Application of lncRNA reference genes and primers in the process of Osmanthus fragrans petal opening and senescence

Abstract

The present invention provides the application of lncRNA internal reference genes and primers in the process of osmanthus petal opening and aging, and belongs to the field of plant molecular biology. The present invention finally selects 17 candidate internal references required for this implementation based on literature review and database search, and designs qRT-PCR primers for internal references. The 17 internal references were evaluated for gene stability by delta-CT, GeNorm, NormFinder and BestKeeper, and the RefFinder website was used to comprehensively analyze the results, and the lncRNA internal reference in the process of osmanthus petal opening and aging, i.e., lnc00229717+RAN1, was obtained. In the process of osmanthus petal opening and aging, lnc00229717+RAN1 is more suitable for quantitative analysis of lncRNA.

Description

Technical Field

[0001] The invention relates to the field of plant molecular biology, and in particular to the application of lncRNA internal reference genes and primers thereof in the process of opening and senescence of osmanthus flower petals. Background Art

[0002] Osmanthus fragrans, as a plant with a strong fragrance, has long been favored in the fields of horticulture, food and medicine. The opening and aging process of its flowers is a profound and complex biological phenomenon. In recent years, long noncoding RNA (lncRNA) has gradually attracted the attention of researchers as an important regulatory factor. lncRNA is a class of RNA molecules with a length of more than 200 nucleotides. Although they do not encode proteins, they play a key role in biological processes such as gene expression regulation, cell signal transduction and chromatin modification.

[0003] During the opening and aging of petals, studies have shown that lncRNA is involved in regulating the expression of a variety of key genes, thereby affecting physiological processes such as cell growth, differentiation, and apoptosis. In order to gain a deeper understanding of the growth and development status and anti-aging mechanism of osmanthus petals, qRT-PCR technology is needed to analyze and verify the expression level of genes. In this process, it is particularly important to ensure the stability and reliability of internal references. However, there is currently a lack of suitable internal reference genes for the study of lncRNA in osmanthus.

[0004] By screening out lncRNAs suitable for this process as internal reference genes, the biological status of osmanthus petals can be more accurately evaluated, providing a reliable basis for further research. This work not only helps to reveal the molecular mechanism of osmanthus petal opening and aging, but also provides theoretical support and practical guidance for the cultivation of osmanthus, the development of new varieties, and the mining of its medicinal value. Summary of the invention

[0005] In view of the above problems existing in the prior art, the purpose of the present invention is to obtain a lncRNA internal reference gene that is stably expressed during the opening and aging of osmanthus petals. The internal reference can meet the requirements of qRT-PCR detection of osmanthus transcriptional expression levels and improve the stability and reliability of osmanthus gene expression analysis research.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] Application of lnc00229717 and RAN1 as lncRNA internal reference genes in osmanthus fluorescence quantitative analysis, the nucleotide sequence of lnc00229717 is shown in SEQ ID NO.1, and the nucleotide sequence of RAN1 is shown in SEQ ID NO.2.

[0008] Furthermore, the present invention provides primers for detecting the lncRNA internal reference gene.

[0009] The primer sequences of lnc00229717 are:

[0010] Forward primer: 5′-CTTAGCCGCCCATCCCA-3′;

[0011] Reverse primer: 5′-CACCCGCATTATCCGTTGA-3′.

[0012] The primer sequence of RAN1 is:

[0013] Forward primer: 5′-AGAACCGACAGGTGAAGGCAA-3′;

[0014] Reverse primer: 5′-TGGCAAGGTACAGAAAGGGCT-3′.

[0015] Furthermore, the present invention relates to the application of the primers in the quantitative fluorescence analysis of sweet osmanthus.

[0016] Furthermore, the application includes: when analyzing between petals of osmanthus at different developmental stages, using lnc00229717 and RAN1 as lncRNA internal reference genes.

[0017] Furthermore, the different developmental periods include the opening and aging process of osmanthus petals.

[0018] Furthermore, the different developmental periods include: the stem stage, the initial flowering stage, the full flowering stage, the late full flowering stage and the shedding stage.

[0019] Furthermore, the application comprises the following steps:

[0020] The cDNA of Osmanthus fragrans was used as a template, lnc00229717 and RAN1 were used as internal reference genes, and the primers were used to perform qRT-PCR detection.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention finally selects 10 lncRNAs with relatively abundant and stable expression and 7 commonly used internal reference genes as candidate internal references through literature review and database search, and screens or designs primer sequences of internal references. The stability of candidate internal references is evaluated by 5 algorithms (delta-CT, geNorm, NormFinder, BestKeeper and RefFinder), and lncRNA internal references suitable for stable expression during the opening and aging of sweet osmanthus petals, i.e., lnc00229717 and RAN1, are obtained, and qRT-PCR primers of internal references are designed. The primers have strong specificity and high amplification efficiency, and can greatly improve the efficiency of qRT-PCR detection of sweet osmanthus genes, and improve the credibility of the test results. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the CQ value of the 17 reference genes screened;

[0024] Figure 2 The geNorm software ranks the expression stability values ​​(M) of 17 internal reference genes. The lower the stability value, the more stable the gene;

[0025] Figure 3 It is the optimal number of internal reference genes determined by geNorm for accurate quantitative analysis;

[0026] Figure 4 Two stable genes, lnc00229717 and RAN1, and unstable genes, TUA5 and EF1B, were used as internal controls, and the expression level of lnc00003036;

[0027] Figure 5 Two stable genes, lnc00229717 and RAN1, and unstable genes, TUA5 and EF1B, were used as internal references, and the expression level of lnc00126603;

[0028] Figure 6 Two stable genes, lnc00229717 and RAN1, and unstable genes, TUA5 and EF1B, were used as internal references, and the expression level of lnc00250780. DETAILED DESCRIPTION

[0029] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0030] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0031] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0032] The test material used in the following examples is a healthy and pest-free Osmanthus fragrans from Huazhong Agricultural University. Petal tissues of the five stages S1 (stalk stage), S2 (early flowering stage), S4 (full flowering stage), S5 (late full flowering stage) and S6 (falling stage) were collected at 10 am. Samples of each stage were replicated three times, quickly frozen in liquid nitrogen, and stored at -80°C for subsequent analysis.

[0033] Example 1

[0034] (1) Screening of candidate reference genes

[0035] 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+), and the 7 candidate reference genes with the highest homology were found. Secondly, 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.

[0036] (2) qRT-PCR primer design

[0037] Primer5 software was used for design, and the parameters were as follows: PCR product length 70-300 bp, melting temperature 58-62°C, CG content 40-60%.

[0038] (3) Total RNA extraction from Osmanthus fragrans

[0039] 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 UV spectrophotometer (Nano Drop 2000, Thermo Scientific, Wilmington, DE, USA).

[0040] (4) Reverse transcription to synthesize cDNA

[0041] Total RNA was used as template. cDNA was synthesized using III RT SuperMix for qPCR (+gDNAwiper) kit (Nanjing Novogene Biotech Co., Ltd.).

[0042] (5) qPCR quantification

[0043] 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 front and back primers (10 μM), and ddH2O was added to 20 uL. The program was as follows: 30 s at 95°C, followed by 40 cycles with parameters of 10 s at 95°C, 30 s at 60°C, and then a melting curve was generated at 60-95°C. Each qRT-PCR analysis was repeated 3 times, and a non-template control was included for each gene.

[0044] (6) Stability assessment

[0045] Calculation of correlation coefficient, slope and amplification efficiency. Take 4 μL of cDNA template from all samples and mix them evenly. Use three replicates of 5-fold dilution cDNA (5-fold, 25-fold, 125-fold, 625-fold, 3125-fold) for qRT-PCR reaction at each concentration to obtain the Ct value at each concentration to establish a standard curve and calculate the correlation coefficient, slope and amplification efficiency (see Table 1). Where E = (5 -1 / slope -1)×100%.

[0046] Four different algorithms, delta-Ct, geNorm, NormFinder and BestKeeper, were used to statistically analyze the expression stability of internal reference genes in different samples. The results were comprehensively analyzed through the RefFinder website to screen out stable internal reference genes.

[0047] Table 1 Gene names, primer sequences, amplification lengths, PCR amplification efficiencies and regression coefficients of 17 internal reference genes in the present invention

[0048]

[0049]

[0050] Note: The primer sequences shown in Table 1 correspond to SEQ ID NO.3 to SEQ ID NO.42 in the sequence table.

[0051] (7) Results

[0052] CT value (CQ value): The CT value is inversely proportional to the gene expression level. The larger the CT value, the lower the gene expression level. Conversely, the smaller the CT value, the higher the gene expression level. Figure 1 As shown in the figure, the average CT values ​​of the 17 reference genes ranged from 8.676 to 28.777, among which 18S had the highest expression and lnc00174850 had a lower expression. The CT value of lnc00229717 had the smallest fluctuation, with a CT value range of 1.571; the CT value of EF1B had the largest fluctuation, with a CT value range of 3.797.

[0053] Delta-CT analysis: The standard deviation average was obtained by delta-CT analysis. The higher the standard deviation average, the worse the gene stability. The results are shown in Table 2. The standard deviation average of lnc00229717 was 0.51, which was the most stable gene; the standard deviation average of EF1B was 1.27, which was the least stable gene.

[0054] Table 2 Delta-CT analysis results

[0055]

[0056] geNorm software analysis: When using geNorm, the raw Ct values ​​are converted to -ΔCt (delta Ct = original Ct value - lowest Ct value in this group) was used for expression stability analysis of internal reference genes. 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, where M = 1.5 is the upper limit ( Figure 2 ). In addition, geNorm can be 2 -ΔCt For the original data, the paired difference value V of two continuous standardized factors is calculated n / V n+1 To determine the appropriate number of internal reference genes, since the paired variation value V2 / V3 of the embodiment is less than 0.15 ( Figure 3 ), therefore, two internal reference genes (lnc00239991 and RAN1) are needed to analyze the relative expression of genes in the opening and senescence of osmanthus petals.

[0057] NormFinder software analysis: When using NormFinder, the raw Ct values ​​were converted to 2 -ΔCt (delta Ct = original Ct value - lowest Ct value in this group) was used for expression stability analysis of internal reference genes. The stability value of candidate internal reference genes was calculated by NormFinder software. The lower the stability value, the better the stability, that is, the gene with the smallest stability value is the most stable gene. The results are shown in Table 3. The stability value of lnc00229717 is 0.125, which is the most stable gene; the stability value of EF1B is 1.207, which is the least stable gene.

[0058] Table 3 NormFinder software analysis results

[0059]

[0060] BestKeeper software analysis: For BestKeeper, the amplification efficiency and original Ct value calculated by the LinRegPCR program based on the original Ct value were used to calculate the coefficient of variation (CV) and standard deviation (SD) of the candidate internal reference gene expression. The smaller the SD value, the more stable the internal reference gene. If the SD value is greater than 1.0, the internal reference gene is considered unstable and should be avoided for gene expression normalization. The results are shown in Table 4. The SD value of EF1B is greater than 1, and the SD values ​​of the other genes are all less than 1. Among them, lnc00249739 is the most stable.

[0061] Table 4 BestKeeper software analysis results

[0062]

[0063] RefFinder website analysis: To verify the accuracy of the internal reference gene screening, we comprehensively ranked the geometric mean of the candidate internal reference genes sorted by delta-Ct, geNorm, NormFinder and BestKeeper under each treatment. The smaller the comprehensive ranking, the better the gene expression stability. The results are shown in Table 5. The stability of the genes is lnc00229717>RAN1>lnc00239991>lnc00067193>lnc00249739>ACT7>lnc00087780>lnc00265419>lnc00042194>18S>lnc00031789>lnc00044331>UBQ4>GAPH>lnc00174850>TUA5>EF1B, among which the geometric mean of lnc00229717 is 1.86, which is the most stable gene; the geometric mean of EF1B is 17.00, which is the most unstable gene.

[0064] Table 5 RefFinder website analysis results

[0065]

[0066]

[0067] (8) Verification of stability of internal reference genes

[0068] Based on our selected throughput sequencing results, (lnc00003036, lnc00126603, and lnc00250780) were used as target genes, and two stable genes, lnc00229717 and RAN1, and unstable genes, TUA5 and EF1B, were used as standards for calculation to confirm the suitability of the candidate genes evaluated in this study. -△△CtMethods The expression of three target genes was calculated. When lnc00229717 and RAN1 were used as internal references, lnc00003036 was highly expressed in S1, lnc00126603 was highly expressed in S2, and lnc00250780 was highly expressed in S1 and S2. When the unstable gene EF1B was used as the internal reference gene, lnc00003036, lnc00126603, and lnc00250780 were highly expressed in S5. When the unstable gene TUA5 was used as the internal reference gene, the trends of lnc00250780, lnc00003036, and lnc00126603 were similar to those when the stable lncRNA was used as the internal reference, but the gene expression levels were significantly lower ( Figure 4 , 5 and 6). Therefore, the selection of appropriate genes has a great influence on the expression trend and expression amount of the target gene.

[0069] 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. Application of lnc00229717 and RAN1 as lncRNA reference genes in fluorescence quantitative analysis of the opening and senescence of Osmanthus fragrans petals, characterized in that: The nucleotide sequence of lnc00229717 is shown as SEQ ID NO.1, the nucleotide sequence of RAN1 is shown as SEQ ID NO.2, and the variety of the osmanthus is Osmanthus fragrans.

2. Use of primers for detecting the lncRNA internal reference gene of claim 1 in fluorescence quantitative analysis of the opening and aging process of sweet osmanthus petals, characterized in that: The primer sequences for lnc00229717 are: Forward primer: 5′-CTTAGCCGCCCATCCCA-3′; Reverse primer: 5′-CACCCGCATTATCCGTTGA-3′; The primer sequence for RAN1 is: Forward primer: 5′-AGAACCGACAGGTGAAGGCAA-3′; Reverse primer: 5′-TGGCAAGGTACAGAAAGGGCT-3′; The variety of osmanthus fragrans is willow-leaf golden osmanthus.

3. The use according to claim 1 or claim 2, characterized in that: The petal opening and aging process includes: the stem stage, the initial flowering stage, the full flowering stage, the late full flowering stage and the shedding stage.

4. The use according to claim 1, characterized in that: The application comprises the following steps: The cDNA of Osmanthus fragrans was used as a template, lnc00229717 and RAN1 were used as internal reference genes, and the primers described in claim 2 were used for qRT-PCR detection.

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