CN109136403B - Reference gene and construction method for Caucasian clover PCR expression analysis - Google Patents

Reference gene and construction method for Caucasian clover PCR expression analysis Download PDF

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CN109136403B
CN109136403B CN201811167483.9A CN201811167483A CN109136403B CN 109136403 B CN109136403 B CN 109136403B CN 201811167483 A CN201811167483 A CN 201811167483A CN 109136403 B CN109136403 B CN 109136403B
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殷秀杰
燕昌江
张鸣宇
贺涛涛
衣琨
李旭
任毅晓
孙博扬
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Northeast Agricultural University
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Abstract

The present invention discloses a kind of reference gene and construction method for Caucasian clover PCR expression analysis, its nucleotide sequence of reference gene is as shown in sequence table SEQ ID No.2；Construction method, as follows: 7 tissue transcript profile data of Caucasian clover are utilized, the reference gene for selecting 7 expression stable, while common 1 traditional reference gene in Trifolium being selected to be used as control；Above-mentioned candidate reference gene is subjected to design of primers, standard PCR amplification is carried out to each cDNA, the amplification efficiency and amplification that obtain primer reach the recurring number of threshold value；The analysis of reference gene stability assessment is carried out to the data of acquisition, finally screens most suitable reference gene and reference gene number under special experiment condition.The reference gene makees object of reference for detecting the variation of Caucasian clover gene expression dose, for correcting applied sample amount and loading existing test error in the process, guarantees the accuracy of experimental result.

Description

Reference gene and construction method for Caucasian clover PCR expression analysis
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of internal reference for Caucasian clover PCR expression analysis Gene and construction method.
Background technique
Gene expression analysis is widely used in life science, research deeply to seeking disease related gene and tune Control mechanism, disclose Secrets Of Life etc. is most important.It is carried out in quantitative gene expression in transcriptional level, such as real time fluorescent quantitative PCR (Quantitative real-time RT-PCR, qRT-PCR), RNA trace (Northern blotting), ribose core Sour enzyme protection analysis (ribonuclease protection assay, RPA), genetic chip (gene chip) etc., to obtain More accurate believable result, requires reference gene and is standardized measurement to the expression of target gene.
Polymerase chain reaction technology (polymerase chain reaction, abbreviation round pcr) is due to having time-consuming The features such as short, special strong, sensitive high, it is widely used in the quantitative analysis of gene expression.Due to round pcr high sensitivity, hold Easily there is false positive, the testing result of false negative, the inside reference that reference gene react as qPCR, with calibration sample amount with The effect of reverse transcription efficiency improves the accuracy of test data.Ideal reference gene should be expressed in all samples Stablize, therefore, using qRT-PCR to target gene carry out relative quantification accuracy depend on using by system verifying, Stable reference gene is expressed, to exclude the false positive being likely to occur in PCR detection process, false negative result.
Caucasian clover (Trifolium ambiguum Bieb.) is that uniquely have subterraneous root in pulse family Trifolium The perennation service life herbage of tiller has flourishing underground root turion, compared with the other plants belonged to, have stronger cold-resistant, Drought resisting and resistance to herding property.Caucasian clover favorable genes and regulated and control network are excavated using the method for gene expression analysis, is to carry out The premise of Trifolium genetic modification of plants.Mostly used in Trifolium gene expression in plants analysis at present is traditional internal reference Gene, these traditional reference genes are simultaneously verified without system, and stability is poor, lead to Trifolium gene expression in plants point The accuracy of analysis is poor.In reality and there is no the ideal reference genes of all constant expression under the conditions of different tests.Especially exist Under Abiotic stress conditions, as the stability of the variation reference gene of environment also will receive different degrees of influence.It makes internal disorder or usurp to grind Functional gene expression under the different tissues of Caucasian clover, different growth and development stages and Abiotic stress conditions, screening Best reference gene or the assortment of genes under specified conditions are very necessary.
Summary of the invention
The purpose of the present invention is to provide a kind of reference gene for Caucasian clover PCR expression analysis and building sides Method, the reference gene do object of reference for detect Caucasian clover gene expression dose variation, for correct applied sample amount and Existing test error, guarantees the accuracy of experimental result during sample.
The purpose of the present invention is realized by following technology: a kind of internal reference base for Caucasian clover PCR expression analysis Cause, nucleotide sequence is as shown in sequence table SEQ ID No.2.
The present invention also has following technical characteristic:
1, a kind of construction method of the reference gene for Caucasian clover PCR expression analysis, as follows:
(1) 7 tissue transcript profile data of Caucasian clover are utilized, the reference gene for therefrom selecting 7 expression stable, Nucleotide sequence such as sequence table SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID Shown in No.6, SEQ ID No.7, SEQ ID No.8, while common 1 traditional reference gene in Trifolium being selected to make For control, nucleotide sequence is as shown in sequence table SEQ ID No.5, totally 8 kinds of candidate reference genes；
(2) above-mentioned 8 kinds candidate reference genes are subjected to design of primers, standard PCR amplification is carried out to each cDNA, is drawn The amplification efficiency of object and amplification reach the recurring number of threshold value；
(3) to the data of acquisition using three reference gene stability assessment software geNorm, NormFinder and BestKeeper is analyzed, and most suitable reference gene and reference gene number under special experiment condition are finally screened；Pass through It is verified using the gene in Caucasian clover root, stem and leaf, the nucleotide sequence as shown in sequence table SEQ ID No.2 Expression stability highest.
2, a kind of application of the reference gene for Caucasian clover PCR expression analysis as described above, qRT-PCR The forward primer sequence of amplimer is as shown in sequence table SEQ ID No.9, reverse primer sequences such as sequence table SEQ ID Shown in No.10.
The present invention has the advantages that and advantage: the C257504 reference gene experiment skill screened through the invention Art is simple, stablizes at Caucasian clover different tissues position and expresses, is the best internal reference of Caucasian clover PCR expression analysis Gene.
1. expression stability is high: the present invention utilizes Caucasian clover transcript profile data, compares base in transcript profile level The expression and stability of cause, have therefrom screened suitable reference gene, and traditional reference gene is in transcriptome analysis Expression and stability is not high.Therefore, the stability for the reference gene that the present invention obtains is better than used at present pass System reference gene.
2. repeated strong: the present invention analyzes expression of the new reference gene at Caucasian clover different tissues position and stablizes Property, therefore, the reference gene that the present invention obtains has wide applicability and repeatability.
3. improving reliability: designed real-time fluorescence quantitative PCR primer specificity is strong, so as to greatly improve Detection efficiency when being detected using real time fluorescent quantitative, and improve the confidence level of testing result.
Detailed description of the invention:
Fig. 1 is the PCR amplification and agarose gel electrophoresis testing result figure of candidate reference gene；
100bp, 25bp, 500bp, 75bp, 1000p, 200bp indicate size 1-c236159,2- of corresponding band in figure c257504,3-c258625,4-c262187,5-JF968419.1,6-c267370,7-c268173,8-c271437；
Fig. 2 is c257504 reference gene PCR amplification and agarose gel electrophoresis inspection in Caucasian clover different tissues Survey result figure；In figure: blade, 2- stem, 3- rootstock, the 4- of 1- root turion plant expand the root turion for having bud root turion section, 5- to expand no bud Section, 6- horizontal branch, 7- are without root turion main root；
Fig. 3 is the expression spirogram in the sequencing of three leaf C261263 gene grass transcript profile of Caucasia；
Fig. 4 is the expression spirogram in the sequencing of three leaf C261427 gene grass transcript profile of Caucasia；
Fig. 5 is relative expression spirogram of the Caucasian clover C261263 gene in candidate reference gene；
Fig. 6 is relative expression spirogram of the Caucasian clover C261427 gene in candidate reference gene.
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited In this.
Embodiment 1
1. sample
Test material is the Caucasian clover for growing third year, is experimental field located at Northeast Agricultural University's experimental field in the school. It is sampled in the Caucasian clover root turion trait expression most apparent root turion strain clump phase, takes show the plant of root turion character respectively The blade of strain, rootstock, expands and has bud root turion section, expands no bud root turion section, horizontal branch and without root turion main root, amount to 7 tissues stem Sample, each sample take 10 plants of mixing, are kept separately 3 secondary pollutants of each tissue by tissue site and repeat to be sequenced, sampling - 80 DEG C of cryogenic freezings save afterwards, spare.
2.RNA is extracted, library construction and transcript profile are sequenced
Extract the RNA of 7 tissue sites respectively with Trizol method, each RNA is both needed to meet A260/A280 1.8~2.0 Between and A260/A230 between 1.8~2.0；And each above-mentioned total serum IgE is required to through integrity detection, in Hiseq 2500 microarray datasets carry out bis- generation of RNA-seq transcript profile sequencing analysis, carry out reverse transcription reaction to each above-mentioned total serum IgE and obtain pair The cDNA answered.
3. transcript profile data screening reference gene
Each tissue site transcript profile data of Caucasian clover are analyzed, in 7 tissue transcript profile data of Caucasian clover It is middle to choose shared nucleotide sequence as follows respectively:
C236159: nucleotide sequence is shown in sequence table SEQ ID No.1；
C257504: nucleotide sequence is shown in sequence table SEQ ID No.2；
C258625: nucleotide sequence is shown in sequence table SEQ ID No.3；
C262187: nucleotide sequence is shown in sequence table SEQ ID No.4；
C267370: nucleotide sequence is shown in sequence table SEQ ID No.6；
C268173: nucleotide sequence is shown in sequence table SEQ ID No.7；
C271437: nucleotide sequence is shown in sequence table SEQ ID No.8；
Using reference gene JF968419.1 common in butch clover as control, nucleotide sequence is shown in sequence table SEQ ID No.5。
1 pcr amplification reaction system table of table
Reagent Dosage (μ L)
ddH2O 6
2×Taq Master Mix 10
Primer1(10μM) 1
Primer2(10μM) 1
cDNA 2
Total volume 20
4. reference gene design of primers
Above-mentioned 8 kinds candidate reference genes are subjected to design of primers and obtain 8 kinds of corresponding primers, and guarantee the expansion of each primer Increase product length control between 80~150bp, standard PCR amplification is carried out to each cDNA with above-mentioned 8 kinds of primers and is obtained often The corresponding pcr amplification product of a cDNA, reaction system are shown in Table 1.Reaction condition setting are as follows: 95 DEG C of initial denaturation 5min then carry out 30 A circulation (95 DEG C of denaturation 15s, the optimum annealing temperature 15s, 72 DEG C of extension 60s of each pair of primer), last 72 DEG C of extensions 5min, instead Amplified production is placed in 4 DEG C of preservations after the completion of answering.It is above-mentioned to the progress detected through gel electrophoresis inspection of each pcr amplification product each to draw Whether object amplifies effective unique band (Fig. 1).
5.qRT-PCR experiment
Primer is diluted to after different multiples, qRT-PCR experiment is carried out to the cDNA of all above-mentioned acquisitions, with copying for primer The logarithm of shellfish number is abscissa, and recurring number Ct value when amplification reaches threshold value is that ordinate draws primer standard curve, qRT- PCR reaction system is shown in Table 2.Reaction condition set 95 DEG C of initial denaturation 5min then carry out 30 circulations (95 DEG C of denaturation 15s, it is each pair of The optimum annealing temperature 15s, 72 DEG C of extension 60s of primer), last 72 DEG C of extensions 5min obtains amplification efficiency and the amplification of primer Reach the recurring number (table 3) of threshold value.
2 qRT-PCR amplification reaction system table of table
Reagent Dosage (μ L)
2×ChamQ Universal SYBR qPCR Master Mix 10
Primer1(10μM) 0.4
Primer2(10μM) 0.4
cDNA 2
ddH2O 7.2
Total volume 20
The primer sequence and its PCR amplification mark sheet of the candidate reference gene of table 3
6. candidate reference gene assessment
To the data of acquisition using three reference gene stability assessment software geNorm, NormFinder and BestKeeper is analyzed, and most suitable reference gene and reference gene number under special experiment condition are finally screened.
(1) stability of above-mentioned candidate reference gene is analyzed using geNorm software, (geNorm software is a certain by calculating Evaluation of the average variation degree M of the Logarithm conversion value of ratio as gene stability, M value are got over two-by-two between gene and other genes Greatly, stability is poorer, conversely, more stable), expressing most stable of candidate reference gene is c271437, followed by c268173 and C258625, most unstable is JF968419.1.
Meanwhile geNorm software can generate the pairing difference value Vn/n+1 of reference gene, and determine that test needs to answer with this Best reference gene number, 0.15 is choice value, such as less than 0.15, then explanation does not need n+1 reference gene and carries out school Positive destination gene expression.This experiment V2/3Less than 0.15, illustrate not needing to introduce the 3rd gene, 2 genes are sufficient, and are considered 2 assortments of genes are unfavorable for simplifying experimental implementation, thus determine individual gene as internal reference because.
(2) circulation when reaching threshold value to amplification efficiency and amplification of the above-mentioned all candidate reference genes in each sample Number calculates gene expression stationary value M with NormFinder software, arrives above-mentioned institute according to obtained all gene expression stationary value M Putting in order for the stability of candidate reference gene is successively: c257504, c258625, c271437, c268173, C262187, c267370, JF968419.1 (control), c236159.Similar to geNorm, the gene for coming front three is it Secondary is c257504, c271437.graph_c3, c258625, and most unstable gene is c236159.
(3) mark of recurring number when reaching threshold value using the amplification that BestKeeper software calculates all candidate reference genes Poisson coefficient R between quasi- difference SD coefficient of variation CV and each candidate reference gene determines candidate reference gene with this Expression stability, candidate reference gene of the standard deviation SD less than 1 are considered as the gene for stablizing expression, and standard deviation SD is smaller, are become Different coefficient CV is smaller, and candidate's reference gene is more stable, and the bigger candidate reference gene of Poisson coefficient R value is more stable；Instead It, more unstable, as shown in Table 4, in above-mentioned candidate's reference gene, c257504 is most stable of candidate internal reference, followed by C268173 and c271437.
The common stability obtained using tri- internal reference screening softwares of geNorm, NormFinder and Best Keeper Best first four candidate gene is successively 1-c257504,2-c271437,3-c268173,4-c258625, common stabilization Property worst two candidate reference genes be JF968419.1 and c236159.
4 geNorm, NormFinder and BestKeeper software integrated-analysis result table of table
7. reference gene stability is verified
Expression stability of the above-mentioned c257504 screened in all samples is analyzed, qRT- is carried out to the cDNA of sample PCR experiment, agarose gel electrophoresis testing result such as Fig. 2.It is relevant by Sucrose Metabolism in root, stem and leaf texture's sample C261263 and hormone signal conduct the expression analysis of relevant C261427 to verify the stability of the reference gene filtered out.? In the expression of C261263 and C261427 gene, when using c257504 as internal reference, expression quantity is normal, expression quantity and transcript profile Expression quantity in sequencing result is more consistent (Fig. 4-5, Fig. 5-6), illustrates that its expression quantity is stablized, so determining gene c257504 The final reference gene obtained as screening.
Sequence table
<110>Northeast Agricultural University
<120>reference gene and construction method of Caucasian clover PCR expression analysis are used for
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 223
<212> DNA
<213>Caucasian clover (Trifolium ambiguum Bieb.)
<400> 1
tgggaatgat gttgaaggaa gcggctcttc cacctctcca gtccttcatc gatggaccat 60
caactgtctt ctgagttgca gtgatagagt ggacggtggt catgagtccc tcaacaattc 120
caaacctgtc gttgataacc ttggcaagtg gagcaaggca gttagtggtg caactagcgt 180
tggaaacaat gttgagatca gacttgtact catgctcatt gac 223
<210> 2
<211> 2366
<212> DNA
<213>Caucasian clover (Trifolium ambiguum Bieb.)
<400> 2
ctaaagcaaa cccattccta ctctagtcta atagcttcta tggaggtacc cagccttaaa 60
ataactagct aggggggaat ttcaaagaat gaaaagtcag attagtagga ttgggaaaag 120
tcaaattaat ccaacatcat tctcagatcg tgtaatttca tcaacctttc tacttagcaa 180
catgcacaga acattgtaaa tatttaaaaa cattcattcc aaacatatta tttccactaa 240
aaaaatgctt tcatcagaaa gctaggcaga tacgtctctg cattgttttt ccacaacact 300
tattgacatg gcagatgaat aagaagctga aaactacagc atgtttgcaa agaaaacaaa 360
taggaaggaa caagggaata aatttatgga cactaactct agagttctaa acttatcctc 420
agtgagatga aaaacaagaa aataatttta tgttgccatc tctacattcc cttgcctatc 480
aaaaagaagg acaaaaccac atgtaaatag ataataagtg gttatcgaac tttagtctga 540
tattctgatc tccactccta aaatgtcctt caccatctca tatgatacaa acgcgagtgc 600
tatggatggg accaccttta cagaattggg aaccaggccc ttgtataatg caccgaatcc 660
ctcgtgtcga acagttttcc taaatgcatc aatcatccca gtatattcaa gtgggacttt 720
gcctcttcca tctccagtta taacagaagc agcatggttc caaccggtca tctgcattct 780
tcgacgaatg acatcaagag ggtaagcgac agtttgtcca atggttccag cagcagcacc 840
acaagccaga cgtgttgtca cactcaactc agaatcctgg actagaccaa atggtctgga 900
tttaatcaac caatctttta gagactcata gacagcaaag ttgagaccca cataaggaac 960
ctttcatgtg atcatatctc tcataactta actgcaccga tttaggttaa aattgactga 1020
taaaattaac agctaaaggg ttggaaaaaa tagatttgtc tcaatagatc agagtgtgtt 1080
tgagggtgta tccttagcat tcctctccaa accatatgct acataaaact aaaagcaatg 1140
aagcaataga tagatgatag acatctacca agatccaata gcaagtggat aagttttatc 1200
acaagactaa tattagtggc aacaataatc agaatttatt cttgggatca agatccctcc 1260
ctcaactgca acttctatgt tgcttttaaa tgcagtaaca acttctcatg aagattgtaa 1320
ataaagcatg ttcatgtttg tcactgtctt taggaatatg aaaatggact catggtcata 1380
attatcttct tgataaatag aagagatatt tcagaaagat cactcacaac tccaattact 1440
gagggaagcc agcccttgta taaagcacgt ggaccttctt ccttaagcac agtagtcaaa 1500
gcatggaaca ttcctctgta ctgataaggg gatttctcag tctgtacagt tatcctgcct 1560
cggaccatat ccatcggata ggttgcagac atagcaatta ttccagcaca tgctccagct 1620
ccaagacgta aaacaggagt cagttgagca tcctcatttc cagtttgctg ctgatacaga 1680
tgaagtatgc ctttagaagc ttgctcataa ctgaagaact ttactgcaga atttgggaca 1740
attcgagcac aattagtacc atttccctta aacagtccac ggaacccttc agttctccat 1800
atatatttga ggccttgtat tgttccattg tattttatgt tatgaggatt ctggacctgc 1860
agcaaaattt tcattcgttc cagcggagca accgctgttc gtgatactcc tcctgcgact 1920
ccaccagcaa cgagagactt gcagatgcta ccgattgcat agttaggagc tttaacaaca 1980
ccttctctag cgagcttcgc ttcttccgcc aaattcacga tcttcgttac agctgattca 2040
ccagttttca catcttccga agccatactt aaataaaatc aataataata ataatttatg 2100
ataaaattaa taaaccctaa cctaaccgga actgaaaaaa ttgaaagaaa aagagtaatt 2160
aattaatcta ggttgccgga gattgagaga tcttagattt tccggtgaat tattagtacg 2220
gcgaggttaa gcgatcattg gtggctgtga aagtagtggt ggtggtggct gcggaagctt 2280
ctagagagag agtgaaagct tacagagacc actaactact tgtatgagcg aagaagagtg 2340
acggcgtttc gttagttatg ctattt 2366
<210> 3
<211> 2670
<212> DNA
<213>Caucasian clover (Trifolium ambiguum Bieb.)
<400> 3
aaaaaaaaac caaaaaaaaa aaaataaaac ttgataactc agataacaat actctgaagc 60
tagttccaat tttttcaatt cccacaaatt aaataattaa tctttcttca cttcttcatc 120
tctctcaccc tttcatttca tgcaattttc tcttatggat ccctttgatt tgattcgtaa 180
agggtttgga tgaagctgtt tggaacttgc ttttactgtt ttcgaagttt atctttgttt 240
tagtcgtttt gtttgtattc tagttaataa tgagtttatc aaagaaaggt ggatccccaa 300
ctgacacgaa actgagctct ccaaacaagg cggtcacttt gaatccaaat gcagcagagt 360
ttattccctt ttccctcaga tctttgccgt ctggaagtac tagctcagta gatgcaacgg 420
caaggcttac aactgctggt tctctaggaa aatcagtttt agatcgatca gaatcatcga 480
tttcaaataa ttctgatgac gaggcccaca attactggcg ttgtcaactc ccggatgata 540
tcactcctga tttcaatgtc attgaagaag atgaacccca aggtcttaac aacctctctt 600
tcgcaggctt atctataaac gatgataatg aatcgtctat gttttcttct accacgggaa 660
gtagatatat accaaacgag cagcaggaat tgtctcatca gcaccttaat ggaaatacct 720
ttgtcgataa gttaaggttt tccaattcaa cctaccggga ggaaccatct tcggcaagct 780
ttttaaattc attggcaaag ccttgggata gacccattgg gaataacaac ctgcatgtta 840
gcggtggtca agacgcagtt gcttatgatg acaatgctag acatggattc ttaaatgatg 900
ttttgactga gaatgctatt gtggatgaga ctgattttaa ccctttggaa tttttagctt 960
ctctattccc tggttttgca tcagaaagcc ttgccgaagt ttactttgcc aatggatgcg 1020
atttacatct gaccattgag atgctcactc agttagaggt gggttgatct tcatgtacca 1080
actaacttgt ggttaaattt tggtttttaa taaagcctct cttcatacgg aatatctttc 1140
tcttagcatg ctgcgatgga aattcatatc ttgcgatatg ttttgataca ctattaaatt 1200
tgaatagatg gaagctaaat gcgtagagca atacttgtgc cttgtttttg cagggatgtt 1260
tccagcatcc cataaagttg tgagatgact ccaacgaaat ccagcttccg gattcaaatc 1320
actgtttcct gaatccttat accctttgca tcatgctcac aggggacctg ataggaactt 1380
atcaggagtt caggactatt aaaatttgat ttgcattatt tggatatata atcttttctt 1440
gttatatgat attgtaatac aaccatgtct caaggttttc tttttcattc catggatgtt 1500
attgtcactt tcctgaatat tttcattttg cttgcatact tggggacatt tatgttacta 1560
tatctacttt ttcctccaaa tttgtagatt caagtcgata gtaatttcgg ccagaaccca 1620
agtccaaaga ctctatcatc tcccaatctg actgcaatgg actttccagc acttgcttca 1680
acaaacgggc agactactac tgcaaaatat gctgcagata atgttcaaca aagtggcaat 1740
ccttacttac cgtctggcaa agaccttctc atgttcaaaa ctagctcttc tattccatct 1800
agaggtgcta ccgattttgc ttcagctgtt agaaaattgg cttctcagga ttctggaata 1860
tggaagtatg atagaaatgg ttctggtgat ccttccactg gatcaagcag gagtttaaat 1920
gttctagcta gtgcttataa tggcggacag gggagaacta actttggtga taggttacaa 1980
aaccgtggtt ctgctcggcc agctcctgtt tggcttgaaa ctggtgacgc tgttggtaaa 2040
tatttatgtt tatatgtttc cttttttgtg atgagttcct tttatactgg tttcctttaa 2100
ctaatttgaa tgacagcaaa tatgtattct gaactacggg aggaggctcg agatcatgca 2160
cgcttgcgta atgcgtattt tgagcaggca cggcaagcct accttattgg caataaggcc 2220
cttgcaaagg agctaagtgc taaagggcaa cttcataaca tgcatatgaa agaagctcac 2280
gggaaagctc aagaatctat ttaccgtcag aggtttgctg ttcctcatta ctatccacta 2340
ttcctatcca acggactttt gatcaaaaca aaaactgtat catgttataa tgagagtata 2400
tatttttcaa cattgttaaa cccaagaaaa tgaactagta tgattttcag ttcaggttca 2460
accatgttaa ccgtttgctt gaatactcaa attgggatga cttcatgtga ttaatttaaa 2520
tcaaataaaa aattatttta aattatttat caaatatgaa aacagtttat tttttcaata 2580
attgttgctt gttcaaccaa gatgaattgg caaattcaac tagtatttgt caatttataa 2640
gattgttttt tattggtcca atggtggtat 2670
<210> 4
<211> 1128
<212> DNA
<213>Caucasian clover (Trifolium ambiguum Bieb.)
<400> 4
catttccatc ctacaatcat ataaaaatta cttagatcat tgcctcactg tgatgcaaag 60
aaatgtgata atagtccata acaaaatata cattaattaa atcatgcgac tatgaccatc 120
ataataatac tttcaaaaat gtcatttcaa cactatctgt catgtattca taagctataa 180
actactttat tttttcaatc ttcattacta aattggtgtc gttattgtta ccttaaggat 240
agtttagaga atctgaataa aaagttattt atcaatgcat tgtctatcca cctacttaga 300
tacagtctct taaatatttt agactactct tcctgtcttc caaagcaacc ttgcattgga 360
aaacagccta tcatccttat cgtgagcata atttcgtagt ttcattaccc ttctaatcat 420
aaacctgaaa cagccatgcc aaacaaaaac atctgctaca tgccaaaaac aatggttaaa 480
aaaaatgtgc acccaaacca ctgatcctta attaaccgtg aaatagtaag aacatcccaa 540
ttcgagaatc acttggggtt tgacggaatt ctgtgactat gcatcaaatc ctaatacttt 600
aaaaaaaaca acaaaaaaaa ataacaaacc ctaaactcac tgacatacta aaacattatt 660
cttgcatcaa acaaaacatt cagcaaagca aactgaatgc atcaaagaac cacgaatcag 720
taagaacacc caaatttgag actcacttgc ggtttgacaa aactatgtga ctatgcatct 780
aatctaatcc ttctactttc aaaaaataaa taacaaagca aacaaaatga caaatcatcg 840
aatcgcatac tgatactcat gtataacact ctcacatagt cgcattaaac aaaaaacaca 900
ccgtatatat aacattatca ttgttacatg gatttaacat atattccaaa tcagcaacaa 960
agggatctaa atagaaaatt aaatttaacc cagtaaaata acaacttaga gattacaaac 1020
atgattaaca ctatttaatg tacaatcttt gatttcaagg actaaaacta acacaaacat 1080
acacaaatcg aatattactt ttcttcttaa tttttttttt tcggaaca 1128
<210> 5
<211> 241
<212> DNA
<213>butch clover (Trifolium repens L)
<400> 5
gacatggaaa agatctggca tcacacattt tacaatgaat tgcgtgttgc tcctgaggag 60
cacccatgct tctaatgagg ctccactcaa cccaaaggcc aacagagaaa agatgaccca 120
aatcatgttc gagaccttta atgtgcctgc catgtatgtg gccattcagg ctgtcctctc 180
cctctatgca agtggtcgta caactggtat tgtcttggat tctggtgatg gtgtgagtca 240
t 241
<210> 6
<211> 3349
<212> DNA
<213>Caucasian clover (Trifolium ambiguum Bieb.)
<400> 6
atgaagtcta ttctacttct agcgatagtg atcctctata gactgattac aaaagttctc 60
acaatccttg agtatagcgt gcgtgtgcat catggctgat atggaaagtc aacaccgagt 120
gtcaacgatt catcagatca gacggtgaag gattaagcaa tatgtaaggg acccaccaca 180
tctaatctaa taaactacat gaaactaatc caaatttcta gggtttcact ttaaaagtag 240
ttcccaaaac ccaacaagca caagcacttt gtactagtga catatccact accaaactat 300
aacaccaaca acatttttca tttcttttct tcacttcttc tagattcttc catctacgcg 360
cactccttca acttcaatca ccggtaagtg caaccttaaa cacatctatc tcctcctcgt 420
acctcggatt catcgatagt ttattttagt acgcaaagtt tttttttttt ttttgtttac 480
ttttagtacc aattatcaat tttgatttag atttagacat agatttgcat ataacttatt 540
gtgtgtgaaa aaaagtgaga gtatggggaa atcagggggt agaagaaaga agggtggtgc 600
tgttgttgtt gacaacaatt cagcttctgc acaaattcca aatggtggtg ttgaattgga 660
ttcctcaatt tttttgaaga aggcacatga aatgaaagaa gaagggaata ggagatttca 720
gagtaaggat tatgctggtg ctcttgaaca ctatgagaat gctcttcgat tgacaccgaa 780
aatgcatcct gatagggctg tttttcacag caatagggct gcttgtttga tgcaaatgaa 840
acctattgat tatgaatctg ttatttctga gtgtactttg gctcttcagg ttcagcctca 900
atttgttcgg gctcttcttc gtagggctcg ggcgtttgag gctgttggta agtatgaatt 960
agctgtgcaa gatgtgcagt tgttgttggc ttctgatcct aatcataagg atgctttgga 1020
tattgctcag aggttgaggg ctgcttttgg gcatcgtcag gaagctcagc aggatctcca 1080
cagtcgacct tctcctgctg cgcttggcgc ttctgctgtc cgtggtgctc ctattgctgg 1140
gttaggtccg tgtttgccag cacggcctgc atcaaagaag ggagcgaatt ctgctgtcgg 1200
gtcggttgtt tctcctaata gcaagataga caagtctcaa aatgttttac tgccaaatga 1260
aaatggtcct gagaccaaga cccagttgcc aaaagttgta ttgaagtctt ttaacaatgg 1320
tcctgcagtg caacctaact cgaaaaatga gaaccagaag gatgttcaca ggcagatttc 1380
ggaggttgca attcggtgta gaccattgaa gcttgtttat gatcatgaca ttagacttgc 1440
ccagatgccg gtgaactgca gtttcagagt actgagagat gtagtaagca aaagatttcc 1500
ttcgtcaaat tcagttctga tcaagtacaa ggatggtgac ggtgatttgg ttactattac 1560
atctacagat gaactcagat tggcagagtc ttttgttgat agctatctgc tgaaggaacc 1620
tgaatcagat aaaagtgatt caatttcggt gctgagacta catattgttg aagttagtcc 1680
ggagcaggag ccacctttgt tggaagaaga ggaagagaaa tttgttgaga atgaagggac 1740
caagggagat gagagtggat ctcattcttc ccttggtgaa tctgtccctg aagttacaga 1800
agttgctgaa gttacagaag ttactgaagt tcctgatact gatgttgata agataataaa 1860
gaaggatgct tcaaaggaaa agacgggtgc cacaggagaa aatgaatgca aagaggtgga 1920
gatggatgat tggttgtttg aatttgctca gcttttccgc tcacatgttg gtattgaccc 1980
agatgctcat attgacttgc atgagcttgg gatggagctt tgttcagagg cacttgaaga 2040
aacagtaaca agtgaggagg ctcaggatct atttgacaag gctgcttcaa aattccagga 2100
ggtggcagct ttggcattct tcaactgggg taatgttcac atgtgtgctg ctaggaagcg 2160
gattcctcta gatgagtcgg ctgggaaaga cgtagtggcg gaacaacttc aagtggctta 2220
tgactgggtc aaagaaaagt attctttggc aagagagaag tacgaggaag cactcttgat 2280
caaaccagat ttctatgagg gacttttggc tctgggccag caacaatttg aaatggccaa 2340
gctccattgg tcttttgcga ttgctaagaa gatagatctt acaagctggg atccaacgga 2400
aactcttcaa ctgtttaaca gtgcggagga gaagatgaaa tctgcaacag atatgtggga 2460
gaagttggag gaacagagag caaaggagct gaaggaccca aatgcaacca agaaagaaga 2520
agtgttgaga agaagaaaga aacagggaag tgctgctgaa ggcgagtctt ctagtgctgg 2580
tggtcaaggt gagatatctc ccgaagaagc tgctgagcaa gcagtggtca tgagatcaca 2640
gattcatctc ttttggggca atatgctttt cgaaaaatcc caagttgaat gcaaattagg 2700
aatggatggt tggaagaaaa atctagatgc tgcaacagag cgctttaagc ttgcaggagc 2760
ttccgaggca gatatttcaa tggttcttaa gaatcattgc tcaaatggag atgcaaaagg 2820
tgatgataaa aaaagtccag agcccacaac ccaacaagac tggtgaacta gagatcaata 2880
aggccaatca agtatgagat cagttatgtg tggctatgaa atctcatttg tttcttttca 2940
gttgtgtcct caatgaacac tctggacaag gaatatactt ggcatcaaga gctcaaaagg 3000
aatgtcttat tttttggaaa tggaaattga aaacttaggg tactttaact agatagctct 3060
gccctttttt tcttttcttt tttctctgaa gttcttggtg atctgatgaa gggagattgc 3120
tttttaacat tatttacaca gttatgaatt tctgatgatc aatttcagtt gctcaaacat 3180
ttttgtcccc tgctttgttc tttgtactgt tgcaatttat tgaggactgt atacttgttc 3240
gactcttgac atttgctttg ttggtcctta ccaatatgct gcatgtattg ttgattttat 3300
ttattaccaa gttataaccc aagttttcta tggttaaaaa aaaaaaaaa 3349
<210> 7
<211> 1513
<212> DNA
<213>Caucasian clover (Trifolium ambiguum Bieb.)
<400> 7
ctctatatat atatatattc tcgtttcacc catctatcac tttcttcctt cactgcactt 60
catcgccgaa ccagagggtg aagctcaaaa atctcttctt caattaatca acgattttat 120
tttatttatt ttattttccc catacgataa tatattattg catcaggttt attatccaat 180
caatttatat atttttcctg aattttcacg tccataatag aaaattaccc cacttatcct 240
aatccactta taagaatgaa aattctctgt cgttaactga tctagggttt ttcaagtttt 300
tgttgaaatc cattgatagg tttcaattag gttttcgtga ataatggcac gtttggttct 360
tccatcgaag actctggctt tttccaggaa gagtctactg ggtttgatct cacctgctcc 420
tcttcagatt agttgcagcg ttaattgttt tcggaagacc tcgaaaagta gtgtcagata 480
tagacatttc cttgcatcag aggttgttct ccgaaagaat tgttgcccat ttaattcttc 540
ggtacagagg gtcaagaaag ctagtaggaa gctaatttgt tcggttgcta ccgaaccaaa 600
gcaatctgaa gaatctaaaa tggcgcagcc aagagaaata ttcttaaagg attacaaaaa 660
gcctgattac tactttgaga ctgtggatct gaaattttcc ttaggagagg agaggacaat 720
agttagttct aaaattgcgg tgtttcctcg cgttgaaggt tctgctcccc cgctagtttt 780
agatggacaa gacatgattt tagtttcagt tcaaattaat ggcaaggcct tgaaggagga 840
agattatcat ttggatgtac gccatctaac aatccaatca cctcctagtg gtaaatatga 900
tcttgaaatt gttaccgaga ttctcccaca gaaaaacaca tcattggagg gactttacaa 960
gtcatctggg aatttctgta ctcaatgtga ggctgaaggc ttccgtaaaa tcacgtatta 1020
tcaggatcgg ccggatataa tggcaaagta tacagttcgc attgaggcag acaaatcact 1080
gtatccagta ttgttgtcta atggaaacct ggctggacag ggtgacttgg agggtggcaa 1140
acattatgct gtttgggaag accccttcaa gaaaccttgt tatctgtttg ccttagtcgc 1200
tgggcaattg gagagcagag atgacacatt tactacccgc tcaggaagga aggtgtccct 1260
taggatctgg acgcctgcag atgacgtacc taagactgca catgctatgt attctctgaa 1320
agcagctatg aagtgggatg aagatgtttt tggacttgaa tatgatctgg atctcttcaa 1380
catcgttgct gtcccagatt ttaatatggg agccatggaa aacaaaagtt tgaatatttt 1440
caattccaag cttgtattgg cttctccaga gactgcttct gatgcagatt atgctgcaat 1500
attaggagtt att 1513
<210> 8
<211> 1869
<212> DNA
<213>Caucasian clover (Trifolium ambiguum Bieb.)
<400> 8
caaatatcga taaaactcat actcatagtt aagacacaag ctaaattttg gtagcagtaa 60
actgcatcag agacgagtta cttttattat tattattatt tgcaggtagt attctcaata 120
ttatacatga ggtctaagta attatgtgaa ctcatatgtc aacataagta aaaaaaaaaa 180
agaagcagca atatatggta tcaattttta aaataaaaaa ttgtcctagt ttcatctcta 240
atccattttc ggacaaaatg ggtagcataa taaaacattt aaaatgcatt caatacatgt 300
gcaataataa aatgcaacaa aaagaataat aatgagggat gaaacaaaag acccaaatgg 360
tttaattcta actttagcca tataggtctt tgtaagtata tattcaaaaa cacatgacaa 420
agtctaaact aagaacctta ttctatgagt actgttaaat ttagaacttg accaaaatca 480
tcataaaaaa tagcacaaaa catacaacaa cctagtgaaa caacattgat tctgcaattc 540
actacaaaac atcctcaaat ttgatcagta agagatggtt gttggttgct cctccagcac 600
tgcttcatgt tggagatcct gaggttgttc tccttgccaa ttccacacaa tgtccttaag 660
tgctggtcta atctcctgtt gcaaaagctt ctgatactcc actgtatcgc catcggactt 720
cttaagttcc acgagatgga agagaggtgt aatctcgaaa atttcggcat caatattcaa 780
agtccctttc cttccttctt ttgaactttc cagcttcaac aaacctccat cttttttcgt 840
aattttcaag cgaagacact tgcaaatttc ttcaaactta gagattataa ttgaagccgg 900
cttgttagat gtaaaccgca tctctttcgt ttgaattgtg tcttcgaaca agctggataa 960
atcaaaacca gatgagaagg agatgatatc aaaggcattt aagttgttgc agggtttggt 1020
ctgttgttgt attgactccg tattagaatc actgccgttc tcggacactt cgaacactcc 1080
atcagcgtgc agagaagcta gttcattttt tccgatatcg ataacaaccg gcttctctaa 1140
ccccttttta aaccaagaat tttccatgat cttggccatt gatatccgag tctttagatt 1200
tggatccaat attctggaca acaaccggcg aacctctagt gcaaaccatt taggaaattt 1260
gacttcccct ttactgattt tcctgtacat ctccatcaga ttttgatcat ggaatggaag 1320
ataaccagcc aatagaacaa acaagattac gccgcacgac caaatatccg ctttgcatcc 1380
ctcatagcct tttcggttta tcacttcagg agcaacatac gcaggcgtac cacatgtagt 1440
gtgaagtaat ccatcctggc acttagattc agaaagggta ctcaaaccaa aatctgaaac 1500
cttcaaatta tcattttcat ccaatagtag attttcgggt ttcaaatctc gatgatacac 1560
acccctgctg tggcagaagt cgactgcact aatcagctgc tgaaaatatc tcctagcaac 1620
gtcaaccttg agctttcctt tagctatctt gttgaagagc tcgccacctt ttgcatactc 1680
cattacaatg aaaattttgg ttttagtcgc cattacctca taaagctcga ccacatttgg 1740
gtgtcttact aatctcatca ctgaaatttc acgcttaatt tgatcaatca tcccaaccct 1800
cagaaccttc tctttatcaa taactttgat agcaacattc atgccgtttt taaggttcct 1860
tgcatgata 1869
<210> 9
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
taatgcaccg aatccctcgt 20
<210> 10
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
gaatgcagat gaccggttgg 20

Claims (3)

1. a kind of application of C257504 gene as the reference gene of Caucasian clover PCR expression analysis, sequence such as sequence Shown in table SEQ ID No.2.
2. a kind of construction method of the reference gene for Caucasian clover PCR expression analysis, which is characterized in that method is such as Under:
(1) 7 tissue transcript profile data of Caucasian clover are utilized, the reference gene for therefrom selecting 7 expression stable, nucleosides Acid sequence such as sequence table SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.6, SEQ Shown in ID No.7, SEQ ID No.8, while common 1 traditional reference gene in Trifolium being selected to be used as control, Nucleotide sequence is as shown in sequence table SEQ ID No.5, totally 8 kinds of candidate reference genes；Caucasian clover 7 tissues Blade, stem, rootstock including root turion plant expand and have bud root turion section, the root turion section for expanding no bud, horizontal branch and without root turion master Root；
(2) above-mentioned 8 kinds candidate reference genes are subjected to design of primers, standard PCR amplification is carried out to each cDNA, obtains primer Amplification efficiency and amplification reach the recurring number of threshold value；
(3) to the data of acquisition using three reference gene stability assessment software geNorm, NormFinder and BestKeeper is analyzed, and most suitable reference gene and reference gene number under special experiment condition are finally screened；Pass through It is verified using the gene in Caucasian clover root, stem and leaf, the nucleotide sequence as shown in sequence table SEQ ID No.2 Expression stability highest.
3. a kind of qRT-PCR method of the reference gene for Caucasian clover PCR expression analysis, which is characterized in that its QRT-PCR amplification forward primer sequence is as shown in sequence table SEQ ID No.9, reverse primer sequences such as sequence table SEQ ID Shown in No.10.
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Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Selection of reference gene from gracilaria lemaneiformis under temperature stress;Yan Ding,et al;《Journal of Applied Phycology》;20150131;第27卷(第3期);1365-1372 *

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