene editing result is shown in figure 5.

Example 3 phenotypic characterization of wheat knockout TaGSR1 Gene progeny

In order to obtain three homologously copied homozygous knockout strains of TaGSR1.2A, TaGSR1.2B and TaGSR1, enzyme digestion amplification polymorphic sequence (Caps) markers are developed aiming at editing sites, and selfed progeny of the gene editing mutant is identified until obtaining the homozygously knocked-out homozygous mutants of TaGSR1.2A, TaGSR1.2B and TaGSR1.

The TaGSR1.2A, TaGSR1.2B and TaGSR1 genes of the gene edition progeny are subjected to PCR amplification by utilizing Caps-2A-F and Caps-2A-R, Caps-2B-F and Caps-2B-R, Caps-2D-F and Caps-2D-R (Table 1) respectively. The PCR reaction system is as follows: KOD-FX NEO buffer: 10 μ L, dNTP (2 mM): 4 μ L, Caps-2A-F (or Caps-2B-F, or Caps-2D-F) (10 μ M): 0.6 μ L, Caps-2A-R (or Caps-2B-R, or Caps-2D-R) (10 μ M): 0.6 μ L, knock-out strain gDNA (about 20 ng/. mu.L): 1 μ L, KOD-FXNEO: 0.4 muL，ddH₂The total amount of O is 20 mu L. The PCR reaction program is: pre-denaturation at 98 ℃ for 2min, and denaturation at 98 ℃ for 12 sec; annealing at 58 ℃ for 20sec, extension at 68 ℃ for 45sec, and reacting for 35 cycles; renaturation at 68 ℃ for 5 min.

5ul of PCR product was subjected to electrophoresis detection, and the PCR result is shown in FIG. 7.

The amplified product is subjected to enzyme digestion by using a restriction enzyme CpoI produced by Saimer Feishel, wherein the enzyme digestion reaction system is as follows: PCR products: 15 μ L, 10 × Buffer Tango: 2 μ L, restriction enzyme CpoI: 0.5. mu.L. The reaction system was digested in a 37 ℃ water bath for 2h, followed by electrophoresis, the results of which are shown in FIG. 8. The homozygous mutant is identified by electrophoresis, the wild type gene can be cut, and the successful sites of gene editing can not be cut. TaGSR1.2A, TaGSR1.2B and TaGSR1 of wild plants can be cut into fragments with the sizes of 696bp +514bp, 729bp +358bp and 869bp +306bp, and gene editing mutants can not be cut, so that a transgenic homozygous three-copy gene knockout strain TaGSR1-ko can be screened out.

Wild type receptor variety JW1, a TaGSR1 overexpression wheat transgenic strain (named as OE-TaGSR1) driven by a maize ubiquitin promoter Ubi and a homozygous knockout strain (named as TaGSR1-ko) of a TaGSR1 gene and homologous copies thereof are planted in a Qingdao school district artificial climate chamber of Shandong university together, and the culture conditions are as follows: illuminating for 16h and darkness for 8 h; the temperature in the daytime is 22 ℃, and the temperature at night is 16 ℃; humidity is 40% -50%; the concentration of CO2 is 500-700 ppm. After seed harvesting, drying in an oven at 30 ℃ for 14 days, photographing seeds of OE-TaGSR1 and TaGSR1-ko by using a Canon high-performance single-lens reflex camera, analyzing and measuring the pictures by using Image-Pro Plus 6.0, and counting the lengths and widths of the seeds of OE-TaGSR1 and TaGSR 1-ko.

The results show that: compared with the wild JW1, the grain length and width of the over-expression strain OE-TaGSR1 are obviously reduced, while the grain length of the mutant strain TaGSR1-ko is obviously increased, and the grain width is not obviously changed (as shown in Table 2 and figure 9). The thousand kernel weight of grains of the transgenic line is measured, the thousand kernel weight of an OE-TaGSR1 overexpression transgenic line is obviously reduced, and the thousand kernel weight of TaGSR1-ko is obviously increased (shown in a table 2). Shows that: the TaGSR1 gene has a negative regulation effect on wheat grain development, and the invention uses agrobacterium to infect wheat young embryo induced callus by constructing a pBUE411-TaGSR1 binary recombinant vector which contains sgRNA and can specifically target the TaGSR1 gene, and specifically edits the TaGSR1 gene to lose the function, thereby obviously increasing the length of wheat grains so as to increase the yield, and providing a new method for cultivating high-yield wheat.

TABLE 2 statistics of the yield traits of homozygous three-copy knockout mutant tagsr1-ko

Note: different lower case superscripts indicate significant differences at the 95% confidence level.

Sequence listing

<110> Shandong university

<120> method for increasing wheat grain length by knocking out grain development related protein TaGSR1 through gene editing

<141> 2021-08-05

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<213> Artificial sequence

<221> nucleotide sequence of expression cassette E1

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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 aagcggcggc ggcgcgtcgt 540

cgccggacat gctgctcgac gagcagcgtt ttagagctag aaatagcaag ttaaaataag 600

gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt tttcgttttg 660

cattgagttt tctccgtcgc atgtttgcag ttttattttc cgttttgcat tgaaatttct 720

ccgtctcatg tttgcagcgt gttcaaaaag tacgcagctg tatttcactt atttacggcg 780

ccacattttc atgccgtttg tgccaactat cccgagctag tgaatacagc ttggcttcac 840

acaacactgg tgacccgctg acctgctcgt acctcgtacc gtcgtacggc acagcatttg 900

gaattaaagg gtgtgatcga tactgcttgc tgct 934

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ctgcagtgca gcgtgacccg gtcgtgcccc tctctagaga taatgagcat tgcatgtcta 60

agttataaaa aattaccaca tatttttttt gtcacacttg tttgaagtgc agtttatcta 120

tctttataca tatatttaaa ctttactcta cgaataatat aatctatagt actacaataa 180

tatcagtgtt ttagagaatc atataaatga acagttagac atggtctaaa ggacaattga 240

gtattttgac aacaggactc tacagtttta tctttttagt gtgcatgtgt tctccttttt 300

ttttgcaaat agcttcacct atataatact tcatccattt tattagtaca tccatttagg 360

gtttagggtt aatggttttt atagactaat ttttttagta catctatttt attctatttt 420

agcctctaaa ttaagaaaac taaaactcta ttttagtttt tttatttaat aatttagata 480

taaaatagaa taaaataaag tgactaaaaa ttaaacaaat accctttaag aaattaaaaa 540

aactaaggaa acatttttct tgtttcgagt agataatgcc agcctgttaa acgccgtcga 600

cgagtctaac ggacaccaac cagcgaacca gcagcgtcgc gtcgggccaa gcgaagcaga 660

cggcacggca tctctgtcgc tgcctctgga cccctctcga gagttccgct ccaccgttgg 720

acttgctccg ctgtcggcat ccagaaattg cgtggcggag cggcagacgt gagccggcac 780

ggcaggcggc ctcctcctcc tctcacggca cggcagctac gggggattcc tttcccaccg 840

ctccttcgct ttcccttcct cgcccgccgt aataaataga caccccctcc acaccctctt 900

tccccaacct cgtgttgttc ggagcgcaca cacacacaac cagatctccc ccaaatccac 960

ccgtcggcac ctccgcttca aggtacgccg ctcgtcctcc cccccccccc ctctctacct 1020

tctctagatc ggcgttccgg tccatggtta gggcccggta gttctacttc tgttcatgtt 1080

tgtgttagat ccgtgtttgt gttagatccg tgctgctagc gttcgtacac ggatgcgacc 1140

tgtacgtcag acacgttctg attgctaact tgccagtgtt tctctttggg gaatcctggg 1200

atggctctag ccgttccgca gacgggatcg atttcatgat tttttttgtt tcgttgcata 1260

gggtttggtt tgcccttttc ctttatttca atatatgccg tgcacttgtt tgtcgggtca 1320

tcttttcatg cttttttttg tcttggttgt gatgatgtgg tctggttggg cggtcgttct 1380

agatcggagt agaattctgt ttcaaactac ctggtggatt tattaatttt ggatctgtat 1440

gtgtgtgcca tacatattca tagttacgaa ttgaagatga tggatggaaa tatcgatcta 1500

ggataggtat acatgttgat gcgggtttta ctgatgcata tacagagatg ctttttgttc 1560

gcttggttgt gatgatgtgg tgtggttggg cggtcgttca ttcgttctag atcggagtag 1620

aatactgttt caaactacct ggtgtattta ttaattttgg aactgtatgt gtgtgtcata 1680

catcttcata gttacgagtt taagatggat ggaaatatcg atctaggata ggtatacatg 1740

ttgatgtggg ttttactgat gcatatacat gatggcatat gcagcatcta ttcatatgct 1800

ctaaccttga gtacctatct attataataa acaagtatgt tttataatta ttttgatctt 1860

gatatacttg gatgatggca tatgcagcag ctatatgtgg atttttttag ccctgccttc 1920

atacgctatt tatttgcttg gtactgtttc ttttgtcgat gctcaccctg ttgtttggtg 1980

ttacttctgc agccctaggc ctactagatg gattacaagg accacgacgg ggattacaag 2040

gaccacgaca ttgattacaa ggatgatgat gacaagatgg ctccgaagaa gaagaggaag 2100

gttggcatcc acggggtgcc agctgctgac aagaagtact cgatcggcct cgatattggg 2160

actaactctg ttggctgggc cgtgatcacc gacgagtaca aggtgccctc aaagaagttc 2220

aaggtcctgg gcaacaccga tcggcattcc atcaagaaga atctcattgg cgctctcctg 2280

ttcgacagcg gcgagacggc tgaggctacg cggctcaagc gcaccgcccg caggcggtac 2340

acgcgcagga agaatcgcat ctgctacctg caggagattt tctccaacga gatggcgaag 2400

gttgacgatt ctttcttcca caggctggag gagtcattcc tcgtggagga ggataagaag 2460

cacgagcggc atccaatctt cggcaacatt gtcgacgagg ttgcctacca cgagaagtac 2520

cctacgatct accatctgcg gaagaagctc gtggactcca cagataaggc ggacctccgc 2580

ctgatctacc tcgctctggc ccacatgatt aagttcaggg gccatttcct gatcgagggg 2640

gatctcaacc cggacaatag cgatgttgac aagctgttca tccagctcgt gcagacgtac 2700

aaccagctct tcgaggagaa ccccattaat gcgtcaggcg tcgacgcgaa ggctatcctg 2760

tccgctaggc tctcgaagtc tcggcgcctc gagaacctga tcgcccagct gccgggcgag 2820

aagaagaacg gcctgttcgg gaatctcatt gcgctcagcc tggggctcac gcccaacttc 2880

aagtcgaatt tcgatctcgc tgaggacgcc aagctgcagc tctccaagga cacatacgac 2940

gatgacctgg ataacctcct ggcccagatc ggcgatcagt acgcggacct gttcctcgct 3000

gccaagaatc tgtcggacgc catcctcctg tctgatattc tcagggtgaa caccgagatt 3060

acgaaggctc cgctctcagc ctccatgatc aagcgctacg acgagcacca tcaggatctg 3120

accctcctga aggcgctggt caggcagcag ctccccgaga agtacaagga gatcttcttc 3180

gatcagtcga agaacggcta cgctgggtac attgacggcg gggcctctca ggaggagttc 3240

tacaagttca tcaagccgat tctggagaag atggacggca cggaggagct gctggtgaag 3300

ctcaatcgcg aggacctcct gaggaagcag cggacattcg ataacggcag catcccacac 3360

cagattcatc tcggggagct gcacgctatc ctgaggaggc aggaggactt ctaccctttc 3420

ctcaaggata accgcgagaa gatcgagaag attctgactt tcaggatccc gtactacgtc 3480

ggcccactcg ctaggggcaa ctcccgcttc gcttggatga cccgcaagtc agaggagacg 3540

atcacgccgt ggaacttcga ggaggtggtc gacaagggcg ctagcgctca gtcgttcatc 3600

gagaggatga cgaatttcga caagaacctg ccaaatgaga aggtgctccc taagcactcg 3660

ctcctgtacg agtacttcac agtctacaac gagctgacta aggtgaagta tgtgaccgag 3720

ggcatgagga agccggcttt cctgtctggg gagcagaaga aggccatcgt ggacctcctg 3780

ttcaagacca accggaaggt cacggttaag cagctcaagg aggactactt caagaagatt 3840

gagtgcttcg attcggtcga gatctctggc gttgaggacc gcttcaacgc ctccctgggg 3900

acctaccacg atctcctgaa gatcattaag gataaggact tcctggacaa cgaggagaat 3960

gaggatatcc tcgaggacat tgtgctgaca ctcactctgt tcgaggaccg ggagatgatc 4020

gaggagcgcc tgaagactta cgcccatctc ttcgatgaca aggtcatgaa gcagctcaag 4080

aggaggaggt acaccggctg ggggaggctg agcaggaagc tcatcaacgg cattcgggac 4140

aagcagtccg ggaagacgat cctcgacttc ctgaagagcg atggcttcgc gaaccgcaat 4200

ttcatgcagc tgattcacga tgacagcctc acattcaagg aggatatcca gaaggctcag 4260

gtgagcggcc agggggactc gctgcacgag catatcgcga acctcgctgg ctcgccagct 4320

atcaagaagg ggattctgca gaccgtgaag gttgtggacg agctggtgaa ggtcatgggc 4380

aggcacaagc ctgagaacat cgtcattgag atggcccggg agaatcagac cacgcagaag 4440

ggccagaaga actcacgcga gaggatgaag aggatcgagg agggcattaa ggagctgggg 4500

tcccagatcc tcaaggagca cccggtggag aacacgcagc tgcagaatga gaagctctac 4560

ctgtactacc tccagaatgg ccgcgatatg tatgtggacc aggagctgga tattaacagg 4620

ctcagcgatt acgacgtcga tcatatcgtt ccacagtcat tcctgaagga tgactccatt 4680

gacaacaagg tcctcaccag gtcggacaag aaccggggca agtctgataa tgttccttca 4740

gaggaggtcg ttaagaagat gaagaactac tggcgccagc tcctgaatgc caagctgatc 4800

acgcagcgga agttcgataa cctcacaaag gctgagaggg gcgggctctc tgagctggac 4860

aaggcgggct tcatcaagag gcagctggtc gagacacggc agatcactaa gcacgttgcg 4920

cagattctcg actcacggat gaacactaag tacgatgaga atgacaagct gatccgcgag 4980

gtgaaggtca tcaccctgaa gtcaaagctc gtctccgact tcaggaagga tttccagttc 5040

tacaaggttc gggagatcaa caattaccac catgcccatg acgcgtacct gaacgcggtg 5100

gtcggcacag ctctgatcaa gaagtaccca aagctcgaga gcgagttcgt gtacggggac 5160

tacaaggttt acgatgtgag gaagatgatc gccaagtcgg agcaggagat tggcaaggct 5220

accgccaagt acttcttcta ctctaacatt atgaatttct tcaagacaga gatcactctg 5280

gccaatggcg agatccggaa gcgccccctc atcgagacga acggcgagac gggggagatc 5340

gtgtgggaca agggcaggga tttcgcgacc gtcaggaagg ttctctccat gccacaagtg 5400

aatatcgtca agaagacaga ggtccagact ggcgggttct ctaaggagtc aattctgcct 5460

aagcggaaca gcgacaagct catcgcccgc aagaaggact gggatccgaa gaagtacggc 5520

gggttcgaca gccccactgt ggcctactcg gtcctggttg tggcgaaggt tgagaagggc 5580

aagtccaaga agctcaagag cgtgaaggag ctgctgggga tcacgattat ggagcgctcc 5640

agcttcgaga agaacccgat cgatttcctg gaggcgaagg gctacaagga ggtgaagaag 5700

gacctgatca ttaagctccc caagtactca ctcttcgagc tggagaacgg caggaagcgg 5760

atgctggctt ccgctggcga gctgcagaag gggaacgagc tggctctgcc gtccaagtat 5820

gtgaacttcc tctacctggc ctcccactac gagaagctca agggcagccc cgaggacaac 5880

gagcagaagc agctgttcgt cgagcagcac aagcattacc tcgacgagat cattgagcag 5940

atttccgagt tctccaagcg cgtgatcctg gccgacgcga atctggataa ggtcctctcc 6000

gcgtacaaca agcaccgcga caagccaatc agggagcagg ctgagaatat cattcatctc 6060

ttcaccctga cgaacctcgg cgcccctgct gctttcaagt acttcgacac aactatcgat 6120

cgcaagaggt acacaagcac taaggaggtc ctggacgcga ccctcatcca ccagtcgatt 6180

accggcctct acgagacgcg catcgacctg tctcagctcg ggggcgacaa gcggccagcg 6240

gcgacgaaga aggcggggca ggcgaagaag aagaagtgag ctcagagctt tcgttcgtat 6300

catcggtttc gacaacgttc gtcaagttca atgcatcagt ttcattgcgc acacaccaga 6360

atcctactga gtttgagtat tatggcattg ggaaaactgt ttttcttgta ccatttgttg 6420

tgcttgtaat ttactgtgtt ttttattcgg ttttcgctat cgaactgtga aatggaaatg 6480

gatggagaag agttaatgaa tgatatggtc cttttgttca ttctcaaatt aatattattt 6540

gttttttctc ttatttgttg tgtgttgaat ttgaaattat aagagatatg caaacatttt 6600

gttttgagta aaaatgtgtc aaatcgtggc ctctaatgac cgaagttaat atgaggagta 6660

aaacacttgt agttgtacca ttatgcttat tcactaggca acaaatatat tttcagacct 6720

agaaaagctg caaatgttac tgaatacaag tatgtcctct tgtgttttag acatttatga 6780

actttccttt atgtaattt 6799

<210> 3

<211> 16248

<212> DNA

<213> Artificial sequence

<221> nucleotide sequence of pBUE411-TaGSR1 binary vector

<222>（1）...（16248）

<400> 3

taaacgctct tttctcttag gtttacccgc caatatatcc tgtcaaacac tgatagttta 60

aactgaaggc gggaaacgac aatctgatcc aagctcaagc tgctctagca ttcgccattc 120

aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg 180

gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca 240

cgacgttgta aaacgacggc cagtgccaag cttcatgaat ccaaaccaca cggagttcaa 300

attcccacag attaaggctc gtccgtcgca caaggtaatg tgtgaatatt atatctgtcg 360

tgcaaaattg cctggcctgc acaattgctg ttatagttgg cggcagggag agttttaaca 420

ttgactagcg tgctgataat ttgtgagaaa taataattga caagtagata ctgacatttg 480

agaagagctt ctgaactgtt attagtaaca aaaatggaaa gctgatgcac ggaaaaagga 540

aagaaaaagc catacttttt tttaggtagg aaaagaaaaa gccatacgag actgatgtct 600

ctcagatggg ccgggatctg tctatctagc aggcagcagc ccaccaacct cacgggccag 660

caattacgag tccttctaaa agctcccgcc gaggggcgct ggcgctgctg tgcagcagca 720

cgtctaacat tagtcccacc tcgccagttt acagggagca gaaccagctt ataagcggag 780

gcgcggcacc aagaagcggc ggcggcgcgt cgtcgccgga catgctgctc gacgagcagc 840

gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 900

ggcaccgagt cggtgctttt ttttttcgtt ttgcattgag ttttctccgt cgcatgtttg 960

cagttttatt ttccgttttg cattgaaatt tctccgtctc atgtttgcag cgtgttcaaa 1020

aagtacgcag ctgtatttca cttatttacg gcgccacatt ttcatgccgt ttgtgccaac 1080

tatcccgagc tagtgaatac agcttggctt cacacaacac tggtgacccg ctgacctgct 1140

cgtacctcgt accgtcgtac ggcacagcat ttggaattaa agggtgtgat cgatactgct 1200

tgctgctaag cttgcatgcc tgcagtgcag cgtgacccgg tcgtgcccct ctctagagat 1260

aatgagcatt gcatgtctaa gttataaaaa attaccacat attttttttg tcacacttgt 1320

ttgaagtgca gtttatctat ctttatacat atatttaaac tttactctac gaataatata 1380

atctatagta ctacaataat atcagtgttt tagagaatca tataaatgaa cagttagaca 1440

tggtctaaag gacaattgag tattttgaca acaggactct acagttttat ctttttagtg 1500

tgcatgtgtt ctcctttttt tttgcaaata gcttcaccta tataatactt catccatttt 1560

attagtacat ccatttaggg tttagggtta atggttttta tagactaatt tttttagtac 1620

atctatttta ttctatttta gcctctaaat taagaaaact aaaactctat tttagttttt 1680

ttatttaata atttagatat aaaatagaat aaaataaagt gactaaaaat taaacaaata 1740

ccctttaaga aattaaaaaa actaaggaaa catttttctt gtttcgagta gataatgcca 1800

gcctgttaaa cgccgtcgac gagtctaacg gacaccaacc agcgaaccag cagcgtcgcg 1860

tcgggccaag cgaagcagac ggcacggcat ctctgtcgct gcctctggac ccctctcgag 1920

agttccgctc caccgttgga cttgctccgc tgtcggcatc cagaaattgc gtggcggagc 1980

ggcagacgtg agccggcacg gcaggcggcc tcctcctcct ctcacggcac ggcagctacg 2040

ggggattcct ttcccaccgc tccttcgctt tcccttcctc gcccgccgta ataaatagac 2100

accccctcca caccctcttt ccccaacctc gtgttgttcg gagcgcacac acacacaacc 2160

agatctcccc caaatccacc cgtcggcacc tccgcttcaa ggtacgccgc tcgtcctccc 2220

cccccccccc tctctacctt ctctagatcg gcgttccggt ccatggttag ggcccggtag 2280

ttctacttct gttcatgttt gtgttagatc cgtgtttgtg ttagatccgt gctgctagcg 2340

ttcgtacacg gatgcgacct gtacgtcaga cacgttctga ttgctaactt gccagtgttt 2400

ctctttgggg aatcctggga tggctctagc cgttccgcag acgggatcga tttcatgatt 2460

ttttttgttt cgttgcatag ggtttggttt gcccttttcc tttatttcaa tatatgccgt 2520

gcacttgttt gtcgggtcat cttttcatgc ttttttttgt cttggttgtg atgatgtggt 2580

ctggttgggc ggtcgttcta gatcggagta gaattctgtt tcaaactacc tggtggattt 2640

attaattttg gatctgtatg tgtgtgccat acatattcat agttacgaat tgaagatgat 2700

ggatggaaat atcgatctag gataggtata catgttgatg cgggttttac tgatgcatat 2760

acagagatgc tttttgttcg cttggttgtg atgatgtggt gtggttgggc ggtcgttcat 2820

tcgttctaga tcggagtaga atactgtttc aaactacctg gtgtatttat taattttgga 2880

actgtatgtg tgtgtcatac atcttcatag ttacgagttt aagatggatg gaaatatcga 2940

tctaggatag gtatacatgt tgatgtgggt tttactgatg catatacatg atggcatatg 3000

cagcatctat tcatatgctc taaccttgag tacctatcta ttataataaa caagtatgtt 3060

ttataattat tttgatcttg atatacttgg atgatggcat atgcagcagc tatatgtgga 3120

tttttttagc cctgccttca tacgctattt atttgcttgg tactgtttct tttgtcgatg 3180

ctcaccctgt tgtttggtgt tacttctgca gccctaggcc tactagatgg attacaagga 3240

ccacgacggg gattacaagg accacgacat tgattacaag gatgatgatg acaagatggc 3300

tccgaagaag aagaggaagg ttggcatcca cggggtgcca gctgctgaca agaagtactc 3360

gatcggcctc gatattggga ctaactctgt tggctgggcc gtgatcaccg acgagtacaa 3420

ggtgccctca aagaagttca aggtcctggg caacaccgat cggcattcca tcaagaagaa 3480

tctcattggc gctctcctgt tcgacagcgg cgagacggct gaggctacgc ggctcaagcg 3540

caccgcccgc aggcggtaca cgcgcaggaa gaatcgcatc tgctacctgc aggagatttt 3600

ctccaacgag atggcgaagg ttgacgattc tttcttccac aggctggagg agtcattcct 3660

cgtggaggag gataagaagc acgagcggca tccaatcttc ggcaacattg tcgacgaggt 3720

tgcctaccac gagaagtacc ctacgatcta ccatctgcgg aagaagctcg tggactccac 3780

agataaggcg gacctccgcc tgatctacct cgctctggcc cacatgatta agttcagggg 3840

ccatttcctg atcgaggggg atctcaaccc ggacaatagc gatgttgaca agctgttcat 3900

ccagctcgtg cagacgtaca accagctctt cgaggagaac cccattaatg cgtcaggcgt 3960

cgacgcgaag gctatcctgt ccgctaggct ctcgaagtct cggcgcctcg agaacctgat 4020

cgcccagctg ccgggcgaga agaagaacgg cctgttcggg aatctcattg cgctcagcct 4080

ggggctcacg cccaacttca agtcgaattt cgatctcgct gaggacgcca agctgcagct 4140

ctccaaggac acatacgacg atgacctgga taacctcctg gcccagatcg gcgatcagta 4200

cgcggacctg ttcctcgctg ccaagaatct gtcggacgcc atcctcctgt ctgatattct 4260

cagggtgaac accgagatta cgaaggctcc gctctcagcc tccatgatca agcgctacga 4320

cgagcaccat caggatctga ccctcctgaa ggcgctggtc aggcagcagc tccccgagaa 4380

gtacaaggag atcttcttcg atcagtcgaa gaacggctac gctgggtaca ttgacggcgg 4440

ggcctctcag gaggagttct acaagttcat caagccgatt ctggagaaga tggacggcac 4500

ggaggagctg ctggtgaagc tcaatcgcga ggacctcctg aggaagcagc ggacattcga 4560

taacggcagc atcccacacc agattcatct cggggagctg cacgctatcc tgaggaggca 4620

ggaggacttc taccctttcc tcaaggataa ccgcgagaag atcgagaaga ttctgacttt 4680

caggatcccg tactacgtcg gcccactcgc taggggcaac tcccgcttcg cttggatgac 4740

ccgcaagtca gaggagacga tcacgccgtg gaacttcgag gaggtggtcg acaagggcgc 4800

tagcgctcag tcgttcatcg agaggatgac gaatttcgac aagaacctgc caaatgagaa 4860

ggtgctccct aagcactcgc tcctgtacga gtacttcaca gtctacaacg agctgactaa 4920

ggtgaagtat gtgaccgagg gcatgaggaa gccggctttc ctgtctgggg agcagaagaa 4980

ggccatcgtg gacctcctgt tcaagaccaa ccggaaggtc acggttaagc agctcaagga 5040

ggactacttc aagaagattg agtgcttcga ttcggtcgag atctctggcg ttgaggaccg 5100

cttcaacgcc tccctgggga cctaccacga tctcctgaag atcattaagg ataaggactt 5160

cctggacaac gaggagaatg aggatatcct cgaggacatt gtgctgacac tcactctgtt 5220

cgaggaccgg gagatgatcg aggagcgcct gaagacttac gcccatctct tcgatgacaa 5280

ggtcatgaag cagctcaaga ggaggaggta caccggctgg gggaggctga gcaggaagct 5340

catcaacggc attcgggaca agcagtccgg gaagacgatc ctcgacttcc tgaagagcga 5400

tggcttcgcg aaccgcaatt tcatgcagct gattcacgat gacagcctca cattcaagga 5460

ggatatccag aaggctcagg tgagcggcca gggggactcg ctgcacgagc atatcgcgaa 5520

cctcgctggc tcgccagcta tcaagaaggg gattctgcag accgtgaagg ttgtggacga 5580

gctggtgaag gtcatgggca ggcacaagcc tgagaacatc gtcattgaga tggcccggga 5640

gaatcagacc acgcagaagg gccagaagaa ctcacgcgag aggatgaaga ggatcgagga 5700

gggcattaag gagctggggt cccagatcct caaggagcac ccggtggaga acacgcagct 5760

gcagaatgag aagctctacc tgtactacct ccagaatggc cgcgatatgt atgtggacca 5820

ggagctggat attaacaggc tcagcgatta cgacgtcgat catatcgttc cacagtcatt 5880

cctgaaggat gactccattg acaacaaggt cctcaccagg tcggacaaga accggggcaa 5940

gtctgataat gttccttcag aggaggtcgt taagaagatg aagaactact ggcgccagct 6000

cctgaatgcc aagctgatca cgcagcggaa gttcgataac ctcacaaagg ctgagagggg 6060

cgggctctct gagctggaca aggcgggctt catcaagagg cagctggtcg agacacggca 6120

gatcactaag cacgttgcgc agattctcga ctcacggatg aacactaagt acgatgagaa 6180

tgacaagctg atccgcgagg tgaaggtcat caccctgaag tcaaagctcg tctccgactt 6240

caggaaggat ttccagttct acaaggttcg ggagatcaac aattaccacc atgcccatga 6300

cgcgtacctg aacgcggtgg tcggcacagc tctgatcaag aagtacccaa agctcgagag 6360

cgagttcgtg tacggggact acaaggttta cgatgtgagg aagatgatcg ccaagtcgga 6420

gcaggagatt ggcaaggcta ccgccaagta cttcttctac tctaacatta tgaatttctt 6480

caagacagag atcactctgg ccaatggcga gatccggaag cgccccctca tcgagacgaa 6540

cggcgagacg ggggagatcg tgtgggacaa gggcagggat ttcgcgaccg tcaggaaggt 6600

tctctccatg ccacaagtga atatcgtcaa gaagacagag gtccagactg gcgggttctc 6660

taaggagtca attctgccta agcggaacag cgacaagctc atcgcccgca agaaggactg 6720

ggatccgaag aagtacggcg ggttcgacag ccccactgtg gcctactcgg tcctggttgt 6780

ggcgaaggtt gagaagggca agtccaagaa gctcaagagc gtgaaggagc tgctggggat 6840

cacgattatg gagcgctcca gcttcgagaa gaacccgatc gatttcctgg aggcgaaggg 6900

ctacaaggag gtgaagaagg acctgatcat taagctcccc aagtactcac tcttcgagct 6960

ggagaacggc aggaagcgga tgctggcttc cgctggcgag ctgcagaagg ggaacgagct 7020

ggctctgccg tccaagtatg tgaacttcct ctacctggcc tcccactacg agaagctcaa 7080

gggcagcccc gaggacaacg agcagaagca gctgttcgtc gagcagcaca agcattacct 7140

cgacgagatc attgagcaga tttccgagtt ctccaagcgc gtgatcctgg ccgacgcgaa 7200

tctggataag gtcctctccg cgtacaacaa gcaccgcgac aagccaatca gggagcaggc 7260

tgagaatatc attcatctct tcaccctgac gaacctcggc gcccctgctg ctttcaagta 7320

cttcgacaca actatcgatc gcaagaggta cacaagcact aaggaggtcc tggacgcgac 7380

cctcatccac cagtcgatta ccggcctcta cgagacgcgc atcgacctgt ctcagctcgg 7440

gggcgacaag cggccagcgg cgacgaagaa ggcggggcag gcgaagaaga agaagtgagc 7500

tcagagcttt cgttcgtatc atcggtttcg acaacgttcg tcaagttcaa tgcatcagtt 7560

tcattgcgca cacaccagaa tcctactgag tttgagtatt atggcattgg gaaaactgtt 7620

tttcttgtac catttgttgt gcttgtaatt tactgtgttt tttattcggt tttcgctatc 7680

gaactgtgaa atggaaatgg atggagaaga gttaatgaat gatatggtcc ttttgttcat 7740

tctcaaatta atattatttg ttttttctct tatttgttgt gtgttgaatt tgaaattata 7800

agagatatgc aaacattttg ttttgagtaa aaatgtgtca aatcgtggcc tctaatgacc 7860

gaagttaata tgaggagtaa aacacttgta gttgtaccat tatgcttatt cactaggcaa 7920

caaatatatt ttcagaccta gaaaagctgc aaatgttact gaatacaagt atgtcctctt 7980

gtgttttaga catttatgaa ctttccttta tgtaattttc cagaatcctt gtcagattct 8040

aatcattgct ttataattat agttatactc atggatttgt agttgagtat gaaaatattt 8100

tttaatgcat tttatgactt gccaattgat tgacaacgaa ttcgtaatca tggtcatagc 8160

tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca 8220

taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct 8280

cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac 8340

gcgcggggag aggcggtttg cgtattggct agagcagctt gccaacatgg tggagcacga 8400

cactctcgtc tactccaaga atatcaaaga tacagtctca gaagaccaaa gggctattga 8460

gacttttcaa caaagggtaa tatcgggaaa cctcctcgga ttccattgcc cagctatctg 8520

tcacttcatc aaaaggacag tagaaaagga aggtggcacc tacaaatgcc atcattgcga 8580

taaaggaaag gctatcgttc aagatgcctc tgccgacagt ggtcccaaag atggaccccc 8640

acccacgagg agcatcgtgg aaaaagaaga cgttccaacc acgtcttcaa agcaagtgga 8700

ttgatgtgat aacatggtgg agcacgacac tctcgtctac tccaagaata tcaaagatac 8760

agtctcagaa gaccaaaggg ctattgagac ttttcaacaa agggtaatat cgggaaacct 8820

cctcggattc cattgcccag ctatctgtca cttcatcaaa aggacagtag aaaaggaagg 8880

tggcacctac aaatgccatc attgcgataa aggaaaggct atcgttcaag atgcctctgc 8940

cgacagtggt cccaaagatg gacccccacc cacgaggagc atcgtggaaa aagaagacgt 9000

tccaaccacg tcttcaaagc aagtggattg atgtgatatc tccactgacg taagggatga 9060

cgcacaatcc cactatcctt cgcaagacct tcctctatat aaggaagttc atttcatttg 9120

gagaggacac gctgaaatca ccagtctctc tctacaaatc tatctctctc gagtctacca 9180

tgagcccaga acgacgcccg gccgacatcc gccgtgccac cgaggcggac atgccggcgg 9240

tctgcaccat cgtcaaccac tacatcgaga caagcacggt caacttccgt accgagccgc 9300

aggaaccgca ggagtggacg gacgacctcg tccgtctgcg ggagcgctat ccctggctcg 9360

tcgccgaggt ggacggcgag gtcgccggca tcgcctacgc gggcccctgg aaggcacgca 9420

acgcctacga ctggacggcc gagtcgaccg tgtacgtctc cccccgccac cagcggacgg 9480

gactgggctc cacgctctac acccacctgc tgaagtccct ggaggcacag ggcttcaaga 9540

gcgtggtcgc tgtcatcggg ctgcccaacg acccgagcgt gcgcatgcac gaggcgctcg 9600

gatatgcccc ccgcggcatg ctgcgggcgg ccggcttcaa gcacgggaac tggcatgacg 9660

tgggtttctg gcagctggac ttcagcctgc cggtaccgcc ccgtccggtc ctgcccgtca 9720

ccgagatttg actcgagttt ctccataata atgtgtgagt agttcccaga taagggaatt 9780

agggttccta tagggtttcg ctcatgtgtt gagcatataa gaaaccctta gtatgtattt 9840

gtatttgtaa aatacttcta tcaataaaat ttctaattcc taaaaccaaa atccagtact 9900

aaaatccaga tcccccgaat taattcggcg ttaattcagt acattaaaaa cgtccgcaat 9960

gtgttattaa gttgtctaag cgtcaatttg tttacaccac aatatatcct gccaccagcc 10020

agccaacagc tccccgaccg gcagctcggc acaaaatcac cactcgatac aggcagccca 10080

tcagtccggg acggcgtcag cgggagagcc gttgtaaggc ggcagacttt gctcatgtta 10140

ccgatgctat tcggaagaac ggcaactaag ctgccgggtt tgaaacacgg atgatctcgc 10200

ggagggtagc atgttgattg taacgatgac agagcgttgc tgcctgtgat caccgcggtt 10260

tcaaaatcgg ctccgtcgat actatgttat acgccaactt tgaaaacaac tttgaaaaag 10320

ctgttttctg gtatttaagg ttttagaatg caaggaacag tgaattggag ttcgtcttgt 10380

tataattagc ttcttggggt atctttaaat actgtagaaa agaggaagga aataataaat 10440

ggctaaaatg agaatatcac cggaattgaa aaaactgatc gaaaaatacc gctgcgtaaa 10500

agatacggaa ggaatgtctc ctgctaaggt atataagctg gtgggagaaa atgaaaacct 10560

atatttaaaa atgacggaca gccggtataa agggaccacc tatgatgtgg aacgggaaaa 10620

ggacatgatg ctatggctgg aaggaaagct gcctgttcca aaggtcctgc actttgaacg 10680

gcatgatggc tggagcaatc tgctcatgag tgaggccgat ggcgtccttt gctcggaaga 10740

gtatgaagat gaacaaagcc ctgaaaagat tatcgagctg tatgcggagt gcatcaggct 10800

ctttcactcc atcgacatat cggattgtcc ctatacgaat agcttagaca gccgcttagc 10860

cgaattggat tacttactga ataacgatct ggccgatgtg gattgcgaaa actgggaaga 10920

agacactcca tttaaagatc cgcgcgagct gtatgatttt ttaaagacgg aaaagcccga 10980

agaggaactt gtcttttccc acggcgacct gggagacagc aacatctttg tgaaagatgg 11040

caaagtaagt ggctttattg atcttgggag aagcggcagg gcggacaagt ggtatgacat 11100

tgccttctgc gtccggtcga tcagggagga tatcggggaa gaacagtatg tcgagctatt 11160

ttttgactta ctggggatca agcctgattg ggagaaaata aaatattata ttttactgga 11220

tgaattgttt tagtacctag aatgcatgac caaaatccct taacgtgagt tttcgttcca 11280

ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 11340

cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 11400

tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 11460

tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 11520

tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 11580

tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 11640

ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 11700

acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 11760

ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 11820

gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 11880

ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 11940

ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga 12000

taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg 12060

cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca 12120

tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc 12180

atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga 12240

cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac 12300

agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg 12360

aaacgcgcga ggcagggtgc cttgatgtgg gcgccggcgg tcgagtggcg acggcgcggc 12420

ttgtccgcgc cctggtagat tgcctggccg taggccagcc atttttgagc ggccagcggc 12480

cgcgataggc cgacgcgaag cggcggggcg tagggagcgc agcgaccgaa gggtaggcgc 12540

tttttgcagc tcttcggctg tgcgctggcc agacagttat gcacaggcca ggcgggtttt 12600

aagagtttta ataagtttta aagagtttta ggcggaaaaa tcgccttttt tctcttttat 12660

atcagtcact tacatgtgtg accggttccc aatgtacggc tttgggttcc caatgtacgg 12720

gttccggttc ccaatgtacg gctttgggtt cccaatgtac gtgctatcca caggaaacag 12780

accttttcga cctttttccc ctgctagggc aatttgccct agcatctgct ccgtacatta 12840

ggaaccggcg gatgcttcgc cctcgatcag gttgcggtag cgcatgacta ggatcgggcc 12900

agcctgcccc gcctcctcct tcaaatcgta ctccggcagg tcatttgacc cgatcagctt 12960

gcgcacggtg aaacagaact tcttgaactc tccggcgctg ccactgcgtt cgtagatcgt 13020

cttgaacaac catctggctt ctgccttgcc tgcggcgcgg cgtgccaggc ggtagagaaa 13080

acggccgatg ccgggatcga tcaaaaagta atcggggtga accgtcagca cgtccgggtt 13140

cttgccttct gtgatctcgc ggtacatcca atcagctagc tcgatctcga tgtactccgg 13200

ccgcccggtt tcgctcttta cgatcttgta gcggctaatc aaggcttcac cctcggatac 13260

cgtcaccagg cggccgttct tggccttctt cgtacgctgc atggcaacgt gcgtggtgtt 13320

taaccgaatg caggtttcta ccaggtcgtc tttctgcttt ccgccatcgg ctcgccggca 13380

gaacttgagt acgtccgcaa cgtgtggacg gaacacgcgg ccgggcttgt ctcccttccc 13440

ttcccggtat cggttcatgg attcggttag atgggaaacc gccatcagta ccaggtcgta 13500

atcccacaca ctggccatgc cggccggccc tgcggaaacc tctacgtgcc cgtctggaag 13560

ctcgtagcgg atcacctcgc cagctcgtcg gtcacgcttc gacagacgga aaacggccac 13620

gtccatgatg ctgcgactat cgcgggtgcc cacgtcatag agcatcggaa cgaaaaaatc 13680

tggttgctcg tcgcccttgg gcggcttcct aatcgacggc gcaccggctg ccggcggttg 13740

ccgggattct ttgcggattc gatcagcggc cgcttgccac gattcaccgg ggcgtgcttc 13800

tgcctcgatg cgttgccgct gggcggcctg cgcggccttc aacttctcca ccaggtcatc 13860

acccagcgcc gcgccgattt gtaccgggcc ggatggtttg cgaccgctca cgccgattcc 13920

tcgggcttgg gggttccagt gccattgcag ggccggcaga caacccagcc gcttacgcct 13980

ggccaaccgc ccgttcctcc acacatgggg cattccacgg cgtcggtgcc tggttgttct 14040

tgattttcca tgccgcctcc tttagccgct aaaattcatc tactcattta ttcatttgct 14100

catttactct ggtagctgcg cgatgtattc agatagcagc tcggtaatgg tcttgccttg 14160

gcgtaccgcg tacatcttca gcttggtgtg atcctccgcc ggcaactgaa agttgacccg 14220

cttcatggct ggcgtgtctg ccaggctggc caacgttgca gccttgctgc tgcgtgcgct 14280

cggacggccg gcacttagcg tgtttgtgct tttgctcatt ttctctttac ctcattaact 14340

caaatgagtt ttgatttaat ttcagcggcc agcgcctgga cctcgcgggc agcgtcgccc 14400

tcgggttctg attcaagaac ggttgtgccg gcggcggcag tgcctgggta gctcacgcgc 14460

tgcgtgatac gggactcaag aatgggcagc tcgtacccgg ccagcgcctc ggcaacctca 14520

ccgccgatgc gcgtgccttt gatcgcccgc gacacgacaa aggccgcttg tagccttcca 14580

tccgtgacct caatgcgctg cttaaccagc tccaccaggt cggcggtggc ccatatgtcg 14640

taagggcttg gctgcaccgg aatcagcacg aagtcggctg ccttgatcgc ggacacagcc 14700

aagtccgccg cctggggcgc tccgtcgatc actacgaagt cgcgccggcc gatggccttc 14760

acgtcgcggt caatcgtcgg gcggtcgatg ccgacaacgg ttagcggttg atcttcccgc 14820

acggccgccc aatcgcgggc actgccctgg ggatcggaat cgactaacag aacatcggcc 14880

ccggcgagtt gcagggcgcg ggctagatgg gttgcgatgg tcgtcttgcc tgacccgcct 14940

ttctggttaa gtacagcgat aaccttcatg cgttcccctt gcgtatttgt ttatttactc 15000

atcgcatcat atacgcagcg accgcatgac gcaagctgtt ttactcaaat acacatcacc 15060

tttttagacg gcggcgctcg gtttcttcag cggccaagct ggccggccag gccgccagct 15120

tggcatcaga caaaccggcc aggatttcat gcagccgcac ggttgagacg tgcgcgggcg 15180

gctcgaacac gtacccggcc gcgatcatct ccgcctcgat ctcttcggta atgaaaaacg 15240

gttcgtcctg gccgtcctgg tgcggtttca tgcttgttcc tcttggcgtt cattctcggc 15300

ggccgccagg gcgtcggcct cggtcaatgc gtcctcacgg aaggcaccgc gccgcctggc 15360

ctcggtgggc gtcacttcct cgctgcgctc aagtgcgcgg tacagggtcg agcgatgcac 15420

gccaagcagt gcagccgcct ctttcacggt gcggccttcc tggtcgatca gctcgcgggc 15480

gtgcgcgatc tgtgccgggg tgagggtagg gcgggggcca aacttcacgc ctcgggcctt 15540

ggcggcctcg cgcccgctcc gggtgcggtc gatgattagg gaacgctcga actcggcaat 15600

gccggcgaac acggtcaaca ccatgcggcc ggccggcgtg gtggtgtcgg cccacggctc 15660

tgccaggcta cgcaggcccg cgccggcctc ctggatgcgc tcggcaatgt ccagtaggtc 15720

gcgggtgctg cgggccaggc ggtctagcct ggtcactgtc acaacgtcgc cagggcgtag 15780

gtggtcaagc atcctggcca gctccgggcg gtcgcgcctg gtgccggtga tcttctcgga 15840

aaacagcttg gtgcagccgg ccgcgtgcag ttcggcccgt tggttggtca agtcctggtc 15900

gtcggtgctg acgcgggcat agcccagcag gccagcggcg gcgctcttgt tcatggcgta 15960

atgtctccgg ttctagtcgc aagtattcta ctttatgcga ctaaaacacg cgacaagaaa 16020

acgccaggaa aagggcaggg cggcagcctg tcgcgtaact taggacttgt gcgacatgtc 16080

gttttcagaa gacggctgca ctgaacgtca gaagccgact gcactatagc agcggagggg 16140

ttggatcaaa gtactttgat cccgagggga accctgtggt tggcatgcac atacaaatgg 16200

acgaacggat aaaccttttc acgccctttt aaatatccgt tattctaa 16248

<210> 4

<211> 612

<212> DNA

<213> wheat (Triticum aestivum L.)

<221> nucleotide sequence encoding protein TaGSR1

<222>（1）...（612）

<400> 4

atggacgcca agggcgcgac ggagagctca aaccctaacc gccgtccagc cagcgtaccg 60

tcggccgccg tggcgtcggc gtcggcgccg acgaagcgca tgctggcgtt ccacttcctg 120

cgcgcgctgt cccggatcca cggcgcggcc ggcccggcga ggcgccgcac gcgcaccatc 180

cgccgcgcgg cctactcctc catggcgcgc gccaccggcc cgcgccgcgc ctggagccgc 240

gcgctgctgc tccaggccca ggcgcgcgcg cggagatcca gggcggagac gtcgaggcgg 300

gccgccgtgc tcgtacggcg gcgcgtcgtc gccggaccgg cggtagcagc agcaccagca 360

ccggcacgcg ccgcttctgt cggcggacag acgtcgtcgg cggctgctat tcgcgcggcg 420

ctggtccctc cgccccctcc ggcgcggcag gcgggggagc cggccaggag cgacgcgctg 480

cggcggctcg tccccggagg cgccgggatg gagtactgca gcctgctgga ggagaccgcc 540

gactacgtcc gctgcctccg cgcgcaggtg cagctcatgc agggcctcgc cgacctcttc 600

tcctgccaat ga 612

<210> 5

<211> 23

<212> DNA

<213> wheat (Triticum aestivum L.)

<221> sgRNA nucleotide sequence of TaGSR1

<222>（1）...（23）

<400> 5

gcggcgcgtcgtcgccggaccgg 23

<210> 6

<211> 22

<212> DNA

<213> wheat (Triticum aestivum L.)

<221> sgRNA nucleotide sequence of TaGSR1

<222>（1）...（22）

<400> 6

atccacggcgcggccggcccgg 22

<210> 7

<211> 23

<212> DNA

<213> wheat (Triticum aestivum L.)

<221> sgRNA nucleotide sequence of TaGSR1

<222>（1）...（23）

<400> 7

aacgccagcatgcgcttcgtcgg 23

<210> 8

<211> 23

<212> DNA

<213> wheat (Triticum aestivum L.)

<221> sgRNA nucleotide sequence of TaGSR1

<222>（1）...（23）

<400> 8

tcccggatccacggcgcggccgg 23

<210> 9

<211> 527

<212> DNA

<213> wheat (Triticum aestivum L.)

<221> TaU3 promoter nucleotide sequence of wheat

<222>（1）...（527）

<400> 9

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 aagcggc 527

<210> 10

<211> 291

<212> DNA

<213> Artificial sequence

<221> nucleotide sequence of terminator T1

<222>（1）...（291）

<400> 10

tttttttttt cgttttgcat tgagttttct ccgtcgcatg tttgcagttt tattttccgt 60

tttgcattga aatttctccg tctcatgttt gcagcgtgtt caaaaagtac gcagctgtat 120

ttcacttatt tacggcgcca cattttcatg ccgtttgtgc caactatccc gagctagtga 180

atacagcttg gcttcacaca acactggtga cccgctgacc tgctcgtacc tcgtaccgtc 240

gtacggcaca gcatttggaa ttaaagggtg tgatcgatac tgcttgctgc t 291

<210> 11

<211> 1992

<212> DNA

<213> corn (Zea mays L.)

<221> nucleotide sequence of ubiquitin promoter Ubi of Zea mays

<222>（1）...（1992）

<400> 11

ctgcagtgca gcgtgacccg gtcgtgcccc tctctagaga taatgagcat tgcatgtcta 60

agttataaaa aattaccaca tatttttttt gtcacacttg tttgaagtgc agtttatcta 120

tctttataca tatatttaaa ctttactcta cgaataatat aatctatagt actacaataa 180

tatcagtgtt ttagagaatc atataaatga acagttagac atggtctaaa ggacaattga 240

gtattttgac aacaggactc tacagtttta tctttttagt gtgcatgtgt tctccttttt 300

ttttgcaaat agcttcacct atataatact tcatccattt tattagtaca tccatttagg 360

gtttagggtt aatggttttt atagactaat ttttttagta catctatttt attctatttt 420

agcctctaaa ttaagaaaac taaaactcta ttttagtttt tttatttaat aatttagata 480

taaaatagaa taaaataaag tgactaaaaa ttaaacaaat accctttaag aaattaaaaa 540

aactaaggaa acatttttct tgtttcgagt agataatgcc agcctgttaa acgccgtcga 600

cgagtctaac ggacaccaac cagcgaacca gcagcgtcgc gtcgggccaa gcgaagcaga 660

cggcacggca tctctgtcgc tgcctctgga cccctctcga gagttccgct ccaccgttgg 720

acttgctccg ctgtcggcat ccagaaattg cgtggcggag cggcagacgt gagccggcac 780

ggcaggcggc ctcctcctcc tctcacggca cggcagctac gggggattcc tttcccaccg 840

ctccttcgct ttcccttcct cgcccgccgt aataaataga caccccctcc acaccctctt 900

tccccaacct cgtgttgttc ggagcgcaca cacacacaac cagatctccc ccaaatccac 960

ccgtcggcac ctccgcttca aggtacgccg ctcgtcctcc cccccccccc ctctctacct 1020

tctctagatc ggcgttccgg tccatggtta gggcccggta gttctacttc tgttcatgtt 1080

tgtgttagat ccgtgtttgt gttagatccg tgctgctagc gttcgtacac ggatgcgacc 1140

tgtacgtcag acacgttctg attgctaact tgccagtgtt tctctttggg gaatcctggg 1200

atggctctag ccgttccgca gacgggatcg atttcatgat tttttttgtt tcgttgcata 1260

gggtttggtt tgcccttttc ctttatttca atatatgccg tgcacttgtt tgtcgggtca 1320

tcttttcatg cttttttttg tcttggttgt gatgatgtgg tctggttggg cggtcgttct 1380

agatcggagt agaattctgt ttcaaactac ctggtggatt tattaatttt ggatctgtat 1440

gtgtgtgcca tacatattca tagttacgaa ttgaagatga tggatggaaa tatcgatcta 1500

ggataggtat acatgttgat gcgggtttta ctgatgcata tacagagatg ctttttgttc 1560

gcttggttgt gatgatgtgg tgtggttggg cggtcgttca ttcgttctag atcggagtag 1620

aatactgttt caaactacct ggtgtattta ttaattttgg aactgtatgt gtgtgtcata 1680

catcttcata gttacgagtt taagatggat ggaaatatcg atctaggata ggtatacatg 1740

ttgatgtggg ttttactgat gcatatacat gatggcatat gcagcatcta ttcatatgct 1800

ctaaccttga gtacctatct attataataa acaagtatgt tttataatta ttttgatctt 1860

gatatacttg gatgatggca tatgcagcag ctatatgtgg atttttttag ccctgccttc 1920

atacgctatt tatttgcttg gtactgtttc ttttgtcgat gctcaccctg ttgtttggtg 1980

ttacttctgc ag 1992

<210> 12

<211> 4101

<212> DNA

<213> Artificial sequence

<221> nucleotide sequence encoded by maize Cas9

<222>（1）...（4101）

<400> 12

gacaagaagt actcgatcgg cctcgatatt gggactaact ctgttggctg ggccgtgatc 60

accgacgagt acaaggtgcc ctcaaagaag ttcaaggtcc tgggcaacac cgatcggcat 120

tccatcaaga agaatctcat tggcgctctc ctgttcgaca gcggcgagac ggctgaggct 180

acgcggctca agcgcaccgc ccgcaggcgg tacacgcgca ggaagaatcg catctgctac 240

ctgcaggaga ttttctccaa cgagatggcg aaggttgacg attctttctt ccacaggctg 300

gaggagtcat tcctcgtgga ggaggataag aagcacgagc ggcatccaat cttcggcaac 360

attgtcgacg aggttgccta ccacgagaag taccctacga tctaccatct gcggaagaag 420

ctcgtggact ccacagataa ggcggacctc cgcctgatct acctcgctct ggcccacatg 480

attaagttca ggggccattt cctgatcgag ggggatctca acccggacaa tagcgatgtt 540

gacaagctgt tcatccagct cgtgcagacg tacaaccagc tcttcgagga gaaccccatt 600

aatgcgtcag gcgtcgacgc gaaggctatc ctgtccgcta ggctctcgaa gtctcggcgc 660

ctcgagaacc tgatcgccca gctgccgggc gagaagaaga acggcctgtt cgggaatctc 720

attgcgctca gcctggggct cacgcccaac ttcaagtcga atttcgatct cgctgaggac 780

gccaagctgc agctctccaa ggacacatac gacgatgacc tggataacct cctggcccag 840

atcggcgatc agtacgcgga cctgttcctc gctgccaaga atctgtcgga cgccatcctc 900

ctgtctgata ttctcagggt gaacaccgag attacgaagg ctccgctctc agcctccatg 960

atcaagcgct acgacgagca ccatcaggat ctgaccctcc tgaaggcgct ggtcaggcag 1020

cagctccccg agaagtacaa ggagatcttc ttcgatcagt cgaagaacgg ctacgctggg 1080

tacattgacg gcggggcctc tcaggaggag ttctacaagt tcatcaagcc gattctggag 1140

aagatggacg gcacggagga gctgctggtg aagctcaatc gcgaggacct cctgaggaag 1200

cagcggacat tcgataacgg cagcatccca caccagattc atctcgggga gctgcacgct 1260

atcctgagga ggcaggagga cttctaccct ttcctcaagg ataaccgcga gaagatcgag 1320

aagattctga ctttcaggat cccgtactac gtcggcccac tcgctagggg caactcccgc 1380

ttcgcttgga tgacccgcaa gtcagaggag acgatcacgc cgtggaactt cgaggaggtg 1440

gtcgacaagg gcgctagcgc tcagtcgttc atcgagagga tgacgaattt cgacaagaac 1500

ctgccaaatg agaaggtgct ccctaagcac tcgctcctgt acgagtactt cacagtctac 1560

aacgagctga ctaaggtgaa gtatgtgacc gagggcatga ggaagccggc tttcctgtct 1620

ggggagcaga agaaggccat cgtggacctc ctgttcaaga ccaaccggaa ggtcacggtt 1680

aagcagctca aggaggacta cttcaagaag attgagtgct tcgattcggt cgagatctct 1740

ggcgttgagg accgcttcaa cgcctccctg gggacctacc acgatctcct gaagatcatt 1800

aaggataagg acttcctgga caacgaggag aatgaggata tcctcgagga cattgtgctg 1860

acactcactc tgttcgagga ccgggagatg atcgaggagc gcctgaagac ttacgcccat 1920

ctcttcgatg acaaggtcat gaagcagctc aagaggagga ggtacaccgg ctgggggagg 1980

ctgagcagga agctcatcaa cggcattcgg gacaagcagt ccgggaagac gatcctcgac 2040

ttcctgaaga gcgatggctt cgcgaaccgc aatttcatgc agctgattca cgatgacagc 2100

ctcacattca aggaggatat ccagaaggct caggtgagcg gccaggggga ctcgctgcac 2160

gagcatatcg cgaacctcgc tggctcgcca gctatcaaga aggggattct gcagaccgtg 2220

aaggttgtgg acgagctggt gaaggtcatg ggcaggcaca agcctgagaa catcgtcatt 2280

gagatggccc gggagaatca gaccacgcag aagggccaga agaactcacg cgagaggatg 2340

aagaggatcg aggagggcat taaggagctg gggtcccaga tcctcaagga gcacccggtg 2400

gagaacacgc agctgcagaa tgagaagctc tacctgtact acctccagaa tggccgcgat 2460

atgtatgtgg accaggagct ggatattaac aggctcagcg attacgacgt cgatcatatc 2520

gttccacagt cattcctgaa ggatgactcc attgacaaca aggtcctcac caggtcggac 2580

aagaaccggg gcaagtctga taatgttcct tcagaggagg tcgttaagaa gatgaagaac 2640

tactggcgcc agctcctgaa tgccaagctg atcacgcagc ggaagttcga taacctcaca 2700

aaggctgaga ggggcgggct ctctgagctg gacaaggcgg gcttcatcaa gaggcagctg 2760

gtcgagacac ggcagatcac taagcacgtt gcgcagattc tcgactcacg gatgaacact 2820

aagtacgatg agaatgacaa gctgatccgc gaggtgaagg tcatcaccct gaagtcaaag 2880

ctcgtctccg acttcaggaa ggatttccag ttctacaagg ttcgggagat caacaattac 2940

caccatgccc atgacgcgta cctgaacgcg gtggtcggca cagctctgat caagaagtac 3000

ccaaagctcg agagcgagtt cgtgtacggg gactacaagg tttacgatgt gaggaagatg 3060

atcgccaagt cggagcagga gattggcaag gctaccgcca agtacttctt ctactctaac 3120

attatgaatt tcttcaagac agagatcact ctggccaatg gcgagatccg gaagcgcccc 3180

ctcatcgaga cgaacggcga gacgggggag atcgtgtggg acaagggcag ggatttcgcg 3240

accgtcagga aggttctctc catgccacaa gtgaatatcg tcaagaagac agaggtccag 3300

actggcgggt tctctaagga gtcaattctg cctaagcgga acagcgacaa gctcatcgcc 3360

cgcaagaagg actgggatcc gaagaagtac ggcgggttcg acagccccac tgtggcctac 3420

tcggtcctgg ttgtggcgaa ggttgagaag ggcaagtcca agaagctcaa gagcgtgaag 3480

gagctgctgg ggatcacgat tatggagcgc tccagcttcg agaagaaccc gatcgatttc 3540

ctggaggcga agggctacaa ggaggtgaag aaggacctga tcattaagct ccccaagtac 3600

tcactcttcg agctggagaa cggcaggaag cggatgctgg cttccgctgg cgagctgcag 3660

aaggggaacg agctggctct gccgtccaag tatgtgaact tcctctacct ggcctcccac 3720

tacgagaagc tcaagggcag ccccgaggac aacgagcaga agcagctgtt cgtcgagcag 3780

cacaagcatt acctcgacga gatcattgag cagatttccg agttctccaa gcgcgtgatc 3840

ctggccgacg cgaatctgga taaggtcctc tccgcgtaca acaagcaccg cgacaagcca 3900

atcagggagc aggctgagaa tatcattcat ctcttcaccc tgacgaacct cggcgcccct 3960

gctgctttca agtacttcga cacaactatc gatcgcaaga ggtacacaag cactaaggag 4020

gtcctggacg cgaccctcat ccaccagtcg attaccggcc tctacgagac gcgcatcgac 4080

ctgtctcagc tcgggggcga c 4101

<210> 13

<211> 571

<212> DNA

<213> Artificial sequence

<221> nucleotide sequence of terminator T2

<222>（1）...（571）

<400> 13

aagcggccag cggcgacgaa gaaggcgggg caggcgaaga agaagaagtg agctcagagc 60

tttcgttcgt atcatcggtt tcgacaacgt tcgtcaagtt caatgcatca gtttcattgc 120

gcacacacca gaatcctact gagtttgagt attatggcat tgggaaaact gtttttcttg 180

taccatttgt tgtgcttgta atttactgtg ttttttattc ggttttcgct atcgaactgt 240

gaaatggaaa tggatggaga agagttaatg aatgatatgg tccttttgtt cattctcaaa 300

ttaatattat ttgttttttc tcttatttgt tgtgtgttga atttgaaatt ataagagata 360

tgcaaacatt ttgttttgag taaaaatgtg tcaaatcgtg gcctctaatg accgaagtta 420

atatgaggag taaaacactt gtagttgtac cattatgctt attcactagg caacaaatat 480

attttcagac ctagaaaagc tgcaaatgtt actgaataca agtatgtcct cttgtgtttt 540

agacatttat gaactttcct ttatgtaatt t 571

Claims (6)

1. a method that utilizes gene editing to knock out the grain development-related protein TaGSR1 to increase the length of wheat grains, it is characterized in that: design the single-stranded guide RNA (sgRNA) of targeting TaGSR1 gene, construct the expression vector that knocks out TaGSR1 gene in wheat body namely The plant binary expression vector pBUE411-TaGSR1; the plant binary expression vector was transformed into common wheat by Agrobacterium to obtain a transgenic wheat with TaGSR1 gene knockout, and the grain length of wheat seeds was increased; wherein the TaGSR1 gene nucleoside The acid sequence is shown in SEQ ID NO.4. 2. The method according to claim 1, wherein the nucleotide sequence of the single-stranded guide RNA targeting the TaGSR1 gene is SEQ ID NO.5, or SEQ ID NO.6, or SEQ ID NO.7, or SEQ ID NO.8; the nucleotide sequence of the plant binary expression vector pBUE411-TaGSR1 is shown in SEQ ID NO.3; the method is to use Agrobacterium to infect wheat embryo callus Organizing method, the specific sgRNA targeting the TaGSR1 gene and the expression element of the Cas9 element contained in the binary recombinant vector pBUE411-TaGSR1 were transferred into wheat, and the TaGSR1 gene was specifically knocked out to obtain a wheat mutant with increased grain length and yield. 3. A binary vector with knockout TaGSR1 gene function, characterized in that: the binary vector is named pBUE411-TaGSR1, and its nucleotide sequence is shown in SEQ ID NO.3; the binary vector pBUE411 -TaGSR1 is composed of expression cassette E1 and expression cassette E2; wherein the nucleotide sequence of expression cassette E1 is shown in SEQ ID NO.1, which from upstream to downstream are: TaU3 promoter from wheat, targeting TaGSR1 gene Single-stranded guide RNA (sgRNA), terminator T1; the nucleotide sequence of the expression cassette E2 is shown in SEQ ID NO. 2, which are sequentially from upstream to downstream: ubiquitin promoter Ubi from maize, maize Cas9 coding sequence , terminator T2. 4. The binary vector with the function of knocking out TaGSR1 gene according to claim 3, characterized in that: the nucleotide sequence of the wheat TaU3 promoter is shown in SEQ ID NO.9; the targeting TaGSR1 gene The nucleotide sequence of the single-stranded guide RNA is SEQ ID NO.5, or SEQ ID NO.6, or SEQ ID NO.7, or SEQ ID NO.8; the nucleotide of the terminator T1 The sequence is shown in SEQ ID NO.10; the nucleotide sequence of the maize ubiquitin promoter Ubi is shown in SEQ ID NO.11; the nucleotide sequence of the maize Cas9 coding sequence is shown in SEQ ID NO.12 ; The nucleotide sequence of the terminator T2 is shown in SEQ ID NO.13. 5. A specific guide single-stranded RNA for obtaining TaGSR1 gene knockout mutant wheat, characterized in that: the nucleotide sequence of the specific guide single-stranded RNA is SEQ ID NO.5, or SEQ ID NO. 6, or SEQ ID NO.7, or SEQ ID NO.8; it belongs to the TaGSR1 coding sequence or the complementary sequence of the TaGSR1 coding sequence, the length is 22 to 23 deoxyribonucleotides, and its 3' end is immediately adjacent to three A deoxynucleotide sequence is NGG, where N is adenine or guanine or thymine or cytosine. 6. The specific guide single-stranded RNA for obtaining TaGSR1 knockout mutant wheat according to claim 5, wherein the nucleotide sequence of the specific guide single-stranded RNA is shown in SEQ ID NO.5 .

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