CN112501183A - Fluorescence quantitative reference gene for different growth time periods of Chinese yam as well as primer and application thereof - Google Patents
Fluorescence quantitative reference gene for different growth time periods of Chinese yam as well as primer and application thereof Download PDFInfo
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Abstract
The invention relates to a fluorescent quantitative reference gene of yam in different growth time periods, a primer and application thereof, belonging to the technical field of yam molecular biology. The reference geneF‑boxThe nucleotide sequence of (A) is shown in SEQ ID NO. 1. Internal reference geneF‑boxThe nucleotide sequence of the PCR amplification primer of (1): the upstream primer is shown as SEQ ID NO. 2; the downstream primer is shown as SEQ ID NO. 3. The invention screens out the reference genes which are relatively stably expressed in different growth periods of the Chinese yam, and provides reference basis for carrying out the research works such as the gene function verification of the growth and development and metabolic mechanism of the Chinese yam in the later period.
Description
Technical Field
The invention belongs to the technical field of molecular biology of yam, and particularly relates to a fluorescent quantitative reference gene for different growth periods of yam, and a primer and application thereof.
Background
The Real-time fluorescent quantitative PCR (quantitative Real-time PCR, qRT-PCR) technology is one of the most common and effective means for researching gene expression in molecular biology, and the product change in the whole PCR reaction process can be monitored in Real time by adding a specific fluorescent group into a PCR reaction system, so that qualitative and quantitative expression analysis of a target gene is achieved. The qRT-PCR has the advantages of strong specificity, high sensitivity, good repeatability, simplicity, high efficiency and the like, but can be influenced by factors such as the quality of RNA, the quality of template cDNA, the specificity of primers, the PCR amplification rate and the like, and proper internal reference genes need to be selected for correction and standardization in the qRT-PCR process. Genes that constitute the cytoskeleton or are involved in basic metabolic activities of cells are often used as reference genes, and these genes are relatively stable in expression. At present, there are many housekeeping genes commonly used, including coding genes for actin (actin), ribosomal RNA (rRNA), transcription elongation factor (EF 1), Tubulin (TUB) and the like.
The reports on the reference genes of yams are few, and only the cloning and expression of an anthocyanin synthase (ANS) gene, an anthocyanin-related gene DaF3H and a flavonol synthase DaFLS1 gene are shown, wherein Actin 2 and Actin 1 are used as the expression analysis of the reference genes, but the stability of the reference genes is not analyzed. The screening of reference genes suitable for different growth periods of the Chinese yam has important significance for researching important genes and protein expression of the Chinese yam. Therefore, how to overcome the defects of the prior art is a problem which needs to be solved in the technical field of the molecular biology of the yam at present.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a fluorescence quantitative reference gene for different growth time periods of Chinese yam, a primer and application thereof. The invention screens out the reference genes which are relatively stably expressed in different growth periods of the Chinese yam, and provides reference basis for carrying out the research works such as the gene function verification of the growth and development and metabolic mechanism of the Chinese yam in the later period.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
reference gene for fluorescence quantification of Chinese yam in different growth time periods, and reference geneF-boxThe nucleotide sequence of (A) is shown in SEQ ID NO. 1.
Further, it is preferable that the reference gene is a gene encoding a polypeptideF-boxThe nucleotide sequence of the PCR amplification primer of (1): the upstream primer is shown as SEQ ID NO. 2; the downstream primer is shown as SEQ ID NO. 3.
The invention also provides a screening method of the fluorescence quantitative reference genes of the Chinese yam in different growth time periods, which comprises the following steps:
s1, selecting 18 gene sequences with E value close to zero (E value =0.0) or zero from the candidate reference genes;
s2, designing primers for the 18 genes screened in the step S2, carrying out PCR amplification by using the primers, and verifying the specificity of the primers;
s3, extracting total RNA of the leaves of the Chinese yam, performing reverse transcription to synthesize first-strand cDNA, performing real-time fluorescent quantitative PCR analysis by adopting a primer designed in S2, calculating Ct value, and analyzing the expression stability of the 18 genes screened in the step S1;
s4, comprehensively determining the most stably expressed reference genes of the Chinese yam with fluorescence quantification in different growth time periods.
Further, it is preferable that, in step S1, 18 genes are F-box, TRX-2, YLS8, HIS, eIF1-1, 40S rRNA-1, eIF1-2, 26S rRNA, TRX-1, ELF, bHLH, 60S rRNA, ACT, 40S rRNA-2, α -TUB, CHC, CKI, eIF 1-3.
Further, it is preferable that, in step S2, the PCR reaction system is 20 μ L: comprises 17.0 mu L Green Mix, 1.0 mu L cDNA, 1.0 mu L10 mu mol/L upstream primer and 1.0 mu L10 mu mol/L downstream primer;
reaction conditions are as follows: pre-denaturation at 95 ℃ for 1 min; then denaturation at 95 ℃ for 10s, annealing at 60 ℃ for 30s, pre-extension at 72 ℃ for 10s, and 30 cycles.
Further, it is preferable that, in step S3, the real-time fluorescence quantitative PCR reaction system is 20 μ L: comprises 10.0 μ L qPCR Master Mix (SYBR Green I), 1.0 μ L cDNA, 0.8 μ L10 μmol/L forward primer, 0.8 μ L10 μmol/L reverse primer, ROX 0.4 μ L, ddH2O 7.0μL；
Reaction conditions are as follows: pre-denaturation at 95 ℃ for 1 min; then denaturation at 95 ℃ for 10s, annealing at 60 ℃ for 30s, pre-extension at 72 ℃ for 10s, and circulation for 40 times.
Further, in step S4, the expression stability of each candidate reference gene is preferably analyzed by the Delta CT, geonorm, Normfinder, bestkeper, Comparative Δ CT method.
The invention also provides application of the fluorescence quantitative reference gene of the Chinese yam in different growth time periods in Chinese yam gene expression analysis.
Compared with the prior art, the invention has the beneficial effects that:
the invention selects Actin gene (Actin, ACT), bHLH13(Basic helix-loop-helix 13), protein kinase I (casein kinase I, CKI), myb (myb family transcription factor), F-box (tube-like F-box protein), eIF1 (acute translation initiation factor), latticed heavy chain protein (CHC), ELF (active eukaryotic initiation factor), histidine family protein (HIS), YLS 8(mitosis protein YLS8), Thioredoxin (TRX), alpha-tubulin gene (alpha-tubulin beta, alpha-TUB), 26S ribosomal RNA (26S ribosomal RNA, 26S rRNA), 40S ribosomal RNA (40S ribosomal RNA, 40S rRNA) and 60S ribosomal RNA (60S ribosomal RNA, 60S rRNA) genes are candidates for different tissues. The stability of candidate genes is evaluated on line by combining qRT-PCR with RefFind (http:// www.ciidirsinaloa.com.mx/RefFinder-master /), the stability of reference genes in different growth time periods of the Chinese yam is analyzed by 18 genes, the reference genes with stable expression in different growth time periods of the Chinese yam are screened out, and reference basis is provided for later-stage research work such as gene function verification of the growth and development and metabolic mechanism of the Chinese yam.
Drawings
FIG. 1 is a PCR amplification gel electrophoresis chart of 18 candidate genes;
FIG. 2 isF-boxThe gene has Plot peaks in different growth time periods of the yam;
FIG. 3 shows the relative expression levels of Ipt 1 and Ipt 5a genes in different growing time of yam.
Detailed Description
The present invention will be described in further detail with reference to examples.
It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The materials or equipment used are not indicated by manufacturers, and all are conventional products available by purchase.
1 materials of embodiment
The method comprises the steps of taking oxtail yam as an implementation material, selecting 1.5-3 g/per complete bulbil every other year, planting the bulbil on a vegetable greenhouse or a plant implementation platform of Yunnan agricultural university, accelerating germination and then planting and managing. Collecting leaves every 5 days after germination, weighing, subpackaging, quick-freezing with liquid nitrogen, and storing in a refrigerator at-80 deg.C for use.
2 reference Gene primer design
Selection of 18 genes including the actin gene based on yam transcriptome CDS dataACT、bHLH13、CKI、eIF1、ELF、F-box、HIS、mybLatticed heavy chain proteinsCHC1、YLS 8、TRX、α-TUB26S ribosomal RNA: (26S rRNA) 40S ribosomal RNA: (40S rRNA) And 60S ribosomal RNA: (60S rRNA) As candidate reference genes, the obtained transcriptome sequences were checked by Blast-N in NCBI, and sequences with E values close to zero or zero were selected for primer design. According to the qRT-PCR Primer design principle, Primer Blast designs a Primer sequence (table 1) on line, and entrusts Kunming Biotechnology Limited company to synthesize a corresponding Primer.
TABLE 1 Yam candidate reference gene qRT-PCR primer sequences
3 extraction of Total RNA and Synthesis of 1 st Strand of cDNA
Reference EastepTMThe Super total RNA extraction kit (Shanghai products) uses the instruction to extract the total RNA of leaves and leaves of Chinese yam at different times (5 d, 10d, 15d, 20d and 25 d). RNA integrity and purity were checked by electrophoresis on a 1.2% agarose gel and RNA concentration was checked using a Denovix DS-11 instrument. cDNA 1 st chain synthesis reference TSINGKE reverse transcription kitGoldenstar TMRT6 cDNA Synthesis Kit instruction, the cDNA obtained was stored in a freezer at-20 ℃.
4 primer specificity PCR verification
Taking the yam leaf cDNA as a template, wherein the PCR reaction system is 20 mu L: includes 17.0. mu.L Green Mix, 1.0. mu.L cDNA, 1.0. mu.L forward primer (10. mu. mol/L), 1.0. mu.L reverse primer (10. mu. mol/L). Reaction conditions are as follows: pre-denaturation at 95 ℃ for 1 min; then denaturation at 95 ℃ for 10s, annealing at 60 ℃ for 30s, pre-extension at 72 ℃ for 10s, and 30 cycles.
5 real-time fluorescent quantitative PCR amplification
The test is carried out by using an ABI Quantstudio real-time fluorescent quantitative PCR system. The qRT-PCR reaction system is 20 μ L: includes 10.0. mu.L qPCR Master Mix (SYBR Green I), 1.0. mu.L cDNA, 0.8. mu.L forward primer (10. mu. mol/L), 0.8. mu.L reverse primer (10. mu. mol/L), ROX 0.4. mu.L, ddH2O7.0. mu.L. Reaction conditions are as follows: pre-denaturation at 95 ℃ for 1 min; then denaturation at 95 ℃ for 10s, annealing at 60 ℃ for 30s, pre-extension at 72 ℃ for 10s, and circulation for 40 times.
6 data processing
Sorting and summarizing RT-qPCR data of the leaves of the Chinese yam at different times through Excel 2010 software; the method comprises the steps of evaluating the expression stability of different internal reference genes by using internal reference gene stability analysis websites (http:// www.ciidirsinaloa.com.mx/RefFinder-master /), including 5 evaluation methods of Delta CT, geonorm, Normfinger, BestKeeper and the compatible Delta Ct method, and specifically analyzing steps and parameter setting data processing and analyzing methods by referring to aging sensitivity and the like (aging sensitivity, Zhang Ru, Qin, etc., lily somatic embryo induction, development, different tissues, real-time quantitative PCR internal reference gene screening [ J ] molecular plant breeding, 2018, 16(15): 4982 and 4990).
7 results
7.1 RNA purity and concentration analysis
After the total RNA of the leaf is extracted, the total RNA of each sample has no degradation and good integrity, the concentration of the total RNA is 476.759 +/-22.937 ng/uL, and A is measured260/A230=1.830～2.327，A260/A280And the samples are satisfactory in terms of 1.908-2.086, so that the RNA integrity of the samples is good and the concentration is high, and the samples can be used for subsequent implementation.
7.2 PCR specificity analysis of reference Gene
Clear and single bands (as shown in figure 1) are obtained after PCR by taking cDNA of leaves as a template and 18 candidate gene primers, which indicates that the primers have good specificity and can be used for qRT-PCR implementation. The detection of the internal reference gene primers of the Chinese yam at different times on a computer only has a single signal peak, and has no phenomena of impurity peaks and nonspecific amplification, which shows that the primers have high accuracy when used for qRT-PCR.
7.3 analysis of expression abundance of reference Gene
And performing RT-qPCR amplification based on the verified primers, and evaluating the expression abundance of the genes in different growth times of the Chinese yam by analyzing the Ct value of the candidate reference gene. Ct values of candidate reference genes of the Chinese yam in different growth times are 22.39-37.52, and differences between the maximum value and the minimum value of Ct are sorted from low to high: 26S < ELF < TRX-1 < 60S < F-box < YLS8 < HIS < TRX-2 < 40S-2 < ACT < eIF1-1 < bHLH < 40S-1 < CHC < eIF1-2 < alpha-TUB < eIF1-3 < CKI, which indicates that the expression abundance of 26S, ELF, TRX-1, 60S and F-box is high. Since the Ct value is inversely related to the expression abundance, the expression abundance is larger as the difference value of the Ct value is smaller.
7.4 stability analysis of reference Gene expression
The stability of the genes was assessed on-line based on RefFind, and the stability values and ranking results for each software are shown in table 2. Reference gene for different growth periods of Chinese yamF-boxThe former six are arranged in 4 evaluation values of Delta CT, geNorm, Normfinder and BestKeeper, and the final Comparative Delta Ct is the first, which indicates that the reference genes are arranged in different growth time periods of the Chinese yamF-boxIs the most stable housekeeping gene.
Table 2 evaluation results of Ct values of reference genes in different growth periods by RefFind
7.5 stability verification of reference Gene expression
HandleF-boxThe fluorescence of the gene is quantified independently, and the stability of the reference gene is verified through the peak height.F-boxThe temperature of the gene melting curve is 79-83 ℃, and the expression height difference of the Plot peak height in different growth periods is small (as shown in figure 2, which illustrates thatF-boxThe stability of the gene is reliable.
7.6 application of reference Gene
Uses yam Ipt 1 gene (SEQ ID NO. 5) and Ipt 5a gene (SEQ ID NO. 4) obtained by homologous cloning of Yunnan agricultural university as target genes,F-boxthe gene is a fluorescence quantitative housekeeping gene, qPCR reaction is carried out, the relative expression quantity of a target gene has a remarkable change trend (as shown in figure 3), and the expression of Ipt 5a gene is higher than that of Ipt 1 gene in the growth time period of yam Ipt 1 and Ipt 5a genes. The results of the example are reliable and the results obtained by screeningF-boxThe reference gene is suitable for real-time fluorescent quantitative analysis of the functional genes of the Chinese yam under different tissue conditions.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Sequence listing
<110> Yunnan university of agriculture
Institute of biotechnology and germplasm resources, Yunnan Academy of Agricultural Sciences
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tcccggtctc cgccggctgc gaagtacgcc tccagctcgt ccaccatacc gtccccaatc 540
atctcatcca cccgccgatc aaggtactcg ccaagcaccg ccgcatcgac atcaacccat 600
aaaaagcagc accgatacct gaaccgctct cgccggacgg acccaaacgg gtcggaccgt 660
ggatcatagt gctccgcaag caacgcgtga atgtacgagt tcgacccgcc ggccagcact 720
gctcgatgac cacgggcagc aatccccgcg atgcacgaac tcgccaggga tcggaaccca 780
cccggcggga gttccccgcc gcaagggtca agctcaccga ggagatgatg cggcacgcca 840
catcgatcag caataggcat cttattcgtg gtgatgtcaa gccctctata aacctggatc 900
ttatcactgt taacaacctc accggaaaac ctagaagcga tgtcgatgga aagcttagac 960
ttgccggtac cggtagcgcc catgatgacg agtacatctt cattccttct cggatgatga 1020
tgatgttgat gttgatgatg atgatgatga aggtagtacc tccgactctg agcacaacag 1080
cgatggtgat ggttatccac agagtcggcg gcgatggtgg atacggaggt ggcggcagcc 1140
tccgcttgga gcgatggacg gaggagtctt gtcgcgttgc agttgatgat acttctagaa 1200
aataaatttt tcatatagtc caatgaatca ccacgactcg ctaaagaaga agatgatgat 1260
gaatggaatg gcaaagagaa gtaatgattg aatggaatgg gttgggtttc gcatgccaag 1320
cgtttgtcag tattcacaga agggaaccaa cagtgaatgc cgttgcgctc gagaccgaga 1380
ccgagaccga gaccgagagc atgagcaggg ctatc 1415
Claims (8)
1. A fluorescence quantitative reference gene for Chinese yam in different growth time periods is characterized in that the reference geneF-boxThe nucleotide sequence of (A) is shown in SEQ ID NO. 1.
2. The reference gene for fluorescence quantification of Chinese yam according to different growth periods of claim 1, wherein the reference gene is characterized in thatF-boxThe nucleotide sequence of the PCR amplification primer of (1): the upstream primer is shown as SEQ ID NO. 2; the downstream primer is shown as SEQ ID NO. 3.
3. The screening method of the fluorescence quantitative reference gene of the Chinese yam in different growth time periods as claimed in claim 1, characterized by comprising the following steps:
s1, selecting 18 gene sequences with E values close to zero or zero from the candidate reference genes;
s2, designing primers for the 18 genes screened in the step S2, carrying out PCR amplification by using the primers, and verifying the specificity of the primers;
s3, extracting total RNA of the leaves of the Chinese yam, performing reverse transcription to synthesize first-strand cDNA, performing real-time fluorescent quantitative PCR analysis by adopting a primer designed in S2, calculating Ct value, and analyzing the expression stability of the 18 genes screened in the step S1;
s4, comprehensively determining the most stably expressed reference genes of the Chinese yam with fluorescence quantification in different growth time periods.
4. The method for screening reference genes for fluorescence quantification of Chinese yam according to claim 3, wherein in step S1, 18 genes are F-box, TRX-2, YLS8, HIS, eIF1-1, 40S rRNA-1, eIF1-2, 26S rRNA, TRX-1, ELF, bHLH, 60S rRNA, ACT, 40S rRNA-2, alpha-TUB, CHC, CKI, eIF 1-3.
5. The method for screening the reference genes for the fluorescence quantification of the Chinese yam in different growth time periods according to claim 3, wherein in the step S2, the PCR reaction system is 20 μ L: comprises 17.0 mu L Green Mix, 1.0 mu L cDNA, 1.0 mu L10 mu mol/L upstream primer and 1.0 mu L10 mu mol/L downstream primer;
reaction conditions are as follows: pre-denaturation at 95 ℃ for 1 min; then denaturation at 95 ℃ for 10s, annealing at 60 ℃ for 30s, pre-extension at 72 ℃ for 10s, and 30 cycles.
6. The method for screening the reference genes for the fluorescence quantification of the Chinese yam in different growth time periods according to claim 3, wherein in the step S3, the real-time fluorescence quantification PCR reaction system is 20 μ L: comprises 10.0 μ L qPCR Master Mix (SYBR Green I), 1.0 μ L cDNA, 0.8 μ L10 μmol/L forward primer, 0.8 μ L10 μmol/L reverse primer, ROX 0.4 μ L, ddH2O 7.0μL；
Reaction conditions are as follows: pre-denaturation at 95 ℃ for 1 min; then denaturation at 95 ℃ for 10s, annealing at 60 ℃ for 30s, pre-extension at 72 ℃ for 10s, and circulation for 40 times.
7. The method for screening the fluorescence quantitative internal reference genes of the Chinese yam according to the different growth time periods of the Chinese yam as claimed in claim 3, wherein in step S4, the expression stability of each candidate internal reference gene is analyzed by using the methods of Delta CT, geNorm, Normfinder, BestKeeper and Comparative Delta Ct.
8. The application of the reference gene for fluorescence quantification of the Chinese yam in different growth periods of the Chinese yam in the Chinese yam gene expression analysis.
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