CN105296651B

CN105296651B - The method that chalcid fly Hsp90 gene expressions are nibbled using fluorescent RT-PCR technology detection Zhou Shi

Info

Publication number: CN105296651B
Application number: CN201510838009.4A
Authority: CN
Inventors: 李敏; 王凤竹; 张新玥; 李婷婷; 朱耿平; 刘强
Original assignee: Tianjin Normal University
Current assignee: Tianjin Normal University
Priority date: 2015-11-26
Filing date: 2015-11-26
Publication date: 2018-07-06
Anticipated expiration: 2035-11-26
Also published as: CN105296651A

Abstract

The invention discloses a kind of methods for nibbling chalcid fly Hsp90 relative expression quantities using fluorescence RT round pcrs detection Zhou Shi.Zhou Shi nibbles the foundation of chalcid fly Hsp90 real-time fluorescence RT PCR detection methods in the present invention, nibbles chalcid fly Hsp90 expression regulations mechanism for research Zhou Shi and its bionomic control lays the foundation.The influence of Hsp90 gene expression amounts in chalcid fly body is nibbled Zhou Shi in the variation of research temperature, result of study can be that scientific utilization natural enemy insect Zhou Shi nibble chalcid fly and provide fundamental basis, this nibbles chalcid fly prevention fall webworms for letting Zhou Shi fly away under which kind of outdoor temperature later and is of great significance.The present invention is provides technology platform in mRNA level in-site to the relative quantitative assay of Hsp90 genes.

Description

The method that chalcid fly Hsp90 gene expressions are nibbled using fluorescent RT-PCR technology detection Zhou Shi

The present invention obtains：State natural sciences fund（31201730）；Tianjin S ＆ T Developmentin High Institutions planning item （20110602）；Tianjin Normal University's doctor's fund（52XB1003）And Tianjin animals and plants resistance key lab open fund Subsidy.

Technical field

The present invention relates to biotechnology, the specificity for being related to nibbling chalcid fly Hsp90 gene expressions for detecting Zhou Shi is drawn In particular object is a kind of method for nibbling chalcid fly Hsp90 gene expressions using fluorescent RT-PCR technology detection Zhou Shi.It is main to use The research of chalcid fly Hsp90 gene expression regulations mechanism and its bionomic control is nibbled in Zhou Shi, result of study can be scientific utilization natural enemy Insect Zhou Shi nibbles chalcid fly progress biological control and provides fundamental basis.

Background technology

Heat shock protein Heat shock protein (Hsp) one kind is widely present in vivo, to inside and outside Environmental change extremely sensitive high conservative protein, the various physiological metabolism approach of wide participation, has become in recent years as biology A hot spot in research field.Different protein families, wherein Hsp90 and temperature can be classified as according to the size of its molecular weight Degree stress irritability is closely related.Temperature is to influence the vital envirment factor of organism existence, only in certain temperature In the range of organism could carry out material energy metabolism to maintain normal physiological activity, the tolerance range beyond temperature can all make life The growth and development of object is stagnated even dead.In general after organism is by high temperature or low temperature stimulation, albumen synthetic quantity has aobvious Variation is write, to enhance degeneration-resistant border ability.Since heat shock protein can keep certain content after stress is eliminated in a period of time Organism is made more preferably to survive, it is believed that it exists adapts to environment important role for organism.

Zhou Shi nibbles chalcid flyChouioiacuneaYang is the important invasive species fall webworms in ChinaHyphantriacunea（Drury）Important parasite, the long 1.1-1.5 mm of body, cluster endoparasitism is in fall webworms pupa In, it is the Natural Enemies factor for inhibiting fall webworms, plays an important role to the harm for controlling fall webworms.Except parasitizing the U.S. Outside white moth, can also parasitize Lepidoptera Lasiocampidae, Lymantriidae, Notodontidae, Geometridae, diamond-back moth section and the Nuscidae of Diptera, Chrysomelidae and ladybirds of Larvaevoridae and coleoptera etc.

It is discharged by fieldC. cuneaIt can achieve the purpose that effectively to prevent fall webworms.However, in the field environment,C. cunea Vigor can be influenced by multiple factors, wherein, temperature can be influenced as a main ecological factorC. cuneaVitality。Chalcid fly is launched in suitable temperature range, can just play better control effect.

Real-time fluorescence PCR technology is the nucleic acid quantitation technique to grow up on the basis of Standard PCR technology.Real-time fluorescence PCR not only realizes leap of the Standard PCR from qualitative to quantitative, but also compared with Standard PCR, and real-time fluorescence PCR technology is by certainly Dynamicization instrument collects fluorescence signal, avoids the subjectivity that naked eyes judge, can further improve sensitivity；Real-time fluorescence PCR skill The totally-enclosed reaction of art post-processes without PCR, avoids polluting, ensure that the reproducibility and reliability of result.At present, extensively should For fields such as molecular biology and medical researches.With the further exploitation of real-time fluorescent PCR reagent case, in recent years, in real time Fluorescence PCR assay is also widely used in animal ecology prevention.

Invention content

The purpose of the present invention is disclose it is a kind of using fluorescent RT-PCR technology detection Zhou Shi nibble chalcid fly Hsp90 gene expressions Method.

To achieve the above object, the technical solution adopted in the present invention is as follows:

A kind of specific primer that chalcid fly Hsp90 gene expressions are nibbled using fluorescent RT-PCR technology detection Zhou Shi is designed, It is characterized in that including meeting the special upstream and downstream primer of Fluorescence PCR feature：Hsp90-F：5'-TAACGATGATGAACAATA -3'; Hsp90-R：5'-AATAGGATAGCCAATGAA-3'；

Specific primer of the present invention quickly detects the method that Zhou Shi nibbles chalcid fly Hsp90 gene expressions, it is characterised in that It carries out as follows：

（1）The detection of the gene is carried out using following primer：

Meet the sequence specific upstream and downstream primer of Fluorescence PCR feature：

Hsp90-F：5'-TAACGATGATGAACAATA-3'

Hsp90-R：5'-AATAGGATAGCCAATGAA-3'

（2）The extraction of total serum IgE：Using various general RNA extraction methods, nibbled in chalcid fly adult from Zhou Shi and extract total serum IgE ；

（3）CDNA is synthesized：Chalcid fly total serum IgE is nibbled as templated synthesis cDNA using Zhou Shi, reaction system is 20 μ L；

（4）Real-time fluorescence PCR：It is above-mentioned using the cDNA of above-mentioned synthesis as template（1）Middle Hsp90-F and Hsp90-R, GADPH-F and GADPH-R is specific primer, carries out real-time fluorescence PCR amplified reaction, and each sample sets 3 parallel pipes, is expanded The Ct values of 3 parallel pipes of gained are averaged after increasing.

（5）Zhou Shi nibbles chalcid fly, and Hsp90 genes relative expression quantity calculates at different temperatures：

After real-time fluorescence PCR, when target gene is close with the amplification efficiency of reference gene, using 2^{Δ Δ CT}Method calculates not Synthermal lower Hsp90 genes relative to internal reference GADPH genes mRNA relative expression quantities.

Detection method of the present invention, wherein every primer is configured to the storage liquid of a concentration of 25 μm of ol/L respectively, Working concentration is 0.5 μm of ol/L.

The present invention further discloses nibble the special of chalcid fly Hsp90 gene expressions using fluorescent RT-PCR technology detection Zhou Shi Property draw prepare detection Zhou Shi nibble chalcid fly adapt to external environment variation in terms of application.The experimental results showed that：Fig. 1 is different temperatures Xia Zhoushi nibbles the relative expression quantity of chalcid fly Hsp90 genes.Either high temperature or low temperature can influence Zhou Shi as seen from the figure Nibble the expression of the Hsp90 in chalcid fly body.In the case of low temperature stress, with the continuous reduction of temperature, Zhou Shi is nibbled in chalcid fly body Hsp90 relative expression quantity in the trend risen, and reach maximum expression quantity at -7 DEG C；In the situation of high temperature stress Under, with the continuous raising of temperature, Zhou Shi nibbles the relative expression quantity of the Hsp90 in small peak body also in the trend risen, but 40 DEG C when reach maximum expression quantity.This in which kind of outdoor temperature decentralization for flying Chouioia cunea chalcid fly prevention fall webworms tool later It is significant.

Specific primer provided by the invention quickly detects the method and the prior art that Zhou Shi nibbles chalcid fly Hsp90 gene expressions The advantageous effect compared is：

（1）The real-time fluorescence RT-PCR technology that the present invention uses is compared with Standard PCR, and real-time fluorescence PCR technology is by certainly Dynamicization instrument collects fluorescence signal, avoids the subjectivity that naked eyes judge, can further improve sensitivity；Real-time fluorescence PCR skill The totally-enclosed reaction of art post-processes without PCR, avoids polluting, ensure that the reproducibility and reliability of result.Real-time fluorescence RT-PCR Technology also eliminates the follow-up tedious steps such as electrophoresis, the quantitative scanning in Standard PCR, substantially reduces experimental period.

（2）The variation of Hsp90 gene expressions under the technique study different temperatures by RT-qPCR, is as a result shown in not The expression that synthermal condition Xia Zhoushi nibbles the Hsp90 genes in chalcid fly body is in a dynamic process, high temperature and low temperature Stress all makes Zhou Shi nibble in chalcid fly body Hsp90 there are one high expression quantity, in 40 DEG C and -7 DEG C internal expression quantity maximums.

（3）Therefore the present invention proposes that having studied Zhou Shi nibbles the variation that chalcid fly adapts to external environment, avoids being forced the factor It to the protection mechanism of the injury of itself, is of great importance for prevention fall webworms, one is provided for green prevention forestry pest A new thinking and technology.

Zhou Shi nibbles chalcid fly heat shock protein Hsp90 genes, the sequence table 1 of cDNA sequence and coded amino acid, table, shown in 2.

Table 1

Table 2

MPADVAMDSTEVETFAFQAEIAQLMSLIINTFYSNKEIFIRELISNSSDALDKIRYESLTDPSKLDSCKELLIKMIP NKNDRTLTFIDSGIGMTKADLVNNLGTIAKSGTKAFMEALQAGADISMIGQFGVGFYSAYLVADKVTVVSKHNDDEQ YIWESSAGGSFTVRPDKGEPLGRGTKIILHIKEDQAEYLEESKIKEIVKKHSQFIGYPIKLVLEKERDKELSEDEEE DDKKDEDKEEDTEKPKIEDVGEDEDEEKSKEEKKKKKKTVKEKYTDEEELNKTKPIWTRNPDDITHEEYGEFYKSLT NDWEDHLAVKHFSVEGQLEFRALLFIPRRAPFDLFENKKRKNNIKLYVRRVFIMDNCEDLIPEYLNFIRGVVDSEDL PLNISREMLQQNKILKVIRKNLVKKCLELFEELAEDKENYKKCYEQFSKNLKLGIHEDSTNRKKLSELLRYHTSASG DEQCSLKDYVGRMKENQKHIYYITGESKDQVANSAFVERVRKRGFEVVYMTEPIDEYVVQQLKEFDGKQLVSVTKEG LELPVDEDEKKKMEEDKTKFENLCKVMKDILDKRVEKVVVSNRLVDSPCCIVTSQYGWTANMERIMKAQALRDTSTM GYMAAKKHLEINPDHPIMDNLRLKAEADKHDKSVKDLVMLLFETALLSSGFSLEDPGVHASRIYRMIKLGLGLDDEE MAVEEEKTENEVPPLEGDAEEASRMEEVD*。

Description of the drawings

Fig. 1 nibbles chalcid fly Hsp90 and expressed under same time different temperatures stress for Zhou Shi to be changed.

Specific embodiment

Illustrate the present invention with reference to embodiment, the scheme of embodiment described here does not limit the present invention, this field it is special Industry personnel spirit according to the invention can make improvements and change, and the such modifications and variations are regarded as at this In the range of invention, the scope of the present invention and essence are defined by the claims；Wherein agents useful for same is commercially available.In the present invention The methods of extraction of described extraction Chouioia cunea total serum IgE, the synthesis of cDNA, real time fluorescent quantitative RT-qPCR is all The technology of this field maturation, kit TransScript RT, TransStart Top Green qPCR SuperMix etc. It can be bought from manufacturer.

Embodiment 1：

Obtain Chouioia cunea Hsp90 gene orders

Experiment material is derived from the Chouioia cunea of indoor secondary culture（Condition of culture：In growth cabinet（PQX- 350H）In, 25 DEG C of temperature, relative humidity 60% ~ 80%, unglazed dark）.The female Zhou Shi after sprouting wings in 24 h is taken to nibble chalcid fly, in Feeler is cut under anatomical lens, is dipped in RNAlater immediately（Ambion companies, AM7020）In；Every white moth Zhou Shi of 200 females is nibbled The feeler of chalcid fly is stored in a 1.5 mL centrifuge tubes, collects 8 pipe samples, and the Cord blood at -20 DEG C, sample preparation altogether Hua Da Gene science Services Co., Ltd is sent after the completion（http://www.genomics.cn/index）Carry out transcript profile sequencing.

Chalcid fly feeler sample is nibbled to Zhou Shi using high-flux sequence platform Illumina HiSeq 2000 and carries out transcript profile Sequencing is spliced into unigene, in conjunction with biological information after being clustered using Trinity softwares to each reading sequence fragment The judgement that software carries out target sequence cDNA overall lengths is learned, the retrieval of Blast homologous sequences is carried out and adds their confirmation, obtain Hsp90 genes The sequence of cDNA sequence and coded amino acid.

Embodiment 2：

The extraction of total serum IgE

It chooses 30 or so Chouioia cuneas and carries out treatment of different temperature, high-temperature process（1h）For 28 DEG C, 32 DEG C, 36 DEG C, 40 DEG C and 42 DEG C；Low-temperature treatment（1h）It is 11 DEG C, 6 DEG C, 1 DEG C, -3 DEG C, -7 DEG C.Product is used or is preserved immediately after processing In -20 DEG C of freezings.

（1）30 or so Chouioia cuneas is taken to be placed in the sterile centrifugation tube of 1.5mL, pour into liquid nitrogen and rapidly with grinding Frotton is fully ground.600 μ L Trizon solution are added in into centrifuge tube, stand 5min.

（2）200 μ L chloroforms are added in into centrifuge tube, acutely vibrate 15s, stand 3min.

（3）12000rpm centrifuges 15min at 4 DEG C, and upper strata colourless aqueous phase is transferred in new sterile centrifugation tube, adds in 400 μ L Isopropanol places 20min, 12000rpm centrifugations 10min at -20 DEG C.

（4）It is careful to outwell liquid in pipe, retain precipitation, add in 600 μ L, 75% ethyl alcohol washing precipitation.At 4 DEG C 7500rpm from Heart 5min repeats an ethyl alcohol washing precipitation operation.

（5）Carefully discard supernatant liquid, drying at room temperature 2min.RNA precipitate is dissolved in 20 μ LRelution Buffer, it must It can 55 DEG C of -60 DEG C of water-bath 10min when wanting.

（6）Gained precipitation is that white moth Zhou Shi nibbles small peak total serum IgE, and product uses immediately or freezen protective is in -70 DEG C.

Embodiment 3：

The synthesis of cDNA

Using Chouioia cunea total serum IgE described in embodiment 2 as template.The 0.2mL centrifuge tubes of a disinfection are taken, are added in Following reaction system（20 μ L systems)：5 μ L, or Random Primer of Total RNA（N9）（0.1μg/μL）1 μ L, 2 × TS 10 1 μ L, RNase-free Water of μ L, TransScript RT/RI Enzyme Mix of Reaction Mix, 3 μ L are mixed It is even.

Reaction condition keeps the temperature 10 minutes for 25 DEG C of water-baths, and 42 DEG C of water-baths keep the temperature 30 minutes, and 85 DEG C are heated at high temperature 5 minutes, with The TransScript retained in centrifuge tube RT is made to lose activity.Product immediately using or be stored in -20 DEG C of freezings.

Embodiment 4：

Quantitative fluorescent PCR reaction system and condition

（1）Design of primers：

The full length sequence of chalcid fly Hsp90 genes is nibbled according to the Zhou Shi, is suitable for using 5 Software for Design of Primer real-time The specific primer of fluorescent PCR detection, primer sequence are as follows：

Hsp90-F：5'-TAACGATGATGAACAATA-3'

Hsp90-R：5'- AATAGGATAGCCAATGAA-3'

PCR products estimated length is 192 bp

Meanwhile the cds sequence designs of chalcid fly GADPH genes are nibbled for real-time according to the Zhou Shi that transcript profile lane database provides The primer of fluorescent PCR internal reference, primer sequence are as follows：

GADPH-F：5'-GAGGTGGTAGAGCCGCTTCC-3'

GADPH-R：5'-TTTAGCTTTGATCGCGTCGT-3'

PCR products estimated length is 154 bp

（2）Real-time fluorescence PCR：RT-PCR analyses are carried out with SYBR Green chimeric fluorescents method.Use CF96X PCR instruments （Bio-Rad）Carry out PCR reactions.Using the cDNA synthesized in above-described embodiment 3 as template, using Hsp90-F and Hsp90-R, GADPH-F and GADPH-R is specific primer, carries out real-time fluorescent PCR amplification reaction, and each sample sets 3 parallel pipes, is expanded The Ct values of 3 parallel pipes of gained afterwards（Fluorescence signal in i.e. each reaction tube reaches the reaction undergone during the thresholding of setting and follows Number of rings）It is averaged.

The setting of real-time fluorescence PCR amplification system is as follows：

The setting of real-time fluorescence PCR Amplification is as follows：

50℃ 120s ；

95℃ 120s；95℃1s ；60 DEG C of 30s (40 cycles)；

（3）Hsp90 genes relative expression quantity calculates Chouioia cunea at different temperatures：

When target gene is close with the amplification efficiency of reference gene, using 2^{Δ Δ CT}Method calculates Hsp90 bases at each temperature Because of the mRNA relative expression quantities relative to reference gene, one-way analysis of variance, Duncan are carried out with 19.0 softwares of SPSS Family name's duncan's new multiple range method examines the significance of difference between different disposal.

Fig. 1 is the relative expression quantity that different temperatures Xia Zhoushi nibbles chalcid fly Hsp90 genes.Either high temperature is also as seen from the figure It is the expression that low temperature can influence the Hsp90 that Zhou Shi is nibbled in chalcid fly body.It is continuous with temperature in the case of low temperature stress It reduces, Zhou Shi nibbles the relative expression quantity of the Hsp90 in chalcid fly body in the trend risen, and reaches maximum expression at -7 DEG C Amount；In the case of high temperature stress, with the continuous raising of temperature, Zhou Shi nibbles the relative expression quantity of the Hsp90 in small peak body In the trend of rising, but reach maximum expression quantity at 40 DEG C.This is for later in which kind of outdoor temperature decentralization style of calligraphy characterized by hollow strokes moth Zhou Shi Chalcid fly prevention fall webworms are nibbled to be of great significance.

SEQUENCE LISTING

<110>Tianjin Normal University

<120>The method that chalcid fly Hsp90 gene expressions are nibbled using fluorescent RT-PCR technology detection Zhou Shi

<160> 6

<170> PatentIn version 3.5

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<212> DNA

<213>Artificial sequence

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taacgatgat gaacaata 18

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gaggtggtag agccgcttcc 20

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<212> DNA

<213>Artificial sequence

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tttagctttg atcgcgtcgt 20

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atgccggctg acgttgcgat ggatagcacc gaagttgaga cttttgcttt tcaagctgaa 60

attgctcagc ttatgagcct tattatcaac accttctact ccaacaaaga aattttcatt 120

cgagaattga tttccaactc atctgatgcc ttggataaaa ttcgttatga atctctcact 180

gatccatcaa agcttgactc ctgcaaagag ctgttgatca aaatgatccc caacaaaaat 240

gaccgaacac tcaccttcat tgactctggt attggtatga caaaagctga cctggtcaac 300

aacttgggta caattgcaaa atcaggcaca aaagctttca tggaagctct gcaggctgga 360

gctgatatat ctatgattgg ccagtttggc gttggtttct actctgctta ccttgttgcc 420

gacaaagtca ctgttgtctc gaaacataac gatgatgaac aatacatctg ggaatcatcg 480

gctgggggat ccttcacggt acgaccagac aagggtgaac ctcttggacg tggtacaaaa 540

atcatattgc acattaagga agatcaggct gaatatctag aagaatcaaa gatcaaggag 600

attgtcaaaa aacactccca gttcattggc tatcctatta aactggttct tgagaaagaa 660

agagataagg aactgagcga ggatgaagaa gaggacgata agaaagatga ggataaggaa 720

gaggatacgg agaagcccaa aattgaagat gttggtgagg atgaagatga agaaaaatct 780

aaagaagaaa agaagaaaaa gaagaagact gtgaaagaga aatacacaga tgaagaggaa 840

cttaacaaaa ccaagcccat ctggaccaga aaccctgatg acatcacaca tgaagaatat 900

ggtgaattct acaagagctt gacaaatgac tgggaagacc atcttgctgt caaacacttt 960

tcagttgagg gtcagcttga gttccgtgct cttcttttca tccctcgccg ggcacctttt 1020

gatctatttg aaaacaagaa gagaaagaac aatatcaaac tctacgtgag aagggtcttc 1080

attatggaca actgtgagga ccttatccca gagtatttga acttcatccg tggtgtggta 1140

gatagcgaag atcttccact taacatttct cgcgagatgc tccagcaaaa taagattctc 1200

aaagtcatca ggaagaatct tgtcaaaaaa tgcttggaac tctttgaaga gctagctgaa 1260

gacaaagaaa actacaagaa atgctacgag caatttagca agaaccttaa gcttggtatc 1320

catgaagata gcaccaatag aaagaagctc tctgaacttc tgcgttatca cacatctgct 1380

tctggcgatg aacagtgctc tctgaaagac tatgttggac gtatgaaaga aaatcagaaa 1440

cacatttatt acatcactgg cgagagcaaa gatcaggtag cgaacagcgc ctttgtcgag 1500

agggtgagga agcgtggctt cgaggttgtt tacatgacag agcccattga tgagtatgta 1560

gtccaacagc tcaaggaatt tgatggcaaa cagctagtct ctgtcacaaa ggagggtctt 1620

gagctgccag tagacgaaga tgaaaagaag aaaatggagg aagacaaaac caagtttgaa 1680

aacctctgca aagtcatgaa ggacatcctt gacaagagag tagagaaggt agttgtatcc 1740

aaccggttgg ttgattctcc atgctgcatc gtcacgtcgc aatatggttg gaccgccaat 1800

atggaaagaa tcatgaaagc tcaagctctc agagacacct ccactatggg ctacatggct 1860

gcaaagaaac acctggaaat caatccagac caccccatca tggacaatct tcgactaaag 1920

gccgaggctg acaaacatga caaatctgtc aaggacttgg tcatgctgct ctttgagact 1980

gctttgctct cgtccggttt ctccttggaa gatccaggtg tacatgcatc cagaatctac 2040

aggatgatca aattgggtct tggcctcgac gacgaggaaa tggccgtaga agaagagaaa 2100

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gtcgactaa 2169

<210> 6

<211> 722

<212> PRT

<213>Zhou Shi nibbles the amino acid sequence of chalcid fly Hsp90 codings

<400> 6

Met Pro Ala Asp Val Ala Met Asp Ser Thr Glu Val Glu Thr Phe Ala

1 5 10 15

Phe Gln Ala Glu Ile Ala Gln Leu Met Ser Leu Ile Ile Asn Thr Phe

20 25 30

Tyr Ser Asn Lys Glu Ile Phe Ile Arg Glu Leu Ile Ser Asn Ser Ser

35 40 45

Asp Ala Leu Asp Lys Ile Arg Tyr Glu Ser Leu Thr Asp Pro Ser Lys

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Leu Asp Ser Cys Lys Glu Leu Leu Ile Lys Met Ile Pro Asn Lys Asn

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Asp Arg Thr Leu Thr Phe Ile Asp Ser Gly Ile Gly Met Thr Lys Ala

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Asp Leu Val Asn Asn Leu Gly Thr Ile Ala Lys Ser Gly Thr Lys Ala

100 105 110

Phe Met Glu Ala Leu Gln Ala Gly Ala Asp Ile Ser Met Ile Gly Gln

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Phe Gly Val Gly Phe Tyr Ser Ala Tyr Leu Val Ala Asp Lys Val Thr

130 135 140

Val Val Ser Lys His Asn Asp Asp Glu Gln Tyr Ile Trp Glu Ser Ser

145 150 155 160

Ala Gly Gly Ser Phe Thr Val Arg Pro Asp Lys Gly Glu Pro Leu Gly

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Arg Gly Thr Lys Ile Ile Leu His Ile Lys Glu Asp Gln Ala Glu Tyr

180 185 190

Leu Glu Glu Ser Lys Ile Lys Glu Ile Val Lys Lys His Ser Gln Phe

195 200 205

Ile Gly Tyr Pro Ile Lys Leu Val Leu Glu Lys Glu Arg Asp Lys Glu

210 215 220

Leu Ser Glu Asp Glu Glu Glu Asp Asp Lys Lys Asp Glu Asp Lys Glu

225 230 235 240

Glu Asp Thr Glu Lys Pro Lys Ile Glu Asp Val Gly Glu Asp Glu Asp

245 250 255

Glu Glu Lys Ser Lys Glu Glu Lys Lys Lys Lys Lys Lys Thr Val Lys

260 265 270

Glu Lys Tyr Thr Asp Glu Glu Glu Leu Asn Lys Thr Lys Pro Ile Trp

275 280 285

Thr Arg Asn Pro Asp Asp Ile Thr His Glu Glu Tyr Gly Glu Phe Tyr

290 295 300

Lys Ser Leu Thr Asn Asp Trp Glu Asp His Leu Ala Val Lys His Phe

305 310 315 320

Ser Val Glu Gly Gln Leu Glu Phe Arg Ala Leu Leu Phe Ile Pro Arg

325 330 335

Arg Ala Pro Phe Asp Leu Phe Glu Asn Lys Lys Arg Lys Asn Asn Ile

340 345 350

Lys Leu Tyr Val Arg Arg Val Phe Ile Met Asp Asn Cys Glu Asp Leu

355 360 365

Ile Pro Glu Tyr Leu Asn Phe Ile Arg Gly Val Val Asp Ser Glu Asp

370 375 380

Leu Pro Leu Asn Ile Ser Arg Glu Met Leu Gln Gln Asn Lys Ile Leu

385 390 395 400

Lys Val Ile Arg Lys Asn Leu Val Lys Lys Cys Leu Glu Leu Phe Glu

405 410 415

Glu Leu Ala Glu Asp Lys Glu Asn Tyr Lys Lys Cys Tyr Glu Gln Phe

420 425 430

Ser Lys Asn Leu Lys Leu Gly Ile His Glu Asp Ser Thr Asn Arg Lys

435 440 445

Lys Leu Ser Glu Leu Leu Arg Tyr His Thr Ser Ala Ser Gly Asp Glu

450 455 460

Gln Cys Ser Leu Lys Asp Tyr Val Gly Arg Met Lys Glu Asn Gln Lys

465 470 475 480

His Ile Tyr Tyr Ile Thr Gly Glu Ser Lys Asp Gln Val Ala Asn Ser

485 490 495

Ala Phe Val Glu Arg Val Arg Lys Arg Gly Phe Glu Val Val Tyr Met

500 505 510

Thr Glu Pro Ile Asp Glu Tyr Val Val Gln Gln Leu Lys Glu Phe Asp

515 520 525

Gly Lys Gln Leu Val Ser Val Thr Lys Glu Gly Leu Glu Leu Pro Val

530 535 540

Asp Glu Asp Glu Lys Lys Lys Met Glu Glu Asp Lys Thr Lys Phe Glu

545 550 555 560

Asn Leu Cys Lys Val Met Lys Asp Ile Leu Asp Lys Arg Val Glu Lys

565 570 575

Val Val Val Ser Asn Arg Leu Val Asp Ser Pro Cys Cys Ile Val Thr

580 585 590

Ser Gln Tyr Gly Trp Thr Ala Asn Met Glu Arg Ile Met Lys Ala Gln

595 600 605

Ala Leu Arg Asp Thr Ser Thr Met Gly Tyr Met Ala Ala Lys Lys His

610 615 620

Leu Glu Ile Asn Pro Asp His Pro Ile Met Asp Asn Leu Arg Leu Lys

625 630 635 640

Ala Glu Ala Asp Lys His Asp Lys Ser Val Lys Asp Leu Val Met Leu

645 650 655

Leu Phe Glu Thr Ala Leu Leu Ser Ser Gly Phe Ser Leu Glu Asp Pro

660 665 670

Gly Val His Ala Ser Arg Ile Tyr Arg Met Ile Lys Leu Gly Leu Gly

675 680 685

Leu Asp Asp Glu Glu Met Ala Val Glu Glu Glu Lys Thr Glu Asn Glu

690 695 700

Val Pro Pro Leu Glu Gly Asp Ala Glu Glu Ala Ser Arg Met Glu Glu

705 710 715 720

Val Asp

Claims

1. the specific primer that chalcid fly Hsp90 gene expressions are nibbled using fluorescent RT-PCR technology detection Zhou Shi is preparing detection Zhou Shi Nibble the application in terms of chalcid fly adapts to external environment variation kit；The wherein described specific primer it be by meeting fluorescence RT- The specific upstream and downstream primer of PCR reaction characteristics：

Hsp90-F：5'-TAACGATGATGAACAATA -3'; Hsp90-R

5'-AATAGGATAGCCAATGAA-3' is formed.