Rice osskp1 gene and application in improving plant type and / or increasing cold tolerance

Abstract

The application belongs to the technical field of plant genetic engineering, and particularly relates to application of a rice OsSKP1 gene in improving plant type and / or improving cold tolerance, wherein the nucleotide sequence of the rice OsSKP1 gene is shown as SEQ ID NO. 1, the coding region sequence of the rice OsSKP1 gene is shown as SEQ ID NO: 2, and the amino acid sequence of the protein encoded by the rice OsSKP1 gene is shown as SEQ ID NO. 3. The application provides a method for reducing the leaf angle of rice and enhancing the cold tolerance by increasing the expression level or protein activity of OsSKP1, and provides a new genetic resource and a molecular breeding target for the coordinated genetic improvement of the ideal plant type and environmental adaptability of rice.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, and particularly relates to the rice OsSKP1 gene and its application in improving plant architecture and / or enhancing cold tolerance. Background Technology

[0002] Rice (Oryza sativa L.) is an important global food crop, and its plant architecture and adaptability to stress are crucial agronomic traits affecting yield potential and stability. Leaf angle determines the canopy structure and light exposure of the rice canopy. Appropriately reducing the leaf angle and maintaining upright leaves is beneficial for constructing a compact plant architecture and improving light utilization potential under canopy conditions, which is one of the important goals of breeding ideal rice plant architectures.

[0003] Some progress has been made in understanding the molecular regulatory mechanisms of leaf angle formation in rice. For example, the brassinosteroid (BR) signaling pathway plays an important role in regulating leaf pulvinus development and leaf angle formation. The OsFLP transcription factor, through synergy with OsBZR1 and OsGSK1, regulates lignin deposition in leaf pulvinus sclerenchyma cells, thereby affecting the size of the leaf angle. The cytokinin oxidase gene OsCKX9 has also been reported to participate in regulating leaf angle development, and the epidermal development-related transcription factors OsZHD1 / OsZHD2 regulate leaf angle formation by affecting the division and expansion of leaf pulvinus epidermal cells. Currently known genes regulating leaf angle are mostly concentrated in hormone signaling pathways or specific transcription factors; many genetic loci and molecular mechanisms regulating leaf angle remain to be explored.

[0004] Cold tolerance in rice is a significant limiting factor for stable yield. Rice is a warm-loving crop and is highly sensitive to low-temperature stress. In some regions, low-temperature damage frequently occurs during the early rice sowing and seedling raising period and the late rice heading and flowering period, often leading to substantial yield reductions. Rice cold tolerance is a complex quantitative trait controlled by multiple genes, involving multiple developmental stages such as budding, seedling, and booting stages, with each stage having its own genetic basis. Cloning and functional analysis of rice cold tolerance genes have been established. For example, the COLD series genes (COLD1, COLD6, etc.) are involved in low-temperature sensing and signal transduction, while the COG series genes (COG1~COG5) positively regulate rice cold tolerance. Among them, COG5 encodes the transcriptional mediator complex subunit Med21, which enhances rice cold tolerance by activating the expression of OsbHLH002 / OsICE1 and downstream cold tolerance genes. Downstream cold tolerance genes such as OsTPP1 and OsDREB1A / B also participate in the low-temperature stress response.

[0005] Furthermore, research on the synergistic regulatory mechanisms of cold tolerance, growth and development, and plant architecture remains weak, and gene resources capable of simultaneously improving plant architecture and cold tolerance are extremely scarce. In existing rice breeding, genes regulating leaf angle and genes regulating cold tolerance have different regulatory directions. Plant architecture improvement often pursues high yield, while cold tolerance improvement focuses on stress resistance. The two are usually carried out as independent breeding goals, lacking molecular targets that can achieve synergistic improvement of plant architecture and cold tolerance. Therefore, there are no reports of the same gene simultaneously negatively regulating leaf angle (i.e., promoting leaf uprightness) and positively regulating cold tolerance.

[0006] The SCF (SKP1-CULLIN-F-box) complex is an important E3 ubiquitin ligase complex in eukaryotes. The SKP1 protein, as the core component of the SCF complex, can participate in the recognition of specific substrate proteins and the regulation of protein stability by binding to different F-box proteins.

[0007] Among them, rice OsSKP1 (S-phase kinase-associated protein 1 or SKP1-like protein, also known as OSK1) is a homolog of rice SKP1. It can interact with the F-box protein FBX206 to form the SCF^FBX206 complex and participate in the regulation of the stability of OsOFP8 and OsOFP19 proteins. However, whether OsSKP1 directly participates in rice plant architecture, especially leaf angle formation, and whether OsSKP1 participates in the rice's response to low-temperature stress, remains unclear. Summary of the Invention

[0008] To address the aforementioned technical issues, this invention provides the rice OsSKP1 gene and its application in improving plant architecture and / or enhancing cold tolerance. It reveals for the first time the dual function of the rice OsSKP1 gene in regulating leaf angle and cold tolerance, and provides a method for simultaneously improving rice plant architecture and cold tolerance. This invention fills the functional gap in the SKP1 family genes regarding leaf angle regulation and low-temperature stress response, providing new gene resources and application strategies for rice molecular design breeding. Further analysis of its function and development of its application in rice molecular design breeding are of significant value for obtaining new rice germplasm with both ideal plant architecture and environmental adaptability.

[0009] The present invention addresses the application of the rice OsSKP1 gene in improving plant architecture and / or enhancing cold tolerance, wherein the nucleotide sequence of the rice OsSKP1 gene is shown in SEQ ID NO.1.

[0010] The coding region sequence of the rice OsSKP1 gene is shown in SEQ ID NO:2.

[0011] The application of the protein encoded by the rice OsSKP1 gene in improving plant architecture and / or enhancing cold tolerance, wherein the amino acid sequence of the protein encoded by the rice OsSKP1 gene is shown in SEQ ID NO.3.

[0012] The OsSKP1 gene described in this invention is derived from rice, and its coding region sequence is used to construct plant overexpression vectors or in vitro protein expression systems.

[0013] The present invention also provides the application of expression cassettes, recombinant expression vectors, or recombinant bacteria containing the rice OsSKP1 gene or its encoded protein in improving plant architecture and / or enhancing cold tolerance, wherein the rice OsSKP1 gene is the rice OsSKP1 gene as described in claim 1; the recombinant expression vector is a recombinant expression vector containing the OsSKP1 gene coding sequence and a plant promoter operably linked to the coding sequence, and the nucleotide sequence of the OsSKP1 gene is shown in SEQ ID NO.1.

[0014] The application of the rice OsSKP1 gene or its encoded protein in the breeding of improved rice plant type and / or cold-resistant transgenic rice and germplasm resources.

[0015] The application is achieved by increasing the expression level of the OsSKP1 gene in rice or increasing the activity of the OsSKP1 protein.

[0016] Increasing the expression level of the OsSKP1 gene or the activity of the OsSKP1 protein can reduce the leaf angle and / or increase the uprightness of rice leaves, and can also reduce the damage to rice plants under low temperature stress, thereby improving the tolerance of rice to low temperature stress.

[0017] The improved plant type is a rice plant type with reduced leaf angle, upright leaves and / or compact plant type; the cold resistance includes, but is not limited to, cold resistance during the seedling stage, cold resistance during the growth and development stage, and the ability to recover growth after low temperature treatment.

[0018] This invention reveals that increasing the expression level of OsSKP1 in rice can reduce the leaf angle, keeping the leaves upright and forming a more compact plant structure; conversely, loss of OsSKP1 function leads to a significant increase in the leaf angle and drooping leaves. Further investigation shows that under low-temperature stress, rice plants lacking OsSKP1 function exhibit more severe leaf wilting, curling, and drooping, while plants overexpressing OsSKP1 show significantly reduced low-temperature damage, indicating that OsSKP1 has a positive regulatory effect on rice cold tolerance.

[0019] Prioritize increasing the expression level of the OsSKP1 gene or the activity of the OsSKP1 protein to enable rice to simultaneously exhibit at least two of the following phenotypes: reduced leaf angle, upright leaves, and enhanced tolerance to low temperature stress.

[0020] In the comprehensive improvement of rice traits, increasing the expression level of OsSKP1 can simultaneously achieve a comprehensive phenotype characterized by reduced leaf angle, upright leaves, compact plant type, and enhanced tolerance to low-temperature stress. OsSKP1 can serve as a molecular target for the synergistic improvement of ideal plant type and cold tolerance in rice, and can be used for molecular design breeding and the creation of superior germplasm in rice.

[0021] The method for increasing the expression level of the OsSKP1 gene described in this invention is selected from at least one of the following: A. Import the OsSKP1 overexpression construct; B. Enhance the activity of the OsSKP1 promoter; C. Using gene editing to alter the cis-regulatory elements of OsSKP1; D. Utilize transcriptional activation systems to enhance OsSKP1 expression; E. Introduce allelic variants that can increase OsSKP1 expression or protein activity.

[0022] The method for improving rice plant architecture and / or enhancing rice cold tolerance using the rice OsSKP1 gene as described above is characterized by comprising the following steps: (1) Construct a recombinant expression vector containing the coding sequence of the OsSKP1 gene and a plant promoter operatively linked to the coding sequence, wherein the nucleotide sequence of the OsSKP1 gene is shown in SEQ ID NO.1; (2) The recombinant expression vector was introduced into recipient rice to overexpress the OsSKP1 gene in rice; (3) Screen transgenic rice lines with increased OsSKP1 gene expression level and / or enhanced OsSKP1 protein activity to obtain rice materials with improved plant type and / or improved cold resistance.

[0023] In the optimization scheme, step (2) introduces methods including using Ti plasmids, plant virus vectors, direct DNA transformation, microinjection, gene guns, electrical conductivity or Agrobacterium-mediated methods; step (3) screens phenotypes including identifying reduced leaf angle, upright leaves, compact plant type and / or enhanced tolerance to low temperature stress.

[0024] Application of the recombinant expression vector in the preparation of rice genetic materials with compact plant type and / or improved cold resistance.

[0025] This invention regulates the leaf angle of rice, constructs a compact rice plant type, and improves the cold tolerance of rice, thereby cultivating rice germplasm that combines a compact plant type and / or cold tolerance, and has the following beneficial effects: (1) This invention clarifies the regulatory role of OsSKP1 in the formation of leaf angle and plant architecture in rice.

[0026] (2) Loss of OsSKP1 function leads to a significant increase in the leaf angle and drooping of rice leaves, while increasing OsSKP1 expression can promote upright leaves and form a compact plant type, indicating that OsSKP1 is an important candidate target for rice plant type improvement.

[0027] (3) This invention found that OsSKP1 participates in the low temperature stress response of rice. Compared with the wild type, the low temperature damage of OsSKP1 loss-of-function materials was significantly more severe, while the low temperature damage of OsSKP1 overexpression materials was reduced, indicating that OsSKP1 can positively regulate the cold tolerance of rice.

[0028] (4) OsSKP1 has the functions of regulating plant type and cold resistance, providing a new molecular target for simultaneously improving rice canopy structure and environmental adaptability, and has important application value in rice molecular breeding.

[0029] This invention provides a method to reduce the leaf angle of rice and enhance cold resistance by increasing the expression level or protein activity of OsSKP1, providing new gene resources and molecular breeding targets for the synergistic genetic improvement of ideal rice plant type and environmental adaptability. Attached Figure Description

[0030] Figure 1 This document presents the positive identification results for the OsSKP1 transgene in this invention (A. Sequencing results of the target site in the OsSKP1 gene CRISPR / Cas9 edited lines. Compared with the wild-type target sequence, osskp1-Cas9-1 has a 2 bp deletion at the target site, and osskp1-Cas9-30 has a 1 bp insertion; boxes indicate mutation sites. B. Expression level analysis of the OsSKP1 overexpression lines. Compared with ZH11, the relative expression levels of OsSKP1 in OsSKP1-OE-21 and OsSKP1-OE-23 were significantly increased. Data are expressed as mean ± SD; *** indicates extremely significant difference (P < 0.001).).

[0031] Figure 2 This invention provides an analysis of OsSKP1 expression (A. qRT-PCR analysis of OsSKP1 gene expression pattern; B. subcellular localization of OsSKP1. OsSKP1-GFP fusion protein and D53-mCherry (a nuclear marker protein) were co-expressed in rice protoplast cells, and fluorescence was observed under a laser scanning confocal microscope. GFP completely overlapped with red fluorescent protein, indicating that OsSKP1 is localized in the cell nucleus. Scale bar: 5 μm).

[0032] Figure 3This invention provides an analysis of the plant morphology of OsSKP1 transgenic rice (A. Comparison of the overall phenotypes of ZH11, osskp1-Cas9, and OsSKP1-OE rice at maturity, scale bar 10 cm; B, C. Close-up observation of the angle between the second leaves of ZH11, osskp1-Cas9, and OsSKP1-OE using a stereomicroscope. B. Scale bar in the figure is 0.5 cm, C. Scale bar in the figure is 0.5 mm. C. Statistical analysis of the flag leaf angle of ZH11, osskp1-Cas9, and OsSKP1-OE. Data are expressed as mean ± standard deviation (n = 15). Student's t-test was used, * P < 0.05, ** P < 0.01.).

[0033] Figure 4 This invention describes the role of OsSKP1 in the BR signaling pathway (where A. the leaf angle response under 24-epiBL treatment was measured using the droplet method; B. leaf angle measurement and analysis. Leaf angle measurements were performed in three biological replicates, with 10 plants per replicate. ** P < 0.01. C. qRT-PCR analysis of the expression levels of BR biosynthesis genes OsBR6ox2 and OsD2 in ZH11, osskp1-Cas9, and OsSKP1-OE plants. * P < 0.05, ** P < 0.01. Three biological replicates were performed. D. qRT-PCR detection of the expression levels of BR catabolism genes CYP734A2 and CYP734A4 in ZH11, osskp1-Cas9, and OsSKP1-OE plants. * P < 0.05, ** P < 0.01. Three biological replicates were performed.).

[0034] Figure 5 Phenotype of OsSKP1 transgenic lines after cold treatment.

[0035] Figure 6 The physiological parameters of the OsSKP1 transgenic material under cold stress in this invention were determined as follows: (A. Conductivity of wild-type ZH11, overexpression lines OsSKP1-OE-21 and OsSKP1-OE-23, and knockout lines osskp1-Cas9-1 and osskp1-Cas9-30 before and after cold treatment; B. Survival rate of seedlings after cold treatment recovery; C. Relative water content; D. Proline content before and after cold treatment; E. Trehalose content. * P < 0.05, ** P < 0.01.) Detailed Implementation

[0036] The present invention will be further described below with reference to specific embodiments:

[0037] Example 1

[0038] Application of the rice OsSKP1 gene in improving plant architecture and enhancing cold tolerance.

[0039] OsSKP1 gene and its encoded protein: The OsSKP1 gene described in this invention is derived from rice, and its genomic DNA sequence is shown in SEQ ID NO:1, its coding region sequence is shown in SEQ ID NO:2, and the amino acid sequence of the encoded OsSKP1 protein is shown in SEQ ID NO:3.

[0040] Application of OsSKP1 in regulating rice plant architecture: By increasing the expression level of the OsSKP1 gene or the activity of the OsSKP1 protein in rice plants, the leaf angle can be reduced, the leaves can be made more upright, and a more compact plant type can be formed.

[0041] The methods for increasing OsSKP1 expression levels include, but are not limited to: (1) Overexpression of OsSKP1 in rice; (2) Introduce a plant expression vector containing the OsSKP1 coding sequence into rice; (3) Improve the transcriptional level of the OsSKP1 gene by using promoter editing; (4) Enhance the expression of the OsSKP1 gene using the CRISPR activation system; (5) Introduction or polymerization of superior allelic variants that can increase OsSKP1 expression or protein activity; (6) Use other molecular breeding methods that can improve the expression level of OsSKP1 gene or the activity of OsSKP1 protein.

[0042] Application of OsSKP1 in improving cold tolerance in rice: Increasing the expression level of OsSKP1 in rice or enhancing the activity of OsSKP1 protein can reduce the damage to rice plants under low temperature stress and improve the tolerance of rice to low temperature stress.

[0043] The cold resistance includes, but is not limited to, cold resistance during the seedling stage, cold resistance during the growth and development stage, and the ability to recover growth after low-temperature treatment.

[0044] Application of OsSKP1 in the comprehensive improvement of rice traits: By increasing the expression level of OsSKP1, a comprehensive phenotype can be obtained simultaneously: reduced leaf angle, upright leaves, compact plant type, and enhanced tolerance to low temperature stress.

[0045] Therefore, OsSKP1 can serve as a molecular target for the synergistic improvement of ideal plant architecture and cold tolerance in rice, and can be used for molecular design breeding and the creation of superior germplasm in rice.

[0046] Compared with wild-type ZH11, the OsSKP1 loss-of-function line osskp1-Cas9 showed a significantly increased leaf angle and drooping leaves; while the OsSKP1 overexpression line showed a decreased leaf angle, upright leaves and compact plant type, indicating that OsSKP1 negatively regulates the leaf angle of rice.

[0047] After low temperature stress, osskp1-Cas9 plants showed severe leaf wilting, curling and drooping, while OsSKP1 overexpressing plants showed less damage, indicating that OsSKP1 positively regulates rice cold tolerance.

[0048] Example 2

[0049] Obtaining OsSKP1 knockout materials: Based on the OsSKP1 gene sequence, CRISPR / Cas9 target sites were designed, and an OsSKP1 gene editing vector was constructed. This vector was then introduced into the rice variety Zhonghua 11 (ZH11) using Agrobacterium (EHA105)-mediated genetic transformation.

[0050] (1) Construct the CRISPR / Cas9 knockout vector of the OsSKP1 gene, named osskp1-Cas9. The specific construction process is as follows: ① Target primer design

[0051] CRISPR / Cas9 vector primers were designed using the online CRISPR-GE website (http: / / skl.scau.edu.cn / ). Target sites with GC content between 45% and 70% and an off-target rate ≤0.6 were selected. The obtained target primers were named osskp1-gRT1 (CGTGACATCATCAATGGCGGGTTTTAGAGCTAGAAAT), osskp1-OsU6aT1 (CCGCCATTGATGATGTCACGCGGCAGCCAAGCCAGCA), osskp1-gRT2 (CTCCTCCACATGATCGAGGAGTTTTAGAGCTAGAAAT), and osskp1-OsU6aT2 (TCCTCGATCATGTGGAGGAGCAACACAAGCGGCAGC), as shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7.

[0052] ② Construction of target sgRNA (single guide RNA) expression cassette

[0053] First round of PCR reaction: Overlapping PCR was used to introduce the target sequence into the sgRNA expression cassette. The first round of PCR reaction was performed, and the reaction procedure is shown in Table 1 below. After the reaction, 3-5 μL of the PCR product was taken and detected by 1.5% agarose gel electrophoresis.

[0054] Table 1. First-round PCR reaction procedure

[0055] Pre-variation 95℃ 30 sec 1 transsexual 95℃ 15 sec annealing 58℃ 15 sec 26 extend 72℃ 20 sec Complete extension 72℃ 1 min 1

[0056] Second round PCR reaction: The product of the first round PCR was used as the template for the second round PCR. The second round of overlapping PCR was performed using universal adapter primers (Pps-GGL: TTCAGAGGTCTCTACCGACTAGTCACGCGTATGGAATCGGCAGCAAA; Pgs-GG2: AGCGTGGGTCTCGTCAGGGTCCATCCACTCCAAGCTC), as shown in SEQ ID NO.8 and SEQ ID NO.9, to add adapters to the target sgRNA expression cassette and construct a complete expression cassette. The reaction procedure was the same as the first round. The PCR product after the reaction was verified by 1.5% agarose gel electrophoresis. When a single target band of the correct size was shown, it was purified and recovered.

[0057] Simultaneous digestion and ligation reaction: The PCR products recovered in the second round were subjected to simultaneous digestion and ligation using BsaI-HF restriction enzyme and T4 DNA ligase (product numbers: R3535, M0202V; NEW ENGLAND BioLabs, respectively). The reaction system is shown in Table 2 below, and the reaction procedure is shown in Table 3 below. After the reaction was completed, the PCR products were verified by 1.5% agarose gel electrophoresis. After confirming the target band, the gel was cut and recovered.

[0058] Table 2. Side-cutting and side-connecting reaction system

[0059] 2×Phanta Max Master Mix (Dye Plus) 15 μL The first-round PCR product of target 1 and U6a 1 μL Universal primer: Pps-GGL 0.5 μL Universal primer: Pgs-GG2 0.5 μL <![CDATA[ddH2O]]> 13 μL

[0060] Table 3. Cutting and Connecting Reaction Procedure

[0061] 10×Cutsmart buffer 1.5 μL 10×T4 buffer 1.5 μL BsaI-HF 0.5 μL T4 DNA ligase 0.5 μL pYLCRISPR / Cas9Pubi-H plasmid 1 μL Second round of U6b recovery products 1 μL Second round of U6b recovery products 1 μL Total volume 8 μL

[0062] ③ Transformation and identification of connecting products

[0063] The ligation product was introduced into *E. coli* DH5α competent cells (catalog number: C502-02, vazyme) using a heat shock method. Colony PCR was performed, and single colonies containing the target gene were picked and inoculated into liquid LB medium containing kanamycin (50 μg / mL) (Table 4) for amplification. Recombinant plasmids were then extracted using a plasmid extraction kit. Sequencing was performed, and the sequences were aligned to complete the construction of the CRISPAR / Cas9 vector.

[0064] Table 4 LB medium

[0065] trypsin 10 g Yeast extract 5 g Sodium chloride 10 g Agar powder (not added when preparing liquid culture medium) 15 g <![CDATA[ddH2O]]> up to 1 L

[0066] (2) Genetic transformation

[0067] ① Rice callus induction and subculture: Plump, mature ZH11 seeds were selected, shelled, and then surface-sterilized and aseptically cleaned before being inoculated onto callus induction medium (Table 5) and cultured at 26°C in the dark for approximately 30 days. Embryogenic callus tissues with good growth and suitable texture were selected and transferred to N6 medium, and subcultured at 28°C in the dark for approximately 2 weeks to provide high-quality recipient material for subsequent infection.

[0068] Table 5 Induction medium formulation

[0069] MS 4.74 g 2,4-D (0.8 mg / mL stock solution) 3.5 mL sucrose 30 g Agar powder 13 g <![CDATA[ddH2O]]> up to 1 L

[0070] ② Agrobacterium culture: Agrobacterium EHA105 carrying the target overexpression vector or CRISPR / Cas9 knockout vector was inoculated into YEP solid medium containing kanamycin (50 μg / mL) for propagation. Single colonies were then picked and inoculated into N6 liquid medium (Table 6) and cultured overnight at 28 ℃ and 200 rpm to allow the bacterial culture to reach OD. 600 It reached approximately 0.8.

[0071] Table 6 Induction medium formulation

[0072] N6 1.995 g sucrose 30 g AS (0.8 mg / mL stock solution, added at 50-55℃ after sterilization) 1 mL Agar powder 13 g <![CDATA[ddH2O]]> up to 1 L

[0073] ③ Agrobacterium infection: Select healthy callus tissues and immerse them in Agrobacterium suspension. Infect the callus tissues with low-speed shaking for 15–20 min. After removing excess bacterial solution, transfer the callus tissues to N6 solid culture medium and co-culture them in the dark at 26 °C for 3 days to promote the transfer of T-DNA into rice cells.

[0074] ④ Agrobacterium removal: After co-culturing, the callus tissue was first thoroughly rinsed with sterile water, and then washed 2–3 times with N6 liquid medium containing 500 mg / L cephalosporin to remove residual Agrobacterium. The washed callus tissue was then placed on sterile filter paper and dried appropriately in a laminar flow hood.

[0075] ⑤ Screening of resistant callus: The treated callus tissue was transferred to N6 selection medium containing cephalosporin (250 mg / L) and hygromycin (50 mg / L) for initial screening. Subsequently, uncontaminated and well-preserved callus tissue was selected for subculture screening. After continuous screening, newly formed milky white resistant callus tissue was selected as subsequent regeneration material.

[0076] ⑥ Resistant callus differentiation, rooting, and transplanting: The resulting resistant callus was transferred to MS medium and cultured in the dark before being transferred to light conditions to promote shoot differentiation. Once the green shoots had grown to about 1–2 cm, they were transferred to 1 / 2 MS medium to induce rooting. After the root system was fully developed, the seedlings were hardened off and finally transplanted into soil to obtain regenerated plants.

[0077] (3) Identification of mutant plants: After obtaining transgenic plants, total DNA was first extracted from the transgenic knockout plants. Target sequencing primers ooskp1-Cas9-cx (F: AACAGTTGGATTCCGTTTCG; R: GGAGATGTTCGGGATCAAGAACGA) were designed at both ends of the target site, as shown in SEQ ID NO. 10 and SEQ ID NO. 11. The fragments were amplified using these primers as templates, and agarose gel electrophoresis was performed. Single bands of the correct size were excised and sent to Qingke Biotechnology for sequencing. Sequence variation analysis was performed using SnapGene software to screen for homozygous and mutant lines. osskp1-Cas9-1 was found to have two bases missing at the target site, and osskp1-Cas9-30 was found to have one base inserted at the target site (e.g., osskp1-Cas9-1). Figure 1 (As shown in A).

[0078] Example 3

[0079] Obtaining rice materials overexpressing OsSKP1: (1) Construction of overexpression vector

[0080] ① Primer design: Download the CDS sequence of the OsSKP1 gene from the National Rice Data Center (RGAP https: / / www.ricedata.cn / gene / ) database; based on the vector multiple cloning site (MCS) and gene CDS sequence information, select appropriate restriction enzyme sites, and design overexpression vector primers OsSKP1-OE (F: GAAATTCGAGCTCTCTAGAACTAGTATGAGCTCATGGCACCTGA; R: CGACTCTAGAGGATCCCCGGGTACCCTAGAAGGCCCATTCATACTCC) using SnapGene and Primer5 software, as shown in SEQ ID NO.12 and SEQ ID NO.13. Send the designed primers to Qingke Biotechnology Co., Ltd. for synthesis.

[0081] ② PCR amplification of the target fragment: RNA was extracted from ZH11 and transcribed into cDNA using reverse transcriptase. This cDNA was used as a template for fragment amplification, and the target fragment was amplified by PCR using a high-fidelity enzyme.

[0082] ③ DNA fragment purification: Gel cutting: Prepare a 1% agarose gel containing Goldview (Catalog No.: GV0010; Biotopped) dye. After electrophoresis at 110V for 25 min, use a gel imaging system to scan the gel and cut out the single PCR bands under UV light, then transfer them to 2.0 mL EP centrifuge tubes for purification and recovery. Recovery: The above agarose gel was purified and recovered using the FastPure® Gel DNA Extraction Mini Kit (catalog number: DC301-01; vazyme).

[0083] ④ Enzyme digestion of the vector plasmid: The PTCK303 plasmid was digested with KpnⅠ and SpeⅠ (catalog numbers: FD0524, ER1251; Thermo, respectively) in a metal bath at 37℃ for 2 h. After terminating the digestion reaction at 65℃, the plasmid was purified by agarose gel chromatography to obtain the linearized vector after digestion and purification.

[0084] ⑤ Linking the target fragment with the linearized vector: The recovered fragments and the digested and recovered backbone vector pTCK303 were ligated using the homologous recombinase ClonExpress® II One Step Cloning Kit (catalog number: C112-01 / 02; vazyme) at 37°C for 30 min.

[0085] ⑥ Transformation of ligation products and identification of positive clones: Immediately transform the above ligation product using pre-prepared competent E. coli (DH5α) cells, and incubate overnight. Single clones were picked from LB plates for positive identification.

[0086] ⑦ Extract plasmids and sequence them: The identified positive clones were cultured and propagated, plasmids were extracted, and sent to Solarbio Science & Technology Co., Ltd. for sequencing. The sequencing results were compared using SnapGene software, and the correctly sequenced plasmids were stored at -20°C for subsequent experiments. Plasmids were extracted using the Plasmid Extraction Mini Kit (catalog number: D1100; Solarbio).

[0087] (2) Genetic transformation

[0088] The genetic transformation process was carried out using the same methods as for knockout lines.

[0089] (3) Positive identification of overexpressing plants

[0090] After obtaining transgenic plants, total RNA was extracted from transgenic rice, reverse transcribed, and the expression level of the target gene was detected by qRT-PCR. The qRT-PCR results showed that, compared with wild-type ZH11, the expression level of the OsSKP1 gene was significantly upregulated in the OsSKP1-OE-21 and OsSKP1-OE-23 lines (e.g., ...). Figure 1 As shown in B).

[0091] Example 4

[0092] Spatiotemporal expression and subcellular localization of OsSKP1: The spatiotemporal expression patterns of the OsSKP1 gene in different tissues were analyzed using qRT-PCR.

[0093] The results showed that OsSKP1 was expressed in all tissues tested, with significantly higher expression levels in young leaves and seeds than in other tissues. Figure 2 A).

[0094] To investigate the subcellular localization characteristics of OsSKP1, an OsSKP1-GFP fusion expression vector was constructed. OsSKP1-GFP was co-transfected with the nuclear marker protein D53-mCherry in rice protoplasts. Confocal microscopy revealed that the green fluorescence signal of GFP and the red fluorescence signal of mCherry were completely co-localized in the cell nucleus, confirming that the OsSKP1 protein is localized in the cell nucleus. Figure 2 B).

[0095] Example 5

[0096] OsSKP1 regulates the angle between rice leaves: Morphological comparison between OsSKP1 transgenic plants and ZH11 mature plants revealed that the osskp1-Cas9 line exhibited a significant increase in leaf angle and drooping leaves; in contrast, the OsSKP1-OE line showed a compact plant type and upright leaves. Figure 3 A). The leaf angle of ZH11 and OsSKP1 transgenic plants was carefully observed and statistically analyzed. At least 10 independent plants were randomly selected from each line. The first to third fully expanded leaves from the top to the bottom of the main stem of each plant were measured. The average value of the three leaves of each plant was taken as the leaf angle phenotypic value of the plant. Each plant was considered as a biological replicate.

[0097] The results showed that, compared with wild-type ZH11, the leaf angles of the osskp1-Cas9-1 and osskp1-Cas9-30 lines increased from approximately 18° to approximately 90°, an increase of approximately 72° (P<0.05); while the leaf angles of the OsSKP1-OE-21 and OsSKP1-OE-23 lines were approximately 16–17°, slightly lower than that of ZH11, but the differences were not statistically significant. Figure 3 B Figure 3 C). These phenotypic data indicate that loss of OsSKP1 function significantly increases the leaf angle in rice, while overexpressing lines generally exhibit a more compact plant type, but their leaf angle is not significantly different from that of ZH11.

[0098] The morphological changes exhibited by OsSKP1 transgenic plants suggest that this gene may be involved in the regulation of the brassinolide (BR) signaling pathway. To verify this hypothesis, BR sensitivity tests were conducted on seedlings of ZH11, osskp1-Cas9, and OsSKP1-OE plants.

[0099] Treatment with exogenous 24-epibrassinol (eBL) revealed that the osskp1-Cas9 mutant exhibited a strong leaf angle response to eBL. Under 100 ng eBL treatment, the leaf angle of the osskp1-Cas9 line was approximately 45°, an increase of about 17° compared to approximately 28° in ZH11 (P<0.05); under 1000 ng eBL treatment, the leaf angle was approximately 92°, an increase of about 32° compared to approximately 60° in ZH11 (P<0.01). Figure 4(A, 4B). Notably, under the 100 ng eBL treatment, the leaf angle of OsSKP1-OE plants was approximately 21–22°, a decrease of about 6–7° compared to ZH11 (P<0.05), while under the 1000 ng eBL treatment, the difference between OsSKP1-OE and ZH11 was not statistically significant. These results suggest that OsSKP1 may influence plant growth and development by regulating the BR signaling pathway.

[0100] Therefore, this invention detected the expression levels of genes related to BR biosynthesis and metabolism in ZH11 and OsSKP1 transgenic plants. qRT-PCR analysis showed that, compared with ZH11, the expression levels of BR biosynthesis genes OsBR6ox2 and OsD2 were significantly upregulated in the osskp1-Cas9 line (P<0.05), while the expression levels of BR catabolic genes CYP734A2 and CYP734A4 were significantly downregulated in the osskp1-Cas9 line (P<0.05). Figure 4 C, Figure 4 D). However, in OsSKP1-OE overexpressing plants, the expression levels of these four genes did not show significant differences compared to the ZH11 wild type. Figure 4 C, Figure 4 D). These results suggest that OsSKP1 may participate in the regulation of the BR signaling pathway by negatively regulating the expression of BR biosynthetic genes.

[0101] BL sensitivity testing methods: Microdroplet method: Following the experimental method of Duan et al. (2006), rice seedlings of uniform size that had germinated and grown for 5-7 days were selected. 1 μL of anhydrous ethanol containing 0, 10, 100, and 1000 ng eBL (catalog number: B8780; Solarbio) was dropped onto the corner of the second leaf, respectively. After culturing for another 3 days, the seedlings were photographed, and the leaf corner size was recorded.

[0102] Example 6

[0103] OsSKP1 participates in the low-temperature stress response of rice: OsSKP1-OE, osskp1-Cas9, and wild-type ZH11 were cultured in rice nutrient solution (product number: KD0052, Beijing Bio-Lab) until the three-leaf stage, then transferred to a 6℃ low-temperature incubator for 7 days of cold treatment, followed by 7 days of recovery at normal temperature. Each line had at least 30 plants, with three biological replicates, and the plant survival rate was counted.

[0104] The experimental results showed that before cold treatment, OsSKP1-OE, ZH11, and osskp1-Cas9 plants all grew well with no significant phenotypic differences. However, after cold treatment, the leaves of osskp1-Cas9 plants showed severe wilting, curling, and drooping, while the damage to ZH11 and OsSKP1-OE plants was relatively mild.

[0105] Survival rate statistics showed that the survival rates of osskp1-Cas9-1 and osskp1-Cas9-30 plants were only 10% and 8%, respectively, significantly lower than those of wild-type ZH11 and OsSKP1-OE lines (Figure 5). These results indicate that the knockout lines significantly reduced the cold tolerance of rice seedlings, suggesting that OsSKP1 may positively regulate the cold stress response of rice.

[0106] Low-temperature stress damages plant cells, disrupting cell membrane integrity and causing significant electrolyte leakage, manifested as a marked increase in electrical conductivity. Simultaneously, root water absorption is inhibited, exacerbating cellular water loss. With prolonged treatment time, the relative water content (RWC) of leaves decreases significantly. Furthermore, to cope with low-temperature stress, plants accumulate osmotic regulators such as proline and soluble sugars (glucose, sucrose, trehalose, etc.) to alleviate cell damage caused by cold stress. Therefore, the degree of electrolyte leakage and the content of osmotic regulators are key indicators for evaluating the cold tolerance of rice.

[0107] To further clarify the role of OsSKP1 in the response to low-temperature stress, the survival rate, ion leakage rate, relative water content, and proline and trehalose content of ZH11, osskp1-Cas9, and OsSKP1-OE plants were measured. After low-temperature treatment, the survival rate of ZH11 was approximately 56%, while that of osskp1-Cas9-1 and osskp1-Cas9-30 lines decreased to approximately 10% and 8%, respectively. In contrast, the survival rates of OsSKP1-OE-21 and OsSKP1-OE-23 lines were approximately 62% and 61%, respectively, both higher than that of ZH11. Figure 6 A). These results indicate that loss of OsSKP1 function significantly reduces the low-temperature survival ability of rice seedlings, while overexpression of OsSKP1 can improve the cold tolerance of plants.

[0108] Under normal conditions, there were no significant differences in ion leakage rate, relative water content, proline, and trehalose content among the different genotypes. After low-temperature treatment, the ion leakage rate of ZH11 increased from approximately 15% to 38%, while that of osskp1-Cas9-1 and osskp1-Cas9-30 increased to approximately 45% and 44%, respectively; in contrast, the ion leakage rates of OsSKP1-OE-21 and OsSKP1-OE-23 were approximately 30% and 32%, respectively, significantly lower than that of ZH11. Figure 6 B). Meanwhile, the relative moisture content of ZH11 decreased from approximately 82% under normal conditions to approximately 51%, osskp1-Cas9-1 and osskp1-Cas9-30 further decreased to approximately 31%, while OsSKP1-OE-21 and OsSKP1-OE-23 remained at higher levels of approximately 67% and 63%, respectively. Figure 6 C).

[0109] Low-temperature treatment also significantly promoted the accumulation of osmotic conditioning substances. The proline content in ZH11 increased from approximately 45 μg·g⁻¹ FW under normal conditions to approximately 86 μg·g⁻¹ FW, while osskp1-Cas9-1 and osskp1-Cas9-30 only accumulated to approximately 71 and 68 μg·g⁻¹ FW, respectively; in contrast, OsSKP1-OE-21 and OsSKP1-OE-23 reached approximately 88 and 89 μg·g⁻¹ FW, respectively. Figure 6 D). Similarly, after low-temperature treatment, the trehalose content of ZH11 was approximately 44 μg·g⁻¹ FW, the two osskp1-Cas9 lines were approximately 37 μg·g⁻¹ FW, and the two OsSKP1-OE lines were approximately 48 and 49 μg·g⁻¹ FW, respectively. Figure 6 E). The above results indicate that OsSKP1 may enhance rice's tolerance to low-temperature stress by maintaining cell membrane stability and plant water status, and by promoting the accumulation of osmotic regulators such as proline and trehalose.

[0110] In summary, OsSKP1 overexpression helps maintain cell membrane stability and tissue water status under low-temperature stress and promotes the accumulation of osmotic regulatory substances, thereby improving the cold tolerance of rice; while knockout of OsSKP1 leads to increased sensitivity of plants to low temperatures. These results indicate that OsSKP1 positively regulates the cold tolerance of rice.

[0111] Further evidence indicates that increasing OsSKP1 expression enhances the cold tolerance of rice, while loss of OsSKP1 function makes rice more sensitive to low-temperature stress. Therefore, OsSKP1 is a positive regulator of rice's response to low-temperature stress.

[0112] The relevant testing methods are as follows: (1) Conductivity measurement: ① The conductivity was measured according to the method in Jia et al. (2022). [3] Weigh 0.3 g of leaflets after 0 days and 5 days of cold treatment at the same time. Clean the leaf surface with deionized water to remove impurities and eliminate ion interference. Transfer the cleaned leaflets to 50 mL centrifuge tubes, add 30 mL of deionized water, and place them on a shaker to shake vigorously at 200 rpm for 4 hours. Measure the initial electrolyte leakage rate L1 using a conductivity meter.

[0113] ② Place the above sample in a boiling water bath and heat for 30 min to ensure complete rupture of the cell membrane and full release of electrolytes. After the sample cools to room temperature, measure the total electrolyte leakage rate L2.

[0114] ③ Calculate the electrolyte leakage rate of the blades according to the formula EL = L1 / L2×100 %

[0115] (2) Determination of proline content: ① Proline extraction: Weigh 0.1 g of samples from 0 days and 5 days of cold treatment, respectively, and cut the samples evenly with scissors before transferring them to 15 mL centrifuge tubes. Add 5 mL of 3% (w / v) sulfosalicylic acid solution, ensuring the samples are completely submerged. Place the centrifuge tubes in a boiling water bath and heat for 30 min, gently shaking every 5 min to ensure thorough extraction. After the samples have cooled to room temperature (25 ± 2℃), collect the supernatant as the crude proline extract.

[0116] ② Colorimetric reaction: Take 1 mL of crude proline extract and a series of proline standard solutions (1, 2, 3, 4, 5, 6 μg / mL), respectively, and add them to a mixture containing 1 mL of 2.5% (w / v) acidic ninhydrin colorimetric solution (prepared with 60 mL glacial acetic acid, 16.4 mL phosphoric acid, 36.6 mL deionized water, and 2.5 g ninhydrin) and 1 mL glacial acetic acid. Vortex to mix. Place the mixture in a boiling water bath and react for 30 min, shaking once every 5 min. After the reaction is complete, cool the sample to room temperature (25 ± 2℃).

[0117] ③ Toluene extraction and spectrophotometric determination: Add 0.5 mL of toluene to 0.75 mL of the colorimetric reaction solution, vortex for 1 min, and let stand for 30 min to allow for complete phase separation. Take the upper toluene phase and measure the absorbance at 520 nm using a microplate reader. Using toluene as a negative control, calculate the proline content in the sample based on the standard curve.

[0118] (3) Determination of trehalose content: Trehalose content was determined using the anthrone-sulfuric acid method. Leaf samples (0.1 g) from 7-day-old seedlings were collected before and after 72 h of cold stress treatment at 6℃. The procedure was performed according to the instructions of the trehalose content assay kit (catalog number: LE-1-221, Grace Biotechnology). The specific experimental steps are as follows: ① Sample preparation: Accurately weigh 0.1 g of leaf samples before and after cold treatment and place them in a sterile mortar. Add 1 mL of extraction buffer and grind until completely homogenized. Transfer the homogenate to a 1.5 mL centrifuge tube and extract at room temperature (25 ± 2℃) with shaking for 30 min. Centrifuge at 8000 g for 10 min and collect the supernatant for later use.

[0119] ② Colorimetric reaction: Adjust the water bath temperature to 95-100℃. Add 150 μL of supernatant to a 1.5 mL EP tube, add 300 μL of reagent one, mix thoroughly, and place in a 95-100℃ water bath for a precise reaction time of 3 min. After the reaction is complete, immediately cool the sample to room temperature.

[0120] ③ Measurement and Analysis: Transfer 200 μL of the cooled reaction solution to a 96-well plate and measure the absorbance at 620 nm using a microplate reader (A measurement). Use an equal volume of extract solution as a blank control (A blank). Calculate ΔA = A measurement - A blank.

[0121] ④ Calculation results: Trehalose content (µg / g fresh weight) = 75.72×(ΔA-0.0033) / W×D (W-sample mass, D-dilution factor).

[0122] (4) Data statistical analysis: This invention utilizes Excel and SPSS 19.0 for statistical analysis of the data, including t-tests, one-way ANOVA, and multiple comparisons. P < 0.05 indicates a significant difference; P < 0.01 indicates a highly significant difference.

[0123] The synergistically improved rice materials obtained through screening, which possess both ideal plant type and cold tolerance, can be self-pollinated to obtain homozygous stable lines, which can be used as intermediate breeding materials for subsequent hybridization or variety selection. These materials can be directly applied to molecular design breeding of rice and have significant breeding value.

[0124] The above embodiments / experimental examples are merely illustrative and not intended to limit the implementation methods. Those skilled in the art will recognize that various variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementation methods. However, obvious variations or modifications derived therefrom remain within the scope of this invention.

[0125] sequence list

[0126] <110> Crop Research Institute (Sichuan Germplasm Resources Center), Sichuan Academy of Agricultural Sciences

[0127] <120> Rice OsSKP1 gene and its application in improving plant architecture and / or enhancing cold tolerance

[0128] <160> 13

[0129] <170> PatentIn version 3.3

[0130] <210> 1

[0131] <211> 751

[0132] <212> OsSKP1

[0133] <213> Artificial sequence

[0134] <400> 1

[0135] aagaaacgaattcaacttaacagttggattccgtttcgaattaacaaggaagcaatcggt

[0136] tcggaataacagttcaaattagatccggattcaattcaattcatcatgcaataaatatat

[0137] gagctcatggcacctgatcggcaatcaatcaccaaacgtgggtcgtctcagagatcgatc

[0138] agcttcagaattcgccgtcgatcaatttagacatctatcagctcgcgtgacatcatcaat

[0139] ggcggcggcggcagaagagaagaacaagaagatgatcaaggtgatcagctccgacgggga

[0140] ggccttcgagatgacggaggcggcggcgagcatgtcgcggatactcctccacatgatcga

[0141] ggacggctgcaccggcgacggcggcgccgggatcactctccccaacgtcgccggcagcgc

[0142] cctcgccaaggtcatcgagtactgcaccaagcacgccatcgccgccgccgaaggtagcag

[0143] cagcagccgcaaggcgaaggaggagctgaagaagttcgacgtggagttcatggaggtggg

[0144] catcgacatgctgtacgacctgatcatggcggccaacttcatgggcgtggagggccttct

[0145] cagcctggcggcgcagcgcacggcggagctgatcaagggcaagtcgccggagcagatcag

[0146] ggagatgttcgggatcaagaacgaccacacgccggaggaggaggagcagatccgcaagga

[0147] gtatgaatgggccttctaggctatcagggtt 751

[0148] <210> 2

[0149] <211> 621

[0150] <212> OsSKP1

[0151] <213> Artificial sequence

[0152] <400> 2

[0153] atgagctcatggcacctgatcggcaatcaatcaccaaacgtgggtcgtctcagagatcga

[0154] tcagcttcagaattcgccgtcgatcaatttagacatctatcagctcgcgtgacatcatca

[0155] atggcggcggcggcagaagagaagaacaagaagatgatcaaggtgatcagctccgacggg

[0156] gaggccttcgagatgacggaggcggcggcgagcatgtcgcggatactcctccacatgatc

[0157] gaggacggctgcaccggcgacggcggcgccgggatcactctccccaacgtcgccggcagc

[0158] gccctcgccaaggtcatcgagtactgcaccaagcacgccatcgccgccgccgaaggtagc

[0159] agcagcagccgcaaggcgaaggaggagctgaagaagttcgacgtggagttcatggaggtg

[0160] ggcatcgacatgctgtacgacctgatcatggcggccaacttcatgggcgtggagggcctt

[0161] ctcagcctggcggcgcagcgcacggcggagctgatcaagggcaagtcgccggagcagatc

[0162] agggagatgttcgggatcaagaacgaccacacgccggaggaggaggagcagatccgcaag

[0163] gagtatgaatgggccttctag 621

[0164] <210> 3

[0165] <211> 206

[0166] <212> OsSKP1

[0167] <213> Artificial sequence

[0168] <400> 3

[0169] msswhlignqspnvgrlrdrsasefavdqfrhlsarvtssmaaaaeeknkkmikvissdg

[0170] eafemteaaasmsrillhmiedgctggdggagitlpnvagsalakvieyctkhaiaaaegs

[0171] sssrkakeelkkfdvefmevgidmlydlimaanfmgvegllslaaqrtaelikgkspeqi

[0172] remfgikndhtpeeeeqirkeyewaf 206

[0173] <210> 4

[0174] <211> 37

[0175] <212> osskp1-gRT1

[0176] <213> artificial sequence

[0177] <400> 4

[0178] cgtgacatcatcaatggcgggttttagagctagaaat 37

[0179] <210> 5

[0180] <211> 37

[0181] <212> osskp1-OsU6aT1

[0182] <213> artificial sequence

[0183] <400> 5

[0184] ccgccattgatgatgtcacgcggcagccaagccagca 37

[0185] <210> 6

[0186] <211> 37

[0187] <212> osskp1-gRT2

[0188] <213> artificial sequence

[0189] <400> 6

[0190] ctcctccacatgatcgaggagttttagagctagaaat 37

[0191] <210> 7

[0192] <211> 36

[0193] <212> osskp1-OsU6aT2

[0194] <213> Artificial sequence

[0195] <400> 7

[0196] tcctcgatcatgtggaggagcaacacaagcggcagc 36

[0197] <210> 8

[0198] <211> 47

[0199] <212> Pps-GGL

[0200] <213> Artificial sequence

[0201] <400> 8

[0202] ttcagaggtctctaccgactagtcacgcgtatggaatcggcagcaaa 47

[0203] <210> 9

[0204] <211> 37

[0205] <212> Pgs-GG2

[0206] <213> Artificial sequence

[0207] <400> 9

[0208] agcgtgggtctcgtcagggtccatccactccaagctc 37

[0209] <210> 10

[0210] <211> 20

[0211] <212> ooskp1-Cas9-cx-F

[0212] <213> Artificial sequence

[0213] <400> 10

[0214] aacagttggattccgtttcg 20

[0215] <210> 11

[0216] <211> twenty four

[0217] <212> ooskp1-Cas9-cx-R

[0218] <213> Artificial sequence

[0219] <400> 11

[0220] ggagatgttcgggatcaagaacga 24

[0221] <210> 12

[0222] <211> 44

[0223] <212> OsSKP1-OE-F

[0224] <213> Artificial sequence

[0225] <400> 12

[0226] gaaattcgagctctctgaactagtatgagctcatggcacctga 44

[0227] <210> 13

[0228] <211> 47

[0229] <212> OsSKP1-OE-R

[0230] <213> Artificial sequence

[0231] <400> 13

[0232] cgactctagaggatccccgggtaccctagaaggcccattcatactcc 47

Claims

1. The application of the rice OsSKP1 gene in improving plant architecture and / or enhancing cold tolerance, characterized by: The nucleotide sequence of the rice OsSKP1 gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that: The coding region sequence of the rice OsSKP1 gene is shown in SEQ ID NO:

2.

3. The application of the protein encoded by the rice OsSKP1 gene in improving plant architecture and / or enhancing cold tolerance, characterized by: The amino acid sequence of the protein encoded by the rice OsSKP1 gene is shown in SEQ ID NO.

3.

4. The application of expression cassettes containing the rice OsSKP1 gene or its encoded protein, recombinant expression vectors, or recombinant bacteria in improving plant type and / or enhancing cold tolerance, characterized in that: The rice OsSKP1 gene is the rice OsSKP1 gene as described in claim 1; the recombinant expression vector is a recombinant expression vector containing the coding sequence of the OsSKP1 gene and a plant promoter operably linked to the coding sequence, and the nucleotide sequence of the OsSKP1 gene is shown in SEQ ID NO.

1.

5. Application of the rice OsSKP1 gene or its encoded protein in the breeding of improved rice plant type and / or cold-resistant transgenic rice and germplasm resources.

6. The application according to any one of claims 1-4, characterized in that: The application is achieved by increasing the expression level of the OsSKP1 gene in rice or increasing the activity of the OsSKP1 protein.

7. The application according to any one of claims 1-4, characterized in that: The plant type is a rice plant type with reduced leaf angle, upright leaves and / or compact plant type; the cold resistance includes but is not limited to cold resistance during seedling stage, cold resistance during growth and development stage and recovery growth ability after low temperature treatment; preferably, the expression level of OsSKP1 gene or the activity of OsSKP1 protein is increased so that rice simultaneously exhibits at least two of the following phenotypes: reduced leaf angle, upright leaves and enhanced tolerance to low temperature stress.

8. The application according to claim 5, characterized in that: The method for increasing the expression level of the OsSKP1 gene is selected from at least one of the following: A. Import the OsSKP1 overexpression construct; B. Enhance the activity of the OsSKP1 promoter; C. Using gene editing to alter the cis-regulatory elements of OsSKP1; D. Utilize transcriptional activation systems to enhance OsSKP1 expression; E. Introduce allelic variants that can increase OsSKP1 expression or protein activity.

9. A method for improving rice plant architecture and / or enhancing rice cold tolerance using the rice OsSKP1 gene as described in claim 1, characterized in that: Includes the following steps: (1) Construct a recombinant expression vector containing the coding sequence of the OsSKP1 gene and a plant promoter operatively linked to the coding sequence, wherein the nucleotide sequence of the OsSKP1 gene is shown in SEQ ID NO.1; (2) The recombinant expression vector was introduced into recipient rice to overexpress the OsSKP1 gene in rice; (3) Screen transgenic rice lines with increased OsSKP1 gene expression level and / or enhanced OsSKP1 protein activity to obtain rice materials with improved plant type and / or improved cold tolerance; preferably, the introduction in step (2) includes methods such as using Ti plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, electrical conductivity or Agrobacterium-mediated transformation; the screening in step (3) includes identifying phenotypes such as reduced leaf angle, upright leaves, compact plant type and / or enhanced tolerance to low temperature stress.

10. The application of the recombinant expression vector as described in claim 4 in the preparation of rice genetic materials with compact plant type and / or improved cold resistance.

Images