CN109182548A - Detect the primer and method of Agasicles hygrophila GR gene expression characteristics - Google Patents
Detect the primer and method of Agasicles hygrophila GR gene expression characteristics Download PDFInfo
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Abstract
The present invention provides the primers and method of detection Agasicles hygrophila GR gene expression characteristics, 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 GR 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 GR gene by fluorescence quantitative with the expression quantity trend of transcript profile, the defense mechanism and GR 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 method of detection Agasicles hygrophila GR 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.
Smell and gustatory system play a crucial role in the existence and reproductive process of insect, and insect passes through it
Sensitive chemocepter identify a variety of different chemical substances complete to look for food, feeding, mating and selection oviposition place
Etc. processes, by some nonvolatile matters in taste perception system identification environment, to distinguish nutriment and avoid
Noxious material.Taste perception nerve is distributed widely in insect body surface, after different nerve cells is stimulated, can cause difference
Behavior reaction.Insect can be made to open lip generation trophic behaviour for example, the gustatory nerve of insect foreleg is stimulated, and stimulated
Gustatory nerve on oopod can then cause mating and the oviposition behavior of insect.
19 bases with 7 transmembrane domains that Clyne etc. (2000) is screened in the portion gene group of drosophila
Cause, by PCR detection discovery wherein have 18 genes be on lip it is specifically expressed, by this 18 special tables of lip
The gene reached is defined as sense of taste gene.The remaining gustatory receptor genes of drosophila are then identified successively again, and drosophila shares 60
A gustatory receptor genes can encode 68 kinds of taste receptor proteins, this be by the gene expression pattern of alternative splicing come
It realizes.The GR gene of other type insects is then also in succession identified to be come out.
Homology between taste receptors is very low, generally 8% ~ 12%.It is identified using the method for bioinformatics
A large amount of gustatory receptor genes are arrived, but the function of most of taste receptors is unclear, it may be possible to because of the expression of GR itself
It measures very low, it is not easy to it be positioned using conventional method.In recent years, the transgenic strain research GR function of drosophila is started with
Can, it is concentrated mainly on the bitterness receptors, sweet receptor, CO of drosophila2Receptor and both sexes trail receptor.Wherein drosophila GR5a
The albumen of coding can identify and perceive trehalose, but it is not the receptor that can uniquely experience trehalose, and GR64f can also be with
Reaction of the drosophila to trehalose is influenced, illustrates that the different taste receptors of insect may combine identical chemical substance.The insect sense of taste
Receptor is lacked in Drosophila Antennapedia nerve except in addition to gustatory organ is expressed, part taste receptors are also expressed in olfactory organ
Gr63a can make drosophila lose identification CO2Ability, silencing or knock out Gr21a gene can also enable drosophila to CO2Perception energy
Power reduces, and when some gene individualism only in Gr63a and Gr21a can not be such that drosophila generates to CO2It is quick
Perception, however only when Gr63a and Gr21a is co-expressed, drosophila just has to CO2Sensing capability, this illustrates Gr63a
It participates in Gr21a collaboration to CO2Perception.Recent studies have shown that the sense of taste of insect and oviposition behavior may have it is close
Relationship, Ryan(2012) research drosophila discovery drosophila front foot Gr66a may be related with oviposition behavior.
Compared with olfactory receptor, the functional study of taste receptors is relatively fewer.The emphasis studied from now on is exactly to difference
Taste receptors carry out the interaction between large-scale functional analysis and taste receptors upstream and downstream albumen in insect,
To deeper into anatomy taste perception nervous system complex network, probe into the generation of Taste Signals, transmitting and terminate machine
Reason will finally illustrate the molecular mechanism of the behaviors such as regulation insect's food-taking, mating.The expression of taste receptors is extremely low, benefit
With in situ hybridization, the methods of fluorescent transgenic protein gene expression can intuitively position the chemistry sense of these Receptor Gene Expressions
By nerve, and transgenic technology and heterologous expression system by be future studies taste receptors function effective means.About lotus
The researchs such as the expression of careless straight chest flea beetle GR gene have not been reported both at home and abroad, therefore use real-time fluorescence quantitative PCR research difference CO2
The expression characterization of the lower Agasicles hygrophila GR gene of concentration processing, the results showed that, with CO2The raising of concentration, female male worm GR base
The expression quantity of cause increases, this is consistent with the variation tendency of transcript profile expression quantity up-regulation, illustrates CO2Concentration raising can influence GR
The expression regulation of gene.Understood fully from molecular level these chemoreceptors function can be more deep announcement insect chemistry sense
The regulatory mechanism received, and the relationship asked with complex behaviors such as insect feeding habits.To utilize genetic engineering means, for special
Gustatory receptor genes design new strategy of insect pest control.Conventional pesticides are easy to produce drug resistance, and effect is unstable, and
And it will cause environmental pollution.Therefore insect lipids are controlled using molecular biology method and carries out control of insect with important
Realistic meaning.
Summary of the invention
The object of the present invention is to provide the primers and method of detection Agasicles hygrophila GR gene expression characteristics, are comprehensive
The different disposal CO for solving the expression characterization of Agasicles hygrophila GR 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 GR gene expression characteristics of design, feature include meeting fluorescent quantitation
The upstream and downstream primer of PCR reaction characteristics:
GR-F:5`-AGAGTGAAGGAAGACCGTAAGATTA-3`
GR-R:5`-ACTGCTATGATGGACCTGATGA-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 GR gene expression characteristics of the present invention, feature is as follows
It carries out:
(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:
GR-F:5`-AGAGTGAAGGAAGACCGTAAGATTA-3`
GR-R:5`-ACTGCTATGATGGACCTGATGA-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 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 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 GR gene can be realized by following steps:
(1) design of primers: soft using DNAMAN according to the sequence for the Agasicles hygrophila GR 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:
GR-F:5`-AGAGTGAAGGAAGACCGTAAGATTA-3`
GR-R:5`-ACTGCTATGATGGACCTGATGA-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 TGRara company® Premix Ex
TaqTMKit carries out.Using the first chain of cDNA of above-mentioned synthesis as template, above-mentioned GR-F, GR-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 GR 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 GR gene.The result shows that with CO2The expression quantity of the raising of concentration, female male worm GR 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 GR 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 gustatory receptor genes, 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 GR gene transcription level of Agasicles hygrophila.
Fig. 1: Agasicles hygrophila GR 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 GR 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 GR 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 GR 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:
GR-F:5`-AGAGTGAAGGAAGACCGTAAGATTA-3`
GR-R:5`-ACTGCTATGATGGACCTGATGA-3`
Agasicles hygrophila GR 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 TGRara 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:
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 supply 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 GR 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 GR 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 GR 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 hygrophila2GR 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 GR 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 GR 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 GR gene expression characteristics are detected
<130> 4
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 25
<212> DNA
<213>artificial sequence
<400> 1
AGAGTGAAGGAAGACCGTAAGATTA 25
<210> 2
<211> 22
<212> DNA
<213>artificial sequence
<400> 2
ACTGCTATGATGGACCTGATGA 22
<210> 3
<211> 18
<212> DNA
<213>artificial sequence
<400> 3
AGATGTCCGCCACCTTCA 18
<210> 4
<211> 24
<212> DNA
<213>artificial sequence
<400> 4
GCCTCTTGGTATTGTTGGTATTCA 24
Claims (2)
1. a kind of fluorescence quantification PCR primer for detecting Agasicles hygrophila GR gene expression characteristics, it is characterised in that: including meeting
The upstream and downstream primer of quantitative fluorescent PCR reaction characteristics:
GR-F:5`-AGAGTGAAGGAAGACCGTAAGATTA-3`,
GR-R:5`-ACTGCTATGATGGACCTGATGA-3`.
2. a kind of quantitative fluorescent PCR side using primer detection Agasicles hygrophila GR gene expression characteristics 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 with following primer
The fluorescent quantitation upstream and downstream primer of the gene:
GR-F:5`-AGAGTGAAGGAAGACCGTAAGATTA-3`
GR-R:5`-ACTGCTATGATGGACCTGATGA-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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| CN111280165A (en) * | 2020-03-04 | 2020-06-16 | 山西农业大学 | Low-temperature preservation method for agasicles hygrophila eggs |
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| CN111280165A (en) * | 2020-03-04 | 2020-06-16 | 山西农业大学 | Low-temperature preservation method for agasicles hygrophila eggs |
| CN111280165B (en) * | 2020-03-04 | 2021-10-29 | 山西农业大学 | Low-temperature preservation method for agasicles hygrophila eggs |
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