LncRNA reference genes and their applications under metal ion stress in Osmanthus fragrans

Abstract

The present invention discloses lncRNA reference genes and applications under metal ion stress of sweet osmanthus, belonging to the field of plant molecular biology. 2+ The best internal references under ion stress were lnc00067193, 18S, and Fe 2+ The best internal references under ion stress were lnc00229717 and ACT7, Al 3+ The optimal internal references for ion stress were lnc00087780 and lnc00265419, and for metal ion stress were lnc00239991 and lnc00067193. These results fill the technical gap of the lack of lncRNA internal references under metal ion stress in Osmanthus fragrans, provide strong support for the quantification of lncRNAs under metal ion stress in Osmanthus fragrans, and provide a theoretical basis for the functional study of lncRNAs in Osmanthus fragrans.

Description

Technical Field

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

[0002] Sweet osmanthus, Osmanthus fragrans, is a woody flower in the Oleaceae family with a unique fragrance and is a common plant in traditional Chinese gardens. Osmanthus trees typically grow 3–10 meters tall, with evergreen, oval or ovate leaves. Osmanthus flowers are small and delicate, with petals in white, pale yellow, or orange-red. Blooming in autumn, its rich aroma is renowned far and wide, earning it the nickname "the fragrance of osmanthus that spreads ten miles." In addition to its ornamental value, osmanthus has medicinal and edible properties and, as a nectar source, attracts many bees. Despite its significant ecological and economic value, relatively little research has examined the physiological and molecular mechanisms of its effects under metal ion stress. In particular, studies examining the expression of lncRNAs (long noncoding RNAs) in osmanthus under metal ion stress have been reported.

[0003] LncRNAs are a class of non-coding RNA molecules longer than 200 nucleotides. Although they do not encode proteins, they play an important role in regulating gene expression, plant growth and development, and stress responses. In plants, lncRNAs have been shown to play a key role in responding to biotic and abiotic stresses, such as drought, salt stress, and metal ion stress. Studying lncRNA expression in Osmanthus fragrans under metal ion stress can reveal its response mechanisms and provide a theoretical basis for improving its stress tolerance. When analyzing lncRNA expression levels using qRT-PCR, selecting appropriate reference genes is crucial for ensuring the accuracy and reliability of experimental results. However, the stability of reference genes can vary across species and stress conditions, necessitating the screening and validation of reference genes for Osmanthus fragrans under metal ion stress. These results are of great significance for revealing the stress response mechanisms of Osmanthus fragrans and improving its stress tolerance. Summary of the Invention

[0004] In view of the deficiencies of the existing technology, the purpose of the present invention is to obtain a lncRNA internal reference that is stably expressed in osmanthus under metal ion stress conditions. The lncRNA can meet the requirements of real-time fluorescence quantitative detection of osmanthus transcriptional expression levels, thereby improving the stability, reliability and efficiency of osmanthus lncRNA expression analysis research.

[0005] In order to achieve the above objectives, the technical solutions adopted by the present invention are as follows:

[0006] lncRNA reference genes under metal ion stress in Osmanthus fragrans, wherein the lncRNA reference genes include at least one of the following (1) to (4);

[0007] (1)Cu 2+ Reference genes lnc00067193 and 18S under ionic stress;

[0008] (2)Fe 2+ Reference genes lnc00229717 and ACT7 under ionic stress;

[0009] (3)Al 3+ Reference genes lnc00087780 and lnc00265419 under ionic stress;

[0010] (4) Reference genes lnc00239991 and lnc00067193 under metal ion stress;

[0011] Among them, the nucleic acid sequence of lnc00067193 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 lnc00229717 is shown in SEQ ID NO.3, the nucleic acid sequence of ACT7 is shown in SEQ ID NO.4, the nucleic acid sequence of lnc00087780 is shown in SEQ ID NO.5, the nucleic acid sequence of lnc00265419 is shown in SEQ ID NO.6, and the nucleic acid sequence of lnc00239991 is shown in SEQ ID NO.7.

[0012] Furthermore, the present invention provides proprietary primers for detecting lnc00067193, the sequences of which are: forward primer 5'-GCATCGGCGATTGTGAGA-3' (SEQ ID NO. 8); reverse primer 5'-AAGCGAAGGTCCGTTTGG-3' (SEQ ID NO. 9).

[0013] The proprietary primers used to detect 18S were: forward primer 5'-CCATAAACGATGCCGACCAG-3' (SEQ ID NO. 10); reverse primer 5'-GCCTTGCGACCATACTCCC-3' (SEQ ID NO. 11).

[0014] The proprietary primers used to detect lnc00229717 were: forward primer 5'-CTTAGCCGCCCATCCCA-3' (SEQ ID NO. 12), and reverse primer 5'-CACCCGCATTATCCGTTGA-3' (SEQ ID NO. 13).

[0015] The proprietary primers used to detect ACT7 are: forward primer 5'-AAATCACTGCCTTGGCTCCTA-3' (SEQ ID NO. 14); reverse primer 5'-GCACTTCCTGTGGACGATAGA-3' (SEQ ID NO. 15).

[0016] The proprietary primers used to detect lnc00087780 were: forward primer 5'-CGAACTGCCCTTATGGTTATTC-3' (SEQ ID NO. 16), reverse primer 5'-CCTGGCGTAGATTCCTTGC-3' (SEQ ID NO. 17).

[0017] The proprietary primers used to detect lnc00265419 were: forward primer 5'-CATTATTGTTACGCCGACCAC-3' (SEQ ID NO. 18) and reverse primer 5'-GATCGTTTAGCCGCTCTTTCT-3' (SEQ ID NO. 19).

[0018] The proprietary primers used to detect lnc00239991 were: forward primer 5′-TTTCTTGGTCGTGTCTTTAGCA-3′ (SEQ ID NO. 20) and reverse primer 5′-CAAGTTGCGGGAGACGTTAT-3′ (SEQ ID NO. 21).

[0019] Furthermore, the present invention also relates to the use of the lncRNA internal reference gene or its proprietary primers in the fluorescence quantitative PCR analysis of lncRNA in Osmanthus fragrans under metal ion stress.

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

[0021] The present invention identified 17 reference genes as candidate genes through literature review, database search and RNA sequencing, and designed primer sequences for 17 internal reference genes. The stability of candidate genes was evaluated by five algorithms (delta-CT, geNorm, NormFinder, BestKeeper and RefFinder), and the stability of Cu2+ was obtained. 2+ The best internal references under ion stress were lnc00067193, 18S, and Fe 2+ The best internal references under ion stress were lnc00229717 and ACT7, Al 3+The optimal internal reference genes under ion stress were lnc00087780 and lnc00265419, and the optimal internal reference genes under metal ion stress were lnc00239991 and lnc00067193. Real-time fluorescence quantitative PCR primers for the internal reference genes were designed. These primers have strong specificity and high amplification efficiency, which can greatly improve the detection efficiency of Osmanthus fragrans lncRNA using real-time fluorescence quantitative detection and enhance the credibility of the detection results. 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 expression.

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

[0024] Figure 3a to Figure 3c : Stable genes and unstable genes were used as internal references, and the expression level of lnc00003036;

[0025] Figure 4a to Figure 4c : Stable genes and unstable genes were used as internal references, and the expression level of lnc00126603;

[0026] Figure 5a to Figure 5c : Stable genes and unstable genes were used as internal references, 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 with good growth and no 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 for 10 minutes, rinsed with distilled water 3 times, and 0.1g 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) Screening of candidate reference genes and design of their primers

[0033] Through literature review and database searches, we performed a local blast comparison (blastVersion 2.4.0+) of reference genes reported for other species against Osmanthus fragrans RNA-seq data to identify seven candidate reference genes with the highest homology. Based on RNA-seq, we selected 10 lncRNAs with relatively high expression levels and fold differences <1.4 as internal references. Primer5 software was used for design, using the following parameters: PCR product length 70-300 bp, melting temperature 58-62°C, and CG content 40-60%. The candidate reference genes and their primer sequences are shown in Table 1.

[0034] (2) Material treatment: In April 2024, the cuttings were sprayed with 3 mM CuSO4·5H2O 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 used for each treatment. Samples were quickly frozen with liquid nitrogen upon collection.

[0035] (3) Total RNA extraction and cDNA preparation: Total RNA from each sample was extracted using the HiPure Plant RNA Mini Kit (Magen). Its integrity, purity, and concentration were determined by 1% agarose gel electrophoresis and a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). Total RNA was used as a template and a RT-PCR premix kit ( cDNA was synthesized using III RT SuperMix for qPCR (+gDNAwiper) R323-01 (Nanjing Novozymes Biotechnology Co., Ltd.). 1 μg of RNA was used each time to synthesize the first-strand cDNA and stored in a −20°C refrigerator until use.

[0036] (4) qRT-PCR quantification

[0037] 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.

[0038] (5) Stability assessment

[0039] Delta-CT analysis: Delta-CT analysis was used to obtain the mean standard deviation. The higher the mean standard deviation, the less stable the gene. Conversely, the lower the mean standard deviation, the more stable the gene. The results are shown in Table 2. The mean standard deviation of lnc00067193 was 0.52, making it the most stable gene. The mean standard deviation of lnc00031789 was 1.50, making it the least stable gene.

[0040] 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 ( Figure 1 ). In addition, according to the geNorm pairwise variation value V n / Vn+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 ), so in Osmanthus fragrans 2+ Only two internal reference genes (18S and lnc00067193) are needed to analyze the relative expression of genes under stress.

[0041] 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's default critical value is 1; when the SD value is greater than 1, the gene is considered unstable. The results are shown in Table 3. The SD value of lnc00031789 is greater than 1, while the SD values ​​of the remaining genes are all less than 1. The gene stability order is UBQ4 > 18S > GAPH > lnc00265419 > lnc00042194 > lnc00067193 > lnc00239991 > RAN1 > EF1B > ACT7 > lnc00174850 > lnc00229717 > lnc00249739 > lnc00087780 > lnc00044331 > TUA5 > lnc00031789, with UBQ4 being the most stably expressed.

[0042] NormFinder software analysis: The stability values ​​of candidate reference genes were calculated using NormFinder software. Higher stability values ​​indicate poorer stability, while lower stability values ​​indicate better stability. In other words, the gene with the smallest stability value is considered the most stable. The results are shown in Table 4. lnc00067193 has a stability value of 0.231, making it the most stable gene; lnc00031789 has a stability value of 1.451, making it the least stable gene.

[0043] 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 lnc00067193 > 18S > lnc00265419 > lnc00239991 > UBQ4 > GAPH > lnc00229717 > lnc00042194 > lnc00249739 > RAN1 > EF1B > lnc00174850 > ACT7 > lnc00044331 > lnc00087780 > TUA5 > lnc00031789. Among them, lnc00067193 + 18S is the most stable gene combination, while TUA5 and lnc00031789 are the least stable genes.

[0044] Example 2

[0045] (1) Screening of candidate reference genes and design of their primers

[0046] Through literature review and database searches, we performed a local blast comparison (blastVersion 2.4.0+) of reference genes reported for other species against the Osmanthus fragrans genome data to identify seven candidate reference genes with the highest homology. Based on RNA-seq, we selected 10 lncRNAs with relatively high expression levels and fold differences <1.4 as internal references. Primer 5.0 software was used for design, using the following parameters: PCR product length 70-300 bp, melting temperature 58-62°C, and CG content 40-60%.

[0047] (2) Material treatment: In April 2024, the cuttings were sprayed with 3 mM FeSO4 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 cycle) and humidity (60%). Samples were then collected at 0, 3, 6, 12, 24, and 72 h after treatment. Three biological replicates were used for each treatment. Samples were quickly frozen with liquid nitrogen upon collection.

[0048] (3) Total RNA extraction and cDNA preparation: Total RNA from each sample was extracted using the HiPure Plant RNA Mini Kit (Magen). Its integrity, purity, and concentration were tested by 1% agarose gel electrophoresis and spectrophotometry (NanoDrop 2000, Thermo Scientific, Wilmington, DE, USA). Using total RNA as a template, a RT-PCR premix kit ( cDNA was synthesized using III RT SuperMix for qPCR (+gDNAwiper) R323-01 (Nanjing Novozymes Biotechnology Co., Ltd.). 1.0 μg of RNA was used each time to synthesize the first-strand cDNA and stored in a −20°C refrigerator until use.

[0049] (4) qRT-PCR quantification

[0050] 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.

[0051] (5) Stability assessment

[0052] Delta-CT analysis: Delta-CT analysis was used to obtain the mean standard deviation. The higher the mean standard deviation, the less stable the gene. Conversely, the lower the mean standard deviation, the more stable the gene. The results are shown in Table 2. The mean standard deviation of lnc00229717 was 0.61, making it the most stable gene. The mean standard deviation of lnc00031789 was 1.89, making it the least stable gene.

[0053] 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 ( 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 ), so in Osmanthus fragrans Fe 2+ Only two internal reference genes (lnc00229717 and lnc00239991) are needed to analyze the relative expression of genes under stress.

[0054] 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's default critical value is 1; when the SD value is greater than 1, the gene is considered unstable. The results are shown in Table 3. The SD values ​​of TUA5 and lnc00031789 are greater than 1, while the SD values ​​of the remaining genes are all less than 1. The order of gene stability is ACT7 > lnc00229717 > RAN1 > lnc00067193 > lnc00044331 > lnc00249739 > GAPH > UBQ4 > lnc00087780 > lnc00239991 > lnc00042194 > lnc00174850 > EF1B > lnc00265419 > 18S > TUA5 > lnc00031789, with ACT7 being the most stably expressed.

[0055] NormFinder software analysis: The stability values ​​of candidate reference genes were calculated using NormFinder software. Higher stability values ​​indicate poorer stability, while lower stability values ​​indicate better stability. The gene with the lowest stability value is considered the most stable. The results are shown in Table 4. lnc00229717 has a stability value of 0.134, making it the most stable gene; lnc00031789 has a stability value of 1.831, making it the least stable gene.

[0056] 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 lnc00229717 > ACT7 > lnc00249739 > lnc00239991 > RAN1 > lnc00044331 > lnc00067193 > UBQ4 > GAPH > lnc00087780 > lnc00265419 > lnc00042194 > EF1B > lnc00174850 > 18S > TUA5 > lnc00031789. The combination of lnc00229717 and ACT7 is the most stable, while TUA5 and lnc00031789 are the least stable.

[0057] Example 3

[0058] (1) Screening of candidate reference genes and design of their primers

[0059] 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 seven candidate reference genes with the highest homology. Based on RNA-seq, we selected 10 lncRNAs with relatively high expression levels and fold differences <1.4 as reference genes. Primer5 software was used for design, using the following parameters: PCR product length 70-300 bp, melting temperature 58-62°C, and CG content 40-60%.

[0060] (2) Material treatment: In April 2024, the cuttings were sprayed with 3 mM AlCl3·6H2O 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 cycle) and humidity (60%). Samples were then collected at 0, 3, 6, 12, 24, and 72 h after treatment. Three biological replicates were used for each treatment. Samples were quickly frozen with liquid nitrogen upon collection.

[0061] (3) Total RNA extraction and cDNA preparation: Total RNA from each sample was extracted using the HiPure Plant RNA Mini Kit (Magen). Its integrity, purity, and concentration were determined by 1% agarose gel electrophoresis and a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). Total RNA was used as a template and a RT-PCR premix kit ( cDNA was synthesized using III RT SuperMix for qPCR (+gDNAwiper, Nanjing Novozymes Biotechnology Co., Ltd.). 1 μg of RNA was used each time to synthesize the first-strand cDNA and stored in a −20°C refrigerator until use.

[0062] (4) qRT-PCR quantification

[0063] 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.

[0064] (5) Stability assessment

[0065] Delta-CT analysis: Delta-CT analysis was used to obtain the mean standard deviation. The higher the mean standard deviation, the less stable the gene. Conversely, the lower the mean standard deviation, the more stable the gene. The results are shown in Table 2. The mean standard deviation of lnc00265419 was 0.76, making it the most stable gene. The mean standard deviation of lnc00031789 was 1.77, making it the least stable gene.

[0066] 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 ( 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 ), so in Osmanthus Al 3+ Only two internal references (lnc00042194 and lnc00239991) are needed to analyze the relative expression of genes under stress.

[0067] 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's default critical value is 1; when the SD value is greater than 1, the gene is considered unstable. The results are shown in Table 3. The SD values ​​of TUA5 and lnc00031789 are greater than 1, while the SD values ​​of the remaining genes are all less than 1. The gene stability order is lnc00087780 > lnc00249739 > lnc00239991 > lnc00265419 > EF1B > UBQ4 > 18S > lnc00042194 > lnc00044331 > GAPH > lnc00067193 > TUA5 > ACT7 > RAN1 > lnc00229717 > lnc00174850 > lnc00031789, with lnc00087780 being the most stably expressed.

[0068] NormFinder software analysis: The stability values ​​of candidate reference genes were calculated using NormFinder software. Higher stability values ​​indicate poorer stability, while lower stability values ​​indicate better stability. In other words, the gene with the smallest stability value is considered the most stable. The results are shown in Table 4. lnc00087780 has a stability value of 0.242, making it the most stable gene; lnc00031789 has a stability value of 1.682, making it the least stable gene.

[0069] 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 lnc00087780 > lnc00265419 > lnc00239991 > lnc00249739 > lnc00042194 > lnc00067193 > UBQ4 > EF1B > lnc00044331 > ACT7 > GAPH > 18S > RAN1 > lnc00229717 > TUA5 > lnc00174850 > lnc00031789. Among them, lnc00087780 + lnc00265419 is the most stable gene combination, while lnc00174850 and lnc00031789 are the least stable lncRNAs.

[0070] Example 4

[0071] (1) Screening of candidate internal controls and application of their primers

[0072] Through literature review and database searches, we performed a local blast comparison (blastVersion 2.4.0+) of reference genes reported for other species against the Osmanthus fragrans genome data to identify seven candidate reference genes with the highest homology. Based on RNA-seq, we selected 10 lncRNAs with relatively high expression levels and fold differences <1.4 as internal references. Primer5 software was used for design, with the following parameters: PCR product length 70-300 bp, melting temperature 58-62°C, and CG content 40-60%.

[0073] (2) Material treatment: In April 2024, the cuttings were sprayed with 3 mM CuSO4·5H2O, 3 mM Alcl3·6H2O, and 3 mM FeSO4 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 cycle) and humidity (60%). Samples were then taken at 0, 3, 6, 12, 24, and 72 h after treatment. Three biological replicates were used for each treatment. The samples were collected and quickly frozen in liquid nitrogen.

[0074] (3) Total RNA extraction and cDNA preparation: Total RNA from each sample was extracted using the HiPure Plant RNA Mini Kit (Magen). Its integrity, purity, and concentration were determined by 1% agarose gel electrophoresis and a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). Total RNA was used as a template and a RT-PCR premix kit ( cDNA was synthesized using III RT SuperMix for qPCR (+gDNA wiper, Nanjing Novozymes Biotechnology Co., Ltd.). 1.0 μg of RNA was used each time for the synthesis of the first-strand cDNA and stored in a −20°C refrigerator until use.

[0075] (4) qRT-PCR quantification

[0076] 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 system 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 reverse 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.

[0077] (5) Stability assessment

[0078] Delta-CT analysis: Delta-CT analysis was used to obtain the mean standard deviation. The higher the mean standard deviation, the less stable the gene. Conversely, the lower the mean standard deviation, the more stable the gene. The results are shown in Table 2. The mean standard deviation of lnc00239991 was 0.70, making it the most stable gene. The mean standard deviation of lnc00031789 was 1.65, making it the least stable gene.

[0079] 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 ( 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 references (lnc00067193 and lnc00239991) were needed to analyze the relative expression of genes under metal ion stress in Osmanthus fragrans.

[0080] 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 SD value of lnc00031789 is greater than 1, while the SD values ​​of the remaining genes are all less than 1. The gene stability order is UBQ4 > 18S > GAPH > lnc00067193 > lnc00239991 > lnc00087780 > ACT7 > lnc00042194 > lnc00265419 > RAN1 > lnc00229717 > lnc00249739 > lnc00044331 > EF1B > lnc00174850 > TUA5 > lnc00031789, with UBQ4 being the most stably expressed.

[0081] NormFinder software analysis: The stability values ​​of candidate reference genes were calculated using NormFinder software. Higher stability values ​​indicate poorer stability, while lower stability values ​​indicate better stability. In other words, the gene with the smallest stability value is considered the most stable. The results are shown in Table 4. lnc00249739 has a stability value of 0.294, making it the most stable gene; lnc00031789 has a stability value of 1.568, making it the least stable gene.

[0082] 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 > lnc00067193 > lnc00249739 > UBQ4 > lnc00265419 > lnc00042194 > ACT7 > lnc00044331 > GAPH > 18S > lnc00229717 > RAN1 > lnc00087780 > EF1B > lnc00174850 > TUA5 > lnc00031789. The most stable gene combination is lnc00239991 + lnc00067193; TUA5 and lnc00031789 are the least stable genes.

[0083] Internal reference gene stability verification

[0084] According to our throughput sequencing results, lnc00003036, lnc00126603 and lnc00250780 were used as target genes (the nucleotide sequences of lnc00003036, lnc00126603 and lnc00250780 are shown in the sequence listing as SEQ ID NO.22-24, and the primer sequences are shown in SEQ ID NO.25-30), and stable genes and two unstable genes were used as internal references for calculation to confirm the applicability of the candidate genes evaluated in this study. -△△Ct Methods The expression of three target genes was calculated.

[0085] When the stable gene is used as the internal reference, the expression pattern and expression level of the target gene are similar; when the unstable gene is used as the internal reference, the expression pattern and expression level of the target gene are quite different ( Figures 3a-5c ). For example: Al 3+ Under stress, when the unstable gene lnc00174850 was used as the internal reference gene, the expression levels of lnc00003036, lnc00126603, and lnc00250780 were significantly high at 12h, and when the unstable gene lnc0031789 was used as the internal reference gene, the expression level of lnc00003036 was significantly low at 24-72h ( Figure 3a 、 Figure 4a 、 Figure 5a Under copper ion stress, when the unstable gene TUA was used as the internal reference gene, the target gene expression level was significantly high from 12 to 72 hours, and when the unstable gene lnc0031789 was used as the internal reference gene, the target gene expression level was significantly low from 12 to 72 hours ( Figure 3b 、 Figure 4b 、 Figure 5bUnder iron stress, when the unstable gene TUA was used as the internal reference gene, the target gene expression level was significantly high from 12 to 72 hours, and when the unstable gene lnc0031789 was used as the internal reference gene, the target gene expression level was significantly low from 12 to 72 hours ( Figure 3c 、 Figure 4c 、 Figure 5c ). Therefore, choosing the right gene has a great influence on the expression level of the target gene.

[0086] Table 1 Primer sequences involved in 3 validated lncRNAs in 17 candidate references

[0087]

[0088] E, amplification efficiency; R 2 ,correlation coefficient

[0089] Table 2Delta-CT analysis results

[0090]

[0091] Table 3 BestKeeper software analysis results

[0092]

[0093]

[0094] Table 4 NormFinder software analysis results

[0095]

[0096] Table 5 RefFinder website analysis results

[0097]

[0098]

[0099] 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. lncRNA reference gene under metal ion stress in Osmanthus fragrans, characterized by: The lncRNA reference genes include the following (1), (2), (3) and (4); (1) Cu 2+ The reference genes lnc00067193 and 18S ; (2) Fe 2+ Reference genes lnc00229717 and ACT7 ; (3) Al 3+ internal reference genes lnc00087780 and lnc00265419 under ionic stress; (4) Reference genes lnc00239991 and lnc00067193 under metal ion stress; Among them, the nucleic acid sequence of lnc00067193 is shown in SEQ ID NO.1, 18S The nucleic acid sequence of lnc00229717 is shown in SEQ ID NO.2, and the nucleic acid sequence of lnc00229717 is shown in SEQ ID NO.

3. ACT7 The nucleic acid sequence of lnc00087780 is shown in SEQ ID NO.4, the nucleic acid sequence of lnc00087780 is shown in SEQ ID NO.5, the nucleic acid sequence of lnc00265419 is shown in SEQ ID NO.6, and the nucleic acid sequence of lnc00239991 is shown in SEQ ID NO.

7.

2. Use of the lncRNA internal reference gene under metal ion stress of Osmanthus fragrans according to claim 1 in fluorescence quantitative PCR analysis of lncRNA in Osmanthus fragrans.

3. Use of the proprietary primers for the lncRNA reference gene under metal ion stress of Osmanthus fragrans according to claim 1 in fluorescence quantitative PCR analysis of lncRNA in Osmanthus fragrans.

4. The use according to claim 3, characterized in that The proprietary primers used to detect lnc00067193 described in claim 1 are: Forward primer 5′-GCATCGGCGATTGTGAGA-3′, reverse primer 5′-AAGCGAAGGTCCGTTTGG-3′; For detecting the 18S The proprietary primers are: Forward primer 5′-CCATAAACGATGCCGACCAG-3′, reverse primer 5′-GCCTTGCGACCATACTCCC-3′; The proprietary primers used to detect lnc00229717 described in claim 1 are: Forward primer 5′-CTTAGCCGCCCATCCCA-3′, reverse primer 5′-CACCCGCATTATCCGTTGA-3′; For detecting the ACT7 The proprietary primers are: Forward primer 5′-AAATCACTGCCTTGGCTCCTA-3′, reverse primer 5′-GCACTTCCTGTGGACGATAGA-3′; The proprietary primers used to detect lnc00087780 described in claim 1 are: Forward primer 5′-CGAACTGCCCTTATGGTTATTC-3′, reverse primer 5′-CCTGGCGTAGATTCCTTGC-3′; The proprietary primers used to detect lnc00239991 described in claim 1 are: Forward primer 5′-TTTCTTGGTCGTGTCTTTAGCA-3′, reverse primer 5′-CAAGTTGCGGGAGACGTTAT-3′; The proprietary primers used to detect lnc00265419 described in claim 1 are: Forward primer 5′-CATTATTGTTACGCCGACCAC-3′, reverse primer 5′-GATCGTTTAGCCGCTCTTTCT-3′.

Images