Rice gene OsGUX1 and its applications

Abstract

The object of the present invention is to provide a gene or protein capable of regulating the development of rice panicles for use in rice molecular breeding. Based on the above object, the present application provides an application of a protein OsGUX1 in regulating the development of rice panicles. The present application has screened a gene OsGUX1 that regulates the development of rice panicles, providing high-quality gene resources for the improvement breeding of rice varieties and greatly promoting the rice breeding process; the panicle development of the rice obtained by constructing a knockout vector and transforming is abnormal, and improving such lines can reduce the rice yield loss; by manipulating the expression level of the gene OsGUX1 to adjust the development process, the development process of rice panicles can also be improved.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and particularly to the rice gene OsGUX1 and its applications. Background Art

[0002] Rice (Oryza sativa L.) is one of the most important food crops in the world, and more than half of the global population takes rice as the staple food. In China, rice accounts for more than half of the total grain output. The yield of rice is directly related to the national food security and the national economy and people's livelihood, and increasing rice yield has always been an important direction in rice research. In the production of rice, with the passage of time and the increase in the number of sowing generations, many high-quality rice varieties tend to experience yield and quality degradation.

[0003] The normal development of the rice panicle is directly related to its yield. The degeneration of the spikelets at the top / base of the panicle will reduce the number of filled grains per panicle, resulting in a decrease in rice yield.

[0004] The currently discovered panicle degeneration includes basal panicle degeneration and apical panicle degeneration. In recent years, some genes causing rice panicle degeneration have been reported. For example, OsALMT7 (Aluminum-activated Malate Transporter) encodes an aluminum-activated malate transporter. The mutation of this gene leads to abnormal malate transport, ultimately resulting in the degeneration of spikelets at the top of the panicle (the degeneration rate is about 22%). OsCIPK31 (Calcineurin B-Like Protein-Interacting Protein Kinase 31) encodes a calcineurin kinase. The mutation of this gene causes excessive accumulation of ROS in the rice panicle, ultimately leading to the degeneration of spikelets at the top (the degeneration rate is about 60%). TUT1 encodes an inhibitor of a cAMP-like receptor protein. After the mutation of this gene, in addition to serious defects in panicle development, it also shows pleiotropic phenotypes such as short roots, reduced plant height, abnormal anther and pollen grain development. SPL6 (Squamosa Promoter-binding Protein-Like 6) encodes a Squamosa promoter-binding protein, inhibits the transcription of the endoplasmic reticulum stress response factor IRE1 in cells, and controls the output intensity of stress signals. The loss of its function leads to the overexpression of IRE1, resulting in the out-of-control output of endoplasmic reticulum stress signals in cells and the overexpression of downstream genes, triggering the senescence and degeneration of apical spikelet cells and the emergence of the "bald" trait of the panicle. DPS1 (Degenerated Panicle and Partial Sterility 1) encodes a protein containing a cystathionine β-synthase (CBS) domain. After the mutation of this gene, the plant shows the degeneration of spikelets at the top of the panicle and the reduction of the fertility of middle spikelets, short and white anthers, no pollen grains, and a significant reduction in the amount of wax and cutin in the anther epidermis. The panicle shows the accumulation of reactive oxygen species (ROS), lower antioxidant activity, and increased programmed cell death.

[0005] Currently, a considerable part of rice degeneration is manifested in apical panicle degeneration. Apical panicle degeneration will cause the grains in the panicle to fail to mature naturally during development, resulting in underfilled or even unfilled grains. In severe cases, it can even reduce rice yield by more than 50%. Apical panicle degeneration is mainly determined by variety characteristics and is also affected by adverse external environments, which is a major problem in rice production. Whether it is apical panicle degeneration in rice affected by variety characteristics or apical panicle degeneration in rice affected by acquired environments, there will be problems with gene or protein function defects at the molecular level. Therefore, whether it is to pre-identify whether rice will have panicle degeneration or to restore the apical panicle development function of rice, it is necessary to explore the functions of apical panicle degeneration genes.

[0006] In addition, on the one hand, there are differences in the understanding of those skilled in the art; on the other hand, when the applicant made this invention, a large number of documents and patents were studied, but due to space limitations, all details and content were not listed in detail. However, this by no means means that this invention does not possess the features of these prior arts. On the contrary, this invention already possesses all the features of the prior arts, and the applicant reserves the right to add relevant prior arts in the background art. Summary of the Invention

[0007] Identifying genes involved in regulating the degeneration of the top part of rice panicles can provide theoretical support for analyzing the molecular mechanism of the formation of rice panicle top degeneration, enriching the regulatory network of panicle development, and carrying out efficient and precise breeding of new rice varieties by molecular breeding techniques. In view of this, the purpose of the present invention is to provide a gene or protein capable of regulating the degeneration of the top part of rice panicles for use in rice molecular breeding.

[0008] Based on the above purpose, the present application provides the use of protein OsGUX1 in inhibiting the degeneration of the top part of rice panicles. The sequence fragment of protein OsGUX1 contains SEQ ID NO:1 (Table 21).

[0009] Another purpose of the present application lies in the use of gene OsGUX1 (LOC_Os01g65780) in inhibiting the degeneration of the top part of rice panicles. The coding sequence fragment of gene OsGUX1 encodes protein OsGUX1 containing SEQ ID NO:1.

[0010] According to a preferred embodiment, the nucleotide sequence of gene OsGUX1 is the nucleotide sequence shown in SEQ ID NO:2 (Table 21) or SEQ ID NO:3 (Table 21).

[0011] Another purpose of the present application lies in providing a method for increasing the yield of rice. The method includes: using genetic engineering means to restore the function of protein OsGUX1 in rice.

[0012] Another purpose of the present application lies in providing a method for increasing the yield of rice. The method includes: using genetic engineering means to overexpress gene OsGUX1 in rice.

[0013] According to a preferred embodiment, the method for restoring the function of protein OsGUX1 in rice includes the following steps:

[0014] Construct a genetic complementary plasmid containing gene OsGUX1;

[0015] Transfer the genetic complementary plasmid into rice to obtain rice with the function of gene OsGUX1 restored.

[0016] According to a preferred embodiment, those of ordinary skill in the art can easily mutate the nucleotide sequence encoding the protein OsGUX1 of the present application by using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 70% or higher identity with the nucleotide sequence of the protein OsGUX1 of the present application, as long as they encode the protein OsGUX1 and the encoded protein OsGUX1 has the function of regulating the panicle development of plants, are derived from the nucleotide sequence of the present application and are equivalent to the sequence of the present application.

[0017] According to a preferred embodiment, the protein for regulating the panicle development of rice in the present application further comprises a fusion protein expressed from a sequence obtained by connecting a tag to the N-terminus and / or C-terminus of the SEQ ID NO:1 sequence.

[0018] Another object of the present application is to provide the application of rice obtained by a method based on restoring the function of the protein OsGUX1 in rice breeding.

[0019] Another object of the present application is to provide the application of rice obtained by a method based on overexpressing the gene OsGUX1 in rice breeding.

[0020] According to a preferred embodiment, the above-mentioned "method for restoring the function of the protein OsGUX1 in rice" or "method for overexpressing the gene OsGUX1 in rice" can achieve the effect of increasing the expression level and / or activity of the above-mentioned protein OsGUX1 in the recipient plant by methods well-known in the art, such as transgenic, multi-copy, promoter modification, regulatory factor modification, etc.

[0021] According to a preferred embodiment, the breeding method includes transgenic, backcross, hybridization, asexual reproduction or self-cross.

[0022] Another object of the present application is to provide a nucleic acid molecule for regulating the panicle development of rice. Preferably, the nucleic acid molecule can be the following DNA molecules:

[0023] B1) A DNA molecule whose coding region is the DNA molecule shown in SEQ ID NO:2;

[0024] B2) A DNA molecule whose nucleotide sequence is the DNA molecule shown in SEQ ID NO:2;

[0025] B3) A DNA molecule whose nucleotide sequence is the DNA molecule shown in SEQ ID NO:3;

[0026] B4) A DNA molecule having 75% or more homology with the nucleotide sequence defined in B1) or B2) or B3), derived from rice and encoding the protein OsGUX1;

[0027] A DNA molecule that hybridizes to the nucleotide sequence defined by B1), B2), or B3), is derived from rice, and encodes the protein OsGUX1.

[0028] According to a preferred embodiment, the nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can be RNA, such as mRNA or hnRNA, etc.

[0029] Another object of the present application is to provide a recombinant vector containing the above nucleic acid molecule.

[0030] According to a preferred embodiment, the recombinant vector containing the above nucleic acid molecule can be a recombinant plasmid obtained by inserting SEQ ID NO:2 or SEQ ID NO:3 into the multiple cloning site of an expression vector. Preferably, the expression vector can be the pCAMBIA2300-35S-eGFP vector.

[0031] Another object of the present application is to provide a plant cell containing the above nucleic acid molecule. Preferably, the plant cell includes Agrobacterium, Escherichia coli, or other cells that can integrate the recombinant vector in their bodies into the DNA of the chromosome of the recipient cell (the cell of the recipient plant).

[0032] Another object of the present application is to provide a molecular marker for rice panicle abortion. The nucleotide sequence of the molecular marker is as shown in SEQ ID NO:2 or SEQ ID NO:3.

[0033] Another object of the present application is to provide the application of the molecular marker for rice panicle abortion in rice breeding. Preferably, the molecular marker for rice panicle abortion is used for the identification of panicle abortion in japonica rice. The molecular marker for rice panicle abortion is used for the identification of panicle abortion in indica rice.

[0034] Another object of the present application is to provide a primer pair for the molecular marker of rice panicle abortion. The nucleotide sequence of the primer pair of the molecular marker is as shown in SEQ ID NO:2 or SEQ ID NO:3.

[0035] The beneficial effects of the present application:

[0036] The experimental results given in Example 1 and Example 2 show that when the gene OsGUX1 is knocked out (or the function of the gene OsGUX1 is inhibited), the rice panicles show the phenomenon of panicle tip abortion (the panicle development length, the number of grains per panicle, the seed setting rate, the number of panicles developed, and the tiller number decrease significantly). From the experimental results given in Example 1 and Example 2, it can be known that the gene OsGUX1 has the function of regulating the development of rice panicles. The phenotypes of the panicle development here include the panicle development length, the number of grains per panicle, the seed setting rate, the number of panicles developed, and the tiller number.

[0037] When the applicant was exploring the gene OsGUX1, it was found that the gene OsGUX1 can be applied to molecular breeding for regulating the development process of rice panicles and the regulation of panicle morphology, and it can also be used for early prediction / identification of rice development status.

[0038] In this application, a series of panicle tip degradation mutants paa967 were obtained by using EMS to mutagenize the maintainer line Yixiang 1B, a backbone parent of hybrid rice. This line showed a panicle tip degradation phenotype in both Sichuan and Hainan bases, and the degradation rate of the florets at the panicle tip was 43.61%.

[0039] Through phenotypic analysis, genetic analysis, gene cloning and expression analysis of this degradation mutant, the gene involved in the regulation of the development of florets at the panicle tip of rice was clarified, laying a foundation for further analyzing the molecular regulation network of floret development in rice panicles and providing gene resources for breeding utilization.

[0040] The gene related to panicle development provided in this application provides high-quality gene resources for the improvement breeding of rice varieties, greatly promoting the rice breeding process; the improvement of the abnormal rice panicle development lines obtained by constructing and transforming knockout vectors can meet the market demand for rice yield. At the same time, in the face of the problem of panicle degradation in existing planted varieties, this application provides a method for pre-judging the growth and development of rice. By understanding the sequence of the gene OsGUX1 from seeds, or the expression level of the gene OsGUX1 or its expressed protein during the plant growth process, it can be determined whether the rice panicle can develop normally. When the expression level of the gene OsGUX1 or its expressed protein decreases to a preset threshold (the expression level during normal rice growth), it indicates that the growth of this rice is restricted, and exogenous assistance for rice panicle development can be considered.

[0041] In actual production, when panicle degradation occurs, breeders can identify whether there is abnormal expression of the gene OsGUX1 by molecular markers or other means to determine whether the panicle degradation behavior is caused by the gene OsGUX1. Breeders can enhance the expression of the gene OsGUX1 by exogenous injection of plasmid rings or other molecular means, or avoid the same panicle degradation problem in rice offspring through cross-breeding means.

[0042] Another object of this application is to provide a method for enhancing rice panicle development. The method includes enhancing the expression levels of the genes OsGS1 and OsGUX1. Another object of this application is to provide a combined identification method based on the genes OsGS1 and OsGUX1.

[0043] Example 3 involves the study of a double-silencing mutant of the OsGS1 gene and the OsGUX1 gene. The relevant research results disclose that the panicle degeneration of the double mutant paa971paa967 line is aggravated (compared with the mutants paa971 and paa967), and at the same time, the trait of plant height change appears, indicating that the effects of the OsGS1 gene and the OsGUX1 gene on panicle development are superimposed. Based on this, the present application proposes a method for enhancing rice panicle development. Brief Description of the Drawings

[0044] Figure 1 For the phenotypic characteristics and agronomic trait statistics of mutants, where (A, H) are plant height; (B, I) are panicle length; (C, D) are grain width; (E, F) are grain length; (G) is the yield per panicle; (J) is the number of effective panicles; (K) is the number of filled grains per panicle; (L) is the degeneration rate; (M) is the 1000-grain weight; (N) is the internode length. Asterisks indicate the significance of differences between samples (*P value < 0.05; **P value < 0.01). The P value is calculated according to Dunnett's one-way analysis of variance. NS indicates no significant difference. Error bars represent the standard error of the mean (SEM). (A) The scale bar is 10 cm; (B - G) The scale bar is 1 cm;

[0045] Figure 2 For the dynamic observation of panicle development and the detection of reactive oxygen species in mutants, where on the left side of each picture is the wild type YX1B, and on the right side is the mutant. (M) is 0.5 cm; (N) is 1 cm; (O) is 3 cm; (P) is 6 cm; (Q) is 9 cm; (R) is 12 cm; (T, U) are DAB staining at a panicle length of 9 cm; (V, W) are NBT staining at a panicle length of 9 cm; Figures (S, U, and W) are magnified views of the red boxes in Figures (Q, T, and V). The scale bar is 1 cm;

[0046] Figure 3 For the paraffin sections of the apical spikelets during the degeneration period of mutants, where (I) is the apical spikelet of the wild type; (J - L) are cross-sections of the apical spikelets of the wild type taken under a stereomicroscope at magnifications of 4× / 0.10, 10× / 0.22, and 100× / 125 respectively; (M) is the apical spikelet of paa967; (N - P) are cross-sections of the degenerated apical spikelets of paa967 taken under a stereomicroscope at magnifications of 4× / 0.10, 10× / 0.22, and 100× / 125 respectively. (I and M) The scale bar is 1 mm, and the others are 100 μm;

[0047] Figure 4Molecular marker linkage analysis of mutants (part), where (A) screening of polymorphic molecular markers for paa967, with 1, 3, and 5 being dominant mixed pools, and 2, 4, and 6 being recessive mixed pools; for molecular markers 1 and 2 in (A), they are 1 - 19; for 3 and 4, they are 1 - 20; for 5 and 6, they are 1 - 21; (B) is molecular marker linkage analysis, where 1 in (B) is YX, 2 is japonica rice 02428, and 3 - 24 are recessive single plants; (C) is the preliminary mapping of paa967;

[0048] Figure 5 Results of gene analysis, where (A) results of Mutmap analysis on chromosome 1; (B - D) analysis of the structure and mutation sites of LOC_Os01g65780, LOC_Os01g66500, and LOC_Os01g66530;

[0049] Figure 6 Schematic diagram of gene expression analysis results, where (A, B) expression analysis of LOC_Os01g66500 in YX1B and paa967; (C) expression analysis of LOC_Os01g66500 in tissue parts; (D, E) expression analysis of LOC_Os01g65780 in YX1B and paa967; (F) expression analysis of LOC_Os01g65780 in tissue parts; (G, H) expression analysis of LOC_Os01g66530 in YX1B and paa967; (I) expression analysis of LOC_Os01g66530 in tissue parts;

[0050] Figure 7 Knockout sites of LOC_Os01g65780 in ZH11;

[0051] Figure 8 Agronomic trait statistical chart of wild - type ZH11 and knockout line OsGUX1 - KO ZH11 ;

[0052] Figure 9 Knockout sites of LOC_Os01g65780 in YX1B;

[0053] Figure 10 Agronomic trait statistical chart of wild - type YX1B and knockout line OsGUX1 - KO YX1B ;

[0054] Figure 11 Tissue expression analysis diagram of OsGUX1 gene;

[0055] Figure 12 Phenotype comparison diagram of double mutant paa971paa967;

[0056] Figure 13Statistical chart of the agronomic traits of double mutant paa971paa967. Detailed implementation mode

[0057] The following is a detailed description with reference to the accompanying drawings.

[0058] The following examples are only used to further explain the beneficial effects of the gene OsGUX1 or the protein OsGUX1 in this application, and the protection scope of this application should not be limited thereby.

[0059] Unless otherwise defined, all scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the description of the present invention are only for describing specific embodiments and do not limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items. For those of ordinary skill in the art, they can understand the specific meaning of the above terms in the present invention according to specific circumstances.

[0060] The experimental procedures described in the following examples are all conventional procedures unless otherwise specified. If not specifically indicated, the examples are carried out under conventional experimental conditions or according to the conditions recommended by the manufacturer's instructions. The raw materials and reagents used in the present invention are all commercially available, and any biological germplasm materials can be provided for scientific research.

[0061] "Identity" refers to the sequence similarity with the natural nucleic acid sequence. "Identity" includes nucleotide sequences having 70% or higher, or 75% or higher, or 85% or higher, or 95% or higher identity with the nucleotide sequence encoding the protein OsGUX1 consisting of the amino acid sequence shown in SEQ ID NO:1 of the present application. Identity can be evaluated by the naked eye or computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0062] This application relates to:

[0063] I. Experimental materials

[0064] The panicle apical abortion mutant paa967 (panicle apical abortion 967) was derived from the indica rice (Oryza sativa L. subsp. Indica) maintainer line YX1B mutagenized by EMS (ethyl methanesulfonate) and obtained in M2. The mutant traits could be stably inherited after continuous self-crossing for multiple generations. Using the mutant paa967 as the female parent and the wild-type YX1B as the male parent, their respective F2 genetic analysis populations were constructed, and then using the japonica rice variety 02428 as the male parent, their respective mapping populations were constructed.

[0065] II. Main reagents

[0066] In this experiment, the Trizol / Tripure Isolation Reagent Roche reagent was purchased from Invitrogen; IIQ Select RT SuperMix for qPCR(+gDNA wiper) was purchased from Nanjing Novoprotein; Phanta Max Super-Fidelity DNA Polymerase was purchased from Nanjing Novoprotein; dNTP Mix was purchased from Nanjing Novoprotein; 2×Bimake TM SYBR Green Master Mix was purchased from bimake; DEPC water (DNase, RNase free) was purchased from Biosharp; 2×Phanta Max Buffer was purchased from Nanjing Novoprotein; 2×inNova PMXHi-Fi Premix(-Dye) was purchased from innovagene biotech; 4×gDNA wiper Mix was purchased from Nanjing Novoprotein; 5×HiScript II Select qRT SuperMix II was purchased from Nanjing Novoprotein; Solution I, Solution II, Solution III were purchased from Shanghai Solarbio Science & Technology Co., Ltd.; HBC Buffer was purchased from Shanghai Solarbio Science & Technology Co., Ltd.; DNA Wash Buffer was purchased from Shanghai Solarbio Science & Technology Co., Ltd.; XP2 BindingBuffer was purchased from Shanghai Solarbio Science & Technology Co., Ltd.; SPW Buffer was purchased from Shanghai Solarbio Science & Technology Co., Ltd.

[0067] III. Main Instruments

[0068] The instruments involved in this application include: RIO-RAD, Gel DOC 1000 agarose gel imager; MICROTEK ScanMaker i800 Plus color image scanner of Zhongjing Technology; Panasonic SIM-F140LBDL ice maker; Analytikjena qTOWER 3 G fluorescence quantitative PCR instrument; Forma TM900 Series -80℃ Ultra - low Temperature Refrigerator; SANFA GHP - 400 Light Incubator; XW - 80A Vortex Mixer; Eppendorf Centrifuge 5804R; Eppendorf Centrifuge 5415D; METTLER TOLEDO AL104 Electronic Balance; LDEX - 50KB Sterilizer.

[0069] IV. Experimental Methods

[0070] 4.1 Agronomic Trait Statistics

[0071] In the summers of 2019 and 2020, the mutant paa967 and the wild type were planted in the Wenjiang Experimental Field of Sichuan Agricultural University. When the plants matured, agronomic traits such as plant height, panicle length, number of effective panicles, number of filled grains per panicle, 1000 - grain weight, etc. were statistically analyzed.

[0072] 4.2 Observation on the Developmental Dynamics of Rice Panicles

[0073] When the rice panicles developed to the stage of the differentiation of the primary and secondary branch primordia, every 1 to 2 days, young seedlings were taken to observe the development of the rice panicles to determine the specific period when abnormal panicle development occurred.

[0074] 4.3 Detection of Superoxide Anions by NBT Staining

[0075] 1) Prepare NBT staining solution (the solution formula is shown in Table 1) and dispense it into 50 - mL centrifuge tubes;

[0076] 2) Use scissors to cut the young rice panicles and immediately put them into the 50 - mL centrifuge tubes containing NBT staining solution;

[0077] 3) Put the centrifuge tubes into a vacuum drying oven, open the tube caps; close the door and exhaust port of the vacuum drying oven, turn on the vacuum pump to pump out the air in the oven. When the reading of the negative pressure gauge reaches - 0.1 MPa, turn off the vacuum pump and maintain the negative pressure for 30 min;

[0078] 4) Open the exhaust port to restore the air pressure in the oven. After the air pressure stabilizes, take out the centrifuge tubes and let them stand at room temperature (25℃) for 60 min;

[0079] 5) Pour out the NBT staining solution, add 95% ethanol to submerge the samples, place the centrifuge tubes in an 80℃ water bath for decolorization, change the 95% ethanol every 10 min. After the green color of the samples has completely faded, take out the samples and take pictures to record the staining results.

[0080] 4.4 Detection of Hydrogen Peroxide by DAB Staining

[0081] 1) Prepare DAB staining solution (the solution formula is shown in Table 1), and immerse the excised young panicles in the DAB staining solution;

[0082] 2) Evacuate the air for 30 min to completely immerse the young panicles in the DAB staining solution;

[0083] 3) Incubate overnight at room temperature;

[0084] 4) Decolorize in 95% ethanol in a water bath at 80 °C and take pictures.

[0085] 4.5 Paraffin sections of rice spikelets

[0086] 1) Sampling and fixation: Cut the spikelets (6 cm or 8 cm) of wild-type and mutant rice, make a small cut at the top, and put them into a centrifuge tube containing 50% (70%) FAA fixative. The volume of the fixative should be no less than 10 times the volume of the material; place the fixative on ice, evacuate the air for 15 min, release the air slowly, and repeat twice until the material sinks to the bottom.

[0087] 2) Fix the samples in FAA fixative for 1 - 2 days, and then transfer the samples to 50% (70%) alcohol for storage.

[0088] 3) Dehydration: Transfer the samples to small bottles for paraffin sectioning. Before the dehydration step, first wash away the formaldehyde and acetic acid in the fixative with ethanol solutions of appropriate concentrations. If fixed with 50% FAA, first replace the fixative with 50% ethanol, generally changing it three times, 30 min each time; if fixed with 70% FAA, first wash away the fixative with 70% ethanol, changing it three times, 30 min each time; dehydrate with gradient ethanol, and the ethanol gradient concentrations are shown in Table 2.

[0089] Table 1

[0090]

[0091] Table 2

[0092] Solution Duration 50% (70%) ethanol 1h 85% ethanol 1h 95% ethanol (containing 1% eosin) 1～2h

[0093] Note: The purpose of adding eosin here is to stain the tissue for convenient embedding and trimming of wax blocks.

[0094] 4) Continue dehydration and clearing, clear with xylene, and the sequence is shown in Table 3:

[0095] Table 3

[0096]

[0097]

[0098] 5) Impregnate with wax, soak in the solutions shown in Table 4 in sequence.

[0099] Table 4

[0100] Solution Temperature Duration 4 / 5 volume of xylene + 1 / 5 volume of paraffin 42℃ 12h 4 / 5 volume of xylene + 1 / 5 volume of paraffin 46℃ 2 days 4 / 5 volume of xylene + 1 / 5 volume of paraffin 46℃ 3 days 4 / 5 volume of xylene + 1 / 5 volume of paraffin 48℃ 3 days 4 / 5 volume of xylene + 1 / 5 volume of paraffin 50℃ 3 days Pure wax 62℃ 2 days Pure wax 62℃ 6h Pure wax 62℃ 6h Pure wax 62℃ 1h

[0101] 6) Embedding: Fold the paper box in advance, melt the new paraffin on the induction cooker, and place it in an incubator at 62 °C for standby; during embedding, first spread a layer of embedding wax, then pour the materials in the glass bottle into the paper box, and arrange the materials evenly. Let the materials cool naturally.

[0102] 7) Wax block trimming: Take out the cooled wax block from the paper box. When turned over, the materials stained with eosin can be seen; use a single-sided blade to trim the materials into a trapezoidal platform with a narrow top and a wide bottom. When trimming the wax block, arrange the materials in the longitudinal or transverse cutting direction according to needs. Among them, the anthers in this experiment need to be cut transversely.

[0103] 8) Adhesion: Before sectioning, the wax block needs to be adhered to a small rectangular wooden block; stick a little broken wax on the blade, heat it on the alcohol lamp, smear it on the small wooden block, and quickly stick the wax block on the wooden block.

[0104] 9) Sectioning: Clamp the small wooden block on the sample stage of the microtome, align the wax block parallel to the blade, adjust the section thickness to 5 μm, rotate the handle of the microtome, and start sectioning; float the wax ribbon in a water bath at 42 °C.

[0105] 10) Slide adhesion: Coat the glass slide with an adhesive; in the ultra-clean bench, first lay a clean A4 paper, place the glass slides face up in two columns on the paper; add 20 μL of 0.1% poly-L-lysine in the center of one column of glass slides, and then place them in a clean section box and dry at 37 °C for 1 h.

[0106] 11) Spreading: Divide the required wax ribbon into small sections, lay it flat on the glass slide, move the glass slide to a spreading table at 42 °C, suck away the excess water with absorbent paper, put it into the section box, and dry at 40 °C.

[0107] 12) Dewaxing and rehydration, the liquid immersion sequence is shown in Table 5.

[0108] Table 5

[0109] Solution Duration Xylene 20 min Xylene 20 min 50% xylene + 50% absolute ethanol 2 min Absolute ethanol 2 min Absolute ethanol 2 min 95% ethanol 1 min 85% ethanol 1 min 70% ethanol 1 min 50% ethanol 1 min 30% ethanol 1 min 15% ethanol 1 min Double-distilled water 1 min Double-distilled water 1 min

[0110] 13) Staining: Stain with 1% eosin for 2 - 3 min.

[0111] 14) Dehydration and clearing: The liquid immersion sequence is shown in Table 6.

[0112] Table 6

[0113] Solution Duration Double-distilled water 1 min Double-distilled water 1 min 15% ethanol 1 min 30% ethanol 1 min 50% ethanol 1 min 70% ethanol 1 min 85% ethanol 1 min 95% ethanol 1 min 1% fast green 1 min Absolute ethanol 1 min Absolute ethanol 1 min Xylene 5 min Xylene 5 min

[0114] 15) Mounting the section: Use forceps to take out the section from xylene, lay it flat on a tissue paper, drop one or two drops of mounting medium (add xylene to the original Canada balsam solution until it is diluted to a light yellow and viscous state), use forceps to pick up a clean cover slip, touch the left edge of the cover slip to the liquid, and gently lower the cover slip; if there are bubbles, use forceps to push the cover slip to drive away the bubbles; blow in the fume hood for 15 min, lay it flat on a slide box, and dry it in an incubator at 37 °C.

[0115] 16) Section: Observe and photograph under a microscope, or store it in a section box for long-term preservation.

[0116] 4.6 Gene mapping

[0117] Extract genomic DNA from the dominant and recessive individuals in the F2 population obtained by crossing the mutant paa967 with 02428. Use 550 pairs of SSR (simple sequence repeat) primers on 12 rice chromosomes for gene mapping. Conduct preliminary mapping using BSA (bulked segregation analysis), that is, use SSR primers with polymorphisms between the two parents (YX1B and 02428) to electrophoretically identify the dominant and recessive DNA pools of the F2 offspring.

[0118] Extract genomic DNA from rice leaves by CTAB method:

[0119] 1) Take about 2 cm long rice leaves and place them in a 1.5 mL centrifuge tube, add liquid nitrogen and grind the leaves thoroughly;

[0120] 2) Add 750 μL of 1.5×CTAB to the above centrifuge tube;

[0121] 3) Incubate in a water bath at 65 °C for 30 min, invert and mix every 5 min;

[0122] 4) Use a pipette to add an equal volume of chloroform / isoamyl alcohol (24:1), invert and mix up and down, let it stand for 10 min and then centrifuge at 10000 rpm for 10 min;

[0123] 5) Use a pipette to aspirate about 400 μL of the supernatant into a new centrifuge tube, add 2 volumes of ice-cold absolute ethanol, place it at -20 °C for 30 min and then centrifuge at 12000 rpm for 15 min;

[0124] 6) Discard the supernatant, add 750 μL of 75% ethanol, centrifuge at 12000 rpm for 10 min;

[0125] 7) Discard the supernatant, add 750 μL of absolute ethanol, centrifuge at 12000 rpm for 10 min;

[0126] 8) Discard the supernatant, place it in a ventilated place to air dry naturally, and dissolve it with 200 μL of ddH2O.

[0127] 4.7 Mutmap Sequencing

[0128] In the F2 backcross population constructed by the mutant paa967 and YX1B, 25 individual plants with the phenotype of panicle degradation were selected to take fresh leaves. Subsequently, the leaves of each individual plant were mixed equally to extract DNA to construct a recessive offspring pool, and sent to Beijing Novogene Bioinformatics Technology Co., Ltd. for sequencing analysis.

[0129] 4.8 Candidate Gene Expression Analysis

[0130] To detect the expression of candidate genes in mutants and wild types, the applicant sampled the panicles of mutants and wild types when the panicle degradation phenotype appeared. The expression of candidate genes was detected by qRT-PCR. The qualified total RNA extracted by Trizol was synthesized into single-stranded cDNA using a quantitative reverse transcription kit. Referring to the instruction manual of 2×SYBR Green qPCR Master Mix reagent of Beijing Biomarker Technologies Corporation and the instruction manual of qTOWER 3 G quantitative PCR instrument of Analytik jena company to perform qPCR detection on the target gene. The samples to be detected and the rice internal reference gene ACTIN were each set with 3 replicates. The relative expression level was calculated using the 2-△△CT method.

[0131] The sampling sites and periods of spatio-temporal qRT-PCR of genes are shown in Table 7 below:

[0132] Table 7

[0133]

[0134]

[0135] The sampling sites and periods of differential qRT-PCR of developing young panicles are shown in Table 8 below:

[0136] Table 8

[0137]

[0138] Note: d represents the panicle length.

[0139] The steps for extracting total RNA from rice by Trizol method are as follows:

[0140] 1) First, pour a small amount of anhydrous ethanol into a clean and dried mortar, then light it to remove RNAase in the mortar by high temperature for sterilization purposes. After the mortar cools to room temperature, grind the sample in it with liquid nitrogen and load it into a 1.5 mL EP tube. Subsequently, add 1000 μL of Trizol reagent;

[0141] 2) After thoroughly mixing the powder and Trizol solution, let it stand at room temperature for 10 min to completely separate the nucleoprotein complex. Add 200 μL of chloroform, shake vigorously for 30 s, and let it stand at room temperature for 5 min;

[0142] 3) Centrifuge the sample tube at 12000 rpm and 4 °C for 15 min to completely dissolve the RNA in the upper aqueous phase. Then transfer the upper aqueous phase into a new EP tube;

[0143] 4) Add an equal volume of chloroform, shake for 20 s, centrifuge at 12000 rpm and 4 °C for 15 min, and transfer the upper aqueous phase into a new EP tube;

[0144] 5) Add an equal volume of isopropanol, let it stand at room temperature for 10 min, centrifuge at 12000 rpm and 4 °C for 10 min to precipitate the RNA to the bottom of the tube;

[0145] 6) Remove the supernatant. The RNA adheres to the bottom of the tube. Add 0.75 mL of 70% ethanol to wash the precipitate, shake well to fully wash the precipitate, centrifuge at 12000 rpm and 4 °C for 5 min, and remove the supernatant;

[0146] 7) Repeat step 6);

[0147] 8) Place the EP tube containing the RNA precipitate in the fume hood. After the alcohol has completely evaporated, add 20 - 50 μL of RNAase Free water to the tube and gently shake to fully dissolve the RNA;

[0148] 9) Take 1 μL and measure the RNA concentration and purity using a spectrophotometer;

[0149] 10) Store the qualified RNA samples obtained at -80 °C in the refrigerator for future use.

[0150] Obtaining reverse-transcribed cDNA:

[0151] According to the operating instructions of the Ⅱ RT SuperMix for qPCR kit, synthesize the first strand of cDNA. The RNA reverse transcription is carried out according to the method of the kit Ⅱ Q RT SuperMix for qPCR. The specific process is as follows:

[0152] 1) Removal of genomic DNA: The 10 μL system is formulated as shown in Table 9 below. Pipette and mix well, at 42 °C for 2 min;

[0153] 2) Reverse transcription: Add 4 μL of 5× to the EP tube after completing the first step Gently pipette and mix SuperMixⅡqRT SuperMixⅡ, and perform reverse transcription. The reaction program is 50°C for 30 min; 85°C for 5 sec;

[0154] 3) Storage: Store the product at -20°C for later use.

[0155] Table 9

[0156]

[0157] Verification by real-time fluorescence quantitative qPCR: Perform qPCR verification on the candidate gene LOC_Os01g65780. The RT-qPCR reaction system is shown in Table 10 below:

[0158] Table 10

[0159] System components Dosage (μL) Template cDNA 1 Primer F+R 2 2×BimakeTM SYBR Green Master Mix 10 <![CDATA[ddH2O]]> 7 Total 20

[0160] The reaction program is shown in Table 11 below:

[0161] Table 11

[0162]

[0163] 4.9 Construction of pYLCRISPR / Cas9 knockout vector

[0164] 1) Selection and synthesis of knockout primers;

[0165] 2) Preparation of vectors pYLCRISPR-Cas9Pubi-H, pYLsgRNA-OsU6a-LacZ, and pYLsgRNA-OsU6b;

[0166] 3) Construction of the sgRNA expression cassette. Use Overlapping PCR to construct the system with pYLsgRNA-OsU6a-LacZ. The components are shown in Table 12 below. Use pYLsgRNA-OsU6a-LacZ as the template for the following reaction:

[0167] Table 12

[0168]

[0169] Construct the sgRNA expression cassette. Use Overlapping PCR to construct the system with pYLsgRNA-OsU6b respectively. The components are shown in Table 13 below. Use pYLsgRNA-OsU6b as the template for the following reaction:

[0170] Table 13

[0171]

[0172]

[0173] The reaction procedures of the above construction systems are the same, as shown in Table 14 below:

[0174] Table 14

[0175]

[0176] Take 1 μL of each of the above two PCR products and dilute them 10-fold with H2O. Take 1 μL as the template and perform the second-round PCR with primers Pps-GGL / Pgs-GG2(U6a) and Pps-GG2 / Pgs-GGR(U6b). Among them,

[0177] The reaction system of pYLsgRNA-OsU6a-LacZ is shown in Table 15 below:

[0178] Table 15

[0179]

[0180] The reaction procedure is as shown in Table 16:

[0181] Table 16

[0182]

[0183] The reaction system of pYLsgRNA-OsU6b is shown in Table 17 below:

[0184] Table 17

[0185]

[0186]

[0187] The reaction procedure is as shown in Table 18:

[0188] Table 18

[0189]

[0190] Then, gel extraction is performed on the target fragments obtained from the above two reactions respectively. The specific steps of gel extraction are as follows:

[0191] a. Put the gels containing the target fragments cut out into 1.5 mL EP tubes respectively, and add XP2 Binding Buffer of equal mass (add 100 mL of XP2 Binding Buffer to 100 mg of gel);

[0192] b. Put the above EP tubes into a 60 °C water bath and turn the EP tubes upside down every three minutes for 3 times;

[0193] c. Transfer the liquid in the above EP tube into DNA Mini Columns, centrifuge at 10,000 rpm for 40 s, and discard the waste liquid in the lower collection tube;

[0194] d. Add 700 μL of XP2 Binding Buffer to DNA Mini Columns, centrifuge at 10,000 rpm for 40 s, and discard the waste liquid in the lower collection tube;

[0195] e. Add 700 μL of SPW Buffer to DNA Mini Columns, centrifuge at 10,000 rpm for 40 s, and discard the waste liquid in the lower collection tube;

[0196] f. Repeat step 1);

[0197] g. Centrifuge the empty column at 13,600 rpm for 40 s, discard the lower collection tube, and place DNA Mini Columns into a new sterilized EP tube and air-dry in a ventilated place;

[0198] h. Add 20 μL of ddH2O preheated at 60 °C to DNA Mini Columns, let stand for 5 min, and centrifuge at 12,000 rpm for 90 s;

[0199] i. Repeat the above step by adding 20 μL of ddH2O preheated at 60 °C to DNA Mini Columns and centrifuge at 12,000 rpm for 30 s;

[0200] j. Discard DNA Mini Columns, and store the target fragment recovered in the EP tube at -20 °C for future use.

[0201] 4) Ligation of the target sgRNA expression cassette and the pYLCRISPR / Cas9 vector. The restriction-ligation reaction system is shown in Table 19 below:

[0202] Table 19

[0203] Components 15 μL SgRNA expression cassette (all gel extraction products in the second round) 1 μL + 1 μL BsaI-HF 0.5 μL T4-DNA-ligase 0.1 μL pYLCRISPR-Cas9Pubi-H plasmid 1 μL 10×T4 Ligase Buffer 1 μL 10×Cutsmart Buffer 25 μL <![CDATA[ddH2O]]> to 15 μL

[0204] The above system is subjected to restriction-ligation using a temperature cycling for about 15 cycles: 37 °C for 5 min; 10 °C for 5 min; 20 °C for 5 min, and finally 37 °C for 5 min. The product is further used for transformation, and the specific steps are as follows:

[0205] a. Take a tube of Trans1-T1 Phage Resistant competent cells and thaw them on ice. Add 10 μL of the above-mentioned ligated PCR product, mix well, and place it in the ice bath for 30 min;

[0206] b. Heat shock in a 42 °C water bath for 30 s, then transfer the EP tube to the ice bath for 2 min without shaking the EP tube;

[0207] c. Add 700 μL of LB liquid medium without antibiotics (refer to the preparation method of LB liquid medium used in "Molecular Cloning: A Laboratory Manual") to the EP tube. After mixing well, place it in a shaker at 37 °C and 200 rpm for 1 h to resuscitate the bacteria;

[0208] d. Take out the EP tube from the shaker, centrifuge at 10000 rpm for 1 min, discard 700 μL of the supernatant, mix the remaining supernatant and bacteria in the EP tube, and spread them on the LB solid medium containing kanamycin;

[0209] e. Invert the plate and place it in an incubator at 37 °C and incubate overnight;

[0210] f. Pick well-grown monoclonal colonies for propagation and plasmid extraction, and send them to Qingke Biotechnology Co., Ltd. for sequencing detection.

[0211] The steps for plasmid extraction are as follows:

[0212] a. Centrifuge the bacterial solution and discard the upper clear liquid;

[0213] b. Add 250 μL of Solution I pre-cooled at 4 °C (for every 10 mL of bacterial solution centrifuged to obtain the bacterial cells, add 250 μL of Solution I), and resuspend;

[0214] c. Add an equal volume of Solution II, gently invert the centrifuge tube but for no more than 5 min;

[0215] d. Add 350 μL of Solution III;

[0216] e. Centrifuge at 10000 rpm for 10 min;

[0217] f. Transfer the supernatant to DNA Mini Columns II, centrifuge at 10000 rpm for 40 s, and pour out the waste liquid in the collection tube;

[0218] g. Add 700 μL of HBC Buffer, centrifuge at 10000 rpm for 40 s, and pour out the waste liquid in the collection tube;

[0219] h. Add 700 μL of DNA Wash Buffer, centrifuge at 10,000 rpm for 40 s, and discard the waste liquid in the collection tube.

[0220] i. Repeat step (h).

[0221] j. Centrifuge the empty column at 13,600 rpm for 40 s, discard the lower collection tube, and place the DNA Mini Columns II into a new sterilized EP tube and air dry in a ventilated place.

[0222] k. Add 20 μL of ddH2O preheated at 60 °C to the DNA Mini Columns II, let it stand for 5 min, and centrifuge at 12,000 rpm for 90 s.

[0223] l. Add 20 μL of ddH2O preheated at 60 °C to the DNA Mini Columns II and centrifuge at 12,000 rpm for 30 s.

[0224] m. Discard the DNA Mini Columns II, and store the plasmid collected in the EP tube at -20 °C for later use.

[0225] 4.10 Genetic transformation of the pYLCRISPR / Cas9 knockout vector

[0226] The detected pYLCRISPR / Cas9 knockout vector was delivered to Wuhan Boyuan Biotechnology Co., Ltd. to construct transgenic knockout lines with YX1B as the background.

[0227] Example 1

[0228] This example involves the phenotypic analysis of the paa967 mutant.

[0229] The mutant paa967 showed a spike phenotype with severe degeneration at the top of the spike ( Figure 1 A, B), and the glume degeneration rate was 43.61% ( Figure 1 L). In addition, the mutant also showed a decrease in plant height ( Figure 1 A, H), a decrease in spike length ( Figure 1 B, I), a significant decrease in the yield per spike ( Figure 1 G, K), no significant changes in grain width, grain length, and the number of effective spikes compared with the wild type ( Figure 1 C-F and J), and the 1000-grain weight was lower than that of YX1B ( Figure 1 M). The decrease in plant height of the mutant was caused by the shortening of the internode length of each node ( Figure 1 N).

[0230] Example 2

[0231] This example involves the observation and analysis of panicle degradation of the gene paa967 mutant.

[0232] (1) Observation of the dynamic development of the mutant panicle and detection of reactive oxygen species

[0233] To determine the specific period when the apical panicle degradation of the mutant occurs, the dynamic development processes of the mutant and wild-type panicles were observed. It was found that when the panicle length of the mutant paa967 was about 8 cm, abnormal development of the apical spikelets began to occur, manifested as the lemma and palea of the spikelets starting to become transparent ( Figure 2 Q, S), and then the apical spikelets gradually withered until they finally degenerated ( Figure 2 R). Before degradation, there were no differences between the wild type and the mutant ( Figure 2 M - P).

[0234] To detect whether there is an accumulation of reactive oxygen species in the apical panicle of the mutant, the young panicles of the wild type and the mutant at the degradation stage were stained with DAB and NBT. It was found that dark brown granules and blue formazan substances could be observed in the degenerated spikelets of the mutant paa967, indicating that there is an accumulation of reactive oxygen species in the degenerated spikelets of the mutant ( Figure 2 T - W).

[0235] (2) Observation of the spikelet tissue sections of the mutant

[0236] To observe the internal tissue structure of the degenerated spikelets, we performed paraffin sectioning on the apical spikelets at the degradation stage. It was observed that the wild-type spikelets could form six normal stamens and both the lemma and palea could develop normally ( Figure 3 J - L); while six stamens were not observed in paa967 ( Figure 3 N - P).

[0237] (3) Genetic analysis and gene mapping of the mutant

[0238] The wild type YX1B and the mutant were reciprocally crossed to construct an F2 segregation population. All the F1 generations of the crosses showed normal panicle phenotypes. The ratio of normal plants / panicle-degenerated plants in the F2 of the segregation population was statistically analyzed. In the segregation population of the mutant paa967, the ratio of normal plants / panicle-degenerated plants conforms to 3:1, indicating that the panicle degradation trait of the mutant is controlled by a single recessive nuclear gene (Table 20). Table 15 shows the statistical results of the segregation ratio of the paa967 mutant trait.

[0239] Table 20

[0240] Hybrid combination Total number of plants Plants with normal phenotype Plants with spikelet degeneration <![CDATA[χ 2 (3:1)]]> paa967 / YX 300 225 75 0

[0241] The mutant paa967 and the japonica rice variety 02428 were crossed to construct an F2 mapping population, and 550 pairs of SSR (Simple Sequence Repeat) primers distributed on 12 rice chromosomes were used for gene mapping. The BSA (Bulked Segregation Analysis) method was used for preliminary mapping, that is, SSR primers with polymorphisms between the two parents were used for PCR on the dominant and recessive DNA pools of the F2 offspring, and a marker linked to the panicle apical degradation trait of paa967 was initially detected on chromosome 1 ( Figure 4 A). Subsequently, linkage analysis was performed on dominant and recessive individual plants using the markers at the corresponding positions ( Figure 4 B). The paa967 mutant gene was initially mapped between St-13 and St-15 with a physical distance of 511 kb ( Figure 4 C).

[0242] (4) Whole-genome resequencing and candidate gene analysis

[0243] In the backcross F2 population of paa967 and the wild type, 25 individual plants with extreme panicle degradation phenotypes were selected to form a DNA pool for whole-genome resequencing.

[0244] Three genes were screened by Mutmap analysis within the initially mapped interval, namely LOC_Os01g65780, LOC_Os01g66500, and LOC_Os01g66530 ( Figure 5 A), and the functions of these three candidate genes are not yet clear. The SNP-index value of LOC_Os01g65780 is equal to 1, and it is annotated as encoding a glycosyltransferase; the SNP-index value of LOC_Os01g66500 is equal to 0.97, and it is annotated as encoding phosphoribosylformylglycinamide synthase; the SNP-index value of LOC_Os01g66530 is equal to 1, encoding ARGOS, and its homologous gene in Arabidopsis thaliana is involved in organ size regulation. The 2755th base of LOC_Os01g65780 mutated from C to T, resulting in the deletion of cysteine at the 221st position encoded by it ( Figure 5 B). The 2109th base of LOC_Os01g66500 mutated from G to A, resulting in the change of the 676th amino acid encoded by it from arginine to lysine ( Figure 5 C). The 310th base of LOC_Os01g66530 mutated from C to T, resulting in the change of the 104th amino acid encoded by it from leucine to phenylalanine ( Figure 5 D).

[0245] (5) Expression analysis of candidate genes

[0246] To investigate the expression characteristics of candidate genes in wild type and mutants during young panicle development, young panicles were collected at panicle lengths of 5 cm, 6 cm, 7 cm, 8 cm, and 9 cm, respectively, and divided into the top, middle, and basal parts of the panicle to analyze the expression differences of candidate genes during panicle development.

[0247] In the mutant paa967, panicle degeneration characteristics began to appear (around 8 cm). Compared with the panicle length of 7 cm, the expression of the three candidate genes LOC_Os01g66500, LOC_Os01g65780, and LOC_Os01g66530 decreased in both YX1B and paa967, and then increased (9 cm); and the expression levels of the three genes were all very low ( Figure 6 A and B, D and E, G and H). LOC_Os01g66500 was mainly expressed in the leaves of rice ( Figure 6 C). LOC_Os01g65780 was mainly expressed in the stems and nodes of rice ( Figure 6 F). LOC_Os01g66530 was mainly expressed in the stems and nodes of rice and the expression was very low ( Figure 6 I).

[0248] (6) Construction of the LOC_Os01g65780 knockout vector

[0249] Using ZH11 and YX1B as wild types, knockout lines of LOC_Os01g65780 were constructed, namely OsGUX1-KO and OsGUX1-KO, respectively.

[0250] ZH11

[0251] a. OsGUX1-KO knockout line

[0252] A target site was selected on the third exon of LOC_Os01g65780 to construct a knockout vector, and finally three homozygous lines (KO-ZH11-1, KO-ZH11-2, and KO-ZH11-3) were obtained, as Figure 7 shown, and the target sequence is shown in SEQ ID NO:4 (this knockout line was purchased from Baige Gene Technology Co., Ltd.).

[0253] Compared with the wild type ZH11, obvious panicle degeneration occurred at the top of the panicles in the three knockout lines. According to the statistics of relevant agronomic traits, compared with the wild type ZH11, the seed width and seed length of the knockout lines did not change, as Figure 8 shown.

[0254] The plant height of wild-type ZH11 was 105.95 cm, and that of the knockout lines was 100.5 cm. The tiller number of wild-type ZH11 was 8, and that of the knockout lines was 6. The panicle length of wild-type ZH11 was 20 cm, and that of the knockout lines was 17 cm. The number of grains per panicle of wild-type ZH11 was 145, and that of the knockout lines was 106. The seed setting rate of wild-type ZH11 was 80%, and that of the knockout lines was 72%. The 1000-grain weight of wild-type ZH11 was 23.13 g, and that of the knockout lines was 21.30 g.

[0255] The relevant results showed that compared with wild-type ZH11, the plant height, tiller number, panicle length, number of grains per panicle, 1000-grain weight, and seed setting rate of the knockout lines decreased.

[0256] YX

[0257] b. OsGUX1-KO knockout lines

[0258] As Figure 9 shown, a target site was selected on the first exon of LOC_Os01g65780 to construct a knockout vector, and finally three homozygous lines (KO-YX-1, KO-YX-2, and KO-YX-3) were obtained.

[0259] Table 21 shows the first-round PCR primers Pps-GGL and Pgs-GG2, and the second-round PCR primers Pps-GG2 and Pgs-GGR used for constructing the knockout vector.

[0260] As Figure 10 shown, compared with wild-type YX, obvious panicle regression occurred at the top of the panicles of the three knockout lines. According to the statistics of relevant agronomic traits, compared with wild-type YX, the seed width and seed length of the knockout lines did not change.

[0261] The plant height of wild-type YX was 108.75 cm, and that of the knockout lines was 101.17 cm. The tiller number of wild-type YX was 7.5, and that of the knockout lines was 6.6. The panicle length of wild-type YX was 28.47 cm, and that of the knockout lines was 20.81 cm. The number of grains per panicle of wild-type YX was 122, and that of the knockout lines was 93. The seed setting rate of wild-type YX was 86.37%, and that of the knockout lines was 71.12%. The 1000-grain weight of wild-type YX was 31.26 g, and that of the knockout lines was 28.63 g.

[0262] The relevant results showed that compared with wild-type YX, the plant height, tiller number, panicle length, number of grains per panicle, 1000-grain weight, and seed setting rate of the knockout lines decreased.

[0263] c. Tissue expression analysis

[0264] The expression levels of LOC_Os01g65780 in mature panicles, stems, sheaths, and leaves of the wild type were detected. The primers used for detecting the expression levels are shown in Table 21 (qPCR-Actin-F, qPCR-Actin-R, qPCR-LOC_Os01g65780-F, qPCR-LOC_Os01g65780-R, qPCR-LOC_Os01g66500-F, qPCR-LOC_Os01g66500-R, qPCR-LOC_Os01g66530-F, qPCR-LOC_Os01g66530-R). As Figure 11 shown in A, LOC_Os01g65780 is mainly expressed in rice leaves.

[0265] As Figure 11 shown in B, the detection of the expression level of LOC_Os01g65780 in panicles of different panicle lengths of the wild type found that with the increase of panicle length, the expression level of LOC_Os01g65780 gradually increased.

[0266] According to the above experiments, it can be known that LOC_Os01g65780 is involved in regulating the development of rice panicles, and it is positively correlated with the development of rice panicles. When the expression level of LOC_Os01g65780 (OsGUX1) in the panicles during the development process decreases, the panicles will show a degradation phenomenon.

[0267] Example 3

[0268] This example relates to a combined detection method for verifying panicle degradation. This example also relates to a method for enhancing panicle development.

[0269] This application also relates to a mutant paa971 with a panicle degradation phenotype, as Figure 12 shown in A. The mutant paa971 is controlled by the OsGS1 gene (LOC_Os02g50240).

[0270] In this application, reciprocal crosses were performed between paa971 and paa967 to construct an F2 segregation population. Investigation found that the two traits conform to the law of independent assortment, that is, normal trait + single mutant trait: double mutant trait is approximately 15:1. Compared with the wild type YX1B, the parents paa971 and paa967, the plant height of the double mutant paa971paa967 is significantly reduced ( Figure 12 A and B), and the panicle degradation is more severe. Compared with the wild type YX1B, the parents paa971 and paa967, the plant height, panicle length, and the number of filled grains per panicle of the double mutant paa971paa967 are all significantly reduced ( Figure 13 A, B, and D); there is no significant difference in the effective tiller number and 1000-grain weight ( Figure 13 C, F); the degradation rate is significantly increased ( Figure 13 E).

[0271] The above results indicate that the OsGS1 gene and the OsGUX1 gene involved in the present application can enhance the panicle development of rice in a way of effect superposition.

[0272] Table 21

[0273]

[0274]

[0275] The present application belongs to the project achievement of the major project of agricultural biological breeding, "Discovery of New Genes for High Yield and Evaluation of Breeding Value", No. 2023ZD0406801.

[0276] It should be noted that the above specific embodiments are exemplary. Those skilled in the art can come up with various solutions inspired by the disclosure of the present invention, and these solutions also fall within the disclosure scope and protection scope of the present invention. Those skilled in the art should understand that the description and drawings of the present invention are illustrative and do not constitute a limitation on the claims. The protection scope of the present invention is defined by the claims and their equivalents. The description of the present invention contains multiple inventive concepts. Expressions such as "preferably", "according to a preferred embodiment" or "optionally" indicate that the corresponding paragraphs disclose an independent inventive concept. The applicant reserves the right to file divisional applications based on each inventive concept. Throughout the text, the features guided by "preferably" are only optional ways and should not be understood as must-be-set. Therefore, the applicant reserves the right to abandon or delete relevant preferred features at any time.

Claims

1. A method for improving rice with degenerated ear top, characterized in that: The method comprises: 1) Detect the expression level / activity of the protein OsGUX1 in rice with degenerated panicle top, or the gene encoding the protein OsGUX1 OsGUX1 The expression level of 2) If the rice panicle top is degenerated, the expression level / activity of the protein OsGUX1 and / or the gene encoding the protein OsGUX1 OsGUX1 The expression level of OsGUX1 in rice with degenerated panicle top is lower than that in wild-type plants. Genetic engineering is used to restore the function of OsGUX1 protein in rice with degenerated panicle top or to overexpress the gene in rice with degenerated panicle top. OsGUX1 ; The amino acid sequence of the protein OsGUX1 is shown in SEQ ID NO: 1, and the gene OsGUX1 The nucleotide sequence is the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO:

3.

2. The method according to claim 1, characterized in that The method for restoring the function of the protein OsGUX1 in rice with degenerated panicle top comprises the following steps: Constructs containing genes OsGUX1 Genetic complementation plasmids; The genetic complementation plasmid was transferred into rice with degenerated panicle top to obtain rice with restored protein OsGUX1 function.

3. A method for detecting rice panicle withdrawal, characterized in that: The method comprises: 1) Detecting the expression level of a molecular marker having a nucleotide sequence such as SEQ ID NO: 2 or SEQ ID NO: 3 in rice; 2) If the expression level of the molecular marker is lower than that of the wild-type plant, the rice is judged to have a panicle retraction phenotype.

4. Use of the method for detecting rice panicle atrophy according to claim 3 in rice breeding.

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