Real-time fluorescent quantitative PCR internal reference gene in plant and its application

Abstract

The present application relates to the technical field of plantago asiatica L. genetic engineering, and specifically provides a plantago asiatica L. real-time fluorescent quantitative PCR internal reference gene and application thereof. The tender leaves, old leaves, stems, flowers and roots of plantago asiatica L. are selected as experimental materials, candidate internal reference genes are screened through plantago asiatica L. transcriptome, then the gene expression stability is analyzed through real-time fluorescent quantitative PCR analysis, melting curve analysis, amplification efficiency analysis and various software (geNorm, NormFinder, BestKeeper, Delta Ct and RefFinder) analysis, and the internal reference genes suitable for gene quantification of different tissue parts of plantago asiatica L., i.e. eIF and Cycl genes, are screened, and the sequences are shown as SEQ ID NO. 1 and SEQ ID NO. 2. The internal reference genes with stable expression are obtained, are suitable for analyzing the expression amount of genes in different parts of plantago asiatica L., make up for the current situation that plantago asiatica L. lacks internal reference genes, and provide a basis for gene expression amount and function analysis in plantago asiatica L.

Description

Technical Field

[0001] This invention belongs to the field of plantago asiatica genetic engineering technology, and relates to plantago asiatica real-time fluorescence quantitative PCR internal reference gene and its application. Background Technology

[0002] Real-time quantitative PCR (qRT-PCR) is a commonly used technique for analyzing gene expression levels. It offers high sensitivity, reproducibility, and stability, and includes both absolute and relative quantification. Absolute quantification calculates gene expression levels by establishing a standard curve, while relative quantification utilizes internal control genes to analyze the expression changes of the target gene relative to the control group. Internal control genes are typically housekeeping genes that are stably expressed in various parts of the plant under different conditions to correct for errors caused by experimental procedures and sample variations. Commonly used internal control genes in plant research include 18S ribosomal RNA (18S rRNA), β-actin (ACT), elongation factor (EF), tubulin (TUB), and eukaryotic translation initiation factor (eIF). However, gene expression stability varies across species, growth stages, and treatment conditions. Therefore, it is necessary to screen for relatively stable internal control genes to analyze gene expression levels in specific species and under specific conditions.

[0003] Plantago asiatica L., a medicinal plant belonging to the Plantaginaceae family, is commonly used for clearing heat and promoting diuresis, cooling the blood, and detoxifying. Plantago possesses various biological activities, with its main active substances including apigenin, luteolin, physostigmine, and verbascoside. Pharmacological studies have shown that plantago seed extract possesses antioxidant and anti-inflammatory activities; furthermore, it also exhibits antiviral and antitumor activities. Currently, research on plantago focuses primarily on the isolation of chemical components and pharmacology, while molecular biology research is relatively lacking. qRT-PCR detection of gene expression levels and subsequent screening of candidate genes is a common method in molecular biology for isolating target genes; however, no internal control genes for plantago have been reported. Therefore, it is necessary to screen stable internal control genes to analyze the expression levels of functional genes in plantago. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the first technical problem this invention aims to solve is to provide a real-time quantitative fluorescence reference gene and dedicated primers for different tissues of *Plantago asiatica*, which can be used for real-time quantitative fluorescence detection of gene expression levels in different tissue sites of *Plantago asiatica*. The second problem this invention aims to solve is to provide the application of the aforementioned reference gene and its primers in real-time quantitative fluorescence detection of *Plantago asiatica*.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0006] The first aspect of the present invention provides a reference gene for real-time quantitative PCR, the reference gene comprising an eIF gene and a Cycl gene; the nucleotide sequence of the eIF gene is shown in SEQ ID NO.1, and the nucleotide sequence of the Cycl gene is shown in SEQ ID NO.2.

[0007] Furthermore, the primers for eIF gene fragment amplification are as follows:

[0008] eIF forward primer 5'-GCCCGCATCTTTCTTGAC-3'

[0009] eIF reverse primer 5'-GCTTCCATCCACTGACCC-3'.

[0010] Furthermore, the primers for amplifying the Cycl gene fragment are as follows:

[0011] Cycl forward primer 5'-CGCTTTCACTGTGGAGAT-3'

[0012] Cycl reverse primer 5'-GTATTGGGACCAGCATTT-3'.

[0013] The second aspect of this invention provides the application of the above-mentioned Plantago asiatica real-time fluorescence quantitative PCR internal reference genes eIF and Cycl in the functional gene expression level analysis of different tissue parts of Plantago asiatica, wherein the different tissue parts include young leaves, old leaves, stems, flowers and roots of Plantago asiatica.

[0014] Furthermore, the reaction system for the real-time quantitative PCR of the caravan is as follows: 5 ng cDNA (1) μL, 0.4 μL each of upstream and downstream primers, 10 μL 2×FastStartUniversal SYBR Green Mix, and enzyme-free water to a volume of 20 μL.

[0015] Furthermore, the reaction procedure for the aforementioned real-time quantitative PCR is as follows: 95℃ for 10 min, 95℃ for 15 s for denaturation, 60℃ for 1 min for extension, 40 cycles.

[0016] The beneficial effects of this invention are:

[0017] This invention screened six candidate internal reference genes using the Plantago asiatica transcriptome. The stability of these candidate genes was evaluated using geNorm, NormFinder, BestKeeper, ΔCt, and RefFinder software. The optimal genes for real-time quantitative PCR (qPCR) in different Plantago asiatica tissues, eIF and Cycl, were obtained. Furthermore, qPCR primers for the internal reference genes were designed. These primers exhibit high specificity and amplification efficiency, addressing the lack of internal reference genes for functional gene expression detection in different Plantago asiatica tissues and improving the efficiency and reliability of qPCR results. Attached Figure Description

[0018] Figure 1 Melting curves for 6 internal reference genes;

[0019] Figure 2 Box plots showing the distribution of Ct values ​​for the five internal reference genes in different tissues of Plantago asiatica;

[0020] Figure 3 The results of geNorm analysis of the expression stability of candidate internal reference genes in Plantago asiatica;

[0021] Figure 4 To analyze the expression levels of G10H and 10HGO genes in different tissues of Plantago asiatica, two relatively stable genes, eIF and Cycl, and an unstable gene, TUA, were used as internal reference genes. Detailed Implementation

[0022] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0023] The main test materials used in the following examples were collected from Yichang City, Hubei Province. Three Plantago asiatica plants with good and uniform growth were randomly selected, and tender leaves, old leaves, stems, flowers, and roots were collected in April 2022. After collection, the materials were flash-frozen in liquid nitrogen, ground into powder, and stored at -80℃.

[0024] Example 1:

[0025] 1. Extraction of total RNA from plant tissues and synthesis of cDNA

[0026] Total RNA was extracted from the samples using the Trizol method, and the RNA concentration was detected using a NanoDrop 2000 micro spectrophotometer. RNA sample integrity was assessed by 1% agarose gel electrophoresis. One μg of qualified RNA sample was used to digest genomic DNA with DNase I, followed by reverse transcription using RevertAid Reverse Transcriptase (ThermoFisherScientific) to synthesize cDNA.

[0027] 2. Selection of internal reference genes and primer design

[0028] Homology searches were performed on the Plantago asiatica transcriptome using internal reference genes reported in Arabidopsis thaliana, resulting in six candidate internal reference genes: eIF (SEQ ID NO.1), Cycl (SEQ ID NO.2), EF (SEQ ID NO.3), 18S (SEQ ID NO.4), TUA (SEQ ID NO.5), and ACT (SEQ ID NO.6). Primers were designed using Primer Premier 5.0 based on the full-length sequences of these candidate internal reference genes and synthesized by Sangon Biotech Co., Ltd. (Table 1). Primer specificity was preliminarily analyzed using conventional PCR and agarose gel electrophoresis. Primers with correct band size, single band, high amplification product content, and no primer dimers were selected. Real-time quantitative PCR was then used to detect primer specificity.

[0029] Table 1. Primer information and amplification parameters for the candidate internal reference gene of *Plantago asiatica*

[0030]

[0031] 3. Real-time quantitative PCR

[0032] Quantitative real-time PCR was performed using Roche FastStart Universal SYBR Green Mix fluorescent dye and an AppliedBiosystems QuantStudio 5 Real-time PCR instrument. A 20 μL reaction solution was prepared according to the manufacturer's instructions, containing 5 ng cDNA (1 μL) as template, 0.4 μL each of upstream and downstream primers, 10 μL of 2×FastStart Universal SYBR Green Mix, and enzyme-free water. The reaction program was 95℃ for 10 min, followed by denaturation at 95℃ for 15 s and extension at 60℃ for 1 min, for 40 cycles. Primer specificity was determined by melting curve analysis. Three biological replicates and three technical replicates were set up for each sample.

[0033] The results of quantitative real-time PCR showed that the melting curves produced by each primer amplification were all single peaks. Figure 1 This indicates that the primers used were for specific amplification; the obtained quantitative Ct value can be used for subsequent internal reference gene stability analysis.

[0034] 4. Plotting the standard curve for the internal reference gene

[0035] cDNA (100 ng / μL) was serially diluted and used as templates for experimental analysis at five concentration gradients (1×, 2.5×, 5×, 20×, 100×). Ct values ​​for each candidate gene at different concentrations were obtained, and each reaction was performed in triplicate. A standard curve was plotted with the logarithm of template dilution on the x-axis and Ct values ​​on the y-axis, yielding the slope (s) and correlation coefficient (R). 2 And according to the formula E = (10 -1 / s The amplification efficiency is calculated as (-1)×100%.

[0036] Table 1 shows the correlation coefficient R of the ACT internal reference gene amplification standard curve. 2 The R value is 0.9393, which is slightly low. The R values ​​of the other five candidates are... 2 The values ​​ranged from 0.9807 to 0.9978, exceeding 98%, indicating a good linear relationship. Except for ACT, the amplification efficiencies of other genes ranged from 95.96% to 106.97%. Based on these results, Cycl, eIF, EF, 18S, and TUA can be used for subsequent qRT-PCR analysis.

[0037] 5. Internal reference gene C t Value Analysis

[0038] C t The value represents the number of cycles required for the fluorescence signal to accumulate to a threshold, and is related to the amount of template initiation; the more template initiation, the higher the C value. t The smaller the value, the better. In different parts of the plantago asiatica (young leaves, old leaves, stems, flowers, roots), the C value of each internal reference gene... t The values ​​differ. Specifically, Cycl's C... t The values ​​were the highest, ranging from 31.132 to 31.663, indicating the lowest Cycl expression level; the 18S C t The values ​​were the lowest, ranging from 23.692 to 27.661, indicating high 18S expression levels. Furthermore, the C of eIF... t The value ranges from 28.945 to 30.694, and the C of EF is... t The value ranges from 27.185 to 31.12, and the C of TUA is... t Values ​​range from 27.871 to 31.007. Figure 2 ).

[0039] 6. Stability analysis of internal reference genes

[0040] The expression stability of five candidate internal reference genes in young leaves, old leaves, stems, flowers, and roots of Plantago asiatica was analyzed using the ΔCt method and software such as geNorm, BestKeeper, NormFinder, and RefFinder.

[0041] (1) geNorm analysis

[0042] The relative expression level ΔCt is obtained by subtracting the minimum Ct value of the internal reference gene from the Ct value of each sample. The relative expression level Q = 2 is then calculated based on ΔCt. -ΔCt The expression stability value M of each internal reference gene was calculated by importing Q into the geNorm software. The threshold for M was 1.5; a higher M value indicated less stable expression of the internal reference gene, and vice versa. The average M value of all internal reference genes was calculated based on the M value of each gene, and the least stable genes were progressively removed. The average M value of the remaining genes was calculated until the two most stable internal reference genes were obtained. Figure 3 The average M value of the five candidate internal reference genes was less than 1.5, indicating relatively stable expression. Among them, eIF and Cycl had the smallest average M value (0.585), indicating that eIF and Cycl were most stable in different regions. TUA, on the other hand, was the least stable, with an M value of 1.116.

[0043] (2) NormFinder Analysis

[0044] Similar to the geNorm software, the NormFinder software calculates stability values ​​based on the relative expression level Q of genes; the smaller the value, the higher the stability. As shown in Table 2, the stability ranking of different internal reference genes in different parts of Plantago asiatica is: eIF>EF>18S>TUA>Cycl. The eIF and EF genes have the highest stability, while Cycl is the least stable gene.

[0045] (3) BestKeeper software

[0046] BestKeeper software calculates the standard deviation (SD) and coefficient of variation (CV) of internal reference genes in different samples based on the Ct values ​​generated by real-time quantitative PCR. Smaller SD and CV values ​​indicate more stable gene expression. As shown in Table 2, BestKeeper's analysis ranked the stability of different internal reference genes as follows: Cycl > eIF > 18S > EF > TUA. Cycl showed the highest stability, while TUA was the least stable.

[0047] (4) Delta Ct analysis

[0048] The ΔCt method analyzes gene expression stability by calculating the mean standard deviation; the smaller the mean standard deviation, the more stable the gene expression. As shown in Table 2, the stability ranking is: eIF > EF > 18S > Cycl > TUA, with eIF being the most stable internal reference gene, followed by EF and 18S.

[0049] (5) RefFinder analysis

[0050] The stability of genes was analyzed using RefFinder software, which combined the sorting results from geNorm, NormFinder, BestKeeper, and the ΔCt algorithm. The results showed that the stability ranking was: eIF > Cycl > EF > 18S > TUA.

[0051] Based on the analysis results of the above software, the eIF gene is most stably expressed in all parts of the plantar fasciculus, followed by Cycl, making it suitable as an internal reference gene.

[0052] Table 2. Analysis results and rankings of internal reference genes geNorm, NormFinder, BestKeeper, ΔCt, and RefFinder from different sites.

[0053]

[0054] Example 2:

[0055] The two internal reference genes, eIF and Cycl, which have stable overall rankings, and the unstable internal reference gene, TUA, were used for validation.

[0056] To verify the stability of the screened internal reference genes, the expression levels of key genes G10H and 10HGO in the cycloene ether terpenoid synthesis pathway of Plantago asiatica were analyzed in different tissue sites.

[0057] The specific primers for the G10H gene are:

[0058] G10H forward primer 5'-CTCCGCTTCCGATAAT-3' (SEQ ID NO.19);

[0059] G10H reverse primer 5'-GCTACGCCACCTGTTT-3' (SEQ ID NO.20).

[0060] The specific primers for the 10HGO gene are:

[0061] 10HGO forward primer 5'-CCATTCGGACCTTCAC-3' (SEQ ID NO.21);

[0062] 10HGO reverse primer 5'-ATCCCACGCCTACTTT-3' (SEQ ID NO.22).

[0063] The results showed that when eIF and Cycl were used as internal reference genes, the expression levels of G10H and 10HGO were basically consistent in different parts of the plant, and the order from high to low was: older leaves > younger leaves > flowers > stems. The expression levels of G10H and 10HGO in roots were extremely low, and they were not detected under the conditions of this study. Figure 4 When TUA is used as an internal reference gene, the relative expression level of the target gene may be overestimated or underestimated. For example, when TUA is used as an internal reference gene, the expression levels of G10H and 10HGO in older leaves are significantly higher than when eIF and Cycl are used as internal reference genes, while the expression level of 10HGO in stems is lower than when eIF and Cycl are used as internal reference genes.

[0064] In summary, the optimal internal reference genes eIF and Cycl selected by this invention have good stability and can be used to detect the expression levels of functional genes in different tissue sites of Plantago asiatica.

Claims

1. The application of real-time quantitative PCR internal reference gene in the analysis of functional gene expression levels in different tissue parts of Plantago asiatica, characterized in that, The different tissue parts include tender leaves, old leaves, stems, flowers, and roots of Plantago asiatica; the internal reference gene includes... eIF Genes and Cycl Genes; the stated eIF The nucleotide sequence of the gene is shown in SEQ ID NO.

1. Cycl The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, eIF The primers for gene fragment amplification are as follows: eIF Forward primer 5'- GCCCGCATCTTTCTTGAC-3'; eIF Reverse primer 5'- GCTTCCATCCACTGACCC-3'.

3. The application according to claim 1, characterized in that, Cycl The primers for gene fragment amplification are as follows: Cycl Forward primer 5'- CGCTTTCACTGTGGAGAT-3'; Cycl Reverse primer 5'-GTATTGGGACCAGCATTT-3'.

4. The application according to claim 1, characterized in that, The reaction system for the aforementioned real-time quantitative PCR is as follows: 5 ng cDNA 1 µL, upstream and downstream primers 0.4 μL each, 10 μL 2×FastStart Universal SYBRGreen Mix, and enzyme-free water to a volume of 20 µL.

5. The application according to claim 1, characterized in that, The reaction procedure for the real-time quantitative PCR described above is as follows: 95℃ for 10 min, 95℃ denaturation for 15 s, 60℃ extension for 1 min, 40 cycles.

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