Fluorescent quantitative reference gene suitable for different flower colors of rhododendron lapponicum as well as primer and application of fluorescent quantitative reference gene

Abstract

The invention belongs to the technical field of fluorescent quantitative PCR (Polymerase Chain Reaction) detection, and discloses a reference gene for fluorescent quantification of Rhododendron laponicum (L.) Wahlenb. With different flower colors as well as a primer and application of the reference gene. The method comprises the following steps: selecting six rhododendron lapponicum varieties with obvious flower color difference, respectively collecting petals in a full-bloom stage, and sequentially carrying out fluorescent quantitative PCR (Polymerase Chain Reaction) analysis, amplification curve and melting curve analysis, LinReg PCR amplification efficiency analysis, BestKeeper, geNorm, NormFinder, Delta Ct and RefFinder gene expression stability analysis to screen out two stably expressed reference genes RlaACT and RlaEF1-alpha suitable for fluorescent quantitative PCR of rhododendron lapponicum with different flower colors. The fluorescent quantitative reference gene which is suitable for different flower colors of rhododendron lapponicum and is stable in expression is obtained, the current situation that no stable reference gene exists in rhododendron lapponicum color variation research is filled up, and molecular biology and genetic breeding research of rhododendron lapponicum is promoted; and data support is provided for accurate quantification of functional genes related to color variation of the alpine azalea, and the stability and accuracy of research can be improved.

Description

Technical Field

[0001] The invention belongs to the field of plant molecular biology, and in particular relates to fluorescence quantitative internal reference genes suitable for alpine rhododendrons with different flower colors and primers and applications thereof. Background Art

[0002] Rhododendron lapponicum (L.) Wahlenb. is an evergreen shrub of the genus Rhododendron L. in the family Ericaceae. It blooms in early spring, with diverse flower shapes and bright colors, and is of great value in landscaping, potted ornamental and ecological restoration. At present, most of the research on alpine rhododendron focuses on resource distribution surveys and breeding of fine varieties, population genetics and evolution, and ecological monitoring, while most of the research on the color changes of alpine rhododendron is only related to its phenotype. The breeding of new varieties of alpine rhododendron is still mainly based on conventional selection and hybrid breeding, which has problems such as long breeding cycle and unstable flower color performance. With the increasing social demand and the further improvement of the level of landscaping, the application of colored flowers in gardens is also becoming more and more extensive. Therefore, if colored flowers are to be further developed, it is necessary to combine advanced scientific and technological means to independently develop and cultivate new varieties with stable traits.

[0003] Flower color is one of the core ornamental traits of Rhododendron alpinum, which directly affects market competitiveness, and the study of the molecular mechanism of flower color formation is the key to directional breeding. The analysis of gene expression using real-time fluorescence quantitative PCR (qRT-PCR) has become the core means to analyze the flower color regulatory network. However, the accuracy of qRT-PCR results is highly dependent on stable reference genes, whose expression needs to remain constant under different tissues, developmental stages and environmental conditions to eliminate experimental errors. At present, molecular research on Rhododendron alpinum is still in its infancy, especially the development and verification of reference genes has not been systematically carried out. Although the commonly used reference genes in other plants (such as Actin, UBQ, GAPDH, etc.) have been borrowed by some studies, due to the complexity of the Rhododendron alpinum genome (such as polyploidy, high repetitive sequences) and the uniqueness of flower color-specific metabolic pathways, these genes may have expression fluctuations in different flower color varieties, resulting in failure of data standardization, thereby affecting the accuracy of target gene detection. For example, flower color formation involves multiple links such as anthocyanin synthesis, transport and vacuolar pH regulation, and the expression dynamics of related genes may interfere with the stability of traditional reference genes. In addition, the differences in genetic background among varieties with different flower colors further increase the difficulty of screening universal reference genes.

[0004] Therefore, we focus on the systematic screening and verification of reference genes of alpine rhododendrons with different flower colors, and screen out reference combinations suitable for the analysis of flower color-related gene expression. This will not only provide reliable technical support for the study of the molecular mechanism of alpine rhododendron flower color, but will also promote its molecular breeding process, help cultivate new and superior flower color varieties that meet market demand, and enhance the international competitiveness of my country's alpine rhododendron industry. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention aims to provide a fluorescence quantitative internal reference gene suitable for different color alpine rhododendrons, which can meet the requirements of real-time fluorescence quantitative detection of transcriptional expression levels of different color alpine rhododendrons, and improve the stability and reliability of gene expression analysis research of different color alpine rhododendrons. Another object of the present invention is to provide a special primer pair for the above-mentioned internal reference gene. Another object of the present invention is to provide an application of the above-mentioned internal reference gene or special primer pair.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides reference genes for fluorescence quantification of Rhododendron azalea with different flower colors, wherein the reference genes are RlaACT gene and / or RlaEF1-α gene, the sequence of the RlaACT gene is shown in SEQ ID NO.1, and the sequence of the RlaEF1-α gene is shown in SEQ ID NO.2.

[0008] By adopting the above technical scheme, the real-time fluorescence quantitative primers designed with RlaACT gene and RlaEF1-α gene as templates not only solve the current situation that there is no internal reference gene in the analysis of functional gene expression pattern of alpine rhododendron with different flower colors, but also the designed real-time fluorescence quantitative primers have strong primer specificity when used for gene expression analysis of alpine rhododendron with different flower colors, thereby greatly improving the detection efficiency of key genes of alpine rhododendron color variation using real-time fluorescence quantitative detection and improving the credibility of the detection results.

[0009] In a second aspect, the present invention further provides a primer set for detecting the internal reference gene in the fluorescence quantification of different flower color alpine rhododendrons described in the first aspect, wherein the primer set includes primer pair 1 of the RlaACT gene and / or primer pair 2 of the RlaEF1-α gene.

[0010] Preferably, the primer pair 1 comprises the following primer sequences:

[0011] RlaACT forward primer 5'-AGCACCCAGTTCTTCTTACA-3' (SEQ ID NO.3)

[0012] RlaACT reverse primer 5'-GGGCATAACCCTCATAGATAG-3' (SEQ ID NO. 4).

[0013] Preferably, the primer pair 2 comprises the following primer sequences:

[0014] RlaEF1-α forward primer 5'-ATGATTCCGACCAAGCCTAT-3' (SEQ ID NO.5)

[0015] RlaEF1-α reverse primer 5′-ATCCTTCTTCTCCACGCTCT-3′ (SEQ ID NO. 6).

[0016] By adopting the above technical solution, the RlaACT gene and the RlaEF1-α gene can be specifically amplified, thereby achieving specific amplification of the internal reference gene.

[0017] In a third aspect, the present invention further provides a detection reagent or kit for the above-mentioned internal reference gene expression amount, wherein the detection reagent or kit comprises the primer set described in the second aspect.

[0018] In a fourth aspect, the present invention also provides the use of the above-mentioned internal reference gene as an internal reference gene in the fluorescence quantification of Rhododendron truncatum with different flower colors.

[0019] In a fifth aspect, the present invention also provides the use of the above primer set or the above detection reagent or kit in the fluorescence quantification of Rhododendron truncatum with different flower colors.

[0020] Furthermore, when using fluorescent quantitative PCR to analyze the transcription of different genes in Rhododendron azalea, a pair of primers was used to detect the internal reference gene RlaACT gene.

[0021] Furthermore, when using fluorescent quantitative PCR to analyze the transcription of different genes in Rhododendron azalea, two pairs of primers were used to detect the internal reference gene RlaEF1-α gene.

[0022] Furthermore, the varieties of alpine rhododendrons with different flower colors are azalea, rhododendron azalea, deer horn rhododendron, azalea, white ice rhododendron and brocade rhododendron.

[0023] Compared with the prior art, the present invention has the following technical effects:

[0024] 1) The present invention screened out for the first time the internal reference genes stably expressed in the petals of alpine rhododendrons with different flower colors (azalea, rhododendron, staghorn rhododendron, azalea, white ice rhododendron and cloud brocade rhododendron), which are RlaACT gene and RlaEF1-α gene, and revealed the sequences of the two internal reference genes. This gene combination can meet the requirements of real-time fluorescence quantitative detection of transcriptional expression levels of alpine rhododendrons with different flower colors, and improves the stability and reliability of gene expression analysis research of alpine rhododendrons with different flower colors.

[0025] 2) The real-time fluorescence quantitative primers designed by the present invention using the RlaACT gene and the RlaEF1-α gene as templates not only solve the current situation that there is no internal reference gene for the analysis of functional gene expression patterns of alpine rhododendrons with different flower colors, but also the designed real-time fluorescence quantitative primers have strong primer specificity when used for gene expression analysis of alpine rhododendrons with different flower colors, thereby greatly improving the detection efficiency of the key genes of alpine rhododendron flower color variation using real-time fluorescence quantitative detection and improving the credibility of the detection results. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 These are sample pictures of alpine rhododendrons with different flower colors, A and G are the inflorescences and flowers of Rhododendron chinense, B and H are the inflorescences and flowers of Rhododendron roxburghii, C and I are the inflorescences and flowers of Rhododendron tatarinowii, D and J are the inflorescences and flowers of Azalea scabra, E and K are the inflorescences and flowers of Rhododendron glabra, and F and L are the inflorescences and flowers of Rhododendron yunjinensis. Bar=1cm.

[0027] Figure 2 This is a gel image of primer specificity detection by agarose gel electrophoresis. The leftmost is Maker, followed by specific bands corresponding to different internal reference genes.

[0028] Figure 3 This is the average Ct value distribution of 11 candidate reference genes in different tissues.

[0029] Figure 4 It is a box plot of the Ct value distribution of 11 candidate internal reference genes.

[0030] Figure 5 This is the result graph of the optimal number of internal reference genes determined by geNorm for accurate quantitative analysis.

[0031] Figure 6 The geNorm software calculates the expression stability of 11 candidate internal reference genes.

[0032] Figure 7 This is a graph showing the stability of the reference gene screened out using the RlaMYB113 gene verification. DETAILED DESCRIPTION

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional experimental methods.

[0034] Unless otherwise specified, the materials and reagents used in the following examples can be purchased from commercial sources.

[0035] The present invention is further described below in conjunction with specific embodiments.

[0036] The main test materials used in the following examples are: The test materials are alpine azaleas with obvious flower color differences planted in the Baihejian azalea germplasm resource bank in Yunhe County. Three plants with good growth and consistent height and growth are randomly selected for sample collection for each variety.

[0037] In April 2024, the complete petals of azalea, rhododendron, azalea, white ice azalea and rhododendron were collected. Figure 1 ), three replicates were collected from each tree in each period, placed in centrifuge tubes, quickly frozen with liquid nitrogen, and stored at -80°C until use.

[0038] Example

[0039] 1. Total RNA extraction from plant tissues and cDNA synthesis

[0040] Total RNA from 6 different color alpine azalea petals was isolated using RNAprep Pure Polysaccharide Polyphenol Plant Total RNAExtrLifACTion Kit (TIANGEN, NanJing, China), and RNA concentration and quality were detected by spectrophotometer (METTLER TOLEDO, Swiss). RNA was detected by 1.0% agarose gel electrophoresis. The preparation of cDNA reverse transcription reaction system for real-time fluorescence relative quantitative PCR analysis was referred to the instructions of One-StepgDNA Removal and cDNA Synthesis SuperMix (Trans, NanJing, China), and 20μL cDNA reverse transcription reaction system was established using 1μg total RNA. After the reaction was completed, it was stored at -20℃ for use.

[0041] 2. Selection of internal reference genes and design of primers

[0042] According to the literature, the main reference genes in other plants were searched and 11 reference genes required for the experiment were finally designed, namely RlaACT (SEQ ID NO.1), RlaEF1-α (SEQ ID NO.2), Rlaα-TUB1 (SEQ ID NO.7), Rlaα-TUB2 (SEQ ID NO.8), RlaDnaJ (SEQ ID NO.9), RlaCDC2 (SEQ ID NO.10), RlaH2A (SEQ IDNO.11), RlaPP2A (SEQ ID NO.12), RlaHIS (SEQ ID NO.13), RlaVHAC (SEQ ID NO.14), and RlaOTU (SEQ ID NO.15).

[0043] First, the full-length sequence and full-length transcript sequence of each candidate gene were determined by comparing with the genome sequence of Rhododendron australis. Primers were designed using Primer Premier 5.0 according to the full-length transcript sequence of the gene, and the primer specificity was identified using the BLAST tool. All primers were synthesized by Sangon Biotech Co., Ltd. (Table 1). Primers were preliminarily screened by ordinary PCR, and the bands of PCR products were observed using agarose gel and gel imaging system to detect product specificity ( Figure 2 ). Primers with correct band size, good band specificity and no primer dimers were selected, and primer specificity was further tested by qRT-PCR. Primers with a single peak, no mixed peaks and no peaks in the negative control were selected as the final primers.

[0044] Table 1 Internal reference genes and their corresponding primers

[0045]

[0046] 3. 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 reverse transcribed cDNA was diluted by a multiple of 5 into 5 gradients (1, 1 / 5, 1 / 25, 1 / 125, 1 / 625) as a template for establishing the standard curve. At the same time, ddH2O was used as a negative control template to detect reagents or human contamination during the experiment. All samples were repeated 3 times to ensure the credibility of the experimental data. qRT-PCR was performed using QIAquant 96 2plex (QIAGEN, Germany), and the amplification efficiency and slope of each candidate gene were obtained using the obtained data results. The fluorescence quantitative PCR amplification efficiency requires that the amplification efficiency of the selected primers be between 90% and 120%.

[0048] 4. Real-time fluorescence quantification

[0049] qRT-PCR was performed using QIAquant 96 2plex (QIAGEN, Germany). Each reaction contained 4 pM of forward and reverse primers, 2 μL of cDNA template diluted at different times, and 10 μL of PowerUp TM SYBR TM Green Master Mix (Thermo Fisher, USA), add ddH2O to 20 μL reaction system. Each reaction system was repeated three times. The qRT-PCR reaction conditions were: 95°C for 3 minutes, followed by 40 cycles of 95°C for 15 seconds, 60°C for 15 seconds, and 72°C for 30 seconds. At the end of each experiment, melting curve analysis was performed at 55-95°C with an increment of 0.3°C for 60 seconds using default parameters. All analyses were repeated three times.

[0050] 5. Data processing

[0051] (1) 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 values ​​to ultimately determine the reference gene with better stability.

[0052] (2) 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 calculate the value of the gene expression stability based on V. n / V n+1 The value is used to determine the number of optimal internal reference genes. n / V n+1 <0.15, the optimal gene of the reference gene combination is the top n genes with high stability ranking. n / V n+1 >0.15, the optimal number of internal reference gene combinations is n+1.

[0053] (3) The NormFinder software uses a model-based approach to obtain the expression stability value of the internal reference gene, and then screens the most suitable internal reference gene based on the size of the stability value. The judgment criterion is that the internal reference gene with the smallest expression stability value is the most suitable internal reference gene.

[0054] (4) The Delta Ct algorithm identifies stable reference genes by comparing the relative expression of "gene pairs" in each sample. If the ΔCt value between two genes remains constant when analyzing different samples, it means that the two genes are stably expressed in these samples. However, if the ΔCt fluctuates, the expression of one or both genes is unstable. Introducing a third, fourth, and fifth gene in the comparison provides more information, and then ranking these genes. Ultimately, you can choose the appropriate reference gene based on your experimental needs.

[0055] (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.

[0056] 6. Results

[0057] (1) RNA extraction quality and primer specificity testing

[0058] OD of each sample 260 / OD 280 OD 260 / OD 230 All of them meet the requirements. The agarose gel electrophoresis showed that the 28S and 18S bands were clear and there was no degradation phenomenon, which met the requirements. The melting curves of the 14 pairs of primers in the real-time fluorescence quantitative analysis had only a single peak, and the electrophoresis detection also had only a single band ( Figure 2 ), indicating that the primers used can specifically amplify the corresponding products of each internal reference gene, there is no primer dimer, and the repeatability of the amplification curve of each sample to be tested is good, the template can be specifically amplified, and the real-time fluorescence quantitative results are accurate and reliable.

[0059] (2) Analysis of Ct values ​​of internal reference genes

[0060] The Ct value is inversely proportional to the gene expression level. The larger the Ct value, the lower the gene expression level. Conversely, the smaller the Ct value, the higher the gene expression level. The average Ct value of the 11 reference genes is between 15.25 and 29.93, among which RlaEF1-α and RlaACT have higher expression levels, while RlaPP2A and RlaHIS have lower expression levels ( Figure 3 , Figure 4 ).

[0061] (3) Software Analysis

[0062] BestKeeper software analysis: BesterKeeper software judges the expression stability of genes based on the standard deviation (SD) and coefficient of variation (CV) of the internal reference gene Ct value, and directly analyzes the gene expression Ct value. The smaller the SD value, the more stable the expression. The default critical value of the program is 1. When the SD value is greater than 1, the gene expression is considered unstable. The analysis results show that the SD values ​​of RlaEF1-α, RlaACT, RlaCDC2, RlaDnaJ, Rlaα-TUB1 and RlaH2A are all less than 1, and the SD values ​​of the remaining genes are all greater than 1. The most stable expression is RlaEF1-α, and the most unstable is RlaHIS (Table 2).

[0063] Delta Ct software analysis: Table 2 lists the 11 candidate reference genes from the most stable to the least stable expression in 6 different alpine rhododendron varieties calculated by the Delta Ct method. The analysis results showed that the stability of the reference genes from high to low was as follows: RlaACT>RlaEF1-α>Rlaα-TUB1>RlaOTU>RlaVHAC>RlaDnaJ>RlaCDC2>Rlaα-TUB1>RlaH2A>RlaPP2A>RlaHIS, among which RlaACT and RlaEF1-α were ideal reference genes.

[0064] NormFinder software analysis: NormFinder software evaluates candidate reference genes based on their stability values. The higher the stability value, the worse the stability. The gene with the smallest stability value is the most stable gene. The value of RlaACT is 0.109, which is the most stable gene, and the stability value of RlaHIS is 0.982, which is the most unstable gene (Table 2).

[0065] Table 2 Analysis results of BestKeeper, Delta Ct and NormFinder software

[0066]

[0067] geNorm software analysis: geNorm software measures gene stability based on the average variation M value. The default cut-off value of the software is 1.5. Genes with values ​​higher than 1.5 are not suitable for use as internal references. The lower the M value, the more stable the gene. In addition, geNorm software also uses the pairwise difference value V of the candidate internal reference gene to determine the stability of the gene. n / V n+1 To determine the appropriate number of internal reference genes, when V n / V n+When 1<0.15, n internal reference genes are used. According to the software, V2 / V3=0.112<0.15, so two internal reference genes should be used for fluorescence quantitative analysis of different flower colors of alpine rhododendron ( Figure 5 ), RlaEF1-α and RlaACT( Figure 6 ).

[0068] RefFinder analysis: The stability of 11 candidate reference genes was analyzed by 4 different programs. Different software used different algorithms, so there were some differences in the ranking of gene stability. In order to provide comprehensive results, the analysis results of the 4 algorithms were further calculated by the comprehensive analysis tool RefFinder to perform a geometric mean, conduct a more authoritative evaluation of the stability of the 11 candidate reference genes, and give the best gene stability ranking. The results showed that when all samples were comprehensively analyzed, the two most stably expressed genes were RlaEF1-α and RlaACT. We found that the expression stability of these two genes in the petals of different flower colors of alpine rhododendron ranked in the top five among the four algorithms. In addition, the comprehensive analysis of the reference genes with the lowest expression stability showed that RlaPP2A and RlaHIS were the two most unstable genes (Table 3).

[0069] Table 3 Comprehensive ranking

[0070]

[0071]

[0072] (4) Verification of stability of internal reference genes

[0073] RlaMYB113 (SEQ ID NO. 16), which regulates anthocyanin biosynthesis, was selected to verify the stability of the selected reference genes RlaEF1-α and RlaACT, and compared with the unstable RlaPP2A and RlaHIS genes. It was found that when the stable RlaEF1-α and RlaACT were selected as reference genes, the expression of RlaMYB113 had similar change trends in alpine rhododendrons of different flower colors. The expression level of RlaMYB113 gene was the highest in azaleas with anthocyanin accumulation, and almost no expression was detected in white Rhododendron baibingensis ( Figure 7 ). However, when RlaPP2A and RlaHIS genes were used as internal references, the expression of RlaMYB113 gene was different and no obvious pattern was found ( Figure 7 ), indicating that the screened reference genes RlaEF1-α and RlaACT are accurate and reliable.

[0074] 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. The internal reference gene in the fluorescence quantification of different flower colors of alpine rhododendron, characterized in that: The internal reference gene is RlaACT gene and / or RlaEF1-α gene, the sequence of the RlaACT gene is shown in SEQ ID NO.1, and the sequence of the RlaEF1-α gene is shown in SEQ ID NO.

2.

2. A primer set for detecting the internal reference gene in the fluorescence quantification of different flower colors of Rhododendron azalea according to claim 1, characterized in that: The primer set includes a primer pair 1 for detecting the RlaACT gene, and / or a primer pair 2 for detecting the RlaEF1-α gene.

3. The primer set according to claim 2, characterized in that The primer pair 1 comprises the following primer sequences: RlaACT forward primer 5'-AGCACCCAGTTCTTCTTACA-3' (SEQ ID NO.3) RlaACT reverse primer 5'-GGGCATAACCCTCATAGATAG-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: RlaEF1-α forward primer 5′-ATGATTCCGACCAAGCCTAT-3′ (SEQ ID NO. 5) RlaEF1-α reverse primer 5′-ATCCTTCTTCTCCACGCTCT-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 2-4.

6. Use of the internal reference gene according to claim 1 as an internal reference gene in fluorescence quantification of Rhododendron azalea with different flower colors.

7. Use of the primer set according to any one of claims 2 to 4 or the detection reagent or kit according to claim 5 in the fluorescence quantification of Rhododendron azalea with different flower colors.

8. The use according to claim 7, characterized in that: When using fluorescent quantitative PCR to analyze the transcription of different genes in Rhododendron azalea, a pair of primers in claim 3 is used to detect the internal reference gene RlaACT gene.

9. The use according to claim 7, characterized in that: When using fluorescent quantitative PCR to analyze the transcription of different genes in Rhododendron azalea, two pairs of primers in claim 4 are used to detect the internal reference gene RlaEF1-α gene.

10. The use according to any one of claims 7 to 9, characterized in that: The varieties of alpine rhododendrons with different flower colors are azalea, rhododendron, deer horn rhododendron, azalea, white ice rhododendron and brocade rhododendron.

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