Osmanthus hormone-treated miRNA internal reference gene, primers, and applications thereof

Abstract

The present invention discloses sweet osmanthus miRNA internal reference genes under hormone treatment, primers, and applications thereof. The optimal miRNA internal reference genes under hormone treatment are obtained: novel33 and ofr-miR159b-3p are the miRNA internal references under ethephon treatment; novel3 and ofr-miR159b-3p are the miRNA internal references under ABA treatment; ofr-miR159b-3p, novel8, novel2, and novel3 are the miRNA internal references under MeJA treatment; and ofr-miR159b-3p, novel8, and novel3 are the miRNA internal references under combined treatment of the three hormones. PCR primers are designed, addressing the current situation where there is no internal reference for sweet osmanthus miRNA fluorescence quantification under different hormone treatments, and providing strong support for the quantitative expression of related miRNAs under different hormone treatments in sweet osmanthus.

Description

Technical Field

[0001] The present invention relates to the technical field of plant molecular biology, and in particular to an osmanthus hormone-treated miRNA internal reference gene, a primer thereof, and an application thereof. Background Art

[0002] Sweet osmanthus (Osmanthus fragrans), belonging to the Oleaceae family, is a common woody ornamental flower known for its rich fragrance and beautiful flowers. The flowering period, fragrance, and color of sweet osmanthus are influenced by multiple factors, among which hormone treatment is an effective regulatory tool. Hormone treatment can influence the growth, development, and flowering process of sweet osmanthus by regulating endogenous plant hormone levels. However, the physiological and molecular mechanisms within plants after hormone treatment are complex and variable, necessitating the need for accurate internal reference for quantitative analysis of miRNA expression.

[0003] When conducting gene expression analysis, selecting appropriate internal reference genes is crucial for ensuring data accuracy and reliability. Internal reference genes are typically genes that are stably expressed under different treatment conditions and are used to correct for technical errors in experiments. However, the expression stability of internal reference genes may vary under different treatment conditions. Therefore, when conducting gene expression studies in osmanthus plants treated with hormones, it is necessary to screen for suitable miRNA internal reference genes to ensure the accuracy and reliability of qRT-PCR data. Hormone treatments induce complex physiological changes within osmanthus plants, including shifts in gene expression patterns. Traditionally used internal reference genes, such as Actin and GAPDH, may become unstable after hormone treatment, making gene internal reference genes unsuitable for miRNA internal references. Therefore, studies specifically targeting osmanthus hormone treatments require experimental screening and validation to identify miRNA internal reference genes that are stably expressed under different hormone treatment conditions. This not only helps to enhance the credibility of research results but also provides a solid foundation for subsequent functional studies of osmanthus miRNAs. Summary of the Invention

[0004] In view of the above-mentioned defects of the prior art, the present invention proposes a miRNA internal reference gene under osmanthus hormone treatment and its primers and applications to solve the problems raised by the above-mentioned background technology.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] Osmanthus hormone-treated miRNA reference gene, the miRNA reference gene includes at least one of the following (1) to (4):

[0007] (1) The miRNA reference genes of Osmanthus fragrans under ethephon treatment were novel33 and ofr-miR159b-3p;

[0008] (2) The internal reference genes of Osmanthus fragrans miRNA under abscisic acid treatment were novel3 and ofr-miR159b-3p;

[0009] (3) The miRNA reference genes of Osmanthus fragrans under methyl jasmonate treatment were ofr-miR159b-3p, novel8, novel2, and novel3;

[0010] (4) The miRNA reference genes of Osmanthus fragrans under the treatments of ethephon, abscisic acid, and methyl jasmonate were ofr-miR159b-3p, novel8, and novel3;

[0011] Among them, the nucleic acid sequence of novel33 is shown as SEQ ID NO.1, the nucleic acid sequence of ofr-miR159b-3p is shown as SEQ ID NO.2, the nucleic acid sequence of novel3 is shown as SEQ ID NO.3, the nucleic acid sequence of novel8 is shown as SEQ ID NO.4, and the nucleic acid sequence of novel2 is shown as SEQ ID NO.5.

[0012] Preferably, the novel33 proprietary primer, the forward primer sequence is

[0013] 5'-ccgcgTTGAACTCGTATGCGAGCGCA-3' (SEQ ID NO. 6).

[0014] Preferably, the sequence of the proprietary primer for ofr-miR159b-3p, the forward primer, is

[0015] 5'-gcgcgcCTTTGGATTGAAGGGAGCTCT-3' (SEQ ID NO. 7).

[0016] Preferably, the novel3 proprietary primer, the forward primer sequence is

[0017] 5'-gcggcggTCAAGATTGGGCAATGAACCA-3' (SEQ ID NO. 8).

[0018] Preferably, the novel8 proprietary primer, the forward primer sequence is

[0019] 5'-gcgccgTTTCCTATTCCTCCCATACCGA-3' (SEQ ID NO. 9).

[0020] Preferably, the novel2 proprietary primer, the forward primer sequence is

[0021] 5'-ccgccgTTTCCTATACCTCCCATACCGA-3' (SEQ ID NO. 10).

[0022] Preferably, the proprietary primers described in novel33, ofr-miR159b-3p, novel3, novel8 and novel2, and the reverse primer are provided by the kit miRcute miRNA qPCR Detection Kit (SYBR Green).

[0023] Preferably, the present invention also relates to the use of the osmanthus hormone-treated miRNA internal reference gene in osmanthus miRNA fluorescence quantitative PCR analysis.

[0024] Preferably, the present invention also relates to the use of the proprietary primers for the osmanthus hormone-treated miRNA internal reference gene in osmanthus miRNA fluorescence quantitative PCR analysis.

[0025] Preferably, the proprietary primers are as described above.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] Through high-throughput sequencing, the present invention ultimately selected nine mature, abundantly expressed, and stably expressed miRNAs and five commonly used internal reference genes as candidate genes, and designed primer sequences for the internal references. Using five algorithms (delta-CT, geNorm, NormFinder, BestKeeper, and RefFinder) to evaluate the stability of the candidate genes, the authors obtained novel33 and ofr-miR159b-3p, suitable for osmanthus fragrans under ethephon treatment; novel3 and ofr-miR159b-3p, suitable for osmanthus fragrans under abscisic acid (ABA) treatment; ofr-miR159b-3p, novel8, novel2, and novel3, suitable for methyl jasmonate (MeJA) treatment; and ofr-miR159b-3p, novel8, and novel3, suitable for osmanthus fragrans under three hormone treatments. Real-time fluorescence quantitative PCR primers for internal reference genes were designed. The primers have strong specificity and high amplification efficiency, which can greatly improve the detection efficiency of osmanthus genes using real-time fluorescence quantitative detection and improve the credibility of the detection results. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the CQ value of the 14 screened miRNA fluorescence quantitative internal reference genes;

[0029] Figure 2 It is the optimal number of internal reference genes determined by geNorm for accurate quantitative analysis;

[0030] Figure 3 This is a ranking diagram of the expression stability values ​​(M) of 14 internal reference genes using geNorm software. The lower the stability value, the more stable the gene expression.

[0031] Figure 4 The stable gene and the unstable gene were used as internal reference genes, and the expression level of ofr-miR166e-5p was used;

[0032] Figure 5 The stable gene and the unstable gene were used as internal reference genes, and the expression level of ofr-miR396b-3p was measured. DETAILED DESCRIPTION

[0033] The technical solution of the present invention will be further described in detail below in conjunction with specific implementation methods.

[0034] Unless otherwise specified, the experimental methods used in the examples of the present invention are all conventional methods.

[0035] Unless otherwise specified, the materials, reagents, etc. used in the examples of the present invention can be obtained from commercial sources.

[0036] The test material used in the following examples is Osmanthus fragrans. Osmanthus fragrans was selected from the "Chang'e" asexual line in good growth condition and free of pests and diseases in Xianning City, Hubei Province as the mother tree. In May 2023, semi-lignified branches of the current year with 2-3 lateral buds were cut as cuttings (12-16 cm). The upper incision was cut flat and the lower incision was cut at a 45° angle. The cuttings were soaked in distilled water for 12 hours, soaked in 1% sodium hypochlorite for 10 minutes, rinsed with distilled water three times, and then treated with 0.1g L -1 Soak GGR rooting powder for 4 hours. The cuttings were planted in the Osmanthus fragrans base in Xianning City, Hubei Province. The soil matrix was a mixed matrix composed of peat, perlite, vermiculite and yellow sand at a ratio of 1:1:1:1, and water was sprinkled twice a week. In April 2024, the Osmanthus fragrans cuttings were treated with 5mM ethephon, 300μM ABA and 300μM MeJA, respectively, with 200mL of treatment solution per seedling. Osmanthus fragrans was exposed to 23°C, a photoperiod (12-h light / 12-h dark cycle) and a light incubator with 60% humidity, and samples were taken at 0, 3, 6, 12, 24 and 72h of extended stress period. Each treatment was repeated three times, frozen with liquid nitrogen, and stored at -80°C for further analysis.

[0037] Example 1

[0038] 1. Screening of candidate reference genes and design of their primers

[0039] Through literature review, database search and high-throughput sequencing, 9 mature, abundantly expressed and stably expressed miRNAs and 5 commonly used reference genes were selected as candidate genes, namely ofr-miR159b-3p, ofr-miR168b-5p, ofr-miR171a-3p, ofr-miR395e, ofr-miR403-3p, novel2, novel3, novel8, novel33, 18S, ACT11, TUA5, U6 and UBQ4.

[0040] 18S, ACT11, TUA5, U6 and UBQ4 internal reference primers were designed using Primer5.0; miRNA upstream primers were based on mature miRNA sequences, with U replaced by T, and the appropriate T was achieved by adding or removing bases at both ends of the primers. m The value is 65°C, the GC content is 40-60%, and the reverse primer is provided by the kit miRcute miRNA qPCR Detection Kit (SYBR Green). The candidate internal control and its primers are shown in Table 1 below:

[0041] Table 1 14 candidate reference genes and their primer sequences

[0042]

[0043]

[0044] Note: E, PCR efficiency; R2, Correlation coefficient

[0045] 2. Total RNA extraction and cDNA preparation from Osmanthus fragrans

[0046] miRNA was extracted using an miRNA isolation kit (Tiangen Biotechnology, Beijing, China), and its integrity, purity, and concentration were determined by 1.0% (w / v) agarose gel electrophoresis and NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA), respectively.

[0047] The synthesized samples were reverse transcribed using the tailing method. Specifically, 0.8 μg of miRNA was used to synthesize the first-strand miRNA cDNA according to the instructions of the miRcute miRNA First-Strand cDNA Synthesis Kit (KR201, Tianjin Biotech, China). The reaction was carried out at 42°C for 60 minutes. The sample was then inactivated by heating at 95°C for 3 minutes. The synthesized cDNA reaction was immediately stored at -20°C.

[0048] 3. qRT-PCR quantification

[0049] A 20 μL PCR reaction system was prepared 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 of each forward and reverse primer (10 μM), 2 μL of 10-fold diluted miRNA first-strand cDNA, and 7.2 μL of RNase-free ddH2O. Three technical replicates were performed for each sample. A negative control without template was run for each candidate reference gene to verify the amplification background.

[0050] qRT-PCR was performed using the Tianlong Gentier 96E system (Tianlong Technology Co., Ltd., Xi'an, China) with the following protocol: initial denaturation at 95°C for 15 minutes, followed by 40 cycles of denaturation at 94°C for 20 seconds and annealing and extension at 60°C for 30 seconds, followed by a 60-95°C melting curve. CT values ​​were obtained by qRT-PCR and are inversely proportional to gene expression: higher CT values ​​indicate lower gene expression, while lower CT values ​​indicate higher gene expression.

[0051] from Figure 1 It can be seen that under abscisic acid (ABA) treatment, the CT value of novel3 changed the least, which was 1.711; under ethephon treatment, the CT value of novel3 changed the least, which was 1.540; under methyl jasmonate (MeJA) treatment, the CT value of novel3 changed the least, which was 1.266; under hormone treatment, the CT value of ofr-miR159b-3p changed the least, which was 2.304.

[0052] 4. Stability assessment

[0053] Four different algorithms, including delta-Ct, geNorm, NormFinder, and BestKeeper, were used to statistically analyze the expression stability of internal reference genes in different samples. The results were comprehensively analyzed using the RefFinder website to screen out stable internal reference genes.

[0054] 4.1 Evaluation Results

[0055] 4.1.1 Delta-CT Analysis

[0056] The standard deviation average was obtained by delta-CT analysis. The higher the standard deviation average, the worse the gene stability. The results are shown in Table 2. Under ethephon treatment, the standard deviation average of novel33 was 0.94, which was the most stable gene; under ABA treatment, the standard deviation averages of novel3 and novel33 were 1.29, which were the most stable genes; under MeJA treatment and hormone treatment, ofr-miR159b-3p was the most stable gene.

[0057] Table 2Delta-CT analysis results

[0058]

[0059]

[0060] 4.1.2 geNorm software analysis

[0061] When using geNorm, convert the raw Ct values ​​by 2 -ΔCt (delta Ct = original Ct value - lowest Ct value in this group) was used to analyze the expression stability of the internal reference gene. The expression stability M value of each candidate internal reference gene was calculated by geNorm software. The larger the M value, the lower the stability, with M = 1.5 being the upper limit. In addition, geNorm can be used to calculate the expression stability of the internal reference gene. -ΔCt For the original data, the paired difference value V of two continuous standardized factors is calculated n / V n+1 To determine the appropriate number of internal reference genes, under ABA and ethephon treatment, since the paired variation value V2 / V3 of the embodiment is less than 0.15 (see Figure 2 ), so only two internal references are needed for relative gene expression analysis in Osmanthus fragrans ABA (novel3 and ofr-miR403-3p); only two internal references are needed for relative gene expression analysis under ethephon treatment (novel33 and ofr-miR395e) (see Figure 3 ). Under MeJA treatment, since the paired variation value V4 / V5 in the embodiment is less than 0.15, under hormone treatment, V3 / V4 is less than 0.15 (see Figure 2 ), therefore, only four internal reference genes (novel8, novel2, ofr-miR159b-3p, and ofr-miR168b-5p) were needed for relative gene expression analysis under Osmanthus fragrans MeJA treatment; three internal reference genes (novel8, novel2, and ofr-miR159b-3p) were needed under Osmanthus fragrans hormone treatment (see Figure 3 ).

[0062] 4.1.3 NormFinder software analysis

[0063] When using NormFinder, convert the raw Ct values ​​by 2 -ΔCt The delta Ct value (original Ct value minus the lowest Ct value in the group) was then used to analyze the expression stability of the reference genes. NormFinder software was used to calculate the stability values ​​of the candidate reference genes. Lower stability values ​​indicate better stability, with the gene with the lowest stability value being the most stable. The results are shown in Table 3. Under ethephon and ABA treatments, the most stable gene was novel33, while under MeJA and hormone treatments, the most stable gene was ofr-miR159b-3p.

[0064] Table 3 NormFinder software analysis results

[0065]

[0066]

[0067] 4.1.4 BestKeeper Software Analysis

[0068] For BestKeeper, the amplification efficiency and raw Ct values ​​calculated by the LinRegPCR program from the raw Ct values ​​were used to calculate the coefficient of variation (CV) and standard deviation (SD) of candidate reference gene expression. Smaller SD values ​​indicate more stable reference genes. If the SD value is >1.0, the reference gene is considered unstable and should not be used for gene expression normalization. As shown in Table 4, under ethephon treatment, the gene stability order was novel3 > U6 > ofr-miR159b-3p > novel33 > ofr-miR171a-3p > novel8 > ofr-miR395e > ofr-miR403-3p > ofr-miR168b-5p > novel2 > 18S > UBQ4 > ACT11 > TUA5, with novel3 being the most stable gene. Similarly, novel3 was the most stable gene under MeJA and hormone treatments. Under ABA treatment, ofr-miR159b-3p was the most stable gene.

[0069] Table 4 BestKeeper software analysis results

[0070]

[0071]

[0072] 4.1.5 RefFinder URL Analysis

[0073] To verify the accuracy of the internal reference gene screening, we used RefFinder to comprehensively rank the stability of candidate internal reference genes. The lower the comprehensive ranking, the more stable the gene expression. The results are shown in Table 5. Under ethephon treatment, the gene stability order was novel33 > ofr-miR159b-3p > ofr-miR395e > novel3 > ofr-miR168b-5p > U6 > novel8 > ofr-miR171a-3p > novel2 > ofr-miR403-3p > 18S > UBQ4 > TUA5 > ACT11. Among them, novel33 + ofr-miR159b-3p was the most stable internal reference combination, and ACT11 was the least stable gene. The stability of genes under ABA treatment was novel3>ofr-miR159b-3p>ofr-miR403-3p>novel33>novel8>U6>novel2>ofr-miR168b-5p>ofr-miR171a-3p>ofr-miR395e>18S>UBQ4>ACT11>TUA5, among which novel3+ofr-miR159b-3p was the most stable internal reference combination, and TUA5 was the most unstable gene. Under MeJA treatment, the stability of genes was ofr-miR159b-3p>novel8>novel2>novel3>ofr-miR403-3p>U6>ofr-miR168b-5p>ofr-miR395e>ofr-miR171a-3p>18S>TUA5>novel33>UBQ4>ACT11, among which ofr-miR159b-3p+novel8+novel2+novel3 was the most stable internal reference combination, and ACT11 was the least stable gene. Under hormone treatment, the stability of genes was ofr-miR159b-3p>novel8>novel3>novel2>ofr-miR403-3p>U6>ofr-miR168b-5p>ofr-miR395e>ofr-miR171a-3p>novel33>18S>TUA5>ACT11>UBQ4, among which ofr-miR159b-3p+novel8+novel3 was the most stable internal reference combination, and UBQ4 was the most unstable gene.

[0074] Table 5 RefFinder website analysis results

[0075]

[0076]

[0077] 4.2 Verification of internal reference gene stability

[0078] Based on our selection throughput sequencing results, ofr-miR166e-5p and ofr-miR396b-3p were selected as target genes to confirm the suitability of the candidate genes evaluated in this study. -△△Ct Methods Calculate the expression of target genes. Under ABA treatment, it was found that when the stable genes novel3 and ofr-miR159b-3p were used as internal references, the change trend of target gene expression was basically the same, that is, the target gene expression level was the highest at 72 hours; however, when the unstable gene TUA was used as the internal reference, the target gene expression level increased significantly at 3 hours, 24 hours, and 72 hours ( Figure 4 a. Figure 5 a). Similarly, under the treatment of ethephon and MeJA, with the stable gene as the internal reference, the change trend of the target gene expression was basically the same. However, when the unstable gene ACT11 was used as the internal reference, the target gene was significantly increased at 24h under the treatment of ethephon ( Figure 4 b. Figure 5 b); The target gene was significantly increased at 6 h after MeJA treatment ( Figure 4 c. Figure 5 c) In summary, the expression profiles and trends of target genes differ when using stable and unstable genes as internal references. Therefore, choosing the right gene can significantly influence target gene expression.

[0079] 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 in the scope of protection of the present invention.

Claims

1. Application of internal reference genes in miRNA fluorescence quantitative PCR analysis under osmanthus hormone treatment, characterized in that, (1) When the hormone is ethephon, the internal reference genes are novel33 and ofr-miR159b-3p; (2) When the hormone was abscisic acid, the reference genes were novel3 and ofr-miR159b-3p; (3) When the hormone was methyl jasmonate, the reference genes were ofr-miR159b-3p, novel8, novel2, and novel3; (4) When the hormones were ethephon, abscisic acid, and methyl jasmonate, the internal reference genes were ofr-miR159b-3p, novel8, and novel3; Among them, the nucleic acid sequence of novel33 is shown as SEQ ID NO.1, the nucleic acid sequence of ofr-miR159b-3p is shown as SEQ ID NO.2, the nucleic acid sequence of novel3 is shown as SEQ ID NO.3, the nucleic acid sequence of novel8 is shown as SEQ ID NO.4, and the nucleic acid sequence of novel2 is shown as SEQ ID NO.

5.

2. Use of the proprietary primers for detecting the internal reference gene according to claim 1 in miRNA fluorescence quantitative PCR analysis under osmanthus hormone treatment, characterized in that: (1) When the hormone is ethephon, the internal reference genes are novel33 and ofr-miR159b-3p; (2) When the hormone was abscisic acid, the reference genes were novel3 and ofr-miR159b-3p; (3) When the hormone was methyl jasmonate, the reference genes were ofr-miR159b-3p, novel8, novel2, and novel3; (4) When the hormones were ethephon, abscisic acid, and methyl jasmonate, the internal reference genes were ofr-miR159b-3p, novel8, and novel3; Among them, the nucleic acid sequence of novel33 is shown in SEQ ID NO.1, the nucleic acid sequence of ofr-miR159b-3p is shown in SEQ ID NO.2, the nucleic acid sequence of novel3 is shown in SEQ ID NO.3, the nucleic acid sequence of novel8 is shown in SEQ ID NO.4, and the nucleic acid sequence of novel2 is shown in SEQ ID NO.5; The reverse primer of the proprietary primer is provided by the kit miRcute miRNA qPCR Detection Kit (SYBRGreen).

3. The use according to claim 2, characterized in that For the proprietary primers used to detect novel33, the forward primer had the sequence 5′-ccgcgTTGAACTCGTATGCGAGCGCA-3′; Proprietary primers for detecting ofr-miR159b-3p: the forward primer had the sequence 5′-gcgcgcCTTTGGATTGAAGGGAGCTCT-3′; For the proprietary primers used to detect novel3, the forward primer had the sequence 5′-gcggcggTCAAGATTGGGCAATGAACCA-3′; For the proprietary primers used to detect novel8, the forward primer had the sequence 5′-gcgccgTTTCCTATTCCTCCCATACCGA-3′; Proprietary primers for novel2 detection: the forward primer has the sequence 5'-ccgccgTTTCCTATACCTCCCATACCGA-3'.

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