Osmanthus fragrans hormone-treated miRNA reference gene as well as primer and application thereof

Abstract

The invention discloses a hormone-treated osmanthus fragrans miRNA reference gene as well as a primer and application thereof. According to the present invention, the optimal miRNA internal reference gene under hormone treatment is obtained, the miRNA internal reference under ethephon treatment is novel33 and ofr-miR159b-3p, the miRNA internal reference under ABA treatment is novel3 and ofr-miR159b-3p, the miRNA internal reference under MeJA treatment is ofr-miR159b-3p, novel8, novel2 and novel3, the miRNA internal reference under co-treatment of the three hormones is ofr-miR159b-3p, novel8 and novel3, and the current situation that the osmanthus fragrans miRNA fluorescent quantitative internal reference does not exist under the treatment of different hormones is filled with the PCR primer; powerful support is provided for expression quantification of related miRNA under treatment of different hormones of osmanthus fragrans.

Description

Technical Field

[0001] The 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) belongs to the Oleaceae family. Sweet Osmanthus is a common woody ornamental flower, known for its rich fragrance and beautiful flowers. The flowering period, fragrance and color of sweet osmanthus are affected by many factors, among which hormone treatment is an effective means of regulation. Hormone treatment can affect the growth, development and flowering process of sweet osmanthus by regulating the level of endogenous hormones in plants. However, the physiological and molecular mechanisms inside the plant after hormone treatment are complex and changeable, so accurate internal references are needed for quantitative analysis of miRNA expression.

[0003] When conducting gene expression analysis, selecting appropriate internal references is the key to ensuring data accuracy and reliability. Internal reference genes are usually some genes that are stably expressed under different treatment conditions, and they are used to correct technical errors in experiments. However, the expression stability of internal reference genes may vary under different treatment conditions. Therefore, when conducting gene expression studies on osmanthus hormone treatment, it is necessary to screen out suitable miRNA internal references to ensure the accuracy and reliability of qRT-PCR data. Hormone treatment can cause complex physiological changes inside osmanthus plants, including changes in gene expression patterns. Traditionally used internal reference genes, such as Actin and GAPDH, may no longer be stable after hormone treatment, and gene internal references are not suitable for miRNA internal references. Therefore, research specifically targeting osmanthus hormone treatment requires experimental screening and verification to find miRNA internal references that are stably expressed under different hormone treatment conditions. This not only helps to improve the credibility of the research results, but also provides a solid foundation for subsequent osmanthus miRNA function studies. Summary of the invention

[0004] In view of the above-mentioned defects of the prior art, the present invention proposes miRNA internal reference genes and primers and applications thereof under osmanthus hormone treatment 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] The miRNA internal reference gene is treated with osmanthus hormone, wherein the miRNA internal 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 internal reference genes of Osmanthus fragrans miRNA under methyl jasmonate treatment were ofr-miR159b-3p, novel8, novel2, and novel3;

[0010] (4) The internal reference genes of Osmanthus fragrans miRNA 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 of 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 application 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 of 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] The present invention uses high-throughput sequencing to finally select 9 mature, relatively abundant and stable miRNAs and 5 commonly used internal reference genes as candidate genes, and designs primer sequences for internal references. The stability of the candidate genes is evaluated by 5 algorithms (delta-CT, geNorm, NormFinder, BestKeeper and RefFinder), and the miRNA internal references novel33 and ofr-miR159b-3p suitable for osmanthus ethephon treatment; the miRNA internal references novel3 and ofr-miR159b-3p suitable for osmanthus under abscisic acid ABA treatment; the miRNA internal references ofr-miR159b-3p, novel8, novel2 and novel3 suitable for osmanthus under methyl jasmonate MeJA treatment; and the miRNA internal references ofr-miR159b-3p, novel8 and novel3 suitable for osmanthus under three hormone treatments are obtained. 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 miRNA fluorescence quantitative internal reference genes screened;

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

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

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

[0032] Figure 5 The stable gene and 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 is further described in detail below in conjunction with specific implementation methods.

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

[0035] Unless otherwise specified, the materials, reagents, etc. used in the embodiments 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 Xianning, Hubei Province, which was in good growth condition and free of pests and diseases 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, the lower incision was cut at 45°, soaked in distilled water for 12 hours, soaked in 1% sodium hypochlorite for 10 minutes, rinsed with distilled water 3 times, and then treated with 0.1g L -1 Soak in GGR rooting powder for 4 hours. The cuttings were planted in the osmanthus base in Xianning City, Hubei Province. The soil matrix was a mixed matrix composed of peat, perlite, vermiculite, and yellow sand in a ratio of 1:1:1:1, and water was sprinkled twice a week. In April 2024, osmanthus cuttings were treated with 5mM ethephon, 300μM ABA, and 300μM MeJA, respectively, with 200mL of treatment solution per seedling. Osmanthus was exposed to 23°C, a photoperiod (12-h light / 12-h dark cycle), and cultured in a light incubator with 60% humidity. 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 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] Primer5.0 was used to design the internal reference primers for 18S, ACT11, TUA5, U6 and UBQ4; the upstream primer of miRNA was based on the mature miRNA sequence, with U replaced by T, and the appropriate T was achieved by adding or removing bases at both ends of the primer. 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 reference 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 1.0% (w / v) agarose gel electrophoresis and NanoDrop2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) were used to detect its integrity, purity, and concentration, respectively.

[0047] The synthesized samples were reverse transcribed by tailing method, i.e., 0.8ug miRNA was synthesized by referring to the instructions of miRcute miRNA First-Strand cDNA Synthesis Kit (KR201, Tiangen Biotech, China). The reaction was carried out at 42°C for 60 minutes; then the sample was incubated at 95°C for 3 minutes for enzyme inactivation reaction. The synthesized cDNA reaction solution was immediately stored at -20°C.

[0048] 3. qRT-PCR quantification

[0049] According to the instructions of miRcute Plus miRNAqPCR Detection Kit (SYBRGreen), a 20 μL PCR reaction system was prepared: 10 μL 2×miRcute Plus miRNAPremix (SYBR&ROX), 0.4 μL each of forward and reverse primers (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, and a negative control without template was added for each candidate internal reference gene to verify the background of amplification.

[0050] qRT-PCR was amplified using the Tianlong Gentier 96E system (Tianlong Technology Co., Ltd., Xi'an, China) with the following procedure: 95°C pre-denaturation for 15 min; followed by 40 cycles of 94°C denaturation for 20 s, 60°C annealing and extension for 30 s; and then a 60-95°C melting curve. The CT value was obtained by qRT-PCR, and the CT value was inversely proportional to the expression level of the gene. The larger the CT value, the lower the expression level of the gene. Conversely, the smaller the CT value, the higher the expression level of the gene.

[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 through the RefFinder website to screen out stable internal reference genes.

[0054] 4.1 Evaluation Results

[0055] 4.1.1delta-CT analysis

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

[0057] Table 2 Delta-CT analysis results

[0058]

[0059]

[0060] 4.1.2geNorm software analysis

[0061] When using geNorm, the raw Ct values ​​are converted by 2 -ΔCt (delta Ct = original Ct value - lowest Ct value in this group) is then used to analyze the expression stability of the internal reference gene. The expression stability M value of each candidate internal reference gene is calculated by geNorm software. The larger the M value, the lower the stability, where M = 1.5 is the upper limit. In addition, geNorm can be used with 2 -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 ), so 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 ​​2 -ΔCt (delta Ct = original Ct value - lowest Ct value in this group) was then used for expression stability analysis of internal reference genes. The stability values ​​of candidate internal reference genes were calculated using NormFinder software. The lower the stability value, the better the stability, that is, the gene with the smallest stability value was the most stable gene. The results are shown in Table 3. Under ethephon and ABA treatments, the most stable gene was novel33, and under MeJA and hormone treatments, the most stable gene was ofr-miR159b-3p.

[0064] Table 3 NormFinder software analysis results

[0065]

[0066]

[0067] 4.1.4BestKeeper Software Analysis

[0068] For BestKeeper, the amplification efficiency and original Ct value calculated by the LinRegPCR program based on the original Ct value were used to calculate the coefficient of variation (CV) and standard deviation (SD) of the expression of the candidate reference gene. The smaller the SD value, the more stable the reference gene. If the SD value is greater than 1.0, the reference gene is considered unstable and should be avoided for gene expression standardization. The results are shown in Table 4. Under ethephon treatment, the stability of the genes was novel3>U6>ofr-miR159b-3p>novel33>ofr-miR171a-3p>novel8>ofr-miR395e>ofr-miR403-3p>ofr-miR168b-5p>novel2>18S>UBQ4>ACT11>TUA5, among which the most stable gene was novel3. Similarly, under MeJA treatment and hormone treatment, the most stable gene was also novel3. Under ABA treatment, the most stable gene was ofr-miR159b-3p.

[0069] Table 4 BestKeeper software analysis results

[0070]

[0071]

[0072] 4.1.5 RefFinder website analysis

[0073] To verify the accuracy of the internal reference gene screening, we used RefFinder to comprehensively rank the stability of the candidate internal reference genes. The smaller the comprehensive ranking, the better the gene expression stability. The results are shown in Table 5. Under the treatment of ethephon, the stability of the genes 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 which novel33+ofr-miR159b-3p was the most stable internal reference combination, and ACT11 was the most unstable 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 most unstable 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 72h; but when the unstable gene TUA was used as the internal reference, the target gene expression level increased significantly at 3h, 24h and 72h ( 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, while when the unstable gene ACT11 was used as the internal reference, the target gene under the treatment of ethephon increased significantly at 24h ( 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 status and expression trend of target genes are different when stable and unstable genes are used as internal references. Therefore, choosing the right gene has a great influence on the expression of the target gene.

[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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Osmanthus fragrans miRNA internal reference gene under hormone treatment, characterized in that, The miRNA reference gene includes at least one of the following (1) to (4): (1) The miRNA reference genes of Osmanthus fragrans under ethephon treatment were novel33 and ofr-miR159b-3p; (2) The internal reference genes of Osmanthus fragrans miRNA under abscisic acid treatment were novel3 and ofr-miR159b-3p; (3) The internal reference genes of Osmanthus fragrans miRNA under methyl jasmonate treatment were ofr-miR159b-3p, novel8, novel2, and novel3; (4) The internal reference genes of Osmanthus fragrans miRNA under the treatments of ethephon, abscisic acid and methyl jasmonate 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. A proprietary primer for detecting novel33 according to claim 1, characterized in that: The sequence of the forward primer was 5′-ccgcgTTGAACTCGTATGCGAGCGCA-3′.

3. A proprietary primer for detecting ofr-miR159b-3p according to claim 1, characterized in that: The sequence of the forward primer was 5′-gcgcgcCTTTGGATTGAAGGGAGCTCT-3′.

4. A proprietary primer for detecting novel3 according to claim 1, characterized in that: The sequence of the forward primer was 5′-gcggcggTCAAGATTGGGCAATGAACCA-3′.

5. A proprietary primer for detecting novel8 according to claim 1, characterized in that: The sequence of the forward primer was 5′-gcgccgTTTCCTATTCCTCCCATACCGA-3′.

6. A proprietary primer for detecting novel2 according to claim 1, characterized in that: The sequence of the forward primer was 5′-ccgccgTTTCCTATACCTCCCATACCGA-3′.

7. The proprietary primer according to any one of claims 2 to 6, characterized in that: The reverse primer was provided by the kit miRcutemiRNA qPCR Detection Kit (SYBR Green).

8. Application of sweet osmanthus miRNA internal reference gene in sweet osmanthus miRNA fluorescence quantitative PCR analysis under hormone treatment according to claim 1.

9. Use of proprietary primers of sweet osmanthus miRNA internal reference gene under hormone treatment according to claim 1 in sweet osmanthus miRNA fluorescence quantitative PCR analysis.

10. The use according to claim 9, characterized in that: The proprietary primer is as shown in any one of claims 2 to 6.

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