Fluorescent quantitative PCR reference gene screening method in different tissues and stress treatment of perilla frutescens

Abstract

The invention relates to the technical field of molecular biology, in particular to a fluorescent quantitative PCR reference gene screening method in different tissues and stress treatment of perilla frutescens, which specifically comprises the following steps: step 1, respectively selecting perilla frutescens leaves, stalks, roots, fruits at different development stages after flowering and perilla frutescens leaves treated by 20% PEG6000 for 0H, 3H, 6H, 9H, 12H and 24H as samples; extracting total RNA (Ribonucleic Acid) of each sample, and carrying out reverse transcription on the RNA to synthesize cDNA (Complementary Deoxyribose Nucleic Acid) as And step 2, screening out candidate reference genes of the purple perilla by using the sequencing data of the early-stage purple perilla transcriptome, namely PfActin, Pf18S, PfEF-1alpha, PfCYP, PfTUA, PfTUB and PfGAPDH respectively, and screening out the candidate reference genes of the purple perilla, namely PfActin, Pf18S, PfEF-1alpha, PfCYP, PfTUA, PfTUB and PfGAPDH. According to the method, the most stable reference genes in perilla leaves, stems, roots, fruits in three development stages and perilla leaves treated by 20% PEG6000 for 0H, 3H, 6H, 9H, 12H and 24H are screened from seven candidate reference genes, and a foundation is laid for deep excavation of functional genes in the later period.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, in particular to a method for screening reference genes for fluorescence quantitative PCR in different tissues of Perilla frutescens and under stress treatment. Background Art

[0002] Perilla frutescens, anciently named Ren, also known as Perilla seed, Perilla frutescens var. acuta and Perilla frutescens var. crispa, etc., belongs to the genus Perilla of the family Labiatae, and is an annual herb with a specific aroma. The seeds, leaves and stems of Perilla frutescens can all be used as medicine or food, and have high edible and medicinal values. At present, 226 components have been isolated from the leaves of Perilla frutescens, which are mainly classified into 7 categories: volatile oils, flavonoids, anthocyanins, phenolic acids, triterpenoids, amino acids and carbohydrates. Modern research shows that the extracts of Perilla frutescens leaves also have effects such as antioxidant, anti-inflammatory, anti-allergic and anti-depressant, and have extremely high nutritional value and economic value. It is also a commonly used antioxidant in food and has important industrial value.

[0003] In recent years, analyzing the metabolic pathways and regulatory mechanisms of functional components of Perilla frutescens from the perspective of molecular biology has become a research hotspot in the field of Perilla frutescens. However, relevant research on the screening of reference genes for Perilla frutescens has not been reported yet. The screening of stably expressed reference genes for real-time fluorescence quantitative PCR of Perilla frutescens plays a fundamental role in further studying the molecular metabolic mechanism of functional components of Perilla frutescens and subsequent biosynthesis. Summary of the Invention

[0004] Aiming at the technical problem of the lack of screening of reference genes for Perilla frutescens in the prior art, the present invention provides a method for screening reference genes for fluorescence quantitative PCR in different tissues of Perilla frutescens and under stress treatment.

[0005] The technical solution adopted by the present invention is: a method for screening reference genes for fluorescence quantitative PCR in different tissues of Perilla frutescens and under stress treatment, specifically including the following steps: Step 1: Respectively select the leaves, stems, roots, fruits at different development stages after flowering of Perilla frutescens and the leaves of Perilla frutescens treated with 20% PEG6000 for 0H, 3H, 6H, 9H, 12H, 24H as samples; extract the total RNA of each sample, and reverse transcribe the RNA into cDNA as the template for amplifying the target gene; Step 2: Utilize the previous transcriptome sequencing data of Perilla frutescens to screen out the candidate reference genes of Perilla frutescens, which are respectively: PfActin , Pf18S , PfEF-1α , PfCYP, PfTUA, PfTUB, PfGAPDH ; Step 3: Design primers for reference genes for real-time fluorescence quantitative PCR with the selected candidate reference gene sequences as templates, and the amplified fragment size of the primers is 150 - 200bp; Step 4. Real-time fluorescence quantitative PCR: Using the cDNA obtained by reverse transcription in Step 1 as a template, perform real-time fluorescence quantitative PCR amplification on the candidate reference genes using the primer pairs in Step 3; perform statistical analysis on the obtained real-time fluorescence quantitative PCR data, and screen out the reference genes and reference gene combinations respectively.

[0006] In one embodiment, in Step 2: The PfActin has a nucleotide sequence as shown in Sequence Listing SEQ ID NO.1; The Pf18S has a nucleotide sequence as shown in Sequence Listing SEQ ID NO.2; The PfEF-1α has a nucleotide sequence as shown in Sequence Listing SEQ ID NO.3; The PfCYP has a nucleotide sequence as shown in Sequence Listing SEQ ID NO.4; The PfTUA has a nucleotide sequence as shown in Sequence Listing SEQ ID NO.5; The PfTUB has a nucleotide sequence as shown in Sequence Listing SEQ ID NO.6; The PfGAPDH has a nucleotide sequence as shown in Sequence Listing SEQ ID NO.7.

[0007] In one embodiment, in Step 3: The primer pair used for amplifying PfActin is SEQ ID NO.8 / SEQ ID NO.9; The primer pair used for amplifying Pf18S is SEQ ID NO.10 / SEQ ID NO.11; The primer pair used for amplifying PfEF-1α is SEQ ID NO.12 / SEQ ID NO.13; The primer pair used for amplifying PfCYP is SEQ ID NO.14 / SEQ ID NO.15; The primer pair used for amplifying PfTUA is SEQ ID NO.16 / SEQ ID NO.17; The primer pair used for amplifying PfTUB is SEQ ID NO.18 / SEQ ID NO.19; The primer pair used for amplifying PfGAPDH is SEQ ID NO.20 / SEQ ID NO.21.

[0008] In one embodiment, before performing the real-time fluorescence quantitative PCR in step four, the specificity of the reference gene primers is first identified by ordinary PCR.

[0009] In one embodiment, in step four, the obtained real-time fluorescence quantitative PCR data is statistically analyzed by three software programs, namely GeNorm, NormFinder, and BestKeeper, to separately screen out the reference genes and reference gene combinations, and then a comprehensive ranking analysis is performed using the RefFinder analysis software.

[0010] The beneficial effects of the present invention are as follows: Compared with the prior art, in the present invention, (1) The present invention screens out the most stable reference genes in perilla leaves, stems, roots, fruits at three developmental stages, and perilla leaves after 20% PEG6000 treatment for 0H, 3H, 6H, 9H, 12H, and 24H from 7 candidate reference genes, laying a foundation for the subsequent in-depth exploration of functional genes and solving the problem of the lack of perilla reference gene screening technology in the prior art; (2) The screened reference genes are applicable to the expression analysis of key genes in perilla, can significantly improve the accuracy of the obtained data, and have wide application, high sensitivity, and good stability; (3) The present invention has a strict screening procedure for reference genes, determines the stable reference genes applicable to different tissues and stress treatments in perilla, and provides a theoretical basis for selecting appropriate reference genes in perilla qRT-PCR analysis. Description of the Drawings

[0011] Figure 1 is the sequence electrophoresis map amplified from perilla cDNA using the primers of 7 reference genes in Example 2; wherein, M: DNA Marker, and the band sizes are in the order from bottom to top as 250bp, 500bp, 750bp, 1000bp, etc., and the genes represented by each lane from left to right are PfActin, Pf18S, PfTUA, PfTUB, PfCYP, PfGAPDH, PfEF-1α ; Figure 2 is the Ct value distribution map of the fluorescence quantitative PCR of 6 reference genes in Example 2; Figure 3 is the melting curve diagram of 6 reference genes in Example 2; Figure 4 is the expression stability value ( M ) and paired difference value ( V ) of 6 candidate reference genes represented by applying the GeNorm program in Example 2, M the smaller the Figure 4 value, the better the stability of the reference gene; wherein Figure 4 (A) Using the data of all samples mixed as the material; Figure 4 (B) Using fruits at three developmental stages as the material;Figure 4 (C) Using leaves, stems and roots as materials; Figure 4 (D) Using leaves under drought treatment as materials; Figure 5 It is the comprehensive ranking obtained by applying the RefFinder program to three statistical analysis methods in Example 2; Figure 5 A Using the data of all samples mixed as materials; Figure 5 B Using fruits at 3 developmental stages as materials; Figure 5 C Using leaves, stems and roots as materials; Figure 5 D Using leaves under drought treatment as materials. Specific implementation manners

[0012] In the description of the present invention, it should be noted that the orientation or positional relationship indicated by the terms "front", "upper", "lower", "left", "right", "vertical", "horizontal", etc. is based on the orientation or positional relationship shown in the drawings. It is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation to the present invention.

[0013] In order to solve the problems existing in the background technology, the present application proposes the following technical solutions: A method for screening reference genes for fluorescence quantitative PCR in different tissues and stress treatments of Perilla frutescens, specifically including the following steps: Step 1: Respectively select leaves, stems, roots of Perilla frutescens, fruits at different developmental stages after flowering, and leaves of Perilla frutescens treated with 20% PEG6000 for 0H, 3H, 6H, 9H, 12H, 24H as samples; extract the total RNA of each sample, reverse transcribe the RNA into cDNA, and use it as the template for amplifying the target gene; Step 2: Utilize the previous Perilla frutescens transcriptome sequencing data to screen out the candidate reference genes of Perilla frutescens, which are respectively: PfActin, Pf18S, PfEF-1α, PfCYP, PfTUA, PfTUB, PfGAPDH In Step 2: The PfActin has a nucleotide sequence as shown in Sequence Listing SEQ ID NO.1; The Pf18S has a nucleotide sequence as shown in Sequence Listing SEQ ID NO.2; The PfEF-1α has a nucleotide sequence as shown in Sequence Listing SEQ ID NO.3; The PfCYP has a nucleotide sequence as shown in Sequence Listing SEQ ID NO.4; The PfTUA has a nucleotide sequence as shown in Sequence Listing SEQ ID NO.5; The PfTUB has a nucleotide sequence as shown in Sequence Listing SEQ ID NO.6; The PfGAPDH nucleotide sequence is shown in SEQ ID NO.7 of the sequence listing.

[0014] Step 3: Using the selected candidate reference gene sequences as templates, design primers for reference genes for real-time fluorescence quantitative PCR, and the amplified fragment size of the primers is 150-200bp; In step 3: The primer pair sequence for amplifying PfActin is SEQ ID NO.8 / SEQ ID NO.9; The primer pair sequence for amplifying Pf18S is SEQ ID NO.10 / SEQ ID NO.11; The primer pair sequence for amplifying PfEF-1α is SEQ ID NO.12 / SEQ ID NO.13; The primer pair sequence for amplifying PfCYP is SEQ ID NO.14 / SEQ ID NO.15; The primer pair sequence for amplifying PfTUA is SEQ ID NO.16 / SEQ ID NO.17; The primer pair sequence for amplifying PfTUB is SEQ ID NO.18 / SEQ ID NO.19; The primer pair sequence for amplifying PfGAPDH is SEQ ID NO.20 / SEQ ID NO.21.

[0015] Step 4: Real-time fluorescence quantitative PCR: Using the cDNA obtained by reverse transcription in step 1 as a template, perform real-time fluorescence quantitative PCR amplification on the candidate reference genes using the primer pairs in step 3; Perform statistical analysis on the obtained real-time fluorescence quantitative PCR data, and respectively screen out the optimal reference genes and reference gene combinations.

[0016] Before performing the real-time fluorescence quantitative PCR in step 4, first identify the specificity of the reference gene primers by ordinary PCR.

[0017] In step 4, perform statistical analysis on the obtained real-time fluorescence quantitative PCR data through three software, namely GeNorm, NormFinder, and BestKeeper, respectively screen out the optimal reference genes and reference gene combinations, and then perform comprehensive ranking analysis using the RefFinder analysis software.

[0018] The explanation of the above technical solution is as follows: The specific screening method used includes the following steps: (1) Select the perilla leaves, stems, roots, fruits at three different developmental stages, and perilla leaves after treatment with 20% PEG6000 for 0H, 3H, 6H, 9H, 12H, and 24H as materials; (2) Use the perilla transcriptome sequencing data to screen out the reference genes of perilla; (3) Using the selected reference gene sequences as templates, design primers for reference genes for real-time fluorescence quantitative PCR; (4) Perform conventional PCR amplification on each cDNA with the primers designed in step (3) above to obtain the amplification efficiency of the primers; (5) Use a 5-fold dilution series (1x, 5x, 25x, 125x, 625x, 3125x) of the mixed cDNA of each perilla sample as a template for real-time fluorescence quantitative analysis; (6) Perform real-time fluorescence quantitative PCR; use three statistical software, GeNorm, NormFinder, and BestKeeper, to statistically analyze the obtained real-time fluorescence quantitative PCR data, respectively screen out the optimal reference genes and reference gene combinations, and then perform comprehensive ranking analysis using the comprehensive analysis software RefFinder.

[0019] Among them, before performing the real-time fluorescence quantitative PCR in step (4), first identify the specificity of the reference gene primers by ordinary PCR (using the DNA polymerase provided by TAKARA: rTaqMix; identification conditions: 95°C for 5 min, 95°C for 30 s, 55°C for 30 s, 72°C for 60 s / kb, 30 cycles; 72°C for 10 min).

[0020] For further design, in step (4), the real-time fluorescence quantitative PCR amplification program is: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 5 s, annealing at 55°C for 30 s, running 40 cycles, and melting curve analysis at 65°C - 95°C.

[0021] For further design, the method for screening reference genes for fluorescence quantitative PCR in different tissues and stress treatments of perilla is as follows: 1) Material collection: Use the perilla leaves, stems, roots, fruits at three developmental stages, and perilla leaves after treatment with 20% PEG6000 for 0H, 3H, 6H, 9H, 12H, and 24H collected from the Donghu Campus of Zhejiang A&F University as experimental materials. After collection, quickly freeze them in liquid nitrogen and store them in a -80°C refrigerator; the fruits at three developmental stages are numbered S1, S2, and S3; 2) RNA extraction and detection: The total RNA of each sample was extracted using the EASYspinPlus Polysaccharide Polyphenol Complex Plant RNA Extraction Kit (Adlai, China) (the extraction method was consistent with the instructions of the kit), and the integrity of the extracted RNA was detected by electrophoresis, 1% agarose gel, TAE buffer, 100V voltage for 20 min, and observed and analyzed using a gel imaging system (Tanon-2500); the purity of the extracted RNA was detected using NanoDropND-2000 (the OD260 / 280 of pure RNA was between 1.8 and 2.2); 3) RNA reverse transcription to cDNA synthesis: After non-denaturing agarose gel electrophoresis, 1 μg of perilla RNA sample was used to synthesize cDNA using SuperScript ® ⅢFirst-Strand Synthesis System Kit (Thermo, USA) was used to reverse transcribe and synthesize cDNA, which was used as a template for target gene amplification; 4) Screening of candidate internal reference genes: Using the transcriptome sequencing data of Perilla frutescens, the homologous genes of the model plant Arabidopsis thaliana were referenced to screen out candidate internal reference genes; 5) Design and detection of specific primers: Using the selected candidate internal reference gene sequence as a template, use VectorNTI software to design the internal reference gene primers for real-time fluorescence quantitative PCR, and the amplified fragment size is between 150-200bp (see Table 1 for details); using the cDNA obtained by reverse transcription of camphor as a template, use ordinary PCR to preliminarily identify the specificity of the primers. After electrophoresis, observe the bands of the PCR product under the gel imaging system, such as Figure 1 The bands shown are of the correct size, single bands, and primers without primer dimers; 6) Establishment of the standard curve of the internal reference gene primer: A 5-fold dilution series (1x, 5x, 25x, 125x, 625x, 3125x) of mixed cDNA of different samples was used as a template for establishing the standard curve, and three technical replicates were designed for each reaction of real-time fluorescence quantitative PCR; 7) Fluorescence quantitative PCR amplification: Using the cDNA obtained by reverse transcription of 12 samples as a template, real-time fluorescence quantitative PCR amplification of the internal reference gene was performed to obtain the corresponding Ct The amplification program is: 95℃3min, 95℃5s, 55℃30s, collecting fluorescence signals for 40 cycles, 95℃5s, melting curve 65℃-95℃. The melting curve signal is collected, and the generated melting curve is a single peak, indicating that the primer specificity is good. The Ct value data is automatically read by the real-time fluorescence quantitative PCR instrument. The distribution range of the Ct value is as follows Figure 2As shown. The Ct value is inversely proportional to the gene expression level. The larger the Ct value, the lower the gene expression level. Conversely, the smaller the Ct value, the higher the gene expression level. Using the obtained fluorescence quantitative PCR results, a standard curve was drawn as shown in Table 1, and the amplification efficiency and slope of each candidate gene were obtained. 8) Evaluate its expression stability using three statistical analysis methods: GeNorm, NormFinder, and BestKeeper. 9) Use the RefFinder program to comprehensively rank the stability of the candidate reference genes obtained by the three statistical analysis methods to obtain the comprehensive results. The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto. Unless otherwise specified, the equipment and reagents used in the examples are all commercially available.

[0022] Example 1: Design of candidate reference genes and their primers: Based on the previous transcriptome data, the present invention analyzed and identified a total of 7 candidate reference genes (Table 1), namely: PfActin, Pf18S, PfEF-1α, PfCYP, PfTUA, PfTUB, PfGAPDH; Among them, the PfActin has a nucleotide sequence as shown in SEQ ID NO.1 in the sequence listing; the Pf18S has a nucleotide sequence as shown in SEQ ID NO.2 in the sequence listing; the PfEF-1α has a nucleotide sequence as shown in SEQ ID NO.3 in the sequence listing; the PfCYP has a nucleotide sequence as shown in SEQ ID NO.4 in the sequence listing; the PfTUA has a nucleotide sequence as shown in SEQ ID NO.5 in the sequence listing; the PfTUB has a nucleotide sequence as shown in SEQ ID NO.6 in the sequence listing; the PfGAPDH has a nucleotide sequence as shown in SEQ ID NO.7 in the sequence listing.

[0023] Use VectorNTI software to design qRT-PCR primers, and the amplified fragment size of the primers is 150-200bp. The primer pair sequence for amplifying PfActin is SEQ ID NO.8 / SEQ ID NO.9; the primer pair sequence for amplifying Pf18S is SEQ ID NO.10 / SEQ ID NO.11; the primer pair sequence for amplifying PfEF-1α is SEQ ID NO.12 / SEQ ID NO.13; the primer pair sequence for amplifying PfCYP is SEQ ID NO.14 / SEQ ID NO.15; the primer pair sequence for amplifying PfTUA is SEQ ID NO.16 / SEQ ID NO.17; the primer pair sequence for amplifying PfTUBThe primer pair sequence is SEQ ID NO.18 / SEQ ID NO.19; used for amplification PfGAPDH The primer pair sequence is SEQ ID NO. 20 / SEQ ID NO. 21. The internal reference gene primers used are shown in Table 1.

[0024] Table 1 qRT-PCR reference genes and their primer sequences.

[0025] Example 2: Stability analysis of candidate reference genes.

[0026] The specific steps include: 1) Total RNA extraction and cDNA synthesis The collected samples (perilla leaves, stems, roots, fruits at three different developmental stages, and leaves treated with drought) were ground into powder with liquid nitrogen. The total RNA of each sample was extracted using the EASYspinPlus Polysaccharide Polyphenol Complex Plant RNA Extraction Kit (Adlai, China). The specific method is described in the instruction manual. The integrity of the extracted RNA was detected by electrophoresis, 1% agarose gel, TAE buffer, electrophoresis at 100V for 20min, and observed and analyzed using a gel imaging system (Tanon-2500); the purity and concentration of the extracted RNA were detected using NanoDropND-2000 (the OD260 / 280 of pure RNA was between 1.8 and 2.2). After non-denaturing agarose gel electrophoresis, 1 μg of perilla RNA sample was taken and analyzed using SuperScript ® ⅢFirst-Strand Synthesis System Kit (Thermo, USA) was used to reverse transcribe and synthesize cDNA as a template for target gene amplification. The specific method is referred to the instruction manual, and the synthesized cDNA was stored in a -20℃ refrigerator. The specific steps of mixing are as follows: First, prepare a total of 20μL of reverse transcription reaction solution in a microtube, including 1μL of ligodTPrimer, 1μL of ntpMixture, and a total amount of RNA below 5μg, supplemented with RNasefreedH 2 0 to 10µL, incubate the microcentrifuge tube containing the mixed solution at 65℃ for 5min, and then quickly cool it on ice to promote RNA denaturation and improve reverse transcription efficiency; prepare the reaction solution in the same microcentrifuge tube, including 10µL RNA and denatured primers, 4µL 5× PrimeScriptⅡBuffer, 0.5µL RNaseInhibitor, 1µL PrimeScriptⅡRTase, and supplement with RNasefree dHO 20 to 20 μL. The reaction conditions for this reaction solution were incubation at 50 °C for 60 min to extend cDNA, incubation at 95 °C for 5 min followed by cooling on ice, and storage at -20 °C in a refrigerator for later use.

[0027] 2) Detection of candidate reference gene specific primers Using the cDNA reverse transcribed from perilla as a template, the primer specificity was initially identified by ordinary PCR. After electrophoresis, the bands of the PCR products were observed under a gel imaging system. If Figure 1 the sizes of the bands were correct as shown, the bands were single, and there were no primer dimers.

[0028] 3) Real-time fluorescence quantitative PCR (qRT-PCR) The total volume of the qRT-PCR reaction system was 25 μL, including 12.5 μL of TB Green Premix Ex Taq (Takara Bio, Beijing), 2 μL of cDNA, 0.5 μL of forward and reverse primers (the 5 candidate reference genes and their primers are shown in Table 1), and 9.5 μL of ddH2O. Each sample had three technical replicates. The qRT-PCR reaction was carried out on a CFX96 real-time PCR instrument (Bio-Rad, USA). Pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 5 s, annealing at 55 °C for 30 s, running for 40 cycles, and melting curve analysis at 65 °C - 95 °C. The Ct value was automatically obtained by the instrument after the reaction, a standard curve was established, and the amplification efficiency of the primers was calculated. Since PfGAPDH the maximum Ct value was close to 40 and the gene expression was extremely unstable, the PfGAPDH gene was removed from the subsequent experiments.

[0029] 4) Analysis of candidate reference gene stability The expression stability of the reference genes was obtained based on a software program in Microsoft Excel 2013. According to the formula Q = 2 -ΔCt , the relative expression level Q of each sample was calculated, where Δ Ct = Ct 样品－ Ct min , Ct min represents the minimum value among all Ct samples. The GeNorm program was used to obtain the average coefficient of variation M value of each reference gene based on the relative expression level Q value, and the genes were ranked according to the size of the M value. The smaller the M value, the higher the gene stability. At the same time, a pairwise variation value V n / n+1To determine the optimal number of reference genes required under the experimental conditions and reduce the bias and fluctuations caused by using a single reference gene. n represents the number of reference genes to be selected, and the paired difference value V n / n+1 <0.15 is the recommended selection threshold (such as Figure 4 ). Different from the calculation method of GeNorm, NormFinder directly evaluates the expression stability of reference genes based on analysis of variance, can determine the degree of variation within and between groups, and calculates the gene expression stability value ( S value), S The smaller the value, the more stable the gene (as shown in Table 2). The BestKeeper program evaluates the stability index of reference genes based on the expression calculated from the standard deviation (SD) and percentage coefficient of variance (CV) values (as shown in Table 3).

[0030] To provide a comprehensive result, the RefFinder software was used to rank the best list of reference genes, Pf18s The gene has the highest comprehensive stability ranking and the best stability (such as Figure 5 ).

[0031] Table 2 NormFinder scores of reference genes in different samples.

[0032] Table 3 Bestkeeper scores of reference genes in different samples.

[0033] In summary, the present invention screens out the most stable reference genes in perilla leaves, stems, roots, fruits at three developmental stages, and perilla leaves after 20% PEG6000 treatment for 0H, 3H, 6H, 9H, 12H, and 24H from 7 candidate reference genes. The data are true and reliable, laying a foundation for further exploration of functional genes in the later stage; the screened reference genes are suitable for the expression analysis of key genes in perilla, can significantly improve the accuracy of the obtained data, and have wide application, high sensitivity, and good stability; the present invention has passed a strict procedure for screening reference genes and determined the reference genes that are stable in different tissues and stress treatments of perilla, providing a theoretical basis for selecting appropriate reference genes in perilla qRT-PCR analysis.

[0034] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for screening internal reference genes for fluorescent quantitative PCR in different tissues and stress treatments of Perilla frutescens, characterized in that: The specific steps include: Step 1, select the leaves, stems, roots, fruits at different developmental stages after flowering, and the leaves of Perilla frutescens treated with 20% PEG6000 for 0H, 3H, 6H, 9H, 12H, and 24H as samples; extract the total RNA of each sample, reverse transcribe the RNA to synthesize cDNA, and use it as a template for target gene amplification; Step 2: Using the previous perilla transcriptome sequencing data, we screened out the candidate internal reference genes of perilla, which are: PfActin, Pf18S, PfEF-1α, PfCYP, PfTUA, PfTUB, PfGAPDH ; Step 3: Using the selected candidate internal reference gene sequence as a template, designing the internal reference gene primers for real-time fluorescence quantitative PCR, wherein the amplified fragment size of the primers is 150-200 bp; Step 4: Real-time fluorescence quantitative PCR: Using the cDNA obtained by reverse transcription in step 1 as a template, the candidate internal reference gene is amplified by real-time fluorescence quantitative PCR using the primers in step 3; The obtained real-time fluorescence quantitative PCR data were statistically analyzed to screen out internal reference genes and internal reference gene combinations.

2. The method for screening internal reference genes of fluorescent quantitative PCR in different tissues and stress treatments of Perilla frutescens according to claim 1, characterized in that: In step 2: Said PfActin The nucleotide sequence is shown in the sequence listing SEQ ID NO.1; Said Pf18S The nucleotide sequence is shown in the sequence listing SEQ ID NO.2; Said PfEF-1α The nucleotide sequence is shown in the sequence listing SEQ ID NO.3; Said PcqI The nucleotide sequence is shown in the sequence listing SEQ ID NO.4; Said Pf The nucleotide sequence is shown in the sequence listing SEQ ID NO.5; Said PfT The nucleotide sequence is shown in the sequence listing SEQ ID NO.6; Said PfGAPDH The nucleotide sequence is shown in the sequence listing SEQ ID NO.

7.

3. The method for screening internal reference genes of fluorescent quantitative PCR in different tissues and stress treatments of Perilla frutescens according to claim 1, characterized in that: In step three: For amplification PfActin The primer pair sequence is SEQ ID NO.8 / SEQ ID NO.9; For amplification Pf18S The primer pair sequence is SEQ ID NO.10 / SEQ ID NO.11; For amplification PfEF-1α The primer pair sequence is SEQ ID NO.12 / SEQ ID NO.13; For amplification PcqI The primer pair sequence is SEQ ID NO.14 / SEQ ID NO.15; For amplification Pf The primer pair sequence is SEQ ID NO.16 / SEQ ID NO.17; For amplification PfT The primer pair sequence is SEQ ID NO.18 / SEQ ID NO.19; For amplification PfGAPDH The primer pair sequence is SEQ ID NO.20 / SEQ ID NO.

21.

4. The method for screening internal reference genes for fluorescent quantitative PCR in different tissues and stress treatments of Perilla frutescens according to claim 1, characterized in that: The method also includes identifying the specificity of the internal reference gene primers by conventional PCR before performing the real-time fluorescence quantitative PCR in step four.

5. The method for screening internal reference genes of fluorescent quantitative PCR in different tissues and stress treatments of Perilla frutescens according to claim 1, characterized in that: In step 4, the real-time fluorescence quantitative PCR data were statistically analyzed using GeNorm, NormFinder, and BestKeeper software to screen out internal reference genes and internal reference gene combinations, and then RefFinder analysis software was used for comprehensive ranking analysis.

Images