CN109295173A - Detect the primer and method of Agasicles hygrophila AK gene expression characteristics - Google Patents

Abstract

The present invention provides the primers and method of detection Agasicles hygrophila AK 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 AK 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 AK gene by fluorescence quantitative with the expression quantity trend of transcript profile, the defense mechanism and AK 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 AK gene expression characteristics

Technical field

The present invention relates to the primers and method of detection Agasicles hygrophila AK 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.

During insect vital movement, arginine kinase (Arginine kinase, AK) is that it is main in vivo Phosphoric acid Clotogen, energy is stored in the height of phosphoarginine by the invertibity reaction between catalysis arginine and ATP by it In energy phosphate bond, or phosphoarginine is decomposed and generates ATP, is one and ATP regeneration, contraction of muscle and intracellular energy fortune Turn etc. directly related important kinases.Since arginine kinase exists only in invertebrate, and the enzyme and its phosphagen Energy metabolic pathways and mammal in it is entirely different, therefore arginine kinase can be used as control one of insect very well Sensitive target.

Since 1998 clone to obtain first insect arginine kinase cDNA sequence from apis mellifera, there is 9 at present A mesh, tens kinds of insects arginine kinase gene cDNA sequence it is identified come out, including Aedes aegypti, American cockroach, silkworm, Bright red blister beetle, Asiatic migrotory locust etc., these genes derive the consistency of the amino acid sequence of expression all 50% or more.

Alonso etc. (2001) to trypanosoma arginine kinase research shows that: in trypanosoma increased logarithmic phase, arginine The Rate activity of kinases remains constant substantially, and the variation of mRNA also very little, illustrates arginine kinase vigor mainly by protein It is adjusted after translation；Simultaneously under starvation, the sustained activity of parasite index build phase, AK increases, thus it is speculated that AK may make For the adjusting control of ability conversion.With RNAi technology by AK gene knockout in phyllotreta striolata body, its energy can be destroyed Metabolic pathway leads to the decline of phyllotreta striolata fertility and death.

The activity expression of arginine kinase will receive the influence of many factors.Pereira etc. (2003) has found in trypanosoma In so that AK overexpression is significantly improved the ability that trypanosoma resists nutrition and pH adverse circumstance, and mention its cell life vigor It is high.In crustacean, arginine kinase is said by one of albumen the most apparent is changed after immunostimulation and virus infection Bright enzyme related to energetic supersession directly or indirectly participates in relevant immune response.To tobacco budworm arginine kinase geneHassAK Studies have shown that not only hot conditions can induce the expression of AK, but also low temperature can equally promote its expression, thus it is speculated thatHassAKBase Because that may be also a kind of anti contravariance related gene.

The researchs such as the expression about Agasicles hygrophila AK gene have not been reported both at home and abroad, therefore use real time fluorescent quantitative PCR studies different CO₂The expression characterization of the lower Agasicles hygrophila AK gene of concentration processing, the results showed that, with CO₂The liter of concentration The expression quantity of height, female male worm AK gene increases, this is consistent with the variation tendency of transcript profile expression quantity up-regulation, illustrates CO₂Concentration Increase the expression regulation that can influence AK gene.Arginine kinase exists only in invertebrate body, and phosphoarginine is The phosphoryl donor of ATP is uniquely effectively formed in insect muscle, this illustrates that the energy metabolic pathways of insect and vertebrate are complete Difference, if can not only open up the frontier of pest molecular regulation using arginine kinase as a target of injurious insect control, and And it is also relatively safer to higher mammal, new method and thinking can be provided for agricultural insect management.

Summary of the invention

The object of the present invention is to provide the primers and method of detection Agasicles hygrophila AK gene expression characteristics, are comprehensive The different disposal CO for solving the expression characterization of Agasicles hygrophila AK 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 AK gene expression characteristics of design, including meet quantitative fluorescent PCR reaction The upstream and downstream primer of feature:

AK-F:5`-GGCTTGGATTCCTCACTTTCTG-3`

AK-R:5`-ATACCACCCTCGGCTTCTG-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 AK 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:

AK-F:5`-GGCTTGGATTCCTCACTTTCTG-3`

AK-R:5`-ATACCACCCTCGGCTTCTG-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 Taq^TM 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.

The more detailed test method of the present invention is as follows:

The real-time fluorescence quantitative PCR detection method of Agasicles hygrophila AK gene can be realized by following steps:

(1) design of primers: soft using DNAMAN according to the sequence for the Agasicles hygrophila AK 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:

AK-F:5`-GGCTTGGATTCCTCACTTTCTG-3`

AK-R:5`-ATACCACCCTCGGCTTCTG-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 Taq^TMKit carries out.Using the first chain of cDNA of above-mentioned synthesis as template, above-mentioned AK-F, AK-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 Taq^TM 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 AK 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 AK gene.The result shows that with CO₂The expression quantity of the raising of concentration, female male worm AK 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 AK 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 arginine kinase gene, this is right Further investigation gene expression characteristics have great importance comprehensively.

(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 AK gene transcription level of Agasicles hygrophila.

Fig. 1: Agasicles hygrophila AK gene by fluorescence quantitative primer Standard PCR product electrophoresis result: product length 175 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 AK 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 AK 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 AK 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:

AK-F:5`-GGCTTGGATTCCTCACTTTCTG-3`

AK-R:5`-ATACCACCCTCGGCTTCTG-3`

Agasicles hygrophila AK gene by fluorescence quantitative PCR product length is 175 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 Taq^TM 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 Taq^TM 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；

(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 AK 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 AK 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 AK 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 hygrophila₂AK 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 AK 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 AK gene in a deep going way Basic data.

SEQUENCE LISTING

<110>Inst. of Plant Protection, fujian Academy of Agricultural Science

<120>primer and method of Agasicles hygrophila AK gene expression characteristics are detected

<130> 4

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 22

<212> DNA

<213>artificial sequence

<400> 1

ggcttggatt cctcactttc tg 22

<210> 2

<211> 19

<212> DNA

<213>artificial sequence

<400> 2

ataccaccct cggcttctg 19

<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 fluorescence quantification PCR primer for detecting Agasicles hygrophila AK gene expression characteristics, it is characterised in that: including meeting The upstream and downstream primer of quantitative fluorescent PCR reaction characteristics:

AK-F:5`-GGCTTGGATTCCTCACTTTCTG-3`,

AK-R:5`-ATACCACCCTCGGCTTCTG-3`.

2. a kind of quantitative fluorescent PCR side using primer detection Agasicles hygrophila AK 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:

AK-F:5`-GGCTTGGATTCCTCACTTTCTG-3`

AK-R:5`-ATACCACCCTCGGCTTCTG-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 Taq^TM 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.