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

Abstract

The invention discloses a miRNA reference gene under metal ion stress of osmanthus fragrans as well as a primer and application of the miRNA reference gene. The optimal reference genes under the stress of Fe < 2 + > are novel33, novel3 and ofr-miR159b-3p, the optimal reference genes under the stress of Al < 3 + > are novel3, ofr-miR395e and novel33, the optimal reference genes under the stress of Cu < 2 + > are novel2, novel8 and ofr-miR159b-3p, and the miRNA reference genes under the stress of metal ions are novel3, ofr-miR159b-3p, novel33, novel2 and ofr-miR395e. The results fill the current situation that the osmanthus fragrans has no miRNA reference gene under the stress of the metal ions, and provide powerful support for expression quantification of related functional genes of the osmanthus fragrans under the stress of the metal ions.

Description

Technical Field

[0001] The invention relates to the field of plant molecular biology, and in particular to miRNA internal reference genes under osmanthus metal ion stress, primers and applications thereof. Background Art

[0002] Osmanthus fragrans is a plant with great ornamental value, known for its fragrant flowers and drought and pollution resistance. Metal ion stress has become 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, triggering a series of physiological and biochemical reactions, which have a negative impact on the growth, development and reproduction of osmanthus.

[0003] MicroRNA (miRNA) is a class of small non-coding RNA molecules that can regulate gene expression by binding to target gene mRNA, thus playing 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 for understanding its tolerance mechanism.

[0004] In miRNA expression analysis, the selection of appropriate internal reference miRNA is the key to ensure the accuracy and reliability of experimental data. Internal reference miRNAs are those miRNAs that are stably expressed under various experimental conditions and are used to correct technical errors in experiments. However, different stress conditions may affect the stability of internal reference miRNAs. Therefore, under metal ion stress, it is necessary to screen out internal reference miRNAs that are stably expressed in Osmanthus fragrans to ensure the accuracy and reliability of fluorescence quantitative PCR (qRT-PCR) data. Summary of the invention

[0005] 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 metal ion stress of osmanthus fragrans to solve the problems raised by the above-mentioned background technology.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The miRNA reference gene under metal ion stress of osmanthus fragrans, wherein the miRNA reference gene comprises at least one of the following (1) to (4):

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

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

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

[0011] (4)Fe 2+ 、Al 3+ and Cu 2+ The miRNA reference genes under the three metal ion stresses were novel3, ofr-miR159b-3p, novel33, novel2, and ofr-miR395e;

[0012] Among them, 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 present invention also relates to the application of the miRNA internal reference gene under osmanthus metal ion stress in osmanthus miRNA fluorescence quantitative PCR analysis.

[0020] Preferably, the present invention also relates to the use of the proprietary primers of the miRNA internal reference gene under osmanthus metal ion stress in osmanthus miRNA fluorescence quantitative PCR analysis.

[0021] Preferably, the application of miRNA internal reference gene under metal ion stress of osmanthus fragrans comprises the following steps: using cDNA of osmanthus fragrans leaves under metal ion stress as a template, using miRNA as an internal reference gene, and performing qRT-PCR detection using proprietary primers of the internal reference gene.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention compares the reference genes of other species with the whole genome data of Osmanthus fragrans by local blast, and then analyzes the conserved domains and ORFs of the genes online through blastx on the NCBI website, and finally selects the reference genes of 5 commonly used miRNAs (18S, ACT11, TUA5, U6 and UBQ4). Secondly, 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 screened as candidate genes by small RNA sequencing. The stability of the above 14 candidate genes was evaluated by 5 algorithms (delta-CT, geNorm, NormFinder, BestKeeper and RefFinder), and the Fe 2+ The best reference genes under stress were novel33, novel3 and ofr-miR159b-3p, Al 3+ The best reference genes under stress were novel3, ofr-miR395e and novel33, Cu 2+The best reference genes under stress were novel2, novel8 and ofr-miR159b-3p, Fe 2+ 、Al 3+ and Cu 2+ The miRNA reference genes under the three metal ion stresses are novel3, ofr-miR159b-3p, novel33, novel2 and ofr-miR395e, and real-time fluorescence quantitative PCR primers of the reference genes were designed. The primers have strong specificity and high amplification efficiency, which can greatly improve the detection efficiency of Osmanthus fragrans genes using real-time fluorescence quantitative detection and improve the credibility of the detection results. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0025] Figure 2 This is the ranking diagram of the stability value (M) of 14 miRNA internal reference expression by geNorm software under different metal ion treatments. The lower the stability value, the more stable the gene expression;

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

[0027] Figure 4 The stable genes and unstable genes under different metal ion treatments were used as internal references, and the expression level of ofr-miR166e-5p;

[0028] Figure 5 The stable genes and unstable genes under different metal ion treatments were used as internal references, and the expression level of ofr-miR396b-3p. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0030] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0031] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0032] The test material used in the following examples is sweet osmanthus. Sweet osmanthus is selected from the "Chang'e" asexual line with good growth and no pests and diseases in Xianning, Hubei Province as the mother tree. In May 2023, cut the semi-lignified branches of the current year with 2-3 lateral buds as cuttings (12-16cm), cut the upper incision flat, cut the lower incision at 45°, soak in distilled water for 12h, soak in 1% sodium hypochlorite for 10min, rinse with distilled water 3 times, and then soak in 0.1g / L GGR rooting powder for 4h. The cuttings were cut into the sweet osmanthus base in Xianning, 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, the cuttings were treated with 3mM CuSO4·5H2O, 3mM AlCl3·6H2O and 3mM FeSO4, respectively, with 200mL of treatment solution per seedling. The Osmanthus fragrans was cultured in a light incubator with a 12-h light / 12-h dark cycle and 60% humidity, and samples were taken at 0, 3, 6, 12, 24 and 72h extended stress period. Each treatment was repeated three times and frozen with liquid nitrogen, stored at -80℃ and further analyzed.

[0033] Example 1

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

[0035] 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).

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

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

[0038] Through literature review and database search, the reference genes used in other species were compared with the osmanthus genome data by local blast comparison (blastVer: 2.4.0+), and the conserved domains and ORFs of the genes were analyzed online by blastx on the NCBI website. Five candidate reference genes required for this experiment were selected and designed, namely 18S, ACT11, TUA5, U6 and UBQ4. Primer 5.0 software was used to design the CDS region of the reference genes, and the parameters were as follows: PCR product length 70-300bp, melting temperature 58-62℃, CG content 40-60%.

[0039] By sequencing the small RNA of Osmanthus fragrans, 9 mature, abundantly expressed and stable miRNAs were selected as candidate genes, namely ofr-miR159b-3p, ofr-miR168b-5p, ofr-miR171a-3p, ofr-miR395e, ofr-miR403-3p, novel2, novel3, novel8 and novel33. 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 reverse primer is provided by the kit miRcute miRNA qPCR Detection Kit (SYBR Green). The candidate reference genes and their primers are shown in Table 1 below:

[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 the Tianlong Gentier 96E system (Tianlong Technology Co., Ltd., Xi'an, China) with the following program: 30 s at 95°C, followed by 40 cycles with parameters of 10 s at 95°C, 30 s at 60°C, and then generating a melting curve at 60-95°C. 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. Each qRT-PCR analysis was performed in triplicate, and a non-template control was included for each gene.

[0046] 4. CT value

[0047] 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.

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

[0049] 5. Stability assessment of internal reference genes

[0050] Analysis of the stability of internal reference genes: using delta-CT, geNorm, NormFinder, BestKeeper and RefFinder algorithms to comprehensively analyze the expression stability of internal reference genes and screen out stable internal reference genes.

[0051] 5.1 Stability test results

[0052] 5.1.1delta-CT analysis

[0053] The standard deviation average value was obtained by delta-CT analysis, 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. 3+ Under Cu stress, novel3 was the most stable gene; 2+ novel2 was the most stable gene under Fe stress and metal ion stress;2+ Under stress, the gene with the best stability is novel33.

[0054] Table 2 Delta-CT analysis results

[0055]

[0056]

[0057] 5.1.2geNorm software analysis

[0058] 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 (see Figure 2 ). In addition, according to the geNorm pairwise variation value V n / V n+1 Analyze and determine the appropriate number of internal reference genes. 3+ Coercion, Cu 2+ Stress, Fe 2+ Under stress, the pairwise variation value V3 / V4 < 0.15 (see Figure 3 ), so only three internal references are needed to analyze the relative expression of genes under these three stresses. Under metal ion stress, the paired variation value V5 / V6 of the embodiment is less than 0.15 (see Figure 3 ), therefore, 5 internal references are needed to analyze the relative expression of genes under metal ion stress in Osmanthus fragrans.

[0059] 5.1.3 NormFinder software analysis

[0060] The stability value of the candidate internal 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. 3+ Under Cu stress, 18S is the most stable gene; 2+ Stress, Fe 2+ Stress and three metal ion stresses, the genes with the best stability were 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 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. 3+ The gene with the most stable expression under metal ion stress was novel33; 2+ The most stable gene under stress was ofr-miR159b-3p; 2+ The most stably expressed gene under stress was novel3, and the most stably expressed 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 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. 3+ Under stress, novel3+ofr-miR395e+novel33 was the most stable internal reference combination; Cu 2+ Under stress, novel2+novel8+ofr-miR159b-3p was the most stable internal reference combination; Fe 2+ Under 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. 3+ Stress, Fe 2+ The most unstable gene under Cu stress and three metal ion stresses, ofr-miR168b-5p is 2+ The most unstable genes under stress.

[0071] Table 5 RefFinder comprehensive analysis results

[0072]

[0073] 6. Stability verification of internal reference genes and target genes

[0074] According to our selected throughput sequencing results, ofr-miR166e-5p and ofr-miR396b-3p were used as target genes, and the obtained stable genes and unstable genes were used as internal references for calculation. -△△Ct Methods The expression of target genes was calculated to confirm the suitability of the candidate genes evaluated in this study.

[0075] like Figure 4 and 5 As shown in Al 3+ Under ion stress, when stable noble3, ofr-miR395e and novel33 were used as internal references, the expression trends of ofr-miR166e-5p and ofr-miR396b-3p were basically the same; however, when the unstable gene TUA5 was used as an internal reference, the target gene expression was significantly upregulated at 72h. 2+ Under Fe stress, the target gene expression level was highest at 12 h when the stable genes novel8, novel2 and ofr-miR159b-3p were used as internal references; while when ofr-miR168b-5p was used as internal reference, the target gene expression level was highest at 24 h. 2+ Under stress, when stable novel33, novel3 and ofr-miR159b-3p were used as internal references, the target gene expression level was highest at 6h; when the unstable gene TUA5 was used as internal reference, the target gene ofr-miR166e-5p was expressed at 72h, and the target gene ofr-miR396b-3p was expressed at 12-72h. Therefore, it is necessary to select appropriate internal references.

[0076] 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. miRNA internal reference gene under metal ion stress of Osmanthus fragrans, characterized in that: The miRNA reference gene includes at least one of the following (1) to (4): (1)Fe 2+ The 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+ The miRNA reference genes under the three metal ion stresses were novel3, ofr-miR159b-3p, novel33, novel2, and ofr-miR395e; Among them, 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. A proprietary primer for detecting novel33 according to claim 1, characterized in that: The forward primer sequence of novel33 was 5′-ccgcgTTGAACTCGTATGCGAGCGCA-3′.

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

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

5. A proprietary primer for detecting ofr-miR395e according to claim 1, characterized in that: The forward primer sequence of ofr-miR395e was 5′-ccggcCTGAAGTGTTTGGGGGAACTC-3′.

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

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

8. Application of miRNA internal reference gene in sweet osmanthus miRNA fluorescence quantitative PCR analysis under sweet osmanthus metal ion stress as claimed in claim 1.

9. Use of the proprietary primers of miRNA internal reference gene under metal ion stress of Osmanthus fragrans as claimed in claim 1 in Osmanthus fragrans miRNA fluorescence quantitative PCR analysis.

10. The use according to claim 9, characterized in that The following steps are involved: qRT-PCR detection was performed using cDNA of Osmanthus fragrans leaves under metal ion stress as a template, miRNA as an internal reference gene, and proprietary primers of the internal reference gene.

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