LncRNA reference genes, primers, and applications under osmanthus hormone treatment

Abstract

The present invention discloses lncRNA internal reference genes for osmanthus hormone treatment, primers, and applications thereof. The present invention obtains the optimal lncRNA internal reference genes for osmanthus under hormone treatment conditions: lnc00239991+18S under ABA treatment, lnc00265419+lnc00249739 under MeJA treatment, lnc00229717+lnc00044331 under ethephon treatment, and lnc00265419+lnc00239991 under multiple hormone treatments. qRT-PCR primers were designed using these genes. This invention fills the technical gap of the lack of internal reference lncRNAs under osmanthus hormone treatment, provides strong support for the quantification of lncRNAs under osmanthus hormone treatment, and provides a theoretical basis for the study of gene function in osmanthus.

Description

Technical Field

[0001] The present invention relates to the field of plant molecular biology, and in particular to a lncRNA internal reference gene under osmanthus hormone treatment, a primer thereof, and an application thereof. Background Art

[0002] Sweet osmanthus (Osmanthus fragrans) is one of my country's ten traditional famous flowers, with a unique fragrance and medicinal value. Hormones play a crucial regulatory role in plant growth and development, and lncRNA, as an important class of transcriptional regulators, is also involved in the regulatory network of plant growth and development. Therefore, studying the internal reference lncRNA of Osmanthus fragrans under hormone treatment conditions, as well as its primers and applications, is of great background significance. Currently, there are no reports on lncRNAs in Osmanthus fragrans under ABA, MeJA, and ethephon treatment conditions. In the process of exploring the differences in lncRNA expression in Osmanthus fragrans under hormone treatment, a stable and reliable internal reference lncRNA is required as a reference for qRT-PCR detection and verification of gene expression levels. Therefore, screening for internal reference lncRNAs stably expressed under ABA, MeJA, and ethephon treatment conditions in Osmanthus fragrans plays a key role in the accuracy of real-time fluorescence quantitative PCR detection results. Summary of the Invention

[0003] In view of the deficiencies in the prior art, the purpose of the present invention is to provide an internal reference of lncRNA under osmanthus hormone treatment, which can meet the requirements of real-time fluorescence quantitative detection of osmanthus transcriptional expression levels and improve the stability, reliability and efficiency of osmanthus gene expression analysis research.

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

[0005] Osmanthus hormone-treated lncRNA reference gene, the lncRNA reference gene includes at least one of the following (1) to (4);

[0006] (1) lncRNA reference genes lnc00239991 and 18S under abscisic acid treatment;

[0007] (2) lncRNA reference genes lnc00265419 and lnc00249739 under methyl jasmonate treatment;

[0008] (3) lncRNA reference genes lnc00229717 and lnc00044331 under ethephon treatment;

[0009] (4) lncRNA reference genes lnc00265419 and lnc00239991 under abscisic acid, methyl jasmonate, and ethephon treatments;

[0010] Among them, the nucleic acid sequence of lnc00239991 is shown as SEQ ID NO.1, the nucleic acid sequence of 18S is shown as SEQ ID NO.2, the nucleic acid sequence of lnc00265419 is shown as SEQ ID NO.3, the nucleic acid sequence of lnc00249739 is shown as SEQ ID NO.4, the nucleic acid sequence of lnc00229717 is shown as SEQ ID NO.5, and the nucleic acid sequence of lnc00044331 is shown as SEQ ID NO.6.

[0011] The present invention further provides proprietary primers for detecting lnc00239991, the sequences of which are:

[0012] Forward primer: 5'-TTTCTTGGTCGTGTCTTTAGCA-3' (SEQ ID NO. 7); reverse primer: 5'-CAAGTTGCGGGAGACGTTAT-3' (SEQ ID NO. 8).

[0013] The sequences of the proprietary primers used to detect the 18S are as follows: forward primer: 5'-CCATAAACGATGCCGACCAG-3' (SEQ ID NO. 9); reverse primer: 5'-GCCTTGCGACCATACTCCC-3' (SEQ ID NO. 10).

[0014] The sequences of the proprietary primers used to detect lnc00265419 are as follows: forward primer: 5'-CATTATTGTTACGCCGACCAC-3' (SEQ ID NO. 11); reverse primer: 5'-GATCGTTTAGCCGCTCTTTCT-3' (SEQ ID NO. 12).

[0015] The proprietary primers used to detect lnc00249739 have the following sequences: forward primer: 5'-TGGACTTGGCTGACCCTTGA-3' (SEQ ID NO. 13); reverse primer: 5'-TTCCAATCTTGCGGACTGAC-3' (SEQ ID NO. 14).

[0016] The proprietary primers used to detect lnc00229717 have the following sequences: forward primer: 5'-CTTAGCCGCCCATCCCA-3' (SEQ ID NO. 15); reverse primer: 5'-CACCCGCATTATCCGTTGA-3' (SEQ ID NO. 16).

[0017] The sequences of the proprietary primers used to detect lnc00044331 are as follows: forward primer: 5'-GGGTTGTGGCGGGTAATTCATCTTG-3' (SEQ ID NO. 17); reverse primer: 5'-GGTAAGGGATGTAATAATGTGCTGA-3' (SEQ ID NO. 18).

[0018] Furthermore, the present invention relates to the use of the osmanthus hormone-treated lncRNA internal reference gene or its primers in osmanthus lncRNA fluorescence quantitative PCR analysis.

[0019] Furthermore, the present invention also relates to the use of the proprietary primers for the lncRNA internal reference gene treated with osmanthus hormone in the fluorescent quantitative PCR analysis of osmanthus lncRNA. The proprietary primers are as shown above.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention screened 17 internal reference genes through literature review, database search, and analysis. The stability of candidate internal reference genes was evaluated using five algorithms (deltaCT, geNorm, NormFinder, BestKeeper, and RefFinder). The authors identified the most stable internal reference genes (18S+lnc00239991) expressed under ABA treatment in Osmanthus fragrans, lnc00265419+lnc00249739 expressed under MeJA treatment in Osmanthus fragrans, lnc00229717+lnc00044331 expressed under ethephon treatment in Osmanthus fragrans, and lnc00265419+lnc00239991 expressed under hormone treatment in Osmanthus fragrans. These internal reference sequences were then revealed, and real-time fluorescence quantitative PCR primers for these internal references were designed. These primers have strong specificity and high amplification efficiency, thereby significantly improving the detection efficiency of Osmanthus fragrans genes using real-time fluorescence quantitative PCR and enhancing the reliability of the detection results. These results provide some basis for the future screening of genes and lncRNA expression analysis under ABA, MeJA and ethephon treatment conditions in Osmanthus fragrans. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : geNorm software ranked the expression stability values ​​(M) of 17 internal reference genes. The lower the stability value, the more stable the gene.

[0023] Figure 2 : geNorm determines the optimal number of reference genes for accurate quantitative analysis;

[0024] Figure 3: Stable genes and two unstable genes used as internal reference genes, and the expression level of lnc00003036;

[0025] Figure 4: Stable genes and two unstable genes used as internal reference genes, and the expression level of lnc00126603;

[0026] Figure 5: Stable genes and two unstable genes used as internal reference genes, and the expression level of lnc00250780. DETAILED DESCRIPTION

[0027] In order to better understand the technical content of the present invention, specific examples are provided below to further illustrate the present invention.

[0028] Unless otherwise specified, the experimental methods used in the examples of the present invention are all conventional methods.

[0029] Unless otherwise specified, the materials, reagents, etc. used in the examples of the present invention can be obtained from commercial sources.

[0030] The test material used in the following examples is Osmanthus fragrans. Osmanthus fragrans was selected from the "Chang'e" asexual line in good growth condition and free of pests and diseases 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 incision was cut flat and the lower incision was cut at a 45° angle. The cuttings were soaked in distilled water for 12 hours, 1% sodium hypochlorite was added for 10 minutes, and the cuttings were rinsed with distilled water three times. The cuttings were then treated with 0.1 g·L -1 Soak in GGR rooting powder for 4 hours. Plant cuttings in an osmanthus fragrans base in Xianning, Hubei Province. The soil matrix is ​​peat, perlite, vermiculite, and yellow sand (1:1:1:1), and water is sprayed twice a week.

[0031] Example 1

[0032] (1) ABA treatment

[0033] In April 2024, Osmanthus fragrans cuttings were evenly sprayed with 300 μM abscisic acid (ABA) until the leaves were completely moistened (200 mL per plant) and cultured in a light incubator at 25°C with the same photoperiod (12 h light / 12 h dark) and humidity (60%). Samples were then collected at 0, 3, 6, 12, 24, and 72 h after treatment. Three biological replicates were performed for each treatment. Upon collection, samples were quickly frozen with liquid nitrogen.

[0034] (2) Total RNA extraction from Osmanthus fragrans leaves

[0035] Total RNA from each sample was extracted according to the instructions of HiPure Plant RNA Mini Kit (Magen), and its integrity, purity, and concentration were determined by 1% agarose gel electrophoresis and spectrophotometry (Nano Drop 2000, Thermo Scientific, Wilmington, DE, USA).

[0036] (3) Reverse transcription to synthesize cDNA

[0037] Using total RNA as template, cDNA was synthesized using III RT SuperMix for qPCR (+gDNAwiper) kit (Nanjing Novozymes Biotechnology Co., Ltd.) and stored in a −20°C refrigerator until use.

[0038] (4) Selection of internal reference genes and design of their primers

[0039] Through literature review and database searches, we performed local blast comparisons (blastVersion 2.4.0+) of previously reported reference genes from other species against the Osmanthus fragrans genome data to identify genes with the highest homology. We then used blastx on the NCBI website to analyze the conserved domains and ORFs of these genes online. We then used Primer5 software to design primers for the CDS regions of these reference genes. Ultimately, we selected and designed seven candidate reference genes. Based on RNA-seq, we selected 10 lncRNAs with relatively high expression levels and fold differences <1.4 as reference genes. The candidate reference genes and their primers are listed in Table 1.

[0040] (5) qRT-PCR quantification

[0041] The protocol was as follows: 95°C for 30 seconds, followed by 40 cycles of 95°C for 10 seconds, 60°C for 30 seconds, and then 60-95°C for melting curve generation. A 20 μL reaction consisted of 10 μL of 2× Taq Pro Universal SYBR qPCR Master Mix, 1 μL of 10-fold diluted cDNA, and 0.8 μL of forward and backward primers (10 μM), brought to 20 μL with ddH2O. Each qRT-PCR analysis was performed in triplicate, and a non-template control was included for each gene.

[0042] (6) Stability assessment

[0043] Internal reference stability analysis uses 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.

[0044] (7) Results

[0045] 1) Delta-CT analysis: Delta-CT analysis was used to determine the mean standard deviation. A higher mean standard deviation indicates poorer gene stability, while a lower mean standard deviation indicates higher gene stability. The results are shown in Table 2. lnc00239991 and lnc00042194 had mean standard deviations of 0.78 and 0.81, respectively, indicating they were the most stable genes. The least stable genes were lnc00031789 and lnc00044331.

[0046] 2) geNorm software analysis: The expression stability M value of each candidate internal reference gene was calculated using 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. lnc00265419 and 18S have the smallest M values ​​and are stable internal references ( Figure 1 ). In addition, according to the geNorm pairwise variation value V n / V n+1 Analysis was performed to determine the appropriate number of internal reference genes, as the paired variation value V2 / V3 < 0.15 ( Figure 2 ), therefore, only two internal reference genes (18S and lnc00265419) were needed for relative gene expression analysis under ABA treatment in Osmanthus fragrans.

[0047] 3) BestKeeper software analysis: The standard deviation (SD) of the candidate internal reference genes was calculated using BestKeeper software. The smaller the SD value, the more stable the expression. The default critical value of the program is 1. When the SD value is greater than 1, the gene expression is considered unstable. The results are shown in Table 3, showing that the SD values ​​of 18S, lnc00229717, lnc00239991, lnc00087780, lnc00265419, ACT7, lnc00249739, TUA5, EF1B, lnc00042194, lnc00174850, UBQ4, and RAN1 are greater than 1, while the SD values ​​of the remaining genes are all less than 1. The gene stability is 18S>lnc00229717, lnc00239991, lnc00087780, lnc00265419, ACT7, lnc00249739, TUA5, EF1B, lnc00042194, lnc00174850, UBQ4, and RAN1. 17>lnc00239991>lnc00087780>lnc00265419>ACT7>lnc00249739>TUA5>EF1B>lnc00042194>lnc00174850>UBQ4>RAN1>GAPH>lnc00067193>lnc00031789>lnc00044331. The most stably expressed genes are 18S and lnc00229717.

[0048] 4) NormFinder Software Analysis: Similar to the geNorm program, NormFinder also first obtains the stability values ​​of reference gene expression and then selects the most suitable reference gene based on the stability values. The reference gene with the smallest stability value is considered the most suitable reference gene. The results are shown in Table 4. lnc00239991 and GAPH, with stability values ​​of 0.198 and 0.209, respectively, are the most stable genes; lnc00031789 and lnc00044331, with stability values ​​of 1.910 and 2.579, are the least stable lncRNAs.

[0049] RefFinder analysis: The geometric mean of the stability rankings obtained from the above-mentioned analyses was calculated using the RefFinder website. A smaller geometric mean indicates more stable expression; conversely, a larger geometric mean indicates more unstable expression. The results are shown in Table 5. The gene stability ranking is lnc00239991 > 18S > lnc00265419 > lnc00229717 > lnc00042194 > EF1B > GAPH > lnc00249739 > lnc00087780 > lnc00174850 > lnc00067193 > ACT7 > RAN1 > TUA5 > UBQ4 > lnc00031789 > lnc00044331. Among them, lnc00239991 and 18S are the most stable gene combination, while lnc00031789 and lnc00044331 are the least stable genes.

[0050] Example 2

[0051] (1) MeJA treatment

[0052] In April 2024, Osmanthus fragrans cuttings were evenly sprayed with 300 μM MeJA until the leaves were completely moistened (200 mL per plant) and cultured in a light incubator at 25°C with a uniform photoperiod (12 h light / 12 h dark) and humidity (60%). Samples were then collected at 0, 3, 6, 12, 24, and 72 h after treatment. Three biological replicates were performed for each treatment. Upon collection, samples were quickly frozen in liquid nitrogen.

[0053] (2) Total RNA extraction from Osmanthus fragrans leaves

[0054] Total RNA from each sample was extracted according to the instructions of HiPure Plant RNA Mini Kit (Magen), and its integrity, purity, and concentration were tested by 1% agarose gel electrophoresis and spectrophotometry (Nano Drop 2000, Thermo Scientific, Wilmington, DE, USA).

[0055] (3) Reverse transcription to synthesize cDNA

[0056] Using total RNA as template, cDNA was synthesized using III RT SuperMix for qPCR (+gDNAwiper) kit (Nanjing Novozymes Biotechnology Co., Ltd.) and stored in a −20°C refrigerator until use.

[0057] (4) Selection of internal reference genes and design of their primers

[0058] Through literature review and database searches, we performed local blast comparisons (blastVersion 2.4.0+) of previously reported reference genes from other species against Osmanthus fragrans RNA-seq data to identify genes with the highest homology. We then used blastx (available on the NCBI website) to analyze the conserved domains and ORFs of these genes online. We then used Primer5 software to design the CDS regions of reference primers, ultimately selecting and designing the seven candidate reference genes. Based on RNA-seq, we selected 10 lncRNAs with relatively high expression levels and fold differences <1.4 as reference genes.

[0059] (5) qRT-PCR quantification

[0060] The protocol was as follows: 95°C for 30 seconds, followed by 40 cycles of 95°C for 10 seconds, 60°C for 30 seconds, and then 60-95°C for melting curve generation. A 20 μL reaction consisted of 10 μL of 2× Taq Pro Universal SYBR qPCR Master Mix, 1 μL of 10-fold diluted cDNA, and 0.8 μL of forward and backward primers (10 μM), brought to 20 μL with ddH2O. Each qRT-PCR analysis was performed in triplicate, and a non-template control was included for each gene.

[0061] (6) Stability assessment

[0062] Internal reference gene stability analysis uses 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.

[0063] (7) Results

[0064] 1) Delta-CT analysis: Delta-CT analysis was used to obtain the mean standard deviation. A higher mean standard deviation indicates poorer gene stability, while a lower mean standard deviation indicates higher gene stability. The results are shown in Table 2. lnc00265419 and lnc00249739 are the most stable genes; the least stable genes are TUA5 and lnc00031789.

[0065] 2) geNorm software analysis: The expression stability M value of each candidate internal reference gene was calculated using 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. 18S and lnc00265419 have the smallest M values ​​and are stable internal references ( Figure 1 ). In addition, according to the geNorm pairwise variation value V n / V n+1 Analysis was performed to determine the appropriate number of internal reference genes, as the paired variation value V2 / V3 < 0.15 ( Figure 2 ), therefore, only two internal references were needed for relative gene expression analysis under Osmanthus fragrans MeJA treatment conditions.

[0066] 3) BestKeeper software analysis: The standard deviation (SD) of the candidate internal reference genes was calculated using BestKeeper software. The smaller the SD value, the more stable the expression. The default critical value of the program is 1. When the SD value is greater than 1, the gene expression is considered unstable. The results are shown in Table 3, showing that the SD values ​​of 18S, lnc00249739, lnc00265419, lnc00239991, lnc00229717, UBQ4, lnc00067193, GAPH, lnc00042194, ACT7, RAN1, EF1B, lnc00087780, and lnc00174850 are greater than 1, while the SD values ​​of the remaining genes are all less than 1. The gene stability is 18S>lnc 00249739>lnc00265419>lnc00239991>lnc00229717>UBQ4>lnc00067193>GAPH>lnc00042194>ACT7>RAN1>EF1B>lnc00087780>lnc00174850>lnc00044331>TUA5>lnc00031789, the most stably expressed are 18S and lnc00249739.

[0067] 4) NormFinder Software Analysis: Similar to the geNorm program, NormFinder also first obtains the stability values ​​of the reference gene expression and then selects the most suitable reference gene based on the stability values. The reference gene with the smallest stability value is considered the most suitable reference gene. The results are shown in Table 4. lnc00265419 and lnc00249739 have stability values ​​of 0.182 and 0.297, respectively, making them the most stable genes. TUA5 and lnc00031789 have stability values ​​of 1.322 and 1.755, respectively, making them the least stable genes.

[0068] 5) RefFinder analysis: The geometric mean of the stability rankings obtained from the above multiple analyses was calculated using the RefFinder website. A smaller geometric mean indicates more stable expression; conversely, a larger geometric mean indicates more unstable expression. The results are shown in Table 5. The gene stability order is lnc00265419 > lnc00249739 > 18S > lnc00239991 > lnc00067193 > lnc00042194 > GAPH > RAN1 > UBQ4 > ACT7 > lnc00229717 > lnc00087780 > lnc00174850 > EF1B > lnc00044331 > TUA5 > lnc00031789. Among them, lnc00265419 and lnc00249739 are the most stable gene combination, while TUA5 and lnc00031789 are the least stable genes.

[0069] Example 3

[0070] (1) Ethephon treatment

[0071] In April 2024, Osmanthus fragrans cuttings were evenly sprayed with 5 mM ethephon until the leaves were completely moistened (200 mL per plant) and incubated in a light incubator at 25°C with a uniform photoperiod (12 h light / 12 h dark) and humidity (60%). Samples were collected at 0, 3, 6, 12, 24, and 72 h after treatment. Three biological replicates were used for each treatment. Upon collection, samples were quickly frozen in liquid nitrogen.

[0072] (2) Total RNA extraction from Osmanthus fragrans leaves

[0073] Total RNA from each sample was extracted according to the instructions of HiPure Plant RNA Mini Kit (Magen), and its integrity, purity, and concentration were determined by 1% agarose gel electrophoresis and spectrophotometry (Nano Drop 2000, Thermo Scientific, Wilmington, DE, USA).

[0074] (3) Reverse transcription to synthesize cDNA

[0075] Using total RNA as template, cDNA was synthesized using III RT SuperMix for qPCR (+gDNAwiper) kit (Nanjing Novozymes Biotechnology Co., Ltd.) and stored in a −20°C refrigerator until use.

[0076] (4) Selection of internal reference genes and design of their primers

[0077] Through literature review and database searches, we performed a local blast comparison (blastVersion 2.4.0+) of previously reported reference genes from other species against the Osmanthus fragrans genome data to identify the genes with the highest homology. We then used blastx (available on the NCBI website) to analyze the conserved domains and ORFs of these genes online. We then used Primer5 software to design the CDS regions of the reference primers, ultimately selecting and designing the seven candidate reference genes. Based on RNA-seq, we selected 10 lncRNAs with relatively high expression levels and fold differences <1.4 as reference genes.

[0078] (5) qRT-PCR quantification

[0079] The protocol was as follows: 95°C for 30 seconds, followed by 40 cycles of 95°C for 10 seconds, 60°C for 30 seconds, and then 60-95°C for melting curve generation. A 20 μL reaction consisted of 10 μL of 2× Taq Pro Universal SYBR qPCR Master Mix, 1 μL of 10-fold diluted cDNA, and 0.8 μL of forward and backward primers (10 μM), brought to 20 μL with ddH2O. Each qRT-PCR analysis was performed in triplicate, and a non-template control was included for each gene.

[0080] (6) Stability assessment

[0081] Internal reference gene stability analysis uses 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.

[0082] (7) Results

[0083] 1) Delta-CT analysis: Delta-CT analysis was used to determine the mean standard deviation. A higher mean standard deviation indicates poorer gene stability, while a lower mean standard deviation indicates higher gene stability. The results are shown in Table 2. lnc00229717 and lnc00044331 had mean standard deviations of 0.55 and 0.58, respectively, indicating they were the most stable genes. TUA5 and lnc00031789 were the least stable genes.

[0084] 2) geNorm software analysis: The expression stability M value of each candidate internal reference gene was calculated using 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. lnc00174850 and EF1B have the smallest M values ​​and are stable internal reference genes ( Figure 1 ). In addition, according to the geNorm pairwise variation value V n / V n+1 Analysis was performed to determine the appropriate number of internal reference genes. Since the paired variation value V2 / V3 of the embodiment was less than 0.15 ( Figure 2 ), therefore, only two internal reference genes were needed to analyze the relative expression of genes under the conditions of ethephon treatment in Osmanthus fragrans.

[0085] 3) BestKeeper Software Analysis: BestKeeper software was used to calculate the standard deviation (SD) of candidate reference genes. Smaller SD values ​​indicate more stable expression. The program defaults to a critical value of 1; when the SD value is greater than 1, the gene is considered unstable. The results are shown in Table 3. The order of gene stability is 18S > lnc00265419 > lnc00067193 > lnc00044331 > lnc00249739 > lnc00229717 > EF1B > lnc00174850 > lnc00042194 > ACT7 > GAPH > lnc00239991 > UBQ4 > lnc00087780 > RAN1 > TUA5 > lnc00031789. 18S and lnc00265419 were the most stably expressed.

[0086] 4) NormFinder Software Analysis: Similar to the geNorm program, NormFinder also first obtains the stability values ​​of the reference gene expression and then selects the most suitable reference gene based on the stability values. The reference gene with the smallest stability value is considered the most suitable reference gene. The results are shown in Table 4. lnc00229717 and lnc00044331 have stability values ​​of 0.133 and 0.265, respectively, making them the most stable genes; TUA5 and lnc00031789 have stability values ​​of 0.979 and 1.850, respectively, making them the least stable genes.

[0087] 5) RefFinder analysis: The geometric mean of the stability rankings obtained from the above multiple analyses was calculated using the RefFinder website. A smaller geometric mean indicates more stable expression; conversely, a larger geometric mean indicates more unstable expression. The results are shown in Table 5. The gene stability order is lnc00229717 > lnc00044331 > lnc00249739 > lnc00067193 > lnc00174850 > EF1B > lnc00265419 > 18S > lnc00239991 > GAPH > lnc00042194 > ACT7 > lnc00087780 > UBQ4 > RAN1 > TUA5 > lnc00031789. lnc00229717 and lnc00044331 are the most stable genes, while TUA5 and lnc00031789 are the least stable genes.

[0088] Example 4

[0089] (1) Hormone treatments, including ABA, MeJA, and ethephon. Samples were collected at 0, 3, 6, 12, 24, and 72 h after treatment. Three biological replicates were performed for each treatment. Samples were collected and quickly frozen in liquid nitrogen.

[0090] (2) Total RNA extraction from Osmanthus fragrans leaves

[0091] Total RNA from each sample was extracted according to the instructions of HiPure Plant RNA Mini Kit (Magen), and its integrity, purity, and concentration were tested by 1% agarose gel electrophoresis and spectrophotometry (Nano Drop 2000, Thermo Scientific, Wilmington, DE, USA).

[0092] (3) Reverse transcription to synthesize cDNA

[0093] Using total RNA as template, cDNA was synthesized using III RT SuperMix for qPCR (+gDNAwiper) kit (Nanjing Novozymes Biotechnology Co., Ltd.) and stored in a −20°C refrigerator until use.

[0094] (4) Selection of internal reference genes and design of their primers

[0095] Through literature review and database searches, we performed a local blast comparison (blastVersion 2.4.0+) of previously reported reference genes from other species against the Osmanthus fragrans genome data to identify the genes with the highest homology. We then used blastx (available on the NCBI website) to analyze the conserved domains and ORFs of these genes online. We then used Primer5 software to design the CDS regions of the reference primers, ultimately selecting and designing the seven candidate reference genes. Based on RNA-seq, we selected 10 lncRNAs with relatively high expression levels and fold differences <1.4 as reference genes.

[0096] (5) qRT-PCR quantification

[0097] The protocol was as follows: 95°C for 30 seconds, followed by 40 cycles of 95°C for 10 seconds, 60°C for 30 seconds, and then 60-95°C for melting curve generation. A 20 μL reaction consisted of 10 μL of 2× Taq Pro Universal SYBR qPCR Master Mix, 1 μL of 10-fold diluted cDNA, and 0.8 μL of forward and backward primers (10 μM), brought to 20 μL with ddH2O. Each qRT-PCR analysis was performed in triplicate, and a non-template control was included for each gene.

[0098] (6) Stability assessment

[0099] Internal reference gene stability analysis uses 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.

[0100] (7) Results

[0101] 1) Delta-CT analysis: Delta-CT analysis was used to obtain the mean standard deviation. A higher mean standard deviation indicates poorer gene stability, while a lower mean standard deviation indicates higher gene stability. The results are shown in Table 2. lnc00239991 is the most stable gene, while lnc00044331 is the least stable gene.

[0102] 2) geNorm software analysis: The expression stability M value of each candidate internal reference gene was calculated using 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. 18S and lnc00265419 have the smallest M values ​​and are stable internal references ( Figure 1 ). In addition, according to the geNorm pairwise variation value V n / V n+1Analysis was performed to determine the appropriate number of internal reference genes. Since the paired variation value V2 / V3 of the embodiment was less than 0.15 ( Figure 2 ), therefore, only two internal reference genes were needed to analyze the relative expression of genes under the conditions of osmanthus hormone treatment.

[0103] 3) BestKeeper Software Analysis: BestKeeper software was used to calculate the standard deviation (SD) of candidate reference genes. 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 3, showing that the stability index was 18S > lnc00265419 > lnc00249739 > lnc00229717 > lnc00239991 > UBQ4 > EF1B > lnc00174850 > lnc00087780 > lnc00042194 > GAPH > RAN1 > ACT7 > lnc00067193 > TUA5 > n00044331 > n00031789, among which 18S and lnc00265419 were the most stably expressed.

[0104] 4) NormFinder Software Analysis: Similar to the geNorm program, NormFinder also first obtains the stability values ​​of the reference gene expression and then selects the most suitable reference gene based on the stability values. The reference gene with the smallest stability value is considered the most suitable reference gene. The results are shown in Table 4. The stability values ​​of lnc00239991 and RAN1 were 0.346 and 0.375, respectively, making them the most stable genes. The stability values ​​of lnc00031789 and lnc00044331 were 1.664 and 1.757, respectively, making them the least stable genes.

[0105] 5) RefFinder analysis: The geometric mean of the stability rankings obtained from the above multiple analyses was calculated using the RefFinder website. A smaller geometric mean indicates more stable expression; conversely, a larger geometric mean indicates more unstable expression. The results are shown in Table 5. The gene stability order is lnc00265419 > lnc00239991 > 18S > RAN1 > lnc00249739 > GAPH > lnc00174850 > lnc00229717 > EF1B > lnc00042194 > lnc00067193 > lnc00087780 > UBQ4 > ACT7 > TUA5 > lnc00031789 > lnc00044331. Among them, lnc00265419 and lnc00239991 are the most stable gene combination, while lnc00031789 and lnc00044331 are the least stable genes.

[0106] Internal reference gene stability verification

[0107] The expression levels of lnc00003036, lnc00126603 (the nucleotide sequences of lnc00003036, lnc00126603, and lnc00250780 are shown in the sequence listing as SEQ ID NOs. 19 to 21, and the primer sequences are shown in SEQ ID NOs. 22 to 27) and lnc00250780 were verified using stable genes and two unstable genes as internal references. It was found that when stable genes were used as internal references, the expression trends of the three target genes were basically the same; however, when unstable genes were used as internal references, the expression trends of lncRNA and stable genes were inconsistent ( Figures 3a to 5c For example, after ABA treatment, when the unstable lnc00044331 was used as an internal reference, the expression of lnc00003036, lnc00126603, and lnc00250780 was significantly upregulated at 6 hours. However, when the unstable lnc00031789 was used as an internal reference, these genes were significantly downregulated from 6 to 72 hours. After ethephon and MeJA treatment, when lnc00031789 was used as an internal reference, the expression of lnc00003036, lnc00126603, and lnc00250780 was downregulated from 3 to 72 hours. However, when TUA was used as an internal reference, these genes were upregulated. In summary, different lncRNA genes used as internal references exhibited different expression trends and levels, indicating the necessity of using stable internal references.

[0108] Table 1 Primer sequences of 17 candidate reference genes and 3 validation genes

[0109]

[0110]

[0111] Table 2Delta-CT analysis results

[0112]

[0113]

[0114] Table 3 BestKeeper software analysis results

[0115]

[0116] Table 4 NormFinder software analysis results

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[0118]

[0119] Table 5 Analysis results of 17 reference genes from RefFinder website

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[0121]

[0122] 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 principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Application of primers for detecting the expression level of internal reference genes in lncRNA fluorescence quantitative PCR analysis under osmanthus hormone treatment conditions, characterized in that: (1) When the hormone is abscisic acid, the internal reference genes are lnc00239991 and 18S; (2) When the hormone is methyl jasmonate, the internal reference genes are lnc00265419 and lnc00249739; (3) When the hormone is ethephon, the internal reference genes are lnc00229717 and lnc00044331; (4) When the hormones are abscisic acid, methyl jasmonate, and ethephon, the internal reference genes are lnc00265419 and lnc00239991; Among them, the nucleic acid sequence of lnc00239991 is shown in SEQ ID NO.1, the nucleic acid sequence of 18S is shown in SEQ ID NO.2, the nucleic acid sequence of lnc00265419 is shown in SEQ ID NO.3, the nucleic acid sequence of lnc00249739 is shown in SEQ ID NO.4, the nucleic acid sequence of lnc00229717 is shown in SEQ ID NO.5, and the nucleic acid sequence of lnc00044331 is shown in SEQ ID NO.

6.

2. The use according to claim 1, characterized in that Forward primer for detecting lnc00239991: 5'-TTTCTTGGTCGTGTCTTTAGCA-3'; reverse primer: 5'-CAAGTTGCGGGAGACGTTAT-3'; Forward primer for detecting the 18S: 5'-CCATAAACGATGCCGACCAG-3'; reverse primer: 5'-GCCTTGCGACCATACTCCC-3'; Forward primer for detecting lnc00265419: 5'-CATTATTGTTACGCCGACCAC-3'; reverse primer: 5'-GATCGTTTAGCCGCTCTTTCT-3'; Forward primer for detecting lnc00249739: 5'-TGGACTTGGCTGACCCTTGA-3'; reverse primer: 5'-TTCCAATCTTGCGGACTGAC-3'; Forward primer for detecting lnc00229717: 5'-CTTAGCCGCCCATCCCA-3'; reverse primer: 5'-CACCCGCATTATCCGTTGA-3'; The forward primer used to detect lnc00044331 was 5'-GGGTTGTGGCGGGTAATTCATCTTG-3'; the reverse primer was 5'-GGTAAGGGATGTAATAATGTGCTGA-3'.

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