Application of rice gene OsFRD4 to regulation and control of rice fertility and mutant of rice gene OsFRD4

Abstract

The invention belongs to the technical field of crop breeding, and relates to application of a rice gene OsFRD4 to regulation and control of rice fertility and a mutant of the rice gene OsFRD4. The coding sequence of the rice gene OsFRD4 is as shown in SEQ ID NO.3, and the amino acid sequence of the protein coded by the rice gene OsFRD4 is as shown in SEQ ID NO.4. The new application of the rice gene OsFRD4 in regulation and control of the rice fertility is found, a constructed rice modified plant containing the modified rice gene OsFRD4 can significantly reduce the rice fertility, and the improvement of the rice fertility character provides a new gene resource for rice breeding. Therefore, a novel molecular breeding approach is provided for cultivating a novel rice sterile line.

Description

Technical Field

[0001] This invention belongs to the field of crop breeding technology, and relates to the use of the rice gene OsFRD4 in regulating rice fertility and its mutants. Background Technology

[0002] Rice (Oryza sativa L.) is one of the world's most important food crops and an important model plant in molecular biology and plant genomics research. In addition, rice is an excellent model system plant for studying basic cellular processes such as pollen germination, pollen tube elongation, and pollen-stigma recognition.

[0003] On the one hand, rice fertility directly affects rice yield; on the other hand, hybrid rice technology, with rice male-sterile lines at its core, fully utilizes heterosis in rice, making significant contributions to increasing rice yield and quality. The discovery of rice male-sterile lines has brought about revolutionary developments in rice breeding. The continued development and utilization of rice male-sterile line resources are of great significance to global food security.

[0004] In recent years, significant progress has been made in rice fertility research. Genetic analysis shows that most rice fertility is regulated by nuclear genes. To date, some key genes involved in rice fertility have been identified and studied, and mutations in these genes lead to the sterile phenotype in rice. However, the regulatory mechanisms of rice fertility are not yet fully understood. Identification of rice sterility genes will contribute to a more comprehensive understanding of the molecular mechanisms of rice sterility and accelerate the process of rice breeding using sterile lines. Summary of the Invention

[0005] The primary objective of this invention is to provide the use of the rice gene OsFRD4 for regulating rice fertility, so as to enable better regulation of rice fertility.

[0006] To achieve this objective, in a basic embodiment, the present invention provides the use of rice gene OsFRD4 (Os04g0559200) for regulating rice fertility, wherein the coding sequence of said rice gene OsFRD4 is shown in SEQ ID NO.3, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.4.

[0007] A second objective of this invention is to provide a modified rice gene, OsFRD4, that can significantly reduce the fertility of rice.

[0008] To achieve this objective, in a basic implementation, the present invention provides a modified rice gene OsFRD4, the coding sequence of which is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0009] A third objective of this invention is to provide a method for constructing a modified rice plant containing the modified rice gene OsFRD4 as described above, so as to obtain a modified rice plant containing the modified rice gene OsFRD4 as described above, which can significantly reduce the fertility of rice.

[0010] To achieve this objective, in a basic implementation scheme, the present invention provides a method for constructing a modified rice plant containing the modified rice gene OsFRD4 as described above. The construction method is based on the principle of CRISPR / Cas9 gene editing, in which a gene carrying the expression of Cas9 protein and an expression vector carrying sgRNA are transferred into a primitive rice plant. After screening and culture, a modified rice plant containing the modified rice gene OsFRD4 as described above is obtained.

[0011] This invention uses a CRISPR / Cas9 gene editing vector to induce a frameshift mutation in the rice gene OsFRD4, thereby causing the protein sequence encoding SEQ ID NO.4 to stop translation prematurely, resulting in the loss of protein function of SEQ ID NO.4.

[0012] Preferably, the construction of the above expression vector may include: screening and designing the OsFRD4 gene editing target sequence, amplifying the rice OsU6a promoter, constructing a gene editing expression cassette, and obtaining the CRISPR / Cas9 gene editing vector of the OsFRD4 gene through steps such as fragment and vector enzyme digestion, T4-DNA ligation, transformation of Escherichia coli DH5α competent cells, single-clone colony PCR identification and vector sequencing.

[0013] Preferably, obtaining the above-mentioned modified rice plants may include: using Agrobacterium-mediated transformation, directly transforming the constructed OsFRD4 gene CRISPR / Cas9 gene editing vector into Nipponbare callus / cells, followed by steps such as hygromycin screening, resistant callus differentiation, and rooting culture to obtain OsFRD4 gene editing vector positive plants. Leaves from each positive plant are collected, and the individual rice genome is isolated using the CTAB method. The obtained OsFRD4 gene-edited positive plants are then analyzed using PCR, agarose gel electrophoresis, target fragment recovery, and sequencing to determine whether they meet the modification requirements. The qualified OsFRD4 gene-edited plants are transplanted to the field, and the fertility of the rice is analyzed and statistically analyzed during the grain-filling stage to determine the changes in fertility of the OsFRD4 gene-edited plants compared to wild-type Nipponbare.

[0014] In a preferred embodiment, the present invention provides a method for constructing a modified rice plant containing the modified rice gene OsFRD4 as described above, wherein the target sequence of the sgRNA is shown in SEQ ID NO.5 or SEQ ID NO.6.

[0015] In a preferred embodiment, the present invention provides a method for constructing a modified rice plant containing the modified rice gene OsFRD4 as described above, wherein the expression vector contains a hygromycin resistance gene and / or a kanamycin resistance gene.

[0016] In a preferred embodiment, the present invention provides a method for constructing a modified rice plant containing the modified rice gene OsFRD4 as described above, wherein the expression vector is transformed into the original rice plant by Agrobacterium-mediated transformation.

[0017] In a preferred embodiment, the present invention provides a method for constructing a modified rice plant containing the modified rice gene OsFRD4 as described above, wherein the original rice plant is selected from one or more of the following japonica rice varieties: Nipponbare, Zhenshan 97, 9311, and Minghui 63.

[0018] In a preferred embodiment, the present invention provides a method for constructing a modified rice plant containing the modified rice gene OsFRD4 as described above, wherein the screening and culture include callus induction, Agrobacterium activation and infection, co-culture, screening and differentiation of resistant callus, rooting culture, identification of positive plants, hardening off, and transplanting to the field.

[0019] A fourth object of the present invention is to provide the use of the modified rice gene OsFRD4 as described above for regulating rice fertility.

[0020] To achieve this objective, in a basic implementation, the present invention provides the use of the modified rice gene OsFRD4 as described above for regulating rice fertility.

[0021] The beneficial effects of this invention are that it discovers a new use of the rice gene OsFRD4 to regulate rice fertility. The modified rice plants containing the modified rice gene OsFRD4 of this invention can significantly reduce rice fertility. Such improvement of rice fertility traits provides new gene resources for rice breeding, thereby providing a new molecular breeding approach for cultivating new sterile rice lines.

[0022] The modified rice gene OsFRD4 and the modified rice plants of this invention are of great significance for studying the regulatory pathways of rice fertility. They provide a novel breeding scheme for the rational and appropriate use of the OsFRD4 gene to cultivate sterile lines and have a very broad application prospect. Attached Figure Description

[0023] Figure 1 is a schematic diagram of the pOsU6a and pCRISPR / Cas9 related plasmid vectors. Figure 1AIn this context, U6apromoter represents the U6a promoter sequence of rice, Insert site is indicated by a black box, representing the insertion location of the target sequence, sgRNA represents the scaffold sequence of the editing vector, and AMP... R This represents the ampicillin resistance gene sequence, AMP. R promoter indicates the promoter used to express the ampicillin resistance gene sequence, Ori indicates the plasmid replication origin, and BsaI and BsmBI are the restriction enzyme sites used to construct the vector; Figure 1B In the text, CaMV 35S promoter is the 35S promoter sequence of cauliflower leaf virus, Hyg... R This is the sequence of the hygromycin resistance gene. CaMV poly(A)signal represents the CaMV transcription termination sequence, KanR represents the kanamycin resistance gene sequence, LB T-DNA repeat and RB T-DNA repeat represent the left and right boundary sequences, respectively, Ubi-promoter represents the Ubiquitin promoter sequence of the maize ubiquitin protein gene, Cas9 represents the Cas9 nuclease gene sequence, NOSterminator represents the NOS transcription termination sequence, and ccdB represents a gene sequence that is toxic to Escherichia coli and is used to improve the screening of positive clones. Figure 1C and 1D This is a schematic diagram showing the insertion of the two target sequences of OsFRD4 into the pOsU6a vector; Figure 1E and 1F This is a schematic diagram showing the insertion of the two OsFRD4 target sequence expression cassettes into the pCRISPR / Cas9 vector.

[0024] Figure 2 This diagram shows the target sequence for OsFRD4 gene editing and the target sequence of OsFRD4 gene-editing positive plants. In the diagram, black boxes represent exons, black lines represent introns, ATG represents the start codon, TAA represents the stop codon, and X represents the deleted base.

[0025] Figure 3 shows the fertility analysis results of plants edited with the OsFRD4 gene. Figure 3A The fertility phenotype of rice plants with OsFRD4 gene editing during the late grain-filling stage, scale bar 10cm; Figure 3B The panicle phenotype of rice plants with OsFRD4 gene editing during the late grain-filling stage, scale bar 1cm; Figure 3A and Figure 3B The left side is WT, the middle is M1-1, and the right side is M2-1; Figure 3CThe fertility statistics of mature OsFRD4 gene-edited plants are presented. The average seed setting rate of WT was 90.11%, that of M1-1 was 0.85%, and that of M2-1 was 0.88%; *: p < 0.05 indicates a significant difference. WT: Nipponbare (wild type); M1-1: Plants obtained after gene editing of OsFRD4 target sequence 1; M2-1: Plants obtained after gene editing of OsFRD4 target sequence 2. Detailed Implementation

[0026] The following examples further illustrate specific embodiments of the present invention. Unless otherwise specified, the experimental methods used in the examples are conventional experimental methods; primer sequence synthesis and DNA fragment sequencing analysis were all performed by Shanghai Sangon Biotech Co., Ltd.

[0027] Example 1: Construction of OsFRD4 gene editing vector and its genetic transformation in rice

[0028] The coding sequence of the rice OsFRD4 gene was obtained from the NCBI database (https: / / www.ncbi.nlm.nih.gov), as shown in SEQ ID NO.3, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.4. Target sequences for gene editing were screened using the online software CRISPR-GE (skl.scau.edu.cn / home / ), and two suitable sequences, SEQ ID NO.5 and SEQ ID NO.6, were selected and named Target-1 and Target-2, respectively. Based on the OsU6a vector restriction enzyme sites, primer sequences were designed for Target-1: the forward primer Target1-F sequence is shown in SEQ ID NO.7, and the reverse primer Target1-R sequence is shown in SEQ ID NO.8. Similarly, primer sequences were designed for Target-2: the forward primer Target2-F sequence is shown in SEQ ID NO.9, and the reverse primer Target2-R sequence is shown in SEQ ID NO.10.

[0029] pOsU6a uses pUC57 as its vector backbone. First, point mutations are performed on the BsmBI and BsaI restriction sites in pUC57 to eliminate these sites. Then, the vector is linearized using the restriction endonuclease EcoRV, and the U6a-sgRNA expression cassette sequence is inserted into the linearized pUC57 sequence, forming the intermediate expression cassette vector for Cas9. The pOsU6a vector structure is shown below. Figure 1A As shown.

[0030] pCRISPR / Cas9 uses pCABMBIA1300 as its backbone. First, a point mutation is performed on the Bsa1 restriction site in pCABMBIA1300 to eliminate this restriction site. Then, the vector is linearized using restriction endonucleases KpnI and HindIII. The Ubiquitin promoter sequence, Cas9 protein sequence, and ccdB sequence are inserted into the linear sequence of pCABMBIA1300, thus constructing the pCRISPR / Cas9 gene editing vector. The pCRISPR / Cas9 vector structure is shown below. Figure 1B As shown.

[0031] First, the primer pairs for target 1 and target 2 were dissolved in sterile ultrapure water to prepare 100 μM stock solutions. Next, target adapters were prepared according to the reaction system in Table 1 (the reaction was carried out in 1.5 mL centrifuge tubes; incubation at 95℃ for 30 s, followed by incubation at room temperature for 5 min, until adapter preparation was complete). Then, the target adapters were incubated with the pOsU6a plasmid vector according to the reaction system in Table 2 (37℃ for 5 min, 10℃ for 1 min, 20℃ for 5 min, for 5 cycles). Finally, the reaction products were transformed into DH5α competent E. coli cells, placed on ice for 30 min, heat-shocked at 42℃ for 30 s, then placed on ice for 2 min, and 500 μL of LB liquid medium was added. The mixture was incubated at 37℃ on a shaker at 220 rpm for 30 min. Finally, the product was evenly spread in LB solid culture dishes containing AMP (50 mg / L). Colony PCR was performed using specific primers (SEQ ID NO.11 and SEQ ID NO.8; or SEQ ID NO.11 and SEQ ID NO.10). Positive single clones were selected for sequencing analysis. The correctly sequenced vectors were named pOsU6a-Target1 and pOsU6a-Target2, and their structures are shown below. Figure 1C and Figure 1D .

[0032] Table 1 Target-Linker Reaction System

[0033]

[0034] Table 2 Reaction system of target linker and pOsU6a plasmid vector

[0035]

[0036] pOsU6a-Target1 and pOsU6a-Target2 were digested with Bsa1, and gene editing expression cassette fragments (approximately 500 bp) were recovered by agarose gel electrophoresis. The recovered fragments were incubated with the pCRISPR / Cas9 vector according to the reaction system in Table 3 (37℃ for 5 min, 10℃ for 1 min, 20℃ for 5 min, 10 cycles). Finally, the reaction products were transformed into competent DH5α *E. coli* cells. Colony PCR was performed using specific primers (SEQ ID NO. 12 and SEQ ID NO. 8; or SEQ ID NO. 12 and SEQ ID NO. 10). Positive single clones were picked and cultured, and plasmids were extracted. Those with correct sequencing were identified as OsFRD4 gene editing vectors, named pCRISPR / Cas9-OsFRD4-Target1 and pCRISPR / Cas9-OsFRD4-Target2, respectively. Their structures are shown below. Figure 1E and Figure 1F .

[0037] Table 3. Reaction system of recovered fragments with pCRISPR / Cas9 plasmid

[0038]

[0039] Based on Agrobacterium-mediated transformation, Nipponbare (wild-type WT) rice was transformed. After callus induction, Agrobacterium activation and infection, co-culture, resistant callus screening and differentiation, rooting culture, positive plant identification, hardening and transplanting to the field, 10 genetically transformed T0 positive rice plants were obtained. The specific operations are as follows.

[0040] 1) Inducing callus

[0041] Select mature and plump rice seeds, remove the husks, add an appropriate amount of 75% ethanol and let stand for 1 minute, then add an appropriate amount of 1% sodium hypochlorite solution and let stand for 15 minutes. Remove the sodium hypochlorite solution, rinse the seeds with sterile water 3-5 times, and place the treated rice seeds in a callus induction medium (composition shown in Table 4A) and place them in a 32℃ plant growth chamber for 7-10 days.

[0042] 2) Agrobacterium activation

[0043] Two days before infection, Agrobacterium EHA105 containing the target gene plasmid vector was streaked on LB solid medium containing 50 mg / L Kan and then cultured at 28°C.

[0044] 3) Preparation, infection, and co-culture of Agrobacterium

[0045] Before infection, Agrobacterium was transferred from LB solid medium to suspension medium (composition shown in Table 4B) and incubated at 28°C and 180 rpm for 3.5 h. Then, the Agrobacterium concentration was adjusted to OD. 600 =0.1-0.2. Transfer the callus tissue induced for 7-10 days into an Agrobacterium suspension, let stand for 1.5 min, discard the Agrobacterium suspension, and use sterile filter paper to absorb the bacterial suspension from the surface of the callus. Cover the callus surface with sterile green plants and let it stand in a clean bench for 30 min. Transfer the callus tissue into a co-culture medium (composition shown in Table 4C) with a layer of sterile filter paper on the surface, first incubate in the dark at 20℃ for 12-14 h, then transfer to a 25℃ incubator for further dark incubation for 2 days.

[0046] 4) Elimination of Agrobacterium

[0047] After co-culturing, the callus tissue was transferred to a sterile Erlenmeyer flask, rinsed three times with sterile water for 30 seconds each time, then rinsed five to six times with sterile water. Finally, it was soaked in sterile water containing 500 mg / L Cn for 30 minutes. The Cn sterile water was removed, and the surface moisture of the callus tissue was blotted dry with sterile filter paper. A layer of sterile filter paper was then placed on the flask and allowed to stand for 1 hour in a laminar flow hood to dry the surface moisture of the callus tissue.

[0048] 5) Screening for resistant callus

[0049] The rinsed callus tissue was placed on the resistance selection medium (composition shown in Table 4D) and cultured at 32°C for 14 days.

[0050] 6) Differentiation of resistant callus

[0051] After 14 days of screening, the resistant callus was transferred to differentiation medium (composition shown in Table 4E) and cultured in a plant growth incubator at 28°C.

[0052] 7) Rooting culture

[0053] When the resistant callus tissue grows to 3-4 cm in length on the differentiation medium, it is transferred to the rooting medium (composition shown in Table 4F) until it grows into a complete plant. After identification by PCR and sequencing, it is ready for use.

[0054] Table 4A Composition of callus induction medium

[0055]

[0056] Add the above reagents to the beaker in sequence. First, add 900 mL of distilled water. Adjust the pH of the culture medium solution to 5.8 with KOH solution (1M). Add distilled water to make up to 1 L. Then, autoclave at 121℃ for 15 min. Dispense the culture medium into petri dishes in a clean bench and let it cool before use.

[0057] Table 4B Composition of Suspension Culture Medium

[0058]

[0059] Add the above reagents to a beaker, first add 200 mL of distilled water, adjust the pH of the suspension culture medium solution to 5.2 with KOH solution (1M), add distilled water to make up to 250 mL, autoclave at 121℃ for 15 min, and add 5 mL of 50% glucose solution and 250 μL of AS stock solution when using.

[0060] Table 4. Composition of C co-culture medium

[0061]

[0062] Add the above reagents to the beaker in sequence. First, add 200 mL of distilled water. Adjust the pH of the co-culture medium solution to 5.6 with KOH solution (1M). Add distilled water to make up to 250 mL. Autoclave at 121℃ for 15 min. Before use, add 5 mL of 50% glucose and 250 μL of AS stock solution.

[0063] Table 4. Composition of the resistance screening medium.

[0064]

[0065] Add the above reagents to a beaker in sequence. First, add 200 mL of distilled water. Adjust the pH of the screening medium to 6.0 with KOH solution (1M). Add distilled water to bring the volume to 250 mL. Autoclave at 121°C for 15 min. When the temperature has cooled to 60°C, add 250 μL of hygromycin (50 mg / mL) and 500 μL of Cn. Pour the mixture into a petri dish on a clean bench and allow it to cool before use.

[0066] Table 4E Differentiation Medium Composition

[0067]

[0068] Add the above reagents to the beaker in sequence, first add 900 mL of distilled water, adjust the pH of the differentiation medium to 5.8 with KOH solution (1M), bring the volume to 1 L, autoclave at 121℃ for 15 min, pour into a culture dish on a clean bench, and let cool before use.

[0069] Table 4. Composition of F-rooting medium

[0070]

[0071] Add the above reagents to the beaker in sequence. First, add 900 mL of distilled water. Adjust the pH of the rooting medium to 5.8 with KOH solution (1M). Add distilled water to make up to 1 L. Autoclave at 121℃ for 15 min. Pour the mixture into a petri dish on a clean bench and let it cool before use.

[0072] 1.MSmax Stock solution

[0073]

[0074] Add the contents to a beaker, add distilled water to dissolve completely, and then bring the volume up to 1L.

[0075] 2.MS min Stock solution

[0076]

[0077]

[0078] Add the contents to a beaker, add distilled water to dissolve completely, and then bring the volume up to 1L.

[0079] 3.N6 max Stock solution

[0080]

[0081] Add the contents to a beaker, add distilled water to dissolve completely, and then bring the volume up to 1L.

[0082] 4.N6 min Stock solution

[0083]

[0084] Add the contents to a beaker, add distilled water to dissolve completely, and then bring the volume up to 1L.

[0085] 5. Fe salt stock solution

[0086] FeSO4·7H2O 2.78g

[0087] EDTANa2·2H2O 3.73g

[0088] Add FeSO4·7H2O and EDTANa2·2H2O to 300mL of distilled water respectively, mix, heat to 70℃ and keep warm for 2h, then dilute the water to 1L and store at 4℃ in the dark for later use.

[0089] 6. Vitamin stock solution

[0090]

[0091] Add the above reagents in sequence, then add distilled water to bring the volume to 1L. Store at 4℃ for later use.

[0092] 7. Kinetin (KT) stock solution

[0093] Weigh 100 mg KT, add 1 mL KOH (1 M) and stir until completely dissolved. Then add distilled water to bring the volume to 100 mL and store at 4°C for later use.

[0094] 8.2,4-Dichlorophenoxyacetic acid (2,4-D) stock solution

[0095] Weigh 100 mg of 2,4-D, add 1 mL of KOH (1 M) and stir until completely dissolved. Then add distilled water to bring the volume to 100 mL and store at 4°C for later use.

[0096] 9. Naphthaleneacetic acid (NAA) stock solution

[0097] Weigh 100 mg NAA, add 1 mL KOH (1 M) and stir until completely dissolved. Then add distilled water to bring the volume to 100 mL and store at 4°C for later use.

[0098] 10. Acetosyringone (AS) stock solution

[0099] Weigh 0.39g AS and dissolve it in 10mL DMSO. Aliquot the solution into 1.5mL centrifuge tubes and store at -20℃ for later use.

[0100] 11. Carbenicillin (Cn)

[0101] Weigh 2.5g Cn, add sterile water to 10mL in a clean bench, dissolve completely, dispense into 1.5mL centrifuge tubes, and store at -20℃ for later use.

[0102] 12. Kanamycin (Kan)

[0103] Weigh 0.5g of Kan, add sterile water to 10mL in a clean bench, dissolve completely, dispense into 1.5mL centrifuge tubes, and store at -20℃ for later use.

[0104] 13.50% glucose solution

[0105] Weigh 50g of glucose, add distilled water, bring the volume to 100mL, autoclave at 121℃ for 15min, and store at 4℃ for later use.

[0106] 14.KOH solution

[0107] Weigh 5.6g of KOH into a beaker, add distilled water to a final volume of 100mL, and store at room temperature for later use.

[0108] 15. LB solid culture medium

[0109] Weigh 10g of trypsin, 5g of yeast extract, and 10g of sodium chloride, add distilled water to a final volume of 1L, autoclave at 121℃ for 15min, and once the solution temperature has dropped to around 60℃, dispense the solution into culture dishes in a clean bench and let it cool before use.

[0110] 8) Identification of positive plants

[0111] Genomic DNA from rice plants was isolated using the CTAB method. Positive plants were identified by PCR using the plant genomic DNA as a template (forward primer sequence as shown in SEQ ID NO.13, reverse primer sequence as shown in SEQ ID NO.14) (denaturation at 95℃ for 2 min, followed by denaturation at 95℃ for 20 s, annealing at 60℃ for 20 s, and extension at 72℃ for 40 s, with a cycle length of 30). The PCR reaction solution was then analyzed by agarose gel electrophoresis to confirm gene-edited positive plants.

[0112] Positive plants transformed by pCRISPR / Cas9-OsFRD4-Target1 and pCRISPR / Cas9-OsFRD4-Target2 were named lines M1 and M2, respectively.

[0113] 9) Hardening off seedlings and transplanting to the field

[0114] Rice seedlings grown in plant culture boxes were unsealed and placed at room temperature for 2 days. Positive seedlings were then transplanted to the field, and seeds were harvested for subsequent experiments.

[0115] Example 2: Molecular identification of OsFRD4 genome sequence modification

[0116] Based on the target sequence information of the OsFRD4 gene, a pair of specific primers were designed approximately 100-200 bp upstream and downstream of the target sequence (the forward primer sequence is shown in SEQ ID NO.15, and the reverse primer sequence is shown in SEQ ID NO.16). Using genomic DNA from Nipponbare (wild type) and the positive plants obtained in Example 1 as PCR templates, PCR amplification was performed on the OsFRD4 target region. Fragment recovery and sequencing analysis were then performed to identify whether the OsFRD4 gene in the T0 generation positive plants had been edited. The results showed that the target site sequence in plant M1-1 was missing 2 bases, while that in plant M2-1 was missing 5 bases. Plants M1 and M2, whose OsFRD4 target sequences had deletions and premature termination of the coding amino acid sequence, were selected as plants for subsequent studies. Progeny separation was performed on the OsFRD4 candidate plants to obtain OsFRD4 gene-edited plants M1-1 and M2-1 without T-DNA for subsequent phenotypic identification.

[0117] The target sequences for OsFRD4 gene editing and the target sequences of OsFRD4 gene-edited positive plants are as follows: Figure 2 As shown.

[0118] Example 3: Fertility detection of OsFRD4 gene-edited plants

[0119] Rice OsFRD4 gene-edited plants (M1-1 and M2-1) and wild-type Nipponbare (WT) were simultaneously planted in the field (100 plants each, 10 rows, 10 plants per row, plant spacing 25 cm, row spacing 30 cm) to observe phenotypic differences between them during the grain-filling period.

[0120] The observation results during the later stage of rice grain filling are as follows: Figure 3A and 3B The statistical results are as follows Figure 3C Comparative and statistical analysis revealed that:

[0121] In terms of fertility, M1-1 and M2-1 had almost no grain filling, showing significantly lower fertility than the wild type Nipponbare. The average seed setting rate of M1-1 plants was 0.85%, a decrease of 99.06% compared to the average seed setting rate of 90.11% for the wild type. The average seed setting rate of M2-1 plants was 0.88%, a decrease of 99.02% compared to the average seed setting rate of 90.11% for the wild type.

[0122] Grain filling rate = number of grains filling / total number of grains.

[0123] This shows that the fertility of plants with the OsFRD4 gene edited was significantly lower than that of wild-type plants, thus proving that the OsFRD4 gene is involved in regulating rice fertility.

[0124] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims and their equivalents, this invention is also intended to include these modifications and variations. The above embodiments or implementations are merely illustrative examples of this invention, and it can also be implemented in other specific ways or forms without departing from its gist or essential characteristics. Therefore, the described embodiments should be considered illustrative rather than limiting in any respect. The scope of this invention should be defined by the appended claims, and any changes equivalent to the intent and scope of the claims should also be included within the scope of this invention.

Claims

1. The use of rice gene OsFRD4 for regulating rice fertility, wherein the coding sequence of rice gene OsFRD4 is shown in SEQ ID NO.3, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.

4.

2. A modified rice gene OsFRD4, characterized in that: The coding sequence of the modified rice gene OsFRD4 is shown in SEQ ID NO.1 or SEQ ID NO.

2.

3. A method for constructing a modified rice plant containing the modified rice gene OsFRD4 as described in claim 2, characterized in that: The construction method described herein is based on the principle of CRISPR / Cas9 gene editing. A gene carrying the expression of Cas9 protein and an expression vector carrying sgRNA are transferred into original rice plants. After screening and cultivation, modified rice plants containing the modified rice gene OsFRD4 described in claim 2 are obtained.

4. The construction method according to claim 3, characterized in that: The target sequence of the sgRNA is shown in SEQ ID NO.5 or SEQ ID NO.

6.

5. The construction method according to claim 3, characterized in that: The expression vector contains a hygromycin resistance gene and / or a kanamycin resistance gene.

6. The construction method according to claim 3, characterized in that: The expression vector was used to transform the original rice plants via Agrobacterium-mediated transformation.

7. The construction method according to claim 3, characterized in that: The original rice plants mentioned are selected from one or more of the following japonica rice varieties: Nipponbare, Zhenshan 97, 9311, and Minghui 63.

8. The construction method according to claim 3, characterized in that: The screening and culture process includes callus induction, Agrobacterium activation and infection, co-culture, screening and differentiation of resistant callus, rooting culture, identification of positive plants, hardening off, and transplanting to the field.

9. The use of the modified rice gene OsFRD4 according to claim 2 for regulating rice fertility.

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