Gene SCE for regulating and controlling heat resistance, participation of gene SCE in heat stress response pathway and application of gene SCE

Abstract

The invention provides a gene SCE for regulating and controlling heat resistance, a heat stress response pathway participated by the gene SCE and application of the gene SCE. The invention discloses a novel gene SCE which encodes a polypeptide with an important biological function, and also discloses a TT1-SCE-Hsp heat stress response pathway in which SCE participates, and an application of the SCE in plant heat resistance or yield trait improvement under heat stress. The invention has important significance on genetic improvement of plant stress resistance characters.

Description

Technical Field

[0001] This invention belongs to the fields of botany and molecular biology; more specifically, this invention relates to a gene SCE that regulates heat tolerance, its participation in heat stress response pathways, and its applications. Background Technology

[0002] Global warming poses a significant threat to food security and human development, with the global average temperature already rising by 1.25°C. This increase is projected to exceed 1.5°C over the next decade. Rice (Oryza sativa) is a crucial staple crop globally; for every 1°C increase in global temperature, rice yields decrease by approximately 3.2%.

[0003] High-temperature stress refers to the phenomenon where plants encounter high temperatures exceeding their normal growth range during their growth process, leading to inhibited or damaged growth and development. In recent years, with the increase in greenhouse gas emissions, global temperatures have continued to rise, and high-temperature environments have had a significant impact on plant life cycles and agricultural production.

[0004] Under high-temperature stress, the physiological and biochemical processes of plants are affected to varying degrees. Plants sense and transmit high-temperature signals through multiple cell signaling pathways under high-temperature stress. This stress triggers a series of changes in gene expression, which are regulated by various signaling pathways. Studies have shown that heat shock proteins are a class of stress proteins induced by high-temperature stress in plants, playing a role in protecting cell structure and function. Furthermore, plants also regulate gene expression by modulating transcription factors and miRNAs under high-temperature stress to cope with the high-temperature environment.

[0005] With the continuous growth of the global population, improving the heat resistance and adaptability of crops has become an urgent problem to be solved. Therefore, it is necessary to explore heat-resistant rice germplasm resources in depth, systematically study the regulatory mechanisms of rice heat resistance, cultivate innovative new crop germplasm, and achieve high and stable crop yields.

[0006] A thorough analysis of the molecular mechanisms by which plants respond to and tolerate heat stress can provide important theoretical basis and genetic resources for breeding new crop varieties with heat resistance.

[0007] Although much scientific research currently focuses on the regulatory network of plant responses to heat stress, including upstream sensors, core regulators and downstream responders, effective regulatory targets are still lacking in this field. Summary of the Invention

[0008] The purpose of this invention is to provide a gene SCE that regulates heat tolerance, its involvement in heat stress response pathways, and its applications.

[0009] In a first aspect of the invention, a method for improving plant heat tolerance or yield under heat stress is provided, comprising: downregulating SCE (SUMO E2-binding enzyme) in plants, thereby improving plant heat tolerance or yield under heat stress; or, regulating the interaction of TT1, SCE, and Hsp (small heat shock protein) in the TT1-SCE-Hsp heat stress response pathway involving SCE in plants, increasing Hsp accumulation (preventing its degradation), and enhancing Hsp-mediated protein folding, thereby improving plant heat tolerance or yield under heat stress; wherein the SCE includes SCE1 or SCE2.

[0010] In one or more embodiments, the improvement of plant yield under heat stress includes: increasing survival rate, increasing grain weight and / or increasing seed setting rate.

[0011] In one or more embodiments, the Hsp-mediated protein folding includes: correcting misfolds to correct folds, or causing an unfolded protein to fold; preferably, causing the protein to fold into its native state.

[0012] In one or more embodiments, the Hsp includes Hsp24.1 or Hsp40.

[0013] In one or more embodiments, modulating the TT1-SCE-Hsp heat stress response pathway includes: (a) upregulating TT1 to promote the interaction between TT1 and SCE (TT1-SCE module), promoting TT1-mediated SCE degradation (degradation via ubiquitinated protein degradation pathway), thereby improving plant heat tolerance or yield under heat stress; or (b) inhibiting the interaction between SCE and Hsp to reduce Hsp SUMOylation modification, increase Hsp accumulation (prevent its degradation), thereby enhancing Hsp-mediated protein folding, thereby improving plant heat tolerance or yield under heat stress.

[0014] In one or more embodiments, downregulating SCE includes downregulating its expression, activity, or stability, preferably including: knocking out or silencing the SCE-coding gene in plants, inhibiting SCE activity, or promoting SCE ubiquitination degradation; preferably, including: gene editing using a CRISPR system to knock out the SCE-coding gene, knocking out the SCE-coding gene by homologous recombination, performing a loss-of-function mutation on SCE in plants containing SCE, silencing SCE with an interfering molecule that specifically interferes with the expression of the SCE-coding gene, or upregulating TT1 to promote its mediated SCE ubiquitination degradation.

[0015] In one or more embodiments, upregulating TT1 includes upregulating its expression, activity, or stability, including: transferring the gene encoding TT1 or an expression construct or vector containing the gene encoding TT1 into a plant; performing a gain-of-function mutation on TT1; promoting TT1 expression by expressing an enhancing promoter or a tissue-specific promoter; or promoting TT1 expression by an enhancer.

[0016] In one or more embodiments, when SCE is reduced, either SCE1 or SCE2 is reduced (not both are reduced).

[0017] In one or more embodiments, the ubiquitination modification of SCE includes K48 ubiquitination.

[0018] In one or more embodiments, the SUMOylation modification sites of Hsp24.1 include K157, K192, K206, and K208.

[0019] In one or more embodiments, the SUMOylation modification sites of Hsp40 include K10, K110, K256, and K314.

[0020] In one or more embodiments, the improvement of plant yield under heat stress includes: maintaining, increasing, or protecting plant yield under high temperature (stress) (to a higher yield than that of unregulated control plants).

[0021] In one or more embodiments, SCE is downregulated by an sgRNA that guides CRISPR gene editing or a reagent capable of forming the sgRNA; preferably, the reagent for forming the sgRNA includes: primers of the sequences shown in SEQ ID NO:17 and SEQ ID NO:18 (targeting SCE1), primers of the sequences shown in SEQ ID NO:19 and SEQ ID NO:20 (targeting SCE1), and primers of the sequences shown in SEQ ID NO:39 and SEQ ID NO:40 (targeting SCE2).

[0022] In another aspect of the invention, the use of an SCE (including exogenous or isolated ones), a TT1-SCE-Hsp heat stress response pathway containing the SCE, or a regulatory molecule thereof, for improving plant heat tolerance or yield under heat stress is provided; preferably, the improvement in plant yield under heat stress includes: improving survival rate, increasing grain weight, and / or increasing seed setting rate; wherein the regulatory molecule is an SCE downregulatory molecule that improves plant heat tolerance or yield under heat stress; or the regulatory molecule is a TT1 upregulatory molecule that promotes the interaction between TT1 and SCE (TT1-SCE module), thereby promoting TT1-mediated SCE degradation, and thus improving plant heat tolerance or yield under heat stress; or the regulatory molecule is an SCE-Hsp interaction inhibitory molecule that reduces Hsp SUMOylation and increases Hsp accumulation, thereby enhancing Hsp-mediated protein folding, and thus improving plant heat tolerance or yield under heat stress; wherein the SCE includes: SCE1 or SCE2.

[0023] In one or more embodiments, the SCE downregulating molecule includes: reagents for knocking out or silencing SCE, reagents for inhibiting SCE activity, and reagents for reducing SCE stability; preferably, it includes: CRISPR gene editing reagents, homologous recombination reagents, or site-directed mutagenesis reagents targeting SCE, wherein the reagents induce loss-of-function mutations in SCE, interfering molecules specifically interfere with the expression of the coding gene for SCE, and reagents for upregulating TT1 to promote its mediated ubiquitination and degradation of SCE (including TT1 upregulating molecules); more preferably, the SCE downregulating molecule includes: CRISPR gene editing-based sgRNA or reagents capable of forming the sgRNA, wherein the reagents for forming the sgRNA include: primers with sequences shown in SEQ ID NO:17 and SEQ ID NO:18 (targeting SCE1), primers with sequences shown in SEQ ID NO:19 and SEQ ID NO:20 (targeting SCE1), and primers with sequences shown in SEQ ID NO:39 and SEQ ID NO:40 (targeting SCE2).

[0024] In one or more embodiments, the TT1 upregulation molecule includes: transferring the coding gene of TT1 or an expression construct or vector containing the coding gene into a plant; performing a gain-of-function mutation on TT1; promoting TT1 expression by expressing an enhancing promoter or a tissue-specific promoter; or promoting TT1 expression by an enhancer.

[0025] In one or more embodiments, the terms "upregulation," "promotion," "enhancement," or "boost" indicate a significant upregulation, promotion, enhancement, or boost, such as an upregulation, promotion, enhancement, or boost of 20%, 40%, 60%, 80%, 90%, or higher.

[0026] In one or more embodiments, the terms "down-regulation," "suppression," "reduction," "weakening," or "attenuation" refer to significant down-regulation, suppression, reduction, weakening, or attenuation, such as down-regulation, suppression, reduction, weakening, or attenuation by 20%, 40%, 60%, 80%, 90%, or lower.

[0027] In one or more embodiments, the loss-of-function mutation includes: causing premature termination of translation of the target protein, thereby causing loss of function; for example, by gene editing, inserting, deleting or mutating bases in the coding gene to cause premature termination of translation of the target protein.

[0028] In one or more embodiments, the plant is or the SCE is derived from plants including the group consisting of: grasses, legumes, cruciferous plants, and solanaceous plants (such as *Solanum* and *Capsicum*); preferably, the plant is or the SCE is derived from plants including the group consisting of: rice (*Oryza sativa*), corn (*Zea mays*), millet (*Setaria italica*), barley (*Hordeum vulgare*), wheat (*Triticum aestivum*), foxtail millet (*Panicum miliaceum*), sorghum (*Sorghum bicolor*), rye (*Secale cereale*), oats (*Avena sativa* L.), short-stalked grass (*Brachypodium distachyum*), soybean (*Glycine max*), potato (*Solanum tuberosum*), tomato (*Solanum lycopersicum*), chili pepper (*Capsicum annuum*), rapeseed (*Brassica napus*), and Arabidopsis thaliana.

[0029] In one or more embodiments, the proteins or genes in the SCE or the TT1-SCE-Hsp thermal stress response pathway containing it include their homologs.

[0030] In one or more embodiments, the SCE includes a cDNA sequence, a genomic sequence (gDNA), or a sequence that is artificially optimized or modified based on them.

[0031] In one or more embodiments, the amino acid sequence of the SCE polypeptide is selected from the group consisting of: (i) a polypeptide with the amino acid sequence shown in SEQ ID NO:4 or 8; (ii) a polypeptide derived from (i) having the regulatory trait function formed by substituting, deleting, or adding one or more (e.g., 1-20, 1-10, 1-5, 1-3) amino acid residues of the amino acid sequence shown in SEQ ID NO:4 or 8; (iii) a polypeptide having the regulatory trait function with an amino acid sequence homology ≥80% (preferably ≥82%, ≥85%, ≥90%, ≥95%, or ≥98%) to the amino acid sequence shown in SEQ ID NO:4 or 8; (iv) an active fragment of a polypeptide with the amino acid sequence shown in SEQ ID NO:4 or 8; or, (v) a polypeptide formed by adding a tag sequence or restriction enzyme site sequence to the N or C end of the polypeptide with the amino acid sequence shown in SEQ ID NO:4 or 8, or by adding a signal peptide sequence to its N end.

[0032] In another aspect of the invention, the use of SCE in plants or the TT1-SCE-Hsp heat stress response pathway containing it is provided for use as a molecular marker for identifying plant heat tolerance or yield under heat stress, or as a molecular marker for targeted screening of plants; preferably, the yield traits under heat stress include: survival rate, grain weight and / or seed setting rate.

[0033] In another aspect of the present invention, a method for selecting or identifying heat tolerance or yield under heat stress in plants is provided, comprising: identifying the expression or sequence characteristics of SCE in a test plant, or identifying the TT1-SCE-Hsp heat stress response pathway in the plant; if the test plant has low or no SCE expression, it is a plant with high heat tolerance or high yield under heat stress; if the test plant has high SCE expression, it is a plant that is heat-sensitive or has low yield under heat stress.

[0034] In one or more embodiments, high expression or high activity refers to a statistically significant increase in expression or activity compared to the average expression or activity of similar or identical plants.

[0035] In one or more embodiments, the low expression or low activity refers to a statistically significant reduction in expression or activity compared to the average expression or activity of similar or identical plants.

[0036] In another aspect of the present invention, a method is provided for screening substances (potential substances) that can improve plant heat tolerance or yield under heat stress, comprising: (1) adding the candidate substance to a system expressing SCE; (2) detecting the system and observing the expression, activity or stability of SCE therein, and if its expression, activity or stability is improved, it indicates that the candidate substance is a substance that can be used to improve plant heat tolerance or yield under heat stress.

[0037] In another aspect of the present invention, a method is provided for screening substances (potential substances) that regulate plant heat tolerance or yield under heat stress, comprising: (1) adding the candidate substance to a system expressing the TT1-SCE-Hsp heat stress response pathway; (2) detecting the system and observing the expression, activity, or interaction of the TT1-SCE-Hsp heat stress response pathway proteins therein, and if it promotes the interaction between TT1 and SCE or inhibits the interaction between SCE and Hsp, then the candidate substance is a substance that can be used to improve plant heat tolerance or yield under heat stress.

[0038] In one or more embodiments, promoting the interaction between TT1 and SCE includes: promoting TT1-mediated SCE degradation (degradation via ubiquitinated protein degradation pathway);

[0039] In one or more embodiments, the inhibition of SCE-Hsp interaction includes: reducing SCE-mediated SUMOylation of Hsp and increasing Hsp accumulation (preventing its degradation).

[0040] In one or more embodiments, a control group is also included to clearly distinguish the differences in SCE expression, activity, or stability between the test group and the control group, or the differences in TT1-SCE interaction, SCE-Hsp interaction, and the control group.

[0041] In one or more embodiments, the candidate substances include (but are not limited to): regulatory molecules (such as upregulatory molecules, small molecule compound gene editing constructs, etc.) designed for SCE, TT1, Hsp proteins or their encoding genes or their upstream or downstream proteins or genes.

[0042] Other aspects of the invention will be apparent to those skilled in the art from the disclosure of this invention. Attached Figure Description

[0043] Figure 11. In vivo and in vitro interaction verification of SCE1 and TT1. (A) Yeast two-hybrid (Y2H) assay to clarify the interaction between SCE1 and TT1. (B) Pull-down assay of TT1 and SCE1 in E. coli. MBP and SCE1 served as controls. Anti-MBP and anti-GST were used to detect the fusion protein signal. (C) Co-IP assay to show the in vivo interaction between SCE1 and TT1. TT1-YFP and SCE1-Flag were co-transfected into rice protoplasts, with YFP and SCE1-Flag serving as negative controls. Immunoprecipitation was performed using anti-GFP antibody, and Western blotting was performed using anti-Flag antibody. (D) SFLC assay to detect the interaction between SCE1 and TT1 in tobacco (N. benthamiana) leaves. nLUC and cLUC represent the N-terminal and C-terminal regions of luciferase, respectively. One positive control and two negative controls were performed on the same tobacco leaf. (E) BiFC assay of TT1 and SCE1 in tobacco cells. H2B-mCherry was used as a nucleoprotein marker. The scale bar is 50 μm.

[0044] Figure 2 Thermotolerant phenotypes of SCE1 gene knockout and overexpression plants at the seedling stage. (A) Three SCE1 gene knockout types were constructed using CRISPR-Cas9 in WYJ background material. (B) Phenotypes of WYJ, sce1-ko, and SCE1-OE seedlings at 14 days of growth were observed before and after heat treatment. The heat treatment environment was 42℃ and relative humidity >90%. The heat treatment time in the left figure was about 20h, and the heat treatment time in the right figure was about 15h. The scale bar was 2cm. (C) qRT-PCR was used to detect the expression level of SCE1 in SCE1-OE. n=3 biological replicates. (D) and (E) are the statistical survival rates of sce1-ko and SCE1-OE, respectively. In (B, D, and E), n=3 biological replicates, with 24 rice plants in each biological replicate. The data in (CE) are mean±SEM, and significant differences were determined by one-way ANOVA and Tukey multiple comparison test (p<0.05).

[0045] Figure 3SCE1 is a ubiquitinated substrate protein. (A) The protein content of endogenous SCE1 in WYJ was detected at 0h, 1h, 2h and 4h after heat treatment. Anti-SCE1 antibody was used to detect the protein content of SCE1, and anti-Actin was used to ensure consistent loading. (B) Protein content of SCE1 in WYJ, tt1-ko and TT1-OE. (C) Protein content of SCE1 in WYJ and tt1-ko. (D) Protein content of endogenous SCE1 in WYJ and tt1-ko under different heat treatment times and 75μM MG132 treatment. (E) Degradation rate of His-SCE1 protein purified by E. coli in leaf milling broth of WYJ and tt1-ko. One group was treated without MG132, and the other group was treated with 75μM MG132. (F) Detection of polyubiquitin chains of SCE1 protein using anti-Ubiquitin, anti-Ubiquitin-K48, and anti-Ubiquitin-K63 antibodies. High molecular weight (HMW) signals above 55 kDa in immunofluorescence assays indicate ubiquitinated SCE1. Black asterisks indicate nonspecific heat-induced proteins.

[0046] Figure 4 Thermotolerant phenotypes of TT1 gene knockout and overexpression plants at the seedling stage. (A) Three TT1 gene knockout types were constructed using CRISPR-Cas9 in WYJ background material. (BC) Phenotypes of WYJ, TT1-OE, and tt1-ko seedlings at 14 days of growth were observed before and after heat treatment. (C) Phenotypes were observed after heat treatment and recovery at room temperature for 7 days. The heat treatment environment was 42℃ and relative humidity >90%. The heat treatment time in the left figure was about 22h, and the heat treatment time in the right figure was about 15h. The scale bar is 2cm. (D) qRT-PCR was used to detect the expression level of TT1 in TT1-OE. n=3 biological replicates. (E) and (F) are the statistical survival rates of TT1-OE and tt1-ko, respectively. (BC, EF) n=3 biological replicates, with 24 rice plants in each biological replicate. The data in (DF) are mean ± SEM, and significant differences were determined by one-way ANOVA and Tukey's multiple comparison test (p < 0.05).

[0047] Figure 5Thermostability phenotypes of SCE1 and TT1 double mutant and double overexpression rice at the seedling stage. (A) Three CRISPR-Cas9 knockout lines of SCE1 and TT1 were constructed in WYJ. (B) Phenotypes of sce1 / tt1-ko and SCE1 / TT1-OE before heat treatment. Scale bar: 2 cm. (C) Phenotypes of sce1 / tt1-ko and SCE1 / TT1-OE after heat treatment. The heat treatment time in the left figure is approximately 20 h, and the heat treatment time in the right figure is approximately 15 h. Scale bar: 2 cm. (DE) Survival rates of sce1 / tt1-ko and SCE1 / TT1-OE were statistically analyzed. n = 2 biological replicates; each replicate included 24 plants. Data are expressed as mean ± SEM, and significant differences were determined by one-way ANOVA and Tukey multiple comparison test (p < 0.05).

[0048] Figure 6 SCE1 participates in the response of SUMOylation modification in vivo under heat stress. (A) SCE1 interacts with SUMO1 in yeast two-hybrid assays. SCE1 interacts with three SUMO E3 ligases in yeast two-hybrid assays. These three SUMO E3 ligases are SIZ1, MMS21, and SIZ2. (B) SFLC assay detects the fluorescent signals of SCE1 interaction with SIZ1, MMS21, and SIZ2 in tobacco cells. (C) SUMOylation modification levels in WYJ, SCE1-OE, and sce1-ko. Anti-AtSUMO1 assay detects SUMO1 modification levels in rice. Protein bands above 70 kDa are considered substrate proteins for SUMOylation modification. (D) SUMOylation modification levels in rice cells during heat treatment from 0 to 20 h. Black pentagrams represent non-specific heat-induced proteins. (E) Changes in SUMOylation modification of SCE1-OE and sce1-ko at different heat treatment time points. (F) Changes in SUMOylation modification of tt1-ko at different heat treatment time points. In (CF), RuBisco is stained with Ponceau S, and RuBisco and Anti-Actin represent the same total protein loading amount. Black pentagrams represent non-specific heat-induced proteins.

[0049] Figure 7Transcriptomic and proteomic analyses revealed that SCE1 influences protein folding and refolding pathways involved in heat stress. (A) Venn plot analysis of differentially expressed genes identified in WYJ and sce1-ko under normal and heat-treated conditions. (B) GSEA analysis of differentially expressed genes in WYJ and sce1-ko after heat treatment. (CD) Enrichment analysis of translation and ribosomal pathways in WYJ and sce1-ko after heat treatment. NES represents the enrichment score normalized to gene size; the green curve represents the running enrichment score of the gene set; the black line represents a gene in the gene set and its corresponding ranking; red indicates a positive correlation with the sce1-ko background after heat treatment; blue indicates a negative correlation with the WYJ background after heat treatment. (E) Heatmap of transcriptomic analysis of WYJ and sce1-ko before and after heat treatment in translation, protein misfolding, and protein refolding enrichment pathways. (FH) Expression analysis of OsCpn60β1 (F), OsCYP20-2 (G), and OsHsp78.4 (H) in WT and sce1-ko before and after heat treatment. n = 3 biological replicates, data are presented as mean ± SEM, and significant differences were determined by one-way ANOVA and Tukey multiple comparison test (p < 0.05). (I) The experimental workflow included: protein extraction, protein purification and digestion, LC-MS / MS, and data analysis. Right figure: Top 25 KEGG pathways enriched by candidate interacting proteins. (JK) SFLC validation of the interactions between SCE1 and Hsp24.1, and between SCE1 and Hsp40.

[0050] Figure 8 Hsp24.1 and Hsp40 are substrate proteins for SUMOylation modification. (A) Hsp24.1 and Hsp40 can undergo SUMOylation modification in E. coli SUMOylation modification experiments. The control group consisted of a single BL21(DE3) E. coli clone containing OsSUMO1, AtE1 (E1 activator), and substrate proteins, but not OsSCE1 (E2 activator). The experimental group consisted of strains containing OsSUMO1, AtE1 (E1 activator), OsSCE1 (E2 activator), and substrate proteins. (B) Potential SUMOylation modification sites for Hsp24.1 and Hsp40 were predicted using GPS-SUMO 2.0. The black asterisks represent the four sites of Hsp24.1, where Hsp24.1-M is a mutation of four lysine residues to arginine residues. The four sites of Hsp40 are arranged in descending order of probability score. (C) In vitro SUMOylation assays showed that the four lysine residues of Hsp24.1 are SUMOylation modification sites. Hsp24.1 showed a SUMOylation modified band, while Hsp24.1-M did not. Blue triangles represent the bands of the background protein, and red triangles represent the bands of the SUMOylated protein.

[0051] Figure 9 Detection of Hsp24.1 protein content in different transgenic materials after heat treatment. (AB) Hsp24.1 protein content in WYJ, sce1-ko, and SCE1-OE under continuous heat treatment. Hsp24.1 protein content was detected using a specific antibody against Hsp24.1. Rubisco staining after Ponceau S staining can be used to represent the same total protein loading amount. (C) Changes in Hsp24.1 expression levels in WYJ and sce1-ko under heat treatment. (DE) Hsp24.1 protein content in WYJ and tt1-ko under continuous heat treatment. Blue triangles represent Hsp24.1 protein, and black stars represent non-specific heat-induced protein. (FG) Degradation rates of purified MBP-Hsp24.1 and MBP-Hsp24.1M proteins from *E. coli* in leaf pulp of WYJ and sce1-ko. Rubisco staining was used as an internal control to ensure equal total protein loading. The figure shows the statistical analysis of the immunoblotting data. (H) Thermoprotective effect of Hsp24.1 on NdeI. The cleavage efficiency of the NdeI endonuclease on DNA fragments was evaluated at different temperatures: 37℃, 40℃, 45℃, and 50℃. The upper band represents uncleaved DNA fragments, and the lower band represents DNA fragments cleaved by NdeI. "-" indicates that no Hsp24.1 protein was added to the reaction system, "+" indicates that Hsp24.1 protein was added, and "+M" indicates that Hsp24.1M protein was added. In (A, B, E, F, G), data are expressed as mean ± standard deviation (n = 3 biological replicates). Two-tailed Student's t-test was used to determine significance. *P < 0.5, **P < 0.01, ns indicates no significance.

[0052] Figure 10Hsp24.1 and Hsp40 gene knockout plants are sensitive to heat stress. (A) Two knockout types of the Hsp24.1 gene in japonica rice ZH11. (B) Phenotypes of WYJ and hsp24.1-ko seedlings at 14 days of growth before and after heat treatment. Scale bar: 2cm. (C) Survival rate of each treatment of hsp24.1-ko seedlings after heat treatment and 7 days of room temperature recovery. (D) Three Hsp40 gene knockout types were constructed using CRISPR-Cas9 in WYJ background material. (E) Photographs of the growth of WYJ and hsp40-ko seedlings at 14 days of growth before and after heat treatment. (F) Survival rate of hsp40-ko after heat treatment. The heat treatment environment was 42℃, relative humidity >90%, heat treatment for 20 hours, and survival rate was measured and photographed after 7 days of room temperature recovery. Scale bar: 2cm. In (C and F), n=3 biological replicates, with 24 rice plants in each replicate. Data are presented as mean ± SEM, and significant differences were determined by one-way ANOVA and Tukey's multiple comparison test (p<0.05).

[0053] Figure 11 Multiple sequence alignment and evolutionary analysis of SCE1. (A) Multiple sequence alignment of SCE1 protein with reported SCE amino acid sequences in the NCBI database. The alignment included SCE1 homologs from rice (OsSCE1, OsSCE2, and OsSCE3), and from species such as Arabidopsis thaliana, soybean (Glycine max), sorghum (Sorghum bicolor), maize (Zeamays), barley (Hordeum vulgare), wheat (Triticum aestivum), millet (Setaria italica), Brachypodium distachyon, potato (Solanum tuberosum), tomato (Solanumlycopersicum), pepper (Capsicum annuum), and rapeseed (Brassica napus). The amino acid sequence alignment of SCE1 protein was performed using DNAMAN software. (B) Phylogenetic tree of SCE1 constructed using MEGA 11. The evolutionary history of SCE1 was inferred using the Neighbor-Joining method and the JTT matrix model, employing 1,000 bootstrap calculations of node support rates. The numbers at the nodes represent the bootstrap values ​​based on these repetitions (representing the confidence level of the phylogenetic tree branch).

[0054] Figure 12Expression patterns and subcellular localization of SCE1 and SCE2. (A) SCE1 expression levels in various plant tissues were detected by qRT-PCR. S, R, L, 1-2cm YP, 5cm YP, 10cm YP, H, and 10-DAF G represent stem, root, leaf, 1-2cm young spikelet, 5cm young spikelet, 10cm young spikelet, glume, and seed 10 days after grain filling, respectively. This experiment had three biological replicates, and the error bar represents SEM. (B) Fluorescence localization signals of YFP and SCE1-YFP in rice protoplasts. Green fluorescence represents YFP expression signal. Red fluorescence represents DsRed-NLS, which is a nuclear localization marker. Purple fluorescence represents the autofluorescence of chloroplasts. The scale bar for the YFP control group was 5 μm, and the scale bar for the SCE1-YFP experimental group was 2 μm. (C) Fluorescence localization signal of SCE2-YFP in rice protoplasts. Green fluorescence represents YFP expression signal. Scale bar 25 μm.

[0055] Figure 13 The SCE2 gene knockout line exhibits strong resistance to heat stress. The phenotypes of sce2-ko seedlings at 14 days of age before and after heat treatment were compared. Survival rates of rice seedlings treated at 42℃ for 18 hours were recorded after a 7-day recovery period at room temperature. The scale bar is 2 cm. There were 3 biological replicates, with 24 rice plants in each replicate. Data are presented as mean ± SEM. Significant differences were determined using one-way ANOVA and Tukey's multiple comparison test (p < 0.05).

[0056] Figure 14 SCE1 and SCE2 have virtually no impact on the growth and development of rice. (A) Plant type of WYJ, sce1-ko, and SCE1-OE at the seedling stage. Scale bar: 10cm. (B) Statistical analysis of plant height and tiller number of WYJ, sce1-ko, and SCE1-OE. (C) Plant type of WYJ, sce2-ko, and SCE2-OE at the seedling stage. Scale bar: 10cm. (D) Statistical analysis of plant height and tiller number of WYJ, sce1-ko, and SCE1-OE. In (B) and (D), n>14 individual plants, data are mean±SEM, and significant differences are determined by two-tailed Student's t-tests. ns represents no significant difference, *** represents P<0.001, and **** represents P<0.0001.

[0057] Figure 15The SCE1 / SCE2 double gene knockout lines exhibit growth disadvantages. (A) The target site of sgRNA in SCE1 and SCE2 is located in the first exon after ATG. (B) SCE1 / SCE2 double gene knockout heterozygous plants do not produce seeds, while SCE2 single gene knockout heterozygous lines can produce seeds normally. (C) The spike and plant types of WYJ and SCE1 / SCE2 double gene knockout heterozygous plants (sce1 / sce2-ko) at the seedling stage.

[0058] Figure 16 1. Yield-related agronomic traits of SCE1 gene knockout lines during seedling stage under high temperature. (A) Ear morphology of WYJ and sce1-ko plants grown under normal conditions. Scale bar: 2.5 cm. (B) Pollen fertility observation of WYJ and sce1-ko plants after heat treatment. Heat treatment was performed in a high-temperature incubator (40℃ / 34℃, 12h daytime / 12h nighttime). Pollen was stained with KI-I2 and observed under a microscope. Scale bar: 200 μm. (C) Ear morphology of WYJ and sce1-ko plants after high-temperature treatment in a field greenhouse. Scale bar: 2.5 cm. (D) Seed setting phenotype of WYJ and sce1-ko plants after high-temperature treatment in a field greenhouse. Scale bar: 2.5 cm. (EF) Seed setting rate, thousand-grain weight, and yield per plant (n>14 individual plants) of WYJ and sce1-ko under normal conditions and after high-temperature treatment in a field greenhouse. (G) Yield measurement of plots after field high-temperature treatment. Each group had 3 plots, with 48 individual plants in each plot. (EG) Data are presented as mean ± SEM. Significant differences were determined by two-tailed Student's t-tests. ns represents no significant difference, ** represents P < 0.01, and **** represents P < 0.0001.

[0059] Figure 17 Schematic diagram of the TT1-SCE-Hsp heat stress response pathway. Top left: Heat stress induces a rapid increase in SUMOylation levels in plants, which then gradually decreases over time in wild-type rice, thereby reducing the heat tolerance of rice. Top right: SCE1 as a negative regulator of heat tolerance. The absence of SCE1 leads to a rapid decrease in high levels of SUMOylation in plants, contributing to the formation of heat-tolerant rice. Bottom diagram shows the molecular mechanism involved by SCE1. SCE1 interacts directly with TT1 and undergoes ubiquitination and protein degradation via the TT1-mediated 26S proteasome. Due to the absence of SCE1, Hsp24.1 and Hsp40 cannot undergo SUMOylation, leading to the accumulation of small heat shock proteins. Increased Hsp24.1 and Hsp40 help to refold unfolded proteins into their native state, ultimately enhancing the heat tolerance of plants. Detailed Implementation

[0060] This invention is the first to study and reveal a novel gene SCE (including SCE1 or SCE2) that encodes a polypeptide with important biological functions. Furthermore, it reveals an SCE-mediated TT1-SCE-Hsp heat stress response pathway and its application in improving plant heat tolerance or yield traits under heat stress.

[0061] This invention reveals for the first time that SCE1, a SUMO E2-binding enzyme, interacts with TT1 and, as a downstream component of TT1, participates in TT1-mediated 26S proteasome degradation. SCE1, as a negative regulator of heat tolerance, is a substrate protein for ubiquitination modification. Furthermore, this invention observes that small heat shock proteins (sHSPs) such as Hsp24.1 and Hsp40 can undergo SCE1-mediated SUMOylation modification, thereby increasing the protein accumulation of Hsp24.1 in the absence of SCE1. This invention further proposes that SCE1-regulated global SUMOylation modification serves as an important signal for responding to high-temperature stress, and a rapid decrease in SUMOylation levels is considered a positive response to increased heat tolerance after the loss of SCE1 gene function. Compared to the wild type, reducing SCE1 levels significantly increased grain yield under high-temperature stress, manifested as increased seed setting rate, pollen viability, and grain filling capacity. The results of this invention reveal the crucial role of SCE1 in TT1-mediated heat tolerance, regulating the abundance and SUMOylation of sHSP proteins, ultimately affecting plant heat tolerance. These findings highlight the significant potential of the TT1-SCE1 module in enhancing crop heat tolerance and its importance for the genetic improvement of plant stress resistance traits.

[0062] the term

[0063] As used in this invention, unless otherwise stated, "heat resistance" refers to "heat resistance under high temperature stress".

[0064] As used herein, the terms “heat resistance capacity / heat resistance”, “heat stress tolerance”, and “heat resistance capacity / heat resistance” are interchangeable.

[0065] As used in this article, the terms "thermal stress response pathway / module", "regulation pathway / module", "control pathway / module" and "signal pathway / module" are interchangeable.

[0066] As used in this invention, the term "(signaling) pathway" refers to a signaling system formed by the mutual constraints or interactions of a series of proteins or genes, which generally leads to the occurrence of certain cellular events.

[0067] As used herein, "plant" refers to a plant whose genome contains the TT1-SCE-Hsp heat stress response pathway of this invention or homologs (homologous genes / homologous polypeptides (proteins)) of genes / proteins involved in this pathway. The plant may include monocotyledonous or polycotyledonous plants; such as: grasses, cruciferous plants, legumes, solanaceous plants, etc.; specifically, grain crops such as millet, wheat, corn, sorghum, and soybeans, and agricultural crops such as vegetables, fruits, flowers, and forage grasses. Preferably, the plant includes grasses. In some preferred embodiments, the plant is a crop, preferably a cereal crop. Preferably, the grasses include rice (Oryza sativa), wheat (Triticum aestivum), and corn (Zea mays). Those skilled in the art should understand that the plants applicable to the technical solutions of this invention are not limited to those listed above, and suitable plants can be determined by identifying the presence of the TT1-SCE-HSP regulatory pathway.

[0068] As used in this invention, "grain" refers to the fruit or seed of a plant, and is also called ear grain in crops such as rice, corn, wheat, and barley.

[0069] As used herein, “homology” refers to a gene or protein derived from a species other than rice that has sequence identity (such as 70-99%, for example, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 98%, more than 99%) with TT1, SCE (including SCE1, SCE2) or Hsp (including Hsp24.1, Hsp40) of the present invention.

[0070] As used herein, "maintaining / increasing yield under high temperature" or "protecting plant yield under high temperature (stress)" refers to the ability of plants to survive and maintain normal growth under high temperature (temperatures higher than their conventional cultivation conditions), and to achieve a relatively normal, essentially normal, or increased yield. In some embodiments, "maintaining / increasing yield under high temperature" means that, after a specific plant is cultivated in a high-temperature environment, its yield is maintained at 30%, 40%, 50%, or 60% of the yield in a "non-high-temperature environment (normal / suitable temperature environment)"; preferably at 70%, 80%, 90%, or 100%; and even more preferably increased to 100%, 105%, 110%, 120%, 130%, 150%, 180%, or 200% of the yield. In other ways, "maintaining / increasing yield at high temperatures" means that after a specific plant is cultivated in a high-temperature environment, its yield is increased to more than 100%, 105%, 110%, 120%, 130%, 150%, 180%, or 200% of the yield of other plant varieties (or unmodified plants) in a high-temperature environment.

[0071] As used herein, “high temperature” or “heat (environment)” refers to a temperature significantly higher than the suitable temperature for plant growth (e.g., 26–30°C for rice, with 28–30°C being the optimal temperature); for example, “high temperature” or “heat (environment)” refers to 35°C or higher, 38°C or higher, 40°C or higher, or 42°C or higher.

[0072] As used herein, “upregulation” includes: promotion, overexpression, enhancement, etc., which are statistically significant or marked upregulation, promotion, enhancement, or enhancement, such as upregulation, promotion, enhancement, or enhancement of 20%, 40%, 60%, 80%, 90%, or higher.

[0073] As used herein, “downregulation” includes: weakening, reducing, lowering, inhibiting; indicating significant downregulation, weakening, lowering, inhibiting, such as downregulation, weakening, lowering, inhibiting or downregulating by 20%, 40%, 60%, 80%, 90% or lower.

[0074] As used herein, "heat tolerance" or "heat capacity" refers to a plant's ability to withstand hot environments. Typically, the heat tolerance of a test plant is determined by comparing it to a control plant under the same temperature conditions. A test plant is generally considered to have better heat tolerance if it has a higher survival rate and longer survival time at higher temperatures.

[0075] As used herein, “high heat tolerance” refers to a statistically significant increase in the heat tolerance of a plant (e.g., a modified plant) compared to the heat tolerance of similar or identical plants, such as an increase in survival rate of 5%, 10%, 20%, 40%, 60%, 80%, 90%, or higher.

[0076] As used herein, “high expression or high activity” means that the expression or activity of a target gene / protein in a specific plant (e.g., a modified plant) is statistically significantly increased compared to the average expression or activity of the same type or plant species, such as by 10%, 20%, 40%, 60%, 80%, 90%, or higher.

[0077] As used herein, “low expression or low activity” means that the expression or activity of a target gene / protein in a particular plant (e.g., a modified plant) is statistically significantly reduced compared to the average expression or activity of the same type or plant species, such as by 10%, 20%, 40%, 60%, 80%, 90%, or less.

[0078] As used herein, "loss-of-function mutations" include those that cause a target protein to lose its function, such as through mutations, deletions, or insertions in key regions of its protein chain. In some cases, the insertion, deletion, or mutation of bases in the gene encoding the target protein causes premature termination of translation.

[0079] As used herein, “gain-of-function mutations” include: enabling the normal expression of a target protein that was previously restricted or not expressed; and in some ways, reverting bases in the target protein coding gene (e.g., reverting to a sequence identical or degenerate to the wild type) to enable the target protein to be expressed again.

[0080] As used herein and as will be understood by those skilled in the art, selecting an appropriate “control plant” is a routine part of experimental design and may include a corresponding wild-type plant or a transgenic plant without the target gene. Control plants are generally the same plant species or even varieties of the same or the same class as the plant being evaluated. Control plants may also be individuals from which the transgenic plant has been lost due to segregation. As used herein, control plants refer not only to whole plants but also to plant parts, including seeds and seed portions.

[0081] SCE

[0082] As used in this invention, the SCE includes SCE1 or SCE2. Preferably, the SCE refers to a polypeptide or its encoding gene having the sequence SEQ ID NO:4 or 8, and also includes sequence variations having the same function as the SCE polypeptide. The encoding gene can be gDNA or cDNA, and may also contain a promoter. For example, the gDNA has the nucleotide sequence shown in SEQ ID NO:2 or 6, the cDNA has the nucleotide sequence shown in SEQ ID NO:3 or 7, and the promoter has the nucleotide sequence shown in SEQ ID NO:1 or 5. The sequence of the encoding gene also includes sequences degenerate with the sequences provided in this invention.

[0083] SCE1 and SCE2 share high homology and are evolutionarily conserved, playing a crucial role in maintaining plant growth and development. Single-gene knockout of either SCE1 or SCE2 does not affect plant growth and development, with single-gene knockout being preferred.

[0084] Variations of the SCE polypeptide include (but are not limited to): deletions, insertions, and / or substitutions of several amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, and even more preferably 1-8 or 1-5); and additions or deletions of one or more amino acids (typically up to 20, preferably up to 10, and more preferably up to 5) at the C-terminus and / or N-terminus. Any protein with high homology to the SCE polypeptide (e.g., 70% or higher homology to the polypeptide sequence shown in SEQ ID NO:4 or 8; preferably 80% or higher; more preferably 90% or higher, such as 95%, 98%, or 99% homology) and having the same function as the SCE polypeptide is also included in this invention. Peptides derived from species other than rice that have high homology to the sequence shown in SEQ ID NO:4 or 8, or that play the same or similar role in the same or similar regulatory pathways, are also included in this invention.

[0085] In this invention, "SCE" also includes its homologs. It should be understood that although SCE obtained from a specific rice species is preferred in this invention, other polypeptides or genes obtained from other species that are highly homologous to the SCE (e.g., having more than 60%, such as 70%, 80%, 85%, 90%, 95%, or even 98% sequence identity) are also within the scope of this invention.

[0086] The polynucleotide (gene) encoding the SCE polypeptide can be a natural gene from a plant or a degenerate sequence thereof.

[0087] Vectors containing the said coding sequence, and host cells genetically engineered using the said vector or polypeptide coding sequence, are also included in this invention. Methods well known to those skilled in the art can be used to construct suitable expression vectors.

[0088] The host cell is usually a plant cell. Plant transformation can generally be achieved using methods such as Agrobacterium-mediated transformation or gene gun transformation.

[0089] TT1-SCE-HSP Thermal Stress Response Pathway

[0090] The TT1-SCE-HSP heat stress response pathway includes (but is not limited to): the SCE gene (and / or its encoded protein), the TT1 gene (and / or its encoded protein), and the HSP gene (and / or its encoded protein).

[0091] As used in this invention, the terms “TT1-SCE-HSP thermal stress response pathway” and “TT1 / SCE / HSP thermal stress response pathway” are used interchangeably.

[0092] The nucleotide sequence of the TT1 (Thermos-Tolerance 1) gene is shown, for example, as LOC_Os03g26970; its protein amino acid sequence is shown, for example, as LOC_Os03g26970 (or Uniprot:Q10KF0). TT1 encodes the α2 subunit of the 26S proteasome, and its mechanism of action is to ensure normal cellular protein metabolism by promptly and effectively clearing the large amounts of denatured proteins produced in cells under high-temperature stress. Currently, the regulatory pathway mediated by TT1 is still unclear in the art, and its upstream and downstream molecules are also unknown.

[0093] As used in this invention, small heat shock proteins (HSPs) are a family of ATP-independent molecular chaperones. Under stress conditions, they can form large oligomers that effectively bind misfolded substrate proteins, protecting them from harmful effects. Heat stress also leads to the over-accumulation of misfolded proteins in the endoplasmic reticulum (ER), inducing ER stress and triggering the unfolded protein response (UPR) in plants.

[0094] Preferably, Hsp24.1 refers to a polypeptide having the sequence SEQ ID NO:12 or its encoding gene, and also includes sequence variations having the same function as the polypeptide. The encoding gene can be gDNA or cDNA, and may also contain a promoter. For example, the gDNA has the nucleotide sequence shown in SEQ ID NO:10, the cDNA has the nucleotide sequence shown in SEQ ID NO:11, and the promoter has the nucleotide sequence shown in SEQ ID NO:9. The sequence of the encoding gene also includes sequences degenerate with those provided by this invention.

[0095] Preferably, Hsp40 refers to a polypeptide having the sequence SEQ ID NO:16 or its encoding gene, and also includes sequence variations having the same function as the polypeptide. The encoding gene can be gDNA or cDNA, and may also contain a promoter. For example, the gDNA has the nucleotide sequence shown in SEQ ID NO:14, the cDNA has the nucleotide sequence shown in SEQ ID NO:15, and the promoter has the nucleotide sequence shown in SEQ ID NO:13. The sequence of the encoding gene also includes sequences degenerate with those provided by this invention.

[0096] When used as targets for artificial regulation or in the creation of screening systems, the proteins or encoding genes mentioned above can be naturally occurring, such as those purified and isolated from mammals; or they can be recombinantly prepared, for example, recombinant proteins can be produced using conventional gene recombination techniques. Furthermore, any variations that do not affect the biological activity of these proteins are acceptable, such as derivatives or variants whose function remains unchanged.

[0097] In the heat stress response pathway of this invention, SCE1, a SUMO E2-binding enzyme, interacts with TT1 and, as a downstream component of TT1, participates in TT1-mediated 26S proteasome degradation. SCE1, as a negative regulator of heat tolerance, is a substrate protein for ubiquitination modification. Furthermore, this invention observed that small heat shock proteins (sHSPs) such as Hsp24.1 and Hsp40 can undergo SCE1-mediated SUMOylation modification, thereby increasing Hsp24.1 protein accumulation in the absence of SCE1. This invention further proposes that SCE1-regulated global SUMOylation modification serves as an important signal for responding to high-temperature stress, and a rapid decrease in SUMOylation levels is considered a positive response to increased heat tolerance after SCE1 gene function loss. Compared to the wild type, reducing SCE1 levels significantly increased grain yield under high-temperature stress, manifested as increased seed setting rate, pollen viability, and grain filling capacity.

[0098] Applications in plant improvement

[0099] Based on the inventor's new discovery, the present invention provides a method for improving plants, the method comprising: regulating the expression, activity, stability or modification (such as ubiquitination or SUMOylation) of SCE or its involved TT1-SCE-HSP heat stress response pathway proteins in plants, thereby regulating plant heat tolerance or yield level under heat stress.

[0100] Based on the results demonstrated by embodiments of the present invention, in vitro and in vivo experiments have shown that SCE1 and TT1 have strong protein-protein interactions, and SCE1 participates in the heat stress regulatory network of TT1. SCE1 and TT1 respond to heat stress in the same genetic pathway, and SCE1 plays a role downstream of TT1. The genetic module composed of SCE1-TT1 provides a new strategy for breeding heat-resistant and high-yielding varieties.

[0101] SCE1 protein is prone to degradation under heat stress; SCE1 protein is modified by ubiquitination, and TT1, as the α2 subunit of the 26S proteasome, promotes the degradation of SCE1 by the 26S proteasome-mediated ubiquitination protein degradation pathway.

[0102] Transcriptomic and proteomic analyses revealed that SCE1 participates in the high-temperature response by regulating protein folding. Strong protein-protein interactions between Hsp24.1 and Hsp40 and SCE1 indicate that SCE1 regulates plant heat tolerance through the stability of proteins such as Hsp24.1 and Hsp40.

[0103] SCE1 functions as a SUMO E2 ligase. SUMOylation in plants is dynamic under heat stress, and SCE1 influences this dynamic process. Maintaining a low level of SUMOylation in plants after heat stress helps enhance their tolerance to heat stress at high temperatures. This can serve as an evaluation indicator of plant tolerance and as a strategy for improving crop heat resistance traits.

[0104] SCE1 can regulate the protein content of Hsp24.1. After heat treatment, the protein abundance of Hsp24.1 is higher in SCE1 gene knockout lines, while the protein abundance is lower in SCE1 gene overexpression lines and TT1 gene knockout lines.

[0105] With the participation of SCE1, Hsp24.1 and Hsp40 can undergo SUMOylation modification, preferably with K157, K192, K206, and K208 being the SUMOylation modification sites for Hsp24.1. The SUMOylation modification sites of Hsp24.1 make the Hsp24.1 protein unstable due to SUMOylation.

[0106] Hsp24.1 and Hsp40 are positive regulators of heat stress. Hsp24.1 acts as a molecular chaperone, protecting substrate proteins and maintaining their normal function under high temperatures. Loss of function in Hsp24.1 and Hsp40 leads to increased susceptibility to heat stress. Overexpression of Hsp24.1 and Hsp40 using biotechnology in other plants significantly increases their resistance to heat stress, thereby increasing yields under high temperatures. Hsp24.1 and Hsp40 provide new genetic resources for breeding heat-tolerant crops.

[0107] The SCE1 gene does not affect plant growth during the seedling or mature stages. Under high-temperature conditions, SCE1 knockout lines exhibit higher seed setting rates, stronger grain filling ability, and higher plot yields. SCE1 provides a new genetic resource for breeding highly heat-resistant crops. SCE1 is a highly conserved gene in plant evolution, widely present in various crops, and has promising application prospects. SCE1 provides a new genetic resource for breeding highly heat-resistant crops, and its application can be applied to a wide variety of plants.

[0108] It should be understood that, after understanding the functions of the SCE and the TT1-SCE-HSP heat stress response pathway (preferably also including its downstream genes), various methods well known to those skilled in the art can be used to regulate the expression or activity of the SCE or to regulate the TT1-SCE-HSP heat stress response pathway. The technical solution of this invention can be applied to molecular design breeding through various pathways.

[0109] In this invention, the upregulating molecules include promoters, agonists, and activators. The terms "upregulation" and "promotion" include "upregulation" and "promotion" of protein activity or protein expression, and these are statistically significant "upregulation" and "promotion." Any substance that can increase the activity of the target protein, improve the stability of the target protein, upregulate the expression of the target gene, or increase the effective duration of the target protein's action is acceptable. These substances can be compounds, small chemical molecules, or biomolecules. The biomolecules can be at the nucleic acid level (including DNA and RNA) or at the protein level.

[0110] In this invention, the downregulating molecule refers to any substance that can reduce the activity and stability of a target protein, downregulate the expression of a target protein, reduce the effective duration of the target protein's action, or inhibit the transcription and translation of a target gene. These substances can all be used in this invention as useful for downregulating targets. They can be compounds, small chemical molecules, or biomolecules. The biomolecules can be at the nucleic acid level (including DNA and RNA) or at the protein level. For example, the downregulating molecule is: an interfering RNA molecule or antisense nucleotide that specifically interferes with the expression of a target gene; or a gene-editing reagent that specifically edits the target gene, etc.

[0111] After understanding the function of the SCE or TT1-SCE-HSP heat stress response pathway, it can be used as a molecular marker for targeted plant screening. This new discovery can also be used to screen for substances or potential substances that can target and regulate plant seed traits or hormone traits by modulating this mechanism.

[0112] Therefore, the present invention provides a method for targeted selection or identification of plants, the method comprising: adding a candidate substance to a system expressing SCE; detecting the system and observing the expression, activity or stability of SCE therein; if its expression, activity or stability is improved, it indicates that the candidate substance is a substance that can be used to improve the heat tolerance or yield of plants under heat stress.

[0113] This invention also provides a method for regulating plant heat tolerance or yield levels under heat stress (potential substances), comprising: adding a candidate substance to a system expressing the TT1-SCE-Hsp heat stress response pathway; detecting the system and observing the expression, activity, or interaction of proteins in the TT1-SCE-Hsp heat stress response pathway; if it promotes the interaction between TT1 and SCE or inhibits the interaction between SCE and Hsp, it indicates that the candidate substance is a substance that can be used to improve plant heat tolerance or yield under heat stress; preferably, promoting the interaction between TT1 and SCE includes: promoting TT1-mediated SCE degradation (degradation via ubiquitinated protein degradation pathway); preferably, inhibiting the interaction between SCE and Hsp includes: reducing SCE-mediated SUMOylation modification of Hsp and increasing Hsp accumulation (preventing its degradation).

[0114] Methods for screening substances that act on proteins or genes or specific regions thereof as targets are well known to those skilled in the art, and these methods can all be used in this invention. The candidate substances can be selected from: peptides, polymeric peptides, peptide-like substances, non-peptide compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, small organic molecules, small inorganic molecules, and nucleic acid sequences, etc. Depending on the type of substance to be screened, those skilled in the art understand how to select an appropriate screening method.

[0115] Through large-scale screening, a class of potential substances that specifically act on the SCE or TT1-SCE-HSP heat stress response pathway can be obtained, which have a regulatory effect on plant heat tolerance and yield traits.

[0116] The present invention is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions, such as those described in J. Sambrook et al., *Molecular Cloning: A Laboratory Manual*, or according to the manufacturer's recommendations.

[0117] Materials and methods

[0118] 1. Gene and protein sequences

[0119] OsSCE1(LOC_Os10g39120):

[0120] >Promoter sequence (SEQ ID NO:1)

[0121]

[0122] Full genome sequence (underlined start and stop codons) (SEQ ID NO:2)

[0123] GGAAAATAATAATACAAGAGACCGAACGACTAGAGATTAGAGAAGGCGGTGCTTCTTCTTCTTTTCCAGAGACTACCAAGCCCAAACCAGCAACCACCCGCCGCCCGCC ATG TGA GGACATTGGCCTAATAAAGTTACTACCCGATTCATCATGCACATCCGCATAGTAGTTTGCAAAATCTCAGATTATTGCACCAGAAAATATCGGATTTTATGGGATATAACCTAAAATGGAATGTTTGAAGAACTGTCCAATATCATTGTGGACTTTTTGTGCTGTCATTACCATTTGTGCTGTGTGAAGAACAGAAATAGCTTTGTCAACTGTTGAAACCGCTTGAGTGATGGATGCTAATTTGGATAACAAATGAACTAAATCCCTGGTCGTACCAACTCTCTTGAAGAGAAAATTGCCTTGTACTGTTTTGCAATTCAACTTGCTTGTGAGTCCTTTCTTCGTGTTTCCATAATTCCATTTATAAAGCGTTAGTGTATCTAATCCAAACCATAA

[0124] >Coding region sequence (SEQ ID NO:3)

[0125] ATGTCGGGAGGGATCGCACGCGGCCGCCTCGCGGAGGAGCGCAAGGCCTGGCGGAAGAACCACCCTCACGGGTTCGTGGCGAAGCCGGAGACGATGGCCGACGGGTCGGCGAACCTCATGATCTGGCACTGCACCATCCCCGGCAAGCAGGGGACCGATTGGGAAGGTGGGTACTACCCTCTTACCCTTCACTTCAGTGAGGACTATCCTAGCAAACCACCCAAGTGCAAGTTCCCACAGGGCTTTTTCCACCCAAATGTCTATCCTTCAGGAACAGTGTGCCTCTCAATTCTTAATGAGGATAGTGGCTGGAGACCTGCTATCACTGTAAAGCAGATCCTTGTTGGAATACAGGACTTGCTTGATCAGCCAAATCCTGCTGATCCTGCACAGACTGACGGTTATCACATTTTTATACAGGACAAACCAGAATATAAGAGGCGTGTTCGTGTTCAGGCCAAGCAGTACCCTGCTTTGCTTTGA

[0126] Protein sequence (SEQ ID NO:4)

[0127] MSGGIARGRLAEERKAWRKNHPHGFVAKPETMADGSANLMIWHCTIPGKQGTDWEGGYYPLTLHFSEDYPSKPPKCKFPQGFFHPNVYPSGTVCLSILNEDSGWRPAITVKQILVGIQDLLDQPNPADPAQTDGYHIFIQDKPEYKRRVRVQAKQYPALL

[0128] OsSCE2(LOC_Os03g03130):

[0129] >Promoter sequence (SEQ ID NO:5)

[0130]

[0131] Full genome sequence (underlined start and stop codons) (SEQ ID NO:6)

[0132] GACTTGCGCCACCCACCCCACCCAAACCCCCACCGCCGGAGGAAGGAGGAGACGCCGCCGCCGCCGCCGCAGCC ATG TGA GAATATTGGCATAGGGACCCATGCTGCACATTCGCTTAGTTCTTGCTAGATCTAAAATGAGTGCCTCTGCAAATATGGATTAGACCAAAATATGACCTGGAAAAAGGGTGCTTTCGGAGTTTGAGAAGTATTATCATCGTTGTGGACTAATGTCTTGTGAGATCCGTCTTGGATGTAAACGGTTTTCTTAACTGTGATTTTTCAACCGTTTGAGTGCTTGCTTTATGGGATAATGTCAAACATTGGTTTGGCAGGCAATCTCAAGAGTATTGCTATTTCTGTGCTAGTCTATATGAAAACCCAGATTAAATTCGACGAAACATATTAGTTTGCAAAAAGATGTGGCAACTTCCATATTTTCTGCATTTTTTTCTGTTTTGATCCGGATCTGTCGGCTATACAGAGGTGAAAATAATGTAAACGTCGGAGGCGTAAGTACACAGTGCACCTGCTTTGCTCAGAATTTCTCTAA

[0133] >Coding region sequence (SEQ ID NO:7)

[0134] ATGTCGGGGGGAATCGCGCGCGGCCGCCTCGCGGAGGAGCGGAAGGCGTGGCGGAAGAACCACCCACACGGTTTCGTCGCCAAGCCGGAGACGTTGGCCGACGGGACGGTCAACCTCATGATCTGGCACTGCACAATCCCCGGCAAGCAAGGGACTGATTGGGAAGGTGGATACTTTCCTCTCACTCTTCATTTCAGTGAGGATTACCCTAGCAAACCTCCCAAGTGCAAGTTCCCACAGGGTTTCTTCCACCCAAATGTCTATCCTTCAGGGACAGTCTGCCTTTCAATTCTTAATGAAGACAGCGGTTGGAGACCTGCTATTACCGTCAAGCAAATTCTTGTTGGAATCCAGGACTTGCTTGATCAGCCTAATCCTGCTGATCCTGCTCAGACCGATGGTTACCATCTTTTTATCCAGGATCCTACGGAATACAAGAGGCGTGTTCGGCTGCAGGCCAAGCAGTATCCTCCGATTGTCTGA

[0135] >Protein sequence (SEQ ID NO:8)

[0136] MSGGIARGRLAEERKAWRKNHPHGFVAKPETLADGTVNLMIWHCTIPGKQGTDWEGGYFPLTLHFSEDYPSKPPKCKFPQGFFHPNVYPSGTVCLSILNEDSGWRPAITVKQILVGIQDLLDQPNPADPAQTDGYHLFIQDPTEYKRRVRLQAKQYPPIV

[0137] OsHsp24.1 (LOC_Os02g52150):

[0138] >Promoter sequence (SEQ ID NO:9)

[0139]

[0140] >Full gene sequence (start codon and stop codon underlined) (SEQ ID NO:10)

[0141] ATTCTCAACGCAAAATCCAAAAGATAAGCACAGTTACGCAGCGAGAGCGAGAGAGGAGTGGAGAGCC AT G GCTTCCATCGTGGCATCGAAGAGGATCCCACTGTTCCGCCTCGTGGAGCAGCTCCTCGCCGCGTCGCCGGCCCAAGGCGCCGCCTCCGCTCTCAGGCCGGTGGCCGTCGCCGGCGGGTCCCGTGCATACAACACCGGCGCGCAGCTCCGCCGGCACGAGAGGGACGAGTCGGACGACGACAGCGGCCGTGGGTACGACACCAGGCGCCCCACACGCGACGCCACCATGCCCGCCTTCTTCTCAGGTAGTCGTCATCTCTCGCCGTGTCAAGCTCATCCGGAGTTTGCACCAATTGATCTCATCAAATCGCTGTCCTGAACATGTTCTCGATTTCCCGATCGATTTGCAGATGTGTTCCGTGATCCGTTCAGCGCGCCGCAGAGCCTCGGGCGGCTGCTGAGCCTCATGGACGACCTGGCGACGCCGGCCGGGCGCGCCGGCGCCGCGACGCTGCGGCGCGGGTGGAACGCGAAGGAGAGCGAGGAGGCGCTGCACCTGAGGGTGGACATGCCGGGCCTGGGGAAGGAGCACGTGAAGGTGTGGGCGGAGCAGAACAGCCTCGTGATCAAGGGCGAGGGGGAGAAGGAGGCCGGCGAGGACGAGGGCGCCGCCCCGGCGAGGTACAGCGGCCGCATCGAGCTCGCGCCGGAGGTGTACAGGATGGACCAGATCAAGGCGGAGATGAAGAACGGCGTGCTCAAGGTGGTCGTGCCCAAGGTGAAGGAGGAGCAGCGCAGGGACGTCTTCCAGGTCAACGTCGAG TAG

[0142] > Coding region sequence (SEQ ID NO:11)

[0143] ATGGCTTCCATCGTGGCATCGAAGAGGATCCCACTGTTCCGCCTCGTGGAGCAGCTCCTCGCCGCGTCGCCGGCCCAAGGCGCCGCCTCCGCTCTCAGGCCGGTGGCCGTCGCCGGCGGGTCCCGTGCATACAACACCGGCGCGCAGCTCCGCCGGCACGAGAGGGACGAGTCGGACGACGACAGCGGCCGTGGGTACGACACCAGGCGCCCCACACGCGACGCCACCATGCCCGCCTTCTTCTCAGGTAATGTGTTCCGTGATCCGTTCAGCGCGCCGCAGAGCCTCGGGCGGCTGCTGAGCCTCATGGACGACCTGGCGACGCCGGCCGGGCGCGCCGGCGCCGCGACGCTGCGGCGCGGGTGGAACGCGAAGGAGAGCGAGGAGGCGCTGCACCTGAGGGTGGACATGCCGGGCCTGGGGAAGGAGCACGTGAAGGTGTGGGCGGAGCAGAACAGCCTCGTGATCAAGGGCGAGGGGGAGAAGGAGGCCGGCGAGGACGAGGGCGCCGCCCCGGCGAGGTACAGCGGCCGCATCGAGCTCGCGCCGGAGGTGTACAGGATGGACCAGATCAAGGCGGAGATGAAGAACGGCGTGCTCAAGGTGGTCGTGCCC AAGGTGAAGGAGGAGCAGCGCAGGGACGTCTTCCAGGTCAACGTCGAGTAG

[0144] > Protein sequence (the amino acids marked with underline are SUMOylation modification sites) (SEQ ID NO:12)

[0145] MASIVASKRIPLFRLVEQLLAASPAQGAASALRPVAVAGGSRAYNTGAQLRRHERDESDDDSGRGYDTRRPTRDATMPAFFSGNVFRDPFSAPQSLGRLLSLMDDLATPAGRAGAATLRRGWNAKESEEALHLRVDMPGLGKEHVKVWAEQNSLVIK GEGEKEAGEDEGAAPARYSGRIELAPEVYRMDQI K AEMKNGVLKVVVP K V K EEQRRDVFQVNVE

[0146] OsHsp40(LOC_Os08g06460):

[0147] >Promoter sequence (SEQ ID NO:13)

[0148]

[0149] >Full gene sequence (start codon and stop codon underlined) (SEQ ID NO: 14)

[0150] CCCTACTCGCTGTCGCCTTCCACCATCTCCGCCACCTTCTCCTTCCCTCCCAAGATCTCTTCTTTGGATGCCGCGCTGAAAGATTCGCTTTCTTGGTTGGTTGGTTGATCGATCCGGTGCCCGGATTTGGGGAGGAATTAAAGGTGGCGCCTTGGCGAAGCAGTAGATTCTTTGATTGCTTGTTTGATTGGTGGTTCTTGAGCGAGAAGAGAGAGACTGATTGATTGGTGGAGAAGCTGTGAGGTGGGGTTTGAAAGGAGGCGCGTTCTTGTGGGGGATTTGGGAAGGGGGGGGGGGGAATTTCGGTCTCGTTGGTTCGGATCTTGTTGGTGTGTAGTAGTAGTAGTTGGGCATGAGCGTCGGGGGAG ATG TGA GCACATTTTCAGAAGCAAATCGACTGTGCCATAGTTCTTGAGATAAACAGACTAATTCAGAGCTCTTGTTCTTCTTCTCCAGTTGGCTTGTACACATAGTTATTTTTCATTTGGAGTTGTTCATCTTTGATCTGCCCCCCTTCTTGGAAAAATAGCAATGTTAATTGATGACCACTACACGTGTAAGCAAAAACACATATCATTACACTTGAAATACAGAGTGCAATGCAATGTACGTTTGGTGAAGCCCCCCTTTTTTGTGTGAATTGCTGTTCTTTGATTTAAAACAAATGAA

[0151] >Coding region sequence (SEQ ID NO:15)

[0152]

[0153] Protein sequence (underlined amino acids are SUMOylation modification sites) (SEQ ID NO:16)

[0154] MGMDYYNVL K VNRNATEEDLKKSYRRMAMKWHPDKNPGDKKKEAEAKFKKISEAYEVLSDPQKRAIYDKYGEEGLKASVDAGASSSMNGNRRFNPRNAEDVFAEFFGSS K PFEGMGRAKSMRFQTEGAGTFGGFGGGNENKFRSYNDSAGTSSSQPRKPPAVETKLPCTLEELYAGSTRKMKISRNVVRPTGQIGTESEILTIDIKPGWKKGTKITFPDKGNEQPNQLPADLVFVIDEKPHDLYTREGNDLLVHQ K IELVDALAGTTVNLKTLDGRDLVIKLTDVVTPGYELAIAKEGMPIVKENGRRGNLRI K FDIVFPKRLSSDQRQNIRKVLGGQTQQQ

[0155] 2. Construction of experimental materials

[0156] The rice (Oryza sativa) genetic material used in the examples comes from two backgrounds: Wuyunjing (WYJ) (Oryza sativa ssp. Japonic) and ZH11 (Oryza sativa ssp. Japonica).

[0157] To verify the genetic characteristics of SCE1, SCE1 overexpression material (SCE1-OE) and SCE2 overexpression material (SCE2-OE) driven by the maize Ubiquitin promoter were constructed.

[0158] The functional characteristics of SCE1 were verified using CRISPR-Cas9 knockout SCE1 knockout materials. Three types of SCE1 knockout lines (sce1-ko) were obtained using the monocotyledonous gene CRISPR-Cas9 gene editing method.

[0159] SCE2 gene knockout material sce2-ko was constructed using CRISPR-Cas9; the SCE1 and SCE2 double gene knockout line sce1 / sce2-ko was also constructed.

[0160] The TT1 gene knockout material tt1-ko was constructed using CRISPR-Cas9, resulting in three types of positive lines.

[0161] To further verify the genetic relationship between SCE1 and TT1, double mutant material of SCE1 and TT1, sce1 / tt1-ko, was obtained by CRISPR-Cas9, and a double overexpression transgenic line of SCE1 and TT1, SCE1 / TT1-OE, was obtained by hybridization.

[0162] To verify the heat tolerance phenotype of the downstream regulatory genes Hsp24.1 and Hsp40 of SCE1, gene knockout lines (hsp40-ko and hsp24.1-ko) of Hsp40 and Hsp24.1 were constructed using CRISPR-Cas9. It was found that there are two types of Hsp24.1 gene knockout lines and three different knockout lines of Hsp40.

[0163] Primers used for gene editing and overexpression are listed in Table 1.

[0164] Table 1

[0165]

[0166]

[0167] In the table, when using CRISPR / Cas9 gene editing technology, two primers are used to prepare annealed sgRNA fragments, which are then introduced into the knockout vector; when target 1 and target 2 are present, sgRNAs targeting both target 1 and target 2 are applied simultaneously.

[0168] 3. Heat treatment during the rice seedling stage

[0169] Seedlings grown hydroponically for 14 days were transferred to a heat treatment chamber at 42℃ and 90% relative humidity for heat treatment. The treatment time was determined based on the observed degree of leaf wilting, generally 18-20 hours. After treatment, the seedlings were allowed to recover for 7 days in an environment of 26℃ for 13 hours during the day and 24℃ for 11 hours in the dark, and photographs were taken for documentation. The survival rate of each material was calculated. A surviving state was defined as an upright stem with at least one green leaf; a semi-surviving state was defined as an upright stem with fewer than one green leaf; and a dead state was defined as a wilted stem with no green leaves. Survival rate = (surviving number + semi-surviving number / 2) / total number of seedlings. Heat treatment was performed on seedlings of sce1-ko, SCE1-OE, sce2-ko, tt1-ko, SCE1 / TT1-OE, sce1 / tt1-ko, hsp24.1-ko, and hsp40-ko, and their heat tolerance phenotypes were statistically analyzed.

[0170] 4. Heat treatment during the rice seedling stage

[0171] To further observe the resistance of SCE1 to heat stress in the field, a heat treatment greenhouse was constructed above rice plants growing in the field until they reached the heading and flowering stage, and the temperature inside the greenhouse was monitored in real time. A high-temperature environment inside the greenhouse was maintained above 38℃ throughout the day, with any period exceeding 4 hours defined as a high-temperature day. These high-temperature days accounted for 71% of the total observation days, thus the greenhouse environment effectively simulated heat stress. After the heat treatment, the agronomic traits of WYJ and sce1-ko materials grown in the field were investigated, including seed setting rate, thousand-grain weight, yield per plant, and plot yield.

[0172] 5. Treatment of rice seedlings with MG132

[0173] To determine whether the target protein in rice would be degraded by the 26S proteasome, 14-day-old rice seedlings were placed in 10 mL centrifuge tubes, and MG132 was added at a concentration of 100 μM. Since MG132 takes time to take effect, the treatment was started 4 hours after treatment and terminated at the designed time point.

[0174] 6. Pollen fertility testing

[0175] To assess pollen viability in the sce1-ko rice variety under heat stress, rice plants were transferred from the field to a heat treatment chamber during the booting stage. The heat treatment conditions were 40°C for 12 hours during the day and 36°C for 12 hours in the dark. The heat treatment lasted approximately 7 days, from the booting stage until the flowering stage. To assess pollen viability, the anthers of both WYJ and sce1-ko materials were gently crushed using tweezers. The crushed anthers released pollen grains, which were then stained with a KI-I2 (0.2% I2, 2% KI) solution. After staining, the released pollen grains were visualized using a scanning electron microscope.

[0176] 7. Verification of protein-protein interactions

[0177] To verify the interaction results between SCE1 and TT1, further verification was performed using yeast two-hybrid (Y2H), pull-down, co-immunoprecipitation (Co-IP), luciferase separation (SFLC), and bimolecular fluorescence complementation (BiFC) experiments.

[0178] To verify the function of SCE1 as an E2-binding enzyme in the SUMOylation process, yeast two-hybrid assay (Y2H) and luciferase separation assay (SFLC) were used to verify the interaction between SCE1 and SUMO1, and the interaction between SCE1 and SUMO E3 ligases SIZ1, SIZ2 and MMS21.

[0179] Meanwhile, the interaction between Hsp24.1 and Hsp40 and SCE1 was verified using the yeast two-hybrid assay (Y2H) and luciferase separation assay (SFLC).

[0180] The primers used for constructing vectors in Y2H, Pull-down, Co-IP, SFLC, and BiFC experiments are shown in Table 2.

[0181] Table 2

[0182]

[0183]

[0184] 8. Subcellular localization

[0185] The complete cDNAs of SCE1 and SCE2 were cloned into the pA7-eYFP vector to generate high concentrations of p35S::-eYFP, p35S::SCE1-eYFP, and p35S::SCE2-eYFP for transient expression in rice protoplasts. The nuclear marker DsRed-NLS was co-expressed in the rice protoplasts. Fluorescence signals were observed using a laser confocal scanning microscope (Zeiss LSM 880 or Leica TSC SP8STED). The green fluorescence signal was YFP, with an excitation wavelength of 488 nm and an emission wavelength of 500-550 nm; the purple fluorescence signal was chlorophyll autofluorescence, with an excitation wavelength of 488 nm and an emission wavelength of 650-750 nm.

[0186] The 5' oligonucleotide primer sequence for constructing the pA7-SCE1-YFP fusion protein expression vector is as follows:

[0187] 5'-CATTTACGAACGATACTCGAGGTCGACATGTCGGGGGGAATCGCG-3'(SEQ ID NO:97)

[0188] The 3' primer sequence is as follows:

[0189] 5'-CCCTTGCTCACCATACTAGTGGATCCAAGCAAAGCAGGGTACTGCTTGG-3'(SEQ ID NO:98)

[0190] The 5' oligonucleotide primer sequence for constructing the pA7-SCE2-YFP fusion protein expression vector is as follows:

[0191] 5'-CATTTACGAACGATACTCGAGGTCGACATGTCGGGAGGGATCGCAC-3'(SEQ ID NO:99)

[0192] The 3' primer sequence is as follows:

[0193] 5'-CCCTTGCTCACCATACTAGTGGATCCGACAATCGGAGGATACTGCTTGG-3'(SEQ ID NO:100)

[0194] 9. SCE1 expression profile analysis and expression level

[0195] RNA was extracted from various tissues of rice, including roots, stems, leaves, spikelets, and glumes 10 days after grain filling. Real-time quantitative PCR was used to determine the expression level of SCE1 in various tissues of rice and after heat treatment.

[0196] The expression level of Hsp24.1 in heat-treated WYJ and sce1-ko was determined by real-time quantitative PCR.

[0197] The 5' oligonucleotide primer sequence for SCE1 expression detection is:

[0198] 5'-CATGCACATCCGCATAGTAG-3'(SEQ ID NO:101)

[0199] The 3' primer sequence is as follows:

[0200] 5'-GGACTCACAAGCAAGTTGAA-3'(SEQ ID NO:102)

[0201] The 5' oligonucleotide primer sequence for Hsp24.1 expression level detection is as follows:

[0202] 5'-TTATCCAATAAGAAATGGCGGCG-3'(SEQ ID NO:103)

[0203] The 3' primer sequence is as follows:

[0204] 5'-GCGATACGAGATCAATCGCATA-3'(SEQ ID NO:104)

[0205] 10. Experiments on SCE1 protein levels and ubiquitination modification under heat treatment.

[0206] To investigate the significance of the SCE1-TT1 interaction, immunoblotting analysis was performed using the SCE1-specific antibody Anti-SCE1 to observe the endogenous SCE1 protein level. To detect SCE1 ubiquitination, SCE1 protein was enriched in SCE1-OE transgenic rice. Anti-ubiquitin, Anti-ubiquitin-K48, and Anti-ubiquitin-K63 antibodies detected diffuse high-molecular-weight protein bands in the SCE1-OE stable transgenic material, and the presence or absence of these bands was used as a criterion for judging whether SCE1 had ubiquitination modification.

[0207] 11. Changes in SUMOylation Modification Patterns in Plants under Heat Stress

[0208] Because SUMO proteins are highly conserved, the Anti-AtSUMO1 / 2 protein derived from Arabidopsis thaliana was used to detect the level of SUMOylation modification in rice. To investigate the plant response to heat stress, samples were taken at 0, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 20 h to detect the level of SUMOylation modification. To further clarify the influence of heat stress on the SUMOylation modification of various SCE1 transgenic materials, samples from WYJ, SCE1-OE, sce1-ko, and tt1-ko were also taken at different locations for immunoassay.

[0209] 12. Transcriptome sequencing analysis

[0210] Total RNA was extracted from 14-day-old WYJ and sce1-ko background seedlings under normal conditions and after heat treatment. Three independent biological replicates were collected in total. RNA concentration, purity, and integrity were assessed before library construction to ensure compliance with required standards. RNA-sequence sequencing was performed with the assistance of Beijing BaiMike Biotechnology Co., Ltd. RNA sequencing libraries were prepared using the Illumina Hieff NGS Ultima Dual-mode mRNA LibraryPrep Kit (Yeasen12301ES24) and sequenced on the Illumina NovaSeq platform. Rigorous quality control assessments were performed on the raw sequencing data. Reads were aligned to the reference rice genome (Oryza_sativa.MSU_v7.0.genome.fa), gene expression levels were quantified, and differential expression analysis was performed using DESeq2 (version 1.30.1). Genes with p-values ​​<0.01 and FoldChange ≥2 were considered differentially expressed genes (DEGs).

[0211] Gene function annotation was performed using the Gene Ontology (GO) database (http: / / www.geneontology.org / ) and the KEGG Pathology database (http: / / www.genome.jp / kegg / ). GO enrichment analysis and KEGG pathway enrichment analysis of differentially expressed genes were performed using R / topGO (version 2.48.0) and R / clusterprofiler (version 4.4.4), respectively. Gene set enrichment analysis (GSEA) was performed using a local version of the GSEA analysis tool (http: / / www.broadinstitute.org / gsea / index.jsp) with the KEGG annotation dataset. Finally, heatmaps of specific gene clusters were generated using the R packageheatmap (version 1.0.12).

[0212] Three representative genes, Cpn60β1, CYP20-2, and Hsp78, were selected from the protein folding and refolding pathway to further verify their expression levels in WYJ and sce1-ko.

[0213] The primer sequence for detecting the 5' oligonucleotide expression level of OsCpn60β1 is as follows:

[0214] 5'-CAAGAAGCTTCAGACTGGAGTA-3'(SEQ ID NO:105)

[0215] The 3' primer sequence is: 5'-TGCAACTGTAACACCATCATTG-3' (SEQ ID NO:106)

[0216] The 5' oligonucleotide primer sequence for OsCYP20-2 expression level is:

[0217] 5'-GAGGGTCGTTATTGGCCTATAC-3'(SEQ ID NO:107)

[0218] The 3' primer sequence is: 5'-CTCCCTGAATCATGAAGTCCTT-3' (SEQ ID NO:108)

[0219] The 5' oligonucleotide primer sequence for OsHsp78.4 expression level is as follows:

[0220] 5'-TGTATCTGGCAAATTTGGTTGG-3'(SEQ ID NO:109)

[0221] The 3' primer sequence is: 5'-CACTCAAGTCCTTGACAATTGG-3' (SEQ ID NO:110)

[0222] 12. LC-MS / MS (Liquid Chromatography-Tandem Mass Spectrometry / Mass Spectrometry)

[0223] LC-MS / MS is liquid chromatography-tandem mass spectrometry / mass spectrometry. First, the concentration of the protein to be loaded onto the instrument is determined. To ensure accurate measurement, one-tenth of the total protein is used for silver staining. The remaining protein solution is denatured in 8M urea for 1 hour, then reduced using 10mM TCEP (Sangon Biotech), followed by alkylation using 50mM CAA (Sigma). The protein is then digested overnight (37°C) with trypsin at one-fiftieth of the total protein mass to cleave it into peptides. The digestion is terminated with 0.1% TFA, and impurities are removed using Pierce™ C18 Spin Columns (ThermoFisher Scientific) to enrich the peptides. The enriched peptides are eluted with a buffer containing 0.1% formic acid and 40% acetonitrile (Sigma) for subsequent loading onto the instrument.

[0224] Peptide enrichment was performed using a Dionex Ultimate 3000RSLCnano chromatography system and a Thermo Orbitrap FusionLumos mass spectrometer (Thermo Fisher Scientific). The enriched peptides were eluted with buffer, enabling efficient sample separation and mass spectrometry analysis. In LC-MS / MS mode, the mass spectrometer first selected specific precursor ions for mass screening, followed by fragmentation. Subsequently, the mass spectrometer collected mass spectrometric data of these fragmented ions. Analysis of this data provided information about protein-protein interactions and complex composition.

[0225] Peptide mixtures were loaded onto a C18 reverse-phase column (25 cm long, 150 μm inner diameter) using buffer A (0.1% formic acid in HPLC-grade water) and eluted linearly with a gradient at a flow rate of 600 nL / min, using buffer B (0.1% formic acid in 80% acetonitrile). Mass spectrometry data were analyzed using Thermo Proteome Discoverer 2.5.0.400 software. Annotation information was obtained from the UniProt database (https: / / www.uniprot.org / blast), and UniProt numbers were mapped to gene numbers using RGAP (http: / / rice.plantbiology.msu.edu / ). Statistical enrichment analysis of genes obtained from IP-MS was performed in the KEGG pathway (http: / / www.genome.jp / kegg / ) using clusterProfiler R software version 4.10.0.

[0226] 13. Detection of SUMOylation Modification of Escherichia coli Proteins

[0227] To verify whether Hsp24.1 and Hsp40 can undergo SUMOylation modification with the participation of SCE1, validation was performed in an *E. coli* SUMOylation modification system. Multiple rice-derived proteins were used to construct a system for identifying substrate protein SUMOylation in *E. coli*. The coding sequences of OsHsp24.1 and OsHsp40 were inserted into the pCDFDuet-Flag vector. Subsequently, the constructed pCDFDuet-OsHsp24.1-Flag and pCDFDuet-OsHsp40-Flag were introduced into *E. coli* strain BL21(DE3) containing pET28-AtSAE1a-His-SAE2 and pACYCDuet-1-MYC-OsSUMO1 or pACYCDuet-1-His-OsSCE1-MYC-OsSUMO1. Similarly, strains expressing Hsp24.1 and Hsp24.1M (containing four mutated SUMO binding sites) were introduced into *E. coli* BL21(DE3) containing pET28-AtSAE1a-His-SAE2 and pACYCDuet-1-MYC-AtSUMO1 or pACYCDuet-1-His-AtSCE1-MYC-AtSUMO1. Bacterial cultures were induced with 0.5 mM IPTG at 37 °C for 12 h. Subsequently, the induced proteins were harvested for Western blot analysis, and SUMO modification was assessed using an anti-Flag antibody.

[0228] The 5' oligonucleotide primer sequence for constructing the pCDFDuet-OsHsp24.1-Flag fusion protein expression vector is as follows:

[0229] 5'-AAGTATAAGAAGGAGATATACATATGATGGCTTCCATCGTGGCA-3'(SEQ ID NO:111)

[0230] The 3' primer sequence is as follows:

[0231] 5'-CTTATCGTCGTCATCCTTGTAATCCTCGACGTTGACCTGGAAGA-3'(SEQ ID NO:112)

[0232] The 5' oligonucleotide primer sequence for constructing the pCDFDuet-OsHsp40-Flag fusion protein expression vector is as follows:

[0233] 5'-AAGTATAAGAAGGAGATATACATATGATGGGGATGGATTACTACAACGTG-3'(SEQ ID NO:113)

[0234] The 3' primer sequence is as follows:

[0235] 5'-CTTATCGTCGTCATCCTTGTAATCCTGCTGCTGAGTTTGCCC-3'(SEQ ID NO:114)

[0236] The 5' oligonucleotide primer sequence for constructing the pACYCDuet-1-His-OsSCE1 fusion protein expression vector is as follows:

[0237] 5'-CCACAGCCAGGATCCGAATTCGATGTCGGGAGGGATCGCAC-3'(SEQ ID NO:115)

[0238] The 3' primer sequence is as follows:

[0239] 5'-GGCCGCAAGCTTGTCGACTTATCAAAGCAAAGCAGGGTACTGCT-3'(SEQ ID NO:116)

[0240] The 5' oligonucleotide primer sequence for constructing the pACYCDuet-1-MYC-OsSUMO1 fusion protein expression vector is as follows:

[0241] 5'-GCTCATTTCTGAAGAGGACTTGATGTCGGCCGCCGG-3'(SEQ ID NO:117)

[0242] The 3' primer sequence is as follows:

[0243] 5'-GCGGTTTCTTACCAGACTCGAGTCAGCCTCCAGTCTGGTGGAG-3'(SEQ ID NO:118)

[0244] 14. Semi-in vitro protein degradation experiment

[0245] After 14 days of growth, total protein was extracted from WYJ, tt1-ko, and TT1-OE seedlings using degradation buffer. The degradation buffer consisted of 25 mM Tris-HCl (pH 7.5), 10 mM NaCl, 10 mM MgCl2, 0.5% Triton X-100, 5 mM DTT, 4 mM PMSF, and 10 mM ATP. Protein concentrations were measured using the Bradford method and normalized to approximately 500 μg total protein. His-SCE1 (pCold-TF-SCE1), a heterologous protein purified from AKTA, was added to each seedling extract (containing 500 μg total protein), with each group containing 200 ng of His-SCE1. The experiment was repeated with the addition of the proteasome inhibitor MG132 (final concentration 40 μM) to the reaction mixture. All experimental groups were incubated at 28°C for 0 h, 1 h, 2 h, and 6 h. The reaction was then immediately terminated by adding 4×SDS loading buffer. All samples were analyzed by SDS-PAGE and Western blotting.

[0246] Cell-free degradation assays for Hsp24.1 and Hsp24.1M were performed using a method similar to that for SCE1. Purified recombinant MBP-Hsp24.1 (pMAL-c5x-Hsp24.1) and MBP-Hsp24.1M (pMAL-c5x-Hsp24.1M) were added to seedling extracts from WYJ and sce1-ko, and analyzed using an anti-MBP antibody.

[0247] The 5' oligonucleotide primer sequence for constructing the His-SCE1 fusion protein expression vector is as follows:

[0248] 5'-GAGCTCGGTACCCTCGAGGGATCCATGTCGGGAGGGATCGCAC-3'(SEQ ID NO:119)

[0249] The 3' primer sequence is as follows:

[0250] 5'-ATTACCTATCTAGACTGCAGTCAAAGCAAAGCAGGGTACTGCT-3'(SEQ ID NO:120)

[0251] The 5' oligonucleotide primer sequence for constructing the MBP-Hsp24.1 / Hsp24.1M-F fusion protein expression vector is as follows:

[0252] 5'-CGGGATCGAGGGAAGGATTTCACATATGATGGCTTCCATCGTGGCA-3'(SEQ ID NO:121)

[0253] The 3' primer sequence is as follows:

[0254] 5'-AGCTTATTTAATTACCTGCAGGGAATTCCTACTCGACGTTGACCTGGAAGA-3'(SEQ ID NO:122)

[0255] 15. Molecular Chaperone Activity Experiment

[0256] The chaperone activities of Hsp24.1 and Hsp24.1M were confirmed using the restriction endonuclease NdeI as a substrate. Purified Hsp24.1 / Hsp24.1M were incubated with NdeI at different temperatures (37–50 °C) for 90 min, with the reaction mixture lacking Hsp24.1 serving as a negative control. Hsp24.1 effectively protected NdeI from heat-induced denaturation, up to 50 °C. Interestingly, compared to Hsp24.1, Hsp24.1M provided only partial protection at 40–50 °C. This suggests that the SUMOylation site in Hsp24.1 is crucial for its chaperone function.

[0257] The 5' oligonucleotide primer sequence for the pET-28a(+) DNA fragment is:

[0258] 5'-TGCCGTAAAGCACTAAATCGGA-3'(SEQ ID NO:123)

[0259] The 3' primer sequence is: 5'-CATTAGGAAGCAGCCCAGTAGT-3' (SEQ ID NO:124)

[0260] Example 1: SCE1 is a negative regulator of heat tolerance in rice.

[0261] In order to gain a deeper understanding of the TT1-mediated thermoregulation pathway, the inventors discovered through in-depth analysis and screening that SCE1 and TT1 can interact.

[0262] SCE1 can be detected by yeast two-hybrid (Y2H), in vitro pull-down assay, fractional luciferase assay (SFLC), bimolecular fluorescence complementation (BiFC), and co-immunoprecipitation (Co-IP). SCE1 is a protein that interacts with TT1, and the interaction between SCE1 and TT1 has been verified in vitro and in vivo. Therefore, the inventors have discovered the functional relationship between TT1 and SCE1 for the first time (see...). Figure 1 ).

[0263] BiFC experiments demonstrated that the signaling pathway for SCE1-TT1 interaction is localized in the cytoplasm and nucleus. Heat treatment of seedling rice revealed that the transgenic knockout line sce1-ko and the overexpression line SCE1-OE exhibited heat-resistant and heat-sensitive phenotypes, respectively. Compared to WT, the sce1-ko line showed a higher survival rate under heat stress, while the SCE1-OE line exhibited a lower survival rate (see...). Figure 2 ).

[0264] These results indicate that SCE1 is a negative regulator of heat tolerance in rice.

[0265] Example 2: TT1 assists SCE1 in the protein degradation of the 26S proteasome.

[0266] With increasing heat treatment time, the abundance of endogenous SCE1 protein decreased. The inventors discovered that SCE1 protein in rice can be ubiquitinated, with K48 ubiquitination being the dominant type and no K63 ubiquitination modification.

[0267] Compared to WYJ, higher SCE1 protein levels were detected in the TT1 knockout line, while lower SCE1 protein levels were found in the TT1 overexpression line (TT1-OE). The 26S proteasome inhibitor MG132 inhibits SCE1 protein degradation (see...). Figure 3 ).

[0268] Endogenous protein assays under heat treatment showed that, compared to WT, the degradation rate of SCE1 protein in the TT1 knockout line was slower under heat stress. Semi-in vitro protein degradation experiments revealed that the degradation rate of SCE1 in WYJ was higher than that in the TT1 knockout line (tt1-ko).

[0269] These results indicate that the instability of the SCE1 protein is related to ubiquitination modification, and that TT1, as the α2 subunit of the 26S proteasome, promotes the degradation of SCE1 by the 26S proteasome-mediated ubiquitination protein degradation pathway (see...). Figure 3 ).

[0270] Example 3: SCE1 is a downstream regulatory gene of TT1.

[0271] To further confirm the genetic relationship between SCE1 and TT1, the inventors constructed various transgenic materials for verification. Compared with WYJ, the tt1-ko line showed increased sensitivity to heat stress, while TT1-OE exhibited an enhanced heat stress tolerance phenotype (see...). Figure 4 The SCE1 / TT1-ko plants exhibited enhanced heat tolerance, consistent with the heat-tolerant phenotype of SCE1-ko; while the SCE1 / TT1-OE plants exhibited reduced heat tolerance, consistent with the heat-tolerant phenotype of SCE1-OE (see [link to relevant documentation]). Figure 5 ).

[0272] These findings suggest that SCE1 and TT1 respond to heat stress in the same genetic pathway, and that SCE1 functions downstream of TT1.

[0273] Example 4: SCE1 regulates the level of SUMOylation modification in plants under heat stress

[0274] In vivo and in vitro experiments have demonstrated that SCE1 interacts with SUMO1, E3 ligases SIZ1, SIZ2, and MMS21 (see [link to study]). Figure 6 ).

[0275] SCE1 influences SUMO-modified substrates; SUMO-binding levels in SCE1-OE were significantly higher than in WYJ, while SUMO-binding levels in sce1-ko were significantly lower than in WYJ. The rapid increase in SUMOylation-modified substrates in plants under heat stress is considered a signal of response to heat stress. The pattern of decline in SUMOylation-modified substrates in plants after 1 hour also has biological significance. High levels of SUMO-bindings tend to decline more rapidly in sce1-ko, while the decline is slower in SCE1-OE and tt1-ko. This rapid decline in SUMOylation modification is a protective mechanism of plants under heat stress (see...). Figure 6 ).

[0276] Example 5: SCE1-targeted regulation of protein folding and refolding

[0277] Transcriptome analysis revealed that heat-treated sce1-ko genes were enriched in genetic pathways related to translation, misfolding, protein folding, protein refolding, and the binding of unfolded proteins. Three representative genes, Cpn60β1, CYP20-2, and Hsp78, showed significantly increased expression levels in heat-treated sce1-ko, suggesting they help rice eliminate unfolded or misfolded proteins more quickly, thereby enhancing its resistance to heat stress (see [link to relevant documentation]). Figure 7 ).

[0278] IP-MS / MS revealed over 1000 proteins that may interact with SCE1. These interacting proteins are enriched in protein processing pathways involving ribosomes, carbon metabolism, proteasomes, oxidative phosphorylation, phagosomes, and the endoplasmic reticulum. Protein processing pathways enriched in the endoplasmic reticulum include small heat shock proteins (sHSPs), including Hsp24.1 and Hsp40 (see...). Figure 7 ).

[0279] SFLC experiments demonstrated protein-protein interactions between Hsp24.1, Hsp40, and SCE1 (see...) Figure 7 In summary, the above results suggest that SCE1 directly or indirectly affects a variety of biological processes at the transcriptional and protein levels, including pathways related to protein folding.

[0280] Example 6: Hsp24.1 participates in the SCE1-regulated thermal stress response pathway

[0281] Based on the SUMOylation modification experiment of E. coli, it was confirmed that Hsp24.1 and Hsp40 are both SUMOylation modified substrate proteins (see...). Figure 8 Furthermore, it indicates that the SUMOylation modification sites for Hsp24.1 are K157, K192, K206, and K208; and the SUMOylation modification sites for Hsp40 include K10, K110, K256, and K314 (see...). Figure 8 After heat treatment, Hsp24.1 protein abundance was higher in SCE1-ko, but lower in SCE1-OE and tt1-ko (see [link to relevant documentation]). Figure 9 Semi-in vitro degradation experiments revealed that SUMOylation of Hsp24.1 affects its protein stability, and loss of SCE1 function prevents the degradation of Hsp24.1 (see...). Figure 9 Molecular chaperone activity experiments demonstrated that Hsp24.1 effectively protects NdeI from high-temperature-induced denaturation, up to 50 °C, and that the SUMOylation site of Hsp24.1 is crucial for its chaperone function (see [link to study]). Figure 9 Both Hsp24.1 and Hsp40 knockout materials are more sensitive to heat stress (see [link]). Figure 10 ).

[0282] These results suggest that Hsp24.1 and Hsp40 are involved in the TT1-SCE1 heat tolerance pathway and positively regulate heat tolerance in rice (see...). Figure 10 ).

[0283] Example 7: SCE1 exhibits evolutionary conservation.

[0284] There are three SUMO E2 sequences in rice: OsSCE1, OsSCE2, and OsSCE3. Multiple sequence alignment showed that OsSCE1 and OsSCE2 were the most homologous, with an amino acid sequence identity of 93.12%. OsSCE3 shared 67.72% sequence identity with OsSCE1 and 70.32% sequence identity with OsSCE2. SCE1 shares homology with multiple species. OsSCE1 shows 85.62% sequence similarity with Arabidopsis thaliana, 87.97% with soybean (Glycine max), 93.75% with sorghum (Sorghum bicolor), 93.75% with maize (Zea mays), 93.67% with barley (Hordeum vulgare), 92.45% with wheat (Triticumaestivum), 95% with millet (Setaria italica), 92.41% with Brachypodium distachyon, 88.07% with potato (Solanum tuberosum), 90.07% with tomato (Solanum lycopersicum), 92.14% with chili pepper (Capsicum annuum), and is homologous with rapeseed (Brassica oleracea). The sequence identity of the napus was 86.25% (see [link to napus sequence]). Figure 11 Phylogenetic analysis revealed that SCE1 is evolutionarily conserved in species including rice (Oryza sativa), Arabidopsis thaliana, soybean (Glycine max), sorghum (Sorghum bicolor), maize (Zea mays), barley (Hordeum vulgare), wheat (Triticum aestivum), millet (Setaria italica), brachypodium distachyon, potato (Solanum tuberosum), tomato (Solanum lycopersicum), pepper (Capsicum annuum), and rapeseed (Brassica napus) (see...). Figure 11 Because SCE1 is highly conserved, this gene also has important applications in heat-resistant breeding of other important crops.

[0285] OsSCE1 and OsSCE2 belong to the same subgroup as AtSCE1, while OsSCE3 belongs to a different subgroup. This suggests that OsSCE3 may have functional differences from OsSCE1 or OsSCE2 (see [link to relevant documentation]). Figure 11 ).

[0286] Example 8: Expression profiling and subcellular localization of SCE1 and SCE2

[0287] RNA was extracted from various tissues of rice, including roots, stems, leaves, spikelets 10 days after grain filling, and glumes. The results showed that SCE1 expression was high in rice leaves and glumes, followed by the expression in roots, stems, and young spikelets at 5 cm and 10 cm in diameter, while expression was low in glumes 10 days after grain filling in young spikelets at 1-2 cm in diameter. This suggests that SCE1 regulates leaf function in response to heat stress (see...). Figure 12 ).

[0288] The inventors constructed pA7-SCE1-YFP and pA7-SCE2-YFP protein expression vectors and transformed them into rice protoplasts. The green fluorescence of SCE1 and SCE2 protein expression was observed in the nucleus and cytoplasm (see...). Figure 12 ).

[0289] Therefore, the gene expression patterns and subcellular localization characteristics of SCE1 and SCE2 are quite consistent.

[0290] Example 9: SCE2 negatively regulates rice's response to heat stress

[0291] The SCE2 gene in the rice genome was knocked out using CRISPR-Cas9, and SCE2-positive transgenic lines were identified by sequencing. After heat treatment with high temperature and humidity, the SCE2 knockout line (sce2-ko) showed strong resistance to heat stress and a significantly increased survival rate (see [link to study]. Figure 13 ).

[0292] Therefore, the homolog of SCE1, SCE2, also negatively regulates the heat tolerance of rice.

[0293] Example 10: Effects of SCE1 and SCE2 on rice growth and development

[0294] SUMO E2 binding enzymes play a crucial role in plant growth and development. The inventors investigated growth-related phenotypes in SCE1 and SCE2 gene knockout lines and ectopic overexpression lines at the heading stage. Under normal growth conditions, plant height and tillering in sce1-ko and SCE1-OE lines showed no significant differences compared to WYJ. Similarly, loss of function of the SCE2 gene (sce2-ko) did not affect agronomic traits such as plant height and tillering in rice. Compared to WYJ, the SCE2 overexpression line (SCE2-OE) had shorter plant height and a slightly increased number of tillers, but this difference was not statistically significant compared to WYJ (see [link to study]. Figure 14 ).

[0295] The inventors constructed sgRNA targets located at the first Exon of SCE1 and SCE2, respectively, to obtain SCE1 and SCE2 double-gene knockout plants. Gene sequencing identified positive knockout lines for SCE1 and SCE2. The SCE1 / SCE2 double-gene knockout T0 generation heterozygous lines exhibited smaller panicles, lack of secondary branches, shorter plant height, and inability to produce grains, demonstrating growth disadvantages and affecting normal rice growth and development. This indicates that the coexistence of SCE1 and SCE2 is crucial for plant growth and development, and that the functions of SCE1 and SCE2 are redundant and can complement each other (see...). Figure 15 ).

[0296] Example 11: SCE1 is a genetic resource for heat-resistant crop breeding.

[0297] Under normal conditions, the SCE1 gene does not affect many agronomic traits of rice, including plant height, tiller number, seed setting rate, thousand-grain weight, and yield per plant (see...). Figure 16 ).

[0298] Under heat stress, the SCE1 gene knockout lines exhibited higher pollen fertility (22.9% increase in seed setting rate), stronger grain filling ability (10.1% increase in thousand-grain weight), higher yield per plant (15.1% increase), and higher plot yield (7.4% increase) (see...). Figure 16 ).

[0299] Therefore, the loss of function of SCE1 can confer heat resistance to rice and significantly reduce the loss of grain yield caused by heat stress, providing a new strategy for cultivating highly heat-resistant crops.

[0300] Example 12: Screening of regulatory molecules

[0301] Test group: rice WYJ cell line, in which the TT1-SCE-Hsp heat stress response pathway involved in the present invention is expressed, and candidate substances are administered;

[0302] Control group: Rice WYJ cell line expressing the TT1-SCE-Hsp heat stress response pathway involved in the present invention, but without being given the candidate substance.

[0303] The TT1-SCE1-Hsp heat stress response pathway was examined and compared in the test and control groups, respectively. If SCE1 expression or activity was statistically increased (e.g., more than 20%) in the test group, TT1-SCE interaction was promoted (including promoting TT1-mediated SCE degradation (via ubiquitinated protein degradation pathway)), and SCE-Hsp interaction was inhibited (including reducing SCE-mediated Hsp SUMOylation modification and increasing Hsp accumulation (preventing its degradation)), then the candidate substance was considered a potential agent for improving plant heat tolerance or yield under heat stress.

[0304] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for improving plant heat tolerance or yield under heat stress, comprising: Downregulating SCE in plants can improve plant heat tolerance or yield under heat stress. or Regulating the interaction of TT1, SCE, and Hsp in the TT1-SCE-Hsp heat stress response pathway involving SCE in plants, enhancing Hsp-mediated protein folding, and thus improving plant heat tolerance or yield under heat stress; The SCE includes either SCE1 or SCE2.

2. The method as described in claim 1, characterized in that, The measures to increase plant yield under heat stress include: improving survival rate, increasing grain weight and / or improving seed setting rate; or The Hsp-mediated protein folding includes: correcting misfolds to correct folds, or causing unfolded proteins to fold; preferably, causing proteins to fold into their native state. Preferably, the Hsp includes Hsp24.1 or Hsp40.

3. The method as described in claim 1, characterized in that, Modulating the TT1-SCE-Hsp thermal stress response pathway includes: (a) Upregulating TT1 to promote the interaction between TT1 and SCE, promote TT1-mediated SCE degradation, thereby increasing plant heat tolerance or yield under heat stress; or (b) Inhibit the interaction between SCE and Hsp to reduce the SUMOylation of Hsp and increase the accumulation of Hsp, thereby enhancing Hsp-mediated protein folding and thus improving plant heat tolerance or yield under heat stress.

4. The method as described in claim 3, characterized in that, Downregulation of SCE includes downregulating its expression, activity, or stability, preferably including: knocking out or silencing the SCE-coding gene in plants, inhibiting SCE activity, or promoting SCE ubiquitination and degradation; preferably including: gene editing using a CRISPR system to knock out the SCE-coding gene, knocking out the SCE-coding gene using homologous recombination, performing loss-of-function mutations on SCE in plants containing SCE, silencing SCE with interfering molecules that specifically interfere with the expression of the SCE-coding gene, upregulating TT1 to promote its-mediated SCE ubiquitination and degradation; or Upregulating TT1 includes upregulating its expression, activity, or stability, including: transferring the gene encoding TT1 or an expression construct or vector containing the gene into plants; performing gain-of-function mutations on TT1; promoting TT1 expression by expressing an enhancing promoter or a tissue-specific promoter; or promoting TT1 expression by an enhancer. Preferably, when SCE is lowered, either SCE1 or SCE2 is lowered.

5. The method as described in claim 4, characterized in that, SCE is downregulated by sgRNA that guides CRISPR gene editing or by a reagent capable of forming the sgRNA; preferably, the reagent for forming the sgRNA includes: primers with the sequences shown in SEQ ID NO:17 and SEQ ID NO:18, primers with the sequences shown in SEQ ID NO:19 and SEQ ID NO:20, and primers with the sequences shown in SEQ ID NO:39 and SEQ ID NO:

40.

6. Use of an SCE, a TT1-SCE-Hsp heat stress response pathway comprising thereof, or a regulatory molecule thereof, for improving plant heat tolerance or yield under heat stress; preferably, the improvement of plant yield under heat stress includes: To improve survival rate, increase grain weight, and / or increase seed setting rate; among which, The regulatory molecule is an SCE downregulatory molecule, which increases plant heat tolerance or yield under heat stress; or The regulatory molecule is a TT1 upregulated molecule, which promotes the interaction between TT1 and SCE, thereby promoting TT1-mediated SCE degradation and thus improving plant heat tolerance or yield under heat stress; or The regulatory molecule is an inhibitory molecule for the interaction between SCE and Hsp, which reduces the SUMOylation of Hsp and increases the accumulation of Hsp, thereby enhancing Hsp-mediated protein folding and thus improving plant heat tolerance or yield under heat stress. The SCE includes either SCE1 or SCE2.

7. The use as described in claim 6, characterized in that, The SCE downregulating molecules include: reagents for knocking out or silencing SCE, reagents for inhibiting SCE activity, and reagents for reducing SCE stability; preferably, they include: CRISPR gene editing reagents, homologous recombination reagents, or site-directed mutagenesis reagents targeting SCE, wherein the reagents induce loss-of-function mutations in SCE, interfering molecules specifically interfere with the expression of the SCE-encoding gene, and reagents that upregulate TT1 to promote its-mediated ubiquitination and degradation of SCE; more preferably, the SCE downregulating molecules include: CRISPR gene editing-based sgRNA or reagents capable of forming the sgRNA, wherein the reagents for forming the sgRNA include: primers with sequences shown in SEQ ID NO:17 and SEQ ID NO:18, primers with sequences shown in SEQ ID NO:19 and SEQ ID NO:20, and primers with sequences shown in SEQ ID NO:39 and SEQ ID NO:40; or The TT1 upregulation molecules include: transferring the TT1 coding gene or an expression construct or vector containing the coding gene into plants; performing gain-of-function mutations on TT1; promoting TT1 expression by expressing an enhancing promoter or a tissue-specific promoter; or promoting TT1 expression by an enhancer.

8. The method as described in any one of claims 1 to 5 or the use as described in any one of claims 6 to 7, characterized in that, The plant is or the SCE is derived from plants including the following group: grasses, legumes, cruciferous plants, and solanaceous plants; preferably, the plant is or the SCE is derived from plants including the following group: rice (Oryza sativa), corn (Zeamays), millet (Setaria italica), barley (Hordeum vulgare), wheat (Triticum aestivum), foxtail millet (Panicum miliaceum), sorghum (Sorghum bicolor), rye (Secale cereale), oats (Avenasativa L.), short-stalked grass (Brachypodium distachyum), soybean (Glycine max), potato (Solanum tuberosum), tomato (Solanum lycopersicum), pepper (Capsicum annuum), rapeseed (Brassicanapus), and Arabidopsis thaliana.

9. The method as described in any one of claims 1 to 5 or the use as described in any one of claims 6 to 7, characterized in that, The amino acid sequence of the SCE polypeptide is selected from the group consisting of: (i) a polypeptide with the amino acid sequence shown in SEQ ID NO:4 or 8; (ii) a polypeptide derived from (i) having the regulatory trait function formed by substituting, deleting or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO:4 or 8; (iii) a polypeptide with the regulatory trait function having ≥80% homology to the amino acid sequence shown in SEQ ID NO:4 or 8; (iv) an active fragment of a polypeptide with the amino acid sequence shown in SEQ ID NO:4 or 8; or, (v) a polypeptide formed by adding a tag sequence or restriction enzyme site sequence to the N or C end of the polypeptide with the amino acid sequence shown in SEQ ID NO:4 or 8, or by adding a signal peptide sequence to its N end.

10. Use of SCE in plants or the TT1-SCE-Hsp heat stress response pathway containing it as a molecular marker for identifying plant heat tolerance or yield under heat stress, or as a molecular marker for targeted plant screening; preferably, the yield trait under heat stress includes: Survival rate, grain weight and / or seed setting rate.

11. A method for selecting or identifying plant heat tolerance or yield under heat stress, comprising: To identify the expression or sequence characteristics of SCE in test plants, or to identify the TT1-SCE-Hsp heat stress response pathway in plants; If the tested plant expresses low levels of SCE or no SCE, it is a plant with high heat tolerance or high yield under heat stress; if the tested plant expresses high levels of SCE, it is a plant that is heat-sensitive or has low yield under heat stress.

12. A method for screening substances that improve plant heat tolerance or yield under heat stress, comprising: (1) Add the candidate substance to the system expressing SCE; (2) Detect the system and observe the expression, activity or stability of SCE therein. If its expression, activity or stability is improved, it indicates that the candidate substance can be used to improve the heat resistance of plants or the yield under heat stress.

13. A method for screening substances that regulate plant heat tolerance or yield under heat stress, comprising: (1) The candidate substances were added to the system expressing the TT1-SCE-Hsp thermal stress response pathway; (2) Detect the system and observe the expression, activity or interaction of the TT1-SCE-Hsp heat stress response pathway proteins. If it promotes the interaction between TT1 and SCE or inhibits the interaction between SCE and Hsp, it indicates that the candidate substance can be used to improve the heat tolerance of plants or the yield under heat stress. Preferably, the promotion of TT1-SCE interaction includes: promoting TT1-mediated SCE degradation; Preferably, the inhibition of the interaction between SCE and Hsp includes: reducing SCE-mediated SUMOylation of Hsp and increasing Hsp accumulation.

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