CN105505923B - The cloning process of tobacco 25S RNA reference gene and its application - Google Patents
The cloning process of tobacco 25S RNA reference gene and its application Download PDFInfo
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Abstract
The invention belongs to technical field of molecular biology, and in particular to tobacco 25S RNA(25S ribosomal RNA) reference gene sequence, cloning process and its application.The partial nucleotide sequence of present invention 25S rna gene obtained can be used as the reference gene of research tobacco functional gene or different tissues, different growth and development stages and the gene expression analysis under hormon treatment conditions.The real-time fluorescence quantitative PCR primer amplification high specificity designed using the reference gene can be improved stability, the reproducibility and reliability of the research of tobacco gene expression analysis.
Description
Technical field
The invention belongs to a kind of internal references needed for technical field of molecular biology more particularly to tobacco gene expression process
Gene 25S RNA.
Background technique
Tobacco (Nicotianatabacum L.) it is Solanaceae Nicotiana, the leaf containing nicotine (Nicotine) was with 1 year
Raw crop.The mankind use about 1500 history of tobacco, as the important source material of tobacco industry, after blade is harvested by
Modulation, classification, working process, are made cigarette, cigar, pipe tobacco, chewing tobacco and snuff etc., thermophilic for people.Tobacco is classical base
Because of one of engineering research mode crop, however, the alternative reference gene and few during analyzing tobacco gene expression
See.Currently, more GAPDH, EF-1a of having is reported in document, and the widely used reference gene in other plant, in tobacco
In explicitly do not cloned and verified.Simultaneously as there is not versatility in the selection of reference gene, i.e., between different plant species
Having differences property of reference gene.This requires using the reference gene in other plant in tobacco, to be opened according to requirement of experiment
Open up necessary estimation of stability and identification.As biochip technology is more and more mature, a large amount of microarray data is in new
The excavation of ginseng gene provides good data basis and platform, provides more extensive space for the selection of reference gene.
Compared with the reference gene that traditional sequence analysis method is obtained, by gene chip expression technology screening to internal reference have
A large amount expression and the characteristic for stablizing expression.
Real-time fluorescence quantitative PCR (real-time quantitative reverse transcription PCR,
It qRT-PCR is) one kind in DNA amplification reaction, product is total after surveying each polymerase chain reaction circulation with fluorescent chemical
The method of amount.It is research means important in present molecular biology, qRT-PCR using qRT-PCR analysis gene expression dose
Have the characteristics that quantitatively accurate, high sensitivity and high throughput.In qRT-PCR, the analysis to target gene relative expression quantity is
It is determined by the homogenization of reference gene.Therefore, the selection of reference gene is the committed step of qRT-PCR.One ideal
Reference gene should be stablize expression in different developmental phases and different tissues organ, and expression quantity not by other it is extraneous because
The influence of element, experiment process condition.So far, especially desirable reference gene is not yet found.Therefore, researcher must combine
Respective experiment condition and sample type select suitable reference gene.
25S ribosomes (25S ribosomal RNA) gene is common reference gene, due to the synthesis of rRNA
Independently of mRNA, gene transcription level stability is more preferable, and therefore, 25S RNA becomes the expression point of many animal-plant genes
The important selecting object of reference gene in analysis.
Summary of the invention
The present invention provides tobacco 25S rna gene partial sequence, can be used as the application of tobacco reference gene.And it provides simultaneously
The specific primer of tobacco 25S rna gene is cloned, the qRT-PCR method based on SYBR Green fluorescent dye technology is established,
To the reference gene as tobacco 25S RNA, to be mentioned using real-time fluorescence quantitative PCR to the research of tobacco gene expression analysis
For useful method.
To achieve the above object, the invention adopts the following technical scheme:
A kind of cloning process of tobacco 25S rna gene sequence, not by 99 to tobacco difference growth and development period
Chip expression data with tissue samples are analyzed, and the coefficient of variation (coefficient for stablizing expression in all samples is filtered out
Of variation, CV) lesser composing type 25S rna gene sequence, with primer pair 25S RNA-F/25S RNA-R progress
PCR amplification obtains positive colony, verifies by plasmid order-checking.
Wherein primer pair sequence is as shown in SEQ ID NO.1/SEQ ID NO.2.
Target fragment obtained such as SEQ ID NO.3, size 459bp.
The reaction condition of the PCR amplification is as follows: 94 DEG C of initial denaturations 4 min, 94 DEG C of denaturation 30 s, 56 DEG C of 30 s of annealing,
72 DEG C of 40 s of extension, totally 25 recycle.Extend 10 min, 4 DEG C of preservations eventually for 72 DEG C again.
The present invention also provides a kind of methods of qRT-PCR amplification tobacco 25S rna gene partial sequence, with 25S RNA's
Based on gene order, according to the principle of qRT-PCR design of primers, a pair of of fluorescent quantitation special primer is designed.Not with tobacco
CDNA with 20 samples under growth and development period, different tissues organ and hormon treatment conditions is template, is utilized
After semiquantitive PCR assesses the stability and reliability of 25S rna gene expression, further progress real time fluorescent quantitative
PCR amplification, fluorescent dye are SYBR Green, wherein the quantitative primer of qRT-PCR, sequence such as SEQ ID NO.4/SEQ ID
Shown in NO.5.
The qRT-PCR amplified reaction program is as follows: in 95 DEG C of 10 min of initial denaturation, first running 95 DEG C again of 40 circulations
15 s, 60 DEG C of 30 s of annealing are denaturalized, are then the specificity for detecting PCR amplification gene, it is bent with dissolution after PCR reaction
Line expands temperature with 0.5 DEG C of amplification and is slowly incremented to 95 DEG C from 65 DEG C, and the time of each amplification is 5 s.
The beneficial effects of the present invention are:
1. the present invention is cloned into tobacco 25S RNA gene for the first time, stability and specificity are via semiquantitive PCR and reality
When fluorescence quantitative PCR detection and verifying；
2.25S RNA gene not only tobacco different tissues organ, different growth and development stage tissue in stablize table
It reaches, and being capable of also stable expression under the conditions of various HORMONE TREATMENTs.25S RNA gene stablizes the characteristic of expression, not for tobacco
The quantitative study that same stage of development, different tissues organ, hormon processing genes are expressed provides new high confidence level
Reference gene；
3. experiments verify that the primer for fluorescence quantitative PCR detection designed by the present invention also can be used for semiquantitive PCR
Quickly detection, and specific amplification and have good stability.
Detailed description of the invention
Fig. 1 is 1% agarose gel electrophoresis figure of tobacco 25S RNA gene magnification in the present invention；
Wherein M is BM2000 Marker, and 1 is tobacco 25S rna gene；
Fig. 2 is the 25S rna gene of the invention PCR amplification in tobacco difference growth and development stage, different tissues organ
1% agarose gel electrophoresis figure；
Fig. 3 is the 1% Ago-Gel electricity of 25S rna gene of the invention in tobacco hormon treatment conditions PCR amplification
Swimming figure；
Fig. 4 is 25S rna gene real-time fluorescence quantitative PCR amplification curve diagram in the present invention；
Fig. 5 is the solubility curve figure of 25S rna gene real-time fluorescence quantitative PCR amplification in the present invention.
Specific embodiment
Present invention will be further explained below with reference to specific examples, these embodiments are merely to illustrate the present invention and do not have to
In limiting the scope of the invention.The test method of actual conditions is not specified in the following example, usually according to normal condition.
A kind of cloning process of the tobacco 25S rna gene sequence of embodiment 1.
1.1 tobacco Total RNAs extractions:
About 100mg tobacco flesh tissue is taken, the every 1mLBB6 of corresponding amount BB6(is added in the rapid grind into powder in liquid nitrogen,
Add 10 μ L beta -mercaptoethanols, matching while using), vortex shakes vigorously and mix well；After being incubated at room temperature 3min, 12000g is centrifuged 2-5 min,
The careful supernatant drawn in centrifuge tube is into the centrifuge tube of RNase-free；0.5 times of volume dehydrated alcohol is added into supernatant, and
It is vortexed and thoroughly mixes, dispersion precipitating, then obtained solution and precipitating are added in centrifugal column together, 30s is centrifuged with 12000g, is abandoned
After falling efflux, adds 500 μ L CB6, room temperature 12000g centrifugation 30s, discard efflux；It is added 80 μ L's to centrifugal column center
I working solution of DNase is placed at room temperature for 15min, and the repetition step is primary, to remove genomic DNA；Add 500 μ L WB6,12000g
It is centrifuged 30s, discards efflux；12000g is centrifuged 2min again, completely removes remaining ethyl alcohol, is being stored at room temperature several minutes, thoroughly
After drying centrifugal column, add 50 μ L RNase-free H2O is stored at room temperature 1min in the center of centrifugal column, and 12000g is centrifuged 2min,
Eluted rna.After 1 μ L primary sample is diluted 10 times or 100 times, 5 μ L is taken to carry out 1% agarose gel electrophoresis detection RNA sample
Integrity detection.And 1 μ LRNA is taken to measure the purity of RNA sample and dense with micro ultraviolet specrophotometer (NanoDrop2000)
Angle value.Qualified samples are placed in -80 DEG C to save for use.
1.2 PCR amplification primer sequences
The upstream primer of 25S RNA is as shown in SEQ ID NO.1；The downstream primer of 25S RNA such as SEQ ID NO.2 institute
Show.
Stablize expression and change in different growth and development period tissue samples according to gene chip expression analysis is obtained
The different lesser 25S RNA gene of coefficient, with 5.0 software design of the Primer amplimer, finally by Beijing six directions Hua Da base
Because Science and Technology Co., Ltd. synthesizes.
1.3 reverse transcriptions (RT)
Using tobacco total serum IgE as template, following examination is sequentially added in the reverse transcription system setting of 25 μ L and reaction process
Agent: 2.0 μ gRNA, 1.5 μ LOligo (dT) Primer (20 μm of ol/L), then plus RNase-free H2O to 14 μ L；70 DEG C of temperature
After bathing 6 min, centrifuge tube is put into ice bath rapidly, sequentially adds the M-MLV 5 × Buffer, the M- of 1.0 μ L of 5.0 μ L
MLV reverse transcriptase, the RNase inhibitor of 1.0 μ L and 4.0 μ LdNTP Mixture, after mixing, 42 DEG C of 75 min of incubation,
After being diluted with water 5 times, -20 DEG C of preservations.
1.4 polymerase chain reactions (PCR)
The cDNA obtained using step 1.3 carries out PCR amplification as primer as template, using the sequence obtained in step 1.2.PCR
Using following 25 μ L reaction system: 50ng template, the forward and reverse primer of 1 μ L, 1 μ L EasyTaq(Quan Shijin biotechnology are limited
Company), add water to 25 μ L.The setting of PCR response procedures are as follows: 94 DEG C of initial denaturation 4min, 94 DEG C of denaturation 30s, 56 DEG C of annealing 30s, 72
DEG C extend 2min, totally 25 circulation, after circulation terminates carry out 72 DEG C eventually extend 10 min, 4 DEG C preservation.PCR product is through 1% agar
Sugared gel electrophoresis comes testing goal segment (Fig. 1).
The clone of 1.5 target gene and sequencing
Gel extraction is carried out to target fragment, DNA fragmentation after purification is connected to pMD18T simple vector carrier
On, it is transferred in competent escherichia coli cell, is detected through the screening of blue hickie and PCR, positive colony is sequenced, is obtained
The nucleotide sequence of the 25S RNA of 459bp (particular sequence is shown in SEQ ID NO.3).
2. tobacco 25S rna gene of embodiment is in different tissues organ and the analysis of stability of different growth and development stage expression
Analysis
The preparation of 2.1 samples
Respectively with tobacco seedling phase root, prosperous growth phase root, maturity period root, full-bloom stage root, stem, leaf, sepal, corolla, column cap, son
The cDNA that the total serum IgE reverse transcription in room, 12 stamen, gynoecium histoorgans obtains is template, specific extracting method detailed in Example 1
In described in 1.1,1.3.
The synthesis of 2.2 RT-qPCR primers
QPCR upstream primer is as shown in SEQ ID NO.4；QPCR upstream primer is as shown in SEQ ID NO.5；
It is synthesized by Beijing Liuhe Huada Genomics Technology Co., Ltd.
2.3 polymerase chain reaction (PCR)
Using the cDNA in step 2.1 as template, pcr amplification reaction is carried out with the primer obtained in 2.2, PCR reaction system:
Successively add 50 ng templates, the forward and reverse primer of 1 μ L, 1 μ L EasyTaq in the system of 25 μ L, adds water to 25 μ L.
PCR response procedures are as follows: 94 DEG C of 4 min of initial denaturation, 94 DEG C of 30 s of denaturation, 60 DEG C of 30 s of annealing, 72 DEG C of 40 s of extension, totally 25 are followed
Ring.Extend 10 min, 4 DEG C of preservations eventually for 72 DEG C again.PCR after reaction, takes 2 μ L PCR products to carry out 1.0% Ago-Gel
Electrophoresis detection, gel imaging observation.
2.4 experimental result
Testing result shows as shown in Fig. 2, the amplification band almost the same, single to 12 brightness in 25S rna gene
It expresses and stablizes in tobacco difference growth and development stage, different tissues organ, while being also shown that the real-time fluorescence that the present invention designs is fixed
Measuring PCR primer has very high specificity.
The stability analysis that 3. tobacco 25S rna gene of embodiment is expressed under hormon treatment conditions
The preparation of the tobacco sample of 3.1 hormons processing
Tobacco seed is placed in culture dish, Hoagland solution is added in h(25/23 DEG C of illumination and dark 12/12) item
It is cultivated under part, until handle the auxin (IAA) of 5 hours: 10 μm ol/L, in witchweed with following hormone when 2 weeks after germination
Ester (GR24), the basic element of cell division (6-BA) and brassinosteroid (BR), the abscisic acid (ABA) of 50 μm of ol/L and gibberellin
(GA) and the methyl jasmonate of 100 μm of ol/L (MeJA), 1.1 methods in strain seedling tobacco reference embodiment 1 that are rounded are extracted
RNA, and cDNA is produced referring to 1.3 methods in embodiment 1.
3.2 polymerase chain reactions (PCR)
Using the cDNA in step 3.1 as template, pcr amplification reaction is carried out with the primer obtained in 2.2, it is detailed with specific method
As described in 2.3 in embodiment 2.
3.3 experimental result
Testing result is as shown in figure 3, when using the real-time fluorescence quantitative PCR primer in embodiment 2 for primer, through 7 kinds
The band almost the same with (untreated) sample brightness is compareed is arrived in amplification in the tobacco sample of HORMONE TREATMENT, to show 25S
Rna gene can stablize expression after the processing of hormon.
The detection of specific real-time fluorescence quantitative PCR of the embodiment 4.25S rna gene in tobacco
The preparation of 4.1 samples
Extract tobacco total serum IgE referring to 1.1 steps in embodiment 1, and referring to 1.4 procedure reverseds in embodiment 1 record at
cDNA。
4.2 quantitative fluorescent PCR
It is quantitative primer with the primer synthesized in 2.2, with Bio-Rad CFX96 fluorescent quantitation using cDNA in 4.1 as template
PCR instrument carries out pcr amplification reaction.Following reagent is sequentially added in the reverse transcription system setting of 25 μ L and reaction process:
12.5 μ L SYBR Green qPCR Mix, 50 ng cDNA are template, and real-time fluorescence quantitative PCR is forward and reverse in example 2
Each 1 μ L(concentration of primer is 10 μm of ol/L), then plus distilled water to 25 μ L, response procedures are as follows: 95 DEG C of 10 min of initial denaturation, first
Run 40 circulations, 95 DEG C of denaturation 15 s, 60 DEG C of 30 s of annealing again.For the specificity for detecting PCR amplification gene, it is reacted in PCR
It is analyzed afterwards with solubility curve, expands temperature and with 0.5 DEG C of amplification be slowly incremented to 95 DEG C from 65 DEG C, the time of each amplification is
5 s, the fluorescence intensity of continuous test sample is to obtain solubility curve.
4.3 experimental result
Reaction result is as shown in figure 4,3 duplicate fluorescent quantitative PCR curves occur in 20 circulations, and stablize good
It is good, show that gene abundance is high and stablizes；Solubility curve result is as shown in Figure 5: Tm value is 83 DEG C, only one is special and sharp
Peak shows that the quantitative amplification primer specificity that the present invention designs is strong, it is existing to amplify without nonspecific products.
Finally show that 25S rna gene expresses stabilization in tobacco, can be used as the reference gene of quantitative tobacco, by this
When reference gene studies tobacco gene expression analysis, it can be improved the reliability and stability of tobacco gene expression analysis research.
Claims (5)
1. a kind of qRT-PCR amplification method of tobacco 25S rna gene, it is characterised in that: using the gene order of 25S RNA as base
Plinth designs 1 pair of fluorescent quantitation special primer, using the cDNA of 10 ~ 30 tobacco samples as template, carries out qRT-PCR amplification, fluorescence
Dyestuff is SYBR Green, wherein the fluorescence quantification PCR primer of qRT-PCR, sequence such as SEQ ID NO.4/SEQ ID NO 5
It is shown.
2. the qRT-PCR amplification method of tobacco 25S rna gene according to claim 1, it is characterised in that: with 25S
Based on the gene order of RNA, according to the principle of real-time fluorescence quantitative PCR design of primers, designs 1 pair of fluorescent quantitation and specifically draw
Object, with the cDNA of 20 tobacco samples under different growth and development periods, different tissues organ and hormon treatment conditions
For template.
3. the qRT-PCR amplification method of tobacco 25S rna gene according to claim 1, it is characterised in that: utilizing half
Quantitative PCR assesses the stability of 25S rna gene expression and reliability carries out qRT-PCR amplification, the sxemiquantitative later
PCR amplification program is as follows: 94 DEG C of initial denaturation 4 min, 94 DEG C of denaturation 30 s, 56 DEG C of annealing 30 s, 72 DEG C of extension 40 s, and totally 25
Circulation, then 72 DEG C of 10 min of whole extension, 4 DEG C of preservations.
4. the qRT-PCR amplification method of tobacco 25S rna gene according to claim 1, it is characterised in that: described
QRT-PCR amplification uses two-step method, i.e., in 95 DEG C of 10 min of initial denaturation, first runs 40 circulations, 95 DEG C of denaturation 15 s again, and 60 DEG C
Anneal 30 s, then be detection PCR amplification gene specificity, PCR reaction after use solubility curve, expand temperature with
0.5 DEG C of amplification is slowly incremented to 95 DEG C from 65 DEG C, and the time of each amplification is 5 s.
5. tobacco 25S rna gene described in claim 1 is in tobacco functional gene or the phase of different development stage gene expression
To the application in horizontal reference gene.
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