CN103820463A - Rhopalosiphum padi GAPDH reference gene partial sequence, cloning method and application - Google Patents

Abstract

The invention relates to a rhopalosiphum padi GAPDH reference gene partial sequence, a cloning method and an application, belongs to the field of molecular biology, and particularly relates to a rhopalosiphum padi reference gene GAPDH with a nucleotide sequence represented in SEQ ID No.1. The obtained nucleotide sequence of the rhopalosiphum padi reference gene GAPDH (999bp) can be used as reference genes for research of rhopalosiphum padi function genes or gene expressions at different development stages as well as relative levels of expressions of viruses in the nucleotide sequence when being used as a virus transmission medium in future.

Description

Rhopalosiphum padi GAPDH reference gene partial sequence, cloning process and application

Technical field

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

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 grain yield by sucking plant nutrition, it is mainly to work the mischief as the vector of gramineous crop and weeds virus disease.Rhopalosiphum padi is propagated yellow dwarf virus with the non-Reproduction methods of persistence, is the advantage biography virus mediator that Lutoevirus section Lutoevirus belongs to multiple barly yellow dwarf virus.

When rhopalosiphum padi thorn absorption food is subject to the plant phloem sap of yellow dwart infection, it has also taken food and has been present in phloem virion.In the middle intestines of vector aphid and/or hindgut and sialisterium, there is receptor-mediated endocytosis exocytosis.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 titre of amboceptor body inner virus and the research of cumulative change report that poison phase and impact pass toxic effect rate, pass all critical periods of poison cycle Field Plants viral prevalence.After in virus entry mediator aphid body, be accompanied by countless genetic expression and be rise or lower, the mode that much albumen gulps down by a kind of dysuria due to the pressure of the fetus is integrated into relevant to virus disseminating.Application reverse transcription real-time quantitative PCR (RT-qPCR), we can promptly evaluate the impact of virus in molecular function and biological procedures and the real-time concentration of virus.

In recent years, the method for relative quantification is comparatively popular in quantitative PCR (qPCR), and it has eliminated between experiment is processed, the systematic error such as aspect 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 guarded in all cells, under normal or abnormal condition, is all continuous expression.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.), finding house-keeping gene becomes one of research emphasis of these complete metamorphosises as reference gene.In the experiment of using RT-qPCR analysis insect gene expression, conventionally use following reference gene, as 18S rRNA, ACT1, EF-1 α, GAPDH, UBI, RPSs, RPLs and TUB etc.For incomplete metamorphosis insect, particularly propagate the aphid of various plants virus, carry out in recent years the research of soybean aphid (Aphis glycines) house-keeping gene.For the variation of monitoring amboceptor rhopalosiphum padi body inner virus plastochondria, 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 that detects two kind of plant viruses in rhopalosiphum padi, this gene is actually and derives from silkworm (Bombyx mori).GAPDH (glyceraldehyde-3-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase) is an enzyme in glycolysis reaction, is made up of molecular weight 146kDa the subunit of 4 30～40kDa.This gene almost in a organized way in high level expressions all, the protein expression amount in allogenic cell or tissue is generally constant, therefore be widely used as the house-keeping gene of real time PCR experimental implementation.But the clone of the GAPDH gene of rhopalosiphum padi and have no report both at home and abroad as being applied in of reference gene.

Summary of the invention

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

And provide clone the Auele Specific Primer of GAPDH of rhopalosiphum padi simultaneously, set up the RT-qPCR method based on SYBR GreenI dyestuff technology, thereby be that the GAPDH of rhopalosiphum padi is as reference gene, utilizing qPCR to rhopalosiphum padi functional gene or as the method that provides use in passing virus mediator virus and breed in vivo the research of variation.

Rhopalosiphum padi GAPDH gene fragment, its nucleotide sequence is as shown in SEQ ID No1.

A kind of cloning process of partial sequence of rhopalosiphum padi GAPDH gene, extract the total RNA of rhopalosiphum padi genome, adopt primer GAPDH to carry out RT-PCR amplification, using product the first chain cDNA as template, carry out pcr amplification with primer pair GAPDH F/GAPDHR, obtain positive colony, finally by sequence verification.Wherein primer sequence is:

GAPDH F:5′-ATGTCAAACATTGGTATCAATGGATTTGG-3′；

GAPDH R:5′-TTTAATCCTTAGATTGCATGTACTTGAT-3′。

The object clip size obtaining is 999bp.

The reaction conditions of described pcr amplification is as follows: 94 ℃ of 3min, and [94 ℃ of 45s, 58 ℃ of 1min, 72 ℃ of 1min] 30 circulations, 72 ℃ extend 10min, 4 ℃ of preservations.

The present invention also provides a kind of method of qPCR amplification GAPDH Gene Partial sequence, the total RNA of extracting rhopalosiphum padi genome, carry out RT reaction take the primer mixture that carries in test kit as primer, then carry out quantitative pcr amplification using product the first chain cDNA as template, fluorescence dye is SYBR Green I, the wherein quantitative primer of the rhopalosiphum padi in quantitative pcr amplification, its sequence is:

QGAPDH F：5′-ACTACTGTTCACGCTACCACCG-3′；

QGAPDH R：5′-GCTGCTTCCTTGACCTTACCTT-3′。

The object clip size obtaining is 272bp.

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

Rhopalosiphum padi GAPDH 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 GAPDH house-keeping gene of rhopalosiphum padi.The corresponding specific quantitative primer of design, set up the RT-qPCR method based on SYBR GreenI dyestuff technology, thereby be that the GAPDH of rhopalosiphum padi is as reference gene, utilizing qPCR to rhopalosiphum padi functional gene or as the method that provides use in passing virus mediator virus and breed in vivo the research of variation.

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

1. the present invention clones the GAPDH gene fragment that obtains rhopalosiphum padi first.

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

The present invention propose first using the GAPDH gene of rhopalosiphum padi as reference gene the quantitative PCR detection at rhopalosiphum padi different development stage;

4. the GAPDH gene of the rhopalosiphum padi that the present invention proposes can relative quantification yellow dwarf virus as reference gene the relative expression level of gene in amboceptor;

5. the detection primer that the present invention proposes has specificity, has optimized pcr amplification program, has improved greatly detection efficiency, has shortened detection time, has improved the confidence level of detected result.

Accompanying drawing explanation

Fig. 1. the PCR electrophorogram of rhopalosiphum padi GAPDH reference gene;

Wherein: M represents DL2000DNA Marker, be followed successively by from top to bottom 2000bp, 1000bp, 750bp, 500bp, 250bp,

100bp; 1～4 swimming lane is to repeat for 4 times.

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

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

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

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

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

Wherein: X-coordinate representative, to the quantized value under the different weaker concns of cDNA template, from left to right represents that cDNA concentration raises successively by gradient; Ordinate zou represents the GAPDH that increases under these weaker concns, R _nexceed threshold value, start to enter the cycle number of Exponential growth stage, i.e. quantitative cycle number Cq, Fitting curve equation is y=-3.416x+30.936(coefficient R ²=1.000), represent that amplification efficiency E is 96.216%.

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

Wherein: X-coordinate representative, to the quantized value under the different weaker concns of cDNA template, from left to right represents that cDNA concentration raises successively by gradient; Ordinate zou represents the GAPDH that increases under these weaker concns, R _nexceed threshold value, start to enter the cycle number of Exponential growth stage, i.e. quantitative cycle number Cq, Fitting curve equation is y=-3.453x+30.962(coefficient R ²=0.999), represent that amplification efficiency E is 94.814%.

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

Wherein: cycle number when X-coordinate represents qPCR amplification GAPDH; Ordinate zou represents the R under corresponding cycle number _nvalue.

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

Wherein: cycle number when X-coordinate represents qPCR amplification GAPDH; Ordinate zou represents the R under corresponding cycle number _nvalue.

Fig. 5 .BYDV-PAV CP gene is there being wing to become the relative expression quantity in aphid;

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

Embodiment

Below in conjunction with specific embodiment, further set forth the present invention, these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition.

The clone of embodiment 1. rhopalosiphum padi GAPDH genes

1.1. the total RNA of rhopalosiphum padi extracts:

Adopt TRIzol(Amion, USA) method extract rhopalosiphum padi (aptery one-tenth aphid) total RNA, concrete grammar is as follows:

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

1.2.PCR amplimer sequence

The upstream primer (SEQ ID NO2) of GADPH: 5 '-ATGTCAAACATTGGTATCAATGGATTTGG-3 ';

The downstream primer (SEQ ID NO3) of GADPH: 5 '-TTTAATCCTTAGATTGCATGTACTTGAT-3 '.

By life work biotechnology (Shanghai), limited-liability company is synthetic.

1.3. reverse transcription (RT)

Take the total RNA(concentration of aphid at 0.05～1 μ g/ μ L) as template, utilize the downstream primer of target gene to synthesize the first chain cDNA.25 μ L reverse transcription systems arrange and reaction process according to the following steps: first add DEPC ddH2O7 μ L, downstream primer (5 μ M) 0.5 μ L, total RNA1 μ L, after of short duration centrifugal mixing, 75 ℃ of temperature bath 10min, are placed in rapidly 5min on ice.DEPCddH ₂O3.5μL，dNTPs(2.5mM)5μL，5×RT reaction buffer5μL，M-MLV(200U/μl2μL，Recombinant RNase Inhibitor(RRI,40U/μL)1μL。After of short duration centrifugal the mixing of mixture, 42 ℃ of temperature are bathed 1h, and 95 ℃ of inactivation 5min, are placed in rapidly on ice.The the first chain cDNA obtaining carries out PCR or-20 ℃ of preservations immediately.

1.4. polymerase chain reaction (PCR)

The the first chain cDNA obtaining take RT carries out pcr amplification as template.All PCR all adopt following 25 μ L reaction systems: ddH2O17 μ L, 10 × EX Taq PCR buffer2.5 μ L, dNTPs(2.5mM) 2 μ L, upstream primer (5 μ M) 0.25 μ L, downstream primer (5 μ M) 0.25 μ L, ExTaq0.5 μ L, cDNA2.5 μ L.PCR response procedures is set to: 94 ℃ of denaturation 3min, and then 30 amplification cycles, each circulation comprises 94 ℃ of sex change 45sec, 56 ℃ of annealing 1min, and 72 ℃ of extension 1min, carry out 72 ℃ and supplement extension 10min after loop ends.PCR product detects testing goal fragment (Fig. 1) through 1% agarose gel electrophoresis.

1.5. the acquisition of goal gene

Target fragment is cut to glue to be reclaimed, DNA fragmentation after purifying is connected on pMD18T simple vector carrier, be transformed in competence Bacillus coli cells, the positive colony detecting through PCR checks order, and obtains the nucleotide sequence (concrete sequence is shown in SEQ ID No.1) of GAPDH.

The detection of the specificity RT-qPCR of embodiment 2.GAPDH gene in two kinds of different worm states of rhopalosiphum padi

2.1. sample preparation

Respectively take two kinds of different worm states of rhopalosiphum padi (have wing become aphid with aptery become aphid) total RNA of extracting is as template, concrete extracting method refers in embodiment 1 described in 1.1.

2.2.qPCR amplimer is synthetic

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

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

By raw work biotechnology (Shanghai) limited-liability company synthetic (PAGE method of purification).

2.3. quantitative fluorescent PCR (RT-qPCR)

Utilize two-step approach to carry out RT-qPCR, move at 7500real time PCR instrument (Applied Biosystems).RT part is utilized the test kit FastQuant RT Kit (with gDNase) of sky, Beijing root (TIANGEN) company, take total RNA of aphid material extraction as template, take the primer mixture (comprising Oigo dT Primer and Random6mers) that carries in test kit as primer carries out RT reaction, obtain the first chain cDNA.The qPCR of recycling based on SYBR Green I dyestuff detects target gene, the specification sheets of SuperReal PreMix Plus (SYBR Green) test kit of reaction system used and procedure reference TIANGEN company.PCR reaction system is 20 μ L, wherein 2 × SuperReal PreMix Plus10 μ L, the each 0.6 μ L of the quantitative primer 10 μ mol/L of upstream and downstream, template cDNA(10ng/ μ L) 2 μ L, 50 × ROX Reference Dye0.4 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,, after 95 ℃ of denaturation 15min, first moves 95 ℃ of 10sec of 40 circulations, 59.2 ℃ of 32sec, 72 ℃ of 32sec, 95 ℃ of 15sec in the solubility curve stage of reruning, 60 ℃ of 1min, 95 ℃ of 30sec, 60 ℃ of 15sec.Obtain 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 to become aphid geneome RNA sample to carry out quantitative pcr amplification, and judge the specificity of amplified production in conjunction with solubility curve.General desirable melting curve should be single peak type curve, if there is two or more peaks, 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 to become aphid substantially all to present unimodality curve, and peak value is all higher than the peak (75 ℃ of left and right) of primer dimer, locate to have started peak at 80 ℃, 83 ℃ to locate peak value the highest, and the peak that falls between 85～86 ℃, illustrates that quantitative amplification primer (SEQ ID No4 and the 5) amplified band the present invention relates to is single, high specificity, does not have non-specific amplification to occur.The present invention can find out from the fluorescent quantitative PCR curve of two groups of different worm states (aptery one-tenth aphid with have wing to become aphid), this increases under the different weaker concns of template to primer, DNA quantity has all experienced typical amplification procedure (comprising baseline, Exponential growth stage, linear phase and plateau), illustrate this to primer can for increase aptery one-tenth aphid with have wing to become the GAPDH gene in two kinds of worm state bodies of aphid.The fit equation of typical curve is respectively y=-3.416x+30.936(coefficient R ²=1.000), and y=-3.453x+30.962(coefficient R ²=0.999), according to formula amplification efficiency E=10 ^{(1/ slope)}-1, draw this to the aptery one-tenth of primer pair aphid with have wing to become the amplification efficiency E of GAPDH gene in two kinds of worm state bodies of aphid to be respectively

96.216% and 94.814%.Illustrate that this is very high to the amplification efficiency of primer, has approached 100%.

Embodiment 3. is as the reference gene that detects barly yellow dwarf virus (BYDV-PAV) CP gene relative expression in aphid body

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 while being 12h, 24h, 36h, 48h, 60h, 72h, 84h and 96h taking food the timed interval.Each timed interval place that takes food, the aphid of every seed wing type is got 8, is combined as a sample extraction RNA.3 repetitions are established in integral experiment.The RNA extracting method of all samples refers in embodiment 1 described in 1.1.

3.2.CP the qPCR amplimer of gene is synthetic

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

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

By raw work biotechnology (Shanghai) limited-liability company synthetic (PAGE method of purification).

3.3.RT-qPCR amplification

The RT-qPCR amplification method of GAPDH gene is shown in embodiment 2 described in 2.3, the GAPDH Cq value (Cq of each sample _re) for relative quantitative assay.The RT-qPCR amplification of CP gene is used primer pair Q CP F and Q CP R, and the RT-qPCR amplification method of the use of other reagent and the response procedures of RT-qPCR and GAPDH gene is in full accord, the CP Cq value (Cq of each sample _target) for relative quantitative assay.

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

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

ΔCq _target=minCq _target-sampleCq _target 1

$Q_{t\arg \mathrm{et}={E_{t\arg \mathrm{et}}}^{{\′}^{\mathrm{\Δ}{\mathrm{Cq}}_{t\arg \mathrm{er}}}}---2}$

ΔCq _re=minCq _re-sampleCq _re 3

$Q_{\mathrm{re}}={E_{\mathrm{re}}}^{{\′}^{\mathrm{\ΔC}{q}_{\mathrm{re}}}}---4$

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

Wherein E'=E+1

Calculate by above-mentioned formula, have wing to become aphid to take food in different time interval and be with malicious plant, the BYDV-PAV CP gene in body with respect to the expression amount of GAPDH as Fig. 5; Aptery one-tenth aphid takes food in different time interval is with malicious plant, the BYDV-PAV CP gene in body with respect to the expression amount of GAPDH as Fig. 6.

3.5. experimental result

Along with aphid (rhopalosiphum padi is aptery become aphid or have wing to become aphid) takes food the growth in the timed interval on malicious oat, the relative expression quantity of BYDV-PAV CP gene also increases gradually, and all starts to be fluctuation status after being 72h taking food the timed interval.This situation with BYDV plant virus the Accumulation and cycling mode in aphid body consistent: raising the poison initial stage (generally raise malicious 48h left and right 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 the poison later stage (after 48h, then raising poison), within part BYDV can be secreted into plant again with the saliva of aphid, this stage viral level slightly reduces; Aphid continues to take food is with malicious plant, and viral level can increase again, and so circulation is gone down.Finally show, GAPDH gene is stable at rhopalosiphum padi expression in vivo, can be used as the reference gene of quantitative aphid body implants virus.

Claims (6)

1. rhopalosiphum padi GAPDH 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 GAPDH gene, extract the total RNA of rhopalosiphum padi genome, adopt primer GAPDH to carry out RT-PCR amplification, using product the first chain cDNA as template, carry out pcr amplification with primer pair GAPDH F/GAPDH R, obtain positive colony, finally by sequence verification.Wherein primer sequence is:

GAPDH F:5′-ATGTCAAACATTGGTATCAATGGATTTGG-3′；

GAPDH R:5′-TTTAATCCTTAGATTGCATGTACTTGAT-3′。

3. cloning process according to claim 2, the reaction conditions of described pcr amplification is as follows: 94 ℃ of 3min, 94 ℃ of 45s, 58 ℃ of 1min, 72 ℃ 1min30 circulation, 72 ℃ of prolongation 10min, 4 ℃ of preservations.

4. the method for a qPCR amplification GAPDH Gene Partial sequence, the total RNA of extracting rhopalosiphum padi genome, carry mix primer as carrying out RT reaction as primer take test kit, then carry out quantitative pcr amplification using product the first chain cDNA as template, fluorescence dye is SYBR Green I, the wherein quantitative primer of the rhopalosiphum padi in quantitative pcr amplification, its sequence is:

QGAPDH F：5′-ACTACTGTTCACGCTACCACCG-3′；

QGAPDH R：5′-GCTGCTTCCTTGACCTTACCTT-3′。

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

6. rhopalosiphum padi GAPDH gene fragment claimed in claim 1 is applied to as reference gene the application that in rhopalosiphum padi functional gene or amboceptor, yellow dwarf virus quantitative gene expression PCR detects.

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