CN116287366A - Screening and application of taro internal reference gene Ce049358 - Google Patents
Screening and application of taro internal reference gene Ce049358 Download PDFInfo
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Abstract
The invention relates to the technical field of plant genes, in particular to screening and application of taro reference genes Ce049358, the invention analyzes candidate genes in a high expression region (FPKM), compared with Ce047468 genes applied in the same day, the expression quantity is higher and more stable, and the taro reference genes Ce049358 obtained by the application can well reflect the expression characteristics of CeAGPL1 in different tissues through analysis, can be used as reference genes for expressing the different tissues of taro, and the designed specific primers can also provide reference basis for subsequent taro gene expression analysis.
Description
[ field of technology ]
The invention relates to the technical field of plant genes, in particular to screening of a taro internal reference gene Ce049358 and application thereof.
[ background Art ]
Taro (Colocasia esculenta) is one of the important food crops at present, tubers of the taro have good edible value, and taro is one of the main agricultural products in Guangxi, but the research on the molecular biology of taro is very little, and the gene research on the taro is very little reported in the current row, and in recent years, along with the continuous development of high-throughput sequencing and molecular biology research, the expression analysis of the gene is gradually applied to the research for revealing the expression and regulation mechanism of the taro gene. Along with the sequencing of the taro genome, the genetic mechanism analysis of taro related characters is accelerated.
Real-time fluorescent quantitative PCR (quantitative Real-time PCR, qRT-PCR) is the main analytical means for detecting gene expression levels at present. Currently, genes commonly used as reference genes in plants include actin (actin), tubulin (TUB), transcription elongation factor genes (elongation factor, EF1A and EF 1B), eukaryotic initiation factors (eukaryotic initiation factor, eIF), ubiquitin-binding enzyme genes (ubquitin-conjugating enzyme, UBC), histones (Histone, H3-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the like.
However, in practical detection, we found that the expression levels of these reference genes are not stable under certain experimental conditions, and different tissues of taro cannot be well expressed, so we need to select stably expressed reference genes by taking materials for different tissues of a sample or different growing and developing time periods.
[ invention ]
In view of the above, it is necessary to provide a reference gene that can perform fluorescence expression analysis on different tissue samples of taro, can analyze the expression of taro, and is stable in expression.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the nucleotide sequence of the taro reference gene Ce049358 and the reference gene Ce049358 is shown in a sequence table SEQ ID NO. 1.
The invention also comprises a primer pair for detecting the taro internal reference gene Ce049358, wherein the upstream sequence of the primer pair is shown in a sequence table SEQ ID NO.2, and the downstream sequence of the primer pair is shown in a sequence table SEQ ID NO. 3.
The invention also comprises application of the taro internal reference gene Ce049358 in fluorescent quantitative expression analysis of different taro tissues.
Further, the taro has different tissues: root, leaf, petiole and bulb of taro.
Furthermore, the upstream sequence of the fluorescent quantitative expression analysis primer pair is shown in a sequence table SEQ ID NO.2, and the downstream sequence is shown in a sequence table SEQ ID NO. 3.
Further, the total 20 μl of the PCR reaction system for fluorescence quantitative expression analysis is: 2X SYBR Green Master Mix 10. Mu.l, 10. Mu.M upstream primer 0.4. Mu.l, cDNA template 1. Mu.l, RNase-free water 8.2. Mu.l; the PCR reaction procedure is that the PCR reaction is performed for 3min at 95 ℃; denaturation at 95℃for 10s, annealing at 57℃for 15s, extension at 72℃for 20s for a total of 45 cycles; fluorescent signals are collected.
The taro internal reference gene Ce049358 provided by the invention is screened by the following steps:
1. after the taro is sowed for 90 days, collecting leaves, petioles, corms and roots of the taro, putting the taro into a 50ml sterile freezing tube, quick-freezing the taro by liquid nitrogen, and then putting the taro into a refrigerator at the temperature of minus 80 ℃ for standby, and extracting sample RNA;
2. reverse transcription is carried out to synthesize corresponding taro tissue cDNA;
3. different primers are designed according to different genes and reacted on a fluorescent quantitative PCR instrument, a dissolution curve is drawn, and stability analysis is carried out, so that the internal reference gene with good stability and good expression effect is obtained.
The invention has the following beneficial effects:
the invention analyzes candidate genes in a high expression region (FPKM), compared with Ce047468 genes applied in the same day, the expression quantity is higher and more stable, the screening of internal reference genes is data of transcript sequencing aiming at different tissues (leaves, petioles, bulbs and roots) of taro, the stability of the expression quantity FPKM value is evaluated by using a variation coefficient CV, and the genes with CV value less than or equal to 0.2 and cDNA less than or equal to 1000bp are selected as the candidate internal reference genes. The qRT-PCR is utilized to analyze the expression stability of 5 candidate genes in different tissues of the taro, three types of reference gene stability analysis software geNorm, normFinder and BestKeeper are utilized to analyze the stability of the 5 candidate reference genes in different tissues and to carry out comprehensive analysis and sequencing, thus obtaining taro reference genes Ce049358, and through verification, ce049358 can well reflect the expression characteristics of CeAGPL1 in different tissues, and the reference genes obtained through screening and the designed specific primers provide reference basis for the subsequent taro gene expression analysis.
[ description of the drawings ]
FIGS. 1-5 are candidate reference genes, respectively: qPCR detection result graphs of Ce007097, ce012507, ce026647, ce041066 and Ce049358 in different tissues; wherein M is Marker DL2000; lanes 1-8 are 2 replicates of leaf-1, leaf-2, petiole-1, petiole-2, root-1, root-2, bulb-1, bulb-2, respectively.
FIGS. 6-10 are candidate reference genes, respectively: amplification plots of Ce007097, ce012507, ce026647, ce041066 and Ce 049358;
FIGS. 11-15 are candidate reference genes, respectively: dissolution profiles of Ce007097, ce012507, ce026647, ce041066 and Ce 049358;
FIG. 16 is a box plot of Ct values for five candidate reference genes in different tissues;
FIG. 17 shows the steady ordering of candidate reference gene expression by gemm software;
FIG. 18 is a histogram of the variation coefficient of the reference gene analyzed by the gemm software;
FIG. 19 is a diagram showing the expression of Ce049358 gene in different tissues of taro.
[ detailed description ] of the invention
In order that the above objects, features and advantages of the invention will be readily understood, a more particular description of the invention will be rendered by reference to the appended drawings. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. The invention may be embodied in many other forms than described herein and similarly modified by those skilled in the art without departing from the spirit or scope of the invention, which is therefore not limited to the specific embodiments disclosed below.
Example 1:
the present example is the acquisition of the reference gene Ce049358, which is obtained by the following method:
1. test materials of this example:
the taro material of the embodiment is taro new variety Gui Henry No.2 selected and bred by the biological technology research institute of Guangxi agricultural academy of sciences. Collecting leaf, petiole, corm and root after 90d sowing, placing into a 50ml sterile freezing tube, quick-freezing with liquid nitrogen, and placing into a refrigerator at-80deg.C for use. Three biological replicates per group of samples.
2. Total RNA extraction, detection and c DNA synthesis:
extracting total RNA of different tissues of the taro by using a small extraction kit (R4165) for total RNA of plant difficult to extract by using a American-based organism, wherein the operation steps are operated according to the instruction book of the kit. The integrity of the extracted RNA was checked using 1.5% agarose gel electrophoresis, while the concentration and quality of RNA was checked using Nanodrop 2100. Using a reverse transcription kit of HiScript II Q RT SuperMix for qPCR (+gDNAwind) from Norpraise (R223-01), 1. Mu.g of total RNA was reverse transcribed into cDNA. The total reaction system was 8. Mu.L, 4 XgDNAwit Mix 2. Mu.L, total RNA 1. Mu.g, and RNase-free water was added to make up to 8. Mu.L. After gentle mixing, reverse transcription reaction is carried out, and the reaction conditions are set as follows: the cDNA was then stored at-20℃in a refrigerator at 50℃for 15min at 85℃for 5 s.
3. Selection of reference genes and design of specific primers:
transcriptome data of different tissues of taro are used, stability of the expression quantity FPKM value is evaluated by using a variation coefficient CV, and genes with CV value less than or equal to 0.2 and cDNA less than or equal to 1000bp are selected as candidate reference genes. From among the candidate reference genes, 5 FPKM genes of 146.54 to 244.90 were selected (Table 1). Specific primers of 5 candidate reference genes were designed by using Primer Premier 6 software, the length of the primers was 131-193 bp, and the primers were synthesized by Beijing qing Biotechnology Co., ltd (Nanning) (Table 2).
TABLE 1 FPKM values of candidate reference genes in different tissues
| Gene name | Root of Chinese character | Blade | Leaf handle | Bulb of ball | Average value of FPKM | Standard deviation/SD | Coefficient of variation/CV |
| 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 |
TABLE 2 primers for 5 candidate internal reference genes of taro
4. qPCR of reference gene:
quantitative fluorescence qRT-PCR analysis was performed using an AnalytikJena qtOWERE2.2 fluorescent quantitative PCR apparatus, germany. The fluorescent quantitative reagent used was a 2X ChamQ Universal SYBR qPCR Master Mix kit of Norfluzab. The reaction system was 20. Mu.L in total, 0.4. Mu.L (10. Mu. Mol/L) for the upstream primer (F) and 0.4. Mu.L (10. Mu. Mol/L) for the downstream primer (R), 1. Mu.L for the cDNA template, 10. Mu.L for SYBR GreenMaster Mix (2X), and 8.2. Mu.L for the RNase-free water. The reaction procedure: pre-denaturation at 95℃for 3min,95℃for 10s; annealing at 57 ℃ for 15s; extending at 72 ℃ for 20s; for 45 cycles. Three biological replicates and three technical replicates were run for each set of samples.
The primers used for each gene during the PCR reaction are shown in Table 2 above:
5. data processing and analysis
The stability of the candidate reference genes is analyzed independently by using software with different algorithms of geNorm, normFinder and BestKeeper. The gemm and NormFinder are those using 2 -△CT The value indicates the expression stability of the reference gene. And BestKeeper evaluates the stability of each candidate reference gene based on its Ct value. And finally, comprehensively sorting by a geometric mean value method.
6. Results and analysis
Detection of total RNA quality of (one) stem
RNA from different tissues (leaf, petiole, bulb and root) was extracted and its quality was assessed. The Agilent 2100/GX is used for detecting the integrity of RNA, and 28S/18S is 1.50-2.06, which shows that the RNA has better integrity and no degradation. The concentration of RNA measured by NanoDrop2100 is 32.9-384.2 ng/. Mu.l, and OD260/280 is 2.08-2.11, which indicates that the purity of RNA is higher.
Specificity analysis of candidate reference genes
The results of the electrophoretic detection of PCR amplified products (FIGS. 1-5) showed that each of the 5 candidate internal reference genes was seen to have a single band consistent with the length of the expected product, and primer dimer was not present. Meanwhile, the amplification curves of 5 candidate genes in the qRT-PCR result are analyzed, and the result shows that the amplification curves are good (figures 6-10); analysis of the lysis curves revealed that the lysis curves were only single peak, indicating a single amplified band, strong specificity, and no non-specific amplification occurred (FIGS. 11-15). Therefore, the designed real-time fluorescent quantitative PCR primer has strong specificity and high amplification efficiency, and can be used for an internal reference primer test of taro fluorescent quantitative PCR.
Ct value analysis of candidate genes
Ct value is an important measure of the abundance of expressed genes. The Ct value is inversely related to the abundance of expression. The expression abundance analysis of 5 reference genes in different taro tissues shows (figure 16), wherein the average Ct value of 5 candidate reference genes is between 18.58 and 24.61, the Ct value of Ce007097 in different taro tissues is between 18.75 and 19.49, and the difference between the maximum value and the minimum value is 0.74 cycles; ct values of Ce012507 in different tissues are 19.75-21.33, and the maximum value and the minimum value are different by 1.58 cycles; ct values of Ce026647 in different tissues are 18.58-19.83, and the maximum value and the minimum value are different by 1.25 cycles; ct values of Ce041066 in different tissues are 22.59-24.61, and the difference between the maximum value and the minimum value is 2.02 cycles; ce049358 has Ct values of 22.09-22.83 in different tissues, with the maximum and minimum values differing by 0.74 cycles.
Stability analysis of reference genes
The study was aided by internal analysis software geNorm, normfinder and BestKeeper software, and the 5 candidate internal genes were evaluated and analyzed for agreement from multiple aspects.
Stability assessment of candidate reference genes by gemm software
The gemum program screens out reference genes with better stability by calculating the M value of the stability of each reference gene, and the judgment standard is that the smaller the M value is, the better the stability of the reference gene is, otherwise, the worse the stability is, and if the M value is larger than 1.5, the reference gene is not considered. According to the expression stability from high to low, ce049358 =ce 026647 (0.1608) > Ce012507 (0.3247) > Ce041066 (0.4094) > Ce007097 (0.5006) is sequentially carried out. All candidate reference genes have an M value of less than 1.5 and are likely to be suitable reference genes. The smaller the M values of Ce049358 and Ce026647, the better the stability (FIG. 17).
The gemm software can also calculate the paired variation V values of the normalization factor after the introduction of 1 new reference gene, called: "pairing variable (Vn/n+1)". The specific number of reference genes can be judged according to the formula. To ensure that a single reference gene does not cause bias or fluctuations, the gemum software ensures the most appropriate number of reference genes under these conditions based on the calculated pairing difference Vn/n+ 1. The critical value in the formula is set to be 0.15, and if Vn/n+ 1 is greater than 0.15, the number of the selected internal reference genes is required to satisfy n+1; if Vn/n+ 1 is less than 0.15, the software experiment requirement can be met when n internal reference genes are selected. The gemm histogram analysis showed that when the V2/3 value (0.133) was selected to be less than 0.15 (FIG. 18), the optimum number of reference gene combinations was 2. Therefore, the most suitable internal reference genes of different tissues of the taro are Ce049358 and Ce026647.
Stability assessment of candidate reference genes by NormFinder software
The NormFinder software program algorithm is similar to the GeNorm program, and is also to obtain stable expression values of the reference genes, screen the most suitable reference genes according to the stable values, and judge that the candidate reference genes with the minimum expression stable values are suitable reference genes. The NormFinder program can compare not only the expression differences of candidate reference genes, but also calculate the variation between sample groups, but the program can only screen out one of the most suitable reference genes.
The evaluation result of the NormFinder software on 5 candidate reference genes shows that the reference genes have stable expression values Ce049358 (0.056) =Ce 026647 (0.056) > Ce041066 (0.324) > Ce012507 (0.330) > Ce007097 (0.403), and the most suitable reference genes in different taro tissues are Ce049358 and Ce026647. The results were substantially identical to those of the gemm software evaluation.
Analysis results of BestKeeper on candidate reference genes
The BestKeeper software calculates sample data to obtain an inter-gene correlation coefficient (r), a Standard Deviation (SD) and a variation Coefficient (CV), and uses the 3 parameters to judge the stability of the internal reference. The higher the correlation coefficient (r), the lower the Coefficient of Variation (CV) and Standard Deviation (SD), the more stable the reference gene is. In addition, when the standard deviation SD is greater than 1, the candidate reference gene expression is considered to be unstable.
According to the sequencing of the correlation coefficient (r), the sequencing of the candidate internal reference genes is Ce026647 (0.998) more than Ce049358 (0.983) more than Ce041066 (0.963) more than Ce012507 (0.749) more than Ce007097 (0.277). According to the SD value sequence, ce049358 (0.226) is larger than Ce007097 (0.244) is larger than Ce026647 (0.379) is larger than Ce CCe012507 (0.431) is larger than Ce041066 (0.529), but all candidate 5 internal reference genes are smaller than 1 in SD value. According to the sequence of the variation Coefficient (CV) values, ce049358 (1.003) is larger than Ce007097 (1.268) is larger than Ce026647 (1.958) is larger than Ce012507 (2.092) is larger than Ce041066 (2.245). The three values of the comprehensive correlation coefficient (r), the variation Coefficient (CV) and the Standard Deviation (SD) are higher, the Ce049358 correlation coefficient is 0.983, the standard deviation is 0.226, and the variation coefficient is 1.003, so that the gene is the most suitable reference gene.
TABLE 3 BestKeeper software analysis of internal control Gene stability values
| Parameters (parameters) | Ce007097 | Ce012507 | Ce026647 | Ce041066 | Ce049358 |
| 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 |
Stability analysis of reference genes
By comparing and analyzing the three reference gene software, the analysis result of BestKeeper, geNorm, normFinder is found to be slightly different. Comprehensively evaluating the analysis result of the 3 types of software in Excel; the smaller the geometric mean, the more stable the reference gene. From table 4, it can be seen that Ce049358 is the most stable reference gene with the smallest geometric mean of the three types of software. Thus, ce049358 is the most suitable reference gene among the 5 candidate genes.
TABLE 4 comprehensive analysis results of three types of software, gemm, normFinder and BestKeeper
| GeNorm ordering | NomFinder ordering | Bestkeeper ordering | Geometric | |
| 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 |
Example 2:
the gene Ce049358 screened out according to example 1 and having the best stability is further verified to ensure that the real and reliable quantitative data are obtained. With Ce049358 as an internal reference gene, 1 gene ADP-glucose pyrophosphorylase large subunit gene (CeAGPL 1) related to bulb starch synthesis was selected, and stability of the selected internal reference gene was verified. The CeAGPL1 gene of different tissues of taro was subjected to qRT-PCR expression analysis by designing primers (Table 5) using Ce049358 as a reference gene and CeAGPL1 as a target gene, and the results show that the reference gene was stably expressed and the variation of the expression level of CeAGPL1 in different tissues was well reflected (FIG. 19). The method comprises the following steps:
respectively extracting cDNA of leaves, petioles, corms and roots of the 90d th taro after sowing as a template for quantitative PCR, taking Ce049358 gene as an internal reference gene, taking a gene ADP-glucose pyrophosphorylase large subunit gene (CeAGPL 1) related to the synthesis of corm starch as a target gene, and designing primers, wherein the primers are shown in Table 5:
TABLE 5 primer sequences of reference gene and target gene
The fluorescent quantitative reaction is carried out on a real-time fluorescent quantitative PCR detector, and the method concretely comprises the following steps:
the total of the PCR reaction system is 20 mu L:2X SYBR Green Master Mix 10. Mu.l, 10. Mu.M upstream primer 0.4. Mu.l, cDNA template 1. Mu.l, RNase-free water 8.2. Mu.l;
the PCR reaction procedure is pre-denaturation at 95 ℃ for 3min; denaturation at 95℃for 10s, annealing at 57℃for 15s, extension at 72℃for 20s for a total of 45 cycles; fluorescent signals are collected.
The reaction results obtained are shown in FIG. 19, which shows that: the internal reference gene Ce049358 is arranged in different tissues of taro: the root, leaf stalk and bulb can stably express, and can well reflect the variation of the expression quantity of CeAGPL1 in different taro tissues.
In conclusion, the gene Ce049358 is found in different tissues of taro: the stable expression of the root, leaf stalk and bulb can be used as fluorescent quantitative reference genes of different tissues of taro, and can lay a foundation for the research of important functional trait genes of taro in future.
The above examples merely represent a few embodiments of the present invention, which are described in more detail and are not to be construed as limiting the scope of the present invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of the invention should be assessed as that of the appended claims.
Claims (5)
1. The taro reference gene Ce049358 is characterized in that the nucleotide sequence of the reference gene Ce049358 is shown in a sequence table SEQ ID NO. 1.
2. The primer pair for detecting the taro reference gene Ce049358 according to claim 1, wherein the upstream sequence of the primer pair is shown in a sequence table SEQ ID NO.2, and the downstream sequence of the primer pair is shown in a sequence table SEQ ID NO. 3.
3. The use of the taro internal reference gene Ce049358 according to claim 1 for fluorescence quantitative expression analysis of different taro tissues.
4. The use according to claim 3, wherein the taro is organized differently as: root, leaf, petiole and bulb of taro.
5. The use according to claim 3, wherein the upstream sequence of the fluorescent quantitative expression analysis primer pair is shown in a sequence table SEQ ID NO.2, and the downstream sequence is shown in a sequence table SEQ ID NO. 3.
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