Internal reference gene combination for real-time fluorescent quantitative PCR (Polymerase Chain Reaction) of indocalamus as well as primer pair combination and application of internal reference gene combination

Abstract

The invention provides a reference gene combination for real-time fluorescent quantitative PCR (Polymerase Chain Reaction) of indocalamus as well as a primer pair combination and application of the reference gene combination, and belongs to the technical field of plant molecular biology. In combination with indocalamus transcriptome data analysis and cross validation of four algorithms, reference genes, MD10B, PP2A, eIF1A, Ite23725, eIF4A, 60S and UBP1, suitable for indocalamus are systematically screened out, optimal reference gene combinations under different experimental conditions (drought, salt, waterlogging stress and different tissues) are refined, and the method is scientific and rigorous and has good application prospects. And a precise tool is provided for gene expression analysis of the indocalamus in different research directions. The screened reference genes show extremely high expression stability in specific stress and tissues after being combined, and the expression stability is far better than that of traditional housekeeping genes such as Actin and UBI. The specific primer has high amplification efficiency, and ensures the accuracy of qRT-PCR results.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular biology, specifically relating to a set of internal reference gene combinations and primer pair combinations and applications for real-time fluorescence quantitative PCR of *Isodon japonicus*. Background Technology

[0002] Bamboo species, particularly those in the genus *Indocalamus*, are bamboo resources of significant ecological and economic value, exhibiting remarkable potential in stress resistance. Real-time quantitative PCR (qRT-PCR) is a crucial technique for studying gene expression patterns, and the accuracy of its results highly depends on the use of stably expressed reference genes for data normalization. Ideally, reference genes should be consistently expressed under various experimental conditions. However, numerous studies have shown that the expression stability of commonly used reference genes (such as Actin, Tubulin, and UBI) varies depending on species, tissue type, and treatment conditions. Using unstable reference genes can lead to biases or even errors in the analysis of target gene expression levels. Currently, for *Indocalamus*, an important bamboo species, there is a lack of research on systematically screening and validating reference genes applicable to various abiotic stresses (such as drought, salinity, and waterlogging) and different tissues across the entire genome. This gap severely restricts the in-depth development of molecular biology research on *Indocalamus*, especially in the analysis of stress resistance mechanisms and the identification of functional genes. Therefore, systematically screening and validating stable reference genes suitable for specific experimental conditions of *Indocalamus* has become an urgent technical problem to be solved in this field. Summary of the Invention

[0003] The purpose of this invention is to provide a set of methods suitable for bamboo (Indocalamus latae). Indocalamus tessellatus Stable internal reference gene combinations, specific primers, screening methods and applications for real-time quantitative PCR (qRT-PCR) analysis under different abiotic stresses and in different tissues.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a set of internal reference gene combinations for real-time quantitative PCR of *Imperata cylindrica*, selected from one or more of (a) to (g): (a) MD10B Genes or genes that have more than 95% similarity to their cDNA sequences and have the same function; (b) PP2A Genes or genes that have more than 95% similarity to their cDNA sequences and have the same function; (c) eIF1A Genes or genes that have more than 95% similarity to their cDNA sequences and have the same function; (d) Ite23725 Genes or genes that have more than 95% similarity to their cDNA sequences and have the same function; (e) eIF4A Genes or genes that have more than 95% similarity to their cDNA sequences and have the same function; (f) 60S Genes or genes that have more than 95% similarity to their cDNA sequences and have the same function; (g) UBP1 Genes or genes that have more than 95% similarity to their cDNA sequences and have the same function; The gene combination is derived from plants of the genus *Indocalamus*.

[0005] Preferably, the MD10B The cDNA sequence of the gene is shown in SEQ ID NO.1; The PP2A The cDNA sequence of the gene is shown in SEQ ID NO.2; The eIF1A The cDNA sequence of the gene is shown in SEQ ID NO.3; The Ite23725 The cDNA sequence of the gene is shown in SEQ ID NO.4; The eIF4A The cDNA sequence of the gene is shown in SEQ ID NO.5; The 60S The cDNA sequence of the gene is shown in SEQ ID NO. 6; The UBP1 The cDNA sequence of the gene is shown in SEQ ID NO.7.

[0006] The present invention also provides a primer pair combination for amplifying the above-mentioned internal reference gene, selected from one or more of (1) to (7); (1) Used to amplify the above MD10B The primer pairs for the gene are shown in SEQ ID NO.75 and SEQ ID NO.76; (2) Used to amplify the above PP2A The primer pairs for the gene are shown in SEQ ID NO.51 and SEQ ID NO.52; (3) Used to amplify the above eIF1A The primer pairs for the gene are shown in SEQ ID NO.47 and SEQ ID NO.48; (4) Used to amplify the above Ite23725 The primer pairs for the gene are shown in SEQ ID NO.61 and SEQ ID NO.62; (5) Used to amplify the above eIF4AThe primer pairs for the gene are shown in SEQ ID NO.49 and SEQ ID NO.50; (6) Used to amplify the above 60S The primer pairs for the gene are shown in SEQ ID NO.55 and SEQ ID NO.56; (7) Used to amplify the above UBP1 The primer pairs for the gene are shown in SEQ ID NO.65 and SEQ ID NO.66.

[0007] The present invention also provides a detection reagent for analyzing real-time quantitative PCR of *Imperata cylindrica* in different adverse environments or different tissues, including the above-mentioned internal reference gene combination or primer pair combination.

[0008] This invention provides the application of the above-mentioned internal reference gene combination, primer pair combination, or the detection reagent in analyzing the expression of functional genes of *Ipomoea aquatica* in adverse environments or different tissues.

[0009] Preferably, the adverse environment includes drought stress, salt stress, or waterlogging stress; the analytical method is real-time quantitative PCR analysis.

[0010] Preferably, when the adverse environment is drought stress, the internal reference gene combination is a combination of items (a) and (b), and the primer pair combination is a combination of items (1) and (2); Or, when the adverse environment is salt stress, the internal reference gene combination is a combination of items (c) and (d), and the primer pair combination is a combination of items (3) and (4); Or, when the adverse environment is waterlogging stress, the internal reference gene combination is a combination of items (b) and (e), and the primer pair combination is a combination of items (2) and (5).

[0011] The internal reference genes used in the gene expression analysis between different tissues were a combination of items (f) and (g), and the primer pair combination was a combination of items (6) and (7).

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Systematic: This invention is the first to combine whole transcriptome data analysis of bamboo and cross-validation of four algorithms: geNorm, NormFinder, BestKeeper and RefFinder, to systematically screen out internal reference genes suitable for bamboo. The method is scientific and rigorous.

[0013] (2) High stability and specificity: The selected internal reference genes (such as MD10B, PP2A, etc.) showed extremely high expression stability under specific stress and tissues, which was far superior to traditional housekeeping genes such as Actin and UBI. Their specific primers had high amplification efficiency, ensuring the accuracy of qRT-PCR results.

[0014] (3) Targeted: This invention refines the optimal combination of internal reference genes under different experimental conditions (drought, salinity, waterlogging stress and different tissues), providing a precise tool for gene expression analysis of *Isodon japonica* in different research directions.

[0015] (4) Practicality: This invention provides a rigorously verified standardized reference for molecular biology research on bamboo, especially in the fields of stress resistance mechanism analysis, functional gene identification and molecular breeding, and has significant application value. Attached Figure Description

[0016] Figure 1 The results of RT-PCR for candidate internal reference genes of *Isodon japonicus* are shown (A represents twenty candidate reference genes, and B represents known stress genes).

[0017] Figure 2 Melting curves of ten candidate internal reference genes.

[0018] Figure 3 Melting curves for ten other candidate internal controls and known stress genes.

[0019] Figure 4 To evaluate the expression stability of twenty candidate reference genes in different stress conditions (A: drought stress; B: salinity stress; C: flooding stress) and different tissues (D) for GeNorm.

[0020] Figure 5 Pairwise variation (V) analysis of 20 reference genes in different tissues.

[0021] Figure 6 For stability evaluation based on NormFinder.

[0022] Figure 7 When using different candidate reference genes in different organs (D) of *Imperata cylindrica* under drought (A), salinity (B), and waterlogging (C) stresses, ItPOD The relative expression level. Detailed Implementation

[0023] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0024] Example 1

[0025] 1. Plant material and stress treatment

[0026] Healthy, vigorously growing bamboo (Inula japonica) Indocalamus tessellatusThe seedlings were collected in March 2024 from Suichang County, Zhejiang Province, China. To ensure genetic consistency, all experimental seedlings were derived from the same mother bamboo rhizome. Healthy, uniformly growing bamboo culms were selected, divided, and transplanted to the greenhouse of Lishui University (119.91°E, 28.47°N) for cultivation. Greenhouse conditions were maintained at a relative humidity of 70±10%, natural light cycle, and an average temperature of 23±1.0℃. Regular watering and fertilization were performed to ensure optimal plant growth. After two months of acclimatization, vigorous individuals were selected for subsequent experiments.

[0027] Drought, salinity, and waterlogging were selected as representative stress conditions. All seedlings were de-watered for two days prior to stress treatment to allow for physiological uniformity.

[0028] Drought stress: simulated by immersing the seedling roots in a 25% PEG 6000 solution.

[0029] Salt stress: simulated by watering with 200 mM NaCl solution.

[0030] Waterlogging stress: Completely immerse the flowerpot in water, ensuring the water level is 5 cm above the soil surface.

[0031] Seedlings with normal irrigation served as a control. Second fully expanded leaves were collected at 0, 5, 12, 24, and 48 hours after the onset of stress. Samples used for organ-specific analysis (including leaves, leaf sheaths, stems, roots, and young shoots) were collected from the same plant. All samples were flash-frozen in liquid nitrogen immediately after collection and stored at -80°C for later use. Each treatment and time point included at least three biological replicates.

[0032] 2. Total RNA extraction and cDNA reverse transcription

[0033] A small amount of plant tissue was thoroughly ground into a fine powder in liquid nitrogen using an RNase-free mortar and pestle. Total RNA was extracted from each sample using the PlantRNA Kit (Omega Bio-tek, USA) according to the manufacturer's instructions. RNA integrity was assessed by 1% agarose gel electrophoresis; samples showing clear 28S and 18S rRNA bands without visible degradation or contamination were considered high-quality RNA. RNA concentration and purity were measured using a NanoDrop micro-spectrophotometer (Thermo Fisher Scientific, USA). Only RNA with an A260 / 280 ratio between 1.8 and 2.2 and an A260 / 230 ratio greater than 2.0 was used for subsequent experiments.

[0034] First-strand cDNA synthesis was performed using the PrimeScript™ FAST RT Reagent Kit with gDNA Eraser (Takara, Japan). 1 μg of total RNA was used per reaction, and genomic DNA was effectively removed according to the manufacturer's protocol. The resulting cDNA was appropriately diluted for subsequent analysis and stored at -20°C until use.

[0035] 3. Identification of candidate internal reference genes and primer design

[0036] Based on unpublished RNA-Seq data from six tissues (roots, stems, mature leaves, young leaves, leaf sheaths, and tender branches) of *Isodon japonicus* in our laboratory, the expression levels of all genes were normalized using TPM. The mean, standard deviation (SD), and coefficient of variation (CV) of the log2 (TPM) values ​​of genes in all samples were calculated. The screening criteria were set as mean log2 (TPM) ≥ 5, SD < 1, and CV ≤ 0.2. A candidate gene pool of 3801 genes with high and stable expression levels in all tissues was selected from the transcriptome.

[0037] Eleven novel genes with the lowest expression variation were selected from these. BS , Ite23725, ARF, UBP1, PEX13, SKA, PNN, RPN8, MD10B, SUGP1, HNRPQ ), and combined with 9 commonly used traditional housekeeper genes ( Actin 7, Tubulin, UBI, eIF1A, eIF4A, PP2A, SAMDC, 60S, CYP Together, they form a candidate internal reference gene set containing 20 genes, as shown in Table 1.

[0038] Table 1 Candidate Genes

[0039] qRT-PCR primers for each candidate internal reference gene were designed using NCBI Primer-BLAST (Table 2), with the following parameters: primer length 18-22 bp, GC content 40-60%, annealing temperature 58-62℃, and amplicon size 80-250 bp.

[0040] 4. Primer specificity verification and qRT-PCR analysis

[0041] Primer specificity was verified by conventional PCR, and the amplified products were analyzed by 2% agarose gel electrophoresis, confirming that they were all single, clear bands of the expected size.

[0042] RT-qPCR analysis was performed using TB Green® Premix Ex Taq™ II (Takara, Japan) on a LightCycler® 96 real-time quantitative PCR system (Roche, Switzerland). Each 20 μL reaction volume contained: 0.6 μL upstream primer, 0.6 μL downstream primer, 10 μL 2× TB Green Premix Ex Taq II, 7.8 μL nuclease-free water, and 1 μL diluted cDNA template. The amplification program was: 95°C pre-denaturation for 30 seconds; followed by 40 cycles of 95°C denaturation for 5 seconds, 60°C annealing / extension for 30 seconds. After amplification, melting curve analysis was performed: 95°C for 1 second, 65°C for 15 seconds, then slowly ramped up to 95°C while continuously monitoring fluorescence. Three technical replicates were set up for each reaction to ensure reliability.

[0043] Before the formal analysis, a standard curve was prepared using serially diluted mixed cDNA templates (undiluted, 1:5, 1:25, 1:125, 1:625) to calculate amplification efficiency (E) and correlation coefficient (R²).

[0044] Table 2 shows that the E values ​​of all candidate genes were between 90% and 105%, and the R... 2 Values ​​between 0.979 and 0.999 indicate high amplification efficiency and good linearity. Melting curve analysis showed that each gene exhibited a single peak within the 80-90℃ range, further confirming the primer specificity.

[0045] Table 2. Primer information for candidate internal reference genes of *Isodon japonica*.

[0046] 5. Results and Analysis

[0047] Specificity analysis of candidate internal control gene primers: The specificity of candidate internal control gene primers was analyzed using PCR technology. Electrophoresis results showed that the amplification products of each primer were single bands of the same size as the expected bands. Figure 1 Meanwhile, qRT-PCR was performed using *Ipomoea aquatica* leaf cDNA as a template. The results showed that the melting curves of all candidate internal reference genes exhibited a single peak. Figure 2 and Figure 3 This indicates that the primers used have good specificity and can be used for subsequent experiments.

[0048] Candidate internal reference gene expression abundance analysis: For each gene, we performed a log2 transformation on the TPM values ​​of all samples. After calculating the average log2 (TPM) value, genes with an average log2 (TPM) value below 5 were removed to exclude low-abundance transcripts that might not be reliably detected or quantified by RT-qPCR. For the remaining genes, the mean, standard deviation (SD), and coefficient of variation (CV) of the log2 (TPM) values ​​were calculated using R (version 4.3.2), where CV was defined as SD divided by the mean. Candidate reference genes must meet the following criteria: standard deviation less than 1 and coefficient of variation ≤ 0.2, indicating relatively stable expression levels.

[0049] Evaluation of expression stability of candidate internal reference genes

[0050] Ct values ​​(cycle thresholds) of 20 candidate internal reference genes were obtained from all samples. The expression stability of each candidate gene under drought, salinity, and waterlogging stresses and in different tissues was evaluated using three algorithms: geNorm, BestKeeper, and Normfinder.

[0051] geNorm analysis: This study used the geNorm algorithm to assess the expression stability of candidate reference genes based on the M-value. Figure 4 The lower the M value, the higher the expression stability. An M value below 1.5 is generally considered a reference gene selection criterion. Under drought stress conditions ( Figure 4 A) down, PP2A and PEX13 It exhibits the highest stability (M=0.15), while UBI has the lowest stability (M=0.93). Salt stress ( Figure 4 B) In the sample, MD10B and Ite23725 It exhibits the best stability (M=0.22). BS The most unstable (M=0.78) is the one experiencing flood stress. Figure 4 During period C), eIF4A and PP2A (M=0.29) has the highest stability, while Ite23725 (M=0.83) and MD10B (M=0.79) showed poor stability. Inter-tissue analysis showed ( Figure 4 D), eIF4A and 60s (M=0.38) is the most stable gene, while Actin7 The expression variability was the greatest (M=1.14).

[0052] We used the geNorm software to calculate the pairwise variance (Vn / n+1) Figure 5A universal threshold of 0.15 was used to assess whether adding a reference gene significantly improved normalization accuracy. Under drought, salt, waterlogging, and different organ conditions, the V² / 3 values ​​were all significantly lower than 0.15. This indicates that accurate qRT-PCR normalization can be achieved using only two reference genes under all test conditions. Figure 5 ).

[0053] BestKeeper analysis: By comparing standard deviation (SD) and coefficient of variation (CV) values, the expression stability of candidate reference genes under different experimental conditions was evaluated. Genes with lower standard deviation and coefficient of variation values ​​showed higher stability.

[0054] Table 3. Ranking of candidate reference genes for *Ipomoea aquatica* under different stresses and in different organs based on BestKeeper.

[0055] As shown in Table 3, under drought stress, MD10B It was identified as the most stable reference gene, followed by PP2A and eIF4A Under salt stress, PP2A It exhibits the highest stability. PEX13 and RPN8 Following closely behind. Under flood stress conditions, eIF4A It demonstrates the most stable level of expression, and PP2A CYP also remained highly stable. Analysis results from different organs showed that... PNN eIF1A and eIF1A rank among the top two most stable reference genes. In contrast, UBI The gene consistently exhibited extremely high standard deviations (SD) and coefficients of variation (CV), showing the worst stability in drought, salt, and waterlogging stress tests. Comparisons between different organs showed that... Actin7 It had the highest SD and CV values, indicating that it was the least stable as a reference gene. The results show that, under specific experimental conditions, MD10B , PP2A, eIF4A and PNN It is a suitable reference gene for qRT-PCR standardization, and UBI and Actin7 Due to its poor stability, it should be avoided.

[0056] Normfinder analysis: NormFinder was used to assess the expression stability of candidate reference genes under different experimental conditions. This method suggests that genes with lower stability values ​​have more consistent expression. Figure 6 As shown, under drought stress, PP2A and MD10B It exhibited the lowest stability value and was therefore identified as the most stable reference gene. Under salt stress, SAMDC and eIF1A It exhibits the highest stability. Similarly, under flood stress, PP2A and Tubulin It once again ranks among the most stable genes. When analyzing gene stability in different tissues... UBP1 and 60S It consistently performs best. In contrast, UBI is unstable under most conditions. It is worth noting that... PP2A It exhibits stable expression under almost all conditions, supporting its applicability as a standardized reference gene in a variety of experimental conditions.

[0057] 6. Overall Stability Ranking

[0058] To obtain a more comprehensive and objective stability assessment, the stability rankings obtained from the three algorithms (geNorm, NormFinder, and BestKeeper) were integrated using the RefFinder online tool. RefFinder generated a comprehensive stability ranking for each candidate gene by calculating the geometric mean.

[0059] Table 4. Ranking of candidate gene stability assessed using RefFinder software

[0060] 7. Stability assessment results

[0061] The comprehensive analysis results of the three algorithms and RefFinder are summarized in Table 5.

[0062] Table 5. Reference genes most stable under different stresses and organs identified by four assessment methods.

[0063] The overall results indicate that the most stable combination of internal reference genes under different experimental conditions is as follows: Drought stress: The most stable internal reference gene is MD10B and PP2A .

[0064] Salt stress: The most stable internal reference gene is eIF1A and Ite23725 .

[0065] Flood stress: The most stable internal reference gene is PP2A and eIF4A .

[0066] Different tissues: The most stable internal reference gene is 60S and UBP1 .

[0067] 8. Internal reference gene verification

[0068] To verify the reliability of the selected internal reference gene, a known stress response gene—the peroxidase gene—was chosen. ItPOD The gene fragment sequence used for qRT-PCR (as shown in SEQ ID NO. 83) was analyzed as the target gene. The expression levels of ItPOD were normalized using the two most stable genes (or their combination) and the least stable gene from the RefFinder ranking under each condition. Figure 7 ).

[0069] The results showed that using stable internal reference genes, such as drought ( Figure 7 A) MD10B and PP2A Salt stress ( Figure 7 B) eIF1A and Ite23725 Flooding Figure 7 C) PP2A and eIF4A Different organizations ( Figure 7 D) 60S and UBP1, When performing normalization, ItPOD The expression patterns showed consistent and biologically expected trends across different stress time points or tissues. Figure 7 Conversely, using unstable internal reference genes (such as...) UBI, Actin7 or BS When normalizing, it will lead to ItPOD Expression levels were significantly overestimated or underestimated, and in some cases, their expression patterns were even altered (e.g., tissue-specific expression trends were reversed). This fully demonstrates that selecting the stable internal reference gene combination determined in this invention is crucial for obtaining accurate and reliable gene expression analysis results.

[0070] Through the aforementioned systematic screening and validation process, this invention has identified specific and stable internal reference gene combinations suitable for real-time quantitative PCR analysis of *Imperata cylindrica* under various abiotic stresses (drought, salinity, and waterlogging) and in different tissues. These internal reference gene combinations provide a reliable standardized tool for subsequent functional gene research, stress response mechanism analysis, and molecular breeding of *Imperata cylindrica*.

[0071] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A combination of reference genes for real-time fluorescent quantitative PCR of Oryza malampuzhaensis, characterized in that, The internal reference gene combination is selected from one or more of (a) to (g): (a) MD10B a gene or a gene having more than 95% similarity to its cDNA sequence and functional equivalence; (b) PP2A Genes or genes that have more than 95% similarity to their cDNA sequences and have the same function; (c) eIF1A Genes or genes that have more than 95% similarity to their cDNA sequences and have the same function; (d) Ite23725 Genes or genes that have more than 95% similarity to their cDNA sequences and have the same function; (e) eIF4A Genes or genes that have more than 95% similarity to their cDNA sequences and have the same function; (f) 60S Genes or genes that have more than 95% similarity to their cDNA sequences and have the same function; (g) UBP1 Genes or genes that have more than 95% similarity to their cDNA sequences and have the same function; The gene combination is derived from plants of the genus *Indocalamus*.

2. The gene combination as described in claim 1, characterized in that, The MD10B The cDNA sequence of the gene is shown in SEQ ID NO.1; The PP2A The cDNA sequence of the gene is shown in SEQ ID NO.2; The eIF1A The cDNA sequence of the gene is shown in SEQ ID NO.3; The Ite23725 The cDNA sequence of the gene is shown in SEQ ID NO.4; The eIF4A The cDNA sequence of the gene is shown in SEQ ID NO.5; The 60S The cDNA sequence of the gene is shown in SEQ ID NO. 6; The UBP1 The cDNA sequence of the gene is shown in SEQ ID NO.

7.

3. A primer pair combination for amplifying the internal reference gene combination of claim 1, characterized in that, The primer pair combination is selected from one or more of (1) to (7); (1) Used to amplify the above MD10B The primer pairs for the gene are shown in SEQ ID NO.75 and SEQ ID NO.76; (2) Used to amplify the above PP2A The primer pairs for the gene are shown in SEQ ID NO.51 and SEQ ID NO.52; (3) Used to amplify the above eIF1A The primer pairs for the gene are shown in SEQ ID NO.47 and SEQ ID NO.48; (4) Used to amplify the above Ite23725 The primer pairs for the gene are shown in SEQ ID NO.61 and SEQ ID NO.62; (5) Used to amplify the above eIF4A The primer pairs for the gene are shown in SEQ ID NO.49 and SEQ ID NO.50; (6) Used to amplify the above 60S The primer pairs for the gene are shown in SEQ ID NO.55 and SEQ ID NO.56; (7) Used to amplify the above UBP1 The primer pairs for the gene are shown in SEQ ID NO.65 and SEQ ID NO.

66.

4. A detection reagent for analyzing real-time quantitative PCR of *Imperata cylindrica* under different adverse environments or in different tissues, characterized in that, Includes the internal reference gene combination of claim 1 or the primer pair combination of claim 3.

5. The application of the internal reference gene combination of claim 1 or 2, the primer pair combination of claim 3, or the detection reagent of claim 4 in analyzing the expression of functional genes of *Ipomoea aquatica* in adverse environments or different tissues.

6. The application as described in claim 5, characterized in that, The adverse environments include drought stress, salt stress, or waterlogging stress; The analytical method used was real-time quantitative PCR.

7. The application as described in claim 5, characterized in that, When the adverse environment is drought stress, the internal reference gene combination is a combination of items (a) and (b), and the primer pair combination is a combination of items (1) and (2). Or, when the adverse environment is salt stress, the internal reference gene combination is a combination of items (c) and (d), and the primer pair combination is a combination of items (3) and (4); Or, when the adverse environment is waterlogging stress, the internal reference gene combination is a combination of items (b) and (e), and the primer pair combination is a combination of items (2) and (5).

8. The application as described in claim 5, characterized in that, The internal reference genes used in the gene expression analysis between different tissues were a combination of items (f) and (g), and the primer pair combination was a combination of items (6) and (7).

Images