CN103805610B - Rhopalosiphum padi EF1-α reference gene partial sequence, cloning process and application thereof - Google Patents

Abstract

The present invention relates to " rhopalosiphum padi EF1-α reference gene partial sequence, cloning process and application " and belong to biology field, particularly, relate to rhopalosiphum padi reference gene EF1-α, its nucleotide sequence is as shown in SEQ ID No.1.The nucleotide sequence of rhopalosiphum padi reference gene EF1-α (837bp) that the present invention obtains can be used as studies rhopalosiphum padi functional gene or from now in different development stage genetic expression, and as the viral reference gene at the relative level of its expression in vivo when passing virus mediator.

Description

Rhopalosiphum padi EF1-α reference gene partial sequence, cloning process and application thereof

Technical field

The invention belongs to technical field of molecular biology, particularly relate to rhopalosiphum padi reference gene EF1-α Gene Partial sequence, cloning process and as the application of reference gene in RT-qPCR.

Background technology

Rhopalosiphum padi (Rhopalosiphum padi L., RP) is under the jurisdiction of Homoptera Aphidiadae (Homoptera:Aphididae).This aphid is distribution in extensive range in the world, is one of important insect of grass.Except directly affecting except grain yield by sucking plant nutrition, it mainly works the mischief as the vector of gramineous crop and weeds virus disease.Rhopalosiphum padi propagates yellow dwarf virus in persistence non-proliferative mode, is the advantage biography virus mediator that Lutoevirus section Lutoevirus belongs to multiple barly yellow dwarf virus.

While rhopalosiphum padi thorn draws the plant phloem sap of food by yellow dwart infection, it has also taken food and has been present in phloem virion.Receptor-mediated endocytosis exocytosis is there is in intestines and/or hindgut and sialisterium in vector aphid.The propagation of yellow dwart in host plant body usually adopts absolute and relative quantitative method, but also not about obtaining the poison phase, connect the poison phase and affect Virus spreading rate, the research of the titre and cumulative change that pass the amboceptor body inner virus of all critical periods of poison cycle Field Plants viral prevalence reports.After in virus entry mediator aphid body, along with countless genetic expression in raising or lowering, much albumen is integrated into relevant to virus disseminating by the mode that a kind of dysuria due to the pressure of the fetus gulps down.Application reverse transcription real-time quantitative PCR (RT-qPCR), we promptly can evaluate the impact of virus and the real-time concentration of virus in molecular function and biological procedures.

In recent years, the method for relative quantification is comparatively popular in quantitative PCR (qPCR), which eliminates between experiment process, the systematic error such as in RNA extraction efficiency and RNA integrity, has been applied to many aspects of molecular studies work.In organism, house-keeping gene (House-keeping gene) is all conservative in all cells, is all continuous expression under normal or abnormal condition.The expression level and change that draw goal gene can be compared by goal gene and house-keeping gene.Therefore, in RT-qPCR experiment, selection is applicable to, believable house-keeping gene is absolutely necessary as reference gene.Along with delivering of the different insect whole genome sequence such as fruit bat (Drosophila melanogaster) and small cabbage moth (Plutella xylostella L.), find house-keeping gene as one of reference gene research emphasis becoming these complete metamorphosises.Analyze in the experiment of insect gene expression using RT-qPCR and usually use following reference gene, as 18S rRNA, ACT1, EF1-α, GAPDH, UBI, RPSs, RPLs and TUB etc.For incomplete metamorphosis insect, particularly propagate the aphid of various plants virus, carried out the research of soybean aphid (Aphis glycines) house-keeping gene in recent years.For the change of monitoring amboceptor rhopalosiphum padi body inner virus gene, determine the relative level of viral gene expression with RT-qPCR, also do not find effectively applicable house-keeping gene.Although there is ACT1 gene in research to be used as the reference gene detecting two kind of plant viruses in rhopalosiphum padi, this gene is actually and derives from silkworm (Bombyx mori).As the nuclear gene of aphid, the partial gene sequence of EF1-α has become the classification foundation of the population genetic diversity of rhopalosiphum padi, but for studying the expression change of other gene, there is not been reported as house-keeping gene.

Summary of the invention

The invention provides rhopalosiphum padi EF1-alpha gene fragment, can be used as the application of rhopalosiphum padi reference gene.

And the Auele Specific Primer of the EF1-α of clone's rhopalosiphum padi is provided simultaneously, set up the RT-qPCR method based on SYBR GreenI dye techniques, thus be the EF1-α of rhopalosiphum padi as reference gene, breed to rhopalosiphum padi functional gene or as biography virus mediator virus the method provided in the research of change utilizing qPCR in vivo.

Rhopalosiphum padi EF1-alpha gene fragment, its nucleotide sequence is as shown in SEQ ID No.1.

A kind of cloning process of partial sequence of rhopalosiphum padi EF1-α gene, extract rhopalosiphum padi genome total serum IgE, primer EF1-α R is adopted to carry out RT-PCR amplification, pcr amplification is carried out using product first chain cDNA as template, with primer pair EF1-α F/EF1-α R, obtain positive colony, finally by sequence verification.Wherein primer sequence is:

EF1-αF：5′-ATTGATATTGCTTTATGGAAATTCG-3′；

EF1-αR：5′-ACCAGGGTGGTTCAATACAATAAC-3′。

The object clip size obtained is 837bp.

The reaction conditions of described pcr amplification is as follows: 94 DEG C of 3min, [94 DEG C of 30s, 56 DEG C of 45s, 72 DEG C of 1min], 30 circulations, and 72 DEG C extend 10min, 4 DEG C of preservations.

The present invention also provides a kind of qPCR method increasing EF1-α Gene Partial sequence, extracting rhopalosiphum padi genome total serum IgE, with the primer mixture carried in test kit (comprising Oigo dT Primer and Random6mers) for primer carries out RT reaction, then quantitative pcr amplification is carried out using product first chain cDNA as template, fluorescence dye is SYBR GreenI, the quantitative primer of the rhopalosiphum padi wherein in quantitative pcr amplification, its sequence is:

QEF1-αF：5′-TAGACGCTATCCTACCCCCCA-3′；

QEF1-αR：5′-GTGAAATCAGCAGCACCCTTG-3′。

The object clip size obtained is 328bp.

Described quantitative pcr amplification adopts two-step approach, namely after 95 DEG C of denaturation 15min, first runs 95 DEG C of 10sec of 40 circulations, 59.2 DEG C of 32sec, 72 DEG C of 32sec, 95 DEG C of 15sec in solubility curve stage of reruning, 60 DEG C of 1min, 95 DEG C of 30sec, 60 DEG C of 15sec.

Rhopalosiphum padi EF1-alpha gene fragment is applied to as reference gene the application that in rhopalosiphum padi functional gene or amboceptor, yellow dwarf virus quantitative gene expression PCR detects.

The present invention, according to the nucleotide sequence of other insect of NCBI, has cloned the partial sequence of the EF1-α house-keeping gene of rhopalosiphum padi.Design corresponding specific quantitative primer, set up the RT-qPCR method based on SYBR GreenI dye techniques, thus be the EF1-α of rhopalosiphum padi as reference gene, breed to rhopalosiphum padi functional gene or as biography virus mediator virus the method provided in the research of change utilizing qPCR in vivo.

Compared with prior art, the present invention has the following advantages:

1. the present invention clones the EF1-alpha gene fragment obtaining rhopalosiphum padi first.

2. the present invention proposes to set up general reference gene first in rhopalosiphum padi quantitative PCR detection;

3. the present invention proposes the EF1-α gene of rhopalosiphum padi first as the quantitative PCR detection of reference gene at rhopalosiphum padi different development stage;

4. the EF1-α gene of rhopalosiphum padi that proposes of the present invention can the relative expression levels of gene in amboceptor of relative quantification yellow dwarf virus as reference gene;

5. the detection primer that the present invention proposes has specificity, optimizes pcr amplification program, improves detection efficiency greatly, shortens detection time, improve the confidence level of detected result.

Accompanying drawing explanation

Fig. 1. the PCR electrophorogram of rhopalosiphum padi EF1-α reference gene;

Wherein: M represents DL2000DNA Marker, is followed successively by 2000bp from top to bottom, 1000bp, 750bp, 500bp, 250bp, 100bp; 1 ~ 4 swimming lane is repeat for 4 times.

The solubility curve (aptery one-tenth aphid) of Fig. 2-1. rhopalosiphum padi EF1-α reference gene;

Wherein: X-coordinate representative is increased the stage at solubility curve, the temperature range from 60 DEG C to 95 DEG C; Ordinate zou represents after SYBRGreen I fluorescence dye is attached to the double-stranded DNA of EF1-α target fragment, enters the solubility curve amplification stage, raises, by the SYBR Green I fluorescent signal value after ROX stdn and R with temperature _nderivative value, peak value refers to R at corresponding temperature _nthe derivative value at the flex point place sharply reduced.

The solubility curve (having wing to become aphid) of Fig. 2-2. rhopalosiphum padi EF1-α reference gene;

Wherein: X-coordinate representative is increased the stage at solubility curve, the temperature range from 60 DEG C to 95 DEG C; Ordinate zou represents after SYBRGreen I fluorescence dye is attached to the double-stranded DNA of EF1-α target fragment, enters the solubility curve amplification stage, raises, by the SYBR Green I fluorescent signal value after ROX stdn and R with temperature _nderivative value, peak value refers to R at corresponding temperature _nthe derivative value at the flex point place sharply reduced.

The typical curve (aptery one-tenth aphid) of Fig. 3-1. rhopalosiphum padi EF1-α reference gene;

Wherein: X-coordinate representative, to the quantized value under the different weaker concn of cDNA template, from left to right represents that cDNA concentration raises successively by gradient; Ordinate zou represents increase under these weaker concns EF1-α, R _nexceed threshold value, start the cycle number entering Exponential growth stage, be i.e. quantitative cycle number Cq, Fitting curve equation is y=-3.392x+24.679(coefficient R ²=0.999), representing amplification efficiency E is 97.157%.

The typical curve (having wing to become aphid) of Fig. 3-2. rhopalosiphum padi EF1-α reference gene;

Wherein: X-coordinate representative, to the quantized value under the different weaker concn of cDNA template, from left to right represents that cDNA concentration raises successively by gradient; Ordinate zou represents increase under these weaker concns EF1-α, R _nexceed threshold value, start the cycle number entering Exponential growth stage, be i.e. quantitative cycle number Cq, Fitting curve equation is y=-3.424x+27.284(coefficient R ²=0.998), representing amplification efficiency E is 95.902%.

The amplification curve (aptery one-tenth aphid) of Fig. 4-1. rhopalosiphum padi EF1-α reference gene;

Wherein: X-coordinate represents cycle number during qPCR amplification EF1-α; The R of ordinate zou representative under corresponding cycle number _nvalue.

The amplification curve (having wing to become aphid) of Fig. 4-2. rhopalosiphum padi EF1-α reference gene;

Wherein: X-coordinate represents cycle number during qPCR amplification EF1-α; The R of ordinate zou representative under corresponding cycle number _nvalue.

Fig. 5 .BYDV-PAV CP gene becomes relative expression quantity in aphid there being wing;

Wherein: X-coordinate representative has wing to become aphid to take food the timed interval of being with malicious plant, and from left to right the time increases successively; Ordinate zou represents the expression amount of BYDV-PAV CP gene relative to EF1-α, and histogram and error line represent each timed interval and locate the relative expression quantity mean value M of 3 repeat samples and the standard error (± SE) of mean value.

The relative expression quantity of Fig. 6 .BYDV-PAV CP gene in aptery one-tenth aphid;

Wherein: X-coordinate represents aptery one-tenth aphid and takes food the timed interval of being with malicious plant, and from left to right the time increases successively; Ordinate zou represents the expression amount of BYDV-PAV CP gene relative to EF1-α, and histogram and error line represent each timed interval and locate the mean value M of the relative expression quantity of 3 repeat samples and the standard error (± SE) of mean value.

Embodiment

Below in conjunction with specific embodiment, set forth the present invention further, these embodiments are only not used in for illustration of the present invention and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually conveniently condition.

The clone of embodiment 1. rhopalosiphum padi EF1-α gene

1.1. rhopalosiphum padi Total RNAs extraction:

Adopt TRIzol(Amion, USA) method extract rhopalosiphum padi (aptery one-tenth aphid) total serum IgE.

According to instrument difference (using the imported product without RNase/DNase) used during ground sample, the leaching process of RNA is: the abundant grind into powder in liquid nitrogen by about 0.05 ~ 0.1g fresh sample or freezing rhopalosiphum padi, be transferred to rapidly in the 1.5mL centrifuge tube that liquid nitrogen freezing crosses, then 1mL TRIzol is added, acutely rock mixing, leave standstill 5min on ice; 4 DEG C, the centrifugal 10min of 12,000rpm; Draw supernatant in new 1.5mL centrifuge tube, add 200 μ l chloroforms, firmly shake 15s, leave standstill 5min on ice.4 DEG C, the centrifugal 10min of 12,000rpm; Upper strata supernatant is transferred to new centrifuge tube, adds 400 μ L chloroform supernatants, acutely rock mixing, leave standstill 5min on ice.4 DEG C, the centrifugal 10min of 12,000rpm; Supernatant is proceeded to new centrifuge tube, adds isopyknic Virahol with supernatant, put upside down mixing gently, 4 DEG C of overnight precipitation; 4 DEG C, the centrifugal 15min of 12,000rpm, removes supernatant; Add 75% ethanol (the DEPC water preparation of sterilizing) of 1mL precooling, washing precipitation.4 DEG C, the centrifugal 5min of 12,000rpm repeats above-mentioned washing step, with thorough wash clean salinity; Abandon supernatant, 4 DEG C, the centrifugal 1min of 12,000rpm, to try one's best sucking-off supernatant with rifle head.In ventilating kitchen, opening up, dry 5 ~ 10min.By the DEPC water dissolution of 50 μ L sterilizings.After 1 μ L primary sample is diluted 10 times or 100 times, get 5 μ L and carry out the integrity detection that 1% agarose gel electrophoresis detects RNA sample.And get purity and the concentration value that 1 μ LRNA NanoDrop-2000 ultraviolet spectrophotometer measures RNA sample.It is stand-by that qualified samples is placed in-70 DEG C of Refrigerator stores.

1.2.PCR amplimer sequence

The upstream primer (SEQ ID NO2) of EF1-α: 5 '-ATTGATATTGCTTTATGGAAATTCG-3 ';

The downstream primer (SEQ ID NO3) of EF1-α: 5 '-ACCAGGGTGGTTCAATACAATAAC-3 '.

Synthesized by Sangon Biotech (Shanghai) Co., Ltd..

1.3. reverse transcription (RT)

For template, the downstream primer of target gene is utilized to synthesize the first chain cDNA with aphid total serum IgE (concentration is at 0.05 ~ 1 μ g/ μ L).25 μ L reverse transcription systems arrange and reaction process according to the following steps: first add DEPC water 7 μ L, downstream primer (5 μMs) 0.5 μ L, total serum IgE 1 μ L, after of short duration centrifugal mixing, 75 DEG C temperature bathe 10min, be placed in rapidly 5min on ice.DEPC water 3.5 μ L, dNTPs (2.5mM) 5 μ L, 5 × RT reaction buffer5 μ L, M-MLV (200U/ μ L) 2 μ L, Recombinant RNaseInhibitor (RRI, 40U/ μ L) 1 μ L.After the of short duration centrifugal mixing of mixture, 42 DEG C of temperature bath 1h, 95 DEG C of inactivation 5min, are placed in rapidly on ice.The the first chain cDNA obtained carries out PCR or-20 DEG C of preservation immediately.

1.4. polymerase chain reaction (PCR)

The the first chain cDNA obtained with RT carries out pcr amplification for template.

PCR adopts following 25 μ L reaction system: ddH ₂o17 μ L, 10 × EX Taq PCR buffer2.5 μ L, dNTPs(2.5mM) 2 μ L, upstream primer (5 μMs) 0.25 μ L, downstream primer (5 μMs) 0.25 μ L, ExTaq0.5 μ L, cDNA2.5 μ L.PCR response procedures is set to: 94 DEG C of denaturation 3min, then 94 DEG C of sex change 30sec, and 56 DEG C of annealing 45sec and 72 DEG C of extension 1min amplification, 35 circulations, carry out 72 DEG C and supplement extension 10min after loop ends.PCR primer carrys out testing goal fragment (Fig. 1) through 1% agarose gel electrophoresis.

1.5. the acquisition of goal gene

Cut glue to target fragment to reclaim, DNA fragmentation after purifying is connected on pMD18T simple vector carrier, be transformed in competence Bacillus coli cells, the positive colony detected through PCR checks order, and obtains the nucleotide sequence (concrete sequence is shown in SEQ ID No.1) of EF1-α.

The detection of the specificity RT-qPCR of embodiment 2.EF1-α gene in the different worm state of rhopalosiphum padi two kinds

2.1. sample preparation

Respectively with two of rhopalosiphum padi kinds of different worm states (have wing become aphid with aptery become aphid) total serum IgE that extracts for template, in concrete extracting method detailed in Example 1 described in 1.1.

2.2.qPCR amplimer synthesis

QPCR amplification upstream primer (SEQ ID NO4): 5 '-TAGACGCTATCCTACCCCCCA-3 ';

QPCR amplification downstream primer (SEQ ID NO5): 5 '-GTGAAATCAGCAGCACCCTTG-3 '.

(PAGE method of purification) is synthesized by Sangon Biotech (Shanghai) Co., Ltd..

2.3.RT-qPCR

Utilize two-step approach to carry out RT-qPCR, run in 7500real time PCR instrument (Applied Biosystems).RT part utilizes the test kit FastQuant RT Kit (with gDNase) of sky, Beijing root (TIANGEN) company, respectively with rhopalosiphum padi have wing become aphid with aptery become the total serum IgE of aphid material extraction for template, carry out RT reaction with the primer mixture carried in test kit (comprising OigodT Primer and Random6mers) for primer, obtain the first chain cDNA.The qPCR recycled based on SYBRGreen I dyestuff detects target gene, the specification sheets of SuperRealPreMix Plus (SYBR Green) test kit of reaction system used and procedure reference TIANGEN company.PCR reaction system is 20 μ L, wherein 2 × SuperReal PreMixPlus10 μ L, the each 0.6 μ L of the quantitative primer of upstream and downstream 10 μm of ol/L, template cDNA(10ng/ μ L) 2 μ L, 50 × ROX ReferenceDye0.4 μ L, with sterilizing distilled water polishing to 20 μ L, all the other carry out to specifications.Quantitative pcr amplification adopts two-step approach, namely after 95 DEG C of denaturation 15min, first runs 95 DEG C of 10sec of 40 circulations, 59.2 DEG C of 32sec, 72 DEG C of 32sec, 95 DEG C of 15sec in solubility curve stage of reruning, 60 DEG C of 1min, 95 DEG C of 30sec, 60 DEG C of 15sec.Experiment obtains melting curve (melting curve) (Fig. 2-1,2-2), quantitatively cycle number (quantification cycle, Cq) (Fig. 3-1,3-2) and amplification curve (amplification plot) (Fig. 4-1,4-2) for post analysis.

2.4. experimental result

The present invention according to quantitative PCR response procedures by the aptery one-tenth aphid of the rhopalosiphum padi of 50ng content with have wing and become aphid total serum IgE sample to carry out quantitative pcr amplification, and judge the specificity of amplified production in conjunction with solubility curve.Generally desirable melting curve should be single peak type curve, if there is two or more peak, has illustrated that the non-specific amplifications such as primer dimer produce.Draw from the solubility curve analysis of primer, aptery one-tenth aphid with have wing and become aphid substantially all to present unimodality curve, and peak value is all higher than the peak value (about 75 DEG C) of primer dimer, peak has been started at 82 DEG C of places, 85 DEG C of place's peak values are the highest, and fall between 87 ~ 88 DEG C peak, illustrates that quantitative amplification primer (SEQ ID No4 and the 5) amplified band that the present invention relates to is single, high specificity, does not have non-specific amplification to occur.The present invention is as can be seen from the fluorescent quantitative PCR curve of two groups of different worm states (aptery one-tenth aphid with have wing become aphid), this increases under the different weaker concns of template to primer, DNA quantity all experienced by typical amplification procedure (comprising baseline, Exponential growth stage, linear phase and plateau), illustrates that this may be used for the EF1-α gene in amplification two kinds of worm state bodies to primer.The fit equation of typical curve is respectively y=-3.392x+24.679(coefficient R ², and y=-3.424x+27.284(coefficient R=0.999) ²=0.998), according to formula amplification efficiency E=10 ^{(-1/ slope)}-1, draw this to primer pair aptery one-tenth aphid with have wing and become the amplification efficiency E of the EF1-α gene in aphid two kinds of worm state bodies to be respectively 97.157% and 95.902%, illustrate that this is very high to the amplification efficiency of primer, close to 100%.

Embodiment 3. is as the reference gene of barly yellow dwarf virus (BYDV-PAV) CP gene in detection zone virulent aphis polypide

3.1. sample preparation

Nontoxic rhopalosiphum padi have wing become aphid with aptery become aphid take food and infect the oat plant of BYDV-PAV, sample respectively when to take food the timed interval be 12h, 24h, 36h, 48h, 60h, 72h, 84h and 96h.Each take food the timed interval place, the aphid of often kind of wing gets 8, is combined as a sample extraction RNA.3 repetitions are established in integral experiment.In the RNA extraction method detailed in Example 1 of all samples described in 1.1.

3.2.CP the qPCR amplimer synthesis of gene

QPCR amplification upstream primer (SEQ ID NO6): 5 '-CGGGGCTGAGGTATTCGTAT-3 ';

QPCR amplification downstream primer (SEQ ID NO7): 5 '-AGGACTTTGAGGCGGATTTG-3 '.

(PAGE method of purification) is synthesized by Sangon Biotech (Shanghai) Co., Ltd..

3.3.RT-qPCR amplification

The RT-qPCR amplification method of EF1-α gene is shown in embodiment 2 described in 2.3, the EF1-α Cq value (Cq of each sample _re) for relative quantitative assay.The RT-qPCR amplification of CP gene uses SEQ ID NO6 and SEQ ID NO7 primer pair, and the RT-qPCR amplification method of the use of other reagent and the response procedures of RT-qPCR and EF1-α gene is completely the same, the CP Cq value (Cq of each sample _target) for relative quantitative assay.

3.4.BYDV-PAV the relative expression quantity of CP gene

In each sample, the relative expression quantity (Relative Quantification, RQ) of CP gene uses Formula Series to calculate.

ΔCq _target=minCq _target-sampleCq _target1

Q _target=E _target'ΔCq _target2

ΔCq _re=minCq _re-sampleCq _re3

Q _re=E _re'ΔCq _re4

$\mathrm{RQ}=\frac{Q_{t\arg \mathrm{et}}}{Q_{\mathrm{re}}}---5$

Wherein E'=E+1

By above-mentioned formulae discovery, have wing to become aphid in different time interval, take food the malicious plant of band, the BYDV-PAV CP gene in body relative to the expression amount of EF1-α as Fig. 5; Aptery one-tenth aphid takes food the malicious plant of band in different time interval, the BYDV-PAV CP gene in body relative to the expression amount of EF1-α as Fig. 6.

3.5. experimental result

Along with aphid (rhopalosiphum padi has wing to become aphid or aptery one-tenth aphid) takes food the growth in the timed interval on the malicious oat of band, the relative expression quantity of BYDV-PAV CP gene also increases gradually, and all take food the timed interval be 72h after start in fluctuation status.This situation and the BYDV plant virus Accumulation and cycling mode in aphid body is consistent: raising poison initial stage (generally raise malicious about 48h aphid and just can pass poison), BYDV accumulates the enteron aisle in aphid gradually, then enter hemolymph and (pair) sialisterium through intestinal walls, this stage virus all shows a rising trend in aphid body; Raise poison later stage (then raising poison after 48h), part BYDV can be secreted within plant with the saliva of aphid again, and this stage viral level slightly reduces; Aphid continues to take food the malicious plant of band, and viral level can increase again, and so circulation is gone down.Finally show, EF1-α gene is stablized at rhopalosiphum padi expression in vivo, can be used as the reference gene of quantitative aphid body implants virus.

Claims (6)

1. rhopalosiphum padi EF1-alpha gene fragment, its nucleotide sequence is as shown in SEQ ID No.1.

2. the cloning process of the partial sequence of a rhopalosiphum padi EF1-α gene, extract rhopalosiphum padi genome total serum IgE, primer EF1-α R is adopted to carry out RT-PCR amplification, pcr amplification is carried out using product first chain cDNA as template, with primer pair EF1-α F/EF1-α R, obtain positive colony, finally by sequence verification; Wherein primer sequence is:

EF1-αF：5′-ATTGATATTGCTTTATGGAAATTCG-3′；

EF1-αR：5′-ACCAGGGTGGTTCAATACAATAAC-3′。

3. cloning process according to claim 2, the reaction conditions of described pcr amplification is as follows: 94 DEG C of 3min; 94 DEG C of 30s, 56 DEG C of 45s, 72 DEG C of 1min, 30 circulations; 72 DEG C extend 10min, 4 DEG C of preservations.

4. the method for a qPCR amplification EF1-α Gene Partial sequence, extracting rhopalosiphum padi genome total serum IgE, carry mix primer for primer with test kit and carry out RT reaction, then quantitative pcr amplification is carried out using product first chain cDNA as template, fluorescence dye is SYBR Green I, the quantitative primer of the rhopalosiphum padi wherein in quantitative pcr amplification, its sequence is:

QEF1-αF：5′-TAGACGCTATCCTACCCCCCA-3′；

QEF1-αR：5′-GTGAAATCAGCAGCACCCTTG-3′。

5. method according to claim 4, described quantitative pcr amplification adopts two-step approach, namely after 95 DEG C of denaturation 15min, first run 95 DEG C of 10sec of 40 circulations, 59.2 DEG C of 32sec, 72 DEG C of 32sec, rerun 95 DEG C of 15sec in solubility curve stage, 60 DEG C of 1min, 95 DEG C of 30sec, 60 DEG C of 15sec.

6. rhopalosiphum padi EF1-alpha gene fragment according to claim 1 is as the application of reference gene in detection rhopalosiphum padi polypide in barly yellow dwarf virus CP gene.