CN105385687B - The cloning process of tobacco 26S RNA reference genes and its application - Google Patents
The cloning process of tobacco 26S RNA reference genes and its application Download PDFInfo
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Abstract
The invention belongs to technical field of molecular biology, and in particular to tobacco 26S RNA（26Sribosomal RNA）The cloning process of reference gene and its application.The partial nucleotide sequence of 26S rna genes that the present invention is obtained can be as the reference gene of the gene expression analysis under research tobacco functional gene or different tissues, different growth and development stages and hormon treatment conditions.The real-time fluorescence quantitative PCR primer amplification high specificity designed using the reference gene can improve 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 26S RNA.
Background technology
Tobacco（Nicotianatabacum L.）For Solanaceae Nicotiana, contain nicotine（Nicotine）Leaf with 1 year
Raw crop.The mankind use the tobacco history of about 1500, 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, alternative reference gene and few during analysis tobacco gene expression
See.At present, 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 ripe, a large amount of microarray data is new interior
The excavation of ginseng gene provides good data basis and platform, and the selection for reference gene provides more wide space.
Compared with the reference gene obtained with traditional sequence analysis method, by gene chip expression technology screening to internal reference have
The characteristic of expression is expressed and stablized to a large amount.
Real-time fluorescence quantitative PCR (real-time quantitative reverse transcription PCR,
QRT-PCR it is) a kind of in DNA amplification reaction, product is total after surveying each PCR cycle with fluorescent chemical
The method of amount.It is research means important in present molecular biology using qRT-PCR analysis gene expression doses, qRT-PCR
There is 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 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.
26S ribosomes（26Sribosomal RNA）Gene is common reference gene, since the synthesis of rRNA is only
MRNA is stood on, gene transcription level stability is more preferable, and therefore, 26S RNA become many animal-plant gene expression analysis
The important selecting object of middle reference gene.
Invention content
The present invention provides tobacco 26S rna gene partial sequences, can be as the application of tobacco reference gene.And it provides simultaneously
The specific primer of tobacco 26S rna genes is cloned, establishes the qRT-PCR methods based on SYBR Green fluorescent dye technologies,
So as to the reference gene as tobacco 26S RNA, to be carried using real-time fluorescence quantitative PCR to the research of tobacco gene expression analysis
For useful method.
To achieve the above object, the present invention uses following technical scheme：
One cloning process for growing tobacco 26S rna gene sequences, by 99 to tobacco difference growth and development period not
With the chip expression data analysis of tissue samples, the coefficient of variation for stablizing expression in all samples is filtered out（coefficient
Of variation, CV）Smaller composing type 26S rna gene sequences are carried out with primer pair 26S RNA-F/26S RNA-R
PCR amplification obtains positive colony, is verified by plasmid order-checking；Wherein primer sequence is such as SEQ ID NO.1/SEQ ID NO.2
It is shown.
The target fragment obtained is as shown in SEQ ID NO.3, size 206bp.
The reaction condition of the PCR amplification is as follows：94 DEG C of pre-degenerations 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 cycles, then 72 DEG C of 10 min of whole extension, 4 DEG C of preservations.
The present invention also provides a kind of method of qRT-PCR amplification tobacco 26S rna gene partial sequences, with 26S 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.With tobacco not
CDNA with 20 samples under growth and development period, different tissues organ and hormon treatment conditions is template, is utilized
After the stability and reliability of semiquantitive PCR assessment 26S rna gene expressions, real time fluorescent quantitative is further carried out
PCR amplification, fluorescent dye be SYBR Green, the quantitative primer of wherein qRT-PCR, sequence such as SEQ ID NO.4/SEQ ID
Shown in NO.5.
The qRT-PCR amplified reaction programs are as follows：In 95 DEG C of 10 min of pre-degeneration, 40 95 DEG C again of cycles are first run
15 s, 60 DEG C of 30 s of annealing are denaturalized, then the specificity for detection PCR amplification gene, with dissolving song after PCR reactions
Line, amplification temperature are slowly incremented to 95 DEG C from 65 DEG C with 0.5 DEG C of amplification, 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 26S RNA genes for the first time, stability and specificity are via semiquantitive PCR and reality
When fluorescence quantitative PCR detection and verification；
2.26S RNA genes not only tobacco different tissues organ, different growth and development stage tissue in stablize table
It reaches, and can also stablize expression under the conditions of various HORMONE TREATMENTs.26S RNA genes stablize the characteristic of expression, for tobacco not
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
Quick detection, and specific amplification and have good stability.
Description of the drawings
Fig. 1：1% agarose gel electrophoresis figure of tobacco 26S RNA gene magnifications in the present invention；
Wherein：M is BM2000 Marker, and 1 is tobacco 26S rna genes；
Fig. 2：The 26S rna genes of present invention PCR amplification in tobacco difference growth and development stage, different tissues organ
1% agarose gel electrophoresis figure；
Fig. 3：The 26S rna genes of the present invention are in the 1% Ago-Gel electricity of tobacco hormon treatment conditions PCR amplification
Swimming figure；
Fig. 4：26S rna genes real-time fluorescence quantitative PCR amplification curve diagram in the present invention；
Fig. 5：The solubility curve figure that 26S rna genes real-time fluorescence quantitative PCR expands in the present invention.
Specific embodiment
With reference to specific embodiment, the present invention is further explained, 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.
Embodiment 1. 1 grows tobacco the clones of 26SRNA genes
1.1 tobacco Total RNAs extractions：
About 100mg tobacco flesh tissues are taken, the rapid grind into powder in liquid nitrogen adds in corresponding amount BB6（Per 1mLBB6,
Add 10 μ L beta -mercaptoethanols, matching while using）, vortex shakes vigorously and mix well；After being incubated at room temperature 3min, 12000g centrifugation 2-5 min,
In the careful supernatant to the centrifuge tube of RNase-free drawn in centrifuge tube；0.5 times of volume absolute ethyl alcohol is added in into supernatant, and
Be vortexed thorough mixing, dispersion precipitation, then obtained solution and precipitation are added in centrifugal column together, centrifuges 30s with 12000g, abandons
After falling efflux, add 500 μ L CB6, room temperature 12000g centrifugation 30s, discard efflux；Add in 80 μ L's to centrifugal column center
I working solutions of DNase are placed at room temperature for 15min, and the repetition step is primary, to remove genomic DNA；Add 500 μ L WB6,12000g
30s is centrifuged, discards efflux；12000g centrifuges 2min again, thoroughly 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, 12000g centrifugation 2min in the center of centrifugal column,
Eluted rna.After 1 μ L primary samples are diluted 10 times or 100 times, 5 μ L is taken to carry out 1% agarose gel electrophoresis detection RNA sample
Integrity detection.And take the micro ultraviolet specrophotometers of 1 μ LRNA（NanoDrop2000）Measure the purity of RNA sample and dense
Angle value.Qualified samples are placed in -80 DEG C to preserve for use.
1.2 PCR amplification primer sequences
The sense primer of 26SRNA is as shown in SEQ ID NO.1；The downstream primer of 26SRNA is as shown in SEQ ID NO.2.
Stablize expression and change in different growth and development period tissue samples according to gene chip expression analysis is obtained
The different smaller 26S RNA genes of coefficient, with 5.0 Software for Design of the Primer amplimer, finally by Beijing six directions Hua Da base
Because Science and Technology Co., Ltd. synthesizes.
1.3 reverse transcription（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 add RNase-free H2O to 14 μ L；70℃
After 6 min of warm bath, centrifuge tube is put into ice bath rapidly, sequentially adds 5 × Buffer of M-MLV of 5.0 μ L, 1.0 μ L's
M-MLV reverse transcriptases, the RNase inhibitor of 1.0 μ L and 4.0 μ LdNTP Mixture, after mixing, 42 DEG C incubate 75
Min, after being diluted with water 5 times, -20 DEG C of preservations.
1.4 PCR（PCR）
Using the cDNA that step 1.3 obtains as template, PCR amplification is carried out as primer using the sequence obtained in step 1.2.PCR
Using following 25 μ L reaction systems：50ng templates, the forward and reverse primer of 1 μ L, 1 μ L EasyTaq（Full formula gold biotechnology is limited
Company）, add water to 25 μ L.PCR response procedures are set as：94 DEG C of pre-degenerations 4min, 94 DEG C of denaturation 30s, 58 DEG C of 30s that anneal, 72
DEG C extension 2min, totally 25 cycle, after circulation terminates carry out 72 DEG C eventually extension 10 min, 4 DEG C preservation.PCR product is through 1% agar
Sugared gel electrophoresis carrys out 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 carriers
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 26S RNA of 206bp（Particular sequence is shown in SEQ ID NO.3）.
2. tobacco 26S rna genes of embodiment are 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 long-term 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 be template, specific extracting method detailed in Example 1
In described in 1.1,1.3.
The synthesis of 2.2 RT-qPCR primers
QPCR sense primers are as shown in SEQ ID NO.4；QPCR sense primers are as shown in SEQ ID NO.5；
It is synthesized by Beijing Liuhe Huada Genomics Technology Co., Ltd.
2.3 PCR（PCR）
Using the cDNA in step 2.1 as template, pcr amplification reaction, PCR reaction systems are carried out with the primer obtained in 2.2：
Add 50 ng templates, the forward and reverse primer of 1 μ L, 1 μ L EasyTaq successively in the system of 25 μ L, add water to 25 μ L.
PCR response procedures are：94 DEG C of 4 min of pre-degeneration, 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 is as shown in Fig. 2, amplification shows that 26S rna genes exist to basically identical, the single band of 12 brightness
It expresses and stablizes in tobacco difference growth and development stage, different tissues organ, while be also shown that the real-time fluorescence that the present invention designs is determined
Measuring PCR primer has very high specificity.
The stability analysis that 3. tobacco 26S rna genes of embodiment are expressed under hormon treatment conditions
The preparation of the tobacco sample of 3.1 hormons processing
Tobacco seed is placed in culture dish, adds in Hoagland solution in illumination and 12/12 h of dark（25/23℃）Item
Cultivated under part, until germination after 2 weeks when with following hormone carry out handle 5 hours：The auxin of 10 μm of ol/L（IAA）, in witchweed
Ester (GR24), the basic element of cell division（6-BA）And brassinosteroid（BR）, the abscisic acid of 50 μm of ol/L（ABA）The gibberellin of sum
（GA）And the methyl jasmonate of 100 μm of ol/L（MeJA）, rounding strain seedling tobacco is with reference to 1.1 methods extraction in embodiment 1
RNA, and produce cDNA with reference in embodiment 1 1.3.
3.2 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 the real-time fluorescence quantitative PCR primer in using embodiment 2 is primer, through 7 kinds
Amplification is arrived with compareing in the tobacco sample of HORMONE TREATMENT（It is untreated）The basically identical band of sample brightness, so as to show 26S
Rna gene can stablize expression after the processing of hormon.
The detection of specific real-time fluorescence quantitative PCR of the 4. 26S rna genes of embodiment in tobacco
The preparation of 4.1 samples
With reference to 1.1 steps extraction tobacco total serum IgE in embodiment 1, and with reference to 1.4 procedure reverseds in embodiment 1 record into
cDNA。
4.2 quantitative fluorescent PCR
Using cDNA in 4.1 as template, using the primer synthesized in 2.2 as quantitative primer, with Bio-Rad CFX96 fluorescent quantitations
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 of primer（A concentration of 10 μm of ol/L）, then add distilled water to 25 μ L, response procedures are：95 DEG C of 10 min of pre-degeneration, first
40 cycles of operation are denaturalized 15 s, 60 DEG C of 30 s of annealing for 95 DEG C again.To detect the specificity of PCR amplification gene, it is reacted in PCR
It is analyzed afterwards with solubility curve, amplification temperature is slowly incremented to 95 DEG C with 0.5 DEG C of amplification from 65 DEG C, and the time of each amplification is
5 s continuously detect the fluorescence intensity of sample to obtain solubility curve.
4.3 experimental result
Reaction result is as shown in figure 4, the fluorescent quantitative PCR curve of 3 repetitions occurs, and stablize good in 20 cycles
It is good, show that gene abundance is high and stablizes；The results are shown in Figure 5 for solubility curve：Tm values are 83 DEG C, only there are one special and sharply
Peak shows that the quantitative amplification primer specificity that the present invention designs is strong, it is existing to be amplified without nonspecific products.
Finally show that 26S rna genes express stabilization in tobacco, it can be as the reference gene of quantitative tobacco, by this
When reference gene studies tobacco gene expression analysis, the reliability and stability of tobacco gene expression analysis research can be improved.
Claims (7)
- A 1. cloning process for growing tobacco 26S rna genes, it is characterised in that：Pass through the chip expression number to Tissues of Tobacco sample According to analysis, 26S rna gene sequences are filtered out, PCR amplification is carried out with primer pair 26S RNA-F/26S RNA-R, obtains the positive Clone, verifies by plasmid order-checking；Wherein primer pair sequence is as shown in SEQ ID NO.1/SEQ ID NO 2；The mesh obtained Segment such as SEQ ID NO.3, size 206bp；The gene nucleotide series are as shown in SEQ ID NO.3；By the chip expression data analysis of 50 ~ 150 different tissues samples to tobacco difference growth and development period, screen Stablize expression and the smaller composing type 26S rna gene sequences of the coefficient of variation in all samples.
- 2. the cloning process of tobacco 26S rna genes according to claim 1, it is characterised in that：By to tobacco difference The chip expression data analysis of 99 different tissues samples in growth and development period.
- 3. the cloning process of tobacco 26S rna genes according to claim 1, it is characterised in that：The PCR amplification it is anti- Answer program as follows：94 DEG C of 4 min of pre-degeneration, 94 DEG C of 30 s of denaturation, 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.
- 4. the qRT-PCR amplification methods for growing tobacco 26S rna genes, it is characterised in that：Using the gene order of 26S RNA as base Plinth designs a pair of of fluorescent quantitation special primer, using the cDNA of 10 ~ 30 tobacco samples as template, carries out qRT-PCR amplifications, glimmering Photoinitiator dye is SYBR Green, the wherein fluorescence quantification PCR primer of qRT-PCR, sequence such as SEQ ID NO.4/SEQ ID Shown in NO.5.
- 5. the qRT-PCR amplification methods of tobacco 26S rna genes according to claim 4, it is characterised in that：With 26S Based on the gene order of RNA, according to the principle of real-time fluorescence quantitative PCR design of primers, 1 pair of fluorescent quantitation of design specifically draws Object, with the cDNA of 20 tobacco samples under different growth and development periods, different tissues organ and hormon treatment conditions For template.
- 6. the qRT-PCR amplification methods of tobacco 26S rna genes according to claim 4, it is characterised in that：Utilizing half QRT-PCR amplifications, the sxemiquantitative are carried out after the stability and reliability of quantitative PCR assessment 26S rna gene expressions PCR amplification program is as follows：94 DEG C of pre-degeneration 4 min, 94 DEG C of denaturation 30 s, 56 DEG C of annealing 30 s, 72 DEG C of extension 40 s, totally 25 Cycle, then 72 DEG C of 10 min of whole extension, 4 DEG C of preservations.
- 7. the qRT-PCR amplification methods of tobacco 26S rna genes according to claim 4, it is characterised in that：Described QRT-PCR amplifications are using two-step method, i.e., in 95 DEG C of 10 min of pre-degeneration, first run 40 cycles 95 DEG C of denaturation 15 s again, 60 DEG C Anneal 30 s, then to detect the specificity of PCR amplification gene, with solubility curve after PCR reactions, amplification 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.
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| CN102816852A (en) * | 2012-08-30 | 2012-12-12 | 浙江中烟工业有限责任公司 | Method for early anticipating alkaloid relative contents of maturation phase leaves of different tobacco varieties |
| CN103642907A (en) * | 2013-11-22 | 2014-03-19 | 贵州省烟草科学研究院 | Tobacco reference genes screened by use of gene chip and combination of real time PCR (polymerase chain reaction) and method thereof |
| CN104032007A (en) * | 2014-06-12 | 2014-09-10 | 广西壮族自治区兽医研究所 | Method for detecting exogenous gene copy number in transgenic tobacco |
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2015
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102816852A (en) * | 2012-08-30 | 2012-12-12 | 浙江中烟工业有限责任公司 | Method for early anticipating alkaloid relative contents of maturation phase leaves of different tobacco varieties |
| CN103642907A (en) * | 2013-11-22 | 2014-03-19 | 贵州省烟草科学研究院 | Tobacco reference genes screened by use of gene chip and combination of real time PCR (polymerase chain reaction) and method thereof |
| CN104032007A (en) * | 2014-06-12 | 2014-09-10 | 广西壮族自治区兽医研究所 | Method for detecting exogenous gene copy number in transgenic tobacco |
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