Reference gene in papaya fluorescent quantitative PCR analysis and its screening method and application

Abstract

The present application belongs to the technical field of plant genetic engineering, and particularly relates to an internal reference gene in papaya fluorescent quantitative PCR analysis and a screening method and application thereof. The present application relates to 18S rRNA, Actin, TUB, GAPDH and EF-1 alpha 5 internal reference genes, and the internal reference genes can be applied to the research on gene expression in different varieties, different periods and different tissues of papaya, and provide more accurate gene expression quantification standards for subsequent experimental research on papaya.

Description

TECHNICAL FIELD

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

[0002] Chaenomles speciosa of Rosaceae is a Chinese traditional medicinal and edible plant with a long history and high economic value. The fruit of C. speciosa is also known as papaya. From the perspective of botany, C. speciosa is a perennial shrub with bisexual flowers and a pyrenoid structure. The development process of the fruit includes the stages of fruit setting, swelling, near maturity, maturity, and post-maturity. The fruit at the near maturity stage can be used as a traditional Chinese medicine, and the fruit at the post-maturity stage can be used for the development of functional foods such as fruit wine and preserved fruit. The branches and leaves of C. speciosa can be used for extraction. C. speciosa is widely cultivated in China, and Anhui Province has been a native producing area since the Song Dynasty. The genetic regulation mechanism and molecular breeding of C. speciosa are particularly important due to its high market application prospect.

[0003] The key prerequisite for studying the genetic regulation mechanism at the molecular level is accurate gene expression quantitative analysis. Real-time fluorescence quantitative PCR (qRT-PCR) has become the primary method for gene expression analysis due to its specificity, high sensitivity, and repeatability. This method requires reference genes (RGs) to normalize the data of target genes. Inappropriate RGs may lead to deviations and low repeatability of the actual gene expression amount. However, due to the particularity of plant components, different conditions of plant materials such as tissues, environments, and varieties can cause different suitable RGs.

[0004] The commonly used internal reference genes at present include tubulin (TUB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ubiquitin (UBI / UBQ) and the like. However, the expression stability of these traditional RGs has not been systematically screened and verified, and only based on the assumption that they have constant expression levels under any conditions, they are applied to the research of various animals and plants. More and more evidence shows that the stability of RGs will fluctuate to a certain extent under different conditions, including different developmental stages, different plant tissues and different growth environments. For example, RGs have a relatively high coefficient of variation (CoV) value (an index for measuring the stability of gene expression, and the lower the CoV value, the more stable the expression) in Arabidopsis seeds and pollen. This shows that the stability of RGs is different under different conditions, and therefore it is necessary to screen the expression stability of RGs under specific experimental conditions. At present, the only research on papaya internal reference genes is the TUB gene reported by the team of the present inventors, which is only applicable to a special harvesting stage, but not applicable to papaya materials with multiple components, multiple varieties and multiple developmental processes.

[0005] Therefore, it is urgent to screen papaya stable internal reference genes of different varieties, different periods and different tissues to meet the molecular level research of papaya. SUMMARY

[0006] The purpose of the present application is to provide internal reference genes in papaya real-time fluorescent quantitative PCR analysis and a screening method and application thereof, which can screen papaya stable internal reference genes of different varieties, different periods and different tissues, and expand the molecular level research of papaya.

[0007] In order to achieve the above purpose, the present application provides the following technical scheme:

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

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

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

[0011] The different varieties are Luohan'gui and Apple Red; the different periods are near-mature period and mature period.

[0012] Preferably, at least one of the TUB or 18S rRNA genes is used as an internal reference gene in the real-time fluorescent quantitative PCR analysis of the fruits of the same variety and different periods of Chaenomeles speciosa;

[0013] The same variety is Luohan'gui or Apple Red; the different periods are near-mature period and mature period.

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

[0015] The same period is near-mature period or mature period; the different varieties are Luohan'gui and Apple Red.

[0016] Preferably, at least one of the GAPDH or Actin genes is used as an internal reference gene in the real-time fluorescent quantitative PCR analysis of the different tissues of the Apple Red variety of Chaenomeles speciosa;

[0017] The different tissues are stems, leaves, flowers and near-mature period fruits.

[0018] The application also provides a specific primer for amplifying the internal reference gene, and the specific primer sequence of the EF-1α gene is shown as SEQ ID NO. 6-7; the specific primer sequence of the 18S rRNA gene is shown as SEQ ID NO. 10-11; the specific primer sequence of the Actin gene is shown as SEQ ID NO. 12-13; the specific primer sequence of the GAPDH gene is shown as SEQ ID NO. 14-15; and the specific primer group sequence of the TUB gene is shown as SEQ ID NO. 16-17.

[0019] The application also provides application of the specific primer in the real-time fluorescent quantitative PCR analysis of Chaenomeles speciosa.

[0020] The application also provides a screening method of the internal reference gene in the real-time fluorescent quantitative PCR analysis of Chaenomeles speciosa, comprising the following steps:

[0021] (1) Sample collection: collect fruit samples of different periods including near-mature period and mature period of the Luohan'gui and Apple Red varieties of Chaenomeles speciosa, and samples of stem, leaf, flower and near-mature fruit tissues of the Apple Red variety of Chaenomeles speciosa, and then freeze the samples in liquid nitrogen and store them in an ultra-low temperature refrigerator;

[0022] (2) Extraction of total RNA and synthesis of cDNA;

[0023] (3) Selection and primer design of candidate internal reference genes: 8 candidate internal reference gene sequences of 18S rRNA, UBQ, Actin, TUB, cytochrome b561, GAPDH, His and EF-1a were screened from the genome database of papaya or related literatures;

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

[0025] (4) qRT-PCR analysis;

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

[0027] (6) Internal reference gene verification: for different varieties and different periods of papaya fruits, 18S rRNA and TUB genes were selected as target genes; for the same variety of papaya fruits at different periods, TUB and 18S rRNA genes were selected as target genes; for the same period of different varieties of papaya, EF-1a and Actin genes were selected as target genes; for different tissues of apple red variety of papaya, GAPDH and Actin genes were selected as target genes; the expression pattern of the target gene was observed, and the reliability of the ranking result of the stability of the internal reference was verified; if the internal reference gene with good stability is consistent with the expression pattern of the target gene, and the internal reference gene with poor stability is inconsistent with the expression pattern of the target gene, it is proved that the ranking result of the stability of the internal reference is reliable;

[0028] The different varieties of papaya are Luohan'gui and apple red; the different periods are near-mature period and mature period; the different tissues are stem, leaf, flower and near-mature fruit.

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

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

[0031] Preferably, the experimental data processing and analysis in the step (5) determines the expression levels of the eight candidate internal reference genes by using the programs of geNorm, NormFinder, BestKeeper and ΔCt, and finally screens the best internal reference gene in combination with the results of the online program RefFinder.

[0032] Advantages of the present application:

[0033] The present application screens suitable stable internal reference genes from different varieties, different periods and different tissues of papaya. The internal reference genes can improve the accuracy and reliability of the verification of gene expression, especially the internal reference genes that can keep stable expression under different experimental treatments, thereby providing more accurate expression quantization standards for subsequent experimental research. BRIEF DESCRIPTION OF DRAWINGS

[0034] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0035] Figure 1 It is a gel electrophoresis diagram of PCR amplification of the eight candidate internal reference genes of papaya;

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

[0037] Figure 3 It is a Ct value distribution diagram of the eight candidate internal reference genes;

[0038] Figure 4 It is a fold line diagram of the geNorm analysis results of the eight candidate internal reference genes; wherein, the experimental objects of a in the diagram are different varieties (LUM and PUM comparison) of papaya, the experimental objects of b are different varieties (LMA and PMA comparison) of papaya, the experimental objects of c are different periods (LUM and LMA comparison) of papaya, the experimental objects of d are different periods (PUM and PMA comparison) of papaya, and the experimental objects of e are different varieties and different periods of papaya;

[0039] Figure 5 It is a fold line diagram of the Normfinder analysis results of the eight candidate internal reference genes; wherein, the experimental objects of a in the diagram are different varieties (LUM and PUM comparison) of papaya, the experimental objects of b are different varieties (LMA and PMA comparison) of papaya, the experimental objects of c are different periods (LUM and LMA comparison) of papaya, the experimental objects of d are different periods (PUM and PMA comparison) of papaya, and the experimental objects of e are different varieties and different periods of papaya;

[0040] Figure 6 The BestKeeper analysis fold line chart of 8 candidate internal reference genes; wherein the experimental object of the chart a is different varieties (LUM and PUM comparison) of papaya, the experimental object of b is different varieties (LMA and PMA comparison) of papaya, the experimental object of c is different periods (LUM and LMA comparison) of papaya, the experimental object of d is different periods (PUM and PMA comparison) of papaya, and the experimental object of e is different varieties and different periods of papaya;

[0041] Figure 7 The RefFinder analysis column chart of 8 candidate internal reference genes; wherein the experimental object of the chart a is different varieties (LUM and PUM comparison) of papaya, the experimental object of b is different varieties (LMA and PMA comparison) of papaya, the experimental object of c is different periods (LUM and LMA comparison) of papaya, the experimental object of d is different periods (PUM and PMA comparison) of papaya, and the experimental object of e is different varieties and different periods of papaya;

[0042] Figure 8 The Ct value distribution box chart of eight candidate internal reference genes in Example 3;

[0043] Figure 9 The geNorm analysis fold line chart of Example 3;

[0044] Figure 10 The Normfinder analysis fold line chart of Example 3;

[0045] Figure 11 The BestKeeper analysis fold line chart of Example 3;

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

[0047] Figure 13 The RefFinder analysis comprehensive thermal stability geometric mean fold line chart of Example 3;

[0048] Figure 14 The relative expression amount and FPKM comparison chart of CHI, PAL and CHS. DETAILED DESCRIPTION

[0049] The present application provides papaya internal reference genes of different varieties, different periods and different tissues. The Luohanbi of the present application is one of the varieties of Xuan papaya. According to the standard of Xuan papaya association, apple red papaya is one of the excellent variant varieties of geographical indication product “Xuan papaya”.

[0050] In the real-time fluorescent quantitative PCR analysis of the different tissues of the apple red variety of Chaenomeles speciosa, at least one of the GAPDH or Actin genes is used as an internal reference gene; the different tissues are stems, leaves, flowers and near-mature fruits.

[0051] In the real-time fluorescent quantitative PCR analysis of the different varieties and different periods of Chaenomeles speciosa fruits, at least one of the 18S rRNA or TUB genes is used as an internal reference gene; the different varieties are Luohan'ei and apple red; the different periods are near-mature period and mature period.

[0052] In the real-time fluorescent quantitative PCR analysis of the same variety and different periods of Chaenomeles speciosa fruits, at least one of the TUB or 18S rRNA genes is used as an internal reference gene; the same variety is Luohan'ei or apple red; the different periods are near-mature period and mature period.

[0053] In the real-time fluorescent quantitative PCR analysis of the same period and different varieties of Chaenomeles speciosa, at least one of the EF-1 alpha or Actin genes is used as an internal reference gene; the same period is near-mature period or mature period; the different varieties are Luohan'ei and apple red.

[0054]

[0055] The specific primers for amplifying the above-mentioned internal reference genes, the specific primer sequence of the EF-1 alpha gene is as shown in SEQ ID NO. 6-7: the specific primer sequence of the 18S rRNA gene is as shown in SEQ ID NO. 10-11: the specific primer sequence of the Actin gene is as shown in SEQ ID NO. 12-13; the specific primer sequence of the GAPDH gene is as shown in SEQ ID NO. 14-15; and the specific primer group sequence of the TUB gene is as shown in SEQ ID NO. 16-17:

[0056] The primers designed in the present application are intended to improve the accuracy and reliability of verifying gene expression, especially under specific experimental conditions. By comparing the expression stability of different internal reference genes, one or more internal reference genes that can maintain stable expression under different experimental treatments are found, thereby providing more accurate gene expression quantification standards for subsequent experimental research.

[0057] In order to further illustrate the present application, the technical solutions provided by the present application are described in detail below in combination with the drawings and examples, but they should not be understood as limiting the scope of protection of the present application.

[0058] The production process, experimental method or detection method involved in the embodiments of the present application, if not specially specified, are all conventional methods in the prior art, and the name and / or abbreviation thereof all belong to the conventional name in the art, which are very clear and explicit in the related application field. The skilled in the art can understand the conventional process steps and apply the corresponding equipment according to the 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 application do not have special restrictions on the source, and are all conventional products that can be purchased through normal commercial channels, or can be prepared according to the conventional methods well known to those skilled in the art.

[0060] Example 1 Screening of internal reference genes

[0061] 1.1 Collection of samples

[0062] The nearly mature period (UM) and mature period (MA) of two varieties of Chaenomeles speciosa (L) and Chaenomeles speciosa (P) were collected, and the stems, leaves, flowers and nearly mature fruits of Chaenomeles speciosa (P) were used as experimental materials. Each sample was set with 3 biological replicates, a total of 24 samples were treated with liquid nitrogen quick freezing and stored in a ultra-low temperature refrigerator at -80℃ (the nearly mature period fruit material of Chaenomeles speciosa variety is abbreviated as LUM, the mature period fruit material of Chaenomeles speciosa variety is abbreviated as LMA, the nearly mature period fruit material of Chaenomeles speciosa variety is abbreviated as PUM, and the mature period fruit material of Chaenomeles speciosa variety is abbreviated as PMA).

[0063] 1.2 Total RNA extraction and cDNA synthesis

[0064] 50-100 mg of different tissue samples of Chaenomeles speciosa were frozen in liquid nitrogen, then put into a ball mill for crushing. Total RNA was extracted according to the reference manual of the RNA extraction kit, and the obtained RNA was measured for its concentration by ultramicro UV spectrophotometer (Denovix DS-11+), and agarose gel electrophoresis experiment was performed, and the sample was stored at -80℃ for standby. The RNA of different tissues of Chaenomeles speciosa was reverse transcribed to synthesize cDNA according to the method in the reverse transcription kit manual, and the cDNA of different tissues was labeled and stored at -20℃ for standby.

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

[0066] Based on the three-generation transcriptome data of different varieties of Chaenomeles speciosa, periods and tissues, and the common candidate internal reference genes in the literature of related plants, 8 commonly used internal reference genes (18S rRNA, UBQ, Actin, TUB, cytochrome b561, GAPDH, His, EF-1α) were selected as candidates. The specific qRT-PCR primers of the candidate internal reference genes were designed using Premier 6.0 software, the primer length was 17-23 bp, Tm was 57.5-62.5℃, the primers were synthesized by General Biotech (Anhui) Co., Ltd., and the primer information of the candidate genes is shown in Table 1.

[0067] Table 1 Primer information of 8 candidate genes

[0068]

[0069]

[0070] To verify the specificity of each candidate internal control gene primer, primers for RT-PCR amplification were designed using software. The amplification system was 20 μL: 0.5 μL each of forward and reverse primers, 10 μL of 2x M5 super TaqPCRMaster Mix, 1 μL of template cDNA, and 8 μL of sterile water. The amplification program was: 95℃ pre-denaturation for 2 min, 94℃ denaturation for 10 s, 53.5–55℃ annealing for 15 s, and 72℃ extension for 20 s, for 35 cycles. Agarose gel electrophoresis was then performed.

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

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

[0073] Select TB Premix Ex Taq TM The II kit was used to monitor changes in fluorescence signals in real time during the PCR reaction process using an MX3000P real-time PCR instrument manufactured by Agilent Technologies, Inc.

[0074] In order to minimize the impact of temperature changes on enzyme activity and reagent stability during the experiment, the entire preparation of the qRT-PCR reaction system was carried out on ice.

[0075] The specific reaction system composition is as follows: 2×Realab Green PCRFastMixture is used as the core component of the reaction, providing a suitable reaction environment for DNA polymerase, and its volume is 10 μl; 0.4 μl of each of the upstream and downstream primers are added to complement the specific regions of the template DNA and guide the DNA polymerase to amplify along the template; 1 μl of cDNA template is added; finally, ddH2O is added to make up the reaction system to 20 μl to ensure that each reaction component reacts fully at the appropriate concentration.

[0076] Reaction conditions of qRT-PCR: 95℃ pre-denaturation for 30 s to make double-stranded DNA fully denatured, preparing for the subsequent primer binding and DNA polymerization reaction; 95℃ denaturation for 5 s to make DNA double-strand denatured again; 60℃ annealing for 30 s, at which temperature the primers can specifically bind to the template DNA to form a stable primer-template complex, and after 40 cycles of repeated amplification. After amplification, a program of 60-95℃ for 15 s is set, mainly for drawing the melting curve, and the specificity of the amplification product can be judged by analyzing the melting curve to exclude the interference of non-specific amplification products.

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

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

[0079] The mixed cDNA of different varieties (Luohan' and Apple Red) of papaya fruits at different periods (near mature period and mature period) was used as a template for real-time fluorescent quantitative PCR amplification of candidate internal reference genes. The obtained Ct values were processed and analyzed by the software of the qRT-PCR instrument, and the melting curves of each candidate internal reference gene were obtained. The results are shown in Figure 2 that a single peak appeared at Tm value greater than 80℃, without dimers and non-specific amplification, and the curves obtained by three repeated samples were well overlapped, indicating that the primers of each candidate internal reference gene were designed correctly.

[0080] 2.2 Analysis of Ct values of internal reference genes

[0081] The cDNA of two varieties of papaya (Luohan' and Apple Red) and the fruits of papaya at near mature period (UM) and mature period (MA) were used as templates for qRT-PCR amplification of each internal reference gene, and the Ct values were obtained. The box plots of each candidate internal reference gene were made by Graphpad software, and the expression level and stability of each candidate internal reference gene were analyzed according to the size and change range of Ct values.

[0082] The results are shown in Figure 3 that the average Ct values of each candidate internal reference gene were between 19.06 and 25.46. Among them, the average Ct value of EF-1α gene was the lowest, indicating that its expression level in different tissues was the highest, and the average Ct value of GAPDH was the highest, indicating that its expression level was the lowest. In addition, in general, the change range of Ct value of Actin was the smallest; in different varieties (LUM and PUM, LMA and PMA), the change range of Ct value of TUB was the smallest; in different periods (LUM and LMA), the change range of Ct value of Actin was the smallest; in different periods (PUM and PMA), the change range of Ct value of His was the smallest. The smallest change range of Ct value indicated the most stable expression.

[0083] 2.3 geNorm analysis

[0084] The Ct values of different candidate reference genes were transformed into relative expression values Q, and the Q values of different candidate reference genes were input into the geNorm macro operation formula to calculate the expression stability value M. The M value data table is shown in Table 2. Ctmin-Ctsample The Ct values were converted into relative expression values Q, and the Q values of different candidate reference genes were input into the geNorm macro operation formula to calculate the expression stability value M. The M value data table is shown in Table 2.

[0085] Table 2 M value table of 8 candidate reference genes

[0086]

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

[0088] 2.4 Normfinder analysis

[0089] The Normdinder algorithm can select a gene with the best stability according to the qRT-PCR results through stability analysis. Like geNorm analysis, the Ct values of the reference genes need to be transformed into relative expression values Q, and then the Normfinder macro operation in the excel table is used to obtain the expression stability value SV of each candidate reference gene. The SV value data table is shown in Table 3.

[0090] Table 3 SV value table of 8 candidate reference genes

[0091]

[0092] The best stability of the internal reference gene is shown alone, and the smaller the SV, the better the stability. The results are shown in Figure 5 Table 3. In different varieties (LUM and PUM comparison) of papaya, Actin ranked first (SV = 0.239), and Cytochrome b561 ranked last (SV = 0.551) (Table 3; Figure 5 a); In different varieties (LMA and PMA comparison) of papaya, 18S rRNA ranked first (SV = 0.157), and UBQ ranked last (SV = 0.653) (Table 3; Figure 5 b); In different periods (LUM and LMA comparison), His was the best choice (SV = 0.108), and EF-1 alpha was the least stable (SV = 0.607) (Table 3; Figure 5 c); In different periods (PUM and PMA comparison), 18S rRNA was the best choice (SV = 0.244), and EF-1 alpha was the least stable (SV = 0.6) (Table 3; Figure 5 d); 18S rRNA had the highest stability in all samples (SV = 0.323), and EF-1 alpha had the lowest stability (SV = 0.604) (Table 3; Figure 5 e).

[0093] 2.5 BestKeeper analysis

[0094] The BestKeeper software calculates the SD (standard deviation) value and CV (coefficient of variation) value of each candidate internal reference gene in different tissues at different periods by the Ct value to evaluate the stability of the internal reference gene. The lower the SD value and CV value of the internal reference gene, the better the stability. Generally, the stability of the internal reference gene is mainly determined by the SD value.

[0095] As shown in Table 4, Actin is suitable for different varieties (LUM and PUM comparison) of papaya, TUB is suitable for different varieties (LMA and PMA comparison), Actin has the highest stability in different periods (LUM and LMA comparison, PUM and PMA comparison) and total samples, Cytochrome b561, GAPDH, and EF-1 alpha are the least stable in different varieties (LUM and PUM comparison), different varieties (LMA and PMA comparison), different periods (LUM and LMA comparison, PUM and PMA comparison), and total samples. The SD value and CV value of different varieties (LUM and PUM comparison), different varieties (LMA and PMA comparison), different periods (LUM and LMA comparison), different periods (PUM and PMA comparison), and total samples are shown in Figure 6 .

[0096] Table 4 SD value table of 8 candidate internal reference genes

[0097]

[0098] 2.6 Delta Ct analysis method

[0099] The average standard deviation (mSD) of the CT value of the internal reference gene in each sample can be used to evaluate the stability of the internal reference gene. As shown in Table 5, the mSD value of Actin is the lowest; from different varieties (LUM and PUM comparison, LMA and PMA comparison), the mSD value of TUB is the lowest; from different periods (LUM and LMA comparison, PUM and PMA comparison), the mSD value of Actin is the lowest; the lower the mSD, the better the stability.

[0100] Table 5 mSD value table of 8 candidate internal reference genes

[0101]

[0102] 2.7 RefFinder comprehensive evaluation analysis

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

[0104] As shown in Table 6, through this method, it is concluded that EF-1α, Actin are relatively stable in different varieties (LUM and PUM comparison) (a), 18S rRNA, TUB are relatively stable in different varieties LMA and PMA comparison) (b); TUB, 18S rRNA are the most suitable choice in different periods (LUM and LMA comparison) (c), 18S rRNA, Actin are the most suitable choice in different periods (PUM and PMA comparison) (d); 18S rRNA, TUB are more stable than other genes in the total sample (e). Figure 7 Figure 7 a), 18S rRNA, TUB are relatively stable in different varieties LMA and PMA comparison) (b); TUB, 18S rRNA are the most suitable choice in different periods (LUM and LMA comparison) (c), 18S rRNA, Actin are the most suitable choice in different periods (PUM and PMA comparison) (d); 18S rRNA, TUB are more stable than other genes in the total sample (e). Figure 7 Figure 7 a), 18S rRNA, TUB are relatively stable in different varieties LMA and PMA comparison) (b); TUB, 18S rRNA are the most suitable choice in different periods (LUM and LMA comparison) (c), 18S rRNA, Actin are the most suitable choice in different periods (PUM and PMA comparison) (d); 18S rRNA, TUB are more stable than other genes in the total sample (e). Figure 7 Figure 7 a), 18S rRNA, TUB are relatively stable in different varieties LMA and PMA comparison) (b); TUB, 18S rRNA are the most suitable choice in different periods (LUM and LMA comparison) (c), 18S rRNA, Actin are the most suitable choice in different periods (PUM and PMA comparison) (d); 18S rRNA, TUB are more stable than other genes in the total sample (e).

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

[0106] 3.1 Internal reference gene primer specificity analysis​​​

[0107] As same as example 1, single peak appeared at Tm value greater than 80℃, no dimer and non-specific amplification, and the curves of three repeats of each sample were well overlapped, indicating that the primer design of each candidate reference gene was correct.

[0108] 3.2 Analysis of Ct value of reference gene

[0109] The 5-fold diluted cDNA of stems, leaves, flowers and nearly mature fruits of apple red variety of papaya was used as template for qRT-PCR amplification of each reference gene, and the Ct value was obtained to make the box plot of each candidate reference gene. The expression level and stability of each candidate reference gene were analyzed according to the size and range of Ct value.

[0110] As shown in the results, Figure 8 the average Ct value of each candidate reference gene was between 20.37 and 30.31. Among them, the average Ct value of GAPDH gene was the lowest, indicating that its expression level was the highest in different tissues, and the average Ct value of 18S rRNA was the highest, indicating that its expression level was the lowest. In addition, the Ct value of GAPDH had the smallest range, indicating that its expression was the most stable.

[0111] 3.3 geNorm analysis

[0112] After qRT-PCR amplification of different tissues of papaya, the obtained Ct value was converted into relative expression value by the formula: Q = 2 Ctmin -Ctsample -ΔCt, and then the Q value of different reference genes was input into the macro operation formula of geNorm to calculate the expression stability value M.

[0113] As shown in the results, Figure 9 the stability of reference gene was negatively correlated with M value, and the stability of eight candidate reference genes was ranked as Actin = GAPDH (M = 0.443) > His (M = 1.24) > EF-1α (M = 1.487) > TUB (M = 1.709) > UBQ (M = 1.922) > 18S rRNA (M = 2.089) > cytochrome b561 (M = 2.697). Among them, the M value of Actin, GAPD, His and EF-1α was less than 1.5, indicating that they were more suitable as reference genes of papaya compared with the other five reference genes. In addition, the further analysis by geNorm showed that V2 / V3 was less than 0.15 in different varieties and different periods, indicating that the number of most suitable normalization genes was 2.

[0114] 3.4 Normfinder analysis

[0115] Normdinder algorithm will select a best stability gene according to the qRT-PCR results. As geNorm analysis, the Ct value needs to be converted into the relative expression value Q, and then the Normfinder macro operation in the excel table is used to obtain the expression stability value SV of each candidate internal reference gene, and the best internal reference gene with the best stability is displayed separately.

[0116] The results are shown in Figure 10 The M value of each candidate internal reference gene is His > GAPDH > Actin > EF-1a > 18S rRNA > UBQ > TUB > cytochrome b561. Normfider algorithm considers that His is the most suitable internal reference gene.

[0117] 3.5 BestKeeper analysis

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

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

[0120] 3.6 Delta Ct analysis method

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

[0122] The results are shown in Figure 12 , and the stability of the candidate internal reference gene is His > GAPDH > Actin > EF-1a > UBQ > 18S rRNA > TUB > cytochrome b561.

[0123] 3.7 RefFinder comprehensive evaluation analysis

[0124] RefFinder comprehensive analysis and evaluation website is a comprehensive sorting and screening of the analysis results of geNorm, Normfinde, Delta-Ct, BestKeeper, that is, based on the ranking of each internal reference gene in each software, assign appropriate weight, calculate the geometric mean to obtain the comprehensive ranking, the lower the comprehensive stability geometric mean, the better the expression stability of the candidate internal reference gene.

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

[0126] Example 4 Application of Internal Reference Genes of Different Varieties of Papaya at Different Stages

[0127] In molecular biology research, in order to determine the stability of the target gene, the best stable internal reference gene is selected for verification analysis according to the above analysis, and the key enzyme gene of papaya involved in flavonoid metabolism is selected as the target gene. The relative expression amount is calculated by qRT-PCR real-time fluorescence quantification, and then compared with the FPKM (Fragments Per Kilobase of exon model per Million mapped fragments) of each thousand base transcription per million mapping fragments. According to the results, a comparison chart is made to verify the stability of the key enzyme gene.

[0128] The key enzyme genes CHI, PAL (PAL-1 marked on the figure), and CHS (CHS-2 marked on the figure) of papaya involved in flavonoid metabolism are selected. The specific qRT-PCR primers of the candidate internal reference genes are designed using Premier6.0 software, the primer length is 18-25 bp, Tm is 58-62℃, the primers are synthesized by Universal Biological (Anhui) Co., Ltd., and the primers are shown in Table 6.

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

[0130]

[0131]

[0132] The key enzyme genes and the best internal reference genes TUB and 18S rRNA of different varieties at different stages are subjected to qRT-PCR real-time fluorescence quantification. After the real-time fluorescence quantitative PCR is completed, the data is processed by the software LC96 of the instrument, and the Ct value of each sample is obtained. The relative expression amount of the key enzyme gene is calculated by the 2-ΔCt method, and then compared with the FPKM value of the key enzyme gene to determine the stability of the selected internal reference gene.

[0133] As shown in the results Figure 14 , the relative expression amount trend is the same as the FPKM trend, which proves that the selected internal reference gene has certain stability.

[0134] Although the above embodiments have been described in detail, it should be understood that these are only some embodiments of the present application, but not all embodiments. Other embodiments can be obtained based on the above embodiments without creativity, and these embodiments all belong to the protection scope of the present application.

Claims

1. Application of EF-1α gene as internal reference gene in real-time fluorescent quantitative PCR analysis of different varieties of Chaenomeles fruit in the same period Chaenomles speciosa ); The nucleotide sequence of the EF-1 alpha gene is shown as SEQ ID NO. 1; The same period is near mature or mature period; The different varieties are Luohanbi and apple red.

2. The specific primers of the internal reference gene in claim 1 are applied in the same period, different varieties of papaya (Carica papaya L.) Chaenomles speciosa ) real-time fluorescent quantitative PCR analysis; The sequence of the specific primer is shown as SEQ ID NO. 6-7; The same period is near mature or mature period; The different varieties are Luohanbi and apple red.

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