LncRNA Reference Genes, Their Primers and Applications under Abiotic Stress Conditions in Osmanthus fragrans

Abstract

The present invention discloses lncRNA internal reference genes, primers thereof and applications under abiotic stress conditions in osmanthus fragrans. The optimal lncRNA internal references in osmanthus fragrans under abiotic stress are screened. The lncRNA internal references under low temperature stress are lnc00249739 + lnc00042194, the lncRNA internal references under salt stress are lnc00239991 + lnc00042194, the lncRNA internal references under drought stress are lnc00042194 + lnc00174850, and the lncRNA internal references under abiotic stress are lnc00042194 + lnc00239991 + lnc00067193 + lnc00265419. qRT-PCR primers are designed with these genes, filling the technical gap of no lncRNA internal reference under abiotic stress in osmanthus fragrans, providing strong support for the expression quantification of related functional genes of lncRNA under abiotic stress in osmanthus fragrans, and providing a theoretical basis for the functional research of lncRNA in osmanthus fragrans.

Description

Technical Field

[0001] The present invention relates to the field of plant molecular biology, and particularly to lncRNA reference genes, primers thereof and applications under abiotic stress conditions in Osmanthus fragrans. Background Art

[0002] Osmanthus fragrans is one of the top ten traditional famous flowers in China. It likes warm and humid climate and is suitable for growing in fertile soil with good drainage. Long non-coding RNA (lncRNA) plays a key regulatory role in the growth, development and stress response of plants. In order to accurately evaluate the expression level of lncRNA under abiotic stress conditions, appropriate internal references must be selected, and these internal references should be stably expressed under stress conditions and not affected by the external environment. Compared with traditional PCR, quantitative real-time polymerase chain reaction (qRT-PCR) has become a rapid and reliable method for quantifying gene transcription levels due to its good repeatability, strong specificity, high sensitivity and high throughput. However, effective qRT-PCR depends on many factors, such as the quality of extracted RNA, the efficiency of reverse transcription, primer specificity and data processing methods, etc., and it is also necessary to use stably expressed internal references to control experimental errors. At present, there is no report on the research of lncRNA reference genes in Osmanthus fragrans under abiotic stress conditions. Therefore, in-depth exploration of the selection of lncRNA internal references, primer design and application research under abiotic stress conditions in Osmanthus fragrans has important theoretical and practical significance for promoting the cultivation and improvement of Osmanthus fragrans. Summary of the Invention

[0003] In view of the deficiencies of the prior art, the purpose of the present invention is to provide lncRNA internal references under abiotic stress conditions in Osmanthus fragrans, which can meet the requirements of real-time fluorescence quantitative detection of the transcriptional expression level of Osmanthus fragrans and improve the stability, reliability and efficiency of gene expression analysis research in Osmanthus fragrans.

[0004] In order to achieve the above purpose, the technical solutions adopted by the present invention are as follows:

[0005] LncRNA reference genes under abiotic stress conditions in Osmanthus fragrans, wherein the lncRNA reference genes include at least one of the following (1) to (4);

[0006] (1) LncRNA reference genes lnc00249739 and lnc00042194 under low temperature stress;

[0007] (2) LncRNA reference genes lnc00239991 and lnc00042194 under salt stress;

[0008] (3) lncRNA reference genes lnc00042194 and lnc00174850 under drought stress;

[0009] (4) lncRNA reference genes lnc00042194, lnc00239991, lnc00067193 and lnc00265419 under low temperature stress, salt stress and drought stress;

[0010] Among them, the nucleic acid sequence of lnc00249739 is shown in SEQ ID NO.1, the nucleic acid sequence of lnc00042194 is shown in SEQ ID NO.2, the nucleic acid sequence of lnc00239991 is shown in SEQ ID NO.3, the nucleic acid sequence of lnc00174850 is shown in SEQ ID NO.4, the nucleic acid sequence of lnc00067193 is shown in SEQ ID NO.5, and the nucleic acid sequence of lnc00265419 is shown in SEQ ID NO.6.

[0011] Furthermore, the present invention provides specific primers for detecting the said lnc00249739, and their sequences are:

[0012] Forward primer: 5’-TGGACTTGGCTGACCCTTGA-3’ (SEQ ID NO.7);

[0013] Reverse primer: 5’-TTCCAATCTTGCGGACTGAC-3’ (SEQ ID NO.8).

[0014] Specific primers for detecting the said lnc00042194, and their sequences are:

[0015] Forward primer: 5’-TCGGCGAAGGGTGAGTAATG-3’ (SEQ ID NO.9);

[0016] Reverse primer: 5’-TGAAGACGACGACGGGATT-3’ (SEQ ID NO.10).

[0017] Specific primers for detecting the said lnc00239991, and their sequences are:

[0018] Forward primer: 5’-TTTCTTGGTCGTGTCTTTAGCA-3’ (SEQ ID NO.11);

[0019] Reverse primer: 5’-CAAGTTGCGGGAGACGTTAT-3’ (SEQ ID NO.12).

[0020] Specific primers for detecting the lnc00174850, and the sequences are as follows:

[0021] Forward primer: 5’-GCTCCCTGTCTCGATATTCATAC-3’ (SEQ ID NO.13);

[0022] Reverse primer: 5’-ATCTGTCGTCAAGCGTTCCT-3’ (SEQ ID NO.14).

[0023] Specific primers for detecting the lnc00067193, and the sequences are as follows:

[0024] Forward primer: 5’-GCATCGGCGATTGTGAGA-3’ (SEQ ID NO.15);

[0025] Reverse primer: 5’-AAGCGAAGGTCCGTTTGG-3’ (SEQ ID NO.16).

[0026] Specific primers for detecting the lnc00265419, and the sequences are as follows:

[0027] Forward primer: 5’-CATTATTGTTACGCCGACCAC-3’ (SEQ ID NO.17);

[0028] Reverse primer: 5’-GATCGTTTAGCCGCTCTTTCT-3’ (SEQ ID NO.18).

[0029] Furthermore, the present invention relates to the application of the lncRNA internal reference gene of the osmanthus under abiotic stress conditions in the fluorescence quantitative PCR analysis of osmanthus lncRNA.

[0030] Furthermore, the present invention relates to the application of the specific primers of the lncRNA internal reference gene of the osmanthus under abiotic stress conditions in the fluorescence quantitative PCR analysis of osmanthus lncRNA. The sequences of the primers are as shown above.

[0031] Compared with the prior art, the beneficial effects of the present invention are:

[0032] Through literature review, database search and analysis, 17 candidate reference gene sequences were screened in the present invention. The stability of candidate reference genes was evaluated by five algorithms (delta-CT, geNorm, NormFinder, BestKeeper and RefFinder). It was determined that the lncRNA reference genes under low temperature stress were lnc00249739 + lnc00042194, the lncRNA reference genes under salt stress were lnc00239991 + lnc00042194, the lncRNA reference genes under drought stress were lnc00042194 + lnc00174850, and the lncRNA reference genes under abiotic stress were lnc00042194 + lnc00239991 + lnc00067193 + lnc00265419. The present invention also designed qRT-PCR primers for reference genes. These primers have strong specificity and high amplification efficiency, which can greatly improve the detection efficiency when detecting Osmanthus fragrans genes by real-time fluorescence quantitative method and improve the credibility of detection results. Brief Description of the Drawings

[0033] Figure 1 : The geNorm software ranks the expression stability values (M) of 17 candidate reference genes; the lower the stability value, the more stable the gene.

[0034] Figure 2 : The geNorm software determines the optimal number of reference genes for accurate quantitative analysis.

[0035] Figure 3: The expression level of lnc00003036 using stable genes and unstable genes as reference genes.

[0036] Figure 4: The expression level of lnc00126603 using stable genes and unstable genes as reference genes.

[0037] Figure 5: The expression level of lnc00250780 using stable genes and unstable genes as reference genes. Detailed Embodiments

[0038] To better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0039] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0040] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can all be obtained from commercial channels.

[0041] The osmanthus used in the following examples was selected from the "Chang'e" clone without pests and diseases and in good growth condition in Xianning City, Hubei Province 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 made flat, and the lower cut was made at a 45° angle. The cuttings were soaked in distilled water for 12 h, then in 1% sodium hypochlorite for 10 minutes, and rinsed 3 times with distilled water, and then soaked in 0.1 g·L -1 GGR rooting powder for 4 h. The cuttings were inserted into the osmanthus base in Xianning City, Hubei Province. The soil substrate was peat, perlite, vermiculite, and yellow sand (1:1:1:1), and the cuttings were watered twice a week.

[0042] Example 1

[0043] (1) Screening of candidate reference genes and primer design

[0044] By consulting literature and searching databases, the reference genes that have been reported for other species were locally blast-aligned (blastVer: 2.4.0+) with the osmanthus whole-genome data to find the 7 candidate reference genes with the highest homology (18S, ACT7, EF1B, GAPH, RAN1, TUA5, and UBQ4). At the same time, based on RNA-seq, 10 lncRNAs with relatively high expression levels and a fold change < 1.4 were selected as reference genes.

[0045] Primer5 software was used for primer design with the following parameters: the length of the PCR product was 70-300 bp, the melting temperature was 58-62 °C, and the GC content was 40-60%. The candidate reference genes and their primers are shown in Table 1 below.

[0046] (2) Material treatment: In April 2024, osmanthus was cultured in a light incubator at 4 °C, a photoperiod of (12-h light / 12-h dark), and 60% humidity, and samples were taken at 3, 6, 12, 24, and 72 h.

[0047] (3) Total RNA extraction of osmanthus: The total RNA of each sample was extracted according to the operation instructions of the HiPure Plant RNA Mini Kit (Magen). Its integrity, purity, and concentration were detected by 1% agarose gel electrophoresis and a spectrophotometer (Nano Drop 2000, Thermo Scientifc, Wilmington, DE, USA).

[0048] (4) cDNA preparation: Using total RNA as a template, cDNA was synthesized using the III RT SuperMix for qPCR (+gDNAwiper) kit (Nanjing Novizan Biotech Co., Ltd.).

[0049] (5) qRT-PCR Quantification: The 20 μL reaction system is as follows: 10 μL of 2× Taq Pro Universal SYBR qPCR Master Mix; 1 μL of cDNA diluted 10-fold and 0.8 μL of forward and reverse primers (10 μM), add ddH2O to 20 μL. The reaction program is as follows: 30 s at 95 °C, followed by 40 cycles with parameters of 10 s at 95 °C, 30 s at 60 °C, and then a melting curve is generated at 60 - 95 °C. Each qRT-PCR analysis is repeated 3 times, and non-template controls are included for each gene.

[0050] (5) Stability Assessment

[0051] Delta-CT analysis: The average value of the standard deviation is obtained through delta-CT analysis. The higher the average value of the standard deviation, the worse the gene stability; conversely, the lower the average value of the standard deviation, the higher the gene stability. The results are shown in Table 2. The average values of the standard deviation of lnc00042194 and GAPH are both 0.66 and 0.72, which are the most stable genes; the average value of the standard deviation of lnc00031789 is 1.62, which is the gene with the worst stability.

[0052] geNorm software analysis: The expression stability M value of each candidate reference gene is calculated by the 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 ( Figure 1 ). This software can also calculate the paired variation V value of the normalization factor after introducing 1 new reference gene, and determine the number of the most suitable reference genes required according to the V n / V n+1 value. The default V value is 0.15. If the V n / V n+1 value < 0.15, the number of the most suitable reference genes is n. Since the paired variation value V2 / V3 of the example < 0.15 ( Figure 2 ), only 2 reference genes (lnc00265419 and lnc00249739) are required for the relative expression analysis of genes under low temperature stress in osmanthus.

[0053] NormFinder software analysis: When using NormFinder, the original Ct values are converted to 2 -ΔCt(Delta Ct = original Ct value - lowest Ct value in this group) was then used for the analysis of the expression stability of reference genes. The stability values of candidate 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 minimum stability value is the most stable gene. The results are shown in Table 3. The stability value of lnc00042194 is 0.212, which is the most stable gene; the stability value of lnc00031789 is 1.527, which is the gene with the worst stability.

[0054] Analysis by BestKeeper software: For BestKeeper, the amplification efficiency calculated by the LinRegPCR program based on the original Ct value and the original Ct value were used to calculate the coefficient of variation (CV) and standard deviation (SD) of the expression of candidate reference genes. The smaller the SD value, the more stable the reference gene. If the SD value > 1.0, it is considered that the reference gene is unstable and should be avoided for gene expression normalization. The results are shown in Table 4. The stability of the genes is lnc00239991 > lnc00249739 > GAPH > 18S > lnc00265419 > lnc00042194 > RAN1 > UBQ4 > EF1B > lnc00067193 > ACT7 > lnc00229717 > lnc00087780 > lnc00044331 > lnc00174850 > TUA5 > lnc00031789, and the most stable one is lnc00239991.

[0055] Analysis by RefFinder website: To verify the accuracy of the screening of reference genes, we comprehensively ranked the geometric means of the sorting results of candidate reference genes by delta-Ct, geNorm, NormFinder, and BestKeeper under each treatment. The smaller the comprehensive ranking, the better the gene expression stability. The results are shown in Table 5. The stability of the genes is lnc00249739 > lnc00042194 > GAPH > lnc00265419 > lnc00239991 > 18S > lnc00067193 > UBQ4 > RAN1 > lnc00229717 > ACT7 > lnc00044331 > lnc00087780 > lnc00174850 > EF1B > TUA5 > lnc00031789, among which lnc00249739 + lnc00042194 is the most stable gene combination; the geometric mean of lnc00031789 is 17.00, which is the most unstable gene.

[0056] Example 2

[0057] (1) Screening of candidate reference genes and design of their primers

[0058] Through literature review and database search, the reference genes reported for other species were locally blast-aligned (blastVer: 2.4.0+) with the Osmanthus fragrans whole-genome data to identify the 7 candidate reference genes with the highest homology. Secondly, based on RNA-seq, 10 lncRNAs with relatively high expression levels and a fold change < 1.4 were selected as references. Primer5 software was used for design with the following parameters: the length of the PCR product was 70 - 300 bp, the melting temperature was 58 - 62 °C, and the CG content was 40 - 60%. The candidate reference genes and their primers are shown in Table 1 below.

[0059] (2) Material treatment: In April 2024, Osmanthus fragrans cuttings were treated with 300 mM NaCl and sampled at 3, 6, 12, 24, and 72 h. Each treatment had three biological replicates, which were frozen with liquid nitrogen and stored at -80 °C.

[0060] (3) Total RNA extraction of Osmanthus fragrans: The total RNA of each sample was extracted according to the operation instructions of the HiPure Plant RNA Mini Kit (Magen). Its integrity, purity, and concentration were detected by 1% agarose gel electrophoresis and a spectrophotometer (Nano Drop 2000, Thermo Scientifc, Wilmington, DE, USA).

[0061] (4) cDNA preparation: Using the total RNA as a template, cDNA was synthesized using the III RT SuperMix for qPCR (+gDNAwiper) kit (Nanjing Novoprotein Biological Technology Co., Ltd.).

[0062] (5) qRT-PCR quantification: The procedure was as follows: 30 s at 95 °C, followed by 40 cycles with parameters of 10 s at 95 °C and 30 s at 60 °C, and then a melting curve was generated at 60 - 95 °C. The 20 μL reaction system was as follows: 10 μL of 2× Taq ProUniversal SYBR qPCR Master Mix; 1 μL of cDNA diluted 10 times and 0.8 μL of forward and reverse primers (10 μM), and ddH2O was added to 20 μL. Each qRT-PCR analysis was repeated 3 times, and each gene included a non-template control.

[0063] (6) Stability assessment

[0064] Delta-CT analysis: The average standard deviation was obtained through delta-CT analysis. The higher the average standard deviation, the worse the gene stability. On the contrary, the lower the average standard deviation, the higher the gene stability. The results are shown in Table 2. The average standard deviations of lnc00239991 and lnc00042194 are 0.76 and 0.78 respectively, which are the most stable genes; the average standard deviation of lnc00031789 is 2.08, which is the gene with the worst stability.

[0065] geNorm software analysis: The expression stability M value of each candidate reference gene was calculated by the geNorm software. The larger the M value, the lower the stability; conversely, the smaller the M value, the higher the stability. Among them, M = 1.5 is the upper limit ( Figure 1 ). This software can also calculate the paired variation V value of the normalization factor after introducing one new reference gene, and determine the number of the most suitable reference genes required according to the V n / V n+1 value. The default V value is 0.15. If the V n / V n+1 value < 0.15, the number of the most suitable reference genes is n. Since the paired variation value V2 / V3 of the example < 0.15 ( Figure 2 ), only 2 reference genes (lnc00042194 and lnc00239991) are required for the relative expression analysis of genes under osmatic stress in osmanthus.

[0066] NormFinder software analysis: When using NormFinder, the original Ct value was converted to 2 -ΔCt (delta Ct = original Ct value - the lowest Ct value in this group) and then used for the expression stability analysis of the reference gene. The stability value of the candidate reference gene was calculated by the 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 minimum stability value is the most stable gene. The results are shown in Table 3. The stability value of lnc00239991 is 0.158, which is the most stable gene; the stability value of lnc00031789 is 1.989, which is the gene with the worst stability.

[0067] BestKeeper software analysis: For BestKeeper, the amplification efficiency calculated by the LinRegPCR program based on the original Ct values and the original Ct values were used to calculate the coefficient of variation (CV) and standard deviation (SD) of the expression of candidate reference genes. The smaller the SD value, the more stable the reference gene. If the SD value > 1.0, the reference gene is considered unstable and should be avoided for gene expression normalization. The results are shown in Table 4, and the gene stability is lnc00042194 > lnc00239991 > 18S > lnc00265419 > lnc00067193 > UBQ4 > lnc00174850 > lnc00087780 > EF1B > RAN1 > GAPH > ACT7 > lnc00229717 > lnc00044331 > lnc00249739 > TUA5 > lnc00031789, and the most stable one is lnc00042194.

[0068] RefFinder website analysis: To verify the accuracy of the screening of reference genes, we comprehensively ranked the geometric means of the sorting results of the candidate reference genes by delta-Ct, geNorm, NormFinder, and BestKeeper under each treatment. The smaller the comprehensive ranking, the better the gene expression stability. The results are shown in Table 4, and the gene stability is lnc00239991 > lnc00042194 > lnc00265419 > lnc00087780 > lnc00067193 > 18S > lnc00044331 > RAN1 > EF1B > lnc00174850 > UBQ4 > lnc00229717 > GAPH > ACT7 > lnc00249739 > TUA5 > lnc00031789, among which the gene combination of lnc00239991 + lnc00042194 is the most stable gene combination; the geometric mean of lnc00031789 is 17.00, which is the most unstable gene.

[0069] Example 3

[0070] (1) Screening of candidate reference genes and primer design

[0071] Through literature review and database search, the reference genes reported for other species were locally blast-aligned (blastVer: 2.4.0+) with the Osmanthus fragrans whole-genome data to identify the 7 candidate reference genes with the highest homology. Based on RNA-seq, 10 lncRNAs with relatively high expression levels and a fold change < 1.4 were selected as references. Primer5 software was used for primer design with the following parameters: the length of the PCR product was 70 - 300 bp, the melting temperature was 58 - 62 °C, and the CG content was 40 - 60%. The candidate reference genes and their primers are shown in Table 1 below.

[0072] (2) Material treatment: In April 2024, Osmanthus fragrans cuttings were treated with 20% PEG-6000, and samples were taken at 3, 6, 12, 24, and 72 h. Each treatment had three biological replicates, which were frozen with liquid nitrogen and stored at -80 °C.

[0073] (3) Total RNA extraction and cDNA preparation of Osmanthus fragrans: The total RNA of each sample was extracted according to the operation instructions of the HiPure Plant RNA Mini Kit (Magen). Its integrity, purity, and concentration were detected by 1% agarose gel electrophoresis and a spectrophotometer (Nano Drop 2000, Thermo Scientific, Wilmington, DE, USA). Using the total RNA as a template, cDNA was synthesized using the III RT SuperMix for qPCR (+gDNA wiper) kit (Nanjing Novoprotein Biological Technology Co., Ltd.).

[0074] (4) qRT-PCR quantification: The 20 μL reaction system was as follows: 10 μL of 2×Taq Pro Universal SYBR qPCR Master Mix; 1 μL of cDNA diluted 10 times and 0.8 μL of forward and reverse primers (10 μM), and ddH2O was added to 20 μL. The reaction program was as follows: 30 s at 95 °C, followed by 40 cycles with parameters of 10 s at 95 °C and 30 s at 60 °C, and then a melting curve was generated at 60 - 95 °C. Each qRT-PCR analysis was repeated 3 times, and each gene included a non-template control.

[0075] (5) Stability assessment

[0076] Delta-CT analysis: The average standard deviation was obtained through delta-CT analysis. The higher the average standard deviation, the worse the gene stability. On the contrary, the lower the average standard deviation, the higher the gene stability. The results are shown in Table 2. The average standard deviations of lnc00042194 and lnc00174850 are 0.89 and 0.96 respectively, which are the most stable genes; the average standard deviation of lnc00249739 is 3.73, which is the gene with the worst stability.

[0077] geNorm software analysis: The expression stability M value of each candidate reference gene was calculated by the geNorm software. The larger the M value, the lower the stability; conversely, the smaller the M value, the higher the stability. Among them, M = 1.5 is the upper limit ( Figure 1 ). This software can also calculate the paired variation V value of the normalization factor after introducing a new reference gene, and determine the number of the most suitable reference genes required according to the V n / V n+1 value. The default V value is 0.15. If the V n / V n+1 value < 0.15, the number of the most suitable reference genes is n. Since the paired variation value V2 / V3 of the example < 0.15 ( Figure 2 ), only 2 reference genes (lnc00067193 and lnc00239991) are required for the relative expression analysis of genes under drought stress in osmanthus.

[0078] NormFinder software analysis: When using NormFinder, the original Ct values were converted to 2 -ΔCt (delta Ct = original Ct value - the lowest Ct value in this group) and then used for the expression stability analysis of reference genes. The stability value of candidate reference genes was calculated by the 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 minimum stability value is the most stable gene. The results are shown in Table 3. The stability value of lnc00042194 is 0.211, which is the most stable gene; the stability value of lnc00249739 is 3.677, which is the gene with the worst stability.

[0079] BestKeeper software analysis: For BestKeeper, the amplification efficiency calculated by the LinRegPCR program based on the original Ct values and the original Ct values were used to calculate the coefficient of variation (CV) and standard deviation (SD) of the expression of candidate reference genes. The smaller the SD value, the more stable the reference gene. If the SD value > 1.0, the reference gene is considered unstable and should be avoided for gene expression normalization. The results are shown in Table 4, and the gene stability is EF1B > lnc00042194 > lnc00174850 > lnc00087780 > lnc00044331 > lnc00239991 > 18S > lnc00229717 > lnc00265419 > lnc00067193 > RAN1 > GAPH > UBQ4 > ACT7 > TUA5 > lnc00031789 > lnc00249739, and the most stable one in expression is EF1B.

[0080] RefFinder website analysis: To verify the accuracy of reference gene screening, we comprehensively ranked the geometric means of the ranking results of candidate reference genes by delta-Ct, geNorm, NormFinder, and BestKeeper under each treatment. The smaller the comprehensive ranking, the better the gene expression stability. The results are shown in Table 5, and the gene stability is lnc00042194 > lnc00174850 > lnc00239991 > EF1B > lnc00067193 > lnc00044331 > lnc00087780 > RAN1 > lnc00265419 > 18S > lnc00229717 > UBQ4 > GAPH > ACT7 > TUA5 > lnc00031789 > lnc00249739, where lnc00042194 + lnc00174850 is the most stable gene combination; the geometric mean of lnc00249739 is 17.00, which is the most unstable gene.

[0081] Example 4

[0082] (1) Screening of candidate genes and primer design

[0083] Through literature review and database search, the reference genes reported for other species were locally blast-aligned (blastVer: 2.4.0+) with the Osmanthus fragrans whole-genome data to identify the 7 candidate reference genes with the highest homology. Based on RNA-seq, 10 lncRNAs with relatively high expression levels and a fold change < 1.4 were selected as references. Primer software was used for design with the following parameters: PCR product length of 70 - 300 bp, melting temperature of 58 - 62 °C, and CG content of 40 - 60%. The candidate reference genes and their primers are shown in Table 1 below.

[0084] (2) Material treatment: In April 2024, Osmanthus fragrans cuttings were treated at 4 °C, and at the same time, the cuttings were treated with 300 mM Nacl and 20% PEG-6000 respectively. Samples were taken at 3, 6, 12, 24, and 72 h. Each treatment had three biological replicates, which were frozen with liquid nitrogen and stored at -80 °C.

[0085] (3) Total RNA extraction and cDNA preparation of Osmanthus fragrans: The total RNA of each sample was extracted according to the operation instructions of the HiPure Plant RNA Mini Kit (Magen). Its integrity, purity, and concentration were detected by 1% agarose gel electrophoresis and a spectrophotometer (Nano Drop 2000, ThermoScientifc, Wilmington, DE, USA). Using the total RNA as a template, cDNA was synthesized using the III RT SuperMix for qPCR (+gDNA wiper) kit (Nanjing Novoprotein Biotechnology Co., Ltd.).

[0086] (4) qRT-PCR quantification: The procedure was as follows: 30 s at 95 °C, followed by 40 cycles with parameters of 10 s at 95 °C and 30 s at 60 °C, and then a melting curve was generated at 60 - 95 °C. The 20 μL reaction system was as follows: 10 μL of 2×Taq ProUniversal SYBR qPCR Master Mix; 1 μL of cDNA diluted 10-fold and 0.8 μL of forward and reverse primers (10 μM), and ddH2O was added to 20 μL. Each qRT-PCR analysis was repeated 3 times, and each gene included a non-template control.

[0087] (5) Stability assessment

[0088] Delta-CT analysis: The average standard deviation was obtained through Delta-CT analysis. The higher the average standard deviation, the worse the gene stability. On the contrary, the lower the average standard deviation, the higher the gene stability. The results are shown in Table 2. The average standard deviations of lnc00042194 and lnc00239991 are 0.89 and 0.94 respectively, which are the most stable genes; the average standard deviation of lnc00249739 is 2.74, which is the gene with the worst stability.

[0089] geNorm software analysis: 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, where M = 1.5 is the upper limit ( Figure 1 ). This software can also calculate the paired variation V value of the normalization factor after introducing a new reference gene, and determine the number of the most suitable reference genes required according to the Vn / Vn+1 value. The default V value is 0.15. If the Vn / Vn+1 value < 0.15, the number of the most suitable reference genes is n. Since the paired variation value V4 / V5 of the example < 0.15 ( Figure 2 ), 4 reference genes (lnc00042194, lnc00239991, lnc00067193, lnc00265419) are required for the relative expression analysis of genes under abiotic stress in osmanthus.

[0090] NormFinder software analysis: When using NormFinder, the original Ct value is converted 2 -ΔCt (Delta Ct = original Ct value - the lowest Ct value in this group) and then used for the expression stability analysis of reference genes. The stability value of candidate reference genes 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. The stability value of lnc00042194 is 0.166, which is the most stable gene; the stability value of lnc00249739 is 2.635, which is the gene with the worst stability.

[0091] BestKeeper software analysis: For BestKeeper, the amplification efficiency calculated by the LinRegPCR program based on the original Ct values and the original Ct values were used to calculate the coefficient of variation (CV) and standard deviation (SD) of the expression of candidate reference genes. The smaller the SD value, the more stable the reference gene. If the SD value > 1.0, the reference gene is considered unstable and should be avoided for gene expression normalization. The results are shown in Table 4, and the gene stability is lnc00042194 > lnc00239991 > 18S > lnc00265419 > lnc00067193 > GAPH > UBQ4 > lnc00229717 > lnc00044331 > lnc00087780 > EF1B > ACT7 > RAN1 > lnc00174850 > TUA5 > lnc00031789 > lnc00249739, and the most stable expression is lnc00042194.

[0092] RefFinder website analysis: To verify the accuracy of the reference gene screening, we comprehensively ranked the geometric means of the sorting results of the candidate reference genes by delta-Ct, geNorm, NormFinder, and BestKeeper under each treatment. The smaller the comprehensive ranking, the better the gene expression stability. The results are shown in Table 5, and the gene stability is lnc00042194 > lnc00239991 > lnc00067193 > lnc00265419 > lnc00044331 > 18S > UBQ4 > GAPH > RAN1 > lnc00087780 > lnc00229717 > EF1B > lnc00174850 > ACT7 > TUA5 > lnc00031789 > lnc00249739, where lnc00042194 + lnc00239991 + lnc00067193 + lnc00265419 is the most stable gene combination; the geometric mean of lnc00249739 is 17.00, which is the most unstable gene.

[0093] Verification of reference gene stability

[0094] According to the RNA-seq results, three differentially expressed lncRNAs (lnc00003036, lnc00126603, and lnc00250780) were used as target genes (the nucleotide sequences of lnc00003036, lnc00126603, and lnc00250780 are shown in Sequence Listing SEQ ID NO. 19 - 21, and the primer sequences are shown in SEQ ID NO. 22 - 27), and different reference genes (combinations) were used as standards for calculation to confirm the applicability of the candidate references evaluated in this study. Using 2 -△△CtThe method calculates the expression levels of three target genes. It is found that when using stable genes as internal references, the change trends of the expression levels of the three target genes are basically the same; however, when using unstable genes as internal references, the three target genes are significantly highly expressed or lowly expressed ( Figures 3a to 5c ), indicating that it is necessary to use stable internal references.

[0095] Table 1 Primer sequences of 17 candidate reference internal genes and 3 validated lncRNAs

[0096]

[0097]

[0098] Table 2 Results of delta-CT analysis

[0099]

[0100]

[0101] Table 3 Results of NormFinder software analysis

[0102]

[0103]

[0104] Table 4 Results of BestKeeper software analysis

[0105]

[0106] Table 5 Results of RefFinder website analysis

[0107]

[0108]

[0109] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. lncRNA internal reference gene under abiotic stress conditions of Osmanthus fragrans, characterized in that: The lncRNA internal reference gene includes at least one of the following (1) to (4); (1) The lncRNA reference genes under low temperature stress are lnc00249739 and lnc00042194; (2) The lncRNA reference genes under salt stress are lnc00239991 and lnc00042194; (3) The lncRNA reference genes under drought stress are lnc00042194 and lnc00174850; (4) The lncRNA reference genes under low temperature stress, salt stress and drought stress are lnc00042194, lnc00239991, lnc00067193 and lnc00265419; The nucleic acid sequence of lnc00249739 is shown in SEQ ID NO.1, the nucleic acid sequence of lnc00042194 is shown in SEQ ID NO.2, the nucleic acid sequence of lnc00239991 is shown in SEQ ID NO.3, the nucleic acid sequence of lnc00174850 is shown in SEQ ID NO.4, the nucleic acid sequence of lnc00067193 is shown in SEQ ID NO.5, and the nucleic acid sequence of lnc00265419 is shown in SEQ ID NO.

6.

2. Use of the lncRNA internal reference gene of Osmanthus fragrans under abiotic stress conditions as described in claim 1 in Osmanthus fragrans lncRNA fluorescence quantitative PCR analysis.

3. Use of the proprietary primers of the lncRNA internal reference gene of Osmanthus fragrans under abiotic stress conditions as claimed in claim 1 in fluorescent quantitative PCR analysis of lncRNA of Osmanthus fragrans.

4. The use according to claim 3, characterized in that: The proprietary primers used to detect lnc00249739 described in claim 1 are: Forward primer: 5′-TGGACTTGGCTGACCCTTGA-3′, reverse primer 5′-TTCCAATCTTGCGGACTGAC-3′; The proprietary primers used to detect lnc00042194 described in claim 1 are: Forward primer: 5′-TCGGCGAAGGGTGAGTAATG-3′, reverse primer 5′-TGAAGACGACGACGGGATT-3′; The proprietary primers used to detect lnc00239991 in claim 1 are: Forward primer: 5′-TTTCTTGGTCGTGTCTTTAGCA-3′, reverse primer 5′-CAAGTTGCGGGAGACGTTAT-3′; The proprietary primers used to detect lnc00174850 described in claim 1 are: Forward primer: 5′-GCTCCCTGTCTCGATATTCATAC-3′, reverse primer 5′-ATCTGTCGTCAAGCGTTCCT-3′; The proprietary primers used to detect lnc00067193 described in claim 1 are: Forward primer: 5′-GCATCGGCGATTGTGAGA-3′, reverse primer 5′-AAGCGAAGGTCCGTTTGG-3′; The proprietary primers used to detect lnc00265419 described in claim 1 are forward primer: 5'-CATTATTGTTACGCCGACCAC-3'; reverse primer 5'-GATCGTTTAGCCGCTCTTTCT-3'.

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