Screening and Application of the Internal Reference Gene Ce009836 in Taro

Abstract

The present invention relates to the field of plant gene technology, and in particular to the screening and application of a taro internal reference gene Ce009836. The present invention is based on transcript sequencing data from different taro tissues obtained by a research group in the early stage. The stability of an expression quantity FPKM value is evaluated using a coefficient of variation (CV). Genes with a CV value of ≤0.2 and a cDNA of ≥1000bp are selected as candidate internal reference genes. The stability of the candidate internal reference genes in different tissues is analyzed using three internal reference gene stability analysis software programs: geNorm, NormFinder, and BestKeeper. Comprehensive analysis and ranking are performed to obtain the taro internal reference gene Ce009836. Verification shows that the internal reference gene can be stably expressed in different taro tissues, including roots, leaves, petioles, and corms. The gene can be used as a fluorescence quantitative internal reference gene for different taro tissues, and can lay a foundation for future research on important functional trait genes of taro.

Description

Technical field

[0001] The present invention relates to the field of plant gene technology, and in particular to the screening and application of taro internal reference gene Ce009836. [Background Technology]

[0002] Taro (Colocasia esculenta), also known as taro, taro root, and hairy taro, is a perennial herbaceous plant of the genus Colocasia in the Araceae family. Taro is a staple food for many people in Africa and the Asia-Pacific islands. Compared to other tubers and root vegetables, taro has a higher nutritional value. Taro is not only edible but also medicinal. It has a sweet, spicy, flat, and smooth taste. Its leaves, flowers, and corms are all used as medicine, with benefits such as tonifying the stomach, tonifying the spleen and stomach, relieving abdominal lumps, and dispersing tuberculosis. It is mainly used to treat swelling, psoriasis, burns, and other conditions. The edible part of taro is the corm, which is a source of protein, carbohydrates, fat, crude fiber, vitamin C, thiamine, riboflavin, and niacin. Starch is the most important component of the taro corm. The carbohydrate content of the taro corm is almost twice that of potatoes, and the protein content is 11% higher than that of yam, cassava, and sweet potato. Given this rich nutritional content, understanding the genetic basis of these nutrients is crucial.

[0003] In recent years, with the continuous development of high-throughput sequencing and molecular biology research, gene expression analysis has gradually been applied to reveal the expression and regulatory mechanisms of taro genes. With the sequencing of the taro genome, the analysis of the genetic mechanisms of taro-related traits has been further accelerated.

[0004] Quantitative real-time PCR (qRT-PCR) is currently the primary analytical method for measuring gene expression levels. The reliability of qRT-PCR results is affected by factors such as initial sample size, RNA integrity, cDNA quality, and amplification efficiency. Therefore, the use of stably expressed internal reference genes is crucial for comparing target gene expression levels across different samples. Proper selection of appropriate internal reference genes for calibration and normalization is crucial for obtaining accurate qRT-PCR results. Currently, genes commonly used as internal reference genes in plants include actin, β-tubulin (TUB), transcription elongation factor genes (EF1A and EF1B), eukaryotic initiation factors (eIFs), ubiquitin-conjugating enzymes (UBCs), histone H3-1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). However, as research deepens, it has been discovered that many previously considered reference genes (such as actin) have unstable expression levels under certain experimental conditions. Therefore, in actual research, it is necessary to sample different tissues or different growth and development time periods to screen for stably expressed 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. This gene can provide an important reference for expression analysis of taro.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] The taro internal reference gene Ce009836, the nucleotide sequence of the internal reference gene Ce009836 is shown in the sequence listing SEQ ID NO.1.

[0008] The present invention also includes a primer pair for detecting the taro internal reference gene Ce009836. The upstream sequence of the primers is shown in the sequence table SEQ ID NO. 2, and the downstream sequence is shown in the sequence table SEQ ID NO. 3.

[0009] The present invention also includes the application of the taro internal reference gene Ce009836 in fluorescence quantitative expression analysis of different taro tissues.

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

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

[0012] Furthermore, the PCR reaction system for the fluorescence quantitative expression analysis is 20 μL, which is: 10 μL of 2X SYBR Green Master 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 procedure is 95°C pre-denaturation for 3 min; 95°C denaturation for 10 s, 57°C annealing for 15 s, and 72°C extension for 20 s for a total of 45 cycles; and the fluorescence signal is collected.

[0013] The taro internal reference gene Ce009836 of the present invention is screened by:

[0014] 1. 90 days after sowing, collect the leaves, petioles, corms, and roots of taro, place them in 50 ml sterile freezing tubes, freeze them in liquid nitrogen, and then store them in a -80°C freezer for RNA extraction.

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

[0016] 3. Design different primers according to different genes and perform reactions on a fluorescent quantitative PCR instrument, draw melting curves, perform stability analysis, and obtain internal reference genes with good stability and expression effects.

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

[0018] The present invention is based on the data of transcript sequencing performed by the research group in the early stage on different taro tissues (leaves, petioles, corms and roots). The stability of the expression amount FPKM value was evaluated using the coefficient of variation CV, and genes with a CV value ≤ 0.2 and a cDNA ≥ 1000bp were selected as candidate internal reference genes. The expression stability of five candidate genes in different taro tissues was analyzed using qRT-PCR. The stability of these five candidate internal reference genes in different tissues was analyzed using three internal reference gene stability analysis softwares: geNorm, NormFinder and BestKeeper, and a comprehensive analysis and ranking was performed to obtain the taro internal reference gene Ce009836. After verification, the internal reference gene can be stably expressed in different taro tissues: roots, leaves, petioles and corms. It can be used as a fluorescence quantitative internal reference gene for different taro tissues, which can lay a foundation for future research on important functional trait genes of taro.

Brief Description of the Drawings

[0019] Figure 1-5Figures 1-8 show the qPCR detection results of candidate reference genes: Ce012579, Ce029238, Ce036387, Ce050340, and Ce009836 in different tissues; M represents Marker DL2000; lanes 1-8 represent two parallel samples of leaf-1, leaf-2, petiole-1, petiole-2, root-1, root-2, corm-1, and corm-2, respectively.

[0020] Figure 6-10 Amplification curves of candidate reference genes: Ce012579, Ce029238, Ce036387, Ce050340, and Ce009836;

[0021] Figure 11-15 These are the melting curves of candidate reference genes: Ce012579, Ce029238, Ce036387, Ce050340, and Ce009836;

[0022] Figure 16 The box plot of Ct values ​​of five candidate internal reference genes in different tissues;

[0023] Figure 17 Stable ranking of candidate reference genes for geNorm software;

[0024] Figure 18 The coefficient of variation histogram of the internal reference gene was analyzed by geNorm software;

[0025] Figure 19 This is a diagram showing the expression of Ce009836 gene in different tissues of taro. [Specific implementation method]

[0026] To make the above-mentioned objects, features, and advantages of the present invention more readily apparent, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings. The following description sets forth numerous specific details to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art may make similar modifications without departing from the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0027] Example 1:

[0028] This example is about obtaining the internal reference gene Ce009836, which is obtained by the following method:

[0029] 1. Test materials of this embodiment:

[0030] The taro material used in this example was Guiyu No. 2, a new taro variety bred by the Institute of Biotechnology, Guangxi Academy of Agricultural Sciences. Leaves, petioles, corms, and roots were collected 90 days after sowing, placed in sterile 50ml cryovials, quickly frozen with liquid nitrogen, and then stored in a -80°C freezer until ready for use. Three biological replicates were used for each sample.

[0031] 2. Total RNA extraction, detection and cDNA synthesis:

[0032] Total RNA from different taro tissues was extracted using the Meiji Biotechnology Difficult Plant Total RNA Mini Kit (R4165), following the kit instructions. The integrity of the extracted RNA was assessed by 1.5% agarose gel electrophoresis, and the concentration and quality of the RNA were determined using a Nanodrop 2100. 1 μg of total RNA was reverse transcribed into cDNA using the Novozymes HiScript II Q RT SuperMix for qPCR (+gDNAwiper) (R223-01) reverse transcription kit. The total reaction volume was 8 μL, containing 2 μL of 4× gDNAwiper Mix and 1 μg of total RNA. The volume was then filled to 8 μL with RNase-free water. After gentle mixing, the reverse transcription reaction was performed using the following reaction conditions: 50°C for 15 min, then 85°C for 5 s. The cDNA was then stored in a -20°C freezer until ready for use.

[0033] 3. Selection of internal reference genes and design of specific primers:

[0034] Using transcriptome data from different taro tissues, we evaluated the stability of expression levels (FPKM) using the coefficient of variation (CV). Genes with CV values ​​≤ 0.2 and cDNA lengths ≥ 1000 bp were selected as candidate internal reference genes. Five genes with FPKM values ​​ranging from 20.80 to 37.00 were selected (Table 1). Specific primers for these five candidate internal reference genes were designed using Primer Premier 6 software. Primers ranged from 109 to 188 bp in length and were synthesized by Beijing Qingke Biotechnology Co., Ltd. (Nanning) (Table 2).

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

[0036] Gene name root blade petiole bulb FPKM average Standard deviation / SD Coefficient of variation / CV Ce012579 23.78 22.46 22.39 21.70 22.58 1.68 0.07 Ce029238 36.57 33.96 37.11 36.85 36.09 1.96 0.05 Ce036387 21.66 20.66 21.61 19.49 20.80 1.24 0.06 Ce050340 37.86 37.88 34.56 37.94 37.00 2.47 0.07 Ce009836 30.29 30.48 31.02 30.43 30.57 1.97 0.06

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

[0038]

[0039]

[0040] 4. qPCR of internal reference genes:

[0041] qRT-PCR analysis was performed using a German Analytik Jena qTOWERE2.2 fluorescence quantitative PCR instrument. The fluorescence quantitative reagent used was the Norwegian 2× ChamQ Universal SYBR qPCR Master Mix kit. The reaction system consisted of 20 μL of upstream primer (F) (10 μmol / L), downstream primer (R) (10 μmol / L), 1 μL of cDNA template, 10 μL of SYBR Green Master Mix (2X), and 8.2 μL of RNase-free water. The reaction procedure was as follows: 95°C initial denaturation for 3 min, 95°C for 10 s, 57°C annealing for 15 s, and 72°C extension for 20 s, for a total of 45 cycles. Three biological replicates and three technical replicates were performed for each sample.

[0042] The primers used for each gene in the above PCR reaction are shown in Table 2 above:

[0043] 5. Data Processing and Analysis

[0044] Three different softwares, geNorm, NormFinder and BestKeeper, were used to analyze the stability of candidate internal reference genes. -△CT The value indicates the expression stability of the internal reference gene. BestKeeper evaluates the stability of each candidate internal reference gene based on its Ct value. Finally, a comprehensive ranking is performed using the geometric mean method.

[0045] 6. Results and Analysis

[0046] (1) Quality detection of total stem RNA

[0047] RNA quality was assessed after extraction from different tissues (leaves, petioles, bulbs, and roots). RNA integrity was assessed using an Agilent 2100 / GX analyzer. The 28S / 18S ratio ranged from 1.50 to 2.06, indicating good RNA integrity and no degradation. RNA concentrations were measured using a NanoDrop 2100 analyzer, ranging from 32.9 to 384.2 ng / μl, with OD260 / 280 values ​​between 2.08 and 2.11, indicating high RNA purity.

[0048] Specificity analysis of candidate reference genes

[0049] PCR amplification product electrophoresis detection results ( Figure 1-5) showed that all five candidate internal reference genes had a single band consistent with the expected product length, and no primer dimers were present. At the same time, the amplification curves of the five candidate genes in the qRT-PCR results were analyzed, and the results showed that the amplification curves were all very good ( Figure 6-10 ); Analysis of the melting curves revealed that the melting curves had only one peak, indicating a single amplified band with strong specificity and no nonspecific amplification ( Figure 11-15 ). Therefore, the designed real-time fluorescence quantitative PCR primers have strong specificity and high amplification efficiency, and can be used for internal reference primer experiments of taro fluorescence quantitative PCR.

[0050] Ct value analysis of candidate genes

[0051] The Ct value is an important measure of gene expression richness. The Ct value is inversely proportional to the expression richness. The expression abundance analysis of five internal reference genes in different taro tissues found that ( Figure 16 ). The average Ct values ​​of the five candidate reference genes ranged from 21.68 to 27.66. With the exception of Ce029238, which had a higher expression level, the overall expression levels of the other four genes were relatively close. Among them, the Ct values ​​of Ce012579 in different tissues ranged from 25.67 to 26.77, with a difference of 1.10 cycles between the maximum and minimum values; the Ct values ​​of Ce029238 in different tissues ranged from 21.68 to 23.39, with a difference of 1.71 cycles between the maximum and minimum values; the Ct values ​​of Ce036387 in different tissues ranged from 26.27 to 27.66, with a difference of 1.39 cycles between the maximum and minimum values; the Ct values ​​of Ce050340 in different tissues ranged from 24.21 to 25.72, with a difference of 1.51 cycles between the maximum and minimum values; and the Ct values ​​of Ce009836 in different tissues ranged from 24.39 to 25.71, with a difference of 1.32 cycles between the maximum and minimum values.

[0052] Stability analysis of internal reference genes

[0053] This study used internal reference analysis software geNorm, Normfinder and BestKeeper software as an aid to evaluate and analyze the above five candidate internal reference genes from multiple aspects.

[0054] Stability assessment of candidate internal reference genes using geNorm software

[0055] The geNorm program screens out reference genes with good stability by calculating the M value of each reference gene's stability. The judgment standard is that the smaller the M value, the better the stability of the reference gene, and vice versa. If the M value is greater than 1.5, it is not considered as a reference gene. In terms of expression stability from high to low, the order is Ce009836 = Ce050340 (0.0868) > Ce029238 (0.1442) > Ce036387 (0.1919) > Ce012579 (0.2734). All candidate reference genes have an M value less than 1.5 and are likely to be suitable reference genes. Among them, Ce009836 and Ce050340 have the smallest M values ​​and better stability ( Figure 17 ).

[0056] The geNorm software can also calculate the paired variation V value of the normalization factor after the introduction of a new internal reference gene, which is called the "paired variable (Vn / n+1)". The specific number of internal reference genes can be determined according to the formula. To ensure that a single internal reference gene does not cause deviation and fluctuation, the geNorm software is based on the calculated paired difference Vn / n+1 to ensure the most appropriate number of internal reference genes under this condition. The critical value in this formula is set to 0.15. If Vn / n+1 is greater than 0.15, the number of selected internal reference genes must meet n+1; if Vn / n+1 is less than 0.15, then when n internal reference genes are selected, the experimental requirements of the software can be met. The geNorm histogram analysis shows that when the V2 / 3 value (0.056) ﹤0.15 ( Figure 18 ), the optimal number of reference gene combinations is 2. Therefore, the most suitable reference genes for different taro tissues are Ce009836 and Ce050340.

[0057] Stability assessment of candidate internal reference genes using NormFinder software

[0058] The NormFinder software algorithm is similar to the geNorm program. It also first determines the stability value of the internal reference gene expression and then selects the most suitable internal reference gene based on the stability value. The candidate internal reference gene with the smallest expression stability value is considered the appropriate internal reference gene. The NormFinder program not only compares the expression differences of candidate internal reference genes but also calculates the variation between sample groups. However, it can only select a single optimal internal reference gene.

[0059] NormFinder software evaluated five candidate reference genes, showing that the expression stability values ​​of the reference genes Ce009836 (0.030) > Ce050340 (0.101) > Ce036387 (0.110) > Ce029238 (0.139) > Ce012579 (0.259). CeRPL28 was the most suitable reference gene for different taro tissues. These results were generally consistent with those evaluated by geNorm software.

[0060] BestKeeper analysis results of candidate internal reference genes

[0061] BestKeeper software calculates sample data to obtain the intergene pairwise correlation coefficient (r), standard deviation (SD), and coefficient of variation (CV). These three parameters are used to determine the stability of the internal reference gene. A higher r, lower CV, and lower standard deviation (SD) indicate a more stable internal reference gene. Furthermore, a standard deviation (SD) greater than 1 indicates that the candidate internal reference gene is unstable.

[0062] According to the correlation coefficient (r), the candidate reference genes were ranked as Ce009836 = Ce029238 (0.994) > Ce036387 (0.981) > Ce050340 (0.977) > Ce012579 (0.913). According to the SD value, Ce012579 (0.449) > Ce009836 (0.489) > Ce050340 (0.494) > Ce036387 (0.602) > Ce029238 (0.622). However, all 5 candidate genes were ranked as Ce009836 = Ce029238 (0.994) > Ce036387 (0.981) > Ce050340 (0.977) > Ce012579 (0.913). The SD values ​​of all reference genes were less than 1. According to the coefficient of variation (CV) values, Ce012579 (1.712) > Ce009836 (1.944) > Ce050340 (1.971) > Ce036387 (2.229) > Ce029238 (2.746). Taking into account the correlation coefficient (r), coefficient of variation (CV), and standard deviation (SD), Ce0009836 was the most suitable reference gene, with a correlation coefficient of 0.994, a standard deviation of 0.489, and a coefficient of variation of 1.944.

[0063] Table 3 Stability values ​​of internal reference genes analyzed by BestKeeper software

[0064] parameter Ce012579 Ce029238 Ce036387 Ce050340 Ce009836 Correlation coefficient (r) 0.913 0.994 0.981 0.977 0.994 Standard deviation (SD) 0.449 0.622 0.602 0.494 0.489 Coefficient of variation (CV) 1.712 2.746 2.229 1.971 1.944

[0065] Stability analysis of internal reference genes

[0066] A comparison and analysis of three internal reference gene software revealed slight discrepancies between the analysis results of BestKeeper, geNorm, and NormFinder. A comprehensive evaluation of the analysis results from the three software programs was performed in Excel; the smaller the geometric mean, the more stable the internal reference gene. Table 4 shows that Ce009836 had the smallest geometric mean across the three software programs, making it the most stable internal reference gene. Therefore, among the five candidate genes, Ce009836 was the most suitable internal reference gene.

[0067] Table 4 Comprehensive analysis results of geNorm, NormFinder and BestKeeper

[0068]

[0069]

[0070] Example 2:

[0071] The gene Ce009836 with the best stability screened out according to Example 1 was further verified to ensure that true and reliable quantitative data were obtained. Using Ce009836 as the internal reference gene, a gene starch branching enzyme (CeSBE2) related to the synthesis of corm starch was selected to verify the stability of the screened internal reference gene. Using Ce009836 as the internal reference gene and CeSBE2 as the target gene, primers were designed (Table 5) to perform qRT-PCR expression analysis on the CeSBE2 gene in different tissues of taro. The results showed that the internal reference gene was stably expressed and could well reflect the changes in the expression level of CeSBE2 in different tissues ( Figure 19 ). The details are as follows:

[0072] cDNA was extracted from leaves, petioles, corms and roots 90 days after sowing of taro respectively as templates for quantitative PCR, Ce009836 gene was used as internal reference gene, and starch branching enzyme (CeSBE2), a gene related to corm starch synthesis, was used as target gene to design primers. The primers are shown in Table 5:

[0073] Table 5 Primer sequences of internal reference genes and target genes

[0074]

[0075] The fluorescence quantitative reaction was performed on a real-time fluorescence quantitative PCR detector as follows:

[0076] The PCR reaction system was 20 μL in total: 2X SYBR Green Master Mix 10 μL, 10 μM upstream primer 0.4 μL, 10 μM upstream primer 0.4 μL, cDNA template 1 μL, and RNase-free water 8.2 μL;

[0077] The PCR reaction program was pre-denaturation at 95°C for 3 min; denaturation at 95°C for 10 s, annealing at 57°C for 15 s, and extension at 72°C for 20 s for a total of 45 cycles; and fluorescence signals were collected.

[0078] After the reaction, use 2 -ΔΔ All data from the CT method are expressed as mean ± standard deviation. Real-time fluorescence quantitative PCR data were statistically analyzed using SPSS 17.0 and Microsoft Office Excel 2007. ANOVA was performed using SPSS 17.0 software, and graphics were prepared using Excel 2010 software.

[0079] The reaction results are as follows Figure 19 As shown, the reference gene Ce009836 can be stably expressed in different taro tissues: roots, leaves, petioles and corms, which can well reflect the changes in the expression level of CeSBE2 in different taro tissues.

[0080] In summary, gene Ce009836 can be stably expressed in different taro tissues: roots, leaves, petioles and corms, and can be used as an internal reference gene for fluorescence quantitative analysis of different taro tissues, laying a foundation for future research on important functional trait genes of taro.

[0081] The above-described embodiments merely illustrate several embodiments of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. Taro internal reference gene Ce009836 , characterized in that, The internal reference gene Ce009836 The nucleotide sequence is shown in the sequence listing SEQ ID NO.

1.

2. Detection of the taro internal reference gene as claimed in claim 1 Ce009836 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. taro internal reference gene as claimed in claim 1 Ce009836 Application in fluorescence quantitative expression analysis of different taro tissues; the taro variety is Guiyu No.

2.

4. The use according to claim 3, characterized in that The different taro tissues are: roots, leaves, petioles and bulbs of taro.

5. The use according to claim 3, characterized in that The fluorescent quantitative expression analysis uses the primer pair as claimed in claim 2.

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