CN112210561A

CN112210561A - Application of OsKEAP1 gene in regulation of rice seed phenotype and germination rate

Info

Publication number: CN112210561A
Application number: CN202011233673.3A
Authority: CN
Inventors: 蒋萌; 舒庆尧; 刘燕华
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2020-11-06
Filing date: 2020-11-06
Publication date: 2021-01-12

Abstract

The invention discloses an application of OsKEAP1 gene in regulation and control of rice seed phenotype and germination rate, wherein the nucleotide sequence of the OsKEAP1 gene is shown as SEQ ID NO. 1; the seed phenotype is that of a brown rice grain. The invention obtains the mutant with the expression level of the OsKEAP1 gene down-regulated by using the CRISPR/Cas9 gene editing technology, finds that the OsKEAP1 gene is related to the phenotype and the germination rate of rice seeds, and can provide a basis for cultivating rice varieties with excellent germplasm.

Description

Application of OsKEAP1 gene in regulation of rice seed phenotype and germination rate

Technical Field

The invention relates to the technical field of plant genetic engineering and rice molecular breeding, and mainly relates to application of an OsKEAP1 gene in regulation and control of rice seed phenotype and germination rate.

Background

Reactive Oxygen Species (ROS) are a generic term for oxygen-containing molecules or ions having strong oxidative activity, and are natural by-products of oxygen-related metabolism in animal and plant cells. The delicate balance that exists between the accumulation of ROS and the antioxidant system that scavenges them is called redox homeostasis (redox homeostatis). In higher plants, ROS are also important signal molecules in salt and drought-related stresses. An imbalance between ROS accumulation and antioxidant clearance systems can lead to Oxidative Stress (OS), which leads to a series of stress reactions such as destruction of intracellular biomacromolecules, inflammatory infiltration in biological tissues, etc.

Cells have developed a variety of classical antioxidant mechanisms for the cytoprotection of oxidative stress. As a key antioxidant pathway, KEAP1(Kelch-like endothelial in-associated protein1) -NRF2(nuclear factor E2 related factor 2, NRF2) plays an important role in regulating intracellular oxidative stress. The KEAP1 is a biosensor sensitive to ROS and electrophilic reagents in a KEAP1-NRF2 system, and the regulation and control of NRF2 realize the regulation and control of downstream cytoprotective genes of the biosensor, wherein the downstream cytoprotective genes mainly code stress protein, metabolic enzyme, antioxidant enzyme, redox balance factor, heterologous biological transport protein and II-phase detoxification enzyme. Plays an indispensable important role in the regulation of redox homeostasis. Although the KEAP1-NRF2 system has been studied extensively in animal systems, the presence of related systems in plants, particularly in rice growth and development and physiological characteristics of rice, has not been reported.

Due to the high accuracy and efficiency of CRISPR/Cas9 mutant generation, the method can be used for researching non-regular target sites (such as UTR and intron sequences in a genome), so that researchers can research partially regulated gene functions instead of lethal knockout of a coding region to generate nonsense mutation. KEAP1 has important oxidative stress regulation as an essential functional protein in the oxidative stress response pathway of animals, but is rarely studied in plants. The UTR region editing can effectively realize partial regulation of gene transcription and translation, and the characteristic can be used for partial regulation of research gene function and avoiding research inconvenience caused by death of important gene knockout. In view of the above background studies, the present application performed targeted editing of the OsKEAP15' UTR region using the CRISPR/Cas9 system to study its biological functions in rice growth and development.

Disclosure of Invention

The invention provides a new application of OsKEAP1 gene in regulation of rice seed phenotype and germination rate, and provides a basis for breeding rice varieties with excellent germplasm.

The specific technical scheme is as follows:

the invention provides application of an OsKEAP1 gene in regulation and control of rice seed phenotype, wherein the nucleotide sequence of the OsKEAP1 gene is shown as SEQ ID No. 1; the seed phenotype is that of a brown rice grain. The gene has the full length of 5658bp, encodes 700 amino acid proteins, and the proteins contain a DCD structure domain which is specific to plants and 5 Kelch structures.

The invention also provides application of the OsKEAP1 gene in regulation and control of rice seed germination rate, wherein the nucleotide sequence of the OsKEAP1 gene is shown as SEQ ID No. 1.

The invention utilizes CRISPR/Cas9 gene editing technology to obtain a mutant with OsKEAP1 gene expression level reduced, and discovers that: the oskeap1 mutation adversely affects seed development, resulting in dark spots and shriveling of the kernels and reduced seed germination.

Compared with the prior art, the invention has the following beneficial effects:

the invention obtains the mutant with the expression level of the OsKEAP1 gene down-regulated by using the CRISPR/Cas9 gene editing technology, finds that the OsKEAP1 gene is related to the phenotype and the germination rate of rice seeds, and can provide a basis for cultivating rice varieties with excellent germplasm.

Drawings

FIG. 1 shows the information of human KEAP1 and rice homologous genes;

wherein, A: information of homologous genes in human KEAP1 and rice; b: gene structure diagrams of human KEAP1 and homologous genes in rice; c: protein conserved domain distribution of human KEAP1 and homologous genes in rice, BTB domain (protein binding, stress response), BACK domain, KELCH domain, DCD domain (plant specific stress response domain).

Fig. 2 is a schematic nucleotide sequence diagram of CRISPR sgRNA sequence targeted editing of the OsKEAP15' UTR region and the mutant at the post-editing target site.

FIG. 3 is a graph of the relative expression of OsKEAP1 in leaf tissue of rice variety Xidao #1 and its two mutants, seedling (A) and heading (B);

wherein OsACTIN is used as an internal reference; data are the average of 3 biological replicates; different letters represent significance at the 0.05 level.

FIG. 4 shows the dehulled rice grain phenotype of wild type West Rice No.1 (WT) and its oskeap1 mutant (A) and the germination rate of dehulled seeds (B) on 1/2MS medium without addition of (C) and with addition of (D) 1. mu.M ABA. Data from different letters on the same observation day were significantly different at the 0.05 level.

Detailed Description

The present invention will be further described with reference to the following specific examples, which are only illustrative of the present invention, but the scope of the present invention is not limited thereto.

Example 1

1. Identification and characterization of KEAP1 homologous gene in rice

The gene information of human KEAP1 and the coded amino acid sequence thereof are searched and obtained from NCBI, and are used as index sequences to be compared with rice genomes in a Gramene database to obtain KEAP1 homologous genes in the rice genomes. The structure of the rice KEAP1 gene and the properties of its encoded protein were analyzed in the KEGG database and RAPDB database, and the amino acid conserved domain was analyzed in the Unipro database and SMART database. Their changes in expression levels after different tissues and hormone treatments of plants were analyzed in riceXPro software.

The amino acid sequences of maize, human and other 10 related species were downloaded from the Gramene database, the OrthoDB database, the NJ method with the boottrp duplication set 1000 as the minimum evolutionary standard, the MEGA7 software was used to construct the KEAP1 orthologous gene phylogenetic tree, and the poisson correction method was used to calculate the evolutionary distance.

As a result: two homologous genes of KEAP1, Os01g0162500 (54%) and Os05g0164900 (46%), were identified in the rice genome with higher coverage (c.v. Nipponbare). In the NCBI database, the former is annotated as encoding a hypothetical KELCH type beta propeller domain containing protein, while the latter is a KELCH repeat type 1 domain containing protein, so the KELCH domains are KEAP 1. However, the amino acid identification scores for rice protein to KEAP1 were only 25.82% and 33.46% (fig. 1A).

Both rice homologous genes were much shorter in length, but had more exons and introns than KEAP 1; both KEAP1 and its rice homologous gene have a longer untranslated region (UTR) at their 3' end (FIG. 1B). The rice KEAP1 homologue had 5 KELCH repeats, whereas KEAP1 had 6 KELCH repeats. At the N-terminus, KEAP1 has a BTB-BACK domain, while the rice homologue has a developmental and programmed cell death (DCD) domain (FIG. 1C).

These characteristics suggest that KEAP1 and its rice homologous gene may retain similar basic functions but respond differently to different developmental and environmental stimuli.

2. Creation of oskeap1 mutant

(1) Gene editing vector construction

The gene editing vector used in the invention is pHUN4c12 based on CRISPR/Cas 9. The first exon of OsKEAP15' UTR is used as a targeted editing region, a CRISPR-P2.0 (http:// CRISPR. hzau.edu.cn/CRISPR2/) is used for designing an editing target, a target sequence with the best specificity and editing efficiency is selected, BsaI recognition site sequences are added at two ends, and forward and reverse complementary DNA single-stranded oligonucleotides P1 and P2 are synthesized.

The positive and negative strand targeting the OsKEAP15' UTR is:

KEAP1-P1：5’-GGCAGGGTGCTACGAAACCCGC-3’(P1)；

KEAP1-P2:5’-AAACGCGGGTTTCGTAGCACCC-3’(P2)；

the DNA forward strand F and reverse strand R oligonucleotides were diluted to 100. mu.M and annealed in TE buffer to form double strands. The annealing reaction system is as follows:

the annealing procedure is as follows: 95 ℃ for 2min, after which the temperature was lowered to 4 ℃ by 0.1 ℃ every 8 sec.

After annealing, the DNA oligo double strand was purified using Axygen DNA gel extraction kit as follows:

(a) adding 300 mu L of buffer solution A to 100 mu L of annealing product and mixing the solution gently;

(b) adding 150 μ L of buffer B, and mixing gently;

(c) transferring the solution to a DNA preparation tube provided by the kit, placing the DNA preparation tube in another 2mL centrifuge tube, and centrifuging the solution at 12,000rpm for 1 min;

(d) the DNA preparation tube was transferred to a new 2mL centrifuge tube, 500. mu. L W1 buffer was added, the tube was centrifuged at 12,000rpm for 30sec, and the supernatant was discarded;

(e) adding 700 mu L W2 buffer solution, centrifuging at 12,000rpm for 30sec, and discarding the supernatant;

(f) repeating the step 5;

(g) centrifuging again at 12,000rpm for 1 min;

(h) transferring the DNA preparation tube to a new 1.5mL centrifuge tube, and placing the centrifuge tube at room temperature;

(i) adding 10-20 μ L eluate, centrifuging at 12,000rpm for 30s, and detecting nucleic acid concentration.

The pHUN4c12 carrier is cut by restriction enzyme BsaI (NEB, Beijing) to form a linearized double strand, and the reaction system is as follows:

after incubation at 37 ℃ for 6-8h, the vector fragment was recovered using the Axygen DNA gel extraction kit (recovery procedure as above).

The purified DNA oligonucleotide duplex was ligated with the linearized pHUN4c12 vector using T4 ligase (Takara, Shanghai). The ligation reaction system is as follows:

after completion of ligation by incubation at 4 ℃ for about 10h, the reaction was directly transformed into DH 5. alpha. E.coli competent cells. Obtaining single clone on LB plate containing kanamycin, picking single clone to cultivate on LB liquid culture medium, checking sequence and obtaining target vector pHUN4c12-OsKEAP 1-UTR.

The above vector was introduced into Agrobacterium (Agrobacterium tumefaciens strain EHA 105) with pHUN4c12-OsKEAP1-UTR by heat shock method, respectively.

(2) Agrobacterium mediated method for culturing transgenic rice

After stannic rice No.1 (japonica rice variety) seeds are soaked in 70% ethanol for 30-60s, the seeds are sterilized in a sterile 50mL centrifuge tube by 1% NaClO disinfectant through oscillation for 30-40min (during the period, the disinfectant can be replaced once if the solution becomes turbid), then the seeds are washed 3-4 times by using sterile water on an ultraclean workbench, transferred to absorbent filter paper and dried for 30-60min, and cultured on a 2N6 callus induction culture medium.

Selecting callus with the size of about 4mm for agrobacterium transformation, and the specific method comprises the following steps: when the OD 600 of the Agrobacterium liquid is 0.8-1.0, 500. mu.L of the supernatant is taken, and 30mL of AAM infected bacteria liquid with 200. mu. mol/L of As is made into bacterial suspension so that the OD 600 of the bacterial liquid is 0.01.

Putting 50-100 rice callus particles into agrobacterium tumefaciens liquid for 5-10min, taking out, placing on sterile filter paper, draining for 30-40min, and performing dark culture on a co-culture medium (shown in table 1) at 25 ℃ for 2.5 days. Then, the callus was transferred to selection medium S1 (Table 2) supplemented with hygromycin (50mg/L) and cephradine (500mg/L) and cultured for 14 days, and the newly grown callus was transferred to second selection medium S2 (hygromycin: 80 mg/L; cephradine: 500mg/L) and cultured continuously to obtain new callus. The new callus growing was transferred to japonica rice differentiation medium (table 3). And transferring the plantlets to a rooting culture medium (table 4) to root and grow into plantlets when the differentiated plantlets grow to about 1 cm. When the plantlet grows to about 7cm, adding tap water to harden the plantlet for 3-5 days, then growing in 1/4MS liquid culture medium for 1 week, and transplanting the plantlet into soil to grow into plant.

TABLE 1 Co-cultivation Medium/liter

Note that MES is ethanesulfonic acid As is acetosyringone

TABLE 2 selection Medium/liter

First screening: cephradine (Cef, 500mg/L) and hygromycin (Hyg, 50 mg/L);

and (3) second screening: cefradine (Cef, 500mg/L) and hygromycin (Hyg, 80mg/L), sterilized and added.

TABLE 3 differentiation Medium/liter

TABLE 4 rooting Medium/liter

As a result: obtaining 12 positive T strains through hygromycin resistance gene detection₀And (5) plant growing. And (4) sequencing a substitution table sample by Sanger to determine the mutation genotype of the mutant single strain. At T₁Class 2 single-base homozygous insertion mutants were detected in the population and were designated oskeap1-1 and oskeap1-2, respectively (FIG. 2).

3. Relative expression of OsKEAP1 in seedling leaf tissue

To assess whether and how both insertion mutations OsKEAP1-1 and OsKEAP1-2 affect expression of OsKEAP1, the abundance of OsKEAP1 transcripts in the mutant and its wild-type parent Xidao #1 was examined using qRT-PCR.

Wherein, the seeds of the paddy variety stannum-rice No.1 are soaked for 48 hours at 30 ℃, then germinate on moist filter paper, and are transferred to a growth chamber for culture after budding. The growth conditions were: the day/night temperature is 24/16 ℃, the photoperiod is 16h, and the irradiance is 300 mu mol.m^-2·s^-1And the relative humidity is 60-70%. Total RNA is extracted from 10-day-old seedlings by using a TIANGEN polysaccharide polyphenol plant total RNA extraction kit (Tiangen, Beijing), and the specific operation steps are as follows：

(a) Wrapping a mortar, a medicine spoon, a 2mL centrifuge tube filled with a broken and oscillated small steel ball and the like required in the RNA extraction process with tinfoil paper, and sterilizing the sterilized pot at 121 ℃ for 30min for 2 times under high temperature and high pressure;

(b) weighing about 100mg of plant tissues, placing the plant tissues in a centrifuge tube filled with small broken and oscillating steel balls, rapidly cooling the plant tissues in liquid nitrogen, and then fully grinding the plant tissues in a grinder at 60Hz for 30 s;

(c) after the leaves are fully ground, 500 mu L of SL buffer solution (added with beta-mercaptoethanol) is added into a centrifuge tube filled with rice tissues, the mixture is evenly mixed by vortex, and the mixture is centrifuged for 90s at 12,000 rpm;

(d) transferring the centrifuged supernatant to a filtration column CS column, centrifuging at 12,000rpm for 90s, and transferring the filtrate in the collection tube to a new 1.5mL RNase-Free centrifuge tube (the cell debris is not touched as much as possible during the suction process);

(e) adding 0.4 times of anhydrous ethanol, mixing, transferring the obtained system to an adsorption column CR3, centrifuging at 12,000rpm for 90s, removing the filtrate, and returning the adsorption column to the collection tube;

(f) the adsorption column was added with deproteinized solution CW1, centrifuged at 12,000rpm for 30s, and the waste liquid was discarded. The adsorption column is put back into the collecting pipe;

(g) removing genome DNA by DNase I working solution;

(h) adding 500 mu L of cleaning solution RW1 added with the recommended volume of absolute ethyl alcohol into a centrifuge tube, centrifuging at 12,000rpm for 30s, and discarding the filtrate;

(i) repeating the step 8 once;

centrifuging at 12,000rpm for 2min, placing adsorption column CR3 in a new 1.5mL RNase-Free centrifuge tube, and adding 40 μ L RNase-Free ddH dropwise to the middle part of the adsorption membrane₂O, standing at room temperature for 2min, centrifuging at 12,000rpm for 90s, removing the adsorption column, and collecting the RNA solution.

cDNA Synthesis cDNA was synthesized using Takara reverse transcription kit (Shanghai), now with gDNA Eraser in buffer to remove excess genomic DNA.

The reverse transcription system is as follows:

PCR program was 37 deg.C, 15 min; 85 ℃ for 5 sec. After the reaction is finished, the product is stored at 4 ℃ for later use.

The results show that OsKEAP1 transcript levels were significantly lower for both mutants at seedling and flowering stages than for Xidao #1 (FIG. 3). In contrast, the mutational effect of OsKEAP1-1 was more profound than OsKEAP1-2, with OsKEAP1 having transcriptional abundances at the seedling stage of only 41.09% (OsKEAP1-1) and 77.39% (OsKEAP1-2) of Xidao #1 (fig. 3A). The mutational effect was more pronounced at anthesis with OsKEAP1 transcript levels of only 29.77% and 50.46% of the wild-type parent Xidao #1 (FIG. 3B).

4. OsKEAP1 downregulation of effects on seed phenotype, seed germination, and ABA sensitivity

The specific test contents are as follows:

taking 100 mutant seeds and parent seeds respectively, soaking in water at 30 ℃ for 48h (changing water once in the period), transferring to wet filter paper for germination, counting the number of the germinated seeds day by day, and repeating for three times.

Selecting 60 well-developed seeds, sterilizing the seeds in 75% ethanol for 30-60s, washing with clean water, sterilizing with 1% NaClO disinfectant for 40min, and washing with sterile water in an ultra-clean bench. Seeds were placed in 1/2MS medium supplemented with 0 and 1. mu.M ABA. Germination rates were then observed and counted daily for 6 days. Repeat 3 times.

The results are shown in fig. 4, and the oskeap1 mutation appears to adversely affect seed development, resulting in dark spots and shriveling of the kernel (fig. 4A). When germination tests were performed with intact rice, the seed germination rates for oskeap1-1 and oskeap1-2 were significantly reduced, being 43.06% and 54.17%, respectively, of the wild-type parent Xidao #1 at day 6 (fig. 4B). To examine whether the reduction in germination rate was associated with black spots and shrunken seeds, the rice was peeled and non-spotted and shrunken brown rice grains (normal-looking grains) were selected for germination test. No significant difference was observed between Xidao #1 and its two oskeap1 mutants grown on 1/2MS medium (fig. 4C), indicating that particles with black spots/shrinkage reduced seed germination.

Sequence listing

<110> Zhejiang university

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 5658

<212> DNA

<213> Rice (Oryza sativa L.)

<400> 1

acgcccaccc atccactcgc accccgcgcc gcaccaacac acgcagaggt ggtgaactgg 60

ttgatcgatg cttgctgctg ctgcgcgtcg aggcggctcc atcggcggca accgctgatc 120

gatcgatcga tcccgcgggt ttcgtagcac ccgagcaggt gagcgagcga gcgatcccct 180

cccgcgcggc cgcgcctcga tttggtcgcg attggtgctg gattttctct ctctctctct 240

ctctctctct acctttcctg tttcggtggg ggttttggtg gggttttgtg gctcgaggtg 300

gtcggggatt tctggggtga tgggagaggg gctcgcgtgg tggtggattg ttcgctggaa 360

tggcggggaa tgctgtgccg tttgggggat tgatgccatg gtgatgggtc gaaatgctgg 420

atggggttga gtttgttcaa gtggttttga attggaatct gtggagtttg gggggaaatg 480

gggaatgggg cttcgagtgc gcaaaggttt tgcatgtgcg gaattgcagc tcgaggtttt 540

cgctggattt aggcgtagct tctgtgcgcg tgtttgttta gcgattttag cagttcgtgg 600

gctggtgccg ccgcgtgttt gaatggttta gcgctttatt taccgttaca gttgatgtct 660

gcggtttttc tggtgattta tgccagtttt tagaggtgaa tcgctgtgtt ttgggggaat 720

tctagagccc agggttgggt ttatgggggc gagattttag ctgttggatt ttaatcctat 780

attgcgagaa gcatgggaga actgcgatcc gtcgtcagag gtgaacgctt tcgaactctg 840

atggttttaa tgtggcagat attgtgaagc ttatggtagc ttcgttaact tgatcgacgt 900

ttttacttgt gctgctcgtg cgttgtggta gatttggctt gtgagagagc aggtggatgg 960

ggtagattca gattacagag tggtttactg catgtagcga tgtgtttgag ttgtaacttg 1020

tgaggctgta agattaaaat gttggcatgc gtggggtttt tgggggtaat attttgtttt 1080

gtttttgttt ttgttttttg ctagtgtttt gacccaatcc atctccccca tcttttttta 1140

gtggaaaacc gctccctgtt ggtactactc aagtaaactt accagaatta caatcctcca 1200

acaaaggaat aataccctgt gctgtttgtt gtgacaactt atctgtacat gatattgcta 1260

gtttgctact gcagtatacc tacatgctca cttatgtttg tttcttttag ataatgtcac 1320

ttatgtttgt gttttgcata atgttgataa tttctcgaag aatgggtgct ggaaagaaga 1380

ctcagaccga gaacgagttc cgtgagctcc cggaaaagga gctaggaggg gtggtctttt 1440

gttgcaataa caacacattc gatgaatgct tcactaaaca gttatttggt tagtaaaagt 1500

cccttttcgc ctgtcctaat ttcatctaaa tgatatgtaa aatccatacg cgtgtgatta 1560

cttttattca agaacattga cattgctaat caacattctt ttgaacttct cgtttaattg 1620

ggaatccatt cgtttgcctt ttgtttgata ggtttgcctc aacgcaatat cttgtatgtg 1680

aagaatgtta aacctggctt gcctctcttt ctgttcaatt atagtaacag acaattgcat 1740

ggcattttca aggctacaag cactggccag cttaatatcg accgatttgc ttggatgtct 1800

gaacagtcta atgatgcaaa gacaaatgca aagacaacac catttcctgc acaggtataa 1860

tagaaacaac atatactggt ctttgttctg gttatagttt cataagcatt gtgagtattt 1920

gtgttcttca ggtccgcttc tctaccagga cagagtgccc tccacttcca gaaagcaaat 1980

acaaaagtgt aatcataaac aattatcgca aggataaacc tagccacttc cgttttgagc 2040

tagaccatcg acaaacaaga gatttgattt ctttgtttct acctgctcct gttcgcgcta 2100

atcaaaacaa acttagtatt ccaaaacctc ctgctaccgc tcatactgtt ccaaatccat 2160

ggaatcgacc tctgccattt cttacagcaa aagcacctgt tgtttctgat aaagtgaaga 2220

gtgaatccaa tgtgaaggat gtggatcagt tcaatgtttc atcacactca catgatattg 2280

ttcctcatac cttgcctgat gtagaagtcg accttgctag cacaagtaca acatcaagga 2340

gcaaccttaa caaagatgct tctggctgtg atgacctggt tgctggtttg atcaaggaag 2400

ataaagaatc tgtggatgat gaccagcatg ctaaaatgga tttaccagta aagctgcaag 2460

agctgtcttc tttacaacag aaggaagcca atttcttgga ggatgctcct gtttctactt 2520

cagctcaaag catacgtcaa gatacacggt ttgctgctac tctccccaaa gattcattta 2580

atgccacatc tcaatgtgac acatcattga aagatacatc ctttgtgcaa tgccatgaat 2640

atgctgaggt gccttaagct attccatccc ttattaccat tttcccttac gcaccatatt 2700

tcagtgctac aacttatata ttttggtgcg atgtttatgg cctgcttttt ggcatttgtc 2760

tgtagctgta tcaaattatc aatgatttat ccaagaagac cgaagaaatg gaaaagatga 2820

aggtactttt cttcttgctt aaaatcagta ggatcttctc ttttaaagaa gaaaaggtat 2880

aaatagatga acccgttttc ctaattggtt ttatctcttg tcagtatgtc tggttttgtg 2940

ggttttaaac catgttcaaa ttttaaccgg aaaaccaaac tagtatatat gtgtttgata 3000

ttgagttaat tacgctgttc aagtgtgcaa actccgatct tgattatggt ttgaacgttc 3060

taggttgatt cagatcaaga aattttgttg ttgaagaaat tggtaaaggt tatggaaaga 3120

aaagttgaac atctggaaca gcagcttgag aaatcgcata gctcttcagc accactcttc 3180

ggtgtaacaa atgatgatgt agaagggcca tcaatactcc taacaggtgg ccataatggc 3240

attaactggc tgtcatccct tgattcatat tgccctgcaa cggacatact agaaactcta 3300

atgccaatga gctcagcccg tgcatatgcg gctgttgcca cattaaagga ccatgtcttc 3360

atttttggtg gttggaatgg cattcgcagt ttgtggtaca acacaggtaa acattcttca 3420

atatgccaaa cgtagtgctc tatataagca agctgttgac catctgcatt tgttttattg 3480

cagtggagtg ctacaacagg ggagccaata agtggatagg attgccctgc ttgaatcatg 3540

agaaagggca tcttgctgga gctaccttga atggtaaaat atttgctatt ggtgggggtg 3600

atgggtctca gtctttttca gaagtagaga tgtttgatcc agcagtgggg aaatggatat 3660

acagtttgtc catgcagcaa cctgtatggt tctacttcta ctcccttata acacttgtat 3720

agagtagaat gtaccagcct ttttggccaa tttaataggg tcctggtctt cgctatgttc 3780

tcactgtgat tttcttgata aggctcaaga tttattagat ggcccaggaa aaaaacatgt 3840

tgtgaagaga ggctgtttga actgcatttc ctataatcac ttacatcttt gggttcttaa 3900

tcttttacaa gatgaatagt gctgctcaca gaacagtttc tactattatt tcaatcattt 3960

cttacgtgtc gtaaatcagt aaaaaagttt gttctcgtgg tgaactatgc tgagtgattt 4020

agaatttaga tagctgtttg agcagtagtg ggcaagctga ctgtgagttg gacattcgct 4080

tgagcactag tcttacaggg ttggttggac catttgcaaa tctaatctaa gctacatttt 4140

gagcacttga ataggcaata aatatttttc aaattcaggc tatttcaaat tatggaatgc 4200

aaattgcaat tcatgtaatt tcatgggatt tatgaggcat atacattgta actttccagt 4260

tcaccttgca tgaaatatta tcccatattt ttaatgtatt tttatttttg accaatatac 4320

ccacctcacc aacatgtatt ttattgttcc agcgatgtgc tcctgctgcg gctgaattaa 4380

atggcgttct ctatgtaatt ggtggttatg atggcaacat gtacttacag ttacgatcca 4440

ccctcaccat tctctccatg ctttacttga ttttgattct gagaccttcc tattgaatga 4500

ttgatcctca tatgcacagg tcagcagaaa ggtatgatcc aagggaaggc ttctggaccc 4560

aacttccacg tatgcggaca agaagaggat cccattcagt agttgtcttg ggggattcac 4620

tgtgagtttc gctcgcttta tctgtgtgtg taatcaacag agaggtcatg tgatttcaaa 4680

aaaacacaca cacagattcc acctggcagt tagatgctac tattagcctt atatcactat 4740

cttttatttg acgtctcgat ataaacctga taatatacga cttaacatcc aacatgggaa 4800

tttcattttc agacatgctc tgggtggcct gaatagaaat accacgtttt ccagcgtaga 4860

gatttttgac acccgtgcca actcatggag aagggggagc ccactcagcg tcccaagagc 4920

acacggatgt gcggttacat tggacggcaa cgcatacctc attggtggta tccaaagcag 4980

tgaagaatac gttgaaactg tgagttcttt cccaagatca ctctctccat catccattgc 5040

tgttgcttca actcgccgaa ctctcacatc cttctctcgt tttattttgt tttgtttttt 5100

atttcacagg ttgaggttta caaggagggc caaggctggt ccatctctgg ttccaaggca 5160

ttcgggaaga gagctttcgc atgcgccgtt gccatttgac aggattgcag aagtgcagat 5220

gaacccccgg gccggttttg tacagcacca gtccccaacc ccgcctcaca gtattacctt 5280

ctctcaagcg aaatattgcc gctcattgct gagcactgac gcctgacggc ccatccattc 5340

tgtgcatccc agtcatacgg tttttacatt ttagaaacaa caccgtagcg agttctggta 5400

tgcgttaact ctgcccgttg ttgtttctca ctgaagatag gtgttgttgg ttgtcacctg 5460

attgtagcgg agcaaactga actgtctaca ggacactagt tacttagaga agttcgaatg 5520

aacaaacttt agctggtgat agcagtacat atgggaggct tggaagctgg tataattttt 5580

gatttttttt ttgaagcaac ttttcaggtt ctgcttgctg ggagcaggcg ggaataatag 5640

ctgcaacaat ttcgccag 5658

<210> 2

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ggcagggtgc tacgaaaccc gc 22

<210> 3

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

aaacgcgggt ttcgtagcac cc 22

Claims

The application of the OsKEAP1 gene in regulation and control of rice seed phenotype is characterized in that the nucleotide sequence of the OsKEAP1 gene is shown as SEQ ID No. 1; the seed phenotype is that of a brown rice grain.
The application of the OsKEAP1 gene in regulation and control of the germination rate of rice seeds is characterized in that the nucleotide sequence of the OsKEAP1 gene is shown as SEQ ID No. 1.