CN112266920A - Application of OsKEAP1 gene in regulation and control of agronomic traits and yield of rice - Google Patents

Abstract

The invention discloses an application of OsKEAP1 gene in regulation and control of agronomic traits and yield of rice, wherein the nucleotide sequence of the OsKEAP1 gene is shown as SEQ ID No. 1; the agronomic characters are plant height, plant type, rice seed setting rate, average ear length and seed number. The invention obtains the mutant with the expression level of the OsKEAP1 gene reduced by using the CRISPR/Cas9 gene editing technology, finds that the OsKEAP1 gene is related to the agronomic characters and the yield of rice, such as the plant height, the plant type, the rice setting percentage, the average ear length, the seed grain number and the like, and can provide a basis for cultivating rice varieties with excellent agronomic characters.

Description

Application of OsKEAP1 gene in regulation and control of agronomic traits and yield of rice

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 agronomic characters and yield.

Background

Reactive Oxygen Species (ROS) are a generic term for oxygen-containing molecules or ions with 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 epithelial hydroxin (ECH) -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 plants, has not been reported to have an effect on rice growth and development and physiological characteristics of rice.

Due to the high accuracy and efficiency of CRISPR/Cas9 mutant generation, the method can be used for researching non-conventional target sites (such as UTR and intron sequences in a genome), so that researchers can research partially regulated gene functions instead of generating nonsense mutation by killing a coding region. 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 OsKEAP 15' 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 and control of rice agronomic traits, and provides a basis for breeding rice varieties with excellent agronomic traits.

The specific technical scheme is as follows:

the invention provides application of an OsKEAP1 gene in regulation and control of rice yield, wherein the nucleotide sequence of the OsKEAP1 gene is shown as SEQ ID No. 1; 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.

Further, the OsKEAP1 gene regulates the yield of rice by controlling the maturing rate of rice.

The invention provides application of an OsKEAP1 gene in regulation and control of rice agronomic traits, wherein the nucleotide sequence of the OsKEAP1 gene is shown as SEQ ID No. 1;

the agronomic characters are plant height, plant type, rice seed setting rate, average ear length and seed number.

The invention utilizes CRISPR/Cas9 gene editing technology to obtain the mutant with OsKEAP1 gene expression level reduced, and agronomic character detection finds that the mutant plant is obviously shorter than the wild type and has poorer fertility, but the spike number of the single plant is similar, the seed number of the single plant is obviously reduced and is only 35.4 percent and 58.6 percent of the wild type, the setting percentage is 45.13 percent and 61.76 percent respectively, and the setting percentage is obviously lower than 93.60 percent of the wild type. Possibly affected by the decrease in seed set rates, the cell yields of oskeap1-1 and oskeap1-2 were reduced by 62.2% and 39.3% compared to wild type. The average ear length of the two mutants was reduced by 22.9% and 7.7% compared to the wild type, respectively.

Further, the OsKEAP1 gene regulates the maturing rate of rice by controlling the fertility of rice pollen.

The invention analyzes the fertility of mutant line and wild pollen, and finds that: the pollen in the mutant has higher proportion of sterile pollen, no starch granules are shown under iodine staining, only yellow pollen shells are typical sterile pollen, which respectively accounts for 15.54 percent and 12.26 percent of the total pollen grains, while the sterile pollen rate of wild plants is only 3.86 percent.

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

the invention obtains the mutant with the expression level of OsKEAP1 gene down-regulated by using CRISPR/Cas9 gene editing technology, finds that the OsKEAP1 gene is related to agronomic characters and yield of rice, such as plant height, plant type, rice seed setting rate, average ear length, seed number and the like, and can provide basis for cultivating rice varieties with excellent agronomic characters.

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 OsKEAP 15' UTR region and the mutant at the post-editing target site.

FIG. 3 is a plant morphology map of Xidao #1(WT) and its oskeap1 mutant.

FIG. 4 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. 5 shows agronomic and yield traits for Xidao #1(WT) and its oskeap1 mutant; wherein denotes P < 0.05; denotes P < 0.01; student's t-test.

FIG. 6 shows pollen fertility of Xidao #1(WT) and its oskeap1 mutant.

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 amino acid sequence coded by the same 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 corn, human and other 10 related species were downloaded from the Gramene database, the OrthoDB database, the NJ method with boottrp repeat 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 KEAP1 homologous genes were identified in the higher coverage rice genome (c.v. nipponbare): os01g0162500 (sequence identity 54%) and Os05g0164900 (sequence identity 46%). In the NCBI database, the former is annotated as encoding a hypothetical protein containing a KELCH type beta propeller domain, while the latter is a protein containing a KELCH repeat type 1 domain, 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 OsKEAP 15' UTR is used as a target editing region, CRISPR-P2.0 (http:// CRISPR. hzau.edu.cn/CRISPR 2/) 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 OsKEAP 15' 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 annealing buffer (Biyun, Shanghai) 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

Xidao #1 (japonica rice variety) seeds are soaked in 70% ethanol for 30-60s, then are sterilized in a sterile 50mL centrifuge tube by shaking with 1% NaClO disinfectant for 30-40min (during which the disinfectant can be replaced once if the solution becomes turbid), then are washed 3-4 times with sterile water on a clean bench, transferred to absorbent filter paper and dried for 30-60min, and are cultured on a 2N6 callus induction 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 used to prepare a 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 further 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, opening the bottle cap, adding tap water, hardening for 3-5 days, growing in 1/4MS liquid culture medium for 1 week, and transplanting to soil to grow into plant.

TABLE 1 Co-cultivation Medium/liter

Note: 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. Taking a representative sample, and sequencing to determine the mutation genotype of the mutant individual. 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 and OsKEAP1, the abundance of OsKEAP1 transcripts in the mutant and its wild-type parent Xidao #1 was examined using qRT-PCR.

Wherein, seeds of a rice variety Xidao #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 adopting 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 centrifugal 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, transferring the filtrate in the collection tube to a new 1.5mL RNase-Free centrifuge tube (the cell debris is not precipitated as much as possible during the aspiration 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) washing solution RW1 with the recommended volume of absolute ethanol was 500. mu.L added to a centrifuge tube, 12,000

Centrifuging at rpm 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 removing residual genomic DNA from the buffer containing gDNA Eraser.

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 of both mutants were significantly lower than Xidao #1 at both seedling and flowering stages (FIG. 4). In contrast, the mutational effect of OsKEAP1-1 was more pronounced 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. 4A). 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. 4B).

4. Effect of oskeap1 mutations on agronomic traits

In the cowpea breeding station of Hangzhou, there is no transgenic T₁Selecting non-transgenic homozygous mutant T from plant₂And carrying out agronomic character detection on the strain and the wild parent Xidao #1 in the field. These two mutant lines and their parent Xidao #1 were grown in three replicates designed at random. Lines 48 (6X 8) per replicate cell. And the agronomic characters such as tillering number, plant height, thousand seed weight, seed setting rate and the like are collected in the mature period of the seeds. Pollen fertility was studied by iodine staining and observed under a microscope. The cell yield is the total yield of grain per iteration.

The 2 mutants of oskeap1 did not differ significantly from the wild type parent before heading. After heading, the mutant plants are obviously shorter than wild plants (figure 3), and have poorer fertility, but the number of ears of each plant is similar. The number of ears per plant of oskeap1-1 and oskeap1-2 was similar to that of the wild type (FIG. 5A), but the plant heights were reduced by 11.8% and 10.0%, respectively, and the differences were all significant (FIG. 5B). The number of seeds per plant was significantly reduced for both mutants, only 35.4% and 58.6% of the wild type (fig. 5C), with results of 45.13% and 61.76%, respectively, significantly lower than 93.60% of the wild type (fig. 5D). Possibly affected by the decrease in seed set rates, the cell yields of oskeap1-1 and oskeap1-2 were reduced by 62.2% and 39.3% compared to wild type (FIG. 5E). The average ear length of the two mutants was reduced by 22.9% and 7.7% compared to the wild type, respectively (fig. 5F).

5. Effect of oskeap1 mutation on pollen development

To clarify the cause of the decrease in the fruit set rates of oskeap1-1 and oskeap1-2, the fertility of mutant and wild-type pollen was further analyzed.

The pollen fertility is investigated by an iodine staining method, and the specific method comprises the following steps:

(a) preparing a 1% iodine-potassium iodide solution: add ddH to 100mL beaker₂About 70mL of O, 8g of potassium iodide was added, and after stirring and dissolving at room temperature, 1g of iodine was added to transfer the solutionTo 100mL volumetric flask, ddH for beaker₂And (4) rinsing twice, pouring rinsing water into the volumetric flask, fixing the volume of the solution to 100mL, and shaking uniformly. The solution is stored in dark place.

(b) Utilizing 1% iodine-potassium iodide solution to investigate pollen fertility, collecting anther experiment: taking a flowering material of rice in a heading period, generally taking the rice ear, namely putting glumes flowering the next day into FAA stationary liquid (worker, Shanghai), respectively taking glumes with uniform growth vigor at the upper part, the middle part and the lower part of the rice ear, and taking a sampling standard of mature anther, wherein the growth length of the anther exceeds 2/3 of the upper and lower lengths in glumes.

(c) Observing pollen fertility by a microscope: placing anthers of one glume flower on the upper, middle and lower parts of the rice ear on a glass slide, dripping 20-40 mu L of 1% iodine-potassium iodide solution on the anthers of the glass slide, lightly pounding the anthers by using the tips of tweezers, releasing pollen grains into the iodine-potassium iodide solution, covering a cover glass, lightly pressing the cover glass by using the tweezers, and observing the shape and dyeing condition of pollen. The 10-fold microscope can be used for counting anther fertility observation statistics. Randomly selecting visual field observation, wherein the visible pollen grains counted in each visual field are not less than 200.

It was found that a higher proportion of sterile pollen appeared in the pollen of both mutants, showing no starch grains under iodine staining, only yellow pollen shells, typical pollen, accounting for 15.54% and 12.26% of the total pollen grains, respectively, whereas the sterile pollen rate of the wild type plants was only 3.86% (fig. 6).

Protein structure analysis of OsKEAP1 revealed that, unlike human KEAP1, which contains one BTB and BACK domain, it contains one DCD domain unique to plants, and 5 KELCH structures (FIG. 1B).

To analyze the conservation of KEAP1 in plants, a KEAP1 homologous gene was further identified in 9 plants such as maize. As a result, the proteins encoded by these homologous genes were found to have a DCD domain and 5-6 KELCH structures, similar in number to the amino acids of OsKEAP1 (FIG. 1). This indicates that the KEAP1 homologous gene is highly conserved in plants.

The OsKEAP1 rice mutant has important influence on the growth and development of rice, and is mainly shown to influence the rice pollen fertility and a series of related agronomic traits. Both oskeap1-1 and oskeap1-2 negatively affected rice growth and development, but the former effect was significantly greater than the latter, consistent with the expression being down-regulated to a greater extent than the latter. These results indicate that the trait deterioration observed in both mutants in this experiment is a manifestation of the oskeap1 mutational effect at the phenotypic level, and not due to other variations such as clonal variations. The CRISPR/Cas9 editing of the 5' UTR region provides an effective way for realizing partial down-regulation of gene expression level but not incomplete knockout through gene editing in the research of important (knockout lethal) gene biological functions so as to partially regulate and control rice-related growth and yield traits.

Sequence listing

<110> Zhejiang university

Application of OsKEAP1 gene in regulation and control of agronomic traits and yield of rice

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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 (4)

1. The application of the OsKEAP1 gene in regulation and control of rice agronomic traits is characterized in that the nucleotide sequence of the OsKEAP1 gene is shown as SEQ ID No. 1;

   the agronomic characters are plant height, plant type, rice seed setting rate, average ear length and seed number.
2. 2. The use of claim 1, wherein the OsKEAP1 gene regulates rice setting rate by controlling rice pollen fertility.
3. The application of the OsKEAP1 gene in regulation and control of rice yield is characterized in that the nucleotide sequence of the OsKEAP1 gene is shown as SEQ ID No. 1.
4. 4. The use of claim 3, wherein the OsKEAP1 gene regulates rice yield by controlling rice seed set percentage.

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