OsGL9 gene, protein for regulating grain length of rice and application thereof

Abstract

This invention relates to the field of plant genetic engineering technology, and discloses a method for regulating rice grain length. OsGL9 Genes, proteins and their applications. OsGL9 The full-length nucleotide sequence of the gene is shown in SEQ ID No. 1, and the CDS nucleotide sequence is shown in SEQ ID No. 2; the amino acid sequence of the OsGL9 protein it encodes is shown in SEQ ID No. 3. This invention also discloses... OsGL9 The application of genes in regulating rice grain length and length-to-width ratio. Methods for improving rice grain length and length-to-width ratio and creating high-quality rice germplasm are also provided, all through knocking out the aforementioned genes. OsGL9 Genetic implementation. This invention provides new genes and technical pathways for improving rice grain shape, contributing to the breeding of high-quality rice.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to the regulation of rice grain length. OsGL9 Genes, proteins and their applications. Background Technology

[0002] Grain shape is a crucial factor influencing rice yield and quality, and improving grain shape to cultivate high-yielding and high-quality rice varieties is an important way to resolve this contradiction. Currently, the molecular mechanisms affecting high yield and quality, especially the inter-gene relationships, are not fully understood. Therefore, conducting cloning and functional studies of grain shape genes, and discovering and aggregating beneficial alleles, is vital for increasing rice yield, improving rice quality, and ensuring food security.

[0003] Rice grain shape depends on grain length and width, with the length-to-width ratio being a crucial factor in appearance quality. In recent years, genetic research on rice grain length has made some progress. Many quantitative trait loci (QTLs) have been located and cloned, such as GS3, GL4, GL3.1, GL7, GLW7, TGW6, Gn1a, and GL3.2. However, most of the cloned grain length genes positively regulate grain length, while genes negatively regulating grain length are still rarely reported. Summary of the Invention

[0004] The purpose of this invention is to provide a method for regulating rice grain length. OsGL9 Genes, proteins and their applications, knockout in rice OsGL9 The gene can produce long-grained rice with better appearance and quality; this not only provides a basis for further elucidating the molecular mechanism of rice grain shape regulation, but also provides new gene and breeding resources for rice breeding.

[0005] To achieve the above objectives, the present invention provides a method for regulating rice grain length. OsGL9 Gene, OsGL9 The full-length nucleotide sequence of the gene is shown in SEQ ID No. 1, and the CDS nucleotide sequence is shown in SEQ ID No. 2.

[0006] Furthermore, the present invention also provides OsGL9 The application of genes in regulating rice grain length and aspect ratio OsGL9 The full-length nucleotide sequence of the gene is shown in SEQ ID No. 1, and the CDS nucleotide sequence is shown in SEQ ID No. 2.

[0007] Furthermore, in application, rice grain length and length-to-width ratio can be regulated, including by overexpressing grain length genes. OsGL9 At the same time, reduce the grain length and length-to-width ratio of rice; knock out the grain length gene. OsGL9 At the same time, increase the length and length-to-width ratio of rice grains.

[0008] This invention also provides the OsGL9 protein, which regulates rice grain length. The amino acid sequence of the OsGL9 protein is shown in SEQ ID No. 3, and the gene encoding the OsGL9 protein is... OsGL9 Gene, OsGL9 The full-length nucleotide sequence of the gene is shown in SEQ ID No. 1, and the CDS nucleotide sequence is shown in SEQ ID No. 2.

[0009] Furthermore, this invention also provides the application of OsGL9 protein in regulating rice grain length and aspect ratio. The amino acid sequence of OsGL9 protein is shown in SEQ ID No. 3, and the gene encoding OsGL9 protein is... OsGL9 Gene, OsGL9 The full-length nucleotide sequence of the gene is shown in SEQ ID No. 1, and the CDS nucleotide sequence is shown in SEQ ID No. 2.

[0010] This invention also provides a method for improving the grain length and length-to-width ratio of rice by knocking out the aforementioned [specific type of gene] in the rice genome. OsGL9 Genes that increase the length and length-to-width ratio of rice grains OsGL9 The full-length nucleotide sequence of the gene is shown in SEQ ID No. 1, and the CDS nucleotide sequence is shown in SEQ ID No. 2.

[0011] This invention also provides a method for creating high-quality rice germplasm, which involves knocking out the aforementioned [unclear] in the rice genome. OsGL9 Gene screening and breeding of high-quality rice germplasm OsGL9 The full-length nucleotide sequence of the gene is shown in SEQ ID No. 1, and the CDS nucleotide sequence is shown in SEQ ID No. 2.

[0012] The present invention describes the regulation of rice grain length OsGL9 The advantages and positive effects of genes, proteins, and their applications are:

[0013] 1. This invention provides a novel grain length gene that was excavated and isolated from common wild rice, *Dysodon yunnanensis* (DY), from Dongxiang, Jiangxi Province. Experiments have shown that... OsGL9 Genes play a role in regulating rice grain length and appearance quality. Overexpression significantly shortens grain length and worsens appearance quality, while knockout mutations significantly increase grain length and improve appearance quality. This not only provides a foundation for further elucidating the molecular mechanisms of rice grain shape but also offers new gene and breeding resources for rice breeding.

[0014] 2. This invention discloses a method for knocking out OsGL9The method of significantly increasing rice grain length and optimizing the length-to-width ratio directly improves the appearance quality of rice, and the mutant trait is stably inherited. It provides a precise and efficient gene editing solution for breeding long-grain high-quality rice, and has outstanding application value.

[0015] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0016] Figure 1 As described in the embodiments of the present invention OsGL9 The image shows the results of gene overexpression material expression level identification. WT represents the rice variety GZX49, and OE-1, OE-2, and OE-3 represent... OsGL9 Transgenic lines with overexpressed genes;

[0017] Figure 2 As described in the embodiments of the present invention OsGL9 Phenotypic diagram of the particle size of gene overexpression material;

[0018] Figure 3 As described in the embodiments of the present invention OsGL9 A bar chart of grain length, grain width, and aspect ratio of gene overexpression materials, where A represents grain length, B represents grain width, and C represents aspect ratio;

[0019] Figure 4 As described in the embodiments of the present invention OsGL9 Image of sequencing results of target site mutations in gene knockout materials;

[0020] Figure 5 As described in the embodiments of the present invention OsGL9 Phenotypic diagram of the particle shape of gene knockout material;

[0021] Figure 6 As described in the embodiments of the present invention OsGL9 A bar chart showing grain length, grain width, and length-to-width ratio of gene knockout materials, where A represents grain length, B represents grain width, C represents length-to-width ratio, ZH11 represents the rice variety Zhonghua 11 (wild type), and KO-1, KO-2, and KO-3 represent... OsGL9 CRISPR gene knockout plants. Detailed Implementation

[0022] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0023] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0024] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the methods of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0025] This invention provides a method for regulating rice grain length. OsGL9 Gene, OsGL9 The full-length nucleotide sequence of the gene is shown in SEQ ID No. 1, and the CDS nucleotide sequence is shown in SEQ ID No. 2.

[0026] In one specific embodiment of the present invention, the gene regulating rice grain length is described. OsGL9

[0027] In one specific embodiment of the present invention, the gene regulating rice grain length is described. OsGL9 The CDS nucleotide sequence is 831 bp in total, and its nucleotide sequence is shown in SEQ ID No. 2.

[0028] In one specific embodiment of the present invention, the gene regulating rice grain length is described. OsGL9The amino acid sequence of the encoded protein is shown in SEQ ID No. 3.

[0029] The amino acid sequence described in this invention was obtained by translating the coding sequence (CDS) using Primer 3 software (http: / / frodo.wi.mit.edu / ), encoding a total of 276 amino acids. The specific sequence is shown in SEQ ID No. 3: MAFRLSNSLLGILNAVTFLLSVPVLGGGIWLATRADGTECERYFSAPVIAFGVFLLLVSLAGLVGACCRVNCLLWFYLVAMFVLIVVLFCFTVFAFVVTNKGAGEAVSGRGYKEYRLGDYSNWLQKRMENSKNWNRIRSCLQDSKVCKKLQDKNWDRTQFFKADLSPLESGCCKPPSSCNFLYVSGTNWTKVPTNSSDPDCNTWVDDGTQLCYNCQSCKAGAVATLKRDWKRVAVVCIVFLVFIVIVYSLGCCAFRNNRRDNRGAYRGAAWKGGYA*.

[0030] This invention also provides a pair of cloned genes regulating rice grain length. OsGL9 The primer pairs include an upstream primer with nucleotide sequences as shown in SEQ ID No. 4 and a downstream primer as shown in SEQ ID No. 5.

[0031] SEQ ID No.4: 5'-ATGGCGTTCCGGCTGAGCAACA-3';

[0032] SEQ ID No. 5: 5'-TCAGGCGTATCCGCCCTTCCAC-3'.

[0033] In one specific embodiment of the present invention, using the genomic DNA of the rice variety Dongye as a template, a PCR amplification system was configured using the primer pair synthesized by a biotechnology company, and PCR amplification was performed to obtain the grain length gene. OsGL9 The full-length sequence. The PCR amplification program for full-length amplification described in this invention includes: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 6 minutes, 35 cycles; and extension at 72°C for 10 minutes.

[0034] In one specific embodiment of the present invention, cDNA was reverse transcribed from RNA of the leaves of the rice variety Dongye as a template, and a PCR amplification system was configured using the aforementioned primer pair. PCR amplification was then performed to obtain the grain length gene. OsGL9The CDS sequence. The PCR amplification program for CDS amplification described in this invention includes: 94℃ pre-denaturation for 5 minutes; 94℃ denaturation for 30 seconds, 55℃ annealing for 30 seconds, 72℃ extension for 1 minute, 30 cycles; 72℃ extension for 7 minutes.

[0035] The present invention also provides a rice grain length regulating gene that includes and expresses the above-mentioned gene. OsGL9 Recombinant expression vectors.

[0036] In one specific embodiment of the present invention, the recombinant expression vector is an overexpression vector, and the base backbone vector of the overexpression vector can be a common vector, such as the pC1300S vector. In one embodiment of the present invention, the grain length gene... OsGL9 The full-length sequence was inserted into the pC1300S vector. Kpn I, BamH The overexpression vector was constructed by cutting the enzyme at the I double restriction site.

[0037] In a specific embodiment of the present invention, the gene regulating rice grain length is preferred. OsGL9 Introducing at both ends of the full-length sequence Kpn I, BamH Homologous recombination linkers flanking the I double restriction site can be designed with primers such as:

[0038] OE-F (SEQ ID No. 6):5'-gagctttcgcgagctcggtaCCATGGCGTTCCGGCTGAGCAACA-3';

[0039] OE-R (SEQ ID No. 7):5'-aggtcgactctagaggatcCTCAGGCGTATCCGCCCTTCCAC-3'.

[0040] In one specific embodiment of the present invention, regulating rice grain length and rice quality includes overexpressing a grain length gene. OsGL9 At times, this shortens the grain length, resulting in a poorer appearance and quality of rice; knocking out the mutant grain length gene... OsGL9 At the same time, the particle length increases and the appearance quality improves.

[0041] The present invention also provides a method for improving the appearance quality of rice, comprising knocking out the aforementioned grain length gene in the plant genome. OsGL9 .

[0042] In one specific embodiment of the present invention, the overexpression can be achieved through genetic transformation, that is, the overexpression vector is transformed into Agrobacterium, and the overexpression vector is transferred into the target plant through Agrobacterium-mediated genetic transformation, thereby obtaining transgenic plants with overexpression. In one specific embodiment of the present invention, the target plant is rice.

[0043] This invention also provides the above-mentioned gene regulating rice grain length. OsGL9 Application in creating high-quality germplasm with superior appearance.

[0044] Using the rice grain length regulation gene described in this invention OsGL9 This can be achieved by regulating the rice grain length gene. OsGL9 Knockout creates germplasm with better appearance quality.

[0045] This invention also provides a method for creating high-quality rice by knocking out the aforementioned rice grain length regulating gene in the genome of the target plant. OsGL9 .

[0046] The present invention does not specifically limit the method of overexpression. In one embodiment, it can be an Agrobacterium-mediated genetic transformation method.

[0047] In this embodiment of the invention, the primers used were synthesized by Shanghai Sangon Biotech, and the sequencing was performed by Shanghai Sangon Biotech. DNA and RNA extraction, PCR, and reagent formulations were performed in accordance with J. Sambrook et al., *Molecular Cloning: A Laboratory Manual* (3rd edition, translated by Jin Dongyan et al., Science Press, 2002).

[0048] The wild rice from Dongxiang in this embodiment of the invention was obtained from the ex-situ conservation nursery of wild rice from Dongxiang, Jiangxi Academy of Agricultural Sciences.

[0049] Example 1

[0050] Gene OsGL9 Clones:

[0051] 1. Genomic DNA was extracted from wild rice from Dongxiang. PCR was performed using primers shown in SEQ ID No. 4 and SEQ ID No. 5. The PCR products were sequenced to obtain the rice genome. OsGL9 The full-length sequence consists of 1892 bases, and the nucleotide sequence shown is SEQ ID No. 1.

[0052] PCR system: DNA (3ng); forward and reverse primers 3μmol each; 2x PCR buffer for KOD FX 10μl; KODFX (1.0U / μl) 0.4μl; 2mM dNTPs 4μl; add water to a total volume of 20μl.

[0053] The PCR program is as follows: 94℃ pre-denaturation for 5 minutes; 94℃ denaturation for 30 seconds, 55℃ annealing for 30 seconds, 72℃ extension for 6 minutes, 35 cycles; 72℃ extension for 10 minutes.

[0054] 2. RNA was extracted from leaves of wild rice in Dongxiang, reverse transcribed into cDNA, and PCR was performed using primers shown in SEQ ID No. 4 and SEQ ID No. 5. The amplified product was 831 bp in size. The obtained PCR product was sequenced to obtain the gene. OsGL9 The coding sequence (CDS) consists of 276 bases, and the nucleotide sequence is shown in SEQ ID No. 2.

[0055] PCR system: 4 μl of reverse transcribed cDNA; 3 μmol each of forward and reverse primers; 10 μl of 2x PCR buffer for KOD FX; 0.4 μl of KOD FX (1.0 U / μl); 4 μl of 2 mM dNTPs; add water to a total volume of 20 μl.

[0056] The PCR program is as follows: 94℃ pre-denaturation for 5 minutes; 94℃ denaturation for 30 seconds, 55℃ annealing for 30 seconds, 72℃ extension for 1 minute, 30 cycles; 72℃ extension for 7 minutes.

[0057] Example 2

[0058] Overexpression OsGL9 Genes cause grain length to decrease and appearance quality to deteriorate:

[0059] 1. Construction of overexpression vectors:

[0060] The rice gene amplified in Example 1 OsGL9 The full-length sequence was obtained, and the pC1300S vector was introduced into the upstream and downstream primers, respectively. Kpn I, BamH Homologous recombination adapters flanking the double restriction enzyme sites were used to design primer pairs OE-F and OE-R, as shown in SEQ ID No. 6 and SEQ ID No. 7.

[0061] Using the Higashino genome obtained in Example 1 as a template, PCR was performed using primers OE-F and OE-R, and the product size was 1892 bp.

[0062] PCR system: DNA (3ng); forward and reverse primers 3μmol each; 2x PCR buffer for KOD FX 10μl; KODFX (1.0U / μl) 0.4μl; 2mM dNTPs 4μl; add water to a total volume of 20μl.

[0063] PCR program: 94℃ pre-denaturation for 5 minutes; 94℃ denaturation for 30 seconds, 55℃ annealing for 30 seconds, 72℃ extension for 1 minute, 30 cycles; 72℃ extension for 7 minutes.

[0064] PCR amplification isolated by homologous recombination OsGL9 Gene fragments are connected to Kpn I, BamH The pC1300S vector was digested with enzyme I. After sequencing and alignment confirmed, it was transformed into Agrobacterium EHA105 to obtain... OsGL9 Gene overexpression vector.

[0065] 2. Obtaining transgenic plants with overexpression:

[0066] Genetic transformation mediated by Agrobacterium-mediated transformation OsGL9 Gene overexpression vectors were transferred into the indica rice variety Ganzaoxian 49 (GZX49). After selection, differentiation, rooting, and hardening, T0 generation transgenic plants were obtained (see Hiei Y, Ohta S, Komari T, Kumashiro T. Efficient transformation of rice (…)). Oryza sativa L.)mediated by agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant Journal (1994, 6: 271-282). Seeds were collected after planting all transgenic materials to complete propagation and obtain stably inherited T1 generation materials. For a given T1 generation transgenic line, if all T2 generation plants obtained after self-pollination are transgenic plants, then the T1 generation transgenic line is a homozygous transgenic line. Three homozygous transgenic lines, OE-1, OE-2, and OE-3, were selected for further analysis.

[0067] 3. Identification of transgenic plants with overexpression:

[0068] (1) Identification of expression level:

[0069] Total RNA was extracted from seedlings at the two-leaf stage, and cDNA was obtained by reverse transcription. Real-time quantitative PCR was then performed for detection. OsGL9 The primers for gene expression are:

[0070] RT-F (SEQ ID No. 8): 5'-ATGGCGTTCCGGCTGAGCAACA-3';

[0071] RT-R (SEQ ID No. 9): 5'-CTCAGGCGTATCCGCCCTTCCAC-3'.

[0072] The reagents used for quantitative analysis were FastStart Universal SYBR Green Master (ROX). The instrument used was a ViiA7 real-time quantitative PCR system from Applied Biosystems, USA. The Ubqtin gene was used as an internal control.

[0073] UBQ-F (SEQ ID No. 17): 5'-AACCAGCTGAGGCCCAAGA-3';

[0074] UBQ-R (SEQ ID No. 18): 5'-ACGATTGATTTAACCAGTCCATGA-3'.

[0075] Overexpression materials OsGL9 The relative expression level of genes, such as Figure 1 As shown, WT represents the rice variety GZX49, and OE-1, OE-2, and OE-3 represent... OsGL9 Transgenic lines with overexpressed genes. OsGL9 In transgenic lines with overexpressed genes OsGL9 The relative expression level of the gene was significantly higher than that of rice GZX49.

[0076] (2) Particle shape phenotype determination:

[0077] Images of the seeds were acquired using a Cano Scan (5600F), and then ImageJ software was used to convert the images into data, including seed length (mm), seed width (mm), and aspect ratio. The specific steps are as follows:

[0078] 1) Use Image-Pro Plus software to connect to the scanner (Cano Scan, 5600F) and fix the pixel size to 300 bpi.

[0079] 2) Select plump seeds (no less than 100) and spread them evenly on the scanner, ensuring that the grains are separated from each other.

[0080] 3) Preview the scanning window, click the scan button, and save the acquired images to a folder. Scan and name the samples to be scanned sequentially, and save them in the same folder.

[0081] 4) Open ImageJ software → Click File → Click Import → Click ImageJ sequence → Locate the folder containing the images and select any image → Open → Check Sort names numerically and Use virtualstack → Click OK.

[0082] 5) Click Image → Click 8-bit → Click Image Adjust → Click Threshold → Check Darkbackground (for black background) or uncheck it (for white background) → Click Set → Enter 70,255 (adjust for black background and white background) → Click OK → Click Apply → Check Black background, leave other settings as default → Click OK.

[0083] 6) Click Process → Click Filters → Click Mean → Enter 0 → Click OK, Yes.

[0084] 7) Click Analyze → Click Set scale → Enter 11.811023622 (this value cannot be used for other resolutions at 300dpi), 1, 1, mm respectively, check Global → Click OK.

[0085] 8) Click Analyze → Click Set measurement → Check Area and Perimeter.

[0086] 9) Click Plugins → Click Measure Roi PA → Enter 5-40 (meaning 5mm) 2 The following and 40mm 2 (Exclude the particles above) → Check Display results, Clear results, Exclude on edges, Include holes → Click OK, Yes.

[0087] 10) Click File → Click Save as to save the data, then manually remove extreme outliers by sorting, and finally use the pivot table function of Excel 2010 to calculate the mean, variance, standard deviation, etc. of each item.

[0088] like Figure 2 As shown, WT represents the rice variety GZX49, and OE-1, OE-2, and OE-3 represent... OsGL9 Transgenic grains with overexpressed genes, scale bar is 1 cm. OsGL9 The transgenic plants with overexpressed genes had significantly smaller grain lengths than rice GZX49 and exhibited poorer appearance and quality.

[0089] like Figure 3 As shown, WT represents the rice variety GZX49, and OE-1, OE-2, and OE-3 represent... OsGL9 Transgenic plants with overexpressed genes. OsGL9 The grain length and length-to-width ratio of transgenic plants with overexpressed genes were significantly smaller than those of rice GZX49.

[0090] Example 3

[0091] Crispr knockout OsGL9 Genes that increase grain length can improve the appearance and quality of rice.

[0092] 1. Construction of CRISPR knockout vector:

[0093] The rice gene amplified according to Example 1 OsGL9 The full-length genome sequence was obtained and designed using the website http: / / skl.scau.edu.cn / targetdesign / OsGL9 The target site for gene knockout was designed as SEQ ID No. 10: 5'-GCTCGCAGTGCGGGCACCAC-3'. Homologous recombination adapters flanking the KpnI restriction site of the pCXUN_CAS9 vector were introduced into the upstream and downstream primers, respectively. The primers were designed as follows:

[0094] U3-F (SEQ ID No. 11): 5'-CCCCTTTCGCCAGGGGTACCgtaattcatccaggtctccaag-3';

[0095] U3-R (SEQ ID No. 12): 5'-TACGAATTCGAGCTCGGTACCgctgtgccgtacgacggtacg-3';

[0096] Crispr-F (SEQ ID No. 13): 5'-GCTCCGCAGTGCGGGCACCACgttttagagctagaaatagcaagtta-3';

[0097] Crispr-R (SEQ ID No. 14): 5'-GTGGTGCCCGCACTGCGAGCgccacggatcatctgcacaactc-3'.

[0098] Using OsU3 as a template, PCR was performed first with primers U3-F and CRISPR-R to obtain PCR product 1, which was 476 bp in size. The PCR program was as follows: 94℃ pre-denaturation for 5 minutes; 94℃ denaturation for 30 seconds, 55℃ annealing for 30 seconds, 72℃ extension for 30 seconds, 30 cycles; 72℃ extension for 7 minutes.

[0099] Using OsU3 as a template, PCR was performed with primers U3-R and Crispr-F to obtain PCR product 2, which was 368 bp in size. The PCR program was as follows: 94℃ pre-denaturation for 5 minutes; 94℃ denaturation for 30 seconds, 55℃ annealing for 30 seconds, 72℃ extension for 30 seconds, 30 cycles; 72℃ extension for 7 minutes.

[0100] Finally, using the mixture of PCR product 1 and PCR product 2 as a template, PCR was performed with primers U3-F and U3-R to obtain the final PCR product, which was 824 bp in size. The PCR program was as follows: 94℃ pre-denaturation for 5 minutes; 94℃ denaturation for 30 seconds, 55℃ annealing for 30 seconds, 72℃ extension for 1 minute, 30 cycles; 72℃ extension for 7 minutes.

[0101] Homologous recombination is used to ligate the final products isolated by PCR amplification into... Kpn The pCXUN_CAS9 vector was digested with enzyme I. After sequencing and alignment confirmed, it was transformed into Agrobacterium EHA105 to obtain... OsGL9 CRISPR gene knockout vector.

[0102] 2. Obtaining CRISPR knockout plants:

[0103] Genetic transformation mediated by Agrobacterium-mediated transformation OsGL9 The CRISPR gene knockout vector was transferred into japonica rice Zhonghua 11 (ZH11). Following the same method as in Example 2, T0 generation transgenic plants were obtained through selection culture, differentiation, rooting, and hardening. All transgenic materials were propagated to obtain stable T1 generation materials with homozygous mutations and no residual Cas9 protein. Three homozygous transgenic lines, KO-1, KO-2, and KO-3, were selected for further analysis.

[0104] 3. Identification of CRISPR knockout plants:

[0105] 1) Genotyping:

[0106] DNA was extracted from seedlings at the two-leaf stage. PCR was performed using primers sq-F and sq-R, yielding a 419 bp amplification product. The PCR product was then sequenced and analyzed. OsGL9 The gene target site editing occurred. The PCR program was as follows: 94℃ pre-denaturation for 5 minutes; 94℃ denaturation for 30 seconds, 55℃ annealing for 30 seconds, 72℃ extension for 30 seconds, 32 cycles; 72℃ extension for 7 minutes. The primer sequences used are as follows:

[0107] sq-F (SEQ ID No. 15): 5'-GGAGAAATCGCCTAACCCTA-3';

[0108] sq-R (SEQ ID No. 16): 5'-GGAACCACACCAAGCCAAGA-3'.

[0109] The sequencing results of the target sites for Crispr knockout materials are as follows: Figure 4 As shown, ZH11 represents the rice variety Zhonghua 11 (wild type), referencing Nipponbare genome version 7.0 (Rice Genome Annotation Project), LOC_Os09g25760. KO-1, KO-2, and KO-3 represent... OsGL9 CRISPR gene knockout line. OsGL9 In CRISPR knockout lines OsGL9 The genes all underwent homozygous mutations at the target sites.

[0110] 2) Particle shape and phenotype determination:

[0111] The method for determining particle shape and phenotype is the same as above.

[0112] like Figure 5 As shown, ZH11 represents the rice variety Zhonghua 11 (wild type), and KO-1, KO-2, and KO-3 represent... OsGL9 CRISPR gene knockout plants. OsGL9 The grain length of plants with the CRISPR gene knockout was significantly greater than that of rice Zhonghua 11, resulting in improved rice appearance and quality.

[0113] like Figure 6 As shown, ZH11 represents the rice variety Zhonghua 11 (wild type), and KO-1, KO-2, and KO-3 represent... OsGL9 CRISPR gene knockout plants. OsGL9 The grain length and length-to-width ratio of the Crispr gene knockout plants were significantly greater than those of Zhonghua 11 rice.

[0114] Therefore, the present invention employs the above-mentioned method for regulating rice grain length. OsGL9 Genes, proteins and their applications, through knockout OsGL9 The gene can significantly increase rice grain length and optimize the length-to-width ratio, directly improving the appearance and quality of rice. It provides new genes and technological pathways for rice grain shape improvement, contributing to the breeding of high-quality rice.

[0115] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. The application of the OsGL9 gene in regulating rice grain length and length-to-width ratio, characterized in that, The full-length nucleotide sequence of the OsGL9 gene is shown in SEQ ID No. 1, and the CDS nucleotide sequence is shown in SEQ ID No. 2; Regulating rice grain length and length-to-width ratio involves overexpressing the grain length gene OsGL9, which reduces rice grain length and length-to-width ratio, and knocking out the grain length gene OsGL9, which increases rice grain length and length-to-width ratio.

2. The application of OsGL9 protein in regulating rice grain length and aspect ratio, characterized in that, The amino acid sequence of the OsGL9 protein is shown in SEQ ID No.

3. The encoding gene of the OsGL9 protein is the OsGL9 gene. The full-length nucleotide sequence of the OsGL9 gene is shown in SEQ ID No. 1, and the CDS nucleotide sequence is shown in SEQ ID No.

2.

3. A method for improving the length and length-to-width ratio of rice grains, characterized in that, Knocking out the OsGL9 gene in the rice genome increases the length and aspect ratio of rice grains. The full-length nucleotide sequence of the OsGL9 gene is shown in SEQ ID No. 1, and the CDS nucleotide sequence is shown in SEQ ID No.

2.

4. A method for creating high-quality rice germplasm, characterized in that, The OsGL9 gene was knocked out in the rice genome to screen and cultivate high-quality rice germplasm. The full-length nucleotide sequence of the OsGL9 gene is shown in SEQ ID No. 1, and the CDS nucleotide sequence is shown in SEQ ID No. 2.

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