Rice gene CDC48E and application thereof

Abstract

The invention belongs to the field of plant molecular biology and genetic engineering, and discloses a rice gene CDC48E and application thereof. The amino acid of the CDC48E encoding protein is as shown in SEQ ID NO: 2, or the CDC48E encoding protein is a functional equivalent protein derived from the SEQ ID NO: 2. The functional research of the CDC48E gene shows that the CDC48E gene is a positive regulatory factor for promoting high environment temperature response growth of plants. The mesocotyl elongation of the cdc48e mutant is obviously inhibited under the condition of 32 DEG C, which indicates that the CDC48E gene plays a key role in regulating and controlling the proliferation and elongation of temperature-dependent cells. The invention also provides a method for improving the temperature adaptability of plants, adjusting the plant type and promoting the yield by using the CDC48E gene, and a gene resource and an application basis are provided for cultivating rice varieties with high environmental temperature adaptability.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular biology and genetic engineering, specifically relating to the rice gene CDC48E and its applications. Background Technology

[0002] Global warming has become a major threat to food security and human development. Global warming includes not only an increase in average temperature, but also the frequent occurrence of extreme heat events worldwide. Predictions indicate that ambient temperatures exceeding 30°C can lead to potential yield losses in major crops. It has been reported that for every 1°C increase in global temperature, the yields of four major crops (wheat, rice, corn, and soybeans) decrease by an average of approximately 6.0%, 3.2%, 7.4%, and 3.1%, respectively. Furthermore, the increase in global warming and extreme heat events may both drive significant domestication responses in plants and affect the growth and reproduction of plants in the terrestrial biosphere. This poses a significant threat to agro-ecosystems, presenting enormous challenges to future agriculture and food security.

[0003] Rice ( Oryza sativa Rice is one of the most important food crops, serving as the staple food for more than 50% of the world's population. Finding rice germplasm adapted to high temperatures has significant practical value.

[0004] The mesocotyl, the tissue between the coleoptile and the radicle attachment point in a seedling, grows only in darkness. Mesocotyl elongation facilitates the emergence of rice seeds from the soil or water surface after germination, a characteristic crucial in direct-seeded rice cultivation. Current rice agricultural production mainly includes two methods: traditional transplanting and direct seeding. Traditional transplanting requires four steps: preparing the seedbed, sowing, raising seedlings, and transplanting. This process is not only labor-intensive and inefficient but also very costly, with labor costs accounting for 60%-70% of the total rice cultivation cost. Direct seeding, on the other hand, involves directly sowing seeds in the soil or water. Compared to traditional transplanting, direct seeding offers numerous advantages, such as eliminating the need for manual labor in seedling cultivation, management, and transplanting; saving resources by eliminating the need for seedbed preparation; and shortening the rice growing cycle by eliminating the need for seedling raising and acclimatization, making it suitable for rice-oilseed rotation. Therefore, direct seeding technology is increasingly favored.

[0005] Currently, the widespread adoption of direct seeding technology still faces several challenges, such as difficulty in seedling emergence in paddy fields and deep-soil dry direct seeding, the vulnerability of seeds to bird consumption in the early stages affecting emergence rates, and the difficulty in controlling weeds growing alongside rice seedlings. Among these, the difficulty in seedling emergence in deep-soil dry direct seeding and paddy field direct seeding is a significant factor limiting the comprehensive adoption of this technology. While paddy field direct seeding and deep-soil dry direct seeding avoid the risk of seed consumption by birds, rice seedlings struggle to emerge from the soil surface or cannot withstand the low-oxygen stress underwater, leading to a lower emergence rate and impacting rice yield. The elongation of the rice mesocotyl under dark conditions or in water can help rice seedlings emerge from the soil or water surface, improving the emergence rate. Furthermore, varieties with longer mesocotyls have stronger emergence ability and are relatively easier to germinate. Experiments show that the mesocotyls of indica rice varieties are generally longer than those of japonica rice. Therefore, identifying genes that regulate mesocotyl elongation is of significant practical value for improving the mesocotyl characteristics of rice varieties and is crucial for the direct seeding promotion of superior japonica rice varieties.

[0006] Existing research has shown that the mesocotyl is a highly temperature-sensitive organ. Mesocotyl elongation is one of the important phenotypic responses of plants to temperature; both high and low temperatures affect mesocotyl elongation, and the elongation rate varies at different temperatures. Therefore, for many superior rice varieties, especially japonica rice, low temperatures severely inhibit mesocotyl elongation during direct sowing in high-latitude, low-temperature regions, thus affecting the direct sowing results. For most rice varieties, mesocotyl elongation generally shows a trend of first increasing and then decreasing with gradually rising temperatures. Furthermore, studies have found that some long-mesocotyl varieties do not grow mesocotyls below 15℃, while under conditions of 15℃-30℃, mesocotyl elongation increases with temperature, but above 30℃, mesocotyl elongation is inhibited.

[0007] Therefore, identifying superior varieties adapted to high environmental temperatures through mesocotyl analysis is an effective means of breeding superior direct-seeded varieties that are not sensitive to temperature. Previous studies have shown that mesocotyl elongation at high environmental temperatures is mainly achieved through cell proliferation and cell elongation, with cell cycle-related genes playing a central role in this process. CDC48E (Cell Division Cycle 48) is a class of cell cycle-related proteins widely found in eukaryotes that inhibit tillering by suppressing cell elongation, but its function in the temperature-adaptive growth of rice remains unclear. Summary of the Invention

[0008] The purpose of this invention is to provide the rice gene CDC48E and its applications.

[0009] To achieve the objectives of this invention, in a first aspect, this invention provides the rice gene CDC48E, which is a gene encoding the following protein (a) or (b): (a) A protein consisting of the amino acid sequence shown in SEQ ID NO:2; or (b) A protein derived from (a) with the sequence shown in SEQ ID NO:2 substituted, deleted or added with one or more amino acids and having the same function.

[0010] The nucleotide sequence of gene CDC48E (including the promoter and 3'UTR sequence) is as follows: i) The nucleotide sequence shown in SEQ ID NO:1; ii) A nucleotide sequence of the nucleotide sequence shown in SEQ ID NO:1 that has been substituted, deleted and / or added with one or more nucleotides and expresses a protein with the same function; iii) A nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO:1 under stringent conditions and expresses a protein with the same function, wherein the stringent conditions are hybridization at 65°C in 0.1×SSPE containing 0.1% SDS or 0.1×SSC containing 0.1% SDS, followed by washing the membrane with the same solution; or, iv) Nucleotide sequences that have more than 90% homology with i), ii) or iii) and express the same functional protein.

[0011] Secondly, the present invention provides biological materials containing the gene, including but not limited to expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria, or transgenic cell lines.

[0012] Thirdly, the present invention provides the application of the gene or biological material containing the gene in regulating plant traits; The trait is selected from at least one of the following: adaptability to environmental temperature, plant type, and / or yield-related traits.

[0013] The application regulates the plant height, tiller number, grain plumpness, or 100-grain weight of the plant by inhibiting the expression of the gene CDC48E or the function of its encoded protein. Furthermore, by inhibiting the expression of the gene CDC48E or the function of its encoded protein, plant height is reduced and lodging resistance is enhanced at low ambient temperatures, without affecting the number of tillers, and grain plumpness and 100-grain weight are increased, thereby increasing potential yield.

[0014] Fourthly, the present invention provides a method for cultivating plants with improved temperature adaptability and / or yield traits, the method comprising: reducing or inhibiting the expression or function of the endogenous CDC48E gene in the plant or its cells.

[0015] Furthermore, the reduction or inhibition is achieved through gene editing technology or RNA interference technology.

[0016] In one specific embodiment of the present invention, the gene editing technology used is CRISPR / Cas9 technology, which achieves the editing of the gene by targeting the gRNA sequence of the endogenous CDC48E gene.

[0017] Preferably, the nucleotide sequence of the gRNA action site is: 5'-GTTGTCAGGAAGAACCTGAGGG-3' (for mutants) cdc48e -C and cdc48e -T design).

[0018] In this invention, the plant is a grass, preferably rice.

[0019] Fifthly, the present invention provides the application of transgenic plants obtained according to the method in plant breeding.

[0020] Breeding methods include, but are not limited to, transgenic, hybrid, backcross, self-cross, or asexual reproduction.

[0021] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: Rice is one of the most important food crops in my country, and its normal adaptive response to environmental temperature is the foundation for its normal growth, development, and maturation. This invention clones a key factor controlling rice's adaptation to high environmental temperatures. Functional studies of the CDC48E gene show that it is a positive regulator promoting plant growth in response to high environmental temperatures. cdc48e The mutant showed significantly inhibited mesocotyl elongation at 32℃, indicating that the CDC48E gene plays a crucial role in regulating temperature-dependent cell proliferation and elongation. This invention provides important guidance for breeding new rice varieties that are lodging-resistant, have high tillering, high yield, and strong temperature adaptability. This invention also provides methods for using the CDC48E gene to improve plant temperature adaptability, regulate plant architecture, and promote yield, providing genetic resources and an application basis for breeding rice varieties with high environmental temperature adaptability. Attached Figure Description

[0022] Picture 1 The following are the mesocotyl cytological phenotypes of ZH11 at 25°C and 32°C in a preferred embodiment of the present invention. (a) Longitudinal section phenotypes of ZH11 mesocotyls at 25°C and 32°C. Scale bar: 100 μm. (b) Cell length and cell number statistics. The number of mesocotyls counted was 3, and the cell number was ≥ 50. A t-test was used for significance analysis; different letters indicate statistically significant differences (…). p < 0.01).

[0023] Picture 2 In a preferred embodiment of the present invention, ZH11 and cdc48eMesocotyl phenotype of mutants. (a) ZH11 and mutants cdc48e -C and cdc48e -T Mesocotyl morphology at 25℃ and 32℃, with arrows indicating the location of the coleoptile knot, and a scale of 1 cm. (b) Statistical analysis of mesocotyl length, with >15 mesocotyls counted. Tukey's test was used for significance analysis, with different letters indicating statistically significant differences. p < 0.05).

[0024] Picture 3 The field phenotypes of mutants under different temperature conditions are shown in the preferred embodiments of the present invention. (a) and (d) ZH11 and mutants cdc48e -C and cdc48e -T Field phenotypes in Hainan (a) and Beijing (d) were measured on a scale bar of 20 cm. Plant height was counted in Hainan (b) and Beijing (e), with a count >15 plants. Tiller number was counted in Hainan (c) and Beijing (f), with a count >15 plants. The Tukey test was used for significance analysis; different letters indicate statistically significant differences. p < 0.05).

[0025] Picture 4 Statistical analysis of agronomic traits in Hainan Province in a preferred embodiment of the present invention. (a) ZH11 and mutants cdc48e -C and cdc48e -T Seed phenotypes were collected in Hainan, with a scale bar of 1 cm. Statistics were compiled on 100-seed weight (b), seed length (c), and seed width (d), with ≥5 counts for 100-seed weight and ≥20 counts for seed length and width. The Tukey test was used for significance analysis; different letters indicate statistically significant differences. p < 0.05).

[0026] Picture 5 Statistical analysis of agronomic traits in Beijing is presented in a preferred embodiment of the present invention. (a) ZH11 and mutants cdc48e -C and cdc48e -T Seed phenotypes were collected in Beijing, with a scale bar of 1 cm. Statistics were compiled on 100-seed weight (b), seed length (c), and seed width (d), with ≥5 counts for 100-seed weight and ≥20 counts for seed length and width. The Tukey test was used for significance analysis; different letters indicate statistically significant differences. p < 0.05). Detailed Implementation

[0027] This invention aims to provide a key gene for regulating the temperature-responsive growth of rice. CDC48E This invention provides genetic resources and a theoretical basis for breeding new rice varieties that are resistant to lodging, have high tillering, high yield, and strong temperature adaptability. Specifically, this invention provides a gene that regulates the rice's response to changes in environmental temperature (i.e., a gene that regulates the rice mesocotyl temperature-responsive growth). CDC48E (Cell Division Cycle 48) Its encoded protein and its applications. Specifically, it involves the utilization of... CDC48E Methods to regulate cell proliferation and elongation to promote the adaptive response of rice to high ambient temperatures.

[0028] The present invention adopts the following technical solution: The inventors successfully constructed and identified the cell cycle-related gene CDC48E (Cell Division Cycle 48) in the context of the japonica rice variety ZH11 using CRISPR / Cas9 gene editing technology, obtaining two allelic mutants with single nucleotide deletions leading to premature termination of protein coding. cdc48e -C and cdc48e -T Both mutants were invalid mutants that terminated prematurely, among which cdc48e -C The C base at position 295 of the CDS sequence is missing. cdc48e -T The base T at position 296 is missing. CDC48E The gene-encoded protein belongs to the AAA-ATPase family and plays a central role in eukaryotic cell cycle regulation and protein homeostasis maintenance. This invention is the first to clearly define... CDC48E It plays an important positive regulatory role in the growth response of rice to changes in environmental temperature.

[0029] Experimental results show that ( Picture 2 ), at 25℃ , cdc48e The mutant's mesocotyl is slightly shorter than that of the wild type, and at a high ambient temperature of 32°C, cdc48e The hypocotyl elongation was significantly inhibited in the mutant, indicating that... CDC48E It participates in maintaining the normal heat-induced growth of the mesocotyl and is a positive regulator of heat-responsive growth in rice, suggesting... CDC48E By regulating the cell cycle process in response to changes in ambient temperature.

[0030] For further evaluation CDC48E To investigate the impact of mutations on field traits of rice, the inventors conducted field planting experiments in Hainan (winter, growing season December to April of the following year) and Beijing (summer, growing season May to October) as a comparison between low ambient temperature (average temperature during the growing season approximately 22℃) and high ambient temperature (average temperature during the growing season approximately 32℃). The results showed ( Picture 3 , Picture 4 , Picture 5 Under low ambient temperature conditions (Hainan). cdc48e The mutant exhibited significantly reduced plant height, fuller grains (significantly increased grain length and weight), and higher 100-grain weight, demonstrating not only an ideal plant type but also improved crop yield. Under high ambient temperature conditions (Beijing), the mutant plant height was significantly greater than the wild type, and the number of tillers was significantly reduced. These results indicate... CDC48E It not only regulates the temperature-responsive growth of the mesocotyl in seedlings, but also affects the plant architecture and yield formation of mature plants.

[0031] Comprehensive analysis shows that moderately reducing CDC48E This function effectively inhibits excessive elongation under low ambient temperatures, reducing plant height and enhancing lodging resistance, while not affecting tiller number, and improving grain plumpness and 100-grain weight, thus increasing potential yield. Therefore, the CDC48E gene, while regulating temperature-dependent growth in rice, provides a new molecular target for improving ideal plant architecture and yield.

[0032] In a first aspect, this invention identifies key genes controlling the temperature-responsive growth of rice. CDC48E The gene encodes the protein shown in SEQ ID NO:2, or a protein derived from SEQ ID NO:2 with one or more amino acid substitutions, deletions, or additions, and having the same function as the protein shown in SEQ ID NO:2. This protein possesses a typical AAA-ATPase domain and participates in cell division, protein turnover, and temperature-dependent growth regulation.

[0033] Secondly, the present invention provides the nucleotide sequence of the CDC48E gene. In a preferred embodiment, the CDC48E gene is the nucleotide sequence shown in SEQ ID NO:1. Those skilled in the art will understand that, in a broader sense, CDC48E includes nucleotide sequences that have more than 90%, preferably more than 99%, homology with the sequence of SEQ ID NO:1 and encode a functionally equivalent protein.

[0034] Thirdly, the present invention provides a protein encoded by the CDC48E gene, said protein being any one of the following: (a) A protein consisting of the amino acid sequence shown in SEQ ID NO:2; (b) A protein derived from SEQ ID NO:2 with one or more amino acid substitutions, deletions or insertions, and having the same function as the amino acid sequence shown in SEQ ID NO:2.

[0035] In a preferred embodiment, the CDC48E protein regulates cell division, promotes tillering under high ambient temperatures, and inhibits plant height, thus having the potential to increase yield while enhancing lodging resistance.

[0036] Fourthly, the present invention provides a recombinant vector containing the CDC48E gene.

[0037] Preferably, the vector includes a gene editing vector, which may contain a specific gRNA sequence targeting the CDC48E CDS region to achieve functional attenuation or knockout.

[0038] Fifthly, the present invention provides a host cell comprising the above-mentioned recombinant vector, wherein the host cell may be selected from Escherichia coli, Agrobacterium, or plant cells for transformation.

[0039] Sixthly, the present invention provides CDC48E The application of genes in breeding crops with ideal plant architecture and temperature adaptability. Through regulation... CDC48E Expression can achieve the following physiological improvements: (1) At low ambient temperatures, the inhibition function maintains normal plant growth, avoids excessive elongation, and increases the number of spikes and potential yield. (2) Increased expression at high ambient temperatures can promote tillering and inhibit plant height, thereby enhancing lodging resistance; (3) Improve grain plumpness and 100-grain weight, and increase yield stability.

[0040] Seventhly, the present invention provides a method for cultivating rice with temperature adaptability and high yield and lodging resistance. This method includes regulating... CDC48E The expression level or function of genes can be transformed through Agrobacterium-mediated transformation or gene gun transformation, thereby obtaining rice varieties with moderate plant height, increased tillering, and higher yield.

[0041] This invention provides a theoretical and material basis for cultivating crop varieties with high environmental temperature adaptability. When the genes of this invention are used to improve the response of rice to high environmental temperature, the following methods can be used: (1) Constructing a gene targeting... CDC48E (1) A vector for gene editing; (2) Transforming the constructed vector into regenerable rice tissues or organs; (3) Cultivating the transformed tissues or organs into plants and screening for gene-mutant plants.

[0042] In summary, this invention is the first to clearly define rice. CDC48E Its crucial role in temperature-dependent growth and yield formation. Regulated through molecular means. CDC48E The expression of this gene provides new genetic resources and application pathways for breeding stable yields and lodging resistance under high environmental temperatures. Specifically, the technical solution adopted in this invention is: The present invention provides an isolated gene for controlling the adaptive response of rice to high ambient temperature, wherein the gene encodes the protein shown in SEQ ID NO:2, or encodes a protein derived from SEQ ID NO:2 by inserting, deleting or substituting one or more amino acids in the amino acid sequence shown in SEQ ID NO:2 and having the same function as the protein shown in SEQ ID NO:2.

[0043] Further, the nucleotide sequence of the gene is as shown in SEQ ID NO:1, or the gene is a nucleotide sequence that has more than 90%, preferably more than 99%, homology with the nucleotide sequence shown in SEQ ID NO:1 and encodes a protein with the same function.

[0044] The present invention also provides a protein encoded by the gene that controls the adaptive response of rice to high ambient temperature, which is the protein shown in SEQ ID NO:2, or a protein derived from SEQ ID NO:2 by inserting, deleting or substituting one or more amino acids in the amino acid sequence shown in SEQ ID NO:2 and having the same function as the protein shown in SEQ ID NO:2.

[0045] The present invention also provides a recombinant vector containing the said gene or a fragment thereof.

[0046] Furthermore, the recombinant vector is a plant expression vector, preferably a vector suitable for expression in rice.

[0047] The present invention also provides a host cell containing the gene or a fragment thereof, or containing the recombinant vector.

[0048] Furthermore, the host cell is selected from bacteria, fungi, or plant cells, preferably from Escherichia coli, Agrobacterium, or plant cells.

[0049] This invention also provides a method for cultivating plants with excellent high environmental temperature adaptability. The method includes using gene editing technology to design gene editing targets using the gene or its fragments as templates and constructing vectors, then transforming the plant cells or tissues, and culturing the transformed plant cells or tissues into… CDC48E Plants with functional deficiencies.

[0050] Furthermore, the transformation is carried out via Agrobacterium-mediated transformation or gene gun transformation.

[0051] In this invention, the plant is a grass, preferably rice.

[0052] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0053] Example 1: Phenotypic analysis of wild-type mesocotyls at different temperatures This invention first determined the temperature-sensitive cellular basis of the mesocotyl in the wild-type (ZH11) morphology using resin sections. Picture 1 This study aimed to provide clues for finding temperature-adapted long mesocotyl germplasm. Samples were prepared using the TECHNOVIT 7100 embedding resin kit. Mesocotyl seedlings grown at 25°C and 32°C for 4 days were sampled, collected, and processed as follows.

[0054] (1) Fixation of materials: Cut 2-3 mm of rice mesocotyl and place it in Carno fixative (ethanol to acetic acid volume ratio of 3:1) for 24-48 hours.

[0055] (2) Dehydration: Discard the fixative and treat the sample with 70%, 85%, 90% and 100% ethanol in sequence at room temperature for 30 minutes each time.

[0056] (3) Pre-permeation: Discard the anhydrous ethanol in the sample, add anhydrous ethanol and Technovit 7100 base solution in equal proportions, and let stand at room temperature for 1-2 hours.

[0057] (4) Penetration: Add 0.1 g of curing agent I to 10 mL of Technovit 7100 base solution as penetrating solution, add it to the sample, and let it stand at room temperature for 12-24 hours.

[0058] (5) Embedding: Cut off the caps of 1.5 or 2 mL centrifuge tubes to use as embedding molds. Add curing agent II to the permeation solution at a ratio of 15:1 and mix by inverting. Add 350 μL to each centrifuge tube cap. Gently place the sample into the centrifuge tube cap with tweezers and adjust the sample position as needed. Let it air dry at room temperature for 6-7 days.

[0059] (6) Slicing: Use a blade to remove the sample from the centrifuge tube cap and trim away any excess resin around the sample. Fix the sample onto the microtome and set the thickness to 4 micrometers.

[0060] (7) Spreading: Add a few drops of double-distilled water to the glass slide, place the cut resin slide on the water droplet to spread it, and place it on a 46°C baking machine to dry.

[0061] (8) Toluidine blue staining: First, prepare the toluidine blue staining solution by mixing 1 g of borax, 100 mL of double-distilled water and 1 g of toluidine blue, heating until dissolved, and then filtering with filter paper; place the glass slide with the resin attached in the staining solution and let it stand for 30 seconds, rinse off the staining solution on the surface of the glass slide with water, and then air dry at room temperature before observing under a microscope.

[0062] Statistical analysis revealed that the temperature-responsive elongation of the mesocotyl is achieved through cell proliferation and elongation.

[0063] Example 2 CDC48E Construction of mutants and mesocotyl phenotypic analysis The cellular basis of mesocotyl thermal elongation was determined using resin sections. Previous studies have reported that cell cycle-related genes are key genes in mesocotyl elongation; we hypothesize that CDC48E may be a gene involved in regulating the temperature response of the mesocotyl. CRISPR / Cas9 Technology Construction CDC48E Allelic mutants that cause premature termination of protein coding due to the deletion of two single nucleotides in a gene. cdc48e -C and cdc48e -T ,in cdc48e -C The C base at position 295 of the CDS sequence is missing. cdc48e -T The base T at position 296 is missing. The specific steps are as follows: (1) Primer design and dilution: Candidate CRISPR sites in the coding region of the target gene were predicted using the CRISPR-P website (http: / / crispr.hzau.edu.cn / CRISPR2 / ). Sequences located in the exon region, close to the 5' end, with high on-target efficiency scores and low off-target probabilities were selected as gene knockout targets. Vector adapters were selected based on the number of target sites in the vector, and primers containing the target sequence and vector adapters were designed (U6aT#-(5'-TCAGGTTCTTCCTGACAACCggcagccaagccagca-3') and gRT#+(5'-GTTGTCAGGAAGAACCTGAgttttagagctagaaat-3')). The primers were diluted in pairs to 1 μM.

[0064] (2) Construction of sgRNA expression cassette: a. First round of PCR: pYLgRNA-OsU6a(From Liu Yaoguang's laboratory at South China Agricultural University, see the literature A Robust CRISPR / Cas9 System for Convenient, High-Efficiency MultiplexGenome Editing in Monocot and Dicot Plants, Ma, Xingliang et al.) Molecular Plant Using the plasmid as a template (Volume 8, Issue 8, 1274-1284) (1:10 dilution), four primers were added to the same reaction. The final concentration of the outer primers UF (5'-CTCCGTTTTACCTGTGGAATCG-3') and gR-R (5'-CGGAGGAAAATTCCATCCAC-3') was 0.1 μM. The final concentration of the inner primers U6aT#-(5'-TCAGGTTCTTCCTGACAACCggcagccaagccagca-3') and gRT#+(5'-GTTGTCAGGAAGAACCTGAgttttagagctagaaat-3') containing the target sequence was 0.2 μM. Add 7.5 μL of 2 × Phanta Mix, 1.5 μL of UF + gR-R primer combination, 3 μL of U6aT#- + gRT#+ primer combination, and 0.5 μL of [unspecified substance] sequentially to a 15 μL reaction system. pYLgRNA-OsU6aTemplate, 2.5 μL ddH2O. After thorough mixing, place in a PCR instrument and react according to the following program: Step 1, denaturation at 95℃ for 30 seconds; Step 2, denaturation at 95℃ for 15 seconds, annealing at 58℃ for 15 seconds, extension at 72℃ for 20 seconds, for 28 cycles; Step 3, complete extension at 72℃ for 5 minutes. b. Second round PCR: Select primers for the second round PCR according to the number of expression cassettes used. Use Pps-GGL (5'-TTCAGAggtctcTctcgACTAGTATGGAATCGGCAGCAAAGG-3') and Pgs-GGR (5'-AGCGTGggtctcGaccgACGCGTATCCATCCACTCCAAGCTC-3') for the F and R ends of the primers, respectively, and dilute the primers in pairs to 1.5 μM. Add 15 μL of 2×Phanta Mix, 3 μL of Pps-GGL + Pgs-GGR primer combination, 1 μL of first-round PCR product (1:10 dilution), and 11 μL of ddH2O sequentially to a 30 μL reaction mixture. After thorough mixing, place the mixture in a PCR instrument and react according to the following program: Step 1: Denaturation at 95℃ for 30 seconds; Step 2: Denaturation at 95℃ for 15 seconds, annealing at 58℃ for 15 seconds, extension at 72℃ for 30 seconds, for 20 cycles; Step 3: Complete extension at 72℃ for 5 minutes. Perform agarose gel electrophoresis on 2 μL of the second-round PCR product to determine product size and specificity.

[0065] (3) Ligation of expression cassette to vector: The sgRNA expression cassette (nucleotide sequence of gRNA target site: 5'-GTTGTCAGGAAGAACCTGAGGG-3') was ligated to the vector using a cut-and-ligate method. pYLCRISPR / Cas9 The vector (from the laboratory of Liu Yaoguang, South China Agricultural University, see the literature A Robust CRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and DicotPlants, Ma, Xingliang et al.) Molecular Plant (Volume 8, Issue 8, 1274-1284). Add 1.5 μL of 10 × Cutsmart Buffer and 0.5 μL of 10 mM ATP sequentially to a 15 μL reaction system. pYLCRISPR / Cas9 0.5 μL of plasmid, 0.5 μL each of the sgRNA expression cassettes constructed in step (2), Bsa0.5 μL of I-HF enzyme (New England Biotechnology Co., Ltd.), 0.1 μL of T4 DNA ligase, and ddH2O to bring the total volume to 15 μL. The reaction was performed according to the following procedure: Step 1: 37℃ for 5 minutes, 10℃ for 5 minutes, 20℃ for 5 minutes, repeated 15 times; Step 2: 37℃ for 5 minutes.

[0066] (4) The ligation product was transformed into E. coli competent cells Trans T1, and the plasmid was extracted and sequenced for identification.

[0067] (5) After the vector construction is completed, transgenic plants are obtained through seed sterilization, callus induction, Agrobacterium infection, screening and differentiation.

[0068] (6) Further positive plant identification, homozygous plant isolation and propagation were carried out on the obtained transgenic plants in the laboratory and field (Hainan, Beijing).

[0069] Mesocotyl phenotype analysis of mutants at 25℃ and 32℃ ( Picture 2 ),Discover cdc48e The mutation significantly suppressed the length of the mesocotyl, and the inhibitory effect was more pronounced at a high ambient temperature of 32℃, indicating that... CDC48E It is a positive factor that regulates the thermal response elongation of the mesocotyl.

[0070] Example 3 Field phenotypic analysis of mutants under different temperature conditions In order to determine cdc48e The field phenotypes in response to temperature were observed in field sowing trials conducted in Beijing during summer (high ambient temperature) and Hainan during winter (low ambient temperature). The results showed ( Picture 3 In Hainan (low ambient temperature), the mutant plant height was significantly dwarfed, while the number of tillers did not change significantly; in Beijing (high ambient temperature), the mutant plant height was significantly increased, while the number of tillers was significantly reduced. This indicates... CDC48E It is a temperature regulator that inhibits plant height and increases tillering under high ambient temperatures, and plays an important role in regulating the ideal plant type of rice and increasing yield.

[0071] Example 4 Statistical analysis of agronomic traits of mutants under different temperature conditions In Hainan (winter, low ambient temperature) Picture 4 ) and Beijing (summer, high ambient temperature) Picture 5 Wild-type and mutant strains were planted separately. Results showed that under Hainan conditions, the mutant strain exhibited significantly increased grain length, grain width, and 100-grain weight; under Beijing conditions, the mutant strain showed only a slight increase in grain length, grain width, and 100-grain weight. (Allelic...) cdc48e -C The grain length and 100-grain weight were significantly increased compared to the wild type. This indicates that... CDC48E It participates in the regulation of agronomic traits of rice in response to temperature changes.

[0072] Example 5 Application Prospects In areas with high ambient temperatures or under summer planting conditions, moderately promote low... CDC48E Its function can inhibit excessive elongation, significantly reduce plant height, thereby enhancing the plant's resistance to lodging, reducing mechanical losses and grain shedding risk under high ambient temperatures, and ensuring population stability and harvest efficiency. In low- or medium-temperature regions, CDC48E Inhibition can suppress plant height and promote grain filling, increase the number of ears and the weight of 100 grains, thereby increasing the overall yield.

[0073] Through the CDC48E Precise control of functions can optimize crop plant type and yield according to the climate characteristics of the cultivation area.

[0074] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

[0075] References: [1] H. Damon Matthews, Seth Wynes, Current global efforts are insufficient to limit warming to 1.5℃.Science376,1404-1409(2022). [2] Schauberger, B., Archontoulis, S., Arneth, A., Balkovic, J.,Ciais, P., Deryng, D. (2017). Consistent negative response of US crops to hightemperatures in observations and crop models. Nat. Commun. 8, 13931. [3] C., S. Piao, [4] Carl J. Bernacchi, Ursula M. Ruiz-Vera, Matthew H. Siebers,Nicholas J. DeLucia, Donald R. Ort. (2023). Short- and long-term warming events on photosynthetic physiology, growth, and yields of field grown crops. Biochem J. 480 (13): 999–1014. [5] Yi Kan, Xiao-Rui Mu, Jin Gao, Hong-Xuan Lin, Youshun Lin. (2023). The molecular basis of heat stress responses in plants. Molecular Plant. 16(10), 1612-1634. [6] Hu Weian (2020). Significance and measures for promoting direct seeding cultivation technology of rice. World Tropical Agriculture Information. 12, 10-11. [7] Wang Yong (2020). High-yield direct seeding cultivation technology for rice. Chinese Agricultural Abstracts - Agricultural Engineering. 32, 78-79. [8] Chen Dong, Mao Bigang, Peng Yan, Shao Ye, Hu Yuanyi, Wu Tianhao, Zhao Bingran (2018). Research progress on the mechanism of mesocotyl elongation in rice. Hybrid Rice 33, 1-6. [9] Yin Biwen, Cao Minglong, Tang Shuju (2020). Advantages, disadvantages and cost analysis of different rice planting methods. Modern Agricultural Science and Technology. 12, 59-60.

[10] Gray WM, Ostin A, Sandberg G, Romano CP, Estelle M (1998) Hightemperature promotes auxin-mediated hypocotyl elongation in Arabidopsis. ProcNatl Acad Sci USA 95:7197–7202.

[11] Chen XW, Yang L, Zhang Y, Gong JF (2005) Effects of lowtemperature at 10 ℃ on some antioxidant enzyme activities andultrastructures of hypocotylar cells in mung bean and garden pea. J PlantPhysiol Mol Biol 31:539–544.

[12] Radford BJ, Henzell RG (1990) Temperature affects the mesocotyland coleoptile length of grain sorghum genotypes. Aust J Agric Res 41:79–87.

[13] Shi L, Zhang XB, Shi YF, Xu X, He Y, Shao G, Huang QN, Wu JL.OsCDC48 / 48E complex is required for plant survival in rice ( Oryza sativa L. ).Plant Mol Biol. 2019 May;100(1-2):163-179。

Claims

1. Rice gene CDC48E, characterized in that, It is a gene encoding either (a) or (b) the following protein: (a) A protein consisting of the amino acid sequence shown in SEQ ID NO:2; or (b) A protein derived from (a) with the sequence shown in SEQ ID NO:2 substituted, deleted or added with one or more amino acids and having the same function.

2. A biomaterial containing the gene of claim 1, characterized in that, The biological materials are expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria, or transgenic cell lines.

3. The application of the gene of claim 1 or the biological material of claim 2 in regulating plant traits; The trait is selected from at least one of the following: adaptability to environmental temperature, plant type, and / or yield-related traits.

4. The application according to claim 3, characterized in that, The application regulates the plant height, tiller number, grain plumpness, or 100-grain weight of the plant by inhibiting the expression of the gene CDC48E or the function of its encoded protein. Furthermore, by inhibiting the expression of the gene CDC48E or the function of its encoded protein, plant height is reduced and lodging resistance is enhanced at low ambient temperatures, without affecting the number of tillers, and grain plumpness and 100-grain weight are increased, thereby increasing potential yield.

5. A method for cultivating plants with improved temperature adaptability and / or yield traits, characterized in that, The method includes: reducing or inhibiting the expression or function of the endogenous CDC48E gene in a plant or its cells.

6. The method according to claim 5, characterized in that, The reduction or inhibition is achieved through gene editing technology or RNA interference technology.

7. The method according to claim 6, characterized in that, The gene editing technology is CRISPR / Cas9 technology, which achieves the editing of the gene by targeting the gRNA sequence of the endogenous CDC48E gene.

8. The method according to claim 7, characterized in that, The nucleotide sequence of the gRNA action site is: 5'-GTTGTCAGGAAGAACCTGAGGG-3'.

9. The method according to any one of claims 5-8, characterized in that, The plant in question is a grass family plant, with rice being the preferred variety.

10. The use of transgenic plants obtained by the method according to any one of claims 5-9 in plant breeding; Breeding methods include transgenic, hybridization, backcrossing, self-pollination, or asexual reproduction.

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