Internal reference gene for gene expression analysis in myricaria laxiflora branches and stems under water flooding stress as well as primer pair and application of internal reference gene

Abstract

The invention provides a reference gene for gene expression analysis in myricaria laxiflora branches and stems under flooding stress and a primer pair and application thereof. The reference gene is a ubiquitin-binding enzyme coding gene, has a nucleotide sequence as shown in SEQ ID NO: 1, is stable in expression in myricaria laxiflora branches and stems under flooding stress, is suitable for being used as the reference gene for gene expression analysis in the myricaria laxiflora branches and stems under flooding stress, and can overcome experimental differences possibly existing in a sample preparation process and a detection process; practical help is provided for accurate identification of the expression quantity of related genes in myricaria laxiflora branches and stems under flooding stress, and a good foundation is laid for analyzing a flooding-resistant regulation mechanism of the myricaria laxiflora branches and stems and better utilizing the flooding-resistant characteristic of the myricaria laxiflora branches and stems.

Description

Technical Field

[0001] The present invention relates to plant biotechnology, and particularly to an internal reference gene, a primer pair thereof, and an application for gene expression analysis in the stem of Myricaria laxiflora under waterlogging stress. Background Art

[0002] Myricaria laxiflora is an upright shrub of the family Tamaricaceae and the genus Myricaria, and is endemic to the Three Gorges Reservoir Area of China. It grows on river beaches and river banks. Due to its long-term adaptation to the waterlogging environment, it has formed the characteristics of shedding leaves and going dormant in summer and autumn under waterlogging, and growing and reproducing in winter and spring after the water recedes. It has strong resistance to flood impact and waterlogging tolerance, and has important application values in aspects such as soil and slope protection in the riparian zone and ecological environment beautification and restoration.

[0003] When the stem of Myricaria laxiflora responds to waterlogging stress, there are two mechanisms: "quiescence" and "escape". In the "quiescence" mechanism, Myricaria laxiflora stops growing under the influence of waterlogging. Therefore, the stem immersed in water elongates little or hardly at all to reduce energy consumption, and Myricaria laxiflora uses the stored energy to recover or regrow after the waterlogging stress is eliminated. In the "escape" mechanism, as the water level rises, the stem of Myricaria laxiflora will elongate to keep the top of the stem above the water level so as to continue to maintain photosynthesis. Therefore, the growth and development of the stem are important traits in the study of the waterlogging tolerance characteristics of Myricaria laxiflora.

[0004] To understand the waterlogging tolerance characteristics of the stem of Myricaria laxiflora more deeply, it is necessary to further understand its molecular mechanism of waterlogging tolerance regulation and identify the key genes responding to waterlogging stress. In related research, the identification and analysis of the relative expression levels of waterlogging tolerance regulation genes in the stem of Myricaria laxiflora are often involved, and it is necessary to simultaneously perform real-time fluorescence quantitative PCR detection on the target gene and the internal reference gene. The stability of the expression of the internal reference gene is crucial for the analysis result of the expression level of the target gene and will directly affect the accuracy of the result. Therefore, screening and identifying relevant internal reference genes is of great significance for the study of functional genes involved in the waterlogging tolerance regulation of the stem of Myricaria laxiflora. Summary of the Invention

[0005] The present invention provides an internal reference gene, a primer pair, a kit, and an application for gene expression analysis in the stem of Myricaria laxiflora under waterlogging stress, which can overcome the possible experimental differences in the sample preparation process and the detection process.

[0006] The present invention provides a method for gene expression analysis in the stem of Myricaria laxiflora under waterlogging stress, which is simple to operate, accurate in results, and convenient for large-scale operation.

[0007] The present invention also provides a method for screening reference genes for gene expression analysis in the stems of Myricaria laxiflora under waterlogging stress, which is scientific, reasonable, simple to operate, and accurate in results, and can screen out reference genes with stable expression in the stem tissues of Myricaria laxiflora under waterlogging stress.

[0008] The present invention provides a reference gene for gene expression analysis in the stems of Myricaria laxiflora under waterlogging stress. Among them, the reference gene is a ubiquitin-conjugating enzyme-encoding gene, and the nucleotide sequence of the reference gene is shown as SEQ ID NO:1.

[0009] The present invention provides an application of a ubiquitin-conjugating enzyme-encoding gene as a reference gene in gene expression analysis of the stems of Myricaria laxiflora under waterlogging stress. Among them, the nucleotide sequence of the ubiquitin-conjugating enzyme-encoding gene is shown as SEQ ID NO:1.

[0010] The present invention provides a primer pair for amplifying a ubiquitin-conjugating enzyme-encoding gene. Among them, the nucleotide sequence of the ubiquitin-conjugating enzyme-encoding gene is shown as SEQ ID NO:1. The primer pair includes a first primer and a second primer. The nucleotide sequence of the first primer is shown as SEQ ID NO:2, and the nucleotide sequence of the second primer is shown as SEQ ID NO:3.

[0011] The present invention provides an application of the above-mentioned primer pair in gene expression analysis of the stems of Myricaria laxiflora under waterlogging stress.

[0012] The present invention provides a kit. Among them, the kit includes the above-mentioned primer pair.

[0013] For the kit as described above, the kit further includes at least one of water and qRT-PCR buffer.

[0014] The present invention provides an application of the above-mentioned kit in gene expression analysis of the stems of Myricaria laxiflora under waterlogging stress.

[0015] The present invention provides a method for gene expression analysis in the stems of Myricaria laxiflora under waterlogging stress. Among them, it includes the following steps:

[0016] Extract the RNA of the stems of Myricaria laxiflora under waterlogging stress and reverse transcribe to obtain a cDNA sample;

[0017] Using the cDNA sample as a template, in the same qRT-PCR amplification system, simultaneously amplify the target gene and the ubiquitin-conjugating enzyme-encoding gene using the primer set designed for the target gene and the above-mentioned primer pair to obtain the Ct value of the target gene and the Ct value of the ubiquitin-conjugating enzyme-encoding gene;

[0018] Analyze the expression level of the target gene according to the Ct value of the target gene and the Ct value of the ubiquitin-conjugating enzyme-encoding gene.

[0019] The method as described above, wherein the treatment time of waterlogging stress is 6 - 48 h.

[0020] The present invention provides a method for screening reference genes for gene expression analysis in the stems of Myricaria laxiflora under waterlogging stress, which includes the following steps:

[0021] Using the coding genes of actin, tubulin, and ubiquitin - conjugating enzyme as candidate reference genes, designing primer pairs for amplifying the candidate reference genes;

[0022] Extracting the RNA of the stems of Myricaria laxiflora under different waterlogging stress times, and reverse - transcribing to obtain cDNA samples; using the cDNA samples as templates, performing qRT - PCR detection with the primer pairs for amplifying the candidate reference genes to obtain the corresponding Ct values of the candidate reference genes under different waterlogging stress times;

[0023] Analyzing the expression stability of the candidate reference genes under waterlogging stress; determining the reference genes according to the corresponding Ct values of the candidate reference genes under different waterlogging stress times and the expression stability of the candidate reference genes under waterlogging stress.

[0024] The present invention provides a reference gene for gene expression analysis in the stems of Myricaria laxiflora under waterlogging stress. This reference gene is the coding gene of ubiquitin - conjugating enzyme, and its nucleotide sequence is as shown in SEQ ID NO:1. It is stably expressed in the stems of Myricaria laxiflora under waterlogging stress and is suitable for use as a reference gene for gene expression analysis in the stems of Myricaria laxiflora under waterlogging stress. It can overcome the possible experimental differences in the sample preparation process and the detection process, and provides practical help for the accurate identification of the expression levels of related genes in the stems of Myricaria laxiflora under waterlogging stress, laying a good foundation for analyzing the waterlogging - tolerance regulation mechanism of the stems of Myricaria laxiflora and better utilizing the waterlogging - tolerance characteristics of Myricaria laxiflora. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the box - and - whisker plot of the Ct values of the candidate reference genes ACT, TUB, and UBC in Example 4;

[0026] Figure 2 It is the melting curve of the candidate reference gene UBC in Example 4. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0027] To enable those skilled in the art to better understand the solution of the present invention, the present invention will be further described in detail below. The following specific embodiments are only used to describe the principles and features of the present invention, and the examples given are only for explaining the present invention and do not limit the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

[0028] In the first aspect of the present invention, a reference gene for gene expression analysis in the stems of Myricaria laxiflora under waterlogging stress is provided. Among them, the reference gene is a ubiquitin-conjugating enzyme-encoding gene, and the nucleotide sequence of the reference gene is shown as SEQ ID NO:1.

[0029] Myricaria laxiflora is an evergreen shrub distributed in the Three Gorges area of the Yangtze River in the subtropical zone and has strong waterlogging tolerance. To analyze the waterlogging tolerance regulation mechanism of Myricaria laxiflora and make better use of its waterlogging tolerance characteristics, it is necessary to perform gene expression analysis on Myricaria laxiflora under waterlogging stress. During the process of gene expression analysis, in order to correct possible experimental differences in sample quality, sample loading amount, and sample loading process, it is necessary to select a suitable reference gene as a calibration standard.

[0030] A reference gene refers to a known reference gene whose expression level is not affected by research conditions and can be constantly expressed among various samples. Generally, since the expression level of housekeeping genes is less affected by environmental factors and can be continuously expressed in almost all tissues and at all growth stages of an organism, housekeeping genes are usually selected as reference genes in gene expression analysis. However, the transcription level of housekeeping genes may also change in some different tissues or under different treatment conditions. In this regard, through long-term research, it has been found that the reference gene with the nucleotide sequence shown as SEQ ID NO:1 is stably expressed in the stems of Myricaria laxiflora under waterlogging stress and is suitable for use as a reference gene for gene expression analysis in the stems of Myricaria laxiflora under waterlogging stress.

[0031] In some embodiments of the present invention, when using the stem tissues of Myricaria laxiflora waterlogged for 6h, 12h, 24h, and 48h as experimental materials for quantitative real-time PCR (qRT-PCR), it can be found that the reference gene with the nucleotide sequence shown as SEQ ID NO:1 has a low Ct value, that is, a high expression abundance, and the average value of its Ct value under different waterlogging durations is 22.36. The Ct value of this reference gene also has a low standard deviation and coefficient of variation under different waterlogging durations, that is, a high expression stability, with a standard deviation of 0.25 and a coefficient of variation of 1.13. At the same time, under the detection of BestKeeper software, geNorm software, and NormFinder software, this reference gene has high stability under different waterlogging durations.

[0032] In the second aspect of the present invention, an application of a ubiquitin-conjugating enzyme-encoding gene as a reference gene in gene expression analysis of the stems of Myricaria laxiflora under waterlogging stress is provided, where the nucleotide sequence of the ubiquitin-conjugating enzyme-encoding gene is shown as SEQ ID NO:1.

[0033] The ubiquitin-conjugating enzyme (UBC) is an enzyme that can execute the second step of the ubiquitination reaction and degrade target proteins through the proteasome. The nucleotide sequence of its encoding gene is as shown in SEQ ID NO:1. In the research, it was found that the ubiquitin-conjugating enzyme encoding gene is stably expressed in the stems of Myricaria laxiflora under waterlogging stress and can be used as an internal reference gene for gene expression analysis in the stems of Myricaria laxiflora under waterlogging stress.

[0034] The third aspect of the present invention provides a primer pair for amplifying the ubiquitin-conjugating enzyme encoding gene. Among them, the nucleotide sequence of the ubiquitin-conjugating enzyme encoding gene is as shown in SEQ ID NO:1. The primer pair includes a first primer and a second primer. The nucleotide sequence of the first primer is as shown in SEQ ID NO:2, and the nucleotide sequence of the second primer is as shown in SEQ ID NO:3.

[0035] In the above technical solution, the first primer is a forward primer and the second primer is a reverse primer.

[0036] Based on the ubiquitin-conjugating enzyme encoding gene with the nucleotide sequence as shown in SEQ ID NO:1, the present invention designed a primer pair for amplifying the ubiquitin-conjugating enzyme encoding gene from the aspects of primer length, GC base content in the primer, and primer annealing temperature. The primer pair of the present invention has excellent accuracy and good specificity. In an embodiment of the present invention, the melting curve of the primer pair has a single melting peak, proving its good specificity.

[0037] The fourth aspect of the present invention provides an application of the above primer pair in gene expression analysis of the stems of Myricaria laxiflora under waterlogging stress.

[0038] The primer pair of the present invention can accurately amplify the ubiquitin-conjugating enzyme encoding gene with the nucleotide sequence as shown in SEQ ID NO:1. Since the ubiquitin-conjugating enzyme encoding gene is stably expressed in the stems of Myricaria laxiflora under waterlogging stress, it can be applied to gene expression analysis of the stems of Myricaria laxiflora under waterlogging stress.

[0039] The fifth aspect of the present invention provides a kit. Among them, the kit includes the above primer pair.

[0040] Since the kit includes the primer pair provided by the third aspect of the present invention, the kit also has the ability to accurately amplify the ubiquitin-conjugating enzyme encoding gene with the nucleotide sequence as shown in SEQ ID NO:1.

[0041] In the above technical solution, the kit further includes at least one of water and qRT-PCR buffer.

[0042] Among them, the water can be sterilized ultrapure water, which can provide the ionic environment and water molecule medium required for the qRT-PCR reaction, enabling the smooth progress of the interactions between various molecules in the reaction. The qRT-PCR buffer can contain at least one of dNTPs, Sso7d fusion polymerase, MgCl 2 , SYBR Green I, and can provide stable performance in rapid cycling within a wide range of reaction conditions, primer concentrations, and temperatures. Among them, dNTPs refer to four free deoxyribonucleoside triphosphates, which are essential raw materials for PCR amplification.

[0043] The sixth aspect of the present invention provides an application of the above-mentioned kit in the gene expression analysis of the stems of Myricaria laxiflora under waterlogging stress.

[0044] The above-mentioned kit includes the primer pair provided by the third aspect of the present invention, which can accurately amplify the ubiquitin-conjugating enzyme-encoding gene with a nucleotide sequence as shown in SEQ ID NO:1, and thus can be applied to the gene expression analysis of the stems of Myricaria laxiflora under waterlogging stress.

[0045] The seventh aspect of the present invention provides a method for gene expression analysis in the stems of Myricaria laxiflora under waterlogging stress, which includes the following steps:

[0046] Extract the RNA of the stems of Myricaria laxiflora under waterlogging stress and reverse transcribe it to obtain a cDNA sample;

[0047] Using the cDNA sample as a template, simultaneously amplify the target gene and the ubiquitin-conjugating enzyme-encoding gene in the same qRT-PCR amplification system using the primer set designed for the target gene and the above-mentioned primer pair to obtain the Ct value of the target gene and the Ct value of the ubiquitin-conjugating enzyme-encoding gene;

[0048] Analyze the expression level of the target gene based on the Ct value of the target gene and the Ct value of the ubiquitin-conjugating enzyme-encoding gene.

[0049] The method for gene expression analysis in the stems of Myricaria laxiflora under waterlogging stress provided by the present invention can overcome the possible experimental differences in the sample preparation process and the detection process, and provides practical help for the accurate identification of the expression levels of related genes in the stems of Myricaria laxiflora under waterlogging stress. At the same time, the method provided by the present invention is simple to operate, the results are accurate, and it is convenient for large-scale operation.

[0050] Among them, how to prepare the tissue materials of the stems of Myricaria laxiflora under waterlogging stress, how to extract the RNA of the materials, and how to reverse transcribe the RNA to obtain a cDNA sample are conventional techniques in the art, and the present invention does not make any limitations.

[0051] In one embodiment of the present invention, the method for preparing the stem tissue material of Myricaria laxiflora under waterlogging stress is as follows: Immerse the whole plant of one-year-old Myricaria laxiflora in water for waterlogging stress treatment, then quickly freeze it with liquid nitrogen and store it in a -80°C refrigerator.

[0052] In one embodiment of the present invention, the method for extracting RNA from the material is as follows: Take 50 - 100 mg of the stem tissue material of Myricaria laxiflora under waterlogging stress, fully grind it in a mortar containing liquid nitrogen until the stem tissue becomes powdery, and then use the Fast Pure Universal Plant Total RNA Isolation Kit produced by Novoprotein to extract the total RNA of the stem tissue.

[0053] In one embodiment of the present invention, the method for reverse transcribing RNA to obtain a cDNA sample is as follows: Use the PrimeScript TM RT reagent Kit with gDNA Eraser produced by TaKaRa to reverse transcribe the extracted total RNA to obtain a cDNA product.

[0054] In addition, for better subsequent qRT-PCR, the concentration of the cDNA product can be detected and regulated to generate a cDNA template more suitable for qRT-PCR. For example, in one embodiment of the present invention, the OD 260 / 280 value of the cDNA product can be measured by ThermoNanoDrop 2000c, and the cDNA product can be diluted at a 10-fold concentration to obtain a cDNA template for subsequent real-time fluorescence quantitative PCR reaction.

[0055] Next, for the target gene, a primer set needs to be designed according to the sequence of the target gene. The method of designing the primer set is a conventional technique in the art, and those skilled in the art can design it according to their needs, and the present invention does not make any limitations.

[0056] Subsequently, using the above cDNA sample as a template, in the same qRT-PCR amplification system, the primer set designed with the target gene and the above-mentioned primer pair are used to simultaneously amplify the target gene and the ubiquitin-conjugating enzyme coding gene, and the amplification curves of the target gene and the ubiquitin-conjugating enzyme coding gene can be obtained, and then the Ct values of the target gene and the ubiquitin-conjugating enzyme coding gene can be obtained. It can be understood that the primer set designed with the target gene will amplify the target gene sequence, and the primer pair provided by the present invention will amplify the ubiquitin-conjugating enzyme coding gene sequence (SEQ ID NO:1).

[0057] Among them, the Ct value (cycle threshold) is the number of amplification cycles corresponding to when the fluorescence signal of the amplification product reaches the set fluorescence threshold during qRT-PCR amplification, and it is a relative measurement of the gene expression abundance in qRT-PCR reactions. However, in addition to gene expression abundance, some experimental factors may also affect the Ct value, thereby causing experimental personnel to make incorrect judgments on the gene expression abundance of the target gene.

[0058] Therefore, the present invention introduces a reference gene that can still be stably expressed in the stems of Myricaria laxiflora under waterlogging stress, namely the ubiquitin-conjugating enzyme encoding gene (SEQ ID NO:1), and uses the Ct value of this gene to analyze the expression level of the target gene.

[0059] For example, in the same qRT-PCR amplification system, the primer set designed using the target gene and the above-mentioned primer pair are used to simultaneously amplify the target gene and the ubiquitin-conjugating enzyme encoding gene. When the Ct value of the ubiquitin-conjugating enzyme encoding gene is between 20 - 25, the Ct value of the target gene can be used to analyze the expression level of the target gene.

[0060] In the above technical solution, the treatment time of waterlogging stress is 6 - 48 h.

[0061] When the waterlogging stress time meets the above range, the method for analyzing gene expression in the stems of Myricaria laxiflora under waterlogging stress provided by the present invention has higher accuracy.

[0062] The eighth aspect of the present invention provides a method for screening a reference gene for analyzing gene expression in the stems of Myricaria laxiflora under waterlogging stress, which includes the following steps:

[0063] Using the actin encoding gene, tubulin encoding gene, and ubiquitin-conjugating enzyme encoding gene as candidate reference genes, design primer pairs for amplifying the candidate reference genes;

[0064] Extract the RNA of the stems of Myricaria laxiflora under different waterlogging stress times, reverse transcribe to obtain cDNA samples; using the cDNA samples as templates, perform qRT-PCR detection with the primer pairs for amplifying the candidate reference genes to obtain the corresponding Ct values of the candidate reference genes under different waterlogging stress times;

[0065] Analyze the expression stability of the candidate reference genes under waterlogging stress; determine the reference gene according to the corresponding Ct values of the candidate reference genes under different waterlogging stress times and the expression stability of the candidate reference genes under waterlogging stress.

[0066] The screening method of the present invention is scientific and reasonable, simple to operate, and accurate in results, and can screen out reference genes that are stably expressed in the stems of Myricaria laxiflora under waterlogging stress.

[0067] Among them, the nucleotide sequence of the ubiquitin-conjugating enzyme encoding gene is as shown in SEQ ID NO:1, and the primer pair sequences designed according to this candidate reference gene are as shown in SEQ ID NO:2 and SEQ ID NO:3; the nucleotide sequence of the actin encoding gene is as shown in SEQ ID NO:4, and the primer pair sequences designed according to this candidate reference gene are as shown in SEQ ID NO:5 and SEQ ID NO:6; the nucleotide sequence of the tubulin encoding gene is as shown in SEQ ID NO:7, and the primer pair sequences designed according to this candidate reference gene are as shown in SEQ ID NO:8 and SEQ ID NO:9.

[0068] Specifically, to better select candidate reference genes, RNA-seq data can be obtained by performing transcriptome sequencing on the stem tissues of Myricaria laxiflora under different waterlogging stress times, and then the FPKM mean values of the reference genes in the Unigene gene database can be obtained based on the RNA-seq data. Finally, the reference genes with relatively stable expression among them are selected as candidate reference genes. In the present invention, the selected reference genes are the actin gene, the tubulin gene, and the ubiquitin-conjugating enzyme gene.

[0069] Next, RNA is extracted from the stem tissues of Myricaria laxiflora under different waterlogging stress times, and cDNA samples are obtained by reverse transcription; using the cDNA samples as templates, qRT-PCR detection is performed using the primer pairs for amplifying the candidate reference genes to obtain the corresponding Ct values of the candidate reference genes under different waterlogging stress times.

[0070] Subsequently, the expression stability of the candidate reference genes under waterlogging stress is analyzed.

[0071] In an embodiment of the present invention, the BestKeeper software is used to compare the standard deviation and coefficient of variation of the Ct values of the candidate reference genes under different waterlogging durations to analyze the expression stability of the candidate reference genes; the geNorm software is used to calculate the M value to analyze the expression stability of the candidate reference genes; the NormFinder software is used to calculate the stability value M to analyze the expression stability of the candidate reference genes.

[0072] Finally, according to the corresponding Ct values of the candidate reference genes under different waterlogging stress times and the expression stability of the candidate reference genes under waterlogging stress, the reference genes are determined. In the present invention, the screened reference gene is the ubiquitin-conjugating enzyme encoding gene (SEQ ID NO:1).

[0073] Hereinafter, the technical solutions of the present application will be further explained and illustrated in combination with specific embodiments. For the experimental methods without specific conditions indicated in the following embodiments, they are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer. The reagents used, unless otherwise specified, are commercially available or can be obtained through public channels.

[0074] The primers or gene sequences involved in the following embodiments are shown in Table 1:

[0075] Table 1

[0076]

[0077] Example 1: Waterlogging stress treatment

[0078] The whole plant of one-year-old Myricaria laxiflora was immersed in water for waterlogging stress treatment. The stem tissues were taken at 6 h, 12 h, 24 h, and 48 h of waterlogging respectively. Each stress treatment was repeated 3 times, and after quick freezing with liquid nitrogen, they were stored in a -80 °C refrigerator.

[0079] Example 2: Transcriptome sequencing (RNA sequencing, RNA-seq)

[0080] Transcriptome sequencing was performed on the stem tissues under different waterlogging durations (6 h, 12 h, 24 h, and 48 h) in Example 1 to obtain RNA-seq data. Based on the RNA-seq data, the FPKM means of the reference genes in the Unigene gene database were obtained, and the reference genes with relatively stable expression were selected as candidate reference genes, namely the genes encoding actin (Actin, ACT), tubulin beta (TUB), and ubiquitin-conjugating enzyme (UBC), as shown in Table 2 for details.

[0081] Table 2

[0082]

[0083] Example 3: RNA extraction and cDNA synthesis

[0084] Take 50 - 100 mg of the stem tissues in Example 1 under different waterlogging durations (6 h, 12 h, 24 h, and 48 h), and grind them thoroughly in a mortar containing liquid nitrogen until the stem tissues become powdery. Use the Fast Pure Universal Plant Total RNA Isolation Kit produced by Novoprotein Scientific Inc. to extract the total RNA of the stem tissues. Subsequently, use the PrimeScriptTM RT reagent Kit with gDNA Eraser produced by TaKaRa to reverse transcribe the extracted total RNA to obtain cDNA products. Measure the OD 260 / 280 value of the cDNA products with a Thermo NanoDrop 2000c, and dilute the cDNA products at a 10-fold concentration to obtain the cDNA templates for subsequent real-time fluorescence quantitative PCR reactions.

[0085] Example 4: Primer synthesis

[0086] Design and synthesize the primer pairs for detecting candidate reference genes by quantitative real-time PCR (qPT-PCR) using primer design software. The length of each primer is 25 bp, the Tm value ranges from 55 °C to 65 °C, and the size of the amplified fragment is between 100 - 300 bp. After performing PCR with the detection primer pairs, use agarose gel electrophoresis to detect the specificity of the PCR products. Select the primer pairs with the correct and unique band sizes for subsequent real-time fluorescence quantitative PCR reactions. The selected primer pairs are shown in Table 1. Among them, the primer shown as SEQ ID NO:2 is the forward primer, the primer shown as SEQ ID NO:3 is the reverse primer, and the primer pair shown as SEQ ID NO:2 and SEQ ID NO:3 can amplify the UBC gene, and its nucleotide sequence is shown as SEQ ID NO:1; the primer shown as SEQ ID NO:5 is the forward primer, the primer shown as SEQ ID NO:6 is the reverse primer, and the primer pair shown as SEQ ID NO:5 and SEQ ID NO:6 can amplify the ACT gene, and its nucleotide sequence is shown as SEQ ID NO:4; the primer shown as SEQ ID NO:8 is the forward primer, the primer shown as SEQ ID NO:9 is the reverse primer, and the primer pair shown as SEQ ID NO:8 and SEQ ID NO:9 can amplify the TUB gene, and its nucleotide sequence is shown as SEQ ID NO:7.

[0087] Example 5: Real-time fluorescence quantitative PCR

[0088] Using the cDNA template obtained in Example 3 and the primer pairs obtained in Example 4, a real-time fluorescence quantitative PCR reaction was performed to obtain an amplification curve. Among them, the total system of the real-time fluorescence quantitative PCR reaction was 20 μL, including 10 μL of SsoAdvanced Universal Green Supermix produced by Bio-Rad, 0.2 μL of forward primer, 0.2 μL of reverse primer, 1 μL of cDNA template, and 8.6 μL of sterilized ultrapure water. The real-time fluorescence quantitative PCR reaction adopted a two-step amplification program: pre-denaturation at 95 °C for 3 min, denaturation at 95 °C for 5 s, annealing at 60 °C for 30 s, for 40 cycles. The cycle threshold (Ct) of the candidate reference genes was obtained according to the amplification curve, and the following analysis was performed based on the Ct values of the candidate reference genes:

[0089] (1) Using Microsoft Excel 2022, the Ct values of the candidate reference genes under different waterlogging durations (6 h, 12 h, 24 h, and 48 h) were statistically analyzed to analyze the expression abundance of the candidate reference genes. Specifically, see Table 3. The smaller the Ct value, the higher the expression abundance of the candidate reference gene; the larger the Ct value, the lower the expression abundance of the candidate reference gene. At the same time, the box plot of the Ct values was made using SPSS 25.0 software. Specifically, see Figure 1 .

[0090] Table 3

[0091]

[0092] As shown in Table 3 and Figure 1 shown, the average Ct values from high to low were TUB, ACT, and UBC, indicating that UBC had the highest average expression abundance, while TUB had the lowest average expression abundance; the range of change in Ct values from large to small was TUB, ACT, and UBC, indicating that UBC had the smallest range of change in Ct values, while TUB had the largest range of change in Ct values.

[0093] (2) Using the BestKeeper software, the standard deviation (SD) and coefficient of variation (CV) of the Ct values of the candidate reference genes under different waterlogging durations (6 h, 12 h, 24 h, and 48 h) were compared to analyze the expression stability of the candidate reference genes. Among them, the smaller the standard deviation and coefficient of variation, the better the expression stability of the candidate reference gene is proven, and vice versa, the worse the expression stability. The default threshold for the SD value is 1.0. When SD < 1, the candidate reference gene is stably expressed; when SD > 1, the candidate reference gene is unstably expressed. Specifically, see Table 4.

[0094] Table 4

[0095] Gene Name Gene Annotation SD Value CV Value ACT Actin 0.33 1.36 TUB Tubulin 0.70 2.81 UBC Ubiquitin Ligase 0.25 1.13

[0096] As shown in Table 4, the SD values and CV values are in descending order as TUB, ACT, and UBC, indicating that under waterlogging stress treatment, the expression of the candidate reference gene UBC is the most stable.

[0097] (3) The geNorm software was used to analyze the expression stability of candidate reference genes based on the M value. The smaller the M value, the better the expression stability of the candidate reference gene, and the larger the M value, the worse the expression stability of the candidate reference gene. The default cut-off value in this experiment was 1.5, that is, candidate reference genes with M values > 1.5 were not suitable for use as reference genes for Myricaria laxiflora under waterlogging stress treatment, while the expression of candidate reference genes with M values ≤ 1.5 was relatively stable. See Table 5 for details.

[0098] Table 5

[0099] Gene Name Gene Annotation M Value ACT Actin 0.275 TUB Tubulin 0.709 UBC Ubiquitin Ligase 0.275

[0100] As shown in Table 5, the candidate reference genes with the lowest M values were ACT and UBC, with M values reaching 0.275, while the M value of TUB was significantly higher, reaching 0.709, indicating that the expression of candidate reference genes ACT and UBC was relatively stable.

[0101] (4) The NormFinder software was used to calculate the stability value M to analyze the expression stability of candidate reference genes. The lower the M value, the better the expression stability of the candidate reference gene, and the larger the M value, the worse the expression stability of the candidate reference gene. See Table 6 for details.

[0102] Table 6

[0103] Gene Name Gene Annotation M Value ACT Actin 0.419 TUB Tubulin 0.908 UBC Ubiquitin Ligase 0.138

[0104] As shown in Table 6, the M values are in descending order as TUB, ACT, and UBC, indicating that under waterlogging stress treatment, the expression of the candidate reference gene UBC is the most stable.

[0105] In addition, after real-time fluorescence quantitative PCR, a melting curve program was carried out by heating from 60 °C to 95 °C at a heating rate of 0.5 °C / s, with 3 biological replicates, and a melting curve graph was obtained. See Figure 2 . Figure 2 is the melting curve graph of the candidate reference gene UBC, indicating that the melting curve peaks of the candidate reference gene UBC of Myricaria laxiflora at different waterlogging durations (6 h, 12 h, 24 h, and 48 h) are all single peaks, and the specificity of the detection primer pair of this candidate reference gene is better.

[0106] In summary, according to the analysis results in the BestKeeper, geNorm and NormFinder software, it can be concluded that the candidate reference gene UBC has the best expression stability and can be applied to the gene expression analysis of the stems of *Myricaria laxiflora* under flooding stress. Therefore, the present invention provides a reference gene for gene expression analysis in the stems of *Myricaria laxiflora* under flooding stress. This reference gene is the coding gene of ubiquitin-conjugating enzyme, and its nucleotide sequence is shown in SEQ ID NO:1. It is stably expressed in the stems of *Myricaria laxiflora* under flooding stress and is suitable for use as a reference gene for gene expression analysis in the stems of *Myricaria laxiflora* under flooding stress. It can overcome the experimental differences that may exist during sample preparation and detection, and provides practical help for the accurate identification of the expression levels of related genes in the stems of *Myricaria laxiflora* under flooding stress, laying a good foundation for analyzing the flooding tolerance regulation mechanism of the stems of *Myricaria laxiflora* and better utilizing the flooding tolerance characteristics of *Myricaria laxiflora*.

[0107] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An internal reference gene for gene expression analysis in branches and stems of Echinops sparsely flowered under flooding stress, characterized in that: The internal reference gene is a ubiquitin-binding enzyme encoding gene, and the nucleotide sequence of the internal reference gene is shown in SEQ ID NO:

1.

2. The use of ubiquitin-binding enzyme encoding genes as internal reference genes in gene expression analysis of Echinops sparsely flowered branches and stems under flooding stress, characterized in that: The nucleotide sequence of the ubiquitin conjugating enzyme encoding gene is shown in SEQ ID NO:

1.

3. A primer pair for amplifying a gene encoding a ubiquitin-binding enzyme, characterized in that: The nucleotide sequence of the ubiquitin conjugating enzyme encoding gene is shown in SEQ ID NO:1, the primer pair includes a first primer and a second primer, the nucleotide sequence of the first primer is shown in SEQ ID NO:2, and the nucleotide sequence of the second primer is shown in SEQ ID NO:

3.

4. Use of the primer pair according to claim 3 in gene expression analysis of Echinops sparsely flowered branches and stems under waterlogging stress.

5. A kit, characterized in that: The kit comprises the primer pair according to claim 3.

6. The kit according to claim 5, characterized in that The kit further comprises at least one of water and qRT-PCR buffer.

7. Use of the kit according to claim 5 or 6 in gene expression analysis of Eupatorium sparseflorum branches and stems under waterlogging stress.

8. A method for analyzing gene expression in branches and stems of Echinops sparsely flowered under flooding stress, characterized in that: The steps include: RNA was extracted from the branches and stems of Echinops sparsely flowered under flooding stress, and reverse transcribed to obtain cDNA samples; Using the cDNA sample as a template, the target gene and the ubiquitin-binding enzyme encoding gene are simultaneously amplified using the primer set designed for the target gene and the primer pair according to claim 3 in the same qRT-PCR amplification system to obtain the Ct value of the target gene and the Ct value of the ubiquitin-binding enzyme encoding gene; The expression level of the target gene is analyzed according to the Ct value of the target gene and the Ct value of the ubiquitin conjugating enzyme encoding gene.

9. The method according to claim 8, characterized in that The treatment time of the waterlogging stress is 6-48h.

10. A method for screening internal reference genes for gene expression analysis in branches and stems of Echinops sparsely flowered under flooding stress, characterized in that: The steps include: Using actin encoding gene, tubulin encoding gene, and ubiquitin conjugating enzyme encoding gene as candidate internal reference genes, designing primer pairs for amplifying the candidate internal reference genes; Extracting RNA from the branches and stems of Echinops sparsely flowered under different flooding stress times, and reversely transcribing to obtain cDNA samples; using the cDNA samples as templates, performing qRT-PCR detection using primer pairs for amplifying the candidate internal reference genes, and obtaining corresponding Ct values ​​of the candidate internal reference genes under different flooding stress times; Analyze the expression stability of candidate reference genes under flooding stress; The internal reference gene is determined according to the corresponding Ct value of the candidate internal reference gene at different flooding stress times and the expression stability of the candidate internal reference gene under flooding stress.

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