A method for screening key genes of rice tolerance to high night temperature based on combined analysis of transcriptome and metabolome and application thereof

Abstract

This invention discloses a method for screening key genes for rice nighttime heat tolerance based on combined transcriptomic and metabolomic analysis and its application. The method includes: applying nighttime heat stress to rice plants during the grain-filling stage; performing transcriptomic sequencing analysis on rice grains subjected to nighttime heat stress and a control group to screen for genes showing significant differential expression after nighttime heat stress treatment; performing metabolomic analysis on rice grains subjected to nighttime heat stress and a control group to screen for metabolites showing differential accumulation after nighttime heat stress treatment; and performing association analysis between the significantly differentially expressed genes and the differentially accumulated metabolites to screen for genes significantly associated with the differentially accumulated metabolites as key regulatory genes related to rice nighttime heat tolerance. These key regulatory genes can serve as molecular markers for screening rice nighttime heat tolerance and can be used to cultivate rice varieties tolerant to nighttime heat.

Description

Technical Field

[0001] This invention relates to the field of rice breeding technology, specifically to a method for screening key genes for rice's tolerance to nighttime high temperatures based on combined transcriptomic and metabolomic analysis, and its application. Background Technology

[0002] With the continued intensification of global warming, high-temperature stress has become one of the important abiotic stress factors affecting the safe production and stable yield of rice. Studies have shown that rice is extremely sensitive to temperature changes during its growth and development, and high-temperature stress can significantly affect its photosynthetic efficiency, nutrient accumulation, grain filling process, and the final yield and quality.

[0003] Existing research primarily focuses on the effects of daytime high temperatures on the physiological and biochemical characteristics and yield traits of rice. However, recent meteorological data and field observations indicate a significant increase in the frequency of nighttime high-temperature events, and their negative impact on rice production is equally significant. Nighttime high temperatures can increase nighttime respiration intensity, disrupt carbon metabolism balance, and interfere with grain filling and material transport processes, leading to decreased thousand-grain weight, deterioration of rice quality, and yield loss. Compared to daytime high temperatures, nighttime high temperatures are usually not accompanied by changes in light intensity, making their effects on crop physiological metabolism and molecular regulatory networks more subtle and their mechanisms more complex. Therefore, nighttime high temperatures have gradually become one of the important environmental factors restricting stable and high rice yields.

[0004] Currently, research on rice under high-temperature stress mainly focuses on physiological responses, transcriptional regulation, and the discovery of heat-tolerant genes under daytime high-temperature conditions. Most related studies are conducted at the whole-plant or single-tissue level. Although some regulatory factors related to high-temperature tolerance have been identified, research on the molecular response mechanisms and key regulatory genes of rice under nighttime high-temperature stress remains limited. Especially under nighttime high-temperature conditions, there is a lack of systematic analysis of the relationship between changes in transcriptional regulation and metabolic regulation, as well as a lack of mature technical solutions that can directly serve the screening and breeding of rice varieties tolerant to nighttime high temperatures.

[0005] Plant hormones play a crucial role in regulating crop growth, development, and stress responses. Among them, cytokinins, as important signaling molecules regulating cell division, delaying senescence, and maintaining source-sink relationships, have key functions in grain filling and maintaining metabolic homeostasis under stress conditions. Studies have shown that the cytokinin signaling pathway may be involved in the response of rice to high-temperature stress. Existing research indicates that type-A response regulators (ARRs) are key negative feedback regulatory elements in cytokinin signaling, among which… OsRR6As a typical type A response regulator, it can be induced to be expressed under cytokinin treatment, light changes and various abiotic stresses, and regulate plant response to cytokinin and stress adaptation under exogenous overexpression conditions (Bhaskar, V., Saradhi, P., & Reddy, AR, 2021). OsRR6 , a type-A response regulator in rice, mediates cytokinin, light and stress responses when over-expressed in Arabidopsis. Plant Physiology and Biochemistry, 166, 238–246.). In contrast, OsRR3 The transcriptional response was weak under short-term exogenous cytokinin treatment, but its overexpression could reduce the plant's sensitivity to cytokinin, suggesting that... OsRR3 The negative feedback regulation of cytokinin signaling is more dependent on specific physiological states or long-term stress conditions. Furthermore... OsRR6 It was significantly upregulated by salt, drought, and low temperature stress (Cheng, X., Jiang, H., Zhang, J., Qian, Y., Zhu, S., & Cheng, B., 2010. Overexpression of type-A rice response regulators, OsRR3 and OsRR5 , results in lower sensitivity to cytokinins. Genetics and Molecular Research, 9(1), 348–359.). Based on the above functional characteristics, OsRR6 It can act as a rapid-response negative feedback regulator, primarily modulating the immediate sensitivity of cytokinin signaling, while OsRR3 It is possible that under adverse or stress conditions, it participates in the fine regulation of cytokinin signaling through low-amplitude or steady-state upregulation. Studies have shown that the cytokinin signaling pathway may be involved in the response of rice to high-temperature stress, with type A response regulators playing a key role. OsRR3 and OsRR6 It has been shown to participate in the negative feedback regulation of cytokinin signaling and exhibits responsive characteristics under various adverse conditions. However, under nighttime high-temperature stress conditions, OsRR3 and OsRR6The transcriptional response characteristics of cytokinins and their relationship with the dynamic changes of endogenous cytokinins are still poorly understood, and their specific regulatory mechanisms during rice grain development remain unclear. Current research largely focuses on single-gene or single-level analyses, lacking comprehensive studies that analyze cytokinin signaling response factors under nighttime high-temperature stress. OsRR3 Also known as OsORR3, OsRR6 Also known as OsORR6, Different names exist in different studies and databases, but they correspond to the same rice gene locus and coding product. For ease of explanation, this application will treat the above-mentioned different names as different names for the same gene, and will use them uniformly thereafter. OsORR3 and OsORR6 Describe it.

[0006] In addition, existing methods for evaluating rice heat tolerance mostly rely on field traits or physiological indicators, which are easily affected by environmental conditions and have poor stability and repeatability. There is a lack of clear, reliable and operable molecular markers for evaluating rice's tolerance to nighttime high temperatures and for molecular breeding, which to some extent restricts the efficient breeding of rice varieties tolerant to nighttime high temperatures.

[0007] Therefore, there is an urgent need to propose a new technical solution that can comprehensively utilize transcriptome data and information on changes in endogenous cytokinin metabolism under nighttime high-temperature stress to systematically screen key regulatory genes closely related to rice's nighttime high-temperature tolerance at the molecular level, and apply them to the evaluation of rice's nighttime high-temperature tolerance and molecular breeding practices, so as to provide scientific basis and technical support for the breeding of rice varieties tolerant to nighttime high temperatures. Summary of the Invention

[0008] To address the problems existing in the prior art, this invention provides a method for screening key genes for rice nighttime heat tolerance based on combined transcriptomic and metabolomic analysis and its application. This method can efficiently and accurately screen regulatory genes closely related to rice tolerance under nighttime heat stress conditions, providing molecular markers for the rapid identification of rice nighttime heat tolerance, and providing scientific basis and technical support for the breeding of rice varieties tolerant to nighttime heat.

[0009] To achieve the above technical objectives, the present invention adopts the following technical solution:

[0010] A method for screening key genes for rice's nighttime heat tolerance based on combined transcriptome and metabolome analysis includes the following steps: Step S1: Apply nighttime high temperature stress treatment to rice plants during the grain-filling stage; Step S2: Obtain rice grains subjected to nighttime high temperature stress treatment, and obtain rice grains grown in the natural environment at the same time as a control group; Step S3: Transcriptome sequencing analysis was performed on rice grains subjected to nighttime high temperature stress and the control group to screen for genes that showed significant differential expression after nighttime high temperature stress treatment; Step S4: Perform metabolomics analysis on rice grains subjected to nighttime high temperature stress and the control group respectively, and screen out metabolites that accumulate differentially after nighttime high temperature stress treatment; Step S5: Perform association analysis between genes with significant differential expression and metabolites with differential accumulation, and screen out genes that are significantly associated with differentially accumulated metabolites as key regulatory genes related to rice's tolerance to nighttime high temperatures.

[0011] Furthermore, the method for treating nighttime high temperature stress specifically involves: from 18:00 to 08:00 the next day, the rice plants are moved into an artificial climate chamber, and the temperature of the artificial climate chamber is set to be 2-6 ℃ higher than the nighttime temperature of the natural environment during the same period; from 08:00 to 18:00 the next day, the rice plants are restored to the natural environment for growth.

[0012] Furthermore, the nighttime high-temperature stress treatment lasted for 15-25 days.

[0013] Furthermore, step S3 includes the following sub-steps: Step S3.1: Extract RNA from rice grains subjected to nighttime high-temperature stress treatment, obtain transcriptome sequencing data, and filter to obtain high-quality sequence fragments; Step S3.2: Locate the high-quality sequence fragment to the rice reference genome and determine the gene in the rice reference genome corresponding to the high-quality sequence fragment; Step S3.3: Using DESeq2 software, quantitative and differential expression analysis of gene expression levels in high-quality sequence fragments of rice grains subjected to nighttime high-temperature stress and the control group was performed to screen out genes with significant differential expression under nighttime high-temperature stress conditions.

[0014] Furthermore, the method for screening genes with significant differential expression is as follows: And the false detection rate (FDR) is less than 0.05. This represents the ratio of gene expression levels under nighttime high-temperature stress conditions to the expression levels of the same gene in the control group.

[0015] Furthermore, the screening method for metabolites exhibiting differential accumulation in step S4 is as follows: significance level and ,in, This indicates the degree of contribution of metabolites in grouping discrimination.

[0016] Furthermore, step S5 includes the following sub-steps: Step S5.1: Construct a gene expression matrix using the normalized expression levels of genes with significant differential expression, and construct a metabolite accumulation matrix using the content of metabolites with differential accumulation. Step S5.2: Calculate the Pearson correlation coefficient between each differentially expressed gene and each differentially accumulated metabolite, and screen out Pearson correlation coefficients. And significance level Gene-metabolite association pairs; Step S5.3: Use the Mantel test to analyze the overall correlation between the gene expression matrix and the metabolite accumulation matrix, and determine the significance level of the Mantel test results. Under the premise of this, genes associated with the metabolites were screened from gene-metabolite association pairs as key regulatory genes related to rice's tolerance to nighttime high temperatures.

[0017] Furthermore, the key regulatory genes related to rice's nighttime heat tolerance in step S5 are: OsORR3 and OsORR6 One or two of them, among which, OsORR3 The amino acid sequence encoding the protein is shown in SEQ ID NO.1. OsORR6 The amino acid sequence encoding the protein is shown in SEQ ID NO.2.

[0018] Furthermore, this invention also provides an application of key genes for rice nighttime heat tolerance based on combined transcriptomic and metabolomic analysis as molecular markers for screening rice nighttime heat tolerance.

[0019] Furthermore, this invention also provides an application of key genes for rice's tolerance to nighttime high temperatures based on combined transcriptomic and metabolomic analysis in the breeding of rice varieties tolerant to nighttime high temperatures.

[0020] Compared with the prior art, the present invention has at least the following beneficial effects: The present invention provides a method for screening key genes for rice's tolerance to nighttime high temperatures. This method combines differential transcriptome expression analysis with quantitative analysis of key metabolites such as cytokinins to construct a correlation analysis system between gene expression changes and metabolic responses. It only screens genes that show significant changes at the transcriptional level and produce significant responses at the metabolic level, thereby effectively reducing false positive results caused by single-mic analysis and improving the accuracy of screening key regulatory genes. This invention identifies key genes for rice's nighttime heat tolerance through combined transcriptomic and metabolomic analysis. OsORR3 and OsORR6It can not only be significantly differentially expressed under high nighttime temperatures, but also be significantly correlated with changes in cytokinin content and have statistical support in the overall expression-metabolism network. It can serve as a stable and reproducible molecular marker. At the same time, it can provide a clear target for molecular design breeding of rice tolerating high nighttime temperatures, and has good application prospects and promotion value. Attached Figure Description

[0021] Figure 1 This is a distribution diagram of dominant and weak particles; Figure 2 This is a schematic diagram comparing the grain length and width of two rice varieties under strong and weak conditions in the control group and under high nighttime temperatures. Figure 3 The bar chart shows the comparison of 100-grain weight between Nanjing 5055 and Yangnong Rice No. 1. In the bar chart, CK-S represents the dominant grains in the control group, CK-I represents the weak grains in the control group, HS represents the dominant grains in the treatment group, and HI represents the weak grains in the treatment group. Figure 4 Principal component analysis plots of the transcriptomes of strong and weak Phenonium 5055 under control and nighttime high temperature conditions; Figure 5 This is a schematic diagram illustrating the correlation between RNA-seq and RT-qPCR results of 20 randomly selected genes from Nanjing 5055. Figure 6 This diagram illustrates the number of differentially expressed genes upregulated and downregulated in the control group and strong and weak grains under high nighttime temperature conditions in Nanjing 5055. Figure 7 A heatmap showing the expression of 10 hormone-related genes in Nanjing 5055 under control and nighttime high temperature conditions; Figure 8 A schematic diagram showing the types and quantities of metabolites that accumulate differently in strong and weak granules under high nighttime temperatures in Nanjing 5055. Figure 9 This is a schematic diagram showing the levels of four differentially accumulated hormones in strong and weak grains of Nanjing 5055 rice under control and nighttime high temperature conditions. Figure 9 In the figure, A represents the accumulation level of cis-zeatin CZ in strong and weak grains. Figure 9 In the figure, B represents the accumulation level of dihydrozeatin DZ in strong and weak grains. Figure 9 C in the figure represents the accumulation level of trans-zeatin TZ in strong and weak grains. Figure 9 D in the figure represents the accumulation level of trans-zeatin riboside TZR in strong and weak granules; Figure 10 for OsORR3 and OsORR6 A schematic diagram illustrating the correlation between expression levels and four cytokinins; Figure 11 for OsORR3and OsORR6 A schematic diagram illustrating the expression levels in strong and weak granules under control and nighttime high-temperature conditions. Figure 11 A in the text is OsORR3 Expression levels in strong and weak granules under control and nighttime high-temperature conditions. Figure 11 B in the text is OsORR6 Expression levels in strong and weak granules under control and nighttime high temperature conditions. Detailed Implementation

[0022] The following is used to illustrate the technical solution of the present invention, but does not constitute a limitation on the scope of protection of the present invention.

[0023] This invention, through combined analysis of transcriptome sequencing, metabolome, and endogenous hormones in rice grains under nighttime high-temperature stress, screened and identified key regulatory genes significantly associated with rice's tolerance to nighttime high temperatures. OsORR3 and OsORR6 .

[0024] Based on the following description of the embodiments, those skilled in the art can understand the technical concept and basic features of the present invention, and, without departing from the spirit of the present invention and the scope of the claims, make appropriate adjustments or substitutions to the experimental conditions, analytical methods and application methods to achieve the screening, evaluation and breeding application of different rice materials' tolerance to nighttime high temperatures.

[0025] Example 1: Screening of key genes for rice's nighttime heat tolerance based on combined transcriptomic and metabolomic analysis Step S1: During the grain-filling stage of rice, apply nighttime high-temperature stress treatment to the rice plants, specifically: The rice plants used in this invention were selected from two widely cultivated japonica rice varieties, Nanjing 5055 and Yangnong Rice No. 1, in the middle and lower reaches of the Yangtze River in China. After routine disinfection and soaking, the seeds were raised under natural conditions and then transplanted into plastic buckets for cultivation and management. Fertilization and water management were carried out according to conventional high-yield cultivation measures.

[0026] Nighttime high-temperature stress treatment was applied to rice starting during the grain-filling stage. The treatment involved moving the experimental materials into an artificial climate chamber from 18:00 to 08:00 the following day, setting the nighttime temperature to be 2-6 °C higher than the ambient nighttime temperature. From 08:00 to 18:00 daily, the materials were returned to their natural growing environment. This nighttime high-temperature stress treatment lasted for 15-25 days. A control group was also included, growing under natural conditions throughout the entire growth period.

[0027] Based on the position of rice grains on the rachis and branches, and differences in grain-filling efficiency, rice grains are divided into dominant grains (S) and weak grains (I), such as... Figure 1 .

[0028] Step S2: Obtain rice grains subjected to nighttime high-temperature stress treatment, and obtain rice grains grown in the natural environment at the same time as a control group. Strong and weak grain samples were collected from both the Nanjing 5055 control group and the treatment group subjected to nighttime high-temperature stress. After being flash-frozen in liquid nitrogen... Store at 80 ℃ for subsequent multi-omics analysis.

[0029] To visually illustrate the impact of nighttime high-temperature stress on rice grain development, the appearance characteristics of mature grains in the treatment and control groups were observed and recorded, such as... Figure 2 Compared with the control group, under nighttime high-temperature stress treatment, there were no significant differences in grain length and width between the dominant grain S and the weakest grain I of Nanjing 5055, while the dominant grain S and weakest grain I of Yangnong 1 showed significant increases in grain length and width. However, the 100-grain weight of both the Nanjing 5055 treatment group and the Yangnong 1 treatment group was significantly lower than that of their respective control groups. Figure 3 As shown in the figure. This result indicates that nighttime high-temperature stress can have a substantial impact on rice grain development, providing phenotypic evidence for subsequent screening of key regulatory genes for nighttime high-temperature tolerance based on combined transcriptomic and metabolomic analysis.

[0030] Step S3: Transcriptome sequencing analysis was performed on rice grains subjected to nighttime high-temperature stress and the control group to screen for genes with significant differential expression after nighttime high-temperature stress treatment; specifically: Step S3.1: RNA was extracted from rice grains subjected to nighttime high-temperature stress treatment, and RNA integrity was rigorously assessed using an Agilent 2100 bioanalyzer. Transcriptome sequencing libraries were constructed using the NEBNext® Ultra™ RNA library preparation kit, and library quality was verified using Illumina HiSeq software. Transcriptome sequencing data were obtained, and high-quality sequence fragments were obtained after quality filtering. Step S3.2: Locate the high-quality sequence fragment to the rice reference genome IRGSP-1.0 and perform sequence alignment using HISAT2 software. This allows for rapid and accurate identification of genes in the rice reference genome corresponding to the high-quality sequence fragment. Before performing differential gene expression analysis, examine the correlation of gene expression levels between samples and assess sample consistency using principal component analysis. Figure 4 The results showed that samples from different treatment groups exhibited a separation trend along the principal component direction, indicating that nighttime high-temperature stress altered the expression characteristics of the grains. To further validate the reliability of the transcriptome data, some key differentially expressed genes were selected for RT-qPCR verification. OsActin As an internal reference gene, through 2 -ΔΔCT The method calculates relative expression levels, and the expression trend is consistent with RNA-seq results, such as... Figure 5As shown.

[0031] Step S3.3: Using DESeq2 software, quantitative and differential expression analysis of gene expression levels in high-quality sequence fragments was performed on rice grains subjected to nighttime high-temperature stress and the control group to screen for genes with significant differential expression under nighttime high-temperature stress. The screening method for genes with significant differential expression is as follows: And the false detection rate (FDR) is less than 0.05. This represents the ratio of gene expression levels under nighttime high-temperature stress conditions to the expression levels of the same gene in the control group.

[0032] like Figure 6 This diagram illustrates the number of differentially expressed genes (upregulated and downregulated) in strong and weak granules of Nanjing 5055 rice under control and nighttime high-temperature conditions. The results show that a large number of differentially expressed genes exist between strong and weak granules under control conditions. After nighttime high-temperature stress treatment, both strong and weak granules showed significant upregulated and downregulated gene counts. Compared to the control conditions, the number of genes upregulated in strong granules was 1125 × 10⁻⁶. 3 The number of units decreased by 1295 × 10 3 The number of weaker particles was increased by 1893 × 10⁻⁶. 3 The number of units decreased by 1373 × 10 3 The number of differentially expressed genes between strong and weak grains decreased under high-temperature stress, indicating that nighttime high-temperature stress significantly affected the transcriptional expression pattern of grains. Furthermore, the number of differentially expressed genes between strong and weak grains decreased under high-temperature stress, specifically, an upregulation of 186 × 10⁶ genes. 3 Individual, down 390×10 3 The presence of these genes indicates that nighttime high-temperature stress altered the differential gene expression patterns between the two types of grains to some extent. Volcano plots were further constructed to visually represent the differentially expressed genes, and KEGG pathway enrichment analysis and GO functional annotation were performed using the BioDeep platform (https: / / www.biodeep.cn). Genes showing significant changes under nighttime high-temperature stress were screened using differential expression analysis. Gene expression levels were standardized using fragments per kilobase of transcript per million mapped reads (FPKM) to eliminate the influence of sequencing depth and gene length differences on expression level calculations. The correlation between gene expression and hormone content was analyzed in conjunction with endogenous hormone assay results. The results showed that nighttime high-temperature treatment significantly affected hormone levels in grains, and some differentially expressed genes were highly correlated with hormone changes. Therefore, these hormone-related genes and their regulatory networks were analyzed in detail, and their expression patterns are as follows: Figure 7As shown, the results indicated that nighttime high-temperature stress significantly affected the expression levels of genes related to some hormone signaling pathways. In dominant grains, all 10 genes reached significant or highly significant levels, while in weak grains, only 2 genes reached significant levels, indicating that the changes caused by nighttime high-temperature stress were more pronounced in dominant grains. Different hormone signaling genes exhibited differentiated expression patterns under nighttime high-temperature stress conditions, suggesting that plant hormone signaling pathways may be involved in the regulatory process of rice grain response to nighttime high-temperature stress.

[0033] Step S4: Metabolomics analysis was performed on rice grains subjected to nighttime high-temperature stress and the control group to screen for metabolites that showed differential accumulation after nighttime high-temperature stress treatment. Specifically, a non-targeted metabolomics method was used to detect metabolites in rice grain samples from the same batch. Approximately 100 mg of frozen-ground sample was extracted with 500 μL of pre-cooled 80% methanol, thoroughly vortexed, sonicated on ice for 10 min, and centrifuged at 4 ℃ and 12000 rpm for 15 min. The supernatant was collected and filtered through a 0.22 μm filter membrane for LC-MS analysis (refer to Dunn et al., 2011; Want et al., 2013). The raw mass spectrometry data were converted to mzXML format using MSConvert and peak detection, filtering, and alignment were performed using XCMS (refer to Smith et al., 2006). Metabolite annotation was based on HMDB, MassBank, LipidMaps, and KEGG databases, with a ppm tolerance of <30 ppm. PCA and OPLS-DA analyses were performed using canonical multivariate methods, and statistical significance was determined by VIP and statistical tests. Using P < 0.05 and VIP > 1 as screening criteria, differentially accumulated metabolites (DAMs) in dominant and weak granules under nighttime high-temperature treatment conditions were obtained, such as... Figure 8 These include: amino acid metabolites, aromatic metabolites, carbohydrate metabolites, fatty acid metabolites, hormone metabolites, nucleotide metabolites, secondary metabolites, steroid metabolites, and vitamin metabolites. Different categories of metabolites showed varying changes under nighttime high-temperature stress. (Combined with...) Figure 7 Transcriptome results showed that different hormone signaling-related genes exhibited differential expression patterns under nighttime high-temperature stress. Further analysis of differentially expressed hormone metabolites revealed that cytokinin metabolites showed the most significant changes, with their content varying across different treatments and grain types. Figure 9As shown, the results indicated that under high-temperature stress, the contents of four cytokinins—zeatin CZ, dihydrozeatin DZ, trans-zeatin TZ, and trans-zeatin nucleoside TZR—were significantly higher in strong grains than in the control group. However, in weak grains, except for zeatin CZ, the contents of the other three cytokinins were significantly lower than in the control group, suggesting that nighttime high-temperature stress treatment may affect grain development through differential regulation of cytokinin metabolic pathways. Functional classification and pathway annotation of differentially metabolites were performed, with a focus on changes in metabolites related to plant hormone synthesis, signal transduction, and stress response.

[0034] Step S5: Association analysis was performed between genes with significant differential expression and cytokinin metabolites to screen for genes significantly associated with cytokinin metabolites as key regulatory genes related to rice's tolerance to nighttime high temperatures. Specifically: Step S5.1: Construct a gene expression matrix using the normalized expression levels of genes with significant differential expression, and construct a metabolite accumulation matrix using the content of cytokinin metabolites; Step S5.2: Calculate the Pearson correlation coefficient between each differentially expressed gene and each cytokinin metabolite, and screen out Pearson correlation coefficients. And significance level Gene-metabolite association pairs; Step S5.3: Use the Mantel test to analyze the overall correlation between the gene expression matrix and the metabolite accumulation matrix, and determine the significance level of the Mantel test results. Under the premise of this, genes associated with metabolites were screened from gene-metabolite association pairs as key regulatory genes related to rice's tolerance to nighttime high temperatures.

[0035] Combining Pearson correlation analysis and Mantel overall correlation test allows for dual validation at both the single-gene-single-metabolite correlation and overall expression-metabolic network correlation levels, enhancing the statistical reliability and stability of screening key regulatory genes related to rice's tolerance to nighttime high temperatures. For example... Figure 10 The combined analysis results showed that changes in cytokinin content in dominant granulocytes were significantly correlated with multiple response regulatory factors, among which, OsORR3 It was significantly correlated with various cytokinins, including trans-zeatin (TZ), dihydrozeatin (DZ), and trans-zeatin nucleoside (TZR), while OsORR6 It was significantly correlated with trans-zeatin nucleoside TZR, indicating that this type of response regulator may be involved in the regulation of seed cytokinin signaling under nighttime high temperature conditions.

[0036] Based on differential expression characteristics of the transcriptome, changes in cytokinin levels in the metabolome, and correlation analysis between the two, a correlation analysis system between gene expression changes and metabolic responses was constructed. This system screens only genes that exhibit significant changes at both the transcriptional and metabolic levels, effectively reducing false positives from single-mic analyses and improving the accuracy of screening key regulatory genes. OsORR3 and / or OsORR6 These are key regulatory genes closely related to rice's tolerance to nighttime high temperatures. The coding sequences and corresponding amino acid sequences of these genes were further obtained based on the rice reference genome IRGSP-1.0 for subsequent analysis and application. OsORR3 The amino acid sequence encoding the protein is shown in SEQ ID NO.1. OsORR6 The amino acid sequence encoding the protein is shown in SEQ ID NO.2. Based on the combined analysis of transcriptomics and metabolomics, it is speculated that the above gene may be involved in the cytokinin-related response process of seed grains under high nighttime temperatures.

[0037] Example 2: OsORR3 and OsORR6 Example of expression level and evaluation of rice's tolerance to nighttime high temperatures To verify OsORR3 and OsORR6 To assess the application value of rice's tolerance to nighttime high temperatures, several rice materials with different genetic backgrounds were selected, and nighttime high temperature stress treatment and normal control treatment were set up during the grain-filling stage. The nighttime high temperature treatment conditions were the same as in Example 1.

[0038] After processing for a certain period of time, seed samples were collected, total RNA was extracted, and detected using real-time quantitative PCR. OsORR3 and OsORR6 The expression level of the gene was calculated for each material under high nighttime temperature conditions, using rice internal reference genes as a control. OsORR3 and OsORR6 The relative expression level. For example... Figure 11 The results showed that different rice materials under nighttime high-temperature stress conditions... OsORR3 and OsORR6 There were significant differences in the expressive responses, with some materials showing... OsORR3 and OsORR6 Expression levels were significantly upregulated under nighttime high-temperature conditions, while in another subset of materials, expression changes were not significant or showed a downward trend. Based on these expression differences, the expression levels under nighttime high-temperature conditions were further analyzed. OsORR3 and OsORR6 Rice materials with higher expression levels were identified as having strong potential for tolerance to nighttime high temperatures, while materials with lower expression levels were identified as having weaker tolerance to nighttime high temperatures.

[0039] The above results indicate that OsORR3and OsORR6 Not only can it be significantly differentially expressed under high nighttime temperatures, but it is also significantly correlated with changes in cytokinin content and has statistical support in the overall expression-metabolism network. It can serve as a stable and reproducible molecular marker. At the same time, it can provide a clear target for molecular design breeding of rice tolerating high nighttime temperatures and provide a simple and rapid technical means for breeding rice varieties tolerant to high nighttime temperatures.

[0040] The above are merely preferred embodiments of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A method for screening key genes for rice's nighttime heat tolerance based on combined transcriptome and metabolome analysis, characterized in that, Includes the following steps: Step S1: Apply nighttime high temperature stress treatment to rice plants during the grain-filling stage; Step S2: Obtain rice grains subjected to nighttime high temperature stress treatment, and obtain rice grains grown in the natural environment at the same time as a control group; Step S3: Transcriptome sequencing analysis was performed on rice grains subjected to nighttime high temperature stress and the control group to screen for genes that showed significant differential expression after nighttime high temperature stress treatment; Step S4: Perform metabolomics analysis on rice grains subjected to nighttime high temperature stress and the control group respectively, and screen out metabolites that accumulate differentially after nighttime high temperature stress treatment; Step S5: Perform association analysis between genes with significant differential expression and metabolites with differential accumulation, and screen out genes that are significantly associated with differentially accumulated metabolites as key regulatory genes related to rice's tolerance to nighttime high temperatures.

2. The method for screening key genes for rice nighttime heat tolerance based on combined transcriptome and metabolome analysis according to claim 1, characterized in that, The specific method for treating nighttime high temperature stress is as follows: from 18:00 to 08:00 the next day, the rice plants are moved into an artificial climate chamber, and the temperature of the artificial climate chamber is set to be 2-6 ℃ higher than the nighttime temperature of the natural environment during the same period; from 08:00 to 18:00 the next day, the rice plants are restored to the natural environment for growth.

3. The method for screening key genes for rice nighttime heat tolerance based on combined transcriptome and metabolome analysis according to claim 2, characterized in that, The nighttime high-temperature stress treatment lasted for 15-25 days.

4. The method for screening key genes for rice nighttime heat tolerance based on combined transcriptome and metabolome analysis according to claim 1, characterized in that, Step S3 includes the following sub-steps: Step S3.1: Extract RNA from rice grains subjected to nighttime high-temperature stress treatment, obtain transcriptome sequencing data, and filter to obtain high-quality sequence fragments; Step S3.2: Locate the high-quality sequence fragment to the rice reference genome and determine the gene in the rice reference genome corresponding to the high-quality sequence fragment; Step S3.3: Using DESeq2 software, quantitative and differential expression analysis of gene expression levels in high-quality sequence fragments of rice grains subjected to nighttime high-temperature stress and the control group was performed to screen out genes with significant differential expression under nighttime high-temperature stress conditions.

5. The method for screening key genes for rice nighttime heat tolerance based on combined transcriptome and metabolome analysis according to claim 4, characterized in that, The screening method for genes with significant differential expression is as follows: And the false detection rate (FDR) is less than 0.

05. This represents the ratio of gene expression levels under nighttime high-temperature stress conditions to the expression levels of the same gene in the control group.

6. The method for screening key genes for rice nighttime heat tolerance based on combined transcriptome and metabolome analysis according to claim 1, characterized in that, The screening method for metabolites with differential accumulation in step S4 is as follows: significance level and ,in, This indicates the degree of contribution of metabolites in grouping discrimination.

7. The method for screening key genes for rice nighttime heat tolerance based on combined transcriptome and metabolome analysis according to claim 1, characterized in that, Step S5 includes the following sub-steps: Step S5.1: Construct a gene expression matrix using the normalized expression levels of genes with significant differential expression, and construct a metabolite accumulation matrix using the content of metabolites with differential accumulation. Step S5.2: Calculate the Pearson correlation coefficient between each differentially expressed gene and each differentially accumulated metabolite, and screen out Pearson correlation coefficients. And significance level Gene-metabolite association pairs; Step S5.3: Use the Mantel test to analyze the overall correlation between the gene expression matrix and the metabolite accumulation matrix, and determine the significance level of the Mantel test results. Under the premise of this, genes associated with the metabolites were screened from gene-metabolite association pairs as key regulatory genes related to rice's tolerance to nighttime high temperatures.

8. The method for screening key genes for rice nighttime heat tolerance based on combined transcriptome and metabolome analysis according to claim 1, characterized in that, The key regulatory genes related to rice's tolerance to nighttime high temperatures in step S5 are: OsORR3 and OsORR6 One or two of them, among which, OsORR3 The amino acid sequence encoding the protein is shown in SEQ ID NO.

1. OsORR6 The amino acid sequence encoding the protein is shown in SEQ ID NO.

2.

9. The application of a key gene for rice nighttime heat tolerance based on combined transcriptome and metabolome analysis as a molecular marker for screening rice nighttime heat tolerance.

10. Application of a key gene for rice's tolerance to nighttime high temperatures based on combined transcriptomic and metabolomic analysis in the breeding of rice varieties tolerant to nighttime high temperatures.

Images