Screening method of pepper reference genes under different hormones or different stresses and application of reference genes
By screening and verifying the pepper intrare genes CaActin1 and CaGAPDH2, the stability of the pepper gene in the pepper gene expression analysis was solved, and the accuracy of qRT-PCR detection was improved. It is suitable for gene expression analysis under different hormones or stress conditions.
Patent Information
- Application Number
- CN202510285086.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-11
- Publication Date
- 2025-07-25
AI Technical Summary
The lack of stable internal reference genes in the prior art is used for pepper gene expression analysis, which has affected the accuracy of qRT-PCR detection results, especially the lack of targeted internal reference genes under different hormones or stress conditions.
Nine candidate intrareal genes were screened and verified. Through fluorescence quantitative PCR and analysis of GeNorm, NormFinder, and BestKeeper software, the most stable intrareal genes CaActin1 and CaGAPDH2 under different hormones or stresses were screened out, and their fluorescence quantitative PCR primers were designed for gene expression level analysis.
The selection of pepper intrareal genes is optimized, the accuracy and stability of qRT-PCR detection is improved, and it is suitable for gene expression analysis under different hormones or stress conditions.
Smart Images
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biological genetic engineering, and particularly relates to a method for screening reference genes in pepper under different hormones or different stresses and the application of the reference genes. Background Art
[0002] Pepper (Capsicum annuum.L) is a very important horticultural crop, which is widely planted and commercially cultivated worldwide.
[0003] In recent years, with the completion of the pepper genome sequencing, functional genomics has become a research hotspot. Gene expression analysis is one of the important branches of functional genomics research. Relevant gene expression analysis is required in the research of pepper growth and development, fruit flavor, seed size, and various abiotic stresses and diseases of pepper. Analysis of gene expression levels helps researchers explore gene functions. In modern molecular biotechnology, techniques available for gene expression analysis include real-time quantitative polymerase chain reaction (qRT-PCR), semi-quantitative PCR, northern blotting, and in situ hybridization, etc. Among them, qRT-PCR has been widely used as a useful research tool for targeted gene expression analysis due to its high sensitivity and reproducibility. However, the accuracy of the results of the qRT-PCR technique may be affected by factors such as the integrity of RNA, the amount of sample added, and the reverse transcription efficiency. Therefore, when performing the analysis of the gene expression pattern by the qRT-PCR technique, it is usually necessary to first select a stably expressed reference gene to correct and standardize the data. Many reports indicate that there is no ideal stable reference gene, and the stability of reference genes varies to a certain extent due to different plant species, growth environments, growth stages, and stress conditions. Selecting stable reference genes plays an important role in the accuracy of the RT-qPCR detection of gene expression results. Common reference gene families include β-tubulin (β3-tubulin), cyclophilin, actin, transcription elongation factor (EF-1α), and α-tubulin, etc., which are commonly used as reference genes in Umbelliferae plants. Currently, there are clearly reported reference genes in many species. However, the reports on pepper reference genes are not yet complete and still need to be further explored. Summary of the Invention
[0004] The technical problem to be solved by the present invention is to overcome the above-mentioned deficiencies and defects in the background art and provide one or more reference genes of pepper to facilitate the study of the expression levels of various genes in pepper.
[0005] To solve the above technical problem, the technical solution proposed by the present invention is as follows:
[0006] A method for screening reference genes in pepper under different hormones or different stresses, comprising the following steps:
[0007] (1) Select nine reference genes, CaGAPDH 1, CaGAPDH 2, CaUbiquitin 1, CaUbiquitin 2, CaTubulin1, CaTubulin 2, CaActin 1, CaActin 2, CaActin 3, as candidate reference genes, and design their fluorescence quantitative PCR primers;
[0008] (2) Treat the pepper with stress or hormone, extract the total RNA of the pepper after stress treatment or hormone treatment, measure the RNA concentration, reverse transcribe the RNA into cDNA, and then use the primers in step (1) to analyze the alternative reference genes by real-time fluorescence quantitative PCR with the cDNA as a template;
[0009] (3) Use GeNorm, NormFinder and BestKeeper software to analyze the stability of the Cq values of the reference genes for the real-time fluorescence quantitative PCR data, so as to screen out the most stably expressed reference genes in the pepper under stress treatment or hormone treatment.
[0010] For the above method for screening reference genes in pepper under different hormones or different stresses, preferably, the hormones include gibberellin and / or auxin; the stresses include at least one of waterlogging, drought, and diseases.
[0011] For the above method for screening reference genes in pepper under different hormones or different stresses, preferably, in step (1), the nucleotide sequences of the forward and reverse primers of the nine reference genes are SEQ ID NO:1-18 in sequence.
[0012] For the above method for screening reference genes in pepper under different hormones or different stresses, preferably, in step (3), the relatively stable reference gene in the pepper under stress treatment is CaGAPDH 2, and the relatively stable reference gene under hormone treatment is CaActin1.
[0013] As the same inventive concept, the present invention provides an application of the CaActin1 gene as a reference gene in pepper under hormone treatment conditions.
[0014] Under different hormone treatment conditions, the CaActin1 gene has relatively stable expression and a suitable expression level, and is suitable for being used as a reference gene in gene expression level analysis.
[0015] Preferably, the hormone is gibberellin and / or auxin.
[0016] As a same inventive concept, the present invention provides an application of the CaGAPDH 2 gene as an internal reference gene for peppers under stress treatment conditions.
[0017] Under different stress treatment conditions, the CaGAPDH 2 gene has relatively stable expression and a suitable expression level, and is suitable for being used as an internal reference gene in gene expression level analysis.
[0018] Whether it is biotic stress (such as diseases) or abiotic stress (waterlogging or drought in soil), the CaGAPDH 2 gene has relatively stable expression and a suitable expression level, and is suitable for being used as an internal reference gene in gene expression level analysis.
[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0020] By using RT-qPCR technology in combination with internal reference gene stability analysis software such as geNorm, NormFinder, BestKeeper and RefFinder, the stable internal reference genes in different tissues are optimized. On this basis, two internal reference genes CaActin1 and GAPDH 2 with the best stability under different hormone treatments and different stresses are further screened out, solving the problem that there is no targeted internal reference gene for peppers under specific treatments. Description of the Drawings
[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to these drawings.
[0022] Figure 1 is the RNA extraction and quality detection of different tissues;
[0023] Figure 2 is the agarose electrophoresis gel bands of 9 candidate genes;
[0024] Figure 3 is the melting curves of 9 candidate genes;
[0025] Figure 4 is the amplification efficiencies of 9 candidate genes;
[0026] Figure 5Stability analysis of cq values of several software internal reference genes: A: Perform Genorm analysis on the fluorescence quantitative data of 9 genes; B: Perform Normfinder analysis on the fluorescence quantitative data of 9 genes; C: Perform Delta ct analysis on the fluorescence quantitative data of 9 genes; D: Perform Bestkeeper analysis on the fluorescence quantitative data of 9 genes; E: cq values of the fluorescence quantitative data of 9 genes.
[0027] Figure 6 Screening of stable internal reference genes under different treatments: A: Comparison of the stability differences of four candidate genes under IAA treatment; B: Comparison of the stability differences of four candidate genes under GA3 treatment; C: Comparison of the stability differences of four genes under waterlogging treatment; D: Comparison of the stability differences of four genes under drought treatment; E: Comparison of the stability differences of four genes under disease inoculation.
[0028] Figure 7 It is the expression heat map of the internal reference gene Actin1 under different treatments. Specific implementation manners
[0029] For the convenience of understanding the present invention, the present invention will be described more comprehensively and carefully below in conjunction with the accompanying drawings of the specification and preferred embodiments, but the protection scope of the present invention is not limited to the following specific embodiments.
[0030] Unless otherwise defined, all professional terms used hereinafter have the same meaning as commonly understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the protection scope of the present invention.
[0031] Unless otherwise specifically stated, various raw materials, reagents, instruments and equipment used in the present invention can be obtained through the market or can be prepared by existing methods.
[0032] Four relatively stable internal reference genes screened in different tissues of peppers in the embodiments of the present invention are: CaTubulin 2, CaActin1, CaGAPDH1, and CaGAPDH2.
[0033] Among the above four genes, the most stable internal reference gene under different hormone treatments was further screened to be CaActin1, and the primers for fluorescence quantification are:
[0034] F: 5'-GAGCTTCGTGTTGCCCCTGA-3';
[0035] R: 3'-CGGTTGTACGACCACTGGCA-5'.
[0036] Among the above four genes, the most stable reference gene under different stress treatments was further screened as CaGAPDH2, and the primers for fluorescence quantitative PCR were:
[0037] F: 5'-CTGCTGCCCACTTGAAGGGT-3';
[0038] R: 3'-GCCAGAGGAGCAAGGCAGT-5'.
[0039] The processes of hormone treatment and stress treatment of materials in the following examples include:
[0040] Hormone treatment of materials:
[0041] Select the whole pepper seedlings at the six-leaf stage and spray 100 μM GA3 and 100 μM auxin on them. After 6 hours of treatment, take the leaves of three different pepper plants and mix them. When extracting RNA, set three technical replicates for each sample, and use this as the experimental material for the selection of reference genes under different hormone treatments.
[0042] Abiotic stress treatment:
[0043] Select another group of pepper seedlings at the six-leaf stage for waterlogging treatment. Take root tissue samples at 0 h and 48 h after treatment respectively. For each sample, take the samples by mixing three pepper plants, extract RNA, and set three technical replicates for each sample. Use this as the experimental material for screening reference genes under waterlogging stress.
[0044] Select another group of pepper seedlings at the six-leaf stage, simulate natural drought conditions for drought treatment (start timing from when there is no water in the plug tray). After 7 days, select the leaves of 3 pepper plants under drought treatment and normal irrigation as mixed samples, extract RNA, and set three technical replicates for each sample. Use this as the experimental material for screening reference genes under drought stress.
[0045] Biotic stress treatment:
[0046] Select another group of pepper seedlings at the six-leaf stage for inoculation with Phytophthora capsici. After 24 hours, take the young leaves of three pepper plants without inoculation and inoculated with the pathogen and mix them, extract RNA, set three technical replicates for each sample, and use this as the experimental material for screening reference genes under disease treatment.
[0047] Example 1: Extraction and quality detection of RNA
[0048] In this example, the roots, stems, leaves of the seedlings at the six-leaf stage of two pepper varieties, Zunla and Zhangshugang, the flowers and ovaries on the day of flowering, and the fruits at the color-changing stage were used as the experimental materials for optimizing reference genes in different tissues.
[0049] Using six-leaf-stage pepper seedlings as materials, RNA was extracted from different tissues. All samples were stored in a -80°C ultra-low temperature freezer for later experiments. The concentration of RNA was measured on a spectrophotometer, and the results are shown in Table 1 (A, B, and C represent three technical replicates of each sample). The results indicate that the RNA was extracted with good quality and there was no obvious degradation.
[0050] Total RNA of pepper was extracted using the Shanghai Promeger Eastep Super Total RNA Extraction Kit, and then synthesized using the TIANGEN Fast King cDNA First Strand Synthesis Kit. The extracted RNA was detected by 1.2% agarose gel electrophoresis, and two clear bands were obtained. The results are as follows Figure 1 (A: represents the sample diagram when sampling different parts of pepper; B: represents the quality of the RNA extracted from each sample).
[0051] Table 1: Concentration determination of each RNA
[0052]
[0053]
[0054] Example 2: Screening of 9 candidate genes, primer design, and detection of primer specificity and amplification efficiency
[0055] In this experiment, 9 genes were screened from the four families of common internal references TUBULIN, Actin, GAPDH, and Ubiquitin. The CDS sequences of the 9 genes were downloaded from the pepper website, and then primers for the 9 genes were designed on the GenScript Pcr Primer Design website (website: https: / / www.genscript.com / tools / pcr-primers-designer). The primer sequences are shown in Table 2 and SEQ ID NO.1 to SEQ ID NO.18 as follows.
[0056] Table 2: Fluorescent quantitative primer design for 9 genes
[0057]
[0058]
[0059] Using the cDNA mixed sample of each tissue as a template, 1.2% agarose gel electrophoresis was performed on 9 candidate internal reference genes, and the PCR product bands were single and the fragment sizes were consistent with the expectations, as Figure 2 shown. As Figure 3As shown, the RT-qPCR melting curves of each candidate reference gene primer showed a single peak, indicating that the primers had high specificity. The amplification efficiency values of each primer were between 90.41% (TUB) and 103.44% (UBQ1), and R2>0.99. As Figure 4 shown, it indicated that the reference gene primers used were all specific and effective, and the experimental data obtained could be used for subsequent analysis.
[0060] Example 3: Analysis of fluorescence quantitative Cq values of 9 genes
[0061] The peppers were subjected to stress treatment or hormone treatment, and the total RNA of the peppers after stress treatment or hormone treatment was extracted. The RNA concentration was measured, and the RNA was reverse transcribed into cDNA. Then, using the cDNA as a template and the primers in Table 2, the alternative reference genes were analyzed by real-time fluorescence quantitative PCR. In this experiment, the Novoprotein fluorescence quantitative system was used for amplification, and the system was as follows:
[0062] Table 3: Fluorescence quantitative amplification system
[0063] Component Volume SYBR Premix Ex Taq 10 μL Primer 0.4 μL each cDNA Template 1 μL <![CDATA[ddH2O]]> 8.2 μL Total 20 μL
[0064] Then, three software programs, Genorm, NormFinder, and Bestkeeper, were used for statistical analysis. Genorm (as Figure 5 A) and NormFinder (as Figure 5 B) needed to use the 2 -ΔΔCt value for analysis, that is, the Cq value was converted into relative expression for analysis (as Figure 5 C); the Bestkeeper software analyzed by inputting the original data (Cq value) (as Figure 5 D). Finally, the three software programs ranked the stability of each candidate gene according to the analysis results, and then, according to the Cq value of each gene (the Cq value of the qualified reference gene should be 19-25), the most suitable four reference genes were finally selected as shown in the following table (Note: The 9 genes CaGAPDH 1, CaGAPDH 2, CaUbiquitin 1, CaUbiquitin 2, CaTubulin 1, CaTubulin 2, CaActin 1, CaActin 2, CaActin 3 in Table 4 were abbreviated as: GAPDH1, GAPDH 2, Ubi1, Ubi2, Tub1, Tub2, Actin 1, Actin 2, Actin 3 in turn).
[0065] Table 4: Final ranking of five analyses
[0066]
[0067] Example 4: Selection of reference genes under different hormones and different stresses
[0068] On the basis of the above screening, reference genes that are stable under the treatments of gibberellin (GA3) and auxin (IAA) were screened from the four relatively stable reference genes. The results of fluorescence quantitative cq value analysis showed that CaTubulin2 showed high stability under the treatment of 100 μM IAA, and CaActin1 showed high stability under the treatment of 100 μM gibberellin, and the Cq values were all less than 25, meeting the requirements for being a reference gene, as Figure 6 shown in AB. However, Tubulin2 has been reported in previous articles. After being verified by the experiments in this article, this gene can still be used as a reference gene under certain hormone treatments after the publication of the new pepper genome. CaActin1 is a new reference gene screened by the applicant.
[0069] On the basis of the above screening, reference genes that are stable under waterlogging, drought and disease treatments were screened from the four relatively stable reference genes. The fluorescence quantitative results showed that CaGAPDH2 showed high stability under waterlogging and drought treatments, and the cq values were all less than 25, meeting the requirements for being a reference gene. When pepper was inoculated with Phytophthora capsici, both CaGAPDH1 and CaGAPDH2 showed high stability, but the Cq value of CaGAPDH1 was greater than 25, so it was not suitable to be a reference. Therefore, CaGAPDH2 can be used as a reference gene under biotic and abiotic stress treatments of pepper, as Figure 6 shown in C D E.
[0070] Example 5: Verification of the stability of reference genes under different treatments
[0071] The expression data of the published genes of pepper under different treatments have been published on the PepperHub website (website: http: / / lifenglab.hzau.edu.cn / PepperHub / Transcriptome / heatmap.php) early. The genes screened in this article can be further verified for their stability on this website. Download the expression data of gene CaActin1 under different treatments on this website, and then use TBtools software to draw a heat map, as Figure 7 (The abscissa represents different stages of root development, and the ordinate represents different hormone treatments. From top to bottom, they represent control, salicylic acid treatment, jasmonic acid treatment, auxin treatment, abscisic acid treatment, gibberellin treatment). The data shows that under different hormone treatments, the overall stability of CaActin1 is still good. There are certain changes in the stability of individual treatment periods in the figure, which is a normal phenomenon. There is no reference gene that is always stable in any part and any state, and individual differences are allowed.
Claims
1. A screening method for reference genes in peppers under different hormones or different stresses, characterized in that, It includes the following steps: (1) Select nine reference genes, CaGAPDH 1, CaGAPDH 2, CaUbiquitin 1, CaUbiquitin 2, CaTubulin1, CaTubulin 2, CaActin 1, CaActin 2, and CaActin 3, as candidate reference genes, and design their fluorescence quantitative PCR primers; (2) Subject the chili peppers to stress treatment or hormone treatment, extract the total RNA of the chili peppers after stress treatment or hormone treatment, measure the RNA concentration, reverse transcribe the RNA into cDNA, and then use the primers in step (1) to analyze the alternative reference genes by real-time fluorescence quantitative PCR with the cDNA as the template; (3) Use GeNorm, NormFinder, and BestKeeper software to analyze the stability of the Cq values of the reference genes for the real-time fluorescence quantitative PCR data, so as to screen out the most stably expressed reference genes in chili peppers under stress treatment or hormone treatment.
2. The screening method of pepper reference genes under different hormones or different stresses according to claim 1, characterized in that, The hormone includes gibberellin and / or auxin; the stress includes at least one of waterlogging, drought, and disease.
3. The screening method of pepper reference genes under different hormones or different stresses as described in claim 1, wherein In step (1), the nucleotide sequences of the forward and reverse primers of the nine reference genes are SEQ ID NO: 1-18 in sequence.
4. The screening method of pepper reference genes under different hormones or different stresses as described in claim 1, characterized in that In step (3), the relatively stable reference gene in chili peppers under stress treatment is CaGAPDH 2, and the relatively stable reference gene under hormone treatment is CaActin1.
5. Application of CaActin1 as a reference gene in chili peppers under hormone treatment conditions.
6. The application according to claim 5, characterized in that The hormone is gibberellin and / or auxin.
7. Application of CaGAPDH 2 as a reference gene in chili peppers under stress treatment conditions.
8. The application according to claim 7, wherein The stress includes biotic stress and abiotic stress.
9. The application according to claim 8, wherein The abiotic stress includes waterlogging or drought.
10. The application according to claim 7, wherein The biotic stress includes disease.