Internal reference gene of ilex rotunda and application thereof

Abstract

This invention relates to the field of molecular biology, specifically to an internal reference gene of whorled holly and its application, and particularly to... Actin2 and / or 28S As an internal reference gene for quantitative analysis of Ilex chinensis genes, the aforementioned Actin2 The nucleotide sequence is shown in SEQ ID NO.1. 28S The nucleotide sequence is shown in SEQ ID NO.2. This invention provides an internal reference gene for analyzing the expression of target genes in samples from different tissues of *Ilex verticillata* at different developmental stages and under different hormone treatments. Actin2 and / or 28S The study also included the detection of primer pairs. This research provides technical support and reliable evidence for the study of gene expression characteristics in whorled holly and subsequent research related to flower buds.

Description

Technical Field

[0001] This invention relates to the field of molecular biology technology, specifically to an internal reference gene of whorled holly and its application. Background Technology

[0002] Whorled holly ( Ilex verticillata Ilex verticillata, also known as North American holly, is a plant belonging to the genus Ilex in the family Aquifoliaceae. It is native to North America and was introduced to my country in 2006. Due to its bright red fruit and long fruiting period (the ornamental period can extend from October to April of the following year), Ilex verticillata is widely used as a potted plant, cut branch, floral material, and landscaping plant, and has extremely high ornamental value.

[0003] Due to a lack of systematic and comprehensive support for related maintenance techniques, the growth of whorled holly in my country is uneven, and the quality of cut branches is far inferior to that of foreign products. This is mainly reflected in the small number of flower buds and sparse fruiting, especially the difficulty in germinating or failing to germinate flower buds at the lower morphological end (difficulty in inducing and forming flower buds), which leads to sparse fruiting or even no fruiting at the lower end of the branches, seriously hindering the further development of whorled holly.

[0004] To address the aforementioned issues, conducting research on the function and expression patterns of genes related to the growth and development of whorled holly at the molecular level is a crucial approach to elucidating its flower bud differentiation and development regulation mechanisms, and ultimately guiding industrial improvement. However, molecular biology research on whorled holly is still in its early stages, and studies on the function and regulatory mechanisms of related genes are rarely reported.

[0005] Real-time quantitative polymerase chain reaction (RT-qPCR) is one of the core technologies for gene expression analysis, boasting advantages such as high sensitivity, high accuracy, high specificity, low cost, and ease of operation. However, in practice, factors such as RNA quality, cDNA synthesis efficiency, and sample loading errors can all affect the accuracy of quantification results. Therefore, it is necessary to standardize and correct the expression level of the target gene using a stable internal reference gene. Ideally, an internal reference gene should be stably expressed in different tissues, developmental stages, and experimental conditions. However, numerous studies have shown that there is no universal internal reference gene that is consistently expressed under all conditions—the expression stability of traditional internal reference genes varies significantly across different species and experimental conditions.

[0006] Therefore, for the species *Ilex whorlatifolia*, screening for specific reference genes that are stably expressed in different tissues and developmental stages is the prerequisite and foundation for conducting gene expression analysis research on this species. Currently, there are no systematic research reports on the screening and evaluation of reference genes in *Ilex whorlatifolia*. Summary of the Invention

[0007] To systematically screen internal reference genes for quantitative real-time PCR of flower buds in different tissues and developmental stages of *Ilex whorlaria*, ensuring stable expression under various conditions and providing a foundation for subsequent research on the molecular mechanisms of *Ilex whorlaria*, this invention proposes an internal reference gene for *Ilex whorlaria* and its application.

[0008] The specific technical solution is as follows: In one aspect, the present invention provides Actin2 and / or 28S As an internal reference gene for quantitative analysis of Ilex chinensis genes, the aforementioned Actin2 The nucleotide sequence is shown in SEQ ID NO.1. 28S The nucleotide sequence is shown in SEQ ID NO.2.

[0009] In one aspect, the present invention also provides the aforementioned Actin2 and / or 28S Application as an internal reference gene for quantitative analysis of Ilex chinensis genes in different tissues.

[0010] Furthermore, the tissues include: roots, stems, and leaves.

[0011] In one aspect, the present invention also provides the aforementioned Actin2 and / or 28S Application as an internal reference gene for quantitative analysis of Ilex henryi genes at different developmental stages.

[0012] Furthermore, the developmental period is the flower bud differentiation period, that is, the stage when the whorled holly transitions from vegetative growth to reproductive growth and the inflorescence primordia begin to differentiate.

[0013] Furthermore, the flower bud differentiation period includes: early stage of flower bud differentiation (S1), inflorescence primordium differentiation period (S2), calyx primordium differentiation period (S3), petal primordium differentiation period (S4), and pistil and stamen primordium differentiation period (S5).

[0014] In one aspect, the present invention also provides the aforementioned Actin2 and / or 28S Application as an internal reference gene for quantitative analysis of Ilex chinensis genes under different hormone treatments.

[0015] Furthermore, the hormone is indole-3-acetic acid, indolebutyric acid, N(-1-naphthyl)-o-carbamoylbenzoic acid, kinetin, abscisic acid, salicylic acid, gibberellin, ethephon, and jasmonic acid.

[0016] In one aspect, the present invention also provides a method for detecting the above. Actin2 and / or 28S The primer pair, the Actin2The nucleotide sequences of the detection primer pairs are shown in SEQ ID NO.29 and SEQ ID NO.30; And / or, the 28S The nucleotide sequences of the detection primer pairs are shown in SEQ ID NO.5 and SEQ ID NO.6.

[0017] In one aspect, the present invention also provides a method for detecting internal reference genes in whorled holly. Actin2 and / or 28S A real-time PCR kit containing the above-mentioned fluorescence. Actin2 Detection primer pairs and / or 28S The detection primer pairs and the reagents for real-time PCR.

[0018] In one aspect, the present invention also provides a real-time quantitative PCR method for detecting the expression level of the internal reference gene of Ilex wilfordii using the above-mentioned kit, comprising the following steps: (1) Total RNA was extracted from Ilex verticillata samples and reverse transcribed into cDNA; (2) Using the cDNA obtained in step (1) as a template, the above-mentioned... Actin2 Detection primer pairs and / or 28S The detection primer pairs were used for real-time quantitative PCR detection.

[0019] Compared with the prior art, the present invention has the following beneficial effects: This invention, starting with different tissues (roots, stems, and leaves), flower buds at different developmental stages, and different hormone treatments in whorled holly, and through extensive data analysis and a rigorous internal reference gene screening procedure, has for the first time identified candidate internal reference genes that are stably expressed in whorled holly. These genes are present at different developmental stages of whorled holly flowers. Actin2 and 28S Gene expression is most stable. The introduction of these internal reference genes can be used for expression analysis of functional genes related to flower bud differentiation in whorled holly, laying a solid foundation for subsequent molecular mechanism analysis, key gene discovery, and molecular design breeding. Attached Figure Description

[0020] Figure 1 Agarose gel electrophoresis image of primers specific to 15 candidate internal reference genes in whorled holly.

[0021] Figure 2A for 18S , 28S , EF-1α , Hsp Melting curves of candidate internal reference genes.

[0022] Figure 2B for CYP , DNAJ , EIF4A , PP2A Melting curves of candidate internal reference genes.

[0023] Figure 2C for PT , TUA , UBQ , Actin2 Melting curves of candidate internal reference genes.

[0024] Figure 2D for TUB , UBC , Actin7 Melting curves of candidate internal reference genes.

[0025] Figure 3 The distribution of Ct values ​​of 15 candidate internal reference genes in different tissues of whorled holly.

[0026] Figure 4 The figure shows the results of GeNorm analysis of 15 candidate internal reference genes in different tissues of holly; among them, Figure 4 In this context, A represents the average stability value (M value) of the 15 candidate internal reference genes. A lower M value indicates more stable expression. Figure 4 In the figure, B represents the pairwise variation (V) calculated by GeNorm, indicating the number of internal reference genes required for qRT-PCR normalization.

[0027] Figure 5 The Ct values ​​are for six candidate internal reference genes under various experimental conditions. Figure 5 In the diagram, A represents flower buds at different developmental stages. Figure 5 In this context, B represents different hormone treatments. Figure 5 In this context, C represents the sum of all samples.

[0028] Figure 6 The mean stability value (M value) and GeNorm ranking of the 6 candidate internal reference genes were determined. Figure 6 In the diagram, A represents flower buds at different developmental stages. Figure 6 In this context, B represents different hormone treatments. Figure 6 In this context, C represents the sum of all samples.

[0029] Figure 7 The paired variants (V) calculated for GeNorm indicate the number of internal reference genes required for qRT-PCR normalization; Figure 7 In the diagram, A represents flower buds at different developmental stages. Figure 7 In this context, B represents different hormone treatments. Figure 7 In this context, C represents the sum of all samples.

[0030] Figure 8 During the differentiation of flower buds of whorled holly IvAGL19 and IvWUS2Gene expression patterns on different reference genes; Figure 8 In this context, A represents the process of flower bud differentiation in whorled holly. IvAGL19 Gene expression patterns on different reference genes; Figure 8 In the text, B represents the process of flower bud differentiation in whorled holly. IvWUS2 Gene expression patterns on different reference genes; the five developmental stages of flower bud differentiation are S1, S2, S3, S4 and S5; error bars represent the mean standard error (n=3). Detailed Implementation

[0031] To better understand the technical content of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the embodiments of this invention are conventional methods. Unless otherwise specified, the materials and reagents used in the embodiments of this invention are commercially available.

[0032] The embodiments described below are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0033] In this invention, the Actin2 The nucleotide sequence is shown in SEQ ID NO.1. 28S The nucleotide sequence is shown in SEQ ID NO.2.

[0034] SEQ ID NO.1:

[0035] SEQ ID NO.2:

[0036] Example 1: Screening of internal reference genes and primer specificity detection 1.1 Sample Collection Different tissue samples (roots, stems, and leaves) were collected from *Ilex whorledii* in the orchard of Zhejiang A&F University. Two-year-old *Ilex whorledii* seedlings in good condition were selected, and flower buds at different developmental stages were collected. Two-year-old seedlings of *Ilex whorledii* (from the greenhouse of Zhejiang A&F University) were sprayed with nine different plant hormones, including indole-3-acetic acid (IAA, 100 μmol·L⁻¹). -1 Indolebutyric acid (IBA, 100 μmol·L⁻¹) -1 N(-1-naphthyl)-o-carbamoylbenzoic acid (NPA, 10 μmol·L⁻¹) -1 Kinetin (KT, 100 μmol·L⁻¹) -1 Abscisic acid (ABA, 100 μmol·L⁻¹) -1 ), salicylic acid (SA, 200 μmol·L) -1 Gibberellin (GA3, 200 μmol·L) -1 Ethephon (ACC, 1 mmol·L) -1 ) and jasmonic acid (MeJA, 100 μmol·L -1 The hormone was applied to the surface of whorled holly leaves, with distilled water serving as a control. The hormone or distilled water was sprayed until the first drop of liquid ran off the leaf surface. Whorled holly leaves were collected at 0 h, 1 h, 3 h, 6 h, and 12 h after treatment. Each sample was tested in triplicate. Samples were immediately stored in liquid nitrogen at -80°C for later use.

[0037] 1.2 Total RNA extraction and reverse transcription cDNA synthesis RNA extraction for each sample was performed according to the instructions of RNAprep Pure polysaccharide polyphenol planttotal RNA extraction kit (DP441; TianGen; China). RNA integrity was assessed by 1.5% agarose gel electrophoresis, and RNA concentration and quality were determined using a NanoDrop 1000 nucleic acid and protein analyzer. cDNA was synthesized according to the instructions of PrimerScript™ RTMaster Mix (PerfeCt Real Time), and the obtained cDNA was stored at -20℃ for subsequent experiments.

[0038] 1.3 RT-qPCR of candidate internal reference genes RT-qPCR amplification was performed on a BIO-RAD CFX96 real-time quantitative PCR instrument. Reaction system: 5 μL TB Green Taq (2×), 2 μL ddH2O, forward and reverse primers (concentration 10 μmol·L⁻¹). -1 0.5 μL of each of the following reagents were added: 2 μL of the diluted cDNA sample to be tested, for a total of 10 μL. The reaction program was as follows: pre-denaturation at 95℃ for 30 seconds, denaturation at 95℃ for 5 seconds, followed by annealing at 60℃ for 30 seconds. This process was repeated 40 times. Finally, melting curves were generated at 55–95℃ to detect primer specificity. Each RT-qPCR reaction was performed in triplicate (biological and technical replicates). Data were acquired using BioRad CFX Manager software.

[0039] 1.4 Screening of internal reference genes and primer design Based on literature related to internal reference genes and transcriptome sequencing data of Ilex verticillata, the following were selected: 18S ( 18S ribosomal RNA ), 28S ( 28S ribosomal RNA ), CYP ( Cyclophilin ), DNAJ ( DnaJ heat shock protein ), EF-1α ( elongation factor 1-α ), Hsp ( Heat shock protein ), EIF4A ( ukaryotic initiation faCtor 4A ), PP2A ( protein phosphatase 2A regulatory subunit A ), PT ( polypyrimidine tract-binding protein ), TUA ( α-tubulin ), TUB ( β-tubulin ), UBC ( ubiquitin-conjugating enzyme ), UBQ ( ubiquitin ), Actin2 ( β-Actin-2 )and Actin7 ( β- Actin-7 Fifteen genes, including [list of genes], were selected as candidate internal control genes. Specific qRT-PCR primers for these candidate internal control genes were designed using Primer Premier 5 software.

[0040] Table 1. Primer information for 15 candidate genes

[0041] 1.5 Primer specificity detection Fifteen candidate internal reference genes were amplified by conventional PCR using cDNA obtained from reverse transcription of Ilex henryi RNA as a template, following the instructions for use of PrimeSTAR® Max DNA Polymerase. The obtained PCR products were detected by agarose gel electrophoresis (1.5%), and the sample melting curves were obtained by RT-qPCR to determine primer specificity.

[0042] Agarose gel electrophoresis results are as follows Figure 1 As shown, all 15 candidate reference genes amplified to the expected size of the target band, and the bands were single, without primer dimers or non-specific amplification, indicating that the screened primers had good specificity. Furthermore, according to the designed reaction system, primers and templates for the 15 candidate genes were added, and RT-qPCR reactions were performed. Figure 2A , Figure 2B , Figure 2C , Figure 2D As shown, the RT-qPCR melting curves of the 15 candidate genes all exhibited a single peak, indicating that the RT-qPCR reaction has high specificity and can be used for subsequent experiments.

[0043] Example 2: Stability analysis of 15 candidate internal reference genes in different tissues of whorled holly 2.1 Ct value analysis The Ct values ​​of 15 candidate internal reference genes in different tissues of whorled holly showed a certain trend. Figure 3 The smaller the Ct value, the higher the gene expression level. The average Ct values ​​of the 15 candidate genes in different tissues of *Ilex wilfordii* roots, stems, and leaves ranged from 19.57 to 25.76. The gene with the highest average expression level... TUB The CT value was 19.57, indicating the gene with the lowest average expression level. UBQ The CT value was 25.76. Furthermore, the fluctuation of the Ct value can intuitively reflect the stability of the candidate internal reference gene. Box plots were drawn based on the Ct values ​​of 15 candidate internal reference genes. Figure 3 The results showed TUA and UBC The Ct value fluctuates the most, indicating the most unstable expression, while Actin2 and Actin7 The Ct values ​​of this group are most concentrated and the expression is most stable.

[0044] 2.2 geNorm Analysis The Ct values ​​of 15 candidate internal reference genes were analyzed in detail using the geNorm software to calculate the relative expression levels (M values) of these genes in different tissues of *Ilex chinensis*. The M value was negatively correlated with the stability of the internal reference gene; that is, the higher the stability of the internal reference gene, the lower its M value. It is worth noting that the upper limit of the M value is 1.5. If the M value > 1.5, it indicates that the gene expression is very unstable and unsuitable as an internal reference gene. Figure 4 As shown in A, in different tissues of whorled holly, the M values ​​of all 15 candidate internal reference genes were below 1.5, indicating that the candidate genes met the basic conditions for being internal reference genes. PT and 28S The gene with the lowest M value, 0.35, indicates the most stable expression.

[0045] The geNorm software can also predict the number of internal reference genes. Because geNorm calculates the standard deviation of the paired expression level ratios of each candidate internal reference gene with other internal reference genes using logarithmic transformation, it is more suitable for accurately quantifying the number of internal reference genes. This software can also calculate the paired variation value V (Variations) of the normalized factor after the introduction of a new gene. n / V n+1 The value of V can determine the optimal number of internal reference genes required. n / V n+1 The default threshold value is 0.15, V n / V n+1 <0.15, where n is the required number of internal reference genes. (From...) Figure 4 Therefore, V 2 / 3 The ratio was 0.146, which is less than 0.15, indicating that in different tissues of whorled holly, PT and 28S The stability of this combination of two internal reference genes meets the requirements.

[0046] 2.3 NormFinder Analysis NormFinder converts the Ct values ​​obtained from RT-qPCR into relative gene expression, further analyzing the degree of intra- and inter-group variation to calculate the M value of each gene. A smaller M value indicates stronger gene expression stability. Table 2 shows the expression levels in different tissues of whorled holly. PT and 18S It has the lowest M value, making it the most stable gene and the best internal reference gene.

[0047] Table 2. M values ​​of 15 candidate internal reference genes in NormFinder analysis Gene name M value Ranking Gene name M value Ranking 0.151 1 0.387 9 0.212 2 0.439 10 0.246 3 0.557 11 0.296 4 0.625 12 0.315 5 0.837 13 0.348 6 0.892 14 0.352 7 0.986 15 0.377 8 2.4 BestKeeper Analysis BestKeeper analyzes the stability of candidate reference genes by calculating the standard deviation (SD), coefficient of variation (CV), and correlation coefficient (r) of the Ct value. The smaller the CV and SD values, the higher the stability of the reference gene. If the SD value > 1, the gene is unstable and unsuitable as a reference gene. The analysis results from BestKeeper software (Table 3) show that among the 15 reference genes… EIF4A It has the lowest SD and CV values, at 0.47 and 2.19 respectively, indicating the highest expression stability. UBQ , EF-1α , Actin7 , TUA and Hsp Genes with an SD value exceeding 1 are considered unstable.

[0048] Table 3. BestKeeper analysis of 15 candidate internal reference genes. Gene name SD CV Ranking 0.47 2.19 0.52 1 0.58 2.70 -0.297 2 0.59 2.55 0.935 3 0.67 3.45 0.874 4 0.73 2.90 0.834 5 0.83 3.33 0.966 6 0.87 3.76 0.917 7 0.91 4.21 0.93 8 0.95 4.00 0.929 9 0.96 4.21 0.909 10 1.01 4.66 0.891 11 1.10 5.24 0.786 12 1.17 4.55 0.905 13 1.39 6.85 0.745 14 1.56 6.71 0.889 15 2.5 RefFinder Analysis The RefFinder program integrates four analysis methods: geNorm, NormFinder, BestKeeper, and DeltaCT. By summarizing the rankings of the results from these four analyses, appropriate weights are assigned to each candidate reference gene, and the geometric mean of these weights is calculated to obtain the overall ranking. A smaller value indicates higher stability of the reference gene. Table 4 shows the overall ranking of the 15 candidate reference genes. PT and 18S These are the two genes with the highest overall stability. Furthermore, due to the geNorm software V... 2 / 3 The ratio is less than 0.15, indicating that in different tissues of whorled holly, PT and 18S The stability of this combination of two internal reference genes meets the requirements.

[0049] Table 4. Expression stability of 15 candidate reference genes in various tissues as analyzed by Ref Finder. Gene name SV Ranking Gene name SV Ranking 1.86 1 8.21 9 2.34 2 8.65 10 3.22 3 9.06 11 3.5 4 10.1 12 5.18 5 12 13 6.04 6 13.47 14 6.24 7 14 15 7.8 8 Example 3: Stability analysis of six candidate internal control genes during flower bud differentiation and hormone response in whorled holly. Based on the stability of 15 candidate internal reference genes in different tissues of whorled holly (Table 4), the 6 genes with the highest stability were selected. PT , 18S , Actin2 , 28S , PP2A and EIF4AThe stability of the flower buds at different stages of differentiation was analyzed.

[0050] 3.1 Ct value analysis The Ct values ​​and trends of the six candidate internal reference genes all showed certain changes at different stages of flower bud differentiation in whorled holly. Figure 5 (A) The results showed that the average Ct values ​​of the six candidate genes ranged from 20.33 to 23.76, and the gene with the highest average expression level was (A). PT The lowest gene is 18S Under different hormone treatments ( Figure 5 In the B group, the average Ct value ranged from 21.07 to 25.17, and the gene with the highest average expression level was [missing information]. Actin2 The lowest gene is 18S In all samples ( Figure 5 The average Ct value ranged from 20.85 to 25.01, with the gene having the lowest Ct value being [missing value]. Actin2 The highest gene count for Ct is 28S .

[0051] 3.2 geNorm Analysis The stable expression of six candidate genes at different developmental stages of flower buds was analyzed using the geNorm software. Figure 6 ),in Actin2 The gene with the lowest M value, 0.565, indicates the most stable expression. 28S Secondly, the value of M is 0.588. EIF4A Gene expression stability was the worst, with the highest M value of 0.961. Under different hormone treatments, Actin2 and 28S The genes with the lowest M values ​​were 0.401 and 0.615, respectively. Figure 6 (B) Considering all sample conditions, Actin2 and 28S The genes exhibited the highest stability, with M values ​​of 0.446 and 0.496, respectively. 18S It has the worst stability, with an M value of 0.683. Figure 6 C in the text). The paired variance of the normalized factor after the introduction of the new gene was calculated using the geNorm software. Figure 7 Under different developmental stages of flowers, different hormone treatments, and comprehensive analysis of all samples, V n / n+1 The ratios were all less than the threshold of 1.5, namely 0.106, 0.105 and 0.133, indicating that the stability of the two internal reference gene combinations met the requirements.

[0052] 3.3 NormFinder Analysis The NormFinder analysis results (Table 5) show that at different developmental stages of the flower buds of whorled holly, Actin2 The gene with the highest expression stability (SV value of 0.155) was [gene name missing], while the least stable gene was [gene name missing]. EIF4A Its SV value was 0.603, which is basically consistent with the results of geNorm. Under different hormone treatments, Actin2 Gene expression was the most stable, with an SV value of 0.112, followed by... 28S The SV value is 0.211. 18S The gene with the highest SV value is the most unstable gene; under all sample conditions, Actin2 Gene expression is most stable, followed by 28S The SV value is 0.212. 18S The gene with the highest SV value, reaching 0.370, is the most unstable gene. The results of NormFinder analysis are basically consistent with those of geNorm.

[0053] Table 5. Norm Finder analysis of the expression stability of six candidate internal reference genes under various experimental conditions.

[0054] 3.4 BestKeeper Analysis The analysis results from BestKeeper software (Table 6) show that at different stages of flower bud differentiation in whorled holly, PT The most stably expressed internal reference gene had the lowest SV value of 0.22, followed by... 18S The SV value is 0.35. EIF4A and PP2A The expression stability was the worst, with an SV value of 0.76. Under different hormone treatments, PT and PP2A Gene expression was most stable, with an SV value of 0.32, while 18S Stability remains the worst; considering all organizational and processing conditions, PT The most stably expressed gene had an SV value of 0.334, followed by... PP2A SV=0.409, the least unstable gene expressed is 18S The SV value is the highest, reaching 0.71.

[0055] Table 6. BestKeeper analysis of the expression stability of the six candidate internal reference genes during flower bud differentiation.

[0056] 3.5 RefFinder Analysis A comprehensive analysis was conducted on the stability of the above six internal reference genes during flower bud differentiation. RefFinder results (Table 7) showed that at different developmental stages of whorled holly flower buds, Actin2 The most stably expressed internal reference gene was identified, with an SV value of 1.73, followed by... 28S The gene has an SV value of 2.21. EIF4A Gene expression stability is the worst. EIF4A In the analysis results of the three software programs above, this gene showed the worst stability. Under different hormone treatments, Actin2 Gene expression is most stable; under conditions that integrate all tissues and treatments, Actin2 It is the most stable gene.

[0057] Table 7. Ref Finder analysis of the expression stability of the six candidate internal reference genes under various experimental conditions.

[0058] Example 4: Stability verification of candidate internal reference genes To verify the reliability of the selected standardized internal reference gene, the following studies were conducted. IvWUS2 and IvAGL19 Expression patterns during flower bud development. Currently, two of the most suitable internal reference genes are used ( Actin2 and 28S Standardization analysis was performed on ) and its combinations. IvAGL19 Expression levels are highest during the S1 stage, and gradually decrease as flower buds develop. Figure 8 (A in the middle). IvWUS2 Expression levels were highest in S2, remained low in S3 and S4, and then increased in S5. Figure 8 (B in the text). However, when using unstable genes... EIF4A When performing normalization, IvAGL19 The expression patterns are significantly different. IvAGL19 Expression levels peaked in S2, then declined in S3, but were lowest in S4 and S5. Although, IvWUS2 Expression levels peaked in S2, but remained high in S3, compared to... Actin2 and 28S The results of analyses using internal reference genes showed significant differences. Therefore, selecting an inappropriate reference gene may lead to biased results. IvWUS2 and IvAGL19 Expression profile analysis showed that the selected reference gene was reliable in terms of qRT-PCR normalization.

Claims

1. Actin2 and / or 28S Its application as an internal reference gene for quantitative analysis of Ilex chinensis genes is characterized by, The Actin2 The nucleotide sequence is shown in SEQ ID NO.

1. 28S The nucleotide sequence is shown in SEQ ID NO.

2.

2. As described in claim 1 Actin2 and / or 28S Application as an internal reference gene for quantitative analysis of Ilex chinensis genes in different tissues.

3. The application according to claim 2, characterized in that, The tissues include: roots, stems, and leaves.

4. As described in claim 1 Actin2 and / or 28S Application as an internal reference gene for quantitative analysis of Ilex henryi genes at different developmental stages.

5. The application according to claim 4, characterized in that, The developmental period referred to is the flower bud differentiation period.

6. As described in claim 1 Actin2 and / or 28S Application as an internal reference gene for quantitative analysis of Ilex chinensis genes under different hormone treatments.

7. The application according to claim 6, characterized in that, The hormones mentioned are indole-3-acetic acid, indole-butyric acid, N(-1-naphthyl)-o-carbamoylbenzoic acid, kinetin, abscisic acid, salicylic acid, gibberellin, ethephon, and jasmonic acid.

8. For detecting the following as described in claim 1 Actin2 and / or 28S The primer pair is characterized in that, The Actin2 The nucleotide sequences of the detection primer pairs are shown in SEQ ID NO.29 and SEQ ID NO.30; And / or, the 28S The nucleotide sequences of the detection primer pairs are shown in SEQ ID NO.5 and SEQ ID NO.

6.

9. Used for detecting genes in the internal reference plants of whorled holly. Actin2 and / or 28S A real-time PCR kit, characterized in that, Includes the claims of claim 8 Actin2 Detection primer pairs and / or 28S The detection primer pairs and the reagents for real-time PCR.

10. A real-time quantitative PCR method for detecting the expression level of the internal reference gene of *Ilex wilfordii* using the kit described in claim 9, characterized in that... Includes the following steps: (1) Total RNA was extracted from Ilex verticillata samples and reverse transcribed into cDNA; (2) Using the cDNA obtained in step (1) as a template, and utilizing the method described in claim 8 Actin2 Detection primer pairs and / or 28S The detection primer pairs were used for real-time quantitative PCR detection.

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