Application of the rice OsFBX22 gene in regulating temperature-sensitive male sterility in rice

Abstract

This invention discloses rice OsFBX22 The application of gene regulation in temperature-sensitive male sterility in rice belongs to the field of genetic engineering technology. This invention utilizes CRISPR-Cas9 gene editing technology to target and knock out rice germ cells. OsFBX22 Genes, successfully obtained casfbx22‑1 homozygous deletion mutants and casfbx22‑2 Both biallelic mutants exhibited a thermosensitive male-sterile phenotype characterized by high-temperature sterility and low-temperature fertility recovery. This demonstrates that rice... OsFBX22 The gene can be used as a novel temperature-sensitive male-sterile line in the production of two-line hybrid rice seeds. This invention provides new germplasm resources and technical approaches for utilizing heterosis in rice.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to rice. OsFBX22 Application of gene regulation in thermosensitive male sterility in rice. Background Technology

[0002] The widespread adoption of hybrid rice technology has made outstanding contributions to increasing grain production in my country. The core of this technology lies in the production of hybrid seeds using male-sterile lines. Among them, the two-line hybrid rice technology based on environment-sensitive male-sterile lines (especially photoperiod- and temperature-sensitive male-sterile lines) has become an important direction in my country's hybrid rice breeding due to its advantages such as free pairing and relatively simplified propagation and seed production procedures. However, the key male-sterile gene resources upon which the two-line male-sterile lines widely used in current production rely are still relatively limited, which to some extent restricts the expansion of genetic diversity in two-line hybrid rice parents and the breeding of new, strong, and vigorous combinations. Therefore, discovering and utilizing novel male-sterile genes and creating new male-sterile lines with stable traits are important ways to break through current breeding bottlenecks and promote the sustainable development of hybrid rice technology.

[0003] At the molecular level, pollen development is a complex biological process precisely regulated by a multi-gene network. E3 ubiquitin ligases, as key components of the ubiquitin-proteasome system, can specifically recognize substrate proteins and mediate their ubiquitination and degradation, thus playing a central regulatory role in many physiological processes such as cell cycle, signal transduction, and programmed cell death. Studies have shown that E3 ubiquitin ligase family genes are specifically or highly expressed at specific stages of rice anther and pollen development (such as meiosis, tapetum degradation, and pollen wall formation), suggesting they may play a crucial role in male fertility regulation. In-depth analysis of the functions of these genes will not only help elucidate the molecular mechanisms of male sterility but also provide possibilities for developing sterile lines based on new gene resources. In particular, if E3 ubiquitin ligase genes whose functions can be induced / inhibited by specific environmental conditions (such as temperature) or exogenous compounds can be identified, it is hoped that "intelligent" sterile lines can be constructed, achieving artificial or environmental control over fertility, thereby further simplifying seed production procedures, reducing production costs, and ensuring seed purity.

[0004] Although the importance of E3 ubiquitin ligases in plant male reproductive development is increasingly recognized, research on E3 ubiquitin ligase genes associated with temperature-sensitive male sterility in rice remains very limited. No E3 ubiquitin ligase genes with well-defined functions and temperature-regulating fertility that can be applied to two-line hybrid rice breeding have been reported. Therefore, discovering novel E3 ubiquitin ligase genes with temperature-sensitive male sterility phenotypes is of significant theoretical and practical value for enriching the genetic resources of two-line hybrid rice and overcoming existing breeding bottlenecks. Summary of the Invention

[0005] This invention aims to solve the technical problems of limited temperature-sensitive male sterility gene resources and insufficient genetic diversity in existing two-line hybrid rice breeding technologies. To this end, this invention provides a rice... OsFBX22 Application of gene regulation in temperature-sensitive male sterility in rice. The rice described... OsFBX22 When a gene loses or reduces its function, it can cause rice to exhibit thermosensitive male sterility, that is, a thermosensitive male sterility phenotype that exhibits high-temperature sterility and low-temperature fertility recovery. Based on the thermosensitive characteristics of this gene loss-of-function mutant, it can be used to create novel thermosensitive male sterile lines and apply them to the production of two-line hybrid rice seeds.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides rice OsFBX22 or rice OsFBX22 The application of genes in any of the following: (1) Rice breeding; (2) Cultivating temperature-sensitive male-sterile rice lines; The amino acid sequence of the rice OsFBX22 is shown in SEQ ID NO.2; the rice OsFBX22 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0007] Optionally, the application involves inhibiting or knocking out the activity of rice OsFBX22 in rice. OsFBX22 Gene.

[0008] Optionally, the rice temperature-sensitive male sterile line exhibits a phenotype of high-temperature sterility, with fertility restored at low temperatures.

[0009] As an optional technical solution, the present invention provides rice OsFBX22 or rice OsFBX22 Application of genes in the breeding of tall rice.

[0010] In this industry, in non-dense planting and low-competition environments (such as experimental fields and rice-fish integrated farming), higher plant height can increase leaf area, increase the total amount of photosynthetic products, and support more grains per panicle, which helps to increase biomass or achieve ecological compound utilization (such as shade fish farming and tourism).

[0011] This invention provides knockout rice OsFBX22 The application of gene-based biomaterials in any of the following: (1) Rice breeding; (2) Cultivating temperature-sensitive male-sterile rice lines; The rice OsFBX22 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0012] Optionally, the biomaterial includes a recombinant vector and recombinant bacteria.

[0013] Optionally, the rice temperature-sensitive male sterile line exhibits a phenotype of high-temperature sterility, with fertility restored at low temperatures.

[0014] This invention provides a method for cultivating a temperature-sensitive male-sterile rice line, comprising knocking out the rice... OsFBX22 The steps of gene generation; the rice OsFBX22 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0015] Optionally, the rice temperature-sensitive male sterile line exhibits a phenotype of high-temperature sterility, with fertility restored at low temperatures.

[0016] Optionally, the knockout method includes CRISPR-Cas9.

[0017] This invention provides a method for producing hybrid rice seeds, comprising the following steps: Knockout rice OsFBX22 Genes were used to obtain a temperature-sensitive male-sterile rice line; Under low temperature conditions, the rice thermosensitive male sterile line was propagated to obtain rice thermosensitive male sterile line seeds; After sowing the seeds of the temperature-sensitive male-sterile rice line, the rice was crossed with the restorer line male parent under high temperature conditions to obtain hybrid rice seeds.

[0018] The present invention discloses the following technical effects: This invention utilizes CRISPR-Cas9 gene editing technology to target and knock out rice. OsFBX22 Genes, successfully obtained casfbx22-1 homozygous deletion mutants and casfbx22-2 Both biallelic mutants exhibited a thermosensitive male-sterile phenotype characterized by high-temperature sterility and low-temperature fertility recovery. This demonstrates that rice... OsFBX22 The gene can be used as a novel temperature-sensitive male-sterile line in the production of two-line hybrid rice seeds. This invention provides new germplasm resources and technical approaches for utilizing heterosis in rice. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 Rice mutant casfbx22 Knockout vector construction results; Figure 2 Here is an electrophoresis diagram of the PCR products; lane 1 is the marker, and lanes 2 and 3 are the PCR products. Figure 3 For CRISPR-Cas9 vector insertion detection (Cas9 primers); among which, casfbx22 -1 and casfbx22 -2 represents two positive plants; Figure 4 Chloroplast genes rbcL Amplification and validation (internal reference gene); among which, casfbx22 -1 and casfbx22 -2 represents two positive plants; Figure 5 This is a field phenotypic comparison of the wild-type ZH11 and the mutant casfbx22 lines at 30℃; from left to right, they are ZH11, casfbx22-1 and casfbx22-2 ; Figure 6 This is a field phenotypic comparison of the wild-type ZH11 and the mutant casfbx22 lines at 23℃; from left to right, they are ZH11, casfbx22-1 and casfbx22-2 ; Figure 7 This is a comparison diagram of pollen fertility between the wild-type ZH11 and the mutant casfbx22 lines at 30℃. Figure 8 This is a comparison diagram of pollen fertility between the wild-type ZH11 and the mutant casfbx22 lines at 23℃. Figure 9 This image shows a comparison of the fruit set of wild-type ZH11 and the mutant casfbx22 lines grown in the field at 30℃; from left to right, they are ZH11, casfbx22-1 and casfbx22-2 ; Figure 10 This image shows a comparison of the fruit set of wild-type ZH11 and the mutant casfbx22 lines grown in the field at 23℃; from left to right, they are ZH11, casfbx22-1 and casfbx22-2 ; Figure 11 Statistical results of seed setting rate of wild-type ZH11 and mutant casfbx22 lines planted in the field at 30℃. Figure 12 The statistical results of the seed setting rate of wild-type ZH11 and mutant casfbx22 lines planted in the field at a low temperature of 23℃. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] rice OsFBX22 The nucleotide sequence of the gene is shown in SEQ ID NO.1, specifically:

[0027] rice OsFBX22 The amino acid sequence of the rice OsFBX22 protein encoded by the gene is shown in SEQ ID NO.2, specifically as follows: MADDGGDGPCDSDYISNLPENVLVTILSLLRLDEAARSTVLSTRWRHLFPYTLLDFRAYALGRDVVAAVNTILAAHPAARVRSFRTGLLYFPPEDDPSVEAWLRDLAGRGVKELSLSFRERWQKIPASLFACTSLKRLHASSCTFPDATQAPVPLAALAEIDLFGVNISEESLGALLSRCTALEHLRMRSMGWCHRIHVRSESLKTLCGCGDFDELLIEHA PNLEQVYGNYMYMRSTHLKVAHAPKLEFLGYLGMSFDTIEIGQSVFTEDDFDIKTLMPSLKTLAIELSYTSEGYINWFMQLLKLFPCLETLYIRSDTWSKVRAAAPGSWDV LRSVPCIDNHLERVVFEVYRGHEWQREMAKFLHGRARFLKAMEFHCQGDKGCSELLGEEWVREQQELLCLDSRASLDARFLFFKGALVNNHHDVSHHEWYKRKYYHYLYNV .

[0028] Example 1: Rice OsFBX22 Construction of CRISPR-Cas9 knockout vector 1. Target site design and primer synthesis Based on rice OsFBX22 The coding sequence of the gene (SEQ ID NO.1) was used to design and synthesize two CRISPR-Cas9 target sites: the target sequence of target site 1 is 5'-CCTCCTCAGGCTCGACGAAG-3' (SEQ ID NO.3); the target sequence of target site 2 is 5'-CTCGCCGAGATCGATCTGTT-3' (SEQ ID NO.4). Oligonucleotide primer pairs with adapters were synthesized for each site. Target 1: U3F: 5'-ggcaCCTCCTCAGGCTCGACGAAG-3' (SEQ ID NO.5); U3R: 5'-aaacCTTCGTCGAGCCTGAGGAGG-3' (SEQ ID NO. 6); Target 2: U6F: 5'-gccgCTCGCCGAGATCGATCTGTT-3' (SEQ ID NO.7); U6R: 5'-aaacAACAGATCGATCTCGGCGAG-3' (SEQ ID NO. 8).

[0029] Note: Lowercase letters are sticky ends formed after BsaI digestion. The U3 vector requires GGC / GCC ends, and the U6a~c vector requires GCCG / CGGC ends.

[0030] 2. Joint annealing The forward and reverse primers for each target site were mixed in equal volumes and diluted to a final concentration of 1 μmol / L (i.e., the final concentrations of both forward and reverse primers were 1 μmol / L). After denaturation at 90°C for 30 seconds, the mixture was allowed to cool naturally to room temperature for annealing to form a double-linked head.

[0031] 3. Construction of intermediate carrier (1) BsaI digestion Take 0.5-1 μg of each of the pYL-U3-gRNA and pYL-U6a-gRNA plasmids (the plasmids have been disclosed in the literature "A Robust CRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants" (Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H, Lin Y, Xie Y, Shen R, Chen S, Wang Z, Chen Y, Guo J, Chen L, Zhao X, Dong Z, Liu YG. A Robust CRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants. Molecular Plant. 2015 Aug 3;8(8):1274-84.), kindly provided by the research group of Academician Liu Yaoguang of South China Agricultural University. The applicant promises to strictly comply with relevant regulations and legally use the plasmid materials from the date of application) and use 5-10 U BsaI was digested at 37℃ for 20 min and then inactivated at 70℃ for 5 min.

[0032] (2) Connection reaction Prepare a 10 μL ligation system: 1 μL 10 × T4 DNA ligase buffer, 12 ng digested vector (pYL-U3-gRNA for U3a expression cassette, pYL-U6a~c-gRNA for U6b expression cassette), 0.1-0.2 μmol / L adapter primers (0.1 μmol / L adapter primers for U3a expression cassette; 0.2 μmol / L adapter primers for U6b expression cassette), add ddH2O to 10 μL, and finally add about 35 U of T4 DNA ligase. Ligate at room temperature (20-25℃) for 20 min.

[0033] 4. gRNA expression cassette amplification (1) First round of PCR (detection of ligation products) Using 2 μL of the ligation product as a template, primers UF: 5'-CTCCGTTTTACCTGTGGAATCG-3' (SEQ ID NO. 9) and gRNA-R: 5'-CGGAGGAAAATTCCATCCAC-3' (SEQ ID NO. 10) were used, with a final concentration of 0.2 μmol / L for each. PrimeSTAR was then used. ® Amplification was performed using Max DNA Polymerase. The total reaction volume was 15 μL, containing 7.5 μL of 2×buffer, 0.18 μL of dNTPs, 0.3 μL of UF, 0.3 μL of gRNA-R, 4.52 μL of ddH2O, 0.2 μL of Phanta Max polymerase, and 2 μL of the ligation product from the previous round. The program was set to 95℃ for pre-denaturation for 1 min, followed by 25 cycles (95℃ denaturation for 10 sec, 60℃ annealing for 15 sec, and 72℃ extension for 10 sec). After PCR, 4 μL of the product was subjected to 1% agarose gel electrophoresis to confirm the correct band size. The remaining PCR product was diluted 20-fold with sterile water and used as a template for the second round of PCR.

[0034] (2) Second round PCR (expression cassette) Using the first-round PCR product diluted to 1 μL as a template, select the appropriate primer combination according to the number of target sites. The reaction system was set to 50 μL for a single target and 30 μL for multiple targets. This example uses a dual-target system, with the following reaction system: 15 μL Buffer, 0.36 μL dNTPs, 0.45 μL each of pps-L (B1′, universal upstream primer for U3 / U6: TTCAGAGGTCTCTCTCGCACTGGAATCGGCAGCAAAGG, SEQ ID NO.11) and pgs-2 (B2, downstream primer for target 1: AGCGTGGGTCTCGTCAGGGTCCATCCACTCCAAGCTC, SEQ ID NO.12) / pps-2 (B2′, upstream primer for target 2: TTCAGAGGTCTCTCTGACACTGGAATCGGCAGCAAAGG, SEQ ID NO.13) and pgs-R (BL, downstream primer for the last target: AGCGTGGGTCTCGCTCGACGCGTATCCATCCACTCCAAGCTC, SEQ ID NO.14), and the first round PCR product 1. 0.45 μL of Phanta Max polymerase was added, and ddH2O was added to bring the total volume to 30 μL, making the total volume 30 μL. The amplification program was 95℃ pre-denaturation for 10 sec, followed by 18-25 cycles (95℃ denaturation for 10 sec, 60℃ annealing for 15 sec, and 68℃ extension for 20 sec). After PCR, agarose gel electrophoresis was performed to confirm the correct band size of each expression cassette. The PCR products were then mixed in equal proportions, excised from the gel, and purified for later use.

[0035] 5. Final carrier construction (cutting and connecting simultaneously) Prepare a 15 μL reaction mixture containing 1.5 μL 10× CutSmart Buffer, 20-70 ng of purified gRNA expression cassette PCR product, 40-60 ng of undigested pYLCRISPR / Cas9Pubi-H plasmid, and 5-10 U BsaI. Add ddH2O to a final volume of 15 μL and digest at 37℃ for 10 min. Then add 1.5 μL 10 mmol / L ATP and 35 U T4 DNA ligase, and perform 10-15 temperature-dependent cycles (each cycle including 37℃ for 5 min, 10℃ for 5 min, and 20℃ for 5 min) to alternate between digestion and ligation, achieving directional insertion of the gRNA expression cassette into the vector. Rice mutants casfbx22 The results of the knockout vector construction are as follows Figure 1 As shown.

[0036] 6. Transformation and Identification (1) The ligation product was transformed into Escherichia coli DH5α competent cells, plated on LB plates containing 25 mg / L kanamycin and 50 mg / L ampicillin, and cultured at 37°C for 16 h.

[0037] (2) Ten single colonies were randomly selected, and 1 μL of bacterial culture was used as templates for PCR amplification using identification primers M13F-47 and U6a-R (Table 1). The total volume of the reaction system was 20 μL, including 10 μL of 2 × Accurate TaqMaster Mix, 1 μL each of the forward and reverse primers (10 pmol / μL), and ddH2O added to 20 μL. The amplification program was 94℃ pre-denaturation for 25 sec; followed by 30 cycles (94℃ denaturation for 25 sec, 58℃ annealing for 25 sec, 72℃ extension for 2 min); and finally, 72℃ extension for 5 min, and storage at 4℃. The PCR products were detected by 1% agarose gel electrophoresis, and positive clones with an expected band size of approximately 750 bp were screened out, such as... Figure 2 As shown.

[0038] Table 1 FBX22 Gene knockout vector E. coli P primer sequence (3) Take 100 μL of bacterial culture from 3-5 positive clones and send it for sequencing to confirm that the target sequence is correctly ligated.

[0039] 8. Agrobacterium-mediated transformation The plasmids that were correctly sequenced and cloned were extracted and transformed into Agrobacterium tumefaciens EHA105 for subsequent genetic transformation in rice.

[0040] Example 2: Genetic transformation of rice 1. Callus induction Select plump, pest-free mature seeds of the japonica rice variety ZH11, remove the husks, and transfer the seeds to sterile Erlenmeyer flasks. On a clean bench, first wash with 75% ethanol for 30 seconds to 1 minute, then discard the ethanol. Next, add 2% sodium hypochlorite solution for sterilization for 15 minutes, repeating twice. Then discard the sodium hypochlorite and rinse 3-4 times with sterile distilled water to remove residual sodium hypochlorite. Remove the seeds, place them on sterile filter paper to air dry, and then inoculate them into induction medium. Incubate in the dark at 25°C for 5-7 days, then remove the sprouts and transfer them to subculture medium. After approximately 20-25 days, select callus tissue derived from the cotyledon scutellum that is dense, granular in texture, and light yellow in color for subculture.

[0041] Induction medium: N6 macroelements + B5 microelements + B5 organics + iron salts + inositol 0.1 g / L + 0.5 g / L L-glutamine + 0.5 g / L L-proline + 0.3 g / L acid-hydrolyzed casein (CH) + 2.5 mg / L 2,4-D + 30 g / L sucrose + 8-8.5 g / L agar powder (add agar powder after adjusting pH); Subculture medium: N6 macroelements + B5 microelements + B5 organics + iron salts + inositol 0.1 g / L + 0.5 g / L L-glutamine + 0.5 g / L L-proline + 0.3 g / L acid-hydrolyzed casein (CH) + 2.5 mg / L 2,4-D + 30 g / L sucrose + 8-8.5 g / L agar powder (add agar powder after adjusting pH); Working concentration of macroelements in 1L N6: Potassium nitrate 2830mg, ammonium sulfate 463mg, potassium dihydrogen phosphate 400mg, magnesium sulfate heptahydrate 185mg, calcium chloride dihydrate 166mg. 10x concentrated stock solution (1L): Weigh out 28.3g of potassium nitrate, 4.63g of ammonium sulfate, 4.0g of potassium dihydrogen phosphate, 1.85g of magnesium sulfate heptahydrate, and 1.66g of calcium chloride dihydrate, dissolve thoroughly in water, and bring the volume to 1L. Use 100mL of this stock solution to prepare 1L of culture medium.

[0042] Working concentration of B5 trace elements in 1L: Potassium iodide 0.75mg, boric acid 3.0mg, manganese sulfate tetrahydrate 10.0mg, zinc sulfate heptahydrate 2.0mg, sodium molybdate dihydrate 0.25mg, copper sulfate pentahydrate 0.025mg, cobalt chloride hexahydrate 0.025mg. 100x concentrated stock solution (1L): Weigh 0.075g potassium iodide, 0.3g boric acid, 1.0g manganese sulfate tetrahydrate, 0.2g zinc sulfate heptahydrate, 0.025g sodium molybdate dihydrate, 0.0025g copper sulfate pentahydrate, and 0.0025g cobalt chloride hexahydrate, dissolve, and bring to a final volume of 1L. Use 10mL of this stock solution to prepare 1L of culture medium.

[0043] Working concentration of organic components in 1L B5: Inositol 100mg, Nicotinic acid 1.0mg, Pyridoxine hydrochloride 1.0mg, Thiamine hydrochloride 10.0mg. 100x concentrated stock solution (1L): Weigh 10.0g inositol, 0.1g nicotinic acid, 0.1g pyridoxine hydrochloride, and 1.0g thiamine hydrochloride, mix well to dissolve, and then bring the volume to 1L. Use 10mL of this stock solution to prepare 1L of culture medium. An additional 0.1g / L of inositol is added to the induction and subculture media, resulting in a final total inositol content of 200mg / L.

[0044] Working concentration of 1L EDTA iron salts: 27.8 mg ferrous sulfate heptahydrate, 37.3 mg disodium EDTA dihydrate. 100x concentrated stock solution (1L): Dissolve 2.78 g ferrous sulfate heptahydrate in 400 mL of pure water; dissolve 3.73 g disodium EDTA dihydrate in 400 mL of pure water (heating can aid dissolution). Slowly mix the ferrous sulfate solution into the EDTA solution while continuously stirring, adjust the pH to 5.5, and add water to a final volume of 1L. Store in a brown container at 4°C, protected from light. Use 10 mL of this stock solution to prepare 1L of culture medium.

[0045] 2. Callus activation and drying From subcultured callus, select healthy tissues with good growth, suitable density, and pale yellow granular texture. Remove parts that are too dark (brown), loosely structured, or transparent. Place the tissues in freshly prepared NB medium and culture at 25°C in the dark for 4-6 days to obtain well-activated and highly viable callus tissues. Using a laminar flow hood, transfer the activated callus tissues to sterilized Erlenmeyer flasks or sterile filter paper petri dishes, seal them with sealing film, and dry them in the dark at 25°C for 0.5-1 days. The starved state of the callus tissues after 0.5-1 days of drying is conducive to infection, thus optimizing the conversion rate.

[0046] 3. Infection and co-culture Successfully transformed Agrobacterium tumefaciens EHA105 was streaked onto YM solid medium containing kanamycin (50 mg / L) and cultured at 28°C for 36-48 h. Single colonies were picked and cultured in YM liquid medium containing kanamycin (50 mg / L) at 28°C with shaking for 36-48 h. 800-1000 µL was then spread onto YM solid medium containing kanamycin (50 mg / L) and cultured at 28°C until the successfully transformed Agrobacterium tumefaciens EHA105 covered the plate.

[0047] Aspirate the Agrobacterium infection solution with a pipette tip and wash it off onto the agar plate. Then, aspirate the solution back into the Agrobacterium infection solution, mix thoroughly, and measure the OD (Organic Discharge). 600 Value, OD 600 The value was controlled between 0.05 and 0.07. The culture was placed in a shaker at 28℃ and 180 rpm for 30 min. The callus tissue was then placed in the infection solution and inoculated for 20 min. The callus tissue was transferred to a culture dish containing sterile filter paper. After drying for 10 min to remove Agrobacterium residue, the culture dish was replaced with new sterile filter paper, and the callus tissue was transferred again. The dish was then dried in a clean bench for 1.5-2 h. The callus tissue was then inoculated into a co-culture solid medium containing filter paper and dried in an open environment for 1-2 h. The dish was sealed with sealing film and cultured at 25℃ in the dark for 2 days.

[0048] 4. Screening for resistant callus Rinse the co-cultured callus twice in a laminar flow hood with a solution containing 300 mg / L cephalosporin and 300 mg / L carbenicillin, 2-3 min each time, then discard the rinsing solution. If the rinsing solution is still cloudy, rinse again until it is clear and transparent. Next, add fresh rinsing solution to the callus and shake on a shaker at 150 rpm for 20-30 min, then discard the rinsing solution. Rinse 2-3 more times in a laminar flow hood, discarding the rinsing solution each time. Place the callus on sterile filter paper plates to air dry, changing the filter paper occasionally, for a total of 30-60 min, until the surface is dry. Place the air-dried callus in selection medium and incubate at 25-26°C in the dark.

[0049] During the selection process, untransformed callus tissue gradually browned and died, while successfully transformed callus tissue continued to grow under selection pressure. Contaminated and browned callus tissue was removed every 10-14 days, and the selection medium was replaced until the selection was completed.

[0050] 5. Differentiation Carefully select resistant callus tissue that is fresh and milky white, inoculate it into differentiation medium, and culture it under light at 28℃ for about 10-14 days. During this period, observe the changes in the callus tissue regularly. If green spots appear on the callus tissue and seedlings grow directly, change the differentiation medium according to the growth status of the green spots and seedlings to prepare for subsequent rooting and strong seedlings.

[0051] 6. Promote root development and strengthen seedlings Observe the resistant callus that has entered differentiation every day. When the seedlings differentiate from the resistant callus to 3-4 cm, carefully remove the browned and dead callus next to the seedlings and insert them into the rooting and seedling strengthening culture medium and culture them under light at 28℃.

[0052] 7. Screening of positive plants (1) Extracting DNA Collect approximately 0.2 g of fresh leaves, place them in a 2 mL explosion-proof centrifuge tube with a steel ball, and rapidly freeze them in liquid nitrogen. While still cold, place the tube in an ultrasonic grinder and grind it into powder. Add 800 μL of SDS micro-extraction buffer and react in a 65℃ metal bath for 30 min. Add chloroform:isoamyl alcohol:ethanol (76:4:20, volume ratio) in equal proportions, gently invert 7-8 times, centrifuge at 12000 rpm for 12 min, discard the supernatant, add an equal volume of isopropanol or twice the amount of ethanol, carefully mix by inverting, centrifuge at 12000 rpm for 5 min, take 0.5 mL of 70% ethanol to double wash the extracted DNA, centrifuge at 1 min intervals, dry at 65℃, and dissolve the DNA in 100 μL of TE buffer or sterile water.

[0053] (2) Identification of positive plants After successfully extracting DNA from resistant plants, primers Cas9-F and Cas9-R, designed for CRISPR / Cas9 knockout vectors, can be used to detect whether the vector has been successfully transferred. Figure 3 ), and amplification of the highly conserved ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit gene in the plant chloroplast genome using primers rbcL-F and rbcL-R (Table 2). rbcL ) is used as an internal reference gene to ensure that there are no quality problems with PCR and DNA. Figure 4 The results show: casfbx22 -1 and casfbx22 Two positive plants were successfully transformed into the CRISPR / Cas9 knockout vector.

[0054] A 20 μL PCR reaction system was used: 10 μL of 2 × GS Taq polymerase chain reaction mixture, 1 μL of 10 μmol / L primers (i.e., both forward and reverse primers were 10 μmol / L, and the volume was 1 μL), 1 μL of DNA template, and ddH2O was added to a final volume of 20 μL. The reaction procedure was as follows: initial stage: 95℃ pre-denaturation for 5 min; 94℃ denaturation stage for 25 sec; 58℃ annealing stage for 25 sec; 72℃ extension stage for 15 sec, for a total of 30-35 cycles; extension at 72℃ for 5 min. Primer sequences are shown in Table 2.

[0055] Table 2 Primer information for identifying positive plants (3) Amplification and sequencing of mutation target sites in positive plants After successfully identifying positive plants, specialized primers need to be designed to detect gene editing. First, find... casfbx22 The target region for gene editing was identified. Based on the DNA sequence of this region, primers were designed to precisely bind upstream and downstream of the target site. The designed primers were used to amplify the DNA samples from positive plant plants using PCR. After amplification, the PCR products were detected by 1% agarose gel electrophoresis. The presence of bands of the expected size was observed under a gel imaging system. If the band position and brightness met expectations, the PCR amplification was successful. After successful detection, the samples were sent to a sequencing company for Sanger sequencing.

[0056] PCR amplification was performed using a 20 μL reaction system: 10 μL 2× Gflex buffer, 0.5 μL Tks Gflex DNA polymerase, 0.6 μL 10 μmol / L primers (i.e., fbx22cas9-F1 and fbx22cas9-R1, both at 10 μmol / L, and used in 1 μL volumes), 0.3 μL template DNA, and ddH2O to a final volume of 20 μL. The reaction conditions were: 94℃ pre-denaturation for 1 min; 98℃ denaturation for 10 sec, 60℃ annealing for 15 sec, and 68℃ extension for 30 sec, for a total of 30-35 cycles. Primer sequences are shown in Table 3.

[0057] Table 3 Primer information for amplification of mutant target sites in positive plants Paste the obtained wild-type reference sequence into the corresponding input box of the DSDecodeM module on the CRISPR-GE webpage (http: / / skl.scau.edu.cn / ). The selected reference sequence must completely overlap with the full length of the sequencing file to be analyzed later. Next, upload the sequencing peak diagram in ab1 format. After uploading the sequencing peak diagram, run the decoding to obtain two allele sequences. Carefully compare them with the initially input reference wild-type sequence to clearly distinguish the mutation patterns of the sequences, which are base deletions and substitutions.

[0058] (4) Obtaining mutants and amplifying and sequencing target sites For the selected positive plants, specific primers were designed for PCR amplification. After Sanger sequencing, the sequence alignment results showed that: casfbx22-1 Both alleles of the mutant have a single base (A) missing at the target site, making it a homozygous deletion mutant. casfbx22-2 The two alleles exhibited a two-base deletion (GA) and a base substitution, respectively, resulting in a biallelic mutant. The two mutants were named... casfbx22-1 and casfbx22-2 . casfbx22 The genotype identification results of the CRISPR / Cas9 editing mutants are shown in Table 4.

[0059] Table 4 casfbx22 Genotyping and Sequence Analysis of CRISPR / Cas9 Editing Mutants 8. Pollen fertility Sowing: Before germination, bake the seeds in a 42℃ oven overnight (this ensures high germination rate and uniformity). Soak them in clean water for 24 hours (8-10 hours in hot summer conditions). After soaking, rinse and air dry for 12 hours. Soak again for 12 hours, drain, and then germinate. Wrap the seeds in a damp cloth or paper towel and place them in a 30℃ incubator. During this period, maintain... Keep moist (but not waterlogged; too much water promotes root growth, too little water promotes sprouting), and turn the seed several times. The seed should sprout from the point where it shows white sprouts to 0.5 cm in diameter (not...). If the seedlings are larger than 0.5 cm, they can be sown.

[0060] Rice growth and development successively goes through the germination and seedling stage, tillering stage, jointing stage, and booting stage. After rice grows to the booting stage, both wild-type ZH11 and mutant varieties are harvested. casfbx22-1, casfbx22-2 Physiologically mature flowers were stained with I2-KI solution for pollen grains. Then, the stained pollen grains were observed and images were acquired using an optical microscope. Pollen grains with black staining and plump shape were identified as fertile. Pollen grains that were poorly developed, collapsed, irregular in structure, and not black were classified as sterile. The research results were then imaged.

[0061] Depend on Figure 5 and Figure 6 It can be seen that the mutant casfbx22 The plant height of the strain was significantly higher than that of the wild type ZH11.

[0062] Depend on Figures 7-12 It can be seen that under the high summer temperatures (30℃) in Guangzhou fields, compared with the wild type, the mutant... casfbx22 The fertility of the mutant was significantly lower than that of the wild type. Under low autumn temperatures (23℃) in a field in Guangzhou, the mutant... casfbx22 Fertility was significantly increased. The above results indicate that... casfbx22 The gene is a thermosensitive male sterility regulatory gene that regulates pollen fertility by responding to changes in environmental temperature.

[0063] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Rice OsFBX22 protein or rice OsFBX22 The application of genes in any of the following: (1) Rice breeding; (2) Cultivating temperature-sensitive male-sterile rice lines; The amino acid sequence of the rice OsFBX22 protein is shown in SEQ ID NO.2; the rice OsFBX22 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The application works by inhibiting or knocking out the activity of rice OsFBX22 in rice. OsFBX22 Gene.

3. The application according to claim 1, characterized in that, The phenotype of the rice thermosensitive male sterile line is high-temperature sterility, with fertility restored at low temperatures.

4. Knock out rice plants OsFBX22 The application of gene-based biomaterials in any of the following: (1) Rice breeding; (2) Cultivating temperature-sensitive male-sterile rice lines; The rice OsFBX22 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

5. The application according to claim 4, characterized in that, The phenotype of the rice thermosensitive male sterile line is high-temperature sterility, with fertility restored at low temperatures.

6. The application according to claim 4, characterized in that, The biomaterials include recombinant vectors or recombinant bacteria.

7. A method for cultivating a temperature-sensitive male-sterile rice line, characterized in that, Including the rice described in the knockout rice OsFBX22 The steps of gene generation; the rice OsFBX22 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

8. The method according to claim 7, characterized in that, The phenotype of the rice thermosensitive male sterile line is high-temperature sterility, with fertility restored at low temperatures.

9. The method according to claim 7, characterized in that, The knockout method includes CRISPR-Cas9.

10. A method for producing hybrid rice seeds, characterized in that, Includes the following steps: Knockout rice OsFBX22 Genes were used to obtain a temperature-sensitive male-sterile rice line; Under low temperature conditions, the rice thermosensitive male sterile line was propagated to obtain rice thermosensitive male sterile line seeds; After sowing the seeds of the temperature-sensitive male-sterile rice line, the rice was crossed with the restorer line male parent under high temperature conditions to obtain hybrid rice seeds.

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