miRNA reference genes, primers, and applications during the opening and senescence of osmanthus petals

Abstract

The present invention provides miRNA internal reference genes and primers thereof and applications during the opening and aging of sweet osmanthus petals, belonging to the field of plant molecular biology. The petals opening and aging process include S1 (spiritual stem stage), S2 (initial flowering stage), S3 (initial flowering stage), S4 (full flowering stage), S5 (late flowering stage) and S6 (petal shedding stage). The present invention screened 14 candidate internal reference genes, evaluated the stability of the candidate genes by 5 algorithms (delta-CT, geNorm, NormFinder, BestKeeper and RefFinder), and obtained miRNA internal references suitable for the opening and aging of sweet osmanthus petals, which are novel33 and ofr-miR395e. The present invention designs real-time fluorescence quantitative PCR primers for internal references, which have strong primer specificity and high amplification efficiency, can greatly improve the detection efficiency when using real-time fluorescence quantitative detection of sweet osmanthus miRNA, and improve the credibility of the test results.

Description

Technical Field

[0001] The present invention relates to the field of plant molecular biology, and in particular to miRNA internal reference genes in the process of opening and senescence of osmanthus flower petals, primers thereof, and applications thereof. Background Art

[0002] Sweet osmanthus (Osmanthus fragrans) is widely loved for its unique fragrance and beautiful flowers. The opening and aging of osmanthus petals are regulated by complex physiological and molecular mechanisms, among which microRNA (miRNA) plays a key role in regulating gene expression. MiRNAs are a class of small, non-coding RNA molecules that can regulate gene expression by binding to target gene mRNAs, thereby influencing plant growth and development, flowering regulation, and aging. Therefore, studying the role of miRNAs in the opening and aging of osmanthus petals will help to reveal their molecular regulatory mechanisms.

[0003] When performing miRNA expression analysis, selecting appropriate internal reference miRNAs is crucial for ensuring data accuracy and reliability. Internal reference miRNAs are miRNAs that are stably expressed under various experimental conditions and are used to correct for technical errors in experiments. During the opening and aging of osmanthus petals, miRNA expression levels can fluctuate significantly. Therefore, traditional internal reference miRNAs may no longer be suitable, necessitating the screening of new, stably expressed internal reference miRNAs tailored to this process. Summary of the Invention

[0004] In response to the above-mentioned problems existing in the prior art, the first technical problem to be solved by the present invention is to provide an internal miRNA reference during the opening and aging of osmanthus petals; the second technical problem to be solved by the present invention is to provide a special primer for the internal miRNA reference during the opening and aging of osmanthus petals; the third technical problem to be solved by the present invention is to provide the application of the above-mentioned internal reference gene or special primer in the fluorescence quantification of osmanthus.

[0005] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0006] The application of novel33 and ofr-miR395e as miRNA reference genes in fluorescence quantitative analysis of the opening and senescence of Osmanthus fragrans petals. The nucleotide sequence of novel33 is shown in SEQ ID NO. 1, and the nucleotide sequence of ofr-miR395e is shown in SEQ ID NO. 2. T in the sequence listing represents the RNA base U.

[0007] Furthermore, the present invention provides primers for detecting the internal reference gene, wherein:

[0008] The primer sequences for novel33 are:

[0009] novel33 forward primer: 5′-ccgcgTTGAACTCGTATGCGAGCGCA-3′ (SEQ ID NO. 3);

[0010] The primer sequences for miR-miR395e are as follows:

[0011] ofr-miR395e forward primer: 5′-ccggcCTGAAGTGTTTGGGGGAACTC-3′ (SEQ ID NO. 4).

[0012] The reverse primer was provided by the miRcute miRNA qPCR detection kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., Beijing, China).

[0013] Furthermore, the present invention also relates to the application of the above primers in fluorescence quantitative analysis of the opening and aging process of osmanthus flower petals.

[0014] Furthermore, the petal opening and aging process includes: the stem stage, the initial flowering stage, the early flowering stage, the full flowering stage, the late flowering stage and the shedding stage.

[0015] Furthermore, the application includes the following steps:

[0016] qRT-PCR detection was performed using cDNA of Osmanthus fragrans petals during opening and senescence as templates, novel33 and ofr-miR395e as internal reference genes, and the aforementioned primers.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention compares internal reference genes (18S, ACT, TUA, U6, and UBQ) used in other species with the whole genome data of Osmanthus fragrans by local blast comparison (blastVer: 2.4.0+) to identify genes with the highest homology. The conserved domains and ORFs of the genes are then analyzed online using blastx on the NCBI website. Nine miRNAs with high expression levels and relatively stable expression during the opening and aging of Osmanthus fragrans petals are selected through small RNA sequencing. The stability of 14 candidate genes is evaluated using five algorithms (delta-CT, geNorm, NormFinder, BestKeeper, and RefFinder). Consequently, novel33 and ofr-miR395e, miRNA internal references suitable for fluorescence quantitative analysis during the opening and aging of Osmanthus fragrans petals, are obtained. Real-time fluorescence quantitative PCR primers for these internal references are designed. These primers have strong specificity and high amplification efficiency, significantly improving the detection efficiency of Osmanthus fragrans genes using real-time fluorescence quantitative analysis and enhancing the reliability of the test results. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the CQ value graph of 14 internal reference genes screened during the opening and senescence of Osmanthus fragrans petals;

[0020] Figure 2 This is the ranking diagram of the expression stability values ​​(M) of 14 internal reference genes using geNorm software;

[0021] Figure 3 The optimal number of internal reference genes for accurate quantitative analysis during the opening and senescence of Osmanthus fragrans petals was determined by geNorm;

[0022] Figure 4 The expression level of ofr-miR166e-5p during the opening and senescence of sweet osmanthus petals is measured using stable and unstable genes as internal reference genes.

[0023] Figure 5 It is the expression level of ofr-miR396b-3p, with stable genes and unstable genes serving as internal reference genes, during the opening and aging of osmanthus petals. DETAILED DESCRIPTION

[0024] In order to better understand the technical content of the present invention, specific examples are provided below to further illustrate the present invention.

[0025] Unless otherwise specified, the experimental methods used in the examples of the present invention are all conventional methods.

[0026] Unless otherwise specified, the materials, reagents, etc. used in the examples of the present invention can be obtained from commercial sources.

[0027] The following examples used a healthy, pest-free Osmanthus fragrans plant from Huazhong Agricultural University as the experimental material. Petal tissue was collected at 10:00 AM at six stages: S1 (stalk stage), S2 (early flowering stage), S3 (early blooming stage), S4 (full blooming stage), S5 (late blooming stage), and S6 (abscissing stage). Samples from each stage were collected using three biological replicates, flash-frozen in liquid nitrogen, and stored at -80°C for subsequent analysis.

[0028] Example 1

[0029] 1. Total RNA extraction and cDNA synthesis

[0030] miRNA was extracted using an miRNA isolation kit (Tiangen Biotechnology, Beijing, China), and its integrity, purity, and concentration were determined by 1% agarose gel electrophoresis and spectrophotometry (NanoDrop 2000, Thermo Scientific, Wilmington, DE, USA).

[0031] The synthesized samples were reverse transcribed using the tailing method. Using total RNA as a template, 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 solution was immediately stored at -20°C until use.

[0032] 2. Selection of internal reference genes and design of their primers

[0033] By performing small RNA sequencing on Osmanthus fragrans, nine mature, abundantly expressed, and stably expressed miRNAs (ofr-miR159b-3p, ofr-miR168b-5p, ofr-miR171a-3p, ofr-miR395e, ofr-miR403-3p, novel2, novel3, novel8, and novel33) were selected. Five commonly used reference genes (18S, ACT11, TUA5, U6, and UBQ4) were selected as candidate genes through literature review and database comparison.

[0034] The upstream miRNA primers were based on mature miRNA sequences, with U replaced by T. Bases were added or removed at both ends to achieve an appropriate Tm of 65°C and a GC content of 40%-60%. The downstream primers were provided by the miRcute miRNA qPCR detection kit (SYBR Green) (Tiangen). Gene primers were designed using Primer5, with a Tm of 58-62°C and a GC content of 40%-60%.

[0035] The 14 candidate reference genes and primer sequences are shown in Table 1.

[0036] Table 1 14 candidate reference genes and primer sequences

[0037]

[0038]

[0039] Note: E, amplification efficiency; R 2 Correlation coefficient.

[0040] 3. qRT-PCR quantification

[0041] qRT-PCR was performed using the Tianlong Gentier 96E system (Tianlong Technology Co., Ltd., Xi'an, China) with the following program: 15 min at 95°C, followed by 40 cycles with parameters of 20 s at 94°C and 30 s at 60°C, followed by generation of a melting curve at 60–95°C.

[0042] The reaction system was as follows: 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 biological and technical replicates were performed for each sample. A negative control without template was run for each candidate internal reference gene to verify background amplification.

[0043] 4. CT value

[0044] The CT value was obtained by qRT-PCR. The CT value was inversely proportional to the gene expression level. The larger the CT value, the lower the gene expression level. Conversely, the smaller the CT value, the higher the gene expression level.

[0045] Gene box plots such as Figure 1As shown in the figure, during the opening and senescence of Osmanthus fragrans petals, the average CT values ​​of the 14 reference genes ranged from 16.444 (18S) to 25.750 (UBQ4), with 18S rRNA expression being the highest and UBQ4 expression being the lowest. novel33 had the smallest CT change (1.000), while UBQ4 had the largest CT change (3.727).

[0046] 5. Stability assessment

[0047] Internal reference gene stability analysis uses five algorithms: delta-CT, geNorm, NormFinder, BestKeeper, and RefFinder to comprehensively analyze the expression stability of internal reference genes and screen for stable internal reference genes.

[0048] 6. Results

[0049] Delta-CT analysis: Delta-CT analysis was used to obtain the standard deviation average. The higher the standard deviation average, the worse the gene stability. Conversely, the lower the standard deviation average, the higher the gene stability. The results are shown in Table 2. During the opening and aging of osmanthus petals, novel33 was the most stable gene, while ofr-miR171a-3p was the least stable gene.

[0050] Table 2Delta-CT analysis results

[0051]

[0052] geNorm software analysis: 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; conversely, the smaller the M value, the higher the stability, where M = 1.5 is the upper limit. In addition, according to the geNorm pairwise variation value V n / V n+1 The appropriate number of internal reference genes was determined by analysis. Since the paired variation value V2 / V3 during the opening and aging of Osmanthus fragrans petals was less than 0.15 ( Figure 2 ), so only two internal reference genes (novel8 and novel2) are needed to analyze the relative expression of genes during the opening and senescence of osmanthus petals ( Figure 3 ).

[0053] NormFinder software analysis: The stability values ​​of candidate reference genes were calculated using NormFinder software. Higher stability values ​​indicate poorer stability, while lower stability values ​​indicate better stability. The gene with the lowest stability value is considered the most stable gene. The results are shown in Table 3. During the opening and senescence of Osmanthus petals, novel33 was the most stable gene, while ofr-miR171a-3p was the least stable.

[0054] Table 3 NormFinder software analysis results

[0055]

[0056] BestKeeper Software Analysis: BestKeeper software was used to calculate the standard deviation (SD) of candidate reference genes. Smaller SD values ​​indicate more stable expression. The program's default threshold is 1; when the SD value is greater than 1, the gene's expression is considered unstable. The results are shown in Table 4. novel33 was the most stably expressed gene during the opening and senescence of osmanthus petals.

[0057] Table 4 BestKeeper software analysis results

[0058]

[0059]

[0060] RefFinder analysis: The geometric mean of the stability rankings obtained from the above-mentioned analyses was calculated using the RefFinder website. A smaller geometric mean indicates more stable expression; conversely, a larger geometric mean indicates more unstable expression. The results are shown in Table 5. The stability of genes during petal opening and senescence in Osmanthus fragrans was novel33 > ofr-miR395e > novel8 > ofr-miR168b-5p > novel2 > ofr-miR159b-3p > U6 > TUA5 > ofr-miR403-3p > novel3 > 18S > UBQ4 > ACT11 > ofr-miR171a-3p. Among them, novel33 and ofr-miR395e were the most stable gene combination, while ofr-miR171a-3p, with a geometric mean of 14.00, was the least stable gene.

[0061] Table 5 RefFinder website analysis results

[0062]

[0063] 7. Verification of internal reference gene stability

[0064] Based on the high-throughput sequencing results, ofr-miR166e-5p and ofr-miR396b-3p were used as target genes, and stable genes and unstable genes were used as standards for calculation to confirm the applicability of the candidate genes evaluated in the present invention. -ΔΔCt Methods The expression of target genes was calculated.

[0065] When the stable genes novel33 and ofr-miR395e were used as internal reference genes, the expression trends of ofr-miR166e-5p and ofr-miR396b-3p were similar, with ofr-miR166e-5p being most expressed in S1 and ofr-miR396b-3p being most expressed in S4. However, when the unstable gene ofr-miRNA171a-3p was used as the internal reference gene, both ofr-miR166e-5p and ofr-miR396b-3p were most expressed in S3 ( Figure 4 、 5 ). Therefore, the selection of appropriate genes has a great influence on the expression level and expression trend of target genes.

[0066] 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 primers for detecting novel33 and ofr-miR395e in quantitative PCR analysis of miRNA expression levels during the opening and aging of sweet osmanthus petals, characterized in that: The nucleotide sequence of novel33 is shown in SEQ ID NO.1, and the nucleotide sequence of ofr-miR395e is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that The primer sequences for novel33 are: novel33 forward primer: 5′-ccgcgTTGAACTCGTATGCGAGCGCA-3′; The primer sequences for miR-miR395e are: ofr-miR395e forward primer: 5′-ccggcCTGAAGTGTTTGGGGGAACTC-3′.

3. The use according to claim 1 or claim 2, characterized in that The petal opening and aging process includes: the stem stage, the initial flowering stage, the early flowering stage, the full flowering stage, the late flowering stage and the petal shedding stage.

4. The use according to claim 1, characterized in that The application comprises the following steps: qRT-PCR detection was performed using the cDNA of osmanthus petals during opening and senescence as a template, novel33 and ofr-miR395e as internal reference genes, and the primers described in claim 2.

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