Hypsizigus marmoreus RT-qPCR reference gene and amplification primer and application thereof

Abstract

The invention provides an internal reference gene of hypsizigus marmoreus RT-qPCR and an amplification primer and application thereof, the internal reference gene is one or more of a pATPase gene, an RGTPase gene and a beta eLYase gene, the transcript sequence of the pATPase gene is shown as SEQ ID NO.1, the transcript sequence of the RGTPase gene is shown as SEQ ID NO.2, and the transcript sequence of the beta eLYase gene is shown as SEQ ID NO.3. The invention further provides an application of the internal reference gene of hypsizigus marmoreus RT-qPCR. The invention also provides a primer for amplifying the reference gene and application of the reference gene in gene expression analysis of hypsizigus marmoreus in different growth and development processes. The reference gene provides reliable reference for gene expression analysis of hypsizigus marmoreus in different growth and development stages, the difference between samples and in the samples caused by reference gene selection is reduced to the maximum extent, and a solid foundation is laid for researching key gene expression in the hypsizigus marmoreus development process.

Description

Technical Field

[0001] The present invention relates to the technical field of gene expression detection of leucopus edodes, and in particular to an internal reference gene of leucopus edodes RT-qPCR and an amplification primer and application thereof. Background Art

[0002] Real-time fluorescence quantitative PCR (RT-qPCR) has become an important tool for evaluating changes in gene expression due to its high accuracy, sensitivity, rapidity and economy, and is widely used in molecular biology research. However, the reliability of RT-qPCR results is affected by many factors, including the quality and quantity of the initial RNA, the efficiency of reverse transcription, the specificity of primers, and the efficiency of amplification. Therefore, the selection of appropriate reference genes is crucial for normalizing experimental data. Traditionally, genes such as β-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) and 18S rRNA are widely used as reference genes. An increasing number of studies have shown that the expression stability of these traditional reference genes is insufficient in different organisms, tissues or experimental conditions, and significant differential expression may occur. For example, the effectiveness of GAPDH as a reference gene depends on the organism, tissue, disease and many other factors. 18S rRNA is also considered unsuitable as a reference gene because of its high transcript abundance (not conducive to the detection of target genes with medium and low transcripts) and high variability. Therefore, it is particularly important to accurately screen and verify reference genes in different tissues, developmental stages and experimental conditions. The use of multiple reference genes can help significantly improve the accuracy of experimental results.

[0003] In the past, the identification of reference genes mainly relied on finding known homologous genes in other species and evaluating them based on the Ct values ​​obtained by RT-qPCR. At the same time, software (such as geNorm, NormFinder, etc.) was used to evaluate their stability. This method limited the discovery of new reference genes. In recent years, with the rapid development of high-throughput sequencing technology, the screening and evaluation of reference genes using transcriptome data has become a cutting-edge method. This method has the advantage of covering the entire genome expression information and can identify a variety of potential reference genes, which is more comprehensive than the traditional Ct value method. In addition, transcriptome data analysis can more accurately evaluate the true expression level of genes in samples, thereby reducing the risk of misselection and avoiding false results caused by poor primer design or sample quality problems. Therefore, the method of screening reference genes based on transcriptome data provides a broader and more accurate basis for gene expression analysis, thereby significantly improving the reliability of experimental results such as RT-qPCR.

[0004] As an important edible fungus, the growth and development of Pleurotus eryngii involves a variety of complex gene expression changes. Accurate gene expression analysis is essential for revealing the physiological mechanisms of Pleurotus eryngii, optimizing cultivation techniques, and genetic improvement. However, the current traditional reference genes have limited stability at different developmental stages of Pleurotus eryngii, which may lead to deviations in experimental results, thereby affecting the effectiveness and repeatability of the research. Therefore, it is urgent to establish a Pleurotus eryngii reference gene system with strong specificity and good stability to ensure the accuracy of gene expression analysis results. This will provide important support for the growth regulation, stress resistance research, and variety improvement of Pleurotus eryngii, and promote the in-depth development of related research. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides internal reference genes for RT-qPCR of Pleurotus eryngii and amplification primers and applications thereof, and the stability of these internal reference genes is better than the common internal reference genes used in existing Pleurotus eryngii.

[0006] In order to achieve the above object, the present invention adopts the following technical solution: The present invention provides an internal reference gene for RT-qPCR of Pleurotus eryngii, wherein the internal reference gene is pATPase Gene, RGTPase Genes and βeLYase One of the genes, pATPase The transcript sequence of the gene is shown in SEQ ID NO.1. RGTPase The transcript sequence of the gene is shown in SEQ ID NO.2. βeLYase The transcript sequence of the gene is shown in SEQ ID NO.3.

[0007] The present invention also provides the use of the above-mentioned internal reference gene in the gene expression analysis of Pleurotus eryngii in different growth and development processes, wherein the internal reference gene is pATPase Gene, RGTPase Gene or βeLYase Gene.

[0008] Above pATPase The nucleotide sequence of the forward primer of the gene is shown in SEQ ID NO.4, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.5.

[0009] Above RGTPase The nucleotide sequence of the forward primer of the gene is shown in SEQ ID NO.6, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.7.

[0010] Above βeLYase The nucleotide sequence of the forward primer of the gene is shown in SEQ ID NO.8, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.9.

[0011] The present invention also provides pATPase Gene, RGTPase Genes and βeLYase The application of genes as internal reference genes in gene expression analysis of enoki mushrooms in different growth and development processes, the pATPase The transcript sequence of the gene is shown in SEQ ID NO.1. RGTPase The transcript sequence of the gene is shown in SEQ ID NO.2. βeLYase The transcript sequence of the gene is shown in SEQ ID NO.3.

[0012] The present invention also provides a method for screening stable internal reference genes in the above-mentioned enoki mushroom, comprising the following steps: (1) obtaining candidate internal reference genes by analyzing and screening transcriptome data of enoki mushrooms in different growth and development processes; (2) Analyze the stability of candidate reference genes using transcriptome data of Pleurotus eryngii; (3) RT-qPCR analysis was used to verify the expression stability of candidate reference genes during the growth and development of Pleurotus eryngii.

[0013] Furthermore, the above step (1) includes: (i) performing differential analysis on the transcriptome data and retaining genes that have no differences between any two periods; (ii) further screening candidate internal reference genes based on the criteria of maximum fold difference (MFC) less than 3 and coefficient of variation less than 0.2; (iii) finally excluding hypothetical genes, genes with unknown functions and highly similar gene families, and finally retaining genes as candidate internal reference genes.

[0014] Furthermore, the above step (2) includes: (i) dividing the expression level of each candidate internal reference gene by the average expression level of the transcriptome data set to which it belongs to obtain the relative expression level of the candidate internal reference gene in the fruiting body; (ii) evaluating the stability of the candidate internal reference gene based on the maximum difference multiple of the candidate internal reference gene in the transcriptome data; (iii) comprehensively evaluating the stability of the candidate internal reference gene based on the variability of the candidate internal reference gene in the transcriptome data; (iv) comprehensively evaluating the stability of the candidate internal reference gene based on 2 and 3; and (v) performing gene function analysis on the candidate internal reference gene.

[0015] Furthermore, the above step (3) includes: (i) analyzing the cycle threshold of the candidate internal reference gene in the RT-qPCR experiment; and (ii) calculating the stability of the candidate internal reference gene by using geNorm and NormFinder software.

[0016] Beneficial effects of the present invention: The method of the present invention is based on the transcriptome data of Pleurotus eryngii at different developmental stages to screen out suitable internal reference genes pATPase Gene, RGTPase Genes and βeLYase These screened internal reference genes all meet the stability thresholds of geNorm and NormFinder analysis software at each growth and development stage of Pleurotus eryngii, showing good stability, providing reliable internal reference support for gene expression analysis of Pleurotus eryngii, and thus providing an important reference for studying the regulatory mechanism of Pleurotus eryngii growth and development stages. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 : Expression change rate of candidate reference genes and existing reference genes; The figure shows the expression change rate of 6 candidate reference genes and 10 existing reference genes in 19 RNA-seq libraries related to the growth and development of Pleurotus eryngii. The relative expression change rate of each gene is obtained by dividing the TPM value of each gene by the average expression level (average TPM value) of the gene in 19 transcriptome data sets.

[0018] Figure 2 : MFC of candidate reference genes and existing reference genes; The figure shows the maximum fold difference (MFC) of 3 candidate reference genes and 10 existing reference genes in 19 RNA-seq libraries related to the growth and development of Pleurotus eryngii. The maximum fold difference of each gene is the ratio of the maximum TPM value to the minimum TPM in the 19 transcriptome datasets of the gene.

[0019] Figure 3 : CV of candidate reference genes and existing reference genes; The figure shows the coefficient of variation (CV) of 3 candidate reference genes and 10 existing reference genes in 19 RNA-seq libraries related to the growth and development of Pleurotus eryngii. The coefficient of variation of each gene is the ratio of the deviation (TPM deviation) of the gene in 19 transcriptome datasets to the average expression level (average TPM value) of the gene in 19 transcriptome datasets.

[0020] Figure 4 :Analysis of expression abundance and variation of candidate reference genes in RT-qPCR analysis; The figure evaluates and analyzes the expression abundance of candidate reference genes in RT-qPCR analysis during the growth and development of Pleurotus eryngii, and the box plot shows the changes in the Ct values ​​of the screened reference genes. The horizontal line in the box plot represents the median of the Ct value in all samples, the upper and lower limits of the box represent the 25th and 75th percentile values ​​of the gene Ct value in the sample, respectively, and the upper and lower bar ends represent the minimum and maximum values ​​of the gene Ct value in the sample.

[0021] Figure 5 : RT-qPCR comprehensive analysis of the expression stability of internal reference genes in Pleurotus eryngii; The figure shows the results of calculating (A) gene stability M and (B) stability Stability using geNorm and NormFinde programs based on the Ct values ​​of candidate reference genes in all samples of Pleurotus eryngii growth and development.

[0022] Figure 6 :based on HmHyd9 Transcriptional changes validate the utility of candidate reference genes; The line charts are pATPase , RGTPase , βeLYase And after combining HmHyd9 Gene expression level. The bar graph shows the TPM value of RNA-seq data. Specific implementation methods The method and apparatus of the present invention are conventional techniques, and all reagents and equipment are purchased conventionally. In order to clearly show the purpose, technical scheme and advantages of the present invention, the following will be described in detail with reference to specific examples. The illustration of these embodiments does not limit the present invention, and any conversion or improvement based on the teachings of the present invention is within the scope of protection. The present invention can be implemented in a variety of ways, and relevant technical personnel can make appropriate improvements without departing from the connotation of the present invention.

[0024] The enoki mushrooms in the following examples were provided by the Fujian Edible Fungi Germplasm Resources Collection and Management Center.

[0025] Example 1 Screening of internal reference genes 1. Screening of reference genes for RT-qPCR during the growth and development of Pleurotus eryngii The specific steps are as follows: S1: The candidate reference genes provided by the present invention are obtained based on transcriptome analysis related to the growth and development of Pleurotus eryngii. A total of 19 transcriptome data from 7 Pleurotus eryngii data sets were differentially analyzed in pairs, and genes that had no difference between any two periods were retained as candidate genes obtained through preliminary screening. Genes with significant differences in different developmental stages indicate that the expression level of the gene is unstable.

[0026] S2: The candidate genes initially screened were filtered, and genes expressed in at least 15 transcriptome data were retained as candidate genes, because the number of data labels indicated that the gene could not be stably expressed and was not suitable as an internal reference gene.

[0027] S3: Candidate reference genes were further screened based on the criteria that the maximum fold difference (MFC; the ratio of the maximum TPM value of a gene to the minimum TPM) was less than 3 and the coefficient of variation (the ratio of the TPM standard deviation of the gene to the TPM mean) was less than 0.2.

[0028] S4: Finally, hypothetical genes, genes with unknown functions, and highly similar gene families were excluded, and the six genes with the smallest coefficient of variation were retained as candidate internal reference genes, which are: pATPase Gene, RGTPase Gene, βeLYase Gene, Clp1 Gene, oeLh Genes and HelC Gene. Its nucleotide sequence is shown in SEQ ID NO.1-3 and SEQ ID NO.10-12.

[0029] By analyzing all transcriptome data, the results are shown in Table 1. The newly screened 6 candidate internal reference genes can meet the above conditions and meet the standards for being internal reference genes.

[0030] Table 1 Details of the expression profiles of candidate reference genes

[0031] 2. Stability analysis of candidate reference genes In order to compare the expression stability of these candidate genes in transcriptome data (RNA-seq), the present invention compares the candidate reference genes with existing reference genes, which include traditional reference genes (traditional reference genes: GAPDH, 18SrRNA, HH3 ) and other reference genes identified in filamentous fungi ( SPRYp, Ras, RNB, VAMP, PP2A and RPL4 ).

[0032] (1) Calculate the expression change rate of the six candidate reference genes and the existing reference gene transcriptome dataset (dividing the expression level of each sample by the average expression level of the transcriptome dataset to which it belongs to obtain the relative expression level of the gene during pulp development). Figure 1 As shown, it shows that in 19 transcriptome databases, the expression of existing reference genes is very unstable, with large fold differences; in contrast, the candidate reference genes show a higher stable expression level ( Figure 1 ).

[0033] (2) Based on the maximum multiple difference of the candidate internal reference gene in the transcriptome data, that is, the size of the MFC value, the candidate internal reference gene and the existing internal reference gene were ranked by expression stability in the transcriptome of Pleurotus eryngii. The smaller the MFC value, the more stable the gene expression and the higher the ranking, indicating that the gene expression difference is smaller. Figure 2 The results show that the top four genes with the smallest expression differences in the growth and development of Pleurotus eryngii are candidate internal reference genes, which are: RGTPase , pATPase, βeLYase and oeLh , their MFCs are all lower than those of the existing reference genes, which indicates that the expression differences and stability of the candidate reference genes are better than those of the existing reference genes.

[0034] (3) Based on the coefficient of variation of the candidate internal reference gene in the transcriptome data, i.e., the size of the CV value, the candidate internal reference gene and the existing internal reference gene were ranked by expression stability in the transcriptome of Pleurotus eryngii. The smaller the CV value, the more stable the gene expression, the higher the ranking, and the more stable the gene. The results are as follows Figure 3 The results show that the top four genes with the smallest coefficient of variation in the growth and development of Pleurotus eryngii are all candidate internal reference genes, which are: pATPase, RGTPase, βeLYase and oeLh , and their CVs were significantly lower than those of the existing internal reference genes, which indicated that the expression stability of these four candidate internal reference genes was better than that of the existing internal reference genes.

[0035] (4) Comprehensively evaluate the stability of candidate reference genes and existing reference genes based on MFC and CV: By sorting by MFC and CV, we found that the stability of candidate reference genes pATPase, RGTPase and βeLYase The ranking of the candidate genes is always in the top three, indicating that the three genes have the best expression stability during the growth and development of Pleurotus eryngii and are the most ideal internal reference genes. oeLh It ranks fourth in both MFC and CV rankings and can be used as an alternative reference gene.

[0036] (5) Gene function analysis of new reference genes The three new reference genes finally obtained ( pATPase, RGTPase and βeLYase ) performed protein structure and function analysis.

[0037] in, pATPase It is the coding gene of P-type ATPase. P-type ATPase couples conformational changes in the process of ion transmembrane transport by forming and decomposing covalent phosphate intermediates, thereby actively transporting ions at the expense of ATP. It is an important enzyme to maintain active cell transport and is essential for all eukaryotes and most prokaryotes. RabGTPase activating protein is a class of proteins containing TBC domains, which can integrate signals between Rab and other small G proteins to ensure the correct sorting, transport and anchoring of vesicles to different intracellular membrane vesicle systems. Beta-eliminatinglyase is a class of enzymes that cut by β-trans elimination. It is mainly involved in the biosynthesis of amino acids and amino acid-derived metabolites. It also exists in the biosynthetic pathway of amino sugars and the synthesis or catabolism of neurotransmitters.

[0038] The newly screened reference genes are similar to existing reference genes in that they all participate in basic biological processes of cells and share highly conserved functions among different species, meeting one of the basic conditions for being ideal reference genes.

[0039] 3. Verify the expression stability of candidate reference genes during the growth and development of Pleurotus eryngii using RT-qPCR (1) The entire growth and development process of Pleurotus eryngii was divided into five stages: mycelium, primordium, young mushroom, elongation, and maturity. RNA was extracted from each stage, referring to the Omega RNA extraction kit (R6827) for details. Then, the RNA was reverse transcribed into cDNA for real-time fluorescence quantitative PCR according to the instructions of TransGen's gold reverse transcription kit (AT341). After extracting total RNA, 1 μL of RNA sample was taken, and the OD260 / 280 and concentration were detected using NanoDrop2000. If the OD260 / 280 value of the sample was between 1.8 and 2.2, it was a qualified sample. Finally, the real-time fluorescence RT-qPCR was performed according to the instructions of TransGen's fluorescence quantitative PCR kit to detect the Ct value. The specific reaction system and reaction procedure are shown in Table 2 below, and the corresponding primer sequences of the candidate internal reference genes are shown in Table 3.

[0040] Table 2 Real-time fluorescence quantitative PCR system and procedures

[0041] Table 3 Primer sequences of candidate internal reference genes

[0042] (2) In order to evaluate the expression abundance of the newly selected internal reference genes, their cycle threshold (Ct value) in the RT-qPCR experiment was analyzed. The lower the Ct value, the higher the gene expression. Figure 4 As shown in the figure, the Ct values ​​of the existing reference genes ranged from 18.89 to 27.65, among which the gene Ras had the largest Ct variation range (difference of 4.39); the smallest was ACTB (difference of 2.12); and the three newly screened reference genes ( pATPase , RGTPase and βeLYase ) were 1.21, 1.87, and 1.52, respectively, indicating that the Ct value variation range of the three newly screened reference genes was small. These results indicate that during the growth and development of Pleurotus eryngii, the expression of the three newly screened reference genes was more stable and had an appropriate expression abundance.

[0043] (3) The RT-qPCR results were analyzed by using the software geNorm and NormFinder to analyze the stability of the candidate internal reference genes of the Pleurotus eryngii. The lower the stability (M) and stability (Stability) values, the more stable the corresponding gene expression. Figure 5 Display, where pATPase , RGTPase and βeLYaseThe stability (M) and stability (Stability) of are the lowest, both in the top three, indicating that the candidate gene is the most stable among the genes evaluated.

[0044] In summary, the present invention firstly screened the genome of Pleurotus eryngii combined with the transcriptome to obtain three genes stably expressed during the growth and development of Pleurotus eryngii, namely pATPase , RGTPase and βeLYase ; Their expression stability was superior to traditional reference genes and reference genes known to be stably expressed in filamentous fungi in transcriptome and RT-qPCR comparative analysis. Further comparison using geNorm and NormFinde programs verified that these three stably expressed genes can be used as reference genes stably expressed during the growth and development of Pleurotus eryngii. The results of this study provide a more useful and reliable reference gene resource for gene expression research on the growth and development of Pleurotus eryngii, which can be used for gene function analysis during the growth and development of Pleurotus eryngii.

[0045] Example 3 Using the growth and development of enoki mushrooms HmHyd9 The expression patterns of the three new reference genes analyzed above verify the practicality of hydrophobic protein, which is an important protein in the primordium formation process of Echinops edulis. hyd9 It is significantly highly expressed during the primordium development period, and transcriptome data also show that compared with the hyphae stage (TPM value is 92), the expression of this gene is significantly increased in the primordium stage (TPM value is 379), and then decreased. HmHyd9 The expression patterns during the growth and development of Pleurotus eryngii verified the reliability and practicality of the novel internal reference genes screened.

[0046] The steps include: Firstly, three new reference genes were used in RT-qPCR analysis. HmHyd9 The relative expression levels of the genes were calculated and then three new reference genes were used in RT-qPCR analysis. HmHyd9 The relative expression levels of the newly selected internal reference genes were calculated to verify the applicability and stability of the newly selected internal reference genes used alone and in combination. Figure 6 Display, three internal reference genes were calculated separately HmHyd9 The expression trend of HmHyd9 The expression trends were similar, and the three newly screened reference genes were combined for calculation. HmHyd9 Similar results were obtained for gene expression levels, indicating that these three genes, whether used alone or in combination, are suitable as internal references for studying gene expression during the growth and development of Pleurotus eryngii.

[0047] The above is only a specific implementation of the present application. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present application. These improvements and modifications should also be regarded as the scope of protection of the present application.

Claims

1. The internal reference gene of RT-qPCR of Pleurotus eryngii is characterized by: The internal reference gene is pATPase Gene, RGTPase Genes and βeLYase One of the genes, pATPase The transcript sequence of the gene is shown in SEQ ID NO.

1. RGTPase The transcript sequence of the gene is shown in SEQ ID NO.

2. βeLYase The transcript sequence of the gene is shown in SEQ ID NO.

3.

2. The use of the internal reference gene as claimed in claim 1 in the gene expression analysis of Pleurotus eryngii in different growth and development processes, characterized in that: The internal reference gene is pATPase Gene, RGTPase Gene or βeLYase Gene.

3. The use according to claim 2, characterized in that: Said pATPase The nucleotide sequence of the forward primer of the gene is shown in SEQ ID NO.4, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.

5.

4. The use according to claim 2, characterized in that: Said RGTPase The nucleotide sequence of the forward primer of the gene is shown in SEQ ID NO.6, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.

7.

5. The use according to claim 2, characterized in that: Said βeLYase The nucleotide sequence of the forward primer of the gene is shown in SEQ ID NO.8, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.

9.

6. pATPase Gene, RGTPase Genes and βeLYase The application of the genes as internal reference genes in the gene expression analysis of enoki mushrooms in different growth and development processes is characterized by: Said pATPase The transcript sequence of the gene is shown in SEQ ID NO.

1. RGTPase The transcript sequence of the gene is shown in SEQ ID NO.

2. βeLYase The transcript sequence of the gene is shown in SEQ ID NO.3.

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