Internal reference genes for real-time fluorescence quantitative PCR analysis of gene expression under heat stress in indica rice and their construction method

Abstract

The present invention discloses a kind of reference gene and construction method of real-time fluorescence quantitative PCR for analyzing gene expression under heat stress of indica rice, and belongs to the field of plant genetic engineering technology. The nucleotide sequences of 3 reference genes of the present invention are shown in sequence table SEQ ID NO.1-10;The flag leaf of II excellent 838 and its parent after high temperature stress is subjected to transcriptome high-throughput sequencing;Select 5 candidate reference genes from the sequencing results, carry out fluorescence quantitative PCR amplification on rice cDNA template, according to the expression difference of candidate genes in different rice samples, screen to the most suitable reference gene;Use the 3 reference genes screened together to calibrate and standardize the expression of target gene, can more accurately detect the expression change of indica rice gene, ensure the sensitivity and accuracy of fluorescence quantitative PCR;Use heat stress response gene to carry out method verification, find that the relative quantitative expression value of each gene in indica rice sample is consistent with the RPKM value and TPM value of sequencing result very well.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and particularly relates to an internal reference gene for real-time fluorescence quantitative PCR for analyzing gene expression under heat stress in indica rice and a construction method thereof. Background Art

[0002] Real-time PCR (Quantitative Real-time PCR) involves adding a fluorescent group to the PCR reaction system. The accumulated fluorescent signal during the DNA amplification reaction allows real-time monitoring of the total product increase after each polymerase chain reaction (PCR) cycle. During the exponential phase of PCR amplification, the Ct value (Cycle Threshold; Ct value refers to the number of cycles required for the fluorescent signal in each reaction tube to reach the threshold) of the template is linearly related to the initial copy number of the template, providing a basis for quantification and enabling real-time monitoring of PCR progress through fluorescence signals. The SYBR Green I method we use involves adding an excess of SYBR fluorescent dye to the PCR reaction system. SYBR dye specifically incorporates into double-stranded DNA, emitting a fluorescent signal. Unincorporated SYBR dye molecules do not emit any fluorescent signal, ensuring that the increase in fluorescent signal is synchronized with the increase in PCR product. SYBR binds only to double-stranded DNA, allowing the specificity of the PCR reaction to be determined using a melting point curve.

[0003] Real-time fluorescence quantitative PCR (RT-qPCR), invented by Applied Biosystems in the United States in 1996, marked a significant leap forward in PCR from qualitative to quantitative analysis, boasting high sensitivity, strong specificity, rapidity, and accuracy. Real-time fluorescence quantitative PCR is a powerful method for analyzing gene expression profiles, enabling analysis of gene expression differences across time periods and between samples subjected to different treatments. In RT-qPCR experiments, internal reference genes are used as controls for data normalization to correct for quantitative variations in the cDNA template (Guierez et al. 2008, Huggette et al. 2005, Vandesopmpele et al. 2002). A good internal reference gene should exhibit consistent expression across samples under different experimental conditions or at different time points. Certain genes, such as GAPDH, β-actin, or rRNA, are commonly used as internal reference genes. However, numerous studies have shown that the expression of these genes varies across different tissues or treatment conditions, making them unsuitable for use as internal reference genes. Therefore, internal reference genes require careful screening based on experimental data.

[0004] 1. Perform total RNA extraction on at least one or two samples per experimental condition or time point to determine their purity and quality (48 samples in my experiment).

[0005] 2. After standardizing the sample concentration, reverse transcription was performed on each sample in the same volume (48 cDNAs).

[0006] 3. Using each cDNA sample as a template, take the same volume for qPCR experiments.

[0007] 4. Compare the C of each gene under different conditions q value.

[0008] The difference in Cq (△Cq) under different conditions or time points should not exceed 0.5.

[0009] Because any specific gene may be affected by different experimental conditions, at least three or four specific internal reference genes located in different metabolic pathways are detected simultaneously in the experiment, and the geometric mean of the genes with the smallest expression differences is performed to accurately normalize the data (Vandesormpele et al 2002).

[0010] Indica rice exhibits superior heat tolerance to japonica rice, and screening for heat tolerance within indica rice varieties increases the chances of obtaining highly heat-tolerant rice varieties. The new medium-season rice combination, II You 838, was successfully developed through traditional hybridization of the sterile indica rice line II-32A with the restorer line Fuhui 838. This combination exhibits excellent heat tolerance during field cultivation, surpassing many rice varieties, including Shanyou 63. Its seed production method has been granted a national patent. With the widespread adoption of II You 838 in warmer regions, its advantages in heat and heat resistance have gradually become apparent. Years of field cultivation have revealed that II You 838 possesses numerous advantages, including high panicle emergence, high tillering rate, strong stems, and excellent rice quality. It also exhibits excellent field stability and tolerance to high and low temperatures, as well as drought. II You 838 is the hybrid rice combination with the largest planting area nationwide after Shanyou 63. Years of field cultivation have accumulated extensive experience in planting, breeding, and pest avoidance. In-depth genetic research on heat tolerance in II You 838, using II You 838 as a genetically improved material, will accelerate the development of heat-tolerant varieties suitable for field cultivation. The flag leaf of rice is the longest-growing leaf, has the highest photosynthetic activity, and transports the most nutrients. It is a critical functional leaf, performing crucial assimilation processes throughout the rice plant's growth period, and its condition is closely linked to high and stable rice yields. Therefore, the physiological and biochemical effects of high temperatures on the flag leaf of rice have been extensively studied. High temperature stress has been found to reduce chlorophyll content, decrease net photosynthetic rate, and decrease RuBPc activity, leading to membrane disruption and reduced levels of osmotic regulatory substances such as soluble sugars. Therefore, analyzing gene expression in the flag leaf of rice under heat stress can reveal the mechanisms of rice's heat stress response.

[0011] Ideal internal reference genes should be constantly expressed under the various experimental factors studied. However, a large number of research results show that any internal reference gene is not constantly expressed under all experimental conditions, and the expression of internal reference genes varies in different types of cells and at different stages of cell growth. Therefore, before carrying out gene expression analysis, the verification of internal reference genes should be carried out, and a relatively stable internal reference gene should be selected to be used for the correction of target gene expression, in the hope of obtaining more realistic and reliable results. Most of what is used in current rice crop gene expression analysis is traditional internal reference genes, and under various different heat stress levels (heat stress levels are much higher than the various situations that can occur under natural conditions), it is difficult to ensure that the stability of internal reference genes will not be affected as the environment changes. In order to study the functional gene expression of indica rice flag leaves under heat stress conditions, it is very necessary to screen the best internal reference genes or gene combinations under specific conditions. Summary of the Invention

[0012] The present invention aims to provide an internal reference gene for real-time fluorescence quantitative PCR for analyzing gene expression under heat stress in indica rice and a method for constructing the same. The present invention uses three verified internal reference genes as reference substances for detecting changes in gene expression levels in indica rice under heat stress, which can more accurately calibrate and standardize the expression levels of target genes, thereby ensuring the sensitivity and accuracy of the fluorescence quantitative PCR method.

[0013] The object of the present invention is achieved through the following technical solution: an application of three genes, LOC_Os03g50885.1, LOC_Os01g12800.1 and LOC_Os02g16709.1, as internal reference genes for real-time fluorescence quantitative PCR analysis of gene expression under heat stress in indica rice, whose nucleotide sequences are shown in sequence listing SEQ ID NO.1-3 respectively.

[0014] The present invention selects three rice varieties, II You 838, its male parent Fu Hui 838, and its female parent II-32A, as materials, uses the digital gene expression profiling sequencing technology developed by Illumina / Solexa to perform high-throughput transcriptome sequencing (transcriptome) of the flag leaves of II You 838 at the flowering stage under different degrees of heat stress, and performs digital gene expression tag profiling (DGE) on the flag leaves of II You 838 and its parents at the flowering stage, and screens the above three genes with constant expression from the gene library obtained by sequencing as internal reference genes.

[0015] The present invention provides a method for constructing an internal reference gene for real-time fluorescence quantitative PCR for analyzing gene expression under heat stress in indica rice, comprising the following steps:

[0016] (1) After the male parent Fuhui 838, the female parent II-32A and the offspring II You 838 rice were subjected to high temperature stress, the flag leaves of II You 838 at the flowering stage were subjected to high-throughput transcriptome sequencing, and the flag leaves of II You 838 and its parents at the flowering stage were subjected to digital expression profile sequencing. Two internal reference genes with stable expression were selected from the 5 transcriptome sequencing results and 12 DGE sequencing results. Their nucleotide sequences are shown in the sequence listing SEQ ID NO.2 and SEQ ID NO.3. At the same time, three traditional internal reference genes commonly used in the genus Oryza were selected as a comparative reference group. Their nucleotide sequences are shown in the sequence listing SEQ ID NO.1, SEQ ID NO.4 and SEQ ID NO.5, for a total of 5 candidate internal reference genes;

[0017] (2) Primers were designed for the five candidate internal reference genes, and fluorescent quantitative PCR reactions were performed on each rice cDNA generated by reverse transcription. The obtained data were analyzed using the internal reference gene stability assessment software Bio-Rad CFX Manager 3.0. The optimal internal reference gene was screened by the M values ​​of the five candidate internal reference genes. The smaller the difference, the more stable the expression value. Finally, the most suitable internal reference genes and the number of internal reference genes under high temperature stress treatment were screened;

[0018] (3) The heat stress response genes in rice were used for verification. Fluorescence quantitative PCR primers for each heat stress response gene were designed, and the rice cDNA template generated by reverse transcription was subjected to fluorescence quantitative PCR reaction. The data were analyzed using Bio-Rad CFXManager 3.0 software. The Pfaffl algorithm was used to obtain the relative quantitative expression value of the heat stress response gene in each rice sample under the joint calibration of the three internal reference genes LOC_Os03g50885.1, LOC_Os01g12800.1 and LOC_Os02g16709.1. The relative quantitative expression value was compared with the RPKM value and TPM value of the high-throughput sequencing results for verification.

[0019] In the present invention, the target gene, i.e., the heat stress response gene, refers to a molecular identification marker for heat tolerance of rice or a molecular identification marker for heat damage response of rice.

[0020] In the present invention, the Illumina HiSeq TM The paired-end (PE) RNA-Seq technology of the 2000 sequencing platform was used to sequence the rice flag leaf transcriptome, and the transcriptome expression profile of the flag leaf of II You 838 rice at the flowering stage after heat stress was constructed to understand the expression of various genes.

[0021] In the present invention, three rice varieties, II You 838, its male parent Fu Hui 838, and its female parent II-32A, were selected as materials. The digital gene expression profiling sequencing technology developed by Illumina / Solexa was used to perform high-throughput sequencing of flag leaves at the flowering stage under different degrees of heat stress, and the gene expression levels in different samples were analyzed.

[0022] In the present invention, the three internal reference genes for stable expression in indica rice real-time fluorescence quantitative RT-PCR under heat stress have amplification primers as follows: the forward primer sequence of LOC_Os03g50885.1 is shown in SEQ ID NO.6 of the sequence listing, and the reverse primer sequence is shown in SEQ ID NO.7 of the sequence listing; the forward primer sequence of LOC_Os01g12800.1 is shown in SEQ ID NO.8 of the sequence listing, and the reverse primer sequence is shown in SEQ ID NO.9 of the sequence listing; the forward primer sequence of LOC_Os02g16709.1 is shown in SEQ ID NO.10 of the sequence listing, and the reverse primer sequence is shown in SEQ ID NO.11 of the sequence listing.

[0023] The technical route of the present invention is: after high temperature stress, the flag leaves of II You 838 at the flowering stage are subjected to high-throughput transcriptome sequencing (transcriptome), and the flag leaves of II You 838 and its parents at the flowering stage are subjected to digital gene expression tag profiling (DGE). From the gene library of the mRNA data set obtained by transcriptome sequencing of all experimental materials in the experiment, genes with constant expression are screened as internal reference genes for real-time PCR. In the sequencing results, the expression levels of the two genes LOC_Os01g12800.1: peroxisomal membrane protein and LOC_Os02g16709.1: peptidase are basically unchanged under different degrees of heat stress in three rice varieties. The expression stability of these two candidate internal reference genes is then evaluated. After the internal reference gene M value of the real-time PCR is measured, if the M value is less than 0.5, it can be used as the internal reference gene for the relative quantitative real-time PCR experiment of rice flag leaves after high temperature stress. Finally, the reliability of the experimental results was verified by comparing the results of quantitative PCR with those of RNA high-throughput sequencing. Three commonly used reference genes in rice reported in the literature were also selected as reference groups for comparison in the experiment: LOC_Os03g50885.1: actin1 (GenBank: AK100267.1), LOC_Os01g45400: ubiquitin, and LOC_Os09g00999.1: 18s rRNA. A total of five candidate reference genes were identified. After verification, it was found that LOC_Os01g12800.1 and LOC_Os02g16709.1 had smaller M values ​​than LOC_Os01g45400 and LOC_Os09g00999.1 reported in the literature, making them more suitable as reference genes for the present invention. Therefore, LOC_Os01g45400 and LOC_Os09g00999.1 were eliminated, and LOC_Os03g50885.1, LOC_Os01g12800.1, and LOC_Os02g16709.1 were selected as internal reference genes.

[0024] The present invention demonstrates that the expression levels of the reference gene meet the requirements under all heat stress conditions across the three rice varieties. Specifically, the expression levels of the reference gene are essentially consistent across all rice samples, with an M value less than 0.5. "Constant" refers to the essentially consistent expression levels across the three rice varieties under different heat stress conditions. "Expression" refers to the expression level of the reference gene, which corresponds to the number of copies of the reference gene transcribed into mRNA in the rice genome.

[0025] The present invention uses three verified internal reference genes (the geometric mean of the three genes) to calibrate the expression level of the target gene, which can more accurately calibrate and standardize the expression level of the target gene. Among them, LOC_Os01g12800.1: peroxisomal membrane protein and LOC_Os02g16709.1: peptidase were selected from the results of 5 transcriptome sequencing and 12 DGE sequencing as internal reference genes for real-time PCR. The expression stability was then evaluated. After the M value of the internal reference genes in real-time PCR was determined to be less than 0.5, the quantitative PCR results were finally compared with the results of RNA high-throughput sequencing to verify the reliability of the experimental results.

[0026] The present invention has the following advantages and beneficial effects:

[0027] 1. The three internal reference genes LOC_Os03g50885.1, LOC_Os01g12800.1 and LOC_Os02g16709.1 screened by the present invention have basically the same expression levels in three rice varieties under different heat stress conditions, and are very accurate and reliable when applied to the real-time fluorescence quantitative RT-PCR method for heat-stressed indica rice.

[0028] 2. The present invention uses internal reference genes to correct the expression of a target gene in the indica rice variety II You 838 and its parents under heat stress, thereby improving the sensitivity and repeatability of fluorescence quantitative PCR. Currently, the use of two or more internal reference genes in fluorescence quantitative PCR for indica rice under heat stress is relatively rare (most literature uses one or two internal reference genes). The present invention uses three genes to correct the expression of the target gene through geometric averaging, which can more accurately correct and standardize the expression of the target gene.

[0029] 3. The present invention uses three verified genes as internal reference genes for the fluorescence quantitative PCR relative quantification method to jointly calibrate and standardize the expression values ​​of each gene in the flag leaf samples of the indica rice II You 838 and its parents. Through this fluorescence quantitative PCR method, the expression level of the target gene of interest (such as rice heat tolerance molecular identification markers or rice heat damage response molecular identification markers, etc.) under heat stress can be quickly and accurately monitored. This fluorescence quantitative PCR method calibrated by three internal reference genes can be used as a rapid rice heat damage identification method, or a heat-tolerant rice molecular breeding identification method. The two genes LOC_Os01g12800.1 and LOC_Os02g16709.1 are the first to be used as internal reference genes for indica rice under heat stress. This method can be extended to other indica rice varieties under heat stress, or other and more rice varieties under heat stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The figure is an agarose gel electrophoresis diagram of total RNA extracted from 5 flag leaf samples of II You 838 rice.

[0031] Figures 2 to 10 The figure shows the comparison of the PCR relative expression values ​​and transcriptome sequencing results of 9 target genes in II8, F and A rice samples on the 0th day, 1st day, 3rd day and 5th day of heat stress of IIyou 838 and its parents, respectively.

[0032] Figure 2 This is a comparison chart of the relative expression values ​​of the target gene LOC_Os03g56540.3 in three samples;

[0033] Figure 3 This is a comparison chart of the relative expression values ​​of the target gene LOC_Os05g44340.1 in three samples;

[0034] Figure 4 This is a comparison chart of the relative expression values ​​of the target gene LOC_Os03g02260.1 in three samples;

[0035] Figure 5 This is a comparison chart of the relative expression values ​​of the target gene LOC_Os04g59150.1 in three samples;

[0036] Figure 6 This is a comparison chart of the relative expression values ​​of the target gene LOC_Os04g08550.1 in three samples;

[0037] Figure 7 This is a comparison chart of the relative expression values ​​of the target gene LOC_Os10g02920.1 in three samples;

[0038] Figure 8 This is a comparison chart of the relative expression values ​​of the target gene LOC_Os03g63750.1 in three samples;

[0039] Figure 9 This is a comparison chart of the relative expression values ​​of the target gene LOC_Os05g45410.1 in three samples;

[0040] Figure 10 This is a comparison chart of the relative expression values ​​of the target gene LOC_Os03g24339.1 in three samples. DETAILED DESCRIPTION

[0041] The present invention will be further described in detail below with reference to the examples, but the embodiments of the present invention are not limited thereto.

[0042] Example 1: High temperature stress treatment of rice materials

[0043] Rice varieties Fuhui 838 (F), II-32A (A), and II You 838 (II8) were provided by the Sichuan Institute of Atomic Energy. Seedlings were sown and raised using conventional methods at the Mianzhu Experimental Base in Chengdu, Sichuan Province. Once the rice reached the booting stage, plants of uniform growth were randomly selected and transplanted into pots with soil, with three plants per pot. The plants were then randomly divided into a heat-treated group and a control group, with two pots per group. After several days of field cultivation, the plants were transferred to an artificial climate chamber for acclimatization two days before flowering. The chamber conditions were set at a daily average temperature of 26°C (32°C from 8:00 AM to 7:00 PM; 22°C from 7:00 PM to 8:00 AM on the second day), 80% humidity, and a 13-h / 11-h light / dark cycle.

[0044] On the day of rice flowering, the heat treatment group was subjected to high temperature stress, and the temperature of the artificial climate chamber was set to a daily average temperature of 37°C (8:00-19:00, 42°C; 19:00-8:00 the next day, 32°C), and other conditions remained unchanged. The artificial climate chamber was set to an 11h / 13h light-dark cycle and 80% humidity. Other conditions such as water and fertilizer for the three rice varieties F, A and II8 were kept consistent. Other conditions such as water and fertilizer for the control and heat treatment groups were also consistent. The sword leaves of the II You 838 heat treatment group and the control group were collected at 19:00 on the 0th, 1st, 3rd and 5th day of heat treatment, immediately frozen with liquid nitrogen, and stored for later use. The sword leaf materials of the II You 838 heat treatment group on the 0th day before heat stress and on the 1st, 3rd and 5th day after heat stress were named T0, T1, T3 and T5, respectively. Total RNA was extracted from the sword leaf materials, and the library was constructed after reverse transcription into cDNA. The library was sequenced using Illumina's HiSeq TM Transcriptome sequencing was performed using the 2000 system sequencing platform.

[0045] At the same time, flag leaves of rice varieties II8, F, and A were collected for DGE sequencing. Flag leaves of II8, F, and A were collected at 19:00 on day 0, day 1, day 3, and day 5 of heat treatment, immediately frozen in liquid nitrogen, and stored for future use. Total RNA was extracted from the flag leaves, reverse transcribed into cDNA, and 12 DGE libraries containing 21 bases were constructed using Illumina's HiSeq. TM High-throughput DGE sequencing was performed using the 2000 system sequencing platform. The flag leaf sample numbers are shown in Table 1 below.

[0046] Table 1 Statistics of rice flag leaf digital gene expression (DGE) sequencing samples

[0047]

[0048] Flag leaves of II8, F, and A rice were also collected for fluorescence quantitative PCR experiments, with each sample containing three biological replicates.

[0049] Example 2: Fluorescence quantitative PCR method

[0050] (1) Main reagents and instruments

[0051] TRIzol reagent from Invitrogen, catalog number 15596-026;

[0052] Takara's reverse transcription kit PrimeScript TM RT reagent Kit with gDNA Eraser (Perfect Real Time), catalog number RR047A;

[0053] Takara Premix Ex Taq TM II, catalog number RR820A;

[0054] Kaiao K5600 ultra-micro spectrophotometer: Beijing Kaiao Technology Development Co., Ltd. (Beijing);

[0055] High-speed refrigerated centrifuge: Eppendorf;

[0056] Fluorescence quantitative PCR instrument CFX-96: Bio-Rad Company.

[0057] (2) Primer design

[0058] First, from the results of five transcriptome sequencing and 12 DGE sequencing, two genes (LOC_Os01g12800.1: peroxisomal membrane protein and LOC_Os02g16709.1: peptide) that were expressed in every sample and whose expression levels remained largely unchanged were selected as internal reference genes for real-time PCR. The sequencing results showed that the expression levels of these two genes remained largely unchanged under different levels of heat stress in the three rice varieties. Three rice internal reference genes reported in the literature were also selected as comparative reference groups in the experiment: LOC_Os03g50885.1: actin1 (GenBank: AK100267.1), LOC_Os01g45400: ubiquitin, and LOC_Os09g00999.1: 18s rRNA. NCBI Primer-Blast was used to design primers for these five genes, ensuring that the target sequences were unique in the rice transcriptome, with lengths ranging from 75 to 200 bp, melting temperatures between 55 and 65°C, and GC contents between 50 and 60%. The primers for each gene are shown in Table 2 below.

[0059] Table 2 Real-time PCR primer statistics

[0060]

[0061] (3) Total RNA extraction from rice flag leaves

[0062] RNase-free consumables and reagents were used in the experiments. All water used was prepared with ddH2O to a 0.1‰ concentration of DEPC water, stored in the dark overnight, and sterilized by wet heat at 121°C for 30 minutes to ensure RNase-free contamination. All glassware was oven-baked at 180°C for at least 4 hours to remove RNase. Plasticware was soaked in 0.5M NaOH for 10 minutes, then thoroughly rinsed with DEPC water and sterilized to remove RNase.

[0063] Total RNA from rice flag leaves was extracted using the TIANGEN RNAsimple total RNA extraction kit. The procedure is as follows:

[0064] (1) Pre-cool the mortar and pestle with liquid nitrogen, then place the rice flag leaf material in the mortar, quickly freeze it with liquid nitrogen, and quickly grind it into powder;

[0065] (2) Collect 50-100 mg of powder in a pre-cooled RNase-free 1.5 mL EP tube, add 450 μL of RL lysis buffer containing β-mercaptoethanol, and vortex for 30 seconds to mix;

[0066] (3) Incubate at 56°C for 3 min to fully lyse the cells;

[0067] (4) Transfer all the solution into the filter column CS, place the CS into a collection tube, centrifuge at 12000 rpm for 5 min at room temperature, and carefully pipette the supernatant from the collection tube into an RNase-free 1.5 mL EP tube;

[0068] (5) Slowly add 0.5 times the volume of the supernatant (about 225 μL) of anhydrous ethanol and mix gently until a precipitate is produced;

[0069] (6) Transfer the solution and precipitate mixture into the adsorption column CR3, place CR3 in a collection tube, centrifuge at 12000 rpm for 60 s at room temperature, discard the waste liquid in the collection tube, and return CR3 to the collection tube;

[0070] (7) Add 350 μL of deproteinized solution RW1 to CR3, centrifuge at 12,000 rpm for 60 s at room temperature, discard the waste liquid in the collection tube, and return the adsorption column CR3 to the collection tube;

[0071] (8) Add 80 μL of DNase I working solution to the central filter membrane of adsorption column CR3 and incubate at room temperature for 15 min.

[0072] (9) Add 350 μL of deproteinized solution RW1 to the adsorption column CR3, centrifuge at 12,000 rpm for 60 s at room temperature, discard the waste liquid in the collection tube, and return the adsorption column CR3 to the collection tube;

[0073] (10) Add 500 μL of ethanol-containing rinse solution RW to CR3, let it stand at room temperature for 2 min, centrifuge at 12,000 rpm for 60 s at room temperature, discard the waste liquid in the collection tube, and return the adsorption column CR3 to the collection tube;

[0074] (11) Add 500 μL of ethanol-containing rinse solution RW to CR3, rinse for the second time, let it stand at room temperature for 2 min, centrifuge at 12,000 rpm for 2 min at room temperature, pour out the waste liquid in the collection tube, put the adsorption column CR3 back into the collection tube, open the EP tube cap, let it stand at room temperature for 5-10 min, and completely dry the remaining rinse solution;

[0075] (12) Place the adsorption column CR3 in a new RNase-free 1.5 mL EP tube, add 50 μL of RNase-free ddH2O to the middle of the filter membrane, and let it stand at room temperature for 2 min.

[0076] (13) Centrifuge at 12000 rpm for 2 min at room temperature to obtain RNA solution.

[0077] The integrity of the obtained RNA was determined by 1.0% agarose gel electrophoresis and the RNA A was measured by UV spectrophotometer. 260 / A 280 quantification and identification of values.

[0078] (IV) Synthesis of first-strand cDNA

[0079] All reagents and consumables used in this experiment are RNase-free. Genomic DNA was removed from approximately 1 μg of total RNA sample in a 20 μl system. The reaction mixture was prepared on ice according to the following ingredients. To ensure accurate reaction preparation, a Master Mix (equivalent to the number of reactions + 2) was prepared for each reaction, then aliquoted into each reaction tube before adding the RNA sample. The reagent system in each reaction tube was as follows: 4.0 μL 5× gDNA Eraser Buffer, 2.0 μL gDNA Eraser, less than 1 μg of total RNA, and RNase-free ddH2O to a volume of 20 μL. The prepared reaction solution was incubated at 42°C for 2 minutes, then terminated at 4°C.

[0080] After genomic DNA removal, the reaction mixture was reverse transcribed to synthesize first-strand cDNA. The reaction mixture was prepared on ice, using the following reagent volumes for a 20 μL system. To ensure accurate reaction preparation, a Master Mix was prepared for each reaction (number of reactions + 2), and 10 μL was aliquoted into each reaction tube. The reverse transcription primers, RT Primer Mix, are a mixture of random hexamers and OligodT Primer, ensuring uniform synthesis of all cDNAs present in the sample. To prepare 10.0 μL of the genomic DNA-removed reaction mixture, add 1.0 μL PrimeScript RT Enzyme Mix I, 1.0 μL RT Primer Mix, 4.0 μL 5× PrimeScript Buffer 2, and 4 μL RNase-free ddH2O to a total volume of 20 μL. After gentle mixing, reverse transcription was immediately performed. Reaction conditions were incubation at 37°C for 15 minutes, reaction at 85°C for 5 seconds, and storage at 4°C.

[0081] (5) Fluorescence quantitative PCR reaction (real-time PCR)

[0082] The first strand of cDNA generated by reverse transcription of each sample was used as a template. The fluorescence quantitative PCR reaction system was 10 μL. The reaction solution was prepared on ice. The volume of each reagent was: 5 μL 5×SYBR Ex TaqII, 2 μL cDNA template, 0.5 μL PCR Forward Primer (10 μM), 0.5 μL PCR Reverse Primer (10 μM), and finally 2 μL ddH2O were added to a 10 μL volume. The experiment was repeated three times with three technical replicates. For each reaction, the Master Mix was prepared first, then 8 μL was aliquoted into each reaction tube. Finally, 2 μL template was added to each reaction tube to ensure the accuracy of the reaction solution preparation.

[0083] (VI) Detection of stable expression of internal reference genes

[0084] Four cDNA templates were randomly selected from all rice flag leaf cDNA samples and adjusted to a consistent concentration of 97.5 μg / mL. Five candidate internal reference genes, including actin, ubiquitin, and 18S ribosomal RNA, reported in the literature, were selected to test their expression stability in rice flag leaf samples after heat stress, along with LOC_Os01g12800.1 and LOC_Os02g16709.1, genes stably expressed in RNA-seq results from II You 838 and its parental rice lines. Four biological replicates were used, and the real-time reaction for each biological replicate included three technical replicates. The data were analyzed using Bio-Rad CFX Manager 3.0 software. The optimal internal reference gene (M value less than 0.5) was screened by the M values ​​of the five internal reference candidate genes. The smaller the M value, the smaller the difference in gene expression between different sample templates, and the more suitable it is as an internal reference gene to calibrate the expression of other target genes.

[0085] (VII) Relative quantitative gene expression analysis

[0086] Twelve heat-stress-responsive rice genes were selected and their relative expression was analyzed in rice samples of II You 838 and its parents subjected to varying degrees of heat stress. Fluorescence-based quantitative PCR reactions were performed on the rice cDNA templates generated by reverse transcription for the 12 heat-stress-responsive genes. Simultaneously, fluorescence-based quantitative PCR reactions were performed in each rice sample using three specific internal reference genes, LOC_Os03g50885.1 (actin), LOC_Os01g12800.1, and LOC_Os02g16709.1, which are involved in three different metabolic pathways. Data were analyzed using Bio-Rad CFX Manager 3.0 software. The Pfaffl algorithm was used to calibrate and normalize the three internal reference genes to obtain relative expression values ​​for the 12 heat-stress-responsive genes for each rice sample.

[0087] Example 3: Transcriptome Sequencing

[0088] (1) Comparison statistics

[0089] Clean reads were aligned to the Nipponbare rice (Oryza sativa L. japonica. cv. Nipponbare) reference genome and gene sequence using SOAPaligner. The proportion of clean reads aligned to the reference genome and gene sequence was counted, and gene sequencing coverage was calculated as the ratio of the number of bases covered by clean reads that aligned only to unique positions in the reference sequence to the total number of bases in the gene coding region. Clean reads with up to five base mismatches were annotated.

[0090] (2) Gene expression normalization

[0091] The number of clean reads corresponding to each gene was counted, and the normalized gene expression level was calculated. The gene expression level was calculated using the RPKM (Reads Per Kb per Million reads) method, and the calculation formula is:

[0092]

[0093] Let RPKM(A) be the expression level of gene A, C be the number of reads uniquely aligned to gene A, N be the total number of reads uniquely aligned to the reference gene, and L be the number of bases in the coding region of gene A. If a gene has multiple transcripts, the longest transcript is used to calculate its sequencing coverage and expression level. The RPKM method eliminates the effects of gene length and sequencing load on gene expression, allowing the calculated gene expression values ​​to be used directly to compare expression differences between samples.

[0094] (3) Rice transcriptome sequencing results

[0095] In the five libraries, the raw reads obtained by sequencing were 25.16-26.53×10 6Clean reads were aligned to the reference genome and reference gene sequences, allowing for five base mismatches. The alignment results are shown in Table 4-1. Across the five libraries, 69.14% to 74.29% of reads mapped exclusively to unique positions on the reference genome, while 50.04% to 53.55% of reads mapped exclusively to unique positions on the reference gene. An average of 17.61% of reads did not map to the reference genome, and 16.26% did not map to the reference gene. For all II You 838 rice flag leaf libraries, over 56% of reads had no mismatches with the reference sequence, indicating high sequencing quality for each sample. Sequencing results have been uploaded to the NCBI database (https: / / www.ncbi.nlm.nih.gov / sra) under accession number SRP168528.

[0096] Table 4-11 Statistics of clean read alignments of You838 rice

[0097] Table 4-1Alignment statistics of the clean reads in the flag leaves of II YOU 838.

[0098]

[0099] Example 4: Digital Expression Sequencing (I) Alignment of Tag Sequences to Reference Genomes

[0100] Based on the Nipponbare rice (Oryza sativa L. japonica. cv. Nipponbare) reference gene database, all CATG sites on mRNA were collected to generate a reference tag database of CATG + 17 bases. All clean tags were aligned with the reference tag database, allowing for a maximum of one base mismatch. Only genes that matched uniquely to a single gene were annotated.

[0101] (2) Gene expression normalization

[0102] The number of Raw Tags, Clean Tags, Distinct Tags, Mapped Tags, Unknown Tags, and Tag Mapped Transcripts corresponding to each gene, as well as their respective proportions, were counted. The TPM (Transcripts per million clean tags) calculation method was used to normalize the clean tags and obtain the standardized gene expression level. The TPM value is the number of raw clean tags contained in each gene / (the total number of clean tags in the sample × 10 6 ).

[0103] (3) Rice digital gene expression profiling results

[0104] The sequencing capacity of 12 rice flag leaf DGE libraries exceeded 3M, and the sequencing results were 3.52-3.72×10 6 Raw Tag, with an average of more than 3.65×10 6 The number of original tags (Distinct RawTag) is 0.31~0.47×10 6 .

[0105] In the rice reference genome, 62,460 genes contain CATG sites, accounting for 94.15% of the total genes. All clean tags were aligned to a reference tag database of CATG+17nt generated for these genes, allowing for a maximum of one base mismatch. A total of 346,256 clean tags were aligned. Of these, 261,810 tags uniquely aligned to a single gene (unambiguous tags), accounting for 75.61% of the aligned clean tags. Across 12 rice flag leaf libraries, 34.32% to 40.86% of clean tags completely aligned to a single gene on the positive strand, with an average of 39.4%. The number of tags completely aligned to a single gene on the positive strand ranged from 16.52% to 29.68%. The number of tags completely aligned to a unique location in the reference genome reached 6.55% to 9.78%. The number of unmatched tags ranged from 6.09% to 9.71%, with an average of 7.6%. The number of unmatched tag species ranged from 10.02% to 17.55%, with an average of 14.454%. Tag alignment statistics indicate high sequencing quality for all samples. The sequencing results have been uploaded to the NCBI database (https: / / www.ncbi.nlm.nih.gov / sra) under accession number SRP168528.

[0106] Example 5: Mutual verification of fluorescent quantitative PCR results and sequencing results

[0107] The relative quantitative expression results of 12 heat stress response genes after correction and standardization by three internal reference genes were verified with the transcriptome sequencing results and DGE sequencing results.

[0108] (1) Fluorescence quantitative PCR results

[0109] (1) After total RNA was extracted from rice flag leaf samples, 2 μL of 5 samples were randomly selected for agarose gel electrophoresis. The electrophoresis results were as follows: Figure 1 : The specific bands of 28S and 18S rRNA in total RNA are neat and in proper proportion, without tailing or smearing. The OD of total RNA was measured on an ultra-micro spectrophotometer. 260 / OD 280 The ratios were all between 1.8 and 2.0. These results indicate that the total RNA in each sample was largely undegraded and of high purity, suitable for subsequent reverse transcription experiments. The total RNA concentration of each sample was adjusted to approximately 200 ng / μL using RNase-free ddH2O. 5 μL of total RNA from each sample was reverse-transcribed to generate first-strand cDNA, which served as a template for quantitative PCR.

[0110] (2) Optimization of fluorescence quantitative PCR reaction conditions and drawing of standard curve

[0111] Fluorescence quantitative PCR reaction conditions after optimization of each primer:

[0112] A. Pre-denaturation at 95°C for 3 min;

[0113] B. Cyclic amplification (40 cycles): denaturation at 95°C for 5 seconds;

[0114] Annealing and extension at 60°C for 30s, with fluorescence signal data collected after each cycle;

[0115] C. Draw the melting curve of the amplified product: change the temperature from 65°C to 95°C at a rate of 0.5°C per 5 seconds.

[0116] (3) The standard curve of each gene primer was drawn using cDNA as a template. The PCR product of each primer was diluted with ddH2O in a 10-fold concentration gradient. 10 -4 to 10 -11 The diluted samples were used as standard templates for real-time fluorescence quantitative PCR amplification and standard curves were drawn. The 10-fold concentration dilution gradient of the 8 templates can fully cover the concentration range of all samples. Each standard curve was drawn by repeating the experiment 3 times, and the standard error SD of the 3 results was <0.25, and the coefficient of variation CV was ≤15%, which met the experimental requirements. The standard curve results of each primer are shown in Table 3 below. The PCR amplification efficiency of all primers is between 95% and 105%, and the confidence level R2 >0.98, which is fully suitable for subsequent experiments.

[0117] Table 3 Standard curve results of real-time PCR for each primer

[0118]

[0119] (4) Expression stability of internal reference genes

[0120] The expression stability verification results of the five internal reference candidate genes in the four randomly selected cDNA templates are shown in Table 4 below. It was found that the M values ​​(normalized △Cq values) of actin, LOC_Os01g12800.1, and LOC_Os02g16709.1 were less than 0.5, indicating that the expression levels of actin, LOC_Os01g12800.1, and LOC_Os02g16709.1 in the flag leaf samples of the three rice varieties were basically stable under different degrees of heat stress and were not affected by heat stress. They can meet the requirements of being used as internal reference genes in the relative quantification of fluorescence quantitative PCR. Therefore, actin, LOC_Os01g12800.1, and LOC_Os02g16709.1 were selected as internal reference genes to calibrate the relative quantitative expression of the target genes. The M values ​​of 18s rRNA and Ubiquitin were high, exceeding 0.5. There were certain differences in the expression levels in the flag leaves of indica rice II You 838 and its parents under different heat stress levels, and they were not suitable as internal reference genes in the samples of this experiment.

[0121] Actin, LOC_Os01g12800.1, and LOC_Os02g16709.1 can be used as internal reference genes in fluorescence-based quantitative PCR relative quantification methods to jointly calibrate and normalize gene expression values ​​in flag leaf samples of the indica rice variety II You 838 and its parents. This fluorescence-based quantitative PCR method can quickly and accurately monitor the expression levels of target genes of interest (such as molecular markers for heat tolerance or heat damage response in rice) under heat stress. This fluorescence-based quantitative PCR method, using three internal reference genes for joint calibration, can be used as a rapid method for heat damage identification in rice or for molecular breeding of heat-tolerant rice. LOC_Os01g12800.1 and LOC_Os02g16709.1 are the first genes to be used as internal reference genes in indica rice under heat stress. This method can be extended to other indica rice varieties under heat stress, as well as to a wider range of heat-stressed rice varieties.

[0122] Table 4 Verification of expression stability of internal reference candidate genes

[0123]

[0124] (III) Mutual verification of real-time PCR results and sequencing results

[0125] (1) The cDNA of the flag leaf material of II You 838 rice without heat treatment on the 0th day was used as a control, and the experimental data were analyzed using Bio-Rad CFX Manager 3.0 software. The Pfaffl method was used to obtain the relative expression values ​​of the 12 target genes in each sample under the common calibration correction of the three internal reference genes actin, LOC_Os01g12800.1 and LOC_Os02g16709.1. The real-time PCR experiment used three biologically significant replicates, and the final result was the average of the three biological replicates. The transcriptome sequencing results of the same gene in each sample were RPKM values. The relative expression values ​​of the same gene in the samples of heat stress on the 1st day, heat stress on the 3rd day and heat stress on the 5th day were divided by the control (day 0) to obtain the expression difference fold value (fold change), and the log2FC value was calculated. The log2FC values ​​of the transcriptome sequencing method and the real-time PCR method were compared, and the results are shown in Table 5 below.

[0126] Table 5 Comparison of Real-time RT-PCR results and transcriptome sequencing results

[0127]

[0128] As shown in Table 5, with the exception of some discrepancies between the 0-day and 3-day results for LOC_Os01g36294.1, which were observed between the real-time PCR and transcriptome data, the expression trends of the remaining genes were consistent. The transcriptome sequencing and real-time PCR results for LOC_Os03g56540.3, LOC_Os05g44340.1, LOC_Os03g02260.1, LOC_Os07g48050.1, LOC_Os10g02920.1, LOC_Os03g63750.1, and LOC_Os05g45410.1 showed excellent concordance. The real-time PCR and transcriptome sequencing results for these genes used in the validation experiments were consistent in the vast majority of samples, demonstrating that the fluorescence quantitative PCR method using the three internal reference genes for calibration is accurate and reliable.

[0129] (2) The cDNA of the flag leaf samples of II You 838 rice and its parents on the 0th day without heat stress was used as a control, and the experimental data were analyzed with Bio-Rad CFX Manager 3.0 software. The Pfaffl algorithm was used to obtain the relative expression values ​​of the 9 target genes in each sample under the common calibration of the three internal reference genes actin, LOC_Os01g12800.1 and LOC_Os02g16709.1. The TPM value was used for the DGE sequencing results of the same gene in each corresponding sample. The real-time PCR relative expression values ​​of the samples of II You 838 and its parents on the 0th day, the 1st day, the 3rd day and the 5th day of heat stress were compared with the TPM value results of the DGE sequencing of the same samples of the same gene. The real-time PCR results and DGE sequencing results of the 9 randomly selected target genes were consistent (see Figures 2 to 10 ); the left vertical axis of each heat stress gene subgraph in the figure is the relative quantitative expression value of fluorescence quantitative PCR (expressed by the column in the figure), the right vertical axis is the TPM value of the DGE sequencing result (represented by the broken line in the figure), and the horizontal axis is the rice samples under four stress levels of three rice varieties. The figure compares and verifies the relative quantitative value of quantitative PCR and the TPM value. The trend of the broken line in each sample is consistent with the height change trend of the column, indicating that this fluorescence quantitative PCR method with three internal reference genes co-calibrated has been successfully established and can accurately detect and analyze the expression level of any rice target gene of interest, facilitating a rapid understanding of the expression changes of each gene in rice heat stress research.

[0130] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention's description and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention. Sequence Listing <110> Sichuan Institute of Atomic Energy <120> Internal reference genes for real-time fluorescence quantitative PCR analysis of gene expression under heat stress in indica rice and their construction method <160> 11 <170> PatentIn version 3.3 <210> 1 <211> 1134 <212> DNA <213> Indica ( Oryza sativa subsp. indica ) <400> 1 ATGGCTGACG CCGAGGATAT CCAGCCCCTC GTCTGCGATA ATGGAACTGG TATGGTCAAG 60 GCTGGGTTCG CCGGAGATGA TGCGCCCAGG GCTGTCTTCC CCAGCATTGT CGGCCGCCCT 120 CGCCACACCG GTGTCATGGT CGGAATGGGC CAGAAGGACG CCTACGTCGG CGACGAGGCG 180 CAGTCCAAGA GGGGTATCTT GACCCTCAAG TACCCCATCG AGCATGGTAT CGTCAGCAAC 240 TGGGATGATA TGGAGAAGAT CTGGCATCAC ACCTTCTACA ACGAGCTCCG TGTGGCCCCG 300 GAGGAGCACC CCGTCCTCCT CACCGAGGCT CCTCTCAACC CCAAGGCCAA TCGTGAGAAG 360 ATGACCCAGA TCATGTTTGA GACCTTCAAC ACCCCTGCTA TGTACGTCGC CATCCAGGCC 420 GTCCTCTCTC TGTATGCCAG TGGTCGTACC ACAGGTATTG TGTTGGACTC TGGTGATGGT 480 GTCAGCCACA CTGTCCCCAT CTATGAAGGA TATGCTCTCC CCCATGCTAT CCTTCGTCTC 540 GACCTTGCTG GGCGTGATCT CACTGATTAC CTCATGAAGA TCCTGACGGA GCGTGGTTAC 600 TCATTCACCA CAACGGCCGA GCGGGAAATT GTGAGGGACA TGAAGGAGAA GCTTTCCTAC 660 ATCGCCCTGG ACTATGACCA GGAAATGGAG ACTGCCAAGA CCAGCTCCTC CGTGGAGAAG 720 AGCTACGAGC TTCCTGATGG ACAGGTTATC ACCATTGGTG CTGAGCGTTT CCGCTGCCCT 780 GAGGTCCTCT TCCAGCCTTC CTTCATAGGA ATGGAAGCTG CGGGTATCCA TGAGACTACA 840 TACAACTCCA TCATGAAGTG CGACGTGGAT ATTAGGAAGG ATCTATATGG CAACATCGTT 900 CTCAGTGGTG GTACCACTAT GTTCCCTGGC ATTGCTGACA GGATGAGCAA GGAGATCACT 960 GCCTTGGCTC CTAGCAGCAT GAAGATCAAG GTGGTCGCCC CTCCTGAAAG GAAGTACAGT 1020 GTCTGGATTG GAGGATCCAT CTTGGCATCT CTCAGCACAT TCCAGCAGAT GTGGATTGCC 1,080 AAGGCTGAGT ACGACGAGTC TGGCCCATCC ATTGTGCACA GGAAATGCTT CTAA 1,134 <210> 2 <211> 669 <212> DNA <213> Indica rice ( Oryza sativa subsp. indica ) <400> 2 ATGCGGCGGC TATGGCGGTG GTACCAGCAG TGCCTGGCCA CCCACCCCGT GCGCACGCAG 60 GTGGTCAGCT CCGGCATCCT CTGGGGCCTC GGCGACATCG GCGCCCAGGC CGTCACCCAC 120 TACTCCGCCC CCGGACGCCC CCGCCACCAC CAGCACCACG CCAAGAATCC TCCCGAGGAT 180 AAAGATAAAG AGTTCAAAAT TGATTGGAAG AGGGTGGGCA TCACAAGCTC ATTTGGATTT 240 GCTTTTGTTG GACCAGTTGG ACATTACTGG TATGAATACT TGGATCGCTT CATCCTGAGG 300 AGATACCAGC CTAAGACCTT CAAATTTGTT GCGTCAAAAG TTGCTGCGGA TGGTCTCCTA 360 TTTGGACCAG TAGATCTTCT CTTGTTCTTC TCATATGTTG GTCTTGCATC AGGAAGGAGT 420 GTAGAGCAGG TGAAGGATGA TGTGAAGAGG GACTTCATTC CTGCTCTAGT TCTAGGGGGA 480 ACCATCTGGC CAGCCGTGCA AATCGCAAAT TTCCGCTTCA TTCCTGTGCG ATATCAGCTC 540 CTTTACGTGA ACCTGTTCTG CCTCTTAGAC AGTTGCTTCT TGTCGTGGAT CGATCAACAA 600 GGAGATGCAC CTTGGAAGCA ATGGTTCACA TCATTCCAGA AAATCGAAGG CCAGAAGGGC 660 AAGGTTTGA 669 <210> 3 <211> 897 <212> DNA <213> Oryza sativa ( Oryza sativa subsp. indica ) <400> 3 ATGTCCTCCA TCCACTTCTT CCCGGCCACC TCCCAGGCCG CCGCCCCGTC GCGCCGCCCC 60 CTCACCAAAC CCTCCCCAAA ACCCCCCGCC GCCACCGCCG CGTACCACCG CGGCGGAGAC 120 AACGAGCCGG TGCTCTTCCC GCGCCTCTTC CTCAGGAGGC GTGCCCGGCC CGCCGCGGCG 180 CCGGCCGAGG CGCCGCCGGT GGGCCCCGAC GGCAGCAGCA GCGGCGGCGG GGGAGGAGGA 240 GGGGAGGGGG GCGGGGGCGG AGGGGACGAC GAGGGATG AGGGACGAG GAAGGGGCTG 300 CTGCCGGAGT GGCTCAGCGT CACGACGGAC GACGCCAAGA CGGTGCTGGC CGCCATCGCC 360 ATCTCGCTCG CCTTCCGCTC CTTCGTCGCC GAGCCGCGCT TCATCCCCTC GCTCTCCATG 420 TTCCCCACCT TCGACGTCGG CGACCGCATT GTCGCCGAGA AGGTCATA CTACTTTAGA 480 AAGCCATGTA TAAATGACAT AGTAATCTTC AAAAGTCCAC CAGTGCTGCA GGAGGTCGGT 540 TATACTGACA ATGATGTTTT CATCAAACGA ATTGTTGCCA GGGAGGGAGA TGTTGTTGAG GTTCATAAAG GAAAACTAGT TGTCAATGGT GAAGTTAGAA ATGAAGAGTT CATTCTTGAG CCACCTTCTT ATGACATGAA CCCAGTGCAA GTACCTGAGA ACTCAGTTTT TGTAATGGGA 720 GATAACCGAA ACAACAGTTA TGATTCACAT GTGTGGGGCC CTCTTCCTTC TAGACATA 780 TTGGGGAGAT CCATTTTCCG GTATTGGCCT CCTGGCCGAA TAGGGCAC TACTACTGAT 840 TGCCTCATCC CTGAGACAAA TCCAAGCTCG CTTATTGATG TCAAACTGGC AAAGTAG 897 <210> 4 <211> 465 <212> DNA <213> Oryza sativa indica ( Oryza sativa subsp. indica ) <400> 4 ATGCAGATCT TCGTGAAGAC CCTGACGGGG AAGACCATCA CGCTGGAGGT GGAGAGCAGC 60 GACACGATCC AGAACGTCAA GGCGAAGGTG CAGGACAAGG AGGGGATCCC GCCGGACCAG 120 CAGCGGCTCA TCTTCGCCGG CAAGCAGCTG GAGGACGGGC GCACGCTGGC CGACTACAAC 180 ATCCAGAAGG AGTCGACGCT GCACCTGGTG CTCCGCCTCC GCGGCGGCCT GAACGTCAAG 240 GTGAGGACGC TCACCGGCAA GGAGATCGAC ATCGACATCG AGATGACGGA CACGGTGGAC 300 AGGATCAAGG AGCGCGTCGA GGAGAGGGAG GGCATCCCGC CCGTGCAGCA GAGGCTCATC 360 TACGGCGGGA AGCAGCTTGC CGACGACAAG ACGGCGCACG ACTACAAGAT CGAGGCTGGC 420 TCCGTCCTCC ACCTCGTCCT CGCTCTCCGA GGCGGGAACT TCTGA 465 <210> 5 <211> 519 <212> DNA <213> Oryza sativa indica ( Oryza sativa [[ID=)40]]subsp. indica ) <400> 5 It should be noted that there seems to be a small error in the original text where "subsp." is split in an unusual way in the translation process above. It should be a continuous "subsp." in the original text. The correct translation should be: TGCCTCATCC CTGAGACAAA TCCAAGCTCG CTTATTGATG TCAAACTGGC AAAGTAG 897 <210> 4 <211> 465 <212> DNA <213> Oryza sativa indica ( Oryza sativa subsp. indica ) <400> 4 ATGCAGATCT TCGTGAAGAC CCTGACGGGG AAGACCATCA CGCTGGAGGT GGAGAGCAGC 60 GACACGATCC AGAACGTCAA GGCGAAGGTG CAGGACAAGG AGGGGATCCC GCCGGACCAG 120 CAGCGGCTCA TCTTCGCCGG CAAGCAGCTG GAGGACGGGC GCACGCTGGC CGACTACAAC 180 ATCCAGAAGG AGTCGACGCT GCACCTGGTG CTCCGCCTCC GCGGCGGCCT GAACGTCAAG 240 GTGAGGACGC TCACCGGCAA GGAGATCGAC ATCGACATCG AGATGACGGA CACGGTGGAC 300 AGGATCAAGG AGCGCGTCGA GGAGAGGGAG GGCATCCCGC CCGTGCAGCA GAGGCTCATC 360 TACGGCGGGA AGCAGCTTGC CGACGACAAG ACGGCGCACG ACTACAAGAT CGAGGCTGGC 420 TCCGTCCTCC ACCTCGTCCT CGCTCTCCGA GGCGGGAACT TCTGA 465 <210> 5 <211> 519 <212> DNA <213> Oryza sativa indica ( Oryza sativa subsp. indica ) <400> 5 ATGCTCGCAG GACCCCCGCC CTCGTGCGGC CGACTGCCGG CTCCCGGGCC CCGTCACTCC 60 ACGGCCGTCC ACGCGCCGTG CCGCCCCAGG CTTCAAGAGA TGCTTGCGCG CTGCTACCCG 120 TCCCACGGGC AGAGGTGCTC GCACACGTCC GCCGCGCCGC GGGCGCCCCA CCGGGCGTCC 180 CGCGGCGGCT CGACGGCGCG AGCGGCGTGG CCTCGCGGCG CCCGGCACCC AAGCGTGCCG 240 GCGCTGCCAA GGCCACCTCG CGCGTGCCAT TGGTCCCGGA TGCCGCCCAC GATACAGGCT 300 CACGGCGGCC CCGCCCCGTG CCTACCCATA AGCGAGATGC TCTCGGAAGA CGACAGCCCG 360 CCCGGCCGCC GCCGTGTCCG CCGCTCCCGA CCCGGGGGCG GCGGCGACGC GCGTCGGACG 420 GCGCGGGCTC GTCGCGGAGG ACGTGCTACC TGGTTGATCC TGCCAGTAGT CATATGCTTG 480 TCTCAAAGAT TAAGCCATGC ATGTGCAAGT ATGAACTAA 519 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <400> 6 CAGCCACACT GTCCCCATCT A 21 <210> 7 <211> 20 <212> DNA<​​​ AGCAAGGTCG AGACGAAGGA 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <400> 8 GAAGAGGGTG GGCATCACAA 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <400> 9 AGACCATCCG CAGCAACTTT 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <400> 10 TTGTCGCCGA GAAGGTTACA 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <400> 11 GCAGCACTGG TGGACTTTTG 20

Claims

1. An application of three genes, LOC_Os03g50885.1, LOC_Os01g12800.1, and LOC_Os02g16709.1, as internal reference genes for real-time fluorescence quantitative PCR analysis of gene expression under heat stress in indica rice, wherein the nucleotide sequences thereof are shown in SEQ ID NOs. 1-3, respectively.

2. The use of the three genes according to claim 1 as internal reference genes for real-time fluorescence quantitative PCR analysis of gene expression under heat stress in indica rice, characterized in that: The forward primer sequence used to amplify LOC_Os03g50885.1 is shown in SEQ ID NO.6 in the sequence listing, and the reverse primer sequence is shown in SEQ ID NO.7 in the sequence listing; the forward primer sequence used to amplify LOC_Os01g12800.1 is shown in SEQ ID NO.8 in the sequence listing, and the reverse primer sequence is shown in SEQ ID NO.9 in the sequence listing; the forward primer sequence used to amplify LOC_Os02g16709.1 is shown in SEQ ID NO.10 in the sequence listing, and the reverse primer sequence is shown in SEQ ID NO.11 in the sequence listing.

3. A method for constructing an internal reference gene for real-time fluorescence quantitative PCR analysis of gene expression under heat stress in indica rice, characterized by: The steps include: (1) After the male parent Fuhui 838, the female parent II-32A and the offspring II You 838 rice were subjected to high temperature stress, the flag leaves of II You 838 at the flowering stage were subjected to high-throughput transcriptome sequencing, and the flag leaves of II You 838 and its parents at the flowering stage were subjected to digital expression profile sequencing. Two internal reference genes with stable expression were selected from the 5 transcriptome sequencing results and 12 DGE sequencing results. Their nucleotide sequences are shown in the sequence listing SEQ ID NO.2 and SEQ ID NO.

3. At the same time, three traditional internal reference genes commonly used in the genus Oryza were selected as a comparative reference group. Their nucleotide sequences are shown in the sequence listing SEQ ID NO.1, SEQ ID NO.4 and SEQ ID NO.5, for a total of 5 candidate internal reference genes; (2) Primers were designed for the five candidate internal reference genes, and fluorescent quantitative PCR reactions were performed on each rice cDNA generated by reverse transcription. The obtained data were evaluated and analyzed for the stability of the internal reference genes using Bio-Rad CFX Manager 3.0 software. The optimal internal reference gene was screened by the M values of the five candidate internal reference genes. The smaller the difference in expression values, the more stable the gene expression. Finally, the most suitable internal reference genes and the number of internal reference genes under high temperature stress treatment were screened; (3) The heat stress response genes in rice were used for verification. Fluorescence quantitative PCR primers for each heat stress response gene were designed, and the rice cDNA template generated by reverse transcription was subjected to fluorescence quantitative PCR reaction. The data were analyzed using Bio-Rad CFXManager3.0 software. The Pfaffl algorithm was used to obtain the relative quantitative expression value of the heat stress response gene of each rice sample under the joint calibration of the three internal reference genes LOC_Os03g50885.1, LOC_Os01g12800.1 and LOC_Os02g16709.

1. The relative quantitative expression value was compared with the RPKM value and TPM value of the high-throughput sequencing results for verification.

4. The method for constructing an internal reference gene for real-time fluorescence quantitative PCR for analyzing gene expression under heat stress in indica rice according to claim 3, characterized in that: The fluorescent quantitative PCR reaction uses the first strand of cDNA generated by reverse transcription of each sample as a template. The fluorescent quantitative PCR reaction system is 10 μL. The reaction solution is prepared on ice. The volume of each reagent is: 5 μL Premix Ex TaqII, 2μL cDNA template, 0.5μL PCR Forward Primer 10μM, 0.5μL PCR Reverse Primer 10μM, and finally add 2μL ddH2O to a volume of 10μL. Repeat the experiment 3 times.

5. The method for constructing an internal reference gene for real-time fluorescence quantitative PCR for analyzing gene expression under heat stress in indica rice according to claim 3 or 4, characterized in that: The fluorescent quantitative PCR reaction conditions are: A. Pre-denaturation at 95°C for 3 min; B. 40 cycles of amplification: denaturation at 95°C for 5 seconds; Annealing and extension at 60°C for 30s, with fluorescence signal data collected after each cycle; C. Draw the melting curve of the amplified product: change the temperature from 65°C to 95°C at a rate of 0.5°C per 5 seconds.

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