CN109182549A - Detect the primer and method of Agasicles hygrophila OR gene transcription level - Google Patents

Detect the primer and method of Agasicles hygrophila OR gene transcription level Download PDF

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CN109182549A
CN109182549A CN201811231572.5A CN201811231572A CN109182549A CN 109182549 A CN109182549 A CN 109182549A CN 201811231572 A CN201811231572 A CN 201811231572A CN 109182549 A CN109182549 A CN 109182549A
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primer
gene
agasicles hygrophila
pcr
cdna
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CN109182549B (en
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傅建炜
何肖云
郑丽祯
李建宇
史梦竹
林凌鸿
王秋月
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Institute Of Quality Standard And Testing Technology For Agro-Products Fujian Academy Of Agricultural Sciences
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Institute Of Quality Standard And Testing Technology For Agro-Products Fujian Academy Of Agricultural Sciences
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes

Abstract

The present invention provides the primers and method of detection Agasicles hygrophila OR gene transcription level, and for primer sequence as shown in SEQ ID NO.1-2, the present invention establishes the female male worm feeding difference CO of Agasicles hygrophila2The fluorescence quantitative PCR method that OR gene expression amount detects after the alternanthera philoxeroides that concentration is cultivated, and the sequencing of transcript profile is carried out, compared with having done to the expression quantity trend of OR gene by fluorescence quantitative with the expression quantity trend of transcript profile, the defense mechanism and OR gene expression characteristics generated when for research Agasicles hygrophila by environment-stress is laid a good foundation.

Description

Detect the primer and method of Agasicles hygrophila OR gene transcription level
Technical field
The present invention relates to the primers and method of detection Agasicles hygrophila OR gene transcription level, belong to biotechnology neck Domain.
Background technique
Agasicles hygrophila category coleoptera, Chrysomelidae are one of main biological and ecological methods to prevent plant disease, pests, and erosion natural enemies of alternanthera philoxeroides.George Vogt had found that Agasicles hygrophila had good control action to alternanthera philoxeroides in 1962 for the first time.Thereafter, the straight chest of lotus grass Flea beetle is widely used in the control of alternanthera philoxeroides on the U.S., Australia and other places, and achieves preferable control efficiency.I State introduced Agasicles hygrophila from the U.S. in 1986, and was applied to the biological control of alternanthera philoxeroides, in Sichuan, Zhejiang, good fortune It builds, the ground such as Guangxi obtain certain success.
Caste is various, is distributed widely in various environment.Some insects are beneficial to the mankind, other insects are serious Influence the production and life of the mankind.Chemical information in the sensitive chemosensing system detection environment of insect utilization, to dominate It the behaviors such as ingests, walk quickly and keep away, breeding.Chemoreception mainly includes smell and the sense of taste, detected by scent volatile materials, and taste perception Water-soluble substances.The chemoreception mechanism of research insect facilitates control of insect, reduces agricultural production loss.
The odorant receptor of insect have two classes, one kind can participate in perception scent molecule, referred to as common odorant receptor, separately One kind is the OR83b, that is, smell co-receptor for not participating in perception scent molecule directly.Common odorant receptor genes are in different insects Conservative between type is generally lower, can perceive scent molecule.OR83b gene compares among different types of insect Conservative, the protein receptor of OR83b family gene coding is referred to as OR83b family receptors, since OR83b receptor can't be single Solely perception smell therefore referred to as atypical olfactory receptor.Compared with common odorant receptor, the insect of each type is only existed A kind of OR83b receptor, but possess diversified common odorant receptor.OR83b receptor widely with common odorant receptor It realizes and co-expresses in the neuron of detected by scent, common olfactory receptor only can the selectively table in Olfactory Receptor Neurons It reaches, and the expression quantity of common odorant receptor is not generally high.The amino acid sequence of these two types of receptors all has 7 transmembrane regions Domain.
Zhao etc. (2011) clones and identifies the OR1 of phyllotreta striolata, belongs to OR83b family, is phyllotreta striolata Certified first smell related gene.Studies have shown that OR83b gene plays key during drosophila smell identification test and odor detection Effect, in drosophila OR22a/b mutant, the neuron for only expressing OR83b receptor does not experience the function of scent molecule, The drosophila strains that OR83b gene is knocked all lose sensing capability to a variety of compounds, and again import OR83b Afterwards, drosophila has restored again to the reaction for trying chemical molecular.Zhao etc. (2011) passes through lineal to phyllotreta striolata OR83b Homologous gene OR1 carries out RNAi and knocks out experiment discovery, after knocking out OR1 gene, selection energy of the flea beetle to host plant Power receives significantly damages.
The researchs such as the expression about Agasicles hygrophila OR gene have not been reported both at home and abroad, therefore use real time fluorescent quantitative PCR studies different CO2The expression characterization of the lower Agasicles hygrophila OR gene of concentration processing, the results showed that, with CO2The liter of concentration The expression quantity of height, female male worm OR gene increases, this is consistent with the variation tendency of transcript profile expression quantity up-regulation, illustrates CO2Concentration Increase the expression regulation that can influence OR gene.The olfactory system of insect is the single-minded and extremely sensitive chemical detection of a height Device, can be with chemical odor molecule specific in environment-identification, and is used as according to this and looks for food, seek a spouse, selecting host and spawning place With the information for hiding natural enemy, therefore carry out odorant receptor research, for verify insect identification scent molecule mechanism have important meaning Justice.
Summary of the invention
The object of the present invention is to provide the primers and method of detection Agasicles hygrophila OR gene transcription level, are comprehensive The different disposal CO for solving the expression characterization of Agasicles hygrophila OR gene and designing2Concentration.
To achieve the above object, the technical solution adopted in the present invention is as follows:
By feeding difference CO2The female male worm of Agasicles hygrophila for the alternanthera philoxeroides that concentration is cultivated respectively adopts 9 and manages to Eppendorf In, it is divided into 3 biology and repeats, totally 12 groups of sample.
The primer of a pair of of detection Agasicles hygrophila OR gene transcription level of design, including meet quantitative fluorescent PCR reaction The upstream and downstream primer of feature:
OR-F:5`-ATCCTTACTCAATTCTGGACTTCC-3`
OR-R:5`-TGCTTATCTCATGCTGGACAA-3`
And the upstream and downstream primer as reference gene:
Tubulin-F: 5`-AGATGTCCGCCACCTTCA-3`
Tubulin-R: 5`-GCCTCTTGGTATTGTTGGTATTCA-3`
The fluorescence quantitative PCR method of detection Agasicles hygrophila OR gene transcription level of the present invention, carries out as follows:
(1) the first chain of cDNA synthesis: extracting the total serum IgE of sample and purifying, uses reverse transcription reagent box to obtain and is with the RNA of extraction The cDNA that template reverse transcription obtains；
(2) Standard PCR detection is carried out to following primer
The fluorescent quantitation upstream and downstream primer of the gene:
OR-F:5`-ATCCTTACTCAATTCTGGACTTCC-3`,
OR-R:5`-TGCTTATCTCATGCTGGACAA-3`.
And the upstream and downstream primer as reference gene:
Tubulin-F:5`-AGATGTCCGCCACCTTCA-3`,
Tubulin-R: 5`-GCCTCTTGGTATTGTTGGTATTCA-3`。
Standard PCR amplification system is 25 μ L:10 × buffer, 2.5 μ L, dNTP 1.0 μ L, 0.5 μ L of upstream primer, downstream 0.5 μ L, 100ng/ μ L cDNA template of primer 1.0 μ L, Ex-TaqE 0.15 μ L, 19.35 μ L of water；
Routine PCR reaction program is as follows: 94 DEG C of 5 min of initial denaturation, then 94 DEG C of 30 s of denaturation, and 56 DEG C of 30 s of annealing, 72 DEG C are prolonged 1 min is stretched, under this condition 35 circulations, last 72 DEG C re-extend 10min；
(3) real-time fluorescence quantitative PCR reacts:
Primer described in step (2) is added as template in the cDNA obtained using step (1), carries out quantitative fluorescent PCR reaction, each 3 repetitions are arranged in sample, are averaged after amplification.
Real-time fluorescence quantitative PCR amplification system are as follows: SYBR® Premix Ex TaqTM12.5 μ L, 0.5 μ L of upstream primer, 0.5 μ L, 100ng/ μ L cDNA of downstream primer, 1.0 μ L, supplies water to 25 μ L；Real-time fluorescence quantitative PCR response procedures are as follows: 95 DEG C 30 s of initial denaturation, then 95 DEG C of 5 s of denaturation, 60 DEG C of 30 s of annealing, 40 circulations.
The more detailed test method of the present invention is as follows:
The real-time fluorescence quantitative PCR detection method of Agasicles hygrophila OR gene can be realized by following steps:
(1) design of primers: soft using DNAMAN according to the sequence for the Agasicles hygrophila OR gene that transcript profile sequencing result obtains Part designs the specific primer for being suitble to fluorescence quantitative PCR detection, and primer sequence is as follows:
OR-F:5`-ATCCTTACTCAATTCTGGACTTCC-3`
OR-R:5`-TGCTTATCTCATGCTGGACAA-3`
Meanwhile the sequence of the Agasicles hygrophila tubulin gene obtained according to transcript profile sequencing result, it is fixed designed for fluorescence The primer of PCR internal reference object is measured, primer sequence is as follows:
Tubulin-F: 5`-AGATGTCCGCCACCTTCA-3`
Tubulin-R: 5`-GCCTCTTGGTATTGTTGGTATTCA-3`
(2) Agasicles hygrophila processing test: in control CO2Concentration (420 μ LL- 1) and high CO2Concentration (750 μ LL- 1) Alternanthera philoxeroides is cultivated in growth cabinet, for raising the female male worm of Agasicles hygrophila, different CO2The female male worm of concentration is respectively adopted 9, into Eppendorf pipe, are divided into 3 biology and repeat, totally 12 groups of samples.It is put into rapidly in liquid nitrogen and fixes after the processing of worm sample, It is saved backup in -80 DEG C.
(3) the first chain of cDNA synthesizes: referring to Quan Shi King CompanyTransZol TMUp Plus RNA Kit kit explanation Book extracts total serum IgE, according to the One-Step gDNA Removal and cDNA Synthesis of TransScript company SuperMix kit specification synthesizes the first chain of cDNA.
(4) real-time fluorescence quantitative PCR reacts: real-time fluorescence quantitative PCR uses the SYBR of TORara company® Premix Ex TaqTMKit carries out.Using the first chain of cDNA of above-mentioned synthesis as template, above-mentioned OR-F, OR-R and Tubulin-F, Tubulin- R is specific primer, carries out quantitative fluorescent PCR program, and each sample sets 3 parallel repetitions, and the parallel Ct value of gained is taken after amplification Average.
Real-time fluorescence quantitative PCR amplification system are as follows: SYBR® Premix Ex TaqTM12.5 μ L, 0.5 μ L of upstream primer, 0.5 μ L, 100ng/ μ L cDNA of downstream primer, 1.0 μ L, supplies water to 25 μ L；
Real-time fluorescence quantitative PCR response procedures are as follows: 95 DEG C of 30 s of initial denaturation, then 95 DEG C of 5 s of denaturation, 60 DEG C of annealing 30 S, 40 circulations.
(5) calculation formula of the Agasicles hygrophila OR gene relative to the expression quantity of tubulin gene are as follows: opposite mRNA table Up to=2-ΔΔCt× 100%, wherein Ct value=target gene Ct value-tubulin Ct value.
(6) Fig. 3 and Fig. 4 is respectively the female male worm feeding difference CO of Agasicles hygrophila2After the alternanthera philoxeroides that concentration is cultivated The differential expression of OR gene.The result shows that with CO2The expression quantity of the raising of concentration, female male worm OR gene increases, and with turn The expression quantity variation tendency of record group is consistent, this provides important for the research that we carry out Agasicles hygrophila OR gene in a deep going way Basic data.
The present invention has the following advantages and effects with respect to the prior art:
(1) the different CO in the present invention2Concentration treatment measures are all suitable for insect overwhelming majority olfactory receptor gene, this is to complete
Face further investigation gene expression characteristics have great importance.
(2) fluorescence quantitative PCR method of efficient quick of the present invention, including quantitative fluorescent PCR program etc., practical used time 50 Min,
The reaction time is substantially reduced compared with the existing technology, the characteristic with efficient quick.
(3) fluorescence quantitative PCR method of the present invention provides Standard PCR electrophoresis result figure, intuitively can efficiently reflect The specificity of primer out.
Detailed description of the invention
Attached drawing is the fluorescence quantitative PCR detection result of the female male worm OR gene transcription level of Agasicles hygrophila.
Fig. 1: Agasicles hygrophila OR gene by fluorescence quantitative primer Standard PCR product electrophoresis result: product length 105 Bp, Marker band are from top to bottom successively are as follows: 2000,1000,750,500,250,100bp.
Fig. 2: Agasicles hygrophila tubulin gene by fluorescence quantitative primer Standard PCR product electrophoresis result: product length is 199 bp, Marker band are from top to bottom successively are as follows: 2000,1000,750,500,250,100bp.
Fig. 3: Agasicles hygrophila female adult (F) feeding difference CO2The difference of OR gene after the alternanthera philoxeroides that concentration is cultivated Expression, cylindricality and left side reference axis are the expression quantity of fluorescent quantitation, and scatterplot and the right reference axis are the expression quantity of transcript profile.
Fig. 4: Agasicles hygrophila male worm (M) feeding difference CO2The difference of OR gene after the alternanthera philoxeroides that concentration is cultivated Expression, cylindricality and left side reference axis are the expression quantity of fluorescent quantitation, and scatterplot and the right reference axis are the expression quantity of transcript profile.
Specific embodiment
Illustrate that the present invention, the scheme of embodiment described here do not limit invention below in conjunction with specific embodiments and drawings, His any changes, modifications, substitutions, combinations, simplifications done without departing from the spirit and principles of the present invention, should be equivalent Substitute mode, be included within the scope of the present invention.
Embodiment 1
The processing of test material
In control CO2Concentration (420 μ LL- 1) and high CO2Concentration (750 μ LL- 1) growth cabinet in cultivate hollow lotus seeds Grass, for raising the female male worm of Agasicles hygrophila, different CO2The female male worm of concentration respectively adopts 9 into Eppendorf pipe, is divided into 3 A biology repeats, totally 12 groups of samples.Worm sample is put into rapidly in liquid nitrogen after collecting and fixes, and saves backup in -80 DEG C.
Embodiment 2
The design of primer
(1) it design of primers: according to the sequence for the Agasicles hygrophila OR gene that transcript profile is sequenced, is set using DNAMAN software The specific primer for being suitble to fluorescence quantitative PCR detection is counted, primer sequence is as follows:
OR-F:5`-ATCCTTACTCAATTCTGGACTTCC-3`
OR-R:5`-TGCTTATCTCATGCTGGACAA-3`
Agasicles hygrophila OR gene by fluorescence quantitative PCR product length is 105 bp, agarose gel electrophoresis results display amplification Product length afterwards is consistent, and is single band, illustrates that designed primer has very strong specificity, suitable for glimmering in real time Fluorescent Quantitative PCR detection, agarose gel electrophoresis results are as shown in Figure 1.
(2) simultaneously, the sequence for the Agasicles hygrophila tubulin gene being sequenced according to transcript profile, designed for glimmering The primer of Fluorescent Quantitative PCR internal reference object, primer sequence are as follows:
Tubulin-F: 5`-AGATGTCCGCCACCTTCA-3`
Tubulin-R: 5`-GCCTCTTGGTATTGTTGGTATTCA-3`
Agasicles hygrophila tubulin gene by fluorescence quantitative PCR product length is 199 bp, and agarose gel electrophoresis results are shown Product length after amplification is consistent, and is single band, illustrates that designed primer has very strong specificity, suitable for real When fluorescence quantitative PCR detection, agarose gel electrophoresis results are as shown in Figure 2.
Embodiment 3
The extraction of total serum IgE
Referring to Quan Shi King CompanyTransZol TMUp Plus RNA Kit kit specification extracts total serum IgE, will with liquid nitrogen Polypide is pulverized, and 1ml is addedTransZol TMUp goes to 1.5 ml and turns in centrifuge tube, after being stored at room temperature 5 min, is added 200 μ L chloroforms/1 mlTransZol TMUp acutely vibrates 30 s, is incubated at room temperature 3 min.4 DEG C of 10000 g centrifugation 15 After min, upper strata aqueous phase (generally < 80%) is moved in new centrifuge tube, the dehydrated alcohol of 1/3 volume is added, is gently mixed by inversion. RNA centrifugal column is placed in 2 ml collecting pipes, upper step mixture is all transferred in centrifugal column, 12000 g, 30 ~ 60 s centrifugation, Mobile phase is abandoned, collecting pipe is reused.500 μ LCB9 are added, 12000 g of room temperature is centrifuged 30 s, abandons mobile phase, adds 500 μ LCB9,12000 g of room temperature are centrifuged 30 s, abandon mobile phase.Diluted 500 μ LWB9 is added, 12000 g are centrifuged 30 s, abandoned stream Dynamic phase adds diluted 500 μ LWB9, and 12000 g are centrifuged 30 s, abandons mobile phase.12000 g are centrifuged 2 min, thoroughly go Except remaining ethyl alcohol, centrifugal column is thoroughly dried being stored at room temperature several minutes.Centrifugal column is put into RNase-free Tube, is added 50 μ LRNase-free Water are stored at room temperature 1 min in the center of centrifugal column, and 12000 g are centrifuged 1 min, eluted rna.Institute RNA be placed in -80 DEG C it is spare.
Embodiment 4
The first chain of cDNA synthesis: according to the One-Step gDNA Removal and cDNA of TransScript company Synthesis SuperMix kit specification step, using the RNA in embodiment 3 as template, reverse transcription generates cDNA first Chain, the specific steps are as follows:
(1) reverse transcription system
(2) 42 DEG C of 30 min of incubation.
(3) 85 DEG C of 5 min of heating, product can be placed on -20 DEG C, -80 DEG C of preservations.
Embodiment 5
Carry out quantitative fluorescent PCR reaction.Real-time fluorescence quantitative PCR uses the SYBR of TORara company® Premix Ex TaqTM Kit carries out.Using the cDNA in embodiment 4 as template, it is anti-that real-time fluorescence quantitative PCR amplification is carried out with the primer in embodiment 2 It answers, each sample sets 3 parallel repetitions, and the average of the parallel Ct value of gained is taken after amplification.
(1) real-time fluorescence quantitative PCR amplification system are as follows: SYBR® Premix Ex TaqTM12.5 μ L, 0.5 μ of upstream primer 1.0 μ L of L, 0.5 μ L, 100ng/ μ L cDNA of downstream primer, supplies water to 25 μ L；
(2) real-time fluorescence quantitative PCR response procedures are as follows: 95 DEG C of 30 s of initial denaturation, then 95 DEG C of 5 s of denaturation, 60 DEG C of annealing 30 s, 40 circulations.
(3) after the completion of real-time fluorescence quantitative PCR, the ratio 2 of relative expression quantity under the conditions of different disposal is calculated according to Ct value-ΔΔCt.Calculation formula of the Agasicles hygrophila OR gene relative to the expression quantity of tubulin gene are as follows: opposite mRNA expression=2-ΔΔCt× 100%, wherein Ct value=target gene Ct value-tubulin Ct value.
Table 1: Agasicles hygrophila female adult (F) feeding difference CO2The C (T) of OR gene after the alternanthera philoxeroides that concentration is cultivated Value, average value, standard deviation, fluorescent quantitation expression quantity and transcript profile expression quantity
Table 2: Agasicles hygrophila male worm (M) feeding difference CO2It is C (T) value of OR gene after the alternanthera philoxeroides that concentration is cultivated, flat Mean value, standard deviation, fluorescent quantitation expression quantity and transcript profile expression quantity
(4) Fig. 3 and Fig. 4 is respectively the female male worm feeding difference CO of Agasicles hygrophila2OR base after the alternanthera philoxeroides that concentration is cultivated The differential expression of cause.The result shows that with CO2The expression quantity of the raising of concentration, female male worm OR gene increases, with transcript profile The variation tendency of expression quantity up-regulation is consistent.This provides important for the research that we carry out Agasicles hygrophila OR gene in a deep going way Basic data.
SEQUENCE LISTING
<110>Fujian Academy of Agricultural Sciences's Agricultural development quality standard and detection technique research institute
<120>primer and method of Agasicles hygrophila OR gene transcription level are detected
<130> 4
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 24
<212> DNA
<213>artificial sequence
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atccttactc aattctggac ttcc 24
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<211> 21
<212> DNA
<213>artificial sequence
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tgcttatctc atgctggaca a 21
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<212> DNA
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agatgtccgc caccttca 18
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<212> DNA
<213>artificial sequence
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gcctcttggt attgttggta ttca 24

Claims (2)

1. a kind of fluorescence quantification PCR primer for detecting Agasicles hygrophila OR gene transcription level, it is characterised in that: including meeting The upstream and downstream primer of quantitative fluorescent PCR reaction characteristics:
OR-F:5`-ATCCTTACTCAATTCTGGACTTCC-3`,
OR-R:5`-TGCTTATCTCATGCTGGACAA-3`.
2. a kind of quantitative fluorescent PCR side using primer detection Agasicles hygrophila OR gene transcription level described in claim 1 Method, it is characterised in that: as follows:
(1) the first chain of cDNA synthesis: extracting the total serum IgE of sample and purifying, uses reverse transcription reagent box to obtain and is with the RNA of extraction The cDNA that template reverse transcription obtains；
(2) Standard PCR detection is carried out to following primer
The fluorescent quantitation upstream and downstream primer of the gene:
OR-F:5`-ATCCTTACTCAATTCTGGACTTCC-3`；
OR-R:5`-TGCTTATCTCATGCTGGACAA-3`；
And the upstream and downstream primer as reference gene:
Tubulin-F: 5`-AGATGTCCGCCACCTTCA-3`；
Tubulin-R: 5`-GCCTCTTGGTATTGTTGGTATTCA-3`；
Standard PCR amplification system is 25 μ L:10 × buffer, 2.5 μ L, dNTP 1.0 μ L, 0.5 μ L of upstream primer, downstream primer 0.5 μ L, 100ng/ μ L cDNA template 1.0 μ L, Ex-TaqE 0.15 μ L, 19.35 μ L of water；
Routine PCR reaction program are as follows: 94 DEG C of 5 min of initial denaturation, then 94 DEG C of 30 s of denaturation, 56 DEG C of 30 s of annealing, 72 DEG C are prolonged 1 min is stretched, under this condition 35 circulations, last 72 DEG C re-extend 10min；
(3) real-time fluorescence quantitative PCR reacts:
Primer described in step (2) is added as template in the cDNA obtained using step (1), carries out quantitative fluorescent PCR reaction, each 3 repetitions are arranged in sample, are averaged after amplification；
Real-time fluorescence quantitative PCR amplification system are as follows: SYBR® Premix Ex TaqTM12.5 μ L, 0.5 μ L of upstream primer, downstream 0.5 μ L, 100ng/ μ L cDNA of primer, 1.0 μ L, supplies water to 25 μ L；
Real-time fluorescence quantitative PCR response procedures are as follows: 95 DEG C of 30 s of initial denaturation, then 95 DEG C of 5 s of denaturation, 60 DEG C of 30 s of annealing, 40 circulations.
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CN104152560A (en) * 2014-08-15 2014-11-19 福建省农业科学院植物保护研究所 Fluorescent quantitative PCR detection kit for transcription level of tubulin gene of Agasicles hygrophila
CN104962630A (en) * 2015-06-30 2015-10-07 福建省农业科学院植物保护研究所 Fluorescence-quantitative PCR detection kit for gene transcription level of agasicles hygrophila actin

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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