Internal reference gene for real-time fluorescent quantitative PCR (Polymerase Chain Reaction) detection of different tissues of bletilla striata in different growth periods

Abstract

The invention discloses reference genes for real-time fluorescent quantitative PCR detection of different tissues of rhizoma bletillae in different growth periods, and belongs to the technical field of plant molecular biology, the reference genes are BsGAPDH and BsEIF, the nucleotide sequence of the BsGAPDH is as shown in SEQ ID NO.1, and the nucleotide sequence of the BsEIF is as shown in SEQ ID NO.2. Three different tissues of rhizoma bletillae in seven growth periods are used as experimental materials, and the reference genes are used for real-time fluorescent quantitative PCR detection of different tissues of rhizoma bletillae in seven growth periods. The reference genes BsGAPDH and BsEIF with stable expression are obtained through fluorescent quantitative PCR (polymerase chain reaction) analysis and gene expression stability analysis by various technologies, and the two genes are stably expressed in different tissues in different growth periods of rhizoma bletillae and can be used as reference genes for researching gene expression in different tissues in different growth periods of rhizoma bletillae. The method can lay a foundation for functional research of genes related to accumulation of traditional Chinese medicinal components of rhizoma bletillae, and has relatively good practical value and scientific research application value.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular biology technology, specifically relating to a fluorescent quantitative internal reference gene for different tissues at different growth stages of Bletilla striata and its application. Background Technology

[0002] Bletilla striata ( Bletilla striata Bletilla striata (Bletilla striata) is a perennial herb belonging to the genus Bletilla of the Orchidaceae family. It is renowned for its numerous significant pharmacological properties, such as promoting wound healing, astringing and hemostasis, anti-inflammatory effects, antioxidant properties, and immunomodulation. These pharmacological activities are considered inseparable from its core active ingredient. Militarine, or 1,4-bis[4-(glucoseoxy)benzyl]-2-isobutylmalate, is widely found in Orchidaceae plants and is the main active ingredient in Bletilla striata. It possesses pharmacological activities including neuroprotection, improvement of cognitive impairment, treatment of lung injury, anticancer, anti-inflammatory, anti-ulcer, and antioxidant effects. The 2020 edition of the Pharmacopoeia of the People's Republic of China added Militarine as an indicator component of Bletilla striata. However, its metabolic patterns and quality formation mechanisms are still unclear, which adversely affects the rational development and utilization of Bletilla striata resources and the improvement of medicinal quality assurance.

[0003] The study of the metabolic patterns and quality formation mechanisms of medicinal components is based on molecular mechanism research, involving the characterization of functional genes. The accumulation of medicinal components in medicinal plants often exhibits spatiotemporal differences, with significant variations across different growth stages and tissues. Quantitative real-time PCR (qRT-PCR), due to its high sensitivity, reproducibility, specificity, speed, and efficiency, has become a primary method for studying gene expression. qRT-PCR results are affected by factors such as RNA quality, reverse transcription efficiency, and reaction procedures; therefore, stable internal reference genes are needed for calibration. The selection of suitable internal reference genes is crucial for generating reliable qRT-PCR data. With the development of molecular biology and the improvement of omics databases, research on functional genes related to the medicinal components of *Bletilla striata* can be greatly facilitated. There is an urgent need for internal reference genes that are stable in expression in different tissues and growth stages of *Bletilla striata*, thereby improving the accuracy of real-time quantitative detection of target genes involved in the metabolism of medicinal components and providing important scientific evidence for the study of functional genes in *Bletilla striata*. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide an internal reference gene suitable for real-time quantitative PCR of different tissues at different growth stages of Bletilla striata. This gene can meet the requirements of real-time quantitative detection of transcriptional expression levels of Bletilla striata at different growth stages in different tissues, thereby improving the stability and reliability of gene expression analysis studies of Bletilla striata at different growth stages in different tissues. Another objective of this invention is to provide an application of the above-mentioned gene-specific primers.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: On the one hand, the present invention provides BsGAPDH and / or BsEIF Application of the internal reference gene in real-time quantitative PCR detection of Bletilla striata at different growth stages and / or in different tissues. BsGAPDH The nucleotide sequence is as described in SEQ ID NO.1. BsEIF The nucleotide sequence is shown in SEQ ID NO.2, and the growth period is June, August, October and December of the first year; February, April and June of the second year; the different tissues are leaves, roots or pseudobulbs.

[0006] In another aspect, the present invention provides the application of specific amplification primers for the internal reference gene in real-time quantitative PCR detection of Bletilla striata at different growth stages and / or different tissues, wherein the primers are at least one of the nucleotide sequences shown in SEQ ID NO. 3 to 6.

[0007] In another aspect, the present invention provides a kit containing the specific amplification primers of the internal reference gene for the application of real-time fluorescence quantitative PCR detection of Bletilla striata at different growth stages and / or different tissues, wherein the kit contains at least one nucleotide sequence as shown in SEQ ID NO. 3 to 6.

[0008] In another aspect, the present invention provides the application of the internal reference gene or its specific amplification primers in detecting the expression levels of target genes related to pharmacodynamic components in different growth stages and / or different tissues of Bletilla striata.

[0009] Preferably, the detection method is real-time quantitative PCR detection.

[0010] Furthermore, this invention provides a real-time quantitative PCR method for detecting target genes in different growth stages and / or different tissues of Bletilla striata, employing... BsGAPDH and / or BsEIF As an internal reference gene, the following steps are included: (1) Design specific amplification primer pairs for the detection of target genes and internal reference genes. BsGAPDH The nucleotide sequence is as described in SEQ ID NO.1. BsEIF The nucleotide sequence is shown in SEQ ID NO.2; (2) Total RNA was extracted from Bletilla striata samples at different growth stages and / or different tissues using a kit and reverse transcribed into cDNA as a template for amplification, and then detected by real-time quantitative PCR.

[0011] Preferred, amplification BsGAPDH The primer pair sequences are shown in SEQ ID NO.3 and 4; amplification BsEIF The primer pair sequences are shown in SEQ ID NO.5 and 6.

[0012] Compared with the prior art, the beneficial effects of this invention are as follows: This invention uses three different tissues from seven growth stages of Bletilla striata as experimental materials. Through quantitative real-time PCR analysis and various techniques, gene expression stability was analyzed to obtain a stable internal reference gene. BsGAPDH and BsEIF These two genes are stably expressed in different tissues at different growth stages of Bletilla striata. They can be used as internal reference genes for studying gene expression in different tissues at different growth stages of Bletilla striata. They can lay the foundation for cross-time and cross-tissue studies on the function of genes related to the accumulation of medicinal components in Bletilla striata, and have good practical and scientific research application value.

[0013] BsGAPDH and BsEIF The gene serves as an internal reference gene for studying gene expression in different tissues and growth stages of Bletilla striata. It not only solves the current situation where there is no suitable internal reference gene in the quantitative PCR detection of Bletilla striata, but also the designed real-time fluorescence quantitative PCR primers can improve the accuracy of gene expression analysis research of Bletilla striata and provide technical support for ensuring the reliability of data screening. Attached Figure Description

[0014] Figure 1 This is an agarose gel electrophoresis image of total RNA extraction in an embodiment of the present invention.

[0015] Figure 2 This is a primer-specific agarose gel electrophoresis image from an embodiment of the present invention, lanes 1-2: BsABC Primer; Lanes 3-4: BsACT Primer; Lanes 5-6: Bsa-TUB Primer; Lanes 7-8: BsEF1a Primer; Lanes 9-10: BsEF2 Primer; Lanes 11-12: BsEIF Primer; Lanes 13-14: BsGAPDH Primer; Lanes 15-16: BsPP2A Primer; Lanes 17-18: BsRPL Primers; lanes 19-21: BsIBMS Primers; lanes 22-24: BsUGT Primers.

[0016] Figure 3 This is a dissolution curve diagram from an embodiment of the present invention, wherein Figure A is... BsGAPDH The gene melting curve, Figure B is... BsEIF Melting curve of a gene.

[0017] Figure 4 This is a diagram showing the results of analyzing the stability of candidate internal reference genes using the delta-Ct method in an embodiment of the present invention.

[0018] Figure 5 This is a graph showing the results of analyzing the stability of candidate internal reference genes using the BestKeeper method in an embodiment of the present invention.

[0019] Figure 6 This is a diagram showing the results of analyzing the stability of candidate internal reference genes using the NormFinder method in an embodiment of the present invention.

[0020] Figure 7 This is a statistical chart showing the results of analyzing the stability of candidate internal reference genes using the GeNorm method in an embodiment of the present invention.

[0021] Figure 8 This is a statistical chart showing the results of using RefFinder to comprehensively evaluate the stability of candidate internal reference genes in this embodiment of the invention.

[0022] Figure 9 The one with the best stability in the embodiments of the present invention BsGAPDH When genes are used as internal controls, they are analyzed in different tissues at different growth stages. BsIBMS and BsUGT Gene expression level diagram.

[0023] Figure 10 In this embodiment of the invention, the stability is secondary. BsEIF When genes are used as internal controls, they are analyzed in different tissues at different growth stages. BsIBMS and BsUGT Relative gene expression levels.

[0024] Figure 11 The one with the worst stability used in this embodiment of the invention BsRPL When genes are used as internal controls, they are analyzed in different tissues at different growth stages. BsIBMS and BsUGT Relative gene expression levels.

[0025] Figure 12 In the embodiments of the present invention BsIBMS and BsUGT Relative expression levels of genes based on transcriptome data, where Figure A represents... BsIBMS The relative expression levels of genes in the transcriptome, as shown in Figure B. BsUGTThe relative expression level of genes.

[0026] Note: Figure 9 , Figure 10 , Figure 11 , Figure 12 In the sample, 01L-07L represents the samples of Bletilla striata leaves taken from the 1st to the 7th sampling, 01R-07R represents the samples of Bletilla striata roots taken from the 1st to the 7th sampling, and 01P-07P represents the samples of Bletilla striata pseudobulbs taken from the 1st to the 7th sampling. Detailed Implementation

[0027] The present invention will now be described in further detail with reference to the embodiments.

[0028] Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in the field or according to the product instructions. Materials or equipment whose manufacturers are not specified are all conventional products that can be obtained by purchase.

[0029] Bletilla striata ( Bletilla striata Bletilla striata is a perennial medicinal plant. The conventional planting period from planting seedlings to forming commercial-grade tubers is 3 years. If higher medicinal efficacy or yield per plant is desired, it can be extended to 4 years. The materials used in the following examples were collected from the leaves, roots and pseudobulbs of Bletilla striata from the Puer base of Puer Liangbao Biotechnology Co., Ltd. From June 2023 to June 2024, samples were collected in the following months: June (vigorous growth period), August (slow growth period), October (seedling collapse-dormancy period), December (seedling collapse-dormancy period), February (germination period), April (flowering-fruiting period), and June (vigorous growth period). Samples were collected every 2 months, with 3 replicates each time. After sampling, all materials were placed in 50mL centrifuge tubes, frozen in liquid nitrogen and stored at -80℃ until further analysis.

[0030] Example 1 1. Total RNA extraction and cDNA synthesis from plant tissues Total RNA was extracted from different tissues (leaves, roots, and pseudobulbs) of *Bletilla striata* at different growth stages (June, August, October, December of the first year, and February, April, and June of the second year) using the Genstar RNA extraction kit. RNA concentration and quality were detected using a micro spectrophotometer. RNA bands were detected by 1.0% agarose gel electrophoresis. The results showed that the quality of all samples met the requirements, and the agarose gel electrophoresis images showed clear 28S and 18S bands with no degradation, thus meeting the requirements. Figure 1 ).

[0031] cDNA for real-time quantitative PCR analysis was obtained by reverse transcription using Bio-Rad iScript™ RT-qPCR reverse transcriptase according to the manufacturer's instructions. A 20 μL cDNA reverse transcription system was prepared using 800 μg total RNA. After the reaction, the cDNA was stored at -20°C for later use.

[0032] 2. Screening of internal reference genes and primer design Nine candidate internal reference genes were screened, namely: BsGAPDH (SEQ ID NO.1) BsEIF (SEQ ID NO.2) BsABC (SEQ ID NO.7) BsACT (SEQ ID NO.8) Bsa-TUB (SEQ ID NO.9) BsEF1a (SEQ ID NO.10) BsEF2 (SEQ ID NO.11) BsPP2A (SEQ ID NO.12) BsRPL (SEQ ID NO.13).

[0033] Primers were designed using the Primer3Plus online primer design tool based on the OFR reading frame of the full-length transcript, and primer specificity was identified using a BLAST tool. Primers were synthesized at Qingke Biotechnology Co., Ltd., and then preliminary screening was performed using conventional PCR. Product specificity was detected using agarose gel electrophoresis. Figure 2 Primers with correct band size, good band specificity, and no primer dimers were selected, and their specificity was further tested by qRT-PCR. Primers with a single peak in their melting curve and no extraneous peaks were selected as the final primers. Figure 3 (Table 1).

[0034]

[0035] 3. Establishment of standard curve for internal reference gene primers Standard curves were established for each internal reference gene primer, and its amplification efficiency was calculated. Mixtures of cDNA from different growth stages and tissues were diluted 5-fold to four gradients (1, 1 / 5, 1 / 25, 1 / 125) to serve as templates for establishing standard curves. qRT-PCR was performed using a Bio-Rad CFX96 real-time PCR instrument. Each reaction included 0.8 μL each of 10 pM forward and reverse candidate reference gene primers, 2 μL of cDNA template diluted at different folds, 10 μL of Bio-Rad SsoAdvanced SYBR® Green reaction premix, and ddH2O to a final volume of 20 μL. Each reaction was performed in triplicate. The qRT-PCR conditions were: 95°C for 30 seconds, followed by 95°C for 15 seconds, 61°C for 30 seconds, for 40 cycles. At the end of each experiment, melting curve analysis was performed using default parameters at 55–95°C in 0.3°C increments for 60 seconds. All analyses were performed in triplicate. The amplification efficiency of the candidate internal reference gene was determined using the obtained data, and the subsequent data were analyzed using the actual amplification efficiency (Table 1).

[0036] 4. Real-time fluorescence quantification qRT-PCR was performed using a Bio-Rad CFX96 real-time PCR instrument. Each reaction included 0.8 μL each of 10 pM forward and reverse candidate reference gene primers, 2 μL of cDNA templates from different tissues at different developmental stages, 10 μL of Bio-Rad SsoAdvanced SYBR® Green reaction premix, and ddH2O to bring the total to 20 μL. Each reaction system was performed in triplicate. The qRT-PCR reaction conditions were the same as above.

[0037] 5. Data Analysis (1) Delta Ct algorithm The ΔCt algorithm analysis ranks candidate genes based on their average standard deviation; a smaller average standard deviation indicates a more stable internal reference gene. Results show that among the nine internal reference genes, BsGAPDH It is the most stable gene among all candidate genes, with the lowest average STDEV value of 2.18; followed by... BsEIF The average STDEV value of the gene was 2.22; while BsRPL The stability was the worst, with the highest average STDEV value of 5.02 (Table 2). Figure 4 ).

[0038]

[0039] (2) BestKeeper software analysis BestKeeper software assesses gene expression stability by comparing the standard deviation (SD), coefficient of variation (CV), and correlation coefficient (r) of Ct values, directly analyzing gene expression Ct values. The analysis results show that the most stable gene expression is... BsACT and BsEIF Genes, the most unstable gene is BsRPL (Table 3, Figure 5 ).

[0040]

[0041] (3) NormFinder software analysis NormFinder software ranks candidate internal reference genes based on analysis of variance and incorporates expression differences between different samples. Higher expression stability values ​​indicate lower stability, with the gene having the lowest stability value being the most stable. The analysis results show that... BsEIF The lowest stability value was 0.93, which was considered the most suitable internal reference gene; the second was... BsGAPDH The gene's stable value is 1.08; meanwhile, BsRPL It is the least stable reference gene, with a stability value of 4.80 (Table 4). Figure 6 ).

[0042]

[0043] (4) Analysis using geNom software The geNorm software measures gene stability based on the mean variance (M-value). The software's default threshold is 1.5; genes with an M-value higher than 1.5 are not suitable for use as internal controls. A lower M-value indicates a more stable gene. Analysis results show... BsGAPDH and BsEF2 The gene with the lowest M value (1.02) has the highest stability; the gene with the lowest stability is... BsRPL The value of M is 2.74 (Table 5). Figure 7 ).

[0044]

[0045] (5) Overall evaluation of RefFinder software Using the online software RefFinder (http: / / blooge.cn / RefFinder / ), the stability of nine candidate internal reference genes in different tissues at different growth stages of Bletilla striata was determined by overall ranking based on the geometric mean of the ranking values ​​from the ΔCt algorithm, geNorm software, NormFinder software, and BestKeeper software. The results showed that, when analyzing all samples, the two genes with the most stable expression were... BsGAPDH and BsEIF These two genes ranked among the top two in expression stability in three out of the four algorithms. Furthermore, a comprehensive analysis of the reference gene with the lowest expression stability showed that... BsRPL is the most Unstable internal reference genes (Table 6, Figure 8 ).

[0046]

[0047] 6. Verification of the stability of the internal reference gene The key upstream genes in the biosynthetic pathway of tetrafida albicans, the main active ingredient in Bletilla striata, were selected. BsIBMS (SEQ ID NO.14) and downstream key genes BsUGT (SEQ ID NO.15) to verify the screened internal reference gene BsGAPDH and BsEIF The stability, and expressed by the most unstable. BsRPL By comparing genes, the results showed that the selected internal reference genes... BsGAPDH and BsEIF When used as an internal reference BsIBMS and BsUGT The trends in gene changes are similar to those in relative expression levels in transcriptome data, but using... BsRPL When these two genes were used as internal controls, their expression patterns differed from the relative expression trends in the transcriptome data, indicating that the selected internal control genes... BsGAPDH and BsEIF It is accurate and reliable. Figure 9 , Figure 10 , Figure 11 , Figure 12 ).

Claims

1. BsGAPDH and / or BsEIF As an internal reference gene, its application in real-time quantitative PCR detection of Bletilla striata at different growth stages and / or in different tissues is characterized by, BsGAPDH The nucleotide sequence is as described in SEQ ID NO.

1. BsEIF The nucleotide sequence is shown in SEQ ID NO.2, and the growth period is June, August, October and December of the first year; February, April and June of the second year; the different tissues are leaves, roots or pseudobulbs.

2. The application of the specific amplification primers for the internal reference gene described in claim 1 in real-time quantitative PCR detection of Bletilla striata at different growth stages and / or in different tissues, characterized in that, The primer is at least one of the nucleotide sequences shown in SEQ ID NO.3 to 6.

3. The application of the kit containing the internal reference gene-specific amplification primers as described in claim 1 in real-time quantitative PCR detection of Bletilla striata at different growth stages and / or in different tissues, characterized in that... The kit contains at least one nucleotide sequence as shown in SEQ ID NO.3 to 6.

4. The application of the internal reference gene or its specific amplification primers as described in claim 1 in detecting the expression levels of target genes related to pharmacodynamic components in different growth stages and / or different tissues of Bletilla striata.

5. The application according to claim 4, characterized in that, The detection method is real-time quantitative PCR.

6. A real-time quantitative PCR method for detecting target genes in different growth stages and / or different tissues of Bletilla striata, characterized in that, use BsGAPDH and / or BsEIF As an internal reference gene, the following steps are included: (1) Design specific amplification primer pairs for the detection of target genes and internal reference genes. BsGAPDH The nucleotide sequence is as described in SEQ ID NO.

1. BsEIF The nucleotide sequence is shown in SEQ ID NO.2; (2) Total RNA was extracted from Bletilla striata samples at different growth stages and / or different tissues using a kit and reverse transcribed into cDNA as a template for amplification, and then detected by real-time quantitative PCR.

7. The method according to claim 6, characterized in that, Amplification BsGAPDH The primer pair sequences are shown in SEQ ID NO.3 and 4; amplification BsEIF The primer pair sequences are shown in SEQ ID NO.5 and 6.

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