Rice large grain gene qrbg1 and application thereof

Abstract

The application belongs to the technical field of biology and relates to a rice large-grain gene qRBG1 and application thereof. The application discloses a rice large-grain gene qRBG1, which has a CDS sequence as shown in SEQ ID NO:1 and a promoter sequence as shown in SEQ ID NO:3, encodes a protein of a signal factor OsBZR5 of rice brassinosteroid signal transduction, and has an amino acid sequence as shown in SEQ ID NO:2. The qRBG1 can be used for regulating rice grain type, improving rice yield and quality, and providing reliable gene resources for design breeding research of rice grain type.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the rice large-grain gene qRBG1 and its applications. Background Technology

[0002] Rice, hailed as the king of grains, possesses numerous health benefits, including replenishing qi, nourishing yin and moistening the lungs, and strengthening the spleen and stomach. It is a staple food for humankind. With the improvement of people's living standards, the demand for rice quality is increasing. Rice grain shape affects the appearance and processing quality of rice. Different grain shapes result in different tastes and nutritional profiles. Rice grain shapes are generally classified into three types based on their length-to-width ratio: long and slender (>3.1), medium-long (2.1–3.0), and short and round (<2.0). Different consumers have different preferences for rice grain shape (Calingacion et al., (2014) Diversity of Global Rice Markets and the Science Required for Consumer-Targeted Rice Breeding. Plos One 9, e85106.). Simultaneously, rice grain shape affects grain weight, which in turn affects yield. To meet the preferences of different groups, it is crucial to cultivate high-yield varieties with different grain shapes according to different needs. To achieve this goal, it is essential to isolate as many grain shape genes as possible and analyze their molecular mechanisms. Summary of the Invention

[0003] In view of this, the present invention provides the rice large-grain gene qRBG1 and its encoded protein, thereby providing a powerful tool for rice transgenic research and molecular marker-assisted selection breeding, and promoting high-yield breeding research of hybrid rice.

[0004] To achieve the above objectives, the present invention adopts the following technical solution:

[0005] The inventors had previously created a multi-grain, three-segment substitution line Z1364 using Nipponbare as the recipient parent and the indica rice restorer line Xihui 18 as the donor through natural mutation (Maetal, (2019) Identification of QTL for kernel number-related traits in a rice chromosome segment substitution line and finemapping of qSP1. The Crop Journal 7, 494-503.). Based on this, this invention uses the F2 population generated from the cross between Nipponbare and Z1364, combined with the MAS method, to breed a large-grain, single-segment substitution line Z499. Figure 1 A) The replacement segment of Z499 is as follows Figure 1As shown in B. The substitution fragments of the single-segment substitution line Z499 are RM10198-RM1329-RM10224-RM6777 on chromosome 1. The shortest substitution length is 50 kb, the estimated length is 0.20 Mb, and the maximum substitution length is 0.35 Mb. Figure 1 B). The substitution fragment of Z499 carries particle length QTL-qGL1, particle width QTL-qGW1, and particle weight QTL-qGWT1, with additive effects of 0.73 mm, 0.15 mm, and 3.24 g, respectively. Figure 2 (CE). Because these three QTLs have multiple effects, this invention collectively refers to them as qRBG1. Scanning electron microscopy statistical analysis of the inner and outer epidermal cells of Z499 and Nipponbare revealed that the large grain phenotype of Z499 is mainly caused by an increase in cell number and size. Then, using Z499 as material, qRBG1 located on chromosome 1 was cloned. qRBG1 is a novel gene, OsBZR5, from the BES1 / BZR1 (Brassinazole-resistant) transcription factor family. This gene exhibits multiple variations in the promoter of Z499; low expression leads to the large grain phenotype in Z499, and this large grain trait is inherited in a single-gene recessive manner. qRBG1, encoding OsBZR5, regulates rice grain development through the BR pathway. Exogenous application of 2,4-epiBL revealed that Nipponbare is sensitive to 2,4-epiBL, while Z499 is insensitive, indicating that qRBG1 plays an important role in the BR response. Therefore, determining the nucleotide sequence (including the promoter and CDS sequence) and encoded protein sequence of the qRBG1 gene will be beneficial for studying the molecular mechanism of large grain development in rice and hybrid rice breeding, and will help to further increase the yield of hybrid rice.

[0006] Specifically, the present invention provides a rice large-grain gene qRBG1, which has a CDS sequence as shown in SEQ ID NO:1 and an amino acid sequence encoded by the gene as shown in SEQ ID NO:2.

[0007] This invention provides the application of the qRBG1 gene in rice breeding, showing that reducing qRBG1 expression can increase rice grain size. Specifically, it increases rice grain length, grain width, and thousand-grain weight.

[0008] This invention also provides the application of the large-grain single-fragment substitution line of the qRBG1 gene in the breeding of large-grain rice varieties. Specifically, the large-grain single-fragment substitution line is bred with a two-line sterile line to breed large-grain rice varieties.

[0009] This invention further provides the application of the qRBG1 gene promoter in rice breeding. By editing the qRBG1 gene promoter of small-grain rice varieties, low expression of the qRBG1 gene can produce new large-grain rice varieties. The promoter sequence of qRBG1 in small-grain rice varieties is shown in SEQ ID NO:3. The promoter sequence of the edited variant qRBG1 is shown in SEQ ID NO:6.

[0010] Based on gene localization, this invention first preliminarily identified the rice large-grain gene qRBG1 as LOC_Os01g08180 through gene prediction, homology search, and gene sequence difference comparison. Subsequently, using the rice large-grain single-fragment substitution line Z499 as material, the full-length cDNA of the mutant gene qRBG1 was cloned. Sequencing results showed that the full-length CDS sequence of the mutant gene qRBG1 was identical to that of the recipient parent, Nipponbare, at 915 bp, consisting of two exons and encoding 304 amino acids. However, compared to the recipient Nipponbare, the large-grain gene qRBG1 exhibited one insertion, multiple substitutions, and deletions at the promoter. Figure 3 The promoter sequence of qRBG1 in the large-grain single-fragment substitution line Z499 of rice is shown in SEQ ID NO:6.

[0011] This invention also cloned the coding sequence of LOC_Os01g08180 from Nipponbare through genetic complementation and overexpressed LOC_Os01g08180 in Z499 using the Ubiquitin promoter. All positive transgenic plants restored the small-grain phenotype of Nipponbare, indicating that LOC_Os01g08180 is the coding gene for the large-grain gene qRBG1. qRBG1 is expressed in all tissues of Nipponbare, including roots, stems, leaves, sheaths, and ears, with the highest expression level at an ear length of 0.5 cm. Compared to Nipponbare, the expression level of qRBG1 in Z499 is downregulated.

[0012] Furthermore, by detecting the expression of BR-related genes in young spikelets of Nipponbare and Z499 rice, it was found that the expression levels of D2 and OsBZR1 were significantly upregulated in Z499 compared to Nipponbare. Among them, D2 encodes a key enzyme in the BR biosynthetic pathway, and its overexpression increases grain size (Fang et al, (2016) SMALL GRAIN 11 controls grain size, grain number and grain yield in rice. Rice 9, 64.). Brassinolide (BR) is an important hormone that is widely involved in regulating plant growth, immunity and other developmental processes. BR signaling is first sensed by the membrane receptor OsBRI1 and the co-receptor OsBAK1, and then through the inactivation of OsGSK2, the transcription factor OsBZR1 accumulates in the cell nucleus, thereby regulating its downstream genes to initiate a series of cellular activities (Li et al, (2018) Control of grain size in rice. Plant Reproduction 31, 237-251). Overexpression of OsBZR1 increases rice grain length, width, and weight (Zhu et al., (2015) Brassinosteroids promote development of rice pollen grains and seeds by triggering expression of Carbon Starved Anther, a MYB domain protein. The Plant Journal 82, 570-581.). Furthermore, the importance of this family of genes, OsBZR1-OsBZR4, in positively regulating rice grain development was also demonstrated by creating four single mutants or higher-order knockout mutants (Liu et al., (2021) Diversification of plant agronomic traits by genome editing of brassinosteroid signaling family genes in rice. Plant Physiology 187, 2563-2576.). In this invention, we discovered a novel mechanism by which qRBG1 regulates large rice grain development through the BR pathway.

[0013] The beneficial effects of this invention are as follows:

[0014] This invention discovers that low expression of qRBG1 can increase rice grain size, and its additive effect can increase grain length, grain width, and thousand-grain weight under the Nipponbare genetic background. On one hand, large-grain single-segment substitution lines of the qRBG1 gene can be crossbred with two-line sterile lines to breed new large-grain rice varieties. On the other hand, the promoter of the qRBG1 gene in small-grain varieties can be edited to induce low expression and produce new large-grain rice varieties. The qRBG1 gene of this invention provides an important genetic resource for molecular breeding of rice grain shape.

[0015] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0016] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein:

[0017] Figure 1 The diagram shows the selection and identification of Z499. A: Flowchart of Z499 selection; B: Replacement fragments and QTLs carried by Z499, GL: grain length, GW: grain width, GWT: weight of 1000 grains.

[0018] Figure 2For the identification of phenotype and grain type QTLs for the single-segment substitution line Z499 containing the qRBG1 gene, A: mature plants of Nipponbare and Z499, proportional length: 10cm; B: grain phenotype of Nipponbare and Z499, proportional length: 1cm; CE: statistical analysis of grain length (C), grain width (D), and thousand-grain weight (E) of Z499, where μ is the mean and ai represents the additive effect of the QTL. The p-value for Z499 represents the probability of a significant difference between Nipponbare and Z499 (t-test, p < 0.05); F: Scanning electron micrograph of the glumes of Nipponbare and Z499 spikelets, with dashed lines indicating the location of the cross-section, scale length: 1 mm; G: Scanning electron micrograph of the outer epidermal cells of Nipponbare and Z499, scale length: 100 μm; H: Scanning electron micrograph of the inner epidermal cells of Nipponbare and Z499, scale length: 100 μm; I: Number of cells in the length and width directions of grains in Nipponbare and Z499; J: Length and width of the inner epidermal cells of Nipponbare and Z499 (n = 50); K: Cross-section of the glumes of Nipponbare and Z499, scale length: 2 mm; L: Enlarged view within the black box of K. Scale bar, 100 μm; M: Number of outer parenchyma cells in the palea and foremilk, as shown in K, specifically the cell layer corresponding to the arrow in L, scale length: 2 mm; N: Area of ​​outer parenchyma cells in the palea. Data are presented as mean ± standard deviation (n = 20). A t-test was used to determine significant differences between wild-type and Z499 (*P < 0.05, **P < 0.001, and ****P < 0.0001).

[0019] Figure 3 This is a sequence comparison diagram of the qRBG1 variant gene and the wild-type gene, showing the variation in the promoter sequence of Z499.

[0020] Figure 4 For the complementation phenotype analysis of qRBG1, A: mature Nipponbare, Z499 and qRBG1 complementary transgenic plants, proportional length: 10cm; B: grain phenotype of Nipponbare, Z499 and qRBG1 complementary transgenic plants, proportional length: 1cm; CE: statistical analysis of grain length (C), grain width (D) and thousand-grain weight (E) of Nipponbare, Z499 and qRBG1 complementary transgenic plants; F: relative expression level of qRBG1 in Nipponbare, Z499 and complementary transgenic lines (COM: ProUbi:qRBG1 transformed in the Z499 background, COM-1, COM-2 and COM-3 represent independent complementary transgenic lines).

[0021] Figure 5Analysis of promoter cis-regulatory elements for gene qRBG1 and its predicted family genes. A: Distribution of cis-regulatory elements in the promoter region of qRBG1 and its predicted family genes; B: Cis-regulatory elements in the promoter region of qRBG1 and its predicted family genes. The numbers in parentheses indicate the number of cis-regulatory elements.

[0022] Figure 6 Multiple sequence alignments of the N-terminus and C-terminus of the gene qRBG1 and its predicted family proteins are shown, with relatively conserved sequences displayed in black boxes.

[0023] Figure 7 Phylogenetic analysis of the gene qRBG1 and its predicted family proteins, including conserved motifs and domains. A: Phylogenetic analysis of qRBG1 and its predicted family proteins from different species. At, Arabidopsis thaliana; Os, rice; Capsella asiatica; FCD, fig; Zm, Zea mays. B: Conserved motif analysis of these proteins in the phylogenetic tree. Three conserved motifs were identified using MEME software. C: Domain analysis of these proteins in the phylogenetic tree.

[0024] Figure 8 The response of Z499 to exogenous 2,4-epiBL is shown in Figure A: Leaf angles after treatment with exogenous 2,4-epiBL in Nip and Z499 plants. (Scale length, 1 cm). Figure B: Statistical analysis of leaf angles in response to 2,4-epiBL.

[0025] Figure 9 The expression levels of BR-related genes in Nipponbare (Nip) and Z499 are shown in Figure 1. A: Relative expression levels of BR biosynthesis-related genes in young spikelets of Nip and Z499. B: Relative expression levels of BR signaling-related genes in young spikelets of Nip and Z499.

[0026] Figure 10 Figure 1 shows the recombinant vector PTCK303-qRBG1. Detailed Implementation

[0027] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0028] Experimental methods not specified in the examples are generally performed under standard conditions, such as those described in Molecular Cloning: A Laboratory Manual (3rd Edition, by J. Sambrook et al., translated by Huang Peitang et al., Science Press, 2002), or as recommended by the manufacturer.

[0029] Materials used in the examples: The rice recipient parental materials, Nipponbare and the large-grain single-fragment substitution line Z499 (qrbg1), were both preserved or cultured in the inventor's laboratory; M-MLV reverse transcriptase, high-fidelity DNA polymerase PFU, Taq DNA polymerase, T4 DNA ligase, restriction endonucleases, pMD19-T vector, Trizol kit, DNA gel extraction kit, plasmid extraction kit, λ-Hind III DNA Marker, and DL5,000 DNA Marker were purchased from TaKaRa; DNA Marker III was purchased from Tiangen Biotech (Beijing) Co., Ltd.; Ampicillin (Amp) and Kanamycin (Kan) were products of Sigma-Aldrich; primer synthesis and DNA sequencing were performed by Shanghai Yingjun Biotechnology Co., Ltd.; other chemical reagents were purchased from Beijing Dingguo Biotechnology Co., Ltd.; Escherichia coli DH5α and Agrobacterium LBA4404 were preserved in the inventor's laboratory.

[0030] The construction process of the single-segment substitution line Z499 used in the example is as follows: Nipponbare was used as the recipient parent and crossed with the donor parent Xihui 18 to produce the F1 generation. This F1 generation was then backcrossed twice with Nipponbare to produce the BC2F1 generation. In the BC2F1 generation, SSR markers covering the entire genome were used to screen for SSR polymorphism markers between parents for marker-assisted selection (MAS). Then, 20 BC2F1 plants were backcrossed with Nipponbare to produce the BC3F1 generation. This was then self-crossed to BC3F8. During this period, each line in each generation was treated with the heterozygous marker MAS. Finally, a rice chromosome segment substitution line Z1364 with a 3-generation substitution fragment was selected. Then, a secondary F2 population was constructed by crossing Nipponbare with Z1364, and QTL mapping was performed using the marker on the substitution fragment. After fine QTL mapping, the single-segment substitution line Z499 for chromosome 1 was further constructed in the F3 generation using MAS. The specific process is as follows: Figure 1 A. The substitution fragments of the single-segment substitution line Z499 are RM10198-RM1329-RM10224-RM6777 on chromosome 1. The shortest substitution length is 50 kb, the estimated length is 0.20 Mb, and the maximum substitution length is 0.35 Mb. Figure 1 As shown in B.

[0031] like Figure 2As shown in Figure A, the single-segment substitution line Z499 and Nipponbare have the same plant type. Compared with the parent Nipponbare, the single-segment substitution line Z499 shows increased grain length, grain width, and thousand-grain weight. Figure 2 As shown in BE. Scanning electron microscopy results showed that, compared with Nipponbare, the single-fragment substitution line Z499 had significantly increased cell numbers in both the longitudinal and transverse directions of the outer epidermis, as well as increased length and width of the inner epidermal cells. Figure 2 As shown in FJ. Paraffin section results showed that, compared with Nipponbare, the number and cell area of ​​the outer thin-walled cells were significantly increased, such as... Figure 2 As shown in KN, the large-particle phenotype of the single-fragment substitution line Z499 is caused by a simultaneous increase in cell proliferation and cell division.

[0032] Based on the cultivated single-fragment substitution line Z499 and fine mapping, this invention first utilizes online gene prediction (http: / / mendel.cs.rhul.ac.uk) and online BLAST alignment (…). http: / / blast.ncbi.nlm.nih.gov / Through gene function complementation analysis, the rice large-grain variant gene qRBG1 was identified as a novel signaling factor OsBZR5 (LOC_Os01g08180) for brassinolide signaling. BES1 / BZR1 family transcription factors regulate plant growth and development through the BR pathway. At least six members are found in Arabidopsis thaliana, and four members, OsBZR1, OsBZR2, OsBZR3, and OsBZR4, have been reported in rice (Baie et al, (2007) Functions of OsBZR1 and 14-3-3 proteins in brassinosteroid signaling in rice. Proceedings of the National Academy of Sciences 104, 13839-13844). OsBZR1 is a core member of the BR signaling pathway. After activation by BR signaling, it usually recognizes and binds to E-box (CANNTG) or BRRE (CGTGC / TG) cis-regulatory elements through its DNA-binding domain to regulate the expression of BR response genes (Shi et al, (2022) BES1 / BZR1 Family Transcription Factors Regulate Plant Development via Brassinosteroid-Dependent and Independent Pathways. International Journal of Molecular Sciences). 23, 10149-10149.). In rice grain development, overexpression of OsBZR1 increases grain length, width, and thickness (Zhu et al, (2015) Brassinosteroids promote development of rice pollen grains and seeds by triggering expression of Carbon StarvedAnther, a MYB domain protein. The Plant Journal 82, 570-581.).Furthermore, the regulation of rice grain development by OsBZR1-OsBZR4 has also been demonstrated by creating four single-mutant or high-order knockout mutants (Liu et al, (2021) Diversification of plant agronomic traits by genome editing of brassinosteroid signaling family genes in rice, Plant Physiology 187, 2563-2576.). In this invention, the large-grain gene qRBG1, located on chromosome 1, was map-based cloned from the large-grain rice single-segment substitution line Z499. qRBG1 encodes a novel gene, OsBZR5, which is different from OsBZR1-OsBZR4 in the BES1 / BZR1 family. In Z499, a promoter mutation in this gene resulted in low expression of qRBG1, leading to increased rice grain size and negative regulation of grain size. The full-length cDNA sequence of the qRBG1 gene in Z499 is identical to that of the recipient parent, Nipponbare, at 915 bp (as shown in SEQ ID NO:1), consisting of two exons and encoding 314 amino acids (as shown in SEQ ID NO:3). However, compared to the recipient Nipponbare, the large-particle qRBG1 gene exhibits one insertion, multiple substitutions, and deletions at its promoter. Figure 3 The mutated qRBG1 promoter sequence is shown in SEQ ID NO:6.

[0033] Subsequently, using the large-grain single-fragment substitution line Z499 as material, this invention cloned the promoter and cDNA of the mutated gene qRBG1, and used bioinformatics methods to study the promoter sequence, nucleotide sequence and encoded protein structure of the wild-type gene qRBG1, and constructed a recombinant plant overexpression vector of the wild-type gene qRBG1 for transgenic research in rice.

[0034] I. Cloning, sequencing, and functional complementation analysis of the variant gene qRBG1

[0035] Based on the rice Nipponbare gene sequence Os01g0176900LOC_Os01g08180 already registered in GeneBank, specific primers were designed using Vector NTI software:

[0036] Upstream primer qRBG1-COM-F-KpnI:

[0037] 5'-TTCTGCAGGATCCCCGGGTACCATGGATCAGGTGGTAGCATCTGG-3' (SEQ ID NO: 4);

[0038] Downstream primer qRBG1-COM-R-SpeI:

[0039] 5'-ACGGAGCTCTCTAGAACTAGTTCAAGAAGTCTGCAGCTTGTCGG-3' (SEQ ID NO: 5).

[0040] Whole seedlings of the rice recipient parent Nipponbare and the large-grain single-fragment substitution line Z499, cultured for two weeks, were rapidly ground into powder in liquid nitrogen. Total RNA was extracted according to the Trizol kit instructions. Electrophoresis results of the obtained total RNA from Nipponbare and the large-grain single-fragment substitution line Z499 showed clear and intact major bands, with a 28S to 18S band brightness ratio of approximately 2:1, indicating that the RNA concentration and purity met the experimental requirements and could be used for double-stranded cDNA synthesis.

[0041] Using total RNA from Nipponbare and the large-particle single-fragment substitution line Z499 as templates, cDNA was obtained by reverse transcription using Oligo(dT) primers according to the M-MLV reverse transcriptase instructions. The synthesized cDNA was then used as a template for PCR amplification using specific primers qRBG1-COM-F-KpnI and qRBG1-COM-R-SpeI and high-fidelity DNA polymerase PFU. The PCR reaction conditions were: 94℃ pre-denaturation for 5 minutes; followed by 94℃ denaturation for 30 seconds, 55℃ annealing for 30 seconds, and 72℃ extension for 1 minute, for a total of 35 cycles; and a final extension at 72℃ for 10 minutes. The RT-PCR products were detected by 1.0% (g / mL) agarose gel electrophoresis, and then purified by gel extraction according to the DNA gel extraction kit instructions. The purified DNA fragments were ligated with the enzyme-digested PTCK303 vector at 50℃ for 30 min using recombinase. The ligation product was transformed into E. coli DH5α competent cells, and positive clones were screened using LB agar plates containing kanamycin. Plasmids were extracted, identified by PCR, and sequenced to obtain the recombinant vector PTCK303-qRBG1. The recombinant vector is as follows: Figure 10 As shown.

[0042] The recombinant vector PTCK303-qRBG1 plasmid obtained above was transformed into Agrobacterium tumefaciens EHA105, resulting in positive Agrobacterium colonies containing the target fragment. These colonies were then transformed into the single-fragment substitution line Z499 mutant. Identification showed that the transgenic plants produced smaller seeds, completely reverting to the small-grained type similar to Nipponbare. Figure 4 (AF). Therefore, it can be determined that the LOC_Os01g08180 gene is a large-particle variant gene.

[0043] II. Bioinformatics Analysis of the Mutant Gene qRBG1

[0044] The 2000bp upstream sequence of the ATG region of the qRBG1 gene was selected as the putative promoter region. Cis-acting elements were identified and retrieved using the online website PlantCARE (http: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ). The obtained information was organized and visualized using TBtools. Open reading frames were identified using the NCBI ORF Finder (http: / / www.ncbi.nlm.nih.gov / gorf / gorf.html). Conserved domains were analyzed using the CDD (conserved domain database) (http: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi). Amino acid sequence alignment and phylogenetic tree generation were performed using MEGA4 software.

[0045] The analysis results of identifying and retrieving cis-acting elements using the online website PlantCARE are as follows: Figure 5 As shown, the promoters of the Arabidopsis and rice BES1 / BZR1 gene family contain multiple cis-regulatory elements related to hormone response, stress response and tissue regulation, and qRBG1, AtBZR1 and OsBZR3 contain seed-specific regulatory elements (RY-element).

[0046] ORF Finder software analysis showed that the variant gene qRBG1 consists of a complete and continuous open reading frame.

[0047] The amino acid sequence alignment results of the qRBG1 encoded protein are as follows: Figure 6 As shown, qRBG1 has high N-terminal homology with proteins encoded by other genes in the BES1 / BZR1 family in rice and Arabidopsis thaliana.

[0048] The phylogenetic tree and CDD analysis results of qRBG1 are as follows: Figure 7 As shown, qRBG1 contains a BES1 conserved structural domain, and qRBG1 and ZmBZR6 are in the same branch and are closely related.

[0049] III. Rice qRBG1 plays an important role in the BR response.

[0050] Different concentrations of 2,4-epiBL were applied to Nipponbare and Z499, and the analytical results are as follows: Figure 8 As shown in the figure, the results indicate that Z499 is less sensitive to BR compared to Nipponbare.

[0051] Changes in BR synthesis and BR-related gene expression levels were analyzed in the young spikelets of Nipponbare and Z499. The results are as follows: Figure 9 As shown in the results, D2 and OsBZR1 were highly expressed in Z499 compared to Nipponbare.

[0052] IV. Application of the rice large-grain variant gene qRBG1

[0053] Rice, hailed as the king of grains, possesses numerous health benefits, including replenishing qi and blood, nourishing yin and moistening the lungs, and strengthening the spleen and stomach. It is a staple food for humankind. Rice grain shape affects its appearance and processing quality, as well as grain weight, which in turn influences yield. To meet the preferences of different populations, it is crucial to cultivate varieties with different grain shapes to meet diverse needs. Low expression of qRBG1 can increase rice grain size; its additive effect can increase grain length by 0.73 mm, grain width by 0.15 mm, and thousand-grain weight by 3.24 g in the Nipponbare genetic background. Therefore, on the one hand, the large-grain single-segment substitution line Z499 of the qRBG1 gene can be crossbred with two-line sterile lines to cultivate new large-grain rice varieties; on the other hand, the promoter of the qRBG1 gene in small-grain varieties can be edited to induce low expression and produce new large-grain rice varieties. The qRBG1 gene of this invention provides an important genetic resource for molecular breeding of rice grain shape. This technology has the advantages of precision, simplicity, speed, and ease of operation.

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. 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 be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. The rice large-grain gene qRBG1, characterized by: The CDS sequence of the gene qRBG1 is shown in SEQ ID NO:1, and the amino acid sequence encoded by the gene is shown in SEQ ID NO:

2.

2. The application of the gene qRBG1 according to claim 1 in rice breeding, characterized in that, Rice grain size can be increased by reducing the expression of the qRBG1 gene.

3. The application of the gene qRBG1 according to claim 1 in increasing rice grain length, grain width, and thousand-grain weight, characterized in that, Increasing rice grain length, grain width, and thousand-grain weight by reducing the expression of the qRBG1 gene.

4. Application of the large-grain single-segment substitution line of gene qRBG1 in the breeding of large-grain rice varieties, characterized in that... Large-grained rice varieties were bred by crossing the large-grained single-fragment substitution line with a two-line sterile line. The CDS sequence of the gene qRBG1 is shown in SEQ ID NO:

1.

5. The application of the qRBG1 gene promoter in rice breeding, characterized in that, By editing the promoter of the qRBG1 gene in small-grain rice varieties, low expression of the qRBG1 gene was achieved, resulting in large-grain rice varieties. The CDS sequence of the qRBG1 gene is shown in SEQ ID NO:1, and the promoter sequence of the qRBG1 gene in small-grain rice varieties is shown in SEQ ID NO:3.

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