CN113862283B

CN113862283B - Application of TGS1 gene in regulation and control of rice grain size and yield

Info

Publication number: CN113862283B
Application number: CN202111139974.4A
Authority: CN
Inventors: 高清松; 赵祥祥; 周海英; 李伟; 张雯霞
Original assignee: Huaiyin Normal University
Current assignee: Huaiyin Normal University
Priority date: 2021-09-27
Filing date: 2021-09-27
Publication date: 2022-03-15
Anticipated expiration: 2041-09-27
Also published as: CN113862283A

Abstract

The invention discloses application of a TGS1 gene in regulation and control of rice grain size and yield, and belongs to the technical field of molecular biology and genetic engineering. According to the invention, a target site is knocked out by designing a coding region of a gene TGS1, a gene knockout mutant is constructed by using a CRISPR/Cas9 technology, and a TGS1 gene in rice is knocked out. Through progeny segregation and detection, homozygous mutants without transgene insertion are obtained. Compared with a control, the grain length, the grain width, the grain thickness and the single plant yield of different mutant grains are obviously increased. In addition, the aspect ratio of the mutant grain was significantly greater than the control, and thus had better appearance quality. Therefore, the application of the TGS1 gene provided by the invention in regulation and control of rice grain size and yield has important application value and can generate good economic benefit.

Description

Application of TGS1 gene in regulation and control of rice grain size and yield

Technical Field

The invention belongs to the technical field of molecular biology and genetic engineering, and relates to application of TGS1 gene in regulation and control of rice grain size and yield.

Background

Rice is one of the most important food crops in the world, and rice is taken as staple food in nearly half of the global population. The method has the advantages of improving the rice yield, meeting the continuously increasing population demand in the world and ensuring the food safety, and has important significance. Rice yield consists of three major elements: the number of ears per plant, the number of grains per ear and the grain weight. Wherein, the grain weight is positively correlated with the grain size, so the grain size is one of the important agronomic traits determining the rice yield. In recent years, a great deal of research carried out by positioning and cloning Quantitative Trait Loci (QTLs) and utilizing a reverse genetics method shows that ubiquitination-mediated signal pathways such as proteasome degradation, phytohormones, G protein signal transduction, MAPK signal transduction and the like, and various transcription regulatory factors participate in the regulation and control of rice grain size (Li N, Xu R, Duan PG, Li Y H.control of grain size in rice plant Reproduction, 2018, 31: 237-.

The transcription regulating factor plays an important role in the growth and development process of plants. GLW7 is a major QTL for controlling rice grain length and grain weight, and encodes a plant-specific transcription factor OsSPL 13. The gene can increase the grain length and the yield of rice by positively regulating the size of glume cells. Meanwhile, the gene also participates in the regulation and control of the development of rice ears. GW8 encodes the SBP family transcription factor OsSPL16, positively regulating cell proliferation. The gene has high expression, promotes glume cell division and grain filling, and further increases grain width and yield. However, the deletion of the 10-bp gene promoter region enables the rice to form a slender seed and a better appearance quality. Interestingly, OsSPL16 can bind to the promoter region of GW7/GL7/SLG7, inhibiting the gene expression. GW7/GL7/SLG7 encodes arabidopsis thaliana LONGIFOLIA homologous protein, and the expression level of the Arabidopsis thaliana LONGIFOLIA homologous protein is up-regulated, so that grains become slender and the appearance quality is improved. Therefore, the OsSPL16-GW7/GL7/SLG7 pathway is adjusted, and the rice yield and quality are expected to be synchronously improved. In addition, GS2/GL2/GLW2/PT2 encodes a GRF family transcription factor OsGRF4, and the expression of the transcription factor OsGRF4 is regulated and controlled by microRNA OsmiR 396. The microRNA target sequence mutation of the gene causes the increase of expression quantity, thereby causing the enlargement of grains and the increase of yield. OsGRF4 can interact with brassinolide signal pathway regulatory factor GSK2, and thus can regulate grain size by influencing brassinolide signal transduction. In addition, OsGRF4 also interacts with the co-activators OsGIF1, 2 and 3. The seeds of the plants over-expressing OsGIF1 become bigger and the grain weight is increased. Although a plurality of molecular pathways and key regulatory factors involved in rice grain size regulation are identified at present, the knowledge of the whole regulatory network is still limited, and new genes are still to be identified.

The CRISPR/Cas9(Clustered regulated short linked genomic templates/CRISPR-associated protein 9) system is a genome-oriented editing system mediated by guide RNA, and is widely applied to genome editing of various organisms.

Disclosure of Invention

Aiming at the problems in the prior art, the technical problem to be solved by the invention is to provide the application of the TGS1 gene in the regulation of the size and the yield of rice grains, namely a method for knocking out the TGS1 gene in rice to increase the size and the yield of the rice grains.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

the application of the TGS1 gene in regulating and controlling the size and yield of rice grains is as follows: the TGS1 gene in rice is knocked out to increase the size or yield of rice grains, wherein the TGS1 gene codes a trihelix family transcription factor, and the nucleotide sequence of the transcription factor is shown as SEQ ID No. 1.

Preferably, the specific method for knocking out the TGS1 gene in rice comprises the following steps: the TGS1 gene in rice is subjected to targeted mutation by using a CRISPR/Cas9 technology.

Preferably, the application comprises the following specific steps:

1) selecting a target fragment from a coding region of a rice grain size control gene TGS 1;

2) synthesizing a linker primer with a cohesive end according to the sequence information of the target fragment, and constructing a CRISPR/Cas9 recombinant vector for TGS1 gene targeting; the recombinant vector comprises an sgRNA expression cassette having the target sequence and a Cas9 nuclease expression cassette capable of being expressed in rice cells;

3) transferring the recombinant vector into an agrobacterium tumefaciens engineering strain, and transforming a conventional rice variety by using an agrobacterium-mediated method to obtain a transgenic rice plant;

4) amplifying and sequencing the genome DNA of the transgenic plant by using the genome primer covering the target site, thereby carrying out T₀Carrying out genotype identification on the generation transgenic plant to obtain homozygous or double allelic mutant;

5) for the above-mentioned T₀Generation of homozygous or biallelic mutants at T₁Planting generation by generation, and using primer pair T₁HPT, sgRNA and Cas9 transformation in progeny plantsThe gene is subjected to PCR detection, and the rice without transgene insertion, with obviously enlarged grains and increased yield can be obtained.

Preferably, in step 1), the target fragment sequence is shown as SEQ ID No. 5.

Preferably, in step 2), the nucleotide sequence of the sgRNA expression cassette is shown in SEQ ID No. 3.

Preferably, in step 2), the nucleotide sequence of the Cas9 nuclease expression cassette is shown as Seq ID No. 4.

Preferably, in step 5), the primer sequences for transgenic amplification of hpt (hygromycin phosphotransferase), sgRNA and Cas9 are:

HPTF：5’-GGGTGTCACGTTGCAAGACC-3’，

HPTR：5’-ATGCCTCCGCTCGAAGTAGC-3’，

sgRNAF：5’-TCCCAGTCACGACGTTGTAA-3’，

sgRNAR：5’-GGCCATTTGTCTGCAGAAT-3’，

Cas9F：5’-CACCATCTACCACCTGAGAA-3’，

Cas9R：5’-CGAAGTTGCTCTTGAAGTTG-3’。

has the advantages that: compared with the prior art, the invention has the advantages that:

the invention provides application of TGS1 gene in regulation of rice grain size and yield, and relates to a method for obtaining transgene-free insertion mutant by targeted mutation of TGS1 gene by using CRISPR/Cas9 technology, wherein T is the gene in the T₁Three pairs of primers are used for detection instead, so that a mutant without transgene insertion can be obtained and is equivalent to a naturally occurring mutant, and the safety risk possibly brought by transgene is avoided. The obtained rice material grains are obviously larger, the aspect ratio is increased, the yield is higher, the appearance quality is better, and the commercial value is higher. The method can be used for quickly and directionally improving the existing rice varieties, thereby greatly shortening the breeding period and saving the time and the cost. Because the targeted mutation is carried out on the target gene, the genetic background of the edited material is not changed, and the original favorable characters of the receptor variety are not changed.

Drawings

FIG. 1 shows the results of sequencing and identification of different mutants in example 1;

FIG. 2 shows T in example 1₁Detecting the result of the generation-free transgene insertion mutant; wherein 1 to 10 each represent a randomly selected T₁The generation plant, WT represents wild type, VC represents vector control, and M represents DNA molecular weight marker;

FIG. 3 shows kernels and brown rice of mutant and Wild Type (WT) plants of example 1 at a scale of 5 mm;

FIG. 4 is the relative expression levels of the TGS1 gene of over-expressed (OE) plants of example 2, P < 0.05;

fig. 5 shows kernels from Wild Type (WT) and over-expressed (OE) plants of example 2 at a scale of 5 mm.

Detailed Description

The invention is further described with reference to specific examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. In the following examples, unless otherwise specified, all experimental procedures were carried out according to conventional methods.

Example 1: method for increasing rice grain size and yield by knocking out TGS1 gene based on CRISPR/Cas9 technology

The sequence of the gene TGS1 is shown in SEQ ID No. 1. Sequence analysis shows that the gene comprises 3 exons, namely the 1 st to 287 th exons (the 1 st exons), the 988 th and 2512 nd exons (the 2 nd exons) and the 2647 th and 3012 nd exons (the 3 rd exons) of the sequence of SEQ ID No. 1.

(1) Construction of recombinant expression vector for TGS1 Gene knockout

A CRISPR/Cas9 gene knockout target site is designed in a TGS1 gene coding region, an antisense chain of a No. 2 exon is targeted, and a target sequence is shown as SEQ ID No. 5. Target sequence primers based on the CRISPR/Cas9 system were synthesized with the following sequences:

TGS1-P1：5’-TGTGGGCGCCGCCGGCCACCCCTG-3’，

TGS1-P2：5’-AAACCAGGGGTGGCCGGCGGCGCC-3’，

wherein the underlined section is the sticky end used for vector ligation and the non-underlined section is the target sequence or its complement.

The maize ZmUBI promoter is connected with hSpCas9 gene (Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F.multiple gene engineering using CRISPR/Cas system science 2013, 339 (6121): 819 and 823), and is inserted into pCAMBIA1300 binary vector to obtain an intermediate vector. The original BsaI cleavage site of the pCAMBIA1300 vector was removed using a point mutation kit (TransGen Biotech). Then, a fragment containing OsU6 promoter, ccdB negative selection marker gene flanked by BsaI cleavage sites, and sgRNA backbone was inserted into the intermediate vector to construct a CRISPR/Cas9 binary vector. The binary vector was stored in E.coli strain DB 3.1.

The annealing procedure was performed on TGS1-P1 and TGS1-P2 to form double stranded DNA with sticky ends as an insert for constructing CRISPR/Cas9 knockout vectors. The above CRISPR/Cas9 binary vector was cleaved with BsaI restriction endonuclease (NEB) at 37 ℃ to obtain a vector backbone fragment. The insert and the vector backbone are ligated with T4 ligase (NEB) to yield a recombinant expression vector targeting the target site. The recombinant expression vector is transferred into escherichia coli for amplification, and the extracted vector plasmid is transferred into agrobacterium tumefaciens strain EHA105 for agrobacterium-mediated rice genetic transformation after being verified to be correct by sequencing (Shanghai Bioengineering Co., Ltd.).

(2) Obtaining and identifying TGS1 gene knockout rice

Referring to the method of Nishimura et al (Nishimura A, Aichi I, Matsuoka M.A protocol for Agrobacterium-mediated transformation in nature Protocols, 2006, 1 (6): 2796-₀Transgenic rice plants are generated.

Extraction of T₀The DNA of a transgenic rice plant is generated, KOD DNA polymerase (TOYOBO) and a genome primer are utilized to amplify a DNA segment containing a target site, and the sequence of an amplification product is determined by Shanghai biological engineering GmbH. The primer sequence is as follows:

TGS1F：5’-TCCGTGTCCGACCCCGACGAGAA-3’，

TGS1R：5’-ACAAGAGGAGCGCCAAGAAGTGC-3’，

sequencing results show that 10 transgenic plants carry target mutations, wherein 2 homozygous mutants and 8 biallelic mutants are obtained.

(3) Acquisition and phenotypic analysis of transgene-free homozygous mutants

Selecting two homozygous T₀The generation mutants were subjected to subsequent analysis. One of the mutants carries a 1bp deletion, designated tgs1-1, and the other mutant carries a 28bp deletion, designated tgs 1-2. The results of mutant sequencing are shown in FIG. 1.

T of the mutant by using HPT, sgRNA and Cas9 primers₁The plant genome DNA is amplified, and T of two mutants₁Mutants without transgene insertion were identified in the generation individual plants (FIG. 2), which shows that the number of T-DNA insertion sites of the transgenic plants obtained by the method is small, and the transgenic plants are easy to be inserted at T₁Mutants without transgene insertion were obtained.

For homozygous mutants without transgene insertion at T₂Planting the plants in generation. The kernel of the mutant plants was significantly larger than the wild type (fig. 3). Wherein, the grain length is respectively increased by 11.3 percent and 9.2 percent, the grain width is respectively increased by 2.2 percent and 1.8 percent, and the grain thickness is respectively increased by 4.8 percent and 2.2 percent (Table 1-1). The thousand seed weight of the mutant is respectively increased by 18.3 percent and 15.2 percent compared with the wild type, and the yield of a single plant is increased by 19.5 percent and 15.8 percent (tables 1-2). The method provided by the invention can obviously increase the grain size and yield of the rice. In addition, the length-width ratio of the mutant kernel was increased by 8.8% and 7.5% respectively (tables 1-2) compared to the wild type, and thus had better appearance quality. The rice material with increased grain size and yield obtained by the invention does not contain transgenic components, avoids the safety risk possibly brought by transgenosis, only changes one gene, and does not affect other agronomic characters of rice varieties.

TABLE 1-1 tgs1 mutant and wild type plant grain phenotypes

	Grain length (mm)	Grain width (mm)	Particle thickness (mm)
				Wild type	7.37±0.16	3.25±0.089	2.28±0.054
tgs1-1	8.20±0.19^***	3.32±0.12^***	2.39±0.067^***
				tgs1-2	8.05±0.17^***	3.31±0.074^***	2.33±0.059^***

Note: the grain length, grain width and grain thickness are the average values of 50 seeds.^***：P＜0.001。

TABLE 1-2 tgs1 mutant and wild type plant grain phenotypes

	Aspect ratio	Thousand Kernel weight (g)	Yield per plant (g)
				Wild type	2.27±0.078	25.53±0.46	34.71±4.97
tgs1-1	2.47±0.075^***	30.21±0.55^***	41.47±6.73^**
				tgs1-2	2.44±0.057^***	29.41±0.79^***	40.18±4.67^**

Note: the aspect ratio is the average of 50 seeds; thousand kernel weight and individual plant yield were the average of 10 individual plants.

^**：P＜0.01；^***：P＜0.001。

Example 2: construction of TGS1 overexpression lines

In order to further verify the function of the gene in regulating the size of rice grains, an over-expression strain of the gene is constructed. The complete coding sequence of TGS1 was amplified using a pair of PCR primers, digested with restriction enzyme Bsa I (NEB), and ligated into the ZmUBI promoter downstream of pEGOEPubi-H vector carrying the hygromycin resistance gene to construct pEGOEPubi-H-TGS1 overexpression transgene vector. The sequence of the pEGOEPubi-H-TGS1 overexpression transgenic vector is shown in SEQ ID NO. 12. The PCR primer sequences are as follows:

TGS1OE-F：

5’-ACTAGGGTCTCGCACCATGCCGCCATTCTCGGCAGCCG-3’，

TGS1OE-R：

5’-ACTAGGGTCTCTACCGTCACTGGACGCAGGCCAAGAATGA-3’，

wherein the underlined part is the recognition site for Bsa I.

With reference to the method of Nishimura et al (Nishimura A, Aichi I, Matsuoka M.A protocol for Agrobacterium-mediated transformation in nature Protocols, 2006, 1 (6): 2796-₀Transgenic rice plants are generated. Will T₀50mg L for generation of transgenic plant seeds^-1Hygromycin is screened to obtain transgenic positive plants, and the transgenic positive plants are transplanted into a field.

Quantitative analysis of the expression level of TGS1 in transgenic lines using a pair of PCR primers revealed a significantly over-expressed strain of TGS 1. The expression of TGS1 was up-regulated by about 4.3 fold in this strain compared to the control (FIG. 4). The heading stage of the over-expression strain was significantly delayed, and the grains were significantly smaller compared to the control (fig. 5). These results demonstrate that TGS1 negatively regulates rice kernel size. The PCR primer sequence for detecting the expression quantity of TGS1 is as follows:

TGS1RT-F：5’-GCAAGGAGAAGTGGGAGAAC-3’，

TGS1RT-R：5’-TCGAGCTGGTGGTAGTAGGG-3’。

sequence listing

<110> Huaiyin college of learning professions

Application of <120> TGS1 gene in regulation and control of rice grain size and yield

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<160> 14

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ccgccgccgg agcaggcggc ggcggcggcg gcggaggagc agctcaacgg gagctcactg 120

gagcacgacg gggtgctggg cggcgaggag ggcgaccggg gcgggtcgtc ggcgggcaac 180

cggtggccgc ggcaggagac gctggcgctg ctcaagatcc ggtcggagat ggacgccgcc 240

ttccgggaag ccgccctcaa gggccccctc tgggaggaag tctccaggtt agctagctac 300

ctaattaact tcttcttaat cccaagaatt aattactcat aatccatctg atgagcatga 360

gcatggccag catggtcaaa attcgagctc aattttggtt agatttgcat gatatatcgg 420

agaattaagc ttgctcggcg cgcggcaata aggtactagc agcttaattg cggcgacgac 480

gagacgagca cagtgaattt cccaattaaa aatgacacac atatcgctct ccctcctccg 540

cttttcggtt cccttttgcc ctttgcgtca cttcatgtat ccagcgccac actgctgatg 600

atgctgttcg tatcagtaat taccctttag cattatataa atatatacat gcttcttcag 660

atccgatgta attaagcatg cagcatcaat cccaagtcca acaattccaa aggggtaaat 720

tatctttcgc tctctaccac ccacatgcag taaaaaaatt ctgtgcaaat ggatatgata 780

tagggtaatt aagtgctaga taaatagatt gttcttttca gtgagagcaa catgcatgta 840

gatgtagcta ggtagtagta gaatgttact agcttagatg catgcatgtg tcgtcgttgc 900

tgcgaacaag ttgtgatggc aacatgaagg ggagatgatg agttgatgac gaggtttaat 960

ttgtggctgc catggcgtgg cgtgcaggaa gctcgcggag atggggtaca agaggagcgc 1020

caagaagtgc cgcgagaagt tcgagaacgt cgacaagtac tacaagcgca ccaaggacgg 1080

ccgcgccggc cgcggcgacg gcaagaccta ccgcttcttc accgagctcg aggccctcca 1140

cggcgccgcc gccgccaccg cgcgcccgcc gccggtgtcc ctcgcgccgg cgcccgtggc 1200

cgtggcgccg ccggccaccc ctgcgggtct ctcggcgttg cgcgtccacg cctcgccgcc 1260

gccgccgcct gtcaagcagc acgccgctcc gccgccgcct gtcatggatg tggcggcgtg 1320

cgtgatgacg atggacgacg tgagcttctc gtcggggtcg gacacggagg agacggcgga 1380

ggagggcggg aagcggaagc ggaggggcgg cggaggcatc ggcggcggcg gcggcggcgg 1440

gaaggcgatg aggatgttcg aggggctgat gcggcaggtg atggagcggc aggaggcgat 1500

gcagcagagg cttctggagg cgatcgagcg gcgcgaccag gagaggatga tccgcgagga 1560

ggcgtggcgg cggcaggagg tggcgcgcct cgcccgcgag caggacgccc tcgcccagga 1620

gcgcgccatt gccgcctccc gcgacgccgc cgtcatctcc ttcatacaga gggtcaccgg 1680

ccagtccatc gccgccgtgc caccgccgcc gctgcagccc acgccggtgg cttccgctgc 1740

accaccacca ccaccgcagc atcatcatca gcaaacacca ccaccgattc aggttcagcc 1800

gcaccacata atgccgatga cgccgcagcc ccagctgcaa ccaccgcagc cgcagagcaa 1860

ggaggccaac accgtcgtcc gggcggcgcc gccgccgcaa gagcagcacg acacggcggc 1920

gtccggcggc ggcggcggcg cgtcgtcgtc gaggtggccg aaggcggagg tgcacgcgct 1980

gatccagctg cgcacagagc tggagacgag gtaccaggac tcggggccca aggggcctct 2040

ctgggaggac atctcggcgg ggatgcggcg gctggggtac agccggagct ccaagagatg 2100

caaggagaag tgggagaaca tcaacaagta cttcaagaag gtgaaggaga gcaacaagaa 2160

gcgccccgag gactccaaga catgccccta ctaccaccag ctcgacgcgc tctaccgcac 2220

caaggcggcc aacgccgccg ccgccgcctc agcctcgccg gctccggcga ccaccaccgt 2280

gcttgccccg gtgccgctct cccagacgcc gccgcacgtc gaccacggcg gcagcaacgg 2340

caatgggaat gggtgggcga gtgccaacaa cggcggcggc ggcagcagct ccgggggcat 2400

gcaaacgaag gctagcaaca atggcacggc gacggccggc gggctgcccg tcgtttcggt 2460

cgccggcggc aacggcaacg gcaacggcgt ggcggcgacg acggataaca aggtaattag 2520

ttaaaaacgc attgcattta actcgtgcag tgtacttgtg cgtgcacagg cacatgcatg 2580

tacgcatcgt tgcctgtacg attcgttctg atcgatcgat gtcacgtatc aaatctgtaa 2640

atttagggat cgaagcaggt gcccgtcgcg aaggagacgg cggggcagag gcagccacag 2700

ccgttggcca tgaaccacaa ctacggcaac gacagaatgg ccgacgacat ggacagcgac 2760

agcatggacg acgacgacga cgacgacgaa tttgacgacg acgaaaacga cgacgacatc 2820

ggcggtggca agatgcaggt gcagtacgag acgtcgtccc atttccaacg gccgcagctg 2880

cagaaccaga acgtcgtcgt cggccggcca aacgcgagcg gcggcggcgg cggtggcgct 2940

ccgacgacac cggcgggccc tccgccgcct gcggcaacaa gtgggacgtc attcttggcc 3000

tgcgtccagt ga 3012

<210> 2

<211> 725

<212> PRT

<213> Oryza sativa

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Met Pro Pro Phe Ser Ala Ala Gly Gly Glu Gly Ala Pro Ser Pro Ile

1 5 10 15

Ser Ser Arg Pro Pro Pro Pro Glu Gln Ala Ala Ala Ala Ala Ala Glu

20 25 30

Glu Gln Leu Asn Gly Ser Ser Leu Glu His Asp Gly Val Leu Gly Gly

35 40 45

Glu Glu Gly Asp Arg Gly Gly Ser Ser Ala Gly Asn Arg Trp Pro Arg

50 55 60

Gln Glu Thr Leu Ala Leu Leu Lys Ile Arg Ser Glu Met Asp Ala Ala

65 70 75 80

Phe Arg Glu Ala Ala Leu Lys Gly Pro Leu Trp Glu Glu Val Ser Arg

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Lys Leu Ala Glu Met Gly Tyr Lys Arg Ser Ala Lys Lys Cys Arg Glu

100 105 110

Lys Phe Glu Asn Val Asp Lys Tyr Tyr Lys Arg Thr Lys Asp Gly Arg

115 120 125

Ala Gly Arg Gly Asp Gly Lys Thr Tyr Arg Phe Phe Thr Glu Leu Glu

130 135 140

Ala Leu His Gly Ala Ala Ala Ala Thr Ala Arg Pro Pro Pro Val Ser

145 150 155 160

Leu Ala Pro Ala Pro Val Ala Val Ala Pro Pro Ala Thr Pro Ala Gly

165 170 175

Leu Ser Ala Leu Arg Val His Ala Ser Pro Pro Pro Pro Pro Val Lys

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Gln His Ala Ala Pro Pro Pro Pro Val Met Asp Val Ala Ala Cys Val

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Met Thr Met Asp Asp Val Ser Phe Ser Ser Gly Ser Asp Thr Glu Glu

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Thr Ala Glu Glu Gly Gly Lys Arg Lys Arg Arg Gly Gly Gly Gly Ile

225 230 235 240

Gly Gly Gly Gly Gly Gly Gly Lys Ala Met Arg Met Phe Glu Gly Leu

245 250 255

Met Arg Gln Val Met Glu Arg Gln Glu Ala Met Gln Gln Arg Leu Leu

260 265 270

Glu Ala Ile Glu Arg Arg Asp Gln Glu Arg Met Ile Arg Glu Glu Ala

275 280 285

Trp Arg Arg Gln Glu Val Ala Arg Leu Ala Arg Glu Gln Asp Ala Leu

290 295 300

Ala Gln Glu Arg Ala Ile Ala Ala Ser Arg Asp Ala Ala Val Ile Ser

305 310 315 320

Phe Ile Gln Arg Val Thr Gly Gln Ser Ile Ala Ala Val Pro Pro Pro

325 330 335

Pro Leu Gln Pro Thr Pro Val Ala Ser Ala Ala Pro Pro Pro Pro Pro

340 345 350

Gln His His His Gln Gln Thr Pro Pro Pro Ile Gln Val Gln Pro His

355 360 365

His Ile Met Pro Met Thr Pro Gln Pro Gln Leu Gln Pro Pro Gln Pro

370 375 380

Gln Ser Lys Glu Ala Asn Thr Val Val Arg Ala Ala Pro Pro Pro Gln

385 390 395 400

Glu Gln His Asp Thr Ala Ala Ser Gly Gly Gly Gly Gly Ala Ser Ser

405 410 415

Ser Arg Trp Pro Lys Ala Glu Val His Ala Leu Ile Gln Leu Arg Thr

420 425 430

Glu Leu Glu Thr Arg Tyr Gln Asp Ser Gly Pro Lys Gly Pro Leu Trp

435 440 445

Glu Asp Ile Ser Ala Gly Met Arg Arg Leu Gly Tyr Ser Arg Ser Ser

450 455 460

Lys Arg Cys Lys Glu Lys Trp Glu Asn Ile Asn Lys Tyr Phe Lys Lys

465 470 475 480

Val Lys Glu Ser Asn Lys Lys Arg Pro Glu Asp Ser Lys Thr Cys Pro

485 490 495

Tyr Tyr His Gln Leu Asp Ala Leu Tyr Arg Thr Lys Ala Ala Asn Ala

500 505 510

Ala Ala Ala Ala Ser Ala Ser Pro Ala Pro Ala Thr Thr Thr Val Leu

515 520 525

Ala Pro Val Pro Leu Ser Gln Thr Pro Pro His Val Asp His Gly Gly

530 535 540

Ser Asn Gly Asn Gly Asn Gly Trp Ala Ser Ala Asn Asn Gly Gly Gly

545 550 555 560

Gly Ser Ser Ser Gly Gly Met Gln Thr Lys Ala Ser Asn Asn Gly Thr

565 570 575

Ala Thr Ala Gly Gly Leu Pro Val Val Ser Val Ala Gly Gly Asn Gly

580 585 590

Asn Gly Asn Gly Val Ala Ala Thr Thr Asp Asn Lys Gly Ser Lys Gln

595 600 605

Val Pro Val Ala Lys Glu Thr Ala Gly Gln Arg Gln Pro Gln Pro Leu

610 615 620

Ala Met Asn His Asn Tyr Gly Asn Asp Arg Met Ala Asp Asp Met Asp

625 630 635 640

Ser Asp Ser Met Asp Asp Asp Asp Asp Asp Asp Glu Phe Asp Asp Asp

645 650 655

Glu Asn Asp Asp Asp Ile Gly Gly Gly Lys Met Gln Val Gln Tyr Glu

660 665 670

Thr Ser Ser His Phe Gln Arg Pro Gln Leu Gln Asn Gln Asn Val Val

675 680 685

Val Gly Arg Pro Asn Ala Ser Gly Gly Gly Gly Gly Gly Ala Pro Thr

690 695 700

Thr Pro Ala Gly Pro Pro Pro Pro Ala Ala Thr Ser Gly Thr Ser Phe

705 710 715 720

Leu Ala Cys Val Gln

725

<210> 3

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<212> DNA

<213> sgRNA expression cassette (Artificial)

<400> 3

ggatcatgaa ccaacggcct ggctgtattt ggtggttgtg tagggagatg gggagaagaa 60

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acgtgcacgg tgagcggccc acagggcgag tgtgagcgcg agaggcggga ggaacagttt 180

agtaccacat tgcccagcta actcgaacgc gaccaactta taaacccgcg cgctgtcgct 240

tgtgtgggcg ccgccggcca cccctggttt tagagctaga aatagcaagt taaaataagg 300

ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttt 348

<210> 4

<211> 6550

<212> DNA

<213> Cas9 nuclease expression cassette (Artificial)

<400> 4

tgcagcgtga cccggtcgtg cccctctcta gagataatga gcattgcatg tctaagttat 60

aaaaaattac cacatatttt ttttgtcaca cttgtttgaa gtgcagttta tctatcttta 120

tacatatatt taaactttac tctacgaata atataatcta tagtactaca ataatatcag 180

tgttttagag aatcatataa atgaacagtt agacatggtc taaaggacaa ttgagtattt 240

tgacaacagg actctacagt tttatctttt tagtgtgcat gtgttctcct ttttttttgc 300

aaatagcttc acctatataa tacttcatcc attttattag tacatccatt tagggtttag 360

ggttaatggt ttttatagac taattttttt agtacatcta ttttattcta ttttagcctc 420

taaattaaga aaactaaaac tctattttag tttttttatt taataattta gatataaaat 480

agaataaaat aaagtgacta aaaattaaac aaataccctt taagaaatta aaaaaactaa 540

ggaaacattt ttcttgtttc gagtagataa tgccagcctg ttaaacgccg tcgacgagtc 600

taacggacac caaccagcga accagcagcg tcgcgtcggg ccaagcgaag cagacggcac 660

ggcatctctg tcgctgcctc tggacccctc tcgagagttc cgctccaccg ttggacttgc 720

tccgctgtcg gcatccagaa attgcgtggc ggagcggcag acgtgagccg gcacggcagg 780

cggcctcctc ctcctctcac ggcacggcag ctacggggga ttcctttccc accgctcctt 840

cgctttccct tcctcgcccg ccgtaataaa tagacacccc ctccacaccc tctttcccca 900

acctcgtgtt gttcggagcg cacacacaca caaccagatc tcccccaaat ccacccgtcg 960

gcacctccgc ttcaaggtac gccgctcgtc ctcccccccc ccccctctct accttctcta 1020

gatcggcgtt ccggtccatg gttagggccc ggtagttcta cttctgttca tgtttgtgtt 1080

agatccgtgt ttgtgttaga tccgtgctgc tagcgttcgt acacggatgc gacctgtacg 1140

tcagacacgt tctgattgct aacttgccag tgtttctctt tggggaatcc tgggatggct 1200

ctagccgttc cgcagacggg atcgatttca tgattttttt tgtttcgttg catagggttt 1260

ggtttgccct tttcctttat ttcaatatat gccgtgcact tgtttgtcgg gtcatctttt 1320

catgcttttt tttgtcttgg ttgtgatgat gtggtctggt tgggcggtcg ttctagatcg 1380

gagtagaatt ctgtttcaaa ctacctggtg gatttattaa ttttggatct gtatgtgtgt 1440

gccatacata ttcatagtta cgaattgaag atgatggatg gaaatatcga tctaggatag 1500

gtatacatgt tgatgcgggt tttactgatg catatacaga gatgcttttt gttcgcttgg 1560

ttgtgatgat gtggtgtggt tgggcggtcg ttcattcgtt ctagatcgga gtagaatact 1620

gtttcaaact acctggtgta tttattaatt ttggaactgt atgtgtgtgt catacatctt 1680

catagttacg agtttaagat ggatggaaat atcgatctag gataggtata catgttgatg 1740

tgggttttac tgatgcatat acatgatggc atatgcagca tctattcata tgctctaacc 1800

ttgagtacct atctattata ataaacaagt atgttttata attattttga tcttgatata 1860

cttggatgat ggcatatgca gcagctatat gtggattttt ttagccctgc cttcatacgc 1920

tatttatttg cttggtactg tttcttttgt cgatgctcac cctgttgttt ggtgttactt 1980

ctgcagccat ggactataag gaccacgacg gagactacaa ggatcatgat attgattaca 2040

aagacgatga cgataagatg gccccaaaga agaagcggaa ggtcggtatc cacggagtcc 2100

cagcagccga caagaagtac agcatcggcc tggacatcgg caccaactct gtgggctggg 2160

ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg 2220

accggcacag catcaagaag aacctgatcg gagccctgct gttcgacagc ggcgaaacag 2280

ccgaggccac ccggctgaag agaaccgcca gaagaagata caccagacgg aagaaccgga 2340

tctgctatct gcaagagatc ttcagcaacg agatggccaa ggtggacgac agcttcttcc 2400

acagactgga agagtccttc ctggtggaag aggataagaa gcacgagcgg caccccatct 2460

tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc taccacctga 2520

gaaagaaact ggtggacagc accgacaagg ccgacctgcg gctgatctat ctggccctgg 2580

cccacatgat caagttccgg ggccacttcc tgatcgaggg cgacctgaac cccgacaaca 2640

gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg ttcgaggaaa 2700

accccatcaa cgccagcggc gtggacgcca aggccatcct gtctgccaga ctgagcaaga 2760

gcagacggct ggaaaatctg atcgcccagc tgcccggcga gaagaagaat ggcctgttcg 2820

gaaacctgat tgccctgagc ctgggcctga cccccaactt caagagcaac ttcgacctgg 2880

ccgaggatgc caaactgcag ctgagcaagg acacctacga cgacgacctg gacaacctgc 2940

tggcccagat cggcgaccag tacgccgacc tgtttctggc cgccaagaac ctgtccgacg 3000

ccatcctgct gagcgacatc ctgagagtga acaccgagat caccaaggcc cccctgagcg 3060

cctctatgat caagagatac gacgagcacc accaggacct gaccctgctg aaagctctcg 3120

tgcggcagca gctgcctgag aagtacaaag agattttctt cgaccagagc aagaacggct 3180

acgccggcta cattgacggc ggagccagcc aggaagagtt ctacaagttc atcaagccca 3240

tcctggaaaa gatggacggc accgaggaac tgctcgtgaa gctgaacaga gaggacctgc 3300

tgcggaagca gcggaccttc gacaacggca gcatccccca ccagatccac ctgggagagc 3360

tgcacgccat tctgcggcgg caggaagatt tttacccatt cctgaaggac aaccgggaaa 3420

agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccctctg gccaggggaa 3480

acagcagatt cgcctggatg accagaaaga gcgaggaaac catcaccccc tggaacttcg 3540

aggaagtggt ggacaagggc gcttccgccc agagcttcat cgagcggatg accaacttcg 3600

ataagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgtac gagtacttca 3660

ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga gggaatgaga aagcccgcct 3720

tcctgagcgg cgagcagaaa aaggccatcg tggacctgct gttcaagacc aaccggaaag 3780

tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat cgagtgcttc gactccgtgg 3840

aaatctccgg cgtggaagat cggttcaacg cctccctggg cacataccac gatctgctga 3900

aaattatcaa ggacaaggac ttcctggaca atgaggaaaa cgaggacatt ctggaagata 3960

tcgtgctgac cctgacactg tttgaggaca gagagatgat cgaggaacgg ctgaaaacct 4020

atgcccacct gttcgacgac aaagtgatga agcagctgaa gcggcggaga tacaccggct 4080

ggggcaggct gagccggaag ctgatcaacg gcatccggga caagcagtcc ggcaagacaa 4140

tcctggattt cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag ctgatccacg 4200

acgacagcct gacctttaaa gaggacatcc agaaagccca ggtgtccggc cagggcgata 4260

gcctgcacga gcacattgcc aatctggccg gcagccccgc cattaagaag ggcatcctgc 4320

agacagtgaa ggtggtggac gagctcgtga aagtgatggg ccggcacaag cccgagaaca 4380

tcgtgatcga aatggccaga gagaaccaga ccacccagaa gggacagaag aacagccgcg 4440

agagaatgaa gcggatcgaa gagggcatca aagagctggg cagccagatc ctgaaagaac 4500

accccgtgga aaacacccag ctgcagaacg agaagctgta cctgtactac ctgcagaatg 4560

ggcgggatat gtacgtggac caggaactgg acatcaaccg gctgtccgac tacgatgtgg 4620

accatatcgt gcctcagagc tttctgaagg acgactccat cgacaacaag gtgctgacca 4680

gaagcgacaa gaaccggggc aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga 4740

tgaagaacta ctggcggcag ctgctgaacg ccaagctgat tacccagaga aagttcgaca 4800

atctgaccaa ggccgagaga ggcggcctga gcgaactgga taaggccggc ttcatcaaga 4860

gacagctggt ggaaacccgg cagatcacaa agcacgtggc acagatcctg gactcccgga 4920

tgaacactaa gtacgacgag aatgacaagc tgatccggga agtgaaagtg atcaccctga 4980

agtccaagct ggtgtccgat ttccggaagg atttccagtt ttacaaagtg cgcgagatca 5040

acaactacca ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc gccctgatca 5100

aaaagtaccc taagctggaa agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc 5160

ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc taccgccaag tacttcttct 5220

acagcaacat catgaacttt ttcaagaccg agattaccct ggccaacggc gagatccgga 5280

agcggcctct gatcgagaca aacggcgaaa ccggggagat cgtgtgggat aagggccggg 5340

attttgccac cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg 5400

aggtgcagac aggcggcttc agcaaagagt ctatcctgcc caagaggaac agcgataagc 5460

tgatcgccag aaagaaggac tgggacccta agaagtacgg cggcttcgac agccccaccg 5520

tggcctattc tgtgctggtg gtggccaaag tggaaaaggg caagtccaag aaactgaaga 5580

gtgtgaaaga gctgctgggg atcaccatca tggaaagaag cagcttcgag aagaatccca 5640

tcgactttct ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc 5700

ctaagtactc cctgttcgag ctggaaaacg gccggaagag aatgctggcc tctgccggcg 5760

aactgcagaa gggaaacgaa ctggccctgc cctccaaata tgtgaacttc ctgtacctgg 5820

ccagccacta tgagaagctg aagggctccc ccgaggataa tgagcagaaa cagctgtttg 5880

tggaacagca caagcactac ctggacgaga tcatcgagca gatcagcgag ttctccaaga 5940

gagtgatcct ggccgacgct aatctggaca aagtgctgtc cgcctacaac aagcaccggg 6000

ataagcccat cagagagcag gccgagaata tcatccacct gtttaccctg accaatctgg 6060

gagcccctgc cgccttcaag tactttgaca ccaccatcga ccggaagagg tacaccagca 6120

ccaaagaggt gctggacgcc accctgatcc accagagcat caccggcctg tacgagacac 6180

ggatcgacct gtctcagctg ggaggcgaca aaaggccggc ggccacgaaa aaggccggcc 6240

aggcaaaaaa gaaaaagtaa ggatcctgat tgatcgatag agctcgaatt tccccgatcg 6300

ttcaaacatt tggcaataaa gtttcttaag attgaatcct gttgccggtc ttgcgatgat 6360

tatcatataa tttctgttga attacgttaa gcatgtaata attaacatgt aatgcatgac 6420

gttatttatg agatgggttt ttatgattag agtcccgcaa ttatacattt aatacgcgat 6480

agaaaacaaa atatagcgcg caaactagga taaattatcg cgcgcggtgt catctatgtt 6540

actagatcgg 6550

<210> 5

<211> 20

<212> DNA

<213> Oryza sativa

<400> 5

ggcgccgccg gccacccctg 20

<210> 6

<211> 24

<212> DNA

<213> TGS1-P1(Artificial)

<400> 6

tgtgggcgcc gccggccacc cctg 24

<210> 7

<211> 24

<212> DNA

<213> TGS1-P2(Artificial)

<400> 7

aaaccagggg tggccggcgg cgcc 24

<210> 8

<211> 23

<212> DNA

<213> TGS1F(Artificial)

<400> 8

tccgtgtccg accccgacga gaa 23

<210> 9

<211> 23

<212> DNA

<213> TGS1R(Artificial)

<400> 9

acaagaggag cgccaagaag tgc 23

<210> 10

<211> 38

<212> DNA

<213> TGS1OE-F(Artificial)

<400> 10

actagggtct cgcaccatgc cgccattctc ggcagccg 38

<210> 11

<211> 40

<212> DNA

<213> TGS1OE-R(Artificial)

<400> 11

actagggtct ctaccgtcac tggacgcagg ccaagaatga 40

<210> 12

<211> 13398

<212> DNA

<213> pEGOEPubi-H-TGS1 overexpression transgenic vector sequence (Artificial)

<400> 12

tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc 60

aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc 120

tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacat 180

gattacgaat tccggccatg cggccgcaag ctgggtgcag cgtgacccgg tcgtgcccct 240

ctctagagat aatgagcatt gcatgtctaa gttataaaaa attaccacat attttttttg 300

tcacacttgt ttgaagtgca gtttatctat ctttatacat atatttaaac tttactctac 360

gaataatata atctatagta ctacaataat atcagtgttt tagagaatca tataaatgaa 420

cagttagaca tggtctaaag gacaattgag tattttgaca acaggactct acagttttat 480

ctttttagtg tgcatgtgtt ctcctttttt tttgcaaata gcttcaccta tataatactt 540

catccatttt attagtacat ccatttaggg tttagggtta atggttttta tagactaatt 600

tttttagtac atctatttta ttctatttta gcctctaaat taagaaaact aaaactctat 660

tttagttttt ttatttaata atttagatat aaaatagaat aaaataaagt gactaaaaat 720

taaacaaata ccctttaaga aattaaaaaa actaaggaaa catttttctt gtttcgagta 780

gataatgcca gcctgttaaa cgccgtcgac gagtctaacg gacaccaacc agcgaaccag 840

cagcgtcgcg tcgggccaag cgaagcagac ggcacggcat ctctgtcgct gcctctggac 900

ccctctcgag agttccgctc caccgttgga cttgctccgc tgtcggcatc cagaaattgc 960

gtggcggagc ggcagacgtg agccggcacg gcaggcggcc tcctcctcct ctcacggcac 1020

ggcagctacg ggggattcct ttcccaccgc tccttcgctt tcccttcctc gcccgccgta 1080

ataaatagac accccctcca caccctcttt ccccaacctc gtgttgttcg gagcgcacac 1140

acacacaacc agatctcccc caaatccacc cgtcggcacc tccgcttcaa ggtacgccgc 1200

tcgtcctccc cccccccccc tctctacctt ctctagatcg gcgttccggt ccatggttag 1260

ggcccggtag ttctacttct gttcatgttt gtgttagatc cgtgtttgtg ttagatccgt 1320

gctgctagcg ttcgtacacg gatgcgacct gtacgtcaga cacgttctga ttgctaactt 1380

gccagtgttt ctctttgggg aatcctggga tggctctagc cgttccgcag acgggatcga 1440

tttcatgatt ttttttgttt cgttgcatag ggtttggttt gcccttttcc tttatttcaa 1500

tatatgccgt gcacttgttt gtcgggtcat cttttcatgc ttttttttgt cttggttgtg 1560

atgatgtggt ctggttgggc ggtcgttcta gatcggagta gaattctgtt tcaaactacc 1620

tggtggattt attaattttg gatctgtatg tgtgtgccat acatattcat agttacgaat 1680

tgaagatgat ggatggaaat atcgatctag gataggtata catgttgatg cgggttttac 1740

tgatgcatat acagagatgc tttttgttcg cttggttgtg atgatgtggt gtggttgggc 1800

ggtcgttcat tcgttctaga tcggagtaga atactgtttc aaactacctg gtgtatttat 1860

taattttgga actgtatgtg tgtgtcatac atcttcatag ttacgagttt aagatggatg 1920

gaaatatcga tctaggatag gtatacatgt tgatgtgggt tttactgatg catatacatg 1980

atggcatatg cagcatctat tcatatgctc taaccttgag tacctatcta ttataataaa 2040

caagtatgtt ttataattat tttgatcttg atatacttgg atgatggcat atgcagcagc 2100

tatatgtgga tttttttagc cctgccttca tacgctattt atttgcttgg tactgtttct 2160

tttgtcgatg ctcaccctgt tgtttggtgt tacttctgca gccaccatgc cgccattctc 2220

ggcagccggc ggcgaggggg cgccgtcgcc gatcagcagc cggccgccgc cgccggagca 2280

ggcggcggcg gcggcggcgg aggagcagct caacgggagc tcactggagc acgacggggt 2340

gctgggcggc gaggagggcg accggggcgg gtcgtcggcg ggcaaccggt ggccgcggca 2400

ggagacgctg gcgctgctca agatccggtc ggagatggac gccgccttcc gggaagccgc 2460

cctcaagggc cccctctggg aggaagtctc caggaagctc gcggagatgg ggtacaagag 2520

gagcgccaag aagtgccgcg agaagttcga gaacgtcgac aagtactaca agcgcaccaa 2580

ggacggccgc gccggccgcg gcgacggcaa gacctaccgc ttcttcaccg agctcgaggc 2640

cctccacggc gccgccgccg ccaccgcgcg cccgccgccg gtgtccctcg cgccggcgcc 2700

cgtggccgtg gcgccgccgg ccacccctgc gggtctctcg gcgttgcgcg tccacgcctc 2760

gccgccgccg ccgcctgtca agcagcacgc cgctccgccg ccgcctgtca tggatgtggc 2820

ggcgtgcgtg atgacgatgg acgacgtgag cttctcgtcg gggtcggaca cggaggagac 2880

ggcggaggag ggcgggaagc ggaagcggag gggcggcgga ggcatcggcg gcggcggcgg 2940

cggcgggaag gcgatgagga tgttcgaggg gctgatgcgg caggtgatgg agcggcagga 3000

ggcgatgcag cagaggcttc tggaggcgat cgagcggcgc gaccaggaga ggatgatccg 3060

cgaggaggcg tggcggcggc aggaggtggc gcgcctcgcc cgcgagcagg acgccctcgc 3120

ccaggagcgc gccattgccg cctcccgcga cgccgccgtc atctccttca tacagagggt 3180

caccggccag tccatcgccg ccgtgccacc gccgccgctg cagcccacgc cggtggcttc 3240

cgctgcacca ccaccaccac cgcagcatca tcatcagcaa acaccaccac cgattcaggt 3300

tcagccgcac cacataatgc cgatgacgcc gcagccccag ctgcaaccac cgcagccgca 3360

gagcaaggag gccaacaccg tcgtccgggc ggcgccgccg ccgcaagagc agcacgacac 3420

ggcggcgtcc ggcggcggcg gcggcgcgtc gtcgtcgagg tggccgaagg cggaggtgca 3480

cgcgctgatc cagctgcgca cagagctgga gacgaggtac caggactcgg ggcccaaggg 3540

gcctctctgg gaggacatct cggcggggat gcggcggctg gggtacagcc ggagctccaa 3600

gagatgcaag gagaagtggg agaacatcaa caagtacttc aagaaggtga aggagagcaa 3660

caagaagcgc cccgaggact ccaagacatg cccctactac caccagctcg acgcgctcta 3720

ccgcaccaag gcggccaacg ccgccgccgc cgcctcagcc tcgccggctc cggcgaccac 3780

caccgtgctt gccccggtgc cgctctccca gacgccgccg cacgtcgacc acggcggcag 3840

caacggcaat gggaatgggt gggcgagtgc caacaacggc ggcggcggca gcagctccgg 3900

gggcatgcaa acgaaggcta gcaacaatgg cacggcgacg gccggcgggc tgcccgtcgt 3960

ttcggtcgcc ggcggcaacg gcaacggcaa cggcgtggcg gcgacgacgg ataacaaggg 4020

atcgaagcag gtgcccgtcg cgaaggagac ggcggggcag aggcagccac agccgttggc 4080

catgaaccac aactacggca acgacagaat ggccgacgac atggacagcg acagcatgga 4140

cgacgacgac gacgacgacg aatttgacga cgacgaaaac gacgacgaca tcggcggtgg 4200

caagatgcag gtgcagtacg agacgtcgtc ccatttccaa cggccgcagc tgcagaacca 4260

gaacgtcgtc gtcggccggc caaacgcgag cggcggcggc ggcggtggcg ctccgacgac 4320

accggcgggc cctccgccgc ctgcggcaac aagtgggacg tcattcttgg cctgcgtcca 4380

gtgacggtga tcctcccgat cgttcaaaca tttggcaata aagtttctta agattgaatc 4440

ctgttgccgg tcttgcgatg attatcatat aatttctgtt gaattacgtt aagcatgtaa 4500

taattaacat gtaatgcatg acgttattta tgaggtgggt ttttatgatt agagtcccgc 4560

aattatacat ttaatacgcg atagaaaaca aaatatagcg cgcaaactag gataaattat 4620

cgcgcgcggt gtcatctatg ttactagatc gggagcaccg gtaaggcgcg ccgtagtgaa 4680

gcttggcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac 4740

ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca 4800

ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atgctagagc agcttgagct 4860

tggatcagat tgtcgtttcc cgccttcagt ttaaactatc agtgtttgac aggatatatt 4920

ggcgggtaaa cctaagagaa aagagcgttt attagaataa cggatattta aaagggcgtg 4980

aaaaggttta tccgttcgtc catttgtatg tgcatgccaa ccacagggtt cccctcggga 5040

tcaaagtact ttgatccaac ccctccgctg ctatagtgca gtcggcttct gacgttcagt 5100

gcagccgtct tctgaaaacg acatgtcgca caagtcctaa gttacgcgac aggctgccgc 5160

cctgcccttt tcctggcgtt ttcttgtcgc gtgttttagt cgcataaagt agaatacttg 5220

cgactagaac cggagacatt acgccatgaa caagagcgcc gccgctggcc tgctgggcta 5280

tgcccgcgtc agcaccgacg accaggactt gaccaaccaa cgggccgaac tgcacgcggc 5340

cggctgcacc aagctgtttt ccgagaagat caccggcacc aggcgcgacc gcccggagct 5400

ggccaggatg cttgaccacc tacgccctgg cgacgttgtg acagtgacca ggctagaccg 5460

cctggcccgc agcacccgcg acctactgga cattgccgag cgcatccagg aggccggcgc 5520

gggcctgcgt agcctggcag agccgtgggc cgacaccacc acgccggccg gccgcatggt 5580

gttgaccgtg ttcgccggca ttgccgagtt cgagcgttcc ctaatcatcg accgcacccg 5640

gagcgggcgc gaggccgcca aggcccgagg cgtgaagttt ggcccccgcc ctaccctcac 5700

cccggcacag atcgcgcacg cccgcgagct gatcgaccag gaaggccgca ccgtgaaaga 5760

ggcggctgca ctgcttggcg tgcatcgctc gaccctgtac cgcgcacttg agcgcagcga 5820

ggaagtgacg cccaccgagg ccaggcggcg cggtgccttc cgtgaggacg cattgaccga 5880

ggccgacgcc ctggcggccg ccgagaatga acgccaagag gaacaagcat gaaaccgcac 5940

caggacggcc aggacgaacc gtttttcatt accgaagaga tcgaggcgga gatgatcgcg 6000

gccgggtacg tgttcgagcc gcccgcgcac gtctcaaccg tgcggctgca tgaaatcctg 6060

gccggtttgt ctgatgccaa gctggcggcc tggccggcca gcttggccgc tgaagaaacc 6120

gagcgccgcc gtctaaaaag gtgatgtgta tttgagtaaa acagcttgcg tcatgcggtc 6180

gctgcgtata tgatgcgatg agtaaataaa caaatacgca aggggaacgc atgaaggtta 6240

tcgctgtact taaccagaaa ggcgggtcag gcaagacgac catcgcaacc catctagccc 6300

gcgccctgca actcgccggg gccgatgttc tgttagtcga ttccgatccc cagggcagtg 6360

cccgcgattg ggcggccgtg cgggaagatc aaccgctaac cgttgtcggc atcgaccgcc 6420

cgacgattga ccgcgacgtg aaggccatcg gccggcgcga cttcgtagtg atcgacggag 6480

cgccccaggc ggcggacttg gctgtgtccg cgatcaaggc agccgacttc gtgctgattc 6540

cggtgcagcc aagcccttac gacatatggg ccaccgccga cctggtggag ctggttaagc 6600

agcgcattga ggtcacggat ggaaggctac aagcggcctt tgtcgtgtcg cgggcgatca 6660

aaggcacgcg catcggcggt gaggttgccg aggcgctggc cgggtacgag ctgcccattc 6720

ttgagtcccg tatcacgcag cgcgtgagct acccaggcac tgccgccgcc ggcacaaccg 6780

ttcttgaatc agaacccgag ggcgacgctg cccgcgaggt ccaggcgctg gccgctgaaa 6840

ttaaatcaaa actcatttga gttaatgagg taaagagaaa atgagcaaaa gcacaaacac 6900

gctaagtgcc ggccgtccga gcgcacgcag cagcaaggct gcaacgttgg ccagcctggc 6960

agacacgcca gccatgaagc gggtcaactt tcagttgccg gcggaggatc acaccaagct 7020

gaagatgtac gcggtacgcc aaggcaagac cattaccgag ctgctatctg aatacatcgc 7080

gcagctacca gagtaaatga gcaaatgaat aaatgagtag atgaatttta gcggctaaag 7140

gaggcggcat ggaaaatcaa gaacaaccag gcaccgacgc cgtggaatgc cccatgtgtg 7200

gaggaacggg cggttggcca ggcgtaagcg gctgggttgt ctgccggccc tgcaatggca 7260

ctggaacccc caagcccgag gaatcggcgt gacggtcgca aaccatccgg cccggtacaa 7320

atcggcgcgg cgctgggtga tgacctggtg gagaagttga aggccgcgca ggccgcccag 7380

cggcaacgca tcgaggcaga agcacgcccc ggtgaatcgt ggcaagcggc cgctgatcga 7440

atccgcaaag aatcccggca accgccggca gccggtgcgc cgtcgattag gaagccgccc 7500

aagggcgacg agcaaccaga ttttttcgtt ccgatgctct atgacgtggg cacccgcgat 7560

agtcgcagca tcatggacgt ggccgttttc cgtctgtcga agcgtgaccg acgagctggc 7620

gaggtgatcc gctacgagct tccagacggg cacgtagagg tttccgcagg gccggccggc 7680

atggccagtg tgtgggatta cgacctggta ctgatggcgg tttcccatct aaccgaatcc 7740

atgaaccgat accgggaagg gaagggagac aagcccggcc gcgtgttccg tccacacgtt 7800

gcggacgtac tcaagttctg ccggcgagcc gatggcggaa agcagaaaga cgacctggta 7860

gaaacctgca ttcggttaaa caccacgcac gttgccatgc agcgtacgaa gaaggccaag 7920

aacggccgcc tggtgacggt atccgagggt gaagccttga ttagccgcta caagatcgta 7980

aagagcgaaa ccgggcggcc ggagtacatc gagatcgagc tagctgattg gatgtaccgc 8040

gagatcacag aaggcaagaa cccggacgtg ctgacggttc accccgatta ctttttgatc 8100

gatcccggca tcggccgttt tctctaccgc ctggcacgcc gcgccgcagg caaggcagaa 8160

gccagatggt tgttcaagac gatctacgaa cgcagtggca gcgccggaga gttcaagaag 8220

ttctgtttca ccgtgcgcaa gctgatcggg tcaaatgacc tgccggagta cgatttgaag 8280

gaggaggcgg ggcaggctgg cccgatccta gtcatgcgct accgcaacct gatcgagggc 8340

gaagcatccg ccggttccta atgtacggag cagatgctag ggcaaattgc cctagcaggg 8400

gaaaaaggtc gaaaagctgt ctttcctgtg gatagcacgt acattgggaa cccaaagccg 8460

tacattggga accggaaccc gtacattggg aacccaaagc cgtacattgg gaaccggtca 8520

cacatgtaag tgactgatat aaaagagaaa aaaggcgatt tttccgccta aaactcttta 8580

aaacttatta aaactcttaa aacccgcctg gcctgtgcat aactgtctgg ccagcgcaca 8640

gccgaagagc tgcaaaaagc gcctaccctt cggtcgctgc gctccctacg ccccgccgct 8700

tcgcgtcggc ctatcgcggc cgctggccgc tcaaaaatgg ctggcctacg gccaggcaat 8760

ctaccagggc gcggacaagc cgcgccgtcg ccactcgacc gccggcgccc acatcaaggc 8820

accctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga 8880

gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc 8940

agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt 9000

gtatactggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatgcgg 9060

tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc 9120

tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 9180

aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 9240

aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 9300

ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 9360

acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 9420

ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 9480

tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 9540

tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 9600

gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 9660

agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 9720

tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 9780

agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 9840

tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 9900

acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgcattct 9960

aggtactaaa acaattcatc cagtaaaata taatatttta ttttctccca atcaggcttg 10020

atccccagta agtcaaaaaa tagctcgaca tactgttctt ccccgatatc ctccctgatc 10080

gaccggacgc agaaggcaat gtcataccac ttgtccgccc tgccgcttct cccaagatca 10140

ataaagccac ttactttgcc atctttcaca aagatgttgc tgtctcccag gtcgccgtgg 10200

gaaaagacaa gttcctcttc gggcttttcc gtctttaaaa aatcatacag ctcgcgcgga 10260

tctttaaatg gagtgtcttc ttcccagttt tcgcaatcca catcggccag atcgttattc 10320

agtaagtaat ccaattcggc taagcggctg tctaagctat tcgtataggg acaatccgat 10380

atgtcgatgg agtgaaagag cctgatgcac tccgcataca gctcgataat cttttcaggg 10440

ctttgttcat cttcatactc ttccgagcaa aggacgccat cggcctcact catgagcaga 10500

ttgctccagc catcatgccg ttcaaagtgc aggacctttg gaacaggcag ctttccttcc 10560

agccatagca tcatgtcctt ttcccgttcc acatcatagg tggtcccttt ataccggctg 10620

tccgtcattt ttaaatatag gttttcattt tctcccacca gcttatatac cttagcagga 10680

gacattcctt ccgtatcttt tacgcagcgg tatttttcga tcagtttttt caattccggt 10740

gatattctca ttttagccat ttattatttc cttcctcttt tctacagtat ttaaagatac 10800

cccaagaagc taattataac aagacgaact ccaattcact gttccttgca ttctaaaacc 10860

ttaaatacca gaaaacagct ttttcaaagt tgttttcaaa gttggcgtat aacatagtat 10920

cgacggagcc gattttgaaa ccgcggtgat cacaggcagc aacgctctgt catcgttaca 10980

atcaacatgc taccctccgc gagatcatcc gtgtttcaaa cccggcagct tagttgccgt 11040

tcttccgaat agcatcggta acatgagcaa agtctgccgc cttacaacgg ctctcccgct 11100

gacgccgtcc cggactgatg ggctgcctgt atcgagtggt gattttgtgc cgagctgccg 11160

gtcggggagc tgttggctgg ctggtggcag gatatattgt ggtgtaaaca aattgacgct 11220

tagacaactt aataacacat tgcggacgtt tttaatgtac tgaattaacg ccgaattaat 11280

tcgggggatc tggattttag tactggattt tggttttagg aattagaaat tttattgata 11340

gaagtatttt acaaatacaa atacatacta agggtttctt atatgctcaa cacatgagcg 11400

aaaccctata ggaaccctaa ttcccttatc tgggaactac tcacacatta ttatggagaa 11460

actcgagctt gtcgatcgac agatccggtc ggcatctact ctatttcttt gccctcggac 11520

gagtgctggg gcgtcggttt ccactatcgg cgagtacttc tacacagcca tcggtccaga 11580

cggccgcgct tctgcgggcg atttgtgtac gcccgacagt cccggctccg gatcggacga 11640

ttgcgtcgca tcgaccctgc gcccaagctg catcatcgaa attgccgtca accaagctct 11700

gatagagttg gtcaagacca atgcggagca tatacgcccg gagtcgtggc gatcctgcaa 11760

gctccggatg cctccgctcg aagtagcgcg tctgctgctc catacaagcc aaccacggcc 11820

tccagaagaa gatgttggcg acctcgtatt gggaatcccc gaacatcgcc tcgctccagt 11880

caatgaccgc tgttatgcgg ccattgtccg tcaggacatt gttggagccg aaatccgcgt 11940

gcacgaggtg ccggacttcg gggcagtcct cggcccaaag catcagctca tcgagagcct 12000

gcgcgacgga cgcactgacg gtgtcgtcca tcacagtttg ccagtgatac acatggggat 12060

cagcaatcgc gcatatgaaa tcacgccatg tagtgtattg accgattcct tgcggtccga 12120

atgggccgaa cccgctcgtc tggctaagat cggccgcagc gatcgcatcc atagcctccg 12180

cgaccggttg tagaacagcg ggcagttcgg tttcaggcag gtcttgcaac gtgacaccct 12240

gtgcacggcg ggagatgcaa taggtcaggc tctcgctaaa ctccccaatg tcaagcactt 12300

ccggaatcgg gagcgcggcc gatgcaaagt gccgataaac ataacgatct ttgtagaaac 12360

catcggcgca gctatttacc cgcaggacat atccacgccc tcctacatcg aagctgaaag 12420

cacgagattc ttcgccctcc gagagctgca tcaggtcgga gacgctgtcg aacttttcga 12480

tcagaaactt ctcgacagac gtcgcggtga gttcaggctt tttcatatct cattgccccc 12540

cgggatctgc gaaagctcga gagagataga tttgtagaga gagactggtg atttcagcgt 12600

gtcctctcca aatgaaatga acttccttat atagaggaag gtcttgcgaa ggatagtggg 12660

attgtgcgtc atcccttacg tcagtggaga tatcacatca atccacttgc tttgaagacg 12720

tggttggaac gtcttctttt tccacgatgc tcctcgtggg tgggggtcca tctttgggac 12780

cactgtcggc agaggcatct tgaacgatag cctttccttt atcgcaatga tggcatttgt 12840

aggtgccacc ttccttttct actgtccttt tgatgaagtg acagatagct gggcaatgga 12900

atccgaggag gtttcccgat attacccttt gttgaaaagt ctcaatagcc ctttggtctt 12960

ctgagactgt atctttgata ttcttggagt agacgagagt gtcgtgctcc accatgttat 13020

cacatcaatc cacttgcttt gaagacgtgg ttggaacgtc ttctttttcc acgatgctcc 13080

tcgtgggtgg gggtccatct ttgggaccac tgtcggcaga ggcatcttga acgatagcct 13140

ttcctttatc gcaatgatgg catttgtagg tgccaccttc cttttctact gtccttttga 13200

tgaagtgaca gatagctggg caatggaatc cgaggaggtt tcccgatatt accctttgtt 13260

gaaaagtctc aatagccctt tggtcttctg agactgtatc tttgatattc ttggagtaga 13320

cgagagtgtc gtgctccacc atgttggcaa gctgctctag ccaatacgca aaccgcctct 13380

ccccgcgcgt tggccgat 13398

<210> 13

<211> 20

<212> DNA

<213> TGS1RT-F(Artificial)

<400> 13

gcaaggagaa gtgggagaac 20

<210> 14

<211> 20

<212> DNA

<213> TGS1RT-R(Artificial)

<400> 14

tcgagctggt ggtagtaggg 20

Claims

The application of TGS1 gene in regulating rice grain size and yield is characterized in that: the TGS1 gene in rice is knocked out to increase the size or yield of rice grains, and the nucleotide sequence of the TGSl gene is shown in SEQ ID No. 1.
2. The use of claim 1, wherein the specific method for knocking out the TGS1 gene in rice is as follows: the TGS1 gene in rice is subjected to targeted mutation by using a CRISPR/Cas9 technology.
3. The application of claim 2, comprising the following steps:

1) selecting a target fragment from a coding region of a rice grain size control gene TGS 1;

2) synthesizing a linker primer with a cohesive end according to the sequence information of the target fragment, and constructing a CRISPR/Cas9 recombinant vector for TGS1 gene targeting; the recombinant vector comprises an sgRNA expression cassette having the target sequence and a Cas9 nuclease expression cassette capable of being expressed in rice cells;

3) transferring the recombinant vector into an agrobacterium tumefaciens engineering strain, and transforming a conventional rice variety by using an agrobacterium-mediated method to obtain a transgenic rice plant;

4) amplifying and sequencing the genome DNA of the transgenic plant by using a genome primer covering the target site, thereby carrying out genotype identification on the T0 generation transgenic plant and obtaining a homozygous mutant;

5) the homozygous mutant is planted in T1 generation, and PCR detection is carried out on HPT, sgRNA and Cas9 transgenes in T1 generation plants by using primers, so that the rice without transgene insertion with increased grain size or yield can be obtained.
4. The use according to claim 3, wherein in step 1) the sequence of the target fragment is SEQ ID No. 5.
5. The use according to claim 3, wherein in step 2), the nucleotide sequence of the sgRNA expression cassette having the target sequence is shown in SEQ ID No. 3.
6. The use according to claim 3, wherein in step 2) the nucleotide sequence of the Cas9 nuclease expression cassette is set forth in SEQ ID No. 4.
7. The use according to claim 3, wherein in step 5), the primer sequences for HPT, sgRNA and Cas9 transgene amplification are:

HPTF：5’-GGGTGTCACGTTGCAAGACC-3’，

HPTR：5’-ATGCCTCCGCTCGAAGTAGC-3’，

sgRNAF：5’-TCCCAGTCACGACGTTGTAA-3’，

sgRNAR：5’-GGCCATTTGTCTGCAGAAT-3’，

Cas9F：5’-CACCATCTACCACCTGAGAA-3’，

Cas9R：5’-CGAAGTTGCTCTTGAAGTTG-3’。