Application of the rice high ambient temperature response gene OsTOGR4

Abstract

This invention discloses a rice high ambient temperature response gene. OsTOGR4 Applications. This invention identifies for the first time a key factor regulating the growth and development of rice plants under high environmental temperatures, and discloses the method of mutation... OsTOGR4 This research aims to develop methods for genetically breeding rice varieties adapted to high environmental temperatures, thereby providing genetic resources and a research foundation for cultivating superior rice varieties adapted to high environmental temperatures.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and more specifically, to a rice high-temperature response gene. OsTOGR4 Applications. Background Technology

[0002] Due to the greenhouse effect, global temperatures are rising continuously, and the climate is constantly changing. Domesticated crops have lost many superior genes inherited from their wild ancestors, resulting in reduced genetic diversity and making them less adaptable to changing natural environments, thus restricting agricultural production. From 2000 to 2009, sorghum and soybean yields in West Africa lost 10-20% and 5-15% respectively due to climate change (Sultan et al., 2019). Affected by temperature, global wheat, rice, maize, and soybean yields will decrease by 6%, 3.2%, 7.4%, and 3.1% respectively if the global average temperature rises by 1°C (Zhao et al., 2017). Temperature change affects food yields, and studying the relationship between plant development and temperature is of significant practical importance for breeding stable and high-yielding crop varieties.

[0003] Plant morphology changes under varying environmental temperatures (Casal and Balasubramanian, 2019), and the series of morphological changes that occur from temperatures below the heat stress point to increases with rising temperatures are collectively referred to as thermomorphogenesis. In Arabidopsis thaliana, the hypocotyl grows rapidly at high temperatures to protect the cotyledons from the warmer soil; the petioles also elongate, and the internodes increase, resulting in a looser plant structure that helps the plant dissipate heat and adapt to the environment (Quint et al., 2016). Rice plant height elongates with increasing environmental temperature (Krishnan et al., 2011), which is a core factor influencing rice plant architecture and yield (Liao et al., 2019). However, to date, research on the molecular mechanisms by which rice regulates plant height in response to environmental temperature is scarce. Summary of the Invention

[0004] The purpose of this invention is to provide a rice high-temperature response gene. OsTOGR4 Applications.

[0005] To achieve the objectives of this invention, in a first aspect, this invention provides a rice high-environment temperature response gene. OsTOGR4 Application in regulating plant sensitivity to high ambient temperatures (i.e., application in regulating plant growth sensitivity to high ambient temperatures).

[0006] In this invention, genes OsTOGR4 The gene encoding either (a) or (b) the following protein:

[0007] (a) A protein consisting of the amino acid sequence shown in SEQ ID NO:2; or

[0008] (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.

[0009] Furthermore, the plant includes grasses, preferably rice.

[0010] Furthermore, by studying the genes in rice that respond to high ambient temperatures... OsTOGR4 The gene was modified to lose its function, thereby reducing the rice's sensitivity to high ambient temperatures (i.e., reducing the rice's growth sensitivity to high ambient temperatures).

[0011] Genome editing technologies, such as CRISPR, TALEN, and ZFN, can be used to edit genes. OsTOGR4 To carry out the renovation.

[0012] Secondly, this invention provides a method for reducing the sensitivity of rice to high ambient temperatures, the method comprising: using genetic engineering techniques to weaken rice genes that respond to high ambient temperatures. OsTOGR4 .

[0013] Furthermore, with genes OsTOGR4 Using CRISPR / Cas9 as the target, a CRISPR / Cas9-based sgRNA sequence was designed. A DNA fragment containing the sgRNA sequence was ligated into a vector carrying CRISPR / Cas9, and rice was transformed or transfected to obtain transgenic rice with the gene function lost.

[0014] Preferably, the original vector for CRISPR / Cas9 was constructed and provided by Baige Gene Technology Co., Ltd., with the serial number SG11588.

[0015] Preferably, the nucleotide sequence of the sgRNA action site is 5'-TGGCACACCGCTGTTAG-3'.

[0016] Furthermore, the transformation or transfection can be performed using Agrobacterium-mediated transformation or gene gun transformation.

[0017] Thirdly, the present invention provides the application of transgenic rice obtained according to the method in plant breeding.

[0018] Furthermore, breeding methods include, but are not limited to, transgenic, hybridization, backcrossing, self-pollination, or asexual reproduction.

[0019] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0020] This invention identifies for the first time a key factor regulating the growth and development of rice plants under high environmental temperatures, and discloses a method for controlling this growth by knocking out... OsTOGR4 Methods to reduce the sensitivity of rice to high ambient temperatures through gene therapy provide genetic resources and a research foundation for breeding superior rice varieties adapted to high ambient temperatures. Attached Figure Description

[0021] Figure 1 In a preferred embodiment of the present invention togr4 Phenotypic comparison with wild-type Zhonghua 11. (a) Mutant togr4 (a) Field phenotypic statistics of wild-type ZH11 in Lingshui, Beijing and Yangzhou. (b) Statistics on seedling height after 2 weeks of growth under different temperature conditions. In the bar charts of (a) and (b), the black bars represent ZH11 and the gray bars represent mutants. togr4 , Numerical values ​​are expressed as mean ± standard error (n ≥ 10), and the scale is 10 cm.

[0022] Figure 2 In a preferred embodiment of the present invention togr4 Cell phenotypes of wild-type Zhonghua 11 at high ambient temperatures. (a) WT and togr4 (b) Longitudinal cellular images of the inverted intersegment at high ambient temperature, scale bar at 100 μm. togr4 Transverse section of tissue in the inverted intersegment at high ambient temperature, scale bar 100 μm. (c) WT and togr4 Leaf epidermal cells at high ambient temperatures. Scale bar is 100 μm. Corresponding charts include: width of the first internode, stem thickness, length, width, area, and total number of cells in the first internode, leaf length, and stomatal cell length. Values ​​are expressed as mean ± standard error; significant differences are indicated as statistically significant. .

[0023] Figure 3 Subcellular localization of proteins OsFIE2 and togr4 in a preferred embodiment of the present invention. (a) Subcellular localization of protein OsFIE2 in rice root cells. The figures show microscopic observations of the roots of the pUb::GFP overexpressing wild-type material (top) and the pUb::OsFIE2-GFP complementary togr4 mutant transgenic line (bottom). Scale bar length: 50 μm. (b) Subcellular localization in rice protoplasts. The fusion protein OsbZIP52-mCherry is used as a nuclear localization marker. Scale bar length: 10 μm. (c) Subcellular localization in tobacco leaves. Scale bar length: 50 μm.

[0024] Figure 4 In a preferred embodiment of the present invention OsTOGR4Gene localization and candidate gene identification. (a) togr4 Phenotypic characteristics of wild-type ZH11 and its F1 hybrids in Yangzhou, scale bar 10 cm. (b) OsTOGR4 Linkage diagram. M1 to M7 are molecular markers, shown in the figure. OsTOGR4 Candidate genes, with black rectangles representing exons and connecting lines representing introns. (c) Recombination frequency of each molecular marker in the linkage map.

[0025] Figure 5 In a preferred embodiment of the present invention OsTOGR4 Gene overexpression vectors.

[0026] Figure 6 In a preferred embodiment of the present invention OsTOGR4 Genes influence certain phenotypes in plant thermomorphogenesis. (a) WT, togr4 The phenotypic characteristics of the complementary materials were observed in the field in Yangzhou, with a scale bar of 10 cm. (b) WT, togr4 and togr4 / OsFIE2 Seedling phenotypes of complementary materials at different culture temperatures, scale bar 10 cm.

[0027] Figure 7 In a preferred embodiment of the present invention togr4 Compared with wild-type medium-flowered 11 OsFIE2 Transcriptional expression analysis of genes. (a) OsFIE2 Transcriptional levels in different rice tissues. (b) Transcriptional levels in leaves, leaf sheaths, and aboveground tissues of rice seedlings at different temperatures over two weeks. OsFIE2 Transcriptional level analysis. Data in (a) and (b) are presented as mean ± standard error, n=3. (By gene) Actin As an internal control, data analysis was performed using the Tukey HSD test (p<0.05), with different letters indicating significant differences. (c) Expression analysis of OsFIE2 protein in the aboveground parts of rice seedlings after two weeks of different temperature treatments. (d) Protein expression analysis of OsFIE2 after heat treatment. OsFIE2-GFP seedlings grown at 25℃ for two weeks were transferred to 35℃ for heat treatment. The upper part of (c) and (d) are protein immunoblotting images, and the lower table is the grayscale analysis of protein expression, with Actin protein as an internal control. Three experiments were replicated, and data analysis was performed using Student's test. .

[0028] Figure 8 This is an example of using CRISPR / Cas9 technology to alter the sensitivity of rice to high ambient temperatures. Among them... osfie2- CRISPR is generated using CRISPR / Cas9 technology. OsFIE2Allelic mutants. (a) Gene OsFIE2 Schematic diagram of allelic mutations. Black boxes represent exons, and triangularly connected segments indicate the mutation sites of the allelic mutants on that gene. Red boxes show the sequencing peaks of the mutation sites. (b) Field plant height and tillering of the two allelic mutant forms. Data n≥15. (c) Phenotypes of the two allelic mutants under temperature treatment. Scale bar length 10 cm, n≥16. Data in (b) and (c) are expressed as mean ± standard error. Data analysis was performed using one-way ANOVA, and significant differences were indicated by asterisks. . Detailed Implementation

[0029] This invention provides a rice gene that responds to changes in environmental temperature. OsTOGR4 ( Thermotolerant Growth Required 4 The invention also provides the gene encoding a protein and functional analogues, a vector containing the nucleotide sequence of the gene, and a host cell containing the nucleotide sequence of the gene or the vector. Furthermore, the invention provides a gene for reducing the sensitivity of rice to high ambient temperatures. OsTOGR4 The method of developing rice varieties that can grow phenotypes close to those at normal temperatures under high environmental temperatures is expected to reduce problems such as lodging and brittle stems caused by rapid growth of rice under high environmental temperatures.

[0030] Using ethyl methanesulfonate (EMS) mutagenesis and genetic screening, a rice plant height mutant that exhibits morphological changes in response to high environmental temperatures was isolated and identified. OsTOGR4 ( Thermotolerant Growth Required 4 ).when togr4 When grown under low ambient temperature conditions (e.g., planted in Hainan from December to April of the following year), its morphological characteristics are very close to those of the wild type; however, when grown under higher ambient temperature conditions (e.g., planted in Yangzhou from May to September), its growth is significantly inhibited, exhibiting a dwarf phenotype. To verify the phenotype, an artificial incubator environment was used to simulate the growth environment. By statistically analyzing the growth phenotypes of rice materials at different temperatures, the morphological characteristics were further confirmed. togr4 Phenotypic response to ambient temperature. Based on the preliminary establishment of the phenotype, key genes regulating the response of rice to changes in ambient temperature were located and cloned through phenotypic screening supplemented by molecular marker identification. OsTOGR4 Phenotypic analysis of the mutant and genetic complementation experiments demonstrated the main function and associated phenotype of the gene. Biochemical experiments determined... OsTOGR4 The content of the protein encoded by the gene shows a decreasing trend with increasing environmental temperature. Based on these experiments, this invention identifies for the first time a seedling height-sensitive gene that regulates the response of rice to high environmental temperatures. OsTOGR4 This gene encodes the OsFIE2 subunit of the rice PRC2 protein complex.

[0031] PRC2 is a polycomb group (PcG) protein composed of four core subunits and several accessory subunits. It regulates chromatin openness by methylating histone H3 at position K27, thus modulating gene silencing and activation at the epigenetic level. It plays a crucial role in early embryonic development, cancer regulation, plant growth and development, and environmental responses in animals (Goodrich et al., 1997; Mozgova and Hennig, 2015; Baileet et al., 2022). In rice, PRC2 contains the methyltransferase subunit OsEZ1, which can trimethylate the Lys residue at position 27 of histone H3, thereby enabling epigenetic regulation. OsFIE2 is one of the core subunits of rice PRC2. It interacts with EZ1 and affects the activity of histone H3 methyltransferase in PRC2, regulating rice plant height, seed development, and grain filling (Nallamilli et al., 2013). It also has an additive effect on the synthesis of some storage proteins and the expression of photosynthetic genes (Liu et al., 2016; Cheng et al., 2020). OsFIE2 The homozygous mutation is lethal (Cheng et al., 2020), and its reduced expression leads to smaller rice seeds, insufficient grain filling, and some seeds failing to achieve normal dormancy (Nallamilli et al., 2013). Mutants osfie2-1 The number of longitudinal cells in the internodes is reduced, the cell layer on the internode side is significantly thinned, and the number and size of large and small vascular bundles, as well as the number and size of cells in the glume, are all decreased (Liu et al., 2016), exhibiting phenotypes such as dwarfing, reduced seed setting rate, floral organ defects, and smaller grains. However, to date, no research has shown that OsFIE2 is involved in the temperature response of rice.

[0032] The core subunit OsTOGR4 / OsFIE2 of the rice PRC2 protein complex identified in this invention participates in the regulation of rice growth and development in response to high ambient temperatures. Normal expression and protein content of this gene ensure normal growth of rice under high ambient temperatures.

[0033] The present invention adopts the following technical solution:

[0034] In a first aspect, this invention identified a plant height gene in rice in response to high ambient temperature. OsTOGR4The gene encodes the protein shown in SEQ ID NO:2, or a protein derived from SEQ ID NO:2 that encodes the amino acid sequence shown in SEQ ID NO:2 with one or more amino acid substitutions, deletions, or additions and has the same function as the protein shown in SEQ ID NO:2. The protein shown in SEQ ID NO:2 consists of 376 amino acids.

[0035] The rice plant height gene in response to high ambient temperature OsTOGR4 The sequence is a separated nucleotide sequence. In a preferred embodiment, the gene controlling the rice's response to high ambient temperatures... OsTOGR4 The nucleotide sequence is shown in SEQ ID NO:1 (the sequence shown in SEQ ID NO:1 also includes a promoter and a 3'UTR sequence). Furthermore, those skilled in the art should understand that, in a broader sense, the rice plant height gene in response to high ambient temperatures... OsTOGR4 The nucleotide sequence 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.

[0036] Secondly, the present invention provides a gene controlling the response of rice to high ambient temperatures. OsTOGR4 The encoded protein is either (a) or (b) below:

[0037] (a) A protein consisting of the amino acid sequence shown in SEQ ID NO:2;

[0038] (b) A protein derived from SEQ ID NO:2 by substituting, deleting or adding one or more amino acids of the amino acid sequence shown in SEQ ID NO:2 and having the same function as the protein shown in SEQ ID NO:2.

[0039] In a preferred embodiment, the gene controlling the rice's response to high ambient temperatures... OsTOGR4 The encoded protein is an isolated protein with the amino acid sequence shown in SEQ ID NO:2.

[0040] Thirdly, the present invention provides a gene comprising the aforementioned gene controlling the response of rice to high ambient temperatures. OsTOGR4 Recombinant carriers.

[0041] Preferably, the recombinant vector contains a foreign nucleotide fragment encoding the amino acid sequence shown in SEQ ID NO:2. More preferably, the recombinant vector contains a foreign nucleotide fragment as shown in SEQ ID NO:1.

[0042] In one embodiment, the plasmid used to construct the recombinant vector may be selected from, but is not limited to, [selections from] [other sources]. pCAMBIA1300 .

[0043] Host cells containing the recombinant vector are also within the scope of this invention. Host cells containing the recombinant vector can be obtained by transforming or transfecting the recombinant vector into cells, for further applications such as amplifying expression vectors, expressing the protein, or obtaining transgenic plants. Cells used for transformation or transfection can be selected from, but are not limited to, bacterial cells, such as Escherichia coli or Agrobacterium cells, fungal cells, such as yeast cells, or plant cells, such as rice cells.

[0044] Fourthly, this invention provides a gene for regulating the high environmental temperature sensitivity of rice. OsTOGR4 Its use in breeding rice varieties that can respond normally and grow under high ambient temperatures.

[0045] Fifthly, the present invention provides a method for breeding rice varieties that can respond and grow normally under high ambient temperatures, the method comprising reducing... OsTOGR4 The content of gene-encoded proteins. Decrease. OsTOGR4 The method for determining the content of gene-encoded proteins can be achieved through the following approach: utilizing... CRISPR / Cas9 Gene editing technology OsTOGR4 Genes are knocked out.

[0046] OsTOGR4 Genes and OsTOGR4 Transformation or transfection of gene editing vectors can be performed using Agrobacterium-mediated transformation or gene gun methods.

[0047] This invention provides a theoretical and material basis for cultivating crop varieties that reduce sensitivity to high ambient temperatures and improve adaptability to high ambient temperatures. When the genes of this invention are used to improve the response of rice to high ambient temperatures, the following methods can be used: (1) Constructing a system for... OsTOGR4 (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 plants that have mutated genes.

[0048] Furthermore, the present invention provides an isolated gene that regulates the sensitivity 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 from the amino acid sequence shown in SEQ ID NO:2 and having the same function as the protein shown in SEQ ID NO:2.

[0049] Furthermore, it 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.

[0050] Furthermore, it is the nucleotide sequence shown in SEQ ID NO:1.

[0051] The present invention also provides the gene OsTOGR4 The encoded isolated protein controlling the high environmental temperature sensitivity of rice 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.

[0052] Furthermore, the protein is the protein shown in SEQ ID NO:2.

[0053] The present invention also provides a product containing the said gene. OsTOGR4 Or a recombinant vector of its fragments.

[0054] Furthermore, the recombinant vector is a plant expression vector, preferably a vector suitable for expression in rice.

[0055] Preferably, the recombinant vector is composed of plasmids. pCAMBIA1300 Build.

[0056] Furthermore, the plant expression vector also contains OsTOGR4 The promoter or Ubiquitin promoter, and the gene sequence encoding the tag protein, such as GFP.

[0057] Furthermore, the host cell is selected from bacteria, fungi, or plant cells, preferably *Escherichia coli* (E. coli). Escherichia coli Agrobacterium ( ) Agrobacterium tumefaciens (or plant cells)

[0058] This invention also provides a method for cultivating plants with excellent high environmental temperature adaptability and insensitivity, the method comprising using gene editing technology to modify the genes... OsTOGR4 Or, using fragments of the gene as templates, design gene editing targets and construct vectors, then transform the cells or tissues of the plant, and cultivate the transformed plant cells or tissues into... OsTOGR4 Functional deficiencies and reduced sensitivity of rice to high ambient temperatures.

[0059] Furthermore, the transformation is carried out via Agrobacterium-mediated transformation or gene gun transformation.

[0060] Furthermore, the plant is a grass family plant, preferably rice.

[0061] The present invention also provides the gene. OsTOGR4 Or the application of the proteins that regulate plant response to high ambient temperature in the cultivation of highly temperature-adapted plants with excellent low sensitivity to high ambient temperature.

[0062] The present invention also provides a method for cultivating rice with excellent high environmental temperature adaptability and low sensitivity to high environmental temperature. The method includes transforming the cells or tissues of the target plant using a host cell containing the plant gene editing vector, and cultivating the transformed cells or tissues to obtain a transgenic plant with the polynucleotide sequence edited.

[0063] Furthermore, the method includes transforming rice cells or tissues using the recombinant vector and cultivating the transformed cells or tissues into plants that overexpress a gene controlling the high environmental temperature sensitivity of rice.

[0064] Furthermore, the transformation is carried out via Agrobacterium-mediated transformation or gene gun transformation, and the plant includes grasses, preferably rice.

[0065] 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.

[0066] Example 1: Isolation of genes controlling high environmental temperature sensitivity in rice

[0067] First, a mutant that exhibits dwarfing under high environmental temperatures was screened from the EMS (Ethylmethylsulfone)-mutated ZH11 japonica rice mutant library. togr4 ( Figure 1 (a). To further determine whether this mutant is sensitive to changes in ambient temperature, we cultured it in the dark for 14 days under three temperature conditions: 25°C / 20°C (25°C for 12 hours and 20°C for 12 hours, simulating day-night cycles), 30°C / 25°C, and 35°C / 30°C. Phenotypic statistical analysis revealed that the seedling height of this mutant was significantly lower than that of the wild type at high ambient temperatures, while the length was similar to that of the wild type at low ambient temperatures, indicating that the seedling height of this mutant is hypersensitive to high ambient temperatures. Figure 1 (b) To further investigate whether the elongation of rice plant height is caused by cell division or cell elongation, we performed resin section analysis on cells from the first internode of the wild type and the mutant. The results showed that the mutant had significantly smaller cells, a significantly lower total cell count, significantly thinner stems, significantly shorter plants, significantly shorter leaf length at the seedling stage, and significantly shorter stomatal length on leaf epidermal cells.Figure 2 (a~c).

[0068] Example 2: Cellular localization of genes controlling high environmental temperature sensitivity in rice

[0069] To explore the cellular function of the protein OsFIE2, we performed subcellular localization experiments on OsFIE2. This was achieved by analyzing the overexpressing transgenic lines... pUb::OsFIE2-GFP Electron microscopy of root tip cells revealed that the expression signal of OsFIE2-GFP protein was distributed in the cytoplasm and nucleus of the cells. Figure 3 (a) To further observe whether the mutated protein togr4 affects subcellular localization, we will carry... OsFIE2-GFP and togr4-GFP The gene was transiently transformed into rice protoplasts using two plasmids. Figure 3 b) and tobacco leaf cells ( Figure 3 In (c), electron microscopy showed that the expression signal of the mutated togr4 protein was still distributed in the cytoplasm and nucleus. These results indicate that OsFIE2 functions in both the cytoplasm and nucleus of the cell, and the localization of the mutated togr4 protein remained unchanged.

[0070] Example 3: Genetic mapping of genes controlling high environmental temperature sensitivity in rice

[0071] In order to study the cause OsTOGR4 The molecular level reasons for phenotypes, we will togr4 It was hybridized with ZH11 and F1 generation plants were constructed. Figure 4 a) F2 generation population was obtained from F1 generation plants, and then the populations of different generations were statistically analyzed. togr4 The number of individual plants with phenotypes similar to ZH11. Based on the diversity of restriction fragment lengths between japonica and indica rice, SSR molecular markers were used to... togr4 Preliminary localization of candidate mutant genes ( Figure 4 (b) Map-based clonal populations are linked on chromosome 8 of rice. Analysis of recombination rates of molecular markers on chromosome 8 revealed significantly reduced recombination efficiency at markers M2 and M3. Figure 4 c) suggests a high degree of linkage in the region between these two molecular markers, thus initially locating the candidate mutant gene to the segment between M2 and M3 on chromosome 8. Since there are approximately 291 genes (1.96 Mb) between M2 and M3, to confirm the mutant gene, we... togr4The entire genome was resequencing and compared with the wild-type genome, revealing only two missense mutation sites between the M2 and M3 regions. One is LOC_Os08g05450, where amino acid 176 is mutated from Ala to Thr. However, since its encoded protein is a transposon and this gene segment is also present on multiple other chromosomes, it is not considered a candidate gene. The other candidate mutant gene is LOC_Os08g04270, where a base substitution occurs at position 419 of the CDS sequence, changing from C to T, resulting in a mutation at position 140 of its encoded protein from threonine (Thr) to isoleucine (Ile). Based on these results, we consider LOC_Os08g04270 as a candidate gene. togr4 The candidate mutant gene encodes an endosperm autogenous gene that is independent of fertilization. OsFIE2 (fertilization-independentendosperm gene 2).

[0072] Example 4 OsTOGR4 Construction of complementary vectors and rice genetic transformation

[0073] From wild-type medium-flower 11 OsTOGR4 The complete gene, which is the nucleotide sequence shown in SEQ ID NO:1 (including its own promoter sequence, genome sequence, and 3' untranslated region sequence), was amplified by DNA polymerase using PCR, and then ligated by restriction enzymes. pCAMBIA1300 Complementary vectors were obtained from the multiple cloning site of the plasmid (purchased from CAMBIA). pUb:: OsTOGR4 ( Figure 5 ).

[0074] Transform the aforementioned constructed complementary carrier to E.coli Positive clones were selected using kanamycin in DH5α competent cells. Plasmids were extracted and sequenced to identify the clones in the vector. OsTOGR4 A sequence-complete positive clone was obtained, and then the plasmid of this positive clone was electrotransformed into EHA105 Agrobacterium competent cells (prepared using conventional methods, referring to *Plant Genetic Engineering*, Wang Guanlin and Fang Hongyun, Science Press, 2nd edition, 2004). Next, the successfully transformed clone was infected with Agrobacterium using... OsTOGR4 Transgenic manipulation is performed on the recipient.

[0075] The procedure for genetic modification is as follows:

[0076] (1) Seed sterilization: Weigh 20-30 g of dried rice seeds, remove the seed coat using a small dehulling machine in the laboratory, soak the dehulled seeds in 70% ethanol for 1 minute, then soak in 30% sodium hypochlorite (the stock solution is 10% available chlorine, add 1 drop of Tween-20 per 50 mL) for 30 minutes, and gently shake on a shaker. Then rinse with sterile water 5-6 times; disinfect once with 30% sodium hypochlorite (without Tween-20) and rinse with sterile water 5-6 times. Transfer the seeds to filter paper in the ultra-clean workbench to air dry.

[0077] (2) Inducing callus: Transfer the seeds to the pre-prepared N6D medium and then culture them in a light incubator at 32℃ and 24 hours of continuous light for 5-7 days until the seeds grow golden yellow callus.

[0078] (3) Agrobacterium infection: Infecting Agrobacterium containing specific plasmids EHA105 Streak the culture onto YEB medium containing rifampicin (Rif 25-50 mg / L) and kanamycin (Kan 50 mg / L), then incubate in the dark at 28°C for 2-3 days. Pick single clones and transfer them to 5-6 mL of liquid YEB medium containing the same antibiotics, then incubate overnight at 28°C (220 rpm) with shaking. Inoculate the bacterial culture at a 1:100 ratio into 50 mL of AAM medium and incubate overnight at 28°C and 220 rpm with shaking until OD (dose elapsed). 600 The concentration is approximately 0.1. Soak healthy callus in Agrobacterium tumefaciens solution for 2 minutes, then quickly remove the callus with tweezers and place it on sterile filter paper to air dry. After the callus has dried, transfer it to N6D-As medium pre-soaked in sterile filter paper (AAM soaking). Wrap the culture dish containing the callus with sealing film and aluminum foil and incubate in the dark at 25°C for 2-2.5 days.

[0079] (4) Screening and Differentiation: First, rinse the callus co-cultured with Agrobacterium tumefaciens 3-4 times in sterile double-distilled water, then rinse 2-3 times with sterile double-distilled water containing carbenicillin (500 mg / L) before soaking for 30 minutes each time, repeating 3-5 times to thoroughly clean the Agrobacterium tumefaciens from the callus surface. After washing, blot the callus with sterile filter paper and air dry. Then transfer it to N6DS medium containing the antibiotics hygromycin B (50 mg / L) and carbenicillin (400 mg / L) and culture it in a light incubator at 32°C with 24-hour continuous light for 2-3 weeks. The callus with good growth was transferred to regeneration RE medium containing hygromycin B (50 mg / L) and carbenicillin (250 mg / L) and cultured in an incubator at 32°C for one month to induce differentiation. The RE medium was changed about every two weeks until green seedlings appeared. The callus that differentiated into green seedlings was transferred to MS medium (30 g / L sucrose) containing hygromycin B (50 mg / L) and carbenicillin (200 mg / L) to induce rooting. When the seedlings grew to a certain size, they were transferred to MS medium without antibiotics for culture.

[0080] The culture media related to the Agrobacterium-mediated transformation of rice are shown in Table 1.

[0081] Table 1. Culture media related to the Agrobacterium-mediated transformation of rice.

[0082]

[0083] Example 5 OsTOGR4 Phenotypic analysis of complementary transgenic plants

[0084] To test OsTOGR4 Can they complement each other? togr4 The phenotype of the positive plants obtained from the transgenic experiment in Example 2 was compared with that of the positive plants. pUb - OsTOGR4-GFP / togr4 With Zhonghua 11 (wild type) and togr4 Dark incubation at different temperatures revealed... pUb - OsTOGR4-GFP It can be restored togr4 Plant height and seedling height ( Figure 6 (a and b). This result illustrates OsTOGR4 It is a key gene that regulates the response of rice plant height to high ambient temperature.

[0085] Example 6: Temperatures at different levels OsTOGR4 Transcriptional level analysis

[0086] In order to determine OsTOGR4 How do we respond to changes in ambient temperature? OsFIE2 Transcriptional levels were analyzed in various tissues and at different temperatures.Figure 7 a). The results are shown in the figure. OsFIE2 It is widely expressed in various tissues of rice, with relatively high expression in the aboveground tissues (mature leaves, leaf sheaths, internodes, and seedling leaves), significantly higher than in young and mature panicles and roots, suggesting... OsFIE2 It may play a major role in the growth and development of the aboveground tissues of rice. Further research is needed to investigate the effects of environmental temperature on... OsFIE2 To investigate the effects of transcription, we treated rice at three different temperatures for two weeks, then sampled seedlings, leaves, leaf sheaths, and the entire aboveground tissue to extract RNA and detect its effects. OsFIE2 Gene transcriptional expression level ( Figure 7 (b) In the leaves OsFIE2 The expression level of [a substance] decreases with increasing ambient temperature, while that in the leaf sheath [is different]. OsFIE2 The expression level of [the substance] did not change significantly with increasing ambient temperature, and remained relatively constant in the overall aboveground tissues. OsFIE2 The expression level of [the substance] was significantly lower at elevated ambient temperatures (30℃ and 35℃) than at 25℃, suggesting that increased temperature reduced [the expression level]. OsFIE2 The transcriptional expression level. Compared with the wild type, the mutant togr4 There was no significant difference in gene expression levels in the leaf sheath tissue, while the mutants showed no significant difference under low ambient temperature. togr4 Gene expression levels in leaves and aboveground tissues were significantly lower than those of ZH11, but showed no significant difference compared to ZH11 under high ambient temperatures. These results suggest that under high ambient temperatures... OsFIE2 Expression levels were not affected by mutations. The above transcriptional expression analysis results indicate that increased environmental temperature inhibited... OsFIE2 Gene expression, and OsFIE2 The mutation had no significant effect on gene expression at high ambient temperatures.

[0087] Example 7 Analysis of OsTOGR4 protein content at different temperatures

[0088] To investigate how the protein OsFIE2 responds to temperature, we selected two complementary transgenic lines, pUb-OsFIE2-GFP-1 and pUb-OsFIE2-GFP-2, and cultured them at different temperatures, extracting proteins from the aboveground tissues for Western blotting (WB). The results showed that the protein content of OsFIE2-GFP was significantly lower at 30℃ and 35℃ than at 25℃, suggesting that high ambient temperature also inhibits the protein level of OsFIE2. [[ID= (c). To further confirm the inhibitory effect of elevated ambient temperature on the OsFIE2 protein, we conducted a heat treatment experiment. pUb-OsFIE2-GFP transgenic seedlings grown at 25℃ for 2 weeks were transferred to 35℃ and sampled at time gradients. Protein was extracted and analyzed by Western blotting (WB). ​(d). The results showed that the protein level of pUb-OsFIE2-GFP decreased significantly with increasing heat treatment time, demonstrating that increased ambient temperature inhibited the protein level of OsFIE2. ​ Transcriptional analysis showed that high ambient temperature inhibited the transcription and expression of OsFIE2.

[0089] Rice is one of the most important food crops in my country, and its normal response to ambient temperature is fundamental to its normal growth, development, and maturation. This invention clones key factors controlling the sensitivity of rice to high ambient temperatures, providing important guidance for improving rice's sensitivity to ambient temperature through rice genetic engineering and molecular breeding.

[0090] Example 8: Using CRISPR / Cas9 technology to reduce the sensitivity of rice to high ambient temperatures

[0091] To investigate the role of the OsFIE2 protein structure in temperature response, we used CRISPR / Cas9 technology to obtain... ​ The OsFIE2 allelic mutant, the mutation type is a three-amino acid substitution mutant ( ​ a). Select wild-type ZH11 and ​ CRISPR was used, along with two other mutants, for phenotypic identification through field and incubator temperature treatments. Analysis of the plant morphology of the field allelic mutants revealed... ​ CRISPR plant height, tillering, and mutants ​ The results were consistent, with both exhibiting similar plant height levels at high ambient temperatures (30℃ and 35℃) as at normal temperatures (25℃), demonstrating a phenotype that is insensitive to high ambient temperatures. ​ b). The temperature treatment in the incubator revealed phenotypic differences consistent with field performance. ​ CRISPR seedlings exhibited a high seedling defect phenotype at high ambient temperatures. ​ c).

[0092] In this embodiment, the original CRISPR / Cas9 vector used in the CRISPR / Cas9 technology was constructed and provided by Baige Gene Technology Co., Ltd., and its serial number is SG11588.

[0093] The nucleotide sequence of the sgRNA action site is 5'-TGGCACACCGCTGTTAG-3'.

[0094] 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.

[0095] References:

[0096] [1]Baile, F., Gómez-Zambrano, Á., and Calonje, M. (2022). Roles of Polycomb complexes in regulating gene expression and chromatin structure inplants. Plant Communications 3, 100267.

[0097] [2]Casal, JJ, and Balasubramanian, S. (2019). Thermomorphogenesis.Annual Review of Plant Biology 70, 321-346.

[0098] [3]Cheng,

[0099] [4]Goodrich, J., Wangsomnuk, P., Martin, M., Long, D., Meyerowitz,E., and Coupland, G. (1997). A Polycomb-group gene regulates homeotic geneexpression in Arabidopsis. Nature 386, 44-51.

[0100] [5]Krishnan, P., Ramakrishnan, B., Reddy, K.R., and Reddy, V.R.(2011). Chapter three - High-Temperature Effects on Rice Growth, Yield, andGrain Quality. In Advances in Agronomy, D.L. Sparks, ed (Academic Press), pp.87-206.

[0101] [6]Liao, Z., Yu, H., Duan, J., Yuan, K., Yu, C., Meng, X., Kou, L.,Chen, M., Jing, Y., Liu, G., Smith, S.M., and Li, J. (2019). SLR1 inhibitsMOC1 degradation to coordinate tiller number and plant height in rice. NatureCommunications 10, 2738.

[0102] [7]Liu, X., Wei, X., Sheng, Z., Jiao, G., Tang, S., Luo, J., and Hu,P. (2016). Polycomb Protein OsFIE2 Affects Plant Height and Grain Yield inRice. PLOS ONE 11, e0164748.

[0103] [8]Mozgova, I., and Hennig, L. (2015). The Polycomb Group ProteinRegulatory Network. Annual Review of Plant Biology 66, 269-296.

[0104] [9]Nallamilli, B.R.R., Zhang, J., Mujahid, H., Malone, B.M., Bridges,S.M., and Peng, Z. (2013). Polycomb Group Gene OsFIE2 Regulates Rice (Oryzasativa) Seed Development and Grain Filling via a Mechanism Distinct fromArabidopsis. PLOS Genetics 9, e1003322.

[0105]

[10] Quint, M., Delker, C., Franklin, K.A., Wigge, P.A., Halliday,K.J., and van Zanten, M. (2016). Molecular and genetic control of plantthermomorphogenesis. Nature Plants 2, 15190.

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[11] Sultan, B., Defrance, D., and Iizumi, T. (2019). Evidence of cropproduction losses in West Africa due to historical global warming in two cropmodels. Scientific Reports 9, 12834.

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[12] Zhao, C., Liu, B., Piao, S., Wang, X., Lobell, DB, Huang, Y.,Huang, M., Yao, Y., Bassu, S., Ciais, P., Durand, J.-L., Elliott, J., Ewert,F., Janssens, I.A., Lin, Litre, E., Maru, Q. P., Müller, C., Peng,S., Peñuelas, J., Ruane, A.C., Wallach, D., Wang, T., Wu, D., Liu, Z., Zhu,Y., Zhu, Z., and Asseng, S. (2017). Temperature increase reduces globalyields of major crops in four independent estimates. Proceedings of theNational Academy of Sciences 114, 9326-9331.

Claims

1. Application of knocking out the rice high environmental temperature response gene OsTOGR4 in reducing the sensitivity of rice to high environmental temperatures; The gene OsTOGR4 is a gene that encodes a protein with the amino acid sequence shown in SEQ ID NO:

2.

2. A method for reducing the sensitivity of rice to high ambient temperatures, characterized in that, The method includes: using genetic engineering techniques to weaken or knock out the rice high environmental temperature response gene OsTOGR4; the gene OsTOGR4 is the same as described in claim 1.

3. The method according to claim 2, characterized in that, Using the OsTOGR4 gene as a target, a CRISPR / Cas9-based sgRNA sequence was designed. A DNA fragment containing the sgRNA sequence was ligated into a vector carrying CRISPR / Cas9, and rice was transformed or transfected to obtain transgenic rice with the gene function lost.

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

5. The method according to claim 3 or 4, characterized in that, The transformation or transfection was performed using Agrobacterium-mediated transformation or gene gun transformation.

6. The application of transgenic rice obtained by the method according to any one of claims 2-5 in rice breeding.

7. The application according to claim 6, characterized in that, Breeding methods include transgenic, hybridization, backcrossing, self-pollination, or asexual reproduction.

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