IncRNA reference gene treated by osmanthus fragrans hormone as well as primer and application of lncRNA reference gene

Abstract

The invention discloses an IncRNA reference gene treated by osmanthus fragrans hormone as well as a primer and application of the IncRNA reference gene. According to the present invention, the optimal lncRNA internal reference of osmanthus fragrans under the hormone treatment condition is obtained, the lncRNA internal reference is lnc00239991 + 18S under the treatment of ABA, the lncRNA internal reference is lnc00265419 + lnc00249739 under the treatment of MeJA, the lncRNA internal reference is lnc00229717 + lnc00044331 under the treatment of ethephon, and the lncRNA internal reference is lnc00265419 + lnc00239991 under the treatment of various hormones, and the qRT-PCR primer is designed by using the genes; the technical blank that no internal reference lncRNA exists under the condition of sweet osmanthus hormone treatment is filled, powerful support is provided for quantification of lncRNA under the condition of sweet osmanthus hormone treatment, and a theoretical basis is provided for sweet osmanthus gene function research.

Description

Technical Field

[0001] The 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 the ten famous traditional flowers in my country, with unique fragrance and medicinal value. In plant growth and development, hormones play an important regulatory role, and lncRNA, as an important type of transcriptional regulatory factor, is also involved in the regulatory network of plant growth and development. Therefore, it is of great background significance to study the internal reference lncRNA of sweet osmanthus under hormone treatment conditions, its primers and applications. At present, there have been no reports on the study of lncRNA under ABA, MeJA and ethephon treatment conditions of sweet osmanthus. In the process of exploring the difference in lncRNA expression under hormone treatment conditions of sweet osmanthus, a stable and reliable internal reference lncRNA is needed as a reference for qRT-PCR detection and verification of gene expression levels. Therefore, screening the internal reference lncRNA stably expressed under ABA, MeJA and ethephon treatment conditions of sweet osmanthus plays a key role in the accuracy of its real-time fluorescence quantitative PCR detection results. Summary of the invention

[0003] In view of the deficiencies in the prior art, the object 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 purpose, the technical solution adopted by the present invention is as follows:

[0005] The lncRNA internal reference gene is treated with osmanthus hormone, wherein the lncRNA internal 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 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.

[0011] The present invention further provides a proprietary primer for detecting the lnc00239991, the sequence of which is:

[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 the lnc00265419 are as follows: forward primer: 5'-CATTATTGTTACGCCGACCAC-3' (SEQ ID NO.11); reverse primer: 5'-GATCGTTTAGCCGCTCTTTCT-3' (SEQ ID NO.12).

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

[0016] The sequences of the proprietary primers used to detect the lnc00229717 are as follows: 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 the 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 application of the proprietary primers for the lncRNA internal reference gene treated with osmanthus hormone in the fluorescence 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 invention screens 17 internal reference genes through literature review, database search and analysis, evaluates the stability of candidate internal references through 5 algorithms (deltaCT, geNorm, NormFinder, BestKeeper and RefFinder), obtains the most stable internal reference 18S+lnc00239991 under ABA treatment of osmanthus, the most stable internal reference lnc00265419+lnc00249739 under MeJA treatment of osmanthus, the most stable internal reference lnc00229717+lnc00044331 under ethephon treatment of osmanthus, and the most stable internal reference gene lnc00265419+lnc00239991 under hormone treatment of osmanthus. The internal reference sequences are disclosed, and real-time fluorescence quantitative PCR primers of the internal references are designed in sequence. The primers have strong specificity and high amplification efficiency, thereby greatly improving the detection efficiency of osmanthus genes detected by real-time fluorescence quantitative detection and improving the credibility of the detection results. These results provide some basis for the 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 internal reference genes for accurate quantitative analysis;

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

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

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

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

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

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

[0030] The test material used in the following examples is osmanthus. Osmanthus was selected from the "Chang'e" asexual line with good growth and no pests and diseases in Xianning City, Hubei Province as the mother tree. In May 2023, semi-lignified branches with 2-3 lateral buds of the current year were cut as cuttings (12-16 cm), the upper incision was cut flat, the lower incision was cut at 45°, soaked in distilled water for 12 hours, 1% sodium hypochlorite for 10 minutes, rinsed with distilled water 3 times, and 0.1 g·L -1 Soak in GGR rooting powder for 4 hours. Plant the cuttings in the sweet-scented osmanthus base in Xianning City, 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, 300 μM abscisic acid (ABA) was evenly sprayed on the Osmanthus fragrans cuttings 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 taken at 0, 3, 6, 12, 24, and 72 h after the treatment conditions. Three biological replicates were performed for each treatment. When collecting samples, they were quickly frozen with liquid nitrogen.

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

[0035] Total RNA of 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 spectrophotometer (Nano Drop 2000, Thermo Scientific, Wilmington, DE, USA).

[0036] (3) Reverse transcription to synthesize cDNA

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

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

[0039] Through literature review and database search, the reference genes reported for other species were compared with the whole genome data of Osmanthus fragrans for local blast comparison (blastVer: 2.4.0+) to find the genes with the highest homology. The conserved domains and ORFs of the genes were analyzed online by blastx on the NCBI website. The CDS region of the reference primers was designed using Primer5 software, and finally the required 7 candidate reference genes were selected and designed. Based on RNA-seq, 10 lncRNAs with relatively high expression levels and a difference fold <1.4 were selected as reference genes. The candidate reference genes and their primers are shown in Table 1.

[0040] (5) qRT-PCR quantification

[0041] The program was as follows: 30 s at 95°C, followed by 40 cycles with 10 s at 95°C, 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 Pro Universal SYBR qPCR Master Mix; 1 μL of 10-fold diluted cDNA and 0.8 μL of forward and backward primers (10 μM), and ddH 2 0 to 20 uL. Each qRT-PCR analysis was performed in triplicate and a non-template control was included for each gene.

[0042] (6) Stability assessment

[0043] For internal reference stability analysis, five algorithms including delta-CT, geNorm, NormFinder, BestKeeper and RefFinder were used 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: The standard deviation average value was obtained by delta-CT analysis. The higher the standard deviation average value, the worse the gene stability. On the contrary, the lower the standard deviation average value, the higher the gene stability. The results are shown in Table 2. The standard deviation average values ​​of lnc00239991 and lnc00042194 were 0.78 and 0.81, respectively, which were the most stable genes. The genes with the worst stability were lnc00031789 and lnc00044331.

[0046] 2) geNorm software analysis: The expression stability M value of each candidate internal reference gene was calculated by geNorm software. The larger the M value, the lower the stability; conversely, the smaller the M value, the higher the stability, where M = 1.5 is the upper limit. 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 to determine the appropriate number of internal reference genes, since the pairwise variation value V 2 / V 3 <0.15( Figure 2 ), therefore, only two internal reference genes (18S and lnc00265419) were needed to analyze the relative expression of genes under ABA treatment in Osmanthus fragrans.

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

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

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

[0050] Example 2

[0051] (1) MeJA treatment

[0052] In April 2024, osmanthus cuttings were evenly sprayed with 300 μM MeJA until the leaves were completely wet (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 taken at 0, 3, 6, 12, 24, and 72 h after treatment. Three biological replicates were performed for each treatment. When collecting samples, they were quickly frozen with liquid nitrogen.

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

[0054] Total RNA of 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 spectrophotometer (Nano Drop 2000, Thermo Scientific, Wilmington, DE, USA).

[0055] (3) Reverse transcription to synthesize cDNA

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

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

[0058] Through literature review and database search, the reference genes reported for other species were compared with the Osmanthus fragrans RNA-seq data by local blast comparison (blastVer: 2.4.0+) to find the genes with the highest homology. The conserved domains and ORFs of the genes were analyzed online by blastx on the NCBI website. The CDS region of the reference primers was designed using Primer5 software, and finally the required 7 candidate reference genes were selected and designed. Based on RNA-seq, 10 lncRNAs with relatively high expression levels and a fold difference of <1.4 were selected as references.

[0059] (5) qRT-PCR quantification

[0060] The program was as follows: 30 s at 95°C, followed by 40 cycles with 10 s at 95°C, 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 Pro Universal SYBR qPCR Master Mix; 1 μL of 10-fold diluted cDNA and 0.8 μL of forward and backward primers (10 μM), and ddH 2 0 to 20 uL. Each qRT-PCR analysis was performed in triplicate and a non-template control was included for each gene.

[0061] (6) Stability assessment

[0062] For the stability analysis of internal reference genes, five algorithms including delta-CT, geNorm, NormFinder, BestKeeper and RefFinder were used 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 standard deviation average value. The higher the standard deviation average value, the worse the gene stability. On the contrary, the lower the standard deviation average value, the higher the gene stability. The results are shown in Table 2. lnc00265419 and lnc00249739 are the most stable genes; the genes with the worst stability are TUA5 and lnc00031789.

[0065] 2) geNorm software analysis: The expression stability M value of each candidate internal reference gene was calculated by geNorm software. The larger the M value, the lower the stability; conversely, the smaller the M value, the higher the stability, where M = 1.5 is the upper limit. 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 to determine the appropriate number of internal reference genes, since the pairwise variation value V 2 / V 3 <0.15( Figure 2 ), therefore, only two internal references were needed to analyze the relative expression of genes under the Osmanthus fragrans MeJA treatment condition.

[0066] 3) BestKeeper software analysis: The standard deviation (SD) of the candidate internal reference genes was calculated by BestKeeper software. The smaller the SD value, the more stable the expression. The default critical value of the program was 1. When the SD value was greater than 1, the gene expression was considered unstable. The results are shown in Table 3, showing that the SD values ​​of 18S, lnc00249739, lnc00265419, lnc00239991, lnc00229717, UBQ4, lnc00067193, GAPH, lnc00042194, ACT7, RAN1, EF1B, lnc00087780, and lnc00174850 were greater than 1, and the SD values ​​of the remaining genes were all less than 1. The stability of the gene was 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 value of the internal reference gene expression, and then selects the most suitable internal reference gene based on the stability value. The judgment standard is that the internal reference gene with the smallest expression stability value is the most suitable internal reference gene. The results are shown in Table 4. The stability values ​​of lnc00265419 and lnc00249739 are 0.182 and 0.297, respectively, which are the most stable genes; the stability values ​​of TUA5 and lnc00031789 are 1.322 and 1.755, which are the genes with the worst stability.

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

[0069] Example 3

[0070] (1) Ethephon treatment

[0071] In April 2024, osmanthus cuttings were evenly sprayed with 5 mM ethephon 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 taken at 0, 3, 6, 12, 24, and 72 h after the treatment conditions. Three biological replicates were performed for each treatment. When collecting samples, they were quickly frozen with liquid nitrogen.

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

[0073] Total RNA of 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 spectrophotometer (Nano Drop 2000, Thermo Scientific, Wilmington, DE, USA).

[0074] (3) Reverse transcription to synthesize cDNA

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

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

[0077] Through literature review and database search, the reference genes reported for other species were compared with the whole genome data of Osmanthus fragrans for local blast comparison (blastVer: 2.4.0+) to find the genes with the highest homology. The conserved domains and ORFs of the genes were analyzed online by blastx on the NCBI website. The CDS region of the reference primers was designed using Primer5 software, and finally the required 7 candidate reference genes were selected and designed. Based on RNA-seq, 10 lncRNAs with relatively high expression levels and a difference fold <1.4 were selected as references.

[0078] (5) qRT-PCR quantification

[0079] The program was as follows: 30 s at 95°C, followed by 40 cycles with 10 s at 95°C, 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 Pro Universal SYBR qPCR Master Mix; 1 μL of 10-fold diluted cDNA and 0.8 μL of forward and backward primers (10 μM), and ddH 2 0 to 20 uL. Each qRT-PCR analysis was performed in triplicate and a non-template control was included for each gene.

[0080] (6) Stability assessment

[0081] For the stability analysis of internal reference genes, five algorithms including delta-CT, geNorm, NormFinder, BestKeeper and RefFinder were used 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 obtain the mean standard deviation. The higher the mean standard deviation, the worse the gene stability. On the contrary, the lower the mean standard deviation, the higher the gene stability. The results are shown in Table 2. The mean standard deviations of lnc00229717 and lnc00044331 were 0.55 and 0.58, respectively, which were the most stable genes. The genes with the worst stability were TUA5 and lnc00031789.

[0084] 2) geNorm software analysis: The expression stability M value of each candidate internal reference gene was calculated by geNorm software. The larger the M value, the lower the stability; conversely, the smaller the M value, the higher the stability, where M = 1.5 is the upper limit. lnc00174850 and EF1B 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 to determine the appropriate number of internal reference genes, due to the paired variation value V of the embodiment 2 / V 3 <0.15( Figure 2 ), therefore, only two internal reference genes were needed to analyze the relative expression of genes under the conditions of osmanthus ethephon treatment.

[0085] 3) BestKeeper software analysis: The standard deviation (SD) of the candidate internal reference genes was calculated by BestKeeper software. The smaller the SD value, the more stable the expression. The default critical value of the program was 1. When the SD value was greater than 1, the gene expression was considered unstable. The results are shown in Table 3. The stability of the genes was 18S>lnc00265419>lnc00067193>lnc00044331>lnc00249739>lnc00229717>EF1B>lnc00174850>lnc00042194>ACT7>GAPH>lnc00239991>UBQ4>lnc00087780>RAN1>TUA5>lnc00031789. The most stable expressions were 18S and lnc00265419.

[0086] 4) NormFinder software analysis: Similar to the geNorm program, NormFinder also first obtains the stability value of the internal reference gene expression, and then selects the most suitable internal reference gene based on the stability value. The judgment standard is that the internal reference gene with the smallest expression stability value is the most suitable internal reference gene. The results are shown in Table 4. The stability values ​​of lnc00229717 and lnc00044331 are 0.133 and 0.265, respectively, which are the most stable genes; the stability values ​​of TUA5 and lnc00031789 are 0.979 and 1.850, which are the genes with the worst stability.

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

[0088] Example 4

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

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

[0091] Total RNA of 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 spectrophotometer (Nano Drop 2000, Thermo Scientific, Wilmington, DE, USA).

[0092] (3) Reverse transcription to synthesize cDNA

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

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

[0095] Through literature review and database search, the reference genes reported for other species were compared with the whole genome data of Osmanthus fragrans for local blast comparison (blastVer: 2.4.0+) to find the genes with the highest homology. The conserved domains and ORFs of the genes were analyzed online by blastx on the NCBI website. The CDS region of the reference primers was designed using Primer5 software, and finally the required 7 candidate reference genes were selected and designed. Based on RNA-seq, 10 lncRNAs with relatively high expression levels and a difference fold <1.4 were selected as references.

[0096] (5) qRT-PCR quantification

[0097] The program was as follows: 30 s at 95°C, followed by 40 cycles with 10 s at 95°C, 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 Pro Universal SYBR qPCR Master Mix; 1 μL of 10-fold diluted cDNA and 0.8 μL of forward and backward primers (10 μM), and ddH 2 0 to 20 uL. Each qRT-PCR analysis was performed in triplicate and a non-template control was included for each gene.

[0098] (6) Stability assessment

[0099] For the stability analysis of internal reference genes, five algorithms including delta-CT, geNorm, NormFinder, BestKeeper and RefFinder were used 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 standard deviation average value, where the higher the standard deviation average value, the worse the gene stability, and conversely, the lower the standard deviation average value, the higher the gene stability. The results are shown in Table 2. lnc00239991 is the most stable gene; the gene with the worst stability is lnc00044331.

[0102] 2) geNorm software analysis: The expression stability M value of each candidate internal reference gene was calculated by geNorm software. The larger the M value, the lower the stability; conversely, the smaller the M value, the higher the stability, where M = 1.5 is the upper limit. 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 to determine the appropriate number of internal reference genes, due to the paired variation value V of the embodiment 2 / V 3 <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: The standard deviation (SD) of the candidate internal reference genes was calculated by BestKeeper software. The smaller the SD value, the more stable the expression. The default critical value of the program was 1. When the SD value was greater than 1, the gene expression was considered unstable. The results are shown in Table 3, showing stability 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 stable.

[0104] 4) NormFinder software analysis: Similar to the geNorm program, NormFinder also first obtains the stability value of the internal reference gene expression, and then screens the most suitable internal reference gene based on the stability value. The judgment standard is that the internal reference gene with the smallest expression stability value is the most suitable internal reference gene. The results are shown in Table 4. The stability values ​​of lnc00239991 and RAN1 are 0.346 and 0.375, respectively, which are the most stable genes; the stability values ​​of lnc00031789 and lnc00044331 are 1.664 and 1.757, which are the genes with the worst stability.

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

[0106] Internal reference gene stability verification

[0107] The stable gene and two unstable genes were used as internal references to verify the expression levels of the selected lnc00003036, lnc00126603 (the nucleotide sequences of lnc00003036, lnc00126603 and lnc00250780 are shown in the sequence list SEQ ID NO.19-21, and the primer sequences are shown in SEQ ID NO.22-27) and lnc00250780. It was found that when the stable gene was used as the internal reference, the change trends of the expression of the three target genes were basically the same; but when the unstable gene was used as the internal reference, the change trends of the expression of the lncRNA and the stable gene were inconsistent ( Figure 3a to Figure 5c ). For example, after ABA treatment, when unstable lnc00044331 was used as an internal reference, the expression of lnc00003036, lnc00126603, and lnc00250780 was significantly upregulated at 6h, and when unstable lnc00031789 was used as an internal reference, these genes were significantly low expressed from 6 to 72h. After ethephon and MeJA treatment, when lnc00031789 was used as an internal reference, the expression of lnc00003036, lnc00126603, and lnc00250780 was low expressed from 3 to 72h, while when TUA was used as an internal reference, these genes were upregulated. In summary, different lncRNA genes have different expression trends and expression levels when used as internal references, indicating that it is necessary to use stable internal references.

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

[0109]

[0110]

[0111] Table 2 Delta-CT analysis results

[0112]

[0113]

[0114] Table 3 BestKeeper software analysis results

[0115]

[0116] Table 4 NormFinder software analysis results

[0117]

[0118]

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

[0120]

[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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Osmanthus hormone treatment lncRNA internal reference gene, characterized in that, The lncRNA internal reference gene includes at least one of the following (1) to (4); (1) lncRNA reference genes lnc00239991 and 18S under abscisic acid treatment; (2) lncRNA reference genes lnc00265419 and lnc00249739 under methyl jasmonate treatment; (3) lncRNA reference genes lnc00229717 and lnc00044331 under ethephon treatment; (4) lncRNA reference genes lnc00265419 and lnc00239991 under abscisic acid, methyl jasmonate, and ethephon treatments; 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. A proprietary primer for detecting lnc00239991 according to claim 1, characterized in that: Forward primer: 5'-TTTCTTGGTCGTGTCTTTAGCA-3'; reverse primer: 5'-CAAGTTGCGGGAGACGTTAT-3'.

3. A proprietary primer for detecting 18S according to claim 1, characterized in that: Forward primer: 5'-CCATAAACGATGCCGACCAG-3'; reverse primer: 5'-GCCTTGCGACCATACTCCC-3'.

4. A proprietary primer for detecting lnc00265419 according to claim 1, characterized in that: Forward primer: 5'-CATTATTGTTACGCCGACCAC-3'; reverse primer: 5'-GATCGTTTAGCCGCTCTTTCT-3'.

5. A proprietary primer for detecting lnc00249739 according to claim 1, characterized in that: Forward primer: 5'-TGGACTTGGCTGACCCTTGA-3'; reverse primer: 5'-TTCCAATCTTGCGGACTGAC-3'.

6. A proprietary primer for detecting lnc00229717 according to claim 1, characterized in that: Forward primer: 5'-CTTAGCCGCCCATCCCA-3'; reverse primer: 5'-CACCCGCATTATCCGTTGA-3'.

7. A proprietary primer for detecting lnc00044331 according to claim 1, characterized in that: Forward primer: 5'-GGGTTGTGGCGGGTAATTCATCTTG-3'; reverse primer: 5'-GGTAAGGGATGTAATAATGTGCTGA-3'.

8. Use of the osmanthus hormone-treated lncRNA internal reference gene according to claim 1 in osmanthus lncRNA fluorescence quantitative PCR analysis.

9. Use of the proprietary primers for the lncRNA internal reference gene treated with osmanthus hormone according to claim 1 in osmanthus lncRNA 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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