LncRNA reference genes, their primers and applications in different tissues of Osmanthus fragrans

Abstract

The present invention discloses lncRNA reference genes in different tissues of Osmanthus fragrans, as well as primers and applications thereof. Through literature review, database search and analysis, and by using five algorithms (delta-CT, GeNorm, NormFinder, BestKeeper, etc.) to evaluate the stability of candidate lncRNAs, the optimal lncRNA reference genes for quantitative fluorescence in different tissues of Osmanthus fragrans were finally obtained (lnc00042194 + lnc00239991 + lnc00067193 + UBQ4 + TUA5 + RAN1). And real-time fluorescence quantitative PCR primers were designed using these lncRNAs. The present invention fills the technical blank of the lack of stable lncRNA reference genes in different tissues of Osmanthus fragrans, provides strong support for the expression quantification and related functional research of lncRNAs in different tissues of Osmanthus fragrans, and thus has important application value.

Description

Technical Field

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

[0002] In the field of plant molecular biology, the research on long non-coding RNA (lncRNA) has been increasingly concerned. LncRNA is a class of RNA molecules with a length exceeding 200 nucleotides. Although it does not encode proteins, it plays an important role in biological processes such as gene expression regulation, chromatin remodeling, and transcriptional regulation. In recent years, with the development of high-throughput sequencing technology, more and more lncRNAs have been identified. However, the research on the functions and regulatory mechanisms of lncRNAs in plants is still relatively insufficient. Osmanthus fragrans is a common woody flowering plant, and its flowers emit a unique fragrance, which is deeply loved by people. However, there are few studies on lncRNAs in Osmanthus fragrans. In order to better understand the expression patterns of lncRNAs in Osmanthus fragrans and their expression levels in different tissues, it is necessary to establish a set of accurate measurement methods. Quantitative real-time PCR (qRT-PCR) is a commonly used molecular biology technique that can quickly and accurately measure the expression levels of RNAs. In this technique, internal references are important reference genes used to correct variations between samples. However, for the research on lncRNAs in Osmanthus fragrans, there is currently a lack of suitable internal reference genes. Therefore, it is necessary to carry out the screening and verification of lncRNA internal references according to the research needs of lncRNAs in different tissues of Osmanthus fragrans. Summary of the Invention

[0003] In view of the deficiencies of the prior art, the purpose of the present invention is to provide lncRNA internal reference genes with stable expression in different tissues of Osmanthus fragrans, and the lncRNA internal reference genes can meet the requirements for real-time fluorescence quantitative detection of the transcriptional expression levels of Osmanthus fragrans genes.

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

[0005] The applications of lnc00042194, lnc00239991, lnc00067193, UBQ4, TUA5 and RAN1 as lncRNA internal reference genes in the fluorescence quantitative PCR analysis of different tissues of osmanthus fragrans. Among them, the nucleotide sequence of lnc00042194 is shown in SEQ ID NO.1, the nucleotide sequence of lnc00239991 is shown in SEQ ID NO.2, the nucleotide sequence of lnc00067193 is shown in SEQ ID NO.3, the nucleotide sequence of UBQ4 is shown in SEQ ID NO.4, the nucleotide sequence of TUA5 is shown in SEQ ID NO.5, and the nucleotide sequence of RAN1 is shown in SEQ ID NO.6.

[0006] Furthermore, the present invention provides primers for detecting the lncRNA internal reference genes, wherein,

[0007] The primer sequences for detecting lnc00042194 are:

[0008] Forward primer: 5’-TCGGCGAAGGGTGAGTAATG-3’;

[0009] Reverse primer: 5’-TGAAGACGACGACGGGATT-3’;

[0010] The primer sequences for detecting lnc00239991 are:

[0011] Forward primer: 5’-TTTCTTGGTCGTGTCTTTAGCA-3’;

[0012] Reverse primer: 5’-CAAGTTGCGGGAGACGTTAT-3’;

[0013] The primer sequences for detecting lnc00067193 are:

[0014] Forward primer: 5’-GCATCGGCGATTGTGAGA-3’;

[0015] Reverse primer: 5’-AAGCGAAGGTCCGTTTGG-3’;

[0016] The primer sequences for detecting UBQ4 are:

[0017] Forward primer: 5’-ACTGCACCCTCCATTTGGT-3’;

[0018] Reverse primer: 5’-TGCCGTTCACGATTAGTTCTC-3’;

[0019] The primer sequences for detecting TUA5 are:

[0020] Forward primer: 5’-ATCATCGCTGACCACTTCTTTG-3’;

[0021] Reverse primer: 5’-GCCATGTATTTCCCGTGTCTT-3’;

[0022] The primer sequences for detecting RAN1 are:

[0023] Forward primer: 5’-AGAACCGACAGGTGAAGGCAA-3’;

[0024] Reverse primer: 5’-TGGCAAGGTACAGAAAGGGCT-3’.

[0025] Furthermore, the present invention relates to the application of the primers in the fluorescence quantitative PCR analysis of different tissues of Osmanthus fragrans.

[0026] Furthermore, the different tissues include roots, stems, leaves, seeds and flowers.

[0027] Furthermore, the application includes the following steps:

[0028] Using the cDNA of different tissues of Osmanthus fragrans as a template, using lnc00042194, lnc00239991, lnc00067193, UBQ4, TUA5 and RAN1 as internal reference genes, and performing qRT-PCR detection using the primers.

[0029] Compared with the prior art, the beneficial effects of the present invention are:

[0030] Through literature review, database search and analysis, the present invention determined 17 reference genes as candidate internal reference genes, screened and obtained the sequences of 17 internal reference genes, and evaluated the stability of candidate internal references through 5 algorithms (delta-CT, geNorm, NormFinder, BestKeeper and RefFinder). Finally, lncRNA internal reference genes suitable for fluorescence quantification of different tissues of Osmanthus fragrans were screened out. The fluorescence quantification internal reference genes are lnc00042194, lnc00239991, lnc00067193, UBQ4, TUA5 and RAN1, and qRT-PCR primers for detecting internal references were designed. The primers have strong specificity and high amplification efficiency, which can greatly improve the detection efficiency of detecting lncRNA of Osmanthus fragrans by real-time fluorescence quantification and improve the credibility of the detection results. Description of the Drawings

[0031] Figure 1 Are the Cq values of the fluorescence quantification of the 17 screened lncRNAs.

[0032] Figure 2 It is the analysis of the expression stability values (M) of 17 reference genes sorted by the geNorm software. The lower the stability value, the more stable the gene.

[0033] Figure 3 It is the number of optimal reference genes determined by geNorm.

[0034] Figure 4 It is the expression level of lnc00003036 using 6 stable genes and unstable genes as reference genes.

[0035] Figure 5 It is the expression level of lnc00126603 using 6 stable genes and unstable genes as reference genes.

[0036] Figure 6 It is the expression level of lnc00250780 using 6 stable genes and unstable genes as reference genes. Detailed implementation manners

[0037] To better understand the technical content of the present invention, specific examples are provided below to further illustrate the present invention.

[0038] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0039] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can all be obtained from commercial channels.

[0040] The test materials used in the following examples: Different tissue samples (roots, stems, leaves, seeds and flowers) collected from osmanthus flowers grown in the natural environment for 15 years. Each treatment has three biological replicates. When collecting samples, they were quickly frozen with liquid nitrogen.

[0041] Example 1

[0042] (1) Screening of candidate reference genes

[0043] Through literature review and database search, the reference genes that have been reported for other species were locally blast aligned (blastVer: 2.4.0+) with the osmanthus full genome data to find the 7 candidate reference genes with the highest homology. Secondly, based on RNA-seq, 10 lncRNAs with relatively high expression levels and a fold change < 1.4 were selected as reference genes. The candidate reference genes and their primers are shown in Table 1.

[0044] (2) qRT-PCR primer design

[0045] The primer design was carried out using Primer5 software, with the following parameters: the length of the PCR product was 70 - 300 bp, the melting temperature was 58 - 62 °C, and the CG content was 40 - 60%.

[0046] (3) Total RNA extraction of osmanthus

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

[0048] (4) cDNA synthesis of osmanthus

[0049] Using total RNA as a template, cDNA was synthesized using III RT SuperMix for qPCR(+gDNAwiper) kit (Nanjing Novoprotein Biological Technology Co., Ltd.).

[0050] (5) qRT-PCR quantification: The 20 μL reaction system was as follows: 10 μL of 2×Taq Pro Universal SYBR qPCR Master Mix; 1 μL of cDNA diluted 10 times and 0.8 μL of forward and reverse primers (10 μM), and ddH2O was added to 20 μL. The reaction procedure was as follows: 30 s at 95 °C, followed by 40 cycles with parameters of 10 s at 95 °C and 30 s at 60 °C, and then a melting curve was generated at 60 - 95 °C. Each qRT-PCR analysis was repeated 3 times, and non-template controls were included for each gene.

[0051] (6) lncRNA internal reference screening: Four different algorithms, delta-Ct, geNorm, NormFinder, and BestKeeper, were used to statistically analyze the expression stability of internal references in different samples; and through comprehensive analysis on the RefFinder website, stable internal reference genes were screened out.

[0052] (7) lncRNA internal reference verification: Using the stable genes lnc00042194, lnc00239991, lnc00067193, UBQ4, TUA5, RAN1 and the unstable genes GAPH and lnc00031789 as internal references, the expression levels of lnc00003036, lnc00126603, and lnc00250780 were calculated.

[0053] Table 1 Primer sequences of 17 candidate reference genes and 3 verified lncRNAs

[0054]

[0055]

[0056] E, PCR efficiency; R 2 , correlation coefficient; the primer sequences shown in Table 1 correspond to SEQ ID NO.7 to SEQ ID NO.46 in the sequence listing in sequence order. SEQ ID NO.47 to SEQ ID NO.49 are the sequences of lnc00003036, lnc00126603 and lnc00250780.

[0057] (8) Results

[0058] 1) CQ value

[0059] The CQ values of 17 internal references are as Figure 1 shown, and the average CQ value ranges from 11.319 (18S) to 32.985 (lnc00174850). Among them, the CQ value of 18S has the smallest change, which is 2.047, and the change of lnc00031789 is the largest, which is 15.664.

[0060] 2) delta-CT analysis

[0061] The average value of the standard deviation is obtained by delta-CT analysis. The higher the average value of the standard deviation, the worse the gene stability. On the contrary, the lower the average value of the standard deviation, the higher the gene stability. The results are shown in Table 2. The average values of the standard deviations of lnc00239991 and lnc00042194 are 1.59, which are the most stable genes; the average value of the standard deviation of lnc00031789 is 4.07, which is the gene with the worst stability.

[0062] Table 2 Results of delta-CT analysis

[0063]

[0064] 2) geNorm software analysis: By converting the cycle threshold (CT) obtained by fluorescence quantitative PCR into relative quantitative data, the average variability M value of the logarithm conversion value of the ratio of a certain gene to other genes in pairs is analyzed by geNorm software, and an M value line graph is drawn ( Figure 2 ). The smaller the M value, the more stable the internal reference gene. The calculation method of geNorm software is not affected by extreme ratios and expression abundance differences between genes, and more than 2 stably expressed internal reference genes can be screened out through this software. Since the pairwise variation value V6 / V7 of the example < 0.15 ( Figure 3), so six reference genes (lnc00042194, lnc00067193, lnc00239991, TUA5, RAN1, and ACT7) are required for the relative expression analysis of genes in different tissues of osmanthus Figure 2 ).

[0065] 3) NormFinder software analysis: When using NormFinder, the original Ct values are converted to 2 -ΔCt (delta Ct = original Ct value - the lowest Ct value in this group) and then used for the expression stability analysis of reference genes. The stability values of candidate reference genes are calculated by NormFinder software. The higher the stability value, the worse the stability; conversely, the lower the stability value, the better the stability, that is, the gene with the smallest stability value is the most stable gene. The results are shown in Table 3. The stability value of lnc00042194 is 0.562, which is the most stable gene; the stability value of lnc00031789 is 3.879, which is the gene with the worst stability (Table 3).

[0066] Table 3 Analysis results of NormFinder software

[0067]

[0068]

[0069] 4) BestKeeper software analysis: BestKeeper evaluates and ranks the stability of candidate genes through the standard deviation SD value. The smaller the SD value, the better the stability of the gene; if SD > 1, the gene is considered unstable. For different tissues of osmanthus, the gene stability is 18S > lnc00265419 > lnc00229717 > lnc00239991 > lnc00174850 > lnc00249739 > lnc00042194 > lnc00067193 > RAN1 > TUA5 > UBQ4 > EF1B > lnc00087780 > ACT7 > lnc00044331 > GAPH > lnc00031789, and the most stable expression is 18S (Table 4).

[0070] Table 4 Analysis results of BestKeeper software

[0071]

[0072] 5) RefFinder website analysis: To verify the accuracy of reference gene screening, we comprehensively ranked the geometric means of the sorting results of candidate reference genes under each treatment by delta-Ct, geNorm, NormFinder, and BestKeeper. The smaller the comprehensive ranking, the better the gene expression stability. The results are shown in Table 5, and the gene stability is lnc00042194 > lnc00239991 > lnc00067193 > UBQ4 > TUA5 > RAN1 > lnc00265419 > lnc00249739 > 18S > ACT7 > lnc00174850 > lnc00229717 > lnc00044331 > EF1B > lnc00087780 > GAPH > lnc00031789, where lnc00042194 + lnc00239991 + lnc00067193 + UBQ4 + TUA5 + RAN1 is the most stable gene combination; while the geometric mean of lnc00031789 is 17.00, which is the most unstable gene.

[0073] Table 5 Results of RefFinder website analysis

[0074]

[0075] (9) Verification of reference gene stability

[0076] Taking (lnc00003036, lnc00126603, and lnc00250780) as target genes and different reference genes (combinations) as standards for calculation to confirm the applicability of the candidate genes evaluated in this study. Calculate the expression of the three target genes using 2 -△△Ct methods. It was found that when the stable genes lnc00042194 + lnc00239991 + lnc00067193 + UBQ4 + TUA5 + RAN1 were used as reference genes, lnc00003036 was highly expressed in roots, lnc00126603 was highly expressed in flowers and roots, and lnc00250780 was highly expressed in leaves. However, when lnc00031789 was used as a reference gene, lnc00003036, lnc00126603, and lnc00250780 were highly expressed in roots and hardly expressed in other tissues; when GAPH was used as a reference gene, lnc00003036 was highly expressed in stems, lnc0012660 was only highly expressed in roots, and lnc00250780 was highly expressed in leaves and stems ( Figure 4 、 5 and 6). In summary, when choosing stable and unstable reference genes, the expression trends of genes are different, indicating that it is necessary to screen reference genes.

[0077] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Application of primers for detecting lncRNA internal reference genes in fluorescent quantitative PCR analysis of lncRNA expression levels in different tissues of Osmanthus fragrans, characterized in that: The lncRNA internal reference genes are lnc00042194, lnc00239991, lnc00067193, UBQ4, TUA5 and RAN1, the nucleotide sequence of lnc00042194 is shown in SEQ ID NO.1, the nucleotide sequence of lnc00239991 is shown in SEQ ID NO.2, the nucleotide sequence of lnc00067193 is shown in SEQ ID NO.3, the nucleotide sequence of UBQ4 is shown in SEQ ID NO.4, the nucleotide sequence of TUA5 is shown in SEQ ID NO.5, and the nucleotide sequence of RAN1 is shown in SEQ ID NO.

6.

2. A primer for detecting the lncRNA internal reference gene according to claim 1, characterized in that: The primer sequences used to detect lnc00042194 are: Forward primer: 5′-TCGGCGAAGGGTGAGTAATG-3′; Reverse primer: 5′-TGAAGACGACGACGGGATT-3′; The primer sequences used to detect lnc00239991 are: Forward primer: 5′-TTTCTTGGTCGTGTCTTTAGCA-3′; Reverse primer: 5′-CAAGTTGCGGGAGACGTTAT-3′; The primer sequences used to detect lnc00067193 are: Forward primer: 5′-GCATCGGCGATTGTGAGA-3′; Reverse primer: 5′-AAGCGAAGGTCCGTTTGG-3′; The primer sequences used to detect UBQ4 are: Forward primer: 5′-ACTGCACCCTCCATTTGGT-3′; Reverse primer: 5′-TGCCGTTCACGATTAGTTCTC-3′; The primer sequences used to detect TUA5 are: Forward primer: 5′-ATCATCGCTGACCACTTCTTTG-3′; Reverse primer: 5′-GCCATGTATTTCCCGTGTCTT-3′; and the primer sequences used to detect RAN1 are: Forward primer: 5′-AGAACCGACAGGTGAAGGCAA-3′; Reverse primer: 5′-TGGCAAGGTACAGAAAGGGCT-3′.

3. The use according to claim 1, characterized in that: The different tissues include roots, stems, leaves, seeds and flowers.

4. The use according to claim 1, characterized in that: The application comprises the following steps: The cDNA of different tissues of Osmanthus fragrans was used as a template, lnc00042194, lnc00239991, lnc00067193, UBQ4, TUA5 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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