CN113604599A - Fluorescent quantitative PCR reference gene stably expressed in different tissues of cinnamomum camphora and application thereof - Google Patents
Fluorescent quantitative PCR reference gene stably expressed in different tissues of cinnamomum camphora and application thereof Download PDFInfo
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Abstract
The invention relates to a fluorescence quantitative PCR reference gene stably expressed in different tissues of cinnamomum camphora and application thereof, belonging to the field of molecular biologyCcEF‑1α、CcEIF‑4α、 CcRPL3、CcACT11、CcTUB5 stably expressed reference genes developed based on cinnamomum camphora transcriptome sequencing data. The invention utilizes qRT-PCR technology, takes camphor leaves, stems and fruits at different development stages as materials, utilizes GeNorm, NormFinder and BestKeeper 3 kinds of software to evaluate gene stability, and finally utilizes RankAggreg to carry out comprehensive ranking to obtain the productCcEF‑1αIs the most stably expressed reference gene in each tissue sample of cinnamomum camphora. The invention provides an effective reference gene tool for screening, separating and expression analysis of the functional genes of cinnamomum camphora in the future, and is suitable for the reference geneThe field is popularized and applied.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a fluorescent quantitative reference gene of different tissues of cinnamomum camphora, and primers and application thereof.
Background
Cinnamomum camphora (L.) PreslCinnamomum camphora) Cinnamomum camphora and Cinnamomum camphora belonging to the genus Cinnamomum (Lauraceae) (Lauraceae)Cinnamomum). In recent years, cinnamomum camphora plays an important role in urban landscaping, and is also an important economic tree species due to its medicinal and aromatic properties. The essential oil or crystals extracted from cinnamomum camphora is a source of food preservatives and additives, and a raw material for the cosmetic and pharmaceutical industries, and has considerable economic value. However, due to the lack of genomic information, the molecular biology research of cinnamomum camphora relies more on the second generation sequencing technology, and we have screened some reference genes by using the transcriptome data of cinnamomum camphora, which will greatly facilitate the further research of cinnamomum camphora gene expression.
Fluorescent quantitative PCR (quantitative Real time PCR, qRT-PCR) is the most common gene expression level quantification technology, and is used in many research fields due to the characteristics of high accuracy, strong specificity, high throughput and the like. In the research of plant reference genes, the expression of homologous reference genes among different species is not constant, and the relative expression quantity of target genes is deviated due to improper selection of the reference genes. Therefore, the selection of a suitable reference gene that is stably expressed under different experimental conditions is critical to the accuracy of the target gene. However, so far, the screening of the cinnamomum camphora reference genes by using transcriptome data has few related reports, so that the invention screens 5 cinnamomum camphora reference genes based on the cinnamomum camphora transcriptome sequence.
Disclosure of Invention
Aiming at the problems, the invention develops the reference genes, selects the stable reference genes and primers thereof for researching the camphor leaf, stem and 6 fruits in different development stages through the camphor transcriptome sequencing database, and provides an effective reference gene tool for screening, separating and expression analysis of camphor functional genes in future.
The invention adopts the following specific scheme:
the invention aims to provide application of 5 internal reference genes in fluorescence quantitative PCR of different tissues of cinnamomum camphora, wherein the 5 internal reference genes areCcEF-1α、CcEIF-4α、CcRPL3、CcACT11、CcTUBThe nucleotide sequence is shown in SEQ ID NO. 1-5. Further, the different tissues include camphor leaves, stems and fruits at different developmental stages.
The invention also aims to provide a reference gene which is developed based on cinnamomum camphora transcriptome sequencing data and stably expressed in different tissues of cinnamomum camphora.
The third object of the present invention is to provide primers for amplifying the above-mentioned reference genes.
The fourth purpose of the invention is to provide a screening method of fluorescence quantitative PCR reference genes in different tissues of cinnamomum camphora, which comprises the following steps:
respectively selecting camphor leaf, stem and fruits at different development stages after blooming as samples; extracting total RNA of each sample, and synthesizing cDNA through reverse transcription of the RNA, wherein the cDNA is used as a template for target gene amplification;
step two, screening out candidate reference genes of the cinnamomum camphora by utilizing early cinnamomum camphora transcriptome sequencing data, wherein the candidate reference genes are respectively as follows:CcEF-1α、CcEIF-4α、CcRPL3、CcACT11、CcTUB(ii) a Wherein, theCcEF-1αThe nucleotide sequence of (A) is shown as SEQ ID NO.1CcEIF-4αThe nucleotide sequence of (A) is shown as SEQ ID NO.2CcRPL3The nucleotide sequence of (A) is shown as SEQ ID NO.3CcACT11The nucleotide sequence of (A) is shown as SEQ ID NO.4CcTUBThe nucleotide sequence of (A) is shown as SEQ ID NO. 5;
step three, the selected candidate internal reference gene sequence is taken as a template to design the entityAn internal reference gene primer of time fluorescence quantitative PCR, wherein the size of an amplification fragment of the primer is 150-200 bp; wherein, for amplificationCcEF-1αThe primer pair has the sequence of SEQ ID NO.6/SEQ ID NO.7 and is used for amplificationCcEIF-4αThe primer pair has the sequence of SEQ ID NO.8/SEQ ID NO.9 and is used for amplificationCcRPL3The primer pair has the sequence of SEQ ID NO.10/SEQ ID NO.11 and is used for amplificationCcACT11The primer pair has the sequence of SEQ ID NO.12/SEQ ID NO.13 and is used for amplificationCcTUBThe sequence of the primer pair is SEQ ID NO.14/SEQ ID NO. 15;
step four, real-time fluorescence quantitative PCR: taking cDNA obtained by reverse transcription in the first step as a template, and carrying out real-time fluorescence quantitative PCR amplification on the candidate reference gene by adopting a primer in the third step; and performing statistical analysis on the obtained real-time fluorescence quantitative PCR data, and respectively screening out the optimal reference gene and the optimal reference gene combination.
As a further optimization of the screening method, before the real-time fluorescent quantitative PCR of the step four, the specificity of the reference gene primer is identified by common PCR.
And in the fourth step, performing statistical analysis on the obtained real-time fluorescent quantitative PCR data through three kinds of software, namely GeNorm software, NormFinder software and BestKeeper software, respectively screening out the optimal internal reference genes and internal reference gene combinations, and then performing comprehensive ranking analysis by using RankAggreg R analysis software.
The difference between the invention and the prior art is that:
(1) the method screens out the most stable internal reference genes in camphor leaf, stem and 6 developmental stage fruits from 5 candidate internal reference genes, has real and reliable data, and lays a foundation for deep excavation of functional genes in the later period;
(2) the screened reference gene is suitable for expression analysis of key genes of cinnamomum camphora, can obviously improve the accuracy of obtained data, and has wide application, high sensitivity and good stability;
(3) the research determines stable reference genes suitable for different tissues of the cinnamomum camphora through a strict reference gene screening program, and provides a theoretical basis for selecting proper reference genes in the cinnamomum camphora qRT-PCR analysis.
Drawings
FIG. 1 is the sequence electrophoresis chart of 5 primers of reference genes in example 2 obtained by amplifying with Cinnamomum camphora cDNA as template; wherein, M: DNA Marker, the order of the size of the bands is 250bp, 500bp, 750bp, 1000bp and the like from bottom to top, and the genes represented by each lane from left to right are respectivelyCcEF-1α、CcEIF-4α、CcRPL3、CcACT11、CcTUB；
FIG. 2 is a Ct value distribution diagram of fluorescent quantitative PCR of 5 reference genes in example 2;
FIG. 3 is a dissolution profile of 5 reference genes in example 2;
FIG. 4 shows the expression stability values of 5 candidate reference genes in example 2 by applying GeNorm program ((C))M) And the pair difference value (V），MThe smaller the value, the better the stability of the reference gene; where FIG. 4 (A) takes as material the data for all sample mixes; FIG. 4 (B) shows fruits at 6 developmental stages as the material; FIG. 4 (C) shows leaves and stems as materials; FIG. 4 (D) is a distribution of the pair difference values under each condition;
FIG. 5 is the composite ranking obtained by applying the RankAggreg R package to the three statistical analysis methods in example 2; FIG. 5A takes as material the data for all sample mixes; FIG. 5B shows fruits at 6 developmental stages as the material; FIG. 5C shows leaves and stems as the material.
Detailed Description
Herein, the following: the camphor tree transcriptome sequencing data comprises the following steps: origin of originhttps://www.ncbi.nlm.nih.gov/sra/ SRX1091098[accn]
GeNorm reference: v andesosample, J., Preter, K.D., Pattyn, F., Poppe, B.B., Roy, N.V., Paepe, A.D., et al, Accurate normalization of real-time quantitative RT-PCR data by geographic visualization of multiple internal control genes [ J ]. Genome biol. 2002, 3, research0031-research0034.
NormFinder reference: andersen, C.L., Jensen, J.L., and Orntoft, T.F. Normal of real-time quantitative transfer-PCR data a model-based variant estimation approach to identification genes Suitd for Normalization, applied to blade and color scanner data sets [ J ] Cancer Res. 2004, 64, 5245 and 5250.
Bestkoeper reference: pfaffl, M.W., TiChopad, A., Prgomet, C., and N euviruses, T.P. Determination of stable housekeeping genes, differential regulated target genes and sample integration, BestKeeper-Excel-based tool using pair-wise solids coatings [ J ] Biotechnol. Lett. 2004, 26, 509-.
The RankAggreg R package is referred to: najafpanah, M.J., Sadeghi, M., and Bakthiazadeh, M.R. Reference genes selection for quantitative real-time PCR using RankAggreg methods in differential properties of Capra hircus. PLoS ONE 2013, 8(12): e83041.
The technical problem solved by the invention is realized by adopting the following technical scheme:
the reference gene of cinnamomum camphoraCcEF-1α、CcEIF-4α、CcRPL3、CcACT11、CcTUB(ii) a The above-mentionedCcEF-1αThe nucleotide sequence of (A) is SEQ ID NO. 1; the above-mentionedCcEIF-4αThe nucleotide sequence of (A) is SEQ ID NO. 2; the above-mentionedCcRPL3The nucleotide sequence of (A) is SEQ ID NO. 3; the above-mentionedCcACT11The nucleotide sequence of (A) is SEQ ID NO. 4; the above-mentionedCcTUBThe nucleotide sequence of (A) is SEQ ID NO. 5.
The method is used for amplificationCcEF-1αThe sequence of the primer pair is SEQ ID NO.6/SEQ ID NO. 7; for amplificationCcEIF-4αThe sequence of the primer pair is SEQ ID NO.8/SEQ ID NO. 9; for amplificationCcRPL3The sequence of the primer pair is SEQ ID NO.10/SEQ ID NO. 11; for amplificationCcACT11The sequence of the primer pair is SEQ ID NO.12/SEQ ID NO. 13; for amplificationCcTUBThe sequence of the primer pair of (1) is SEQ ID NO.14/SEQ ID NO. 15.
The real-time fluorescent quantitative PCR screening method of the camphor reference gene comprises the following steps:
(1) selecting camphor leaf, stem and 6 fruits in different development stages as materials;
(2) screening out the reference gene of the cinnamomum camphora by utilizing cinnamomum camphora transcriptome sequencing data;
(3) designing a reference gene primer of real-time fluorescent quantitative PCR by taking the selected reference gene sequence as a template;
(4) performing conventional PCR amplification on each cDNA by using the primers designed in the step (3) to obtain the amplification efficiency of the primers;
(5) performing real-time fluorescence quantitative analysis by using a 5-fold dilution series (1 x, 5x, 25x, 125x, 625x and 3125 x) of the mixed cDNA of each sample of cinnamomum camphora as a template;
(6) carrying out real-time fluorescence quantitative PCR; and performing statistical analysis on the obtained real-time fluorescence quantitative PCR data through three statistical software of GeNorm, NormFinder and BestKeeper, respectively screening out the optimal reference gene and reference gene combination, and then performing comprehensive ranking analysis through comprehensive analysis software RankAggreg R.
Preferably, before the real-time fluorescent quantitative PCR in the step (4), the specificity of the primers of the reference gene is identified by ordinary PCR (using DNA polymerase supplied by TAKARA: rTaq Mix; identification conditions: 95 ℃ 5min, 95 ℃ 30s, 55 ℃ 30s, 72 ℃ 60s/kb, 30 cycles; 72 ℃ 10 min).
Preferably, in the step (4), the real-time fluorescent quantitative PCR amplification procedure is: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 5s, annealing at 55 ℃ for 30s, running for 40 cycles, and analysis of dissolution curve at 65-95 ℃.
Preferably, the real-time fluorescent quantitative PCR screening method of the reference gene of the cinnamomum camphora comprises the following specific steps:
1) collecting materials: the leaves, stems and fruits of cinnamomum camphora at 6 developmental stages collected in the school zone of east lake of agriculture and forestry university at Zhejiang are taken as experimental materials, and the collected materials are quickly frozen in liquid nitrogen and stored in a refrigerator at minus 80 ℃. The fruit numbers of 6 developmental stages are S1, S2, S3, S4, S5 and S6;
2) RNA extraction and detection: extracting total RNA (the extraction mode is consistent with the content in the kit specification) of each sample by adopting an EASYspin Plus polysaccharide polyphenol complex plant RNA extraction kit (Edley, China), detecting the integrity of the extracted RNA by utilizing electrophoresis, carrying out electrophoresis for 20min under the voltage of 1% agarose gel and TAE buffer solution and observing and analyzing by utilizing a gel imaging system (Tanon-2500); performing purity detection on the extracted RNA by using NanoDrop ND-2000 (OD 260/280 of pure RNA is between 1.8 and 2.2);
3) synthesizing cDNA by RNA reverse transcription: after detection by non-denaturing agarose gel electrophoresisTaking 1. mu.g of Cinnamomum camphora RNA sample and using SuperScript®III, reverse transcription Synthesis of cDNA by a First-Strand Synthesis System kit (Thermo, USA) as a template for target gene amplification;
4) screening candidate reference genes: screening out various candidate reference genes by utilizing camphor transcriptome sequencing data and referring to homologous genes of a model plant arabidopsis thaliana;
5) designing and detecting a specific primer: taking the screened candidate internal reference gene sequence as a template, and designing an internal reference gene primer of real-time fluorescence quantitative PCR by utilizing Vector NTI software, wherein the size of an amplified fragment is between 150 and 200bp (see table 1 in detail); the cDNA obtained by the reverse transcription of the camphor is taken as a template, and the specificity of the primer is preliminarily identified by utilizing the common PCR. Observing bands of the PCR products under a gel imaging system after electrophoresis, wherein each band is correct in size, single and has no primer of a primer dimer as shown in figure 1;
6) establishing a reference gene primer standard curve: using 5-fold dilution series (1 x, 5x, 25x, 125x, 625x, 3125 x) of mixed cDNAs of different samples as templates for establishing standard curves, and designing 3 technical repeats for each reaction of real-time fluorescence quantitative PCR;
7) fluorescent quantitative PCR amplification: taking cDNA obtained by reverse transcription of 8 samples as a template, carrying out real-time fluorescence quantitative PCR amplification on the internal reference gene to obtain correspondingCtA value; the amplification procedure was: collecting fluorescence signals for 40 cycles at 95 ℃ for 3min, 95 ℃ for 5s and 55 ℃ for 30s, and obtaining a dissolution curve at 95 ℃ for 5s and 65-95 ℃. And collecting a melting curve signal, wherein the generated melting curve is a single peak, which indicates that the specificity of the primer is good, the Ct value data is automatically read by a real-time fluorescence quantitative PCR instrument, and the distribution range of the Ct value is shown in figure 2. The Ct value is inversely proportional to the expression quantity of the gene, the larger the Ct value is, the lower the expression quantity of the gene is, and conversely, the smaller the Ct value is, the higher the expression quantity of the representative gene is; drawing a standard curve by using the obtained fluorescent quantitative PCR result, and obtaining the amplification efficiency and the slope of each candidate gene as shown in table 1;
8) evaluating the expression stability of the gene by using 3 statistical analysis methods of GeNorm, NormFinder and BestKeeper;
9) carrying out comprehensive statistical ranking on the stability of the candidate internal reference genes obtained by the 3 statistical analysis methods by using a RankAggreg R program package to obtain a comprehensive result;
the invention will be further described with reference to specific embodiments, but the scope of the invention is not limited thereto; the equipment and reagents used in the examples are, unless otherwise specified, conventionally available commercially.
Example 1: candidate reference gene and primer design thereof
Based on the pre-transcriptome data, 5 candidate reference genes were co-analyzed and identified in this study (table 1), which are:CcEF-1α、CcEIF-4α、CcRPL3、CcACT11、CcTUB(ii) a The above-mentionedCcEF-1αThe nucleotide sequence of (A) is SEQ ID NO. 1; the above-mentionedCcEIF-4αThe nucleotide sequence of (A) is SEQ ID NO. 2; the above-mentionedCcRPL3The nucleotide sequence of (A) is SEQ ID NO. 3; the above-mentionedCcACT11The nucleotide sequence of (A) is SEQ ID NO. 4; the above-mentionedCcTUBThe nucleotide sequence of (A) is SEQ ID NO. 5. Designing qRT-PCR primers by using Vector NTI software, wherein the size of amplified fragments of the primers is 150-200 bp; the method is used for amplificationCcEF-1αThe sequence of the primer pair is SEQ ID NO.6/SEQ ID NO. 7; for amplificationCcEIF-4αThe sequence of the primer pair is SEQ ID NO.8/SEQ ID NO. 9; for amplificationCcRPL3The sequence of the primer pair is SEQ ID NO.10/SEQ ID NO. 11; for amplificationCcACT11The sequence of the primer pair is SEQ ID NO.12/SEQ ID NO. 13; for amplificationCcTUBThe sequence of the primer pair of (1) is SEQ ID NO.14/SEQ ID NO. 15. The primers for the reference gene used are shown in Table 1.
TABLE 1 qRT-PCR reference genes and their primer sequences.
Example 2: and (5) analyzing the stability of the candidate reference gene.
The method specifically comprises the following steps:
1) total RNA extraction and cDNA Synthesis
The collected samples (camphor leaves, stems, fruits of 6 different developmental stages) were ground into powder with liquid nitrogen. Complex plant RNA extraction kit (Edeland) adopting EASYspin Plus polysaccharide polyphenolChina) to extract the total RNA of each sample, and the specific method refers to the instruction. Detecting the integrity of the extracted RNA by electrophoresis, carrying out electrophoresis for 20min by using 1% agarose gel and TAE buffer solution under the voltage of 100V, and observing and analyzing by using a gel imaging system (Tanon-2500); the purity and concentration of the extracted RNA were determined by using NanoDrop ND-2000 (OD 260/280 of pure RNA was between 1.8-2.2). After detection by non-denaturing agarose gel electrophoresis, 1. mu.g of Cinnamomum camphora RNA sample was applied to SuperScript®III First-Strand Synthesis System kit (Thermo, USA) reverse transcription Synthesis cDNA, as the target gene amplification template. The synthesized cDNA was stored in a freezer at-20 ℃ according to the instructions. The mixing steps are as follows: firstly, preparing 20 muL total amount of reverse transcription reaction liquid in a microtube, wherein the total amount of the reverse transcription reaction liquid comprises 1 muL Oligo dT Primer and 1 muL Dntp mix, and RNA with the total amount of below 5 mug is supplemented by RNase free dH2Culturing the micro centrifugal tube filled with the mixed solution at 65 ℃ for 5min, and then placing the micro centrifugal tube on ice for rapid cooling to promote RNA denaturation and improve reverse transcription efficiency; configuring a reaction solution in the same micro centrifugal tube, wherein the reaction solution comprises 10 muL RNA and a denatured primer, 4 muL 5 XPrmeScript II Buffer, 0.5 muL RNase Inhibitor, 1 muL PrmeScript II Rtase, and RNase free dH supplement2And O to 20 mu L, incubating the reaction solution at 50 ℃ for 60min to extend the cDNA, incubating at 95 ℃ for 5min, cooling on ice, and storing in a refrigerator at-20 ℃ for later use.
2) Detection of candidate reference gene specific primers
The cDNA obtained by the reverse transcription of the camphor is taken as a template, and the specificity of the primer is preliminarily identified by utilizing the common PCR. After electrophoresis, the bands of the PCR products were observed under a gel imaging system, and as shown in FIG. 1, each band was of the correct size, the band was single, and there was no primer of the primer dimer.
3) Real-time fluorescent quantitative PCR (qRT-PCR)
The overall qRT-PCR reaction was 25. mu.L, including 12.5. mu.L TB Green Premix Ex Taq (Baori Hospital, Beijing), 2. mu.L cDNA, 0.5. mu.L forward and reverse primers (5 candidate reference genes and their primers see Table 1) and 9.5. mu.L ddH2O, with three technical replicates per sample. The qRT-PCR reaction was performed on a CFX96 real-time PCR instrument (Bio-Rad, USA) with pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 5s, annealing at 55 ℃ for 30s, running for 40 cycles, and analysis of dissolution curve at 65-95 ℃. The Ct value is automatically obtained by an instrument after the reaction is finished, a standard curve is established, and the amplification efficiency of the primer is calculated.
4) Candidate reference gene stability analysis
The expression stability of the reference gene is obtained based on a software program of Microsoft Excel 2013. According to the formula Q =2-ΔCtCalculating the relative expression Q of each sample, whereinCt=Ct Sample- Ct min，Ct minMeans all ofCtMinimum in the sample. The GeNorm program is to obtain the average variation degree of each reference gene by using the Q value of the relative expression quantityMValue according toMThe values were sorted for gene stability,Mthe smaller the value, the higher the gene stability. At the same time, a pair variance (pair variance) V is providedn/n+1The optimum number of the internal reference genes required under the experimental conditions is determined so as to reduce the deviation and fluctuation caused by using a single internal reference gene. n represents the number of reference genes to be selected and the pairing difference value Vn/n+1<0.15 is the threshold value of the suggested selection (see fig. 4). Unlike GeNorm's calculation method, NormFinder directly evaluates the expression stability of reference genes based on analysis of variance, can determine the variation degree between groups, and calculates the gene expression stability value ((SA value),Sthe smaller the value, the more stable the gene (see Table 2). Whereas the bestkoeper program evaluated the stability index of the reference gene based on expressions calculated from Standard Deviation (SD) and percent Covariance (CV) values (see table 3).
To provide a comprehensive result, RankAggreg software was used to order the best list of reference genes,CcEF-1αthe comprehensive stability of the genes is ranked first, and the stability is the best (see figure 5).
TABLE 2 reference gene NormFinder scores for different samples.
Table 3 reference gene Bestkeeper scores in different samples.
It should be noted that the above-mentioned embodiments illustrate rather than limit the scope of the invention, which is defined by the appended claims. It will be apparent to those skilled in the art that certain insubstantial modifications and adaptations of the present invention can be made without departing from the spirit and scope of the invention.
SEQUENCE LISTING
<110> Zhejiang agriculture and forestry university, Living State and Biam Biotechnology Ltd
<120> fluorescence quantitative PCR reference gene stably expressed in different tissues of cinnamomum camphora and application thereof
<130> 1
<160> 15
<170> PatentIn version 3.3
<210> 1
<211> 1344
<212> DNA
<213> Cinnamomum camphora
<400> 1
atgggtaaag agaaggttca catcaacatt gtcgtcattg gacatgtcga ctccggtaag 60
tccaccacta ctggtcatct catctacaag cttggaggca ttgacaagcg tgtgattgag 120
aggttcgaga aggaggctgc tgaaatgaac aagcggtcat tcaagtatgc ctgggttctt 180
gacaagctca aggctgagcg tgagcgtggt atcactattg atattgcctt gtggaaattt 240
gagactacaa agtattactg cactgttatt gatgcccctg gccatcgtga ctttatcaag 300
aacatgatta cgggaacttc tcaggcggat tgtgctgtcc ttatcattga ctccaccact 360
ggtggttttg aagctggaat ctccaaagat ggccagaccc gtgagcatgc tttacttgct 420
ttcactcttg gagtcaagca gatgatctgt tgttgcaaca agatggatgc cacaaccccc 480
aagtactcca aggcacggta tgatgaaatt ataaaggagg tctcatccta tttaaagaag 540
gttggttaca acccagacaa gattcctttt gtacccatct ctgggtttga gggtgacaac 600
atgattgaga ggtccaccaa tctggactgg tacaagggac caacactcct cgaggccctt 660
gacatgatcc aggagcccaa gaggcccaca gacaagcctc ttcgtctccc tcttcaggat 720
gtttacaaga ttggtggcat tgggactgtc cctgttggcc gtgtcgagac tggcattctc 780
aagcctggta tggttgtcac atttggtcct actggtctca ccactgaagt taagtcagtg 840
gagatgcacc atgaggctct tcaagaggca ttaccaggtg ataatgttgg ctttaatgtg 900
aagaacgttg ctgtcaagga tatcaagcgt gggtttgttg cctccaactc caaggatgac 960
cctgccaggg aggcggctaa tttcacttct caggttatca tcgtgaacca ccttggtcag 1020
attggcaatg ggtatgctcc agttctcgac tgccacacca gccacattgc tgtgaagttt 1080
gctgagatcc ttaccaagat tgatagacgg tctggcaagg agcttgagaa ggagcccaag 1140
ttcttgaaga atggcgacgc agggtttgtt aagatgattc cgaccaagcc catggttgtc 1200
gagaccttct ctgcataccc gccacttgga cgatttgccg tgagagacat gcgacagacg 1260
gttgctgttg gagttatcaa gagcgtggag aagaaggatc caactggagc caaggtcact 1320
aaatctgctg caaagaaggg caag 1344
<210> 2
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<213> Cinnamomum camphora
<400> 2
atggcgggat tggcaccaga aggatcacag ttcgatgctc gtcaatatga tgccaaaatg 60
actgaattgc ttacaacaga tggccaagat tttttcacat cctatgatga agtttatgag 120
agtttcgact ctatgggtct gcaggagaac cttcttaggg gcatttatgc ttatggtttt 180
gagaagccat ctgcaatcca acaaaggggt attgtgccct tctgcaaggg acttgatgtc 240
attcagcagg cacaatctgg gactggcaag acagcaactt tctgctctgg aattctgcag 300
cagctagatt atgccctagt tgaatgtcag gccttggttc tcgctccaac tcgagagctt 360
gcacaacaaa ttgaaaaggt tatgagggca cttggtgatt atcttggtgt gaaggttcat 420
gcttgtgtag gtggtactag tgtccgtgaa gatcaacgca ttctttccag tggtgttcat 480
gttgttgttg gcactcctgg tcgtgttttt gacatgctgc gaagacagtc cctgcgtccc 540
gactacatta agatgtttgt actggatgag gctgacgaaa tgctttctcg aggatttaag 600
gatcagatat atgatatctt ccagcttctc ccgtcaaaag ttcaggttgg tgtcttctct 660
gctacgatgc cgcctgaggc gcttgaaatc actcgcaagt tcatgaacaa gcctgtgagg 720
attctggtga agagagatga gctcacccta gagggtatca agcagtttta tgtgaatgtt 780
gaaaaggaag aatggaagtt ggagacccta tgtgacctct acgagacatt ggccattaca 840
cagagtgtaa tatttgtgaa cactcgacgg aaggttgact ggctgactga taagatgcgc 900
agccgagacc acacagtctc tgccacccac ggagatatgg atcaaaataa tagggatata 960
attatgcgtg agttccgatc tggatcctca cgtgtactta tcacaactga tctgttggcc 1020
cgtggcattg atgtccagca ggtatcactt gtcataaatt atgatctgcc gactcagccg 1080
gaaaactatc tccatcgtat tggacgtagt ggtcgttttg gaaggaaggg tgttgcaatc 1140
aactttgtta ctcgtgatga tgaaaggatg ctgtttgata ttcagaaatt ctacaatgtg 1200
gtgatagagg agctgccatc aaatgttgct gacctcctc 1239
<210> 3
<211> 894
<212> DNA
<213> Cinnamomum camphora
<400> 3
atggcggtta ctgtcgcggg atgttggtgc tccatgtcat ttcccatgat ctcctactcc 60
cggcagagtg tatttcctgc tcgatctcct tcccttgctc gagtgaccac accgctaacc 120
ttaaccaaga catccctcca gtacagtggt tcaccctcgt ggacaggttt ggaagcaaat 180
gggtataggt ttggatatgg gtggaggatg aagagaagaa tcacaaccat gagctatgaa 240
gctggaattg gtgtcatggg aactaagttg ggcatgatga cttactttga gccggggggg 300
aaggtggtgc ctgtgactgt tgtcggtttc cgtgagggca acattgtcac tcaagtcaag 360
actcttgcta ctgatggata tgacgcagtc caggttggct accggagggt tcgagacaag 420
aagcttacca aaggggagct tggccatctc aacaagattg gtgccattcc tatgaggcat 480
cttcaggagt accggctggt gtcgattgat ggctttgagc ccaaccagaa gctcctccca 540
gaggaagtct tcaaggaagg agaccttgtt gatgttgctg gcacgtccat tggaaaggga 600
ttccaaggtg gaattaagag gcataatttc catcgcggac cgatgacgca tggttccaag 660
agtcaccgag ccattggatc tattggtgcc ggaacaacgc caggccgtgt atataaaggt 720
aagaagatgc caggtaggat gggagggacg aaaacaaaaa tccgcaagtt gcagattgta 780
aaaatagaca gtgagctccg tgttttcatg atcaagggtg cagtccctgg taagccaggt 840
aatcttcttc gcataacacc agcaaaaatt gtcggcaaga acataccaaa gaat 894
<210> 4
<211> 1131
<212> DNA
<213> Cinnamomum camphora
<400> 4
atggcagatg cagaggacat tcagcctctt gtttgtgata atggaacagg aatggtcaag 60
gctgggtttg ctggagatga tgcgccaagg gctgttttcc ctagcattgt gggtcgtcca 120
cgtcacactg gtgtgatggt tggtatgggt cagaaagatg catatgtagg agatgaagct 180
cagagtaaga gaggtatctt aaccctgaaa tacccaattg agcatggtat tgtgagcaac 240
tgggatgaca tggagaagat atggcatcac acattctaca acgagttgcg agtggccccg 300
gaagagcacc ccgttctcct cacggaggca cctcttaatc ctaaggccaa ctgtgagaag 360
atgactcaga tcatgtttga aacattcaat actcctgcta tgtatgttgc cattcaggct 420
gttctctccc tttatgccag tggccgcaca actggtattg tgctggactc tggggatggt 480
gtcagccaca cggtccctat ctatgaaggc tatgcacttc cacatgctat cctgcgtctt 540
gacctggcag gtcgcgacct aactgatgcc cttatgaaaa tactgacaga acgtgggtat 600
tccttcacca cgactgcaga gcgtgaaatt gtgagggaca tgaaggaaaa gctgtcatac 660
atcgcacttg actatgagca ggagctggaa actgccaaga ccagctcttc tgtggagaag 720
agctatgagc tgcctgatgg acaagtgatc actattggag ccgagcgttt ccgttgccct 780
gaggtcctct tccagccatc gatgattgga atggaagctg cagggataca tgagaccaca 840
tacaactcta tcatgaagtg tgatgtggat ataaggaagg atttgtatgg aaatattgtt 900
cttagcggtg ggtccaccat gttccctggt attgctgaca ggatgagcaa ggagatcaca 960
gcactggcac ctagcagcat gaagatcaag gttgttgcac cccctgagag gaagtacagt 1020
gtgtggatag gaggctctat tttggcatcg ctcagcacct ttcagcagat gtggattgcg 1080
aaggcggagt atgatgagtc tgggccatca attgtacaca ggaaatgctt c 1131
<210> 5
<211> 1338
<212> DNA
<213> Cinnamomum camphora
<400> 5
atgagagaaa tcctccacat tcaaggcggg cagtgcggaa accagatcgg atccaagttc 60
tgggaggtcg tctgcgacga gcacggcata gatccgacgg gtcgctacac cggaacctcg 120
gatctgcagc tggagcgcgt caatgtctac tacaacgagg cttcctgcgg acgattcgtt 180
ccaagagctg tgctcatgga tctggagcca ggaaccatgg acagcgtacg gacgggcccg 240
tacggacaga tcttccggcc cgacaacttc gtgttcgggc agtcgggcgc cgggaacaac 300
tgggcaacgg ggcactacac cgagggagct gagctcatcg attcggtgct cgatgtcgtc 360
aggaaagagg ccgagaactg cgattgcctt caagggtttc aggtgtgcca ctcgcttggt 420
ggtggaactg gatctgggat gggaaccttg ctgatatcaa agatcaggga agagtatcct 480
gatcggatga tgctcacatt ctcggtgttt ccgtcgccta aggtttctga tactgtggtt 540
gagctgtaca atgccactct ctctgttcat cagctggtgg agaatgcaga tgagtgcatg 600
gtgttggaca atgaggctct gtatgatatc tgcttcagaa ctctgaagct aaccactccc 660
agctttgggg atctaaatca cttgatatct gcaaccatga gtggtgtcac ctgctgcctg 720
aggttccctg gacagctgaa ctcagacctc cgaaagcttg ctgtcaattt gattcccttt 780
ccccgtctcc acttctttat ggttgggttt gctcccctaa cttcgcgtgg ctctcagcag 840
tatcgtgccc tgacggtccc tgagcttacc cagcagatgt gggattcgaa aaacatgatg 900
tgtgctgctg accctcgcca tggacggtac ttgactgcct cggccatgtt ccgtggcaaa 960
atgagcacca aggaggttga tgagcagatg atcaatgtgc agaataagaa ctcgtcttac 1020
tttgttgaat ggattccaaa taatgtgaag tcaagcgtct gtgacatccc accaaagggg 1080
ctttccatgg catcaacctt cattggcaat tctacctcga tccaggagat gttccgcagg 1140
gtgtcggagc agttcactgc catgttcagg aggaaggcct tcttgcattg gtacacagga 1200
gagggaatgg atgagatgga gttcacagaa gctgagagca acatgaacga tctggtgtct 1260
gaataccagc agtaccagga tgcaactgct gatgaggagg gtgagtatga ggatgaggaa 1320
gaagaggtcc aggatatg 1338
<210> 6
<211> 25
<212> DNA
<213> Artificial Synthesis
<400> 6
tactgcactg ttattgatgc ccctg 25
<210> 7
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 7
gagtgaaggc aagcaaagca tgc 23
<210> 8
<211> 25
<212> DNA
<213> Artificial Synthesis
<400> 8
tggttttgag aagccatctg caatc 25
<210> 9
<211> 25
<212> DNA
<213> Artificial Synthesis
<400> 9
ggcctgacat tcaactaggg cataa 25
<210> 10
<211> 24
<212> DNA
<213> Artificial Synthesis
<400> 10
ggtttggata tgggtggagg atga 24
<210> 11
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 11
<210> 12
<211> 25
<212> DNA
<213> Artificial Synthesis
<400> 12
accctgaaat acccaattga gcatg 25
<210> 13
<211> 25
<212> DNA
<213> Artificial Synthesis
<400> 13
ttctcacggt tggccttagg attaa 25
<210> 14
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 14
<210> 15
<211> 24
<212> DNA
<213> Artificial Synthesis
<400> 15
tgagctcagc cccctcagtg tagt 24
Claims (7)
1. The application of the reference gene in fluorescence quantitative PCR of different tissues of cinnamomum camphora is characterized in that: the internal reference gene isCcEF-1α、CcEIF-4α、CcRPL3、CcACT11、CcTUB；
The above-mentionedCcEF-1αThe nucleotide sequence of (A) is shown as a sequence table SEQ ID NO. 1;
the above-mentionedCcEIF-4αThe nucleotide sequence of (A) is shown as a sequence table SEQ ID NO. 2;
the above-mentionedCcRPL3The nucleotide sequence of (A) is shown as a sequence table SEQ ID NO. 3;
the above-mentionedCcACT11The nucleotide sequence of (A) is shown as a sequence table SEQ ID NO. 4;
the above-mentionedCcTUBThe nucleotide sequence of (A) is shown in a sequence table SEQ ID NO. 5.
2. Use according to claim 1, characterized in that: the different tissues comprise camphor leaves, stems and fruits at different development stages.
3. The reference gene which is developed based on cinnamomum camphora transcriptome sequencing data and stably expressed in different tissues of cinnamomum camphora is characterized in that: the internal reference gene isCcEF-1α、CcEIF-4α、CcRPL3、CcACT11、CcTUB；
The above-mentionedCcEF-1αThe nucleotide sequence of (A) is shown as a sequence table SEQ ID NO. 1;
the above-mentionedCcEIF-4αThe nucleotide sequence of (A) is shown as a sequence table SEQ ID NO. 2;
the above-mentionedCcRPL3The nucleotide sequence of (A) is shown as a sequence table SEQ ID NO. 3;
the above-mentionedCcACT11The nucleotide sequence of (A) is shown as a sequence table SEQ ID NO. 4;
the above-mentionedCcTUBThe nucleotide sequence of (A) is shown in a sequence table SEQ ID NO. 5.
4. The primer for reference gene according to claim 3, characterized in that:
for amplificationCcEF-1αThe sequence of the primer pair is SEQ ID NO.6/SEQ ID NO. 7;
for amplificationCcEIF-4αThe sequence of the primer pair is SEQ ID NO.8/SEQ ID NO. 9;
for amplificationCcRPL3The sequence of the primer pair is SEQ ID NO.10/SEQ ID NO. 11;
for amplificationCcACT11The sequence of the primer pair is SEQ ID NO.12/SEQ ID NO. 13;
for amplificationCcTUBThe sequence of the primer pair of (1) is SEQ ID NO.14/SEQ ID NO. 15.
5. The screening method of the fluorescence quantitative PCR reference gene in different tissues of cinnamomum camphora is characterized in that: the method comprises the following steps:
respectively selecting camphor leaf, stem and fruits at different development stages after blooming as samples; extracting total RNA of each sample, and synthesizing cDNA through reverse transcription of the RNA, wherein the cDNA is used as a template for target gene amplification;
step two, screening out candidate reference genes of the cinnamomum camphora by utilizing early cinnamomum camphora transcriptome sequencing data, wherein the candidate reference genes are respectively as follows:CcEF-1 α、CcEIF-4α、CcRPL3、CcACT11、CcTUB(ii) a Wherein, theCcEF-1αThe nucleotide sequence of (A) is shown as SEQ ID NO.1CcEIF-4αThe nucleotide sequence of (A) is shown as SEQ ID NO.2CcRPL3The nucleotide sequence of (A) is shown as SEQ ID NO.3CcACT11The nucleotide sequence of (A) is shown as SEQ ID NO.4CcTUBThe nucleotide sequence of (A) is shown as SEQ ID NO. 5;
thirdly, designing a reference gene primer of the real-time fluorescence quantitative PCR by taking the selected candidate reference gene sequence as a template, wherein the amplification fragment size of the primer is 150-200 bp; wherein, for amplificationCcEF-1αThe primer pair has the sequence of SEQ ID NO.6/SEQ ID NO.7 and is used for amplificationCcEIF-4αThe primer pair has the sequence of SEQ ID NO.8/SEQ ID NO.9 and is used for amplificationCcRPL3The primer pair has the sequence of SEQ ID NO.10/SEQ ID NO.11 and is used for amplificationCcACT11The primer pair has the sequence of SEQ ID NO.12/SEQ ID NO.13 and is used for amplificationCcTUBThe sequence of the primer pair is SEQ ID NO.14/SEQ ID NO. 15;
step four, real-time fluorescence quantitative PCR: taking cDNA obtained by reverse transcription in the first step as a template, and carrying out real-time fluorescence quantitative PCR amplification on the candidate reference gene by adopting a primer in the third step; and performing statistical analysis on the obtained real-time fluorescence quantitative PCR data, and respectively screening out the optimal reference gene and the optimal reference gene combination.
6. The screening method according to claim 5, wherein: before the real-time fluorescent quantitative PCR of the step four, the specificity of the reference gene primer is identified through common PCR.
7. The screening method according to claim 5, wherein: and in the fourth step, performing statistical analysis on the obtained real-time fluorescence quantitative PCR data through three kinds of software, namely GeNorm software, NormFinder software and BestKeeper software, respectively screening out the optimal reference genes and reference gene combinations, and then performing comprehensive ranking analysis by using RankAggreg R analysis software.
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