CN112575010A - Reference gene for fluorescence quantification of different tissues of Chinese yam as well as primer and application thereof - Google Patents
Reference gene for fluorescence quantification of different tissues of Chinese yam as well as primer and application thereof Download PDFInfo
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Abstract
The invention relates to a reference gene for fluorescence quantification of different tissues of Chinese yam, and primers and application thereof, and belongs to the technical field of molecular biology of Chinese yam. The nucleotide sequence of the reference gene CKI-2 is shown in SEQ ID NO. 1. The nucleotide sequence of the PCR amplification primer of the reference gene CKI-2 is as follows: the upstream primer is shown as SEQ ID NO. 2; the downstream primer is shown as SEQ ID NO. 3. The invention selects genes such as protein kinase I, myb, F-box, actin gene, EFl, bHLH13, eIF1, gridding heavy chain protein and the like as candidate genes of different tissues. The stability of the candidate gene is evaluated by combining qRT-PCR with RefFind, an internal reference gene with stable expression in different tissues of the Chinese yam is screened out, and a reference basis is provided for the later-stage development of research works such as the gene function verification of the growth and development and metabolic mechanism of the Chinese yam.
Description
Technical Field
The invention belongs to the technical field of molecular biology of yam, and particularly relates to a reference gene for fluorescence quantification of different tissues of yam, and a primer and application thereof.
Background
One of the most conventional and effective means for gene expression research is the Real-time fluorescent quantitative PCR (qRT-PCR) technology, and by adding a specific fluorescent group into a PCR reaction system, the product change in the whole PCR reaction process can be monitored in Real time, so that qualitative and quantitative expression analysis of a target gene is achieved. However, qRT-PCR is affected by factors such as RNA quality, template cDNA quality, primer specificity and PCR amplification rate, and proper internal reference genes need to be selected for correction and standardization in the qRT-PCR reaction process. 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 screening work of reference genes of different species, varieties and tissues has been carried out at home and abroad, and researches show that the expression of the reference genes is specific, and the reference genes suitable for all different test conditions do not exist.
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 Actin2 and Actin1 are used as the expression analysis of the reference genes, but the stability of the reference genes is not analyzed. The screening of the reference genes suitable for different tissues of the Chinese yam has important significance for researching important genes and protein expression of the Chinese yam.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a reference gene for fluorescence quantification of different tissues of Chinese yam, a primer and application thereof. The invention screens out the reference genes which are expressed more stably in different tissues 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:
a fluorescence quantitative internal reference gene for different tissues of Chinese yam is characterized in that the nucleotide sequence of the internal reference gene CKI-2 is shown as SEQ ID NO. 1.
Further, preferably, the nucleotide sequence of the PCR amplification primer of the reference gene CKI-2 is: 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 reference genes for fluorescence quantification of different tissues of Chinese yam, which comprises the following steps:
s1, selecting 12 gene sequences with E value close to zero (E value =0.0) or zero from the candidate reference genes;
s2, designing primers for the 12 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 stem tip, the leaf, the root and the bouquet 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 a Ct value, and analyzing the expression stability of the 12 genes screened in the step S1;
s4, comprehensively determining the most stably expressed reference genes for fluorescence quantification of different tissues of the Chinese yam.
Further, in step S1, it is preferable that the 12 genes are ACT-1, ACT-2, bHLH13, CKI-1, CKI-2, CHC-1, CHC-2, EFI, eIF1, F-box-1, F-box-2, myb.
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, the mixture is denatured at 95 ℃ for 10s, annealed at 60 ℃ for 30s and stretched at 72 ℃ for 10s, and circulated for 30 times.
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, and extension at 72 ℃ for 10s, and the cycle is repeated 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 reference gene for fluorescence quantification of different tissues of the Chinese yam in gene expression analysis of the Chinese yam.
Compared with the prior art, the invention has the beneficial effects that:
the invention selects 12 genes such as protein kinase I (CKI), myb (myb family transcription Factor), F-box (tub-like F-box protein), Actin gene (Actin, ACT), EFl (infection Factor l), bHLH13(Basic helix-loop-helix 13), eIF1 (acute transcription initiation Factor) and latticed heavy chain protein (CHC) as candidate genes of different tissues. The stability of the candidate gene is evaluated on line by combining qRT-PCR with RefFind (http:// www.ciidirsinaloa.com.mx/RefFinder-master /), and the reference gene with more stable expression in different tissues of the Chinese yam is screened out, so that a reference basis is provided for the later-stage development of research works such as the gene function verification of the growth development and metabolic mechanism of the Chinese yam.
Drawings
FIG. 1 is a PCR amplification gel electrophoresis chart of 12 candidate genes;
FIG. 2 isCKI-2Plot peaks of the genes in different tissues of the Chinese yam;
FIG. 3 shows the relative expression levels of Ipt 1 and Ipt 5b genes in different tissues of Dioscorea opposita.
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 4 different tissues of the stem tip, the leaves, the roots and the bouquet of the Chinese yam in the vegetable greenhouse; the plant is applied to the pearl skin after sprouting on the table, the underground tuber in the growth period, 6 tissues are weighed, subpackaged and then quick-frozen by liquid nitrogen, and stored in a refrigerator at the temperature of minus 80 ℃ for later use.
2 reference Gene primer design
Selection of 12 genes including actin gene based on CDS data of yam transcriptomeACT、bHLH13、CKI、EFl、eIF1、F-box、mybAnd clathrin heavy chain proteinCHC1As 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 design principle of qRT-PCR primers, Primer Blast designing primer sequence on line (Table 1), and entrusting Kunming Optimak biotechnology Limited to synthesize 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 different tissues (pearl skin, stem tip, leaf, bouquet, tuber and root) of Chinese yam at different times. 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, and extension at 72 ℃ for 10s are performed for 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, and extension at 72 ℃ for 10s, and the cycle is repeated 40 times.
6 data processing
Sorting and summarizing RT-qPCR data of different organizations of the Chinese yam by 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 by referring to crusted and the like ((crusted, Zhang Ru, Qiu and the like, lily somatic embryo induction, development and different tissues, real-time quantitative PCR internal reference gene screening [ J ] molecular plant breeding, 2018, 16(15): 4982 and 4990.)) data processing and analyzing methods.
7 results
7.1 RNA purity and concentration analysis
After the total RNA of the yam tissue 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
The cDNA of the leaf is taken as a template, and clear and single bands are obtained after PCR of 12 candidate gene primers (as shown in figure 1), which shows that the primer specificity is good and can be used for qRT-PCR implementation. The detection of the internal reference gene primers of different tissues of the Chinese yam 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 tissues of the Chinese yam by analyzing the Ct value of the candidate reference gene. Ct values of the candidate reference genes of different tissues of the Chinese yam are 22.48-39.39, and the difference values of the maximum value and the minimum value of the Ct values are sorted from low to high: CKI-2 < EFI < bHLH13 < eIF1 < MYB < F-box-2 < ACT-2 < F-box-1 < ACT-1 < CKI-1 < CHC1-2 < CHC1-1, which indicates that the expression abundance of CKI-2, EFI, bHLH13, eIF1 and MYB is high, and the expression abundance of CKI-2 is highest.
Because the Ct value is in negative correlation with the expression abundance, namely the expression abundance is larger when the Ct difference is smaller, the Ct minimum value of CKI-2 is relatively stable in different tissues of the Chinese yam, and the expression abundance is higher when the difference is minimum.
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. Internal reference genes in different tissues of Chinese yamCKI-2The evaluation values of Delta CT, geNorm, Normfinder and BestKeeper 4 are ranked first, and the final Comparative Delta Ct is also first, which indicates that the reference genes are in different tissues of Chinese yamCKI-2Is the most stable housekeeping gene.
Table 2 evaluation results of Ct values of reference genes in different tissues by RefFind
7.5 stability verification of reference Gene expression
HandleCKI-2The fluorescence of the gene is quantified independently, and the stability of the reference gene is verified through the peak height.CKI-2The temperature of the gene melting curve is 78-84 ℃, and the expression height difference of the Plot peak height in different tissues is small (as shown in figure 2), which indicates thatCKI-2The stability of the gene is reliable.
7.6 application of reference Gene
Uses yam Ipt 1 gene (SEQ ID NO. 4) and Ipt 5b gene (SEQ ID NO. 5) obtained by homologous cloning of Yunnan agricultural university as target genes,CKI-2the gene is a fluorescence quantitative housekeeping gene, qPCR reaction is carried out, the relative expression of a target gene has a significant change trend (as shown in figure 3), the Ipt 1 gene of the Chinese yam is highest in the bead skin, and the Ipt 5b gene is best in leaf expression. The results of the example are reliable and the results obtained by screeningCKI-2The 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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tggttccaac tccttcattg aagaactcgt cgacggtgaa aacaaggagt ttagagccaa 600
gtacgaatgt tacttccttt gggtcgacgt cgacagtgac ctgctgcacg agttcgtgag 660
cccggaatct gattgcacgc gcgggatacg taggtccatc ggtgtgcccg agatgcaaga 720
ctacttctgt gccgaagcgt ccggcgccga ctcagacacg ctggctttgc ttctcggtaa 780
ggctatcgat gacatgaagg ctcatacgtg caggctaacg tgcggacagc tcttgaaaat 840
caataggttg aggttggagt gtggttggga gcttaacagg gtggatgcaa gcaaggtgtt 900
cgacggaagt tctagctggg aggagatagt cttgaagccg agctttgcgc tcgtgcagcg 960
cttcatggac ggtgaactgc cggaaaaaat ccgtgccggt gctgatgtcg atgtggttgt 1020
ggacttcgcg gcgttggggg cctcagcgta gcgtgtgcgt gtgcgcgtgt gtgaggagtt 1080
agtgcgtgga gccattgccg cacgcacgtg tgtcttagag ctaacccgtg ctgaatgatg 1140
atatatatta tattgttata taaatataaa ttataaatga tatatataat gtgtggtgtt 1200
ttaatgattt gtactaagag agtacatttt gggaggtttc tttaaaaaaa aaaaaaaagc 1260
cctatagtga gtcgaatctc acgctcttc 1289
Claims (8)
1. A fluorescence quantitative internal reference gene for different tissues of Chinese yam is characterized in that the nucleotide sequence of the internal reference gene CKI-2 is shown as SEQ ID NO. 1.
2. The reference gene for fluorescence quantification of different tissues of Chinese yam according to claim 1, wherein the nucleotide sequence of the PCR amplification primer of the reference gene CKI-2 is as follows: 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 reference gene for fluorescence quantification of different tissues of Chinese yam as claimed in claim 1, which is characterized by comprising the following steps:
s1, selecting 12 gene sequences with E values close to zero or zero from the candidate reference genes;
s2, designing primers for the 12 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 stem tip, the leaf, the root and the bouquet 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 a Ct value, and analyzing the expression stability of the 12 genes screened in the step S1;
s4, comprehensively determining the most stably expressed reference genes for fluorescence quantification of different tissues of the Chinese yam.
4. The method for screening reference genes for fluorescence quantification of different tissues of Chinese yam according to claim 3, wherein in step S1, 12 genes are ACT-1, ACT-2, bHLH13, CKI-1, CKI-2, CHC-1, CHC-2, EFI, eIF1, F-box-1, F-box-2 and myb.
5. The method for screening the reference genes for fluorescence quantification of different tissues of Chinese yam according to claim 3, wherein 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, the mixture is denatured at 95 ℃ for 10s, annealed at 60 ℃ for 30s and stretched at 72 ℃ for 10s, and circulated for 30 times.
6. The method for screening reference genes for fluorescence quantification of different tissues of Chinese yam according to claim 3, wherein in 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, and extension at 72 ℃ for 10s, and the cycle is repeated 40 times.
7. The method for screening reference genes for fluorescence quantification of different tissues of Chinese yam according to claim 3, wherein in step S4, the expression stability of each candidate 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 different tissues of Chinese yam in the analysis of the gene expression of Chinese yam according to claim 1.
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