Application of TT2-SCT1 / SCT2-WR2 regulatory pathway in heat stress tolerance of plants

Abstract

The application provides application of a TT2-SCT1 / SCT2-WR2 regulation channel in plant heat stress tolerance. The application first discloses a new signal channel involved in plant heat tolerance: a TT2-SCT1 / SCT2-WR2 signal channel; wherein, TT2 is a negative regulation factor for regulating the heat tolerance of plants or protecting (maintaining) the yield of plants in Gramineae under high temperature, SCT1 / SCT2 are also negative regulation factors for regulating the heat tolerance of plants or protecting (maintaining) the yield of plants in Gramineae under high temperature, and WR2 is a positive regulation factor for playing a role, which is beneficial to maintaining the homeostasis in plants. The application has important theoretical significance for genetic improvement of plant traits.

Description

Technical Field

[0001] This invention belongs to the fields of botany and agriculture, and more specifically, this invention relates to the application of the TT2-SCT1 / SCT2-WR2 regulatory pathway in plant heat stress tolerance. Background Technology

[0002] Under natural conditions, various adverse environments arise due to different geographical locations, climates, and human activities, exceeding the tolerance range for normal plant growth and development, causing damage and even death to plants. These harmful environments are called adversity or stress. Adversity includes, but is not limited to, freezing, low temperatures, high temperatures, drought, salinity, excessive soil moisture, and diseases. Although plants are affected by adversity, they resist it through physiological responses. However, if the damage exceeds the plant's tolerance range and its own repair capacity, the damage becomes irreversible, and the plant will suffer harm or even die.

[0003] In recent years, with the continuous growth of the population and the intensification of human activities, global warming has become a major global problem. It is well known that crops are sensitive to climate change. Climate change can cause large-scale crop yield reductions, potentially leading to serious food security issues. As the global average temperature gradually rises, it will cause varying degrees of yield reductions in major food crops such as wheat and rice.

[0004] Improving the heat tolerance of plants has always been a crucial research topic in this field. There is an urgent need to explore more effective ways to enhance plant heat tolerance. Rice, a member of the Poaceae family, is one of the world's most important food crops, feeding more than half of the world's population. It is also one of the most important monocotyledonous model plants in scientific research. Understanding how rice adapts to increasingly high temperatures and applying genetic engineering to cultivate superior rice varieties with good adaptability to environmental temperature changes will provide new strategies for rice production and application in this field, and will have profound significance for promoting the continuous and steady increase of rice yield. At the same time, the discovery and molecular mechanism research of rice's environmental temperature adaptability genes will also provide new ideas and references for the breeding of other crops. Summary of the Invention

[0005] The purpose of this invention is to provide the application of the TT2-SCT1 / SCT2-WR2 regulatory pathway in plant heat stress tolerance.

[0006] In a first aspect of the invention, a method is provided to enhance the heat tolerance of gramineous plants or protect (maintain) the yield of gramineous plants under high temperatures, comprising: regulating the TT2-SCT1 / SCT2-WR2 signaling pathway in plants; preferably, the regulation comprises: (a) downregulating the expression or activity of TT2; (b) downregulating the expression or activity of SCT1 / SCT2; and / or (c) blocking the regulation of WR2 by SCT1 / SCT2 to maintain WR2 homeostasis.

[0007] In one or more embodiments, maintaining WR2 homeostasis means that WR2 is at normal expression levels / activity.

[0008] In one or more embodiments, maintaining the steady state of WR2 means that WR2 is not affected by changes in calcium ion flow caused by thermal environment (thermal induction / thermal stimulation).

[0009] In one or more embodiments, maintaining WR2 homeostasis means that WR2 is not regulated by SCT1 and / or SCT2 (calmodulin interacts with SCT1 / SCT2, thereby negatively regulating WR2).

[0010] In one or more embodiments, in (a), the expression or activity of TT2 is downregulated, thereby reducing the TT2 response to heat-induced changes, reducing intracellular calcium concentration under heat stimulation, and regulating the interaction between calmodulin and SCT1.

[0011] In one or more embodiments, in (b), the expression or activity of SCT1 / SCT2 is downregulated, thereby downregulating the regulation of WR2 by SCT1 / SCT2 and maintaining WR2 homeostasis (normal expression level / activity).

[0012] In one or more embodiments, in (c), for plants that are induced to have low or no WR2 expression, WR2 homeostasis is maintained by replenishing WR2 to the normal level, or by altering the SCT1 / SCT2 binding region of the WR2 promoter region so that it does not interact with SCT1 / SCT2.

[0013] In one or more embodiments, in (a), downregulating the expression or activity of TT2 includes: knocking out or silencing the gene encoding TT2 in a plant, or inhibiting the activity of TT2; preferably, it includes: silencing TT2 with an interfering molecule that specifically interferes with the expression of the gene encoding TT2, knocking out the gene encoding TT2 by gene editing using a CRISPR system, knocking out the gene encoding TT2 by homologous recombination, or performing a loss-of-function mutation on TT2 in a plant containing TT2.

[0014] In one or more embodiments, in (b), downregulating the expression or activity of SCT1 / SCT2 includes: knocking out or silencing the coding gene for SCT1 / SCT2 in a plant, or inhibiting the activity of SCT1 / SCT2; preferably, it includes: silencing SCT1 / SCT2 with an interfering molecule that specifically interferes with the expression of the coding gene for SCT1 / SCT2, knocking out the coding gene for SCT1 / SCT2 by gene editing using a CRISPR system, knocking out the coding gene for SCT1 / SCT2 by homologous recombination, or performing a loss-of-function mutation on SCT1 / SCT2 in a plant containing SCT1 / SCT2.

[0015] In one or more embodiments, in (c), the reintroduction of WR2 includes: transferring the encoding gene of WR2 or an expression construct or vector containing the encoding gene into a plant; mutating the WR2 promoter to eliminate the SCT1 / SCT2 binding site; promoting WR2 expression with a constitutive promoter or a tissue-specific promoter; or promoting WR2 expression with an enhancer.

[0016] In one or more embodiments, in (a), downregulating the expression or activity of TT2 includes: knocking out the coding gene for TT2, thereby expressing an inactive protein or not expressing the protein; preferably, mutating nucleotide C to A at position 165 of the coding gene for TT2, thereby forming a TGA and prematurely terminating the expression of TT2 protein, or knocking out positions 1 to 40 and positions 85 to 90 of the coding gene for TT2; inserting a nucleotide at position 19 and knocking out positions 87 to 90; knocking out nucleotide 20 and mutating nucleotide A to T at position 91, thereby causing a frameshift in the coding region of TT2, thereby forming a stop codon and prematurely terminating the expression of TT2 (TT2). KO ).

[0017] In one or more embodiments, in (b), downregulating the expression or activity of SCT1 / SCT2 includes: knocking out positions 267 to 268 of the SCT1 coding gene; knocking out positions 219 to 253; inserting one nucleotide at position 222, thereby causing a frameshift in the coding region of SCT1 and resulting in premature termination; knocking out nucleotides 218-248 of the SCT2 coding gene; inserting one nucleotide at position 231; and knocking out nucleotides 229-231, thereby causing disruption or frameshift in the coding region of SCT2 (SCT1 / SCT2-knockout).

[0018] In another aspect of the present invention, the use of a regulator of the TT2-SCT1 / SCT2-WR2 signaling pathway or thereof is provided for enhancing the heat tolerance of gramineous plants or protecting (maintaining) the yield of gramineous plants under high temperatures; wherein the regulator comprises: (a) a downregulator that downregulates the expression or activity of TT2; (b) a downregulator that downregulates the expression or activity of SCT1 / SCT2; and / or (c) a reagent that blocks the regulation of WR2 by SCT1 / SCT2 to maintain WR2 homeostasis (normal expression level / activity).

[0019] In one or more embodiments, the downregulatory agent for downregulating the expression or activity of TT2 includes: a reagent that knocks out or silences TT2, a reagent that inhibits the activity of TT2; preferably, it includes: an interfering molecule that specifically interferes with the expression of the gene encoding TT2, a CRISPR gene editing reagent, a homologous recombination reagent, or a site-directed mutagenesis reagent targeting TT2, wherein the reagent performs a loss-of-function mutation on TT2.

[0020] In one or more embodiments, the downregulators that downregulate the expression or activity of SCT1 / SCT2 include: agents that knock out or silence SCT1 / SCT2, agents that inhibit the activity of SCT1 / SCT2; preferably, they include: interfering molecules that specifically interfere with the expression of the genes encoding SCT1 / SCT2, CRISPR gene editing reagents, homologous recombination reagents, or site-directed mutagenesis reagents targeting SCT1 / SCT2, wherein the reagents induce loss-of-function mutations in SCT1 / SCT2.

[0021] In one or more embodiments, the reagent for maintaining WR2 homeostasis includes a reagent for replenishing WR2; preferably, it includes the coding gene for WR2 or an expression construct containing the coding gene; or a reagent for performing gain-of-function mutations on WR2.

[0022] In one or more embodiments, the grasses include cereal crops, or the TT2-SCT1 / SCT2-WR2 signaling pathway or its pathway genes are derived from cereal crops; preferably, the grasses include (but are not limited to): rice (Oryza sativa), maize (Zea mays), millet (Setaria italica), barley (Hordeum vulgare), wheat (Triticum aestivum), sorghum (Panicum miliaceum), sorghum (Sorghumbicolor), rye (Secale cereale), oats (Avena sativa L.), and brachypodium distachyum.

[0023] In one or more embodiments, the TT2, SCT1, SCT2, or WR2 includes a cDNA sequence, a genomic sequence (gDNA), or a sequence that is artificially optimized or modified based on them.

[0024] In one or more embodiments, the rice is selected from the group consisting of indica rice and japonica rice.

[0025] In one or more embodiments, the proteins or genes in the TT2-SCT1 / SCT2-WR2 signaling pathway include their homologs.

[0026] In one or more embodiments, the amino acid sequence of the TT2 polypeptide is selected from the group consisting of: (i) a polypeptide with the amino acid sequence shown in SEQ ID NO:5; (ii) a polypeptide derived from (i) having the regulatory trait function formed by substituting, deleting, or adding one or more (e.g., 1-20, 1-10, 1-5, 1-3) amino acid residues of the amino acid sequence shown in SEQ ID NO:5; (iii) a polypeptide having the regulatory trait function with an amino acid sequence homology ≥80% (preferably ≥85%, ≥90%, ≥95%, or ≥98%) to the amino acid sequence shown in SEQ ID NO:5; (iv) an active fragment of a polypeptide with the amino acid sequence shown in SEQ ID NO:5; or, (v) a polypeptide formed by adding a tag sequence or restriction enzyme site sequence to the N or C end of the polypeptide with the amino acid sequence shown in SEQ ID NO:5, or by adding a signal peptide sequence to its N end.

[0027] In one or more embodiments, the amino acid sequence of the SCT1 polypeptide is selected from the group consisting of: (i) a polypeptide with the amino acid sequence shown in SEQ ID NO:10; (ii) a polypeptide derived from (i) having the regulatory trait function formed by substituting, deleting, or adding one or more (e.g., 1-20, 1-10, 1-5, 1-3) amino acid residues of the amino acid sequence shown in SEQ ID NO:10; (iii) a polypeptide having the regulatory trait function with an amino acid sequence homology ≥80% (preferably ≥85%, ≥90%, ≥95%, or ≥98%) to the amino acid sequence shown in SEQ ID NO:10; (iv) an active fragment of a polypeptide with the amino acid sequence shown in SEQ ID NO:10; or, (v) a polypeptide formed by adding a tag sequence or restriction enzyme site sequence to the N or C end of the polypeptide with the amino acid sequence shown in SEQ ID NO:10, or by adding a signal peptide sequence to its N end.

[0028] In one or more embodiments, the amino acid sequence of the SCT1 polypeptide is selected from the group consisting of: (i) a polypeptide with the amino acid sequence shown in SEQ ID NO:10; (ii) a polypeptide derived from (i) having the regulatory trait function formed by substituting, deleting, or adding one or more (e.g., 1-20, 1-10, 1-5, 1-3) amino acid residues of the amino acid sequence shown in SEQ ID NO:10; (iii) a polypeptide having the regulatory trait function with an amino acid sequence homology ≥80% (preferably ≥85%, ≥90%, ≥95%, or ≥98%) to the amino acid sequence shown in SEQ ID NO:10; (iv) an active fragment of a polypeptide with the amino acid sequence shown in SEQ ID NO:10; or, (v) a polypeptide formed by adding a tag sequence or restriction enzyme site sequence to the N or C end of the polypeptide with the amino acid sequence shown in SEQ ID NO:10, or by adding a signal peptide sequence to its N end.

[0029] In one or more embodiments, the amino acid sequence of the SCT2 polypeptide is selected from the group consisting of: (i) a polypeptide with the amino acid sequence shown in SEQ ID NO:14; (ii) a polypeptide derived from (i) having the regulatory trait function formed by substituting, deleting, or adding one or more (e.g., 1-20, 1-10, 1-5, 1-3) amino acid residues of the amino acid sequence shown in SEQ ID NO:14; (iii) a polypeptide having the regulatory trait function with an amino acid sequence homology ≥80% (preferably ≥85%, ≥90%, ≥95%, or ≥98%) to the amino acid sequence shown in SEQ ID NO:14; (iv) an active fragment of a polypeptide with the amino acid sequence shown in SEQ ID NO:14; or, (v) a polypeptide formed by adding a tag sequence or restriction enzyme site sequence to the N or C end of the polypeptide with the amino acid sequence shown in SEQ ID NO:14, or by adding a signal peptide sequence to its N end.

[0030] In one or more embodiments, the amino acid sequence of the WR2 polypeptide is selected from the group consisting of: (i) a polypeptide with the amino acid sequence shown in SEQ ID NO:18; (ii) a polypeptide derived from (i) having the regulatory trait function formed by substituting, deleting, or adding one or more (e.g., 1-20, 1-10, 1-5, 1-3) amino acid residues of the amino acid sequence shown in SEQ ID NO:18; (iii) a polypeptide having the regulatory trait function with an amino acid sequence homology ≥80% (preferably ≥85%, ≥90%, ≥95%, or ≥98%) to the amino acid sequence shown in SEQ ID NO:18; (iv) an active fragment of a polypeptide with the amino acid sequence shown in SEQ ID NO:18; or, (v) a polypeptide formed by adding a tag sequence or restriction enzyme site sequence to the N or C end of the polypeptide with the amino acid sequence shown in SEQ ID NO:18, or by adding a signal peptide sequence to its N end.

[0031] In another aspect of the invention, the use of the TT2-SCT1 / SCT2-WR2 signaling pathway and the pathway genes therein is provided for use as molecular markers for identifying the heat tolerance of grass plants or the yield of grass plants under high temperatures.

[0032] In another aspect of the invention, the use of the TT2-SCT1 / SCT2-WR2 signaling pathway and the pathway genes therein is provided as a molecular marker for targeted screening of grasses that have heat tolerance or maintain yield at high temperatures.

[0033] In another aspect of the present invention, the use of the TT2-SCT1 / SCT2-WR2 signaling pathway and the pathway genes therein is provided for screening substances that improve the heat tolerance of gramineous plants or maintain yield at high temperatures.

[0034] In another aspect of the invention, a method for targeted selection of grass plants is provided, comprising: identifying the TT2-SCT1 / SCT2-WR2 signaling pathway and the expression or sequence characteristics or interactions of pathway genes therein in a test plant; if the test plant has one or more characteristics selected from the group consisting of: low expression or no expression of TT2 in the test plant; low expression or no expression of SCT1 / SCT2 in the test plant; and stable WR2 in the test plant.

[0035] In one or more embodiments, the method for selecting plants can be carried out at high temperature or at room temperature.

[0036] In one or more embodiments, the low expression or low activity refers to a statistically significant reduction in expression or activity compared to the average expression or activity of similar or identical plants.

[0037] In one or more embodiments, high expression or high activity means a statistically significant increase in expression or activity compared to the average expression or activity of similar or identical plants.

[0038] In another aspect of the present invention, a method is provided for screening substances (potential substances) that improve the heat tolerance of gramineous plants or maintain yield at high temperatures, comprising: (1) adding candidate substances to a system expressing the TT2-SCT1 / SCT2-WR2 signaling pathway; (2) detecting the system and observing the expression or activity of the TT2-SCT1 / SCT2-WR2 signaling pathway proteins therein: if the expression or activity of TT2 is reduced, then the candidate substance is a substance that improves the heat tolerance of gramineous plants or maintains yield at high temperatures; if the expression or activity of SCT1 and / or SCT2 is reduced, then the candidate substance is a substance that improves the heat tolerance of gramineous plants or maintains yield at high temperatures; if the addition of the candidate substance promotes the maintenance of WR2 homeostasis, then the candidate substance is a substance that improves the heat tolerance of gramineous plants or maintains yield at high temperatures.

[0039] In one or more embodiments, a control group is also included to clearly distinguish the difference in expression or activity of the target gene / protein between the test group and the control group.

[0040] In one or more embodiments, the candidate substances include (but are not limited to): regulatory molecules (such as upregulators, small molecule compound gene editing constructs, etc.) designed to target TT2, SCT1, SCT2 proteins or their encoding genes or their upstream or downstream proteins or genes.

[0041] In another aspect of the invention, a downregulator of the TT2-SCT1 / SCT2-WR2 signaling pathway gene is provided for improving the heat tolerance of gramineous plants or maintaining yield at high temperatures. This downregulates the expression of TT2, SCT1 and / or SCT2 using a CRISPR gene editing reagent (such as a construct / expression vector).

[0042] In one or more embodiments, the CRISPR gene-editing reagent targets position 165 of the TT2 coding gene, causing a mutation of nucleotide C to A, thereby forming a TGA and prematurely terminating TT2 protein expression; or knocking out positions 1 to 40 and positions 85 to 90 of the TT2 coding gene; inserting a nucleotide at position 19; knocking out positions 87 to 90; knocking out nucleotide 20; and mutating nucleotide A to T at position 91, thereby causing a frameshift in the TT2 coding region, thus forming a stop codon and prematurely terminating TT2 expression. KOOr targeting the expression or activity of SCT1 / SCT2 includes: knocking out positions 267 to 268 of the SCT1 coding gene; knocking out positions 219 to 253; inserting one nucleotide at position 222, thereby causing a frameshift in the coding region of SCT1 and resulting in premature termination; knocking out nucleotides 218-248 of the SCT2 coding gene; inserting one nucleotide at position 231; knocking out nucleotides 229-231, thereby causing disruption or frameshift in the coding region of SCT2 (SCT1 / SCT2-knockout).

[0043] In another aspect of the invention, a construct (expression vector) or plant cell, tissue or organ is provided, wherein the construct contains an exogenous downregulator of the TT2-SCT1 / SCT2-WR2 signaling pathway gene described above, which is used to improve the heat tolerance of grasses or maintain yield at high temperatures.

[0044] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0045] Figure 1 Map-based cloning and transgenic verification of TT2.

[0046] (A) Heat resistance phenotypes of HPS32 replacement line and recurrent parent HJX (42℃, RH>90%, 24hr), scale bar 4cm. (B) Heat resistance phenotypes of recombinant individuals with four local fragments (42℃, RH>90%, 24hr). (C) The TT2 site was defined within 25.22 kb; black arrows indicate ORF; the right-hand bar chart represents the survival rate of the corresponding recombinant individuals, n=3 samples. (D) Knockout of TT2 in the context of Chinese japonica rice resulted in superior heat stress tolerance compared to the control HJX, scale bar 4cm. (E) In NIL-TT2... HPS32 Overexpression of TT2 in the context of NIL-TT2 caused loss of heat stress tolerance, scale bar 4cm. (F) In NIL-TT2 HPS32 In the context of complementary full-length TT2 gDNA, its heat stress tolerance is lost. Scale bar 4cm. (G)NIL-TT2 HJX and NIL-TT2 HPS32 Phenotypic identification at maturity (13hr, 38℃, daytime / 11hr, 35℃, nighttime; 12 days, recovery 7 days), scale bar 10cm. (H) Comparison of grain length between different transgenic lines. (I) TT2 KO / HJX、pUbi:TT2 HJX / NIL-TT2 HPS32 pTT2:TT2 HJX / NIL-TT2 HPS32Survival rates were recorded after heat treatment and a 7-day recovery period, with n=6 samples. Each sample contained 24 independent plants. Data were expressed as Mean ± SD. Student's t-test was used for significance testing, with * indicating P < 0.05 (significant difference) and ** indicating P < 0.01 (extremely significant difference); or one-way ANOVA was used, with the same letter indicating P > 0.05. In the figure, the normal condition is a temperature of 28℃ (the same applies below).

[0047] Figure 2 TT2 HPS32 The replacement segment is from tropical japonica rice.

[0048] (A) The origin of the substitution fragment in the substitution line HPS32 was determined based on chr3:16662761–17212811, 550 kb. Data were obtained from the rice 3000 genome. The inventors indicated the fragment origin of HPS32 by the base difference frequency at each SNP site under different backgrounds. (B) The frequency differences of two bases at key SNP sites in different rice subspecies are presented as a bar chart. (C) Identification of heat resistance phenotypes in 10 tropical japonica rice species (GP5, 7, 55, 86, 112, 536, 629, 640, 641, and 642), scale bar, 4 cm. (D) Identification of heat resistance phenotypes in 6 temperate japonica rice species (WYJ:Wuyunjing 7; KY131:Kongyu131; ZH11:Zhonghua11; Jiahua1; NIP:Nipponpare; and Xiushui09). Scale bar, 4cm.

[0049] Figure 3 TT2 HPS32 Protecting rice yield under heat stress.

[0050] (A) Ear phenotype of NILs (13hr, 38℃, daytime / 11hr, 35℃, nighttime; 12 days), scale bar 3cm. (B) NIL-TT2 HJX and NIL-TT2 HPS32 Yield per plant under normal conditions and heat stress. (C, D)NIL-TT2 HJX and NIL-TT2 HPS32 Grain set rate and thousand-grain weight under normal conditions and heat stress. (E) NIL-TT2 after heat treatment. HJX and NIL-TT2 HPS32I2-KI staining, scale bar 0.1 mm. (F) Agronomic traits of NILs under normal conditions were investigated. Plant height (n=48), grain length (n=15), grain width (n=15), number of grains per ear (n=~26), and number of ears per plant (n=~26) were measured as Mean±SD, with NIL-TT2 as the criterion. HJX For comparison, a one-way ANOVA test was used to determine the significance of the difference. The same letter indicates P>0.05.

[0051] Figure 4 TT2 is involved in the regulatory pathways related to wax synthesis.

[0052] (A) Gene set enrichment analysis based on KEGG annotation (listing the top ten by NES score), where ES is the enrichment score and NES is the enrichment score after gene set size normalization. For gene set enrichment analysis of the keratin, cork ester, and wax synthesis pathways, green indicates the enrichment score of this gene set; black lines represent each gene in this gene set and their corresponding ordinal position; and red lines represent the enrichment score of NIL-TT2 under heat stress. HPS32 A positive correlation is shown; the blue line represents the correlation between NIL-TT2 and heat stress. HJX (A) Positive correlation. (B) Cluster analysis under different treatment conditions and different NIL backgrounds. FKPM values ​​were converted to log2(fpkm+1) and presented on the vertical axis. The changes in FPKM values ​​of each gene under different samples and treatments are shown by the gray line, and the blue line represents the average FPKM. (C) KEGG annotation analysis for subcluster 2 and subcluster 3. (D) Transcriptome analysis of differential expression of wax-related genes among near-isogenic lines under heat treatment at different times. This figure covers a total of 49 wax-related genes. (E) The heatmap shows the expression of wax-related DEGs among near-isogenic lines after heat treatment. There are 3 biological replicates, and each biological replicate contains 24 independent rice seedlings. (F) Differences in wax components among different treatment conditions, i.e., different near-isogenic lines. Wax components were extracted from the samples and analyzed by GC-MS. The heatmap shows wax metabolites with significant differences. (G) Scanning electron microscopy observation of the waxy crystal state on the leaf surface of different near-isogenic lines under treatment times of 0, 12, 20, and 24 hours. Scale bar: 1 μm. The right figure shows the corresponding heat-resistant phenotype under different treatment times and after 7 days of recovery. Scale bar: 5 cm. (H) qPCR identification of heat-induced changes in OsWR2 expression.

[0053] Figure 5 OsWR2 acts downstream of the TT2-regulated heat resistance pathway.

[0054] (A) In NIL-TT2 HPS32 (B) Knockout of OsWR2 in the context of NIL-TT2, compared with the knockout result of wild type. HPS32 OsWR2 knockout transgenic plants (wr2 / NIL-TT2) in the context of HPS32 ) and NIL-TT2 HPS32 (C) Survival statistics of transgenic plants and their wild-type plants under heat stress. (D) Toluidine blue staining to characterize the permeability of the plant epidermis. Leaves of 12-day-old seedlings were stained in TB solution for 1 hour under heat conditions, and then excess staining was washed off. The darker the color, the stronger the permeability of the epidermis. Scale bar, 2cm.

[0055] Figure 6 SCT1 is a promoter that transcribes the factor OsWR2.

[0056] (A) Homologous gene evolution analysis of SCT1 in crops (rice, wheat, maize, soybean) and Arabidopsis thaliana. (B) In vitro chip-qPCR experiments demonstrated that the Halo-SCT1-NT protein can bind to the promoter region of OsWR2. This CG1-like element is located approximately 293 bp upstream of the OsWR2 transcription start site. The black line indicates the region amplified by qPCR. The enrichment fold was used as an internal control by the input, and the empty Halo vector protein served as a control. (C) Gel migration assays demonstrated that SCT1 / SCT2 can bind to CG1-like cis elements. The sequences of normal and mutant probes are shown at the top of the figure. Each probe is labeled with cy5 at the 5' end. The binding specificity was detected by adding unlabeled cold probes (5x, 25x, 50x). (D) Transient expression of SCT1-YFP, SCT2-YFP, and CaM-mCherry in tobacco. Scale bar: 20 μm.

[0057] Figure 7 SCT1 negatively regulates wax synthesis and rice heat resistance.

[0058] (A) Heat resistance phenotype of sct1 / sct2 mutants and wild-type ZH11 recovered for 7 days after heat treatment. Scale bar, 5 cm. (B) Heat resistance phenotype of sct1 / sct2 mutants and ZH11 during the grain-filling stage, recovered for 5 days after 7 days of treatment at 37℃ / 33℃. Scale bar, 10 cm. (C) SCT1 / SCT2 knockout in the ZH11 background, comparison of knockout genomic DNA with wild-type. (D) Survival rate statistics of gene-treated plants and corresponding wild-type plants after heat treatment, 3 samples, each containing 24 independent rice seedlings. Data are Mean ± SD, one-way ANOVA analysis, P>0.05 is indicated by the same letter. (E) qPCR detection of OsWR2 expression patterns in sct1 / sct2 mutants and wild-type ZH11 under normal conditions and heat stress, 3 biological replicates. Each sample contained 24 independent rice seedlings. Data are Mean ± SD, Student's t-test analysis, and p-values ​​after analysis are labeled on the figure. (F) Transcriptome sequencing data: KEGG-based analysis showed that wax-related regulatory pathways were enriched in differentially expressed genes between mutants and wild-type under untreated conditions, with 3 biological replicates. Each biological replicate contained 24 independent rice seedlings. (G) Under heat treatment conditions, GC-MS showed the changes in wax content and composition between the sct1 / sct2 mutant and wild-type ZH11, with 4 biological replicates.

[0059] Figure 8 TT2 is involved in regulating intracellular calcium signaling under heat stimulation.

[0060] (A) SIET was used to detect changes in calcium flux in the root mesophyll zone (n=6) and aboveground mesophyll cells (n=6) of rice under heat stimulation. Data are Mean±SEM, with NIL-TT2 as the index. HJX (B) Quantitative statistical analysis of the calcium flow velocity change curve of NILs under thermal stimulation conditions as measured by SIET, using the integral (net area of ​​the curve) over time. Data are Mean ± SD, with NIL-TT2 as the control. HJX (C) Statistical analysis of maximum calcium influx in rice under heat stimulation using SIET. Data are Mean ± SD, expressed as NIL-TT2. HJX (D) Dynamic changes in intracellular calcium induced by root and intermediate heat, based on NES-YC3.6 transgenic material. Intracellular calcium was assessed using a normalized fluorescence intensity ratio (cpVenus / ECFP), data are mean ± SEM, with NIL-TT2 as the control. HJX For control. (E) Peak area (with Y=1 as baseline). (F) Peak value (cpVenus / ECFP). n=6, data are Mean±SD, with NIL-TT2. HJX(G) Dynamic changes in intracellular calcium concentration induced by heat in the whole seedling, based on transgenic material of UBQ10-aequorin. n = 21–22, data are Mean ± SEM, with NIL-TT2 as the main indicator. HJX For control. (H) Integral calcium concentration curve, with the resting calcium concentration before treatment as the baseline, the net peak area under the curve was calculated, and the data are Mean ± SD. n = 21–22. (I) Δ[Ca 2+ ] cyt peak-rest ([Ca 2+ ] cyt peak -[Ca 2+ ] cyt rest Significance analysis of the quantitative results. Data are Mean±SD, n=21~22.

[0061] Figure 9 CaM / SCT1 responds to calcium signaling by negatively regulating the expression of downstream OsWR2.

[0062] (A) Yeast two-hybrid assay: Empty vector AD, truncated SCT1 (ΔCaMBD) (1-670aa), full-length SCT1, and CaM-BD were co-transformed into the Y2HGold yeast strain. Empty vector AD served as a negative control. (B) SFLC assay to verify the interaction between SCT1 and CaM in tobacco, with empty vector serving as a negative control. (C) Pull-down assay to detect the interaction between SCT1-CaMBD and GST-CaM or GST-Hal3, with GST-Hal3 serving as a negative control. Antibodies for GST and MBP were used for Western blot detection. (D) SPR assay to detect the interaction between SCT1 and CaMBD under conditions with and without calcium or EGTA. The baseline signal value represents the intensity of the interaction between the two proteins, and the KD value is calculated based on the signal curves of CaM at three gradient concentrations. (E) Gel migration assay of the CaMBD core region protein peptide in SCT1: The peptide synthesized from amino acids 897-917 of SCT1 was selected. A gradient molar ratio of peptide to CaM was set, and the experiment was performed using 0.1 mM CaCl2 and 2 mM EGTA electrophoresis buffer. Samples were separated by non-denaturing PAGE and stained with Coomassie brilliant blue. In the mutated peptide region, valine at position 900 and tryptophan at position 907 were mutated to arginine. (F) Transcriptional regulation of SCT1 by OsWR2: Five copies of the 16 bp OsWR2 promoter region (containing the ACGCGC element) were used as a reporter vector. CaM inhibited the transcriptional activity of SCT1 in a CaM concentration-dependent manner. The LUC / REN ratio was normalized to 1 when transformed into the empty vector effector. (G) Mutant CaM 1234 Lacking the ability to bind calcium, it also failed to inhibit the transcriptional activation activity of SCT1. (H) Inhibition of Ca2+ in rice protoplasts by lanthanum ion treatment 2+ Dual fluorescence detection experiments under normal and heat treatment conditions. The reporter system was 5 copies of UAS, and the effector was Gal4-SCT1. (I) Expression patterns of OsWR2 under normal and heat treatment conditions, as well as under treatment with water, magnesium chloride, and lanthanum chloride. 100 μM MgCl2 was used as a negative control, and two different concentrations of LaCl3 were used to inhibit calcium channels. Detailed Implementation

[0063] This invention is the first to study and reveal a novel signaling pathway involved in plant heat tolerance: the TT2-SCT1 / SCT2-WR2 signaling pathway. TT2 acts as a negative regulator of plant heat tolerance or the protection (maintenance) of yield in gramineous plants under high temperatures. SCT1 / SCT2 also act as negative regulators of plant heat tolerance or the protection (maintenance) of yield in gramineous plants under high temperatures. WR2, on the other hand, is a positive regulator that plays a beneficial role in maintaining homeostasis in plants. This invention has significant theoretical implications for the genetic improvement of plant traits.

[0064] the term

[0065] As used herein, the terms “heat resistance capacity / heat resistance”, “heat stress tolerance” and “heat resistance capacity / heat resistance” are used interchangeably.

[0066] As used in this article, the terms "signaling pathway" and "regulatory pathway" are interchangeable.

[0067] As used herein, "plant" refers to a plant whose genome contains the TT2-SCT1 / SCT2-WR2 signaling pathway of the present invention or homologs (homologous genes / homologous polypeptides (proteins)) of genes / proteins involved in this signaling pathway. The plant may include monocotyledonous or polycotyledonous plants, such as: grasses, cruciferous plants, legumes, etc. Preferably, grasses are included. In some preferred embodiments, the plant is a crop, preferably a cereal crop. Preferably, the grasses include rice (Oryza sativa), wheat (Triticum aestivum), and maize (Zea spp.). Examples include: rice, sorghum, maize, barley, wheat, oats, and rye. Those skilled in the art will understand that the plants applicable to the technical solutions of the present invention are not limited to those listed above, and suitable plants can be determined by identifying the presence of the TT2-SCT1 / SCT2-WR2 signaling pathway.

[0068] As used herein, "maintaining yield at high temperatures," "protecting the yield of gramineous plants at high temperatures," or "maintaining the yield of gramineous plants at high temperatures" refers to the survival and normal growth of plants in a high-temperature environment (temperatures higher than their conventional cultivation conditions), resulting in a relatively normal or essentially normal yield. In some embodiments, "maintaining yield at high temperatures" means that, after a specific plant is cultivated in a high-temperature environment, its yield is maintained at more than 30%, 40%, 50%, or 60% of the yield in a "non-high-temperature environment (normal / suitable temperature environment)," and preferably at more than 70%, 80%, or 90% of the yield in a "non-high-temperature environment."

[0069] As used herein, “high temperature” or “heat (environment)” refers to a temperature significantly higher than the suitable temperature for plant growth (e.g., 26–30°C for rice, with 28–30°C being the optimal temperature); for example, “high temperature” or “heat (environment)” refers to 35°C or higher, 38°C or higher, 40°C or higher, or 42°C or higher.

[0070] As used in this article, in the term "TT2-SCT1 / SCT2-WR2 signal path", "SCT1 / SCT2" means "SCT1 and SCT2" or "SCT1 or SCT2". SCT1 and / or SCT2 are highly homologous and have a certain degree of functional redundancy.

[0071] As used herein, “upregulation” includes: promotion, overexpression, enhancement, etc., which are statistically significant or marked upregulation, promotion, enhancement, or enhancement, such as upregulation, promotion, enhancement, or enhancement of 20%, 40%, 60%, 80%, 90%, or higher.

[0072] As used herein, “downregulation” includes: weakening, reducing, lowering, inhibiting; indicating significant downregulation, weakening, lowering, inhibiting, such as downregulation, weakening, lowering, inhibiting or downregulating by 20%, 40%, 60%, 80%, 90% or lower.

[0073] As used herein, "heat tolerance" refers to a plant's ability to withstand hot environments. Typically, the heat tolerance of a test plant is determined by comparing it to a control plant under the same temperature conditions. A test plant is generally considered to have better heat tolerance if it has a higher survival rate and longer survival time at higher temperatures.

[0074] As used herein, “high heat tolerance” refers to a statistically significant increase in the heat tolerance of a plant (e.g., a modified plant) compared to that of similar or identical plants, such as an increase in survival rate of 5%, 10%, 20%, 40%, 60%, 80%, 90%, or higher.

[0075] As used herein, “high expression or high activity” means that the expression or activity of a target gene / protein in a specific plant (e.g., a modified plant) is statistically significantly increased compared to the average expression or activity of the same type or plant species, such as by 10%, 20%, 40%, 60%, 80%, 90%, or higher.

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

[0077] As used herein, "loss-of-function mutations" include those that cause a target protein to lose its function, such as through mutations, deletions, or insertions in key regions of its protein chain. In some approaches, gene editing involves the insertion, deletion, or mutation of bases in the gene encoding the target protein, causing premature termination of translation of the target protein.

[0078] As used herein, “gain-of-function mutations” include: enabling the normal expression of a target protein that was previously restricted or not expressed; and in some ways, reverting the expression of the target protein by reversing bases in the target protein coding gene (e.g., reverting to a sequence identical or degenerate to the wild type) through gene editing.

[0079] As used herein and as will be understood by those skilled in the art, selecting an appropriate “control plant” is a routine part of experimental design and may include a corresponding wild-type plant or a transgenic plant without the target gene. Control plants are generally the same plant species or even varieties of the same or the same class as the plant being evaluated. Control plants may also be individuals from which the transgenic plant has been lost due to segregation. As used herein, control plants refer not only to whole plants but also to plant parts, including seeds and seed portions.

[0080] TT2-SCT1 / SCT2-WR2 signaling pathway

[0081] In this invention, the term "(signaling) pathway" refers to a signaling system formed by the mutual restraint or interaction of a series of proteins or genes, which generally leads to the occurrence of certain cellular events. The TT2-SCT1 / SCT2-WR2 signaling pathway includes (but is not limited to): the TT2 (THERMOTOLERANCE 2) gene (and / or its encoded protein), SCT1 / SCT2 (Ca... 2+ Sensing transcription factor 1 / 2 (and / or its encoded protein), WR2 gene (and / or its encoded protein).

[0082] The full-length gene sequence of TT2 is shown in SEQ ID NO:1, its coding region sequence is shown in SEQ ID NO:3, and its protein sequence is shown in SEQ ID NO:6.

[0083] The full-length sequence of the SCT1 gene is shown in SEQ ID NO:7, for example; its coding region sequence is shown in SEQ ID NO:9, for example; and its protein amino acid sequence is shown in SEQ ID NO:10, for example.

[0084] The full-length sequence of the SCT2 gene is shown in SEQ ID NO:11; its coding region sequence is shown in SEQ ID NO:13; and its protein amino acid sequence is shown in SEQ ID NO:14.

[0085] The full-length sequence of the WR2 gene is shown in SEQ ID NO:15, for example; its coding region sequence is shown in SEQ ID NO:17, for example; and its protein amino acid sequence is shown in SEQ ID NO:18, for example.

[0086] When used as targets for artificial regulation or in the creation of screening systems, the proteins or encoding genes mentioned above can be naturally occurring, such as those purified and isolated from mammals; or they can be recombinantly prepared, for example, recombinant proteins can be produced using conventional gene recombination techniques. Furthermore, any variations that do not affect the biological activity of these proteins are acceptable, such as derivatives or variants whose function remains unchanged.

[0087] The variant forms of the signaling pathway proteins described above are also included in this invention, including (but not limited to): deletions, insertions, and / or substitutions of several amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, and even more preferably 1-8 or 1-5), and additions or deletions of one or more amino acids (typically up to 20, preferably up to 10, and more preferably up to 5) at the C-terminus and / or N-terminus. Any protein with high homology to the described polypeptide (e.g., 70% or higher homology to the polypeptide sequence shown in SEQ ID NO:4; preferably 80% or higher; more preferably 90% or higher, such as 95%, 98%, or 99% homology) and having the same function as the polypeptide is also included in this invention. Peptides derived from species other than rice that have high homology with the peptide sequence or play the same or similar role in the same or similar regulatory pathways are also included in this invention.

[0088] Homologs of the signaling pathway genes / proteins described above are also included in this invention. It should be understood that while this invention preferably studies corresponding signaling pathway genes / proteins obtained from specific rice species, other polypeptides or genes obtained from other species that are homologous to the aforementioned signaling pathway genes / proteins (e.g., having more than 60%, such as 70%, 80%, 85%, 90%, 95%, or even 98% sequence identity) are also within the scope of this invention.

[0089] The present invention also includes a truncated mutant form of the TT2 polypeptide, wherein the gene sequence encoding it is mutated at position 165 corresponding to SEQ ID NO:3, forming a sequence as shown in SEQ ID NO:4, thereby significantly truncating the TT2 polypeptide mutant. The truncated mutant does not possess the function of the wild-type TT2 polypeptide, and plants containing this mutation exhibit significantly altered heat tolerance, transforming into heat-tolerant plants.

[0090] The polynucleotide (gene) encoding the polypeptide can be a natural gene from a plant, or it can be a degenerate sequence or a codon-optimized sequence.

[0091] Vectors containing the said coding sequence, and host cells genetically engineered using the said vector or polypeptide coding sequence, are also included in this invention. Methods well known to those skilled in the art can be used to construct suitable expression vectors.

[0092] The host cell is usually a plant cell. Transformation of plants can generally be achieved using methods such as Agrobacterium-mediated transformation or gene gun transformation, for example, the leaf disc method or rice embryo transformation; Agrobacterium-mediated transformation is preferred. Transformed plant cells, tissues, or organs can be regenerated into plants using conventional methods, thereby obtaining plants with altered traits compared to the wild type.

[0093] Plant Transformation

[0094] In this invention, the term "plant" includes plants that express TT2 or contain the TT2-SCT1 / SCT2-WR2 signaling pathway. According to knowledge in the art, plants possessing TT2 or containing the TT2-SCT1 / SCT2-WR2 signaling pathway inherently possess the mechanism of action claimed in this invention, and can achieve the technical effects claimed in this invention.

[0095] In a specific embodiment of the present invention, a set of chromosome segment replacement lines (CSSLs) was constructed using a heat-resistant rice variety as the donor parent and the high-yielding Huajingxian 74 (HJX) as the recurrent parent. Through heat resistance phenotype identification and screening of the CSSLs, a heat-resistant line, HPS32, was selected. The inventors crossed HPS32 with HJX to construct an F2 localization population and discovered a QTL locus that significantly affects rice heat resistance, which was named TT2. Using map-based cloning, the TT2 gene controlling the heat resistance phenotype in rice was successfully located. Sequencing revealed that TT2 originated from the HPS32 parent. HPS32A mutation at nucleotide position 165 of the gene, from C to A, leads to premature termination of the TGA gene, thus encoding a truncated protein. Transgenic identification confirmed that TT2 is indeed a functional gene controlling heat tolerance in rice. Sequence alignment analysis confirmed that TT2... HPS32 The locus is derived from a tropical japonica rice variety. TT2 encodes a γ subunit of a G protein. The inventors constructed and bred a near-isogenic line, NIL-TT2, carrying the TT2 gene locus from HPS32. HPS32 And its corresponding control NIL-TT2 HJX The inventors conducted heat resistance assessments on the mature phenotypes of NILs and found that TT2... HPS32 High temperatures can protect rice yield, especially the seed setting rate, ultimately resulting in a 54.6% increase in yield compared to the control. TT2 HPS32 It weakens heat-induced transcriptional responses and differentially expressed genes are enriched in pathways related to wax synthesis. Studies at the level of wax metabolism have revealed that heat affects NIL-TT2... HJX Induced wax levels decreased, but in NIL-TT2 HPS32 Moderate heat does not induce a decrease in wax content, thus enhancing heat resistance. Transgenic analysis of WR2, an important heat-responsive gene positively regulating wax content, revealed that in heat-resistant NIL-TT2... HPS32 Knocking out WR2 results in the loss of its thermostable phenotype. Through analysis of the WR2 promoter elements, the inventors screened a transcription factor, SCT1. Further research revealed that SCT1 can act as a calcium sensor, its transcriptional regulatory activity is influenced by calmodulin, and the interaction between calmodulin and SCT1 is affected by intracellular calcium concentration. TT2 participates in the increase of intracellular calcium flux under heat stimulation; loss of function of TT2... HPS32 This process is weakened. SCT1 can sense differences in intracellular calcium concentration induced by heat stimulation in the context of different NILs and ultimately reflect them in the regulation of WR2 transcription levels.

[0096] SCT1 / SCT2 can act as a calcium ion sensor; changes in intracellular calcium ion concentration are recognized and bound by CaM, which records the signal and forms Ca2+. 2+ The form of -CaM promotes the interaction between CaM and SCT1 and inhibits the transcriptional activity of SCT1, thereby leading to a decrease in the expression of downstream genes such as OsWR2, which in turn regulates the content of waxes and heat resistance.

[0097] Therefore, the inventors have elucidated a regulatory pathway from extracellular heat stimulation to intracellular calcium signaling, and the decoding and transduction of calcium signaling into a transcriptional response, which ultimately affects the regulatory pathway at the level of wax metabolism, thereby regulating heat resistance.

[0098] Based on the inventor's new discovery, the present invention provides a method for improving plants, the method comprising: regulating the TT2-SCT1 / SCT2-WR2 signaling pathway in plants, thereby regulating the heat tolerance or yield of plants under high temperature.

[0099] On the one hand, a method is provided to enhance the heat tolerance of grass plants or protect (maintain) the yield of grass plants under high temperatures, including downregulating the expression or activity of TT2.

[0100] On the other hand, a method is provided to enhance the heat resistance of grass plants or protect (maintain) the yield of grass plants under high temperatures, including downregulating the expression or activity of SCT1 / SCT2.

[0101] On the other hand, a method is provided to enhance the heat resistance of gramineous plants or protect (maintain) the yield of gramineous plants under high temperature, including blocking the regulation of WR2 by SCT1 / SCT2 to maintain WR2 homeostasis.

[0102] It should be understood that, once the function of the TT2-SCT1 / SCT2-WR2 signaling pathway (optionally including its upstream and downstream genes) is known, various methods well known to those skilled in the art can be used to regulate the TT2-SCT1 / SCT2-WR2 signaling pathway. For example, various methods well known to those skilled in the art can be used to reduce or eliminate TT2 expression.

[0103] In this invention, the upregulators of the TT2-SCT1 / SCT2-WR2 signaling pathway protein or its encoding gene include promoters, agonists, and activators. The terms "upregulation" and "promotion" include "upregulation" and "promotion" of protein activity or protein expression, and these are statistically significant. Any substance that can increase the activity of the signaling pathway protein, improve the stability of the signaling pathway protein, upregulate the expression of the signaling pathway gene, increase the effective duration of the signaling pathway protein, or increase the phosphorylation / activation level of each protein can be used in this invention as a substance useful for upregulating TT2 or the signaling pathway. These substances can be compounds, small chemical molecules, or biomolecules. The biomolecules can be at the nucleic acid level (including DNA and RNA) or at the protein level.

[0104] In this invention, the downregulator of the TT2-SCT1 / SCT2-WR2 signaling pathway protein or its encoding gene refers to any substance that can reduce the activity of the TT2-SCT1 / SCT2-WR2 signaling pathway protein, reduce the stability of the signaling pathway protein or its encoding gene, downregulate the expression of other signaling pathway proteins, reduce the effective duration of the signaling pathway protein, inhibit the transcription and translation of the signaling pathway gene, or reduce the phosphorylation / activation level of each protein. These substances can all be used in this invention as substances useful for downregulating TT2. They can be compounds, small chemical molecules, or biomolecules. The biomolecules can be at the nucleic acid level (including DNA and RNA) or at the protein level. For example, the downregulator is: an interfering RNA molecule or antisense nucleotide that specifically interferes with the expression of the signaling pathway gene; or a gene editing reagent that specifically edits TT2, etc.

[0105] This invention also provides a method for downregulating TT2 or SCT1 / SCT2 in the TT2-SCT1 / SCT2-WR2 signaling pathway in plants, including targeted mutation, gene editing, or gene recombination to achieve downregulation. For TT2, as a more specific embodiment, any of the above methods can be used to convert TT2 into its truncated form, thereby altering plant traits. As a more specific embodiment, a CRISPR / Cas9 system is used for gene editing to knock out or downregulate the target gene. Suitable sgRNA target sites lead to higher gene editing efficiency; therefore, suitable target sites can be designed and identified before gene editing. After designing specific target sites, in vitro cell activity screening is required to obtain effective target sites for subsequent experiments.

[0106] As another embodiment of the present invention, a method for downregulating TT2 expression in plants is provided, comprising: (1) transferring an interfering molecule that interferes with TT2 gene expression into plant cells, tissues, organs, or seeds to obtain plant cells, tissues, organs, or seeds transformed with the interfering molecule; (2) regenerating plants from the plant cells, tissues, organs, or seeds transformed with the interfering molecule obtained in step (1). Preferably, the method further comprises: (3) selecting plant cells, tissues, or organs transformed with the vector; and (4) regenerating plants from the plant cells, tissues, or organs in step (3).

[0107] This invention reveals that WR2 plays a positive regulatory role in the aforementioned signaling pathway, with its promoter region interacting with SCT1 / SCT2 and thus being transcriptionally regulated by the latter. To maintain the stable function of WR2, the regulation of WR2 by SCT1 / SCT2 can be blocked; alternatively, the SCT1 / SCT2 binding region of the WR2 promoter region can be altered to prevent its interaction with SCT1 / SCT2. With such manipulation, changes in intracellular calcium flow under thermal stimulation will no longer affect the normal function of WR2 and will not affect the synthesis of waxes.

[0108] Plant targeted screening or selective screening

[0109] Having learned about the TT2-SCT1 / SCT2-WR2 signaling pathway and the functions of its genes / proteins, these can be used as molecular markers for targeted plant screening. This new discovery can also be used to screen for substances or potential substances that can regulate plant heat tolerance or yield at high temperatures by modulating this mechanism.

[0110] Therefore, the present invention provides a method for targeted selection or identification of plants, the method comprising: identifying the TT2-SCT1 / SCT2-WR2 signaling pathway and the expression or sequence characteristics or interactions of pathway genes therein in a test plant; if the test plant has one or more characteristics selected from the group below, the test plant is a gramineous plant with heat tolerance or maintaining yield at high temperatures: the test plant has low or no expression of TT2; the test plant has low or no expression of SCT1 / SCT2; the test plant maintains a steady state of WR2.

[0111] This invention provides a method for screening substances (potential substances) that can improve the heat resistance of gramineous plants or maintain yield at high temperatures, comprising: (1) adding candidate substances to a system expressing the TT2-SCT1 / SCT2-WR2 signaling pathway; (2) detecting the system and observing the expression or activity of the TT2-SCT1 / SCT2-WR2 signaling pathway proteins therein: if the expression or activity of TT2 decreases, then the candidate substance is a substance that can improve the heat resistance of gramineous plants or maintain yield at high temperatures; if the expression or activity of SCT1 and / or SCT2 decreases, then the candidate substance is a substance that can improve the heat resistance of gramineous plants or maintain yield at high temperatures; if the addition of the candidate substance promotes the maintenance of WR2 homeostasis, then the candidate substance is a substance that can improve the heat resistance of gramineous plants or maintain yield at high temperatures.

[0112] Methods for screening substances that act on proteins or genes or specific regions thereof as targets are well known to those skilled in the art, and these methods can all be used in this invention. The candidate substances can be selected from: peptides, polymeric peptides, peptide-like substances, non-peptide compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, small organic molecules, small inorganic molecules, and nucleic acid sequences, etc. Depending on the type of substance to be screened, those skilled in the art understand how to select an appropriate screening method.

[0113] Through large-scale screening, a class of potential substances that specifically act on the TT2-SCT1 / SCT2-WR2 signaling pathway or its pathway genes / proteins can be obtained, which have a regulatory effect on plant heat tolerance and yield traits.

[0114] 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, 3rd Edition, Science Press, 2002, or according to the manufacturer's recommendations.

[0115] Materials and methods

[0116] 1. Experimental materials and localization cloning

[0117] The inventors constructed a chromosome segment replacement line (CSSL) using a heat-tolerant rice variety as the donor parent and the high-yielding heat-sensitive variety Huajingxian 74 as the recurrent parent. A line with excellent heat resistance, HPS32, was then screened and backcrossed with Huajingxian 74 to purify the background and generate the F1 generation. Self-crossing yielded a segregating F2 population for subsequent mapping. First, by observing the segregation of the F2 generation, the TT2 gene was initially mapped to the short arm of rice chromosome 3 using map-based cloning, between molecular markers Caps-0 and InDel8956. Then, using 7820 F2 rice plants, the inventors finely mapped it to between molecular markers Caps-3 and Caps-4. This candidate region is approximately 25.22 kb. Comparison with online Nipponbare reference sequences revealed three candidate genes in this region: LOC_Os03g29370, Os03g0407400, and LOC_Os03g29389. By designing primers to amplify the full-length genome sequences of these three genes and comparing the differences between wild-type and mutant sequences, it was discovered that the 165th base in the coding region of the candidate gene Os03g0407400 was mutated from C to A, leading to a premature termination mutation at amino acid position 55. Therefore, the inventors hypothesized that the candidate gene Os03g0407400 is TT2. Simultaneously, a small segment of the HPS32 chromosome (carrying TT2) was constructed using BC5F2 generation plants.HPS32 Other near-isogenic lines with a predominantly HJX genetic background include NIL-TT2. HPS32 and its control NIL-TT2 HJX The primer sequences used are shown in Table 1 below.

[0118] Table 1. Primers used in construction

[0119]

[0120] 2. Transgenic complementation verification, overexpression and complementation verification, and CRISPR / Cas9 gene editing

[0121] To further confirm that Os03g0407400 is the cause of TT2 HPS32 For phenotypic genes, the inventors constructed a 2.5kb Os03g0407400 promoter, full-length cds, and a 500bp 3'UTR derived from wild-type HJX74 into the pCAMBIA1300 complementary vector. Genetic transformation was performed using *Agrobacterium tumefaciens* EHA105-mediated rice seed-induced callus transformation, and transgenic positive lines were screened. Additionally, the inventors constructed a vector that overexpressed the TT2 gene (full-length cds from wild-type TT2) driven by the maize ubiquitin promoter. Gene knockout vectors were designed using CRISPR / Cas9 gene editing technology for the knockout of target genes (TT2, SCT1 / SCT2, OsWR2). All genetic experiments were conducted using *Agrobacterium tumefaciens* EHA105-mediated rice seed-induced callus transformation. Transgenic positive lines were obtained through genetic screening, stable T2 generation transgenic plants were obtained, and their phenotypes were examined. The primer sequences used for vector construction are shown in Table 2.

[0122] Table 2. Primers used in construction

[0123]

[0124] Construction method:

[0125] pCAMBIA1300 complementary vector: Using the rice Huajingxian genome as a template, amplification was performed using the corresponding primers in Table 2, and the amplification products were cloned into pCAMBIA1300.

[0126] pCAMBIA1301 overexpression vector: Using rice cDNA as a template, amplification was performed using the corresponding primers in Table 2, and the amplification product was cloned into pCAMBIA1300.

[0127] CRISPR / Cas9 TT2 knockout vector: After the vector is introduced into the cell, it knocks out positions 1 to 40 and positions 85 to 90 of the TT2 coding gene; inserts a nucleotide at position 19 and knocks out positions 87 to 90; knocks out nucleotide position 20; and mutates nucleotide A to T at position 91, which causes a frameshift in the TT2 coding region, thereby forming a stop codon and prematurely terminating the expression of TT2.

[0128] CRISPR / Cas9 SCT1 / SCT2 knockout vector: After being introduced into cells, this vector knocks out positions 267 to 268 and positions 219 to 253 of the SCT1 coding gene; inserts one nucleotide at position 222, resulting in a frameshift and premature termination of the SCT1 coding region; it also knocks out nucleotides 218-248 of the SCT2 coding gene; inserts one nucleotide at position 231; and knocks out nucleotides 229-231, resulting in damage or frameshift of the SCT2 coding region.

[0129] CRISPR / Cas9 OsWR2 knockout vector: After this vector is introduced into cells, nucleotides 148-169 and 526 of the OsWR2 coding gene are knocked out; a nucleotide is inserted at position 166, and nucleotide 526 is knocked out; nucleotides 165 and 522-526 are knocked out, resulting in a frameshift of the OsWR2 protein coding region, which in turn forms a stop codon prematurely and terminates OsWR2 expression prematurely.

[0130] 3. Calcium detection experiment under heat stimulation

[0131] TT2 encodes a γ subunit of a G protein. Based on existing research, the inventors hypothesized that TT2 might be involved in the regulation of calcium signaling under heat stimulation. Therefore, three experiments based on different principles were used to detect changes in extracellular calcium flux, intracellular calcium signaling, and intracellular calcium concentration under heat stimulation. Figure 8The Scanning Ion-Selective Electrode Technique (SIET) is widely used for non-destructive detection of extracellular ion flow rate changes under different treatment conditions in plants. Negative values ​​indicate influx, and positive values ​​indicate efflux. By monitoring changes in the velocity and direction of extracellular ions, changes in calcium signal under heat stimulation can be determined. Differences in calcium flow among NILs under heat stimulation are assessed by integrating the velocity curve with time to obtain the flow rate per unit area and the maximum calcium flow. Using stable transgenic rice material NES-YC3.6 (a calcium ion sensor), changes in intracellular calcium levels under heat stimulation can be detected. The principle is based on fluorescence resonance energy transfer (FRET). When intracellular calcium levels increase, the distance between the donor CFP and the acceptor YFP is shortened, thus enhancing the YFP (FRET) signal activated by CFP. Ultimately, the intracellular calcium ion level is directly represented by the YFP / CFP ratio. Transgenic plants stably inoculated with UBQ10-Aequorin under different NIL backgrounds were used to monitor changes in intracellular calcium concentration under heat stimulation. The principle is to use bioluminescence to detect intracellular calcium concentration and finally obtain the relative calcium concentration through the corresponding calculation formula.

[0132] 4. Protein-protein interaction detection

[0133] To demonstrate the interaction between SCT1 and CaM, in vitro protein interaction verification was performed using yeast two-hybrid technology. Both the CaM and SCT1 genes were constructed into pGBKT7 and pGADT7 vectors, and then co-transformed into Y2HGOLD competent yeast strains, respectively. After growing on SD media with two, three, and four deficiencies for 3 days, the size and color of the bacterial colonies were used to determine whether the three genes interacted pairwise. Based on the results of the yeast two-hybrid method, the inventors found that SCT1 and CaM can interact. To confirm the reliability of this result, further verification was performed in tobacco using the Split Firefly Luciferase Complementation assay. The coding regions of the SCT1 and CaM genes were constructed at the N-terminus and C-terminus of fireflyluciferase, respectively. These were then transformed into Agrobacterium tumefaciens GV3101, and subsequently co-transformed into tobacco in pairs. After 48 hours of dark incubation, the luciferase substrate was uniformly transferred to the leaf surface, and then imaged using a CCD imager with a cooling module. The results showed that SCT1 and CaM do indeed interact. The inventors further used tobacco as a co-localization method to determine the specific subcellular sites of interaction. The inventors linked the coding region of the SCT1 gene to a Venus vector and CaM to an mCherry vector. The results showed that SCT1 and CaM co-localized in the cell nucleus. The inventors constructed the N-termini of TT2 and SCT1 into prokaryotic expression vectors, pmal-c5x (MBP-SCT1-CaMBD) and pGEX4T-2 (GST-CaM), respectively, and transformed them into competent E. coli expression cells. Expression was induced for approximately 24 hours at 10-16°C with IPTG. The protein was then purified using MBP-affinity beads, and the ability to pull down the GST fusion protein was tested. The experimental results showed that GST-CaM could be pulled down by MBP-SCT1-CaMBD, thus proving the interaction between SCT1 and CaM. The primer sequences used for vector construction are shown in Table 3 below.

[0134] Table 3. Primers used in construction (amplification template: rice cDNA)

[0135]

[0136] 5. EMSA and in vitro Chip-qPCR

[0137] SCT1 encodes a transcription factor that binds to calmodulin. Therefore, the inventors transformed *E. coli* using the pMal prokaryotic expression vector in vitro, purified the MBP-SCT1-NT fusion protein, and synthesized a 50 bp DNA strand with a Cy5 chemiluminescent group (from the promoter region of OsWR2) in vitro. This strand was then bound to the corresponding protein and a cold probe, followed by TBE-PAGE and non-denaturing gel electrophoresis, and photographed using a gel electrophoresis apparatus. The inventors discovered that SCT1-NT can bind to the promoter region of OsWR2. The inventors also conducted in vitro Chip-qPCR experiments, expressing the empty Halo protein and Halo-SCT1-NT protein, respectively. These were then incubated with fragmented DNA against an HJX background. The proteins were purified using Halo magnetic beads, and the purified samples were subjected to qPCR to determine the binding of SCT1 to the OsWR2 promoter region. The experimental results showed that a region containing the CG1 motif within the OsWR2 promoter region was significantly enriched by SCT1 protein, thus demonstrating that SCT1 can interact with the OsWR2 promoter region. Figure 6 The primer / probe sequences used in the construction are shown in Table 4 below.

[0138] Table 4. Primers / Probes Used in Construction (Template: Rice Genome)

[0139]

[0140] 6. Surface plasmon resonance (SPR) and gel migration assays to examine protein interaction strength under different calcium environments.

[0141] A protein sensor chip was fabricated by immobilizing the SCT1-CaMBD protein on a carboxyl sensor chip (Nicoya, SCA0815). The analyte protein (CaM) was injected at a flow rate of 20 mL / min into different HBS buffers (10 mM HEPES, 150 mM NaCl, 0.01% Tween), and Ca was added. 2+ Alternatively, use EGTA, and then record the response curve. When testing multiple proteins on the same chip, unless otherwise specified, the channels should be cleaned before injecting the next protein. Accurate kinetic data are obtained using at least three gradient analyte concentrations (1.05, 2.10, 4.20 μmol / L CaM). Analyze data generated via OpenSPR using TraceDrawer. TMA suitable mathematical model is selected, and the affinity binding constant (KD value) is calculated. A smaller KD value indicates a stronger interaction between the two proteins. Experimental results show that EGTA inhibits the interaction between CaM and SCT1-CaMBD, while calcium enhances this interaction. In gel migration experiments, a 21-amino acid peptide was synthesized in vitro, incubated with calmodulin, and separated using a non-denaturing gel. Electrophoresis buffers were prepared in both calcium and EGTA environments, and changes in gel migration were used to determine the interaction between the two proteins. The results indicate that calcium is crucial for the interaction between SCT1 and CaM. Figure 9 ).

[0142] 7. Cytological examination

[0143] NIL-TT2 HJX and NIL-TT2 HPS32 The wax content changed significantly after heat treatment. Therefore, the inventors observed the structure of surface wax crystals before and after heat treatment using scanning electron microscopy. Leaves of near-isogenic lines before and after treatment were rapidly frozen in liquid nitrogen and fixed by freeze-drying. The samples were then observed under a field emission scanning electron microscope. Before treatment, NIL-TT2... HJX and NIL-TT2 HPS32 There was no difference in the arrangement and distribution density of the surface lamellar wax crystals, but after 20 hours of treatment, NIL-TT2 was clearly noticeable. HJX The plate-like crystals in NIL-TT2 became sparser after 24 hours of treatment. HJX The epidermis was severely damaged, and the waxy papillae were shrunken, thus concluding that TT2 can participate in the regulation of waxy components under heat stress. Figure 4 Toluidine blue (TB) staining, a water-soluble dye, is used to quickly observe the content of surface waxes in plants. When the surface wax content decreases, the TB solution quickly penetrates the plant, resulting in enhanced staining. Experimental results show that, against the background of OsWR2 knockout, TBO staining is deeper than its corresponding wild type, thus proving that OsWR2 knockout reduces the surface wax content. Figure 5 ).

[0144] 8. Dual-fluorescence transcriptional activity detection

[0145] To understand Ca more precisely 2+How does CaM regulate the transcriptional activity of SCT1? The inventors first constructed the coding regions of SCT1 and CaM into a pGreen-62sk vector, respectively. A 16 bp segment containing a CGL element from the OsWR2 promoter was tandemly repeated five times and constructed into an 0800 vector as a reporter vector (Pro: 5xCGL: LUC). Rice leaf sheath cells were prepared into protoplasts, and the above vectors were co-transferred into rice protoplasts via PEG. After incubation at room temperature for 16 hours, the results were analyzed. SCT1 is a positive regulator of transcriptional activity. The inventors co-transferred different concentrations of CaM into the protoplasts along with SCT1, and then detected the transcriptional activity of SCT1. The results showed that with the addition of CaM, the transcriptional activity of SCT1 was inhibited, and this process was CaM concentration-dependent. Figure 9 By mutating the calcium-binding region of CaM, a CaM lacking calcium-binding capacity can be obtained. 1234 CaM 1234 While calcium cannot inhibit SCT1 transcriptional activity, it plays a crucial role in the transcriptional regulation of CaM-SCT1. Treatment of protoplasts with lanthanum chloride (a calcium ion channel inhibitor) showed that reducing intracellular calcium ions could, to some extent, alleviate the inhibitory effect of CaM on SCT1 transcriptional activity.

[0146] The SCT1 promoter region was constructed into a DBD-GAL4 vector as an effector vector, and CaM was constructed into a pGreen-62sk vector as a plus vector. A 7x UAS-driven luciferase vector was selected as the reporter system, and 35S-driven renilla was used as an internal control. These different vector combinations were transformed into rice protoplasts to observe the effect of CaM on SCT1 transcriptional activity under different La treatments. The results showed that CaM significantly inhibited SCT1 transcriptional activity, but this inhibitory effect was somewhat relieved under La treatment. The primer sequences used for vector construction are shown in Table 5 below.

[0147] Table 5. Primers used in construction

[0148]

[0149] 9. Treatment of abiotic stress

[0150] For temperature treatment, each sample was first germinated and allowed to grow at a normal temperature of 28℃ for 12 days. Then, it was subjected to a corresponding temperature treatment (42℃). After treatment, the samples were allowed to recover for 20-24 hours, and the phenotype was observed after 7 days of recovery. The survival rate was then statistically analyzed.

[0151] 10. Sequence Information

[0152] The full-length sequence of the TT2 gene is shown in SEQ ID NO:1 (Source: HJX):

[0153]

[0154] The TT2 promoter sequence is shown in SEQ ID NO:2 (Source: Rice HJX):

[0155]

[0156] TT2 HJX The coding region sequence is as shown in SEQ ID NO:3 (source: rice HJX):

[0157] >ATGGCAATGGCGGcggcgccccggcccaagtcgccgccggcgccgcccgacccatgcggccgccaccgcctccagctcgccgtcgacgcgctccaCCGCGAGATCGGATTCCTCGAGGGTGAAATAAATTCAATCGAAGGGATCCACGCTGCCTCCAGATGCTGCAGAGAGGTTGACGAATTCATCGGAAGAACTCCTGATCCATTCATAACGATTTCATCGGAGAAGCGAAGTCATGATCATTCTCACCACTTCTTGAAGAAGTTTCGCTGTTTGTGCAGAGCAAGTGCGTGCTGCCTCAGCTACCTCTCCTGGATctgctgctgcagcagcgccgccggcggctgctcatcctcctcctcctccttcaacctcaagaggccgagctgctgctgcaactgcaactgcaactgctgctgctcctcctcctcctcATGTGGGGCGGCGTTAACGAAGAGTCCGTGTCGCTGCCGCCGCCGCAGCTGCTGCTGCCGTCGCTGCTGCTGCGGCGGCGTCGGCGTCCGCGCGTGCGCGAGCTGCAGCTGCTCCCCGCCGTGCGCGTGCTGCGCGCCGCCGTGCGCGGGATGCTCGTGCCGCTGCACCTGCCCGTGCCCGTGCCCCGGCGGCTGCTCCTGCGCGTGCCCGGCGTGCAGGTGCTGCTGCGGCGTCCCTCGTTGCTGCCCCCCCTGCTTGTGA

[0158] TT2 HPS32 The coding region sequence is as shown in SEQ ID NO:4 (source: rice HPS32): The 165th nucleotide C→A mutation is marked with an underline, resulting in premature termination:

[0159] >ATGGCAATGGCGGcggcgccccggcccaagtcgccgccggcgccgcccgacccatgcggccgccaccgcctccagctcgccgtcgacgcgctccaCCGCGAGATCGGATTCCTCGAGGGTGAAATAAATTCAATCGAAGGGATCCACGCTGCCTCCAGATGCTG A AGAGAGGTTGACGAATTCATCGGAAGAACTCCTGATCCATTCATAACGATTTCATCGGAGAAGCGAAGTCATGATCATTCTCACCACTTCTTGAAGAAGTTTCGCTGTTTGTGCAGAGCAAGTGCGTGCTGCCTCAGCTACCTCTCCTGGATctgctgctgcagcagcgccgccggcggctgctcatcctcctcctcctcctccttcaacctcaagaggccgagctgctgctgcaactgcaactgcaactgctgctcctcctcctcctcctcATGTGGGGCGGCGTTAACGAAGAGTCCGTGTCGCTGCCGCCGCCGCAGCTGCTGCTGCCGTCGCTGCTGCTGCGGCGGCGTCGGCGTCCGCGCGTGCGCGAGCTGCAGCTGCTCCCCGCCGTGCGCGTGCTGCGCGCCGCCGTGCGCGGGATGCTCGTGCCGCTGCACCTGCCCGTGCCCGTGCCCCGGCGGCTGCTCCTGCGCGTGCCCGGCGTGCAGGTGCTGCTGCGGCGTCCCTCGTTGCTGCCCCCCCTGCTTGTGA

[0160] TT2 HJX The protein sequence of is as shown in SEQ ID NO:5 (source: rice HJX):

[0161] >MAMAAAPRPKSPPAPPDPCGRHRLQLAVDALHREIGFLEGEINSIEGIHAASRCCREVDEFIGRTPDPFITISSEKRSHDHSHHFLKKFRCLCRASACCLSYLSWICCCSSAAGGCSSSSSSFNLKRPSCCCNCNCNCCCSSSSSCGAALTKSPCRCRRRSCCCRRCCCGGVGVRACASCSCSPPCACCAPPCAGCSCRCTCPCPCPGGCSCACPACRCCCGVPRCCPPCL

[0162] TT2 HPS32 The protein sequence is as shown in SEQ ID NO:6 (source: rice HPS32): amino acid position 55 is changed to a terminator.

[0163] >MAMAAAPRPKSPPAPPDPCGRHRLQLAVDALHREIGFLEGEINSIEGIHAASRC

[0164] SCT1 Nipponbare The full-length gene sequence is shown in SEQ ID NO:7 (Source: Nipponbare rice (NIP)):

[0165]

[0166] SCT1 Nipponbare The promoter sequence is as follows: SEQ ID NO:8 (Source: Nipponbare rice):

[0167]

[0168] SCT1 Nipponbare The coding region sequence is as shown in SEQ ID NO:9 (Source: Nipponbare rice):

[0169]

[0170] SCT1 Nipponbare The protein sequence is shown in SEQ ID NO:10 (Source: Nipponbare rice):

[0171]

[0172] SCT2 Nipponbare The full-length gene sequence is shown in SEQ ID NO:11 (Source: Nipponbare rice):

[0173]

[0174] SCT2 Nipponbare The promoter sequence is as shown in SEQ ID NO:12 (Source: Nipponbare Rice):

[0175]

[0176] SCT2 Nipponbare The coding region sequence is as shown in SEQ ID NO:13 (Source: Nipponbare rice):

[0177]

[0178] SCT2 Nipponbare The protein sequence is shown in SEQ ID NO:14 (Source: Nipponbare rice):

[0179]

[0180] OsWR2 HJX The full-length gene sequence is shown in SEQ ID NO:15 (Source: Rice HJX):

[0181]

[0182] OsWR2 HJX The promoter sequence is as shown in SEQ ID NO:16 (Source: Rice HJX):

[0183]

[0184] OsWR2 HJX The coding region sequence of

[0185] >atgggacagtcgaagaagaagttccgcggagtcaggcagcgccactggggctcctgggtctccgagatcaggcaccctctccttaagaggagggtgtggctgggtacctttgagacggcggaggaggcggcgcgggcgtacgacgaggccgccatcctgatgagcggccgcaacgccaagaccaacttcccggtcgcgaggaacgccacgggggagctcacaccggcggctgcggtggcagggcgggatggccgtgtcggcggcggcagcggcagctcgtcctcaatgacggccaacggcggcgggaacagcctgtctcagatcctcagcgccaagctccgcaagtgctgcaagacgccgtcgccgtcgctcacctgcctccgcctcgacccggagaagtcccacattggcgtctggcagaagcgcgccggcgcacgcgctgactccagctgggtcatgaccgtcgagctcaacaaggacacggccgtgtcgtcggctgcgaaggtggcagcagcaacagcagcgtcgtccagcgaccagccgactccgagtgacagcacagtcacaacgacgtccacgtccaccacgggctcgccgtcgccaccacctccggcaatggacgacgaggagaggatcgcgctgcagatgatcgaggagctgctgggcaggagcggcccgggctcgccgtcacatgggctgctgcacggtggtgaaggtagcctcgtcatctga

[0186] OsWR2 HJX The protein sequence of

[0187] >MGQSKKKFRGVRQRHWGSWVSEIRHPLLKRRVWLGTFETAEEAARAYDEAAILMSGRNAKTNFPVARNATGELTPAAAVAGRDGRVGGGSGSSSSMTANGGGNSLSQILSAKLRKCCKTPS PSLTCLRLDPEKSHIGVWQKRAGARADSSWVMTVELNKDTAVSSAAKVAAATAASSSDQPTPSDSTVTTTSTSTTGSPSPPPPAMDDEERIALQMIEELLGRSGSGSPHGLLHGGEGSLVI

[0188] Example 1: Natural site TT2 with high tolerance to high temperatures HPS32 The acquisition and cloning of genes that control their phenotype

[0189] To explore more superior heat-resistant rice germplasm resources and study the molecular regulatory mechanisms of stress resistance formation, the inventors constructed a set of replacement lines covering the entire rice chromosome segment using heat-resistant cultivated rice varieties as donor parents and the widely promoted high-yielding variety Huajingxian 74 (HJX) as the recurrent parent. A replacement line with stable heat resistance, HPS32, was identified from this set. Figure 1 A). HPS32 was crossed with the recurrent parent HJX to construct a BC5F2 localization population. Multiple generations of localization were performed on 7820 individuals, and the heat resistance phenotype of recombinant individuals with several local segment replacements was identified. Figure 1 B) The inventors ultimately located TT2 (THERMOTOLERANCE 2) within a 25.22kb range and discovered that one gene within it contains a SNP in both HPS32 and HJX, and that this SNP causes premature termination of protein translation in HPS32. Figure 1 C). To identify candidate genes for TT2, TT2 was knocked out in the HJX background. KO HJX, which is intolerant to heat stress, exhibits a phenotype with significantly enhanced heat resistance, and its survival rate is as high as about 90%. Figure 1 D, Figure 1 I).

[0190] In NIL-TT2 HPS32 Overexpression of TT2(pUbi:TT2) from the HJX background in the background HJX / NIL-TT2 HPS32 This can restore NIL-TT2. HPS32 The phenotype (changing it from a heat-resistant phenotype to a heat-sensitive phenotype) Figure 1 E, Figure 1 I). In NIL-TT2HPS32 In this context, the system switches to TT2 driven by its own bootloader. HJX Full-length genomic DNA (pTT2:TT2) HJX / NIL-TT2 HPS32 Similarly, it can complement NIL-TT2. HPS32 The heat-resistant phenotype resulted in a highly significant decrease in survival rate compared to the control. Figure 1 F, Figure 1 I).

[0191] Through sequence alignment analysis, the inventors confirmed that TT2 HPS32 The locus originates from tropical japonica rice varieties. Tropical japonica rice containing this locus exhibits strong heat resistance, while the presence of this locus is lower in temperate japonica rice, thus exhibiting weaker heat resistance. Figure 2 AD).

[0192] Therefore, the inventors successfully located and cloned a QTL that regulates rice heat stress tolerance. TT2 is a negative regulator of rice heat stress tolerance. When TT2 function is lost, rice will have a strong heat stress tolerance.

[0193] Example 2, TT2 HPS32 Sites protect rice yield under heat stress

[0194] The inventors identified the heat resistance of mature plants from near-isogenic lines and found that NIL-TT2 showed resistance under high-temperature treatment. HPS32 It exhibits strong tolerance to heat stress, and green leaf tissue can still be observed after treatment, maintaining an upright plant shape. In contrast, NIL-TT2 HJX This manifests as the withering of the entire plant, leaf curling and chlorosis, and a series of other heat-sensitive phenotypes. Figure 1 GH).

[0195] The inventors observed that, compared to NIL-TT2, the ear of grain... HPS32 NIL-TT2 HJX The phenotype of obvious ear development abnormality was observed. Figure 3 A, E), and the seed setting rate was significantly reduced. Under normal conditions without heat stress, there were no differences among NILs in terms of yield per plant, seed setting rate, and number of panicles per plant; when rice was treated with high temperatures throughout the entire grain-filling period, it was clearly observed that high temperatures caused NIL-TT2 HJX The severe yield reduction was mainly due to a significant decrease in seed setting rate; however, in NIL-TT2... HPS32 In rice, the loss of TT2 function reduced the decline in yield, mainly by protecting the seed setting rate. After heat treatment, NIL-TT2... HJXThe fruit setting rate is only about 40%, however, NIL-TT2 HPS32 It will remain at around 75%. Figure 3 C). Under normal conditions, NIL-TT2 HJX and NIL-TT2 HPS32 There was no significant difference in yield per plant, but after heat treatment, NIL-TT2... HPS32 Compared to NIL-TT2 HJX Production increased by approximately 55.6% ( Figure 3 B), which reduces yield loss due to heat treatment. Figure 3 D). Agronomic traits of each plant were investigated under normal conditions, such as... Figure 3 F.

[0196] The above results prove that TT2 HPS32 It plays an important role in protecting rice yield under heat stress.

[0197] Example 3: TT2 encodes the γ subunit of a G protein, which participates in regulating changes in wax content under heat stress through OsWR2.

[0198] Transcriptome data demonstrate that, regardless of whether heat-induced gene upregulation or heat-induced gene downregulation occurs, NIL-TT2... HPS32 The trend of change is always weaker than that of NIL-TT2 HJX ( Figure 4 Therefore, the inventors discovered that the loss of TT2 function inhibited the heat-induced transcriptional response in rice to some extent. Using KEGG enrichment analysis, the inventors found that differentially expressed genes in near-isogenic lines after heat treatment were enriched in the wax synthesis pathway (BC). Figure 4 A). Through transcriptome sequencing at different time points, the inventors discovered that in NIL-TT2 HJX Most wax genes showed a downregulation trend after 1 hour of heat treatment, and the downregulation trend became more pronounced with increasing heat treatment time. However, in NIL-TT2... HPS32 During the treatment, these genes remained largely unchanged throughout the treatment period, or exhibited characteristics similar to NIL-TT2. HJX Compared to the weakening downward trend ( Figure 4 These results show that heat stress induces the downregulation of wax-related genes in rice, but loss-of-function TT2 prevents this downregulation. Metabolomics data demonstrate that under normal conditions, NIL-TT2... HJX and NIL-TT2 HPS32 There was no significant difference between them. However, after heat treatment, the inventors were able to observe that in NIL-TT2... HJX The wax content showed a significant decrease, but in NIL-TT2 HPS32The phenomenon of thermally induced decrease in the waxy component was suppressed. Figure 4 F). Electron microscopic observation of the waxy crystals on the surface of rice also supports the above conclusions, TT2. HPS32 Under heat stress, TT2 reduces the heat-induced decrease in wax content, and plays an important role in the regulation of rice wax content under heat stress. Figure 4 G). The inventors identified an important transcription factor, OsWR2, that positively regulates wax synthesis in wax-related genes altered by heat between NILs. Figure 4 H).

[0199] In NIL-TT2 HPS32 OsWR2-knockout plants (wr2 / NIL-TT2) were constructed in this context. HPS32 The inventors discovered that knocking out OsWR2 complements NIL-TT2. HPS32 The phenotype of [the organism] significantly reduced its survival rate. Figure 5 Therefore, OsWR2 acts downstream of the TT2 thermoregulatory pathway.

[0200] Example 4: SCT1 directly binds to the OsWR2 promoter

[0201] Through analysis of the binding elements of the OsWR2 promoter, the inventors discovered a CG1-like binding element, which they named SCT1 (Sensing Ca). 2+ Transcription Factor 1) and SCT2 ( Figure 6 A). SCT1, as a transcription factor, has the function of binding to promoters and regulating the transcription level of target genes. The inventors demonstrated through EMSA (gel migration assay) and in vitro Chip-qPCR experiments that SCT1 can bind to the promoter region of OsWR2. Figure 6 BD). SCT2 can also bind (EMSA experimental verification, Figure 6 C) SCT1 and SCT2 are functionally redundant, so the inventors mutated both genes.

[0202] These results suggest that SCT1 can directly bind to the OsWR2 promoter and regulate OsWR2 expression.

[0203] Example 5: SCT1 negatively regulates rice wax synthesis and heat stress tolerance under heat stress.

[0204] SCT1 / SCT2-knockout transgenic lines (sct1 / sct2) were constructed using the Zhonghua 11 gene. Heat resistance testing showed that sct1 / sct2 plants exhibited stronger heat stress tolerance than Zhonghua 11, and their survival rate was significantly higher than that of wild-type Zhonghua 11. The mutation in sct1 / sct2 also enabled rice to acquire the ability to withstand high temperatures at maturity. Figure 7 (AD). qPCR experiments showed that the expression level of OsWR2 in wild-type Zhonghua 11 was significantly downregulated by heat, but this downregulated expression was suppressed in the sct1 / sct2 mutant, exhibiting similarity to NIL-TT2. HPS32 Consistent expression patterns under thermal stress Figure 7 E). KEGG analysis and GC-MS results showed that the content of various waxy metabolites in wild-type Zhonghua 11 was significantly lower than that in the sct1 / sct2 mutant after heat treatment. Figure 7 FG).

[0205] Therefore, the above experiments demonstrate at both the transcriptional and metabolic levels that SCT1 / SCT2 is a transcription factor that negatively regulates wax content under heat stress and ultimately negatively regulates the heat tolerance of rice.

[0206] Example 6: TT2 participates in the regulation of intracellular calcium signaling under heat stress

[0207] G proteins have been shown to participate in calcium signaling under stress in previous studies. The inventors used SIET non-invasive electrophysiological technology, the FRET-based NES-YC3.6 calcium reporter system, and the jellyfish fluorescent protein bioluminescence-based calcium concentration detection system to detect the differences in calcium signaling between near-isogenic lines under heat stimulation. The inventors detected calcium under heat stimulation in different regions of the roots, different regions of the leaves, or the whole seedling.

[0208] The results showed that NIL-TT2 HJX The increase in intracellular calcium concentration after heat stress was significantly higher than that of NIL-TT2. HPS32 ( Figure 8 AI) indicates that the TT2 is functioning normally. HJX TT2 is essential for the induction of intracellular calcium signaling under heat stress, but its function is lost. HPS32 It inhibited intracellular calcium signaling under heat stress.

[0209] Example 7: SCT1 can sense calcium signals and negatively regulate the heat-induced transcriptional response of OsWR2 in a calcium-dependent manner via CaM.

[0210] Protein-protein interaction experiments demonstrated that SCT1 can interact with calmodulin (CaM), and that the interaction between CaM and SCT1 is calcium ion-dependent. Figure 9 (AG). Dual-fluorescence transcriptional activity assays demonstrated that SCT1 is a transcriptional activator, but with the addition of CaM, SCT1 transforms from a transcriptional activator into a transcriptional repressor, and this process is CaM concentration-dependent. This is further demonstrated by the co-transfer of a mutant form of CaM that cannot bind calcium ions. 1234 At that time, CaM 1234 It cannot exert an inhibitory effect on SCT1 transcriptional activity. However, by blocking calcium ion channels through chlorination, the inhibitory effect of CaM on SCT1 transcriptional activity can be relieved to some extent. Figure 9 H).

[0211] The inventors verified this process through in vivo experiments, using qPCR to detect the effects of heat treatment and lanthanum chloride alternating treatment on rice plants in NIL-TT2. HJX The thermally induced downregulation of OsWR2 was attenuated, and this attenuation was concentration-dependent on lanthanum ions. Figure 9 I).

[0212] Therefore, SCT1 / SCT2 (based on analysis, SCT2 also plays a role) can act as a calcium ion sensor. Changes in intracellular calcium ion concentration are recognized and bound by CaM, which records the signal and forms Ca2+. 2+ The form of -CaM promotes the interaction between CaM and SCT1, and enhances the activity of SCT1 / SCT2 transcriptional repression, which in turn leads to a decrease in the expression of downstream genes such as OsWR2, thereby regulating the content of waxes and heat resistance.

[0213] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims. sequence list <110> Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences <120> Application of the TT2-SCT1 / SCT2-WR2 regulatory pathway in plant heat stress tolerance <130> 215420 <160> 70 <170> SIPOSequenceListing 1.0 <210> 1 <211> 5609 <212> DNA <213> Oryza sativa L. <400> 1 gatcatcc attatcggaa cttcggagtg acatggcaat ggcggcggcg ccccggccca 60 agtcgccgcc ggcgccgcc gacccatgcg gccgccaccg cctccagctc gccgtcgacg 120 cgctccaccg cgagatcgga ttcctcgagg tacaatctat ctctatctgt ctatatcact 180 accattcata ctccttcgat cttgcttcaa aaaaaaaa tatatatttc ctacttcata 240 ttcatataca cacgtacggc ttgctatctg tcgaattgtt tgcttctgca tgcatgcatc 300 actctcattg taagttttc ccagcttaaa accactcctt ttatcttcgt tcttcttcct 360 tcttgttttt ttttaaaaaa acaacaactc atttaatctt catatagtgt atcatgcatc 420 atttgcttct ttgatcagtt ccccaaaaac tgctcctctc ttcccagcca aataattaaa 480 cttaagcaaa caagcaagtt gaactgatga tccaataaaa caaaacaaaa ccgatcgaat 540 aggaagtcaa tggcatatac cagctgctat agctgaagcc acgaatgctt agcttagctc 600 tagtcgatcc ctgttgactg ttcaacacac tgcactaaca caccagttaa tgagctgatt 660 aattaaacca ttaaatgagc ttaacgggcg gctagcttct tcctctgggc ccgtgccgat 720 cgtaccatcg gtttgcgcgt ccctccacct aaactctctg ccttgttatt cccttgcct 780 agtactacat gcatttgcat catcatccca tcaccaatag tactacgttt caactggatt 840 ttggtggtgt cataccatat catatttggt tttgttctct agtttactcc tacattagtc 900 tctagcggtt ttgtggagta ctaaagaaaa caactaatca gccagggttt aacgtttaat 960 cggttggtgg ttttgttaat taatttcatc tactatttta gacttcacag gtcttcgagc 1020 tataagcatc gattgccatg catcaatcga tgctggtcca cgctagtttc tgagttctga 1080 ctagctctct taattgtgct ttgacctact ttaattaatt aaccagtggc tgcgtcactc 1140 attgaccaac attgtcatgt tacccggact gatttttttt ttctttaaaa aaacaccgga 1200 tatattatta gttagtgtat atatatgtct gctcaagaag cgcatgcata tagttctcg 1260 tcaaacaaaa aatgtactgt atgctcaaag catctgtttt ggaattgtca tattcgcctt 1320 tataattaaa ataattaaaa tggtgatgcc cagcttttttt tttcctccaa taatttattt 1380 attggcttga ttcctgtgc tattaggatt aaactactc cgttttaatt agcaccattt 1440 ttaaagcttc taaaattaac ctaagtaag tacgacagta cttgctgtct agctttaaat 1500 gttttggggtg ttaaaatatc cctcagacat cacctgaaaa gttgacaggc taaacacatg 1560 cccatctccc tcgtttactt aaatttaatc gaacaaca ctgtatatat atttcttgca 1620 gggtgaaata attcaatcg aagggatcca cgctgcctcc agatgctgca gagagtaagc 1680 cagcctgctg tttctttg tactacttcc atttctctc gtctttactc taccatgca 1740 ttcacaaaat attacktactt acccagtttt ttgatcatga acttgaccg tattcttttt 1800 aagcaacttc aaaaatag gtttaaatgc aaatgttatg tacaccattg attataaaac 1860 cttggcaat gaagtaaa aaccagcaca tttaatttct gaacgttggg agtattatta 1920 ttttatatc ttttactc atttaatcat agtatcgtgc aagctttg agtgtatta 1980 ggttgcttaa ggtaaaaaaa tgtaactagg ttacatttag tactaactg aacatttaat 2040 tagtaatgtt tcgttaagta actgtaattt caatgcatgc atgtcctccc gtaagagcaa 2100 gttaatagt atagccaact actagctcca attattat agacaatcta atagctcatt 2160 catacaataa ttacatacta cactattaat atctgatccc acctgtcata cacatactgc 2220 atttggagt ccgtgctata gctgactaca aatctatagt ccgctgctct tctctcttt 2280 tattatctc cttaaaatat gtttgcagct ggcttatagc ctgctattgt acctgctctg 2340 aaaatagtgc agagactgtt caaaaagtca ttgcacaata actattcaca tggaactgtg 2400 aaaagtatat attggaactt actagctaga tccttttggg aacatgggaa aagccaagtc 2460 acgtgtggaa tccctattcc ctgtgttctt cagctagaag agtgaaaata atgtactact 2520 actatacgga gatgaaatta cagcaggagc agaaagcggg aaaaaaactt taaatcaatt 2580 aaacaaacct ctctctgcaa aattcaacac cagcaacgaa caactcatca agttcttgtg 2640 tttgtaccg gccggtaact aattgttgtt tgcataagcg aaacggtata tttgcaaaca 2700 aaaaatatt tatgaataaa acttttatat acatgttctt aattatctca aaaaaaggt 2760 tgaaaaataa acttcgatga aaaaatctca aaatcaattc caaatttatg gtgaaaattt 2820 taaatttgt ctgataaca taagtataag CAAAAAAAAA agTAagCA tgtcactatg 2880 ttaatggtat tgtatatat accttgagt ttctgtctgt actttaggag tacatgctac 2940 gaacatgttt tcttggctc tattcgttg gattacttg cgtattgtgg gccagacgcc 3000 tggtgaactt cgtcgattgt gtggactaat taagctcacc tgaataagt ggttgaaac 3060 aaggctaaag atgattttta tcatggattt tggcttgga atttttgt agggttgacg 3120 aattcatcgg aagactcct gatccattca taacgatgta tggattttca ggtcgagaat 3180 ttgtctttaa cttcgcacga ctgttatttt tttctttt aattctctgt ttacaagcag 3240 3300 acttcattcc cggatcttaa tgtatatatg catatctgca ctgtgctaat tggtgtacac 3360 attatgtgat catcagtcca agttaattat tactacaa actgaacta taacactag 3420 aaatatgta acttgcaaag tacatattga atcaggtt catatataga actccaccctg 3480 cagatttctt ccaatatata tatgctgtca ccatgttttc acttgtcacc tagtacacct 3540 ttgactggga gactttcctt gatgatcgac gtggtcatat tcttcagat gatttaattt 3600 cagatagaaa aaatattgt ttacttagtt tctctccttc agtaagag atgtgcaaga 3660 ccagcgatca aactatatga actgttcgtt tcatgataaa aaaaacatga tatggaataa 3720 ctaggtgatt cacatataa tggctgataa tccctcgtttt caggagatac catcagtttt 3780 ctactttct acttttctcc atgttcttt ttcatgttt gaggtggatc ggagcttgta 3840 ttagatgttt gctcagctca attattgctg cagatttccc tatatagcct ccactgtata 3900 tatactccct ccgattccat aatttaatga tattttgaac aatgacgctg tctccaaaat 3960 attackttca ctttgttttc ctattataat atacaata aaaaaaatac attack 4020 ttcttataaa tagtttcaa haaatcta tatatgttgt tatatactc ttttaaacta 4080 atatttttta aagttatagt aaagttaca aaagttggac ctcaacatg tataaaacgt 4140 cgagaattgt atatctgatc aaccaataat ttgtagtgca ttgtcttaaa aaaattgcat 4200 ttctagctat gtatctagat attackacaga accaggtgaa atcacatttt atttttactg 4260 gaccacgaac tcattgttta attacttcca gccttgcact aaataacaat aattgaacct 4320 ggcatcacct gcacaattaa tttggacaca aagtaaacat gaatgcaaca tactcgttt 4380 catattgttt gttggtgtag gatttaaatt ttgtgtcaaa atacttgccg ttcttactac 4440 attcttaagc actttgagaa ctaaccttct cttctaccc ttcatcaata cagtcatact 4500 aactaattgg ctcttatgcc ttgaaaaact aattaggatg tatttaatga gggtaaccat 4560 gtaatctgcc accagtgaat gcaattttgg ttcaaaattt cgggcccccg ccctaaaaag 4620 tcattatctc gatagaattt ttttgaattt agtcaaaatt tattcaaatt tagccaaatt 4680 gtgttaaatt tcaaataatt tcagtctaaa aagtgctgaa aatcccgaaa tttaggttct 4740 accgaaatgg ccggaaattt tcagcgaaaa tcaaacatga aaaccttgtc tgccacattt 4800 gttagtttgt gtaaagattt gctagaacga taagtaattt gaagcggatg tatattatat 4860 atcccacaaa accatcaact tgttaattac atgtatattt gtgcatgatg ctttcaccac 4920 tttgtggttg ttaacgatta aatcacacgt tttattttct cacagctgtt tgtgcagagc 4980 aagtgcgtgc tgcctcagct acctctcctg gatctgctgc tgcagcagcg ccgccggcgg 5040 ctgctcatcc tcctcctcct cctccttcaa cctcaagagg ccgagctgct gctgcaactg caactgcaac tgctgctcct cctcctcctc ctcatgtggg gcggcgttaa cgaagagtcc gtgtcgctgc cgccgccgca gctgctgctg ccgtcgctgc tgctgcggcg gcgtcggcgt 5220 ccgcgcgtgc gcgagctgca gctgctcccc gccgtgcgcg tgctgcgcgc 5280 gggatgctcg tgccgctgca cctgcccgtg cccgtgcccc ggcggctgct cctgcgcgtg 5340 cccggcgtgc aggtgctgct gcggcgtccc tcgttgctgc cccccctgct tgtgatcgat5400 cgatcgattg agcgaagctg cactgattgg ttaattaatt agttctcgat gatgatcgat cgagctgcgc gcgtacttaa ttagctagct aggttctggt gttaattagt tcctcatcga tgcatatgtt gattgccttg ctctgcttgc ggttatctgt aatttggctt tgctgccatg 5580 atgagtacgt gattgctgat ttattttac <210> 2 <211> 2643 <212> DNA <213> Oryza sativa L. <400> 2 actttttaac caacgtttga ccactcgtat tattcaaaaa tttatgtaa atataaaaat 60 acttatgtca tgcttaaaga acatttgatg ataaatcaag tcacaataaa ataaattata 120 attacaaatt ttttttgaat aagacgaaag gtcaaacgtt tgtaaaaaag tcaacggcgt 180 catacattaa aatacggagg gagtacataa tactccatcc atttttttag tacgacatta 240 tttagttaaa atgtggaaaa aacttagaga aagtagacta taaaaaggtt gaatggtgca 300 aagtaagcat agctaaactg gttttttatg ttacttgttg tggaccctat ctacttggat 360 tcaaggttta aacttggtat gggtgcacgt attacggct aattattttt aaatggtagg 420 tgatgtactg ttgacagcga gacgctctca tgatgacttt atcaatctaa acatatatat 480 gttggtgcaa ctttttcaaa ggtacttaat tcaaaggata ggatatttat gtatgagttt 540 gttcataaga acgaatatgc cttgataggt tgtgcatggg tatctataag agtgagtgtg 600 tgtataat gatagggtgt gtgtgtgttt ttataagaac gactacgcgc atctctatat 660 ttcggtacat cccgggtatg ccttctctaa aaatgtcttg tgctaaagtt gaagacaact 720 tctaaggaag gtatctctca tcttccttgt gtgtccctac tagaaaatat ttttacaggc 780 ggccaaaata tttttttttg caggcgtcca taacctccgc ctacacccaa agtcatgcaa 840 aaatcaataa tttttgtagg tggctaaatc cacccgtttg cgaaaatatt ttcagcgtaa 900 agaaaattcg ccgccctcag ccctacccac cgccgccctc cccttcggcc ggatctaggg 960 ttatggtttt gggaggaaga gggaggggag gggagctgcg tcaccatcac tgaatctggt 1020 aggtgagagc gccgccatga gctcaccggt gctgccgccg acctcctgcc tacgtgccgc 1080 cttctccgct gcaagtcgcc gctgctgccc tccccacgac acagctggat ccgggagggg 1140 agggcaggga aggttcgctg tcgccgccgc caccctccct acgacgtagc cagatatggg 1200 tggagaggta agcgccgccg ccgctgcaca acccacgatg ccgccgctgc acaacccgcg 1260 atgccgtcgg ccggatttgg gaggtggagc gtcgccgccc tccgccccgc gccatgtctg 1320 aggaaagaga gcgatgggag cgccgccggc caccaccgtc cccccttccc cctccaacca 1380 gatctgggag gaagggaggg agggagggga gccatcacgt cgttgggagt agatgccgcc 1440 accgccctcc ccctccctgc ggccaccaca ctggggtgca ccgccaccgc taggtagccg 1500 cctctggctg gatccacgcc gaggagagga aggagagggga gagggagagg gaggggagg 1560 gagatgtgga agttgactta gaaaatttg acgcccggtg gttttaaga tactttacct 1620 ttttgcaggc ggatttatta aagaggtccg tctgcaaaaa taatgatatt ttcacggtcg 1680 gacctcttaa gatatctaca tgtagaaatc tatatttttt tacttaaatt actgccaggg 1740 ctttgcctcg tagtataatt ttcataaaaa ctcaaaatat ttacaaatta caaaagagga 1800 aggaaggaga aaaacttata aagattacaa tgtttgtaac caatcaaaaa ctatctgctt 1860 taaaggatct ttcattctac acatatgaag aagaacttct tttctaaacg atattcttca 1920 tgaaccaaag gagggaagct cattccgaaa attttccca tttctctgct cgtaggcgga 1980 taactatctc atctcacggt tgttattttt gtaggtggct atttggcttc cagagaccta 2040 ctcgtccggg aaaaagaaat gccagcatct agaaaatag ttttcttagt agagtgttgt 2100 gagcgtctgc gatcgtggta aagaaagcaa aaaaggtgaa ggacgagtgg catgatacat 2160 atgggaaagc tgtagacttt gacccttgac tactcgttgg aagtgtgcgt ctgcatgcat 2220 tattgaacgg ctctgatccc cgcggcgcag cggatcgggg tcatgtccgg atgggcatat 2280 cgacgagaag gatccgtccc cgacaatctt tcaaggcccg tgcccccgtc cctcctctcc 2340 tctgcgcctt tccatcatca tttacgccca accccaacac atgtacattt cccttggctt 2400 gcttccggag aagaaaagag cggccatcca ctccactctc cactctctcc cttccatcat 2460 tacttgccca aaaacggcaa tcccctcccc tccatctcca tgtgctcttc cacctagctc 2520 cgccattcaa agcaaagcac caagcttttg cctctccctc taccatgcct gccccctata 2580 catagctgct gcaccgtctc tcttcataaa tatactagta ggagtagcag agctcatcac 2640 ttc 2643 <210> 3 <211> 696 <212> DNA <213> Oryza sativa L. <400> 3 atggcaatgg cggcggcgcc ccggcccaag tcgccgccgg cgccgcccga cccatgcggc 60 cgccaccgcc tccagctcgc cgtcgacgcg ctccaccgcg agatcggatt cctcgagggt 120 gaaataaatt caatcgaagg gatccacgct gcctccagat gctgcagaga ggttgacgaa 180 ttcatcggaa gaactcctga tccattcata acgatttcat cggagaagcg aagtcatgat 240 cattctcacc acttcttgaa gaagtttcgc tgtttgtgca gagcaagtgc gtgctgcctc 300 agctacctct cctggatctg ctgctgcagc agcgccgccg gcggctgctc atcctcctcc 360 tcctccttca acctcaagag gccgagctgc tgctgcaact gcaactgcaa ctgctgctgc 420 tcctcctcct cctcatgtgg ggcggcgtta acgaagagtc cgtgtcgctg ccgccgccgc 480 agctgctgct gccgtcgctg ctgctgcggc ggcgtcggcg tccgcgcgtg cgcgagctgc 540 agctgctccc cgccgtgcgc gtgctgcgcg ccgccgtgcg cgggatgctc gtgccgctgc 600 acctgcccgt gcccgtgccc cggcggctgc tcctgcgcgt gcccggcgtg caggtgctgc 660 tgcggcgtcc ctcgttgctg ccccccctgc ttgtga 696 <210> 4 <211> 699 <212> DNA <213> Oryza sativa L. <400> 4 atggcaatgg cggcggcgcc ccggcccaag tcgccgccgg cgccgcccga cccatgcggc 60 cgccaccgcc tccagctcgc cgtcgacgcg ctccaccgcg agatcggatt cctcgagggt 120 gaaataaatt caatcgaagg gatccacgct gcctccagat gctgaagaga ggttgacgaa 180 ttcatcggaa gaactcctga tccattcata acgatttcat cggagaagcg aagtcatgat 240 cattctcacc acttcttgaa gaagtttcgc tgtttgtgca gagcaagtgc gtgctgcctc 300 agctacctct cctggatctg ctgctgcagc agcgccgccg gcggctgctc atcctcctcc 360 tcctcctcct tcaacctcaa gaggccgagc tgctgctgca actgcaactg caactgctgc 420 tcctcctcct cctcctcatg tggggcggcg ttaacgaaga gtccgtgtcg ctgccgccgc 480 cgcagctgct gctgccgtcg ctgctgctgc ggcggcgtcg gcgtccgcgc gtgcgcgagc 540 tgcagctgct ccccgccgtg cgcgtgctgc gcgccgccgt gcgcgggatg ctcgtgccgc 600 tgcacctgcc cgtgcccgtg ccccggcggc tgctcctgcg cgtgcccggc gtgcaggtgc 660 tgctgcggcg tccctcgttg ctgccccccc tgcttgtga 699 <210> 5 <211> 231 <212> PRT <213> Oryza sativa L. <400> 5 Met Wing Met Wing Wing Wing Pro Arg Pro Lys Ser Pro Wing Pro Wing 1 5 10 15 Asp Pro Cys Gly Arg His Arg Leu Gln Leu Ala Val Asp Ala Leu His 20 25 30 Arg Glu Gly Phe Leu Glu Gly Glu Ile Asn Ser Ile Glu Gly Ile 35 40 45 His Ala Ala Ser Arg Cys Cys Arg Glu Val Asp Glu Phe Ile Gly Arg 50 55 60 Thr Pro Asp Pro Phe Ile Thr Ile Is Glu Lys Arg Is Asp 65 70 75 80 His Ser His Phe Leu Lys Lys Phe Arg Cys Leu Cys Arg Ala Ser 85 90 95 Ala Cys Cys Leu Ser Tyr Leu Ser Trp Ile Cys Cys Ser Ser Ala 100 105 110 Ala Gly Gly Cys Ser Ser Ser Ser Ser Phe Asn Leu Lys Arg Pro 115 120 125 Ser Cys Cys Cys Asn Cys Asn Cys Asn Cys Cys Cys Ser Ser Ser Ser 130 135 140 Ser Cys Gly Ala Ala Leu Thr Lys Ser Pro Cys Arg Cys Arg Arg Arg 145 150 155 160 Ser Cys Cys Cys Arg Arg Cys Cys Cys Gly Gly Val Gly Val Arg Ala 165 170 175 Cys Ala Ser Cys Ser Cys Ser Pro Pro Cys Ala Cys Cys Ala Pro Pro 180 185 190 Cys Ala Gly Cys Ser Cys Arg Cys Thr Cys Pro Cys Pro Cys Pro Gly 195 200 205 Gly Cys Ser Cys Ala Cys Pro Ala Cys Arg Cys Cys Cys Gly Val Pro 210 215 220 Arg Cys Cys Pro Pro Cys Leu 225 230 <210> 6 <211> 54 <212> PRT <213> Oryza sativa L. <400> 6 Met Ala Met Ala Ala Ala Pro Arg Pro Lys Ser Pro Pro Ala Pro Pro 1 5 10 15 Asp Pro Cys Gly Arg His Arg Leu Gln Leu Ala Val Asp Ala Leu His 20 25 30 Arg Glu Ile Gly Phe Leu Glu Gly Glu Ile Asn Ser Ile Glu Gly Ile 35 40 45 His Ala Ala Ser Arg Cys 50 <210> 7 <211> 7873 <212> DNA <213> Oryza sativa L. <400> 7 tgcgtgcggt gtccaattgt tctccctctc gcctcgcccc cccaagcaac accgccacct 60 accgtctctc gcgtcgaacc cccgcacaaa acccccgcg aaatctcgcc cggatcgcc 120 ggggaaaggg aggtggtggg agcccgctcg cgatccggga ggaggagg gagcggtgaa 180 tctgctggcc ggcgcgcgcg cgggcgggcg ggttgttcgc gatggcggag gggcggcgct 240 acgcgatcgc gccgcagcta ggtgagggcc ggtctttgtg ggctctgaga ttttcctttt 300 tcttcccccg gtttgtgctc atctgcgtgc gttctgtggt ttgattggtg gatttacgaa 360 gcggctggct ttggtcaacg cctcggtct tgttttggtt agttcctag tactccggtt 420 gcgttccggg tggtggagtt tgtggcgcgg aagccggtgg agctgggagt tttgttggcc 480 ctgttcgttc ggcgcgggat ttggaatttg ggtttggagt ctactgattg cgtgggtggt 540 tggaaacttg gggagttcgt cggaattttt cgtgttcctc tgtcggaatt gtacggagtt 600 tttggtggcc acaggagttg aggtgatgat gcaaagcaga gattccggtc cggtctctaa 660 gactgagcgt tctggactct ggagtgatcg ggaactgttg caactgctct aaaggattgg 720 gaattgtatt tgtgttctgg agatgggctt ggacctaaaa gttggcttag atgcgtagta 780 atttgaaaaa aaagggcgta attctttctc atttacaaat atattcggtt gtctaactct 840 900 gttacgtccg actgaaattt gtgaaatact taagaattac aggagtttcc gtattgctcc 960 tgaacctcca aataggcctc caagtatgat ctccacgagt actgttgtct tataattttt 1020 tgtcacaaaa gtcgcatgtt tattattttt aagcagttga catatacccc tgacctaact 1080 atgttgtttt tggcgggttc attcgttaac ttgtactatg ctgtttaaga acataaaaacg 1140 tgggtgtgtg attttcagtt atcatgctac cctgcccaaa cataaaacat ttgccacttg 1200 atcaaaagcc tatacacctt ctcatttttt aattgtattt cctggcccct tgtgttatca 1260 actttaccatc ctttaatctt tcaattacac tatgctcttt tcctttaatc aatccccttt 1320 tgatcagtta tgattttgtg gtttgcacat ttgatttact actgtcttca ctctcaacac 1380 ctgtgattgc ctcaattacg tacacaaagg atttgataaa gtgtctataa gtaatcagtt 1440 taaggctttg tttttgtttt tttgtatctt actaatattt tgtatccatg gctttaggtg 1500 gttcactttt cttgtttgat cggaaggtcc tgagatattt caggaaggac ggtcataatt 1560 ggaggaagaa aagagatgga aagacagtca aagaagccca tgaaaggtta aaggtaagta 1620 aaaaaattag tattacaaat ttcttctgcg atagtacctt acagcatttg tttaaaaggg 1680 tttctatact ggcagtccag tactctttgt atattaagat aatgactttt ttatttataa 1740 atttagtctg gaagcattga tgtgcttcat tgctactatg ctcatgggga ggagaatata 1800 aatttccaaa ggaggagtta ttggatgttg gaggagtaag tgaactattg ttgatcaatt 1860 attttgcttt tgtactactc tgtgcatctg ctgctcaaag gcacaataac aagtaactaa 1920 tagcatgtgt tcataatgca gcttatatgc atgcacgatg acacaaaaaa caattagttg 1980 atatataatc attttttttg tttttcaggg attacatgca cattgtactt gtacattatc 2040 tggaagtcaa ggttgcacct aatcctgcta tgattgcagt ttattagtt atctgttcat 2100 cttatattg aatttaaatc ttatatacat ctcttatagg agtggaaagc ttcatataaa gtaaattgtt taatagtatt atcgattttc tcgtaaaact acctcatcat gttgattgat agctggcttt tgatgctata caaaattgtt tgtgatccac atttcactgg acccacgtgt ttggtgatat ttttatgtta aaccagtagc taaacaacat atatggcagt tacagaactg ctttagcacc atgatatgtt aagttagtcc tagtgttcc cttctgatat tattaatagt gtggtgaata ttgaaaacag acaatatttt ttaaaacagc agcaagtctt ttagtaaaat 2520. sightseeing sightseeing sightseeing aacattgtcc sightseeing tttttaacta gtctcaggag ggcaccatat gtaatatata tcttatgata aacatattaa atttactata ttaacattta acattgttta atttacattt gaggacctat tatggaaacc ctctgtttgt gttggattta gtggtagtt taacgctctt gcttgctttt ttctaccttg gttacctaat ttctttgttg tcttcacgtt tattttcttt catctaag ctgaagtttc atttcctgca catatatggg tatgaccatc aggctggaaa attaagctct cgtagtacgg gatatgatga tgtactgcaa gcatctcatg cggatagccc tttaagtca ttgccttcac 2880 aaactaccga aggaaagc tcagttagcg gataggccctc tgaatgat gaacagaat 2940 caggtagagc tgtttagcca cctccttctg ttctgcataa agtccattaa atagtttca 3000 ctgatacata ttccaactat acacagcata tctaaagcaa aattcggtgc agcagatatt 3060 tattcgggag gagccagata caaccttc tctcggatgc ggcagcatga aaatggaggt 3120 ggatctgtga ttgatgattc tatattcagc tcttatgttc ctgcgtcatc tgtaggcaag 3180 tggcattaag tataatcttc tcattatgaa cctaagttga atacatgt aagattttat 3240 aaatttatat ttttaaaaa atgttcaggc agttacag gattacaggc agttacaggc 3300 atacaggct tctattctca tggtcagat aacttaccg tggttcttaa cgagtcagac 3360 cttgggactg catttaatgg ccctaatagt cagtttgatc tatcattatg catgaagca 3420 atgaaacctg acaaagggac ccatcaatt cctctttacc aagctcctgt tccctccgaa 3480 cagtctcctt ttacaggagg cccaggaatt gatctttca catttgatga agtatacaac 3540 aatggactga gtatcagga tgtagatggt gatgatacag atggagaac tccatggcag 3600 gtattgtgca tgcatttg tattgcaaat aaaagctc tctcgtggta aaaaggcata 3660 gccatattgg tagatt ttgaataca gtattgacaa cagtagcagc tgcccttcag 3720 taagagaaaa aagagtcaa accttttaaa tatgttgtca ttttggtagg aaactccata 3780 tccattgaga aaaaaaaaa gattgctcg aagttttata atgatttct tattaaaaat 3840 atgcctggct ggagtgtgtt aaatatgtt tattttgtt cgccaagttg tgttttgaga 3900 accttgttt gtgaaatgga gtttaccatt gcctggatgg atttggacaa gtactccctc 3960 cgttttttaa tagatgacgc cgttgacttt ttctcacatg tttgaccatt cgtcttattc 4020 aaaaaattta cgtaattata atttattg tttagagttg tttatcact cataatactt 4080 taagtgtgat tatatctta tacatttgca aaaaaattttt gataagacg aatggccaaa 4140 catgtgagaa aaagtcaacg gcgtcatcta ttaaaaaacg gaggtagtac ttgacaagtg 4200 aaaactgaa cacaatcgaa taacccata ttatgtactc gatgaatact attgaagtttt 4260 aataatgtca agagtcttac atattgtttt tactattgcg attaactact gttgttcctc 4320 aaaattccca tattgaacct tgaatgtttt aagacaaaaa tgacgtattc acaaggtaaa 4380 attagatgaa agtataggga aaaggtgcaa aattatgatg ttaaagagat attgcacagc 4440 taacaaactg tagagatgta gcccaggtcc atgatttgtt caggtttaaa gtctggaatg 4500 gacttatggt attgtctggt ttactgga catagctacg gattttaggg tcttccaatt 4560 ggcatatata tgtgtatata tctttataag cgagcgcctg gttttgtttt ttgatggata 4620 aaactacttc tctttgttgt cttgctagca cttgagatct tcgcaagttt atattgttga 4680 ttgttttgca tactgcactt aattgcagct tcatgagttt catcttacta gtattgtact 4740 atatatatat atatatataa tatttgtca ggattctttt tggttttggt acataactga 4800 gaagctactg aatttcagat tccaaatgca agtggtacat ttgctacagc ggacagcttt 4860 caacaaaatg ataaaacttt ggaggaagcc attaattacc ctctcttgaa aactcaatca 4920 tctagtcttt ctgatatcat aaaggacagt tttaagaaaa atgacagttt cactagatgg 4980 atgagcaaag agcttgctga agtagatgat tctcaaatca catctagttc tggagtgtac 5040 tggaacagtg aagaagctga caatatcatt gaagcatcaa gcagtgatca gtatactctg 5100 ggaccagtgc ttgcacaaga tcagctgttt actatagttg atttttctcc aacctggaca 5160 tatgcaggtt caaagactag ggtatgtctt ctgaaaaccc accattggtg ccttttgaga 5220 tttatttttt ttgacgcaag atgcctttga gattggaggt cttggtagtg tcatcggatt 5280 gtccttgtag ctcagagtga attactgtt gcttacgaat ttctcgtata ctgtaggtat 5340 ttattaaagg taatttctta agttctgatg aagttaagag attaaaatgg tcatgcatgt 5400 ttggagaatt tgaagtccca gctgagatta tagcagatga tactcttgta tgtcattctc 5460 cctcacacaa gcctggcagg gtccctttct atgtgacttg ctccaataga ttggcctgca 5520 gtgaagtacg ggagtttgat ttccgacctc agtacatgga tgctccaagt ccacttggtt 5580 ctacaaacaa aatatatctc cagaagcgtc ttgataaact attgtctgtg gagcaggatg 5640 agatccagac aactctgtct aaccccacga aggagattat tgatttgagc aagaagataa 5700 gttcattgat gttgacaat gttgattggt ctgagttgct aaagttggct gttgataatg agcctgccac agcctgcc tccttcaaaa ccgcatc 5820. agcctgccac atatctggct tctccataaa gtagggatg gaggcaaagg tcccagtatg ttagatgagg 5880 aggggcaggg tgtgcttcat ttggcagctg ccctagggta cgattgggca ataagaccaa 5940 caatagctgc tggtgtgaat ataaacttca gagatgctca tggatggact gcactccact gggctgcatt ttgtggaagg taattgctca gagtttattt attttttt tttttttt gctttatatg tattaaactt gtttgacctg ttgtgcagag agcggacagt cgttgcactt 6120. atagcactag gtgcagctcc tggagctgta acagacccaa caccaagttt cccttcagga agcacaccag ctgatcttgc atcagccaac ggacacaagg gaatctctgg tttcctagca gagtcttctt taacaagtca tctgcagact ctcaatctaa aggaagccat gcgtagcagt gcaggagaaa tatctggtct acctggcatt gtaaatgttg ctgatagaag cgcctcgcct ttggcagttg aaggccatca gactggatct atgggagatt cactaggtgc tgttcgtaat gccgcccaag ctgctgcccg gatataccaa gtttttagga tgcaatcctt ccagagaaaa 6480 caagcagttc agtatgagga tgaaaatggt gcgatatcag atgagcgtgc catgtcactc 6540 ttgtctgcta aaccatccaa acctgcacag cttgatcctc tgcatgctgc tgcgactcga 6600 atacaaaaca agttccgtgg atggaaggga agaaaagaat ttctgcttat tagacagcga 6660 atcgtcaaga tccaggtatg gatgttattg tatgatgata attcacctaa atgttttatg 6720 tattctgaaa gctccagatt aatgttttag acaagattgc cagtgtttta aagatcttct 6780 gctaccgaat tctattttag ctttgatgaa ccataagaaa ttatacttag ttattaaatt 6840 gaatgtgagt tttttcctta ttgagaagcc tcttttcaca gtagaggata tgaatcacat 6900 aattatgttt aacaaacact tacatgcttt gtgactgtat tatagcacta taagtataat 6960 tgatgatttc tttgtcgtcg ttattttctg cttgctaggc gcatgtgcga ggtcaccaag 7020 tgagaaagca ttatcgcaaa ataatttggt ctgttgggat agtggagaag gttatattgc 7080 gctggaggcg acgaggtgct gggttgcgtg ggtttcgacc cacagaaaat gcagtcacag 7140 agagtactag cagcagcagt ggcaacgtga cccaaaacag acctgccgag aatgactatg 7200 atttcttaca agaaggacgg aagcagactg aagaaaggct acagaaagct cttgccagag 7260 tgaaatccat ggttcaatac ccagatgcca gggatcagta ccagaggatt ttgacggtgg 7320 taactaaaat gcaggaatcc caggtactat catgctttca gttttggaat attagagtga 7380 caactgaact aaaattagac aggcaattca tggaatgatt gctgaattct ttactgctaa 7440 taggctatgc aagaaaagat gctcgaggag tcgacagaga tggatgaagg ccttttgatg 7500 agcgaattca aagaactgtg ggatgacgac atgccaacgc ctggatattt ctaggatcta 7560 acgtcttgtg tatgcgcttg taaattactt ccaggtcttt gtacatagca ttaccaaatc 7620 gataacctgt agttgcattg ctaaattttt gtatgttttg aagtttggtt ggtacaataa 7680 tttttgtatg cgttttgtac agtaattctt ctgtaaaatg tatcaaaatg atgtagctcg 7740 tgaaaagctt cagatagttg gagaagctag gtctagaatc tgcagatagt ttgtgtaagg 7800 aaaaatgttt aatccttgct atgttttgac tacggtgtct cgagttcaaa cggttgtcca 7860 ggctgacagc gtc 7873 <210> 8 <211> 2497 <212> DNA <213> Oryza sativa L. <400> 8 ttttttataa ttcgtatttt tattgttgtt agatgataag atatgattaa tattttatgc 60 gtgacttgtt ttttaattt ttttcataa ttttcaaat aagacggacg gtcaaacgtt 120 agacacggaa acctgggttt gtcttttttt ttaaacggaa atagtacgtg agattgtgag 180 cagtgccgag ggaagagctg tagcctgtag aggcaagaaa aagctgaaga aagacgagga 240 tattaggcgc agagtctccg tagactgtcg cacaactcta catgcgggct actgccgcat 300 gatgctcacc ttccaaccat ccgtccctaa tctctaatct tttcaagacg ctgagtaaaa 360 aagctggact gctggatgct gcttaattag tggttgcaaa ttgcaatctg ccgaaccacg 420 aactcaaaac gacatgtaga ttactactag tccccggatg agatgatcac atcccccatc 480 ttaactactc catccgcaag caactgcttt ctcctctcca actttcacgg actgaaagat 540 cctttagaaa ggcgaaaata ataggcaaaa caaagcacat attattatat attaattaa 600 gtatttgcta atttttttat aaatagatt atatgtttt taagcaactt tctaatatat 660 ttttttaaca aacgtattgt ttacaattt aaaaaacgtg cacgcgaaaa atatgagagg 720 aaggttggga aagggagga acgaaggag cctaagcgat aaggagctgg tggtggtgag 780 atagaatat tagcctccct aacatgatgg atcgatggtt ggtggggact atcacaggt 840 gtggccacga aagttaaagg tgtgtctggt ctttttttt taagtttttta agtatacaa 900 cacacatttg agtattaaa tagagactaa tgataaaaaaattacagat tctgcctgta 960 aactgcaaga cgaatttatt aaacatatt atccatcac tagtaatgt ttactgtata 1020 tcacattgtc aaatcatgac gtattagat tcacatt cgtctcgtaa tttacatgta 1080 aactgtgtaa ttggtttt tttctatatt taatgtttca tatatatgta caaatttg 1140 atgtaatgtt ttggccaaa atattctgga tctaagaag gcctaaagtt ggcagcaggg 1200 gtggtcaggt ggtcaggtgg tcagatattc atattagcga tgtgccgatg aggccattgg 1260 gggcggagca agcgtaagt gagacggtga ggccggcgtg cggcaggcgc ccaccgggtg 1320 aggcgaggcg tgggcgagac cgcgaccgga cggcgacagc ggcgaaggac cgaggaatag 1380 catggatatg tactatgtgc gccgcacga agcgctatcg ttgtgggatc tggaccatct 1440 gaagattctg gggaattatg ccttcttggg ccatgctaat cttcttttt aacgaattga 1500 tagtctattt tgcattgaat aagcaaatag tctcaatcca agggcttgtt taattggcaa 1560 aaaatttgc ccatacatat cacatcagat atatggacac acatttgaat tattaaatgt 1620 agtctaataa caaaacaaat tacagattct gccagaaaac cgcgagacga atttattaag 1680 ctaattaatc tgtcattagc aaatatttaa tgtagcacca cattatcaaa tcctgacata 1740 attaggctta aaagattcgt ctcgcaattt tctggcgaac tatgtgattg ttttttttct 1800 acatttaata tctcatatat gtgttcaaac attcggtgcg acagcgtgga aaattttgtt 1860 tgggaactaa acatgtccta aatttgaaca agtccggttt tctcccccag cgctttagat 1920 aattactgat tgtgtgtttg gccaggaact agggctgatg cgccgtcaaa ataagggttt 1980 ctaaatttga aagcagaaaa gatctaggat actatccaaa tgcagaaaat gcaaaactag 2040 gcattgtgtg gaacgcataa atttcataag atttactgtg cctttttgta agaaagtttc father accggaat tatttttcca father agtggtttac gtcgcttctg tcaataaaca cactttgtga tggttgattt ggtgttagac ttttcctcat tagcttgtgt ttgtgtagtt fathers fathers atctggcacg gtttcggaat agacaggaga gcttaactgg cagagctcgc cgttgaaacc cttttggcgc 2400. ctccatcaac cgtccacgtc gtcttcggcc tcactcacca accgaaacgg acggcccaga tcgtcccatt tctcgcgggg ggcgaaccg ccatttcgcc caaactctcc ctcttccttc 2460 cttctagtag ccgctttgcc cgccgatgat gcgtgcg 2497 <210> 9 <211> 3090 <212> DNA <213> Oryza sativa L. <400> 9 atggcggagg ggcggcgcta cgcgatcgcg ccgcagctag atattgagca gatactgaag gaggctcaac gccgatggtt acgtccgact gaaatttgtg aaatacttaa gaattacagg agtttccgta ttgctcctga acctccaaat aggcctccaa gtggttcact tttcttgttt gatcggaagg tcctgagata tttcaggaag gacggtcata attggaggaa gaaaagagat 240 ggaaagacag tcaaagaagc ccatgaaagg ttaaagtctg gaagcattga tgtgcttcat 300 tgctactatg ctcatgggga ggagaatata aatttccaaa ggaggagtta ttggatgttg 360 gaggaggatt acatgcacat tgtacttgta cattatctgg aagtcaaggc tggaaaatta 420 agctctcgta gtacgggaca tgatgatgta ctgcaagcat ctcatgcgga tagcccttta 480 agtcaattgc cttcacaaac taccgaagga gaaagctcag ttagcggaca ggcctctgaa 540 tatgatgaaa cagaatcaga tatttattcg ggaggagcca gatacaactc tttctctcgg 600 atgcggcagc atgaaaatgg aggtggatct gtgattgatg attctatatt cagctcttat 660 gttcctgcgt catctgtagg cagttatcaa ggattacagg ctacagcacc taatacaggc 720 ttctattctc atggtcaaga taacttaccc gtggttctta acgagtcaga ccttgggact 780 gcatttaatg gccctaatag tcagtttgat ctatcattat ggattgaagc aatgaaacct 840 gacaaaggga cccatcaaat tcctctttac caagctcctg ttccctccga acagtctcct 900 tttacaggag gcccaggaat tgaatctttc acatttgatg aagtatacaa caatggactg 960 agtatcaagg atgtagatgg tgatgataca gatggagaaa ctccatggca gattccaaat 1020 gcaagtggta catttgctac agcggacagc tttcaacaaa atgataaaac tttggaggaa 1080 gccattaatt accctctctt gaaaactcaa tcatctagtc tttctgatat cataaaggac 1140 agttttaaga aaaatgacag tttcactaga tggatgagca aagagcttgc tgaagtagat 1200 gattctcaaa tcacatctag ttctggagtg tactggaaca gtgaagaagc tgacaatatc 1260 attgaagcat caagcagtga tcagtatact ctgggaccag tgcttgcaca agatcagctg 1320 tttactatag ttgatttttc tccaacctgg acatatgcag gttcaaagac tagggtattt 1380 attaaaggta atttcttaag ttctgatgaa gttaagagat taaaatggtc atgcatgttt 1440 ggagaatttg aagtcccagc tgagattata gcagatgata ctcttgtatg tcattctccc 1500 tcacacaagc ctggcagggt ccctttctat gtgacttgct ccaatagatt ggcctgcagt 1560 gaagtacggg agtttgattt ccgacctcag tacatggatg ctccaagtcc acttggttct 1620 acaaacaaaa tatatctcca gaagcgtctt gataaactat tgtctgtgga gcaggatgag 1680 atccagacaa ctctgtctaa ccccacgaag gagattattg atttgagcaa gaagaataagt 1740 tcattgatga tgaacaatga tgattggtct gagttgctaa agttggctga tgataatgag 1800 cctgccacag atgataagca agatcagttc cttcaaaacc gcattaagga aaaattgcat 1860 atctggcttc tccataaagt aggggatgga ggcaaaggtc ccagtatgtt agatgaggag 1920 gggcagggtg tgcttcattt ggcagctgcc ctagggtacg attgggcaat aagaccaaca 1980 atagctgctg gtgtgaatat aaacttcaga gatgctcatg gatggactgc actccactgg 2040 gctgcatttt gtggaagaga gcggacagtc gttgcactta tagcactagg tgcagctcct 2100 ggagctgtaa cagacccaac accaagtttc ccttcaggaa gcacaccagc tgatcttgca 2160 tcagccaacg gacacaaggg aatctctggt ttcctagcag agtcttcttt aacaagtcat 2220 ctgcagactc tcaatctaaa ggaagccatg cgtagcagtg caggagaaat atctggtcta 2280 cctggcattg taaatgttgc tgatagaagc gcctcgcctt tggcagttga aggccatcag 2340 actggatcta tgggagattc actaggtgct gttcgtaatg ccgcccaagc tgctgcccgg 2400 atataccaag tttttaggat gcaatccttc cagagaaaac aagcagttca gtatgaggat 2460 gaaaatggtg cgatatcaga tgagcgtgcc atgtcactct tgtctgctaa accatccaaa 2520 cctgcacagc ttgatcctct gcatgctgct gcgactcgaa tacaaaacaa gttccgtgga 2580 tggaagggaa gaaaagaatt tctgcttatt agacagcgaa tcgtcaagat ccaggcgcat 2640 gtgcgaggtc accaagtgag aaagcattat cgcaaaataa tttggtctgt tgggatagtg 2700 gagaaggtta tattgcgctg gaggcgacga ggtgctgggt tgcgtgggtt tcgacccaca 2760 gaaaatgcag tcacagagag tactagcagc agcagtggca acgtgaccca aaacagacct 2820 gccgagaatg actatgattt cttacaagaa ggacggaagc agactgaaga aaggctacag 2880 aaagctcttg ccagagtgaa atccatggtt caatacccag atgccaggga tcagtaccag 2940 aggattttga cggtggtaac taaaatgcag gaatcccagg ctatgcaaga aaagatgctc 3000 gaggagtcga cagagatgga tgaaggcctt ttgatgagcg aattcaaaga actgtgggat 3060 gacgacatgc caacgcctgg atatttctag 3090 <210> 10 <211> 1029 <212> PRT <213> Oryza sativa L. <400> 10 Met Ala Glu Gly Arg Arg Tyr Ala Ile Ala Pro Gln Leu Asp Ile Glu 1 5 10 15 Gln Ile Leu Lys Glu Ala Gln Arg Arg Trp Leu Arg Pro Thr Glu Ile 20 25 30 Cys Glu Ile Leu Lys Asn Tyr Arg Ser Phe Arg Ile Ala Pro Glu Pro 35 40 45 Pro Asn Arg Pro Pro Ser Gly Ser Leu Phe Leu Phe Asp Arg Lys Val 50 55 60 Leu Arg Tyr Phe Arg Lys Asp Gly His Asn Trp Arg Lys Lys Arg Asp 65 70 75 80 Gly Lys Thr Val Lys Glu Ala His Glu Arg Leu Lys Ser Gly Ser Ile 85 90 95 Asp Val Leu His Cys Tyr Tyr Ala His Gly Glu Glu Asn Ile Asn Phe 100 105 110 Gln Arg Arg Ser Tyr Trp Met Leu Glu Glu Asp Tyr Met His Ile Val 115 120 125 Leu Val His Tyr Leu Glu Val Lys Ala Gly Lys Leu Ser Ser Arg Ser 130 135 140 Thr Gly His Asp Asp Val Leu Gln Ala Ser His Ala Asp Ser Pro Leu 145 150 155 160 Ser Gln Leu Pro Ser Gln Thr Thr Glu Gly Glu Ser Ser Val Ser Gly 165 170 175 Gln Ala Ser Glu Tyr Asp Glu Thr Glu Ser Asp Ile Tyr Ser Gly Gly 180 185 190 Ala Arg Tyr Asn Ser Phe Ser Arg Met Arg Gln His Glu Asn Gly Gly 195 200 205 Gly Ser Val Ile Asp Asp Ser Ile Phe Ser Ser Tyr Val Pro Ala Ser 210 215 220 Ser Val Gly Ser Tyr Gln Gly Leu Gln Ala Thr Ala Pro Asn Thr Gly 225 230 235 240 Phe Tyr Ser His Gly Gln Asp Asn Leu Pro Val Val Leu Asn Glu Ser 245 250 255 Asp Leu Gly Thr Ala Phe Asn Gly Pro Asn Ser Gln Phe Asp Leu Ser 260 265 270 Leu Trp Ile Glu Ala Met Lys Pro Asp Lys Gly Thr His Gln Ile Pro 275 280 285 Leu Tyr Gln Ala Pro Val Pro Ser Glu Gln Ser Pro Phe Thr Gly Gly 290 295 300 Pro Gly Ile Glu Ser Phe Thr Phe Asp Glu Val Tyr Asn Asn Gly Leu 305 310 315 320 Ser Ile Lys Asp Val Asp Gly Asp Asp Thr Asp Gly Glu Thr Pro Trp 325 330 335 Gln Ile Pro Asn Ala Ser Gly Thr Phe Ala Thr Ala Asp Ser Phe Gln 340 345 350 Gln Asn Asp Lys Thr Leu Glu Glu Ala Ile Asn Tyr Pro Leu Leu Lys 355 360 365 Thr Gln Ser Ser Ser Leu Ser Asp Ile Ile Lys Asp Ser Phe Lys Lys 370 375 380 Asn Asp Ser Phe Thr Arg Trp Met Ser Lys Glu Leu Ala Glu Val Asp 385 390 395 400 Asp Ser Gln Ile Thr Ser Ser Ser Gly Val Tyr Trp Asn Ser Glu Glu 405 410 415 Ala Asp Asn Ile Ile Glu Ala Ser Ser Ser Asp Gln Tyr Thr Leu Gly 420 425 430 Pro Val Leu Ala Gln Asp Gln Leu Phe Thr Ile Val Asp Phe Ser Pro 435 440 445 Thr Trp Thr Tyr Ala Gly Ser Lys Thr Arg Val Phe Ile Lys Gly Asn 450 455 460 Phe Leu Ser Ser Asp Glu Val Lys Arg Leu Lys Trp Ser Cys Met Phe 465 470 475 480 Gly Glu Phe Glu Val Pro Ala Glu Ile Ile Ala Asp Asp Thr Leu Val 485 490 495 Cys His Ser Pro Ser His Lys Pro Gly Arg Val Pro Phe Tyr Val Thr 500 505 510 Cys Ser Asn Arg Leu Ala Cys Ser Glu Val Arg Glu Phe Asp Phe Arg 515 520 525 Pro Gln Tyr Met Asp Ala Pro Ser Pro Leu Gly Ser Thr Asn Lys Ile 530 535 540 Tyr Leu Gln Lys Arg Leu Asp Lys Leu Leu Ser Val Glu Gln Asp Glu 545 550 555 560 Ile Gln Thr Thr Leu Ser Asn Pro Thr Lys Glu Ile Ile Asp Leu Ser 565 570 575 Lys Lys Ile Ser Ser Leu Met Met Asn Asn Asp Asp Trp Ser Glu Leu 580 585 590 Leu Lys Leu Ala Asp Asp Asn Glu Pro Ala Thr Asp Asp Lys Gln Asp 595 600 605 Gln Phe Leu Gln Asn Arg Ile Lys Glu Lys Leu His Ile Trp Leu Leu 610 615 620 His Lys Val Gly Asp Gly Gly Lys Gly Pro Ser Met Leu Asp Glu Glu 625 630 635 640 Gly Gln Gly Val Leu His Leu Ala Ala Ala Leu Gly Tyr Asp Trp Ala 645 650 655 Ile Arg Pro Thr Ile Ala Ala Gly Val Asn Ile Asn Phe Arg Asp Ala 660 665 670 His Gly Trp Thr Ala Leu His Trp Ala Ala Phe Cys Gly Arg Glu Arg 675 680 685 Thr Val Val Ala Leu Ile Ala Leu Gly Ala Ala Pro Gly Ala Val Thr 690 695 700 Asp Pro Thr Pro Ser Phe Pro Ser Gly Ser Thr Pro Ala Asp Leu Ala 705 710 715 720 Ser Ala Asn Gly His Lys Gly Ile Ser Gly Phe Leu Ala Glu Ser Ser 725 730 735 Leu Thr Ser His Leu Gln Thr Leu Asn Leu Lys Glu Ala Met Arg Ser 740 745 750 Ser Ala Gly Glu Ile Ser Gly Leu Pro Gly Ile Val Asn Val Ala Asp 755 760 765 Arg Ser Ala Ser Pro Leu Ala Val Glu Gly His Gln Thr Gly Ser Met 770 775 780 Gly Asp Ser Leu Gly Ala Val Arg Asn Ala Ala Gln Ala Ala Ala Arg 785 790 795 800 Ile Tyr Gln Val Phe Arg Met Gln Ser Phe Gln Arg Lys Gln Ala Val 805 810 815 Gln Tyr Glu Asp Glu Asn Gly Ala Ile Ser Asp Glu Arg Ala Met Ser 820 825 830 Leu Leu Ser Ala Lys Pro Ser Lys Pro Ala Gln Leu Asp Pro Leu His 835 840 845 Ala Ala Ala Thr Arg Ile Gln Asn Lys Phe Arg Gly Trp Lys Gly Arg 850 855 860 Lys Glu Phe Leu Leu Ile Arg Gln Arg Ile Val Lys Ile Gln Ala His 865 870 875 880 Val Arg Gly His Gln Val Arg Lys His Tyr Arg Lys Ile Ile Trp Ser 885 890 895 Val Gly Ile Val Glu Lys Val Ile Leu Arg Trp Arg Arg Arg Gly Ala 900 905 910 Gly Leu Arg Gly Phe Arg Pro Thr Glu Asn Ala Val Thr Glu Ser Thr 915 920 925 Ser Ser Ser Ser Gly Asn Val Thr Gln Asn Arg Pro Ala Glu Asn Asp 930 935 940 Tyr Asp Phe Leu Gln Glu Gly Arg Lys Gln Thr Glu Glu Arg Leu Gln 945 950 955 960 Lys Ala Leu Ala Arg Val Lys Ser Met Val Gln Tyr Pro Asp Ala Arg 965 970 975 Asp Gln Tyr Gln Arg Ile Leu Thr Val Val Thr Lys Met Gln Glu Ser 980 985 990 Gln Ala Met Gln Glu Lys Met Leu Glu Glu Ser Thr Glu Met Asp Glu 995 1000 1005 Gly Leu Leu Met Ser Glu Phe Lys Glu Leu Trp Asp Asp Asp Met Pro 1010 1015 1020 Thr Pro Gly Tyr Phe 1025 <210> 11 <211> 7283 <212> DNA <213> Oryza sativa L. <400> 11 accccgaaaa atcccccaca aaaaaaagaa aaaaaaaccc aaaccccctc gcgcggaggc 60 cgagagagat cccccgctcc gctccgatcc ccaggcgtgc gcgcgtgcgg ggctcgccgc 120 gcgtcgcatg gtggcggcgc ggccgccgga gcacacgcct cggcctcgat accccccacc 180 acagccatgg cggcggcggc ggacgcgcgg cgcttcgcgg tcgtgccgca gctaggtgag 240 cataacacgc gcggggtttg ggcggagagc cgcggctgcg tcgccgtcgc tctggtccgg 300 atgggggcac gccgcgcgtg tcgcgcgggg gtgctttgtc ggctcgttcc gatctggtgt 360 ggtggatgga tcatggatgg acaggggagg gggggtcgtc gttggtcgcg gtcaacgcgc 420 cggtcgtggt tacctcgcgg ctattgcttg gggggggggg ggggtggggg tagcggaggg 480 attggtagag ctgtagtgga tttttttttt gttttgtttg gcgttgtgga agatttttcg 540 ttcgattctg ctgcttcgag tgttggtgta gatcgggctc ctacggattg gtttcatttc 600 tcggtgatag tctctctgtt tttttagtcc attgctccag tgattattac tgttctagta 660 aatgaaatgc ggtgtcgttg catttagcgt tgaatggagt aagtattggg ctggattttg 720 gagcttggac tgcagcgcgc acaatttagt ttcgtgcgta gagcagttgg ttgtactact 780 ttttttggtt cagtcatgct tttgttcccc tttcgaaacg aggggatcat gttggccggc 840 ctgcagttct ccacttgtgt ggacaagagt gttagtagta tcggttggtt ctgaaagaga 900 attttgttat gtgatgatgc tgtctatgat ttttttttt tgagaaatat ggtgcactgt 960 ttaactgtcg ctgactctgt gatctataaa cagatattgc gcagatactg aaggaggctc 1020 aacaacgatg gttgcgtcct gctgaaattt gcgaaatact taaaaactac aaaagtttcc 1080 gtattgcacc agaaccacca aacaggcctc aaagtaggat acttgtctcc ttaattaaat 1140 gttgctctaa tttgttgtgc tttgtattg aggttatttt tatcggtgaa tcatcataat 1200 taaaaataat catttgaaaa aaatactagg agaaacatga aatttgattg catgctaatt 1260 tgtgaaaata tctttaacct taataatatg aagttctgaa cttctatctt atagctgcct 1320 gcatgtatta tggtagtttt agcttgttat ttaccttttg tgccacgttg tattcctgtt 1380 taatttctgt gtgcatcata agtaaataaa acatactacc tccgtttttt ttatgtatga 1440 cgccgttgac ttttcacata atgtttgacc attcatcatt ttcaaaaatt tagtataaat 1500 atgtagaagt atagttaag atttagtttt ctttgatggt aaaaacaagctc acaaaaat 1560 aaatgacatt tattatata tattttgaa window gatcaacgt cacgtcaaa 1620 gccaacaa tcatagatca aacattaaaaaacggaggta gtatattatta ttactggggct 1680 ccttatgact gttacaatgg aacactgtc aggctgcatt agtgaggtat caatgcat 1740 ttcttttacc atcaagagt tattctattc tcttgactt gatgcctttc cttcatgaaa 1800 aaaatactg tattgcacat aaaacagat gtactggtat gtgaatttaa aaatctactt 1860 tttattgacc tgatcgttta gttgcacac tagtaacag tcactttc tcattacc 1920 attack gcatgcatgt aatgaagtt gataaagtgc ctataccatc ctcaagttca 1980 atatttctga tcattcttgc cctgcctagg tggctcgctt tcctattg atcggaaagt 2040 attgagatac tttaggagg acggccataa tggaggag aaaaaggatg gaagactgt 2100 caaagaagca catgaaaggc tgaaagtaag tcacttgcg ttatgtagtg atttattac 2160 aatgtgaata agactcactg ttgtttgcca atccagtctg gaagtattga tgtgcttcac 2220 tgctactatg cacatggaga agagaatgag aacttccaaa ggaggactta ctggatgttg 2280 gaagagtaag ccgaattctt ccaaaaatat tacatgttcg tttatgttat tagcaatgct 2340 ataattgttc tgagcctatg aggctatgaa cacaaattct tttcagagg cgcataaatg 2400 aacacacttg catgtagagt ttgcatacag aattttagtt atttgcattt ggataacctt 2460 atagagttac ccttgctttt gcagggattt tatgcacatt gttcttgtac attatcttga 2520 gaccaaggta ttactgaact ctcctatctt tcacttaatg attagcttat atctacctta 2580 atctacattc catctttcgg tatccaaaag ttacatgcat tgtttagcgc cataatgttt 2640 gattcctttt tctggcgctt tcattttttt tattgtggac tttatttatt ttaattttt 2700 attgaccaaa tactagtact atattgcacc actgtgctac gaaattgtag tactatgatt 2760 atttagtttt gatgattgat attcacataa tttgggatat cacttatgta tgctttcgtg 2820 cttgctagca atctttacat gcatttttca gaaatacaca ttttcctttt gtatttgtac 2880 ttaaggtggt gcttgttacg tgtaatgatt tttgaattaa ttatatgtca gctaccaatt 2940 ccacgataca gaaaacagcg gtatgatacc aactctagga gttacatgat attgacctga 3000 cttaatgtaa gtctcatatt agaattaga taaatattag gtgttaacat ctgatttttt 3060 ttgtagtatc attatataat gataatgaat atgtcttaa aattagacgg aacagtgtgt 3120 aattttttc atttgtatag ttggactag ttttagaac agagtgctat tattttctga 3180 tttatctgct tagtgaattt tcattcattt ccatttccat ttctgct caagtcagtt 3240 ttcttatttc acattgtga tagaattcct cattgcctgt tgaagagc ttttcctct 3300 cttgttttgc tcttgtaaga agtcacttaa ccctcattgc atcgtgctat gcgactatga 3360 acttcagggt gggaaatctc gtactagggg cacaatgac atgcatcag cagctgttat 3420 ggatagtcca ttaagtcaat taccttcaca aactatagat ggtgaaagct cacttagtgg 3480 acagttctct gatatgaag agggagaatc agtaaaaat gttggctgtc ttctcggtt 3540 atctataatg ataatgaaaa tactacacag aaaaaaatag ttcactgat acatattcca 3600 actatgcaca gcgtatctaa agcaaaattc ggtgcagcag atgttttc aggaggaacc 3660 ggataccact ctttcactca gatgcagcag cagcaaaatg gaattggacc tgtgactgat 3720 gcttctatgt tcagttctcg tgttctgct tcatccatag gtaattaaca agagaaatac 3780 gcatgtggtt tgaacctaag tgaagtcgat attaacttct tttgtcacat gatttcaggt 3840 aattatcagg gacaacatgc tatgggacat acaacaaact tctattctag tagtcaacat 3900 gattcacctc ttgttctcag tgatccaaac cttgaacttg caaataatgg acatgagtca 3960 ttgtggaatg gggttatgaa acctgatgaa gggactgttc aaatgactca tctacaacct 4020 cctgttcatc ctgagcaagg catgttcacc acagaaggcc aaggggttga atatttgaca 4080 ttcgatgaag tatattctga tggactcagt ctgaaggata taggtgctgc aggtgctgat 4140 gtagaaccat tctggcaggt atgtgcaagt tttgtaatag atgctaaaat attgtcctgt 4200 tgtaaagtta atagttgctc tcatatggta attcttcccc atattgtgcg ctaaaaatca 4260 aagtttcaac tctcatatgg taattcttcc ctatattggc gctcttggtg cttttgtctg 4320 gtttagtttt cttcaaacag ctatctaaca gaagccattg catttcagct ctctagtgct 4380 actgctgata tatctgcaac agagacagc gtccacaaa atgatggatc tctaggggca 4440 gctattggct tcccatttt gaaaactcag tcatccaatc tatctgacat cctgaaagac 4500 agctttaaga aatctgacag ttcaga tggatgagca agagcttct tgatgttgag 4560 gattcgcaaa ttcagtctag ctcaggggca tactggaaca ctgaagaggc agatagtatc 4620 atcgaagcat caagccgtga gccactggat cagttcaccg tggcccctat ggttttgcaa 4680 gaccagctat tcagtatagt tgattttcg ccaagctgga catagcagg ctcgaagacc 4740 aaggtatagt tttctgatgg tgaaactttt cagcttttt tttgttga aattccagct 4800 aaatagtgt tctccaggtg ctcacaaata tgttgtagc aatgtaggtt ttagttactg 4860 gtagattctt acatgctaat gaagtcacgg agagatgcaa gtggtcatgc atgtttggag 4920 aagttgaaat tcaagcggag atttcagcag atgggactct tcgattt tctcccccgc 4980 aaaccagg cagagtccct ttctatgtca cctgttccaa caggttagcc tgtagtgaag 5040 tacgtgagtt tgaatttcga ccaagtgact ctcaatacat ggatgctcct agcccgcttg 5100 gtgcaaccaa caaagtttat ttccagatac gtcttgacaa cctattgtcc ctggggccag 5160 5220 ttagttcact gttggcaaac aatgatgagt ggtccaagtt acttaaattg gctgatgata 5280 atgagcctct tagccatgat cagcaggatc agtatgctga aaacttaatt aaagaaaat 5340 tgcatgtctg gcttctccat aaagtaggcg atggtggcaa aggacccagt gtgttagatg 5400 atgaggggct gggtgttctt catcttgcag ctgcccttgg ttatgattgg gccataaggc 5460 caactgttac tgctggtgtg aaataaatt tcagagattt tcatggatgg actgcattac 5520 attgggctgc attttgtggc aggtaagtga ctaaaatggt gaattaggct aagagtatgt 5580 tttttcttta atttttgaat attggacagt aaatactctt tgatctatgt tgcagagaac 5640 ggactgtagt agcacttatt gcactgggag cagctcctgg agctttgaca gatccacatc 5700 ctaattaccc tgctgaaagc acaccagcag accttgcatc tgctaacgga cataagggca 5760 tctctggttt cctggcagag tcctctctga caagtcatct tcaagccctc aatctgaagg 5820 aagccaatat gtctgaaata tctggtctac ctggtattgg agatgtaact gagagaaatg 5880 catctcagcc tgcaattgga gattccttag gtgctgttcg aaatgccgct caagctgctg 5940 ctcgaatata tcaagttttc agggtgcaat ccttccagag gaaaagcg gtccaatatg 6000 agggtgacaa aggtggcata tcggacgaac acgcactttc actcctgtct atgaagccat 6060 ccaagtcagg ccaagcttgat cccctgcatg ctgcagcatc tcgtatacaa ataaatata 6120 ggggatggaa ggggagaaag gaatttcttc tcttcagaca acgaattgtg aagatccagg 6180 tactgtatat tatatatatt gaaatccaac agataattta gtttcccata tttctcaata 6240 gttgcattgc tcttatcaaa attttgtttg tctgccactg ggaattaa tgaacacctg 6300 gatggatatt aagatcaaga gtactatagt gggataatct ttgttttacc gtctaatccg 6360 tcattggcta atgctgatat atatgttat atatcttttt gctggctagg ctcatgtgcg 6420 6480 agttatattg cgctggaggc gaaaggc tgggttacgt ggttttaggc ctacagaagg 6540 tgcaatagag agcagcagtg gtggcacaag tagcaatttg gtcaaagata aacctgctgg 6600 agatgattat gatttcttgc aagaaggacg gaagcaaact gaagaaaggc tgcagaaagc 6660 tcttgccaga gtgaagtcca tggttcaata cccagaagca agggatcagt accaaaggat 6720 tttgaatgtt gttcgaaaaa tgcaagagtc tcaggtaatg tcctggttcc ctataaacaa 6780 gtagcattaa gcgttgctgt tatgcttac tctgcaactt ttatccaaat ggaacactta 6840 cgccgtttgt ccctgctgat atgacgacag accgtgcaag aaaagatttt agacgaatca 6900 accgagatgg atgaaggtga tttcatgagt gaattcaagg agctgtggga tgatgataca 6960 ccactccctg gttatttcta gtgttctaga ctaactggtg gtgtctctgt aaatctcttc 7020 tcttcagttc tgctgaattg acacctgccg ataacccaaa ctctcaacat agcatgaata 7080 gcatgatctt aaaatcttga atgcttggat ttggattcag ttgtggtatt cttgctttta 7140 tgttatagcg ttatgtacag tagaatctta tttatcatt gaaggagcca tgtatatgca 7200 gtatgaaact aatgcagaaa gttggtggtt gtatcatttg aattcaaagc tgcatgtgtg 7260 ctaagcaatt tcttgcatta gag 7283 <210> 12 <211> 2446 <212> DNA <213> Oryza sativa L. <400> 12 ccgacacagt gtcagccggt cagaccgcgg gctggccggt ccgaccgctc gatcaccgcc 60 ggtctgaccg gcaacccctg cccggtctga ccggaccaca taaaatcaca gcagtatcct 120 gtaaattcac ctgtaaatcc aatcatctcc aaaattactt tgtgaataaa ttccaaatac 180 aaaaccaata atctccaatg cccaattgtt catcacagaa tataatcaa aaacaccttt 240 gattttacat acaatcttcg taggattgtc catccactgt gaattttaaa ggaaaggata 300 agattcaatc atttatttta aaaggcttt attaaaaataa ttctcataaa attgaaatcc 360 tcaaattatc ctatgggaag attccatcca taccagaccg aaaccataaa ataccacaag 420 agtttactat agcaacatca aaacttccaa tctatcccac aaaatttccc ttttccttat 480 gaaaaaaatt tgctaattct acaattaccc ttatcgtctt cccttgcta caccgaacaa 540 agaagtccct agacaggttt ctcgaggtcg aaggttggtc cgcaccgccg ttgaaggatg 600 agctcaagcg ccatcgaaga tgctacccaa gaggagcttg atccacaccg tagacactag 660 gataggcttg aacgttgcca ccgctgccaa gatgtggtcg aaccacacca ttactgccta 720 gaatgaggtt gatccacact gcaattaccc acaataagtt caagcgccac ctttgttgca 780 tggagtgatc ctgaccgatg acaatgacat tgcttgggac aagcttgatc tatggcgacg 840 ccgctgggag gcttgcctga gtccatgtcg ctggccattg ctcttggatg agctttgacc 900 aacggtagca gggaggagaa gggcaagagt gtcattgcac tttgttcagt tgtactataa 960 attagaaccc tccaaaaatg tgatattta tgttttttga acaaatcttc gtggtatgga 1020 tgaaaatttc atatcgtata tattacatac ggatatcaac agacttaaag tgagtactct 1080 ttgattgcta tttttccatt taatttgagc acgttttcca aacggctaag cactacgttc 1140 ccttaaagag ccttttatgt agaagttttt aaaaaataat ataatttttc aagtttataa 1200 tagttaatgt ttaattaata atatgctaat aacctctctt attttgcaca cccttaataa 1260 gctcagctaa taaactagat cgaactcagg cacttatgtg taggctatag cactgctatt 1320 ttcatactcc ttcgtccca aaaayagagg cctcatcttt ttttattact tatgcttatc 1380 agtcaaattt tgaatttca accttaaata tggagttgat ttggggtttt ttcattgtag 1440 tttatttgt agtctttgct tttagatcgt taagacacg catataaaag tattattac 1500 aaattatttt tcatttgcaa atatgccaat aagccaacg atggggatga agttcatttt 1560 tcatctatac cacacatacc ataccaac agaaagact atatagtcc cactttgtca 1620 aatccaaatg caatatttt tctttttata tattcccatg catttatttt atattttcac 1680 attackcccga tgaaacgatt gtttgaaat aaatgtattt gggacaacg gaaggaaaa 1740 aaaaatctaa tttggacg gagggattag tgatggttc acggcgcaggt gcggtgttgg 1800 agtgcggctt cttgtcggct gccccaccaaaaga agctcccag ttcccaccgtc 1860 ctgcctgcag gatatgtccg gagcgatcac actgcactta gctagtgtct tattttcctt 1920 ttttatttt ttcactcac atactcttt atccacacgc atattttca gacggctaaa 1980 caatacgtttt tttttaaagt tttctataga aaagctattt taaaaaatca tattaatcta 2040 ttttcaaact ttttagttaa tactaaatta ttcacgcgtt aatgagtaac atttttttac 2100 gagctccacc tcctggactc agactaaagg gctcattgtt tgcttatgct tataaatcaa 2160 agtttaaatt ttaaaactta atttaaatt taaatttttg gtttttccat cgcagtttat 2220 tttaaagtat tctctgtaaa atcattaaaa acacgtacat gaaagtttta ctcgtaggaa 2280 atatgaataa gctaaacaat gacacgtaaa actcaggggc aaaaccgtct ttctcctcat 2340 ccagctacag tcatccagag acagcctgcc tcagctccac ccgttcccat cgatccatcc 2400 ccgaaaccag cgagtgaaaa catccctcct ccctcctttt catttc 2446 <210> 13 <211> 3072 <212> DNA <213> Oryza sativa L. <400> 13 atggcggcgg cggcggacgc gcggcgcttc gcggtcgtgc cgcgctaga tattgcgcag 60 atactgaagg aggctcaaca acgatggttg cgtcctgctg aaatttgcga ataacttaaa 120 aactacaaaa gtttccgtat tgcaccagaa ccaccaaaca ggcctcaaag tggctcgctt 180 ttcctatttg atcggaaagt attgagatac tttaggaagg acggccataa ttggaggaag 240 aaaaaggatg gaaagaactgt aaagaagca catgaaaggc tgaaatctgg aagtattgat 300 gtgcttcact gctactatgc acatggagaa gagatgaga acttccaaag gagacttac 360 tggatgttgg aagaggattt tatgcacatt gttcttgtac attatcttga gaccaagggt 420 gggaaatctc gtactagggg caacaatgac atgcatcaag cagctgtttat ggatagtcca 480 ttaagtcaat taccttcaca aactagat ggtgaaagct cacttagtgg acagttctct 540 gaatatgaag aggcagaatc agatgtttat tcaggagagaa ccggatacca ctctttcact 600 cagatgcagc agcagcaaaa tggaattgga cctgtgactg atgcttctat gttcagttct 660 cgtgtttctg cttcatccat aggtaattat cagggacaac atgctatggg acatacaaca 720 aacttttatt ctagtagtca acatgattca cctcttgttc tcagtgatcc aaaccttgaa 780 cttgcaaata atggacatga gtcattgtgg aatggggtta tgaaacctga tgaagggact 840 gttcaaatga ctcatctaca acctcctgtt catcctgagc aaggcatgtt caccacagaa 900 ggccaagggg ttgaatattt gacattcgat gaagtatatt ctgatggact cagtctgaag 960 gatataggtg ctgcaggtgc tgatgtagaa ccattctggc agctctctag tgctactgct 1020 gatatatctg caacagagaa cagcgtccaa caaaatgatg gatctctagg ggcagctatt 1080 ggcttcccat ttttgaaaac tcagtcatcc aatctatctg acatcctgaa agacagcttt 1140 aagaaatctg acagttttac aagatggatg agcaaagagc ttcttgatgt tgaggattcg 1200 caaattcagt ctagctcagg ggcatactgg aacactgaag aggcagatag tatcatcgaa 1260 gcatcaagcc gtgagccact ggatcagttc accgtggccc ctatggtttt gcaagaccag 1320 ctattcagta tagttgattt ttcgccaagc tggacatatg caggctcgaa gaccaaggtt 1380 ttagttactg gtagattctt acatgctaat gaagtcacgg agagatgcaa gtggtcatgc 1440 atgtttggag aagttgaaat tcaagcggag atttcagcag atgggactct tcgatgttat 1500 tctcccccgc ataaaccagg cagagtccct ttctatgtca cctgttccaa caggttagcc 1560 tgtagtgaag tacgtgagtt tgaatttcga ccaagtgact ctcaatacat ggatgctcct 1620 agcccgcttg gtgcaaccaa caaagtttat ttccagatac gtcttgacaa cctattgtcc 1680 ctggggccag atgtgtacca agctactata actaacccca gtaaggagat gatcgacttg 1740 next to you ttagttcact gttggcaaac aatgatgagt ggtccaagtt acttaaattg 1800 gctgatgata atgagcctct tagccatgat cagcaggatc agtatgctga aaacttaatt 1860 aaggaaaaat tgcatgtctg gcttctccat aaagtaggcg atggtggcaa aggacccagt 1920 gtgttagatg atgaggggct gggtgttctt catcttgcag ctgcccttgg ttatgattgg 1980 gccataaggc caactgttac tgctggtgtg aaataaatt tcagagattt tcatggatgg 2040 actgcattac attgggctgc attttgtggc agagaacgga ctgtagtagc acttattgca 2100 ctgggagcag ctcctggagc tttgacagat ccacatccta attaccctgc tgaaagcaca 2160 ccagcagacc ttgcatctgc taacggacat aagggcatct ctggtttcct ggcagagtcc 2220 tctctgacaa gtcatcttca agccctcaat ctgaaggaag ccaatatgtc tgaaatatct 2280 ggtctacctg gtattggaga tgtaactgag agaaatgcat ctcagcctgc aattggagat 2340 tccttaggtg ctgttcgaaa tgccgctcaa gctgctgctc gaatatatca agttttcagg 2400 gtgcaatcct tccagaggaa acaagcggtc caatatgagg gtgacaaagg tggcatatcg 2460 gacgaacacg cactttcact cctgtctatg aagccatcca agtcaggcca gcttgatccc 2520 ctgcatgctg cagcatctcg tatacaaaat aaatataggg gatggaaggg gagaaaggaa 2580 tttcttct tcagacaacg aattgtgaag atccaggctc atgtgcgagg ccaccaggtg 2640 aggaagcatt atcggaaaat agtttggtct gttgggatag tggagaaagt tatattgcgc 2700 tggaggcgaa gaagggctgg gttacgtggt tttaggccta cagaaggtgc aatagagagc 2760 agcagtggtg gcacaagtag caatttggtc aaagataaac ctgctggaga tgattatgat 2820 ttcttgcaag aaggacggaa gcaaactgaa gaaaggctgc agaaagctct tgccagagtg 2880 aagtccatgg ttcaataccc agaagcaagg gatcagtacc aaaggatttt gaatgttgtt 2940 tcgaaaatgc aagagtctca gaccgtgcaa gaaaagattt tagacgaatc aaccgagatg 3000 gatgaaggtg atttcatgag tgaattcaag gagctgtggg atgatgatac accactccct 3060 ggttattct ag 3072 <210> 14 <211> 1023 <212> PRT <213> Oryza sativa L. <400> 14 Met Ala Ala Ala Ala Asp Ala Arg Arg Phe Ala Val Val Pro Gln Leu 1 5 10 15 Asp Ile Ala Gln Ile Leu Lys Glu Ala Gln Gln Arg Trp Leu Arg Pro 20 25 30 Ala Glu Ile Cys Glu Ile Leu Lys Asn Tyr Lys Ser Phe Arg Ile Ala 35 40 45 Pro Glu Pro Pro Asn Arg Pro Gln Ser Gly Ser Leu Phe Leu Phe Asp 50 55 60 Arg Lys Val Leu Arg Tyr Phe Arg Lys Asp Gly His Asn Trp Arg Lys 65 70 75 80 Lys Lys Asp Gly Lys Thr Val Lys Glu Ala His Glu Arg Leu Lys Ser 85 90 95 Gly Ser Ile Asp Val Leu His Cys Tyr Tyr Ala His Gly Glu Glu Asn 100 105 110 Glu Asn Phe Gln Arg Arg Thr Tyr Trp Met Leu Glu Glu Asp Phe Met 115 120 125 His Ile Val Leu Val His Tyr Leu Glu Thr Lys Gly Gly Lys Ser Arg 130 135 140 Thr Arg Gly Asn Asn Asp Met His Gln Ala Ala Val Met Asp Ser Pro 145 150 155 160 Leu Ser Gln Leu Pro Ser Gln Thr Ile Asp Gly Glu Ser Ser Leu Ser 165 170 175 Gly Gln Phe Ser Glu Tyr Glu Glu Ala Glu Ser Asp Val Tyr Ser Gly 180 185 190 Gly Thr Gly Tyr His Ser Phe Thr Gln Met Gln Gln Gln Gln Asn Gly 195 200 205 Ile Gly Pro Val Thr Asp Ala Ser Met Phe Ser Ser Arg Val Ser Ala 210 215 220 Ser Ser Ile Gly Asn Tyr Gln Gly Gln His Ala Met Gly His Thr Thr 225 230 235 240 Asn Phe Tyr Ser Ser Ser Gln His Asp Ser Pro Leu Val Leu Ser Asp 245 250 255 Pro Asn Leu Glu Leu Ala Asn Asn Gly His Glu Ser Leu Trp Asn Gly 260 265 270 Val Met Lys Pro Asp Glu Gly Thr Val Gln Met Thr His Leu Gln Pro 275 280 285 Pro Val His Pro Glu Gln Gly Met Phe Thr Thr Glu Gly Gln Gly Val 290 295 300 Glu Tyr Leu Thr Phe Asp Glu Val Tyr Ser Asp Gly Leu Ser Leu Lys 305 310 315 320 Asp Ile Gly Ala Ala Gly Ala Asp Val Glu Pro Phe Trp Gln Leu Ser 325 330 335 Ser Ala Thr Ala Asp Ile Ser Ala Thr Glu Asn Ser Val Gln Gln Asn 340 345 350 Asp Gly Ser Leu Gly Ala Ala Ile Gly Phe Pro Phe Leu Lys Thr Gln 355 360 365 Ser Ser Asn Leu Ser Asp Ile Leu Lys Asp Ser Phe Lys Lys Ser Asp 370 375 380 Ser Phe Thr Arg Trp Met Ser Lys Glu Leu Leu Asp Val Glu Asp Ser 385 390 395 400 Gln Ile Gln Ser Ser Ser Gly Ala Tyr Trp Asn Thr Glu Glu Ala Asp 405 410 415 Ser Ile Ile Glu Ala Ser Ser Arg Glu Pro Leu Asp Gln Phe Thr Val 420 425 430 Ala Pro Met Val Leu Gln Asp Gln Leu Phe Ser Ile Val Asp Phe Ser 435 440 445 Pro Ser Trp Thr Tyr Ala Gly Ser Lys Thr Lys Val Leu Val Thr Gly 450 455 460 Arg Phe Leu His Ala Asn Glu Val Thr Glu Arg Cys Lys Trp Ser Cys 465 470 475 480 Met Phe Gly Glu Val Glu Ile Gln Ala Glu Ile Ser Ala Asp Gly Thr 485 490 495 Leu Arg Cys Tyr Ser Pro Pro His Lys Pro Gly Arg Val Pro Phe Tyr 500 505 510 Val Thr Cys Ser Asn Arg Leu Ala Cys Ser Glu Val Arg Glu Phe Glu 515 520 525 Phe Arg Pro Ser Asp Ser Gln Tyr Met Asp Ala Pro Ser Pro Leu Gly 530 535 540 Ala Thr Asn Lys Val Tyr Phe Gln Ile Arg Leu Asp Asn Leu Leu Ser 545 550 555 560 Leu Gly Pro Asp Val Tyr Gln Ala Thr Ile Thr Asn Pro Ser Lys Glu 565 570 575 Met Ile Asp Leu Ser Lys Lys Ile Ser Ser Leu Leu Ala Asn Asn Asp 580 585 590 Glu Trp Ser Lys Leu Leu Lys Leu Ala Asp Asp Asn Glu Pro Leu Ser 595 600 605 His Asp Gln Gln Asp Gln Tyr Ala Glu Asn Leu Ile Lys Glu Lys Leu 610 615 620 His Val Trp Leu Leu His Lys Val Gly Asp Gly Gly Lys Gly Pro Ser 625 630 635 640 Val Leu Asp Asp Glu Gly Leu Gly Val Leu His Leu Ala Ala Ala Leu 645 650 655 Gly Tyr Asp Trp Ala Ile Arg Pro Thr Val Thr Ala Gly Val Asn Ile 660 665 670 Asn Phe Arg Asp Phe His Gly Trp Thr Ala Leu His Trp Ala Ala Phe 675 680 685 Cys Gly Arg Glu Arg Thr Val Val Ala Leu Ile Ala Leu Gly Ala Ala 690 695 700 Pro Gly Ala Leu Thr Asp Pro His Pro Asn Tyr Pro Ala Glu Ser Thr 705 710 715 720 Pro Ala Asp Leu Ala Ser Ala Asn Gly His Lys Gly Ile Ser Gly Phe 725 730 735 Leu Ala Glu Ser Ser Leu Thr Ser His Leu Gln Ala Leu Asn Leu Lys 740 745 750 Glu Ala Asn Met Ser Glu Ile Ser Gly Leu Pro Gly Ile Gly Asp Val 755 760 765 Thr Glu Arg Asn Ala Ser Gln Pro Ala Ile Gly Asp Ser Leu Gly Ala 770 775 780 Val Arg Asn Ala Ala Gln Ala Ala Ala Arg Ile Tyr Gln Val Phe Arg 785 790 795 800 Val Gln Ser Phe Gln Arg Lys Gln Ala Val Gln Tyr Glu Gly Asp Lys 805 810 815 Gly Gly Ile Ser Asp Glu His Ala Leu Ser Leu Leu Ser Met Lys Pro 820 825 830 Ser Lys Ser Gly Gln Leu Asp Pro Leu His Ala Ala Ala Ser Arg Ile 835 840 845 Gln Asn Lys Tyr Arg Gly Trp Lys Gly Arg Lys Glu Phe Leu Leu Phe 850 855 860 Arg Gln Arg Ile Val Lys Ile Gln Ala His Val Arg Gly His Gln Val 865 870 875 880 Arg Lys His Tyr Arg Lys Ile Val Trp Ser Val Gly Ile Val Glu Lys 885 890 895 Val Ile Leu Arg Trp Arg Arg Arg Arg Ala Gly Leu Arg Gly Phe Arg 900 905 910 Pro Thr Glu Gly Ala Ile Glu Ser Ser Ser Gly Gly Thr Ser Ser Asn 915 920 925 Leu Val Lys Asp Lys Pro Ala Gly Asp Asp Tyr Asp Phe Leu Gln Glu 930,935,940 Gly Arg Lys Gln Thr Glu Glu Arg Leu Gln Lys Ala Leu Ala Arg Val 945 950 955 960 Lys Ser Met Val Gln Tyr Pro Glu Ala Arg Asp Gln Tyr Gln Arg Ile 965,970,975 Leu Asn Will Be Lys Met Gln Glu Serve Gln Thr Will Gln Glu Lys 980,985,990 Ile Leu Asp Glu Ser Thr Glu Met Asp Glu Gly Asp Phe Met Ser Glu 995 1000 1005 Phe Lys Glu Leu Trp Asp Asp Thr Pro Leu Pro Gly Tyr Phe 1010 1015 1020 <210> 15 <211> 1575 <212> DNA <213> Oryza sativa L . <400> 15 atcttagcca cattgaacgc acgctacact gcaagaagg caagtatagg ggtggcaggt 60 agtttatatt cactggaaac cgagaacttc actcctcca gcactccaca ctaggaggat 120 gctcactgtc tgactgactg actggctgct gctgctgctc attccttc ctactctgcc 180 tgcctcctct gctgtgtcct tgggagaatt cttcagggac agtatccctg cagaggtgag 240 atcatgggac agtcgaagaa gaagttccgc ggagtcaggc agcgccactg gggctcctgg 300 gtctccgaga tcaggcaccc tctcctgtaa gtctctatct cactagtgat gatcactagc 360 tagctatgtt gctctgatgt gttatgccac tgtcgtgata gatacatttt gatggatagc 420 tagtggttct ggtttgttgt tgctttcttt ctttttttct ttctctttca tggtatgtta 480 gtagctaact caaggtgctt gggtgcgtgc atgcatgaat gatggatgat gttttttttg 540 ctacgtgcgt tgtggcagta agaggagggt gtggctgggt acctttgaga cggcggagga 600 ggcggcgcgg gcgtacgacg aggccgccat cctgatgagc ggccgcaacg ccaagaccaa 660 cttcccagtc gcgaggaacg ccacggggga gctcacaccg gcggctgcgg tggcagggcg 720 ggatggccgt gtcggcggcg gcagcggcag ctcgtcctca atgacggcca acggcggcgg 780 gaacagcctg tctcagatcc tcagcgccaa gctccgcaag tgctgcaaga cgccgtcgcc 840 gtcgctcacc tgcctccgcc ttgacccgga gaagtcccac attggcgtct ggcagaagcg 900 cgccggcgca cgcgctgact ccagctgggt catgaccgtc gagctcaaca aggacacggc cgtgtcgtcg gctgcgacgg tggcagcagc aacagcagtg tcgtccagcg accagccgac tccgagtgac agcacagtca caacgacgtc cacgtccacc acgggctcgc cgtcgccacc acctccggca atggacgacg aggagaggat cgcgctgcag atgatcgagg agctgctggg 1140 caggagcggc ccgggctcgc cgtcacatgg gctgctgcac ggtggtgaag gtagcctcgt catctgaatt ccggaagaac aggaagaaa ttgaaaatgc aaggttaaaa cagcatgatc aggtcaccat ctaagatcaa ggatctggta gggtggttgg tgcacaggca gttaagattg ctacatatga tagtatatc tctattacta ctacatatcc agcttaatta ggaacaatt father ttactactgt gtgaagtga gctgtgtatt father ttacttgtat gtacaagacg tccccgtcat tatagtcata ctggtgaaag ctctgctatg tatcaacgtc atcagagatc agactacata father gtgtcaactt ctaataaaag fatheraaaa cattgtaatt gtgta <210> 16 <211> 2670 <212> DNA <213> Oryza sativa L . <400> 16 agtcatcaac aaataataat aactagcatg atggcccgcg cagattgcgc ggctagcatc 60 attatatttt ctctcatata atagcatata tgttttca ttattattatt caaatatatt 120 aaaatgacaa catatttta aattttgcaa taactttaca aaactactaa tgtgtatat 180 tcatattgca ttttatatac gtgttagtta ttaattattt ttaagataaa attttagtta 240 tttgtaaatt atatatattc atatatggac tctagactcg tctttaata tttcttttttt 300 ctgtaaattg tatttctata tagactata gttctctctc taatattatt tattttatt 360 tctgaaattt tattattct aattgtattt ctatgtggac tctaactca tctttcaata 420 ttatttaatt tttaatttcg aatttagtt acttctaaat tgtattccta tattagcatt 480 aaactcttct tcccattttttcttaatttc gatttttaac tatttgtaaa ttgtattttt 540 atacggactc taaacctac ttatttt attatgtttta ttccaattt tagttagttt 600 taagttttcta tatggactct attackctact tctaatattc cttatttta attccgaatt 660 tcagttattt cctaattgta ttctacatg gactctatac tctacttcta atattcctta 720 ttttaattc cgaatttcag ttatttccta attgtatttc tatatggact ccggtctcct 780 cttctaatat tccttatttt ttaattccga atttcagcta tttcctaatt gtatttctat 840 atggactccg gtctccctt ctaatattcc ttattttt attccgaatt taagctattt 900 ctaaattgta tttctatatg gactctgctt tttctttc tccgattaat gtgagaattt 960 ctaggccatg agagcgaacg tggaggctcc ttttctatt cctttaatta tataatagat 1020 aataatt tagatcca tcctattctt gaagtgtggc agtagatgat gaggttgttg 1080 attackacta gtataatca tacctgtcac atattgtga aggtttgagc tagagggacc 1140 cttaagattt tcttccttga atcgctagtc ctagcatttg tactgacaat gtatatatga 1200 tcagtgtggt gtactagcgt ttatagtgct cacctgtcaa cggtttttac tggacgacag 1260 gtcctgacag tccttgcaa ccaccacaaa cctgcctgca cttttccatg taaaaaaagg 1320 agtgcgtgca tgtgtacgac gagcagcagc agagaagct tccatccctg caggaactca 1380 ctgacacacg tatctgtgtg gagccgatgg aactgtaaaa gcacccggac ggaagcccca 1440 ttcaattcga tcggccggac aaacaatggc aacgaactca ctccgccatt aactccgttt 1500 caccttttta cggagagaag agcaagttct tctgctgctg caatgctgtg tgcgccttcc 1560 ttgccgatgc gccatggatg gatggatgat tggctgcttg attggggcag ttattgcagg 1620 ccaactgctc tgtcgaaaga gtgtccctcg gaaagtggct ttagaagttg gagtaaacct 1680 tgcagtgcat ggcatacata cagcatggag tggtattacc ttggttacga acgcccatca 1740 agtccatttc tggagtagta cacactgtcc aagcttcagt gtttgtacct ggaaagaaat 1800 gcacattagc catatgcgca aggcaaggca aggcattgag caaagttaga ttcatcagtt 1860 actagtttag ctagttagca catacacact actggacaat gactagcgag tgagcttagc 1920 tagctgatcg gcgacaagtg acaaccagct agttagcaat aagagctggt gatggttagc 1980 tcatctcagt tcatccttag gttttcctcc agtgaaggcc ataaatgagg tcgccaaggg 2040 cgtaggtggg ggagtttatt accggcgtac tggcagagaa ggtggttatg ccgctcgcca 2100 cagttcaccg acagcccacc ccagcagccc atgagggccc cccatcctct ttccatctat 2160 ctctctcttc cactaatctc accaaagagt cttcttcact tccttctcta ggcttattaa 2220 cagcttattt ctgatctctc acatgttcag tttatctca tccctcctgc tagatattac 2280 tgttgctggc cccaaccacc ctgcatggat tacaacacaa gtttatcagt accactgtgg 2340 ttcaaagtag tgtatgtact ggactcgtgg agttatgacc acaaggaaat tgaccagaaa 2400 gttaattaca atgactgaag gtgtgtactc acttggtcct ggcagagtag aaggagagta 2460 caccaactag atctgaaccc tggtgtgcta ctgtaccaaa aggctaaggc cactgacaga 2520 ggagatacgt gacagacaag tcccagttga gttactgtcc aacccccaaa tctaaccctc 2580 ttctaccacc acccccactc gtgcgccaca gcgctctcac acgcgcgcca cactcatgcc 2640 atgtatgtct atatatatgt gcatctatat 2670 <210> 17 <211> 732 <212> DNA <213> Oryza sativa L. <400> 17 atgggacagt cgaagaagaa gttccgcgga gtcaggcagc gccactgggg ctcctgggtc 60 tccgagatca ggcaccctct ccttaagagg agggtgtggc tgggtacctt tgagacggcg 120 gaggaggcgg cgcgggcgta cgacgaggcc gccatcctga tgagcggccg caacgccaag 180 accaacttcc cggtcgcgag gaacgccacg ggggagctca caccggcggc tgcggtggca 240 gggcgggatg gccgtgtcgg cggcggcagc ggcagctcgt cctcaatgac ggccaacggc 300 ggcgggaaca gcctgtctca gatcctcagc gccaagctcc gcaagtgctg caagacgccg 360 tcgccgtcgc tcacctgcct ccgcctcgac ccggagaagt cccacattgg cgtctggcag 420 aagcgcgccg gcgcacgcgc tgactccagc tgggtcatga ccgtcgagct caacaaggac 480 acggccgtgt cgtcggctgc gaaggtggca gcagcaacag cagcgtcgtc cagcgaccag 540 ccgactccga gtgacagcac agtcacaacg acgtccacgt ccaccacggg ctcgccgtcg 600 ccaccacctc cggcaatgga cgacgaggag aggatcgcgc tgcagatgat cgaggagctg 660 ctgggcagga gcggcccggg ctcgccgtca catgggctgc tgcacggtgg tgaaggtagc 720 ctcgtcatct ga 732 <210> 18 <211> 243 <212> PRT <213> Oryza sativa L. <400> 18 Met Gly Gln Ser Lys Lys Lys Phe Arg Gly Val Arg Gln Arg His Trp 1 5 10 15 Gly Ser Trp Val Ser Glu Ile Arg His Pro Leu Leu Lys Arg Arg Val 20 25 30 Trp Leu Gly Thr Phe Glu Thr Ala Glu Glu Ala Ala Arg Ala Tyr Asp 35 40 45 Glu Ala Ala Ile Leu Met Ser Gly Arg Asn Ala Lys Thr Asn Phe Pro 50 55 60 Val Ala Arg Asn Ala Thr Gly Glu Leu Thr Pro Ala Ala Ala Val Ala 65 70 75 80 Gly Arg Asp Gly Arg Val Gly Gly Gly Ser Gly Ser Ser Ser Ser Met 85 90 95 Thr Ala Asn Gly Gly Gly Asn Ser Leu Ser Gln Ile Leu Ser Ala Lys 100 105 110 Leu Arg Lys Cys Cys Lys Thr Pro Ser Pro Ser Leu Thr Cys Leu Arg 115 120 125 Leu Asp Pro Glu Lys Ser His Ile Gly Val Trp Gln Lys Arg Ala Gly 130 135 140 Ala Arg Ala Asp Ser Ser Trp Val Met Thr Val Glu Leu Asn Lys Asp 145 150 155 160 Thr Ala Val Ser Ser Ala Ala Lys Val Ala Ala Ala Thr Ala Ala Ser 165 170 175 Ser Ser Asp Gln Pro Thr Pro Ser Asp Ser Thr Val Thr Thr Thr Ser 180 185 190 Thr Ser Thr Thr Gly Ser Pro Ser Pro Pro Pro Pro Ala Met Asp Asp 195 200 205 Glu Glu Arg Ile Ala Leu Gln Met Ile Glu Glu Leu Leu Gly Arg Ser 210 215 220 Gly Pro Gly Ser Pro Ser His Gly Leu Leu His Gly Gly Glu Gly Ser 225 230 235 240 Leu Val Ile <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <400> 19 tgttcatcgc agaagaagaa 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <400> 20 ccagtttatc ctccactgtt 20 <210> 21 <211> 21 <212> DNA <213> Artificial Sequence <400> twenty one ctcccatccc aataatcctc a 21 <210> twenty two <211> twenty one <212> DNA <213> Artificial Sequence <400> twenty two atgcttgaca acaggatacc a 21 <210> twenty three <211> 20 <212> DNA <213> Artificial Sequence <400> twenty three aggatgcggt cttaaaaagt 20 <210> twenty four <211> 20 <212> DNA <213> Artificial Sequence <400> twenty four gttggacaag ggtaaagtca 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <400> 25 ttctatccgt cgtcttttgg 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <400> 26 attacggagt gtgtacagtg 20 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <400> 27 tgcagggtga aataaattca 20 <210> 28 <211> twenty one <212> DNA <213> Artificial Sequence <400> 28 tgcttaaaaa gataacggtc a 21 <210> 29 <211> 49 <212> DNA <213> Artificial Sequence <400> 29 aattcgagct cggtacccgg ggatccttac aaattttttt tgaataaga 49 <210> 30 <211> 47 <212> DNA <213> Artificial Sequence <400> 30 caagcttgca tgcctgcagg tcgacccaaa ggcaaccaag tcatttg 47 <210> 31 <211> 35 <212> DNA <213> Artificial Sequence <400> 31 tgcagcccgg gatccatggc aatggcggcg gcgcc 35 <210> 32 <211> 36 <212> DNA <213> Artificial Sequence <400> 32 tccacccatc aattgcaagc aggggggggca gcaacg 36 <210> 33 <211> twenty four <212> DNA <213> Artificial Sequence <400> 33 ggcatggcaa tggcggcggc gccc 24 <210> 34 <211> twenty four <212> DNA <213> Artificial Sequence <400> 34 aaacgggcgc cgccgccatt gcca 24 <210> 35 <211> twenty three <212> DNA <213> Artificial Sequence <400> 35 gccgacgcgc tccaccgcga gat 23 <210> 36 <211> twenty three <212> DNA <213> Artificial Sequence <400> 36 aaacatctcg cggtggagcg cgt 23 <210> 37 <211> twenty three <212> DNA <213> Artificial Sequence <400> 37 gccggaagga cggtcataat tgg 23 <210> 38 <211> twenty three <212> DNA <213> Artificial Sequence <400> 38 aaacccaatt atgaccgtcc ttc 23 <210> 39 <211> twenty three <212> DNA <213> Artificial Sequence <400> 39 ggcagaccaa cttcccggtc gcg 23 <210> 40 <211> twenty three <212> DNA <213> Artificial Sequence <400> 40 aaaccgcgac cgggaagttg gtc 23 <210> 41 <211> twenty three <212> DNA <213> Artificial Sequence <400> 41 gccgctcctg ggtctccgag atc 23 <210> 42 <211> twenty three <212> DNA <213> Artificial Sequence <400> 42 aaacgatctc ggagacccag gag 23 <210> 43 <211> 47 <212> DNA <213> Artificial Sequence <400> 43 atggccatgg aggccgaatt catggcggac cagctcaccg acgagca 47 <210> 44 <211> 43 <212> DNA <213> Artificial Sequence <400> 44 tgcggccgct gcaggtcgtc acttggccat catgacctta acg 43 <210> 45 <211> 48 <212> DNA <213> Artificial Sequence <400> 45 atggccatgg aggccagtga attcatggcg gaggggcggc gctacgcg 48 <210> 46 <211> 43 <212> DNA <213> Artificial Sequence <400> 46 tgcagctcga gctcgatgga tctagaaata tccaggcgtt ggc 43 <210> 47 <211> 48 <212> DNA <213> Artificial Sequence <400> 47 atggccatgg aggccagtga attcatggcg gaggggcggc gctacgcg 48 <210> 48 <211> 43 <212> DNA <213> Artificial Sequence <400> 48 tgcagctcga gctcgatgga tctatctgaa gtttatattc aca 43 <210> 49 <211> 46 <212> DNA <213> Artificial Sequence <400> 49 acgcgtcccg gggcggtacc atggcggacc agctcaccga cgagca 46 <210> 50 <211> 45 <212> DNA <213> Artificial Sequence <400> 50 cgaaagctct gcaggtcgac tcacttggcc atcatgacct taacg 45 <210> 51 <211> 40 <212> DNA <213> Artificial Sequence <400> 51 acgggggacg agctcggtac catggcggag gggcggcgct 40 <210> 52 <211> 40 <212> DNA <213> Artificial Sequence <400> 52 gcgtacgaga tctggtcgac gaaatatcca ggcgttggca 40 <210> 53 <211> 45 <212> DNA <213> Artificial Sequence <400> 53 tttcatttgg agagaacacg gagctcatgg cggaggggcg gcgct 45 <210> 54 <211> 44 <212> DNA <213> Artificial Sequence <400> 54 tcctcctcct cctcctccgg atccgaaata tccaggcgtt ggca 44 <210> 55 <211> 51 <212> DNA <213> Artificial Sequence <400> 55 aacacggggg actgagctcg gtaccatggc ggaccagctc accgacgagc a 51 <210> 56 <211> 58 <212> DNA <213> Artificial Sequence <400> 56 gtcgacagat cctccaccac cactacctcc acctcccttg gccatcatga ccttaacg 58 <210> 57 <211> 53 <212> DNA <213> Artificial Sequence <400> 57 cgggatcgag ggaaggattt cacatatgat gatataccaa gtttttagga tgc 53 <210> 58 <211> 48 <212> DNA <213> Artificial Sequence <400> 58 agctttattta attacctgca gggaattcct agaaatatcc aggcgttg 48 <210> 59 <211> 44 <212> DNA <213> Artificial Sequence <400> 59 ctggttccgc gtggatccat ggcggaccag ctcaccgacg agca 44 <210> 60 <211> 47 <212> DNA <213> Artificial Sequence <400> 60 cgatgcggcc gctcgagtcg actcacttgg ccatcatgac cttaacg 47 <210> 61 <211> 53 <212> DNA <213> Artificial Sequence <400> 61 cgggatcgag ggaaggattt cacatatgat ggcggagggg cggcgctacg cga 53 <210> 62 <211> 55 <212> DNA <213> Artificial Sequence <400> 62 agctttattta attacctgca gggaattcct aataagagct gaatatagaa tcatc 55 <210> 63 <211> 50 <212> DNA <213> Artificial Sequence <400> 63 cgtgcgccac agcgctctca cacgcgcgcc acactcatgc catgtatgtc 50 <210> 64 <211> 42 <212> DNA <213> Artificial Sequence <400> 64 cgtgcgccac agcgctctca ccacactcat gccatgtatg tc 42 <210> 65 <211> 47 <212> DNA <213> Artificial Sequence <400> 65 gccgctctag aactagtgga tccatggcgg aggggcggcg ctacgcg 47 <210> 66 <211> 45 <212> DNA <213> Artificial Sequence <400> 66 tcgataagct tgatatcgaa ttcctagaaa tatccaggcg ttggc 45 <210> 67 <211> 49 <212> DNA <213> Artificial Sequence <400> 67 gccgctctag aactagtgga tccatggcgg accagctcac cgacgagca 49 <210> 68 <211> 48 <212> DNA <213> Artificial Sequence <400> 68 tcgataagct tgatatcgaa ttctcacttg gccatcatga ccttaacg 48 <210> 69 <211> 46 <212> DNA <213> Artificial Sequence <400> 69 attgacgagt acggtgggat ccatggcgga ggggcggcgc tacgcg 46 <210> 70 <211> 45 <212> DNA <213> Artificial Sequence <400> 70 tcgataagct tgatatcgaa ttcctagaaa tatccaggcg ttggc 45

Claims

1. A method of enhancing the heat tolerance or protecting the yield of a grass plant at high temperature, comprising: Modulating TT2-SCT1 and SCT2-WR2 signaling pathway in a plant; The plant is rice, and the modulating comprises: (a) down-regulating expression or activity of TT2; the amino acid sequence of TT2 is shown as SEQ ID NO: 5; (b) down-regulating expression or activity of SCT1 and SCT2; the amino acid sequence of SCT1 is shown as SEQ ID NO: 10; the amino acid sequence of SCT2 is shown as SEQ ID NO: 14; and / or (c) blocking the regulation of WR2 by SCT1 and SCT2, and maintaining WR2 homeostasis; the amino acid sequence of WR2 is shown as SEQ ID NO:

18.

2. The method of claim 1, wherein, In (a), the down-regulating expression or activity of TT2 reduces the response of TT2 to heat induction, reduces intracellular calcium concentration under heat stimulation, and modulates the interaction between calmodulin and SCT1. In (b), the down-regulating expression or activity of SCT1 and SCT2 down-regulates the regulation of WR2 by SCT1 and SCT2, and maintains WR2 homeostasis. In (c), for a plant with heat-induced low expression or no expression of WR2, WR2 homeostasis is maintained by complementing WR2 to a normal amount, or by changing the SCT1 and SCT2 binding region of the WR2 promoter region so that it does not interact with SCT1 and SCT2.

3. The method of claim 2, wherein, In (a), the down-regulating expression or activity of TT2 comprises knocking out or silencing the coding gene of TT2 in the plant, or inhibiting the activity of TT2.

4. The method of claim 3, wherein, In (a), it comprises silencing TT2 with an interference molecule that specifically interferes with the expression of the coding gene of TT2, knocking out the coding gene of TT2 with a CRISPR system, knocking out the coding gene of TT2 with a homologous recombination method, or performing loss-of-function mutation on TT2 in a plant containing TT2.

5. The method of claim 2, wherein, In (b), the down-regulating expression or activity of SCT1 and SCT2 comprises knocking out or silencing the coding gene of SCT1 and SCT2 in the plant, or inhibiting the activity of SCT1 and SCT2.

6. The method of claim 5, wherein, In (b), it comprises silencing SCT1 and SCT2 with an interference molecule that specifically interferes with the expression of the coding gene of SCT1 and SCT2, knocking out the coding gene of SCT1 and SCT2 with a CRISPR system, knocking out the coding gene of SCT1 and SCT2 with a homologous recombination method, or performing loss-of-function mutation on SCT1 and SCT2 in a plant containing SCT1 and SCT2.

7. The method of claim 2, wherein, In (c), the complementing WR2 comprises transferring the coding gene of WR2 or an expression construct or vector containing the coding gene into the plant, mutating the promoter of WR2 itself to eliminate the binding site of SCT1 and SCT2, promoting the expression of WR2 with a constitutive promoter or a tissue-specific promoter, or promoting the expression of WR2 with an enhancer.

8. The method of claim 1, wherein, In the (a), the down-regulating the expression or activity of TT2 comprises: knocking out the coding gene of TT2, so as to express an inactive protein or not express a protein; wherein the nucleotide C at the 165th position of the TT2 coding gene is mutated into A, thereby forming TGA to terminate the expression of TT2 protein in advance, or the 1st to 40th position of the TT2 coding gene is knocked out, the 85th to 90th position is knocked out; one nucleotide is inserted at the 19th position, the 87th to 90th position is knocked out; the 20th nucleotide is knocked out, the nucleotide A at the 91st position is mutated into T, thereby causing a frame shift in the coding region of TT2, thereby forming a stop codon to terminate the expression of TT2 in advance.

9. The method of claim 1, wherein, In the (b), the down-regulating the expression or activity of SCT1 and SCT2 comprises: knocking out the 267th to 268th position of the SCT1 coding gene; knocking out the 219th to 253rd position; inserting one nucleotide at the 222nd position, thereby causing a frame shift in the coding region of SCT1 to generate an early termination; knocking out the 218-244th nucleotide of the SCT2 coding gene; inserting one nucleotide at the 231st position; knocking out the 229-231st nucleotide, thereby causing the coding region of SCT2 to be damaged or frame shifted.

10. Use of a TT2-SCT1 and SCT2-WR2 signaling pathway or a modulator thereof for enhancing heat tolerance or protecting yield of a plant under high temperature; the plant is a rice; wherein, The modulator comprises: (a) a down-regulator of the expression or activity of TT2; the amino acid sequence of the TT2 is shown as SEQ ID NO: 5; (b) a down-regulator of the expression or activity of SCT1 and SCT2; the amino acid sequence of the SCT1 is shown as SEQ ID NO: 10; the amino acid sequence of the SCT2 is shown as SEQ ID NO: 14; and / or (c) an agent for blocking the regulation of WR2 by SCT1 and SCT2 and maintaining the homeostasis of WR2; the amino acid sequence of the WR2 is shown as SEQ ID NO:

18.

11. Use according to claim 10, characterized in that, The down-regulator of the expression or activity of TT2 is an agent for knocking out or silencing TT2, an agent for inhibiting the activity of TT2, comprising: an interference molecule specifically interfering with the expression of the coding gene of TT2, a CRISPR gene editing agent, a homologous recombination agent or a site-directed mutagenesis agent for TT2, which causes loss-of-function mutations in TT2; and / or The down-regulator of the expression or activity of SCT1 and SCT2 is an agent for knocking out or silencing SCT1 and SCT2, an agent for inhibiting the activity of SCT1 and SCT2, comprising: an interference molecule specifically interfering with the expression of the coding gene of SCT1 and SCT2, a CRISPR gene editing agent, a homologous recombination agent or a site-directed mutagenesis agent for SCT1 and SCT2, which causes loss-of-function mutations in SCT1 and SCT2; and / or The agent for maintaining the homeostasis of WR2 is a complementing agent for WR2, comprising the coding gene of WR2 or an expression construct containing the coding gene; an agent for gain-of-function mutations in WR2.

12. Use of a TT2-SCT1 and SCT2-WR2 signal pathway and a pathway gene therein, for: as a molecular marker for identifying a plant of the family Poaceae with heat tolerance or yield under high temperature; or as a molecular marker for directional screening of a plant of the family Poaceae with heat tolerance or yield under high temperature; or for screening of a substance for improving heat tolerance or yield under high temperature of a plant of the family Poaceae; wherein, the plant of the family Poaceae is rice; the amino acid sequence of the TT2 is as shown in SEQ ID NO: 5, the amino acid sequence of the SCT1 is as shown in SEQ ID NO: 10, the amino acid sequence of the SCT2 is as shown in SEQ ID NO: 14, and the amino acid sequence of the WR2 is as shown in SEQ ID NO:

18.

13. A method of selecting a grass plant by orientation, characterized by, The method comprises: identifying the expression or sequence characteristics or interaction of the TT2-SCT1 and SCT2-WR2 signal pathways and the pathway genes therein in a test plant; and if the test plant has one or more characteristics selected from the following group, the test plant is a plant of the family Poaceae with heat tolerance or yield under high temperature: the test plant has low or no expression of TT2; the test plant has low or no expression of SCT1 and SCT2; the test plant has stable WR2; the plant of the family Poaceae is rice; the amino acid sequence of the TT2 is as shown in SEQ ID NO: 5, the amino acid sequence of the SCT1 is as shown in SEQ ID NO: 10, the amino acid sequence of the SCT2 is as shown in SEQ ID NO: 14, and the amino acid sequence of the WR2 is as shown in SEQ ID NO:

18.

14. A method for screening of a substance for improving heat tolerance or yield under high temperature of a plant of the family Poaceae, comprising: (1) adding a candidate substance to a system expressing the TT2-SCT1 and SCT2-WR2 signal pathways; (2) detecting the system to observe the expression or activity of the TT2-SCT1 and SCT2-WR2 signal pathway proteins therein: if the expression or activity of TT2 is reduced, the candidate substance is a substance for improving heat tolerance or yield under high temperature of a plant of the family Poaceae; if the expression or activity of SCT1 and / or SCT2 is reduced, the candidate substance is a substance for improving heat tolerance or yield under high temperature of a plant of the family Poaceae; if the addition of the candidate substance promotes the stable state of WR2, the candidate substance is a substance for improving heat tolerance or yield under high temperature of a plant of the family Poaceae; the plant of the family Poaceae is rice; the amino acid sequence of the TT2 is as shown in SEQ ID NO: 5, the amino acid sequence of the SCT1 is as shown in SEQ ID NO: 10, the amino acid sequence of the SCT2 is as shown in SEQ ID NO: 14, and the amino acid sequence of the WR2 is as shown in SEQ ID NO:

18.

15. A down-regulator of TT2-SCT1 and SCT2-WR2 signal pathway genes for improving heat tolerance or maintaining yield at high temperature of a plant in the family Poaceae, which is a CRISPR gene editing reagent that down-regulates the expression of TT2, SCT1 and SCT2; the plant in the family Poaceae is rice; the amino acid sequence of the TT2 is shown as SEQ ID NO: 5, the amino acid sequence of the SCT1 is shown as SEQ ID NO: 10, the amino acid sequence of the SCT2 is shown as SEQ ID NO: 14, and the amino acid sequence of the WR2 is shown as SEQ ID NO: 18; the CRISPR gene editing reagent targets the 165th position of the TT2-encoding gene, mutates the nucleotide C into A, and then forms TGA to prematurely terminate the expression of the TT2 protein; or knocks out the 1st to 40th position of the TT2-encoding gene, knocks out the 85th to 90th position; inserts a nucleotide at the 19th position, knocks out the 87th to 90th position; knocks out the 20th nucleotide, mutates the nucleotide A at the 91st position into T, and then causes a frame shift in the TT2-encoding region, and then forms a stop codon to prematurely terminate the expression of the TT2; or targets the expression or activity of the SCT1 and SCT2, including: Knocking out the 267th to 268th of the SCT1 coding gene; knocking out the 219th to 253th; inserting 1 nucleotide at the 222th, which further causes the coding region of SCT1 to be frame-shifted and to produce a premature termination; knocking out the 218th to 244th nucleotide of the SCT2 coding gene; inserting a nucleotide at the 231th; knocking out the 229th to 231th nucleotide, which further causes the coding region of SCT2 to be destroyed or frame-shifted.

16. A construct comprising an exogenous down-regulator of the TT2-SCT1 and SCT2-WR2 signal pathway genes for improving the heat tolerance or maintaining yield of a plant under high temperature according to claim 15, wherein the plant is rice, the amino acid sequence of the TT2 is shown as SEQ ID NO: 5, the amino acid sequence of the SCT1 is shown as SEQ ID NO: 10, the amino acid sequence of the SCT2 is shown as SEQ ID NO: 14, and the amino acid sequence of the WR2 is shown as SEQ ID NO: 18.

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