Application of OsvWA36 gene in regulating grain type and yield of rice

Abstract

This invention discloses OsvWA36 The application of genes in regulating rice grain shape and yield falls under the field of agricultural bioengineering technology. OsvWA36 The application of genes in regulating rice grain shape and yield, the OsvWA36 The nucleotide sequence of the gene is shown in SEQ ID NO: 1. This invention provides a new option for increasing rice grain length and yield; the application is to... OsvWA36 Genes are integrated into rice through overexpression vectors, increasing rice grain length and yield, which has significant application value for rice breeding.

Description

Technical Field

[0001] This invention belongs to the field of agricultural bio-genetic engineering technology, specifically involving OsvWA36 Application of genes in regulating rice grain shape and yield. Background Technology

[0002] Rice is one of the world's most important food crops, and its yield is directly related to food security. Grain size and morphology (grain shape) are core factors determining rice yield, and also affect the appearance quality and market value of rice. Therefore, identifying key genes that regulate rice grain shape and elucidating their mechanisms of action is a crucial foundation for achieving high-yield and high-quality rice breeding. Currently, the research and breeding applications of rice grain shape regulating genes face the following limitations:

[0003] First, the regulatory mechanisms are limited to traditional pathways. Most cloned grain shape genes, such as GS3 and GW5, primarily function in traditional hormone signal transduction or cell cycle regulation pathways. While these pathways are important, our understanding of the grain shape regulatory network remains incomplete. In recent years, liquid-liquid phase separation, as a novel mechanism that efficiently regulates key physiological processes through the aggregation of biomolecules into membrane-free organelles, has shown significant importance in plant and animal development. However, its role in rice grain development and grain shape regulation has not yet been reported, making it difficult for current technologies to overcome the regulatory limitations of traditional pathways.

[0004] Second, there is insufficient coordinated regulation of multiple physiological processes. Ideal grain development is the result of the precise coordination of multiple physiological processes, including cell division, cell expansion, cell wall construction, and nutrient transport and deposition. Currently known genes mostly regulate only a specific process, such as promoting cell division or affecting hormone levels, making it difficult to achieve coordinated optimization of these multiple processes. This singular regulation easily leads to the paradox of increased grain size but decreased quality, such as empty or shriveled grains, increased chalkiness, or insufficient firmness, failing to achieve synergistic improvement in both yield and quality.

[0005] Third, molecular breeding lacks efficient and precise markers. Although marker-assisted selection has been widely used in rice breeding, the existing research on the association between natural variations in many grain shape genes and traits is not in-depth enough, and there is a lack of molecular markers that are universally effective in breeding populations and closely linked to superior grain shapes. This leads breeders to still rely heavily on phenotypic selection in practice, which is time-consuming, inefficient, and makes it difficult to accurately aggregate multiple minor grain shape genes, thus restricting the breeding efficiency of high-yielding and high-quality rice varieties.

[0006] Therefore, discovering key genes for grain shape / yield with novel regulatory mechanisms, synergistic effects on multiple physiological pathways, and clear natural variations can not only improve the theory of genetic regulation of rice grain shape, but also provide key technical support for breaking through existing breeding technology bottlenecks and cultivating new rice varieties that combine high yield and high quality. This has significant theoretical research value and agricultural practical significance. Summary of the Invention

[0007] This invention aims to provide OsvWA36 The application of genes in regulating rice grain shape and yield provides a new option for increasing rice grain length and yield. This application involves... OsvWA36 Genes are integrated into rice through overexpression vectors, increasing rice grain length and yield, which has significant application value for rice breeding.

[0008] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0009] OsvWA36 The application of genes in regulating rice grain shape and yield, the OsvWA36 The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0010] Preferably, the OsvWA36 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:2.

[0011] Preferred, by increasing OsvWA36 Gene expression level or OsvWA36 Gene-encoded protein activity or content increases rice grain length and yield.

[0012] The present invention also provides an overexpression vector for increasing rice grain length and yield, the overexpression vector comprising the... OsvWA36 Gene.

[0013] The present invention also provides a strain that increases rice grain length and yield, the strain comprising the overexpression vector described above.

[0014] The present invention also provides a method for cultivating rice plants with increased grain length and yield, the method comprising the following steps:

[0015] Introducing and expressing the above into rice receptors OsvWA36 Genes were used to obtain transgenic rice plants with grain length and yield higher than those of the rice recipient.

[0016] Preferably, the import is achieved by... OsvWA36 The gene was linked downstream of the rice promoter to construct an overexpression vector, which was then transformed into rice using Agrobacterium-mediated transformation.

[0017] This invention also provides a method for cultivating rice plants with increased grain length, the method comprising modifying the rice plants as described above using gene editing technology. OsvWA36 Genes, Enhancement OsvWA36 The ability of gene-encoded proteins to separate into liquid and liquid phases.

[0018] The present invention also provides a DNA molecular marker for detecting rice grain length traits, the molecular marker being based on the... OsvWA36 Design of single nucleotide polymorphism sites in the gene coding region that are associated with grain length traits.

[0019] This invention also provides a method for breeding rice varieties with a target grain length, comprising the following steps: detecting the target grain length of the rice using the molecular markers as described above. OsvWA36 Gene haplotypes: Select rice varieties with haplotypes associated with the target grain length for breeding.

[0020] Compared with the prior art, the present invention has the following advantages and technical effects:

[0021] 1. This invention discloses OsvWA36 The application of genes in regulating rice grain shape and yield, through the construction of osvwa36 Phenotypic verification of mutants, complemented lines, and overexpression lines confirmed... OsvWA36 It is a key gene that positively regulates rice grain length. osvwa36 The mutant grain length was significantly reduced compared to the wild type. The grain length of the reintroduced lines could be restored to the level of the wild type, and the grain length of the overexpressed lines was further increased compared to the wild type. This regulatory effect is directly related to the thousand-grain weight (grain length is the core influencing factor of thousand-grain weight), providing a clear target for improving rice yield. Compared with the traditional grain shape improvement that relies on the aggregation of multiple genes, the regulatory target is more precise and the yield gain is more predictable.

[0022] 2. Revealing the basis of cellular regulation, providing precise direction for granule shape improvement, and confirmed by scanning electron microscopy experiments. osvwa36 The mutant showed inhibited elongation of glume epidermal cells, which was clearly evident. OsvWA36 Grain length is affected by regulating the elongation process of glume epidermal cells.

[0023] 3. Tissue-specific high expression avoids negative effects from non-target tissues, as confirmed by qRT-PCR and Western-Blot experiments. OsvWA36 It is highly expressed in the panicle (a key tissue for grain development) of rice, but its expression level is low in vegetative organs such as roots, stems, and leaves.

[0024] 4. A novel regulatory mechanism for liquid-liquid phase separation (LLPS) was discovered, overcoming the limitations of traditional pathways. This was confirmed by in vitro protein aggregate observation, in vivo fluorescence fusion experiments, and FRAP experiments. OsvWA36The LLPS mechanism mediates the formation of dynamic, reversible membrane-free aggregates through the IDR (intrinsic disorder region). This mechanism provides a novel pathway for rice grain shape regulation, breaking the traditional framework of existing technologies that rely on hormone signals (auxin, cytokinin) or cell cycle genes. Furthermore, the dynamic nature of LLPS enables "flexible regulation" of grain development, avoiding abnormal grain development caused by rigid regulation and improving the adaptability and stability of regulation.

[0025] 5. Uncovering natural variation sites to empower efficient molecular breeding: Haplotype analysis of the 3K rice genome population revealed... OsvWA36 Eleven natural haplotypes related to grain length were found in the coding region. Among them, the grain length of rice samples carrying Hap6 was significantly shorter than that of haplotypes such as Hap1 and Hap4. This finding can be directly converted into molecular markers for rapid screening of rice germplasm resources and targeted aggregation of superior grain shape traits. Compared with traditional breeding methods, it can shorten the grain shape improvement cycle and greatly improve breeding efficiency.

[0026] In summary, this invention not only clarifies OsvWA36 Its core role in rice grain shape and yield regulation reveals a novel LLPS regulatory mechanism and multi-pathway synergistic mode. Its tissue specificity, dynamic regulation and availability of natural variation make it an ideal target for high-yield and high-quality rice breeding. It has important practical significance for solving the problems of "low precision, quality defects and long breeding cycle" in existing grain shape improvement, and provides key technical support for ensuring food security.

[0027] 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

[0028] Figure 1 for OsvWA36 The expression pattern diagram, in which, Figure 1 In this context, A represents the tissues of clove B (DXB) during its seedling and reproductive growth stages. OsvWA36 Graph of qRT-PCR analysis of transcripts Figure 1 In this context, B represents the Western blot analysis of different tissues during DXB development. OsvWA36 The results of protein abundance are shown in the figure.

[0029] Figure 2 for OsvWA36 Subcellular localization map, in which, Figure 2 A in the image is a confocal microscope image, showing... OsvWA36 -GFP is distributed on the cell nucleus. Figure 2 B in the image is a confocal microscope image, showing... OsvWA36 -GFP and FM4-64 stained membrane systems are located differently;

[0030] Figure 3 for OsvWA36 Liquid-liquid phase separation (LLPS) can occur both in vitro and in vivo, among which, Figure 3 In this context, A represents purified recombinant His- OsvWA36 Results of in vitro LLPS assay of protein, Figure 3 B in the text refers to the body. OsvWA36 The dynamic formation process of GFP, i.e., transient expression of 35S:: OsvWA36 -GFP aggregates in tobacco leaf epidermal cells Figure 3 C in the text is OsvWA36 Analysis results of fluorescence recovery after photobleaching (FRAP) of GFP condensates. Figure 3 D in the figure represents the quantitative result of FRAP recovery kinetics;

[0031] Figure 4 for osvwa36 Grain type analysis diagrams of mutant and control plants, in which, Figure 4 In this context, A represents wild-type (WT) and osvwa36 Representative images of mutant grain length. Figure 4 B in the text represents wild-type (WT) and osvwa36 Representative images of the mutant grain width. Figure 4 C in the text represents wild type (WT) and osvwa36 Statistical analysis of grain length in mutant seeds. Figure 4 In this context, D stands for wild-type (WT) and osvwa36 Statistical analysis of grain width in mutant seeds. Figure 4 E in the text represents WT and osvwa36 Thousand-grain weight (TGW) of the mutant;

[0032] Figure 5 for osvwa36 Analysis of glume cell length and lignin deposition in mutants, among which, Figure 5 A in the figure represents WT and osvwa36 Image of spikelets before flowering in the mutant. Figure 5 B in the text represents WT and osvwa36 Scanning electron microscopy (SEM) images of the outer and inner epidermal cells of the mutant glume. Figure 5 C in the text represents WT and osvwa36 Statistical analysis of the length of epidermal cells in the glume of mutants. Figure 5 D in the figure represents WT and osvwa36 Statistical analysis of the width of the outer epidermal cells of the glume in mutants. Figure 5 E in the text represents WT and osvwa36 Statistical analysis of the length of epidermal cells inside the glume of mutants. Figure 5 F in the figure represents WT and osvwa36 Statistical analysis of the width of epidermal cells inside the glume of mutants. Figure 5G in the figure represents the detection of WT and pyrogallol-hydrochloric acid staining. osvwa36 Lignin deposition (magenta) in spikelets before and 1 day after flowering in mutants.

[0033] Figure 6 for OsvWA36 A diagram showing the grain shape analysis of a gene-complemented line, in which... Figure 6 In this context, A represents wild-type (WT). osvwa36 Representative images of grain length from mutants and genetically complementary lines. Figure 6 B in the text represents wild type (WT). osvwa36 Representative images of grain width from mutants and genetically complementary lines. Figure 6 C in the text represents wild type (WT). osvwa36 Statistical analysis of grain length in mutants and genetically complementary lines. Figure 6 The D in the text represents the wild type (WT). osvwa36 Statistical analysis of grain width in mutants and genetically complementary lines;

[0034] Figure 7 Background of ZH11 OsvWA36 A diagram showing the genetic validation results of gene regulation of grain length function. Figure 7 A in the text is ZH11. osvwa36ΔIDR Mutants (CR-1, CR-2) and OsvWA36 Representative images of grain length from overexpression (OE) lines. Figure 7 B in the text is ZH11. osvwa36 IDR Mutants (CR-1, CR-2) and OsvWA36 Representative images of grain width from overexpression (OE) lines. Figure 7 In the figure, C represents the statistical analysis results of grain length. Figure 7 In the figure, D represents the statistical analysis results of grain width;

[0035] Figure 8 for OsvWA36 The results of the gene natural variation analysis are shown in the figure. Figure 8 In the diagram, A represents a comparative analysis of Hap1 and Hap6. Figure 8 B in the figure is a comparative analysis diagram of Hap4 and Hap6. Detailed Implementation

[0036] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0037] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0038] Source of experimental materials:

[0039] Wild type (WT): The high-quality maintainer line indica rice variety “Dingxiang B” from Guangxi was selected. In addition, genetic materials with the background of “Zhonghua 11 (ZH11)” were also selected for further functional verification.

[0040] 3K rice genome population materials: Data are from public databases ( https: / / v1.rmbreeding.cn / Genotype / haplotype ), select containing OsvWA36 Whole set 3K-RG germplasm of coding region sequences were analyzed using GL from RFGBphenotype.

[0041] Molecular cloning reagents: Trizol reagent (Invitrogen), reverse transcription kit (Invitrogen), high-fidelity DNA polymerase (vazyme), restriction endonucleases (EcoRI, BamHI, NEB), homologous recombinase (vazyme).

[0042] Vectors: CRISPR / Cas9 vector, overexpression vector (pCAMBIA1300-35S-GFP), prokaryotic expression vector (pET-28a).

[0043] Antibody: OsvWA36 Polyclonal antibodies (Pujian Biotechnology (Wuhan) Co., Ltd.), rabbit-derived antibodies, actin antibodies (Proteintech), His antibodies (Proteintech)

[0044] In this invention, unless otherwise specified, all other test materials and instruments are conventional test materials in the field and can be purchased through commercial channels.

[0045] Example 1

[0046] Seeds were disinfected with 75% ethanol for 30 seconds, then with 2.5% sodium hypochlorite for 20 minutes, rinsed 5 times with sterile water, and then... Germination in darkness for 2 days, then transferred to Kimura B nutrient solution (pH 5.5) and placed in an incubator (28°C). Photoperiod Dark, light intensity 300 mol m -2 s -1 Cultivate at 70% humidity, and transplant to the field after the seedling stage (with conventional water and fertilizer management).

[0047] Microbial culture: Escherichia coli (DH5α) BL21) in LB medium at 37°C Shaking culture (200 rpm); Agrobacterium (GV3101) in LB medium at 28°C Shaking culture (180 rpm).

[0048] 1. osvwa36 Creation of mutants and transgenic lines:

[0049] CRISPR / Cas9 mutants ( osvwa36 ) Build:

[0050] sgRNA design: based on OsvWA36 The coding region sequence (MSU_Locus: LOC_Os11g45990) was used to design sgRNA1 and sgRNA2 using CRISPR-P 2.0 tools. The sgRNA1 sequence is shown in SEQ ID NO:3 and the sgRNA2 sequence is shown in SEQ ID NO:4.

[0051] SEQ ID NO: 3: 5'-GATTGTGGGGAAGATGGGTGTGG-3'.

[0052] SEQ ID NO: 4: 5'-GCATAAGCGCATGCCACCACGG-3'.

[0053] Vector construction: The sgRNA1 and sgRNA2 sequences were cloned into... The BsaI restriction site of the vector, transformed into DH5α Competent cells were selected, and positive clones were sequenced for verification.

[0054] Agrobacterium transformation: The correct recombinant vector was transformed into Agrobacterium GV3101 and sent to Baige Gene Technology (Jiangsu) Co., Ltd. for transformation of syringe B.

[0055] Mutant identification: DNA was extracted from the leaves of regenerated plants and identified using primers F and R, where the F sequence is shown in SEQ ID NO:5 and the R sequence is shown in SEQ ID NO:6.

[0056] SEQ ID NO:5:ACTGCTCCAGTTTTCCTTTGAA.

[0057] SEQ ID NO: 6: TGGTATTCTAGCACGGAGGAGT.

[0058] Perform PCR amplification, sequence the amplification products, and screen for homozygous mutants (e.g., vw-1: 1bp deletion; vw-2: 2bp deletion).

[0059] (5) The background of the 11 yuan spent on ordering from Baige Gene Technology (Jiangsu) Co., Ltd. osvwa36Mutants were identified and determined.

[0060] 2. OsvWA36 Construction of complemented and overexpressed lines

[0061] (1) Carrier construction: OsvWA36 The coding region sequence was cloned into the pRHVcGFP vector (driven by the ubiquitin promoter), fused with a GFP tag, and verified by sequencing.

[0062] (2) Obtaining transgenic lines: Following the Agrobacterium-mediated rice transformation method described above, the complementation vector was transferred into the transgenic line. osvwa36 The mutant was overexpressed into "Zhonghua 11" and positive lines were obtained through hygromycin selection. qRT-PCR was then used for verification. OsvWA36 The expression level was determined using primers qRT-PCR-F and qRT-PCR-R, wherein the qRT-PCR-F sequence is shown in SEQ ID NO:7 and the qRT-PCR-R sequence is shown in SEQ ID NO:8.

[0063] SEQ ID NO:7: TATAGCTCCGGCTTGTTGGA.

[0064] SEQ ID NO:8:AAGGTGTGGACGGGGTACTT.

[0065] OsvWA36 The gene is the nucleotide sequence shown in SEQ ID NO:1.

[0066] SEQ ID NO:1:

[0067]

[0068] OsvWA36 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:2.

[0069] SEQ ID NO:2:

[0070] .

[0071] Example 2

[0072] OsvWA36 Analysis of expression patterns:

[0073] 1. qRT-PCR detection of tissue expression specificity

[0074] (1) Sample collection: Roots (3-leaf stage), leaves (3-leaf stage), and panicles (young panicle stage and flowering stage) of Lilac B rice (a high-quality maintainer line in Guangxi) were selected. Each tissue was biologically replicated three times and flash-frozen in liquid nitrogen at -80°C. save.

[0075] (2) RNA extraction and reverse transcription: Total RNA was extracted using Trizol reagent, and RNA purity was detected using Nanodrop 2000. =1.8-2.0), 1% agarose gel to verify RNA integrity; 1% agarose gel was used to verify RNA integrity; 1% agarose gel was used to verify RNA integrity. gRNA is reverse transcribed into cDNA.

[0076] (3) qRT-PCR reaction: OsActin was used as an internal control. The primers included OsActin-F and OsActin-R. The OsActin-F sequence is shown in SEQ ID NO:9 and the OsActin-R sequence is shown in SEQ ID NO:10.

[0077] SEQ ID NO:9: GAGTATGATGAGTCGGGTCCAG.

[0078] SEQ ID NO: 10: ACACCAACAATCCCAAACAGAG.

[0079] OsvWA36 Specific primers include OsvWA36 -F and OsvWA36 -R, perform real-time quantitative PCR (instrument: CFX96, Bio-Rad), reaction system (20 L): SYBR Premix Ex Taq II 10 L, 0.4g each of upstream and downstream primers. L, cDNA 2 L, ddH2O 7.2 L; Reaction procedure: 95 Pre-denaturation 30s, 95 Transgender 5s, 60 Annealing for 30 seconds, 40 cycles, and melting curve analysis to verify specificity. The OsActin-F sequence is shown in SEQ ID NO:11, and the OsActin-R sequence is shown in SEQ ID NO:12.

[0080] SEQ ID NO: 11: TATAGCTCCGGCTTGTTGGA.

[0081] SEQ ID NO: 12: AAGGTGTGGACGGGGTACTT.

[0082] (4) Result Calculation: Using The relative expression level was calculated using the method, and the results are as follows: Figure 1 As shown in A in the diagram.

[0083] Depend on Figure 1 As can be seen from A, OsvWA36 is expressed at the highest level in the spike, especially in the spike one day after flowering (1DAF), where the expression level is 45 times that in the leaves during the booting stage, while the expression level is lower in the roots and stems.

[0084] Western blotting to detect protein accumulation:

[0085] (1) Protein extraction: Take 0.5 g of each of the above tissue samples, add 1 mL of RIPA protein extraction buffer (containing 1 mM PMSF and a mixture of protease inhibitors), grind in an ice bath, and centrifuge at 12000 rpm for 15 min (4 ), take the supernatant.

[0086] (2) Protein quantification and electrophoresis: Protein concentration was determined by BCA method, and 30 μL of protein was taken. g protein sample added 5 SDS loading buffer, 95 Denaturation for 5 min; 10% SDS-PAGE gel electrophoresis (stacking gel 80V, separating gel 120V) until bromophenol blue reaches the bottom of the gel.

[0087] (3) Transfer and immunoassay: The gel protein was transferred to a PVDF membrane (200mA, 90min), and blocked with 5% skim milk for 1h (room temperature); Add OsvWA36 Polyclonal antibody (1:2000 dilution), 4 Incubate overnight; wash the membrane three times with TBST (10 min each time), add HRP-labeled goat anti-rabbit secondary antibody (1:5000 dilution), and incubate at room temperature for 1 h; after washing the membrane three times with TBST, develop using an ECL chemiluminescence kit (instrument: ChemiDoc XRS+, Bio-Rad), the results are as follows. Figure 1 As shown in B in the diagram.

[0088] Depend on Figure 1 From B, we can know that OsvWA36 The protein accumulates in a high amount in the ear, especially in the ear one day after flowering (1DAF), and is basically consistent with the mRNA expression pattern.

[0089] Example 3

[0090] Subcellular localization:

[0091] (1) Utilizing overexpression OsvWA36 Leaf sheath sections were prepared from transgenic plantlets of the -GFP fusion gene (OE-VW-2) and stained with DAPI or FM4-64.

[0092] (2) Fluorescence observation: GFP fluorescence signal, DAPI signal, and RFP signal were observed using laser confocal microscopy. The results are as follows: Figure 2 As shown.

[0093] The results show: OsvWA36 -GFP forms dotted green fluorescent aggregates in the cytoplasm, and some of these dotted aggregates are expressed in the nucleus (purple). Figure 2 In section A), these dot-like aggregates do not overlap with the red fluorescence of the FM4-64 membrane staining agent. Figure 2 (B in the middle).

[0094] Example 4

[0095] OsvWA36 Validation of the liquid-liquid phase separation (LLPS) characteristics:

[0096] 1. In vitro LLPS validation (observation of recombinant protein aggregates)

[0097] (1) Expression and purification of recombinant proteins: OsvWA36 The coding region was cloned into the pET-28a vector (His tag) and transformed into E. coli BL21(DE3); single colonies were picked and inoculated into LB medium (containing 50 g of LB broth). Kanamycin), 37 Incubate until OD600 = 0.6, then add 0.5 mM IPTG, 16 Induction for 16 h; bacterial cells were collected, sonicated (300W power, 3s on, 5s off, 30min total), recombinant protein was purified by Ni-NTA affinity chromatography column (GE Healthcare), and purity was verified by SDS-PAGE (purity >90%).

[0098] (2) LLPS condition optimization: the purified OsvWA36 -His protein (final concentration 2) M, 4 M and 8 M) was dissolved in Tris-HCl buffer (20 mM, pH 7.0) containing 0.2 M NaCl and incubated at room temperature for 30 min. Aggregate formation was observed using a laser confocal microscope (LSM 980, Zeiss) (excitation wavelength 488 nm, emission wavelength 520 nm). The number of aggregates was counted using ImageJ. The results are shown below. Figure 3 As shown in A in the diagram.

[0099] Depend on Figure 3 As can be seen from A, under the conditions of 0.2M NaCl and pH 7.0, 8 M has the highest efficiency in forming OsvWA36-His protein aggregates (30-40 per field of view).

[0100] 2. In vivo LLPS validation (observation of fluorescent fusion protein)

[0101] (1) Integration carrier transformation: OsvWA36 -GFP fusion gene (vector pRHVcGFP - OsvWA36 -GFP was transformed into Nicotiana benthamiana using the Agrobacterium-mediated transformation method described above, and observed after 48 h of expression.

[0102] (2) Fluorescence observation: GFP fluorescence signal was observed using a laser confocal microscope, indicating that the punctate aggregates were non-membrane structures and exhibited movement, fusion, and separation. Figure 3 (B in the text) confirms the in vivo LLPS characteristics.

[0103] 3. Fluorescent Recovery After Fluorescence (FRAP) Assay:

[0104] (1) FRAP operation: Select the above OsvWA36 -GFP aggregates were bleached using a laser confocal microscope in bleaching mode (488nm laser, 50% power), and fluorescence recovery images were then captured every 10 seconds for 60 seconds.

[0105] (2) Recovery rate calculation: ImageJ software was used to analyze the fluorescence intensity of the bleached area. With the fluorescence intensity before bleaching as 100%, the relative fluorescence intensity at different time points was calculated. The results are as follows: Figure 3 C and Figure 3 D in the middle.

[0106] Depend on Figure 3 C and Figure 3 As indicated by D in the data, the fluorescence recovery rate reached 50%-60% 60 seconds after bleaching, confirming... OsvWA36 The aggregates have dynamic recovery capabilities and are membrane-free dynamic aggregates.

[0107] Example 5

[0108] OsvWA36 Functional verification of particle shape regulation:

[0109] Determination of Particle Shape and Yield Related Indicators

[0110] (1) Material cultivation: wild type (WT), osvwa36 mutant ( vw-1 , vw-2 30 plants of each type were planted in the field according to a randomized block design and managed with conventional water and fertilizer.

[0111] (2) Index determination: Harvest the main ear at maturity, select 30 plump kernels from each line, and use the Wanshen SC-S system to determine the kernel length and width; select 1000 plump kernels and use an electronic balance (accuracy 0.001g) to determine the thousand-kernel weight.

[0112] (3) Results analysis: Statistical analysis was performed using GraphPad Prism 9 software, and t-tests were used to compare the differences among the strains. The results are as follows: Figure 4 As shown.

[0113] Depend on Figure 4 It can be seen that, osvwa36 The mutant's grain length was significantly shorter than that of the WT. Figure 4 A and Figure 4 (C in the text), the thousand-grain weight was significantly reduced ( Figure 4 (E in the text).

[0114] Example 6

[0115] Observation of epidermal cell morphology and analysis of lignin deposition in glumes:

[0116] 1. Morphological observation of glume epidermal cells (scanning electron microscopy)

[0117] (1) Sample preparation: Select WT (clove B) samples before flowering and osvwa36 Mutant glumes, 1 mm from the middle section. 2mm fragments were immediately placed in 2.5% glutaraldehyde fixative (prepared with 0.1M phosphate buffer, pH 7.2). After a fixed 12-hour interval, the sample was sent to Wuhan Sewell Company for scanning electron microscopy and analysis.

[0118] (2) Statistics: Five fields of view were selected for each sample. ImageJ software was used to count the length, width and number of cells per field of view of epidermal cells. The results are as follows: Figure 5 As shown.

[0119] Depend on Figure 5 B- Figure 5 From F in the equation, we can see that... osvwa36 The length of the glume epidermal cells in the mutant was significantly shorter than that in the WT, confirming that cell elongation was inhibited.

[0120] 2. Lignin deposition analysis of glumes

[0121] (1) Sample preparation: WT (clove B) samples were selected before flowering and one day after flowering. osvwa36 Preparation of freehand sections from mutant spikelets;

[0122] (2) Stain with phloroglucinol-hydrochloric acid, observe and photograph immediately with a stereomicroscope, and the results are as follows. Figure 5 As shown in G.

[0123] Depend on Figure 5 From G, we can know that osvwa36 The mutant showed greater lignin deposition (magenta) in spikelets before and one day after flowering compared to the wild type. Figure 5 G).

[0124] Example 7

[0125] OsvWA36 Functional verification of particle shape regulation:

[0126] Determination of Particle Shape and Yield Related Indicators

[0127] (1) Material planting: Clove B, osvwa36 mutant ( vw-1 30 plants each of the supplementary line (Comp) and the replacement line (Comp) were planted in the field according to a randomized block design and managed with conventional water and fertilizer.

[0128] (2) Index determination: Harvest the main ear at maturity, select 30 plump grains from each line, and use the Wanshen SC-S system to determine the grain length and grain width.

[0129] (3) Results analysis: Statistical analysis was performed using GraphPad Prism 9 software, and t-tests were used to compare the differences among the strains. The results are as follows: Figure 6 As shown.

[0130] Depend on Figure 6 It can be seen that, osvwa36 The mutant grain length was significantly shorter than that of syringe B; the grain length of the reintroduced lines was restored to the WT (syringe B) level.

[0131] Example 8

[0132] OsvWA36 Functional verification of particle shape regulation:

[0133] Determination of Particle Shape and Yield Related Indicators

[0134] (1) Material planting: ZH11, osvwa36 mutant ( CR-1, CR-2 Thirty plants each of the overexpression lines (OE-VW-2 and OE-VW-3) were planted in the field according to a randomized block design and managed with conventional water and fertilizer.

[0135] (2) Index determination: Harvest the main ear at maturity, select 30 plump grains from each line, and use the Wanshen SC-S system to determine the grain length and grain width.

[0136] (3) Results analysis: Statistical analysis was performed using GraphPad Prism 9 software, and t-tests were used to compare the differences among the strains. The results are as follows: Figure 7 As shown.

[0137] Depend on Figure 7 It can be seen that, osvwa36 The mutant grain length was significantly shorter than that of the WT, while the grain length of the overexpression line was increased compared to that of the WT.

[0138] Example 9

[0139] OsvWA36 Natural haplotype analysis and molecular marker design:

[0140] 1. Haplotype typing: Data is sourced from a public database (https: / / v1.rmbreeding.cn / Genotype / haplotype), selecting those containing... OsvWA36 Whole set 3K-RG germplasm of coding region sequences was analyzed using GL from the RFGB phenotype, and the results are as follows. Figure 8 As shown.

[0141] Depend on Figure 8 It can be seen that, OsvWA36 Rice samples carrying Hap6 had significantly shorter grain lengths than haplotypes such as Hap1 and Hap4.

[0142] 2. Molecular marker design: CAPS markers were designed targeting the differential SNP sites between Hap6 and Hap1, which can be used for rapid screening of haplotype materials with excellent particle size.

[0143] This implementation method systematically verifies the results by constructing mutants and transgenic lines, combined with molecular biology, cell biology, and genetic experiments. OsvWA36 The positive regulation function of grain length and the LLPS regulation mechanism were investigated, clarifying their application value in improving rice grain shape / yield; at the same time, natural haplotypes were discovered and molecular markers were designed, providing an operable technical means for efficient molecular breeding.

[0144] 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 OsvWA36 gene in increasing rice grain length, characterized by: The nucleotide sequence of the OsvWA36 gene is shown in SEQ ID NO:1; The amino acid sequence of the protein encoded by the OsvWA36 gene is shown in SEQ ID NO:2; Rice grain length can be increased by upregulating the expression level of the OsvWA36 gene or the activity or content of the protein encoded by the OsvWA36 gene.

2. A method for cultivating rice plants with increased grain length, characterized in that, The method includes the following steps: By introducing and expressing the OsvWA36 gene as described in claim 1 into a rice recipient, transgenic rice plants with grain lengths greater than those of the rice recipient were obtained. The introduction was carried out by linking the OsvWA36 gene downstream of the rice promoter to construct an overexpression vector, and then transforming rice using Agrobacterium-mediated transformation.

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