CN112813181B - Reference gene suitable for gene expression analysis of different tissues of cigar and application thereof - Google Patents
Reference gene suitable for gene expression analysis of different tissues of cigar and application thereof Download PDFInfo
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Abstract
The invention relates to an internal reference gene suitable for gene expression analysis of different tissues of cigars and application thereof. The expression stability of the ten candidate reference genes including RPL8, RPL14A, RPS16, UBC27, UBC28, UBC35, AC10, EIF2, PSAP and CPI in different tissues (roots, stems, leaves, calyces, corolla, stigma, ovary, stamen, pistil and seeds) of the cigar in different development stages is analyzed through three analysis software of geNorm, NormFinder and BestKeeper. The results show that the RPL14A and/or UBC27 genes are stably expressed in different tissues during different developmental stages of the cigar, namely the RPL14A and/or UBC27 genes can be used as reference genes for fluorescence quantitative PCR of different tissues of the cigar. The invention further provides qRT-PCR primers of the RPL14A gene and the UBC27 gene, and lays a foundation for accurate quantitative analysis of gene expression of different tissues of cigars.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to an internal reference gene suitable for gene expression analysis of different tissues of cigars and application thereof.
Background
Cigars (Nicotiana tabacum L) are round or square smoking cigarettes made of tobacco leaves, and are special tobacco products. The cigar is a pure natural tobacco product, is made by rolling original tobacco leaves after the processes of modulation (air drying and air curing), fermentation and alcoholization, has pure and strong fragrance, comfortable taste and small content of main harmful ingredients (tar and salt and alkali), thereby having little harm to human body, wide market prospect and considerable economic value at present. According to the application in tobacco products, the cigar raw material tobacco leaves are generally called to be divided into outer wrappers, inner wrappers and core leaves in China, namely eggplant coats, eggplant covers and eggplant cores. Among them, the eggplant coat is the coat of cigars, which is the most beautiful and expensive in cigars and is the only important part to be seen by consumers.
At present, the high-quality cigar tobacco leaves are mainly produced in the countries of Cuba, America, Ecuador, Indonesia and the like in the world. The quality of cigar coats produced in China is far from reaching the high-quality level, so that the shortage of high-quality cigar coat raw materials in China seriously restricts the rapid development of the cigar industry in China. The lack of good varieties and the degeneration of the existing varieties become the main problems of eggplant-coated tobacco leaves. With the rapid development of molecular biology and genetics, the research and improvement of the existing varieties become possible from the perspective of eggplant genes.
The gene expression level analysis by using real-time fluorescent quantitative PCR (qRT-PCR) is an important research means in modern molecular biology, and has the characteristics of accurate quantification, high sensitivity, high flux and the like. In qRT-PCR, analysis of the relative expression amount of a target gene is mainly determined by normalization of an internal reference gene, and therefore, it is very critical to select an appropriate internal reference gene. The ideal reference gene should be stably expressed in different developmental stages and different tissues and organs, and the expression level is not influenced by other external factors and experimental conditions. The use of a reference gene is required in the analysis of cigar coat gene expression.
Generally, housekeeping genes are widely involved in basic biochemical reactions of organisms, and thus are often used as reference genes. Currently, there are commonly used reference genes such as ACTIN family (ACTIN), Ribosomal protein gene (RPL), transcription Elongation factor gene (Elongation factors 1alpha, EF-1 alpha), polyubiquitin gene (UBQ), glyceraldehyde-3-phosphate dehydrogenase Gene (GAPDH), and β -tubulin gene (TUB). However, the expression of these widely used reference genes is not stable under different plant varieties and different experimental conditions; so far, there are few reports on the reference gene screening of cigar. Therefore, the selection of reference genes in cigars remains blind.
Disclosure of Invention
The invention aims to provide an internal reference gene suitable for gene expression analysis of different tissues of cigars, wherein the internal reference gene is RPL14A gene or UBC27 gene, has good stability in different tissues of cigars in different growth periods, and lays a foundation for quasi-quantitative analysis of transcription expression levels of functional genes of cigars.
The second purpose of the invention is to provide the application of the RPL14A gene or UBC27 gene as a reference gene in qRT-PCR detection of gene transcription expression levels of different tissues of cigars.
In order to achieve the purpose, the invention adopts the following technical scheme:
in one aspect, the invention provides an internal reference gene suitable for gene expression analysis of different tissues of cigars, wherein the internal reference gene is RPL14A gene or UBC27 gene, and the nucleotide sequence of the RPL14A gene is shown as SEQ ID No.1 in a sequence table; the nucleotide sequence of the UBC27 gene is shown as SEQ ID NO.2 in the sequence table.
Further, the amplification primers of the RPL14A gene are as follows:
F：ATGCCGTTCAAGAGGTTCG
R：TTAAGCAGCGGTCTTCTTAAG
the amplification primers of the UBC27 gene are as follows:
F:ATGGTGGACTTGGCAAG
R：CTAGCCAGAGCACAGCTTTTC
on the other hand, the invention provides application of the RPL14A gene and/or the UBC27 gene as reference genes in qRT-PCR detection of gene transcription expression levels of different tissues of cigars.
Specific primers for the RPL14A gene were:
F：TCCTCCGCAGCTTCACAA(SEQ ID NO.3)
R：TTTATCTGGCTCCTCACC(SEQ ID NO.4)
specific primers for the UBC27 gene were:
F:CTGGACTGAAACTTTTGC(SEQ ID NO.5)
R：TGGCTTCCAAAGTACTCCT(SEQ ID NO.6)
preferably, a forward primer with a nucleotide sequence shown as SEQ ID NO.3 and a reverse primer with a nucleotide sequence shown as SEQ ID NO.4 are adopted to carry out specific amplification on the RPL14A gene; the UBC27 gene is specifically amplified by using a forward primer with a nucleotide sequence shown as SEQ ID NO.5 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 6.
Preferably, the qRT-PCR is carried out by sequentially adding the following reagents into a 20 mu L reaction system: 10 μ L of 2 XSSYBR I Master, 0.5 μ L of each of the upstream and downstream primers, 50ng of cDNA, plus ddH 2 O to 20. mu.L.
Preferably, the reaction procedure of qRT-PCR is as follows: pre-denaturation at 94 ℃ for 30 s; denaturation at 94 ℃ for 5s, annealing at 60 ℃ for 10s, and elongation at 72 ℃ for 10s, for 45 cycles.
The invention has the following beneficial effects:
the present invention analyzes the expression stability of ten candidate internal reference genes including ribosomal protein genes (RPL8, RPL14A and RPS16), ubiquitin-conjugating enzyme genes (UBC27, UBC28 and UBC35), actin family gene (AC101), transcription elongation factor gene (EIF2), sphingolipid activating protein element (PSAP) and Cysteine Protease Inhibitor (CPI) in different tissues (root, stem, leaf, calyx, corolla, stigma, ovary, stamen, pistil and seed) during different development stages of cigar based on the transcriptome sequencing data of the previous stage and by three analysis software of geNorm, NormFinder and bestkoeper. The result shows that the RPL14A and UBC27 genes are stably expressed in different tissues of cigar in different development periods, namely the RPL14A or UBC27 genes can be used as reference genes of qRT-PCR to normalize the expression level of target genes, and the problem that the genes of different tissues of cigar lack proper reference genes in the aspect of gene expression analysis is solved. In addition, the internal reference genes RPL14A and UBC27 provided by the invention are both derived from transcriptome data of cigars, and compared with the internal reference genes obtained by a traditional homologous comparison method, the internal reference screened by the transcriptome data has the characteristics of high-amount expression and stable expression.
The invention also provides application of the RPL14A gene or the UBC27 gene as a reference gene in qRT-PCR detection of gene transcription expression levels of different tissues of the cigar, and the application can be used for expression analysis of key genes in the cigar development process and can obviously improve the accuracy of obtained data. The invention further provides qRT-PCR primers of the RPL14A gene and the UBC27 gene, and the primers have no non-specific amplification and high specificity amplification efficiency.
Drawings
FIG. 1 is a melting curve of 10 candidate reference genes of cigar;
FIG. 2 is a standard curve of each candidate reference gene primer of cigar during qRT-PCR experiment;
FIG. 3 shows Ct values of qRT-PCR in all samples for the reference gene candidate for cigar.
Detailed Description
The present invention will be further described with reference to specific embodiments, but the scope of the present invention is not limited thereto; the equipment and reagents used in the examples are all conventionally commercially available unless otherwise specified.
Example 1 screening of candidate reference genes
1. Screening of candidate reference genes of cigar
10 gene sequences with high and consistent expression of roots, stems and leaves are selected from transcriptome sequencing data (uploaded to Chinese nucleic acid database https:// bigd.big.ac.cn/gsa. retrieval number: CRA003020) to serve as candidate reference genes (Table 1). Primer design was performed using Primer Premier 5.0 (table 2).
TABLE 1 information on candidate reference genes
| Name of Gene | Description of genes | Seq ID |
| RPL8 | 60S ribosomal protein L8 | transcript_19714 |
| RPL14A | 60S ribosomal protein L14-1 | transcript_22219 |
| RPS16 | 40S ribosomal protein S16 | transcript_22287 |
| UBC27 | Ubiquitin-conjugating enzyme E2 27 | transcript_20180 |
| UBC28 | Ubiquitin-conjugating enzyme E2 28 | transcript_20220 |
| UBC35 | Ubiquitin-conjugating |
transcript_21000 |
| EIF2 | Eularotic translation initiation factor 2subunit beta | transcript_19181 |
| CPI | Cysteine proteinase inhibitor | transcript_22854 |
| AC101 | Actin-101 | transcript_16513 |
| PSAP | Prosaposin | transcript_19206 |
2. Sampling of different tissues of cigar and total RNA extraction
Tissues of cigars used in the experiment are collected in 2019, and the seedling stage (roots, stems and leaves), the vigorous growth stage (roots, stems and leaves), the full-bloom stage (roots, stems, leaves, calyx, corolla, stigma, ovary, stamen and pistil) and seeds of the cigars are respectively taken. After harvesting, the roots were carefully washed with clean water and blotted dry with filter paper. All samples were frozen immediately after collection with liquid nitrogen and stored in a-80 ℃ freezer.
Taking 100mg of different tissues of cigars, quickly grinding the tissues into powder in liquid nitrogen, and extracting RNA of the different tissues of the cigars by using a plant RNA extraction kit. mu.L of the original sample RNA was diluted 10-fold or 100-fold, and 5. mu.L of the diluted sample was subjected to 1% agarose gel electrophoresis to examine the integrity of the RNA sample. And 1. mu.L RNA was used to determine the purity and concentration of RNA samples using a micro-UV spectrophotometer (NanoDrop 2000). And (4) preserving qualified samples at-80 ℃ for later use.
3. Reverse Transcription (RT)
Taking total RNA of each tissue of cigar as a template, and sequentially adding the following reagents in the setting of a reverse transcription system of 20 mu L and the reaction process: mu.g RNA, 1. mu.L Oligo (dT) Primer (20. mu. mol/L), plus RNase-free H 2 O to 13 μ L; after incubation at 65 ℃ for 10min, the centrifuge tube was quickly placed in an ice bath, 4. mu.L of Transcriptor Reverse Transcriptase 5. multidigiffer, 0.5. mu.L of Detector RNase Inhibitor, 2. mu.L of Deoxynucleotide Mix and 0.5. mu.L of Transcriptor Reverse Transcriptase were added again, and after slowly mixing, incubation at 55 ℃ for 30min, incubation at 85 ℃ for 5min, and the centrifuge tube was quickly incubatedPlacing into ice bath, adding water to dilute 5-10 times, and storing at-20 deg.C.
4. Polymerase Chain Reaction (PCR)
And (4) taking the cDNA obtained in the step (3) as a template, designing primers for cloning 10 candidate genes, wherein the cloning primers are shown in a table 2.
TABLE 2 cloning primers for candidate reference genes
The following 20. mu.L reaction system was used for PCR: 50ng of template, 1. mu.L of forward and reverse primers, 1. mu.L of Hifi taq enzyme (all-purpose gold Biotechnology Co., Ltd.), and water to 20. mu.L.
The PCR reaction program is set as follows: pre-denaturation at 94 deg.C for 4min, denaturation at 94 deg.C for 30s, annealing at 56 deg.C for 30s, and extension at 72 deg.C for 1min, for 30 cycles, final extension at 72 deg.C for 10min, and storing at 4 deg.C. The PCR product was subjected to 1% agarose gel electrophoresis to detect the desired fragment.
5. Cloning and sequencing of the Gene of interest
And (3) cutting the target fragment and recovering the gel, connecting the purified DNA fragment to a pMD18-T simple vector, transferring the DNA fragment into an escherichia coli competent cell DH5 alpha, detecting by PCR, and sequencing the positive clone.
Example 2 candidate reference Gene primer design and qRT-PCR
The sequence of the gene is obtained by a method combining third-generation sequencing with transcriptome sequencing, and then a specific qPCR primer is designed, wherein the sequence of a candidate reference gene primer is shown in a table 3. 10 samples (root, stem, leaf, calyx, corolla, stigma, ovary, stamen, pistil) and seed) were mixed in equal amount as template, and diluted according to 5-fold gradient, that is, the concentration of each template was 1 and 10 respectively -1 、10 -2 、10 -3 、10 -4 In total, 5 gradients were used for fluorescent quantitative PCR and plottingA standard curve was prepared (as shown in fig. 2).
RT-qPCR amplification reaction is carried out by using a Roche Light Cycler 96 real-time fluorescent quantitative PCR instrument, and the following reagents are sequentially added into a 20 mu L reaction system: 10 μ L of 2 XSSYBR I Master, 0.5 μ L of each of the upstream and downstream primers, 50ng of cDNA plus ddH 2 O to 20. mu.L. The PCR reaction program is: pre-denaturation at 94 ℃ for 30 s; denaturation at 94 ℃ for 5s, annealing at 60 ℃ for 10s, and elongation at 72 ℃ for 10s, for 45 cycles.
After the reaction is complete, the cycle threshold (CT value, as shown in FIG. 3) is calculated by 2 -ΔΔCT The method calculates the relative expression level of the reference gene. The amplification efficiency of the primers was calculated using the formula (10-1/K-1) × 100%. Where E is the amplification efficiency and K is the slope of the standard curve. Each reaction was set to 3 replicates and the data obtained was analyzed by Excel and the results are shown in table 3.
TABLE 3 candidate reference Gene qPCR primers and related information
As can be seen from the table, the correlation coefficient R of the standard curve of 10 candidate reference genes 2 More than 0.981, the amplification efficiency is between 91% and 107%, the dissolution curves only generate single melting peaks, and no non-specific amplification interference exists, so that the linear analysis and the amplification efficiency of each candidate internal reference gene meet the requirements.
As can be seen from FIG. 1, the average C of 10 candidate reference genes t The value is between 21.39(RPL8) and 29.93(PSAP), and the expression graduation distribution range is wide. Among the 10 candidate reference genes, the expression levels of UBC28, UBC35 and AC101 genes were more enriched, and the average C of all samples t Values are 24.00, 24.66 and 25.21, respectively; the expression level of the RPL8 gene was most dispersed, with a maximum of 27.51, a minimum of 21.39, and an average Ct value of 24.4.
Example 3 stability analysis of candidate reference genes
To screen for the best reference genes, the stability of each candidate reference gene was evaluated by analysis using the three software GeNorm, NormFinder and Bestkeeper. Wherein, the general software of GeNorm is C t The value is original data, expression stability of the candidate reference gene is judged by calculating expression stability values (M) of the candidate reference gene in different samples, the larger the M value is, the lower the gene stability is, and conversely, the smaller the M value is, the higher the gene stability is. Generally, when the M value is 1.5 or less, the gene expression is considered to be stable, and the gene is suitable for use as an internal reference gene. Among the 10 candidate genes, RPL14A and UBC27 were the most stable, PSAP the least stable.
NormFinder software is similar to geNorm, also C t The value is original data, the expression stability is judged by calculating the expression stability value of the candidate reference gene, and the smaller the stability value is, the more suitable the value is for serving as the reference gene. From the results of NormFinder, UBC27 and RPL14A were also the most stable, in substantial agreement with the results of the analysis by the geNorm software.
Bestkeeper software average C in samples according to different candidate reference genes t Value calculation the Standard Deviation (SD), correlation coefficient (r) and variation Coefficient (CV) of the pairing between two genes, and finally selecting the reference gene with good stability by comparing the values. The larger the correlation coefficient, the smaller the standard deviation and the coefficient of variation, the better the stability of the reference gene. When SD>1, the expression of the reference gene is unstable. The results of the Bestkeeper software analysis showed that RPL14A and UBC27 were the most stable, consistent with the results of the geonorm and NormFinder analyses.
TABLE 4 stability analysis of the three software
In conclusion, through various evaluation and analysis methods, two optimal reference genes suitable for the gene expression analysis of cigars in different tissues are finally screened out, namely RPL14A and UBC 27.
<110> Zhengzhou tobacco institute of China tobacco general company
<120> reference gene applicable to gene expression analysis of different tissues of cigar and application thereof
<160> 42
<170> PatentIn version 3.5
<210> 1
<211> 402
<212> DNA
<213> cigar (Nicotiana tabacum L.)
<221> RPL14A Gene
<400> 1
atgccgttca agaggttcgt ggagattgga agggtagcct taatcaacta cggaaaggaa 60
tatggcaagc tggtcgttat agtcgatgtc atcgaccaaa atagggctct tgttgatgct 120
ccagacatgg tgaggagcca gattaacttc aagagacttt cactcacaga tatcaaaatt 180
gacatcaaaa gaatcccaaa gaagaagacc ttggtcgagg ctatggaagc tgctgatgtg 240
aagaccaagt gggagaacag ttcatgggga aggaagttga tagtgcagaa gaggagggct 300
gcacttaacg attttgacag gttcaagctt atgttggcaa agattaagag ggccggagtt 360
gtgagacagg agcttgcaaa gcttaagaag accgctgctt aa 402
<210> 2
<211> 579
<212> DNA
<213> cigar (Nicotiana tabacum L.)
<221> UBC27 Gene
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atggtggact tggcaagggt acaaaaggag ctgcaagagt gcaacagaga tgttggggtt 60
tcaggaataa gtgtaaccct taaaggtgac agtctcactc acttgattgg tacaatccct 120
ggtcctcttg gtactcctta tgaaggtggt tctttcaaga tcgatatcac tcttactgat 180
ggctacccat tcgagcctcc aaaaatgaga tttgccacaa aagtttggca tcctaatata 240
agtagccaaa gtggagcaat atgcctagac atcctgaaag accagtggag cccagcgcta 300
actctcaaga cagctctcct ttctatacaa gcattactct ctgctcctga acctgatgat 360
ccacaagatg cagttgttgc acaacagtat ctcagagacc atcagacctt tgttggcaca 420
gctcgttact ggactgaaac atttgcgaaa acacttggca ccgaggagaa gatacaaaag 480
cttgtggaaa tggggtttcc agaagctcaa gtgaggagta ctttggaagc cacaggttgg 540
gatgaaaaca tggcacttga aaagctgtgc tctggctag 579
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<221> qRT-PCR primer F for RPL14A gene
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<221> RPL8 Gene cloning primer F
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<221> UBC28 Gene cloning primer F
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<221> UBC28 Gene cloning primer R
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<221> UBC35 Gene cloning primer R
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<221> EIF2 Gene cloning primer F
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<221> AC101 Gene cloning primer F
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<221> RPL8 Gene qRT-PCR primer F
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gacggtctca aatccgatc 19
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tagtgtcaag ccaaaggaaa c 21
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gaatatgagg agcttctgg 19
<210> 36
<211> 19
<212> DNA
<213> Artificial sequence
<221> EIF2 gene qRT-PCR primer R
<400> 36
tgtgccttca cgaagaact 19
<210> 37
<211> 18
<212> DNA
<213> Artificial sequence
<221> qRT-PCR primer F for CPI gene
<400> 37
<210> 38
<211> 18
<212> DNA
<213> Artificial sequence
<221> qRT-PCR primer R for CPI gene
<400> 38
<210> 39
<211> 18
<212> DNA
<213> Artificial sequence
<221> qRT-PCR primer F for AC101 gene
<400> 39
<210> 40
<211> 18
<212> DNA
<213> Artificial sequence
<221> qRT-PCR primer R for AC101 gene
<400> 40
<210> 41
<211> 19
<212> DNA
<213> Artificial sequence
<221> PSAP Gene qRT-PCR primer F
<400> 41
ctgagagaaa tatcgagag 19
<210> 42
<211> 19
<212> DNA
<213> Artificial sequence
<221> PSAP Gene qRT-PCR primer R
<400> 42
gtggatcagt atgttcgtc 19
Claims (4)
1.RPL14AGenes orUBC27The gene is used as an internal reference gene in the application of qRT-PCR detection of gene transcription expression levels of different tissues of cigars; the describedRPL14AThe nucleotide sequence of the gene is shown as SEQ ID NO.1 in the sequence table; the above-mentionedUBC27The nucleotide sequence of the gene is shown as SEQ ID NO.2 in the sequence table.
2. The use according to claim 1, characterized in that a forward primer with a nucleotide sequence as shown in SEQ ID No.3 and a reverse primer pair with a nucleotide sequence as shown in SEQ ID No.4 are usedRPL14ACarrying out specific amplification on the gene; or a forward primer with a nucleotide sequence shown as SEQ ID NO.5 and a reverse primer pair with a nucleotide sequence shown as SEQ ID NO.6UBC27The gene is specifically amplified.
3. The use of claim 2, wherein the qRT-PCR comprises the following reagents added sequentially to a 20 μ L reaction: 10 μ L of 2 XSSYBR I Master, 0.5 μ L of each of the upstream and downstream primers, 50ng of cDNA plus ddH 2 O to 20. mu.L.
4. The use of claim 2, wherein the qRT-PCR reaction procedure is: pre-denaturation at 94 ℃ for 30 s; denaturation at 94 ℃ for 5s, annealing at 60 ℃ for 10s, and extension at 72 ℃ for 10s, for 45 cycles.
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| CN113832158A (en) * | 2019-07-08 | 2021-12-24 | 中国农业科学院植物保护研究所 | Reference gene RPL11 sequence stably expressed under treatment of sitobion avenae E-beta-farnesene solution, cloning method and application |
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