Gene ATT2 for synergistically regulating yield and alkali-heat tolerance and application thereof

Abstract

The invention provides a gene ATT2 for synergistically regulating yield and alkali-heat tolerance and application of the gene ATT2. In the invention, an alkali-heat resistant QTLs, which is called ATT2 (ALKALI-THERMAL TOLERANCE 2), is cloned and positioned, and is called ATT2 (ALKALI-THERMAL TOLERANCE 2). In a normal field environment, through overexpression of ATT2 (including a promoter and a genomic sequence thereof or a coding sequence thereof), the active gibberellin level can be properly increased, and meanwhile, the yield of gramineous plants can be increased. In an alkaline field environment, the yield under alkali stress is increased by overexpressing ATT2. The ATT2 can improve the yield under normal conditions and alkali-heat stress.

Description

Technical Field

[0001] This invention belongs to the field of plant science and technology, and more specifically, this invention relates to an ATT2 gene that synergistically regulates yield and alkali-heat tolerance and its applications. Background Technology

[0002] With the rapid growth of the global population and the challenges of climate change, improving crop yields and resilience has become a crucial task for modern agriculture. Especially under abiotic stresses such as salinization and heat stress, improving crop varieties to adapt to these environmental pressures is particularly urgent. Data from the Food and Agriculture Organization of the United Nations shows that more than 1.1 billion hectares of arable land globally are affected by salinization, with 40% of the soil being alkaline. Furthermore, the global average temperature has risen by 1.2°C since 1900 and is projected to continue rising over the next decade, posing a serious threat to crop yields.

[0003] Cereal crops are important agricultural products, and how to grow more crops on limited arable land has always been a focus of agricultural research. Studying methods to regulate crop plant architecture and optimize crop cultivation is crucial. Rice, in particular, has long been an important food crop and a staple food on the Chinese table; grain shape regulation is a key research area for breeding experts. Against this backdrop, developing new green rice varieties (GRVs) with stress resistance and high yield is vital for ensuring future food security. Rice yield is determined by various factors, including the number of effective tillers, the number of grains per panicle, and grain weight. Identifying superior loci in rice that control grain shape will help to better and faster utilize these loci to cultivate higher-yielding rice germplasm.

[0004] In the field of crop breeding, although some genes have been closely associated with stress resistance or yield traits, finding genes with specific characteristics and developing new plant varieties with further phenotypic improvements remains a goal pursued as people's demands increase. Alkali and heat tolerance traits in cereal crops are quantitative traits controlled by multiple gene loci. These gene loci are not yet fully understood in this field. Obtaining control loci and elucidating the molecular mechanisms of alkali and heat tolerance in crops through multifaceted research will help construct materials related to alkali and heat tolerance, thereby enhancing crop stress resistance. Summary of the Invention

[0005] The purpose of this invention is to provide a gene ATT2 that synergistically regulates yield and alkali-heat tolerance, and its applications.

[0006] In a first aspect of the present invention, a method for improving the traits of grass plants is provided, comprising: introducing ATT2 genomic DNA (gATT2) or introducing an ATT2 coding region gene into the grass plant; wherein the ATT2 genomic DNA is derived from indica rice; wherein the improved grass plant traits include: increasing yield, increasing alkali tolerance, increasing heat tolerance, or increasing plant height.

[0007] In one or more preferred embodiments, the increased yield includes: increasing ear length, increasing the number of grains per ear, increasing the seed setting rate, or increasing grain length.

[0008] In one or more preferred embodiments, the introduction of ATT2 genomic DNA or ATT2 coding region gene into a gramineous plant includes: introducing an expression construct or vector containing ATT2 genomic DNA or ATT2 coding region gene into the plant.

[0009] In one or more preferred embodiments, the ATT2 genomic DNA includes: an ATT2 promoter and a gene region (located downstream of the promoter).

[0010] In one or more preferred embodiments, the promoter has the nucleotide sequence shown in SEQ ID NO:2; a polynucleotide capable of hybridizing with the polynucleotide sequence shown in SEQ ID NO:2 under stringent conditions and having the same driving expression function; or a polynucleotide having 49% or more (preferably 50%, 60%, 65%, 70%, 75%, 80%, or 85%; more preferably 90% or more; more preferably 95% or more; such as 98% or 99% or more) homology with the polynucleotide sequence shown in SEQ ID NO:2 and having the same driving expression function.

[0011] In one or more preferred embodiments, the gene region of the ATT2 genomic DNA has the nucleotide sequence shown in SEQ ID NO:1 or 3; a polynucleotide that can hybridize with the polynucleotide sequence shown in SEQ ID NO:1 or 3 under stringent conditions and has the same driving expression function; or a polynucleotide that has 49% or more (preferably 50%, 60%, 65%, 70%, 75%, 80%, or 85%; more preferably 90% or more; more preferably 95% or more; such as 98% or more or 99% or more) identity with the polynucleotide sequence shown in SEQ ID NO:1 or 3 and has the same driving expression function.

[0012] In one or more preferred embodiments, the indica rice includes: Asian cultivated rice Longtepu (LTP).

[0013] In one or more preferred embodiments, the ATT2 coding region gene has the nucleotide sequence shown in SEQ ID NO:3; a polynucleotide that can hybridize with the polynucleotide sequence shown in SEQ ID NO:3 under stringent conditions and has the same driving expression function; or a polynucleotide that is 49% or more (preferably 50%, 60%, 65%, 70%, 75%, 80% or 85% or more; more preferably 90% or more; more preferably 95% or more; such as 98% or 99% or more) homologous to the polynucleotide sequence shown in SEQ ID NO:3 and has the same driving expression function.

[0014] In one or more preferred embodiments, the promoter or gene region includes homologous genes that have the same function.

[0015] In one or more preferred embodiments, the promoter does not include the promoter of the nucleotide sequence shown in SEQ ID NO:6 (i.e., a promoter not derived from BART).

[0016] In one or more preferred embodiments, the gene region of the ATT2 genomic DNA does not include the promoter of the nucleotide sequence shown in SEQ ID NO:5 (i.e., a gene region not derived from BART).

[0017] In one or more preferred embodiments, the upstream of the ATT2 coding region gene does not include the promoter of the nucleotide sequence shown in SEQ ID NO:5.

[0018] In one or more preferred embodiments, the protein encoded by ATT2 (ATT2) comprises: (a) a protein or homologous protein of the amino acid sequence shown in SEQ ID NO:4; (b) a protein derived from (a) having the protein function of (a) formed by substitution, deletion or addition of one or more (e.g., 1-20; preferably 1-10; more preferably 1-5) amino acid residues of the amino acid sequence shown in SEQ ID NO:4; (c) a protein having the protein function of (a) and having an amino acid sequence that is at least 49% (preferably 50%, 60%, 65%, 70%, 75%, 80% or 85%; more preferably 90%; more preferably 95%; e.g., 98% or 99%) identical to the amino acid sequence defined in (a); or (d) a fragment of SEQ ID NO:4 having the protein function of (a).

[0019] In one or more preferred embodiments, the method involves introducing ATT2 genomic DNA or ATT2 coding region genes from indica rice into japonica rice.

[0020] In another aspect of the invention, the use of isolated ATT2 genomic DNA or ATT2 coding region gene or expression constructs or vectors containing the same is provided for improving traits of gramineous plants, wherein the ATT2 genomic DNA is derived from indica rice; wherein the improved gramineous plant traits include: increasing yield, increasing alkali tolerance, increasing heat tolerance, or increasing plant height; preferably, the increased yield includes: increasing panicle length, increasing number of grains per panicle, increasing seed setting rate, and increasing grain length.

[0021] In one or more preferred embodiments, the ATT2 genomic DNA includes: an ATT2 promoter and a gene region (located downstream of the promoter).

[0022] In one or more preferred embodiments, the protein encoded by the ATT2 genomic DNA is GA20 oxidase 1, which regulates the heat tolerance, alkali tolerance (alkali-heat stress tolerance), yield or plant height of grasses by controlling the synthesis of gibberellins.

[0023] In one or more preferred embodiments, the grass species includes cereal plants, or the ATT2 or its homologs are derived from cereal plants; preferably, the grass species includes (but is not limited to): grass species (such as, but not limited to, rice, wheat, millet, foxtail millet, corn, sorghum, millet, barley, rye, oats, and sedge).

[0024] In another aspect of the invention, the use of Gramineae ATT2 genomic DNA (gATT2) is provided for use as a molecular marker for identifying plant traits; wherein the plant traits include: yield, alkali tolerance, heat tolerance, or plant height; preferably, wherein the yield trait includes: spike length, number of grains per spike, seed setting rate, or grain length.

[0025] In one or more preferred embodiments, when identifying plant traits, the ATT2 genomic DNA of a grass plant is analyzed, the ATT2 genomic DNA comprising: an ATT2 promoter and a gene region (located downstream of the promoter); if the promoter and gene region have the promoter and gene region sequences defined above, then the grass plant has an improved trait; if the promoter and gene region do not have the promoter and gene region sequences defined above, then the grass plant does not have an improved trait; the improved trait includes: increased yield, increased alkali tolerance, increased heat tolerance, or increased plant height.

[0026] 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.

[0027] In another aspect of the invention, a separated polynucleotide is provided, comprising: an ATT2 promoter and a gene region (located downstream of the promoter), wherein the promoter and gene region have the promoter and gene region sequences defined above.

[0028] In another aspect of the invention, a cell, tissue, or organ of a grass plant is provided, comprising: exogenous ATT2 genomic DNA or an ATT2 coding region gene, or an expression construct or vector containing therein; wherein the ATT2 genomic DNA is derived from indica rice and has the promoter and gene region sequences defined above, and the ATT2 coding region gene has the sequences defined above.

[0029] In one or more preferred embodiments, the plant cells, tissues, or organs do not have the ability to reproduce into adult plants.

[0030] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0031] Figure 1 Fine mapping of ATT2. (A) Fine mapping diagram of ATT2. The numbers between each molecular marker indicate the number of exchanges obtained, and the n value indicates the number of individual plants used in the F2 population. In the chromosome pattern diagram, the white part represents the fragment from the recurrent parent Lontepus, and the black part represents the fragment from the donor parent wild rice O. barthii. C1 indicates that the fragment is completely derived from Lontepus, and C2 indicates that the fragment is completely derived from O. barthii. R1-R3 indicate that the fragment is derived from O. barthii exchange homozygous lines. (B) Phenotypes of recombinant exchange individuals with different target O. barthii wild rice fragments under high temperature (42.5℃, RH>90%, 20-30 h) and alkali (65mM sodium bicarbonate, 7-14 days) treatment; scale bar 3cm. (C) and (D) represent the survival rates of C1-C2 and R1-R3 lines under heat and alkali treatments, respectively, in the ATT2 fine mapping (n = 8 biological replicates, each consisting of 24 rice seedlings). Data are expressed as Mean ± SD; significant differences were determined by Statix ​​8.0 analysis and post-hoc paired multiple comparison test (LSD < 0.05).

[0032] Figure 2 Identification of heat and alkali tolerance phenotypes and survival rates of near-isogenic ATT2 lines. (AC) Near-isogenic line NIL-ATT2 LTP and NIL-ATT2 BARTPhenotypic (A) and survival rates (B and C) under alkaline and high-temperature stress: Alkaline treatment (65 mM sodium bicarbonate) for 14 days; High-temperature treatment (42°C, RH > 90%) for 28 hours. Scale bar: 10 cm. Data are presented as mean ± standard deviation. n = 3 replicates, each consisting of 24 rice seedlings. Two-tailed t-tests were used to determine significance, with **P < 0.01 and ***P < 0.001.

[0033] Figure 3 ATT2 promoter sequence analysis. ATT2 promoter sequence analysis in near-isogenic lines. Red boxes indicate regulatory element variations caused by genetic polymorphism. Promoter alignment was performed using Clustal Omega software, and regulatory elements were predicted from the PlantCARE database.

[0034] Figure 4 Analysis of ATT2 exon variations. Analysis of ATT2 coding region variations in near-isogenic lines.

[0035] Figure 5 Alkali-thermal phenotypic identification of knockout and reintroduction transgenic materials. (A) NIL-ATT2 BART and NIL-ATT2 BART The background contains information from NIL-ATT2. LTP Relative expression of ATT2 in transgenic complementary lines of ATT1 genomic DNA; (B) att2(NIL-ATT2) LTP Background) Gene mutation sites and types; (CE) NIL-ATT2 BART The background contains information from NIL-ATT2. LTP Phenotypic (C) and survival (D, E) of transgenic complementary lines of (FH)att2(NIL-ATT2) genomic DNA (gATT2) under alkaline and high-temperature stress; LTP (Background) Phenotypic (F) and survival (G, H) of mutants under alkaline and high-temperature stress. Alkaline treatment: 65 mM sodium bicarbonate for 14 days; high-temperature treatment: 42°C, RH > 90% for 28 hours. Scale bar: 10 cm. Data are mean ± standard deviation (n = 3 replicates). Significance was determined using a two-tailed t-test, **p < 0.01, ***p < 0.001.

[0036] Figure 6 near-isogenic line NIL-ATT2 LTP and NIL-ATT2 BART Agronomic traits were investigated under normal conditions. (A) Near-isogenic line NIL-ATT2 LTP and NIL-ATT2 BARTPlant type at maturity (scale bar, 1m) and ear type (scale bar, 10cm). (BC) NIL-ATT2 under normal field conditions LTP and NIL-ATT2 BART Statistical analysis of single-plant yield (B) and plot yield (C), n=20 plants or n=3 plots (180 plants per plot). (DK) NIL-ATT2 under normal field conditions. LTP and NIL-ATT2 BART Statistical analysis was performed on plant height (D), tiller number (E), panicle length (F), number of grains per panicle (G), seed setting rate (H), thousand-grain weight (I), grain length (J), and grain width (K). n = 20 plants. Values ​​in BK represent the mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences in Student's t-tests; ns, no statistically significant difference.

[0037] Figure 7 near-isogenic line NIL-ATT2 LTP and NIL-ATT2 BART Phenotypic characteristics of alkali treatment in the field. (AL) near-isogenic line NIL-ATT2 LTP and NIL-ATT2 BAR The following were comparisons of plant type (upper part, scale bar, 1m) and ear type (lower part, scale bar, 10cm) after alkali treatment in the field: yield per plant (B), plot yield (C), yield loss (D), plant height (E), number of tillers (F), ear length (G), number of grains per ear (H), seed setting rate (I), 1000-grain weight (J), and grain length (K) and width (L). Data in BL are presented as mean ± standard deviation. For n = 20 plants, a two-tailed t-test was used to test for significance. *p < 0.05, **p < 0.01, ***p < 0.001, ns: no significant difference was observed.

[0038] Figure 8 Near-isogenic line NIL-ATT2 under high temperature stress LTP and NIL-ATT2 BART Impact on rice yield. (AL) near-isogenic line NIL-ATT2 LTP and NIL-ATT2 BARTThe plant type (upper part, scale bar, 1m) and ear type (lower part, scale bar, 10cm) after field heat treatment were compared (A), yield per plant (B), plot yield (C), yield loss (D), plant height (E), number of tillers (F), ear length (G), number of grains per ear (H), seed setting rate (I), 1000-grain weight (J), and grain length (K) and width (L). Data for BL are presented as mean ± standard deviation. For n = 20 plants, a two-tailed t-test was used to test for significance. *p < 0.05, **p < 0.01, ***p < 0.001, ns: no significant difference was found.

[0039] Figure 9 Construction of ATT2 knockout and overexpression materials in the green revolution rice variety ZH11. (A) Background att2 gene mutation sites and types in ZH11; (B) Relative expression of ATT2 in ZH11 (WT) and OE-ATT2.

[0040] Figure 10 Agronomic traits of ZH11 background att2 knockout and ATT2 overexpression materials under normal conditions were investigated. (A) Plant type (scale bar, 1m) and panicle type (scale bar, 10cm) of ZH11 background att2 knockout and ATT2 overexpression materials under normal conditions. (BK) Statistical analysis of single plant yield (B), plot yield (C), plant height (D), number of tillers (E), panicle length (F), number of grains per panicle (G), seed setting rate (H), thousand-grain weight (I), grain length (J), and grain width (K) of ZH11 background att2 knockout and ATT2 overexpression materials under normal field conditions. n = 10 plants. Values ​​in CK represent mean ± standard deviation. The same letter indicates no significant difference. Significant differences were determined by Statix ​​8.0 analysis and post-hoc paired multiple comparison test (LSD < 0.05).

[0041] Figure 11 OE-ATT2 ZH11 (ATT2 overexpression) Field alkali treatment phenotype of transgenic lines. (AL)OE-ATT2 ZH11 The plant type (upper part, scale bar, 10cm) and panicle type (lower part, scale bar, 10cm) of the strains after alkali treatment in the field were compared (A), expression level (B), yield per plant (C), plot yield (D), plant height (E), number of tillers (F), panicle length (G), number of grains per panicle (H), seed setting rate (I), thousand-grain weight (J), grain length (K), and grain width (L). Data in BL are presented as mean ± standard deviation. n = 15 plants, n = 3 plots (180 plants per plot). A two-tailed t-test was used to test for significance. *p < 0.05, **p < 0.01, ***p < 0.001, ns: no significant difference.

[0042] Figure 12 Conservation analysis of ATT2. (A) Conservation analysis of ATT2 in monocotyledonous and dicotyledonous plants. (B) Phylogenetic tree of ATT2 constructed in MEGAX 5.0 using the Neighbor-joining method. Detailed Implementation

[0043] Through in-depth research, the inventors cloned and located an alkali-heat tolerant QTL, named ATT2 (ALKALI-THERMAL TOLERANCE 2). In normal field environments, overexpression of ATT2 moderately increases gibberellin activity and simultaneously increases plant yield. In alkaline field environments, overexpression of ATT2 increases plant yield under alkaline stress. ATT2 can improve rice yield under both normal conditions and alkali-heat stress.

[0044] As used herein, "improved traits" refer to improved plant characteristics. In this invention, these characteristics mainly include: plant yield, plant heat tolerance, or plant alkali tolerance. Preferably, the yield traits include: ear length, number of grains per ear, seed setting rate, and / or grain length.

[0045] As used herein, terms such as “improvement of plant traits,” “improved traits,” “improved plant traits,” and “trait improvement” can be used interchangeably. They refer to statistically significant changes in the traits or characteristics of plants modified by the technical solution of this invention compared to unmodified plants (such as wild-type plants), resulting in beneficial agronomic traits.

[0046] As used herein, "plant" includes plants that express the ATT2 protein or plants whose genome contains the ATT2 gene. According to knowledge in the art, plants expressing ATT2 possess the mechanism of action claimed in this invention and can achieve the technical effects claimed in this invention. The plant can be a monocotyledonous or dicotyledonous plant. In some preferred embodiments, the plant is a crop, preferably a cereal crop, which is a crop with grains (ears). In some preferred embodiments, the "cereal crop" can be a grass (Poaceae); preferably, the grass (Poaceae) includes, but is not limited to: rice, wheat, millet, foxtail millet, corn, sorghum, foxtail millet, barley, rye, oats, and *Brachys edulis*.

[0047] 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.

[0048] As used herein, “high temperature (stress)” or “heat (environment) (stress)” refers to a temperature significantly higher than the optimal temperature for plant growth (e.g., 26–30°C for grasses, preferably 28–30°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.

[0049] As used herein, “alkaline (stress)” or “alkaline (environment) (stress)” refers to a pH level significantly higher than that suitable for plant growth (e.g., 5.8–7.5 for grasses, with 6.0–7.0 being the optimal soil pH); for example, “alkaline (stress)” is 7.0 or higher, 7.2 or higher, 7.5 or higher, 7.8 or higher, or 8.0 or higher.

[0050] As used herein, the terms “upregulate,” “increase,” “enhance,” “promote,” “enhance,” “enhance,” 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 significantly, compared to a control plant, control gene, or control protein as defined herein.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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 polypeptides in their natural state within living cells are not isolated and purified, but the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances present in their natural state.

[0055] 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.

[0056] In this invention, unless otherwise specified, ATT2 refers to a gene having ATT2 genomic DNA (gATT2) or an ATT2 coding region gene (but without a promoter from LARP), and also includes sequence variations having the same function as the ATT2 polypeptide, as well as a promoter. For example, the gDNA has the nucleotide sequence shown in SEQ ID NO:1 (LTP source), the corresponding cDNA has the nucleotide sequence shown in SEQ ID NO:3, and the promoter has the nucleotide sequence shown in SEQ ID NO:2. The gene sequence also includes sequences degenerate with those provided in this invention.

[0057] In this invention, ATT2 LTP With ATT2 BART These are genes from LTP and BART located at the same locus.

[0058] Variations of the ATT2 polypeptide 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 additions or deletions of one or more amino acids (typically up to 20, preferably up to 10, and more preferably up to 5) at the C-terminus and / or N-terminus. Any protein with high homology to the ATT2 polypeptide (e.g., 50% or higher, 60% or higher, 70% or higher homology to the polypeptide sequence shown in SEQ ID NO:4; 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 ATT2 polypeptide is also included in this invention. Peptides derived from species other than rice that have high homology to the sequence shown in SEQ ID NO:4, or that play the same or similar roles in the same or similar regulatory pathways, are also included in this invention.

[0059] In this invention, the ATT2 gene / protein (including the gene promoter) also includes its homologs. It should be understood that while this invention preferably studies genes obtained from a specific rice species, other genes obtained from other species that are homologous to ATT2 (e.g., having 49% or more, 60% or more, more particularly 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, or even 98% or more sequence identity) are also within the scope of this invention.

[0060] The polynucleotide (gene) of ATT2 can be a natural gene from a plant or a degenerate sequence of it.

[0061] The present invention also relates to a vector containing the aforementioned polynucleotide, and a host cell generated by genetic engineering using the aforementioned vector.

[0062] In this invention, the ATT2 genomic DNA or coding sequence can be inserted into a recombinant expression vector. 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 an expression vector is that it typically contains 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.

[0063] 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.

[0064] Transforming 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.

[0065] In a specific embodiment of the present invention, the full-length gene sequence of ATT2, the rice alkali-heat stress tolerance and yield control gene, includes the ATT2 promoter and gene region (located downstream of the promoter) sequence, as well as the molecular mechanism by which ATT2 co-regulates rice alkali-heat stress tolerance and yield. ATT2 LTP and ATT2 BART The natural variation sites of the gene, and ATT2 LTP and ATT2 BART Naturally occurring variant sites confer alkali-heat tolerance and yield phenotypes on rice.

[0066] According to the analysis of specific embodiments of the present invention, ATT2 encodes GA20 oxidase 1, which regulates rice tolerance to alkali-heat stress and yield by controlling gibberellin synthesis.

[0067] Analysis of specific embodiments of the present invention, ATT2 from Lontaup LTP It has excellent application prospects compared to NIL-ATT2. BART NIL-ATT2 LTP Under normal field conditions, yield increases are around 15%, while in artificially simulated alkaline soil environments, yield increases further to around 30%, and under artificially simulated heat stress environments, yield increases further to around 20%. Overexpression of ATT2 in Green Revolution varieties results in approximately 20% yield increases under normal field conditions, and approximately doubling of yield in artificially simulated alkaline soil environments. Therefore, the ATT2 gene locus is an important genetic resource for breeding high-yield, highly alkaline-tolerant, and heat-tolerant rice. CRISPR / Cas9 gene editing technology can be used to modify the promoter or coding region of the ATT2 gene, or to... LTP Gene loci are introduced into the current main cultivated varieties through hybridization to improve the alkali resistance, heat resistance and yield of crop varieties, thereby cultivating new high-yielding and stress-resistant crop varieties (including but not limited to rice, wheat, corn, sorghum, millet, soybeans, etc.).

[0068] Based on the inventor's new discovery, this invention provides a method for improving plants, the method comprising: introducing ATT2 genomic DNA (gATT2) or an ATT2 coding region gene into a gramineous plant; wherein the ATT2 genomic DNA is derived from indica rice; wherein the improved gramineous plant traits include: increased yield, increased alkali tolerance, increased heat tolerance, or increased plant height. The increased yield includes: increased panicle length, increased number of grains per panicle, increased seed setting rate, or increased grain length.

[0069] It should be understood that, once the function of the ATT2 genomic DNA or the ATT2 coding region gene is known, various methods well-known to those skilled in the art can be used to regulate the expression or activity of the ATT2 genomic DNA or the ATT2 coding region gene. For example, various methods well-known to those skilled in the art can be used to overexpress the ATT2 genomic DNA or the ATT2 coding region gene.

[0070] The present invention also provides a method for upregulating the expression of ATT2 genomic DNA or ATT2 coding region gene in plants, the method comprising: transferring ATT2 genomic DNA or ATT2 coding region gene or expression construct or vector containing said gene into plants.

[0071] As a preferred embodiment of the present invention, a promoter for specific expression is provided, which is the promoter of the ATT2 gene, and has been isolated for the first time. Preferably, it is a 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.

[0072] 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.

[0073] The technical solution of this invention can be applied to molecular design breeding through various pathways. ATT2 is a conserved gene in plant evolution, widely present in various crops, and has great application prospects.

[0074] After learning about the function of ATT2, 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 regulate plant traits such as seed shape, tiller number, and yield by modulating this mechanism.

[0075] 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.

[0076] 1. Experimental materials and localized cloning

[0077] A set of chromosome segment replacement lines (CSSLs) were constructed using African wild rice (O. barthii) as the donor parent and Asian cultivated rice LTP (Oryza sativas sp. indica) as the recipient parent. These lines were used to locate and clone QTLs regulating alkali and heat stress in rice. Through initial mapping, the inventors identified a QTL in the replacement lines that commonly regulates alkali and heat tolerance in rice, named ATT2. ATT2 was finely mapped using molecular markers in 20,030 plants, narrowing the candidate region to a 47.25 kb region on chromosome 3. A near-isogenic line of ATT2, NIL-ATT2 derived from indica rice, was then constructed. LTP The survival rate under alkali-heat stress was significantly higher than that of NIL-ATT2 derived from African wild rice. BART Candidate genes for ATT2 were identified by combining gene sequencing and expression level detection for further analysis.

[0078] The molecular marker primers used for localization are shown in Table 1.

[0079] Table 1

[0080] 5' oligonucleotide primer 3' primer M3-1 5'-GGCAACATATAATACATACACCC-3'(SEQ ID NO:21) 5'-TTGTGAGCTGCATCCTCC-3'(SEQ ID NO:22) M3-2 5'-TCACGCATTAAATTCGAGAA-3'(SEQ ID NO:23) 5'-ACTCAAACACTTCTATCATCGA-3'(SEQ ID NO:24) M3-3 5'-GGACTATCCGATTTTCAGTGA-3'(SEQ ID NO:25) 5'-AACATTGCTTCAGATTGAGC-3'(SEQ ID NO:26) M3-4 5'-ACTCTAAGCAGTTTGGGTAG-3'(SEQ ID NO:27) 5'-AAAGTGGCAAAGAACAGATG-3'(SEQ ID NO:28) M3-5 5'-CCTTTCTCTCCATCCCGA-3'(SEQ ID NO:29) 5'-CGAGTGATAGGTCCGTCT-3'(SEQ ID NO:30) M3-6 5'-TAGTACTACCCTGCTTTGCT-3'(SEQ ID NO:31) 5'-TTCGTTTGTGGTTGGGTC-3'(SEQ ID NO:32) M3-7 5'-TTGGCTTGCTAGGGAGTAT-3'(SEQ ID NO:33) 5'-AGGTATAATGCCTCCCCTTT-3'(SEQ ID NO:34) M3-8 5'-GCAACTTGTTTTGAGAGACA-3'(SEQ ID NO:35) 5'-AATGCACTCCCTAACTTCTC-3'(SEQ ID NO:36) M3-9 5'-CGGCGGTTCTCTGTATTTGT-3'(SEQ ID NO:37) 5'-AAAGCAAAGGCCAGTGAAGA-3'(SEQ ID NO:38) M3-10 5'-CCATCCCTTTCACATTCACT-3'(SEQ ID NO:39) 5'-CTCCCCTAGCTACCTTCTTA-3'(SEQ ID NO:40) M3-11 5'-CCAGAAAATGAACTAGAAGCC-3'(SEQ ID NO:41) 5'-GGGCTCCAATAAGTGTGTAT-3'(SEQ ID NO:42) M3-12 5'-TTCTGCAAACTCCACATTTC-3'(SEQ ID NO:43) 5'-TAGTACATCGGGAGTAGAGG-3'(SEQ ID NO:44)

[0081] ATT2 LTP Genome sequence (SEQ ID NO:1): Italicized underlines represent start and stop codons.

[0082]

[0083] ATT2 LTP Promoter sequence (SEQ ID NO:2):

[0084] >GGTTGTCCAGTCATCAATCTGGTGGGGCACAGGCGCTGGGGTGTTTTGTAGTTTCTACTTGAGTAAATTGCATAAACGTGAATTTCACGTTTTAAAAATCAAACATGTGAAGTGGTTGTCTAAATCTCGACGTAGCGTCATGTCGCAGTACAACAACACTTTTGCAACGCTAGCCAAAGAAAAAAAAAACTTGCATTTAGATAACCCTTGCATGACCAAGATAAAATTGGCCCAAGTGAAAAACTCAGTACTACATAAATAATTTTCGCAAATGGCCAAAACTCATTTTTGGATATGGTATTTTGACGTGTGGGCATCACAGGTGCACGTGAAAACCCATTTTCACATGTGGATCTGTTGAGGAGGCTGCTTGCAAAACTGATTTTTGCAGGCAGGCAAGTAAGGACTTTCGACTAGAACGATTTTTTTTTTAAAAAAAAAGAATAAACACTACAGATCCAAAACCCTATAGCTAGGGTAAGCCGCATGTGGCCGCGGCCGTGCTGGGTACAAAGAGCAGAGTGGGCTAAGCAAATACCGGGTTGTGGCACCATCCCTTTCACATTCACTCGCTCTTGATATCTTTCTCTCTCATGGAAAAGAAGAGATAAGTAATTTTAGATAGAGAGAGAGAGAGAGTTAAGAAGGTAGCTAGGGGAGCGAGCGAGGTTGATGCCGTGATCGATCGATCGATCTGTTGGCGCAGCGTGTATATAAGGGCGGGAAGGGGAGTGAGAGAGAGCAGCAGCTAGCTAGCCGCGGTCGGTCGATCCAGCTGCTGGGGATGAATACTTAGTTAGCTCGGAGCTAGCTACTAATGGATGATATACTTATGCTAGTTAGTTAAATACAGTTATTAGTTAGTTGTAGGTTGCATCTATCATATCTCCATCGGTTAATTAATTGATTGATAGCTAGATTATCAACAATTA

[0085] ATT2 LTP Coding region sequence (SEQ ID NO:3):

[0086]

[0087] ATT2 LTP The protein sequence (SEQ ID NO:4)

[0088] >MSMVVQQEQEVVFDAAVLSGQTEIPSQFIWPAEESPGSVAVEELEVALIDVGAGAERSSVVRQVGEACE RHGFFLVVNHGIEAALLEEAHRCMDAFFTLPLGEKQRAQRRAGESCGYASSFTGRFASKLPWKETLSFRYSSAGDEEGEEGVGEYLVRKLGAEHGRRLGEVYSRYCHEMSRLSLELMEVLGESLGIVGDRRHYFRRFFQRNDSIMRLNYYP ACQRPLDTLGTGPHCDPTSLTILHQDHVGGLEVWAEGRWRAIRPRPGALVVNVGDTFMALSNARYRSCLHRAVVNSTAPRRSLAFFLCPEMDTVVRPPEELVDDHHPRVYPDFTWRALLDFTQRHYRADMRTLQAFSDWLNHHRHLQPTIYS

[0089] ATT2 BART Genome sequence (SEQ ID NO:5): Bold underlined marks indicate naturally occurring variant sites, and italic underlined marks represent start and stop codons.

[0090]

[0091] ATT2 BART Promoter sequence (SEQ ID NO:6): Bold underlined markers indicate naturally occurring variant sites.

[0092] ATT2 BART The coding region sequence (SEQ ID NO:3):

[0093]

[0094] ATT2 BART The protein sequence (SEQ ID NO:4):

[0095] >MSMVVQQEQEVVFDAAVLSGQTEIPSQFIWPAEESPGSVAVEELEVALIDVGAGAERSSVVRQVGEACE RHGFFLVVNHGIEAALLEEAHRCMDAFFTLPLGEKQRAQRRAGESCGYASSFTGRFASKLPWKETLSFRYSSAGDEEGEEGVGEYLVRKLGAEHGRRLGEVYSRYCHEMSRLSLELMEVLGESLGIVGDRRHYFRRFFQRNDSIMRLNYYP ACQRPLDTLGTGPHCDPTSLTILHQDHVGGLEVWAEGRWRAIRPRPGALVVNVGDTFMALSNARYRSCLHRAVVNSTAPRRSLAFFLCPEMDTVVRPPEELVDDHHPRVYPDFTWRALLDFTQRHYRADMRTLQAFSDWLNHHRHLQPTIYS

[0096] 2. Identification of alkali- and heat-stress phenotypes in rice

[0097] Alkali stress treatment: First, rice seedlings were cultured for 12 days, at which point they reached the one-leaf-one-heart stage. Then, sodium bicarbonate was added to a 1×Yoshida nutrient solution to adjust the sodium bicarbonate concentration to 65 mM. The seedlings were placed in this alkali treatment solution for 7 to 14 days, depending on the seedlings' tolerance, with the solution changed every four days. The alkali treatment was stopped when the rice leaves began to yellow, curl, and wither. Afterward, the seedlings were returned to normal growing conditions for 7 to 14 days to observe their recovery. At the end of the experiment, the seedlings were photographed and the survival rate was recorded.

[0098] Heat stress treatment: After rice seedlings were cultured for 12 days and reached the one-leaf-one-heart stage, they were placed in a heat treatment chamber. The temperature of the heat treatment chamber was set at 42℃, the humidity was maintained at 90%, and the treatment time was 20 to 30 hours, adjusted according to the heat tolerance of the seedlings. After heat treatment, the seedlings were restored to normal growth conditions for 7 to 14 days to assess their recovery. At the end of the experiment, photos were taken and the survival rate was recorded.

[0099] 3. CRISPR / Cas9 gene editing, overexpression, and genetic complementation

[0100] To validate the candidate gene, NIL-ATT2 was constructed. BART The background contains information from NIL-ATT2. LTP Transgenic complementary lines of ATT2 genomic DNA (gATT2) were developed, and NIL-ATT2 was constructed using CRISPR / Cas9 technology. LTP Background: att2 mutant. Simultaneously, two knockout target sites were designed in the ATT2 gene coding region and constructed into a CRISPR / Cas9 vector for target gene knockout. A p35S::ATT2 plasmid was constructed to overexpress ATT2, and genetic transformation was performed in the ZH11 background. Genetic transformation was carried out using Agrobacterium tumefaciens EHA105-mediated mature embryo transformation of rice. Transgenic positive lines were screened, planted in the field, and phenotypes were observed in the transgenic T2 generation.

[0101] NIL-ATT2 BART The background contains information from NIL-ATT2. LTP Primers were used to construct a transgenic complementary vector of ATT2 genomic DNA (gATT2) (the sequence amplified by this primer pair includes ATT2). LTP Promoter sequence and genome sequence, including sequences of SEQ ID NO:2 and SEQ ID NO:1; amplified product length is 2kB):

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

[0103] 5'-ACGAATTCGAGCTCGGTACC TAATTGCGTGGTTGACATGC-3'(SEQ ID NO:7)

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

[0105] 5'-GCAGGTCGACTCTAGAGGATCCTTTACACATGCATGCATCAT-3'(SEQ ID NO:8)

[0106] NIL-ATT2 LTP Primers used to construct the att2 CRISPR / Cas9 knockout vector in the background:

[0107] The 5' oligonucleotide primer sequence for target 1 is as follows:

[0108] 5'-GGTTAACCACGGCATCGAGG-3'(SEQ ID NO:9)

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

[0110] 5'-CCTCGATGCCGTGGTTAACC-3'(SEQ ID NO:10)

[0111] The 5' oligonucleotide primer sequence for target 2 is as follows:

[0112] 5'-GCAGGAGGTGGTGTTCGACG-3'(SEQ ID NO:11)

[0113] The 3' primer sequence is as follows:

[0114] 5'-CGTCGAACACCACCTCCTGC-3'(SEQ ID NO:12)

[0115] Primers constructed using CRISPR / Cas9 knockout vectors in the ZH11 context:

[0116] The 5' oligonucleotide primer sequence for target 1 is as follows:

[0117] 5'-gccgCCCGTCGCAGTTCATATGGC-3'(SEQ ID NO:13)

[0118] The 3' primer sequence is as follows:

[0119] 5'-aaacGCCATATGAACTGCGACGGG-3'(SEQ ID NO:14)

[0120] The 5' oligonucleotide primer sequence for target 2 is as follows:

[0121] 5'-ggcaCCACTCGACACGCTGGGCAC-3'(SEQ ID NO:15)

[0122] The 3' primer sequence is as follows:

[0123] 5'-aaacGTGCCCAGCGTGTCGAGTGG-3'(SEQ ID NO:16)

[0124] Primers constructed using the p35S::ATT2 vector under the ZH11 background:

[0125] The 5' oligonucleotide primer sequence is as follows:

[0126] 5'-CATTTGGAGAGGACAGGGTACCATGAGCATGGTGGTGCAGCAGG-3'(SEQ ID NO:17)

[0127] The 3' primer sequence is as follows:

[0128] 5'-TAGTGTCGACTCTAGAGGATCCGGAGTATATTGTTGGTTGCAGG-3'(SEQ ID NO:18)

[0129] When using the above primers for CRISPR / Cas applications, two primers are used to prepare an annealed sgRNA fragment, which is then introduced into the knockout vector. If both target 1 and target 2 are present, sgRNAs targeting both target 1 and target 2 are used simultaneously. For overexpression (OE), two primers are used to amplify the target gene from the genome, which is then introduced into the overexpression vector.

[0130] 4. Field agronomic trait survey of near-isogenic lines

[0131] In a farm in Songjiang District, Shanghai, a study was conducted targeting the near-isogenic line NIL-ATT2. LTP and NIL-ATT2 BART This field planting trial aimed to evaluate the agronomic traits and yield performance of two near-isogenic lines. Experimental design and methodology: Three replicates were set up for each material to ensure data reliability and statistical significance. Approximately 300 rice plants were planted in each replicate to fully represent the phenotypic differences of the genotypes. At maturity, plant type and panicle type were photographed for subsequent morphological analysis. When all rice grains turned yellow, marking maturity, 20 individual plants from the near-isogenic lines were collected for detailed agronomic trait measurements. Measured traits included plant height, number of tillers, panicle length, number of grains per panicle, seed setting rate, thousand-grain weight, grain length, and grain width; these traits are key indicators for evaluating rice yield and quality.

[0132] Yield determination: 180 individual plants were randomly selected from the field to form a yield measurement plot to examine the plot area and total yield. The yield per unit area of ​​the plot was calculated. To ensure the accuracy of the experiment, water, fertilizer, and other management practices in the field were kept consistent throughout the growing season to minimize the impact of environmental factors on the experimental results.

[0133] 5. Field Alkali Treatment Experiment

[0134] The study was conducted at a specific experimental site in Songjiang District, Shanghai—an alkaline soil cement pool—to simulate and study the growth response of rice under alkaline soil conditions. Soil that had been subjected to sodium bicarbonate-induced alkali stress in the previous year was used as the experimental soil. Before transplanting the rice seedlings, the soil was thoroughly tilled to ensure it was loose and uniform. After tilling, the soil was irrigated to maintain a water level of at least 5 cm to facilitate seedling transplantation. Rice seedlings were transplanted into the prepared alkaline soil cement pool, using the same fertilizer management methods as for normally growing rice fields. Starting from the rice tillering stage, the alkaline soil was sampled regularly to monitor soil electrical conductivity (EC), pH, and redox potential. If the pH fell below 9.0, sodium bicarbonate was promptly added to maintain the soil pH above 9.0, simulating a continuous alkali stress environment. The alkali stress treatment continued until rice maturity to comprehensively assess the impact of alkali stress on the entire rice growth cycle.

[0135] 6. Field heat treatment experiment

[0136] In a rice heat stress experiment conducted at Songjiang Farm in Shanghai, a special treatment method was used to simulate rice growth under high-temperature conditions. When the rice reached the heading stage, just before entering the grain-filling stage, a period particularly sensitive to temperature changes, a thin film was placed over the rice plants, and a greenhouse was constructed using a spliced ​​steel frame structure to control the internal environment. The greenhouse was closed every morning, allowing the greenhouse effect to allow the temperature inside to rise naturally. When the temperature inside reached above 45 degrees Celsius, the sides of the greenhouse were opened to allow heat dissipation and a temperature drop. This treatment was continued throughout the grain-filling stage until the rice matured. This treatment simulated the effects of high-temperature stress on the grain-filling and ripening processes of rice.

[0137] 7. Investigation of agronomic traits of rice after alkali stress and heat stress

[0138] Following field and greenhouse alkali treatments and field heat stress treatments, a detailed agronomical trait assessment was conducted on mature rice to evaluate its growth performance and yield potential under different stress conditions. After maturity, individual plants were collected from the treatment areas, and a series of agronomic traits were measured, including but not limited to yield per plant, plant height, number of tillers, panicle length, number of grains per panicle, seed setting rate, thousand-grain weight, grain length, and grain width. Yield measurement plots were randomly distributed in the field, consisting of an appropriate number of plants, to ensure the comprehensiveness and accuracy of the data. The area of ​​each yield measurement plot and the total yield within each plot were examined in detail, and the rice yield per unit area was determined through precise measurement and calculation.

[0139] 8. ATT2 Conservatism Analysis

[0140] To determine the conservation of ATT2, BLAST analysis was performed on the ATT2 protein sequence (https: / / blast.ncbi.nlm.nih.gov / ) to obtain the corresponding homologous gene sequences. Sequence alignment analysis was then performed using MEGA 10.0, and a phylogenetic tree was constructed. The phylogenetic tree was constructed using the NJ neighbor-joining method with 1000 bootstrap values.

[0141] Example 1: Cloning of a gene

[0142] First, quantitative trait loci (QTLs) related to alkali and heat tolerance in rice were cloned to breed rice varieties adapted to extreme environmental conditions. A series of chromosome segment replacement lines (CSSLs) were constructed using African wild rice (Oryza barthii) as donor parents and the Asian cultivated indica rice variety Longtepu (LTP) as recurrent parents. Phenotypic identification of these materials for alkali and heat stress tolerance was performed, and QTL mapping analysis for alkali and heat tolerance was conducted using molecular markers. Based on fine mapping, a key alkali and heat tolerance QTL gene, ATT2, was located on rice chromosome 3, specifically within a 47.25 kb interval, between molecular markers M3-8 and M3-10. Figure 1 ).

[0143] Through in-depth analysis of the alkali tolerance and heat tolerance phenotypes of near-isogenic lines (NILs), the inventors discovered that NIL-ATT2, which carries the ATT2 gene locus derived from African wild rice (BART), is similar to... BART In contrast, NIL-ATT2 carrying the ATT2 gene locus from indica (LTP) rice LTP The survival rate was significantly higher under alkaline and heat treatment conditions. Figure 2 This indicates that the identified candidate region does indeed contain the gene ATT2, which can simultaneously enhance alkali tolerance and heat tolerance. LTP It has stronger resistance to adverse conditions.

[0144] Furthermore, the inventors used the rice gene prediction website (http: / / rice.plantbiology.msu.edu / ) to predict and analyze candidate genes within the ATT2 candidate region. A total of 18 candidate genes were identified within this region, of which 10 had functional annotations, 4 encoded expressed proteins, and the other 4 were transposons and retrotransposons (Table 2). Notably, this region contains a key enzyme in gibberellin synthesis—GA20 oxidase 1 (LOC_Os03g63970). Previous studies have shown that gibberellin synthesis and signal transduction are closely related to rice's response to abiotic stresses.

[0145] Table 2. Genes representing candidate regions for ATT2

[0146]

[0147]

[0148] Sequence alignment analysis of candidate genes revealed that the LOC_Os03g63970 gene is involved in NIL-ATT2. BART The starter region contains numerous mutations, while the coding region has no mutations, compared to NIL-ATT2. LTP ( Figure 3 and Figure 4 These findings suggest that variations in the promoter region of ATT2 in near-isogenic lines are the primary cause of the differences in alkali and heat tolerance phenotypes, thus LOC_Os03g63970 is highly likely to be a candidate gene for ATT2.

[0149] To further confirm that the LOC_Os03g63970 gene is a candidate gene for ATT2, NIL-ATT2 was constructed. BART The background contains information from NIL-ATT2. LTP Transgenic complementary lines of ATT2 genomic DNA (gATT2) were obtained, and NIL-ATT2 was also obtained using CRISPR / Cas9 technology. LTP The background is a mutant line of att2. Among them, LOC_Os03g63970 has two mutation types, including a 1bp deletion and a 1bp insertion. Alkali-heat resistance was identified in the complementary and mutant lines. Figure 5 The results showed that att2(NIL-ATT2) LTP The tolerance to both alkaline and thermal stress of the α-type ... LTP NIL-ATT2 BART The background contains information from NIL-ATT2. LTP The transgenic complementary lines of ATT2 genomic DNA showed significantly higher tolerance to both alkaline and heat stress than NIL-ATT2. BART ( Figure 5 This indicates that the LOC_Os03g63970 gene is a candidate gene for ATT2, which is involved in regulating the rice's alkali-heat stress response.

[0150] Therefore, by screening and identifying chromosome segment replacement lines, the QTL-ATT2, which regulates rice alkali and heat tolerance, was located on chromosome 3, and the ATT2 gene controlling its phenotype was cloned.

[0151] Example 2: ATT2 regulation of rice yield under normal conditions, alkaline stress, and heat stress

[0152] NIL-ATT2 under normal field conditions LTP and NIL-ATT2 BART Field yield and yield traits were investigated. Compared to NIL-ATT2... BART NIL-ATT2 LTP It showed a 19.50% increase in yield per plant and a 15.42% increase in yield per plot. Figure 6 Further investigation into agronomic traits revealed that, compared to NIL-ATT2... BART In comparison, NIL-ATT2 LTP Plant height, main panicle length, and number of grains per panicle all increased significantly. NIL-ATT2 showed a significant increase in tiller number, grain length, and grain width. LTP and NIL-ATT2 BART There is no difference between them. Figure 6 Therefore, under normal conditions, enhancing the function of ATT2 in Green Revolution rice varieties can increase the number of grains per panicle and the thousand-grain weight, as well as increase the yield per rice plant and the yield per plot.

[0153] The near-isogenic line NIL-ATT2 of ATT2 LTP and NIL-ATT2 BART Planted in alkaline soil, yield and related agronomic traits were investigated at maturity. Under alkaline stress, compared to its near-isogenic line NIL-ATT2... BART NIL-ATT2 LTP At maturity, it showed a 25.88% increase in yield per plant and a 26.5% increase in yield per plot. Compared with NIL-ATT2... BART In comparison, NIL-ATT2 LTP Plant height, main panicle length, and number of grains per panicle increased significantly. However, NIL-ATT2 showed a significant increase in tiller number, grain length, and width. LTP and NIL-ATT2 BART There is no difference between them. Figure 7 Therefore, under alkaline stress, NIL-ATT2 LTP It can reduce yield loss caused by alkali stress.

[0154] NIL-ATT2, a near-isogenic line of ATT2 LTP and NIL-ATT2 BART High-temperature field treatment was conducted during the grain-filling stage, and yield and related agronomic traits were investigated during the maturity stage. Figure 8 Under high temperature stress, compared to its near-isogenic line NIL-ATT2, BART NIL-ATT2LTP It showed a 41.52% increase in yield per plant and a 23.65% increase in yield per plot. Figure 8 Further investigation into agronomic traits revealed that, compared to NIL-ATT2... BART In comparison, NIL-ATT2 LTP Plant height, main panicle length, number of grains per panicle, and seed setting rate increased significantly. Figure 8 Therefore, NIL-ATT2 LTP It can reduce yield loss caused by heat stress and increase heat resistance during the ripening period.

[0155] Example 3: Overexpression of ATT2 can increase rice yield under normal conditions and under alkaline stress.

[0156] To further illustrate how ATT2 can increase rice yield in Green Revolution rice varieties, the inventors constructed a series of ATT2 knockout and overexpression lines in the genetic background of Zhongzhonghua 11 (ZH11, a semi-dwarf Green Revolution rice variety).

[0157] The gene (ATT2) that overexpresses the sequence SEQ ID NO:3 in Zhonghua 11. LTP The coding region sequence), and the primers are as follows:

[0158] 5' oligonucleotide primer sequence:

[0159] CATTTGGAGAGGACAGGGTACCATGAGCATGGTGGTGCAGCAGG(SEQ ID NO:19)

[0160] The 3' primer sequence is as follows:

[0161] TAGTGTCGACTCTAGAGGATCCGGAGTATATTGTTGGTTGCAGG(SEQ ID NO:20)

[0162] Field trials were conducted on these lines under normal conditions. Results showed that, compared to the wild-type ZH11, ATT2 overexpression lines resulted in a 22.66%–30.88% increase in yield per plant and an 18.77%–20.35% increase in plot yield. Figure 9 , Figure 10 Conversely, compared to wild-type plants, the ATT2 knockout mutant resulted in reduced yield. Figure 9 , Figure 10 Further agronomic trait analysis showed that the higher yield of ATT2 overexpression lines was mainly due to increased grains per ear and increased ear length. Figure 9 , Figure 10For other traits, the ATT2 knockout mutant showed increased tiller number and decreased plant height compared to the wild type, while the ATT2 overexpression lines showed the opposite. Figure 9 , Figure 10 In the ATT2 gene knockout mutant, the decrease in kernel width and kernel length leads to a decrease in thousand-kernel weight. Figure 9 , Figure 10 ATT2 overexpression lines had no effect on thousand-grain weight, but increased grain length and decreased grain width. There was no significant difference in seed setting rate among ATT2 knockout mutants, ATT2 overexpression lines, and wild-type plants. Figure 9 , Figure 10 These results indicate that ATT2 is crucial for regulating rice yield, providing a novel target for molecular design breeding.

[0163] To further verify whether overexpressing ATT2 in "Green Revolution" rice varieties can reduce yield loss caused by alkaline stress, the "Green Revolution" rice variety Zhonghua 11 and rice materials overexpressing ATT2 under the background of Zhonghua 11 were planted in alkaline soil. Yield and related agronomic traits at maturity were investigated. The results showed that, compared with Zhonghua 11, lines overexpressing ATT2 under field alkaline stress could increase single-plant yield by 80.67%-88.88% and plot yield by 77.96%-100.92%. Figure 11 Further analysis revealed that overexpression of ATT2 under alkali stress significantly increased plant height, number of grains per ear, and grain length, as well as the seed setting rate. Figure 11 ).

[0164] These results demonstrate that overexpression of ATT2 under alkaline stress can reduce yield loss, further proving that ATT2 not only increases rice yield under normal conditions by moderately increasing the active gibberellin content in "Green Revolution" rice varieties, but also maintains a strong yield-increasing effect under alkaline stress. ATT2 can be used as a gene for strong stress resistance to breed new alkaline-resistant varieties, and its large-scale application in alkaline soils in the future can help ensure food security.

[0165] Example 4: ATT2 increases rice yield and alkali-heat tolerance, and has application value in other crops.

[0166] The ATT2 gene positively regulates rice yield, tolerance to alkali stress, and heat stress. To further reveal the conservation of ATT2 in different species, phylogenetic trees were constructed and protein sequence alignment analysis was performed to verify the conservation of ATT2 in multiple plants.

[0167] Phylogenetic analysis revealed that ATT2 is conserved in various crops (including but not limited to wheat (71%), maize (75% homology), and sorghum (49%)), providing a potential molecular target for future genetic engineering to improve the ATT2 gene in crops to increase their yield and stress tolerance. Figure 12 ).

[0168] In summary, the conservation of the ATT2 gene in various crops and its potential application value in improving rice yield and stress resistance provide new perspectives and strategies for crop genetic improvement.

[0169] 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 traits of grasses, comprising: Introduce ATT2 genomic DNA or ATT2 coding region genes into grasses; The ATT2 genomic DNA is derived from indica rice; wherein the improved traits of the grass plant include: increased yield, increased alkali resistance, increased heat resistance, or increased plant height.

2. The method as described in claim 1, characterized in that, The increased yield includes: increasing ear length, increasing the number of grains per ear, increasing the seed setting rate, or increasing grain length.

3. The method as described in claim 1, characterized in that, The introduction of ATT2 genomic DNA or ATT2 coding region gene into grass plants includes: introducing an expression construct or vector containing ATT2 genomic DNA or ATT2 coding region gene into the plant.

4. The method according to any one of claims 1 to 3, characterized in that, The ATT2 genomic DNA includes: the ATT2 promoter and gene regions; Preferably, 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 49% homology with the polynucleotide sequence shown in SEQ ID NO:2 and has the same driving expression function. Preferably, the gene region of the ATT2 genomic DNA has the nucleotide sequence shown in SEQ ID NO:1 or 3; a polynucleotide that can hybridize with the polynucleotide sequence shown in SEQ ID NO:1 or 3 under stringent conditions and has the same driving expression function; or a polynucleotide that has more than 49% identity with the polynucleotide sequence shown in SEQ ID NO:1 or 3 and has the same driving expression function. Preferably, the indica rice includes: Asian cultivated rice Longtepus.

5. The method according to any one of claims 1 to 3, characterized in that, The ATT2 coding region gene has the nucleotide sequence shown in SEQ ID NO:3; a polynucleotide that can hybridize with the polynucleotide sequence shown in SEQ ID NO:3 under stringent conditions and has the same driving expression function; or a polynucleotide that has more than 49% homology with the polynucleotide sequence shown in SEQ ID NO:3 and has the same driving expression function.

6. The use of the isolated ATT2 genomic DNA or the ATT2 coding region gene, or expression constructs or vectors containing it, for the purpose of improving traits in gramineous plants, wherein the ATT2 genomic DNA is derived from indica rice; wherein, The improved traits of the grasses include: increasing yield, increasing alkali resistance, increasing heat resistance, or increasing plant height; preferably, the increased yield includes: increasing ear length, increasing the number of grains per ear, increasing the seed setting rate, and increasing grain length.

7. The use as described in claim 6, characterized in that, The ATT2 genomic DNA includes: the ATT2 promoter and gene regions; Preferably, 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 49% homology with the polynucleotide sequence shown in SEQ ID NO:2 and has the same driving expression function. Preferably, the gene region of the ATT2 genomic DNA has the nucleotide sequence shown in SEQ ID NO:1 or 3; a polynucleotide that can hybridize with the polynucleotide sequence shown in SEQ ID NO:1 or 3 under stringent conditions and has the same driving expression function; or a polynucleotide that has more than 49% identity with the polynucleotide sequence shown in SEQ ID NO:1 or 3 and has the same driving expression function. Preferably, the ATT2 coding region gene has the nucleotide sequence shown in SEQ ID NO:3; a polynucleotide that can hybridize with the polynucleotide sequence shown in SEQ ID NO:3 under stringent conditions and has the same driving expression function; or a polynucleotide that has more than 49% homology with the polynucleotide sequence shown in SEQ ID NO:3 and has the same driving expression function.

8. The method as described in any one of claims 1 to 5 or the use as described in any one of claims 6 to 7, characterized in that, The grass family includes cereal plants, or the ATT2 or its homologs are derived from cereal plants; preferably, the grass family includes: grass family plants.

9. Uses of ATT2 genomic DNA in grasses (Poaceae family) as molecular markers for identifying plant traits; among which, The plant traits mentioned include: yield, alkali tolerance, heat tolerance, or plant height; preferably, the yield traits include: ear length, number of grains per ear, seed setting rate, or grain length.

10. The use as described in claim 9, characterized in that, When identifying plant traits, the ATT2 genomic DNA of grasses is analyzed. The ATT2 genomic DNA includes an ATT2 promoter and a gene region. If the promoter and gene region have the promoter and gene region sequence defined in claim 4, the grass has an improved trait. If the promoter and gene region do not have the promoter and gene region sequence defined in claim 4, the grass does not have an improved trait. The improved traits include increased yield, increased alkali tolerance, increased heat tolerance, or increased plant height.

11. Isolated polynucleotides, including: ATT2 promoter and gene region, wherein the promoter and gene region have the promoter and gene region sequence defined in claim 4.

12. A cell, tissue, or organ of a grass plant, comprising: The ATT2 genomic DNA or ATT2 coding region gene, or an expression construct or vector containing it; the ATT2 genomic DNA is derived from indica rice and has the promoter and gene region sequence defined in claim 4, and the ATT2 coding region gene has the sequence defined in claim 5.

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