MiRNA internal reference gene and primer thereof under metal ion stress of osmanthus fragrans and application thereof

Abstract

The application discloses miRNA internal reference genes under metal ion stress of Osmanthus fragrans, primers and application thereof. 2+ The optimal internal reference genes under Fe stress are novel33, novel3 and ofr-miR159b-3p. 3+ The optimal internal reference genes under Cu stress are novel3, ofr-miR395e and novel33. 2+ The optimal internal reference genes under Cu stress are novel3, ofr-miR395e and novel33. The optimal internal reference genes under metal ion stress are novel3, ofr-miR159b-3p, novel33, novel2 and ofr-miR395e. The results fill the present situation that there is no miRNA internal reference gene under metal ion stress of Osmanthus fragrans, and provide strong support for expression quantification of relevant functional genes of Osmanthus fragrans under metal ion stress.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant molecular biology, in particular to miRNA reference genes under metal ion stress of Osmanthus fragrans and primers and applications thereof. BACKGROUND

[0002] Osmanthus fragrans is a plant with high ornamental value, known for its fragrant flowers and drought tolerance and pollution resistance. Metal ion stress becomes an important factor affecting the growth and development of Osmanthus fragrans. Metal ions such as copper ions (Cu 2+ ), iron ions (Fe 2+ ) and aluminum ions (Al 3+ ) can enter the plant body through soil and water sources, triggering a series of physiological and biochemical reactions, and having a negative impact on the growth, development and reproduction of Osmanthus fragrans.

[0003] microRNA (miRNA) is a small non-coding RNA molecule that can regulate gene expression by binding to target mRNA, and plays an important role in plant growth and development, stress response and signal transduction. Studies have shown that miRNA plays a key regulatory role in the process of plant response to metal ion stress. Therefore, revealing the expression pattern of miRNA in Osmanthus fragrans under metal ion stress is of great significance to understand its tolerance mechanism.

[0004] In miRNA expression analysis, selecting appropriate reference miRNA is the key to ensuring the accuracy and reliability of experimental data. Reference miRNA is a miRNA that is stably expressed under various experimental conditions, used to correct technical errors in experiments. However, different stress conditions may affect the stability of reference miRNA. Therefore, under metal ion stress, it is necessary to screen reference miRNA that is stably expressed in Osmanthus fragrans to ensure the accuracy and reliability of qRT-PCR data. SUMMARY

[0005] In view of the above defects of the prior art, the present application proposes miRNA reference genes under metal ion stress of Osmanthus fragrans and primers and applications thereof to solve the problems raised in the background art.

[0006] To achieve the above purpose, the present application provides the following technical solutions:

[0007] The miRNA reference genes under metal ion stress of Osmanthus fragrans include at least one of the following (1) to (4):

[0008] (1) Fe 2+The miRNA internal reference genes under stress are novel33, novel3 and ofr-miR159b-3p;

[0009] (2) Al 3+ The miRNA internal reference genes under stress are novel3, ofr-miR395e and novel33;

[0010] (3) Cu 2+ The miRNA internal reference genes under stress are novel2, novel8 and ofr-miR159b-3p;

[0011] (4) Fe 2+ , Al 3+ and Cu 2+ The miRNA internal reference genes under stress of three metal ions are novel3, ofr-miR159b-3p, novel33, novel2 and ofr-miR395e;

[0012] The nucleic acid sequence of novel33 is shown as SEQ ID NO. 1; the nucleic acid sequence of novel3 is shown as SEQ ID NO. 2; the nucleic acid sequence of ofr-miR159b-3p is shown as SEQ ID NO. 3; the nucleic acid sequence of ofr-miR395e is shown as SEQ ID NO. 4; the nucleic acid sequence of novel2 is shown as SEQ ID NO. 5; and the nucleic acid sequence of novel8 is shown as SEQ ID NO. 6.

[0013] Preferably, the forward primer sequence of novel33 is 5'-ccgcgTTGAACTCGTATGCGAGCGCA-3' (SEQ ID NO. 7).

[0014] Preferably, the forward primer sequence of novel3 is 5'-gcggcggTCAAGATTGGGCAATGAACCA-3' (SEQ ID NO. 8).

[0015] Preferably, the forward primer sequence of ofr-miR159b-3p is 5'-gcgcgcCTTTGGATTGAAGGGAGCTCT-3' (SEQ ID NO. 9).

[0016] Preferably, the forward primer sequence of ofr-miR395e is 5'-ccggcCTGAAGTGTTTGGGGGAACTC-3' (SEQ ID NO. 10).

[0017] Preferably, the forward primer sequence of novel2 is 5'-ccgccgTTTCCTATACCTCCCATACCGA-3'(SEQ ID NO. 11).

[0018] Preferably, the forward primer sequence of novel8 is 5'-gcgccgTTTCCTATTCCTCCCATACCGA-3'(SEQ ID NO. 12).

[0019] Preferably, the application also relates to the application of the miRNA reference genes of Osmanthus fragrans under metal ion stress in the fluorescence quantitative PCR analysis of miRNA of Osmanthus fragrans.

[0020] Preferably, the application also relates to the application of the specific primers of the miRNA reference genes of Osmanthus fragrans under metal ion stress in the fluorescence quantitative PCR analysis of miRNA of Osmanthus fragrans.

[0021] Preferably, the application of the miRNA reference genes of Osmanthus fragrans under metal ion stress comprises the following steps: taking the cDNA of the leaf of Osmanthus fragrans under metal ion stress as a template, taking miRNA as a reference gene, and using the specific primers of the reference gene to perform qRT-PCR detection.

[0022] Compared with the prior art, the application has the beneficial effects that:

[0023] The application finally selects five commonly used miRNA reference genes (18S, ACT11, TUA5, U6 and UBQ4) by performing local blast comparison of the reference genes of other species with the whole genome data of Osmanthus fragrans, and then performing online analysis of the conserved domains and ORF of the genes on the blastx of the NCBI website. Secondly, nine mature and relatively abundant and stable miRNAs (ofr-miR159b-3p, ofr-miR168b-5p, ofr-miR171a-3p, ofr-miR395e, ofr-miR403-3p, novel2, novel3, novel8 and novel33) are screened as candidate genes through small RNA sequencing. The stability of the above-mentioned 14 candidate genes is evaluated through five algorithms (delta-CT, geNorm, NormFinder, BestKeeper and RefFinder), and the best reference genes of Fe 2+ The best reference genes under stress are novel33, novel3 and ofr-miR159b-3p, Al 3+ The best reference genes under stress are novel3, ofr-miR395e and novel33, Cu 2+The optimal reference genes under stress are novel2, novel8 and ofr-miR159b-3p, Fe 2+ , Al 3+ and Cu 2+ The miRNA reference genes under stress of the three metal ions are novel3, ofr-miR159b-3p, novel33, novel2 and ofr-miR395e, and the real-time fluorescent quantitative PCR primers of the reference genes are designed, the primers are high in specificity and amplification efficiency, can greatly improve the detection efficiency of the real-time fluorescent quantitative detection of the osmanthus fragrans genes, and improve the credibility of the detection results. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is a CQ value graph of 14 miRNA references under different metal ion treatments;

[0025] Figure 2 is a graph of the expression stability values (M) of 14 miRNA references sorted by the geNorm software under different metal ion treatments; the lower the stability value, the more stable the gene expression;

[0026] Figure 3 is the optimal reference gene number determined by the geNorm for accurate quantitative analysis under different metal ion treatments;

[0027] Figure 4 is the expression amount of ofr-miR166e-5p under different metal ion treatments, with stable genes and unstable genes as references;

[0028] Figure 5 is the expression amount of ofr-miR396b-3p under different metal ion treatments, with stable genes and unstable genes as references. DETAILED DESCRIPTION

[0029] The technical solutions of the present application will be further described in detail in combination with specific embodiments.

[0030] The experimental methods used in the embodiments of the present application are all conventional methods if no special description is given.

[0031] The materials, reagents and the like used in the embodiments of the present application can be obtained from commercial channels if no special description is given.

[0032] The test material used in the following examples is Osmanthus fragrans. Osmanthus fragrans is a clone of "Chang'e" selected from Hubei Xianning City with good growth state and no disease and insect pests 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 cut was flat cut, the lower cut was 45° bevel cut, distilled water immersion 12h, 1% sodium hypochlorite immersion 10min, distilled water washing 3 times, then 0.1g / L GGR rooting powder immersion 4h. The cuttings were inserted into the Osmanthus fragrans base in Hubei Xianning City, the soil matrix was a mixture of grass charcoal, perlite, vermiculite and yellow sand in a ratio of 1:1:1:1, and water was sprayed twice a week. In April 2024, 3mM CuSO4·5H2O, 3mM AlCl3·6H2O and 3mM FeSO4 were used to treat the cuttings, 200mL treatment solution per plant. The light period (12-h light / 12-h dark cycle) and 60% humidity of the light incubator were used to culture, and the stress period was extended to 0, 3, 6, 12, 24 and 72h for sampling. Each treatment has three biological replicates, and liquid nitrogen freezing treatment is used, stored at-80℃ and further analyzed.

[0033] Example 1

[0034] 1. Total RNA extraction and cDNA synthesis of Osmanthus fragrans

[0035] The miRNA was extracted using an miRNA isolation kit (Tiangen Biotech, Beijing, China). Its integrity, purity and concentration were detected by 1% agarose gel electrophoresis and spectrophotometer (NanoDrop 2000, Thermo Scientifc, Wilmington, DE, USA).

[0036] Using 0.8μg of total RNA as a template, the synthesis of the first strand of miRNA cDNA was carried out according to the instructions of miRcute miRNA First-Strand cDNA Synthesis Kit (KR201, Tiangen Biotech, China), and stored in a-20℃ refrigerator for later use.

[0037] 2. Selection of internal reference genes and design of primers

[0038] The reference genes for other species were compared with the Osmanthus fragrans genome data by literature review and database search, and then the conservative domain and ORF of the genes were analyzed online by blastx on the NCBI website. Five candidate reference genes were selected and designed, which were 18S, ACT11, TUA5, U6 and UBQ4. The CDS region of the reference genes was designed by Primer 5.0 software, and the parameters were as follows: the length of PCR product was 70-300 bp, the melting temperature was 58-62℃, and the CG content was 40-60%.

[0039] Nine mature and stable miRNAs were selected from the Osmanthus fragrans small RNA sequencing as candidate genes, which were ofr-miR159b-3p, ofr-miR168b-5p, ofr-miR171a-3p, ofr-miR395e, ofr-miR403-3p, novel2, novel3, novel8 and novel33. The upstream primer of the miRNA was based on the mature miRNA sequence, and U was replaced by T. The appropriate Tm value was 65℃, the GC content was 40-60%, and the reverse primer was provided by the kit miRcute miRNA qPCR Detection Kit (SYBR Green). The candidate reference genes and their primers are shown in Table 1:

[0040] Table 1 14 candidate reference genes and primer sequences

[0041]

[0042]

[0043] Note: E: amplification efficiency; R 2 : correlation coefficient

[0044] 3. qRT-PCR quantification

[0045] qRT-PCR was performed using Tianlong Gentier 96E system (Tianlong Science and Technology Co., Ltd., Xi'an, China) for amplification, and the program was as follows: 30 s at 95 °C, followed by 40 cycles of 10 s at 95 °C and 30 s at 60 °C, and then generating a melting curve at 60-95 °C. The reaction system was as follows: 2xmiRcute Plus miRNA Premix (SYBR & ROX) 10 μL, forward and reverse primers (10 μM) 0.4 μL each, 10-fold diluted miRNA first strand cDNA 2 μL, and RNase-Free ddH2O 7.2 μL. Each qRT-PCR analysis was performed in triplicate, and each gene included a non-template control.

[0046] 4、CT value

[0047] The CT value was obtained by qRT-PCR, and the CT value was inversely proportional to the expression amount of the gene. The greater the CT value, the lower the expression amount of the gene, and vice versa, the smaller the CT value, representing the higher expression amount of the gene.

[0048] The CT value of novel33 changed the least (1.756) under Al 3+ treatment; the CT value of ofr-miR159b-3p changed the least (1.101) under Cu 2+ treatment; the CT value of novel3 changed the least (1.375) under Fe 2+ treatment; and the CT value of ofr-miR171a-3p changed the least, which was 3.164, under the treatment of the three metal ions (see Figure 1 ).

[0049] 5、Evaluation of stability of internal reference genes

[0050] The stability of internal reference genes was analyzed by using delta-CT, geNorm, NormFinder, BestKeeper, and RefFinder algorithms to comprehensively analyze the expression stability of internal reference genes. The stable internal reference genes were screened.

[0051] 5.1 Stability test results

[0052] 5.1.1 delta-CT analysis

[0053] The standard deviation average was obtained by delta-CT analysis, and the higher the standard deviation average, the worse the stability of the gene, and vice versa, the lower the standard deviation average, the higher the stability of the gene. The results are shown in Table 2. Under Al 3+ stress, novel3 was the most stable gene; under Cu 2+ stress and metal ion stress, novel2 was the most stable gene; and under Fe2+ The most stable gene under stress is novel33.

[0054] Table 2 delta-CT analysis results

[0055]

[0056]

[0057] 5.1.2 geNorm software analysis

[0058] The expression stability M value of each candidate reference gene was calculated by geNorm software, the larger the M value, the lower the stability; on the contrary, the smaller the M value, the higher the stability, and M = 1.5 is the upper limit (see Figure 2 ). In addition, the appropriate number of reference genes was determined according to the geNorm pair-wise variation value Vn / n+1 n n+1 analysis. Since Al 3+ stress, Cu 2+ stress, Fe 2+ stress, the pair-wise variation value V3 / V4<0.15 (see Figure 3 ) under stress, so only 3 reference genes are needed for the relative expression analysis of genes under these three stresses. While under metal ion stress, the pair-wise variation value V5 / V6<0.15 (see Figure 3 ) of the embodiment, so 5 reference genes are needed for the relative expression analysis of genes under metal ion stress of Osmanthus fragrans.

[0059] 5.1.3 NormFinder software analysis

[0060] The stability value of the candidate reference gene was 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. Under Al 3+ stress of Osmanthus fragrans, 18S is the most stable gene; under Cu 2+ stress, Fe 2+ stress, and three metal ion stresses, the most stable genes are novel2, ofr-miR395e, and ofr-miR395e, respectively.

[0061] Table 3 NormFinder software analysis results

[0062]

[0063]

[0064] 5.1.4 BestKeeper software analysis​

[0065] 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 program default threshold value was 1. When the SD value was greater than 1, it was considered that the gene expression was unstable. The results are shown in Table 4. 3+ The most stable gene under metal ion stress was novel33; the most stable gene under Cu 2+ stress was ofr-miR159b-3p; and the most stable gene under Fe 2+ stress was novel3, and the most stable gene under metal ion stress was novel33.

[0066] Table 4 BestKeeper software analysis results

[0067]

[0068]

[0069] 5.1.5 RefFinder website analysis

[0070] The stability ranking obtained by the above-mentioned multiple analyses was geometrically averaged by the RefFinder website. The smaller the geometric mean value, the more stable the expression; on the contrary, the larger the geometric mean value, the more unstable the expression. The results are shown in Table 5. Under Al 3+ stress, novel3+ofr-miR395e+novel33 was the most stable internal reference combination; under Cu 2+ stress, novel2+novel8+ofr-miR159b-3p was the most stable internal reference combination; under Fe 2+ stress, novel33+novel3+ofr-miR159b-3p was the most stable internal reference combination; under three metal ion stresses, novel3+ofr-miR159b-3p+novel33+novel2+ofr-miR395e was the most stable internal reference combination. TUA5 was the most stable gene under Al 3+ stress, Fe 2+ stress and three metal ion stresses. 2+

[0071] Table 5 RefFinder comprehensive analysis results

[0072]

[0073] 6、Internal reference genes and target genes are combined to verify stability ​

[0074] According to our choice of flux sequencing results, ofr-miR166e-5p and ofr-miR396b-3p are taken as target genes, and the obtained stable genes and unstable genes are taken as internal references for calculation, respectively, and 2 -△△Ct The expression of target genes is calculated to confirm the suitability of the candidate genes evaluated in the present study.

[0075] As shown in Figure 4 and 5 , under Al 3+ ion stress, the expression trends of ofr-miR166e-5p and ofr-miR396b-3p are basically consistent when stable novle3, ofr-miR395e and novel33 are taken as internal references; and when unstable gene TUA5 is taken as internal reference, the target genes are significantly up-regulated at 72h. Under Cu 2+ stress, when stable genes novel8, novel2 and ofr-miR159b-3p are taken as internal references, the expression of target genes is the highest at 12h; and when ofr-miR168b-5p is taken as internal reference, the expression of target genes is the highest at 24h. Under Fe 2+ stress, when stable genes novel33, novel3 and ofr-miR159b-3p are taken as internal references, the expression of target genes is the highest at 6h; and when unstable gene TUA5 is taken as internal reference, the expression of target gene ofr-miR166e-5p is the highest at 72h, and the expression of target gene ofr-miR396b-3p is the highest at 12-72h. Therefore, it is necessary to select appropriate internal references.

[0076] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A miRNA reference gene under metal ion stress of Osmanthus fragrans, characterized in that, The miRNA internal reference gene comprises at least one of the following (1)-(4): (1) Fe 2+ miRNA reference genes under stress were novel33, novel3 and ofr-miR159b-3p; (2) Al 3+ The miRNA reference genes under stress were novel3, ofr-miR395e and novel33. (3) Cu 2+ The miRNA reference genes under stress were novel2, novel8 and ofr-miR159b-3p. (4) Fe 2+ , Al 3+ and Cu 2+ Three miRNA reference genes under metal ion stress of novel3, ofr-miR159b-3p, novel33, novel2 and ofr-miR395e; The nucleic acid sequence of novel33 is shown as SEQ ID NO. 1; the nucleic acid sequence of novel3 is shown as SEQ ID NO. 2; the nucleic acid sequence of ofr-miR159b-3p is shown as SEQ ID NO. 3; the nucleic acid sequence of ofr-miR395e is shown as SEQ ID NO. 4; the nucleic acid sequence of novel2 is shown as SEQ ID NO. 5; and the nucleic acid sequence of novel8 is shown as SEQ ID NO.

6.

2. The application of the miRNA internal reference gene under metal ion stress of Osmanthus fragrans in the fluorescence quantitative PCR analysis of miRNA of Osmanthus fragrans according to claim 1.

3. The application of the specific primer of the miRNA internal reference gene under metal ion stress of Osmanthus fragrans in the fluorescence quantitative PCR analysis of miRNA of Osmanthus fragrans according to claim 1.

4. The use according to claim 3, wherein the compound is ###0002### The method comprises the following steps: The cDNA of the leaf of Osmanthus fragrans under metal ion stress is used as a template, the miRNA is used as an internal reference gene, and the specific primer of the internal reference gene is used for qRT-PCR detection; The specific primer for detecting novel33 is 5'-ccgcgTTGAACTCGTATGCGAGCGCA-3'; The specific primer for detecting novel3 is 5'-gcggcggTCAAGATTGGGCAATGAACCA-3'; The specific primer for detecting ofr-miR159b-3p is 5'-gcgcgcCTT TGGATTGAAGGGAGCTCT-3'; The specific primer for detecting ofr-miR395e is 5'-ccggcCTGAAGTGTTTGGGGGAACTC-3'; The specific primer for detecting novel2 is 5'-ccgccgTTTCCTATACCTCCCATACCGA-3'; The specific primer for detecting novel8 is 5'-gcgccgTTTCCTATTCCTCCCATACCGA-3'.

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