Screening and application of taro internal reference gene Ce049358

Abstract

This invention relates to the field of plant gene technology, and in particular to the screening and application of the taro reference gene Ce049358. This invention analyzes candidate genes in the high expression region (FPKM). Compared with the Ce047468 gene applied for on the same day, the expression level is higher and more stable. Analysis shows that the taro reference gene Ce049358 obtained in this application can well reflect the expression characteristics of CeAGPL1 in different tissues and can be used as a reference gene for expressing different tissues of taro. The designed specific primers can also provide a reference for subsequent taro gene expression analysis.

Description

[Technical Field]

[0001] This invention relates to the field of plant gene technology, and in particular to the screening and application of the taro internal reference gene Ce049358. [Background Technology]

[0002] Taro (Colocasia esculenta) is an important food crop, and its tubers have high nutritional value. It is also a major agricultural product of Guangxi province. However, current research on the molecular biology of taro is limited, and there are few reports on its gene research. In recent years, with the continuous development of high-throughput sequencing and molecular biology research, gene expression analysis has been increasingly applied to reveal the mechanisms of taro gene expression and regulation. The sequencing of the taro genome has further accelerated the analysis of the genetic mechanisms of taro-related traits.

[0003] Quantitative real-time PCR (qRT-PCR) is currently the main analytical method for detecting gene expression levels. Genes commonly used as internal reference genes in plants include actin, β-tubulin (TUB), elongation factors (EF1A and EF1B), eukaryotic initiation factor (eIF), ubiquitin-conjugating enzyme (UBC), histone (H3-1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

[0004] However, in actual testing, we found that the expression levels of these internal reference genes were not stable under certain experimental conditions and could not be well expressed in different tissues of taro. Therefore, we need to collect samples from different tissues or at different growth and development stages to screen out stable internal reference genes. [Summary of the Invention]

[0005] In view of the above, it is necessary to provide an internal reference gene that can be used for fluorescence expression analysis of different tissue samples of taro, and that can analyze the expression of taro and is stably expressed.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] Taro reference gene Ce049358, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] The present invention also includes a primer pair for detecting the taro reference gene Ce049358, wherein the upstream sequence of the primer pair is shown in SEQ ID NO.2 and the downstream sequence is shown in SEQ ID NO.3.

[0009] The present invention also includes the application of the taro internal reference gene Ce049358 in the quantitative fluorescence expression analysis of different taro tissues.

[0010] Furthermore, the different tissues of the taro are: the root, leaves, petioles, and corms.

[0011] Furthermore, the upstream sequence of the fluorescent quantitative expression analysis primer pair is shown in SEQ ID NO.2, and the downstream sequence is shown in SEQ ID NO.3.

[0012] Furthermore, the PCR reaction system for the quantitative expression analysis consisted of 20 μL of the following: 10 μL of 2X SYBR GreenMaster Mix, 0.4 μL of 10 μM upstream primer, 0.4 μL of 10 μM upstream primer, 1 μL of cDNA template, and 8.2 μL of RNase-free water. The PCR reaction program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 10 s, 57℃ annealing for 15 s, and 72℃ extension for 20 s, for a total of 45 cycles; fluorescence signals were then collected.

[0013] The screening method for the taro internal reference gene Ce049358 of this invention is as follows:

[0014] 1. Ninety days after taro sowing, collect taro leaves, petioles, corms and roots, put them into 50ml sterile cryovials, flash freeze them in liquid nitrogen and then store them in a -80℃ freezer for later use, and extract RNA from the samples.

[0015] 2. Reverse transcription synthesizes the corresponding taro tissue cDNA;

[0016] 3. Design different primers for different genes and perform the reaction on a real-time PCR instrument. Plot the melting curve and perform stability analysis to obtain internal reference genes with good stability and good expression effect.

[0017] The present invention has the following beneficial effects:

[0018] This invention analyzes candidate genes in the high-expression region (FPKM). Compared with the Ce047468 gene applied for on the same day, the expression level is higher and more stable. The screening of internal reference genes is based on transcript sequencing data from different taro tissues (leaves, petioles, corms, and roots). The stability of expression levels in FPKM values ​​is evaluated using the coefficient of variation (CV). Genes with a CV value ≤ 0.2 and cDNA ≥ 1000 bp are selected as candidate internal reference genes. The expression stability of five candidate genes in different taro tissues is analyzed using qRT-PCR. The stability of these five candidate internal reference genes in different tissues is analyzed using three internal reference gene stability analysis software programs: geNorm, NormFinder, and BestKeeper. After comprehensive analysis and ranking, the taro internal reference gene Ce049358 is obtained. After verification, Ce049358 can well reflect the expression characteristics of CeAGPL1 in different tissues. The screened internal reference gene and the designed specific primers provide a reference for subsequent taro gene expression analysis. [Attached Image Description]

[0019] Figure 1-5 The images show the qPCR detection results of candidate internal reference genes Ce007097, Ce012507, Ce026647, Ce041066, and Ce049358 in different tissues; where M is the Marker DL2000; lanes 1-8 are two parallel samples of leaf-1, leaf-2, petiole-1, petiole-2, root-1, root-2, bulb-1, and bulb-2, respectively.

[0020] Figure 6-10 The amplification curves of the candidate internal reference genes Ce007097, Ce012507, Ce026647, Ce041066 and Ce049358 are shown below.

[0021] Figure 11-15 The melting curves of the candidate internal reference genes Ce007097, Ce012507, Ce026647, Ce041066 and Ce049358 are shown below.

[0022] Figure 16 Box plots of Ct values ​​for five candidate internal reference genes in different tissues;

[0023] Figure 17 The expression stability of candidate internal reference genes was determined using the geNorm software.

[0024] Figure 18 Analyze the coefficient of variation of internal reference genes using geNorm software (bar chart);

[0025] Figure 19 This figure shows the expression of the Ce049358 gene in different tissues of taro.

Detailed Implementation Methods

[0026] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0027] Example 1:

[0028] This example demonstrates the acquisition of the internal reference gene Ce049358, which was obtained through the following method:

[0029] I. Test materials for this embodiment:

[0030] The taro material used in this embodiment was the new taro variety Guiyu No. 2, bred by the Institute of Biotechnology, Guangxi Academy of Agricultural Sciences. Leaves, petioles, corms, and roots were collected 90 days after sowing, placed in 50ml sterile cryovials, flash-frozen in liquid nitrogen, and then stored at -80℃ for later use. Three biological replicates were used for each sample group.

[0031] II. Total RNA Extraction, Detection, and cDNA Synthesis:

[0032] Total RNA was extracted from different taro tissues using the MigiBio Total RNA Extraction Kit (R4165), following the kit's instructions. The integrity of the extracted RNA was assessed using 1.5% agarose gel electrophoresis, and RNA concentration and quality were assessed using a Nanodrop 2100. 1 μg of total RNA was reverse transcribed into cDNA using the Novizan HiScript II Q RT SuperMix for qPCR (+gDNAwiper) (R223-01) reverse transcription kit. The total reaction volume was 8 μL, with 2 μL of 4×gDNAwiper Mix, 1 μg of total RNA, and RNase-free water added to bring the volume to 8 μL. After gentle mixing, the reverse transcription reaction was performed at 50°C for 15 min, followed by 85°C for 5 s. The cDNA was then stored at -20°C for later use.

[0033] III. Selection of internal reference genes and design of specific primers:

[0034] Transcriptome data from different taro tissues were used to assess the stability of expression levels (FPKM values) using the coefficient of variation (CV). Genes with a CV value ≤ 0.2 and cDNA ≥ 1000 bp were selected as candidate internal control genes. From these candidate internal control genes, five genes with FPKM values ​​ranging from 146.54 to 244.90 were selected (Table 1). Specific primers for the five candidate internal control genes were designed using Primer Premier 6 software. Primer lengths ranged from 131 to 193 bp, and the primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. (Nanning) (Table 2).

[0035] Table 1. FPKM values ​​of candidate internal reference genes in different tissues

[0036] Ce007097 165.09 111.89 171.01 142.82 146.54 25.22 0.17 Ce012507 219.61 189.70 184.93 192.57 195.17 18.60 0.10 Ce026647 183.39 209.44 203.31 177.16 193.99 18.21 0.09 Ce041066 147.45 139.47 151.61 151.05 147.39 8.82 0.06 Ce049358 233.16 274.09 285.52 183.87 244.90 46.67 0.19

[0037] Table 2 Primers for 5 candidate internal reference genes of taro

[0038]

[0039] IV. qPCR of the internal reference gene:

[0040] Quantitative real-time PCR analysis was performed using an AnalytikJena qTOWERE 2.2 PCR instrument (Germany). The 2×ChamQ Universal SYBR qPCR Master Mix kit from Novizan was used for quantitative PCR. The total reaction volume was 20 μL, containing 0.4 μL (10 μmol / L) upstream primer (F), 0.4 μL (10 μmol / L) downstream primer (R), 1 μL cDNA template, 10 μL SYBR GreenMaster Mix (2X), and 8.2 μL RNase-free water to make up the volume. The reaction program was: 95℃ pre-denaturation for 3 min, 95℃ for 10 s; 57℃ annealing for 15 s; 72℃ extension for 20 s; for a total of 45 cycles. Each sample group had three biological replicates and three technical replicates.

[0041] The primers used for each gene in the above PCR reaction are shown in Table 2 above:

[0042] V. Data Processing and Analysis

[0043] The stability of candidate internal reference genes was analyzed individually using three software programs with different algorithms: geNorm, NormFinder, and BestKeeper. geNorm and NormFinder utilize 2... -△CT The Ct value represents the expression stability of the internal reference gene. BestKeeper evaluates the stability of each candidate internal reference gene based on its Ct value. Finally, the geometric mean method is used for comprehensive ranking.

[0044] VI. Results and Analysis

[0045] (I) Quality detection of total RNA in stems

[0046] RNA was extracted from different tissues (leaves, petioles, corms, and roots) and its quality was evaluated. RNA integrity was assessed using an Agilent 2100 / GX analyzer; a 28S / 18S ratio of 1.50–2.06 indicated good RNA integrity and no degradation. The RNA concentration measured by a NanoDrop 2100 ranged from 32.9 to 384.2 ng / μl, with OD260 / 280 values ​​between 2.08 and 2.11, indicating high RNA purity.

[0047] Specificity analysis of candidate internal reference genes

[0048] PCR amplification product electrophoresis detection results ( Figure 1-5 The results showed that all five candidate internal control genes exhibited a single band consistent with the expected product length, and primer dimers were absent. Furthermore, analysis of the amplification curves of the five candidate genes in the qRT-PCR results showed that the amplification curves were all very good. Figure 6-10 Analysis of the melting curves revealed that each curve exhibited only a single peak, indicating a single amplification band with high specificity and no non-specific amplification. Figure 11-15 Therefore, the designed real-time quantitative PCR primers have high specificity and high amplification efficiency, and can be used as internal reference primers for taro real-time quantitative PCR.

[0049] Ct value analysis of candidate genes

[0050] The Ct value is an important measure of gene expression richness. The Ct value is inversely proportional to the degree of gene expression richness. Analysis of the expression abundance of five internal reference genes in different tissues of taro revealed (…). Figure 16 The average Ct values ​​of the five candidate internal reference genes ranged from 18.58 to 24.61. Specifically, the Ct values ​​of Ce007097 in different tissues ranged from 18.75 to 19.49, with a difference of 0.74 cycles between the maximum and minimum values; the Ct values ​​of Ce012507 in different tissues ranged from 19.75 to 21.33, with a difference of 1.58 cycles between the maximum and minimum values; the Ct values ​​of Ce026647 in different tissues ranged from 18.58 to 19.83, with a difference of 1.25 cycles between the maximum and minimum values; the Ct values ​​of Ce041066 in different tissues ranged from 22.59 to 24.61, with a difference of 2.02 cycles between the maximum and minimum values; and the Ct values ​​of Ce049358 in different tissues ranged from 22.09 to 22.83, with a difference of 0.74 cycles between the maximum and minimum values.

[0051] Stability analysis of internal reference genes

[0052] This study used the internal reference analysis software geNorm, Normfinder, and BestKeeper as auxiliary tools to conduct a consistent evaluation and analysis of the above five candidate internal reference genes from multiple perspectives.

[0053] Stability assessment of candidate internal reference genes using geNorm software

[0054] The geNorm program screens internal reference genes for those with good stability by calculating the M value of each gene's stability. The criterion is that the smaller the M value, the better the stability of the internal reference gene; conversely, the larger the M value, the worse the stability. If the M value is greater than 1.5, it is not considered as an internal reference gene. In order of expression stability from highest to lowest, Ce049358 = Ce026647 (0.1608) > Ce012507 (0.3247) > Ce041066 (0.4094) > Ce007097 (0.5006). All candidate internal reference genes have an M value less than 1.5, making them potentially suitable. Among them, Ce049358 and Ce026647 have the smallest M values ​​and the best stability. Figure 17 ).

[0055] The geNorm software can also calculate the paired variation V value of the normalized factor after introducing a new internal reference gene, called "paired variable (Vn / n+1)". The specific number of internal reference genes can be determined based on the formula. To ensure that a single internal reference gene does not cause bias or fluctuation, the geNorm software uses the calculated paired difference Vn / n+1 to guarantee the optimal number of internal reference genes under these conditions. The critical value in this formula is set at 0.15. If Vn / n+1 is greater than 0.15, then the number of selected internal reference genes should be n+1; if Vn / n+1 is less than 0.15, then selecting n internal reference genes will meet the experimental requirements of the software. geNorm histogram analysis shows that when the V² / 3 value (0.133) < 0.15... Figure 18 The optimal number of internal reference genes is two. Therefore, the most suitable internal reference genes for different taro tissues are Ce049358 and Ce026647.

[0056] Stability assessment of candidate internal reference genes using NormFinder software

[0057] The NormFinder software program algorithm is similar to the geNorm program. It first obtains the stable expression value of the internal reference gene, and then selects the most suitable internal reference gene based on the size of the stable value. The criterion for selection is that the candidate internal reference gene with the smallest stable expression value is the suitable internal reference gene. The NormFinder program can not only compare the expression differences of candidate internal reference genes, but also calculate the variation between sample groups. However, this program can only select one most suitable internal reference gene.

[0058] Evaluation results of five candidate internal reference genes using NormFinder software showed that the stable expression values ​​of the internal reference genes were Ce049358 (0.056) = Ce026647 (0.056) > Ce041066 (0.324) > Ce012507 (0.330) > Ce007097 (0.403). The most suitable internal reference genes for different tuber tissues were Ce049358 and Ce026647. These results are largely consistent with the evaluation results from the geNorm software.

[0059] BestKeeper's analysis results of candidate internal control genes

[0060] BestKeeper software calculates the correlation coefficient (r), standard deviation (SD), and correlation coefficient (CV) between genes from sample data, and uses these three parameters to determine the stability of the internal reference gene. The higher the correlation coefficient (r) and the lower the coefficient of variation (CV) and standard deviation (SD), the more stable the internal reference gene is. Furthermore, when the standard deviation (SD) is greater than 1, the candidate internal reference gene is considered unstable.

[0061] Based on correlation coefficient (r), the candidate internal reference genes were ranked as Ce026647 (0.998) > Ce049358 (0.983) > Ce041066 (0.963) > Ce012507 (0.749) > Ce007097 (0.277). Based on SD value, the ranking was Ce049358 (0.226) > Ce007097 (0.244) > Ce026647 (0.379) > Ce012507 (0.431) > Ce041066 (0.529), but the SD values ​​of all five candidate internal reference genes were less than 1. Based on the coefficient of variation (CV) values, Ce049358 (1.003) > Ce007097 (1.268) > Ce026647 (1.958) > Ce012507 (2.092) > Ce041066 (2.245). Considering the correlation coefficient (r), CV, and standard deviation (SD), Ce049358, with a correlation coefficient of 0.983, a standard deviation of 0.226, and a CV of 1.003, is the most suitable internal reference gene.

[0062] Table 3. Stability values ​​of internal reference genes analyzed by BestKeeper software.

[0063] Correlation coefficient (r) 0.277 0.749 0.998 0.963 0.983 Standard deviation (SD) 0.244 0.431 0.379 0.529 0.226 Coefficient of variation (CV) 1.268 2.092 1.958 2.245 1.003

[0064] Stability analysis of internal reference genes

[0065] Comparison and analysis of three internal reference gene analysis software programs revealed slight differences in the results obtained from BestKeeper, geNorm, and NormFinder. A comprehensive evaluation of the results from the three programs was performed in Excel; a smaller geometric mean indicates a more stable internal reference gene. Table 4 shows that Ce049358 has the smallest geometric mean across the three programs, making it the most stable internal reference gene. Therefore, among the five candidate genes, Ce049358 is the most suitable internal reference gene.

[0066] Table 4. Comprehensive Analysis Results of Three Software Programs: geNorm, NormFinder, and BestKeeper

[0067] Ce007097 5 5 2 3.68 Ce012507 3 4 4 3.63 Ce026647 1 1 3 1.44 Ce041066 4 3 5 3.91 Ce049358 1 1 1 1.00

[0068] Example 2:

[0069] The gene Ce049358, which exhibited the best stability as selected in Example 1, was further validated to ensure reliable quantitative data. Using Ce049358 as an internal reference gene, a gene related to bulb starch synthesis, the ADP-glucose pyrophosphorylase large subunit gene (CeAGPL1), was selected to validate the stability of the selected internal reference gene. Primers were designed using Ce049358 as the internal reference gene and CeAGPL1 as the target gene (Table 5) to analyze the expression of CeAGPL1 in different taro tissues using qRT-PCR. The results showed that the internal reference gene was stably expressed and could well reflect the changes in CeAGPL1 expression levels in different tissues. Figure 19 The details are as follows:

[0070] cDNA was extracted from leaves, petioles, corms, and roots of taro on day 90 post-planting as templates for quantitative PCR. The Ce049358 gene was used as an internal reference gene, and primers were designed with the ADP-glucose pyrophosphorylase large subunit gene (CeAGPL1), a gene related to corm starch synthesis, as the target gene. Primers are shown in Table 5.

[0071] Table 5 Primer sequences for internal reference genes and target genes.

[0072]

[0073] The fluorescence quantitative reaction was performed on a real-time quantitative PCR instrument, as follows:

[0074] The total PCR reaction volume was 20 μL: 10 μL 2X SYBR Green Master Mix, 0.4 μL 10 μM upstream primer, 0.4 μL 10 μM upstream primer, 1 μL cDNA template, and 8.2 μL RNase-free water;

[0075] The PCR reaction program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 10 s, 57℃ annealing for 15 s, and 72℃ extension for 20 s, for a total of 45 cycles; fluorescence signals were collected.

[0076] Use 2 after the reaction is complete -ΔΔ All data from the CT method are presented as mean ± standard deviation. Statistical analysis of real-time quantitative PCR data was performed using SPSS 17.0 and Microsoft Office Excel 2007. Analysis of variance was performed using SPSS 17.0 software, and graphs were generated using Excel 2010 software.

[0077] The reaction results obtained are as follows Figure 19 As shown, the internal reference gene Ce049358 can be stably expressed in different taro tissues: roots, leaves, petioles and corms, and can well reflect the changes in the expression level of CeAGPL1 in different taro tissues.

[0078] In summary, the gene Ce049358 can be stably expressed in different taro tissues: roots, leaves, petioles, and corms. It can serve as a quantitative fluorescent internal reference gene for different taro tissues and lay the foundation for future research on genes of important functional traits in taro.

[0079] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. Taro Gene Ce049358 Its characteristics are, The internal reference gene Ce049358 The nucleotide sequence is shown in SEQ ID NO.1 of the sequence listing.

2. Detection of the taro reference gene as described in claim 1 Ce049358 The primer pair is characterized in that, The upstream sequence of the primer is shown in SEQ ID NO.2, and the downstream sequence is shown in SEQ ID NO.

3.

3. The taro reference gene as described in claim 1 Ce049358 Its application in the quantitative fluorescence expression analysis of different tissues of taro is characterized by... The taro variety mentioned is Guiyu No.

2.

4. The application according to claim 3, characterized in that, The different tissues of the taro are: the root, leaves, petioles and corms.

5. The application according to claim 3, characterized in that, The upstream sequence of the fluorescent quantitative expression analysis primer pair is shown in SEQ ID NO.2, and the downstream sequence is shown in SEQ ID NO.3.

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