Osmanthus abiotic stress miRNA internal reference gene, its primers and applications

Abstract

This invention discloses the miRNA reference genes for abiotic stress in Osmanthus fragrans, their primers, and applications. The optimal miRNA reference genes under abiotic stress were screened and obtained. Under drought stress, the miRNA references were ofr-miR168b-5p, novel8, and ofr-miR159b-3p; under low temperature stress, the miRNA references were ofr-miR159b-3p and ofr-miR403-3p; under salt stress, the miRNA references were novel3 and novel8; and under abiotic stress, the miRNA references were ofr-miR159b-3p, novel8, ofr-miR403-3p, and novel2. qRT-PCR primers were also designed, filling the gap in the lack of miRNA references under abiotic stress conditions in Osmanthus fragrans and providing strong support for the quantitative expression of relevant miRNAs under abiotic stress conditions in Osmanthus fragrans.

Description

Technical Field

[0001] This invention relates to the field of plant molecular biology, and in particular to the miRNA internal reference gene for abiotic stress in Osmanthus fragrans, its primers, and applications. Background Technology

[0002] Osmanthus fragrans, belonging to the Oleaceae family, is one of China's ten traditional famous flowers. Abiotic stressors such as drought, heat, low temperature, and salt damage trigger a series of physiological and metabolic responses in plants. However, there are currently no reports on miRNAs in Osmanthus fragrans under abiotic stresses, including drought, low temperature, and salt stress. Research on differential gene expression under abiotic stresses involves analyzing and verifying gene expression levels using real-time quantitative PCR (qPCR). Stable and reliable internal reference genes are needed; therefore, screening for stably expressed miRNAs under abiotic stress conditions is crucial for accurate qPCR results. Summary of the Invention

[0003] To address the aforementioned deficiencies in existing technologies, this invention proposes an internal reference gene for abiotic stress in Osmanthus fragrans, its primers, and their applications, thereby resolving the problems raised in the background.

[0004] To achieve the above objectives, the present invention provides the following technical solution:

[0005] Osmanthus abiotic stress miRNA reference gene, wherein the miRNA reference gene includes at least one of (1) to (4):

[0006] (1) The miRNA internal reference genes under drought stress are ofr-miR168b-5p, novel8 and ofr-miR159b-3p;

[0007] (2) Under low temperature stress, the miRNA internal reference genes are ofr-miR159b-3p and ofr-miR403-3p;

[0008] (3) Under salt stress, the miRNA internal reference genes are novel3 and novel8;

[0009] (4) The miRNA reference genes under drought stress, low temperature stress and salt stress are ofr-miR159b-3p, novel8, ofr-miR403-3p and novel2;

[0010] The nucleic acid sequences of ofr-miR168b-5p are shown in SEQ ID NO.1; the nucleic acid sequence of novel8 is shown in SEQ ID NO.2; the nucleic acid sequence of ofr-miR159b-3p is shown in SEQ ID NO.3; the nucleic acid sequence of ofr-miR403-3p is shown in SEQ ID NO.4; the nucleic acid sequence of novel3 is shown in SEQ ID NO.5; and the nucleic acid sequence of novel2 is shown in SEQ ID NO.6.

[0011] Preferably, the proprietary primers for ofr-miR168b-5p have a forward primer sequence of 5'-ccgcTCGCTTGGTGCAGGTCGGGAA-3' (SEQ ID NO.7).

[0012] Preferably, the proprietary primers of novel8 have the following forward primer sequence:

[0013] 5'-gcgccgTTTCCTATTCCTCCCATACCGA-3' (SEQ ID NO. 8).

[0014] Preferably, the proprietary primers for ofr-miR159b-3p have a forward primer sequence of 5'-gcgcgcCTTTGGATTGAAGGGAGCTCT-3' (SEQ ID NO.9).

[0015] Preferably, the proprietary primers for ofr-miR403-3p have the following forward primer sequence:

[0016] 5'-cgccgccgTTAGATTCACGCACAAACTCG-3' (SEQ ID NO. 10).

[0017] Preferably, the proprietary primers of novel3 have the following forward primer sequence:

[0018] 5'-gcggcggTCAAGATTGGGCAATGAACCA-3' (SEQ ID NO. 11).

[0019] Preferably, the proprietary primers of novel2 have the following forward primer sequence:

[0020] 5'-ccgccgTTTCCTATACCTCCCATACCGA-3' (SEQ ID NO. 12).

[0021] Preferably, this application also relates to the application of the Osmanthus abiotic stress miRNA internal reference gene in Osmanthus miRNA real-time PCR analysis.

[0022] Preferably, this application also relates to the application of proprietary primers for the Osmanthus abiotic stress miRNA internal reference gene in Osmanthus miRNA real-time PCR analysis, the proprietary primers being as described above.

[0023] Compared with the prior art, the beneficial effects of the present invention are:

[0024] This application involved local BLAST alignment (BLASTVer: 2.4.0+) of reference genes used in other species with the whole genome data of Osmanthus fragrans, followed by online analysis of conserved domains and ORFs using BLASTX on the NCBI website. Five commonly used reference genes (18S, ACT11, TUA5, U6, and UBQ4) were ultimately selected. Nine mature and relatively abundant and stable miRNAs (ofr-miR159b-3p, ofr-miR168b-5p, ofr-miR171a-3p, ofr-miR395e, ofr-miR403-3p, novel2, novel3, novel8, and novel33) were obtained through small RNA sequencing, and primer sequences for the reference genes were designed. The stability of candidate internal controls was evaluated using five algorithms (delta-CT, geNorm, NormFinder, BestKeeper, and RefFinder). A real-time quantitative PCR primer for the internal control gene was obtained, suitable for various abiotic stresses (drought, low temperature, and salt stress) on Osmanthus fragrans. Primers for this internal control gene were designed; these primers exhibit high specificity and amplification efficiency, significantly improving the detection efficiency and reliability of Osmanthus fragrans miRNA detection using real-time quantitative PCR. Attached Figure Description

[0025] Figure 1 This is a graph showing the CQ values ​​of 14 internal reference genes screened under various abiotic stress treatments;

[0026] Figure 2 This is a diagram showing the optimal number of internal reference genes for accurate quantitative analysis under various abiotic stress treatments determined by geNorm.

[0027] Figure 3 This is a ranking chart of the expression stability values ​​(M) of 14 internal reference genes under various abiotic stress treatments using the geNorm software.

[0028] Figure 4 The expression levels of ofr-miR166e-5p were measured using stable and unstable genes as internal reference genes under various abiotic stress treatments.

[0029] Figure 5The expression levels of ofr-miR396b-3p are measured using stable and unstable genes as internal reference genes under various abiotic stress treatments. Detailed Implementation

[0030] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0031] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0032] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0033] The test material used in the following examples was Osmanthus fragrans. The Osmanthus fragrans were selected from the "Chang'e" clonal line, which was in good condition and free from pests and diseases, in Xianning City, Hubei Province, as the mother tree. In May 2023, semi-lignified branches with 2-3 lateral buds from the current year were taken as cuttings (12-16cm). The upper cut was made horizontally, and the lower cut was made at a 45° angle. The cuttings were soaked in distilled water for 12 hours, then soaked in 1% sodium hypochlorite for 10 minutes, rinsed three times with distilled water, and then soaked in 0.1g / L GGR rooting powder for 4 hours. The cuttings were then planted in the Osmanthus fragrans base in Xianning City, Hubei Province. The soil substrate was a mixed substrate composed of peat moss, perlite, vermiculite, and yellow sand in a 1:1:1:1 ratio, and watered twice a week. In April 2024, Osmanthus fragrans cuttings were cultured in a light incubator with a photoperiod of 12-hlight / 12-hdarkcycle and 60% humidity. The cuttings were treated with 4°C, 300 mM NaCl, and 20% PEG-6000, respectively. Samples were collected at extended stress periods of 0, 3, 6, 12, 24, and 72 hours. Each treatment was performed in triplicate and was cryopreserved in liquid nitrogen at -80°C for further analysis.

[0034] Example 1

[0035] 1. Total RNA extraction and cDNA synthesis from Osmanthus fragrans

[0036] miRNA was extracted using an miRNA isolation kit (Tiangen Biotech, Beijing, China). Integrity, purity, and concentration were determined by 1.0% (w / v) agarose gel electrophoresis and a spectrophotometer (NanoDrop2000, ThermoScientific, Wilmington, DE, USA).

[0037] The synthesized samples were analyzed using a tailing method for reverse transcription: 0.8 μg of miRNA was used to synthesize the first strand of miRNA-cDNA according to the instructions of the miRcute miRNAFirst-Strand cDNA Synthesis Kit (KR201, Tiangen Biotech, China). The reaction was carried out at 42°C for 60 min; subsequently, the sample was inactivated at 95°C for 3 min. The synthesized cDNA reaction solution was immediately stored at -20°C.

[0038] 2. Selection of internal reference genes and design of their primers

[0039] Nine mature, highly expressed, and stable miRNAs (ofr-miR159b-3p, ofr-miR168b-5p, ofr-miR171a-3p, ofr-miR395e, ofr-miR403-3p, novel2, novel3, novel8, and novel33) were selected from small RNA sequencing of Osmanthus fragrans. Five commonly used internal reference genes (18S, ACT11, TUA5, U6, and UBQ4) were screened through literature review and database comparison. Primers for 18S, ACT11, TUA5, U6, and UBQ4 were designed using Primer 5.0. The upstream primers for the miRNAs were based on mature miRNA sequences, with U replaced by T, and appropriate T values ​​were achieved by adding or removing bases at both ends of the primers. m The temperature was 65℃, the GC content was 40-60%, and the reverse primer was provided by the miRcute miRNA qPCRDetection Kit (SYBR Green).

[0040] Table 1. 14 candidate reference genes and primer sequences

[0041]

[0042] Note: E: amplification efficiency; R2: correlation coefficient

[0043] 3. qRT-PCR quantification

[0044] Prepare a 20 μL PCR reaction mixture according to the miRcute Plus miRNA qPCR Detection Kit (SYBR Green) instructions: 10 μL of 2×miRcute Plus miRNA Premix (SYBR & ROX), 0.4 μL each of forward and reverse primers (10 μM), 2 μL of miRNA first-strand cDNA diluted 10-fold, and 7.2 μL of RNase-free ddH2O. Perform three technical replicates for each sample. Include a negative control (without template) for each candidate internal control gene to check for amplification background.

[0045] qRT-PCR was performed using a Tianlong Gentier 96E system (Tianlong Technology Co., Ltd., Xi'an, China). The program was as follows: pre-denaturation at 95℃ for 15 min; followed by 40 cycles of denaturation at 94℃ for 20 s and annealing extension at 60℃ for 30 s; then melting curve analysis was performed at 60-95℃. CT values ​​were obtained from qRT-PCR. CT values ​​are inversely proportional to gene expression levels; a higher CT value indicates lower gene expression, and vice versa.

[0046] Low temperature stress Figure 1 Under a) conditions, novel3 showed the smallest CT change, at 1.086; drought stress ( Figure 1 Under b) conditions, the CT change value of novel3 was the smallest, at 0.961; salt stress ( Figure 1 c) and abiotic stress ( Figure 1 Under condition d), the CT change value of novel8 is the smallest, at 2.772.

[0047] 4. Stability assessment

[0048] Internal reference gene stability analysis was performed using five algorithms: delta-CT, geNorm, NormFinder, BestKeeper, and RefFinder. Stable internal reference genes were then screened out.

[0049] 4.1 Delta-CT Analysis

[0050] The mean standard deviation was obtained through delta-CT analysis. A higher mean standard deviation indicates lower gene stability, while a lower mean standard deviation indicates higher gene stability. The results are shown in Table 2. Under drought treatment, ofr-miR168b-5p was the most stable gene; under low temperature stress and abiotic stress, ofr-miR159b-3p was the most stable gene; and under salt stress, novel2 was the most stable gene. TUA5 was the least stable gene under low temperature stress, salt stress, and abiotic stress; while under drought stress, UBQ4 was the least stable gene.

[0051] Table 2. Delta-CT Analysis Results

[0052]

[0053] 4.2geNorm Software Analysis

[0054] When using geNorm, the original Ct value is converted to 2. -ΔCt (delta Ct = original Ct value - lowest Ct value in this group) was then used for expression stability analysis of the internal reference gene. The expression stability M value of each candidate internal reference gene was calculated using geNorm software. A larger M value indicates lower stability; conversely, a smaller M value indicates higher stability, where M = 1.5 is the upper limit. Furthermore, geNorm can... -ΔCt Using the original data, the paired difference value V between two continuous standardized factors was calculated. n / V n+1 To determine the appropriate number of internal reference genes, since the pairwise variation value V2 / V3 of the Osmanthus fragrans examples under salt and low temperature stress was <0.15 ( Figure 2 Under drought stress, the pairwise variance V3 / V4 of the examples was <0.15. Figure 2 Under abiotic stress, the paired variance V4 / V5 < 0.15. Figure 2 Therefore, only two internal reference genes (novel3 and ofr-miR403-3p) are needed under salt stress, only two internal reference genes (ofr-miR159b-3p and ofr-miR403-3p) are needed under low temperature stress, three internal reference genes (novel8, novel2, and novel3) are needed under drought stress, and four internal reference genes (ofr-miR159b-3p, novel8, ofr-miR403-3p, and novel2) are needed under abiotic stress. Figure 3 ).

[0055] 4.3 NormFinder Software Analysis

[0056] When using NormFinder, convert the original Ct value to 2.-ΔCt The delta Ct (original Ct value - lowest Ct value in this group) was then used for expression stability analysis of the internal reference genes. The stability values ​​of candidate internal reference genes were calculated using NormFinder software; higher stability values ​​indicated lower stability, and vice versa. The gene with the lowest stability value was considered the most stable. The results are shown in Table 3. ofr-miR168b-5p, U6, novel8, and ofr-miR159b-3p showed the best stability under drought, low temperature, salt stress, and abiotic stress, respectively.

[0057] Table 3. NormFinder Software Analysis Results

[0058]

[0059]

[0060] 4.4 BestKeeper Software Analysis

[0061] For BestKeeper, the amplification efficiency calculated by the LinRegPCR program based on the original Ct value and the original Ct value were used to calculate the coefficient of variation (CV) and standard deviation (SD) of candidate internal reference gene expression. The smaller the SD value, the more stable the internal reference gene. If the SD value > 1.0, the internal reference gene is considered unstable and should be avoided for gene expression normalization. The results are shown in Table 4. The most stable gene under drought stress, salt stress, and abiotic stress was novel3, while the most stable gene under low temperature stress was ofr-miR159b-3p.

[0062] Table 4. BestKeeper Software Analysis Results

[0063]

[0064] 4.5 RefFinder URL Analysis

[0065] To verify the accuracy of the internal reference gene screening, we used RefFinder to perform a comprehensive ranking of the candidate internal reference genes under each treatment. The lower the comprehensive ranking, the better the gene expression stability.

[0066] The results are shown in Table 5. The stability of genes under drought stress was as follows: ofr-miR168b-5p>novel8>ofr-miR159b-3p>novel2>novel3>ofr-miR403-3p>U6>novel33>ofr-miR395e>ofr-miR171a-3p>18S>ACT11>TUA5>UBQ4, among which miR168b-5p+novel8+miR159b-3p was the most stable gene combination. The stability of genes under low temperature stress was as follows: ofr-miR159b-3p > ofr-miR403-3p > novel33 > U6 > ofr-miR395e > novel3 > novel8 > ofr-miR168b-5p > novel2 > ofr-miR171a-3p > 18S > UBQ4 > ACT11 > TUA5, among which ofr-miR159b-3p + ofr-miR403-3p was the most stable gene combination. The stability of genes under salt stress was novel3>novel8>novel2>ofr-miR159b-3p>ofr-miR403-3p>novel33>U6>18S>ofr-miR395e>ofr-miR168b-5p>ACT11>ofr-miR171a-3p>UBQ4>TUA5, among which novel3+novel8 was the most stable internal control combination. Under abiotic stress, the gene stability was as follows: ofr-miR159b-3p>novel8>ofr-miR403-3p>novel2>novel3>novel33>ofr-miR168b-5p>U6>ofr-miR395e>ofr-miR171a-3p>18S>ACT11>UBQ4>TUA5, with the most stable gene combination being ofr-miR159b-3p+novel8+ofr-miR403-3p+novel2.

[0067] Table 5. RefFinder URL Analysis Results

[0068]

[0069] 5. Verification of the stability of the internal reference gene

[0070] Based on our selected high-throughput sequencing results, ofr-miR166e-5p and ofr-miR396b-3p were used as target genes. Stable and unstable genes under various abiotic stresses were used as criteria for calculation to confirm the applicability of the candidate genes evaluated in this study. (Using 2...) -△△Ct The method calculates the expression of target genes.

[0071] Under low-temperature stress, using stable ofr-miR159b-3p and ofr-miR403-3p as internal controls, the expression trends of ofr-miR166e-5p and ofr-miR396b-3p were similar: ofr-miR166e-5p was highly expressed at 0h and 72h, while ofr-miR396b-3p was highly expressed at 0h. However, when the unstable gene TUA5 was used as an internal control, ofr-miR166e-5p and ofr-miR396b-3p were significantly highly expressed from 6 to 12h. Figure 4 a, Figure 5 a). Under drought stress, using stable ofr-miR168b-5p, novel8, and ofr-miR159b-3p as internal controls, the expression trends of ofr-miR166e-5p and ofr-miR396b-3p were similar, indicating that the target gene was highly expressed at 3 hours; however, when the unstable gene UBQ4 was used as an internal control, the target gene was significantly highly expressed at 72 hours. Figure 4 b、 Figure 5 b). Under salt stress, when stable genes novel3 and novel8 were used as internal controls, the target gene expression level was highest at 3 hours, while when the unstable gene TUA5 was used as an internal control, the target gene expression level was highest at 12 hours. Figure 4 c. Figure 5 c). Therefore, choosing the right gene does have a significant impact on the expression level and expression of the target gene.

[0072] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of the internal reference gene in the quantitative real-time PCR analysis of miRNAs under abiotic stress in Osmanthus fragrans, characterized in that, (1) When the abiotic stress is drought stress, the internal reference genes are ofr-miR168b-5p, novel8 and ofr-miR159b-3p; (2) When the abiotic stress is low temperature stress, the internal reference genes are ofr-miR159b-3p and ofr-miR403-3p; (3) When the abiotic stress is salt stress, the internal reference genes are novel3 and novel8; The nucleic acid sequence of ofr-miR168b-5p is shown in SEQ ID NO.1; the nucleic acid sequence of novel8 is shown in SEQ ID NO.2; the nucleic acid sequence of ofr-miR159b-3p is shown in SEQ ID NO.3; the nucleic acid sequence of ofr-miR403-3p is shown in SEQ ID NO.4; and the nucleic acid sequence of novel3 is shown in SEQ ID NO.

5.

2. The application of the proprietary primers for detecting the internal reference gene described in claim 1 in the quantitative real-time PCR analysis of miRNAs under abiotic stress in Osmanthus fragrans, characterized in that, (1) When the abiotic stress is drought stress, the internal reference genes are ofr-miR168b-5p, novel8 and ofr-miR159b-3p; (2) When the abiotic stress is low temperature stress, the internal reference genes are ofr-miR159b-3p and ofr-miR403-3p; (3) When the abiotic stress is salt stress, the internal reference genes are novel3 and novel8; The nucleic acid sequence of ofr-miR168b-5p is shown in SEQ ID NO.1; the nucleic acid sequence of novel8 is shown in SEQ ID NO.2; the nucleic acid sequence of ofr-miR159b-3p is shown in SEQ ID NO.3; the nucleic acid sequence of ofr-miR403-3p is shown in SEQ ID NO.4; and the nucleic acid sequence of novel3 is shown in SEQ ID NO.

5.

3. The application according to claim 2, characterized in that, The proprietary primers used to detect ofr-miR168b-5p have the sequence 5'-ccgcTCGCTTGGTGCAGGTCGGGAA-3' for the forward primer; The proprietary primers used to detect novel8 have a forward primer sequence of 5'-gcgccgTTTCCTATTCCTCCCATACCGA-3'; The proprietary primers used to detect ofr-miR159b-3p have the forward primer sequence 5'-gcgcgcCTTTGGATTGAAGGGAGCTCT-3'; The proprietary primers used to detect ofr-miR403-3p have the forward primer sequence 5'-cgccgccgTTAGATTCACGCACAAACTCG-3'; The proprietary primers used to detect novel3 have a forward primer sequence of 5'-gcggcggTCAAGATTGGGCAATGAACCA-3'. The reverse primers for the proprietary primers were provided by the miRcute miRNA qPCR Detection Kit (SYBRGreen).

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