Rice gene OsCWIN5 and its application

Abstract

The purpose of the present invention is to provide a gene or protein that can regulate rice panicle type for use in rice molecular breeding. Based on the above purpose, the present application provides an application of a protein OsCWIN5 in regulating rice panicle type. The present application provides an application of a protein OsCWIN5 in regulating rice plant height. The present application screened a gene OsCWIN5 that regulates rice plant height and panicle type, providing high-quality gene resources for rice variety improvement and breeding, and greatly promoting the rice breeding process; the rice panicle type improved strain obtained by constructing a knockout vector transformation can meet the market demand for rice yield. At the same time, for some ornamental rice, the rice strain obtained by overexpression vector transformation has the characteristics of beautiful appearance; by manipulating the expression level of the gene OsCWIN5 to adjust the development process, the rice plant type and development time can also be improved.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, in particular to a rice gene OsCWIN5 and an application thereof. Background Art

[0002] Rice is one of China's three major grain crops. According to the public data of the "China Rice and Rice Industry Market Research and Development Prospects Planning Report 2023-2029" released by Zhiyan Consulting, China's annual demand for rice is stable at more than 200 million tons. The components of rice yield include effective panicles, number of panicles and grain weight. The effective panicle is the number of effective panicles per unit area. The number of panicles is determined by the number of primary stalks, the number of secondary stalks, and the number of effective spikelets on the primary and secondary stalks, while the grain weight is determined by the length, width, thickness and filling degree of the rice grains. These three elements complement each other and jointly determine the yield of rice.

[0003] At the same time, the panicle type of rice (including the number of panicles and panicle length) has an important relationship with rice nutrient consumption, pests and diseases, etc. The different panicle structures of rice will affect the consumption of nutrients during the growth and development of grains. Generally speaking, varieties with large panicles may require more nutrients to support their growth and development, so they may absorb more nutrients from the grains. Varieties with small panicles may require fewer nutrients, so the nutrient content in their grains may be higher. The panicle structure of rice also affects its susceptibility to pests and diseases. Some pests and diseases may choose to reproduce on specific panicles. For example, rice planthoppers prefer to lay eggs on panicles with compact and stick-shaped panicles and soft rice stalks. Therefore, those varieties with compact panicles and soft rice stalks may be more susceptible to rice planthoppers. Varieties with different panicle types may have different resistance to certain pests and diseases. For example, varieties with small panicles of the Wan sickle type have stronger disease resistance, mainly because their loose structure reduces the living and hiding space for pests and diseases.

[0004] According to previous studies, rice panicle type is a quantitative trait controlled by multiple genes, and there are complementary and cumulative effects between the genes controlling the panicle type traits. At present, some cloned genes for rice panicle type include OsCKX2, LAX1, PLA2, etc. How to coordinate the relationship between rice plant type, panicle type and rice yield and quality is the key research goal of rice breeders. Collecting, creating and identifying rice panicle type-related mutants and cloning and analyzing related genes, and using them for actual rice panicle type improvement, is an effective way to cultivate new varieties, "expand the pool", ensure food security, and improve rice quality.

[0005] In addition, on the one hand, there are differences in understanding among those skilled in the art; on the other hand, the applicant studied a large number of documents and patents when making the present invention, but due to space limitations, not all details and contents are listed in detail. However, this does not mean that the present invention does not have the characteristics of these prior arts. On the contrary, the present invention already has all the characteristics of the prior art, and the applicant reserves the right to add relevant prior art to the background technology. Summary of the invention

[0006] Identifying genes that regulate panicle type in rice can provide theoretical support for analyzing the molecular mechanism of panicle type formation, enriching the regulatory network of panicle type change, and using molecular breeding technology to carry out efficient and accurate breeding of new rice varieties. In view of this, the purpose of the present invention is to provide a gene or protein that can regulate rice panicle type for use in rice molecular breeding.

[0007] Based on the above purpose, the present application provides an application of protein OsCWIN5 in regulating rice panicle shape. The present application also provides an application of protein OsCWIN5 in regulating rice plant height. The amino acid sequence of the protein encoded by gene OsCWIN5 is shown in SEQ ID NO:1.

[0008] Another object of the present application is to provide an application of the gene OsCWIN5, wherein the application is as follows a1) or a2): a1) regulating rice panicle shape; a2) regulating rice plant height.

[0009] Preferably, the gene OsCWIN5 encodes the protein OsCWIN5 in the above-mentioned use.

[0010] According to a preferred embodiment, the nucleotide sequence of the gene OsCWIN5 is the nucleotide sequence shown in SEQ ID NO:2 or SEQ ID NO:3.

[0011] Another object of the present application is to provide a method for increasing rice yield, which comprises: weakening or knocking out the gene OsCWIN5 in rice by genetic engineering.

[0012] According to a preferred embodiment, the method for increasing rice yield comprises the following steps:

[0013] Using the gene OsCWIN5 as the target gene, the sgRNA sequence based on CRISPR / Cas9 was designed;

[0014] Connect the DNA fragment containing the sgRNA sequence to the vector carrying CRISPR / Cas9;

[0015] The vector is transferred into rice to obtain rice with OsCWIN5 gene function deleted.

[0016] According to a preferred embodiment, the nucleotide sequence of the sgRNA action site is 5'-CCGGGATGGCCTGTGGAGGATGG-3'.

[0017] According to a preferred embodiment, a person skilled in the art can easily mutate the nucleotide sequence encoding the protein OsCWIN5 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 OsCWIN5 of the present application are all derived from the nucleotide sequence of the present application and are equivalent to the sequence of the present application as long as they encode the protein OsCWIN5 and the encoded protein OsCWIN5 has the function of regulating plant height or panicle type.

[0018] According to a preferred embodiment, the protein for regulating rice plant height or panicle type of the present application also comprises a fusion protein expressed by a sequence obtained by connecting a tag to the N-terminus or / and C-terminus of the sequence of SEQ ID NO: 1.

[0019] Another object of the present application is to provide a method for regulating the expression of the gene OsCWIN5, which comprises applying gibberellin or auxin to rice tissue.

[0020] According to a preferred embodiment, the rice tissue is leaf.

[0021] Another object of the present application is to provide an application of rice obtained by the method of regulating the expression level of the gene OsCWIN5 in rice breeding.

[0022] According to a preferred embodiment, the above-mentioned "method for regulating the expression level of the gene OsCWIN5" can achieve the effect of increasing / decreasing the expression level and / or activity of the above-mentioned protein OsCWIN5 in the recipient plant through methods well known in the art such as transgenics, multiple copies, changing promoters, regulatory factors, etc.

[0023] According to a preferred embodiment, the breeding method comprises transgenic, backcrossing, hybridization, asexual reproduction or selfing.

[0024] According to a preferred embodiment, the rice panicle type comprises panicle length, number of primary branches, number of secondary branches and / or number of grains per panicle.

[0025] Another object of the present application is to provide a nucleic acid molecule containing the sequence of SEQ ID NO: 2 or SEQ ID NO: 3. The nucleic acid molecule is used to regulate the panicle shape and / or plant height of rice. Preferably, the nucleic acid molecule can be the following DNA molecule:

[0026] B1) the coding region is a DNA molecule shown in SEQ ID NO: 2;

[0027] B2) a DNA molecule whose nucleotide sequence is shown in SEQ ID NO: 2;

[0028] B3) The nucleotide sequence is a DNA molecule shown in SEQ ID NO: 3

[0029] B4) a DNA molecule having 75% or more homology with the nucleotide sequence defined in B1) or B2) or B3), which is derived from rice and encodes the protein OsCWIN5;

[0030] B5) is a DNA molecule that hybridizes with the nucleotide sequence defined in B1) or B2) or B3), and is derived from rice and encodes the protein OsGATA6.

[0031] 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.

[0032] Another object of the present application is to provide a recombinant vector containing the above nucleic acid molecule.

[0033] 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 the expression vector. Preferably, the expression vector can be pBWA(V)HS, pCAMBIA2300-35S vector.

[0034] Another object of the present application is to provide a plant cell containing the above nucleic acid molecule. Preferably, the plant cell comprises Agrobacterium, Escherichia coli or other cells capable of integrating the recombinant vector in the body into the DNA of the chromosome of the recipient cell (cell of the recipient plant).

[0035] Beneficial effects of this application:

[0036] According to relevant experiments, when the gene OsCWIN5 is overexpressed in rice, the plant height of the overexpression strain is significantly lower than that of the wild type. At the same time, the number of grains per panicle, panicle length, number of primary stalks, number of secondary stalks and / or number of grains per panicle are significantly reduced.

[0037] Furthermore, knocking out the gene OsCWIN5 in rice resulted in a knockout strain with a significantly higher plant height than the wild type, and a significantly increased number of grains per panicle, panicle length, number of primary stalks, number of secondary stalks, and / or number of grains per panicle. Based on this, it can be seen that the gene OsCWIN5 involved in the present application can significantly affect the plant height and panicle type of rice.

[0038] According to the research on the regulatory mechanism of gene OsCWIN5 in the present application, the applicant has found at the application level that hormones can affect the expression of gene OsCWIN5 in leaves at an early stage. For rice plants with defects in gene OsCWIN5 screened by molecular markers at an early stage, the application of exogenous hormones can increase the probability of their normal growth and reduce the risk of yield reduction.

[0039] At the same time, the study also screened proteins that interact with the gene OsCWIN5, which also indicates that regulating the expression of the gene OsCWIN5 includes controlling its interacting proteins GF14D and GF14F.

[0040] The present application screened out a gene OsCWIN5 that regulates rice plant height and panicle type, providing high-quality gene resources for rice variety improvement and breeding, and greatly promoting the rice breeding process; the rice panicle type improved strains obtained by constructing a knockout vector transformation can meet the market demand for rice yield. At the same time, for some ornamental rice, the rice strains obtained by overexpression vector transformation have beautiful characteristics; by manipulating the expression level of the gene OsCWIN5 to adjust the development process, the plant type and development time of rice can also be improved.

[0041] The present application screens a mutant strain that regulates rice panicle type and plant height in a mutant library, and obtains genetic resources that affect rice panicle type and plant height through related research. This genetic resource enables breeders to identify whether the gene OsCWIN5 has abnormal expression through molecular markers or other means when rice with abnormal panicle type / plant height changes occurs, so as to determine whether the panicle type change or plant height change behavior is caused by the gene OsCWIN5. Breeders can enhance the expression of the gene OsCWIN5 by exogenous injection of plasmid rings or other molecular means, or avoid the problem of the same panicle type change or plant height change in rice offspring through hybrid breeding. Another purpose of the present application is to provide an application of rice protein OsCWIN5 that interacts with protein GF14D or protein GF14F in regulating rice panicle type or regulating rice plant height. The amino acid sequence of rice protein OsCWIN5 is shown in SEQ ID NO: 1. The amino acid sequence of protein GF14D is shown in SEQ ID NO: 5. The amino acid sequence of protein GF14F is shown in SEQ ID NO: 6.

[0042] According to the experimental results involved in Example 3, it can be seen that protein GF14F interacts with protein OsCWIN5. Protein OsCWIN5 interacts with protein GF14D. The above results show that protein GF14D / protein GF14F can regulate the activity of protein OsCWIN5 by binding to protein OsCWIN5, thereby regulating the function of OsCWIN5. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Phenotypic analysis of OsCWIN5 knockout and overexpression plants, including: (A) Phenotypes of ZH11, CWIN5-KO, and CWIN5-OE plants at maturity; (B) Grain shape phenotypes of ZH11, CWIN5-KO, and CWIN5-OE; (C) Ear shape diagram of ZH11, CWIN5-KO, and CWIN5-OE; (D I) Comparison of main agronomic traits of ZH11, CWIN5-KO, and CWIN5-OE (D) Plant height; (E) Ear length; (F) Grain width; (G) Grain length;

[0044] Figure 2 The subcellular localization of OsCWIN5 protein in Nicotiana benthamiana and rice protoplasts;

[0045] Figure 3 The purified protein OsCWIN5-GST was induced to express in E. coli DE3;

[0046] Figure 4 OsCWIN5 can catalyze sucrose hydrolysis (A) Total protein extraction from panicle tissue of OsCWIN5 overexpressing strain, (B) Protein expression detection in overexpressing strain CWIN5-OE, (C) Purified CWIN5 catalyzes sucrose hydrolysis in vitro;

[0047] Figure 5 The kinetic curves of CWIN5 enzyme (A) catalytic enzyme activity of purified CWIN5-His protein in the pH range of 3.5 to 7.5 at a substrate (sucrose) concentration of 50 mM, (B) catalytic enzyme activity of purified CWIN5-His protein measured in the sucrose substrate concentration range of 0 to 400 mM at pH 4.5;

[0048] Figure 6 The changes of cell wall sucrose invertase activity during the development of young spikelets;

[0049] Figure 7 is the expression level of OsCIN1, OsCIN4, OsCIN7, and OsCWIN5 in ZH11, KO, and OE strains;

[0050] Figure 8 OsCWIN5 expression is induced by gibberellins and auxins. (A) gibberellins and (B) auxins can induce OsCWIN5 expression, while (C) cytokinins and (D) methyl jasmonate have no significant effect on OsCWIN5 expression.

[0051] Fig. 9 To show the expression of OsCWIN5 induced by gibberellin (GA), (A) GMS staining of the treated seedlings, (B) local leaves after gibberellin (GA) treatment;

[0052] Fig.10 The expression of OsCIN1 and OsCIN7 is induced by gibberellins and auxins;

[0053] Fig.11 The changes of sucrose invertase activity in ZH11 and KOs seedlings at the three-leaf stage after auxin and gibberellin treatment. (A) Changes of sucrose invertase activity in ZH11 and KOs seedlings after auxin treatment. (B) Changes of sucrose invertase activity in ZH11 and KOs seedlings after gibberellin treatment.

[0054] Fig.12 Analysis of differentially expressed genes in ZH11 and KOs, (A) differentially expressed genes in 3 cm young ears, (B) differentially expressed genes in 8 cm young ears, (C) differentially expressed genes in 10 cm young ears, (D) differentially expressed genes in 15 cm young ears;

[0055] Fig.13 Differentially expressed genes are enriched in starch and sugar metabolism pathways and MAPK signal transduction pathways. (A) Differentially expressed genes are enriched in starch and sugar metabolism pathways; (B) Differentially expressed genes are enriched in MAPK signal transduction pathways;

[0056] Fig.14 To verify the results of transcriptome analysis using RT-qPCR technology, (A) OsAP2-29, (B) OsMKKK70, (C) OsMKKK62, (D) OsM4PK7, (E) OsMST3, (F) OsMST4, (G) OsMST6, and (H) OsSUS3 were differentially expressed in ZH11 and CWIN5-KO;

[0057] Fig.15 In vitro interaction between OsCWIN5 and GF14D and GF14F, (A) CWIN5 total protein IP, (B) CWIN5 CoIP silver staining;

[0058] Fig.16 To verify the interaction between OsCWIN5 and GF14D and GF14F, (A) OsCWIN5 and GF14D yeast two-hybrid control group, (B) OsCWIN5 and GF14D yeast two-hybrid experimental group, (C) OsCWIN5 and GF14F yeast two-hybrid control group, (D) OsCWIN5 and GF14F yeast two-hybrid experimental group, (E) OsCWIN5 and GF14D in vitro pull-down control group, (F) OsCWIN5 and GF14D in vitro pull-down group, (G) OsCWIN5 and GF14D in vitro pull-down control group, (H) OsCWIN5 and GF14D in vitro pull-down experimental group.

[0059] Fig.17 Phenotypes of interacting protein gene mutants: (A) gf14d grain shape, (B) gf14d thousand-grain weight, (C) gf14f grain shape, (D) gf14f thousand-grain weight;

[0060] Fig.18 Schematic diagram of the knockout site of the knockout plant in this application. DETAILED DESCRIPTION

[0061] The following is a detailed description with reference to the accompanying drawings.

[0062] The following examples are only used to further explain the beneficial effects of the gene OsCWIN5 or protein OsCWIN5 in the present application, and should not be used to limit the protection scope of the present application.

[0063] Unless otherwise defined, all scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more related listed items. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0064] The experimental procedures described in the following examples are all conventional procedures unless otherwise specified. Unless otherwise specified, the examples are based on conventional experimental conditions or 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 material can be provided to the outside for scientific research.

[0065] "Identity" refers to sequence similarity with a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 70% or more, or 75% or more, or 85% or more, or 95% or more identity with the nucleotide sequence of the protein OsCWIN5 encoding the amino acid sequence shown in SEQ ID NO:1 of the present application. Identity can be evaluated by the naked eye or by 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.

[0066] This application relates to:

[0067] 1. Experimental Materials

[0068] The knockout strains CWIN5-KO, OsGF14D-KO, and OsGFl4F-KO in the ZH11 background were purchased from BioGene Technology Co., Ltd., and the wild-type ZH11 was also provided by the company. In this experiment, the CWIN5-OE overexpression vector was constructed and sent to Boyuan Company for transformation into ZH11. All materials were planted in the Wenjiang Experimental Base of Sichuan Agricultural University in summer and in the Lingshui South Breeding Base of Sichuan Agricultural University in Hainan in winter.

[0069] 2. Main Reagents

[0070] In this experiment, TRIzol was purchased from Invitrogen; high-fidelity PCR mix kit, reverse transcription kit, and qPCR kit were purchased from Nanjing Novozymes; DNA purification kit, BCA protein quantification kit, and plasmid mini- and maxi-extraction kits were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; yeast culture medium SD Base, defective amino acids, yeast plasmid mini-extraction kit, and yeast inserted gene detection kit were purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd.; GUS chemical staining reagent was purchased from Beijing Coolbo Technology Co., Ltd.; primer synthesis and small fragment sequencing were completed by Chengdu Qingke Zixi Biotechnology Co., Ltd., Shenggong Biotechnology (Shanghai) Co., Ltd., and Zhejiang Youkang Biological Co., Ltd.

[0071] 3. Main instruments

[0072] The instruments involved in this application include: RIO-RAD, Gel DOC 1000 molecular imager; BIO-RAD-ChemiDocXRS+ molecular imager; BIO-RAD-PDS-1000 gene gun; MICROTEK ScanMaker i800 Plus, color image scanner; Panasonic SIM-F140LBD ice maker; Analytikjena qTOWER3G fluorescence quantitative PCR instrument; Forma TM 900Series-80℃ ultra-low temperature refrigerator; SANFA GHP-400 light incubator; XW-80A vortex mixer; Eppendorf Centrifuge 5804R centrifuge; Eppendorf Centrifuge 5415D centrifuge; METTLERTOLEDO AL104 electronic balance; LDEX-50KB sterilizer; Thermo Fisher JXL-100 metal bath; Shanghai Huxi JY88-IIN ultrasonic crusher; Shanghai Tianneng VE-60 vertical gel electrophoresis instrument; VE180 membrane transfer instrument; Thermo Scientific TM Varioskan TM LMX microplate reader.

[0073] IV. Experimental Methods

[0074] 4.1 Agronomic trait statistics

[0075] Twenty wild-type and mutant rice plants at normal maturity were selected in the field, and their agronomic traits such as grain length, grain width, and 1000-grain weight were investigated.

[0076] 4.2 Vector construction

[0077] 4.2.1 Overexpression vector construction

[0078] The CDS sequence of the gene OsCWIN5 (SEQ ID NO: 2) was amplified using primers containing a linker, and the target fragment was recovered using a gel recovery kit. The fragment was ligated to the pBWA(V)HS vector, and then subjected to recombination reaction, E. coli transformation, single clone picking, plasmid extraction, and sequencing verification.

[0079] The target fragment amplification system is shown in Table 1.

[0080] Table 1

[0081]

[0082]

[0083] The amplification reaction program is shown in Table 2.

[0084] Table 2

[0085]

[0086] 4.2.2 Construction of GUS fusion protein vector

[0087] The promoter sequence of gene OsCWIN5 (SEQ ID NO. 4) was amplified using primers containing double restriction sites, the amplified fragment was recovered by the kit and inserted into the pCAMBIA1391 vector, transformed into E. coli, single clones were picked, plasmids were extracted, and sequenced for verification.

[0088] 4.2.3 Construction of subcellular localization vector

[0089] The CDS sequence (SEQ ID NO. 3) of the gene OsCWIN5 was amplified using primers containing double restriction sites, and the target fragment was recovered by a gel recovery kit. The fragment was inserted into the pCAMBIA2300-GFP vector, transformed into Escherichia coli, single clones were picked, plasmids were extracted, and sequenced for verification (the universal primers were 2300-CWIN5-GFP-F and 2300-CWIN5-GFP-R).

[0090] 4.2.4 Construction of prokaryotic protein expression vector

[0091] The CDS sequence of the gene OsCWIN5 was amplified using primers containing double restriction sites, and the target fragment was recovered by a gel recovery kit. The fragment was inserted into the pGEX-6P-1 vector, transformed into Escherichia coli, single clones were picked, plasmids were extracted, and sequencing was performed for verification.

[0092] 4.3 Paraffin sections

[0093] The experimental method refers to the rice tissue paraffin sections described on pages 223-227 of Zhang Qifa's version of "Rice Experimental Methods" (Bio-protocol).

[0094] 4.4 Hormone treatment

[0095] Seeds of ZH11 and OsCWIN5 knockout strains (KO1, KO2, and KO3) were selected, rinsed with distilled water, soaked in distilled water for 24 hours, hydroponically cultured in nutrient solution, and placed in a greenhouse for normal culture until the three-leaf stage.

[0096] Seedlings with consistent growth were selected and moved into containers of the same size. Each plant was immersed in a nutrient solution containing 100 μmol / L auxin and gibberellin. RNA was extracted after 2 h and 4 h of treatment and reverse transcribed into cDNA. RT-qPCR was used to detect changes in gene expression and to measure changes in sucrose invertase activity in leaf tissues.

[0097] 4.5 GUS staining

[0098] Take the mature leaves, stems, panicles, spikelets, and grains at the filling stage of the identified GUS transgenic plants for GUS staining. After 24 to 36 hours of treatment under light-proof conditions, use FAA fixative (brand: Kanos) for decolorization and observe and take pictures. For the GUS staining method, see Coolable GUS Staining Kit.

[0099] 4.6 Subcellular localization

[0100] 4.6.1 Agrobacterium-mediated expression of foreign proteins in tobacco

[0101] Transform the pCAMBIA2300-35S-CWIN5-EGFP vector into Agrobacterium GV3101 and screen using a plate containing rifampicin resistance. After culturing at 28°C for 72 hours, pick the positive monoclonal colonies and transfer them to 1 mL of culture medium containing the corresponding antibiotics, and culture them overnight at 28°C in a shaker at 250 r / min. Transfer the bacterial solution to 10 mL of rifampicin and kanamycin resistance culture medium and continue to culture until OD600 = 1.0.

[0102] After the culture was completed, the cells were centrifuged at 4000 rpm for 10 min, the supernatant was discarded, the cells were resuspended with 10 mmol / L MgCl2 to a value of 0D600 = 1.0, 100 mmol / L ASA was added to the resuspended solution, and the suspension was allowed to stand.

[0103] Select Nicotiana benthamiana that has not yet bloomed, use a syringe without a needle to absorb the bacterial solution, and inject the bacterial solution into the leaves from the back.

[0104] The cells were cultured in the dark for 48 to 72 hours and the fluorescence was observed under a fluorescence microscope.

[0105] 4.6.2 Rice protoplast preparation and transformation

[0106] a. Albino seedling cultivation:

[0107] The rice seeds were hydroponically grown in a dark incubator at 28°C. After turning white, they were sown in a culture dish covered with filter paper and cultured in a dark incubator at 28°C for 10 to 15 days.

[0108] b. Preparation of protoplasts:

[0109] Use a blade to cut the albino seedlings into small pieces of 0.5 mm and transfer the cut tissue pieces into a conical flask containing enzyme reaction solution at 0.25 kg / cm 2 The pressure was evacuated for 1 hour, and the enzymatic reaction was carried out at 50 rpm / min for 4 hours at 28°C in the dark.

[0110] After the enzymatic hydrolysis reaction is completed, the liquid is filtered through a filter, and 30 mL of W5 solution is added, centrifuged at 150 g / min for 5 min, and the supernatant is discarded. The complete protoplast precipitate is light green.

[0111] Add 30 mL of W5 solution to resuspend the protoplasts, centrifuge at 150 g / min for 5 min, discard the supernatant, and repeat once.

[0112] c. Transformation of protoplasts:

[0113] Pipette the plasmid to be transformed into a 2 mL enzyme-free tube, ensuring that the amount of the plasmid is between 5 and 10 μg.

[0114] Resuspend the protoplasts with MMg and pipette 200 μL into each centrifuge tube. After thorough mixing, add 200 μL PEG4000, mix well by pipetting, and place at 28°C for 15 min.

[0115] Add 1.5 mL of W5 solution to resuspend, centrifuge and discard the supernatant. Repeat once.

[0116] Add 400 μL of W5 solution, incubate at 28°C in the dark for 16-24 h, and observe the fluorescence under a fluorescence microscope.

[0117] The formulas of W5 solution, MMg, PEG 4000, and enzymatic reaction solution are shown in Table 3.

[0118] Table 3

[0119]

[0120]

[0121] 4.6.3 Subcellular localization in onion epidermis

[0122] a. Washing gold powder:

[0123] Weigh 5 mg of 0.6 μm gold powder into a 1.5 mL sterile centrifuge tube, add 1 mL of 70% ethanol, and vortex thoroughly for 5 minutes. Place on ice for 15 minutes, centrifuge instantly, and discard the supernatant. Repeat this step 3 times.

[0124] Add 2 mL of ddH2O, vortex for 3 min, centrifuge briefly, discard the supernatant, and add 100 μL of 50% sterile glycerol.

[0125] b. Plasmid embedding:

[0126] Add 1000 ng of plasmid into the enzyme-free tube, place it on a vortex and mix it continuously. Pipette 4 μL / pipette the gold powder in the shaking into the enzyme-free tube and continue shaking for 10 min.

[0127] Add 8.5 μL of 2.5 M / L CaCl2 solution to the enzyme-free tube, and immediately add 4 μL of 0.1 M spermidine. Mix thoroughly.

[0128] Place on ice for 10 min. Centrifuge briefly and discard the supernatant.

[0129] Add 30 μL of 70% ethanol to each tube, vortex for 1 min, centrifuge briefly, discard the supernatant, repeat twice, wash twice with anhydrous ethanol, discard the supernatant, and add 10 μL of anhydrous ethanol to prepare for gene gun.

[0130] After the completion of the gene gun bombardment, the cells were cultured in the dark at 28°C for 16 h, and then the fluorescence was observed under a fluorescence microscope.

[0131] 4.7 Invertase activity assay

[0132] Tissue protein extraction: Grind the young spikelet tissue to be tested in liquid nitrogen. After grinding into powder, add 1 mL of protein extract, grind the extract and tissue powder evenly, transfer to a 2 mL enzyme-free tube, then clean the mortar with 1 mL of protein extract, and then transfer the extract to an enzyme-free tube. Add 15 μL of PMSF and 7.5 μL of DTT to each tube of tissue extract. After thorough mixing, centrifuge at 4°C, 12000 r / min for 10 minutes in a refrigerated centrifuge, aspirate the supernatant to a new 2 mL enzyme-free tube, and place on ice for testing.

[0133] Take 40 μL of protein extract supernatant into a new 2 mL enzyme-free tube, add 360 μL of the analysis solution for the required invertase, and prepare a blank control by adding 360 μL of sodium acetate buffer. Place in a 30°C metal bath for 1 hour.

[0134] After the reaction is completed, 60 μL Tris-HCl pH 8.0 is added to the reaction solution for measuring CWINs and VINs to make the reaction solution alkaline, while the CINs reaction solution does not need to be treated. Place it in an 85℃ metal bath for 3 minutes. When the reaction solution cools to room temperature, add 460 μL chloroform, mix thoroughly, and centrifuge in a refrigerated centrifuge at 4℃, 12000r / min for 10 minutes.

[0135] Pipette 140 μL of supernatant into a new tube without enzyme, add 380 μL of glucose assay solution, and place in a 30°C metal bath for 30 min. When the reaction solution cools to room temperature, use a spectrophotometer to measure the absorbance at OD340 nm, and then calculate the enzyme activity based on the standard curve.

[0136] The reagent ratios are shown in Tables 4 and 5. The glucose determination solution configuration is shown in Table 6, which details the dosage for a single sample.

[0137] Table 4

[0138]

[0139]

[0140] Table 5

[0141]

[0142] Table 6

[0143] Reagents Single sample (μL) Glucose analysis solution 380 110mM ATP 3.8 110mM NADP 3.8 HK 0.4M G6P 0.4M

[0144] 4.8 Protein silver staining

[0145] Experimental reagents:

[0146] Ethanol, glacial acetic acid, sodium acetate, sodium thiosulfate, silver nitrate, sodium carbonate, glycine or EDTA sodium salt, deionized water, formaldehyde.

[0147] Experimental steps:

[0148] (1) Fixation: Measure 100 mL of ethanol and 25 mL of glacial acetic acid, add deionized water to 250 mL, so that the final concentration reaches 40% ethanol and 10% glacial acetic acid. Immerse the gel plate in the above-prepared fixative and fix for more than 30 minutes.

[0149] (2) Water washing: Soak in deionized water.

[0150] (3) Sensitization: Measure 75 mL of ethanol, 17 g of sodium acetate (or 28.2 g of sodium acetate trihydrate), and 0.5 g of sodium thiosulfate, and add deionized water to make the volume 250 mL. Pick up the fixed gel plate and immerse it in the above-prepared sensitization solution for 30 minutes.

[0151] (4) Washing: Soak in deionized water.

[0152] (5) Silver staining: 0.625 g of AgNO3 and 100 μL of 37% formaldehyde (added before use) were weighed and deionized water was added to make the final volume 250 mL. The gel plate was immersed in the above-prepared silver staining solution for 30 min.

[0153] (6) Water washing: Soak in deionized water.

[0154] (7) Color development: Measure 6.25 g Na2CO3 and 50 μL 37% formaldehyde (added before use), add deionized water to a final volume of 250 mL. Immerse the colloid in the above-prepared liquid for 2 to 15 minutes until the bands are visible, then remove the gel.

[0155] (8) Termination: Measure 3.65 g of EDTA sodium salt or 1 g of glycine, add deionized water to the final volume of 250 mL, and soak the colloid.

[0156] (9) Storage: 1% glacial acetic acid, 4°C.

[0157] 4.9 Western Blotting

[0158] (1) Prepare 12% protein separation gel and 5% protein concentration gel, the formula is as follows:

[0159] Separation gel: H2O 6.6 mL; 30% polyacrylamide 8 mL; Tris (pH: 8.8) 5 mL; 10% SDS 0.2 mL; 10% APS 0.2 mL; TEMED 0.008 mL.

[0160] Concentrating gel: H20 4.1 mL; 30% polyacrylamide 1 mL; Tris (pH: 6.8) 0.75 mL; 10% SDS 0.06 mL; 10% APS 0.06 mL; TEMED 0.006 mL.

[0161] (2) Preparation of protein samples: Add the test samples to a final concentration of 1× SDS denatured protein loading buffer, incubate at 100°C for 5 min, cool to room temperature, and start spotting, 10 μL per lane.

[0162] (3) Constant voltage protein electrophoresis, constant voltage 100 V electrophoresis.

[0163] (4) Transfer:

[0164] The PVDF membrane was pretreated by using tweezers to pick up the membrane and soak it in methanol for 15 seconds, then transferring it to ddH2O for washing, and then transferring the membrane to transfer buffer on ice for equilibration for 20-30 minutes.

[0165] The gel was processed by cutting away the concentrated gel and carefully transferring the separation gel to transfer buffer on ice for 10 min.

[0166] Use the sandwich method (foam cotton-filter paper-PVDF membrane-PAGA gel-filter paper-foam cotton) to sandwich the PVDF membrane and PAGE gel in order, and then put them into a wet transfer apparatus with a current of 250mA and a voltage of 400V.

[0167] After the transfer is completed, prepare the blocking solution. Prepare 5% skim milk with 1×TBST solution and place it on ice for later use. After the transfer, place the membrane in the milk blocking solution, shake it gently on a shaker, and block it at room temperature for 1.5 hours or at 4℃ overnight.

[0168] (5) Antibody immunization: When incubating with the primary antibody, prepare 20 mL of blocking solution according to the same formula as the blocking solution, then add the antibody (antibody to the protein to be detected or antibody to the relevant tag) according to the concentration specified in the instructions attached to the antibody. After mixing, quickly transfer the PVDF membrane in the blocking solution to the primary antibody, shake on a shaker, and hybridize at room temperature for two hours.

[0169] After the primary antibody incubation was completed, the membrane was removed from the primary antibody and washed with TBST solution for 10 min / time for 6 times.

[0170] When incubating with the secondary antibody, prepare 20 mL of blocking solution according to the same formula as the blocking solution, select the corresponding secondary antibody according to the type of primary antibody, and then add the corresponding proportion (e.g. 2 μL, 1:10000) of the secondary antibody according to the attached instructions, mix well, add the washed membrane to the secondary antibody solution, shake on a shaker, and hybridize at room temperature for 2 hours. Remove the membrane from the secondary antibody and wash the membrane with TBST solution for 10 minutes / time, 6 times.

[0171] (6) Development: Spread plastic wrap on the table, then place the PVDF membrane on the plastic wrap, wipe the water on the surface of the PVDF membrane with a small piece of filter paper, prepare the colorimetric substrate under dark conditions (under a safe red light), take 1 mL of the two reagents in the kit and mix them in equal volumes, then add them to the surface of the PVDF membrane, then evenly apply the colorimetric substrate, cover the membrane once with plastic wrap, and image with a bioimager.

[0172] 5 Yeast two-hybrid

[0173] (1) One-to-one interaction verification was performed between the bait protein plasmids AD-GF14D (universal primers are pGADT7-GF14D-F and pGADT7-GF14D-R) and AD-GF14F (universal primers are pGADT7-GF14F-F and pGADT7-GF14F-R) and the capture protein plasmid BK-CWIN5 (universal primers are pGBKT7-CWIN5-F and pGBKT7-CWIN5-R): the bait protein plasmid and the capture protein plasmid were co-transfected into Y2HGold yeast competent cells, and finally the co-transfected cells were spread on SD / -Ade-His-Leu-Trp / X-α-Gal / AbA plates and cultured in a 30°C incubator for 3 to 5 days. If positive clones grew out, it indicated interaction.

[0174] Example 1

[0175] This example relates to phenotypic analysis of OsCWIN5 knockout and overexpression strains.

[0176] To identify the function of the gene OsCWIN5, the present application designed a CRISPR / Cas9 knockout mutant OsCWIN5-KO in the ZH11 background. Fig.18 shown.

[0177] In terms of plant shape, the overall plant height of the loss-of-function mutant was higher than that of the wild-type ZH11 ( Figure 1 A and 1C). It was found that the plant height, number of primary branches, number of secondary branches, and number of grains per ear changed between CWIN5-KO and CWIN5-OE.

[0178] To further explore the regulatory effect of OsCWIN5 on panicle type, the CDS sequence of OsCWIN5 was connected to the pCAMBIA2300-2×His-3×Flag vector, and after recombination reaction and competent transformation, the pCAMBIA2300-2×His-3×F1ag-CWIN5 overexpression vector was obtained, which was transformed into the wild-type ZH11 to obtain the OsCWIN5 transgenic positive strain CWIN5-OE. qRT-PCR verification showed that the expression levels of 10 strains were significantly upregulated.

[0179] The plant type observation of the above 10 strains revealed that the plant height of the overexpression strains was significantly lower than that of the wild type. Figure 1 C shows that the average plant height of the wild type is 105.9 cm, the average plant height of the knockout mutant is 111.1 cm, and the average plant height of the overexpression mutant is 101.1 cm. The spikelets of the overexpression strain and the wild type strain at the filling stage and the waxy stage were collected to analyze the agronomic traits. The results show that Figure 1 B, 1D-G show that the mutants have significant changes in plant height, number of grains per ear, number of primary branches and number of secondary branches compared with the wild type. Specifically, the average number of grains per ear of the wild type is 97.1, the average number of grains per ear of the knockout mutant is 85.2, and the average number of grains per ear of the overexpression mutant is 72.1; the average number of primary branches of the wild type is 9, the average number of primary branches of the knockout mutant is 8.8, and the average number of primary branches of the overexpression mutant is 5.4; the average number of secondary branches of the wild type is 36.6, the average number of secondary branches of the knockout mutant is 35.7, and the average number of secondary branches of the overexpression mutant is 21.6; the average ear length of the wild type is 20.0 cm, the average ear length of the knockout mutant is 20.3 cm, and the average ear length of the overexpression mutant is 20.1 cm.

[0180] Through relevant agronomic traits, it can be seen that the number of primary branches, secondary branches, number of grains per ear and plant height of the overexpression mutant are significantly reduced compared with those of the wild type and knockout mutant.

[0181] The results showed that OsCWIN5 is a gene that negatively regulates rice panicle shape. The panicle shape of CWIN5-OE and CWIN5-KO changed significantly, and the overexpression plants showed a significant decrease in the number of branched panicles and the number of grains on the branched panicles.

[0182] Example 2

[0183] This example relates to the analysis of the endogenous regulatory mechanism of the gene OsCWIN5.

[0184] (1) Subcellular localization of OsCWIN5

[0185] To explore the subcellular localization of the protein OsCWIN5, we ligated the CDS sequence of OsCWIN5 to the plasmid pCAMBIA2300-35S-EGFP to construct a CWIN5-GFP fusion protein, which was then transformed into Nicotiana benthamiana and rice protoplasts. Fluorescence microscopy revealed that OsCWIN5 may be localized in the cytoplasm and plasma membrane ( Figure 2 ).

[0186] (2) Prokaryotic induction of OsCWIN5 protein

[0187] OsCWIN5 is functionally annotated as a cell wall sucrose invertase, which irreversibly hydrolyzes sucrose into glucose and fructose at the cell wall. The reaction products enter the cell through the apoplast pathway or the symplast pathway and participate in subsequent biological activities. In order to verify the catalytic function of the protein encoded by this gene, the present application connected the CDS de-terminant codon sequence of OsCWIN5 to the plasmid pGEX-6P-1 containing a GST tag (the universal primers are pGEX-6P-1-CWIN5-F and pGEX-6P-1-CWIN5-R). After recombination transformation, monoclonal colony picking, and plasmid extraction and sequencing verification, the pGEX-6P-1-CWIN5 protein expression vector was obtained and transferred into the competent Escherichia coli (DE3) for fusion expression. The results showed that the optimal induction conditions were 1 mol / L IPTG concentration, 28°C temperature, and 6h. Under these conditions, OsCWIN5 protein was induced, and after lysis and purification, WB verification showed that the recombinant protein showed a band of about 70 kDa on SDS-PAGE, which was similar to the predicted size of OsCWIN5 protein ( Figure 3 ).

[0188] (3) Identification of the catalytic function of OsCWIN5

[0189] To confirm the catalytic function of OsCWIN5 protein, the present application connected the OsCWIN5 sequence to pBWA(V)HS, and added His and Flag tags to the vector to obtain the vector pBWA(V)HS-6×His-CWIN5-3×F1ag. The vector was transferred into the wild-type ZH11 to obtain the OsCWIN5 overexpression strain carrying the tag. The panicle tissue of the transgenic plant was taken, the total protein was extracted, and the target protein OsCWIN5 was obtained by purification using a nickel column. The purified protein was concentrated using an ultrafiltration centrifuge tube and quantified to 20 ng / mL. Three reaction solutions containing sucrose at different pH values ​​(pH=4.5, pH=4.7, pH=7.0) were added to the protein solution, respectively, and reacted at 37°C for 30 minutes, and then 125 μL of 3,5-dinitrosalicylic acid reagent (DNS) was added, and the reaction was carried out at 95°C for 10 minutes for color development. When the reaction solution was completely cooled to room temperature, the absorbance of the reaction solution was measured at 540 nm.

[0190] The results showed that when pH = 4.5, the reaction solution had a strong color reaction, and as the reaction time increased, the absorbance of the reaction solution at 540nm gradually increased. The above results indicate that the protein encoded by OsCWIN5 has the function of catalyzing the hydrolysis of sucrose ( Figure 4 ).

[0191] (4) OsCWIN5 enzyme kinetic analysis

[0192] Through bioinformatics analysis (https: / / web.expasy.org / compμte_pi / ), the isoelectric potential of OsCWIN5 was predicted to be 4.9. Using 0.01 mol / L sodium acetate solution as the reaction solution and the sucrose substrate concentration of 50 mmol / L, 10 pH gradients (3.0, 3.5, 4.25, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5) were set to measure the changes in the enzyme activity of OsCWIN5 in this pH range. The results showed that the optimal pH of OsCWIN5 was 4.5. When pH>5.5, the enzyme activity dropped sharply until pH>6.5 and pH<3.5, when it almost completely lost its activity. This experiment proves that OsCWIN5 is an acidic sucrose invertase ( Figure 5 A).

[0193] pH = 4.5 was selected as the optimal pH of OsCWIN5, and the enzyme kinetic curve of OsCWIN5 was drawn. With sucrose as the substrate, the Km value of OsCWIN5 was 60.25 mM. In the range of sucrose concentration of 0 to 150 mmol / L, the enzyme activity increased with the increase of sucrose concentration ( Figure 5 B).

[0194] (5) Determination of enzyme activity in young spike tissue

[0195] OsCWIN5 encodes a cell wall sucrose invertase. This application determined the enzymatic activity of the cell wall sucrose invertase in young panicle tissues with high expression of OsCWIN5. The results showed that in the 2-3 cm period of the young panicle, the enzymatic activity of the cell wall sucrose invertase in the knockout mutant CWIN5-KO was significantly higher than that of the wild type. The enzyme activity decreased compared with the early stage of heading in the young panicle at the 10 cm stage, but the enzymatic activity of the cell wall sucrose invertase in CWIN5-KO was still significantly higher than that of ZH11. In the late stage of heading, the enzyme activity decreased significantly compared with the early stage of heading, but the enzyme activity of CWIN5-KO was still slightly higher than that of the wild type ZH11. Throughout the development of the young panicle, the enzymatic activity of the cell wall sucrose invertase in the knockout strain CWIN5-KO was higher than that of the wild type ZH11 ( Figure 6 ). In summary, after the OsCWIN5 gene function was lost, the activity of cell wall sucrose invertase in young panicle tissue increased, thus affecting the development of young panicle.

[0196] At the same time, the expression levels of three genes encoding cell wall sucrose invertase, OsCIN1, OsCIN4, and OsCIN7, in the young panicle were quantitatively analyzed. It was found that the expression levels of OsCIN1 and OsCIN7 in the young panicle tissue of the knockout strain CWIN5-KO were significantly increased, and significantly decreased in CWIN5-OE ( Figure 7 In summary, after the loss of OsCWIN5 function, the expression levels of other cell wall sucrose invertase genes OsCIN1 and OsCIN7 in the same family increased significantly to compensate for its function, thereby leading to an increase in the total cell wall sucrose invertase activity in young panicle tissues.

[0197] (6) OsCWIN5 expression is regulated by hormones

[0198] In the present application, 100 μmol / L auxin (3-IAA), gibberellin (GA), cytokinin (6-BA) and methyl jasmonate (MeJA) were applied to ZH11 three-leaf seedlings for short-term hormone treatment, and RNA was extracted from the treated leaves and roots for RT-qPCR analysis.

[0199] like Figure 8 As shown in A, under gibberellin (GA) treatment, there was almost no expression in the wild type roots, but 2 hours after treatment, the expression level of OsCWIN5 in its leaves reached 6.44, and 4 hours after treatment, the expression level of OsCWIN5 in the leaves reached 10.82.

[0200] like Figure 8 As shown in B, under auxin (IAA) treatment, there was almost no expression in the wild-type roots, but after the initial treatment, the OsCWIN5 expression level in its leaves was 1.012; 2 hours after treatment, the OsCWIN5 expression level in the leaves reached 4.64, and 4 hours after treatment, the OsCWIN5 expression level in the leaves reached 6.44.

[0201] like Figure 8 As shown in C and 8D, the expression level of OsCWIN5 in the roots and leaves of the wild type did not change significantly under the treatment of cytokinin (6-BA) and methyl jasmonate (MeJA).

[0202] The results showed that the expression level of OsCWIN5 increased after GA and IAA treatment in leaves, and the change in expression level was more significant after GA treatment. There was no significant change in the expression level of OsCWIN5 in roots.

[0203] By analyzing the promoter response element of OsCWIN5, it was found that there was a gibberellin (GA) response element CARE upstream of its promoter. In order to more intuitively observe the regulation of gibberellin (GA) on the expression of the OsCWIN5 gene, the present application subjected the three-leaf stage OsCWIN5:GUS seedlings to low-concentration and long-term hormone treatment, stained the treated seedlings with GUS, and observed and photographed them after decolorization with FAA. Fig. 9 shown.

[0204] The results showed that after GA treatment, the expression of OsCWIN5 was upregulated, and its expression increased with the increase of GA concentration, especially in the developing young tissues. This experiment further proved that gibberellin (GA) induced the expression of OsCWIN5.

[0205] Through quantitative analysis of OsCIN1 and OsCIN7 in ZH11 and CWIN5-KO seedlings after hormone treatment, it was found that the expression levels of genes were affected to varying degrees after induction by IAA and GA. The expression level of OsCIN1 in CWIN5-KO increased more significantly after induction by IAA and GA than that in ZH11 ( Fig.10 ).like Fig.10 As shown in A, after 2 h of treatment, the relative expression levels of OsCIN1 and OsCIN7 in the wild type were 0.67 and 3.16, respectively.

[0206] like Fig.10 As shown in B, after 2 h of treatment, the relative expression levels of OsCIN1 and OsCIN7 in the wild type were 1.62 and 3.72, respectively.

[0207] like Fig.10 As shown in C, after 2 h of treatment, the relative expression levels of OsCIN1 and OsCIN7 in the CWIN5-KO group were 3.89 and 2.16, respectively.

[0208] like Fig.10 As shown in D, after 2 h of treatment, the expression levels of OsCIN1 and OsCIN7 in the CWIN5-KO group were 3.59 and 1.68, respectively.

[0209] To further explore the effect of hormones on sucrose invertase activity, the present application determined the activities of cytoplasmic sucrose invertase, vacuolar sucrose invertase and cell wall sucrose invertase in seedling tissues before and after IAA and GA treatment. Fig.11 A and Fig.11 As shown in B.

[0210] (7) OsCWIN5 transcriptome analysis

[0211] To further explore the endogenous regulatory network of OsCWIN5, this application conducted a genome-wide gene expression analysis of 1-3 cm, 7 cm, 10 cm, and 15 cm panicles of wild-type ZH11 and knockout mutant CWIN5-KO, and compared the gene expression differences between the wild type and knockout mutant. After the loss of OsCWIN5 gene function, 143 genes were significantly upregulated and 73 genes were significantly downregulated in 1-3 cm panicles; 367 genes were significantly upregulated and 91 genes were significantly downregulated in 7 cm panicle tissues; 402 genes were significantly upregulated and 9 genes were significantly downregulated in 10 cm panicle tissues; 58 genes were significantly upregulated and 313 genes were significantly downregulated in 15 cm panicle tissues ( Fig.12 ).

[0212] Functional enrichment analysis was performed on the differentially expressed genes between wild-type and knockout strains.

[0213] The differentially expressed genes were mainly enriched in starch and sugar metabolism pathways, MAPK signaling pathways, etc. Fig.13 ), and the related genes were verified by RT-qPCR. The related genes include CWIN5 (amplification primers are qCWIN5-F and qCWIN5-R), MADSS55 (amplification primers are MADSS55-F and MADSS55-R), OsAP2-29 (amplification primers are OsAP2-29-F and OsAP2-29-R), MPK3 (amplification primers are MPK3-F and MPK3-R), OsAMY3D (amplification primers are OsAMY3D-F and OsAMY3D-R), OsAPK9 (amplification primers are OsAPK9-F and OsAPK9-R), OsMKKK70 (amplification primers are OsMKKK70-F and OsMKKK70-R), OsMKKK62 (amplification primers are OsMKKK62-F and Os MKKK62-R), OsMPK7 (amplification primers are OsMPK7-F and OsMPK7-R), OsMST3 (amplification primers are OsMST3-F and OsMST3-R), OsMST4 (amplification primers are OsMST4-F and OsMST4-R), OsMST6 (amplification primers are OsMST6-F and OsMST6-R), SUS3 (amplification primers are SUS3-F and SUS3-R), OsGLN2 (amplification primers are OsGLN2-F and OsGLN2-R), OsOXO3 (amplification primers are OsOXO3-F and OsOXO3-R), and Os4BGLU12 (amplification primers are Os4BGLU12-F and Os4BGLU12-R).

[0214] The results showed that the expression levels of monosaccharide transporters OsMST3, OsMST4, OsMST6, sucrose synthase SUS3, OsMKKK70, OsMKKK62, and OsMPK7 were all increased in the knockout strains ( Fig.14 ).

[0215] Example 3

[0216] This example relates to the analysis of the gene regulatory mechanism of the gene OsCWIN5.

[0217] (1) Screening of CWIN5-interacting proteins

[0218] The effect of OsCWIN5 on rice panicle type is likely to be regulated by other genes. In this application, total proteins from the panicle tissue of CWIN5-OE plants were used for co-immunoprecipitation (Co-IP) to explore candidate proteins that may interact with the CWIN5 bait protein. Western-blog analysis of total proteins showed that the overexpression strain successfully expressed the CWIN5 recombinant protein, and differential protein bands were found in the SDS-PAGE gel electrophoresis of the pulled-down proteins, indicating that there may be proteins in the total proteins of young panicles that interact with CWIN5. The differential protein bands were recovered from the gel and sent to Ouyi Biotech for mass spectrometry analysis, from which four possible candidate differential proteins were screened out: GF14D, GF14F, ribosomal protein L24, glutathione S-transferase ( Fig.15 ).

[0219] (2) Verification of CWIN5-interacting proteins

[0220] The results of one-to-one verification by yeast two-hybrid showed that there was an interaction between CWIN5 and GF14D and GF14F ( Fig.16 superior).

[0221] The CDS of GF14D and CDS of GF14F (SEQ ID NO.6) were fully connected to pET-28a, and the pET-28a-GF14D and pET-28a-GF14F vectors were transformed into competent E. coli, and GF14D-His and GF14F-His proteins were obtained by induction purification. In vitro pull-down was used to prove that protein CWIN5 (universal primers are pET-28a-CWIN5-GST-F and pET-28a-CWIN5-GST-R) can interact with protein GF14D (SEQ ID NO.5) and protein GF14F (SEQ ID NO.6). Fig.16 Down).

[0222] (3) Phenotypic analysis of interacting protein genes

[0223] To verify the gene function of the candidate interacting proteins, this experiment designed OsGF14D and OsGF14F knockout strains gf14d and gfI4f with wild-type ZH11 as the background, and conducted phenotypic investigations. The results showed that the grain width of gf14d did not change significantly, but the grain length increased; the grain width of gJ14f increased significantly, and the grain length was slightly longer than that of the wild type ( Fig.17 ).

[0224] Table 6

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231] This application is a result of the major agricultural biobreeding project "Mining new high-yield genes and breeding value evaluation" 2023ZD0406801.

[0232] It should be noted that the above-mentioned specific embodiments are exemplary, and those skilled in the art can come up with various solutions inspired by the disclosure of the present invention, and these solutions also belong to the disclosure scope of the present invention and fall within the protection scope of the present invention. Those skilled in the art should understand that the present invention specification and its drawings are illustrative and do not constitute limitations on the claims. The scope of protection of the present invention is defined by the claims and their equivalents. The present invention specification contains multiple inventive concepts, such as "preferably" indicating that the corresponding paragraph discloses an independent concept, and the applicant reserves the right to file a divisional application based on each inventive concept. Throughout the text, the features guided by "preferably" are only an optional method and should not be understood as being required. Therefore, the applicant reserves the right to abandon or delete the relevant preferred features at any time.

Claims

1. Application of protein OsCWIN5, characterized in that: The amino acid sequence of the protein OsCWIN5 is shown in SEQ ID NO: 1, and the application is to regulate rice plant height.

2. The use of gene OsCWIN5, characterized in that: The gene OsCWIN5 encodes the protein OsCWIN5 in the application of claim 1, and the application is to regulate rice plant height.

3. The use according to claim 2, characterized in that: The nucleotide sequence of the gene OsCWIN5 is the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO:

3.

4. A method for regulating rice plant height, characterized in that: The method comprises: using genetic engineering means to weaken or knock out the gene OsCWIN5 in the application as claimed in claim 2 or 3 in rice.

5. The method according to claim 4, characterized in that The method comprises the following steps: Using the gene OsCWIN5 as the target gene, the sgRNA sequence based on CRISPR / Cas9 was designed; Connecting the DNA fragment containing the sgRNA sequence to the vector carrying CRISPR / Cas9; The vector is transferred into rice to obtain rice with OsCWIN5 gene function deleted.

6. The method according to claim 5, characterized in that The nucleotide sequence of the sgRNA action site is 5'-CCGGGATGGCCTGTGGAGGATGG-3'.

7. Use of the rice obtained by the method according to any one of claims 4 to 6 in plant breeding.

8. The use according to claim 7, characterized in that: The plant breeding methods include transgenic, hybridization, backcrossing, selfing or asexual reproduction.

9. The use of rice protein OsCWIN5 interacting with protein GF14D or protein GF14F in regulating the growth of rice plants, characterized in that: The amino acid sequence of the rice protein OsCWIN5 is shown in SEQ ID NO:1, the amino acid sequence of the protein GF14D is shown in SEQ ID NO:5, and the amino acid sequence of the protein GF14F is shown in SEQ ID NO:6.

Images