CN109022598A - Detect the primer and its method of Agasicles hygrophila CP gene expression characteristics - Google Patents
Detect the primer and its method of Agasicles hygrophila CP gene expression characteristics Download PDFInfo
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Abstract
The present invention provides the primer and its method of detection Agasicles hygrophila CP gene expression characteristics, primer includes two pairs, and sequence establishes the female male worm feeding difference CO of Agasicles hygrophila as shown in SEQ ID NO.1-4, using the primer2The fluorescence quantitative PCR method that CP 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 CP gene by fluorescence quantitative with the expression quantity trend of transcript profile, the defense mechanism and CP gene expression characteristics generated when for research Agasicles hygrophila by environment-stress is laid a good foundation.
Description
Technical field
The present invention relates to the primers and its method of detection Agasicles hygrophila CP gene expression characteristics, 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.
Insect cuticle albumen (cuticular proteins, CP) is structural proteins, it and chitin form insect together
Resist the cuticula of external environment barrier.The joint effects insect cuticle protein gene such as environment, hormone, transcription factor and introne
Expression, make CP gene have period and tissue specificity.CP gene is considered as studying insect molting and abnormal regulation
Mechanism and the important models for understanding modification of the insect growth phase cuticula in biochemistry, physical chemistry and structure, by
It is more and more to pay attention to.
The environment-stress such as drying, hyperosmosis, seasonal photoperiod make insect generate many biochemical reactions, and thus
Cause the activation and silencing of a series of related genes, to reflect insect to the tolerance of environment-stress.Therefore, epidermis conduct
Insect resists the first line of defence of external environment, when insect is by environmental conditions such as drying stress, osmotic stress, seasonal photoperiods
When influence, the expression regulation function of CP will make respective reaction.Zhang etc. (2008) research discovery is dried potato first
Worm can induce CP gene expression, and CP gene is not expressed when humidity is handled, they also found that insecticide azinphos-methyl can be lured highly
The expression of colorado potato bug epidermal protein gene is led, and the insect is made to generate drug resistance.Le Trionnaire etc. (2007) exists
Pea aphid head is identified by daytime long 59 different transcripts for shortening regulation, wherein having 19 is to encode not
Thus same CP explains that the photoperiod can influence regulation of the expression regulation CP expression in other words by photoperiodic signal of CP, and
It may be related to the development of the epidermis of insect.
The researchs such as the expression about Agasicles hygrophila CP gene have not been reported both at home and abroad, therefore use real time fluorescent quantitative
PCR studies the female male worm feeding difference CO of Agasicles hygrophila2The expression characterization of CP gene after the alternanthera philoxeroides that concentration is cultivated, knot
Fruit shows with CO2The expression quantity of the raising of concentration, female male worm CP gene increases, the change of this and the up-regulation of transcript profile expression quantity
Change trend is consistent, illustrates CO2Concentration increases the expression regulation that can influence CP gene.This in depth carries out the straight chest of lotus grass to be comprehensive
The research of flea beetle CP gene provides basic data, further discloses the inherent law of CP gene expression regulation, thus promote CP with
The relation mechanism of insectean metamorphosis development.
Summary of the invention
The purpose of the present invention is to provide the primers and its method of detection Agasicles hygrophila CP gene expression characteristics.
The difference that it is another object of the present invention to design to fully understand the expression characterization of Agasicles hygrophila CP gene
Handle CO2Concentration.
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 CP gene expression characteristics of design, including meet quantitative fluorescent PCR reaction
The upstream and downstream primer of feature:
CP-F:5`-TGTAGCAACGGGAGCAGATA-3`；
CP-R:5`-TACGCCGCACCTGTATCATA-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 CP gene expression characteristics 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:
CP-F:5`-TGTAGCAACGGGAGCAGATA-3`
CP-R:5`-TACGCCGCACCTGTATCATA-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 as follows:
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 is as follows:
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.
The more detailed test method of the present invention is as follows:
The real-time fluorescence quantitative PCR detection method of Agasicles hygrophila CP gene can be realized by following steps:
(1) design of primers: soft using DNAMAN according to the sequence for the Agasicles hygrophila CP 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:
CP-F:5`-TGTAGCAACGGGAGCAGATA-3`
CP-R:5`-TACGCCGCACCTGTATCATA-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 Takara company® Premix Ex
TaqTMKit carries out.Using the first chain of cDNA of above-mentioned synthesis as template, above-mentioned CP-F, CP-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 is as follows:
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 annealing 30
S, 40 circulations.
(5) calculation formula of the Agasicles hygrophila CP 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 CP gene.The result shows that with CO2The expression quantity of the raising of concentration, female male worm CP 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 CP 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 epidermal protein 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 substantially reduces the reaction time 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 CP gene transcription level of Agasicles hygrophila.
Fig. 1: Agasicles hygrophila CP gene by fluorescence quantitative primer Standard PCR product electrophoresis result: product length 102
Bp,
Marker band is 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 is 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 CP 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 CP 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.
The design of 2 primer of embodiment
(1) it design of primers: according to the sequence for the Agasicles hygrophila CP 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:
CP-F:5`-TGTAGCAACGGGAGCAGATA-3`
CP-R:5`-TACGCCGCACCTGTATCATA-3`
Agasicles hygrophila CP gene by fluorescence quantitative PCR product length is 102 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.
The extraction of 3 total serum IgE of embodiment
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 Takara 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 is as follows:
(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 CP 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 feeding difference CO2Concentration cultivate alternanthera philoxeroides after CP gene C (T) value,
Average value, standard deviation, fluorescent quantitation expression quantity and transcript profile expression quantity
Table 2: Agasicles hygrophila male worm feeding difference CO2It is C (T) value of CP gene after the alternanthera philoxeroides that concentration is cultivated, average
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 hygrophila2CP 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 CP 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 CP gene in a deep going way
Basic data.
SEQUENCE LISTING
<110>Xuan China is opened
<120>primer and its method of Agasicles hygrophila CP gene expression characteristics are detected
<130> 4
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>artificial sequence
<400> 1
tgtagcaacg ggagcagata 20
<210> 2
<211> 20
<212> DNA
<213>artificial sequence
<400> 2
tacgccgcac ctgtatcata 20
<210> 3
<211> 18
<212> DNA
<213>artificial sequence
<400> 3
agatgtccgc caccttca 18
<210> 4
<211> 24
<212> DNA
<213>artificial sequence
<400> 4
gcctcttggt attgttggta ttca 24
Claims (2)
1. a kind of quantitative fluorescent PCR specific primer for detecting Agasicles hygrophila CP gene expression characteristics, it is characterised in that: institute
Stating primer includes the upstream and downstream primer for meeting quantitative fluorescent PCR reaction characteristics:
CP-F:5`-TGTAGCAACGGGAGCAGATA-3`,
CP-R:5`-TACGCCGCACCTGTATCATA-3`；
And the upstream and downstream primer as reference gene:
Tubulin-F:5`-AGATGTCCGCCACCTTCA-3`,
Tubulin-R: 5`-GCCTCTTGGTATTGTTGGTATTCA-3`。
2. a kind of fluorescent quantitation using the detection Agasicles hygrophila CP gene expression characteristics of specific primer described in claim 1
PCR method, characterized by the following steps:
(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 using following primer
The fluorescent quantitation upstream and downstream primer of the gene:
CP-F:5`-TGTAGCAACGGGAGCAGATA-3`,
CP-R:5`-TACGCCGCACCTGTATCATA-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 TaqTM 12.5 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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