CN105420244B - Zhou Shi nibbles cDNA full length sequences and the application of chalcid fly heat shock protein Hsp90 - Google Patents

Abstract

The invention discloses cDNA full length sequences and application that Zhou Shi nibbles chalcid fly heat shock protein Hsp90,2169 bp of the sequence contains complete open reading frame, encodes 722 amino acid.By real time fluorescence quantifying PCR method, the variation of Chouioia cunea Hsp90 gene expressions under different temperatures is studied, the results showed that, high temperature and low temperature stress can all induce Zhou Shi to nibble chalcid fly Hsp90 expression quantity to increase, and at 40 DEG C and -7 DEG C, expression quantity is maximum.The present invention can nibble chalcid fly for scientific utilization natural enemy insect Zhou Shi and provide molecular basis.

Description

Zhou Shi nibbles cDNA full length sequences and the application of chalcid fly heat shock protein Hsp90

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 invention belongs to field of molecular biotechnology, research Zhou Shi nibbles the cDNA full length sequences of chalcid fly heat shock protein Hsp90 The variation expressed with Hsp90 under different temperatures.

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 biology in recent years 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 environmental factor of organism existence, only in certain temperature Organism could carry out material energy metabolism to maintain normal physiological activity in range, and the tolerance range beyond temperature can all make life The growth and development of object is stagnated even dead.In general organism by high temperature or low temperature stimulation after, 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 set more preferably to survive, it is believed that it exists adapts to environment important role for organism.

Zhou Shi nibbles chalcid flyChouioia cuneaYang is the important invasive species fall webworms in ChinaHyphantria cunea（Drury）Important parasite, the long 1.1-1.5 mm of body, cluster endoparasitism is to inhibit beautiful in fall webworms pupa The Natural Enemies factor of the white moth of state, the harm to controlling fall webworms play an important role.In addition to parasitizing fall webworms, may be used also Parasitize the Lasiocampidae of Lepidoptera, Lymantriidae, Notodontidae, Geometridae, diamond-back moth section and Diptera Nuscidae, Larvaevoridae and elytrum Purpose Chrysomelidae and ladybirds etc..

It is discharged by fieldC. cuneaIt can achieve the purpose that effectively to prevent fall webworms.However, in the field environment,C. cuneaVigor 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.The present invention passes through transcription Group sequencing a, it was recently reported that Zhou Shi nibbles the cDNA full length sequences of chalcid fly heat shock protein Hsp90, which can be further investigation Zhou Shi The temperature tolerance mechanism for nibbling chalcid fly lays the foundation.By real time fluorescence quantifying PCR method, white moth Zhou Shi under different temperatures is studied The variation of chalcid fly Hsp90 gene expressions is nibbled, result of study can nibble chalcid fly for scientific utilization natural enemy insect Zhou Shi and provide fundamental basis.

Invention content

The purpose of the present invention is report one Zhou Shi nibble chalcid fly heat shock protein gene Hsp90 genes cDNA complete sequences and its For the urgent influence of temperature association.

The technical solution adopted by the present invention is that acquisition Zhou Shi is sequenced by transcript profile to nibble chalcid fly heat shock protein gene Hsp90 bases The complete sequence of cause, the variation of Hsp90 gene expressions under the technique study different temperatures by RT-qPCR.

The invention discloses following technology contents thus：

Zhou Shi nibbles the nucleotide sequence of chalcid fly heat shock protein Hsp90,2169 bp of the sequence, is read containing complete opening Frame has nucleotide sequence shown in SEQ ID NO.1.Corresponding amino acid sequence is as shown in SEQ ID NO.2；The sequence 722 amino acid are encoded, molecular weight is 83.3 KDa, isoelectric point 5.01.

The nucleotides sequence that chalcid fly heat shock protein Hsp90 is nibbled the present invention further discloses Zhou Shi is listed in for detecting prevention U.S. The application of the white moth disease aspect of state.The prevention fall webworms refer to：Hsp90 gene expressions are regulated and controled by temperature, pass through detection The expression of Hsp90 genes can learn the tolerance situation that Zhou Shi nibbles chalcid fly for temperature stress, and chalcid fly is being nibbled less than Zhou Shi It bears to discharge chalcid fly under limiting temperature, can preferably play the purpose of prevention fall webworms.By real-time fluorescence quantitative PCR side Method studies the variation of Chouioia cunea Hsp90 gene expressions under different temperatures, and result of study can be scientific utilization natural enemy elder brother Worm Zhou Shi nibbles chalcid fly and provides fundamental basis.

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

Sequence table

Zhou Shi disclosed by the invention nibbles the advantages of cDNA full length sequences of chalcid fly heat shock protein Hsp90 and is：

（1）The present invention is the cDNA complete sequences that at home and abroad first public Zhou Shi nibbles chalcid fly Hsp90 genes, which can be The temperature tolerance mechanism that further investigation Zhou Shi nibbles chalcid fly lays the foundation.

（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 so that Zhou Shi is nibbled in chalcid fly body Hsp90 there are one high expression quantity, in 40 DEG C and -7 DEG C internal expression quantity maximums.Therefore originally The it is proposed of invention has studied Zhou Shi and nibbles the variation that chalcid fly adapts to external environment, avoids the guarantor for being forced the injury of factor pair itself Protection mechanism is of great importance for prevention fall webworms, and a new thinking and technology is provided for green prevention forestry pest.

Description of the drawings

Fig. 1 is that Zhou Shi nibbles chalcid fly Hsp90 expression variations under same time different temperatures stress.

Specific implementation mode

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 extraction of the extraction Chouioia cunea total serum IgE, the synthesis of cDNA, the methods of real time fluorescent quantitative RT-qPCR are 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 to each reading sequence fragment using Trinity softwares 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）It takes 30 or so Chouioia cuneas to be placed in the sterile centrifugation tube of 1.5mL, pours into liquid nitrogen and rapidly with grinding Frotton is fully ground.Centrifuge tube is added in 600 μ L Trizon solution, stands 5min.

（2）Centrifuge tube is added in 200 μ L chloroforms, acutely vibrates 15s, stands 3min.

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

（4）It is careful to outwell liquid in pipe, retain precipitation, 600 μ L, 75% ethyl alcohol washing precipitation is added.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 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 is that 25 DEG C of water-baths keep the temperature 10 minutes, 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 RT retained in centrifuge tube are 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：According to the full length sequence of gained Hsp90 genes, it is suitable for using 5 Software for Design of Primer real When fluorescent PCR detection specific primer, primer sequence is as follows：

（2）Real-time fluorescence PCR：RT-PCR analyses are carried out with SYBR Green chimeric fluorescent methods.Use CF96X PCR instruments （Bio-Rad）Carry out PCR reactions.Using the cDNA synthesized in above-described embodiment 3 as template, use is above-mentioned（1）In it is designed special Property primer, carry out real-time fluorescent PCR amplification reaction, each sample sets 3 parallel pipes, the Ct values of 3 parallel pipes of gained after amplification （Fluorescence signal in i.e. each reaction tube reaches the reaction cycle number undergone when the thresholding of setting）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)；

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^-ΔΔCTMethod calculates Hsp 90 at each temperature The mRNA relative expression quantities of gene carry out one-way analysis of variance, DuncanShi duncan's new multiple range methods with 19. 0 softwares of SPSS The significance of difference between inspection 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>Zhou Shi nibbles cDNA full length sequences and the application of chalcid fly heat shock protein Hsp90

<160> 2

<170> PatentIn version 3.5

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<211> 2169

<212> DNA

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gagctgccag tagacgaaga tgaaaagaag aaaatggagg aagacaaaac caagtttgaa 1680

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

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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

85 90 95

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

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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

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

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

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

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

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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 (2)

1. a kind of coding Zhou Shi nibbles the nucleic acid of chalcid fly heat shock protein Hsp90,2169 bp of the sequence, for SEQ ID NO.1 institutes Show, contain complete open reading frame, encodes 722 amino acid；The corresponding amino acid sequence such as SEQ ID of wherein described nucleic acid Shown in NO.2；722 amino acid of the sequential coding, molecular weight are 83.3 KDa, isoelectric point 5.01.

2. a kind of coding Zhou Shi as described in claim 1 nibbles the detection reagent of the nucleic acid of chalcid fly heat shock protein Hsp90 for examining Survey the application in terms of prevention fall webworms；The wherein described prevention fall webworms refer to：Hsp90 gene expressions are regulated and controled by temperature, By detecting the expression of Hsp90 genes, the tolerance situation that Zhou Shi nibbles chalcid fly for temperature stress can be learnt, less than week Family name nibbles chalcid fly and bears to discharge chalcid fly under limiting temperature, can preferably play the purpose of prevention fall webworms.