Application of rice high environment temperature response gene OsTOGR4

Abstract

The invention discloses an application of a rice high environment temperature response gene OsTOGR4. According to the invention, a key factor for regulating and controlling the growth and development of a rice plant at the high environment temperature is identified for the first time, and a method for cultivating the rice adapting to the high environment temperature by mutating the OsTOGR4 gene is disclosed, so that gene resources and a research basis are provided for cultivating excellent rice varieties adapting to the high environment temperature.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and in particular to a rice high environmental temperature response gene. OsTOGR4 application. Background Art

[0002] Affected by the greenhouse effect, global temperatures continue to rise and the climate continues to change. Domesticated crops have lost many of the excellent genes from their wild ancestors, their genetic diversity has decreased, and they are difficult to adapt to the changing natural environment, which restricts agricultural production. From 2000 to 2009, sorghum and soybeans in West Africa lost 10-20% and 5-15% of their yields respectively due to climate change (Sultan et al., 2019). Affected by temperature, the global wheat, rice, corn and soybeans will also 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 changes affect food yields, and studying the relationship between plant development and temperature is of great practical significance for breeding stable and high-yield crop varieties.

[0003] When the ambient temperature changes, the plant morphology will change (Casal and Balasubramanian, 2019), and the series of changes in plant morphology starting from the temperature below heat stress and occurring as the temperature rises are collectively referred to as thermal morphogenesis. The hypocotyl of Arabidopsis accelerates growth under high ambient temperature to protect the cotyledons from the higher temperature soil; the petiole will also elongate, and the distance between the leaves will increase, forming a looser plant shape, which helps the plant to dissipate heat and adapt to the environment (Quint et al., 2016). The plant height of rice will elongate as the ambient temperature rises (Krishnan et al., 2011), which is a core factor affecting rice plant shape and yield (Liao et al., 2019). However, so far, there is little research on the molecular mechanism by which rice regulates plant height in response to ambient temperature. Summary of the invention

[0004] The purpose of the present invention is to provide a rice high environmental temperature response gene OsTOGR4 application.

[0005] In order to achieve the purpose of the present invention, in the first aspect, the present invention provides a rice high environmental temperature response gene OsTOGR4 Application in regulating the sensitivity of plants to high ambient temperature (i.e., application in regulating the growth sensitivity of plants under high ambient temperature).

[0006] In the present invention, gene OsTOGR4 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 equivalent functions, wherein one or more amino acids are substituted, deleted or added to the sequence shown in SEQ ID NO: 2.

[0007] Furthermore, the plant includes a grass plant, preferably rice.

[0008] Furthermore, by regulating the high temperature response gene of rice OsTOGR4 The gene is modified to lose its function, thereby reducing the sensitivity of rice to high ambient temperatures (i.e. reducing the sensitivity of rice growth to high ambient temperatures).

[0009] Genome editing technologies such as CRISPR, TALEN, and ZFN can be used to modify genes. OsTOGR4 Carry out transformation.

[0010] In a second aspect, the present invention provides a method for reducing the sensitivity of rice to high ambient temperature, the method comprising: using genetic engineering means to weaken the high ambient temperature response gene of rice OsTOGR4 .

[0011] Furthermore, the gene OsTOGR4 The sgRNA sequence based on CRISPR / Cas9 is designed as the target, the DNA fragment containing the encoding sgRNA sequence is connected to the vector carrying CRISPR / Cas9, and the rice is transformed or transfected to obtain the transgenic rice with the gene function missing.

[0012] Preferably, the original vector of CRISPR / Cas9 is constructed and provided by BioGene Corporation, with the number SG11588.

[0013] Preferably, the nucleotide sequence of the sgRNA action site is 5'-TGGCACACCGCTGTTAG -3'.

[0014] Furthermore, the transformation or transfection can be performed by Agrobacterium-mediated method or gene gun method.

[0015] In a third aspect, the present invention provides the use of the transgenic rice obtained according to the method in plant breeding.

[0016] Further, breeding methods include but are not limited to transgenic, hybridization, backcrossing, selfing or asexual reproduction.

[0017] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: The present invention identifies for the first time a key factor regulating rice plant growth and development under high ambient temperature and discloses a method for knocking out the key factor.OsTOGR4 A method to genetically reduce the sensitive growth of rice in high ambient temperatures, thereby providing genetic resources and research basis for breeding high-quality rice varieties that can adapt to high ambient temperatures. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 In the preferred embodiment of the present invention togr4 Phenotypic comparison with the wild type Zhonghua11. (a) Mutant togr4 (a) and (b) Field phenotypic statistics of wild-type ZH11 in Lingshui, Beijing, and Yangzhou. (b) Statistics of plant height of seedlings grown for 2 weeks under different temperature conditions. The black bars in the bar graphs in (a) and (b) represent ZH11, and the gray bars represent mutants. togr4 , The values ​​are expressed as mean ± standard error (n ≥ 10), and the scale bar is 10 cm.

[0019] Figure 2 In the preferred embodiment of the present invention togr4 Cell phenotypes of wild-type Zhonghua11 under high ambient temperature. (a) WT and togr4 Longitudinal section of the tissue of the first internode under high ambient temperature, scale bar is 100 μm. (b) WT and togr4 Cross-section of the tissue of the first internode under high ambient temperature, scale bar is 100 μm. (c) WT and togr4 Leaf epidermal cells under high ambient temperature. The scale is 100 μm. The corresponding charts include: the width of the first internode, the thickness of the stem, the length, width, area and total number of cells in the first internode, the length of the leaf blade and the length of the stomatal cells in the leaf epidermis. The values ​​are expressed as mean ± standard error, and significant differences are expressed as .

[0020] Figure 3 The subcellular localization of proteins OsFIE2 and togr4 in the preferred embodiment of the present invention. (a) Subcellular localization of protein OsFIE2 in rice root cells. The figure shows microscopic observation of the roots of pUb::GFP overexpressing wild-type material (top) and pUb::OsFIE2-GFP complemented togr4 mutant transgenic strain (bottom). The scale bar is 50 μm long. (b) Subcellular localization in rice protoplasts. The fusion protein OsbZIP52-mCherry is used as a nuclear localization marker. The scale bar is 10 μm long. (c) Subcellular localization in tobacco leaves. The scale bar is 50 μm long.

[0021] Figure 4 In the preferred embodiment of the present invention OsTOGR4 Gene location and candidate gene identification. (a) togr4, wild-type ZH11, and the F1 generation of hybrid plants in Yangzhou. The scale bar is 10 cm. (b) OsTOGR4 Linkage map. M1 to M7 are molecular markers, shown in the figure OsTOGR4 Candidate genes, black rectangles represent exons, and the connecting line segments in the middle represent introns. (c) Recombination frequency of each molecular marker in the linkage map.

[0022] Figure 5 In the preferred embodiment of the present invention OsTOGR4 Gene overexpression vector.

[0023] Figure 6 In the preferred embodiment of the present invention OsTOGR4 Genes that affect some phenotypes of plant thermomorphogenesis. (a) WT, togr4 Field phenotypes of WT and complementary materials planted in Yangzhou, with a scale of 10 cm. (b) WT, togr4 and togr4 / OsFIE2 Seedling phenotypes of complementary materials at different culture temperatures, the scale is 10 cm.

[0024] Figure 7 In the preferred embodiment of the present invention togr4 Compared with wild type Zhonghua11 OsFIE2 Gene transcription expression analysis. (a) OsFIE2 Analysis of transcriptional levels in different tissues of rice. (b) Analysis of transcriptional levels in leaves, sheaths and aboveground tissues of rice seedlings at different temperatures for two weeks OsFIE2 Transcription level analysis of genes. Data in (a) and (b) are expressed as mean ± standard error, n = 3. Actin The data were analyzed using Tukey HSD test (p<0.05) as the internal reference. Different letters indicate significant differences. (c) Expression analysis of the aboveground protein OsFIE2 in rice seedlings after two weeks of temperature treatment. (d) Protein expression analysis of OsFIE2 after heat treatment. OsFIE2-GFP seedlings grown at 25°C for two weeks were transferred to 35°C for heat treatment. The upper part of (c) and (d) are protein immunoblot images, and the lower table is the grayscale analysis of protein expression, with protein Actin as the internal reference. The experiment was repeated three times, and the data were analyzed using Student's test. .

[0025] Figure 8 This is an example of using CRISPR / Cas9 technology to change the sensitivity of rice to high environmental temperature. osfie2- CRISPR is generated using CRISPR / Cas9 technology OsFIE2 Allelic mutant. (a) Gene OsFIE2Schematic diagram of allelic mutations. The black wireframe represents the exon, and the line segments connected by triangles indicate the mutation site of the allelic mutant on the gene. The red wireframe shows the sequencing peak graph of the mutation site. (b) Plant height and tillering of two allelic mutant forms in the field. Data n≥15. (c) Phenotypes of two allelic mutants under temperature treatment. Scale bar length 10 cm, n≥16. The data in (b) and (c) are expressed as mean ± standard error, and the data were analyzed by one-way analysis of variance. The significant differences are indicated by asterisks, respectively. . DETAILED DESCRIPTION

[0026] The invention provides a rice gene that responds to environmental temperature changes OsTOGR4 ( Thermotolerant Growth Required 4 ), the encoded protein and functional analogs of the gene, a vector containing the nucleotide sequence of the gene, and a host cell containing the nucleotide sequence of the gene or the vector. The present invention also provides a method for reducing the sensitivity of rice to high environmental temperature by using the gene OsTOGR4 The method of cultivating rice varieties that can adapt to high ambient temperatures and have a growth phenotype close to that at normal temperatures is expected to reduce the problems of lodging and brittle stems caused by the rapid growth of rice at high ambient temperatures.

[0027] Using ethyl methanesulfonate (EMS) mutagenesis and genetic screening, we isolated and identified a rice plant height mutant that undergoes morphological changes in response to high ambient temperature. OsTOGR4 ( Thermotolerant Growth Required 4 ).when togr4 When planted under low ambient temperature conditions (for example, planted in Hainan from December to April of the following year), its morphological characteristics are very close to those of the wild type; when grown under higher ambient temperature conditions (for example, planted in Yangzhou from May to September), its growth is significantly inhibited, showing a dwarf phenotype. To verify the phenotype, an artificial incubator environment was used to simulate the growth environment, and the growth phenotypes of rice materials at different temperatures were statistically analyzed to further confirm the togr4 Phenotypes that respond to environmental temperature. Based on the preliminary establishment of the phenotype, the key genes that regulate rice's response to environmental temperature changes were located and cloned through phenotypic screening supplemented by molecular marker identification. OsTOGR4 Through phenotypic analysis of mutants and genetic complementation experiments, the main function and associated phenotypes of the gene were confirmed. OsTOGR4 The protein content encoded by the gene showed a decreasing trend as the ambient temperature increased. Based on these experiments, the present invention identified for the first time a seedling height-sensitive gene that regulates rice's response to high ambient temperature. OsTOGR4 , which encodes the OsFIE2 subunit of the rice PRC2 protein complex.

[0028] PRC2 is a polycomb group (PcG) protein, which consists of four core subunits and some accessory subunits. It modulates the open state of chromatin by methylating K27 of histone H3 in the form of a complex, regulating the silencing and activation state of genes at the epigenetic level, and plays an important role in early embryonic development, cancer regulation, plant growth and development, and environmental response in animals (Goodrich et al., 1997; Mozgova and Hennig, 2015; Bailey et al., 2022). In rice, PRC2 contains the subunit OsEZ1 with methyltransferase function, which can trimethylate the 27th Lys residue of histone H3, thereby performing epigenetic regulation. OsFIE2 is one of the core subunits of rice PRC2. It binds and interacts with EZ1, affects the histone H3 methyltransferase activity of PRC2, regulates rice plant height, seed development and grain filling (Nallamilli et al., 2013), and has an additive effect on the synthesis of some storage proteins and the expression regulation of photosynthesis genes (Liu et al., 2016; Cheng et al., 2020). OsFIE2 Homozygous mutations are lethal (Cheng et al., 2020), and reduced expression levels can cause rice seeds to become smaller, insufficiently fill, and some seeds to fail to enter normal dormancy (Nallamilli et al., 2013). osfie2-1 The number of longitudinal cells in the internodes decreased, the lateral cell layer of the internodes became significantly thinner, and the number of large and small vascular bundles and the number and size of cells in the glume decreased (Liu et al., 2016), showing phenotypes such as dwarfing, reduced fruiting rate, floral organ defects, and smaller grains. However, so far, no studies have shown that OsFIE2 is involved in the response of rice to temperature.

[0029] The core subunit OsTOGR4 / OsFIE2 of the rice PRC2 protein complex identified in the present invention is involved in the growth and development regulation of rice in response to high ambient temperature. The normal expression and protein content of the gene ensure the normal growth of rice under high ambient temperature.

[0030] The present invention adopts the following technical solution: In the first aspect, the present invention identifies a rice plant height gene that responds to high ambient temperature. OsTOGR4The gene encodes the protein shown in SEQ ID NO: 2, or encodes a protein derived from SEQ ID NO: 2, which has the same function as the protein shown in SEQ ID NO: 2 after one or more amino acids are substituted, deleted or added to the amino acid sequence shown in SEQ ID NO: 2. The protein shown in SEQ ID NO: 2 consists of 376 amino acids.

[0031] The rice plant height gene that responds to high ambient temperature OsTOGR4 is an isolated nucleotide sequence. In a preferred embodiment, the gene controlling the response of rice to high ambient temperature OsTOGR4 The nucleotide sequence of is shown in SEQ ID NO: 1 (SEQ ID NO: 1 also includes the promoter and 3'UTR sequence). In addition, those skilled in the art should understand that, in a broader sense, the rice plant height gene that responds to high ambient temperature OsTOGR4 The nucleotide sequence is a nucleotide sequence that has 90% or more, preferably 99% or more homology with the nucleotide sequence shown in SEQ ID NO: 1 and encodes a protein having the same function.

[0032] In a second aspect, the present invention provides a gene for controlling the response of rice to high ambient temperature. OsTOGR4 The encoded protein is the following (a) or (b): (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2; (b) A protein derived from SEQ ID NO: 2 by which one or more amino acids are substituted, deleted or added to the amino acid sequence of SEQ ID NO: 2 and which has the same function as the protein of SEQ ID NO: 2.

[0033] In a preferred embodiment, the gene controlling the response of rice to high ambient temperature OsTOGR4 The encoded protein is an isolated protein, and its amino acid sequence is shown in SEQ ID NO:2.

[0034] In a third aspect, the present invention provides a gene comprising the control rice response to high environmental temperature. OsTOGR4 of the recombinant vector.

[0035] Preferably, the exogenous nucleotide fragment contained in the recombinant vector encodes the amino acid sequence shown in SEQ ID NO: 2. More preferably, the exogenous nucleotide fragment contained in the recombinant vector is shown in SEQ ID NO: 1.

[0036] In one embodiment, the plasmid used to construct the recombinant vector can be selected from but not limited to pCAMBIA1300 .

[0037] Host cells containing the recombinant vector are also within the scope of the present invention. The host cells containing the recombinant vector can be obtained by transforming or transfecting the recombinant vector into cells, so as to be further used for amplifying the expression vector, expressing the protein or obtaining transgenic plants. The cells used for transformation or transfection can be selected from, but not limited to, bacterial cells, such as Escherichia coli cells or Agrobacterium cells, fungal cells, such as yeast cells, or plant cells, such as rice cells, etc.

[0038] In a fourth aspect, the present invention provides a gene for regulating the high environmental temperature sensitivity of rice. OsTOGR4 Use in breeding rice varieties that can respond and grow normally under high environmental temperatures.

[0039] In a fifth aspect, the present invention provides a method for cultivating a rice variety that can respond and grow normally at high environmental temperatures, the method comprising reducing OsTOGR4 The amount of protein encoded by the gene. OsTOGR4 The method of gene encoding protein content can be achieved by the following method: using CRISPR / Cas9 Gene editing technology OsTOGR4 Gene knockout.

[0040] OsTOGR4 Genes and OsTOGR4 Transformation or transfection of gene editing vectors can be performed by Agrobacterium-mediated or gene gun-mediated methods.

[0041] The present invention provides a theoretical and material basis for cultivating crop varieties that reduce sensitivity to high ambient temperature and improve adaptability to high ambient temperature. When the gene of the present invention is used to improve the response of rice to high ambient temperature, the following methods can be used: (1) construct a OsTOGR4 (1) constructing a vector for gene editing; (2) transforming the constructed vector into regenerable rice tissues or organs; and (3) culturing the transformed tissues or organs into plants and screening for plants with gene mutations.

[0042] Furthermore, the present invention provides an isolated gene for regulating rice sensitivity to high environmental temperature, wherein the gene encodes the protein shown in SEQ ID NO:2, or encodes a protein derived from SEQ ID NO:2, which has the same function as the protein shown in SEQ ID NO:2 after one or several amino acids are inserted, deleted or replaced in the amino acid sequence shown in SEQ ID NO:2.

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

[0044] Furthermore, it is the nucleotide sequence shown in SEQ ID NO:1.

[0045] The present invention also provides a OsTOGR4 The encoded isolated protein for controlling rice's high environmental temperature sensitivity is the protein shown in SEQ ID NO: 2, or a protein derived from SEQ ID NO: 2, which has one or several amino acids inserted, deleted or replaced in the amino acid sequence shown in SEQ ID NO: 2 and has the same function as the protein shown in SEQ ID NO: 2.

[0046] Furthermore, the protein is the protein shown in SEQ ID NO:2.

[0047] The present invention also provides a method comprising the gene OsTOGR4 or a recombinant vector containing a fragment thereof.

[0048] Furthermore, the recombinant vector is a plant expression vector, preferably a vector suitable for expression in rice.

[0049] Preferably, the recombinant vector is composed of a plasmid pCAMBIA1300 Build.

[0050] Furthermore, the plant expression vector also comprises OsTOGR4 Promoter or Ubiquitin promoter, and a gene sequence encoding a tag protein, such as GFP.

[0051] Furthermore, the host cell is selected from bacteria, fungi or plant cells, preferably Escherichia coli ( Escherichia coli ), Agrobacterium ( Agrobacterium tumefaciens ) or plant cells.

[0052] The present invention also provides a method for cultivating a plant with high environmental temperature adaptability having excellent high environmental temperature insensitivity, the method comprising using gene editing technology to modify the gene OsTOGR4 or its fragments as templates to design gene editing targets and construct vectors, then transform cells or tissues of the plant, and cultivate the transformed plant cells or tissues into OsTOGR4 Loss of function and reduced sensitivity of rice to high ambient temperature.

[0053] Furthermore, the transformation is performed by Agrobacterium-mediated method or gene gun method.

[0054] Furthermore, the plant is a grass plant, preferably rice.

[0055] The present invention also provides the gene OsTOGR4Or the use of the protein for regulating the plant's response to high ambient temperature in cultivating plants with excellent high ambient temperature adaptability and low sensitivity to high ambient temperature.

[0056] The present invention also provides a method for cultivating rice with excellent adaptability to high environmental temperatures and low sensitivity to high environmental temperatures, the method comprising transforming 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 in which the polynucleotide sequence is edited.

[0057] Furthermore, the method comprises using the recombinant vector to transform rice cells or tissues, and cultivating the transformed cells or tissues into plants that overexpress genes that control the high environmental temperature sensitivity of rice.

[0058] Furthermore, the transformation is performed by Agrobacterium-mediated method or gene gun method, and the plant includes a grass plant, preferably rice.

[0059] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present 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.

[0060] Example 1 Isolation of genes controlling rice sensitivity to high ambient temperature First, a mutant with dwarfed plant height under high ambient temperature was screened from the EMS (Ethylmethylsulfone)-induced japonica rice ZH11 mutant library. togr4 ( Figure 1 , a). To further determine whether the 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 12h and 20°C 12h, simulating diurnal cycle conditions), 30°C / 25°C, and 35°C / 30°C. Phenotypic statistical analysis found that the seedling height of the mutant was significantly lower than that of the wild type at high ambient temperature, while the length was similar to that of the wild type at low ambient temperature, indicating that the seedling height of the mutant was hypersensitive to high ambient temperature ( 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 the cells of the first internode of the wild type and mutant. The results showed that the cells of the mutant were significantly smaller, the total number of cells was significantly reduced, the stems were significantly thinner, the plants were significantly shorter, the leaf length in the seedling stage was significantly shortened, and the stomata on the leaf epidermal cells were significantly shortened ( Figure 2 , a~c).

[0061] Example 2 Cytological localization of genes controlling sensitivity to high ambient temperature in rice In order to explore the function of protein OsFIE2 at the cellular level, we conducted subcellular localization experiments on OsFIE2. pUb::OsFIE2-GFP Electron microscopy was performed on the root tip cells of the rats and it was found that the expression signal of OsFIE2-GFP protein was distributed in the cytoplasm and nucleus of the cells ( Figure 3 , a). In order to further observe whether the mutated protein togr4 affects the subcellular localization, we carried OsFIE2-GFP and togr4-GFP Two plasmids carrying the gene were transiently transformed into rice protoplasts ( Figure 3 , b) and tobacco leaf cells ( Figure 3 , c), electron microscopy showed that the expression signal of togr4 protein was still distributed in the cytoplasm and nucleus after mutation. The above results indicate that OsFIE2 acts in both the cytoplasm and nucleus of cells, and the localization of togr4 protein does not change after mutation.

[0062] Example 3 Genetic Location of Rice High Ambient Temperature Sensitivity Control Gene To study the OsTOGR4 The molecular causes of the phenotype will be togr4 and ZH11 were hybridized and the F1 generation plants were constructed ( Figure 4 , a), and then the F2 generation population was obtained through the F1 generation plants, and then the populations were counted. togr4 Based on the diversity of restriction fragment lengths between japonica and indica rice, SSR molecular markers were used to identify togr4 Preliminary positioning of candidate mutant genes ( Figure 4 , b). The positional clonal population was linked on chromosome 8 of rice. By analyzing the recombination rate of molecular markers on chromosome 8, it was found that the recombination efficiency at the molecular markers M2 and M3 positions was significantly reduced ( Figure 4 , c), suggesting that the segment between the two molecular markers is highly linked, so the candidate mutant gene is initially located in the segment between M2 and M3 of chromosome 8. Since there are 1.96Mb between M2 and M3, nearly 291 genes. In order to confirm the mutant gene, we togr4The whole genome of LOC_Os08g05450 was resequenced and compared with the wild-type genome, and it was found that there were only two gene loci with missense mutations between the M2 and M3 regions. One of them was LOC_Os08g05450, and the amino acid at position 176 of the gene mutated from Ala to Thr, but because its encoded protein was a transposon and there were also fragments of this gene on other chromosomes, this gene was not considered a candidate gene. Another candidate mutant gene was LOC_Os08g04270, and the base 419 of the CDS sequence of this gene underwent a base substitution from C to T, resulting in the mutation of the amino acid at position 140 of its encoded protein from threonine (Thr) to isoleucine (Ile). Based on the above results, we selected LOC_Os08g04270 as togr4 Candidate mutant genes encoding endosperm-autonomous genes independent of fertilization OsFIE2 (fertilization-independentendosperm gene 2).

[0063] Example 4 OsTOGR4 Construction of complementary vector and genetic transformation of rice From the wild type flower 11 OsTOGR4 The complete gene, which is the nucleotide sequence shown in SEQ ID NO: 1 (including the promoter sequence, genomic sequence and 3' non-coding region sequence), is amplified by PCR using DNA polymerase, and then ligated to pCAMBIA1300 The complementary vector was obtained from the multiple cloning site of the plasmid (purchased from CAMBIA) pUb:: OsTOGR4 ( Figure 5 ).

[0064] The constructed complementation vector was transformed into E.coli In DH5α competent cells, positive clones were screened using kanamycin. Plasmids were extracted and sequenced to obtain the cloned vector. OsTOGR4 The sequence of the positive clone is completely correct, and then the plasmid of the positive clone is electroporated into EHA105 Agrobacterium competent cells (refer to "Plant Genetic Engineering", Wang Guanlin, Fang Hongjun, Science Press, 2004, 2nd edition, prepared by conventional methods). Next, the transformed clone is infected with Agrobacterium to OsTOGR4 Perform transgenic manipulation for the recipient.

[0065] The genetic modification operation method is as follows: (1) Seed sterilization: Weigh 20-30 g of dried rice seeds and remove the seed husks using a small laboratory huller. Soak the hulled seeds in 70% ethanol for 1 minute, then soak them in 30% sodium hypochlorite (the stock solution is 10% effective chlorine, add 1 drop of Tween-20 for every 50 mL) for 30 minutes and shake them gently on a shaker. Then rinse them with sterile water 5-6 times; then disinfect them once with 30% sodium hypochlorite (without Tween-20) and rinse them with sterile water 5-6 times. Transfer the seeds to filter paper in the clean bench and air dry them.

[0066] (2) Inducing callus: Transfer the seeds to the N6D medium prepared in advance, and then culture them in a light incubator at 32°C and 24 hours of continuous light for 5-7 days until the seeds grow golden callus.

[0067] (3) Agrobacterium infection: Agrobacterium containing a specific plasmid is introduced into EHA105 Streak culture on YEB medium containing antibiotics rifampicin (Rif 25-50 mg / L) and kanamycin (Kan 50 mg / L), then place in an oven at 28°C for dark culture for 2-3 days; pick a single clone strain into 5-6 mL of liquid YEB medium containing the same resistance, and shake it overnight in a shaker at 28°C (220 rpm). Inoculate the bacterial liquid into 50 mL of AAM medium at a ratio of 1:100, and shake and culture overnight in a shaker at 28°C and 220 rpm until OD 600 About 0.1; soak the callus in good condition in the Agrobacterium solution for 2 minutes, then quickly take out the callus with tweezers and place it on sterile filter paper to air dry. After the callus is air-dried, transfer it to N6D-As medium covered with sterile filter paper (soaked in AAM) in advance, wrap the culture dish containing the callus with sealing film and tin foil, and culture it in the dark at 25℃ for 2-2.5 days.

[0068] (4) Screening and differentiation: First, rinse the callus co-cultured with Agrobacterium in sterile double distilled water for 3-4 times, then rinse it 2-3 times with sterile double distilled water containing carboxybenzyl (500 mg / L) and then soak it for 30 minutes each time, repeat 3-5 times, and try to clean the Agrobacterium on the surface of the callus. Use sterile filter paper to absorb the water of the washed callus and air-dry it, then transfer it to N6DS medium containing antibiotics hygromycin B (50 mg / L) and carboxybenzyl (400 mg / L), and culture it in a light incubator at 32°C with 24 hours of continuous light for 2-3 weeks. The calli with good growth were transferred to regeneration RE medium containing hygromycin B (50 mg / L) and carbobenzyl (250 mg / L), and cultured in a 32°C incubator for 1 month to induce differentiation. The RE medium was replaced approximately every two weeks until green seedlings appeared. The calli that differentiated into green seedlings were transferred to MS (sucrose 30 g / L) medium containing hygromycin B (50 mg / L) and carbobenzyl (200 mg / L) to induce rooting. When the seedlings grew to a certain size, they were transferred to MS medium without antibiotics for culture.

[0069] The culture medium related to rice transformation by Agrobacterium infection is shown in Table 1.

[0070] Table 1 Culture media related to rice transformation by Agrobacterium infection Example 5 OsTOGR4 Phenotypic analysis of complemented transgenic plants To test OsTOGR4 Can they be complementary? togr4 The positive plants obtained from the transgenic experiment in Example 2 pUb - OsTOGR4-GFP / togr4 Compared with Zhonghua11 (wild type) and togr4 Dark culture was performed at different temperatures and the results were analyzed. pUb - OsTOGR4-GFP Can be restored togr4 Plant height and seedling height ( Figure 6 , a and b). This result shows OsTOGR4 It is a key gene that regulates the response of rice plant height to high ambient temperature.

[0071] Example 6 at different temperatures OsTOGR4 Transcriptional analysis To determine OsTOGR4 How to respond to changes in ambient temperature? OsFIE2 Transcript levels were analyzed in various tissues and at different temperatures ( Figure 7 , a). The results are shown in the figure. OsFIE2It is widely expressed in various rice tissues, and its expression is relatively high in aboveground tissues (mature leaves, leaf sheaths, internodes and seedling leaves), which is significantly higher than that in young panicles, mature panicles and roots, suggesting that OsFIE2 It may play a major role in the growth and development of rice aboveground tissues. OsFIE2 To investigate the effects of temperature on rice transcription, we placed rice at three temperatures for 2 weeks and then sampled seedlings, leaves, leaf sheaths, and the entire aerial tissues to extract RNA and test OsFIE2 The transcriptional expression level of the gene Figure 7 , b). In leaves OsFIE2 The expression level of OsFIE2 The expression level of OsFIE2 The expression level of OsFIE2 Compared with the wild type, the mutant togr4 There was no significant difference in gene expression levels in the leaf sheath tissues of the mutants, but at low ambient temperature togr4 The gene expression levels in leaves and aboveground tissues were significantly lower than those in ZH11, and there was no significant difference compared with ZH11 under high ambient temperature. These results suggest that under high ambient temperature, OsFIE2 The expression level was not affected by the mutation. The above transcriptional expression analysis results showed that the increase in ambient temperature inhibited OsFIE2 Gene expression, and OsFIE2 The mutation had no significant effect on gene expression under high ambient temperature.

[0072] Example 7 Analysis of OsTOGR4 protein content at different temperatures In order to explore how the protein OsFIE2 responds to temperature, we selected two complementary transgenic lines, pUb-OsFIE2-GFP-1 and pUb-OsFIE2-GFP-2, cultured them at different temperatures, extracted proteins from aboveground tissues, and then performed immunoblotting (WB). The results showed that the protein content of OsFIE2-GFP at 30℃ and 35℃ was significantly lower than that at 25℃, suggesting that high ambient temperature also inhibited the protein level of OsFIE2 ( Figure 7 , c). To further confirm the inhibitory effect of elevated ambient temperature on the protein OsFIE2, we conducted a heat treatment experiment. The pUb-OsFIE2-GFP transgenic seedlings grown at 25°C for 2 weeks were transferred to 35°C and sampled over a time gradient. The protein was extracted and tested by WB ( Figure 7, d). The results showed that as the heat treatment time increased, the protein level of pUb-OsFIE2-GFP decreased significantly, proving that the increase in ambient temperature inhibited the protein level of OsFIE2. OsFIE2 Transcriptional level analysis results showed that high ambient temperature inhibited the transcription and expression of OsFIE2.

[0073] Rice is one of the most important food crops in my country, and its normal response to normal ambient temperature is the basis for normal growth, development and maturity. The present invention cloned the key factors that control the sensitivity of rice to high ambient temperature, providing important guidance for improving the sensitivity of rice to ambient temperature through rice genetic engineering and molecular breeding.

[0074] Example 8 Using CRISPR / Cas9 technology to reduce the sensitivity of rice to high ambient temperature To explore the role of OsFIE2 protein structure in temperature response, we used CRISPR / Cas9 technology to obtain osfie2CRISPR OsFIE2 allele mutant, the mutation type is a mutant with three amino acid substitutions ( Figure 8 , a). Select wild-type ZH11 and osfie2 CRISPR, combined with two other mutants, phenotype identification was performed by field and incubator temperature treatment. The plant type of the field allele mutant was analyzed and the results showed that osfie2 Plant height, tillering and mutants using CRISPR togr4 The performance was consistent, and both showed similar plant height levels under high ambient temperatures (30℃ and 35℃) as under normal temperature conditions (25℃), showing a phenotype that was insensitive to high ambient temperature ( Figure 8 , b). The phenotypic differences found in the incubator temperature experiment were consistent with those in the field. osfie2 CRISPR seedlings showed a high ambient temperature seedling defect phenotype ( Figure 8 , c).

[0075] In this embodiment, the CRISPR / Cas9 original vector used in the CRISPR / Cas9 technology was constructed and provided by BioGene, Inc., with the code number SG11588.

[0076] The nucleotide sequence of the sgRNA action site is 5'-TGGCACACCGCTGTTAG -3'.

[0077] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

[0078] References: [1]Baile, F., Gómez-Zambrano, Á., and Calonje, M. (2022). Roles of Polycomb complexes in regulating gene expression and chromatin structure in plants. Plant Communications 3, 100267. [2]Casal, J.J., and Balasubramanian, S. (2019). Thermomorphogenesis. Annual Review of Plant Biology 70, 321-346. [3]Cheng, X., Pan, M., E, Z., Zhou, Y., Niu, B., and Chen, C. (2020). Functional divergence of two duplicated Fertilization Independent Endosperm genes in rice with respect to seed development. The Plant Journal 104, 124-137. [4]Goodrich, J., Wangsomnuk, P., Martin, M., Long, D., Meyerowitz, E., and Coupland, G. (1997). A Polycomb-group gene regulates homeotic gene expression in Arabidopsis. Nature 386, 44-51. [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. [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. [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. [8]Mozgova, I., and Hennig, L. (2015). The Polycomb Group ProteinRegulatory Network. Annual Review of Plant Biology 66, 269-296. [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.

[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.

[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.

[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, E., Maru, Litre, 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. Rice high temperature response genes OsTOGR4 Application in regulating plant sensitivity to high ambient temperature; The gene OsTOGR4 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 equivalent functions, wherein one or more amino acids are substituted, deleted or added to the sequence shown in SEQ ID NO:

2.

2. The use according to claim 1, characterized in that: The plants include grass plants, preferably rice.

3. The use according to claim 2, characterized in that: High temperature response gene in rice OsTOGR4 The gene is modified to change its function, thereby reducing the sensitivity of rice to high ambient temperatures.

4. A method for reducing the sensitivity of rice to high ambient temperature, characterized in that: The method comprises: using genetic engineering means to weaken or knock out the high environmental temperature response gene of rice OsTOGR4 The gene OsTOGR4 As described in claim 1.

5. The method according to claim 4, characterized in that Gene OsTOGR4 The sgRNA sequence based on CRISPR / Cas9 is designed as the target, the DNA fragment containing the encoding sgRNA sequence is connected to the vector carrying CRISPR / Cas9, and the rice is transformed or transfected to obtain the transgenic rice with the gene function missing.

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

7. The method according to claim 5 or 6, characterized in that: The transformation or transfection is carried out by Agrobacterium-mediated method or gene gun method.

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

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

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