Application of the rice LOC_Os01g67410 gene in plant response to high ambient temperature

Abstract

This invention discloses the application of the rice LOC_Os01g67410 gene in plant response to high ambient temperature. PLT5 It is a negative regulatory factor for deep-seeded rice seedlings. The length of internodes and mesocotyls during the seedling stage... plts The mutant showed no significant difference in germination speed and germination rate under shallow sowing conditions compared to the wild type, but under deep sowing conditions, it germinated faster and had a higher germination rate than the wild type. Furthermore, although a warm environment of 32℃ promoted earlier and faster germination time for both the wild type and the mutant, the germination rate of the wild type decreased significantly at 32℃. plts The mutant was still able to maintain a high germination rate. plts The mutant's tolerance to deep sowing and strong emergence performance suggest its important application value in dry direct sowing, especially in high-temperature summer direct sowing production. This can be achieved by finding mutants in natural populations or cultivars. PLTs The superior haplotype will be beneficial for breeding rice varieties suitable for deep sowing.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and more specifically, to the application of the rice LOC_Os01g67410 gene in plant responses to high ambient temperatures. Background Technology

[0002] With the rapid development of industry and agriculture, labor migration, reduced agricultural irrigation water use, and climate change, rice production faces severe challenges. Direct seeding of rice, achieved by mechanically sowing dried seeds directly into the soil at a certain depth, allows the seeds to utilize soil moisture for germination and growth, thus addressing some of the limitations on rice production. However, currently promoted rice varieties are mainly bred for transplanting, and the poor tolerance of most varieties to deep sowing also restricts the widespread adoption of direct seeding technology. Internode elongation and mesocotyl elongation during the seedling stage are key forces driving emergence of rice seedlings under deep sowing conditions, and the elongation of these two organs is influenced by ambient temperature, contributing to deep-sown rice's ability to cope with climate change. However, the molecular mechanisms underlying the temperature-responsive elongation of internodes and mesocotyls during the rice seedling stage have not yet been reported. Therefore, exploring the molecular mechanisms of temperature-responsive growth in rice seedling internodes and mesocotyls, identifying key regulatory genes, and understanding their emergence potential under deep sowing conditions are crucial for the promotion of direct seeding technology. Summary of the Invention

[0003] The purpose of this invention is to provide the application of the rice LOC_Os01g67410 gene in plant response to high ambient temperature.

[0004] The concept of this invention is as follows: First, physiological experiments are conducted to understand the tissue and cellular changes in the elongation of internodes and mesocotyls in response to temperature during the rice seedling stage. RNA-seq sequencing is performed on internodes and mesocotyls grown at different temperatures to screen for differentially expressed genes specific to temperature responses in these tissues. Further, ATAC-seq data is combined to compare the epigenetic differences between the two tissues, identifying potential regulatory genes at both omics levels. Simultaneously, the two omics data are combined to identify potential transcription factors and mine potential downstream regulatory genes, constructing a gene expression regulatory network. Based on the analysis results, CRISPR / Cas9 gene editing is performed on the selected potential regulatory genes and transcription factors of internodes and mesocotyls during the seedling stage to obtain corresponding mutants and verify their functions in rice heat-responsive growth and deep sowing. This invention deepens the understanding of the heat-responsive growth of internodes and mesocotyls in rice seedlings at the chromosomal and transcriptional levels, providing clues for further understanding the regulatory mechanisms of internodes and mesocotyls in heat-responsive growth. The discovery of multiple key regulatory genes will facilitate the application of internodes and mesocotyls in direct seeding.

[0005] To achieve the objectives of this invention, in a first aspect, this invention provides the application of the rice LOC_Os01g67410 gene (PLT5 gene) in plant responses to high environmental temperatures, specifically including any of the following applications:

[0006] 1) Regulate the thermal response growth of internodes and mesocotyls in plant seedlings;

[0007] 2) Regulate deep sowing of plants under high ambient temperature.

[0008] AP2 / EREBP transcription factors can integrate metabolic, hormonal, and environmental signals to regulate plant growth in stress adaptation and retrograde signals, and are an important part of the gene regulatory network.

[0009] The PLT5 gene is numbered in the Rice Genome Database (https: / / rice.uga.edu / index.shtml) as follows: LOC_Os01g67410 ( AP2 / EREBP transcription factor BABY BOOM, PLT5 ).

[0010] Furthermore, the regulation is negative regulation.

[0011] The plants include grasses, preferably rice, and more preferably direct-seeded rice.

[0012] Secondly, the present invention provides a method for promoting the growth of internodes and mesocotyls in rice seedlings under high ambient temperatures, the method comprising: using genetic engineering techniques to weaken or knock out the PLT5 gene in rice.

[0013] Thirdly, the present invention provides a method for promoting deep-sown rice seedlings under high ambient temperatures, the method comprising: using genetic engineering techniques to weaken or knock out the PLT5 gene in rice.

[0014] Furthermore, the gene can be weakened or knocked out using gene editing technologies such as CRISPR, TALEN, or ZFN.

[0015] In one specific embodiment of the present invention, using the PLT5 gene as a target, a CRISPR / Cas9-based sgRNA sequence is designed, and a DNA fragment containing the encoding the sgRNA sequence is ligated into a vector carrying CRISPR / Cas9, which is then transformed into rice to obtain transgenic rice with the gene function lost.

[0016] Preferably, the nucleotide sequence of the sgRNA action site is 5'-CATGAGGGGATCGGAGCTCTCGG-3' (SEQ ID NO:12).

[0017] Expression vectors carrying the target gene can be introduced into plant cells 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, 2). nd Edition).

[0018] Fourthly, the present invention provides the application of transgenic rice obtained according to the method in plant breeding.

[0019] Breeding methods include, but are not limited to, transgenic, hybridization, backcrossing, self-pollination, or asexual reproduction.

[0020] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0021] This invention is the first to discover that PLT transcription factors of the AP2 / EREBP family have great application value in direct-seeded rice. PLT5 It is a negative regulatory factor for deep-seeded rice seedlings. The length of internodes and mesocotyls during the seedling stage... plts The mutant showed no significant difference in germination speed and germination rate under shallow sowing conditions compared to the wild type, but under deep sowing conditions, it germinated faster and had a higher germination rate than the wild type. Furthermore, while a warm environment of 32℃ promoted earlier germination time and faster germination speed for both the wild type and the mutant, and when the germination rate of the wild type decreased significantly at 32℃... plts The mutant was still able to maintain a high germination rate. plts The mutant's tolerance to deep sowing and strong emergence performance suggest its important application value in dry direct sowing, especially in high-temperature summer direct sowing production. This can be achieved by finding mutants in natural populations or cultivars. PLTs The superior haplotype will be beneficial for breeding rice varieties suitable for deep sowing. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the internodes and mesocotyls of deep-sown rice seedlings in a preferred embodiment of the present invention.

[0023] Figure 2 In a preferred embodiment of the present invention, rice seedlings grown deep-sown specifically produce internodes and mesocotyls during the seedling stage.

[0024] Figure 3In a preferred embodiment of the present invention, high ambient temperature promotes the elongation of internodes and mesocotyls during the seedling stage. Note: Statistical analysis of four phenotypes of NJ6 rice seedlings grown for 4-6 days at four different temperatures. The four phenotypes are internode (Inter), mesocotyl (Mesocotyl), coleoptile (Cole), and full-length above seed (Flas). Values ​​represent mean ± standard error, n ≥ 25.

[0025] Figure 4 In a preferred embodiment of the present invention, cell expansion mediates the heat-responsive growth of seedling internodes. Note: AB represents the cytological phenotypes of seedling internodes of indica rice A and japonica rice B at two temperatures. Seedling internode cells from indica rice NJ6 grown at 30℃ and 32℃ for 4 days, and japonica rice NIP and DJ grown at 25℃ and 32℃ for 7 days were used for statistical analysis. The sample size was ≥3, and the cell count was ≥100. Student's method was used. t-test Significance analysis was performed; the observation method for internode cells in indica rice seedlings in the figure was Calcofluor tissue staining, and for japonica rice, it was resin semi-thin section; the scale bar is 100 μm.

[0026] Figure 5 This illustrates the alignment of RNA-seq data with the reference genome in a preferred embodiment of the present invention. Note: Inter (seedling internode), Meso (mesocotyl), Sdl (photogenerated seedling), "Inter4-30" represents the transcriptome of seedling internodes grown at 30°C for 4 days, and "DJ-25-Inter" represents the transcriptome of DJ seedling internodes grown at 25°C. "_1 / _2" represent two biological replicates.

[0027] Figure 6 In a preferred embodiment of the present invention, cell expansion-related pathways were significantly enriched in the thermal response transcriptome of seedling internodes. Note: A represents the number of temperature-responsive differentially expressed genes (TDEGs) in seedling internodes and light-grown seedlings. Inter (seedling internodes), Sdl (light-grown seedlings), and "Inter4_30" represent the transcriptome of seedling internodes grown at 30°C for 4 days. Seedling internode material was obtained from deep-sown rice seedlings, and seedling material was obtained from the aboveground tissue of shallow-sown rice seedlings that had been exposed to light. B shows the GO enrichment analysis of all seedling internode TDEGs.

[0028] Figure 7In a preferred embodiment of the present invention, the thermal response process of seedling internodes progresses progressively with the number of growth days. Note: A shows the differential analysis of TDEGs in seedling internodes at different growth stages. B shows the expression trend analysis of common TDEGs in seedling internodes across three growth days. The values ​​in the radar chart represent the fold change in TDEG expression levels between two temperatures, i.e., Log2 (Foldchange); where blue shading indicates that expression is inhibited with increasing temperature in tissues at days 4, 5, and 6, red shading indicates that expression is induced with increasing temperature, and gray shading indicates inconsistent temperature response changes across the three growth days. C shows the GO enrichment analysis of common TDEGs in seedling internodes across three growth stages. DE shows the heatmap D and GO analysis E of expression levels of specific TDEGs in seedling internodes at different developmental stages. Z-score represents the number of standard deviations between the gene expression level of each sample and the mean gene expression level.

[0029] Figure 8 In a preferred embodiment of the present invention, cell proliferation and expansion act together on the thermoresponsive growth of the mesocotyl. Note: AB represents the cytological phenotype of the mesocotyl of indica rice A and japonica rice B at two temperatures. Internode cells from seedlings of indica rice NJ6 grown at 25℃ and 30℃ for 5 days, and japonica rice NIP and DJ grown at 25℃ and 32℃ for 6 days were used for statistical analysis. The sample size was ≥ 3, and the cell count was ≥ 100. Student's method was used. t-test Significance analysis was performed; the mesodermal cells in the figure were observed using Calcofluor tissue staining; the scale bar is 100 μm.

[0030] Figure 9 In a preferred embodiment of the present invention, cell proliferation and expansion-related pathways were significantly enriched in the thermoresponsive transcriptome of the mesocotyl. Note: A represents the number of TDEGs in the mesocotyl and light-grown seedlings. Mesocotyl (mesocotyl), the mesocotyl material was obtained from deep-sown rice seedlings. B represents the GO enrichment analysis of all mesocotyl TDEGs.

[0031] Figure 10 In a preferred embodiment of the present invention, the thermal response process of the mesocotyl progresses progressively with the number of growth days. Note: A shows the differential analysis of TDEGs in the mesocotyl at different growth stages. B shows the expression trend analysis of common TDEGs in the mesocotyl across three growth days. C shows the GO enrichment analysis of common TDEGs in the mesocotyl across three growth stages. DE shows the heatmap D of expression levels of specific TDEGs in the mesocotyl at different developmental stages and the GO enrichment analysis E. The Z-score represents the number of standard deviations between the gene expression level of each sample and the mean gene expression level.

[0032] Figure 11In the preferred embodiment of this invention, the thermal response transcriptomes of internodes and mesocotyls during the seedling stage showed high similarity. Note: A is RNA-seq principal component analysis. B is correlation analysis of gene expression levels across all transcriptome samples. C is the number of TDEGs in internodes, mesocotyls, and photogrown seedlings. D is GO enrichment analysis of common TDEGs in internodes and mesocotyls during the seedling stage. E is a comparative analysis of TDEGs in internodes, mesocotyls, and photogrown seedlings.

[0033] Figure 12 In a preferred embodiment of the present invention, internodes and mesocotyls in the seedling stage each possess their own unique heat-responsive genes. Note: A shows the GO enrichment analysis of TDEGs specific to internodes and mesocotyls in the seedling stage. BC shows the expression levels of organ-specific TDEGs in internodes and mesocotyls in the seedling stage, as well as the expression levels of TDEGs specific to seedlings under light, in heat-sensitive conditions. C shows the expression level of tissue-specific genes. Gene expression levels (TPM) in the same organ at all temperatures differ from those in other organs by a fold increase of ≥ 5, and the differentially expressed genes are organ-specific temperature-responsive genes. D shows the verification of the relative expression levels of tissue-specific genes. qRT-PCR values ​​are the mean ± standard deviation (n = 3), and RNA-seq shows the mean of two biological replicates. Z-score represents the number of standard deviations between the gene expression levels of each sample and the mean gene expression levels per row.

[0034] Figure 13 This section presents a quality characteristic analysis of accessible chromatin regions (ACRs) in rice seedling internodes and mesocotyls during the preferred embodiment of the present invention. Note: A shows the fragment size distribution of accessible chromatin regions (ACRs) in seedling internodes and mesocotyls grown at two temperatures. The horizontal axis represents the fragment size after Tn5 transposase digestion, and the vertical axis represents the percentage distribution of different fragment sizes. B shows the ATAC-seq signal spectra and heatmaps of all samples from seedling internodes and mesocotyls. The upper figure shows the characteristic curve of read enrichment on the genome; the lower figure shows the heatmap of read distribution characteristics near genes. C shows Spearman correlation analysis of ACRs in all samples from seedling internodes and mesocotyls.

[0035] Figure 14This is a correlation analysis of chromatin-accessible regions and their neighboring gene expression levels in a preferred embodiment of the present invention. Note: A shows the distribution of ACRs in different regions of the genome. Transcription start site (TSS): ACRs located within -1000 bp to +50 bp of the gene transcription start site; Transcription termination site (TES): ACRs located within -50 bp to +1000 bp of the gene transcription termination site; Intron and exon: ACRs located in the intron and exon regions of the gene; Intergenic region: All ACRs in other regions. The number and proportion of ACRs are marked in the figure. B shows the correlation between different ACR categories and gene expression levels.

[0036] Figure 15 This is a negative regulatory factor for internode thermal response elongation during the seedling stage in a preferred embodiment of the present invention. Note: A is a comparative analysis of TDEGs and TAAGs specifically expressed during internodes in rice seedlings. Genes with common differential expression in both omics are marked in red. BC is a mutant phenotypic analysis of genes regulating internode thermal response elongation during the seedling stage. The figure shows the target location of the edited gene and the editing status of the mutant in the CRISPR / cas9 gene editing system, and the IGV plot shows the changes in ATAC-seq peak and gene expression of the corresponding gene when the internode response temperature changes during the seedling stage; the internode lengths of background-related materials of indica rice NJ6 grown for 6 days and japonica rice ZH11 grown for 7 days under deep sowing conditions were used for statistical analysis, with values ​​= mean ± standard deviation (n ≥ 20), using Student's method. t-test Perform significance analysis; scale bar is 10 mm.

[0037] Figure 16 This is a negative regulator of mesocotyl thermal response elongation in a preferred embodiment of the present invention. Note: A is a comparative analysis of mesocotyl TDEGs and TAAGs. Genes with common differential expression in both omics are marked in red. BC is a mutant phenotypic analysis of genes regulating mesocotyl thermal response elongation. The figure shows the target location and mutation form of the edited gene, as well as the changes in ATAC-seq peak and gene expression level of the corresponding gene during the mesocotyl temperature response process. Mesocotyls of deep-sown rice seedlings grown for 6 days were used for statistical analysis, values ​​= mean ± standard deviation (n ≥ 20), using Student's algorithm. t-test Perform significance analysis; scale bar is 10mm.

[0038] Figure 17In a preferred embodiment of the present invention, multiple transcription factor families were enriched in the TACRs of heat-responsive genes in internodes and mesocotyls during the seedling stage. Note: A represents transcription factors targeting TACRs in internodes and mesocotyls obtained by HOMER analysis. Values ​​in the heatmap are -log10 (p-value). Z-score represents the number of standard deviations between the mean of each sample data and the mean of the row data. B represents transcription factors involved in regulating the expression of temperature-responsive genes in internodes and mesocotyls during the seedling stage, enriched by FIMO method. Score 1-3 represent ACRs of all TDEGs in the three growth stages; Score 3 represents ACRs of all TDEGs in the three growth stages; when the ratio is >1.5, the transcription factor cluster is considered significantly enriched. C represents information on the significantly enriched transcription factor clusters. D is an interaction network diagram of AP2 / EREBP and SPL transcription factors with open chromatin regions of chromatin of genes specifically regulating internodes and mesocotyls during the seedling stage. The yellow and blue arrows target TACRs of temperature-responsive genes in seedling internodes and mesocotyls, respectively.

[0039] Figure 18 In a preferred embodiment of the present invention, the AP2 / EREBP transcription factor is a common negative regulator of internode and mesocotyl thermal response growth during the seedling stage. Note: A represents... PLTs The target sites and mutation types of gRNAs in gene mutants. BD has 3. PLTs Functional analysis of genes in the thermal response elongation of internodes and mesocotyls during seedling growth. Statistical analysis was performed on internodes during seedling growth at 7 days and mesocotyls during growth at 6 days in rice seedlings grown at 25℃ and 32℃. Values ​​are expressed as mean ± standard deviation (n ≥ 20). Student's score was used. t-test Perform a significance test; the scale bar is 10 mm.

[0040] Figure 19 In a preferred embodiment of the present invention, AP2 / EREBP is a negative regulatory factor for deep-seeded rice seedlings. Note: AB PLTs The dynamic emergence of the gene mutant under shallow sowing (A) and deep sowing (B) conditions was studied. The soil covering depth for shallow sowing was 2 cm, and the soil covering depth for deep sowing was 6 cm. Emergence was recorded daily for two consecutive weeks, with n = 24.

[0041] Detailed Implementation

[0042] This invention analyzes the differences in tissue and cellular behavior in the temperature-responsive growth of internodes and mesocotyls during the seedling stage through physiological experiments and cytological observations. It also mines potential key transcription factors and regulatory genes through combined analysis of transcriptomics and chromatin accessibility sequencing. Furthermore, it uses genetics, biochemistry, and molecular biology to elucidate the molecular mechanisms by which key genes participate in the heat-responsive growth of the two organs. Finally, it verifies the emergence potential of related genes under deep sowing conditions through emergence experiments, providing gene resources for breeding rice varieties suitable for direct seeding in dry conditions.

[0043] 1. Molecular mechanisms of heat-responsive growth in rice

[0044] 1.1 Thermal response growth characteristics of internodes and mesocotyls in rice seedlings

[0045] 1.1.1 The elongation characteristics of internodes and mesocotyls during the seedling stage are the driving force for emergence of deep-sown rice seedlings.

[0046] In the process of direct-seeded rice production, rice seeds are generally sown at a certain depth in the soil to allow them to absorb moisture from the soil for germination and growth. For successful emergence, the seedling tip needs a strong pushing force to lift the apical meristem to the soil surface. According to relevant research, different grasses exhibit different phenotypic plasticities in adapting to deep sowing. Based on their emergence strategies, they can be divided into three categories: when seeds are buried deep in the soil, the mesocotyl of corn and sorghum lifts the apical meristem to the soil surface; the mesocotyl of wheat seedlings does not elongate under deep sowing conditions, but the apex is raised to the soil surface through the elongation of the first internode; while the mesocotyl and first internode of oats and rice both elongate under deep sowing conditions, helping the seedlings to emerge quickly (Hoshikawa, 1969).

[0047] The mesocotyl refers to the portion between the coleoptile node and the radicle attachment point in a seedling. Figure 1 The mesocoaxial axis is a crucial channel connecting seeds and the ground. Varieties with long mesocoaxial axes exhibit excellent emergence performance under deep sowing conditions. In rice, the emergence rate of rice varieties with long mesocoaxial axes is positively correlated with their deep sowing rate; long mesocoaxial rice varieties have significantly higher emergence rates than medium and short mesocoaxial varieties. For example, Turner et al. (1982) compared the mesocoaxial elongation of different rice varieties with their emergence rates at different sowing depths, finding that varieties with longer mesocoaxial axes had higher emergence rates and faster emergence speeds. Chung (2010) compared the emergence potential of 128 different genotype rice varieties under deep sowing conditions, finding that the japonica weedy rice WD-3 had the longest mesocoaxial axis and the highest emergence rate. Alibu et al. (2012) compared different genotype rice varieties and found that only genotypes with significantly elongated mesocoaxial axes could emerge from deep soil. Therefore, mesocoaxial elongation provides the driving force for deep sowing and emergence.

[0048] The first internode refers to the internode tissue between the coleoptile node and the next higher node in a seedling, which is referred to as the seedling internode in this invention. Figure 1 Internode elongation during the seedling stage facilitates deep-seeded rice seedling emergence. In rice, an analysis of the emergence motive forces across different germplasm resources revealed a significant positive correlation between internode elongation and emergence rate when soil cover depth was increased, suggesting that internode elongation during the seedling stage provides a driving force for deep-seeded rice seedling emergence (Yang Qing, 2016). Therefore, internode elongation during the seedling stage is a key driving force for deep-seeded rice seedling emergence. In conclusion, the elongation characteristics of internodes and mesocotyls during the seedling stage help enhance the soil-penetrating force of deep-seeded rice seedlings in dry direct seeding, thus improving problems such as low emergence rate and uneven emergence in dry direct seeding.

[0049] Deep-sown rice exhibits specific elongation characteristics of internodes and mesocotyls during the seedling stage. To explore the morphological differences between shallow-sown and deep-sown rice seedlings, we sowed uniformly germinated indica rice seeds of Nanjing 6 (NJ6) on the surface of water-saturated vermiculite and at a depth of 10 cm, respectively, and grew them in a 32℃ incubator. Morphological observation of the seedlings revealed that in deep-sown seedlings, elongation occurred between the scutellum and coleoptile internodes, forming the mesocotyl, while elongation also occurred between the coleoptile internode and the first internode, forming the first internode. Typically, the first internode elongated more significantly than the mesocotyl. In shallow-sown rice seedlings, seedling height was mainly manifested by leaf elongation, while internode tissue accumulated at the seed base without significant elongation. Furthermore, deep-sown seedlings showed faster seedling height elongation than shallow-sown seedlings. Figure 2 Next, we compared the growth of the first internode and mesocotyl of different rice varieties under deep sowing conditions. The results showed that the mesocotyl of all rice varieties elongated under deep sowing conditions. In some varieties, the first internode did not elongate, but the second internode did (e.g., ZH11 and NIP), while in others, both the first and second internodes elongated (e.g., NJ6 and DJ). Therefore, we collectively refer to the first or second internodes of different rice varieties that elongated under deep sowing conditions as seedling internodes. Figure 2 C). In summary, the rapid elongation of internodes and mesocotyls during the rice seedling stage under deep sowing conditions significantly increases the overall seedling height, suggesting that these two organs may provide the driving force for seedling emergence from the soil in deep-sown seedlings.

[0050] 1.1.2 The elongation growth of internodes and mesocotyls during the seedling stage is affected by ambient temperature.

[0051] Internodes and mesocotyls in rice seedlings exhibit heat-responsive growth characteristics. Temperature is one of the key factors affecting seed germination and emergence. To verify whether the elongation of internodes and mesocotyls in rice seedlings is affected by ambient temperature, uniformly germinating indica rice NJ6 seeds were sown in 10 cm deep water-saturated vermiculite and placed in dark incubators at four temperatures (25℃, 28℃, 30℃, and 32℃) for 4–6 days. Statistical analysis was then performed on the height of internodes, mesocotyls, coleoptiles, and seedlings above the seed. Figure 3 The results showed that seedling height, internodes, and mesocotyls all elongated rapidly with increasing temperature during the 4-6 day growth period, while the elongation of coleoptiles was less affected by temperature. This indicates that the heat-responsive elongation of internodes and mesocotyls during the seedling stage may be the main reason for the increased seedling height. Furthermore, we found that internode elongation during the seedling stage was influenced by both temperature and growth time. For example, when temperatures rose within a small range of 25℃ to 28℃, the elongation trend of internodes after 4 days of growth was weak, while the temperature-responsive elongation trend of internodes after 5 or 6 days was more rapid, indicating that both growth time and temperature changes affect internode elongation. Mesocotyls elongated slightly with growth time, but the elongation was small, indicating that changes in ambient temperature are one of the main factors affecting mesocotyl growth. In contrast, coleoptiles only elongated in response to increased ambient temperature after 4 days of growth. These results indicate that the elongation of internodes and mesocotyls during the seedling stage is temperature-sensitive, gradually elongating with increasing temperature.

[0052] 1.2 Thermoresponsive growth of internodes during the seedling stage is mainly achieved through cell expansion.

[0053] To further understand the cellular basis of internodes during heat-responsive growth in seedlings, we compared and analyzed the cortical cells of internodes grown at lower and higher ambient temperatures. First, we performed cellular analysis on internodes of 4-day-old indica rice seedlings (NJ6) that rapidly elongated after a small temperature increase from 30℃ to 32℃. The results showed that the cell length and width of internodes grown at 32℃ significantly expanded compared to 30℃, leading to a significant increase in the cell length-to-width ratio and theoretical cell area. Although the number of cells per unit length decreased significantly, the total cell number remained unchanged. Figure 4(A) indicates that the internodes of NJ6 seedlings promote rapid elongation of internode tissue under high ambient temperatures by causing rapid cell expansion. Simultaneously, to understand whether there are differences in the cellular behavior of internode heat response growth among different rice varieties, we observed the internode cells of two japonica rice varieties, NIP and DJ. The results showed that compared to 25℃, the internode cells of NIP and DJ seedlings became longer and wider at 32℃, with significantly increased cell length-to-width ratio and theoretical cell area, and significantly reduced cell number per unit length. The total cell number in NIP did not differ significantly between the two temperatures, but the cell number in DJ showed an increasing trend (…). Figure 4 (B) indicates that japonica rice mainly promotes internode elongation during the seedling stage through cell expansion, while cell division in DJ may further drive the thermal response growth of internodes during the seedling stage. These results suggest that cell expansion is the main cause of thermal response elongation of internodes during the seedling stage.

[0054] 1.3 Cell expansion-related genes undergo significant changes during the heat-responsive growth of internodes in seedlings.

[0055] To understand the gene expression changes in internodes during heat-responsive growth in seedlings, we performed transcriptome analysis on internodes sampled at 25℃ and 32℃ for 4, 5, and 6 days. No significant growth was observed in internodes grown at 25℃ for 4 days; therefore, we selected internodes grown at 30℃ that showed significant elongation compared to those grown at 32℃ for transcriptome library construction. Furthermore, to differentiate the heat response differences of internode organs under light and dark conditions, we incorporated transcriptome data from rice seedlings grown for 2 weeks in incubators at 25℃, 30℃, and 35℃ for comparative analysis. First, we performed quality control on the raw transcriptome data, aligning the sequenced reads with the rice genome. Oryza sativa MSU v7.0 (Ouyang et al., 2006). Statistically, the alignment rate of reads generated from sequencing each sample ranged from 86.41% to 90.72%. Figure 5 This indicates that the RNA-seq sequencing quality is good. Next, based on the following screening strategy: a fold change in gene expression between samples ≥ 2 and a q-value < 0.05, gene expression in the same organ at two temperatures was compared and analyzed, and the obtained temperature-differentially expressed genes were termed Thermoresponsive differentially expressed genes (TDEGs).

[0056] First, we compared and analyzed the TDEGs of internodes at the same growth stage. 5,156, 7,052, and 10,352 TDEGs were obtained in heat-responsive elongation internodes at 4, 5, and 6 days of growth, respectively. The number of TDEGs gradually increased with the extension of growth time, indicating that the changes in gene expression in internodes at the seedling stage became increasingly sensitive to temperature increases with growth time. Figure 6 A). We also compared the thermal responses of internodes in seedlings grown in the dark with those grown in the light. The results showed that in seedlings grown in the light, the number of genes upregulated and downregulated at increasing temperature were similar, while in internodes during the seedling stage, the number of upregulated genes was higher than the number of downregulated genes across the three growth days. This suggests that the elongation of the thermal response in internodes during the seedling stage may be a process of gene expression activation. Figure 6 A). Next, we performed GO functional enrichment analysis on TDEGs from internodes during the seedling stage. The results showed that genes involved in cell wall and microtubule skeleton composition and auxin signaling response were significantly enriched ( Figure 6 (B) indicates that differential expression of heat-responsive genes involved in cell wall and microtubule skeleton remodeling may promote heat-responsive growth in seedling internodes by mediating cell expansion during the seedling stage.

[0057] 1.4 The thermal response transcriptome of internodes during the seedling stage exhibits developmental time dependence.

[0058] Furthermore, we considered the influence of growth time on the thermal response elongation trend of internodes during the seedling stage. Figure 3 Furthermore, the number of TDEGs gradually increases with the extension of growth time. Figure 6 A), and then explored its thermal response regulation mechanism at the time level. First, the TDEGs of seedling internodes at three growth days were compared and analyzed. The results showed that 1,298, 969 and 4,142 TDEGs specifically responded to the temperature in the seedling internodes at 4, 5 and 6 days of growth, respectively. Most TDEGs were shared between adjacent days, while 2,021 TDEGs were shared across the three growth days (A). Figure 7 A). Gene expression trend analysis of TDEGs shared across three growth days revealed that the expression patterns of TDEGs shared across the three growth days were largely consistent during the seedling internodes. 47.5% of TDEGs were upregulated during heat-responsive growth, 40.7% were downregulated, and only a small number (11.8%) showed different expression trends across different growth days. Figure 7 B). GO enrichment analysis showed that microtubule cytoskeleton and cell wall biogenesis-related processes were significantly enriched in TDEGs common to the three growth days of the seedling internodes (B). Figure 7(C) indicates that these pathways are crucial in the growth and development of internodes and their thermal elongation during the seedling stage. Furthermore, by enriching TDEGs specific to different growth stages using GO, we found an interesting developmental progression in the temperature response of seedling internodes over time. Specifically, biological processes related to nucleic acid metabolism and gene expression were enriched on day 4, functional pathways such as membrane lipid metabolism, proteasomes, and organelles were enriched on day 5, and microtubule skeleton and cell wall formation processes were significantly enriched on day 6. Figure 7 These results indicate that rice seedlings at the 4-6 day stage mobilize relevant genes at the molecular, cellular, and subcellular levels, and then at the tissue level, to participate in their heat-responsive growth and development.

[0059] 1.5 Thermoresponsive growth of the mesocotyl is determined by both cell proliferation and expansion.

[0060] Meanwhile, to further understand the cellular basis of mesocotyls during heat-responsive growth, we compared and analyzed the cortical cells of mesocotyls in indica rice NJ6 grown at lower and higher ambient temperatures. The results showed that the cell length, width, length-to-width ratio, theoretical cell area, and number of cells per unit length of mesocotyls did not change significantly after 5 days of growth at 25℃ and 30℃, but the total cell number at 30℃ was significantly higher than that at 25℃. Figure 8 A) indicates that cell proliferation mediates the thermoresponsive growth of mesocotyls in indica rice NJ6. To verify this result, we simultaneously observed changes in mesocotyl cells during thermoresponsive growth in japonica rice NIP and DJ. Figure 8 (B) Among them, in Japonica rice NIP, the elongation and narrowing of mesocotyl cells at 32℃ led to a decrease in the theoretical cell area, but the total cell number increased significantly. In Japonica rice DJ, the length and width of mesocotyl cells both increased significantly at high ambient temperatures, and the cell length-to-width ratio and theoretical cell area both increased significantly, along with a significant increase in the total cell number. This result indicates that the mesocotyl of Japonica rice undergoes rapid elongation at high ambient temperatures through cell proliferation, and this cell elongation further promotes its thermal response elongation. These results suggest that the thermal response elongation of the mesocotyl is mainly achieved through cell proliferation, and due to the diversity of rice varieties, cell expansion further promotes the thermal response growth of the Japonica rice mesocotyl.

[0061] 1.6 Transcriptome changes related to cell proliferation and expansion promote mesocotyl thermal elongation.

[0062] Next, we explored the heat-responsive growth mechanism by performing transcriptome sequencing on mesocotyls grown for different days at two different temperatures. First, we compared and analyzed the TDEGs of mesocotyls grown for the same duration. In heat-responsive mesocotyls grown for 4, 5, and 6 days, we obtained 7,774, 2,072, and 2,950 TDEGs, respectively. The number of TDEGs decreased with increasing growth time, indicating that changes in mesocotyl gene expression are more sensitive to temperature increases in the early stages of growth. Figure 9 A). Meanwhile, compared with seedlings exhibiting heat-responsive growth under light, we found that unlike high ambient temperatures which activate more gene expression in seedling internodes, high ambient temperatures tend to suppress more mesocotyl gene expression, suggesting that heat-responsive growth of mesocotyls may be a process of gene expression desuppression. Figure 9 A). Next, we performed GO functional enrichment analysis on all mesocotyl TDEGs. The results showed that genes involved in cell cycle, DNA replication, microtubule cytoskeleton, and cell wall composition were significantly enriched (A). Figure 9 (B) indicates that changes in the expression of relevant TDEGs may promote the rapid proliferation and expansion of mesocotyl cells during heat-responsive growth, thereby promoting heat-responsive elongation growth of the mesocotyl.

[0063] 1.7 The mesocotyl thermoresponsive transcriptome also exhibits developmental time dependence.

[0064] To further investigate the time-dependent regulatory mechanism of mesocotyl thermal response during growth, we first compared and analyzed the TDEGs of mesocotyls at three growth days. The results showed that 5,391, 695, and 690 TDEGs, respectively, specifically responded to temperature changes in mesocotyls at growth days 4, 5, and 6. Similar to internodes in the seedling stage, mesocotyls at adjacent growth days shared most of their TDEGs, with 544 TDEGs shared across the three growth days. Figure 10 A). Further gene expression trend analysis of TDEGs shared across the three growth days revealed that the expression patterns of TDEGs in the mesocotyl were largely consistent across the three growth days. 23.7% of TDEGs were upregulated during heat-responsive growth, 56.1% were downregulated, and 20.2% showed different expression trends across different growth days. Figure 10 B). Similar to internodes in the seedling stage, cell wall biogenesis-related processes were significantly enriched in TDEGs common to all three growth days of the mesocotyl (B). Figure 10(C) indicates that this pathway is crucial in the growth and development of both organs and in their heat-responsive elongation. Furthermore, GO enrichment analysis of TDEGs specific to different growth stages revealed a heat-responsive developmental process in the mesocotyl that differed from that in the seedling internodes. Translational and protein metabolism processes were enriched on day 4, while secondary metabolic processes were enriched in temperature-responsive genes specific to days 5 and 6. Figure 10 (DE). The above results indicate that the heat-response transcriptome of the rice mesocotyl also exhibits developmental time dependence, and its heat response during growth days 4–6 encompasses related biological processes from primary to secondary metabolism.

[0065] 1.8 The temperature response transcriptomes of internodes and mesocotyls during the seedling stage showed high similarity.

[0066] Meanwhile, we compared and analyzed the thermal response transcriptomes of internodes and mesocotyls during the seedling stage to explore the similarities and differences between the two organs in thermal response growth. First, the results of principal component analysis (PCA) and correlation heatmaps showed that samples from internodes and mesocotyls during the seedling stage had higher correlations, and samples from both organs clustered together, unlike those from seedlings grown under light which were dispersed. Figure 11 The results suggest that the differentially expressed genes in internodes and mesocotyls of seedlings grown under dark conditions are more similar compared to those grown under light. Secondly, TDEGs in internodes, mesocotyls, and seedlings grown under light were compared and analyzed. The results showed that a total of 13,473, 9,519, and 7,942 TDEGs were obtained in internodes, mesocotyls, and shared by both at the three growth days, respectively. Fewer TDEGs were detected in seedlings grown under light (s3,649), and only 1,748 TDEGs were shared by all three. Figure 11 (C) This result further illustrates that TDEGs in seedling internodes and mesocotyls have certain similarities. Next, we performed GO functional annotation on TDEGs shared by the two dark-growing organs and by tissues growing under both light and dark conditions. We found that processes such as ribosomes, translation, polypeptide synthesis and metabolism, and microtubule scaffold remodeling were significantly enriched in the TDEGs shared by the two dark-growing organs. Cell wall biogenesis and metabolic processes were enriched in the TDEGs shared by all three organs. Figure 11 The expression changes of genes involved in the basic rate-limiting steps of cell growth and development, such as ribosome biogenesis and polypeptide synthesis, indicate that changes in the expression of genes in seedling internodes and mesocotyls induce rapid elongation in response to increased temperature. Changes in the expression of genes involved in cell expansion promote the thermal response growth of the two dark-growing organs and light-growing seedlings.

[0067] 1.9 Heat-responsive genes specific to internodes and mesocotyls during seedling stage

[0068] Comparative analysis of TDEGs from internodes, mesocotyls, and seedlings grown under light yielded 4,894 internode-specific and 1,390 mesocotyl-specific TDEGs, which were then subjected to GO enrichment analysis. Internode-specific TDEGs were enriched for biological pathways related to various RNA metabolism and modification, while cell wall recombination-related genes were significantly enriched in mesocotyl-specific TDEGs. Figure 12 A) indicates that the two organs have different heat-responsive growth mechanisms. Simultaneously, by screening genes whose mean TPM of the same organ differed from other organs by ≥ 5 fold and exhibited temperature-specific expression differences, we obtained 237 seedling internode-specific genes, 429 mesocotyl-specific genes, and 728 light-dependent seedling-specific genes. Figure 12 B). GO enrichment analysis showed that TDEGs specifically expressed in internodes during seedling growth were enriched in pathways related to cell wall biogenesis and auxin response. TDEGs specifically expressed in mesocotyls were mainly involved in processes related to cell wall biogenesis. Meanwhile, TDEGs specific to light-sensitive heat-responsive seedlings were enriched in processes related to photosynthesis, enzyme activity, and redox reactions. This indicates that both organs growing in darkness and seedlings growing in light possess their own specific heat-responsive growth mechanisms. Figure 12 C). To verify the reliability of the two organ-specific TDEGs selected, we reprocessed a new batch of biological samples and selected three TDEGs that were specifically expressed in the internodes during the seedling stage. LOC_Os10g27280 , LOC_Os12g44270 and LOC_Os09g24840 and in mesocotyl-specific expression LOC_Os10g31620 , LOC_Os10g01930 and LOC_Os10g31540 Gene expression levels were detected by qRT-PCR. The results showed that the gene expression trends of the six selected genes were basically consistent in both RNA-seq and qRT-PCR. Figure 12 (D) indicates that our RNA-seq data and the tissue-specific genes screened are reliable. These results suggest that internodes and mesocotyls in seedlings possess their own unique heat-responsive genes, and that differential expression of these genes mediates their heat-responsive elongation growth.

[0069] 1.10 Thermoresponsive growth-related transcriptional regulatory network of internodes and mesocotyls during seedling stage

[0070] To further investigate the heat-responsive growth mechanism of internodes and mesocotyls during the seedling stage, we used Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq) to map the whole genome of accessible chromatin regions (ACRs) in the internodes and mesocotyls of 6-day-old indica rice NJ6 seedlings. First, we assessed the quality of all ACRs obtained from all samples. The results showed that the size of ACR fragments was mainly distributed between 30 and 900 bp; simultaneously, due to the periodicity of nucleosome arrangement, most fragments were concentrated between 100 and 250 bp. Figure 13 A). Secondly, analysis of the distribution density of ACRs in genes and neighboring genes across all samples revealed that ACRs exhibited a similar distribution pattern in all samples, and these ACRs were mainly enriched at gene transcription start sites (TSS). Figure 13 (B) This indicates that the upstream regulatory regions of these genes are open, allowing some transcription factors to dock with these regions and thus regulate the expression of neighboring genes. Next, we calculated the correlation between different samples. The correlation between the two biological replicates was high, with a correlation coefficient exceeding 95%. Figure 13 C). Statistically, over 32,000 high-resolution ACRs were obtained in each sample. The majority of ACRs (~70%) were located in the proximal region, including approximately 35% upstream of the transcription start site (TSS), approximately 15% in introns and exons, and approximately 10% downstream of the transcription termination site (TES); a minority (~28%) were located in the distal region or intergenic region. Figure 13 A). Meanwhile, we found that the proportion of ACRs in the proximal regions of genes in internodes and mesocotyl samples grown at 32℃ was significantly higher than that at 25℃, especially the number of ACRs located in exon regions was significantly increased. Figure 14A). ACRs located in different regions of the genome are often associated with gene expression levels. For example, distal ACRs are widely used to predict putative enhancers in plants, while proximal ACRs are closely associated with gene expression (Tian et al., 2021). Therefore, we conducted a comprehensive analysis of gene expression data and chromatin accessibility obtained from transcriptomics. The results showed that the number of genes containing only a single class of ACRs was the highest. The expression abundance of genes containing only ACRs in the TSS, Exon, and Intron regions was significantly higher than that of genes containing only TES and Intergenic regions. Fewer genes were detected to contain multiple classes of ACRs, but genes containing multiple classes of ACRs had higher expression levels, and a very small number of genes containing five classes of ACRs had the highest expression abundance. Overall, ACRs in the proximal TSS region of genes contributed the most to gene expression. Figure 14 B). The above results indicate that the chromatin-accessible regions of internodes and mesocotyls during the rice seedling stage affect the expression abundance of neighboring genes.

[0071] Simultaneously, we compared and analyzed organ-specific TDEGs expressed in the transcriptome and TAAGs from ATAC-seq, identifying overlapping genes as key regulatory genes for heat-responsive elongation of internodes or mesocotyls during seedling stages. Through comparative analysis, we identified 154 key regulatory genes for heat-responsive elongation of internodes and 41 key regulatory genes for heat-responsive elongation of mesocotyls during seedling stages. Figure 15 A and Figure 16 A). Further selection of genes was used to obtain loss-of-function mutants via CRISPR / Cas9 gene editing technology, and the thermal response elongation phenotypes of two organs were observed. First, the gene encoding the AAA-type ATPase family protein was... LOC_Os12g10670 mutant im1 and encoding terpene synthase LOC_Os03g31430 mutant im2 The internode length during the seedling stage was not significantly different from that of the wild type at 25℃, but was significantly longer than that of the wild type at 32℃. Furthermore, the internode elongation trend during the seedling stage in both groups was significantly increased compared to the wild type. Figure 15 (BC) indicates LOC_Os12g10670 and LOC_Os03g31430 Negative regulation of internode elongation in seedlings during thermal response. Secondly, it encodes transferase family proteins. LOC_Os10g01930 mutant mm1The mesocotyl was significantly longer than the wild-type at 25°C, encoding a glycine-rich cellwall structural protein 2 precursor. LOC_Os10g31540 mutant mm2 The mesocotyls of the mutants were significantly shorter than those of the wild type at 25℃, while the mesocotyls of both mutants were significantly longer than those of the wild type at 32℃, and the thermal response growth trend of the mesocotyls was slightly increased compared to the wild type. Figure 16 (BC) indicates LOC_Os10g01930 and LOC_Os10g31540 Negative regulation of mesocotyl thermal response elongation. These results collectively confirm the reliability of the key regulatory genes screened through combined RNA-seq and ATAC-seq analysis in regulating the thermal response growth of both organs.

[0072] Because ACRs contain specific cis-acting elements, they allow trans-acting factors to dock and thus regulate gene expression (Tian et al., 2021). Therefore, we sought to identify key transcription factors regulating the expression of heat-responsive genes in internodes and mesocotyls during the seedling stage. First, we used the HOMER (Hypergeometric Optimization of Motif Enrichment) tool to enrich transcription factors in TACRs of internodes and mesocotyls during the rice seedling stage. The results showed that transcription factor families such as SPL, MYB, bHLH, C2H2, Zf, and Homeobox were significantly enriched in rTACRs of both internodes and mesocotyls during the seedling stage; among them, rTACRs of internodes recruited MADS-box transcription factors, while rTACRs of mesocotyls recruited transcription factor families such as TCP, bZIP, NAC, and WRKY; at the same time, we found that the AP2 / EREBP transcription factor family was recruited in iTACRs of both internodes and mesocotyls during the seedling stage (iTACRs). Figure 17(A) This result indicates that TACRs recruited a rich number of transcription factors to regulate TDEG expression during the heat-responsive growth process in both organs. Next, we further used the FIMO (Find Individual Motif Occurrences) tool to scan the ACRs of TDEGs in both organs, enriching the transcription factors involved in the regulation of TDEG expression. Based on the similarity of transcription factor binding to target motifs, all enriched transcription factor families were divided into 44 clusters, each containing one or more transcription factor families. The results showed that transcription factor clusters 2, 8, 10, 23, 26, 35, 40, and 41 were significantly enriched in upregulated TDEGs induced by high temperature in seedling internodes, while clusters 21 and 30 were enriched in downregulated TDEGs suppressed by high temperature in seedling internodes. Compared with seedling internodes, upregulated TDEGs in the mesocotyl recruited only cluster 40, while downregulated TDEGs recruited only cluster 21. Figure 17 B). Because the 2nd, 8th, and 10th transcription factor clusters, which were significantly enriched in the up-regulated TDEGs of seedling internodes, all contained AP2 / EREBP transcription factors, and the 21st transcription factor cluster containing SPL was recruited in the down-regulated TDEGs of both organs (…). Figure 17 C), and both gene families were significantly enriched in both transcription factor enrichment modes, suggesting that these two transcription factor families are crucial for the heat-responsive growth of internodes and mesocotyls during the seedling stage. Further analysis of the interaction networks between the AP2 / EREBP and SPL transcription factor families and key regulatory genes in internodes or mesocotyls during the seedling stage revealed that key regulatory genes in internodes were linked to both transcription factor families via different types of TACRs at a higher rate than those in mesocotyls. Specifically, the AP2 / EREBP transcription factor family tethered to TACRs of 14 regulatory genes in internodes and 7 in mesocotyls, while the SPL transcription factor family tethered to TACRs of 26 internodes and 2 in mesocotyls. Figure 17 (D) indicates that the AP2 / EREBP and SPL transcription factor families are crucial in the thermoresponsive growth regulatory network of internodes and mesocotyls during the seedling stage.

[0073] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0074] Example 1: AP2 / EREBP family negatively regulates the thermal response growth of internodes and mesocotyls during seedling stage

[0075] The PLETHORA (PLT) transcription factors of the AP2 / EREBP family play an important role in plant growth and development (Aida et al., 2004; Scheres & Krizek, 2018; Hao et al., 2023). In this invention, we found that the AP2 / EREBP transcription factor family may regulate gene expression by binding to ACRs of TDEGs in seedling internodes. Therefore, we further investigated the role of AP2 / EREBP transcription factor family genes in the heat-responsive growth of seedling internodes.

[0076] choose LOC_Os01g67410 ( AP2 / EREBP transcription factor BABY BOOM, PLT5 ), LOC_Os02g40070 ( AP2-likeethylene-responsive transcription factor PLETHORA 2, PLT3 ) LOC_Os04g55970 ( AP2-like ethylene-responsive transcription factor AINTEGUMENTA, PLT1 Gene editing mutants were constructed and their thermal response growth phenotypes in rice seedling internodes and mesocotyls were validated.

[0077] In this invention, LOC_Os04g55970 ( PLT1 The genomic nucleotide sequence, CDS sequence, and amino acid sequence of the encoded protein of the ) are shown in SEQ ID NO:1-3, respectively; LOC_Os02g40070 ( PLT3 The genomic nucleotide sequence, CDS sequence, and amino acid sequence of the encoded protein of the ) are shown in SEQ ID NO:5-7, respectively; LOC_Os01g67410 (PLT5) The genomic nucleotide sequence, CDS sequence, and amino acid sequence of the encoded protein are shown in SEQ ID NO:9-11, respectively.

[0078] First, we used CRISPR / Cas9 technology to... PLT1 , PLT3 and PLT5 Gene editing was performed, resulting in three loss-of-function mutants: one with a deletion of two bases and another with a deletion of one base. plt1 Both have a missing base and an inserted base. plt3 and plt5 mutant ( Figure 18 (A) These editing methods all lead to premature termination of protein translation.

[0079] The target site selection for CRISPR / Cas9 target genes is as follows:

[0080] LOC_Os04g55970 ( PLT1 ): 5′-TGAGATCGGCCGGTATAACGTGG-3′ (SEQ ID NO: 4)

[0081] LOC_Os02g40070 ( PLT3 ): 5′-AGAGAATCCGCAGCCTCACCAGG-3′ (SEQ ID NO: 8)

[0082] LOC_Os01g67410 ( PLT5 ): 5′-CATGAGGGGATCGGAGCTCTCGG-3′ (SEQ ID NO: 12)

[0083] The primers for identifying the relevant mutants are as follows (SEQ ID NO:13-18):

[0084] plt1 -F:5′-AGCAGCCCGTGTTTGGAATA-3′

[0085] plt1 -R: 5′-GAAGAGCTGTGAACGCAAGC-3′

[0086] plt3 -F:5′-TACCTCCCCCGTATCAGCTT-3′

[0087] plt3 -R: 5′-GCATGCATACCTTGTGCCTC-3′

[0088] plt5 -F:5′-GTCTGCTTCAACATCCCCCA-3′

[0089] plt5 -R: 5′-AATCTCCTTGCGACCACCAG-3′

[0090] Next, we observed the growth phenotype of the relevant mutants during the seedling stage internode heat response. The results showed that three... plts The internode length of the mutant seedlings was not significantly different from that of the wild type at 25℃, but was significantly longer than that of the wild type at 32℃, and also became more sensitive to increases in ambient temperature. Figure 18 (BD), indicating PLT1 , PLT3 and PLT5 Negative regulation of internode thermal response growth during the seedling stage. Simultaneously, three... plts The results of the mesocotyl thermoresponsive growth phenotype of the mutants showed that the mesocotyls were significantly longer than those of the wild type at 32℃, and the thermoresponsive trend was also correspondingly increased. Figure 18(BD), indicating that these 3 PLTs The gene inhibits the heat-responsive growth of the mesocotyl. These results indicate that the AP2 / EREBP transcription factor is involved in the heat-responsive growth process of internodes and mesocotyls during the seedling stage. PLT1 , PLT3 and PLT5 All of them are negative regulators of the thermal response growth of internodes and mesocotyls during the seedling stage.

[0091] To determine the impact of internode and mesocotyl elongation during the seedling stage on rice emergence potential, we studied deep-sown and shallow-sown rice seedlings. plts Statistical analysis was performed on the emergence rate of the mutants at two temperatures. First, under a shallow sowing condition of 2 cm, three... plts The germination rate was similar to that of the wild type at 25℃; however, at 32℃, the germination rate of all three species... plts The germination rate of both types was significantly higher than that of the wild type. Figure 19 A). Secondly, under the condition of a seeding depth of 6 cm, plt1-1 The germination rate of the mutant was similar to that of the wild type at 25℃. plt3-1 and plt5-1 The germination rate of the mutant was slightly higher than that of the wild type; at 32℃, the germination rates of both the wild type and the mutant decreased significantly, while the germination rates of the three mutants were... plts The mutant still maintains a higher germination rate compared to the wild type. Figure 19 B), indicating PLT1 , PLT3 and PLT5 All of these were negative regulatory factors for deep-sown rice seedling emergence. Simultaneously, we found that under both deep and shallow sowing conditions, the emergence rate of all materials grown at 32℃ was significantly accelerated, and... plt3-1 and plt5-1 The germination rate of both types is faster than that of the wild type. Figure 19 The results (AB) indicate that high ambient temperatures accelerate seedling emergence, and plants with significantly elongated internodes and mesocotyls during the seedling stage emerge even faster. These results suggest that having both longer internodes and mesocotyls during the seedling stage can accelerate the emergence of deep-sown seedlings and increase the seedling emergence rate, indicating that the driving force for rice seedling emergence is the combined elongation of internodes and mesocotyls during the seedling stage.

[0092] The characteristics of internode and mesocotyl elongation during the seedling stage are of great significance for the promotion of direct seeding technology in rice. This invention discovers that transcription factors involved in the heat-responsive growth of internodes and mesocotyls during the seedling stage show considerable application potential in improving the seedling emergence rate of deep-seeded rice. These genetic resources may help to breed more rice varieties suitable for direct seeding in dry conditions.

[0093] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for promoting the growth of internodes and mesocotyls in rice seedlings under high ambient temperatures, characterized in that, The method includes: using genetic engineering techniques to knock out the LOC_Os01g67410 gene in rice; The amino acid sequence of the protein encoded by the LOC_Os01g67410 gene is shown in SEQ ID NO:

11.

2. A method for promoting deep-sown rice seedlings under high ambient temperatures, characterized in that, The method includes: using genetic engineering techniques to knock out the LOC_Os01g67410 gene in rice; The amino acid sequence of the protein encoded by the LOC_Os01g67410 gene is shown in SEQ ID NO:

11.

3. The method according to claim 1 or 2, characterized in that, The gene was knocked out using CRISPR, TALEN, or ZFN gene editing technologies.

4. The method according to claim 3, characterized in that, Using the LOC_Os01g67410 gene as a target, a CRISPR / Cas9-based sgRNA sequence was designed. A DNA fragment containing the sgRNA sequence was ligated into a vector carrying CRISPR / Cas9, and rice was transformed to obtain transgenic rice with the gene function lost.

5. The method according to claim 4, characterized in that, The nucleotide sequence of the sgRNA action site is 5'-CATGAGGGGATCGGAGCTCTCGG-3'.

6. The application of transgenic rice obtained by the method according to any one of claims 1-5 in rice breeding with high-temperature deep seeding.

7. The application according to claim 6, characterized in that, Breeding methods include transgenic, hybridization, backcrossing, self-pollination, or asexual reproduction.

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