Use of rice gene OsFER2 for regulating rice plant type and its mutant

Abstract

The present invention belongs to the technical field of crop breeding and relates to the use of the rice gene OsFER2 for regulating rice plant type and its mutants. The coding sequence of the rice gene OsFER2 is shown in SEQ ID NO. 3, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO. 4. The present invention has discovered a new use of the rice gene OsFER2 for regulating rice plant type. The constructed rice plants containing the modified rice gene OsFER2 of the present invention can significantly reduce rice plant height and tillering. Such improvements in rice plant height and tillering traits provide new genetic resources for rice breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of crop breeding, and relates to the use of the rice gene OsFER2 for regulating rice plant type and its mutants. Background Art

[0002] Rice (Oryza sativa L.) is a staple crop for half of the world's population.

[0003] In modern agriculture, plant height is a key agronomic trait of crops. The rice "Green Revolution" focused on breeding shorter, higher-yielding rice varieties. The semi-dwarf gene sd-1 plays a crucial role in modern rice breeding, as reducing plant height can effectively prevent lodging.

[0004] Rice tillering is also an important agronomic trait that affects rice yield. The number of effective tillers determines the number of rice panicles, and the number of panicles is one of the three factors affecting rice yield. Therefore, an appropriate number of effective rice tillers is conducive to high rice yield.

[0005] In summary, regulating rice plant height and tiller number is helpful for the breeding of high-yield rice varieties. Summary of the Invention

[0006] The primary purpose of the present invention is to provide a method for regulating rice plant type by using the rice gene OsFER2, so as to better regulate rice plant type.

[0007] To achieve this object, in a basic embodiment, the present invention provides the use of the rice gene OsFER2 (Os12g0106000) for regulating rice plant type, wherein the coding sequence of the rice gene OsFER2 is shown in SEQ ID NO. 3, and the amino acid sequence of the protein encoded by it is shown in SEQ ID NO. 4.

[0008] The second object of the present invention is to provide a modified rice gene OsFER2, which can significantly reduce the plant height and tillering of rice.

[0009] To achieve this object, in a basic embodiment, the present invention provides a modified rice gene OsFER2, the coding sequence of which is shown as SEQ ID NO. 1 or SEQ ID NO. 2.

[0010] A third object of the present invention is to provide a method for constructing a modified rice plant containing the modified rice gene OsFER2 as described above, so as to construct a modified rice plant containing the modified rice gene OsFER2 as described above, wherein the modified rice plant can significantly reduce the plant height and tillering of rice.

[0011] To achieve this objective, in a basic embodiment, the present invention provides a method for constructing a modified rice plant containing the modified rice gene OsFER2 as described above. The construction method is based on the principle of CRISPR / Cas9 gene editing, and an expression vector carrying a gene expressing the Cas9 protein and carrying sgRNA is transferred into the original rice plant. After screening and cultivation, the modified rice plant containing the modified rice gene OsFER2 as described above is obtained.

[0012] The present invention uses a CRISPR / Cas9 gene editing vector to cause a frameshift mutation in the rice gene OsFER2, thereby stopping the translation of the protein sequence encoded by SEQ ID NO. 4 prematurely, resulting in the loss of protein function of SEQ ID NO. 4.

[0013] Preferably, the construction of the above-mentioned expression vector may include: screening and designing the OsFER2 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 OsFER2 gene through the steps of fragment and vector enzyme digestion, T4-DNA ligation, transformation of Escherichia coli DH5α competent cells, single clone colony PCR identification and vector sequencing.

[0014] Preferably, obtaining the modified rice plants may include: directly transforming the constructed CRISPR / Cas9 gene-editing vector for the OsFER2 gene into callus tissue / cells of Zhenshan 97 using Agrobacterium-mediated transfection. Plants positive for the OsFER2 gene-editing vector are obtained through hygromycin selection, callus differentiation, and rooting culture. Leaves from each positive plant are collected, and the individual rice genome is isolated using the CTAB method. PCR, agarose gel electrophoresis, target fragment recovery, and sequencing analysis are performed to determine whether the OsFER2 gene-edited plants meet the transformation requirements. OsFER2 gene-edited plants that meet the requirements are transplanted into the field. Upon reaching maturity, the plant height and tillering are analyzed and counted to determine the differences in plant height and tillering between the OsFER2 gene-edited plants and wild-type Zhenshan 97.

[0015] In a preferred embodiment, the present invention provides a method for constructing a modified rice plant containing the modified rice gene OsFER2 as described above, wherein the target sequence of the sgRNA is shown as SEQ ID NO. 5 or SEQ ID NO. 6.

[0016] In a preferred embodiment, the present invention provides a method for constructing a modified rice plant containing the modified rice gene OsFER2 as described above, wherein the expression vector contains a hygromycin resistance gene and / or a kanamycin resistance gene.

[0017] In a preferred embodiment, the present invention provides a method for constructing a modified rice plant containing the modified rice gene OsFER2 as described above, wherein the expression vector is transformed into the original rice plant by Agrobacterium-mediated method.

[0018] In a preferred embodiment, the present invention provides a method for constructing a modified rice plant containing the modified rice gene OsFER2 as described above, wherein the original rice plant is selected from one or more of Zhenshan 97, 9311, Minghui 63, and japonica rice Nipponbare.

[0019] In a preferred embodiment, the present invention provides a method for constructing modified rice plants containing the modified rice gene OsFER2 as described above, wherein the screening and cultivation include callus induction, Agrobacterium activation and infection, co-cultivation, resistant callus screening and differentiation, rooting culture, positive plant identification, seedling hardening and field transplanting.

[0020] The fourth object of the present invention is to provide the use of the modified rice gene OsFER2 as described above for regulating rice plant height and tillering.

[0021] To achieve this object, in a basic embodiment, the present invention provides the use of the modified rice gene OsFER2 as described above for regulating rice plant height and tillering.

[0022] The present invention has the beneficial effect of discovering a new use of the rice gene OsFER2 for regulating rice plant architecture. The constructed rice plants containing the modified rice gene OsFER2 can significantly reduce rice plant height and tillering. Such improvements in rice plant height and tillering provide a new genetic resource for rice breeding.

[0023] The modified rice gene OsFER2 and the modified rice plants of the present invention are of great significance for studying the regulatory pathways of rice tillering and plant height, and provide a new breeding scheme for rationally and appropriately utilizing the OsFER2 gene to cultivate rice varieties with short stems and appropriate tillering numbers, which has very broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of pOsU6a and pCRISPR / Cas9 related plasmid vector maps. Figure 1 In A, U6apromoter represents the U6a promoter sequence of rice, Insert site is indicated by a black box, which is the insertion position of the target sequence, sgRNA represents the scaffold sequence of the editing vector, AMP RIndicates the ampicillin resistance gene sequence, AMP R promoter represents the promoter used to express the ampicillin resistance gene sequence, Ori represents the plasmid replication origin, and BsaI and BsmBI are the restriction enzyme sites used to construct the vector; Figure 1 In B, CaMV 35S promoter is the 35S promoter sequence of cauliflower virus, Hyg R is the hygromycin resistance gene sequence, CaMV poly (A) signal represents the CaMV transcription terminator sequence, KanR represents the kanamycin resistance gene sequence, LB T-DNA repeat and RB T-DNA repeat represent the left and right border sequences, respectively, Ubi-promoter represents the Ubiquitin promoter sequence of the maize ubiquitin gene, Cas9 represents the nuclease Cas9 gene sequence, NOSterminator represents the NOS transcription terminator sequence, and ccdB represents a gene sequence toxic to Escherichia coli, used to improve positive clone screening; Figure 1 C and 1D are schematic diagrams of the insertion of two target sequences of OsFER2 into the pOsU6a vector; Figure 1 E and 1F are schematic diagrams of the two target sequence expression cassettes of OsFER2 inserted into the pCRISPR / Cas9 vector respectively.

[0025] Figure 2 Schematic diagram of the target sequence of OsFER2 gene editing and the target sequence of OsFER2 gene editing-positive plants. In the figure, black boxes represent exons, black lines represent introns, ATG represents the start codon, TGA represents the stop codon, and X represents the missing base at that location.

[0026] Figure 3 shows the phenotype, plant height, and tillering results of OsFER2 gene-edited plants. Figure 3A The phenotypic observation results of rice in the late grain filling stage are shown in the figure. The scale bar is 15 cm. Figure 3B The statistical analysis results of rice plant height in the late grain filling period are as follows: the average plant height of WT is 99.3 cm, the average plant height of M1-1 is 89.6 cm, and the average plant height of M2-1 is 89.1 cm; Figure 3C The statistical analysis results of rice tillering in the late grain filling period are as follows: the average tiller number of WT is 19.3, the average tiller number of M1-1 is 3.4, and the average tiller number of M2-1 is 3.5; Figure 3D The phenotypic observation results of rice during tillering period are shown in the figure. The scale bar is 7 cm. Figure 3E The statistical analysis results of rice plant height during the tillering period are as follows: the average plant height of WT is 85.1 cm, the average plant height of M1-1 is 35.2 cm, and the average plant height of M2-1 is 37.4 cm; Figure 3FThe statistical analysis results of tillering in rice tillering period are as follows: the average tillering number of WT is 9.5, the average tillering number of M1-1 is 2.1, and the average tillering number of M2-1 is 1.8. Figure 3B 、 3C , 3E, and 3F. *: Significant difference at p < 0.05. WT: Zhenshan 97 (wild type); M1-1: plant obtained by gene editing of OsFER2 target sequence 1; M2-1: plant obtained by gene editing of OsFER2 target sequence 2. DETAILED DESCRIPTION

[0027] The following examples further illustrate the specific embodiments of the present invention. Unless otherwise specified, the experimental methods involved in the examples are conventional experimental methods, and the primer sequence synthesis and DNA fragment sequencing analysis were all completed by Shanghai Sangon Biotechnology Co., Ltd.

[0028] Example 1: Construction of OsFER2 gene editing vector and its genetic transformation in rice

[0029] The coding sequence of the rice OsFER2 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 CRISPR-GE software (skl.scau.edu.cn / home / ). Two suitable sequences, SEQ ID NOs. 5 and 6, were selected and designated as Target-1 and Target-2, respectively. Primer sequences for Target-1 were designed based on the restriction sites of the OsU6a vector. The forward primer Target1-F is shown in SEQ ID NO. 7, and the reverse primer Target1-R is shown in SEQ ID NO. 8. Primer sequences for Target-2 were designed. The forward primer Target2-F is shown in SEQ ID NO. 9, and the reverse primer Target2-R is shown in SEQ ID NO. 10.

[0030] pOsU6a uses pUC57 as the vector backbone. First, the BsmBI and BsaI restriction sites contained in pUC57 are point mutated to eliminate these two restriction sites. Then, the vector is linearized using the restriction endonuclease EcoRV, and the U6a-sgRNA expression cassette sequence is inserted into the pUC57 linearized sequence to form the Cas9 expression cassette intermediate vector. The pOsU6a vector structure is as follows: Figure 1 As shown in A.

[0031] pCRISPR / Cas9 is based on pCABMBIA1300. First, the Bsa1 restriction site contained in pCABMBIA1300 is mutated 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 pCABMBIA1300 linear sequence to form the pCRISPR / Cas9 gene editing vector. The pCRISPR / Cas9 vector structure is as follows: Figure 1 As shown in B.

[0032] First, dissolve the primer pairs for target 1 and target 2 in sterile ultrapure water to prepare a 100 μM stock solution. Next, prepare the target adapter according to the reaction system in Table 1 (reactions were performed in 1.5 mL centrifuge tubes; incubate at 95°C for 30 seconds and then at room temperature for 5 minutes to complete adapter preparation). Then, incubate the target adapter with the pOsU6a plasmid vector according to the reaction system in Table 2 (37°C for 5 minutes, 10°C for 1 minute, and 20°C for 5 minutes, for 5 cycles). Finally, transform the reaction products into competent DH5α Escherichia coli, incubate on ice for 30 minutes, heat shock at 42°C for 30 seconds, and then incubate on ice for 2 minutes. Add 500 μL of LB liquid medium and incubate at 37°C on a shaker at 220 rpm for 30 minutes. Finally, spread the product evenly on a LB solid culture plate containing 50 mg / L AMP. 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), and positive single clones were picked for sequencing analysis. The vectors with correct sequencing results were named pOsU6a-Target1 and pOsU6a-Target2, and their structures are shown in the following table. Figure 1 C and Figure 1 D.

[0033] Table 1 Target linker reaction system

[0034] name volume Target-F (100 μM) 1 μL Target-R (100 μM) 1 μL <![CDATA[ddH2O]]> 8μL

[0035] Table 2 Reaction system of target linker and pOsU6a plasmid vector

[0036] name volume T4 DNA ligase buffer 2μL T4 DNA ligase 1μL (10U) Cut smart buffer 2μL BsmB 1μL (10U) Target adapter 1 μL pO6 1 μL <![CDATA[ddH2O]]> 12 μL

[0037] Bsa1 was used to digest pOsU6a-Target1 and pOsU6a-Target2, and the gene editing expression cassette fragments (about 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°C 5 min, 10°C 1 min, 20°C 5 min, and 10 cycles). Finally, the reaction products were transformed into DH5α competent Escherichia coli. 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), and positive single clones were picked for shaking. The plasmids were extracted and sequenced correctly, which were the OsFER2 gene editing vectors, named pCRISPR / Cas9-OsFER2-Target1 and pCRISPR / Cas9-OsFER2-Target2, respectively. Their structures are shown in the following table. Figure 1 E and Figure 1 F.

[0038] Table 3 Reaction system of recovered fragments and pCRISPR / Cas9 plasmid

[0039] name volume T4 DNA ligase buffer 2μL T4 DNA ligase 1μL (10U) Cut smart buffer 2μL BsaI 1μL (10U) Recycling Fragments 8μL pCRISPR / Cas9 Plasmid 1 μL <![CDATA[ddH2O]]> 5μL

[0040] Zhenshan 97 (wild type WT) was transformed based on the Agrobacterium-mediated method. After callus induction, Agrobacterium activation and infection, co-cultivation, resistant callus screening and differentiation, rooting culture, positive plant identification, seedling hardening and field transplanting, 10 genetically transformed T0 rice positive plants were obtained. The specific operations are as follows.

[0041] 1) Induced wound healing

[0042] Select mature and plump rice seeds, remove the husks, add an appropriate amount of 75% ethanol and let it stand for 1 min, then add an appropriate amount of 1% sodium hypochlorite solution and let it stand for 15 min. Remove the sodium hypochlorite solution and rinse the seeds with sterile water 3-5 times. Place the treated rice seeds on callus induction medium (composition see Table 4A) and place them in a 32°C plant growth chamber for 7-10 days.

[0043] 2) Agrobacterium activation

[0044] Two days before infection, Agrobacterium EHA105 containing the target gene plasmid vector was streaked onto LB solid medium containing 50 mg / L Kan and cultured at 28°C.

[0045] 3) Agrobacterium configuration, infection, and co-cultivation

[0046] Before infection, transfer the Agrobacterium from LB solid medium to suspension medium (composition see Table 4B), culture at 28°C, 180 rpm for 3.5 h, and then adjust the Agrobacterium concentration to OD 600 =0.1-0.2. Transfer callus tissue after 7-10 days of induction into the Agrobacterium suspension and allow to stand for 1.5 minutes. Discard the Agrobacterium suspension and absorb the suspension from the surface of the callus with sterile filter paper. Cover the callus surface with sterilized green plant material and let it rest in a clean bench for 30 minutes. Transfer the callus tissue to co-culture medium (composition see Table 4C) covered with a layer of sterile filter paper. Incubate in the dark at 20°C for 12-14 hours, then transfer to a 25°C incubator and continue in the dark for 2 days.

[0047] 4) Eliminate Agrobacterium

[0048] After co-cultivation, transfer the callus to a sterile Erlenmeyer flask and rinse with sterile water three times for 30 seconds each, then rinse again with sterile water 5-6 times. Finally, soak in sterile water containing 500 mg / L Cn for 30 minutes. Remove the Cn sterile water, blot the surface of the callus with sterile filter paper, cover with a layer of sterile filter paper, and let it stand in a clean bench for 1 hour to dry the surface of the callus.

[0049] 5) Screening of resistant calli

[0050] The rinsed callus was placed on resistance screening medium (composition see Table 4D) and cultured at 32°C for 14 days.

[0051] 6) Differentiation of resistant callus

[0052] After 14 days of screening, the resistant calli were transferred to differentiation medium (composition see Table 4E) and cultured in a plant growth chamber at 28°C.

[0053] 7) Rooting culture

[0054] When the resistant callus grows into 3-4 cm regenerated seedlings on the differentiation medium, it is transferred to the rooting medium (composition see Table 4F) and grown into complete plants. After PCR and sequencing identification, it is set aside.

[0055] Table 4A Callus induction medium composition

[0056] name volume <![CDATA[N6 max Stock Solution]]> 100mL <![CDATA[N6 min Stock Solution]]> 10mL Fe salt stock solution 10mL Vitamin stock solution 10mL 2,4-D stock solution 2.5mL Proline 0.6g hydrolyzed casein 0.8g sucrose 30g Plant Gel 3g

[0057] Add the above reagents to a beaker in sequence. First, add 900 mL of distilled water. Adjust the pH value of the culture medium solution to 5.8 with KOH solution (1 M). Add distilled water to 1 L, then sterilize under high pressure at 121°C for 15 min. Dispense into culture dishes on a clean bench and cool for use.

[0058] Table 4B Suspension culture medium composition

[0059] name Volume or mass N6max stock solution 12.5mL N6min stock solution 1.25mL Fe salt stock solution 2.5mL Vitamin stock solution 10mL Proline 0.5g Hydrolyzed tyrosine 0.2g 2,4-D stock solution 0.625mL sucrose 9g

[0060] Add the above reagents to a beaker, first add 200 mL of distilled water, adjust the pH value of the suspension culture solution to 5.2 with KOH solution (1 M), add distilled water to 250 mL, and sterilize by autoclaving at 121°C for 15 min. When using, add 5 mL of 50% glucose solution and 250 μL of AS stock solution.

[0061] Table 4C Co-culture medium composition

[0062] name Volume or mass <![CDATA[N6 max Stock Solution]]> 12.5mL <![CDATA[N6 min Stock Solution]]> 1.25mL Fe salt stock solution 1.25mL Vitamin stock solution 2.5mL Proline 0.15g Hydrolyzed tyrosine 0.2g 2,4-D stock solution 0.625mL sucrose 7.5g

[0063] The above reagents were added to a beaker in sequence. 200 mL of distilled water was added first. The pH value of the co-culture medium solution was adjusted to 5.6 with KOH solution (1 M). The volume was then made up to 250 mL with distilled water. The solution was autoclaved at 121°C for 15 min. Before use, 5 mL of 50% glucose and 250 μL of AS stock solution were added.

[0064] Table 4D Resistance screening medium composition

[0065] name Volume or mass <![CDATA[N6 max Stock Solution]]> 25mL <![CDATA[N6 min Stock Solution]]> 2.5mL Fe salt stock solution 2.5mL Vitamin stock solution 2.5mL Proline 0.15g Hydrolyzed tyrosine 0.2g 2,4-D stock solution 0.625mL sucrose 7.5g agar powder 2g

[0066] Add the above reagents to a beaker in sequence, first add 200 mL of distilled water, adjust the pH value of the screening medium to 6.0 with KOH solution (1 M), add distilled water to 250 mL, sterilize under high pressure at 121 ° C for 15 min, add 250 μL of hygromycin (50 mg / mL) and 500 μL of Cn when cooled to 60 ° C, pour into a culture dish under the clean bench, and cool for use.

[0067] Table 4E Differentiation medium composition

[0068] name Volume or mass <![CDATA[N6 max Stock Solution]]> 100mL <![CDATA[N6 min Stock Solution]]> 10mL Fe salt stock solution 10mL Vitamin stock solution 10mL KT stock solution 2mL NAA stock solution 0.2mL Proline 0.6g Hydrolyzed tyrosine 0.8g sorbitol 30g sucrose 30g Plant Gel 3g

[0069] Add the above reagents to the beaker in sequence, first add 900 mL of distilled water, adjust the pH value of the differentiation medium to 5.8 with KOH solution (1 M), make up to 1 L, sterilize by high pressure at 121 ° C for 15 min, pour into the culture dish on the clean bench, and cool for use.

[0070] Table 4F Rooting medium composition

[0071] name Volume or mass <![CDATA[MS max Stock Solution]]> 50mL <![CDATA[MS min Stock Solution]]> 5mL Fe salt stock solution 5mL Vitamin stock solution 5mL sucrose 20g Plant Gel 3g

[0072] Add the above reagents to a beaker in sequence. First, add 900 mL of distilled water. Adjust the pH value of the rooting medium to 5.8 with KOH solution (1 M). Add distilled water to 1 L. Sterilize under high pressure at 121 °C for 15 min. Pour into a culture dish on a clean bench and cool for use.

[0073] 1.MS max stock solution

[0074] NH4NO3 16.5g <![CDATA[KH2PO4]]> 1.7g <![CDATA[KNO3]]> 19g <![CDATA[MgSO4·7H2O]]> 3.7g <![CDATA[CaCl2·2H2O]]> 4.4g

[0075] Add them to the beaker in sequence, add distilled water until they are completely dissolved, and then adjust the volume to 1L.

[0076] 2.MS min stock solution

[0077] MnSO4·4H2O 2.23g <![CDATA[ZnSO4·7H2O]]> 0.86g <![CDATA[H3BO3]]> 0.62g KI 0.083g <![CDATA[Na2MoO4·2H2O]]> 0.025g <![CDATA[CoCl2·6H2O]]> 0.0025g <![CDATA[CuSO4·5H2O]]> 0.0025g

[0078] Add them to the beaker in sequence, add distilled water until they are completely dissolved, and then adjust the volume to 1L.

[0079] 3.N6 max stock solution

[0080] KNO3 28.3g <![CDATA[KH2PO4]]> 4.0g <![CDATA[(NH4)2SO4]]> 4.63g <![CDATA[MgSO4·7H2O]]> 1.85g <![CDATA[CaCl2·2H2O]]> 1.66g

[0081] Add them to the beaker in sequence, add distilled water until they are completely dissolved, and then adjust the volume to 1L.

[0082] 4.N6 min stock solution

[0083] MnSO4·4H2O 0.44g <![CDATA[ZnSO4·7H2O]]> 0.15g <![CDATA[H3BO3]]> 0.16g KI 0.08g

[0084] Add them to the beaker in sequence, add distilled water until they are completely dissolved, and then adjust the volume to 1L.

[0085] 5.Fe salt stock solution

[0086] FeSO4·7H2O 2.78g <![CDATA[EDTANa2·2H2O]]> 3.73g

[0087] Add FeSO4·7H2O and EDTANa2·2H2O to 300 mL of distilled water respectively, mix, heat to 70℃ and keep warm for 2 h, make up to 1 L with distilled water, and store at 4℃ in the dark until use.

[0088] 6. Vitamin reserve solution

[0089] niacin 0.1g <![CDATA[VB6]]> 0.1g <![CDATA[VB1]]> 0.1g Glycine 0.2g Inositol 10g

[0090] Add the above reagents in sequence, add distilled water to make up to 1L, and store at 4℃ for later use.

[0091] 7. Kinetin (KT) stock solution

[0092] Weigh 100 mg of KT, add 1 mL of KOH (1 M) and stir until completely dissolved. Then add distilled water to make up to 100 mL and store at 4°C until use.

[0093] 8. 2,4-Dichlorophenoxyacetic acid (2,4-D) stock solution

[0094] Weigh 100 mg of 2,4-D, add 1 mL of KOH (1 M), stir until completely dissolved, then add distilled water to make up to 100 mL and store at 4°C until use.

[0095] 9. Naphthaleneacetic acid (NAA) stock solution

[0096] Weigh 100 mg of NAA, add 1 mL of KOH (1 M) and stir until completely dissolved. Then add distilled water to make up to 100 mL and store at 4°C until use.

[0097] 10. Acetosyringone (AS) stock solution

[0098] Weigh 0.39 g of AS and dissolve it in 10 mL of DMSO. Aliquot the solution into 1.5 mL centrifuge tubes and store at -20°C until use.

[0099] 11. Carbenicillin (Cn)

[0100] Weigh 2.5 g of Cn and add sterile water to 10 mL in a clean bench. After complete dissolution, divide the solution into 1.5 mL centrifuge tubes and store at -20°C until use.

[0101] 12. Kanamycin (Kanamycin)

[0102] Weigh 0.5 g of Kan, add sterile water to 10 mL in a clean bench, and after complete dissolution, divide the solution into 1.5 mL centrifuge tubes and store at -20°C for later use.

[0103] 13. 50% glucose solution

[0104] Weigh 50 g of glucose, add distilled water, dilute to 100 mL, sterilize by autoclaving at 121°C for 15 min, and store at 4°C until use.

[0105] 14. KOH solution

[0106] Weigh 5.6 g of KOH and add it to a beaker. Add distilled water to make up to 100 mL and store at room temperature until use.

[0107] 15. LB solid medium

[0108] Weigh 10 g of trypsin, 5 g of yeast extract, and 10 g of sodium chloride, add distilled water to make up to 1 L, and sterilize under high pressure at 121°C for 15 min. When the solution temperature drops to about 60°C, dispense into culture dishes on a clean bench and cool for later use.

[0109] 8) Identification of positive plants

[0110] Rice genomic DNA was isolated using the CTAB method. Positive plants were identified by PCR (forward primer sequence: SEQ ID NO. 13, reverse primer sequence: SEQ ID NO. 14) using this plant genomic DNA as the reaction template (denaturation at 95°C for 2 min, followed by denaturation at 95°C for 20 s, annealing at 60°C for 20 s, and extension at 72°C for 40 s, with 30 cycles). The PCR reaction mixture was analyzed by agarose gel electrophoresis to identify gene-edited plants. Positive plants transformed with pCRISPR / Cas9-OsFER2-Target1 and pCRISPR / Cas9-OsFER2-Target2 were designated M1 and M2, respectively.

[0111] 9) Hardening and transplanting seedlings into the field

[0112] Remove the sealing film from rice seedlings grown in a plant incubator and place them at room temperature for 2 days. Transplant the positive seedlings into the field and harvest the seeds for subsequent experiments.

[0113] Example 2: Genotyping of OsFER2-positive edited plants

[0114] Based on the target sequence information of the OsFER2 gene, two pairs of specific primers were designed approximately 100-200 bp upstream and downstream of the target sequence (the forward primer sequence of the first pair is shown in SEQ ID NO. 15, and the reverse primer sequence is shown in SEQ ID NO. 16; the forward primer sequence of the second pair is shown in SEQ ID NO. 17, and the reverse primer sequence is shown in SEQ ID NO. 18). Using genomic DNA from Zhenshan 97 (wild type) and the positive plants obtained in Example 1 as PCR templates, PCR amplification of the OsFER2 target region was performed. Fragments were then recovered and sequenced to determine whether the OsFER2 gene in the T0 generation positive plants had been edited. Results showed that the target site sequence of the M1-1 plant had a two-base deletion, while the target site sequence of the M2-1 plant had a one-base deletion. Plants M1 and M2, which had a deletion in the OsFER2 target sequence and premature termination of the encoded amino acid sequence, were used for subsequent studies. The progeny of the OsFER2 candidate plants were separated to obtain T-DNA-free OsFER2 gene-edited plants M1-1 and M2-1 for subsequent phenotypic identification.

[0115] The target sequences of the OsFER2 gene editing and the target sequences of the OsFER2 gene editing positive plants are as follows: Figure 2 shown.

[0116] Example 3: Phenotypic Identification of OsFER2 Gene-Edited Plants

[0117] Rice OsFER2 gene-edited plants and wild-type ZS97 (WT) were planted simultaneously in the field (100 plants each, 10 rows, 10 plants per row, 25 cm plant spacing, 30 cm row spacing), and their phenotypic differences were observed throughout the maturity period.

[0118] In the late filling stage of rice, the observation results are as follows Figure 3A , the statistical results are as follows Figure 3B 、 Figure 3C After comparison and statistical analysis, we found that:

[0119] In terms of plant height, M1-1 and M2-1 were significantly lower than the wild type ZS97. The average plant height of M1-1 was 89.6 cm, which was 9.77% lower than the wild type's average plant height of 99.3 cm. The average plant height of M2-1 was 89.1 cm, which was 10.27% lower than the wild type's average plant height of 99.3 cm.

[0120] In terms of tiller number, M1-1 and M2-1 were significantly lower than the wild type ZS97. The average tiller number of M1-1 was 3.4, which was 82.38% lower than that of the wild type (19.3). The average tiller number of M2-1 was 3.5, which was 81.87% lower than that of the wild type (19.3).

[0121] During the tillering stage of rice, the observation results are as follows Figure 3D , the statistical results are as follows Figure 3E 、 Figure 3F After comparison and statistical analysis, we found that:

[0122] In terms of plant height, M1-1 and M2-1 were significantly lower than the wild type ZS97. The average plant height of M1-1 was 35.2 cm, which was 58.6% lower than the wild type's average plant height of 85.1 cm. The average plant height of M2-1 was 37.4 cm, which was 56.1% lower than the wild type's average plant height of 85.1 cm.

[0123] In terms of tiller number, M1-1 and M2-1 were significantly lower than the wild type ZS97. The average tiller number of M1-1 was 2.1, which was 77.9% lower than the average tiller number of the wild type (9.5). The average tiller number of M2-1 was 1.8, which was 81.1% lower than the average tiller number of the wild type (9.5).

[0124] It can be seen that the plant height and tiller number of OsFER2 gene-edited plants are significantly lower than those of wild-type plants, which proves that the OsFER2 gene is involved in regulating rice plant height and tillering.

[0125] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these changes and variations. The above embodiments or implementation methods are merely illustrative of the present invention, and the present invention may also be implemented in other specific ways or other specific forms without departing from the gist or essential features of the present invention. Therefore, the described implementation methods should be regarded as illustrative and not restrictive in any respect. The scope of the present invention should be described by the appended claims, and any changes that are equivalent to the intent and scope of the claims should also be included within the scope of the present invention.

Claims

1. Use of the rice gene OsFER2 for reducing rice plant height and reducing the number of rice tillers, wherein the coding sequence of the rice gene OsFER2 is shown in SEQ ID NO. 3, and the amino acid sequence of the protein encoded by it is shown in SEQ ID NO. 4, and the use is achieved by deleting the protein function of SEQ ID NO. 4.

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