Application and method of gene OsSV5 and protein coded by gene OsSV5 in regulation and control of rice seed vigor

Abstract

The invention relates to the field of plant seed biology and rice breeding, in particular to application of a gene OsSV5 and protein coded by the gene OsSV5 to regulation and control of rice seed vigor and a method. According to the application of the gene OsSV5 in regulation and control of the activity of the rice seeds, the OsSV5 gene can positively regulate and control the activity of the rice seeds, the whole genome sequence of the OsSV5 gene is as shown in SEQ ID NO: 1, and the CDS sequence of the OsSV5 gene is as shown in SEQ ID NO: 2. The invention also provides application of the protein coded by the gene in regulating and controlling the activity of rice seeds and a method for screening or cultivating high-activity rice seeds. In the application and the method, the expression level of the gene is positively correlated with the activity of rice seeds.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant seed biology and rice breeding, and particularly relates to a gene OsSV5 and an application and a method of a protein encoded by the gene in regulating rice seed vigor. BACKGROUND

[0002] Seed vigor refers to the sum of the germination and emergence rate of seeds, the potential of plant growth, the ability of stress resistance and the production potential under suitable environmental conditions. Seed vigor not only reflects the physiological state and potential production capacity of seeds, but also is a standard for measuring the quality of seeds. Rice varieties with high vigor can improve seed germination and field seedling growth under stress conditions, guarantee grain yield, and are more suitable for mechanized seeding and seedling raising, thereby saving production costs. Improving seed vigor is one of the key strategies for improving crop productivity and adaptability. However, there are few genes related to seed vigor in rice, and the molecular mechanisms are unclear.

[0003] Therefore, in-depth analysis of the genetic basis of seed vigor, systematic exploration of quantitative trait loci (QTL) and key genes related to rice seed vigor can provide novel gene resources and technical paths for molecular design breeding. SUMMARY

[0004] The technical problem to be solved by the present application is to overcome the deficiencies and defects mentioned in the background, and to provide a gene OsSV5 and an application and a method of a protein encoded by the gene in regulating rice seed vigor.

[0005] The technical scheme is that the gene OsSV5 in regulating rice seed vigor, OsSV5 The gene positively regulates rice seed vigor, and the whole genome sequence is shown as SEQ ID NO: 1, and the CDS sequence is shown as SEQ ID NO: 2.

[0006] In an embodiment, the knockout OsSV5 The gene reduces the seed vigor of rice, so that the rice vigor phenotype is significantly reduced relative to the wild type.

[0007] In an embodiment, OsSV5 The phenotype of the gene positively regulating rice seed vigor includes the germination rate, germination index, germination potential, root length and / or bud length of rice seeds.

[0008] Based on the same inventive concept, an application of a protein encoded by the gene in regulating rice seed vigor is provided, and the gene is the above-mentioned OsSV5 The amino acid sequence of the protein is shown as SEQ ID No. 3.

[0009] Based on the same inventive concept, a method for screening or breeding high-vigor rice seeds is provided, which detects whether the haplotype of the gene in the variety is Hap3. OsSV5 Based on the same inventive concept, a method for screening or breeding high-vigor rice seeds is provided, which detects whether the haplotype of the gene in the variety is Hap3.

[0010] Compared with the prior art, the present application has the following beneficial effects: The present application provides a gene OsSV5 In the application of regulating the seed vigor of rice, the expression level of the gene is positively correlated with the seed vigor of rice, and knocking out the coding gene can significantly reduce the seed vigor of rice, which is significantly lower than the wild type in germination rate, germination index, and root length and shoot length after germination, indicating that the seed vigor of rice is significantly reduced. The present application provides a method for screening or breeding high-vigor rice seeds, which detects whether the haplotype of the gene in the variety is Hap3. OsSV5 The present application provides a gene BRIEF DESCRIPTION OF DRAWINGS

[0011] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings described below are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0012] Figure 1 For OsSV5 Schematic diagram of construction of gene editing vector, wherein A: gel electrophoresis imaging of first round PCR result; B: gel electrophoresis imaging of second round PCR result; C: peak graph comparison identification of target 1 sequencing result; D: peak graph comparison identification of target 2 sequencing result.

[0013] Figure 2 For Nipponbare as background OsSV5 Schematic diagram of standard germination test of seed of gene knockout mutant; wherein A: wild type and OsSV5 Rice seedling phenotype of standard germination test of seed of gene knockout mutant on day 7, scale = 1 cm; B: wild type and OsSV5 Rice germination of standard germination test of seed of gene knockout mutant on day 7, scale = 2 cm; C: wild type and OsSV5 Bar chart of germination rate of standard germination test of seed of gene knockout mutant (n = 3); D: wild type and OsSV5 Bar chart of germination energy of standard germination test of seed of gene knockout mutant (n = 3); E: wild type and OsSV5Figure 6: Standard germination test of knockout mutant seeds. A: Germination index of standard germination test of knockout mutant seeds (n=3); B: Wild type vs. knockout mutant seeds; C: Vigor index of standard germination test of knockout mutant seeds (n=3); D: Wild type vs. knockout mutant seeds; E: Root length of standard germination test of knockout mutant seeds (n=10); F: Wild type vs. knockout mutant seeds; G: Shoot length of standard germination test of knockout mutant seeds; H: Wild type vs. knockout mutant seeds. OsSV5 Figure 6: Standard germination test of knockout mutant seeds. A: Germination index of standard germination test of knockout mutant seeds (n=3); B: Wild type vs. knockout mutant seeds; C: Vigor index of standard germination test of knockout mutant seeds (n=3); D: Wild type vs. knockout mutant seeds; E: Root length of standard germination test of knockout mutant seeds (n=10); F: Wild type vs. knockout mutant seeds; G: Shoot length of standard germination test of knockout mutant seeds; H: Wild type vs. knockout mutant seeds. OsSV5 Figure 6: Standard germination test of knockout mutant seeds. A: Germination index of standard germination test of knockout mutant seeds (n=3); B: Wild type vs. knockout mutant seeds; C: Vigor index of standard germination test of knockout mutant seeds (n=3); D: Wild type vs. knockout mutant seeds; E: Root length of standard germination test of knockout mutant seeds (n=10); F: Wild type vs. knockout mutant seeds; G: Shoot length of standard germination test of knockout mutant seeds; H: Wild type vs. knockout mutant seeds. OsSV5 Figure 6: Standard germination test of knockout mutant seeds. A: Germination index of standard germination test of knockout mutant seeds (n=3); B: Wild type vs. knockout mutant seeds; C: Vigor index of standard germination test of knockout mutant seeds (n=3); D: Wild type vs. knockout mutant seeds; E: Root length of standard germination test of knockout mutant seeds (n=10); F: Wild type vs. knockout mutant seeds; G: Shoot length of standard germination test of knockout mutant seeds; H: Wild type vs. knockout mutant seeds. P <0.05, ** indicates P <0.01, *** indicates P <0.001, *** indicates P <0.0001, ns indicates no significant difference.

[0014] OsSV5 Figure 7: Expression level of gene in different tissues of Nipponbare; A: qPCR analysis of gene in different tissues of Nipponbare; B: GUS protein staining of gene in different tissues of Nipponbare; the part stained blue indicates Figure 3 Figure 7: Expression level of gene in different tissues of Nipponbare; A: qPCR analysis of gene in different tissues of Nipponbare; B: GUS protein staining of gene in different tissues of Nipponbare; the part stained blue indicates OsSV5 Figure 7: Expression level of gene in different tissues of Nipponbare; A: qPCR analysis of gene in different tissues of Nipponbare; B: GUS protein staining of gene in different tissues of Nipponbare; the part stained blue indicates P <0.05; B: GUS protein staining, the part stained blue indicates OsSV5 Figure 7: Expression level of gene in different tissues of Nipponbare; A: qPCR analysis of gene in different tissues of Nipponbare; B: GUS protein staining of gene in different tissues of Nipponbare; the part stained blue indicates

[0015] OsSV5 Figure 8: Sequence of Hap3 haplotype of gene and sequence of SEQ ID NO: 2. Figure 4 Figure 8: Sequence of Hap3 haplotype of gene and sequence of SEQ ID NO: 2.

[0016] OsSV5 Figure 8: Sequence of Hap3 haplotype of gene and sequence of SEQ ID NO: 2. Figure 5 Figure 8: Sequence of Hap3 haplotype of gene and sequence of SEQ ID NO: 2. DETAILED DESCRIPTION

[0017] In order to facilitate the understanding of the present application, the present application will be described in more detail below in conjunction with the accompanying drawings and preferred embodiments, but the scope of protection of the present application is not limited to the following specific embodiments.

[0018] Unless otherwise defined, all the professional terms used herein have the same meaning as commonly understood by those skilled in the art. The professional terms used herein are only for the purpose of describing the specific embodiments and are not intended to limit the scope of protection of the present application.

[0019] Unless otherwise specified, the various raw materials, reagents, instruments and equipment used in the present application can be purchased from the market or can be prepared by existing methods.

[0020] The application of a kind of regulation and control of rice seed vigor gene and its encoded protein will be described in detail below with examples and experimental data.

[0021] Example 1 This example uses Crispr-Cas9 technology to edit the target gene, obtain OsSV5 gene knockout mutant lines. OsSV5

[0022] (1) Preparation of early work According to the design principles of gene CRISPRY / Cas9 gene editing vector target, using online tool CRISPR-PV2.0 (http: / / crispr.hzau.edu.cn / CRISPR2 / ), combined with OsSV5 the base sequence of the gene, the sgRNA of the gene was designed. When selecting primers, ensure that the GC content of the primers is between 50% and 60%, the off-target rate is less than 100 (the lower the better), there are no 4 T bases in the target site, and the target site is selected on the conserved domain of the gene. And use online tool NCBI to detect the specificity of the target, synthesis. The primer sequences of target 1 and 2 are shown in the following table: OsSV5 Table 1 Primer sequences of target 1 and 2 OsSV5

[0023] The primer sequences of M-F, gR-R, Pps-GGL and Pgs-GGR are the construction of Crispr-Cas9 universal primers, and the sequences are shown in the following table: Table 2 Primer sequences of M-F, gR-R, Pps-GGL and Pgs-GGR

[0024] The strain pYLCRISPR / Cas9-MTmono stored at-80℃ in the laboratory was thawed on ice, and then transferred to LB solid medium containing 50 μg / mL Kan+. The CRISPR / gRNA vector strain was thawed on ice and then transferred to LB solid medium containing 100 μg / mL Amp+. Both were cultured overnight in a constant temperature incubator at 37.5℃. Single colonies were picked and inoculated in LB liquid medium containing the corresponding antibiotics for expansion, and the plasmid was extracted.

[0025] (2) Construction of sgRNA expression cassette Adapter primer: 1 μL of 2 μM adapter primer was placed in a PCR tube, 90℃, 30s, room temperature annealing.​​​

[0026] Table 3 grna expression cassette ligation reaction system

[0027] The reaction procedure is 37 ℃ for 5 min, 20 ℃ for 5 min, 5 cycles, and two rounds of nested PCR reaction are performed immediately after the PCR is completed.

[0028] The first round of PCR reaction system is shown in the following table: Table 4 First round of PCR reaction system

[0029] The above two PCR reactions are performed simultaneously, and the reaction procedure is 95 ℃, 1 min, then 25 cycles of 95 ℃, 10 s, 60 ℃ annealing for 15 s, 68 ℃ extension for 30 s, 68 ℃ continuous extension for 5 min, and an appropriate amount of PCR product is taken for electrophoresis observation.

[0030] The second round of PCR reaction system is shown in the following table: Table 5 Second round of PCR reaction system

[0031] The reaction procedure is 94 ℃ for 2 min, 94 ℃ for 10 s, 60 ℃ for 30 s, 68 ℃ for 50 s, 25 cycles, then 68 ℃ for 5 min. The PCR amplification product is electrophoresed in 1% agarose, and the target fragment is purified using a PCR product gel recovery kit for use as a double vector and sgRNA expression cassette end-to-end ligation reaction system as shown in the following table: Table 6 Double vector and sgRNA expression cassette end-to-end ligation reaction system

[0032] The end-to-end ligation reaction procedure is 37 ℃ for 5 min, 10 ℃ for 5 min, 20 ℃ for 5 min, a total of 13 cycles; finally 20 ℃ for 5 min, and finally the CRISPR / Cas9 gene knockout vector is obtained.

[0033] (3) Agrobacterium plasmid transformation The GV3101 Agrobacterium competent cells stored at -80 ℃ are partially thawed on ice, and then inserted into ice to thaw when they are in ice water mixture; Take 3 μL of the constructed CRISPR / Cas9 gene knockout vector into a 1.5 mL EP tube containing 50 μL of Agrobacterium competent cells, mix well, and sequentially stand for 5 min on ice, 5 min in liquid nitrogen, 5 min in a 28°C water bath, and 5 min in an ice bath for recovery; Add 700 μL of LB liquid culture and incubate at 28°C for 3 h; after sufficient incubation, collect the bacteria at 6000 rpm for 1 min, take 100 μL and mix well on LB medium containing the corresponding antibiotics, and invert in a 28°C constant temperature incubator for 3 d.

[0034] Select a single colony and add it to LB liquid medium containing kanamycin (Kan) and rifampicin (Rif), and place it in a 28°C shaker at a speed of 200 rpm for 3 d.

[0035] (4) Transgenic rice transformation Use Nipponbare as the genetic receptor material, and send the Agrobacterium liquid after transformation to Wuhan Boyuan Biotechnology Co., Ltd. for genetic transformation: use mature rice embryos as materials, induce rice embryonic callus as genetic transformation receptor material, and use Agrobacterium carrying OsSV5 the gene vector to infect the receptor material, insert T-DNA into the genome, and obtain independent resistant callus through corresponding antibiotic selection, further differentiation and regeneration, and obtain transgenic positive plants.

[0036] (5) Genotype identification of gene knockout transgenic rice, use gene knockout transformation seedlings as templates, design primers to detect Cas9 and target points, respectively, for PCR amplification, and the primer sequences are shown in the following table: Table 7 Target-1-F, Target-1-R, Target-2-F, Target-2-R primer sequences

[0037] PCR amplification: the target fragment is amplified by PCR method, and the PCR reaction system is shown in the following table: Table 8 PCR reaction system

[0038] PCR reaction program: 98°C for 3 min, 98°C for 15 s, 60°C for 15 s, 72°C for 30 s, 72°C for 5 min, 16°C for 10 min, 35 cycles. After electrophoresis of the PCR products, the remaining PCR stock solution and corresponding candidate gene identification primers were observed and sent to Hunan Branch of Beijing Qingke Biotechnology Co., Ltd. for sequencing. SeqMap was used to compare the sequencing results, and homozygous lines with base mutations were selected as CRISPR / Cas9 gene-edited transgenic rice for seed vigor phenotypic identification.

[0039] Example 2 This embodiment performs OsSV5 Seed viability assessment of knockout lines (1) Standard germination experiment, OsSV5 Knockout strains and wild-type Nipponbare strains were planted simultaneously in a field with the same water and fertilizer conditions. Seeds from each strain with the same maturity were harvested, naturally dried, and then the viability of the rice seeds was assessed through a germination experiment. One hundred plump seeds of uniform maturity were randomly selected, submerged in distilled water, and placed in a 37 ℃ incubator for 1 day. They were then evenly placed on germination paper moistened with distilled water, and then placed in seed germination petri dishes in a light incubator (30 ℃, 16 hours light / 8 hours darkness). Three replicates were set up, and the germination status of the seeds was observed and photographed daily for 7 consecutive days. Germination rate, germination index, germination potential, vigor index, root length, and shoot length were then examined as rice seed vigor indicators.

[0040] (2) Statistical analysis of seed vigor-related phenotypic data Germination was defined as the radicle exceeding the length of the entire seed and the plumule exceeding half the length of the seed. Germination was recorded for 7 consecutive days after placement in the incubator. The germination rate was calculated as the percentage of germination on day 7 divided by the total number of seeds. Vigor index = seedling growth × germination index; Germination potential = number of seeds germinating at peak germination / number of tested seeds × 100%. Seed storage tolerance was measured by the difference between the initial germination rate and the germination rate after aging; the larger the difference, the worse the storage tolerance. The germination index was calculated using the formula GI = ∑Gt / Dt, where Gt is the daily germination count, Dt is the number of germination days, and ∑ is the sum. On day 7 of germination, representative rice seedlings with uniform germination were selected to measure root and shoot lengths.

[0041] (3) Germination phenotype record For wild-type Nipponbare and two OsSV5 Germination phenotypes were compared between gene knockout lines (sv5-1 and sv5-2). Two... OsSV5 Germination potential, germination rate, germination index, and vigor index of gene knockout lines (sv5-1, sv5-2), as well as root length and shoot length, were significantly reduced compared to wild-type Nipponbare, indicating...OsSV5 Knockout of the gene has a significant impact on seed vigor. The results are shown in OsSV5 OsSV5 The gene positively regulates rice seed vigor, and loss of function leads to reduced rice seed vigor.

[0042] Example 3 This example analyzes Figure 2 the expression level of the gene in each tissue of Nipponbare.

[0043] (1) The expression level of the gene in each tissue of Nipponbare was detected by fluorescence quantitative PCR (qRT-PCR) technology. OsSV5

[0044] 1. Extraction of RNA from each part of rice and reverse transcription Reagents for extracting RNA from rice plants: "Seed RNA extraction solution" is used in combination with Trizol or RNA extraction kit, and corresponding experimental protocols are adopted according to the part of the extracted RNA. The specific experimental steps are as follows: Take about 0.1 g of rice seeds in an RNase-Free 2.0 mL centrifuge tube, add 300 μL of seed RNA extraction solution, grind into homogenate with a grinding rod, and shake well to mix; add 300 μL of phenol solution ((phenol PH=8.0): chloroform volume ratio=1:1), shake well to mix; centrifuge at 13000 rpm for 5 min at room temperature, and transfer the supernatant to a centrifuge tube.

[0045] Trizol method extraction: after adding 1 mL Trizol, shake well to mix, and let stand at room temperature for 20 min, then add 200 μL of chloroform, shake well to mix again; centrifuge at 1300 rpm for 10 min at room temperature, take 700 μL of supernatant to a new centrifuge tube, and add an equal volume of isopropanol to mix well, centrifuge at 13000 rpm for 10 min at room temperature, discard the supernatant; add 1 mL of 75% ethanol (DEPC treated) for cleaning, centrifuge at 13000 rpm for 5 min at room temperature, discard the supernatant; blow dry on a clean bench, then add 50 μL of ddH2O to dissolve.

[0046] 2. Reverse transcription of rice RNA The operation is carried out according to the instructions of Gen Star's RNA reverse transcription kit, and the reaction system is shown in the following table: Table 9 Reaction system of Gen Star's RNA reverse transcription kit

[0047] ​​The reaction procedure is: after mixing and short centrifugation, 37 ℃ reaction for 5 min in a PCR instrument; in the above reaction tube, directly add other required components for reverse transcription, and perform the first strand cDNA synthesis step, and the reaction system is shown in the following table: Table 10 Reverse transcription reaction system

[0048] After mixing, centrifugation for 10 s, incubation at 42 ℃ for 15-50 min using a PCR instrument; inactivation of StarScript II Enzyme Mix after heating at 85 ℃ for 5 min; the cDNA obtained after the reaction can be measured for concentration using an ultramicro spectrophotometer, and then placed on ice for subsequent experiments or frozen.

[0049] (2) Real-time fluorescent quantitative PCR The cDNA of the roots, stems, leaves and seeds of the rice variety Nipponbare extracted in the above experiment is used as a PCR template, and the expression level of the endogenous gene of rice Eef1a is used as a reference. The gene expression amount is investigated by relative quantification method, and the relative expression level is evaluated by ΔΔCt calculation method. Each sample is set with three repeats, and the experiment is repeated three times. The primer sequences used for quantification are shown in the following table: Table 11 eEF1a-F, eEF1a-R, OsSV5 -F, OsSV5 -R primer sequences

[0050] The kit involved in the experiment and the qRT-PCR instrument are Roche FastStart Universal SYBR Green Master (ROX) and ABI ViiA7 Real-time PCR System, respectively. The qRT-PCR reaction system is shown in the following table: Table 12 qRT-PCR reaction system

[0051] The reaction condition of qRT-PCR is: 95 ℃ reaction for 10 min, 95 ℃ reaction for 10 s, 60 ℃ reaction for 40 s, 40 cycles.

[0052] (3) Detection by GUS staining OsSV5 Expression of the gene in each tissue of Nipponbare.

[0053] 1. Extraction of rice genomic DNA This study used rice leaves as experimental material and extracted total rice DNA using CTAB. Fresh rice leaves approximately 3 cm in size were placed in 2.0 mL centrifuge tubes, and steel balls were added. After quick-freezing in liquid nitrogen, the leaves were ground into powder. 600 µL of 2% CTAB (preheated to 65 °C) was added, and the mixture was vortexed and incubated at 65 °C for 30 min. 700 µL of chloroform was added, and the mixture was centrifuged at 12000 rpm for 8 min. The supernatant was transferred to a 1.5 mL EP tube (pre-filled with 700 µL of isopropanol), vortexed, and incubated at 4 °C for 20 min. The tube was centrifuged at 12000 rpm for 8 min, and the supernatant was discarded (the white flocculent material should not be poured out). At this point, the DNA adhered to the bottom of the centrifuge tube. 600 µL of 75% ethanol was added, and the flocculent material was agitated. The tube was centrifuged at 12000 rpm for 5 min. Washing with 75% ethanol was repeated. After draining the ethanol and allowing it to air dry, add 50 µL of ultrapure water to fully dissolve the DNA. Measure the concentration using an ultra-micro spectrophotometer and store at -80 °C for long-term storage.

[0054] 2. PCR amplification OsSV5 Gene promoter fragments Gene design using online software Primer3Plus OsSV5 The promoter fragment PCR amplification primers were determined, and primer specificity was detected using the BLAST tool of NCBI online tool. The primer sequences are shown in the table below: Table 13 Primer sequences for Sv5-qi-F and Sv5-qi-R

[0055] PCR amplification was performed using genomic DNA from Nipponbare rice as a template, and the reaction was carried out using DNA amplification enzyme from Nanjing Novizan Biotechnology Co., Ltd. The reaction volume was 50 µL, and the specific components are shown in the table below: Table 14 PCR amplification reaction system

[0056] The reaction procedure was as follows: pre-denaturation at 95 °C for 5 min, denaturation at 95 °C for 15 s, annealing at 55 °C for 20 s, extension at 72 °C for 40 s, 35 cycles, followed by extension at 72 °C for 5 min, and storage at 16 °C.

[0057] 3. Recovery of PCR amplification products All the amplification products from the above PCR reaction were spotted onto a 1% agarose gel. After electrophoresis for 20 min, the separated target band was observed and recovered using a gel imaging system. The recovered band was then placed in a 2.0 mL centrifuge tube and extracted using the Magen Agarose Gel DNA Extraction Kit.OsSV5 The specific steps for gene recovery are as follows: The gel fragment containing the target gene was transferred to a 2.0 mL centrifuge tube, and an equal volume of Binding Buffer was added until the target gel fragment was completely submerged. The mixture was incubated in a 55 °C water bath for 15 min to allow the DNA gel to completely dissolve, then cooled to room temperature. The gel solution was transferred to a centrifuge column and centrifuged at 12000 rpm for 1 min. The filtrate was discarded, and 750 µL of SPW Buffer was added, followed by centrifugation at 12000 rpm for 1 min. The filtrate was discarded, and the centrifuge column was recycled, running empty at 12000 rpm for 2 min. The column was transferred to a new 1.5 µL centrifuge tube, and 20 µL of ddH₂O was added to the center of the adsorption membrane. After standing at room temperature for 2 min, the solution was centrifuged at 12000 rpm for 1 min. The concentration of the resulting solution was measured using a micro spectrophotometer and stored at -20 °C for later use.

[0058] 3. OsSV5 Construction of the gene GUS vector The Pro35::GUS fusion vector was constructed using homologous recombination. The selected vector was pCAMBIA1381Xa, which has a set of multiple cloning sites. Analysis was performed... OsSV5 The restriction enzyme sites within the gene are selected only if they exist on the vector and not in the genome. OsSV5 Homologous recombination primers were designed using the two vector-compatible restriction enzyme sites, EcoI and BamHI, on the gene fragment. The synthesized homologous recombination primers were used to amplify and recover the purified target fragment. The primer sequences are shown in the table below: Table 15 Primer sequences of B-Sv5-qi-F and H-Sv5-qi-R

[0059] The vector was digested using the corresponding restriction endonucleases for the EcoI and BamHI restriction sites. The reaction mixture (50 µL) was prepared in triplicate, as shown in the table below. Table 16 Endonuclease Digestion Reaction System

[0060] The reaction conditions were as follows: after reacting in a water bath at 37 ℃ for 2.5 h, all the product was spotted onto a 1% agarose gel for electrophoresis. The products from the three tubes were then mixed and purified to create the target linearized vector. The purified DNA fragment was ligated with the pCAMBIA1381Xa digested empty vector. The reaction system is shown in the table below. Table 17 Reaction system for ligating DNA fragments with pCAMBIA1381Xa digested empty vector

[0061] Reaction condition: After 1 h water bath at 50 °C, use Trelief.5a of Beijing Chengke Biological Technology Co., Ltd. Hunan Branch to take the competent cells out of the -80 °C environment, dissolve them on ice, then add 20 μΐ of the ligation product, mix gently, stand on ice for 5 min, then use a metal water bath at 42 °C for 45 s, quickly put it on ice after the heat shock is over, stand for 2 min (to ensure the transformation efficiency, do not shake it during the standing process); add 500 μΐ of pre-packaged sterile liquid LB liquid medium, mix thoroughly, then recover it in a 37 °C, 200 rpm shaker for 15 min; take 200 μΐ of the recovered liquid and plate it on LB solid medium containing the corresponding antibiotic, and incubate it in a 37 °C incubator overnight. After 16 h of culture, pick a single colony from the plate, add LB liquid medium containing the corresponding antibiotic, and culture for no more than 24 h. The turbidity of the LB liquid medium can be used as a rough estimate.

[0062] Use the purchased plasmid extraction kit (NO. omega; REF: D6943-02) to extract the completed culture of the chromobacterium plasmid. The specific steps for plasmid extraction are as follows: Transfer the completed 5 ml bacterial liquid culture into 2 ml EP tubes in two batches, centrifuge at 10,000 rpm at room temperature for 60 s to collect the bacterial cells and discard the upper LB liquid medium, add 250 μΐ of bacterial lysis solution (Solution I / RNaseA), and invert the mixture several times to completely suspend the bacterial cells; Add 250 μΐ of Solution II to the resuspended bacterial mixture after lysis, mix well by inverting the mixture 10 times. Then add 350 μΐ of Solution III, invert it 10 times to form a white flocculent bacterial precipitate. Centrifuge at 12,000 rpm at room temperature for 10 min using a horizontal centrifuge; Use a 1 ml pipette to transfer the supernatant after centrifugation to a DNA binding column with a collection tube, centrifuge at 12,000 rpm at room temperature for 1 min using a horizontal centrifuge, and discard the filtrate in the collection tube; Put the DNA binding column back into the collection tube, add 500 μΐ of HBC Buffer diluted with isopropyl alcohol in advance, centrifuge at 12,000 rpm at room temperature for 1 min, and discard the filtrate in the collection tube; Put the binding column back into the collection tube, in order to elute the mixed proteins and other organic substances, add 700 μΐ of DNA Wash Buffer diluted with anhydrous ethanol in advance for elution, centrifuge at 12,000 rpm at room temperature for 60 s, and discard the filtrate in the collection tube; After centrifuging for 60 seconds, reassemble the binding column into the collection tube and centrifuge at 12,000 rpm to remove any residual alcohol from the column and prevent damage to the plasmid DNA. Finally, place the binding column containing the target strain plasmid into a new 1.5 mL centrifuge tube, add 35 µL of Elution Buffer to the column matrix, let stand for 1 min, and centrifuge horizontally at 13,000 rpm for 60 seconds to elute the plasmid DNA. The extracted DNA plasmid concentration was measured using a microspectrophotometer (Beijing Kai'ao Technology Development Co., Ltd., K5600). The plasmid was then stored at 20℃ for an extended period, and 10 µL of plasmid was sent to the Hunan branch of Beijing Qingke Biotechnology Co., Ltd. for sequencing. OsSV5 The plasmid with the correct gene was sent to Wuhan Boyuan Biotechnology Co., Ltd. for genetic transformation using NPB as the background material.

[0063] 4. GUS staining of rice plants First, the X-gluc solvent from the GUS staining kit (Coollabo, SL7160) was mixed with X-Gluc powder to prepare a concentrated chromogenic solution. This concentrated solution was then mixed with buffer to prepare the GUS staining solution (prepare fresh before use; the stock solution can be stored at -80℃ for long-term storage). The specific staining steps are as follows: The prepared plant tissues and seeds were immersed in the GUS staining solution, and a vacuum was applied for 20 minutes to ensure thorough staining. Afterward, they were placed in a 37℃ oven for 1 hour, or even overnight. The stained plant tissues were then destained using 70% alcohol, repeated three times, until the negative control became colorless. The presence of blue spots on the plant tissues and seed embryos, observed visually, indicates the presence of GUS activity in these tissues, confirming the presence of GUS. OsSV5 The gene is expressed in this tissue. The stained material was then observed and photographed under a stereomicroscope (ZEISS, Smart zoom 5) to obtain local images of the stained tissue. The results are as follows: OsSV5 As shown, GUS staining results revealed its expression characteristics in tissues: roots > leaves > stems, with the lowest expression level in rice stems. This result is consistent with the results of quantitative real-time PCR (qPCR) analysis. Because... Figure 3 Higher expression characteristics in roots and seeds, and OsSV5 The function of regulating rice seed vigor is consistent, providing evidence at the expression level for its participation in seed vigor-related regulation.

[0064] Example 4 This embodiment analyzes OsSV5 Genetically modified rice seeds with excellent vigor haplotypes.

[0065] Data was selected based on data published on the database website of Huazhong Agricultural University (http: / / ricevarmap.ncpgr.cn / ). OsSV5 All non-synonymous mutant SNP sites in the upstream 2 Kb promoter region, exons, introns, and downstream 1 Kb promoter region of the gene were screened. SNP sites with a primary allele frequency ≤80% and a DELF frequency ≤8% were selected, resulting in three different haplotypes (≥20 materials). Germination rate, germination index, germination potential, vigor index, and storage tolerance phenotypic data from existing 3K rice core germplasm measurements were also analyzed. (See [link to relevant documentation]). OsSV5 One-way ANOVA Figure 4 The differences in seed vigor phenotypic values ​​among the three haplotypes were used to ultimately select Hap3 as the most viable seed vigor phenotypic type. OsSV5 A haplotype with excellent seed viability. See also: OsSV5 , Figure 5 The key sequence characteristic of the Hap3 haplotype of the gene is that the nucleotide sequence shown in SEQ ID NO: 2 contains specific alleles at the following single nucleotide polymorphism (SNP) sites: GGCGA at site 67 (relative to the start codon ATG) (G for other haplotypes); T at site 603; C at site 664; and C at site 702. When all of the above SNP sites are detected to contain the specific alleles, the gene is determined to be the Hap3 haplotype.

[0066] In this invention, the relevant nucleotide and amino acid sequences are as follows: SEQ ID NO:1: SEQ ID NO: 2: SEQ ID NO:3: MYMDAFGWSAPAAPCQPSCPGGGDDDDDVLLAAVLGASFELHSLVDGGGNGAAGAVRSDDAYGLDVDLPSHQMSLLRCQDGLSALHGDASPTAAAAAFLDSVDVLPVPAIAGATHDDGGLLDRFAFPNVAETTTVQAAASNTAFSGYSSNTTGGGNISSGESNTYTEVASTPCAVSTTTTTTALPPSKRKLPEKYPVVGTSPTTKTTT TSETAAERRSTKRGAGGSSSITFGGGCHGAGAAAALLGYGRGYEPDTEAIAQVKEMIYRAAAMRPVTLGGPASASDPSSRPPPPPQRPRRRKNVRISSDPQTVAARLRRERVSERLRVLQRLVPGGSKMDTATMLDEAASYLKFLKSQLEALETLGNGNGNGNLLHHGYYTGSRNATATAATGSSNSTVLAFGRDGLAGFVKSNRNLQL* Among them, SEQ ID NO:1 is OsSV5 The full-length gene sequence, SEQ ID NO:2 is OsSV5 The cDNA sequence of a gene, its structure is as follows: OsSV5 As shown in A, this gene is located on chromosome 5 of rice, and its gene number in the rice gene database is LOC_Os05g46370 (Os05g0541400). SEQ ID NO:3 is... Figure 1 OsSV5 The protein sequence of a gene.

[0067] The above are merely preferred embodiments of the present invention. It should be noted that the present invention is not limited to the above embodiments. For those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. gene OsSV5 In the use for regulating the seed vigor of rice, characterized in that, OsSV5 The gene positively regulates rice seed vigor, and the whole genome sequence is shown as SEQ ID NO: 1, and the CDS sequence is shown as SEQ ID NO:

2.

2. Use according to claim 1, characterized in that, knockout OsSV5 Genetic reduction of seed vigor in rice results in a significant reduction of the rice vigor phenotype relative to wild type.

3. Use according to claim 1, characterized in that, OsSV5 Phenotypes of the upregulation of the gene on the seed vigor of rice include the germination rate, germination index, germination potential, root length and / or shoot length of rice seeds.

4. Use of a gene-encoded protein in regulating the seed vigor of rice, characterized in that, The gene is as claimed in claim 1 OsSV5 The gene, the amino acid sequence of the protein is shown as SEQ ID No.

3.

5. A method of screening or breeding high vigour rice seeds, characterised by, Detecting varieties in OsSV5 whether the haplotype of the gene is Hap3.

Images