MiRNA reference gene in Osmanthus fragrans petal opening and senescence process as well as primer and application of miRNA reference gene

Abstract

The invention provides a miRNA reference gene in the opening and senescence process of sweet osmanthus petals and a primer and application of the miRNA reference gene, and belongs to the field of plant molecular biology. The petal opening and senescence process comprises S1 (the pedicel period), S2 (the initial flowering period), S3 (the initial stage of full-bloom), S4 (the full-bloom period), S5 (the later stage of full-bloom) and S6 (the petal falling period). According to the invention, 14 candidate reference genes are screened, and the stability of the candidate genes is evaluated through five algorithms (delta-CT, geNorm, NormFinder, BestKeeper and RefFinder), so that miRNA internal references, namely novel33 and ofr-miR395e, which are suitable for the opening and senescence processes of the sweet-scented osmanthus petals are obtained. According to the invention, the real-time fluorescent quantitative PCR primer of the internal reference is designed, the primer has strong specificity and high amplification efficiency, the detection efficiency of real-time fluorescent quantitative detection of sweet osmanthus miRNA can be greatly improved, and the credibility of the detection result is improved.

Description

Technical Field

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

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

[0003] When performing miRNA expression analysis, selecting appropriate internal reference miRNA is the key to ensuring data accuracy and reliability. Internal reference miRNAs are those miRNAs that are stably expressed under various experimental conditions and are used to correct technical errors in experiments. During the opening and aging of osmanthus petals, the expression level of miRNA may change significantly. Therefore, traditional internal reference miRNAs may no longer be applicable, and new, stably expressed internal reference miRNAs need to be screened for this process. Summary of the invention

[0004] In view of the above problems existing in the prior art, the first technical problem to be solved by the present invention is to provide miRNA internal references in the process of opening and aging of osmanthus petals; the second technical problem to be solved by the present invention is to provide special primers for miRNA internal references in the process of 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 genes or special primers in osmanthus fluorescence quantification.

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

[0006] Application of novel33 and ofr-miR395e as miRNA reference genes in fluorescence quantitative analysis of the opening and senescence of sweet osmanthus 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 table represents the base U of RNA.

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

[0008] The primer sequences of novel33 are:

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

[0010] The primer sequences of ofr-miR395e are as follows:

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

[0012] The reverse primer was provided by the kit miRcute miRNAqPCR 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 sweet osmanthus 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 comprises the following steps:

[0016] qRT-PCR detection was performed using the cDNA of osmanthus petals during opening and aging as a template, 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 invention compares the internal reference genes (18S, ACT, TUA, U6 and UBQ) used in other species with the whole genome data of sweet osmanthus fragrans locally (blastVer: 2.4.0+), finds the gene with the highest homology, and then performs online analysis on the conservative domain and ORF of the gene through blastx on the NCBI website. Through small RNA sequencing, 9 miRNAs with high expression and relatively stable expression in the process of opening and aging of sweet osmanthus fragrans petals are selected. The stability of 14 candidate genes is evaluated through 5 algorithms (delta-CT, geNorm, NormFinder, BestKeeper and RefFinder), and the miRNA internal references suitable for fluorescence quantitative detection in the process of opening and aging of sweet osmanthus fragrans petals are obtained: novel33 and ofr-miR395e, and the real-time fluorescence quantitative PCR primers of the internal reference are designed. The primers have strong specificity and high amplification efficiency, can greatly improve the detection efficiency when the real-time fluorescence quantitative detection of sweet osmanthus fragrans genes is adopted, and the credibility of the detection results is improved. 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 petals;

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

[0021] Figure 3 It is the optimal number of internal reference genes used for accurate quantitative analysis during the opening and senescence of osmanthus petals determined by geNorm;

[0022] Figure 4 The expression level of ofr-miR166e-5p in the process of the opening and senescence of the osmanthus petals, with stable genes and unstable genes as internal reference genes;

[0023] Figure 5 It is the expression level ofr-miR396b-3p, with stable genes and unstable genes 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 embodiments are provided below to further illustrate the present invention.

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

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

[0027] The following examples use a healthy and pest-free Osmanthus fragrans from Huazhong Agricultural University as the test material. Petal tissues of the six stages S1 (stalk stage), S2 (early flowering stage), S3 (early flowering stage), S4 (full flowering stage), S5 (late flowering stage) and S6 (falling stage) were collected at 10 a.m. Samples of each stage were used in three biological replicates, quickly 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 detected by 1% agarose gel electrophoresis and spectrophotometer (NanoDrop 2000, Thermo Scientific, Wilmington, DE, USA).

[0031] The synthesized samples were reverse transcribed by tailing method. The total RNA was used as template and 0.8ug miRNA was used to synthesize the first strand of miRNA cDNA according to the instructions of miRcute miRNA First-Strand cDNA Synthesis Kit (KR201, Tiangen Biotech, China). The reaction was carried out at 42°C for 60min; then the sample was incubated at 95°C for 3min for enzyme inactivation reaction. The synthesized cDNA reaction solution was immediately stored in a -20°C refrigerator for later use.

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

[0033] By sequencing small RNA in Osmanthus fragrans, 9 mature, abundantly 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. Five commonly used reference genes (18S, ACT11, TUA5, U6 and UBQ4) were screened as candidate genes through literature review and database comparison.

[0034] The upstream primer of miRNA is based on the mature miRNA sequence, with U replaced by T, and the bases are added or subtracted at both ends of the primer to achieve a suitable Tm value of 65°C and a GC content of 40%-60%. The downstream primer is provided by the kit miRcute miRNAqPCR detection kit (SYBR Green) (Tiangen). The gene primer is designed using Primer5, so that the Tm is 58-62°C and the GC content is 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 replicates and 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.

[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 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), among which 18S rRNA had the highest expression level and UBQ4 had a lower expression level. novel33 had the smallest CT change value (1.000), while UBQ4 had the largest CT change value (3.727).

[0046] 5. Stability assessment

[0047] Analysis of the stability of internal reference genes: using five algorithms including delta-CT, geNorm, NormFinder, BestKeeper and RefFinder to comprehensively analyze the expression stability of internal reference genes and screen out stable internal reference genes.

[0048] 6. Results

[0049] Delta-CT analysis: The standard deviation average was obtained by delta-CT analysis. The higher the standard deviation average, the worse the gene stability. On the contrary, 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, and ofr-miR171a-3p was the least stable gene.

[0050] Table 2 Delta-CT analysis results

[0051]

[0052] geNorm software analysis: 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; 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 Analysis was performed to determine the appropriate number of internal reference genes. Since the paired variation value V2 / V3 during the opening and aging of osmanthus petals was <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 by NormFinder software. The higher the stability value, the worse the stability. On the contrary, the lower the stability value, the better the stability. That is, the gene with the smallest stability value is the most stable gene. The results are shown in Table 3. During the opening and senescence of osmanthus petals, novel33 is the most stable gene, while ofr-miR171a-3p is the least stable gene.

[0054] Table 3 NormFinder software analysis results

[0055]

[0056] BestKeeper software analysis: The standard deviation (SD) of the candidate internal reference genes was calculated by BestKeeper software. The smaller the SD value, the more stable the expression. The default critical value of the program was 1. When the SD value was greater than 1, the gene expression was considered unstable. The results are shown in Table 4. novel33 was the most stable gene in the process of opening and aging of osmanthus petals.

[0057] Table 4 BestKeeper software analysis results

[0058]

[0059]

[0060] RefFinder website analysis: The geometric mean of the stability rankings obtained from the above 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 during the opening and aging of Osmanthus petals is novel33>ofr-miR395e>novel8>ofr-miR168b-5p>novel2>ofr-miR159b-3p>U6>TUA5>ofr-miR403-3p>novel3>18S>UBQ4>ACT11>ofr-miR171a-3p, among which novel33 and ofr-miR395e are the most stable gene combination, while the geometric mean of ofr-miR171a-3p is 14.00, which is the most unstable gene.

[0061] Table 5 RefFinder website analysis results

[0062]

[0063] 7. Verification of internal reference gene stability

[0064] According to 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 Calculate the expression of target genes.

[0065] When the stable genes novel33 and ofr-miR395e were used as reference genes, the expression trends of ofr-miR166e-5p or ofr-miR396b-3p were similar, with ofr-miR166e-5p being the most expressed in S1 and ofr-miR396b-3p being the most expressed in S4. However, when the unstable gene ofr-miRNA171a-3p was used as the 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 the target gene.

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

Claims

1. Application of novel33 and ofr-miR395e as miRNA reference genes in fluorescence quantitative analysis of the opening and aging process 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. A primer for detecting the internal reference gene according to claim 1, characterized in that: The primer sequences of novel33 are: novel33 forward primer: 5′-ccgcgTTGAACTCGTATGCGAGCGCA-3′; The primer sequence of ofr-miR395e is: ofr-miR395e forward primer: 5′-ccggcCTGAAGTGTTTGGGGGAACTC-3′.

3. The primer according to claim 2, characterized in that The reverse primer was provided by the miRcute miRNA qPCR detection kit.

4. Use of the primers described in claim 2 in fluorescence quantitative analysis of the opening and aging process of sweet osmanthus petals.

5. The use according to claim 1 or claim 4, 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.

6. The use according to claim 1, characterized in that: The application comprises the following steps: The cDNA of osmanthus petals in the process of opening and aging is used as a template, novel33 and ofr-miR395e are used as internal reference genes, and the primers described in claim 2 are used for qRT-PCR detection.

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