Internal reference gene in papaya fluorescent quantitative PCR (Polymerase Chain Reaction) analysis as well as screening method and application of internal reference gene

Abstract

The invention belongs to the technical field of plant gene engineering, and particularly relates to a reference gene in papaya fluorescent quantitative PCR (Polymerase Chain Reaction) analysis as well as a screening method and application thereof. The invention relates to five reference genes including 18S rRNA, Actin, TUB, GAPDH and EF-1 alpha, and the reference genes can be applied to research on gene expression of papaya in different varieties, different periods and different tissue ranges, so that a more accurate gene expression quantitative standard is provided for subsequent papaya experimental research.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and particularly relates to an internal reference gene in papaya fluorescence quantitative PCR analysis, a screening method and an application thereof. Background Art

[0002] Chaenomles speciosa, a plant of the genus Chaenomles in the Rosaceae family, is a long-standing Chinese medicinal and edible plant with high economic value. Its fruit is also known as papaya (hereinafter referred to as papaya instead of the botanical name). Botanically, papaya is a perennial shrub with bisexual flowers and a pear-like structure. The fruit develops through stages including fruit set, swelling, near-ripening, maturation, and post-ripening. The near-ripening stage is used as a medicinal plant; the post-ripening stage can be developed into functional foods such as fruit wine and preserved fruit; and the branches and leaves can be used for extraction. Papaya is widely cultivated in China, with Anhui Province having been an authentic production area since the Song Dynasty. Given the papaya's high market potential, its genetic regulation mechanisms and molecular breeding are particularly important.

[0003] The key prerequisite for studying gene regulation mechanisms at the molecular level is accurate quantitative analysis of gene expression. Due to its specificity, high sensitivity, and reproducibility, real-time fluorescence quantitative PCR (qRT-PCR) has become the primary method for gene expression analysis. This method requires internal reference genes (RGs) to average the target gene data. Inappropriate RGs may lead to deviations in the actual gene expression and low reproducibility. However, due to the specificity of plant component composition, different conditions of plant materials such as tissue, environment, and variety will result in different suitable internal reference genes.

[0004] Commonly used internal reference genes include tubulin (TUB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and ubiquitin (UBI / UBQ). However, the expression stability of these traditional RGs has not been systematically screened and validated. Instead, they have been applied to studies of various plants and animals based solely on the assumption that they maintain constant expression levels under all conditions. Increasing evidence indicates that the stability of RGs fluctuates to some extent under different conditions, including developmental stages, plant tissues, and growth environments. For example, RGs in Arabidopsis seeds and pollen exhibit high coefficient of variation (CoV) values ​​(a measure of gene expression stability; lower CoV values ​​indicate more stable expression). This suggests that RGs exhibit variable stability under different conditions, necessitating the screening of RG expression stability under specific experimental conditions. Currently, the only study on a papaya internal reference gene is the TUB gene reported by our group. This gene is only applicable to specific harvest stages and is not suitable for papaya materials with multiple components, varieties, and developmental stages.

[0005] Therefore, there is an urgent need to screen stable internal reference genes of papaya of different varieties, different periods and different tissues to meet the needs of papaya molecular level research. Summary of the Invention

[0006] The purpose of the present invention is to provide an internal reference gene for papaya fluorescence quantitative PCR analysis, a screening method and an application thereof. The present invention can screen out stable internal reference genes of papaya of different varieties, different periods and different tissues, and expand the molecular level research of papaya.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides the use of EF-1α, Actin, TUB, 18S rRNA or GAPDH gene as an internal reference gene in papaya real-time fluorescence quantitative PCR analysis;

[0009] The nucleotide sequence of the EF-1α is shown in SEQ ID NO.1; the nucleotide sequence of the Actin is shown in SEQ ID NO.2; the nucleotide sequence of the TUB is shown in SEQ ID NO.3; the nucleotide sequence of the 18S rRNA is shown in SEQ ID NO.4; and the nucleotide sequence of the GAPDH is shown in SEQ ID NO.5.

[0010] Preferably, when analyzing papaya fruits of different varieties and different periods in the real-time fluorescence quantitative PCR analysis, at least one of the 18S rRNA or TUB gene is used as an internal reference gene;

[0011] The different varieties are Luo Han Qi and Apple Red; the different periods are near-maturity period and maturity period.

[0012] Preferably, when analyzing papaya fruits of the same variety but at different stages in the real-time fluorescence quantitative PCR analysis, at least one of the TUB or 18S rRNA gene is used as an internal reference gene;

[0013] The same variety is Luo Han Qi or Apple Red; the different periods are the near-maturity period and the maturity period.

[0014] Preferably, when analyzing papaya fruits of different varieties at the same period in the real-time fluorescence quantitative PCR analysis, at least one of the EF-1α or Actin genes is used as an internal reference gene;

[0015] The same period is the near-maturity period or the maturity period; the different varieties are Luo Han Qi and Apple Red.

[0016] Preferably, when analyzing different tissues of the apple red variety of papaya in the real-time fluorescence quantitative PCR analysis, at least one of the GAPDH or Actin genes is used as an internal reference gene;

[0017] The different tissues are stems, leaves, flowers and nearly mature fruits.

[0018] The present invention also provides specific primers for amplifying the internal reference gene according to claim 1, wherein the specific primer sequences of the EF-1α gene are shown in SEQ ID NOs. 6-7; the specific primer sequences of the 18S rRNA gene are shown in SEQ ID NOs. 10-11; the specific primer sequences of the Actin gene are shown in SEQ ID NOs. 12-13; the specific primer sequences of the GAPDH gene are shown in SEQ ID NOs. 14-15; and the specific primer set sequence of the TUB gene is shown in SEQ ID NOs. 16-17.

[0019] The present invention also provides application of the specific primers in real-time fluorescence quantitative PCR analysis of papaya.

[0020] The present invention also provides a method for screening internal reference genes in real-time fluorescence quantitative PCR analysis of papaya, comprising the following steps:

[0021] (1) Sample collection: Fruit samples of the papaya varieties Luohanna and Pingguohong at different stages, including near-mature and mature stages, as well as samples of the stem, leaf, flower, and near-mature fruit tissues of the Pingguohong variety of papaya, were collected. The samples were quickly frozen with liquid nitrogen and stored in an ultra-low temperature refrigerator.

[0022] (2) Total RNA extraction and cDNA synthesis;

[0023] (3) Selection of candidate internal reference genes and primer design: Eight candidate internal reference gene sequences, including 18SrRNA, UBQ, Actin, TUB, cytochrome b561, GAPDH, His, and EF-1α, were screened from the papaya genome database or relevant literature;

[0024] The nucleotide sequence of EF-1α is shown in SEQ ID NO.1; the nucleotide sequence of Actin is shown in SEQ ID NO.2; the nucleotide sequence of TUB is shown in SEQ ID NO.3; the nucleotide sequence of 18S rRNA is shown in SEQ ID NO.4; and the nucleotide sequence of GAPDH is shown in SEQ ID NO.5.

[0025] (4) qRT-PCR analysis;

[0026] (5) Experimental data processing and analysis;

[0027] (6) Verification of internal reference genes: For papaya fruits of different varieties and different periods, 18S rRNA and TUB genes were selected as target genes; for papaya fruits of the same variety at different periods, TUB and 18S rRNA genes were selected as target genes; for papaya fruits of different varieties at the same period, EF-1α and Actin genes were selected as target genes; for different tissues of the apple red variety of papaya, GAPDH and Actin genes were selected as target genes; the expression pattern of the target gene was observed to verify the reliability of the ranking results of the internal reference expression stability; if the expression pattern of the internal reference gene with good stability is consistent with that of the target gene, and the expression pattern of the gene with poor stability is inconsistent with that of the target gene, then the ranking results of the internal reference expression stability are reliable;

[0028] The different varieties of papaya are Luo Han Navel and Apple Red; the different periods are the near-mature period and the mature period; and the different tissues are stems, leaves, flowers and nearly-mature fruits.

[0029] Preferably, the reaction system for qRT-PCR analysis in step (4) is: 10 μl of DNA polymerase; 0.4 μl of upstream and downstream primers, 1 μl of cDNA template, and ddH2O is added to make up the reaction system to 20 μl;

[0030] The reaction conditions of the qRT-PCR were as follows: pre-denaturation at 95°C for 30 s, denaturation at 95°C for 5 s, and annealing at 60°C for 30 s, for a total of 40 cycles.

[0031] Preferably, in the step (5), the experimental data processing and analysis utilizes geNorm, NormFinder, BestKeeper and ΔCt software to measure the expression levels of 8 candidate internal reference genes, and finally the best internal reference gene is comprehensively screened out in combination with the results of the online program RefFinder.

[0032] Beneficial effects of the present invention:

[0033] The present invention screened suitable stable internal reference genes based on three conditions: different papaya varieties, different periods, and different tissues. These internal reference genes can improve the accuracy and reliability of verifying gene expression, especially those that maintain stable expression under different experimental treatments, thereby providing more accurate expression quantification standards for subsequent experimental studies. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0035] Figure 1 The figure is the gel electrophoresis of PCR amplification of 8 candidate internal reference genes;

[0036] Figure 2 is the melting curve of 8 candidate internal reference genes of papaya;

[0037] Figure 3 The Ct value distribution diagram of 8 candidate internal reference genes;

[0038] Figure 4 The figure shows the line graph of the geNorm analysis results of 8 candidate internal reference genes; among them, the experimental subjects in figure a are papayas of different varieties (comparison between LUM and PUM), the experimental subjects in figure b are papayas of different varieties (comparison between LMA and PMA), the experimental subjects in figure c are papayas of different periods (comparison between LUM and LMA), the experimental subjects in figure d are papayas of different periods (comparison between PUM and PMA), and the experimental subjects in figure e are papayas of different varieties at different periods.

[0039] Figure 5 The figure shows the line graph of the Normfinder analysis results of 8 candidate internal reference genes; among them, the experimental subjects in figure a are papayas of different varieties (comparison between LUM and PUM), the experimental subjects in figure b are papayas of different varieties (comparison between LMA and PMA), the experimental subjects in figure c are papayas of different periods (comparison between LUM and LMA), the experimental subjects in figure d are papayas of different periods (comparison between PUM and PMA), and the experimental subjects in figure e are papayas of different varieties at different periods.

[0040] Figure 6 The following is a line graph of BestKeeper analysis of 8 candidate internal reference genes; among them, the experimental subjects in figure a are papayas of different varieties (comparison between LUM and PUM), the experimental subjects in figure b are papayas of different varieties (comparison between LMA and PMA), the experimental subjects in figure c are papayas of different periods (comparison between LUM and LMA), the experimental subjects in figure d are papayas of different periods (comparison between PUM and PMA), and the experimental subjects in figure e are papayas of different varieties and periods.

[0041] Figure 7 The RefFinder analysis histograms of eight candidate internal reference genes are shown in Figure a. The experimental subjects are different varieties of papaya (comparison between LUM and PUM), different varieties of papaya (comparison between LMA and PMA), different periods of papaya (comparison between LUM and LMA), different periods of papaya (comparison between PUM and PMA), and different varieties of papaya (comparison between PUM and PMA).

[0042] Figure 8 This is a box plot of the Ct value distribution of the eight candidate internal reference genes in Example 3;

[0043] Figure 9 This is a line graph of the geNorm analysis of Example 3;

[0044] Figure 10 This is the Normfinder analysis line graph of Example 3;

[0045] Figure 11 This is the BestKeeper analysis line chart of Example 3;

[0046] Figure 12 The mSD value line graph of eight candidate internal reference genes;

[0047] Figure 13 This is a line graph showing the geometric mean of the comprehensive thermal stability of the RefFinder analysis in Example 3;

[0048] Figure 14 The figure shows the comparison of the relative expression levels of CHI, PAL and CHS and FPKM. DETAILED DESCRIPTION

[0049] The present invention provides reference genes for papayas of different varieties, different periods, and different tissues. The Luohan navel described in the present invention is a type of Xuan papaya variety 1. According to the standards of the Xuan papaya Association, Apple Red papaya is one of the superior variants of the geographical indication product "Xuan papaya."

[0050] In the real-time fluorescence quantitative PCR analysis of the present invention, when analyzing different tissues of the apple red variety of papaya, at least one of the GAPDH or Actin genes is used as an internal reference gene; the different tissues are stems, leaves, flowers and nearly mature fruits.

[0051] In the real-time fluorescence quantitative PCR analysis of the present invention, when analyzing papaya fruits of different varieties and different periods, at least one of the 18S rRNA or TUB gene is used as an internal reference gene; the different varieties are Luo Han Qi and Ping Guo Hong; and the different periods are the near-maturity stage and the maturity stage.

[0052] In the real-time fluorescence quantitative PCR analysis of the present invention, when analyzing papaya fruits of the same variety but different stages, at least one of the TUB or 18S rRNA gene is used as an internal reference gene; the same variety is Luo Han Qi or Ping Guo Hong; and the different stages are the near-maturity stage and the mature stage.

[0053] In the real-time fluorescence quantitative PCR analysis of the present invention, when analyzing papayas of different varieties at the same period, at least one of the EF-1α or Actin gene is used as an internal reference gene; the same period is the near-maturity period or the mature period; and the different varieties are Luo Han Qi and Ping Guo Hong.

[0054]

[0055] The specific primers used in the present invention to amplify the above-mentioned internal reference genes are as shown in SEQ ID NOs. 6-7 for the specific primer sequences of the EF-1α gene; SEQ ID NOs. 10-11 for the specific primer sequences of the 18S rRNA gene; SEQ ID NOs. 12-13 for the specific primer sequences of the Actin gene; SEQ ID NOs. 14-15 for the specific primer sequences of the GAPDH gene; and SEQ ID NOs. 16-17 for the specific primer set of the TUB gene.

[0056] The primers designed in this paper are designed to improve the accuracy and reliability of gene expression verification, especially under specific experimental conditions. By comparing the expression stability of different reference genes, one or more reference genes that maintain stable expression under different experimental treatments can be identified, providing a more precise standard for gene expression quantification for subsequent experimental studies.

[0057] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0058] Unless otherwise specified, the production processes, experimental methods or detection methods involved in the embodiments of the present invention are all conventional methods in the prior art, and their names and / or abbreviations are conventional names in the field and are very clear and unambiguous in the relevant fields of use. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment to implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0059] The various instruments, equipment, raw materials or reagents used in the embodiments of the present invention are not particularly limited in their sources and are all conventional products that can be purchased through regular commercial channels or prepared according to conventional methods well known to those skilled in the art.

[0060] Example 1 Screening of internal reference genes

[0061] 1.1 Sample collection

[0062] Two papaya varieties, Luohanqi (L) and Pingguohong (P), were collected at their near-maturity (UM) and near-maturity (MA) stages. Stems, leaves, flowers, and near-maturity fruits of Pingguohong (P) were used as experimental materials. Three biological replicates were set for each sample, for a total of 24 samples. After processing, the samples were quickly frozen in liquid nitrogen and stored at -80°C. (Hereinafter, the near-maturity fruit materials of the Luohanqi variety are represented by the abbreviations LUM, the mature fruit materials of the Luohanqi variety are represented by the abbreviations LMA, the near-maturity fruit materials of the Pingguohong variety are represented by the abbreviations PUM, and the mature fruit materials of the Pingguohong variety are represented by the abbreviations PMA.)

[0063] 1.2 Total RNA extraction and cDNA synthesis

[0064] 50-100 mg of papaya tissue samples were taken from different papaya tissues and quickly frozen in liquid nitrogen, then pulverized in a ball mill. Total RNA was extracted according to the instructions of the RNA extraction kit. The resulting RNA concentration was measured using an ultra-micro UV spectrophotometer (Denovix DS-11+) and subjected to agarose gel electrophoresis. Samples were stored at -80°C until further use. RNA from different papaya tissues was reverse transcribed into cDNA according to the instructions of the reverse transcription kit. The cDNA from different tissues was labeled and stored at -20°C until further use.

[0065] 1.3 Primer design and PCR amplification of candidate internal reference genes

[0066] Based on the three-generation transcriptome data of different papaya varieties, periods, and tissues and the common candidate internal reference genes in the literature of related plant families and genera, eight commonly used internal reference genes (18S rRNA, UBQ, Actin, TUB, cytochrome b561, GAPDH, His, and EF-1α) were selected as candidates. Specific qRT-PCR primers for the candidate internal reference genes were designed using Premier 6.0 software. The primers were 17-23 bp in length and had a Tm of 57.5-62.5°C. The primers were synthesized by General Biotechnology (Anhui) Co., Ltd. The primer information of the candidate genes is shown in Table 1.

[0067] Table 1 Primer information of 18 candidate genes

[0068]

[0069]

[0070] To verify the specificity of each candidate internal reference gene primer, software was used to design primers for RT-PCR amplification. The amplification system consisted of 20 μl of the following: 0.5 μl of each upstream and downstream primer, 10 μl of 2x M5 super TaqPCR Master Mix, 1 μl of template cDNA, and 8 μl of sterile water. The amplification protocol was as follows: 95°C pre-denaturation for 2 min, 94°C denaturation for 10 s, 53.5-55°C annealing for 15 s, and 72°C extension for 20 s, for 35 cycles. Agarose gel electrophoresis was then performed.

[0071] Agarose gel electrophoresis results Figure 1 As shown, conventional PCR amplification was performed with the cloning primers of eight candidate internal reference genes of papaya using the cDNA obtained by reverse transcription of papaya RNA as a template. The amplified products were subjected to gel electrophoresis experiments to obtain a single band with bp between 80-400, which was consistent with the expected results.

[0072] 1.4 Real-time fluorescence quantitative PCR of internal reference genes

[0073] Choose TB Premix Ex Taq TM II kit, and the changes in fluorescence signals during the PCR reaction were monitored in real time using the MX3000P fluorescence quantitative PCR instrument produced by Agilent Technologies, Inc., USA.

[0074] During the experimental operation, in order to minimize the impact of temperature changes on enzyme activity and reagent stability in the reaction system, the qRT-PCR reaction system was configured on ice throughout the entire process.

[0075] The specific reaction system consists of: 10 μl of 2×Realab Green PCRFastMixture, the core component of the reaction, provides a suitable reaction environment for the DNA polymerase; 0.4 μl of each upstream and downstream primer is added to complementarily bind to specific regions of the template DNA, guiding the DNA polymerase to amplify along the template; 1 μl of the cDNA template is added; and finally, ddH2O is added to make up the reaction system to 20 μl to ensure that each reaction component is fully reacted at the appropriate concentration.

[0076] qRT-PCR reaction conditions include: pre-denaturation at 95°C for 30 seconds to fully unwind the double-stranded DNA, preparing for subsequent primer binding and DNA polymerization; denaturation at 95°C for 5 seconds to further unwind the DNA double strands; annealing at 60°C for 30 seconds, at which the primers specifically bind to the template DNA, forming a stable primer-template complex. Amplification is repeated for 40 cycles. After amplification, a program is set to 60-95°C for 15 seconds, primarily for plotting a melting curve. Analysis of the melting curve can be used to determine the specificity of the amplified product and eliminate interference from nonspecific amplification products.

[0077] Example 2 Analysis of internal reference genes and primers

[0078] 2.1 Analysis of primer specificity of internal reference gene

[0079] Using mixed cDNA of papaya fruits from different varieties (Luo Han navel and Ping Guohong) at different stages (near maturity and maturity) as templates, real-time fluorescence quantitative PCR amplification of candidate internal reference genes was performed. The obtained Ct values ​​were processed and analyzed using the software provided by the qRT-PCR instrument to obtain the melting curve of each candidate internal reference gene. The results are shown in Figure 2. Figure 2 As shown in the figure, a single peak appeared when the Tm value was greater than 80℃, without dimers and non-specific amplification, and the curves obtained by three replicates of each sample had good coincidence, indicating that the primers of each candidate internal reference gene were correctly designed.

[0080] 2.2 Ct value analysis of internal reference genes

[0081] Using cDNA from papaya Luohanumi (L) and Apple Red (P) varieties and papaya fruits at the near-maturity (UM) and maturity (MA) stages as templates, qRT-PCR amplification of each reference gene was performed to obtain Ct values. Graphpad software was used to create box plots of each candidate reference gene, and the expression level and stability of each candidate reference gene were analyzed based on the size and variation range of the Ct values.

[0082] The results are as follows Figure 3 As shown, the average Ct values ​​of the candidate reference genes ranged from 19.06 to 25.46. EF-1α had the lowest average Ct value, indicating the highest expression level across tissues. GAPDH had the highest average Ct value, indicating the lowest expression level. Furthermore, Actin had the smallest Ct value range overall. Among different varieties (LUM and PUM, LMA and PMA), TUB had the smallest Ct value range. Among different stages (LUM and LMA), Actin had the smallest Ct value range. And among different stages (PUM and PMA), His had the smallest Ct value range. The smallest Ct value range indicates the most stable expression.

[0083] 2.3geNorm analysis

[0084] After qRT-PCR amplification of papaya fruits of different varieties (Luohan navel and Apple red) at different developmental stages (near maturity and maturity), the obtained Ct values ​​were calculated using the formula: Q = 2 Ctmin-Ctsample The Ct value was converted into a relative expression value Q, and the obtained Q values ​​of different internal reference genes were input into the geNorm macro calculation formula to calculate the expression stability value M. The obtained M value data table is shown in Table 2.

[0085] Table 2 M values ​​of 8 candidate internal reference genes

[0086]

[0087] The stability of the reference gene is negatively correlated with the M value. The smaller the M value, the higher the expression stability. Figure 4 As shown, UBQ / EF-1α is the most suitable reference gene combination for different varieties of papaya (LUM and PUM comparison) ( Table 2 ; Figure 4 a), Actin / Cytochrome b561 was the most suitable reference gene combination for different varieties of papaya (comparison of LMA and PMA) ( Table 2 ; Figure 4 b), 18SrRNA / Cytochrome b561 was the most suitable reference gene combination for papaya at different stages (LUM and LMA comparison) ( Table 2 ; Figure 4 c), Actin / Cytochrome b561 was the most suitable reference gene combination for papaya at different stages (comparison between PUM and PMA) ( Table 2 ; Figure 4 d), 18S rRNA / TUB was the most suitable reference gene combination for different varieties and different stages of papaya ( Table 2 ; Figure 4 e). In addition, further analysis using geNorm showed that the V2 / V3 ratio was less than 0.15 in all varieties and at different stages, indicating that the most suitable number of genes for normalization is 2.

[0088] 2.4Normfinder analysis

[0089] The Normdinder algorithm uses stability analysis based on qRT-PCR results to select the most stable gene. Similar to geNorm analysis, the Ct values ​​of the reference genes need to be converted into relative expression values ​​(Q). Then, using the Normfinder macro in Excel, the expression stability values ​​(SV) of each candidate reference gene are calculated. The resulting SV value data table is shown in Table 3.

[0090] Table 3 SV values ​​of 8 candidate internal reference genes

[0091]

[0092] The reference gene with the best stability will be displayed separately. The smaller the SV, the better the stability. Figure 5 As shown, Actin ranked first (SV = 0.239) and Cytochrome b561 ranked last (SV = 0.551) among different varieties (comparison of LUM and PUM) of papaya ( Table 3 ; Figure 5 a); Among the different varieties of papaya (comparison between LMA and PMA), 18S rRNA ranked first (SV = 0.157), and UBQ ranked last (SV = 0.653) (Table 3; Figure 5 b); His was the best choice (SV = 0.108) and EF-1α was the most unstable (SV = 0.607) at different stages (comparison between LUM and LMA) (Table 3; Figure 5 c); 18S rRNA was the best choice (SV = 0.244) and EF-1α was the most unstable (SV = 0.6) at different stages (PUM and PMA comparison) (Table 3; Figure 5 d); 18S rRNA had the highest expression stability among all samples (SV = 0.323), while EF-1α had the lowest stability (SV = 0.604) (Table 3; Figure 5 e).

[0093] 2.5 BestKeeper Analysis

[0094] BestKeeper software evaluates the stability of reference genes by calculating the SD (standard deviation) and CV (coefficient of variation) values ​​of each candidate reference gene from its Ct value in different tissues and at different time periods. Lower SD and CV values ​​indicate better stability. Generally speaking, the stability of a reference gene is primarily determined by its SD value.

[0095] The results are shown in Table 4. Actin is suitable for different varieties of papaya (LUM and PUM comparison), TUB is suitable for different varieties (LMA and PMA comparison), and Actin has the highest stability in samples collected at different stages (LUM and LMA comparison, PUM and PMA comparison) and the total sample. Cytochrome b561, GAPDH, and EF-1α are the least stable in different varieties (LUM and PUM comparison), different varieties (LMA and PMA comparison), different stages (LUM and LMA comparison, PUM and PMA comparison), and the total sample, respectively. The SD and CV values ​​of different varieties (LUM and PUM comparison), different varieties (LMA and PMA comparison), different stages (LUM and LMA comparison), different stages (PUM and PMA comparison), and the total sample are shown in the line graphs. Figure 6 shown.

[0096] Table 4 SD values ​​of 8 candidate internal reference genes

[0097]

[0098] 2.6 Delta Ct analysis

[0099] The mean standard deviation (mSD) of the internal reference gene CT value across samples can be used to evaluate the stability of the reference gene. As shown in Table 5, Actin had the lowest mSD value. TUB had the lowest mSD value across varieties (LUM vs. PUM, LMA vs. PMA), and Actin had the lowest mSD value across time periods (LUM vs. LMA, PUM vs. PMA). Lower mSD values ​​indicate better stability.

[0100] Table 5 mSD values ​​of 8 candidate internal reference genes

[0101]

[0102] 2.7 RefFinder Comprehensive Evaluation and Analysis

[0103] The RefFinder comprehensive analysis and evaluation website performs comprehensive ranking and screening based on the analysis results of geNorm, Normfinde, ΔCt, and BestKeeper. That is, based on the ranking of each reference gene in each software and algorithm, it assigns appropriate weights to each reference gene and calculates the geometric mean to obtain a comprehensive ranking. The lower the geometric mean of comprehensive stability, the better the expression stability of the candidate reference gene.

[0104] like Figure 7 As shown in the results, the expression of EF-1α and Actin in different varieties (LUM and PUM) was relatively stable ( Figure 7 a), 18S rRNA and TUB expression was relatively stable in different varieties of LMA and PMA ( Figure 7 b); TUB and 18S rRNA are the most suitable choices in different periods (comparison between LUM and LMA) ( Figure 7 c), 18S rRNA and Actin are the most suitable choices in different stages (PUM and PMA comparison) ( Figure 7 d); 18S rRNA and TUB were more stable than other genes in the total samples ( Figure 7 e).

[0105] Example 3 Verification and analysis of reference genes in different tissues of papaya of the same variety

[0106] 3.1 Analysis of primer specificity of internal reference gene

[0107] As in Example 1, a single peak appeared when the Tm value was greater than 80°C, with no dimer or nonspecific amplification, and the curves obtained by three replicates of each sample had good coincidence, indicating that the primers of each candidate internal reference gene were correctly designed.

[0108] 3.2 Ct value analysis of internal reference genes

[0109] Using 5-fold diluted cDNA from the stems, leaves, flowers, and nearly mature fruits of the Apple Red papaya variety as templates, qRT-PCR amplification was performed for each reference gene. Ct values ​​were calculated and boxplots were created for each candidate reference gene. The expression level and stability of each candidate reference gene were analyzed based on the magnitude and range of the Ct values.

[0110] The results are as follows Figure 8 As shown, the average Ct values ​​of the candidate reference genes ranged from 20.37 to 30.31. GAPDH had the lowest average Ct value, indicating the highest expression level across tissues. 18S rRNA had the highest average Ct value, indicating the lowest expression level. Furthermore, GAPDH had the smallest range of Ct values, indicating the most stable expression.

[0111] 3.3geNorm analysis

[0112] After qRT-PCR amplification of different papaya tissues, the obtained Ct values ​​were calculated using the formula: Q = 2 Ctmin -Ctsample Convert it into relative expression value, and then input the Q value of different internal reference genes into the geNorm macro calculation formula to calculate the expression stability value M.

[0113] The results are as follows Figure 9 As shown, the stability of the reference genes was negatively correlated with the M value. The stability ranking of the eight candidate reference genes was Actin = GAPDH (M = 0.443) > His (M = 1.24) > EF-1α (M = 1.487) > TUB (M = 1.709) > UB Q (M = 1.922) > 18S rRNA (M = 2.089) > cytochrome b561 (M = 2.697). Among them, Actin, GAPD, His, and EF-1α had M values ​​less than 1.5, indicating that they were more suitable as reference genes for papaya than the other five reference genes. Furthermore, further analysis using geNorm showed that the V2 / V3 ratio was less than 0.15 across different varieties and different growth stages, indicating that the most suitable number of genes for normalization is 2.

[0114] 3.4 Normfinder analysis

[0115] The Normdinder algorithm selects the most stable gene based on the qRT-PCR results. Similar to geNorm analysis, the Ct value is converted to a relative expression value (Q). Using the Normfinder macro in Excel, the expression stability value (SV) for each candidate reference gene is calculated. The reference gene with the highest stability is displayed separately.

[0116] The results are as follows Figure 10 As shown in the figure, the M values ​​of the candidate internal reference genes are His > GAPDH > Actin > EF-1α > 18SrRNA > UBQ > TUB > cytochrome b561. The Normfider algorithm believes that His is the most suitable internal reference gene.

[0117] 3.5 BestKeeper Analysis

[0118] BestKeeper directly uses the Ct values ​​of each candidate reference gene in different tissues to calculate its SD (standard deviation) and CV (coefficient of variation) values. The lower the SD and CV values ​​of the reference gene, the better the stability.

[0119] The results are as follows Figure 11 As shown, Actin is the most stable gene and UBQ is the least stable gene.

[0120] 3.6 Delta Ct analysis

[0121] The Delta Ct analysis method calculates the average standard deviation of the fluorescence quantitative results of each candidate internal reference gene in different tissues of papaya. The lower the calculated average standard deviation, the more stable the expression of the internal reference gene in different tissues of papaya.

[0122] The results are as follows Figure 12 As shown in the figure, the stability of candidate reference genes is His>GAPDH>Actin>EF-1α>UBQ>18S rRNA>TUB>cytochrome b561.

[0123] 3.7 RefFinder Comprehensive Evaluation and Analysis

[0124] The RefFinder comprehensive analysis and evaluation website comprehensively ranks and screens the analysis results of geNorm, Normfinde, Delta-Ct, and BestKeeper. That is, based on the ranking of each reference gene in each software, it assigns appropriate weights to them and calculates the geometric mean to obtain a comprehensive ranking. The lower the geometric mean of comprehensive stability, the better the expression stability of the candidate reference gene.

[0125] The results are as follows Figure 13As shown in the figure, the stability of candidate reference genes analyzed by this method is GAPDH>Actin>His>EF-1α>18S rRNA>UBQ>TUB>cytochrome b561.

[0126] Example 4 Application of internal reference genes of papaya of different varieties and different periods

[0127] In molecular biology research, to determine the stability of the target gene, the internal reference gene with the best stability was selected for verification analysis based on the above analysis. The key enzyme gene involved in flavonoid metabolism in papaya was selected as the target gene. The relative expression level was calculated by real-time fluorescence quantification through qRT-PCR, and then compared with the fragments per kilobase of transcript per million mapped reads FPKM (Fragments Per Kilobase of exonmodel per Million mapped fragments). A comparison chart was made based on the results to verify the stability of the key enzyme gene.

[0128] The key enzyme genes CHI, PAL (labeled PAL-1 on the figure), and CHS (labeled CHS-2 on the figure) involved in flavonoid metabolism in papaya were selected, and specific qRT-PCR primers for the candidate internal reference genes were designed using Premier 6.0 software. The primer length was 18-25 bp, and the Tm was 58-62°C. The primers were synthesized by General Bio (Anhui) Co., Ltd., and the primers are shown in Table 6.

[0129] Table 6 Primer information of 3 flavonoid key enzyme genes

[0130]

[0131]

[0132] qRT-PCR was performed on key enzyme genes and the optimal internal reference genes, TUB and 18S rRNA, obtained from different varieties and at different times. After the completion of the real-time fluorescence quantitative PCR, the data was processed using the instrument's built-in software, LC96, to obtain the Ct value for each sample. The relative expression levels of key enzyme genes were calculated using the 2-ΔCt method and compared with the FPKM values ​​of the key enzyme genes to determine the stability of the selected internal reference genes.

[0133] The results are as follows Figure 14 As shown in the figure, the trend of the relative expression level is the same as that of FPKM, which proves that the selected reference gene has a certain stability.

[0134] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. Application of EF-1α, Actin, TUB, 18S rRNA, or GAPDH genes as internal reference genes in real-time fluorescence quantitative PCR analysis of papaya; The nucleotide sequence of the EF-1α is shown in SEQ ID NO.1; the nucleotide sequence of the Actin is shown in SEQ ID NO.2; the nucleotide sequence of the TUB is shown in SEQ ID NO.3; the nucleotide sequence of the 18S rRNA is shown in SEQ ID NO.4; and the nucleotide sequence of the GAPDH is shown in SEQ ID NO.

5.

2. The application according to claim 1, characterized in that When analyzing papaya fruits of different varieties and different periods in the real-time fluorescence quantitative PCR analysis, at least one of the 18S rRNA or TUB gene is used as an internal reference gene; The different varieties are Luo Han Qi and Apple Red; the different periods are near-maturity period and maturity period.

3. The application according to claim 1, characterized in that When analyzing papaya fruits of the same variety but at different times in the real-time fluorescence quantitative PCR analysis, at least one of the TUB or 18S rRNA gene is used as an internal reference gene; The same variety is Luo Han Qi or Apple Red; the different periods are the near-maturity period and the maturity period.

4. The application according to claim 1, characterized in that When analyzing papaya fruits of different varieties at the same period in the real-time fluorescence quantitative PCR analysis, at least one of the EF-1α or Actin gene is used as an internal reference gene; The same period is the near-maturity period or the maturity period; the different varieties are Luo Han Qi and Apple Red.

5. The application according to claim 1, characterized in that: When analyzing different tissues of the apple red variety of papaya in the real-time fluorescence quantitative PCR analysis, at least one of the GAPDH or Actin genes is used as an internal reference gene; The different tissues are stems, leaves, flowers and nearly mature fruits.

6. A specific primer for amplifying the internal reference gene according to claim 1, characterized in that: The specific primer sequences of the EF-1α gene are shown in SEQ ID NOs.6-7; the specific primer sequences of the 18S rRNA gene are shown in SEQ ID NOs.10-11; the specific primer sequences of the Actin gene are shown in SEQ ID NOs.12-13; the specific primer sequences of the GAPDH gene are shown in SEQ ID NOs.14-15; and the specific primer set sequences of the TUB gene are shown in SEQ ID NOs.16-17.

7. Use of the specific primers according to claim 6 in real-time fluorescence quantitative PCR analysis of papaya.

8. A method for screening internal reference genes in real-time fluorescence quantitative PCR analysis of papaya, characterized in that: The following steps are involved: (1) Sample collection: Fruit samples of the papaya varieties Luohanna and Pingguohong at different stages, including near-mature and mature stages, as well as samples of the stem, leaf, flower, and near-mature fruit tissues of the Pingguohong variety of papaya, were collected. The samples were quickly frozen with liquid nitrogen and stored in an ultra-low temperature refrigerator. (2) Total RNA extraction and cDNA synthesis; (3) Selection of candidate internal reference genes and primer design: Eight candidate internal reference gene sequences, including 18SrRNA, UBQ, Actin, TUB, cytochrome b561, GAPDH, His, and EF-1α, were screened from the papaya genome database or relevant literature; The nucleotide sequence of EF-1α is shown in SEQ ID NO.1; the nucleotide sequence of Actin is shown in SEQ ID NO.2; the nucleotide sequence of TUB is shown in SEQ ID NO.3; the nucleotide sequence of 18S rRNA is shown in SEQ ID NO.4; and the nucleotide sequence of GAPDH is shown in SEQ ID NO.

5. (4) qRT-PCR analysis; (5) Experimental data processing and analysis; (6) Internal reference gene verification: For papaya fruits of different varieties and different periods, 18S rRNA and TUB genes were selected as target genes; for papaya fruits of the same variety at different periods, TUB and 18S rRNA genes were selected as target genes; for papaya fruits of different varieties at the same period, EF-1α and Actin genes were selected as target genes; for different tissues of papaya of the Apple Red variety, GAPDH and Actin genes were selected as target genes; Observe the expression pattern of the target gene and verify the reliability of the ranking results of the internal reference expression stability. If the expression pattern of the internal reference gene with good stability is consistent with that of the target gene, and the expression pattern of the gene with poor stability is inconsistent with that of the target gene, then the ranking results of the internal reference expression stability are reliable. The different varieties of papaya are Luo Han Navel and Apple Red; the different periods are the near-mature period and the mature period; and the different tissues are stems, leaves, flowers and nearly-mature fruits.

9. The screening method according to claim 8, characterized in that The reaction system for qRT-PCR analysis in step (4) is as follows: 10 μl of DNA polymerase; 0.4 μl of each of the upstream and downstream primers, 1 μl of the cDNA template, and ddH2O is added to make up the reaction system to 20 μl; The reaction conditions of the qRT-PCR were as follows: pre-denaturation at 95°C for 30 s, denaturation at 95°C for 5 s, and annealing at 60°C for 30 s, for a total of 40 cycles.

10. The screening method according to claim 8, characterized in that In the step (5), the experimental data were processed and analyzed using geNorm, NormFinder, BestKeeper and ΔCt software to measure the expression levels of 8 candidate internal reference genes, and finally the best internal reference genes were comprehensively screened out in combination with the results of the online program RefFinder.

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