Application of plant AP2 / EREBP family transcription factor PLT1 gene

Abstract

The invention discloses an application of a plant AP2 / EREBP family transcription factor PLT1 gene. PLT1 is a negative regulation factor for deep-sowing seedling growing of rice. The seedling emergence speed and the seedling emergence rate of the plts mutant with the internode length and the mesocotyl length in the seedling stage are not obviously different from those of the wild type under the shallow sowing condition, but the seedling emergence speed and the seedling emergence rate of the plts mutant are higher than those of the wild type under the deep sowing condition; meanwhile, although the warm environment at 32 DEG C promotes the emergence time of the wild type and the mutant to be advanced and the emergence speed to be accelerated, when the emergence rate of the wild type is greatly reduced at 32 DEG C, the plts mutant can still keep a relatively high emergence rate. The deep sowing resistance and strong seedling emergence performance of the plts mutant prompt that the plts mutant has important application value in dry direct seeding, especially in dry direct seeding production at high temperature in summer. The excellent haplotype of PLTs is found in a natural population or a cultivated variety, so that the cultivation of a rice variety suitable for deep sowing is facilitated.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, in particular to the application of plant AP2 / EREBP family transcription factor PLT1 gene. Background Art

[0002] With the rapid development of industry and agriculture in my country, labor transfer, reduced agricultural irrigation water, and climate change have brought severe challenges to rice production. The dry direct seeding technology of rice can solve the problem of limiting rice production to a certain extent by sowing dry seeds directly into the soil at a certain depth by machine, so that the seeds can use the water in the soil to germinate and grow. However, the rice varieties currently promoted are mainly bred for the purpose of transplanting, and the poor tolerance of most rice varieties to deep sowing also limits the promotion of dry direct seeding technology of rice. Among them, the elongation of internodes and mesocotyls in the seedling stage is the force for rice seedlings to emerge from the soil under deep sowing conditions, and the elongation growth of the two organs is affected by the external environmental temperature, which helps deep-sown rice to cope with climate change. However, there are currently no reports on the molecular mechanism of the elongation of internodes and mesocotyls in response to temperature in the rice seedling stage. Therefore, it is of great significance to explore the molecular mechanism of internodes and mesocotyls in response to temperature growth in the rice seedling stage, discover key regulatory genes, and their emergence potential under deep sowing conditions for the promotion of dry direct seeding technology. Summary of the invention

[0003] The purpose of the present invention is to provide application of plant AP2 / EREBP family transcription factor PLT1 gene.

[0004] The present invention is conceived as follows: First, through physiological experiments, we understand the changes in the tissue and cytological levels of the elongation of internodes and mesocotyls in response to temperature in rice seedlings, perform RNA-seq sequencing on internodes and mesocotyls in seedlings grown at different temperatures, and screen for differentially expressed genes in tissue-specific temperature responses; further combine ATAC-seq data to compare the epigenetic differences of the two tissues, and obtain potential regulatory genes at two omics levels; at the same time, the two omics data are combined to identify potential transcription factors and explore potential downstream regulatory genes, and construct a gene expression regulatory network. According to the analysis results, we performed CRISPR / cas9 gene editing on the selected potential regulatory genes and transcription factors of internodes and mesocotyls in seedlings, and obtained corresponding mutants to verify their functions in the heat-responsive growth and deep sowing of rice. The present invention deepens the understanding of the heat-responsive growth of internodes and mesocotyls in rice seedlings at the chromosome and transcription levels, and provides clues for further understanding the regulatory mechanism of internodes and mesocotyls in seedlings in response to heat. At the same time, the multiple key regulatory genes discovered will help the application of internodes and mesocotyls in seedlings in direct sowing.

[0005] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides any of the following applications of the plant AP2 / EREBP family transcription factor PLT1 gene: 1) Regulate the thermal response growth of internodes and mesocotyls in plant seedlings; 2) Regulate the high ambient temperature to allow plants to germinate deeply.

[0006] AP2 / EREBP transcription factors can integrate metabolism, hormones and environmental signals to regulate plant growth in stress adaptation and retrograde signals, and are an important part of the gene regulatory network.

[0007] The number of PLT1 gene in the rice gene database (https: / / rice.uga.edu / index.shtml) is LOC_Os04g55970 ( AP2-like ethylene-responsive transcription factor AINTEGUMENTA, PLT1 ).

[0008] Furthermore, the regulation is negative regulation.

[0009] The plant includes a grass plant, preferably rice, and more preferably direct-seeded rice.

[0010] In a second aspect, the present invention provides a method for promoting the growth of internodes and mesocotyls of rice seedlings under high environmental temperature, the method comprising: weakening or knocking out the PLT1 gene in rice by genetic engineering means.

[0011] In a third aspect, the present invention provides a method for promoting rice seeding under high environmental temperature, the method comprising: weakening or knocking out the PLT1 gene in rice by genetic engineering means.

[0012] Furthermore, the gene can be weakened or knocked out by using CRISPR, TALEN or ZFN gene editing technology.

[0013] In a specific embodiment of the present invention, the PLT1 gene is used as a target, an sgRNA sequence based on CRISPR / Cas9 is designed, a DNA fragment containing the encoding sgRNA sequence is connected to a vector carrying CRISPR / Cas9, and rice is transformed to obtain transgenic rice with a functional deficiency of the gene.

[0014] Preferably, the nucleotide sequence of the sgRNA action site is 5'-TGAGATCGGCCGGTATAACGTGG -3' (SEQ ID NO: 4).

[0015] The expression vector carrying the target gene can be introduced into plant cells by conventional biotechnology methods such as Ti plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, etc. (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).

[0016] In a fourth aspect, the present invention provides the use of the transgenic rice obtained according to the method in plant breeding.

[0017] Breeding methods include, but are not limited to, transgenic, hybridization, backcrossing, selfing, or asexual reproduction.

[0018] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: The present invention is the first to discover that the PLT transcription factor of the AP2 / EREBP family has great application value in direct seeding of rice. PLT1 It is a negative regulatory factor for rice seedling growth. plts The mutant had no significant difference in germination speed and germination rate under shallow sowing conditions compared with the wild type, but had faster germination and higher germination rate under deep sowing conditions compared with the wild type. At the same time, although the warm environment of 32°C caused the germination time of the wild type and mutant to be earlier and the germination speed to be faster; and when the germination rate of the wild type was greatly reduced at 32°C, plts The mutant was still able to maintain a high germination rate. plts The deep seeding resistance and strong germination performance of the mutant suggest that it has important application value in dry direct seeding, especially in high temperature summer dry direct seeding. PLTs The superior haplotypes will be beneficial for breeding rice varieties suitable for deep sowing. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a schematic diagram of the internodes and mesocotyls of deep-sown rice seedlings at the seedling stage in a preferred embodiment of the present invention.

[0020] Figure 2 In a preferred embodiment of the present invention, deep-sown rice seedlings specifically produce internodes and mesocotyls in the seedling stage.

[0021] Figure 3In the preferred embodiment of the present invention, high ambient temperature promotes the elongation of internodes and mesocotyls in the seedling stage. Note: Four phenotypes of NJ6 rice deep-sown seedlings grown for 4 to 6 days at four temperatures were counted. The four phenotypes were internodes (Inter, seedling internode), mesocotyl (Meso, mesocotyl), coleoptile (Cole, coleoptile) and full-length above seed (Flas, full-length above seed). The values ​​represent the mean ± standard error, n ≥ 25.

[0022] Figure 4 The cell expansion mediates the heat response growth of internodes in the seedling stage in the preferred embodiment of the present invention. Note: AB is the cytological phenotype of internodes in the seedling stage of indica rice A and japonica rice B at two temperatures. The internode cells of indica rice NJ6 grown at 30℃ and 32℃ for 4 days, and the internode cells of japonica rice NIP and DJ grown at 25℃ and 32℃ for 7 days were used for statistical analysis. The number of statistical samples was ≥3, the number of cells was ≥100, and the Student's t-test The significance analysis was performed; the observation method of internode cells in indica rice seedling stage was Calcofluor tissue staining, and that of japonica rice was resin semi-thin section; the scale bar is 100 μm.

[0023] Figure 5 This is the comparison of RNA-seq data with reference genome in a preferred embodiment of the present invention. Note: Inter (seedling internode), Meso (mesocotyl), Sdl (light-grown seedling), "Inter4-30" means the transcriptome of seedling internode grown at 30°C for 4 days, "DJ-25-Inter" means the transcriptome of DJ seedling internode grown at 25°C. "_1 / _2" are two biological replicates respectively.

[0024] Figure 6 The cell expansion-related pathways in the preferred embodiment of the present invention are significantly enriched in the thermoresponsive transcriptome of the internodes at the seedling stage. Note: A is the number of temperature-responsive differentially expressed genes (TDEGs) in the internodes at the seedling stage and in the seedlings grown under light. Inter (seedling internode), Sdl (light-grown seedlings), "Inter4_30" represents the transcriptome of the internodes at the seedling stage grown for 4 days at 30°C. The seedling internode materials come from deep-sown rice seedlings, and the seedling materials come from the aboveground tissues of shallow-sown rice seedlings that have been illuminated. B is the GO enrichment analysis of all internodes at the seedling stage TDEGs.

[0025] Figure 7The thermal response process of the internodes in the seedling stage in the preferred embodiment of the present invention progresses layer by layer with the number of growing days. Note: A is the difference analysis of the TDEGs in the internodes of the seedling stage at different growth periods. B is the expression trend analysis of the common TDEGs in the internodes of the seedling stage at three growing days. The values ​​in the radar chart are the difference multiples of the expression of TDEGs at two temperatures, that is, Log2 (Foldchange); the blue shadows are suppressed expression in the tissues of 4, 5, and 6 days with the increase of temperature, the red shadows are induced expression with the increase of temperature, and the gray shadows are inconsistent in the temperature response changes in the three growing days. C is the GO enrichment analysis of the common TDEGs in the internodes of the seedling stage in the three growth periods. DE is the expression heat map D and GO analysis E of the specific TDEGs in the internodes of the seedling stage at different developmental stages. Z-score represents the number of standard deviations between the gene expression of each sample and the mean gene expression of the row.

[0026] Figure 8 The cell proliferation and expansion act together on the heat response growth of the mesocotyl in the preferred embodiment of the present invention. Note: AB is the cytological phenotype of the mesocotyl of indica rice A and japonica rice B at two temperatures. The internode cells 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 number of statistical samples was ≥ 3, the number of cells was ≥ 100, and the Student's t-test Significance analysis was performed; the observation method of mesocotyl cells in the figure was Calcofluor tissue staining method; the scale bar is 100 μm.

[0027] Fig. 9 In the preferred embodiment of the present invention, cell proliferation and expansion-related pathways are significantly enriched in the thermoresponsive transcriptome of the mesocotyl. Note: A is the number of TDEGs in the mesocotyl and light-grown seedlings. Meso (mesocotyl), the mesocotyl material comes from deep-sown rice seedlings. B is the GO enrichment analysis of all mesocotyl TDEGs.

[0028] Fig.10 The thermal response process of the mesocotyl in the preferred embodiment of the present invention progresses layer by layer with the number of growth days. Note: A is the difference analysis of the TDEGs of the mesocotyl at different growth periods. B is the expression trend analysis of the common TDEGs of the mesocotyl at three growth days. C is the GO enrichment analysis of the common TDEGs of the mesocotyl at three growth periods. DE is the expression heat map D of the specific TDEGs of the mesocotyl at different developmental stages and the GO enrichment analysis E. Z-score represents the number of standard deviations between the gene expression of each sample and the mean gene expression of the row.

[0029] Fig.11The thermoresponsive transcriptomes of internodes and mesocotyls in the seedling stage in the preferred embodiment of the present invention have high similarity. Note: A is the principal component analysis of RNA-seq. B is the correlation analysis of gene expression levels of all transcriptome samples. C is the number of TDEGs in internodes, mesocotyls and light-grown seedlings at the seedling stage. D is the GO enrichment analysis of common TDEGs in internodes and mesocotyls at the seedling stage. E is the comparative analysis of TDEGs in internodes, mesocotyls and light-grown seedlings at the seedling stage.

[0030] Fig.12 In the preferred embodiment of the present invention, internodes and mesocotyls in the seedling stage have their own unique heat-responsive genes. Note: A is the GO enrichment analysis of internodes and mesocotyls-specific TDEGs in the seedling stage. BC is the expression heat map B of TDEGs specifically expressed in internodes and mesocotyls in the seedling stage and specifically expressed in seedlings under light and its GO enrichment analysis C. The difference multiple of the mean value of gene expression (TPM) of the same organ at all temperatures compared with other organs is ≥ 5, and the differentially expressed genes regulated by temperature are organ-specific temperature-responsive genes. D is the relative expression verification of tissue-specific genes. The values ​​of qRT-PCR are mean ± standard deviation (n = 3), and RNA-seq shows the average of two biological replicates. Z-score represents the number of standard deviations between the gene expression of each sample and the mean value of the gene expression of the row.

[0031] Fig.13 This is a quality feature analysis of accessible chromatin regions in internodes and mesocotyls of rice seedlings in a preferred embodiment of the present invention. Note: A is the fragment size distribution of accessible chromatin regions (ACRs) of internodes and mesocotyls of seedlings grown at two temperatures. The horizontal axis is the fragment size after Tn5 transposase cutting, and the vertical axis represents the distribution percentage of different fragment sizes. B is the ATAC-seq signal spectrum and heat map of all samples of internodes and mesocotyls of seedlings. The upper figure is the characteristic curve of the enrichment of reads on the genome; the lower figure is the heat map of the distribution characteristics of reads near genes. C Spearman correlation analysis of ACRs of all samples of internodes and mesocotyls of seedlings.

[0032] Fig.14This is an analysis of the correlation between the chromatin accessible region and the expression level of its adjacent genes in a preferred embodiment of the present invention. Note: A is the distribution of ACRs in different regions of the genome. Transcription start site (TSS): ACRs located in the range of -1000 bp to +50 bp from the transcription start site of the gene; Transcription termination site (TES): ACRs located in the range of -50 bp to +1000 bp from the transcription termination site of the gene; Introns and exons: ACRs located in the introns and exons of the gene; Intergenic regions: All ACRs in other regions. The number and proportion of ACRs are marked in the figure. B is the correlation between different ACRs categories and gene expression levels.

[0033] Fig.15 It is a negative regulatory factor for the elongation of internodes in response to heat in the seedling stage in a preferred embodiment of the present invention. Note: A is a comparative analysis of TDEGs and TAAGs specifically expressed in rice internodes in the seedling stage. The common differential genes of the two omics are marked in red. BC is a mutant phenotypic analysis of genes regulating elongation of internodes in response to heat in the seedling stage. The figure shows the target position of the edited gene and the editing status of the mutant in the CRISPR / cas9 gene editing system, and the IGV graph shows the changes in ATAC-seq peaks and gene expression of the corresponding genes when the internodes in the seedling stage respond to temperature changes; the internode length of the seedlings of the indica rice NJ6 background-related materials grown for 6 days under deep sowing conditions and the japonica rice ZH11 background-related materials grown for 7 days were used for statistical analysis, and the value = mean ± standard deviation (n ≥ 20), using the Student's t-test The significance level was analyzed; the scale bar is 10 mm.

[0034] Fig.16 It is a negative regulatory factor for the elongation of the mesocotyl in response to heat in a preferred embodiment of the present invention. Note: A is a comparative analysis of mesocotyl TDEGs and TAAGs. The common differential genes of the two groups are marked in red. BC is a mutant phenotype analysis of the mesocotyl thermal response elongation regulatory gene. The figure shows the target location and mutant form of the edited gene, as well as the changes in ATAC-seq peak and gene expression of the corresponding gene during the mesocotyl temperature response. The mesocotyl of deep-sown rice seedlings grown for 6 days was used for statistical analysis, and the value = mean ± standard deviation (n ≥ 20), using Student's t-test The significance level was analyzed; the scale bar is 10 mm.

[0035] Fig.17In the preferred embodiment of the present invention, multiple transcription factor families are enriched in the TACRs of heat-responsive genes in internodes and mesocotyls at the seedling stage. Note: A is the transcription factor targeting TACRs of internodes and mesocotyls at the seedling stage obtained by HOMER analysis. The value in the heat map is -log10 (p-value). Z-score represents the number of standard deviations between the data of each sample and the mean of the row data. B is the transcription factor enriched by the FIMO method that is involved in regulating the expression of temperature-responsive genes in internodes and mesocotyls at the seedling stage. Score 1~3 are the ACRs of all TDEGs in the three growth periods; Score 3 is the ACRs of all TDEGs in the three growth periods; when the ratio is >1.5, the transcription factor cluster is considered to be significantly enriched. C is the information of the significantly enriched transcription factor cluster. D is the interaction network diagram of AP2 / EREBP and SPL transcription factors with the chromatin open regions of internode and mesocotyl-specific regulatory genes at the seedling stage. Yellow and blue arrows indicate TACRs targeting temperature-responsive genes in internodes and mesocotyls at the seedling stage, respectively.

[0036] Fig.18 In the preferred embodiment of the present invention, the AP2 / EREBP transcription factor is a common negative regulatory factor for the heat response growth of internodes and mesocotyls in the seedling stage. PLTs The target location and mutation form of the gRNA of the gene mutant. BD is 3 PLTs Functional analysis of genes in heat-responsive elongation of internodes and mesocotyls in seedlings. Statistical analysis was performed on internodes of rice seedlings grown for 7 days and mesocotyls grown for 6 days at 25℃ and 32℃. Values ​​are mean ± SD (n ≥ 20) using Student's t-test Significance test was performed; scale bar is 10 mm.

[0037] Fig.19 In the preferred embodiment of the present invention, AP2 / EREBP is a negative regulatory factor for deep seeding and germination of rice. PLTs Dynamic emergence of gene mutants under shallow sowing A and deep sowing B. The depth of soil covering was 2 cm for shallow sowing and 6 cm for deep sowing. Seedling emergence was counted every day for 2 consecutive weeks, n = 24.

[0038] DETAILED DESCRIPTION

[0039] The present invention analyzes the differences in tissue and cell behavior of internodes and mesocotyls in response to temperature growth during the seedling stage through physiological experiments and cytological observations, explores potential key transcription factors and regulatory genes through joint analysis of transcriptome and chromatin accessibility sequencing, and further analyzes the molecular mechanism of key genes involved in the thermal response growth of the two organs using genetics, biochemistry and molecular biology. In addition, the emergence potential of related genes under deep sowing conditions is verified through seedling emergence experiments, providing genetic resources for breeding rice varieties suitable for dry direct sowing.

[0040] 1. Molecular mechanism of heat-responsive growth in rice 1.1 Growth characteristics of internodes and mesocotyls in response to heat in rice seedlings 1.1.1 The elongation characteristics of internodes and mesocotyls during the seedling stage are the driving force for deep-sown rice seedlings to emerge from the soil In the process of direct seeding of rice, rice seeds are generally sown in a certain depth of soil layer in order to dry the seeds and absorb water from the soil for germination and growth. In order to successfully break through the soil and emerge, the top of the seedling is required to have a strong soil-penetrating force to lift the apical meristem to the soil surface. According to relevant research, different grass plants have different phenotypic plasticity in adapting to deep sowing. According to their strategies for breaking through the soil and emerging, they can be divided into three categories: when the seeds are buried deep in the soil, the mesocotyls of corn and sorghum will lift their apical meristems to the soil surface; the mesocotyls of wheat seedlings will not elongate under deep sowing conditions, but will be elongated through the first internode to raise the top to the soil surface; while the mesocotyls and first internodes of oats and rice can be elongated when sown in deep layers, helping the seedlings to quickly emerge from the soil (Hoshikawa, 1969).

[0041] The mesocytic axis refers to the part between the coleoptile node and the radicle attachment point of the seedling. Figure 1 ). The mesocotyl is an important channel connecting the seed and the ground. Varieties with long mesocotyls have good germination performance under deep sowing conditions. For rice, rice varieties with long mesocotyls are positively correlated with their deep sowing germination rates, and the germination rates of rice varieties with long mesocotyls are significantly higher than those of medium and short mesocotyls. For example, Turner et al. (1982) compared the mesocotyl elongation of different rice varieties with their germination rates at different sowing depths and found that varieties with long mesocotyls had higher germination rates and faster germination speeds. Chung (2010) compared the germination potential of 128 rice varieties with different genotypes under deep sowing conditions and found that the japonica weedy rice WD-3 had the longest mesocotyl and the highest germination rate. Alibu et al. (2012) compared rice varieties with different genotypes and found that only genotypes with significantly elongated mesocotyls could extend from deep soil. It can be seen that mesocotyl elongation provides a driving force for deep sowing germination.

[0042] The first internode refers to the internode tissue between the seedling coleoptile node and the next higher node, which is referred to as the seedling internode in the present invention ( Figure 1 ). The elongation of internodes in the seedling stage helps the deep sowing and germination. In rice, the power source of germination was analyzed by different germplasm resources. When the soil cover was deep, the elongation length of internodes in the seedling stage was significantly positively correlated with the germination rate, suggesting that the elongation growth of internodes in the seedling stage can provide a force for the deep sowing and germination of rice (Yang Qing, 2016). It can be seen that the elongation of internodes in the seedling stage is the key power source for the deep sowing and germination of rice. In short, the elongation characteristics of internodes and mesocytic axes in the seedling stage can help improve the soil-topping force of deep-sown rice seedlings in dry direct seeding, and improve the problems of low germination rate and uneven germination in deep sowing in dry direct seeding.

[0043] Deep-sown rice has the characteristic of specific elongation of internodes and mesocotyls in the seedling stage. In order to explore the morphological differences between shallow-sown and deep-sown rice seedlings, we sowed the indica rice Nanjing No. 6 (NJ6) seeds with consistent germination on the surface of water-saturated vermiculite and at a depth of 10 cm and grew them in a 32°C incubator. Through morphological observation of the seedlings, we found that: the deep-sown seedlings elongated between the scutellum node and the coleoptile node to form the mesocytic axis, and at the same time, the coleoptile node and the first node elongated to form the first internode, and usually the first internode had a greater elongation than the mesocytic axis; the seedling height of shallow-sown rice seedlings was mainly manifested in the elongation of the leaves, while the stem internode tissue accumulated at the base of the seed without obvious signs of elongation; and the seedling height of deep-sown seedlings was elongated faster than that of shallow-sown seedlings ( Figure 2 , AB). 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 different rice varieties elongated under deep sowing conditions. The first internode of some rice varieties did not elongate while the second internode elongated (such as ZH11 and NIP), and the first and second internodes of some varieties elongated (such as NJ6 and DJ). Therefore, we collectively refer to the first or second internodes of seedlings of different rice varieties that elongated under deep sowing conditions as seedling internodes ( Figure 2 C). In summary, internodes and mesocotyls of rice seedlings elongated rapidly under deep sowing conditions, significantly increasing the overall seedling height, suggesting that these two organs may provide the driving force for deep sowing seedlings to emerge from the soil.

[0044] 1.1.2 The elongation of internodes and mesocotyls during the seedling stage is affected by environmental temperature Internodes and mesocotyls in the seedling stage have heat-responsive growth characteristics. Temperature is one of the key factors affecting seed germination and emergence. In order to verify whether the elongation of internodes and mesocotyls in the rice seedling stage is affected by environmental temperature, we sowed NJ6 indica rice seeds with consistent germination in water-saturated vermiculite at a depth of 10 cm and placed them in dark incubators at four temperatures of 25°C, 28°C, 30°C, and 32°C for 4 to 6 days. We also conducted a statistical analysis of the heights of internodes, mesocotyls, coleoptiles, and seedlings above the seeds ( Figure 3 ). The results showed that seedling height, internodes and mesocotyls in the seedling stage all elongated rapidly with the increase of temperature at 4-6 days of growth, while the elongation of coleoptiles was less affected by temperature, indicating that the elongation of internodes and mesocotyls in the seedling stage in response to heat may be the main reason for the increase in seedling height. In addition, we found that the elongation of internodes in the seedling stage was affected by both temperature and growth time; for example, when the temperature increased in a small range of 25℃ to 28℃, the elongation trend of internodes in the seedling stage of 4 days was weak, while the elongation trend of internodes in the seedling stage of 5 or 6 days in response to temperature was faster, indicating that the growth time and temperature changes jointly affected the elongation of internodes in the seedling stage. The mesocotyl elongated slightly with the growth time, but the elongation amplitude was small, indicating that the change of environmental temperature was one of the main factors affecting the growth of mesocotyls. In contrast, the coleoptile only elongated in response to the increase of environmental temperature at 4 days of growth. The above results show that the elongation of internodes and mesocotyls in the seedling stage are both temperature sensitive and gradually elongate with the increase of temperature.

[0045] 1.2 Heat response growth of internodes during the seedling stage is mainly achieved through cell expansion To further understand the cytological basis of seedling internodes during heat-responsive growth, we conducted comparative analysis of the cortical cells of seedling internodes grown in lower and higher ambient temperatures. First, we conducted cytological analysis of the internodes of the indica rice NJ6 seedlings that rapidly jointed after a temperature increase in a small range of 30°C to 32°C for 4 days. The results showed that the length and width of the internode cells of the seedlings grown at 32°C were significantly expanded compared to those at 30°C, which significantly increased the cell aspect ratio and theoretical cell area. Although the number of cells per unit length was significantly reduced, the total number of cells did not change ( Figure 4 A), indicating that the internodes of NJ6 in the seedling stage promote the rapid elongation of its internode tissue under high ambient temperature by causing rapid cell expansion. At the same time, in order to understand whether there are differences in the cytological behavior of heat-responsive growth of internodes in seedlings of different rice varieties, we observed the internode cells of two japonica rice varieties, NIP and DJ, in the seedling stage. The results showed that compared with 25°C, the internode cells of NIP and DJ in the seedling stage became longer and wider at 32°C, the cell aspect ratio and theoretical cell area increased significantly, and the number of cells per unit length decreased significantly. There was no significant difference in the total number of cells in NIP at the two temperatures, but the number of cells in DJ showed an increasing trend ( Figure 4 B), indicating that japonica rice mainly promotes internode elongation in the seedling stage through cell expansion, and cell division may exist in DJ to further promote the heat response growth of internodes in the seedling stage. The above results show that cell expansion is the main reason for the heat response elongation of internodes in the seedling stage.

[0046] 1.3 Significant changes in cell expansion-related genes during heat response growth of seedling internodes In order to understand the changes in gene expression in seedling internodes during heat-responsive growth, we sampled seedling internodes grown at 25°C and 32°C for 4, 5, and 6 days for transcriptome analysis. Among them, the seedling internodes grown at 25°C for 4 days showed no obvious growth, so we chose the 30°C seedling internodes that were significantly elongated relative to 32°C for transcriptome library construction. At the same time, in order to distinguish the differences in thermal response of seedling internode organs grown under light conditions and dark conditions, we introduced the transcriptome data of rice seedlings grown in light incubators at 25°C, 30°C, and 35°C for 2 weeks for joint comparative analysis. First, we performed quality control on the raw transcriptome data and aligned the reads obtained by sequencing with the rice genome. Oryza sativa MSU v7.0 (Ouyang et al., 2006). According to statistics, the alignment rate of reads generated by sequencing each sample ranged from 86.41% to 90.72% ( Figure 5 ), indicating that the RNA-seq sequencing quality was good. Next, based on the following screening strategy: the gene expression difference between samples was ≥ 2 and q-value < 0.05, the gene expression of the same organ at two temperatures was compared and analyzed, and the obtained temperature differentially expressed genes were called thermoresponsive differentially expressed genes (TDEGs).

[0047] First, we compared and analyzed the TDEGs of internodes in seedlings with the same growth time. 5,156, 7,052, and 10,352 TDEGs were obtained in the heat-responsive elongated internodes of seedlings at 4, 5, and 6 days of growth, respectively. The number of TDEGs increased gradually with the extension of growth time, indicating that the changes in gene expression in seedling internodes gradually became more sensitive to the increase in temperature with the growth time ( Figure 6 A). At the same time, we compared the thermal response of internodes in dark-grown seedlings with that of seedlings grown under light. The results showed that the number of genes upregulated and downregulated in response to temperature increase in seedlings grown under light was similar, while the number of upregulated genes in internodes during the three growth days was higher than the number of downregulated genes, suggesting that the thermal response elongation of internodes during seedlings may be a process in which gene expression is activated ( Figure 6 A). Next, we performed GO functional enrichment analysis on internode TDEGs in the seedling stage. The results showed that genes involved in cell wall and microtubule skeleton composition and auxin signal response were significantly enriched ( Figure 6 B), indicating that the differential expression of heat-responsive genes involved in cell wall and microtubule cytoskeleton reorganization may promote heat-responsive growth of internodes in the seedling stage by mediating cell expansion in internodes.

[0048] 1.4 The heat-responsive transcriptome of seedling internodes is developmental time-dependent Furthermore, we investigated the effect of heat response of internodes on the elongation trend of seedlings ( Figure 3 ), and the number of TDEGs showed a gradual increasing trend with the extension of growth time ( Figure 6 A), and then explore its thermal response regulation mechanism at the temporal level. First, the TDEGs of seedling internodes at three growing days were compared and analyzed. The results showed that 1,298, 969 and 4,142 TDEGs responded specifically to temperature in seedling internodes at 4, 5 and 6 days of growth, respectively. Most of the TDEGs were shared between adjacent days, and 2,021 TDEGs were shared between 3 growing days ( Figure 7 A). Gene expression trend analysis of TDEGs shared by three growth days revealed that the expression patterns of TDEGs shared by seedling internodes on three growth days were basically consistent, 47.5% TDEGs were upregulated during heat response growth, 40.7% TDEGs were downregulated, and only a few 11.8% TDEGs had different expression trends on different growth days ( Figure 7 B). GO enrichment analysis results showed that microtubule skeleton and cell wall biogenesis-related processes were significantly enriched in the TDEGs shared by the three growth days of the internodes in the seedling stage ( Figure 7 C), indicating that these pathways are crucial in the growth and development of internodes in the seedling stage and their elongation in response to heat. In addition, by enriching GO for TDEGs specific to different growth times, we found that the response of internodes in the seedling stage to temperature with the growth time showed an interesting developmental process. Among them, biological processes related to nucleic acid metabolism and gene expression were enriched on the 4th day, functional pathways such as membrane lipid metabolism, proteasome and organelles were enriched on the 5th day, and microtubule skeleton and cell wall formation processes were significantly enriched on the 6th day ( Figure 7 , DE). These results indicate that the internodes of rice seedlings at the 4-6 day old stage mobilize relevant genes from the molecular level, cellular and subcellular level, and then to the tissue level to participate in its heat response growth and development process.

[0049] 1.5 Thermoresponsive growth of mesocotyls is determined by both cell proliferation and expansion At the same time, in order to further understand the cytological basis of mesocotyl growth in thermal response, we compared and analyzed the cortical cells of mesocotyls of indica rice NJ6 grown at lower and higher ambient temperatures. The results showed that there was no significant change in the length, width, aspect ratio, theoretical cell area, and number of cells per unit length of mesocotyls grown at 25°C and 30°C for 5 days, but the total number of cells at 30°C was significantly higher than that at 25°C ( Figure 8A), indicating that cell proliferation mediates the thermoresponsive growth of mesocotyls in indica rice NJ6. To verify this result, we simultaneously observed the changes in mesocotyl cells during thermoresponsive growth in japonica rice NIP and DJ ( Figure 8 B). Among them, the elongation and narrowing of the mesocotyl cells of japonica rice NIP at 32°C resulted in a decrease in the theoretical cell area, but a significant increase in the total number of cells; in japonica rice DJ, the length and width of the mesocotyl cells increased significantly at high ambient temperatures, the cell aspect ratio and theoretical cell area increased significantly, and the total number of cells increased significantly. This result indicates that the mesocotyl of japonica rice promotes its rapid elongation at high ambient temperatures through cell proliferation, and at the same time, it needs to further promote its thermal response elongation through the action of cell elongation. The above results show 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 mesocotyl of japonica rice.

[0050] 1.6 Transcriptome changes associated with cell proliferation and expansion promote mesocotyl elongation in response to heat Next, we performed transcriptome sequencing on mesocotyls grown for different days at two temperatures to explore their thermoresponsive growth mechanism. First, we compared and analyzed the TDEGs of mesocotyls grown for the same growth time. 7,774, 2,072, and 2,950 TDEGs were obtained in thermoresponsive mesocotyls grown for 4, 5, and 6 days, respectively. The number of TDEGs decreased with the extension of growth time, indicating that changes in mesocotyl gene expression were more sensitive to temperature increases in the early stages of growth ( Fig. 9 A). At the same time, compared with the seedlings growing in response to heat under light, we found that unlike the high ambient temperature that activated more internode gene expressions in the seedling stage, the high ambient temperature tended to inhibit more mesocotyl gene expressions, suggesting that the heat-responsive growth of the mesocotyl may be a process in which gene expression is de-repressed ( Fig. 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 skeleton and cell wall composition were significantly enriched ( Fig. 9 B), indicating that changes in the expression of related TDEGs may promote the rapid proliferation and expansion of mesocotyl cells during thermoresponsive growth, thereby promoting the thermoresponsive elongation growth of the mesocotyl.

[0051] 1.7 The thermoresponsive transcriptome of the mesocotyl is also developmental time-dependent The regulatory mechanism of mesocotyl thermal response growth at the temporal level was further explored. First, the TDEGs of mesocotyls at three growth days were compared and analyzed. The results showed that 5,391, 695, and 690 TDEGs were expressed in mesocotyls at 4, 5, and 6 days of growth, respectively, in response to temperature; similar to internodes in the seedling stage, mesocotyls on adjacent days shared most of the TDEGs, and 544 TDEGs were shared in 3 growth days ( Fig.10 A). Further analysis of the gene expression trend of the TDEGs shared by the three growth days showed that the expression patterns of the TDEGs shared by the mesocytic axes on the three growth days were basically consistent, 23.7% of the TDEGs were up-regulated during the heat response growth process, 56.1% of the TDEGs were down-regulated, and 20.2% of the TDEGs had different expression trends on different growth days ( Fig.10 B). Similar to internodes in the seedling stage, cell wall biogenesis-related processes were significantly enriched in the TDEGs shared by the three growth days of the mesocotyl ( Fig.10 C), indicating that this pathway is crucial in the growth and development of the two organs and the thermal response elongation. At the same time, through GO enrichment analysis of TDEGs specific to different growth times, a thermal response development process different from that of internodes in the seedling stage was found in the mesocotyl. Among them, translation and protein metabolism processes were enriched on the 4th day, and secondary metabolism processes were enriched in temperature-responsive genes specific to the 5th and 6th days ( Fig.10 The above results indicate that the thermal response transcriptome of rice mesocotyl is also developmental time-dependent, and its thermal response during 4-6 days of growth covers relevant biological processes from primary metabolism to secondary metabolism.

[0052] 1.8 The temperature-responsive transcriptomes of internodes and mesocotyls at the seedling stage are highly similar At the same time, we conducted a comparative analysis of the thermoresponsive transcriptomes of internodes and mesocotyls in the seedling stage to explore the similarities and differences between the two organs in thermoresponsive growth. First, the results of principal component analysis (PCA) and correlation heatmaps showed that samples of internodes and mesocotyls in the seedling stage were more correlated, and samples of the two organs were able to cluster together, while they were dispersed in seedlings grown under light ( Fig.11, AB), suggesting that the differentially expressed genes in internodes and mesocotyls of seedlings grown under dark conditions were more similar than those in seedlings grown under light. Secondly, the TDEGs of internodes, mesocotyls and seedlings grown under light were compared and analyzed. The results showed that during the three growth days, a total of 13,473, 9,519 and 7,942 TDEGs were obtained in internodes, mesocotyls and both of them, respectively, while fewer TDEGs were detected in seedlings grown under light (s3,649), and only 1,748 TDEGs were shared by the three ( Fig.11 C), which further indicates that the TDEGs of internodes and mesocotyls in the seedling stage have certain similarities. Next, we annotated the TDEGs shared by the two dark-growing organs and the TDEGs shared by light and dark-growing tissues with GO functions, and found that processes such as ribosomes, translation, peptide synthesis and metabolism, and microtubule cytoskeleton reorganization were significantly enriched in the TDEGs shared by the two dark-growing organs, and cell wall biogenesis and metabolism-related processes were enriched in the TDEGs shared by the three organs ( Fig.11 , DE), suggesting that changes in the expression of genes involved in the basic rate-limiting steps of cell growth and expansion, such as ribosome biogenesis and polypeptide synthesis, prompt internodes and mesocytoids in the seedling stage to sense the temperature increase and elongate rapidly; while changes in the expression of genes involved in cell expansion jointly promote the thermal response growth of the two dark-grown organs and light-grown seedlings.

[0053] 1.9 Heat-responsive genes specific to internodes and mesocotyls at the seedling stage GO enrichment analysis was performed on 4,894 internode-specific and 1,390 mesocotyl-specific TDEGs obtained by comparative analysis of TDEGs of internodes, mesocotyls and light-grown seedlings. Among them, internode-specific TDEGs in the seedling stage were enriched with biological pathways related to multiple RNA metabolism and modification, while cell wall reorganization-related genes were significantly enriched in mesocotyl-specific TDEGs ( Fig.12 A), indicating that the two organs have different thermal response growth mechanisms. At the same time, we screened the genes whose TPM mean of the same organ differed by ≥ 5 times compared with the rest of the organs and whose expression was different under temperature, and obtained 237 internode-specific expression in the seedling stage, 429 mesocotyl-specific expression, and 728 seedling-specific expression TDEGs under light ( Fig.12 B). GO enrichment analysis results showed that cell wall biogenesis and auxin response-related pathways were enriched in TDEGs specifically expressed in internodes at the seedling stage, and mesocotyl-specific TDEGs were mainly involved in cell wall biogenesis-related processes, while processes related to photosynthesis, enzyme activity, and redox reactions were enriched in TDEGs specific to seedlings grown in light and in response to heat, indicating that the two organs grown in the dark and seedlings grown in the light have their own specific thermal response growth mechanisms ( Fig.12C). In order to verify the credibility of the TDEGs specifically expressed in the two organs, we reprocessed a new batch of biological samples and selected three TDEGs specifically expressed in internodes of seedlings. LOC_Os10g27280 , LOC_Os12g44270 and LOC_Os09g24840 , and mesocotyl-specific expression LOC_Os10g31620 , LOC_Os10g01930 and LOC_Os10g31540 qRT-PCR gene expression detection was performed. The results showed that the gene expression trends of the selected 6 genes in RNA-seq and qRTPCR were basically consistent ( Fig.12 D), indicating that our RNA-seq data and the selected tissue-specific genes are reliable. The above results show that internodes and mesocytic axes at the seedling stage have their own unique heat-responsive genes, and the heat-responsive differential expression of these genes mediates their heat-responsive elongation growth.

[0054] 1.10 Transcriptional regulatory network related to heat response growth of internodes and mesocytic axes at the seedling stage To further investigate the mechanism of heat-responsive growth of internodes and mesocotyls at the seedling stage, we used Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq) to map the genome-wide accessible chromatin regions (ACRs) of internodes and mesocotyls of 6-day-old indica rice NJ6 seedlings. First, we performed a quality assessment on the ACRs obtained from all samples, and the results showed that the size of ACRs fragments was mainly distributed between 30 and 900 bp; at the same time, due to the periodicity of nucleosome arrangement, it can be seen that most of the fragments were concentrated between 100 and 250 bp ( Fig.13 A). Secondly, the distribution density of ACRs in genes and adjacent genes of all samples was analyzed, and it was found that ACRs showed similar distribution patterns in all samples, and these ACRs were mainly enriched in the transcription start site (TSS) of genes ( Fig.13 B), indicating that the upstream regulatory regions of these genes are open, and some transcriptional regulatory factors can dock with this region to 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 of more than 95% ( Fig.13C). According to statistics, more than 32,000 high-resolution ACRs were obtained in each sample, of which most ACRs (~70%) were located in the proximal region of the gene, including about 35% 2 kb upstream of the transcription start site (TSS), about 15% in introns and exons, and about 10% about 2 kb downstream of the transcription termination site (TES); and a few (~28%) were located in the distal region or intergenic region ( Fig.13 A). At the same time, we found that the proportion of ACRs in the proximal region of genes in internode and mesocotyl samples grown at 32°C was significantly higher than that at 25°C, especially the number of ACRs located in the exon region increased significantly ( Fig.14 A). ACRs located in different regions of the genome are often correlated with gene expression levels. For example, distal ACRs are widely used to predict putative enhancers in plants, while proximal ACRs are closely related to gene expression (Tian et al., 2021). Therefore, we conducted a comprehensive analysis of the gene expression data and chromatin accessibility obtained from the transcriptome. The results showed that the number of related genes containing only a single category of ACRs was the largest, and the expression abundance of related genes with ACRs only in the TSS, Exon and Intron regions was significantly higher than that of TES and Intergenic only; fewer genes were detected to contain multiple categories of ACRs at the same time, but the related genes containing multiple categories of ACRs had higher expression levels, and the very few related genes containing 5 categories of ACRs at the same time had the highest expression abundance; Overall, ACRs in the proximal TSS region of the gene contributed the most to gene expression ( Fig.14 B). The above results indicate that the chromatin accessible regions in internodes and mescotyls at the rice seedling stage affect the expression abundance of their neighboring genes.

[0055] At the same time, we compared and analyzed the organ-specifically expressed TDEGs in the transcriptome and the TAAGs of ATAC-seq, and the overlapping genes between the two were considered to be the key regulatory genes for the elongation of internodes or mesocotyls in response to heat in the seedling stage. Through comparative analysis, we obtained 154 key regulatory genes for internodes and 41 key regulatory genes for the elongation of mesocotyls in response to heat in the seedling stage ( Fig.15 A and Fig.16A). We further selected some genes and used CRISPR / Cas9 gene editing technology to obtain loss-of-function mutants to observe the thermal response elongation phenotype of the two organs. First, the gene encoding the AAA-type ATPase family protein LOC_Os12g10670 Mutants im1 and terpene synthase LOC_Os03g31430 Mutants im2 The internode length of the seedlings of was not significantly different from that of the wild type at 25°C, but was significantly longer than that of the wild type at 32°C. In addition, the internode elongation trend of the two seedlings in response to heat was significantly increased compared with that of the wild type ( Fig.15 , BC), indicating LOC_Os12g10670 and LOC_Os03g31430 Negatively regulates internode heat response elongation growth in seedling stage. Secondly, the gene encoding transferase family protein LOC_Os10g01930 Mutants mm1 The mesocotyls were significantly longer than those of the wild type at 25°C, and the gene encoding the glycine-rich cellwall structural protein 2 precursor LOC_Os10g31540 Mutants mm2 The mesocotyls of the two mutants were significantly shorter than those of the wild type at 25°C, and significantly longer than those of the wild type at 32°C. The thermal response growth trend of the mesocotyls was slightly increased compared with that of the wild type ( Fig.16 , BC), indicating LOC_Os10g01930 and LOC_Os10g31540 Negative regulation of mesocotyl elongation growth in response to heat. The above results confirm the reliability of the key regulatory genes screened by RNA-seq and ATAC-seq joint analysis in regulating the thermal response growth of the two organs.

[0056] Since ACRs contain specific cis-acting elements, they allow trans-acting factors to dock and regulate gene expression (Tian et al., 2021). Therefore, we tried to find the key transcription factors that regulate the expression of heat-responsive genes in internodes and mesocotyls at the seedling stage. First, we used the HOMER (Hypergeometric Optimization of Motif Enrichment) tool to enrich the transcription factors in TACRs of rice seedling internodes and mesocotyls. 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 at the seedling stage; among them, rTACRs of internodes at the seedling stage also recruited MADS-box transcription factors, and 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 at the seedling stage ( Fig.17 A), the results show that TACRs in the heat response growth of the two organs recruited very abundant transcription factors to regulate the expression of TDEGs. Next, we further used the FIMO (Find Individual Motif Occurrences) tool to scan the ACRs of TDEGs in the two organs and enrich the transcription factors involved in the regulation of TDEGs expression. According to the similarity of the transcription factor binding target motifs, all enriched transcription factor families were divided into 44 clusters, each containing one or more transcription factor families. The results showed that the 2nd, 8th, 10th, 23rd, 26th, 35th, 40th and 41st transcription factor clusters were significantly enriched in genes induced by high temperature and upregulated in seedling internodes (up-regulated TDEGs), and the 21st and 30th clusters were enriched in TDEGs inhibited by high temperature and downregulated in seedling internodes (s down-regulated TDEGs); compared with seedling internodes, up-regulated TDEGs in mescots only recruited the 40th transcription factor cluster, while down-regulated TDEGs only recruited the 21st transcription factor cluster ( Fig.17 B). Since the 2nd, 8th and 10th transcription factor clusters significantly enriched in the up-regulated TDEGs in the seedling internodes all contain AP2 / EREBP transcription factors, and the down-regulated TDEGs in both organs recruited the 21st transcription factor cluster containing SPL ( Fig.17C), and these two gene families were significantly enriched in both transcription factor enrichment methods. We believe that these two transcription factor families are essential for the heat-responsive growth of internodes and mesocotyls in the seedling stage. Further mining of the interaction network between the AP2 / EREBP and SPL transcription factor families and key regulatory genes in internodes or mesocotyls in the seedling stage revealed that the proportion of key regulatory genes in internodes in the seedling stage connected to the two transcription factor families through different categories of TACRs was higher than that in the mesocotyls. The AP2 / EREBP transcription factor family was docked with TACRs of 14 internodes and 7 mesocotyl regulatory genes in the seedling stage, while the SPL transcription factor family was docked with TACRs of 26 internodes and 2 mesocotyls in the seedling stage ( Fig.17 D), indicating that the AP2 / EREBP and SPL transcription factor families are crucial in the heat-responsive growth regulatory network of internodes and mescots during the seedling stage.

[0057] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present 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.

[0058] Example 1 AP2 / EREBP family negatively regulates heat-responsive growth of internodes and mesocotyls at the seedling stage 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 the present invention, we found that the AP2 / EREBP transcription factor family may regulate gene expression by binding to the ACRs of internode TDEGs in the seedling stage, so we further explored the role of AP2 / EREBP transcription factor family genes in internode heat response growth in the seedling stage.

[0059] 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 )Genes were constructed and gene-edited mutants were used to verify the heat-responsive growth phenotypes of internodes and mesocotyls in rice seedlings.

[0060] In the present invention, LOC_Os04g55970 ( PLT1 )'s genomic nucleotide sequence, CDS sequence and amino acid sequence of its encoded protein are shown in SEQ ID NOs: 1-3, respectively; LOC_Os02g40070 ( PLT3)'s genomic nucleotide sequence, CDS sequence and amino acid sequence of its encoded protein are shown in SEQ ID NOs: 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 NOs: 9-11, respectively.

[0061] First, we used CRISPR / Cas9 technology to PLT1 , PLT3 and PLT5 Gene editing was performed and functional loss mutants of three genes were obtained, one missing two bases and the other missing one base. plt1 , both have 1 base missing and 1 base inserted plt3 and plt5 Mutants ( Fig.18 A) These editing forms all lead to premature termination of protein translation.

[0062] The target site of the CRISPR / cas9 target gene is selected as follows: LOC_Os04g55970 ( PLT1 ): 5′-TGAGATCGGCCGGTATAACGTGG-3′ (SEQ ID NO: 4) LOC_Os02g40070 ( PLT3 ): 5′-AGAGAATCCGCAGCCTCACCAGG-3′ (SEQ ID NO: 8) LOC_Os01g67410 ( PLT5 ): 5′-CATGAGGGGATCGGAGCTCTCGG-3′ (SEQ ID NO: 12) The primers for identifying the relevant mutants are as follows (SEQ ID NO: 13-18): plt1 -F:5′-AGCAGCCCGTGTTTGGAATA-3′ plt1 -R: 5′-GAAGAGCTGTGAACGCAAGC-3′ plt3 -F:5′-TACCTCCCCCGTATCAGCTT-3′ plt3 -R: 5′-GCATGCATACCTTGTGCCTC-3′ plt5 -F:5′-GTCTGCTTCAACATCCCCCA-3′ plt5-R: 5′-AATCTCCTTGCGACCACCAG-3′ Next, we observed the internode heat response growth phenotypes of the relevant mutants at the seedling stage. plts The internodes of the mutant seedlings were not significantly different from those of the wild type at 25°C, but were significantly longer than those of the wild type at 32°C, and the mutant also became more sensitive to rising ambient temperatures ( Fig.18 , BD), indicating PLT1 , PLT3 and PLT5 Negative regulation of internode heat response growth in the seedling stage. plts The results of the mutant mesocotyl thermal response growth phenotype showed that the mesocotyls were significantly longer than those of the wild type at 32°C, and the thermal response trend also increased accordingly ( Fig.18 , BD), indicating that these three PLTs The above results indicate that AP2 / EREBP transcription factors are involved in the heat response growth of internodes and mesocotyls in the seedling stage. PLT1 , PLT3 and PLT5 Both are negative regulatory factors of thermal response growth of internodes and mesocotyls in the seedling stage.

[0063] To determine the effects of internode and mesocotyl elongation on rice seedling emergence potential, we conducted a double-batch experiment on rice seedlings from deep and shallow sowing. plts The emergence rates of mutants at two temperatures were statistically analyzed. First, under the shallow sowing condition of 2 cm, three plts The germination rates of the three species were close to those of the wild type at 25°C; however, at 32°C, plts The germination rates of Fig.19 A). Secondly, under the condition of 6 cm deep sowing, plt1-1 The emergence rate of the mutant was close to that of the wild type at 25°C. plt3-1 and plt5-1 The germination rate of the wild type and mutant was slightly higher than that of the wild type. At 32℃, the germination rates of the wild type and mutant were greatly reduced, while the germination rates of the three plts The mutant still maintained a higher seedling emergence rate than the wild type ( Fig.19 B), indicating PLT1 , PLT3 and PLT5 All of them are negative regulatory factors for rice seeding under deep sowing. At the same time, we found that the seedling emergence speed of all materials grown at 32°C was significantly accelerated under deep and shallow sowing conditions, and plt3-1 and plt5-1 The emergence speed of Fig.19, AB), indicating that high ambient temperature can accelerate the emergence of seedlings, and plants with significantly elongated internodes and mesocotyls at the seedling stage emerge more quickly under high ambient temperature. The above results show that having longer internodes and mesocotyls at the seedling stage can accelerate the emergence of deep-sown seedlings and increase the deep-sown seedling emergence rate, suggesting that the driving force for rice seedlings to emerge from the soil comes from the joint elongation of internodes and mesocotyls at the seedling stage.

[0064] The elongation characteristics of internodes and mesocotyls in the seedling stage are of great significance for the promotion of rice dry direct seeding technology. The present invention found that the relevant transcription factors involved in the heat response growth of internodes and mesocotyls in the seedling stage showed considerable application prospects in improving the seedling rate of deep seeding of rice. These gene resources may help breed more rice varieties suitable for dry direct seeding.

[0065] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. Any of the following applications of plant AP2 / EREBP family transcription factor PLT1 gene: 1) Regulate the thermal response growth of internodes and mesocotyls in plant seedlings; 2) Controlling high ambient temperature for deep sowing of plants; The number of the PLT1 gene in the rice gene database is LOC_Os04g55970.

2. The use according to claim 1, characterized in that: The regulation is negative regulation.

3. The use according to claim 1 or 2, characterized in that: The plants include grass plants, preferably rice.

4. A method for promoting the growth of internodes and mesocotyls of rice seedlings at high ambient temperature, characterized in that: The method comprises: using genetic engineering means to weaken or knock out the PLT1 gene in rice; the PLT1 gene is the same as that described in claim 1.

5. A method for promoting rice seeding under high ambient temperature, characterized in that: The method comprises: using genetic engineering means to weaken or knock out the PLT1 gene in rice; the PLT1 gene is the same as that described in claim 1.

6. The method according to claim 4 or 5, characterized in that: CRISPR, TALEN or ZFN gene editing technology is used to weaken or knock out the gene.

7. The method according to claim 6, characterized in that Taking the PLT1 gene as the target, a sgRNA sequence based on CRISPR / Cas9 was designed, and a DNA fragment containing the sgRNA sequence was connected to a vector carrying CRISPR / Cas9 to transform rice, thereby obtaining transgenic rice with a functional deficiency of the gene.

8. The method according to claim 7, characterized in that The nucleotide sequence of the sgRNA action site is 5'-TGAGATCGGCCGGTATAACGTGG -3'.

9. Use of the transgenic rice obtained by the method according to any one of claims 4 to 8 in plant breeding.

10. The use according to claim 9, characterized in that: Breeding methods include transgenic, hybridization, backcrossing, selfing or asexual reproduction.

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