Domestication gene tt5 for regulating heat tolerance of plants and application thereof

Abstract

The application provides a domestication gene THERMO TOLERANCE 5 (TT5) for regulating heat tolerance of plants and application thereof. In the application, a temperature-dependent Oryza minuta chromosome substitution line material for regulating rice quality and seed setting is identified, a QTL capable of polyfunctionally protecting yield and quality under high temperature is located and cloned by a method of map-based cloning, and the QTL is TT5. TT5 is a positive regulation transcription factor for heat tolerance, polyfunctionally regulates starch synthesis and storage protein accumulation, and endows high temperature tolerance. Cloning of TT5 has important significance for guiding de novo domestication of wild rice, and rapid domestication of wild rice can be realized by adding or erasing methylation of a genome.

Description

Technical Field

[0001] This invention belongs to the fields of botany and biotechnology, and more specifically, this invention relates to a domestication gene TT5 that regulates plant heat tolerance and its application. Background Technology

[0002] Temperature is a major environmental factor affecting plant growth and development, and it also severely limits crop yield. With the continuous growth of the global population and industrialization, the problem of global warming is becoming increasingly serious.

[0003] To address global warming, the Paris Climate Agreement proposed limiting temperature increases to below 2°C. However, assessments predict that average temperatures will still rise by 2.6-3.1°C by 2100, severely threatening crop production and food security. For every 1°C increase in average annual temperature, yields of rice, wheat, corn, and other grain crops will decrease by approximately 3% to 8%, seriously threatening food production.

[0004] Therefore, exploring heat-resistant genetic resources and improving the heat tolerance of important crops is of great significance for achieving high and stable crop yields.

[0005] On the other hand, in-depth analysis of the molecular mechanisms by which plants respond to high-temperature stress and develop high-temperature tolerance can provide important theoretical basis and genetic resources for breeding new crop varieties with high-temperature tolerance. Summary of the Invention

[0006] The purpose of this invention is to provide a domestication gene TT5 that regulates plant heat tolerance and its application.

[0007] In a first aspect of the invention, a method is provided to improve the heat stress tolerance and yield of grass plants, comprising: increasing the expression or activity of TT5 in grass plants; wherein TT5 is a protein with the amino acid sequence shown in SEQ ID NO:6 (TT5 HGX74 Proteins or their equivalent variants.

[0008] In one or more preferred embodiments, the increase in yield includes: promoting (including maintaining) grain filling, increasing (including maintaining) the number of branches (including the number of secondary branches) under heat stress, improving (including maintaining) fertility / seed setting rate, improving (including maintaining) grain starch and storage protein content, increasing (including maintaining) thousand-grain weight, and improving (including maintaining) yield per plant.

[0009] In one or more preferred embodiments, the heat stress tolerance is the plant's tolerance to high temperature stress.

[0010] In one or more preferred embodiments, the high temperature is a temperature above 38°C.

[0011] In one or more preferred embodiments, the output is the output under thermal stress.

[0012] In one or more preferred embodiments, the improvement of heat stress tolerance and yield in grasses includes: improving heat stress tolerance and yield in plants with low or no TT5 expression.

[0013] In one or more preferred embodiments, the isofunctional variants of TT5 include conserved variants of TT5.

[0014] In one or more preferred embodiments, the TT5 is located in the cell nucleus and is specifically expressed in the ear, hull, and grain.

[0015] In one or more preferred embodiments, TT5 enhances or maintains the transcriptional levels of downstream genes, including the following group, under heat stress to regulate the accumulation of grain starch or storage proteins: GS2, AGPL4, and GluA1-L1; preferably, TT5 binds to CArG-box motifs in the promoters of the downstream genes, thereby enhancing the transcriptional level.

[0016] In one or more preferred embodiments, the enhancement of TT5 expression or activity in gramineous plants includes: introducing an expression construct or vector containing TT5 genomic DNA or TT5 coding region DNA into the plant; preferably, driving the expression of the TT5 genomic DNA or TT5 coding region DNA (non-ectopic expression) with a spike, glume, or grain-specific expression promoter; more preferably, driving the expression of the TT5 genomic DNA or TT5 coding region DNA with a TT5 promoter (a natural promoter or its isofunctional variant).

[0017] In one or more preferred embodiments, the increase of TT5 expression or activity in gramineous plants includes: performing a gain-of-function mutation on the TT5 expressed by the plant; preferably, mutating TT5 that is mutated and has low or lost function to obtain a protein with the amino acid sequence shown in SEQ ID NO:6 or a homofunctional variant thereof.

[0018] In one or more preferred embodiments, increasing the expression or activity of TT5 in grasses includes: optimizing or modifying the promoter region of the TT5 gene using gene editing technology (such as CRISPR / Cas9-based technology) to improve the transcription level of TT5 and increase the expression of TT5.

[0019] In one or more preferred embodiments, increasing the expression or activity of TT5 in grass plants includes: introducing the TT5 allele into currently cultivated plant varieties through hybridization to increase the expression or activity of TT5.

[0020] In one or more preferred embodiments, the promoter has the nucleotide sequence shown in SEQ ID NO:2; a polynucleotide that can hybridize with the polynucleotide sequence shown in SEQ ID NO:2 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:2 and has the same driving expression function.

[0021] In one or more preferred embodiments, the TT5 protein encoded by the TT5 genomic DNA or TT5 coding region DNA is selected from the group consisting of: (i) a protein having the amino acid sequence shown in SEQ ID NO:6; (ii) a protein having the regulatory trait function with an amino acid sequence that is ≥80% homology to the amino acid sequence shown in SEQ ID NO:6 (preferably ≥85%, ≥90%, ≥95%, or ≥98%); (iii) a protein 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:6; 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:6, or by adding a signal peptide sequence to its N end.

[0022] In one or more preferred embodiments, the TT5 protein is encoded by the nucleotide sequence shown in SEQ ID NO:4.

[0023] In one or more preferred embodiments, the TT5 protein is encoded by the nucleotide sequence shown in SEQ ID NO:1.

[0024] In one or more preferred embodiments, the promoter or gene region includes homologous genes that have the same function.

[0025] In one or more preferred embodiments, the promoter does not include the promoter of the nucleotide sequence shown in SEQ ID NO:3 (i.e., a promoter not derived from Y261).

[0026] In another aspect of the invention, the use of TT5 or its expression constructs or vectors for improving heat stress tolerance and yield in gramineous plants is provided, wherein TT5 is a protein with the amino acid sequence shown in SEQ ID NO:6 (TT5 HGX74Proteins or their equivalent variants; preferably, the increase in yield includes: promoting (including maintaining) grain filling, increasing (including maintaining) the number of branches, improving (including maintaining) fertility / seed setting rate, increasing (including maintaining) grain starch and storage protein content, increasing (including maintaining) thousand-grain weight, and increasing (including maintaining) yield per plant under heat stress.

[0027] In one or more preferred embodiments, the grass species includes cereal plants, or the TT5 or its homologs are derived from cereal plants; preferably, the grass species includes (but is not limited to): rice, wheat, millet, foxtail millet, corn, sorghum, foxtail millet, barley, rye, oats, and sedge.

[0028] In another aspect of the invention, the use of TT5 in grasses is provided as a molecular marker for identifying plant traits; wherein the plant traits include: heat stress tolerance and yield; preferably, the yield traits include: grain filling under heat stress, number of branches, fertility / seed setting rate, grain starch and storage protein content, thousand-grain weight, and yield per plant.

[0029] In one or more preferred embodiments, when identifying plant traits, the protein, genomic DNA, coding region DNA sequence and / or promoter of TT5 in grass plants are analyzed. If the TT5 protein has the characteristics (sequence) defined above, the grass plant has heat stress tolerance; if the promoter and gene region do not have the characteristics defined above, the grass plant has low heat stress tolerance.

[0030] In one or more preferred methods, nucleic acid sequences are identified using methods including sequencing, PCR amplification, restriction enzyme digestion analysis, probe methods, hybridization, microarray methods, and allele polymorphism analysis.

[0031] In another aspect of the invention, a method for rapid domestication of grasses is provided, comprising erasing the methylation of the promoter region of TT5 using a gene-editing-based demethylation system to guide the de novo domestication of wild varieties; preferably, the methylation erasure of the promoter region is achieved using a CRISPR-dCas9-TET1cd demethylation system.

[0032] In another aspect of the invention, a cell, tissue, or organ of a grass plant is provided, comprising: exogenous TT5 genomic DNA or TT5 coding region DNA, or an expression construct or vector containing therein; preferably, the exogenous TT5 genomic DNA or TT5 coding region DNA is operatively linked to a TT5 promoter (a natural promoter or a variant thereof), the promoter driving the expression of the TT5 genomic DNA or TT5 coding region DNA; wherein the TT5 genomic DNA, TT5 coding region DNA, or promoter has the sequence defined above.

[0033] In one or more preferred embodiments, the plant cells, tissues, or organs do not have the ability to reproduce into adult plants.

[0034] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0035] Figure 1 Preliminary mapping of linkage segments and genetic background analysis of TT5. (a) Genotype of CSSL(TT5); (b) Grain phenotypes of replacement lines Y261 and HGX74.

[0036] Figure 2 TT5 localization cloning. (a) Fine localization of TT5 and candidate genes; (b) NIL-TT5 HGX74 and NIL-TT5 Y261 Plant type, scale bar 10cm; (c) Natural variation site in the coding region of candidate gene MADS8; (de) Candidate genes SPL18 and MADS8 in NIL-TT5 HGX74 and NIL-TT5 Y261 Expression levels in NIL-TT5 were measured, with data presented as mean ± SEM, n = 3. A two-tailed t-test was used for significance testing, and no significant difference was found in ns. (f) NIL-TT5 expression was examined for two consecutive years. HGX74 and NIL-TT5 Y261 phenotype of grain endosperm.

[0037] Figure 3 Different transgenic constructions of TT5. (a) NIL-TT5 Y261 Background restoration (COM) TT5 HGX74(a) Plant grain phenotype, scale bar 2cm; (b) Grain phenotype of ZH11 background TT5 knockout (KO) homozygous plants, scale bar 2cm; (cd) Plant type of ZH11 background TT5 knockout and overexpression, scale bar 10cm; (ef) Ear type of ZH11 background TT5 knockout and overexpression, scale bar 2cm; (g) Expression level determination of ZH11 background overexpression plants.

[0038] Figure 4 TT5 expression patterns and subcellular localization of TT5. (ab)pTT5 HGX74 (c) GUS vector construction and staining results of various plant tissues; NIL-TT5 HGX74 Expression levels of TT5 in grains 7 days after fertilization under heat stress; subcellular localization of (de)TT5 protein in rice protoplasts and tobacco leaves.

[0039] Figure 5 NIL-TT5 HGX74 and NIL-TT5 Y261 Grain filling rate determination and genetic analysis of TT5. (ab)NIL-TT5 HGX74 and NIL-TT5 Y261 Statistical analysis of grain phenotypes and grain dry weight at different stages; (c) NIL-TT5 HGX74 NIL-TT5 Y261 and the phenotype of seeds from heterozygous plants; (d)NIL-TT5 HGX74 and NIL-TT5 Y261 Phenotypes of seeds from direct and reciprocal crosses of plants.

[0040] Figure 6 NIL-TT5 HGX74 and NIL-TT5 Y261 The agronomic traits of NIL-TT5 were investigated. (a) NIL-TT5 HGX74 and NIL-TT5 Y261 (b) Comparison of kernel length; NIL-TT5 HGX74 and NIL-TT5 Y261 Comparison of spikelet type and secondary branches in Songjiang, Shanghai; (ck)NIL-TT5 HGX74 and NIL-TT5 Y261 In the statistical analysis of agronomic traits such as tiller number, plant height, ear length, grain width, grain length, number of primary branches, number of secondary branches, thousand-grain weight, and yield per plant, the data were expressed as mean ± SEM, n>=10. The two-tailed t-test was used to test the significance of differences. The results showed no significant differences (n ns), ***P<0.001, ****P<0.0001.

[0041] Figure 7NIL-TT5 at different temperatures HGX74 and NIL-TT5 Y261 A comparison of yield performance. (a) NIL-TT5 HGX74 and NIL-TT5 Y261 (a) Comparison of single-plant yield in the field and after high-temperature treatment; (b) NIL-TT5 after high-temperature treatment HGX74 and NIL-TT5 Y261 Phenotype; (cd) Temperature records of greenhouse high-temperature treatment and field ambient temperature records; (e) NIL-TT5 HGX74 and NIL-TT5 Y261 Grains under field and high temperature conditions at 28℃ / 22℃; (f) NIL-TT5 HGX74 and NIL-TT5 Y261 Ear type under high temperature conditions of 28℃ / 22℃; (gi)NIL-TT5 HGX74 and NIL-TT5 Y261 The seed setting rate of grains under 28℃ / 22℃, field and high temperature conditions was statistically analyzed. Data are mean ± SD, n = 10. A two-tailed t-test was used for significance testing. **P < 0.01, ****P < 0.0001; (jk)NIL-TT5 HGX74 and NIL-TT5 Y261 Yield per plant (n=15) and yield per plot (n=4) in the field and in high-temperature field conditions were measured. The data are mean ± SD. One-way ANOVA was used to test the significance.

[0042] Figure 8 NIL-TT5 HGX74 and NIL-TT5 Y261 Pollen fertility observation and yield comparison of TT5 knockout plants. (ab)NIL-TT5 HGX74 and NIL-TT5 Y261 Pollen KI-I2 staining was performed at 28℃ / 22℃ in the field and under high-temperature conditions. Data are mean ± SD, n = 12. Two-tailed t-test was used to test for significance. No significant difference was found (ns), **P < 0.01; (c) Yield per plant of ZH11 and TT5 knockout plants in the field and after high-temperature treatment in Shanghai, n = 15; (de) Temperature records of TT5 knockout greenhouse high-temperature treatment and field ambient temperature records.

[0043] Figure 9 Determination of starch content and storage protein content in TT5 grains, and urea swelling experiment. (ab)NIL-TT5 HGX74 and NIL-TT5 Y261(c) Determination of the swelling volume of rice flour under 0-9M urea; (d) SDS-PAGE detection of NIL-TT5 HGX74 and NIL-TT5 Y261 Storage protein content in (dg) NIL-TT5 HGX74 and NIL-TT5 Y261 The total starch and total protein content of ZH11 and TT5-KO materials were detected. e and g data were mean ± SEM, n = 3. The significance of the differences was tested using a two-tailed t-test. *P < 0.05, **P < 0.01, ****P < 0.0001. f data were mean ± SEM, n = 3. The significance of the differences was tested using a one-way ANOVA.

[0044] Figure 10 Scanning electron microscopy and transmission electron microscopy observation of TT5 grains. (a) NIL-TT5 HGX74 and NIL-TT5 Y261 (a) SEM images of starch granules at 28℃ / 22℃ and high temperature; (b) Transmission electron microscopy observation of NIL-TT5. HGX74 and NIL-TT5 Y261 (c) Starch granule morphology of 10DAF grains at 28℃ / 22℃ and at 28℃ / 22℃ and in the field; (d) Starch granule morphology of ZH11 and TT5-KO materials in the field under scanning electron microscopy.

[0045] Figure 11 The main pathways regulated by TT5 and their expression levels were determined. (a) NIL-TT5 HGX74 and NIL-TT5 Y261 Grain transcriptome differential gene heatmap; (bc) GO enrichment analysis and KEGG enrichment pathway; (df) qRT-PCR validation of NIL-TT5 HGX74 and NIL-TT5 Y261 Differential gene expression levels were measured using mean ± SEM, n = 3. A two-tailed t-test was used to assess the significance of the differences. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0046] Figure 12TT5 binds to the promoter regions of genes such as AGPL4 to activate transcription. (a) GO enrichment analysis of differentially expressed genes by DAP-seq results; (be) DAP-seq results revealed that TT5 binds to gene loci such as GS2, AGPL4, RCN2, and GIF1; (fh) Dual-fluorescence reporter system was used to detect the trans-activation of genes such as GS2, AGPL4, RCN2, and GIF1 by TT5; (ij) CUT&RUN-qPCR was used to detect the enrichment of TT5 binding to gene loci such as AGPL4 and GluA1-L1. Data are mean ± SD, n = 3. The significance of difference was tested using a two-tailed t-test, and ***P < 0.001; (k) EMSA was used to verify the binding of TT5 to AGPL4; (l) Morphology of grains of the glua1-l1 mutant under scanning electron microscopy.

[0047] Figure 13 TT5 Y261 The promoter exhibits a high level of methylation. (ab)pTT5 Y261 (c) GUS vector construction and staining results of various plant tissues, scale bar is 2cm; (d) Chop-PCR detection of TT5 Y261 Promoter methylation level; (di) TT5 detection by bisulfite conversion method Y261 Methylation of promoter regions P4 and P5 segments, dg, methylation type in leaves, hi, methylation type in young spikelets.

[0048] Figure 14 TT5 is highly selected during domestication. (a) TT5 coding region variation is mainly present in wild rice; (bd) Chop-PCR and bisulfite conversion method were used to detect the degree of methylation in wild rice; (e) Statistical analysis of whole-genome methylation data showed the degree of TT5 promoter methylation in wild rice and modern cultivated indica and japonica rice.

[0049] Figure 15 CRISPR-dCas9-TET1cd system erases TT5 Y261 Promoter methylation. (a) Schematic diagram of the CRISPR-dCas9-TET1cd system vector; (bc) Detection of TT5 in transgenic T1 plants by bisulfite conversion method. Y261 Methylation of the P5 fragment in the -dcas9 promoter.

[0050] Figure 16Heat resistance identification of TT5 indica and japonica haplotype materials and heat resistance identification of TT5 transgenic plants. (a) Plant architecture of SN-155 and SN-115, scale bar 10 cm; (bc) Ear type of SN-155 and SN-115 under normal and high temperature conditions, scale bar 2 cm; (d) qRT-PCR detection of TT5 expression level in SN-155 and SN-115 plants, data are mean ± SD, n = 3, two-tailed t-test was used for significance test, **P < 0.01; (e) qRT-PCR detection of TT5 expression level in pTT5:TT5-3F plants, data are mean ± SD, n = 3, two-tailed t-test was used for significance test, **P < 0.01; (fg) Comparison of plant architecture and yield per plant of pTT5:TT5-3F plants, scale bar 10 cm, data are mean ± SD, n = 10, two-tailed t-test was used for significance test. t-test for significant difference, ns, no significant difference, **P<0.01; (h) pTT5:TT5-3F plant yield determination in plots under high temperature treatment, data are mean±SD, n=3, two-tailed t-test for significant difference, *P<0.05.

[0051] Figure 17 TT5 amino acid sequence homology analysis. Multiple sequence alignment analysis of TT5 homology in Arabidopsis thaliana, maize, soybean and other plants. Detailed Implementation

[0052] Based on in-depth research and analysis, the inventors identified a common wild rice chromosome replacement line material that regulates rice quality and grain filling in a temperature-dependent manner. They located and cloned the QTL that can protect yield and quality in multiple ways at high temperatures using map-based cloning, and named it THERMO TOLERANCE 5 (TT5).

[0053] TT5 encodes a MIKCc-type MADS-box gene that maintains plant yield and quality under high temperatures by enhancing transcriptional levels through binding to the promoter regions of target genes such as GS2, AGPL4, and GluA1-L1. As a novel domestication gene, TT5 exhibits continuously decreasing promoter methylation levels during domestication, significantly improving plant heat tolerance. TT5 is crucial for maintaining seed setting and grain filling in plants under high temperatures. The highly methylated promoter of the TT5 allele from common wild rice silences its own expression, leading to decreased seed setting rate, slower grain filling rate, and a significant reduction in thousand-grain weight and yield per plant under high-temperature stress. Simultaneously, the grains exhibit significant endosperm powderiness. In contrast, the TT5 allele from indica rice Huajingxian 74 confers heat tolerance during the heading stage, maintaining seed setting and grain filling by sustaining the transcriptional levels of genes such as GS2, AGPL4, and GluA1-L1. The TT5 allele promoter from the indica rice variety Huajingxian 74 was used to drive TT5 expression in the japonica rice variety Zhonghua 11, resulting in upregulation of TT5 transcription and significantly improving heat tolerance and yield under high temperatures. These results indicate that TT5, as a transcription factor positively regulating heat tolerance, pleiotropically regulates starch synthesis and storage protein accumulation, conferring high-temperature tolerance. Meanwhile, cloning TT5 is of great significance for guiding the de novo domestication of wild rice, enabling rapid domestication through gene editing techniques to add or remove methylation in the genome.

[0054] As used herein, "optimized traits" or "improved traits" refer to characteristics that positively regulate plants. In this invention, these traits mainly include: heat stress tolerance and yield. Preferably, yield traits include: grain filling, number of branches (including the number of secondary branches), fertility / seed setting rate under heat stress, increased (including maintained) grain starch and storage protein content, increased (including maintained) thousand-grain weight, and increased (including maintained) yield per plant.

[0055] As used herein, 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 a statistically significant change 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 this change forms a beneficial agronomic trait.

[0056] As used herein, “plant” includes plants that express TT5. HGX74 The protein or its homologous protein containing TT5 in the plant or genome HGX74 Plants that express TT5 genes or their homologous genes. Based on knowledge in this field, [the following is an example / specification]: HGX74Plants containing homologous proteins of similar function possess the mechanism of action claimed in this invention and can achieve the technical effects claimed in this invention. The plants can 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" can be grasses; preferably, the grasses include, but are not limited to: rice, wheat, millet, foxtail millet, corn, sorghum, foxtail millet, barley, rye, oats, and *Bruguiera gymnorhiza*.

[0057] 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.

[0058] As used herein, “high temperature (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 35°C or higher, 38°C or higher, 40°C or higher, or 42°C or higher.

[0059] As used herein, the terms “enhance,” “increase,” “upregulate,” “enlarge,” “promote,” “strengthen,” etc., are interchangeable and, in their application, should mean an increase of 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 significant, compared to a control plant, control gene, or control protein as defined herein.

[0060] 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.

[0061] As used 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.

[0062] As used herein, a “promoter” or “promoter region” refers to a nucleic acid sequence that is typically located upstream (5' end) of a target gene sequence and guides the transcription of the nucleic acid sequence into mRNA. Generally, a promoter or promoter region provides recognition sites for RNA polymerase and other factors necessary for proper transcription initiation. In this document, the promoter or promoter region includes variants of the promoter, obtained through methods such as insertion or deletion of regulatory regions, random or site-directed mutagenesis, etc.

[0063] As used in this article, "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 particular sequence is "exogenous" to the cell or organism into which it is inserted.

[0064] As used in this article, "isolated" means that a substance has been separated from its original environment (in the case of a natural substance, the original environment is the natural environment). For example, polynucleotides and proteins in their natural state within living cells are not isolated and purified, but the same polynucleotides or proteins are isolated and purified if they are separated from other substances present in their natural state.

[0065] In this invention, the TT5 protein can be a protein having the amino acid sequence shown in SEQ ID NO:6, and also includes its conserved variants. In this invention, a "conserved variant protein" refers to a protein that substantially retains the same biological function or activity as the stated protein. A "conserved variant protein" can be (i) a protein with one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) substituted, where 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).

[0066] The term "conserved variant protein" 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.

[0067] Unless otherwise specified, the TT5 HGX74 Genes refer to those with TT5 HGX74 Genomic DNA (gTT5) HGX74 ) gene, or TT5 HGX74 The coding region genes also include those with TT5 HGX74 Sequence variations with the same function as the protein also include promoters. The gene sequence also includes sequences degenerate with the sequences provided in this invention.

[0068] In this invention, the TT5 HGX74 With TT5 Y261 Genes located at the same locus from indica rice HGX74 and the indica rice replacement line Y261.

[0069] The TT5 HGX74 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 variation related to TT5 described herein... HGX74 Proteins with high homology (e.g., 50%, 60%, or 70% homology to the protein sequence shown in SEQ ID NO: 6; preferably 80% or higher; more preferably 90% or higher, such as 95%, 98%, or 99% homology) and possessing TT5 HGX74Proteins with the same function as the protein are also included in this invention. Proteins derived from species other than rice that have 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.

[0070] In this invention, the TT5 HGX74 Genes / proteins (including gene promoters) also include their homologs. It should be understood that while rice from a specific species is preferably studied in this invention, homologs from other species are also included. HGX74 Other homologous genes (such as those with more than 49%, 60%, more particularly 70%, 80%, 85%, 90%, 95%, or even 98% sequence identity) are also within the scope of this invention.

[0071] The TT5 HGX74 Polynucleotides (genes) can be natural genes from plants or their degenerate sequences.

[0072] The present invention also relates to a vector containing the aforementioned polynucleotide, and a host cell generated by genetic engineering using the aforementioned vector.

[0073] In this invention, the TT5 HGX74 Genomic DNA or coding sequences can be inserted into recombinant expression vectors. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses, or other vectors well-known in the art. In short, any plasmid and vector can be used as long as it can replicate and remain stable within the host. An important characteristic of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translation control elements. Preferably, the expression vector may also selectively contain resistance elements, selection elements, or reporter gene elements, such as Bar or GUS.

[0074] When the aforementioned polynucleotide is expressed in higher eukaryotic cells, the insertion of an enhancer sequence into the vector will enhance transcription. An enhancer is a cis-acting factor of DNA, typically consisting of approximately 10 to 300 base pairs, that acts on the promoter to enhance gene transcription. Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. Plant transformation can be performed using methods such as Agrobacterium-mediated transformation or gene gun transformation, including spraying, leaf disc transformation, and rice embryo transformation.

[0075] In a specific embodiment of the present invention, the rice heat tolerance and rice quality control gene TT5 was discovered. TT5 has pleiotropic effects, regulating the synthesis of rice starch and storage proteins, as well as regulating rice heat tolerance and yield. The present invention provides TT5... HGX74 and TT5 Y261Genes, including TT5 HGX74 It imparts heat resistance, panicle type, and yield phenotypes to rice.

[0076] The inventors' research results show that TT5 regulates the development of rice panicles, and the decrease in its transcription level significantly reduces the reduction of secondary branches in rice, resulting in a decrease in the number of grains per panicle; at the same time, TT5 regulates the plant architecture and panicle development of rice in a dose-effect manner.

[0077] The inventors' research showed that TT5 is a positive regulator of rice's heat tolerance. Knocking out the TT5 gene significantly weakened the plant's heat tolerance, resulting in a significant decrease in seed setting rate under high temperatures, and also causing the endosperm of the grains to become powdery. Using TT5... HGX74 Self-promoted TT5 expression can significantly improve the grain setting rate of rice under high temperatures and reduce yield loss. Preferably, instead of ectopic expression, the expression of the TT5 genomic DNA or TT5 coding region DNA is driven by panicle, glume, or grain-specific promoters (including their own promoters), which is more conducive to positive regulation of the trait.

[0078] The inventors' research shows that the rice TT5 gene encodes a MIKCc-type MADS-box transcription factor, which is located in the cell nucleus and is specifically highly expressed in the anthers, panicles, and grains, but not in leaves and other parts. Its expression level is not induced by high temperature. TT5 is involved in maintaining pollen fertility under high temperature; knocking out the TT5 gene makes pollen extremely sensitive to high temperatures. TT5 is crucial for maintaining starch synthesis and storage protein synthesis in rice grains under high temperature; loss of TT5 function leads to abnormal starch grain structure, decreased starch content, and increased storage protein content, affecting rice quality and weight under high temperature conditions.

[0079] The inventor's research results show that TT5 HGX74 Rice starch synthesis and storage protein accumulation are regulated by maintaining the transcriptional levels of downstream genes such as GS2, AGPL4, and GluA1-L1 under high temperatures. Meanwhile, loss of function of TT5 induces upregulation of HSP transcription levels. TT5 enhances transcriptional levels by binding to CArG-box motifs in the promoters of downstream genes such as GS2, AGPL4, and GluA1-L1.

[0080] Based on the inventor's new discovery, a method for improving plants is provided, the method comprising: increasing the expression or activity of TT5 in grasses; wherein TT5 is a protein with the amino acid sequence shown in SEQ ID NO:6 (TT5 HGX74Proteins or their isofunctional variants; wherein the increase in yield includes: promoting (including maintaining) grain filling, increasing (including maintaining) the number of branches (including the number of secondary branches) under heat stress, improving (including maintaining) fertility / seed setting rate, improving (including maintaining) grain starch and storage protein content, increasing (including maintaining) thousand-grain weight, and improving (including maintaining) yield per plant.

[0081] It should be understood that, once the function of TT5 is known, its expression or activity can be modulated using a variety of methods well known to those skilled in the art.

[0082] This invention also provides a method for upregulating TT5 expression in plants. The method includes (but is not limited to): introducing an expression construct or vector containing TT5 genomic DNA or TT5 coding region DNA into plants; preferably, driving the expression of the TT5 genomic DNA or TT5 coding region DNA (non-ectopic expression) with a spike, glume, or grain-specific expression promoter; more preferably, driving the expression of the TT5 genomic DNA or TT5 coding region DNA with a TT5 promoter (a natural promoter or its isofunctional variant); or, performing a gain-of-function mutation on the TT5 expressed by the plant; preferably, mutating a TT5 that has variations and low or lost function to obtain a protein with the amino acid sequence shown in SEQ ID NO:6 or its isofunctional variant; or using gene editing technology (such as CRISPR / Cas9-based technology) to optimize or modify the promoter region of the TT5 gene to improve the transcription level of TT5 and increase TT5 expression; or introducing the TT5 allele into currently cultivated plant varieties through hybridization to increase TT5 expression or activity.

[0083] As a preferred embodiment of the present invention, a promoter for specific expression is provided, which is the promoter of the TT5 gene, and has been isolated for the first time. Preferably, it is the promoter of the nucleotide sequence shown in SEQ ID NO:2, which contributes to the improvement of traits in grasses. The present invention also includes variant promoters or promoter fragments having the same driving expression function as the promoter of the nucleotide sequence shown in SEQ ID NO:2. Polynucleotide hybridization is a technique well known to those skilled in the art, and the hybridization characteristics of a particular pair of nucleic acids indicate their similarity or identity. The present invention also relates to polynucleotides that are hybridizable to the polynucleotides described in the present invention under stringent conditions. The present invention also includes nucleic acids having 50% or more (preferably 60%, 70%, 80%, more preferably 90%, and most preferably 95%) identity with any promoter sequence of the present invention, said nucleic acid also having the function of guiding the specific expression of a target gene. "Identity" refers to the level of similarity (i.e., sequence homology, similarity, or identity) between two or more nucleic acids according to the percentage of positions identical.

[0084] The promoter and / or the target gene sequence that drives its expression may be contained in a recombinant vector. This invention also includes recombinant vectors containing the promoter and / or the target gene sequence.

[0085] The technical solution of this invention can be applied to molecular design breeding through various pathways. TT5 is a conserved gene in plant evolution, widely present in various crops, and has great application prospects.

[0086] After understanding the function of TT5, it 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 and regulate heat stress tolerance and yield by modulating this mechanism.

[0087] The inventors' research shows that TT5 is a highly selected domestication gene. During the domestication selection process, the TT5 allele with low methylation levels was continuously selected, gradually changing from the highly methylated ancestor of wild rice to the low-methylated modern cultivated rice. During the domestication selection process, the transcriptional level of TT5 also gradually increased from transcriptional silencing in wild rice.

[0088] In modern cultivated rice, the coding region of TT5 is almost highly conserved, while promoter variation shows significant differentiation between indica and japonica rice. The TT5 allele from indica rice exhibits greater heat tolerance than that from japonica rice due to promoter variation. Y261 Promoter methylation can be erased by the CRISPR-dCas9-TET1cd demethylation system. The cloning of TT5 is of great significance for guiding the de novo domestication of wild rice. Gene editing technology can be used to add or erase methylation of the genome to achieve rapid domestication of wild rice.

[0089] TT5 from Asian cultivated rice HGX74 Alleles have promising applications and are crucial for maintaining grain filling and development in rice under high temperatures, effectively protecting yield. Simultaneously, the cloning of TT5 provides an important genetic resource for improving rice quality. In the future, CRISPR / Cas9 gene editing technology can be used to optimize or modify the promoter region of the TT5 gene to further enhance its transcriptional level, or to... HGX74 Alleles are introduced into the current main cultivated varieties through hybridization to improve the plant's high temperature tolerance and yield, thereby cultivating new high-yielding and stress-resistant crop varieties (including but not limited to rice, wheat, corn, sorghum, millet, soybeans, etc.).

[0090] 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.

[0091] 1. Experimental materials and localized cloning

[0092] This invention utilizes a chromosome substitution line constructed using common wild rice as the donor parent and the Asian cultivated indica rice variety Huagngxian 74 (HGX74) as the recipient parent for phenotypic evaluation and heat tolerance assessment. Preliminary mapping identified endosperm powderiness in the substitution line Y261 (indica) with a significant temperature dependence. The QTL regulating rice heat tolerance was named TT5, and it was backcrossed with the recipient parent HGX74 to construct an F2 population. TT5 was finely mapped using molecular markers, narrowing the candidate region to a 25.7 kb area on chromosome 9. The candidate gene for TT5 was preliminarily identified using qRT-PCR.

[0093] Y261 is a chromosome replacement line produced by crossing common wild rice with Huajingxian 74. Y261 contains a segment of chromosome 9 from common wild rice.

[0094] The molecular marker primers used for localization are shown in Table 1.

[0095] Table 1

[0096] 5' oligonucleotide primer 3' primer M504 5'-TTGGAAGGTAGAACTGTTGT-3'(SEQ ID NO:8) 5'-ACTAGAACCCTTGAAAGCTG-3'(SEQ ID NO:9) M507 5'-CAAATGTAATCTTGGCCCAC-3'(SEQ ID NO:10) 5'-TGGGACACGTACTCAATTAG-3'(SEQ ID NO:11) M601 5'-GTCATAGTTTACTGCTGCCT-3'(SEQ ID NO:12) 5'-TTCAGATGAAGGAAAGCTTT-3'(SEQ ID NO:13) M603 5'-AGTACGTATATGTGGATGCA-3'(SEQ ID NO:14) 5'-ATACACCGAGAGATCGTCAT-3'(SEQ ID NO:15) M606 5'-GGGGAAAATAATAATGGCGAT-3'(SEQ ID NO:16) 5'-ATGTAACGTCAGTGCTTAGG-3'(SEQ ID NO:17) M608 5'-CTGAAGACGTGAACAGTTTG-3'(SEQ ID NO:18) 5'-ATCAGACAGCCACAGATTAC-3'(SEQ ID NO:19) M701 5'-TATGTCAGTGATACGGAACC-3'(SEQ ID NO:20) 5'-AATGTGGTCAGGTAAGCATT-3'(SEQ ID NO:21) M703 5'-ATCACACTGTTCTTGCTTCT-3'(SEQ ID NO:22) 5'-GAGAAAACGTTGCAAAGTGA-3'(SEQ ID NO:23) M705 5'-ACACGGAGATGATAAAGTTTG-3'(SEQ ID NO:24) 5'-GCTAGCACCCAAACAAAATC-3'(SEQ ID NO:25) M78 5'-TGGGCATCGAGAAATGTAAA-3'(SEQ ID NO:26) 5'-GGCACACAGACAAAGGATAA-3'(SEQ ID NO:27) M79 5'-TGCATCCCTTAACTATCGAC-3'(SEQ ID NO:28) 5'-CCTCTCGTCTATTTGAGTCTGT-3'(SEQ ID NO:29) M7720 5'-TTTGCATATAACACCCTGC-3'(SEQ ID NO:30) 5’-CTACTGTACCAACCACGAAT-3’(SEQ ID NO:31) M7818 5’-CCCACCAATCAAACCAATTC-3’(SEQ ID NO:32) 5’-AAGCCTCGAAATCAGCAAAA-3’(SEQ ID NO:33) C7065 5’-TCTTCATCTTCGTTCCATCC-3’(SEQ ID NO:34) 5’-ATCGATCATTGCACGTACTC-3’(SEQ ID NO:35) M7038 5’-TGCTATTACACCATGGATCG-3’(SEQ ID NO:36) 5’-TGCATGGTAAGTTGTACTCC-3’(SEQ ID NO:37) M7854 5’-TGGTATCATATCCTCGTTCG-3’(SEQ ID NO:38) 5’-GGCTTTAGTCATGAAAACGG-3’(SEQ ID NO:39) M7861 5’-TTTTAAACCCCTCATCCCC-3’(SEQ ID NO:40) 5’-GATGAATCAACCAATCTCGC-3’(SEQ ID NO:41) M7073 5’-GTCCAGCCCATTTGTTTGGTAC-3’(SEQ ID NO:42) 5’-GCACGAGTATGGAAGGCTAGTT-3’(SEQ ID NO:43)

[0097] Primers used for qRT-PCR are shown in Table 2.

[0098] Table 2

[0099] 5'-terminal oligonucleotide primer 3'-terminal primer qTT5 5’-GCAAGCTCTACGAGTTCTGC-3’(SEQ ID NO:44) 5’-AAATTTTCCACCCGTGCCTT-3’(SEQ ID NO:45) qSPL18 5’-ACAAGTCAACAAGGGACAAGGTT-3’(SEQ ID NO:46) 5’-GGGTGGTGATGATGGCTGTAATA-3’(SEQ ID NO:47)

[0100] 2. CRISPR / Cas9 gene editing, overexpression, and genetic complementation

[0101] To further validate the TT5 candidate gene, CRISPR / Cas9 technology was used to knock out the target gene LOC_Os09g32948. sgRNAs targeting exons 1 and 7 of LOC_Os09g32948 were designed and constructed into a CRISPR / Cas9 vector to obtain homozygous knockout plants. TT5 expression was driven by the constitutive strong promoter UBI (pUbi). HGX74Used to obtain overexpression lines. Genetic complementation was performed using TT5. HGX74 Approximately 2.6 kb of sequence preceding the translation start site serves as TT5's own promoter to drive TT5. HGX74 CDS expression. Simultaneously, in the context of Japonica rice ZH11, expression was achieved via TT5. HGX74 Self-starter driver TT5 HGX74 CDS expression enables in situ TT5 expression and upregulates transcription levels.

[0102] Primers used to construct CRISPR knockout or overexpression vectors are shown in Table 3.

[0103] Table 3

[0104]

[0105] 3. GUS staining to detect TT5 expression patterns

[0106] GUS transgenic positive plants from different growth stages were dissected and isolated. The isolated tissues were fixed with 90% acetone at 4°C for 20 min, then the acetone was washed off. The samples were then immersed in a pre-prepared GUS staining solution, and vacuum was applied three times, each time for 10-15 min, to ensure complete immersion. The samples were then placed at 37°C and observed periodically. After staining, the staining solution was discarded, and the samples were destained with 95% ethanol. Once the green color had completely faded, the samples were observed and photographed.

[0107] The 5' oligonucleotide primer sequence for constructing the pCAMBIA1300-GUS vector is as follows:

[0108] 5'-gtcgactctagaggatccTGGAGAGAAATTTTCTGCTCACTTGAA-3' (SEQ ID NO: 56);

[0109] The 3' primer sequence is as follows:

[0110] 5'-cagatctaccatggtaccCACCCTCCCTCTCCCCAT-3' (SEQ ID NO: 57).

[0111] 4. Subcellular localization

[0112] Subcellular localization of TT5 was observed using vigorously growing Nicotiana benthamiana plants (approximately 4 weeks old). Plants containing 35S::TT5 were analyzed. HGX74The GFP expression plasmid was transformed into Agrobacterium strain GV3101, and subcellular localization observation of TT5 was achieved through Agrobacterium-mediated transient expression in tobacco leaves. After 48-72 hours of dark incubation, the inner epidermis of the injected tobacco leaves was harvested for fluorescence imaging. Subcellular observation of rice protoplasts was achieved by transfecting TT5... HGX74 The coding region was constructed into the pA7-YFP vector. Protoplasts were isolated using HGX74 seedlings cultured under sterile conditions for 2 weeks. The purified and concentrated expression plasmid was then used to achieve transient expression of TT5 via PEG-mediated transformation of rice protoplasts. After 14 hours of dark incubation, fluorescence imaging was performed using a laser confocal microscope. Primers used to construct the expression vector are shown in Table 4.

[0113] Table 4

[0114]

[0115] 5. Transcriptome sequencing and qRT-PCR detection

[0116] Transcriptome sequencing was used to explore the molecular mechanisms by which TT5 maintains rice yield and quality under high temperatures. NIL-TT5 rice grown in Songjiang, Shanghai was selected. HGX74 and NIL-TT5 Y261 Seven days after fertilization, the grains of the fertilized plant were used as experimental material. After being ground into powder using liquid nitrogen, they underwent subsequent transcriptome sequencing. A total of 990 differentially expressed genes were detected, including NIL-TT5. Y261 A total of 742 genes were significantly downregulated and 248 genes were significantly upregulated. The differentially expressed genes were mainly involved in responses to heat and oxidative stress, as well as post-embryonic development and carbohydrate metabolism. These differentially expressed genes were detected by qRT-PCR. Primers used for qRT-PCR are shown in Table 5.

[0117] Table 5

[0118] 5'-terminal oligonucleotide primer 3'-terminal primer qAGPL4 5’-GACTACAATGTGCAGGCCTATCTA-3’(SEQ ID NO:62) 5’-ATAGAAGTTTGGCGACTGATCTGT-3’(SEQ ID NO:63) qGS2 5’-GCAGAGTCAACTCATTACTGAAGC-3’(SEQ ID NO:64) 5’-CTCTGAACCTTTGAAAGTGAGCTG-3’(SEQ ID NO:65) qGluA1-L1 5’-GACCGACGAGGAGATGATGAG-3’(SEQ ID NO:66) 5'-ATCTTGATGAGCTTGAGGACGG-3'(SEQ ID NO:67) qSUS5 5'-AGAACGGCTGACAGATTTACA-3'(SEQ ID NO:68) 5'-TCTTGTTCTGGCCATACCACTC-3'(SEQ ID NO:69) qSUS7 5'-AATGCAATGTGGAGCTTGAACC-3'(SEQ ID NO:70) 5'-ATTATCAAGTCCGGTTTGCCCT-3'(SEQ ID NO:71) qAUX2 5'-GTTTACAGCAGCGTCAATACTG-3'(SEQ ID NO:72) 5'-GCACCAAAATCACACGTAATGT-3'(SEQ ID NO:73) qGIF1 5'-ACCACTGATCTCATTCAGCAGTAT-3'(SEQ ID NO:74) 5'-TACATGAGATTGTGCTGGAGCTTA-3'(SEQ ID NO:75) qPro13B.17 5'-TTGCAATCAGCTGCGTTTCAACT-3'(SEQ ID NO:76) 5'-GGTCGTTTTGGGACGTGACA-3'(SEQ ID NO:77) qPro14 5'-TCATGTTTTAGCTGTGTTTC-3'(SEQ ID NO:78) 5'-CCATAGTACCTAGGGTAGATAC-3'(SEQ ID NO:79)

[0119] 6. Scanning electron microscopy and transmission electron microscopy observation of rice grains

[0120] To further observe the microstructure of the endosperm powdering in the grains, mature grains from different transgenic materials were selected, and the differences in microstructure were observed using scanning electron microscopy. NIL-TT5 was also selected. HGX74 and NIL-TT5 Y261 Ultrathin sections were prepared from seeds 10 days after fertilization and mature seeds. The structure of starch granules in the seeds was further observed using transmission electron microscopy.

[0121] 7. Determination of starch, storage protein, and other contents in rice grains

[0122] The total starch content of the grains was tested using a starch content test kit (BC0705) manufactured by Solarbio. In addition to SDS-PAGE analysis, the total protein content in different materials was further quantified using a Kjeldahl nitrogen analyzer.

[0123] 8. In vitro DNA affinity purification sequencing and dual-fluorescence reporter system

[0124] DAP-seq uses NIL-TT5 HGX74 DNA was extracted and a library was constructed from leaf tissue, and TT5 was used to extract DNA. HGX74 A Halo-tag in vitro expression plasmid was constructed, and protein expression and purification were performed using a wheat germ system. TT5 was captured by binding and enriching the protein with the library. HGX74 The combined DNA sequence was used. The dual-luciferase reporter system was illustrated using GluA1-L1 as an example: a promoter fragment containing 2.3 kb before the GluA1-L1 translation start site was constructed and inserted before the Luciferase reporter gene to drive Luciferase expression. Simultaneously, the 62SK-TT5 vector was constructed as an effector, which uses the tobacco mosaic virus 35S promoter to drive TT5 expression. Different plasmid combinations were transiently transformed into rice protoplasts for expression. After 14 hours of incubation in the dark, the rice protoplasts were lysed using the Dual-luciferase reporter assay system (Promega), and the ratio of Luciferase to Ren luciferase was measured. The data were then normalized. 62SK-EV was used as a negative control.

[0125] The primers used to construct the expression vector are shown in Table 6.

[0126] Table 6

[0127]

[0128] 9. EMSA assay and in vivo DNA binding assay

[0129] To verify the binding of TT5 to downstream target genes, TT5 was expressed and purified in vitro. HGX74The coding region sequence was constructed into the pMal-c5x vector and transformed into *E. coli* Rossta(DE3). After IPTG-induced protein expression, bacterial cells were collected. The supernatant was separated by ultracentrifugation after sonication and purified by AKTA to obtain the MBP-TT5 fusion protein. EMSA experiments were conducted using the AGPL4 promoter as an example: based on DAP-seq results, DNA sequences containing CArG-boxes in the AGPL4 promoter region were selected. Probes approximately 70 bp in length containing Cy5 labels and cold probes without Cy5 labels were designed. The binding of TT5 to the DNA fragment was verified by detecting the binding of the MBP-TT5 fusion protein to the probes and the competitive binding of the cold probes. In addition, the inventors also transiently expressed TT5 using rice protoplasts. HGX74 Cell nuclei were collected and the TT5-binding DNA fragments were captured using the cut run (Vazyme) method, and further validated by qPCR. Primers used to construct the expression vector and qPCR primers are shown in Table 7.

[0130] Table 7

[0131]

[0132] 10. Detection of Chop-PCR and DNA methylation

[0133] Use from NIL-TT5 HGX74 and NIL-TT5 Y261 DNA was extracted from plant leaves and then subjected to Chop-PCR. DNA from NIL-TT5 was used. HGX74 and NIL-TT5 Y261 DNA from plant leaves and young panicle tissues underwent bisulfite transformation. The bisulfite-transformed DNA was amplified by PCR using specific primers and ligated into a T-plasmid. Methylation levels of the P4 and P5 fragments of the TT5 promoter were detected by first-generation sequencing. In addition to detecting methylation levels in some wild and modern cultivated rice varieties, population methylation analysis also utilized whole-genome methylation sequencing files of different rice varieties from published public databases to further analyze the selective role of TT5 during domestication. Primers used for DNA methylation detection are shown in Table 8.

[0134] Table 8

[0135]

[0136] 11. Sequence Information

[0137] TT5 HGX74 Gene sequence: Yellow highlighted and underlined represent the start codon and stop codon (SEQ ID NO:1)

[0138] >AGCTTTCCCCTCTCTTTCGCTTCGCGAGATTGGTTGATTCATCTCGCGATTGATCGAGCTCGAGCGGCGGTGAGGTGAGGTGGAGGAGGAGGA GGAGGAGATCGGG ATG TGA ACTCCTGAAGGCCGATGCGACAACCAATAAAAACGGATGTGACGACACAGATCAAGTCGCACCATTAGATTGATCTTCTCCTACAAGAGTGAGACTAGTAATTCCGTGTTTGTGTGCTAGCGTGTTGAAACTTTTCTGATGTGATGCACGCACTTTTAATTATTATTAAGCGTTCAAGGACTAGTATGTGGTATAAAAGGCCGTACGTGACAGCCTATGGTTATATGCTGCACAAAAACTACGTATGGTACAGTGCAGTGCCTGTACATTTCATAATTTGCGGTAAAGTTTATTGACTATATATCCAGTGTGTCAAATATAATAAAATGTCGAGGTTTAATTACCA

[0139] TT5 HGX74 Promoter sequence (SEQ ID NO:2):

[0140]

[0141] TT5 Y261 Promoter sequence (SEQ ID NO:3):

[0142]

[0143] TT5 HGX74 Coding region sequence (SEQ ID NO:4):

[0144] >ATGGGGAGAGGGAGGGTGGAGCTGAAGAGGATCGAGAACAAGATCAACAGGCAGGTGACGTTCGCGAAGCGGAGGAATGGGC TGCTCAAGAAGGCGTACGAGCTCTCCGTGCTCTGCGACGCCGAGGTCGCCCTCATCATCTTCTCCAACCGCGGCAAGCTCTACGAGTTCTGCAGCGGCCAAAGCATGACCAGAACTTTGGAAAGATACCAAAAATTGAGTTATGGTGGGCCAGATACTGCAATACAGAACAAGGAAAATGAGTTAGTGCAAAGCAGCCGCAATGAGTACCTCAAACTGAAGGCACGGGTGGAAAATTTACAGAGGACCCAAAGGAATCTTCTTGGTGAAGATCTTGGGACACTTGGCATAAAAGAGCTAGAGCAGCTTGAGAAACAACTTGATTCATCCTTGAGGCACATTAGATCCACAAGGACACAGCATATGCTTGATCAGCTCACTGATCTCCAGAGGAGGGAACAAATGTTGTGTGAAGCAAATAAGTGCCTCAGAAGAAAACTGGAGGAGAGCAACCAGTTGCATGGACAAGTGTGGGAGCACGGCGCCACCCTACTCGGCTACGAGCGGCAGTCGCCTCATGCCGTCCAGCAGGTGCCACCGCACGGTGGCAACGGATTCTTCCATTCCCTGGAAGCTGCCGCCGAGCCCACCTTGCAGATCGGGTTTACTCCAGAGCAGATGAACAACTCATGCGTGACTGCCTTCATGCCGACATGGCTACCCTGA

[0145] TT5 Y261 Coding region sequence: The underlined and bolded sites indicate natural variation sites (SEQ ID NO:5)

[0146] >ATGGGGAGAGGGAGGGTGGAGCTGAAGAGGATCGAGAACAAGATCAACAGGCAGGTGACGTTCGCGAAGCGGAGGAATGGGC TGCTCAAGAAGGCGTACGAGCTCTCCGTGCTCTGCGACGCCGAGGTCGCCCTCATCATCTTCTCCAACCGCGGCAAGCTCTACGAGTTCTGCAGCGGCCAAAGCATGACCAGAACTTTGGAAAGATACCAAAAAT G CAGTTATGGTGGGCCAGATACTGCAATACAGAACAAGGAAAATGAGTTAGTGCAAAGCAGCCGCAATGAGTACCTCAAACTGAAGGCACGGGTGGAAAATTTACAGAGGACCCAAAGGAATCTTCTTGGTGAAGATCTTGGGACACTTGGCATAAAAGAGCTAGAGCAGCTTGAGAAACAACTTGATTCATCCTTGAGGCACATTAGATCCACAAGGACACAGCATATGCTTGATCAGCTCACTGATCTCCAGAGGAGGGAACAAATGTTGTGTGAAGCAAATAAGTGCCTCAGAAGAAAACTGGAGGAGAGCAACCAGT TGCA TGCATGGACAAGTGTGGGAGCACGGCGCCACCCTACTCGGCTACGAGCGGCAGTCGCCTCATGCCGTCCAGCAGGTGCCACCGCACGGTGGCAACGGATTCTTCCATTCCCTGGAA TT GCTGCCGCCGAGCCCACCTTGCAGATCGGGTTTACTCCAGAGCAGATGAACAACTCATGCGTGACTGCCTTCATGCCGACATGGCTACCCTGA

[0147] TT5 HGX74 Protein sequence (SEQ ID NO:6)

[0148] >MGGRGRVELKRIENKINRQVTFAKRRNGLLKKAYELSVLCDAEVALIIFSNRGKLYEFCSGQSMTRTLERYQKLSYGGPDTAI QNKENELVQSSRNEYLKLKARVENLQRTQRNLLGEDLGTLGIKELEQLEKQLDSSLRHIRSTRTQHMLDQLTDLQRREQMLCEAN KCLRRKLEESNQLHGQVWEHGATLLGYERQSPHAVQQVPPHGGNGFFHSLEAAAEPTLQIGFTPEQMNNSCVTAFMPTWLP.

[0149] TT5 Y261 The protein sequence (SEQ ID NO:7)

[0150] >MGRGRVELKRIENKINRQVTFAKRRNGLLKKAYELSVLCDAEVALIIFSNRGKLYEFCSGQSMRTLERYQK C SYGGPDTAI QNKENELVQSSRNEYLKLKARVENLQRTQRNLLGEDLGTLGIKELEQLEKQLDSSLRHIRSTRTQHMLDQLTDLQRREQMLCEAN KCLRRKLEESNQLH AWTSVGARRHPTRLRAAVASCRPAGATARWQRILPF PGI AAAEPTLQIGFTPEQMNNSCVTAFMPTWLP.

[0151] Example 1: Localization of heat-resistance-related QTL-TT5 and cloning of the TT5 gene

[0152] This invention utilizes a chromosome substitution line constructed using common wild rice material (Oryza rufipogon) as the donor parent and the Asian cultivated indica rice variety Huajingxian 74 (HGX74) as the recipient parent for phenotypic evaluation and heat resistance assessment. Among them, the substitution line Y261 exhibits endosperm powdering and a significant temperature dependence. Figure 1-2Two years of phenotypic observation confirmed that the phenotype of floury endosperm is regulated by temperature. To explore the molecular mechanism of temperature regulation of floury endosperm, it was recrossed with the recipient parent HGX74 to construct an F2 segregating population, and the QTL controlling rice heat tolerance located in this region was named TT5. TT5 was initially located at the posterior end of the long arm of chromosome 9. Through multi-season fine mapping, the candidate region of TT5 was finally narrowed down to a 25.7 kb region (between molecular markers M7854 and C7065). This region contains only two predicted genes, SPL18 (LOC_Os10g11980) and MADS8 (LOC_Os09g32948). No variation was detected in the coding region of gene SPL18, and its expression level did not change significantly. However, gene MADS8 was located in NIL-TT5. Y261 The expression level is almost undetectable in various tissues, and its coding region contains SNP / Indel variations. Insertion and deletion of Indels will lead to frameshift mutations in the subsequent amino acid sequence. Figure 2 Therefore, MADS8 is suggested to be a candidate gene for TT5.

[0153] To further confirm the accuracy of TT5 localization and identify candidate genes for TT5, genetic validation was performed in both indica and japonica rice backgrounds. In the japonica rice variety Zhonghua 11 (ZH11), the MADS8 gene was knocked out using the CRISPR / Cas9 gene editing system, and a MADS8 gene model was also constructed. HGX74 Overexpression of MADS8 resulted in plants with no significant difference in plant architecture, but they exhibited high-temperature sensitivity, significantly reduced fertility, and endosperm mealy texture. Genetic complementation results showed that in the indica rice variety NIL-TT5... Y261 complementary MADS8 in the genetic background HGX74 The full-length CDS sequence successfully rescued NIL-TT5 in transgenic positive plants. Y261 Powdered endosperm phenotype of grains Figure 3 Indica rice variety NIL-TT5 Y261 It is essentially equivalent to a mutant with reduced / missing TT5 function.

[0154] The above results indicate that a QTL-TT5 that regulates heat tolerance and rice quality under high temperature conditions has been successfully located and cloned. It is located at the posterior end of the long arm of rice chromosome 9. TT5 positively regulates the heat tolerance of rice and is crucial for maintaining grain filling and grain filling in rice under high temperature conditions.

[0155] Example 2: TT5 is specifically highly expressed in the ear and grain and localized in the cell nucleus.

[0156] To investigate the tissue expression patterns of TT5, TT5 was cloned in the Zhonghua 11 background.HGX74 Approximately 2.5 kb of sequence preceding the translation start site served as the TT5 self-promoter to drive GUS gene expression. The expression sites of the GUS gene in transgenic plants were then examined. GUS activity analysis showed that TT5 was significantly expressed in the ear, glumes, and kernels, but almost undetectable in other parts. Figure 4 Meanwhile, qRT-PCR results also confirmed that TT5 is mainly expressed in the ear, glumes, and kernels. Figure 2 ).

[0157] Given that TT5 regulates heat tolerance in rice, the transcriptional level of TT5 was examined under different high-temperature treatments. The results showed that no significant changes were detected in the transcriptional level of TT5.

[0158] To further investigate the subcellular localization of TT5, TT5 was transiently expressed in both rice protoplast and tobacco systems. TT5 exhibited clear nuclear localization, significantly overlapping with nuclear localization marker signals. Figure 4 ).

[0159] The above results indicate that TT5 expression is spatiotemporally specific, mainly expressed in the ear and grains, and its subcellular localization is nuclear localization. Its transcription level is not induced by heat.

[0160] Example 3: TT5 affects grain filling rate and functions as a maternal imprinting gene.

[0161] To further investigate the effects of TT5 on grain development, NIL-TT5 was studied in Songjiang, Shanghai. HGX74 and NIL-TT5 Y261 Phenotypic analysis of grains from different growth stages was conducted, and grain dry weight was recorded. Results showed that NIL-TT5... Y261 The grains showed obvious insufficient grain filling. At 18 and 24 DAF, a relatively obvious powdery endosperm could be observed around the embryo. Extending the harvest time to 44 days could not save the grain development defects.

[0162] Dominance-recession analysis of TT5 revealed that grain development defects occurred only in homozygous NIL-TT5. Y261 As can be seen, the grain development of heterozygous plants is related to NIL-TT5. HGX74 Consistent.

[0163] NIL-TT5 HGX74 and NIL-TT5 Y261 The results of the forward and reverse crosses show that when NIL-TT5 is used... Y261 When used as the female parent, the grain development is defective, with a more pronounced mealy endosperm. Figure 5 ).

[0164] The above results indicate that TT5 regulates the grain filling rate, and TT5 from wild rice... Y261 The alleles show slow grain filling and significant powdery endosperm in the grains. This trait is recessive and regulated by maternal effects.

[0165] Example 4, NIL-TT5 HGX74 and NIL-TT5 Y261 Trait analysis

[0166] For NIL-TT5 HGX74 and NIL-TT5 Y261 An investigation of yield-related agronomic traits revealed no significant differences between the two species in plant height, tiller number, panicle length, grain length, and grain width, but significant differences were observed in the number of secondary branches, thousand-grain weight, and yield per plant. Figure 6 NIL-TT5 HGX74 It is characterized by a significantly increased number of secondary branches, a significantly increased thousand-grain weight, and a significantly increased yield per plant.

[0167] These results indicate that TT5 positively regulates the thousand-grain weight and yield per plant in rice, and downregulation of TT5 transcription levels leads to reduced rice yield. (NIL-TT5) HGX74 and NIL-TT5 Y261 They showed significant differences in agronomic traits such as the number of secondary branches, thousand-grain weight, and yield per plant.

[0168] Example 5, NIL-TT5 HGX74 and NIL-TT5 Y261 Grain development phenotypes are regulated by temperature, resulting in significant differences in yield.

[0169] During the positioning process, NIL-TT5 Y261 The resulting grain development defects showed significant differences between Songjiang, Shanghai and Sanya, Hainan. In the colder Hainan, NIL-TT5... Y261 The proportion of abnormal grain development in the region remains at around 30%, while in Shanghai, where high temperatures are frequent, the proportion is over 70%. This difference is caused by high temperature stress.

[0170] To further verify whether TT5 participates in the response to high temperature, three environmental conditions were set up in Shanghai: 28℃ / 22℃, Songjiang area of ​​Shanghai, and high temperature in a greenhouse (38℃) to observe the effect of temperature on TT5 grain development. NIL-TT5 grains were treated with high temperature in the field and greenhouse in Songjiang, Shanghai. Y261 A distinctly floury endosperm was observed in the grains, and the extent of the floury endosperm increased significantly with increasing temperature. At 28℃ / 22℃, NIL-TT5... Y261 Grains and NIL-TT5 HGX74There was no significant difference in grain size, and no abnormalities in endosperm development were observed. High temperature also affected NIL-TT5. HGX74 and NIL-TT5 Y261 The fruit setting rate. Under conditions of 28℃ / 22℃, NIL-TT5 Y261 The fruit setting rate can be maintained at around 90%, while in Songjiang, Shanghai, the fruit setting rate is around 70%, showing a significant decrease. Under high-temperature treatment in greenhouses, NIL-TT5... Y261 The fruit set rate plummeted, to only about 40%. High temperatures caused NIL-TT5... Y261 Pollen fertility is reduced.

[0171] Yield measurement results showed that NIL-TT5 in the field HGX74 and NIL-TT5 Y261 There is already a significant difference between the yield of individual plants and the yield of NIL-TT5. Y261 The yield per plant is around 20g, while NIL-TT5 HGX74 The yield per plant is around 30g, due to the relatively hot weather in Songjiang, Shanghai. After heat treatment, NIL-TT5... Y261 The yield per plant decreased significantly. Figure 7 KI-I2 staining of pollen revealed that NIL-TT5 was reduced under high-temperature treatment. Y261 Pollen fertility was significantly reduced. High-temperature treatment of ZH11 and TT5 knockout in a japonica rice background yielded consistent results: TT5 knockout in a japonica rice background exhibited stronger heat sensitivity and a significantly reduced seed setting rate. High temperatures in Shanghai significantly reduced seed setting and grain filling in TT5 knockout plants. Figure 8 NIL-TT5 HGX74 It can maintain a high settling rate at high temperatures.

[0172] The above results indicate that TT5, as a positive regulator of rice heat tolerance, participates in the response to high temperatures. The loss of TT5 function leads to extreme heat sensitivity in rice, which significantly affects rice yield under high temperatures.

[0173] Example 6, NIL-TT5 HGX74 and NIL-TT5 Y261 Starch and storage protein content determination and physicochemical property testing of NIL-TT5 in fields in Songjiang, Shanghai. HGX74 and NIL-TT5 Y261 The total starch content of the grains showed a significant difference between the two. (NIL-TT5) Y261 The total starch content was significantly reduced, and the starch content of both was further reduced after heat treatment compared with that in the field (normal field).

[0174] The total starch content of the grains at 28 / 22℃ was also tested, and no difference was found between the two.

[0175] The starch content of TT5 knockout plants in the Japonica rice background was also tested. The results were consistent with those of the NIL population. The total starch content decreased significantly after TT5 knockout.

[0176] The urea swelling experiment revealed that NIL-TT5 Y261 The rice noodles are difficult to expand, which indicates that while the starch content is reduced, the physicochemical properties of the starch also change significantly.

[0177] The storage protein content of mature grains was determined using a Kjeldahl nitrogen analyzer. The results showed that NIL-TT5... Y261 The grain storage protein levels of TT5 knockout plants were higher, and high temperature exacerbated the further accumulation of storage protein.

[0178] SDS-PAGE results showed that NIL-TT5 Y261 The content of glutenin (Glu) decreased significantly, while the content of prolamin (Pro) increased significantly. Figure 9 ).

[0179] These results indicate that TT5 is involved in the accumulation of starch and storage proteins at high temperatures, and leads to changes in the physicochemical properties of starch.

[0180] Example 7, NIL-TT5 HGX74 and NIL-TT5 Y261 Microscopic morphology observation of grains

[0181] To further investigate the effects of high temperature on grain development at the microscopic level, the structure of starch granules in the grains was observed. Scanning electron microscopy results showed that under conditions of 28℃ / 22℃, NIL-TT5... HGX74 and NIL-TT5 Y261 The starch granules in the seeds are neatly arranged, dense, and regularly shaped, with no obvious abnormalities in starch accumulation. Under high-temperature treatment in a greenhouse, NIL-TT5... Y261 The grains exhibit significant starch accumulation defects, with abnormally shaped and varying sizes of starch granules, mostly spherical, loosely arranged, and containing large gaps between granules. Under transmission electron microscopy, NIL-TT5... Y261 The abnormal starch granule structure of the grains was more pronounced. The grains of TT5 knockout plants exhibited characteristics similar to those of NIL-TT5. Y261 A consistent, loose, irregular microstructure. NIL-TT5 HGX74 The starch granules in the seeds are neatly arranged, dense, and regularly shaped, resulting in high starch accumulation. Figure 10 ).

[0182] These results indicate that NIL-TT5 HGX74 and NIL-TT5 Y261 The starch granule structure of grains varies significantly, and this variation is regulated by temperature.

[0183] Example 8: TT5 participates in the response to high temperature and regulates starch and storage protein-related genes.

[0184] To elucidate the function of TT5 in regulating endosperm development under high temperatures, transcriptome analysis was performed on 7DAF grains. NIL-TT5 Y261 A total of 742 genes were significantly downregulated and 248 genes were significantly upregulated.

[0185] GO analysis revealed that differentially expressed genes were primarily involved in responses to heat and oxidative stress, as well as post-embryonic development and carbohydrate metabolism. These differentially expressed genes are involved in numerous biochemical metabolic pathways, such as phenylpropane synthesis, starch and sucrose metabolism, fatty acid metabolism, and plant hormone signaling. This suggests that TT5 is involved in heat perception, specifically in NIL-TT5... Y261 The transcriptional levels of many HSPs were induced to be upregulated.

[0186] In addition, among the differentially expressed genes, many genes that positively regulate grain development are present in NIL-TT5. Y261 Significant downregulation was observed. Among them, GS2 positively regulated grain weight and rice yield, while AGPL4, which encodes the fourth large subunit of adenosine diphosphate glucose pyrophosphorylase, affected starch synthesis. Many other cell cycle protein genes were also significantly downregulated.

[0187] The inventors also noted significant changes in many storage proteins and precursors. Figure 11 ).

[0188] These results suggest that TT5 plays an important role in responding to heat stress and regulating the synthesis of starch and storage proteins.

[0189] Example 9: TT5-regulated expression of GS2, AGPL4, and GluA1-L1 to maintain grain filling and rice quality at high temperatures. TT5 was captured using in vitro DNA affinity purification and sequencing technology (DAP-seq). HGX74 The combined gene sequence. Combined analysis of DAP-seq and RNA-seq revealed that TT5... HGX74 TT5 can bind to CArG-box motifs in the promoter regions of differentially expressed genes such as GS2 and AGPL4. GO enrichment analysis using DAP-seq showed that TT5 is involved in the regulation of starch synthesis-related genes. These results suggest that genes such as GS2, AGPL4, and GluA1-L1 are regulated by TT5.

[0190] To verify this hypothesis, a plasmid related to the dual-luciferase reporter system was constructed, and TT5 was validated by transiently transforming rice protoplasts. HGX74 Transactivation of genes such as GS2, AGPL4, and GluA1-L1 was performed. Using the tobacco mosaic virus 35S promoter to drive TT5 expression as an effector, a promoter fragment containing approximately 2.3 kb before the translation initiation site of genes such as GluA1-L1 was inserted before the Luciferase reporter gene, enabling it to drive Luciferase as a reporter gene. Different plasmid combinations were transiently transformed into rice protoplasts. After 14 hours of incubation in the dark, protoplasts were lysed using the Dual-luciferase reporter assay system (Promega), and the activities of two luciferases, Luc and Ren, were measured. The Luc / Ren ratio was calculated, and the data were normalized. Statistical results showed that, compared to the 62SK-EV negative control, TT5 significantly enhanced the transcriptional levels of genes such as GS2, AGPL4, and GluA1-L1.

[0191] The binding of TT5 to the promoter region of AGPL4 was also verified using EMSA experiments. To detect TT5 in vivo... HGX74 The enrichment levels of genes such as GS2, AGPL4, and GluA1-L1 were assessed using transient expression of TT5 in rice protoplasts. HGX74 Cell nuclei were collected for cut & run-qPCR experiments. qPCR results showed that TT5... HGX74 CArG-box motifs can bind to the promoter regions of genes such as GS2, AGPL4, and GluA1-L1. Homozygous knockout plants of GluA1-L1 were also generated to study the effect of GluA1-L1 on rice quality. The glua1-l1 mutant exhibited similarities to NIL-TT5. Y261 Consistent powdery endosperm phenotype. Therefore, this invention demonstrates biochemically and genetically that GluA1-L1 is a downstream gene of TT5. Figure 12 ).

[0192] The above results indicate that TT5 regulates gene transcription levels by binding to CArG-box motifs in the promoter regions of genes such as GS2, AGPL4, and GluA1-L1, thereby maintaining grain filling and rice quality under high temperatures.

[0193] Example 10, TT5 Y261 The expression of TT5 is affected by DNA methylation, leading to transcriptional silencing. The methylation level of TT5 has been highly selectively controlled during the long process of domestication.

[0194] To further explore TT5 Y261The molecular mechanisms underlying transcriptional repression were first analyzed, focusing on miRNA regulation of TT5. Y261 The possibility of predicting TT5. Y261 The sequence changes did not generate new miRNA regulatory sites, thus ruling out the possibility of miRNA regulating gene expression. To verify the possibility of epigenetic regulation, TT5 was cloned. Y261 A promoter sequence approximately 2.5 kb prior to the translation start site was used to drive GUS gene expression and transgenic plants were obtained. GUS activity analysis results indicated that TT5… Y261 It is clearly expressed in the ear, glumes, and kernels.

[0195] These results indicate that TT5 Y261 The regional variation was not the root cause of TT5 expression suppression; the inventors hypothesized that promoter methylation led to expression silencing. Therefore, Chop-PCR was used for qualitative analysis of NIL-TT5. HGX74 and NIL-TT5 Y261 The methylation level between them. After digestion with two methylation-sensitive restriction enzymes, HpaII and MspI, NIL-TT5 Y261 The genome of TT5 can amplify obvious DNA bands, indicating that TT5 Y261 The promoter region of TT5 exhibits a high level of methylation. To quantify the differences in methylation levels within the TT5 promoter region, NIL-TT5 was analyzed. HGX74 and NIL-TT5 Y261 Genomic DNA from plant leaves and young spikelets underwent bisulfite transformation to quantitatively detect differences in specific DNA methylation types. Methylation detection of the P4 and P5 sequences in the TT5 promoter revealed differences in NIL-TT5 methylation. Y261 TT5 Y261 The promoters exhibit high levels of DNA methylation, particularly the CG type. Figure 13 The above results indicate that TT5 Y261 The transcriptional silencing is due to promoter hypermethylation, not promoter sequence variations.

[0196] Analysis of the conservation of the coding region sequence of TT5 reveals that TT5 is relatively conservation in evolution. Y261 The variant types of the two indels in the coding region are mainly found in wild rice, due to NIL-TT5. Y261 The TT5 allele from common wild rice is primarily epigenetically regulated. Therefore, it is inferred that TT5 is a domestication gene, and its promoter region methylation level was highly selected during domestication.

[0197] Therefore, the inventors used Chop-PCR to detect the methylation level in the AA genome of wild rice. The results showed that the TT5 promoter region exhibited a high degree of methylation in common wild rice, while the TT5 promoter in modern cultivated rice showed a low level of methylation. Figure 14 ).

[0198] Simultaneously, the inventors also developed a CRISPR-dCas9-TET1cd demethylation system to erase NIL-TT5. Y261 Neutralization was used to design eight sgRNAs targeting TT5. Y261 Methylation sites in the P4 and P5 sequences of the promoter. DNA methylation level detection in transgenic positive T1 plants revealed that the CRISPR-dCas9-TET1cd system successfully erased some TT5. Y261 Methylation in promoters Figure 15 ).

[0199] The above results indicate that TT5 underwent strong selection during domestication. Lower-level methylated TT5 alleles were continuously selected and preserved, gradually forming the TT5 alleles in modern cultivated rice. Cloning TT5 is of great significance for guiding the de novo domestication of wild rice. Methylation of the genome can be erased using gene editing technologies such as the CRISPR-dCas9-TET1cd system or added using the CRISPR-dCas9-MQ1V system, enabling rapid domestication of wild rice and rapid improvement of agronomic traits.

[0200] Example 11: In modern cultivated rice, the TT5 promoter haplotype can be divided into two types: indica and japonica. The indica haplotype can confer more significant heat resistance to rice. In situ enhancement of TT5 transcription level can significantly improve the heat resistance of rice.

[0201] Since the TT5 coding region sequence has only undergone a few SNP changes in modern cultivated varieties and no major variations have been observed, NIL-TT5 in wild rice... Y261 The coding variation is a rare variation. Considering that TT5 is a domesticated gene and its transcriptional level is affected by the promoter DNA methylation level, a low TT5 transcriptional level significantly affects rice yield and rice quality. Therefore, mining promoter variations of TT5 to find highly expressed TT5 alleles can effectively improve rice quality and heat tolerance.

[0202] The inventors performed haplotype analysis on the promoter region of TT5, and the results showed that TT5 exhibits significant differentiation between indica and japonica rice. TT5 haplotypes can be divided into seven haplotypes, with haplotypes I and III being the most prevalent, occurring most frequently in japonica and indica rice, respectively. Specifically, haplotype I accounts for 99.26% in japonica rice and haplotype III accounts for 77.22% in indica rice.

[0203] To verify the potential of haplotypes I and III in production applications, replacement lines (SN-115 and SN-155) containing the TT5 fragment of indica and japonica rice were selected for high-temperature tolerance evaluation in rice. High-temperature treatment results showed that the replacement line SN-115 containing the TT5 fragment of indica rice exhibited a more pronounced heat-tolerant phenotype than varieties containing the japonica rice fragment, significantly protecting rice yield under high temperatures.

[0204] Meanwhile, by driving TT5 expression through the TT5 allele promoter of the indica rice variety Huajingxian 74, the transcriptional level of TT5 was upregulated, and high-temperature treatment in the plot significantly increased rice yield. Figure 16 Multiple sequence alignment of the TT5 amino acid sequence revealed that TT5 is relatively conserved in major crops, showing 89.20% sequence identity with the maize homology MADS27, 74.86%, 86.56%, 87.35%, and 89.20% sequence identity with soybean, barley, wheat, and sorghum, respectively, and 60.56% sequence identity with Arabidopsis thaliana AtSEP3. Figure 17 ).

[0205] Given the high conservation of TT5, it suggests that TT5 and its homologs also have important application value for heat-resistant breeding and improvement of storage protein content in other important crops.

[0206] 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 the heat stress tolerance and yield of gramineous plants, comprising: Increase the expression or activity of TT5 in grasses; wherein TT5 is a protein with the amino acid sequence shown in SEQ ID NO:6 or a functional variant thereof.

2. The method as described in claim 1, characterized in that, The increased yield includes: promoting grain filling, increasing the number of branches under heat stress, improving fertility / seed setting rate, increasing grain starch and storage protein content, increasing thousand-grain weight, and increasing yield per plant.

3. The method as described in claim 2, characterized in that, The TT5 is located in the cell nucleus and is specifically expressed in the ear, hull, and grain; or The TT5 enhances or maintains the transcriptional levels of downstream genes, including the following groups, under heat stress to regulate the accumulation of grain starch or storage proteins: GS2, AGPL4, and GluA1-L1; preferably, TT5 binds to the CArG-box motif in the promoters of the downstream genes, thereby enhancing the transcriptional level.

4. The method as described in claim 1, characterized in that, The increase in TT5 expression or activity in grasses includes: An expression construct or vector containing TT5 genomic DNA or TT5 coding region DNA is introduced into plants; preferably, the expression of the TT5 genomic DNA or TT5 coding region DNA is driven by a spike, glume, or grain-specific expression promoter; more preferably, the expression of the TT5 genomic DNA or TT5 coding region DNA is driven by a TT5 promoter; or A gain-of-function mutation is performed on TT5 expressed in plants; preferably, a variant of TT5 with decreased or lost function is mutated to obtain a protein with the amino acid sequence shown in SEQ ID NO:6 or a homologous variant thereof; or Gene editing technology can be used to optimize or modify the promoter region of the TT5 gene to increase the transcription level of TT5 and increase its expression; or The TT5 allele was introduced into the currently cultivated plant varieties through hybridization to increase the expression or activity of TT5.

5. The method as described in claim 1, characterized in that, The promoter has the nucleotide sequence shown in SEQ ID NO:2; a polynucleotide that can hybridize with the polynucleotide sequence shown in SEQ ID NO:2 under stringent conditions and has the same driving expression function; or a polynucleotide that has more than 75% homology with the polynucleotide sequence shown in SEQ ID NO:2 and has the same driving expression function; or The TT5 protein encoded by the TT5 genomic DNA or TT5 coding region DNA is selected from the group consisting of: (i) a protein with the amino acid sequence shown in SEQ ID NO:6; (ii) a protein with the amino acid sequence having ≥80% homology to the amino acid sequence shown in SEQ ID NO:6 and having the regulatory trait function; (iii) a protein derived from (i) having the regulatory trait function formed by substituting, deleting, or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID NO:6; 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:6, or by adding a signal peptide sequence to its N end.

6. Use of TT5 or its expression constructs or vectors for improving heat stress tolerance and yield in grasses, wherein TT5 is a protein with the amino acid sequence shown in SEQ ID NO:6 or an isofunctional variant thereof; preferably, the improvement in yield includes: Under heat stress, it promotes grain filling, increases the number of branches, improves fertility / seed setting rate, increases grain starch and storage protein content, increases thousand-grain weight, and increases yield per plant.

7. The method as described in any one of claims 1-5 or the use as described in claim 6, characterized in that, The grasses include cereal plants, or the TT5 or its homologs are derived from cereal plants; preferably, the grasses include: rice, wheat, millet, foxtail millet, corn, sorghum, foxtail millet, barley, rye, oats, and two-stalked short-stalked grass.

8. Uses of TT5 in grasses (Poaceae): as molecular markers for identifying plant traits; among which, The plant traits mentioned include: heat stress tolerance and yield; preferably, the yield traits include: grain filling under heat stress, number of branches, fertility / seed setting rate, grain starch and storage protein content, thousand-grain weight, and yield per plant.

9. The use as described in claim 8, characterized in that, When identifying plant traits, the protein, genomic DNA, coding region DNA sequence and / or promoter of TT5 in grass plants are analyzed. If the grass plant has the characteristics defined in claim 5, then the grass plant has heat stress tolerance; if the promoter and gene region do not have the characteristics defined in claim 5, then the grass plant has low heat stress tolerance.

10. A method for rapid domestication of grasses, comprising methylation erasure of the promoter region of TT5 by a gene-editing-based demethylation system to guide the de novo domestication of wild varieties; preferably, the methylation erasure of the promoter region is achieved by a CRISPR-dCas9-TET1cd demethylation system.

11. A cell, tissue, or organ of a grass plant, comprising: The exogenous TT5 genomic DNA or TT5 coding region DNA, or an expression construct or vector containing it; preferably, the exogenous TT5 genomic DNA or TT5 coding region DNA is operatively linked to a TT5 promoter, which drives the expression of the TT5 genomic DNA or TT5 coding region DNA; wherein the TT5 genomic DNA, TT5 coding region DNA, or promoter has the sequence defined in claim 5.

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