CN109182552A - Detect the primer and method of Agasicles hygrophila CSP gene expression characteristics - Google Patents

Detect the primer and method of Agasicles hygrophila CSP gene expression characteristics Download PDF

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CN109182552A
CN109182552A CN201811232459.9A CN201811232459A CN109182552A CN 109182552 A CN109182552 A CN 109182552A CN 201811232459 A CN201811232459 A CN 201811232459A CN 109182552 A CN109182552 A CN 109182552A
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primer
csp
agasicles hygrophila
cdna
gene
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郑丽祯
傅建炜
何肖云
林凌鸿
王秋月
李建宇
史梦竹
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Institute of Plant Protection of FAAS
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Abstract

The present invention provides the primers and method of detection Agasicles hygrophila CSP 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 CSP 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 CSP gene by fluorescence quantitative with the expression quantity trend of transcript profile, the defense mechanism and CSP 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 CSP gene expression characteristics
Technical field
The present invention relates to the primers and method of detection Agasicles hygrophila CSP 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 passes through long-term evolution, and develop complicated, sensitive out and selective chemoreception mechanism.Insect relies on Chemoreception is completed to exchange with extraneous chemical information, and the feeding, mating, female of insect are instructed by identifying these information It lays eggs and hides the behaviors such as natural enemy, insect is to the chemical sensor that the identification of semiochemical is usually by insect.McKenna A kind of soluble protein, referred to as OS-D family protein are had found in Drosophila Antennapedia first Deng using abatement cDNA technology (olfactory specific-D)；Pikielny is identified and is obtained -10 egg of another albumin A in Drosophila Antennapedia It is white.It due to participating in the chemical information in perception environment, and is distributed in the chemocepter of insect, because of referred to herein as chemoreception PROTEIN C SPs, the CSPs of subsequent various insects are found successively.
CSPs is a kind of albumen with compared with low relative molecular mass, relative molecular mass substantially 13k Da, composition For the amino acid number of CSP between 100 ~ 115,2 level structures have 6 alpha-helixes, the Conserved cysteines of CSPs Site contains only 4, this 4 conservative cysteines can form 2 disulfide bond, and the evolutionary conservatism of CSPs compares Height is not only kept between the different CSPs of same insect compared with high homology, and different mesh and it is not equal between homology Also higher homology is maintained, such as the homology of Asiatic migrotory locust and desert locust CSPs are 50% ~ 60%, Lepidoptera and straight The homology of wing mesh CSPs is 37% ~ 50%.The expression and distribution position of CSPs is very extensive, and insect CSPs is removed to express in feeler In addition, it can also be expressed at other positions, such as sexual gland, labipalp, beak, foot etc..The CSPs of silkworm and black cutworm is through grinding Studying carefully discovery can express in sexual gland, and the CSP4 of bollworm and tobacco budworm can be expressed in beak.Therefore, insect CSPs is expressed Than wide, type is more various, and distribution is commonplace in inter-species kind, these features are conducive to adapt to insect in external environment The chemical stimulation of varied complexity undertakes more complicated chemoreception function.
Insect CSPs assumes responsibility for diversified physiological function, comprising: and it (1) combines and transports different lyophilised ligands, this It is the most basic function of insect CSPs.Briand etc. shows that the ASP3c in Apis mellifera feeler can using fluorescence Binding experiment With bound fat and esters, but not in conjunction with general scent molecule and pheromones；(2) possess part olfactory function, immune group Change display, the CSP1-5 of desert locust can be expressed in the lymph of antennal sensilla as OBPs, therefore many researchers Think that these CSPs may have part olfactory function concurrently；(3) insect circadian rhythm is adjusted, such as PEB III may result in drosophila It develops into smell and weakens saltant type；(4) immunization, PEB III biological rhythm of adjustable insect but also can not only join With tissue repair and identify outside invading object；(5) insect growth is adjusted, Guo etc. is using RNA interference method the study found that east Several CSPs and one of sub- migratory locustsTakeout Mutually turn between gene coordinated regulation Asiatic migrotory locust gregarious phase and type of living scattered Change.
The researchs such as the expression about Agasicles hygrophila CSP gene have not been reported both at home and abroad, therefore fixed using real-time fluorescence It measures PCR and studies different CO2The expression characterization of the lower Agasicles hygrophila CSP gene of concentration processing, the results showed that, with CO2Concentration It increases, the expression quantity of female male worm CSP gene increases, this is consistent with the variation tendency of transcript profile expression quantity up-regulation, illustrates CO2It is dense Degree increases the expression regulation that can influence CSP gene.Knot of the related gene as target, according to molecule is experienced by insect chemistry Figure drug is matched in structure design, is future in control of insect research field to research and develop pest Behavioral interference compound of new generation Direction of advance.The chemoreception gene for studying Agasicles hygrophila can be established for the research of Agasicles hygrophila chemoreception mechanism Determine molecular basis, is the function and Agasicles hygrophila of subsequent further investigation Agasicles hygrophila chemoreception GAP-associated protein GAP Smell mechanism lays the foundation.
Summary of the invention
The purpose of the present invention is detecting the primer and method of Agasicles hygrophila CSP gene expression characteristics, to fully understand The expression characterization of Agasicles hygrophila CSP gene and the different disposal CO designed2Concentration.
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 CSP gene expression characteristics of design, including meet quantitative fluorescent PCR reaction The upstream and downstream primer of feature:
CSP-F:5`-GCAGCCAAAAGCAGAAAGAAG-3`
CSP-R:5`-ATCCTCATCATCGCCTCTAAGT-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 CSP 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:
CSP-F:5`-GCAGCCAAAAGCAGAAAGAAG-3`
CSP-R:5`-ATCCTCATCATCGCCTCTAAGT-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 CSP gene can be realized by following steps:
(1) design of primers: according to the sequence for the Agasicles hygrophila CSP gene that transcript profile sequencing result obtains, DNAMAN is used Software design is suitble to the specific primer of fluorescence quantitative PCR detection, and primer sequence is as follows:
CSP-F:5`-GCAGCCAAAAGCAGAAAGAAG-3`
CSP-R:5`-ATCCTCATCATCGCCTCTAAGT-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 CSP-F, CSP-R and Tubulin-F, Tubulin-R is specific primer, carries out quantitative fluorescent PCR program, and each sample sets 3 parallel repetitions, gained is taken after amplification The average of parallel Ct value.
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, 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 CSP 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.
(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 CSP gene.The result shows that with CO2The expression quantity of the raising of concentration, female male worm CSP gene increases, and with The expression quantity variation tendency of transcript profile is consistent, this provides weight for the research that we carry out Agasicles hygrophila CSP gene in a deep going way The basic data wanted.
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 chemosensory protein gene, this Have great importance to comprehensive further investigation gene expression characteristics.
(2) fluorescence quantitative PCR method of efficient quick of the present invention, including quantitative fluorescent PCR program etc., practical used time 55 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 CSP gene transcription level of Agasicles hygrophila.
Fig. 1: Agasicles hygrophila CSP gene by fluorescence quantitative primer Standard PCR product electrophoresis result: product length 192 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 CSP 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 CSP 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) design of primers: according to the sequence for the Agasicles hygrophila CSP gene that transcript profile is sequenced, DNAMAN software is used The specific primer for being suitble to fluorescence quantitative PCR detection is designed, primer sequence is as follows:
CSP-F:5`-GCAGCCAAAAGCAGAAAGAAG-3`
CSP-R:5`-ATCCTCATCATCGCCTCTAAGT-3`
Agasicles hygrophila CSP gene by fluorescence quantitative PCR product length is 192 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 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:
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；
(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 CSP 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 CSP 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 CO2Concentration cultivate alternanthera philoxeroides after CSP gene C (T) value, 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 hygrophila2CSP 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 CSP gene increases, with transcript profile Expression quantity up-regulation variation tendency it is consistent.This provides weight for the research that we carry out Agasicles hygrophila CSP gene in a deep going way The basic data wanted.
SEQUENCE LISTING
<110>Inst. of Plant Protection, fujian Academy of Agricultural Science
<120>primer and method of Agasicles hygrophila CSP gene expression characteristics are detected
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<170> PatentIn version 3.3
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Claims (2)

1. a kind of fluorescence quantification PCR primer for detecting Agasicles hygrophila CSP gene expression characteristics, it is characterised in that: including symbol Close the upstream and downstream primer of quantitative fluorescent PCR reaction characteristics:
CSP-F:5`-GCAGCCAAAAGCAGAAAGAAG-3`,
CSP-R:5`-ATCCTCATCATCGCCTCTAAGT-3`.
2. a kind of quantitative fluorescent PCR side using primer detection Agasicles hygrophila CSP 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:
CSP-F:5`-GCAGCCAAAAGCAGAAAGAAG-3`,
CSP-R:5`-ATCCTCATCATCGCCTCTAAGT-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 denaturation 30 s, 56 DEG C of 30 s of annealing, 72 DEG C extend 1 Min, 35 recycle under this condition, and 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.
CN201811232459.9A 2018-10-22 2018-10-22 Detect the primer and method of Agasicles hygrophila CSP gene expression characteristics Pending CN109182552A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
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