High-temperature-resistant novel gene tt4 and application thereof in high-temperature-resistant breeding of plants

Abstract

The application provides a high-temperature-resistant new gene TT4 and application of the gene in high-temperature-resistant plant breeding. The gene has the following functions: enhancing the heat-resistant capacity of plants or increasing the yield of plants in a hot environment; serving as amylase to increase the starch content in plant grains in a hot environment; and serving as adenylyl cyclase to generate cyclic nucleotides (cAMP) with ATP as a substrate. The application provides a new implementation for optimizing the phenotype of plants.

Description

Technical Field

[0001] This invention belongs to the field of agriculture; more specifically, this invention relates to the novel heat-resistant gene TT4 and its application in heat-resistant plant breeding. Background Technology

[0002] Plant growth and development are closely related to the environment, with temperature being a crucial ecological factor influencing plant physiological processes. All physiological and biochemical processes occurring throughout a plant's life cycle require specific temperature conditions within its environment. In recent years, with continuous population growth and intensified human activities, global warming has become a major global issue. Agricultural yields are closely linked to the environment; extreme temperatures caused by global warming lead to reduced crop production, severely threatening food security and sustainable development.

[0003] High-temperature stress can trigger a series of physiological and biochemical reactions in plants, such as increased cell membrane permeability and cytoplasmic extravasation; increased reactive oxygen species content, intensified membrane lipid peroxidation, and malondialdehyde accumulation; abnormal transcription in the cell nucleus, inhibition of protein processing and translation, and loss of enzyme activity. It is easy to imagine that these changes in physiological and biochemical reactions will affect plants from growth delay to organ damage and even plant death, severely restricting the growth and development of food crops and fruits and vegetables, posing a serious challenge to agricultural production. However, plants under stress do not merely passively endure damage; they also actively regulate and adapt. It is this continuous adaptation process to stress that has enabled plants to develop sophisticated and complex stress resistance systems to adapt to adverse environments over a long period of evolution. Therefore, further research into the mechanisms of heat resistance in plants is one of the important ways to understand the relationship between plants and the environment and to cultivate new resistant varieties, with significant theoretical and applied implications. The damage caused by high temperatures to plants is multifaceted, and the plant response to abiotic stress is a complex process involving multiple signaling pathways and multi-gene regulation.

[0004] Rice, a member of the Poaceae family, is one of the world's most important food crops, feeding more than half of the world's population. It is also one of the most important monocotyledonous model plants in scientific research. Understanding how rice adapts to increasingly hot environments and applying genetic engineering to breed superior rice varieties with good adaptability to environmental temperature changes will provide new strategies for rice production and have profound significance for promoting the continuous and steady increase of rice yield. At the same time, the discovery and molecular mechanism research of rice's environmental temperature adaptability genes will also provide new ideas and references for the breeding of other crops. Summary of the Invention

[0005] The purpose of this invention is to provide a novel heat-resistant gene TT4 and its application in heat-resistant plant breeding.

[0006] In a first aspect of the invention, there is provided the use of a heat tolerance control gene THERMOTOLERANCE 4 or an expression unit thereof for: (a) enhancing plant heat tolerance or increasing plant yield under hot conditions; (b) acting as an amylase (or exercising amylase activity) to increase the amount of starch in plant grains under hot conditions; said starch comprising total starch or amylopectin; or, (c) acting as an adenylate cyclase (or exercising adenylate cyclase activity) to produce cyclic nucleotides (cAMP) using ATP as a substrate.

[0007] In one or more embodiments, the improvement of plant yield under thermal conditions includes improving the following under thermal conditions: seed setting rate, thousand-grain weight, grain length, yield per plant, and yield per plot.

[0008] In one or more embodiments, the expression unit includes: THERMOTOLERANCE 4 expression cassette, expression construct, or expression vector.

[0009] In one or more embodiments, the cAMP enhances the heat tolerance of plants.

[0010] In one or more embodiments, the heat resistance control gene THERMOTOLERANCE 4 is located in the chloroplasts of the cell and is diffusely distributed. Under heat conditions, it aggregates and becomes droplets.

[0011] In another aspect of the invention, a method for optimizing plant phenotype is provided, comprising: increasing the expression or activity of the heat tolerance control gene THERMOTOLERANCE 4 in a plant; wherein the optimization of plant phenotype includes: (a) enhancing the heat tolerance of the plant or increasing the yield of the plant under hot conditions; (b) increasing the amount of starch in the seeds of the plant under hot conditions; said starch includes total starch or amylopectin; or (c) generating cyclic nucleotides using ATP as a substrate.

[0012] In one or more embodiments, the optimization of plant phenotype includes optimizing plant phenotype in plants in which the heat tolerance control gene THERMOTOLERANCE 4 is expressed at low levels or not at all.

[0013] In one or more embodiments, the thermal (thermal stress) environment or high temperature is a temperature above 30°C, above 32°C, above 34°C, above 35°C, above 36°C, above 38°C, above 40°C, or above 42°C.

[0014] In one or more embodiments, the enhancement or improvement includes maintaining or restoring, i.e., reducing the losses caused by the thermal environment.

[0015] In one or more embodiments, the enhancement of the expression or activity of the heat tolerance control gene THERMOTOLERANCE 4 in plants includes introducing expression units containing the genomic DNA of the heat tolerance control gene THERMOTOLERANCE 4 or the coding region DNA of the heat tolerance control gene THERMOTOLERANCE 4 into the plant.

[0016] In one or more embodiments, the expression of the genomic DNA of the thermostability control gene THERMOTOLERANCE 4 or the coding region DNA of the thermostability control gene THERMOTOLERANCE 4 is driven by an expression promoter (chloroplast expression promoter).

[0017] In one or more embodiments, expression of the thermodynamic control gene THERMOTOLERANCE 4 genomic DNA or the coding region DNA of the thermodynamic control gene THERMOTOLERANCE 4 is driven by the promoter (natural promoter or its equivalent variant).

[0018] In one or more embodiments, the enhancement of the expression or activity of the heat tolerance control gene THERMOTOLERANCE 4 in plants includes: in plants where the heat tolerance control gene THERMOTOLERANCE 4 is mutated or has low function, performing a gain-of-function mutation on the corresponding gene to obtain a functional heat tolerance control gene (e.g., making positions 257, 604, and 781 of the protein it encodes conserved as V, G, and L); preferably, the gain-of-function mutation is performed using gene editing technology (such as CRISPR / Cas9-based technology) or site-directed mutagenesis technology.

[0019] In one or more embodiments, increasing the expression or activity of the heat tolerance control gene THERMOTOLERANCE 4 in plants includes: introducing the allele of the heat tolerance control gene THERMOTOLERANCE 4 into the main plant variety through hybridization, thereby increasing the expression or activity of the heat tolerance control gene THERMOTOLERANCE 4.

[0020] In one or more embodiments, the heat tolerance control gene THERMOTOLERANCE 4 includes genes derived from grasses, genes derived from cruciferous plants, or their homologs (homologous genes) or isofunctional variants (conserved variants).

[0021] In one or more embodiments, the heat tolerance control gene THERMOTOLERANCE 4 encodes a protein selected from the group consisting of: (i) a protein with the amino acid sequence shown in SEQ ID NO:8 (rice-derived, TT4).HP21 (i) a protein derived from (i) having the regulatory function, wherein the amino acid sequence is ≥80% homology (preferably ≥85%, ≥90%, ≥95% or ≥98%) with positions 257, 604, and 781 being conserved to be V, G, and L; (ii) a protein derived from (i) having the regulatory function, wherein the amino acid sequence shown in SEQ ID NO:8 is formed by substitution, deletion or addition of one or more (e.g., 1-20, 1-10, 1-5, 1-3) amino acid residues, and positions 257, 604, and 781 are conserved to be V, G, and L; or (iv) a protein formed by adding a tag sequence or restriction enzyme site sequence to the N or C end of the protein with the amino acid sequence shown in SEQ ID NO:8, or by adding a signal peptide sequence to its N end.

[0022] In one or more embodiments, the heat resistance control gene THERMOTOLERANCE 4 encodes a protein selected from the group consisting of: (i) a protein with the amino acid sequence shown in SEQ ID NO:12 (maize source, ZmTT4) or SEQ ID NO:16 (Arabidopsis source, AtTT4); (ii) a protein derived from (i) having the regulatory function, with an amino acid sequence having ≥80% homology (preferably ≥85%, ≥90%, ≥95%, or ≥98%) to the amino acid sequence shown in SEQ ID NO:12 or SEQ ID NO:16; (iii) a protein derived from (i) having the regulatory function, formed by substituting, deleting, or adding one or more (e.g., 1-20, 1-10, 1-5, 1-3) amino acid residues to the amino acid sequence shown in SEQ ID NO:12 or SEQ ID NO:16; or, (iv) a protein with the regulatory function shown in SEQ ID NO:12 or SEQ ID NO:16. The protein with the amino acid sequence shown in NO:16 is formed by adding a tag sequence or restriction enzyme site sequence to the N or C end of the protein, or by adding a signal peptide sequence to its N end.

[0023] In one or more embodiments, the nucleotide sequence of the heat resistance control gene THERMOTOLERANCE 4 is as shown in SEQ ID NO:6, SEQ ID NO:11, SEQ ID NO:15 or their degenerate sequences.

[0024] In one or more embodiments, the heat resistance control gene THERMOTOLERANCE 4 includes its isofunctional variants or conserved variants.

[0025] In one or more embodiments, the promoter has the nucleotide sequence shown in SEQ ID NO:5, SEQ ID NO:2, SEQ ID NO:10 or SEQ ID NO:14; a polynucleotide that can hybridize with the polynucleotide sequence shown in SEQ ID NO:5, SEQ ID NO:2, SEQ ID NO:10 or SEQ ID NO:14 under stringent conditions and has the same driving expression function; or a polynucleotide that is 75% or more (preferably 80% or more, more preferably 90% or more, more preferably 95% or more, such as 98% or more or more) homologous to the polynucleotide sequence shown in SEQ ID NO:5, SEQ ID NO:2, SEQ ID NO:10 or SEQ ID NO:14 and has the same driving expression function.

[0026] In one or more embodiments, the promoter or gene region includes their homologous genes that have the same function.

[0027] In one or more embodiments, the plant includes the following group or the heat tolerance control gene THERMOTOLERANCE 4 or its homologs are derived from the following group: monocotyledonous plants or dicotyledonous plants; preferably, the plant includes: food crops, vegetables, fruits, flowers, and forage grasses; preferably, the plant includes: cereal plants, cruciferous plants, and legumes; preferably, the plant includes (but is not limited to): grasses (such as, but not limited to, rice, wheat, millet, foxtail millet, corn, sorghum, millet, barley, rye, oats, and sedge), and cruciferous plants (such as, but not limited to, plants of the genus *Mucor*).

[0028] In another aspect of the invention, isolated THERMOTOLERANCE 4 protein is provided, comprising proteins selected from the group consisting of: (i) proteins with the amino acid sequence shown in SEQ ID NO:8 (rice-derived, TT4). HP21(i) a protein derived from (i) having the regulatory function, wherein the amino acid sequence is ≥80% homology (preferably ≥85%, ≥90%, ≥95% or ≥98%) with positions 257, 604, and 781 being conserved to be V, G, and L; (ii) a protein derived from (i) having the regulatory function, wherein the amino acid sequence as shown in SEQ ID NO:8 is formed by substitution, deletion or addition of one or more (e.g., 1-20, 1-10, 1-5, 1-3) amino acid residues, with positions 257, 604, and 781 being conserved to be V, G, and L; or (iv) a protein formed by adding a tag sequence or restriction enzyme site sequence to the N or C end of the protein with the amino acid sequence shown in SEQ ID NO:8, or by adding a signal peptide sequence to its N end.

[0029] In one or more embodiments, the nucleotide sequence of the heat resistance control gene THERMOTOLERANCE 4 is as shown in SEQ ID NO:6, SEQ ID NO:11, SEQ ID NO:15 or their degenerate sequences.

[0030] In another aspect of the invention, a plant cell, tissue, or organ is provided, comprising: exogenous thermotolerance control gene THERMOTOLERANCE 4 genomic DNA or coding region DNA, or expression units containing therein.

[0031] In one or more embodiments, the exogenous thermoresistance control gene THERMOTOLERANCE 4 genomic DNA or coding region DNA is operatively linked to a THERMOTOLERANCE 4 promoter (a natural promoter or its equivalent variant), which drives the expression of the THERMOTOLERANCE 4 genomic DNA or coding region DNA.

[0032] In one or more embodiments, the plant cells, tissues, or organs are not adult plants and do not have the ability to reproduce into adult plants.

[0033] In another aspect of the invention, the use of the plant heat tolerance control gene THERMOTOLERANCE 4 is provided for analyzing plant phenotypes; wherein the plant phenotypes include: heat tolerance or plant yield under heat conditions; starch content in plant grains under heat conditions; and cyclic nucleotide content.

[0034] In one or more embodiments, the protein, genomic DNA, and coding region DNA (e.g., expression) of the plant heat tolerance control gene THERMOTOLERANCE 4 are analyzed. If the plant possesses the characteristics (sequences) defined above, then the plant has heat tolerance or relatively high yield, relatively high starch content in grains, and relatively high cyclic nucleotide content under hot conditions. If the plant does not possess the characteristics defined above, then the plant has low heat tolerance or relatively low yield, relatively low starch content in grains, and relatively low cyclic nucleotide content (preferably, compared with control plants under the same conditions, which may be corresponding wild-type plants or corresponding transgenic plants without the target gene).

[0035] In one or more embodiments, nucleic acid sequence analysis is performed using methods including sequencing, PCR amplification, restriction enzyme digestion analysis, probe analysis, hybridization, microarray analysis, and allele polymorphism analysis.

[0036] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0037] Figure 1 TT4 localization cloning and transgenic verification.

[0038] (A) TT4 fine localization, identification of heat resistance phenotype in parents and 5 fragment replacement recombinant individuals. Black indicates replacement fragments from HP21, and white indicates fragments from HJX. R represents recombinant individuals, and C represents control individuals.

[0039] (B) Identification of heat tolerance phenotype in seedlings after knocking out the three genes LOC_Os06g12450, LOC_Os06g12455, and LOC_Os06g12460 in the background of Zhonghua 11 (ZH11).

[0040] (C)NIL-TT4 HP21 TT4 knockout in the background KO / NIL-TT4 HP21 Overexpression of TT4 from African rice in the HJX context HP21 (pUbi:TT4 HP21 -GFP-HJX) seedling heat tolerance phenotype identification. Survival rate was recorded after heat treatment and 7 days of recovery, n=3 samples. Each sample contained 24 independent plants. Data are expressed as Mean±SD. One-way ANOVA was used to test for significance, with P>0.05 indicating the same letter.

[0041] Figure 2 ,TT4 HP21 The site protects rice yield under heat stress.

[0042] NILs plants were treated in a greenhouse for 31 days. The average daily temperature from 6:00 AM to 6:00 PM (33.8℃) was higher than the normal temperature (30.4℃), with the highest daily temperature exceeding the normal growth conditions by 7.2℃. The plant type, ear phenotype, and yield after treatment are shown in the diagram above. Quantitative analysis was performed on NILs, including 1000-grain weight, seed setting rate, grain length, number of grains per ear, and yield per plant. n=30 plants per plot, n=3 plots, each containing 96 plants. Scale bar: 5cm. Data are Mean ± SD, expressed as NIL-TT4. HJX For comparison, Student's t-test was used to determine the significance of the difference.

[0043] Figure 3 TT4 affects starch synthesis and is located in chloroplasts.

[0044] (A) Changes in starch content and amylopectin ratio in seeds of near-isogenic lines before and after heat treatment. Student's t-test was used to assess the significance of differences, with three biological replicates.

[0045] (B) Subcellular localization of TT4 in rice protoplasts and stable transgenic materials. Scale bar: protoplast (left), 5 μm; rice root (top right), 10 μm; electron microscopy (bottom right), 1 μm.

[0046] (C) Positioning of TT4 before and after instantaneous tobacco heat treatment. Scale bar, 5 μm.

[0047] Figure 4 TT4 has adenylate cyclase activity.

[0048] (A) GSEA analysis of differentially expressed genes by RNA-seq before and after heat treatment in near-isogenic TT4. Left panel: 12 pathways enriched by differentially expressed genes among NILs under untreated conditions (qval<0.25); Right panel: the top 6 enriched pathways when NES is positive and the top 6 enriched pathways when NES is negative under heat treatment conditions (qVal<0.05).

[0049] (B)NIL-TT4 HP21 Compared to NIL-TT4 HJX Difference analysis based on GO enrichment analysis after heat treatment.

[0050] (C) Screening and enrichment pathway annotation of rice genes containing AC functional domains ([RKS]x[DE]x{9,11}[KR]x{1,3}[DE]).

[0051] (D) Evolutionary analysis of 8 homologous family proteins of TT4 in rice (top figure), evolutionary genes of TT4 homologs in different species (bottom figure).

[0052] (E)Analysis of the conservation of TT4 and its homologs in the AC functional domain, where arrows indicate four key amino acid sites and positions 627-711aa indicate potential ATP-binding sites.

[0053] (F) In vivo and in vitro verification of the AC enzyme activity of TT4. Left figure: TT4 calculated using the Michaelis-Menten equation. HJX and TT4 HP21 The maximum enzyme activity rate Vmax and the dissociation constant Km with the substrate are shown in the figure. The middle figure shows the difference in endogenous cAMP content between NILs before and after heat treatment, detected by an ELISA kit. The right figure shows the difference in endogenous cAMP content between knockout plants and wild-type plants, detected by ELISA. n=3 biological replicates (A, B, F), each containing 24 seedlings, are presented as Mean ± SD. The significance of differences was assessed using Student's t-test or one-way ANOVA, with P>0.05 indicated by the same letter.

[0054] Figure 5 ,TT4 HP21 Enhance the heat resistance of corn.

[0055] Maize overexpression of TT4 derived from rice HP21 The gene induced high-temperature tolerance, and the heat tolerance of maize seedlings is shown in the figure above. Note: The scale bar is 5cm; one-way ANOVA was used for significance testing, and the same letter indicates P>0.05. Detailed Implementation

[0056] This invention discloses a heat tolerance control gene, THERMOTOLERANCE 4, abbreviated as TT4. This gene has the following functions: enhancing plant heat tolerance or increasing plant yield under hot conditions; acting as an amylase to increase the starch content in plant seeds under hot conditions; and acting as an adenylate cyclase to produce cyclic nucleotides (cAMP) using ATP as a substrate.

[0057] In this invention, "plant" includes plants that express THERMOTOLERANCE 4 protein or its homologous protein, or plants whose genome contains the THERMOTOLERANCE 4 gene or its homologous gene. Based on knowledge in the art, plants expressing THERMOTOLERANCE 4 or its homologous protein possess the mechanism of action claimed in this invention and can achieve the technical effects claimed in this invention.

[0058] The plants mentioned may include food crops, vegetables, fruits, flowers, and forage grasses. The plants may be monocotyledonous or dicotyledonous. In some preferred embodiments, the plants are crops, preferably "cereal crops," which are crops with grains (ears). In some preferred embodiments, the "cereal crops" may be grasses (Poaceae); preferably, the grasses include, but are not limited to: rice, wheat, millet, foxtail millet, corn, sorghum, foxtail millet, barley, rye, oats, and short-stalked grass.

[0059] In this invention, terms such as "optimization / improvement of plant traits", "optimized / improved traits", "optimized / improved plant traits", and "trait optimization / improvement" can be used interchangeably. They refer to the statistically significant changes in the traits or characteristics of the plant modified by the technical solution of this invention compared to the plant before modification (such as wild-type plants), and these changes form beneficial agronomic traits.

[0060] In this invention, the "optimized traits" or "improved traits" refer to characteristics that positively regulate plants. These characteristics mainly include: heat tolerance, plant yield under hot conditions, starch content in plant seeds under hot conditions, and cyclic nucleotide content. Preferably, the yield-related traits include: seed setting rate, thousand-seed weight, seed length, yield per plant, and plot yield.

[0061] 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.

[0062] In this invention, "high temperature," "heat (stress)," or "heat (environment) (stress)" refers to temperatures significantly higher than the optimal temperature for plant growth (e.g., 18–30°C for grasses, preferably 20–32°C (e.g., 22–30°C or 22–28°C)); for example, "high temperature" or "heat (environment)" refers to temperatures above 30°C, above 32°C, above 34°C, above 35°C, above 36°C, above 38°C, above 40°C, or above 42°C.

[0063] In this invention, overexpression, 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, such as an increase of 10%, 20%, 40%, 60%, 80%, 90%, or higher.

[0064] In this invention, the terms “enhance,” “improve,” “increase,” “upregulate,” “promote,” etc., are interchangeable and, in their application, should mean at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, preferably at least 15% or 20%, more preferably 25%, 30%, 50%, 80%, 100%, or more significantly, an improvement compared to a control plant, control gene, or control protein as defined herein.

[0065] In this invention, "exogenous" or "heterogeneous" refers to the relationship between two or more nucleic acid or protein sequences from different sources. For example, if the combination of a promoter and a target gene sequence is not naturally occurring, then the promoter is exogenous to the target gene. A specific sequence is "exogenous" to the cell or organism into which it is inserted.

[0066] Regarding "control plants," selecting appropriate control plants is a routine part of experimental design. These can include corresponding wild-type plants or transgenic plants without the target gene. Control plants are generally the same plant species or even varieties of the same species or class as the plant being evaluated. Control plants can also be individuals from transgenic plants that have lost their transgenic components due to segregation. As used in this article, control plants refer not only to whole plants but also to plant parts, including seeds and seed portions.

[0067] In a specific embodiment of this invention, the heat-resistant African cultivated rice variety HP21 was used as the donor parent, and the high-yielding and high-quality Huajingxian 74 (HJX) was used as the recurrent parent to construct a chromosome segment replacement line (CSSL) (genetic background: Huajingxian 74). Through heat resistance phenotype identification and screening of the CSSL, a heat-resistant line HPS59 was selected. HPS59 was crossed with HJX to construct an F2 localization population, and a QTL locus significantly affecting plant heat resistance was identified and named TT4. Using map-based cloning, the TT4 gene controlling the plant's heat resistance phenotype was successfully located. Sequencing revealed that TT4 originated from the HP21 parent. HP21 The nucleotide C at position 770 of the gene was mutated to T, the nucleotide A at position 1810 to G, and the nucleotide T at position 2341 to C. This resulted in mutations in the protein: alanine at position 257 was replaced by valine, serine at position 604 by glycine, and proline at position 781 by leucine. Transgenic identification confirmed that TT4 is indeed a functional gene positively controlling plant heat tolerance. TT4 encodes a soluble starch synthase II and should have the function of regulating quality characteristics of rice, such as gelatinization temperature. A near-isogenic line, NIL-TT4, carrying the TT4 gene locus from HP21, was constructed and bred. HP21 And its corresponding control NIL-TT4 HJXHeat resistance was assessed by evaluating the mature phenotype of NILs. Three years of field greenhouse heat treatment revealed that TT2... HP21 High temperatures can protect plant yield, especially seed setting rate, ultimately resulting in yield increases of 64.1% (2021), 46.1% (2023), and 36.94% (2024) compared to the control. TT4 is located in chloroplasts, and the inherent disordered region of amino acids 1-314 at the front of its sequence allows it to transition from a diffuse to an aggregated state under high-temperature conditions. HJX and TT4 HP21 The protein showed no difference in morphological transformation induced by heat. Since the inherent enzymatic activity of TT4 (soluble starch synthase II) cannot explain its excellent heat resistance throughout the plant's growth cycle, sequence alignment revealed an adenylate cyclase functional domain at amino acid positions 418-434 of TT4, which is highly conserved in TT4 homologs. Liquid chromatography-tandem mass spectrometry and enzyme-linked immunosorbent assay (ELISA) further confirmed that TT4 possesses adenylate cyclase activity both in vitro and in vivo, catalyzing the conversion of the substrate ATP to the second messenger molecule cAMP, thereby achieving signal transduction and participating in the regulation of heat resistance. Furthermore, it was found that TT4… HJX and TT4 HP21 There was no difference in the activity of adenylate cyclase between the proteins. RNA and protein quantification experiments confirmed that TT4... HP21 At the same mRNA level, its protein content is higher than that of TT4. HJX This explains the differences in heat tolerance among NILs. The heat tolerance of TT4 in other species (such as Arabidopsis and maize) has been further confirmed, thus demonstrating that the broad-spectrum heat tolerance of TT4 can be achieved in multiple species, which is of great significance for breeding high-quality and high-yield crop varieties adapted to high temperatures.

[0068] Based on the above-mentioned new research results of the present invention, the TT4 includes the following main functions:

[0069] TT4 is a positive regulator of heat stress tolerance, and overexpression of TT4 in plants is beneficial. HP21 Genes that significantly enhance plant heat tolerance can be introduced into crop varieties using transgenic technology to improve heat resistance, while knocking out the TT4 gene in crops significantly reduces heat tolerance. For example, African cultivated rice varieties contain TT4. HP21 The site exhibits strong heat resistance, and the TT4 gene from Asian rice can be selected using molecular marker selection and hybridization breeding methods. HP21 The TT4 gene was introduced into Asian rice varieties to enhance their heat resistance. The TT4 gene plays a crucial role in plant adaptation to environmental temperature; genetic modification of this gene can improve crop stress resistance and yield.

[0070] The TT4 gene encodes a soluble starch synthase, and in a preferred manner, in TT4 HP21 The coding region of the site contains a mutation where nucleotide C at position 770 is changed to T, nucleotide A at position 1810 is changed to G, and nucleotide T at position 2341 is changed to C, thus encoding an enhanced TT4. HP21 Proteins that produce a heat-resistant phenotype, TT4 HP21 These sites have the function of enhancing heat resistance and can be applied to breeding improvement to enhance crop heat resistance.

[0071] TT4 plays an important role in the starch and amylopectin content of kernels before and after heat treatment. In the preferred method, TT4 is included. HP21 The starch and amylopectin content of the plants increased after high-temperature treatment.

[0072] TT4 is located in chloroplasts and is diffusely distributed. High-temperature treatment causes it to aggregate into droplets, which may be involved in regulating heat resistance.

[0073] In addition to its own amylase activity, TT4, as a part-time protein, also has adenylate cyclase (AC) activity. It can use ATP as a substrate to produce cAMP, which acts as a signal transduction molecule and activates a series of downstream genes.

[0074] In this invention, a “conservative variant” refers to a protein that substantially retains the same biological function or activity as the protein. A “conservative variant” may be (i) a protein in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) a protein having substituent groups in one or more amino acid residues; or (iii) a protein formed by the fusion of a mature protein with another compound; or (iv) a protein formed by the fusion of an additional amino acid sequence into the protein sequence (such as a leader sequence, secretory sequence, or sequence used to purify the protein, or a proteomic sequence).

[0075] The “conservative variant” may include (but is not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and additions or deletions of one or more amino acids (e.g., up to 50, more preferably up to 20 or 10, more preferably up to 5) at the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein. Similarly, adding one or more amino acids at the C-terminus and / or N-terminus generally does not alter the function of the protein. The present invention also provides analogs of the said protein. These analogs may differ from the natural protein in amino acid sequence, in the form of modifications that do not affect the sequence, or both.

[0076] Unless otherwise specified, the TT4 HP21 Genes refer to those with TT4 HP21 Genomic DNA (gTT4) HP21 ) gene, or TT4 HP21 The coding region genes also include those with TT4 HP21 Sequence variations with the same function as the protein also include promoters. The gene sequence also includes sequences degenerate with those provided in this invention. The TT4... HP21 With TT4 HJX These are genes located at the same locus.

[0077] The TT4 HP21 Protein variations also include (but are not limited to): deletions, insertions, and / or substitutions of several amino acids (typically 1-100 or 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, and even more preferably 1-8 or 1-5), and the addition or deletion 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 described protein (e.g., 50% or higher, 60% or higher, 70% or higher homology to the protein sequence shown in SEQ ID NO:6; preferably 80% or higher homology; more preferably 90% or higher homology, such as 95%, 98%, or 99% homology) and having the same function as the described protein is also included in this invention. Proteins derived from species other than rice that share high homology with the sequence shown in SEQ ID NO:6, or that play the same or similar roles in the same or similar regulatory pathways, are also included in this invention.

[0078] In this invention, TT4 HP21Genes / proteins (including gene promoters) also include their homologs. It should be understood that although this invention preferably studies genes obtained from specific species such as rice, maize, or Arabidopsis, other genes obtained from other species that are homologous to them (e.g., having more than 49%, 60%, more particularly 70%, 80%, 85%, 90%, 95%, or even 98% sequence identity) are also within the scope of this invention.

[0079] The high-temperature adaptability gene TT4 (full-length genomic DNA and cDNA) has high homology in other crops and will have similar heat-resistant functions in other crops. Therefore, it is important to protect the full-length genomic DNA and cDNA sequences of the TT4 gene in rice, as well as the homologous genes in other food crops such as wheat and corn, and other agricultural crops such as vegetables, fruits, flowers, and forage grasses.

[0080] The TT4 HP21 Polynucleotides (genes) can be natural genes from plants or their degenerate sequences.

[0081] The present invention also relates to a vector containing the aforementioned polynucleotide, and a host cell generated by genetic engineering using the aforementioned vector.

[0082] It should be understood that, once the function of TT4 is known, its expression or activity can be modulated using a variety of methods well known to those skilled in the art.

[0083] The technical solution of this invention can be applied to molecular design breeding through various pathways. Analysis shows that TT4 is a conserved gene in plant evolution, widely present in various crops, and has great application potential.

[0084] TT4 can be used as a molecular marker for targeted screening of plants. This new discovery can also be used to screen for substances or potential substances that can target heat stress tolerance and yield by modulating this mechanism.

[0085] The present invention will be 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, Science Press, or according to the manufacturer's recommendations.

[0086] Materials and methods

[0087] 1. Experimental materials and localized cloning

[0088] Using heat-tolerant African rice varieties as donor parents and the high-yielding heat-sensitive variety Huajingxian 74 as a recurrent parent, a set of chromosome segment replacement lines (CSSL) was constructed. A line with excellent heat resistance, HPS59, was then screened and obtained. HPS59 was then backcrossed with Huajingxian 74 to purify the background and generate the F1 generation. Self-pollination yielded a segregating F2 population for subsequent mapping. First, by observing the segregation of the F2 generation, map-based cloning was used to initially map the TT2 gene to the short arm of rice chromosome 6, between molecular markers InDel2667 and InDel2753. Then, using 8920 F2 rice plants, the gene was finely mapped to the region between molecular markers InDel2694 and InDel2705. This candidate region is approximately 13.75 kb. By comparing the online reference genome sequence of Nipponbare, three candidate genes were identified in this region: LOC_Os06g12450, LOC_Os06g12455, and LOC_Os06g12460. Primers were designed to amplify the full-length genome sequences of these three genes. Comparison of wild-type and mutant sequences revealed that candidate gene LOC_Os06g12450 had three SNPs causing amino acid substitutions, LOC_Os06g12455 had one SNP causing an amino acid substitution, and LOC_Os06g12460 had three SNPs causing amino acid substitutions.

[0089] Simultaneously, using BC5F2 generation plants, a segment of chromosome 6 containing a small HP21 chromosome (located between InDel2694 (6735000) and InDel2721 (6802500) on chromosome 6, carrying TT4) was constructed. HP21 Other genetic backgrounds include the near-isogenic line NIL-TT4 (HJX). HP21 and its control NIL-TT4 HJX (carrying TT4) HJX Other genetic backgrounds are also HJX.

[0090] The 5' oligonucleotide primer sequence for InDel2667 is as follows:

[0091] 5'-GGTCAGGTTTTGTAGTTTTCC-3'(SEQ ID NO:17)

[0092] The 3' primer sequence is as follows:

[0093] 5'-TATGAGCGTGACGTGTAATT-3'(SEQ ID NO:18)

[0094] The 5' oligonucleotide primer sequence for InDel2673 is as follows:

[0095] 5'-AAAAGAAGAAAGTAGCGCGA-3'(SEQ ID NO:19)

[0096] The 3' primer sequence is as follows:

[0097] 5'-GTTGCAACGGGATAGACATA-3'(SEQ ID NO:20)

[0098] The 5' oligonucleotide primer sequence for InDel2685 is as follows:

[0099] 5'-CGACAAATGCAACAAACAAC-3'(SEQ ID NO:21)

[0100] The 3' primer sequence is as follows:

[0101] 5'-AGAATACACTGGATGGAAGC-3'(SEQ ID NO:22)

[0102] The 5' oligonucleotide primer sequence for InDel2694 is as follows:

[0103] 5'-CCAATGTCTTTAGGTTTGATTA-3'(SEQ ID NO:23)

[0104] The 3' primer sequence is as follows:

[0105] 5'-TCATACCCTTCCATTTTCAT-3'(SEQ ID NO:24)

[0106] The 5' oligonucleotide primer sequence for InDel2701 is as follows:

[0107] 5'-CCTTTGCACAACCGAAATC-3'(SEQ ID NO:25)

[0108] The 3' primer sequence is as follows:

[0109] 5'-GATGACATGCAAATATCGGT-3'(SEQ ID NO:26)

[0110] The 5' oligonucleotide primer sequence for InDel2705 is as follows:

[0111] 5'-TGTCAGCGAATCGATATAGC-3'(SEQ ID NO:27)

[0112] The 3' primer sequence is as follows:

[0113] 5'-GCAAATTCTATGCTGTCACC-3'(SEQ ID NO:28)

[0114] The 5' oligonucleotide primer sequence for InDel2721 is as follows:

[0115] 5'-GGTATCTTGCAACACCTTCT-3'(SEQ ID NO:29)

[0116] The 3' primer sequence is as follows:

[0117] 5'-CTTTTACCCCTTGACCTTCA-3'(SEQ ID NO:30)

[0118] The 5' oligonucleotide primer sequence for InDel2753 is as follows:

[0119] 5'-AGTTAGAAGCTGGAAAGTGC-3'(SEQ ID NO:31)

[0120] The 3' primer sequence is as follows:

[0121] 5'-CACTGCAACCGGGATTAC-3'(SEQ ID NO:32)

[0122] 2. Transgenic validation, overexpression, and CRISPR / Cas9 gene editing

[0123] To further confirm that LOC_Os06g12450 is the cause of TT4 HP21 To assess the phenotype, a gene overexpression vector was constructed that overexpressed the full-length cds of the HP21-derived TT4 gene using a maize ubiquitin promoter. Gene knockout vectors were designed using CRISPR / Cas9 gene editing technology to knock out the target gene (TT4). All genetic experiments were conducted using the Agrobacterium tumefaciens EHA105-mediated callus transformation method mediated by rice seeds. Transgenic positive lines were obtained through genetic screening, and stable T2 generation transgenic plants were obtained and their phenotypes were examined.

[0124] The 5' oligonucleotide primer sequence for constructing the pCOMBIA1300 complementary vector is as follows:

[0125] 5'-aattcgagctcggtacccggggatccCCTCTTGCTAATCTTTTGGTAATGGC-3' (SEQ ID NO: 33)

[0126] The 3' primer sequence is as follows:

[0127] 5'-caagcttgcatgcctgcaggtcgacAGATCTGTACAGCTCGTCCATGCCGTGA-3'(SEQ IDNO:34)

[0128] The 5' oligonucleotide primer sequence for the U3 fragment constructed using the CRISPR / Cas9 knockout TT4 vector is as follows:

[0129] 5'-GGCATGCTCCGGCAATTCCATGTA-3'(SEQ ID NO:35)

[0130] The 3' oligonucleotide primer sequence for the U3 fragment constructed using the CRISPR / Cas9 knockout TT4 vector is as follows:

[0131] 5'-AAACTACATGGAATTGCCGGAGCA-3'(SEQ ID NO:36)

[0132] The 5' oligonucleotide primer sequence for the U6a fragment constructed using the CRISPR / Cas9 knockout TT4 vector is as follows:

[0133] 5'-GCCGCGCCGGAAGGCTAGCCCTA-3'(SEQ ID NO:37)

[0134] The 3' oligonucleotide primer sequence for the U6a fragment constructed using the CRISPR / Cas9 knockout TT4 vector is as follows:

[0135] 5'-AAACTAGGGCTAGCCTTCCGGCG-3'(SEQ ID NO:38)

[0136] The 5' oligonucleotide primer sequences for constructing the Pun1301 overexpression vector are as follows:

[0137] 5'-cttctgcaggtcgactctagaggatccATGTCGTCGGCCGTCGTCGCGTCATCCA-3'(SEQ IDNO:39)

[0138] The 3' oligonucleotide primer sequences for constructing the Pun1301 overexpression vector are as follows:

[0139] 5'-gctcctcgcccttgctcacggtaccCCATTGGTACTTGGCCTTGACAAGGACC-3'(SEQ IDNO:40)

[0140] The 5' oligonucleotide primer sequence for constructing the P3300 overexpression vector is as follows:

[0141] 5'-ctgcaggtcgactctagaggatccATGTCGTCGGCCGTCGTCGCGTCATCCA-3'(SEQ ID NO:41)

[0142] The 3' oligonucleotide primer sequence for constructing the P3300 overexpression vector is as follows:

[0143] 5'-gaacgatcggggaaattcgagctctcaTTTCGAACCCGGCGTACCGCCCTTA-3'(SEQ ID NO:42)

[0144] The 5' oligonucleotide primer sequence for the U6a fragment constructed using the CRISPR / Cas9 knockout LOC_Os06g12455 vector is as follows:

[0145] 5'-gccgTAGGGACCACCACACAGTGC-3'(SEQ ID NO:43)

[0146] The 3' oligonucleotide primer sequence for the U6a fragment constructed using the CRISPR / Cas9 vector knockout LOC_Os06g12455 is as follows:

[0147] 5'-aaacGCACTGTGTGGTGGTCCCTA-3'(SEQ ID NO:44)

[0148] The 5' oligonucleotide primer sequence for the U3 fragment constructed using the CRISPR / Cas9 vector with LOC_Os06g12455 knockout is as follows:

[0149] 5'-GGCATTGAGAAAAACTAGTTGCC-3'(SEQ ID NO:45)

[0150] The 3' oligonucleotide primer sequence for the U3 fragment constructed using the CRISPR / Cas9 vector knockout LOC_Os06g12455 is as follows:

[0151] 5'-aaacGGCAACTAGTTTTTCTCAA-3'(SEQ ID NO:46)

[0152] The 5' oligonucleotide primer sequence for the U6a fragment constructed using the CRISPR / Cas9 vector knockout LOC_Os06g12460 is as follows:

[0153] 5'-GCCGAGCAGCTTGCGGACTATCG-3'(SEQ ID NO:47)

[0154] The 3' oligonucleotide primer sequence for the U6a fragment constructed using the CRISPR / Cas9 vector knockout LOC_Os06g12460 is as follows:

[0155] 5'-AAACCGATAGTCCGCAAGCTGCT-3'(SEQ ID NO:48)

[0156] The 5' oligonucleotide primer sequence for the U3 fragment constructed using the CRISPR / Cas9 vector with LOC_Os06g12460 knockout is as follows:

[0157] 5'-GGCATGCAAGTCTTGGGGTAATAA-3'(SEQ ID NO:49)

[0158] The 3' oligonucleotide primer sequence for the U3 fragment constructed using the CRISPR / Cas9 vector with LOC_Os06g12460 knockout is as follows:

[0159] 5'-AAACTTATTACCCCAAGACTTGCA-3'(SEQ ID NO:50)

[0160] 3. Starch content detection

[0161] The aboveground parts of transgenic plant seedlings and seeds of near-isogenic types were obtained separately for starch content detection. The starch content was detected using the Grace company's linear / branched / total starch content (enzymatic method) kit (catalog number: G0548F). The specific procedure was as follows: 1-5g of sample was dried (50℃) to constant weight, ground, and sieved (e.g., through a 0.5mm sieve) to obtain a uniform powder. 10mg of the powder sample was placed in a 2mL EP tube, 0.5mL of DMSO was added, and the sample was vortexed to disperse and suspend it in the liquid. The tube was then boiled in a water bath for about 2 minutes until the sample was dispersed and dissolved. High-speed vortexing was followed by another 15 minutes of boiling in a water bath (with intermittent vortexing every 2-3 minutes to ensure complete dispersion and dissolution). The tube was then removed and allowed to cool at room temperature for about 5 minutes. 1mL of anhydrous ethanol was added, and the tube was immediately vortexed at high speed. Another 0.5mL of anhydrous ethanol was added, and the tube was inverted repeatedly. The tube was allowed to stand for 5 minutes, then centrifuged at 5000rpm at room temperature for 5 minutes. The supernatant was discarded, and the precipitate was retained. Add 1 mL of DMSO to the precipitate and vortex to mix. Incubate in a boiling water bath for 15 min (with intermittent vortexing every 2-3 min to ensure complete dispersion and dissolution of the sample). Allow to cool naturally for 5 min, then centrifuge at 3000 rpm at room temperature for 5 min. Take 0.1 mL of the supernatant into a new 2 mL EP tube, and add 0.9 mL of reagent diluent to obtain the test solution. Take 200 μL of the test solution and add 100 μL of reagent II. Mix by inversion and let stand for 1 hour. Centrifuge at 14000 rpm at room temperature for 10 min. Take 75 μL of the supernatant and add 175 μL of reagent III. Boil at 95-100℃ for 5 min, then incubate at 40℃ for 5 min. Add 50 μL of reagent IV, mix well, incubate at 40℃ for 30 min, then centrifuge at 8000 rpm at room temperature for 5 min. Take 160 μL of the supernatant and add 80 μL of reagent V and 560 μL of reagent VI. Mix well and incubate at 40℃ in the dark for 20 min. Calculate the amylose content by reading the absorbance at 510 nm. Add 300 μL of reagent three and 50 μL of reagent four to 40 μL of the test solution. Incubate at 40°C for 30 min. Then, take 160 μL and add 80 μL of reagent five and 560 μL of reagent six. Mix well and incubate at 40°C in the dark for 20 min. Read the absorbance at 510 nm to calculate the total starch content. The total starch content minus the amylose content is the amylopectin content.

[0162] 4. Localization detection of protoplasts and living root subcellular structures

[0163] Subcellular localization detection of rice protoplast transient transformation. Rice seedlings were aseptically cultured for 12-15 days at 28℃ / 25℃ for 13 hours in light / 11 hours in dark. Leaves were removed, leaving only the green tissue of the leaf sheaths. The protoplasts were cut into 0.3-0.5 mm segments using a single-edged blade and transferred to a 0.6M mannitol solution on ice in the dark, ensuring complete protection from light until all leaves were removed. The mannitol solution was gently removed, and freshly prepared enzyme digest was added. The mixture was vacuum-sealed for 10-15 minutes in the dark, and then gently shaken at 65 rpm at room temperature for 4-5 hours. The enzyme digest was gently poured out and W5 was added. The mixture was gently shaken and rinsed 3-5 times. The protoplasts were filtered through a 40 μm nylon mesh into 50 ml tubes and centrifuged at 100 g for 2 minutes. The brake speed was adjusted to 0 during the rise and 1 during the fall (i.e., slow acceleration and slow deceleration). The supernatant was discarded, and the protoplasts were slowly washed with W5, repeating twice. Discard the supernatant. Slowly add 6-8 mL of MMG, adhering to the tube wall, and gently shake to mix the solid at the bottom. Using a cut pipette tip (to prevent excessive pressure from breaking the protoplasts), divide the protoplasts into 200 μL portions. For each sample, slowly add 25 μg of plasmid DNA along the tube wall (adhering to the wall) by rotating the pipette tip. Slowly add freshly prepared PEG, adhering to the wall, and gently invert to mix. Incubate at room temperature in the dark for 10-20 min. Slowly add 4 volumes of W5 solution, adhering to the wall, and gently invert to mix. Centrifuge at 100 rcf for 2 min. Aspirate the supernatant using a cut pipette tip. Repeat 3 times. Slowly add 1 mL of W5, adhering to the wall, and gently invert to resuspend. Incubate horizontally in a 24°C incubator in the dark for 12-16 hours. Observe under a Leica SP8 ultra-high resolution laser confocal microscope.

[0164] In vivo root subcellular localization detection. pUBI::TT4 HP21 -GFP / HJX rice seeds were dehulled and disinfected with 75% sodium hypochlorite solution for 45 seconds, then with 33% sodium hypochlorite solution for 15 minutes. After washing with sterilized ddH2O 5-6 times, they were sown on 1 / 2 MS medium and cultured for 3-4 days. The root tips of the seedlings were then immersed in FM4-64 staining solution for 1 minute and observed using a Leica SP8 ultra-high resolution laser confocal microscope.

[0165] 5. Immunoassay with colloidal gold

[0166] The experiment used pUBI::TT4 HP21Immunogold labeling assays were performed on GFP / HJX rice materials. First, rice samples were taken from both room temperature culture conditions and after high-temperature treatment (42℃, 24h). Leaves were rapidly cut into strips 2mm long and 1mm wide and quickly fixed using 4% (v / v) glutaraldehyde diluted in 0.1M PBS (pH 7.2). After fixation, the samples were washed three times with PBS and placed in 2% (v / v) osmium tetroxide (PBS diluted) at room temperature for 1h. Subsequently, the samples were washed three times with ultrapure water, dehydrated with anhydrous ethanol, and then embedded in LR white resin for several days. The embedded samples were then prepared into ultrathin sections of 80-100nm thickness using a Leica Ultracut R microtome and diamond scalpel, and collected on a gold grid. The prepared samples can be stored long-term in a desiccator. Before labeling, the gold grid was treated with 10% H2O2 for 10 min, washed with ultrapure water, and fixed with 5% (w / v) BSA (1xTBST dilution) at room temperature for 2 h. After drying the filter paper, it was incubated at room temperature for 4 h with antibody dilution buffer containing anti-GFP as needed. The antibody was diluted with 1% (w / v) BSA (1xTBST dilution) at a dilution ratio of 1:30. After incubation, it was washed three times with 1xTBST, dried on the filter paper, and transferred to antibody dilution buffer containing anti-mouse IgG antibody (whole molecule 10-nm colloidal gold) (Sigma, G7652) at a dilution ratio of 1:30. It was incubated at room temperature for 1 h, washed three times with 1xTBST, dried on the filter paper, and then stained in 2% (w / v) uranyl acetic acid for 3 min. It was then quickly transferred to lead citrate for staining for 2 min. Finally, it was rinsed three times with ultrapure water and dried on the filter paper for preservation or microscopic examination. The tests were performed using a Hitachi HT7650 TEM transmission electron microscope at a voltage of 80 kV.

[0167] 6. Changes in the location of tobacco cells after heat treatment.

[0168] Agrobacterium GV3101 containing the target plasmid was mixed with tobacco injection buffer (10 mM MES, 10 mM MgCl2, 50 μM acetylsylcholine) and slowly and evenly injected into tobacco leaves. After culturing at a suitable temperature for 3 days, samples were taken and observed under a Leica SP8 super-resolution laser confocal microscope. To observe changes in subcellular localization after heat treatment of tobacco in vivo, the injected tobacco leaves were first perforated and then treated in a metal bath at 42°C heated to ddH2O for the required time before being observed under a 63X objective lens in SP8.

[0169] 7. AC enzyme activity detection

[0170] In vivo enzyme activity assay. Rice seedlings grown at 28℃ for 12 days were treated accordingly, then sampled and ground in liquid nitrogen. The samples were then analyzed using the Cayman Cylic AMP ELISA Kit (catalog number: 581001). Approximately 0.2g of sample was added to 400ul of 5% TCA aqueous solution, mixed, and lysed on ice for 15min. The mixture was then centrifuged at 1500g for 15min at 4℃. The supernatant was transferred to a new 1.5ml EP tube, and 5 volumes of water-saturated ether were added. The tube was vortexed for 10s and allowed to stand for phase separation. The upper ether phase was removed. This process was repeated three times. After removing the tube, the ether residue was evaporated at 70℃. The acetylated standard and sample were then added to the ELISA plate, and buffer was added. The plate was incubated at 4℃ for 18h. The reaction solution in each well was discarded. Each well was washed 5 times with 200ul of wash buffer. 200ul of Ellman's Reagent was added to each well, and the plate was incubated at room temperature for 45-90min. The absorbance at 412nm was read, and the cAMP content of each sample was calculated.

[0171] In vitro enzyme activity assay. The target protein with the GST tag was expressed and purified using an insect expression system. A 100 μL reaction mixture was prepared containing: 10 mM Tris-HCl buffer (pH 7.6), 1 mM MgCl2, 1 mM MnCl2, 1 mM IBMX (3-isobutyl-1-methylxanthine), 1 mM ATP, 1 mM DTT, and 5 μg protein. The reaction was carried out at 30 °C for 45 min, boiled at 99 °C for 10 min, and then terminated by adding 300 μL of pre-cooled methanol. The mixture was centrifuged at 16,000 rpm for 20 min at 4 °C, and the supernatant was collected for LC-MS / MS analysis of cAMP production.

[0172] 8. Treatment of abiotic stress

[0173] For high-temperature treatment of rice, each sample was first soaked and germinated. After growing at the normal temperature of 28℃ for 12 days, it was subjected to high-temperature treatment (42℃). After 7 days of recovery, the phenotype was observed and the survival rate was statistically analyzed.

[0174] For high-temperature treatment of rice during the booting stage, rice seedlings are transplanted to the field and, after they grow normally to the booting stage, high-temperature treatment is carried out in plastic greenhouses (treatment time > 20 days, average daytime maximum temperature > 40℃). After the seeds mature, agronomic traits such as seed setting rate, yield per plant, and plot yield are examined.

[0175] For high-temperature treatment of maize, plump maize seeds were sown in nutrient soil and cultured at 28℃ / 25℃ for 12-15 days until the second leaf stage, 16 hours of light / 8 hours of dark. The seeds were then transferred to a treatment chamber (42℃) for high-temperature treatment. After 7 days of recovery following treatment, the phenotype was observed and the survival rate was statistically analyzed.

[0176] Example 1: Gene Discovery

[0177] To explore more superior heat-resistant rice germplasm resources and study the molecular regulatory mechanisms of stress resistance formation, the inventors used the heat-resistant African cultivated rice variety HP21 as the donor parent and the widely promoted high-yielding and high-quality variety Huajingxian 74 (HJX) as the recurrent parent to construct a set of replacement lines covering the entire rice chromosome segment. From this, a replacement line HPS59 with stable heat resistance was identified. HPS59 was crossed with the recurrent parent HJX to construct a BC5F2 mapping population. Through multi-generation mapping of 8920 individuals and identification of the heat resistance phenotype of 5 recombinant individuals with local fragment exchanges, TT4 (THERMOTOLERANCE4) was finally mapped to a range of 13.75 kb. It was found that there were three SNPs between HPS59 and HJX. Specifically, LOC_Os06g12450 had 3 SNPs causing amino acid substitutions, LOC_Os06g12455 had 1 SNP causing amino acid substitutions, and LOC_Os06g12460 had 3 SNPs causing amino acid substitutions.

[0178] To identify candidate genes for TT4, three genes—LOC_Os06g12450 (450KO), LOC_Os06g12455 (455KO), and LOC_Os06g12460 (460KO)—were knocked out in the Zhonghua 11 (ZH11) genus. Only the knockout of LOC_Os06g12450 reduced the heat resistance of ZH11, demonstrating that LOC_Os06g12450 contains the gene encoding TT4. Figure 1 As shown in A and B.

[0179] Overexpression of TT4 derived from African rice in the HJX background of heat stress intolerance HP21 (pUbi:TT4 HP21 -GFP-HJX) exhibits a significantly enhanced heat resistance phenotype, and its survival rate reaches approximately 50%, such as Figure 1 As shown in C.

[0180] NIL-TT4, which has strong resistance to heat stress HP21 TT4 knockout in the background KO / NIL-TT4 HP21 It can lower NIL-TT4 HP21 The heat-resistant phenotype (changing it from a heat-resistant phenotype to a heat-sensitive phenotype), such as Figure 1 As shown in C. Therefore, the QTL TT4, which regulates rice heat stress tolerance, was successfully located and cloned. TT4 is a positive regulator of rice heat stress tolerance. When TT4 from African cultivated rice was introduced... HP21 At this time, it will endow rice with a stronger tolerance to heat stress.

[0181] In summary, a natural site, TT4, with high tolerance to high temperatures was obtained using map-based cloning. HP21 They also identified the TT4 gene that controls its phenotype.

[0182] Rice TT4 HJX The full-length gene sequence is shown in SEQ ID NO:1; TT4 HJX The promoter sequence is as follows: SEQ ID NO:2; TT4 HJX The coding region sequence is as shown in SEQ ID NO:3.

[0183] Rice TT4 HP21 The full-length gene sequence is shown in SEQ ID NO:4; TT4 HP21 Promoter sequences such as SEQ ID NO:5; TT4 HP21 The coding region sequence is as follows: SEQ ID NO:6: TT4 HJX The protein sequence is shown in SEQ ID NO:7; TT4 HP21 The protein sequence is shown in SEQ ID NO:8.

[0184] Example 2, TT4 HP21 Sites protecting rice yield under heat stress

[0185] The heat resistance of mature plants from near-isogenic lines was identified, and high-temperature treatment in a field greenhouse revealed that NIL-TT2... HP21 It exhibited strong tolerance to heat stress, and the morphology of its plants did not change significantly, mainly in the ear: compared to NIL-TT4 HJX NIL-TT4 HP21 It exhibits a significantly high seed setting rate phenotype, such as Figure 2 As shown.

[0186] Quantitative analysis showed that high temperature caused NIL-TT4 HJX The severe yield reduction, with decreased seed setting rate and thousand-grain weight, severely damaged the yield per plant and the yield per plot. However, in NIL-TT4 HP21 Medium, TT4 HP21 It protects rice yield, mainly by protecting the seed setting rate. After heat treatment, NIL-TT4 HJX The fruit set rate is only about 21.4%, however, NIL-TT4 HP21 It will remain around 43.0%, such as Figure 2 As shown.

[0187] Under normal conditions, NIL-TT4 HJX and NIL-TT4 HP21There was no significant difference in yield per plant or yield per plot; however, after heat treatment, NIL-TT4... HP21 Compared to NIL-TT4 HJX The output of the community increased by approximately 46.1%, such as Figure 2 As shown.

[0188] The above results prove that TT2 HP21 It plays an important role in protecting rice yield under heat stress.

[0189] Example 3: Amylase activity of TT4

[0190] The inventors' analysis also showed that the protein encoded by TT4 is a soluble starch synthase, and it functions within chloroplasts. TT4 affects the synthesis of medium-length branched chains in amylopectin, thereby regulating rice quality. Under normal conditions, the total starch content among NILs remains unchanged, but after high-temperature treatment in a greenhouse, NIL-TT4... HP21 The total starch and amylopectin content in the kernels were significantly higher than those in NIL-TT4. HJX . TT4 HJX The activity of starch synthase was higher than that of TT4 after high temperature stress. HP21 Some decrease, such as Figure 3 As shown in Figure A, TT4 positively regulates the content of total starch and amylopectin in rice grains.

[0191] In vivo imaging of protoplasts and roots of transgenic materials showed that TT4 was located in the chloroplasts of the cells, and TT4 HJX With TT4 HP21 There was no difference in subcellular localization. Immunogold assays on transgenic materials further confirmed that TT4 is located in chloroplasts. Under normal temperature conditions, TT4 is diffusely distributed in chloroplasts. After high-temperature heat treatment, TT4 begins to aggregate in chloroplasts, forming a dot-like arrangement, such as... Figure 3 As shown in B and C.

[0192] Example 4: Adenylate cyclase activity of TT4

[0193] The inventors' analysis also showed that TT4 regulates multiple downstream pathways and possesses novel adenylate cyclase (AC) activity. Seedling transcriptome data from NILs showed that TT4 participates in the regulation of multiple signaling pathways, including ATP-binding pathways, covering chromatin remodeling, RNA processing, protein translation, and metabolism, such as... Figure 4As shown in A and B. A single starch synthase located at the downstream end of signal regulation is insufficient to trigger so many downstream responses, therefore it is speculated that TT4 may possess novel enzymatic activity. Adenylate cyclase (AC) produces cyclic nucleotides (cAMP) using ATP as a substrate. Its functional domain is mostly found in part-time proteins, and the cAMP it mediates can act as a signal molecule to activate downstream responses. Through genome-wide matching of the AC functional domain ([RKS]x[DE]x{9,11}[KR]x{1,3}[DE]), a total of 13,050 genes carrying the AC functional domain were screened. Enrichment analysis revealed that it is enriched in the starch binding and amylopectin formation pathways, which contain TT4, such as... Figure 4 As shown in C.

[0194] Sequence analysis further revealed that the AC functional domain is highly conserved in TT4 and its two homologous proteins in rice, as well as in crops such as Arabidopsis thaliana, maize, and sorghum, suggesting that TT4 possesses AC activity. Figure 4 As shown in D and E.

[0195] The full-length maize ZmTT4 gene is shown in SEQ ID NO:9; the ZmTT4 promoter sequence is shown in SEQ ID NO:10; the ZmTT4 coding region sequence is shown in SEQ ID NO:11; and the ZmTT4 protein sequence is shown in SEQ ID NO:12.

[0196] The full-length sequence of Arabidopsis thaliana AtTT4 is shown in SEQ ID NO:13; the AtTT4 promoter sequence is shown in SEQ ID NO:14; the AtTT4 coding region sequence is shown in SEQ ID NO:15; and the AtTT4 protein sequence is shown in SEQ ID NO:16.

[0197] In vitro expression of TT4 protein and subsequent gas chromatography-tandem mass spectrometry (GC-MS) experiments confirmed that the expressed TT4 protein possesses adenylate cyclase activity, producing cyclic nucleotides (cAMP) using ATP as a substrate. Michaelis-Menten equations showed that TT4… HJX The maximum enzyme rate was 18.98 fmol / min / μg protein, and the dissociation constant Km was 0.2318 mM; TT4 HP21 The maximum enzyme rate was 16.77 fmol / min / μg protein, and the dissociation constant Km was 0.1903 mM. Figure 4 As shown in F.

[0198] Immunoenzyme-linked immunosorbent assay (ELISA) confirmed that NIL-TT4 HP21 The endogenous cAMP content in the middle liposome is higher than that in NIL-TT4. HJX ,TT4 KO / NIL-TT4 HP21 The cAMP content in it was significantly lower than that in NIL-TT4 HP21 ,like Figure 4 As shown in F.

[0199] This result confirms that TT4 possesses adenylate cyclase activity, NIL-TT4 HP21 The endogenous cAMP content in the middle liposome is higher than that in NIL-TT4. HJX cAMP content is NIL-TT4 HP21 The reason for its stronger heat resistance.

[0200] Example 5, TT4 HP21 Imparting high-temperature heat resistance to corn

[0201] Through genetic engineering, TT4, derived from African cultivated rice, is... HP21 Overexpression of TT4 derived from rice was confirmed in maize by Western blot experiments. HP21 Transcription and translation can proceed normally in the maize system, such as Figure 5 As shown.

[0202] Heat tolerance assessment of soil-grown corn seedlings confirmed that TT4 HP21 The B104 plant exhibits a significantly stronger heat tolerance phenotype compared to the wild-type B104, such as... Figure 5 As shown, this confirms that TT4 can still enhance heat tolerance in other species, laying a solid foundation for the future application of this gene in other crops.

[0203] sequence

[0204] Rice TT4 (including full-length gene, promoter, coding region, and protein)

[0205] TT4 HJX Full-length gene sequence (SEQ ID NO:1):

[0206]

[0207] CGTCGCTTCGGTTCGGTTCCGGCTCGATCGGGGAGGTTCCTCGGGGCGACC

[0208]

[0209] TT4 HJX Promoter sequence (SEQ ID NO:2):

[0210]

[0211] TT4 HJX The coding region sequence (SEQ ID NO:3):

[0212]

[0213] TT4 HP21 Full-length gene sequence (SEQ ID NO:4):

[0214]

[0215] TT4 HP21 Promoter sequence (SEQ ID NO:5):

[0216]

[0217] TT4 HP21 Coding region sequence, with the mutation sites marked in bold and underlined (SEQ ID NO:6):

[0218]

[0219] TT4 HJX Protein sequence of (SEQ ID NO:7):

[0220] MSSAVVASSTTFLVALASSASRGGPRRGRVVGVAAPPALLYDGRAGRLALRAPPPPRPRPRRRDAGVVRRADDGENEAAVERAGEDDDEEEEFSSGAWQPPRSRRGGVGKVLKRRGTVPPVGRYGSGGDAARVRGAAAPAPAPTQDAASSKNGALLSGRDDDTPASRNGSVVTGADKPAAATPPVTITKLPAPDSPVILPSVDKPQPEFVIPDATAPAPPPPGSNPRSSAPLPKPDNSEFAEDKSAKVVESAPKPK A TRSSPIPAVEEETWDFKKYFDLNEPDAAEDGDDDDDWADSDASDSEIDQDDDSGPLAGENVMNVIVVAAECSPWCKTGGLGDVAGALPKALARRGHRVMVVVPRYGDYAEAQDVGIRKYYKAAGQDLEVKYFHAFIDGVDFVFIDAPLFRHRQDDIYGGNRQEIMKRMILFCKAAVEVPWHVPCGGVPYGDGNLVFLANDWHTALLPVYLKAYYRDNGMMQYTRSVLVIHNIAYQGRGPVDEFPYMELPEHYLDHFKLYDPVGGEHANIFGAGLKMADRVVTVSPGYLWELKTTEGGWGLHDIIRENDWKMNGIVNGIDYREWNPEVDVHLQSDGYANYTVASLDS SKPRCKAALQRELGLEVRDDVPLIGFIGRLDGQKGVDIIGDAMPWIAGQDVQLVLLGSGRRDLEVMLQRFEAQHNSKVRGWVGFSVKMAHRITAGADVLVMPSRFEPCGLNQLYAMAYGTVPVVHAVGGLRDTVSAFDPFEDTGLGWTFDRAEPHKLIEALGHCLETYRKYKESWRG F QVRGMSQDLSWDHAAELYEEVLVKAKYQW

[0221] TT4 HP21 The protein sequence, with mutation sites marked in bold underlined (positions 257, 604, and 781) (SEQ ID NO: 8):

[0222] Maize TT4 (ZmTT4) (including full-length gene, promoter, coding region, and protein)

[0223] Full length of ZmTT4 gene (SEQ ID NO:9):

[0224]

[0225] ZmTT4 promoter sequence (SEQ ID NO:10):

[0226] CAATCTGTTGAAACCGAGCTCACGGGCTAGGCCAACTGGACTCCCGACACCGCCGACGCAACCCAAGGATCACCCAAAACTTCCACCTCAGACCCCAACCCCCTCCATGGCCCCATAACTAGAGAAACAACAACACTACGTCCCTACGAAACGCAAGCAAGCCATAGAAAGATGTCCCACAGAGAAAGGGAGGAGGGAGGAGGAGGAAAACAAATTTCAATCAGGATTTTGTGCCGCCGCCGCGATGAAGGTTGCCGTCGGTGGAACATGTCAACTGTTGACGGTTTTTTTTTTCGAGAGATGGAACTTGCTTTTCTCTTTTTTTGAAACATGGGATTCCTGCAGTCTGCTCTTTACCGTGTCCACGGCACCATCCTCCGCCGTCGGGTGTAGCCGTCTAGCCT

[0227] ZmTT4 coding region sequence (SEQ ID NO:11):

[0228]

[0229] ZmTT4 protein sequence (SEQ ID NO:12):

[0230] MLTFQLSDQTLHPFDHASFQGTARLSAVRCPPRALRSRAALPAARPRAHWTSFRPAPIDPAAMSSAAVSSSSSTFFLALASASPGGRRRARVGSSPFHTGASLSFAFWAPPSPPRAPRDAALVRAEAEAGGKDAPPERSGDAARLPRARRNAVSKRRDPLQPVGRYGSATGNTARTGAASCQNAALADVEIKSIVAAPPTSIVKFPAPGYRMILPSGDIAPETVLPAPKPLHESPAVNSVPPVSIPNPDTDGIAALAEKKYAQVDGDSNGIAPPTVEPLVQEATWDFKKYIGFDEPDEAKDDSRVGADDAGSFEHYGDNDSGPLAGENVMNVIVVAAECSPWCKTGKNITLSGFFQNCFSFEFDGVTDVVVPRYGDYVEAFDMGIRKYYKAAGQDLEVNYFHAFIDGVDFVFIDAPLFRHRQDDIYGGSRQEIMKRMILFCKVAVEGRGPVDEFPYMDLPEHYLQHFELYDPVGGEHANIFAAGLKMADRVVTVSRGYLWELKTVEGGWGLHDIIRSNDWKINGIVNGIDHQEWNPKVDVHLRSDGYTNYSLETLDAGKRQCKAALQRELGLEVRDDVPLLGFIGRLDGQKGVDIIGDAMPWIAGQDVQLVMLGTGRADLERMLQHLEREHPNKVRGWVGFSVPMAHRITAGADVLVMPSRFEPCGLNQLYAMAYGTVPVVHAVGGLRDTVAPFDPFSDAGLGWTFDRAEANKLIEALRHCLDTYRNYEESWKSLQARGMSQDLSWDHAAELYEDVLVKAKYQW.

[0231] AtTT4 sequence (including the full length of the above gene, promoter, coding region and protein)

[0232] Full length sequence of AtTT4 (SEQ ID NO:13):

[0233]

[0234] AtTT4 promoter sequence (SEQ ID NO:14)

[0235] ATTATTCAATAAAGTTTTATTCCGGAGTTATGCTAAAGAAAAGAGTGTCGATATCTGTATTTTGTGGACGATTTAGATTACGAAGAAAATCTCGAGGAATAAAAATTTATGTGGCGAGAGATCAATTGCATCATAATATTATGCTCATGAATCACGCGAGAGTTTATGGTGATTTCGCTCATTGTGTCCCCATTCTCTCTTCTTTTGTTTGATTCCTCCTCCCTCTTGGTTAACAACAACTTCACTCACAAAGCATTCCCTTTTTTATTTTTATCGCTACTCTCCGACAAAGCCTTCTTCTTTGGATCATCTAGTCTAGGTTCGGTTTTCTCAATTAGCTCTGTTCGATTTCGCGGATGAAACTTGTTCAAGTTTGTGTCTGATCTGCTCTGCTCTGCTCATACTTAAAAAAGGAAACTTTATTTTTCTGTAA

[0236] AtTT4 coding region sequence (SEQ ID NO:15)

[0237]

[0238] AtTT4 protein sequence (SEQ ID NO: 16):

[0239] MASVAESSFPLLCQIKTQRRINSSTLRHSRVSYHDLPSGSLSFRSRSFVLGHRCKCVSRVEASGSDDDEPEDALQATIDKSKKVLAMQRNLLHQIAERRKLVSSIKESTPDLDDAKASSKQESASSVNANTDATKKEIMDGDANGSVSPSTYGKSSLSKEPEAKTFSPSTESLKNRKQSSASVISSSPVTSPQKPSDVATNGKPWSSVVASSVDPPYKPSSVMTSPEKTSDPVTSPGKPSKSRAGAFWSDPLPSYLTKAPQTSTMKTEKYVEKTPDVASSETNEPGKDEEKPPPLAGANVMNVILVAAECAPFSKTGGLGDVAGALPKSLARRGHRVMVVVPRYAEYAEAKDLGVRKRYKVAGQDMEVMYFHAFIDGVDFVFIDSPEFRHLSNNIYGGNRLDILKRMVLFCKAAVEVPWYVPCGGVCYGDGNLAFIANDWHTALLPVYLKAYYRDHGIMKYTRSVLVIHNIAHQGRGPVDDFSYVDLPSHYLDSFKLYDPVGGEHFNIFAAGLKAADRVLTVSHGYSWEVKTLEGGWGLHNIINENDWKFRGIVNGIDTQEWNPEFDTYLHSDDYTNYSLENLHIGKPQCKAALQKELGLPVRPDVPLIGFIGRLDHQKGVDLIAEAVPWMMSQDVQLVMLGTGRPDLEEVLRQMEHQYRDKARGWVGFSVKTAHRITAGADILLMPSRFEPCGLNQLYAMNYGTIPVVHAVGGLRDTVQQFDPYSETGLGWTFDSAEAGKLIHALGNCLLTYREYKESWEGLQRRGMTQDLSWDNAAEKYEEVLVAAKYHW

[0240] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively 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 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. Furthermore, all documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference.

Claims

1. The use of a heat tolerance control gene THERMOTOLERANCE 4 or its expression unit for: (a) Enhance the heat resistance of plants or increase plant yield under hot conditions; (b) As an amylase, to increase the amount of starch in plant seeds under heat conditions; said starch includes total starch or amylopectin; (c) As an adenylate cyclase, it produces cyclic nucleotides using ATP as a substrate.

2. The use as described in claim 1, characterized in that, The improvement of plant yield under thermal conditions includes increasing the following aspects under thermal conditions: seed setting rate, thousand-seed weight, seed length, yield per plant, and yield per plot; or The expression units include: THERMOTOLERANCE 4 expression cassettes, expression constructs, or expression vectors; or The cAMP enhances the heat tolerance of plants; Preferably, the heat tolerance control gene THERMOTOLERANCE 4 is located in the chloroplasts of the cell, is diffusely distributed, and aggregates into droplets under hot conditions.

3. A method for optimizing plant phenotypes, comprising: Enhancing the expression or activity of the heat tolerance control gene THERMOTOLERANCE 4 in plants; wherein the optimized plant phenotype includes: (a) Enhance the heat resistance of plants or increase plant yield under hot conditions; (b) Increasing the starch content in plant seeds under thermal conditions; said starch includes total starch or amylopectin; (c) Circular nucleotides are produced using ATP as a substrate.

4. The method as described in claim 3, characterized in that, The method of enhancing the expression or activity of the heat tolerance control gene THERMOTOLERANCE 4 in plants includes: introducing an expression unit containing the genomic DNA of the heat tolerance control gene THERMOTOLERANCE 4 or the coding region DNA of the heat tolerance control gene THERMOTOLERANCE 4 into the plant; preferably, driving the expression of the genomic DNA of the heat tolerance control gene THERMOTOLERANCE 4 or the coding region DNA of the heat tolerance control gene THERMOTOLERANCE 4 with an expression promoter; more preferably, driving the expression of the genomic DNA of the heat tolerance control gene THERMOTOLERANCE 4 or the coding region DNA of the heat tolerance control gene THERMOTOLERANCE 4 with a heat tolerance control gene THERMOTOLERANCE 4 promoter.

5. The method as described in claim 3, characterized in that, The method of increasing the expression or activity of the heat tolerance control gene THERMOTOLERANCE 4 in plants includes: in plants where the heat tolerance control gene THERMOTOLERANCE 4 is mutated or has low function, performing a gain-of-function mutation on the corresponding gene to obtain a functional heat tolerance control gene; preferably, the gain-of-function mutation is performed using gene editing technology or site-directed mutagenesis technology.

6. The method as described in claim 3, characterized in that, The method of increasing the expression or activity of the heat tolerance control gene THERMOTOLERANCE 4 in plants includes: introducing the allele of the heat tolerance control gene THERMOTOLERANCE 4 into the main plant variety through hybridization, thereby increasing the expression or activity of the heat tolerance control gene THERMOTOLERANCE 4.

7. The use as described in any one of claims 1-2 or the method as described in any one of claims 3-6, characterized in that, The heat tolerance control gene THERMOTOLERANCE 4 includes genes derived from grasses, genes derived from cruciferous plants, or their homologs or functional variants. Preferably, the thermotoughness control gene THERMOTOLERANCE 4 encodes a protein selected from the group consisting of: (i) a protein with the amino acid sequence shown in SEQ ID NO:8; (ii) a protein derived from (i) having the regulatory function, with an amino acid sequence having ≥80% homology to the amino acid sequence shown in SEQ ID NO:8 and with positions 257, 604, and 781 conserved as V, G, and L; (iii) a protein derived from (i) having the regulatory function, formed by substituting, deleting, or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO:8 and with positions 257, 604, and 781 conserved as V, G, and L; or (iv) a protein formed by adding a tag sequence or restriction enzyme site sequence to the N or C terminus of the protein with the amino acid sequence shown in SEQ ID NO:8, or by adding a signal peptide sequence to its N terminus; or Preferably, the thermotoughness control gene THERMOTOLERANCE 4 encodes a protein selected from the group consisting of: (i) a protein with the amino acid sequence shown in SEQ ID NO:12 or SEQ ID NO:16; (ii) a protein derived from (i) having the regulatory function, with an amino acid sequence having ≥80% homology to the amino acid sequence shown in SEQ ID NO:12 or SEQ ID NO:16; (iii) a protein derived from (i) having the regulatory function, formed by substituting, deleting, or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO:12 or SEQ ID NO:16; or (iv) a protein formed by adding a tag sequence or restriction enzyme site sequence to the N or C end of the protein with the amino acid sequence shown in SEQ ID NO:12 or SEQ ID NO:16, or by adding a signal peptide sequence to its N end.

8. The use as described in any one of claims 1-2 or the method as described in any one of claims 3-6, characterized in that, The plant includes the following group or the heat tolerance control gene THERMOTOLERANCE 4 or its homologs are derived from the following group: monocotyledonous plants or dicotyledonous plants; preferably, the plant includes: food crops, vegetables, fruits, flowers, and forage grasses; preferably, the plant includes: cereals, cruciferous plants, and legumes; preferably, the plant includes: grasses and cruciferous plants.

9. An isolated THERMOTOLERANCE 4 protein, comprising proteins selected from the group consisting of: (i) a protein with the amino acid sequence shown in SEQ ID NO:8; (ii) a protein derived from (i) having the regulatory function, with an amino acid sequence having ≥80% homology to the amino acid sequence shown in SEQ ID NO:8 and with positions 257, 604, and 781 conserved as V, G, and L; (iii) a protein derived from (i) having the regulatory function, formed by substituting, deleting, or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO:8 and with positions 257, 604, and 781 conserved as V, G, and L; or, (iv) a protein formed by adding a tag sequence or restriction enzyme site sequence to the N or C terminus of the amino acid sequence shown in SEQ ID NO:8, or by adding a signal peptide sequence to its N terminus; or, The nucleotide sequence of the heat resistance control gene THERMOTOLERANCE 4 is shown in SEQ ID NO:6, SEQ ID NO:11, SEQ ID NO:15 or their degenerate sequences.

10. A plant cell, tissue, or organ, comprising: The exogenous thermoturbation control gene THERMOTOLERANCE 4 genomic DNA or coding region DNA, or expression units containing it; Preferably, the exogenous thermoresistance control gene THERMOTOLERANCE 4 genomic DNA or coding region DNA is operatively linked to the THERMOTOLERANCE 4 promoter, which drives the expression of the THERMOTOLERANCE 4 genomic DNA or coding region DNA.

11. The application of the plant heat tolerance control gene THERMOTOLERANCE 4: for analyzing plant phenotypes; among which, The plant phenotypes mentioned include: heat tolerance or plant yield under heat conditions; starch content in plant seeds under heat conditions; and cyclic nucleotide content. Preferably, the analysis of the protein, genomic DNA, and coding region DNA of the plant heat tolerance control gene THERMOTOLERANCE 4 reveals that if the plant possesses the characteristics defined in claim 7, then the plant has heat tolerance or relatively high yield under hot conditions, relatively high starch content in the grain, and relatively high content of cyclic nucleotides; if the plant does not possess the characteristics defined in claim 7, then the plant has low heat tolerance or relatively low yield under hot conditions, relatively low starch content in the grain, and relatively low content of cyclic nucleotides.

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