Paeonia suffruticosa flowering phase regulation PCR detection reference gene, primer, screening method and application

Abstract

The invention discloses a peony flowering phase regulation PCR detection reference gene, a primer, a screening method and application, and belongs to the technical field of plant molecular biology, the reference gene is RPL17, and the nucleotide sequence is shown as Seq ID No. 25 in a sequence table. And specific primers for amplifying the reference gene RPL17 are shown as Seq ID No. 17 and Seq ID No. 18 in a sequence table. The gene can meet the requirements of qRT-PCR (quantitative reverse transcription-polymerase chain reaction) technology for detecting the expression level of related genes of white peony varieties in the peony development process and in different flowering periods, and the stability and the accuracy for detecting the expression of the related genes of the white peony varieties in the peony development process and in different flowering periods are improved; the difference between samples and the difference inside the samples caused by selection of the reference genes are reduced to the maximum extent, and a solid foundation is laid for later analysis of expression modes of key genes related to peony flowering phase regulation by using qRT-PCR (quantitative reverse transcription-polymerase chain reaction).

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant molecular biology, and particularly relates to a PCR detection reference gene for regulating the flowering period of peony, primers, a screening method and application. BACKGROUND

[0002] Peony (Paeonia suffruticosa Andrews.) is a perennial deciduous shrub of Sect. Moutan DC. of Paeonia L. of Paeoniaceae, and is a unique original biological species resource in China. The flowering period of peony is short and concentrated, and under natural conditions, it is only 50-60 days from germination to flower fading, 3-5 days for a single flower, and 10-15 days for a group of flowers. Moreover, most varieties are medium-flowering varieties, and the proportion of early and late flowering varieties is small. These parameters directly affect the ornamental value and economic value of peony.

[0003] Gene expression research is one of the important means to explore functional genes. Known housekeeping genes include tubulin, actin, 18S ribosomal RNA (18S rRNA), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). However, in recent years, many studies have found that these reported housekeeping genes are not stable, and these genes have differential expression in different treatments or different tissues, which makes the function of some housekeeping genes as internal reference genes questionable. Therefore, in order to eliminate the bias caused by different factors on the expression analysis results, it is necessary to select appropriate internal reference genes as a reference for the change of gene expression level. In the test, when testing the expression difference of a certain gene in different samples, the original concentration of each sample, the reverse transcription efficiency, the specificity of the designed primer, and the amplification efficiency should be consistent. However, in the actual operation process, these conditions cannot be met at the same time. The internal reference gene is relatively stable in the expression of each species tissue, and is mainly used to correct the errors that cannot be overcome by the operator when adding samples, so as to ensure the reliability of the test results. Therefore, it is necessary to select appropriate internal reference genes to correct errors and analyze the relative expression amount in the test.

[0004] An ideal internal reference gene should have consistent expression level and expression amount between different treatments and different tissues. However, due to the high genetic diversity between different peony varieties, the gene expression pattern and stability of the internal reference gene are also different. The applicable internal reference genes of different peony varieties are obviously different, and even the applicable internal reference genes of the same peony variety, different tissues, and different treatment conditions are different. Therefore, it is necessary to screen targeted internal reference genes in the study of peony flowering period regulation, especially to screen stable internal reference genes in the development process of different flowering periods of peony flowers for white peony varieties. SUMMARY

[0005] The technical problem to be solved by this invention is to provide a method and application for PCR detection of internal reference genes, primers, and screening methods for regulating peony flowering period, thereby improving the stability and accuracy of detecting the expression of related genes in white peony varieties during the peony flower development process and different flowering periods, and laying a solid foundation for subsequent research and identification of the characteristics and functions of genes related to peony flowering period regulation.

[0006] The technical solution adopted by the present invention to solve the above-mentioned technical problems is: a reference gene for detecting peony flowering period regulation by fluorescence quantitative PCR, which is used to detect the gene expression level of peony organs and tissues at different flowering periods. The reference gene is RPL17, and its nucleotide sequence is shown as Seq ID No. 25 in the sequence listing.

[0007] The peony mentioned is a white peony variety.

[0008] The organ or tissue mentioned is a flower bud or petal.

[0009] The specific primers for amplifying the internal reference gene RPL17 are shown in the sequence listing as Seq ID No. 17 and Seq ID No. 18.

[0010] The method for screening the internal reference gene includes the following steps: (1) Select petals of white peony varieties that grow uniformly, are free from pests and diseases, and have different flowering periods. After the samples are picked, they are quickly placed in a liquid nitrogen tank for quick freezing, and then stored in an ultra-low temperature freezer at -80 ℃. (2) Extract total RNA from peony petals, and use 1% agarose gel electrophoresis to detect the integrity of the extracted RNA. Use a UV-Vis spectrophotometer to detect the integrity, concentration and purity of the extracted RNA. (3) Remove genomic DNA from the sample and reverse transcribe to synthesize cDNA; (4) Candidate reference genes were selected from the peony transcriptome data, including 18S rRNA, Actin, EF1-α, e1F-5A, GAPDH, RNA-polymerase, RPL17, Tubulin-α, Tubulin-β, Ubiquitin, LTP and UDC; (5) The optimal internal reference gene was determined by real-time quantitative PCR analysis, primer amplification efficiency analysis and expression stability analysis.

[0011] The reaction system for the real-time quantitative PCR analysis consisted of a total volume of 20 μL, including 2.0 μL of template (≤100 ng), 0.8 μL of qPCR forward primer (10 μmol), 0.8 μL of qPCR reverse primer (10 μmol), 10.0 μL of 2×TB Green Premix Ex Taq II (Tli RNaseH Plus), and 8.4 μL of RNase-free ddH2O. The reaction program was as follows: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s; 60℃ annealing for 30 s, for a total of 45 cycles, with each reaction repeated 3 times.

[0012] In the primer amplification efficiency analysis, the primer amplification efficiency was determined by serially diluting cDNA 10-fold. 2 -10 6 The standard curve obtained after reacting with the desired concentration determines the primer amplification efficiency E = (10) for each candidate internal reference gene. [-1 / slope] -1)×100%, calculated using the slope of the corresponding standard curve. Three technical replicates were performed for each dilution gradient.

[0013] The expression stability of 12 candidate internal reference genes was analyzed using the geNorm and NormFinder programs.

[0014] This invention also provides the application of internal reference genes in real-time quantitative PCR analysis of genes related to the regulation of peony flowering period.

[0015] This invention also provides the application of specific primers in the preparation of a real-time fluorescence quantitative PCR kit for regulating peony flowering period.

[0016] The beneficial effects of this invention are as follows: Based on the analysis of peony RNA-Seq sequencing data, the expression stability of each candidate internal reference gene in peony varieties at different flowering stages was evaluated and analyzed using software, and the target gene was used for verification. Finally, RPL17 was selected as the more stable internal reference gene in the qRT-PCR experiment for peony flowering period regulation-related genes. This gene can meet the requirements of qRT-PCR technology for detecting the expression levels of related genes in white peony varieties at different flowering stages during the peony flower development process, improving the stability and accuracy of detecting the expression of related genes in white peony varieties at different flowering stages during the peony flower development process, and minimizing the differences between and within samples caused by the selection of internal reference genes. This lays a solid foundation for the subsequent analysis of the expression patterns of key genes related to peony flowering period regulation using qRT-PCR. Attached Figure Description

[0017] Figure 1The following are the flower characteristics of white peony varieties with different flowering periods during their full bloom in the embodiments of the present invention: (a): Paeonia suffruticosa 'Doukou Nianhua', (b): Paeonia ostii 'FengDan', (c): Paeonia suffruticosa 'Yulou Diancui', (d): Paeonia suffruticosa 'LianHe', (e): Paeonia suffruticosa 'Baiwang Shizi'; Figure 2 The image shows the agarose gel electrophoresis results of the candidate internal reference genes in the method of this invention, where: 1: 18S rRNA, 2: Actin, 3: EF1-α, 4: elF-5A, 5: GAPDH, 6: LTP, 7: UDC, 8: RNA-polymerase, 9: RPL17, 10: Tubulin-α, 11: Tubulin-β, 12: Ubiquitin; Figure 3 This is the melting curve of the candidate internal reference gene in the method of this invention; Figure 4 The results of the geNorm program calculation of the stability of candidate internal reference genes in peonies at different flowering stages in the method of this invention; Figure 5 To determine the optimal number of internal reference genes in peonies at different flowering stages in the method of this invention; Figure 6 The figure shows the results of the expression verification analysis of the target gene PsFRL5 in the seven developmental stages of different peony varieties in the method of this invention, where: A: RPL17, B: Tubulin-α, C: 18S rRNA. Detailed Implementation

[0018] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings and specific embodiments. The specific contents listed in the following embodiments are not limited to the technical features necessary to solve the technical problem of the present invention. Furthermore, the listed embodiments are merely a part of the present invention, and not all embodiments.

[0019] To obtain the desired internal reference gene, this invention uses the following method for internal reference gene screening: 1. Test materials Experimental materials were preserved in the Peony Germplasm Resource Nursery of Henan University of Science and Technology. White peony varieties with uniform growth, good development, and no pests or diseases were selected, including three early-flowering varieties: *Paeonia suffruticosa* 'Doukou Nianhua', *Paeonia ostii* 'FengDan', and *Paeonia suffruticosa* 'Yulou Diancui', and two late-flowering varieties: *Paeonia suffruticosa* 'LianHe' and *Paeonia suffruticosa* 'Baiwang Shizi'. Petal samples were obtained from each variety at different flowering stages, with the peak flowering period of each variety as follows: Figure 1 As shown. Each sample contained 3 biological replicates. All samples were rapidly placed in liquid nitrogen tanks for flash freezing after collection, and then stored in an ultra-low temperature freezer at -80 °C for subsequent experimental use.

[0020] 2. Extraction of total RNA and synthesis of cDNA Total RNA extraction from peony petals was performed according to the instructions of the Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (TIANGEN, Beijing). Subsequently, the RNA was extracted using 1% agarose gel electrophoresis and NanoDrop gel electrophoresis. TM The integrity, concentration, and purity of the extracted RNA were determined using a One micro-volume UV-Vis spectrophotometer. RNA samples that passed the tests were treated with a 2U gDNA Eraser at 42°C for 2 minutes to remove genomic DNA. PrimeScript was used. TM The RT reagent kit with gDNAEraser (Perfect Real Time) (TaKaRa, Japan) was used for reverse transcription to prepare a sufficient amount of cDNA to meet the needs of the selected gene template.

[0021] 3. Selection of candidate internal reference genes and primer design Based on the previous peony RNA-Seq sequencing results of this study, 12 candidate internal reference genes with high and stable expression levels were selected from the obtained unpublished peony transcriptome data: 18S rRNA, Actin, EF1-α, e1F-5A, GAPDH, RNA-polymerase, RPL17, Tubulin-α, Tubulin-β, Ubiquitin, LTP, and UDC. Specific primers were designed using Primer Premier 5.0 software (Table 1), and the nucleotide sequences are shown in the sequence listing, from Seq ID No. 1 to Seq ID No. 24. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0022] 4. Real-time quantitative PCR reaction qRT-qPCR reactions were performed using the TB Green™ Premix Ex Taq™ II (TliRNaseH Plus) kit from TaKaRa (Japan). The total reaction volume was 20 μL, including 2.0 μL of template (≤100 ng), 0.8 μL of qPCR Forward Primer (10 μmol), 0.8 μL of qPCR Reverse Primer (10 μmol), 10.0 μL of 2×TB Green Premix Ex Taq II (Tli RNaseH Plus), and 8.4 μL of RNase-Free ddH2O. Three technical replicates were performed for each sample, following the prescribed program: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s; 60℃ annealing for 30 s; 45 cycles. Each reaction was repeated three times.

[0023] 5. Processing and Analysis of Experimental Data 5.1 Specificity test of internal reference gene primers The primers for 12 candidate internal reference genes were subjected to specificity testing. After temperature gradient PCR analysis, the suitable annealing temperature for each candidate gene was determined to be 60 ℃. The PCR amplification products were then detected using a 1% agarose gel electrophoresis. The PCR products of the 12 candidate genes were clearly visible and consistent with the expected length, all between 140-250 bp, without any impurities or primer dimers. Figure 2 The melting curve of each gene shows a single peak. Figure 3 ).

[0024] 5.2 Standard Curve and Amplification Efficiency Analysis The amplification efficiency of each primer was analyzed using a standard curve. The amplification efficiency of the primers was determined by serially diluting cDNA 10-fold. 2 -10 6 The standard curve obtained after reacting with the desired concentration determines the primer amplification efficiency E = (10) for each candidate internal reference gene. [-1 / slope] -1)×100%, calculated using the slope of the corresponding standard curve. Three technical replicates were performed for each dilution gradient. Primer amplification efficiencies ranged from 190% to 209%, with a linear correlation coefficient R0. 2 >0.97 (Table 2). This demonstrates that all primers exhibit good specificity and amplification efficiency, meeting the basic requirements of qRT-PCR and can be used for subsequent experiments.

[0025] Table 1 Primer sequences of candidate internal reference genes Table 2. Correlation coefficients and amplification efficiency of the standard curves for candidate internal reference genes. 5.3 Screening of the most suitable internal reference gene for regulating peony flowering period 5.3.1 Analysis using geNorm software When screening internal reference genes for peony varieties at different flowering stages, the stability (M value) of the 12 candidate internal reference genes was as follows: UDC > Actin > RNA-polymerase > 18S rRNA > GAPDH > Ubiquitin > Tubulin-β > LTP > RPL17 > elF-5A > EF1-α = Tubulin-α ( Figure 4 At this point, the most stable genes are EF1-α and Tubulin-α, while UDC is the least stable gene.

[0026] Further analysis using the geNorm program to determine the pairwise differences yielded a V² / 3 value of 0.128 for peony varieties at different flowering stages. Figure 5 Both experimental groups showed values ​​less than the default threshold of 0.15, indicating that no third internal reference gene was needed for correction. Therefore, the optimal internal reference genes for peonies at different flowering stages, calculated by the geNorm program, are two genes: Tubulin-α and EF1-α.

[0027] 5.3.2 NormFinder Software Analysis The NormFinder program uses the same selection criteria as the geNorm program, choosing the internal reference gene with the lowest stability score as the optimal internal reference gene for the selected treatment. However, the NormFinder program can only select one optimal internal reference gene. According to calculations using the NormFinder program (Table 3), for peony varieties with different flowering periods, the most stable expression among the candidate internal reference genes is RPL17, followed by elF-5A, while the least stable expression is UDC. The stability scores of the 12 candidate internal reference genes in this experimental group are, in descending order: UDC > Actin > GAPDH > Tubulin-β > LTP > Ubiquitin > RNA-polymerase > EF1-α > Tubulin-α > 18S rRNA > elF-5A > RPL17. The genes ranked highest by the NormFinder program are basically the same as those calculated by the geNorm program, only the order differs.

[0028] Table 3. Calculation results of the NormFinder program As shown in Table 3, RPL17 had the lowest score and the most stable expression in the NormFinder analysis. Tubulin-α and EF1-α ranked 4th and 5th in stability, respectively, and their scores differed significantly from RPL17. However, in the geNorm analysis, RPL17 ranked 3rd, and its M value was not significantly different from that of EF1-α and Tubulin-α, which ranked 1st. Therefore, based on comprehensive evaluation, RPL17 was deemed the most stable and selected as the most suitable internal reference gene for white peony varieties at different flowering stages.

[0029] 5.4 Verification of gene stability of peony internal reference To further verify the stability of the optimal internal reference gene, we selected the important gene PsFRL5, which is related to flowering period regulation, for verification during the flowering development stages of different peony varieties (early-flowering variety MU, early-flowering variety FD, and late-flowering variety LH). The results are as follows: Figure 6 A and Figure 6 As shown in B. During different peony varieties at different developmental stages, when RPL17 and Tubulin-α, which have the best stability, were used as internal controls, the expression pattern of the target gene PsFRL5 was consistent, while when 18S rRNA, which has the worst stability, was used... Figure 6 C) The expression pattern is different when used as an internal reference.

[0030] Therefore, this invention selects RPL17 as the internal reference gene for real-time quantitative PCR detection of peony flowering period regulation. It is used to detect gene expression levels in petals, flower buds, and other peony organ tissues at different flowering stages of white peony varieties. The nucleotide sequence of the internal reference gene RPL17 is shown in Seq ID No. 25 of the sequence listing. Specific primers for amplifying the internal reference gene RPL17 are shown in Seq ID No. 17 and Seq ID No. 18 of the sequence listing. Using the internal reference gene RPL17 for real-time quantitative PCR analysis of genes related to peony flowering period regulation can improve the stability and accuracy of detecting gene expression related to peony development and different flowering stages in white peony varieties. The specific primers for the internal reference gene RPL17 can be used to prepare a real-time quantitative PCR kit for peony flowering period regulation, facilitating related detection and laying a solid foundation for subsequent analysis of the expression patterns of key genes related to peony flowering period regulation using qRT-PCR.

[0031] The above description of specific embodiments is only for the purpose of helping to understand the technical concept and core idea of ​​the present invention. Although specific preferred embodiments have been used to describe and illustrate the technical solutions, they should not be construed as limiting the present invention itself. Those skilled in the art can make various changes in form and detail without departing from the technical concept of the present invention. These easily conceived changes or substitutions should all be covered within the protection scope of the present invention.

Claims

1. A quantitative real-time PCR method for detecting internal reference genes during peony flowering period regulation, used to detect gene expression levels in peony organs and tissues at different flowering stages, characterized by: The internal reference gene is RPL17, and its nucleotide sequence is shown in Seq ID No. 25 in the sequence listing.

2. The internal reference gene as described in claim 1, characterized in that: The peony mentioned is a white peony variety.

3. The internal reference gene as described in claim 1, characterized in that: The organ or tissue mentioned is a flower bud or petal.

4. Specific primers for amplifying the internal reference gene of claim 1, characterized in that: The specific primers for amplifying the internal reference gene RPL17 are shown in the sequence listing as Seq ID No. 17 and Seq ID No.

18.

5. The method for screening internal reference genes as described in claim 1, characterized in that: Includes the following steps: (1) Select petals of white peony varieties that grow uniformly, are free from pests and diseases, and have different flowering periods. After the samples are picked, they are quickly placed in a liquid nitrogen tank for quick freezing, and then stored in an ultra-low temperature freezer at -80 ℃. (2) Extract total RNA from peony petals, and use 1% agarose gel electrophoresis to detect the integrity of the extracted RNA. Use a UV-Vis spectrophotometer to detect the integrity, concentration and purity of the extracted RNA. (3) Remove genomic DNA from the sample and reverse transcribe to synthesize cDNA; (4) Candidate reference genes were selected from the peony transcriptome data, including 18S rRNA, Actin, EF1-α, e1F-5A, GAPDH, RNA-polymerase, RPL17, Tubulin-α, Tubulin-β, Ubiquitin, LTP and UDC; (5) The optimal internal reference gene was determined by real-time quantitative PCR analysis, primer amplification efficiency analysis and expression stability analysis.

6. The screening method as described in claim 5, characterized in that: The reaction system for the real-time quantitative PCR analysis consisted of a total volume of 20 μL, including 2.0 μL of template (≤100 ng), 0.8 μL of qPCR forward primer (10 μmol), 0.8 μL of qPCR reverse primer (10 μmol), 10.0 μL of 2×TB Green Premix Ex Taq II (Tli RNaseH Plus), and 8.4 μL of RNase-free ddH2O. The reaction program was: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s; 60℃ annealing for 30 s, for a total of 45 cycles, with each reaction repeated 3 times.

7. The screening method as described in claim 5, characterized in that: In the primer amplification efficiency analysis, the primer amplification efficiency was determined by serially diluting cDNA 10-fold. 2 -10 6 The standard curve obtained after reacting with the desired concentration determines the primer amplification efficiency E = (10) for each candidate internal reference gene. [-1 / slope] -1)×100%, calculated using the slope of the corresponding standard curve. Three technical replicates were performed for each dilution gradient.

8. The screening method as described in claim 5, characterized in that: The expression stability of 12 candidate internal reference genes was analyzed using the geNorm and NormFinder programs.

9. The application of the internal reference gene as described in claim 1 in real-time quantitative PCR analysis of genes related to the regulation of peony flowering period.

10. The application of the specific primers as described in claim 4 in the preparation of a real-time fluorescence quantitative PCR kit for regulating peony flowering period.

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