CN113717982B - A method for increasing wheat grain length by knocking out the grain development-related protein TaGSR1 by gene editing - Google Patents

Abstract

The invention discloses a method for increasing the length of wheat grains by utilizing gene editing and knocking out a grain development related protein TaGSR1, which is to design single-stranded guide RNA of a targeted TaGSR1 gene and construct a plant binary expression vector pBUE411-TaGSR1 for knocking out the TaGSR1 gene in the wheat body; 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 grain length of wheat seeds; wherein the nucleotide sequence of the TaGSR1 gene is shown as SEQ ID NO. 4. Experiments prove that the transgenic wheat with TaGSR1 gene knocked out realizes the increase of the grain length of wheat seeds and the improvement of thousand kernel weight. The technical scheme of the invention provides a feasible method for realizing rapid breeding of wheat and improving the wheat yield by utilizing a genetic engineering technology, and has important breeding application value and wide market application prospect.

Description

A method of knocking out the grain development-related protein TaGSR1 by gene editing to increase wheat seed grain length method

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 the grain development-related protein TaGSR1 by gene editing; it belongs to the technical field of plant genetic engineering and molecular breeding.

Background technique

Wheat is the second largest food crop in the world, providing 20% carbohydrates and 23% protein for humans. Human food security is faced with problems such as the reduction of arable land, global warming, continuous population growth, and increased environmental pollution. How to increase wheat production under limited resource conditions is of great significance to food security.

Brassinolide (BR), as the sixth plant hormone in plants, plays a very important role in the processes of cell elongation and division, leaf senescence, plant morphogenesis, and plant stress resistance. Due to the extensive role of BR in the process of plant growth and development, its application in agricultural production has attracted more and more attention. BRI1 (Brassinosteroid-insensitive1) is a BR receptor located on the cell membrane. After binding to BR, it will phosphorylate with the co-receptor BAK1 (BRI1-associated receptor) to initiate downstream signal transduction, and then phosphorylate BSK/CDG1 kinase, phosphorylate The BSK/CDG1 further activates the downstream BSU1 (BRI1-suppressor 1) through phosphorylation, and BSU1 acts as a phosphatase, and dephosphorylates BIN2 (Brassinosteroid-insensitive 2) to disable BIN2, a negative regulator in the BR signaling pathway. activity, thereby regulating the expression of downstream genes.

The applicant's research found that TaGSR1 (Grain Size Regulator 1 of Triticum aestivum L.) is a transcription factor downstream of BR signaling, and has the function of regulating wheat grain size. This regulatory function provides a very important theoretical basis for the study of BR growth and development and high-yield breeding in wheat. However, after searching, it was found that the coding sequence of the wheat TaGSR1 gene and its encoded grain development-related protein TaGSR1, as well as the method of using gene editing to knock out the grain development-related protein TaGSR1 to increase the length of wheat grains, have not been reported.

Contents of the invention

In view of the deficiencies in the prior art, the problem to be solved by the present invention is to provide a method for increasing the length of wheat grains by knocking out the grain development-related protein TaGSR1 by gene editing.

The method of using gene editing to knock out the grain development-related protein TaGSR1 of the present invention to increase the length of wheat grains is characterized in that: a single-stranded guide RNA (sgRNA) targeting the TaGSR1 gene is designed, and an expression vector for knocking out the TaGSR1 gene in wheat is constructed. The plant binary expression vector pBUE411-TaGSR1; the plant binary expression vector is transformed into common wheat by Agrobacterium, and the transgenic wheat with TaGSR1 gene knockout is obtained, so as to increase the grain length of wheat seeds; wherein the nucleoside of the TaGSR1 gene The acid sequence is shown in SEQ ID NO.4.

Wherein, in the above method: 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, or the sgRNA that meets the described characteristics; preferably the sgRNA of the nucleotide sequence shown in SEQ ID NO.5; the nucleotide sequence of the plant binary expression vector pBUE411-TaGSR1 is as shown in SEQ ID NO.3 The method further uses the method of using Agrobacterium to infect the wheat immature embryo callus, and the specific sgRNA targeting the TaGSR1 gene contained in the binary recombinant vector pBUE411-TaGSR1 and the expression element of the Cas9 element are transferred into wheat, and the specificity By knocking out the TaGSR1 gene, wheat mutants with increased grain length and yield were obtained.

The present invention also discloses a binary vector with the function of knocking out the TaGSR1 gene, which is characterized in that: the binary vector is named pBUE411-TaGSR1, and its nucleotide sequence is shown in SEQ ID NO.3; the binary vector The meta-vector pBUE411-TaGSR1 is composed of expression cassette E1 and expression cassette E2; the nucleotide sequence of expression cassette E1 is shown in SEQ ID NO.1, and its sequence from upstream to downstream is: TaU3 promoter from wheat, targeting The single-stranded guide RNA (sgRNA) of TaGSR1 gene, terminator T1; The nucleotide sequence of expression cassette E2 is shown in SEQ ID NO.2, and it is from upstream to downstream in sequence: ubiquitin promoter Ubi from maize, maize Cas9 coding sequence, terminator T2.

Wherein: the nucleotide sequence of the wheat TaU3 promoter is shown in SEQ ID NO.9; 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 terminator T1 is as shown in SEQ ID NO.10; the nucleotide sequence of the maize ubiquitin promoter Ubi The sequence 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.

The present invention further discloses 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, with a length of 22 to 23 deoxyribonucleotides, and its 3' end The last three deoxynucleotide sequences are NGG, where N is adenine or guanine or thymine or cytosine.

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

The method for increasing the grain length of wheat by gene editing and knocking out the grain development-related protein TaGSR1 provided by the present invention is to construct the expression vector pBUE411-TaGSR1 with the function of knocking out the TaGSR1 gene, and transform it into common wheat to realize the knockout of the TaGSR1 gene, Increased grain length of wheat seeds was achieved by reducing the expression of the TaGSR1 gene. The nucleotide sequence of the coding sequence of the wheat TaGSR1 gene provided by the present invention is shown in SEQ ID NO.4, which is published for the first time. The wheat TaGSR1 is a transcription factor downstream of BR signal, and has the function of regulating wheat grain size.

The outstanding effect of the invention is: by designing the guide single-stranded RNA (sgRNA) of the TaGSR1 gene, an expression vector capable of knocking out the TaGSR1 gene in wheat is constructed; wherein the sgRNA sequence can target and destroy the exons of the TaGSR1 gene region, the targeting domain sequence of the sgRNA sequence is selected from the coding sequence of the TaGSR1 gene, which can specifically target and destroy the coding region of the TaGSR1 gene to effectively reduce the protein level of TaGSR1. Through the genetic transformation of wheat, the TaGSR1 gene knockout progeny was obtained, and the wheat mutant with increased grain length and yield was obtained. Experiments confirmed that the transgenic wheat with TaGSR1 gene knockout achieved increased grain length and thousand-grain weight of wheat seeds. The technical scheme of the invention provides a practical method for realizing rapid wheat breeding and increasing wheat yield by using genetic engineering technology, and has important breeding application value and broad market application prospect.

Description of drawings

Figure 1 is the amplified electrophoresis diagram of the TaGSR1 gene of Shixin 828 wheat.

Among them: Lane 1 is Transgen 2K plus marker, and Lane 2 is the target band of TaGSR1.

Fig. 2 The TaGSR1 gene coding region of wheat variety Shixin 828.

Where: the underlined sequence is the sgRNA sequence used for gene editing.

Fig. 3 Sequencing results of pBUE411-TaGSR1 binary expression vector.

Where: the underline is sgRNA, and the rest of the sequence is the vector backbone sequence of vector pBUE411.

Fig. 4 Transformation and identification results of T0 generation wheat transgenic lines.

Among them: Lane 1 is Transgen 2K plus marker, Lane 2 is the amplification result of recombinant plasmid pBUE411-TaGSR1, Lane 3-15 is the identification result of T0 transgenic wheat lines, and Lane 16 is the amplification result of wild type JW1.

Fig. 5 Sequencing results of the tagsr1-ko strain with gene editing in all three homologous copies.

Fig. 6 The protein coding results of the strain tagsr1-ko with gene editing in all three homologous copies.

Figure 7 The results of three-copy specific amplification of the tagsr1-ko strain.

Among them: 1 is the Transgen 2K plus marker, 2 is the specific PCR fragment for detecting the editing of TaGSR1.2A, 3 is the specific PCR fragment for detecting the editing of TaGSR1.2B, and 4 is the specific PCR fragment for detecting the editing of TaGSR1.

Fig. 8 Restriction identification results of TaGSR1 gene-edited offspring.

Among them: 1 is the Transgen 2K marker, 2 to 6 are the enzyme digestion results of three homologous copies of different selfed progeny, 7 is the enzyme digestion result of three homologous copies of wild-type JW1, and the box indicates the three homologous copies of the plant. Both source copies were edited and were homozygous knockout lines.

Fig.9 Grain size comparison of OE-TaGSR1 overexpression transgenic and homozygous gene knockout lines tagsr1-ko.

Among them: A&B, compared with wild-type JW1 wheat, the grain length and width of OE-TaGSR1 overexpression lines were significantly reduced; C&D, compared with wild-type JW1 wheat, the homozygous gene knockout line tagsr1-ko had significantly reduced grain length increase, but no significant change in grain width. Bar = 1 cm.

Detailed ways

The content of the present invention will be described in detail below in conjunction with specific drawings and embodiments. The examples described below are only preferred implementation modes of the present invention. It should be noted that the following descriptions are only for explaining the present invention, and are not intended to limit the present invention in any form. Any simple modifications, equivalent changes and modifications all fall within the scope of the technical solutions of the present invention.

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

The test materials used in the following examples were purchased from conventional biochemical reagent companies unless otherwise specified.

The high-fidelity enzyme required for PCR amplification is KOD-FX NEO (Toyobo); the restriction endonuclease BsaI and T4 ligase required for Gibson assembly were purchased from NEB Company; The gel extraction kit and plasmid extraction kit required for recovery were purchased from Thermo Fisher Scientific. Inorganic salts required for medium preparation were purchased from Sinopharm, and vitamins and antibiotics were purchased from Sigma. The plant CRISPR/Cas9 gene editing vector is pBUE411, which contains the wheat U3 promoter TaU3 to activate sgRNA. Cas9 mimics the characteristics of higher GC content at the 5' end of the grass gene, and is a plant codon-optimized gene designed and synthesized . The plasmid pBUE411 is publicly available from China Agricultural University, and its sequence is known, with a length of 17430 bp. The Escherichia coli strain used in the present invention is E.coli Transgen5α, which was purchased from Beijing Quanshijin Company; the primers used were synthesized by Qingdao Qingke Zixi Biotechnology Co., Ltd., and the relevant primer sequences are shown in Table 1:

Table 1: Primers used in the present invention

Wheat variety JW1 used in the present invention is a new germplasm with good tissue culture ability self-selected by Shandong Academy of Agricultural Sciences Crop Research Institute, and the public can obtain it from Shandong Academy of Agricultural Sciences Crop Research Institute; Shixin 828 is Shijiazhuang Wheat New Germplasm. Breeding in 2001 by the Variety New Technology Research Institute through the 8-generation pedigree breeding method, and passed the approval of the Hebei Variety Approval Committee in 2005. It is a wheat variety with excellent traits such as cold resistance, drought resistance, and lodging resistance. Acquired by Feng Seed Industry Co., Ltd.

Example 1 Construction of expression vector

1. Design of sgRNA targeting TaGSR1

In order to design an sgRNA capable of editing the coding region of the TaGSR1 gene, the sequence of the TaGSR1 gene was first amplified in the wheat variety Shixin 828. The primers used were TaGSR1-F and TaGSR1-R, and the PCR reaction system was: KOD-FXNEO buffer : 25 μL, dNTP (2mM): 10 μL, TaGSR1-F (10 μM): 1.5 μL, TaGSR1-F (10 μM): 1.5 μL, Shixin 828gDNA (50ng/μL): 1 μL, KOD-FX NEO: 1 μL, ddH ₂ O make up 50 μL. The PCR reaction program was: pre-denaturation at 98°C for 2 min, denaturation at 98°C for 12 sec; annealing at 58°C for 20 sec, extension at 68°C for 25 sec, and 35 cycles of reaction; renaturation at 68°C for 5 min.

The PCR results are shown in Figure 1. Lane 1 is the Transgen 2K plus marker, and Lane 2 is the target band of TaGSR1.

The PCR product was sent to Qingdao Qingke Zixi Biotechnology Co., Ltd. for sequencing. The sequence of the TaGSR1 gene in Shixin 828 wheat is shown in Figure 2, and the Ensembl Plants database (http://plants.ensembl.org/index.html) The sequence of the gene was 93.79% similar in wheat. The gene has a full length of 612 bases, does not contain introns, and its nucleotide sequence is shown in SEQ ID NO.4.

Find a suitable targeting site in the coding region of the TaGSR1 gene through the website CRISPRdirect (http://crispr.dbcls.jp/), find a 20bp sequence fragment before the PAM structure and set it as the target sequence. In this example, one of the sgRNAs is used as Example knockout experiment, its nucleotide sequence is GCGGCGCGTCGTCGCCGGACCGG, shown in SEQ ID NO.5, that is, the underlined sequence in the TaGSR1 gene sequence shown in Figure 2 is the sgRNA sequence targeting the TaGSR1 gene, and the CGG at the 3' end of the sgRNA is PAM sequence.

2. Obtaining fragments containing sgRNA

The two primers TaGSR1-gR1-F and TaGSR1-gR1-R (Table 1) used to construct the pBUE411-TaGSR1 binary expression vector were synthesized by Qingdao Qingke Zixi Biotechnology Co., Ltd. Phosphorylate the two primers and directly anneal to form double strands. The reaction system is: TaGSR1-gR1-F (10μM): 4μL, TaGSR1-gR1-R (10μM): 4μL, 10×T4 PNKbuffer: 1.5μL, PNK : 1 μL, ATP: 1 μL, ddH ₂ O make up 15 μL. PCR instrument reaction: 37°C, 30min; 95°C, 5min; ramp to 25°C at 5°C/min.

3. The pBUE411 vector is connected with sgRNA

Use Gibson assembly to complete the connection between pBUE411 and sgRNA. The specific reaction system: pBUE411 plasmid (100ng/μL): 2 μL, the product fragment of step 2: 2 μL, 10×NEB T4 Buffer: 1.5 μL, 10×BSA: 1.5 μL, BsaI: 1 μL, T4 Ligase: 1 μL, ddH ₂ O make up 15 μL. The reaction system was reacted in a 37°C water bath for 5 hours to obtain the ligation product of the pBUE411 vector and sgRNA.

4. Transformation and identification

Transform the ligation product into Escherichia coli, specifically: add the ligation product to E. coli competent Transgen 5α in an ice bath for 20 min, heat shock at 42°C for 60 sec, and ice bath for 2 min, add no anti-LB, put it in a shaker at 37°C for 1 h, Then spread it on LB (containing kanamycin) plate with a spreader, culture it upside down at 37°C until the clones grow out, pick 3 single clones for sequencing, and use pBUE411-F and pBUE411-R as primers.

The sequencing results (as shown in Figure 3) detected the target sequence of the sgRNA, and detected the TaU3 promoter sequence upstream of the target sequence. The sequencing results showed that the expression cassette E1 containing the sgRNA was successfully assembled into the pBUE411 binary expression vector, proving the CRISPR of TaGSR1 The /Cas9 gene editing vector, the recombinant binary expression vector pBUE411-TaGSR1, was successfully constructed. The nucleotide sequence of the recombinant binary expression vector pBUE411-TaGSR1 is shown in SEQ ID NO.3.

Example 2 Obtaining and identification of transgenic offspring

1. TaGSR1 transgenic offspring obtained

Transform the recombinant binary expression vector pBUE411-TaGSR1 constructed in Experimental Example 1 into Agrobacterium EHA105 competent cells, specifically: add the recombinant binary expression vector pBUE411-TaGSR1 into Agrobacterium EHA105 competent cells for 5 minutes in ice bath, and quickly freeze in liquid nitrogen 5min, heat shock reaction at 37°C for 5min, then ice-bathed for 5min, added LB without anti-antibody, put it in a shaker at 28°C for 2h, and then spread it on LB (containing rifampicin, streptomycin and kanamycin ) plate, cultured upside down at 28°C until clones grow out. Pick a single clone and inoculate it in the LB medium containing the corresponding resistance, shake the bacteria at 28°C, 160rpm, and cultivate for 24 hours.

The JW1 wheat seeds about 15 days after pollination were taken, and the young embryos were peeled off. Agrobacterium Suspension Take 1ml of bacterial solution in a 1.5ml centrifuge tube, add 1.4ul of acetosyringone (0.1M) and mix well. Add the prepared bacterial solution to infect for 5 minutes, put it on the co-cultivation medium, and incubate in the dark at 23°C for 3 days. After co-cultivation, they were cultured in the dark at 25°C for 5 days on rest medium. Transfer the callus to the screening medium 1, seal the culture dish with a parafilm, and culture it in the dark at 25.5°C for 2 weeks. After the callus was cut, it was transferred to the screening medium 2, and the petri dish was sealed again with a parafilm, and the culture was continued in the dark at 25.5°C for 2 weeks in an incubator. Two weeks after callus excision selection, resistant calli exhibiting green bud spots were transferred to regeneration medium. Seal the culture dish and put it in a light/dark (16h/8h) incubator at 25°C for 2 weeks. After 2 weeks of regrowth, transfer healthy seedlings to new resistant regrowth boxes. When the seedlings grow to a certain size, samples can be taken for testing.

The various mediums involved in the above-mentioned genetic transformation of wheat and their preparations can be found in the following documents:

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

The young leaves of regenerated wheat were taken, the genomic DNA was extracted by CTAB method, and the primers on the two vectors of BUE-DF1 and BUE-DR1 were used for PCR identification. The PCR reaction program was: 2×PCR master mix: 10 μL, pBUE411-F (10 μM) : 0.5 μL, pBUE411-R (10 μM): 0.5 μL, gDNA (50 ng/μL): 1 μL, ddH ₂ O: 8 μL. The PCR reaction program was: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 sec; annealing at 58°C for 30 sec, extension at 72°C for 30 sec, and 32 cycles of reaction; renaturation at 72°C for 5 min. The results are shown in Figure 4, the bands represent the successfully transformed T0 wheat transgenic lines, and a total of 12 positive transgenic lines were obtained.

2. Identification of TaGSR1 transgene knockout offspring

Wheat is an allohexaploid, the TaGSR1 gene is a copy on chromosome 2D, and the homologous copies on chromosome 2A and 2B are TaGSR1.2A and TaGSR1.2B. The DNA sequence homology of the three copies is high, and the amino acid sequence With a similarity of 88.6%, it is necessary to detect the gene editing of three homologous copies at the same time. In this experiment, the Hi-TOM gene editing site detection kit purchased from Xi'an Qingxue Biotechnology Co., Ltd. was used. The kit completed the high-throughput library construction process by PCR, and directly analyzed the multiple samples and multiple sites with Hi-TOM online software. Point variation information. TaGSR1.2A, TaGSR1.2B, and TaGSR1 were simultaneously amplified using the specific primers on both sides of the target sequence (Seq-F and Seq-R in Table 1), and the amplified products were sent to Xi'an Qingxue Biotech after building a library Technology Limited Sequencing.

The gene editing results of the target genes wheat TaGSR1.2A, TaGSR1.2B and TaGSR1 genes are shown in Figure 5, and the encoded amino acid sequences are shown in Figure 6. After comparison, the frameshift of the mutant protein tagsr1.2a started from the 112th amino acid, the frameshift of the protein tagsr1.2b started from the 111th amino acid, and the frameshift of the protein tagsr1 started from the 109th amino acid. The results showed that the transformation of sgRNA and Cas9 elements was successful and functioned, and the TaGSR1 gene was edited, resulting in three protein frameshift mutations of TaGSR1.2A, TaGSR1.2B and TaGSR1. loss of function.

Among them: the first round of PCR reaction system: 1 μL of wheat plant leaf DNA transfected with pBUE411-TaGSR1 gene as template, 10 μL of 2×Taq Master Mix in the kit, each of Seq-F and Seq-R (Table 1) (10 μM) 0.5μL, make up the volume to 20μL with Nucleasefree Water. The PCR reaction conditions were: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 s, annealing at 64°C for 30 s, and extension at 72°C for 20 s, a total of 32 cycles; finally, extension at 72°C for 5 min. After PCR, 5 μL of agarose gel electrophoresis was used to detect the PCR product to ensure the presence of the target product and good specificity. Afterwards, the second round of PCR reaction was carried out. 12 μL of Hi-TOM Mix in the kit was added, 1 μL was added using the first round of PCR product as a template, and the volume was made up to 20 μL with Nuclease-free Water. PCR reaction program: denaturation at 94°C for 2 min; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 25 s, a total of 33 cycles; finally extension at 72°C for 5 min. The amplified products were mixed for gel recovery, and the gel recovery products were the samples for library construction and sequencing, which were then sent to Xi’an Qingxue Biotechnology Co., Ltd. for sequencing. The gene editing results are shown in Figure 5.

Example 3 Phenotypic identification of wheat offspring with TaGSR1 gene knockout

In order to obtain homozygous knockout lines with three homologous copies of TaGSR1.2A, TaGSR1.2B and TaGSR1, an enzyme-cut amplified polymorphic sequence (Caps) marker was developed for the editing site, and the selfing of gene editing mutants Progeny were identified until a homozygous mutant for TaGSR1.2A, TaGSR1.2B and TaGSR1 simultaneous knockout was obtained.

TaGSR1.2A, Caps-2A, TaGSR1.2B and TaGSR1 genes were amplified by PCR. The PCR reaction system is: KOD-FX NEO buffer: 10 μL, dNTP (2mM): 4 μL, Caps-2A-F (or Caps-2B-F, or Caps-2D-F) (10 μM): 0.6 μL, Caps-2A -R (or Caps-2B-R, or Caps-2D-R) (10μM): 0.6μL, gene knockout strain gDNA (about 20ng/μL): 1μL, KOD-FX NEO: 0.4μL, ddH ₂ O Make up 20 μL. The PCR reaction program was: pre-denaturation at 98°C for 2 min, denaturation at 98°C for 12 sec; annealing at 58°C for 20 sec, extension at 68°C for 45 sec, and 35 cycles of reaction; renaturation at 68°C for 5 min.

5 ul of the PCR product was taken for electrophoresis detection, and the PCR result is shown in Figure 7.

The amplified product was digested with the restriction endonuclease CpoI produced by Thermo Fisher. The enzyme digestion reaction system was: PCR product: 15 μL, 10×Buffer Tango: 2 μL, restriction endonuclease CpoI: 0.5 μL. The reaction system was digested in a water bath at 37° C. for 2 hours, and then detected by electrophoresis. The results are shown in FIG. 8 . Homozygous mutants were identified by electrophoresis, the wild-type gene could be cut, and the successfully edited sites could not be cut. TaGSR1.2A, TaGSR1.2B and TaGSR1 of wild-type plants can be cut into 696bp+514bp, 729bp+358bp, 869bp+306bp fragments, while gene editing mutants cannot be cut, so that transgene homozygous can be screened Three-copy gene knockout line tagsr1-ko.

The wild-type recipient variety JW1, the TaGSR1 overexpression wheat transgenic line driven by the maize ubiquitin promoter Ubi (named OE-TaGSR1), and the homozygous knockout line of the TaGSR1 gene and its homologous copy (named tagsr1- ko), were planted together in the artificial climate chamber of Qingdao Campus of Shandong University, and the cultivation conditions were: 16 hours of light, 8 hours of darkness; daytime temperature 22°C, night temperature 16°C; humidity 40%-50%; CO2 concentration 500ppm-700ppm. After the seeds were harvested, they were dried in an oven at 30°C for 14 days, and the seeds of OE-TaGSR1 and tagsr1-ko were photographed with a Canon high-performance SLR camera, and the images were analyzed and measured with Image-Pro Plus 6.0, and OE-TaGSR1 was counted , the length and width of the tagsr1-ko grain.

The results showed that compared with the wild-type JW1, the grain length and width of the overexpression line OE-TaGSR1 were significantly reduced, while the grain length of the mutant line tagsr1-ko was significantly increased, but the grain width had no significant change (Table 2, Figure 9 shown). The thousand-grain weight of the grains was measured, and the thousand-grain weight of the OE-TaGSR1 overexpression transgenic line was significantly decreased, and the thousand-grain weight of tagsr1-ko was significantly increased (Table 2). It shows that the TaGSR1 gene has a negative regulatory effect on the development of wheat grains. The present invention constructs the pBUE411-TaGSR1 binary recombinant vector containing sgRNA and can specifically target the TaGSR1 gene, and uses Agrobacterium to infect the callus induced by young wheat embryos. Specifically editing the TaGSR1 gene to disable its function significantly increases the grain length of wheat and increases yield, providing a new method for breeding high-yielding wheat.

Table 2 Statistics of yield traits of homozygous three-copy knockout mutant tagsr1-ko

Note: Superscripts with different lowercase letters indicate significant differences at the 95% confidence level.

Sequence List

<110> Shandong University

<120> A method to increase wheat grain length by knocking out the grain development-related protein TaGSR1 by gene editing

<141> 2021-08-05

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ccacattttc atgccgtttg tgccaactat cccgagctag tgaatacagc ttggcttcac 840

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gtattttgac aacaggactc tacagtttta tctttttagt gtgcatgtgt tctccttttt 300

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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 catcccaacac 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 tcctggaca 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 aattcaagg 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 aagagattg 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 cggcaggggag 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 cacggggccag 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

ttattataata 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

ctcaccctgttgtttggtgttacttctgca 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 atcgagggggg 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 gactccatg 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

accacgagg 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 accacctgc 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 agttccccaga taagggaatt 9780

agggttccta tagggtttcg ctcatgtgtt gagcatataa gaaaccctta gtatgtattt 9840

gtatttgtaa aatacttcta tcaataaaat ttctaattcc taaaaccaaa atccagtact 9900

aaaatccaga tcccccgaat taattcggcg ttaattcagt acattaaaaa cgtccgcaat 9960

gtgtttattaa 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 tcaggggagga 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

gggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 11700

acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 11760

ggtaagcggc agggtcggaa caggagagcg cacgaggggag 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

accccagcgcc 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 ttgattaat 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 cgttccccctt 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 aactttgaga 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 tccccacctcg 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 tttgcagttttattttccgt 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> Maize (Zea mays L.)

<221> Nucleotide sequence of maize ubiquitin promoter Ubi

<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 tccattagg 360

gtttagggtt aatggttttt atagactaat ttttttagta catctatttt attctatttt 420

agcctctaaa ttaagaaaac taaaactcta ttttagtttttttatttaat 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 encoding 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 acccggaca 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 tcaggagggag 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 aggagacta 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 gggtccccaga tcctcaagga gcacccggtg 2400

gagaacacgc agctgcagaa tgagaagctc tacctgtact acctccagaa tggccgcgat 2460

atgtatgtgg accagagct 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 gacggggggag atcgtgtggg acaagggcag ggatttcgcg 3240

accgtcagga aggttctctc catgccacaa gtgaatatcg tcaagaagac agggtccag 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 (3)

1. A method for increasing the length of wheat grains by utilizing gene editing knockout grain development related protein TaGSR1 is characterized in that: design targetingTaGSR1Construction of single stranded guide RNA of Gene in knockout wheatTaGSR1Plant binary expression vector pBUE411-TaGSR1 of the gene; targeting of plant binary expression vector pBUE411-TaGSR1 contained by using agrobacterium to infect wheat immature embryo callusTaGSR1The single-stranded guide RNA of the gene and the expression element of Cas9 are transferred into wheat, and the specific knockout is carried outTaGSR1Gene, obtainTaGSR1The transgenic wheat with the gene knocked out realizes the increase of the grain length of wheat seeds; wherein saidTaGSR1The nucleotide sequence of the gene is shown as SEQ ID NO. 4.

2. The method for increasing grain length in wheat using gene editing knockout grain development related protein TaGSR1 according to claim 1, wherein: the targetingTaGSR1The nucleotide sequence of the single stranded guide RNA of the gene is SEQ ID NO.5.

3. The method for increasing grain length in wheat using gene editing knockout grain development related protein TaGSR1 according to claim 1, wherein: the nucleotide sequence of the plant binary expression vector pBUE411-TaGSR1 is shown as SEQ ID NO. 3.