Internal reference gene for gene expression analysis in myricaria laxiflora leaves under 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 leaves under flooding stress and a primer pair and application thereof. The reference gene is a ubiquitin-binding enzyme coding gene, the nucleotide sequence of the reference gene is shown as SEQ ID NO: 1, and the reference gene is stably expressed in myricaria laxiflora leaves under water logging stress, is suitable for being used as the reference gene for gene expression analysis in the myricaria laxiflora leaves under water logging 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 the related genes in the myricaria laxiflora leaves under flooding stress, and a good foundation is laid for analyzing a flooding-resistant regulation mechanism of the myricaria laxiflora leaves and better utilizing the flooding-resistant characteristic of the myricaria laxiflora.

Description

Technical Field

[0001] The present invention relates to plant biotechnology, and particularly to a reference gene, a primer pair thereof and an application for gene expression analysis in Myricaria laxiflora leaves 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 after the water recedes in winter and spring. 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. Therefore, carrying out the functional research on the genes related to waterlogging tolerance regulation in Myricaria laxiflora and analyzing the waterlogging tolerance regulation mechanism of Myricaria laxiflora can provide an important theoretical basis for the utilization of waterlogging tolerance-related resources in Myricaria laxiflora.

[0003] In the functional research on the genes related to waterlogging tolerance regulation in Myricaria laxiflora, the identification and analysis of the relative expression levels of genes are often involved, and it is necessary to simultaneously perform real-time fluorescence quantitative PCR detection on the target gene and the reference gene. The stability of the reference gene expression is crucial for the analysis result of the target gene expression level and will directly affect the accuracy of the result. Therefore, screening and identifying relevant reference genes is of great significance for the research on the functional genes involved in waterlogging tolerance regulation in Myricaria laxiflora leaf tissues. Summary of the Invention

[0004] The present invention provides a reference gene, a primer pair, a kit and an application thereof for gene expression analysis in Myricaria laxiflora leaves under waterlogging stress, which can overcome the experimental differences that may exist in the sample preparation process and the detection process.

[0005] The present invention provides a method for gene expression analysis in Myricaria laxiflora leaves under waterlogging stress, which is simple to operate, has accurate results and is convenient for large-scale operation.

[0006] The present invention also provides a method for screening a reference gene for gene expression analysis in Myricaria laxiflora leaves under waterlogging stress, which is scientific and reasonable, simple to operate and has accurate results, and can screen out a reference gene with stable expression in Myricaria laxiflora leaves under waterlogging stress.

[0007] The present invention provides a reference gene for gene expression analysis in Myricaria laxiflora leaves under waterlogging stress, wherein the reference gene is a ubiquitin-conjugating enzyme-encoding gene, and the nucleotide sequence of the reference gene is as shown in SEQ ID NO:1.

[0008] The present invention provides an application of a ubiquitin-conjugating enzyme encoding gene as an internal reference gene in gene expression analysis of Myricaria laxiflora leaves under waterlogging stress, wherein the nucleotide sequence of the ubiquitin-conjugating enzyme encoding gene is as shown in SEQ ID NO: 1.

[0009] The present invention provides a primer pair for amplifying a ubiquitin-conjugating enzyme encoding gene, wherein 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.

[0010] The present invention provides an application of the above primer pair in gene expression analysis of Myricaria laxiflora leaves under waterlogging stress.

[0011] The present invention provides a kit, wherein the kit includes the above primer pair.

[0012] The kit as described above, wherein the kit further includes at least one of water and qRT-PCR buffer.

[0013] The present invention provides an application of the above kit in gene expression analysis of Myricaria laxiflora leaves under waterlogging stress.

[0014] The present invention provides a method for gene expression analysis in Myricaria laxiflora leaves under waterlogging stress, which includes the following steps:

[0015] Extract the RNA of Myricaria laxiflora leaves under waterlogging stress and reverse transcribe to obtain a cDNA sample;

[0016] 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 with the primer set designed for the target gene and the above primer pair to obtain the Ct value of the target gene and the Ct value of the ubiquitin-conjugating enzyme encoding gene;

[0017] 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.

[0018] The method as described above, wherein the treatment time of waterlogging stress is 6 - 48 h.

[0019] The present invention further provides a method for screening an internal reference gene for gene expression analysis in Myricaria laxiflora leaves under waterlogging stress, which includes the following steps:

[0020] Using the actin encoding gene, the tubulin encoding gene, and the ubiquitin-conjugating enzyme encoding gene as candidate internal reference genes, design primer pairs for amplifying the candidate internal reference genes;

[0021] Extract the RNA of Myricaria laxiflora leaves under different waterlogging stress times, and reverse transcribe to obtain cDNA samples; using the cDNA samples as templates, perform qRT-PCR detection with primer pairs for amplifying candidate reference genes to obtain the corresponding Ct values of the candidate reference genes under different waterlogging stress times;

[0022] 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.

[0023] The present invention provides a reference gene for gene expression analysis in Myricaria laxiflora leaves under waterlogging stress. This reference gene encodes a ubiquitin-conjugating enzyme, and its nucleotide sequence is shown in SEQ ID NO:1. This reference gene is stably expressed in Myricaria laxiflora leaves under waterlogging stress and is suitable for use as a reference gene for gene expression analysis in Myricaria laxiflora leaves under waterlogging stress. It can overcome the possible experimental differences during sample preparation and detection processes, and provides practical help for the accurate identification of the expression levels of related genes in Myricaria laxiflora leaves under waterlogging stress, laying a good foundation for analyzing the waterlogging tolerance regulation mechanism of Myricaria laxiflora leaves and better utilizing the waterlogging tolerance characteristics of Myricaria laxiflora. Description of the Drawings

[0024] Figure 1 It is the box plot of the Ct values of candidate reference genes ACT, TUB, and UBC in Example 4;

[0025] Figure 2 It is the melting curve of candidate reference gene UBC in Example 4. Detailed Embodiments

[0026] 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 listed are only used to describe the principles and features of the present invention, and the examples given are only used to explain 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.

[0027] In the first aspect of the present invention, a reference gene for gene expression analysis in Myricaria laxiflora leaves under waterlogging stress is provided, wherein the reference gene is a gene encoding a ubiquitin-conjugating enzyme, and the nucleotide sequence of the reference gene is shown in SEQ ID NO:1.

[0028] Myricaria laxiflora is an evergreen shrub distributed in the Three Gorges area of the Yangtze River in the subtropical zone, with strong waterlogging tolerance characteristics. To analyze the regulatory mechanism of waterlogging tolerance in Myricaria laxiflora and make better use of its waterlogging tolerance characteristics, it is necessary to conduct gene expression analysis on Myricaria laxiflora under waterlogging stress. During the process of gene expression analysis, in order to correct the possible experimental differences in sample quality, sample loading amount, and sample loading process of the analysis samples, it is necessary to select appropriate internal reference genes as correction standards.

[0029] Internal reference genes refer to known reference genes whose expression levels are not affected by research conditions and can be constantly expressed among various samples. Generally, since the expression levels of housekeeping genes are less affected by environmental factors and can be continuously expressed in almost all tissues and at all growth stages of organisms, housekeeping genes are usually selected as internal reference genes in gene expression analysis. However, the transcriptional levels 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 internal reference gene with the nucleotide sequence shown in SEQ ID NO:1 is stably expressed in the leaves of Myricaria laxiflora under waterlogging stress and is suitable for use as an internal reference gene for gene expression analysis in the leaves of Myricaria laxiflora under waterlogging stress.

[0030] In some embodiments of the present invention, when using the leaves of Myricaria laxiflora waterlogged for 6h, 12h, 24h, and 48h as experimental materials for real-time fluorescence quantitative PCR (qRT-PCR), it can be found that the internal reference gene with the nucleotide sequence shown in 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 22.81. The Ct value of this internal 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 0.39 and a coefficient of variation of 1.71. At the same time, under the detection of BestKeeper software, geNorm software, and NormFinder software, this internal reference gene has high stability under different waterlogging durations.

[0031] The second aspect of the present invention provides an application of a ubiquitin-conjugating enzyme encoding gene as an internal reference gene in the gene expression analysis of the leaves of Myricaria laxiflora under waterlogging stress, wherein the nucleotide sequence of the ubiquitin-conjugating enzyme encoding gene is as shown in SEQ ID NO:1.

[0032] 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 leaves of Myricaria laxiflora under waterlogging stress, and can be used as an internal reference gene for gene expression analysis in the leaves of Myricaria laxiflora under waterlogging stress.

[0033] 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.

[0034] Among them, the first primer is a forward primer, and the second primer is a reverse primer.

[0035] 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 aspects such as 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.

[0036] The fourth aspect of the present invention provides an application of the above primer pair in gene expression analysis of the leaves of Myricaria laxiflora under waterlogging stress.

[0037] 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, and because the ubiquitin-conjugating enzyme encoding gene is stably expressed in the leaves of Myricaria laxiflora under waterlogging stress, it can also be applied to gene expression analysis in the leaves of Myricaria laxiflora under waterlogging stress.

[0038] The fifth aspect of the present invention provides a kit. Among them, the kit includes the above primer pair.

[0039] Since the kit includes the primer pair provided in 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.

[0040] In the above technical solution, the kit further includes at least one of water and qRT-PCR buffer.

[0041] 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, MgCl2, and 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.

[0042] The sixth aspect of the present invention provides an application of the above-mentioned kit in the gene expression analysis of Myricaria laxiflora leaves under waterlogging stress.

[0043] 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 Myricaria laxiflora leaves under waterlogging stress.

[0044] The seventh aspect of the present invention provides a method for gene expression analysis in Myricaria laxiflora leaves under waterlogging stress, which includes the following steps:

[0045] Extract the RNA of Myricaria laxiflora leaves under waterlogging stress and reverse transcribe it to obtain a cDNA sample;

[0046] 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;

[0047] 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.

[0048] The method for gene expression analysis in Myricaria laxiflora leaves 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 Myricaria laxiflora leaves 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.

[0049] Among them, how to prepare the Myricaria laxiflora leaf material under waterlogging stress, how to extract the RNA of the material, 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 restrictions.

[0050] In one embodiment of the present invention, the method for preparing the Myricaria laxiflora leaf material under waterlogging stress is as follows: Immerse the whole one-year-old Myricaria laxiflora plant in water for waterlogging stress treatment, then quickly freeze it with liquid nitrogen and store it in a -80°C refrigerator.

[0051] In one embodiment of the present invention, the method for extracting RNA from the material is as follows: Take 50 - 100 mg of the Myricaria laxiflora leaf material under waterlogging stress, grind it thoroughly in a mortar containing liquid nitrogen until the leaf tissue becomes powdery, and then use the Fast Pure Universal Plant Total RNA Isolation Kit produced by Novizan Biotech Co., Ltd. to extract the total RNA of the leaf tissue.

[0052] 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 Company to reverse transcribe the extracted total RNA to obtain a cDNA product.

[0053] 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 Thermo NanoDrop 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.

[0054] 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, which is not limited in the present invention.

[0055] 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 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 value of the target gene and the Ct value of 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).

[0056] 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 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 misjudge the gene expression abundance of the target gene.

[0057] Therefore, the present invention introduces an internal reference gene that can still be stably expressed in the leaves 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.

[0058] For example, in the same qRT-PCR amplification system, the primer pair 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 18-25, the Ct value of the target gene can be used to analyze the expression level of the target gene.

[0059] In the above technical solution, the treatment time of waterlogging stress is 6-48 h.

[0060] When the waterlogging stress time meets the above range, the method for analyzing gene expression in the leaves of Myricaria laxiflora under waterlogging stress provided by the present invention has higher accuracy.

[0061] The eighth aspect of the present invention provides a method for screening an internal reference gene for analyzing gene expression in the leaves of Myricaria laxiflora under waterlogging stress, which includes the following steps:

[0062] Using the actin-encoding gene, tubulin-encoding gene, and ubiquitin-conjugating enzyme-encoding gene as candidate internal reference genes, design primer pairs for amplifying the candidate internal reference genes;

[0063] Extract the RNA of the leaves of Myricaria laxiflora under different waterlogging stress times, and reverse transcribe to obtain cDNA samples; use the cDNA samples as templates, and perform qRT-PCR detection using the primer pairs for amplifying the candidate internal reference genes to obtain the corresponding Ct values of the candidate internal reference genes under different waterlogging stress times;

[0064] Analyze the expression stability of the candidate internal reference genes under waterlogging stress; determine the internal reference gene according to the corresponding Ct values of the candidate internal reference genes under different waterlogging stress times and the expression stability of the candidate internal reference genes under waterlogging stress.

[0065] The screening method of the present invention is scientific and reasonable, simple to operate, and accurate in results, and can screen out internal reference genes that are stably expressed in the leaves of Myricaria laxiflora under waterlogging stress.

[0066] Among them, the nucleotide sequence of the actin-encoding gene is shown in SEQ ID NO:4, and the primer pair sequences designed according to this candidate reference gene are shown in SEQ ID NO:5 and SEQ ID NO:6; the nucleotide sequence of the tubulin-encoding gene is shown in SEQ ID NO:7, and the primer pair sequences designed according to this candidate reference gene are shown in SEQ ID NO:8 and SEQ ID NO:9; the nucleotide sequence of the ubiquitin-conjugating enzyme-encoding gene is shown in SEQ ID NO:1, and the primer pair sequences designed according to this candidate reference gene are shown in SEQ ID NO:2 and SEQ ID NO:3.

[0067] Specifically, to better select candidate reference genes, RNA-seq data can be obtained by first performing transcriptome sequencing on Myricaria laxiflora leaves under different waterlogging stress times, then obtaining the FPKM mean values of the reference genes in the Unigene gene database based on the RNA-seq data, and finally selecting the reference genes with relatively stable expression among them as candidate reference genes. In the present invention, the selected candidate reference genes are the actin gene, the tubulin gene, and the ubiquitin-conjugating enzyme gene.

[0068] Next, RNA is extracted from Myricaria laxiflora leaves 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.

[0069] Subsequently, the expression stability of the candidate reference genes under waterlogging stress is analyzed.

[0070] In one 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.

[0071] 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).

[0072] Hereinafter, the technical solutions of the present application will be further explained and illustrated in conjunction with specific embodiments.

[0073] For the experimental methods without specific conditions in the following examples, they are usually 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 examples are shown in Table 1:

[0075] Table 1

[0076]

[0077]

[0078] Example 1: Submergence stress treatment

[0079] The whole plant of one-year-old Myricaria laxiflora was immersed in water for submergence stress treatment. The newly emerged young leaf tissues were collected at 6 h, 12 h, 24 h, and 48 h of submergence respectively. Each stress treatment was repeated 3 times, and after quick-freezing with liquid nitrogen, they were stored in a -80 °C refrigerator.

[0080] Example 2: Transcriptome sequencing (RNA sequencing, RNA-seq)

[0081] RNA-seq data were obtained by transcriptome sequencing of the leaf tissues under different submergence durations (6 h, 12 h, 24 h, and 48 h) in Example 1. The FPKM means of the reference genes in the Unigene gene database were obtained based on the RNA-seq data. The reference genes with relatively stable expression were selected as candidate reference genes, namely Actin (ACT) gene, Tubulin beta (TUB) gene, and Ubiquitin-conjugating enzyme (UBC) gene, as shown in Table 2 for details.

[0082] Table 2

[0083]

[0084] Example 3: RNA extraction and cDNA synthesis

[0085] Take 50 - 100 mg of leaf tissues from 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 leaf tissues become powdery. Then, use the Fast Pure Universal Plant Total RNA Isolation Kit produced by Novoprotein to extract the total RNA of the leaf tissues. Subsequently, use the PrimeScript TM RT reagent Kit with gDNA Eraser produced by TaKaRa to reverse-transcribe the extracted total RNA to obtain cDNA products, and measure the OD 260 / 280 value of the cDNA products, and dilute the cDNA products at a 10-fold concentration to obtain the cDNA templates for subsequent real-time fluorescence quantitative PCR reactions.

[0086] Example 4: Primer synthesis

[0087] Design and synthesize the primer pairs for quantitative real-time PCR (qRT-PCR) detection of candidate reference genes using primer design software. Each primer is 25 bp in length, with a Tm value ranging from 55 °C to 65 °C, and the amplified fragment size is between 100 - 300 bp. After PCR using the detection primer pairs, use agarose gel electrophoresis to detect the specificity of the PCR products, and 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.

[0088] Example 5: Real-time fluorescence quantitative PCR

[0089] Using the cDNA template obtained in Example 3 and the primer pair obtained in Example 4, a real-time fluorescence quantitative PCR reaction was carried out 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. According to the amplification curve, the cycle threshold (Ct) of the candidate reference gene was obtained, and the following analysis was carried out based on the Ct value of the candidate reference gene:

[0090] (1) Use Microsoft Excel 2022 to statistically analyze the Ct values of the candidate reference genes under different waterlogging durations (6 h, 12 h, 24 h, and 48 h) to analyze the expression abundance of the candidate reference genes. 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. Specifically, see Table 3. At the same time, use SPSS 25.0 software to make a box plot of the Ct values, specifically see Figure 1 .

[0091] Table 3

[0092]

[0093]

[0094] As shown in Table 3 and Figure 1 shown, the average Ct values from high to low are TUB, ACT, and UBC, indicating that UBC has the highest average expression abundance, while TUB has the lowest average expression abundance; the range of Ct value changes from large to small is ACT, TUB, UBC, indicating that UBC has the smallest range of Ct value changes, while ACT has the largest range of Ct value changes.

[0095] (2) Use the BestKeeper software to compare 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) 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 proved, otherwise the expression stability is worse. The default threshold of 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.

[0096] Table 4

[0097] Gene Name Gene Annotation SD Value CV Value ACT Actin 0.60 2.56 TUB Tubulin 0.47 1.99 UBC Ubiquitin Ligase 0.39 1.71

[0098] As shown in Table 4, the SD values and CV values are ACT, TUB, and UBC from high to low, indicating that under the waterlogging stress treatment, the expression of the candidate reference gene UBC is the most stable.

[0099] (3) Use the geNorm software to analyze the expression stability of candidate reference genes according to the M value. The smaller the M value, the better the expression stability of the candidate reference gene; the larger the M value, the worse the expression stability of the candidate reference gene. The default cut-off value in this experiment is 1.5, that is, candidate reference genes with M values > 1.5 are not suitable as reference genes for Myricaria laxiflora under waterlogging stress treatment, while candidate reference genes with M values ≤ 1.5 have relatively stable expression. See Table 5 for details.

[0100] Table 5

[0101] Gene Name Gene Annotation M Value ACT Actin 0.336 TUB Tubulin 0.413 UBC Ubiquitin Ligase 0.336

[0102] As shown in Table 5, the candidate reference genes with the lowest M values are ACT and UBC, with an M value of 0.336, while the M value of TUB is significantly higher, reaching 0.413, indicating that the expression of the candidate reference genes ACT and UBC is relatively stable.

[0103] (4) Use the NormFinder software 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; the larger the M value, the worse the expression stability of the candidate reference gene. See Table 6 for details.

[0104] Table 6

[0105] Gene Name Gene Annotation M Value ACT Actin 0.383 TUB Tubulin 0.396 UBC Ubiquitin Ligase 0.168

[0106] As shown in Table 6, the M values are UBC, ACT, and TUB from high to low, indicating that under the waterlogging stress treatment, the expression of the candidate reference gene UBC is the most stable.

[0107] In addition, after the 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 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.

[0108] In summary, according to the analysis results of 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 Myricaria laxiflora leaves under waterlogging stress. Therefore, the present invention provides a reference gene for gene expression analysis in Myricaria laxiflora leaves under waterlogging stress. This reference gene is a ubiquitin-conjugating enzyme-encoding gene, and its nucleotide sequence is as shown in SEQ ID NO:1. It is stably expressed in Myricaria laxiflora leaves under waterlogging stress and is suitable for use as a reference gene for gene expression analysis in Myricaria laxiflora leaves under waterlogging 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 Myricaria laxiflora leaves under waterlogging stress, laying a good foundation for analyzing the waterlogging tolerance regulation mechanism of Myricaria laxiflora leaves and better utilizing the waterlogging tolerance characteristics of Myricaria laxiflora.

[0109] 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 on 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 leaves 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 leaves of Echinops sparsely flowered 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 described in claim 3 in gene expression analysis of leaves of Echinops sparsely flowered 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 leaves of Echinops sparsely flowered under waterlogging stress.

8. A method for analyzing gene expression in leaves of Echinops sparsely flowered under flooding stress, characterized in that: The steps include: The RNA of leaves of Echinops sparsely flowered was extracted under flooding stress, and the cDNA samples were obtained by reverse transcription. 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 leaves 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 leaves of Echinops sparsely flowered branches 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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