Use of rice gene OsLG4 for regulating rice grain shape and its mutants

Abstract

The present invention belongs to the technical field of crop breeding and relates to the use of the rice gene OsLG4 for regulating rice grain shape and its mutants. The coding sequence of the rice gene OsLG4 is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by it is shown in SEQ ID NO.2. The present invention has discovered a new use of the rice gene OsLG4 for regulating rice grain shape. The constructed rice modified plants containing the modified rice gene OsLG4 of the present invention can significantly increase the grain length and yield traits of rice grains. Such improvements in rice grain shape and yield 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 OsLG4 for regulating rice grain shape and its mutants. Background Art

[0002] As a staple food crop, rice (Oryza sativa L.) feeds more than half of the world's population. To meet the growing food demand driven by a growing global population, increasing rice yields has become a crucial step. Although global rice production has increased significantly in recent decades, this is primarily due to improvements in agricultural technology and the increased use of fertilizers and pesticides. These practices have adverse environmental impacts, necessitating reductions in their use and the implementation of alternative environmental strategies. Currently, genetic improvement of rice yield remains insufficient, given projected population growth, shrinking arable land, and climate change. To address the increased food demand driven by future population growth, new rice varieties with higher yield potential must be developed in the future. One strategy involves identifying novel, superior genes that determine rice grain size and weight.

[0003] Rice yield mainly depends on the number of rice panicles, the number of grains per panicle and grain weight. Rice grain shape is the main determinant of rice grain weight and is composed of grain length, width and thickness. The length and width of rice grains are determined by the number and cell length of cells in the lemma, while the thickness is mainly affected by the grain filling process. However, the existing rice grain shape and grain weight are the most suitable grain shape and grain weight established during the evolution of rice, which in turn improves the adaptability and survival rate of offspring and has a relatively narrow evolutionary range. Among them, rice grain shape is a complex quantitative trait determined by multiple genes and factors. It has been the focus of research and breeding since the beginning of modern agriculture.

[0004] Rice grain shape is a crucial trait in rice domestication and has become a key breeding target. Current research on genes regulating rice grain shape has not yet established a comprehensive regulatory network. Therefore, identifying and studying more genes regulating rice grain shape will help further understand how rice regulates grain shape, ultimately improving rice appearance and yield. Summary of the Invention

[0005] The primary purpose of the present invention is to provide a use of the rice gene OsLG4 for regulating rice grain shape, so that it can be better used for regulating rice grain shape.

[0006] To achieve this object, in a basic embodiment, the present invention provides the use of the rice gene OsLG4 (LOC_Os04g42770) for regulating rice grain shape, wherein the coding sequence of the rice gene OsLG4 is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by it is shown in SEQ ID NO.2.

[0007] The second object of the present invention is to provide a modified rice gene OsLG4 that can significantly increase the grain length, 1000-grain weight and yield of rice.

[0008] To achieve this object, in a basic embodiment, the present invention provides a modified rice gene OsLG4, the coding sequence of which is shown in SEQ ID NO. 3 or SEQ ID NO. 4.

[0009] The third object of the present invention is to provide a method for constructing a modified rice plant containing the modified rice gene OsLG4 as described above, so as to construct a modified rice plant containing the modified rice gene OsLG4 as described above, which can significantly increase the grain length, 1000-grain weight and yield of rice.

[0010] 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 OsLG4 as described above. The construction method is based on the principle of CRISPR / Cas9 gene editing, and an expression vector carrying a gene expressing 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 OsLG4 as described above is obtained.

[0011] The present invention uses a CRISPR / Cas9 gene editing vector to cause a frameshift mutation in the rice gene OsLG4, thereby stopping the translation of the protein sequence encoded by SEQ ID NO.2 prematurely, resulting in the loss of protein function of SEQ ID NO.2.

[0012] Preferably, the construction of the above-mentioned expression vector may include: screening and designing the OsLG4 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 OsLG4 gene through the steps of fragment and vector enzyme digestion, T4-DNA ligation, transformation of Escherichia coli DH5α competent cells, monoclonal colony PCR identification and vector sequencing.

[0013] Preferably, obtaining the modified rice plants may include: directly transforming the constructed CRISPR / Cas9 gene-editing vector for the OsLG4 gene into callus tissue / cells of japonica rice Zhonghua 11 using the Agrobacterium-mediated method. Plants positive for the OsLG4 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 OsLG4 gene-edited plants meet the transformation requirements. OsLG4 gene-edited plants that meet the requirements are transplanted into the field. At maturity, grain length, 1000-grain weight, and yield are analyzed and statistically analyzed to demonstrate changes in grain length, 1000-grain weight, and yield of the OsLG4 gene-edited plants compared to wild-type japonica rice Zhonghua 11.

[0014] In a preferred embodiment, the present invention provides a method for constructing a modified rice plant containing the modified rice gene OsLG4 as described above, wherein the target sequence of the sgRNA is shown as 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 OsLG4 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 OsLG4 as described above, wherein the expression vector is transformed into the original rice plant by Agrobacterium-mediated method.

[0017] In a preferred embodiment, the present invention provides a method for constructing a modified rice plant containing the modified rice gene OsLG4 as described above, wherein the original rice plant is japonica rice Zhonghua 11.

[0018] In a preferred embodiment, the present invention provides a method for constructing modified rice plants containing the modified rice gene OsLG4 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 transplanting into the field.

[0019] The fourth object of the present invention is to provide the use of the modified rice gene OsLG4 as described above for regulating rice grain shape and / or yield.

[0020] To achieve this object, in a basic embodiment, the present invention provides the use of the modified rice gene OsLG4 as described above for regulating rice grain shape and / or yield.

[0021] The beneficial effect of the present invention is that the present invention has discovered a new use of the rice gene OsLG4 for regulating rice grain shape. The constructed rice modified plants containing the modified rice gene OsLG4 of the present invention can significantly increase the grain length and yield traits of rice grains. Such improvement of rice grain shape and yield provides a new genetic resource for rice breeding.

[0022] The modified rice gene OsLG4 and the modified rice plants containing it of the present invention are of great significance for studying rice grain shape and yield regulation pathways, and provide a new breeding scheme for rationally and appropriately utilizing the OsLG4 gene to cultivate large-grain and high-yield rice varieties, which has very broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] 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 R Indicates 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 OsLG4 into the pOsU6a vector; Figure 1 E and 1F are schematic diagrams of the two target sequence expression cassettes of OsLG4 inserted into the pCRISPR / Cas9 vector respectively.

[0024] Figure 2 Schematic diagram of the target sequence for OsLG4 gene editing and the edited sequence of OsLG4-positive plants. Black boxes represent exons, black lines represent introns, ATG represents the start codon, TGA represents the stop codon, bold text and green fill indicate insertions or deletions, and red text indicates the PAM sequence.

[0025] Figure 3 The results of the grain phenotype, grain shape and yield-related traits of OsLG4 gene-edited plants are shown in Figure 2. Figure 3 A is the observation result of grain length phenotype of mature plants, scale bar is 10 mm; Figure 3 B is the statistical result of grain length of mature plants; Figure 3 C is the statistical result of 1000-grain weight of mature plants; Figure 3 D is the statistical result of single plant yield of mature plants; Figure 3 E is the statistical result of plant height. Figure 3 In B, 3C, 3D, and 3E, *: significant difference at p < 0.05. WT: japonica rice Zhonghua 11 (wild type); M1-1: plant obtained after gene editing of OsLG4 target sequence 1; M2-1: plant obtained after gene editing of OsLG4 target sequence 2. DETAILED DESCRIPTION

[0026] 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.

[0027] Example 1: Construction of OsLG4 gene editing vector and its genetic transformation in rice

[0028] The coding sequence of the rice OsLG4 gene was obtained from the NCBI database (https: / / www.ncbi.nlm.nih.gov) as shown in SEQ ID NO. 1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO. 2. Target sequences for gene editing were screened using the online CRISPR-GE software (skl.scau.edu.cn / home / ). Two suitable sequences, SEQ ID NO. 5 and SEQ ID NO. 6, were ultimately selected and designated as Target-1 and Target-2, respectively. Based on the restriction enzyme cleavage sites of the OsU6a vector, primer sequences were designed for Target-1. 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.

[0029] 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 vector structure is as follows: Figure 1 As shown in A.

[0030] pCRISPR / Cas9 is based on pCABMBIA1300. First, the Bsa1 restriction site contained in pCABMBIA1300 is point 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. Its vector structure is as follows: Figure 1 As shown in B.

[0031] First, the primer pairs for target 1 and target 2 were dissolved in sterile ultrapure water to prepare a 100 μM stock solution. Next, the target adapters were prepared according to the reaction system in Table 1 (the reaction was performed in a 1.5 mL centrifuge tube; incubation was performed at 95°C for 30 seconds and then at room temperature for 5 minutes to complete the adapter preparation). The target adapters were then incubated 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, the reaction products were transformed into DH5α competent E. coli, incubated on ice for 30 minutes, heat-shocked at 42°C for 30 seconds, and then incubated on ice for 2 minutes. 500 μL of LB liquid medium was added and incubated at 37°C on a shaker at 220 rpm for 30 minutes. Finally, the product was evenly plated on a LB solid culture plate 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), and positive single clones were picked for sequencing analysis. The vectors with correct sequencing results were named pOsU6a-Target1 and pOsU6a-Target2, and their vector structures were as follows: Figure 1 C and Figure 1 D.

[0032] Table 1 Target linker reaction system

[0033]

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

[0035]

[0036] 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 for 5 min, 10°C for 1 min, 20°C for 5 min, and 10 cycles of reaction). Finally, the reaction products were transformed into DH5α Escherichia coli competent 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), and positive single clones were picked for shaking. The plasmids were extracted and the correct sequencing was the OsLG4 gene editing vector, named pCRISPR / Cas9-OsLG4-Target1 and pCRISPR / Cas9-OsLG4-Target2, respectively. The vector structures are as shown below. Figure 1 E and Figure 1 F.

[0037] Table 3 Reaction system of recovered fragments and pCRISPR / Cas9 vector

[0038]

[0039] Based on the Agrobacterium-mediated method, japonica rice Zhonghua 11 (wild type WT) was transformed. 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.

[0040] 1) Induced wound healing

[0041] Mature, plump rice seeds were selected and the husks removed. An appropriate amount of 75% ethanol was added and allowed to stand for 1 min. Then, an appropriate amount of 1% sodium hypochlorite solution was added and allowed to stand for 15 min. The sodium hypochlorite solution was removed and the seeds were rinsed 3-5 times with sterile water. The treated rice seeds were placed on callus induction medium (composition see Table 4A) and placed in a 32°C 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 onto LB solid medium containing 50 mg / L Kan and cultured at 28°C.

[0044] 3) Agrobacterium configuration, infection, and co-cultivation

[0045] Before infection, the Agrobacterium in LB solid medium was transferred to suspension medium (composition see Table 4B), cultured at 28°C, 180 rpm for 3.5 h, and then the Agrobacterium concentration was adjusted 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 callus surface with sterile filter paper. Cover the callus surface with sterile green plant material and let it rest in a clean bench for 30 minutes. Transfer the callus tissue to co-cultivation 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.

[0046] 4) Eliminate Agrobacterium

[0047] 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.

[0048] 5) Screening of resistant calli

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

[0050] 6) Differentiation of resistant callus

[0051] After 14 days of screening, the resistant calli were transferred to differentiation medium (composition see Table 4E) and cultured in a 28°C plant growth chamber.

[0052] 7) Rooting culture

[0053] When the resistant callus grows to 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.

[0054] Table 4A Callus induction medium composition

[0055]

[0056]

[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, and 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]

[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]

[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 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]

[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 at 121 ° C autoclave 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 on the clean bench, and cool for use.

[0067] Table 4E Differentiation medium composition

[0068]

[0069]

[0070] 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 under high pressure at 121 ° C for 15 min, pour into the culture dish on the clean bench, and cool for use.

[0071] Table 4F rooting medium composition

[0072]

[0073] 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.

[0074] 1.MS max stock solution

[0075]

[0076] Add them to the beaker in sequence, add distilled water until they are completely dissolved, and then adjust the volume to 1L.

[0077] 2.MS min stock solution

[0078]

[0079]

[0080] Add them to the beaker in sequence, add distilled water until they are completely dissolved, and then adjust the volume to 1L.

[0081] 3.N6 max stock solution

[0082]

[0083] Add them to the beaker in sequence, add distilled water until they are completely dissolved, and then adjust the volume to 1L.

[0084] 4.N6 min stock solution

[0085]

[0086] Add them to the beaker in sequence, add distilled water until they are completely dissolved, and then adjust the volume to 1L.

[0087] 5.Fe salt stock solution

[0088] FeSO4·7H2O 2.78g

[0089] EDTANa2·2H2O 3.73g

[0090] 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.

[0091] 6. Vitamin reserve solution

[0092]

[0093]

[0094] Add the above reagents in sequence, add distilled water to make up to 1L, and store at 4℃ for later use.

[0095] 7. Kinetin (KT) stock solution

[0096] 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.

[0097] 8.2,4-Dichlorophenoxyacetic acid (2,4-D) stock solution

[0098] Weigh 100 mg of 2,4-D, 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.

[0099] 9. Naphthaleneacetic acid (NAA) stock solution

[0100] 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.

[0101] 10. Acetosyringone (AS) stock solution

[0102] 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.

[0103] 11. Carbenicillin (Cn)

[0104] 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.

[0105] 12. Kanamycin (Kanamycin, Kan)

[0106] 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.

[0107] 13.50% glucose solution

[0108] 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.

[0109] 14.KOH solution

[0110] 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.

[0111] 15.LB solid medium

[0112] 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, divide the solution into culture dishes on a clean bench and cool it for later use.

[0113] 8) Identification of positive plants

[0114] Rice plant genomic DNA was isolated using the CTAB method, and positive plants were identified by PCR (forward primer sequence such as SEQ ID NO. 13, reverse primer sequence such as SEQ ID NO. 14) using plant genomic DNA as the reaction template (first denaturation at 95°C for 2 minutes, followed by denaturation at 95°C for 20 seconds, annealing at 60°C for 20 seconds, and extension at 72°C for 40 seconds, with a total of 30 cycles). The PCR reaction solution was then analyzed by agarose gel electrophoresis to identify gene-edited plants. Positive plants transformed with pCRISPR / Cas9-OsLG4-Target1 and pCRISPR / Cas9-OsLG4-Target2 were designated M1 and M2, respectively.

[0115] 9) Hardening and transplanting seedlings into the field

[0116] 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.

[0117] Example 2: Molecular Identification of OsLG4 Genomic Sequence Modification

[0118] Based on the target sequence information of the OsLG4 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 of primers 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 of primers is shown in SEQ ID NO. 17, and the reverse primer sequence is shown in SEQ ID NO. 18). Using genomic DNA from japonica rice Zhonghua 11 (wild type) and the positive plants obtained in Example 1 as PCR templates, PCR amplification of the OsLG4 target region was performed. Fragments were then recovered and sequenced to determine whether the OsLG4 gene in the T0 generation positive plants was edited. The results showed that the M1-1 plant had a single base insertion at the target site sequence, while the M2-1 plant had a single base deletion at the target site sequence. Plants M1 and M2, which had base insertions or deletions in the OsLG4 target site sequence and premature termination of the encoded amino acid sequence, were used as plants for subsequent studies. The progeny of the OsLG4 candidate plants were separated to obtain T-DNA-free OsLG4 gene-edited plants M1-1 and M2-1 for subsequent phenotypic identification.

[0119] The target sequence of the OsLG4 gene editing and the edited sequence of the OsLG4 gene editing positive plant are as follows: Figure 2 shown.

[0120] Example 3: Grain shape-related detection of OsLG4 gene-edited plants

[0121] The OsLG4 gene-edited rice plants and the wild-type japonica rice Zhonghua 11 (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 the differences between them were observed throughout the growth period.

[0122] During the rice maturity period, the observation results are as follows Figure 3 A. Statistical results are as follows Figure 3 B. Figure 3 C. Figure 3 D. Figure 3 E. After comparison and statistical analysis, it was found that:

[0123] In terms of grain length, M1-1 and M2-1 were significantly larger than the wild type japonica rice Zhonghua 11. The average grain length of M1-1 was 8.11 mm, which was a significant increase of 7.85% compared with the wild type's average grain length of 7.52 mm. The average grain length of M2-1 was 8.09 mm, which was a significant increase of 7.58% compared with the wild type's average grain length of 7.52 mm.

[0124] In terms of 1000-grain weight, M1-1 and M2-1 were significantly larger than the wild type japonica rice Zhonghua 11. The average 1000-grain weight of M1-1 (27.60 g) was significantly increased by 6.03% compared with the wild type's average 1000-grain weight of 26.03 g. The average 1000-grain weight of M2-1 (27.43 g) was significantly increased by 5.38% compared with the wild type's average 1000-grain weight of 26.03 g.

[0125] In terms of single-plant yield, M1-1 and M2-1 were significantly greater than the wild-type japonica rice Zhonghua 11. The average single-plant yield of M1-1 was 44.15 g, a significant increase of 6.80% over the wild-type average single-plant yield of 41.34 g; the average single-plant yield of M2-1 was 43.62 g, a significant increase of 5.52% over the wild-type average single-plant yield of 41.34 g.

[0126] In terms of plant height, there was no significant difference between M1-1 and M2-1 and the wild type japonica rice Zhonghua 11. The average plant height of WT was 109.43 cm, the average plant height of M1-1 was 110.09 cm, and the average plant height of M2-1 was 109.23 cm.

[0127] It can be seen that the grain shape and yield traits of OsLG4 gene-edited plants are significantly higher than those of wild-type plants, which proves that the OsLG4 gene is involved in regulating rice grain shape and yield.

[0128] 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 OsLG4 for increasing rice grain length, wherein the coding sequence of the rice gene OsLG4 is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by it is shown in SEQ ID NO.2, and the use is achieved by deleting the protein function of SEQ ID NO.2.

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