miRNA reference genes in different tissues of Osmanthus fragrans and their primers and applications

Abstract

The present invention discloses miRNA internal reference genes of different tissues of sweet osmanthus and primers and applications thereof, and belongs to the field of plant molecular biology. The present invention finally selects 14 required candidate internal references according to literature review, database search and small RNA sequencing, and designs real-time fluorescence quantitative PCR primers for internal references. The stability of candidate internal references is evaluated by 5 algorithms (delta-CT, geNorm, NormFinder, BestKeeper and RefFinder), and the best miRNA internal references for fluorescence quantification of different tissues of sweet osmanthus are obtained, which are novel2 and ofr-miR395e. The present invention fills the technical gap of no miRNA fluorescence quantification internal reference in different tissues of sweet osmanthus, provides strong support for the quantitative expression of functional miRNA related to different tissues of sweet osmanthus, and thus has important application value.

Description

Technical Field

[0001] The present invention relates to the field of plant molecular biology, and in particular to miRNA internal reference genes of different tissues of osmanthus fragrans, primers thereof and applications thereof. Background Art

[0002] Osmanthus fragrans, a plant of the Oleaceae family, is widely distributed in countries such as China, Japan, and Vietnam. As an evergreen shrub or small tree, osmanthus is renowned for its beautiful flowers and rich fragrance, holding a prominent place in horticultural and ornamental plants. Osmanthus not only holds a significant position in traditional Chinese culture but is also widely used in food, spices, medicine, and cosmetics.

[0003] In plant molecular biology research, internal reference genes are a type of genes that are stably expressed in different tissues, at different growth stages, and under different environmental conditions, and are often used for standardization of gene expression analysis. The selection of internal references is crucial for the accuracy and reliability of gene expression studies. Currently, there are few reports on the study of miRNAs in different tissues of Osmanthus fragrans. In the process of exploring the differences in miRNA expression in different tissues of Osmanthus fragrans, stable and reliable miRNA internal references are needed as references to perform real-time fluorescence quantitative PCR detection and verify the expression levels of miRNAs. Therefore, screening for stably expressed miRNA internal references in different tissues of Osmanthus fragrans plays a key role in the accuracy of its real-time fluorescence quantitative PCR detection results. Summary of the Invention

[0004] In view of the deficiencies in the prior art, the object of the present invention is to provide a miRNA reference gene stably expressed in different tissues of Osmanthus fragrans. The miRNA reference gene can meet the requirements of real-time fluorescence quantitative detection of Osmanthus fragrans gene transcription expression levels, thereby improving the stability, reliability and efficiency of Osmanthus fragrans miRNA expression analysis research.

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

[0006] The application of novel2 and ofr-miR395e as internal reference genes in the quantitative PCR analysis of miRNA in different tissues of Osmanthus fragrans. The nucleic acid sequence of novel2 is shown in SEQ ID NO. 1; the nucleic acid 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 also provides primers for detecting the internal reference gene, wherein:

[0008] The primer sequences of novel2 are as follows:

[0009] novel2 forward primer: 5′-ccgccgTTTCCTATACCTCCCATACCGA-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 relates to the application of the above primers in the analysis of miRNA fluorescence quantitative PCR in different tissues of Osmanthus fragrans.

[0014] Furthermore, the different tissues include roots, leaves, seeds and flowers.

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

[0016] qRT-PCR detection was performed using cDNA from different Osmanthus tissues as templates, novel2 and ofr-miR395e as internal reference genes, and the above-mentioned primers.

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

[0018] The present invention is to consult literature, database search and small RNA sequencing, and determine 14 reference genes as candidate genes.By 5 kinds of algorithms (delta-CT, geNorm, NormFinder, BestKeeper and RefFinder), the stability of candidate internal reference is assessed.Finally, the miRNA internal reference gene that is applicable to sweet osmanthus different tissue fluorescence quantification is screened out, and this fluorescence quantification internal reference is novel2 and ofr-miR395e, and the real-time fluorescence quantitative PCR primer of internal reference gene is designed, and this primer has strong specificity, and amplification efficiency is high, can greatly improve the detection efficiency when adopting real-time fluorescence quantitative detection sweet osmanthus miRNA, and improve the credibility of testing result. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the CQ value of the 14 screened miRNA fluorescence quantitative internal reference genes;

[0020] Figure 2This is the result graph of geNorm software ranking the expression stability values ​​(M) of 14 internal reference genes. The lower the stability value, the more stable the gene expression.

[0021] Figure 3 It is geNorm that determines the optimal internal parameters for accurate quantitative analysis;

[0022] Figure 4 Two stable genes, novel2 and ofr-miR395e, and one unstable gene, ofr-miR171a-3p, were used as internal controls, and the expression level of ofr-miR166e-5p was used;

[0023] Figure 5 Two stable genes novel2 and ofr-miR395e and one unstable gene ofr-miR171a-3p were used as internal controls, and the expression level of ofr-miR396b-3p was used. 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 were prepared using Osmanthus fragrans as the test material. Different tissue samples (roots, leaves, seeds, and flowers) were collected from 15-year-old Osmanthus fragrans trees grown in a natural environment. Three biological replicates were used for each treatment. Upon collection, samples were rapidly frozen with liquid nitrogen.

[0028] Example 1

[0029] 1. Screening of candidate reference genes and design of their primers

[0030] By performing small RNA sequencing on Osmanthus fragrans, we selected 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). Five commonly used reference genes (18S, ACT11, TUA5, U6, and UBQ4) were identified as candidate genes through literature review and genome-wide searches of Osmanthus fragrans.

[0031] qRT-PCR primer design parameters are as follows: The upstream miRNA primer is based on the mature miRNA sequence, with U replaced by T. Bases are added or removed at both ends to achieve an appropriate Tm of 65°C and a GC content of 40-60%. The downstream primer is 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%.

[0032] The 14 candidate reference genes and primer sequences are shown in Table 1.

[0033] 2. Total RNA extraction and cDNA preparation

[0034] 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 spectrophotometry (Nano Drop 2000, Thermo Scientific, Wilmington, DE, USA).

[0035] The synthesized samples were reverse transcribed using the tailing method. Specifically, 0.8 μg of miRNA was synthesized as the first-strand miRNA cDNA according to the instructions of the miRcute miRNA First-Strand cDNA Synthesis Kit (Tiangen). The reaction was performed at 42% for 60 minutes; then, the sample was inactivated at 95% for 3 minutes. The synthesized cDNA reaction was immediately stored at -20°C.

[0036] 3. qRT-PCR quantification

[0037] Amplification was performed using the Tianlong Gentier 96E system (Tianlong Technology Co., Ltd., Xi'an, China) with the following program: initial denaturation at 95°C for 15 min, followed by 40 cycles of denaturation at 94°C for 20 s and annealing and extension at 60°C for 30 s, followed by a 60–95°C melting curve.

[0038] The 20 μL 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 the amplification background.

[0039] 4. Stability assessment

[0040] Four different algorithms, including delta-Ct, geNorm, NormFinder, and BestKeeper, were used to statistically analyze the expression stability of internal references in different samples. The results were comprehensively analyzed using the RefFinder website to screen out stable internal references.

[0041] 5. Results

[0042] 1) CQ value

[0043] The CQ values ​​of the 14 internal references are as follows Figure 1 As shown in the figure, the average CQ values ​​ranged from 16.569 (ofr-miR159b-3p) to 28.447 (TUA5). Among them, the CQ value of ofr-miR168b-5p changed the least, at 1.718, while that of TUA5 changed the most, at 11.766.

[0044] 2) Delta-CT analysis

[0045] The delta-CT analysis obtained the standard deviation average value, where the higher the standard deviation average value, the worse the gene stability, and conversely, the lower the standard deviation average value, the higher the gene stability. The results are shown in Table 2. The standard deviation average value of ofr-miR395e is 1.67, which is the most stable gene; the standard deviation average value of ofr-miR168b-5p is 3.54, which is the least stable gene.

[0046] 3) geNorm software analysis

[0047] The cycle threshold (CT) obtained by fluorescence quantitative PCR was converted into relative quantitative data, and the average variation M value of the logarithmic transformation value of the pairwise ratio of a gene to other genes was analyzed using geNorm software, and an M value line graph was drawn ( Figure 2 The smaller the M value, the more stable the reference gene. The calculation method of geNorm software is not affected by the extreme ratios and expression abundance differences between genes. The software can be used to screen out more than two stably expressed reference genes. Since the pairwise variation value V2 / V3 < 0.15 ( Figure 3 ), therefore, only two internal references (novel2 and ofr-miR159b-3p) were needed to analyze the relative expression of genes in different tissues of Osmanthus fragrans.

[0048] 4) NormFinder software analysis

[0049] When using NormFinder, convert the raw Ct values ​​by 2 -ΔCtThe delta Ct value (delta Ct = original Ct value minus the lowest Ct value in the group) was then used to analyze the expression stability of the reference genes. The stability values ​​of the candidate reference genes were calculated using NormFinder software. Higher stability values ​​indicate lower stability, while lower stability values ​​indicate better stability. The gene with the lowest stability value was considered the most stable. The results are shown in Table 3. Ofr-miR395e had a stability value of 0.428, making it the most stable gene; ofr-miR168b-5p had a stability value of 3.289, making it the least stable gene.

[0050] 5) BestKeeper Software Analysis

[0051] BestKeeper evaluates and ranks the stability of candidate genes using their standard deviation (SD). A smaller SD value indicates greater gene stability; if SD > 1, the gene is considered unstable. The most stably expressed gene in different Osmanthus tissues is ofr-miR168b-5p (Table 4).

[0052] 6) RefFinder website analysis

[0053] The geometric mean of the stability rankings obtained from the above-mentioned multiple analyses was calculated using the RefFinder website. The smaller the geometric mean, the more stable the expression; conversely, the larger the geometric mean, the more unstable the expression. The results are shown in Table 5. The stability of genes is novel2>ofr-miR395e>ofr-miR159b-3p>novel33>18S>novel3>UBQ4>ofr-miR168b-5p>U6>ACT11>ofr-miR403-3p>novel8>TUA5>ofr-miR171a-3p, among which novel2 and ofr-miR395e are the most stable gene combination, while ofr-miR171a-3p is the least stable gene.

[0054] 6. Verification of internal reference gene stability

[0055] The present invention uses (ofr-miR166e-5p and ofr-miR396b-3p) as target genes and different reference genes (combinations) as standards for calculation to confirm the applicability of the candidate genes evaluated in this study. -△△CtThe target gene expression was calculated using the method. It was found that when the stable genes novel2 and ofr-miR395e were used as internal references, the target gene expression levels and change trends were basically the same, that is, ofr-miR166e-5p and ofr-miR396b-3p were expressed in seeds at the highest level, followed by leaves or flowers, and in roots at the lowest level. However, when the unstable gene ofr-miR171-3p was used as an internal reference, the target gene expression trend was different from that of the stable gene internal reference ( Figure 4 and 5 ), namely ofr-miR166e-5p and ofr-miR396b-3p, which were expressed at the lowest levels in flowers. Therefore, it is necessary to screen for stable miRNA internal controls.

[0056] Table 1 14 candidate reference genes and primer sequences

[0057]

[0058] E, PCR efficiency; R 2 , correlation coefficient

[0059] Table 2 Delta-CT analysis results

[0060]

[0061]

[0062] Table 3 NormFinder software analysis results

[0063]

[0064] Table 4 BestKeeper software analysis results

[0065]

[0066]

[0067] Table 5 RefFinder website analysis results

[0068]

[0069] 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 novel2 and ofr-miR395e as internal reference genomes in the quantitative PCR analysis of miRNA in different tissues of Osmanthus fragrans, characterized by: The nucleic acid sequence of novel2 is shown in SEQ ID NO.1; the nucleic acid sequence of ofr-miR395e is shown in SEQ ID NO.2, and the different tissues include roots, leaves, seeds and flowers.

2. A primer set for detecting the internal reference genome according to claim 1, characterized in that: The primer sequences for novel2 are as follows: novel2 forward primer: 5′-ccgccgTTTCCTATACCTCCCATACCGA-3′; The primer sequences for miR-miR395e are as follows: ofr-miR395e forward primer: 5′-ccggcCTGAAGTGTTTGGGGGAACTC-3′.

3. The primer set according to claim 2, wherein The reverse primer was provided by the miRcute miRNAqPCR detection kit.

4. Use of the primer set according to claim 3 in quantitative PCR analysis of miRNA in different tissues of Osmanthus fragrans.

5. The use according to claim 1, characterized in that The application comprises the following steps: qRT-PCR detection was performed using cDNA from different Osmanthus tissues as templates, novel2 and ofr-miR395e as internal reference genomes, and the primer set described in claim 2.

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