CN113604599B - Fluorescent quantitative PCR reference gene stably expressed in different tissues of Cinnamomum camphora and application thereof - Google Patents

Abstract

The invention relates to a fluorescent quantitative PCR reference gene stably expressed in different tissues of Cinnamomum camphora and application thereof, belonging to the field of molecular biology, wherein the reference gene isCcEF‑1α、CcEIF‑4α、 CcRPL3、CcACT11、CcTUBIs 5 stably expressed internal reference genes developed based on the Cinnamomum camphora transcriptome sequencing data. The invention uses qRT-PCR technology, takes camphor leaves, stems and fruits at different development stages as materials, uses GeNorm, normFinder, bestKeeper kinds of software to evaluate gene stability, and finally uses Rankaggreg to comprehensively rank to obtainCcEF‑1αIs the reference gene which is most stably expressed in each tissue sample of the camphor. The invention provides an effective reference gene tool for screening, separating and expressing analysis of the camphor functional genes in the future, and is suitable for popularization and application in the reference gene field.

Description

Fluorescent quantitative PCR reference gene stably expressed in different tissues of Cinnamomum camphora and application thereof

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a fluorescent quantitative internal reference gene of different tissues of camphor, and a primer and application thereof.

Background

Cinnamomum camphora (L.) PreslCinnamomum camphora) Also called camphor tree, black camphor belonging to genus LauraceaeCinnamomum). In recent years, cinnamomum camphora plays an important role in urban landscaping, and in addition, cinnamomum camphora is also an important economic tree species due to its medicinal and aromatic properties. Essential oils or crystals extracted from Cinnamomum camphora are sources of food preservatives and additives, as well as raw materials for the cosmetic and pharmaceutical industries, having 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 technique, and we have screened some reference genes using the transcriptome data of Cinnamomum camphora, which will greatly promote the further research of Cinnamomum camphora gene expression.

Fluorescent quantitative PCR (quantitative Real time PCR, qRT-PCR) is the most commonly used technique for quantifying the level of gene expression, and is used in many research fields due to its high accuracy, high specificity, high throughput and other characteristics. In research of plant reference genes, the expression of homologous reference genes among different species is not constant, and the selection of unsuitable reference genes leads to deviation of the relative expression amount of target 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, few reports are about the screening and developing of the Cinnamomum camphora reference genes by utilizing transcriptome data, so that 5 Cinnamomum camphora reference genes are screened based on Cinnamomum camphora transcriptome sequences.

Disclosure of Invention

Aiming at the problems, the invention provides an effective reference gene tool for screening, separating and expression analysis of the functional genes of the camphor tree in the future by carrying out reference gene development, selecting camphor tree leaves, stems and 6 stable reference genes and primers thereof for researching fruits at different development stages through a camphor tree transcriptome sequencing database.

The invention adopts the following specific scheme:

the invention aims at providing fluorescent quantification of 5 internal reference genes in different tissues of Cinnamomum camphoraUse in PCR 5 of the internal reference genesCcEF-1α、CcEIF-4α、CcRPL3、CcACT11、CcTUBThe nucleotide sequence is shown as SEQ ID NO. 1-5. Further, the different tissues include camphor leaves, stems and fruits at different stages of development.

The second object of the invention is to provide an internal reference gene which is developed based on the sequence data of the Cinnamomum camphora transcriptome and stably expressed in different tissues of Cinnamomum camphora.

The third object of the present invention is to provide a primer for amplifying the above-mentioned reference gene.

The invention aims at providing a screening method of fluorescent quantitative PCR reference genes in different tissues of Cinnamomum camphora, comprising the following steps:

step one, respectively selecting the fruits of the camphor leaves, stems and different development stages after flowering as samples; extracting total RNA of each sample, synthesizing cDNA by RNA reverse transcription, and taking the total RNA as a template for amplifying a target gene;

step two, screening out candidate internal reference genes of the camphor tree by utilizing the sequencing data of the pre-camphor tree transcriptome, wherein the candidate internal reference genes are respectively:CcEF-1α、CcEIF-4α、CcRPL3、CcACT11、CcTUBthe method comprises the steps of carrying out a first treatment on the surface of the Wherein the saidCcEF-1αThe nucleotide sequence of (2) is shown as SEQ ID NO.1, theCcEIF-4αThe nucleotide sequence of (2) is shown as SEQ ID NO.2, theCcRPL3The nucleotide sequence of (2) is shown as SEQ ID NO.3, theCcACT11The nucleotide sequence of (2) is shown as SEQ ID NO.4, theCcTUBThe nucleotide sequence of (2) is shown as SEQ ID NO. 5;

step three, designing an internal reference gene primer of real-time fluorescence quantitative PCR by taking the selected candidate internal reference gene sequence as a template, wherein the amplified fragment size of the primer is 150-200bp; wherein, for amplification ofCcEF-1αThe primer pair sequence of (2) is SEQ ID NO.6/SEQ ID NO.7, and is used for amplificationCcEIF-4αThe primer pair sequence of (2) is SEQ ID NO.8/SEQ ID NO.9, and is used for amplificationCcRPL3The primer pair sequence of (1) is SEQ ID NO.10/SEQ ID NO.11 for amplificationCcACT11The primer pair sequence of (1) is SEQ ID NO.12/SEQ ID NO.13, and is used for amplificationCcTUBThe primer pair sequence of (a) is SEQ ID NO.14/SEQ ID NO.15;

step four, real-time fluorescence quantitative PCR: carrying out real-time fluorescence quantitative PCR amplification on candidate internal reference genes by using the cDNA obtained by reverse transcription in the step one as a template and adopting the primer pair in the step three; and carrying out statistical analysis on the obtained real-time fluorescence quantitative PCR data, and respectively screening out the optimal internal reference genes and the combination of the internal reference genes.

As a further optimization of the screening method, the specificity of the internal reference gene primer was first identified by ordinary PCR before the real-time fluorescent quantitative PCR of step four was performed.

In the fourth step, the obtained real-time fluorescence quantitative PCR data are subjected to statistical analysis by using three kinds of software GeNorm, normFinder, bestKeeper to respectively screen out the optimal internal reference genes and the combination of the internal reference genes, and then comprehensive ranking analysis is performed by using RankAggreg R analysis software.

The invention differs from the prior art in that:

(1) The invention screens out the most stable reference genes in the camphor leaves, stems and 6 fruits in the development stages from 5 candidate reference genes, the data is true and reliable, and a foundation is laid for deep excavation of functional genes in the later stage;

(2) The screened internal reference genes are suitable for the expression analysis of the camphor critical genes, can obviously improve the accuracy of the obtained data, and have wide application, high sensitivity and good stability;

(3) The research determines stable reference genes applicable to different tissues of the camphor through strict screening reference gene programs, and provides a theoretical basis for selecting proper reference genes in qRT-PCR analysis of the camphor.

Drawings

FIG. 1 is a sequence electrophoresis chart obtained by amplifying 5 internal reference gene primers in example 2 by using Cinnamomum camphora cDNA as a template; wherein M: DNA Marker, the sequence of the band size is 250bp, 500bp, 750bp, 1000bp and the like from bottom to top, and genes represented from left to right in each lane are respectivelyCcEF-1α、CcEIF-4α、CcRPL3、CcACT11、CcTUB；

FIG. 2 is a graph showing Ct value distribution of fluorescent quantitative PCR for 5 reference genes in example 2;

FIG. 3 is a graph showing the dissolution profile of 5 reference genes in example 2;

FIG. 4 shows the expression stability values of 5 candidate reference genes using the GeNorm program in example 2M) Pairing difference valueV），MThe smaller the value, the better the stability of the reference gene; wherein fig. 4 (a) uses data mixed by all samples as materials; FIG. 4 (B) with 6 stage fruits as material; FIG. 4 (C) is a view of leaves and stems; fig. 4 (D) is a pairing difference value distribution under each condition;

FIG. 5 is a comprehensive ranking of three statistical analysis methods using the RankAggreg R package in example 2; FIG. 5A is a graph of data mixed for all samples; FIG. 5B is a 6 stage fruit of development as material; fig. 5C uses leaves and stems as materials.

Detailed Description

Herein, the following is the case: the Cinnamomum camphora transcriptome sequencing data: source(s)https://www.ncbi.nlm.nih.gov/sra/ SRX1091098[accn]

GeNorm reference: v-andesompele, J., pre, K.D., pattyn, F., poppe, B., roy, N.V., paepe, A.D., et al Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes [ J ]. Genome biol 2002, 3, research0031-research0034.

NormFinder reference: andersen, C.L., jensen, J.L., and Orntoft, T.F. Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon Cancer data sets [ J ]. Cancer Res. 2004, 64, 5245-5250.

BestKeeper reference: pfaffl, M.W., tichopad, A., prgomet, C., andNEUvirs, T.P. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity, bestKeeper-Excel-based tool using pair-wise correlations [ J ]. Biotechnol. Lett. 2004, 26, 509-515.

RankAggreg R package see: najafpanah, M.J., sadeghi, M. and Bakhtiarizadeh, M.R. Reference genes selection for quantitative real-time PCR using RankAggreg method in differenttissues of Capra hircus. PLoS ONE.2013, 8 (12): e83041.

The technical problems solved by the invention are realized by adopting the following technical scheme:

an internal reference gene of camphor, which isCcEF-1α、CcEIF-4α、CcRPL3、CcACT11、CcTUBThe method comprises the steps of carrying out a first treatment on the surface of the The saidCcEF-1αThe nucleotide sequence of (2) is SEQ ID NO.1; the saidCcEIF-4αThe nucleotide sequence of (2) is SEQ ID NO.2; the saidCcRPL3The nucleotide sequence of (2) is SEQ ID NO.3; the saidCcACT11The nucleotide sequence of (2) is SEQ ID NO.4; the saidCcTUBThe nucleotide sequence of (2) is SEQ ID NO.5.

The method is used for amplificationCcEF-1αThe primer pair sequence of (a) is SEQ ID NO.6/SEQ ID NO.7; for amplification ofCcEIF-4αThe primer pair sequence of (a) is SEQ ID NO.8/SEQ ID NO.9; for amplification ofCcRPL3The primer pair sequence of (a) is SEQ ID NO.10/SEQ ID NO.11; for amplification ofCcACT11The primer pair sequence of (1) is SEQ ID NO.12/SEQ ID NO.13; for amplification ofCcTUBThe primer pair sequence of (C) is SEQ ID NO.14/SEQ ID NO.15.

The real-time fluorescence quantitative PCR screening method for the internal reference gene of the camphor tree comprises the following steps:

(1) Selecting camphor leaves, stems and 6 fruits at different development stages as materials;

(2) Screening out internal reference genes of the Cinnamomum camphora by utilizing Cinnamomum camphora transcriptome sequencing data;

(3) Designing an internal reference gene primer of real-time fluorescence quantitative PCR by taking the selected internal reference gene sequence as a template;

(4) Carrying out conventional PCR (polymerase chain reaction) 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 5-fold dilution series (1 x,5x,25x,125x,625x,3125 x) of mixed cDNA of each sample of Cinnamomum camphora as template;

(6) Performing real-time fluorescent quantitative PCR; and (3) carrying out statistical analysis on the obtained real-time fluorescence quantitative PCR data through three pieces of statistical software GeNorm, normFinder, bestKeeper, respectively screening out optimal internal reference genes and internal reference gene combinations, and then carrying out comprehensive ranking analysis through comprehensive analysis software RankAggreg R.

Preferably, the specificity of the internal reference gene primer is identified by conventional PCR (using DNA polymerase: rTaq Mix supplied by TAKARA; identification conditions: 95℃for 5min,95℃for 30s,55℃for 30s,72℃for 60s/kb,30 cycles; 72℃for 10 min) before performing the real-time fluorescent quantitative PCR in said step (4).

Preferably, in the step (4), the real-time fluorescent quantitative PCR amplification procedure is: pre-denaturation at 95℃for 3min; denaturation at 95℃for 5s, annealing at 55℃for 30s, running 40 cycles, dissolution profile analysis at 65℃to 95 ℃.

Preferably, the real-time fluorescence quantitative PCR screening method for the internal reference gene of the camphor tree comprises the following specific steps:

1) And (3) material collection: the leaves, stems and 6 fruits of Cinnamomum camphora in the Donghu province of Zhejiang agricultural and forestry university are used as experimental materials, and the fruits are quickly frozen in liquid nitrogen after being collected and stored in a refrigerator at the temperature of minus 80 ℃. The fruit numbers of the 6 development stages are S1, S2, S3, S4, S5 and S6;

2) RNA extraction and detection: extracting total RNA of each sample by using an EASYspin Plus polysaccharide polyphenol complex plant RNA extraction kit (Edley, china) (the extraction mode is consistent with that in the specification of the kit), detecting the integrity of the extracted RNA by electrophoresis, performing electrophoresis for 20min at 100V voltage by using a TAE buffer solution, and observing and analyzing by using a gel imaging system (Tanon-2500); the purity of the extracted RNA is detected by using NanoDrop ND-2000 (OD 260/280 of the pure RNA is between 1.8 and 2.2);

3) RNA reverse transcription to synthesize cDNA: after detection by non-denaturing agarose gel electrophoresis, 1. Mu.g of Cinnamomum camphora RNA sample was taken and SuperScript was used ^® III First-Strand Synthesis System kit (Thermo, USA) for reverse transcription synthesis of cDNA as template for amplification of target gene;

4) Screening candidate reference genes: screening out candidate internal reference genes by utilizing the sequence data of the Cinnamomum camphora transcriptome and the homologous genes of the reference model plant Arabidopsis thaliana;

5) Designing and detecting a specific primer: designing internal reference gene primers of real-time fluorescence quantitative PCR by using the screened candidate internal reference gene sequences as templates and utilizing Vector NTI software, wherein the size of amplified fragments is 150-200bp (see Table 1 for details); the cDNA obtained by reverse transcription of the camphor tree is used as a template, and the specificity of the primer is initially identified by using common PCR. After electrophoresis, observing the bands of the PCR product under a gel imaging system, wherein the sizes of the bands are correct as shown in figure 1, the bands are single, and primers of primer dimer are not present;

6) Establishing an internal reference gene primer standard curve: taking a 5-fold dilution series (1 x,5x,25x,125x,625x,3125 x) of mixed cDNA of different samples as a template for establishing a standard curve, and designing 3 technical repeats for each reaction of the real-time fluorescence quantitative PCR;

7) Fluorescent quantitative PCR amplification: performing real-time fluorescent quantitative PCR amplification on the reference genes by taking cDNA (complementary deoxyribonucleic acid) obtained by reverse transcription of 8 samples as a template to obtain corresponding genesCtA value; the amplification procedure was: fluorescent signals are collected for 40 cycles at 95 ℃ for 3min,95 ℃ for 5s and 55 ℃ for 30s, and dissolution curves are 65-95 ℃. And (3) collecting melting curve signals, wherein the generated melting curve is a single peak, which indicates that the primer specificity is good, ct value data is automatically read by a real-time fluorescent quantitative PCR instrument, and the distribution range of Ct values is shown in figure 2. The Ct value is inversely proportional to the expression level of the gene, and the larger the Ct value is, the lower the expression level of the gene is, whereas the smaller the Ct value is, the higher the expression level of the gene is; drawing a standard curve by using the obtained fluorescent quantitative PCR result, and obtaining the amplification efficiency and slope of each candidate gene as shown in table 1;

8) Evaluating the expression stability by using GeNorm, normFinder, bestKeeper statistical analysis methods;

9) The stability of candidate internal reference genes obtained by 3 statistical analysis methods is comprehensively counted and ranked by using a RankAggreg R program package, and a comprehensive result is obtained;

the invention is further described in connection with the following detailed description, but the scope of the invention is not limited thereto; the equipment and reagents used in the examples were all conventionally commercially available unless otherwise specified.

Example 1: candidate reference gene and primer design thereof

Based on the prior transcriptome data, 5 candidate internal reference genes were co-analyzed and identified in this study (table 1), respectively:CcEF-1α、CcEIF-4α、CcRPL3、CcACT11、CcTUBthe method comprises the steps of carrying out a first treatment on the surface of the The saidCcEF-1αThe nucleotide sequence of (2) is SEQ ID NO.1; the saidCcEIF-4αThe nucleotide sequence of (2) is SEQ ID NO.2; the saidCcRPL3The nucleotide sequence of (2) is SEQ ID NO.3; the saidCcACT11The nucleotide sequence of (2) is SEQ ID NO.4; the saidCcTUBThe nucleotide sequence of (2) is SEQ ID NO.5. Designing qRT-PCR primer with Vector NTI software, wherein the amplified fragment size of the primer is 150-200bp; the method is used for amplificationCcEF-1αThe primer pair sequence of (a) is SEQ ID NO.6/SEQ ID NO.7; for amplification ofCcEIF-4αThe primer pair sequence of (a) is SEQ ID NO.8/SEQ ID NO.9; for amplification ofCcRPL3The primer pair sequence of (a) is SEQ ID NO.10/SEQ ID NO.11; for amplification ofCcACT11The primer pair sequence of (1) is SEQ ID NO.12/SEQ ID NO.13; for amplification ofCcTUBThe primer pair sequence of (C) is SEQ ID NO.14/SEQ ID NO.15. The internal reference gene primers are shown in Table 1.

TABLE 1 qRT-PCR reference genes and primer sequences thereof.

Example 2: and (5) analyzing the stability of the candidate reference genes.

The method specifically comprises the following steps:

1) Total RNA extraction and cDNA Synthesis

The collected samples (camphor leaves, stems, 6 fruits at different development stages) were ground into powder with liquid nitrogen. Total RNA of each sample was extracted using an EASYspin Plus polysaccharide polyphenol complex plant RNA extraction kit (Edley, china), and the specific method is referred to in the specification. The integrity of the extracted RNA was checked by electrophoresis, 1% agarose gel, TAE buffer, electrophoresis at 100V for 20min, and observation and analysis by gel imaging system (Tanon-2500); the purity and concentration of the extracted RNA were checked using NanoDrop ND-2000 (OD 260/280 of pure RNA was between 1.8 and 2.2). After detection by non-denaturing agarose gel electrophoresis, 1. Mu.g of Cinnamomum camphora RNA sample was taken and SuperScrip was usedt ^® The III First-Strand Synthesis System kit (Thermo, USA) was used for reverse transcription to synthesize cDNA as a template for amplification of the target gene. Specific methods referring to the instructions, the synthesized cDNA was stored in a-20℃refrigerator. The mixing comprises the following specific steps: firstly, preparing RNA with total volume of 20 mu L, including 1 mu L L Oligo dT Primer,1 mu L Dntp Mixture and total volume of below 5 mu g, into a microtube, and supplementing RNase free dH ₂ O to 10 mu L, incubating a microcentrifuge tube filled with the mixed solution at 65 ℃ for 5min, and then placing the microcentrifuge tube on ice for rapid cooling to promote RNA denaturation and improve reverse transcription efficiency; preparing a reaction solution in the same microcentrifuge tube, wherein the reaction solution comprises 10 mu L of RNA and a denaturation primer, 4 mu L of 5 XPrmeScript II Buffer,0.5 mu L of L RNase Inhibitor,1 mu L of PrmeScript II Rtase and RNase free dH supplementation ₂ O to 20 mu L, incubating the reaction solution at 50 ℃ for 60min to extend cDNA, incubating at 95 ℃ for 5min, cooling on ice, and storing in a refrigerator at-20 ℃ for standby.

2) Detection of candidate reference gene specific primers

The cDNA obtained by reverse transcription of the camphor tree is used as a template, and the specificity of the primer is initially identified by using common PCR. After electrophoresis, the bands of the PCR products were observed under a gel imaging system, and each band was the correct size as shown in FIG. 1, with a single band and no primer of primer dimer.

3) Real-time fluorescence quantitative PCR (qRT-PCR)

The total qRT-PCR reaction was 25. Mu.L, including 12.5. Mu. L TB Green Premix Ex Taq (Takara Shuzo, beijing), 2. Mu.L cDNA, 0.5. Mu.L forward and reverse primers (5 candidate reference genes and their primers are shown in Table 1) and 9.5. Mu.L ddH2O, three technical replicates per sample. qRT-PCR reactions were performed on a CFX96 real-time PCR instrument (Bio-Rad, USA), pre-denatured at 95℃for 3min; denaturation at 95℃for 5s, annealing at 55℃for 30s, running 40 cycles, dissolution profile analysis at 65℃to 95 ℃. Ct values are automatically obtained by the instrument after the reaction is completed, 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 in a software program based on Microsoft Excel 2013Obtaining the product. According to the formula q=2 ^-ΔCt Calculating the relative expression quantity Q of each sample, wherein deltaCt=Ct _Sample- Ct _min ，Ct _min Representing allCtMinimum in the sample. The GeNorm program is to obtain the average variation degree of each reference gene relative to the Q value of the expression quantityMAccording to the value ofMThe values and sizes are subjected to gene stability sorting,Mthe smaller the value, the higher the gene stability. At the same time, a paired variation value (pairwise variation) V is provided _n/n+1 The optimum number of reference genes required under the experimental conditions was determined to reduce the bias and fluctuations caused by the use of a single reference gene. n represents the number of reference genes to be selected, and the paired difference value V _n/n+1 <0.15 is the threshold value of the recommended selection (see fig. 4). Different from the GeNorm calculation method, normFinder directly evaluates the expression stability of the reference gene based on variance analysis, can determine the variation degree in and among groups, and calculates the gene expression stability value #SA value),Sthe smaller the value, the more stable the gene (see Table 2). While the bestdeeper program evaluates the stability index of the reference gene based on the expression calculated from Standard Deviation (SD) and percent Covariance (CV) values (see table 3).

In order to provide a comprehensive result, rankAggreg software was used to rank the optimal list of reference genes,CcEF-1αthe overall stability of the gene is ranked first and the stability is best (see FIG. 5).

Table 2 differs from the NormFinder scores for the reference genes.

Table 3 shows the Bestkeeper scores for the reference genes.

It should be noted that the above-mentioned embodiments are to be understood as illustrative, and not limiting, the scope of the invention, which is defined by the appended claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made to the present invention without departing from its spirit or scope.

SEQUENCE LISTING

<110> Zhejiang university of agriculture and forestry, changzhou biological technology Co., ltd

<120> fluorescent quantitative PCR reference gene stably expressed in different tissues of Cinnamomum camphora and application thereof

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<160> 15

<170> PatentIn version 3.3

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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> Synthesis

<400> 6

tactgcactg ttattgatgc ccctg 25

<210> 7

<211> 23

<212> DNA

<213> Synthesis

<400> 7

gagtgaaggc aagcaaagca tgc 23

<210> 8

<211> 25

<212> DNA

<213> Synthesis

<400> 8

tggttttgag aagccatctg caatc 25

<210> 9

<211> 25

<212> DNA

<213> Synthesis

<400> 9

ggcctgacat tcaactaggg cataa 25

<210> 10

<211> 24

<212> DNA

<213> Synthesis

<400> 10

ggtttggata tgggtggagg atga 24

<210> 11

<211> 20

<212> DNA

<213> Synthesis

<400> 11

gccctcacgg aaaccgacaa 20

<210> 12

<211> 25

<212> DNA

<213> Synthesis

<400> 12

accctgaaat acccaattga gcatg 25

<210> 13

<211> 25

<212> DNA

<213> Synthesis

<400> 13

ttctcacggt tggccttagg attaa 25

<210> 14

<211> 20

<212> DNA

<213> Synthesis

<400> 14

gttcccaggg ccgtgctcat 20

<210> 15

<211> 24

<212> DNA

<213> Synthesis

<400> 15

tgagctcagc cccctcagtg tagt 24

Claims (3)

1. The application of the reference gene in the fluorescence quantitative PCR of different tissues of the camphor tree is characterized in that: the reference gene isCcRPL3The method comprises the steps of carrying out a first treatment on the surface of the The saidCcRPL3The nucleotide sequence of (2) is shown as SEQ ID NO.3 of the sequence table.

2. The use according to claim 1, characterized in that: the different tissues comprise camphor leaves, stems and fruits at different development stages.

3. The use according to claim 1, characterized in that: the primers for amplifying the reference genes are as follows:

for amplification ofCcRPL3The primer pair sequences of (a) are SEQ ID NO.10 and SEQ ID NO.11.