Reference genes of Aesculus hippocastanum and Aesculus carthamus as well as primers and application of reference genes

Abstract

The invention discloses a reference gene for real-time fluorescence quantification of Aesculus hippocastanum and Aesculus carthamus, and a special primer and application thereof, and belongs to the technical field of plant molecular biology. According to the invention, different tissues and flowers at different development stages of Aesculus hippocastanum and Aesculus carthamus are taken as experimental materials, and two reference genes GAPDH and UBC with stable expression are screened through real-time fluorescent quantitative PCR (Polymerase Chain Reaction) analysis, melting curve and amplification efficiency analysis, and geNorm, NormFinder, BestKeeper, Delta Ct and RefFinder gene expression stability analysis in sequence. The reference gene GAPDH and / or UBC is suitable for expression analysis of flower color synthetic genes or other functional genes of Aesculus hippocastanum and Aesculus carthamus, fills up the current situation that there is no reference gene in Aesculus hippocastanum and Aesculus carthamus, can provide data support for accurate quantification of the functional genes of the Aesculus hippocastanum and Aesculus carthamus, and significantly improves the stability, accuracy and reliability of the obtained data.

Description

Technical Field

[0001] The invention relates to the field of plant molecular biology, and in particular to fluorescence quantitative internal reference genes of Aesculus hippocastanum and Aesculus hippocastanum, primers thereof and applications thereof. Background Art

[0002] Aesculus Aesculus L.) contains more than 30 species, Aesculus hippocastanum ( A. hippocastanum Aesculus hippocastanum is a species of Aesculus hippocastanum, a relic of the Tertiary period. It is found only in the Balkans. It is a large-leaved tree that can reach a height of 36 meters. Its flowers are white with red rhyme spots and its fruits are thorny. It is one of the most common ornamental trees and street trees in European towns, parks and woodlands. At the same time, the seeds of Aesculus hippocastanum have high medicinal value and have anti-inflammatory, vascular protection, anti-edema, analgesic and antioxidant properties. A. carnea Hayne is a species of Aesculus hippocastanum and Aesculus hippocastanum ( A. pavia Linn.) The flower is pink and has a very high ornamental value in gardens. Compared with Aesculus hippocastanum, there is less research on Aesculus hippocastanum. Studies have shown that the flowers of Aesculus hippocastanum and Aesculus hippocastanum contain 5 anthocyanin components, but the anthocyanin content of Aesculus hippocastanum is higher, with pelargonidin-3-o-glucoside as the main anthocyanin in the total anthocyanins. Therefore, the flower color formation of Aesculus hippocastanum is mainly related to the type and content of anthocyanins. It is crucial to analyze the flower color formation mechanism of Aesculus hippocastanum through multi-omics analysis of anthocyanin synthesis pathways and mining key genes for flower color. Screening and identifying the optimal internal reference required for the expression of key genes for flower color is the premise and basis for gene expression and functional analysis.

[0003] Quantitative Real-Time PCR (qRT-PCR) is a common method for detecting gene expression levels. It is accurate, highly specific, and highly sensitive. It is often used to study the expression patterns of genes in different species, tissues, and developmental stages. To ensure the accuracy of the quantitative results, it is necessary to use appropriate reference genes to correct the expression of the target gene. However, there are great differences in universal reference genes in different species, different tissues, and different physiological conditions. Therefore, it is necessary to screen the best reference genes with stable expression according to the species, organ tissue, and experimental purpose of the study. However, the reference genes for expression in European horse chestnut or red horse chestnut tissues have not been reported, which limits gene expression analysis and functional research. Therefore, the use of qRT-PCR to screen the best reference genes in different tissues and flowers at different developmental stages has important application value and significance. Summary of the invention

[0004] Based on this, the object of the present invention is to provide an internal reference gene, detection primer and application of different tissues and flowers of Aesculus hippocastanum and Aesculus hippocastanum in different developmental stages, wherein the internal reference gene can be stably expressed in different tissues and flowers of Aesculus hippocastanum and Aesculus hippocastanum in different developmental stages.

[0005] The technical solutions to achieve the above-mentioned invention objectives include the following:

[0006] In a first aspect of the present invention, a reference gene of flowers of different tissues and different developmental stages of Aesculus hippocastanum and Aesculus hippocastanum is provided, characterized in that the reference gene is a GAPDH gene and / or a UBC gene, the nucleotide sequence of the GAPDH gene is shown in SEQ ID NO.1, and the nucleotide sequence of the UBC gene is shown in SEQ ID NO.2.

[0007] The second aspect of the present invention provides a primer set for detecting the internal reference gene described in the first aspect of the present invention, wherein the primer set includes a primer pair 1 for detecting the GAPDH gene and / or a primer pair 2 for detecting the UBC gene.

[0008] Preferably, the primer pair 1 comprises the following primer sequences:

[0009] GAPDH forward primer 5'-GTTTGGCATTGTTGAGGGTCTC-3' (SEQ ID NO.3)

[0010] GAPDH reverse primer 5'-TGATGTTGAAGGAAGCAGCTCT-3' (SEQ ID NO. 4).

[0011] Preferably, the primer pair 2 comprises the following primer sequences:

[0012] UBC forward primer 5'-GGAATGCCGTTATATTTGGGCC-3' (SEQ ID NO.5)

[0013] UBC reverse primer 5'-ATCCGAGAAACAAAACGCACTG-3' (SEQ ID NO. 6).

[0014] By adopting the above technical solution, the GAPDH gene and the UBC gene can be specifically amplified, and the accurate amplification of the internal reference gene can be achieved.

[0015] The third aspect of the present invention provides a detection reagent or kit for the expression level of the above-mentioned internal reference gene, wherein the detection reagent or kit comprises the primer set of the internal reference gene described in the first aspect.

[0016] A fourth aspect of the present invention provides an application of the above-mentioned internal reference gene as an internal reference gene in fluorescent quantitative PCR of flowers of different tissues and different developmental stages of Aesculus hippocastanum and Aesculus hippocastanum.

[0017] The fifth aspect of the present invention provides an application of the above primer set or the above detection reagent or kit in fluorescent quantitative PCR of flowers of different tissues and different developmental stages of Aesculus hippocastanum and Aesculus hippocastanum.

[0018] Furthermore, when using fluorescent quantitative PCR to analyze the flowers of different tissues and developmental stages of Aesculus hippocastanum and Aesculus hippocastanum, a pair of primers was used to detect the internal reference gene GAPDH gene.

[0019] Furthermore, when using fluorescent quantitative PCR to analyze the flowers of different tissues and developmental stages of Aesculus hippocastanum and Aesculus hippocastanum, two pairs of primers were used to detect the internal reference gene UBC gene.

[0020] Furthermore, the different tissues of Aesculus hippocastanum and Aesculus hippocastanum are leaves, buds, stems, inflorescences and bracts.

[0021] Furthermore, the flowers of Aesculus hippocastanum and Aesculus hippocastanum at different developmental stages include bud stage, early flowering stage and full flowering stage.

[0022] Compared with the prior art, the present invention has the following technical effects:

[0023] 1) The present invention screens out for the first time the internal reference genes stably expressed in different tissues and flowers of Aesculus hippocastanum and Aesculus chinensis at different developmental stages as GAPDH gene and / or UBC gene, and simultaneously reveals the sequences of the two internal reference genes, and the gene combination can meet the requirements of real-time fluorescence quantitative detection of transcriptional expression levels of flowers of different tissues and developmental stages of Aesculus hippocastanum and Aesculus chinensis, improves the stability and reliability of gene expression analysis and research, and provides strong support for the accurate quantification of functional gene expression. In addition, the two internal reference gene sequences suitable for qRT-PCR of Aesculus hippocastanum and Aesculus chinensis provided by the present invention are both from the transcriptome sequences of Aesculus hippocastanum and Aesculus chinensis, and have the advantages of good specificity and high stability compared with the universal internal reference genes on other species.

[0024] 2) The real-time fluorescence quantitative primers designed with GAPDH gene and / or UBC gene as templates in the present invention not only solve the current situation that there is no internal reference gene for the expression pattern analysis of relevant functional genes of Aesculus hippocastanum and Aesculus hippocastanum, but also the designed real-time fluorescence quantitative primers have strong primer specificity when used for gene expression analysis of flowers of different tissues and different developmental stages of Aesculus hippocastanum and Aesculus hippocastanum, thereby improving the efficiency of fluorescence quantitative detection of gene expression and the credibility of the detection results. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 These are flowers of Aesculus hippocastanum (Ah1 - Ah3, representing the bud stage, early flowering stage, and full flowering stage) and Aesculus hippocastanum (Ac1 - Ac3, same as above) at different developmental stages.

[0026] Figure 2 This is a gel image of primer specificity detection by agarose gel electrophoresis. The leftmost side is the marker, and the numbers 1-12 represent the specific bands of different candidate internal reference genes, 1: HIS3; 2: Rab7; 3: EF1γ; 4: GAPDH; 5: UBC; 6: TUB; 7: CYP; 8: RPL13; 9: AQP; 10: EIF5A; 11: ADF; 12: UBQ.

[0027] Figure 3 is the melting curve of the candidate internal reference gene PCR product.

[0028] Figure 4 : is a box plot of the Ct value distribution of candidate reference genes, where A is the Ct value distribution of the reference genes in different tissues of Aesculus hippocastanum and Aesculus hippocastanum; B is the Ct value distribution of the reference genes in flowers at different developmental stages.

[0029] Figure 5 The expression stability analysis of candidate internal reference genes in different tissues of Aesculus hippocastanum and Aesculus hippocastanum. A to E are the stability values ​​of 11 candidate internal reference genes based on geNorm, NormFinder, BestKeeper, Delta Ct and RefFinder methods, respectively; F is the pairwise variation (V) analysis by geNorm software.

[0030] Figure 6 The expression stability analysis of candidate reference genes in flowers of Aesculus hippocastanum and Aesculus hippocastanum at different developmental stages. A~E are the stability values ​​of 11 candidate reference genes based on geNorm, NormFinder, BestKeeper, Delta Ct and RefFinder methods, respectively; F is the pairwise variation (V) analysis by geNorm software.

[0031] Figure 7 This is a graph showing the stability of the reference genes screened by verifying the C4H, CHI, DFR and MYB4 genes in Aesculus hippocastanum and Aesculus hippocastanum during flowering (blooming period).

[0032] Figure 8This is the result of using the WRKY75 gene to verify the stability of the selected reference gene in different tissues of Aesculus hippocastanum and Aesculus hippocastanum. Ah represents Aesculus hippocastanum, Ac represents Aesculus hippocastanum; B, bud; S, stem; L, leaf; F, inflorescence.

[0033] Fig. 9 This is a graph showing the results of using the HY5 gene to verify the stability of the selected reference gene in the undeveloped flowers of Aesculus hippocastanum and Aesculus hippocastanum. DETAILED DESCRIPTION

[0034] The present invention is further described below in conjunction with specific embodiments. The equipment and reagents used in the following embodiments can be purchased commercially. The test materials are Aesculus hippocastanum and Aesculus hippocastanum planted in the Aesculus hippocastanum germplasm resource nursery in Linyi City, Shandong Province, with consistent tree age, growth environment, soil and water and fertilizer management. Three plants with good growth and consistent height and growth were randomly selected for sample collection, and leaves, buds, stems, inflorescences and bracts of Aesculus hippocastanum and Aesculus hippocastanum were collected. Flowers of Aesculus hippocastanum and Aesculus hippocastanum at different developmental stages were collected multiple times, including the bud stage, initial flowering stage, full flowering stage ( Figure 1 ). Each sample was sampled three times for biological replication and mixed, and the sample was quickly frozen in liquid nitrogen and stored in a -80°C refrigerator for later use.

[0035] Example 1

[0036] 1. Materials and Methods

[0037] 1.1 Extraction of plant total RNA and synthesis of cDNA

[0038] According to the instructions of RNA prep Pure Polysaccharide Polyphenol Plant Total RNA Extraction Kit (TIANGEN, Beijing, China), total RNA was extracted from all test samples and genomic DNA contamination was removed by DNA enzyme treatment. The concentration and quality of total RNA were measured by Nanodrop 2000 ultra-micro UV spectrophotometer, and the integrity of total RNA was detected by 1.2% agarose gel electrophoresis. The RNA OD260 / OD280 and OD260 / OD230 of each test sample met the requirements, and agarose gel electrophoresis showed that the 28S and 18S bands were clear and there was no degradation, which met the requirements.

[0039] The cDNA reverse transcription reaction system for real-time fluorescence quantitative PCR analysis was prepared according to the instructions of SynScript III RTSuperMix for qPCR (Beijing Qingke Biotechnology Co., Ltd., Beijing), and 1 μg of total RNA was used to establish a 20 μL cDNA reverse transcription reaction system. After the reaction was completed, it was stored at -20°C for future use.

[0040] 2.1 Selection of candidate internal reference genes and primer design

[0041] According to the transcriptome data of Aesculus hippocastanum and Aesculus hippocastanum, 12 candidate internal reference genes were selected with the gene expression FPKM>60 and the FPKM value difference between samples less than 2 as the screening criteria, namely GAPDH (SEQ ID NO.1), UBC (SEQ ID NO.2), UBQ (SEQ ID NO.7), AQP (SEQ ID NO.8), HIS3 (SEQ ID NO.9), ADF (SEQ ID NO.10), RPL13 (SEQ ID NO.11), TUB (SEQ ID NO.12), EF1γ (SEQ ID NO.13), Rab7 (SEQ ID NO.14), EIF5A (SEQ ID NO.15), CYP (SEQ ID NO.16). The FPKM values ​​of the 12 internal reference genes were relatively stable in all samples (Table 1), where FPKM refers to the fragments per kilobase of transcription per million mapped reads, which can reflect the expression level of the gene. The larger the FPKM value, the higher the expression level of the gene. Then, quantitative specific primers were designed in Primer 3 Plus (https: / / www.primer3plus.com / ) according to the sequences of the internal reference genes. At the same time, primers were designed for anthocyanin synthesis genes trans-cinnamate 4-hydroxylase C4H (Trans-cinnamate 4-hydroxylase), chalcone isomerase CHI (Chalcone isomerase), dihydroflavonol-4-reductase DFR (Dihydroflavonol 4-reductase) and regulatory transcription factors MYB4, HY5, and WRKY75 (Table 2) to verify the stability of the internal reference genes. Primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. First, primers were preliminarily screened by ordinary PCR, and the bands of PCR products were observed using 1% agarose gel and gel imaging system to detect product specificity. Primers with correct band size, good band specificity, and no primer dimers were selected. Primer specificity was further detected by qRT-PCR, and primers with a single peak graph, no miscellaneous peaks, and no peaks in the negative control were selected as the final primers.

[0042] Table 1 FPKM values ​​of candidate reference genes

[0043]

[0044] Table 2 Primer information of internal reference genes and verification genes

[0045]

[0046] 3.1 Establishment of the standard curve of internal reference gene primers

[0047] For each pair of primers of the internal reference gene, a separate standard curve was prepared to calculate the amplification efficiency of the corresponding primers. The cDNA stock solution was diluted by 5 times into 5 gradients (1 / 5, 1 / 25, 1 / 125, 1 / 625, 1 / 3125) as a template for establishing the standard curve. At the same time, ddH2O was used as a negative control. All samples were repeated 3 times to ensure the credibility of the experimental data. qRT-PCR was performed using Roche Light Cycler 480, and a standard curve was drawn based on the data results. The slope (k) and correlation coefficient r were obtained based on the standard curve. 2 , the amplification efficiency was calculated using the formula E = (10 -1 / k -1) × 100%. Real-time fluorescence quantification requires that the amplification efficiency of the selected primers be between 90% and 110%.

[0048] 4.1 Real-time fluorescence quantification

[0049] qRT-PCR was performed using Roche Light Cycler 480, using the ArtiCanATM SYBR qPCR Mix quantitative kit [Beijing Qingke Biotechnology Co., Ltd., Beijing]. The total reaction system was 10 μL: SYBR qPCR Mix 5 μL, upstream and downstream primers (10 μmol / L) 0.2 μL each, cDNA template (reverse transcription cDNA product diluted 10 times) 1 μL, sterile distilled water 3.6 μL. Each reaction system was repeated three times.

[0050] The qRT-PCR reaction program is: 95℃ for 1 minute, followed by 95℃ for 10 seconds, 60℃ for 10 seconds for a total of 35 cycles. After the cycle is completed, a melting curve is drawn. The temperature is slowly increased from 60℃ to 97℃, and the fluorescence signal is collected 5 times for each 1℃ increase in temperature. Each reaction is repeated 3 times. After the PCR is completed, the corresponding threshold cycle number, i.e., the Ct value, is obtained.

[0051] 5.1 Data processing

[0052] (1) The geNorm software uses the pairwise comparison method to calculate the gene expression stability M value. The judgment standard is that when the M value is lower than 1.5, the lower the M value, the higher the stability, and vice versa. The software can also calculate the average pairwise variation V value of the normalization factor after introducing a new internal reference gene, and determine the number of optimal internal reference genes required based on the Vn / Vn+1 value. If Vn / Vn+1<0.15, it means that the stability of the n internal reference genes is good, and there is no need to add the n+1th gene.

[0053] (2) NormFinder software uses a model-based approach to obtain the stable expression value of the internal reference gene, and then screens the most suitable internal reference gene based on the stable value. The judgment criterion is that the internal reference gene with the smallest stable expression value is the most suitable internal reference gene.

[0054] (3) BestKeeper uses repeated pairwise correlation analysis, correlation analysis, and regression analysis to calculate the standard deviation (SD) and coefficient of variation (CV) of the Ct value, and then compares the sizes of each value to ultimately determine the reference gene with better stability.

[0055] (4) The Delta Ct algorithm identifies stably expressed genes by comparing the relative expression levels of “gene pairs” in each sample and determines the stability of genes based on the mean standard deviation (STDEV) of the expression level of each gene.

[0056] (5) The RefFinder algorithm can comprehensively rank the results obtained by the above four algorithms, assign an appropriate weight to each candidate reference gene, and calculate the geometric mean of its weight. By assigning points and comprehensively ranking, the most stable reference gene can be screened out.

[0057] 2. Results and Analysis

[0058] 2.1 Analysis of primer specificity and amplification efficiency

[0059] The specificity of the primers was analyzed by agarose electrophoresis and melting curve analysis. The fragments amplified by the 12 pairs of internal reference gene primers were consistent with the expected length and were single bands ( Figure 2 ), the melting curves of 11 internal reference genes all showed a single peak. Since the melting curve peak of the AQP gene was not single, the primers for this gene were discarded ( Figure 3 The standard curve was used to analyze the amplification efficiency of each primer. The amplification efficiency of the primers was between 90.00% and 102.34%, and the linear correlation coefficient r 2 >0.98 (Table 3). It can be seen that all 11 pairs of primers have good specificity and amplification efficiency, meet the basic requirements of qRT-PCR, and can be used for subsequent experiments.

[0060] Table 3 Amplification characteristics of internal reference gene primers

[0061]

[0062] 2.2 Analysis of internal reference gene expression levels

[0063] The Ct values ​​of 11 candidate internal reference genes in different tissues and different flowering stages of A. hippocastanum and A. chinensis were summarized to analyze the stability of their expression abundance at the transcriptional level. In different tissues of A. hippocastanum and A. chinensis, the mean Ct values ​​of the 11 internal reference genes ranged from 21.48 to 26.04, among which the expression levels of HIS3, GAPDH, and UBC genes were higher, and the differences were smaller ( Figure 4 A). In the flowers of Aesculus hippocastanum and Aesculus hippocastanum at different flowering stages, the mean Ct values ​​of the 11 reference genes ranged from 19.75 to 24.22, with the smallest being UBQ (Ct=19.75) and the largest being EF1γ (Ct=24.22) ( Figure 4 B).

[0064] 2.3 Software Analysis

[0065] The geNorm software evaluates the expression stability of each candidate gene based on the gene expression stability value (M). The smaller the M value, the higher the stability. In different tissues of Aesculus hippocastanum and Aesculus hippocastanum, CYP, EIF5A and UBC were the most stable, while UBQ was the least stable ( Figure 5 A). In flowers at different developmental stages, the top three most stable ones are GAPDH, CYP, and UBC, and the least stable one is UBQ ( Figure 6 A). geNorm software can also determine the appropriate number of internal reference genes based on the gene pair difference value Vn / Vn+1. When Vn / Vn+1< 0.15, n internal reference genes are used. In different tissues and flowers of Aesculus hippocastanum and Aesculus hippocastanum, the software calculated V 2 / 3 >0.15, V 3 / 4 <0.15, indicating that the combination of three reference genes can ensure stability ( Figure 5 F, Figure 6 F).

[0066] NormFinder software uses expression stability (SV) to evaluate internal reference genes. The lower the SV value, the more stable the expression of the gene. The analysis results showed that in different tissues of Aesculus hippocastanum and Aesculus hippocastanum, GAPDH had the highest stability, followed by UBC and CYP, and the worst stability was UBQ ( Figure 5 B). In flowers at different developmental stages, GAPDH has the highest stability, followed by EIF5A and UBC, while HIS3 has the lowest stability ( Figure 6 B).

[0067] BestKeeper uses the Ct values ​​of the internal reference gene in different samples to analyze its coefficient of variation (CV) and standard deviation (SD). The smaller the coefficient of variation and standard deviation, the better the stability of the candidate internal reference gene. In different tissues of Aesculus hippocastanum and Aesculus hippocastanum, Rab7 has the best stability, followed by EF1γ, and UBQ has the worst stability ( Figure 5 C). For flowers at different developmental stages, EF1γ has the best stability, followed by GAPDH, and TUB has the worst stability ( Figure 6 C).

[0068] The Delta Ct algorithm determines the stability of genes based on the mean standard deviation (STDEV) of the expression of each internal reference gene. The results showed that among the flowers of different tissues and developmental stages of Aesculus hippocastanum and Aesculus hippocastanum, GAPDH had the smallest STDEV, followed by UBC. This indicates that GAPDH is the most stable, followed by UBC ( Figure 5 D, Figure 6 D).

[0069] The results obtained by the above four software analyses were different. Therefore, based on the analysis results of the four software, the geometric mean was calculated using RefFinder software to obtain the ranking of the comprehensive index of internal reference gene stability. In different tissues of Aesculus hippocastanum and Aesculus hippocastanum, the top three in the comprehensive ranking were GAPDH, UBC, and CYP ( Figure 5 E), and the results of Delta Ct and NormFinder software analysis were the same, with UBQ and ADF having the worst expression stability. In flowers at different developmental stages, the top three reference genes were GAPDH, UBC, and EIF5A ( Figure 6 E). It can also be seen that HIS3 and UBQ have the worst expression stability.

[0070] Through the ranking of the above reference gene stability comprehensive index, combined with the geNorm pairwise difference value to obtain the best three internal parameters, GAPDH, UBC, and CYP are the best candidate internal references in different tissues of Aesculus hippocastanum and Aesculus hippocastanum; GAPDH, UBC, and EIF5A are the most suitable candidate internal references in flowers at different developmental stages. In summary, GAPDH and UBC are the most stably expressed internal references in different tissues and flowers at different developmental stages of Aesculus hippocastanum and Aesculus hippocastanum, and UBQ is the most unstable internal reference.

[0071] 2.4 Verification of stability of internal reference genes

[0072] The anthocyanin biosynthesis pathway genes C4H (trans-cinnamate 4-hydroxylase), CHI (chalcone isomerase), DFR (dihydroflavonol 4-reductase) and transcription factors MYB4, WRKY75 and HY5 were selected to verify the stability of the selected reference genes in the flowers of Aesculus hippocastanum and Aesculus hippocastanum, and the most unstable reference gene UBQ was used for comparison. The results showed that with GAPDH, UBC and GAPDH+UBC combination, GAPDH+UBC+EIF5A combination as internal references, C4H, CHI, DFR and MYB4 genes were expressed in Aesculus hippocastanum ( A. carnea, safflower) was higher than that of Aesculus hippocastanum ( A. hippocastanu, In white flowers, it was significantly upregulated in red flowers, and the gene expression patterns were consistent. At the same time, the expression patterns of the four genes were consistent with the transcriptome sequencing data (FPKM) ( Figure 7 ), further confirming the reliability of the data. With unstable UBQ as the internal reference, the expression levels of the four anthocyanin genes in safflower were low. For example, with UBQ as the internal reference, the C4H gene was downregulated in safflower, and the expression pattern changed; while the expression levels of CHI, DFR and MYB4 genes were not significantly different between safflower and white flowers ( Figure 7 ), which is inconsistent with the actual results, strongly indicating that inappropriate internal reference genes will lead to deviations in gene expression levels and affect the accuracy of the test results.

[0073] The WRKY75 and HY5 genes were used for verification in different tissues and flowers of Aesculus hippocastanum and Aesculus hippocastanum. In different tissues, the expression patterns of WRKY75 gene were consistent in buds, stems, leaves and inflorescences when GAPDH, UBC, GAPDH+UBC combination and GAPDH+UBC+CYP combination were used as internal references, while the expression patterns of WRKY75 were different when UBQ, which has poor stability, was used as internal reference ( Figure 8 In flowers at different developmental stages, the expression patterns of HY5 gene were consistent when GAPDH, UBC, GAPDH+UBC combination, and GAPDH+UBC+EIF5A combination were used as internal references, but the expression pattern changed when UBQ was used as the internal reference (Figure 9).

[0074] In summary, in the flowers of different tissues and developmental stages of A. hippocastanum and A. chinensis, the relatively stable reference genes GAPDH, UBC and GAPDH+UBC combination were selected. Compared with the three reference gene combinations (GAPDH+UBC+CYP / EIF5A), the expression patterns of the target genes were the same, and the expression levels were slightly different, but the differences were not significant. Considering the experimental cost and sample consumption, the use of GAPDH and / or UBC is a better choice. In addition, in species with close genetic relationships, the conservation of the same type of genes is relatively higher. Therefore, the stable reference genes GAPDH and UBC finally selected in this study can not only be used for the expression analysis of related genes of A. hippocastanum and A. chinensis, but also provide a basis and reference for the selection of reference genes for gene expression analysis of other species of the genus Aesculus.

[0075] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. Reference genes for fluorescence quantification of flowers of different tissues and developmental stages of Aesculus hippocastanum and Aesculus hippocastanum, characterized in that: The internal reference gene is the GAPDH gene and / or the UBC gene. The nucleotide sequence of the GAPDH gene is shown in SEQ ID NO.1, and the nucleotide sequence of the UBC gene is shown in SEQ ID NO.

2.

2. A primer set for detecting the internal reference gene according to claim 1, wherein the primer set comprises a primer pair 1 for detecting the GAPDH gene and / or a primer pair 2 for detecting the UBC gene.

3. The primer set according to claim 2, characterized in that The primer pair 1 comprises the following primer sequences: GAPDH forward primer 5'-GTTTGGCATTGTTGAGGGTCTC-3' (SEQ ID NO.3) GAPDH reverse primer 5'-TGATGTTGAAGGAAGCAGCTCT-3' (SEQ ID NO. 4).

4. The primer set according to claim 2, characterized in that The primer pair 2 includes the following primer sequences: UBC forward primer 5'-GGAATGCCGTTATATTTGGGCC-3' (SEQ ID NO.5) UBC reverse primer 5'-ATCCGAGAAACAAAACGCACTG-3' (SEQ ID NO. 6).

5. A detection reagent or kit for the internal reference gene expression level according to claim 1, characterized in that: The detection reagent or kit comprises the primer set according to any one of claims 2 to 4.

6. Application of the internal reference gene of claim 1 as an internal reference gene in fluorescence quantification of flowers of different tissues and different developmental stages of Aesculus hippocastanum and Aesculus hippocastanum.

7. Use of the primer set described in any one of claims 2 to 4 or the detection reagent or kit described in claim 5 in the fluorescence quantification of flowers of different tissues and different developmental stages of Aesculus hippocastanum and Aesculus hippocastanum.

8. The use according to claim 7, characterized in that: When using fluorescent quantitative PCR to analyze the flowers of different tissues and different developmental stages of Aesculus hippocastanum and Aesculus hippocastanum, a pair of primers in claim 3 is used to detect the internal reference gene GAPDH gene.

9. The use according to claim 7, characterized in that: When using fluorescent quantitative PCR to analyze the flowers of different tissues and different developmental stages of Aesculus hippocastanum and Aesculus hippocastanum, two pairs of primers in claim 4 are used to detect the UBC gene, an internal reference gene.

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