Use of hsfa1 protein or its encoding gene in regulating hypocotyl growth in plants

Abstract

This invention relates to the field of plant genetic engineering technology, specifically to the application of HSFA1 protein or its encoding gene in regulating plant mesocotyl growth. This invention provides the application of the plant HSF family transcription factor HSFA1 in regulating the temperature-responsive growth of plant mesocotyls. HSFA1 is a negative regulator of the high-temperature response of mesocotyls. Under high environmental temperatures, the mesocotyl length of HSFA1 knockout mutants is significantly longer than that of the wild type, exhibiting greater sensitivity to high environmental temperatures. This has important application value in direct-seeded rice production under high-temperature conditions, providing new genetic resources and methods for breeding rice varieties suitable for direct seeding.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to the application of HSFA1 protein or its encoding gene in regulating plant mesocotyl growth. Background Technology

[0002] The mesocoaxial is an organ located between the base of the radicle and the coleoptile node of a rice seedling. It elongates between the coleoptile node and the point of attachment of the radicle, providing the power to push the stem tip out of the soil surface. It has an important influence on the uniform emergence of rice seedlings in direct seeding. Varieties with longer mesocoaxial have stronger emergence ability. Therefore, the length of the mesocoaxial is a key trait in direct seeding rice cultivation.

[0003] Temperature is one of the main factors affecting mesocotyl elongation. Currently, multiple genes have been identified that control mesocotyl elongation by influencing cell elongation and division, meaning that mesocotyl elongation is controlled by multiple genes. However, the relationships between these genes and their roles in the mesocotyl's response to temperature are unclear, and research on the molecular regulatory network of mesocotyl temperature response is almost nonexistent. Therefore, identifying key regulatory genes in rice mesocotyl temperature response and studying their effects on mesocotyl temperature-responsive growth is of great significance for the promotion of direct seeding cultivation technology. Summary of the Invention

[0004] This invention provides the application of HSFA1 protein or its encoding gene in regulating plant mesocotyl growth.

[0005] To identify key regulatory genes in the hypocotyl temperature response of rice, this invention first employs a multi-omics approach combining transcriptomics, proteomics, and chromatin accessibility analysis to study the early temperature response process of the hypocotyl and identify key regulatory transcription factors, constructing a transcription factor regulatory network. Furthermore, gene editing targeting these key regulatory factors is performed, and phenotypic identification and functional analysis of gene knockout mutants are conducted.

[0006] Specifically, the present invention provides the following technical solutions.

[0007] In a first aspect, the present invention provides any of the following applications of the HSFA1 protein, its encoding gene, or biological material containing said encoding gene: (1) Regulating the temperature response of plant mesocotyls to growth; (2) Regulate direct seeding of plants under high ambient temperature.

[0008] In this invention, regulating the temperature-responsive growth of the mesocotyl refers to controlling the growth of the mesocotyl in response to temperature changes. Preferably, it involves regulating the temperature-responsive growth of the mesocotyl during the seedling stage.

[0009] This invention discovers that the HSF family transcription factor HSFA1 has the function of regulating the temperature-responsive growth of plant mesoderm, and regulating the expression and / or activity of HSFA1 can significantly affect the temperature-responsive growth of plant mesoderm.

[0010] In this invention, the high ambient temperature refers to an ambient temperature higher than the suitable temperature for the plant. For rice seedlings, the high ambient temperature is preferably above 30℃, more preferably 30-45℃, even more preferably 30-38℃, and even more preferably 31-33℃.

[0011] Preferably, the regulation is negative regulation.

[0012] The HSF family plays a major role in plant heat stress response, with HSFA1 being a core regulator and an important part of the gene regulatory network. Sequences of HSFA1 and its encoding genes from different plants are available in publicly available databases. The HSFA1 gene is identified as LOC_Os03g63750 (HEAT SHOCK TRANSCRIPTION FACTOR, HSFA1) in the Rice Gene Database (https: / / rice.uga.edu / index.shtml).

[0013] In this invention, the HSFA1 has the amino acid sequence shown in SEQ ID NO.3.

[0014] Preferably, the amino acid sequence of HSFA1 is shown in SEQ ID NO.3.

[0015] SEQ ID NO.3: MEAAVAAAAAAAGAVTTAVAPPPGAAVSNGVATAPPPFLMKTYEMVDDPATDAVVSWGPGNNSFVVWNTPEFARDLLPKYFKHSNFSSFVRQLNTYGFRKVDPDRWEFANEGFLRGQKHLLKTINR RKPTHGNNQVQQPQLPAAPVPACVVGKFGMEEEIEMLKRDKNVLMQELVRLRQQQQTTDHQLQTLGKRLQGMEQRQQQMMSFLAKAMHSPGFLAQFVQQNENSRRRIVASNKKRRLPKQDGSLDSE SASLDGQIVKYQPMINEAAKAMLRKILKLDSSHRFESMGNSDNFLLENYMPNGQGLDSSSSTRNSGVTLAEVPANSGLPYVATSSGLSAICSTSTPQIQCPVVLDNGIPKEVPNMSAVPSVPKAVAP GPTDINILEFPDLQDIVAEENVDIPGGGFEMPGPEGVFSLPEEGDDSVPIETDEILYNDDTQKLPAIIDSFWEQFLVASPLSVDNDEVDSGVLDQKETQQGNGWTKAENMANLTEQMGLLSSHHTG.

[0016] In this invention, the coding gene sequence of HSFA1 can be obtained from the amino acid sequence of HSFA1. Due to the degeneracy of codons, the nucleotide sequence of the coding gene of HSFA1 is not unique. All genes that can encode the above-mentioned HSFA1 are within the protection scope of this invention.

[0017] In this invention, the biological material includes recombinant DNA, expression cassettes, transposons, vectors, microorganisms, plant cells, plant tissues, or plants. The vectors include plasmid vectors, viral vectors, artificial chromosomes, etc. The microorganisms include bacteria, fungi, and viruses. Bacteria include, but are not limited to, *Escherichia coli* and *Agrobacterium*. The plants include transgenic plants.

[0018] In this invention, the plant includes plants of the Poaceae family.

[0019] Preferably, the plant is rice. The rice may be a direct-seeded rice variety.

[0020] In a second aspect, the present invention provides any of the following applications of HSFA1 protein expression and / or activity reduction, HSFA1 protein inhibitors, or biological materials containing said inhibitors: (1) Promotes the growth of plant mesocotyls under high ambient temperatures; (2) Promote direct seeding of plants under high ambient temperature.

[0021] This invention has found that reducing the expression and / or activity of transcription factor HSFA1 can significantly promote the growth of the mesocotyl in plants under high ambient temperatures, significantly increase the length of the mesocotyl, and thus promote direct seeding of plants.

[0022] The aforementioned high ambient temperature refers to an ambient temperature higher than the suitable temperature for the plant. For rice seedlings, the high ambient temperature is preferably above 30℃, more preferably 30-45℃, even more preferably 30-38℃, and even more preferably 31-33℃.

[0023] In this invention, the inhibitor of HSFA1 protein refers to substances that can inhibit the expression and / or activity of HSFA1 protein, including but not limited to nucleic acid molecules (such as siRNA, miRNA, shRNA, sgRNA used in CRISPR technology, etc.) and proteins that can inhibit the expression and / or activity of HSFA1 protein.

[0024] In this invention, the reduction in the expression and / or activity of the HSFA1 protein can be achieved by weakening or knocking out the encoding gene of HSFA1.

[0025] In the above applications, the plants include grasses (Poaceae).

[0026] In some embodiments of the present invention, the plant is rice. The rice may be a direct-seeded rice variety.

[0027] Thirdly, the present invention provides a method for promoting the growth of rice mesocotyls under high ambient temperatures, the method comprising: reducing the expression and / or activity of HSFA1 protein in rice.

[0028] Preferably, the mesocotyl growth refers to mesocotyl growth during the seedling stage. Promoting mesocotyl growth manifests as promoting mesocotyl elongation, i.e., increasing the length of the mesocotyl.

[0029] At high ambient temperatures, reducing the expression and / or activity of HSFA1 protein in rice can significantly promote mesocotyl elongation during the seedling stage.

[0030] Fourthly, the present invention provides a method for promoting direct seeding of rice at high ambient temperatures, the method comprising: reducing the expression and / or activity of HSFA1 protein in rice.

[0031] At high ambient temperatures, reducing the expression and / or activity of HSFA1 protein in rice can significantly promote the elongation of the mesocotyl during the seedling stage, thereby promoting direct seeding of rice.

[0032] Fifthly, the present invention provides a method for improving the sensitivity of rice mesocotyl growth to high environmental temperature response, the method comprising: reducing the expression and / or activity of HSFA1 protein in rice.

[0033] In the above method, the high ambient temperature is preferably an ambient temperature higher than the suitable temperature for rice seedlings, preferably higher than 30℃, more preferably 30-45℃, more preferably 30-38℃, and even more preferably 31-33℃.

[0034] In the above method, the HSFA1 protein has the amino acid sequence shown in SEQ ID NO.3; Preferably, the amino acid sequence of the HSFA1 protein is shown in SEQ ID NO.3.

[0035] In the above method, the reduction of HSFA1 protein expression and / or activity in rice is preferably achieved by weakening or knocking out the coding gene of HSFA1.

[0036] The weakening or knockout of the HSFA1 protein encoding gene can be achieved using genetic engineering techniques. Optional genetic engineering techniques include, but are not limited to, CRISPR, TALEN, or ZFN gene editing technologies.

[0037] Preferably, the method includes: knocking out the gene encoding the HSFA1 protein in rice using CRISPR / Cas9 technology to obtain transgenic rice.

[0038] In some embodiments of the present invention, the HSFA1 gene is used as a target, a CRISPR / Cas9-based sgRNA sequence is designed, a DNA fragment containing the encoding the sgRNA sequence is ligated into a vector carrying CRISPR / Cas9, and introduced into rice to obtain transgenic rice with the gene function missing.

[0039] Preferably, the nucleotide sequence of the sgRNA action site is 5'-TGTACCTGCTTGCGTAGAAGTGG-3' (SEQ ID NO.4).

[0040] Among the above methods, the vector can be introduced into rice using conventional biotechnological methods such as Ti plasmids, plant virus vectors, direct DNA transformation, microinjection, and electroporation (Weissbach, 1998, Method for Plant Molecular Biology VIII, Academy Press, New York, pp. 411-463; Geiserson and Corey, 1998, Plant Molecular Biology, 2nd Edition).

[0041] In a sixth aspect, the present invention provides the application of rice with reduced expression and / or activity of HSFA1 protein in rice mesocotyl growth regulation breeding or direct seeding breeding.

[0042] The breeding methods mentioned include, but are not limited to, transgenic, hybrid, backcross, self-cross, or asexual reproduction.

[0043] Preferably, the rice with reduced expression and / or activity of the HSFA1 protein is transgenic rice. The reduced expression and / or activity of the HSFA1 protein can be achieved by weakening or knocking out the HSFA1 gene.

[0044] In some embodiments of the present invention, the gene encoding the HSFA1 protein in rice is knocked out using CRISPR / Cas9 technology to obtain the transgenic rice.

[0045] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: This invention is the first to discover the significant application value of the HSFA1 transcription factor from the HSF family in direct-seeded rice cultivation. HSFA1 is a negative regulator of the high-temperature response of the rice mesocotyl. Under suitable temperatures, the mesocotyl length of the HSFA1 gene knockout hsfa1 mutant is not significantly different from that of the wild type. However, under high temperatures, the mesocotyl length of the hsfa1 mutant is significantly longer than that of the wild type, indicating a greater sensitivity to high temperatures. The long mesocotyl performance of the hsfa1 mutant under high temperatures suggests its important application value in direct-seeded rice production during the hot summer months. Finding superior haplotypes of hsfa1 in natural populations or cultivars will be beneficial for breeding rice varieties suitable for direct-seeding. The key regulatory transcription factor for the high-temperature response of the mesocotyl provided by this invention facilitates in-depth research on the temperature-responsive growth of the rice mesocotyl at the chromosomal and transcriptional levels, supports further understanding of the temperature-responsive growth regulation mechanism of the seedling mesocotyl, and promotes the application of seedling mesocotyl regulation in direct-seeding cultivation technology. Attached Figure Description

[0046] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0047] Figure 1In Example 1 of this invention, the expression of mesocotyl translation-related genes changed during early temperature treatment. (A) PCA analysis of mesocotyl gene expression at various temperature treatment times. (B) Number of TDEGs in response to high / low temperatures at different temperature treatment times in the mesocotyl. The identification criteria for TDEGs were Fold Change > 2 and padj < 0.05. (C) Intersection analysis and GO enrichment analysis of all low-temperature response TDEGs and high-temperature response TDEGs in the mesocotyl at 1 min and 5 min temperature treatment times.

[0048] Figure 2 In Example 1 of this invention, the expression of mesocotyl proteins changed during the early temperature response. (A) Distribution of peptide ion mass deviation. (B) Protein quantification results. (CD) Identification and GO enrichment analysis of temperature-responsive differentially expressed proteins (TDEPs) of mesocotyls under 5 min high temperature (C) and low temperature (D) treatments. The identification criteria for TDEPs were Fold Change > 1.2 and pvalue < 0.05. (EF) Intersection analysis and expression change analysis of TDEPs and TDEGs in mesocotyls under high temperature (E) and low temperature (F). In the scatter plot, each point represents a gene, and the horizontal and vertical axes represent the degree of change in RNA expression in gene RNA-seq and the degree of change in protein expression in TMT, respectively. The degree of expression change is represented by log2FC. Red dots represent genes that are TDEPs but not TDEGs.

[0049] Figure 3In Example 1 of this invention, the chromatin accessibility of the mesocotyl is more significantly affected by high temperature. (A) Analysis of the distribution characteristics of reads near genes. The upper figure shows the characteristic curve of read distribution near genes. The horizontal axis represents the normalized gene length, and the vertical axis represents the range from 3 kb before the transcription start site (TSS) to 3 kb after the transcription termination site (TES) for each annotated gene. The vertical axis represents the enrichment degree of reads. The lower figure is a characteristic heatmap of read distribution near genes. Each row represents a gene, and different colors represent the enrichment degree of reads. (B) Distribution of accessible chromatin regions (ACRs) on the genome. (C) PCA analysis of ACRs under different temperature treatment times. (DE) The number of thermoresponsive ACRs (TACRs) in response to high temperature (D) and low temperature (E) under different temperature treatment times of the mesocotyl. TACRs are classified into repressed TACRs (rTACRs) and induced TACRs (iTACRs). The criteria for identifying TACRs are Fold Change > 2 and padj < 0.05.

[0050] Figure 4 This is the screening of key regulatory factors for the mesocotyl temperature response in Example 1 of the present invention. (A) Intersection analysis of transcription factors of genes related to open chromatin at different temperatures under different treatment times. The key regulatory factors for the mesocotyl temperature response screened are in red. (B) GO enrichment analysis of transcription factors regulating high and low temperature responses under different treatment times. (C) Motif enrichment analysis of all transcription factors identified in A.

[0051] Figure 5 In Example 2 of this invention, HSFA1 is a negative regulator of mesocotyl temperature-responsive elongation. (A) GRNA target location and mutation form of OsHSFA1 gene mutant. (B) Functional analysis of OsHSFA1 regulating mesocotyl temperature-responsive elongation. Statistical analysis was performed on the mesocotyl length of rice seedlings grown at 25℃ and 32℃ for 5 days. The values ​​are expressed as mean ± standard error (n≥25). The significance was tested using a t-test (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001); FC (Fold Change) represents the ratio of mesocotyl length at 32℃ to that at 25℃; the scale bar is 1 cm.

[0052] Figure 6 This is a diagram illustrating the regulatory network of HSFA1, a key transcription factor in the mesocotyl temperature response, as described in Example 2 of this invention. Note: (A) Intersection analysis of TDEGs and TAAGs at all high and low temperature treatment times. (B) Regulatory network diagram of HSFA1 and its downstream regulatory genes, where red nodes indicate upregulation of target genes and blue nodes indicate downregulation of target genes. The colored letters on the left represent the HSFA1 binding motif.

[0053] Figure 7 This is Example 2 of the present invention, showing the temperature-sensitive changes of HSFA1, a key regulator of mesocotyl temperature response. (A) Expression of HSFA1 in various organs of rice seedlings. The average gene expression of all samples treated at different times under the optimal temperature of 28℃ was selected as the gene expression value in the organ, expressed as log2(TPM+1). (B) qPCR results showing the average expression value of HSFA1 in the sample replicates under temperature treatments of 5 min, 20 min, and 1 h. (C) Minimum Free Energy (MFE) curves of RNA secondary structure predicted at HSFA1 5'-UTRs and stem-loop structures predicted near AUG at 5'-UTR. A 40-base sliding window was used for prediction. The horizontal axis of the curve represents the position of the sliding window at 5'-UTR, and the vertical axis represents the MFE of the corresponding sliding window. The sliding window with minimum MFE near AUG was selected to display the RNA stem-loop structure. (D) Prediction of Intrinsically Disordered Region (IDR). The peptides above the red line can form disordered regions. (E) Prion-like Domain (PrD) prediction is performed. The horizontal axis represents the amino acid position, and the vertical axis represents the prediction score. The red line represents the Prd score, and the black line represents the non-Prd score. The sum of the two is 1. The peptides above the black line have prion-like structures. Detailed Implementation

[0054] This invention utilizes multi-omics technologies combined with genetic analysis to identify key regulatory genes in the mesocotyl temperature response, providing a molecular basis and genetic resources for breeding direct-seeded rice varieties adapted to temperature changes. In specific embodiments, this invention employs a multi-omics approach combining transcriptomics, proteomics, and chromatin accessibility analysis to study the early temperature response process of the mesocotyl, identify key regulatory transcription factors, construct a transcription factor regulatory network, and perform CRISPR / Cas9 gene editing on key factors. Phenotypic identification and functional analysis are then conducted on gene knockout mutants.

[0055] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0056] Example 1: Discovery of key regulatory genes for mesocotyl temperature response 1. Expression of mesocotyl translation-related genes in response to early temperature changes To investigate the earlier temperature response of the hypocotyl, a shorter temperature treatment time of 1 min was selected. Specifically, germinating ZH11 japonica rice seeds (approximately 1 mm in diameter) were cultured for 5 days in darkness at a suitable ambient temperature of 28℃. Mesocotyls were then subjected to short-term temperature treatments of 1 min and 5 min at 28℃, 4℃, and 45℃, respectively. Samples were rapidly frozen in liquid nitrogen and subjected to transcriptome sequencing. Remaining samples were stored at -80℃ for later use. In the RNA-seq experiment investigating the temperature response of rice seedling organs, PCA analysis showed good intra-sample reproducibility and large inter-sample distance in the mesocotyl transcriptome data. Figure 1 The results (A) indicate that temperature treatment caused significant changes in mesocotyl gene expression. The high- and low-temperature responses (TDEGs) of mesocotyls at different temperature treatment times were identified and visualized using a volcano plot. The results showed that TDEGs were relatively low in mesocotyls at 1 min of temperature treatment, but increased significantly at 5 min (…). Figure 1 The presence of B indicates that mesocotyl gene expression just begins to respond to temperature changes within 1 min of temperature treatment, and by 5 min, a significant number of genes have responded to temperature changes. To further analyze the process involved by temperature-responsive genes in the mesocotyl, a comparative analysis was first performed on all high-temperature and low-temperature TDEGs of the mesocotyl at 1 min and 5 min of temperature treatment. This revealed 1491 high-temperature specific TDEGs, 2102 low-temperature specific TDEGs, and 1002 shared TDEGs between high and low temperatures. Figure 1 Further GO enrichment of these TDEGs revealed that both high- and low-temperature TDEGs were enriched in ion transport and stress response pathways. Translation pathways were enriched in all three groups of TDEGs. Furthermore, protein ubiquitination was enriched at high temperatures, and ribosome assembly was enriched at low temperatures. Figure 1 (C). The above results indicate that the expression of mesocotyl translation-related genes responds to early temperature changes, meaning that the mesocotyl may also participate in the early temperature response at the protein level.

[0057] 2. Mesocotyl responds to early temperature changes at the protein level. The preceding analysis of mesocotyl RNA-seq suggests that the mesoderm may also participate in the early temperature response at the protein level. Therefore, to investigate protein changes during the early temperature response of the mesoderm, tandem mass tagging (TMT) was employed. First, proteins were extracted from mesocotyl samples treated with 5 min of high and low temperatures (parallel to RNA-seq), followed by proteolytic digestion and peptide tagging. Tandem mass spectrometry was then used to quantify the proteins in different samples. The peptide mass deviation was mainly within 10 ppm. Figure 2 The A result indicates that the peptide identification results are accurate and reliable. A total of 577,991 spectra were identified, of which 62,832 spectra could be matched with known spectra. These detected spectra were assigned to 27,803 peptides, of which 23,392 were unique peptides. Ultimately, 5,534 proteins were identified, and 5,530 proteins were quantified. Figure 2 B). Differential expression analysis was performed on quantitative proteins, and 467 temperature-responsive differentially expressed proteins (TDEPs) were identified at high temperatures. GO enrichment analysis showed that these TDEPs were related to translation, lipid metabolism, stress response, and energy activity. Figure 2 (C). 483 TDEPs were identified at low temperatures. GO enrichment analysis showed that these TDEPs were involved in processes similar to those of high-temperature TDEPs, including translation, lipid metabolism, stress response, and energy activity. Figure 2 (D). Comparative analysis of TDEPs and TDEGs revealed very few overlapping genes, with 434 genes found at high temperatures. Figure 2 E), there are 440 at low temperatures ( Figure 2 The F genes respond to temperature at the protein level rather than the transcriptional level; these genes are marked in red in the scatter plot. Figure 2 The results indicate that the mesocotyl responds to early temperature changes at the protein level, and that some genes respond to temperature changes only at the protein level rather than the transcriptional level.

[0058] 3. The chromatin openness of the mesocotyl is more sensitive to high temperatures. Changes in gene expression are typically mediated by transcription factors binding to a conserved DNA motif. When a transcription factor binds to a gene motif, the chromatin near the motif becomes more open. Therefore, to investigate the transcriptional regulatory mechanisms of gene expression changes, this study employed Assay for Targeting Accessible-Chromatin with high-throughout sequencing (ATAC-seq). First, mesoderms treated parallel to RNA-seq at 1 min and 5 min of high and low temperatures were analyzed. The Tn5 transposase was used to identify and cleave accessible chromatin regions (ACRs). The cleaved sequences were enriched and sequenced to analyze the temperature response changes of mesoderms at the chromosome level. Analysis of the distribution density of ACRs across all samples revealed that ACRs were primarily enriched at transcription start sites (TSS). Figure 3 Further analysis of the distribution of ACRs across the genome revealed that most ACRs (~75%) were located in promoter regions, while a minority (~25%) were located in untranslated regions, exons, introns, within 300 bytes downstream of the gene, and in distal intergenic regions. Figure 3 (B) This aligns with the pattern that promoter region chromatin is more open, and transcription factors mainly bind to the TSS. PCA analysis showed good intra-sample reproducibility and large inter-sample distance in the mesocotyl chromatin accessibility group data. Figure 3 The results (C) indicate that temperature treatment significantly altered the chromatin openness of the mesocotyl. Thermoresponsive chromatin receptors (TACRs) of the mesocotyl under different temperature treatment times were identified. TACRs were classified into repressed TACRs (rTACRs) and induced TACRs (iTACRs), and the results were visualized using a volcano plot. The results show ( Figure 3(Figures D and E) High-temperature treatment for 1 min resulted in a significant number of TACRs, including 1079 iTACRs and 18 rTACRs. At 5 min, the number of TACRs increased significantly, with 4599 iTACRs and 638 rTACRs (Figure 3D). The number of iTACRs far exceeded that of rTACRs, indicating that high-temperature induction made the mesocotyl chromatin more open. Low-temperature treatment for 1 min resulted in no TACRs, and at 5 min, only 110 TACRs were observed, including 13 iTACRs and 97 rTACRs. Figure 3 (E) The number of rTACRs is much greater than that of iTACRs. Unlike the high-temperature response, low-temperature induced chromatin becomes more closed. These results indicate that the chromatin openness of the mesocotyl responds differently to high and low temperatures, and is more sensitive to high temperatures. This is consistent with previous findings that gene expression in rice seedlings is more sensitive to high temperatures.

[0059] 4. Multi-omics joint analysis to discover key regulatory genes of mesocotyl temperature response Since ACRs can bind to transcription factors, thereby regulating the expression of downstream genes, this study aimed to identify key regulatory genes in the mesocotyl temperature response. HOMER software was used to enrich DNA motifs of high-temperature and low-temperature response TACRs of the mesocotyl under different temperature treatment times. Using a p-value < 0.01 as the identification criterion, the enriched motifs were matched to rice transcription factors using BLASTP software. The results showed that 84 transcription factors regulated mesocotyl gene expression in response to high-temperature changes at 1 min, 155 transcription factors regulated mesocotyl gene expression in response to high-temperature changes at 5 min, and 23 transcription factors regulated mesocotyl gene expression in response to low-temperature changes. Figure 4 A total of 191 transcription factors were identified that regulate the temperature response of the mesoderm. Most of these transcription factors, which exerted a regulatory effect after 1 min of high-temperature treatment, also exerted a regulatory effect after 5 min of high-temperature treatment. GO enrichment analysis of these 191 transcription factors revealed that they were mainly related to temperature stress, hormonal response, and RNA regulation. Figure 4 (B). These 191 transcription factors collectively bind to 99 DNA motifs. Enrichment analysis of the DNA binding motifs of these transcription factors revealed that they were mainly enriched in family-type transcription factors such as HSF, ARF, and HSF (B). Figure 4 The presence of C indicates that these transcription factor families play a major regulatory role in the early temperature response of the mesocotyl. Furthermore, four transcription factors were found to be involved in the regulation of both high and low temperature responses in the early mesocotyl. Figure 4The figure (A) is indicated in red, with three of them being heat shock factors: OsHSFA2E, OsHSFA2B, and OsHSFA1. This further emphasizes the important role of the HSF family in regulating the mesocotyl temperature response, suggesting that they may be key regulatory genes for the mesocotyl temperature response.

[0060] Example 2 OsHSFA1 Negative regulation of mesocotyl elongation in response to high ambient temperature 1. Functional identification of HSFA2 Heat shock transcription factors (HSFs) play a crucial role in regulating plant responses to biotic and abiotic stresses (Jacob, Hirt et al. 2017). Plant HSFs are divided into three main subfamilies: HSFA, HSFB, and HSFC. Among them, HSFA-type factors are the main positive regulators of heat stress (Fragkostefanakis, Mesihovic et al. 2016). This invention identified three HSFA-type factors, among which HSFA1 is the core regulator in plant heat stress response and regulates the HSFA2 gene, meaning HSFA1 is located upstream in the regulatory network (Peng, Jaeger et al. 2025). To investigate the role of HSFA2 in regulating hypocotyl elongation in rice in response to temperature, CRIPSR / Cas9 technology was used to analyze... OsHSFA1 Gene editing was performed on the ZH11 japonica rice to verify the phenotypic response of the mutant to temperature-responsive growth of the rice mesocotyl.

[0061] In this invention, LOC_Os03g63750 ( HSFA1 The genomic nucleotide sequence (including the promoter and 3'UTR sequence), CDS sequence and the amino acid sequence of the encoded protein are shown in SEQ ID NO.1-3, respectively.

[0062] Two allelic loss-of-function mutants were obtained using CRIPSR / Cas9 gene knockout technology, among which, hsfa1 -1 The deletion of 2 bases causes the protein to change from the 151st amino acid and ultimately terminates translation prematurely; hsfa1 -2 results in the deletion of 7 bases, causing the protein to change from the 149th amino acid and ultimately leading to premature termination of translation. Figure 5 A).

[0063] The target sites for CRISPR / Cas9 are selected as follows: LOC_Os03g63750 ( HSFA1): 5′-TGTACCTGCTTGCGTAGAAGTGG-3′ (SEQ ID NO. 4).

[0064] The primers for identifying the relevant mutants are as follows (SEQ ID NO.5-6): hsfa1 -F: 5′-GTGCTATTTACATTAGCCCTTC -3′; hsfa1 -R: 5′-TAGCAACTATTCTTCTCTGCT -3′.

[0065] Next, genetic studies were conducted on the function of the genes using the aforementioned mutants. The results showed that... hsfa1 -1 and hsfa1 -2 At 25℃, the mesocotyl was not significantly different from the wild type, but at 32℃, it was significantly longer than the wild type, and the FC value was significantly greater than that of the wild type. Figure 5 (B) Explanation hsfa1 The sensitivity of mesocotyl elongation to temperature increases. These results indicate that... OsHSFA1 It plays an important role in regulating the temperature response of the mesocotyl and exerts a negative regulatory effect on the elongation of the mesocotyl temperature response.

[0066] 2. Regulatory network of key transcription factors in mesocotyl temperature response To investigate which downstream genes HSFA1 regulates to exert its effect on temperature-responsive mesocotyl elongation, an intersection analysis was performed on TDEGs and TAAGs under all high- and low-temperature treatments. This analysis revealed 4600 TDEGs and 3337 TAAGs, sharing 588 genes. Figure 6 The chromatin open regions and expression of these 588 genes (A) all respond to temperature changes and are regulated by transcription factors. Motifs of the promoter regions of these 588 genes were scanned using FIMO software to construct a regulatory network of the HSFA1 transcription factor. The results showed that HSFA1 regulates 73 downstream mesocotyl temperature-responsive genes, of which 21 were downregulated and 52 were upregulated. These genes are mainly associated with lower-level transcription factors, metabolic processes, stress responses, and heat shock proteins. Figure 6 (B) and related.

[0067] 3. HSFA1 may sense temperature changes through its stem-ring structure and phase transition. The above results indicate that HSFA1 This gene plays an important role in regulating the temperature-responsive elongation of the mesocotyl. To further analyze whether this gene is a mesocotyl organ-specific expression factor, its expression in various organs of rice seedlings was analyzed. It was found that it is highly expressed in the mesocotyl, followed by the meristem and leaves. Figure 7 (A) Hint HSFA1 It likely plays a regulatory role primarily within the mesocotyl. To investigate how HSFA1 senses temperature changes and transmits temperature signals to downstream temperature-responsive genes to regulate mesocotyl temperature-responsive elongation, qPCR was first used to analyze its gene expression. The results showed that expression levels increased at 5 min, 20 min, and 1 h. Figure 7 Under temperature treatment (B), the expression of this gene was not affected by temperature, indicating that... HSFA1 It does not sense temperature changes at the transcriptional level. Studies have shown that stem-loop structures formed at the RNA 5'-UTR can sense temperature changes and thus affect translation. Therefore, to predict the RNA secondary structure at the HSFA1 5'-UTR, referring to existing methods for predicting 5'-UTR secondary structure (Chung, Balcerowicz et al. 2020), a 40-base sliding window was used to scan the gene 5'-UTR. The minimum free energy (MFE) of the RNA secondary structure in each window was predicted, and MFE curves were plotted. It was found that a stable stem-loop structure can form in HSFA1 at a window 40 bases upstream of AUG. Figure 7 The presence of an intrinsically disordered region (IDR) and a prion-like domain (PrD) in HSFA1 suggests that the protein content may be altered by sensing temperature through the RNA stem-loop structure. Studies have shown that proteins can sense temperature changes through phase transitions. Therefore, IDR and PrD predictions were performed on HSFA1, revealing the presence of an IDR region (…). Figure 7 D), but no PrD region ( Figure 7 The E value suggests that HSFA1 may undergo a phase transition response temperature change.

[0068] The characteristics of mesocotyl elongation during the rice seedling stage are of great significance for the promotion of direct seeding technology for rice. This invention discovers... HSFA1 This gene resource, which regulates the temperature-responsive growth of the mesocotyl during the seedling stage, has promising applications in the breeding of suitable direct-seeding rice varieties.

[0069] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Any of the following applications of the HSFA1 protein, its encoding gene, or biological material containing said encoding gene: (1) Regulating the temperature response of plant mesocotyls to growth; (2) Regulate direct seeding of plants under high ambient temperature.

2. The application according to claim 1, characterized in that, The regulation mentioned is negative regulation.

3. The application according to claim 1 or 2, characterized in that, The HSFA1 protein has the amino acid sequence shown in SEQ ID NO.3; And / or, the plant includes grasses, preferably rice.

4. Any of the following applications of decreased expression and / or activity of HSFA1 protein, an inhibitor of HSFA1 protein, or biological material containing said inhibitor: (1) Promotes the growth of plant mesocotyls under high ambient temperatures; (2) Promote direct seeding of plants under high ambient temperature.

5. The application according to claim 4, characterized in that, The HSFA1 protein has the amino acid sequence shown in SEQ ID NO.3; And / or, the plant includes grasses, preferably rice.

6. A method for promoting the growth of rice mesocotyls under high ambient temperatures, characterized in that, The method includes: reducing the expression and / or activity of HSFA1 protein in rice.

7. A method for promoting direct seeding of rice at high ambient temperatures, characterized in that, The method includes: reducing the expression and / or activity of HSFA1 protein in rice.

8. A method for improving the sensitivity of rice mesocotyl growth to high ambient temperature response, characterized in that, The method includes: reducing the expression and / or activity of HSFA1 protein in rice.

9. The method according to any one of claims 6 to 8, characterized in that, The HSFA1 protein has the amino acid sequence shown in SEQ ID NO. 3; And / or, the reduction of HSFA1 protein expression and / or activity in rice is achieved by weakening or knocking out the HSFA1 coding gene.

10. Application of rice with reduced expression and / or activity of HSFA1 protein in mesocotyl regulation breeding or direct seeding breeding of rice.

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