CN104131109A - Fluorescent quantitation PCR method for detecting expression characteristic of agasicles hygrophila HSP70 gene - Google Patents
Fluorescent quantitation PCR method for detecting expression characteristic of agasicles hygrophila HSP70 gene Download PDFInfo
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Abstract
The invention discloses a fluorescent quantitation PCR method for detecting an expression characteristic of an agasicles hygrophila HSP70 gene. Based on the establishment of the fluorescent quantitation PCR method for treating the agasicles hygrophila HSP70 gene under different temperatures, the foundation for researching generation mechanisms of the expression characteristic and the heat resistance of the agasicles hygrophila HSP70 gene is laid.
Description
Technical field
The present invention relates to a kind of fluorescence quantitative PCR method that detects Agasicles hygrophila heat shock protein 70 (HSP70) gene expression characteristics, particularly differing temps (37 DEG C-45 DEG C) is processed the lower efficient quick fluorescence quantitative PCR method that detects Agasicles hygrophila HSP70 gene expression amount.
Background technology
Agasicles hygrophila belongs to Coleoptera, and Chrysomelidae is one of main biological and ecological methods to prevent plant disease, pests, and erosion natural enemy of alternanthera philoxeroides.George Vogt has found first that in 1962 Agasicles hygrophila has good control action kou to alternanthera philoxeroides.Thereafter, Agasicles hygrophila is widely used in the control of alternanthera philoxeroides on the ground such as the U.S., Australia, and has obtained good prevention effect.China introduces Agasicles hygrophila in 1986 from the U.S., and is applied to the biological control of alternanthera philoxeroides, in Sichuan, the ground such as Zhejiang, Fujian, Guangxi obtains certain success.
In China, each worm state of Agasicles hygrophila presents corresponding population Changing Pattern with the variation in season in different habitats, particularly high temperature season in full summer, Agasicles hygrophila population quantity fast-descending, easily cause population crash in its in summer, be unfavorable for the biological control of summer high temperature alternanthera philoxeroides in season, and the Agasicles hygrophila population of tropic countries (as Thailand) can breed by normal existence in hot environment, thereby be necessary to study the molecule mechanism that Agasicles hygrophila thermotolerance produces, understand the dependence rule of growing to temperature of Agasicles hygrophila.
Insect can produce heat shock protein(HSP) (heat shock protein, HSP) in body in the time being subjected to heat stress or other inductions, and normal protein synthesis is suppressed.Heat shock protein(HSP) is found in 1962 by Ritossa the earliest in insect.According to the structure of heat shock protein(HSP), function, several extended familys such as being divided into HSP100, HSP90, HSP70, HSP60, HSP20 vary in size, wherein HSP70 is the most conservative in organism and most important heat-shock protein family, stress can highly being induced in cell of nearly all biology.Along with the development of bio-science technology, the research of heat shock protein(HSP) has obtained very large development in recent years.Because insect heat shock protein(HSP) participates in heat-resisting reaction process, therefore study insect HSPs and stable on heating relation, contribute to understand insect grow and temperature between dependency.Research about Agasicles hygrophila HSP70 gene clone and expression has no report both at home and abroad, therefore adopt the expression characterization of Agasicles hygrophila HSP70 gene under real-time fluorescence quantitative PCR research treatment of different temperature, the research of on the one hand comprehensively in depth carrying out Agasicles hygrophila HSP70 gene for us provides basic data, make on the other hand its alternative gene as Agasicles hygrophila breeding, the Agasicles hygrophila kind strong for seed selection in future temperature capacity provides possibility.
Summary of the invention
One of object of the present invention is the different treatment temperature designing for the expression characterization of overall understanding Agasicles hygrophila HSP70 gene.
Another object of the present invention is to provide a kind of real time fluorescence quantifying PCR method of efficient quick detection Agasicles hygrophila HSP70 gene expression amount.
For achieving the above object, the technical solution adopted in the present invention is as follows:
By 5 groups of Agasicles hygrophilas respectively at 37 DEG C, 39 DEG C, 41 DEG C, 43 DEG C and 45 DEG C heat shock process after 1.5 h and recover 1 h at 26 DEG C, each sample does 3 parallel repetitions.
The primer that designs a pair of detection Agasicles hygrophila HSP70 gene expression characteristics, its its feature comprises the upstream and downstream primer that meets quantitative fluorescent PCR reaction characteristics:
HSP70-F：5`- GCAGGTTGATTATCTGCGTATGTT -3`、
HSP70-R：5`- ATTGAGACAGCAGGAGGAGTTATG -3`；
And as the upstream and downstream primer of reference gene:
Tubulin-F: 5`-CAGGTGAAGGTATGGACGAAAT-3`、
Tubulin-R: 5`-GCCTCTTGGTATTGTTGGTATTC-3`；
The fluorescence quantitative PCR method of detection Agasicles hygrophila HSP70 gene expression characteristics of the present invention, its feature is carried out as follows:
(1) cDNA the first chain is synthetic: extract total RNA the purifying of sample, the RNA that adopts reverse transcription test kit to obtain extracting is the cDNA that template reverse transcription obtains;
(2) following primer is carried out to conventional PCR detection
The fluorescent quantitation upstream and downstream primer of this gene:
HSP70-F：5`- GCAGGTTGATTATCTGCGTATGTT -3`
HSP70-R：5`- ATTGAGACAGCAGGAGGAGTTATG -3`
And as the upstream and downstream primer of reference gene:
Tubulin-F: 5`-CAGGTGAAGGTATGGACGAAAT-3`
Tubulin-R: 5`-GCCTCTTGGTATTGTTGGTATTC-3`
Conventional pcr amplification system is as follows:
Conventional PCR response procedures is as follows: 94 DEG C of denaturation 5 min, and then 94 DEG C of sex change 30sec, 56 DEG C of annealing 30sec, 72 DEG C are extended 1min, circulate 35 times, and last 72 DEG C are extended 10min again;
(3) real-time fluorescence quantitative PCR reaction:
The cDNA obtaining taking step (1), as template, adds the described primer of step (2), carries out the reaction of quantitative fluorescent PCR program, and each sample arranges 3 repetitions, after amplification, averages.
Real-time fluorescence quantitative PCR amplification system is as follows:
Real-time fluorescence quantitative PCR response procedures is as follows: 95 DEG C of denaturation 3 min, and then 95 DEG C of sex change 10sec, 60 DEG C of annealing 30sec, circulate 40 times.
The more detailed test method of the present invention is as follows:
The real-time fluorescence quantitative PCR detection method of Agasicles hygrophila HSP70 gene can be realized by following steps:
(1) design of primers: the sequence total length of the Agasicles hygrophila HSP70 gene obtaining according to clone, use DNAMAN software design to be applicable to the Auele Specific Primer of fluorescence quantitative PCR detection, primer sequence is as follows:
HSP70-F：5`- GCAGGTTGATTATCTGCGTATGTT -3`
HSP70-R：5`- ATTGAGACAGCAGGAGGAGTTATG -3`
Meanwhile, the sequence total length of the Agasicles hygrophila tubulin gene obtaining according to clone, is designed for the primer of quantitative fluorescent PCR internal reference thing, and primer sequence is as follows:
Tubulin-F: 5`-CAGGTGAAGGTATGGACGAAAT-3`
Tubulin-R: 5`-GCCTCTTGGTATTGTTGGTATTC-3`
(2) Agasicles hygrophila is processed test: gather Agasicles hygrophila adult in Eppendorf pipe in room temperature (26 DEG C), have each 3 of male and female Agasicles hygrophila, totally 5 groups of samples in each pipe.By 5 groups of Agasicles hygrophilas respectively at 37 DEG C, 39 DEG C, 41 DEG C, 43 DEG C and 45 DEG C heat shock process after 1.5 h and recover 1 h at 26 DEG C, each sample does 3 parallel repetitions.Worm sample is put into rapidly liquid nitrogen after processing fixing, saves backup in-70 DEG C.
(3) cDNA the first chain is synthetic: the Total RNA Kit test kit specification sheets with reference to E.Z.N.A extracts total RNA, according to the One-Step gDNA Removal and cDNA Synthesis SuperMix test kit specification sheets of TransScript company, synthetic cDNA the first chain.
(4) real-time fluorescence quantitative PCR reaction: real-time fluorescence quantitative PCR adopts the SYBR Premix Ex Taq of Takara company
tMtest kit carries out.Taking above-mentioned synthetic cDNA the first chain as template, above-mentioned HSP70-F, HSP70-R and Tubulin-F, Tubulin-R are Auele Specific Primer, carry out quantitative fluorescent PCR program, and each sample is established 3 parallel repetitions, gets the mean number of the parallel Ct value of gained after amplification.
Real-time fluorescence quantitative PCR amplification system is as follows:
Real-time fluorescence quantitative PCR response procedures is as follows: 95 DEG C of denaturation 3 min, and then 95 DEG C of sex change 10sec, 60 DEG C of annealing 30sec, circulate 40 times.
(5) Agasicles hygrophila HSP70 gene with respect to the calculation formula of the expression amount of tubulin gene is: mrna expression=2 relatively
-Δ Δ Ct× 100%, wherein Ct value=target gene Ct value-tubulin Ct value.
(6) Fig. 3 is that the expression of Agasicles hygrophila HSP70 gene after differing temps heat shock changes.Result shows, along with the rising of heat-shock temperature, the expression amount of HSP70 gene all raises gradually, has reached climax in the time of 41 DEG C, is 159 times of blank, and after this, along with the rising of temperature, the expression amount of HSP70 gene declines.This provides important basic data for the research that we carry out Agasicles hygrophila HSP70 gene in a deep going way.
The present invention has following advantage and effect with respect to prior art:
(1) the treatment of different temperature measure in the present invention, is all suitable for insect overwhelming majority heat shock protein gene, and this has great importance to comprehensive further investigation gene expression characteristics.
(2) fluorescence quantitative PCR method of efficient quick of the present invention, comprises quantitative fluorescent PCR program etc., and actual used times 55 min, has shortened the reaction times greatly with respect to prior art, has the characteristic of efficient quick.
(3) fluorescence quantitative PCR method of the present invention, provides conventional PCR electrophoresis result figure, can intuitively reflect efficiently the specificity of primer.
Brief description of the drawings
1500,1000,900,800,700,600,500,400,300,200,100bp Fig. 1: the conventional PCR product of Agasicles hygrophila HSP70 gene by fluorescence quantitative primer electrophoresis result: product length is 105 bp, and Marker band is followed successively by from top to bottom:.
1500,1000,900,800,700,600,500,400,300,200,100bp Fig. 2: the conventional PCR product of Agasicles hygrophila tubulin gene by fluorescence quantitative primer electrophoresis result: product length is 88 bp, and Marker band is followed successively by from top to bottom:.
Fig. 3: the differential expression of Agasicles hygrophila HSP70 gene after differing temps heat shock.
Specific embodiments
Below in conjunction with specific embodiment and brief description of the drawings the present invention; the scheme of embodiment described here; do not limit invention; other are any does not deviate from change, the modification made under spirit of the present invention and principle, substitute, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.
Embodiment 1
The processing of test materials
Gather Agasicles hygrophila adult in Eppendorf pipe in room temperature (26 DEG C), have each 3 of male and female Agasicles hygrophila in each pipe, each sample does 3 parallel repetitions, totally 5 groups of samples.By 5 groups of Agasicles hygrophilas respectively at 37 DEG C, 39 DEG C, 41 DEG C, 43 DEG C and 45 DEG C heat shock process after 1.5 h and recover 1 h at 26 DEG C, remaining 1 group is blank group, does not do heat shock processing.Worm sample is put into rapidly liquid nitrogen after processing fixing, saves backup in-70 DEG C.
Embodiment 2
The design of primer
(1) design of primers: the sequence total length of the Agasicles hygrophila HSP70 gene obtaining according to clone, use DNAMAN software design to be applicable to the Auele Specific Primer of fluorescence quantitative PCR detection, primer sequence is as follows:
HSP70-F：5`- GCAGGTTGATTATCTGCGTATGTT -3`
HSP70-R：5`- ATTGAGACAGCAGGAGGAGTTATG -3`
Agasicles hygrophila HSP70 gene by fluorescence quantitative PCR product estimates that length is 105bp, agarose gel electrophoresis result shows that the product length after amplification is consistent, and be single band, illustrate that designed primer has very strong specificity, be suitable for real-time fluorescence quantitative PCR and detect, agarose gel electrophoresis result as shown in Figure 1.
(2) simultaneously, the sequence total length of the Agasicles hygrophila tubulin gene obtaining according to clone, is designed for the primer of quantitative fluorescent PCR internal reference thing, and primer sequence is as follows:
Tubulin-F: 5`-CAGGTGAAGGTATGGACGAAAT-3`
Tubulin-R: 5`-GCCTCTTGGTATTGTTGGTATTC-3`
Agasicles hygrophila tubulin gene by fluorescence quantitative PCR product estimates that length is 88 bp, agarose gel electrophoresis result shows that the product length after amplification is consistent, and be single band, illustrate that designed primer has very strong specificity, be suitable for real-time fluorescence quantitative PCR and detect, agarose gel electrophoresis result as shown in Figure 2.
Embodiment 3
The extraction of total RNA
Total RNA Kit test kit specification sheets with reference to E.Z.N.A extracts total RNA, polypide is pulverized with liquid nitrogen, add 1ml RNA solo Reagent, forwarding 1.5 ml to leaves in core barrel, room temperature leaves standstill after 2 ~ 3 min, add 200 ul chloroform/1 ml RNA solo Reagent, vortex vibrates 15 s, 10 min on ice.After 4 DEG C of 13000 centrifugal 15 min of g, move upper strata water (general <80%) and, in new centrifuge tube, add the dehydrated alcohol (room temperature) of 1/3 volume, whirlpool 15 s.RNA Combining bolt is placed in 2 ml collection tubes, upper step mixture is all proceeded in Combining bolt, 10000 g, 30 ~ 60 s are centrifugal, abandon moving phase, reuse collection tube.Add 300ul RNA wash Buffer I, room temperature is centrifugal, 10000 g, and 30 ~ 60 s, abandon moving phase.Add 400 ul RNA wash Buffer I, room temperature is placed 5 min, centrifugal, abandons moving phase again.The 500 ul RNA wash Buffer II that add dilution, 1000 g 30 ~ 60 s are centrifugal, abandon moving phase.13000 g 2 min skies from.RNA Combining bolt is proceeded to 1.5 clean ml centrifuge tubes, add 30 ul DEPC water of 70 DEG C of preheatings, dissolve RNA, room temperature leaves standstill 5 min, centrifugal 1 min of 13000 g.The RNA of gained can be used for reverse transcription experiment.
Embodiment 4
CDNA the first chain is synthetic: according to the One-Step gDNA Removal and cDNA Synthesis SuperMix test kit specification sheets step of TransScript company, taking the RNA in embodiment 3 as template, reverse transcription generates cDNA the first chain, and concrete steps are as follows:
(1) reverse transcription system
(2) 42 DEG C of incubation 30 min.
(3) 85 DEG C of heating 5 min, product can be placed on-20 DEG C ,-70 DEG C preservations.
Embodiment 5
Carry out the reaction of quantitative fluorescent PCR program.Real-time glimmering amount PCR adopts the SYBR Premix Ex Taq of Takara company
tMtest kit carries out.Taking the cDNA in embodiment 4 as template, carry out real-time fluorescence quantitative PCR amplified reaction with the primer in embodiment 2, each sample is established 3 parallel repetitions, gets the mean number of the parallel Ct value of gained after amplification.
(1) real-time fluorescence quantitative PCR amplification system is as follows:
(2) real-time fluorescence quantitative PCR response procedures is as follows: 95 DEG C of denaturation 3 min, and then 95 DEG C of sex change 10sec, 60 DEG C of annealing 30sec, circulate 40 times.
(3) after real-time fluorescence PCR completes, the ratio 2 of relative expression quantity under the time period of setting according to Ct value calculating different treatment condition
-Δ Δ Ct.Agasicles hygrophila HSP70 gene with respect to the calculation formula of the expression amount of tubulin gene is: mrna expression=2 relatively
-Δ Δ Ct× 100%, wherein Ct value=target gene Ct value-tubulin Ct value.
(4) Fig. 3 is that the expression of Agasicles hygrophila HSP70 gene after differing temps heat shock changes.Result shows, along with the rising of heat-shock temperature, the expression amount of HSP70 gene all raises gradually, has reached climax in the time of 41 DEG C, is 159 times of blank, and after this, along with the rising of temperature, the expression amount of HSP70 gene declines.This provides important basic data for the research that we carry out Agasicles hygrophila HSP70 gene in a deep going way.
The foregoing is only preferred embodiment of the present invention, all equalizations of doing according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
SEQUENCE LISTING
<110> Inst. of Plant Protection, fujian Academy of Agricultural Science
<120> detects the fluorescence quantitative PCR method of Agasicles hygrophila HSP70 gene expression characteristics
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<170> PatentIn version 3.3
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gcaggttgat tatctgcgta tgtt 24
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attgagacag caggaggagt tatg 24
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<212> DNA
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caggtgaagg tatggacgaa at 22
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Claims (2)
1. an Auele Specific Primer that detects Agasicles hygrophila HSP70 gene expression characteristics, is characterized in that: comprise the upstream and downstream primer that meets quantitative fluorescent PCR reaction characteristics:
HSP70-F：5`- GCAGGTTGATTATCTGCGTATGTT -3`、
HSP70-R：5`- ATTGAGACAGCAGGAGGAGTTATG -3`；
And as the upstream and downstream primer of reference gene:
Tubulin-F: 5`-CAGGTGAAGGTATGGACGAAAT-3`、
Tubulin-R: 5`-GCCTCTTGGTATTGTTGGTATTC-3`。
2. a fluorescence quantitative PCR method that detects Agasicles hygrophila HSP70 gene expression characteristics, is characterized in that, comprises the following steps:
(1) Agasicles hygrophila is processed test: gather Agasicles hygrophila adult in Eppendorf pipe 26 DEG C of room temperatures, have each 3 of male and female Agasicles hygrophila, totally 5 groups of samples in each pipe; By 5 groups of Agasicles hygrophilas respectively at 37 DEG C, 39 DEG C, 41 DEG C, 43 DEG C and 45 DEG C heat shock process after 1.5 h and recover 1 h at 26 DEG C, each sample does 3 parallel repetitions;
(2) cDNA the first chain is synthetic: extract total RNA the purifying of sample, the RNA that adopts reverse transcription test kit to obtain extracting is the cDNA that template reverse transcription obtains;
(3) following primer is carried out to conventional PCR detection:
The fluorescent quantitation upstream and downstream primer of this gene:
HSP70-F：5`- GCAGGTTGATTATCTGCGTATGTT -3`、
HSP70-R：5`- ATTGAGACAGCAGGAGGAGTTATG -3`；
And as the upstream and downstream primer of reference gene:
Tubulin-F: 5`-CAGGTGAAGGTATGGACGAAAT-3`、
Tubulin-R: 5`-GCCTCTTGGTATTGTTGGTATTC-3`；
Conventional pcr amplification system is as follows:
Conventional PCR response procedures is as follows: 94 DEG C of denaturation 5 min, and then 94 DEG C of sex change 30sec, 56 DEG C of annealing 30sec, 72 DEG C are extended 1min, circulate 35 times, and last 72 DEG C are extended 10min again;
(4) real-time fluorescence quantitative PCR reaction:
The cDNA obtaining taking step (2), as template, adds the described primer of step (3), carries out the reaction of quantitative fluorescent PCR program, and each sample arranges 3 repetitions, after amplification, averages;
Real-time fluorescence quantitative PCR amplification system is as follows:
Real-time fluorescence quantitative PCR response procedures is as follows: 95 DEG C of denaturation 3 min, and then 95 DEG C of sex change 10sec, 60 DEG C of annealing 30sec, circulate 40 times.
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| 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 |
| CN105274239A (en) * | 2015-11-19 | 2016-01-27 | 浙江省农业科学院 | Method for screening reference genes for chilo suppressalis under temperature stress and application of reference genes |
| CN108998548A (en) * | 2018-09-19 | 2018-12-14 | 福建省农业科学院植物保护研究所 | Detect the primer and method of Agasicles hygrophila TnT gene expression characteristics |
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| CN109055507A (en) * | 2018-09-19 | 2018-12-21 | 福建省农业科学院植物保护研究所 | Detect the primer and method of Agasicles hygrophila EF gene expression characteristics |
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| CN109182548A (en) * | 2018-10-22 | 2019-01-11 | 福建省农业科学院农业质量标准与检测技术研究所 | Detect the primer and method of Agasicles hygrophila GR gene expression characteristics |
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