Application and method of gene osSV5 and protein encoded by gene in regulating rice seed vigor

Abstract

The application relates to the field of plant seed biology and rice breeding, and particularly relates to a gene OsSV5 and application of a protein encoded by the gene in regulating rice seed activity and a method. OsSV5 application of the gene in regulating rice seed activity, OsSV5 The gene positively regulates rice seed activity, the whole genome sequence of the gene is shown as SEQ ID NO: 1, and the CDS sequence is shown as SEQ ID NO: 2. Application of a protein encoded by the gene in regulating rice seed activity and a method for screening or cultivating high-activity rice seeds are also provided. In the application and the method, the expression level of the gene is positively correlated with rice seed activity.

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:

[0011] 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

[0012] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the following embodiment or prior art description will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0013] Figure 1 To 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.

[0014] Figure 2 To Nipponbare as background OsSV5 Schematic diagram of standard germination test of seed of gene knockout mutant; wherein A: wild type and OsSV5 Phenotype of rice seedling on the 7th day of standard germination test of seed of gene knockout mutant, scale = 1 cm; B: wild type and OsSV5 Rice germination on the 7th day of standard germination test of seed of gene knockout mutant, 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 OsSV5Germination potential bar graph of standard germination test of gene knockout mutant seeds (n=3); E: wild type and OsSV5 Germination index bar chart of standard germination test of gene knockout mutant seeds (n=3); F: wild type and OsSV5 Gene knockout mutant seeds standard germination test viability index bar chart (n=3); G: wild type and OsSV5 Root length bar graph of standard germination test of gene knockout mutant seeds (n=10); H: wild type and OsSV5 A bar graph showing the shoot length in a standard germination test of seeds from gene knockout mutants. express P <0.05, express P <0.01, express P <0.001, express P <0.0001, ns indicates no significant difference.

[0015] OsSV5 for Figure 3 Schematic diagram of gene expression levels in various tissues of Nipponbare; where A: qPCR analysis. OsSV5 Gene expression levels in roots, stems, leaves, and seeds; letters a, b, and c in the figure indicate significant differences. P <0.05; B:GUS protein staining, the part stained blue indicates OsSV5 The gene is highly expressed in this region.

[0016] OsSV5 For genes Figure 4 The diagram illustrates the base differences between gene structures and haplotypes, showing the differences in germination rate (GR), germination index (GI), and storage tolerance (SS) among different haplotypes; the higher the SS value, the less resistant the rice seeds are to storage.

[0017] OsSV5 For genes Figure 5 A comparison diagram of the sequence of the Hap3 haplotype and the sequence of SEQ ID NO: 2. Detailed Implementation

[0018] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0019] Unless otherwise defined, all terms used in the disclosure, including technical and scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art. The term "including" as used herein is used expansively and is intended to encompass the terms "including", "having", and the like. The term "consisting of" is intended to mean the limiting of the scope of a claim to the recited elements named. The term "consisting essentially of" means that the claim encompasses the recited elements named in the claim, and any additional unrecited elements that do not materially affect the basic and novel characteristics of the compositions or methods. The use of the terms "step" and / or "steps" does not imply that the disclosed steps are necessarily performed in sequence. The use of the terms "first", "second", "third", etc. does not imply any priority, unless otherwise specifically stated.

[0020] Unless otherwise specified, the various raw materials, reagents, instruments and equipment used in the present application can be purchased on the market or prepared by existing methods.

[0021] The application of a kind of regulation and control rice seed vigor gene and its encoding protein will be described in detail below with examples and experimental data.

[0022] Example 1

[0023] In this embodiment, Crispr-Cas9 technology is used to edit the gene OsSV5 , and obtain OsSV5 gene knockout mutant lines.

[0024] (1) Preparation of early work

[0025] According to the design principles of gene CRISPRY / Cas9 gene editing vector target, online tool CRISPR-PV2.0 (http: / / crispr.hzau.edu.cn / CRISPR2 / ) is used, combined with the base sequence of the gene, the sgRNA of the gene is designed. When selecting primers, ensure that the GC content of the primer 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, and synthesize. The primer sequences of target 1 and 2 are shown in the following table: OsSV5 OsSV5 OsSV5

[0026] Table 1 Primer sequences of target 1 and 2

[0027]

[0028] The primer sequences of M-F, gR-R, Pps-GGL and Pgs-GGR are universal primers for constructing Crispr-Cas9, and the sequences are shown in the following table:

[0029] Table 2 Primer sequences of M-F, gR-R, Pps-GGL and Pgs-GGR

[0030]

[0031] ​​​The strain pYLCRISPR / Cas9-MTmono stored in the laboratory-80℃ was thawed on ice, then transferred to LB solid medium containing 50 μg / mL Kan+, and the CRISPR / gRNA vector strain was thawed on ice and transferred to LB solid medium containing 100 μg / mL Amp+. Both were cultured overnight in a constant temperature incubator at 37.5℃, and single colonies were inoculated in LB liquid medium containing the corresponding antibiotics for expansion and plasmid extraction.

[0032] (2) Construction of sgRNA expression cassette

[0033] Linker primer: 1 μL of 2 μM linker primer was placed in a PCR tube, 90℃ for 30 s, and then annealed at room temperature.

[0034] Table 3 grna expression cassette ligation reaction system

[0035]

[0036] The reaction program was as follows: 37℃ for 5 min, 20℃ for 5 min, 5 cycles, and two rounds of nested PCR reaction were performed immediately after PCR.

[0037] The first round of PCR reaction system is shown in the following table:

[0038] Table 4 First round of PCR reaction system

[0039]

[0040] The above two PCR reactions were performed simultaneously, and the reaction program was as follows: 95℃ for 1 min, then 25 cycles of 95℃ for 10 s, 60℃ for 15 s, and 68℃ for 30 s, followed by 68℃ for 5 min. Take an appropriate amount of PCR product for electrophoresis observation.

[0041] The second round of PCR reaction system is shown in the following table:

[0042] Table 5 Second round of PCR reaction system

[0043]

[0044] The reaction procedure is: 94 ℃, 2 min, 94 ℃, 10 s, 60 ℃, 30 s, 68 ℃, 50 s, 25 cycles, then 68 ℃ extension for 5 min. The PCR amplification product above is electrophoresed in 1% agarose to observe, and the target fragment is purified by a PCR product gel recovery kit for use as a double vector and sgRNA expression cassette. The ligation reaction system is shown in the following table:

[0045] Table 6 Ligation reaction system of double vector and sgRNA expression cassette

[0046]

[0047] The 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 constructed.

[0048] (3) Agrobacterium plasmid transformation

[0049] The GV3101 Agrobacterium competent cells stored at -80 ℃ are partially thawed on ice, and then inserted into ice to thaw when in ice water mixture;

[0050] 3 μL of the CRISPR / Cas9 gene knockout vector that has been constructed is placed in a 1.5 mL EP tube containing 50 μL of Agrobacterium competent cells, mixed, and then sequentially placed on ice for 5 min, liquid nitrogen for 5 min, 28 ℃ water bath for 5 min, and ice bath for 5 min for recovery;

[0051] 700 μL of LB liquid culture is added, and cultured at 28 ℃ for 3 h; after sufficient time, the bacteria are collected at 6000 rpm for 1 min, and 100 μL is taken and mixed and plated on LB medium containing the corresponding antibiotics, and incubated in a 28 ℃ constant temperature incubator for 3 d.

[0052] A single colony is selected and then added to LB liquid medium containing kanamycin (Kan) and rifampicin (Rif), and cultured in a 28 ℃ shaker at a speed of 200 rpm for 3 d.

[0053] (4) Transgenic rice transformation

[0054] Nipponbare is used as the genetic receptor material, and the bacteria liquid after Agrobacterium transformation is sent to Wuhan Boyuan Biotechnology Co., Ltd. for genetic transformation: mature rice embryos are used as the material, and rice embryonic callus is induced as the genetic transformation receptor material, and the CRISPR / Cas9 gene knockout vector is used to transform the rice embryos. OsSV5Agrobacterium infection of the receptor material of the gene vector, T-DNA is inserted into the genome, and independent resistant callus is obtained through the selection of corresponding resistant antibiotics, and the transgenic positive plants are obtained by further differentiation and regeneration.

[0055] (5) Genotype identification of gene knockout transgenic rice, using gene knockout transformed seedlings as templates, primers for detecting vector Cas9 and target points were designed respectively, PCR amplification was carried out, and the primer sequences are shown in the following table:

[0056] Table 7 Target-1-F, Target-1-R, Target-2-F, Target-2-R primer sequences

[0057]

[0058] PCR amplification: the target fragment is amplified by PCR method, and the PCR reaction system is shown in the following table:

[0059] Table 8 PCR reaction system

[0060]

[0061] PCR reaction program: 98°C 3 min, 98°C 15 s, 60°C 15 s, 72°C 30 s, 72°C 5 min, 16°C 10 min, 35 cycles. After electrophoresis of the PCR product, the remaining PCR stock solution and the corresponding candidate gene identification primer were observed, and were sent to Beijing Genesee Biotechnology Co., Ltd. Hunan Branch for sequencing. Using SeqMap to compare the sequencing results, selecting homozygous lines with base mutations as CRISPR / Cas9 gene editing transgenic rice for seed vigor phenotype identification.

[0062] Example 2

[0063] This example is carried out OsSV5 Seed vigor identification of knockout lines

[0064] (1) Standard germination experiment, 100 seeds of each line were placed in a 9 cm Petri dish, and 10 ml of sterilized water was added. The Petri dish was covered with a piece of sterile filter paper, and then placed in a 28°C incubator for 7 days. The number of germinated seeds was counted every day, and the germination rate was calculated. OsSV5Knockout 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.

[0065] (2) Statistical analysis of seed vigor-related phenotypic data

[0066] 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.

[0067] (3) Germination phenotype record

[0068] 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 Gene knockout has a significant impact on seed vigor. The results are as follows: OsSV5 As shown, OsSV5 Genes that positively regulate rice seed vigor, and loss of their function leads to reduced rice seed vigor.

[0069] Example 3

[0070] This embodiment analyzes Figure 2 Gene expression levels in various tissues of Japanese Haruhi.

[0071] (1) Detection using quantitative real-time PCR (qRT-PCR) technology OsSV5 Gene expression levels in various tissues of Japanese Haruhi.

[0072] 1. Extraction and reverse transcription of rice RNA from different parts

[0073] Reagents for extracting rice plant RNA: "Seed RNA extraction solution" is combined with Trizol or RNA extraction kit, and corresponding experimental protocols are adopted according to the parts of the extracted RNA. The specific experimental steps are as follows:

[0074] About 0.1 g of rice seeds were taken in an RNase-Free 2.0 mL centrifuge tube, 300 μL of seed RNA extraction solution was added, and it was ground into homogenate with a grinding rod, and shaken well to mix; 300 μL of phenol solution ((phenol PH = 8.0): chloroform volume ratio = 1:1) was added, and shaken well to mix; centrifuged at 13000 rpm for 5 min at room temperature, and the supernatant was transferred to a centrifuge tube.

[0075] Trizol method extraction: after adding 1 mL Trizol and shaking well, it was placed at room temperature for 20 min, then 200 μL of chloroform was added, and it was shaken well again; centrifuged at 1300 rpm for 10 min at room temperature, 700 μL of supernatant was taken to a new centrifuge tube, and an equal volume of isopropanol was added to mix well, centrifuged at 13000 rpm for 10 min at room temperature, and the supernatant was discarded; 1 mL of 75% ethanol (DEPC treated) was added for washing, centrifuged at 13000 rpm for 5 min at room temperature, and the supernatant was discarded; after blowing dry on a clean bench, 50 μL of ddH2O was added to dissolve it.

[0076] 2. Reverse transcription of rice RNA

[0077] The instructions of Gen Star's RNA reverse transcription kit were followed to operate, and the reaction system is shown in the following table:

[0078] Table 9 Reaction system of Gen Star's RNA reverse transcription kit

[0079]

[0080] The reaction program is: after mixing and short centrifugation, react at 37 ℃ for 5 min in a PCR instrument; other required components for reverse transcription are directly added in the above reaction tube, and the first strand cDNA synthesis step is carried out, and the reaction system is shown in the following table:

[0081] Table 10 Reverse transcription reaction system

[0082]

[0083] After mixing, centrifuge for 10 s, incubate at 42 ℃ for 15-50 min using a PCR instrument; deactivate StarScript II Enzyme Mix after heating at 85 ℃ for 5 min; after the reaction is completed, the cDNA obtained can be measured for concentration using an ultramicro spectrophotometer, and then placed on ice for subsequent experiments or frozen for preservation.

[0084] (2) Real-time fluorescent quantitative PCR

[0085] The cDNA of the roots, stems, leaves and seeds of the rice variety Nipponbare extracted in the above experiment was used as a PCR template, and the expression level of the endogenous gene of rice Eef1a was used as a reference. The gene expression amount was investigated by using a relative quantitative method, and the relative expression level was evaluated by using a ΔΔCt calculation method. Three replicates were set for each sample, and the experiment was repeated three times. The primer sequences used for quantification are shown in the following table:

[0086] Table 11 eEF1a-F, eEF1a-R, OsSV5 -F, OsSV5 -R primer sequences

[0087]

[0088] The kits and qRT-PCR instruments involved in the experiment were 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:

[0089] Table 12 qRT-PCR reaction system

[0090]

[0091] The reaction conditions of qRT-PCR were as follows: 95 ℃ for 10 min, 95 ℃ for 10 s, 60 ℃ for 40 s, 40 cycles.

[0092] (3) Detection by GUS staining OsSV5 Expression of the gene in each tissue of Nipponbare.

[0093] 1. Extraction of rice genomic DNA

[0094] 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.

[0095] 2. PCR amplification OsSV5 Gene promoter fragments

[0096] 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:

[0097] Table 13 Primer sequences for Sv5-qi-F and Sv5-qi-R

[0098]

[0099] 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:

[0100] Table 14 PCR amplification reaction system

[0101]

[0102] 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.

[0103] 3. Recovery of PCR amplification products

[0104] 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 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:

[0105] 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.

[0106] 3. OsSV5 Construction of the gene GUS vector

[0107] 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:

[0108] Table 15 Primer sequences of B-Sv5-qi-F and H-Sv5-qi-R

[0109]

[0110] 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.

[0111] Table 16 Endonuclease Digestion Reaction System

[0112]

[0113] Reaction conditions: after 2.5 h of reaction in a 37 °C water bath, all were put into 1% agarose gel for electrophoresis reaction, and 3 tubes of product were mixed for recovery and purification of the linearized vector. The purified DNA fragment was ligated with pCAMBIA1381 Xa enzyme-digested empty carrier, and the reaction system is shown in the following table:

[0114] Table 17 DNA fragment and pCAMBIA1381 Xa enzyme-digested empty carrier ligation reaction system

[0115]

[0116] Reaction conditions: after 1 h of reaction in a 50 °C water bath, Trelief.5α of Beijing Qianke Biological Technology Co., Ltd. Hunan Branch was used, the competent cells were taken out at a storage temperature of -80 °C, dissolved on ice, and then 20 µL of the ligation product was added, gently mixed, and then placed on ice for 5 min. Then a metal water bath was used for 45 s of 42 °C water bath, and after heat shock, the cells were quickly placed on ice for 2 min (to ensure transformation efficiency, do not shake during the standing process); 500 µL of pre-packaged sterile liquid LB liquid medium was added, mixed thoroughly, and then recovered in a 37 °C, 200 rpm shaker for 15 min; 200 µL of the recovered liquid was plated on LB solid medium containing the corresponding antibiotic, and incubated in a 37 °C incubator overnight. After 16 h of culture, single colonies were picked from the plate and cultured in LB liquid medium containing the corresponding antibiotic, and the culture time was not more than 24 h, which could be roughly judged according to the turbidity of the LB liquid medium.

[0117] The plasmid of the completed culture was extracted using a purchased plasmid extraction kit (NO. omega; REF: D6943-02). The specific steps for plasmid extraction are as follows:

[0118] The 5 ml of completed culture was transferred into 2 ml EP tubes in two portions, centrifuged at 10000 rpm for 60 s at room temperature to collect the bacterial cells, and the upper LB liquid medium was discarded. 250 µL of bacterial lysis solution (Solution I / RNaseA) was added, and the bacterial cells were completely suspended by inverting several times;

[0119] 250 µL of Solution II was added to the resuspended bacterial mixture after lysis, and mixed well by inverting 10 times. Then 350 µL of Solution III was added, and after inverting 10 times, a white flocculent bacterial precipitate was formed. The horizontal centrifuge was used for centrifugation at a speed of 12000 rpm for 10 min at room temperature;

[0120] Transfer the supernatant after centrifugation to the DNA binding column with a collection tube using a 1ml pipette, and centrifuge at 12000 rpm for 1 min at room temperature using a horizontal centrifuge, and discard the filtrate in the collection tube;

[0121] Put the DNA binding column back into the collection tube, add 500µL of HBC Buffer diluted in advance with isopropanol, and centrifuge at 12000 rpm for 1 min at room temperature, and discard the filtrate in the collection tube;

[0122] Put the binding column back into the collection tube, and to elute the organic substances such as mixed proteins, add 700µL of DNA Wash Buffer diluted in advance with anhydrous ethanol, and centrifuge at 12000 rpm for 60s at room temperature, and discard the filtrate in the collection tube;

[0123] Empty, put the binding column back into the collection tube, and centrifuge the empty column at 12000 rpm for 60s, and dry the residual alcohol in the column to prevent damage to the plasmid DNA; finally, put the binding column containing the plasmid of the target strain into a new 1.5mL centrifuge tube, add 35µL Elution Buffer to the column matrix, stand for 1 min, and centrifuge at 13000 rpm for 60s, and elute the plasmid DNA;

[0124] Measure the concentration of the extracted DNA plasmid using a ultramicro spectrophotometer (Beijing Kai Ao Technology Development Co., Ltd., K5600), and then store the DNA plasmid at 20℃ for a long time, and take 10µL of the plasmid to Beijing Qianke Biological Technology Co., Ltd. Hunan Branch for sequencing, and send the plasmid containing OsSV5 the correct gene to Wuhan Boyuan Biological Technology Co., Ltd. for genetic transformation with NPB as the background material.

[0125] 4. GUS staining of rice plants

[0126] First, mix the X-gluc solvent in the GUS staining kit (Coolai, SL7160) with X-Gluc dry powder to form a color developing liquid concentrate, and mix the color developing liquid concentrate and buffer to form a GUS staining solution (freshly prepared, color developing liquid stock solution stored at -80℃ for a long time). The specific staining steps are as follows: soak the prepared plant tissues and seeds in the GUS staining solution, and extract vacuum for 20 min to allow the staining solution to fully penetrate the cell tissues. Then incubate it in a 37℃ oven for 1h or overnight. After staining the plant tissue parts containing color, use 70% alcohol for decolorization treatment, repeat 3 times, until the negative control becomes colorless. Observe the blue spots on the plant tissue parts and seed embryos under the naked eye, and consider that the tissue part has GUS activity, which proves OsSV5The 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.

[0127] Example 4

[0128] This embodiment analyzes OsSV5 Genetically modified rice seeds with excellent vigor haplotypes.

[0129] 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 in other haplotypes); T at site 604; C at site 665; and C at site 703. When all of the above SNP sites are detected to contain the specific alleles, the gene is determined to be the Hap3 haplotype.

[0130] In this invention, the relevant nucleotide and amino acid sequences are as follows:

[0131] SEQ ID NO:1:

[0132]

[0133] SEQ ID NO: 2:

[0134]

[0135] SEQ ID NO:3:

[0136] MYMDAFGWSAPAAPCQPSCGPGGDDDDDVLLAAVLGASFELHSLVDGGGNGAAGAVRSDDAYGLDVDLPSHQMSLLRCQDGLSALHGDASPTAAAAAFLDSVDVLPVPAIAGATHDDGGLLDRFAFPNVAETTTVQAAASNTAFSGYSSNTTGGGNISSGESNTYTEVASTPCAVSTTTTTTALPPSKRKLPEKYPVVGTSPTTKTTTTSETAAERRSTKRGAGGSSSITFGGGCHGAGAAAALLGYGRGYEPDTEAIAQVKEMIYRAAAMRPVTLGGPASASDPSSRPPPPPQRPRRKNVRISSDPQTVAARLRRERVSERLRVLQRLVPGGSKMDTATMLDEAASYLKFLKSQLEALETLGNGNGNGNLLHHGYYTGSRNATATAATGSSNSTVLAFGRDGLAGFVKSNRNLQL

[0137] SEQ ID NO:1 is the full-length sequence of the gene, SEQ ID NO:2 is the cDNA sequence of the gene, and SEQ ID NO:3 is the protein sequence of the gene. OsSV5 OsSV5 The gene is located on chromosome 5 of rice, and the gene number in the rice gene database is LOC_Os05g46370 (Os05g0541400). OsSV5 Figure 1 OsSV5

[0138] The above is only a preferred embodiment of the present application, and it should be noted that the present application is not limited to the above-mentioned embodiments. For those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application should also be considered within the protection scope of the present application.​​​

Claims

1. A method of screening high vigour rice seeds, characterized in that, Among the rice varieties tested OsSV5 Whether the haplotype of the gene is Hap3, the CDS sequence of the OsSV5 gene is shown in SEQ ID NO:

2. The key sequence characteristics of the Hap3 haplotype of the OsSV5 gene are that, in the nucleotide sequence shown in SEQ ID NO: 2, position 67 is GGCGA, position 604 is T, position 665 is C, and position 703 is C. When all of the above specific alleles are detected, the gene is determined to be the Hap3 haplotype. The phenotype of rice seed vigor is germination rate, germination potential, and germination index.

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