Osmanthus fragrans abiotic stress miRNA reference gene as well as primer and application thereof

Abstract

The invention discloses a sweet osmanthus abiotic stress miRNA reference gene as well as a primer and application thereof, the optimal miRNA reference gene under abiotic stress is obtained through screening, the miRNA internal reference under drought stress is ofr-miR168b-5p, novel8 and ofr-miR159b-3p, the miRNA internal reference under low temperature stress is ofr-miR159b-3p and ofr-miR403-3p, the miRNA internal reference under salt stress is novel8 and novel3, the miRNA internal reference under abiotic stress is ofr-miR159b-3p, novel8, ofr-miR403-3p and novel2, a qRT-PCR (quantitative reverse transcription-polymerase chain reaction) primer is designed, and the miRNA reference gene under abiotic stress is obtained through screening. The current situation that no miRNA internal reference exists under the condition of abiotic stress of osmanthus fragrans is filled up, and powerful support is provided for expression quantification of related miRNA under the condition of abiotic stress of osmanthus fragrans.

Description

Technical Field

[0001] The invention relates to the technical field of plant molecular biology, and in particular to an osmanthus abiotic stress miRNA internal reference gene and primers thereof and applications thereof. Background Art

[0002] Sweet osmanthus, belonging to the Oleaceae family, is one of the top ten traditional famous flowers in China. When abiotic stress factors such as drought, heat damage, low temperature, and salt damage act on plants, they will cause a series of physiological and metabolic reactions in the plant body. However, there have been no reports on the study of miRNA under abiotic stress in sweet osmanthus, which includes drought stress, low temperature stress, and salt stress. In the process of studying the difference in gene expression in sweet osmanthus under abiotic stress, the use of real-time fluorescence quantitative technology to analyze and verify the expression level of genes requires stable and reliable internal reference genes. Therefore, screening for miRNA internal references that are stably expressed under abiotic stress conditions in sweet osmanthus is a key factor in the accuracy of real-time fluorescence quantitative results. Summary of the invention

[0003] In view of the above-mentioned defects of the prior art, the present invention proposes an osmanthus abiotic stress miRNA internal reference gene and primers and applications thereof to solve the problems raised by the above-mentioned background technology.

[0004] In order to achieve the above object, the present invention provides the following technical solutions:

[0005] Osmanthus fragrans abiotic stress miRNA internal reference gene, the miRNA internal reference gene includes at least one of (1) to (4):

[0006] (1) The miRNA reference genes under drought stress were ofr-miR168b-5p, novel8, and ofr-miR159b-3p;

[0007] (2) miRNA reference genes under low temperature stress conditions were ofr-miR159b-3p and ofr-miR403-3p;

[0008] (3) miRNA reference genes under salt stress conditions were novel3 and novel8;

[0009] (4) The miRNA reference genes under drought stress, low temperature stress and salt stress were ofr-miR159b-3p, novel8, ofr-miR403-3p and novel2;

[0010] The nucleic acid sequence of ofr-miR168b-5p is shown as SEQ ID NO.1; the nucleic acid sequence of novel8 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-miR403-3p is shown as SEQ ID NO.4; the nucleic acid sequence of novel3 is shown as SEQ ID NO.5; and the nucleic acid sequence of novel2 is shown as SEQ ID NO.6.

[0011] Preferably, the forward primer sequence of the proprietary primer of ofr-miR168b-5p is 5'-ccgcTCGCTTGGTGCAGGTCGGGAA-3' (SEQ ID NO. 7).

[0012] Preferably, the novel8 proprietary primer, the forward primer sequence is

[0013] 5'-gcgccgTTTCCTATTCCTCCCATACCGA-3' (SEQ ID NO. 8).

[0014] Preferably, the forward primer sequence of the proprietary primer of ofr-miR159b-3p is 5'-gcgcgcCTTTGGATTGAAGGGAGCTCT-3' (SEQ ID NO. 9).

[0015] Preferably, the proprietary primer of ofr-miR403-3p, the forward primer sequence is

[0016] 5'-cgccgccgTTAGATTCACGCACAAACTCG-3' (SEQ ID NO. 10).

[0017] Preferably, the novel3 proprietary primer, the forward primer sequence is

[0018] 5'-gcggcggTCAAGATTGGGCAATGAACCA-3' (SEQ ID NO. 11).

[0019] Preferably, the novel2 proprietary primer, the forward primer sequence is

[0020] 5'-ccgccgTTTCCTATACCTCCCATACCGA-3' (SEQ ID NO. 12).

[0021] Preferably, the present application also relates to the use of the osmanthus abiotic stress miRNA internal reference gene in osmanthus miRNA fluorescence quantitative PCR analysis.

[0022] Preferably, the present application also relates to the use of the proprietary primers of the osmanthus abiotic stress miRNA internal reference gene in osmanthus miRNA fluorescence quantitative PCR analysis, and the proprietary primers are as shown above.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] This application used the reference genes for other species to perform local blast comparison with the whole genome data of Osmanthus fragrans (blastVer: 2.4.0+), and then used blastx on the NCBI website to perform online analysis of the conserved domains and ORFs of the genes, and finally selected 5 commonly used reference genes (18S, ACT11, TUA5, U6 and UBQ4). Through small RNA sequencing, 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) candidate genes were obtained, and the primer sequences of the reference genes were designed. The stability of candidate internal references was evaluated by five algorithms (delta-CT, geNorm, NormFinder, BestKeeper and RefFinder), and fluorescent quantitative miRNA internal references suitable for Osmanthus fragrans under various abiotic stresses (drought stress, low temperature stress and salt stress) were obtained. Real-time fluorescent quantitative PCR primers for internal reference genes were designed. The primers have strong specificity and high amplification efficiency, which can greatly improve the detection efficiency of Osmanthus fragrans miRNA using real-time fluorescent quantitative detection and improve the credibility of the detection results. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is the CQ value graph of 14 reference genes screened under various abiotic stress treatments;

[0026] Figure 2 It is the optimal number of internal reference genes for accurate quantitative analysis under each abiotic stress treatment determined by geNorm;

[0027] Figure 3 It is the ranking diagram of the stable expression values ​​(M) of 14 internal reference genes under various abiotic stress treatments by geNorm software;

[0028] Figure 4 is the expression level of ofr-miR166e-5p, which is the stable gene and unstable gene as internal reference gene under each abiotic stress treatment;

[0029] Figure 5The stable genes and unstable genes under various abiotic stress treatments were used as internal reference genes, and the expression level of ofr-miR396b-3p. DETAILED DESCRIPTION

[0030] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0031] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0032] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0033] 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, Osmanthus fragrans was placed in a light incubator with a photoperiod (12-hlight / 12-hdarkcycle) and 60% humidity, and exposed to 4°C, 300mM NaCl, and 20% PEG-6000 to treat the Osmanthus fragrans cuttings, and samples were taken at 0, 3, 6, 12, 24, and 72h of extended stress period. Each treatment was repeated three times, frozen with liquid nitrogen, and stored at -80°C for further analysis.

[0034] Example 1

[0035] 1. Total RNA extraction and cDNA synthesis from Osmanthus fragrans

[0036] miRNA was extracted using an miRNA isolation kit (Tiangen Biotechnology, Beijing, China), and 1.0% (w / v) agarose gel electrophoresis and spectrophotometer (NanoDrop 2000, Thermo Scientific, Wilmington, DE, USA) were used to detect its integrity, purity and concentration, respectively.

[0037] The synthesized samples were reverse transcribed by the tailing method, i.e., 0.8ug miRNA was synthesized 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 at -20°C.

[0038] 2. Selection of internal reference genes and design of primers

[0039] By sequencing the small RNA of 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, novel33) were selected, and 5 commonly used internal reference genes (18S, ACT11, TUA5, U6 and UBQ4) were screened by literature review and database comparison. The internal reference primers of 18S, ACT11, TUA5, U6 and UBQ4 were designed using Primer5.0; the upstream primer of miRNA was based on the mature miRNA sequence, and U was replaced by T, and the bases were added or subtracted at both ends of the primer to achieve the appropriate T. m The value was at 65°C, the GC content was at 40-60%, and the reverse primer was provided by the kit miRcute miRNA qPCR Detection Kit (SYBR Green).

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

[0041]

[0042] Note: E: amplification efficiency; R2: correlation coefficient

[0043] 3. qRT-PCR quantification

[0044] According to the instructions of miRcute Plus miRNA qPCR Detection Kit (SYBRGreen), a 20 μL PCR reaction system was prepared: 10 μL 2×miRcute Plus miRNA Premix (SYBR&ROX), 0.4 μL each of forward and reverse primers (10 μM), 2 μL of 10-fold diluted miRNA first-strand cDNA, and 7.2 μL of RNase-Free ddH2O. Three 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.

[0045] qRT-PCR was amplified using the Tianlong Gentier 96E system (Tianlong Technology Co., Ltd., Xi'an, China) with the following procedure: 95°C pre-denaturation for 15 min; followed by 40 cycles of 94°C denaturation for 20 s, 60°C annealing and extension for 30 s; and then a 60-95°C melting curve. The CT value was obtained by qRT-PCR, and the CT value was inversely proportional to the expression level of the gene. The larger the CT value, the lower the expression level of the gene. Conversely, the smaller the CT value, the higher the expression level of the gene.

[0046] Low temperature stress ( Figure 1 a), novel3 had the smallest CT change value, which was 1.086; under drought stress ( Figure 1 b), novel3 had the smallest CT change value, which was 0.961; salt stress ( Figure 1 c) and abiotic stresses ( Figure 1 d), novel8 has the smallest CT change value of 2.772.

[0047] 4. Stability assessment

[0048] 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.

[0049] 4.1delta-CT analysis

[0050] The standard deviation average was obtained by delta-CT analysis, where the higher the standard deviation average, the worse the gene stability, and conversely, the lower the standard deviation average, the higher the gene stability, as shown in Table 2. Under drought treatment, ofr-miR168b-5p was the most stable gene; under low temperature stress and abiotic stress, ofr-miR159b-3p was the most stable gene; under salt stress, novel2 was the most stable gene. TUA5 was the least stable gene under low temperature stress, salt stress and abiotic stress; and the least stable gene under drought stress was UBQ4.

[0051] Table 2 Delta-CT analysis results

[0052]

[0053] 4.2geNorm software analysis

[0054] When using geNorm, the raw Ct values ​​are converted by 2 -ΔCt (delta Ct = original Ct value - lowest Ct value in this group) is then used to analyze the expression stability of the internal reference gene. 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, geNorm can 2 -ΔCt For the original data, the paired difference value V of two continuous standardized factors is calculated n / V n+1 To determine the appropriate number of internal reference genes, since the paired variation value V2 / V3 of the embodiment of Osmanthus fragrans under salt stress and low temperature stress is less than 0.15 ( Figure 2 ), the paired variation value V3 / V4 of the embodiment under drought stress <0.15 ( Figure 2 ), the pairwise variance under abiotic stress V4 / V5<0.15( Figure 2 ). Therefore, only two internal reference genes (novel3 and ofr-miR403-3p) are needed under salt stress, only two internal reference genes (ofr-miR159b-3p and ofr-miR403-3p) are needed under low temperature stress, three internal reference genes (novel8, novel2, and novel3) are needed under drought stress, and four internal reference genes (ofr-miR159b-3p, novel8, ofr-miR403-3p, and novel2) are needed under abiotic stress ( Figure 3 ).

[0055] 4.3 NormFinder software analysis

[0056] When using NormFinder, convert the raw Ct values ​​2-ΔCt (delta Ct = original Ct value - lowest Ct value in this group) was then used for expression stability analysis of internal reference genes. The stability values ​​of candidate internal reference genes were calculated by NormFinder software. The higher the stability value, the worse the stability. Conversely, 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. Ofr-miR168b-5p, U6, novel8 and ofr-miR159b-3p are the genes with the best stability under drought, low temperature, salt stress and abiotic stress, respectively.

[0057] Table 3 NormFinder software analysis results

[0058]

[0059]

[0060] 4.4BestKeeper Software Analysis

[0061] For BestKeeper, the amplification efficiency and original Ct value calculated by LinRegPCR program based on the original Ct value were used to calculate the coefficient of variation (CV) and standard deviation (SD) of the candidate reference gene expression. The smaller the SD value, the more stable the reference gene. If the SD value is greater than 1.0, the reference gene is considered unstable and should be avoided for gene expression standardization. The results are shown in Table 4. The most stable expression under drought stress, salt stress and abiotic stress is novel3, and the most stable expression under low temperature stress is ofr-miR159b-3p.

[0062] Table 4 BestKeeper software analysis results

[0063]

[0064] 4.5 RefFinder website analysis

[0065] To verify the accuracy of the internal reference gene screening, we used RefFinder to comprehensively rank the candidate internal reference genes under each treatment. The smaller the comprehensive ranking, the better the gene expression stability.

[0066] The results are shown in Table 5. The stability of genes under drought stress was ofr-miR168b-5p>novel8>ofr-miR159b-3p>novel2>novel3>ofr-miR403-3p>U6>novel33>ofr-miR395e>ofr-miR171a-3p>18S>ACT11>TUA5>UBQ4, among which miR168b-5p+novel8+miR159b-3p was the most stable gene combination. The stability of genes under low temperature stress was ofr-miR159b-3p>ofr-miR403-3p>novel33>U6>ofr-miR395e>novel3>novel8>ofr-miR168b-5p>novel2>ofr-miR171a-3p>18S>UBQ4>ACT11>TUA5, among which ofr-miR159b-3p+ofr-miR403-3p was the most stable gene combination. The stability of genes under salt stress was novel3>novel8>novel2>ofr-miR159b-3p>ofr-miR403-3p>novel33>U6>18S>ofr-miR395e>ofr-miR168b-5p>ACT11>ofr-miR171a-3p>UBQ4>TUA5, among which novel3+novel8 was the most stable internal reference combination. Under abiotic stress, the stability of genes was ofr-miR159b-3p>novel8>ofr-miR403-3p>novel2>novel3>novel33>ofr-miR168b-5p>U6>ofr-miR395e>ofr-miR171a-3p>18S>ACT11>UBQ4>TUA5, among which the most stable gene combination was ofr-miR159b-3p+novel8+ofr-miR403-3p+novel2.

[0067] Table 5 RefFinder website analysis results

[0068]

[0069] 5. Verification of internal reference gene stability

[0070] Based on our selected throughput sequencing results, ofr-miR166e-5p and ofr-miR396b-3p were used as target genes, and stable genes and unstable genes in various abiotic stresses were used as standards for calculation to confirm the suitability of the candidate genes evaluated in this study. -△△Ct Methods Calculate the expression of target genes.

[0071] Under low temperature stress, with stable ofr-miR159b-3p and ofr-miR403-3p as internal references, the expression trends of ofr-miR166e-5p and ofr-miR396b-3p were similar, that is, ofr-miR166e-5p was highly expressed at 0h and 72h, and ofr-miR396b-3p was highly expressed at 0h; when the unstable gene TUA5 was used as an internal reference, ofr-miR166e-5p and ofr-miR396b-3p were significantly highly expressed at 6-12h ( Figure 4 a. Figure 5 a). Under drought stress, when stable ofr-miR168b-5p, novel8 and ofr-miR159b-3p were used as internal references, the expression trends of ofr-miR166e-5p and ofr-miR396b-3p were similar, that is, the target gene was highly expressed at 3h; when the unstable gene UBQ4 was used as the internal reference, the target gene was significantly highly expressed at 72h ( Figure 4 b. Figure 5 b). Under salt stress, when the stable genes novel3 and novel8 were used as internal references, the target gene was expressed at the highest level at 3h, while when the unstable gene TUA5 was used as internal reference, the target gene was expressed at the highest level at 12h ( Figure 4 c. Figure 5 c). Therefore, choosing the right gene does have a significant impact on the amount and expression of the target gene.

[0072] 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. Osmanthus fragrans abiotic stress miRNA internal reference gene, characterized in that: The miRNA reference gene includes at least one of (1) to (4): (1) The miRNA reference genes under drought stress were ofr-miR168b-5p, novel8, and ofr-miR159b-3p; (2) miRNA reference genes under low temperature stress conditions were ofr-miR159b-3p and ofr-miR403-3p; (3) miRNA reference genes under salt stress conditions were novel3 and novel8; (4) The miRNA reference genes under drought stress, low temperature stress and salt stress were ofr-miR159b-3p, novel8, ofr-miR403-3p and novel2; The nucleic acid sequence of ofr-miR168b-5p is shown as SEQ ID NO.1; the nucleic acid sequence of novel8 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-miR403-3p is shown as SEQ ID NO.4; the nucleic acid sequence of novel3 is shown as SEQ ID NO.5; and the nucleic acid sequence of novel2 is shown as SEQ ID NO.

6.

2. A proprietary primer for detecting ofr-miR168b-5p according to claim 1, characterized in that: The sequence of the forward primer was 5′-ccgcTCGCTTGGTGCAGGTCGGGAA-3′.

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

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

5. A proprietary primer for detecting ofr-miR403-3p according to claim 1, characterized in that: The forward primer sequence was 5′-cgccgccgTTAGATTCACGCACAAACTCG-3′.

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

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

8. Use of the sweet osmanthus abiotic stress miRNA internal reference gene according to claim 1 in sweet osmanthus miRNA fluorescence quantitative PCR analysis.

9. Use of the proprietary primers of the sweet osmanthus abiotic stress miRNA internal reference gene according to claim 1 in sweet osmanthus miRNA fluorescence quantitative PCR analysis.

10. The use according to claim 9, characterized in that: The proprietary primers are as shown in claims 2 to 7.

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